m-

- : -
FAO FOOD AND NUTRITION PAPER 14/7

manuals
Of
food quality control

7. food analysis:
general techniques,
additives,
contaminants,
and composition

prepared by fao with the support of the

swedish international development authority (sida)

FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS

Rome, 1986
 The designations employed and the presentation
of material in this publication do not imply the
expression of any opinion whatsoever on the
part of the Food and Agriculture Organization
of the United Nations concerning the legal
status of any c o u n t r y , territory, city or area or
of its authorities, or concerning the delimitation
of its frontiers or boundaries.

M-87
ISBN 92-5-102399-9

A l l rights reserved. No part of this publication may be reproduced,

stored in a retrieval system, or transmitted in any f o r m or by any means,
electronic, mechanical, photocopying or otherwise, w i t h o u t the prior
permission of the copyright owner. Applications for such permission,
w i t h a statement of the purpose and extent of the reproduction, should
be addressed t o the Director, Publications Division, Food and Agriculture
Organization of the United Nations, Via delle Terme di Caracalla, 00100
Rome, Italy.

© FAO 1986
 FOREWORD

The control of food safety and quality Is an Integral part of n a t i o n a l

programmes for development. National food control systems are designed to
protect the health and welfare of the consumer, to promote the development of
trade in food and food products, and to protect the interests of the fair and
honest food p r o d u c e r , p r o c e s s o r or m a r k e t e r against d i s h o n e s t and unfair
competition. Emphasis is placed on the prevention of chemical and biological
hazards which result from contamination, adulteration or simple mishandling of
foods. Also important are the maintenance of general food quality and the
control of the use of food additives and food processing procedures.

In order to establish a workable food control system, a national government

must :

1. Enact food control legislation.

2. Promulgate regulations to enforce that legislation.

3. Create an agency to conduct the enforcement.

4. Establish food inspection and analysis staff within the agency or

agencies concerned.

5. Provide physical facilities including a food control laboratory.

To assist the national governments of developing countries in this process, FAO,

with the support of the Swedish International Development Authority (SIDA) has
published the series Manuals of Food Quality Control. These are incorporated as
part of the FAO Food and Nutrition Paper Series No. 14, and include:

No. 14/1 The Food Control Laboratory

No. 14/2 Additives, Contaminants, and Techniques (out of print)

No. 14/3 Commodities (out of print)

No . 14/4 Microbiological Analysis

No. 14/5 Food Inspection

No. 14/6 Food for Export

No. 14/7 Food Analysis: General Techniques, Additives,

Contaminants, and Composition

No. 14/8 Food Analysis: Quality, Adulteration, and Tests of Identity

In addition, FAO, WHO and UNEP jointly have published many guidelines and other
documents designed to further assist developing countries in forming adequate
food control systems. These publications include:

Methods of Sampling and Analysis of Contaminants in Food -

A Report of the Second Joint FAO/WHO Expert Consultation,
Rome - 1978

Guidelines for Establishing or Strengthening National Food

C o n t a m i n a t i o n M o n i t o r i n g P r o g r a m m e s - FAO Food Control
Series No. 5 - 1979

iii
 •

Guidelines for the Study of Dietary Intakes of Chemical

Contaminants - WHO Offset Publication No. 87 - 1985

Guide to Codex Recommendations concerning Pesticide

R e s i d u e s , Part 2 - Maximum Limits for Pesticide Residues,
Second Preliminary Issue - Rome - 1985

Recommended Practices for the Prevention of Mycotoxins in

F o o d , Feed and their P r o d u c t s - FAO Food and N u t r i t i o n
Paper No. 10, Rome - 1979

Food Standards, Codes of Practice and Methods of Analysis

Recommended by the Codex Alimentarius Commission - Joint
FAO/WHO Food Standards Programme (several titles)

Food Additive Evaluations and Specifications of Purity and

I d e n t i t y - R e p o r t s and M o n o g r a p h s of the Joint FAO/WHO
Expert Committee on Food Additives (several titles)

The above publications, and others, are available to persons and organizations.
FAO is also interested in receiving comments regarding this volume and
suggestions for future improvement. Please send to:

The Chief
Food Quality and Standards Service
Food Policy and Nutrition Division
Food Agriculture Organization of the
United Nations
Via delle Terme di Caracalla
00100 Rome, Italy

FAO wishes to acknowledge the generous support of the Swedish International

Development Authority (SIDA), in the preparation of this volume, and the efforts
of M r . J. W e a t h e r w a x and M r . P.G. M a r t i n who were r e s p o n s i b l e for the
preparation of the text.

iv
 SPECIAL NOTE

The methods and analytical procedures described in this

Manual are designed to be carried out by properly trained
personnel in a suitably equipped laboratory. In common with
many laboratory procedures, the methods quoted frequently
involve hazardous materials.

For the correct and safe execution of these methods it is

essential that laboratory personnel follow standard safety
procedures for the handling of hazardous materials.

W h i l e the g r e a t e s t care has b e e n e x e r c i s e d in the

preparation of this information, FAO expressly disclaims
a n y l i a b i l i t y to u s e r s of t h e s e p r o c e d u r e s for
consequential damages of any kind arising out of or
connected with their use.

The methods are also not to be regarded as official because

of their inclusion in this Manual. They are simply methods
which have been found by experience to be usable in the
average laboratory.

v
 CONTENTS

1. SCOPE OF THIS MANUAL OF FOOD ANALYSIS 1

2. SAMPLE PREPARATION TECHNIQUES

2.1 Subsampling 2
2.2 Compositing 2
2.3 Chopping, Grinding, Mixing 3
2.4 Freezing and Thawing 3
2.5 Reserve Storage 3

3. HANDLING TECHNIQUES

3.1 Sources of Error 5

3.2 Weighing and Solid Transfers 5
3.3 Liquid and Semi-Solid Transfers 6
3.4 Use of Volumetric Ware 8
3.5 Calibration of Volumetric Ware 9
3.6 Care and Use of Standards 10
3.7 Text References 10

4. GENERAL ANALYSIS TECHNIQUES

4.1 Heating and Drying 12

4.2 Ashing and Digestion 12
4.3 Extraction 15
4.4 Distillation 16
4.5 Titration 19
4.6 Text References 21

5. DETERMINATIVE TECHNIQUES

5.1 Paper Chromatography (PC) 22

5.2 Thin-Layer Chromatography (TLC) 26
5.3 Gas-Liquid Chromatography (GLC) 31
5.4 High Performance Liquid Chromatography (HPLC) 33
5.5 Spectrophotometry 33
5.6 Refractometry 38
5.7 Microscopy 38
5.8 Text References 48

6. FOOD ADDITIVE METHODS

6.1 Preservatives

Discussion 53
Analysis - Benzoic Acid (Quantitative Method) 54
Benzoic Acid (Qualitative Identification) . . . . 57
Parabens (Semi-Quantitative Method) 58
Sorbic Acid 60
Sulphur Dioxide 62
Formaldehyde 65

6.2 Antioxidants

Discussion 67
Analysis - Gallates 68
" BHA 70
- BHT 71
- General TLC Method 73

vii
 6.3 Colours

Discussion 75
Analysis - Water-Soluble (Wool Dye Extraction) 76
Water-Soluble (Polyamide Separation) 77
Water-Soluble (Quinoline Extraction) 80
- Water-Soluble (TLC) 84
- Water-Soluble (PC) 91
Oil-Soluble (isolation and Identification) . . . 93
Colour Identificastion by Spectra 97

6.4 Other Additives

Discussion 115
Analysis - Emulsifiers (TLC Method) 118
Saccharin 120
Artificial Sweeteners 122
Mono-Sodium Glutamate 124

6.5 Text References 126

7. CONTAMINANT RESIDUE METHODS

7.1 Pesticides
Discussion 136
Analysis - Residues in Fruits and Vegetables 139
Residues in Milk and Oilseeds 142
Residues in Dry Low-Fat Foods 145
Residues in Fatty Foods . . 147
Residue Identity Confirmation (using TLC) . . . . 151
Residue Identity Confirmation (using PC) . . . . 154

7.2 Metals
Discussion 157
Dry Ashing 157
Acid Digestion 158
Analysis - Lead (Colourimetric Method) 163
- Lead and Cadmium (Atomic Absorption Method) . . . 167
Arsenic (Colourimetric Method) 169
Mercury (Organic Residues) 172
Mercury (Inorganic Residues) 174
Tin (Colourimetric Method) 178
Copper (Colourimetric Method) 180
Metals Screening Test (Lead, Copper, Zinc
and Others) 182

7.3 Mycotoxins
Discussion 185
Af latoxin 185
Analysis - Aflatoxins (TLC Method) 188
Aflatoxin Bj Confirmation of Identity 192
Aflatoxin (Mini-column Method) 194
- Aflatoxin M (TLC Method) 197

7.4 Text References 200

viii
8. COMPOSITIONAL ANALYSIS METHODS

8.1 Moisture
Discussion 203
Analysis - Moisture (Air Oven M e t h o d ) 205
- Moisture (Toluene Distillation M e t h o d ) 206
Moisture (Vacuum Oven M e t h o d ) 208

8.2 Fat
Discussion 210
Analysis - Crude Fat 212
- Fat (Weibull-Stoldt M e t h o d ) 214
- Total Lipids 216
- Total Fat (Chloroform-Methanol Method) 218

8.3 Protein
Discussion 220
Analysis - Crude Protein (Kjeldahl Macro M e t h o d ) 221
- Crude Protein (Semi-Micro M e t h o d ) 224

8.4 Ash
Discussion 227
Analysis - Total Ash 228

8.5 Other Constituents

Discussion 229
Analysis - Crude Fibre 230
- Salt (Volhard M e t h o d ) 233
- Starch 235

8.6 Text References 236

APPENDIX - Abbreviations used in the Manual 237

ix
 1. SCOPE OF THIS MANUAL OF FOOD ANALYSIS

This Manual includes discussions of analytical techniques as well as analytical

m e t h o d s g e n e r a l l y a p p l i c a b l e to all foods. S p e c i f i c m e t h o d s used o n l y w i t h
c e r t a i n foods are c o v e r e d in the M a n u a l , "Food A n a l y s i s : G e n e r a l Q u a l i t y ,
A d u l t e r a t i o n , Identity."

The techniques discussions cover all physical manipulations during analysis as

well as how to effectively use analytical equipment, instruments and glassware.
P r o p e r t e c h n i q u e is all i m p o r t a n t for a n a l y t i c a l r e s u l t s to be c o n s i s t e n t ,
reproducible and valid. The manipulation techniques shown will keep error at a
m i n i m u m if followed exactly. It is therefore recommended that they be adopted,
even if another technique has been previously used.

The g e n e r a l m e t h o d s are those for a d d i t i v e s , c o n t a m i n a n t r e s i d u e s , and

compositional (proximate) analysis in foods. The following precautions apply
to all of the procedures:

1. Use o n l y d i s t i l l e d w a t e r or the e q u i v a l e n t . (Deionized water is o f t e n

suitable).

2. Use the best grade of reagent chemicals available and purify if necessary.

3. Follow the m e t h o d i n s t r u c t i o n s e x a c t l y , as m a n y of the p r o c e d u r e s are

empirical.

4. Use all laboratory safety procedures and equipment.

The r e f e r e n c e c i t a t i o n s at the end of a p p r o p r i a t e s e c t i o n s g i v e s b o t h the

r e f e r e n c e s listed in the text, as w e l l as s o m e g e n e r a l r e f e r e n c e s w h i c h may
provide background information.

The first e d i t i o n of this M a n u a l w a s w r i t t e n in 197 7 by Mr. P e t e r G. M a r t i n

p r e s e n t l y of L y n e , M a r t i n and R a d f o r d , P u b l i c A n a l y s t s , R e a d i n g , B e r k s h i r e ,
England. The p r e s e n t revised e d i t i o n has b e e n p r e p a r e d w i t h Mr. M a r t i n ' s
support and assistance by Mr. John R. Weatherwax, retired Laboratory Director
for the U n i t e d S t a t e s Food and Drug A d m i n i s t r a t i o n , Los A n g e l e s , C a l i f o r n i a ,
USA.

1
 2. SAMPLE PREPARATION TECHNIQUES

2.1 SUBSAMPLING

The w a y in w h i c h a s a m p l e is taken for a n a l y s i s is the first of a series of

potential sources of error in food analysis. Some liquid foods are reasonably
homogeneous, but solid and semi-solid foods are always heterogeneous. It must
be assumed that the attribute for which the food is being examined, is unevenly
distributed throughout the sample.

The l a b o r a t o r y u s u a l l y has no c o n t r o l over the field s a m p l i n g of the food

p r o d u c t and m u s t a s s u m e that the p o r t i o n r e c e i v e d for a n a l y s i s is
r e p r e s e n t a t i v e of the lot of food s a m p l e d . (Note that field s a m p l i n g is
covered in detail in Chapter 3 of FAO Food and Nutrition Paper 14/5, Manual of
Food Quality Control #5 - Food Inspection). The laboratory sample as received
m a y be b a g g e d , p a c k a g e d , tinned or b o t t l e d and m o s t o f t e n i n c l u d e s m u l t i p l e
units. The l a b o r a t o r y a n a l y s t m u s t first d e c i d e w h a t type of s u b s a m p l i n g
should be d o n e on the s a m p l e r e c e i v e d . The a n a l y s t m a y m a k e the s u b s a m p l e s
r e p r e s e n t a t i v e or s e l e c t i v e d e p e n d i n g on w h a t is to be d e t e r m i n e d in the
analysis. If the analyst merely wishes to prove that something exists without
worrying about its relationship to the whole, then a selective sample is taken.
An example would be spotty decomposition or mould contamination. The sample
could be inspected visually or by odour and only the suspect parts removed for
examination. Most analyses, however, involve comparison to a standard for the
whole, so that representative portions must be taken.

The t a k i n g of a r e p r e s e n t a t i v e s a m p l e is o b v i o u s l y the m o r e d i f f i c u l t of the

two. A liquid food (e.g. m i l k ) g e n e r a l l y need only be w e l l m i x e d or s h a k e n
before subsampling. S e m i - s o l i d foods are t h o s e c o n t a i n i n g a solid m a t e r i a l
plus a large portion of free liquid. Examples include many canned foods. In
the e v e n t that the solid or the liquid are to be a n a l y z e d i n d i v i d u a l l y , they
are separated using a sieve or filter and individually mixed for subsampling.
W h e n b o t h solid and liquid p h a s e s are to be a n a l y z e d as a u n i t , it is o f t e n
advisable to blend or otherwise homogenize the two before subsampling.

Solid samples can be of three general types, namely finely divided (e.g. whole
cereal grains or flour), an aggregate (e.g. solid mixtures such ps sausage), or
a whole unit (e.g. an entire fruit). Finely divided dry products can be mixed
for subsampling using commercial portioning equipment such as a Jones Divider,
or by s p r e a d i n g the s a m p l e over a large s u r f a c e , q u a r t e r i n g w i t h a s t r a i g h t -
edge and mixing opposite quarters. The two mixed halves can be recombined and
the process repeated one or more times to make the subsample portion even more
representative. An aggregate solid sample is probably the most difficult as it
c o n s i s t s of d i f f e r e n t food m a t e r i a l s u s u a l l y w i t h d i f f e r e n t physical
properties. The c h a l l e n g e is to take a s u b s a m p l e h a v i n g a c o m p o s i t i o n
representing an average of the food sampled. This most often requires that the
a g g r e g a t e food be c h o p p e d or g r o u n d b e f o r e m i x i n g and s u b s a m p l i n g . A
d i s c u s s i o n of this can be found in S e c t i o n 2.3. The w h o l e unit s a m p l e can be
m o s t e a s i l y s u b s a m p l e d by taking a r e p r e s e n t a t i v e p o r t i o n of the food. This
could be a q u a r t e r of a f r u i t , a p i e c e of loin from a w h o l e fish or o t h e r
similar sectioning.

In summary, a selective subsample consists only of suspect portions and ignores

the remainder of the sample. A representative subsample, however, must as best
possible represent an average of the whole sample.

2.2 COMPOSITING

A composite is defined as an admixture of two or more portions of a substance.

A c o m p o s i t e is f o r m e d by first s u b s a m p l i n g t w o or m o r e p o r t i o n s of the s a m e
food. An example would be subsampling several individual cans from the same
food lot. These subsamples are then combined and mixed so that a portion taken
of the composite would be representative of the whole. A composite is simply a

2
p h y s i c a l a t t e m p t to a v e r a g e the n o r m a l v a r i a t i o n b e t w e e n i n d i v i d u a l s a m p l e
units or p o r t i o n s . It is m o s t u s e f u l w h e n the a n a l y t i c a l r e s u l t m u s t be
compared to a standard or requirement involving the entire food product.

As the c o m p o s i t e is to be r e p r e s e n t a t i v e , the s u b s a m p l e s of the i n d i v i d u a l

s a m p l e u n i t s m u s t not o n l y be t a k e n c o r r e c t l y (see S e c t i o n 2.1), but m u s t all
be approximately the same size, weight or volume. Given correct subsampling,
the only r e m a i n i n g p r o b l e m is to m a k e the c o m p o s i t e r e a s o n a b l y u n i f o r m and
representative. T h i s m a y i n v o l v e c h o p p i n g and g r i n d i n g as w e l l as p h y s i c a l
mixing.

2.3 CHOPPING, GRINDING, MIXING

The w e l l e q u i p p e d food a n a l y s i s l a b o r a t o r y should h a v e a v a r i e t y of s a m p l e

preparation equipment including mechanical choppers, mincers, grinders,
blenders and a h a m m e r or similar mill.

The type of mechanical processing equipment selected will depend on the food
p r o d u c t to be t r e a t e d . The a n a l y s t m u s t also k e e p in m i n d that m e c h a n i c a l
g r i n d e r s , m i l l s , etc. u s u a l l y g e n e r a t e heat d u r i n g the p r o c e s s i n g . T h i s can
possibly change the sample composition, such as for fatty foods where the heat
may be sufficient to partially melt the fat. In such cases, hand chopping and
mixing may be the best procedure. In other instances, the sample may have to
be frozen before grinding. The analyst must judge the best method for himself,
depending on the kind of food and the substance for which it is to be analyzed.

The moisture content of a food also plays an important role in determining the
food p r o c e s s i n g p r o c e d u r e or e q u i p m e n t to use. Dry foods can g e n e r a l l y be
m i l l e d , w h i l e m o i s t foods can be c h o p p e d , m i n c e d or g r o u n d . V e r y m o i s t and
liquid foods can be blended. The home food processors now available are very
useful for many products.

If no m e c h a n i c a l p r o c e s s i n g e q u i p m e n t is a v a i l a b l e , then of c o u r s e hand
processing must be done. The tools used include knives, graters and choppers.
When a sample is processed by hand, it must be sufficiently finely divided to
permit proper mixing and later subsampling of the mixture.

The analyst must always keep in mind that proper sample preparation is not only
to gain a representative portion for analysis, but is also to prevent change in
the sample which may result in a biased analytical result.

2.4 FREEZING AND THAWING

Freezing is often the only way to prevent a change in a food before analysis or
for r e s e r v e s t o r a g e . Examples include foods for d e c o m p o s i t i o n a n a l y s i s or
foods w h i c h w e r e s a m p l e d w h i l e frozen. It w a s m e n t i o n e d in S e c t i o n 2.3 that
some foods must be frozen before grinding or other processing.

The single m o s t i m p o r t a n t p r o b l e m in h a n d l i n g frozen food s a m p l e s is p r o p e r

thawing before analysis. T h a w i n g m u s t take p l a c e in such a m a n n e r that the
c o m p o s i t i o n of the food r e m a i n s u n c h a n g e d . T h a w i n g should be done s l o w l y
w i t h o u t heat and in a c l o s e d c o n t a i n e r to p r e v e n t m o i s t u r e loss by d r y i n g or
gain by c o n d e n s a t i o n . A n y s e p a r a t e d liquid m u s t be m i x e d b a c k in the t h a w e d
product before subsampling for analysis.

2.5 RESERVE STORAGE

The r e s e r v e p o r t i o n of a food s a m p l e m u s t be m a i n t a i n e d in s t o r a g e so that

there is v e r y l i t t l e or no c h a n g e from the o r i g i n a l a n a l y s i s . I d e a l l y , the
reserve portion analyzed at a future time will give a result equivalent to the
original.

3
T h e s t o r a g e c o n t a i n e r is v e r y i m p o r t a n t . T h e c o n t a i n e r s h o u l d p r o t e c t the
p r o d u c t a g a i n s t m o i s t u r e l o s s or g a i n , and p h y s i c a l d a m a g e s u c h as a t t a c k by
vermin. In some cases the container must be h e r m e t i c a l l y sealed to prevent air
oxidat ion.

For dry storage at room temperature, glass or metal containers with appropriate
c l o s u r e s are u s u a l l y sufficient. R i g i d p l a s t i c c o n t a i n e r s w o u l d be a s e c o n d
choice. Plastic and paper bags are not as useful for dry storage because they
are easily broached by insects or mechanically. The analyst should also keep
in mind that c o m m e r c i a l l y canned foods can deteriorate in storage and should
check such stored items periodically.

Glass, rigid plastic, or thick plastic bags can be used for frozen storage. If
g l a s s or r i g i d p l a s t i c is u s e d , s p a c e m u s t be a l l o w e d in the c o n t a i n e r for
expansion of the ice so as not to break the container. If thin plastic bags or
paper is used, the product m a y lose m o i s t u r e due to drying while frozen.

It is not advisable to store a reserve portion in a refrigerator for any great

l e n g t h of t i m e , u n l e s s the c o n t a i n e r is a i r - t i g h t . E v e n if the s t o r e d food
d o e s not o t h e r w i s e d e t e r i o r a t e , it m a y b e c o m e m o u l d y u n l e s s protected.
Preservatives m a y be added only if they do not affect the desired analysis.

The amount of food product to be kept as a reserve depends on any legal as well
as a n a l y t i c a l r e q u i r e m e n t s . A r e a s o n a b l e r e s e r v e w o u l d be t h a t a m o u n t
n e c e s s a r y to do t h r e e a n a l y s e s . M o r e s h o u l d be k e p t if s t o r a g e s p a c e is
available.

The above reserve storage precautions also apply to overnight or longer storage
while the analysis is being performed.

4
 3. HANDLING TECHNIQUES

3.1 SOURCES OF ERROR

E r r o r is d e f i n e d in one d i c t i o n a r y as a "....deviation f r o m a c c u r a c y or
c o r r e c t n e s s ; a mistake...." M o s t e x p e r i e n c e d a n a l y t i c a l c h e m i s t s w i l l a g r e e
that p o s s i b l e s o u r c e s of e r r o r in a n a l y s i s s e e m to be u n l i m i t e d . On a
practical level, h o w e v e r , m o s t error is controllable.

M a n y t e x t s d i v i d e e r r o r into t w o c a t e g o r i e s , n a m e l y r a n d o m and s y s t e m a t i c .
Random errors are considered to be unsuspected and nonreproducib le errors w h i c h
are b e y o n d c o n t r o l . T h e i r v e r y r a n d o m n e s s , h o w e v e r , p e r m i t s use of the
techniques of statistics and p r o b a b i l i t y to e v a l u a t e a n a l y t i c a l r e s u l t s . On
the o t h e r h a n d , s y s t e m a t i c e r r o r s are t h o s e w h i c h a r i s e f r o m d e f i n i t e (and
often k n o w n ) causes and which are controllable. Systematic errors result in
b i a s in the a n a l y s i s r e s u l t . T h i s b i a s can be p o s i t i v e or n e g a t i v e and w h e n
c o n s i d e r i n g the e f f e c t of a k n o w n e r r o r it is u s e f u l to d e t e r m i n e its
direction. This is especially true when an attempt is made to find the major
source of error w h i c h m a y have caused an incorrect result. For example, if the
r e s u l t is i n c o r r e c t and is u n u s u a l l y h i g h , l o o k for s o u r c e s of c o n t r i b u t i n g
errors which m i g h t be something like a low potency standard w h i c h would give a
calculated high analytical result.

Systematic error can be grouped into five general sources:

1. A n a l y s t - this i n c l u d e s all h u m a n e r r o r such as i n c o r r e c t handling

technique, calculation blunders and simple errors in judgement.

2. Equipment and glassware - includes malfunctions, incorrect calibrations or

u s e , etc.

3. Reagents and standards - includes contamination, low potency,

interferences, etc.

4. Environment - includes the effects of temperature, h u m i d i t y , light, etc.

5. Method - includes specificity, applicability, sensitivity, etc.

N o t e that all of the a b o v e s y s t e m a t i c e r r o r s are c o n t r o l l a b l e . The k e y to

c o n t r o l , of c o u r s e , is the a n a l y s t . A properly trained analyst who thinks
critically about the analysis and who considers and corrects sources of error,
will provide the m o s t accurate analytical result.

The f o l l o w i n g s e c t i o n s (3.2 - 3.6) d i s c u s s s o m e of the p h y s i c a l and handling

aspects of analysis and controlling possible error.

3.2 WEIGHING AND SOLID TRANSFERS

The f i r s t step in m o s t a n a l y s e s is w e i g h i n g the s a m p l e p o r t i o n t a k e n for

analysis. Even liquid foods are g e n e r a l l y w e i g h e d rather than portioned
volumetrically.

T h e r e are t w o c o m m o n w e i g h i n g t e c h n i q u e s used in a l a b o r a t o r y . T h e s e are

d i r e c t and by d i f f e r e n c e . To w e i g h d i r e c t l y , a c o n t a i n e r t a r e w e i g h t is
determined, the sample is added and the container plus sample reweighed. This
g i v e s an a c c u r a t e s a m p l e w e i g h t b u t the m a j o r d r a w b a c k is the n e c e s s i t y to
q u a n t i t a t i v e l y t r a n s f e r the s a m p l e f r o m the tared c o n t a i n e r to a n o t h e r
receptacle. T h i s can be d o n e m o s t e a s i l y by r i n s i n g u s i n g an a p p r o p r i a t e
solvent. T h e t e c h n i q u e to do this u s i n g a g l a s s rod is i l l u s t r a t e d in F i g u r e

5
 Figure 3.1

Tip of the
rod should
extend about
1 inch beyond
the spout

The u s u a l m e t h o d of w e i g h i n g is by d i f f e r e n c e . The s a m p l e and c o n t a i n e r are

first weighed and then are reweighed after removal of the sample. This is the
m o s t convenient procedure and generally does not require quantitative transfer
techniques. If, h o w e v e r , the s a m p l e is to be t r a n s f e r r e d to a n a r r o w - n e c k
container such as a v o l u m e t r i c flask, then quantitative rinsing m u s t be done as
in F i g u r e 3.2.

The weighing bottle and contents are

carefully weighed before and after
removal of the sample

Figure 3.2
Wash the sample into
the flask from only one
side, for otherwise the
sample may clog the
funnel

(b)

3.3 LIQUID A N D SEMI-SOLID TRANSFERS

Most liquid transfers are done using volumetric or other pipettes. Pipettes
are designed either to deliver a set v o l u m e or to contain a volume. Volumetric
p i p e t t e s are d e s i g n e d to d e l i v e r and s h o u l d n e v e r be b l o w n out. The proper
t e c h n i q u e for v o l u m e t r i c p i p e t t e s is i l l u s t r a t e d in F i g u r e 3.3. R e m e m b e r to
n e v e r p i p e t t e by m o u t h , a l w a y s use a s a f e t y b u l b or v a c u u m tube. N o t e that
drainage time for a grade A pipette is marked on its' side.

6
 Fill pipet to
one inch aberre
the graduation Wipe the tip
mark with a clean
cloth Keeping vertical,
drain to gradu-
ation mark.
Throw away the
excess solution

After bulk of liquid

has been discharged,
allow a 10-second
drainage period. Touch
the tip to the side of
Allow the pipet the flask. Wash down
to drain verti- the sides of the flask
cally

The liquid remaining in the

tip of the pipet should not be
blown out. The pipet was
calibrated for this amount to
remain

Figure 3.3

P i p e t t e s d e s i g n e d 'to c o n t a i n ' m u s t h a v e t h e l a s t l i q u i d b l o w n o u t . T h e s e
pipettes are not as accurate as v o l u m e t r i c but can be used w h e r e o n l y m o d e r a t e
v o l u m e a c c u r a c y is required. Often they are c a l i b r a t e d in m u l t i p l e u n i t s such
as a 10 m l pipette divided into 1 m l i n c r e m e n t s .

W h e n a l i q u i d t r a n s f e r is m a d e f r o m an i t e m such as a b e a k e r , then the

technique using a glass rod illustrated in Figure 3.4 should be used.

Figure 3.4

7
F i l t r a t i o n is c o m m o n l y u s e d to s e p a r a t e l i q u i d s f r o m s o l i d s . F l u t e d f i l t e r
p a p e r is o f t e n a v a i l a b l e , b u t if n o t a v a i l a b l e , the t e c h n i q u e to p r e p a r e a
f l u t e d f i l t e r i l l u s t r a t e d in F i g u r e 3.5 s h o u l d be u s e d . T h e f i l t e r p a p e r is
first folded in half and again in quarters, and opened up as shown in (a). The
e d g e 2,1 is t h e n folded on to 2,4 and e d g e 2,3 on to 2,4, p r o d u c i n g , w h e n the
p a p e r is o p e n e d , n e w f o l d s at 2,5 and 2,6. T h e f o l d i n g is c o n t i n u e d , 2,1 to
2,6 and 2,3 to 2,5, thus p r o d u c i n g f o l d s at 2,7 and 2,8 r e s p e c t i v e l y (b);
f u r t h e r 2,3 to 2,6 g i v i n g 2,9, and 2,1 to 2,5 g i v i n g 2,10 (c). The final
operation consists in making a fold in each of the eight segments - b e t w e e n 2,3
and 2,9, b e t w e e n 2,9 and 2,6, etc. - in a d i r e c t i o n o p p o s i t e to the f i r s t
s e r i e s of f o l d s , i.e., the f o l d s are m a d e o u t w a r d s i n s t e a d of i n w a r d s as at
first. The r e s u l t is a fan a r r a n g e m e n t (d), and u p o n o p e n i n g , the f l u t e d
filter paper (e) is obtained.

3.4 OSE OF VOLÜMETRIC WARE

The proper use of volumetric pipettes was discussed in Section 3.3. Burettes
and their use will be discussed in Section 4.5.

The volumetric flask is indispensible for accurate analtyical work, but only if
p r o p e r l y u s e d and c l e a n e d . When filling a volumetric flask, avoid
i n c o r p o r a t i o n of air and r e s u l t a n t f o a m i n g . F i l l u n t i l the b o t t o m of the
m e n i s c u s rests on the calibration line. For m o s t accurate work, r e m e m b e r that
the v o l u m e t r i c f l a s k w a s c a l i b r a t e d at 20°C, so that if it is f i l l e d at a
d i f f e r e n t t e m p e r a t u r e , a t e m p e r a t u r e c o r r e c t i o n m u s t be m a d e . T h i s is
discussed in Section 3.5.

A l l v o l u m e t r i c g l a s s w a r e , such as f l a s k s , p i p e t t e s , etc., m u s t be k e p t
scrupulously clean. Washing with detergent solutions is often not enough, and
in fact can aggravate the problem by leaving detergent residues if not rinsed
properly. One of the b e s t c l e a n i n g m e t h o d s is to t r e a t the v o l u m e t r i c w a r e
w i t h d i c h r o m a t e c l e a n i n g s o l u t i o n , s o a k for s o m e t i m e , r i n s e w i t h d i s t i l l e d
water then alcohol. D i c h r o m a t e c l e a n i n g s o l u t i o n c a n be m a d e by c a r e f u l l y
mixing 800 ml concentrated sulphuric acid with 500 ml of a saturated aqueous
solution of potassium dichromate. The cleaning solution can be reused until it
develops a greenish tinge. At that point it should be discarded.

Another, less rigorous, cleaning solution is 15% trisodium phosphate in water.

A w a r m solution of this (about 70°C) has been shown to be very effective.

8
3.5 CALIBRATION OF V O L U M E T R I C WARE

Most volumetric glassware is calibrated at 20°C. Where the ambient temperature

is higher, such g l a s s w a r e will deliver less than the expected weight. In the
case of w a t e r and d i l u t e a q u e o u s s o l u t i o n s , this a m o u n t s to a b o u t 0.02% per
degree.

Pipettes m a y be re-calibrated by filling with distilled water at the desired

t e m p e r a t u r e , and a l l o w i n g the w a t e r to r u n i n t o a t a r e d w e i g h i n g b o t t l e ,
capping and re-weighing. For example, a 25 ml pipette delivers 24.8266 g water
at 30°C; the density of water at 30°C = 0.987623, therefore v o l u m e delivered =
24.8266/0.987623 = 24.96 ml. If the calibration weighing is repeated three or
m o r e t i m e s , an e s t i m a t e c a n be m a d e of the p i p e t t i n g e r r o r and the v o l u m e
delivered at the new temperature recorded for the pipette to the correct n u m b e r
of significant figures.

Volumetric flasks and other volumetric containers are difficult to recalibrate.

It is usually sufficient to correct the v o l u m e from the temperature of use to
the calibration temperature (20°C). The following table gives the correction
to various observed v o l u m e s of w a t e r , (measured at the designated t e m p e r a t u r e s )
to g i v e the v o l u m e at the s t a n d a r d t e m p e r a t u r e , 20°C. It is a s s u m e d that the
v o l u m e s are measured in volumetric glassware having a coefficient of cubical
expansion of 0.000025 per degree centigrade. The table is applicable to dilute
aqueous solutions having the same coefficient of expansion as water.

R e c a l i b r a t i o n of v o l u m e t r i c w a r e is n o t n e e d e d for m o s t r o u t i n e w o r k and in
fact, is usually only done w h e n absolute exactness is required.

Measurement Rated capacity of glassware in millilitres at 20°C

Temperature

(°C) 2,000 1,000 500 400 300 250 150

(Correction in milliliters to give volume of water at 20°C)

15 + 1.54 +0.77 +0 .38 +0.31 +0.23 +0.19 +0 .12

16 + 1.28 + .64 + .32 + .26 + .19 + .16 + .10
17 + .99 + .50 + .25 + .20 + . 15 + . 12 + .07
18 + .68 + .34 + .17 + .14 + .10 + .08 + .05
19 + .35 + .18 + .09 + .07 + .05 + .04 + .03

21 - .37 - .18 -
.09 - .07 —
.06 _ .05 _ .03
22 - .77 - .38 - . 19 - .15 - . 12 -
. 10 - .06
23 -1.18 - .59 - .30 - .24 - .18 - .15 -
.09
24 -1.61 - .81 - .40 - .32 - .24 - .20 -
. 12
25 -2.07 -1.03 — .52 - .41 - .31 - .26 - .15

26 -2.54 -1.27 -
.64 —
.51 _ .38 _ .32 . 19
27 -3.03 -1.52 - .76 - .61 - .46 - .38 - .23
28 -3.55 -1.77 - .89 - .71 - .53 -
.44 -
.27
29 -4.08 -2.04 - 1 .02 - .82 - .61 - .51 - .31
30 -4.62 -2.31 -1 . 16 .92 .69 .58 — .35

î abo ve t a b l e is u s ed to cor r e c t the vo 1 u m e o f c e r t a i n s t a n d a r d acid and

i so l u t i o n s to 20 °C, m o r e a c c u r a t e r e s u l t s w i l l be o b t a i n e d if the
ical v a l u e s of the a b o v e c o r r e c t i o n s a r e i n c r e a s e d by the p e r c e n t a ges
belo w:

9
 Normality
Solution
N N/2 N/10

Nitric acid 50 25 6
Sulphuric acid 45 25 5
Sodium hydroxide 40 25 5
Potassium hydroxide 40 20 4

3.6 CARE AND USE OF STANDARDS

The term 'standards' includes all reagents and materials used as a reference
and against w h i c h s a m p l e s are m e a s u r e d . There are two broad classes of
standards, n a m e l y p r i m a r y and secondary. A p r i m a r y standard is one w h o s e
composition and purity is established by a recognized national or international
authority. (Examples are the U. S. National Bureau of S t a n d a r d s and the
European C o m m u n i t y Bureau of Reference). P r i m a r y s t a n d a r d s are often very
expensive and/or very difficult to obtain. Therefore, many laboratories make
do with secondary standards and compare these to the primary where possible.
Secondary standards are usually the daily working standards of the laboratory
and m o s t often are the purest form available to the l a b o r a t o r y on a routine
basis.

All standards should be:

1. Stored separate from other reagents and materials.

2. Stored under proper conditions.

3. Used in a manner to prevent contamination.

4. Periodically checked to confirm their stability and purity.

The above would also apply to standard solutions. These r e q u i r e m e n t s are

b a s i c a l l y c o m m o n sense, but unless they are c o m p l i e d w i t h , a standard m a y
quickly lose its validity. Separate storage is needed to control and identify
which materials are standards and which are not, so that everyday reagents are
not a c c i d e n t a l l y used as reference standards. Proper storage is o b v i o u s l y
n e c e s s a r y to prevent early d e t e r i o r a t i o n . Proper use includes using clean
i m p l e m e n t s and never returning unused standard to the container. Periodic
checking is the final and probably m o s t i m p o r t a n t r e q u i r e m e n t . Short lived
organic m a t e r i a l s have to be frequently checked. Even e x t r e m e l y stable
inorganics and metals should be checked on a routine (though infrequent) basis.

3.7 TEXT REFERENCES

None

Further Reading

GARFIELD, F.M. 1984. Quality Assurance Principles for Analytical Laboratories.

AOAC.

G A R F I E L D , F.M., P A L M E R , M. & S C H W A R T Z M A N , G. 1980. Optimising Chemical

Laboratory Performance Through the Application of Quality Assurance Principles.
AOAC.

INHORN, S.L. 1978. Quality A s s u r a n c e Practices for Health Laboratories.

American Public Health Association.

CAULCUTT, R. & BODDY, R. 1983. Statistics for Analytical Chemists. Chapman &
Hall.

10
YOUDEN, W.J. & STEINER, E.H. 1975. The Statistical Manual of the AOAC. AOAC.

1982. Technical Drawings for Glassware. ISO - 6414.

ISO TC48 has published over fifty specifications for laboratory glassware and
related apparatus.

11
 4. GENERAL ANALYSIS TECHNIQUES

4.1 HEATING AND DRYING

Heating devices commonly used in a laboratory include:

Localized heating Overall heating

Hot plates Gravity convection oven

Mantles Mechanical convection oven
Steam baths Vacuum oven
Water baths
Burners
Heating tape

T w o a s p e c t s are i m p o r t a n t w h e n u s i n g a l o c a l i z e d h e a t i n g d e v i c e , spot
overheating and accurate temperature control. All electrical heating devices
are, or can b e , rhe o s t a t i c a 11 y c o n t r o l l e d . T h e r e f o r e , they are u s e f u l over a
broad range from warming reaction vessels to distillations. Baths, however,
are usually limited to evaporation duty as they have no flexibility. Burners
should only be used when nothing else is available (and flammable solvents are
not in use), or when extremely hot local heating is required (such as charring
before ashing).

Ovens are indispensable for uniform sample drying by heat. Some precautions
must be observed, however, to ensure best operation:

1. Do not overload an oven. Oven efficiency decreases dramatically when

it is charged with too many units at one time.

2. Do not try to dry e x t r e m e l y w e t m a t e r i a l s . It is u s u a l l y best to

evaporate on a steam bath first, then use the oven.

3. C h e c k (and r e c o r d ) the oven t e m p e r a t u r e f r e q u e n t l y to e n s u r e it is

operating correctly.

4. Never leave the oven door open for any great length of time. If this
happens, close the door and re-equilibrate the temperature before use.

5. Ensure that the oven conforms to an appropriate performance standard,

e.g. B.S. 2648(1).

The other means of laboratory drying is by use of desiccators. Self-indicating

silica gel is probably the most convenient desiccant. Once pink, it should be
dried in the oven until blue again. Stronger desiccants such as concentrated
sulphuric acid and phosphorus pentoxide may be required on occasion. Magnesium
p e r c h l o r a t e is not only a p o w e r f u l d e s i c c a n t but also a p o w e r f u l o x i d i s i n g
agent and must therefore be used with proper precautions.

D e s i c c a t o r lids w i t h o u t taps m u s t be left s l i g h t l y ajar w h e n hot c r u c i b l e s ,

etc. are put in the d e s i c c a t o r , o t h e r w i s e a p a r t i a l v a c u u m is f o r m e d as the
crucibles cooi. The lid may then become unremovable for many hours. Where the
lid has a tap, this must be opened extremely carefully to prevent dispersal of
the m a t e r i a l to be w e i g h e d . L i g h t and f r i a b l e ash m u s t be in a closed
receptacle to avoid this.

4.2 ASHING AND DIGESTION

Ashing is the process of removing organic matter by heat (usually in a muffle

or o t h e r f u r n a c e ) , to 1 eave a i n o r g a n i c r e s i d u e , T h i s is d o n e e i t h e r to
measure the total inorgan ics or to simplify analysis of trace materials such as
heavy metals.

12
S p e c i f i c ashing p r o c e d u r e s are c o v e r e d within individual analytical methods.
H o w e v e r , t h e r e a r e s o m e g e n e r a l c o m m e n t s and p r e c a u t i o n s :

1. D r y a s h i n g is a p p l i c a b l e to the d e t e r m i n a t i o n of m o s t c o m m o n m e t a l s
in o r g a n i c m a t t e r , e x c e p t i n g v o l a t i l e m e t a l s l i k e m e r c u r y . Substances amenable
to t h i s m e t h o d m u s t b e c h a r r e d s l o w l y a n d t h e c a r b o n o x i d i s e d g e n t l y a n d
completely. L o s s of m e t a l s by v o l a t i l i s a t i o n or by c o m b i n a t i o n w i t h the
m a t e r i a l of t h e c o n t a i n e r m u s t b e a v o i d e d b y w o r k i n g a t t h e l o w e s t p o s s i b l e
temperature. P a r t i c u l a r c a r e m u s t b e e x e r c i s e d w h e n l a r g e a m o u n t s of h a l o g e n s
are p r e s e n t in e i t h e r c o v a l e n t or i o n i c f o r m . It h a s b e e n r e p o r t e d t h a t l o s s e s
of c e r t a i n m e t a l s (e.g. z i n c , t i n or a n t i m o n y ) o c c u r w h e n d r y a s h i n g is c a r r i e d
o u t in the p r e s e n c e of h a l i d e s ; s u c h l o s s e s can b e m i n i m i s e d b y e n s u r i n g t h a t
an a l k a l i n e a s h r e m a i n s , for e x a m p l e b y a d d i n g c h a l k .

2. Dry ashing usually requires little attention. L a r g e r a m o u n t s of

m a t e r i a l can be dealt w i t h m o r e c o n v e n i e n t l y than b y acid d i g e s t i o n by r e p e a t e d
a d d i t i o n of f r e s h m a t e r i a l to a l r e a d y a s h e d m a t e r i a l a n d r e - a s h i n g . Lower
blank values are generally achieved than w i t h acid d i g e s t i o n .

3. T h e d r y a s h i n g p r o c e d u r e is of p a r t i c u l a r a d v a n t a g e w h e n the u s e of
s u l p h u r i c a c i d is o b j e c t i o n a b l e . F o r i n s t a n c e , for t h e d e t e r m i n a t i o n of l e a d
i n m a t e r i a l s c o n t a i n i n g a p p r e c i a b l e q u a n t i t i e s of the a l k a l i n e e a r t h s , w h o s e
s u l p h a t e s occlude lead s u l p h a t e .

4. O n e of the p r o b l e m s w i t h d r y a s h i n g is t h a t it is s o m e t i m e s d i f f i c u l t
to o b t a i n c o m p l e t e e x t r a c t i o n of the m e t a l b e i n g d e t e r m i n e d f r o m s o m e i g n i t e d
residues. E x c e s s i v e h e a t i n g also m a k e s a n u m b e r of m e t a l l i c compounds
i n s o l u b l e (e.g., t h o s e o f t i n ) . S o m e f l o u r p r o d u c t s m a y g i v e a d a r k m e l t in
w h i c h c a r b o n p a r t i c l e s are t r a p p e d and w i l l n o t b u r n .

5. T h e s l o w i g n i t i o n of s o m e o r g a n i c m a t e r i a l s c a n c a u s e t h e e v o l u t i o n
of p o i s o n o u s or n o x i o u s f u m e s , so a l l a s h i n g o p e r a t i o n s m u s t be c a r r i e d o u t in
well ventilated fume hoods.

A s h i n g m a y b e c o n d u c t e d w i t h or w i t h o u t an a s h - a i d , d e p e n d i n g on c i r c u m s t a n c e .
A s h - a i d s i n c l u d e 5 0 % m a g n e s i u m n i t r a t e s o l u t i o n or c o n c e n t r a t e d s u l p h u r i c a c i d .
T h e y are u s e d to p r e v e n t v o l a t i l i z a t i o n of c o n s t i t u e n t s of i n t e r e s t d u r i n g the
ashing process. I n d i v i d u a l m e t h o d s w i l l s t a t e h o w a n d w h e n a s h - a i d s a r e to b e
used .

A c i d d i g e s t i o n is the o t h e r m e a n s c o m m o n l y u s e d to d e s t r o y o r g a n i c m a t t e r in
o r d e r to d e t e r m i n e a n i n o r g a n i c c o n s t i t u e n t . S u l p h u r i c a n d n i t r i c a c i d s a r e
u s u a l l y the d i g e s t i o n a c i d s of c h o i c e , s o m e t i m e s in c o n j u n c t i o n w i t h a
catalyst. I n s p e c i a l c a s e s , p e r c h l o r i c a c i d is a l s o u s e d , b u t is v e r y
d a n g e r o u s to w o r k w i t h and s a f e t y p r e c a u t i o n s m u s t b e f o l l o w e d c l o s e l y (2).

O n e d i f f i c u l t y w i t h a c i d d i g e s t i o n for m e t a l r e s i d u e a n a l y s i s is t h e l i k e l i h o o d
of c o n t a m i n a t i o n . A l l g l a s s w a r e m u s t be p r e v i o u s l y c l e a n e d w i t h s u l p h u r i c and
n i t r i c a c i d s and r i n s e d t h o r o u g h l y w i t h d i s t i l l e d w a t e r b e f o r e u s e . All acids
and o t h e r r e a g e n t s m u s t b e f r e e of the a n a l y t e m e t a l . S o m e of the m o r e c o m m o n l y
u s e d r e a g e n t s a r e n o w a v a i l a b l e in g r a d e s s u i t a b l e f o r f o o d a n a l y s i s . They
s h o u l d n o t b e t r a n s f e r r e d f r o m t h e l e a d - f r e e - g l a s s b o t t l e in w h i c h t h e y a r e
supplied. Even w h e n these reagents are u s e d , reagent blank d e t e r m i n a t i o n s w i l l
be n e c e s s a r y . No s e p a r a t e i n s t r u c t i o n s a r e g i v e n in i n d i v i d u a l m e t h o d s for
r e a g e n t b l a n k s , but the b l a n k s m u s t be p r e p a r e d w i t h the s a m e q u a n t i t i e s of
r e a g e n t s as are u s e d in the t e s t s .

W h e n o r g a n i c m a t t e r is h e a t e d w i t h m i x t u r e s of c o n c e n t r a t e d a c i d s in K j e l d a h l
flasks experience has s h o w n that d e c o m p o s i t i o n w i l l take place m o s t e f f i c i e n t l y
w h e n s o m e m e a n s f o r p a r t i a l r e f l u x o f t h e h o t a c i d s is p r o v i d e d , as b y a n
e x t e n s i o n to t h e n e c k o f t h e f l a s k . W h e n m a n y o x i d a t i o n s a r e i n p r o g r e s s a t
t h e s a m e t i m e , it is a d v i s a b l e to t r a p t h e f u m e s , d i l u t e t h e m w i t h w a t e r a n d
d i s p o s e of t h e m d o w n t h e d r a i n s r a t h e r t h a n i n t o the a t m o s p h e r e . The following

13
description of suitable apparatus is given for guidance only: Kjeldahl flasks
- These should be m a d e of borosilicate glass (100 to 250 m l n o m i n a l capacity)
fitted with an extension to the neck by means of a standard ground joint. The
extension serves to condense fumes into an acid-fume condenser and carries a
tap f u n n e l t h r o u g h w h i c h the r e a g e n t s are i n t r o d u c e d (see F i g u r e 4.1). Each
f l a s k s h o u l d be s u p p o r t e d in a c i r c u l a r h o l e in a c e r a m i c or o t h e r s h e e t , and
the h o l e s h o u l d be of such a d i a m e t e r that the f l a s k r e c e i v e s no d i r e c t h e a t
from the burner above the level of the acid.

Fume condenser

Modified Kjeldahl flask (open type).

Dimensions are for a flask of 150 ml capacity

Figure 4.1

In the Kjeldahl digestion rack and acid-fume condenser (Figure 4.2), a current
of water is kept flowing through the condenser. Removal of acid fumes can also
be a s s i s t e d by c o n n e c t i n g the u p p e r o u t l e t to a w a t e r p u m p . G a s h e a t i n g is
preferable. The flasks, not being rigidly clamped, are easily handled when it
is necessary to deal with vigorous reactions or excessive frothing, by m e a n s of
tongues or suitable finger-guards.

Acid-fume condenser and modified Kjeldahl flask. A. water inlet; B, con-

nection to pump ; C, wide outlet to waste ; D, tap with wide outlet to prevent clogging with
solid matter

Figure 4.2

14
4.3 EXTRACTION

All extractions, whether liquid-liquid or 1iquid-solid, are partitionings of a

material between two phases. In a liquid-liquid extraction, the partitioning
is governed basically by the relative solubility of the extractant in the two
liquids. In a liquid-solid extraction, this is complicated further by possible
physical occlusion of the extractant within some inert solid material.

M o s t l i q u i d - s o l i d e x t r a c t i o n s are e x h a u s t i v e , w h e r e a m a t e r i a l is to be
completely extracted. An example would be fat from a meat sample. Continuous
e x t r a c t o r s such as a S o x h l e t u n i t (see F i g u r e 4.3) can be used. When using a
c o n t i n u o u s e x t r a c t o r , the solid s a m p l e m u s t be f i n e l y d i v i d e d (to p r e v e n t
o c c l u s i o n ) and s u f f i c i e n t e x t r a c t i v e s o l v e n t m u s t be used to p r o v i d e a good
s i p h o n i n g a c t i o n plus e n o u g h in the r e s e r v o i r flask to p r e v e n t going to
dryness. However, if too fine, such as full-cream milk powder, channeling can
occur through the mass and extraction may be incomplete.

T h e r e are c o n t i n u o u s 1 i q u i d - 1 i q u i d e x t r a c t o r s , but the u s u a l l a b o r a t o r y

extractions are done using separatory funnels. As at least one of the liquids
in a separatory funnel is likely to be volatile, there may be internal pressure
build-up during the extraction process. This should be normally vented through
the funnel tip after inversion unless the method specifies the top.

If emulsion formation is a problem, it is often sufficient to gently invert the

funnel several times. Partitioning of the extractant between the two liquids
takes place at the interface and the mixing of the two liquids does not have to
be violent for partitioning to take place.

Condenser

Figure 4.3

Thimble

Soxhlet
extractor

P a r t i o n i n g of a s u b s t a n c e is r a r e l y c o m p l e t e in one e x t r a c t i o n . For this

reason multiple extractions are often necessary for quantitative results. This
can be illustrated by the partitioning of butyric acid between water and ether.
If 4 gm of the acid w e r e d i s s o l v e d in 500 m l of w a t e r and e x t r a c t e d once w i t h
500 m l e t h e r , then the acid w o u l d p a r t i t i o n 3 gm into the e t h e r w i t h 1 gm

15
r e m a i n i n g in the w a t e r (see F i g u r e 4.4). If, h o w e v e r , t w o e x t r a c t i o n s w e r e
done using two 250 m l portions of ether (to total 500 ml), then a total of 3.36
gm of acid is partitioned into the ether (see Figure 4.5).

SINGLE EXTRACTION DOUBLE EXTRACTION

Figure 4.4 Figure 4.5

T h e r e f o r e , by u s i n g the s a m e t o t a l e x t r a c t i o n v o l u m e in a s e r i e s of
e x t r a c t i o n s , r a t h e r t h a n all at o n c e , m o r e q u a n t i t a t i v e results can be
obtained.

If a s u b s t a n c e is n a t u r a l l y a c i d i c or a l k a l i n e , this f e a t u r e can be used to

a f f e c t the d e g r e e of p a r t i t i o n i n g b e t w e e n a w a t e r s o l u t i o n and an o r g a n i c
solvent. T h i s is d o n e by pH c o n t r o l of the w a t e r s o l u t i o n . For e x a m p l e , a
water soluble organic acid can be retained in the water by m a k i n g it alkaline,
w h i l e o t h e r m a t e r i a l s are b e i n g e x t r a c t e d into an o r g a n i c s o l v e n t such as
ether. When desired, the water solution can then be m a d e acidic, freeing the
organic acid to be extracted into the ether. This is the basis for removal of
interfering substances by double extraction.

4.4 DISTILLATION

Another technique for purifying a substance, is distillation. There are three

general types of distillation c o m m o n l y used in a laboratory, simple, steam and
fractionation.

S i m p l e d i s t i l l a t i o n c o n s i s t s of h e a t i n g a l i q u i d s o l u t i o n to a t e m p e r a t u r e
where a portion of the liquid becomes vapor, w h i c h is then condensed on a cold
surface and collected. If the liquid solution is a m i x t u r e , it is important to
r e m e m b e r that the constituents of the vapor formed on heating will depend on
the r e l a t i v e v a p o r p r e s s u r e s of the i n d i v i d u a l l i q u i d m i x t u r e c o n s t i t u e n t s .
For e x a m p l e , if a solid is d i s s o l v e d in a l i q u i d , the l i q u i d c a n u s u a l l y b e
d i s t i l l e d in r e l a t i v e l y p u r e f o r m , l e a v i n g the solid b e h i n d b e c a u s e of its
extremely low vapor pressure. Distillation of m i s c i b l e liquids such as dilute
spirits is another example. Ethanol has a higher vapor pressure than water and
therefore will distill first from a mixture of the two. However, ethanol-water
mixtures containing 95.6% of the alcohol are an example of the phenomena of co-
distillation. T h e y f o r m w h a t is t e r m e d an a z e o t r o p i c m i x t u r e and w i l l c o -
d i s t i l l at the s a m e t e m p e r a t u r e and can t h e r e f o r e not be s e p a r a t e d by s i m p l e
distillation. An azeotrope is a mixture of constant c o m p o s i t i o n , the same in
the v a p o u r as in the l i q u i d p h a s e . H o w e v e r , the c o m p o s i t i o n is p r e s s u r e -
dependent. Dilute ethanol/water solutions cannot give rise to a vapour m o r e
c o n c e n t r a t e d t h a n 95.6% e t h a n o l . H o w e v e r , as d i s t i l l a t i o n p r o c e e d s , the

16
concentration of ethanol in the vapour will exceed that in the liquid until all
the ethanol is removed and the boiling-point of the liquid has risen to that of
pure water.

In the case of two immiscible liquids, they both contribute the vapour pressure
that they would if the other liquid w a s absent. Thus this vapour pressure will
rise to that of the surrounding atmosphere at a lower temperature than would
the v a p o u r p r e s s u r e of e i t h e r l i q u i d a l o n e . T h i s is the b a s i s of the u s e f u l
s e p a r a t i o n t e c h n i q u e of s t e a m d i s t i l l a t i o n , u n d e r w h i c h e v e n s o l i d s s u c h as
b e n z o i c and s o r b i c a c i d s c a n be m a d e s u f f i c i e n t l y v o l a t i l e to d i s t i l . A
typical steam distillation unit is shown in Figure 4.6. It consists of a steam
g e n e r a t o r , s u c h as a l a r g e f l a s k w i t h a w i r e h e a t e r , a s a m p l e f l a s k and a
condenser. The a m o u n t of s t e a m g e n e r a t e d m u s t be c a r e f u l l y c o n t r o l l e d to
p r e v e n t e x c e s s i v e f o a m i n g in the s a m p l e f l a s k . T h e long g l a s s t u b e in the
generator flask serves to equalize minor pressure changes, and should extend
about three feet above the flask.

Figure 4.6

If t h e a m o u n t of a v a i l a b l e s a m p l e is s m a l l , t h e n a s e m i - m i c r o steam
d i s t i l l a t i o n a p p a r a t u s such as a M a r k h a m u n i t can be used (see F i g u r e 4.7).
The sample (in solution) is placed in the inner jacket via the funnel, which is
then s t o p p e r e d and the s t e a m is i n t r o d u c e d to the o u t e r j a c k e t . The steam
passas through the solution and exits into the condenser.

17
Fractional distillation is used usually to separate a liquid m i x t u r e having two
or more substances with close boiling points. The technique can also be used
to prevent loss of partially v o l a t i l e m a t e r i a l s w h i l e e v a p o r a t i n g a s o l v e n t .
An example is the Kuderna-Danish evaporators fitted with a Snyder column, used
in pesticide residue analysis. The important part of a fractional distillation
apparatus is the column. The fractionating c o l u m n is designed to allow vapour
containing a greater concentration of the m o r e volatile constituent to b e c o m e
separated from the next most volatile constituent sufficiently well that the
p u r e s u b s t a n c e d i s t i l s o v e r c o m p l e t e l y at the f i r s t b o i l i n g - p o i n t . The
t e m p e r a t u r e of the l i q u i d b e i n g d i s t i l l e d m u s t t h e n be r a i s e d u n t i l the
boiling-point of the second component is reached. In this w a y a succession of
f r a c t i o n s can be o b t a i n e d , e a c h a m o r e or less p u r e c o m p o n e n t of the i n i t i a l
mixture. Some examples of fractionating columns are given in Figure 4.8.

Figure 4.8

18
4.5 TITRATION

T i t r a t i o n s are among the most f r e q u e n t l y p e r f o r m e d of a n a l y t i c a l t e c h n i q u e s in

a laboratory. C o n s i s t e n t and a c c u r a t e r e s u l t s d e p e n d on p r o p e r c a r e o f t h e
b u r e t t e , c o r r e c t t e c h n i q u e d u r i n g the t i t r a t i o n and a c c u r a t e r e a d i n g of the
burette.

The b u r e t t e can be c l e a n e d u s i n g d i c h r o m a t e c l e a n i n g s o l u t i o n by i n v e r t i n g the

b u r e t t e i n t o a b e a k e r of c l e a n i n g s o l u t i o n and a p p l y i n g a vacuum to the b u r e t t e
t i p to d r a w the s o l u t i o n i n t o the b u r e t t e . U s e the s t o p c o c k to s t o p the
p r o c e s s when the b u r e t t e is f u l l . When c l e a n the b u r e t t e should d e l i v e r a
w a t e r s o l u t i o n w i t h o u t drops a d h e r i n g to the i n n e r s u r f a c e .

Some b u r e t t e s n o w h a v e t e f l o n stopcocks. T h e s e m u s t n o t be g r e a s e d , u n l i k e
ground glass stopcocks which m u s t be p e r i o d i c a l l y c l e a n e d and g r e a s e d as
i l l u s t r a t e d in F i g u r e 4 . 9 .

Remove stopcock

Bad, cloudy
A dark background shows the
transparency of a well-greased
stopcock. The plug should turn
easily. Grease should not clog
slightly- the bore (Q

Figure 4.9

T i t r a t i o n t e c h n i q u e i s s i m p l e and s t r a i g h t f o r w a r d b u t n e e d s p r a c t i c e to
perfect. The s t o p c o c k i s m a n i p u l a t e d w i t h the h a n d a r o u n d the b a r r e l o f the
burette. T h i s p e r m i t s t h e a n a l y s t to k e e p t h e s t o p c o c k s e a t e d by t u g g i n g
gently while turning. The o t h e r hand s w i r l s the r e c e i v i n g f l a s k . T h i s is
p i c t u r e d in F i g u r e 4 . 1 0 .

19
Note that the burette tip m u s t extend into the flask to prevent spattering. If
any d r o p s of t i t r a n t a d h e r e to the flask w a l l , t h e y m u s t be w a s h e d d o w n w i t h
distilled water.

N e a r the end p o i n t , it is o f t e n n e c e s s a r y to s p l i t a d r o p from the burette.

The technique to do this is illustrated in Figure 4.11.

W h e n the e n d p o i n t is r e a c h e d , the b u r e t t e c a n be a c c u r a t e l y r e a d by u s e of a
black strip on a white card as background. This is illustrated in Figure 4.12.

Figure 4.12

20
4.6 TEXT REFERENCES

1. British Standards Institution, 1955. Performance Requirements for

Electrically-heated Laboratory Drying Ovens.

2. Analtyical Methods Committee (U.K.), 1959. Analyst, 84, 214-216.

Further Reading

GROB, R.L. 1977. Modern Practice of Gas Chromatography Wiley.

LITEANU, C. 1980. Statistical Theory & Methodology of Trace Analysis Wiley.

MIKES, 0. 1979. Laboratory Handbook of Chromatographic & Allied Methods

Wiley.

MILLER, J.N. & MILLER, J.C. 1984. Statistics in A n a l y t i c a l Chemistry Wiley.

SNYDER L . R . & KIRKLAND, J.J. Introduction to Modern Liquid Chromatography. 2nd

Ed. Wiley.

TOUCHSTONE, J.C. & DOBBINS, M.F. 1978. Practice of Thin Layer Chromatography
Wiley.

VARMA, A. CRC H a n d b o o k of Atomic Absorption Analysis Vol I & II CRC Press.

Z E I F , M. & MITCHELL, J.W. 1976. Contamination Control in Trace Element

A n a l y s is Wiley.

ZWEIG, G. & SHERMA, J. CRC H a n d b o o k of Chromatography CRC Press

21
 5. DETERMIHATIVE TECHNIQUES

5.1 PAPER CHROMATOGRAPHY (PC)

Paper chromatography is a type of partition chromatography, so called because

s u b s t a n c e s a r e s e p a r a t e d a c c o r d i n g to t h e i r s o l u b i l i t y b e t w e e n the a q u e o u s
phase bound to the paper (the stationary phase) and the w a t e r - i n s o l u b l e organic
phase (the mobile phase) w h i c h travels up (or d o w n ) the paper. Alternatively,
the paper can be completely dried and impregnated with a non-polar substance
s u c h as l i q u i d p a r a f f i n or o l i v e o i l , and an a q u e o u s s o l v e n t u s e d as m o b i l e
phase. This is called "reverse-phase" chromatography.

If the a t m o s p h e r e of the l a b o r a t o r y is v e r y dry the r e s i d u a l m o i s t u r e on the

p a p e r m a y be so l o w that the a n a l y t e c a n n o t d i s t r i b u t e i t s e l f e f f i c i e n t l y
b e t w e e n the two phases. Separation will not be o p t i m a l if equilibrium is not
attained. For this r e a s o n , n o n - p o l a r s o l v e n t s are u s u a l l y the m o b i l e p h a s e .
A l s o , b e f o r e c h r o m a t o g r a p h y , the p a p e r c a n be h e l d for s e v e r a l h o u r s in an
atmosphere saturated with the vapour of the water-rich phase so that it attains
equilibrium. Another method is to m o i s t e n the paper w i t h a fine spray or hold
it in steam for a few minutes.

Polar organic compounds such as ethylene glycol or d i m e t h y l - f o r m a m i d e m a y also

be u s e d as s t a t i o n a r y p h a s e s i n s t e a d of w a t e r or a q u e o u s m i x t u r e s . Not all
s e p a r a t i o n s by p a p e r c h r o m a t o g r a p h y r e l y on p a r t i t i o n . for e x a m p l e , s o m e
colours can be separated using a 2.5% sodium chloride solution as solvent. In
t h i s c a s e the c o l o u r s are r e t a r d e d in d i f f e r i n g d e g r e e s b y a b s o r p t i o n on the
cellulose.

P a p e r c h r o m a t o g r a p h y can be d o n e w i t h the m o b i l e p h a s e e i t h e r a s c e n d i n g or
descending. Figure 5.1 illustrates t h i s s h o w i n g a s c e n d i n g c h r o m a t o g r a p h y in
(a) (mobile phase moves up by capillary action) and descending in (b) (mobile
phase m o v e s d o w n by gravity).

Paper Paper
Support - Support
Eluent
Samples —
Reservoir
Spotted
Paper here
Paper —

1
• i
. • •' 11
Samples - --Eluent
Spotted s . .
here V < Reservoir

(a) <b>
Figure 5.1

In t h i s s e c t i o n w e w i l l d i s c u s s t y p e s of p a p e r and solvents used as w e l l as

techniques of sample application and development.

Selection of Paper

W h a t m a n p a p e r no. 1 or e q u i v a l e n t is n o r m a l l y u s e d . F a s t e r d e v e l o p m e n t is
o b t a i n e d on p a p e r s such as W h a t m a n no. 4 or Sch 1 e i c h e r - S c h u l 1 no. 4 0 a .
Preparative work m a y be carried out on W h a t m a n 3 M M , Schleicher-Schull no. 2071 ,
etc. S o m e p r o p e r t i e s of the m o r e i m p o r t a n t c h r o m a t o g r a p h i c p a p e r s are as
follows (taken from Hais and Macek (1)):

22
 Rate of
Thickness Movement Weight
Paper (mm) (hours)(*) (g/m2) Characteristics

Whatman
No. Í 0.16 15 - 16 85 - 90 Standard paper
No. 2 0.18 17 95 - 100 Standard paper
No. 3 0.36 11 185 Preparative paper
No. 4 0.19 9 90 - 95 Fast
No. 3MM 0.31 11 180 Preparative paper
No. 31ET 0.50 4 190 Very fast
No. 540 0.15 17 85 - 90 W a s h e d , hardened

Schleicher-Schull
Nr. 2040a 0.18-0.19 7 85 - 90 Fast
Nr. 2040b(M) 0.22-0.24 12 120 - 125 Med ium
Nr. 2043a 0.18-0.19 20 90 - 95 Standard Paper
Nr. 2043(MG1) 0.22-0.24 15 120 - 125 Standard Paper
Nr. 2071 0.65-0.70 23 600 - 700 Preparative paper

Filtrak-Hieder-
Schlag
FN 1 9 90 Fast
FN 2 15 125 Standard paper
FN 3 16 90 Preparative paper
FN 8 9 180 Preparative papei

Ederol
Nr. 202 16 120 Standard paper
Nr. 208 26 120 Slow
Nr. 225 180 Preparative paper

Macherey-Nagel
Nr. 2212 0.21 19 120 Washed
Nr. 214 0.28 14 140 Standard paper
Nr. 260 0.25 16 90 Standard paper

Leningradskaya
bumaga B 18 85 Standard paper

Munktell
Chr 100 95 - 100 Standard paper
ELE 130 130 Standard paper

Eaton Dikeman
Mo. 048 0.18 88 Very fast
No. 248 0.18 88 Standard paper
No. 613 0.14 70 Standard paper
No. 320 2.54 700 Preparative paper

(*) Descending chromatography using (4+1+5) butanol-acetic acid-water.

23
Selection of Solvent

T h e f o l l o w i n g t a b l e g i v e s a list of s o l v e n t s in o r d e r of p o l a r i t y , s t a r t i n g
with the m o s t polar (water), and proceeding to the least (paraffin oil):

Dieletric Dieletric
Solubility* Constant** Solubility* Constant**
Solvent (a) (b) (c) Solvent (a) (b) (c)

Water N/A 81.1 n-Butyl acetate 0.5 (25°) 5

Formamide Miscible 84 Di-isopropoxy-
Formic acid do 58.5 me thane
Acetonitrile do 38.8 Dipropoxymethane
Methanol do 31.2 Ni trome thane 39
Acetic acid do 6.3 n-Butyl bromide
Ethanol do 25.8 isoPropyl ether 0.2
isoPropanol do 26 n-Butyl butyrate
Acetone do 21.5 n-Propyl bromide 0.3
n-Propanol do 22.2 n-Butyl ether
Dioxane do 3 Methylene
Dimethylformamide do 3.15 Chloride 2
Tetrahydrofuran do Chloro forra 1 (16°) 5.1
t-Butanol do Dichloroethane 0.6
2-Butanol 12.5 Bromobenzene 0.05 (30°) 5.4
Methyl ethyl Trichloroethane
ke tone 35.3 (10°) 18 Ethyl bromide 0.9
Cyclohexanone 2.4 (31°) 18.2 Ben zene 0.08 (22°) 2. 24
Phenol 6.7 (16°) 9.7 Propyl chloride 0.27
n-Butanol 7.9 19.2 Xrichloroethylene 0.1
Cyclohexanol 5.7 (15°) 15 Toluene 0.05 (16°) 2.3
isoAmyl alcohol 2.7 (22°) Tetrachloromethane 0.08 2.25
n-Amyl alcohol 2.7 (22°) 16 Carbon disulphide 2.6
Benzyl alcohol 4 (17°) 13 Decalin 2.13
Ethyl acetate 8.6 6.1 Cyclopentane
n-Hexano1 0.6 Cyclohexane
2-Col1 id ine 3.5 Hexane 0.01 (15°) 1.88
Ether 7.5 4.4 Heptane 0.005(15°) 1.97
Diethoxymethane Paraffin oil

* - ml of water in 100 ml of solvent

** - at room temperature

(a) Values from 'Handbook of Chemistry and Physics' (2).

(b) U n l e s s n o t e d , the g i v e n v a l u e r e p r e s e n t s the s o l u b i l i t y at 20°C.
Other temperatures are given in parenthesis.
(c) Values from 'Taschenbuch fur Chemiker und Physiker' (3).

T h e c h o i c e of s o l v e n t is not e n t i r e l y a m a t t e r of g u e s s w o r k . T h e c a r b o n to
oxygen (C/0) ratio of the analytes is some guide. If it is very low, b e t w e e n 1
and 2, a polar mobile phase is used. For values b e t w e e n 2 and 5, use solvents
of i n t e r m e d i a t e p o l a r i t y and o v e r 5 n o n - p o l a r s o l v e n t s s u c h as h y d r o c a r b o n s .
In principle, like is used with like, a more polar solvent mixture being used
to separate m o r e polar substances.

The Rj: value is the ratio of the distance travelled by the spot divided by the
d i s t a n c e t r a v e l l e d by the s o l v e n t f r o n t . If t h i s v a l u e is too l o w u s i n g the
s o l v e n t c h o s e n , r e p e a t w i t h a m o r e l i p o p h i l i c one. If the R f v a l u e s are too
low even with a very polar mixture such as n-butanol:acetic acid:water (4:1:5)
(the P a r t r i d g e m i x t u r e ) a s i n g l e p h a s e s y s t e m m a y be t r i e d s u c h as a 2.5%
sodium chloride solution or aqueous solutions of acids, alcohols or a m m o n i a .
If this fails, an alternative technique must be used. If the R f values are too
h i g h e v e n w i t h a n o n - p o l a r s o l v e n t , s e p a r a t i o n m a y be a t t e m p t e d u s i n g a
r e v e r s e d - p h a s e t e c h n i q u e , or u s i n g a p o l a r o r g a n i c s o l v e n t as the s t a t i o n a r y
phase.

24
I d e a l l y , the s u b s t a n c e s b e i n g s e p a r a t e d s h o u l d h a v e R f v a l u e s in the r a n g e
0.15-0.85. V a l u e s of 0.00 and 1.00, t h o u g h t h e y c a n s o m e t i m e s be u s e d for
separation, are not suitable for characterisation of any substance.

Addition of polar solvents (for example DMF, acetic acid, or ethanol) to non-
polar solvents (for example chlorinated hydrocarbons or hydrocarbons) may have
a n u m b e r of b e n e f i c i a l e f f e c t s , s u c h as i n c r e a s i n g the s o l u b i l i t y of the
substance in both phases and suppressing absorption on the paper. The result
is rounder spots and higher R^ values.

Spots may be elongated (tailing or trailing) for a n u m b e r of reasons including

dissociation, poor solubility, absorption on the paper, and poor equilibration.
Run a c h r o m a t o g r a m w i t h s p o t s of d i f f e r e n t c o n c e n t r a t i o n . If the m o r e
c o n c e n t r a t e d s p o t s t a i l w o r s e , b u t the f r o n t e d g e h a s the s a m e R^ for all
concentrates, the tailing is due to poor solubility and a better solvent must
be found. If the m o r e concentrated spots tail worse and also have front edges
or centres of higher R f , the overall shape being that of a comet, the cause is
e i t h e r d i s s o c i a t i o n or a b s o r p t i o n . V e r y s l i g h t d i s s o c i a t i o n and c o m p l e t e
d i s s o c i a t i o n g i v e rise to c o m p a c t s p o t s , it is o n l y if it is p a r t i a l that
tailing occurs. T h i s is a p r o b l e m w i t h s u b s t a n c e s s u c h as c o l o u r s . It is
s o l v e d by e i t h e r s u p p r e s s i n g the d i s s o c i a t i o n , or m a k i n g it c o m p l e t e . For
e x a m p l e , t h e d i s s o c i a t i o n of a c i d i c s u b s t a n c e s c a n b e s u p p r e s s e d by
c h r o m a t o g r a p h i n g t h e m in an a c i d i c s o l v e n t (the u s u a l c h o i c e ) or can be m a d e
c o m p l e t e b y u s e of an a l k a l i n e s o l v e n t . A l t e r n a t i v e l y , the paper m a y be
impregnated with a buffer that will m a i n t a i n a pH at w h i c h the dissociation is
shifted in one direction or the other. Adsorption can be a problem in single-
p h a s e s y s t e m s , b u t if t a i l i n g o c c u r s in the u s u a l t w o - p h a s e s y s t e m it is
usually due to poor equilibration.

Application of Test Solutions

Sample and standard spots are applied to the paper at about 2 cm intervals and
about 3 cm from the bottom edge for ascending development. It is convenient to
d r a w a p e n c i l l e d m a r k a c r o s s the p a p e r as the s p o t s m u s t all be in an e x a c t
straight line. Solutions are applied from capillary tubes or a micro-syringe.
The size of the spot s h o u l d be as s m a l l as p o s s i b l e , no m o r e t h a n 3 or 4 m m
across. T h i s is a c c o m p l i s h e d by a p p l y i n g the t e s t s o l u t i o n in s m a l l v o l u m e
increments and gently drying between applications. Drying can be done using
warm air from a hair dryer or similar device.

Development and Chromatography

For a s c e n d i n g d e v e l o p m e n t , the s p o t t e d p a p e r is s u s p e n d e d in a p r e v i o u s l y
e q u i l i b r a t e d t a n k , w i t h the b o t t o m e d g e of the p a p e r i m m e r s e d in the m o b i l e
p h a s e to a d e p t h of a b o u t 1 cm. T h e t a n k s h o u l d be in an a r e a free from
d r a u g h t s , d i r e c t s u n l i g h t and s o u r c e s of h e a t . The t a n k m a y be l i n e d w i t h
filter paper soaked in the mobile phase in order to hasten equilibration. The
c h r o m a t o g r a m is a l l o w e d to d e v e l o p at l e a s t 10 cm ( c o n s i d e r a b l y m o r e is
n e c e s s a r y for s o m e s e p a r a t i o n s ) . D r y i n g and r e d e v e l o p m e n t m a y r e s u l t in
improved separation. The p a p e r is f i n a l l y r e m o v e d and d r i e d . If the
substances are heat-labile, leave to air dry. The stability of the substances
may be affected by the solvent used; for example phenolic solvents increase the
loss of peptides and amino-acids that may occur during drying.

C o m p l e t e d r y i n g b e t w e e n m u l t i p l e d e v e l o p m e n t s is n o t e s s e n t i a l , b u t it is
important in two-dimensional development, where remains of the first solvent
m a y lead to u n r e p r o d u c ib 1e Rf v a l u e s . If v i s u a l i s a t i o n is l i k e l y to b e
affected by pH, any basic or acidic solvents used must be completely removed.
Papers that have been treated (e.g. with buffers) should be air-dried, so that
they do not buckle.

Descending development has been used for the separation of sugars and colours,
among other groups. The p a p e r is h e l d in a t r o u g h by a h e a v y g l a s s rod or
other m e a n s , and the trough filled with mobile phase.

25
T w o - d i m e n s i o n a l chromatography is used for the separation of complex mixtures
such as amino-acids. One spot only is placed in a corner of the paper and the
c h r o m a t o g r a m is developed. The paper is removed, dried, turned through 90° and
developed with a different m o b i l e phase. S t a n d a r d s m u s t be on a s e p a r a t e
paper. This is often the only way to separate closely related compounds.

Visualisation

The developed paper is sprayed with a suitable reagent, or dipped in a solution

of it in a t r o u g h . M a n y of the r e a g e n t s a r e t o x i c to a g r e a t e r or l e s s e r
extent and the fineness of the spray increases the likelihood of inhalation, so
the s p r a y i n g s h o u l d be d o n e in a f u m e h o o d w i t h an e f f i c i e n t e x h a u s t s y s t e m .
The spray should be kept some distance from the paper so that larger drops are
lost and o n l y the f i n e s t r e a c h the p a p e r , g i v i n g a m o r e e v e n c o l o u r
development. T h e r e are m a n y t y p e s of g l a s s s p r a y b o t t l e s commercially
available. It is advisable to use a c o m m e r c i a l unit rather than attempting to
construct one, as they generally deliver a more uniform spray.

Spots m a y also be visualised by exposing the paper to vapours such as a m m o n i a

or acetic acid as well as iodine. This may s o m e t i m e s be a m e a n s of rescuing a
p a p e r t h a t h a s b e e n s p r a y e d w i t h o u t c o m p l e t e r e m o v a l of an a c i d i c or b a s i c
solvent .

Pain (4) describes an improved method using iodine. The dry paper is sprayed
on both sides with a l u m i n i u m sulphate solution (20% w/v of the hydrate) and re-
dried. It is then left in i o d i n e v a p o u r at l e a s t t h r e e h o u r s and p r e f e r a b l y
overnight. Excess iodine is removed by leaving the paper suspended one hour,
and then it is s p r a y e d w i t h 0.5% s t a r c h s o l u t i o n . S p o t s m a y a p p e a r as d a r k
b l u e on a l i g h t b l u e b a c k g r o u n d . L i p i d s , a l k a l o i d s , and a m i n o - a c i d s can be
visualised in this way.

5.2 THIN-LAYER CHROMATOGRAPHY (TLC)

M a n y of the r e m a r k s u n d e r the s e c t i o n on p a p e r c h r o m a t o g r a p h y a l s o a p p l y to
thin-layer, although the separation principle involved is quite different. In
thin-layer chromatography, the substances are absorbed to a greater or lesser
extent on the solid layer and the principal m e c h a n i s m of separation is elution
from it by the mobile solvent. H o w e v e r , in reversed-phase TLC, the principle
of separation is similar to that in paper chromatography.

As was done in the above section on paper chromatography, this section on TLC
w i l l d i s c u s s the m a t e r i a l s u s e d and t h e i r p r e p a r a t i o n , a l o n g w i t h s a m p l e
application and development.

Selection of TLC Layers and Supports

The m o s t c o m m o n l y used m a t e r i a l s are s i l i c a g e l , a l u m i n a , p o l y a m i d e and

c e l l u l o s e p o w d e r s b u t o t h e r s are s o m e t i m e s u s e d , s u c h as p o l y c a r b o n a t e ,
m a g n e s i u m s i l i c a t e , etc. T h e l a y e r m u s t be s u p p o r t e d on g l a s s , a l u m i n i u m
s h e e t , p l a s t i c or o t h e r i n e r t m a t e r i a l . A l u m i n i u m or p l a s t i c a r e m o s t
convenient if the plates are purchased already prepared, as they can be cut by
s c i s s o r s and t h u s u s e d m o r e e c o n o m i c a l l y . Such plates usually give m o r e
satisfactory results than ones prepared in the laboratory and are therefore to
be p r e f e r r e d . The l e t t e r G a f t e r the n a m e of the s u b s t a n c e i n d i c a t e s t h a t
calcium sulphate has been added to it as a binder, traditionally at the level
of 13%. The letter H indicates that no binder has been added. the symbol
i n d i c a t e s that the t h i n layer f l u o r e s c e s at 254 nm due to i n c o r p o r a t i o n of a
fluorescing a g e n t .

Preparation of Glass Plates

Spreading devices are available commercially. The operating instructions must

be followed. A slurry is prepared from water and the powder to be used (about
2 + 1 by w e i g h t for s i l i c a gel). ( A l t e r n a t i v e l y the p o w d e r can be m i x e d w i t h

26
an organic solvent). Silica gel slurries are best h o m o g e n i z e d in a b l e n d e r for
a c o u p l e of m i n u t e s . If t h i s is d o n e , it m a y b e n e c e s s a r y to i n c r e a s e t h e
a m o u n t of w a t e r s l i g h t l y to o b t a i n a s l u r r y of the r i g h t c o n s i s t e n c y . The
g l a s s p l a t e s , w h i c h m u s t b e on a p e r f e c t l y f l a t s u r f a c e s u c h as a p l a t e
l e v e l l e r , a r e fed i n t o the s p r e a d e r t h r o u g h a g a t e w h i c h h a s b e e n set w i t h a
feeler gauge to the a p p r o p r i a t e thickness. Glass sheets, 20 x 20 cm or 5 x 20
cm are n o r m a l l y u s e d . 0.25 m m is a s u i t a b l e l a y e r t h i c k n e s s for r o u t i n e
applications. A n a n d a r a m a n et al (5) d e s c r i b e a s i m p l y c o n s t r u c t e d glass
spreader .

An a p p l i c a t i o n device can be constructed using zinc oxide plaster tape. Most

b r a n d s of z i n c o x i d e p l a s t e r a r e a b o u t 0.25 m m t h i c k . P l a c e f i v e 20 x 2 0 c m
p l a t e s on a p e r f e c t l y f l a t s u r f a c e b u t t i n g a g a i n s t e a c h o t h e r in a l i n e and
h o l d t h e m in p l a c e by p l a s t e r t a p e a l o n g t h e t w o l e n g t h s a n d o n e e n d o f t h e
line, allowing the p l a s t e r to o v e r l a p the g l a s s b y a b o u t 0.5 c m . See
i l l u s t r a t i o n in Figure 5.2.

1 cm plaster strip,
half on glass

n /

Glass rod

Figure 5.2

A s l u r r y (e.g. 3 0 g m s i l i c a g e l + 70 m l w a t e r is e n o u g h f o r f i v e 20 x 20 c m
p l a t e s ) is p o u r e d o n t o the p l a t e s f r o m a b e a k e r in the l e f t h a n d and a s t o u t
glass rod (about 30 m m long) is m o v e d across the p l a t e s i m m e d i a t e l y behind the
beaker. After a little p r a c t i c e the slurry can be poured on, and the rod m o v e d
along at the correct speed so that by a rapid, s m o o t h a c t i o n all the plates are
evenly coated. I n s t e a d of u s i n g t a p e a l o n g the p l a t e e d g e s , c e l l o t a p e or
scotch tape m a y be wound around the glass rod at t w o p o i n t s about 19 c m apart
and the rod d r a w n a l o n g the p l a t e s j u s t b e h i n d the s l u r r y as the l a t t e r is
poured. If this m e t h o d is used, it is a d v i s a b l e to hold the plates in place in
s o m e other way.

L a y e r s m a y a l s o be s u p p o r t e d o n m i c r o s c o p e s l i d e s . The dry clean slide is

dipped into the slurry, a i r - d r i e d and the layer on one side b r u s h e d off.

F o r s o m e s e p a r a t i o n s , it is n e c e s s a r y t h a t the l a y e r be i m p r e g n a t e d w i t h a
b u f f e r , a n o n - p o l a r s u b s t a n c e o r s o m e o t h e r a i d to s e p a r a t i o n . For e x a m p l e ,
plates i m p r e g n a t e d w i t h silver nitrate (argentation c h r o m a t o g r a p h y ) are u s e f u l
in s e p a r a t i n g s a t u r a t e d f a t t y a c i d s f r o m u n s a t u r a t e d , as t h e l a t t e r f o r m
a d d u c t s w i t h the s i l v e r n i t r a t e and h e n c e t r a v e l m o r e s l o w l y . Impregnation
with a non-polar substance and subsequent u s e of a p o l a r solvent for
d e v e l o p m e n t is c a l l e d r e v e r s e - p h a se c h r o m a t o g r a p h y . If t h e p l a t e is b e i n g
p r e p a r e d in t h e l a b o r a t o r y , t h e s u b s t a n c e w i t h w h i c h t h e l a y e r is to b e
i m p r e g n a t e d is incorporated in the slurry instead of using pure solvent. The
l a y e r o n c o m m e r c i a l p l a t e s is u s u a l l y f i r m e n o u g h so t h a t t h e y c a n be d i p p e d
i n t o a s o l u t i o n of the s u b s t a n c e in a t r a y and t h e n r e d r i e d or r e - a c t i v a t e d .
Some care m a y be n e e d e d to ensure that i m p r e g n a t i o n occurs evenly.

27
Drying and Activation of Prepared Plates

Prepared plates must be air-dried until they can be moved without damaging the
layer. Plates of silica gel and a l u m i n a are then activated by oven-heating and
m a y be s t o r e d in a d e s i c c a t o r . S i l i c a gel m a y be a c t i v a t e d by h e a t i n g 20
m i n u t e s at 110°C or at 100°C. A l u m i n a is generally activated by heating at the
same temperatures for varying periods. A l w a y s avoid over-heating.

The a c t i v i t y of a l u m i n a on a p r e p a r e d p l a t e m a y be d e t e r m i n e d as f o l l o w s :
P r e p a r e a d y e m i x t u r e of 3 0 m g of p - a z o b e n z e n e a n d 2 0 m g e a c h o f p -
aminoazobenzene, p-methoxyazobenzene, Sudan Red and Sudan Y e l l o w dissolved in
carbon tetrachloride and diluted to 50 ml with the same solvent, Add 0.02 ml
of this solution to the plate and develop in carbon tetrachloride. Water m u s t
be rigidly excluded. The activity of the alumina is determined by comparison
of the Rf values of the dyes with the values in the following table:

Grade of Alumina according to

Dye Brockmann and S c h o d d e r ( 6 )

II III IV V

Azobenzene 0.59 0.74 0.85 0.95

p-Methoxyazobenzene 0.16 0.49 0.65 0.89
Sudan Yellow 0.01 0.25 0.57 0.78
Sudan Red 0.00 0.10 0.33 0.56
p-Aminoazobenzene 0.00 0.03 0.08 0.19

Application of Test Solutions

At the top of the p l a t e s c r a t c h a s i m p l e c o d e w i t h a pin so that t h e r e is no

c o n f u s i o n a b o u t the i d e n t i t y of the s p o t s a f t e r v i s u a l i s a t i o n . Solutions
s h o u l d be a p p l i e d at 2 cm i n t e r v a l s and a b o u t 1.5 cm f r o m the e d g e of the
plate. S p o t p a r a l l e l to w h e r e the l a y e r e x t e n d s to the b a s e e d g e . If the
l a y e r is f i r m e n o u g h , p r i c k s m a y be m a d e at 2 cm i n t e r v a l s in a s t r a i g h t line
w i t h the aid of a p i n and a r u l e r . The s o l u t i o n s are t h e n s p o t t e d in b e t w e e n
the p r i c k m a r k s , j u d g i n g by eye that the s p o t s are in a s t r a i g h t line. It is
p r e f e r a b l e , and e s s e n t i a l if the l a y e r s a r e l o o s e , to u s e a t r a n s p a r e n t
template with raised edges so that it stands over the plate, and having a row
of h o l e s on o n e s i d e t h r o u g h w h i c h the s o l u t i o n s m a y be s p o t t e d . Do n o t s p o t
s a m p l e s too n e a r the e d g e of the p l a t e , as the R^ w i l l be a l t e r e d (the e d g e
effect). It is because of this effect that the few m i l l i m e t r e s of the layer on
each of what will b e c o m e the two vertical sides of the plate should be r e m o v e d ,
if p r e s e n t . T h i s is c o n v e n i e n t l y d o n e by r u n n i n g a l o n g the e d g e a c l e a n c o r k
in w h i c h a r i g h t a n g l e of the r e q u i r e d l e n g t h h a s b e e n cut. T r y to k e e p the
spot l e s s t h a n 2 m m a c r o s s . T h i s is e a s i l y d o n e in q u a l i t a t i v e w o r k , as v e r y
fine c a p i l l a r i e s m a y be d r a w n and u s e d . T i n y s p o t s a b o u t 0.5 m m a c r o s s g i v e
considerably better results than those from spots a few m i l l i m e t r e s across, and
e x t r a c a r e is w o r t h w h i l e . If a s e m i - q u a n t i t a t i v e r e s u l t is r e q u i r e d , the
v o l u m e applied m u s t be measured with a m i c r o - l i t r e syringe such as the H a m i l t o n
t y p e , an A g l a m i c r o s y r i n g e , or a g r a d u a t e d m i c r o cap i 1 1 a r y ( O x f o r d type or a
m i c r o c a p ) (See F i g u r e 5.3). U s u a l l y , o n e to five m i c r o l i t r e s are a p p l i e d b u t
it may be possible to apply up to 50 microlitres without the layer breaking up.
It is p r e f e r a b l e to u s e a s t r o n g e r s o l u t i o n r a t h e r t h a n try to s p o t t h e s e
l a r g e r a m o u n t s . . T h e c o n c e n t r a t i o n of s t a n d a r d s to be u s e d d e p e n d s on the
s e n s i t i v i t y of the m e t h o d of d e t e c t i o n , b u t a m i c r o l i t r e or t w o of a 1%
solution is c o m m o n l y used. If the method of detection is fairly sensitive, a
0.1% solution m a y be better. The capillary containing the solution is touched
against the layer so that liquid is absorbed without the surface being spoiled.
It is m o r e difficult to contain the size of the spot w h e n measured v o l u m e s are
being applied. A s m a l l p l a s t i c jet a t t a c h e d to the end of the s y r i n g e or
c a p i l l a r y m a y h e l p in p l a c i n g a s m a l l e r v o l u m e o n t h e p l a t e at e a c h
application. The drying of the spot can be hastened by use of a hair-dryer or
o t h e r s o u r c e of h o t air. The d r o p s h o u l d n o t be a l l o w e d to d r y as it l e a v e s

28
the s y r i n g e , or the a n a l y t e m i g h t c o l l e c t at the tip and not r e a c h the l a y e r .
The s y r i n g e s h o u l d be r i n s e d w i t h a m i c r o l i t r e or t w o of s o l v e n t and t h i s
a p p l i e d to the s a m e s p o t , but if the s u b s t a n c e h a s b e e n d e p o s i t e d on the
outside of the tip, rinsing with solvent m a y succeed only in leaving it behind
in the s o l v e n t b e a k e r . If g l a s s p l a t e s are b e i n g u s e d , it m a y be m o r e
satisfactory to keep the plate on a w a r m surface rather than use a blower.

Hamilton syringe

Agla microsyringe pipette

Micropipette

Figure 5.3

It is b e t t e r , w h e r e p o s s i b l e , to d i s s o l v e the a n a l y t e and s t a n d a r d s in a
volatile solvent such as acetone, methanol or chloroform. Diethyl ether is too
v o l a t i l e for m o s t p u r p o s e s , w a t e r n o t q u i t e v o l a t i l e e n o u g h , so t h a t g r e a t e r
care is required to restrict the size of the spots.

The R.£ v a l u e of a s u b s t a n c e w i l l a l t e r to s o m e e x t e n t w i t h c o n c e n t r a t i o n so
even if only qualitative results are required it is important to add amounts of
sample and standard as closely similar as possible. A satisfactory procedure
is to apply several standards spots, some containing more and some less of the
analyte than the sample spot. It is advisable to apply the least amount that
gives a recognisable spot.

A number of devices are on the market that automate the spotting of the sample
to a greater or lesser extent. They achieve a m o r e regular application of the
solutions than is possible m a n u a l l y and therefore better results, but are not
e s s e n t i a l for r o u t i n e w o r k . I m p r o v e d r e s u l t s c a n be o b t a i n e d by a p p l y i n g a
line or a band of solution to the plate rather than a spot. This is difficult
normally, but works very well with one of the mechanical applicators available
commercially.

Rf values on TLC plates are not reproducible and constant as they depend upon
factors such as temperature, fineness of absorbent, relative h u m i d i t y , activity
of the layer and so on. It is therefore convenient to use a 'marker' substance
and calculate R^ values relative to it.

Development and Chromatography

The chromatogram is developed in a tank with a close-fitting lid and containing

s o l v e n t to a d e p t h of a b o u t 0.5 cm for a 20 x 20 or 5 x 20 cm p l a t e . The
s o l v e n t s h o u l d be left in the t a n k for at l e a s t an h o u r b e f o r e r u n n i n g the
chromatogram. The w a l l s of the tank m a y be lined with filter paper soaked in
the d e v e l o p i n g s o l v e n t in o r d e r to b r i n g the v a p o u r to e q u i l i b r i u m m o r e
q u i c k l y . The tank s h o u l d be p l a c e d in an a r e a f r e e f r o m d r a f t , h a v i n g an e v e n
temperature and away from direct sunlight.

29
S m a l l s c r e w - c a p j a r s , l i p l e s s b e a k e r s c o v e r e d with watchglasses and similar
containers m a y be used for plates cut from a l u m i n i u m or plastic sheets, or for
l a y e r s on m i c r o s c o p e s l i d e s . T h e s t a i n i n g jars used in h i s t o l o g y are a l s o
satisfactory for the latter.

T h e s o l v e n t is a l l o w e d to run up the p l a t e to 10 cm a b o v e the s t a r t i n g line

(for 20 cm high plates) or further if desired. The 10 cm line m a y be marked on
t h e s i d e of t h e p l a t e w i t h a p i n b e f o r e t h e r u n is s t a r t e d . Various
m o d i f i c a t i o n s m a y be of use. For e x a m p l e , a s i n g l e s p o t m a y be p l a c e d in the
corner of a plate, which can be developed in one solvent, dried, turned through
90° and developed in another solvent (two-dimensional TLC). In this technique
s e v e r a l p l a t e s h a v e to be run if s t a n d a r d s are i n c l u d e d . In o r d i n a r y o n e -
dimensional development a certain i m p r o v e m e n t is obtained if the c h r o m a t o g r a m
is r u n , d r i e d , and then run a g a i n in the s a m e s o l v e n t ( m u l t i p l e d e v e l o p m e n t ) .
Alternatively, stepwise development m a y be carried out with different solvents.
The chromatogram is run a few centimetres in the least polar solvent, r e m o v e d ,
d r i e d , and t h e n r u n f u r t h e r in the s o l v e n t of n e x t p o l a r i t y and so on. The
r e m a r k s on p o l a r i t y , c h o i c e of s o l v e n t and i m p r o v e m e n t of s e p a r a t i o n u n d e r
" p a p e r c h r o m a t o g r a p h y " a l s o a p p l y to s e p a r a t i o n s on t h i n l a y e r s . If l o o s e
layers without binder are used these are so delicate that development must be
carried out with the plate close to horizontal.

Overspotting

If s a m p l e and s t a n d a r d a p p e a r to be i d e n t i c a l , a f r e s h p l a t e s h o u l d be
prepared, including sample, standard and sample w i t h standard spotting on top
of it. If the t w o t r a v e l t o g e t h e r , and this can be r e p e a t e d w i t h t w o f u r t h e r
solvents, it m a y be assumed that standard and sample are identical. Exceptions
are rare, e.g. the colours Ponceau SX and Allura Red.

Visualisation

After removing the c h r o m a t o g r a m from the tank and drying, it should be examined
u n d e r u l t r a v i o l e t l i g h t , b o t h s h o r t e r w a v e (254 n m ) and l o n g e r w a v e (365 n m ) .
If a f l u o r e s c o r has b e e n used that f l u o r e s c e s u n d e r the s h o r t e r w a v e , m a n y
s u b s t a n c e s s h o w up as d a r k s p o t s w h e r e t h e y h a v e q u e n c h e d the f l u o r e s c e n c e .
T h e s e s p o t s m a y be l i g h t l y c i r c u m s c r i b e d w i t h a pin to m a r k t h e i r p o s i t i o p
before spraying. A f t e r s p r a y i n g ( w h i c h u s e s the s a m e d e v i c e s as m e n t i o n e d
u n d e r " P a p e r c h r o m a t o g r a p h y " ) the p l a t e m a y r e q u i r e h e a t i n g b e f o r e s p o t s
appear. S o m e t i m e s the plate can be sprayed with a succession of reagents, as
with Korbelak's method for the detection of artificial sweeteners.

Useful general visualising agents include iodine. A f e w c r y s t a l s c a n be

scattered on the bottom of a large desiccator and the plate left in the vapour
for h a l f an h o u r or so ( d e p e n d i n g on a m b i e n t t e m p e r a t u r e ) . Most organic
c o m p o u n d s show up. The charring action of concentrated sulphuric acid m a y also
be u t i l i s e d . The p l a t e is s p r a y e d w i t h the c o n c e n t r a t e d acid and h e a t e d to
110-140°C. Inclusion of 0.5% vanillin or salicylaldehyde gives colours in the
cold with m a n y substances, and other colours on heating. It is less hazardous
to s p r a y w i t h acid a m m o n i u m s u l p h a t e s o l u t i o n ( 5 0 % ) and h e a t the p l a t e (for
e x a m p l e on a h o t p l a t e in a f u m e c u p b o a r d ) . The a m m o n i a is d r i v e n o f f and
s u l p h u r i c acid r e m a i n s . P h o s p h o r i c acid can a l s o be u s e d in the s a m e w a y .
Another useful general reagent is weak potassium p e r m a n g a n a t e solution, either
acid or alkaline.

30
5.3 GAS-LIQUID CHROMATOGRAPHY (GLC)

A gas-liquid chromatograph is shown schematically in Figure 5.4.

Pressure
Regulator, Oven ^

JtH
¿•lOTnrPfP"
'1 i 1 J Flow
Sample Column Detector Meter
Injection
Chamber
Carrier I I
Gas |___J
Tank Amplifier Recorder

Figure 5.4

A s o l u t i o n o f t h e s a m p l e is i n j e c t e d o n to t h e c o l u m n a n d s w e p t t h r o u g h it b y
an i n e r t c a r r i e r gas (the m o b i l e p h a s e ) . For p a c k e d c o l u m n s the s t a t i o n a r y
p h a s e is a l i q u i d a d s o r b e d o n a f i n e s o l i d s u p p o r t m a t e r i a l . The t e m p e r a t u r e
o f t h e c o l u m n m u s t b e s u f f i c i e n t to v o l a t i l i s e t h e s a m p l e a n d a l l o w it to
partition b e t w e e n the m o b i l e and s t a t i o n a r y phases. The g r e a t e r the s o l u b i l i t y
of a s u b s t a n c e in t h e l i q u i d p h a s e t h e s l o w e r it t r a v e l s . In a m i x t u r e , the
v a r i o u s c o m p o n e n t s w i l l t h e r e f o r e s e p a r a t e d e p e n d i n g on their i n t e r a c t i o n w i t h
the l i q u i d s t a t i o n a r y p h a s e . W h e n the c o m p o u n d s leave the c o l u m n , they are
s e n s e d by the d e t e c t o r a n d a r e r e c o r d e d as a t r a c e on a m o v i n g g r a p h . In t h e
case of a c a p i l l a r y c o l u m n , the liquid f o r m s a t h i n f i l m on the w a l l s of a v e r y
n a r r o w t u b e a n d n o s o l i d s u p p o r t m a t e r i a l is r e q u i r e d .

G a s c h r o m a t o g r a p h s a r e e x p e n s i v e a n d it is i m p o r t a n t t h a t t h e a n a l y t i c a l s t a f f
be w e l l t r a i n e d in t h e i r u s e . T h i s t r a i n i n g is o f t e n o f f e r e d b y the
manufacturer. T h i s s h o u l d b e a c o n s i d e r a t i o n w h e n a n e w i n s t r u m e n t is b e i n g
purchased.

There are several potential problems which become important when operating gas
chromatographs:

1. V o l t a g e s u p p l y m u s t be s t a b l e . M a n y GLC units have b u i l t - i n voltage

stabilizers. If n o t , a s t a b i l i z e r ( u s u a l l y a t r a n s f o r m e r to s m o o t h o u t surges)
m u s t be i n s t a l l e d b e t w e e n the i n s t r u m e n t and the v o l t a g e s o u r c e .

2. The c a r r i e r gas m u s t be pure e n o u g h for r o u t i n e w o r k . If t h e p u r i t y

is u n k n o w n , i t i s b e s t t o u s e a m o l e c u l a r s i e v e b e t w e e n t h e g a s t a n k a n d t h e
instrument. The instrument m a n u f a c t u r e r can usually r e c o m m e n d a correct sieve.
T h i s o n l y a p p l i e s to g a s e s u s e d as a c a r r i e r . O t h e r g a s e s s u c h as h y d r o g e n
u s e d in a f l a m e i o n i z a t i o n d e t e c t o r , d o n o t n e e d t h e s a m e p u r i t y r e q u i r e m e n t .

3. It is g e n e r a l l y b e s t to u s e g l a s s f o r c o l u m n m a t e r i a l as metal
c o l u m n s o f t e n c a t a l y z e d e c o m p o s i t i o n r e a c t i o n s w h i l e the s a m p l e is going
t h r o u g h the c o l u m n .

T h e e f f i c i e n c y o f a G L C c o l u m n is b a s e d u p o n t h e n u m b e r o f t h e o r e t i c a l p l a t e s
it c o n t a i n s . T h e f o l l o w i n g e x a m p l e c a l c u l a t i o n of t h e t h e o r e t i c a l p l a t e s u s e s
the s e p a r a t i o n of s t e a r a t e and o l e a t e :

Number of t h e o r e t i c a l plates, n, (or the efficiency):

=I6
[¿f]2
31
The resolution, R, is given by, (see Figure 5.5):

2D
+ w2

where :

dR is the r e t e n t i o n d i s t a n c e , m e a s u r e d in m m , from the start to the

m a x i m u m of the peak for m e t h y l stearate.

W ^ , and W 2 , are the w i d t h s , in m m , of the p e a k s for m e t h y l s t e a r a t e and

m e t h y l oleate, measured b e t w e e n the points of intersection of the tangents
at the inflexion points of the curve with the base line.

D is the distance b e t w e e n the respective peak m a x i m a for m e t h y l stearate

and oleate.

Figure 5.5

Some additional precautions to keep in mind while operating a gas

chromatograph:

1. A l w a y s h a v e the i n j e c t i o n p o r t at l e a s t 10°C h i g h e r t h a n the c o l u m n

temperature. This assures the sample and solvent will be properly volatilized.

2. ' Change injection septa frequently as they are prone to leak because
of age and/or frequent piercing.

3. Check the carrier gas flow rate at least once per day.

4. C h e c k for g a s l e a k s ( w i t h s o a p s o l u t i o n ) e a c h t i m e g a s connections
are re-fitted.

5. Do not tighten brass swage-lock type fittings excessively as they m a y

be damaged.

The b e s t r o u t i n e d e t e c t o r to use in food a n a l y s i s is the F I D or flame

ionization detector. T h i s d e t e c t o r " s e e s " any c o m p o u n d w h i c h w i l l i g n i t e in
the FID flame. This, of course, includes all organic m a t t e r , so that the major
d r a w b a c k of the FID is its non-selectivity. Selective detectors are useful as
they ignore everything except what they are designed to detect. As e x a m p l e , a
selective nitrogen detector will "see" only nitrogen containing c o m p o u n d s and
w i l l be b l i n d to all o t h e r s . T h i s m e a n s that m a n y p r e - s e p a r a t i o n s or
purifications do not have to be done w h e n a selective detector is used.

32
5-4 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Liquid chromatography is a very useful technique to separate compounds which

are not e a s i l y s e p a r a b l e by o t h e r m e a n s . E x a m p l e s i n c l u d e i o n i c s u b s t a n c e s ,
large m o l e c u l e s and m a t e r i a l s which are readily degraded by heat. H o w e v e r , by
its very nature, liquid chromatography is a slow process as low flow rates give
the b e s t s e p a r a t i o n e f f i c i e n c y . T h i s is d u e in g r e a t p a r t to the v e r y s l o w
d i f f u s i o n r a t e of a s o l u t e in the s o l u t i o n b e i n g c h r o m a t o g r a p h e d . This
diffusion rate is m o r e than 50 times slower in a liquid than in a gas.

M o d e r n H P L C i n s t r u m e n t s h a v e o v e r c o m e this d i f f i c u l t y b y the d e v e l o p m e n t of
columns containing very small particles. This increases the rate of diffusion
d r a m a t i c a l l y a n d p e r m i t s s e p a r a t i o n s n e a r l y as q u i c k l y as w i t h g a s
chromatography.

A basic isocratic HPLC unit can be considered to consist of:

1. An

2. A

3. A

4. A

In gas chromatography, the liquid sample is injected directly into the carrier
gas s t r e a m . T h i s d i r e c t i n j e c t i o n is i m p r a c t i c a l w i t h H P L C b e c a u s e of the
t r e m e n d o u s l y h i g h p r e s s u r e the c a r r i e r l i q u i d is u n d e r . I n s t e a d , a l o o p is
u s e d w h i c h h a s a r e s e r v o i r w h e r e the s a m p l e is i n j e c t e d . T h i s is t h e n
introduced into the carrier liquid stream without loss of pressure.

H P L C p u m p s m u s t be c a p a b l e of o p e r a t i o n up to at l e a s t 6 0 0 0 p s i and d e l i v e r a
l i q u i d f l o w w i t h o u t s u r g e s or p u l s e s . T h e u s u a l H P L C p u m p is m e c h a n i c a l and
delivers a constant flow rate, with the pressure dependent on the resistance of
the l i q u i d t h r o u g h the c o l u m n . T h e r e is a l s o the gas d i s p l a c e m e n t t y p e H P L C
p u m p w h i c h d e l i v e r s c o n s t a n t p r e s s u r e , w i t h the f l o w r a t e d e p e n d i n g on
resistance. Either are usable.

Small particle size for column packings are the key to rapid HPLC separations.
An e x a m p l e is 10 m i c r o n s i z e p o r o u s s i l i c a g e l p a r t i c l e s . Even smaller
particles can be used, but these are often bonded on 30-50 m i c r o n glass beads
to form a large surface area without restricting- the c o l u m n flow. Such coated
beads are called pellicular beads and are especially useful if it is desired to
coat them further with a thin layer of a liquid stationary phase. See Gray (7)
for a discussion of columns used in food analysis.

The two m o s t useful HPLC detectors, in food analysis, are the ultraviolet and
the differential refractometer. The ultraviolet detector consists of a f l o w -
through m i c r o cell in a UV spectrophotometer. C o m m e r c i a l UV detectors range
from one fixed w a v e l e n g t h ( u s u a l l y 254 m m ) to v a r i a b l e w a v e l e n g t h over the
w h o l e UV s p e c t r u m . As m o s t c a r r i e r l i q u i d s do n o t a b s o r b in the U V , t h e s e
d e t e c t o r s are v e r y u s e f u l for c o m p o u n d s h a v i n g s o m e UV a b s o r p t i o n . Some
spectrophotometric detectors include the visible w a v e l e n g t h s as well as the UV,
thus permitting HPLC analyses of food colours among others.

The d i f f e r e n t i a l r e f r a c t o m e t e r m o n i t o r s the d i f f e r e n c e in r e f r a c t i v e index

between the pure carrier liquid and the carrier plus sample.

5.5 SPECTROPHOTOMETRY

W h e n l i g h t is p a s s e d t h r o u g h a l i q u i d s o m e of the r a d i a t i o n m a y be a b s o r b e d .
The a m o u n t a b s o r b e d v a r i e s w i t h w a v e l e n g t h and w i t h t h e i d e n t i t y and
concentration of t h e s o l u t e . T h i s is t h e b a s i s f o r a l l absorption
spectrophotometry. As long as the l i g h t i n t e n s i t y is s t r o n g e n o u g h to p a s s

33
t h r o u g h w i t h o u t all being a b s o r b e d , its actual i n t e n s i t y is i m m a t e r i a l . It is
t h e d i f f e r e n c e in i n t e n s i t y b e t w e e n i n c i d e n t a n d t r a n s m i t t e d l i g h t t h a t is
measured. M a c L e o d (8) p o i n t e d o u t t h a t a b s o r p t i o m e t r y is n o t p a r t i c u l a r l y
s e n s i t i v e as it o n l y m e a s u r e s d i f f e r e n c e s in l i g h t i n t e n s i t y . H o w e v e r , the
s e n s i t i v i t y achieved is a d e q u a t e for m o s t n e e d s in r e g u l a t o r y food analysis.

The a m o u n t of a b s o r b a n c e of r a d i a t i o n d e p e n d s u p o n the n u m b e r of m o l e c u l e s in
the l i g h t p a t h and h e n c e d e p e n d s on the d e p t h o f the m e a s u r i n g c e l l and the
c o n c e n t r a t i o n of the solution. This is an e x p r e s s i o n of the B e e r - L a m b e r t L a w ,
w h i c h m a y be put in f o r m u l a form as:

A = log I e ct
E

where: A = absorbance
I = intensity of incident light
E = i n t e n s i t y of light e m e r g i n g from the solution
t = molar absorptivity
c = c o n c e n t r a t i o n in g - m o l / l i t r e
t = thickness in cm.

The scale on the spectrophotometer may also give readings as

t r a n s m i t t a n c e (T):

100 E
%T =

The m o l a r a b s o r p t i v i t y (e) is equal to the a b s o r b a n c e of a s o l u t i o n c o n t a i n i n g

1 g - m o l / l i t r e in a cell of 1 cm thickness. The a b s o r p t i v i t y of a s u b s t a n c e is
the a b s o r b a n c e of a s o l u t i o n c o n t a i n i n g 1 g/litre in a cell of 1 cm thickness.

F r o m the f o r m u l a A = fct it is seen that for an a b s o r b i n g s p e c i e s in a cell of

g i v e n t h i c k n e s s , a plot of A against c is a straight line. For any p a r t i c u l a r
s u b s t a n c e this m a y be true only over a c e r t a i n n a r r o w range. It is p r e f e r a b l e
to use o n l y the straight part of the c a l i b r a t i o n curve w h e n e v e r p o s s i b l e .

A c l a s s i c e x a m p l e of
e x a m ined in the the u l t r a v i o l e t w a v e l e n g t h s . B e n z e n e is t r a n s p a r e n t in the
v i s i b l e , b u t h a s a v e r y c h a r a c t e r i s t i c a b s o r p t i o n s p e c t r u m in t h e UV (see
F i g u r e 5. 6).

61 -
O
J

B < 'nzene
I /

.
0
Solvent cyclohexa ne \

1 ! l I t
220 240 2 80 280 200

Wavelength, nm

Figure 5-6

34
A b s o r p t i o n s p e c t r o p h o t o m e t r y ( a b s o r p t i o m e t r y ) is done in two m a i n wavelength
regions. These are, ultraviolet (approximately 200 nm to 350 n m ) and visible
( a p p r o x i m a t e l y 350 nm to 700 nm). S p e c t r o p h o t o m e t e r s are e i t h e r s i n g l e or
double beam. In a single-beam instrument, the sample and reference (or blank)
solutions are read alternately and the differing absorption values subtracted
from each other. A double-beam instrument does this a u t o m a t i c a l l y by splitting
the light f r o m the m o n o c h r o m ator b e t w e e n the s a m p l e and b l a n k , so that the
difference in absorption is subtracted electronically. This can be illustrated
by the schematic diagram in Figure 5.7.

Blank

Chopper 1 Chopper 2

Figure 5.7

One of the key parts of a spectrophotometer is the m o n o c h r o m a t o r . It takes the

source light, divides it into different wavelengths using a prism or grating,
and focuses light of a specific wavelength on the sample cell. This is shown
in F i g u r e 5.8.

Figure 5.8

In s c a n n i n g s p e c t r o p h o t o m e t e r s , the p r i s m or g r a t i n g is r o t a t e d to g i v e a
continuous spectrum of wavelengths through the sample cell.

The a b s o r p t i o n c e l l (also c a l l e d c u v e t t e ) m u s t be k e p t s c r u p u l o u s l y c l e a n .
Optical surfaces should not be touched, as oil smudges are difficult to remove.
P u r i t y of s o l v e n t s u s e d for s a m p l e s and for c l e a n i n g is i m p o r t a n t for the
p r o t e c t i o n of the c e l l s ; o n l y d i s t i l l e d w a t e r or s p e c t r o p h o t o m e t r i c g r a d e
solvents should be used. As soon as possible after use, cells should be rinsed
and soaked in distilled water. For cleaning, use a solution of mild detergent
c o n t a i n i n g no l a n o l i n e , o i l s or i n s o l u b l e m a t t e r . Ultrasonic cleaning
e q u i p m e n t can be used to a d v a n t a g e in m a i n t a i n i n g c e l l c l e a n l i n e s s and is
e s p e c i a l l y h e l p f u l in r e m o v i n g m a t e r i a l s w h i c h h a v e b e e n d e p o s i t e d on c e l l
walls. Avoid prolonged contact with alkalies or hot, concentrated acids. Do
not u s e a b r a s i v e s or o t h e r a g e n t s w h i c h m i g h t m a r the p o l i s h e d s u r f a c e s . A
brush or other device capable of scratching cells should never be used. Avoid
blowing cells with air to dry them; it is better to speed evaporation by m e a n s
of s u c t i o n . C e l l s s h o u l d not be c l e a n e d in c h r o m i c acid. R i n s i n g in a c e t o n e
or alcohol often aids drying.

35
Glass cells are satisfactory d o w n to about 350 nm. B e l o w this they are opaque
and silica cells m u s t be used. Small differences will be found b e t w e e n cells.
E v e n " m a t c h e d p a i r s " m a y be e x a c t l y m a t c h e d o n l y o v e r t w o or t h r e e s h o r t
wavelength ranges. When using a double-beam instrument, it is useful to check
the matching of the available cells at the wavelengths of interest. If there
is a d i f f e r e n c e , m a k e s u r e the s a m p l e c e l l h a s a p o s i t i v e a b s o r p t i o n w h e n
compared to the blank or reference. Record this value and subtract it later.

All s o l v e n t s h a v e a l o w e r w a v e l e n g t h l i m i t or " c u t - o f f " w h e r e t h e y b e c o m e

opaque to light. S o m e limits are given b e l o w for c o m m o n solvents. The table
is taken from MacLeod (8):

Cut-off Cut-off
Solvent point (nm) Solvent point (nm)

acetone 330 ether 220

acetonitrile 210 ethyl acetate 260
benzene 280 glycerol 220
carbon disulphide 380 heptane 210
carbon tetrachloride 265 hexane 210
chloroform 245 methanol 210
cyclohexane 210 2-methylbutane 210
cyclopentane 210 methylcyclohexane 210
1,2-dichloroethane 230 nitromethane 380
dichloromethane 235 pentane 210
N,N-dimethy1 formamide 270 toluene 285
dioxane 220 m-xylene 290

Substances tend to give more detailed spectra in less polar solvents, w h i c h are
thus to be preferred provided the solubility of the substance permits this. If
water has to be used, it should be r e m e m b e r e d that the spectrum m a y vary with
pH.

I n f r a r e d s p e c t r o p h o t o m e t r y is u s e f u l p r i m a r i l y as a m e a n s to i d e n t i f y an
organic compound. Q u a n t i t a t i v e IR is p o s s i b l e b u t v e r y d i f f i c u l t . The
i n f r a r e d " f i n g e r p r i n t " of a c o m p o u n d is not c o n f i r m a t i o n of a b s o l u t e p u r i t y ,
b u t is a g o o d p r e l i m i n a r y i d e n t i f i c a t i o n . It is n o t o f t e n u s e d in food
analysis because of the difficulty in purifying substances to obtain matching
IR s p e c t r a .

A t o m i c absorption spectroscopy depends on the absorption by the a t o m s in the

flame, of light of a specific wavelength emitted by a lamp or other source, of
the s a m e e l e m e n t as that b e i n g d e t e r m i n e d . T h u s i n t e r f e r e n c e is g e n e r a l l y
rare. One type of interference is caused by absorption by m o l e c u l e s and other
non-atomic species, absorbing continuously over a range of wavelength. This is
u s u a l l y o n l y of p r a c t i c a l i m p o r t a n c e b e l o w a b o u t 2 5 0 n m n e a r the l i m i t of
d e t e c t i o n of the e l e m e n t and in s o l u t i o n s c o n t a i n i n g h i g h c o n c e n t r a t i o n s of
salts. The difficulty is resolved by use of a deuterium or hydrogen background
corrector, which measures the non-atomic absorption and subtracts it from the
total s i g n a l .

S a t i s f a c t o r y r e s u l t s are o b t a i n e d o n l y if the s a m p l e s o l u t i o n is p r o p e r l y
p r e p a r e d , and f r e s h l y - p r e p a r e d s t a n d a r d s are t r e a t e d in the s a m e w a y . The
v i s c o s i t y and s u r f a c e t e n s i o n of the s o l u t i o n a s p i r a t e d a f f e c t s the r a t e of
uptake through the nebulizer and hence the signal. The standards m u s t be in a
c a r r i e r l i q u i d w i t h the s a m e p h y s i c a l p r o p e r t i e s . D i l u t e s o l u t i o n s of
s t a n d a r d s q u i c k l y d e t e r i o r a t e and s h o u l d be p r e p a r e d f r e s h e a c h d a y f r o m
concentrates containing at least 1000 m g / 1 unless it has been established that
the dilute standards are stable. For example, Moody et al (9) have shown that
addition of about 1 ppm of gold tetrachloride stabilises very dilute m e r c u r y
s o l u t i o n s in 0.5M H N O in g l a s s . The s t a n d a r d s o l u t i o n s h o u l d be a s p i r a t e d

36
after every h a l f - d o z e n or so of s a m p l e s to c h e c k that the i n s t r u m e n t r e s p o n s e
has not c h a n g e d . If t h e e l e m e n t b e i n g d e t e r m i n e d h a s b e e n e x t r a c t e d i n t o
solvent, s t a n d a r d s m u s t be treated in the s a m e way.

M a t r i x i n t e r f e r e n c e s , d u e to u n a v o i d a b l e d i f f e r e n c e s in v i s c o s i t y , s u r f a c e
t e n s i o n or o t h e r p r o p e r t i e s b e t w e e n t e s t s o l u t i o n and s t a n d a r d , m u s t be
o v e r c o m e by use of the m e t h o d of additions. If m a t r i x i n t e r f e r e n c e is absent,
results by direct a s p i r a t i o n and by the m e t h o d of a d d i t i o n s w i l l be identical.
T h e m e t h o d i n v o l v e s a d d i n g k n o w n a m o u n t s of s t a n d a r d to a l i q u o t s of t e s t
solution and plotting signal against c o n c e n t r a t i o n . Suppose a test s o l u t i o n is
t h o u g h t to c o n t a i n a b o u t 3 m ic r o g r a m s / m 1 o f l e a d (Pb). P i p e t t e 5 m l of t h i s
s o l u t i o n i n t o e a c h o f f o u r 10 m l g r a d u a t e d f l a s k s . A d d 0, 2, 3, 4 m l
r e s p e c t i v e l y of a standard lead s o l u t i o n of 5 m i c r o g r a m s / m l . and d i l u t e all the
flasks to 10 ml. M a k e a plot of a b s o r b a n c e against c o n c e n t r a t i o n (see Figure
5.9) .

Absorbance

Figure 5.9

T h e c o n c e n t r a t i o n of l e a d in t h e d i l u t e d t e s t s o l u t i o n is r e a d f r o m the
n e g a t i v e a b s c i s s a as 1.3 m i c r o g r a m s / m l . S i n c e t h i s w a s d e r i v e d f r o m 5 m l of
test s o l u t i o n diluted to 10 ml. the c o n c e n t r a t i o n in the o r i g i n a l test s o l u t i o n
w a s 2.6 m i c r o g r a m s P b / m l . T h e a m o u n t s o f a d d e d s t a n d a r d s o l u t i o n s h o u l d be
j u d g e d so t h a t t h e c a l i b r a t i o n c u r v e is at an a n g l e f a i r l y c l o s e to 45°, in
o r d e r to m a i n t a i n p r e c i s i o n . P r e c i s i o n is a l s o r e d u c e d b y t h e s m a l l v o l u m e s
p i p e t t e d , but this is o f t e n u n a v o i d a b l e in p r a c t i c e and should be a l l e v i a t e d by
use of Grade A pipettes. There are other i n t e r f e r e n c e s , such as i o n i s a t i o n or
f o r m a t i o n o f t h e r m a l l y s t a b l e c h e m i c a l c o m p l e x e s , b u t t h e s e w i l l n o t be
covered. M o s t s t a n d a r d t e x t s on a t o m i c a b s o r p t i o n s p e c t r o s c o p y include such
informat ion.

37
5.6 REFRACTOMETRY

The r e f r a c t i v e index (n) of a m e d i u m is the r a t i o of the speed of light in

v a c u o to its speed in the m e d i u m . It is m e a s u r e d by the r a t i o of the size of
the a n g l e of i n c i d e n c e to the size of the angle of r e f r a c t i o n w h e r e a ray of
light p a s s e s from air into the liquid. U n l e s s o t h e r w i s e s p e c i f i e d , the
refractive index is reported for the mean wave-length of the D lines of sodium.
For most refTactometers in c o m m o n use, the observation is made in white light
but is corrected to sodium light by a compensating device in the instrument.

The r e f r a c t o m e t e r is m o s t u s e f u l for the e x a m i n a t i o n of o i l s and of s u g a r

s o l u t i o n s (e.g. as s o l u b l e s o l i d s in fruit j u i c e p r e p a r a t i o n s ) . It is
important to keep the prisms scrupulously clean. The scale should be checked
occasiona 1 1y against water and alpha-bromonaphthalene or alpha-
chloronaphthalene. The refractive index of liquids changes quite appreciably
with temperature (and wavelength of the impinging light) and this must be borne
in mind when checking the scale or testing samples.

In g e n e r a l , r e f r a c t i v e index r e a d i n g s are taken at 20°C but fats w h i c h are

solid at that temperature are tested at 40°C, 60°C, or 80°C (usually the lowest
of these t e m p e r a t u r e s above the m e l t i n g - p o i n t ) . The space b e t w e e n the t w o
p r i s m s m u s t be c o m p l e t e l y filled w i t h the l i q u i d u n d e r test. Take a r e a d i n g
after the t e m p e r a t u r e has r e m a i n e d s t e a d y for at least five m i n u t e s . The
t e m p e r a t u r e should be w i t h i n 2°C of the c h o s e n s t a n d a r d t e m p e r a t u r e . A
c o r r e c t i o n can be m a d e u s i n g : n c = n'l + (tl -t)F w h e n t 1 is g r e a t e r than t,
and n ü = n fc l - ( t - t ^ F w h e n t 1 is less t h a n t. For fats, F = 0.00035 n e a r 20°C
and 0.00036 at 40°C and above.

5.7 MICROSCOPY

M i c r o s c o p y is an e x t e n s i v e s u b j e c t , m u c h of w h i c h can o n l y be learned by
experience. The p r a c t i c e of it r e q u i r e s a g r e a t d e a l of p a t i e n c e and care.
Samples must always be compared with reference specimens. There are diagrams
and photomicrographs in a number of publications, but they should on no account
be the o n l y r e f e r e n c e for a p o s i t i v e i d e n t i f i c a t i o n . H o w e v e r , they are
invaluable for showing the diagnostic features for which the less experienced
m i c r o s c o p i s t should look, and for m a k i n g t e n t a t i v e i d e n t i f i c a t i o n s so that
reference material can be obtained if not already available. In this way they
may considerably ease and speed up the w o r k . A l t h o u g h they s h o w s o m e
diagnostic features, they do not necessarily show them all, nor the proportions
and variations of the different elements which may be present. These will only
be seen by examining a number of different specimens, or different slides from
a representative specimen. It is therefore very important for the laboratory
to build up a collection of reference material.

It is m o s t i m p o r t a n t in m i c r o s c o p y to set up the m i c r o s c o p e p r o p e r l y . It
should be p l a c e d on a b e n c h or t a b l e l o w e n o u g h to e n a b l e the o b s e r v e r to be
seated and look c o m f o r t a b l y d o w n the m i c r o s c o p e w i t h o u t h a v i n g to tilt it.
This is less of a problem with modern microscopes in which the tube is usually
at an angle of 45° to the stage. T h e r e m u s t be a b r i g h t s o u r c e of light w i t h
its o w n c o n d e n s e r so that the b e a m can p a s s e i t h e r d i r e c t l y , as w i t h m o s t
m o d e r n m i c r o s c o p e s , or by r e f l e c t i o n from a m i r r o r , t h r o u g h the m i c r o s c o p e
condenser to the object stage.

Best results will be obtained if the light and various parts of the microscope
are aligned and adjusted for critical illumination. First of all, check that
the A b b e c o n d e n s e r is c e n t r a l l y p l a c e d . To do this, m a k e a s m a l l ink dot on
the c e n t r e of the top lens of the Abbe c o m b i n a t i o n and focus the dot w i t h a
low-power objective. Bring the dot to the centre of the field by adjusting the
centering screws of the substage. Clean off the ink dot. Place a slide with a
s m a l l o b j e c t on the stage and focus a l o w - p o w e r o b j e c t i v e on the o b j e c t . If
the l a m p has a d i f f u s e r , this should be r e m o v e d , or a s h a r p o b j e c t such as a
n e e d l e placed in front of it so that the n e e d l e or the l a m p f i l a m e n t is seen
t h r o u g h the m i c r o s c o p e , w h i c h is f o c u s s e d on one or the o t h e r by v e r t i c a l

38
m o v e m e n t of the s u b s t a g e . A f t e r f o c u s s i n g r e p l a c e the d i f f u s e r . If n o n e Is
a v a i l a b l e , the s u b s t a g e m a y be m o v e d u n t i l the l a m p f i l a m e n t is just out >f
f o c u s so that the f i l a m e n t i m a g e d o e s n o t i n t e r f e r e w i t h the v i e w of the
object. On m a n y m o d e r n microscopes, the lamp is in the base of the instrument
and is u n l i k e l y to be out of a l i g n m e n t , b u t if the l a m p is s e p a r a t e and the
stage lit via a m i r r o r , the alignment m u s t be checked. To do this, remove the
objective and eyepiece lenses, place a dark glass s o m e w h e r e b e t w e e n the lamp
and the e y e , and a d j u s t the m i r r o r of the m i c r o s c o p e , the l a m p and the l a m p
c o n d e n s e r so that the l a t t e r a p p e a r s t h r o u g h the tube of the m i c r o s c o p e as a
clear round evenly-illuminated circle. It is important that the objective is
filled with an even light. Provided the lamp and m i c r o s c o p e r e m a i n in the same
p o s i t i o n , the l a m p c o n d e n s e r n e e d be f o c u s s e d and a l i g n e d o n l y w h e n set up
initially. R e m o v a l of l e n s e s s h o u l d be d o n e o n l y if n e c e s s a r y , as d u s t m a y
collect in parts of the optical system, obscuring the image, and be difficult
to remove. Once the various parts of the instrument have been aligned and the
l i g h t f o c u s s e d , it o n l y r e m a i n s to a d j u s t the iris. A f t e r f o c u s s i n g the
o b j e c t , r e m o v e the e y e p i e c e , c l o s e the iris to s o m e e x t e n t and a d j u s t it so
that it b e c o m e s c e n t r a l l y p l a c e d if it is n o t so a l r e a d y . A d j u s t the i r i s so
that a p p r o x i m a t e l y the o u t e r third of the field of v i e w is c o v e r e d . Replace
the e y e p i e c e . The w h o l e f i e l d of v i e w s h o u l d be i l l u m i n a t e d and t h i s m a y
r e q u i r e m o v i n g the l a m p c l o s e r to the m i r r o r for l o w e r p o w e r o b j e c t i v e s . If
the i n t e n s i t y of l i g h t c a n b e v a r i e d , t h i s s h o u l d be a d j u s t e d u n t i l m a x i m u m
contrast is obtained. The microscope is n o w ready for use.

Underneath the iris will n o r m a l l y be found an annulus w h i c h will swing out on

its p i v o t . A p i e c e of p o l a r o i d can be i n s e r t e d h e r e for l o o k i n g at s p e c i m e n s
by p o l a r i s e d l i g h t . A s e c o n d p i e c e of p o l a r o i d i n s e r t e d in the e y e p i e c e is
r o t a t e d (or the w h o l e e y e p i e c e r o t a t e d ) u n t i l the s t a g e a p p e a r s dark. With
b i n o c u l a r m i c r o s c o p e s , it is u s u a l l y p o s s i b l e to i n s e r t a s i n g l e p i e c e of
p o l a r o i d in the u n i f i e d part of the i n s t r u m e n t . A r t i c l e s that s h o w up u n d e r
polarised light, such as starch, calcium oxalate and sand w i l l either appear as
d a r k s p o t s on a b r i g h t b a c k g r o u n d or a l t e r n a t i v e l y as b r i g h t s p o t s on a d a r k
background depending on the r o t a t i o n . Sand p a r t i c l e s a p p e a r m u l t i - c o l o u r e d ,
easily distinguishing them from glass, which shows as dull as the background,
a l t h o u g h its o u t l i n e can u s u a l l y be d i m l y seen. P o l a r o i d l e n s e s m a y be
purchased in shops selling camera equipment. Alternatively the plastic lenses
from sunglasses m a y be used. They can be m a d e to shape by cutting under water
with scissors. E a c h cut s h o u l d be m a d e s t r a i g h t as the p l a s t i c m a y s p l i t if
one tries to shape a curve at one cutting.

The m i c r o s c o p e m u s t be c o n s i d e r e d as a h i g h p r e c i s i o n i n s t r u m e n t , not o n l y
during service, but during periods of inactivity as well. The instrument m u s t
be k e p t free f r o m d u s t . A c a m e l ' s - h a i r b r u s h is s u i t a b l e for this p u r p o s e .
The e x p o s e d s u r f a c e s of o b j e c t i v e , e y e p i e c e and c o n d e n s e r l e n s e s , as w e l l as
the r e f l e c t i n g m i r r o r m a y be c l e a n e d w i t h l e n s - c l e a n i n g p a p e r or s o f t , good
quality linen. Silk m u s t not be used. W h e n balsam solution or i m m e r s i o n oils
c o n t a m i n a t e lens s u r f a c e s , a soft c l o t h or lens p a p e r m o i s t e n e d w i t h x y l e n e
should b e u s e d for c l e a n i n g . A l c o h o l m u s t n o t be u s e d for t h i s p u r p o s e as it
has a marked solvent action upon lens-mounting cements. Constant cleaning with
x y l e n e w i l l h a v e the s a m e r e s u l t s . O b j e c t i v e s m u s t n e v e r be t a k e n a p a r t in
order to clean inside lens surfaces. They should never need it. Eyepieces on
occasion do need unscrewing for cleaning purposes, but great care is necessary
and the fingers should not touch the lens surface as the resultant greasy film
deposited is very difficult to remove.

W h e n the m i c r o s c o p e is n o t in u s e , the l o w - p o w e r o b j e c t i v e s h o u l d be left

turned around towards the stage and the l o w - p o w e r eyepiece in position. Other
l e n s e s s h o u l d be put a w a y in their c o n t a i n e r s . D a m a g e to o b j e c t i v e s is far
c o m m o n e r than it need be, the chief cause being the racking d o w n of the tube
s h a r p l y on to the s l i d e . In the case of n o n - i m m er s i on l e n s e s the f o l l o w i n g
p r o c e d u r e m u s t be a d o p t e d : B e f o r e l o o k i n g i n t o the e y e p i e c e , the o b j e c t i v e s
should be lowered carefully until the front lens almost touches the coverglass

39
or o b j e c t u n d e r e x a m i n a t i o n , then w h i l e c a r e f u l l y l o o k i n g i n t o the e y e p i e c e f o r
the a p p e a r a n c e o f an i m a g e , the t u b e i s s l o w l y e l e v a t e d by m e a n s of the c o a r s e
adjustment. F i n a l f o c u s s i n g is then o b t a i n e d b y means of the f i n e a d j u s t m e n t .
The d i v e r s i t y of structures found among plant cells is an aid in the
i d e n t i f i c a t i o n of the p l a n t s from w h i c h f o o d s are d e r i v e d . In the case of
p o w d e r e d f o o d s s u c h as g r o u n d s p i c e s , m i c r o s c o p i c a l e x a m i n a t i o n n o t o n l y
a s s i s t s i n i d e n t i f y i n g t h e p o w d e r , b u t a l s o m a y e n a b l e t h e a n a l y s t to d e t e c t
the p r e s e n c e of a d u l t e r a n t s . For e x a m p l e , s a w d u s t (a common adulterant)
c o n s i s t s l a r g e l y o f s u p p o r t i v e and c o n d u c t i n g t i s s u e ( v e s s e l s , tracheids,
fibres and sieve-tubes) and these elements are likely to be easily
d i s t i n g u i s h a b l e u n d e r the m i c r o s c o p e from the e l e m e n t s c h a r a c t e r i s t i c of most
spices. When e x a m i n i n g food m i c r o s c o p i c a l l y , i t i s i m p o r t a n t to l o o k a t
s e v e r a l s l i d e s i n o r d e r to a s s e s s t h e r e l a t i v e p r o p o r t i o n s o f t h e d i f f e r e n t
e l e m e n t s p r e s e n t , as w e l l as s e a r c h f o r d i a g n o s t i c features.

I t is not p o s s i b l e to d e a l a d e q u a t e l y w i t h the m i c r o s c o p y o f f o o d s i n s e v e r a l
pages. A f e w s i m p l e d i a g r a m s a n d a s h o r t g l o s s a r y a r e g i v e n h e r e so t h a t t h e
b a s i c t e r m s and the c r u d e m o r p h o l o g y may be u n d e r s t o o d . T h o s e w i s h i n g to h a v e
a better understanding of p l a n t anatomy are r e f e r r e d to E s a u ' s , "Plant
An a t o m y " ( 9 ) and " A n a t o m y of S e e d P1 a n t s " ( 1 0 ) . T h e r e follows a brief
d e s c r i p t i o n of the s t r u c t u r e of the l e a f , seed and f r u i t . Although these are
the p a r t s of p l a n t s m o s t c o m m o n l y u s e d as f o o d t h a t r e q u i r e microscopical
e x a m i n a t i o n , other parts are also u s e d , for example bark (cimmamon, c a s s i a ) ,
r o o t s ( g i n g e r , t u r m e r i c ) , f l o w e r b u d s ( c l o v e s ) , s t i g m a t a and u p p e r p a r t s of the
styles ( s a f f r o n ) , etc.

F i g u r e 5 . 1 0 i l l u s t r a t e s the t r a n s v e r s e s e c t i o n t h r o u g h the m i d r i b of a tea l e a f

(Camellia s inens is). F i g u r e 5.11 is the same t r a n s v e r s e s e c t i o n , showing
elements in the cortex. Both illustrations are from the "Textbook of
Pharmacognosy" by Wallis(12).

Epidermis

Stele or vascular tissue ( d e r i v e d f r o m

the cambium, in its simplest f o r m ,
a cylinder one cell wide which produces
xylem on the inside and phloem on
the outside)

Ground tissue or c o r t e x

Figure 5.10

40
 dorsal surface

cuticle
epidermal layer
palisade layer
sTclerenchymatous idioblast
sclerenchyma
vessels showing spiral
and annular thickening
sieve tissue (phloem)
s toma cell containing chloroplasts
vessels in the rystals of calcium oxalate
wood (xylem)

hypoderma, a layer beneath the

epidermis distinguishable from
the surrounding tissue in some
ventral surface (the area near
plants
the vein may be referred to as
neural)
Figure 5.11

The following is a glossary of terms used in plant m i c r o s c o p y :

D o r s a l and V e n t r a l : For those leaves with a distinct upper and lower surface,
they may be referred to as dorsal and ventral respectively.

Epidermis: This is usually composed of a single layer of cells.

Cuticle: Composed of cutin, a w a x on the outer surface of the epidermal cells

making them more or less impervious to water. The cuticle is prominent on some
leaves as a b r o w n , s o m e w h a t crinkled layer.

Parenchyma: This is tissue from any part of the plant composed of cells which
have not differentiated to any special shape or c o m p o s i t i o n and are therefore
approximately rectangular in cross-section and are generally thin-walled. Such
cells m a y be described as isodiametric, i.e. all diagonals are of equal length.
(See F i g u r e 5.12).
intercellular
wall in surface view spaces

a thin-walled type of parenchyma, with regularly

shaped cells and schizogenous intercellular spaces,
from petiole of celery

Figure 5.12

Palisade layer: T h i s is o n e or t w o c e l l s t h i c k and o c c u r s u n d e r one or b o t h

epidermises. The c e l l s are t y p i c a l l y t a b u l a r ( p o l y g o n a l p r i s m s ) , w i t h no
intercellular s p a c e s .

41
Mesophyll: This includes all p a r e n c h y m a t o u s tissue b e t w e e n the two
epidermises, including the palisade layer and the spongy m e s o p h y l l which has a
characteristic open structure. The chloroplasts are found in m e s o p h y l l cells.

T r i c h o m e s (hairs): These are also often characteristic, and are most c o m m o n l y

found on leaves, but not confined to them. There are m a n y forms of trichomes,
some of which are shown in Figure 5.13 below.

Trichomes. A, simple hair from Cistus leaf. At its base is a compartment formed by deposition of a siliceous
wall. B, uniseriate hair from Saintpaulia leaf. C, D, tufted hair from leaf of cotton (Gossypium). E, stellate hair from leaf of
alkali mallow (Sida). F, dendroid hair from lavender leaf (Lavandula). G, short multicellular hair from leaf of potato (So-
lanum). H, I, peltate scale from leaf of olive (Olea). J, bicellular hair from stem of Pelargonium. K-M, cotton (Gossypium)
Epidermal hairs from seed (K) in young stage (L) and mature, with secondary walls (M). N, water vesicle of Mesem-
bryanthemum. O - O , hairs in three stages of development from leaf of soybean (Glycine).

Figure 5.13
Stomata: T h e s e are s p e c i a l c e l l s in the e p i d e r m a l l a y e r t h r o u g h w h i c h the
p l a n t r e g u l a t e s e x c h a n g e of w a t e r and g a s e s . A m o n g the d i c o t y l e d o n s , four
distinct types of stoma occur which are classified according to their spatial
relationship w i t h surrounding cells. They are (see Figure 5.14):

(a) Ranunculaceous or anomocytic

(b) Cruciferous or anisocytic
(c) Rubiaceous or paracytic
(d) Caryophyllaceous or diacytic

Epidermis in surface views illustrating patterns formed by guard cells and

neighboring cells.

Figure 5.14

Stomata m a y be above, b e l o w or level w i t h surrounding cells. They occur on the

aerial parts of plants, but more on leaves, especially the undersurface.

Idioblast: The term used to describe any cell radically different from those
n e a r it, e.g. c e l l s w h i c h c o n t a i n c a l c i u m o x a l a t e c r y s t a l s . Calcium oxalate
crystals show up well b e t w e e n crossed polaroids. They are c o m m o n l y found in
many parts of plants.

Pro8enchy»a: This n a m e may be applied to tissue m a d e up of cells in which the

l e n g t h g r e a t l y e x c e e d s the b r e a d t h . T h e y u s u a l l y h a v e p o i n t e d e n d s and
thickened walls.

Vessels: A type of cell t h r o u g h w h i c h w a t e r is c o n d u c t e d in the p l a n t . They

are found as long wide tubes with thickened walls and no living contents, the
e n d - w a l l s u s u a l l y p a r t l y or t o t a l l y b r o k e n d o w n , p e r f o r a t e d s e p t a s o m e t i m e s
o c c u r r i n g at i n t e r v a l s , o f t e n at an a n g l e to the t u b e w a l l s . Helically
thickened vessels are often prominent at the extreme ends of the veins in leaf
tissue. Water is conducted through the vessels in the living plant. Vessels
occur in the xylem.

43
Sieve tubes: These are also long, wide tubes, but the walls are not thickened.
The contents m a y still be alive, although restricted to near the walls, and the
cells are separated by sieve-plates. Sieve-tubes have alongside them companion
cells with dense granular contents. Nutrients are transported through sieve-
tubes in the living plant. Sieve-tubes and companion cells occur in the phloem
(see F i g u r e 5.15).

Cn
C=3
K
G H

Secondary wall structure in primary tracheary elements. T h e second-

ary thickenings are as follows: A, annular; B, partly annular, partly helical; C.
heiical; D, reticulate; E, scalariform-reticulate; F, pitted; G and H, annular in
an unstretched element ( G ) , and in a stretched element ( H ) . A-F, from an
Avena seedling; G and H, from a Zea seedling. (A-F, X450; G , H, X420.
A-F, drawn from photomicrographs in Goodwin, 1942.)

Figure 5.15

Fibres: F i b r e s m a y be f r o m the x y l e m , or f r o m o t h e r t i s s u e s ( e x t r a x y l a r y
fibres). These latter may arise from a n u m b e r of tissues, p h l o e m , cortex and
pericycle. The exact phylogenetic status of these fibres is usually of little
i n t e r e s t to the a n a l y s t . P e r i v a s c u l a r f i b r e s d e s c r i b e t h o s e that o c c u r in a
r i n g a r o u n d the v a s c u l a r s y s t e m , and if t h i s r i n g a r i s e s f r o m the p e r i c y c l e ,
m a y be called pericyclic. Cortical and phloem fibres arise from adjacent areas
and do n o t n e e d to be d i s t i n g u i s h e d for our p u r p o s e s . One m a y a l s o see
extraxylary fibres described as bast or bast fibres (see Figure 5.16).

44
 tracheids
Illustration of the main lines of specialization of the tracheary ele-
ments and fibers. E-G, long tracheids from primitive woods. E and F have
circular bordered pits; G has elongated bordered pits in scalariform arrangement.

Figure 5.16

Pits: T h e s e are a r e a s on t r a c h e i d s , vessels and sieve-tubes where no

thickening has taken place. (See Figure 5.16).

Tracheids: This is different from a vessel in that the e n d - w a l l s of the cell

are not perforated. Tracheids occur in xylem tissue. The conduction of water,
although generally in vessels, m a y occur through tracheids, w h i c h consist of
single c e l l s , p r o s e n c h y m a t o u s and u s u a l l y w i t h b o r d e r e d p i t s or s p i r a l
thickening. T r a c h e i d s are found in l e a v e s and in w o o d . Coniferous wood
c o n s i s t s of long t r a c h e i d s w h i c h look r a t h e r like f i b r e s w i t h b o r d e r e d pits.
Thus it is a f a i r l y s i m p l e m a t t e r to c l a s s i f y s a w d u s t as f r o m s o f t or h a r d
wood.

45
Sclerenchyma: In this type of c e l l , l i g n i n h a s b e e n d e p o s i t e d in and a r o u n d
the c e l l - w a l l . The t i s s u e is i d e n t i f i e d by b e i n g s t a i n e d s c a r l e t with
p h 1 o r o g 1 u c i n o 1 and h y d r o c h l o r i c acid. Sclereids (stone-cells) and fibres are
c o m m o n forms. The presence of stone cells accounts for the "gritty" feeling of
the c e n t e r of p e a r s . F i b r e s l a r g e l y a c c o u n t for the m e c h a n i c a l s t r e n g t h of
plants and their fibrous derivatives, such as flax, cotton, sisal.

Collenchyna: T h i s is y o u n g t i s s u e in w h i c h the c e l l s b e c o m e t h i c k e n e d , e v e n
though still alive. The thickening takes a characteristic form.

Cell Inclusions: These include calcium oxalate, starch, aleurone (protein)

grains and oil droplets. Aleurone grains are characteristic of oil-containing
seeds and the term is also applied to the small protein-containing granules of
leguminous seeds and cereals.

Secretory Cells: These are often useful in diagnosis, the laticiferous (latex
or inilk producing) cells of Euphorbiaceae being examples.

Starches: These tend to be characteristically different in different plants.

The Seed: A seed d e v e l o p s f r o m the f e r t i l i z e d o v u l e s i t u a t e d at the b a s e of

the flower. The ovule contains a zygote, which develops into the e m b r y o , and a
p r i m a r y endosperm nucleus from which the endosperm is formed. Several layers
(or coats) surround the ovule: the outer seed coat is called the testa and the
inner is the tegumen, with parenchyma tissue b e t w e e n them called the nucellus.
Structures which m a y be detectable on the outer surface of the seed are: The
m i c r o p y l e , w h i c h is a small aperture through which air and w a t e r enter the seed
and the hilum, which is the scar of the stalk by w h i c h the seed w a s attached to
the f r u i t . An a r i l is a f l e s h y c o v e r i n g w h i c h , in s o m e s e e d s , d e v e l o p s f r o m
the hilum. A c o m m o n example is m a c e , the aril covering a nutmeg. An arillode
is a similar fleshy covering but which arises from around the m i c r o p y l e , as in
cardamom. T h e r e is g r e a t d i v e r s i t y in the form of s e e d s , d e p e n d i n g in p a r t
u p o n w h e t h e r t h e s e v a r i o u s t i s s u e s a c t u a l l y d e v e l o p and at w h a t r a t e . A
d i a g r a m m a t i c r e p r e s e n t a t i o n is g i v e n in F i g u r e 5.17 of a seed in w h i c h all
these tissues/structures are shown.

Figure 5.17

The various seed parts (as represented in Figure 5.17) are:

E m b r yo: The e m b r y o p o s s e s s e s the r u d i m e n t s of the p l a n t into w h i c h it w i l l

develop, that is, the cotyledon or cotyledons, which s o m e t i m e s contain storage
m a t e r i a l and o f t e n h a v e the c e l l u l a r s t r u c t u r e of the l e a v e s into w h i c h they
w i l l d e v e l o p ; a h y p o c o t y l w h i c h d e v e l o p s i n t o a r o o t and an e m b r y o n i c s h o o t ,
the p l u m u l e , which develops into the stem.

46
Endosperm: G e n e r a l l y c o m p o s e d of ce 1 1 u l o s e - w a 1 1 e d p a r e n c h y m a c e l l s w h i c h
c o n t a i n food, such as the p r o t e i n r e s e r v e s , a l e u r o n e g r a i n s . They are only
found in seeds and are therefore diagnostic.

Perisperm: The perisperm usually consists of thin-walled parenchymatous cells

containing abundant starch grains, as in cardamom and pepper.

Testa: The seed coat structure is often very characteristic and may he useful
in i d e n t i f y i n g p o w d e r s . U s u a l l y it c o n s i s t s of four l a y e r s : the e p i d e r m i s ,
the p i g m e n t l a y e r , the s c l e r e n c h y m a t o u s layer and the n u t r i e n t layer of the
testa.

1. The Epiderm is is variously developed in different seeds and may take

the following forms:

A p a l i s a d e layer w i t h cells in the form of p o l y g o n a l prisms,

e g. s o y b e a n .

b. A layer of sclereids, e.g. capsicum.

c. A layer of scattered sclereids, e.g. almond.

d. A mucilaginous layer, e.g. mustard.

e. A trichome-bearing layer, e.g. cotton •

f. A layer of thin-walled prosenchyma, e •g. cardamom.

g- Stomata occur only occasionally, but do appear in cotton

2. The P i g m e n t L a y e r . In all c o l o u r e d seeds, pigment is d e p o s i t e d in

one of the layers of the testa.

3. The Sclerenchymatous Layer. In the majority of seeds, the cells of

one or more of the layers of the testa have thickened walls.

4. The Nutrient Layer of the Testa. Generally several cells thick, and
consists of thin-walled parenchyma, which eventually becomes flattened and is
full of starch.

The F r u i t : The ovary wall or the walls of the carpels (which enclosed ovules
in the f l o w e r ) d e v e l o p to form a p e r i c a r p w h i c h acts as a case for the seeds
and is called a fruit. The fruit can e i t h e r be dry as in c a r a w a y s e e d s , or
fleshy as in a p p l e or c h e r r y . The p e r i c a r p d i f f e r e n t i a t e s into e n d o c a r p ,
mesocarp and exocarp (see Figure 5.18).

47
F r u i t s m a y be s i m p l e ( f r o m one c a r p e l , e.g. p r u n e , o l i v e ) , or a g g r e g a t e
( r a s p b e r r y ) , or c o m p o u n d ( f r o m a n u m b e r of f l o w e r s , e.g. fig, m u l b e r r y , red
pepper).

The epicarp and endocarp m a y consist of only an epidermal layer and the epicarp
m a y h a v e a few s t o m a t a . The e p i c a r p of p e p p e r and c u b e b c o n s i s t of the
e p i d e r m i s p l u s h y p o d e r m a l l a y e r of s c l e r e i d s and p a r e n c h y m a . It is the
e n d o c a r p of u m b e l l i f e r o u s f r u i t s t h a t f o r m s the p a r q u e t r y l a y e r w h i c h is
important in their identification. In citrus fruits the flavedo or rind forms
the epicarp, the white tissue or albedo forms the m e s o c a r p and the edible flesh
is composed of modified hairs derived from the endocarp. The m e s o c a r p m a y be
s u c c u l e n t as in t a m a r i n d s or p i t h y as in c o l o c y n t h or it m a y be c o m p o s e d of a
spongy parenchyma as in lobelia.

Preparing Specimens and Clearing Agents

S t a r c h y s p e c i m e n s s u c h as f l o u r s a r e u s u a l l y m o u n t e d in a l c o h o l or
m e t h a n o 1 : g 1 y c e r o 1 (1:1). If it is n e c e s s a r y to p r e p a r e s e c t i o n s of s t a r c h y
r e f e r e n c e s p e c i m e n s such as c e r t a i n s e e d s , it is c o n v e n i e n t to s o a k t h e m
overnight in glycerol to soften the specimen. Starch granules gradually swell
in water. Water is therefore not a suitable m e d i u m in w h i c h to examine them.
N o n - s t a r c h y p o w d e r s m a y be m o u n t e d in a d r o p of w a t e r for a p r e l i m i n a r y
examination. It is important to take only a tiny amount of the sample so that
individual particles are completely separated from each other. If the sample
is too heterogeneous to allow all of the different elements present to appear
on one slide, it is better to prepare several slides rather than try to crowd a
confusing mass of the sample on a single slide.

Cellulosic elements in plants are resistant to solvents such as chloral hydrate

and dilute sodium hydroxide solution and it is therefore convenient to boil a
small amount of the sample with one of these clearing agents. Chloral hydrate
s o l u t i o n is u s u a l l y p r e p a r e d by d i s s o l v i n g 7 g of the s o l i d in 5 m l of w a t e r .
2% s o d i u m h y d r o x i d e is a l s o e f f e c t i v e . If the a m o u n t of c e l l u l o s i c m a t e r i a l
p r e s e n t in a s a m p l e is s m a l l it m a y be n e c e s s a r y to c e n t r i f u g e the c l e a r e d
s p e c i m e n and e x a m i n e the d e p o s i t . Schulze's m a c e r a t i o n fluid (potassium
chlorate dissolved in concentrated nitric acid) is effective in breaking woody
tissue into its component cells. The specimen is usually left to soak in the
l i q u i d at r o o m t e m p e r a t u r e . S o d i u m h y p o c h l o r i t e s o l u t i o n (or a s o l u t i o n of
bleaching p o w d e r ) is useful in lightening the colour of m a t e r i a l such as tea.
Some solutions are used diagnostically. Iodine solution (in aqueous Kl) gives
a blue colour with starch cells and a b r o w n colour with aleurone grains. Fat-
soluble dyes such as Sudan III and Sudan Blue are absorbed by any oil globules
p r e s e n t in the s p e c i m e n ( e x a m i n e d in a l c o h o l ) . Hydrochloric acid and
phloroglucinol (1% in alcohol) stain lignin red. Corallin soda stains m u c i l a g e
pink.

5.8 TEXT REFERENCES

1. H A I S , I.M. & M A C E K , K. 1 963. Paper Chromatography; A C o m p r e h e n s i v e

Treatise. P u b l i s h i n g H o u s e of the C z e c h o s l o v a k A c a d e m y of S c i e n c e s ,
Vodickova 40, 11229 Prague and Academic Press.

2. H O D G M A N , C.D., W E A S T , R.C. & S E L B Y , S.M. 1960. Handbook of C h e m i s t r y &

Physics 43rd Edition. CRC Press Inc.

3. D ' A N S , J. & L A X , E. 1949. Taschenbuch fur Chemiker und Physiker.

Berlin, Springer.

4. PAIN, S.C. 1959. Journal of Chromatography, 2, 433.

5. A N A N D A R A M A N , S., S H A N K A R A C H A R Y A , N.B., N A T A R A J A N , C.P., & D A M O D A R A N , N.P.

1977. Laboratory Practice, 26_, (11 ), 870.

48
6. BROCKMANN, H. & SCHODDER, H. 1941. Chemische Berichte, 74, 73.

7. GRAY, R.A.C. 1978. Laboratory Equipment Digest, 16, 85.

8. M a c L E O D , A.J. 1973. I n s t r u m e n t a l M e t h o d s of F o o d A n a l y s i s . Paul Elek

(Scientific Books) Ltd. n o w Granada Publishing Ltd. (o.o.p.)

9. MOODY, B. & LE FLEUR, D. 1974. Accuracy in Trace Analysis, 1: Sampling,

S a m p l e H a n d l i n g , A n a l y s i s ; P r o c e e d i n g s of the 7th M a t e r i a l s R e s e a r c h
S y m p o s i u m , National Bureau of Standards special publication 422, USA.

10. EASU, K. 1962. Plant Anatomy. Wiley.

11. EASU, K. 1960. Anatomy of Seed Plants. Wiley.

12. WALLIS, T.E. 1962. Text Book of Pharmacognosy. Churchill.

Further Reading

The b i b l i o g r a p h y g i v e n h e r e i n c l u d e s t e x t s r e l a t i n g to m a t e r i a l s o t h e r t h a n
foods, as such materials m a y have to be identified w h e n present as adulterants
in samples. Information relating to microscopy tends to be s o m e w h a t scattered,
and some of the better texts are n o w out of print (marked o.o.p. below).

General

EWING, G.W., 1975. Instrumental Methods of Chemical Analysis. McGraw-Hill.

W I L L A R D , A.J., M E R R I T T , L.L., and D E A N , J.A. 1 9 74. Instrumental Methods of

Anal ys i s . Van I'ostrand, London.

Liquid Chromatography

B R I S T O W , P.A. 1976. Liquid Chromatography in P r a c t i c e . HETP Publications,

W i l m s l o w (England).

H A M I L T O N , R.J. & S E W E L L , P.A. 1978. I n t r o d u c t i o n to H i g h P e r f o r m a n c e Liquid

Chromatography. Chapman & Hall, London.

J O H N S O N , E.C. & S T E V E N S O N , R. 1978. Basic Liquid Chromatography. Varian

Aerograph, Walnut Creek (California).

S N Y D E R , L.R. & K I R K L A N D , J.J. 1979. Introduction to Modern Liquid

Chromatography. Wiley, New York.

MACRAE, R. 1981. Journal of Food Technology, 16 1-11.

MACRAE, R. (Ed). 1982. HPLC in Food Analysis. Academic Press, London.

Gas Chromatography

AMBROSE, D. 1971. Gas Chromatography. Butterworth, London.

D I C K E S , G.J. & N I C H O L A S , P.V. 1976. Gas Chromatography in Food Analysis.

Butterworth, London.

49
G R O B , R.L. (Ed). 1977. Modern Practice of Gas Chromatography. Wiley
Interscience , New York.

M c N A I R , H.M. & B O N E L L I , E.J. 1969. Basic Gas Chromatography. Varian

Aerograph, Walnut Creek (California).

Atomic Absorption Spectroscopy

C A N T L E , J.E. (Ed). 1982. Atomic Absorption Spectrometry. Elselvier,

Amsterdam.

P R I C E , W.J. 1974. Analytical Atomic Absorption Spectrometry. Heyden, London.

R E Y N O L D S , R.J. & A L D O N S , K. 1970. Atomic Absorption Spectroscopy: A

Practical Guide. Griffin, London.

Thin-Layer Chromatography

KIRCHNER, J.G. 1978. Thin Layer Chromatography. Wiley, New York.

STAHL, E. (Ed). 1969. Thin Layer Chromatography. George Allen & Unwin,
London.

G A A L , 0 . , M E D G Y E S I , G.A., V E R E C Z K E Y , L. 1980. Electrophoresis in the

S e p a r a t i o n of B i o l o g i c a l M a c r o m o l e c u l e s . Wiley, Interscience, New York.

SHAW, D.J. 1969. Electrophoresis. Academic Press, London.

Immunoassays and ELISA

V O L L E R , A., B A R L E T T , A. & B I D W E L L , D. 1981. Immunoassays for the 8 0 ' s . MTP

Près s .

V O L L E R , A., B I D W E L L , D.E. & B A R T L E T T , A. 1979. The Enzyme Linked

Immunosorbent A s s a y (ELISA). MTP Press.

Infra—red Spectroscopy

C R O S S , A.D. & A L A N - J O N E S , R. 1969. An Introduction to P r a c t i c a l Infra-Red

Spectroscopy. Butterworth, London.

W I L L I A M S , D.H. & F L E M I N G , I. 1966. Spectroscopic Methods in O r g a n i c Analysis.

McGraw-Hill, New York.

Electrochemical

DELAHAY, P. 1958. New Instrumental Methods in E 1 e c t r o c h e m i str

Interscience, New York.

50
Microscopy

J A C K S O N , B.P. & S N O W D E N , D.W. 1974. Powdered Vegetable Drugs. Stanley

Thornes, London.

MASON, C.W. 1983. Handbook of Chemical Microscopy. Wiley, New York.

WALLIS, T.E. 1965. Analytical Microscopy. Churchill, London.

GREENISH & COLLIN. 1904. An Anatomical Atlas of Vegetable Powders. Churchill

o.o.p. (some of the m o n o g r a p h s are r e p r i n t e d in the r e l e v a n t p a r t s of this
Manual).

K U R T Z O'D.L. & H A R R I S , K.L. 1962. Micro-analytical Entomology for Food

Sanitation Control. Association of Official Analytical Chemists.

V A U G H N , J.G. 1970. The S t r u c t u r e and U t i l i z a t i o n of O i l s e e d s . C h a p m a n and

Hall.

BURRELLS, W. 1961. Industrial Microscopy in Practice. Fountain Press.

W R E N , R.C. 1975. P o t t e r ' s N e w C y c l o p a e d i a of B o t a n i c a l D r u g s a n d

P r e p a r a t ions. H e a l t h S c i e n c e P r e s s (gives a lot of u s e f u l i n f o r m a t i o n on
morphology, but not microscopy, of many plants).

WINTON & WINTON. 1932-1939. The Structure and Composition of F o o d s . 4

volumes, Wiley, o.o.p.

C O M M I S S I O N G E N E R A L E D ' U N I F I C A T I O N DES M E T H O D E S D ' A N A L Y S E S . 1966. Annexes

Microphotographique aux Methodes Officielles d'Analyse. 2 Vols., Paris.

CLAUSE, E.P. 1961. Pharmacognosy. 4 Ed. Lea and Febiger.

YANNGKEN. 1948. Textbook of Pharmacognosy. 6th Ed.

D e t a i l s r e l a t i n g to s o m e h e r b s and spices still used as b o t h c o n d i m e n t s and

drugs will be found in various pharmacopoeias or their companion volumes such
as the British Pharmaceutical Codex.

P E A R S O N , D. 1981. The C h e m i c a l A n a l y s i s of F o o d s . Churchill Livingstone,

(includes drawings of some herbs and beverages).

KENT-JONES, D.W. & A M O S , A.J. 1957. Cereal Chemistry. The Northern

Publishing Co. 5th Ed.

PARKINSON, S.T. & FIELDING, W.L. 1930. The Microscopic Examination of Cattle
Foods. Invicta Press, Ashford, Kent, England.

HAMENCE, J. HUBERT. 1966. The Determination of the Composition or Purity of

A n i m a l F e e d i n g S t u f f s by M i c r o s c o p y . J o u r n a l of the A s s o c i a t i o n of P u b l i c
A n a l y s t s 4(1), 15-20.

PRANDL. 1961. Die Histologische Analyse von Wurstwaren. 0. Rostger, Munich.

R A D L E Y , J.A. 1 97 6. E x a m i n a t i o n and A n a l y s i s of S t a r c h and S t a r c h Products.

Applied Ipience.

51
W H I T E , G.W. & SHENTON are p u b l i s h i n g e x c e l l e n t r e v i e w s of the literature
r e l a t i n g to f o o d m i c r o s c o p y in t h e J o u r n a l of the A s s o c i a t i o n of Public
A n a l y s t s ( J A P A ) , as f o l l o w s :

General History JAPA 1.(3), 74-80 ( 1 9 7 0 )

General Texts & Reviews JAPA 12(1) 27-31 ( 1 9 7 4 )
Cereals & Flours JAPA 12.(3) 77-81 ( 1 9 7 4 )
Starch & Gluten JAPA 13(1) 34-38 ( 1 9 7 5 )
E g g s & Egg P r o d u c t s JAPA 13(4) 135-138 ( 1 9 7 5 )
Fats & Oils JAPA 14(3) 113-117 ( 1 9 7 6 )
Milk & Milk Powder JAPA 15(1) 33-37 ( 1 9 7 7 )
Sugars JAPA 15(4) 141-144 ( 1 9 7 7 )
Water JAPA 17.(2) 79-81 ( 1 9 7 9 )
Bakery & Allied Products JAPA 17(3) 111-114 ( 1 9 7 9 )
Beverages JAPA 18(2) 63-66 ( 1 9 8 0 )
Chocolate & Confectionery JAPA 18(4) 129-132 ( 1 9 8 0 )
Dairy Products JAPA 19(3) 105-111 ( 1 9 8 1 )
Butter & Margarine JAPA 20(2) 51-54 ( 1 9 8 2 )
Fruits & Nuts JAPA 20(1) 25-30 ( 1 9 8 2 )
General Kitchen Products JAPA 20(3) 99-101 ( 1 9 8 2 )
Meat & Fish JAPA 21(1) 29-33 ( 1 9 8 3 )
Vegetable Products JAPA 21(3) 93-98 ( 1 9 8 3 )

52
 6- FOOD ADDITIVE METHODS

6.1 PRESERVATIVES

Preservatives can be generally defined as chemicals inhibiting or reducing the

g r o w t h of b a c t e r i a or o t h e r m i c r o b i a l flora. S u b s t a n c e s w h i c h h a v e an
a n t i o x i d a n t f u n c t i o n m a y also serve as p r e s e r v a t i v e s , but are d e a l t w i t h in
another section.

N a t i o n a l l e g i s l a t i o n r e l a t i n g to p r e s e r v a t i v e s is e x t r e m e l y v a r i e d but in
g e n e r a l , it is u s u a l l y n e c e s s a r y to carry out q u a l i t a t i v e t e s t s in o r d e r to
ensure that non-permitted preservatives are absent and quantitative analysis to
ensure that permitted preservatives do not exceed any legal limit there may be.

In a d d i t i o n to the c h e m i c a l p r e s e r v a t i v e s d i s c u s s e d h e r e , it should not be

forgotten that salts, especially c o m m o n salt itself, and acids such as vinegar,
sodium diacetate and fruit acids all have a preservative action. Non-specific
a n a l y s i s such as ash and a c i d i t y d e t e r m i n a t i o n s are u s u a l l y s u f f i c i e n t to
establish that their use has not been excessive. Exception can reasonably be
taken to the presence of an excessive amount of alum.

One of the m o s t c o m m o n p r e s e r v a t i v e s used for canned or b o t t l e d foods is

benzoic acid, usually as its sodium, potassium or calcium salt. Benzoic acid
retards the growth of moulds and yeasts and works best in food products having
an acid pH of 2.5 to 4.0. Its effectiveness decreases dramatically when the pH
rises a b o v e 5.0.

A group of effective benzoic acid derivatives are compounds c o m m o n l y referred

to as "parabens". T h e s e are m e t h y l , e t h y l or p r o p y l p - h y d r o x y - b e n z o a t e s .
Their main usefulness is that they are effective over a wider range of pH than
benzoic acid and are quite stable.

Sorbic acid ( 2 , 4 - h e x a d i e n o i c a c i d ) is o f t e n used in d a i r y p r o d u c t s such as

cheese to inhibit mould growth, as it does not affect the lactobacilli present.
Like benzoic, it is added as the sodium, potassium or calcium salt.

A n o t h e r w i d e l y used p r e s e r v a t i v e is s u l p h u r d i o x i d e . It is added to foods

primarily as the sulphite, bisulphite or metabisulphite of sodium, potassium or
calcium. It is p r i m a r i l y a p r e s e r v a t i v e , but h a s a n t i o x i d a n t p r o p e r t i e s as
well and is often used to prevent browning.

Analytical methods for other chemicals having preservative action, which are
used only with specific foods, are given in the Manual, "Food Analysis: General
Q u a l i t y , A d u l t e r a t i o n , Identity". T h e s e i n c l u d e n i t r i t e / n i t r a t e in m e a t
products and boric acid in fish.
 BENZOIC ACID
(Quantitative M e t h o d )

PRINCIPLE

B e n z o i c acid m a y be e x t r a c t e d f r o m a l i q u i d s a m p l e u s i n g c h l o r o f o r m , a f t e r
saturating with s a l t , or m a y be s t e a m d i s t i l l e d . P e r m a n g a n a t e is u s e d to
destroy unwanted acidic substances. Spectrophotometry is the preferred method
to q u a n t i f y , b u t the e x t r a c t e d b e n z o i c acid can be t i t r a t e d u s i n g s t a n d a r d
sodium hydroxide solution (1 ml of 0.05 N NaOH = 0.0072 gm sodium benzoate).,

APPARATUS

1. Separatory funnels.

2. Steam distillation apparatus.

3. Spectrophotometer, preferably double beam.

REAGENTS

1. Ortho-phosphoric acid.

2. Sodium chloride.

3. 1 N sodium hydroxide.

4. 0.1 N sodium hydroxide.

5. 5% potassium permanganate.

6. Sodium sulphite.

7. Diethyl ether/petroleum ether BP 40-60° (1:1).

8. 10% sodium hydroxide.

9. Dilute hydrochloric acid (1+3).

10. Chloroform.

11. Diethyl ether.

•12. Benzoic acid standard in ether (5 mg/100 ml).

PROCEDURE

Solvent Extraction

M i x sample thoroughly, grinding if solid or semi-solid. Transfer 150

m l or 150 g to a 500 m l volumetric, flask, add enough sodium chloride
to s a t u r a t e the w a t e r in the s a m p l e , m a k e a l k a l i n e to l i t m u s p a p e r
w i t h 3.0 X s o d i u m h y d r o x i d e s o l u t i o n and d i l u t e to v o l u m e with
saturated sodium chloride solution. Shake thoroughly and let stand
for at least two hours, shaking frequently and filter. If the sample
c o n t a i n s l a r g e a m o u n t s of f a t , p o r t i o n s of w h i c h m a y c o n t a m i n a t e
f i l t r a t e , add a f e w m l of 10% s o d i u m h y d r o x i d e to f i l t r a t e and
extract with ether before proceeding with the determination.

For p r o d u c t s c o n t a i n i n g a l c o h o l , m a k e 2 5 0 m l of s a m p l e a l k a l i n e to
litmus paper with 10% sodium hydroxide and evaporate on a steam bath
to a b o u t 100 m l . T r a n s f e r to a 2 5 0 m l v o l u m e t r i c f l a s k , add 30g

54
s o d i u m c h l o r i d e and s h a k e u n t i l d i s s o l v e d . D i l u t e to 2 5 0 m l w i t h
saturated sodium chloride solution, let stand for at least 2 hours,
shaking frequently and filter.

For salted or dried products such as fish, wash 50 g of ground sample

into a 500 m l volumetric flask with water. M a k e slightly alkaline to
litmus paper with 10% sodium hydroxide solution and dilute to v o l u m e
with water. Let s t a n d for at l e a s t 2 h o u r s , s h a k i n g f r e q u e n t l y and
filter. Pipette as large a measured v o l u m e of filtrate as possible (
300 m l ) into a second 500 m l volumetric flask and add 30 g of sodium
chloride for each 100 ml of solution. Shake until salt dissolves and
d i l u t e to v o l u m e w i t h s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n . Mix
thoroughly and filter off precipitated protein and other extraneous
matter.

Pipette 100-200 ml of filtrate into a separating funnel. Neutralise

to l i t m u s w i t h d i l u t e h y d r o c h l o r i c a c i d and add 5 m l e x c e s s . With
salted fish, protein usually precipitates with the acid but this does
not i n t e r f e r e w i t h the e x t r a c t i o n . Extract carefully with
c h l o r o f o r m , u s i n g s u c c e s s i v e p o r t i o n s of 70, 50, 40 and 30 m l . To
avoid f o r m a t i o n of e m u l s i o n , s h a k e c a u t i o u s l y e a c h t i m e u s i n g a
rotary motion. The chloroform layer usually separates readily after
s t a n d i n g for a f e w m i n u t e s . If an e m u l s i o n f o r m s , b r e a k it b y
stirring the chloroform layer with a glass rod, by drawing off into a
s e c o n d s e p a r a t o r and g i v i n g one or t w o s h a r p s h a k e s f r o m o n e end of
s e p a r a t o r to the o t h e r , or by c e n t r i f u g i n g for z. f e w m i n u t e s . As
this is a p r o g r e s s i v e e x t r a c t i o n , c a r e f u l l y d r a i n as m u c h c l e a r
chloroform as possible after each extraction, but do not drain any of
the emulsion. If this precaution is taken, the chloroform layer need
not be washed.

Transfer the combined chloroform extracts to a porcelain evaporating

dish, rinse container several times with a few m l of chloroform, and
evaporate to dryness at room temperature in a current of dry air.

Steam Distillation

P l a c e 3 0 - 1 0 0 g s a m p l e in a 5 0 0 m l s t e a m d i s t i l l a t i o n f l a s k . Add
s u f f i c i e n t w a t e r a n d an e x c e s s of s a l t (40 g / 1 0 0 m l ) . Make
d i s t i n c t l y acid w i t h o r t h o p h o s p h o r i c acid and rapidly steam distil
5 0 0 m l i n t o a f l a s k c o n t a i n i n g 10 m l of 1 N s o d i u m h y d r o x i d e . Wash
d o w n the c o n d e n s e r w i t h 25 m l of 0.1 N s o d i u m h y d r o x i d e into the
receiving flask. Evaporate the alkaline distillate d o w n to about 20
m l on a s t e a m b a t h . A l l o w to cool to a b o u t 45°C and add p o t a s s i u m
p e r m a n g a n a t e solution until a pink colour persists after stirring.
Decolourise with sodium sulphite and add sufficient dilute sulphuric
acid to d i s s o l v e the p r e c i p i t a t e d m a n g a n e s e d i o x i d e and m a k e the
liquid acid.

T r a n s f e r to a s t o p p e r e d s e p a r a t o r y f u n n e l , s a t u r a t e w i t h salt (33
g/'lOO m l w a t e r ) and extract the benzoic acid w i t h four successive 15
m l p o r t i o n s of e t h e r / l i g h t p e t r o l e u m . C o m b i n e and t r a n s f e r all
e x t r a c t s to a b e a k e r and e v a p o r a t e o f f the s o l v e n t by m e a n s of a
current of dry air, while holding the beaker in a water bath at 30°C.

Determination

D i s s o l v e the r e s i d u e from e i t h e r the s o l v e n t e x t r a c t i o n or s t e a m

d i s t i l l a t i o n in s u f f i c i e n t d i e t h y l e t h e r to m a k e a s o l u t i o n of 5 g
equivalent of s a m p l e per 100 ml. (This a s s u m e s a sample
concentration of 0.1% benzoic acid. Adjust v o l u m e s if the expected
amount differs from this.)

55
 Scan both the sample and benzoic standard solutions between 240 and
300 nm, using ether as the reference in each case.

Record the absorbances at the maxima of 272 nm and the two minima of
267.5 and 276.5 nm. Also compare the overall scans of both standard
and sample. They should have identical shapes, even though they may
differ in magnitude.

CALCULATION

ppm benzoic acid = -AiL x (.05)(1000)(V)

where :
As = absorbance of the sample solution at 272 nm minus
the average of the a b s o r b a n c e s at 267.5 and
276.5.

Ax = absorbance of the standard solution calculated as

for the sample.

V = ml of ether used to dissolve sample residue.

w = g equivalent of sample in final solution.

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 24.024-.028.

 BENZOIC ACID
(Qualitative Identification)

PRINCIPLE

The UV scan of benzoic acid, used in the quantitative procedure, is distinctive

but is not an a b s o l u t e i d e n t i f i c a t i o n . In the event that c o n f i r m a t i o n of
identification is needed, use the following thin-layer chromatography method.

APPARATUS

1. Thin-layer apparatus including tanks, plates and spotting

pipettes.

2. Viewing cabinet with shortwave (254 nm) UV light.

REAGENTS

1. Kieselguhr G.

2. Silica gel GF 254.

3. n-Hexane: acetic acid solution (96 + 4).

4. Benzoic acid standard solution (1 mg/ml in ether).

PROCEDURE

Prepare thin-layer plates as follows: In 250 ml conical flask, mix

10 g each of k i e s e l g u h r G and silica gel GF 254. Add 45 m l of w a t e r
and shake 30 seconds. Set applicator at 0.25 mm. Apply mixture to 5
glass plates. Air dry for 10 minutes and dry in an oven at 100°C for
1 hour.

D i s s o l v e the residue extracted from the s a m p l e (see " Q u a n t i t a t i v e

M e t h o d " ) in ether (not m o r e than 2 ml). Spot 50 V 1 s a m p l e s o l u t i o n ,
using 10 y 1 syringe (5x) on prepared plate. Also, spot 50 p 1 benzoic
acid standard solution. Spot s a m p l e ( s ) and standard about 2.5 cm
from b o t t o m edge of plate and 4 cm a p a r t , b e g i n n i n g 2.5 cm in from
side edge. Draw a line across the plate 10 cm above the base line.

Place plate in c h r o m a t o g r a p h i c tank c o n t a i n i n g 2 5 0 - 3 0 0 ml m o b i l e

solvent (n-hexane:acetic (96 + 4)). Develop chromatogram for 10 cm.
Remove from tank and air dry for 5 minutes.

Observe the plate under UV s h o r t w a v e light (254 nm). S a m p l e and

standard benzoic acid appear as dark b l u e - p u r p l e spots on light
fluorescent background. W i t h a sharp point (e.g. pin or p e n c i l )
circle spots from edge of each spot. (This gives exact location of
each acid w h e n plate is r e m o v e d from the light). C a l c u l a t e and
compare RF value of standard and sample solution.

INTERPRETATION

If the sample streaks or otherwise does not present a discrete spot, the sample
residue m a y have to be further purified by d i s s o l v i n g in an a l k a l i n e aqueous
solution, extracting with chloroform (discarding the chloroform), then making
distinctly acid (at least pH 4) and re-extracting with chloroform. Evaporate
the chloroform, dry the residue overnight in a desiccator and repeat the thin-
layer identification analysis.

REFERENCE

Official M e t h o d s of A n a l y s i s of the AOAC, 1984, 20.029-.033.

57
 PARABENS
(Semi-quantitative Method)

PRINCIPLE

"Parabens" are p-hydroxybenzoates. This method determines their presence using

thin-layer chromatography. T h e i r c o n c e n t r a t i o n can be s e m i - q u a n t i t a t i v e 1 y
estimated by comparing the sample to standards of differing concentration.

APPARATUS

1. Separatory funnels.

2. Thin-layer chromatography apparatus.

3. Viewing cabinet with shortwave (254 nm) UV light.

REAGENTS

1. Mobile solvent-toluene¡methanol : acetic acid (90:16:8).

2. 2% s t a n d a r d s o l u t i o n s in e t h e r of methyl, ethyl and propyl

hydroxy-benzoates and the free acid.

3. Deniges reagent: m i x 5 g yellow mercuric oxide w i t h 40 ml water,

c o o l in i c e / s a l t and v e r y s l o w l y add f r e e z i n g - c o 1 d concentrated
sulphuric acid (20 m l ) with stirring. Add another 40 m l of water.

4. TLC plate coated w i t h silica gel containing a pigment

fluorescent under short w a v e UV.

5. Sulphuric acid 10% by volume.

6. 2% sodium nitrite solution, freshly prepared.

7. Sodium sulphate.

8. Methanol.

PROCEDURE

Add 5 m l 10% s u l p h u r i c acid to 10 g of food and g r i n d w i t h s o d i u m

s u l p h a t e in a m o r t a r u n t i l the s a m p l e is dry. Add a b o u t as m u c h
sodium sulphate again. G r i n d the p o w d e r w i t h s m a l l successive
quantitites of ether and decant the ether through a filter paper into
a flask. Evaporate the ether at as low a temperature as possible and
d i s s o l v e the r e s i d u e in 0.5 m l of m e t h a n o l . S p o t 20 m i c r o l i t r e s of
sample and about the same amount of the 2% standards on the TLC plate
and run the c h r o m a t o g r a m in the toluene:methanol:acetic acid solvent.

p - H y d r o x y b e n z o a t e s s h o w b l a c k u n d e r s h o r t - w a v e UV. Interfering
substances m a y be present so caution should be used in interpreting
the r e s u l t s . M a r k any q u e n c h e d a r e a s l i g h t l y w i t h a p i n , and s p r a y
lightly with Deniges reagent. p-Hydroxybenzoates give a white spot,
visible by its different reflectivity from the background. Heat at
100°C for five minutes and spray lightly w i t h fresh 2% sodium nitrite
solution. Any spots b e c o m e red.

INTERPRETATION

If t h e o b j e c t o f t h e t e s t is to c o n f i r m t h a t t h e a m o u n t s o f a n y p-
hydroxybenzoates present are below a legal m a x i m u m , the quantities of standards
s p o t t e d can be c h o s e n to c o r r e s p o n d to that m a x i m u m . S a m p l e s p o t s of l o w e r
i n t e n s i t y are t a k e n to i n d i c a t e c o m p l i a n c e . If q u a n t i t a t i v e a n a l y s i s is

58
necessary the method of Thackray and Hewlett (1) m a y be followed. Recoveries
are about 85% except with certain samples such as coffee essence (about 50%).
D i c k e s d i s c u s s e s f a l s e p o s i t i v e s o b t a i n e d b y this m e t h o d . The solvent
e x t r a c t i o n is n e c e s s a r y as, u n l i k e b e n z o i c and s o r b i c a c i d , the free acid is
not steam-volatile and the esters are only partially so.

REFERENCE

Dickes, G.T., 1965. Journal of the Association of Public Analysts _3, 73-75.

59
 SORBIC ACID

PRINCIPLE

This analysis involves steam d i s t i l l a t i o n of the free acid, o x i d a t i o n to

malonaldehyde using dichromate and reaction with thiobarbituric acid to form a
red complex. This is determined spectrophotometrically at 532 nm.

APPARATUS

1. Steam distillation apparatus.

2. Volumetric flasks, 50 ml, 100 ml, 500 ml, 1 litre.

3. Boiling water bath.

4. Spectrophotometer.

REAGENTS

1. Magnesium sulphate, heptahydrate .

2. Sulphuric acid solutions, 1 N and 0.01 N.

3. Sodium hydroxide solution, 1 N.

4. D i c h r o m a t e solution: m i x equal v o l u m e s of 0.3 N H 2 S O 4 and a

solution of 0.5 g ^ C ^ O y in one litre of water. Prepare fresh as
needed.

5. Thiobarbituric acid (TBA) solution: dissolve 0.5 g TBA in 25 ml

w a t e r + 20 m l 0.5 N NaOH. Add 11 m l 1 N HC1 and dilute to 100 m l
with water.

PROCEDURE

Weigh 50 g sample into a 1 litre steam distillation flask. Add 100 g

MgSOA'yH^O and 100 m l 1 N H^SO^. Place 10 m l 1 N NaOH in the steam
distillation apparatus receiver. Steam distill rapidly. (Note - do
not heat the distilling flask). Collect about 450 ml distillate in
about 30 min.

Cool and transfer the distillate to a 500 ml v o l u m e t r i c flask. Add

15 m l 1 N H 2 S 0 ^ and m a k e to v o l u m e w i t h water. Mix. Pipette 2 ml
into a test tube and add 2 ml of the d i c h r o m a t e solution. Heat in a
boiling water bath for 5 min. Then cool. Add 2 ml TBA solution and
heat in a boiling water bath 10 min. Cool rapidly and transfer to a
50 m l v o l u m e t r i c flask w i t h water. M a k e to v o l u m e w i t h w a t e r .
M e a s u r e the absorbance of the solution at 532 nm using a 1 cm
cuvette, and water as the reference.

Prepare a standard curve as f o l l o w s : D i s s o l v e 1.0 g of sorbic acid

in a small volume of 1 N NaOH and dilute to 1 litre with water. This
is the Stock Solution (1 m g / m l ) . Prepare a blank and four w o r k i n g
standard solutions by first pipetting 25.0 ml of the stock solution
to a 500 ml volumetric flask (50 p g / m l ) and diluting to volume with
w a t e r . Next, pipette 0.0, 10.0, 20.0, 50.0 and 80.0 of this solution
into five 100 ml v o l u m e t r i c flasks and dilute to v o l u m e with w a t e r
(range 0, 5, 10, 25 and 40 p g / m l ) . Pipette 2 m l of each of the
working standards and blank into five test tubes and continue as in
the above p r o c e d u r e , starting at a d d i t i o n of d i c h r o m a t e . Plot
absorbance vs yg sorbic acid for a standard curve. ( yg sorbic = 0,
10, 20, 50, 80 in the 2 ml aliquots).

60
 CALCULATION

Sorbic acid ppm = A x

where :
A = Jig sorbic acid corresponding to the sample
absorbance, taken from the standard curve.
S = sample weight in g.

REFERENCE

Carr, W. and S m i t h , G.A., 1964. Journal of the American Pharmaceutical

Association 1_ (2), 37-43.

61
 SULPHUR DIOXIDE

PRINCIPLE

Sulphur dioxide, in solution as sulphurous acid, inhibits the growth of mold,

y e a s t s and a e r o b i c b a c t e r i a . It a l s o p r e v e n t s the b r o w n i n g of f r u i t s and
vegetables due to enzymic reactions. It assists in conserving V i t a m i n C, but
d e s t r o y s V i t a m i n B l , by c l e a v a g e of the m o l e c u l e . The a n a l y s i s of s u l p h u r
dioxide in foods involves distillation using nitrogen as a carrier, collection
in an oxidizing solution (hydrogen peroxide), thus converting sulphurous acid
to s u l f u r i c , and a t i t r a t i o n of the acid. T w o t r a p s are u s e d for d i s t i l l a t e
collection to ensure all sulphur dioxide is removed from the nitrogen carrier
stream. T h i s m e t h o d u s e s p h o s p h o r i c i n s t e a d of h y d r o c h l o r i c for i n i t i a l
acidification in order to slow the release of sulphur dioxide.

APPARATUS

1. Distillation apparatus (see Figure).

2. Nitrogen gas (from a cylinder, with regulator).

3. Burette.

REAGENTS

1. Methanol.

2. Hydrogen peroxide solution, 3.0% (w/v): d i l u t e 10 m l of 30%

H2O2 t 0 100 m l with water. Prepare daily.

3. Mixed indicator solution: m i x e q u a l v o l u m e s of 0.03% m e t h y l

red (in e t h a n o l ) and 0.05% m e t h y l e n e b l u e (also in e t h a n o l ) and
filter.

4. Sodium hydroxide solution, 0.01 N.

5. Phosphoric acid, concentrated (88%).

PROCEDURE

I n t r o d u c e s a m p l e and d i s t i l l e d w a t e r into the d i s t i l l a t i o n f l a s k .

The a m o u n t of e a c h is d e p e n d e n t cn the e x p e c t e d s u l p h u r d i o x i d e
content, as noted below:

Expected S02 (ppm) Sample weight (g) Water volume (ml)

10 50 20
10 100 25 30
100 10 40

Add 50 ml methanol and mix by swirling. Introduce 20 ml of the H 2 0 o ,

50 m l distilled water and a few drops of the mixed indicator into the
conical receiving flask. Add a few drops of 0.01N NaOH to produce a
green color. I n t r o d u c e 10 m l of n e u t r a l i z e d H 2 O 2 s o l u t i o n to the
second receiver. (Make sure the receiving tubes are b e l o w the level
of the l i q u i d in e a c h trap). C o n n e c t the n i t r o g e n and a d j u s t the
f l o w to a b o u t one b u b b l e per s e c o n d . Add 15 m l H-jPO^ to the
distillation flask and quickly attach the condenser. Heat rapidly to
a boil, then reduce heat and s i m m e r for 30 minutes. Detach receivers
( r i n s i n g the t u b e s w i t h w a t e r in the p r o c e s s ) and c o m b i n e t h e i r
contents. Titrate the H 2 S 0 4 using 0.01N NaOH to a green endpoint.

62
 CALCULATION

Sulphur dioxide (ppm) = (V) (N) (32) (1000)

S

Where: V = ml of NaOH used to titrate.

N = normality of NaOH.
S = g of sample.

INTERPRETATION

Note that products containing significant amounts of sulphur compounds, such as

garlic, onion, m u s t a r d and cabbage, m a y give false p o s i t i v e s using this
analysis.

REFERENCE

Tanner, H., 1963. Mitt. Geb. L e b e n s m itt. U. Hyg. 54, 158 (This is an EEC
official method.)
Sulphur Dioxide Distillation Apparat

64
 FORMALDEHYDE

PRINCIPLE

The use of f o r m a l d e h y d e in food is prohibited or severely restricted in most

countries, but it may be illegally used from time to time. The method involves
distillation of the f o r m a l d e h y d e , reaction w i t h c h r o m o t r o p i c acid and
spectrophotometrie determination.

APPARATUS

1. Spectrophotometer.

2. Distillation apparatus (e.g. macro-Kjeldahl) .

REAGENTS

1. Chromotropic acid solution: Prepare a saturated solution (about

0.5%) of l , 8 - d i h y d r o x y n a p h t h a l e n e - 3 , 6 - d i s u l p h o n i c acid in 60% v/v
sulphuric acid. Prepare the latter by adding 60 ml of concentrated
sulphuric acid slowly, with stirring and cooling, to 40 ml of water.
Use only a freshly prepared solution of chromotropic acid.

2. Standard formaldehyde solution: Dilute 1 ml of 35% formaldehyde

solution to 1 litre with water. To check the exact strength add 100
ml by pipette to 25 m l 0.1 N NaOH and 10 ml n e u t r a l i z e d 3% H 2 0 2 in a
conical flask, cover the mouth of the flask and heat on a steam bath
for 5 m i n u t e s , shaking occasionally. Cool and titrate the excess
alkali with standard acid.

25
g/L formaldehyde - ~0^tre x 0.1 x x 30.0264

Prepare standards containing 0 - 1 5 micrograms/ml of formaldehyde.

3. Orthophosphoric acid, 10% v/v.

PROCEDURE

To 100-200 ml water in a distillation flask add 10-20 g or m o r e of

s a m p l e , a c c u r a t e l y w e i g h e d , and 5 m l of 10% p h o s p h o r i c acid (or
enough to acidify the sample + 5 ml). Distil slowly a total of about
90 ml into a conical flask, the end of the condenser tube being kept
below the surface (a little water may be put in at the start and the
flask tilted). Dilute the distillate to exactly 100 ml. To a series
of tubes, each containing 5 ml of c h r o m o t r o p i c acid s o l u t i o n , add 1
ml of each standard solution, m i x i n g thoroughly. Leave the tubes,
lightly stoppered or capped, in a boiling waterbath for 15 minutes,
cool and leave to stand 30 minutes. Determine the absorbance at 565
nm, using a spectrophotometer. Construct a standard graph and read
off the concentration in the distillate from the graph.

CALCULATION
100
yg/g in the food = pg/ml in distillate x weight of sample

This test may be used as only qualitative, omitting the preparation

of standards. Other qualitative tests w i l l be found in standard
texts. Results are likely to be low due to incomplete recovery as a
result of polymerization and decomposition after incorporation of the
formaldehyde into the food.

65
REFERENCE ïgfteflJLàMlÔi

Kleinert , T. and Srepel, E., 1948. Mikrochem. ¿3, 328-332.

imiaiim

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.bis» ai oso- ria i o s * sbO

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M ,„H1Í:V SVÍ3BÍ j fsUB vioo »» fessa 3d 3893

6.2 ANTIOXIDANTS

A n t i o x i d a n t s by d e f i n i t i o n are d e s i g n e d to r e t a r d d e t e r i o r a t i o n of a food
through the a c t i o n of a t m o s p h e r i c o x y g e n . They m a y a l s o i n c i d e n t a l l y h a v e a
preservative action, but are used primarily for their antioxidant function.

A n t i o x i d a n t s c o m m o n l y found in fats, oils and the fatty f r a c t i o n s of foods

i n c l u d e the g a l l a t e s ( m a i n l y p r o p y l , o c t y l and d o d e c y l ) , butylated
h y d r o x y a n i s o l e (BHA), and b u t y l a t e d h y d r o x y t o l u e n e (BHT). T o c o p h e r o l s and
their esters, usually acetate or p a l m i t a t e , may a l s o be present.
Nordihydroguaiaretic acid, ascorbyl palmitate, ethoxyquin, 2,4,5-
t r i h y d r o x y b u t y r o p h e n o n e , guaiacol from guaiac resin, terti ary-buty 1
hydroquinone (TBHQ), thiodiprop ionic acid and its dilauiryl and distsaryl ôsùsits
and 4 - h y d r o x y m e t h y 1 - 2 , 6 - d i - t e r t - b u t y 1 p h e n o 1 ( 4 - h y d r o x y B H T ) m a y be found
o c c a s i o n a l l y e i t h e r as a r e s u l t of i n c o r p o r a t i o n in food or m i g r a t i o n from
packaging material. Most of them are permitted in some countries up to certain
limits. Many amines and aminophenols such as diphenyl paraphenylenediamine are
very e f f e c t i v e a n t i o x i d a n t s , used in other p r o d u c t s such as g a s o l i n e and
rubber. However, they should not be found in food.

If a permitted antioxidant is found by a qualitative test it may be necessary

to establish that it is present at a level below a legal limit. If the amount
is w e l l b e l o w the l i m i t a s e m i - q u a n t i t a t i v e test by TLC m a y be e n o u g h to
establish that the sample gives a spot of less colour intensity than that of a
standard a m o u n t c o r r e s p o n d i n g to the legal l i m i t . O t h e r w i s e a q u a n t i t a t i v e
method must be used.

Q u a n t i t a t i v e tests for a n t i o x i d a n t s are described in the Official Methods of

A n a l y s i s of the A O A C (1984) and by A m a t o (2) and T a k e s h i t a , S a k o g a m i & Itoh
(3). C a n t a f o r (4) has r e v i e w e d the m e t h o d o l o g y . TLC of a n t i o x i d a n t s is
described by Schneider (5) and EEC method 992/VI/71.

Quantitative methods for gallates, BHA & BHT are described in the publication
of the Association of Public Analysts (U.K.)(6). Page & Kennedy (7) describe a
GLC-EC m e t h o d for BHA, TBHQ and PG. The c o l o r i m e t r i c m i c r o e s t i m a t i o n of
antioxidants is detailed by Jayaraman, Vasundhara and Panhar (8). The papers
by L a m b and W o l l e r (9), H o l d t (10) and G r o e b e l and W e s s e l s (11) are also
useful. GLC determination of ethoxyquin in apples is described by Wirell (12).

For qualitative methods for BHA see Anglin, Mahan and Chapman (13), Amato (2)
and Dilli and Roberts (14) who describe a spectrofluorimetric technique. For
BHT see S a h a s r a b u n d h e (15), S e l m e c i and A c z e l (16), and for the t w o t a k e n
together see K e e n and G r e e n (17), Sato and K a w a m i r a (18), T a k a h a s h i (19) and
Hartman and Rose (20), the two latter describing GLC methods. Gallates may be
determined by the method of Cassidy and Fisher (21), or the GLC method of Wachs
and G a s s m a n n (22). B e r k and B i e l e c k i (23) d e s c r i b e the d e t e r m i n a t i o n of
antioxidants in essential oils. The I U P A C - A O A C liquid c h r o m a t o g r a p h y m e t h o d
was first described by Page (24)(25).

67
 GALLATES

PRINCIPLE

The gallates are extracted with 95% m e t h a n o l . An aliquot of the m e t h a n o l i c

extract is treated with acetone and finely powdered ammonium sulphate and the
blue colour compared with standards.

APPARATUS

1. Spectrophotometer or colorimeter.

REAGENTS

1. 95% methanol. Mix 95 ml methanol with 5 ml water.

2. Calcium carbonate, finely divided.

3. Acetone.

4. Ammonium sulphate, finely powdered.

5. Standard n-propyl, n-octyl or n-dodecyl gallate. Dissolve 0.100

g in 95% m e t h a n o l and dilute to 100 ml (1 m g / m l ) . Prepare dilute
standards in the range 5-50 mg/L.

PROCEDURE

Vigorously shake 10 g of warm liquid sample or melted fat with 25 ml

of 95% m e t h a n o l for one m i n u t e in a c e n t r i f u g e tube. Place in a
w a t e r - b a t h at 40-50°C and a l l o w to separate for about 15 m i n u t e s
(separation into clear layers is u n l i k e l y and unnecessary). Pour
the upper layer into a 50 ml volumetric flask. Repeat the extraction
w i t h 20 ml of 95% m e t h a n o l , again transfer the upper layer to the
flask and dilute to the mark. Add 1 g of c a l c i u m c a r b o n a t e , shake
and filter through a paper ( W h a t m a n No. 1 or equivalent), r e j e c t i n g
the first few ml. of filtrate. The amount of c a l c i u m carbonate is
not critical but must be enough to ensure a clear filtrate.

Pipette 10 ml of filtrate and add 1 ml of acetone and about 10 mg of

finely divided ammonium sulphate. Shake for one minute. After half
an hour measure the absorbance of the blue colour at 580 nm in a 1 cm
cell and c o m p a r e w i t h the colour developed from 10 m l of dilute
standard in 95% methanol treated similarly.

CALCULATION

Absorbance values for the three commonest gallate antioxidants are as

follows :

Approximate molar
absorbance using absorptivity
10 ppm solution molecular (absorbance
in above test absorptivity weight x M. Wt)

Propyl
gallate 0.1777 17.68 212 3749

Octyl
gallate 0.140 14.01 282 3952

Dodecyl
gallate 0.116 11.55 338 3905

68
 The c l o s e s i m i l a r i t y of the molar a b s o r p t i v i t i e s means that r e s u l t s
may, w i t h o u t s e r i o u s e r r o r , be e x p r e s s e d in mg of the g a l l a t e used as
standard per k i l o g r a m of sample even i f another g a l l a t e or a m i x t u r e
i s present.

Gallates in mg/kg of sample = sample a b s o r b a n c e

standard a b s o r b a n c e

x standard in m g / L x 50/10

Enough s t a n d a r d s should be prepared to bracket the sample con-

centration.

REFERENCE

Cassidy, W. and F i s h e r , A.J., 1960. A n a l y s t _85, 295-7.

69
 BRA

PRINCIPLE

BHA (butylated hydroxyanisole) is extracted from a sample with 95% methanol and
reacted with Gibb's reagent to form a stable indophenol colour.

APPARATUS

1. Spectrophotometer.

REAGENTS

1. 95% v/v Methanol.

2. 0.5% Sodium tetraborate decahydrate.

3. 2,6-Dichloro-p-benzoquinone-4-chloroimine (Gibb's reagent) 0.01%

in 95% methanol.

4. n-Butanol.

5. BHA standard, 25 mg/L in 95% Methanol.

PROCEDURE

Shake 10 g of w a r m liquid s a m p l e w i t h 25 m l 9 5 % m e t h a n o l for one

m i n u t e in a c e n t r i f u g e tube. P l a c e in a w a t e r b a t h at 40-50°C and
let separate for 15 minutes (separation into clear layers is unlikely
and u n n e c e s s a r y ) . Pour the u p p e r layer into a 50 m l v o l u m e t r i c
flask. Repeat the extractions with 20 ml of 95% methanol and again
transfer the upper layer to the flask. Dilute the flask to the mark
with 95% methanol.

P i p e t 2 m l of this e x t r a c t , add 2 m l of 95% m e t h a n o l , 8 m l of b o r a x

s o l u t i o n and 2 m l of G i b b ' s r e a g e n t . A f t e r 15 m i n u t e s d i l u t e to
e x a c t l y 20 m l w i t h n - b u t a n o l . P r e p a r e a b l a n k u s i n g 2 m l of 95%
m e t h a n o l and a s t a n d a r d u s i n g the 25 m g / L BHA s o l u t i o n . Read
absorbances at 610 nm.

CALCULATION
A A
sample ~ blank
BHA in sample, in mg/L = 50
A
^standard ~ blank x 25 x 10
INTERPRETATION

The test may be made more sensitive by taking 4 ml of the methanol extract and
omitting the 2 ml of 95% methanol. Other antioxidants, particularly gallates,
r e d u c e the i n t e n s i t y of the c o l o u r . For e x a m p l e , 200 m g / L of p r o p y l g a l l a t e
r e d u c e s the c o l o u r f r o m 200 m g / L of BHA to about one h a l f u n d e r the s t a n d a r d
conditions of the test (concentrations calculated on the original sample). A
table of corrections to be applied can be found in the referenced method.

REFERENCE

A s s o c i a t i o n of P u b l i c A n a l y s t s (U.K.). 1962. The D e t e c t i o n and Determination

of Antioxidants in Food.

70
 BHT

PRINCIPLE

BHT (butylated hydroxytoluene) is steam-distilled, and determined by the colo

reaction with o-dianisidine and sodium nitrite.

APPARATUS

1. Steam-distillation apparatus and steam generator.

2. Bath at 160°C. A one-litre beaker half-full of paraffin wax is

convenient. An oil-bath may also be used.

3. Squibb-type separatory funnels of low actinic glass, or painted

black. 60 ml.

4. 10 ml volumetric flasks of low actinic glass or painted black.

REAGENTS

1. Chloroform.

2. Magnesium chloride solution. Dissolve 100 g of the hexahydrate

in 50 ml water.

3. o-Dianisidine solution. Dissolve 0.25 g in 50 ml methanol, add

100 mg activated charcoal, shake for 5 minutes and filter. Mix 40 ml
of this clear s o l u t i o n w i t h 60 ml of IN HC1. P r e p a r e d a i l y and
protect from light.

4. Sodium nitrite, 0.3% in water.

5. S t a n d a r d s o l u t i o n of BHT, 500 m g / L . D i s s o l v e 50 mg in m e t h a n o l
and d i l u t e to 100 m l w i t h m e t h a n o l . Prepare working standards
containing 1-5 mg/L by diluting with 50% v/v methanol.

PROCEDURE

Add 15 ml of the magnesium chloride solution to the distilling flask,

add 5g of s a m p l e or less ( p r e f e r a b l y c o n t a i n i n g a b o u t 0.4 m g BHT).
P r e h e a t the bath for the d i s t i l l i n g flask to 160°Cjf 10. A d j u s t the
steam generator to distill about 4 ml of water per minute. Maintain
these conditions throughout the distillation. Connect the condenser
and the s t e a m g e n e r a t o r to the d i s t i l l i n g f l a s k , and i m m e d i a t e l y
immerse the latter in the bath. Steam distillation must be vigorous.
C o l l e c t the first 100 m l of the d i s t i l l a t e in a 200 m l v o l u m e t r i c
flask containing 50 ml of methanol. Disconnect the distilling flask
from the s t e a m g e n e r a t o r and r e m o v e the d i s t i l l i n g f l a s k from the
bath. When the mouth of the condenser has cooled, disconnect it from
the distilling flask and drain the water from the jacket. Wash the
condenser with 5 ml portions of methanol adding the washings to the
volumetric flask. Cool to room temperature and adjust the volume to
200 ml with methanol. Mix.

Thoroughly clean and dry three 60 ml Squibb-type separatory funnels

of low a c t i n i c g l a s s or painted b l a c k and m a r k t h e m B, S, and X
respectively. Into funnel B p i p e t t e 25 ml of 50% m e t h a n o l (v/v);
into funnel S pipette 25 ml of standard containing 1-3 p g of BHT per
m l ; and into funnel X p i p e t t e 25 ml of the 50% m e t h a n o l s o l u t i o n
( d i s t i l l a t e ) of the s a m p l e . To each funnel add 5 m l of d i a n i s i d i n e
solution, stopper the funnel and carefully mix the contents. Then to
each funnel add 2 m l of 0.3% s o d i u m n i t r i t e s o l u t i o n , s t o p p e r the
funnels and thoroughly mix the contents. Let stand for 10 minutes,

71
 then add 10 m l of c h l o r o f o r m to each f u n n e l . E x t r a c t the c o l o u r e d
complex by vigorously shaking the funnels for 30 seconds. Let stand
for 2-3 minutes to allow the two layers to separate completely.

Mark three 10 ml volumetric flasks of low actinic glass, B, S, and X

respectively. P i p e t t e 2 m l of a b s o l u t e m e t h a n o l into each flask.
F r o m the c o r r e s p o n d i n g funnel d r a w off a s u f f i c i e n t v o l u m e of the
chloroform (bottom layer) to reach the mark on the flask. Mix well.
Read the a b s o r b a n c e of each s o l u t i o n in a s u i t a b l e c o l o r i m e t e r or
spectrophotometer set at 520 nm using a mixture of 2 ml methanol and
8 ml of chloroform as a blank. Avoid any unnecessary exposure of the
solutions to light and air. 50 p g BHT should give an absorbance of
a b o u t 0.39 in a 1 cm cell. R e c o v e r y s h o u l d be 97 +_ 2%.

CALCULATION
A
, s ~ Ab , 200
BHT in sample, mg/kg = x p g/ml standard x
A x - Ajj g sample

INTERPRETATION

If e x t r a c t i o n w i t h 9 5 % m e t h a n o l is u s e d as an a l t e r n a t i v e to s t e a m
d i s t i l l a t i o n , only about h a l f of the BHT is e x t r a c t e d so that d o u b l i n g the
r e s u l t o b t a i n e d b y the o - d i an i s i d i n e r e a c t i o n g i v e s an a p p r o x i m a t e l y
quantitative answer. An extraction using acetonitrile is quantitative for BHT.
Santoquin is the only reported interference.

REFERENCE

A s s o c i a t i o n of P u b l i c A n a l y s t s (U.K.). 1962. The D e t e c t i o n and Determination

of Antioxidants in Food.

72
 ANTIOXIDANTS
(General Thin-Layer Chromatography Method)

PRINCIPLE

BHA, BHT and other antioxidants are extracted from oil or fat and separated
thin-layer chromatography. This method is semi-quantitative and the amounts
the various antioxidants can be estimated by comparing with standards.

APPARATUS

1. Separatory funnels, 250 ml.

2. Rotary vacuum evaporator.

3. Thin-layer plate spreader and developing tanks.

4. Spray bottle.

REAGENTS

1. Petroleum ether (40° - 60°BR).

2. Acetonitrile.

3. Methanol.

4. Silica Gel.

5. Citric acid, 1% in methanol.

6. Developing solution - (2+2 + 1) p e t r o l e u m ether .'benzene ¡glacial

acetic acid.

7. Antioxidant standards - 0.1% in methanol.

8. Gibbs Reagent - 2,6-dichloro-p-benzoquinone-4-chloroimine, 0.5%

in ethanol.

PROCEDURE

W e i g h 10 g oil or fat and dissolve in 80 ml pet. ether. Transfer to

a separatory funnel using about 20 ml pet. ether to wash. Add 25 ml
acetonitrile and extract by inverting funnel several times. (Violent
shaking m a y form an emulsion). Drain the a c e t o n i t r i l e into the
rotary v a c u u m flask. Repeat the extraction with three m o r e 25 ml
portions of acetonitrile. Combine all extracts in the flask.
Evaporate using vacuum at not over 40°C. Dissolve extract with 2 ml
methanol.

Prepare 0.25 mm TLC plates using a slurry of 30 g silica gel G and 60

ml 1% citric acid solution. Air dry the prepared plates, then heat
in an air oven for one hour at 130°C. Store the dried plates in a
desiccator until required.

Line the developing tank with fil ter paper. Add solvent, close and
allow to e q u i l i b r a t e 1-2 hours in the dark. Spot 10 U l and 20 pi
portions of the sample extract on the prepared plate. Also spot 2, 5
and 7 pi portions of appropriate antioxidant standards of interest
(corresponding to 2, 5 and 7 Mg).
 Develop the plate until the solvent front reaches 1 cm from the top.
R e m o v e from the tank, air dry and then spray w i t h Gibb's Reagent.
The following antioxidants will appear as coloured spots at about the
R„ noted:

Antioxidant R Color
F
Propyl gallate 0.12 Brown
Octyl gallate 0.22 Brown
Dodecyl gallate 0.27 Brown
BHA 0.62 Brown-red
BHT 0.89 Brown-violet

Compare standard and sample spot size and intensity.

CALCULATION

If a semi-quantitative estimation is needed, compare the sample and

standard spots (interpolate if the sample falls between two standards
in intensity).

1000 x
ppm antioxidant = P ? standard
5 x y g sample.

REFERENCE

E g a n , H., Kirk, R.S. and S a w y e r , R., 1981. Pearson's Chemical Analysis

Foods, 8th Ed., 88.

74
6.3 COLOURS

Colouring m a t t e r s in food m a y be n a t u r a l or s y n t h e t i c , and m a y be a c i d i c ,

neutral, or basic. Also, they may be conveniently classified as oil-soluble or
water-soluble. They must be separated from the food before identification is
attempted. Once s e p a r a t i o n is a c h i e v e d by d y e i n g on to w o o l , q u i n o l i n e
extraction, use of a polyamide column, or other means identification is carried
out by paper or thin-layer chromatography and confirmed by examination of the
spectral absorption and by chemical tests. It should be possible to establish
w h e t h e r or not a c o l o u r b e l o n g s to those g e n e r a l l y p e r m i t t e d b u t the
i d e n t i f i c a t i o n of a n o n - p e r m i t t e d c o l o u r m a y w e l l p r e s e n t considerable
difficulty. P r i o r i t y should be g i v e n to t e s t i n g s a m p l e s for a few c o m m o n
colours w h i c h are g e n e r a l l y r e g a r d e d as o b j e c t i o n a b l e , m a i n l y on g r o u n d s of
c a r c i n o g e n i c i t y ( s u s p e c t e d or c o n f i r m e d ) , i n c l u d i n g a u r a m i n e , the m a g e n t a s
(fuchsin, rosaniline), crystal violet, methyl yellow and the rhodamines. Some
of these are b a s i c d y e s and t h e r e f o r e their p r e s e n c e w i l l be s h o w n by w o o l
taking up the dye from ammoniacal solution. Some can occur as sulphonates and
are t h e r e f o r e a c i d i c w a t e r - s o l u b l e f o r m s . P r e c a u t i o n s for the use of
c a r c i n o g e n i c s u b s t a n c e s s h o u l d be t a k e n w i t h s t a n d a r d s o f s u s p e c t e d
c a r c i n o g e n i c c o l o u r s . A m a r a n t h is b r o k e n d o w n into n a p h t h i o n i c acid and 2 -
naphthol-3,6-disulphonic acid in the presence of salt and sugars. Erythrosine
loses four atoms of iodine, forming fluorescein by the electrolytic action of
tin and the latter dye may therefore sometimes be found in canned food. Some
dyes occur as the c a l c i u m or a l u m i n i u m "lakes", w h i c h can be d e c o m p o s e d to
release the free dye by treatment with hydrochloric acid.

Great care must be taken in identifying a food colour which does not appear to
be one in common use or on a permitted list. The presence of subsidiary dyes,
impurities, co-extracted material from the food or too rigorous an extraction
procedure may cause decomposition of the dye, and a resultant change of shade
and Rj value during paper or thin-layer chromatography or interference in the
spectral curve. For e x a m p l e , Red 2G w i l l d e c o m p o s e to Red 10B in b o i l i n g
dilute acid s o l u t i o n and Y e l l o w R F S is c o n v e r t e d to a n o t h e r dye by the w o o l -
dyeing e x t r a c t i o n p r o c e d u r e . Red 40 b e h a v e s s i m i l a r l y to P o n c e a u SX. It is
therefore i m p o r t a n t in c a s e s of d o u b t to m i x dye and s t a n d a r d and c h e c k that
they cannot be s e p a r a t e d on paper or t h i n - l a y e r by at least three s o l v e n t s
selected for their different polarities and acidities. Also examine UV-visible
spectra, and c a r r y out c h e m i c a l tests to c o n f i r m i d e n t i t y . S p e c t r a m u s t be
compared with authentic dye specimens, preferably more than one, derived from
different sources.

A given colour has often been given different names by different manufacturers.
There are even c a s e s of t w o d i f f e r e n t d y e s b e i n g g i v e n the s a m e n a m e by
different manufacturers. A particular dye is identified unequivocally by its
Colour Index n u m b e r . Food c o l o u r s p r e s e n t l y or at one t i m e p e r m i t t e d in the
EEC have numbers starting at E100. Under U.S. legislation, colouring matters
have been given numbers preceded by letters which indicate the permitted use.
Although in most cases the dye.has subsequently been prohibited under U.S. law,
it m a y still be i d e n t i f i e d in this w a y . For e x a m p l e , F.D. & C. Blue No. 1
indicates that the dye may be used in foods, drugs and cosmetics. Ext. D. & C.
Red 9 w a s once p e r m i t t e d for e x t e r n a l use only in d r u g s and c o s m e t i c s . The
trivial n a m e s m a y i n c l u d e any n u m b e r s or letters in order to s p e c i f y a
particular dye. For example, Phloxine J and Phloxine ZB1 refer to Colour Index
No. 45405 while Phloxine JN, Phloxine B and several more suffix letters refer
to C. I. No. 4 5 4 1 0 .
 WATER-SOLUBLE COLOURS
(Wool Dye E x t r a c t i o n )

PRINCIPLE

A c i d i c c o l o u r d y e s are a l l o w e d to dye l e n g t h s of w h i t e d e f a t t e d w o o l from

dilute acetic acid solution. The colour is stripped from the wool w i t h dilute
a m m o n i a s o l u t i o n and e v a p o r a t e d to s m a l l v o l u m e . T h e c o l o u r i n g m a t t e r is
identified by an appropriate method. The dyeing and stripping m a y be repeated
w i t h f r e s h w o o l as a p u r i f i c a t i o n step. B a s i c c o l o u r s are dyed o n t o w o o l in
the presence of a m m o n i a and stripped from the wool by dilute acetic acid.

REAGENTS

1. White defatted wool. Extract white wool in a Soxhlet extractor

w i t h p e t r o l e u m e t h e r for 2 - 3 h o u r s to r e m o v e fat. Do n o t f u r t h e r
h a n d l e the w o o l , as it m a y a b s o r b g r e a s e f r o m the skin. S q u e e z e as
d r y as p o s s i b l e and l e a v e on a s t e a m b a t h i m m e r s e d in 5% a m m o n i a
solution for an hour to remove petroleum ether, then hang the wool up
to dry. Store in a w i d e - m o u t h capped jar.

2. 5% a m m o n i a solution. Dilute 5 ml concentrated ammonia to 100 ml

with water.

3. A m m o n i a - s o lvent m i x t u r e . Mix equal proportions of 3N a m m o n i a

solution, acetone and ethanol.

PROCEDURE

If necessary, for liquid samples, acidify slightly with acetic acid

or K H S O 4 (e.g. 30 m l s a m p l e + 5 m l 10% K H S O ^ ) . If the l i q u i d
contains alcohol it should be evaporated first to r e m o v e the alcohol
and r e - d i l u t e d . For w a t e r - s o l u b l e f o o d s s u c h as p r e s e r v e s and
s w e e t s , d i s s o l v e a s u i t a b l e w e i g h t in 30 m l 5 % a m m o n i a in 7 0 %
e t h a n o l , l e a v e s e v e r a l h o u r s and c e n t r i f u g e . For g e l a t i n - b a s e d
foods, use 5% a m m o n i a in 90% ethanol. In either case, evaporate to a
t o t a l v o l u m e of 30 m l and add 5 m l 10% K H S O ^ . For h i g h - p r o t e i n
s a m p l e s soak o v e r n i g h t in 1% a m m o n i a s o l u t i o n , c e n t r i f u g e and
acidify. For h i g h - f a t f o o d s s u c h as m e a t p r o d u c t s , d e - f a t w i t h
petroleum ether before applying one of the procedures given above.

For b a s i c d y e s , add 1 0 - 2 0 cm of w o o l to an a m m o n i a c a l s o l u t i o n ,
either separately prepared or at the appropriate stage of one of the
extraction procedures above. If the wool absorbs any colour leave it
in contact with the warm solution until the colour is transferred to
the w o o l . R e m o v e the w o o l f r o m the s o l u t i o n , r i n s e , s t r i p off the
dye with 1% acetic acid, make the solution just alkaline with a m m o n i a
and d y e a f r e s h p i e c e of w o o l . S t r i p a g a i n w i t h a c e t i c a c i d and
g e n t l y e v a p o r a t e to a s m a l l v o l u m e . C a r r y out appropriate
identification t e s t s .

For acidic, dyes, add wool to the solution acidified w i t h acetic acid
or 10% KHSO4 solution. Wash the wool thoroughly w i t h w a r m water and
t h e n w a r m w i t h 5 m l a m m o n i a - s o l v e n t m i x t u r e for a b o u t 5 m i n u t e s .
R e m o v e the w o o l , g e n t l y e v a p o r a t e the s o l u t i o n to d r y n e s s and
dissolve the residue in a drop or two of water. Conduct appropriate
identification t e s t s .

REFERENCE

A s s o c i a t i o n of P u b l i c A n a l y s t s . 1960. S e p a r a t i o n and I d e n t i f i c a t i o n of Food

Colours Permitted by the Colouring Matters in Food Regulation (1957). London

76
 WATER-SOLUBLE COLOURS
(Polyamide Separation)

PRINCIPLE

The sample is defatted, if necessary, and the colour extracted using a suitable
solvent chosen according to the nature of the foodstuff. The colour extract is
adsorbed on a polyamide column, washed with various solvents and eluted with a
mixture of acetone and ammonia.

APPARATUS

1. C h r o m a t o g r a p h i c t u b e s , 300 m m , 250 m m x 15 m m and 200 m m x 10

m m , all fitted with a glass stopcock.

2. Oven set at 100-102°C.

3. Water-bath.

4. Soxhlet continuous extraction apparatus and thimbles.

5. Glass evaporating basin.

REAGEHTS

1. Acetone.

2. Acetone-ammonia solution: mix 40 ml of acetone, 9 ml of water

and 1 m l of a m m o n i a s o l u t i o n (SG = 0.880). This s h o u l d be f r e s h l y
prepared.

3. Chloroform.

4. Methanol-ammonia solution: mix 90 ml of methanol, 5 ml of water

and 5 ml of ammonia solution.

5. Petroleum ether, boiling range 40-60°C.

6. Acetic acid, glacial.

7. P o 1 y o x y e t h y lene s o r b i t a n m o n o - o l e a t e s o l u t i o n : m i x 1 ml of
p o l y o x y e t h y l e n e s o r b i t a n m o n o - o l e a t e ("Tween" 80) w i t h 99 m l of
water.

8. Methanol-water-tetramethylammonium hydroxide mixture (40+9+1).

9. Chloroform-absolute ethanol-water-formic acid mixture (100+90+

10 + 1) .

10. Hydrochloric acid, 0.5N.

11. Hydrochloric acid, 0.1N.

12. P o l y a m i d e staple fibre: n y l o n 66, 3.3 g per 10,000 m. of fibre

obtained from Macherey, Nagel and Co., 5161 Duren, Wertstrasse 628,
Federal Republic of Germany.

13. P o l y a m i d e p o w d e r for c o l u m n c h r o m a t o g r a p h y : MN-CC 6 from

Macherey, Nagel and Co., grain size, 0.16 m m .

14. Celite 545.

15. Sand, acid washed.

77
PROCEDURE

For water soluble products (jellies, jams, sweets):

W e i g h a b o u t 5 g of the s a m p l e i n t o a b e a k e r , a d d 50 m l of w a t e r a n d
w a r m the b e a k e r on a w a t e r - b a t h u n t i l the w a t e r - s o l u b l e m a t e r i a l is
dissolved. A c i d i f y the m i x t u r e w i t h g l a c i a l acetic a c i d .

Place a p l u g of p o l y a m i d e staple fibre in t h e e n d of the

c h r o m a t o g r a p h i c t u b e a n d a d d a s u s p e n s i o n of p o l y a m i d e p o w d e r in
w a t e r to the t u b e to g i v e a c o l u m n a p p r o x i m a t e l y 20 m m h i g h . Rinse
t h e w a l l s o f t h e t u b e w i t h a s m a l l v o l u m e o f a c e t o n e to a i d t h e
s e t t l i n g of the p o l y a m i d e and t h e n p l a c e sand on t o p of the p o l y a m i d e
to f o r m a l a y e r a b o u t 6 m m d e e p .

P o u r the h o t e x t r a c t t h r o u g h the c o l u m n . W a s h the c o l u m n w i t h six 10

m l p o r t i o n s of h o t w a t e r f o l l o w e d b y t h r e e 5 m l v o l u m e s of a c e t o n e
( s l i g h t a i r p r e s s u r e m a y b e u s e d , if n e c e s s a r y ) . E l u t e t h e c o l o u r s
w i t h t h e m i n i m u m v o l u m e of a c e t o n e - a m m o n i a s o l u t i o n , r e j e c t i n g t h e
c o l o u r - f r e e l i q u i d t h a t is e l u t e d a t f i r s t . R e m o v e t h e a m m o n i a b y
b l o w i n g air o v e r the s u r f a c e of the l i q u i d and t h e n e v a p o r a t e the
s o l u t i o n to a b o u t a q u a r t e r o f t h e o r i g i n a l v o l u m e o n a w a t e r b a t h .
A d d w a t e r to g i v e t h e o r i g i n a l e l u a t e v o l u m e a n d a d j u s t t h e p H to 5 - 6
w i t h h y d r o c h l o r i c a c i d 0.5N.

P r e p a r e a p o l y a m i d e c o l u m n in a 2 0 0 m m c h r o m a t o g r a p h i c t u b e a s
described above. A d d the f i n a l s o l u t i o n o b t a i n e d a b o v e to t h i s
c o l u m n and w a s h w i t h f i v e 5 m l p o r t i o n s of h o t w a t e r . E l u t e the d y e s
w i t h the m i n i m u m v o l u m e of a c e t o n e - a m m o n i a s o l u t i o n . R e m o v e the
a m m o n i a in the s a m e w a y as b e f o r e and e v a p o r a t e the s o l u t i o n to n e a r
d r y n e s s on a s t e a m - b a t h . D i s s o l v e t h e r e s i d u e in a f e w d r o p s of 0.1N
h y d r o c h l o r i c a c i d and p r o c e e d w i t h i d e n t i f i c a t i o n . If e r y t h r o s i n e is
s u s p e c t e d , d i s s o l v e the r e s i d u e in w a t e r .

For bakery products (cakes, cake p o w d e r s , pastries):

W e i g h a b o u t 5 g of the c h o p p e d s a m p l e i n t o a g l a s s e v a p o r a t i n g b a s i n
a n d p l a c e t h e b a s i n in a d r y i n g o v e n a t 1 0 0 ° f o r 30 m i n u t e s . Cool
a n d a d d s u f f i c i e n t p e t r o l e u m e t h e r to c o v e r t h e d r i e d s a m p l e ( a b o u t
30 m l ) a n d s t i r t h e m i x t u r e . A l l o w t h e s o l i d to s e t t l e a n d d e c a n t
the p e t r o l e u m e t h e r . Repeat this p r o c e d u r e t w i c e m o r e and then a l l o w
the r e s i d u a l p e t r o l e u m e t h e r to e v a p o r a t e . G r i n d the s a m p l e g e n t l y
so as to f o r m a c o a r s e p o w d e r , a d d 4 g of C e l i t e 5 4 5 a n d m i x .

P l a c e a p l u g o f p o l y a m i d e s t a p l e f i b r e in t h e e n d o f a 2 5 0 m m
c h r o m a t o g r a p h y t u b e a n d t r a n s f e r t h e p o w d e r e d s a m p l e to t h e t u b e .
P o u r 30 m l o f a c e t o n e o n t o t h e t o p o f t h e c o l u m n a n d , w h e n t h e
s o l v e n t h a s p e r c o l a t e d the w h o l e l e n g t h of t h e c o l u m n , a p p l y s l i g h t
a i r p r e s s u r e to aid u n i f o r m p a c k i n g . D i s c a r d the e l u a t e . Carefully
p o u r 50 m l of the m e t h a n o 1 - w a t e r - t e t r a m e t h y 1 a m m o n i u m hydroxide
solution through column. ( S l i g h t air p r e s s u r e m a y be used if
necessary). A d j u s t t h e p H o f t h e e l u a t e to a p p r o x i m a t e l y 6 b y t h e
a d d i t i o n of d i l u t e h y d r o c h l o r i c acid 0.5N. A d d 5 m l o f t h e 1%
p o l y o x y e t h y l e n e s o r b i t a n m o n o - o l e a t e s o l u t i o n and e v a p o r a t e the
s o l u t i o n to a b o u t a q u a r t e r o f t h e o r i g i n a l v o l u m e o n a w a t e r - b a t h
w i t h the aid of a c u r r e n t of air b l o w n o v e r the s u r f a c e of the
liquid. A d d w a t e r to g i v e the o r i g i n a l e l u a t e v o l u m e and a l l o w the
s o l u t i o n to c o o l .

P r e p a r e a p o l y a m i d e c o l u m n i n a 2 0 0 m m c h r o m a t o g r a p h i c t u b e as
b e f o r e . A d d the s o l u t i o n of e x t r a c t e d a c e t o n e . W a s h f i v e t i m e s w i t h
5 m l p o r t i o n s of c h l o r o f o r m - a b s o l u t e e t h a n o l — w a t e r — f o r m i c acid
s o l u t i o n , t h r e e t i m e s w i t h 5 m l of a c e t o n e and f i n a l l y t h r e e t i m e s
w i t h 10 m l o f w a t e r . E l u t e the d y e s w i t h the m i n i m u m v o l u m e of

78
 a c e t o n e - a m m o n i a s o l u t i o n , r e j e c t i n g the e l u a t e u n t i l the dyes are
eluted. R e m o v e t h e a m m o n i a in t h e c o l o u r e d e l u a t e b y b l o w i n g a
c u r r e n t of air o v e r the s u r f a c e of the l i q u i d and e v a p o r a t e the
s o l u t i o n to a b o u t a q u a r t e r of t h e o r i g i n a l v o l u m e on a w a t e r b a t h .
A d d w a t e r to g i v e the o r i g i n a l v o l u m e of e l u a t e and a d j u s t the pH to
a b o u t 6 w i t h h y d r o c h l o r i c a c i d 0.5N.

A d d the a c i d i f i e d s o l u t i o n to a p o l y a m i d e c o l u m n c o n t a i n e d in a 2 0 0
m m chromatographic tube. W a s h t h e c o l u m n w i t h t h e s a m e v o l u m e s of
s o l v e n t s as d e s c r i b e d in the p r e v i o u s p a r a g r a p h . E l u t e the d y e s w i t h
a m i n i m u m v o l u m e of a c e t o n e - a m m o n i a s o l u t i o n . R e m o v e t h e a m m o n i a a n d
e v a p o r a t e to n e a r d r y n e s s on a s t e a m - b a t h . D i s s o l v e the r e s i d u e in a
f e w d r o p s of 0.1N h y d r o c h l o r i c a c i d a n d p r o c e e d w i t h a p p r o p r i a t e
identification. If e r y t h r o s i n e is s u s p e c t e d , d i s s o l v e t h e r e s i d u e in
water.

For meat products:

W e i g h 25 g of s a m p l e and p l a c e on a f l a t s u r f a c e , c h o p up the s a m p l e
w i t h a k n i f e , a d d 5 g of a c i d - w a s h e d s a n d a n d g r i n d t h e m i x t u r e to a
paste. A d d 10 g o f C e l i t e a n d m i x w i t h a p a l e t t e k n i f e u n t i l a
h o m o g e n e o u s m i x t u r e is o b t a i n e d . T r a n s f e r t h e m i x t u r e to a t h i m b l e ,
p l a c e the t h i m b l e in a S o x h l e t e x t r a c t o r and e x t r a c t w i t h c h l o r o f o r m
f o r 2 h o u r s . R e m o v e t h e s a m p l e f r o m t h e t h i m b l e a n d p l a c e it in a n
e v a p o r a t i n g b a s i n to a l l o w the r e s i d u a l c h l o r o f o r m to e v a p o r a t e .

P l a c e a p l u g of p o l y a m i d e s t a p l e f i b r e in t h e e n d of a 3 0 0 m m
c h r o m a t o g r a p h i c t u b e and add the p o w d e r e d s a m p l e to the t u b e , t a p p i n g
the c o l u m n g e n t l y to aid p a c k i n g . P a s s m e t h a n o 1 - a m m o n i a s o l u t i o n
t h r o u g h the c o l u m n u n t i l all of the d y e s are e l u t e d . (Slight air
p r e s s u r e m a y b e u s e d if n e c e s s a r y ) . A d d 5 m l o f 1% p o l y o x y e t h y l e n e
s o r b i t a n m o n o - o l e a t e and e v a p o r a t e the s o l u t i o n to a b o u t a q u a r t e r of
the o r i g i n a l v o l u m e o n a w a t e r - b a t h , w i t h the a i d of a c u r r e n t of a i r
b l o w n over the s u r f a c e of the l i q u i d . A d d w a t e r to r e s t o r e t h e
e l u a t e to i t s o r i g i n a l v o l u m e a n d a d j u s t t h e p H of t h e s o l u t i o n to 6
w i t h h y d r o c h l o r i c a c i d 0.5N.

P r e p a r e a p o l y a m i d e c o l u m n in a c h r o m a t o g r a p h i c t u b e in the s a m e w a y
a s b e f o r e . P o u r t h e s o l u t i o n of t h e d y e s t h r o u g h t h e c o l u m n . Wash
the c o l u m n t h r e e t i m e s w i t h 10 m l v o l u m e s o f w a t e r , t w i c e w i t h 5 m l
v o l u m e s of a c e t o n e , t w i c e w i t h 5 m l of a c h l o r o f o r m - a b s o l u t e e t h a n o l -
w a t e r - f o r m i c a c i d m i x t u r e and t w i c e w i t h 5 m l of a c e t o n e . E l u t e the
d y e s w i t h t h e m i n i m u m v o l u m e of a c e t o n e - a m m o n i a s o l u t i o n r e j e c t i n g
the e l u a t e u n t i l the d y e s are e l u t e d . R e m o v e the a m m o n i a b y b l o w i n g
a c u r r e n t of a i r o v e r the s u r f a c e of t h e l i q u i d a n d t h e n e v a p o r a t e
t h e s o l u t i o n to a b o u t a q u a r t e r o f t h e o r i g i n a l v o l u m e o n a w a t e r -
bath. A d d w a t e r t o r e s t o r e t h e e l u a t e to i t s o r i g i n a l v o l u m e a n d
a d j u s t the pH to a p p r o x i m a t e l y 6 w i t h 0.5N H C 1 .

P r e p a r e a p o l y a m i d e c o l u m n in a 2 0 0 m m c h r o m a t o g r a p h i c t u b e a s
before. A d d the a c i d i f i e d e l u a t e to t h i s c o l u m n and w a s h the c o l u m n
as d e s c r i b e d p r e v i o u s l y . E l u t e the d y e s w i t h t h e m i n i m u m v o l u m e of
a c e t o n e - a m m o n i a s o l u t i o n . R e m o v e the a m m o n i a by a c u r r e n t of air
o v e r t h e s u r f a c e o f t h e l i q u i d a n d e v a p o r a t e t h e s o l u t i o n to n e a r
dryness. D i s s o l v e the r e s i d u e in a f e w d r o p s of 0.1N h y d r o c h l o r i c
acid and p r o c e e d w i t h a p p r o p r i a t e i d e n t i f i c a t i o n . If e r y t h r o s i n e is
s u s p e c t e d d i s s o l v e the r e s i d u e in w a t e r .

REFERENCE

L e h m a n n , G., C o l l e t , P., H a h n , H . G . a n d A s h w o r t h , M . R . F . 1 9 7 0 . J o u r n a l of the

AOAC ¿3, 1182.

79
 WATER-SOLUBLE COLOURS
(Quinoline E x t r a c t i o n )

PRINCIPLE

T h e s a m p l e ( d e f a t t e d if n e c e s s a r y ) is e x t r a c t e d b y q u i n o l i n e in a b u f f e r e d
m e d i u m at pH 3. The q u i n o l i n e s o l u t i o n is d i l u t e d w i t h d i e t h y l e t h e r and
extracted successively by water, by a m m o n i a and hydrochloric solutions, and by
chloroform. The c o l o u r i n g m a t t e r s are s e p a r a t e d in o n e or o t h e r of t h e s e
phases, according to their group affinities.

In the c a s e of p r o d u c t s m a n u f a c t u r e d f r o m m e a t (the p r o t e i n c o n s t i t u e n t s of
which are likely to absorb the colouring matters to a high degree) extraction
by q u i n o l i n e is p r e c e d e d by t r e a t m e n t u s i n g a l i q u i d i o n - e x c h a n g e r e s i n
(Amber1ite LA 2).

APPARATUS

1. Water bath.

2. Homogenizer.

3. C e n t r i f u g e w i t h g r o u n d - g l a s s s t o p p e r e d t u b e s of v o l u m e 50 m l ,
height not less than 20 cm and external diameter about 2 cm.

4. Separating funnels.

5. Specimen tubes, wide-necked, volume approximately 7 ml.

6. Pestle and mortar.

7. Oven, set at 100 - 102°C.

REAGENTS

1. B u f f e r s o l u t i o n (pH 3): D i s s o l v e in d i s t i l l e d w a t e r 2 g of
sodium acetate (CI^COONa.SHjO), add 25 m l of glacial acetic acid and
dilute to 1 litre with distilled water.

2. Quinoline (a light yellow colour does not prohibit its use).

3. Strong ammonia solution.

4,. Diethyl ether (free from peroxides).

5. A m m o n i a , 10% solution: Dilute 40 ml concentrated ammonia to 100

m l with water.

6. Hydrochloric acid, IN.

7. Chloroform.

8. Ethanol, 96%.

9. Ethanol, 70% (v/v).

10. Acetic acid, glacial.

11. Acetone.

12. Sea sand (silver sand) washed in hydrochloric acid and calcined.

80
»

13. "Celite" in powder form.

14. Amberlite LA 2 (BDH, Rohm and Haas, or equivalent).

PROCEDURE

The a m o u n t s of s a m p l e , a u x i l i a r y s u b s t a n c e s and solvents may be

modified depending on the nature of the sample and the intensity of
its colouring.

For f o o d s with a high sugar content, alcoholic drinks and dairy

products :

H o m o g e n i z e solid f o o d s , take 10 g and m i x t h o r o u g h l y w i t h 40 m l of

b u f f e r s o l u t i o n (pH 3). P l a c e 30 m l of t h i s m i x t u r e i n t o a
centrifuge tube. For liquid foods, place 10 ml of the sample into a
centrifuge tube and add 20 m l of buffer solution.

Next add 10 ml of quinoline, shake vigorously, then centrifuge for 5

m i n u t e s at 1 2 0 0 r p m . W i t h d r a w and d i s c a r d the a q u e o u s s o l u t i o n as
completely as possible by suction using a Pasteur pipette. Shake the
quinoline phase with 20 m l of water, centrifuge and reject the upper
aqueous phase. Repeat this washing if the quinoline phase is cloudy.
Filter using a dry filter-paper and collect the filtrate in another
c e n t r i f u g e tube.

Add to the q u i n o l i n e s o l u t i o n 30 m l of d i e t h y l e t h e r and 1 m l of

water. Shake vigorously and centrifuge. Repeat extractions w i t h 1
ml aliquots of water until no colour is visible in the aqueous phase.
Combine all coloured aqueous extracts in a s p e c i m e n tube and retain
the quinoline-ether solution.

If the combined aqueous extract is coloured, shake it several times

with a little diethyl ether. Discard the ether after separation of
the p h a s e , t h e n m a k e the s o l u t i o n just acid w i t h a c e t i c acid and
e v a p o r a t e on a w a t e r b a t h u n t i l the v o l u m e is r e d u c e d to a b o u t 0.5
ml. Proceed with identification.

S h a k e the e t h e r / q u i n o l ine s o l u t i o n w i t h 10 m l of h y d r o c h l o r i c acid

IN. C o l o u r i n g of the acid a q u e o u s p h a s e i n d i c a t e s t h e p r e s e n c e of
basic colours. S u c h c o l o u r i n g is red in the c a s e of r h o d a m i n e B.
C e n t r i f u g e , r e m o v e the e t h e r q u i n o l i n e p h a s e as c o m p l e t e l y as
p o s s i b l e and d i s c a r d it. S h a k e the c o l o u r e d acid a q u e o u s s o l u t i o n
w i t h 2 m l of c h l o r o f o r m , a l l o w to s e p a r a t e , centrifuging if
n e c e s s a r y , and d i s c a r d the a q u e o u s p h a s e . Wash the c h l o r o f o r m
s o l u t i o n t w i c e w i t h 1 m l of w a t e r , d i s c a r d the a q u e o u s p h a s e a f t e r
e a c h w a s h and t r e a t the c h l o r o f o r m s o l u t i o n as the a q u e o u s e x t r a c t
above.

If the ether/quinoline phase remains coloured (which m a y indicate the

presence.of erythrosine, eosine, phloxine and/or certain natural
c o l o u r i n g s ) add 1 m l of a m m o n i a and s h a k e v i g o r o u s l y . R e p e a t the
e x t r a c t i o n s w i t h 2 m l a l i q u o t s of 10% a m m o n i a u n t i l no c o l o u r is
v i s i b l e in the a q u e o u s p h a s e . C o m b i n e all of the c o l o u r e d a m m o n i a
e x t r a c t s in a s p e c i m e n tube and t r e a t the s a m e as the a q u e o u s
extract, above.

Dilution with ether converts certain colours into a colourless form

which remains in solution in the ether/quinoline. If r h o d a m i n e B is
the o n l y red c o l o u r i n g m a t t e r p r e s e n t in the s a m p l e , the loss in
c o l o u r of the q u i n o l i n e p h a s e on d i l u t i o n w i t h e t h e r c o n f i r m s t h a t
this colour is present.

81
For food with a high starch or protein content (with the exception of
products manufactured from meat, or containing egg yolks):

If the food has a h i g h fat c o n t e n t , r e m o v e the fat as f o l l o w s : Put

10 g of the sample into a mortar. Grind with 3 g of silver sand, 6-7
g of " C e l i t e " and 30 to 40 m l of a c e t o n e . D e c a n t the liquid and
f i l t e r t h r o u g h a fluted f i l t e r paper. R e t u r n the r e s i d u e to the
m o r t a r , t r i t u r a t e w i t h 3 0 - 4 0 m l of a c e t o n e , d e c a n t and f i l t e r as
before. Repeat twice more. L e a v e the d e f a t t e d r e s i d u e to dry at
room temperature and then complete drying in the oven for 30 minutes.
Grind the r e s i d u e to a p o w d e r . For l o w fat foods dry if n e c e s s a r y
and grind to a powder.

Put 2 g of the d r i e d , p u l v e r i z e d food in a m o r t a r , add 15 m l of

quinoline which has been p r e v i o u s l y s a t u r a t e d w i t h w a t e r . Extract
the colouring matter as completely as possible by grinding the sample
with quinoline. Add 20 m l of b u f f e r s o l u t i o n and g r i n d again.
T r a n s f e r the m i x t u r e to a c e n t r i f u g e tube and shake v i g o r o u s l y .
C e n t r i f u g e for 10 m i n u t e s at 1200 r p m and r e j e c t the u p p e r a q u e o u s
phase. If an e m u l s i o n f o r m s at the level b e t w e e n the p h a s e s , it
should be d r a w n off w i t h the a q u e o u s phase. Add 20 m l of w a t e r ,
shake vigorously, centrifuge for 10 minutes at 1200 rpm. Discard the
aqueous phase as completely as possible by use of suction and filter
the cake and quinoline under vacuum through a sintered glass crucible
of p o r o s i t y G3 into a c e n t r i f u g e tube. P a s s 5 m l of w a t e r t h r o u g h
the filter and add a f u r t h e r 5 m l to the total f i l t r a t e , shake the
q u i n o l i n e and w a t e r v i g o r o u s l y and c e n t r i f u g e . D r a w off the l o w e r
q u i n o l i n e layer and filter it t h r o u g h a dry f i l t e r paper into a
centrifuge tube.

Add 30 m l of d i e t h y l ether and 1 m l of w a t e r to the q u i n o l i n e

filtrate, shake vigorously and centrifuge. If colour is present in
the a q u e o u s phase t r a n s f e r it to a fresh c e n t r i f u g e tube. Repeat
extractions with 1 ml aliquots of water until no colour is visible in
the a q u e o u s e x t r a c t s . Add 10 m l of q u i n o l i n e and 20 m l of b u f f e r
s o l u t i o n pH 3 to the c o m b i n e d c o l o u r e d a q u e o u s e x t r a c t s , shake
v i g o r o u s l y and c e n t r i f u g e . D r a w off the l o w e r q u i n o l i n e layer and
t r a n s f e r it to a c e n t r i f u g e tube. Add 30 m l of d i e t h y l ether.
E x t r a c t the c o l o u r s into w a t e r or 10% a m m o n i a r e s p e c t i v e l y . Wash
s e p a r a t e l y the c o m b i n e d w a t e r and c o m b i n e d a m m o n i a e x t r a c t s as
described above for high sugar foods.

Preserved fruits and fruits in syrup:

R e m o v e the s u r f a c e sugar from the f r u i t s w i t h a m i n i m u m of w a t e r .

B r e a k d o w n the s a m p l e into s m a l l p i e c e s , take 10 g, add 100 m l of
e t h a n o l 70% and heat in a w a t e r b a t h u n t i l the c o l o u r i n g d i s s o l v e s .
D e c a n t the liquid and e v a p o r a t e the e t h a n o l to a v o l u m e of a b o u t 10
ml. T r a n s f e r to a c e n t r i f u g e tube u s i n g 20 m l of b u f f e r s o l u t i o n ,
add 10 m l of q u i n o l i n e and then p r o c e e d as a b o v e for h i g h s u g a r
food s .

Meat products :

Chop f i n e l y 10 g of sample. D e f a t the s a m p l e as d e s c r i b e d a b o v e

using petroleum ether in place of acetone. Grind the dried defatted
meat with a solution of Amberlite LA2 (1.25 m l ) in quinoline (15 ml).
Add 5 ml of water and grind; leave for 1 hour, grinding occasionally.

If s o m e s u p e r n a t a n t liquid is a p p a r e n t d e c a n t it t h r o u g h a c o t t o n -
w o o l plug into a c e n t r i f u g e tube. Grind the r e s i d u e w i t h 4 x 5 m l
a l i q u o t s of q u i n o l i n e , d e c a n t i n g the s u p e r n a t a n t liquid after each
washing through the cotton-wool plug. Centrifuge the residue (2500
rpm) and filter the supernatant liquid through the cotton-wool plug.

82
 If the ground m i x t u r e is a paste, (e.g. as with luncheon m e a t ) with
no supernatant liquid, grind with 15 ml of quinoline. Transfer the
m i x t u r e to a centrifuge tube and centrifuge (2500 rpm). Filter the
supernatant liquid through a cotton-wool plug. Wash out the mortar
w i t h 5 ml of quinoline into the centrifuge tube. Stir the contents
of the tube and centrifuge (2500 rpm). Filter through the cotton-
wool plug.

Add 20 ml of buffer pH 3 to the combined filtrates from either of the

above, shake and centrifuge. D r a w off the q u i n o l i n e and filter
through c o t t o n - w o o l into a tapered centrifuge tube. Add 30 ml of
diethyl ether + 2 ml of ammonia 10%; shake and centrifuge. Transfer
the aqueous phase to another centrifuge tube. Repeat the extractions
with ammonia until no colour is visible in the aqueous phase. To the
combined ammonia extracts add 20 ml of buffer + 10 ml of quinoline;
shake and centrifuge. Draw off the quinoline and filter through
c o t t o n - w o o l into a tapered centrifuge tube. Add 30 ml of diethyl
ether and extract w i t h 2 m l aliquots of 10% a m m o n i a , shaking and
centrifuging each time, until the final ammonia extract contains no
colour. Combine the coloured ammonia extracts in a specimen tube and
treat as described for aqueous extracts, under high sugar foods.

REFERENCE

Mottier, M. and Potterat, M., 1953. Travaux de Chimie Alimentaire et d'Hygiene

44, 293.

83
 WATER-SOLUBLE COLOURS
(Thin-Layer Chromatography)

PRINCIPLE

The e x t r a c t e d dye is i d e n t i f i e d by thin-layer chromatography using cellulose

and silica gel. The dyes are grouped according to a code depending on their Rj
values in different solvents. This is of assistance in identifying some of the
dyes not usually encountered in permitted lists.

APPARATUS

1. T h i n - l a y e r c h r o m a t o g r a p h i c a p p a r a t u s , including spreaders for

the p r e p a r a t i o n of thin l a y e r s 0.25 m m t h i c k on 200 x 200 m m g l a s s
plates, chromatographic development tanks and 5 yl pipettes.

REAGENTS

1. Cellulose powder. M i c r o c r y s t a 11ine cellulose (available from

Applied Science Laboratories Inc. or equivalent). Prepare plates as
follows: shake 20 g c e l l u l o s e p o w d e r w i t h 60 m l m e t h a n o l for 3
m i n u t e s and b l e n d at h i g h speed for 30 s e c o n d s . spread onto plates
and dry in an o v e n at 80°C. A l t e r n a t i v e l y , use p r e p a r e d p l a t e s as
purchased.

2. R e f e r e n c e dye s o l u t i o n s . 0.1% in w a t e r .

3. Chromatographic solvents. All s o l v e n t m i x t u r e s s h o u l d be

f r e s h l y p r e p a r e d . The f o l l o w i n g s o l v e n t m i x t u r e s m a y be used w i t h
cellulose plates:

a. Trisodium citrate (2g), water (85 ml), ammonia (15 ml).

b. t-Butanol: propanoic acid:water (50:12:3).

c. Trisodium citrate (2g), hexamine (5g), water (50 ml).

d. 2-Methylpropan-l-ol: water :ethanol: ammonia (25:25:50:2).

e. Propan-l-ol: ethyl acetate :water (6:1:3).

f. Butan-l-ol: water:glacial acetic acid (20:12:10).

g. Hydrochloric acid:water (23:77).

h. Butan-l-ol:water : pyridine :ethanol (4:5:2:2).

i. Ethylmethylketone: acetone :water : ammonia (70:30:30:0.5).

j. Butan-l-ol: water :ethanol:quinoline (4:5:3:2).

The following solvent mixtures may be used with silica gel plates:

k. Propan-2-ol: ammonia (4:1).

1. Propan-2-ol: ammonia (85:15).

m. Methano1 : chloroform : water :quinoline (4:2:2:2).

n. Methanol :chloroform :quinoline (4:4:2).

o. Propan-2-o1 :chloroform:water:diethylamine (50:25:20:15).

84
 PROCEDURE

P l a c e 2 s p o t s of 1 - 2 p i of the dye s o l u t i o n o n t o e a c h o f four

c e l l u l o s e p l a t e s at a d i s t a n c e of at l e a s t 20 m m f r o m the e d g e and
b o t t o m of the p l a t e . A l s o spot on the p l a t e s 1 - 2 p i o f a s o l u t i o n
of Orange G and a solution of Amaranth as reference spots and place a
spot of a m i x t u r e of O r a n g e G and A m a r a n t h on top of one of the
s a m p l e spots. D r y the s p o t s by p l a c i n g the p l a t e s in an o v e n at
105°C for 5-10 minutes. Develop the cooled plates in solvents a, b,
c and d for a l e n g t h of r u n of a b o u t 150 m m at r o o m t e m p e r a t u r e .
Remove the plates from the tanks and allow them to air dry. When the
plates are dry rule lines across so as to divide the plates into the
following sections: code A: spots travelling above Orange G; code
B: s p o t s t r a v e l l i n g w i t h O r a n g e G; c o d e C: s p o t s t r a v e l l i n g b e l o w
Orange G but above Amaranth; code D: spots travelling with A m a r a n t h ;
code E: spots travelling b e l o w Amaranth.

Check whether the sample has affected the development characteristics

of O r a n g e G and A m a r a n t h and if so m a k e a l l o w a n c e for t h i s w h e n
d i v i d i n g the p l a t e into s e c t i o n s . O b s e r v e w h i c h s e c t i o n the s p o t s
from the sample solution appear in for each plate and w r i t e d o w n all
possible composite codes for each spot by listing the code individual
l e t t e r s in the o r d e r - s o l v e n t a, s o l v e n t b, s o l v e n t c, s o l v e n t d.
C o m p a r e the c o d e s w i t h the list g i v e n in T a b l e I and h e n c e o b t a i n a
preliminary identification of the dyes. W h e n two or m o r e spots are
similar in colour, cross code the dyes so that all possible dyes are
o b t a i n e d f r o m T a b l e I. A l s o if a d y e is v i s i b l e in o n e s o l v e n t b u t
not in another then this indicates that the dye is masked by another
dye and so all c o d e s for s p o t s in t h a t s o l v e n t m u s t be u s e d in
c o n s t r u c t i n g the c o m p o s i t e c o d e s . A f u r t h e r i d e n t i f i c a t i o n of the
d y e s m a y be o b t a i n e d by c a l c u l a t i n g the R f and R x ( w i t h r e s p e c t to
O r a n g e G ) v a l u e s and r e f e r r i n g to the T a b l e s II-V. This will
e l i m i n a t e s o m e of the d y e s o b t a i n e d f r o m T a b l e I. A l l Rf and R x
values are calculated by measuring to the leading edge of the spots.

Confirm identity of the sample dye by chromatography on a plate w i t h

s t a n d a r d s p o t s of the s u s p e c t e d c o l o u r s u s i n g s u i t a b l e s o l v e n t s .
A l s o o v e r s p o t s p o t s of the s a m p l e s o l u t i o n w i t h s p o t s of the
suspected dyes. The u n k n o w n dye is i d e n t i f i e d by g i v i n g a s i n g l e
spot with the correct standard while all the other standards should
give rise to double spots. If the sample contains several dyes, more
than o n e s o l v e n t m a y be n e c e s s a r y for c o m p l e t e c o n f i r m a t i o n of the
identity of. the dyes.

INTERPRETATION

In c o m p i l i n g the t a b l e of c o d e s for the d y e s , s l i g h t v a r i a t i o n s in the

d e v e l o p m e n t c h a r a c t e r i s t i c s of the d y e s h a v e b e e n t a k e n i n t o a c c o u n t so t h a t
some dyes occur under a number of different codes. B r o w n FK, Chocolate B r o w n
FB and C h o c o l a t e B r o w n H T h a v e n o t b e e n i n c l u d e d in t h i s t a b l e as t h e y s t r e a k
in the s o l v e n t s u s e d . If the s t a n d a r d d y e s , O r a n g e G and A m a r a n t h , act
d i f f e r e n t l y w h e n o v e r - s p o t t e d on the s a m p l e f r o m w h e n t h e y a r e s p o t t e d
s e p a r a t e l y on the p l a t e , then the s p o t s in the s a m p l e s h o u l d be c o d e d t w i c e ,
once u s i n g the s t a n d a r d s in the s a m p l e to d i v i d e up the p l a t e and o n c e u s i n g
the s t a n d a r d s p o t t e d s e p a r a t e l y to d i v i d e up the p l a t e . By this m e a n s all
p o s s i b l e d y e s w i l l be i n c l u d e d b u t a n u m b e r of t h e s e w i l l be r e j e c t e d on the
basis of c o l o u r and R f v a l u e . H o w e v e r , do not d i s c o u n t d y e s w h i c h c o u l d g i v e
rise to the c o l o u r of the spot (e.g. an o r a n g e c o l o u r e d s p o t m a y be a red and
yellow dye superimposed).

As m o s t p r o b l e m s a r i s e from the p o s s i b i l i t y of a red and y e l l o w d y e b e i n g

together in the m i x t u r e , the s e p a r a t i o n of the r e d s , o r a n g e s and y e l l o w s are
set out in Table VI. All R f and R values have been calculated by measuring to
the l e a d i n g e d g e of a spot as t h i s w a s found to be m o r e r e l i a b l e for s p o t s

85
which t a i l . When c o n f i r m i n g the i d e n t i t y of a dye by r u n n i n g i t w i t h s t a n d a r d
d y e s i t i s u s e f u l to o b s e r v e t h e p l a t e u n d e r UV l i g h t o f 2 5 4 nm a n d 3 5 0 nm as
some of the dyes f l u o r e s c e .

T h e f o l l o w i n g m i x t u r e s of d y e s c o u l d n o t be s e p a r a t e d i n a n y o f t h e s o l v e n t s
tried: C h o c o l a t e Brown HT and C h o c o l a t e Brown FB, Ponceau 3R and Ponceau MX,
V i o l e t 5BN and V i o l e t BNP. C h o c o l a t e Brown HT can be t e n t a t i v e l y d i s t i n g u i s h e d
f r o m C h o c o l a t e B r o w n FB by r u n n i n g i n s o l v e n t o , on s i l i c a g e l . Chocolate
Brown HT produces two spots and a s t r e a k from the s p o t t i n g l i n e . The two spots
t r a v e l h i g h e r than the s t r e a k from C h o c o l a t e Brown FB.

C o - e x t r a c t i v e s may a f f e c t the r u n n i n g c h a r a c t e r i s t i c s of v a r i o u s dyes but by

o v e r - s p o t t i n g the sample w i t h the s u s p e c t e d dyes in the f i n a l c o n f i r m a t i o n any
i r r e g u l a r i t i e s should not a f f e c t the i d e n t i f i c a t i o n of the d y e s t u f f .

REFERENCE

Hoodless, R.A., Thomson, J. and A r n o l d , J.E. 1971. Journal of Chromatography

56., 3 3 2 .
 Table I

(Codes for colours chromatographed using solvents a, b, c and d).

Code Possible Colour Code Possible Colour Code Possible Colour

AAAA Blue VRS CCCC Acid Yellow EABA Auramine

Brilliant Blue Orange GGN Methyl Violet
Light Green Yellowish Ponceau 4R Rhodamine B
AAAB Brilliant Blue Red 2G EACA Auramine
Fast Green Sunset Yellow Eo s ine
Green S CCCD Acid Yellow Erythrosine
Light Green Yellowish CCDC Red 2G Chrysoidine
Patent Blue V DAAA Rhodamine B Orange I
Yellow 2G Violet 6B Orange RN
AAAC Yellow 2G DABA Rhodamine B EACB Chrysoidine
AABA Scarlet GN DACA Orange RN Eos ine
AACA Scarlet GN DACB Chrysoin S Erythrosine
ABAA Brilliant Blue DACC Chrysoin S Orange I
Light Green Yellowish Orange RN EACC Chrysoin S
ABAB Brilliant Blue DADA Orange RN Orange RN
Light Green Yellowish DADC Orange RN EADB Orange I
ABAC Acid Magenta DBCA Naphthol Yellow Quinoline
ABAD Acid Magenta DBCB Orange GGN Yellow
ACAC Acid Magenta Sunset Yellow EADC Orange RN
ACAD Acid Magenta DCCA Orange RN Quinoline
ACCD Tartrazine DCCB Orange GGN Yellow
ADCD Ponceau 6R Sunset Yellow EAEB Quinoline
Tartrazine DCCC Acid Yellow Yellow
ADCE Ponceau 6R Orange GGN EAEC Quinoline
AECD Ponceau 6R Orange RN Yellow
AECE Ponceau 6R Sunset Yellow EBCB Fast Red E
BAAA Guinea Green B DCCD Acid Yellow EBCC Fast Red E
Violet 5BN Orange GGN EBCD Carmoisine
Violet BNP Sunset Yellow EBCE Carmois ine
BBBB Orange G DCDA Orange RN EBDB Fast Red E
BCBC Ponceau 4R DCDC Orange RN Quinoline
BCCC Ponceau 4R DCDD Red 6B Yellow
BCCD Tartrazine DCDE Red 6B EBDC Fast Red E
BDCD Tartrazine DCED Red 6B Quinoline
CAAA Guinea Green B DCEE Red 6B Yellow
Violet BNP DDDD Amaranth EBDD Carmoisine
Violet 5BN Red 6B EBDE Carmois ine
Violet 6B DDDE Red 6B EBEB Quinoline
CACB Chrysoin S DDEE Red 6B Yellow
CACC Chrysoin S DEDD Red 6B EBEC Quinoline
CBCA Naphthol Yellow S DEDE Red 6B Yellow
CBCB Orange GGN DEED Red 6B ECCB Fast Red E
Sunset Yellow DEEE Red 6B ECCC Fast Red E
CCBC Ponceau 4R EAAA Auramine Indigo Carmine
CCCB Acid Yellow Methyl Violet Orange RN
Orange GGN Rhodamine B Red 10B
Sunset Yellow Violet 6B

87
 Table 1 (continued)

Code Possible Colour Code Possible Colour Code Possible Colour

ECCD Carmois ine ECDE Carmoisine EDDE Red 6B

Indigo Carmine Red 6B Red 10B
Red 10B Red 10B EDED Black 7984
ECCE Carmoisine ECEC Bordeaux B Black PN
Red 10B Ponceau 3R Red 6B
ECDA Orange RN Ponceau MX EDEE Black 7984
ECDB Fast Red E Ponceau SX Black PN
ECDC Bordeaux B ECED Ponceau SX Red 6B
Fast Red E Red 6B Red FB
Indigo Carmine ECEE Red 6B EEDD Red 6B
Orange RN EDCC Indigo Carmine EEDE Red 6B
Ponceau 3R Red 10B EEED Black 7984
Ponceau MX EDCD Indigo Carmine Black PN
Ponceau SX Red 10B Red 6B
Red 10B EDCE Red 10B EEEE Black 7984
ECDD Carmoisine EDDC Indigo Carmine Black PN
Indigo Carmine Red 10B Red 6B
Ponceau SX EDDD Indigo Catmine Red FB
Red 6B Red 6B Indanthrene
Red 10B Red 10B Blue

Table II

(Rf and R x with respect to Orange G values for red colours).

Colour Approximate Rj values Approximate Revalues

Index
Colour Number Solvent Solvent

a b c d e £ h i k « b c d e £ h i k

Amaranth- 46185 0.6 0,3 0.5 0.6 0.4 0.2 0.6 0.4 0.4 0.8 0.4 0.5 0.8 0.7 0.4 0.8 0.4 0.9
Bordeaux B 16180 0.2 0.6 0.4 0.6 0.5 0.6 0.7 1.0 0.4 0.3 0.9 0.4 0.8 0.9 1.0 1.0 1.0 0.9
Carmoisine 14720 0.3 0.7 0.6 0.5 0.7 0.6 0.8 0.9 0.4 0.4 1.1 0.6 0.7 1.1 1.0 1.1 0.9 0.9
Eosine 45380 0.2 1.0 0.7 0.8 1.0 1.0 0.9 1.0 0.6 0.3 1.5 0.7 1.1 1.6 1.7 1.3 1.0 1.4
Erythros ine 45430 0.1 1.0 0.7 0.9 1.0 1.0 0.9 1.0 0.7 0.2 1.5 0.7 1.2 1.6 1.7 1.3 1.0 1.6
Fast Red E 16045 0.4 0.7 0.6 0.7 0.6 0.5 0.8 1.0 0.4 0.6 1.0 0.6 1.0 1.0 0.9 1.1 1.0 0.9
Ponceau 3R 16155 0.2 0.6 0.4 0.6 0.5 0.5 0.8 0.9 0.3 0.3 0.9 0.4 0.8 0.7 0.9 1.1 0.9 0.7
Ponceau 4R 16255 0.7 0.5 0.9 0.6 0.4 0.3 0.7 0.6 0.2 1.0 0.6 1.0 0.8 0.7 0.5 1.0 0.6 0.4
Ponceau 6R 16290 0.8 0.2 0.8 0.4 0.3 0.1 0.6 0.2 0.1 1.1 0.2 0.8 0.5 0.4 0.2 0.8 0.2 0.2
Ponceau MX 16150 0.2 0.7 0.5 0.6 0.5 0.5 0.8 0.9 0.4 0.3 0.9 0.5 0.8 0.9 0.9 1.1 0.9 0.8
Ponceau SX 14700 0.4 0.7 0.5 0.6 0.5 0.5 0.8 0.9 0.4 0.6 0.9 0.5 0.8 0.9 0.9 1.1 0.9 0.8
Red 2G 18050 0.6 0.6 0.7 0.6 0.4 0.5 0.7 0.9 0.4 0.8 0.8 0.7 0.8 0.7 0.9 1.0 0.9 0.9
Red 6B 18055 0.4 0.3 0.4 0.5 0.4 0.2 0.6 0.5 0.4 0.6 0.4 0.4 0.7 0.7 0.4 0.8 0.5 0.9
Red 10B 17200 0.2 0.5 0.6 0.6 0.4 0.3 0.7 0.8 0.4 0.3 0.6 0.6 0.8 0.7 0.5 1.0 0.8 0.9
Red FB 14780 0.0 0.3 0.1 0.2 0.4 0.2 0.7 0.4 0.6 0.0 0.4 0.1 0.3 0.7 0.4 1.0 0.4 1.3
Rhodamine B 45170 0.5 1.0 0.9 1.0 1.0 1.0 0.9 1.0 0.8 0.7 1.5 1.0 1.3 1.6 1.7 1.4 1.0 1.8
Scarlet GN 14815 0.9 0.7 0.9 0.8 0.8 0.6 0.8 1.0 0.5 1.1 0.9 1.0 1.1 1.2 1.0 1.1 1.0 1.2

88
 Table III

(Rf and R with respect to O r a n g e G values for yellow and orange colours.)

Colour Approximate R f values Approxiaate t x value

Index
Colour fluaber Solvent Solvent

a b c d e f S h k a b c d e £ 8 h k

Auramine 41000 0.3 1.0 0.9

1.0 0.9 1.0 - 0.8 0; 80.4 1.4 1.0
1.6 1.4 1.9 — 1.3 1.8
Acid Yellow 13015 0.7 0.6 0.6
0.9 0.6 0.5 0.7 0.7 0.4 0.8 0.9 1.0
0.9 0.9 0.9 1.1 1.0 1.0
Chrysoidine 11270 0.1 0.8 0.7
0.9 0.8 0.9 0.1 0.9 0.8 0.2 1.2 0.7
1.6 1.2 1.7 0.2 1.4 1.8
Chrysoin S 14270 0.5 0.8 0.8
0.6 0.9 0.7 0.4 0.8 0.5 0.6 1.2 0.9
0.9 1.3 1.4 0.7 1.2 1.0
Orange G 16230 0.8 0.7 0.9
0.6 0.7 0.5 0.7 0.7 0.4 1.0 1.0 1.0
1.0 1.0 1.0 1.0 1.0 1.0
Orange GGN 15980 0.6 0.7 0.8
0.6 0.6 0.5 0.2 0.7 0.4 0.8 1.0 0.9
1.0 1.0 1.0 0.3 1.0 1.0
Orange I 14600 0.4 0.8 0.8
0.7 0.9 0.7 0.1 0.8 0.5 0.6 1.2 0.9
1.1 1.3 1.4 0.2 1.2 1.2
Orange RN 15970 0.4 0.9 0.7
0.6 0.6 0.5 0.1 0.7 0.7 0.5 1.2 0.8
0.9 0.8 0.9 0.2 1.0 1.6
0.5 0.8 0.9 0.8 0.8 0.7 1.3 1.4 1.5 1.2
Quinoline 47005 0.1 0.7 0.4 0.6 0.7 0.6 0.1 0.6 0.7 0.1 1.0 0.5 1.0 1.1 1.2 0.1 0.9 1.6
Yellow 0.3 0.7 0.4 1.0
Sunse t
Yellow 15985 0.6 0.7 0.8 0.6 0.6 0.5 0.2 0.7 0.4 0.7 0.9 0.9 1.0 0.9 1.0 0.4 1.0 1.0
Tartrazine 19140 0.8 0.4 0.8 0.4 0.5 0.3 0.4 0.5 0.3 2.0 0.6 0.8 0.6 0.7 0.6 0.6 0.7 0.6
Yellow 2G 18965 0.9 0.8 1.0 0.6 0.8 0.6 0.9 0.7 0.4 1.1 1.2 1.1 1.0 1.2 1.2 1.5 1.0 1.0

Table IV

(Rf and R with respect to O r a n g e G values for brown and violet colours.)

Colour Approximate R^ value* Approxiaate R x value

Index
Colour Huaber Solvent Solvent

a b c d e k n o a b c d e k n o

Brown FK N/A S S S 0.6 S 0.7 S 0.4 S S S 0.9 S 1.1 S 0.6

0.8 S 0.5 1.3 S 0.7
Chocolate Brown FB N/A S S S S S 0.0 0.0 S S S S S S 0.0 0.0 S
Chocolate Brown HX 20285 s s S S S 0.0 0.0 S S S S S S 0.0 0.0 S
Acid Magenta 42685 1.0 0.4 0.9 0.6 0.5 0.2 - - 1.4 0.7 1.1 0.8 0.8 0.3 - -

0.6 1.0 0.9

Methyl Violet 42535 s 1.0 0.8 1.0 1.0 0.8 0.8 0.7 S 1.5 1.0 1.4 1.6 1.3 1.9 1.9
0.9 0.9 1.1 1.4
Violet BNP 0.7 0.8 0.9 0.9 0.9 0.5 0.4 0.5 1.0 1.3 1.1 1.3 1.4 0.7 1.1 0.7
Violet 5BN 42650 0.7 0.8 0.9 0.9 0.9 0.5 0.4 0.5 1.0 1.3 1.1 1.3 1.4 0.7 1.1 0.7
Violet 6B 42640 0.6 0.8 1.0 1.0 0.9 0.5 0.4 0.6 0.8 1.3 1.2 1.4 1.4 0.8 1.1 0.9
0.6 0.5 0.9 1.2

Note: S " streak on plate.

89
 Table V

(Rf and R x with respect to O r a n g e G v a l u e s for green, blue and black colours.)

Colour Approximate R^ values Approximate R e v a l u e s

Index
Colour Number Solvent Solvent

a b c d e k 1 • j a b c d e k 1 • j

Fast Green
FC 42053 0.9 0.8 1.0 0.8 0.8 0.1 0. 1 0.8 0.8 1.2 1.1 1.1 1.0 1.1 0.5 0.1 1.0 1.0
Green S 44090 0.9 0.8 0.9 0.8 0.8 0.1 0. 1 0.7 0.8 1.2 1.1 1.0 1.0 1.2 0.4 0.2 0.9 1.0
Guinea
Green B 42085 0.7 0.9 1.0 1.0 0.9 0.3 0. 3 0.8 0.9 0.9 1.2 1.1 1.3 1.3 1.1 1.4 1.0 1.2
Light Green
Ye 1lowish 42095 0.9 0.8 1.0 0.9 0.8 0.3 0. 1 0.8 0.8 1.1 1.1 1.1 1.2 1.1 0.9 0.5 1.0 1.0
Blue VRS 42045 0.9 0.9 1.0 0.9 0.9 0.3 0. 2 0.8 0.9 1.1 1.2 1.1 1.2 1.3 1.1 1.0 1.0 1.2
Brilliant Blue
FCF 42090 0.9 0.8 1.0 0.9 0.8 0.3 0. 2 0.8 0.8 1.1 1.1 1.1 1.2 1.1 1.0 0.7 1.0 1.0
Indigo 73015 0.2 0.4 0.5 0.6 0.5 0.3 0. 2 0.8 0.7 0.3 0.6 0.6 0.8 0.7 1.0 1.0 1.0 0.9
C a m ine 0.3 0.4
Patent BlueV 42051 0.9 0.9 1.0 0.9 0.9 0.1 0. 0 0.8 0.9 1.2 1.2 1.1 1.1 1.3 0.2 0.0 1.0 1.2
Black 7984 27755 0.2 0.3 0.2 0.4 0.4 0.1 0. 0 0.7 0.5 0.2 0.4 0.2 0.6 0.5 0.4 0.0 0.9 0.7
Black PN 28440 0.4 0.3 0.2 0.4 0.4 0.1 0. 0 0.7 0.5 0.4 0.4 0.2 0.6 0.5 0.4 0.0 0.9 0.9
Table VI

( S e p a r a t i o n of r e d s , o r a n g e s and y e l l o w s u s i n g four s o l v e n t m i x t u r e s . ) (The

l i s t i n g is in o r d e r of d e c r e a s i n g R^. T h o s e c o l o u r s in b r a c k e t s do n o t
c o m p l e t e l y s e p a r a t e from each other.)

Solvent 3a Solvent 3b Solvent 3c Solvent 3d

Yellow 2G Eos ine Yellow 2G Rhodamine B

Scarlet GN Erythrosine Rhodamine B .Auramine
Tartrazine Rhodamine B Auramine Chrysoidine
Ponceau 6R Auramine Orange G .Orange RN
Orange G Orange RN "Scarlet GN Erthyrhrosine
Ponceau R4 Chrysoin S Acid Yellow Eosine
"Acid Yellow Chrysoidine Chrysoin S Scarlet GN
Red 2G Orange I Naphthol Naphthol
Orange GGN Yellow 2G Yellow S Yellow S
Sunset Yellow Scarlet GN Eos ine Orange I
Naphthol Naphthol Erythrosine Orange G
Yellow S Yellow S Ponceau '4R Yellow 2G
'Orange RN Orange G Ponceau 6R "Fast Red E
Amaranth Carmois ine Orange GGN Acid Yellow
.Chrysoin S Quinoline Orange I Chrysoin S
Orange I Yellow Orange PN Orange GGN
Rhodamine B Fast Red E Sunset Yellow Quinoline
Red 6B Orange GGN Tartra zine Yellow
Ponceau SX Sunset Yellow Amaran th .Sunset Yellow
Fast Red E 'Bordeaux B Carmois ine Bordeaux B
Carmo i s ine Ponceau 3R Fast Red E Ponceau 3R
Bordeaux B Ponceau MX Ponceau SX Ponceau 4R
Auramine Ponceau SX Red 2G Ponceau MX
Quinoline Acid Yellow Red 10 B Ponceau SX
. Yellow Orange RN .Chrysoidine Red 2G
Eos ine Ponceau 4R Bordeaux B Red to B
Ponceau 3R .Red 2G Ponceau 3R .Orange RN
Ponceau MX "Red 10 B Ponceau MX "Amaranth
Red 10 B Tartrazine Quinoline Carmoisine
Erythrosine Amaranth Yellow .Tar trazine
Chrysoidine Ponceau 6R Red 6B Ponceau 6R
Quinoline Red 6B Red FB Red 6B
Yellow .Red FB Red FB
Red FB

90
I

WATER-SOLUBLE COLOURS
(Paper Chromatography)

PRINCIPLE

The e x t r a c t e d c o l o u r s are i d e n t i f i e d by p a p e r c h r o m a t o g r a p h y and c o m p a r i s o n

with standards. Identity is confirmed for some dyes by reducing the dye on the
paper w i t h t i t a n o u s c h l o r i d e s o l u t i o n . T h i s s p l i t s the m o l e c u l e at the a z o -
group.

APPARATUS

1. Development tank for paper chromatography suitable for holding

sheets of paper 200 x 260 m m .

2. Paper filter sheets for chromatography, 200 x 260 mm.

3. Microcapillary pipettes, 2 microlitres.

4. Filter papers, diameter 7 cm coarse grain, e.g. W h a t m a n No. 4.

5. Absorbent cotton-wool.

6. Sintered glass crucible, porosity G3.

REAGENTS

1. Reference colours.

2. Elution solvents (prepare as required):

a. Hydrated tri-sodium citrate (2g):Ammonia (20ml):water

(80ml).

b. 2.5% ( w / v ) h y d r a t e d trisodium citrate:colourless pyridine

(20:1).

c. n - B u t ano 1 : e t h a n o 1 : water : a m m o n i a (100:21:42:2).

d. n-Butano 1 :ethanol : water ¡ammonia (50:25:25:10) (for descend-

ing c h r o m a t o g r a p h y , s a t u r a t e the c h r o m a t o g r a p h y t a n k in
advance for 24 hours).

e. n-Butanol:ethanol: water (50:26:24).

f. n-Butano 1 : wa ter :col our le s s py r id ine : e t h a n o 1 ( 4 0 : 4 0 : 2 0 : 1 0 )

(for descending chromatography, saturate the chromatography
tank in advance for 24 hours).

g. t - B u t a n o 1: 0.4% (w/v) potassium ch1 o r i d e : p r o p i o n ic acid

(50:50:240).

3. Sulphanilic acid (4-aminobenzenesulphonic acid).

4. Naphthionic acid (4-aminonaphthalene-l-sulphonic acid).

5. Metanilic acid (3-aminobenzenesulphonic acid).

6. 2,4-Dimethyl-5-aminobenzenesulphonic acid.

7. 2,5-Diaminobenzenesulphonic acid.

8. Solution of 15 percent (w/v) titanium trichloride.

91
 9. n-Propanol.

10. Ammonia solution, with 25 percent (w/v) of NH^.

11. p-Dimethylaminobenzaldehyde Reagent: dissolve 1 g of p-

d i m e t h y l a m i n o b e n z a l d e h y d e in 20 ml of e t h a n o l 9 6 % , add 180 ml of n-
b u t a n o l and 30 ml of c o n c e n t r a t e d h y d r o c h l o r i c acid.

12. Petroleum ether, boiling range 40-60°C.

PROCEDURE

S p o t t h r e e c h r o m a t o g r a p h y p a p e r s w i t h the u n k n o w n c o l o u r e x t r a c t .
A l s o spot a p p r o p r i a t e r e f e r e n c e c o l o u r s . D e v e l o p each by a s c e n d i n g
c h r o m a t o g r a p h y u s i n g e l u t i o n s o l v e n t s a or b f o r the f i r s t p a p e r , c
or d f o r t h e s e c o n d and e i t h e r e , f or g f o r t h e t h i r d p a p e r .

W i t h the e x c e p t i o n of orange GGN, sunset y e l l o w FCF and Ponceau SX, a

c o l o u r i s o l a t e d from a sample and a r e f e r e n c e c o l o u r are c o n s i d e r e d
as i d e n t i c a l i f t h e i r c h r o m a t o g r a p h i c b e h a v i o u r s (Rf v a l u e , colour,
s h a d e ) i s t h e same a f t e r e l u t i o n b y t h r e e e l u t i o n s o l v e n t s w h i c h
comply w i t h the c o n d i t i o n s m e n t i o n e d above. When the c o l o u r s orange
G G N , s u n s e t y e l l o w FCF and P o n c e a u SX a r e p r e s e n t , identification
must be c o n f i r m e d by the s u p p l e m e n t a r y p r o c e d u r e d e s c r i b e d b e l o w .

S p o t a c h r o m a t o g r a p h y p a p e r w i t h t h e s o l u t i o n s o f t h e c o l o u r to b e
i d e n t i f i e d , t h e r e f e r e n c e c o l o u r and s u l p h a n i l i c a c i d , n a p h t h i o n i c
a c i d , m e t a n i l i c a c i d , 2 , 4 - d i m e t h y l '- 5 - a m i n o b e n z e n e s u l p h o n i c a c i d and
2 , 5-diaminobenzenesulphonic acid.

D r y the s p o t s u s i n g a c u r r e n t o f w a r m a i r . O v e r s p o t the dry spots

w i t h 6 m i c r o l i t r e s o f a m i x t u r e o f 2 ml o f t i t a n i u m trichloride
s o l u t i o n and 9 ml of d i s t i l l e d w a t e r . I n most c a s e s t h i s t r e a t m e n t
r e s u l t s i n d e c o l o r i z a t i o n of t h e s t a i n s . I f t h i s is not the c a s e ,
r e p e a t t h i s t r e a t m e n t u n t i l d e c o l o r i z a t i o n has been c o m p l e t e d . Dry
the s t a i n s thus t r e a t e d w i t h a c u r r e n t of warm a i r .

D e v e l o p b y d e s c e n d i n g c h r o m a t o g r a p h y at 1 5 - 2 0 ° C , f o r at l e a s t 16
h o u r s , u s i n g a m i x t u r e of n-propanol (2 p a r t s by v o l u m e ) and ammonia
(1 p a r t b y v o l u m e ) . D r y the c h r o m a t o g r a m i n a i r . Spray with a
s o l u t i o n of p - d i m e t h y l a m i n o b e n z a l d e h y d e . Compare the r e a c t i o n s g i v e n
by the c o l o u r s to be i d e n t i f i e d w i t h those of the r e f e r e n c e c o l o u r s
and s u l p h o n i c a c i d s . Orange GGN g i v e s a y e l l o w s t a i n w i t h the same
Rf v a l u e s as m e t a n i l i c a c i d , sunset y e l l o w FCF g i v e s a y e l l o w s t a i n
w i t h t h e s a m e R f v a l u e as s u l p h a n i l i c a c i d a n d P o n c e a u SX g i v e s a
yellow stain with the same R f v a l u e as 2 , 4 - d i m e t h y 1 - 5 - a m i n o -
benzenesulphonic a c i d .

U n d e r the same o p e r a t i n g c o n d i t i o n s , the f o l l o w i n g r e a c t i o n s a r e

o b t a i n e d from other c o l o u r s : Y e l l o w s t a i n w i t h the same R^ v a l u e as
naphthionic acid: Fast red E , a z o r u b i n e , a m a r a n t h , c o c h i n e a l red A,
Ponceau 6R. Y e l l o w s t a i n w i t h the same R^ v a l u e as s u l p h a n i l i c a c i d :
A c i d y e l l o w G, c h r y s o i n , t a r t r a z i n e , b r i l l i a n t b l a c k BN. Red s t a i n
w i t h t h e s a m e R f v a l u e as 2 , 5-d i a m i n o b e n z e n e su 1 p h o n i c a c i d : Acid
y e l l o w G.

REFERENCE

EEC Document 1867/VI/72/F/Rev.1.

 OIL-SOLUBLE COLOURS
(Isolation and Identification)

PRINCIPLE

The food fat (in the unaltered state or extracted from the food) is dissolved
in petroleum ether. The solution is subjected to chromatography on a column of
aluminium oxide, and the colouring matters undergo elution by means of several
elution solvents. The eluates are evaporated to dryness under vacuum and the
residues (subjected to saponification if need be) are taken up in diethyl ether
and identified by thin-layer c h r o m a t o g r a p h y , benzene being used as elution
solvent. Adequate safety precautions m u s t be taken w h e n using benzene.
Toluene is an alternate solvent but use must be validated.

APPARATUS

1. Balance.

2. Aluminium dish of low form, diameter 7 cm.

3. Drying chamber, set at 60°C.

4. Filter papers, diameter 7 cm.

5. Filter papers, coarse, in sheets.

6. Soxhlet apparatus, with accessories.

7. Water bath.

8. Graduated test-tubes, 10 ml, 25 ml, 50 ml, 100 ml and 250 ml.

9. Beakers, 50 ml.

10. Chromatography tube 20 cm x 1 cm diameter with a tap.

11. Absorbent cotton-wool.

12. Volumetric pipette, 2 ml.

13. Round-bottom flask, 100 ml, with standard ground-glass joint.

14. Rotary evaporator.

15. Bunsen burner or electrically heated plate.

16. Condenser.

17. Separating funnels, 250 ml.

18. Development tank for TLC capable of holding plates 200 x 200 cm.

19. TLC plates 200 x 200 c m , coated w i t h a layer of silica gel G.

Prepare as f o l l o w s : W e i g h 30 g of silica gel G in a 300 ml conical
flask, equipped with g r o u n d - g l a s s stopper. Add 60 m l of distilled
water, stopper the flask and homog enize the contents for 1 minute by
shaking. Spread the suspension by means of a spreader onto 5 plates
in such a w a y as to obtain a layer of 0.25 m m thickness (in the wet
state). A l l o w the plates to dry in air for o n e - h a l f hour and keep
them in a drying-chamber at 60°C at least overnight until they are to
be used.

20. Microcapillary pipettes of 2 micro-litres, Desaga or equivalent.

93

t
21. Spray bottle .

22. Oven, set at 100°C.

REAGENTS

1. Sea sand, washed in h y d r o c h l o r i c acid and calcinated.

2. Ethanol, 96%.

3. Petroleum ether, boiling range 40-60°C.

4. B a s i c a l u m i n i u m o x i d e ( W o e l m or equivalent), activated for 1

hour at 4 0 0 ° C i m m e d i a t e l y b e f o r e u s e .

5. Benzene (or substitute solvent).

6. Acetone.

7. M i x t u r e of p e t r o l e u m e t h e r and a c e t o n e , 9 8 : 2 by v o l u m e . Measure
e x a c t l y b y p i p e t t i n g 2 m l o f p e t r o l e u m e t h e r f r o m a f i l l e d 1 0 0 ml
f l a s k and r e p l a c i n g i t w i t h 2 ml o f a c e t o n e .

8. M i x t u r e of petroleum ether and a c e t o n e , 1 : 1 by v o l u m e . Measure

25 ml of p e t r o l e u m e t h e r and 25 ml o f a c e t o n e , b y g r a d u a t e d cylinder
and m i x .

9. M i x t u r e of a c e t o n e and e t h a n o l , 4 : 1 by v o l u m e . Measure 40 ml of
a c e t o n e and 10 ml of e t h a n o l by g r a d u a t e d c y l i n d e r and m i x .

10. Ammonia (25% m/m NH3).

11. M i x t u r e o f e t h a n o l and a m m o n i a , 2 : 1 by v o l u m e . M e a s u r e 4 0 ml o f
e t h a n o l a n d 2 0 ml o f a m m o n i a 0 . 9 1 0 , u s i n g a g r a d u a t e d c y l i n d e r and
mix.

12. S o l u t i o n of e t h a n o l i c p o t a s s i u m h y d r o x i d e 0 . 5 N . W e i g h 14 g o f
p o t a s s i u m h y d r o x i d e , d i s s o l v e i n 5 0 0 ml o f e t h a n o l . Keep in the
dar k .

13. Diethyl ether, free from peroxides.

14. Anhydrous magnesium sulphate.

15. Sulphuric acid 8N.

16. Solutions of the reference colours, 0.5% in e t h a n o l or

chloroform. D i s s o l v e 5 0 mg o f e a c h r e f e r e n c e c o l o u r i n 10 m l of
e t h a n o l , e x c e p t c a r o t e n e w h i c h must be d i s s o l v e d i n c h l o r o f o r m .

17. Mixture of n-hexane and ethyl acetate, 9:1 by volume.

18. Carr-Price Reagent. W e i g h 25 g o f a n t i m o n y t r i c h l o r i d e i n a

c o n i c a l f l a s k w i t h a g r o u n d g l a s s s t o p p e r and d i s s o l v e i n 75 ml
c h l o r o f o r m , t a k i n g c a r e to e x c l u d e m o i s t u r e .

PROCEDURE

W e i g h 5 to 1 0 g o f t h e s a m p l e i n an a l u m i n i u m d i s h c o n t a i n i n g s a n d ,
a d d 5 t o 1 0 ml e t h a n o l , s t i r , then l e a v e the m i x t u r e in the oven
overnight. T r a n s f e r the c o n t e n t s o f the d i s h to a t h i m b l e or f i l t e r
p a p e r and e x t r a c t w i t h p e t r o l e u m e t h e r f o r 4 h o u r s in a S o x h l e t .
R e m o v e f r o m t h e a p p a r a t u s and e v a p o r a t e t h e p e t r o l e u m e t h e r o v e r a
water bath.

94
D i s s o l v e 0.5 g of the r e s i d u e or 0.5 g of the oil or fat to be
examined in 10 m l of petroleum ether in a 50-ml beaker. Place a plug
of c o t t o n - w o o l in a c h r o m a t o g r a p h y tube and p u s h this d o w n to just
ab ove the tap. Fill the tube with a suspension of a l u m i n i u m oxide in
benzene so as to obtain a column 10 cm in height. Elute the benzene,
t a k i n g c a r e to see t h a t the c o l u m n d o e s n o t b e c o m e dry. R i n s e the
c o l u m n w i t h 50 m l of p e t r o l e u m e t h e r or u n t i l all the b e n z e n e h a s
been removed. D i s c a r d the b e n z e n e and r i n s i n g s . Add the c o l o u r
solution and regulate the speed of elution to about 1 ml per minute.
R i n s e the c o l u m n w i t h 100 m l of p e t r o l e u m e t h e r . Do n o t a l l o w the
c o l u m n to b e c o m e dry. Discard the eluate.

Elute carotenes with 50 m l of the mixture of petroleum ether/acetone.

Collect the eluate in a 100 ml round-bottomed flask. Evaporate under
partial v a c u u m , using a rotary evaporator or a current of nitrogen,
w i t h the f l a s k over a w a t e r b a t h . T a k e up the r e s i d u e in 1 m l of
diethyl ether and proceed w i t h identification as below. Do not allow
the column to b e c o m e dry.

Elute amino-aniline colours with 50 ml of the mixture of petroleum

e t h e r / a c e t o n e 1:1. C o l l e c t the e l u a t e in a 100 m l r o u n d - b o t t o m e d
flask. Evaporate under partial vacuum using a rotary evaporator or
by a c u r r e n t of n i t r o g e n , w i t h the f l a s k on a w a t e r b a t h . T a k e up
the residue in 1 ml of diethyl ether and proceed with identification.
Do not allow the column to run dry.

E l u t e h y d r o x y - a n i l i n e c o l o u r s w i t h 50 m l of the a c e t o n e / e t h a n o 1
mixture. C o l l e c t the e l u a t e in a 100 m l r o u n d - b o t t o m flask.
Evaporate to dryness under vacuum using a rotary evaporator or on a
w a t e r - b a t h in a c u r r e n t of n i t r o g e n . T a k e up the r e s i d u e w i t h 1 m l
of diethyl ether and proceed with identification. Avoid drying the
column.

Eilute bixine and hydroxy-aniline colours which m a y still r e m a i n on

the c o l u m n w i t h 50 m l of the m i x t u r e of e t h a n o l / a m m o n i a (2:1).
Collect the eluate in a 100 ml round-bottomed flask. Evaporate under
partial vacuum using a rotary evaporator or in a current of nitrogen,
w i t h the f l a s k on a w a t e r b a t h . T a k e up the r e s i d u e in 1 m l of
diethyl ether and proceed with identification.

Note that a change of colour of the aluminium c o l u m n to a red-violet

shade after the e t h a n o l / a m m o n i a mixture has been added indicates the
presence of curcumin in the sample.

The presence of residual oil or fat in any of the above fractions can
hinder identification by thin-layer chromatography. In this case, it
is recommended to saponify the lipids present as indicated below.

Add to e a c h of the r e s i d u e s 50 m l of e t h a n o l i c p o t a s s i u m h y d r o x i d e
s o l u t i o n and s o m e f r a g m e n t s of p u m i c e - s t o n e . B o i l for 45 m i n u t e s
under reflux. Cool and transfer the solutions into separate funnels
u s i n g 100 m l of w a t e r . C a r e f u l l y e x t r a c t the a q u e o u s p h a s e of e a c h
(if it does not contain bixine) once with 50 m l and twice with 25 m l
of diethyl ether. Wash the ethereal extracts three times, using 25
m l of water each time.

Dry each ether extract with anhydrous m a g n e s i u m sulphate. Evaporate

u n d e r p a r t i a l v a c u u m u s i n g a r o t a r y e v a p o r a t o r or a c u r r e n t of
nitrogen. T a k e up the r e s i d u e in 1 m l of d i e t h y l e t h e r and p r o c e e d
with identification.

Conduct identification by thin-layer chromatography as follows:

95
 P l a c e 4 m i c r o l i t r e s (or i f need b e , a l a r g e r q u a n t i t y ) of each of the
e t h e r s o l u t i o n s from above u s i n g a m i c r o c a p i 1 l a r y p i p e t t e , along an
i m a g i n a r y l i n e 2 . 5 cm a w a y f r o m the e d g e o f the p l a t e . Space the
spots at i n t e r v a l s of 2 cm. In the same w a y , p l a c e 2 m i c r o l i t r e s of
s o l u t i o n s of r e f e r e n c e c o l o u r s . D e v e l o p the p l a t e w i t h b e n z e n e in a
tank s a t u r a t e d w i t h vapours of t h i s s o l v e n t . A l l o w to m i g r a t e over a
d i s t a n c e of 17 cm. T h e s e p a r a t i o n o f the d i f f e r e n t c o l o u r s may be
f a c i l i t a t e d by l e a v i n g the p l a t e to dry in a i r a f t e r d e v e l o p m e n t , and
by d e v e l o p i n g a g a i n w i t h b e n z e n e . I n c e r t a i n c a s e s , i t is u s e f u l to
repeat this procedure. (To s e p a r a t e S u d a n I f r o m S u d a n I I , d e v e l o p
w i t h the m i x t u r e of n - h e x a n e / e t h y l a c e t a t e . )

Examine the p l a t e and i d e n t i f y the c o l o u r s , c o m p a r i n g the Rf v a l u e s

of the spots from the e x t r a c t s w i t h those of the r e f e r e n c e s o l u t i o n s .
A f t e r e x a m i n a t i o n , p l a c e the p l a t e in a tank c o n t a i n i n g enough Carr-
P r i c e r e a g e n t to s a t u r a t e the t a n k w i t h i t s v a p o u r u n t i l t h e p l a t e
becomes v i s i b l y w e t . A b l u e s t a i n a p p e a r i n g in the s u s p e c t e d b i x i n
f r a c t i o n i n d i c a t e s the p r e s e n c e of b i x i n . Heat the p l a t e for ten
m i n u t e s to 100°C. The b l u e s t a i n turns red-brown.

IHTERPKETATION

B i x i n is t r a n s f o r m e d by s a p o n i f i c a t i o n into n o r b i x i n . I f the s u s p e c t e d b i x i n
f r a c t i o n has been s a p o n i f i e d , a c c o u n t must be t a k e n of t h i s during
c h r o m a t o g r a p h i c e x a m i n a t i o n , so that when c o m p a r i s o n i s made of the Rf v a l u e s
o f t h e s p o t s w i t h t h o s e o f t h e c o n t r o l s , i t i s u s e f u l to p r e p a r e a r e f e r e n c e
s o l u t i o n of n o r b i x i n .

As the c a r o t e n o i d s are l i k e l y to o x i d i s e , it is n e c e s s a r y to spot the f r a c t i o n s

c o n t a i n i n g t h e m on to t h e c h r o m a t o g r a p h i c p l a t e as q u i c k l y as possible,
p r e f e r a b l y under a current of carbon d i o x i d e or n i t r o g e n .

O i l Y e l l o w GG and O i l Y e l l o w XP may be e n c o u n t e r e d and can be d i s t i n g u i s h e d as

follows: T h e f o r m e r i s a m i x t u r e o f a p h e n o l i c a z o and b i s a z o d y e , b o t h
c o m p o n e n t s o f w h i c h a r e e x t r a c t a b l e f r o m e t h e r by c a u s t i c a l k a l i . This
b e h a v i o u r d i s t i n g u i s h e s it from O i l Y e l l o w X P , a p y r a z o l o n e d y e , and from many
other oil-soluble colours. It a l s o means that i t cannot be s e p a r a t e d from an
o i l y m i x t u r e by s a p o n i f i c a t i o n f o l l o w e d by e x t r a c t i o n of the non-saponifiable
f r a c t i o n , w h i c h i s a u s e f u l means of i s o l a t i n g many other c o l o u r s of t h i s t y p e .
H o w e v e r , i f a s o l u t i o n of the f a t i n e t h e r i s e x t r a c t e d w i t h a q u e o u s s o d i u m
h y d r o x i d e b e f o r e s a p o n i f i c a t i o n t h i s d i f f i c u l t y i s l a r g e l y o v e r c o m e , as O i l
Y e l l o w GG w i l l then be removed.

Oranges may be examined for O i l Orange XO by removing the p e e l and s o a k i n g it

i n a 5 0 : 5 0 m i x t u r e o f a c e t o n e and e t h e r , d e c a n t i n g t h e c o l o u r e d s o l v e n t and
e v a p o r a t i n g it n e a r l y to d r y n e s s . The r e s i d u e is then taken up w i t h a m i x t u r e
of w a t e r and ether and t r a n s f e r r e d to a s e p a r a t i n g f u n n e l where i t is s h a k e n .
A f t e r s e p a r a t i o n the a q u e o u s l a y e r i s d i s c a r d e d . The e t h e r i s w a s h e d w i t h a
d i l u t e s o l u t i o n of c a u s t i c soda and then w i t h w a t e r u n t i l n e u t r a l . I t is d r i e d
w i t h anhydrous sodium s u l p h a t e and then e v a p o r a t e d . The r e s i d u e is taken up in
p e t r o l e u m e t h e r ( b o i l i n g r a n g e 4 0 ° - 6 0 ° C ) a n d i s r u n t h r o u g h a 0 . 5 cm b y 4 cm
column of BDH c h r o m a t o g r a p h i c a l u m i n a or e q u i v a l e n t g r a d e . The column is w e l l
washed w i t h p e t r o l e u m e t h e r and then w i t h p e t r o l e u m e t h e r c o n t a i n i n g 5 p e r c e n t
of d i e t h y l e t h e r , w h i c h causes the dye to t r a v e l down the column as a red band.
T h i s c o l o u r e d f r a c t i o n is c o l l e c t e d , the s o l v e n t is removed and the r e s i d u e is
d i s s o l v e d in c h l o r o f o r m for e x a m i n a t i o n in a s p e c t r o p h o t o m e t e r . I n t h i s way
c o r r e s p o n d e n c e w i t h t h e s t a n d a r d c u r v e c a n be o b t a i n e d r i g h t i n t o t h e u l t r a -
violet .

REFERENCE

EEC Document 1867/VI/72/F/Rev.1.

 COLOUR IDENTIFICATION BY SPECTRA

If, after separation and attempted identification by chromatography, the colour

appears to be a n o n - p e r m i t t e d one, it must be identified before an adverse
report can be given. The first step is to cut out the paper spot or scrape the
spot off the TLC plate and dissolve the dye in a suitable solvent, w a t e r or
acetone-alcohol. A useful device for the removal of spots from TLC plates is
described by Sahasrabudhe in JAOAC 47 1964 (889). After c e n t r i f u g i n g or
filtration, remove organic solvents by gentle evaporation and dissolve in 0.1 N
NaOH or adjust the strength of an aqueous solution to 0.1 N with alkali and
read the spectrum from about 210 nm to 650 nm. This solution, or a fresh one,
should be adjusted to 0.1 N with h y d r o c h l o r i c acid and the r e a d i n g s repeated.
The plotted curves may be compared with those reproduced below.

The following spectra were taken from "Separation and Identification of Food
Colours P e r m i t t e d by the Colouring M a t t e r in Food R e g u l a t i o n s 1957", by the
Association of Public Analysts (U.K.), 1960.

Included with each spectra are the usual n a m e , the Colour Index N u m b e r ,
occasionally the EEC n u m b e r (in parenthesis), and s y n o n y m s and other n a m e s
(also in parentheses).

Absorption curve for Quinoline Yellow.

Absorption curve for Fast Yellow AB.

 ¡N/I0 NaOH

S I mg/100 mi

Napthol Yellow

C.I. 10316
(FD&C Y e l l o w no. 1 & 2)

\ N/10 N a O H

W a v e l e n g t h , mp

Tsrtrazine

C.I. 19140 (E102 )

(FD&C Yellow no. 5)

98
 08

YELLOW RY I mg/100 ml

Yellow RY

C.I. 14330

Wavelength, mjj

99
 Sunset Tellow FCF

C . I . 1 5 9 8 5 (El 1 0 )
(FD&C Yellow n o . 6)

400 500
Wavelength, mp

1-2

Oil Yellow XP and GG è

g 10
v
C . I . 12740 1
•B 0 8
Q.
o
0-6

00 225 250 275 300 325 350 400 450 500 550 600 650
Wavelength(nanometers)
Absorption curve for permitted oil-soluble colours Oil Yellow G G and Oil Yellow X P
at concentrations of 2 mg per 100 ml of light petroleum.

100
 20

Chrysoin S

C.I. 14270 (E103)

(Chrysoin SGX)

Neutral

500

Wavelength (nanometres) Chrysoine S

Absorption curves for Chrysoine S

Orange GGH

C.I. 15980 (E111)

(Orange GGL or GGP)

200 300 400 500 600

Wavelength (nanometres) Orange GGN
Absorption curves for Orange G G N

101
 Orange G

C.I. 16230
(D & C Orange no.

400
Wavelength, mp

102
 Yellow 2G

C.I. 18965

400
Wavelength, my

INDIGO CARMINE I mg/100 ml

Indigo Carmine

C . I . 73015 (E132)
(FD&C Blue n o . 2)
(Indigoline)

W a v e l e n g t h , mp

103
 Scarlet GH

C.I. 14815 (E125)

Wavelength (nanometres) Scarlet G N

Absorption curves for Scarlet GN

Ponceau 6R

C.I. 16290 (E126 )

( P o n c e a u 6M)

200 300 400 500 600

Wavelength (nanometres) Ponceau 6 R

Absorption curves for Ponceau 6R

104
 ERYTHROSINE 0-5 mg/100 ml

<\
I \
Erythrosine

C.I. 45430 (E127)

(Erythrosine BS)
N/10 NaOH
(FD&C Red n o . 3)

N/10 HCI

•«00
W a v e l e n g t h , my

REO 6B 1mg/100 ml

\
Red 6B \
\
\
\
C . I . 18055 — \

(Ext. D & C Red no. 1)

N/10 HCl\\
UN/10 >laOH
/*"v
/ \
' \
\
\ \
/
' ^ / VN
/
• / "^-v^N/10 HCl\
X v \
f /
/ /
/ /
/ /
N/IONaOH v \
\ \

1 1 1 ! I ' l l . 1 1 1 1 1 1 1 1
W a v e l e n g t h , mp

105
 0'8|
1
1
1
RED FB mg/100 ml
1
1

1
I
(
- \
\ \
\
\ \ \N/I0 NaOH
y
\ \ / 1 / / \ \
\ \ / / \ \ Red FB
\ \ // \ \
//
_ \ \ // \ \n/IO NaOH
\ \ / \ \
\ \ C.I. 14780
\\ ' \/ \ \ \
\ \ \\
\ \ / \ '/
N/10 \ K¿/ \\
\ \\
\ \\
\ /
\\
\_
N/10 HCl\i
. 1 1 1 1 t i l l 1 I 1 1
200 •400 1 1 1 1
Wavelength, mjj

Red 2G

C . I . 18050
( E x t . D & C Red no. 11)

•wo
W a v e l e n g t h , mjj

106
 F a s t Red E

C.I. 16045

400
Wavelength, mjj

Red 10B

C . I . 17200
(D & C Red n o . 33)

•400
Wavelength, m>j

107
 Ponceau 4R

C.I. 16185 (E123)

(FD&C Red n o . 2)

Amaranth

C . I . 16255 (E124)
(Brilliant Scarlet 4R)
(Cochineal Red A )

108
 Ponceau SX

C . I . 14700
(FD&C Red 110. 4)

Wavelength, mjj

C A R M O I S I N E I mg/100 ml
Carmoisine

C . I . 14720 ( E 1 2 2 )
( E x t . D & C Red n o . 1 0 )

Wavelength, mjj

109
 Ponceau MX

C.I. 16150
(Ponceau 2R and R R )
(D & C R e d n o . 5 )

POMC AU 3R
\N/IO HCI
Ponceau 3R n
V /
C . I . 16155 \ \N/I0 NaO
\ \
(FD&C Red n o . 1) \ *
\ \
\ \
\l
\\
\ W ~ \
\
- \ \
\
\
\
\ \
y / X \
• / \ \
• /
/ N/10 N a O H \ \
\\

N/10 H C l V x
I ! 1 1 ! 1 I 1 l i l i i i i K;
Wavelength, mp

110
 Green S

C.I. 44090 (E142)

(Lissamitie Green)
(Acid Brilliant
Green BS)
(Wool Green B S )

400 500
Wavelength. mjj

Blue VRS

C.I. 42045

«J0 500
Wavelength, m/j

111
 Absorption curve for Patent Blue V .

20 r

• 1 mg per 100 ml of buffer

18 •
0-5 mg per 100 ml of 0-1 N NaOH
1 mg per 100 ml of 0-1 N HCI buffer
1-6 •

Brilliant Blue FCF

1-4 •
>

C.I. 42090 i 12 •
(FD&C Blue no. 1) Î
(D & C Blue no. 4) J 10 •
°
a 08-

0-6 •

0 4 •

0 2 •

200 400 500 700

Wavelength (nm)

Absorption curve for Brilliant Blue F C F .

112
 0-8

0-7 CHOCCLATE BROWN HT 1 {m

/I00 ml

0-5
Chocolate Brown
k
l\
\\ C.I. 20285
\ \ (Food Brown 3)
0-3 \\ N/10 NaOH
\ \ / \ (Reddish Brown)
\ \ / \
\ N. /
/
/
,— \
\
\
0-2
/ N/IO H3<
\ \
\ \
\ \
0-1- \
\
\
\
S
\
» ' , ^
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
K » «lo a» 6C10 7C a
Wavelength, m>i

0-8,

Black PN
BLACK PN

C.I. 28440 (E151 )

(Food Black 1)

I
0-5(— IJ N/10 NaOH
6- \
lc I
3
a.
O

0*1
N/10 NaOH

J I I L iserJ i I L T® I I I I I I J I I I
5» W
Wavelenjth, mp

113
 VIOLET BNP I mg 100 ml

Violet BNP

C.I. 42580
(Acid Violet 21)

Wavelength, mjj

2 mg per 100 m l of buffer

Violet 6B 1-8 2 mg per 100 ml of 0-1 N H C I
C.I. 42640 No reading with N a O H soin
(FD&C Violet no. 1) 1-6
(D & C Violet no. 1) ! Ai \
1-4
i1
1
i r
i
^HCI
l i
- i i
i i
i \ / \ ^^-buffer
0-8 - u \

0-6

0-4

0-2
l\ ' i i V - - . I \ i i i /
/ \ i--- 1 i
300 400 500 700
Wavelength (nm)

Absorption curve for Violet 6B.

114
6.4 OTHER ADDITIVES

P r e s e r v a t i v e s , a n t i o x i d a n t s and c o l o u r s are the m o s t c o m m o n food a d d i t i v e s .

H o w e v e r , t h e r e are m a n y o t h e r a d d i t i v e s w h i c h are i n c o r p o r a t e d i n t o food to
improve appearance, taste and physical character.

Generally speaking, thickeners, emulsifiers and stabilizers used in food are of

v e r y l o w t o x i c i t y and t h e i r use is not r e s t r i c t e d on t h i s b a s i s . However,
their u s e is p r o h i b i t e d in a n u m b e r of c o u n t r i e s in p r o d u c t s s u c h as f l o u r
c o n f e c t i o n e r y , s u g a r c o n f e c t i o n e r y , p r e s e r v e s , fruit juices and soft drinks,
meat and m e a t products and dairy products. For this reason, qualitative tests
for their detection m a y be required.

E x t r a c t i o n f r o m c o n f e c t i o n e r y and i d e n t i f i c a t i o n by e l e c t r o p h o r e s i s on
c e l l u l o s e a c e t a t e and IR s p e c t r a of a n u m b e r of t h i c k e n e r s are d e s c r i b e d b y
A e r n y and M i s e r e z (26). T h o s e d e a l t w i t h are s t a r c h , c a r r a g e e n a n , a l g i n a t e ,
a g a r , m e t h y l and c a r b o x y m e t h y l c e l l u l o s e and the g u m s of g u a r , t r a g a c a n t h ,
a c a c i a and c a r o b . TLC and IR of s o m e e m u l s i f i e r s is d e s c r i b e d b y S r e b r n i k -
Friszman and Charon (27). Kroller (28) has published a series of papers on the
d e t e c t i o n of e m u l s i f i e r s . G r a h a m and c o - w o r k e r s (29)(30)(31) p u b l i s h e d a
number of papers on the determination of carrageenan, alginate and other g u m s
in food. Martelli and Proserpio (32) describe a rapid TLC method for alginates
and p e c t i n s in food. A r t a u d et al (33) h a v e p u b l i s h e d the f i r s t of w h a t is
i n t e n d e d to be a s e r i e s of p a p e r s on the a n a l y s i s of n a t u r a l g u m s . T h e p a p e r
d e s c r i b e s the a n a l y s i s of g u a r g u m by h y d r o l y s i s and GLC of the silyl
d e r i v a t i v e s of the a l c o h o l s p r o d u c e d by r e d u c t i o n of the s u g a r s w i t h s o d i u m
borohydride. The p a p e r on s o m e n a t u r a l g u m s by J a c o b s and J a f f e (34) is a l s o
still u s e f u l . P o l y g l y c e r o 1 s can be i d e n t i f i e d by the TLC of t h e i r a c e t a t e s
(Dallas (35)). Seher (36) published a series of papers on the analysis of non-
ionic surface active agents dealing with the IR spectra of partial glycerides.
G e r n e r t (37) u s e d T L C to d e t e c t the g l y c e r i d e s of l a c t i c , t a r t a r i c and c i t r i c
acids in food. Stearoyl lactates m a y be detected by the TLC method of Regula
(38). For d e t e r m i n a t i o n of p o l y s o r b a t e 60 in a n u m b e r of f o o d s , u s i n g a
g r a v i m e t r i c f i n i s h w i t h b a r i u m p h o s p h o m o 1 y b d a t e , see S m u l l i n et al (39).
Murphy and Scott (40) describe the d e t e r m i n a t i o n of polyoxyethylene e m u l s i f i e r s
by TLC after column clean-up. Monosaccharides obtained by hydrolytic cleavage
of po 1 y s a c c h a r i d e - c o n t a i n i n g stabilizers have been separated gas
c h r o m a t o g r a p h i c a l 1 y v i a t h e i r a l d o n i t r i l a c e t a t e s ( M e r g e n t h a l e r and S c h e r z
(41)).

By far the most c o m m o n l y found permitted artificial sweetener at the present

time is saccharin (2 - s u 1 p h o b e n z o ic imide). Cyclamates (salts of
cyclohexylsulphamic acid) have also been used, and still are medicinally. 5-
Nitro-l-propoxyaniline (P-400) and d u l c i n ( p - e t h o x y p h e n y l u r e a ) are g e n e r a l l y
prohibited but m a y be encountered.

S a c c h a r i n can be d e t e r m i n e d by gas c h r o m a t o g r a p h y ( U l t e r h a l t (42), H a s h i r o

(43)), ultra-violet s p e c t r o p h o t o m e t r y ( Y o z a w a (44), H u s s e i n (45)), h i g h - s p e e d
liquid c h r o m a t o g r a p h y ( T a n a k a (46)) f l u o r i m e t r y ( N a k a m u r a , 47)), m o l e c u l a r
e m i s s i o n c a v i t y a n a l y s i s ( B e l c h e r (48)) and c o l o r i m e t r y ( T a n a k a (49)). Doun
(50) u s e s m e t h y l i o d i d e to f o r m the v o l a t i l e m e t h y l d e r i v a t i v e p r i o r to G L C ,
thus avoiding use of the s o m e w h a t hazardous diazomethane.

Makita (51) treats the saccharin in aqueous alkali with the

t r i c h l o r o m e t h y l t h i o l and d e t e r m i n e s b y GLC. An i o n - s e 1 e c t i v e electrode
sensitive to saccharin has been developed (Hazemoto (52)).

Cyclamate m a y be determined in fruit juices and drinks by the v o l u m e t r i c method

of Davies (53). M e a d o w s (54) describes a volumetric method for cyclamates in
sweetening tablets. H a r r i s o n and C o o k (55) d e s c r i b e a c o l o r i m e t r i c m e t h o d
suitable for both types of sample. Colorimetric m e t h o d s are also described by
D a w a n a (56) and M a t s u i (57) and G L C m e t h o d s by G r o e b e l and W e s s e l s (58) and
M o r i (59).

115
T a k e s h i t a (60)(61) uses c o l u m n and TLC to detect s a c c h a r i n , c y c l a m a t e and
dulcin in a v a r i e t y of foods. N a g a s a w a et al (62) use TLC alone. Das et al
(63)(64) describe TLC and PC methods. Sasaki et al (65) describe the detection
of dulcin by PC.

A number of polyols (naturally occurring polyhydric alcohols) are used in food,

e s p e c i a l l y that intended for diabetics. The m o s t i m p o r t a n t m e m b e r s of this
class are sorbitol and m a n n i t o l and, more recently, xylitol. If used to
replace sugar they will be present in quite substantial proportions as they are
less sweet. They are also used as h u m e c t a n t s and se que s t ran t s . S o m e s i m p l e
peptides and their d e r i v a t i v e s are s w e e t , for e x a m p l e A s p a r t a m e (sodium
aspartyl phenylalanine methyl ester), and Suosan, N-(p-nitrophenyl carbamoyl)-
beta-alanine sodium salt. A review has been published by British Food Journal
77 5-6 (1 975 ) on a n u m b e r of sugar substitutes including neohes perid in
dihydrochalcone and some dipeptides. Ammonium glycyrrhizinate, from liqurice,
is another sweetening agent, more usually found in medicinal products. This
may be determined by GLC (Larry (66)).

Sorbitol may be determined by GLC of the trimethylsilyl derivatives (Blum and

Koehler (67), Jones, et al (68) and W i l l i a m s and M a r t i n (69)). Polyhydric
alcohols react with borax to form r e l a t i v e l y strong acids w h i c h can be
titrated. This is the basis of the classical m e t h o d . Sorbitol m a y also be
reacted with sodium periodate and the excess determined iodometrically (Van Os
and Elena (70)). Graham (71) suggests a c o l o r i m e t r i c m e t h o d based on the
K o m a r o w s k y r e a c t i o n , the colour being formed in the presence of a cyclic
aldehyde, thiourea, concentrated sulphuric acid and heat. Konig (72) describes
a GLC method for determining cyclamate, saccharin and dulcin simultaneously.

A very large n u m b e r of additives are n o w accepted for use in foods for a w i d e

variety of purposes. The additives m a y change, react with the food or
d i s a p p e a r during processing and storage so that analysis m a y be d i f f i c u l t or
impossible. In many cases the chemical in question is already present in the
food but m o r e is added during processing. E n f o r c e m e n t of food law does not
necessarily require the detection or determination of additives, such as those
known to be present in amounts accepted as innocuous or for which no limit is
prescribed. S o m e additives can be d e t e r m i n e d or the a m o u n t present deduced
from general m e t h o d s of analysis such as acidity or ash. The Food A d d i t i v e s
and Contaminants Commission of IUPAC (73) reviewed analytical methods available
for some flour i m p r o v e r s , a n t i o x i d a n t s and p r e s e r v a t i v e s . W a l k e r (74)
discusses the role of chromatography in the detection and determination of food
additives. Clark and Robinson (75) describe the determination of surfactants
in food.

Most flavouring materials were originally derived from plants in the form of
extracts and d i s t i l l a t e s such as e s s e n t i a l oils. Some of these are n o w m a d e
synthetically. They are used in only small a m o u n t s and w e r e g e n e r a l l y
considered safe until recently, but now doubts are expressed about a few. For
example, there is evid ence that safrole and iso — safrole are carcinogenic. The
Food Additives and Contaminants Committee of the U.K. Ministry of Agriculture,
1976 Fisheries and Food Report on the Review of Flavourings in Food serves as a
useful i n t r o d u c t i o n to legislative and m a n u f a c t u r i n g c o n s i d e r a t i o n s . The
m a t t e r has been considered by the W o r k i n g Party on Natural and A r t i f i c i a l
Flavouring S u b s t a n c e s , a subsidiary body of the S u b - C o m m i t t e e on the Health
Control of Foodstuffs of the Council of Europe (EEC). There was a symposium on
f l a v o u r i n g s in food in 1976, reported in " I n t e r n a t i o n a l F l a v o u r s and Food
Additives", Nov/Dec 1976 issue. The FAO/WHO Expert Committee on Food Additives
11th Meeting (1967) details acceptable daily intakes for 35 flavours.

Monosodium glutamate belongs to a group of compounds that function as flavour

enhancers. Ingestion of quite large amounts results in a temporary headache
and "hot flushes". F e r n a n d e z - F l o r a s , Johnson and Blomquist (76)(77) reported
on a method studied collaboratively, using separation on an ion-exchange column
followed by a Sorensen formaldehyde titration. Baily and Swift (78) describe a

116
paper c h r o m a t o g r a p h y m e t h o d , and Gal and Schilling (79) one using GLC.
Coppola, Christie and Hanna (80) describe a rapid m e t h o d using a short ion-
exchange column with the determination conducted fluorimetrically.

, : J - T " •'•;•> HT} - Í? " . N

•
 EMULSIFIERS
(Thin-layer Chromatography Method)

PRINCIPLE

E m u l s i f i e r s , together with fat, are extracted by blending with chloroform and

methanol. On adding a calculated amount of water, the chloroform containing
fat and emulsifier separates. The chloroform layer is evaporated to dryness
and e m u l s i f i e r s p r e s e n t e x t r a c t e d w i t h m e t h a n o l . The m e t h a n o l e x t r a c t is
examined using thin-layer chromatography (TLC).

APPARATUS

1. TLC equipment, including Polygram Sil G. plates (manufactured by

Macherey Nagel and Co., 5161 Buren, Wertstrasse 628, Federal Republic
of Germany;.

2. Short-wave U.V. lamp, 254 nm.

3. Rotary film evaporator.

REAGENTS

1. Chloroform.

2. Methanol.

3. Magnesium chloride solution, 2N.

4. 1,2-Dichloroethane.

5. Cyclohexane.

6. Butan-2-one.

7. Glacial acetic acid.

8. Dibromofluorescein, 0.02 percent in 96 % ethanol.

9. TLC Solvent 1: Pipette 12 ml M e O H into a dry 200 m l volumetric

flask and dilute to the m a r k with 1,2-dichlorethane.

10. TLC Solvent 2: Pipette 4 m l water and 16 m l acetic acid into a

dry 200 m l v o l u m e t r i c flask. Add f r o m a m e a s u r i n g c y c l i n d e r 80 m l
dry butan-2-one and dilute to the m a r k with cyclohexane (this solvent
separates b e l o w about 15°C).

PROCEDURE

I n t o a d r y m a c e r a t o r g o b l e t , put a b o u t 20 g s a m p l e , 40 m l C H C L 3 , 80
m l M e O H a n d 4.0 m l 2 N M g C L 2 s o l u t i o n . M a c e r a t e a b o u t 2 m i n . Add a
further 40 ml CHCI3 and macerate about 2 m i n more. Filter (Whatman
No. 1 f i l t e r p a p e r or e q u i v a l e n t ) . A reasonably clear filtrate
should be obtained. Wash the goblet with CHCI3 and pass through the
filter. Transfer the filtrate to a 250 ml separator. Add sufficient
w a t e r to m a k e a t o t a l of 72 m l t a k i n g into a c c o u n t the m o i s t u r e
content of the sample. M i x thoroughly and allow to stand until the
C H C l o has c o m p l e t e l y s e p a r a t e d and is c l e a r (takes at l e a s t one
hour;. Run off the C H C I 3 into a tared r o u n d - b o t o m e d flask.
E v a p o r a t e on a w a t e r b a t h and d r y at 100°C to o b t a i n an e s t i m a t e of
the fat and e m u l s i f i e r c o n t e n t . Add s u f f i c i e n t M e O H to g i v e a 10
percent solution. H e a t to b o i l i n g on a w a t e r b a t h . S t o p p e r and
s h a k e the flask. U n s t o p p e r and h e a t a g a i n to b o i l i n g t h e n a l l o w to
s t a n d and cool for a b o u t 10 m i n u t e s u n t i l the u n d i s s o l v e d fat h a s

118
 settled. Decant off the methanol into a tared flask, evaporate on a
w a t e r b a t h , t h e n d r y at 100°C to g i v e the w e i g h t of e m u l s i f i e r
concentrate. D i s s o l v e c o n c e n t r a t e in C H C l 3 / M e O H 1:1 to g i v e a ten
percent s o l u t i o n .

TLC plates supplied as 20 x 20 cm need washing to remove fluorescent

impurities. This is done by developing them with C H C l 3 / M e O H 1:1 in
an u n l i n e d tank for a b o u t 24 h o u r s . D r y at 100°C for a b o u t h a l f an
hour. The plate is then cut into quarters. The cut edges are tidied
b y r e m o v i n g a 3 m m w i d e s t r i p of s i l i c a gel u s i n g a r u l e r and r a z o r
blade. The origin is placed 8 cm from both cut edges (see diagram).
In this w a y d i s t o r t i o n due to e d g e e f f e c t s is m i n i m i z e d . The
prepared plates can be stored at 100°C until required since they are
robust enough to be stacked on top of each other.

cut edge

8 cm

origin

1
—.— 8 cm — —

0.5 y 1 of emulsifier solution is spotted at the origin in as small a

spot as p o s s i b l e . T h e spot is d r i e d at 100°C for 10 m i n u t e s . The
p l a t e s can be b e n t so that t h e y can be r u n in a l a r g e g l a s s jar.
A b o u t 25 m l of s o l v e n t is n e e d e d and d e v e l o p m e n t t a k e s a b o u t 20
m i n u t e s each way. The p l a t e s s h o u l d be r e m o v e d as s o o n as the
s o l v e n t f r o n t has r e a c h e d the top. T h e f i r s t s o l v e n t is r e m o v e d at
105°C in v a c u o for 10 m i n u t e s b e f o r e r u n n i n g in the s e c o n d s o l v e n t .
T h e s e c o n d s o l v e n t a l s o m u s t be r e m o v e d u n d e r the s a m e c o n d i t i o n s
otherwise it interferes with spot location. The spots are located by
s p r a y i n g w i t h a 0.02% d i b r o m o f l u o r e s c e i n s o l u t i o n in 96 % e t h a n o l
until pale orange. Dry b r i e f l y at 100°C and v i e w u n d e r U.V. l i g h t .
It o f t e n h e l p s to r e s p r a y the p l a t e l i g h t l y and a l l o w to d r y u n d e r
the UV. The plates can be written on with soft pencil and copied on
a photocopier.

INTERPRETATION

Of the e m u l s i f i e r s g l y c e r y l m o n o s t e a r a t e , distilled monoglycer ide, propylene

glycol monostearate, sorbitan monostearaete, sorbitan tristearate,
p o l y o x y e t h y l e n e s o r b i t a n m o n o s t e a r a t e , polyoxyethylene sorbitan tristearate,
lactic acid esters of monoglycerides, acetic acid esters of monoglycerides and
p o l y g l y c e r o l e s t e r s , o n l y g l y c e r y l m o n o s t e a r a t e and l a c t i c acid e s t e r s of
monoglycerides are indistinguishable. Many e m u l s i f i e r s give several spots so
that mixtures m a y be difficult to identify.

The presence of polyoxyethylene emulsifiers can be confirmed by adding a m m o n i u m

c o b a l t o t h i o c y a n a t e in 50 p e r c e n t e t h a n o l to the e m u l s i f i e r c o n c e n t r a t e in
chloroform-methanol. A b l u e c o l o u r is p r o d u c e d in the o r g a n i c l a y e r if
polyoxyethylene compounds are present.

It is e s s e n t i a l to run s t a n d a r d c o m m e r c i a l e m u l s i f i e r preparations as
comparisons. Identification should be regarded as tentative.

119
 SACCHARIN

PRINCIPLE

The saccharin is extracted from the acidified s a m p l e , and reacted with

phenothiazine to form a coloured material which is extracted into xylene and
determined spectrophotometrically.

APPARATUS

1. Separatory funnels.

2. Water bath.

3. Spectrophotometer.

REAGENTS

1. Sulphuric acid, 10%.

2. Diethyl ether.

3. Sodium bicarbonate solution, 1%.

4. Hydrochloric acid.

5. Ethanol , 50% and absolute.

6. Cupric acetate solution, 10%.

7. Phenothiazine solution, 5%.

8. Xylene.

9. Anhydrous sodium sulphate.

10. Saccharin standard solution in ether (0.5 mg/ml).

PROCEDURE

W e i g h 50 g sample and add 30 ml water. Acidify w i t h 10% sulphuric

acid and add 5 ml excess. Place in a separatory funnel and extract
twice with 100 ml portions of diethyl ether. Collect the ether
extracts in a second separatory funnel and extract twice with 25 ml
portions of 1% sodium bicarbonate. Collect the bicarbonate extracts
in a third separatory funnel and discard the ether layer. Acidify
the bicarbonate extract s with 10% hydrochloric acid. Extract twice
with 30 ml ether. Discard the acid layer and wash the ether extracts
with 10 ml distilled water. Transfer to a 100 ml flask and evaporate
to a small volume at 40°C.

Transfer the saccharin residue in the flask to a 2 x 16 cm test tube

using 5 m l of 50% ethanol. Add 1 ml cupric acetate solution, 1 m l
phenothiazine solution, and 3 ml absolute ethanol.

Place the tube and contents in a water bath at 65-70°C for 5 minutes
shaking occasionally. Cool and transfer the solution to a 50 ml
separatory funnel, using 2 m l of absolute ethanol. Add 5 m l x y l e n e
and 15 ml water. Shake v i g o r o u s l y for 5 - 1 0 m i n u t e s . Separate the
xylene layer and dry with 1 gm anhydrous sodium sulphate.

120
 Pipette 1, 2 and 4 ml of the saccharin standard solution into three
test tubes. Evaporate to a small volume, add 5 ml of 50% ethanol and
continue as with the sample starting with addition of cupric acetate
solution.

D e t e r m i n e the absorbance of the sample and three standards in 1 cm

cells at 510 nm using xylene as the reference. Prepare a standard
curve.

CALCULATION

Saccharin ppm = — x 1000

50

where: w = mg saccharin in sample solution read from the standard

curve.

REFERENCE

Tanka, A., Nose, N., Suzuki, T., K o b a y a s h i , S. and W a t a n a b e , A., 1977. Analyst
102, 367-70.

121
 ARTIFICIAL SWEETENERS

PRINCIPLE

The food is e x t r a c t e d w i t h e t h y l a c e t a t e . The c o n c e n t r a t e d e x t r a c t is

subjected to thin-layer chromatography on silica gel and the spots visualized.
Saccharin, cyclamate, 5-nitro-2-propoxyaniline (P-4000) and p-ethoxyphenylurea
(dulcin) are detected. The method is designed primarily for beverages.

APPARATUS

1. Apparatus for thin-layer chromatography.

2. Short-wave (254 n m ) UV lamp.

REAGENTS

(Note: Prepare solutions fresh on day of use.)

1. Developing solvent - n-butanol:alcohol:ammon i a : w a t er (40:4:1:9,

by volume).

2. C h r o m o g e n i c a g e n t s - (1) b r o m i n e in CCl^, 5% by v o l u m e (great

c a r e m u s t be t a k e n w i t h this r e a g e n t as b r o m i n e is e x t r e m e l y
corrosive and toxic and CCl^ is very toxic); (2) 0.25% fluorescein in
dimethylformamide-alcohol (1+1); (3) 2% N-l-naphthyl-ethylenediamine.
2HC1 in alcohol.

3. Standard mixture - 50 mg Ca cyclamate, 10 mg Na saccharin, 4 mg

d u l c i n , and 4 m g P - 4 0 0 0 in 10 m l d i l u t e a l c o h o l (1 + 1). 5jil = 25 p g
c y c l a m a t e , 5yg s a c c h a r i n , lyg d u l c i n , and 2pg P - 4 0 0 0 . W a r m s o l u t i o n
to dissolve dulcin if necessary. Avoid skin contact with P-4000.

4. S i l i c a gel. A d s o r b o s i l - 1 (Applied Sciences Labs., State

College, PA 16801) or silica gel (Merck).

5. Sulphuric acid solution, 50% (v/v).

6. Petroleum ether.

7. Ethyl acetate.

8. Ammonium hydroxide :water : alcohol (5:5:10).

PROCEDURE

D e c a r b o n a t e a c a r b o n a t e d b e v e r a g e by r e p e a t e d s h a k i n g and p o u r i n g .
To 50 m l s a m p l e in 125 m l s e p a r a t o r , c a u t i o u s l y add 10 m l H 2 S 0 ^
(1+1). Cool, extract with two 50 ml portions petroleum ether (shake
gently, but thoroughly) and discard petroleum ether. To the aqueous
layer, cautiously add 5 ml 50% NaOH solution (w/w), cool, and extract
w i t h t w o 50 m l p o r t i o n s e t h y l a c e t a t e . (Use 60 m l for c o l a s a m p l e s
to p r e v e n t e m u l s i o n s ) . F i l t e r the e x t r a c t s t h r o u g h e t h y l a c e t a t e -
w a s h e d c o t t o n into a b e a k e r or f l a s k w i t h a p o u r i n g lip. Evaporate
to 5 - 1 0 m l on a s t e a m b a t h (using an air c u r r e n t ) and t r a n s f e r to a
graduated tube. (Do not let the solution evaporate to dryness before
transfer. Sweeteners may be difficult to redissolve). Evaporate the
s o l u t i o n in the g r a d u a t e d tube to d r y n e s s on a s t e a m b a t h w i t h a
current of air. Dilute to 2.5 ml with NH^OH: water:alcohol (5:5:10)
and m i x t h o r o u g h l y . (Any i n s o l u b l e r e s i d u e in the t u b e w i l l not
interfere with detection).

Slurry 35 g Adsorbosil with 50 ml water or 30 g silica gel H with 75-

80 m l w a t e r and a p p l y as a 0.25 m m layer to five 20 x 20 cm p l a t e s .

122
 Dry the plates for more than an hour at room temperature. Do not dry
in the oven. Do not store in a desiccator. Score the layer 5 m m
from each side edge and r e m o v e a 5 m m band of adsorbent from the
b o t t o m edge of the layer. Use the plates w i t h i n 36 hours of
preparation.

Line the developing tank with adsorbent paper. Pour 25 ml of

developing solvent into the tank, w e t t i n g the paper. Place a V-
shaped trough in the tank and add 25 ml of developing solvent to the
trough. (Alternatively, put the developing solvent in the tank to
about 1 cm). Place a lid on the tank, seal and let stand for about
1/2 hour to saturate the tank atmosphere.

H a r k a TLC plate at the side edges only, 2.5 cm from the b o t t o m to

designate a spotting line. M a r k a dotted line 10 cm above the
spotting line. Spot a total of 5 p i each of standard and sample
(Level 1). Dilute sample to 5 ml with NH^OHrwater: ethanol (5:5:10)
and spot 5 y l (Level 2). Place spots at least 2 cm apart and 2 cm
from the edges. Spot 1 yl at a time and use a warm-air blower to dry
the spot between applications to restrict the spot diameter. Use the
same technique to spot sample and standard. (Total v o l u m e spotted
should not be m o r e than 5 yl). Use m i x e d standard rather than
superimposed single standards.

Place the plate in the tank and develop to the 10 cm line (about one
hour). Dry the plate in a fume hood until the layer is no longer
translucent (about 10 minutes). V i e w under s h o r t w a v e (254 n m ) UV.
Outline any fluorescent saccharin spot at R^ about 0.5. (Spot may be
crescent-shaped if a large amount of cyclamate is present). In the
f u m e h o o d , s p r a y c h r o m o g e n i c a g e n t s (1) and (2), l i g h t l y to
m o d e r a t e l y , in i m m e d i a t e succession, until the c y c l a m a t e standard
appears as a pink spot at R^ about 0.3 to 0.4. P - 4 0 0 0 is a b r o w n -
pink spot at Rf about 0.85. Spray c h r o m o g e n i c agent (3) on a plate
until the background pink fades to light y e l l o w . The contrast of
c y c l a m a t e and P - 4 0 0 0 i m p r o v e s and at Rf about 0.7 dulcin appears.
. The dulcin spot may be brown-pink or blue, depending on the condition
of spray reagents and the concentration of the sweetener. The plate
m a y be resprayed with c h r o m o g e n i c agent (3) to restore contrast if
the pink background reappears.

INTERPRETATION

Certain experimental parameters were found to be critical for optimum results.

The m o i s t u r e content of the TLC plates is very i m p o r t a n t in obtaining good
separation of saccharin and cyclamates. The plates must be freshly prepared
and dried w i t h o u t heat or desiccation. L i m i t e d studies indicate that
commercially-prepared TLC plates or sheets are not suitable.

A number of potential interferences are removed from beverages by the petroleum

ether prewash. The aqueous solution must be strongly acidified, otherwise any
P - 4 0 0 0 present w i l l be extracted by the p e t r o l e u m ether. Gentle shaking
prevents excessive e m u l s i o n s . Partial n e u t r a l i z a t i o n of the acidity of the
aqueous phase prior to ethyl acetate extraction resulted in a higher recovery
of dulcin and P-4000.

Specifying a technique for applying the chromogenic sprays is rather difficult.

Detection has been accomplished on some plates with light spraying but at other
times, heavier spraying was needed. The best practice is to spray until the
standards are clearly detected. Occasionally, standards fail to show up on a
plate but a repeat experiment with a fresh plate is usually successful.

REFERENCE

Korbelak, T., 196S. Journal of the AOAC 52, 487.

123
 MOHOSODIUM GLUTAMATE

PRINCIPLE

The m o n o s o d i u m glutamate is isolated from the sample using an ion exchange

resin. The glutamic acid is then titrated potentiometrically.

APPARATUS

1. Chromatographic column - 500 x 22 mm 0D tube, 30 ml bed volume

with Dowex 50 W - X8 (H+ form) 100-200 mesh.

2. pH meter.

3. Burette.

REAGENTS

1. Activated Carbon - Darco G-60.

2. Acetone.

3. Hydrochloric acid, 1 volume + water, 2.5 volumes.

4. 0.8 N Hydrochloric acid.

5. IN Hydrochloric acid.

6. 50% Sodium hydroxide.

7. 0.1 N Sodium hydroxide.

8. Formaldehyde.

PROCEDURE
•

For products in dry form, reduce about 40g to powder in a mortar and
w e i g h 10 g of sample into a 250 ml beaker. For undiluted condensed
soups or canned green beans, homogenise entire undiluted content of
can in blender and weigh 20 g of sample into a 250 ml beaker. For
c o n s o m m e type (clear, condensed) soup w e i g h 20 g into a 250 ml
beaker.

Dilute the sample to about 70 ml with water at room temperature and

mix until all water-soluble substances are in solution (about 15
minutes). Add 6 g of activated carbon and mix thoroughly, (For
products containing starch, also add 60 ml of acetone to precipitate
starch and to aid sample solution). Let stand 30 minutes (or
overnight). Filter with vacuum through a 60 ml coarse, fritted glass
funnel containing an asbestos pad, wash flask and residue with six 25
ml portions of water or (if acetone was added) w i t h six 25 ml
portions of acetone-water (1+1).

Collect filtrate and washings in a 400 ml beaker, add 2 drops

hydrochloric acid (1 + 2.5) and evaporate on a steam bath to about 40
ml. (Hydrochloric acid prevents conversion of glutamic acid to
pyrrolidone carboxylic acid). Quantitatively transfer to a 50 ml
volumetric flask, dilute to volume with water and mix.

Transfer 25 ml of extract to prepared column and adjust flow to about

0.5 ml/minute. After all the solution enters the resin, wash column
wall with about 10 ml of water. Let wash pass into the resin. Add
120 ml 0.8 N hydrochloric acid and maintain flow rate (0.8 N HCL will
elute any serine, threonine and aspartic acid). After all the 0.8 N

124
 h y d r o c h l o r i c acid p a s s e s into the r e s i n , add 170 ml of 1 N
hydrochloric acid and adjust the flow rate to b e t w e e n 25 and 30
drops/min to elute glutamic acid. Collect eluate in a 400 ml beaker.
(Any glycine present will elute after 200 ml of IN hydrochloric
acid). Nearly neutralise with 50% sodium hydroxide. Neutralize 25
ml of 37% formaldehyde to pH 7 with 0.1 N sodium hydroxide and adjust
p o t e n t i o m e t r i c a l l y to pH 7 with 0.1 N sodium hydroxide and add to
prepared neutral sample. Mix for 10 minutes on a m a g n e t i c stirrer
and titrate potentiometrically to pH 8.9 with 0.1 N sodium hydroxide.

Determine blank by titrating to pH 8.9 a m i x t u r e of 25 ml of

neutralised formaldehyde and 170 ml of neutralised IN hydrochloric
ac id.

CALCULATION

% Glutamic acid = (S-B) x N x 0.147 x 100/W

Where: S = ml NaOH used to titrate sample, B = ml NaOH used to

titrate blank, N = normality of NaOH and W = weight of sample.

% M.S.G. = % Glutamic acid x 1.15 (anhydrous).

REFERENCE

Fèrnandez-Flores, E., Johnson, A.R. and Blomquist, V.H., 1969. Journal of the
AO AC 5 2 , 1131-32.

125
6.5 TEXT REFERENCES

1. T H A C K R A Y , G . B . & H E W L E T T , A. 1964. Journal of the A s s o c i a t i o n of Public

A n a l y s t s , _2_ 13-17.

2. AMATO, F. 1968. Industria Alimentaria, 7 (12) 81-83.

3. TAKESHITA, R., SAKAGANNI, Y. & ITOH, M. 1969. Journal of Hygenic

Chemistry, 15 ( 2 ) 77-83.

4. C A N T A F O R A , A. 1976. Rivista della Societa Italiana di Scienza dell

'Alimentazione, _5_ ( 7 ) 87-92.

5. SCHNEIDER, W. 1970. Seifen-Ole-Fette-Wachse, 96 (16) 559-61.

6. ASSOCIATION OF P U B L I C A N A L Y S T S (UK). 1962. The Detection and

Determination of A n t i o x i d a n t s in Food.

7. PAGE, B.O. & KENNEDY, B.P. 1976. Journal of t h e A s s o c i a t i o n of Official

A n a l y t i c a l C h e m i s t s , 59 ( 6 ) 1 2 0 8 - 1 2 1 2 .

8. JAYARAMAN, S. V A S U N D H A R A , T.S. & PARIHAR, D.B. 1976. Mikrochimica

Acta I I ( 3 / 4 ) 3 6 5 - 3 6 9 .

9. LAMB, E. & WOLLER, R. 1976. Deutsche Lebensmittel-Rundschau, 72_(8) 276-9.

10. HOLDT, D. 1971. Chimie Analytique, 53 (12) 776-782.

11. GROEBEL, W. & WESSELS, A. 1973. Deutsche Lebensmittel-Rundschau, 69 (12)

453-459.

12. WIRELL, B. 1976. Analyst, 101 883-886.

13. A N G L I N , C . , M A H O N , H. & C H A P M A N , R. 1956. Journal of Agricultural and

Food C h e m i s t r y , _4_ 1 0 1 8 .

14. DILLI, S. & ROBERTS, K. 1977. Analyst, 102 201-205.

15. SAHASRABUDHE, M.R. 1964. Journal of the Association of Official

A g r i c u l t u r a l C h e m i s t s , 47 8 8 9 .

16. SELMECI, G. & ACZEL, A. 1975. Elelmiszervizsgalati Kozlemenyek, 21 (5/6)

298-305.

17. KEEN, G. & GREEN, M.S. 1976. Journal of the Association of Public
Analysts, 365-369, 1975 99-102.

18. SATO, Y. & K A W A M I R A , T. 1972. J o u r n a l o f the Food H y g i e n i c Society of

Japan (Shokuhin Eiseigaku Z u s s h i ) , 13 ( 1 ) 53-56.

19. TAKAHASHI, D.M. 1970. Journal of the Association of Official Analytical

C h e m i s t s , 53 ( 1 ) 39-43.

20. HARTMAN, K.T. & ROSE, L.C. 1970. Journal of the American Oil Chemists
S o c i e t y , 47_ ( 1 ) 7 - 1 0 .

21. CASS I D Y , W. & F I S H E R , A.J. 1960. A n a l y s t , 85 295.

22. W A C H S , W. & G A S S M A N N , L. 1970. Deutsche Lebensmittel-Rundschau, 66 (2)

37-38.

23. BERK, E. & BIELECKI, F. 1973. Ernahoungswirtschaft, 11 852-854.

126
24. P A G E , B.D. 1979. Journal of the Association of Official Analytical
C h e m i s t s , 62 1 2 3 9 - 1 2 4 6 1 ~ ~

25. PAGE,. B.D. 1983. Journal of the Association of Official Analytical

C h e m i s t s , 66 (3) 7 2 7 - 7 4 5 .

26. AERNY, J. & M I S E R E Z , A. 1973. Mitteilungen aus dem Gebiete der

L e b e n s m i t t e l u n t e r s u c h u n g und H y g i e n e . 64 (1) 1 3 9 - 5 1 .

27. S R E B R N I K - F R I S Z M A N , S. & C H A R O N , C., 1 9 7 0 . M i t t e i l u n g e n a u s d e m Gebiete

d e r L e b e n s m i t t e l u n t e r s u c h u n g u n d H y g i e n e , 61 ( 3 / 4 ) 2 2 0 - 5 4 .

28. KROLLER, E. 1974. Fette, Seifen, Anstrichmittel, 76, ( 1 1 ) 498-500.

29. GRAHAM, H.D. 1969. Journal of D a i r y S c i e n c e , 52. (6) 883.

30. GRAHAM, H.D. 1970. Journal of F o o d S c i e n c e , 35 (4) 494-98.

31. GRAHAM, H.D. 1971. J o u r n a l of D a i r y S c i e n c e , 54 (11) 1622-28.

32. M A R T E L L I , A. & PROSERPIO, G. 1973. Scienza e Tecnologia degli Alimenti.

1 ( 6 ) 367-69.

33. A R L A U D , J. E I T I E N N E , J. & C A S , M . 1975. Annales des Falsifications et de

l ' E x p e r t i s e C h i m i q u e , 68 ( 7 2 5 ) 9 - 2 7 .

34. J A C O B S , H.B. & J A F F E , L. 1931. Industrial and E n g i n e e r i n g Chemistry,

A n a l y t i c a l E d i t i o n , _3_ (2) 210-12.

35. DALLAS, M.S. 1970. Journal of C h r o m a t o g r a p h y , 48 (2) 225-30.

36. SEHER, A. 1969. Fette, Seifen, Anstrichmittel, 71 (2), 138-44.

37. G E R N E R T , F. 1968. Zeitschrift fur L e b e n s m i t te 1 unt er s u c h u n g und

F o r s c h u n g , 138 (4) 2 1 6 - 2 0 .

38. REGULA, E. 1975. Journal of C h r o m a t o g r a p h y , 115 (2) 639-44.

39. SMULLIN, C.F. 1970. J o u r n a l of the A m e r i c a n Oil C h e m i s t s Society, 48 (1)

18-20.

40. MURPHY, J. & SCOTT, C. 1969. A n a l y s t , 94 (1119) 481-83.

41. M E R G E N T H A L E R , E. & S C H E R Z , H. 1976. Zeitschrift fur Lebensmitte1-

u n t e r s u c h u n g u n d F o r s c h u n g , 162. (1) 2 5 - 9 .

42. U N T E R H A L T , B. 1975. Zeitschrift fur Lebensmitteluntersuchung und

F o r s c h u n g , 159 (3) 1 6 1 - 6 4 .

43. H O S H I N O , Y., S U Z U K I , T., K I K U C H I , Y., N O S E , N . & W A T A N A B E , A. 1975.

J o u r n a l o f t h e F o o d H y g i e n i c S o c i e t y o f J a p a n , 16 (3) 1 8 2 - 8 6 .

44. Y A Z A W A , Y., N A K A M U R A , M . , W A T A N A B E , K., K I R I G A W A , T., W A T A B E , A., S U Z U K I ,

Y . & K A W A M U R A , T . 1 97 5. J o u r n a l o f t h e F o o d H y g i e n i c S o c i e t y o f J a p a n ,
16.(4) 264-70.

45. HUSSEIN, M.M., J A C I N , H. & R O D R I G U E S , F.B. 1976. Journal of Agricultural

and Food C h e m i s t r y , 24 (1) 3 6 - 4 0 .

46. T A N A K A , Y., I K E B E , K., T A N A K A , R. & K U N I T A , N . 1975. J o u r n a l of the Food

H y g i e n i c S o c i e t y o f J a p a n , 16 (5) 2 9 5 - 3 0 0 .

47. NAKAMURA, Y. 1975. Journal of the Food H y g i e n i c Society of J a p a n , 16 (6)

368-74.

127
48. B E L C H E R , R., B O G D A N S K I , S.L., SHEIKH, R. A. & T O W N S H E N D , A. 1 9 7 6.
Analyst, 101 562-65.

49. TANAKA, A., NOSE, N., SUZUKI, T., K O B A Y A S H I , S. & W A T A N A B E , A. 1 977.

Analyst, 102 367-71.

50. DOUN, R.J. 1971. Journal of the Association of O f f i c i a l Analytical

Chemists, 54 (5) 1140-45.

51. M A K I T A , M., Y A M A M O T O , S. & Y O S H I H A R A , K. 1973. Journal of Hygienic

Chemistry, 19 (6) 297-301.

52. HAZEMOTO, M. & KOBATAKE, Y. 1974. Journal of the Association of Official

Analytical Chemists, 57 (5) 1205-08.

53. DAVIE S, A.M.C. 1966. Journal of the A s s o c i a t i o n of Public A n a l y s t s , 4_

11-14.

54. M E A D O W S , G. 1968. Journal of the A s s o c i a t i o n of Public A n a l y s t s , 6 97-

100.
55. H A R R I S O N , A. & COOK, A. 1969. Journal of the A s s o c i a t i o n of Public
Analysts, 7 42-6.

56. K A W A N A , K., W A D A , Y., T A K A H A S H I , T. & K A N N O , S. 1971. Journal of the

Food Hygienic Society of Japan, 12 (1) 33-9.

57. MATSUI, T. 1972. Journal of Hygienic Chemistry, ^ ( 5 ) 324-28.

58. GROEBEL, W. & WESSELS, A. 1972. Deutsche Lebensmittel-Rundschau, 68 (12)

393-97.

59. M O R I , M., SUZUKI, T., K A K I Z A K I , K., SUZUKI, K. & K A W A H A R A , N. 1970.

Canners Journal, 49 (2) 155-60.

60. T A K A S H I T A , R., S A K A G A M I , Y. & Y A M A S H I T A , T. 1969. J o u r n a l of Hygienic

Chemistry, 15 (2) 66-71.

61. TAKASHITA, R. 1972. Journal of Chromatography, 66 (2) 283-93.

62. N A G A S A W A , K., Y O S H I D O M E , H. & ANRYU, K. 1970. Journal of Chromato-

graphy, 52_ ( 1 ) 173-76.

63. DAS, D.K., M A T H E W , T.V. & M I T R A , S.N. 1970. J o u r n a l of Chromatography,

5_5_ (2) 354-56.

64. DAS, D.K. & M A T H E W , T.V. 1969. Journal of the I n s t i t u t i o n of C h e m i s t s ,

India, 41 (5) 1972-93.

65. SASAKI, IKURA, Z. & Y O K O T S U K A , T. 1969. Seasoning S c i e n c e , 16 (2) 6 - 1 0 .

66. LARRY, D., FULLER, M.J. & H A R R I L L , P.G. 1970. Journal of the A s s o c i a t i o n
of Official Analytical Chemists, 53 (4) 698-700.

67. BLUM & KOEHLER, 1968. Gas Chromatography, _6_120.

68. JONES, H.D., S M I T H , D.M. & S A H A S R A B U D H E , M. 1966. Journal of the

Association of Official Analytical Chemists, 49, 1183.

69. WILLIAMS & MARTIN, 1967. Horticultural Science, 2 68.

70. VAN OS & ELENA, 1968. Pharmacie Weekblad., 103 205.

71. GRAHAM, H.D. 1965. Journal of Food Science, 30, 846.

128
72. KONIG, H. 1971. Zeitschrift fur Analytische Chemie, 255 (2) 123-25.

73. I N T E R N A T I O N A L UNION OF PURE AND APPLIED C H E M I S T R Y , FOOD A D D I T I V E S AND

CONTAMINANTS COMMISSION, 1971. Pure and Applied C h e m i s t r y , 26 (1) 77-
120. '

74. WALKER, R. 1972. Proceedings of the Society for Analytical Chemistry, 9

(4) 96-7. -

75. CLARK, J.F. & R O B I N S O N , W.P. 1971. Journal of the A s s o c i a t i o n of Public

— —
A n a l y s t s , 8 (1). "

76. F E R N A N D E Z - F L O R E S , E., JOHNSON, A.R. & B L O M Q U I S T , V.H. 1969. Journal of

the Association of Official Analytical Chemists, 52 (6) 1131-32.

77. F E R N A N D E Z - F L O R E S , E., JOHNSON, A.R. & B L O M Q U I S T , V.H. 1969. Journal of

the Association of Official Analytical Chemists, 52 (4) 744-46.

78. BAILEY, B.W. & S W I F T , H.L. 1970. Journal of the A s s o c i a t i o n of Official

Analytical Chemists, 53 (6) 1268-69.

79. GAL, S. & SCHILLING, P. 1972. Zeitschrift fur Lebensmittelunter suchung

und Forschung, 148 (1) 18-22.

80. COPPOLA, E.D., CHRISTIE, S.N. & HANNA, J.G. 1975. Journal of the
Association of Official Analytical Chemists, 58 (1) 58-61.

Farther Reading

Preservatives, General

FOGDEN, E., FRYER, M. & URRY, S. 1974. Journal of the A s s o c i a t i o n of Public

Analysts, 12 93-101.

GOSSELE, J.A.W. & S R E B R N I K - F R I S Z M A N , S. 1974. M e d e l i n g e n van de Faculteit

Landbouwwetenschappen Rijksuniversiteit Gent, 39 (1) 50-58.

PRAHL, L., ENGST, R. & J A R M A T Z , E. 1968. N a h r u n g , 12_(8) 845-51.

NAGASAWA, K., YOSHIDOME, H. & TAKESHITA, R. 1969. Journal of Chromatography,

43. (4) 473-79.

C L E M E N T , J., PRESSEL, L. Van, K E Y M E N T E N , S. Van. 1969. Zeitschrift fur

Analytische Chemie, 248 (3/4) 182.

TJAN, G.H. & K O N T E R , T. 1972. Journal of the Association of Official

Analytical Chemists, 55 (6) 1223-1225.

ENGST, R., PRAHL, L. & JARMATZ,. E. 1969. N a h r u n g , 13 (5) 417-26.

RANGONE, R. & AMBROSIA, J. 1970. Journal of Chromatography, 50 (3) 436-441.

L E H M A N N , G. & LUTZ, I. 1970. Zeitschrift fur L e b e n s m i t t e l - u n t e r s u c h u n g und

Forschung, 144 (5) 318-21.

COURTIAL, W. 1970. Deutsche Lebensmittel-Rundschau, 66 (7) 220-23.

TAÑI, T. & KANNO, S. 1970. Journal of the Food H y g i e n i c Society of Japan

(Shokuhin Eiseigaku Zasshi), 11 (1) 23-27.

RO, H.S. 1972. Korean Journal of Food Science and Technology. _4_ (1) 24-28.

129
F U J I W A R A , M., M A T S U M U R A , I. & I T O K A W A , T . 1 9 7 1 . J o u r n a l of the F o o d Hygienic
S o c i e t y of J a p a n ( S h o k u h i n E i s e i g a k u Z a s s h i ) , 12 (1) 4 0 - 4 6 .

Y O S H I D A , M., T A K E N C H I , T. & Y O S H I I , H. 1 9 6 8 , p u b l 1 9 6 9 . A n n u a l R e p o r t s of the

Food Research Institute Aichi Prefecture (Aichi-ken Shokuhin Kogyu Shikenjo
N e n p o ) , _9 8 8 - 9 2 .

RAJAMA, J. & M A K E L A , P. 1973. Journal of C h r o m a t o g r a p h y , 76 (1) 199-205.

D U D E N , R., F R I C K E R , A., C A L V E R L E Y , R., P A R K , K.H. & R I O S , V.M. 1 9 7 3.

Z e i t s c h r i f t fur L e b e n s m i t t e l u n t e r p u c h u n g u n d F o r s c h u n g , 151 (1) 2 3 - 2 5 .

TAKEMURA, I. 1971. Japan Analyst (Bunseki Kagaku), 20 (1) 61-64.

D A E N E N S , P. & L A R U E L L E , L. 1973. Journal of the Association of Official

A n a l y t i c a l C h e m i s t s , 56 (6) 1 5 1 5 - 1 5 1 7 .

FURIA, T.E. 1981. H a n d b o o k of F o o d A d d i t i v e s 2nd E d . CRC.

B U G L I O , B. 1968. Journal of t h e A s s o c i a t i o n of O f f i c i a l Analytical Chemists,

5 1 (6) 1 2 7 8 .

BONDUELLE, C. & L U Q U E T , F.M. 1971. Revue Laitiere F r a n ç a i s e , 475 477-8.

C A R R , W . & S M I T H , G.A. 1964. J o u r n a l of t h e A s s o c i a t i o n of P u b l i c A n a l y s t s , _2_

37.

C A R R U T H E R S , A., H E A N E Y , R.K. & O L D F I E L D , J.F.T. 1965. International Sugar

Journal, 364-366.

F A W C E T T , R., T A M E , D.A. & J O H N S O N , T.E. 1976. J o u r n a l of the A s s o c i a t i o n of

P u b l i c A n a l y s t s , 14 2 3 - 2 5 .

F U D G E , R . & T R U M A N , R.W. 1973. Journal of the A s s o c i a t i o n of Public Analysts,

1JL_ 1 9 - 2 7 .

GANTENBEIN, W . M . & K ^ R A S Z , A.B. 1969. Journal of the Association of

A n a l y t i c a l C h e m i s t s , 52 (4) 7 3 8 .

GOSSELE, J.A.W. & SREBRNIK-FRISZMAN, S. 1966. Journal of Chromatography, 23

305.

G R E E N , L.F. 1976. F o o d C h e m i s t r y , 1 (2) 1 0 3 - 1 2 4 .

G R E E N , M.S. 48-50. J o u r n a l of the A s s o c i a t i o n of P u b l i c A n a l y s t s , _8_ 4 8 - 5 0 .

G R U Ñ E , A . & M O B B E , V. 1967. Riechstaffe Aromen Kooperpflagemittel, 17 501.

G U A G N I N I , O.A. & V O N E S C H , E.E. 1959. Anales de l'Asociacion quimica

A r g e n t i n a , 4 7 (1) 4 1 .

D Y E R , B., T A Y L O R , G. & H A M E N C E , J.H. 1941. A n a l y s t , 66 9-12.

H A M I L T O N , J.E. 1976. Journal of the Association of Official Analytical

C h e m i s t s , 59 (2) 2 8 4 - 8 .

INSTITUTE OF F O O D T E C H N O L O G I S T S , U S A . Expert Panel on Food Safety and

Nutrition. 1975. F o o d T e c h n o l o g y , 29 (10) 1 1 7 - 1 2 0 .

J O I N T E X E C U T I V E C O M M I T T E E ON FOOD A D D I T I V E S 17th R e p o r t . Toxicological

E v a l u a t i o n of C e r t a i n Food A d d i t i v e s w i t h a R e v i e w of G e n e r a l P r i n c i p l e s cTf
S p e c i f i c a t i o n s , FA0 R o m e .

L U C K , E. 1970. Australian Food M a n u f a c t u r e , 39 (8) 28 & 32.

130
M A N N I N G , P.B., COULTER, T. & J E N N E S O , R. 1968. Journal of Dairy Science, 51
(1 1) 1725-30.

M A T S U M 0 T 0 , S., N I S H I M A , T., NAGASE, M. & K U R A U C H I , M. 1969. Annual Report of

Tokyo Metropolitan Research Laboratory of Public Health. 21 63-65.

McKAY, A.J. 1974. ^ Australian Journal of Dairy Technology, 29 (1) 34-36.

M I S H I M A , T. & M A T S U M O T O , S. 1969. Annual Report of Tokyo Metropolitan

Research Laboratory of Public Health, 21 59-62.

RAMMALL, L.G. & JOERIM, M.M. 1972. Journal of Dairy Research, 39 (1) 89-94.

SCHULLER, P.L. & VEEN, E. 1967. Journal of the Association of Official

Analytical Chemists, 50 1127-1145.

SHELLEY, R.M., S A L W I N , H. & H O R O W I T Z , W. 1963. Journal of the A s s o c i a t i o n of

Official Analytical Chemists, 46 486.

W A L K E R , G.H., GREEN, M.S. & FENN, C.E. 1964. Journal of the A s s o c i a t i o n of

Public Analysts, _2_ (2-4) & 3 (1965) 83-92.

WOIDICH, H., GRAVER, H. & GALINOVSKY, E. 1967. Zeitschrift fur Lebensmittel-

untersuchung und Forschung, 133 317.

ZONNEVELD, H. 1975. Journal of Science, Food & Agriculture, 26 (7) 879-888.

M A X S T A D T , J.J. & P O L L M A N , R.M. 1980. Journal of the A s s o c i a t i o n of Official

Analytical Chemists, 63 (3) 667-674.

GERTZ, C. & HERRMANN, K. 1983. Deutsch Lebenson-Rundschau, 79 (10) 331-334.

CHAKRAVORTY, K.L. 1981. Journal of the Association of Public Analysts, 19 (4)

131-133.

LOPEZ, J. & SIMAL, J. 1983. Anales Bromatologia, 34 (1) 123-131.

HILD, J. & GERTZ, C. 1980. Zeitschrift fur Lebensmitteluntersuchung und

Forschung, 170 (2) 103-114.

FAUGERE, J.G. 1979. Annales F a l s i f i c a t i o n et E x p e r t i s e C h i m i q u e , 72 (774)

227-232.

VALDEHITA, M.T., C A R B A L L I D O , A. & G A R C I A - M O R E N O del RIO, M.C. 1979. Anales

Bromatologia, 31 (1) 31-37.

C0NACHER, H.B.S. & PAGE, B.D. 1979. Journal of C h r o m a t o g r a p h i c S c i e n c e , 17

(4) 188-195.

TILBURY, R.H. 1980. Developments in Food Preservatives - 1, Applied Science.

Sulphur Dioxide

MONIER-WILLIAMS, G.W. 1927. Analyst, 52 343, 415.

JENNINGS, N., BURTON, N.G., CROSBY, N.T. & A L L I S T O N , T.G. 1978. Journal of
the Association of Public Analysts, 16 (2) 59-70.

KEARSLEY, M.W. & THANOS, A. 1982. Journal of the A s s o c i a t i o n of Public

Analysts, 20 (4) 145-152.

131
Sorbic Acid

TAME, D.A. 1980. Journal of the Association of Public Analysts, 18 (2) 59-61.

L A R S S O N , B.K. 1983. Journal of the Association of Official Analytical

Chemists, 66 (3) 775-780.

M O D I , G., C H I T I , F. & F I O R E N T I N O , P. 1982. B o l l e t i n C h i m i c a U n i o n e Italiana

Laboratoria Provinciale Parte Scienzia, 33 (52) 143-151.

Antioxidants

GERTZ, C. & HERRMANN, K. 1983. Zeitschrift fur Lebensmitteluntersuchung und

Forschung, 177 (3) 186-192.

PUJOL FORN, M. , 1981. Circular Farmacéutica, _39_ (270) 5-28.

ALARY, J., GROSSET, C. & COEUR, A. 1982. Annales Pharmaceutiques Françaises,

40_ (4) 3 0 1 - 3 0 9 .

G A L E N S A , R. & S C H A E F E R , F.I. 1982. D e u t s c h e L e b e n s m i 1 1 e 1 - R u n d s c h a u , 78 (7)

258-261.

Colours

The C o l o u r Index 3rd E d i t i o n (1971), w i t h a 2 - v o l u m e r e v i s i o n in 1975, p l u s

additions and amendments, gives details of chemical formulae, manufacturers and
many technical properties of thousands of dyes, including those used in food.
It is a v a i l a b l e from "The S o c i e t y of D y e r s and C o l o u r i s t s " , P.O. Box 2 4 4 , D e a r
House, Piccadilly, Bradford, Yorkshire, UK. The names of manufacturers will be
found on pp. 5175-5197 of the Revised Third Edition 1975, Vol. 5. Some further
selected references are:

Colours, General

THALER & SOMMER. 1953. Zeitschrift fur Lebensmitteluntersuchung und

Forschung, 97 345-65, 441-6.

P A L L O T T I , G., B E N C I V I N G A , B. & B O N I F A Z I , M. 1976. I n d u s t r i A l i m e n t a i r i , 1_5

(5) 75-79.

QUINTANALLA SAEZ, J.A. 1969. Alimentaria, _6_ (24) 3, 5-9, 11-13 and 15-29.

B L A K E , C.J. 1982. S c i e n t i f i c T e c h n i c a l S u r v e y - B r i t i s h Food Manufacturing

Industries Research Association, No,. 130.

Quantitative Colour Determination

G R A I C H E N , C. 1975. Journal of the Association of Official Analytical

Chemists, 58 (2) 278-282.

WALTHIER, J. & JENEY, 0. 1968. Szappan Kozmetik, 17 (4) 114-17.

B A N E R J E E , S.K., M A T H E W , T.V. & M I T R A , S.N. 1969. Research and Industry.

C.S.I.R., I n d i a . _14_(2) 87-89.

132
M A T H E W , T.V., B A N E R J E E , S.K., M U K H E R J E E , A.K. & M I T R A , S.N. 1969. Research
a n d I n d u s t r y C . S . I . R . , I n d i a , 1_4_(3) 1 4 0 - 4 2 .

G R A I C H E N , C. & M O L I T O R , J.C. 1963. Journal of the A s s o c i a t i o n of Official

A g r i c u l t u r a l C h e m i s t s , 4 6 (6) 1022-1029.

S C L A R , R.N. & F R E E M A N , K.A. 1955. Journal of the Association of Official

A g r i c u l t u r a l C h e m i s t s , 38 (3) 796-890.

G R A I C H E N , C., S C L A R , R.N., E T T E L S T E I N , N . & F R E E M A N , K.A. 1955. Journal of

t h e A s s o c i a t i o n o f O f f i c i a l A g r i c u l t u r a l C h e m i s t s , 38 (3) 7 9 2 - 7 9 5 .

S T E U E R L E , H. 1979. Zeitschrift fur L e b e n s m it t e l u n t e r s u c h u n g und Forschung,

1 6 9 (6) 4 2 9 - 4 3 4 .

Tests for I n d i v i d u a l Colours

H O W A R D , F.J. 1965. Journal of the A s s o c i a t i o n of Public Analysts, 3 137.

P E A R S O N , D. 1967. Journal of the A s s o c i a t i o n of Public Analysts, 5 37.

T O E N N E S E N , H.H. & K A R L S E N , J. 1983. J o u r n a l of C h r o m a t o g r a p h y , 259 (2) 3 6 7 -

371.

BRITISH STANDARDS INSTITUTION. BS 4 5 8 3 Part 13 1983.

C O R R A D I , C., M I C H E L I , G. & S P R O C A T I , G. 1981. Industria Alimentaria, 20 (9)

624-629.

Detection of Colours in V a r i o u s Foods

McNEAL, J.W. 1976. Journal of the Association of Official Analytical

C h e m i s t s , 59 (3) 5 7 0 - 5 7 7 .

RIZVI, S.R.H. 1972. Science and Industry, P a k i s t a n , _9_ ( 3 / 4 ) 226-231.

L E H M A N N , G., C O L L E T , P. & M O R G A N , M . 1970. Zeitschrift fur Lebensmitte1-

u n t e r s u c h u n g u n d F o r s c h u n g , 143 (3) 1 9 1 - 9 5 .

Food Additives Analytical Techniques

Liquid Chromatography

B O L E Y , N.P., C R O S B Y , N.T. & R O G E R , P. 1979. A n a l y s t , 104 472-473.

B R I C O U T , J. 1978. A n n a l e s d e la N u t r i t i o n et de l ' A l i m e n t a t i o n , 32 (5) 947-

955 .

S A L A G O I T Y - A U G U S T E , M.H., BERTRAND, A. & SUDRAUD, P. 1983. Sciences des

A l i m e n t s , _3_ (1) 1 2 7 - 1 3 6 .

133
Thin Layer Chromatography

LEPRI, L., DESIDER, P.G. & COAS, V. 1978. Journal of Chromatography, 161 279-
286.

OMORI, T. 1980. Bunseki Kagaku, _29_(3) 189-193.

V L A D I M I R S K A Y S , A.L., S N I T K O V A K A Y A , T.M. & V E R B I L O V , A.A. 1976. Gigiena i

Sanitariya, _4_ 54-55.

DICKES, G.J. 1965. Journal of the Association of Public Analysts, _3_49.

PEARSON, D. 1973. Journal of the Association of Public Analysts, 11. 135.

CHAPMAN, W.B. & O A K L A N D , D. 1968. Journal of the Association of Public

Analysts, 6 124.

ALDRED, J.B. 1965. Journal of the Association of Public Analysts, _3_ 79.

T U R N E R , T.D. & JONES, B.E. 1971. Journal of P h a r m a c y and P h a r m a c o l o g y , 23

(10) 806-807.

PEARSON, D. 1964. Journal of the Association of Public Analysts, 13 103-108.

S P A U L D I N G , R.C. 1964. Journal of the A s s o c i a t i o n of Public A n a l y s t s , 2 (4)

111 .

PEARSON, D. 1966. Journal of the Association of Public Analysts, 4 (3) 61.

CHIANG, H.C. 1969. Journal of Chromatography, 40 (1) 189-90.

CHIANG, H.C. & LIN, S.L. 1969. Journal of C h r o m a t o g r a p h y , 44.(1) 203-4.

G R A H A M , R.J.T. & NYA, A.E. 1969. Journal of C h r o m a t o g r a p h y , 43_ (4) 547-50.

L E H M A N N , G., HAHN, H.G., COLLET, P., SE I F F E R T - E I S T E R , B. & M O R A N , M. 1970.

Zeitschrift fur Lebensmitteluntersuchung und Forschung, 143 (4) 256-63.

LEHMANN, G. & COLLET, P. 1970. Zeitschrift fur Lebensmitteluntersuchung und

F o r s c h u n g , 143 (6) 4 1 8 - 2 0 , 144 (2) 104-6 & 107-9 (1970).

HAYES, W.P., N Y A K Ü , N.Y. & THORBURN BURNS, D. 1972. Journal of

Chromatography, 71 (3) 585-587.

H O O D L E S S , R.A., P I T M A N , K.G., S T E W A R T , T.E., T H O M S O N , J. & A R N O L D , J.E. 1971.

Journal of Chromatography, 54 (3) 393-404.

H O O D L E S S , R.A., T H O M S O N , J. & A R N O L D , J.E. 1971. Journal of C h r o m a t o g r a p h y ,

56 (2) 332-337.

T A K E S H I T A , R., ITOH, N. & S A K A G A M I , Y. 1971. Journal of C h r o m a t o g r a p h y , 57

(3) 437-440.

T A K E S H I T A , R., Y A M A S H I T A , T. & ITOH, N. 1972. Journal of C h r o m a t o g r a p h y , 73

(1) 173-182.

RAI, J. 1971. Chromatographia, £ (5) 211-213.

P U T T E M A N S , M.L., DRYON, L. & M A S S A R T , D.L. 1982. Journal of the A s s o c i a t i o n

of Official Analytical Chemists, 65 (3) 730-744.

P R A S A D , C.A.K., RAO, M.V., N A G A R A J A , K.V. & KAPUR, O.P. 1983. Indian Food_
Packer , 3_7_(2) 74-77.
Emulsifiers

A I T Z E T M U E L L E R , K., B O E H R S , M. & ARZBERGER, E. 1 979 . Feite, Se i fen

Anstrichmittel, 81_ (11) 436-441. "

D I E F F E N B A C H E R , A. & BRACCO, U. 1978. Journal of the A m e r i c a n Oil Chemists

Society, 55 (9) 642-6.

RUSOM, T. & HOFFMEYER, L. 1978. Journal of the American Oil Chemists Society,
55. (9) 649-52. ~

CONACHER, H.B.S. & PAGE, B.D. 1979. Journal of C h r o m a t o g r a p h i c S c i e n c e , 17

(4) 188-195.

BRUTORF, U.P., B R U S C H W E I L E R , H. & M A R T I N , E. 1982. Zeitschrift fur L e b e n s -

mitteluntersuchung und Forschung, 175 (1) 25-28.

GARTI, N., W E L L M E R , E., A S E R I N , A. & SARIG, S. 1983. J o u r n a l of the A m e r i c a n

Oil Chemists Society, 60 (6) 1151-1154.

SLACK, P.T., & PORTER, D.C. 1983. C h e m i s t r y and Industry (London), 5.12.83.
(23) 896-897.

H I B B E R T , H.R. 1968. The Detection and D e t e r m i n a t i o n of Eiaulsifiers and

Stabilizers in Foods, The British Food M a n u f a c t u r i n g Industries Research
Association, No. 54.

ENDEAN, M.E. 1974. The Detection of T h i c k e n e r , S t a b i l i z e r s and G u m s in

Foodstuffs, The British Food Manufacturing Industries Research Association, No.
82.

Thickeners

PREUSS, A. & THIER, H.P. 1983. Lebensmittelchemie Gerichtliche Chemie, 37 (4)

96-97.

SCHAEFER, H., SCHERZ, H. 1983. Zeitschrift fur Lebensmitteluntersuchung und

Forschung, 177 (3) 193-195.

PECHANEK, U., BLAICHER, G., P F A N N H A U S E R , W. & W O I D I C H , H. 1982. Journal of

the Association of Official Analytical Chemists, 65 (3) 745-752.

135
 7. CONTAMINANT RESIDUE METHODS

7.1 PESTICIDES

T h e r e a r e h u n d r e d s of p e s t i c i d e s in u s e t h r o u g h o u t t h e w o r l d . However, most
c o u n t r i e s h a v e s p e c i f i e d b y law or r e g u l a t i o n w h i c h p e s t i c i d e s m a y be used on
their foods. In t h i s c o n t e x t , t h e t e r m " p e s t i c i d e s " m e a n s i n d u s t r i a l c h e m i c a l s
u s e d to k i l l i n s e c t s ( i n s e c t i c i d e s ) , d e s t r o y u n w a n t e d p l a n t s ( h e r b i c i d e s ) a n d
to p r e v e n t m o u l d and m i l d e w ( f u n g i c i d e s ) . By far the g r e a t e s t a m o u n t of
' p e s t i c i d e s ' u s e d a r e i n s e c t i c i d e s ; so t h e a n a l y t i c a l m e t h o d s g i v e n h e r e w i l l
t a r g e t t h e s e c o m p o u n d s a n d f u n g i c i d e s to a l e s s e r e x t e n t .

In m o s t c a s e s , the l a b o r a t o r y w i l l n o t h a v e d a t a on w h i c h p e s t i c i d e s w e r e u s e d
on a p a r t i c u l a r food s a m p l e . T h i s is e v e n m o r e t r u e f o r i m p o r t e d foods.
T h e r e f o r e , there m u s t be close c o o p e r a t i o n b e t w e e n the l a b o r a t o r y and the
inspectorate staff. For d o m e s t i c a l l y g r o w n f o o d s , the i n s p e c t o r a t e should be
a b l e to d e t e r m i n e a t l e a s t w h i c h p e s t i c i d e s a r e b e i n g u s e d in t h e h a r v e s t a r e a .
This i n f o r m a t i o n w o u l d p r o v i d e a s t a r t i n g p o i n t for the l a b o r a t o r y a n a l y s i s .

For the p u r p o s e s of r e s i d u e a n a l y s i s , p e s t i c i d e s m a y be d i v i d e d into four

g r o u p s - c h l o r i n a t e d h y d r o c a r b o n c o m p o u n d s to w h i c h the electron-capture
detector responds, organo-phosphorus c o m p o u n d s for w h i c h the specific
p h o s p h o r u s d e t e c t o r s are the m o s t a p p r o p r i a t e , c a r b o t h i o a t e s (thio-carbamates,
o f t e n of m e t a l s s u c h as z i n c a n d m a n g a n e s e ) , a n d a m i s c e l l a n e o u s g r o u p w h i c h
i n c l u d e s c o m p o u n d s of d i v e r s e c h e m i c a l t y p e s s u c h as q u i n t o z e n e and
dichloropropionic acid. T h e t e r m c h l o r i n a t e d h y d r o c a r b o n is i n t e n d e d h e r e to
c o v e r o n l y t h e c o m m o n l y e n c o u n t e r e d o r g a n o - c h l o r i n e i n s e c t i c i d e s such as D D T
and its d e r i v a t i v e s , the v a r i o u s i s o m e r s of BHC and the c h l o r i n a t e d p o l y c y c l i c
c o m p o u n d s s u c h as A l d r i n a n d H e p t a c h l o r .

T h e i n i t i a l p e s t i c i d e r e s i d u e a n a l y s i s o f a f o o d is b y n e c e s s i t y a b r o a d
s c r e e n i n g e x a m i n a t i o n for c o m p o u n d s of a g i v e n t y p e (i.e. o r g a n o - c h l o r i n e s ) .
Such screening m e t h o d s are called "multiresidue" procedures. A multiresidue
a n a l y s i s c a n b e d i s c u s s e d in f o u r d i s t i n c t s t e p s :

1. Preparation

T h e i n i t i a l p r e p a r a t i o n o f t h e f o o d s a m p l e is as i m p o r t a n t as a n y of t h e o t h e r
residue analysis steps. T w o things are critical. F i r s t , r e m o v e and d i s c a r d
any inedible portions. T h i s c o u l d b e an o u t e r s k i n or p o d , an i n n e r s e e d or
p i t , or a n y t h i n g e l s e t h a t is o b v i o u s l y i n e d i b l e . Never discard portions which
a r e m e r e l y u n d e s i r a b l e ( s u c h as t h e w i l t e d o u t e r l e a v e s o f a l e a f y v e g e t a b l e ) .
N e x t , c h o p or g r i n d , a n d m i x so t h a t t h e p o r t i o n t a k e n f o r a n a l y s i s is as
r e p r e s e n t a t i v e as p o s s i b l e .

2. Extraction

In this s t e p , the p e s t i c i d e r e s i d u e s and s o l u b l e food m a t e r i a l are e x t r a c t e d

into an a p p r o p r i a t e s o l v e n t . T h e i n s o l u b l e m a t e r i a l ( s u c h as f i b r e or
c e l l u l o s e ) is r e m o v e d b y f i l t r a t i o n . T h e s e l e c t i o n o f t h e s o l v e n t is c r u c i a l .
P e s t i c i d e s r a n g e w i d e l y in p o l a r i t y f r o m the n o n - p o l a r (fat or w a x s o l u b l e )
c h l o r i n a t e d h y d r o c a r b o n s to t h e p o l a r ( m o r e w a t e r s o l u b l e ) o r g a n o - p h o s p h a t e s or
carbamates. The s o l v e n t u s e d m u s t b r i d g e the g a p b e t w e e n t h e s e p o l a r i t i e s and
b e a b l e to s i m u l t a n e o u s l y e x t r a c t a l l r e s i d u e s . A c e t o n e h a s b e e n f o u n d to w o r k
v e r y w e l l in t h i s r e s p e c t . A s e c o n d p r o b l e m o f t h e e x t r a c t i o n p r o c e s s is t h a t
the s o l v e n t m u s t p h y s i c a l l y c o n t a c t the site of the r e s i d u e . If the r e s i d u e
w a s o n l y o n t h e f o o d o u t e r s u r f a c e , t h e n a s i m p l e w a s h i n g or t u m b l i n g w i t h the
s o l v e n t w o u l d s u f f i c e . In p r a c t i c e , h o w e v e r , t h e r e s i d u e m u s t b e a s s u m e d to
have been i n c o r p o r a t e d into the food m a t r i x . It is t h e r e f o r e n e c e s s a r y to
b l e n d t h e f o o d w i t h the s o l v e n t to g a i n t h e i n t i m a t e c o n t a c t n e e d e d to e n s u r e
quantitative extraction.

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 3. Isolation

T h i s is o f t e n r e f e r r e d to as the " c l e a n - u p " in p u b l i s h e d r e s i d u e m e t h o d s . It

is where the solubilized pesticide residues are isolated and the other soluble
i n t e r f e r i n g m a t e r i a l s are r e m o v e d . T h e r e are s e v e r a l w a y s to do t h i s , w h i c h
i n c l u d e p a r t i t i o n i n g b e t w e e n s o l v e n t s , a d s o r p t i o n on a s o l i d m a t e r i a l or
o u t r i g h t d e s t r u c t i o n of the i n t e r f e r e n c e s . A l l m u l t i r e s i d u e m e t h o d s e m p l o y
some form of partitioning. Most also use adsorption as the preferred m e a n s to
remove interferences. O n l y a f e w e m p l o y d r a s t i c d e s t r u c t i o n m e t h o d s as the
residues m u s t be able to survive and not m a n y are that chemically stable.

4. Detection

The t e c h n i q u e that o f f e r s the m o s t v e r s a t i l i t y and g r e a t e s t s e n s i t i v i t y for

p e s t i c i d e r e s i d u e a n a l y s i s is g a s c h r o m a t o g r a p h y . High p e r f o r m a n c e liquid
chromatography has some applicability to residues which are heat sensitive and
d e g r a d e d u r i n g gas c h r o m a t o g r a p h i c analysis. T h i n - l a y e r and paper
chromatography are most useful for confirmation of identity after the residue
has b e e n t e n t a t i v e l y i d e n t i f i e d by g a s c h r o m a t o g r a p h y . Some c o m m o n gas
chromatograph detectors are:

a. Electron Capture

The advantages of this detector are its low m a i n t e n a n c e requirements and high
s e n s i t i v i t y for h a l o g e n a t e d c o m p o u n d s . H o w e v e r , it is a l s o r e s p o n s i v e to
compounds that have electronegative groups such as carbonyls or double bonds.
Lovelock(l) notes that if the detector is used in a dc m o d e and not in a pulsed
mode, its performance can also be affected by nonelectronegative compounds that
survive the isolation procedure.

b. Flame Photometric

T h i s d e t e c t o r is r e l a t i v e l y s p e c i f i c to e i t h e r o r g a n o - p h o s p h o r u s or o r g a n o -
sulfur compounds, depending on the light filter that is used. The detector has
a l i n e a r r e s p o n s e r a n g e of a b o u t four d e c a d e s of c o n c e n t r a t i o n for o r g a n o -
p h o s p h a t e s b u t h a s a r e s p o n s e that is p r o p o r t i o n a l to the s q u a r e of the
concentration of organo-sulphur compounds. It is therefore most useful for the
d e t e r m i n a t i o n of o r g a n o - p h o s p h o r u s c o m p o u n d s . H o w e v e r , c a r e s h o u l d be
e x e r c i s e d in the i n t e r p r e t a t i o n of the c h r o m a t o g r a m s of s a m p l e s w i t h a h i g h
sulphur background w h e n organo-phosphorus compounds are being determined. The
p r e s e n c e of l a r g e a m o u n t s of s u l p h u r c a n n o t be e f f e c t i v e l y f i l t e r e d out and
could t h e r e f o r e p r o d u c e p e a k s that m a y be m i s i n t e r p r e t e d as p h o s p h o r u s
compound s.

c. Alkali Flame

There are several c o m m e r c i a l versions of the alkali flame detectors. Some use
d i f f e r e n t a l k a l i s a l t s in the d e t e c t o r , and o n e d o e s n o t u s e a f l a m e at all
but heats an alkali bead to the desired operating temperature. The detectors
are m o r e responsive to compound s that contain phosphorus or nitrogen, with a
g r e a t e r s e n s i t i v i t y for p h o s p h o r u s . T h e i r p e r f o r m a n c e can be a f f e c t e d to
v a r y i n g d e g r e e s , by r e s i d u a l a m o u n t s of h a l o g e n a t e d s o l v e n t s in the s a m p l e
extract, depending on the commercial detector used.

d. Electroconductivity

Design improvements by Hall(2) and Anderson(3) have resulted in

e l e c t r o c o n d u c t i v i t y d e t e c t o r s c a p a b l e of d e t e r m i n i n g n a n o g r a m s of o r g a n o -
nitrogen compounds and subnanogram amounts of organo-sulphur and organo-halogen
compounds. The effluent from the gas chromatographic c o l u m n is either oxidized
or reduced to produce a species that becomes ionic when dissolved in a suitable
s o l v e n t . O r g a n o - n i t r o g e n c o m p o u n d s are r e d u c e d to a m m o n i a . Organo-sulphur
compounds are oxidized to the sulphur oxides. Organo-halogen c o m p o u n d s can be
o x i d i z e d or r e d u c e d to f o r m the h y d r o g e n h a l i d e s . T h e s p e c i f i c i t y of the

137
d e t e c t o r s is attained by the use of scrubbers that r e m o v e other reaction by-
products and by the selection of an a p p r o p r i a t e c o n d u c t i n g solvent. The
detector is very reliable and usual m a i n t e n a n c e g e n e r a l l y consists only of
replacement of the conducting solvent every few weeks.

Residue analysis is c o m p l e x but is further c o m p l i c a t e d by the fact that

p e s t i c i d e s are often degraded by o x i d a t i o n or h y d r o l y s i s . This can occur in
the environment by the action of sunlight and air, and in plants or animals by
enzymatic and other metabolic processes. The d e g r a d a t i o n p r o d u c t s are often
generically referred to as "metabolites". Many times the metabolite is less
toxic than the original c o m p o u n d and m a y be b i o l o g i c a l l y inactive. In some
cases, h o w e v e r , the m e t a b o l i t e s are equally or even m o r e toxic than the
original. An example of the latter is Heptachlor Epoxide which is considerably
m o r e toxic than Heptachlor. The residue analyst m u s t be a w a r e of the m o r e
commonly found metabolites which do have biological toxicity (such as DDE and
DDD from DDT).

A n o t h e r c o m p l i c a t i n g factor in residue analysis is the potential presence of

environmental contaminants which are not pesticides, but which may interfere in
analysis. The best e x a m p l e s are polych1 orinated b i p h e n y l s (PCB), c h e m i c a l s
commonly used as a heat exchange fluid for cooling transformers or processing
machinery. PCBS are very stable and once in the e n v i r o n m e n t w i l l stay there
indefinitely. They are a mixture of chlorinated compounds and usually appear
as a series of peaks on a gas chromatogram.

Sometimes peaks are detected which can best be described as natural artifacts.
They are sometimes referred to as "crop peaks" and consist of some natural food
ingredient which is not removed by the isolation or clean-up process.

The possible presence of m e t a b o l i t e s , contaminants and crop peaks underscore

the need to c o n f i r m the identity of any pesticide r e s i d u e found. Thin-layer
and paper c h r o m a t o g r a p h y can be used, but m a y not be s e n s i t i v e enough to
confirm very low level residues. In such cases it is usually sufficient to gas
chromatograph the sample and standard on two or three columns having differing
polarities. If the sample and standard have the same relative retention time
on all of the columns, it can be assumed that they are probably the same.

M a k e sure that the p e s t i c i d e standards used for residue a n a l y s i s have not

u n d e r g o n e d e g r a d a t i o n t h e m s e l v e s while in storage. Chlorinated hydrocarbons
like DDT and BHC are generally quite stable, but m a n y o r g a n o - p h o s p h a t e s and
c a r b a m a t e s are unstable and need refrigerated storage as w e l l as frequent
checks on their stability. Also r e m e m b e r that dilute w o r k i n g standards are
e v e n m o r e p r o n e to d e g r a d a t i o n , as w e l l as c o n c e n t r a t i o n by s o l v e n t
evaporation. Make fresh working standards frequently.

The sensitivity of modern GLC detectors requires extreme care be taken to use
only the purest solvents, reagents and gases. N i t r o g e n c a r r i e r gas can be
purified by inserting a m o l e c u l a r sieve b e t w e e n the cylinder and the
instrument. Solvents, however, are purified by redistillation (from glass). A
quick check of a solvent's suitability is to e v a p o r a t e 300 ml to 5 ml and
inject 5 yl of this into a gas c h r o m a t o g r a p h . There should be no e x t r a n e o u s
peaks and the baseline should not raise m o r e than 1 m m for up to one hour.

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 RESIDUES IN FRUITS AND VEGETABLES

PRINCIPLE

This m e t h o d is a p p l i c a b l e to o r g a n o - c h l o r i n e , o r g a n o - p h o s p h o r u s and o r g a n o -
n i t r o g e n p e s t i c i d e s , as w e l l as a f e w h y d r o c a r b o n p e s t i c i d e s . The p e s t i c i d e
r e s i d u e s are e x t r a c t e d into a c e t o n e and after p a r t i t i o n i n g , the o r g a n o -
p h o s p h o r o u s and o r g a n o - n i t r o g e n p e s t i c i d e s are d e t e r m i n e d d i r e c t l y u s i n g an
a l k a l i - f l a m e GLC d e t e c t o r . I n t e r f e r e n c e s are r e m o v e d u s i n g F l o r i s i l b e f o r e
determining organo-chlorine compounds using electron capture detection.

APPARATUS

1. High speed blender - Waring b l e n d e r or equivalent. (explosion

proof).

2. Chromatographic tube with stopcock, 22 mm x 300-400 mm.

3. K u d e r n a - D a n i s h (KD) c o n c e n t r a t o r , 500 ml with 10 or 15 ml

graduated receiving tubes and Snyder column.

4. Separatory funnels, 1 L.

5. Gas chromatograph with electron capture and flame photometric or

alkali-flame detectors, and nitrogen carrier gas.

6. GLC C o l u m n s , g l a s s , 6 feet (or 2 m ) x 4 m m ID, p a c k e d w i t h the

f o l l o w i n g liquid loads on 8 0 - 1 0 0 m e s h C h r o m o s o r b W (HP): ( n i t r o g e n
flow rate and column temperature also indicated).

a. 2% D E G S ( D i e t h y 1 e n e g 1 y c o 1 s u c c i n a t e ) at 165°C and 60
m l / m i n N2.

b. 3% OV-lOl at 200 °C and 120 ml/min.

c. 10% OV-lOl at 200 °C and 120 ml/min.

d. 10% OV-lOl and 15% OV-210 at 200°C and 120 ml/min.

7. Steam bath.

REAGENTS

1. Acetone

2. Methylene chloride

3. Petroleum ether

4. Ethyl ether

(Note: all solvents must be distilled from glass or equivalent).

5. Florisil, 60-100 mesh - activate by heating at 130°C for 5

hours.

6. Sodium sulphate, anhydrous, granular.

7. G l a s s w o o l - rinse w i t h a c e t o n e and e t h a n o l s e v e r a l t i m e s and

dry - washed glass wool will be somewhat brittle.

8. Sodium chloride, granular.

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PROCEDURE

C h o p or b l e n d f r u i t s and v e g e t a b l e s and m i x t h o r o u g h l y . W e i g h 100g

c h o p p e d or b l e n d e d s a m p l e i n t o h i g h - s p e e d b l e n d e r j a r , add 200 m l
a c e t o n e , and b l e n d 1 m i n at h i g h s p e e d . Do n o t add C e l i t e . Filter
w i t h s u c t i o n t h r o u g h 12 c m B u c h n e r f u n n e l f i t t e d w i t h s h a r k s k i n
p a p e r , c o l l e c t i n g e x t r a c t in 5 0 0 m l s u c t i o n f l a s k .

P l a c e 80 m l s a m p l e e x t r a c t in 1 L s e p a r a t o r y f u n n e l , a n d a d d 1 0 0 m l
p e t . e t h e r and 100 m l M e C ^ . Shake vigorously 1 m i n . Transfer lower
a q u e o u s l a y e r to s e c o n d 1 L s é p a r a t o r y f u n n e l . Dry upper organic
l a y e r o f f i r s t s e p a r a t o r y f u n n e l b y p a s s i n g t h r o u g h a b o u t 3.4 c m
N a 2 S 0 ^ s u p p o r t e d o n w a s h e d g l a s s w o o l in a 10 c m f u n n e l , c o l l e c t i n g
in 5 0 0 m l K u d e r n a - D a n i s h c o n c e n t r a t o r f i t t e d w i t h r e c e i v i n g t u b e . To
s e p a r a t o r y f u n n e l w i t h a q u e o u s p h a s e a d d 7g N a C l a n d s h a k e v i g o r o u s l y
30 sec u n t i l m o s t of the N a C l is d i s s o l v e d . A d d 100 m l M e C l 2 , s h a k e
1 m i n , and dry l o w e r organic phase through s a m e N a 2 S 0 ^ . Extract
a q u e o u s p h a s e w i t h a d d i t i o n a l 100 m l M e C ^ and d r y as a b o v e . Rinse
N a 2 S 0 ^ w i t h ca 50 m l M e C ^ . Add b o i l i n g chips and attach Snyder
c o l u m n on K u d e r n a - D a n i s h c o n c e n t r a t o r and s t a r t e v a p o r a t i o n s l o w l y b y
placing only receiver tube into s t e a m . After 100-150 ml has
e v a p o r a t e d , t h e c o n c e n t r a t o r m a y b e e x p o s e d to m o r e s t e a m . When
l i q u i d l e v e l i n h o t c o n c e n t r a t o r t u b e is c a 2 m l , a d d 10 m l a c e t o n e
a n d r e c o n c e n t r a t e to c a 2 m l . R e p e a t a b o v e c o n c e n t r a t i o n w i t h t w o
a d d i t i o n a l 10 m l p o r t i o n s of a c e t o n e . D o n o t a l l o w s o l u t i o n to go to
dryness.

A n a l y s i s of O r g a n o - p h o s p h o r u s and O r g a n o - n i t r o g e n Compounds

A d j u s t t h e v o l u m e in t h e c o n c e n t r a t o r t u b e t o 7 m l w i t h a c e t o n e .
I n j e c t 5 y 1 of t h i s s o l u t i o n ( e q u i v a l e n t to a b o u t 20 m g s a m p l e ) i n t o
gas c h r o m a t o g r a p h using p h o s p h o r u s d e t e c t o r and c o l u m n c o n d i t i o n s
( a ) , ( b ) o r ( c ) . C o l u m n (d) m a y b e u s e d to c o n f i r m r e s i d u e i d e n t i t y .

A n a l y s i s of O r g a n o - c h l o r i n e Compounds

D i l u t e 7 m l s o l u t i o n in c o n c e n t r a t o r t u b e to 10 m l w i t h a c e t o n e .
T r a n s f e r to a 1 0 0 m l g l a s s - s t o p p e r e d g r a d u a t e d c y l i n d e r using
p e t r o l e u m e t h e r to r i n s e . D i l u t e to 1 0 0 m l w i t h p e t r o l e u m e t h e r ,
stopper and m i x w e l l .

P r e p a r e a F l o r i s i l c o l u m n b y p l a c i n g 10 c m a c t i v a t e d F l o r i s i l i n a
chromatographic tube. Top w i t h 1 cm anhydrous sodium sulphate.
P l a c e a K D c o n c e n t r a t o r u n d e r the c o l u m n to c o l l e c t the e l u a t e .

W e t F l o r i s i l w i t h 50 m l p e t . e t h e r . E l u t e at a b o u t 5 m l / m i n . Add
e n t i r e s a m p l e s o l u t i o n ( 1 0 0 m l ) to F l o r i s i l a n d c o n t i n u e e l u t i o n .
W h e n t h e t o p of t h e s a m p l e s o l u t i o n e n t e r s t h e s o d i u m s u l p h a t e , a d d
2 0 0 m l 1 5 % e t h y l e t h e r in p e t . e t h e r . E l u t e u n t i l the liquid has
d r a i n e d from the c o l u m n .

E v a p o r a t e and c o n c e n t r a t e to 4 m l . I n j e c t 5 p i ( e q u i v a l e n t to a b o u t
34 m g s a m p l e ) i n t o a g a s c h r o m a t o g r a p h u s i n g e l e c t r o n capture
d e t e c t o r and c o l u m n c o n d i t i o n s (b) or (c). C o l u m n s (a) or (d) m a y b e
u s e d for c o n f i r m a t i o n .

A n a l y s i s of H y d r o c a r b o n Compounds

Inject 5 p i ( a b o u t 34 m g s a m p l e e q u i v a l e n t ) i n t o a g a s c h r o m a t o g r a p h
using flame i o n i z a t i o n d e t e c t o r a n d c o l u m n c o n d i t i o n s (c).

140
 CALCULATION

mg sample = (100)(80)
U 1 injected (200 + W - 10)(V)

where: 100 = initial g sample

80 = ml taken for analysis
200 = ml acetone
W = ml water in 100 g sample
10 = acetone/water concentration factor
V = ml of final solution (7 or 4 )

(Note: As a first a p p r o x i m a t i o n , W = 85 for m o s t f r u i t s and

vegetables. W a t e r c o n t e n t of r a w p r o d u c e m a y be found in food
composition tables.)

REFERENCE

L u k e , M.A., F r o b e r g , J.E. and M a s u m o t o , H.T., 1975. J o u r n a l of the A s s o c i a t i o n

of Official Analytical Chemists _58, 1020-1026.

141
 RESIDUES IH MILK AND OILSEEDS

PRINCIPLE

M i l k is b l e n d e d w i t h a l u m i n i u m oxide and a c e t o n itri 1 e / w a t e r . The fat is

a d s o r b e d on the A I 2 O 3 and f a t - s o l u b l e p e s t i c i d e s p a r t i t i o n from the fat,
d i r e c t l y into the C H 3 C N . O i l s e e d s are treated the s a m e w a y after g r i n d i n g .
The m e t h o d is a p p l i c a b l e to o r g a n o - c h l o r i n e p e s t i c i d e s . It is v e r y rapid
compared to procedures where the fat is first extracted and then examined for
residues.

APPARATUS

1. High speed blender, Waring or equivalent (explosion proof).

2. Food grinder with 2 mm screen.

3. Separatory funnels, 1 L.

4. Kuderna-Danish (KD) concentrator, 500 ml, with 15 ml graduated

receiving tube, and a Snyder column.

5. Chromatographic tube with stopcock, 22 mm x 300 - 400 mm.

6. Gas chromatograph with e l e c t r o n capture detector and nitrogen

carrier gas at 120 ml/min.

7. GLC column, glass, 6 feet (or 2 m ) x 4 mm ID, packed with:

a. 3% OV-17 on 80-100 mesh Chromosorb W(HP)

b. 2% OV-lOl on 100-120 mesh Chromosorb W(HP)

8. Steam bath.

REAGENTS

1. Acetonitrile.

2. Petroleum ether.

3. Ethyl ether.

(Note: All solvents must be distilled from glass or equivalent).

4. Aluminium oxide, 80-200 mesh - for each g, wash with 10 m l 90%

ethanol, then 10 ml hexane, drain and dry on a steam bath.

5. Florisil, 60-100 mesh - activate by heating at 130°C for 5

hours.

6. Sodium sulphate, anhydrous, granular - heat to 600°C for 1 hour,

cool and store in desiccator.

7. Glass wool - rinse with ethanol several times and dry.

8. Saturated sodium chloride solution.

142
PROCEDURE

Analysis of Milk (Containing 4% or Less Fat)

W e i g h 50 g m i l k into b l e n d e r c u p . A d d 20 g a l u m i n i u m o x i d e , 25 m l
distilled w a t e r and 280 m l a c e t o n i t r i l e . Blend 2 m i n at h i g h speed.
W a i t for solids to s e t t l e , then filter s u p e r n a t a n t l i q u i d u s i n g
vacuum.

M e a s u r e 2 5 0 m l of f i l t r a t e a n d t r a n s f e r to 1 L s e p a r a t o r y f u n n e l .
A d d 1 0 0 m l p e t . e t h e r a n d s h a k e 3 0 s e c . A d d 10 m l s a t u r a t e d N a C l
solution and 500 m l water. Shake 1 m i n and let layers separate.

Drain lower aqueous layer into second 1 L separatory funnel. Add 100
m l pet. e t h e r to s e c o n d f u n n e l a n d s h a k e 1 m i n . L e t l a y e r s s e p a r a t e
and d r a i n and discard l o w e r a q u e o u s layer.

C o m b i n e p e t . e t h e r layer in s e c o n d f u n n e l w i t h p e t . e t h e r in t h e
first. Wash the combined pet. ether with t w o successive 100 m l
portions water. Drain and discard the w a t e r w a s h e s .

Dry the pet. ether by passing through anhydrous Na^SO^ and collect in
a KD concentrator. C o n c e n t r a t e to 10 m l .

Prepare a Florisil column by placing 10 c m F l o r i s i l in a

c h r o m a t o g r a p h i c tube. Top with 1 cm anhydrous N a 2 S 0 ^ . Place a KD
c o n c e n t r a t o r under the c o l u m n to c o l l e c t the e l u a t e .

W e t F l o r i s i l w i t h 50 m l p e t . e t h e r . E l u t e at about 5 m l / m i n . A d d
a l l o f s a m p l e c o n c e n t r a t e (10 m l ) u s i n g p e t . e t h e r t o r i n s e . When
the top of the s a m p l e s o l u t i o n e n t e r s the s o d i u m s u l p h a t e , add 2 0 0 m l
6% e t h y l e t h e r in p e t . e t h e r . Elute until the liquid has drained
from the c o l u m n . Place a second KD concentrator under the c o l u m n and
e l u t e w i t h 2 0 0 m l 15% e t h y l e t h e r in p e t . e t h e r .

C o n c e n t r a t e b o t h i n d i v i d u a l l y , o n a s t e a m b a t h to 4 m l . I n j e c t 5 y l
(equivalent to a b o u t 45 m g of s a m p l e ) of each into a gas
c h r o m a t o g r a p h u s i n g c o l u m n (a) or (b). ( O n e c o u l d b e u s e d for
analysis and the other for confirmation). The u s e of 6 and 15%
e l u t i o n s is to s e p a r a t e t h e p e s t i c i d e s D i e l d r i n a n d E n d r i n f r o m D D E
and A l d r i n . A single e l u t i o n of 15% e t h y l e t h e r could be u s e d , to
e l u t e a l l p e s t i c i d e s at o n c e .

Analysis of Oilseeds

G r i n d t h e o i l s e e d s to pass a 2 m m s c r e e n t h r e e t i m e s . Weigh a
portion containing n o t m o r e than 2 g fat into a blender c u p . A d d 20
g A 1 2 0 3 and 350 m l of a c e t o n i t r i l e - w a t e r (80+20).

Continue as above for m i l k starting with "...Blend 2 min...".

CAL C ULATIOHS

For milk:

mg sample (50)(250)
p 1 injected (344)(V)

where: 50 = g sample
250 = ml taken for analysis
344 = CH3CN/water factor
V = m l of final solution

143
 For oilseeds:

mg sample _ (W)(250)
Hi injected (350)(V)

where: W = g sample
250 = ml taken for analysis
350 = CHgCN/water factor
V = ml of final solution

REFERENCE

Luke, M.A. and Doose, G.M., 1984. Bulletin of Environmental Contamination and
Toxicology 32, 651-656.

144
 RESIDUES IH DRY, LOW-FAT FOODS

PRINCIPLE

"Dry" p r o d u c t s are d e f i n e d as h a v i n g less than 10% w a t e r , such as dried

vegetables, spices and pulses. The problem with analysis of such products has
been the difficulty of extraction of the residues from the sample matrix. This
d i f f i c u l t y w a s solved by use of a w a t e r / a c e tone m i x t u r e . The m e t h o d is
d e s i g n e d for r e l a t i v e l y l o w - f a t food p r o d u c t s but can be used for foods
c o n t a i n i n g up to 10% fat. The F l o r i s i l i s o l a t i o n step m u s t be used w h e n the
food contains above 2-3% fat.

APPARATUS

1. High speed b l e n d e r - Waring blender or e q u i v a l e n t (explosion

proof).

2. Separatory funnel, 1 L.

3. K u d e r n a - D a n i s h (KD) c o n c e n t r a t o r 500 m l w i t h 10 m l graduated

receiving tube and Snyder column.

4. Chromatographic tube with stopcock, 22 mm x 300-400 mm.

5. Gas chromatograph with electron capture and flame photometric or

alkali flame detectors and nitrogen carrier gas.

6. G L C c o l u m n , g l a s s , 6 feet (or 2 m ) x 4 m m ID p a c k e d w i t h 3% 0 V -
101 on 8 0 - 1 0 0 m e s h c h r o m o s o r b W(AW). N i t r o g e n f l o w rate of 120
m l / m i n and column temperature of 200°C.

REAGENTS

1. Acetone

2. Methylene chloride

3. Petroleum ether

4. Ethyl ether

(Note: all solvents must be distilled from glass or equivalent.)

5. Activated carbon

6. Celite 545 (diatomaceous earth)

7. Magnesium oxide, 200 mesh, adsorptive grade

8. Florisil, 60-100 mesh - activate by heating at 130°C for 5

hours.

9. Sodium sulphate, anhydrous, granular.

10. Sodium chloride

PROCEDURE

Grind s a m p l e to pass 20 m e s h . W e i g h 15 g into b l e n d e r cup and add

350 m l a c e t o n e + w a t e r m i x t u r e (65+35). Blend at h i g h speed 2 m i n .
Filter and transfer 80 ml filtrate to 1 L separatory funnel.

145
 Add 100 ml m e t h y l e n e chloride and 100 ml p e t r o l e u m ether. Shake
v i g o r o u s l y 1 min. A l l o w layers to separate and transfer the l o w e r
aqueous layer to a second 1 L funnel.

Dry the upper organic layer by passing through sodium sulphate and
collect in a KD concentrator.

Add 7 g sodium chloride to the second funnel. Extract with 2/100 ml

portions of m e t h y l e n e chloride by shaking 1 min. Dry each lower
organic phase by passing through sodium sulphate and collect all in
the KD concentrator.

Rinse the sodium sulphate with 50 ml m e t h y l e n e chloride and add to

the KD. Concentrate the c o m b i n e d e x t r a c t s to 2 ml. Add 2/10 m l
portions acetone and reconcentrate to 2 ml after each addition.

This extract can be injected directly into the gas chromatograph, or

can be further cleaned using the carbon or Florisil columns below.

Carbon Coluam

Prepare by placing 2.5 cm Celite in a column. Then add 6 g of a

carbon mixture containing carbon + Celite + magnesium oxide (1 + 4 +
2). T a m p d o w n firmly and wash with 25 ml m e t h y l e n e chloride. Use
air pressure to elute through the column. Discard the wash and place
a KD under the column.

Add the sample extract to the c o l u m n using small portions of

methylene chloride to transfer. When the sample solution enters the
c o l u m n , elute with 200 ml acetone + m e t h y l e n e chloride (2 + 1).
Concentrate as above.

Florisil Column

Prepare and use as described in the m e t h o d , "Residues in M i l k and

Oilseeds".

CALCULATIONS

mg sample _ (15) (80)

yl injected (350) (V)

where: 15 = g sample
80 = ml taken for analysis
350 = acetone/water
V = ml of final solution

REFERENCE

Luke, M.A. and Doose, G.M., 1983. Bulletin of Environmental Contamination and
Toxicology, 30. 110-116.

146
 RESIDUES IN FATTY FOODS

PRINCIPLE

T h i s p r o c e d u r e is o n l y a p p l i c a b l e to f a t - s o l u b l e , n o n - p o l a r p e s t i c i d e s . Th
m e t h o d i n v o l v e s f i r s t e x t r a c t i n g the fat, t h e n i s o l a t i n g the p e s t i c i d
residues, by partitioning and adsorption chromatography.

APPARATUS

1. H i g h s p e e d b l e n d e r - W a r i n g b l e n d e r or e q u i v a l e n t (explosion-
proof)

2. Separatory funnels, 125 ml and 1 L.

3. K u d e r n a - D a n i s h (KD) c o n c e n t r a t o r , 5 0 0 m l w i t h 10 m l receiving
tube and Snyder column.

4. Chromatographic tube with stopcock, 22 mm x 300-400 mm.

5. Gas chromatograph with e l e c t r o n capture detector and nitrogen

c a r r i e r gas.

6. GLC column, glass, 6 feet (or 2 m ) x 4 m m ID, packed with 3% 0V-

101 on 80-100 m e s h Chromosorb W (AW). Nitrogen flow rate 120 m l / m i n
and column temperature of 200°C.

7. Steam bath

8. Water bath

REAGENTS

1. Acetonitrile

2. Petroleum ether

3. Ethyl ether

(Note: all solvents must be distilled from glass or equivalent.)

4. Sodium sulphate, anhydrous, granular

5. Ethanol

6. Florisil, 60-100 mesh - activate by heating at 130°C for 5

hours.

7. Sodium (or potassium) oxalate

8. Sodium chloride

9. Sodium hydroxide

PROCEDURE

Isolate fat (at least 3 g) as follows:

A n i m a l and V e g e t a b l e f a t s and o i l s : If solid, w a r m until liquid and

filter through dry filter paper.

Butter: W a r m to a b o u t 50°C u n t i l the fat s e p a r a t e s and d e c a n t the

fat through a dry filter.

147
Cheese: P l a c e 2 5 - 1 0 0 g ( s u f f i c i e n t to p r o v i d e 3 g f a t ) of d i c e d
s a m p l e , a b o u t 2 g of o x a l a t e and 100 m l e t h a n o l in a h i g h - s p e e d
b l e n d e r and b l e n d 2 - 3 m i n u t e s . (if e x p e r i e n c e w i t h the p r o d u c t
indicates that e m u l s i o n s will not be broken by centrifuging, add 1 m l
water/2 g sample before blending). P o u r into a 5 0 0 m l c e n t r i f u g e
bottle. Add 50 m l of e t h y l e t h e r and s h a k e v i g o r o u s l y 1 m i n u t e .
T h e n add 50 m l of p e t r o l e u m e t h e r and s h a k e v i g o r o u s l y 1 m i n u t e .
C e n t r i f u g e a b o u t 5 m i n at 1 5 0 0 r p m . S i p h o n o f f the s o l v e n t l a y e r
using a wash bottle device prepared as follows: Use tube similar to
d e l i v e r y t u b e of o r d i n a r y w a s h b o t t l e b u t w i t h i n t a k e end b e n t up
into U-shape in opposite direction to outlet end, with opening 6 - 1 2
m m higher than bottom of U, cut off horizontally. (Avoid excessive
constriction when bending). Set d e l i v e r y t u b e l o o s e l y e n o u g h in
s t o p p e r t h a t it can be r a i s e d or l o w e r e d . In o p e r a t i n g , a d j u s t
o p e n i n g of U b e n d to a b o u t 3 m m a b o v e s u r f a c e of a q u e o u s l a y e r and
b l o w ether layer off by gently b l o w i n g through m o u t h p i e c e tube
i n s e r t e d in a d j a c e n t h o l e in s t o p p e r . S i p h o n into a 1 L s e p a r a t o r
containing 500-600 m l of water and 30 m l of saturated salt solution.
Re-extract the aqueous residue twice, shaking vigorously with 50 ml
p o r t i o n s of e t h y l e t h e r - p e t r o l e u m e t h e r (1 + 1). C e n t r i f u g e and
s i p h o n off the s o l v e n t l a y e r into the s e p a r a t o r a f t e r each
extraction. M i x the combined extracts and water cautiously. Drain
and discard the water layer. Rewash the solvent layer twice with 100
m l portions of water, discarding the water each time. (If e m u l s i o n s
form, add about 5 ml of saturated sa.lt solution to the solvent layer
or include it with the water wash). Pass the ether solution through
a c o l u m n of a n h y d r o u s s o d i u m s u l p h a t e 50 m m h i g h in a t u b e and
c o l l e c t the e l u a t e in a 4 0 0 m l b e a k e r . W a s h the c o l u m n w i t h s m a l l
p o r t i o n s of p e t r o l e u m e t h e r and e v a p o r a t e the s o l v e n t f r o m the
combined extracts on a water-bath at 100°C w i t h the assistance of a
current of air.

Fish: W e i g h 25-50 g thoroughly ground and mixed sample into a high-

speed b l e n d e r . (If the fat c o n t e n t is k n o w n or c a n be e s t i m a t e d ,
adjust the sample size so that a m a x i m u m of about 3 g of fat will be
extracted). Add 100 g of anhydrous sodium sulphate to combine w i t h
water present and disintegrate the sample. Alternately blend and mix
w i t h a spatula until the sample and sulphate are well mixed. Scrape
d o w n the sides of the blender jar and break up caked material with a
spatula. Add 150 m l of petroleum ether and blend at high speed for 2
minutes. D e c a n t the s u p e r n a t a n t p e t r o l e u m e t h e r t h r o u g h a 12 cm
Buchner funnel fitted with 2 sharkskin papers into a 500 m l suction
flask. S c r a p e d o w n the s i d e s of the b l e n d e r jar and b r e a k up the
caked m a t e r i a l w i t h a spatula. Re-extract the residue in the blender
jar with two 100 ml portions of petroleum ether, blending for 2 m i n
each time. (After blending 1 m i n , stop the blender, scrape d o w n the
s i d e s of the jar and b r e a k up c a k e d m a t e r i a l w i t h a s p a t u l a , then
c o n t i n u e b l e n d i n g for 1 m i n ) . S c r a p e d o w n the s i d e s of the b l e n d e r
jar and b r e a k up c a k e d m a t e r i a l b e t w e e n e x t r a c t i o n s . D e c a n t the
s u p e r n a t a n t p e t r o l e u m e t h e r f r o m r e p e a t e d b l e n d i n g s t h r o u g h the
B u c h n e r f u n n e l and c o m b i n e w i t h the first e x t r a c t . A f t e r the last
b l e n d i n g , t r a n s f e r the r e s i d u e f r o m the b l e n d e r jar to the B u c h n e r
funnel and rinse the blender jar and material in the Buchner funnel
with several portions of petroleum ether. Pour the combined extracts
t h r o u g h a 4 0 m m c o l u m n of a n h y d r o u s s o d i u m s u l p h a t e in a t u b e , and
c o l l e c t t h e e l u a t e in a 5 0 0 m l K D c o n c e n t r a t o r w i t h a p l a i n
c o l l e c t i o n tube. W a s h the f l a s k and c o l u m n w i t h s m a l l p o r t i o n s of
p e t r o l e u m e t h e r and e v a p o r a t e m o s t of the p e t r o l e u m e t h e r f r o m the
combined extracts and rinses. Transfer the concentrated fat extract
to a tared beaker using small amounts of petroleum ether. Evaporate
the p e t r o l e u m e t h e r on a w a t e r - b a t h at 100°C u n d e r a c u r r e n t of d r y
air.

148
O t h e r f a t t y f o o d s: w e i g h into b l e n d e r jar a m o u n t e s t i m a t e d to
p r o v i d e ca 3 g of fat. (Refer to t a b l e s of a v e r a g e c o m p o s i t i o n , if
necessary.) In s e p a r a t e c o n t a i n e r m i x a m o u n t of s o d i u m s u l p h a t e
e q u a l to 2.5 x e s t i m a t e d w e i g h t of w a t e r in s a m p l e w i t h 100 m l
p e t r o l e u m e t h e r , and t r a n s f e r to b l e n d e r jar. M i x at m e d i u m speed
for 3 min. Allow solids to settle and decant petroleum ether through
m e d i u m p o r o s i t y f i l t e r p a p e r into tared 5 0 0 m l e r l e n m e y e r flask to
which a few boiling chips had been added before weighing. Add 100 m l
petroleum ether to residue in blender jar, m i x at m e d i u m speed for 1
m i n , allow solids to settle and decant petroleum ether through filter
to c o m b i n e w i t h a b o v e f i l t r a t e . T r a n s f e r solid r e s i d u e in b l e n d e r
jar to filter paper, fold paper over solids, and squeeze paper gently
a g a i n s t side of f u n n e l w i t h s p a t u l a to r e c o v e r as m u c h s o l v e n t as
possible. Evaporate petroleum ether on a water bath. Dry flask and
w e i g h flask plus contents. D e t e r m i n e a m o u n t of fat b y s u b t r a c t i n g
tare weight of flask.

D e t e r m i n e the p e r c e n t fat in the food f r o m the w e i g h t of the

extracted fat. (This will be used later in the final calculation.)

W e i g h 3 g of fat into a 125 m l s e p a r a t o r and add p e t r o l e u m e t h e r so

that the total v o l u m e of fat and petroleum ether is 15 ml. Add 30 m l
of acetonitrile saturated with petroleum ether. Shake vigorously 1
min, allow the layers to separate and drain the acetonitrile into a 1
L s e p a r a t o r c o n t a i n i n g 6 5 0 m l w a t e r , 40 m l s a t u r a t e d salt s o l u t i o n
and 100 inl of petroleum ether. Extract the remaining petroleum ether
in the 125 m l s e p a r a t o r w i t h 3 a d d i t i o n a l 30 m l p o r t i o n s of
acetonitrile saturated with petroleum ether, shaking vigorously 1 m i n
each tine. Combine all the extracts in the 1 L separator.

Hold the 1 L separator in a horizontal position and m i x thoroughly

3 0 - 4 5 s e c o n d s . Let the l a y e r s s e p a r a t e and d r a i n the a q u e o u s p h a s e
into a s e c o n d 1 L s e p a r a t o r . A d d 100 m l of p e t r o l e u m e t h e r to the
s e c o n d s e p a r a t o r , s h a k e v i g o r o u s l y 15 s e c o n d s and let the l a y e r s
separate. D i s c a r d the a q u e o u s p h a s e , c o m b i n e the p e t r o l e u m e t h e r
with that in the first separator and wash with two 100 ml portions of
water. Discard the washings and draw off the petroleum ether layer
through a 50 m m column of anhydrous sodium sulphate into a 500 ml KD
concentrator. R i n s e the s e p a r a t o r and t h e n the c o l u m n w i t h t h r e e
a p p r o x i m a t e l y 10 m l p o r t i o n s of p e t r o l e u m e t h e r . E v a p o r a t e the
combined extract and washings to about 10 ml.

Prepare a columm of Florisil 10 cm high (after settling) and topped

w i t h a b o u t 1 cm of a n h y d r o u s s o d i u m s u l p h a t e . W e t the c o l u m n w i t h
4 0 - 5 0 m l of p e t r o l e u m e t h e r . P l a c e the KD e v a p o r a t o r ( w i t h a
g r a d u a t e d c o l l e c t i o n tube u n d e r the c o l u m n ) to r e c e i v e the e l u a t e .
T r a n s f e r the p e t r o l e u m e t h e r c o n c e n t r a t e to the c o l u m n and let it
pass through at 5 m l / m i n . Rinse containers w i t h two approximately 5
ml portions of petroleum ether, pour the rinsings on to the column,
r i n s e the w a l l s of the tube w i t h a d d i t i o n a l s m a l l p o r t i o n s of
p e t r o l e u m ether. E l u t e at a b o u t 5 m l / m i n w i t h 200 m l of 6% e t h y l
e t h e r in pet. e t h e r . C h a n g e r e c e i v e r s and e l u t e w i t h 2 0 0 m l of 15%
ethyl ether in pet. ether.

C o n c e n t r a t e e a c h e l u a t e to a s u i t a b l e d e f i n i t e v o l u m e in the KD
concentrators. The 6% eluate contains chlorinated pesticides such as
A l d r i n , B H C , D D E , D D D , o',p'- and p , p ' - D D T , H e p t a c h l o r , H e p t a c h l o r
E p o x i d e , L i n d a n e , M e t h o x y c h 1 or, M i r e x and P e r t h a n e and i n d u s t r i a l
c h e m i c a l s such as p o l y c h l o r i n a t e d b i p h e n y l s (PCB). It is u s u a l l y
suitable for GLC directly. If further clean-up is necessary, repeat
the F l o r i s i l c l e a n - u p u s i n g a n e w c o l u m n . T h e 15% e l u a t e c o n t a i n s
the chlorinated pesticides Dieldrin and Endrin. In those cases where
the 15% e l u a t e m u s t be f u r t h e r c l e a n e d , d e s t r u c t i o n of the f a t t y
material can be accomplished by saponification, as follows:

149
 Transfer the concentrated eluate to an 125 ml glass-stoppered flask,
rinsing with petroleum ether and evaporate just to dryness. Add 20
ml of 2% ethanolic sodium hydroxide and reflux 30 minutes under an
air condenser. Transfer to an 125 ml separator and rinse the flask
with three 10 ml portions of petroleum ether, transfering each to the
separator. Add 20 ml of water and shake vigorously. Drain the
aqueous layer into a second separator containing 20 ml of petroleum
ether. Shake vigorously, allow to separate, discard the aqueous
layer and add petroleum ether to the first separator. Wash the
combined petroleum ether extracts with three 20 ml portions of
aqueous ethanol (1 + 1). (if the initial aqueous alcohol wash causes
heavy emulsions, use water only for additional washes.) Discard the
wash. Dry the petroleum ether layer through a 50 m m column of
anhydrous sodium sulphate rinsing with petroleum ether. Concentrate
the solution using a KD concentrator, to suitable volume (2-4 ml) for
gas chromatography.

Inject 5 p 1 of the final cleaned extract into the gas chromatograph

and compare to known standards.

CALCULATIONS

On a fat basis:

mg fat _ (W)
pi injected (V)

where :
W = g fat taken for analysis
V = ml of final solution

On a whole food basis:

mg sample _ (W)
pi injected (V)(%)

where :
W = g fat taken for analysis
V = ml of final solution
% = percent fat in the sample expressed as a
decimal (i.< . 12% fat = 0.12)

REFERENCES

Pesticide Analytical Manual, Vol. I, U.S. Food and Drug Administration,

Washington, D.C.

Official Methods of Analysis of the AOAC, 1984, 29.001-.002, 29.008-.018.

150
 RESIDUE IDENTITY CONFIRMATION
(Using Thin-Layer C h r o m a t o g r a p h y )

PRINCIPLE

See 5.2 of this M a n u a l for a g e n e r a l discussion of TLC t e c h n i q u e , includ

preparation of plates.

APPARATUS

1. TLC apparatus including glass plates, developing tanks, spotting

pipettes and TLC spray flasks.

2. Forced draft oven.

3. UV viewing cabinet.

REAGENTS

1. Aluminium oxide, neutral G or equivalent.

2. Developing solvents for c h l o r i n a t e d pesticides: (a) n - H e p t a n e

(b) n-Heptane containing 2% acetone.

3. Chromogenic agent for chlorinated pesticides: Dissolve 0.100 g

silver nitrate in 1 ml water, add 20 ml of 2-phenoxyethanol, dilute
to 2 0 0 m l w i t h a c e t o n e , add a v e r y s m a l l d r o p of 3 0 % H 2 0 2 and m i x .
S t o r e in d a r k o v e r n i g h t and d e c a n t into a s p r a y bottle." Discard
after 4 days.

4. Developing solvents for phosphated pesticides: (a) I m m o b i l e ; 15

or 20% N , N - d i m e t h y l f o r m a m i d e (DMF) in ether. Dilute 75 or 100 m l of
DMF to 500 ml with ether 'and mix. (b) Mobile; methylcyclohexane.

5. C h r o m o g e n i c a g e n t s for p h o s p h a t e d p e s t i c i d e s : (a) S t o c k d y e
solution; dissolve 1 g tetrabromophenolphthalein ethyl ester in 100
m l acetone. (b) Dye solution; dilute 10 m l of the stock dye solution
(a) to 50 m l w i t h a c e t o n e . (c) S i l v e r n i t r a t e s o l u t i o n ; d i s s o l v e
0.5g in 25 m l w a t e r and d i l u t e to 100 m l w i t h a c e t o n e . (d) C i t r i c
acid solution; dissolve 5 g of granular citric acid in 50 ml of water
and dilute to 100 m l with acetone.

PROCEDURE

Preparation of TLC Plates

P r e p a r e p l a t e s as d e s c r i b e d in C h a p t e r 5.2 of this M a n u a l , u s i n g a
s l u r r y of 30 g a l u m i n i u m o x i d e and 50 m l w a t e r . W h e n m a k i n g the
s l u r r y , be sure to s h a k e o n l y m o d e r a t e l y for 45 s e c o n d s . Violent
shaking produces bubbles, resulting in "pock-marked" layers. (Note:
Suspensions that contain adsorbents with binders set rapidly and the
entire operation from preparation of slurry to final coating m u s t be
completed within 2 minutes.) T h i s a m o u n t is s u f f i c i e n t for a b o u t
five p l a t e s .

Let the plates air dry for 15 min, then 30 m i n in a forced-draft oven
a t 80°C. C o o l t h e p l a t e s in a d r y a r e a . E x a m i n e the p l a t e s
c a r e f u l l y in t r a n s m i t t e d and r e f l e c t e d light for i m p e r f e c t i o n s or
i r r e g u l a r i t i e s in c o a t i n g . D i s c a r d a n y p l a t e s on w h i c h the l a y e r
shows extensive rippling or mottling.

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Pre-wa8hing of Plates

S c r a p e 1 cm of a d s o r b e n t off the e d g e of the p l a t e w i t h a r a z o r

blade. P o u r 15 m l of 50% a q u e o u s a c e t o n e i n t o a m e t a l t r o u g h i n s i d e
a c h r o m a t o g r a p h i c t a n k . C u t o u t a 2 x 20 cm s t r i p of W h a t m a n N o . 1
filter p a p e r , w e t w i t h solvent and place over the s c r a p e d - o f f area
w i t h 6 m m of t h e p a p e r o v e r l a p p i n g t h e a d s o r b e n t l a y e r . P l a c e t h e
p l a t e in the c h r o m a t o g r a p h i c t a n k , s e a l t h e t a n k w i t h m a s k i n g t a p e
and d e v e l o p the p l a t e w i t h 5 0 % a q u e o u s a c e t o n e to w i t h i n 4 c m of the
top (75-90 m i n u t e s ) . R e m o v e the plate from the t a n k , detach the
f i l t e r p a p e r w i c k , i n v e r t t h e p l a t e a n d d r y in a f u m e h o o d f o r 5
minutes. D r y t h e p l a t e f o r 4 5 m i n u t e s a t 80°C. R e m o v e the plate
f r o m t h e o v e n , c o o l and s t o r e in a d e s i c c a t o r . Use the p r e p a r e d
plates within 1 week.

Sample Application

M a r k the p l a t e w i t h a p e n c i l on b o t h s i d e s 4 cm f r o m the b o t t o m . The

i m a g i n a r y l i n e b e t w e e n t h e t w o p o i n t s i n d i c a t e s s a m p l e s p o t t i n g or
origin line. D r a w a l i n e , r e m o v i n g the l a y e r c o m p l e t e l y a c r o s s the
p l a t e 14 c m f r o m the b o t t o m e d g e . This line r e p r e s e n t s the solvent
front after d e v e l o p m e n t . O n the l o w e r e d g e of a d s o r b e n t s t a r t i n g 2
c m in f r o m t h e l e f t s i d e of t h e p l a t e m a k e 18 m a r k s w i t h a p e n c i l at
1 cm i n t e r v a l s . ( F e w e r m a r k s a t l o n g e r i n t e r v a l s m a y b e u s e d , if
desired.) The m a r k s s e r v e as h o r i z o n t a l guides to sample
application. I d e n t i t y of s a m p l e s a n d s t a n d a r d s m a y b e e t c h e d i n t o
a d s o r b e n t l a y e r d i r e c t l y a b o v e t h e s e m a r k s at t h e t o p of t h e p l a t e .

U s e the s h a d o w c a s t b y a s t r o n g l i g h t s o u r c e on a s t r a i g h t e d g e
s u p p o r t e d a b o u t 2 c m a b o v e t h e p l a t e to s h o w a s t r a i g h t l i n e a c r o s s
the p a p e r . A l i g n the s h a d o w on the t w o 4 c m m a r k s on e i t h e r e d g e of
t h e p l a t e . T h e l i n e of t h e s h a d o w a n d t h e 18 m a r k s s e r v e as v e r t i c a l
a n d h o r i z o n t a l g u i d e s r e s p e c t i v e l y for a p p l i c a t i o n of the s a m p l e .

For o p t i m u m s e m i - q u a n t i t a t i v e d e t e r m i n a t i o n , s p o t p o r t i o n s of the
s a m p l e s as f o l l o w s :

For Chlorinated pesticides: A d j u s t t h e a l i q u o t to g i v e a r e s i d u e

spot w i t h i n the r a n g e 0.005-0.1 p g . Spot s t a n d a r d s and standard
m i x t u r e s o f 0 . 0 0 5 , 0 . 0 1 , 0 . 0 2 , 0 . 0 5 , 0.1 a n d 0.2 p g . Sample spots
o v e r 0.2 g a r e d i f f i c u l t to d e t e r m i n e q u a n t i t a t i v e l y a n d u n d e r 0.005
pg m a y b e d i f f i c u l t to s e e . S p o t a l l 6% F l o r i s i l e l u a t e s on o n e
p l a t e a n d 15% F l o r i s i l e l u a t e s o n a n o t h e r p l a t e .

For Phosphated pesticides: A d j u s t a l i q u o t s of s a m p l e a n d s t a n d a r d s

to g i v e s p o t s w i t h i n t h e r a n g e 0.1-0.5 p g . Spot all Florisil eluates
on the s a m e p l a t e . R o n n e l , E t h i o n and C a r b o p h e n o t h i o n are not
resolved. S p o t s t a n d a r d s of e a c h of t h e s e s e p a r a t e l y . Spot
D i a z i n o n , M e t h y l P a r a t h i o n and M a l a t h i o n s e p a r a t e l y or as m i x t u r e .

T h e t o t a l v o l u m e of s a m p l e e x t r a c t s p o t t e d s h o u l d b e l e s s t h a n 10 p i ,
if p o s s i b l e , and s p o t t i n g s h o u l d b e d o n e r e p e a t e d l y w i t h 1, 2 or 3 p i
spotting pipettes. Use s e p a r a t e p i p e t t e s for each s a m p l e e x t r a c t
and s t a n d a r d . F o r b e s t r e s u l t s , k e e p the s i z e of t h e s p o t s as s m a l l
as p o s s i b l e .

Chromatography

Chlorinated pesticides: P l a c e a f i l t e r paper liner and a m e t a l

t r o u g h in t h e t a n k . P r e s a t u r a t e t h e l i n e r b y p o u r i n g 75 m l o f
d e v e l o p i n g s o l v e n t i n t o the b o t t o m of the t a n k 30 m i n u t e s b e f o r e
d e v e l o p i n g the p l a t e . P r e - s a t u r a t i o n d e c r e a s e s the d e v e l o p m e n t t i m e
a n d i m p r o v e s u n i f o r m i t y of the R f v a l u e s . F o r p l a t e s s p o t t e d w i t h 6%
F l o r i s i l e l u a t e s , p o u r 50 m l of n - h e p t a n e i n t o t h e t r o u g h . P l a c e the

152
 lower edge of the plate in the metal trough with the top of the plate
leaning against the side of the tank. Place a glass plate on the
tank and seal with m a s k i n g tape. For plates spotted with the 15%
F l o r i s i l e l u a t e s , u s e a c e t o n e and n - h e p t a n e (2 + 9 8 ) as the
developing solvent.

Phosphate*! p e s t i c i d e s : Place a liner and a metal trough in the tank.

Pour 50 ml of m e t h y l c y c lohexane into the trough and 75 ml into the
b o t t o m of the tank. Q u i c k l y fill a dipping tank to w i t h i n 4 - 5 cm
from the top w i t h i m m o b i l e solvent. Invert the plate and dip w i t h
the uncoated side touching the back w a l l of the tank to prevent the
front w a l l from scraping the adsorbent layer during the dipping
operation. Dip the plate just to the spotting line, r e m o v e and
i m m e d i a t e l y place in the m e t a l trough w i t h the top p o r t i o n of the
plate leaning against the side of the tank. Place a glass plate on
the tank and seal with masking tape.

Visualization of Residue Spots

W h e n the solvent front just reaches the pencil line 10 cm above the
"spotting" line remove the plate and dry in a hood for 5 minutes.

For chlorinated pesticides: Support the plate on one side and spray
fairly heavily with silver nitrate reagent, using lateral motions of
the spray bottle. Spray until the plate appears translucent or
soaked with reagent. Underspraying will result in poor sensitivity.
After spraying, dry the plate in a hood for 15 min, then immediately
place under a source of ÜV light for viewing. Expose the plate to UV
light until the spot for the standard of lowest c o n c e n t r a t i o n
appears. 5 ng of m o s t chlorinated organic p e s t i c i d e s should be
visible after about 15-20 m i n of exposure. E x p o s u r e times of 30
minutes generally will not harm the plates. For best results place
the plates 8 cm from the b o t t o m edge of the lamps. This distance
should be adjusted based on experience.

For phosphated pesticides: Immediately spray the plate heavily and

u n i f o r m l y w i t h dye solution, using lateral m o t i o n s of the spray
flask. The plate should be vivid blue after spraying. Overspray the
plate lightly and u n i f o r m l y w i t h silver n i t r a t e solution. At this
p o i n t the p l a t e s h o u l d be b l u i s h p u r p l e and s p o t s s h o u l d be
discernible. After 2 m i n u t e s overspray the plate m o d e r a t e l y and
uniformly with citric acid solution. After spraying, thiophosphate
pesticides should i m m e d i a t e l y appear as vivid blue or purple spots
against a yellow background. The colour of the spots reaches maximum
intensity about 5-10 m i n after spraying w i t h citric acid. After
about 10 min the background begins to change from yellow to greenish
blue, m a s k i n g the spots. At this point, r e s p r a y i n g the plate w i t h
citric acid solution changes the background to yellow again and makes
the spots stand out as w e l l as or better than before. E v a l u a t e the
c h r o m a t o g r a m 10 m i n u t e s after respraying. The blue spots fade
c o m p l e t e l y and irreversibly 30-40 m i n u t e s after the t i m e of the
original citric acid spraying.

REFERENCE

Official M e t h o d s of Analysis of the AOAC, 1984, 29.003, 29.005-.006, 29.019-

.027 .

153
 RESIDUE IDEHTITY CONFIRMATION
(Using Paper Chromatography)

PRINCIPLE

S e e 5.1 of this Manual for a general discussion of paper chromatography

technique.

APPARATUS

1. Developing tanks with glass tops.

2. Spotting pipettes.

3. UV viewing cabinet with short and long wave lamps.

4. Chromatographic paper, Whatman N o . 1 or e q u i v a l e n t , 20x20cm.

REAGENTS

Aqueous System

1. Immobile solvent: d i s s o l v e 2.5 m l m i n e r a l o i l (or c o r n , soya or

c o t t o n s e e d ) in e t h y l e t h e r a n d d i l u t e t o 5 0 0 m l with ether.

2. Mobile solvents:

a . 75% acetone in water

b. 75% 2 - m e t h o x y e t h a n o l in water

c. 75% m e t h a n o l in water

d. 40% pyridine in water

Non-aqueous System

1. Immobile solvents:

a. D i l u t e 175 m l of N,N ' - d i m e t h y 1 f o r m a m i d e ( D M F ) to 5 0 0 ml

with ethyl ether.

b. Dilute 50 ml of 2-phenoxyethanol to 500 ml with ethyl

e ther.

2. Mobile solvent - 2, 2, 4-trimethylpentane

Chromogenic Reagent

P l a c e 1.7 g o f s i l v e r n i t r a t e in a 2 0 0 m l v o l u m e t r i c f l a s k , d i s s o l v e
i n 5 m l of w a t e r , a d d 10 m l o f 2 - p h e n o x y e t h a n o l , a n d d i l u t e to v o l u m e
with acetone. (Add 1 s m a l l d r o p of 3 0 % h y d r o g e n p e r o x i d e s o l u t i o n to
t h e f l a s k j u s t b e f o r e d i l u t i n g to m a r k . )

PROCEDURE

Preparation of Paper

W i t h a h a r d p e n c i l , r u l e a n o r i g i n l i n e 2.5 c m f r o m t h e b o t t o m e d g e
a n d m a k e a d o t a t 8 - 1 0 e v e n l y s p a c e d p o s i t i o n s , w i t h e n d d o t s 2.5 c m
f r o m the s i d e s of the p a p e r . W i t h a p e n c i l , m a r k a t e s t n u m b e r or
other identification below each dot. W a s h papers for a q u e o u s s y s t e m s
s e v e r a l t i m e s w i t h d i s t i l l e d w a t e r and d r y b e f o r e u s e . ( P a p e r s for
n o n - a q u e o u s s y s t e m s need not be washed.)

154
Paper (especially washed paper) must be dry. (All air-dried papers
s h o u l d be f u r t h e r d r i e d 30 m i n u t e s at 1 0 0 - 1 1 0 ° C b e f o r e use.) If an
a p p r e c i a b l e a m o u n t of m o i s t u r e is in the p a p e r , it c a n n o t a b s o r b
enough i m m o b i l e solvent solution, and high R f values result, as well
as f a i n t , i n d i s t i n c t c h r o m a t o g r a m s . A p p a r e n t air d r y n e s s is n o t
a d e q u a t e , d r y i n g in a f o r c e d - d r a f t o v e n is n e c e s s a r y . Once dried,
the p a p e r m a y be k e p t in o r d i n a r y d r y s t o r a g e w i t h o u t adverse
results. T h i s m o i s t u r e e f f e c t is m o s t c r i t i c a l w i t h n o n - a q u e o u s
systems.

(Note : o n c e p a p e r c h r o m a t o g r a p h y is s t a r t e d , s p o t , d e v e l o p , s p r a y
with chromogenic agent and expose to UV light without delay; do not
i n t e r r u p t overnight.)

Sample Application

Take a portion of the cleaned sample extract equivalent to 2-5 y g of

the pesticide residue to be confirmed. Evaporate under a gentle air
stream at room temperature just to dryness. (Prolonged drying will
r e s u l t in l o s s of p e s t i c i d e . ) W a s h d o w n the s i d e s of the t u b e w i t h
0.5 ml of e t h e r , e v a p o r a t e and a g a i n w a s h d o w n w i t h 0.1-0.2 m l of
ether. Evaporate, take up the residue (which is usually not visible)
with 0.03-0.04 ml ether and transfer to one of the dots on the origin
line of a c h r o m a t o g r a p h i c p a p e r u s i n g a 1 y 1 p i p e t t e . Repeat until
all the r e s i d u e is p l a c e d on one spot. L e t the spot d r y a f t e r e a c h
application to restrict its size. Wash the tube again with 0.03-0.04
m l of e t h e r and t r a n s f e r to the s a m e spot. T r a n s f e r the s t a n d a r d
s o l u t i o n s of k n o w n p e s t i c i d e s to o t h e r d o t s on the s a m e p a p e r
adjacent to the sample spots. Use additional pipettefuls to increase
the a m o u n t of p e s t i c i d e on a spot (one m i c r o l i t r e c o n t a i n i n g o n e
m i c r o g r a m of standard). For identification place several compounds
on separate dots on the same paper as the unknown.

Chromatography

A f t e r the s a m p l e s and s t a n d a r d s are s p o t t e d on the p a p e r , put 50 m l

of the m o b i l e solvent in the tank trough. Fill the dipping tank w i t h
i m m o b i l e solvent. H o l d i n g the p a p e r by the b o t t o m u s i n g a s p r i n g
clip, i m m e r s e the paper top d o w n w a r d s into the i m m o b i l e solvent just
to the o r i g i n l i n e , then i m m e d i a t e l y r e m o v e it. For an a q u e o u s
s y s t e m , h a n g the p a p e r to dry 2 - 3 m i n u t e s . W h i l e the p a p e r is
d r y i n g , clip a g l a s s rod ( w h i c h acts as a s u p p o r t for the p a p e r in
the t a n k ) to the top of the p a p e r o p p o s i t e the o r i g i n line. When
using the non-aqueous system, after dipping the paper in the i m m o b i l e
solvent, place it in the mobile solvent in the chromatography tank as
quickly as possible without allowing time for drying. (As the ether
e v a p o r a t e s it m a y c o n d e n s e m o i s t u r e on to the p a p e r and this
interferes with the ability of pesticides to dissolve in the i m m o b i l e
solvent.) Excessive h u m i d i t y and temperature tend to result in high
Rf values and faint, indistinct chromatograms. Hang the paper in the
tank so that the end near the origin dips about 1 cm into the trough
filled with m o b i l e solvent. P l a c e a g l a s s p l a t e on top of the tank
and seal with masking tape.

Visualization of Residue Spots

W h e n the m o b i l e s o l v e n t h a s r i s e n up the p a p e r to w i t h i n 2.5 cm of

the top (2 to 4 hours, depending on the solvent system used), unseal
the tank, m a r k the s o l v e n t front and h a n g the p a p e r up u n t i l it
a p p e a r s dry. U n i f o r m l y s p r a y the d r y p a p e r w i t h the c h r o m o g e n i c
r e a g e n t (do not s p r a y so h e a v i l y that it r u n s d o w n the p a p e r ) . Dry
the paper until m o s t of the solvent is removed and expose both sides
to UV light until reduced silver spots are developed. (Darkening of
the c h r o m a t o g r a m background during storage m a y be largely prevented

155
 by w a s h i n g the f i n i s h e d c h r o m a t o g r a m , a f t e r exposure to UV l i g h t , as
follows: S u s p e n d the paper from a g l a s s rod w i t h 3 or 4 c l i p s and
t h o r o u g h l y p l a y a g e n t l e stream of d i s t i l l e d w a t e r on both s i d e s of
the s h e e t . Let the suspended papers hang u n t i l d r y . P a p e r s are v e r y
f r a g i l e when w e t . )

I t i s a d v i s a b l e to e v a l u a t e c h r o m a t o g r a m s b e f o r e w a s h i n g them.
C o m p a r e l o c a t i o n , s i z e and i n t e n s i t y o f s p o t s f r o m u n k n o w n s w i t h
those from s t a n d a t r d s , identify and e s t i m a t e pesticides semi-
quantitatively. A l w a y s chromatograph knowns and unknowns on the same
paper.

REFERENCE

Official Methods of A n a l y s i s of the AOAC, 1984, 29.004, 29.007, 29.028.

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7.2 METALS

Analysis of m e t a l residues in foods is a unique challenge to the laboratory.

Organic food contaminant residues, such as pesticides, mycotoxins and others,
are isolated for analysis using techniques usually involving partitioning or
chromatography. M e t a l r e s i d u e s , h o w e v e r , c a n n o t be i s o l a t e d without
destruction of the entire organic m a t r i x . This often presents serious
problems.

Another difficulty, common to all residues, is non-uniformity of the residue

distribution through the food product. For example, the U.S. National Canners
Association have shown the impossibility of accurately determining the level of
lead in canned corned beef w i t h o u t analyzing the entire contents of the can.
Sampling difficulties may be of far more importance than analytical errors in
causing discrepancies in results. Contamination from grinding m a c h i n e r y m a y
also invalidate analyses of some m e t a l s , p a r t i c u l a r l y iron, tin and lead.
Al so, concentration of a contaminant may be distributed according to particle
size of the food. Tea "dust" has been found to contain several times the
amount of lead, iron, zinc and copper found in leaves retained on a 30 m e s h
sieve.

The destruction of the organic matrix of a food is accomplished by oxidation of

the c a r b o n a c e o u s m a t e r i a l w h i l e adding energy, usually in the form of heat.
The two most common systems are dry ashing (air oxidation at high temperatures)
and acid digestion (chemical oxidation at moderate temperatures). The ashing
system for a specific m e t a l w i l l be given in the individual method for that
metal. However, a discussion of dry ashing and acid digestion is given below.

Dry Ashing

Dry ashing is applicable to the determination of most common metals, usually

with the exception of m e r c u r y , arsenic and tin. Substances a m e n a b l e to this
method must be charred slowly, and the carbon oxidised gently and completely.
Loss of m e t a l s by v o l a t i l i s a t i o n or by c o m b i n a t i o n w i t h the m a t e r i a l of the
container must be avoided by w o r k i n g at the lowest possible t e m p e r a t u r e .
Particular care mut be exercised when large amounts of halogens are present in
either covalent or ionic form. It has been reported that losses of certain
metals (e.g. zinc, tin or antimony) occur when dry ashing is carried out in the
presence of halides. Such losses can be minimized by ensuring that an alkaline
ash remains.

Dry ashing usually requires little attention. Larger amounts of material can
be dealt with more conveniently than by acid decomposition by repeated addition
of fresh m a t e r i a l to already ashed m a t e r i a l and r e - c a l c i n i n g . It is of
particular advantage w h e n the use of sulphuric acid is o b j e c t i o n a b l e . For
e x a m p l e , for the d e t e r m i n a t i o n of lead in m a t e r i a l s containing appreciable
quantities of the alkaline earths, whose sulphates occlude lead sulphate. Dry
ashing also avoids the use of large quantities of reagents and the potentially
high blank values than can result from their use.

However, it is sometimes difficult to obtain complete extraction of the metal

being d e t e r m i n e d from certain ignited residues, and e x c e s s i v e heating m a k e s
certain metallic compounds (e.g., those of tin) insoluble.

Certain flour products give a dark melt in which carbon particles are trapped
and will not burn.

For foods having a relatively bulky ash straight ashing without an ash-aid is
usually sufficient. The technique is as follows:

Weigh accurately a suitable quantity of the well mixed sample in a tared silica
or p l a t i n u m dish. Heat first by m e a n s of a gentle f l a m e , such as that of an
Argand burner, to volatilise as m u c h as possible of the organic m a t t e r , then
transfer the dish to a temperature-controlled muffle furnace, at a temperature

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preferably not exceeding 420°C. M a k e sure the food emits only smoke during the
flaming, and does not catch fire. This is especially true of fatty foods which
will give copious smoke. Catching fire results in uncontrolled oxidation and
p o s s i b l e l o s s of the m e t a l r e s i d u e s . As an a l t e r n a t i v e to h e a t i n g w i t h an
A r g a n d b u r n e r , the s a m p l e m a y be p l a c e d in a cool m u f f l e f u r n a c e (100°C to
150°C) and the t e m p e r a t u r e r a i s e d to a b o u t 420°C and left o v e r n i g h t . It is an
a d v a n t a g e to d r a w a s l i g h t c u r r e n t of air o v e r the s a m p l e d u r i n g the e a r l y
stages of the ignition to prevent carbon and tarry m a t t e r from being deposited
inside the furnace.

M o s t s u b s t a n c e s can be ashed in a r e a s o n a b l e t i m e at a t e m p e r a t u r e as l o w as
42C°C if heated overnight, and such low temperatures are to be preferred. The
t i m e can be r e d u c e d by s p r e a d i n g the m a t e r i a l in a l a y e r o v e r the d i s h . The
t e n p e r a t u r e r e q u i r e d w i l l v a r y a c c o r d i n g to the m a t e r i a l b e i n g a s h e d . In
general the ash must not be sintered or fused, but with substances containing
m u c h ash, higher temperatures are required.

If it is suspected that all the carbon has not been r e m o v e d , cool the ash, add
a slight excess of dilute hydrochloric or diluted nitric acid (1 + 2), w a r m on
a s t e a m - b a t h , and n o t e w h e t h e r a n y c o l o u r is e x t r a c t e d or w h e t h e r o r g a n i c
m a t t e r is still present. If so, evaporate the mixture to dryness on the steam-
b a t h , and g e n t l y char the r e s i d u e o v e r a s m a l l f l a m e u n t i l all the o r g a n i c
m a t t e r h a s b e e n d e s t r o y e d , or b e t t e r , r e p e a t the i g n i t i o n at a h i g h e r
temperature or for a longer period.

If a food h a s v e r y l i t t l e a s h , or the m e t a l r e s i d u e is r e l a t i v e l y v o l a t i l e ,
t h e n an a s h - a i d m u s t be u s e d . One of the c o m m o n a s h a i d s is a 50% m a g n e s i u m
nitrate solution. To p r e p a r e a s o l u t i o n free f r o m m e t a l l i c c o n t a m i n a n t s ,
a d j u s t the pH of a 50% s o l u t i o n of m a g n e s i u m n i t r a t e to 9.5 w i t h a m m o n i u m
hydroxide, using thymol blue as indicator, and shake with successive portions
of dithizone solution in chloroform until the dithizone layer r e m a i n s green.

W h e n u s i n g a s h - a i d , add a b o u t 2 - 5 m l of a s h - a i d s o l u t i o n to the s a m p l e . The

solution and sample must be well mixed. The stirring rod m a y be wiped w i t h a
s m a l l p i e c e of f i l t e r p a p e r or c o t t o n w o o l as long as h a n d s are t h o r o u g h l y
clean and a similar quantity of paper or wool is included in the blank. Dry to
r e m o v e m o i s t u r e a n d a s h as b e f o r e . M a t e r i a l s s u c h as m i l k a n d some
confectionery can be charred without ignition by adding them a little at a time
to a dish heated over a burner or hotplate. Oil and fat must be "smoked" away
by h e a t i n g at a b o u t 350°C. C o v e r i n g the f l o o r of the m u f f l e w i t h a s h e e t of
refractory material such as silica causes most of the heating to take place by
radiation from the walls and ceiling and facilitates this.

Acid Digestion

D r y a s h i n g is p r e f e r a b l y c o n d u c t e d in a f u m e h o o d . H o w e v e r , f u m e h o o d s are
m a n d a t o r y for acid digestions. The use of fume hoods constructed entirely of
n o n - f l a m m a b l e m a t e r i a l s is r e c o m m e n d e d , as there have been instances of w o o d e n
fume hoods taking fire after a long period of use w i t h perchloric acid. On the
other hand, m a n y wooden-framed fume hoods have been in use for perchloric acid
e v a p o r a t i o n o v e r a p e r i o d of y e a r s w i t h o u t a n y u n t o w a r d c o n s e q u e n c e s . The
hazard of using wooden fume hoods depends on the adequacy of ventilation, and
the exposure of the wood to fumes from digestion vessels. In a k n o w n instance
of c o n f l a g r a t i o n , the use of p e r c h l o r i c acid w a s h i g h and the f u m e h o o d w a s
constructed of unprotected soft wood, which was subjected to considerable heat
from an electric hot-plate.

It is suggested that, when a wooden-framed fume hood is used:

a. The wood should be teak.

b. The hood should have an efficient draught to prevent condensation of

the acid vapours.

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 c. T h e fans s h o u l d be left on for a w h i l e a f t e r d i g e s t i o n s have been
completed in order to clear residual fumes.

d. The w o o d w o r k should be washed d o w n with water at frequent intervals

and should be treated occasionally w i t h liquid paraffin wax.

e. The w o o d s h o u l d be e x a m i n e d o c c a s i o n a l l y . A s p l i n t e r p l a c e d on a
h o t - p l a t e w i l l i n d i c a t e w h e t h e r or n o t the w o o d h a s b e c o m e u n d u l y
inflammable.

f. The wood should be made non-absorbent by coating with an epoxy

compound.

A satisfactory hood can be constructed from epoxy-resin-glass fibre laminates,

but before any particular material is used a small fragment should be treated
w i t h hot p e r c h l o r i c acid and its r e s i s t a n t p r o p e r t i e s c o n f i r m e d . Vitreous
tiles provide the m o s t suitable material for the bases of fume hoods. Dirt and
d u s t m u s t not be a l l o w e d to a c c u m u l a t e in f u m e h o o d s or f u m e d u c t s u s e d for
p e r c h l o r i c acid. A f u m e h o o d in w h i c h p e r c h l o r i c a c i d is u s e d s h o u l d n o t be
c o n n e c t e d to an o r d i n a r y c h i m n e y flue. R e a d i l y o x i d i s a b l e c e m e n t s , such as
those m a d e with litharge and glycerine, m u s t not be used to seal cracks in fume
hoods. Pastes of Alundum cement and sodium silicate or similar m a t e r i a l s are
satisfactory. F u m e h o o d s i n c o r p o r a t i n g w a t e r c o o l e d b a f f l e s and s c r u b b i n g
c h a m b e r s and c o n s t r u c t e d of m a t e r i a l s such as P V C are a v a i l a b l e and m a y be
safely used with perchloric acid provided the m a n u f a c t u r e r ' s r e c o m m e n d a t i o n s
are followed.

It is a b s o l u t e l y e s s e n t i a l to use r e a g e n t s and d i s t i l l e d w a t e r h a v i n g v e r y
little or no m e t a l content. One reason is that concentrated mineral acids are
generally used in amounts several times that of the sample. Some of the m o r e
c o m m o n l y used acids and reagents are n o w available in grades suitable for food
analysis. They should not be transferred from the lead-free glass bottles in
which they are supplied. However, even when these reagents are used, reagent
blank determinations will be necessary. Blanks m u s t be prepared with the same
q u a n t i t i e s of r e a g e n t s as a r e u s e d in the t e s t s . A l s o , all g l a s s or s i l i c a
w a r e m u s t be t h o r o u g h l y c l e a n e d w i t h s u l p h u r i c and n i t r i c a c i d s and then
thoroughly washed with distilled water i m m e d i a t e l y before use to ensure that it
does not yield traces of metals under the conditions of test.

Acid digestion takes place m o s t efficiently w h e n there is some form of reflux

a c t i o n d u r i n g the h e a t i n g . T h i s can be a c c o m p l i s h e d b y u s e of a r e f l u x
c o n d e n s e r (as in the o r g a n i c m e r c u r y a n a l y s i s ) , b u t u s e of a K j e l d a h l f l a s k
(with its long neck) is usually sufficient. The generation of hazardous fumes
during the digestion is always a problem. If many digestions are being done at
the s a m e t i m e , the f u m e hood m a y not be a b l e to h a n d l e the f u m e s . One m e t h o d
of fume control involves constructing a fumé collector as follows: cut a 100
mm diameter PVC pipe to fit horizontally in an available fume hood. Cap both
e n d s w i t h PVC c a p s and e p o x y s e a l e r . D r i l l a s e r i e s of h o l e s a l o n g the p i p e
(about 140 m m b e t w e e n c e n t r e s ) w h i c h w i l l l o o s e l y a c c o m m o d a t e the n e c k of a
K j e l d a h l flask. D r i l l a s i n g l e h o l e at o n e end to a l l o w a b r a s s (not c o p p e r )
or P V C tube of s i z e to be a t t a c h e d w i t h e p o x y and c o n n e c t e d to v a c u u m t u b i n g .
The vacuum tubing would go to a water aspirator attached to a faucet in a sink.
W i t h the f a u c e t o n , a s u f f i c i e n t v a c u u m is f o r m e d to d r a w the f u m e s out and
dispose of them by mixing with water in the aspirator.

D i g e s t i o n of o r g a n i c m a t t e r is u s u a l l y d o n e u s i n g s u l p h u r i c acid as the
solutional material. Nitric acid is the most c o m m o n oxidant, with perchloric
and hydrogen peroxide used for difficult foods. M e t a l or m e t a l oxide catalysts
m a y a l s o b e u s e d to e n h a n c e the o x i d a t i o n . A c a u t i o n s h o u l d be n o t e d if the
s a m p l e c o n t a i n s l a r g e a m o u n t s of a l k a l i n e e a r t h m e t a l s . Their insoluble
s u l p h a t e s w i l l o f t e n a b s o r b or o c c l u d e t r a c e m e t a l s s u c h as lead. In s u c h
e v e n t s , s u l p h u r i c acid c a n n o t be used and n i t r i c and p e r c h l o r i c a c i d s are
necessary. Extreme care m u s t be taken when perchloric acid is used, to avoid
explosions or overly rapid oxidations.

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W h e n s a m p l i n g b e v e r a g e s , it is o f t e n b e s t to t a k e a p o r t i o n o f t h e b e v e r a g e
e q u i v a l e n t to a b o u t 5 g of s o l i d s . If i n d o u b t , t h i s c a n b e d e t e r m i n e d b y
e v a p o r a t i o n of a k n o w n a m o u n t of the b e v e r a g e . A n y s a m p l e ( s u c h as a b e v e r a g e )
c o n t a i n i n g a l a r g e p r o p o r t i o n of w a t e r , m u s t b e b o i l e d d o w n to a s m a l l e r b u l k
(after a d d i n g n i t r i c a c i d ) , b e f o r e a d d i n g the s u l p h u r i c acid and a d d i t i o n a l
nitric.

W h e n a l e s s r e a c t i v e f o o d is to b e d i g e s t e d , a d d o n l y n i t r i c a c i d f i r s t a n d
h e a t u n t i l the r e a c t i o n s u b s i d e s , then add the s u l p h u r i c acid and c o n t i n u e
digestion.

If a f o o d is e x t r e m e l y r e a c t i v e to n i t r i c , a d d t h e a c i d in d i l u t e (1 + 2 ) f o r m
a n d w a r m g e n t l y u n t i l t h e i n i t i a l v i g o r o u s r e a c t i o n is o v e r . O f t e n , a t t h i s
p o i n t a s p o n g y , t a r r y c a k e is f o r m e d . C o o l the m i x t u r e , p o u r o f f the a c i d i n t o
a b e a k e r , and w a s h the t a r r y r e s i d u e w i t h a s m a l l a m o u n t of d i s t i l l e d w a t e r
( t h r e e or f o u r 1 - m l p o r t i o n s ) , a d d i n g t h e w a s h i n g s to t h e a c i d l i q u o r in t h e
b e a k e r . A d d 8 m l o f s u l p h u r i c a c i d to t h e t a r r y r e s i d u e , a g i t a t e t o d i s p e r s e
the c a k e , and i n t r o d u c e n i t r i c a c i d , d r o p b y d r o p , w i t h w a r m i n g if n e c e s s a r y ,
until vigorous oxidation ceases. R e t u r n t h e o r i g i n a l a c i d l i q u o r to t h e f l a s k ,
and b o i l u n t i l the s o l u t i o n j u s t b e g i n s to d a r k e n , t h e n c o n t i n u e the d i g e s t i o n .
A b l a n k m u s t be p r e p a r e d . W h e n s e v e r a l s a m p l e s are b e i n g d i g e s t e d , they and
t h e b l a n k m u s t e v e n t u a l l y r e c e i v e the s a m e a m o u n t s of a c i d so t h a t a n y m e t a l l i c
i m p u r i t i e s in t h e r e a g e n t s c a n c e l o u t .

R e m e m b e r t h a t for an e f f e c t i v e d i g e s t i o n a s m a l l , b u t n o t e x c e s s i v e , a m o u n t of
free n i t r i c acid m u s t be p r e s e n t t h r o u g h o u t the d i g e s t i o n . Continue adding
s m a l l a m o u n t s o f n i t r i c as n e c e s s a r y u n t i l t h e s o l u t i o n f a i l s to d a r k e n o n
p r o l o n g e d h e a t i n g to f u m i n g (5 to 10 m i n u t e s ) . T h e c r i t e r i o n of c o m p l e t i o n of
o x i d a t i o n is t h a t t h e f i n a l s o l u t i o n is f u m i n g w h e n h o t a n d c o l o u r l e s s w h e n
c o l d e r , b u t if m u c h i r o n is p r e s e n t the s o l u t i o n w i l l b e p a l e y e l l o w in c o l o u r ,
f r e q u e n t l y w i t h a g r a n u l a r p r e c i p i t a t e s o l u b l e on d i l u t i o n . A l l o w to c o o l
s o m e w h a t , d i l u t e t h e s o l u t i o n w i t h 10 m l of d i s t i l l e d w a t e r ( t h i s s h o u l d g i v e a
c o l o u r l e s s s o l u t i o n , o r a f a i n t l y y e l l o w o n e if i r o n is p r e s e n t ) , a n d b o i l
g e n t l y to f u m i n g . A l l o w t h e s o l u t i o n to c o o l a g a i n , add a f u r t h e r 5 m l of
d i s t i l l e d w a t e r , a n d b o i l g e n t l y to f u m i n g . F i n a l l y , c o o l , and d i l u t e the
s o l u t i o n w i t h 5 m l of d i s t i l l e d w a t e r . T h e d i g e s t i o n is n o w c o m p l e t e .

W h e n p e r c h l o r i c a c i d is u s e d , it c a n c o n s i d e r a b l y r e d u c e the a m o u n t of n i t r i c
a c i d r e q u i r e d a n d c o m p l e t e the o x i d a t i o n in a s h o r t e r t i m e , p r o v i d e d t h a t t h e
p r e s e n c e of c h l o r i d e is n o t d e t r i m e n t a l to the p r o c e d u r e for d e t e r m i n a t i o n of
the m e t a l s . It is m o s t i m p o r t a n t t o r e a d t h e s a f e t y p r e c a u t i o n s r e l a t i n g t o
p e r c h l o r i c acid before boiling it. S o m e i n p o r t a a t p r o p e r t i e s and u s e s of
perchloric acid are:

1. P e r c h l o r i c a c i d is c o m p l e t e l y s t a b l e u n d e r o r d i n a r y s t o r a g e
c o n d i t i o n s in c o n c e n t r a t i o n s of l e s s t h a n 8 5 % . T h e c o n c e n t r a t i o n s n o r m a l l y
s u p p l i e d are 6 0 % and 7 2 % .

2. T h e a z e o t r o p i c m i x t u r e w i t h w a t e r c o n t a i n s 72.5% of p e r c h l o r i c a c i d
a n d b o i l s a t 203°C a t 7 6 0 m m p r e s s u r e , so t h a t t h e e v a p o r a t i o n o f a n a q u e o u s
s o l u t i o n of p e r c h l o r i c a c i d can n e v e r p r o d u c e an a c i d of d a n g e r o u s l y h i g h
concentration.' If, h o w e v e r , m e t a l l i c salts are p r e s e n t , the m i x t u r e should not
b e e v a p o r a t e d to d r y n e s s o v e r an o p e n f l a m e .

3. P e r c h l o r i c acid v a p o u r and i n f l a m m a b l e gases form v i o l e n t l y explosive

m i x t u r e s , and c a r e s h o u l d b e t a k e n to a v o i d t h e i r f o r m a t i o n .

4. H o t 6 0 t o 7 2 % p e r c h l o r ic a c i d is a p o w e r f u l o x i d i s i n g a g e n t a n d
o x i d i s e s a l l f o r m s o f o r g a n i c m a t t e r , b u t it l o s e s i t s o x i d i s i n g p r o p e r t i e s
e n t i r e l y w h e n cooled and d i l u t e d w i t h w a t e r . The c o n s t i t u t i o n and p r o p e r t i e s
of a n y m a t e r i a l m u s t be t a k e n i n t o c o n s i d e r a t i o n b e f o r e t r e a t m e n t with
p e r c h l o r i c a c i d , w h e t h e r t h e m a t e r i a l b e v e g e t a b l e or a n i m a l m a t t e r or a p u r e
chemical. G e n e r a l l y , the s p e e d of the r e a c t i o n c a n b e e a s i l y c o n t r o l l e d , b u t

160
samples containing alcohol, glycerol or other substances that form esters
should not be heated with perchloric acid or perchloric acid mixtures, except
under previously well tried conditions.

5. When possible, nitric acid should be present during an oxidation by

perchloric acid. It should be added before the perchloric acid and definitely
before e v a p o r a t i o n to fumes. The effect of nitric acid is to m o d e r a t e the
reaction by oxidising the more reactive components at lower temperatures. It
should be remembered, however, that heterocyclic substances containing nitrogen
are as a rule not readily oxidised by nitric acid, and the p o s s i b i l i t y of
delayed reactions should not be overlooked.

6. Some inorganic m a t e r i a l s , such as h y p o p h o s p h i t e s and tervalent

antimony compounds, also tend to form explosive mixtures with perchloric acid
w h e n hot. A large excess of nitric acid should a l w a y s be present w h e n
oxidising inorganic salts.

7. Perchloric acid should not n o r m a l l y be used for oxidising organic

materials with which it is highly reactive but immiscible, as the reaction is
localised in the zone of contact. It should therefore not be used for
oxidising m a t e r i a l s containing m u c h fat until any excess of fat has been
removed, since local over-heating cannot be controlled and the temperature may
rise dangerously. The o x i d a t i o n of other substances such as sulphur is,
however, slower and readily controlled.

8. When perchloric acid is used as a dehydrating agent as, for example,

in the determination of silica, with subsequent filtration, the residue must be
thoroughly washed with dilute h y d r o c h l o r i c acid before the filter paper is
ignited.

9. The use of a face shield is strongly advised when perchloric acid is

being used in laboratory procedures, as well as protective clothing.

10. Perchloric acid is supplied, and should be stored, in glass-stoppered

bottles. To prevent the possibility of hazard in the event of b r e a k a g e ,
perchloric acid should be kept apart from organic c h e m i c a l s and reducing
substances, especially alcohol, glycerol and h y p o p h o s p h i t e s . Bottles of
perchloric acid should not be kept on w o o d e n shelves or b e n c h e s , since acid
trapped in the stopper joints may spill on to the benches and these may at some
future time b e c o m e heated and ignite. The bottles should stand in glass or
porcelain dishes or on c e r a m i c or other n o n - f l a m m a b l e and n o n - a b s o r b e n t
benches, preferably in such a position that the acid can easily be washed away
in the event of breakage or spillage.

11. Any acid spilled should be diluted with water. Swabs used to wipe it
up should be, if possible, of wool waste or some other non-flammable material
and not of cellulose.

12. Perchloric acid that has b e c o m e discoloured should be diluted with

water and washed away.

When either perchloric or 30% hydrogen peroxide are used, the food is normally
digested in great part using nitric/sulphuric acids before addition of the more
vigorous oxidizing agents. As example, perchloric acid or hydrogen peroxide in
small a m o u n t s (0.5 m l ) can be added after the s a m p l e has b e e n digested to a
pale yellow solution using nitric. The digestion is then continued until the
solution is colourless and w h i t e f u m e s are obtained. The w h i t e fumes are
sulphur trioxide and indicate that all nitric (or other) oxidation products are
gone. Cooling and adding 10 ml w a t e r and d i g e s t i n g to w h i t e fumes again
(twice) will eliminate all nitrogen oxides.

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In the event that s u l p h u r i c acid m a y not be u s e d , then n i t r i c acid m u s t be
added to the food (about 25 m l for e a c h 2 g), and the m i x t u r e b o i l e d for a b o u t
30 m i n . Cool and add 15 ml 60% p e r c h l o r i c . Boil g e n t l y u n t i l f u m e s a p p e a r .
Do not boil to dryness as this can be dangerous.

The a d v a n t a g e s of u s i n g c o n c e n t r a t e d h y d r o g e n p e r o x i d e are the a v o i d a n c e of

nitric acid fumes and the low reagent blank values. Oxidation is catalyzed by
various metal compounds, especially their oxides and hydroxides and by precious
metals. It is accelerated by alkaline conditions.

Hydrogen peroxide should be stored and used under such conditions that, through
spillage, it does not come into contact with combustible materials. Spilled
p e r o x i d e s o l u t i o n should be w a s h e d a w a y at once w i t h w a t e r , or w i p e d up w i t h
dilute a m m o n i a solution, as higher concentrations are produced on evaporation,
and s p o n t a n e o u s i g n i t i o n of i n f l a m m a b l e m a t e r i a l s can r e s u l t . R u b b e r or
plastic gloves should be worn when handling the reagent. In contact with the
skin it i m m e d i a t e l y p r o d u c e s " w h i t e burns". A n y b u r n s m u s t be w a s h e d
immediately with water or dilute p o t a s s i u m p e r m a n g a n a t e s o l u t i o n , o t h e r w i s e
they w i l l b e c o m e p a i n f u l and m a y cause b l i s t e r i n g . Eye p r o t e c t i o n is
e s s e n t i a l , and o x i d a t i o n p r o c e d u r e s should be c a r r i e d out b e h i n d a s a f e t y
screen in a fume hood.

For the purposes of oxidation, 50% or 30% hydrogen peroxide must always be used
in c o n j u n c t i o n w i t h a s u f f i c i e n t a m o u n t of s u l p h u r i c acid. E x p l o s i o n s h a v e
b e e n p r o d u c e d d e l i b e r a t e l y by e v a p o r a t i n g large v o l u m e s (about 50 m l ) of
peroxide with small volumes (less than 5 m l ) of sulphuric acid. Though noisy,
the explosions did not produce mechanical fracture of the glassware involved.

The f o l l o w i n g table g i v e s s o m e v o l u m e s of 50% p e r o x i d e and sulphuric acid

required for some selected food products:

Sulphuric acid fuming when Sulphuric acid not fuming

hydrogen peroxide added when hydrogen peroxide added

Minutes for Minutes for

complete complete
Food H2O2111I ^SO^ml digestion H2O21JÍI l^SO^ml digestion

Soya Bean Oil 15 to 20 10 15 to 20 10 to 15 5 20

Cabbage 5 to 10 10 5 to 10 5 5 10
Cheese 15 to 20 10 10 10 to 15 5 10 to 15
Meat 10 to 15 10 10 10 to 15 5 10

162
 LEAD
(Colorimetrie Method)

PRINCIPLE

Lead may occur naturally in some foods in small amounts, but is usually present
as a c o n t a m i n a n t . Lead can e n t e r food f r o m e n v i r o n m e n t or f r o m c o n t a c t w i t h
p r o c e s s i n g e q u i p m e n t or s t o r a g e c o n t a i n e r s h a v i n g lead-containing materials
(these could be solders, paints, ceramic glazes, etc.) Dust in the laboratory
a t m o s p h e re or s e t t l e d on s u r f a c e s m a y c o n t a i n l e a d , e s p e c i a l l y if the
l a b o r a t o r y is n e a r a r o a d c a r r y i n g a l o t of v e h i c u l a r t r a f f i c . The
determination should be carried out in diffuse light as bright sunlight tends
to decompose dithizone and dithizonates.

A c h o i c e m u s t be m a d e b e t w e e n a s h i n g and acid d i g e s t i o n . Both m e t h o d s w o r k

satisfactorily on most foods as long as certain ground rules are followed. If
the ash c o n t e n t is v e r y l o w and the s a m p l e Í3 to be a s h e d , a s h - a i d s h o u l d be
added. This is because lead is strongly retained on silicates, whether those
in the sample or the w a l l s of the silica or porcelain dish used. A l u m i n i u m and
c a l c i u m o x i d e s act as a m a t r i x t h r o u g h w h i c h the lead c a n m o r e e a s i l y r e m a i n
dispersed. F o o d s c o n t a i n i n g r e l a t i v e l y h i g h a m o u n t s of s i l i c a t e s s h o u l d be
d i g e s t e d w i t h acid. S a m p l e s h i g h in c a l c i u m s h o u l d n o t be d i g e s t e d w i t h
s u l p h u r i c acid as the c a l c i u m s u l p h a t e s o c c l u d e lead. High chloride levels
may lead to loss of lead by volatilisation. Although sodium chloride cannot be
r e s p o n s i b l e for loss in this w a y , a m m o n i u m c h l o r i d e can. (The c h l o r i d e s of
a m m o n i u m , m a g n e s i u m and c a l c i u m all c a u s e l o s s of zinc on a s h i n g . ) Samples
high in chloride m a y be digested in acid. Alternatively, add dilute sulphuric
and e v a p o r a t e s l o w l y so that c h l o r i d e is lost as h y d r o c h l o r i c a c i d at a r o u n d
100°C and then ash in the normal way.

This method involves destruction of organic matter by dry ashing and separation
of the lead from interfering substances. The lead is extracted into chloroform
as the red-coloured dithizonate, and is determined spectrophotometrically.

APPARATUS

1. Ashing dishes, porcelain or silica.

2. Muffle furnace.

3. Separatory funnels.

4. Volumetric flasks.

5. Spectrophotometer.

REAGENTS

1. A m m o n i a - c i t r a t e - dissolve 400 g citric acid in ca 800 ml water.

Add N H ^ O H u n t i l a l k a l i n e to p h e n o l red (pH 8.4). D i l u t e to 1 l i t r e .
Purify by extracting with dithizone solution.

2. Hydroxylamine hydrochloride s o l u t i o n - d i s s o l v e 20 g N H 2 0 H . H C 1
in w a t e r to m a k e 65 m l . Add a f e w d r o p s of m e t a - c r e s o l p u r p l e
indicator. Add NH^OH until the solution turns y e l l o w (pH 2.8). Add
an excess of 1% diethyldithiocarbamate solution. Extract with CHCI3
until the yellow colour is gone. Add HC1 until the solution is pink
(pH 1.2). Make to a total volume of 100 ml with water.

3. P o t a s s i u m c y a n i d e s o l u t i o n - d i s s o l v e 100 g K C N in w a t e r and
m a k e to 1 litre. Add 10 g M g O and b o i l g e n t l y for 30 m i n . Cool,
filter using vacuum and dilute to 1 litre with water. NOTE: Cyanide
m u s t be h a n d l e d w i t h g r e a t c a r e , e x p e c i a l l y by t h o s e w h o are not
sensitive to the odor.

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4. Dithizone (diphenylthiocarbazone) solution - Extract (in a
s e p a r a t o r y f u n n e l ) 1 l i t r e of CHCI3 w i t h 100 ml w a t e r c o n t a i n i n g 0.5
g N H 2 0 H . H C 1 and m a d e a l k a l i n e to p h e n o l r e d u s i n g N H ^ O H . D r a i n and
c o l l e c t C H C I 3 and i m m e d i a t e l y a d d 5 ml e t h a n o l ( a s a s t a b i l i z e r ) .
D i s s o l v e 3 0 mg d i t h i z o n e in t h i s CHCI3. Keep in refrigerator.
E x t r a c t w i t h 1% HNO3 j u s t b e f o r e u s e .

5. Ammonia - cyanide s o l u t i o n - dissolve 10 g KCN in one litre

NH40H. (See ' N O T E ' above.)

6. pH 3 . 4 b u f f e r s o l u t i o n - d i l u t e 9.1 ml HNOo to 5 0 0 ml w i t h w a t e r
and a d j u s t pH to 3 . 4 w i t h N H ^ O H . Add 25 m l 0 . 4 N KH p h t h a l a t e a n d 5
ml o f 0 . 2 N H C 1 . M a k e to 1 l i t r e w i t h w a t e r . C h e c k w i t h pH m e t e r
before using. A d j u s t pH to 3.4 w i t h NH^OH, i f n e c e s s a r y .

7. Lead standard s o l u t i o n - d i s s o l v e 3 . 1 9 7 g lead n i t r a t e ( d r i e d at

1 0 0 ° C ) i n 1% HNO3 and d i l u t e to 1 l i t r e w i t h 1% H N O 3 . This is the
stock s o l u t i o n (2 mg P b / m l ) . P r e p a r e the w o r k i n g s t a n d a r d s o l u t i o n
(2 p g P b / m l ) by d i l u t i n g 1 . 0 ml o f t h e s t o c k to 1 l i t r e u s i n g pH 3 . 4
b u f f e r solution.

PROCEDURE

W e i g h 20-40 g (to n e a r e s t 0 . 1 g) sample i n t o a s h i n g d i s h . Dry

s a m p l e s s e v e r a l h o u r s or o v e r n i g h t in 120°C f o r c e d - d r a f t oven.
( S a m p l e m u s t be a b s o l u t e l y d r y to p r e v e n t s p a t t e r i n g i n f u r n a c e . )
P l a c e samples i n f u r n a c e set at 250°C. Slowly r a i s e temperature (50°
i n c r e m e n t s ) to 3 5 0 ° C and h o l d at t h i s t e m p e r a t u r e u n t i l smoking
ceases. I n c r e a s e t e m p e r a t u r e to 5 0 0 ° C i n a b o u t 7 5 ° C increments
( s a m p l e m u s t n o t i g n i t e ) . A s h 16 hr ( o v e r n i g h t ) at 5 0 0 ° C . Remove
from f u r n a c e and a l l o w to c o o l . Ash should be w h i t e and e s s e n t i a l l y
C - f r e e . I f ash s t i l l c o n t a i n s e x c e s s c a r b o n p a r t i c l e s ( i . e . ash is
grey r a t h e r than w h i t e ) , then wet w i t h minimum amount w a t e r f o l l o w e d
b y d r o p w i s e a d d i t i o n o f n i t r i c a c i d ( 0 . 5 - 3 m l ) . D r y on a h o t p l a t e .
T r a n s f e r to the f u r a n c e at 2 5 0 ° C , s l o w l y i n c r e a s e the t e m p e r a t u r e to
5 0 0 ° C and c o n t i n u e h e a t i n g 1-2 h r . R e p e a t t h e a c i d t r e a t m e n t and
a s h i n g i f n e c e s s a r y to o b t a i n C-free r e s i d u e .

D i s s o l v e r e s i d u e in 5 ml IN n i t r i c a c i d w a r m i n g on s t e a m b a t h or
h o t p l a t e 2-3 min to a i d s o l u t i o n . F i l t e r , i f n e c e s s a r y , i n t o 100 ml
volumetric flask. Repeat w i t h two 5 ml p o r t i o n s IN a c i d , f i l t e r and
a d d w a s h i n g s to o r i g i n a l f i l t r a t e . D i l u t e to m a r k w i t h I N a c i d .
P r e p a r e d u p l i c a t e reagent b l a n k s for s t a n d a r d s and s a m p l e s , i n c l u d i n g
any a d d i t i o n a l w a t e r and a c i d , i f used for sample a s h i n g . (Note: do
n o t " a s h " n i t r i c a c i d i n f u r n a c e , s i n c e l e a d c o n t a m i n a n t w i l l be
lost. Dry n i t r i c in a s h i n g d i s h on steam bath or hot p l a t e , and then
proceed. )

Shake the f l a s k t h o r o u g h l y , then p i p e t t e 50 ml to a s e p a r a t o r y f u n n e l

c o n t a i n i n g 15 ml of the a m m o n i a - c i t r a t e s o l u t i o n . Make a l k a l i n e to
phenol red (pH 8 . 4 ) using a m m o n i a , then c o o l . Add 5 ml KCN s o l u t i o n ,
1 ml N H 2 O H . H C I s o l u t i o n a n d 5 ml d i t h i z o n e s o l u t i o n . E x t r a c t by
shaking.

I f d i t h i z o n e l a y e r remains g r e e n , there is l i t t l e or no lead p r e s e n t .

D r a i n the d i t h i z o n e in a second s e p a r a t o r y f u n n e l and c o n t i n u e from
the w a t e r wash step b e l o w .

I f the d i t h i z o n e is red or r e d d i s h - g r e e n , d r a i n i n t o a second f u n n e l

and r e - e x t r a c t w i t h 5 ml p o r t i o n s o f f r e s h d i t h i z o n e u n t i l one
remains green. Combine a l l e x t r a c t s in the same s e p a r a t o r y f u n n e l .
W a s h t h e c o m b i n e d d i t h i z o n e e x t r a c t s w i t h 5 0 ml w a t e r . D r a i n the
d i t h i z o n e into a t h i r d s e p a r a t o r y f u n n e l . D i s c a r d the w a t e r w a s h .

164
 A d d 5 0 m l p H 3.4 b u f f e r s o l u t i o n t o t h e d i t h i z o n e i n t h e t h i r d
funnel, and extract by shaking. (This w i l l strip the lead from the
d i t h i z o n e into the b u f f e r s o l u t i o n . The dithizone should turn green.
If it d o e s n o t t h e n b i s m u t h is p r e s e n t - s e e b e l o w . )

D i s c a r d the d i t h i z o n e l a y e r . (If b i s m u t h is p r e s e n t , e x t r a c t t h e
b u f f e r s o l u t i o n w i t h t w o 5 m l p o r t i o n s of f r e s h d i t h i z o n e and d i s c a r d
them.)

A d d 20 m l of the a m m o n i a - c y a n i d e s o l u t i o n to the b u f f e r . Pipette

10.0 m l f r e s h d i t h i z o n e s o l u t i o n i n t o t h e f u n n e l w i t h t h e b u f f e r , a n d
shake 1 min. (Note - release p r e s s u r e t h r o u g h the s t o p p e r , rather
than the stopcock.) P l a c e a s m a l l p l u g o f c o t t o n in t h e s t e m o f t h e
separatory funnel. D r a i n the d i t h i z o n e t h r o u g h the cotton,
d i s c a r d i n g the first 1-2 m l , into a 1 cm c u v e t t e .

D e t e r m i n e the a b s o r b a n c e at 5 1 0 n m v s f r e s h d i t h i z o n e s o l u t i o n a s t h e
reference. M a k e a p r o c e d u r a l b l a n k d e t e r m i n a t i o n by r u n n i n g an
a n a l y s i s u s i n g all r e a g e n t s , but w i t h o u t the s a m p l e .

P r e p a r e a s t a n d a r d c u r v e as f o l l o w s : P i p e t t e 0 . 0 , 5.0, 1 0 . 0 , 15.0
a n d 2 5 . 0 m l o f t h e l e a d w o r k i n g s t a n d a r d (2 y g P b / m l ) i n t o f i v e
separatory funnels. A d d p H 3.4 b u f f e r to e a c h f u n n e l to m a k e a t o t a l
v o l u m e o f 50 m l in e a c h . N e x t a d d 2 0 m l o f t h e ammonia-cyanide
s o l u t i o n to e a c h f u n n e l a n d c o n t i n u e t h e a n a l y s i s f r o m "...Pipette
10.0 m l f r e s h d i t h i z o n e . . . " . R e c o r d the a b s o r b a n c e s and p r e p a r e a
standard c u r v e p l o t t i n g a b s o r b a n c e vs lead c o n c e n t r a t i o n .

CALCULATION

= A x 100
Lead ppm s 50

where :
A = y g of lead c o r r e s p o n d i n g to t h e s a m p l e absorbance
(taken from the standard curve)

S = Weight of sample in g

INTERPRETATION

Lead recovery, when added to a l e a d - f r e e food, should be at l e a s t 80% in the

10-40 yg range.

A n y s u b s t a n c e s r e m a i n i n g u n d i s s o l v e d , e i t h e r a f t e r d i g e s t i o n , or o n m a k i n g t h e
d i g e s t a l k a l i n e , a r e l i k e l y to c a u s e l o w r e s u l t s , d u e to o c c l u s i o n of l e a d in
the p r e c i p i t a t e . T h e r e f o r e , if a s a m p l e c o n t a i n s m o r e c a l c i u m o r m a g n e s i u m
p h o s p h a t e s t h a n c a n b e h e l d in s o l u t i o n b y a m m o n i u m c i t r a t e (e.g. c a c a o
p r o d u c t s , t e a , s a r d i n e s ) the l e a d m u s t b e s e p a r a t e d f r o m t h e p h o s p h a t e b e f o r e
c o m p l e t i n g the c o l o r i m e t r i c d e t e r m i n a t i o n .

A d d i t i o n of h i g h e r a m o u n t s of c i t r a t e t h a n t h o s e r e c o m m e n d e d m a y l e a d to
i n c o m p l e t e e x t r a c t i o n of the l e a d . T i n in e x c e s s o f a b o u t 1 5 0 m g / k g m a y r e s u l t
in a m i l k y s u s p e n s i o n o f S n 0 2 i n t h e a s h s o l u t i o n o r p r e c i p i t a t e w h e n t h e
d i g e s t is m a d e a l k a l i n e . In e i t h e r c a s e t h e i n t e r f e r e n c e m u s t b e r e m o v e d .

Removal of Excess Phosphate (Society of A n a l y t i c a l Chemistry Method):

To the s o l u t i o n p r e p a r e d f r o m the a s h or d i g e s t , a d d 2 d r o p s of m e t h y l red

indicator. M a k e j u s t a l k a l i n e a n d a d d a f u r t h e r 10 m l . W a r m to 50°C a n d a d d 2
m l of 20% sodium iodide solution. R e d u c e any l i b e r a t e d i o d i n e w i t h 2 m l of
1.25% s o d i u m m e t ab i s u 1 ph i t e s o l u t i o n . ( i o d i n e o x i d i s e s d i t h i z o n e ) . C o o l and
t r a n s f e r to a s e p a r a t i n g f u n n e l a n d a d j u s t t h e v o l u m e t o 5 0 t o 75 m l (to b r i n g
t h e a c i d c o n c e n t r a t i o n to N w i t h r e s p e c t o f h y d r o c h l o r i c a c i d ) . A d d 10 m l o f

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1% d i e t h y l a m m o n i u m diethyl carbodithioate reagent in chloroform by pipette and
shake the funnel vigorously for 30 seconds. Allow the layers to separate and
transfer the chloroform layer to a 100 ml flask. Wash the aqueous layer twice
with small amounts of chloroform without mixing and add these washings to the
flask. Repeat the extraction with 10 ml of carbodithioate reagent and add the
s e c o n d e x t r a c t to the m a i n e x t r a c t . D i s c a r d the a q u e o u s l a y e r . To the
c o m b i n e d e x t r a c t s add 2.0 m l of d i l u t e d s u l p h u r i c acid and e v a p o r a t e the
chloroform. A d d 0.5 m l of p e r c h l o r i c a c i d to the r e s i d u a l s o l u t i o n and h e a t
until fumes are evolved and the fuming solution is clear and colorless. Cool
the s o l u t i o n , add 10 m l of w a t e r and 5 m l of 5 N h y d r o c h l o r i c a c i d , b o i l for 1
m i n u t e , c o o l , and then t r a n s f e r to a 100 m l v o l u m e t r i c f l a s k u s i n g 1 N n i t r i c
acid. D i l u t e to v o l u m e u s i n g 1 N n i t r i c . P i p e t t e 50 m l i n t o a s e p a r a t o r y
f u n n e l c o n t a i n i n g 15 m l of the a m m o n i a - c i t r a t e s o l u t i o n and c o n t i n u e the
analysis from there.

Removal of Large Amounts of Tin (AOAC Method):

A f t e r an a l m o s t C - f r e e a s h is o b t a i n e d , add 1 5 - 2 0 m l 4 0 % redistilled
hydrobromic acid. If nitrates were used as ash aids, cover the crucible with a
watch glass and heat on a steam bath until b r o m i n e evolution d i m i n i s h e s , then
rinse the watch glass with water and bring to the boil to complete expulsion
of b r o m i n e . (This p r o c e s s d e s t r o y s u n d e c o m p o s e d n i t r a t e s ) . Add m o r e H B r if
necessary to dissolve ash and examine che solutions for clearness. If there is
an i n s o l u b l e r e s i d u e of S n 0 2 , add 5 0 - 1 0 0 m g p u r e T i n to the s i m m e r i n g H B r
s o l u t i o n of the ash and let it d i s s o l v e . ( M e t a l l i c tin is the b e s t a g e n t to
bring ignited SnÛ2 into solution. To be effective, the ash solution m u s t be in
reduced state. F e 2 Û 3 s o m e t i m e s b e c o m e s " n o b l e " d u r i n g a s h i n g and d i s s o l v e s
w i t h difficulty, but treatment with metallic tin also brings it into solution.
Treatment with tin is necessary only with contents of badly corroded cans.)

W h e n the s o l u t i o n of a s h is free f r o m m i l k i n e s s d u e to SnC>2j add 20 m l 6 0 %

perchloric acid, oxidize the mixture with few ml H B r - B r 2 m i x t u r e , and then add
a d d i t i o n a l l y 15 m l of the r e a g e n t a l i t t l e at a t i m e , w h i l e the s o l u t i o n is
e v a p o r a t e d to i n c i p i e n t f u m e s (ca 150°C) on a h o t p l a t e . R e p e a t w i t h an
a d d i t i o n a l 10 m l p o r t i o n of the H B r - B ^ m i x t u r e if 100 m g Sn w a s u s e d to
d i s s o l v e the ash. (Hot p e r c h l o r i c h e l p s k e e p ash s a l t s in s o l u t i o n and w i t h
B r 2 holds Sn as volatile SnBr^). W h e n HBr and B r 2 are c o m p l e t e l y volatilized,
c o o l , and take up w i t h hot w a t e r (200 m l m a y be n e c e s s a r y if m u c h c h l o r a t e is
present). F i l t e r o f f a n y s m a l l a m o u n t s of d e h y d r a t e d S ÍO2 > e x t r a c t r e s i d u e
twice with 5 ml hot 20% HCl-20% citric acid reagent and hot water and isolate
lead by dithizone extraction.

REFERENCES

Manual for Food C a n n e r s and Processors, Vol. 2, U.S. National Canners

Association, 1976.

Official Methods of Analysis of the AOAC, 1984, 25.126.

166
 LEAD AND CADHIOM
(Atomic Absorption Method)

PRINCIPLE

Lead and c a d m i u m are e x t r a c t e d from a s o l u t i o n of the s a m p l e ash. Standards

are treated in the same way and both sample and standard extracts are aspirated
in the flame of an atomic absorption spectrophotometer.

APPARATUS

(Before u s e , all i t e m s of g l a s s w a r e and s i l i c a d i s h e s should be

immersed in 5% hydrochloric acid for several hours, and then rinsed
with double-distilled water.)

1. Lipped silica dishes, volume about 30 ml.

2. Graduated 5 ml and 1 ml pipettes.

3. 25-ml volumetric flasks with plastic stoppers.

4. Atomic absorption spectrophotometer, with recorder. (The

operating conditions for lead and cadmium, using an Atomspek H1170,
(Hilger and Watts) are:

Lead Cadmium

Lamp current (mA) 6 5

Wavelength (nm) 217.0 228.9
Slit width ( m ) 100 45
Burner height (mm) 9 9
Acetylene (litre m i n - ^ at 5 psig) 0.75 0.75
Air (litres m i n - * at 30 psig) 2 2
Scale expansion 1.0 0

REAGENTS

1. Water: Double-distilled, using silica-sheathed elements.

2. Nitric acid, low-in-lead quality.

3. Hydrochloric acid, low-in-lead quality.

4. Diethylammonium diethylcarbodithioate (DDCD): A 1% w/v solution

in m e t h y l i s o b u t y l k e t o n e . The s o l u t i o n m a y be kept for s e v e r a l
weeks without deterioration.

5. Methyl isobutyl ketone (MIBK) solvent saturated with water.

6. Ascorbic acid freshly prepared, 10% aqueous solution.

7. S t a n d a r d s o l u t i o n of lead and c a d m i u m : A 2% w / v s o l u t i o n of
h y d r o c h l o r i c acid c o n t a i n i n g 10 p p m of lead and 2 p p m of c a d m i u m ,
prepared from appropriate salts.

PROCEDURE

Dry ash 10g of homogenised sample at 450-500°C in a silica dish until

the ash is grey or white. Moisten with diluted nitric acid/water (1
+ 9) and b r i e f l y r e - a s h if n e c e s s a r y . T r e a t the ash w i t h 5 m l of
water followed by 5 ml of hydrochloric acid and evaporate to dryness
on a steambath. Add 1.0 ml of hydrochloric acid and 3-5 ml of water,
stirring with a glass rod, filter into a 25-ml volumetric flask and
m a k e up to the m a r k w i t h dish and rod r i n s i n g s . Prepare aqueous

167
 s t a n d a r d s in 2 5 - m l calibrated flasks using 0, 0.10, 0.20, 0.50 and
1.00 ml of the standard lead/cadmium solution. To each flask add 1.0
ml of hydrochloric acid and make up to the mark with water.

To the blank, standards and s a m p l e s add 0.5 m l of ascorbic acid

solution (to prevent stannous i n t e r f e r e n c e ) and invert to m i x the
contents. Then, without delay, add 1.50 ml of DDCD solution, stopper
and shake the flasks for 30 seconds. When the phases have separated,
tap the flasks on the bench to remove solvent globules caught on the
sides of the flasks and aspirate the organic layers into the
spectrophotometer first for cadmium and then for lead. Aspirate MIBK
solvent before and after each sample or standard in order to
e s t a b l i s h the baseline. C o n s t r u c t c a l i b r a t i o n g r a p h s of the
responses to the standards.

CALCULATIONS

C o m p a r e the sample absorbance to the appropriate standards (after

subtracting the blank) and calculate the amounts of lead and cadmium
present.

INTERPRETATION

E x t r a c t i o n of lead and c a d m i u m from 0.5 N HC1 by DDCD in M I B K is a convenient

a l t e r n a t i v e to the c o l o r i m e t r i c p r o c e d u r e . If an a t o m i c a b s o r p t i o n
spectrophotometer is not available, the lead and cadmium may be extracted with
D D C D / M I B K , using a s o m e w h a t larger v o l u m e and r e p e a t i n g the e x t r a c t i o n w i t h
fresh DDCD to ensure c o m p l e t e extraction. The organic phase m u s t be
e v a p o r a t e d , digested in the m i n i m u m a m o u n t of acid, and the d e t e r m i n a t i o n
c o m p l e t e d with dithizone. C a l c i u m , m a g n e s i u m and p h o s p h a t e are u n l i k e l y to
cause difficulties of precipitation in 0.5 N hydrochloric acid.

Of the e l e m e n t s likely to be present in food d i g e s t s , only iron, copper and

zinc could interfere in this determination. These elements are usually masked
by forming their cyanide complexes at pH 8.5. In this method it has been found
necessary to eliminate completely the interference from iron by reducing any
ferric to ferrous with ascorbic acid prior to the extraction step.

E x p e r i m e n t s have indicated that the lead-DDCD c o m p l e x is stable for several

h o u r s , w h i l e that of c a d m i u m is subject to slight d e c o m p o s i t i o n (judged by a
decrease of 10 to 20 percent in the i n s t r u m e n t response). For this reason
cadmium is best determined first and the lead determination completed within
three hours of extraction.

REFERENCES

Roschnik, R.K., 1973. Analyst 98.» 596-604.

Official Methods of Analysis of the AOAC, 1984, 25.061-.065.

168
 ARSENIC
(Colorimetrie Method)

PRINCIPLE

The s a m p l e is d i g e s t e d using acid. Dry a s h i n g should not be used b e c a u s e of

the v o l a t i l i t y of a r s e n i c . The a r s e n i c is c o n v e r t e d to A s 2 0 3 d u r i n g the
digestion process. This is then reduced to a r s i n e (AsHj) by the h y d r o g e n
g e n e r a t e d by acid and zinc. The a r s i n e is r e a c t e d w i t h a s o l u t i o n of s i l v e r
d i e t h y l d i t h i o c a r b a m a t e to form a red c o m p l e x . T h i s is m e a s u r e d u s i n g a
spectrophotometer at 522 m m .

APPARATUS

1. Acid digestion flasks.

2. Volumetric flask, 250 ml.

3. Spectrophotometer.

4. Generator apparatus - as shown.

Use 5 0 - 6 0 m l w i d e - m o u t h b o t t l e s of u n i f o r m c a p a c i t y and d e s i g n as
generators and fit each by means of a perforated stopper with a glass
tube which broadens into a section 10 x 60 -70 m m . Place a small wad
of glass wool in the constricted bottom end of the tube and add 3.5-4
g sand, taking care to h a v e s a m e a m o u n t in each tube. M o i s t e n the
sand w i t h 10% lead a c e t a t e s o l u t i o n and r e m o v e e x c e s s by light
suction. Clean the sand when necessary by treatment (do not remove
sand from t u b e ) w i t h n i t r i c f o l l o w e d by w a t e r r i n s e and s u c t i o n .
Treat with lead acetate solution. If sand has dried through disuse,
c l e a n and r e m o i s t e n as d i r e c t e d . C o n n e c t the tube by m e a n s of a
r u b b e r s t o p p e r , glass tube, and r u b b e r s l e e v e to a bent c a p i l l a r y
tube (7 m m o u t s i d e d i a m e t e r , 2 m m i n s i d e d i a m e t e r ) t a p e r e d at the
l o w e r end such that it slides into the r u b b e r s l e e v e to form a g a s -
t i g h t seal and can also be p l a c e d in the n e c k of a 25 m l v o l u m e t r i c
flask. The upper end of the tube e x p a n d s into a trap of about 10 m l
c a p a c i t y tapered by a B19 j o i n t , about a q u a r t e r full w i t h s m a l l
glass beads. Clean the trap between determinations without removing
beads by flushing with water followed by nitric acid, soaking for 30
m i n u t e s and u n t i l the n i t r i c acid b e c o m e s c o l o u r l e s s . R e m o v e all
traces of acid with water, rinse with acetone and dry with a current
of air by applying suction to the tip of the trap.

169
REAGENTS

1. Nitric acid, concentrated.

2. Sulphuric acid, concentrated.

3. Ammonium oxalate solution, saturated.

4. Sea s a n d - c l e a n sand by treating with concentrated n i t r i c acid

and then r i n s e w i t h d i s t i l l e d w a t e r to r e m o v e a l l t r a c e s of a c i d ( a t
least 5 washings).

5. Lead acetate solution - 10% Pb(OAc)2.3H20 in water.

6. Hydrochloric acid, concentrated.

7. P o t a s s i u m i o d i d e s o l u t i o n - 15% K I in water - keep in the dark

and d i s c a r d when s o l u t i o n turns y e l l o w .

8. Stannous chloride solution - 15% SnCl2.2H20 in HC1.

9. Silver diethylcarbodithioate reagent - 0.5 g dissolved in

p y r i d i n e and made to 1 0 0 ml w i t h p y r i d i n e . S t o r e i n an amber b o t t l e .
(The r e a g e n t i s s t a b l e f o r s e v e r a l m o n t h s at room t e m p e r a t u r e . )

10. Zinc, granulated or small pellets.

11. Arsenious oxide (As2Û3) standard solution - dissolve 1.32 g

A s 2 0 3 ( e q u i v a l e n t to 1 . 0 0 g A s ) i n 2 5 ml 2 0 % N a O H s o l u t i o n . Dilute
to 1 l i t r e w i t h w a t e r . T h i s i s t h e s t o c k s o l u t i o n (1 m g / m l ) . The
w o r k i n g s o l u t i o n is made by d i l u t i n g 1 ml o f the s t o c k s o l u t i o n to 1
l i t r e w i t h w a t e r (1 p g / m l ) .

PROCEDURE

W e i g h 25 g s a m p l e i n t o d i g e s t i o n f l a s k . Add 4 0 ml e a c h o f n i t r i c and
sulphuric acids. Digest with heat. Add a s m a l l a m o u n t o f n i t r i c
w h e n e v e r the d i g e s t t u r n s b r o w n or d a r k e n s . Continue digestion until
organic matter is gone. The final solution should be nearly
colorless. S t o p d i g e s t i o n when w h i t e S O 3 fumes a p p e a r .

Cool and add 75 ml w a t e r . N e x t add 25 ml s a t u r a t e d ammonium o x a l a t e

(to c o m p l e t e r e m o v a l of n i t r o g e n o x i d e s ) . H e a t to e v a p o r a t e the
water and c o n t i n u e h e a t i n g u n t i l w h i t e S O 3 f u m e s are f o r m e d a g a i n .
Cool and t r a n s f e r to a 2 5 0 ml v o l u m e t r i c w i t h w a t e r . Make to v o l u m e
with water.

P i p e t t e 5 ml to a g e n e r a t o r b o t t l e a n d add 3 0 ml w a t e r . Add ( w h i l e
s w i r l i n g ) 5 ml H C 1 , 2 ml K I s o l u t i o n and 8 d r o p s of the SnCl2
solution. L e t s t a n d 15 m i n . Add 4 . 0 ml o f the s i l v e r c o l o u r r e a g e n t
to t h e t r a p o f t h e g e n e r a t o r a p p a r a t u s . Add 4 g z i n c to t h e b o t t l e
and q u i c k l y a t t a c h t h e s c r u b b e r t u b e a n d t r a p a s s e m b l y , . Let react
for 30 min. T r a n s f e r t h e s i l v e r r e a g e n t f r o m t h e t r a p to a c u v e t t e
a n d d e t e r m i n e t h e a b s o r b a n c e a t 5 2 2 nm v s w a t e r . (Note - cuvette
s h o u l d be c a p p e d . )

P r e p a r e a s t a n d a r d c u r v e as f o l l o w s : Pipet 0.0, 1.0, 3.0, 6.0, 10.0

a n d 1 5 . 0 ml o f t h e w o r k i n g s t a n d a r d (1 y g A s / m l ) i n t o s i x g e n e r a t o r
bottles. M a k e e a c h to a t o t a l v o l u m e o f 35 ml w i t h w a t e r . Proceed
as a b o v e . Prepare a standard curve, p l o t t i n g absorbance vs y g As.

170
 CALCULATIONS

Read y g As corresponding to sample absorbance, from standard curve.

Jig As x 250
Arsenic ppm = g sampie x 5

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 25.041-.044.

171
 MERCURY
(Organic Residues)

PRINCIPLE

Organic m e r c u r y is c o m m o n l y found in the flesh of fish. It is passed up the

food chain from prey to predator and is c u m u l a t i v e l y stored by each host.
Therefore, the larger predator fish at the top of the food chain have the most
likelihood of high residues of organic mercury. This method acid digests the
fish using heat, oxidizing conditions and a catalyst in order to break the
m e r c u r y from its organic bond. The r e s u l t a n t m e r c u r i c ion is reduced to
elemental mercury with stannous chloride. The mercury is then aerated from the
solution as a vapor into a .chamber through w h i c h light at the m e r c u r y
wavelength is passing. The resultant light absorbance is measured.

APPARATUS

1. Digestion flask and reflux condenser.

2. Volumetric flasks and pipettes.

3. M e r c u r y A n a l y z e r , C o l e m a n or equivalent (see d i a g r a m below),

Many atomic absorption s p e c t r o p h o t o m e t e r s have a cold v a p o u r
accessory tor mercury analysis.

16 G a g e Teflon Tubing

REAGENTS

1. Vanadium pentoxide (V2O5) powder.

2. Sulphuric - Nitric acid mixture (1 + 1).

3. Hydrogen peroxide (l^C^), 30%.

4. Diluting solution - mix 58 ml HNO3 and 67 ml H 2 S 0 ^ w i t h ca 500

ml water. Dilute to 1 litre.

5. R e d u c i n g s o l u t i o n - M ix 50 m l H 2 S 0 4 w i t h 3 0 0 m l w a t e r .
Cool and dissolve 15 g NaCl, 15 g h y d r o x y l a m i n e HC1 and 25 g S n C L 2 .

172
 PROCEDURE

W e i g h 5 g prepared fish into digestion flask. Add some boiling

chips, 10-20 m g V 2 0 5 and 20 m l H 2 S 0 4 - H N 0 3 acid m i x t u r e . Connect
flask to cond enser and heat to produce a gentle boil in about 5 min.
Continue heating w i t h a strong boil for about 20 min. The solution
should be clear. Ignore any fat globules floating on top.

Remove heat and wash down condenser with ca 15 ml water. Add 2 drops
H 2 0 2 and wash into flask with another 15 ml w a t e r . Cool flask to
room temperature and disconnect condenser. Transfer digest into 100
ml volumetric flask using water to wash. Make to volume with water.

Pipette 20 ml into aeration bottle and add 80 ml diluting solution,

Add 20 ml reducing solution and i m m e d i a t e l y aerate. Read d i r e c t l y
from Analyzer meter. (Analyzer should be calibrated at 0.5 yg Hg).

CALCULATION

Read the micrograms mercury directly from the Analyzer Meter. This
is also the ppm as 1 g sample equivalent is in the final solution.

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 25.134-.137.

173
 MERCURY
(Inorganic R e s i d u e s )

PRINCIPLE

The sample is digested in nitric and sulphuric acids under reflux. The m e r c u r y
is i s o l a t e d by d i t h i z o n e e x t r a c t i o n , c o p p e r is r e m o v e d and the c o l o u r of the
m e r c u r y dithizonate compared with standards.

APPARATUS

1. R e f l u x a p p a r a t u s (see D i a g r a m ) . "A" is a b o u t 200 m l c a p a c i t y

and about 50 m m outside diameter. Friedricks condenser or equivalent
s h o u l d be used. "B" h a s a c a p a c i t y of a b o u t 75 m l .

2. Spectrophotometer.

REAGENTS

1. M e r c u r y standard: (Add 8 ml hydrochloric acid per litre to all

s t a n d a r d s b e f o r e d i l u t i n g to v o l u m e . ) D i s s o l v e 0.1354g m e r c u r i c
chloride in 100 ml water (equivalent to lgHg/L). Dilute to a working
standard of 2 Jjg/ml.

2. Chloroform: D i s t i l on a h o t w a t e r b a t h , c o l l e c t i n g the
distillate in 10 m l absolute alcohol per litre of distillate. Shake
the receiver intermittently during distillation.

3. Dithizone: 100 mg/L in redistilled chloroform. Dilute as

re quired.

4. Sodium thiosulphate solution: 1.5%. Prepare on day of use.

5. Sodium hypochlorite solution: P r e f e r a b l y w i t h 5% a v a i l a b l e

chlorine content. Check by titration. S t o r e in the r e f r i g e r a t o r
w h e n n o t in use. Certain p r e p a r a t i o n s i n t e n d e d for h o u s e h o l d u s e
contain traces of mercury. If these are used, check the blank value
and discard the reagent if 0.1 p g / m l Hg or m o r e is present.

6. Dilute acetic acid, 30% V/V.

7. Hydroxylamine hydrochloride solution: 20% w/v. Extract with

dilute dithizone until the chloroform layer remains green, remove the
excess dithizone with chloroform and filter.

PROCEDURE

W e i g h an a m o u n t of s a m p l e c o n t a i n i n g less than 10 g of d r y m a t t e r .
Digestion m u s t be almost complete, otherwise residual organic m a t t e r
m a y c o m b i n e w i t h m e r c u r y and p r e v e n t or h i n d e r e x t r a c t i o n w i t h
dithizone. Oxidizing materials in the digest m u s t also be destroyed
otherwise the d i t h i z o n e is d e c o m p o s e d and m e r c u r y is not
quantitatively extracted. C a r e f u l h e a t i n g of the d i g e s t d u r i n g
sample preparation is necessary because of the volatility of m e r c u r y
compounds. The a c i d i t y of the f i n a l d i g e s t ( a f t e r partial
n e u t r a l i z a t i o n w i t h a m m o n i a ) b e f o r e e x t r a c t i o n s h o u l d be a b o u t 1 N
a n d n o t m o r e t h a n 1.2 N. Do n o t u s e s i l i c o n e g r e a s e in t h e
stopcocks.

Fresh Fruits and Vegetables and Beverages

Place the weighed sample in the digestion flask w i t h 6 glass beads,

c o n n e c t the a s s e m b l y and a d d , t h r o u g h the d r o p p i n g f u n n e l , 20 m l
nitric acid. P a s s w a t e r t h r o u g h the c o n d e n s e r as r a p i d l y as

174
practicable. With the stopcock to the Soxhlet portion open and that
of the s e p a r a t o r c l o s e d , h e a t the f l a s k g e n t l y . The original
reaction m u s t not proceed with such violence that evolved N 0 2 carries
d i g e s t v a p o u r s t h r o u g h the c o n d e n s e r and c a u s e s l o s s of m e r c u r y .
After the initial reaction is complete, apply heat so that the digest
just r e f l u x e s . If the m i x t u r e d a r k e n s , add n i t r i c a c i d d r o p w i s e
t h r o u g h the f u n n e l as n e e d e d . N o t e the t o t a l v o l u m e of n i t r i c acid
used so that a blank can be prepared identically. Continue refluxing
h a l f an h o u r or u n t i l the d i g e s t d o e s n o t c h a n g e c o n s i s t e n c y , t h e n
cool.

Slowly add 20 ml of cold nitric-sulphuric acids m i x t u r e (1+1) (or 10

m l if the d r y w e i g h t of s a m p l e is l e s s t h a n 5g). Heat w i t h a small
flame, adding nitric acid dropwise as needed to dispel darkening of
the d i g e s t . C o n t i n u e h e a t i n g u n t i l f i b r o u s m a t e r i a l (fruit s k i n ,
c e l l u l o s e , etc.) is a p p a r e n t l y d i g e s t e d . T u r n the s t o p c o c k of the
S o x h l e t u n i t so that w a t e r and acid t h a t d i s t i l s is t r a p p e d and
continue heating. L e t the d i g e s t b e c o m e d a r k b r o w n (not b l a c k )
b e f o r e a d d i n g f u r t h e r i n c r e m e n t s of n i t r i c acid. F a t s and w a x e s
c a n n o t be t o t a l l y d i g e s t e d by the hot a c i d s u n d e r r e f l u x , so no
attempt should be m a d e to effect complete digestion. W h e n all except
fat and wax is in solution, let the digest cool, and cautiously drain
the w a t e r and a c i d s i n t o the m a i n d i g e s t . C o o l and p o u r 2 x 25 m l
w a t e r t h r o u g h the c o n d e n s e r and m o d i f i e d S o x h l e t . R e m o v e the
r e a c t i o n f l a s k , c h i l l u n d e r w a t e r or by s u r r o u n d i n g w i t h ice to
s o l i d i f y fats and w a x e s and f i l t e r off the i n s o l u b l e m a t t e r on a
small pledget of glass wool. Rinse the reaction flask and filter pad
successively with two 10 ml portions of water. R e m o v e the modified
S o x h l e t u n i t and w a s h it and the f l a s k w i t h h o t w a t e r to r e m o v e
insoluble material. Pour hot water through the condenser to remove
volatile fats and oils. Discard all the washings.

C o n n e c t the flask c o n t a i n i n g the f i l t e r e d s a m p l e s o l u t i o n to.the

a s s e m b l e d a p p a r a t u s , h e a t and c o l l e c t w a t e r and a c i d s in the trap.
C o m p l e t e the d i g e s t i o n , u s i n g s m a l l a d d i t i o n s of n i t r i c acid as
needed. In the f i n a l s t a g e of d i g e s t i o n , a d j u s t the h e a t u n t i l the
digest s i m m e r s and the acid vapours do not rise above the curved half
of the c o n d e n s e r . C o n t i n u e h e a t i n g for 15 m i n u t e s a f t e r the last
addition of nitric acid. The digest should n o w be colourless or pale
yellow. Let the d i g e s t c o o l , d r a i n the t r a p p e d l i q u i d s c a r e f u l l y
i n t o t h e r e a c t i o n f l a s k and a d d 2 x 50 m l w a t e r t h r o u g h t h e
condenser. R e f l u x s o l u t i o n u n t i l all N O 2 is e x p e l l e d f r o m the
apparatus. Add 5 ml 4 0 % urea s o l u t i o n and r e f l u x for 15 m i n u t e s .
The digest should be colourless or pale yellow.

Dried Fruit, Cereal, Seeds and Grains

Dilute the sample w i t h 50 m l of water before adding the nitric acid,

and proceed as above.

Meats, Fish and Similar Material

Conduct the initial digestion carefully to avoid foam (caused by the

h i g h fat and p r o t e i n c o n t e n t of t h e s e m a t e r i a l s ) f r o m r e a c h i n g the
condenser. Add 20 ml nitric to the sample, swirl the flask and leave
to stand h a l f an h o u r b e f o r e h e a t i n g . Add 25 m l w a t e r and h e a t
c a u t i o u s l y w i t h a s m a l l f l a m e m o v e d o v e r the u n d e r s u r f a c e of the
f l a s k u n t i l t h e i n i t i a l r e a c t i o n and e x c e s s i v e f o a m i n g ceases.
Complete the digestion as for fresh fruit.

T r e a t m e n t of D i g e s t : T i t r a t e 1 m l of the p r e p a r e d s a m p l e s o l u t i o n
with standard alkali. Add to the w h o l e d i g e s t the a m o u n t of
concentrated a m m o n i a solution to reduce the acidity to IN. Swirl the
f l a s k d u r i n g the a d d i t i o n of the a m m o n i a to a v o i d l o c a l e x c e s s .

175
S o l u t i o n m u s t n o t b e a l l o w e d to b e c o m e a l k a l i n e as a m m o n i a - m e r c u r y
c o m p l e x e s w o u l d be f o r m e d . T r a n s f e r the s o l u t i o n to a 5 0 0 m l
s e p a r a t o r . A d d 10 m l 4 m g / L d i t h i z o n e s o l u t i o n and s h a k e v i g o r o u s l y
one m i n u t e . If t h e d i t h i z o n e l e v e l r e m a i n s g r e e n , l e s s t h a n 5 p g of
m e r c u r y is p r e s e n t . L e t the l a y e r s s e p a r a t e and d r a i n t h e c h l o r o f o r m
l a y e r q u i c k l y i n t o a s e c o n d s e p a r a t o r c o n t a i n i n g 25 m l 0.1N H C 1 a n d 5
m l of h y d r o x y a m m o n i u m c h l o r i d e s o l u t i o n . A s m a l l a m o u n t o f o x i d i z i n g
m a t e r i a l m a y still be p r e s e n t , w h i c h could d e s t r o y the d i t h i z o n e on
l o n g c o n t a c t ( p r e v e n t i n g the e x t r a c t i o n of m e r c u r y ) so the c h l o r o f o r m
l a y e r m u s t b e r u n o f f as s o o n as it s e p a r a t e s . R e p e a t the e x t r a c t i o n
w i t h 2 x 5 m l of d i t h i z o n e s o l u t i o n , t r a n s f e r r i n g the c h l o r o f o r m
l a y e r to the s e c o n d s e p a r a t o r e a c h t i m e . If t h e f i r s t e x t r a c t i o n
i n d i c a t e s m o r e t h a n 5 m i c r o g r a m s of H g u s e m o r e d i t h i z o n e a c c o r d i n g
to t h e f o l l o w i n g t a b l e , u n t i l ( a f t e r s h a k i n g v i g o r o u s l y for 1 m i n u t e )
the c h l o r o f o r m l a y e r r e m a i n s g r e e n . D r a i n the c h l o r o f o r m l a y e r i n t o
t h e s e c o n d s e p a r a t o r c o n t a i n i n g 0.1N H C 1 a n d a g a i n e x t r a c t the s a m p l e
w i t h 2 x 10 m l of m g / L d i t h i z o n e s o l u t i o n , d r a i n i n g e a c h s u c c e s s i v e
e x t r a c t into the second s e p a r a t o r .

Mercury Range Dithizone V o l u m e of

(micrograms) Concentration (mg/L) Dithizone (ml)

0 - 1 0 6 5
10 - 50 10 25
50 - 100 10 40

S h a k e the c o n t e n t s of the s e c o n d s e p a r a t o r v i g o r o u s l y 1 m i n u t e and

d r a i n the c h l o r o f o r m l a y e r i n t o a t h i r d s e p a r a t o r c o n t a i n i n g 50 m l of
0.1N H C 1 . S h a k i n g t h e d i t h i z o n e e x t r a c t w i t h d i l u t e a c i d in t h e
s e c o n d s e p a r a t o r r e m o v e s e n t r a i n e d o r g a n i c m a t t e r . W i t h m e a t and
o t h e r m a t e r i a l s of h i g h p r o t e i n c o n t e n t the a q u e o u s l a y e r is u s u a l l y
l i g h t y e l l o w b e c a u s e of n i t r a t e d organic compounds. Small amounts
are carried into the third separator w h e r e they are d e s t r o y e d by
chlorine. E x t r a c t the s o l u t i o n in the s e c o n d s e p a r a t o r w i t h 1 - 2 m l
c h l o r o f o r m a n d t r a n s f e r t h e o r g a n i c l a y e r to t h e t h i r d s e p a r a t o r .

To the third s e p a r a t o r add 3 m l 30% a c e t i c acid and the a p p r o p r i a t e

v o l u m e and c o n c e n t r a t i o n of d i t h i z o n e s o l u t i o n i n d i c a t e d by the
table. P r o c e e d as for s t a n d a r d s , m e a s u r i n g the a b s o r b a n c e A at 4 9 0
n m and d e t e r m i n i n g the n u m b e r of m i c r o g r a m s of m e r c u r y p r e s e n t f r o m
the standard c u r v e .

T o t h e c o n t e n t s o f t h e t h i r d s e p a r a t o r a d d 2 m l o f 1.5% s o d i u m
t h i o s u l p h a t e s o l u t i o n , s h a k e v i g o r o u s l y 1 m i n u t e and l e a v e to
separate. D r a i n o f f t h e c h l o r o f o r m as c o m p l e t e l y a s p o s s i b l e a n d
discard. ( C o p p e r , if p r e s e n t , is r e m o v e d a s i t s d i t h i z o n a t e ) .
E x t r a c t a g a i n w i t h 1 - 2 m l o f c h l o r o f o r m , d r a i n c a r e f u l l y and d i s c a r d .
A d d 3.5 m l o f 5 % s o d i u m h y p o c h l o r i t e s o l u t i o n ( o r e n o u g h o f a
d i f f e r e n t c o n c e n t r a t i o n to p r o v i d e 1 7 5 m g a v a i l a b l e c h l o r i n e ) to
d e c o m p o s e the m e r c u r y t h i o s u l p h a t e c o m p l e x and o x i d i s e the e x c e s s
t h i o s u l p h a t e , and shake v i g o r o u s l y 1 minute. Add 5 ml of
h y d r o x y l a m i n e h y d r o c h l o r i d e s o l u t i o n b y p i p e t t e , t a k i n g c a r e to w e t
b o t h the s t o p p e r and the n e c k of the s e p a r a t o r . S h a k e v i g o r o u s l y 1
m i n u t e . B l o w any r e m a i n i n g c h l o r i n e gas out of the s e p a r a t o r by a
g e n t l e jet of a i r . S t o p p e r the s e p a r a t o r and s h a k e v i g o r o u s l y 1
minute. It is i m p e r a t i v e t h a t a l l h y p o c h l o r i t e is r e d u c e d . Trace
a m o u n t s r e m a i n i n g w o u l d o x i d i z e t h e d i t h i z o n e a d d e d l a t e r , to a
y e l l o w o x i d a t i o n p r o d u c t w h i c h w o u l d b e m e a s u r e d as m e r c u r y . Extract
the s o l u t i o n w i t h 2 - 3 m l of c h l o r o f o r m , d r a i n o f f t h e o r g a n i c l a y e r
c a r e f u l l y and d i s c a r d . T h e f i n a l a q u e o u s s o l u t i o n so o b t a i n e d s h o u l d
be colourless.

176
 Prepare a standard curve in the required range 1-10, 1-50 or 1-100
m i c r o g r a m s of m e r c u r y using a blank, the standard at the top of the
range and 4 intermediate standards. Add each standard and the blank
to s e p a r a t o r s e a c h c o n t a i n i n g 50 m l 0.1N H C 1 . Add 5 m l
hydroxyammonium hydrochloride reagent and 5 ml chloroform and shake
v i g o r o u s l y one m i n u t e . Let the layers s e p a r a t e , drain off the
chloroform, being careful to remove all droplets of it, and discard.
Add 3 m l 30% acetic acid and the a p p r o p r i a t e v o l u m e of d i t h i z o n e
solution, shake v i g o r o u s l y one m i n u t e and let the layers separate.
The acetic acid aids in stabilizing the mercuric dithizonate. Insert
a cotton pledget into the stem of the separator and collect the
dithizone extract (discarding the first m l ) in a 1 cm cell. Read
i m m e d i a t e l y at 4 9 0 n m . B o t h d i l u t e d i t h i z o n e and m e r c u r i c
d i t h i z o n a t e are s o m e w h a t unstable. Plot a b s o r b a n c e (A) against
micrograms of mercury.

CALCULATIONS

Read the m i c r o g r a m s of m e r c u r y for the s a m p l e a b s o r b a n c e , from the

standard curve. Divide by the grams of sample taken to obtain ppm.

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 25.138-.145.

L-K*»»^

177
 TIH
(Colorimetric Method)

PRINCIPLE

The s a m p l e ( c o n t a i n i n g up to 1 mg of tin) is d i g e s t e d in n i t r i c and s u l p h u r i c

a c i d s and the d i g e s t d i l u t e d to a k n o w n v o l u m e . An a l i q u o t is n e u t r a l i z e d
u s i n g 2 , 4 - d i n i t r o p h e n o 1 as i n d i c a t o r , the solution acidified with a standard
a m o u n t of acid and the c o l o u r w i t h q u e r c e t i n d e v e l o p e d in 50% e t h a n o l .
Comparison of the colour is made with standard solutions developed at the same
time.

APPÂRARUS

1. Spectrophotometer, preferably with 2 and 4-cm cells.

2. D i g e s t i o n f l a s k s of the K j e l d a h l type. If s i l i c a f l a s k s and a

M e k e r b u r n e r are u s e d , the d i g e s t i o n t i m e can be g r e a t l y r e d u c e d
(e.g. 5 g of m e a t can be d i g e s t e d in 15 m i n u t e s ) .

REAGEHTS

1. Nitric acid (SG 1.42).

2. Sulphuric acid (SG 1.84).

3. 2,4-Dinitrophenol, 0.1% in 50 v/v aqueous alcohol.

4. Sodium carbonate solution, 10% w/v in water.

5. Hydrochloric acid, 2.5N.

6. Thiourea, saturated aqueous solution.

7. Quercetin, 0.2 w/v in ethanol.

8. Ethanol, 95%.

PROCEDURES

Transfer a known weight of the comminuted sample, containing not more

t h a n 1 0 0 0 m i c r o g r a m s of tin, to a d i g e s t i o n f l a s k ( p r e f e r a b l y
silica). Add 10 ml of c o n c e n t r a t e d n i t r i c acid a n d , after 10
minutes, add 5 ml of concentrated sulphuric acid. Boil the mixture
v i g o r o u s l y and, w h e n c h a r r i n g c o m m e n c e s , add s m a l l a m o u n t s of
c o n c e n t r a t e d n i t r i c acid. C o n t i n u e the o x i d a t i o n in this m a n n e r
u n t i l c h a r r i n g c e a s e s and then boil u n t i l w h i t e f u m e s of s u l p h u r
trioxide appear. Cool the m i x t u r e , (if s i l i c a f l a s k s are used this
is effected instantaneously by immersion in cold water).

Add 20 ml of water and transfer the digested solution quantitatively

to a 50 m l v o l u m e t r i c flask. D i l u t e to v o l u m e w i t h w a t e r and m i x .
Pipette 2 ml of this solution into another 50 ml volumetric flask and
add 0.2 ml of 2,4-dinitrophenol solution. Add 10% sodium carbonate
s o l u t i o n d r o p w i s e w i t h c o n t i n u a l a g i t a t i o n of the flask u n t i l the
first a p p e a r a n c e of a y e l l o w c o l o u r . D i s c h a r g e this c o l o u r by the
d r o p w i s e a d d i t i o n of 2.5N h y d r o c h l o r i c acid and i m m e d i a t e l y add a
further 5.0 ml of the acid. Add 3 ml of thiourea solution and 5.0 ml
of q u e r c e t i n r e a g e n t , f o l l o w e d by 25 m l of e t h a n o l ; d i l u t e to 50 m l
with water and mix. After 30 minutes, measure the absorbance in a 4 -
cm cell at 4 3 7 nm a g a i n s t a r e a g e n t b l a n k s o l u t i o n p r o d u c e d in the
same manner as the sample (2-cm cells are preferable if the tin level
is high).

178
 Prepare a standard curve as follows. Prepare a standard tin solution
by dissolving 0.0500 g of pure tin in 50 ml of boiling concentrated
sulphuric acid. Cool and c a u t i o u s l y add this to 120 ml of w a t e r ,
with further cooling and transfer to a 200 ml calibrated flask. Make
up to the m a r k w i t h 25% v/v sulphuric acid (1 ml = 25 m i c r o g r a m s of
tin). To a s e r i e s of d i g e s t i o n f l a s k s c o n t a i n i n g 5 m l of
concentrated sulphuric acid and 10 ml of c o n c e n t r a t e d nitric acid,
add 0, 1.0, 2.0, 3.0 and 4.0 ml of the standard tin solution. Boil
until the nitric acid is expelled and white fumes apppear. Cool, add
20 ml of water and proceed w i t h the colour d e v e l o p m e n t exactly as
above. Construct the standard curve of absorbance against micrograms
of tin.

CALCULATIOUS

Tin ppm = -A-

W

where :
A = pg of tin on standard curve corresponding to
sample absorbance

W = g sample

REFERENCE

Kirk and Pocklington, 1969. Analyst 94, 71-74.

179
 COPPER
(Colouriaetric Method)

PRINCIPLE

S o d i u m d i e t h y l d i t h i o c a r b a m a t e is u s e d to f o r m a c o l o u r e d c o m p l e x w h i c h is t h e n
e x t r a c t e d w i t h c h l o r o f o r m f r o m a n a m m o n i c a l s o l u t i o n (pH 8.5) c o n t a i n i n g E D T A
to p r e v e n t i n t e r f e r e n c e b y o t h e r m e t a l s . T h e a b s o r b a n c e o f t h e c o m p l e x is
m e a s u r e d at 4 4 0 n m .

APPARATUS

1. Spectrophotometer.

REAGENTS

1. Nitric acid, concentrated.

2. Hydrochloric acid, concentrated.

3. C i t r i c a c i d , 6 0 % w / v : D i s s o l v e 60 g o f c i t r i c a c i d in 70 m l of
w a t e r and d i l u t e to 100 m l .

4. Ammonium hydroxide.

5. D i s o d i u m e t h y l e n e d i a m i n e tetra-acetic acid ( E D T A ) 1% : Dissolve

5 g o f t h e s a l t i n w a t e r to d i l u t e t o 5 0 0 m l .

6. Chloroform.

7. S t a n d a r d c o p p e r s o l u t i o n (0.5 m g / m l ) : B o i l 0 . 5 0 g o f c o p p e r in
20 m l (1+1) n i t r i c a c i d , c o o l and m a k e u p to 1L.

8. D i l u t e c o p p e r s t a n d a r d s o l u t i o n : D i l u t e 10 ml of the above
c o n c e n t r a t e d s t a n d a r d s o l u t i o n t o 5 0 0 m l to o b t a i n 10 p g / m l . (Make
f r e s h daily.)

9. Sodium diethylcarbodithioate solution: 0.1% aqueous solution.

10. Anhydrous sodium sulphate, granular.

11. T h y m o l b l u e i n d i c a t o r - 0.1% in water (add sufficient 0.1N NaOH

to c h a n g e t h e c o l o u r to b l u e .

PROCEDURE

A s h 2 0 g o f s a m p l e a t a b o u t 600°C i n a s i l i c a d i s h . E x t r a c t t h e a s h
b y a d d i n g 10 m l of a s o l u t i o n of h y d r o c h l o r i c - n i t r i c - w a t e r (2+1+3)
a n d m a k e u p to 1 0 0 m l w i t h w a t e r . T r a n s f e r a s u i t a b l e a m o u n t of
a l i q u o t into a 125 m l s e p a r a t i n g funnel and c a r r y out the
d e t e r m i n a t i o n as in t h e s t a n d a r d c u r v e p r e p a r a t i o n b e l o w .

Preparation of Standard Curve

A d d 2 0 m l w a t e r to e a c h o f s i x 1 2 5 m l s e p a r a t o r y f u n n e l s . P i p e t t e 0 ,
1 . 0 , 2 . 0 , 3 . 0 , 4.0 a n d 5.0 m l r e s p e c t i v e l y , o f t h e d i l u t e standard
s o l u t i o n into the six f u n n e l s .

A d d 5 m l of c i t r i c a c i d and 5 m l of E D T A s o l u t i o n s to e a c h f u n n e l .
A d d 2 d r o p s of t h y m o l b l u e to e a c h a n d t h e n a m m o n i u m h y d r o x i d e u n t i l
the s o l u t i o n t u r n s g r e e n or b l u e - g r e e n . N e x t , a d d 10 m l o f 0.1%
sodium diethylcarbodithioate. F i n a l l y , a d d 10 m l o f c h l o r o f o r m a n d

180
 shake for about 1 min. Allow the two layers to separate and transfer
the bottom layer into a 50 ml volumetric flask (filter through some
cotton in a funnel, to remove water drops).

Repeat the e x t r a c t i o n twice w i t h 15 m l portions of c h l o r o f o r m (or

until the chloroform is colourless). Combine the extracts and dilute
to the mark with chloroform.

M e a s u r e the absorbance at 440 nm in a 1 - c m cell against a reagent

blank carried through the procedure. Construct the standard curve by
plotting absorbance against concentration.

CALCULATION

Copper
H
ppm = (100)
(W) (V)

Where :
A = pg copper from standard curve, corresponding to
the sample absorbance.

W = g sample

V = ml of sample extract used.

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 25.066-.071.

181
 METALS SCREENING TEST
(Lead, Copper, Zinc and Others)

PRINCIPLE

The sample is ashed or digested in acid and the solution obtained is tested for
lead u s i n g d i t h i z o n e and for c o p p e r u s i n g s o d i u m d i e t h y l c a r b o d i t h i o a t e .
Another aliquot of the solution is tested with dithizone in chloroform, and a
p o s i t i v e r e s u l t is o b t a i n e d in the p r e s e n c e of zinc and o t h e r m e t a l s such as
c a d m i u m , bismuth, copper, mercury, nickel, silver, thallium and tin. Samples
giving positive results certainly require further examination by a specific and
m o r e a c c u r a t e and p r e c i s e m e t h o d . If the p o s i t i v e r e s u l t in the zinc test
p r o v e s to be due to a n o t h e r e l e m e n t , this m u s t be i d e n t i f i e d and q u a n t i f i e d .
T h i s s c r e e n i n g test does not o b v i a t e the need to test for toxic m e t a l s o t h e r
than lead, c o p p e r and zinc. A l t h o u g h m e r c u r y w o u l d r e a c t p o s i t i v e l y in the
r e a c t i o n for zinc it is l i k e l y to h a v e b e e n l a r g e l y lost d u r i n g the a s h i n g or
wet digestion used.

APPARATUS

1. Muffle furnace or apparatus for digestion using Kjeldahl flasks.

REAGENTS

1. Ash aid S o l u t i o n : D i s s o l v e 50 g a m m o n i u m n i t r a t e , and 50g

p o t a s s i u m s u l p h a t e in 250 m l w a t e r . C h e c k for a b s e n c e of m e t a l s .
Use 2 m l as an ash aid.

2. Ammonium citrate, 10%.

3. Hydroxylamine HC1, 1%.

4. Potassium cyanide, 5%.

5. Dithizone solution, 0.02% prepared from 0.2% solution in

chloroform.

6. Sodium diethylcarbodithioate, 0.1%.

7. Concentrated nitric and sulphuric acids as required for

digestion .

8. Standard metals solutions. Prepare aqueous solutions using pure

salts in the following concentrations:

Lead - 1 mg/ml
Copper - 0.1 mg/ml
Zinc - 0.1 mg/ml

9. Mixed m e t a l s standard solution: P r e p a r e by d i l u t i n g the

f o l l o w i n g v o l u m e of the a b o v e s o l u t i o n s to a total of 500 m l in one
flask: Lead - 1 ml, copper - 100 ml and zinc - 50 ml.

10. Hydrochloric acid, approximately 3N.

PROCEDURE

Prepare the sample solution by either acid digestion or dry ashing as

follows :

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Digestion

Weigh an appropriate amount of sample into a Kjeldahl flask and add

10 m l n i t r i c acid. Boil over s m a l l f l a m e u n t i l v o l u m e in the flask
is reduced to 2-3 ml or until mixture becomes viscous. Do not allow
c h a r r i n g to occur. A l l o w to cool, then add 10 m l c o n c e n t r a t e d
s u l p h u r i c a c i d , m i x and boil u n t i l w h i t e f u m e s are e v o l v e d or
charring occurs, but prevent overheating. Treat blank similarly.

Place 25 ml nitric acid into a tap funnel and drop the acid into the
b o i l i n g liquid in flask u n t i l the s o l u t i o n b e c o m e s c o l o u r l e s s or
nearly colourless. Stop h e a t i n g and c a l c u l a t e v o l u m e of n i t r i c
added. Where more than one sample is being tested, make the volume
of n i t r i c in each flask (including the b l a n k ) up to the h i g h e s t
volume of nitric required. Boil down until white fumes are evolved.
A l l o w to c o o l , add 10-15 ml w a t e r and a g a i n boil d o w n u n t i l w h i t e
f u m e s are e v o l v e d . R e p e a t the b o i l i n g d o w n p r o c e d u r e after the
addition of a second 10—15 ml water.

Cool, t r a n s f e r s o l u t i o n u s i n g m e t a l - f r e e d i s t i l l e d w a t e r to 100 m l
v o l u m e t r i c flask. Add 10 m l 3N HC1 to K j e l d a h l flask, b r i n g to the
b o i l , t r a n s f e r to the v o l u m e t r i c f l a s k , and r i n s e w i t h 1 m l 3N HC1.
Cool and d i l u t e to 100 ml. T r e a t the b l a n k s i m i l a r l y t h r o u g h o u t .
Solutions of sample and blank containing 10% sulphuric (with a little
HC1) are thus obtained. Aliquots of these solutions are used for the
estimations of toxic metals.

Ashing

W e i g h 10 g of s a m p l e into a s i l i c a dish. Add 2 m l of a s h - a i d

s o l u t i o n , t h o r o u g h l y m i x , dry and ash at 500°C. D i s s o l v e in 2 m l of
3N H C 1 , boil and d i l u t e to 20 ml. T r a c e s of c a r b o n need not be
filtered.

Test for Lead

To four c l e a n test tubes (150 x 25 m m ) i n d i v i d u a l l y add: (1)

appropriate volume of sample solution; (2) same volume of digestion
or ash blank solution; (3) same volume of 10% sulphuric acid; and (4)
same as (3) plus 5 ml mixed standard metal solution. In a series of
s a m p l e s only one of each of (2), (3) and (4) is n e c e s s a r y .

To each tube add 2 m l 10% w/v citric acid and mix; 0.3 ml thymol blue
i n d i c a t o r and m i x ; c o n c e n t r a t e d a m m o n i a to full b l u e c o l o u r of
indicator and mix; 2 ml potassium cyanide and mix; 10 ml chloroform;
0.6 ml 0.02% w / v d i t h i z o n e ( f r e s h l y p r e p a r e d ) . S h a k e e a c h tube
v i g o r o u s l y 1 0 - 2 0 s e c o n d s a n d a l l o w l a y e r s to s e p a r a t e . The
C o l o r i m e t r i c B l a n k should be g r e e n and the S t a n d a r d p u r p l e - r e d but
not full red. Record the c o l o u r of (1) and (2) as p r o p o r t i o n s of
(4). W h e r e a colour s h o w s no d i f f e r e n c e from (3) record "less than
0.1 t i m e s Standard". If c o l o u r is m o r e red than (4) r e p e a t the
estimation on a smaller aloquot.

(Note : Dithizone solutions may vary in strength, and the proportion

of dithizone solution given above may be insufficient to give correct
c o l o u r . The standard m u s t be s l i g h t l y p u r p l e i n d i c a t i n g a s l i g h t
excess of unchanged dithizone, and not a full red colour. If further
dithizone is required to produce a satisfactory coloured Standard the
same amount must be added to all tubes.)

(Note: For 50 m l a l i q u o t s , w h i c h are s o m e t i m e s r e q u i r e d for foods

with low lead limits, the above solutions and additions and shaking
should be m a d e in a 100 ml flask. A l l o w the l a y e r s to s e p a r a t e ,
d e c a n t 10-15 m l of a q u e o u s layer to w a s t e , and pour r e m a i n d e r of

183
l i q u i d into 150 x 25 m m t u b e s for c o m p a r i s o n of c o l o u r s of c h l o r o f o r m
l a y e r . T h i s p r o c e d u r e is o n l y n e c e s s a r y for 50 m l a l i q u o t s , as 25 m l
a l i q u o t and r e a g e n t can n o r m a l l y be a c c o m m o d a t e d in the 150 x 25 m m
stoppered tubes.)

T e s t for Copper

To f o u r c l e a n t e s t t u b e s (150 x 25 m m ) i n d i v i d u a l l y a d d : (1)
a p p r o p r i a t e v o l u m e of s a m p l e s o l u t i o n ; (2) s a m e v o l u m e of d i g e s t i o n
or ash b l a n k s o l u t i o n ; (3) s a m e v o l u m e of 10% s u l p h u r i c a c i d ; and (4)
s a m e as (3) p l u s 5 m l m i x e d s t a n d a r d m e t a l s o l u t i o n . In a s e r i e s of
s a m p l e s , o n l y o n e e a c h o f ( 2 ) , ( 3 ) a n d ( 4 ) is n e c e s s a r y .

To e a c h t u b e a d d 2 m l 10% w / v c i t r i c a c i d and m i x ; 0.3 m l t h y m o l b l u e

s o l u t i o n and m i x ; a m m o n i a to a full b l u e c o l o u r of i n d i c a t o r and m i x
(3N or c o n c e n t r a t e d d e p e n d i n g u p o n a m o u n t of a c i d to b e n e u t r a l i z e d
but use same r e a g e n t for all t u b e s ) ; 1 m l of 0.1% sodium
d i t h y l c a r b o d i t h i o a t e ; and 10 m l c h l o r o f o r m .

S h a k e e a c h t u b e v i g o r o u s l y f o r 1 0 - 2 0 s e c o n d s a n d a l l o w l a y e r s to
s e p a r a t e . R e c o r d t h e c o l o u r o f (2) a s p r o p o r t i o n o f (4). W h e n t h e
c o l o u r s h o w s n o d i f f e r e n c e f r o m ( 3 ) r e c o r d " l e s s t h a n 0.1 t i m e s
S t a n d a r d " . If c o l o u r is g r e a t e r t h a n S t a n d a r d , r e p e a t o n a s m a l l e r
aliquot.

T e s t for Z i n c a n d O t h e r Metals

T o f o u r c l e a n t e s t t u b e s ( 1 5 0 x 25 m m ) a d d : (1) a p p r o p r i a t e v o l u m e
o f s a m p l e s o l u t i o n ; (2) s a m e v o l u m e o f d i g e s t i o n o r a s h b l a n k
s o l u t i o n ; ( 3 ) s a m e v o l u m e o f 1 0 % s u l p h u r i c a c i d ; a n d ( 4 ) s a m e as ( 3 )
plus 1 m l mixed standard m e t a l solution. In a s e r i e s of s a m p l e s o n l y
o n e e a c h o f ( 2 ) , ( 3 ) a n d ( 4 ) is n e c e s s a r y .

To e a c h t u b e a d d : 2 m l 1 0 % w / v c i t r i c a c i d a n d m i x ; 0.3 m l t h y m o l
b l u e s o l u t i o n and m i x ; c o n c e n t r a t e d a m m o n i a to full b l u e of i n d i c a t o r
a n d m i x ; 15 m l c h l o r o f o r m ; a n d 1.5 m l 0.2% w / v d i t h i z o n e ( f r e s h l y
prepared). S h a k e e a c h tube v i g o r o u s l y 1 0 - 2 0 s e c o n d s and a l l o w l a y e r s
to s e p a r a t e . T h e (3) t u b e s h o u l d b e g r e e n a n d (4) s h o u l d b e p u r p l e
red b u t n o t full r e d . R e c o r d c o l o u r of (1) and (2) as p r o p o r t i o n s of
(4) t o 0.1. W h e r e a c o l o u r s h o w s n o d i f f e r e n c e f r o m (3) r e c o r d as
" l e s s t h a n 0.1 t i m e s s t a n d a r d " . If c o l o u r is m o r e r e d t h a n ( 4 )
r e p e a t on a s m a l l e r a l i q u o t .

(Note : This test w i l l include other m e t a l s ; b i s m u t h , c a d m i u m ,

c o p p e r , m e r c u r y , n i c k e l , s i l v e r , t h a l l i u m and t i n . If t h e " z i n c "
i n d i c a t e d b y this e s t i m a t i o n is less t h a n the l i m i t p r e s c r i b e d t h e n
t h e t r u e z i n c f i g u r e is c e r t a i n l y b e l o w the l i m i t . )

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7.3 Mycotoxins

P o i s o n o u s m o u l d m e t a b o l i c p r o d u c t s a r e g e n e r i c a l l y r e f e r r e d to as " m y c o t o x i n s " .
S e v e r a l a r e k n o w n c a r c i n o g e n s or m u t a g e n s in a d d i t i o n to t h e i r t o x i c i t y »

M o u l d g r o w t h on f o o d s is v e r y c o m m o n , e s p e c i a l l y in w a r m , h u m i d c l i m a t e s . It
c a n o c c u r in the f i e l d , or in s t o r a g e a f t e r h a r v e s t . M o u l d i n f e c t i o n of f o o d s
s u c h as g r a i n s , s e e d s a n d n u t s is o f t e n l o c a l i z e d i n p o c k e t s , e s p e c i a l l y i n
bulk s t o r a g e . F r e q u e n t and a d e q u a t e s a m p l i n g for t e s t , t h e r e f o r e b e c o m e s a
necessity. T h e s a m p l i n g p r o b l e m a n d a s o l u t i o n is d i s c u s s e d b y W h i t a k e r et a l
(4), u s i n g a f l a t o x i n in g r o u n d n u t s as t h e e x a m p l e .

A l l food s a m p l e s s u s p e c t e d of b e i n g c o n t a m i n a t e d w i t h m y c o t o x i n s , m u s t be
handled with care. U s e d i s p o s a b l e g l o v e s a n d p r o t e c t i v e m a s k s if g r i n d i n g the
food creates d u s t . When handling pure mycotoxin reference m a t e r i a l , use
e x t r e m e c a r e , p r e f e r a b l y in a h o o d or m i c r o b i o l o g i c a l g l o v e b o x . Treat any
s p i l l a g e of t o x i n w i t h a 5% s o l u t i o n of s o d i u m h y p o c h l o r i t e and r i n s e a l l
e x p o s e d g l a s s w a r e w i t h a 1% s o l u t i o n of t h e b l e a c h b e f o r e w a s h i n g in the u s u a l
m a n n e r . The b l e a c h destroys the toxin b y o x i d a t i o n . D e c o n t a m m i n a t i o n m e t h o d s
a r e d i s c u s s e d b y S t o l o f f a n d T r a g e r (5).

Aflatoxin

A f l a t o x i n is p r o b a b l y the m o s t c o m m o n a n d w i d e l y k n o w n m y c o t o x i n c o n t a m i n a n t .
It is p r o d u c e d b y t h e m o u l d s A s p e r g i l l u s f l a v u s a n d A s p e r g i l l i s p a r a s i t i c u s .
I n f a c t t h e n a m e is a c o m p o s i t e w o r d d e r i v e d f r o m 'A. f l a v u s t o x i n 1 . Foods
w h i c h are c o m m o n l y a f f e c t e d include all nuts ( e s p e c i a l l y g r o u n d n u t s and tree
n u t s s u c h as p i s t a c h i o s and B r a z i l s ) , c o t t o n s e e d , c o p r a , r i c e , m a i z e , w h e a t
g r a i n , s o r g h u m , p u l s e s , figs and oilseed c a k e s . U n r e f i n e d v e g e t a b l e oils m a d e
f r o m c o n t a m i n a t e d s e e d s or n u t s , u s u a l l y c o n t a i n s a f l a t o x i n . However,
a f l a t o x i n is d e s t r o y e d in t h e r e f i n i n g p r o c e s s , so t h a t r e f i n e d o i l s a r e s a f e .
It is an u n f o r t u n a t e f a c t t h a t in m a n y c o u n t r i e s , u n r e f i n e d v e g e t a b l e o i l s a r e
u s e d in m u c h g r e a t e r q u a n t i t i e s t h a n r e f i n e d , b e c a u s e of t h e s o m e t i m e s l a r g e
d i f f e r e n c e in c o s t . In s u c h a r e a s , u n r e f i n e d o i l s s h o u l d b e g i v e n h i g h
p r i o r i t y for f r e q u e n t r o u t i n e t e s t i n g .

T h e r e are six a f l a t o x i n s of a n a l y t i c a l i n t e r e s t . F o u r o c c u r in f o o d s a n d t w o
as m e t a b o l i t e s i n t h e m i l k o f a n i m a l s w h o h a v e b e e n f e d c o n t a m i n a t e d f e e d .
T h e i r c h e m i c a l s t r u c t u r e s are n o t e d b e l o w :

AFLATOXIN BI AFLATOXIN B2

AFLATOXIN GJ AFLATOXIN G2

185
 AFLATOXIN B ^ (WATER A D D U C T , H E M I A C E T A L )

A f l a t o x i n s B ^ , B 2 , G ^ a n d G 2 r e f e r to t o x i n s w h i c h f l u o r e s c e b l u e (B) or g r e e n
(G) u n d e r u l t r a v i o l e t l i g h t and a r e s e p a r a b l e by t h i n - l a y e r chromatography.
Note that the o n l y s t r u c t u r a l d i f f e r e n c e b e t w e e n B and G toxins is the
i n c l u s i o n of an o x y g e n in t h e e y e l o p e n t a n o n e r i n g . A l f a t o x i n B ^ is b y f a r t h e
m o s t c o m m o n l y found of the four t o x i n s .

A f l a t o x i n s M ^ and M 2 r e p r e s e n t the t o x i n s Bj and B 2 w h i c h h a v e b e e n m e t a b o l i z e d

and c o n v e r t e d w i t h i n the b o d y of a l a c t a t i n g a n i m a l . T h e i r f i n d i n g in m i l k led
to t h e i r ' M ' d e s i g n a t i o n . T h e o b v i o u s s t r u c t u r a l d i f f e r e n c e b e t w e e n B a n d M is
the a d d i t i o n of the h y d r o x y l g r o u p .

A f l a t o x i n B 2 ¿ ^ is a h e m i a c e t a l d e r i v a t i v e o f B ^ and is c o m m o n l y called the

"water adduct". It d o e s n o t o c c u r n a t u r a l l y a n d is o n l y u s e d as a m e a n s to
c o n f i r m B^ i d e n t i t y .

Aflatoxins pure reference material are obtainable from the following

i n t e r n a t i o n a l s o u r c e s (taken from O f f i c i a l M e t h o d s of A n a l y s i s of the A O A C ,
1984, 2 6 . 0 0 5 ) ( l i s t is alphabetical):

1. Aldrich Chemical C o . , PO Box 355, Milwaukee, WI 53201 USA.

2. Applied Science Division, Milton Roy Co., 2051 Waukegan Rd.,

Deerfie.ld, IL 6 0 0 1 5 U S A .

3. Calbiochem-Behring, PO Box 12087, San D i e g o , CA 92112 USA.

4. C. R o t h , Postfach 1387, 7500 Karlsruhe 1, F e d e r a l Democratic Republic

of Germany.

5. Eureka Laboratories, Inc., 215 26th St., Sacramento, CA 95816 USA.

6. ICN K and K Laboratories Inc., 121 Express St, Plainview, NY 11803

USA.

7. Makor Chemicals Ltd., Box 6570, Jerusalem, 91060 Israel.

8. Myco Lab Co., P. 0. Box 321, Chesterfield, MO 63017 USA.

9. RFR Corp., 1 Main St., H o p e , RI 02831 USA.

10. Rijksinstitut voor de Vo1ks gezondheid, P. 0. Box 1, 3 720 BA

Bilthoven, The Netherlands.

186
 11. Senn C h e m i c a l s , Laboratorium Guido A. S e n n , Postfach 2, CH-8157
Dielsdorf, Switzerland.

12. Sigma Chemical Co., S u p e l c o Inc., P. 0. Box 14509, St. Louis, MO

63178 USA.

13. TCI Tridom Chemical Inc., H a u p p a u g e , NY 11787, USA.

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 AFLATOXINS
(Thin Layer Chromatography Method)

PRINCIPLE

This m e t h o d m a y be used for the detection and d e t e r m i n a t i o n of aflatoxin Bj,

and aflatoxins B2, G^ and G23 if needed, in a wide variety of foods, including
nuts, grains, oilseeds, and mixed feeds. The limit of determination can be the
very low ng/g range, dependent on whether and which optional cleanup procedures
are used.

Aflatoxins are extracted from the finely ground food with aqueous methanol and
partitioned to methylene chloride. For some products, such as cottonseed and
mixed feeds w h i c h contain interfering p i g m e n t s , an optional cleanup of the
aqueous methanol extract by treatment with metals is done before the partition.
If necessary, the aflatoxins are further separated from most interferences by
silica gel column chromatography. Separation of the aflatoxins for detection
and quantitation is by thin layer chromatography.

APPARATUS

1. C h r o m a t o g r a p h i c c o l u m n s , 300 m m x 10 m m ID w i t h stopcock and

either medium pore frit disc or glass wool plug.

2. T h i n - l a y e r c h r o m a t o g r a p h y apparatus including plates, spotting

pipettes, developing tanks and a UV light viewing chamber.

3. Flask shaker apparatus.

4. Warm air blower (such as hair dryer).

REAGENTS

(Note: All solvents must be procured and stored in glass containers;

p l a s t i c l i n e r s or c o a t i n g u s e d in m e t a l c o n t a i n e r s contain
extractives that interfere with analysis.)

1. Acetic acid

2. Acetone

3. Acetonitrile

4. Benzene

5. Chloroform

6. Ethyl ether

7. Formic acid

8. Isopropanol

9. Methanol

10. Methylene chloride (dichloromethane)

11. Petroleum ether

12. Toluene

13. D i a t o m a c e o u s earth filter aid - Acid washed Hyflo SuperCel

(Johns Manville Corp., Greenwood Plaza, Denver, CO 80217, USA)

188
14. Silica gel - Particle size 80-230 mesh

15. Sodium c h l o r i d e solution, 100 g/L

16. Sodium sulphate, anhydrous, granular

17. Zinc a c e t a t e - a l u m i n i u m c h l o r i d e solution - 200 g Zn(OAc), and 5

g A I C I 3 to make 1 L w i t h w a t e r

18. Aflatoxin standards - prepare a mixed working standard to

c o n t a i n 0 . 5 y g / m l e a c h of a f l a t o x i n s Bj^ and G j a n d 0 . 1 jjg/m 1 e a c h B2
and G 2 •

PROCEDURE

P r e p a r e the food by g r i n d i n g or comminuting to pass a 20 mesh s c r e e n .

U s e as l a r g e a s a m p l e as p o s s i b l e depending on t h e available
preparation equipment. ( T h i s s h o u l d be as much as 10 k i l o s if
possible. The l a r g e r t h e s a m p l e s i z e , t h e g r e a t e r t h e p o t e n t i a l to
d e t e c t pockets of c o n t a m i n a t i o n ) .

W e i g h 50 g sample in g l a s s - s t o p p e r e d e r l e n m e y e r f l a s k , and add 200 ml

methanol:water (85:15). S h a k e v i g o r o u s l y no l e s s t h a n 3 0 m i n ( u s e
mechanical shaker). F i l t e r through medium p o r o s i t y p a p e r . Collect
4 0 ml f i l t r a t e from m i d f l o w , o r , i f o p t i o n a l c l e a n u p i s u s e d , c o l l e c t
1 ml f i l t r a t e from s t a r t of f l o w .

Optional c l e a n - u p to r e m o v e interfering plant pigments from

c o t t o n s e e d or mixed a n i m a l f e e d s : t r a n s f e r 100 ml above f i l t r a t e to
2 5 0 ml b e a k e r . Add 2 0 ml Z n ( O A c ) 2 " A l C l 3 s o l u t i o n and 8 0 ml w a t e r .
S t i r and l e t s t a n d at l e a s t 5 m i n to f l o c c u l a t e . Mix in 5 g f i l t e r
aid and f i l t e r through medium p o r o s i t y paper. C o l l e c t 80 ml f i l t r a t e
from m i d f l o w .

T r a n s f e r e i t h e r the 4 0 ml or the 8 0 ml f i l t r a t e s f r o m a b o v e to a
separatory funnel. Add 4 0 ml s o d i u m c h l o r i d e s o l u t i o n and 25 ml
petroleum ether. S h a k e 1 m i n , l e t l a y e r s s e p a r a t e and t r a n s f e r
b o t t o m l a y e r to s e c o n d s e p a r a t o r y funnel. Discard top layer.
( R e p e a t t h i s p a r t i t i o n for h i g h fat c o n t e n t f o o d s ) . Add 25 ml
m e t h y l e n e c h l o r i d e to the s e c o n d f u n n e l . Shake 1 m i n , let layers
s e p a r a t e and d r a i n bottom l a y e r into 125 ml e r l e n m e y e r f l a s k . Repeat
the e x t r a c t i o n w i t h a second p o r t i o n m e t h y l e n e c h l o r i d e and combine
w i t h the f i r s t . Add s e v e r a l b o i l i n g chips and e v a p o r a t e the combined
m e t h y l e n e c h l o r i d e e x t r a c t s a l m o s t to d r y n e s s on a steam b a t h . Cool,
and c o v e r w i t h a l u m i n i u m f o i l , and h o l d f o r e i t h e r t h i n layer
c h r o m a t o g r a p h y or c o l u m n c h r o m a t o g r a p h y ( i f n e e d e d f o r further
c l e a n u p of e x t r a c t ) .

If further column chromatography c l e a n - u p i s n e e d e d to r e m o v e

i n t e r f e r e n c e s , proceed as f o l l o w s : P r e p a r e a column by s l u r r y i n g 2 g
( a b o u t 5 ml v o l u m e ) of 8 0 - 2 3 0 m e s h s i l i c a g e l w i t h 10 ml e t h e r -
p e t r o l e u m e t h e r ( 3 + 1 ) in a s m a l l b e a k e r and p o u r i n g i n t o the column.
Wash any r e s i d u a l s i l i c a gel into the column u s i n g 5 ml of the ( 3 + 1 )
mixed e t h e r s . A f t e r the g e l s e t t l e s , top the g e l p a c k i n g w i t h 1.5 g
sodium sulphate. Drain the s o l v e n t to the t o p o f t h e sodium
sulphate. D i s s o l v e r e s i d u e i n 3 ml m e t h y l e n e c h l o r i d e , and t r a n s f e r
s o l u t i o n to column. R i n s e f l a s k t w i c e w i t h 1 ml p o r t i o n s m e t h y l e n e
c h l o r i d e and add r i n s i n g s to column. E l u t e e x t r a c t through column no
f a s t e r than 1 m l / m i n . Add s u c c e s s i v e l y to c o l u m n 3 ml p e t r o l e u m
e t h e r , 3 ml e t h y l e t h e r and 3 ml m e t h y l e n e c h l o r i d e . Elute through
columns no f a s t e r than 3 m l / m i n . Discard eluates. Add 6 ml
m e t h y l e n e c h l o r i d e ¡ a c e t o n e ( 9 + 1 ) to c o l u m n . Elute solvent through
column ( p r e f e r a b l y w i t h no vacuum) no f a s t e r than 1 m l / m i n . Collect
e l u a t e in a small v i a l from time of s o l v e n t a d d i t i o n to c e s s a t i o n of

189
flow. Add a few b o i l i n g chips and e v a p o r a t e e l u a t e just to d r y n e s s
on s t e a m b a t h u n d e r a s t r e a m of n i t r o g e n . Cap v i a l , w r a p in foil,
and hold for thin layer chromatography.

U n c a p v i a l c o n t a i n i n g e x t r a c t r e s i d u e , a d d 0.2 m l benzene-
a c e t o n i t r i l e (98+2), r e s e a l v i a l , and shake v i g o r o u s l y , p r e f e r a b l y
w i t h V o r t e x type m i x e r to d i s s o l v e r e s i d u e . On a thin layer p l a t e
score a line in s i l i c a gel layer to d i v i d e p l a t e into 2 e q u a l 10x20
cm sections. Also score lines perpendicular to the first score line
and at 1 cm intervals across the plate to produce 1 cm wide channels
for s p o t t i n g and d e v e l o p m e n t . A l o n g one 20 cm d i m e n s i o n , on an
imaginary line 3 cm from bottom edge of a scored plate, spot 4 centre
c h a n n e l s w i t h 2.5, 5.0, 7.5 and 10 p i of the m i x e d aflatoxin
reference standard. In the remaining channels spot 2-10 p i of each
sample extract and superimpose 5 p i of aflatoxin standard on 1 spot
of each of the sample extracts.

Develop spotted plate until solvent reaches score line that divides
p l a t e into 2 p a r t s (ca 20 min). Use an u n e q u i 1 i b r a t e d tank w i t h
s o l v e n t c o m b i n a t i o n p r e v i o u s l y s e l e c t e d from the f o l l o w i n g for
o p t i m u m r e s o l u t i o n of a f l a t o x i n s f r o m e a c h o t h e r and s u b s t r a t e
interferences: (order of aflatoxin R j from top is Bj, B2, Gj, G 2 )

a. Chloroform:acetone (9+1)
b. Chioro form:acetone : water (88 + 12 + 1.5)
c. Chloroform : acetone :isopropanol: water (88 + 12 + 1.5 + 1)
d. Chloroform:isopropanol (99+1)
e. Benzene:methanol: acetic acid (90+5+5)
f. Ether¡methanol:water (96+3+1)

(Note : Polar component of each combination may be adjusted to change

a b s o l u t e Rf. I n c r e a s e the p r o p o r t i o n for h i g h e r R f , d e c r e a s e to
lower Rf).

Remove the plate from tank and evaporate solvent from the plate in a
hood at room temperature. Examine plate under long-wave UV light in
d a r k e n e d r o o m or a c a b i n e t . L o o k in c h a n n e l s w i t h reference
standards for clean separation of 4 blue fluorescent spots. Look in
sample channels without superimposed standard for fluorescent spots
that c o i n c i d e in hue and d e v e l o p m e n t p o s i t i o n w i t h reference
s t a n d a r d s in a d j a c e n t c h a n n e l s . Each s amp 1 e-p1us - standard
f l u o r e s c e n t spot should p r o d u c e a m o r e i n t e n s e f l u o r e s c e n c e than
corresponding spot from the sample alone, and should show no sign of
separating spots (evidence of dissimilar compounds).

C o m p a r e the i n t e n s i t y of each f l u o r e s c e n t spot p r e s u m e d to be an

aflatoxin, with corresponding spots of reference standards alone in
four centre channels. Look for an intensity match. Interpolate, if
sample intensity lies between intensities of two standards. If spot
from s a m p l e is too i n t e n s e to m a t c h h i g h e s t s t a n d a r d , e s t i m a t e
relative intensities, dilute sample by this factor, and
rechromatograph.

CALCULATION

The mixed standard spots of 2.5, 5.0, 7.5 and 10 p 1 are equivalent to
the following ng aflatoxins respectively:

B : and G x - 1.25, 2.5, 3.75, 5.0

B 2 and G 2 - .25, .5, .75, 1.0

190
 T h e s a m p l e s p o t s are e q u i v a l e n t to 0.05 g s a m p l e / y l of e x t r a c t
spotted. T h e r e f o r e , if s a m p l e s p o t s of 2 to 10 pi a r e m a d e , t h e s e
w o u l d be e q u i v a l e n t to 0.1 to 0.5 g.

Therefore :
W

Aflatoxin ppb (ng/g) = Q Q5 y

where :
W = ng a f l a t o x i n w i t h c l o s e s t i n t e n s i t y match to
sample spot V.
V = pi of sample spot closest to W.

Calculate for each different aflatoxin in a given sample spot and add
together so that total aflatoxins are reported. (For example: found
10 ppb B j , 3 ppb B2 and no G^ or G 2 - R e p o r t as 13 p p b t o t a l
aflatoxins).

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 26.001-.013.

191
 AFLATOXIN B 1

CONFIRMATION OF IDENTITY

PRINCIPLE

Some food products may give a blue fluorescent spot similar in Rj. to aflatoxin
Bj (the m o s t c o m m o n l y found aflatoxin). This m e t h o d p r o v i d e s a m e a n s to
c o n f i r m the B^ identity by preparing the h e m i a c e t a l B a n d identifying it
using two-dimensional thin-layer chromatography.

APPARATUS

1. T h i n - l a y e r c h r o m a t o g r a p h y apparatus including plates, spotting

pipettes, developing tanks and a UV light viewing chamber.

2. Warm air blower (such as a hair dryer).

REAGENTS

1. Silica gel for TLC plates.

2. Trifluoroacetic acid (TFA).

3. Developing solvent A: ethyl ether-methanol-water (96+3+1).

4. Developing solvent B: c h l o r o f o r m - a c e t o n e - i s o p r o p a n o l (85+12.5

+2.5).

5. Aflatoxin Bj working standard of 0.5 y g B^/ml.

PROCEDURE

Prepare silica gel TLC plates as in usual aflatoxin analysis. Score

a plate for two-dimensional chromatography, as shown in the figure
below. (The distances given on the figure are for a 10 cm x 10 cm
plate. For a 20 cm x 20 cm, they would double to 3 c m , 13 c m , 3 cm
and 1 cm respectively).

I*

\ STANDARD
SPOTS

64

SAMPLE SPOT \
X
/
JOvil 1»,

— 1 4 6", >
14 •< > 4

On the: sample spot, apply sufficient s a m p l e extract to give an

estimated 5 ng aflatoxin Bj. On both standard spots, apply 10 p i of
the Bj working standard. Overspot all three spots with 2 p i each of
t r i f l u o r o a c e t i c acid. Place plate in a dark area for 5 m i n to
c o m p l e t e the reaction. E v a p o r a t e residual TFA using w a r m air (TFA
must be completely dry before continuing).

192
 D e v e l o p p l a t e in d i r e c t i o n 1 u s i n g S o l v e n t A u n t i l the s o l v e n t f r o n t
r e a c h e s the l o w e r s c o r e d l i n e . R e m o v e t h e p l a t e f r o m t h e t a n k and
dry w i t h warm a i r or in a oven for a few m i n u t e s .

T u r n p l a t e 9 0 ° and d e v e l o p i n d i r e c t i o n 2 u s i n g S o l v e n t B , again
u n t i l the solvent front reaches the l o w e r scored l i n e . Remove from
t h e t a n k and d r y .

E x a m i n e the d r y p l a t e u s i n g l o n g w a v e UV l i g h t i n a c a b i n e t . The
sample and s t a n d a r d s should have a new b l u e f l u o r e s c e n t spot of
at an Rj. o f a b o u t 0 . 3 . The s a m p l e B 2 ¿ s p o t s h o u l d l i e at an
i n t e r s e c t i o n of i m a g i n a r y l i n e s p r o j e c t e d from the two s t a n d a r d
s p o t s , p e r p e n d i c u l a r to the d i r e c t i o n of each d e v e l o p m e n t .

REFERENCE

Official Methods of A n a l y s i s of the AOAC, 1984, 26.083.

193
 AFLATOXINS
(Mini-column M e t h o d )

PRINCIPLE

The s a m p l e is e x t r a c t e d w i t h a c e t o n e - w a t e r , i m p u r i t i e s are r e m o v e d , and the

aflatoxins extracted into chloroform, The chloroform extract is passed through
a c o l u m n c o n t a i n i n g c a l c i u m s u l p h a t e , F l o r i s i l , a l u m i n a and s i l i c a gel. The
a f l a t o x i n s r e m a i n i n g at the top of the F l o r i s i l layer are c o m p a r e d w i t h
standards under longwave UV.

APPARATUS

1. Food blender, or shaker.

2. Ultra-violet lamp, long-wave (365 nm). Intensity at least 430

m i c r o - w a t t s / c m ^ at 15 cm.

3. Minicolumn. Borosilicate tubing 6 m m ID x 190 m m long, tapered

at one end to about 2 mm.

4. 5 ml syringe with 5" needle.

5. Filter paper, 24 cm, Whatman No. 4 or equivalent.

REAGENTS

1. Chloroform (technical grade is adequate).

2. Acetone (technical grade is adequate).

3. 0.2 N sodium hydroxide (8 g/L).

4. Ferric chloride solution. M i x 20 g a n h y d r o u s f e r r i c c h l o r i d e

with 300 ml of water. Alternatively, dilute 33 ml of a 60% solution
to 300 m l .

5. Copper carbonate, basic (cupric hydroxide carbonate) solid.

6. Diatomaceous earth.

7. Sulphuric acid, 0.03% v/v.

8. Potassium hydroxide/potassium chloride wash solution. Dissolve

1.12g KOH + 10 g KC1 in water and dilute to 1 litre.

9. Sodium sulphate, anhydrous.

10. Florisil 100-200 mesh for column chromatography.

11. S i l i c a gel 60, 0.063-0.200 m m , n e u t r a l for column chroma-

tography, Activity 2-3 (Brockmann and Schodder).

12. Calcium sulphate anhydrous, 20-40 mesh.

13. Alumina, activity 1 (Brockmann and Schodder), 80-200 mesh,

neutral.

14. A f l a t o x i n B j or m i x e d a f l a t o x i n s s t a n d a r d with a final

concentration of 2 y g / m l of each aflatoxin.

15. Glass wool.

16. Elution solvent. Chloroform-acetone (9+1).

194
PROCEDURE

Prepare one minicolumn per sample as follows: Block the tapered end
of a m i n i c o l u m n w i t h a s m a l l p l e d g e t of g l a s s w o o l , and fill the
c o l u m n as g i v e n in the d i a g r a m , t a p p i n g the c o l u m n after each
addition. Place a s m a l l p l e d g e t of g l a s s w o o l on top and p r e s s
l i g h t l y into place w i t h a glass rod. (Note: dry each of the
adsorbents for 2 hours at 110°C before use.)

8-10 anhydrous calcium sulphate

8-10 mm neutral alumina
16-20 mm silica gel
8-10 mm Florisil
8-10 mm anhydrous calcium sulphate
pledget

A l s o p r e p a r e a r e f e r e n c e m i n i c o l u m n as above. To this add 5 pi of

the standard solution which has been dissolved in 1 ml of chloroform.
Use a syringe for the addition. As the solvent sinks to the surface
of the column, add 3 ml of the chloroform-acetone (9+1) and allow to
d r a i n by g r a v i t y . If dried and sealed and kept in the d a r k in a
f r e e z e r , the c o l u m n w i l l keep 2 - 3 m o n t h s , but r e p e a t e d e x p o s u r e to
ultraviolet light diminishes the intensity of the fluorescence, which
is a p p a r e n t at the top of the F l o r i s i l layer. T h i s f l u o r e s c e n c e is
due to the aflatoxins.

N e x t , m i x 50 g s a m p l e and 250 m l a c e t o n e + w a t e r (85 + 15) in a b l e n d e r

at high speed for 3 minutes or agitate on a mechanical shaker for 45
minutes. F i l t e r t h r o u g h a 24 cm W h a t m a n No. 4 f i l t e r p a p e r into a
graduated cylinder. T r a n s f e r 150 m l of the f i l t r a t e to a 400 m l
b e a k e r , add about 3 g of b a s i c c o p p e r c a r b o n a t e and m i x w e l l . To a
500 m l b e a k e r add e x a c t l y 170 m l of 0.2N N a O H and 30 m l ferric
c h l o r i d e s o l u t i o n and m i x w e l l . Pour the m i x t u r e of f i l t r a t e and
basic copper carbonate into this, add 150 ml (use a 150 m l beaker as
a s c o o p ) of d i a t o m a c e o u s earth and m i x w e l l . Filter through a
Whatman No. 4 or equivalent filter paper.

T r a n s f e r e x a c t l y 150 m l of f i l t r a t e to 500 m l s e p a r a t o r , add 150 m l

of 0.03% s u l p h u r i c acid s o l u t i o n and 10 m l c h l o r o f o r m . Shake
vigorously 2 minutes and allow to separate. Transfer the lower layer
to a 125 m l s e p a r a t o r , add 100 m l p o t a s s i u m h y d r o x i d e / p o t a s sium
c h l o r i d e w a s h s o l u t i o n , s w i r l g e n t l y h a l f a m i n u t e and a l l o w to
separate. If an e m u l s i o n is f o r m e d , d r a i n it into a l i p l e s s
graduated cylinder, add about 1 g anhydrous sodium sulphate, stopper,
shake half a minute and allow to separate. It is not important that
the chloroform layer be completely clear. If the emulsion is still
not b r o k e n , w a s h it w i t h 50 m l 0.03% s u l p h u r i c acid in a s e p a r a t o r .
F i n a l l y , c o l l e c t at least 3 ml of the c h l o r o f o r m layer in a 10 ml
graduated cylinder.

By m e a n s of a 5 m l s y r i n g e w i t h a 5 in n e e d l e , t r a n s f e r 2 ml of the
chloroform extract to a minicolumn. Allow to drain by gravity. The
solvent may be forced through the dry column at a rate not exceeding
10 c m / m i n u n t i l it r e a c h e s the tip, by a p p l i c a t i o n of g e n t l e air or
inert gas pressure to the top of the column. Then allow to drain by
gravity. W h e n c h l o r o f o r m r e a c h e s the c o l u m n s u r f a c e , add 3 m l of
e l u t i o n s o l v e n t ch 1 o r o f o r m + a c e t o n e (9 + 1) and a l l o w to d r a i n by
gravity.

E x a m i n e the s a m p l e and r e f e r e n c e c o l u m n s side by side in a UV

cabinet. The l a t t e r has a b l u e f l u o r e s c e n t band a b o u t 2.5 cm from
the b o t t o m , at the top of the F l o r i s i l layer. A s i m i l a r blue
fluorescent band in the sample column is presumptive evidence of the

195
 presence of aflatoxins. S o m e samples s h o w a faint white y e l l o w or
brown fluorescence, but if there is no definite bluish tint there are
no aflatoxins.

CALCULATIONS

The reference m i n i c o l u m n contains 10 ng of each aflatoxin. The

extract from 50 x 150/250 x 150/350 = 13 g is shaken with chloroform.
The extract contains acetone, so although only 10 ml of chloroform is
added, the non-aqueous layer is about 13 ml. The 2 ml of chloroform
applied to the minicolumn therefore contains aflatoxins from about 2
g of sample and sample columns showing fluorescence greater than that
of the r e f e r e n c e are l i k e l y to c o n t a i n o v e r 5 n g / g ( p p b ) of
aflatoxin. The sample column may be retained so that the presumptive
aflatoxin can be eluted and examined by thin layer chromatography.

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 26.014-.019.

 AFLATOXIH M
(Thin-layer Chromatography Method)

PRINCIPLE

A f l a t o x i n M is e x t r a c t e d f r o m m i l k or m i l k p r o d u c t s and i s o l a t e d b y
partitioning and later column chromatography. Final determination is by thin-
layer chromatography.

APPARATUS

1. Explosion-proof blender with 1 L jar and cover.

2. Chromatographic tubes, 22 mm (ID) x 500 mm with stopcock.

3. Thin-layer chromatography apparatus, complete.

REAGEHTS

1. Acetone

2. Benzene

3. Chloroform

4. Ethyl ether

5. Hexane

6. Methanol

7. Isopropanol

8. Lead acetate solution - dissolve 200 g lead acetate trihydrate

in water, add 3 ml acetic acid and dilute to 1 L.

9. Sodium sulphate solution, saturated - dissolve 150 g anhydrous

sodium sulphate in 500 ml water. (Prepare 2 or 3 days in advance of
analysis).

10. Diatomaceous earth.

11. C e l l u l o s e - W h a t m a n CF-11 or CF-1 p o w d e r - soak 4 hr in hot

chloroform, filter and wash with chloroform, and air dry. Check each
prepared lot for M recovery using a standard M solution through the
method.

12. Aflatoxin M standard solution, 0.5 yg/ml.

PROCEDURE

Weigh fluid or powdered milk or butter into blender jar and add the
a m o u n t of w a t e r i n d i c a t e d in the table b e l o w . A l s o add 10 g
d i a t o m a c e o u s earth and 300 ml a c e t o n e . For c h e e s e , w e i g h then cut
into small cubes and add to the water, acetone and diatomaceous earth
already in the blender jar.

197
T a b l e of w a t e r addition:

Sample volume ml water

Milk product or w e i g h t to b e a d d e d

Fluid milk 100 ml 10

Powdered milk 10 g 100
Blue cheese 50 g 80
Ricotta cheese 50 g 65
Cheddar cheese 50 g 80
Butter 50 g 90

B l e n d 3 m i n t h e n f i l t e r t h r o u g h f o l d e d W h a t m a n 2V p a p e r (or
e q u i v a l e n t ) into a 500 m l graduated cylinder. T r a n s f e r 275 m l into a
6 0 0 m l b e a k e r c o n t a i n i n g 20 m l lead a c e t a t e s o l u t i o n . R i n s e the
t r a n s f e r c y l i n d e r w i t h 2 0 0 m l w a t e r a n d a d d to t h e b e a k e r . Stir and
l e t s t a n d 5 m i n for p r e c i p i t a t i o n to t a k e p l a c e . A d d 10 m l s a t u r a t e d
s o d i u m s u l p h a t e a n d 10 g d i a t o m a c e o u s e a r t h . Stir and filter into a
500 m l c y l i n d e r .

T r a n s f e r 3 5 0 m l f i l t r a t e to a 5 0 0 m l s e p a r a t o r and add 100 m l h e x a n e

(for b u t t e r or a n n a t t o c o l o u r e d c h e e s e , u s e e t h e r ) . Shake vigorously
1 m i n and let s t a n d . D r a i n l o w e r a q u e o u s layer into 600 m l b e a k e r .
D i s c a r d u p p e r h e x a n e or e t h e r l a y e r . Pour aqueous layer back into
the s a m e s e p a r a t o r . R i n s e the b e a k e r w i t h 50 m l 5% s o d i u m c h l o r i d e
s o l u t i o n a n d a d d to t h e s e p a r a t o r . A d d 100 m l c h l o r o f o r m a n d s h a k e 1
min. Let s t a n d , then drain lower c h l o r o f o r m layer into a 600 ml
b e a k e r a n d r e - e x t r a c t t h e a q u e o u s l a y e r w i t h a n a d d i t i o n a l 50 m l
chloroform. C o m b i n e t h e c h l o r o f o r m e x t r a c t s . D i s c a r d the a q u e o u s
layer.

T r a n s f e r t h e c h l o r o f o r m e x t r a c t s b a c k to t h e s e p a r a t o r . R i n s e t h e
b e a k e r w i t h 100 m l 5% s a l t s o l u t i o n a n d a d d to the s e p a r a t o r . Shake
1 m i n and d r a i n l o w e r c h l o r o f o r m l a y e r t h r o u g h 5 cm of a n h y d r o u s
sodium sulphate into a 400 ml beaker. D i s c a r d the a q u e o u s p h a s e .
E v a p o r a t e c h l o r o f o r m on a s t e a m b a t h u n d e r a g e n t l e s t r e a m of
n i t r o g e n ( v a c u u m e v a p o r a t i o n m a y be u s e d - do n o t o v e r h e a t the d r y
extract).

Prepare a c h r o m a t o g r a p h i c c o l u m n by first inserting a glass w o o l plug

in the b o t t o m , t h e n a d d i n g a s l u r r y of 10 g c e l l u l o s e p o w d e r in 70 m l
m e t h a n o l - w a t e r (7 + 3 ) . W a s h the w a l l s of the c o l u m n w i t h m o r e
m e t h a n o 1 - w a t e r a n d d r a i n the s o l v e n t u n t i l it r e a c h e s the t o p of t h e
c é l l u l o s e . N e x t add 100 m l h e x a n e and d r a i n o f f 50 m l . P l a c e a p l u g
o f g l a s s w o o l o n t o p o f c e l l u l o s e , t a m p f i r m l y to p a c k ( p r e v e n t s
c h a n n e l i n g ) . D r a i n r e m a i n i n g h e x a n e to t o p of c e l l u l o s e .

D i s s o l v e the dry e x t r a c t w i t h 1 m l c h l o r o f o r m and add 2 m l b e n z e n e

a n d 10 m l h e x a n e . T r a n s f e r to t h e p r e p a r e d c o l u m n . W a s h t h e e m p t y
s a m p l e b e a k e r w i t h 5 m l h e x a n e - b e n z e n e ( 3 + 1 ) a n d add to c o l u m n .
D r a i n c o l u m n u n t i l s a m p l e s o l u t i o n r e a c h e s t o p of g l a s s w o o l . Add a
w a s h of 150 m l h e x a n e - b e n z e n e (3+1) and then 150 m l h e x a n e - e t h e r
( 2 + 1 ) (let the f i r s t d r a i n to the t o p of the g l a s s w o o l b e f o r e a d d i n g
the second). Discard all c o l u m n w a s h e s .

E l u t e a f l a t o x i n M w i t h 200 m l h e x a n e - c h 1 o r o f o r m (1+1) and c o l l e c t

e l u a t e in a 1 5 0 m l b e a k e r . E v a p o r a t e on a s t e a m b a t h as b e f o r e .
D i s s o l v e r e s i d u e in a s m a l l a m o u n t o f c h l o r o f o r m a n d transfer
q u a n t i t a t i v e l y to a s m a l l g l a s s v i a l . E v a p o r a t e to d r y n e s s on a
steam bath using nitrogen.

198
 Add 100 pi chloroform (microlitre syringe is convenient) to the vial.
Cap and shake 1 m i n to d i s s o l v e r e s i d u e . P r e p a r e T L C p l a t e s as for
regular aflatoxin analysis. Spot two 20 y 1 sample spots plus 2, 4,
6, 8 and 10 p i M s t a n d a r d . Spot a 4 p i M s t a n d a r d over one s a m p l e
spot to serve as a c o n t r o l . D e v e l o p the p l a t e u s i n g i s o p r o p a n o l -
acetone-chloroform (5+10 + 85). Visualize the spots using longwave UV
light in a cabinet.

CALCULATION

By f o l l o w i n g t h e a b o v e m e t h o d e x a c t l y , the following sample

equivalents will be in the final extract:

Fluid milk 47.6 ml

Powdered milk 4.8 g
Cheeses and butter 23.8 g

Aflatoxin M ppb S x 0.5 x 100

W x 20

where :
S = pi of standard spot equivalent to sample

W = sample equivalent from above.

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 26.090-.094.

199
7.4 TEXT REFERENCES

1. LOVELOCK, J.E. 1963. Analytical Chemistry 35 474-81.

2. HALL, R.C. 1974. Journal of Chromatographic Science 12 152-160.

3. A N D E R S O N , R.J. & H A L L , R.C. 1980. A m e r i c a n L a b o r a t o r y 12 (2) 108-24.

4. W H I T A K E R , T.B., D I C K E N S , J.W. & M O N R O E , R.J. 1974. Journal of the

American Oil Chemists Society 51 214-218.

5. S T O L O F F , L. & T R A G E R , W. 1965. J o u r n a l of the A s s o c i a t i o n of Official

Agricultural Chemists 48 681-2.

Further Reading

Pesticide Residues

GETZ, M. 1980. Paper and Thin Layer Chromatographic Analysis of Environmental

Toxicants. Heyden & Son Ltd., London, N W 4 2JQ UK.

ERNEY, D.R. 1983. Rapid Screening Procedure for P e s t i c i d e s and

P o l y c h l o r i n a t e d B i p h e n y l s in F i s h : C o l l a b o r a t i v e Study. J o u r n a l of the
Association of Official Analytical Chemists 66 969-73.

ÜS Food and Drug A d m i n i s t r a t i o n . Pesticide Analytical Manual. Item No.

NTISUB/C/118. National Technical I n f o r m a t i o n Service, 5285 Port Royal Road,
Springfield, VA 22161, USA.

S H E R M A , J. & B E R O Z A , M. 1981. M a n u a l of A n a l y t i c a l Q u a l i t y C o n t r o l for

P e s t i c i d e s and R e l a t e d C o m p o u n d s in H u m a n and E n v i r o n m e n t a l S a m p l e s . US
Environmental Protection Agency, Research Triangle Park. NC 27711, USA.

VETTORAZZI, G. 1979. International Regulatory Aspects for Pesticide Chemicals

Vol. I T o x i c i t y P r o f i l e s . Vol. II T a b l e s and B i b l i o g r a p h y . CRC P r e s s , Boca
R a t o n , FL 3 3 4 3 1 , USA.

W H O T A S K FORCE. 1976. E n v i r o n m e n a l H e a l t h C r i t e r i a for P o 1 y c h 1 o r i n a t e d

B i p h e n y l s and T e r p h e n y l s . E n v i r o n m e n t a l H e a l t h C r i t e r i a 2. World Health
Organisation, Geneva, Switzerland.

ZWEIG, G. (Ed.) Analytical Methods of Pesticides and Plant Growth Regulators,

Vols 1-13, Academic Press.

ANSON MOYE, H. 1981. Analysis of Pesticide Residues, Wiley.

Metal Residues

WHO TASK GROUP. 1977. Environmental Health Criteria for Lead. Environmental
Health Criteria 3. World Health Organisation, Geneva, Switzerland.

JOINT FAO/WHO EXPERT COMMITTEE ON FOOD ADDITIVES. 1972. Evaluation of Certain

Food Additives and the Contaminants Mercury, Lead and Cadmium.

World Health Organisation Technical Report Series No. 505, World Health
Organisation, Geneva, Switzerland.

200
SKOGERBOE, R.K. 1982. The Analytical Blank: Sources and Effects on Lead
Analyses. Journal of the Association of Official Analytical Chemists 65 957-
64. ~~

Mycotoxin Residues

L I N S E L L , C.A. 1982. C a r c i n o g e n i c i t y of M y c o t o x i n s . In Env i r o n m e n t a l

C a r c i n o g e n s , Selected M e t h o d s of A n a l y s i s , Vol 5, Some M y c o t o x i n s . IARC
Science P u b l i c a t i o n 44, International A g e n c y for R e s e a r c h on Cancer, L y o n ,
France 3-14.

SCHULLER, P.L., Van E G M O N D , H.P. & STOLOFF, L. 1983. L i m i t s and R e g u l a t i o n s

on Mycotoxins. In Proc. of the International Symposium on Mycotoxins. NAGUIB,
K., NAGUIB, M., PARK, D.L. & P O H L A N D , A.E. (Eds). Gen'l. O r g a n i s a t i o n for
G o v e r n m e n t Printing Offices, Cairo, Egypt. 111-129. And in abbreviated form
b y : S C H U L L E R , P.L., S T O L O F F , L. & v a n E G M O N D , H.P. 1982. L i m i t s and
Regulations. In Environmental Carcinogens, Selected Methods of Analysis, Vol
5, Some M y c o t o x i n s . IARC Science P u b l i c a t i o n 44. I n t e r n a t i o n a l A g e n c y for
Research on Cancer, Lyon, France. 107-16.

STOLOFF, L. (Ed). 1980. Mycotoxins Methodology Association of Official

Analytical Chemists, Arlington, VA 22209, USA 8-9.

D I C K E N S , J.W. & W H I T A K E R , T.B. 1982. S a m p l i n g and S a m p l e P r e p a r a t i o n . In

E n v i r o n m e n t a l Carcinogens, Selected M e t h o d s of A n a l y s i s , Vol. 5, Some
Mycotoxins. IARC Science Publication 44. International Agency for Research on
Cancer, Lyon, France 17-32.

WHO TASK GROUP. 1979. E n v i r o n m e n t a l Health Criteria for M y c o t o x i n s .

E n v i r o n m e n t a l Health Criteria II. World Health Organisation, Geneva,
Switzerland.

McKINNEY, J.D. 1984. Analyst Performance with Aflatoxin Methods as Determined

by the AOCS Smalley Check Sample P r o g r a m . Journal of the A s s o c i a t i o n of
Official Analytical Chemists 67 25-32.

RODRICKS, J.V., H E S S E L T I N E , C.W. & M E H L M A N , M.A. 197 7. M y c o t o x i n s in H u m a n

and Animal Health. Pathotox Publishers Inc., Illinois, USA.

Food Contamination Monitoring

FOOD & AGRICULTURE ORGANISATION & WORLD HEALTH ORGANISATION. 1979. Guidelines
for E s t a b l i s h i n g or Strengthening National Food C o n t a m i n a t i o n M o n i t o r i n g
Programmes. FAO Food Control Series No. 5. World Health Organisation, Geneva,
Switzerland.

WORLD HEALTH ORGANISATION. 1979. S u m m a r y Report of Data Received from

Collaborating Centres for Food Contamination Monitoring Stage 1-1977 under the
joint FAO/WHO Food and Animal Feed Contamination Monitoring Programme, Phase
II.

NATIONAL FOOD ADMINISTRATION. 1982. Summary and Assessment of Data Received

from the F A O / W H O Collaborating Centres for Food C o n t a m i n a t i o n M o n i t o r i n g ,
Uppsala, Sweden.

Expert Consultation on the Joint F A O / W H O Food C o n t a m i n a t i o n Monitoring

Programme (Rome, 7-11 Oct 1974) Identification of Contaminants to be Monitored
and R e c o m m e n d a t i o n s on Sampling Plans and M e t h o d o l o g y . Report FAO-ESN:
MON/74.21. Food and Agriculture O r g a n i s a t i o n of the United N a t i o n s , R o m e ,
Italy.

201
JOINT F A O / W H O EXPERT CONSULTATION. R o m e 27 Feb - 3 Mar 1978. M e t h o d s of
Sampling and Analysis of Contaminants in Food. Document No. ISB 92/5/100572/9.
Food and Agriculture Organisation of the United Nations, Rome, Italy.

Environmental Pollutants - Selected Analytical Methods (Scope 6) Butterworths

1975.

S C H O E N T A L , R. (Ed). Dietary Influences on C a n c e r - Traditional & Modern, CRC

Press.

GRIMMER, G. (Ed). Environmental Carcinogens: Polycyclic Aromatic Hydrocarbons

CRC Press.

GILBERT, J. 1984. Analysis of Food Contaminants Elsevier.

CROSBY, N.T. 1981. Food Packaging Materials Elsevier.

B A R T L E , K.D., L E E , M.L. & W I S E , S.A. M o d e r n A n a l y t i c a l M e t h o d s for

Environmental Polycyclic Aromatic Compounds Royal Society of Chemistry, London.

202
 8. COMPOSITIOHAL ANALYSIS METHODS

Compositional analysis is more c o m m o n l y r e f e r r e d to as " p r o x i m a t e " a n a l y s i s .

It refers to the determination of the major constituents of a food, and is used
to assess if the food is within normal compositional parameters, or has somehow
b e e n adulterated.

The four usual constituents measured for compositional analysis are:

Moisture
Fat
Protein
Ash

Other constituents such as fibre and added substances such as salt and starch
also m a y be d e t e r m i n e d to g i v e f u r t h e r i n f o r m a t i o n on food c o m p o s i t i o n ,
especially for processed foods.

8.1 Moisture

W a t e r is o f t e n one of the m a j o r c o n s t i t u e n t s in a food. It is held to other

constituents by physical and chemical forces of diverse nature and strength and
its determination is often subject to a number of inaccuracies. The variety of
w a y s in w h i c h w a t e r is p r e s e n t in food i n c l u d e : as a s o l v e n t or d i s p e r s i o n
m e d i u m , as mono- or polymolecular layers or in capillaries, held by molecular
forces, or combined as water of hydration, either as true hydrates or held by
h y d r o g e n b o n d i n g w i t h i n p r o t e i n and p o l y s a c c h a r i d e m o l e c u l e s . W a t e r is not
released from these different states with equal ease and therefore different
and to some extent arbitrary methods are used with different foods.

The t w o m o s t c o m m o n m o i s t u r e d e t e r m i n a t i o n s are d i r e c t d r y i n g and co-

distillation. C h e m i c a l m e t h o d s such as K a r l - F i s c h e r t i t r a t i o n w i l l not be
discussed here.

In d i r e c t d r y i n g , the p r e s e n c e of other v o l a t i l e s u b s t a n c e s and c h e m i c a l

reactions leading to the creation of water and other volatile substances will
cause false h i g h r e s u l t s . F a l s e l o w r e s u l t s m a y o c c u r due to i n c o m p l e t e
r e m o v a l of w a t e r u n d e r the d r y i n g c o n d i t i o n s c h o s e n . If d r y i n g is c o n t i n u e d
for too long a period there m a y be an a c t u a l i n c r e a s e in w e i g h t due to
oxidation. The r e s u l t w i l l also v a r y w i t h the e x t e n t to w h i c h the cell
structure is broken down as well as the particle size. There are many possible
sources of error. For routine moisture determinations at ambient pressure, use
an oven with air circulation. Dishes of aluminium, nickel, stainless steel or
platinum are suitable. Moisture determinations carried out in silica tend to
be less accurate as moisture may be picked up during cooling prior to weighing.
On the other hand, it is sometimes convenient to determine the ash subsequently
in which case silica, porcelain or platinum must be used. A well-fitting lid
is e s s e n t i a l for s a m p l e s that are h y g r o s c o p i c o n c e d r i e d (such as m i l k and
cereals). The lid is placed on the dish just prior to the r e m o v a l of the
latter from the oven. The cooling desiccator must contain effective desiccant,
s e l f - i n d i c a t i n g silica gel being c o n v e n i e n t . An h o u r or t w o in the oven is
s u f f i c i e n t to r e g e n e r a t e this. D i s h e s m u s t be left in the d e s i c c a t o r long
e n o u g h for the m o i s t u r e film on the o u t s i d e of the dish to r e a c h e q u i l i b r i u m
with the surroundings. This may take 30-40 minutes. Samples that contain fat
or can form an i m p e r v i o u s skin are dried on sand. The sand is p r e p a r e d by
b o i l i n g w i t h 50% h y d r o c h l o r i c acid u n t i l t h e r e is no f u r t h e r e x t r a c t and
washing free of chloride with water.

Standard methods for moisture determination by oven drying stipulate drying at

a certain temperature either for a specified time or until constant weight is
achieved. In the latter c a s e , the s a m p l e is r e m o v e d after a f e w h o u r s ,
w e i g h e d , and r e t u r n e d to the o v e n for a p p r o x i m a t e l y one h o u r p e r i o d s u n t i l
successive weighings do not differ by more than a small amount (0.5 mg, 1 mg, 2

203
m g or 5 m g v a r y i n g w i t h the m e t h o d and the m a t e r i a l ) . E v e n if t h e d i f f e r e n c e s
e x c e e d t h e s p e c i f i e d v a l u e i t is p r o b a b l y n o t w o r t h w h i l e t o c o n t i n u e d r y i n g
o n c e the w e i g h t s in e a c h s u c c e s s i v e h e a t i n g a r e n o t d e c r e a s i n g s i g n i f i c a n t l y .

D r y i n g u n d e r v a c u u m at r e d u c e d p r e s s u r e is u s e d for p r o d u c t s s u c h as f r u i t a n d
v e g e t a b l e s w h e r e c h e m i c a l r e a c t i o n s and the p r e s e n c e of v o l a t i l e s u b s t a n c e s
w o u l d g i v e r i s e to s e r i o u s e r r o r s at a h i g h e r t e m p e r a t u r e . V a c u u m m u s t be
o b t a i n e d and t h e n w a t e r r e l e a s e d s l o w l y e n o u g h so t h a t l o s s f r o m t h e d i s h d o e s
not occur. T h e r e m u s t b e a d r y a i r - b l e e d p a s s i n g t h r o u g h the o v e n . T h e a i r is
t a k e n f r o m a c y l i n d e r or m a y be c o n v e n i e n t l y d r i e d b y b u b b l i n g through
concentrated sulphuric acid. T w o p r e s s u r e s a r e c o m m o n l y u s e d , a r o u n d 100 m m
H g , o r a r o u n d 2 5 m m H g . U n d e r t h e S . I . s y s t e m o f u n i t s p r e s s u r e is e x p r e s s e d
as p a s c a l s (Pa). 2 5 m m H g is e q u i v a l e n t t o 3.3 k P a . In s o m e m e t h o d s , t h e
p r e s s u r e is e x p r e s s e d as m i l l i b a r s (1000 m i l l i b a r s e q u a l 7 6 0 m m H g ) .

T h e r e a r e a n u m b e r of r a p i d t h e r m o g r a v i m e t r i c m e t h o d s for d e t e r m i n i n g m o i s t u r e .
T h e s e h a v e o f t e n b e e n d e v e l o p e d in r e s p o n s e to the n e e d s of i n d u s t r y for r a p i d
quality control procedures. P r o v i d e d r e s u l t s are r e p r o d u c i b l e , c o r r e c t i o n s can
be m a d e for k n o w n i n a c c u r a c i e s . I n f r a - r e d m o i s t u r e t e s t e r s and the B r a b e n d e r
m o i s t u r e o v e n for f l o u r s a m p l e s a r e e x a m p l e s of e q u i p m e n t u s e d for t h i s t y p e of
method.

A z e o t r o p i c c o - d i s t i l l a t i o n is o f t e n u s e d for f o o d s c o n t a i n i n g v o l a t i l e o i l s . A
w a t e r - i m m i s c i b l e o r g a n i c s o l v e n t ( o f t e n t o l u e n e ) is b o i l e d w i t h t h e f o o d a n d
w a t e r v a p o u r d i s t i l s o v e r , b e i n g c o l l e c t e d in a g r a d u a t e d t r a p . T h e p r o c e s s
t e n d s to b e i n c o m p l e t e so t h a t r e s u l t s m a y b e l o w . The organic solvent should
b e s h a k e n w i t h a s m a l l q u a n t i t y of w a t e r and d i s t i l l e d p r i o r to u s e . T h e u p p e r
p a r t s of a p p a r a t u s m a y b e w r a p p e d in an i n s u l a t i n g m a t e r i a l in o r d e r to o b t a i n
a more even heating. A s p i r a l c o p p e r w i r e p a s s e d d o w n the c o n d e n s e r w i l l
d i s l o d g e a n y w a t e r a d h e r i n g to the w a l l s . O c c a s i o n a l a d d i t i o n o f 5 m l p o r t i o n s
of t o l u e n e d o w n the c o n d e n s e r m a y a l s o a s s i s t in t h i s . R e f l u x i n g is c o n t i n u e d
u n t i l t h e w a t e r l e v e l in t h e r e c e i v e r r e m a i n s u n c h a n g e d f o r 30 m i n u t e s a n d the
r e a d i n g t a k e n 2 0 m i n u t e s o r so a f t e r r e m o v i n g t h e s o u r c e o f h e a t . The
d e t e r m i n a t i o n s h o u l d b e c a r r i e d o u t in a f u m e h o o d d u e to the t o x i c n a t u r e of
the s o l v e n t s and the f o r m of h e a t i n g m u s t n o t b e a n a k e d f l a m e , d u e to the f i r e
risk. If d i f f i c u l t y is e x p e r i e n c e d w i t h d r o p l e t s o f w a t e r a d h e r i n g to t h e
w a l l s of the c o n d e n s e r , the c o o l i n g w a t e r m a y be t u r n e d o f f for a f e w m i n u t e s
b e f o r e the end of the d i s t i l l a t i o n . Cleaning the a p p a r a t u s , i n c l u d i n g the
condenser, in c h r o m i c a c i d p r i o r t o u s e is a l s o h e l p f u l . The rate of
d i s t i l l a t i o n s h o u l d be a b o u t 100 d r o p s p e r m i n u t e , i n c r e a s i n g to a b o u t 2 0 0
d r o p s per m i n u t e n e a r the end of the h e a t i n g p e r i o d . H e a t i n g for an h o u r or
l o n g e r m a y b e n e c e s s a r y to d i s t i l a l l the w a t e r .

T h e r e s i s t a n c e of f o o d s to the p a s s a g e of an e l e c t r i c c u r r e n t v a r i e s w i t h the
a m o u n t of w a t e r p r e s e n t . T h e e f f e c t h a s b e e n u s e d as t h e b a s i s for t h e d e s i g n
of s e v e r a l m o i s t u r e m e t e r s . M o i s t u r e m e t e r s d e p e n d i n g on the c o n d u c t i v i t y
p r i n c i p l e h a v e m e a n s for c o m p r e s s i n g t h e s a m p l e in a c h a m b e r of d e f i n i t e s i z e
to a c h i e v e a d e q u a t e l y c o n s i s t e n t r e s u l t s . The area of c o n t a c t b e t w e e n the
c o n d u c t i n g p a r t i c l e s a n d h e n c e t h e p a r t i c l e s i z e h a v e an i m p o r t a n t e f f e c t in
the c o n d u c t i v i t y of the m a s s . T h e m o i s t u r e a l s o h a s to b e e v e n l y d i s t r i b u t e d
t h r o u g h o u t t h e f o o d in o r d e r to o b t a i n r e l i a b l e r e s u l t s .

204
 MOISTURE
(Air Oven Method)

PRINCIPLE

The sample is dried at 100-102°C for 16-18 hours or at 125°C for 2-4 hours in a
forced draft air oven. The loss in w e i g h t is reported as m o i s t u r e . This
method is applicable to meat and fish, and other products. If the product is a
cereal, heat for one hour at 130°C. If it is a dried vegetable or tea, heat in
a v a c u u m oven for five hours at 100°C using 100 m m of m e r c u r y . If it is a
spice or is expected to contain volatile oils, use the toluene d i s t i l l a t i o n
method .

APPARATUS

1. Forced draft air oven maintained at appropriate temperature.

2. Moisture dishes.

3. Desiccator containing absorbing material (e.g. Silica gel).

4. Analytical balance.

PROCEDURE

Reduce sample to fine form and mix well. Weigh accurately 3-4 g of
the sample (in d u p l i c a t e ) into m o i s t u r e dishes. (Sample should be
spread evenly across the dish and weighed as rapidly as possible to
minimize loss of moisture). Dry the sample for 16-18 hours at 100-
102°C, or for 4 hours at 125°C. (Care m u s t be exercised that drying
oven is not overloaded or samples w i l l be i n s u f f i c i e n t l y dried and
lower results will be obtained.) After the drying is c o m p l e t e ,
remove samples from the oven and place in desiccator. Cool to room
temperature (for about 30 minutes) and weigh accurately.

CALCULATION

Moisture (%) = " C> x 100

Where A = sample weight in g.

B = weight of dish + sample prior to drying

C = weight of dish + sample after drying

(B-C) = loss in weight of sample after drying

REFERENCE

Official Methods of Analysis of the A0AC, 1984, 24.003.

205
 MOISTURE
(Toluene Distillation Method)

PRINCIPLE

T h i s m e t h o d is a p p l i c a b l e for m o s t d r i e d p r o d u c t s w i t h a m o i s t u r e c o n t e n t
g r e a t e r t h a n 1%. It h a s b e e n f o u n d v e r y u s e f u l for the d e t e r m i n a t i o n of
m o i s t u r e in m i l k p o w d e r s and in g r a i n p r o d u c t s . T h e m e t h o d is of s p e c i a l
importance w h e n it is desired to determine water in spices or other foods which
c o n t a i n v o l a t i l e o i l s . T o l u e n e is a d d e d to a s a m p l e in a f l a s k and h e a t e d to
boiling. T h e w a t e r and t o l u e n e b o i l o f f as an a z e o t r o p i c m i x t u r e . Upon
cooling the water and toluene separate and the water is collected in a special
t r a p and m e a s u r e d . B e n z e n e is u s e d i n s t e a d of t o l u e n e on red p e p p e r s , c h i l i
powder and paprika which tend to decompose at 100°C to release water.

APPARATUS

1. Balance - approximately 500 g capacity and 0.1 g or better

sensitivity.

2. Condenser - Liebig, 500 mm in length.

3. C o n d e n s e r b r u s h - o v e r a l l l e n g t h 24", l e n g t h of b r i s t l e t u f t 3
diameter of tuft.

4. Distillation traps - Dean and Stark or Bidwell-Stelling type (5

ml graduated in 0.1 divisions).

5. Electric heater - with rheostat control to m a i n t a i n a

distillation rate of a p p r o x i m a t e l y 4 d r o p s / s e c u n d e r c o n d i t i o n s of
the test.

6. Flask - Erlenmeyer or round bottom, 300 ml narrow-mouth.

REAGEHTS

1. Toluene (technical grade) (Blank determinations should be made.)

PROCEDURE

Accurately w e i g h an appropriate amount of sample (see b e l o w ) and as

q u i c k l y as p o s s i b l e t r a n s f e r to a c l e a n , d r y 3 0 0 m l E r l e n m e y e r or
round bottom flask.

Moisture Estimate Sample Size

5% 50 g

5 - 10% 30 g

10 - 25% 20 g

I m m e d i a t e l y pour e n o u g h t o l u e n e to c o v e r s a m p l e c o m p l e t e l y ( a b o u t
100-150 ml). Rinse d o w n any particles on the side of the flask when
i n t r o d u c i n g the t o l u e n e . S w i r l the flask w i t h s a m p l e t h o r o u g h l y .
Connect the flask to the distillation trap and the condenser. Make
s u r e that all j o i n t s are t i g h t . F i l l r e c e i v i n g tube w i t h t o l u e n e ,
p o u r i n g it t h r o u g h the top of the c o n d e n s e r . H e a t the c o n t e n t s to
boiling but make sure that the sample does not scorch on the bottom
of the f l a s k (see that cold w a t e r is c i r c u l a t i n g through the
condenser). The a m o u n t of h e a t s h o u l d be so r e g u l a t e d that the
t o l u e n e w i l l c o n d e n s e i n t o the t r a p at a r a t e of 4 d r o p s / s e c . This

206
 rate is m a i n t a i n e d throughout the r e m a i n d e r of the d i s t i l l a t i o n .
(Most of the w a t e r in the s a m p l e w i l l pass over to the trap w i t h i n
the first 15 min).

W h e n all the water is a p p a r e n t l y in the trap (usually about 45 min

after distillation has begun) and without interrupting distillation,
dislodge water droplets in the condenser tube by means of a condenser
brush. While the brush is in the upper port of the condenser, flush
the tube with about ten ml of toluene. If any water adheres to the
side of the tube (trap), rub it d o w n by m e a n s of a long, stiff wire.
Read the m o i s t u r e level in the trap to the nearest half a scale
division (0.05 ml). Continue the d i s t i l l a t i o n for an a d d i t i o n a l 30
m i n to see if more water distils over. If so, repeat w a s h i n g - d o w n
process and note the water level in the trap. If this reading agrees
w i t h the p r e v i o u s r e a d i n g w i t h i n 0.05 ml w a t e r in the t r a p ,
d i s c o n t i n u e the d i s t i l l a t i o n . (For s o m e p r o d u c t s , a 75 m i n
distillation period is not sufficient. If readings fail to agree as
required, continue the d i s t i l l a t i o n for a d d i t i o n a l 15 m i n periods
until successive results agree within a 1/2 scale division). Record
the amount of water collected in the trap.

CALCULATION

% moisture in sample = V x 100

Where: V = volume of water in trap (in ml)

S = sample weight (g)

INTERPRETATION
It is e x t r e m e l y i m p o r t a n t to clean the condenser and d i s t i l l a t i o n trap
thoroughly in order to m i n i m i z e the adherence of water to the glass surface
during distillation. If e q u i p m e n t is dirty or greasy, d i f f i c u l t y will be
experienced in dislodging water droplets and results may be unreliable. Wash
glass e q u i p m e n t by soaking in and scrubbing with a solution of suitable
d e t e r g e n t ; then rinse w i t h clean water. If this fails to r e m o v e dirt and
grease, soak glassware overnight in chromic acid cleaning solution and rinse
equipment well with clean water and dry thoroughly. Distillation traps require
thorough cleaning after each determination and condensers usually once a week.

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 7.004-.005.

207
 MOISTORE
(Vacuum Oven Method)

PRINCIPLE

T h i s m e t h o d is a p p l i c a b l e e s p e c i a l l y to p r o c e s s e d v e g e t a b l e s . The g r o u n d or
comminuted sample is first partially dried, by one of three techniques, then is
vacuum dried.

APPARATUS

1. S i e v e w i t h square o p e n i n g 2.8 x 2.8 cm (11.2 x 11.2 for t o m a t o ) ,

20 cm diameter if total contents of sample less than 1.5 kilos, 30 cm
if more.

2. Food chopper or blender.

3. Water-bath or forced draft oven.

4. V a c u u m oven at 70°C w i t h t e m p e r a t u r e variation on different

parts of the shelf not exceeding about 2°C.

PROCEDURE

For foods c o m p o s e d of solid and liquid p o r t i o n s (e.g., canned

v e g e t a b l e s ) if only the solid p o r t i o n is r e q u i r e d for a n a l y s i s ,
t h o r o u g h l y grind the d r a i n e d food in a m o r t a r or food c h o p p e r .
H o w e v e r , if a c o m p o s i t e of solid and liquid p o r t i o n is r e q u i r e d ,
grind the entire contents of the can in a mortar or food chopper. In
all cases, thoroughly mix the portion used and store the remainder in
a glass-stoppered container.

For c o m m i n u t e d p r o d u c t s ( t o m a t o j u i c e , t o m a t o c a t s u p , s t r a i n e d
vegetables) thoroughly shake the u n o p e n e d c o n t a i n e r to i n c o r p o r a t e
any sediment. Transfer entire contents to large glass or porcelain
dish, and mix thoroughly, continuing stirring 1 minute. Transfer the
well-mixed sample to a glass-stoppered container and shake or stir
thoroughly each time before removing portions for analysis.

To a flat-bottomed metal dish with a tight-fitting cover, add about

15 m g of d i a t o m a c e o u s e a r t h fi 1 1 e r - a i d / s q u a r e c m , dry a b o u t 30
m i n u t e s at 110°C, cool in d e s i c c a t o r , w e i g h , and to each dish add a
sample of such size that the dry residue will be over 9 but less than
30 m g / s q cm. W e i g h as r a p i d l y as p o s s i b l e to avoid m o i s t u r e loss.
Mix with filter-aid and distribute uniformly over the bottom of the
d i s h , d i l u t i n g w i t h w a t e r if n e c e s s a r y to f a c i l i t a t e d i s t r i b u t i o n .
B r i n g the s a m p l e to a p p a r e n t d r y n e s s ( r e m a i n i n g m o i s t u r e n o t m o r e
than about 50% dry solids) by one of the following methods:

(a) P l a c e s a m p l e s on a b o i l i n g w a t e r - b a t h and remove when

samples reach apparent dryness.

(b) P l a c e s a m p l e s in a f o r c e d - d r a f t o v e n at 70°C. The o v e n

m u s t h a v e rapid air c i r c u l a t i o n and e n o u g h e x c h a n g e w i t h
o u t s i d e air to r e m o v e the m o i s t u r e r a p i d l y . E x a m i n e the
d i s h e s at i n t e r v a l s of 30 m i n u t e s and r e m o v e as soon as
they reach apparent dryness.

(c) P l a c e s a m p l e s in a v a c u u m oven at 70°C w i t h the v a c u u m

l e a k - v a l v e left p a r t l y o p e n to a l l o w a rapid f l o w of air
t h r o u g h the oven at 310 m m Hg. E x a m i n e the d i s h e s at 30
m i n u t e i n t e r v a l s and r e m o v e w h e n they r e a c h a p p a r e n t
dryness.

208
 Place the partially dried samples in a vacuum oven with the bottoms
of the dishes in direct contact w i t h the shelf. M e a s u r e the
temperature of the oven by a thermometer in direct contact with the
shelf. A d m i t dry air to the oven at a rate of 2 - 4 bubb 1 e s / s econd
by bubbling through sulphuric acid. Dry the s a m p l e s for 2 hours at
69 - 71 °C (oven m a y be as low as 65°C at the start of the d r y i n g , but
m u s t reach 69 - 71°C before the end of the first h o u r ) at a pressure
of 50 m m Hg. (As the dried sample will absorb an appreciable amount
of moisture on standing over most desiccating agents, cover quickly
and w e i g h as soon as possible after the s a m p l e reaches room
temperature.)

CALCULATION

% total solids = weight of dry residue x 10Q

weight of sample taken

The "water capacity" of a rigid container such as a can or glass jar

is the v o l u m e of distilled w a t e r at 20°C w h i c h the c o n t a i n e r w i l l
hold when completely filled. In the case of cans, the lid is cut off
without removing or altering the height of the double seam. The can
is weighed when clean and dry, filled with distilled water at 20°C to
within 4.8 mm vertical distance below the top and weighed again. The
w e i g h t in g r a m s is taken to be equal to the v o l u m e in ml (the error
being less than 0.2%). The total solids m a y be expressed as a
percentage of the water capacity by use of the following formula:

Total solids as % total solids in product x net contents

% of water capacity ~ water capacity

REFERENCE

Official Methods of Analysis of the AOAC, 1984, 14.002-.003.

209
8.2 Fat

T h e f a t t y m a t e r i a l o r " c r u d e f a t " in f o o d s is f r e q u e n t l y d e t e r m i n e d by
e x t r a c t i o n of the d r i e d food w i t h d i e t h y l e t h e r or p e t r o l e u m e t h e r or of an
a q u e o u s m i x t u r e of the food b y a m i x t u r e of t h o s e t w o s o l v e n t s , f r o m a l k a l i in
the R o s e - G o t t l i e b m e t h o d and f r o m a c i d in t h e W e i b u l 1 - S t o 1dt and S c h m i d -
B o n d z y n s k i - R a t z l a f f m e t h o d s . O n e w o r k e r r e c o m m e n d e d u s e of c h l o r o f o r m / m e t h a n o l
m i x t u r e s in t h e p r e s e n c e of w a t e r . T h i s m e t h o d g i v e s s i g n i f i c a n t l y h i g h e r
r e s u l t s in s o m e c a s e s , f o r e x a m p l e in r a w l e a n b e e f . T h i s is p a r t l y b e c a u s e
c h l o r o f o r m / m e t h a n o l is a m o r e e f f e c t i v e s o l v e n t for p h o s p h o l i p i d s a n d s o m e of
the o t h e r f a t t y s u b s t a n c e s c o m m o n l y f o u n d in s m a l l a m o u n t s . In t h e u s e of t h i s
m e t h o d t h e p r e s e n c e of a h i g h p o l a r i t y s o l v e n t ( w a t e r , m e t h a n o l ) is i m p o r t a n t
to d i s r u p t h y d r o g e n b o n d s a n d r e l e a s e f a t b o u n d to p r o t e i n o r c a r b o h y d r a t e .
T h e p r o c e d u r e w a s o r i g i n a l l y i n t e n d e d for t h e e x t r a c t i o n of t o t a l l i p i d s f r o m
animal tissues. A l t h o u g h t r i g l y c e r i d e s and p h o s p h o l i p i d s a r e e x t r a c t e d f r o m
p r o c e s s e d f o o d , c h l o r i n e and s i m i l a r c o m p o u n d s w i l l n o t be e x t r a c t e d w i t h o u t
p r i o r s p l i t t i n g w i t h h y d r o c h l o r i c a c i d as t h e y r e m a i n i n c o r p o r a t e d in t h e
protein matrix.

D r y i n g of the s a m p l e p r i o r to e x t r a c t i o n w i t h d i e t h y l e t h e r or p e t r o l e u m e t h e r
m a y c a u s e f o r m a t i o n of a " s k i n " t h a t r e t a r d s e x t r a c t i o n . T h i s c a n s o m e t i m e s b e
a v o i d e d b y d r y i n g the s a m p l e o v e r sand and t r a n s f e r r i n g the d r y m a t e r i a l and
the s a n d to t h e e x t r a c t i o n a p p a r a t u s . P l a n t cells c o n t a i n i n g fat w i l l r e t a i n
t h i s f a t , so t h a t it is n e c e s s a r y to d i s r u p t the c e l l s b y a b r a s i o n of t h e c e l l
w a l l s w i t h s a n d a f t e r the r e m o v a l of f r e e f a t a n d c o n t i n u e t h e e x t r a c t i o n for a
f u r t h e r p e r i o d of t i m e . T h i s p r o c e d u r e m a y a l s o a u g m e n t the a m o u n t of fat
o b t a i n e d f r o m s a m p l e s in w h i c h t h e f a t is b o u n d to o t h e r c o n s t i t u e n t s s u c h as
protein. D a m p d i e t h y l e t h e r d i s s o l v e s s u g a r s and o t h e r m a t e r i a l s to s o m e
e x t e n t a n d for t h i s r e a s o n , m e t h o d s c o n s i s t of e i t h e r e x t r a c t i o n of t h e d r i e d
s a m p l e b y p e t r o l e u m e t h e r or e x t r a c t i o n of t h e s a m p l e in w a t e r b y a m i x t u r e of
d i e t h y l e t h e r a n d p e t r o l e u m e t h e r i n o r d e r to r e d u c e t h e e x t r a c t i o n o f n o n -
f a t t y c o m p o n e n t s . E x t r a c t i o n o f t h e d r i e d m a t e r i a l w i t h p e t r o l e u m e t h e r is
c o m m o n l y u s e d f o r f a t d e t e r m i n a t i o n in m e a t p r o d u c t s , c e r e a l s a n d o t h e r l o w -
s u g a r f o o d s t h a t are d r y or e a s i l y d r i e d . T h e e x t r a c t i o n m a y b e c a r r i e d o u t in
the c o l d ( S o x h l e t ) or h o t ( B o l t o n , G o l d f i s c h and B a i l e y - W a l k e r e x t r a c t o r s ) .
D i r e c t e x t r a c t i o n of the d r y m a t e r i a l m a y g i v e l o w r e s u l t s as the p r o t e i n
m a t r i x h a s to b e d e s t r o y e d b y a c i d or a l k a l i .

T h e R o s e - G o t t l i e b m e t h o d is u s e d for f o o d s c o n t a i n i n g p r o t e i n and s u g a r s u c h as
m i l k a n d m i l k p r o d u c t s ; t h e p r o t e i n is d i s s o l v e d b y a m m o n i a a n d n o t b y a c i d
( w h i c h a c t s on s u g a r s y i e l d i n g e t h e r - s o 1 u b l e p r o d u c t s ) . T h e m e t h o d d e p e n d s
u p o n e x t r a c t i o n f r o m an e t h a n o l i c a m m o n i a m i x t u r e b y m i x e d d i e t h y l and
petroleum ethers. T h e e t h a n o l i c a m m o n i a s t a b i l i z e s the p r o t e i n p r e s e n t . The
s a m e s o l v e n t s a r e u s e d in the S c h m i d - B o n d z y n s k i - R a t z l a f f m e t h o d , b u t p r o t e i n is
first d e c o m p o s e d by hydrochloric acid. T h e m e t h o d is u s e d for m a t e r i a l s s u c h
as c h e e s e b u t a n u n d u e a m o u n t of c h a r r i n g o c c u r s in s a m p l e s h i g h in s u g a r . The
W e i b u l l - S t o l d t m e t h o d i n v o l v e s d i g e s t i o n of t h e s a m p l e in d i l u t e acid,
f i l t e r i n g and d r y i n g the fat t o g e t h e r w i t h any other m a t e r i a l r e t a i n e d by the
f i l t e r p a p e r , c o m p l e t i o n b e i n g b y e x t r a c t i o n of t h e p a p e r in a S o x h l e t
a p p a r a t u s w i t h p e t r o l e u m e t h e r . T h e m e t h o d is t h e b a s i s o f t h a t o f t h e O I C C
f o r e s t i m a t i o n o f f a t in c h o c o l a t e a n d w a s f o u n d p r e f e r a b l e to t h e S c h m i d -
B o n d z y n s k i - R a t z l a f f a n d R o s e - G o t t l i e b m e t h o d s for t h e d e t e r m i n a t i o n of f a t in
b a b y food.

T h e f a t t y m a t e r i a l in food d o e s n o t c o n s i s t o n l y of f a t t y a c i d t r i g l y c e r i d e s ,
a l t h o u g h t h e s e n o r m a l l y f o r m b y far the l a r g e r p a r t . S m a l l q u a n t i t i e s of m o n o -
and d i g l y c e r i d e s m a y b e p r e s e n t e i t h e r n a t u r a l l y or as a d d e d e m u l s i f i e r s . Many
o f t h e e m u l s i f i e r s u s e d i n f o o d a r e p a r t l y o r w h o l l y s o l u b l e in t h e s o l v e n t s
u s e d to e x t r a c t f a t f r o m s a m p l e s . S t e r o l s ( s u c h as c h o l e s t e r o l and beta-
s i t o s t e r o l ) and p h o s p h o l i p i d s and their d e r i v a t i v e s ( s u c h as lecithin,
p h o s p h a t i d y l i n o s i t o l and o t h e r i n o s i t i d e s ) and p h o s p h a t i d y l d e r i v a t i v e s of
a m i n o - c o m p o u n d s ( s u c h as s e r i n e and e t h a n o 1 a m i n e ) a r e a l s o p a r t l y or w h o l l y
e x t r a c t a b l e u n d e r the e x p e r i m e n t a l c o n d i t i o n s of s o m e c o m m o n l y u s e d m e t h o d s .
In m o s t c a s e s t h e e r r o r so i n t r o d u c e d is n o t l a r g e a s t h e p r o p o r t i o n o f f a t t y

210
m a t e r i a l other than triglycerides is very low. E x c e p t i o n s include egg yolk,
the lipids of w h i c h contain only a little over 60% of t r i g l y c e r i d e s , the
remainder being mainly lecithin, cephalin and cholesterol. Triglycerides may
not be completely extracted by hexane, hot isopropanol or ethylene dichloride.
Methods of fat determination have been received by Hannant (1) and Carter (2).
There is a useful discussion in Pearson (3), pp 20-23.

211
 CRUDE FAT

PRINCIPLE

Crude fat can be determined by extracting the dried ground food material with
anhydrous ethyl ether or petroleum ether (BP 40°-60°C) in a continuous
extraction apparatus of the Soxhlet type. The solvent is then removed from the
extract by evaporation and the residue is weighed and reported as fat.

APPARATUS

1. Soxhlet extraction apparatus.

2. Extraction thimbles (Whatman cellulose, single thickness,

3 OmmX10 Omm).

3. Filter paper. (Whatman No. 42).

4. Desiccator.

5. Evaporating dish.

6. Water-bath.

REAGENTS

1. Ethyl ether, anhydrous.

PROCEDURE

Accurately weigh 3-4 g of sample (in a fine form) into a thimble

lined with a circle of filter paper. Place thimble and contents into
a 50 ml beaker and dry in a mechanical convection oven for 6 hours at
100-102°C or for 1-1/2 hours at 125°C. (Note: the sample m u s t be
free of m o i s t u r e , otherwise some water-soluble material will be
extracted and reported as fat. However, it is important to avoid
excessive drying of samples, and to dry samples at 125°C or below to
prevent possible oxidation of fat.)

Transfer thimble and contents to extraction apparatus. Rinse beaker

several times with ethyl ether, adding rinsing to the apparatus.
Extract the sample contained in the thimble with ethyl ether in a
Soxhlet extraction apparatus for 6-8 hours at a condensation rate of
at least 3-6 drops per second. At the completion of the extraction,
transfer the fat extract from the extraction flask into a pre-weighed
evaporating dish with several rinsings of ethyl ether. Place the
evaporating dish in a fume hood and with the fan on, evaporate off
the ethyl ether until no odour of it is detectable.

Dry the dish and contents in a mechanical convection oven for 30

m i n u t e s at 100°C. Remove from the oven, cool in a desiccator and
weigh dish plus contents.

CALCULATION

(w x 100
Crude fat (ether extract) % = 2 ~

Where: Wj = weight of empty evaporating dish

W£ = weight of evaporating dish + contents after

drying

S = sample weight in g.

212
REFERENCES

Official Methods of Analysis of the AOAC, 1984, 7.060-.062.

Stubbs, G. and More, A., 1919, Analyst 44, 125.

213
 FAT
(Weibull—Stoldt Method)

PRINCIPLE

T h e s a m p l e is b o i l e d w i t h h y d r o c h l o r i c a c i d , f i l t e r e d a n d t h e f i l t e r w a s h e d ,
d r i e d and e x t r a c t e d i n t o a t a r e d f l a s k w i t h p e t r o l e u m e t h e r . T h e s o l v e n t is
e v a p o r a t e d and the r e s i d u e w e i g n e a . A D i a n K s h o u l d De r u n f o r t h e m o s t
accurate work.

APPARATUS

1. 250 ml conical flask.

2. Soxhlet extractor or similar.

REAGENTS

1. Petroleum eth er , b o i l i n g r a n g e 4 0 - 6 0 °C.

2. H y d r o c h l o r i c a c i d , 33% v / v . Dilute 100 m l of concentrated

h y d r o c h l o r i c a c i d w i t h 200 m l of w a t e r .

PROCEDURE

A c c u r a t e l y w e i g h an a m o u n t of the s a m p l e p r e f e r a b l y c o n t a i n i n g m o r e
t h a n 0.1 g o f f a t , b u t p r e f e r a b l y n o t m o r e t h a n 3 g o f d r y m a t t e r ,
into a 250 m l c o n i c a l f l a s k . Add 50 m l of d i l u t e h y d r o c h l o r i c acid
and a few g l a s s b e a d s and cover the flask w i t h a w a t c h g l a s s . Boil
for an h o u r , m a i n t a i n i n g the v o l u m e b y the a d d i t i o n of w a t e r . Add
150 m l of h o t w a t e r , a b o u t 1 g of d i a t o m a c e o u s e a r t h (e.g. " H y f l o -
s u p e r c e l " ) or a b o u t 1 0 0 c m ^ o f f i l t e r p a p e r t o r n i n s m a l l p i e c e s .
F i l t e r t h r o u g h a w e t t e d f l u t e d 15 c m W h a t m a n 5 4 0 p a p e r or e q u i v a l e n t
( u s e d d o u b l e if p r e f e r r e d ) . A g l a s s f i l t e r m a y b e u s e d to a d v a n t a g e .
H a s h t h e f l a s k a n d w a t c h g l a s s t h o r o u g h l y w i t h h o t w a t e r and p a s s t h e
v a s h i n g s t h r o u g h the f i l t e r . D r y the f l a s k and w a t c h g l a s s in the
o v e n . W a s h the f i l t e r w i t h h o t w a t e r u n t i l the f i l t r a t e is a c i d - f r e e
( t e s t w i t h b l u e l i t m u s p a p e r ) . D r y the f i l t e r p a p e r o n a w a t c h g l a s s
at 120°C (60°C o v e r n i g h t if p i e c e s of f i l t e r p a p e r w e r e a d d e d ) .

U s e t o n g s to p l a c e t h e f i l t e r p a p e r in a t h i m b l e . T r a n s f e r the
t h i m b l e to a S o x h l e t e x t r a c t o r . The flask m a y c o n t a i n a few anti-
b u m p i n g g r a n u l e s , w h i c h m u s t have been included w h e n the flask was
weighed. R i n s e the c o n i c a l f l a s k , w a t c h g l a s s and t o n g s with
p e t r o l e u m e t h e r and add the r i n s i n g to the S o x h l e t and a d d s u f f i c i e n t
e x t r a s o l v e n t t o b r i n g t h e t o t a l q u a n t i t y o f s o l v e n t to o n e a n d a
h a l f to t w o t i m e s the c a p a c i t y of the e x t r a c t o r . H e a t at s u c h a r a t e
t h a t at l e a s t 4 2 0 m l of p e t r o l e u m e t h e r h a s c y c l e d t h r o u g h t h e
e x t r a c t o r in 4 h o u r s . R e m o v e the f l a s k and e v a p o r a t e o f f the s o l v e n t
u s i n g an e m p t y S o x h l e t or s o l v e n t - r e c o v e r y u n i t for the b u l k a n d
c o m p l e t i n g the e v a p o r a t i o n on a w a t e r - b a t h , t a k i n g c a r e t h a t h e a t i n g
is n o t so a b r u p t as to c a u s e l o s s b y v i g o r o u s b o i l i n g or s p a t t e r i n g .
F i n a l l y d r y i n t h e o v e n o n e h o u r a t 1 0 2 +_ 2°C. D r y to c o n s t a n t
w e i g h t ( s u c c e s s i v e w e i g h i n g s do not d i f f e r by m o r e than 1 m g ) .
E x t r a c t t h e p a p e r in t h e S o x h l e t f o r o n e h o u r w i t h f r e s h s o l v e n t
u s i n g a s e c o n d w e i g h e d f l a s k to c h e c k t h a t e x t r a c t i o n is c o m p l e t e .
I f t h e c h e c k r e s i d u e e x c e e d s 2 m g c o n t i n u e e x t r a c t i o n u n t i l it is
complete.

214
 CALCULATION

% fat = weight of residue x 10Q

weight of sample taken

If a second extraction provided a weighable residue the total weight

of residue is used for the calculation.

REFERENCE

ISO 1443-1973.

215
 TOTAL LIPIDS

PRINCIPLE

Total lipids comprise the total amount of fats (free and bound) and fat soluble
substances, expressed in percent by weight obtained after acid hydrolysis of
the sample, followed by extraction with hexane.

Lipids in protein and carbohydrate complexes are released by hydrochloric acid

treatment, in the presence of ethanol and formic acid. Ethyl formate (which is
p r o d u c e d in s i t u ) d i s s o l v e s the l i p i d s and b o t h are then e x t r a c t e d by h e x a n e .
They are determined gravimetrically after evaporating the solvent. The method
is a p p l i c a b l e to c e r e a l s , c e r e a l p r o d u c t s ( i n c l u d i n g m i l l e d and b a k e d g o o d s )
and animal feeds.

APPARATUS

1. Analytical balance, accurate to 0.1 mg. Range to 200 g.

2. Grinding mill.

3. Hand sieve, 0.400 mm.

4. Electric water-bath 75°C _+ 1.

5. Extractor flask described in the figure with condenser (standard

taper joints 29/32).

6. Round flask, 250 ml with ground glass joint.

7. Distillation apparatus with electric heater (rotary evaporator

under vacuum preferably).

8. Magnetic stirrer with a Teflon-coated magnet.

9. Nitrogen gas.

(Note : A 160 ml extractor flask with standard taper joint 24/29 may
be preferred for products with a lipids content exceeding 3%. Use 4
g of sample and half of the reagent volumes given in the method).

REAGENTS

1. Ethanol 95%.

2. Hydrochloric acid - water (50 + 20 v/v).

3. Formic acid 99%.

4. Hexane, BR 68-70°C (less than 0.001g residue per 100 ml).

PROCEDURE

The p a r t i c l e size of the s a m p l e should be such that less than 5%

r e s i d u e r e m a i n s on a 0.4 m m sieve. Grind if n e c e s s a r y . M i x the
ground sample to obtain m a x i m u m homogeneity.

W e i g h 8 g of the s a m p l e into the e x t r a c t o r f l a s k ( w i t h m a g n e t i c

stirrer) and spread the sample out on the bottom of the flask. Add 8
m l e t h a n o l and p l a c e the f l a s k on the s t i r r e r in o r d e r to o b t a i n a
homogeneous slurry as far as possible. Add 8 ml of formic acid and 12
m l of hydrochloric acid, then homogenize with the stirrer. Place the
e x t r a c t o r flask (with a c o n d e n s e r ) in the w a t e r - b a t h for 15 m i n ,
s t i r r i n g the s u s p e n s i o n by s w i r l i n g from t i m e to t i m e . R e m o v e the

216
condenser, cool the flask and place it on the magnetic stirrer. Add
20 ml ethanol and stir the m i x t u r e , then add 50 m l h e x a n e , stir at
m a x i m u m speed for 5 min. Decant the two phases. Pour the hexane
into the round flask and the aqueous phase r e m a i n i n g into the side
arm of the extractor flask. Rinse the neck of the extractor flask
with a few drops of hexane. Introduce another 30 ml of hexane, stir
the mixture 5 min. Repeat the extraction twice.

E v a p o r a t e the solvent preferably under reduced p r e s s u r e using a

rotary evaporator. Immediately after evaporation direct a stream of
n i t r o g e n for 10 m i n onto the surface of the lipids contained in the
round flask. Weigh the extracted lipids.

CALCULATION

The total lipids content of the m o i s t m a t t e r ( m m ) is calculated on

the following basis:

Lipids content (% mm) = lipids (g) x 100

weight of sample (g)

The reduction to dry matter basis (dm):

Lipids content (% dm) = lipids (g) x 100

weight of dry matter (g)

The difference between duplicate determinations should not exceed 2%,

otherwise a further determination should be carried out.

ml.

Available from Ets. Bercauverre S.A., 3 rue R o l l i n , 75005 Paris,

France.

217
 TOTAL FAT
(Chloroform-Methanol Method)

PRINCIPLE

The sample is digested using an enzyme and the released fat is extracted into
chloroform-methanol. The fat is weighed after e v a p o r a t i o n of the e x t r a c t i n g
solvent.

APPARATUS

1. Blender, semi-micro.

2. Digestion tube, 50 ml.

3. Water bath.

4. Centrifuge.

REAGENTS

1. Methanol.

2. Chloroform.

3. E n z y m e solution: 2% T a k a - d i a s t a s e in 0.5 M sodium acetate:

(Prepare as needed) Mix 25 ml of Parke-Davis Taka-diastase digestant
w i t h 200 ml of 2.5 M sodium acetate solution in a 1 L v o l u m e t r i c
flask, dilute to volume ami mix.

PROCEDURE

Accurately weigh 5 g of well minced sample (3 g if more than 10% fat)

into a 50 ml d i g e s t i o n tube, add 30 ml of e n z y m e solution and m i x .
Place tube and contents in a water bath at 50°C w i t h o c c a s i o n a l
mixing for one hour. Quantitatively transfer this mixture to a semi-
micro blending assembly with 80 ml of methanol followed by 40 ml of
chloroform. Cover and blend for two minutes. Remove cover, add 40 ml
chloroform. Cover and blend for an additional 30 seconds. Transfer
extract to a 250 polythene centrifuge bottle and centrifuge at 5000
rpm for 10 m i n u t e s to clarify the bottom c h l o r o f o r m layer. (if
s a m p l e extract is stored at 20°C for two h o u r s , c l a r i f i c a t i o n w i l l
occur for m o s t products and centrifugat ion is not necessary).
Transfer ca 20 ml aliquot of the clear c h l o r o f o r m layer to a tared
100 ml beaker. (Care should be taken to prevent any of the aqueous
phase from entering the beaker). Evaporate to dryness on a bath in a
fume hood. Dry residue in beaker for 30 minutes at 101°C and cool on
bench top. D e t e r m i n e residue w e i g h t and c a l c u l a t e p e r c e n t a g e of
total fat.

CALCULATION

total fat = residue weight x 4 x 100

sample weight

It is imperative that the volumes of chloroform, methanol and water

in the initial and final steps of the e x t r a c t i o n be kept in the
p r o p o r t i o n s 1 : 2 : 0.8 and 2 : 2 : 1.8 respectively. This is
n e c e s s a r y to ensure a q u a n t i t a t i v e e x t r a c t i o n of total fat in the
initial step and to prevent c o n t a m i n a t i o n of the c h l o r o f o r m layer
with methanol and water in the final biphasic step.

218
REFERENCES

Official Methods of Analysis of the AOAC, 1984, 43.275-.277.

Bligh E.G. & Dyer, W.J., 1959, Canadian Journal of Biochemistry and Physiology
•J ' y / l i t

219
8.3 Protein

The crude protein content of foods is traditionally determined by the organic

n i t r o g e n c o n t e n t u s i n g the K j e l d a h l m e t h o d . This is still an a c c e p t a b l e
p r o c e d u r e and has the a d v a n t a g e of not r e q u i r i n g e x t e n s i v e e q u i p m e n t or
sophisticated instrumentation. A table of c a l c u l a t i o n f a c t o r s to c o n v e r t
organic nitrogen to crude protein is given below.

Conversion Factor Table

Food Factor

Cereal s
Wheat whole meal or flour 5.83
Wheat flour (low or medium extraction) 5.70
Macaroni, spaghetti, wheat pastes 5.70
Wheat bran 6.31
Rice (all products) 5.95
Rye (all products) 5.83
Barley (all products) 5.83
Oats (all products) 5.83
Pulses, Nuts and Seeds
Groundnuts 5.46
Soya bean (all products) 5.71
Almonds 5.18
Brazil nuts 5.46
Coconut 5.30
Chestnuts 5.30
Seeds (sesame, safflower, sunflower) 5.30
Other seeds 5.30
Milk and Dairy Products
Milk (all-fresh or dry) 6.38
Cheeses (all) 6.38
Butter (and margarines) 6.38
Other Foods 6.25

220
 CRUDE PROTEIN
(Kjeldahl Macro Method)

PRINCIPLE

The s a m p l e is d i g e s t e d in s u l p h u r i c acid in the p r e s e n c e of a c a t a l y s t . The

n i t r o g e n from p r o t e i n and s o m e o t h e r c o n s t i t u e n t s is c o n v e r t e d to a m m o n i u m
sulphate. The a m m o n i a is d i s t i l l e d into s t a n d a r d acid after the d i g e s t has
b e e n m a d e a l k a l i n e . The p e r c e n t a g e of n i t r o g e n is c a l c u l a t e d and the r e s u l t
converted to "crude protein" by multiplication by a factor (usually 6.25).

APPARATUS

1. Kjeldahl flasks, borosilicate glass, 300, 500 or 800 ml.

2. Distillation unit (see figure).

REAGENTS

1. S u l p h u r i c acid. Very high purity is not essential but the

nitrogen should be less than 0.0005%.

2. Catalyst: m a n y c o m b i n a t i o n s of c a t a l y s t h a v e b e e n u s e d , for
e x a m p l e 10 g p o t a s s i u m s u l p h a t e and 0.5 g m e r c u r i c o x i d e per
digestion. If mercury is used, sodium sulphide or thiosulphate must
be added later to decompose mercury a m m o n i u m compounds. The use of
mercury should be avoided where possible. 1000 g potassium sulphate,
30 g c o p p e r s u l p h a t e p e n t a h y d r a t e and 30 g t i t a n i u m d i o x i d e g r o u n d
together or mixed in a ball mill or powder mixer gives an effective
s u b s t i t u t e . A l t e r n a t i v e l y , r e a d y p r e p a r e d t a b l e t s m a y be o b t a i n e d
from suppliers.

3. Saturated sodium hydroxide. This should be prepared in a stout

polythene bucket or other suitable container. (The solution attacks
glass w h i c h should t h e r e f o r e not be used). P l a c e about 3 l i t r e s of
w a t e r (tap w a t e r m a y be u s e d ) in the b u c k e t and add w i t h c o n s t a n t
stirring 2.5 kg of technical grade sodium hydroxide. After this has
dissolved add water until the 5-litre mark is reached. (The bucket
m a y be c a l i b r a t e d at this level b e f o r e u s e , if desired). The
s o l u t i o n should be left at least 24 h o u r s for s o d i u m c a r b o n a t e to
settle, and the s u p e r n a t a n t used for the d e t e r m i n a t i o n . Eye
p r o t e c t i o n should be w o r n d u r i n g the p r e p a r a t i o n of the c a u s t i c
solution. It is c o n v e n i e n t to c a r r y out the o p e r a t i o n in a sink
half-full of water to cool the solution during the initial stages.

4. 0.1N hydrochloric acid.

5. 2% boric acid solution.

6. M e t h y l red i n d i c a t o r . 0.1 g is d i s s o l v e d in 18.6 m l of 0.02N

NaOH and diluted to 250 ml with distilled water. Screened methyl red
m a y be used. D i s s o l v e 0.016 g m e t h y l red and 0.083 g b r o m o c r e s o l
green in 100 ml of neutral 96% ethanol.

7. 0.1 N sodium hydroxide solution.

PROCEDURE

P l a c e a b o u t 1 g of s a m p l e , a c c u r a t e l y w e i g h e d , in the K j e l d a h l
d i g e s t i o n flask. M o i s t s a m p l e s such as s a u s a g e s are c o n v e n i e n t l y
w e i g h e d on a tared filter p a p e r , on a w a t c h g l a s s and put in the
flask w r a p p e d in the paper. The w e i g h t of s a m p l e t a k e n should be
such as to n e u t r a l i z e about 20 m l 0.1 N acid (i.e., c o n t a i n about
0.03 g of n i t r o g e n ) . Add 25 m l of s u l p h u r i c acid and 10 g of

221
 catalyst and digest in a fume hood, slowly at first to prevent undue
frothing. C o n t i n u e to d i g e s t for at least 45 m i n u t e s after the
d i g e s t has b e c o m e a clear pale green. O n l y 30 - 40 m i n u t e s m a y be
used for the total digestion in routine control where speed is more
i m p o r t a n t than a c c u r a c y . L e a v e u n t i l c o m p l e t e l y c o o l , and r a p i d l y
add 100 - 200 ml water. Mix and transfer to the distillation flask,
r i n s e the d i g e s t i o n flask 2 or 3 t i m e s and add the r i n s i n g s to the
bulk.

Add 80 - 85 ml of s a t u r a t e d s o d i u m h y d r o x i d e s o l u t i o n from a
m e a s u r i n g c y l i n d e r so that a m m o n i a is not lost. If a f t e r s h a k i n g ,
the d i g e s t does not turn b l u e due to c o p p e r h y d r o x i d e , it i n d i c a t e s
that insufficient alkali had been added. Distil into 25 ml of 0.1 N
h y d r o c h l o r i c acid c o n t a i n i n g a f e w d r o p s of m e t h y l red i n d i c a t o r .
Alternatively distil into 50 ml of 2% boric acid containing screened
m e t h y l red. The b o r i c acid is n e u t r a l to this i n d i c a t o r and the
alkaline a m m o n i u m borate formed is titrated directly with a 0.1 N HC1
w h i c h is then the o n l y s t a n d a r d s o l u t i o n r e q u i r e d . The e x a c t
s t r e n g t h of the b o r i c acid is not i m p o r t a n t . If i n s u f f i c i e n t
standard acid has been added it is permissible to pipette a further
quantity into the flask provided this is done as soon as the solution
s h o w s signs of b e c o m i n g a l k a l i n e . The acid m a y tend to suck b a c k
into the c o n d e n s e r at the b e g i n n i n g and end of the d i s t i l l a t i o n .
This is easily avoided by using an Allihn condenser and by adjusting
the tube on the end of the c o n d e n s e r so that it is o n l y just b e l o w
the level of the acid, and the suction is broken by the liquid rising
up the c o n d e n s e r . D i s t i l u n t i l the c o n t e n t s of the flask "bump".
Titrate the excess acid with 0.1N NaOH.

(Note: If rubber stoppers are used, the flask is inclined and sodium
h y d r o x i d e s o l u t i o n c a r e f u l l y added d o w n the side of the n e c k , so as
to f o r m a layer u n d e r the d i l u t e d s u l p h u r i c acid. The s t o p p e r s on
the splashhead may then be wetted with a few drops of water and the
apparatus connected together. Not until then is the flask shaken so
as to mix the contents. Distillation is commenced immediately).

CALCULATION

% nitrogen in sample = 14 x — - — x 0.1 x — - —

1000 100

Where: V = ml of 0.1 N acid added - ml of 0.1 N NaOH used to

neutralize the ammonia nitrogen).

W = g of sample.

INTERPRETATION

N x 6.25 = crude p r o t e i n . 6.25 is a g e n e r a l factor s u i t a b l e for p r o d u c t s in

w h i c h the p r o p o r t i o n s of s p e c i f i c p r o t e i n s is not w e l l d e f i n e d . For f a c t o r s
for selected foods see the table under 8.3.

A m m o n i a and .rnmonium salts and u r e a w o u l d be i n c l u d e d but are not n o r m a l l y

present in significant amounts. Nitrates, nitrites and nitroso compounds are
not converted to a m m o n i u m salts by this method. Nitrogen from amino-acids and
such a d d i t i v e s or c o n s t i t u e n t s as g l u t a m a t e , g u a n y l a t e ( f l a v o u r - e n h a n c e r s ) ,
creatine and creatinine (from meat extracts) is also determined by the method.
Crude protein calculated from the percentage of nitrogen will be erroneously
high in samples containing compounds of higher average percentage nitrogen than
protein. S p e c i f i c m e t h o d s for g l u t a m a t e , c r e a t i n e and c r e a t i n i n e should be
used.

The s o d i u m or p o t a s s i u m s u l p h a t e is added to r a i s e the t e m p e r a t u r e and thus

speed the digestion. In most methods the volume of acid added initially is 2-

222
1/2 times the weight of catalyst. In fact, it is the ratio towards and at the
end of the d i g e s t i o n w h i c h is i m p o r t a n t and s h o u l d be a b o u t 1:1, m o r e acid
possibly resulting in incomplete digestion and less acid in loss of nitrogen.
Assuming that fat requires 10 ml per g for digestion and carbohydrate 4 m l per
g, the amount of acid to be added can be a p p r o x i m a t e l y estimated. Digests that
go quite solid on cooling should be discarded.

Distillation Apparatus

REFERENCES

Official Methods of Analysis of the AOAC, 1984, 2.055-.057.

L a k i n , A.L., 1 9 7 8 , D e v e l o p m e n t s in Food Analysis Techniques-1. King, R.D.

(Ed.), A p p l i e d S c i e n c e .

223
 CRUDE PROTEIN
(Semi-Micro M e t h o d )

PRINCIPLE

The sample protein is digested using sulphuric acid and copper as a catalyst.
Sodium sulphate is added to raise the boiling point. The resultant ammonia is
released by adding alkali and is steam distilled into a standard acid solution.
A residual titration of the acid determines the account of ammonia distilled.
This is then calculated as crude protein using an appropriate factor.

APPARATUS

1. Tecator Digestion System 6 (1007 Digester) and tubes or

equivalent.

2. Markham Steam Distillation Apparatus. (See figure).

3. Burettes.

4. Pipettes.

Markham semi-micro Kjeldahl distillation apparatus

(by courtesy of Quickfit and Quartz, Ltd.).

>

Yuen and Pollard's modification of the Markham semi-micro

Kjeldahl distillation apparatus, incorporating splash-head.
A = steam inlet; B = water outlet; C = safety outlet

224
REAGENTS

1. Catalyst - 1 part copper sulphate plus 10 parts sodium sulphate.

Grind to powder in a mortar.

2. Concentrated sulphuric acid.

3. Sodium hydroxide solution (40%): Dissolve 40 g NaOH in water

and make to 100 ml.

4. 0.1 N sulphuric acid solution.

5. 0.02 N sodium hydroxide solution (standardize vs the 0.1 N acid

solut ion).

6. Screened methyl red/methylene blue indicator: Mix equal parts

of aqueous 0.2% methyl red and aqueous 0.1% methylene blue.

PROCEDURE

Accurately weigh (by difference), 2 to 3 g of sample into the Tecator

digestion tube. Add 15 g of catalyst and 35 ml of concentrated
sulphuric acid. Digest (using Tecator Digestion System) until
solution is clear (i.e. no more visible particles of sample).

A l l o w the tube and contents to cool to room temperature. Transfer

the clear digest solution into a 100 ml volumetric flask with several
rinsings of distilled water and make up to the mark when solution has
cooled (Precaution: Heat is liberated in the process, therefore cool
flask in cold water bath). Pipette 5 ml and transfer to the steamed-
out M a r k h a m Distillation apparatus through the funnel of the
apparatus .

Stopper funnel, add 5 ml of 40% sodium hydroxide to the funnel.

Attach a 100 ml distillation receiving flask containing 5 ml of 0.1 N
acid with about 6-8 drops of screened mixed indicator added. (Make
sure the tip of the receiver is below the surface of the acid in the
flask.) When steam reaches the condenser, slowly run down the 40%
sodium hydroxide solution until the digest turns brown. Distil for 5
minutes.

Lower the distillation flask and rinse down the condenser with
distilled water. Titrate the distillate with 0.02N sodium hydroxide
solution to a permanent green end-point. Carry out a blank
titration by titrating 5 ml of 0.1 N acid with 0.02N base.

CALCULATION

Total Nitrogen % = (B - S) x 1.4007 x N x 2Q

Sample Weight g

Where: B = ml of NaOH solution used for blank

S = ml of NaOH solution used for sample
N = Normality of NaOH

1.4007 = meq. wt. of nitrogen (includes factors of 100 for %)

20 = Dilution factor (5 ml of digest used out of 100 ml)

Crude Protein % = total nitrogen % x protein conversion

factor (see table in 8.3).

225
REFERENCES

Markham, R., 1942, Biochemical Journal ¿6, 790.

Hoskins, J.L., 1944, Analyst 69, 271.

Yuen, S.H. and Pollard, A.G., 1953, Journal of the Science of Food and
Agriculture _4, 490.

226
8.4 Ash

The ash of a food represents its inorganic residue after the organic
c a r b o n a c e o u s portion and other v o l a t i l e s have been oxidized and evaporated
away. Ash often can serve as a m e a s u r e of a d u l t e r a t i o n of a food. For
e x a m p l e , a higher ash level than expected could indicate addition of
adulterants having a higher inorganic level than the food. Conversely, a low
ash finding could m e a n d i l u t i o n of the food w i t h m a t e r i a l having a low
inorganic level.

Some foods have unique ashing problems, many of which were discussed in the
section on metals analysis. For example, for high fat foods the fat should be
carefully smoked off w i t h o u t ignition, by using a heat l a m p or b u r n e r , or at
the lip of an open muffle furnace.

For foods w h i c h do not ash s a t i s f a c t o r i l y using the general m e t h o d , break up

the pieces with a thick platinum wire. Addition of a few drops of water to the
cooled dish, evaporation and re-ashing may be enough to obtain a white ash from
samples such as flour. If this is also inadequate, cool the dish, add a few ml
of water and filter through an ashless paper. Return the paper and contents to
the dish, dry and re-ash. As a last resort, the dish m a y be cooled and a few
drops of 10% nitric acid or 50% ammonium nitrate added, evaporated and ashing
continued.

227
 TOTAL ASH

PRINCIPLE

The ash of a foodstuff is the inorganic residue remaining after the foodstuff
is ignited until it is carbon free (i.e. after the organic m a t t e r has been
burnt away), usually at a temperature not exceeding red heat. The ash obtained
is not n e c e s s a r i l y of exactly the same c o m p o s i t i o n as the m i n e r a l m a t t e r
present in the original food as there m a y be losses due to v o l a t i l i z a t i o n or
some other interaction between constituents. The ash figure can be regarded as
a general measure of quality and often is a useful indication of identity.

APPARATUS

1. Porcelain dish.

2. Drying oven.

3. Muffle furnace.

PROCEDURE

Weigh 5 g of the sample into a weighed porcelain dish. Dry at 100°C

for 3 - 4 hrs in a m e c h a n i c a l c o n v e c t i o n oven. R e m o v e the p o r c e l a i n
dish from the oven. Do an initial carbonization by placing dish over
a b u n s e n flame. Heat gently until the contents turn black (for
sugars and sugar products add a few drops of pure olive oil and heat
until swelling stops).

Transfer the dish and contents to a muffle furnace and ignite at 500
to 600°C until free from carbon (residue appears g r e y i s h - w h i t e )
(about 8 hr). R e m o v e from m u f f l e oven and m o i s t e n this first ash
w i t h a few drops of water. (This is to expose bits of unashed
carbon).

R e - d r y in oven at !00°C for 3 - 4 hrs, and re-ash at 500-600°C for

another hour. R e m o v e from m u f f l e furnace, allow to cool for a
moment, place in a desiccator until cool, and weigh. Calculate and
express results as % Ash.

C ALC ULAT I OH

Ash % = <B-C> * 100

A

Where: A = sample weight in g.

B = wt. in g of dish and contents after drying.

C = wt. in g of empty dish.

REFERENCE

Official M e t h o d s of Analysis of the AOAC, 1984, 14.006, as well as other

references under other commodity headings.

228
8.5 Other Constituents

The previously discussed food constituents (moisture, fat, protein and ash) can
be c o n s i d e r e d to be ' n a t u r a l ' or n o n - a d d e d . C r u d e f i b r e is a l s o in t h i s
category. It generally represents that portion of a food which is not used by
the b o d y . It c a n a l s o i n d i c a t e a d u l t e r a t i o n by h i g h - f i b r e m a t e r i a l s s u c h as
wood saw-dust.

O t h e r c o n s t i t u e n t s such as salt ( s o d i u m c h l o r i d e ) and s t a r c h m a y be a n o r m a l

p a r t of a f o o d , b u t are m o s t o f t e n a d d e d . An e x a m p l e is c e r e a l ( s t a r c h )
p r o d u c t s a d d e d to p r o c e s s e d m e a t f o o d s as a f i l l e r , or salt p r e s e n t as a
seasoning or a preservative.

229
 CRUDE FIBRE

PRINCIPLE

C r u d e fibre is the o r g a n i c r e s i d u e left after the d e f a t t e d m a t e r i a l has b e e n

treated with boiling dilute sulphuric solution, boiling dilute sodium hydroxide
solution, dilute hydrochloric acid, alcohol and ether.

APPARATUS

1. 1-litre conical flasks.

2. Buchner funnel and flask, funnel to take 11 cm papers, and

vacuum pump.

3. Hot plate.

4. Silica or porcelain crucibles.

5. Muffle furnace.

REAGENTS

1. Petroleum ether.

2. Sulphuric acid, 0.255 N, standardized.

3. Sodium hydroxide solution, carbonate-free , 0.313 N,

standardized.

4. Ethanol, 95%.

5. Diethyl ether.

6. 1% hydrochloric acid.

PROCEDURE

Accurately weigh 3 g of sample into a 1-litre conical flask. If the

oil c o n t e n t is over 1%, add p e t r o l e u m e t h e r , s w i r l , l e a v e to stand
and c a r e f u l l y d e c a n t and r e p e a t t w i c e m o r e , p r e f e r a b l y l e a v i n g the
l a s t q u a n t i t y of s o l v e n t in c o n t a c t o v e r n i g h t , w i t h a s m a l l
w a t c h g l a s s over the m o u t h of the c o n i c a l . D e c a n t the s o l v e n t
c a r e f u l l y a v o i d i n g loss of p a r t i c l e s of fibre and w a r m g e n t l y to
r e m o v e visible solvent. A l t e r n a t i v e l y e x t r a c t the s a m p l e on the
S o x h l e t in a t h i m b l e or h a r d e n e d paper that w i l l not c o n t r i b u t e
fibre .

Add 200 ml of boiling 0.255 N acid and place the flask on a hot plate
or over a B u n s e n b u r n e r so as to r e t u r n the s o l u t i o n to the b o i l as
quickly as possible. Mark the glass at the liquid surface. Place a
f u n n e l of a b o u t 8 - 1 0 cm d i a m e t e r in the m o u t h to d i m i n i s h
evaporation. As soon as the liquid b o i l s , c o n t r o l the h e a t i n g so
that gentle ebullition is maintained and continue for 3 0 + 2 minutes.
(If f r o t h i n g is e x c e s s i v e , add a drop of a n t i f o a m . ) Add b o i l i n g
w a t e r to m a i n t a i n the v o l u m e if n e c e s s a r y . S w i r l o c c a s i o n a l l y to
remove solids from the sides of the flask.

P r e p a r e a B u c h n e r flask and funnel c o n n e c t e d via a trap to a v a c u u m

pump. P l a c e a W h a t m a n 11-cm No. 52 p a p e r or e q u i v a l e n t in the
funnel, fill with hot water. At the end of the boiling period remove
the flask from the h e a t , leave to settle a f e w m o m e n t s and d e c a n t
t h r o u g h the B u c h n e r f u n n e l , a p p l y i n g g e n t l e s u c t i o n such that the
funnel is not permitted to empty completely until most of the flask

230
 contents are transferred. If the solution is maintained well-mixed
instead of d e c a n t e d , the fibre p a r t i c l e s m a y b l o c k the filter
opposite the funnel holes. Increase suction as necessary. Rinse the
conical flask with near-boiling water so as to transfer all trace of
acid t h r o u g h the filter. R e m o v a l of all solid p a r t i c l e s is not
essential. (Since p l a s t i c w a s h b o t t l e s s o f t e n w h e n c o n t a i n i n g hot
liquids, it may be preferred to use a glass one, blowing by mouth to
p r o v i d e the n e c e s s a r y p r e s s u r e . ) A f t e r r i n s i n g the acid f r o m the
w a l l s of the c o n i c a l flask and d r a i n i n g into the f u n n e l , w a s h the
filter l i b e r a l l y w i t h hot w a t e r and d r a i n . T u r n off the v a c u u m .
R a i s e the edge of the paper w i t h a s p a t u l a , and lay flat on the side
of the funnel, the latter being in the neck of the flask.

Pour a m e a s u r e d 200 m l of near b o i l i n g 0.313 N b a s e s o l u t i o n into a

washbottle with a fine jet and use this solution to wash the residue
on the paper into the flask, then w a s h any p a r t i c l e s of fibre from
the B u c h n e r funnel into the flask. Pour any u n u s e d b a s e s o l u t i o n
into the flask, bring to the boil as quickly as possible and maintain
gentle ebullition for 30 +_ 2 minutes. Filter through an 11- or 12.5-
cm rapid h a r d e n e d paper (e.g. W h a t m a n 5 4 1 ) in an o r d i n a r y c o n i c a l
filter funnel. As soon as the b u l k of the fibre and s.olution are
transferred, wash the filter paper with enough 1% hydrochloric acid
to m a k e the p a p e r and c o n t e n t s acid. T h i s is u s u a l l y o b v i o u s by a
change in colour, but check with an indicator paper at the funnel tip
if necessary. Use water to transfer any remaining particles from the
conical flask to the paper and wash the paper with water until acid-
free, then w a s h w i t h a l c o h o l and d i e t h y l e t h e r u n t i l s u b s t a n t i a l l y
all the water is removed. (This last stage must be carried out away
from any naked flame). Leave the residue to air-dry and then remove
and open the paper and c a r e f u l l y t r a n s f e r the r e s i d u e to a tared
clean and incinerated crucible with the aid of a spatula. Take care
not to include fibres from the filter paper but to i n c l u d e all
residue. Dry in the oven, cool and weigh. Incinerate at 500°C, cool
and w e i g h . The loss in w e i g h t r e p r e s e n t s the fibre c o n t e n t . The
fibre residue from the test consists mainly of cellulose, with some
lignin, but not all of the cellulose is determined.

CALCULATION

% fibre = Loss in weight from incineration x ^QQ

Weight of sample before defatting

INTERPRETATION

T h i s is an e m p i r i c a l m e t h o d and r e s u l t s d e p e n d on a d h e r e n c e to the exact

conditions of test. The results vary with the particle size of the sample. The
d i f f i c u l t y of r e d u c i n g the f i b r o u s part of the s a m p l e to as fine a state as
desirable leads to error both from nonhomogeneity and diminished attack by the
acid and a l k a l i . The u s e f u l n e s s of the test o f t e n lies in c o n f i r m i n g the
absence of fibrous adulterants such as sawdust and the test may be reported as
"fibre negligible" or "fibrous adulterants not detected" or even omitted from
the report, as the important point is that the analyst has satisfied himself as
to the genuineness of the sample. On the other hand, if it is necessary to use
a quantitative result, as for a sample claiming to be wholemeal flour and low
in f i b r e , then 3 or 4 d e t e r m i n a t i o n s at least should be d o n e . The s a m p l e
should be very carefully mixed and even then there is inherent uncertainty in
the result.

231
REFERENCES

Official Methods of Analysis of the AOAC, 1984, 7.066-.070.

ISO 5498-1981. Agricultural Food P r o d u c t s - D e t e r m i n a t i o n of Crude Fibre

Content - General Method.

The Feeding Stuffs (Sampling and A n a l y s i s ) R e g u l a t i o n s , 1982, S.I. 1982 No.

1144 HMSO.

232
 SALT
(Volhard Method)

PRINCIPLE

To d e t e r m i n e salt, an a n a l y s i s is m a d e for total c h l o r i d e s and the r e s u l t is

expressed as sodium chloride. The method involves the digestion of the sample
w i t h c o n c e n t r a t e d n i t r i c acid and o x i d a t i o n of any r e m a i n i n g o r g a n i c m a t t e r
with potassium permanganate. The d i s s o l v e d c h l o r i d e s in the s a m p l e are
p r e c i p i t a t e d w i t h e x c e s s silver n i t r a t e as silver c h l o r i d e , and the e x c e s s
silver ions backtitrated with thiocyanate using ferric alum as the indicator.
W h e n all the e x c e s s silver ions h a v e r e a c t e d w i t h t h i o c y a n a t e , any slight
e x c e s s of t h i o c y c n a t e w i l l r e a c t w i t h ferric a l u m to form a r e d - c o l o u r e d
c o m p l e x (ferric t h i o c y a n a t e ) at the e n d - p o i n t . An e x c e s s of t h i o c y a n a t e m a y
react with the precipitated silver chloride, since the solubility product of
s i l v e r t h i o c y a n a t e is 0.01 t h a t of s i l v e r c h l o r i d e . The a d d i t i o n of
nitrobenzene, diethyl ether or acetone overcomes this difficulty by coating the
precipitated silver chloride and thereby withdrawing it from reaction with the
thiocyanate solution. Nitrobenzene is toxic and its use is best avoided.

APPARATUS

1. Pipettes and burettes.

2. Laboratory glassware.

REAGENTS

1. Ferric alum indicator: saturated aqueous solution of reagent

grade ferric ammonium sulphate (I2H2O).

2. S i l v e r n i t r a t e s o l u t i o n , 0.20 N - d i s s o l v e 17.04 g of silver

nitrate, (previously dried at 110°C) in distilled water and dilute to
1 litre. Standardize (using excess silver nitrate solution) against
0.10 N s o d i u m c h l o r i d e (5.845 g per litre).

3. P o t a s s i u m t h i o c y a n a t e s o l u t i o n (0.100N) - d i s s o l v e 9.72 g of
reagent grade potassium thiocyanate in distilled water, and dilute to
one litre.

4. Concentrated nitric acid.

5. Nitric acid (1+1).

6. Nitrobenzene.

7. Potassium permanganate solution (5%).

PROCEDURE

A c c u r a t e l y w e i g h a p p r o p r i a t e a m o u n t (about 2-3 g) of s a m p l e into a

300 m l E r l e n m e y e r flask. (For s h r i m p p a s t e or o t h e r h i g h l y s a l t e d
f o o d s , w e i g h about 5 g into 250 m l v o l u m e t r i c flask and d i g e s t for
a b o u t one h o u r in w a t e r b a t h to d i s s o l v e the salt. Filter through
No. 1 f i l t e r paper. T a k e a p p r o p r i a t e a m o u n t of this f i l t r a t e ) (e.g.
for s h r i m p p a s t e , take 10 ml). Add 25 m l of 0.100N s i l v e r n i t r a t e
s o l u t i o n , s w i r l flask u n t i l s a m p l e and s o l u t i o n are in i n t i m a t e
contact. Add 15 ml of concentrated nitric acid. (The silver nitrate
s o l u t i o n m u s t be added f i r s t , f o l l o w e d by the c o n c e n t r a t e d n i t r i c
acid. The o r d e r of a d d i t i o n e n s u r e s c o m p l e t e p r e c i p i t a t i o n of the
chlorides. If n i t r i c acid is added f i r s t , loss of c h l o r i d e by
v o l a t i l i z a t i o n as h y d r o g e n c h l o r i d e could o c c u r , since h y d r o g e n
chloride has a far greater vapour pressure than nitric acid.)

233
 B o i l in a f u m e h o o d u n t i l d i s s o l v e d and add p o t a s s i u m p e r m a n g a n a t e
s o l u t i o n (5%) u n t i l c o l o u r d i s a p p e a r s and the s o l u t i o n b e c o m e s
colourless. ( S h o u l d too m u c h p o t a s s i u m permanganate be accidently
a d d e d , c o l o u r r e m o v a l c a n be e f f e c t e d by the a d d i t i o n of s m a l l
quantities of sugar). Add about 15 to 25 ml of distilled w a t e r , boil
for 5 m i n s . C o o l and d i l u t e to a b o u t 150 m l w i t h d i s t i l l e d w a t e r .
Add about 1 ml nitrobenzene, 2 ml of ferric alum indicator and shake
v i g o r o u s l y to c o a g u l a t e the p r e c i p i t a t e d s i l v e r c h l o r i d e . Titrate
the excess silver nitrate with 0.100 N potassium thiocyanate solution
to a permanent light b r o w n end-point.

C a r r y out a b l a n k t i t r a t i o n c o n t a i n i n g 25.0 m l of 0.100N s i l v e r

n i t r a t e s o l u t i o n , 2 m l f e r r i c a l u m i n d i c a t o r and 15 m l of ( 1 + 1 )
nitric acid with 0.100N potassium thiocyanate.

CALCULATION

Salt (as % N a C l ) = (B-T)(N)(5.85)

S

Where :
B = ml of potassium thiocyanate solution used
for titration of blank.

T = ml of potassium thiocyanate solution used

for sample titration.

N = Normality of potassium thiocyanate.

S = Sample weight (in g).

5.85 = milliequivalent weight of sodium chloride

(including the factor of 100%).

For h i g h l y s a l t e d f o o d s r e q u i r i n g d i l u t i o n , m u l t i p l y the above

calculation by 250/ml aliquot taken for analysis.

REFERENCE

O f f i c i a l M e t h o d s of A n a l y s i s of the A O A C , 1 9 8 4 , 32.033, and o t h e r references

under other c o m m o d i t y headings.

234
 STARCH

PRINCIPLE

C e r e a l is o f t e n added to m e a t p r o d u c t s as a b i n d e r . This m e t h o d for the

e s t i m a t i o n of s t a r c h in m e a t p r o d u c t s d e p e n d s on the s o l u t i o n of the p r o t e i n
plus other n i t r o g e n o u s , fatty and salt c o m p o n e n t s of m e a t in an a l c o h o l i c
s o l u t i o n of p o t a s s i u m h y d r o x i d e . The fat is s a p o n i f i e d and the p r o t e i n
hydrolysed. S p i c e s , c e l l u l o s e and starch r e m a i n as a s e d i m e n t . The c e r e a l
starch is then dissolved in hydrochloric acid, re-precipitated with ethanol and
determined gravimetrically.

APPARATUS

1. Centrifuge and tubes.

2. Steam bath.

REAGENTS

1. 95% ethanol.

2. 8% alcoholic potassium hydroxide solution : 40 g KOH dissolved

in 300 ml of 95% ethanol and diluted to 500 m l w i t h 95% ethanol.

3. Hydrochloric acid-water (1+1).

PROCEDDRE

Add about 50 m l of 8% a l c o h o l i c KOH s o l u t i o n to 10.0 g of s a m p l e and

d i g e s t on a s t e a m b a t h for 20 m i n u t e s w i t h o c c a s i o n a l s t i r r i n g .
Transfer to c e n t r i f u g e tubes. E n s u r e tubes are b a l a n c e d and
centrifuge for 5 minutes at 2000 rpm.

Decant and discard the supernatant. Wash the sediment with 25 ml of

95% ethanol, stirring thoroughly. Centrifuge for 5 minutes at 2000
r p m . D e c a n t and discard s u p e r n a t a n t . Add 50 m l of HC1 (1 + 1). M i x
thoroughly to dissolve the cereal starch.

Transfer 25 ml of clear solution to a 150 ml beaker containing 75 ml

of 95% e t h a n o l . M i x w e l l and let stand o v e r n i g h t . (Cover b e a k e r
w i t h a w a t c h g l a s s to p r e v e n t e x t r a n e o u s m a t t e r from e n t e r i n g . )
Filter through a weighed dried filter paper that has been pre-washed
w i t h 95% e t h a n o l . W a s h filter paper and c o n t e n t s w i t h t w o 25 m l
p o r t i o n s of 95% e t h a n o l . Dry for 30 m i n u t e s (or u n t i l c o n s t a n t
weight) at 75°C and weigh.

CALCULATION

Starch, % = (100)(A-B)(2)

Where: A = g of filter paper + contents after drying.

B = g of dried filter paper.

S = Sample weight in g.

2 = Correction factor.

235
8.6 TEXT REFERENCES

1. HANNANT, G. 1982. Journal of the Association of P u b l i c Analysts 20

177-123.

2. C A R T E R , J.R. 1977. In C h a r a c t e r i s a t i o n of Oils and F a t s , S y m p o s i u m

P r o c e e d i n g s No. 28, Food R e s e a r c h A s s o c i a t i o n ( B F M I R A ) , L e a t h e r h e a d ,
Engand.

3. E G A N , H., K I R K , R.S. & S A W Y E R , R. 1981. Pearson's Chemical Analysis of

Food s 8th Ed., Churchill, Livingstone.

Further Reading

RECHCIGL, M. (Ed). CRC Handbook of N u t r i t i v e Value of P r o c e s s e d Food CRC

Press .

Composition of F o o d s , A g r i c u l t u r e H a n d b o o k No. 8 ( R e v i s i o n s o f ) US D e p t . of
Agriculture, Washington.

236
 APPENDIX

Abréviations Used in the Manual

Units of Measure

A = absorbance

g = gram 3
kg = kilogram (10 g^
mg = milligram (10_(.g)
ju.g = microgram (10 g g)
ng = nanogram (10 g)

L = litre
ml = millilitre (10 '¡L)
1^1 = microlitre (10 L )

m = metre _2
cm = centimetre (10_^m)
mm = millimetre (10 m)
in = inch(es) (25 mm)

hr = hour(s)
min = minute(s)
sec = second(s)

rpm = revolutions per minute

o
C = degrees Celsius (centigrade)

.. distance spot move d

R ration of: — —
f distance solvent moved

Descriptive Units

AR = analytical reagent (grade)

BR = boiling range

EDTA = ethylene diamine tetracetic acid

G/G = ground glass

IU = International Units

MW = molecular weight

% = parts per hundred (percent)

ppm = parts per million
ppb = parts per billion

max = maximum
min = minimum
ave = average

M = molar
N = normal

ID = interior diameter
0D = outside diameter

237
m/m = m a s s i nn m a s s
m/v = m a s s 4- v o l u m e
v/v = v o l u m e in v o l u m e

Analytical Techniques

AAS = atomic absorption spectrophotometry

GLC « gas-liquid chromatography
HPLC = high performance liquid chromatography
PC = paper chromatography
TLC = thin-layer chromatography
RI = refractive index
SG = specific gravity
IR = infrared
UV » ultraviolet
vis m visible

Organizations and Agencies

AACC = A m e r i c a n A s s o c i a t i o n of C e r e a l C h e m i s t s
AOAC = A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l C h e m i s t s
AOCS = American Oil Chemists Society
BP = British Pharmacopoeia
BS = British Standards
CAC = Codex Alimentarius Commission
EEC = European Economic Community
ICC = I n t e r n a t i o n a l A s s o c i a t i o n for C e r e a l S c i e n c e and T e c h n o l o g y
ICUMSA = I n t e r n a t i o n a l C o m m i s s i o n f o r U n i f o r m M e t h o d s of S u g a r A n a l y s i s
ISO = International Standardization Organization
IUPAC = I n t e r n a t i o n a l U n i o n of P u r e a n d A p p l i e d C h e m i s t r y
OIV = O f f i c e I n t e r n a t i o n a l de la V i g n e et du V i n

238
FAO TECHNICAL PAPERS

FAO FOOD AND NUTRITION PAPERS:

1. Review of food consumption surveys, 1977
Vol. 1 — Europe, North America, Oceania, 1977 (E*)
Vol. 2 — Far East, Near East, Africa, Latin America, 1979 (E*)
2. Report of the joint FAO/WHO/UNEP conference on mycotoxins, 1977 (E* F* S*)
3. Report of the joint FAO/WHO expert consultation on the rôle of dietary fats and oils in human nutrition, 1977
(E* F* S*)
4. JECFA specifications for identity and purity of thickening agents, anticaking agents, antimicrobials, antioxidants and
emulsifiers, 1978 (E*)
5. Guide to JECFA specifications, 1978 (E* F*)
5 Rev. 1. Bibliography of food consumption surveys, 1984 (E*)
6. The feeding of workers in developing countries, 1978 (E* S*)
7. JECFA specifications for identity and purity of food colours, enzyme preparations and other food additives, 1978
(E* F*)
8. Women in food production, food handling and nutrition, 1978 (E* F* S*)
9. Arsenic and tin in foods: reviews of commonly used methods of analysis, 1979 (E*)
10. Prevention of mycotoxins, 1979 (E* F* S*)
11. The economic value of breast-feeding, 1979 (E*)
12. JECFA specifications for identity and purity of food colours, flavouring agents and other food additives, 1979 (E* F*)
13. Perspective on mycotoxins, 1979 (E* S*)
14. Manuals of food quality control
1 — Food control laboratory, 1979 (E*)
2 — Additives, contaminants, techniques, 1979 (E* F*)
3 —Commodities, 1979 (E*)
4 — Microbiological analysis, 1979 (E* F* S*)
5 — Food inspection, 1981 (E*) (Revised edition, 1984)
6 — Food for export, 1979 (E*)
7 — Food analysis: general techniques, additives, contaminants, and composition, 1986 (E*)
8 — Food analysis: quality, adulteration, and tests of identity, 1986 (E*)
15. Carbohydrates in human nutrition, 1980 (E* F* S*)
16. Analysis of food and nutrition survey data for developing countries, 1980 (E* S*)
17. JECFA specifications for identity and purity of sweetening agents, emulsifying agents, flavouring agents and other
food additives, 1980 (E*)
18. Bibliography of food consumption surveys, 1981 (E*)
18 Rev. 1. Bibliography of food consumption surveys, 1981 (E*)
19. JECFA specifications for identity and purity of carrier solvents, emulsifiers and stabilizers, enzyme preparations,
flavouring agents, food colours, sweetening agents and other food additives, 1981 (E*)
20. Legumes in human nutrition, 1982 (E* F*)
21. Mycotoxin surveillance — a guideline, 1982 (E*)
22. Guidelines for agricultural training curricula in Africa, 1982 (E*)
23. Management of group feeding programmes, 1982 (E* F*)
24. Evaluation of nutrition interventions, 1982 (E*)
25. JECFA specifications for identity and purity of buffering agents, salts, emulsifiers, thickening agents, stabilizers,
flavouring agents, food colours, sweetening agents and miscellaneous food additives, 1982 (E* F*)
26. Food composition tables for the Near East, 1983 (E*)
27. Review of food consumption surveys — 1981,1983 (E*)
28. JECFA specifications for identity and purity of buffering agents; extraction solvents; flavouring agents; and miscella-
neous food additives, 1983 (E*)
29. Post-harvest losses in quality of foodgrains, 1983 (E*)
30. FAO/WHO food additives data system, 1984 (E*)
30 Rev. 1, FAO/WHO food additives data system, 1985 (E*)
31/1. JECFA specifications for identity and purity of food colours, 1984 (E* F*)
31/2. JECFA specifications for identity and purity of food additives, 1984 (E* F*)
32. Residues of veterinary drugs in foods, 1985 (E/F/S*)
33. Nutritional implications of food aid: an annotated bibliography, 1985 (E*)
34. Specifications for identity and purity of certain food additives. (E*** F***)
35. Review of food consumption surveys 1985,1986 (E*)
36. Guidelines for can manufacturers and food canners, 1986 (E*)

Availability: April 1986

E — English Available
F — French Out of print
S — Spanish In preparation

The FAO Technical Papers can be purchased locally through FAO Sales Agents or directly from Distri-
bution and Sales Section, FAO, Via delle Terme di Caracalla, 00100 Rome, Italy.
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