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UNIVERSITY OF MALAYSIA TERENGGANU

FACULTY OF FISHERIES AND FOOD SCIENCES

PRACTICAL GUIDE

STM 3233 CHEMICAL

ANALYSIS OF FOOD
 STM 3233 – Chemical Analysis of Food

PRACTICE 1

DETERMINATION OF MOISTURE AND DRY MATERIALS

1.1 Introduction to Oven Drying Method

The oven drying method is a thermogravimetric method (loss during drying) in which
the food sample is dried for a certain period (up to a constant sample weight) at a
constant temperature. Moisture content is determined by weighing the sample
before and after drying and determining the resulting weight difference.

Most moisture determination methods involve drying the sample at atmospheric

pressure and high temperature (135℃). This method can result in the loss of volatile
fatty acids (easily converted into gas) and some types of sugar will be destroyed at
high temperatures (>70℃). Therefore, several other methods can be used:

(a) Drying in the absence of air (vacuum) at a temperature of 95-100℃, the

rate of loss of volatile fatty acids and some types of sugar will be reduced.
(b) Distillation with toluene.
(c) Drying in vacuum without heating over sulfuric acid.

1.1.1 Apparatus

Oven 110℃, crucible, crucible lid, tongs and desiccator.

1.1.2 Procedure

A. Weigh the crucible and sample

1. Weigh the crucible and sample to four decimal places (balance sensitivity
0.0001 g (or 0.1 mg).
2. Clear (tare) balance reading (0.0000 g) before weighing the sample.
3. Use the same scale throughout the analysis (before and after the drying
process).
4. Use tongs to lift the crucible and its lid.
5. When weighing dry samples from the desiccator (after drying), weigh as soon
as possible because the sample readily absorbs moisture.

B. Method
1. Label the crucible using a 2B pencil.
2. Dry the lid and bowl inside the oven at 100-110°C for 30 minutes.
3. Cool the lid and crucible in the desiccator for 20-30 minutes.
*If the crucibile has been dried, label the crucible using a 2B pencil.
 STM 3233 – Chemical Analysis of Food

4. Weigh the empty crucible accurately. Record the weight of the crucible (W1).
5. Weigh accurately 2 grams of sample into the crucible. Record the weight of the
sample (W2).
6. Dry the crucible containing the sample in an oven at 105℃ (the lid of the crucible
is not tightly closed) for 16 hours (overnight).
** For dry food items (such as pet food pellets) need to be dried for 6
hours.
For moist material (muscle tissue) it needs to be dried for 24 hours
until the weight is constant.
7. After drying, close the lid, cool in a desiccator and weigh the crucible containing
the dried sample (without the lid). Record the weight of the crucible and dry
sample.
8. Calculate the weight of the dry sample.
9. Calculation:

dry sample weight (g)

Dry materials (%) = × 100
original sample weight (2g)

Moisture (%) = 100 – Dry matter (%)

𝐎𝐫𝐢𝐠𝐢𝐧𝐚𝐥 𝐬𝐚𝐦𝐩𝐥𝐞 𝐰𝐞𝐢𝐠𝐡𝐭 (𝐠)−𝐃𝐫𝐲 𝐬𝐚𝐦𝐩𝐥𝐞 𝐰𝐞𝐢𝐠𝐡𝐭

Moisture percentage (%) wet base = × 𝟏𝟎𝟎
𝐎𝐫𝐢𝐠𝐢𝐧𝐚𝐥 𝐬𝐚𝐦𝐩𝐥𝐞 𝐰𝐞𝐢𝐠𝐡𝐭 (𝟐𝐠)

1.2 Infrared Heating Method (Infrared)

1.2.1 Apparatus

Moisture Balance(Infra Red)

Samples: nuts, biscuits, bread and so on.

1.2.3 Procedure:

1. Place 2 g of the sample on the tray of the infrared balance and level the
sample and do not compact it.
2. "Turn it on" (switch on) infrared balance device (light on) and set the heating
temperature at 120℃. Record the change in weight loss every 2 minutes until
the reading does not change.
3. Then cool down for a while and do it again to get an accurate reading by
calculating the average of the two readings.
4. Plot a graph of weight loss (Y Axis) against time (X Axis).

Question:
Is the percentage of moisture by drying using infra-red found to be the same as
the percentage of moisture through oven drying?
 STM 3233 – Chemical Analysis of Food

PRACTICE 2

DETERMINATION OF ASH

Introduction

Ash is the inorganic part of food (remains after water and organic matter are lost
through the burning process)

Apparatus

Crucible, Muffle Furnace and a desiccator.

Sample

Beans, nuggets, crackers and so on.

Procedure

2.1 Total Ash Content in Method Food

1. Label the crucible before drying.
2. If the crucible has been dried, label the crucible with a pencil after weighting
the empty crucible.
3. *Use the same scale during the experiment.
4. Dry the crucible and the lid in the oven at 100℃ (30 minutes). Then cool in a
desiccator and weigh accurately, uncovered.
5. Weigh 5 g of the sample into a dried crucible.
6. Put the crucible inside muffle furnace temperature of 550℃for 24 hours.
7. Remove the sample from the muffle furnace and cool the sample in a
desiccator.
8. After cooling, weigh and calculate the percentage of ash remaining in the
crucible.
9. Calculation

Ash Percent (%) = ash weight (g) × 100

original sample weight (g)

 STM 3233 – Chemical Analysis of Food

PRACTICE 3

DETERMINATION OF CRUDE FAT CONTENT

STANDARD OPERATING PROCEDURE FOR LABTEC ST310

SAMPLE PREPARATION
*Weigh 1.00 g sample into filter paper, fold the filter paper, then put into a thimble
*Fill in solvent in aluminum cup: 40 ml of petroleum ether

OPERATING THE EQUIPMENT

Press the "MAINS" / POWER key to switch on the control unit
↓
Switch on the cooling unit
↓
Move each knob to BOILING position
↓
Attach thimbles to adapters, fasten the thimbles onto the magnet
↓
Lower the knob to RINSING position (The thimble will now hang just below the
condenser level)
↓
Insert the filled extraction cups using the cup holder
↓
Lower the handle ensuring that the safety hook engages.
The cups are now clamped into the condensers
↓
Start the program on the Control Unit by pressing the Ф key.
The buzzer signal starts when the set temperature minus 5°C is reached
↓
Move the extraction mode knobs to BOILING position, then press ''TIMER'' (Make
sure the condenser valves are open!)
The buzzer signal starts when the countdown reaches zero
↓
Move the extraction mode knobs to RINSING position, then press ''TIMER'' Buzzer
signal starts when the countdown reaches Zero
↓
After RINSING, close the condenser valves by turning a quarter turn
↓
Press ''TIMER'' to start the RECOVERY (Last traces of solvent will be collected in the
condenser). Buzzer signal starts when the recovery countdown reaches Zero
↓
Press ''TIMER'' to start PRE-DRYING
↓
 STM 3233 – Chemical Analysis of Food

After PRE-DRYING, take out the cups with the cup holder. Move them to oven for
drying
↓
Take out thimbles *CAUTION - BEWARE OF THE HOT PLATE SURFACE
↓
Stop the cooling water supply and turn the "POWER" off

Percentage of crude fat (%) = (W₃ - W₂)/ W₁ ×100%

W₁ = weight of the sample (g)

W₂ = weight of the extraction cup (g)
W₃ = weight
 STM 3233 – Chemical Analysis of Food

PRACTICE 4

DETERMINATION OF CRUDE PROTEIN –

KJELDAHL METHOD

Introduction

Protein determination by the Kjeldahl method requires heating the sample with
concentrated sulfuric acid and a catalyst. Carbon oxidation process will occur and
nitrogen will become ammonium sulfate (digestion process). Ammonium
concentration can be determined by the titration method. This method determines
the percentage of nitrogen content. The nitrogen content will then be multiplied by
a conversion factor value (F).

Use of the Kjeltec® System

The system consists of:

1. Digestor DS6 (with Exhaust)

2. Kjeltec 2100 Distilling Unit

Test material

Kjeltabs Cu 3.5 Catalyst, Concentrated H2SO4, 4% Boric Acid (4 g H2BO3 in 100 ml

hot distilled water), Glass Beads/Antifoaming Agent (H2O2), 40% NaOH (40 g NaOH
in 100 ml distilled water), Bromocresol Green Indicator (0.25 g methyl red + 0.25 g
bromocresol green
– make 100 ml with ethanol), 0.1N HCl

Procedure

1. Weigh accurately 1.0 g (accuracy 0.1 mg/0.0001 g) of sample in a digestion

tube (250 ml).
2. Add 2 Kjeltabs Cu 3.5 catalyst tablets to the tube.
3. Add 12 ml of concentrated H2SO4 carefully, and shake the tube gently to wet
the sample with the acid.
4. Connect the Exhaust system to the tube (which has been placed on the rack)
and make sure the aspirator system can start working.
5. Lock racks and Exhaust systems to the Digester DS6 heater Block (which has
been preheated at 420℃) to start the acidic digestion process.
6. After almost 5 minutes, turn off the aspirator system so that acid fumes can
form only at the top of the Exhaust system.
7. The digestion process continues until a clear green/blue solution forms in the
sample tube (usually takes 30-60 minutes – depending on the type of sample)
8. Once the digestion process is complete, remove the rack from the tube (make
 STM 3233 – Chemical Analysis of Food

sure the Exhaust system is still connected to the tube) and cool the tube
vertically for 10-20 minutes.
9. Carefully add 75 ml of distilled water to the cooled tube. This step continues
with the distillation process
10. To prepare the receiving solution (result of sample distillation) – Add the
receiving solution (25 ml of 4% Boric Acid with 10 drops of green Bromocresol
Indicator) to a 250 ml conical flask.
11. The receiving solution is then placed into the Kjeltec 2100 distillation unit.
12. Place the tube (from step 9) into the distillation unit and close the safety door.
13. Automatically flow 50 ml of 40% NaOH into the tube.
14. The distillation process is allowed to run for 4 minutes until a clear green color
is formed on the receiving solution (distillation result).
15. Titrate the distillate with standard 0.1N HCl solution until the clear green color
changes to blue/grey. Record the volume (ml) of titrant used.

Note:
Determination of the blank (Blank) should be carried out for each analysis. The blank
contains only 12 ml of concentrated H2SO4 and 2 Kjeltabs Cu 3.5 catalyst tablets
(without sample) and has to go through the same procedure (steps 4 to 15).

Calculation

(T−B) ×HCl (mol) ×14.007 (g/mol N) × 100

Nitrogen content (%) = Weight of sample (g)×1000 (𝑚𝑙)

Crude Protein Content (%) = N (%) × F

T = Titration volume of the sample

B = Titrate volume of blank
N = Normality of HCl/acid
F = Protein factor; 6.25/5.7/6.38 depending on the type of sample. (wheat
and produce=5.7, milk and produce=6.38, other food (assuming it contains
10% N) = 6.25, gelatin=5.5)
 STM 3233 – Chemical Analysis of Food

DETERMINATION OF CRUDE PROTEIN

7.1 INTRODUCTION

Most of the nitrogen (N) in food is in the form of proteins (contained in

amino groups, NH2 in amino acid molecules). In general protein consists
of about 50%C, 22%O2, 7%H and 16%N (depending on the sample).

Through proximate analysis in the determination of crude protein

content, the sample will go through a heating process with concentrated
sulfuric acid with the help of a catalyst (this process is known as
digestion). Carbon oxidation process occurs and nitrogen becomes
ammonium sulfate. Then the concentration of nitrogen in the sample is
determined through the titration method. Assuming that the protein
contains 16%N, % protein is calculated by multiplying the determined
N concentration in the sample by 6.25.

7.2 APPARATUS AND TESTING MATERIALS

i. Digestive units set

ii. Distillation unit set
iii. Nitration unit
iv. Digital electronic scale (sensitivity 0.1 mg
or 0.0001 g)
v. Concentrated Sulfuric Acid
vi. Catalyst material (1 part CuSO4+ 9 parts
K2SO4) orcatalyst tablet
vii. Boric Acid (4%)
viii. Mixed indicator

Methyl red (0.1% in 95% ethanol) : 1 section

Bromocresol Green (0.1% in 95% ethanol) : 3 section
Distilled water : 4 section

ix. Sodium Hydroxide, NaOH (40% weight/volume)

x. 0.01N HCl standard solution
 STM 3233 – Chemical Analysis of Food

7.3 METHOD

7.3.1 Digestion

i. Weigh accurately approximately 0.2 g of sample

(record as W) and place in a 100ml Kjeldahl digestion tube.
Prepare a tube without sample to be used as a blank.

ii. Add 5 ml of concentrated sulfuric acid (H2SO4) and

half catalyst tablets in all tubes including tubes that are not
sampled (blank). Shake the tube thoroughly so that the
sample is wetted with acid.

iii. Place the digestion tube into the holder rack and
transfer it to the heating block on the digestion system at
once.

iv. Connect the exhaust system to each digestion tube

and lock securely in the holder rack.

v. Activate the ‟scrubber‟ system switch and make sure it works

before starting the digestion process.

vi. Activate the digestion system switch and start

digestion by adjusting the temperature control knob. White
smoke will form in the tube while digestion is taking place.
This gas is acidic and highly toxic, avoid inhaling it and
keep the scrubber system running throughout the
digestion process so that it can be treated.

vii. Continue digestion until all samples are dissolved

and a clear green/blue solution is formed. Usually it takes
around 30-60 minutes – depending on the type of sample.

viii. After the digestion process is over, move the rack

and cool the tube vertically for 10 – 20 minutes. Make sure
the exhaust system is still connected to the tube
throughout the cooling process.
 STM 3233 – Chemical Analysis of Food

7.3.2 Distillation

i. Prepare the receiving solution by placing 30 ml of

Boric Acid (4%) in a 250 ml conical flask (collecting flask)
and adding 8 drops of the mixed indicator solution. This
receiving solution is red in color.

ii. Place the collecting flask below the tip of the

condenser with the tip of the condenser below the surface
of the receiving solution.

iii. Activate the Kjeldahl Buchii distillation apparatus

and pump 40 ml of distilled water and 30 ml of
concentrated NaOH (40%) into the distillation tube that
already contains the sample. (The addition of distilled
water and NaOH can be done manually if the distiller is not
equipped with a pump system)

iv. Adjust the steam knob to the active (“on‟) position

and let the distillation process take place for about 5
minutes. Next, lower the collecting beaker so that the
distillate can drip for 1 minute. The solution in the
collecting beaker will change color from red to dark
green/blue.

v. Remove the receiving flask for the titration process.

vi. Do the same distillation process to the empty tube

(blank)

7.3.3 Titration

i. Titrate the distillate in a collecting beaker (method

7.3.2 step v) with 0.1N HCl until the dark green/blue color
changes to red. The final point of the titration is when the
green/blue color starts to change to red – under the correct
conditions the solution is gray in color - record the volume
of HCl used as T.

ii. Perform the same titration process on the distillate

from the blank - record the volume of HCl used as B.
 STM 3233 – Chemical Analysis of Food

7.4 CALCULATION

% N in the sample = (T-B) x N x 14.007 X 100

W

% Protein in the sample =%NxF

Where: T = Volume of HCl used in the titration of sample distillation (ml)

B = Volume of HCl used in the titration of the blank

distillate (ml)
N = Normality of HCl
W = Sample weight (mg)
F = Protein Factor; 6.25 / 5.7 / 6.38

7.5 PRECAUTIONARY MEASURES

It should be remembered that the digestion of the sample in the

determination of protein content uses concentrated sulfuric acid which
can cause injury. Handle it under the fume chamber with care. During
the digestion process this acid smoke will form and it is very dangerous.
Ensure that it is not released by ensuring that the exhaust system is
installed perfectly and that the scrubber unit is working properly. The fume
vaporizing solution should be blue. Replace it if it has turned clear.

Note the following points to ensure that the analysis performed is error-
free:

i. Weigh the sample accurately (record up to four

decimal points displayed on the screen of the electronic
scale used). Carelessness in sample weighing may affect
the accuracy of the results.
ii. One of the most important aspects of protein
determination is the accuracy of the nitration process.
Proceed with caution and repeat if excessive nitration is
suspected. A color change from deep green to red during
the nitration process may occur for samples with low
protein content. For such cases, perform the nitration
using more dilute HCl (lower N normality).
 STM 3233 – Chemical Analysis of Food

iii. Always rinse the distillation unit thoroughly every

time you finish distilling a sample. Imperfect rinsing will
affect the accuracy of subsequent sample results.

7.6 RESULT DATA OF CRUDE PROTEIN DETERMINATION ANALYSIS

i. Sample:

ii. Recorder :

Replicate
Things Average
1 2

Sample Weight (W)

Volume of HCL Used in

Sample Titration (T)

Volume of HCl Used In

Titration blank (B)
 STM 3233 – Chemical Analysis of Food

PRACTICE 5

DETERMINATION OF CRUDE FIBER

6.1 INTRODUCTION

Crude fiber in food is composed of a combination of cellulose and other elements

such as lignin and pentosan as well as several types of polysaccharides that are
insoluble in hot liquid acid/alkali. Through proximate analysis, determination of raw
fiber is done by heating the sample in dilute acid followed by dilute alkali. The
remaining materials (residue) after going through the process are considered
insoluble by hot acids and alkalis and consist of fibers (organic) and ash (inorganic).
Next, the determination of the amount of ash in the resulting residue is done by
heating the residue at a high temperature to burn all the organic matter. The fiber
content is calculated from the difference in the weight of the residue with the
determined weight of the ash.

However, the presence of fat in the sample may interfere with the accuracy and
smoothness of the process. Therefore, it is better if this analysis uses a sample that
has gone through the fat extraction process (or de-fat process) first.

6.2 APPARATUS AND TESTING MATERIALS

i. Oven
ii. Desiccator
iii. Digital electronic scale (sensitivity 0.1 mg or 0.0001 g)
iv. Muffle furnace
iv. ANKOM Fiber Analyzer Set200
v. Filter bag(Filter bags)– F57 or F58
vi. Heat mount(Heat sealer)
vii. Solvent and acid resistance marking pen
viii. Sulfuric Acid (H2SO4) – 0.255N
ix. Sodium Hydroxide (NaOH) – 0.313N

6.3 METHOD

i. Weigh all dried filter bags (W1) and record accurately. Label the filter
bags to be used (depending on the number of samples/replicates with
an additional bag as a blank). Take another filter bag to use as a blank.
ii. Weigh out 0.95-1.00 g of the sample (W2) and put it in a filter bag.
Avoid placing the sample too close to the mouth of the filter bag to
allow it to seal perfectly.

*Leave the vacuum bag without a sample.

 STM 3233 – Chemical Analysis of Food

iii. Using a heat sealer, seal the mouth of the filter bag perfectly.

*Please use enough heat and sealing time (about 2 seconds) to get a
perfect sealed but not melt the filter bag.

iv. Extract the fat in the sample by immersing all bags (including vacuum
bags) used in sufficient organic solvent (petroleum ether) for 10
minutes. Drain and allow the filter bag to air-dry. Disperse the sample
in the filter bag by shaking and flicking it to avoid lumps.

v. Place a maximum of 24 filter bags (including vacuum bags)

inSuspender Bag. Place 3 bags on each tray properly and match with
other 9 trays available regardless of the number of samples available
(do not place the bag on the topmost of tray). Insert Suspender Bag
in the ANKOM200 Fiber Analyzer chamber (vessel). and put a weight
on it to keep it submerged.

vi. Add sulfuric acid (H2SO4– 0.255N) into the ANKOM200 Fiber Analyzer
chamber (vessel). For analysis of less than 20 bags, the minimum
volume of acid required is 1500 ml. An additional 100ml/bag should be
made when the sample exceeds 20. The maximum volume is 1900-
2000 ml.

vii. Turn on (On) “Agitate‟ and “Heat‟ buttons and make sure the
Suspender Bag moves perfectly. Close and tighten the lid of the
ANKOM200 Fiber Analyzer chamber (vessel) perfectly. Extract the
sample for 40 minutes.

viii. After the extraction period is over, turn off “Agitate” and “Heat”
buttons. Open the drain valve slowly to remove the acid and hot
dissolved material before opening the chamber cover. *Never open the
lid without removing the acid completely first.

ix. Open the chamber cover and close the drying valve again. Add 1900ml
of hot water (50-90°C) and "agitate" for 5 minutes. Repeat this rinsing
process with hot water twice.

*This process of rinsing with hot water can be done either with:
1) close the chamber lid and turn on the heating button (heat
) or
2) leaving the chamber lid open and without turning on the heating
button (heat)

x. Repeat steps vi, vii and viii using sodium hydroxide (0.313N NaOH) as
an extracting solution.
 STM 3233 – Chemical Analysis of Food

xi. Do the rinsing process with hot water as in step ix and repeat three
times.

xii. After the rinsing process is complete, remove the filter bag and press
gently to remove excess water inside.

xiii. Dry thoroughly in the oven (oven) at a temperature of 102±2°C

(usually takes around 2-4 hours)

xiv. Remove the bag from oven and cool to room temperature in a
desiccator and weigh accurately (W3).

xv. Put the bag into the crucible (perfectly dried) that has been weighed
(W4) and carry out the ashing process in a muffle furnace for 2 hours
at a temperature of 600±15°C.

xvi. Remove the crucible from the muffle furnace, cool in a dessicator and
weigh accurately (W5).

6.4 CALCULATION

𝑊𝑥−(𝑊1 ×𝐶1)
Crude Fiber (%) = × 100
W2

Where: W1 = Weight of filter bag

W2 = Weight of sample
Wx = Weight of organic matter (difference weight
before and after ashing)
= (W3 + W4) - W5
C1 = Ash corrected blank bag factors
W3∗+W4∗ −W5
= W1
PRECAUTIONARY MEASURES

Note the following points to ensure that the analysis performed is error-free:

i. The accuracy of the analysis depends on the accuracy of the scales.

To get accurate results make sure all weighings are recorded
accurately (record up to four decimal points displayed on the screen
of the electronic scale used).
ii. The pore size of the filter bag is 25 microns. Samples that are too fine
will come out and will cause errors in the analysis results. It can usually
be detected when the value of C1 is greater than 1.
iii. Always use forceps to handle any apparatus that has been dried (or
after going through the ashing process) because moisture from your
hands may affect the accuracy of the results obtained. After removing
 STM 3233 – Chemical Analysis of Food

the desiccator, weigh it immediately so that moisture absorption from

the air can be reduced.
 STM 3233 – Chemical Analysis of Food

PRACTICE 6

DETERMINATION OF LIPID RANCIDITY

INTRODUCTION

The term rancidity refers to the off-odor and off-flavour resulting from lipolysis
(hydrolytic rancidity) or lipid oxidation (oxidative rancidity). Lipolysis is the
hydrolysis of triglyceride molecules into free fatty acid and glycerol. Because of their
volatility, hydrolysis of short-chained fatty acids can result in off-odor. Lipolysis is
commonly determined using acid value or percentage of free fatty acid.

Lipid oxidation or oxidative rancidity (also called autoxidation) occurs in bulk fats
and oils proceeds via a self-sustaining free radical mechanism that produces
hydroperoxides (initial or primary products) that will undergo scission to form
various products including aldehyde, ketones, organic acids and hydrocarbons (final
or secondary products). Measuring the current status of a fat or oil in regard to lipid
oxidation can be achieved using such procedures as peroxide value, p-anisidine
value, hexanal measurement and the thiobarbituric acid (TBA) test. Some of these
procedures have been modified (especially with respect to sample size) for use in
biological tissue assays.

The purpose of this laboratory exercise is to observe the effects of trace minerals
and antioxidants on the course of lipid oxidation and to become familiar with
different procedures utilized to evaluate the extent of lipid oxidation that has
occurred in vegetable oil samples. This laboratory exercise will extend until the next
scheduled laboratory session and will require an analysis to be conducted during
the week to be scheduled with the instructor and laboratory assistants followed by
a final analysis of the samples at the next week's scheduled laboratory session.

6.1 Sample Preparation for Lipids Oxidation (Effect of

storagetemperature on lipid oxidation of oils)

1) Palm oil (bottle packaging)

2) Palm oil (plastic packaging)
3) Rice bran oil
4) Sunflower oil
5) Corn oil
6) Canola oil

Procedure:

1) Place 20 ml of the oil into each of three (3) beakers:

a. Beaker 1: Control – Keep at room temperature.
b. Beaker 2: Heat the oil at 40°C in an oven for 2 days.
c. Beaker 3: Heat the oil at 60°C in an oven for 2 days.
2) Do a peroxide value analysis.
 STM 3233 – Chemical Analysis of Food

Peroxide value

1) Weigh 5 ± 0.05 g sample into a 250 ml Erlenmeyer flask. Perform the

experiment in duplicate.
2) Add 30 ml of acetic acid and chloroform solution (3:2 v/v) (Do this in the
fume hood).
3) Swirl the flask until the sample is dissolved and add 0.5 ml saturated
potassium iodide (KI) solution.
4) Allow the solution to stand with occasional swirling for one minute and then
add 30 ml of distilled water.
5) Slowly titrate with 0.01 N sodium thiosulfate (Na2S2O3), adding it with
constantand vigorous shaking.
6) Continue titrating until the color changes to light yellow (Take note: titration
beyond the supposed endpoint will result in no changes to blue color after
the soluble starch indicator is added).
7) Add a few drops of 10% soluble starch indicator which will give a blue color.
8) Continue titrating with Na2S2O3until the blue color just disappears.
9) Do the same procedure as above (step 1-8) for blank (with no addition of fat
sample).

Calculation:

Peroxide value (mEq peroxide / kg fat) =

(S−B)×N ×1000
Sample weight (g)

S = sample titration, ml
B = blank titration, ml
N = Normality of Na2S2O3 solution
References

1) Horwitz, W. (2002),41.1.16 AOAC Official Method 965.33, Peroxide value of

oils and fats. Official Methods of Analysis of AOAC International, 17Thed.,
Gaithersberg, MD: AOAC International.

2) Nawar, WW (1996),Lipids. In: Fennema, OR, editor. Food Chemistry. 3rded.

New York: Marcel Dekker, pp. 225 – 319.
 STM 3233 – Chemical Analysis of Food

PRACTICE 7

DETERMINATION OF ASCORBIC ACID (VITAMIN C)

CONTENT

Introduction

Vitamin C consists of 2 biologically active substances, L-ascorbic acid and L-

dehydroascorbic acid. The natural form commonly found in food is L-ascorbic acid
but there is also L-dehydroascorbic acid. The analysis of ascorbic acid in food is
usually done by using the oxidation reaction of ascorbic acid with a redox indicator
such as 2, 6 dichlorophenolindophenol.

The chemical estimation of ascorbic acid depends on its ability to reduce the redox
indicator, 2, 6 dichlorophenolindophenol (DCPIP). Ascorbic acid is extracted from
food and nitration of ascorbic acid with an indicator is performed in the presence
of acids such as metaphosphoric acid or oxalic acid. The acid is used to maintain
the correct acidity and is suitable for neutralization and to prevent the auto-
oxidation of ascorbic acid.

Apparatus

Conical flask (150 ml), Burette, Pipette, Glass wool

Sample

Fruit juice

Test material

1. Dissolve the dye/dye standard: Dissolve 0.05 g of 2, 6

dichlorophenolindophenol in 100 ml of distilled water and filter.
2. Ascorbic acid standard solution: 0.05 g of ascorbic acid in 60 ml of 20%
metaphosphoric acid and dilute to 250 ml.
3. Acetone.

Procedure

A. Standardization of dye solution 2, 6 dichlorophenolindophenol

1. Pipette 10 ml of ascorbic acid standard solution into a conical flask.
2. Titrate with the dye solution until a pale and permanent pink color is obtained
(15 seconds). Record the ml value of the titrant.
3. Calculate the concentration of ascorbic acid as mg of ascorbic acid equivalent
to 1 ml of dye solution.
 STM 3233 – Chemical Analysis of Food

B. Sample preparation and determination of ascorbic acid

1. Pipette 50 ml of fruit juice (which has been filtered) into a 100 ml volumetric
flask.
2. Add 25 ml of 20% metaphosphoric acid and make the volume to 100 ml with
distilled water.
3. Pipette 10 ml of the solution into a conical flask and add 2.5 ml of acetone.
4. Titrate with the dye solution until a pale and permanent pink color is obtained
(15 seconds). Record the ml value of the titrant.
5. Calculate the ascorbic acid content in the sample as mg/100 g sample and IU
(International Unit).

CALCULATION
1. Dye standardization: to express mg of ascorbic acid equivalent to ml of dye
(C)
Prepared standard ascorbic acid solution = 0.05 g/250 mL
or

mg/mL
Therefore, 10 mL of a standard ascorbic acid solution contains
mg of ascorbic acid
Volume of dye reacted with mg of ascorbic acid
=_ mL
So, C= mg ascorbic acid/ mL dye (to be used in subsequent
calculations)

2. Calculation of ascorbic acid in the sample

Prepared juice = 50 mL/100 mL (diluted sample)

So, 10 mL of the diluted sample contains _ mL (original
sample used)
10 mL of diluted sample react with mL dye (V) 10
mL blank solution reacts with _ mL dye (B)
mg of ascorbic acid / 100 mL sample = (VB) x C x (1/mL original sample
used) x 100

*Can also use the following formula:

mg ascorbic acid/g or ml sample = (V – B) x C x (DF/WT)
where
C = mg of ascorbic acid / ml of dye
V/B = ml dye used for titration of diluted sample / Blank
DF = dilution factor (final diluted volume for sample dilution/diluted volume
used in titration)
WT = sample weight, g
 STM 3233 – Chemical Analysis of Food

PRACTICE 8

DETERMINATION OF REDUCING SUGAR

(SOMOGYI METHOD)

Introduction

Apart from the colorimetric technique, reducing sugars can also be analyzed through
the titrimetric technique. This method is suitable for extracting neutral or nearly
neutral sample that are free of protein. Sweets usually contain sucrose as the main
sweetening agent and for analysis purposes, sucrose (not reducing sugar) needs to
be hydrolysed first to its monosaccharide components and the amount of reducing
sugar will then be determined. The Somogyi method is based on the reaction of
reducing sugars with an alkaline cupric tartrate solution followed by iodimetric
titration. Reducing sugars reduce Cu (II) ions to Cu (I) ions when boiled. The
cuprous oxide formed is further oxidized back to Cu (II) by the iodine released from
the reaction of KIO3 with KI under acidic conditions. Excess I2 is titrated with
thiosulfate solution. The difference between the titration value of the blank and the
titration value of the sample is the total amount of iodine required to re-oxidize Cu
(I) ions.

The equation
Reducing sugar + Cu2+ Cu2O + oxidized sugar

IO3- + 5I- + 6H+ I2 + 3H2O

Cu2O + 2H+ + I2 2Cu2+ + 2I- + H 2O

I2 + 2S2O2-3 2I- + S4O2-6

Apparatus

Boiling water container, Burette (50 ml), volumetric flask (100 ml), test tube, glass
ball - 15 mm diameter, beaker

Sample

Candy does not crystallize like Hudson.

Test material

Cupric test material - KI

Dissolve the following chemicals one by one in 800 ml of distilled water in the order
given: 7.5 g Cu4SO2.5H2O; 25 g of KNa (C4H6O6).4H2O (potassium sodium tartrate);
20 g of Na2CO3; 20 g NaHCO3 and 5.0 g KI. Weigh accurately 0.7850 g of KIO3 and
 STM 3233 – Chemical Analysis of Food

dissolve in 20-30 ml of distilled water. Add this solution to the solution above and
mix thoroughly. Transfer the solution to a 1000 ml volumetric flask and dilute to
volume with distilled water.

Sodium thiosulfate, 0.1 M (standardization of sodium thiosulfate using potassium

dichromate)Dissolve 25 g of Na2S2O3.5H2O with distilled water and dilute to 100 ml.
Standardize with a 0.1 M dichromate solution (4.903 g K2Cr2O7 in 1 liter of distilled
water) as follows; Pipette 25 ml of 0.1 M dichromate solution into a 250 ml conical
flask, add 25 ml of 0.5 H2SO4 and 3 g of KI. Mix and leave for 1 minute and titrate
with thiosulfate solution. Add the starch indicator before the entire iodine color
disappears and continue the titration until the blue color disappears. Use the results
obtained to make 0.01 M thiosulfate.

Starch indicator solution

Weigh 10 g of soluble starch and make a starch suspension using 50 ml of distilled
water. Add this suspension to 950 ml of boiling water and stir gently. Let the solution
boil for 3 minutes while stirring. Cool and add 1 g of ZnCl2 as a preservative.

Indicator solution

Phenolphthalein stock solution

Glucose

Dissolve 1 g of anhydrous glucose with 100 ml of distilled water and mix

thoroughly. (1g/100ml = 1000mg/100ml = 10mg/ml)

Solution H2SO4 0.5M

Procedure

Sample preparation
1. Prepare a 0.2% candy solution by dissolving 0.2 g of finely chopped candy in
100 ml of distilled water (in a 100 ml volumetric flask).
2. To hydrolyze non-monosaccharide sugars to the monosaccharide form, pipette
5 ml of sugar solution into a 50 ml beaker and add 10 ml of H2SO4 (0.5 M)
solution. Transfer to a boiling water container and heat for 10 minutes (Note 1).
3. Then, cool the hydrolysis product under running water. Add 2 drops of
phenolphthalein indicator solution and neutralize with 1 M NaOH solution until
the hydrolysis product turns a pale red color.
4. Transfer the neutralized hydrolyzate into a 50 ml volumetric flask quantitatively
and dilute to volume with distilled water.

Determination of reducing sugars

1. Dilute 5 ml of glucose stock solution to 100 ml with distilled water to make
glucose standard solution.
 STM 3233 – Chemical Analysis of Food

2. Pipette 0, 1.0, 2.0, 3.0, 4.0, and 5.0 ml standard aliquots of glucose into 6
separate test tubes.
3. Make the above volume to 5 ml by adding distilled water to an appropriate
volume using a pipette.
4. Pipette 5 ml of the neutral hydrolysis product into the other 2 test tubes.
5. To all test tubes, add 5 ml cupric test material - KI and mix thoroughly.
6. Cover the test tube with a marble and simultaneously put all the test tubes into
the boiling water (Note 1).
7. Boil for 15 minutes.
8. Remove the test tube, cool under running water and add 5 ml of 0.5 M H2SO4
solution. Mix thoroughly and leave for 1-2 minutes.
9. Titrate the liberated Iodine with 0.01 M sodium thiosulfate solution until the
Iodine color almost disappears.
10. Add a few drops of starch indicator and continue the titration until the blue
color of the Iodine-starch complex disappears.
11. Record the volume of sodium thiosulfate required to react with Iodine
completely.

Calculation
1. The difference between the blank titration value and the other titration values is
the amount of iodine required to oxidize the Cu (I) ions brought down by the
reducing sugar.
2. Construct a standard curve by plotting on graph paper or using Microsoft Excel
the titration value difference against the amount of reducing sugar (mg).
3. Get the amount of reducing sugar in the sample extract that corresponds to the
titration value of the hydrolysis product from the standard curve. Calculate the
concentration of reducing sugar (mg/ml) in the hydrolysis product.
4. Next, calculate the reducing sugar content (g/100 g) in the original sample.

Note
1. The sucrose hydrolysis procedure does not need to be performed if the desired
value is for the available reducing sugar content in the sample.
2. If there is a blue to brown color change during boiling, you need to use a more
dilute hydrolysis product.