GENERAL FOOD ANALYSIS

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Table of Contents
INTRODUCTION TO FOOD ANALYSIS.................................................................................................4
BASIC LABORATORY TECHNIQUES..................................................................................................5
1-1 CONCENTRATION OF SOLUTIONS............................................................................................5
1-2. Preparation of standard 0.1N-NaOH solution...............................................................................7
1-3. Preparation of standard 0.1N HCL solution..................................................................................8
2.0. APPROXIMATE ANALYSIS.............................................................................................................9
2-1.Determinaton of moisture content.....................................................................................................9
2-1.a. Oven drying method..................................................................................................................9
2-1.b. Vacuum oven method.............................................................................................................10
2-1.c. Moisture determination by distillation method........................................................................10
2-1.d. Chemical moisture determination method (Karl Fischer titration method)..............................12
2-2. DETERMINATION OF CRUDE FAT BY SOXHLET METHOD...................................................15
2-3. DETERMINATION OF CRUDE FIBRE BY HENNENBERG-STOHMANN METHOD...............18
2-4. DETERMINATION OF ASH............................................................................................................20
2-4.1.Ash-rapid (magnesium acetate) method...................................................................................20
2-5. DETERMINATION OF CRUDE PROTEIN CONTENT BY SEMI- MICRO KJELDHAL
METHOD..................................................................................................................................................23
3.0 . SPECIFIC VALUE FOR FATS AND OILS.....................................................................................25
3-1. Saponification value.......................................................................................................................25
3-2. Determination of iodine value.......................................................................................................27
3-3. Determination of peroxide value (po. v.)......................................................................................29
3-4. Determination of acid value...........................................................................................................31
4.0. ACIDITY...........................................................................................................................................32
4-1. Determination of total titratable acidity..........................................................................................32
5.0. VITAMINS........................................................................................................................................34
5-1. Determination of vitamin c (ascorbic acid) by indophenol method................................................34
6.0. PREPARATION OF REAGENTS.....................................................................................................36
7.0. INSTRUMENTAL ANALYSIS.........................................................................................................40
7-1. Spectrophotometric methods..........................................................................................................40

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7-2. Colorimetry....................................................................................................................................42
7-3. Rheometry......................................................................................................................................43

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INTRODUCTION TO FOOD ANALYSIS
Investigations in food science and technology often require determination of food composition
and characteristics.

Food Analysis- it’s the study of a technique and methods used in the laboratory in analyzing
different food material

-is the disciplinary field of developing application and study analytical procedure for the food
component /constituent .

Standard in food quality -is the set control parameter required to be a cord with during the
production of the product.

Quality is the degree of excellency or acceptability

Trends and demands of consumers, the food industry and the national and international
regulations challenge food scientists as they work to monitor food composition and ensure
quality and safety of food.

All food products require analysis as part of a quality management program, throughout the
development, production and after the product is in the market. The characteristics of foods i.e.
chemical composition, physical and sensory properties are used to answer specific questions for
regulatory purposes and typical quality control.

The nature of the sample and the specific reason for analysis dictates the choice of analytical
method. Speed, precision and accuracy are the key factors in this choice. The success of any
analytical method relies on the proper selection and preparation of the food sample, carefully
performing the analysis and doing the appropriate calculations and interpretation of the data.

The chemical components of food can be evaluated by various methods which include:-
chemical, physical and sensory.

There are two type of analysis

 Proximate/qualitative analysis
 Ultimate quantitative analysis

The purpose of practical in food chemistry and food analysis is mainly to solve qualitative and
quantitative problems in food stuff.

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 In qualitative analysis, the aim is to determine which constituents are present in the food stuff
e.g mineral ,protein

while the quantitative analysis determines how much of a desired component is present in food
stuff.

Before the start of analysis, several considerations have to be made:-

 The kind of food sample.

 The accuracy and precision demanded.
 Whether the constituents of interest are major or minor in the sample.
 Possible interferences.
 The number of samples to be analyzed.
 The separations required.
 The equipment and apparatus used.

It is important to choose the method and procedure of analysis or measurements that is proper
for solving the problems given which is often dictated by the availability of chemicals and
equipment in the laboratory. The student should fully understand the principle that each method
is based on.

There are six basic types of techniques used in food analysis:-

 Gravimetric methods
 Volumetric methods(titrimetric)
 Spectrophotometry
 Atomic absorption
 Chromatography.

The first two are chemical methods (mass and volume measurements) while the rest are
instrumental methods. The instrumental methods are more sensitive to accuracy than gravimetric
and volumetric methods. The methods can also be classified as either relative or absolute.
However, most methods are relative meaning that they require comparison against some solution
of known concentration. e.g. in titrimetric method ,the analyte is reacted with a solution of a
reagent in a known stoichiometric ratio.

Most instrumental methods are relative, instruments register a signal due to some physical
property of the solution, and for example spectrophotometer measures the fraction of
electromagnetic radiation from a light source that is absorbed by the sample. The fraction must
be related to the analyte concentration by comparison against the fraction absorbed by a known
concentration of the analyte.

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 A standard procedure for calibrating instruments is to prepare a working curve known as
analytical calibration curve by plotting the measured signal as a function of the analyte
concentration.

Role of analyst

 To decide the best method and procedure for a particular analysis for a given food
 Provide means to protect consumers against toxicity fraud and cheating
 Determine the level of additives or acceptable additives
 Checking compliance or quality standard
 Evaluating new processing method for making food product
 Formulating and developing of new product
 Assessment and maintenance of food quality

Factor that determines method of analytical to used

 Specific reason for analysis

 Nature of sample
 Characteristic of method it self
 The immediate result and accuracy
 Cost of equipment
 Training of personnel

Application of food analysis

 In quality control
 Official laboratory
 Research institute eg DAIRY BOARD, TEA, KMRIE
 College’s lab
 Education

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 METHODS OF FOOD ANALYSIS

BASIC LABORATORY TECHNIQUES

1-1 CONCENTRATION OF SOLUTIONS

1-1. a. One gram equivalent

This refers to the grams of substance which release or accept one mole of hydrogen ion
(H ) or electron (e-).
+

One gram equivalent (e q) = molecular weight

n
+
Where n = no. of H which can be released from one molecule of the substance.
1-1. b. Normality
This refers to the number of gram equivalent which is contained in one liter of solution.
Therefore (1N) solution contains one gram equivalent of substance per one liter.
Normality = number of gram equivalent/volume of solution in one liter.
1-1. c. Factor (f)
This is one kind of correction coefficient. For example, when 0.1023 Normal of solution
is prepared, it is usually labeled 0.1N solution.
Factor = 1.023, meaning that the exact value of 0.1023 normal is given by multiplying the
factor of 1.023 to 0.1N.
Factor = no. of grams taken in practice
A gram equivalent of solute x approx. normality you want
1-1. d. Weight percent

Refers to the grams of substance contained in 100g of sample.

In case of a unit volume (W/V) it is the grams of solute in 100ml solution.

1-1. e. Parts per million (ppm)

Refers to the grams of solute contained in a million grams of solution. e.g. milligrams per
kilogram.

ppm = grams of substance x 106 = milligrams of substance

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 Grams of sample kilograms of sample

In case of solution

ppm = grams of solute x 106 = milligrams of solute/liters of solution.

Grams of solvent

1ppm = 1mg/1l

1-1. f. Parts per billion (ppb)

Refers to grams of solute contained in a billion grams of solution e.g micro grams per
kilogram.

ppb = grams of substance x 109 = micrograms of substance/kilograms of sample

Grams of sample

Ppb = grams of solute x 109 = micrograms of substance/liters of solution.

Grams of solvent

1-2. Preparation of standard 0.1N-NaOH solution

1-2 a. Reagents
 Oxalic acid
 Saturated NaOH solution
 Phenolphthalein solution
1-2 b. Procedure.

1-2 b. 1 Preparation of standard 0.1N oxalic acid

Oxalic acid has a molecular weight of 126.08 and it can release 2H + hence a gram equivalent is
63.04.
Weigh 6.3g of oxalic acid in a 1000ml volumetric flask and fill up with distilled water and mix
well.
1-2.b.2. Calculation.
Factor (f) = weight of oxalic acid taken/equivalent weight of oxalic acid x normality
1-2.b.3. Preparation of 0.1N NaOH

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 NaOH has a molecular weight of 40g which is required to prepare a 1N solution in
1000ml.therefore a 0.1N solution will require 4g of NaOH in 1000ml of distilled water.
1-2.b.4. Standardization of 0.1N NaOH (to obtain a factor of 0.1N NaOH)
 Take 20ml of oxalic acid in to a conical flask
 Add 2drops of phenolphthalein
 Titrate against NaOH until a pink color appears and persists for 30seconds.
 Carry out three times and calculate the average.
1-2.b.5. Calculation of the factor
N a x f a x V a = N b x f b x V b where:-
 N a – normality of acid(0.1)
 F a – factor of acid
 V a – volume of acid(20ml)
 N b – normality of base(0.1)
 F b –factor of base
 V b –volume of base
1-3. Preparation of standard 0.1N HCL solution
1-3 a. Reagents
 Sodium carbonate (anhydrous).
 Concentrated HCL solution
 0.2% methyl red solution
1-3.b. Procedure
1-3.b.a Preparation of standard 0.1N sodium carbonate (anhydrous Na2Co3)
The molecular weight of sodium carbonate is 105.998, a gram equivalent is 53g hence 0.1g
equivalent is 5.3g.
Weigh 5.3g of sodium carbonate in to a 1000ml volumetric flask and fill up with distilled water
then mix well.
1-3.b.b Calculation
Factor (f) = weight of sodium carbonate taken/equivalent weight x normality
1-3.b.c. Preparation of 0.1N HCL
Concentrated HCL is approximately 12N, take and dilute it 120 times to get 0.1N.

1-3.b.d. Standardization of 0.1N HCL solution (to obtain a factor of 0.1N HCL)

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  Take 20ml of 0.1N sodium carbonate into a conical flask
 Add 2 drops of methyl red
 Titrate against 0.1N HCL until a red color appears
 Carry out three times and calculate the average.
1-3 b.e. Calculation of factor
N a x f a x V a = N b x f b x V b where:-
 N a – normality of acid
 F a – factor of acid
 V a – volume of acid
 N b – normality of base
 F b – factor of base
 V b – volume of base
SAMPLING TECHNIQUES
Sampling -is the study of relationship of sample drawn from population or lot
Sample -it’s a part of the fraction of the population
Population- is a collection of all individual
Lot-number of unit in one batch
Field sample-the material collected which consist of same sample unit
Test sample-a unit taken from sample directly for analysis
Sample plan -it’s the flexible part of a lot
Types of sample
 Raw material
 Process control sample
 Finished product sample
 Complaint sample
 Competitors sample
 Buying sample
preparation of sample

Important of sampling
 Save time and avoid wastage for convenience

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  Its less costly and accurate
 Its confirm safety in all areas

Step involve in sampling

 Select and prepare sample
 Perform the analysis
 Calculate and interpreted the result
Method of sampling /sampling techniques
 random sampling (probability sampling)
 stratified sampling
 systematic sampling
 clustered sampling

sample preparation and preservation

Physico-chemical methods

i) mechanical methods
ii) sensory methods
iii) microbiological methods
iv) texture evaluation

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Microbiological methods

Texture evaluation

2.0. APPROXIMATE ANALYSIS

2 -1.Determinaton of moisture content

It is one of the most fundamental and important analytical procedures performed on food
products. The dry matter that remains after moisture removal is commonly referred to as total
solids. The analytical value is of great economic importance to both the food producer and
consumer, of great significance is the effect of moisture on the stability of foods. The most
commonly used methods for moisture determination in food include:-
Determination of moisture content
i) Drying method
ii) Distillation method
iii) chemical methods eg Karl Fischer method
iv) Instrumental method/ physical methods.

2-1.a. Oven drying method

The food material is heated under specified conditions of time and temperature, the loss of
weight is taken as a measure of the moisture content of the sample.

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 Sample: dry cereals (wheat flour, rice, maize)
Materials
 Oven
 Moisture dishes
 Weighing balance
 Spatula
 desiccator
Procedure
 Grind the sample to powder (to pass a no. 30 sieve) and store in air tight container.
 Dry the moisture dish(stainless steel/crucible) and its lid in an oven at 105-110 0c for
1-2 hours ,then cool to room temperature in a desiccator
 Weigh the dish and its lid accurately (w0).
 Take about 5g of the sample in to the moisture dish.
 Weigh the sample + container and the lid accurately (w1).
 Heat at 1050c for 12 hours while the lid is open (whereby the weight is taken after
every 2hrs until a constant weight is attained).
 Close the lid and cool to room temperature in a desiccator, weigh accurately(w 2)
 Calculate the moisture content of the sample
% moisture = weight of moisture evaporated x 100
Weight of the sample

= w1-w2 x 100
W1-w0
Liquid sample: milk
Procedure
 Put a stainless steel moisture dish in an oven at 98-1000c
 Dry for 1-2 hours, put on a lid in the oven
 Remove the dish to a desiccator and cool to room temperature for (30-45 minutes)
 Weigh exactly up to 0.1mg with a direct reading chemical balance
 Record the weight of the dish
 Repeat the procedure as given below
 Give a constant weight(w0g)
 Put approximately 3ml of the sample in to the previously weighed dish
 Put on the lid and weigh(w1g)
 Take off the lid
 Heat the contents in the dish on a water bath to evaporate the liquid
 Put the dish and the lid in an oven at a fixed temperature of 98-1000c
 Dry for 3hrs to a constant weight

 Cool to room temperature in a desiccator(30-45min)

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  Weigh exactly up to 0.1mg (w2g)
Calculation
% moisture = w1-w2 x 100
W1-w0
2-1.b. Vacuum oven method
Materials:-
 Moisture dishes
 Vacuum oven at 650c
 desiccator
Procedure
 weigh accurately 5g of the sample in a dish
 put the dish and contents in a vacuum oven maintained at 65 0c and at a pressure not
exceeding 100mmHg with an air through flow
 Dry for 2hours
 Cool in a desiccator and weigh
 Continue this operation until the moisture content agree within 0.05%
 Calculate the moisture content in %

2-1.c. Moisture determination by distillation method

Principle
Water is released from the sample by continuous distillation with an immiscible solvent. The
water is collected in a suitable receiver where it is allowed to settle so that its volume can be
measured while the solvent overflows in to the distilling flask.
Reagent
Xylene or toluene
Sample:- milk

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Materials
 Distillation apparatus
 Round bottomed flasks
 Pipette
 Measuring cylinder
Procedure
 Take 5ml of the sample in a 250ml boiling flask so that the top of the flask may not be
stained with sample
 Add 75ml of xylene or toluene
 Connect a receiver with the volumetric tube to a boiling flask and a condenser
 Start to heat
 Adjust heat so that the water droplets may fall from the tip of the condenser at a rate of 2-
3 drops per second at first
 After the increase of volume of the distilled water in the volumetric tube, it becomes
conspicuous (after 30-45 minutes from the start), then increase heat gradually.
 Adjust heat so that water droplets may fall from a tip of a condenser at a rate of 4-5 drops
per second
 When no more water droplets fall from the tip of the condenser pour vigorously a small
amount of xylene into the condenser from the top with a squeeze bottle with fine tip to
rinse a drop of water on the wall of the condenser
 Continue distillation for a few minutes until no more water droplets can be seen falling in
to the volumetric tube
 Stop heating and remove the receiver with a volumetric tube
 Cool the part of a volumetric tube until it reaches room temperature
 Read and estimate the volume of distilled water to the nearest 0.01ml

Calculations
% moisture = volume of distilled water x 100
Weight of the sample

2-1.d. Chemical moisture determination method (Karl Fischer titration method)

Objective
Determine the moisture content of milk powder and corn flour by the Karl Fischer (KF) method.
Principle
When the sample is titrated with the KF reagent, which contains iodine and sulfur dioxide, the
iodine is reduced by sulfur dioxide in the presence of water from the sample. The water react

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stoichiometric ally with the KF reagent. The volume of KF reagent required to reach the
endpoint of the titration (visual, conductometric, or colorimetric) is directly related to the amount
of water in the sample.
Reagents
 KF reagent
 Methanol, anhydrous
 Sodium tartrate dihydrate, 1 g, dried at 150°C for 2 h
Hazards, Cautions, and Waste Disposal
Use the anhydrous methanol in an operating hood since the vapors are harmful and it is toxic.
Otherwise, adhere to normal laboratory safety procedures. Use appropriate eye and skin
protection. The KF reagent and anhydrous methanol should be disposed of as hazardous wastes.
Supplies
 Corn flour
 Graduated cylinder, 50 ml
 Milk powder
 2 Spatulas
 Weighing paper
Equipment
 Analytical balance, 0.1 g sensitivity
 Drying oven
 KF titration unit (e.g., from Barnsted Themaline, Berkeley, CA, Aquametry Apparatus)

Procedure
Instructions are given as for a non-automated unit, and for analysis in triplicate. If using an
automated unit, follow instructions of the manufacturer.
I. Apparatus Set Up
Assemble titration apparatus and follow instructions of manufacturer. The titration apparatus
includes the following: burette; reservoir for reagent; magnetic stirring device; reaction/titration
vessel; electrodes; and circuitry for dead stop endpoint determination.
Note that the reaction/titration vessel of the KF apparatus (and the anhydrous methanol within
the vessel) must be changed after analyzing several samples (exact number depends on type of
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sample). Remember that this entire apparatus is very fragile. To prevent contamination from
atmospheric moisture, all openings must be closed and protected with drying tubes.
ii. Standardizing Karl Fischer Reagent
The KF reagent is standardized to determine its water equivalence. Normally, this needs to be
done only once a day or when changing the KF reagent supply.
1. Add approximately 50 ml of anhydrous methanol to reaction vessel through the sample
port.
2. Put the magnetic stir bar in the vessel and turn on the magnetic stirrer.
3. Remove the caps (if any) from drying tube. Turn the burette stopcock to the filling
position. Hold one finger on the air-release hold in the rubber bulb and pump the bulb to
fill the burette. Close the stopcock when the KF reagent reaches the desired level (at
position 0.00 ml) in the burette.
4. Titrate the water in the solvent (anhydrous methanol) by adding enough KF reagents to
just change the color of the solution from clear or yellow to dark brown. This is known as
the KF endpoint. Note and record the conductance meter reading. (You may titrate to any
point in the brown KF zone on the meter, but make sure that you always titrate to that
same endpoint for all subsequent samples in the series.) Allow the solution to stabilize at
the endpoint on the meter for at least 1 min before proceeding to the next step.
5. Weigh, to the nearest milligram, approximately 0.3 g of sodium tartrate dihydrate,
previously dried at 150°C for 2 h.
6. Fill the burette with the KF reagent then titrate the water in the sodium tartrate dihydrate
sample. Record the volume (ml) of KF reagent used.
7. Calculate the KF reagent water (moisture) equivalence (KFReq) in mg H2O/ml:

iii. Titration of Sample

1. Prepare samples for analysis and place in reaction vessel as described below.
If samples are in powder form:
– Use an analytical balance to weigh out approximately 0.3 g of sample, and record the exact
sample weight (S) to the nearest milligram.
– Remove the conductance meter from the reaction vessel then transfer your sample to the
reaction vessel through the sample port immediately. (Use an extra piece of weighing paper to

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form a cone-shaped funnel in the sample port then pour your sample through the funnel into the
reaction vessel.)
– Put the conductance meter and stopper back in the reaction vessel. The color of the solution in
the vessel should change to light yellow and the meter will register below the KF zone on the
meter.
If any samples analyzed are in liquid form:
– Use a 1-ml syringe to draw up about 0.1 ml of sample. Weigh the syringe with sample on an
analytical balance and record the exact weight (S1) to the nearest milligram.
– Inject 1–2 drops of sample into the reaction vessel through the sample port then weigh the
syringe again (S0), to the nearest milligram.
– Sample weight (S) is the difference of S1 and S0.
S = S1 – S0
– Put the stopper back in the sample port of the reaction vessel. The color of the solution in the
vessel should change to light yellow and the meter will register below the KF zone on the meter.
2. Fill the burette then titrate the water in the sample. Record the volume (ml) of KF reagent
used.
3. After titrating several samples (exact number depends on the nature of the sample), it is
necessary to start with fresh methanol in a clean reaction vessel. Record the volume (ml) of KF
reagent used for each titration.
Data and Calculations
Calculate the moisture content of the sample as follows:

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 2-2. DETERMINATION OF CRUDE FAT BY SOXHLET METHOD
An accurate and precise quantitative and qualitative analysis of lipids in food components is
important for accurate nutritional labeling. Determining of whether the food meets the standards
of identity and to ensure the products meet manufacturing specifications.

Lipids are soluble in organic solvents and insoluble in water which is the analytical property
used as the basis of separation of lipids from proteins, water and carbohydrates in foods.

For semi- continuous solvent extraction (soxhlet), the solvent builds up in the extraction
chamber for 5-10 minutes and completely surrounds the sample and then siphons back to the
boiling flask. The fat content is measured by weight loss of the sample or by weight of the fat
removed.

Sample:- meat

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2-2.a. Apparatus

 Analytical balance
 Continuous extraction apparatus(soxhlet type) with an extraction flask of about 150ml
 Clock glass or petri dish-diameter not less than 80mm
 Conical flask-250ml
 Cotton wool – defatted
 Extraction thimble-made of filter paper and defatted
 Desiccator-containing an efficient desiccant
 electrically heated drying oven –adjusted to operate at 1050c
 water bath
 meat mincer
 fluted filter paper

Reagents

 blue litmus paper

 boiling stone
 Hydrochloric acid-approximately 4N solution. Dilute 100ml of concentrated hydrochloric
acid (specific gravity 1.19) with 200ml of water and mix. For the solvent, the residue on
complete evaporation shall not exceed 0.002g/100ml.

2-2.b. Preparation of the sample

 proceed from a representative sample of 200g

 Render the sample uniform by passing it at least twice through the meat mincer and
mixing.
 Keep it in a completely filled air-tight container and store it in such a way that
deterioration and change in composition are prevented.
 Analyze the sample as soon as possible but in any case within 24hrs.

2-2.c. Procedure

 Weigh 3-5g of the minced sample to the nearest 0.001g in to a 150ml conical flask.
 Dry the flask of the extraction apparatus in an oven at 1050c for 1hour.
 Allow the flask to cool in a desiccator.
 Weigh the flask to the nearest 0.001g.
 Add 50ml of HCL to the test portion.
 Cover the conical flask with a small watch glass.
 Heat the conical flask on asbestos wire gauze by means of a glass burner until the
contents begin to boil.
 Continue boiling for 1hr and shake occasionally.
 Add 150ml of hot water.

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  Moisten a fluted filter paper held in a glass funnel with water.
 Pour the hot contents of the flask on to the filter.
 Wash the flask and the wash glass thoroughly three times with hot water in to the filter.
 Wash the filter with hot water until the washings do not affect the color of the blue litmus
paper.
 Put the filter paper on the clock glass or petri-dish.
 Dry for 1hr in an oven at 1050c.
 Allow to cool.
 Roll up the filter paper and insert it in to the extraction thimble.
 Remove any traces of fat from the clock glass or petri-dish. Using cotton wool moistened
with the solvent and also transfer the cotton wool to the thimble.
 Place the thimble in the extraction apparatus.
 Pour the extraction solvent in to the flask of the extraction apparatus from the top of the
condenser.
 Wash the inside of the conical flask used for the disintegration with HCL and the
covering clock glass with a portion of the extraction solvent and add it to the extraction
flask from the top of the condenser.
 The total solvent quantity should be one and a half to two times the capacity of the
extraction tube of the apparatus.
 Heat the extraction flask for 4hours on the heated water bath.
 After extraction, remove the flask containing the liquid from the extraction apparatus and
evaporate the solvent using the heated water bath.
 Evaporate the last traces of the solvent on the water bath, using air blowing if possible.
 Dry the extraction flask for 1hour in the drying oven at 1050c
 After cooling to room temperature in a desiccator, weigh to the nearest 0.001g.
 Repeat this process until the results of two successive weighing do not differ by more
than 0.1% of the sample weighed.
 Cary out two determinations on the same prepared sample.

2-2.d. Calculations:

Total fat of the sample (%) = w1-w2 x 100

W0

Where:-

 W2 – weight of the extraction flask with the dried fat(g)

 W1 – weight of the empty extraction flask with boiling stones(g)
 W0 – weight of the minced sample(g)

The difference between the results of two determinations carried out simultaneously should not
be greater than 0.5g of total fat per 100g of sample.

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Sample : cereals and beans(grind to pass no.30 sieve and store in air-tight bottle).

2-2.e. Procedure

 Heat the extraction flask in an oven at 105-1100c for 1hr.

 Cool to room temperature and weigh (w0).
 Weigh 5g of the sample accurately.
 Transfer it in to an extraction thimble and stopper with defatted cotton wool.
 Dry at 105-1100c in an oven for 1hr.
 Place the thimble in the extraction apparatus.
 Put ethyl ether up to 2/3 of the flask.
 Set up the apparatus and start extraction (extract for 8hours).
 Control the temperature so that 80 condensed drops fall down on the thimble per minute.
 After extraction, when the ether goes down to the flask pick the thimble out with forceps
quickly.
 Set up the apparatus again and after ether comes up collect it (redistill and use it again).
 On the water bath evaporate ether until the ether smell disappears.
 Heat the flask in an oven at 105-1100c for 1hr.
 Cool to room temperature in a desiccator.
 Weigh accurately (wo).

Repeat until the weight is coming up again and use the lowest weight as constant weight (w).
After all water is evaporated, fats start to be oxidized and weight increases.

2-2.f. Calculation

Fat % = weight of fat extracted x 100

Weight of the sample

2-3. DETERMINATION OF CRUDE FIBRE BY HENNENBERG-STOHMANN

METHOD.
Dietary fiber is the indigestible component of food stuff. Adequate consumption of dietary
fiber from a variety of foods helps protect against colony cancer and helps to keep blood lipids
within the normal range thereby reducing the risk of obesity, hypertension and cardiovascular
disease in general. Its determination is also important for labeling requirements.

Sample: cereals and beans.

22
Reagents

 Dilute sulphuric acid – 1.25% (w/v) accurately prepared.

 Sodium hydroxide solution – 1.25% (w/v) accurately prepared.
 Ethyl-alcohol – 95% by volume.
 Petroleum ether - distilling below 600c.
 1% hydrochloric acid.

2-3.a. Preparation of the sample

Grind the sample to pass No. 30 sieve and store in tightly stoppered bottle.

2-3.b. Procedure

 Weigh 2g of the sample.

 Extract with petroleum ether free from fat on a soxhlet apparatus.
 Transfer to 500ml conical flask.
 Pour 200ml of boiled 1.25% sulphuric acid to the flask and boil for 30minutes under
reflux condenser (rotate the flask frequently, taking care to prevent the material from
remaining on the sides of the flask)
 Filter the digest with Pyrex glass filter (crucible type) under a slight vacuum.
 Wash the insoluble matter with boiling water until the washings are free from acid.
 Boil 200ml of 1.25% sodium hydroxide solution.
 Wash the residue on the filter paper back in to the original flask by means of a wash
bottle containing boiling sodium hydroxide solution.
 Boil for 30minutes under reflux condenser.
 Remove the flask from the heat and allow it to stand for one minute.
 Then filter it immediately through Pyrex glass filter.
 Wash the insoluble matter first with boiling water then 1% hydrochloric acid and finally
with boiling water until free from acid.
 Wash it twice with alcohol and three times with diethyl ether.
 Transfer the insoluble matter to the porcelain crucible by means of a wash bottle
containing boiling water.
 Dry the crucible and residue in the oven at 1000c to a constant weight (for about 1hour).
 Cool to room temperature in a desiccator and weigh (w1).
 Incinerate the residue in the crucible in a muffle furnace at 450-500 0c until all the
carbonaceous matter is burnt.
 After the temperature comes down to 2000 c, transfer the crucible to a desiccator.

 Cool in a desiccator and weigh (w2).

23
2-3.c. Calculations

Crude fiber % = (w1-w2) x 100

Where:-

 S – sample weight
 W1 – weight of the crucible and residue before ashing (g)
 W2 - weight of the crucible and residue after ashing (g)

2-4. DETERMINATION OF ASH

Ash refers to the inorganic residue remaining after either ignition or complete oxidation of
organic matter in food stuff. Ash content represents the total mineral content in foods and its
determination is important for nutritional evaluation and it is the first step in preparing a food
sample for elemental analysis. Two major types of ashing are used:- dry ashing and wet ashing.

Dry ashing involves use of a muffle furnace capable of maintaining temperatures of between
500-6000 c. water and volatiles are vaporized and inorganic substances are burned in presence of
oxygen in air to carbon dioxide and oxides of nitrogen. Most minerals are converted to oxides,
sulphates, phosphates, chlorides and silicates. The method must be used if ashing is a
preliminary step for specific elemental analysis.

Wet ashing involves oxidizing organic substances by using acids and oxidizing agents or their
combination. Minerals are solubilized without volatilization.

2-4.1.Ash-rapid (magnesium acetate) method

Reagent

Alcoholic magnesium acetate solution: dissolve 15mg of magnesium acetate(Mg(c 2H3o2)4H2) in

150ml of water and 2ml of glacial acetic acid and make up to 1liter with methyl alcohol.

2-4.1.a. Apparatus/equipment

 Desiccator
 Weighing balance
 Spatula
 Ash dish
 Muffle furnace
 Bunsen burner

24
Sample: wheat flour.

2-4.1.b. Procedure

 Put the porcelain ash dish in an electric muffle furnace maintained at 700 0c and heat for
3-4 hrs.
 Remove the dish, cool in a desiccator to room temperature and weigh.
 Reheat and weigh to a constant weight (w1) g.
 Weigh 3g (+0.01gm) of the sample in to the ash dish and weigh (w2) g.
 Pipette 3ml of alcoholic magnesium acetate solution in such a way that it will spread
evenly over the surface and wet the sample
 After 5minutes heat the contents in the dish with a small flame of burner until smoke
from sample disappears.
 Place the dish in a muffle furnace at 700 0c and heat until incineration is complete (3-
4hrs).
 Stop heating, cool to 2000c and remove the dish and cool in a desiccator to room
temperature and weigh (w3) g.

Liquid sample:

 Weigh in an evaporation dish and dry it up (about 20g)

 Transfer to a previously heated and cooled crucible (w0) with a small amount of water.
 Dry in an oven at 1050c and weigh(w1)
 Then proceed with incineration in a muffle furnace at 550-6000c (2-5 hours)
 Stop heating, remove the dish from the furnace at 2000c to a desiccator
 Cool to room temperature and weigh (w2)

High moisture content sample (fruits, vegetables, fish and meat).

 Weigh in a previously heated and cooled crucible (w0)

 Dry at 1050c in an oven and weigh (w1).
 Proceed with incineration at 550-6000c for 2-5 hours.
 Cool to room temperature in a desiccator and weigh (w2).

2-4.1.c. Calculation

Crude ash % = w2 – w0 x 100

W1 – w0

Where:-

 W2 is weight after ashing.

 W1 is weight before ashing.
25
  W0 is weight of the crucible.

Method 2

Sample

Cereals and beans (grind until they pass no.30 sieve).

Procedure

(A). Obtain a constant weight of containers by heating at 5500c-6000c, cool to room temperature
and weigh accurately (w0).

Repeat until the difference becomes less than 1.0mg (for 30 minutes)

(B). Pre-treatment

1. Liquid sample:-
 Weigh in an evaporation dish and dry it up.
 Transfer to a crucible with small amount of water.
 Dry in an oven at 1050c.
2. High moisture content samples (fruits, vegetables, fish and meat).
 Weigh in a crucible and dry at 1050c or dry and weigh in crucible
3. The sample that is swelled up by heating (cereal flour)
 After weighing in a crucible, char the sample by flame
4. Fats and oils.
 After weighing in a crucible, put the heat on.
(c) Incineration
 Take 2-5g of the sample in to the container (liquid sample 20g).
 Weigh the sample with the container accurately (w1).
 Heat at 5500c-6000c for 2-5 hours.
 Cool to room temperature in a desiccator (close the lid).
 Weigh accurately (w2). Repeat until the difference becomes less than 1.0mg (for 30
minutes heating).

NB. If some black matters (carbon) remain after incineration, carry out the following procedure:-

 Wet ash with small amount of distilled water.

 Crash ash and remained carbon with a glass rod.
 Wash down ash attached on the tip of glass rod in to the crucible with small amount of
distilled water.
 Dry in an oven and incinerate in a muffle furnace oven again.

26
(D). calculation.

Crude ash % = weight of ash remained x 100

Weigh of the sample

= weigh after ashing (w2) – weigh of the crucible (w0) x 100

Weight before ashing (w1)—weight of the crucible (w0)

2-5. DETERMINATION OF CRUDE PROTEIN CONTENT BY SEMI- MICRO

KJELDHAL METHOD.
2-5.1. Principle

In the Kjeldhal procedure, proteins and other inorganic components in a sample are digested
with sulphuric acid in presence of catalysts. The total organic nitrogen is converted to
ammonium sulphate. The digest is neutralized with alkali and distilled in to boric acid
solution. The boric acid anions formed are titrated with standardized acid which is converted
to nitrogen in the sample. The results of the analysis represent the crude protein content of
the food since nitrogen also comes from non-protein components. A reagent blank should be
run to subtract reagent nitrogen from the sample nitrogen.

Reagents

 Concentrated sulphuric acid

 40% sodium hydroxide.
 Mixed indicator
 4% boric acid.
 Standard 0.02N HCL.
 Mixed catalyst (Titanium oxide, copper sulphate, potassium sulphate).

Sample

Cereals and beans

2-5.2. Procedure

Standardization of 0.02N – HCL

Refer to 1-3 and prepare 0.02N HCL and standardize with 0.02N Na2 CO3.

Digestion.

 Weigh about 1g of the sample accurately.

27
  Transfer to a digestion tube.
 Add 1g of the mixed catalyst (potassium sulphate – 5g copper sulphate -0.5g,
titanium oxide – 0.5g).
 Add 10ml of Concentrated Sulphuric acid and rotate the tube until sulphuric acid
soaks in to the sample.
 Carry out a blank without sample at the same time.
 Place the tubes in to the digestion block which is set at 400 0c and heat for 1-1.5hours
until the color changes from brown to clear green. After the digest assumes a
translucent appearance, it has to be digested for additional 30minutes.
 Cool the digest to about 50-600c.

Distillation.

 The content of the flask are liquefied with VE water which helps the absorption of
the heat formed during the solution and neutralization process.
 After neutralization, steam is blown to heat up the sample to the boiling point and
ammonia gas is released.
 The ammonia gas is collected in 4% boric acid (50ml), the distillation is done
until about 50ml of the distillate has collected into the receiver flask.

The ammonia produced reacts with boric acid to give ammonium borate which is directly
titrated with 0.02N hydrochloric acid.

Calculation

% Nitrogen = {C x (V—VBL) x M x 100%}/E

Where:-

C = concentration of titration solution (mole/ml)

V = consumption of titration solution

VBL = consumption of titration solution for the blank

M = molar mass nitrogen in g/mole

E = initial sample weight

Protein content % = % N x F

F = conversion factor

28
3.0 . SPECIFIC VALUE FOR FATS AND OILS

3-1. Saponification value

Saponification is the hydrolysis of fats and oils under basic conditions to afford a glycerol and a
salt of the corresponding fatty acid. (Soap making)

Saponification number is the no. of milligrams of potassium hydroxide required to neutralize the
fatty acids resulting from the complete hydrolysis of 1g of fat. It is used to determine refining
loss by manufacturers.

Reagents

3-1.a. Standardization of Hydrochloric Acid.

 Methyl red indicator.

 Anhydrous sodium carbonate.

3-1.b. Apparatus

 Conical flasks
 Reflux condenser.
 Water bath.
 Burette.
 Pipette.
 Weighing balance.
 Dropper/spatula.

3-1.c. Sample determination

 Alcoholic potassium hydroxide (0.5N).

 Phenolphthalein indicator.
 Standard 0.5N HCL solution.

Sample

Fats and oils

3-1.d. Procedure

3-1.d.1 Standardization of 0.5N HCl by sodium carbonate.

 Refer to 1-3.b.d. and prepare 0.5N HCl and standardize it with 0.5N Na2CO3.

29
3-1.d.2. Sample determination

 Take the weight of a conical flask. (w1).

 Weigh about 2g of the sample.
 Weigh the flask with the sample (w2).
 Add exactly 25ml of 0.5N alcoholic potassium hydroxide to the flask.
 Pipette 25ml of 0.5N alcoholic KOH to another flask for the blank.
 Boil both the sample and the blank under reflux for 30minutes.
 Add 2drops of phenolphthalein and titrate with 0.5N HCl until the pink color disappears.

3-1.d.3. Calculation

Molecular weight of KOH = 56.1, therefore

0.5N – KOH = 28.05g of KOH/liter = 28.05mg/ml

Saponification value (S.V) = (B –A) x f x 28.05

Weight of fat taken (w2 – w1)

Where:-

 B = volume of 0.5N HCl required for the blank (ml).

 A = volume of 0.5N HCl required for the sample (ml).
 F = factor of 0.5N HCl.

NOTE: Saponification value is an indication of the average molecular weight of fat and the value
is constant depending on the kind of oil.

3-1.d.4. Principle

A fat is hydrolyzed by alkali (KOH) to produce a glycerol and the alkali salt of the fatty acid
(soaps). And the excess KOH is back titrated with standard hydrochloric acid.

3-1.d.5. Reference

S.V and I.V of fats and oils.

30
Animal fats plant oils

Name S.V I.V Name S.V I.V

Butter fat 210-245 25-47 corn oil 187-198 117-123

Cow fat 109-202 32-47 cotton seed 189-199 88-121

Pork fat 193-202 46-70 olive oil 185-197 75-90

Sheep fat 192-198 31-47 soya bean 188-196 114-138

Coconut oil 245-271 7-16

3-2. Determination of iodine value

Iodine value refers to the measure of the degree of unsaturation defined as the grams of iodine
absorbed per 100g of the sample.

3-2.a. Principle

A measured quantity of fat /oil dissolved in solvent is reacted with a measured excess amount
of iodine which reacts with the carbon – carbon double bonds.

After a solution of potassium iodide is added to reduce iodine chloride to free iodine, the
liberated iodine is titrated with a standardized solution of sodium thiosulphate using a starch
indicator. The amount of iodine that reacted with the double bonds is used to calculate the iodine
value.

Halogen is added to carbon double bond of fat.

-CH = CH + ICl (from wij’s solution) -CHI – CH-Cl

And excess of ICl is decomposed by potassium iodide

ICl + KI KCl + I2

The liberated iodine is titrated with standard Sodium Thiosulphate solution

2Na2S203 + I2 2NaI + Na2S406

31
3-2.b. Reagents

 1% starch solution(freshly prepared)

 10% potassium iodide (KI)solution
 0.1N potassium dichromate solution(K2Cr2O7)
 0.1N sodium thiosulphate solution(Na2S2O3.5H2O)
 Carbon tetrachloride(CCl4)
 Wij’s solution
 Concentrated HCl

Sample

Fats and oils

3-2.c. Procedure

3.2.c.1. Standardization of 0.1N sodium thiosulphate

 Take 10ml of 10% KI in a flask and add 5ml of concentrated HCl.

 Pipette 25ml of 0.1N K2Cr207 and mix well.
 Add 100ml of distilled water and mix well.
 Titrate with 0.1N Na2S203.
 Just before yellow color disappears, add a few drops of starch solution.
 Continue to titrate until the color changes from blue to green.
 Carry out a blank test without 0.1N K2Cr207(the end point is yellow to colorless)

3.2 .c.2. Sample determination

 Weigh the sample container(w1)

 Weigh about 2g of the sample to the flask
 Weigh the sample and the container (w2).
 Add 10ml of CCl4 and dissolve.
 Carry out blank test at the same time.
 Pipette 25ml of wij’s solution.
 Stopper the flask and place in a dark place for 1hour.
 Add 20ml of 10% KI and 100ml of distilled water and mix well.
 Titrate with 0.1N Na2S203.
 When the color is changed to faint yellow, add a few drops of starch solution.
 Continue to titrate until the blue color is disappeared.

3-2.c.3. Calculation
32
1 mole of Na2S203 reacts with ½ mole I2 = 126.9g of I2, therefore

2ml of 0.1N Na2S203 reacts with 126.9 x 10-4 of iodine

Iodine value = (B – A) x f x 126.9 x 10-4 x 100

Weight of fat (w2-w1)

Where:-

 B = volume of 0.1N Na2S203 required for the blank

 V = volume of 0.1N Na2S2O3 required for the sample
 f = factor of 0.1N Na2S2O3

Note: iodine value is the amount of halogen expressed in grams of iodine that is absorbed by
100g of a fat or oil

3-3. Determination of peroxide value (po. v.)

It is defined as the milli equivalent of peroxide per kilogram of fat as determined in a titration
procedure to measure the amount of peroxide or hydro peroxide groups.it is the milligram
equivalent of iodine that is isolated from KI by 1kg of fat or oil.

3-3. a. Principle

To a known amount of fat or oil, excess potassium iodide is added which reacts with the
peroxides in the sample. The liberated iodine is titrated with standardized sodium thiosulphate
using a starch indicator. The calculated amount of potassium iodide required to react with the
peroxide present is used to determine the peroxide value.

3-3. b. Reagents

 Acetic acid-chloroform mixture(3:2)

 Saturated potassium iodide solution(freshly prepared)
 0.01N sodium thiosulphate solution(Na2S203.5H20)
 1% starch solution

Sample

Fats and oils

33
3-3.c. Procedure

3-3.c.1. Standardization of 0.01N sodium thiosulphate

Refer to 3-2.c.1. Of iodine determination

3-3.c.2. Sample determination

 Weigh the sample container(w1)

 Weigh about 2g of the sample
 Weigh the sample and the container(w2)
 Add 25ml of acetic acid- chloroform mixture and dissolve
 Carry out a blank test at the same time
 Add 1ml of saturated KI solution and mix
 Place in a dark place for 10 minutes
 Add 30ml of water and mix well
 Add about 1ml of starch solution and titrate with 0.01N Na 2S2O3 until the blue color
disappears.

3-3.c.3. Calculation

Peroxide value (Po.V) = (A – B ) x f x 10

Weight of fat (w2-w1)

Where:-

A = volume of 0.01N Na2S203 required for the sample

B = volume of 0.01N Na2S203 required for the blank

F = factor of 0.01N Na2S203

34
3-4. Determination of acid value
Acid value is the number of milli grams of potassium hydroxide that is required to neutralize
free fatty acids in 1g of fat.

It expresses the amount of free fatty acid which is the final product of oxidation. Therefore this
value is a barometer of rancidity of fats or oils.

3-4.a. Reagents

 0.1N- alcoholic potassium hydroxide(KOH)

 Phenolphthalein indicator
 Benzene-ethanol(2:1) mixture(neutralized with 0.1N alcoholic KOH just before using)
 Oxalic acid(for standardization of 0.1N alcoholic KOH)

Sample

Fats and oils

3-4.b. Procedure

3-4.b.1. Standardization of 0.1N alcoholic KOH (refer to 1-2.b.4 )

3-4.b.2. Sample determination

 Weigh the sample container(flask)(w1)

 Weigh about 2g of the sample to the flask
 Weigh the sample and the container(w2)
 Add 100ml of benzene- ethanol mixture and dissolve
 Add phenolphthalein and titrate with 0.1N KOH until pink color appears

3-4.b.3. Calculation

Acid value (A.V) = 5.611 x f x A

Weight of fat (w2-w1)

Where:-

A = volume of 0.1N alcoholic KOH required for the sample

F = factor of 0.1N alcoholic KOH

35
4.0. ACIDITY

4-1. Determination of total titratable acidity

The assay of titratable acidity is a volumetric method that uses a standard solution and most
commonly the indicator phenolphthalein.

In the titration, a standard solution of sodium hydroxide reacts with the organic acids present
in the sample. The normality of sodium hydroxide solution, the volume used and the volume of
the test sample are used to calculate the titratable acidity, expressing it in terms of predominant
acid present in the sample

Reagents

 Standard 0.1N – NaOH

 Phenolphthalein indicator
 Oxalic acid ( for standardization of 0.1N NaOH )

Samples

Fruits (homogenize the sample by a blender or mortar and pestle)

4-1.a. Procedure

4-1.b. Standardization of 0.1N NaOH (refer to 1-2)

4-1.c. sample determination

 Weigh the container

 Take about 25g of the sample
 Weigh the sample and the container accurately
 Transfer to a flask with 50ml of hot water
 Boil for 30minutes under reflux condenser
 Cool and make the volume to 250ml with freshly boiled and cooled water
 Mix well and filter the sample solution
 Pipette 10-100ml of the filtrate
 Add phenolphthalein and titrate with 0.1N sodium hydroxide (if the solution is coloring,
titrate until a pH of 8.3 using a pH meter).

4-1.d. Calculation

T.T.A = titration volume x f x F x 250/V x 100/s

 f = factor of 0.1N NaOH

 F = factor of principle acid(see below)

36
  V = ml of the sample solution taken for titration
 S = g of the sample taken

Note: T.T.A is the amount of acid expressed in grams of the principal acid in 100g of sample.

The principal acid in most fruits is citric acid

CH2-COOH

HO-C-COOH

CH2-COOH Molecular weight = 192.14 equivalent weight = 64.04

1mole of NaOH reacts with 1/3 mole of citric acid, therefore :- 1000ml of 1N citric acid reacts
with 64.04g of citric acid and 1ml of 0.1N NaOH reacts with 0.0064g of citric acid.

4-1.e. Factors of principal acid

Principal acid Factor (F) Sample ( food)

Citric acid 0.0064 Citrus fruits ,strawberry ,pineapple, tomato,
Passion fruits and melon
Lactic acid 0.0090 Milk products
Acetic acid 0.0060 Vinegar
Malic acid 0.0067 Apple, peach, cherry, apricot, unripe fruits
Tartaric acid 0.0070 Grape

5.0. VITAMINS.

5-1. Determination of vitamin c (ascorbic acid) by indophenol method.

5-1.a. Principle

37
Ascorbic acid reduces the indicator dye to a colorless solution. At the end point of titrating an
ascorbic acid containing sample with dye, excess unreduced dye is a rose pink color in the acid
solution.

The titer of the dye can be determined using a standard ascorbic acid solution. Food samples in
the solution can be titrated with the dye and the volume for titration used to calculate the
ascorbic acid content.

Reagents

 10 % Trichloro acetic acid (TCA) solution.

 Standard ascorbic acid solution (1 mg/ml).
 Standard indophenol solution.

Sample

Fresh fruits and vegetables (remove the inedible part and cut in some pieces and start the
procedure immediately).

5-1.b. Procedure

5-1.b.1. Standardization of indophenol solution

1. Pipette 5ml of TCA solution to 3flasks.

2. Add 2ml of standard ascorbic acid solution to the flasks.
3. Titrate with indophenol solution until pink color appears.
4. Pipette 7ml of TCA solution to 3 flasks for the blank.
5. Add same volume of water as the indophenol solution used in 3. Above.
6. Titrate with indophenol solution until pink color appears.

5-1.b.2. Calculation

Mg of ascorbic acid equivalent to 1.0ml of indophenol solution (C) =

mg of ascorbic acid in 2ml of standard solution

Titer of indophenol solution

5-1.b.3. Sample determination

1. Weigh about 5g of the sample accurately.

38
 2. Grind the sample in a mortar with acid washed sand and TCA solution.
3. Transfer the ground sample in to a 100ml volumetric flask quantitatively by using TCA
solution and fill up with TCA solution.
4. Mix well and filter immediately.
5. Take 10ml of the sample solution to flask.
6. Titrate with indophenol solution until pink color appears.
7. Pipette 10ml of TCA solution to flask for the blank.
8. Add same volume of water as indophenol solution used in 6.
9. Titrate with indophenol solution until pink color appears.

5-1.b.4. Calculation

Vitamin C content (mg/100g) = (A- B) x C x 100 x 1 x 100

10 S

 A = volume in ml of indophenol solution used for the sample

 B = volume in ml of indophenol solution used for the blank
 C = mass in grams of ascorbic acid equivalent to 1.0ml of standard indophenol solution.
(5-1.b.2. Calculation)
 S = weight of the sample taken (g).

6.0. PREPARATION OF REAGENTS

Phenolphthalein

1g of phenolphthalein is dissolved in 100ml of 95% ethanol

Methyl red indicator

0.1g of methyl red are dissolved in 100ml of water

Mixed indicator

0.5g of bromocresol green and 0.1g of methyl red are dissolved in 100ml of 95% ethanol.

Fehling solution 1

34.639g of copper sulphate are dissolved in water and made up to 500ml and filtered.

Fehling solution 2

39
173g of sodium potassium tartarate (KNaC4H4O6.4H2O) and 50g of NaOH are dissolved in water
and made up to 500ml.

Tollen’s reagent (ammonia silver nitrate solution)

2N ammonia water are added to 0.2N silver nitrate solution(AgNO 3) until the faint brown
precipitate is formed initially and dissolved again.(prepare freshly).

Iodine solution

5g of iodine and 10g of potassium iodide (KI) are dissolved in 200ml water

Saturated neutral lead acetate solution

About 3g of neutral lead acetate are dissolved in water and made up to 10ml (30% solution).

Standard invert sugar solution

4.750g of pure sucrose are dissolved in 90ml of water and 5ml of concentrated HCL are added.
Afterwards incubated at 65±10C for 20 minutes in a water bath, cooled and diluted to 500ml in a
volumetric flask with water. Accurately 25ml of this solution is neutralized with 10% NaOH and
made up to 100ml in a volumetric flask with water.

This solution contains 2.5mg of invert sugar per 1ml

Methylene blue solution

1g of methylene blue is dissolved in 100ml of water.

Samogy A Solution.

1. 90g of sodium potassium tartarate (KNaC 4H4O6. H2O) and 255g of Na3PO4.l2H2O are
dissolved in 700ml of water.
2. 30g of CuSO4.5H2O are dissolved in 100ml water.
3. 3.5g of KIO3 are dissolved in small amount of water.
4. 1, 2, and 3 are mixed and made up to one liter with water.

Samogy B Solution.

90g of potassium oxalate and 20g of KI are dissolved in water and made up to one liter.

Samogy C Solution

2N H2SO4
40
Samogy D Solution

0.05N Na2S2O3.5H2O (12g/l).

0.5N alcoholic potassium hydroxide

32g of KOH pellets are dissolved in 32ml of water and diluted to one liter with ethanol in a
volumetric flask.

The solution is allowed to stand for a day and filtered.

The filtered solution are put in rubber stoppered bottle and stored at a dark place.

1% starch solution

1g of soluble starch is added to 100ml cold water and stir thoroughly, and heat to boil.

10% - Potassium Iodide Solution.

10g of potassium iodide is dissolved in water and made up to 100ml.

0.1N Potassium Dichromate Solution

4.9037g of potassium dichromate is weighed out in to volumetric flask and dissolved in water to
be made up to 1000ml.1/6 mole of potassium dichromate; (K 2Cr2O7) is equivalent to one gram
atom (one mole) of iodine.

I.K2Cr2O7 + 14HCl +6KI 2crcl3 + 8Kcl + 7H2O+ 3I2

0.1N Sodium Thiosulphate Solution

25g of Na2S2O3. 5H2O is dissolved in water to be made up to one liter

Saturated KI solution

13g of KI is dissolved in 7ml of hot water and cooled.

0.01N Sodium Thiosulphate Solution.

Take 0.1N sodium thiosulphate and dilute 10 times.

0.1N alcoholic potassium hydroxide

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100ml of 0.5N alcoholic potassium hydroxide and dilute to 500ml in a volumetric flask with
ethanol.

10 % Trichloro acetic acid (TCA) solution

100g of TCA is dissolved in one liter of water.

Standard ascorbic acid (1mg/ml)

0.1g of pure ascorbic acid are weighed accurately and made up to 100ml in a volumetric flask
with 10% TCA solution.(pure ascorbic acid powder is stored in a desiccator kept away from
sunlight).

Standard indophenol solution

1. 42mg of sodium bicarbonate are dissolved in 50ml of water.

2. 50mg of sodium 2,6 dichlorophenol indophenol (indophenol sodium) are dissolved in
solution 1 completely, it is then diluted to 200ml with water and filtered in a glass
stoppered brown bottle. Keep the solution in a refrigerator.

Every time just before experiment, standard indophenol solution must be standardized.
Indophenol solution can be stored 2 weeks only.

Indophenol powder is stored in a desiccator over soda lime.

1N Hcl solution

Concentrated HCl is diluted 10 times (83ml/liter).

1M phosphoric acid

115g of phosphoric acid is made up to 1liter in a volumetric flask with water.

0.05N sodium chloride

About 2.92g of NaCl is weighed out to the nearest 0.1mg (W 1g) and dissolved in water to be
made up to 1000ml.

Factor of 0.05N sodium chloride (f) = W1

2.9225

0.05N silver nitrate

Exactly 8.5g of silver nitrate is taken in to 1000ml volumetric flask and dissolved in water to be
made up to the mark and keep in a brown bottle.
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0.3% 0- phenanthroline

0.3g of o- phenanthroline is dissolved in 100ml of water at 700c, stored in dark cold place.

Iron standard solution

1. 1000ppm solution
3.512g of F3 (NH4)2 .6H2o is dissolved in about 300ml of water and add 5ml of
concentrated HCL.
Transfer to a 500ml flask and made up to the mark with water.

2. 100ppm solution

10ml of Fe 1000ppm solution is pipetted in to a 100ml volumetric flask and made up to the
mark.

Bromo phenol blue (BPB) indicator.

0.05g of BPB is dissolved in 1.5ml of 0.05N- NaOH by grinding in a mortar and then 120ml of
water are added to dilute.

25% sodium citrate

50g of sodium citrate (Na3C6H5O7.2H2O are dissolved in 200ml of water.

0.5M methanolic potassium hydroxide

2.8g of KOH are dissolved in 100ml of methanol.

7.0. INSTRUMENTAL ANALYSIS

7-1. Spectrophotometric methods

Spectroscopic methods normally measure the interaction between electromagnetic radiations
and analyte atoms/molecules. Spectroscopy deals with the production, measurement, and
interpretation of spectra arising from the interaction of electromagnetic radiation with matter.
There are many different spectroscopic methods available for solving a wide range of analytical
problems.

The methods differ with respect to the species to be analysed (such as molecular or atomic
spectroscopy), the type of radiation–matter interaction to be monitored (such as absorption,
emission, or diffraction), and the region of the electromagnetic spectrum used in the analysis.

A ray of light can either interact with molecules in substance or pass through. When
electromagnetic radiation passes through a substance some of it is absorbed by the interactions
43
and some of it is transmitted. The degree of absorption depends on the concentration of the
atoms.

When a light of intensity I o is made incident upon a closed medium, with a length b which
contains atoms of concentration C. the intensity of light will be reduced to I. the decrease in
radiant energy of a beam of monochromatic light is proportional to the intensity of the beam and
the amount of absorbing species and the thickness of the path. This is known as the Beer’s
lambert law.

The relationship will be I = I0 e-kbc or -log I/Io = Kbc = absorbance. The term –log I/Io is called
absorbance.

The equation tells us that absorbance is linearly proportional to concentration of the atoms.

The concentration of the elements in an unknown solution can be determined from the
absorbance by means of a calibration curve using standards of known concentration.

Determination of tannins in tea leaves using UV-visible spectrophotometer

Reagents

 Folin dennis reagent

 Sodium carbonate

 Tannic acid

Equipment.

 UV- Vis spectrophotometer

 Homogenizer.

Methodology.

The tannin content is determined by Folin Denis colorimetric method described by Kirk and
Sawyer (1998).

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Preparation of the sample

About 5g of the sample is dispersed in distilled water (20ml) and shaken. The mixture is allowed
to stand for about 30minutes at 280c before it is filtered through a Whatman No. 42 filter paper.

Preparation of standards

1. Standard tannin solutions containing 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg\ml tannic acid are
prepared (50ml).

2. 2ml of the sample extract and tannic acid standards are dispersed in 50ml volumetric
flasks.

3. 2ml of Folin dennis reagent is added to each flask followed by 2.5ml of saturated sodium
carbonate solution.

4. The content of each flask is made to 50ml with distilled water and allowed to incubate at
280c for 90minutes.

5. Distilled water is put in a separate flask for the blank to calibrate the instrument to zero.

Their respective absorbance is measured in a UV-Vis Spectrophotometer.

Mg/100g = Y/m × 1000

SW 100

Where:-

 Y = absorbance.

 M = gradient of the standard curve.

 SW = sample weight.

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7-2. Colorimetry
Colorimetry is the science of color measurement which is widely employed in commerce,
industry and the laboratory to express color in numerical terms and to measure color differences
between specimens.

Procedure

The color of the sample will be determined using a color meter. The color of the juice will be
measured at four regions along the midsection spaced 90 0 apart. The reflected color L*, a* and
b* values will be determined directly, results will be tabulated in terms of L* values and
calculated hue angle, Chroma values E values.

Notes:

 The three attributes of color are hue, Chroma, and value

 Hue describes a visual sensation according to which an area appears to be similar to one
or proportions of two of the perceived colors red, yellow, green and blue. The hue angle
is thus the actual color.

 Chroma describes the intensity of a fundamental color with respect to the amount of
white light in the background.

 Value is an indication of the color lightness i.e. color intensity changing from white to
dark.

Chroma value = (a* + b*) 0.5

Hue angle = tan-1(b*/a*)

Color difference ( E) = { (L*0 – L*)2 + (a*0 – a*)2 + (b*0 – b*)2}0.5

L* =value (0= black = high value, 100 = white)

Stage is taken as the stage 0 in color calculations.

The results will be expressed in tables and data discussed with emphasis on the color difference
between different concentrations of juices.

Example:

Sample L* a* b* Chroma Hue angle E

1 70.88 -18.3 36.8 41.1 116.4

46
2 40.2 26.8 24.55 36.34 42.51 56

7-3. Rheometry
Rheology is the study of deformation (of solids and semi-solids) and flow( of fluids).generally
rheological properties are important in the design of food processing operations and have a
bearing on food stability, functionality, acceptability and quality control. Fluid flow can either be
Newtonian or non-Newtonian. Newtonian fluids exhibit a linear relationship between shear rate
and shear stress, that is to say, the viscosity (shear stress divide by shear rate) of a Newtonian
fluid is a single value invariant of the shear rate. Non-Newtonian fluids show non-linear
relationship between shear stress and shear rate. Therefore the viscosity of a Non-Newtonian
fluid is depended on the shear rate. For some fluids, viscosity may decrease with increase in
shear rate (shear thinning) e.g. yoghurt, tomato ketchup. In other fluids, viscosity increases with
increase in shear rate (shear thickening) e.g. cassava starch slurry. The viscosity of a Non-
Newtonian fluid is specified at a given shear rate and is called apparent viscosity. Most fluids
and semi-solid foods exhibit shear thinning behavior. However certain foods like mayonnaise
require a minimum shear stress called the yield stress to initiate flow. Once the yield stress is
exceeded, flow properties may be reminiscent of Newtonian systems. Such foods may exhibit
both elastic and viscous properties and they are said to be viscoelastic

Objectives
 To determine the apparent or coefficient of viscosity of fluid foods using a single cylinder
viscometer.
 To determine the variation of viscosity with rotational speed.
Materials
 Rheometer
 Yoghurt
 Fruit juice concentrate
 Milk
 250 and 500ml glass cylinders
 Mercury in glass thermometer

Method

47
  With the help of a lab technician, assemble the Rheometer on a flat surface and ensure
that it is horizontal by observing the foam level.
 Fix a suitable probe and ensure the scale pointer is reading zero (tap gently on the sides to
zero the pointer).
 Pour suitable quantity of sample (sufficient to immerse the probe up to the indicated
mark) in to the beaker and set it directly below the probe.
 Clamp the lever and leave it in position as you lower the probe in to the sample
 Set the rotational speed N
 Turn the synchronous motor to ON
 Unclamp the lever, whereupon the indicator needle shifts with respect to the rotational
dial
 Allow the indicator needle to stabilize(one or two rotations depending on the sample
 Clamp the lever and turn OFF the motor, ensuring the scale remains in view
 Read and record the torque(M) as indicated by the pointer on the scale
 Remove the probe, wash and re-attach
 Change the rotational speed and repeat the experiment in duplicate for each of the
samples
 Set the speed at a constant value and vary the shearing time
 Record the change in torque with shearing time for each of the experiments
 After finishing the experiments, thoroughly clean the apparatus, return the indicator
needle to the zero position and clamp the lever
Results and discussion
 Present your results graphically
 Discuss the variation of viscosity or torque with shear rate for each of the samples. Is the
samples Newtonian, shear thinning or shear thickening?

48