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Laboratory Manual: University College Sedaya International
Laboratory Manual: University College Sedaya International
Laboratory Manual
Food Chemistry
MF 203
May 2015
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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)
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:
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
- Grinder (which permit food samples to be grounded with minimum heating)
- Porcelain crucible
- Dessicator
- Ventilated oven
- Analytical balance
- Halogen moisture analyzer
Oven method:
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Moisture by weight (%) = (m1 – m2) x 100%
(m1 – m0)
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Experiment 2: Determination of protein
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
- 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.
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
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
- 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.
5. Place the dish in cold muffle furnace and bring the temperature to 550ºC.
7. Remove dish, cool in dessicator and weigh soon after attaining room temperature.
Calculation:
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Experiment 4: Determination of Fat
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
- 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.
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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
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:
Procedure:
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.
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.
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.
Table 2:
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Experiment 9 Determination of Vitamin A
Introduction
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.
Procedure:
1. Prepare a series of standard solutions as shown below. Dilute standards in volumetric flask
with deionised water.
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
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.
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.
d) Determine the concentration of metal ions in the sample from standard curves.
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