TABLE OF CONTENTS

No Topic Time/Hour Date

Dr Wan Zunairah binti Wan Ibadullah
1 General safety rules in the lab and sampling procedure 3 W1
https://www.youtube.com/watch?v=VRWRmIEHr3A (safety 22 Mar 2021
rules in chemistry lab) (Dr WanZ)
2 Determination of Moisture Content W2
Determination of Ash Content 29 Mar 2021
https://www.youtube.com/watch?v=ZZ9qgQ9SbSM (Dr WanZ)
(Moisture content)

3 Determination of crude protein 3 W3

5 Apr 2021
(Dr WanZ)
4 Determination of crude fat 3 W4
https://www.labtube.tv/video/MTA0Njc4 (Soxhlet method) 12 Apr 2021
https://www.youtube.com/watch?v=24qWVLm7hhw (AOAC (Dr WanZ)
method)
Dr Mohd Sabri Pak Dek
5 Determination of crude fiber 3 W5
19 Apr 2021

(Dr Sabri)
6 Sugar profile in various beverages 3 W6
26 Apr 2021
(Dr Sabri)
7 Peroxidase activity in vegetables 3 W7
3 May
8 Determination of ascorbic acid 3 W8
17 May 2021
(Dr Sabri)
Dr Muhamad Hafiz Abd Rahim
9 Determination of calcium in milk 3 W9
24 May 2021
(Dr Sabri)
10 Determination of tannin in tea 3 W10
31 May 2021
(Dr Hafiz)

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11 Determination of acid benzoic in chilli sauce 3 W11
7 Jun 2021
(Dr Hafiz)
12 Effect of processing on food component 3 W12
14 Jun 2021
(Dr Hafiz)

Introduction to Laboratory Safety

As in any chemistry laboratory, you will be working with hazardous substances. Strong acids
and bases, flammable organic solvents, toxic compounds, open flames, and angry professors
usually are present. Learn to recognize the different types of hazards that are present in
each type of technique. Don't forget the long-term hazards while thinking about immediate
dangers.

Hazards associated with analytical chemistry include those you would expect
because you are handling chemicals and therefore face substances that could be toxic,
carcinogenic, explosive, flammable, or corrosive. Some chemicals even have bad smells.
Control of these hazards is based around isolation and containment. Depending upon the
potential danger, the use of closed vessels, fume hoods, protective clothing, and breathing
masks may not be enough. However, there are dangers that are not associated with
chemical reactivity. Burns from hot plates or boiling liquids are frequent. Fingers are cut by
broken glass.

We are in a state of flux at the moment; the rules and regulations governing
laboratory safety are being changed rapidly. There is more concern on the part of both the

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workers and the regulators about possible hazards. The most important ingredient in
laboratory safety, however, is not rules and regulations but common sense.

Here are a number of ''rules" that are, I hope, simply common sense. Please observe
them at all times, and please make sure others do so as well.

1. Wear protective clothing. A laboratory apron or coat is a first line of defense against
stains and acids and bases and may save a $40 sweater. SAFETY GOGGLES will
protect your eyes against corrosive fumes and splashes. SAFETY GOGGLES AND LAB
COATS ARE REQUIRED AT ALL TIMES.
2. Do NOT wear contact lenses in a chemical laboratory. Chemical splashes and even
fumes are absorbed by the eye moisture and carried behind the lens. The lens then
holds the chemical in better contact with the eye for a longer period of time. Once
the chemical begins to react, there is a very strong reflexive action that clamps the
eyelids together, making it impossible to remove the lens or flush the eye. (THE
ONLY FIRST AID FOR A CHEMICAL IN THE EYE IS TO FLUSH WITH LOTS OF DISTILLED
WATER!) Unfortunately, however, this flushing is NOT effective if the patient is
wearing a contact lens.
3. Keep flammables covered and in the fume hood. When using a solvent to extract fat,
for example, all operations must be done under the hood except when the vessel is
closed tightly. Flammables should be properly stored and kept in small containers.
Proper flammable storage is equipped with means of preventing ignition, such as
flame stops, anti-sparking devices, and adequate ventilation. Proper flammable
storage also is equipped with fire containment devices, such as fireproof doors and
explosion blowout panels. By assuming there will be a fire someday, one can easily
provide for much greater safety.
4. NO EATING, DRINKING, CHEWING GUM OR SMOKING IN THE LABORATORY.
5. Bunsen burners should be kept away from flammables such as wooden shelves or
lab notebooks. Never reach across a flame and never leave a burner unattended. Be
sure the GAS is turned OFF COMPLETELY when you are finished.
6. Do not point test tubes, Babcock bottles, or any other narrow mouthed container at
either yourself or anyone else. Heating can blow out any liquid in the neck, such as
over a burner or by adding acid.
7. Add the acid TO the water when diluting. (Acids like to get things “wet”, but they
don’t like to get “wet” themselves). Add the base to the water when diluting. Less
heat and violence is generated this way. Even if you wished to dilute only slightly a
concentrated acid, you should measure out the appropriate amount of water and
slowly pour the acid into it. The receiving container should be in a plastic or lead-
lined sink and the container should be made of metal or heat resistant glass such as
Pyrex These precautions are necessary because considerable amounts of heat are
involved when diluting concentrated acids and bases.
8. Promptly wash off acids or bases spilled on skin or clothes with large quantities of
WATER. Rinses with sodium bicarbonate for acids and with vinegar (dilute acetic
acid) for bases should not be used until AFTER lots of water has been used because
you don't want a violent reaction on top of everything else. Besides, the floor
needed mopping anyway.

3
 9. Clean up all spills promptly. This means right now, not when you have finished the
next task. Again, it is first flush with water then with the appropriate neutralizing
agent and again with water. Sweep up solids and broken glass. Broken glass is to be
disposed of in the broken glass container…not in the general trash.
10. Keep your work area clean and orderly. Clean up as you work. Allow only absolutely
essential books and papers to be on the lab bench. Specifically, book packs and coats
may not be kept in the laboratory other than near the large table.

Guidelines for Report Preparation and Submission

1. The cover page of the report should contain the following information:
a. Title
b. Session (8-11 am or 12 – 3.00 pm) and group number
c. Group member who attended the laboratory practical (the name of any member
who did not attend the laboratory session need not be included. You may choose to
omit the name of a member who did not contribute in the preparation of the report)
d. Date of laboratory practical

2. Each report should contain the following:

a. Abstract (summary of the objective, method and results obtained. Do not include
discussion and references)
b. Introduction including the objective of experiment
c. Materials and Methods (especially if modifications in procedure have been made)

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 d. Results and Discussion. Do not include calculations and refer to food composition
tables whenever possible to compare the results you had obtained. Provide reasons
that might have caused differences in values eg. Different variety of sample used etc.
(Tables must have title above, while figures and graphs must have title below)
e. Conclusion
f. Answers to questions provided in the laboratory manual, or by the coordinators
g. References must be cited in the text and listed in the Reference at the end of the
report (Use the style of the Journal of Food Science in your reference list and in
citations in the text available at www.sciencedirect.com).
h. Appendices (all raw data and experimental calculations must be included)

3. A data should be presented in one form only. If you choose to tabulate your data, do not
present the data again in the form of a graph, and vice versa. A graph may be plotted
using any suitable software.

4. Report must be submitted to the course coordinator two weeks after the laboratory
practical has been conducted.

5. Each group submits one report only.

6. Plagiarism: Plagiarism in practical reports will not be tolerated. A ZERO mark will result
for any report found to be plagiarised from another student’s report. This other
student’s report will also be given a zero mark. Plagiarism particularly applies to copying
whole sections of text from another person’s report. You are encouraged to collaborate
on data analysis, particularly on the required calculations, but the written report must
be your own work.

5
Laboratory Report Assessment Criteria

6
7
 Practical Skills Evaluation Assessment Criteria

Up to maximum of 1-5 % per indicator (to be evaluated in each practical session):

1. attendance at all laboratory sessions;

2. amount of effort put into laboratory practical exercises;
3. amount of preparation undertaken prior to entering the laboratory;
4. quality of performance in the laboratory;
5. confidence in the laboratory;
6. student-student interaction in the laboratory;
7. student-staff interaction in the laboratory.

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 Learning outcomes

Upon successful completion of the course students should have the ability to:

1. Explain the principles behind the analytical techniques used in food analysis.
2. Search the scientific literature and find appropriate instrumental analytical
procedures for analysis of food products.
3. Determine when specific techniques are required, and be able to evaluate and
present data generated using these techniques.
4. Evaluate the desired information for a food or food component and determine the
appropriate technique to generate this information.
5. Demonstrate proficiency in the understanding and applications of various analytical
techniques.
6. Cooperate with other in performing analytical tests.

9
TITLE: DETERMINATION OF MOISTURE CONTENT

PURPOSE: to comprehend the importance of determining moisture content in related to the

quality of food.

LEARNING OUTCOMES:
1. Understand the importance of moisture in food preservation and compositional
analysis.
2. Demonstrate diverse techniques for different sample characteristics.
3. To collect, analyze and evaluate the experimental data.

INTRODUCTION
The moisture (or total solids) content of foods is important to food manufacturers for a
variety of reasons. Moisture is an important factor in food quality, preservation, and
resistance to deterioration. Determination of moisture content also is necessary to calculate
the content of other food constituents on a uniform basis (i.e., dry weight basis). The dry
matter that remains after moisture analysis is commonly referred to as total solids.
While moisture content is not given on nutrition label, it must be determined to calculate
total carbohydrate content. Moisture content of foods can be determined by a variety of
methods, but obtaining accurate and precise data is commonly a challenge. Moisture
determination can be done via few methods such as evaporation (loss on drying) methods
(ex. forced draft oven, microwave oven, vacuum oven and microwave oven), chemical
reaction method (ex. Karl Fisher titration) and using instrumental method [nuclear magnetic
resonance (NMR) and near infrared (NIR)]. The carious methods of analysis have different
applications, advantages, and disadvantages. If the ash content also is to be determined, it is
often convenient to combine the moisture and ash determination.

Oven Drying Method

Principle of Method
Sample with known weight are dried in oven at 105°C to constant weight, and the loss of
weight is used to calculate the moisture content of the sample.

MATERIALS AND PROCEDURES

MATERIALS
Sample given

APPARATUS
Crucible (or similar porcelain or metal dishes)
Drying oven at 101-105°C (or vacuum oven at 68-72°C)
Analytical balance, 0.1 mg sensitivity
Glass rod
Desiccator
Tongs
Spatula

10
PROCEDURES
Preliminary Steps:
NOTE: Handle the crucible with tongs to avoid introducing extra moisture and fat from
fingertips.

1. Set the oven temperature to 105 °C and keep the temperature constant.
2. Clean and dry the crucible and its cover in the oven for a minimum of 30 mins.
3. After 30 mins, remove the crucible with tongs and cool them in a desiccator until they
reach room temperature (approximately 20 minutes).
4. Place the crucible and its cover on a balance and weigh rapidly and accurately.
5. Return the crucible and its cover in the dessicator.
6. Repeat the weighing procedure at least twice until a constant reading is obtained.
7. Weigh 2-5 g of sample into crucible mentioned above.
8. Place the crucible together with the cover and sample into the oven and leave for at
least 7 hrs.
9. Take out the covered crucible using tongs and place directly in the dessicator.
10. After cool, weigh the crucible together with the cover and sample.
11. Repeat step 10 to constant weight.

RESULT AND DISCUSSION

Calculation
Moisture content is calculated based on percentage of wet-weight

% of wet-weight = a - b x 100
a

where; a = weight of sample used

b = dry weight of sample

Data reporting
a. Table 1 is a summary of the moisture content in the sample. Stated mean and
standard deviation.

Discussion
- Effect of moisture content in food system
- How the amount of moisture (based on the data) can affect the quality
characteristics of the food
- Stated any reference values from previous research (books, journals or articles). If
the experimental values deviate why and if not deviate why. Gives justification or
evidence.

QUESTIONS
1. Based on the method above, what will affect the rate and efficiency of the moisture
removal?
2. Why is constant weight important?

11
 3. List and describe briefly 2 methods other than the oven drying method that may be
used to determine the moisture content of your sample.
4. Explain briefly the correct weighing procedure or your sample.

REFERENCES (3-4 references)

12
TITLE: DETERMINATION OF ASH CONTENT

PURPOSE: to understand the importance of minerals content in food, analysis and nutrient
requirement.

LEARNING OUTCOMES:
1. Demonstrate the ability to select suitable ashing procedure based on the limitations.
2. Demonstrate correlation of ash content with food quality, proximate analysis and
nutrient intake.
3. Demonstrate the sample preparation for different sample characteristics.
4. To collect and analyze the experimental data.

INTRODUCTION
Ash is the inorganic residue which is remained after burning process of organic compounds
in food at temperature 500 to 700ºC. Amount and composition of ash depends on methods
of ashing for respective foods. Ash content can be used to identify any authenticity in food
and also act as basic food component.
There are three methods in determination of ash i.e. dry, wet and conductometric methods.
Dry method is normally used for all kinds of food but not suitable for trace metals such as
arsenic, mercury or lead. All non-water soluble, water soluble and non-acid soluble ash can
be determined using this method. Meanwhile, wet ashing method is more suitable to be
used to obtain ash which will be used to determine trace metals and metallic poison.
Conductometric is normally used for sugar and high sugar products such as syrup, honey
and drinks.

Principles
Samples in liquid form or samples with high moisture content need to be dry prior to
analyses. Known weight samples are burned to white ash without black particles. Crucibles
containing ash are cooled in desiccators and weighed.

MATERIALS AND PROCEDURES

MATERIALS
Sample

APPARATUS
Muffle furnace at 550oC
Crucible
Thong
Desiccator
spatula

PROCEDURES
1. Place the crucibles for 1 hour in the oven at 105oC.
2. Cool the crucibles in the desiccators.
3. Weigh the crucibles rapidly and accurately.

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 4. Weigh sample (3 - 5g) in the crucibles. Use bigger portion of sample (5 – 10g) for
liquid sample.
5. Insert crucible together with the sample into the muffle furnace at 550oC.
6. Burn at least 2 hours or until no black particle present to obtain permanent weight.
7. Cool the crucibles and ash in the desiccators.
8. Weigh the crucibles together with the ash.

RESULT AND DISCUSSION

Calculation
% Ash = (weight of ash + weight of crucibles) - (weight of crucible) x 100

Weight of sample

Change to % ash based on dry weight of sample if % of moisture in the sample is known

Data reporting
a. Table 1 is summary of ash content in the sample. Stated the mean and standard
deviation.

Discussion
- Find reference values for your sample (journals, previous researches and books).
Gives evidence to defence your experimental value.
- Discuss the importance of minerals in the proximate analysis and nutrient uptake.
- How the minerals of the food varies.

QUESTIONS
1. Why is the burning process of sample need to be continued when black particles of
carbon still present?
2. What is the purpose of placing the crucible in the oven at 105oC? Give other
alternative that have the same purpose.
3. How food industries meet the mineral requirement for the food that they produced.
4. What type of desiccant that being used in this analysis? How you handle your
desiccant to give good performance?

REFERENCES (3-4 references)

14
TITLE: DETERMINATION OF CRUDE PROTEIN

PURPOSE: to comprehend the appropriate selection of method for determining protein in

food systems.

LEARNING OUTCOMES:
1. Demonstrate the ability to differentiate various method and safety on handling
hazard chemicals.
2. Able to understand and explain the reaction during the experiment.
3. To collect, analyze and evaluate the experimental data.
4. To collaborate with companion students in lab and making report efficiently.

INTRODUCTION
Proteins are polymers of amino acids, the majority of which are α-amino acid having the
general formula NH2CHRCOOH, and may thus be distinguished from fats and carbohydrate
in being the only macronutrient in foods to contain nitrogen. The presence of nitrogen in
proteins is often used as the basis of the estimation of proteins in foods. Protein content can
be determined with the analyses of any protein component like carbon or nitrogen, amino
acid group or peptide chains. Few methods are available for the determination of crude
protein but the analyses using nitrogen (N) content are the most prominent and widely used
in Kjeldahl method.

Principle
The organic part of food is digested by nitrogen-free concentrate H2SO4, where nitrogen
from the protein is being reduced and transformed to ammonium bisulphate formation.
Then, concentrate NaOH is being added to the digestion to release Ammonia (NH3). Free
ammonia is then being distilled into hydrochloric (HCl) or boric acid (HNO3). If HCl is being
used, determination of ammonia is being carried out through reverse titration. However, if
boric acid is being used, ammonia is determined through direct titration. Percentage of
protein in the sample is obtained by multiplying the appropriate conversion factor with
Nitrogen percentage.

MATERIALS AND PROCEDURES

MATERIALS
Concentrated sulphuric acid (nitrogen-free)
Mix catalyst: 96% sodium sulphate anhydrous + 3.5% cuprum sulphate + 0.5% selenium
dioxide
Sodium hydroxide solution: 45% (w/v)
Distilled water
Indicator solution: 0.2% methyl red + 0.1% methylene blue in 96% ethanol
0.5N HCL (for reverse titration) or 2% boric acid (for direct titration)
0.5N NaOH (for reverse titration) or 0.05N H2SO4 (for direct titration)
Sample given

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APPARATUS
Micro Kjeldahl flask
Spatula
Heater Distiller
Conical flask
Burette
Pipette
Pipette pump
Measuring cylinder

PROCEDURES
1. Weigh accurately 0.15g dry sample (Note 1) (Micro Kjeldahl method). For macro
Kjeldahl method, 1 to 2g of sample is used. Place sample in micro Kjeldahl test tube.
For blank, no sample is used.
2. Add 0.8g of mixed catalyst (8g for macro Kjeldahl method).
3. Add 2.5 ml (25ml for macro Kjeldahl method) concentrated sulphuric acid and swirl
the tube gently to mix the content.
4. Heat the tube slowly on a heating coil under fume hood. Boil the content until the
solution becomes clear and gives blue-green colour. (1h for macro Kjeldahl method).
5. Cool flask to 40oC (Note 2).
6. Add 10ml distilled water (200ml for macro Kjeldahl method) and transfer the
digested product into distillation tube.
7. Add slowly 10ml (50ml for macro Kjeldahl method) 45% NaOH solution to separate
the two layers of solution. Fixed distillation tube to the condenser neatly.
8. In a conical flask (for distillation product), add 10ml 2% boric acid (50ml for macro
Kjeldahl method) and add few drops of indicator (Refer Alternative B if HCl is used
during distillation).
9. Place conical flask at the distillate platform and immerse the tip of distillation tube
into the acid solution.
10. Mix the content of distillation flask by swirl it gently. Purge steam into the flask.
11. Let the ammonia solution being distilled into the conical flask (distillation product)
for about 120ml (150ml for macro Kjeldahl method).
12. After mixing the distillation product by swirl the flask gently, titrate unreacted boric
acid with 0.05N H2SO4 until neutral.
13. Repeat the same procedure for blank.

Alternative B
Alternative B is the procedure to follow when HCl is used to react with ammonia during
distillation. Repeat step 9 to 14 using 0.5N NaOH for the titration to substitute 0.05N H2SO4.
Follow the same procedure for blank. Calculation is as below but H2SO4 is changed with
NaOH.

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RESULT AND DISCUSSION
Calculation
Weight of sample =W
Volume of H2SO4 to titrate boric acid = Is
Volume of H2SO4 to titrate blank = Ib
Normality of H2SO4 =N
Therefore, % of nitrogen = (ls-lb) x N x 1.4
W
% protein = % nitrogen x conversion factor

See the conversion factor in Table 1.

Data reporting
a. Table 1 is a summary of protein content in the sample. Stated the mean and
standard deviation.

Discussion
- Find reference values (journals, books etc). Compare and give justification. Why the
data cannot be claimed as accurate?
- Stated random and systematic errors in the experiment. How to overcome.
- Find the roles of protein as part of your sample in terms of quality and health.
- What are the limitations of the method that is used?

QUESTIONS
1. Why the blank is used in this experiment?
2. Where is the error if estimation of protein percentage is lower than the expected
value?
3. Why method like Kjeldahl and Dumas are used in determining protein content?
4. What is the purpose of HCl or boric acid in the conical flask?

Notes
1. Sample to use in Kjeldahl method cannot be too moist due to high moisture will lead
to frothing. However, few authors suggested that liquid can also be used as sample.
For micro Kjeldahl method, 1 to 2ml is needed. If the sample is high in fat, decrease
the sample weight by 20 to 25%.
2. The content in the flask cannot be left cold for condensation will occur.

REFERENCES (3 – 4 references)
1. Tee et al. (1996) Nutrient Analyses of Foods.
2. James, C. S. (1995) Analytical Chemistry of Foods. Blackie Academic & Professional

17
Table 1: Factors for converting nitrogen to protein.

Foodstuff Conversion factor

Cereals
Wheat, hard, medium or soft
Whole meal or flour or bulgur 5.83
Flour, medium or low extraction 5.70
Macaroni,spaghetti,wheat pastes 5.70
Bran 6.31
Rice
Husked or brown (only hulls }
removed) } 5.95
Home-pounded,undermilled, }
Parboiled Milled, white
Rye }
Whole meal, dark flour }
Flour, medium extraction } 5.83
Flour, light low extraction
Barley }
Whole seed, except hulls and }
groats
Pearled, light or dark }
Oats
Oatmeal, rolled oats
Pulses, nuts and seeds
Groundnuts 5.46
Soybean, seeds, flour or products 5.71
Treenuts
Almonds 5.18
Brazilnut 5.46
Coconuts(outer husk removed)
Old, ripe, in shell }
Chesnuts }
Fresh, dry } 5.30
Treenuts, other }
Seeds
Sesame, safflower, sunflower
Milk and cheese
Milk, all species, fresh or dry }
Cheese, hard or soft } 6.38
Whey cheese }

Oils and fats

Margarine (either vegetable or }
animal) } 6.38
Butter

Other foods 6.25

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TITLE: DETERMINATION OF CRUDE FAT

PURPOSE: to understand the importance of fat in food system and analysis.

LEARNING OUTCOMES:
1. Demonstrate the suitable method to extract amount fat from food system.
2. Able to describe the advantages and disadvantages of fat in food system.
3. To collect, analyze and evaluate the experimental data.

INTRODUCTION
Fat is the natural component in food. It serves to give sensory characteristics and added
value to food. Determination of crude fat is normally used for quality control either in
processing or commodity classification like meat, fish, cereals and legumes. Few developed
methods can be used for crude fat determination i.e. Soxhlet, Gerber, Babcock, Bligh-Dyer,
Werner-Schmidt and Rose-Gottlieb. Soxhlet method is widely used in food commodity
especially for solid food.
This method can only be used to determine free fat if the solvent used for extraction is non
polar organic solvent such as petroleum ether or n-hexane. Total fat content (bound and
free) can be determined if mixed solvent like chloroform-methanol is used in appropriate
ratio. Gerber and Babcock method is suitable for food product or commodity in the form of
emulsion of oil in water like milk, dairy products (cheese, cream), mayonnaise and salad
dressing. This method does not include phospholipids content in food. Bligh-Dyer method is
more suitable for fish and fish products or other food commodities which contain high
saturated fat and water (around 70%). Other than for the determination of crude fat
content, this method can also be used for extraction of fat for characterization. Werner-
Schmidt method can be used for dairy products with less sugar like yoghurt. For fat
determination in high sugar food like condensed m ilk, chocolate and ice cream, R ose-
Gottlieb method can be used.
Generally, the achievement of crude fat determination in food can be affected by high
water content (except in Bligh-Dyer method) or absolutely low water content (e.g. less than
11% of or cereals and legumes), carbohydrate and protein.

Principle
A known weight of food is placed in a porous thimble and inserted in Soxhlet reflux
apparatus and the extracting solvent (petroleum ether) is placed in a dried, weighed
distillation flask. The solvent is then heated, when it volatilizes, and it is collected, after
condensing in a container housing the porous thimble. The solvent then mixes with the
food, dissolves out the fat and eventually siphons back into the original distillation flask. The
process is then repeated continuously for a period of about 8 hrs, after which it is assumed
that all the fat has been extracted from the food and present in solution in the distillation
flask. Removal of the solvent leaves the fat as a residue. The flask is reweighed and the
increase in flask weight taken as the weight of fat present in the original food.

MATERIALS AND PROCEDURES

MATERIALS
Solvent petroleum ether
Sample given

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APPARATUS
Soxhlet apparatus
Distillation/round bottom flask
Cotton wool
Thimble
Beaker
Measuring cylinder
Spatula
Desiccator

PROCEDURES
1. Dry sample following procedure. Record the dry weight of sample.
2. Weigh dry sample (5g) in the thimble. Insert the thimble into Soxhlet apparatus.
3. Weigh round bottom flask accurately and pour 200ml petroleum ether.
4. Connect Soxhlet to the reflux and round bottom flask and reflux sample continuously
for about 8 hrs. Make sure the water content of the water bath is sufficient and add
if necessary and control the temperature of the heater.
5. Be extra cautious in making sure that the solvent is not evaporated or dry during
refluxing. If so, add more petroleum ether.
6. After 8 hrs, evaporate petroleum ether from the round bottom flask using rotary
evaporator.
7. Place the round bottom flask into oven (105oC) for 15 mins.
8. Then, cool the round bottom flask in desiccators.
9. Weigh round bottom flask together with the fat extracted.
10. Repeat step 7-9 until constant weight.

RESULT AND DISCUSSION

Calculation
Calculate fat content in percentage of dry basis sample:
Weight of sample (g) =W
Weight of round bottom flask (g) = -------
Weight of round bottom flask + oil (g) = -------
Weight of oil (g) =M

% of oil in sample = M x 100

W

Data reporting
1. Table 1 is a summary of fat content of the sample. Stated mean and standard
deviation.

Discussion
- Find the reference values (journals, articles etc). Compared with experimental value
and give justifications.

20
 - Importance of fat in food quality and daily life.
- Advantages and disadvantages of the fat.

QUESTIONS
1. What were the advantages of using the Soxhlet extraction method rather than the
Goldfish extraction method?
2. If the fat content measured here differed from that reported on the nutrition label,
how might this be explained?
3. Why the dry sample is used in the experiment?
4. How to handle the hazardous chemical in this experiment correctly?

REFERENCES (3-4 references)

1. Bligh, E. G. And Dyer, W. J. (1959) A rapid method of total lipid extraction and
purification. Can J. Biochem. Physiol. 37(8): 911-917.
2. James, C.E. (1995) Theory of analytical methods for specific food constituents. In
Analytical Chemistry of Foods. Blackie Academic & Professional, London. pp 37-66.
3. Pearson, D. (1976) General Methods. In The Chemical Analysis of Foods. Longman
Group Limited. London. pp 6-26.
4. Pomeranz, Y. And Meloan, C. E. (1978) Lipids in Food Analysis. Theory and Practice
(Rev Ed.). AVI Publishing Co. Connecticut, USA. pp 618-667.

21
TITLE: DETERMINATION OF CRUDE FIBER

PURPOSE: to comprehend the determination of crude fibre in food systems.

LEARNING OUTCOMES:
1. Demonstrate the ability to handle the equipment and chemical efficiently.
2. Demonstrate the advantages and disadvantages of various methods.
3. To collect, analyze and evaluate the experimental data.

INTRODUCTION
Crude fiber is defined as an organic food residue which has been hydrolyzed by acid (eg;
sulphuric acid and hydrochloric acid) and aqueous alkaline (eg; sodium hydroxide). Crude
fiber method measures cellulose and lignin and therefore there is no specific component
being determined.
There are two common ways to determine crude fiber. The first method is using sulphuric
acid followed by sodium hydroxide to hydrolyze the food sample. This method is suitable for
all types of food. The second method is using detergent, which is basically the
determination of the vegetative component that is insoluble and cannot be metabolized by
proteolytic and amylolitic enzyme. This method is suitable for by-product of cereal industry.
Overall, in determining the crude fiber, the sample characteristics should be taken into
consideration such as particle size, defatted or undefatted sample, the rate of heat given to
the sample to achieve the boiling point, boiling rate and filtration rate.
Crude fiber can be used to evaluate nutrition, manufacturer performance and the degree of
fruit senescence.

Principle
The sample, which have been weighed and grind (optional), will be hydrolyzed with an
aqueous sulphuric acid. Then, the sample residue will be rinsed with the water and dried in
oven for 3hrs at 105oC. The sample will be cooled in dessicator and the step will be repeated
for several times until constant weight obtained. The remaining residue, will be ashed at
550oC in a muffle furnace until completed. The ash is weighed after it is have cooled down
to room temperature. The weight of crude fiber is the differences between the weight of
dried sample residue to the weight of ash.

MATERIALS AND PROCEDURES

MATERIALS
Sulphuric acid: 0.25N
Sodium hydroxide: 0.313N
Alcohol
Sample given

APPARATUS
Muffle furnace set at 550oC
Buchner funnel (or glass funnel)
Spatula
Heater
Conical flask

22
Desiccator
Oven set at 105oC
Crucible
Filter paper
Condenser
Ashless filter paper No. 541
Aspirator pump

PROCEDURES
1. Dry the crucible and ashless filter paper in the oven for 1hr at 105oC. Cool both
crucible and ashless filter paper in dessicator. Calculate the weight of the filter
paper.
2. Weigh 2g of defatted sample into 500ml conical flask.
3. Add 200ml boiled sulphuric acid and dissolve the sample using spatula.
4. Attach the conical flask to the reflux condenser.
5. Boil the sample for 30mins. Turn the conical flask every 5mins to make sure that the
sample is always in contact with the acid (if it is condensed, top-up with distilled
water until the initial level of the acid).
6. Filter the hydrolyzed mixture through filter paper Whatman No.541.
7. Rinse the residue with boiled distilled water to remove the acid from the filtrate.
8. Place back the residue to its conical flask and add 200ml boiled sodium hydroxide
(NaOH).
9. Attach to the condenser and boil for another 30mins.
10. Filter the hydrolyzed mixtures that have been boiled through dried and weighed
filter paper (1). Rinse the residue with boiled distilled water until the filtrate is free
from alkaline. Rinse again with small amount of alcohol and then drain it.
11. Place the residue and the filter paper into the crucible and dry in oven at 105oC.
12. Place the crucible in the muffle furnace at 550oC and burn completely.
13. Place the crucible into the dessicator. Calculate the constant weight.

RESULT AND DISCUSSION

Calculation
Weight of sample =W
Weight of filter paper without ash =K
Weight of crucible + filter paper + dried residue =S
Weight of crucible + ash =A

% crude fiber = (S – K) – A x 100

W
Data reporting
1. Table 1 is a summary of fat content in the sample. Stated mean and standard
deviation.

23
Discussion
- Find reference values (journals, articles etc). Why the reference values are differ
from the experimental value? Stated the reasons.
- How useful the data of crude fibre? Why?
- Gives limitations from the crude fibre values.
- Importance of sample preparation in the experiment.

QUESTIONS
1. How to prepare 0.25N of sulphuric acid and 0.313N sodium hydroxide? What is
the purpose of those chemicals?
2. Why the hydrolyzed sample must be in-contact with the hydrolyze agent?
3. What is the difference of crude fibre and dietary fibre? Gives example.
4. Why the ashless filter paper No. 541 is used in this experiment?

REFERENCES (3-4 references)

1. Lees, R. (1968). Crude fibre. In Laboratory Handbook of Method of Food Analysis.
Leonard Hill Books. London, pp109.
2. Meloan, CE and Pomeranz, Y (1980). Crude Fiber. In Food Analysis Laboratory
Experiments. 2nd Edition. AVI Publishing Company. Connecticut, pp 94-96.
3. Osborne, DR and Voogt, P (1978). The analysis of nutrients in foods. Academic
Press London, New York, San Francisco, pp 151-153.

24
TITLE: DETERMINATION OF CARBOHYDRATES (BY DIFFERENCE) AND ENERGY

PURPOSE: to comprehend the calculation and the role of carbohydrate and energy in
analysis.

LEARNING OUTCOMES:
1. Demonstrate the ability to calculate the carbohydrate and energy for different foods.
2. Demonstrate the ability to understand the function and properties of carbohydrate
and energy.

The determination of carbohydrate is by calculation:

% Carbohydrate = 100% - percentages of moisture, ash, protein, fat and fiber

Energy content of the food is determined according to Regulation 18B of the Malaysian
Food Act 1983 (Act 281) & Regulations.

25
TITLE: DETERMINATION OF REDUCING SUGAR USING THE SOMOGYI-NELSON METHOD

PURPOSE: to comprehend the importance of reducing sugar in food quality.

LEARNING OUTCOMES:
1. Demonstrate the ability to select suitable method for different characteristics of the
sample.
2. Understand the reaction behind the experiment.
3. To collect, tabulate and analyze the experimental data.
4. To collaborate the experimental data with the theory.

INTRODUCTION
The most often used method to determine amounts of reducing sugars is the Somogyi-
Nelson method. This method is based on reduction of Cu2+ ions to Cu+ ions by reducing
sugars. The Cu+ ions then reduce an aresenomolybdate complex, which is prepared by
reacting ammonium molybdate and sodium arsenate in sulphuric acid. Reduction of the
aresenomolybdate complex produces an intense, stable, blue colour that is measured
spectrophotometrically. This reaction must be used with a standard curve the sugar(s) being
determine or with D-glucose.

MATERIALS AND PROCEDURES

MATERIALS
Low-alkalinity copper reagent – Sodium potassium tartarate, KNa(C4H4O6).4.H2O (Rochelle
salt) (40g) and anhydrous Na2HPO4 (28g) are dissolved in about 700ml of water. Add 100ml
of 1M NaOH. A solution of 10% cupric sulphate pentahydrate in water (80ml) is added in
small amounts with stirring (make sure the cupric sulphate is dissolved before adding more).
Add 180g anhydrous NaSO4 in small amounts and dilute to 1L. After 1 day of standing, the
clear supernatant solution is used.

Aresenomolybdate reagent – To 25 g of ammonium molybdate in 450 ml of water is added

21 ml of 96% sulphuric acid, followed by 3.0 g of disodium hydrogen arsenate heptahydrate
dissolved in 25 ml of water. The mixed solution is incubated 25 h at 37ºC and stored in a
glass-stoppered brown bottle.

Standard glucose solution – Dilute stock solution containing 1 mg/ml (w/v) glucose in a
saturated benzoic acid solution to give a standard solution of 100 μg/ml and 300 μg/ml.
Carbonated drinks
Distilled water

APPARATUS
Beaker
Pipette
Pipette pump
Pastuer pipette
Volumetric flask 250ml, 100ml and 10ml
Heater

26
Test tube
Cuvette
Spectrophotometer at 520nm

PROCEDURES
A. Sample preparation
1. Shake the bottle containing the carbonated drink.
2. Pour 100 ml of the drink into a 200 ml beaker.
3. Transfer the drink from one beaker to another beaker. Repeat this procedure until
no gas remains in the drink.
4. Pipette 5.0 ml of the degassed sample into a 250 ml volumetric flask and dilute to
volume. This is called solution A.
5. Pipette 10 ml of solution A into a 100 ml beaker and add 50 ml distilled water.
Neutralize the solution. Transfer the solution into a 100 ml volumetric flask and
dilute to volume. This is solution B

B. Determination of reducing sugar

1. Prepare a blank and standard solution containing between 0 and 450 μg glucose.
This can be done as follows:

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7

Glucose solution
(100 μg/ml) - 0.5 1.0 1.5 2.0 - -
(mL)
Glucose solution
(300 μg/ml) - - - - - 1.0 1.5
(mL)
Distilled water
2.0 1.5 1.0 0.5 - 1.0 0.5
(mL)

2. A sample solution containing not more than 2 ml of solution B is made. Make a series
of dilution and make it up to volume of 2 ml of distilled water.
3. To all samples, blanks and standard sugar solutions add 2 ml copper reagent and mix
the contents well.
4. Samples, blanks and standard sugar solutions are heated 10 min in a vigorously
boiling water bath and then cooled under running water for 5 min.
5. Add 1 ml of arsenomolybdate reagent to all tubes and mix well.
6. When all the cuprous oxide is dissolved after mixing, the solution is diluted to the 10
ml using a volumetric flask and then allowed to stand at least 15 min, but not more
than 40 min.

Absorbance is read at 520 nm using a spectrophotometer. The average absorbance of a

blank is subtracted from the average absorbance of the samples; then the sugar content is
computed from a curve previously established with sugar solutions.

27
RESULT AND DISCUSSION
Calculation
i. Concentration of standard solution,

M 1V 1 = M 2V 2
M1 = concentration of glucose solution before dilution
M2 = concentration of glucose after dilution
V1 = volume of glucose solution
V2 = final volume
ii. Amount of sugar in the sample,
X2 = X1 x DF of solution A x DF of solution B x DF of solution in tube
X1 = concentration of sugar from the standard curve
DF = dilution factor

Data reporting
1. Table 1 is a summary of average absorbance and sugar content of the sample.

Discussion
- Stated the reference value for the sample. Why the value is different with the
experimental data? Give justifications based on the food compositions and quality of
the food. From your justifications what factors that can contribute to it? How to
control?
- Gives recommendations on the suitability or limitation of the method.
- Stated your opinion to consumer based on your experimental data.

QUESTIONS
1. You and your group have been provided with a solution comprising 0.51mg/ml
glucose, 1.22mg/ml fructose, 0.26mg/ml lactose and 1.07mg/ml sucrose. Calculate
the total concentration of reducing sugar in the solution assuming no error of
analysis had occurred during the analysis. Give justification for your answer.
2. Can a reducing sugar method be used to calculate the glucose content in a starch
hydrolysate solution? Explain your answer.
3. How to prepare standard solution of 300µg/ml?
4. Explain briefly the principle of spectrophotometer.

REFERENCES (3-4 references)

1. Southgate, D.A.T (1976). Determination of food carbohydrates. Appl. Science Publ.
Ltd. London.

28
TITLE: DETERMINATION OF ASCORBIC ACID IN FRUITS

PURPOSE: to estimate the content of ascorbic acid and its stability towards blanching.

LEARNING OUTCOMES:
1. Demonstrate the ability to examine the stability of ascorbic acid.
2. Demonstrate understanding on properties of ascorbic acid.
3. Demonstrate the ability to do the experiment systematically.
4. To collect, tabulate, analyze and evaluate the experimental data.

INTRODUCTION
Vitamin C is consist of two biological active component, L-ascorbic acid and L-
dehydroascorbic acid. The natural state is commonly found in food is L-ascorbic acid but
there is also little a small amount of L-dehydroascorbic acid found. The analysis of ascorbic
acid in food is usually being done using the reaction of ascorbic acid oxidation together with
the redox indicator such as 2,6-dichlorophenolindophenol. However, this procedure does
not determine the dehydroascorbic acid, which have more than 80% vitamin C activity.

Principles
Chemical estimation of ascorbic acid depends on its ability to reduce the redox indicator
(colouring) 2,6-dichlorophenolindophenol. Ascorbic acid is extracted from food and titrated
with the indicator in the presence of acid like metaphosphoric or oxalic acid. These acids are
used to preserve the correct acidity for reaction and to avoid auto oxidation of ascorbic acid.

MATERIALS AND PROCEDURES

MATERIALS
Friuts
Distilled water
2,6-dichlorophenolindophenol solution: Dissolve 0.2g in 100ml hot distilled water, filter
solution and dilute 10 times.
Standard solution of ascorbic acid: Dissolve 0.1mg/ml ascorbic acid in 2% metaphosphoric
acid
Oxalic acid solution: 0.5% (v/v)

APPARATUS
Conical flasks 100ml
Burette
Pipette
Beaker 500ml
Filter funnel and cotton wool
Blender
Measuring cylinder: 250ml
Retort stand

29
PROCEDURES
A. Standardization of redox indicator (dye) solution 2,6-dichlorophenolindophenol
1. Pipette 10ml ascorbic acid standard solution in a 100ml conical flask.
2. Titrate with the dye solution until a permanent faint pink colour is obtained. Record
the volume of dye solution required 10ml of ascorbic acid.
3. Calculate the volume (ml) of dye solution required to oxidize 1mg ascorbic acid.

B. Sample preparation and determination of ascorbic acid content

1. Blend 40g of raw fruits in 200ml oxalic acid for 2 mins.
2. Filter through cotton wool, rinse with some oxalic acid and determine the volume of
filtrate (Vf) using a 250ml measuring cylinder.
3. Pipette 10ml of filtrate (Vs) into a 100ml conical flask and titrate with the dye
solution (titre).
4. Calculate the ascorbic content in the sample in mg/100g sample.

RESULT AND DISCUSSION

Calculation
Dye factor = amount of ascorbic acid in 10ml standard solution
Vol. of dye to oxidize 1mg ascorbic acid

Ascorbic acid content (mg/100g) = volume of titration (ml) x dye factor (mg/ml) x Vf x 100
Weight of sample (g) x Vs

Data reporting
Table 1 is a summary of the amount of ascorbic acid in fruits.

Discussion
- Which sample give high amount of ascorbic acid? Did varies in amount of ascorbic
acid differ in the characteristics of the fruits? What characteristics that related to the
ascorbic acid?
- How to maintain the ascorbic acids in fruits? Why it is important?

QUESTIONS
1. What is the function of metaphosphoric acid and oxalic acid?
2. What is the effect of additives like sulphur dioxide in samples on the estimation of
ascorbic acid using 2,6-dichlorophenolindophenol?
3. Give one method which can be used to estimated ascorbic acid in food. (please you’re
your reference)

30
REFERENCES (3-4 references)
1. Christie, A.A and Wiggins, R.A (1978). Development in Vitamin Analysis. In
“Developments of Food Analysis Techniques”, ed. R.D. King, Appl. Sci. Publ., pp. 18-
23.
2. Pearson, D (1976). The Chemical Analysis of Foods, 7th edn., Churchill Livingstone,
Edinburgh.
3. Tannembaum, S.R., Young, V.R and Archer, M.C (1985). Vitamins and Minerals. In
“Food Chemistry”, ed. O.R. Fennema, 2nd Edn., Marcel Dekker, N.Y pp 488-493.

31
TITLE: ENZYMATIC BROWNING IN FRUITS & VEGETABLES

PURPOSE: to comprehend the polyphenoloxidase (PPO) activity towards food quality.

LEARNING OUTCOMES:
1. Demonstrate the inhibition of browning enzyme by various substances.
2. Demonstrate the effect of chemical structure of substrate on PPO activity.
3. To collect and analyze the experimental data.

INTRODUCTION
Most fruits and plant tissues when bruished, cut or damaged rapidly darken. This enzyme
catalysed reaction is usually undesirable from the point of view of consumer acceptance. In
some instances however, such as the manufacture of tea, coffee or cider, this darkening is a
desirable part of the process. The enzymes responsible for these browning reactions are
known as polyphenoloxidases. They catalyse the oxidation of phenolic substrates to
quinones which subsequently polymerize to give brown/black pigments.

For oxidative browning to occur, three components must be present, namely, enzyme,
substrate and oxygen. A number of methods of inhibiting enzymatic browning are used
commercially and domestically. These include destruction of the enzyme by
heat,elimination of oxygen, control of pH and the use of chemical inhibitors.

MATERIALS AND PROCEDURES

MATERIALS
Sample given
Distilled water
Phosphate buffer pH 7
P-Phenylene Diamine
Pyrogallol
Quinol
Resorcinol
Catechol
Ascorbic Acid
Sodium Metabisulphite
Diethyldithiocarbamate

APPARATUS
Knife
Chopping board
Measuring cylinder 250ml
Blender
Conical flask 250ml
Test tube
Filter funnel
Pipette 5ml

32
PROCEDURES
1. Peel and cut a large potato, apple or pear under running water.
2. Weigh approximately 100g into a blender and add 200ml of phosphate buffer pH 7.0
3. Homogenise at maximum speed for 30 s and filter through a Whatman filter paper
No.4 into a clean conical flask.
4. Stopper and store.

Substrate specificity
1. Compare the browning (qualitatively) with that of a blank by mixing 0.2ml of the
enzyme extract with 5ml of the following solutions:
i) water
ii) 10-3 M p-phenylene diamine
iii) 10-3 M pyrogallol
iv) 10-3 M quinol
v) 10-3M resorcinol
vi) 10-3M catechol
2. Allow to stand for 10 min and relate substrate structure to polyphenoloxidase (PPO)
activity of the sample.

Inhibition of browning
1. Compare the effectiveness of browning inhibition using 5ml of 10-3 M and 10-2 M
catechol solutions containing 0.2ml of enzyme axtract with 0.2ml of the following
solutions:
i) water
ii) 10-2 M ascorbic acid
iii) 10-2 M sodium metabisulphite
iv) 10-2 M diethyldithiocarbamate

RESULT AND DISCUSSION

Data reporting
1. Table 1 is a summary of polyphenoloxidase (PPO) with substrate specificity.
2. Table 2 is a summary on the effectiveness of substances toward browning inhibition.

Discussion
- Which substrate affected the PPO activity? Why?
- Which substance is the most effective to inhibit the browning enzyme? Why?
- Did the substance affect other food properties if being added? How? It is safe to be
eaten? Did it have limit?
- Which substance did not give any change? Why?
- Did any factors contribute to the reactions?

33
QUESTIONS
1. Describe the chemical reaction catalysed by PPO.
2. List the group of organism that produce PPO.
3. Describe the correct way of sample preparation in this experiment? Why the process is
very crucial?
4. Explain the function of phosphate buffer in this experiment.

REFERENCES (3-4 references)

34