University College Sedaya International

Laboratory Manual

Food Chemistry
MF 203

May 2015

Asst. Prof. Dr. Nyam Kar Lin

Contents Page

Safety and Assessment 3

Experiment 1: Determination of Moisture. 4

Experiment 2: Determination of protein. 5

Experiment 3: Determination of Ash. 7

Experiment 4: Determination of Fats. 8

Experiment 5: Analysis of lipid – Iodine Value 13

Experiment 6: Analysis of lipid – Peroxide Value 14

Experiment 7: Quantitative determination of sugar in honey. 10

Experiment 8: Determination of lactose concentration in milk (Nelson Method). 14

Experiment 9: Determination of Vitamin A. 17

Experiment 10: Trace metals in baby food. 18

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Safety
1. Before attempting an unfamiliar procedure you must seek the guidance of the instructor.
2. The usual prohibition of smoking and eating in the laboratory applies.
3. Unauthorized experiments are forbidden.
4. Footwear and eye protection is required. In addition a laboratory coat as recommended.

Assessment

Laboratory work will be assessed on written reports. The written report should include

1. The result (included calculation) presented in a format that is relevant to the exercise.
[3 m]
2. A discussion, the objectives being to interpret the result, that is, to explain why they
occurred and to put them into perspective. [8 m]
3. Conclusion [2 m]
4. Reference is any were consulted. [2 m]
5. Question, if any.

Three important attributes of a good report are: accuracy, brevity and clarity.

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Experiment 1: Determination of moisture (Drying Oven Method)

Objective: To determine the moisture content in food samples

Introduction

Moisture content in food can be measured using air ovens. Weighed samples are placed in an oven
at a specified temperature over a time limit until they reach constant weight. Then the moisture
content can be determined by using formula listed below:

Moisture by weight (%) = (m1 – m2) x 100%

(m1 – m0)

Where,
m0 is the mass, in grams of empty dish and its lid
m1 is the mass, in grams, of the dish and its lid and the test portion before drying
m2 is the mass, in grams, of the dish and its lid and the test portion after drying

The result is being expressed to an accuracy of one decimal place. The method of analysis
used to determine the moisture content does not discriminate against other components that easily
evaporate (such as certain organic acids) and disappear from the food sample during the procedure.

Material & Method

Material
- Grinder (which permit food samples to be grounded with minimum heating)
- Porcelain crucible
- Dessicator
- Ventilated oven
- Analytical balance
- Halogen moisture analyzer

Oven method:

1. Grind sample as finely as possible or homogenize in a blender.

2. Dry a porcelain crucible with lid for 3 hours in an oven at 105 oC.
3. Cool crucible in a dessicator and mass of the crucible without lid was measured using electronic
balance soon after it has attained room temperature.
4. The sample was weighted for approximately ± 5 gram into the crucible.
5. Crucible with sample was put on its lid in the oven at the temperature of 105 oC and being left for
3 hours.
6. Crucible with sample and lid were being removed from oven and placed in the dessicator,
allowing them to cool to the ambient temperature.
7. Mass of crucible and sample without lid was measured.
8. The percentage of moisture content of sample was calculated using the formula.

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 Moisture by weight (%) = (m1 – m2) x 100%
(m1 – m0)

Halogen moisture analyzer method:

1. Turn on the halogen moisture analyzer.

2. Open the heating module.
3. Place the empty sample pan into the sample pan handler.
4. Place the sample pan handler into the draft shield. Ensure that the tongue of the pan handler
fits exactly in the slot of the draft shield. The sample pan must lie flat in the pan holder.
5. Close the heating module. This sets the balance installed in the Moisture Analyze to zero.
6. Open the heating module and place the specimen sample in the sample pan.
7. Close the heating module and the instrument then automatically begins the drying process and
measuring process.
8. When drying is complete, an audio signal sounds. You can now read off the moisture content
of your sample in the display.

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Experiment 2: Determination of protein

Objective: To determine the protein content in food samples

Introduction

The kjeldahl method, which essentially determines the total nitrogen content of a foodstuff, is used.

The foodstuff is oxidized by heating with nitrogen-free concentrated sulphuric acid in a long-
necked digestion flask, in the presence of a catalyst (usually copper sulphate or selenium). In this
digestion process, nitrogen in the sample is converted to ammonium sulphate. After making alkaline
with concentrated sodium hydroxide solution, the ammonia is steam-distilled and trapped in
saturated boric acid solution. This is then titrated with a standard solution of hydrochloric acid, the
volume of acid used giving an indication of amount of ammonia liberated from the foodstuff.
Nitrogen content is hence determined.

To obtain protein content, the amount of nitrogen in the sample is multiplied by a specific
factor, 6.25 being usually used since most proteins contain 16% nitrogen. Results so obtained from
this method are often called “crude protein” content. It should be noted that these figures for protein
content often include some non-protein nitrogenous substances.

Material & Method

Material
- Sulphuric acid, concentrated
- Copper sulphate, catalyst
- 40%, Sodium hydroxide
- 4%, Boric acid
- Methyl red and bromocresol green indicator
- 0.1M Hydrochloric acid (HCl)

Method

1. Weigh accurately about 0.5 g of the homogenized sample (analytical balance) on a folded
nitrogen free weighing paper, roll the paper and its contents and drop it directly into the
bottom of a dry Kjeldahl digestion flask.

2. Add two pellets of catalyst, 12 ml of concentrated sulphuric acid and a few boiling chips to
prevent bumping. Perform a blank determination at the same time incorporating all reagents
including the nitrogen free weighing paper.

3. Heat the mixture in Kjeldahl digestor (electric coil heating block) in a fume cupboard.

4. Set up the digesting programme to 100% heating power for 20 min and 70% heating power
for 60 min and start the digestion. Digestion is complete when the sample turns to clear
solution. Further digestion is needed when black precipitate still remains.

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 5. Allow the flask to cool and cautiously dilute the mixture with 70 ml water, followed by 50ml
40% sodium hydroxide.

6. To the receiving flask add 50ml of 4% boric acid, and five drops of methyl red and
bromocresol green indicator each.

7. Connect the digesting flask and receiving flask to the Kjeldahl distilling unit for distillation for
5 min.

8. After distillation, receiving flask was removed to titrate with 0.1M HCl.

Calculate the percent protein using:

Sample blank Molarity
Total titre - titre x 1000 HCl x 14 x 100
Nitrogen % = sample mass

Protein % = Total nitrogen % x Relevant factor

In your report make reference to the significance of the 6.25 factor in the above calculation.

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Experiment 3: Determination of ash

Objective: To determine the ash content in food sample

Introduction

The ash of a foodstuff is the inorganic residue remaining after the organic matter has been burnt
away. Hence ash content can be determined by incinerating a known quantity of foodstuff,
previously dried, until constant weight is obtained. Ashing should not be done at temperature
exceeding 650oC, at which temperatures inorganic salts like alkali chlorides will volatilize. Moreover,
a portion of the ash will fuse and enclose some carbon, preventing them from being ignited. Ash
contents of fresh food rarely exceed 5%, although some processed foods can have ash contents as
high as 12%, e.g., dried beef.

Material & Method

Material
- Spatula
- Dessicator
- Oven
- Hotplate
- Porcelain crucible
- Analytical balance
- Muffle furnace

Method

1. Dry a shallow porcelain crucible in an oven at 105ºC for 3 hours. Cool it in a dessicator and
weigh soon after it has attained room temperature.

2. Weight accurately 0.5g of the homogenized food sample into the porcelain crucible.

3. The sample was then preheated on a hotplate. This is to remove any moisture such as fat
or water contain in the sample.

4. The sample was heated until it has ceased smoking.

5. Place the dish in cold muffle furnace and bring the temperature to 550ºC.

6. Ash the sample until a whitish or grayish ash is obtained.

7. Remove dish, cool in dessicator and weigh soon after attaining room temperature.

8. Calculate the total ash content of the food sample.

Calculation:

(g) ash per 100g sample = weight of ash (g) x 100

weight of sample (g)

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Experiment 4: Determination of Fat

Objective: To determine the fat content in food sample

Introduction

Fat and oils are characterized by their extreme insolubility in water, very slightly soluble in alcohol,
and by the readiness with which they are dissolved by ethyl ether, petroleum ether and carbon
tetrachloride. Since small amounts of substances other than fat may be included in the ether extract,
the result is generally referred to as “crude fat”.

The Soxhlet extraction technique is one of the extraction techniques used to determine the
fat content in food by isolating fats using organic solvents. The thimbles are widely used in Soxhlet
extraction units providing a safe, convenient and efficient method of solvent extraction of solids and
semi-solids. The anti-bumping granules is involved in this experiment to provide nuclei on which gas
bubbles can grow and leading to smooth boiling rather than bumping which can be hazardous. The
solvents that are generally used are extremely volatile and inflammable. The extraction procedure
must therefore be carried out with extreme caution.

Material & Method

Material
- Hexane, methanol & chloroform
- Anti-bumping granules & Degreased cotton wool
- Soxhlet extractor, 250 ml round bottom flask, Extraction thimble & Rotary evaporator

Procedure:

1. Determine the mass of round-bottom flask (250 ml) containing a few pieces of anti-bumping
granules.

2. Accurately weigh 3 g of each sample (previously dried at 105ºC for one hour) into Soxhlet
thimbles and seal with degreased cotton wool.

3. Set up the Soxhlet extraction apparatus on a steam bath or heating mantle using 150 ml
hexane in round bottom flasks.

4. Heat the apparatus for 10 times cycle then disconnect the Soxhlet extractor and allow the
solvent to evaporate gently in a rotary evaporator.

5. When all solvent has evaporated (no more bubbles forming in the solution), transfer flask
into an oven at 70oC for 10 min to dry the extract.

6. Transfer immediately into a dessicator to cool and weigh. Calculate the percent fat in the
cereal from the mass of material held in the flask.

% fat in sample = F x 100

Weight, in g, of sample taken
Where F = weight of fat in sample
= (weight of flask + fat) – (weight of flask)

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Experiment 5: Analysis of lipid – Iodine Value
Materials
A) Iodine Value

Reagents
 S1- sample 1 oil
 S2 – sample 2 oil
 B - blank
 Carbon tetrachloride
 Wij’s solution
 0.1M standard Sodium Thiosulfate, Na2S2O3 solution
 10% Potassium iodide solution
 Starch indicator
Apparatus
 Conical flask
 Pipette
 Burette
 Aluminium foil paper

B) Peroxide value

Reagents
 S1- sample 1 oil
 S2 – sample 2 oil
 S3 – sample 3 oil
 B - blank
 Acetic acid-chloroform solution
 Saturated potassium iodide solution
 0.01M sodium thiosulfate, Na2S2O3 solution
 Starch solution
Apparatus
 Conical flasks

Methods
A) Iodine Value
B) Label 3 conical flasks with B (blank), S1 (sunflower oil) and S2 (palm oil).
C) Weigh 0.3g of cooked oil and 0.3g of fresh oil into conical flask S1 and S2 respectively.
D) Add 10ml of carbon tetrachloride solvent into the conical flask B, S1 and S2 respectively.
E) Dispense 25ml of Wij’s solution in conical flask B, S1 and S2 respectively.
F) Store the conical flask B, S1 and S2 in dark at 25 oC ± 5oC after covering them with
aluminium foil paper.
G) Remove the conical flask B, S1, S2 from dark after 35±5minutes.
H) Add 20mL of 10% Potassium iodide solution and 100mL of distilled water into the conical
flask B,S1 and S2.
I) Titrate the solution in the conical flask B, S1, S2 with 0.1M standard Sodium Thiosulfate,
Na2S2O3 solution until the pale yellow colour of solution almost disappears. Shake the
conical flask constantly and vigorously during titration.

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 J) Add few drops of starch indicator into the solution and continue the titration until the blue
colour of solution just disappears.
K) Repeat steps 6 to 9 by replacing the conical flask B with conical flask S1 and S2.
L) Record the titration of conical flask B, S1 and S2 and use formula to calculate the iodine
value.

IV = (B-S) x N x 12.69

Experiment 6: Analysis of lipid - Peroxide Value

1. Measure 5g±0.05g of fresh oil (S1) and fried oil (S2) and exposure oil (S3) into three
different conical flasks.
2. Add 30mL of acetic acid-chloroform solution into each conical flask, including the blank
conical flask.
3. Swirl the mixed solution within each conical flask to dissolve.
4. Add 0.5mL of saturated potassium iodide solution, KI into each conical flask.
5. Shake mixture within conical flask for 1 minute.
6. Add 30mL of distilled water into each conical flask.
7. Add 1mL of starch indicator into each conical flask.
8. Continue the titration with 0.1 M Na2S2O3until the blue colour of solution just
disappears.Record the amount of 0.01M Na 2S2O3 in titration process and calculate the
peroxide value for fresh oil, exposure oil and cooked oil.

PV = S x N X 1000

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Experiment 7: Quantitative determination of sugars in honey and milk

Reagents:

i) Enzyme solution (Enzymatic Glucose Reagent)

1) Glucose oxidase
2) Peroxidase
3) 4-aminoantipyrine
4) 4-hydroxybenzoic acid
5) Phosphate buffer
6) pH 7.5±0.1 at 20ºC

ii) Modified Harding B Solution

1) NaHCO3
2) Na2CO3
3) Potassium Oxalate
4) Sodium Potassium Tartrate

Procedure:

Part A: Enzymatic determination of glucose in honey (Glucose oxidase Method)

i. Prepare the following solutions by using pipette:

Reagent Test Tube Number

1(standard) 2(Sample) 3(Blank)
Enzyme solution 1.5ml 1.5ml 1.5ml
Glucose solution (50μg/mL) 0.1ml - -
Honey solution (150 mg /L) - 0.1ml -
Distilled water - - 0.1ml

ii. Stopper each test tube and thoroughly mix the contents.
iii. Incubate the tubes at 37ºC for 10 minutes.
iv. Cool the tubes and within 30 minutes measure the absorbance of the solutions at 500
nm by using 1.5mL cuvettes.

Results and Discussion:

i. From the results of this experiment and those of Part B, calculate the concentration of
glucose and fructose in honey.
ii. Discussion should include a) chemical reactions (glucose oxidase method), b)
advantages of this method, c) composition of honey.

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Part B : Determination of reducing sugar in honey by the Arsenomolybdic Acid Reagent (Nelson
Method)

i. Prepare the solutions specified in the Table 1, using a pipette for all volume measurements.
ii. Add 2.0 ml Modified Harding B solution to each test tube.
iii.Mix and plug each tube with a piece of cotton wool (to reduce aerial oxidation of Cu+ ions)
iv. Heat the tubes in a boiling water bath for exactly 10 minutes, then allow the tubes to cool in
cold water.
v. Add 6ml of Arsenomolybdic acid reagent to each tube and shake well over a period of five
minutes (to remove CO₂).
vi. Measure the absorbance of each solution at 600 nm.

Results and Discussion:

i. Prepare a standard curve and use it to determine the concentration of reducing sugars
(expressed as g /100 g honey).
ii. Discussion should include a) chemical reactions in Nelson method, b) disadvantages of
this method.

Table 1
Blank Glucose Std. Tubes Honey
Reagent 1 2 3 4 5 6 Sample
Std. Glucose (50μg/ml) in isotonic - 0.25ml 0.5ml 0.75ml 1.0ml 1.5ml 2.0ml -
Na₂SO₄ - CuSO₄ solution
Isotonic Na₂SO₄ - CuSO₄ solution 2.0ml 1.75ml 1.5ml 1.25ml 1.0ml 0.5ml - -
Honey (40 mg honey/L) in isotonic - - - - - - - 2.0 ml
Na₂SO₄ - CuSO₄ solution
Modified Harding B Solution 2.0 2.0 ml 2.0ml 2.0 ml 2.0ml 2.0ml 2.0ml 2.0 ml
ml
Stoppered all tubes with cotton and put in boiling waterbath for exactly 10 min.
Remove tubes and put in cool immediately in running water.
Arsenomolybdic Acid Reagent 6.0ml 6.0ml 6.0ml 6.0ml 6.0ml 6.0ml 6.0ml 6.0ml
Read absorbance at 600 nm.

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Experiment 8 Determination of lactose concentration in milk (Nelson Method).

Sample preparation:

i. Treat 1 ml of fat-free milk in a 100ml volumetric flask with 2 ml of 10% sodium tungstate
solution.
ii. Add slowly and with constant shaking 2 ml of 0.3 M H₂SO₄.
iii. Make the solution up to 100 ml with water and allow it to stand for five minutes to allow for
complete precipitation of the protein.
iv. Filter the mixture.
v. Prepare the solutions (as Table 2) using a pipette for all volume measurements.
vi. To each tube add 2 ml of Modified Harding B solution and heat in a boiling water bath for
exactly 10 minutes.
vii. Cool the tubes in water, and with thorough mixing, add 6ml of Arsenomolybdic acid reagent.
viii. After 3 minutes measure the absorbance of the solution at 600 nm.

Results and Discussion:

i. Calculate the concentration (mg/mL) of lactose in the milk.
ii. In this experiment, the Beer-Lambert Law is obeyed for the concentration of lactose.
So, the lactose concentration in milk can be determined by comparison of its light
absorbance with that of a solution of known lactose concentration.

c = A therefore Aunknown = Cunknown

εl Astandard Cstandard

Table 2:

Reagent Blank Std. Sample

Isotonic Na₂SO₄ - CuSO₄ solution 2.0 ml - 1.9 ml
Std. Lactose (50μg/ml) in isotonic Na₂SO₄ - CuSO₄ - 2.0 ml
solution
Milk Filtrate - - 0.1 ml
Modified Harding B Solution 2.0 ml 2.0 ml 2.0 ml
Stoppered all tubes with cotton and put in boiling waterbath for exactly 10 min.
Remove tubes and put in cool immediately in running water.
Arsenomolybdic Acid Reagent 6.0 ml 6.0 ml 6.0 ml
Read absorbance at 600 nm.

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Experiment 9 Determination of Vitamin A

Objective: To determine the vitamin A content using the spectrophotometric method

Introduction

β-Carotene is a strongly-colored red-orange pigment abundant in plants and fruits. It is an organic

compound and chemically is classified as a hydrocarbon and specifically as a terpenoid
(isoprenoid), reflecting its derivation from isoprene units.

Carotene is the substance in carrots, pumpkins and sweet potatoes that colors them orange and is
the most common form of carotene in plants. When used as a food coloring, it has the E number
E160a.

β-Carotene is a non-polar compound, so it is separated with a non-polar solvent such as hexane.

Being highly conjugated, it is deeply colored, and as a hydrocarbon lacking functional groups, it is
very lipophilic. Spectrophotometry method is widely used technique for the analysis of β-carotene in
commercial products.

Methods and materials

Apparatus:

volumetric flask (10 ml)

test tubes wrapped with aluminium foil
B-carotene standard solution (1000 ppm)
Unknown B-carotene solution
Hexane

Procedure:

1. Prepare a series of standard solutions as shown below. Dilute standards in volumetric flask
with deionised water.

Stock B- Total volume Concentration

carotene (1000 (ml) (ppm)
ppm)
Blank 10
0.25 ml 10
0.50 ml 10
1.00 ml 10

2. Pipette 1 ml of the unknown sample into the volumetric flask and top up to
10 ml with deionised water.

3. The absorbance of the filtrate was measured spectrophotometrically at 450 nm (= λmax for β-
carotene in hexane) using hexane as a blank.

4. Calculate the amount of B-carotene in the unknown solution from the standard
curve.

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Experiment 10: Determine Trace Metals (Cadmium) in Baby Foods

Procedure:

1. Sample preparation

a) Weigh 5 g of the sample and transfer to 100 ml conical beaker.

b) Add 35mL of 6 M HCI and boil over the Bunsen Burner for 15 minutes.

c) Cool and adjust the mass of the digest to 50 g by adding deionised water.

d) Aliquot of the digest was centrifuged at 4400 rpm for 10 minutes.

2. Determination of metal ions

a) A series of Cadmium standard solutions covering the appropriate range (2mg/L, 1mg/L,
0.5mg/L, 0.25mg/L, 0.125 mg/L) was given.

b) Measure the absorbance of Cadmium standard solutions and sample (prepared from
step 1) by using Atomic Absorption Spectrophotometer.

c) Plot standard curves, absorbance of Cadmium versus concentrations of Cadmium

standard solutions.

d) Determine the concentration of metal ions in the sample from standard curves.

Discussion should include the following:

a. the possible origin(s) of trace metals in foods;

b. the suitability of the sample preparation;
c. the biochemical role of the two elements (iron and cadmium) in nutrition, pollution or
toxicology
d. How the sensitivity of this method could be improved?

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