Week 1. FST 3112 Lab. Manual FST3112-2012-2013-2 PDF

CHEMICAL ANALYSIS OF FOOD
(FST 3112)
A PRACTICAL MANUAL
Practical Manual for FST 3112 Faculty of Food Science and Technology, UPM
TABLE OF CONTENTS
1 Introduction: Laboratory safety; Guidelines for report preparation

and submission; Laboratory report assessment and criteria; Practical
skills evaluation assessment criteria; Learning outcomes
2 Proximate Analysis
Determination of Moisture Content
Determination of Ash Content
Determination of Crude Protein
Determination of Fat Content
Determination of Crude Fiber (Dietary fiber analysis will be
demonstrated)
Determination of Carbohydrate (by difference)
3 Carbohydrate Analysis
Determination of reducing sugar using The Somogyi-Nelson
Method.
4 Protein Analysis
Determination of soluble protein using the modified Lowry’s
method
Determination of amino acids using HPLC (Demo)
5 Lipid Analysis
Determination of the free fatty acids and acid value of fats and oils
Determination of iodine value of fats and oils
Determination of saponification value of fats and oils
Determination of fatty acids using GC (Demo)
6 Acidity Analysis
Determination of pH, titratable acidity and total soluble solids
7 Vitamin Analysis
Determination of Vitamin C
8 Mineral Analysis
Determination of calcium using AAS (Demo)
9 Enzyme Inhibition
Effect of blanching on peroxidase activity
10 Case Study
11 Appendix
MSDS
Chemical Analysis of Foods – A Practical manual 2

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 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.
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. Experiment number and 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)
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
http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1750-3841).
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.

Laboratory Report Assessment Criteria


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.

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.

PROXIMATE ANALYSIS

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
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?
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)

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
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.
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 550 oC.
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.

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?

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.
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 (NH 3). 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
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

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. Continue boiling for
another 10 mins (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 H 2SO4 is
changed with NaOH.

Calculation
Weight of sample =W
Volume of NaOH to titrate HCl = Is
Volume of NaOH to titrate blank = Iu
Normality of NaOH =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

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,s paghetti,w heatp astes 5.70
Bran 6.31
Rice
Husked or brown (only hulls removed) }
Home-pounded,undermilled, parboiled } 5.95
Milled, white }
Rye
Whole meal, dark flour }
Flour, medium extraction }
Flour, light low extraction } 5.83
Barley
Whole seed, e,xcept 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) }
Butter } 6.38
Other foods 6.25

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.
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
Solvent petroleum ether
Sample given
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.
RESULTS 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.
- 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?

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.

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.
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
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
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 105 oC. 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 105 oC.
12. Place the crucible in the muffle furnace at 550oC and burn completely.

13. Place the crucible into the dessicator. Calculate the constant weight.

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.
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?

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.

TITLE: DETERMINATION OF DIETARY FIBRE BY THE ENGLYST ENZYMATIC METHOD
PURPOSE: to comprehend the importance of determining dietary fibre in foods.
LEARNING OUTCOMES:
1. To familiarize the method in dietary fibre determination.
2. Demonstrate the ability to differentiate the component of dietary fibre.
3. Demonstrate the ability to do the calculation for dietary fibre.
INTRODUCTION
Principle
Dietary fibre is measured by NSP (non-starch polysaccharides) by first defatting the food
sample if necessary, then removing starch enzymatically, after solubilisation and
gelatinisation, and estimating NSP as the sum of constituent sugars released by acid
hydrolysis of the remaining polysaccharides, the sugars being measured calorimetrically.
By various modifications to the procedure, values may be obtained for:

(a) Total dietary fibre (as total NSP)
(b) Soluble fibre (soluble NSP)
(c) Insoluble fibre (insoluble NSP)
(d) Resistant starch

MATERIALS
Ethanol absolute
85% ethanol
12 M sulphuric acid (66% v/v)
3.9 M sodium hydroxide solution (39 g sodium hydroxide per 250 ml)
Sodium potassium tartarate
Acetone
Dimethyl sulphoxide (DMSO)
10% acetic acid
50% saturated benzoic acid solution. Prepare a saturated solution of benzoic acid. Filter
off the required volume of saturated solution and dilute 1:1 (v/v with water. The final
solution is stable for up to 2 months)
0.1 M sodium acetate buffer. (dissolved 13.6 g sodium acetate trihydrate,
CH3COONa.3H2O, in volumetric flask and making up to 1 L with 50% benzoic acid. Adjust
this 0.1 M solution of sodium acetate to pH 5.2 by thedrop wise addition of 10% acetic
acid. Add 4 ml of 1 M calcium chloride per litre of this buffer solution to stabilise and
activate enzymes.

Pullulanase. (100 units per mL, (E.C 3.2.1.4) – Boehringer 108944 – is used. Dilute
pullulanase 1:100 (50 μl made up to 5 ml) with 0.1 M sodium acetate buffer
immediately before use.
α- amylase. α- amylase (EC 3.2.1.1; Pancrex V capsules, approximately 9000 BP units α-
amylase per capsule, Paines and Byrne Ltd. Greenford. Middlesex, UK). (empty 2 α-
amylase capsules into a centrifuge tube, add a magnetic stirrer and 9 ml water, and
vortex mix. Further mix for 10 min on a magnetic stirrer and the centrifuge at 1500 g for
10 min. use the supernatant solution as the solution of α- amylase. Prepare immediately
before use.
2 M sulphuric acid. (50 ml conc. Sulphuric acid to 400 ml distilled water)
Dinitrosalicylate solution. (dissolve 10 g powdered 3,5-Dinitrosalicylic acid in 300 ml hot
distilled water, add 16.0 g sodium hydroxide and 300 g sodium potassium tartarate, cool
and make up to 1 L with distilled water. Keep for 2 days in a dark bottle before use. The
solution is stable at least 6 months.
0.2 M phosphate buffer, pH 7.0
(i) 14.2 g disodium hydrogen orthophosphate dissolved in 500 ml of dist. water
(ii) 15.6 sodium dihydrogen orthophosphate dissolved in 500 ml of dist. water.
Adjust the pH solution (i) to 7.0 using solution (ii).
Standard sugar solution
Standard used are dependent on the nature of food samples being analyzed:
(a) Standard A – fruit and vegetables

500 mg arabinose
1000 mg glucose
500 mg galacturonic acid
(b) Standard B – foods in general

2000 mg glucose
(c) Standard C – cereals

600 mg arabinose
800 mg xylose
600 mg glucose
For each particular standard required, dissolved the sugar(s) in 50% saturated benzoic
acid solution and make up to 500 ml with 50% saturated benzoic acid solution to
provide a stock solution. To prepare standards take 1.00, 2.00, 3.00 and 4.00 ml of the
stock solution and make up to 4.00 ml with the 50% saturated benzoic acid solution.
Add 4.00 ml 2 M sulphuric acid to give standards of 0.5, 1.0, 1.5 and 2.0 mg total
sugar(s) per ml in 1 M sulphuric acid. The stock sugar standards are stable up to 2
months and the working standards for up to 1 week.

APPARATUS
Volumetric flasks
Water bath
Pipettes
Hot plate and stirrer
Magnetic stirrer
Centrifuge
Centrifuge tubes (50 to 60 ml capacity, with screw tops)
Vortex mixer
Spectrophotometer at 530 nm
Cuvettes
Ovens or incubators at 35oC and 42oC
PROCEDURES
1. Sample preparation. Prepare 1 to 3 samples depending on the nature of the data
required for the particular food in relation to:
(i) Total NSP (soluble NSP + insoluble NSP)
(ii) Individual values of soluble NSP and insoluble NSP
(iii) Resistant starch
Use as many replicates as it practically feasible. Weigh accurately, between 50 to 1000

mg, depending on the water and NSP content of the sample, of non-dried food sample
(to give not more than 300 mg of dry, matter; 300 mg is normally adequate for most
food and 100 mg of very high fibre foods such as bran) into a 50 ml screw cap tubes.
2. Fat extraction/drying of wet samples. For dry, low-fat foods proceed directly to step
3. if the food contains 15% or more water and if the fat content of the food exceeds
5%, add 40 ml acetone, mix on a magnetic stirrer for 30 min, centrifuge and remove
as much as possible of the supernatant by aspiration without disturbing the
sediment. Place the tube in a beaker of water at 65-70ºC on a stirrer/hot plate and
mix for 2-3 min until the residue appears dry.
3. Dispersion and hydrolysis of starch
(a) Total NSP. Add 2 ml DMSO, cap the tube, and mix on a magnetic stirrer for about 2
min. place the tube in a beaker of boiling water on a stirrer/hot plate and mix for 1 hour.
(Gel formation may occur which may prevent movement of the stirrer, but this will not
affect the procedure). Remove the tube and without cooling, add 8 ml 0.1 M sodium
acetate buffer, pH 5.2 pre-equilibrated at 50ºC and vortex mix.
Leave the tubes at room temperature or in a water bath at about 35ºC until the
contents has cooled to between 30 and 40ºC. Immediately, add 0.5 ml α- amylase
solution followed by 0.1 ml of pullulanase solution and vortex mix. (Do not mix the

enzyme solution beforehand). Cap the tube and incubate at 42ºC for about 16 hours or
overnight, mixing continuously or intermittently for the first hour.
Add 40 ml ethanol, mix well by inversion and leave for 1 h at room temperature.
Centrifuge at 1500 g for 10 min and then remove as much as possible of the supernatant
by decantation or aspiration without disturbing the residue. Wash the residue twice
using 50 ml 85% ethanol each time. Mix by inversion, and then use a magnetic stirrer to
suspend the residue. Centrifuge and remove the supernatant liquid as before.
Add 40 ml acetone to the washed residue, stir for 5 min and then centrifuge at 1500 g
for 10 min. Remove the supernatant liquid by aspiration and discard it. Place the tube in
a beaker of water at 65-70ºC on a stirrer/hot plate and mix the contents for 2-3 min
until the residue appears dry.
(b) Soluble and insoluble NSP. Proceed as for total NSP in (a), but make the following
modifications to the procedure.
After the 16 h or overnight treatment with 0.5 ml of α- amylase solution followed by 0.1
ml of pullulanase solution, do not add 40 ml ethanol, instead add 40 ml 0.2 M
phosphate buffer pH 7.0. Place the capped tubes in a boiling water bath for 30 min,
mixing at least three times during this period. Removes the tubes to room temperature
and leave for 10 min. Centrifuge at 1500 g for 10 min and then remove as much as
possible of the supernatant by decantation or aspiration without disturbing the residue.
Add approximately 10 ml water and vortex mix. Make to approximately 50 ml with

water, mix by inversion and then use the magnetic stirrer to form a suspension of the
residue. Centrifuge at 1500 g for 10 min and then remove as much as possible of the
supernatant by decantation or aspiration without disturbing the residue. Repeat this
stage of the process using 50 ml of absolute ethanol.
Add 40 ml acetone to the washed residue, stir for 5 min and then centrifuge at 1500 g
for 10 min. Remove the supernatant liquid by aspiration and discard it. Place the tube in
a beaker of water at 65-70ºC on a stirrer/hot plate and mix the contents for 2-3 min
until the residue appears dry.
(c) Resistant starch. Proceed as for procedure (a) for total NSP above but omit the
addition of DMSO and the 8 ml of acetate buffer. Instead, add 10 ml of acetate buffer
to the sample and then proceed as for (a) by vortex mixing, placing in a water bath at
42ºC for 2-3 min, removing, adding 0.5 M α- amylase, etc.
4. Acid hydrolysis of NSP. The procedure for acid hydrolysis is common to all estimation,
i.e. samples (a), (b) and (c) above. For each sample, add 2 ml 12 M
sulphuric acid to the dried residue and immediately disperse if by vortex mixing or
magnetic stirring. Leave at 35°C for 1 h with occasional or continuous mixing to disperse

cellulose. Rapidly add 22ml water, cap the tube, and mix. Place in a boiling water bath
for 2h, stirring continuously and then cool to room temperature.
5. Colorimetric determination of NSP. Take a suitable number of labeled test tubes. In

the first place 1 ml of the blank solution (0 mg sugar per 100ml), in the next four place
respectively 1 ml of each of the sugar standards and in the remaining test-tubes place
1ml of the food hydrolysate(s) to be measured. To each test tube add 0.5 ml of the
standard 0.05 g per 100 ml glucose solution and 0.5 ml of 3.9M sodium hydroxide
solution and vortex mix. Add 2 ml dinitrosalicylate solution to each tube and vortex
mix. Place all tubes, simultaneously, in to briskly boiling water baths and leave for 10
min. Cool under water to room temperature. Add 20 ml distilled water to each tube and
mix well by inversion. Measure the absorbance of each tube at 530nm against the zero
sugar blank.
Note: Reagents for performing a more rapid version of the above Englyst procedure are
available in a kit form from Novo Nordisk Bioindustries UK Ltd, Farnham, Surrey, UK. The
procedures for using the kit, developed at the Medical Research Council Dunn Clinical
Nutrition Centre, are similar to those outlined above for the Englyst method but allow
for a more rapid determination by reducing the times required for various stages of the
determination. The kit contains the enzymes amylase, pancreatin, pullulanase and
pectinase, and instructions for the use of the kit including the following generalized
modifications to the original full method described above.
(i) The time of treatment with dimethyl sulphoxide is reduced from 1 h to 30 min.
(ii) The time of treatment with the enzymes amylase, pancreatin and pullulanase is
reduced to 40 min compared with 16-18h with amylase and pullulanase in the full
method.
(iii) Ethanol treatment times are reduced from 1 h to 30 min
(iv) The time of acid hydrolysis of the residue from enzymic digestion is reduced from 3h
to lh.

Calculation
Prepare a calibration graph of absorbance against concentration of sugar in g per 100m l
(0.05, 0.1, 0.15 and 0.2 g sugar per 100ml) and calculate the total NSP, insoluble NSP,
soluble NSP and resistant starch as follows. The values for NSP are given by the equation:
NSP = Atx Vt x 100 x 0.89

As x Wt
where: At = absorbance of test solution, Vt, = 24 (total volume of test solution), As =

absorbance (from graph) corresponding to 0.1 g sugar per 100ml, Wt= weight of food
sample, in mg, for sample, and 0.89 = correction factor to compensate for losses of
monosaccharides during acid hvdrolvsis.

Thus, for total NSP, the value for the absorbance of the test solution from sample (a) is
used. For insoluble NSP, the value for the absorbance of the test solution from sample
(b) is used. Soluble NSP is obtained as the difference between total NSP and insoluble
NSP. Resistant starch is obtained as the difference between the value obtained for total
NSP in (a) and that obtained for total NSP in (c).
QUESTIONS
1. Define dietary fiber and give the constituents that compose dietary fibre.
2. Compare and contrast the AOAC method and Englyst-Cummings method for
determination of total dietary fiber. Consider principles, procedures, applications,
and advantages and disadvantages.
3. Explain how both gravimetric and chemical methods allow differentiation of soluble,
in soluble, and total dietary fiber.
4. Describe the principles involved in the analysis of total dietary fiber.
5. Differentiate between total soluble fiber and total insoluble fiber.

1. l. AOAC International (1997). Official Methods of Analysis, l6th ed., 3 rd revision,
Method 991.43. AOAC International, Gaithersburg, Maryland.
2. Bennink, M.R (1998). Fiber analysis. In Food Analysis, ed. Nielsen, S.S. Aspen
Publ.,Inc. Gaithersburg, Maryland.
3. James, C.S (1995). Analytical chemistry of foods. Blackie Academic &Professional,
Glasgow.
4. Prosky, L., Asp, N,-G., Schweizer T.F., de Vries, J .W and Furda, I (1988).
Determination of insoluble, soluble and total dietary fiber in foods and food
products: Inter-laboratory study. J. Assoc. Off, Anal. Chem. 71: l0l7-1023.

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.

CARBOHYDRATE ANALYSIS

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
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
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.

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.


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

PROTEIN ANALYSIS

TITLE: DETERMINATION OF SOLUBLE PROTEIN USING THE MODIFIED LOWRY’S

METHOD
PURPOSE: to estimate the content of soluble protein in fish after extraction with NaCl
solution.
LEARNING OUTCOMES:
1. Demonstrate the ability to select method based on protein characteristics.
2. Demonstrate the ability to extract and quantitate the soluble protein in the sample.
3. To collect, tabulate and analyze and evaluate the experimental data.
4. To collaborate with companion students to do the experiment efficiently.
INTRODUCTION
The Lowry method (Lowry et al., 1951; Eggstein and Kreutz, 1967) determines the
protein content in a sample after extraction of the protein in a soluble form. The limit of
the sensitivity of this method is 10 μg/ml soluble proteins. It is the most frequently used
method of protein determination in the world. Despite that, the method has an obvious
weakness as it only measures concentration of the protein that is soluble in the solvent
used. In general, the degree of protein solubility depends on factors like types of
solvent, ionic strength, pH, temperature and others. Under the same extraction
conditions (example, pH and same solvent), the heterogeneity of protein in sample can
reduce the degree of protein solubility. Types of protein like globulin, albumin and etc.,
will have different degrees of solubility although in the same solvent.
Principle
This method is based on the formation of colour by (1) the Biuret reaction between
protein and Cu (II) ion under alkaline conditions (2) reduction of the
phosphomolybdicphosphotugstic reagent (in Reagent D) by tyrosine and tryptophan
which is present in the Cu(II)-protein complex to give a bluish-green colour. The
intensity of the coloured formed is directly related to the amount of the protein in the
extract and is measured at a wavelength of 750 nm. The sensitivity limit of this method
is 10 – 300 μg/ml protein.

MATERIALS
Sample given
1% NaCl (w/v) solution
Bovine serum albumin, BSA solution, (1 mg/ml, freshly prepared)
Reagent A: 2% Na2CO3 in 1 N NaOH solution.
Reagent B: 0.5% CuSO4.5H2O in 1% sodium citrate solution.
Reagent C: Mix 1 ml reagent B with 50 ml reagent A to give an alkaline cupric solution.
Freshly made on the day used.

Reagent D: Dilute the Folin – Ciocalteau reagent (commercial reagent) with distilled
water to get and acidity of 1 N (determine the acidity by titration with 1 N NaOH until
final endpoint with phenolphthalein). Alternatively dilute Folin – Ciocalteau reagent in a
ratio of 1:1 with distilled water before use. The reagent must be discarded if the colour
turns greenish.
APPARATUS
High speed centrifuge at 4oC, centrifuge bottles 250ml
Spectrophotometer at 750nm and plastic cuvette 1cm
Test tubes
Pipettes 1.0ml, 2.0ml, 5.0ml, 10ml
Pipette pump
Conical flasks 250ml
Vortexer
Ice container/ bucket
Pastuer pipette
Spatula
Measuring cylinder 100ml
Aluminium foil
Blender
Knife
Chopping board
PROCEDURES
Extraction of soluble protein from sample
1. Prepare a representative sample using a suitable procedure.
2. Weigh 5 g of representative sample on an aluminium foil.
3. Blend the sample with 100 ml 1% NaCl solution for 1 minute.
4. Transfer the contents in a centrifuge tube and centrifuge for 10 minutes at 10
000 rpm at 4ºC.
5. Tilt the supernatant into a measuring cylinder and record the volume of protein
extract.
6. Transfer to a conical flask and store in ice until required.
Construction of calibration curve

1. Prepare a series of protein standard solutions as follows:
1 2 3 4 5 6 7 8 9
BSA solution
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.40 0.50
(1 mg/ml)
Distilled
water 1.00 0.95 0.90 0.85 0.80 0.75 0.70 0.60 0.50
(ml)

2. Add 5.0 ml reagent C to all test tubes, mix using a vortexer and leave at room
temperature for 10 minutes.
3. After 10 minutes, add 0.5 ml reagent D to all test tubes and mix as fast as
possible with a vorterxer. Leave for 30 minutes before reading the absorbance at
750 nm. (You can leave the mixture for up to 2 hours before measuring the
absorbance).
4. Construct a calibration curve by plotting absorbance, A, at 750 nm against the
amount of protein standard (μg) in a graph paper.
Determination of protein concentration in the extract

1. Add 0.05, 0.10, 0.20 and 0.30 ml protein extract in different test tubes.
2. Prepare a blank by adding 1.0 ml distilled water in another test tube.
3. Determine the protein content in all test tubes following steps 2 and 3 in Part B.
Measure the absorbance in all test tubes at 750 nm.
If you find the absorbance when using the 0.30 ml extract to be <0.2, repeat the
determination of protein content using a higher volume of extract but not more
than 0.5 ml.
However, if the absorbance when using 0.05 ml protein extract more than the
highest absorbance in the calibration curve, repeat protein determination using
a diluted extract (record the dilution factor used).
4. Get the amount of protein contained in 0.05, 0.10, 0.20 and 0.30 ml protein
extract from the calibration curve.
5. Calculate the average protein concentration in the sample in mg/ml extract.
6. Calculate the concentration of protein in the sample in g/100g.

Calculation
i. Concentration of BSA solution,
M1V1= M2V2
M1 = concentration of BSA solution
M2 = concentration of BSA with distilled water
V1 = volume of BSA solution
V2 = volume of BSA solution with distilled water
ii. protein concentration in the sample in mg/mL,
M1V1= M2V2
M1 = protein concentration from the graph
M2 = protein concentration in protein extract
V1 = volume of protein extract with distilled water
V2 = volume of protein extract
iii. concentration of protein in the sample in g/ 100g,

Sample weight, g = x1
Total extract volume of protein, ml = x2
Amount of protein in total extract, g = x3
Protein concentration in the sample (g/100g) = x1 x 100

X3
Data reporting
1. Table 1 is a summary of average protein concentration extract (mg/ml) and
concentration of protein in the sample (g/100g).
Discussion
- How do you compare your experimental data with your reference value?
- What component that might be undetected based on your sample? Why?
- How do you prepare your sample? Why the sample preparation is crucial to your
data?
- Why it is important to know the amount of protein in your food?
QUESTIONS
1. Give another suitable method that can analyze your sample. Give reasons for your
answer.
2. Why is the conversion factor from Kjeldahl nitrogen to protein different from various
foods, and how is the factor 6.25 obtained?
3. Differentiate and explain the chemical basis of the Kjeldahl, Biuret, Lowry and Bradford
techniques that can be used to quantitate proteins in quality control and research.
4. How to pepare 1% NaCl (w/v) solution? What is the usage of NaCl in this experiment?

1. Meloan, C.E and Pomeranz, Y (1980). Nitrogenous Compounds In Food Analysis
Laboratory Experiments. 2nd Edition. AVI Publishing Co., Connecticut, U.S.A. pp.
102- 110.
2. Osborne, D.R and Voogt, P (1978). The analysis of nutrients in foods. Academic
Press. London, New York, San Francisco.
3. Pearson, D (1976). The chemical analysis of foods. Churchill Livingstons.
Edinburgh, London & New York.
4. Pomeranz, Y. and Meloan, C.E (1978). Nitrogenous compounds. In food analysis

theory and practice (Rev. Ed.) AVI Publishing Co., Connecticut, U.S.A. pp. 668-
691.

TITLE: DETERMINATION OF AMINO ACIDS USING HPLC (DEMO)
PURPOSE: to understand the importance of determining amino acids composition in

food systems.
LEARNING OUTCOMES:
1. Demonstrate the ability to understand the principle of HPLC.
2. Understand the importance and reaction conditions of sample preparation.
INTRODUCTION
High performance liquid chromatography (HPLC) has many applications in food
chemistry. Food components that have been analyzed with HPLC include vitamins,
amino acids, pigments and organic acids. Many food-related HPLC analyses utilize
reversed phase chromatography rather than normal phase chromatography. HPLC is
also widely used in many industries like Pharmaceuticals, Forensics and etc. HPLC can be
defined as a liquid pumped under high pressure through a tube packed with coated
particles to separate a complex mixture into its component compounds. The difference
between the techniques is basically on the sample that not necessary to be volatile, the
column that is shorter and the operating temperatures that are more lower.
Information of amino acids in foods is very important because human cannot produce
the essentials amino acids by themselves. Before such analysis, the sample must be
solubilized and free from all particulate matter, so centrifugation and filtration are
common procedures. Solid phase extraction is also commonly used in sample
preparation to remove interfering compound from the sample matrix prior to HPLC
analysis. Qualititation and quantitation are generally involves by comparing the area and
the height of the sample with the standard. The results are usually expressed in mg/g or
ml/g of food sample.

MATERIALS
Sample
6N HCl
Nitrogen gas
Deionised water
Redrying solution (methanol + water + triethylamine )
Derivatization reagent [methanol + triethylamine + water + phenylisocyanate (PITC)]
α-amino butaric acid (AABA) (0.2578g make up to 1L of 0.1N HCl)
Mobile phase
APPARATUS
Analytical balance
Test tube
Vortex mixer

Oven at 110oC
Volumetric flask
Filter paper
0.2µm Cellulose nitrate membrane filter paper
Vial
Syringe
Pipette
Liquid chromatography unit
PROCEDURES
Sample preparation
a. Sample hydrolysis
Weight *0.25g of sample in a close test tube
Add 15 ml 6N HCl and vortex
Put in oven at 110oC for 24 hour. Then let it cool
Add 10ml AABA (internal standard)
Pour in the 50ml volumetric flask and made up with deionised water
Filter with the filter paper (throw 5-11ml of upper layer)
Hydrolysis sample can be kept for 4 weeks at -20oC
*0.25g is determined by 4/16 = 0.25g. The 16 value is the percentage of protein

of the sample. So, the sample weight is based on the percentage of the protein.
b. Preparing redrying solution

Methanol + Water + Triethylamine (2 : 2 : 1)
c. Derivatization reagent
Methanol + Triethylamine + Water + Phenylisocyanate (PITC) (7 : 1 : 1 : 1)

d. Preparing sample and standard (Derivation)

Hydrolysis sample must be filter first with 0.2µm cellulose nitrate membrane
filter
10µl sample is put in a vial
Add 10µl standard in a vial
Dry under vacuum for 30mins
Add 20µl redrying solution to the sample and standard
Vortex the mixture
Dry under vacuum for 30 mins
Add 20µl derivatization reagent and vortex
Leave at room temperature for 20mins
Vacuum back for 30mins until dry
**Sample and standard can be kept for 4 weeks
Sample injection/standard
Sample and standard must mix 100µl sample diluent (mobile phase) and vortex before
inject to HPLC
Inject volume: Standard - 8µl
Blank - 8µl
Sample - 20µl
Detector: PDA

Data reporting
a. Table 1 is a summary of amino acids composition and the concentration of the
amino acids of the sample.
Calculation
RRF (Reference Retention Factor) = Area of AABA (from standard)
Area of AA (from standard)

Original concentration (mg/g)
= RRF x area of peak of sample x DF X molecular weight of sample

Area of AABA in sample x weight of sample
Discussion
- If your experimental data is the same with your reference value, what are your
evidences and if the values are different, what are your justifications?
- What is the purpose of sample preparation in this experiment? Why is it
important?
- What are the advantages and disadvantages of your method?
- What is your recommendation based on your data?
QUESTIONS
1. Why was it is important to filter and degas the mobile phase and the samples?
2. How you make sure your data is accurate? What factors need to be control?
3. How to prepare the 6N HCl?
4. How is the “reversed-phase” HPLC used here different from “normal-phase” with
regard to stationary phase and mobile phase and order of elution?

1. Christian. Gary D. (2004). Analytical Chemistry (6th Edition). United States,
America. Wiley International Edition.
2. Nielsen. S. Suzanne. (2003). Food Analysis Laboratory Manual. New York. Kluwer
Academic/ Plenum Publishers.

LIPID ANALYSIS

TITLE: DETERMINATION OF THE FREE FATTY ACIDS AND ACID VALUE OF FATS AND OILS
PURPOSE: to comprehend the effects of free fatty acid in quality of fats and oils.
LEARNING OUTCOMES:
1. Demonstrate the ability to examine FFA in food systems.
2. Demonstrate the reaction conditions that lead to the formation of FFA.
INTRODUCTION
Measures of fat acidity normally reflect the amount of fatty acids hydrolyzed from
triacylglycerols. Free fatty acid (FFA) is the percentage by weight of a specified fatty acid
(e.g., percent oleic acid). Acid value is defined as the mg of KOH necessary to neutralize
the free acids present in 1 g of fat or oil. FFA content is calculated using specific
molecular weight of fatty acid. The molecular weight used depends on the type of fat or
oil.
Type of Fat or Oil Specified as Molecular weight

Coconut, palm oil Lauric acid 200
kernel and similar oils.
Palm oil
Other oils Palmitic acid 256
Oleic acid 282
In the addition to the free fatty acids, acid phosphates and amino acids also can
contribute to acidity. In samples containing no acids other than fatty acids, FFA and acid
value may be converted from one to another using a conversion factor.
%FFA (as oleic acid) x 1.99 = acid value
Acid value conversions factors for lauric and palmitic are 2.81 and 2.19 respectively.
Applications
In crude fat, FFA or acid value estimates the amount of oil that will be lost during
refining steps designed to remove fatty acids. A high acidity level in refined fats means a
poorly refined fat or fat breakdown after storage or use. However, if a fat seems to have
a high amount of FFAs, it may be attributable to acidic additives (e.g. citric acid added as
a metal chelator) since any acid will precipitate in the reaction. If the fatty acids
liberated are volatile, FFA or acid value may be measure of hydrolytic rancidity.

MATERIALS
Sample given
Solvent: Mix 95 % (v/v) ethanol with isopropanol in a ratio 1:1 (v/v). The solvent should
be neutralized with 0.1 N sodium hydroxide before use

Sodium hydroxide: 0.1 N or 0.5 N standardized solution

Phenolphthalein indicator: 1 % solution in 95 % (v/v) ethanol
APPARATUS
Conical flasks, 250 mL
Burette, 25 mL
Measuring cylinders, 250 mL
Retort stand
Pastuer pipette
PROCEDURES
The sample weight and normality of sodium hydroxide used depends on the type of fat
or oil. Please refer to the following table:
Type of sample Sample weight Normality of Alkali

Refined oil 20 0.1
Crude oil 10 0.1
Acidic oil 4 0.5
1. Weigh the recommended amount of fat sample in a 250 mL conical flask.

2. Liquefy the fat with 50 mL solvent.
3. To the liquid fat sample, phenolphthalein indicator is added. The sample is then
titrated with sodium hydroxide (the end-point is reached when the indicator
changes to pink for at least 10 seconds).

Calculation
Weight of sample (g) = w
Volume of sodium hydroxide solution required = v
Normality of sodium hydroxide = N
Molecular weight of fatty acid = g/mol = M
Acid value = 56.1 N v
w
FFA (%) = vNM
10w
Data reporting
a. Table 1 is a summary for FFA and acid value of the sample. Stated the mean and
standard deviation.

Discussion
- Which samples changed in color the fastest and why? Which samples give slower
change in color and why? Did the samples make any difference between each other?
Why?
- How to control the samples during the experiment? What factors need to be control?
QUESTIONS
1. Describe the factors that may increase the free fatty acid value of a fat or oil.
2. In crude fat, FFAs are naturally present, but they are removed during processing.
Why the FFAs are being removed?
3. How to prepare 0.1N and 0.5N sodium hydroxide?
4. Explain briefly the proper technique for titration.

1. Cocks, L.V and van Rede, C. (1966). Laboratory Handbook for Oil and Fat Analysts.
Academic Press, London, pp. 109-113.
2. Standard Methods for the Analysis of Oils, fats and derivatives, 6th edn. (1979). Ed.
C. Paguot, Pergamon Press, Oxford, pp. 66-68.
3. AOCS Official Method Cd 1-25 (1998). Official Methods and recommended Practices
of the AOCS. 5th Edn. American oil Chemists’ Society, Champaign, Illinois.

TITLE: DETERMINATION OF IODINE VALUE OF FATS AND OILS
PURPOSE: to comprehend the importance of determining iodine value for fat and oils.
LEARNING OUTCOMES:
1. Demonstrate the ability to understand the reaction conditions behind the
experiment.
2. Demonstrate the ability to handle the hazardous chemicals efficiently.
3. To collect, calculate, analyze and evaluate the experimental data.
INTRODUCTION
Iodine value is defined as the number of grams of halogen, expressed as iodine that can
be absorbed by 100 g o the fat or oil. It is a measure of the degree of unsaturation of the
sample.
The fat or oil is treated with an excess Wij’s solution, which contains iodine
monochloride, and this reacts with the unsaturated part(s) of the fat or oil molecule:
-CH=CH- + ICI -CGI-CHCl + ICl
(Excess Wij’s solution) (Unreacted Wij’s solution)
Iodine is then liberated from the unreacted Wij’s solution by the addition of potassium
iodide:
ICl + KI KCl + I2
The amount of the iodine liberated is determined by titration with standard sodium
thiosulphate:
2Na2S2O3 + I2 Na2S4O6 + 2NaI

MATERIALS
Wij’s solution (iodine monochloride solution)
Fat or oil sample
Cyclohexane
10% Potassium iodide solution
0.25M Sodium thiosulphate solution
Starch indicator
APPARATUS
Conical flasks
Burette
Retort stand

Pastuer pipette
Pipette
Pipette pump
Measuring cylinder 250ml
PROCEDURES
1. Weight accurately 0.1 – 0.3g of the fat or oil sample (see table 1 to determine the actual
weight required) into the conical flask.
2. Dissolve a fat in 20ml of cyclohexane.
3. Run in 25ml Wij’s solution from a dispenser.
4. Simultaneously, prepare a blank sample as above but omitting the fat or oil.
5. Mix well and allow the samples to stand in the dark for about 30mins.
6. At the end of this time, add to each flask 20ml of 10% potassium iodide solution and
followed with 100ml of distilled water.
7. Titrate the liberated iodine with 0.25M sodium thiosulphate solution to a pale yellow
colour.
8. Then, add 2 – 3 drops of starch solution and continue the titration to the colourless end-
point making sure that the flask is well shaken to remove all traces of colour.
Table 1. Weight of fat/oil required for analysis based on *expected iodine value
Expected Iodine Value Weight of sample (g)

<5 3.00
5-20 1.00
21-60 0.34
61-80 0.25
81-130 0.15
>130 0.10
* You will know the weight range of your sample only if you know the expected iodine
value of the sample. You should, therefore, obtain the information first from
appropriate references.

Calculation
Calculate the iodine value of the fat or oils given that the difference between the
volume of thiosulphate used in the blank and in the test sample gives the amount
equivalent to the iodine absorbed by the fat or oil, so that:
1 mL 0.25M sodium thoisulphate solution = 0.03175 g iodine and thus:
Iodine value = (B – T) x 100/W
Where: B= blank titrate of 0.25M sodium thiosulphate, T = sample titrate of 0.25M
sodium thiosulphate, and W = weight in grams of sample of fat or oil.

Data reporting
a. Table 1 is a summary of different oils with expected value of iodine value and
experimental value of iodine value. Stated the mean and standard deviation.
Discussion
- Compare your experimental data with reference value. Gives explanations if your
experimental data is out of the range and vice versa.
- What factors that may affect your sample? How you control the factors? If you
applied in industry, why it is important to control those factors?
- How is your quality characteristics of your sample based on iodine value?
QUESTIONS
1. What does a high value versus a low iodine value tell you about the nature of the
sample?
2. Did your experiment suitable to determine iodine value of processed foods?
Gives explanation for your answer.
3. How do you handle your hazardous chemicals in this experiment?
4. What is other chemical that can replace cyclohexane? Why do you think the
chemical is suitable?
REFERENCES
2. Standard Methods for the Analysis of Oils, fats and derivatives, 6th edn. (1979). Ed.
C. Paguot, Pergamon Press, Oxford, pp. 66-68.

DETERMINATION OF SAPONIFICATION VALUE OF FATS AND OILS
PURPOSE: to understand the importance of determining the saponification value of

vegetable oils.
LEARNING OUTCOMES:
1. Demonstrate the ability to examine saponification value and its effects to the
food quality.
3. To collaborate with companion student on making the experiment and report
efficiently.
INTRODUCTION
The reaction between alkali and fat or fatty acids is the basis for two important
analytical determinations for fat that includes acid value and saponification value
determinations. The reaction between the fat and alkali to produce glycerol and salt or
alkali metal soap is known as saponification reaction. The saponification reaction is as
follows:
C3H5(OOCR)3 + 3 KOH C3H5(OH)3 + 3 KOOCR

Triacylglycerol Alkali Glycerol Alkali metal soap
The saponification value measures the average molecular weight of fatty products. It is a
characteristic used in identification of fats and oils. This method can be used for animal
and vegetable fats.
Definition
Saponification value is the number of mg of potassium hydroxide (KOH) required to
saponify 1 g of fat or oil.
Principle
Saponification value is determined through the reaction between fats or oils with
alcoholic potassium hydroxide solution followed by titration in the presence of excess
KOH with hydrochloric acid.

MATERIALS
Hydrochloric acid: 0.5 N aqueous solution standardized accurately.
Potassium hydroxide: 0.5 M solution in 95% (v/v) ethanol (Note 1).
Phenolphthalein: 1% solution in 95% (v/v) ethanol.
Sodium sulphate anhydrous

Sample given
APPARATUS
Conical flasks, 100 mL, 250 mL x 2
Pipette and burette 50 mL each
Reflux condenser 30 cm in length (minimum)
Heater
Filter funnel and Filter paper Whatman No. 1
Pastuer pipette
Pipette pump
Retort stand
PROCEDURES
1. Weigh accurately to 1 mg, 5 g of fat or oil sample that has been melted in a 250 mL
conical flask. Filter using Whatman No. 1 filter paper containing some anhydrous
sodium sulphate.
2. Pipette 50 mL of 0.5 M alcoholic KOH solution into a 250 mL conical flask containing
the fat sample.
3. For the control, pipette 50 mL of 0.5 M alcoholic KOH solution into a clean 250 mL
conical flask.
4. Connect the reflux condenser to the flask. Heat and when the ethanol boils, shake
the flask until the fat dissolves.
5. Boils the solution for 30 minutes.
6. Cool the solution and titrate with 0.5 M hydrochloric acid until end point is reached
using phenolphthalein as the indicator. (The end point is reached when there is a
change from pink to the original colour.)
Note
Alcoholic KOH solution used should be colourless. The solution can be prepared as
follows:
Boil 1 litre of ethanol 95% under reflux with 8 g KOH and 5 g aluminum pellets. Boil
for 1 hour and distill ethanol. Dissolve the amount of KOH required in the distilled
ethanol. Leave the solution for a few days and separate the clear supernatant from
the calcium carbonate precipitate. The supernatant solution is stable and does not
turn yellow when left or heated.

Calculation
i. Mass (g) fat or oil used =w=
ii. Volume (mL) of hydrochloric acid required for sample = vs =

iii. Volume (mL) of ydrochloric acid required for blank = vb =

iv. Molarity of hydrochloric acid =M=
Saponification = 56.1 M (vb – vs)

w
Data reporting
a. Table 1 is a summary of saponification value for the sample. Stated the mean and
standard deviation
Discussion
- Which sample showed the highest and lowest saponification value? Why?
- What is the importance of saponification value towards food quality? How the
value affected the food properties?
- Which aspects that need to be controlled during the experiment to get accurate
and consistent result?
QUESTIONS
1. In the saponification value determination, why the blank volume higher than
that of the sample?
2. What is meant by unsaponifiable matter in lipid samples? Give an example of
such a type of compound.
3. What chemical and physical properties of fat and oil does the saponification
value signify?
4. Explain the best way of cleaning glassware used in this experiment.
REFERENCES

TITLE: DETERMINATION OF FATTY ACIDS USING GAS CHROMATOGRAPHY (DEMO)
PURPOSE: to comprehend the importance of fatty acids profile in foods.
LEARNING OUTCOMES:
1. Demonstrate the ability to utilize two methods to prepare methyl esters from
fatty acids in food oils.
2. Demonstrate the ability to understand the principle of gas chromatography.
INTRODUCTION
Gas chromatography (GC) has many applications in the analysis of food products. GC has
been used for determination of fatty acids, triglycerides, cholesterol, gases, water,
alcohols, pesticides, flavor compounds and many more. Information about fatty acids
profile on food is important for nutrition labeling, which involves the measurement of
not only total fat but also saturated, unsaturated and monosaturated fat. Gas
chromatography is an ideal instrument to determine (qualitatively and quantitatively)
fatty acid profile or fatty acid composition of a food product. This usually involves
extracting the lipids and analyzing them using capillary gas chromatography. Before
such analysis, triacylglycerols and phospholipids are saponified and the fatty acids
liberated are esterified to form fatty acid methyl esters (FAMEs) so that the volatility is
increased. Two methods of sample preparation for FAMEs determination will be used in
this experiment (1) sodium methoxide method and (2) boron trifluoride (BF 3) method
(AOAC method 969.33). In the sodium methoxide method, sodium methoxide is used as
a catalyst to interesterify fatty acid. This method is applicable to saturated and
unsaturated fatty acid containing from 4 to 24 carbon atoms. In the BF 3 method, lipids
are saponified, and fatty acids are liberated and esterified in presence of BF3 catalyst for
further analysis. This method is applicable to common animal and vegetable oils and fats
and fatty acids. Lipids that cannot be saponified are not derivatived and, if present in
large amount, may interfere with subsequent analysis. This method is not suitable for
preparation of methyl esters of fatty acids containing large amount of epoxy,
hydroperoxy, aldehyde, ketone, cyclopropyl and cyclopentyl groups and conjugated
polyunsaturated and acetylenic compounds because of partial or complete destruction
of these groups.

MATERIALS
Boron trifluoride (BF3)- methanol 14% solution
Hexane (GC grade)
Methanolic sodium hydroxide 0.5 N (Dissolve 2g of NaOH in 100 ml of methanol)
Oils: Olive, palm, corn, virgin coconut oil
Reference standard (FAME 25 mg is dissolved in 10 ml hexane)
Sodium methoxide, 0.5 M solution in methanol
Sodium chloride, saturated

Sodium sulfate, anhydrous granular
APPARATUS
Boiling flask, 100ml with water-cooled condenser for saponification and esterification
Pasteur pipette
Syringe
Vials or sample bottle with tight-seal cap
Analytical balance
Centrifuge
Vortex mixer
Gas chromatography unit
PROCEDURES
Preparation of Methyl Esters
Method A: Preparation of Methyl Esters by BF3
1. Add 500 mg sample to 100 ml boiling flask. Add 8 ml methanolic NaOH solution
and boiling chip
2. Attach condenser and reflux until fat globules disappear (about 5-10 min)
3. Add 9 ml BF3 solution through condenser and continue boiling for 2 min
4. Add a5 ml hexane through condenser and boil for 1 more min.
5. Remove the boiling flask and add 15 ml saturated NaCl solution
6. Stopper flask and shake vigorously for 15s while solution is still tepid.
7. Add additional saturated NaCl solution to float hexane solution into neck of flask
8. Transfer 1 ml upper hexane solution into a small bottle and add anhydrous
Na2SO4 to remove H2O
Method B: Preparation of Methyl Esters by Sodium methoxide method

1. Using a Pasteur pipette to transfer, weigh 100 mg of sample oil to the nearest
0.1 mg into a vial or small bottle with a tight-sealing cap
2. Add 5 ml of hexane to the vial and vortex briefly to dissolve lipid.
3. Add 250 µl of sodium methoxide reagent, cap the vial tightly, and vortex for 1
min, pausing every 10 s.
4. Add 5 ml of saturated NaCl solution to the vial, cap the vial and shake vigorously
for 15 s. Let sit for 10 min.
5. Remove the hexane layer and transfer to a vial containing a small amount
Na2SO4. Do not transfer any interfacial precipitate or any aqueous phase.
6. Allow the hexane phase containing the methyl esters to sit in contact with
Na2SO4 for at least 15 min prior to analysis.
7. Transfer the hexane phase to a vial or small bottle for subsequent GC analysis.
(hexane solution can be stored in the freezer)

Injection of standards and samples into GC

1. Rinse syringe three times with hexane, and three times with the reference
standard mixture (25 mg of reference standard FAME dissolved in 10 ml
hexane). Inject 1 µl of standard solution, remove syringe from injection port,
then press start button. Rinse syringe again three times with solvent.
2. Rinse syringe three times with hexane and three times with the sample
solution prepared by method A. Inject 1 µl of sample solution, remove
syringe from injection port, then press start button. Rinse syringe again three
times with solvent.

Calculation
Concentration of fatty acid (%) = Area of individual fatty acid x 100
Total area of fatty acids
Data reporting
a. Table 1 is a summary of fatty acids present in the sample with the amount in
percentage.
Discussion
- How closed is your experimental data with standard? Which FA does not in your data?
Why? Which FA has the highest percentage and which FA gives the lowest percentage?
Did the values same with the reference values? Give recommendations based on your
data.
- How to get good chromatogram? What factors that may affect the resolution of the
peak?
QUESTIONS
1. During your analysis, sometimes the peak that should present is not in the
chromatogram. How this situation can occur?
2. Explain briefly the principle of GC.
3. What is the difference in preparation of FAME using Boron Trifluoride and
Sodium Methoxide?

ACIDITY ANALYSIS

TITLE: DETERMINATION OF pH, TITRATABLE ACIDITY AND TOTAL SOLUBLE SOLIDS
PURPOSE: to determine pH, titratable acidity and total soluble solids of fruits.
LEARNING OUTCOMES:
1. Demonstrate the ability to comprehend the technical and theoretical part of acidity
analysis.
3. To collaborate with companion students to do the experiment efficiently.
INTRODUCTION
The physical characteristics of foods that are commonly analyzed include pH, titratable
acidity and total soluble solid. pH and titratable acidity are two concepts in food analysis
that are related i.e. both reflect the acidity characteristics of a food. Both these
characteristics use different methods and each exerts individual effects on food. pH
shows the H+ concentration, and in food less than 3% food acid is capable of being
ionized to H+. The organic acids that contribute to the formation of H + include citric acid,
tartaric acid, ascorbic acid and oxalic acid. In aqueous solution, H+ exists as the
hydronium ion, H3O+. The concentration of H3O+ ion can influence the ability of
microorganism growth in a food while this activity is not dependent on the titratable
acidity of that food. Titratable acidity shows the concentration of the total acid found in
a food and better reflects the flavor characteristic of food than pH. The quantity is
determined by titration of acid until finish using a standard base solution. Currently, pH
is measured using a pH meter.
Solids soluble in a food or drink can result of the presence water soluble components
like sugar, organic acid, water soluble vitamin and etc. Soluble solids content can
influence consumer acceptability of a beverage. Usually, soluble solids content in a
beverage is tightly controlled because this value will affect among others, the body and
mouth feel of that beverage. The total soluble solids content can be measured using a
refractometer and reported as ○Brix.

MATERIALS
0.1 M NaOH solution
Fresh distilled water
Sample given
APPARATUS
Blender
Refractometer (Model Otago ATC-1, 0-30 ○Brix)
Knife

Chopping board
Filter funnel and Whatman No.1 filter paper
50 mL beaker
250 mL conical flask
100 mL volumetric flask
25 mL burette
pH meter and standard buffer solutions pH 4 and 7
Tissue paper
Aluminum foil
50 mL bulb pipette
100 mL measuring cylinder
Pasteur pipette
Retort stand
PROCEDURES
A. Determination of pH and titratable acidity
1. Calibrate pH meter using standard buffer solutions at pH 4 and 7.
2. Prepare a burette containing 0.1 M NaOH solution.
3. Homogenized 10 g representative sample with 50 mL fresh distilled water.
4. Filter homogenate through Whatman No. 1 filer paper into a 100 mL volumetric
flask. Wash the residue with fresh distilled water, collect the filtrate in the same
volumetric flask. Make up volume to 100 mL (Vj).
5. Pour 20 mL homogenate into a 50 mL beaker and determine pH using a calibrated
pH meter.
6. Determine the titratable acidity for by titrating 50 mL homogenate (Vt) with 0.1 M
NaOH solution. Measure volume of base (titre) required to make the pH of the
homogenate to pH 8.1 (end-point).
7. Titratable acidity for is defined as % citric acid.
B. Total soluble solids

1. Pipette homogenate using a Pasteur pipette and put a drop on to the surface of the
refractometer window that has been dried with a tissue paper.
2. Read the brix value (total soluble solids) of the homogenate by looking in the
refractometer through eyepiece.
3. Calculate the true total soluble solids.


Calculation
a. % citric acid = titre (mL) x base concentration (M) x Vj x miliequivalent acid x 100
Vt x sample weight (g)
Where Vj is the total volume of the homogenate, Vt is the volume of homogenate used
for titration and the miliequivalent of citric acid is 0.064
b. Total soluble solids (○Brix) = Reading value (○Brix) x dilution factor
Where the factor in this case is x 10, i.e., 10 g samples made up to 100 mL
Note
Titratable acidity of a sample is normally expressed as % dominant acid in a sample. For
example, if it is known that dominant acid in the sample is malic acid, use the
miliequivalent of this organic acid.
Data reporting
a. Table 1 is a summary of pH, titratable acidity and total soluble solids of the sample.
Stated the mean and standard deviation.
Discussion
- Based on your experimental data, what are the quality characteristics that related to
your sample? Did the data related to each other? How they can be related? Explain.
- Based on the data, how do you control the values to meet the optimum quality of your
sample?
- Can this method be applied in industry? What are the advantages and disadvantages of
the method?
- Did your sample preparation crucial? Give justifications for your answer.
QUESTIONS
1. What caused the colour changes in the sample titrated with 0.1N NaOH solution? How
would you recommend the endpoint in the titration of tomato juice?
2. The electrode of your pH meter has a slow response time due to heavily used for a
variety of solutions high in protein, lipids, and minerals. Give the specific
recommendations on cleaning the electrode.
3. How do you calibrate the refractometer? Why the surface of the refractometer window
need to be dried before used?
4. Why the homogenate sample need to be used during the total soluble solid
determination?


1. Ranggana, S (1997). Manual of analysis of fruits and vegetable products. New
Delhi: Tata Mcgraw-Hill.

VITAMIN ANALYSIS

TITLE: DETERMINATION OF VITAMIN C
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 made up of two active biological components that include L-ascorbic acid
and L-dehydroascorbic acid. The natural and main form commonly found in food is L-
ascorbic acid but there is also little L-dehydroascorbic acid. Ascorbic acid analysis in food
is usually done using the reaction between ascorbic acid oxidation with redox indicator
such as 2,6-dichlorophenolindophenol. Unfortunately, this procedure is not capable of
determining dehydroascorbic acid content that represents more or less 80% of vitamin
C activity shown by ascorbic acid. The diagram below shows the reaction between
ascorbic acid and 2,6-dichlorophenolindophenol.
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
Sample given
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 x 2; 500ml
Burette: 50ml
Pipette: 10ml
Beaker 500ml
Filter funnel and cotton wool
Blender
Measuring cylinder: 250ml
Retort stand
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. Obtain a representative sample from the sample given using the most
appropriate sampling method.
2. Weigh and homogenize 40g of the representative sample in 200ml oxalic acid for
3 x 10 seconds.
3. Filter through cotton wool, wash with some oxalic acid and determine the
volume of filtrate (Vf) using a 250ml measuring cylinder.
4. Pipette 10ml of filtrate (Vs) into a 100ml conical flask and titrate with the dye
solution (titre).
5. Calculate the ascorbic content in the sample in mg/100g sample.

C. Effect of blanching time on ascorbic acid content

1. Boil 200ml distilled water in a 500ml beaker.
2. Weigh 40g of representative sample and add to the boiling water. Blanch for 30
seconds.
3. Remove the blanched sample and extract the ascorbic acid in the sample with
oxalic acid as described in Section B. Determine the total volume of extract and
the ascorbic acid content of the sample.
4. Repeat step 2 but blanch the sample for 1, 3, 5 and 10 minutes. Determine the
total volume of extract and residual ascorbic acid content in the blanch samples.
5. Compare results obtained with the ascorbic acid content of sample that was not
blanched (control).
6. Discuss your results.

Calculation
Dye factor = amount of ascorbic acid in 10ml standard solution

Vol. of dye to oxidize 1mg ascorbic acid
Ascorbic acid content (mg/100g) = titre (ml) x dye factor (mg/ml) x Vf x 100
aliquot for extraction (g) x Vs
Data reporting
a. Figure 1 is a plot of ascorbic acid content vs. time.
Discussion
- Which sample gives the highest amount of ascorbic acid and which give the
lowest value? What is the trend for the graph? Why?
- Which parts that are crucial in this experiment? Why? How to control?
- Did the blanching method suitable? What method that suitable to replace it?
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. What method would you use to determine the vitamin C content of a highly
coloured sample (e.g. roselle extract). State reference used.
4. Explain in detail how you would prepare 100 mL of 0.1 mg ascorbic acid/mL in 2%
metaphosphoric acid.


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.

MINERAL ANALYSIS

TITLE: DETERMINATION OF CALCIUM (Ca) CONTENT IN MILK USING ATOMIC

ABSORPTION SPECTROSCOPY (AAS) – DEMO
PURPOSE: to comprehend the importance of concentration in specific minerals in foods.
LEARNING OUTCOMES:
1. Demonstrate the understanding on principle of AAS.
2. Demonstrate a broad, experimental overview of the diverse techniques for the
determination of metals.
INTRODUCTION
PRINCIPLE
Atomic absorption spectroscopy detects the presence of certain metals based on the
absorption of UV or visible radiation by free atoms in the gaseous state. The sample is
drawn up through a capillary tube and is sprayed as a fine mist into a flame. The flame
breaks the molecules into free atoms in the gaseous state, and the atoms absorb
radiation from a beam of light that is directed into the flame. The concentration of the
analyte in solution is determined by the amount of light energy that is absorbed by the
atoms in the sample. We will study flame atomic absorption spectroscopy by analyzing
a milk sample for calcium. After sample preparation, the calcium is atomized in the
flame and quantified based on adsorption of a beam of radiation from a calcium hollow
cathode lamp. The amount of calcium is calculated based on a standard curve.

MATERIALS
Milk
24% (w/v) Trichloroacetic acid
Calcium standard solutions (0, 2, 4, 6, 8, 10, 12, 16 ppm)
5% Lanthanum oxide (99.9% rare earth content)
APPARATUS
Large plastic funnel
35-ml New plastic vials with lids attached (3 per sample)
Whatman #541 filter paper
10-ml bottle dispenser
50-ml bottle dispenser
Analytical balance accurate to .0001
Top loading balance (capacity to 1000gm)
Micropipette 1ml
Pipette tips

Hazards
1. Wear safety glasses when using the atomic absorption spectrophotometer. Do
not directly look at the flame, furnace, or hollow cathode lamps without proper
eye ware protection.
2. Check to see if the burner head is level, drain tube is securely connected, and
that the fuel line connections are tight.
3. Check acetylene tank. Do not use if tank pressure falls to below 75 psig.
4. Acetylene line pressure from cylinder to instrument should never exceed 15 psi.
5. When done for the day, close gas cylinder valves but bleed off the remaining
gases in line while exhaust fan is ON.
6. Only have a knowledgeable person change the acetylene tank.
7. Drain vessels:
 There should be a 6-inch loop filled with water in the waste drain tube.
 Waste drain carboy should be emptied and filled with 5 inches of water when
switching from organic to aqueous solutions.
 The waste drain carboy will contain approximately 1.4% TCA, 0.02 % lanthanum
chloride, and 0.03% milk along with any other chemicals other people have used.
Environmental safety has said it is ok to dump this down the sink while running
water.
8. If the glow plug does not turn red when the ignition switch is pressed, then
something is not correctly assembled in the burner unit. Check to see if the pin
on the hold-down cable is properly inserted in the safety interlock at the base of
the burner. Also check that the waste drain system is properly installed. If the
accessory drain interlock is installed, the gases will not ignite unless the drain
interlock is connected.
9. NEVER LEAVE THE FLAME UNATTENDED.
PROCEDURES
1. Assign one student to prepare two sample blanks. The blanks are used to
monitor any background calcium or possibly detect some calcium contamination.
Follow the same procedure except add 0.75ml of high quality distilled water
rather than milk. The blanks should not exceed 0.030. If they do, the TCA may
be contaminated.

2. Obtain one 35-ml vial and label it as sample.
3. Weigh the sample vial. Record the weight and all other weights to the 4 th
decimal place.
4. Using the micropipette set at 75 and a clean tip, pipette 0.75 ml of the milk
sample into the pre-weighed vial.
5. Immediately reweigh the vial and record the weight. The target weight range of
the milk sample should be 0.7 to 0.8 gm.
6. Using a 50-ml bottle dispenser, add 29.5 ml of 12% TCA to the sample vial.
Immediately reweigh the vial and record the weight.
7. Cap the vial and invert twice to mix TCA and sample.
8. Wait 30 minutes to allow the TCA to interact with the sample.
9. Obtain a second clean 35-ml vial and label it as filtrate. (It is not necessary to
weigh this vial.)
10. After 30 minutes, filter the milk sample through Whatman #541 filter paper into
the vial labeled as filtrate. The filtrate should be free of any particulate matter.
If not, filter again.
11. Obtain a third clean 35-ml vial and label it as solution. Weigh the vial and record
the weight to the 4th decimal place.
12. Use a clean10-ml volumetric, pipette and transfer 10 ml of filtrate into the vial
labeled as solution. Immediately reweigh the vial and record the weight.
13. Using the micropipette set at 40, and a clean tip, pipette 0.40 ml of lanthanum
oxide into the solution vial.
14. Using a 10-ml bottle dispenser, add 9.6 ml of distilled water to the solution vial.
Weigh the vial and record the weight.
15. Cap the vial and invert a few times to mix the solution. You are now ready to
analyze your sample!
16. Discard the extra 12% TCA and filtrate into the TCA waste bottle.
17. Follow directions for operation of the atomic absorption spectrophotometer.


Calculations
1. Prepare a standard curve by plotting concentration (x) vs. absorbance (y) and use
linear regression to determine the equation of the line.
2. Use the equation of the line to calculate concentration of milk in ppm.
3. Calculate the % calcium (see example problem below).
Formulas
1. ppm of ca2+ in milk: [((x ppm) * (df1) * (df2)]
2. (x ppm) is obtained from linear regression analysis on standard curve
3. Dilution factor (df1) = [(weight of milk + weight of TCA) / (weight of milk)]
4. Dilution factor (df2) = [(weight of filtrate, lanth oxide & H2O) / (weight of filtrate)]
5. To convert ppm calcium to percent calcium, multiply by 0.0001.
Example calculations of percent calcium in milk:

Linear regression analysis of the standard curve gives the equation y = .0537x + .029.
The absorbance of a milk sample is 0.798 and using the standard curve the
calculated calcium concentration is 14.32 ppm
Weight of milk (g ) = 0.8246

Weight of TCA (g) = 31.0016
Weight of filtrate (g) = 10.515
Weight of filtrate, lanth oxide & H2O (g) = 20.4186
df1 = [(weight of milk + weight of TCA) / (weight of milk)]

df1 = [(0.8246 + 31.0016) / 0.8246] = 38.5959
df2 = [(weight of filtrate, lanth oxide & H2O) / (weight of filtrate)]

df2 = (20.4186 / 10.515) = 1.9419
Conc. of calcium: ppm fm standard curve x df1 x df2

14.32 x 38.5959 x 1.9419 = 1073 ppm calcium in milk
To convert ppm calcium to percent calcium, multiply by 0.0001

1073 .0001 = .1073 %
Data reporting
1. Table 1 is a summary of calcium concentration in milk. Stated the mean and
standard deviation.

Discussion
- Compare your result with reference value. Was the calcium content typical of
milk? Did any factors affect the value? How?
- Importance of calcium in daily life.
QUESTIONS
1. Error usually occurred during the analysis. How to minimize error in this
analysis?
2. Explain briefly on how the result is figured out.
3. Give the correct procedure on preparation of your sample.
4. How to prepare 2ppm and 16ppm of calcium solution from the standard given.

ENZYME INHIBTION

TITLE: EFFECT OF BLANCHING ON PEROXIDASE ACTIVITY
PURPOSE: to determine the importance of inactivation to peroxidase enzyme in fruit

and vegetable
LEARNING OUTCOMES:
1. Demonstrate the ability to examine peroxidase activity in food systems and its
effect to the food quality.
2. To observe and analyze the experimental data.
INTRODUCTION
Fresh fruit and vegetables contain many active enzymes that cause post-harvest
deterioration in quality and nutritional value. This deterioration occurs even when the
products are frozen. Thus fruits and vegetables are usually blanched before freezing or
canning.
The thermal stabilities of enzymes vary widely. Therefore, blanching conditions need to
target at most heat-resistant ezymes. Peroxidase is one of the most heat-stable
enzymes in plants. Thus it is a good indicator for blanching adequacy, since heat
treatments sufficient to inactivate peroxidase also inactivate most other enzymes.
Blanching consists of immersing the material in hot water or steam for a short period of
time. Advantages of blanching are the "spoilage" enzymes are destroyed, the material is
cleaned, air is removed and the product is softened making packing easier. However,
some water soluble nutrients are lost during blanching.
This experiment studies the effect of temperature on the adequacy of blanching by

assaying for peroxidase.

MATERIALS
Sample given
Guaiacol (1% v/v in 95% ethanol)
Hydrogen peroxide (0.5% v/v)
Sand
Distilled water
APPARATUS
Beakers, 600 ml
Slotted spoon
Knife
Mortar and pestle
Graduated cylinder, l0 ml
Pipettes, 1ml

Heat plate
Test tubes
Paper towel
PROCEDURES
1. Blanch a few pieces of potato and apples as follows: Bring 300 ml distilled water
to boiling in a 600 ml beaker. Submerge pieces of each sample in the boiling
water for 2 min. Remove and submerge in cold water to cool. Place on paper
towel.
2. Assay the potato and apple before and after blanching as follows:
a. Cut a piece of sample weighing about 5g into small pieces.
b. Transfer the sample to a mortar containing a small quantity of sand. Add
about 5ml distilled water and grind for 2 to 3 min.
c. Add another 5ml of water, and transfer contents to a test tube.
d. Add 1ml 1% guaiacol solution and 1 ml 0.5% hydrogen peroxide. Mix by
inverting the tube.
e. Peroxidase activity is indicated by development of a reddish colour. If no
colour develops in 3.5min, consider the product adequately blanched.

Data reporting
a. Table 1 is a summary of peroxidase activity before and after blanching.
Discussion
- Which samples are darker? Why? Which samples did not change in colour?
Why?
- What factors that affect the process? How to control it? What is the effect to the
quality of foods?
- What are the advantages and disadvantages of blanching? Give other method
that can give the same result with good quality.
QUESTIONS
1. Describe the chemical reaction catalysed by peroxidase.
2. List the groups of organism that produce peroxidase.
3. Describe the correct way of sample preparation in this experiment? Why the
process is very crucial?
4. Explain the function of guaiacol solution and hydrogen peroxide in this
experiment.

1. Fox, P.F (ed) (1992). Food Enzymology. Elsevier Applied

CASE STUDY

CASE STUDY
Select your case study and have your topic and methods approved by the instructor.
Apply the principles of the scientific method covered throughout this course. Once the
topics and methods are verbally approved, the plans should formalize in writing for a
grade. The following should be included in your proposal.
Research proposal
 Title
 Background: review literature relevant to the project. Justify the methods you
selected. Are they standard methods? Give the purpose of your project.
 Approach: Give methods – what you plan to do and how. Be specific. Include
procedures and recipes and their source. Quantities of ingredients must be in
metric units
 Work plan: Plan each step – what you are going to do each week and the
preparations required prior to the laboratory period. Replicate analysis at least
two times
 Supplies needed: Turn in supply sheets with your proposal. List item and amount
and when needed along with any specification (brand, etc.).
Written presentation
 Your report must be typed, spell-checked and neat. Use a technical writing style.
Avoid the use of first person, contractions, and colloquial and literary styles. Use
proper grammar.
 Title should be descriptive but not excessively long.
 An abstract is a one-paragraph summary of problem, methods and main findings.
 Introduction: This section should state the problem being studied with sufficient
background to fully understand the project. This will likely require a discussion of
a chemical process learned in class. This section may also include a review of
methods available to test your sample and an explanation for your selected
approach. This section should include a statement of the purpose of the project.
This statement should come at the end of this section.
 Methods
o Give subheadings
o Give your overall design then specific procedures/assays/formulas.
o Give sufficient detail so that the project can be repeated by someone else
(eg. Include settings, any important temperature, ph controls, equipment
type, sample preparation, etc)
o Discuss replication and sampling
 Discussion
o If calculation are used in creating data, sample calculation should be
provided here or in the Appendix

o Give scientific and literature-based explanations and potential sources of

error in interpreting results. Discussions without sufficient citations from
literature will result in substantial point deductions.
o Give suggestions for further work
 Results: Summarize data in tables and figures using complete titles that can be
understood without reference to text (including type of product, if relevant). The
text must refer to each table and figure numbers chronologically.
 References
o Use the style of the Journal of Food Science in your reference list and in
citations in the text. Avoid direct quotations of references. Paraphrase
source-do not plagiarize!
o Limited use of general textbooks is acceptable. Emphasize original journal
articles. The literature available on selected topic should be well
represented
 Appendix: Include raw data of experiments performed
Project evaluation
You will be awarded marks for the following:
Group work (5/30):

Problem well thought out
Used class time to best advantage
Careful planning for each day
Attendance and cooperativeness
Written report (15/30)

Abstract
Introduction
Justification
Material and Methods
Results
Discussion
References
Presentation (10/30)

Week 1. FST 3112 Lab. Manual FST3112-2012-2013-2 PDF

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Week 1. FST 3112 Lab. Manual FST3112-2012-2013-2 PDF

Uploaded by

Copyright:

Available Formats

CHEMICAL ANALYSIS OF FOOD

1 Introduction: Laboratory safety; Guidelines for report preparation

Chemical Analysis of Foods – A Practical manual 2

Introduction to Laboratory Safety

1. Wear protective clothing. A laboratory apron or coat is a first line of defense

Chemical Analysis of Foods – A Practical manual 3

Chemical Analysis of Foods – A Practical manual 4

Guidelines for Report Preparation and Submission

2. Each report should contain the following:

5. Each group submits one report only.

Chemical Analysis of Foods – A Practical manual 5

to collaborate on data analysis, particularly on the required calculations, but the

Chemical Analysis of Foods – A Practical manual 6

Laboratory Report Assessment Criteria

Chemical Analysis of Foods – A Practical manual 7

Chemical Analysis of Foods – A Practical manual 8

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;

Chemical Analysis of Foods – A Practical manual 9

Chemical Analysis of Foods – A Practical manual 10

Chemical Analysis of Foods – A Practical manual 11

TITLE: DETERMINATION OF MOISTURE CONTENT

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

Oven Drying Method

MATERIALS AND PROCEDURES

Chemical Analysis of Foods – A Practical manual 12

RESULT AND DISCUSSION

where; a = weight of sample used

Chemical Analysis of Foods – A Practical manual 13

REFERENCES (3-4 references)

Chemical Analysis of Foods – A Practical manual 14

TITLE: DETERMINATION OF ASH CONTENT

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

MATERIALS AND PROCEDURES

Chemical Analysis of Foods – A Practical manual 15

RESULT AND DISCUSSION

REFERENCES (3-4 references)

Chemical Analysis of Foods – A Practical manual 16

TITLE: DETERMINATION OF CRUDE PROTEIN

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

MATERIALS AND PROCEDURES

Chemical Analysis of Foods – A Practical manual 17

Chemical Analysis of Foods – A Practical manual 18

RESULT AND DISCUSSION

Chemical Analysis of Foods – A Practical manual 19

2. James, C. S. (1995) Analytical Chemistry of Foods. Blackie Academic &

Chemical Analysis of Foods – A Practical manual 20

Table 1: Factors for converting nitrogen to protein.

Foodstuff Conversion factor

Other foods 6.25

Chemical Analysis of Foods – A Practical manual 21

TITLE: DETERMINATION OF CRUDE FAT

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

Chemical Analysis of Foods – A Practical manual 22

MATERIALS AND PROCEDURES

RESULTS AND DISCUSSION

% of oil in sample = M x 100

Chemical Analysis of Foods – A Practical manual 23

REFERENCES (3-4 references)

Chemical Analysis of Foods – A Practical manual 24

TITLE: DETERMINATION OF CRUDE FIBER