ANALYTICAL METHODS TO MEASURE THE

CONSTANTS OF FATS AND OILS

1. Acid Value
2. Saponification Value
3. Iodine Value
4. Gas Chromatographic Analysis for Fatty Acids
5. Liquid Chromatography
6. Cholesterol Determination
 1. Acid Value

Number of mgs of KOH required to neutralize the Free

Fatty Acids in 1 g of fat.

ml of KOH x N x 56
AV = = mg of KOH
Weight of Sample
The acid value (AV) is a common parameter in the specification of
.fats and oils

weight of KOH in mg needed to neutralize the organic acids present

in 1g of fat : a measure of the free fatty acids (FFA) present in the
fat or oil. An increment in the amount of FFA in a sample of oil or
fat indicates hydrolysis of triglycerides

Such reaction occurs by the action of lipase enzyme and it is and

indicator of inadequate processing and storage conditions (i.e., high
temperature and relative humidity, tissue damage). The source of
the enzyme can be the tissue from which the oil or fat was extracted
or it can be a contaminant from other cells including
microorganisms. Besides FFA, hydrolysis of triglycerides produces
.glycerol
 2. Saponification Value

Saponification - hydrolysis of ester under alkaline

condition.

O
H2 C OH
H2 C O C R O
O +
+ 3 KOH HC OH 3 R C OK
HC O C R
O
H2 C OH
H2 C O C R
?What are Saponifiable Lipids

Lipids that can be hydrolyzed either by Heat, •

,Alkaline or Acid solutions
:The hydrolyzed products usually include •
,Fatty Acids (salts of fatty acids) •
Glycerol, and in some cases other molecular •
,components contained in the lipid
,Examples: • Neutral fats, • Phospholipids •
?What are Non-Saponifiable Lipids

Non-Saponifiable lipids are those lipids that •

,cannot be hydrolyzed
•
:Examples
,Terpenes •
Steroids •
Fat-soluble Vitamins •
Saponification Number of a Lipid

Saponification Number is the number of •

milligrams of KOH that is needed to Saponify
;one gram of Fat

Since each molecule of fat regardless of its •

size requires 3 molecules of KOH to Saponify
it, the Saponification number also indicates the
;number of molecules of fat in one gram of fat
 2. Saponification Value Determination

Saponification # --mgs of KOH required to saponify 1 g of fat.

1. 5 g in 250 ml Erlenmeyer.
2. 50 ml KOH in Erlenmeyer.
3. Boil for saponification.
4. Titrate with HCl using phenolphthalein.
5. Conduct blank determination.

56.1(B -S) x N of HCl

SP# =
Gram of Sample

B - ml of HCl required by Blank.

S - ml of HCl required by Sample.
Saponification Value of Fats and Oils

Fat Saponification #

Milk Fat 210-233

Coconut Oil 250-264

Cotton Seed Oil 189-198

Soybean Oil 189-195

Lard 190-202
 3. Iodine Number

Number of iodine (g) absorbed by 100 g of oil.

Molecular weight and iodine number can calculate the

number of double bonds. 1 g of fat adsorbed 1.5 g of
iodine value 150.
 Iodine Value Determination

Iodine Value = (ml of Na2S2O3 volume for blank - ml of Na2S2O3

volume for sample)  N of Na2S2O3  0.127g/meq  100
Weight of Sample (g)

CH CH + ICl CH CH
Iodine chloride Cl I

Excess unreacted ICl

ICl + KI KCl + I2

I2 + 2 Na2 S 2 O3 Na2 S 4 O6 + 2 NaI

 METHODS
.Arrange all the reagent solutions prepared and the requirements on the table
Pipette out 10ml of fat sample dissolved in chloroform to an iodination flask
."labeled as “TEST
Add 20ml of Iodine Monochloride reagent in to the flask. Mix the contents in the
.flask thoroughly
.Then the flask is allowed to stand for a half an hour incubation in dark
.Set up a BLANK (add 10ml Chloroform to an empty flask)
Add to the BLANK, 20ml of Iodine Monochloride reagent and mix the contents
.in the flask thoroughly
.Incubate the BLANK in dark for 30 minutes
Mean while, Take out the TEST from incubation after 30 minutes and add 10 ml
.of potassium iodide solution into the flask
.Rinse the stopper and the sides of the flask using 50 ml distilled water
Titrate the “TEST” against standardized sodium thiosulphate solution until a
.pale straw colour is observed
Add about 1ml starch indicator into the contents in the flask, a purple colour is
.observed
.Continue the titration until the color of the solution in the flask turns colourless
The disappearance of the blue colour is recorded as the end point of the
.titration
.'Similarly, the procedure is repeated for the flask labeled ‘Blank
. Record the endpoint values of the BLANK
 Iodine Numbers of Triglycerides

Fatty Acids # of Double-bonds Iodine #

Palmitoleic Acid 1 95

Oleic Acid 1 86

Linoleic Acid 2 173

Linolenic Acid 3 261

Arachidonic Acid 4 320

Iodine Value

High iodine value means…..

 Peroxide value (PV)

Peroxide Value is commonly used to determine the

rancidity of a sample containing fat or oil subject to
.oxidation
In general fresh oils have a peroxide value of >10 mEq/Kg
while peroxide values in the 30-40 mEq/Kg range are
.generally associated with a rancid taste
 4. GC Analysis for Fatty Acids

1. Extract fat.
2. Saponify (hydrolysis under basic condition).
3. Prepare methyl ester (CH3ONa).
4. Chromatography methyl ester.
5. Determine peak areas of fatty acids.
Fatty acids are identified by retention time.
6. Compare with response curve of standard.
 Fatty Acids Methyl Esters:

Response

18:1

14

18:3 21:1 24
16 18:2 20
18 22

Time

GC condition: 10% DEGS Column (from supelco)

Column temperature 200C.
 5. TRIGLYCERIDE ANALYSIS BY LIQUID
CHROMATOGRAPHY

Soybean Oil
Solvent CH3CN/HF
Column 84346 (Waters Associates)

RESPONSE

RETENTION TIME
Oleate-containing triglycerides in olive oil

Fatty Acid Total Acyl Carbons: Equivalent Carbon

Composition Unsaturation Number

OL2 54:5 44

O2L 54:4 46

OPL 52:3 46

O3 54:3 48

OSL 54:3 48

O2P 52:2 48

O2S 54:2 50

OPS 52:1 50

OS2 54:1 52
 6. CHOLESTEROL DETERMINATION

Enzymatic Determination: Cholesterol Oxidase

Cholesterol Oxidase
etc. + H2 O2
HO O

CH3O OCH3 CH3O OCH3

H2 O2 + H2N NH2 Peroxidase NH
+ H2 O
HN

0-Dianisidine Oxidized 0-Dianisidine

(Colorless) (Brown color)At 440 nm
 Total Cholesterol Methodologies

Cholesterol-Ester-Hydrolase
Cholesterol ester Free
cholesterol
Cholesterol Oxidase

H2O2Free cholesterol

Peroxidase

H2O2 + Chromogen Colored

Chromogen

23
 Absorption
at 440 nm

 g/ml Cholesterol

Cholesterol by GLC
1. Prepare cholesterol butyrate.
2. Analyze by GLC.
time in GC - 15 min.
sensitivity - 10-7 g.
SPECTROMERTIC ABSORPTION STANDARD CURVE
OF CHOLESTEROL

Absorption
at 440 nm

 g/ml Cholesterol

Cholesterol by GLC
1. Prepare cholesterol butyrate.
2. Analyze by GLC.
time in GC - 15 min.
sensitivity - 10-7 g.
Peroxide value
Anisidine value
Thiobarbituric value
 LIPID CONTENT ANALYSES

1. Gravimetric Method
(1) Wet extraction - Roese Gottliegb & Mojonnier.
(2) Dry extraction - Soxhlet Method.

2. Volumetric Methods (Babcock, Gerber Methods)

1. Gravimetric Method
 (2) DRY EXTRACTION - SOXHLET
METHOD.

Sample in thimble is continuously extracted with ether

using Soxhlet condenser. After extraction, direct
measurement of fat
- evaporate ether and weigh the flask.

Indirect measurement - dry thimble and weigh thimble and

sample.
SOXHLET METHOD.
 2. Volumetric Method (Babcock, Gerber Methods)

Theory:
1. Treat sample with H2SO4 or detergent.
2. Centrifuge to separate fat layer.
3. Measure the fat content using specially calibrated bottles.

Methods:
1. Known weight sample.
2. H2SO4 - digest protein, liquefy fat.
3. Add H2O so that fat will be in graduated part of bottle.
4. centrifuge to separate fat from other materials completely.
 REACTIONS OF FATS

Hydrolytic Rancidity:

1. Triglyceride -> Fatty acids

Specially C4 butyric acid (or other short chain fatty
acids) are the real problem.

2. By lipase.
 LIPID OXIDATION (peroxidation)

Major flavor problems in food during storage are mainly

due to the oxidation of lipid.

Lipid Oxidation - free radical reactions.

1. Initiation.
2. Propagation.
3. Termination.
Pentane Formation from Linolenic Acid
14 13 12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH 2 CH CH CH 2 COOH
n
Initiation (metal)
- H.

12 11 10 9
CH3 (CH 2 )3 CH 2 .
CH CH CH CH CH CH 2 n COOH
Propagation + O2
12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH CH CH CH 2 nCOOH
O
Propagation
.
O + H.

12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH CH CH- CH 2 n COOH
O
Hydroperoxide
Decomposition O _ .OH
H

12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH CH CH CH 2 n COOH
.
O

O
CH .
12 11 10 9
CH3 (CH 2 )3 2 + H C CH CH CH CH CH 2 n COOH

Termination + H .
CH3 (CH 2 )3 CH 3
Pentane
Initiation: The step in which a fatty acid radical is produced. The
most notable initiators in living cells are
reactive oxygen species (ROS), such as OH· and HOO·, which
combines with a hydrogen atom to make water and a fatty acid
radical
Propagation: The fatty acid radical is not a very stable molecule
, so it reacts readily with molecular oxygen, thereby creating a
peroxyl-fatty acid radical. This radical is also an unstable
species that reacts with another free fatty acid, producing a
different fatty acid radical and a lipid peroxide, or a cyclic
peroxide if it had reacted with itself. This cycle continues, as the
.new fatty acid radical reacts in the same way
When a radical reacts with a non-radical, it always produces
another radical, which is why the process is called a "chain
."reaction mechanism

The radical reaction stops when two radicals react and produce
a non-radical species. This happens only when the
concentration of radical species is high enough for there to be a
.high probability of collision of two radicals

Living organisms have different molecules that speed up termination by neutralizing

free radicals and, therefore, protecting the cell membrane. One important such
vitamin C antioxidant is vitamin E. Another important antioxidant is

The end products of lipid peroxidation are reactive aldehydes,

such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE)
 ANALYSIS OF FLAVOR QUALITY & STABILITY OF OIL

1. Peroxide Value

O O
A. KI + CH 3 C OH HI + CH 3 C OK

B. ROOH + 2 HI I2 + H2O + ROH

C. I2 + 2 Na2 S 2 O3 2 NaI + Na2 S4 O6

Peroxide Value = ml of Na2S2O3  N  1000

(milliequivalent peroxide/kg of sample) Grams of Oil
2. Active Oxygen Method (AOM)

Determined the time required to obtain certain

peroxide value under specific experimental conditions.
The larger the AOM value (i.e. the longer time for
oxidation), the better the flavor stability of the oil.
Rancidity testing determines the level of oxidation in a sample

Oxidation generates a sequence of breakdown products, starting

with primary oxidation products (peroxides, dienes, free fatty
acids) then secondary products(carbonyls, aldehydes, trienes)
.and then tertiary products
.
When lipids (fats and oils) go rancid, its nutritional value is
.compromised, and the lipids will take on a rancid taste and odor

Proper rancidity testing is an essential component in determining

the shelf life of the product
Rancidity may occur in vegetable oil upon storage
especially when it contains high content of fatty
acid or fatty oils. The decomposed components
such as free fatty acids, peroxides, low molecular
weight aldehydes and low molecular weight of
ketones are produced. This would result in
distinctive smell and affect the quality of the
.vegetable oil samples

In view of this, acid value which is defined as the

number of mg of potassium hydroxide required to
neutralize the free acid in 1g of fat, fatty oil or other
related substances is determined to assess the
rancidity of the vegetable oil samples. Sodium
.hydroxide may also be used
Oxidative Rancidity

Oxidation of lipids is a chemical change in food, feed, and ingredients

that may impact flavor, aroma, nutritional quality, and texture. The
chemicals produced from oxidation of lipids are responsible for rancid
flavor and aroma. Oxidized fats can interact with proteins and
.carbohydrates causing changes in texture

Lipid oxidation occurs in three phases: and initiation phase, propagation

.phase, and termination phase

During initiation phase peroxides and hydroperoxides are the

predominant reaction. These reactions will initiate a series of reactions
during propagation that lead to increase concentrations of aldehydes,
.ketones, and hydrocarbons

During termination phase saturated aldehydes are the main reaction

product. The products of each of these phases increase and decrease
.over time making it difficult to quantitatively measure lipid oxidation
Pentane Formation from Linolenic Acid
14 13 12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH 2 CH CH CH 2 COOH
n
Initiation (metal)
- H.

12 11 10 9
CH3 (CH 2 )3 CH 2 .
CH CH CH CH CH CH 2 n COOH
Propagation + O2
12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH CH CH CH 2 nCOOH
O
Propagation
.
O + H.

12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH CH CH- CH 2 n COOH
O
Hydroperoxide
Decomposition O _ .OH
H

12 11 10 9
CH3 (CH 2 )3 CH 2 CH CH CH CH CH CH 2 n COOH
.
O

O
CH .
12 11 10 9
CH3 (CH 2 )3 2 + H C CH CH CH CH CH 2 n COOH

Termination + H .
CH3 (CH 2 )3 CH 3
Pentane
Important Components to Rancidity
:Shelf-life Testing

Peroxide Value (PV)

.p-Anisidine (p-AV)
TBA Rancidity (TBAR)
.Free Fatty Acids (FFA
.Oxidative Stability Index (OSI)
Peroxide Value (PV) testing determines the amount of
peroxides in the lipids. Peroxides are the initial indicators of
lipid oxidation. (Peroxides will continue to react and produce
.)secondary products such as aldehydes, see p-AV

p-Anisidine (p-AV) testing indicates the amount of aldehydes in

the lipids. This test is often paired with PV, as aldehydes are
the secondary indicators of oxidation. Aldehydes can produce
.strong objectionable flavors and odors at relatively low levels

TBA Rancidity (TBAR) also measures aldehydes (primarily

malondialdhyde) created during the oxidation of lipids. This
analysis is primarily useful for low-fat samples, as the whole
.sample can be analyzed rather than just the extracted lipids
.
Free Fatty Acids (FFA) testing determines the amount of
fatty acids that have been liberated from their triglyceride
structure. Free fatty acids can produce strong flavors and
odors at relatively low levels. Free fatty acids are hydrolytic
rancidity (not oxidation) products, and can be caused by
.microbial activity

Oxidative Stability Index (OSI) indicates how resistant a

sample is to oxidation. The samples are subjected to heat
while air (containing oxygen) is streamed through it. This
process accelerates the oxidation process causing most oils
to go rancid quickly. The samples are monitored and the
time required for the sample to become rancid is
determined. Samples that require longer times to become
.rancid, and more stable
peroxide value : indicate the extent to which an oil sample has
.undergone primary oxidation

Oils with a high degree of unsaturation are most susceptible to

autoxidation. The best test for autoxidation (oxidative rancidity)
is determination of the peroxide (intermediates in the
.autoxidation reaction) value

Autoxidation is a free radical reaction involving oxygen that

leads to deterioration of fats and oils which form off-flavours
.and off-odours

Peroxide value, concentration of peroxide in an

oil or fat, is useful for assessing the extent to which spoilage

Detection of peroxide gives the initial evidence of rancidity in

.unsaturated fats and oils
 The peroxide value is defined as

.”the amount of peroxide oxygen per 1 kilogram of fat or oil“

Expressed in units of milliequivalents, or mmol/kg

note: 1 milliequivalents = 0.5 mmol; because 1 mEq of O2(

.)mmol/2=0.5 mmol of O2, where 2 is valence 1=

Peroxide values of fresh oils are less than 10

;milliequivalents/kg

Rancid taste of oils is noticeable when the peroxide value is

.between 30 and 40 milliequivalents/kg
Peroxide value: determined by measuring the amount of iodine
which is formed by the reaction of peroxides (formed in
.fat or oil) with iodide ion

I− + H2O + HOOH -> HOH + 2OH− + I2 2

Note that the base produced in this reaction is taken up by the

excess of acetic acid present. The iodine liberated is titrated with
.sodium thiosulphate
−
2S2O32− + I2 -> S4O62− + 2 I

The acidic conditions (excess acetic acid) prevents formation of

hypoiodite (analogous to hypochlorite), which would interfere with
.the reaction
.
Starch solution is used as the indicator used in this reaction.
Amylose forms a blue to black solution with iodine and is
.colourless where iodine is titrated

A precaution that should be observed is to add the starch

indicator solution only near the end point (the end point is near
when fading of the yellowish iodine colour occurs) because at
high iodine concentration starch is decomposed to products
.whose indicator properties are not entirely reversible
p-anisidine test determines the extent of secondary oxidation

p-Anisidine reacts with secondary oxidation products such

as aldehyde and ketones in fats and oils to form products that
.absorb at 350 nm wavelength of light

Analysis Methods

The chemical analysis method for p-Anisidine Value

determines the amount of aldehydes (principally 2-alkenals and
2,4-dienals) in animal and vegetable oils and fats by reaction of
.these compounds with the p-Anisidine

This reaction highlights the concentration of the quantity of

aldehydes and ketones, giving the dimension of the secondary
.oxidation of the fat matrices
p-anisidine test determines the extent of secondary oxidation

This method is particularly good at detecting unsaturated

aldehydes, which are the ones that are most likely to generate
unacceptable flavors, thus useful in food
quality testing

The lower the p-Anisidine Value, the better the quality of fats
.and oils analyzed

Depending on the market the values required vary: for fish oils
the p-Anisidine value must be lower than 30, in other sectors
.is instead required less than 10 AV

P-anisidine test: http://www.nus.edu.sg/nurop/2009/FoS/14th%20NUROP

%20Congress_FoS/Food%20Science%20&%20Technology/Lee%20Chiew
%20Yi_U052917M.pdf
 Anisidine value
The p–anisidine value is defined by convention as 100 times the
optical density measured at 350 nm in a 1 cm cuvette of a
solution containing 1.00 g of the oil in 100 mL of a mixture of
solvent and reagent
3. Thiobarbituric acid (TBA) Test.
To determine the rancidity degree (lipid oxidation) of meat
or fish product.
HS N OH O O
+ C CH 2 C
N H H
OH

HS N OH HO N SH
+ 2 H2O
N N
CH CH CH
OH OH
Colored Pigment
Thiobarbituric acid is an organic compound and a heterocycle. It is used as a
reagent in assaying malondialdehyde(the TBARS assay of lipid peroxidation)

Thiobarbituric acid reactive substances - TBARS - are formed

as a byproduct of lipid peroxidation (i.e. as degradation
products of fats) which can be detected by the TBARS
.assay using thiobarbituric acid as a reagent

Because reactive oxygen species (ROS) have extremely short

half-lives, they are difficult to measure directly. Instead, what
can be measured are several products of the damage produced
.by oxidative stress, such as TBARS

Assay of TBARS measures malondialdehyde (MDA) present in

the sample, as well as malondialdehyde generated from lipid
hydroperoxides by the hydrolytic conditions of the reaction

Thiobarbituric acid reactive substances )TBARS(

YOUTUBE vidoes

:P-anisidine
https://www.youtube.com/watch?v=R1PSWRl9fLQ

Peroxide value
https://www.youtube.com/watch?v=KnKMQ-nK9zg

TBA rancidity test

https://www.youtube.com/watch?v=gAvLMwT0F5g