A LABORATORY MANUAL

ON

ANALYSIS OF MILK LIPIDS (GHEE)

Dr. Vivek Sharma Dr. Sumit Arora

Senior Scientist Senior Scientist

Dr. Darshan Lal Dr. (Mrs.) Balbir Kaur Wadhawa

Principal Scientist Principal Scientist

DAIRY CHEMISTRY DIVISION

NATIONAL DAIRY RESEARCH INSTITUTE
(I.C.A.R)
KARNAL – 132001 (HARYANA), INDIA
2011-2012
No. part of this publication can be reproduced without the prior
permission of Director, National Dairy Research Institute.

Cover Design: Vivek Sharma

Published by: Director,

National Dairy Research Institute
(Deemed University), Karnal – 132001, Haryana, INDIA
Tel: 91-0184-2252800, Fax: 91-0184-2250042

Printed at: Intech Printers & Publishers

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 PREFACE

The fat content of milk is of utmost economic importance because

milk is sold on the basis of fat. Milk fat is a source of fat soluble vitamins

A, D, E & K and essential fatty acids linoleic and arachidonic acids,

phospholipids and cholesterol. Today most undergraduates and

technicians working in the area of dairy processing require a working

knowledge of dairy chemistry and especially the practical aspect of the

subject. The manual therefore should be suitable for many

BSc/BTech/MSc/MTech students.

The manual consists of detailed information on several

procedures including necessary reagents and apparatus essential for the

analysis of milk lipids/ghee in addition to theoretical principles and

information on them. Evidently, this work is a compilation of different

well established methods of analysis recorded in different official

publications, textbooks etc. However, majority of the methods described

here have actually been followed by the authors themselves and by their

students.

YEAR-2012 Dr. Vivek Sharma

Dr. Sumit Arora
Dr. Darshan Lal
Dr. (Mrs). B.K. Wadhwa
 Acknowledgement

Support and encouragement from Director, National Dairy Research

Institute, Karnal is thankfully acknowledged. The authors are also

thankful to Joint Director (A) and Registrar, Dairy Science College, NDRI

for providing funds under the scheme on strengthening the post graduate

educational programme at ICAR institutes.

 CONTENTS

S.No Name of Experiment Page

No
1 Determination of Butyro Refractometer (B.R) reading of 1-3
ghee.
2 Determination of slip point of ghee. 4-5
3 Determination of Free Fatty Acid (FFA) value in ghee. 6-9
4 Determination of peroxide value in ghee. 10 -15
5 Determination of thiobarbituric acid (TBA) value in ghee. 16 -17
6 Determination of total carbonyl content in ghee by flask 18 - 20
method.
7 Determination of Iodine Value of ghee by the Wijs’ method. 21 - 26
8 Estimation of unsaponifiable matter in ghee. 27 -29
9 Colorimetric estimation of cholesterol in ghee by indirect 30 - 35
method.
10 Colorimetric estimation of total cholesterol in ghee by direct 36 - 39
method.
11 Estimation of cholesterol in ghee using enzymatic diagnostic 40 - 42
kit
12 Determination of Reichert Meissl (RM) and Polenske (PV) 43 - 48
Values of ghee.
13 Estimation of BHA content in ghee. 49 - 53
14 Determination of Vitamin D in ghee. 54 - 56
15 Estimation of vitamin A in ghee. 57 - 60
16 Determination of Vitamin E in ghee. 61- 65
17 Estimation of phospholipids in ghee and ghee- residue. 66 - 70
18 Analysis of fatty acids of ghee by Gas liquid 71 - 79
Chromatography.
Experiment 1: Determination of Butyro Refractometer (B.R) reading of
ghee.

General: Ghee exhibits a B.R reading about 43 at 40ºC, corresponding to a

refractive index of 1.4545. Most vegetable oils have a B.R value of about 65 i.e.
refractive index of 1.4754.
In the homologous series of saturated fatty acids viz; butyric to stearic
acid, refractive index increases steeply among the lower numbers and flattens
out at higher chain lengths. A double bond elevates the refractive index;
hence stearic acid (saturated) has lower refractive index than oleic acid
(unsaturated, monoenoic), which in turn has lower values than linoleic acid
(unsaturated, dienoic). An increase in B.R. in ghee is caused by a decrease in
lower chain fatty acids and by an increase in either higher chain or
unsaturated fatty acids.
B.R. reading is indirectly proportional to temperature i.e

∞ 1
BR
Temperature

The B.R reading decreases with a rise and increases with a fall in temperature.

Therefore, if the temperature is not exactly at 40°C, correction factor is added

to the observed reading for each degree above or subtracted for each degree
below 40°C, where :
correction factor = 0.55 BR / ºC
Normally the temperature of observation shall not deviate by more than ±
2°C.
Determination of B.R. reading can be used to check the adulteration of milk
fat, particularly if adulterated with vegetable oils.

Principle: Butyro Refractometer reading (BR reading) measures the index of

refraction between air and liquid fat, and varies with the nature of fat. The

1
instrument is calibrated in Butyro- Refractometer degrees instead of the usual
absolute refractive indices, the two values being inter-convertible as follows:
BR in ghee = 42 + Factor
i.e Factor = (observed refractive index – 1.4538)

Refractive index = 1.4538 + Factor (observed BR – 42)

i.e Factor = (observed BR – 42)

Apparatus and Glass ware:

Butyro Refractometer, Circulatory water bath maintained at 40ºC, Glass rod,
Beaker, Tissue paper.

Circulatory water bath

Chemicals and Reagents:

Ethyl alcohol or toluene or petroleum ether
Procedure:
i) Take the sample fat in a beaker and melt. Filter the molten fat through a
Whatman No 1 filter paper to remove any debris.
ii) Set the temperature of the instrument at 40 + 1ºC by adjusting the
thermostat of circulatory water bath.
iii) Clean the prism of the instrument with cotton plug/ tissue paper
moistened with Ethyl alcohol or toluene or petroleum ether and allow it to
dry. Repeat cleaning step between readings.

2
iv) Place 1-2 drops of sample on the lower prism. Close the prisms and wait
for 2-5 minutes to ensure that the sample has attained the constant
temperature of the instrument. Now, adjust mirror until it gives sharpest
reading. If reading remains indistinct after running constant temperature
water through instrument for sometime, test sample is unevenly distributed
on prism surfaces.
Calibration of the Instrument:
The instrument is calibrated with a glass prism of known refractive index. The
correctness of the instrument shall be tested before taking readings by
carrying out tests with fluid of known refractive index. The fluid can be water
at 20°C whose theoretical refractive index is 1.3330 at 20°C. Usually suppliers
of the instrument also give the standard fluid of known B.R reading for this
purpose.
Light Source:
Normal day light or if the refractometer is equipped with a compensator, a
tungsten lamp. The preferred light source for accurate results is the
monochromatic light of a wave length of 589.3 mµ ( The mean of the D- lines
of Sodium).
Observations:
S.No Type of fat Observed B.R at Corrected B.R at
40ºC 40ºC

3
Experiment 2: Determination of slip point of ghee.

Principle: Anhydrous milk fat (ghee) consists of a heterogeneous mixture of

triglycerides. Hence, it does not have a sharp melting point. The melting point
of milk fat ranges from -40º C to 40º C. Therefore, for practical purposes, it is
possible to obtain a temperature range at which a column of fat in a capillary
tube starts melting and when it melts completely. The temperature at which
ghee just starts melting is known as slip point.

Apparatus and Glass ware: Melting point tubes (Capillary tubes – thin
walled, uniformly bored, open at ends, length 50-60mm, internal diameter 0.8-
1.1 mm and outer diameter 1.2 – 1.5mm.
Thermometer, melting point apparatus (Beaker with a side tube heating
arrangement), Gas burner or spirit lamp.

Procedure:
i) Melt the given fat sample and filter through filter paper to remove any
suspended impurities or particles and last traces of moisture.
ii) Mix the sample thoroughly. Insert a clean melting point tube in the molten
fat in such a manner that a column of fat of 10mm length is formed.
iii) Place the filled melting point tube in a horizontal position in a freezer for
at least 1 hr.
iv) Fill water at 10ºC in the beaker of melting point apparatus. Hang the
thermometer in such a way so that the bulb of the thermometer is dipped in
water at least 30mm below the surface of the water.
v) Take out the melting point tube from the freezer and attach it to the
thermometer in such a manner that the lower end of the melting point tube is
even with the bulb of the thermometer.
vi) Now, start heating the water in the beaker gently and increase the
temperature to 25ºC @ 2ºC/ min. After attaining 25ºC further increase the

4
temperature @ 0.5ºC/min. The melting point tube is observed constantly for
any change in the appearance of fat column and any upward movement in
the column of fat. The temperature at which fat column slips is noted as slip
point “S”.
vii) Note down the temperature at which ghee sample starts melting as “T1”
and “T2” when ghee melts completely.

Observations:
S. No Sample Observed temperature ºC

Initial temp Final Melting Slip

point
(T1). temp. temperature
Range
(T2)

5
Experiment 3: Determination of Free Fatty Acid (FFA) value in ghee.

General: Free fatty acids (FFA) are the unbound form of fatty acids present in
the oil or fat samples. These have significance for determining the quality of
the milk fat because they increase the fat’s susceptibility to oxidation and can
contribute to bitter or soapy flavours. Presence of more FFA in any fat rich
product indicates the occurrence of hydrolytic rancidity. Free fatty acids in a
fat sample (or fat extracted from a milk product or food product) can be
determined by titration. The FFA value is then expressed as % of a fatty acid
predominant in the product being tested. Frequently, values are expressed as
% oleic acid for butter fat, tallow or soybean oil. For coconut oil or other oils
that contain high levels of short chain fatty acids, FFA may be expressed as %
lauric acid. The value is a measure of the amount of fatty acids which have
been liberated by hydrolysis from the glycerides due to the action of moisture,
temperature and/or lypolytic enzyme lipase. Fresh ghee has FFA content in
the vicinity of 0.2% oleic acid. As per FSSAI standards the maximum
permitted FFA level in ghee is 3% oleic acid.

Principle of the method

Oils and fats can be directly used for determining FFA after taking a
representative sample from the whole lot. However, for milk product /food
sample the lipids are first extracted with the help of solvents. The solvent is
then evaporated and lipids are then dissolved in neutralized alcohol. The
contents are then warmed and titrated with an alkali using phenolphthalein
as an indicator until a pinkish color appears.

Reactions:

O O
‫װ‬ ‫װ‬
R – C – OH + NaOH R – C – ONa + H2O
(FFA)

6
Apparatus and Glass ware:

Water bath (boiling), Beaker 250 ml capacity, Conical Flask (250ml).

Chemicals and Reagents:

Ethanol (Ethyl alcohol):- 95% ethanol.

Phenolphthalein indicator solution: - Weigh 1.0 gm phenolphthalein and
place the powder in a 100 ml volumetric flask containing about 50ml of 95%
ethanol. Stopper and shake vigorously for a few minutes, then add 20ml more
ethanol and shake until a clear solution is formed and make the volume to 100
ml.

Neutralized ethanol: Titrate 95% ethanol with a few drops of standardized

alkali in the presence of phenolphthalein indicator until a faint pink colour is
obtained that persists for 15 – 30 sec.

Standard aqueous potassium hydroxide or sodium hydroxide solution (0.1

or 0.5 N): The solution should be colourless and stored in an amber color
glass bottle.

Procedure:
i) Mix the melted fat thoroughly before weighing. Filter it using Whatman No
1 filter paper to get rid of any suspended particles.

ii) Weigh accurately about 5 to 10 g of molten sample in a 250 ml conical flask

and add 50 ml to 100 ml of freshly neutralized ethanol and about one ml of
phenolphthalein indicator solution.

iii) Boil the mixture for about five minutes on a boiling water bath and titrate
while hot against standard alkali solution shaking vigorously during the
titration.

7
Note: The weight of the fat taken for the estimation and the strength of the alkali used
for titration shall be such that the volume of alkali required for the titration does not
exceed 10 ml.

Calculation:

FFA ( % oleic acid) = ml NaOH used X Normality of NaOH X 2.82

Weight of sample

Derivation:
1L of 1.0N sodium hydroxide ≡ 1L of 1.0N Oleic acid.

Mol. weight of Oleic acid = 282.

Hence
1N oleic acid contains 282gm of oleic acid /L of solution

Hence
1 ml of 0.1N sodium hydroxide ≡ 282
------------------------
1000 X10
= 0.0282 gm of oleic acid.

Suppose “T” ml of 0.1N NaOH is used for W gm of sample

Then,
T ml of 0.1N sodium hydroxide ≡ 0.0282 X T gm of oleic acid.

So
W gm of sample contains = 0.0282T gm of oleic acid.
1 gm of sample contains = (0.0282T)/W gm of oleic acid.
100 gm of sample contains = [(0.0282T)/W]X100

% FFA = 2.82T/W

8
Observations:

S.No Sample Volume of 0.1N NaOH used Weight of

/Blank sample % FFA

Initial Final ( gm)

Vol. Used
reading reading.

9
Experiment 4: Determination of peroxide value in ghee.

General: Peroxides (R-OOH) are the primary reaction products formed

during the initial stages of oxidation, and therefore peroxide value gives an
indication of the progress of lipid oxidation. One of the most commonly used
methods to determine peroxide value utilizes the ability of peroxides to
liberate iodine from potassium iodide.

Definition: Peroxide value is the measure of hydroperoxides formed during

oxidation of lipids. It is expressed in many ways (i) as mmol active oxygen (i.e
peroxide)/ 2Kg sample. (ii) as meq active oxygen / Kg sample (iii) as mmol
active oxygen/ Kg sample and (iv) number of milligram-equivalents of
oxygen / Kg sample. (v) ml of 0.002 N sodium thiosulfate per g of sample.

The recent unit of expression is mmol active oxygen (i.e peroxide)/ 2Kg
sample. Other units can be converted to this value by following means:

mmol active oxygen/ 2Kg sample = i) 1 X meq active oxygen/Kg sample

ii) 0.5 X mmol active oxygen/ Kg

sample
iii) 8 X milligram-equivalents of
oxygen per kilogram of
anhydrous milk fat.

Principle of the method

The lipid is dissolved in a suitable organic solvent and an excess of potassium

iodide is added. Once the reaction has completed, the amount of peroxides
that has reacted can be determined by measuring the amount of iodine
formed. This is done by titration with sodium thiosulfate and a starch
indicator. The amount of sodium thiosulfate required for titration in the
reaction is related to the concentration of peroxides in the original sample.

10
Reaction:

ROOH + KI (in excess) ROH + KOH + I2

I2 + starch + 2Na2S2O3 2NaI + starch + Na2S4O6

(blue colour) (colourless)

Apparatus and Glass ware: Glass stoppered Erlenmeyer flask (250 ml), Glass
pipette (1-2 ml), Graduated class-A glass burette (10 or 25 ml), Oven 100 –
110 º C.

Chemicals and Reagents:

Sodium thiosulfate pentahydrate (Na2S2O3.5H2O, M. Wt= 248.17) AR grade.

Sodium Carbonate (Na2CO3).

Potassium Iodate (KIO3) as primary standard. Before use heat the primary
standard in an oven maintained at 110º C / 1hr and then cool in a desiccators.

Potassium iodide (KI), iodate free

Hydrochloric acid (6.0 M)

0.1 N Sodium thiosulfate solution: Add 25.0 gm of sodium thiosulfate

pentahydrate into previously boiled & cooled distilled water. Stir the contents
until all crystals are dissolved. Make up the volume to 1000.0 ml. Store the
contents in a dark coloured airtight bottle with a guard tube filled with soda
lime. This is approximate 0.1 N solution of sodium thiosulfate. After storing
the solution for about two weeks, filter, if necessary, and standardize as
follows:

Weigh accurately 0.10 - 0.15 gm of the primary standard into a 250 ml

Erlenmeyer flask. Dissolve the contents in 75 ml of distilled water and add 2.0

11
gm of iodate free potassium iodide. Then add 2.0 ml of 6.0 M HCl and titrate
immediately with 0.1 N sodium thiosulfate solution until the solution
becomes yellow, shaking constantly and vigorously during titration. Add 0.5
ml of starch indicator solution. Blue / violet color will appear. Continue
titration with (0.1N) sodium thiosulfate drop wise until the blue/violet colour
disappears. Record the total volume of added sodium thiosulfate solution.
Repeat the titration twice/thrice to record the exact volume of (0.1N) sodium
thiosulfate used. Carry out the blank titration in a similar manner, without
taking the primary standard.

Calculate the normality of the sodium thiosulfate solution as follows:

N= ( 28.037XW)/(S-B)

Where:

W= weight in grams of KIO3

S= Volume (ml) of sodium thiosulfate required to titrate the sample

B= Volume (ml) of sodium thiosulfate required to titrate the blank

0.002 N Sodium thiosulfate solution: Prepare freshly by diluting the

previously prepared 0.1N Sodium thiosulfate solution

Saturated Potassium iodide solution: Boil some water for about 5 min and
allow cooling. To a portion, add enough potassium iodide to ensure a
saturated solution (10.0 gm KI in 6.0 ml water). Store it overnight to ensure
that un-dissolved crystals remain there. To test stability, dilute 0.5 ml
saturated KI solution in 30.0 ml of 3:2 (V/V) acetic acid/ chloroform solution
and then add 2 drops of 1% starch indicator solution. If a violet color appears
that requires more than 1drop of 0.1N sodium thiosulfate solution to
discharge, discard the iodine solution and prepare a fresh one.

12
1% (W/V) starch indicator solution: Make a paste of 1.0 gm soluble starch in
30.0 ml of water. Transfer the paste to 80.0 ml of boiling water and heat until a
clear solution is formed. Cool the contents and store in a tight stoppered
bottle. It can be stored for 2-3 weeks at 4ºC.

Mixed Solvent: 2:1 (V/V) glacial acetic acid/ chloroform solution

Procedure:

i) Accurately weigh 1.0 ± 0.05 gm of molten fat into a 250 ml glass stoppered
Erlenmeyer flask or weigh suitable quantity of sample in such a manner that
the amount of 0.002 N sodium thiosulfate solution used in titration does not
exceed 10 ml.

ii) Add 20.0 ml of mixed solvent and swirl flask until fat is dissolved. Add 1
gm of solid potassium iodide (KI) or 0.5 ml of saturated solution of potassium
iodide. Heat the contents of the flask to boiling within 30 seconds preferably
on steam bath. As solvent vapours begin to escape from the hole of the
stopper, close the hole with finger or glass rod of the size of the hole. Now,
place the flask quickly under tap water to cool the contents. Add 30 ml of
distilled water to the flask and shake the contents gently.

iii) Gradually add 0.002N standardized sodium thiosulfate solution from a

graduated burette and titrate until yellow colour has almost disappeared.
Shake the contents constantly and vigorously. Add 0.5 ml of starch indicator
solution to the above flask. Violet color will appear in the solution. Continue
titration, shaking flask vigorously to liberate all iodine from the chloroform
layer. Towards the end point, add sodium thiosulfate solution dropwise until
the violet colour disappears. Record the total added sodium thiosulfate
volume. Conduct a simultaneous blank, without fat.

Calculations: Peroxide value (PV) as miliequivalents of peroxide oxygen per

Kg of sample is :

13
Peroxide Value = (S-B)X N X 1000 X 8

Expressed as meq active oxygen / Kg sample.

Where,

S= Volume (ml) of sodium thiosulfate required in titration of sample

B= Volume (ml) of sodium thiosulfate required in titration of blank.

N= exact normality of sodium thiosulfate solution.

W= Weight (gm) of sample.

Results may be expressed in milli molecules of oxygen per Kg of fat, where

peroxide value is divided by 16.

Results may be expressed in milli equivalents of oxygen per Kg of fat, where

peroxide value is divided by 8.

BIS method of expressing the peroxide value is by calculating the amount of

0.002 N sodium thiosulfate used in the titration. From peroxide value, the
quality of ghee can be judged as follows:

Peroxide value in terms of volume in ml Quality of ghee

of 0.002 N sodium thiosulfate used per
gm of fat
< 1.5 Very good
1.6 – 2.0 Good
2.1 – 2.5 Fair
2.6 – 3.5 Poor
3.6 – 4.0 Not acceptable
Limitations: Peroxide value is one of the most widely used tests to check the
oxidative rancidity of milk fat. However, there are a number of problems with
the use of peroxide value as an indicator of lipid oxidation. Firstly, peroxides

14
are primary products that are broken down in the latter stages of lipid
oxidation. Thus, a low value of peroxide value may represent either the initial
or final stages of oxidation. Secondly, the results of the procedure are highly
sensitive to the conditions used to carry out the experiment, and so the test
must always be standardized. This technique is an example of a measurement
of the increase in concentration of primary reaction products,

Note: If high oxidation is suspected then less quantity of sample can be used ( 0.3 –
2.0 gm)

Observations:
S. No Sample/ Volume of 0.002N Sodium Inference
Blank thiosulfate used ( Quality of the sample)

Initial Final
Vol. used
reading reading

15
 Experiment No 5. Determination of Thiobarbituric acid (TBA) value in
ghee.

General: TBA is the most widely used test for measuring the extent of lipid
oxidation. This test is preferred due to its simplicity and is reported to
correlate well with sensory scores. The test is very sensitive and useful
method for quantifying lipid oxidation. As a result of secondary oxidation of
polyunsaturated fatty acids, malonaldehyde is formed in relatively small
amounts. Malonaldehyde (MDA) reacts with thiobarbituric acid to yield a red
pigment which is quantified at 532 nm. Other products of lipid oxidation such
as saturated aldehydes, 2-alkenals and 2,4- alkadienals also react with the
TBA reagent.
Principle: The test is based on the condensation of two molecules of TBA with
one molecule of malonaldehyde resulting in the formation of a red colored
complex (chromagen) in acid medium, which has absorption maxima at
532nm. Quantitatively it may be determined by using a standard of acetal
tetraethoxy propane, which hydrolyzes under acidic conditions to generate a
source of unstable malonaldehyde.
Chemical Reaction:

+ 2H2O

Thiobarbutyric acid Malonaldehyde TBA- MA complex

(TBA (MA) ( Red color complex

Apparatus and Glass ware: Water bath, Glass stoppered test tubes,
volumetric flask (100ml), Glass rod, Funnel.

16
Chemicals and Reagents:
i) Carbon tetrachloride (CCl4) density 1.59.
ii) Glacial Acetic acid.
ii) TBA reagent: Take 0.76 gm of TBA. Transfer it in a 100ml volumetric flask.
Add some distilled water and try to dissolve it by warming on a water bath
and filter. Take the filtrate and mix with glacial acetic acid in a ratio of 1:1.

Procedure:
i) Weight accurately 3.0 gm of molten fat in a glass stoppered test tube.
ii) To this add 10.0ml of carbon tetrachloride solvent and 10.0 ml of TBA
reagent.
iii) Shake the contents vigorously giving about 125 oscillations/min. for 4
minutes. Leave the test tube undisturbed to obtain clear two layers separated.
iv) Remove about 5.0 ml aqueous portion of the separated upper layer with
the help of a pipette and transfer to a test tube.
v) Incubate the contents in a boiling water bath for 30 minutes.
vi) Simultaneously carry out a blank test and take reading at 532 nm. using a
spectrophotometer.

17
Experiment 6: Determination of total carbonyl content in ghee by flask
method.

General: Total carbonyl content is also a measure of oxidative rancidity in

fats. Fresh milk fat should have a value of 4-8 μmol/gm of fat. Carbonyls are
responsible for pleasant flavour of milk fat. In case of cow milk fat carbonyl
value is slightly lower than buffalo milk fat. Volatile carbonyls are formed
from degradation of hydro -peroxides.

Principle: Carbonyls on reacting with 2,4-dinitrophenyl hydrazine give

corresponding dinitrophenyl hydrazones of yellow colour. The intensity of
colour is proportional to the concentration of carbonyls present in a given fat
sample. These hydrazones have their absorption maxima at 340 nm (UV-
range). Depending upon absorbance the carbonyl content is calculated.
Chemicals and reagents: Orthophosphoric acid (87%), 2,4-
dinitrophenylhydrazine, Cellite- 545, carbonyl free benzene and carbonyl free
hexane.

Chemical Reaction:

NHNH2 NHN= C
NO2 NO2
+ C=O + H2O
NO2 NO2
2,4- Dinitrophenyl Carbonyl 2,4- Dinitrophenyl Water
Hydrazine Hydrazone

Apparatus and Glass ware:

Pestle and Mortar, Spectrophotometer, Glass column, Glass rod having
flattened ends, Flasks 25ml capacity.

18
Procedure:
A. Preparation of carbonyl free hexane/ Benzene: Take 1.0 liter of hexane/
Benzene in a round bottom flask. Add 5.0 gm of 2, 4-dinitrophenyl
hydrazine and 1 gm of TCA ( To provide acidic pH). Reflux the
contents for 3-4 hrs. Distill the contents and collect the distillate.
B. Take 6.0 ml of 87% orthophosphoric acid in a pestle & mortar. To this
add 0.5gm of 2, 4-dinitrophenyl hydrazine (2, 4 -DNPH) and ground
with the help of mortar till its dissolution. Add 10 ml of distilled water
and reground to get a clear solution (on addition of water some DNPH
may precipitate).
C. Preparation of column material:
i) Activate adsorbent Cellite - 545 by heating at 120ºC for 12 hrs.
ii) Add activated Cellite - 554 to the contents of step B and grind to get a
homogeneous mass.
iii) Fix a glass column to a proper stand. Put some glass wool at the base
of the column. Add a few ml of carbonyl free hexane and close the
opening of the column. Now transfer the above homogeneous mass to
glass column in 4-5 installments, each time packing the material with
glass rod having a flattened end. Drain out extra hexane.
iv) Add 50 ml of carbonyl free benzene on the top of the column and
flush the column with air pressure. Carbonyl free benzene is used to
remove excess of DNPH. Continue washing till colourless effluent
starts coming out of the column and wait till the removal of excess
hexane. The column is ready to be used in flask method.

Flask method:

1. Take about 0.1gm molten fat in a 25.0 ml conical flask. Add 0.5ml of
carbonyl free hexane and dissolve the fat. Add 0.50 gm of column
material and keep the contents at room temperature over night.
2. After keeping over night, add 9.5 ml of carbonyl free hexane.

19
 3. Take reading at 340nm. If the absorbance is more than 0.9, dilute the
contents by mixing 1.0 ml of solution to 9.0 ml of carbonyl free hexane.
Absorbance should be in the range of 0.2-0.8.
4. Prepare a corresponding blank.

Calculations:
Extinction coefficient (E) = D/ C X d
Where
D = Absorbance
C = Conc. of absorbing species in moles/liter
d = length of light path in solution. (e.g. 1.0 cm)

Then
E= D/C or C= D/E moles/liter
For milk fat E= 22,500
So
C= D X 1 X 10-6 µmoles/ml
22500 X 1000

= D/22.5 μ moles/ml

If, W gm of fat was taken and diluted to V ml.

Then, Conc. of total carbonyl = (D/22.5) X (V/W) μ moles/gm of fat.

Observations:

Weight of sample (W) =

Absorbance (D) =

20
Experiment 7: Determination of Iodine Value of ghee by the Wijs’ method.

General: The iodine value (IV) gives a measure of the degree of unsaturation of
a lipid. The higher the iodine value, the greater the number of C=C double
bonds. The iodine value is normally used to know the degree of unsaturation
of oils, and to follow processes such as hydrogenation and oxidation that
involve changes in the degree of unsaturation. One of the most commonly
used methods for determining the iodine value of lipids is "Wijs’ method".

Definition: The iodine value is expressed as the grams of iodine absorbed per
100g of lipid.

Principle: The lipid to be analyzed is weighed and dissolved in a suitable

organic solvent, to which a known excess of iodine mono chloride (ICl) is
added. Some of the ICl reacts with the double bonds in the unsaturated lipids,
while the rest remains un-reacted. The amount of ICl that has reacted is
determined by measuring the amount of ICl remaining after the reaction has
gone to completion ( ICl reacted =ICl excess - ICl remaining ). The amount of ICl
remaining is determined by adding excess potassium iodide in the solution to
liberate iodine, and then titrating with a sodium thiosulfate (Na2S2O3)
solution in presence of starch to determine the concentration of iodine
released. Initially, starch is added to the solution that contains the iodine and
the solution turns dark blue. The solution is then titrated with a sodium
thiosulfate solution of known normality. As long as there is any I2 remaining
in the solution it stays blue, but once all of the I2 has been consumed in the
reaction it turns colorless. Thus, a change in appearance of solution from blue
to colorless can be used as the end-point of the titration.

The concentration of C=C in the original sample can therefore be calculated

by measuring the amount of sodium thiosulfate needed to complete the
titration. The higher the degree of unsaturation more will be the iodine
absorbed and higher will be the iodine value.

21
Reaction:

R-CH=CH-R + IClexcess R-CHI-CHCl-R + IClremaining

IClremaining + 2KI KCl + KI + I2

I2 + starch + 2Na2S2O3 2NaI + starch + Na2S4O6

( Blue Colour) (Colourless)

Apparatus and glassware

Analytical balance (sensitivity 0.1 mg.),

Erlenmeyer flask (iodine flask) with ground glass stopper

(300–500 ml), Graduated burettes (50.0 ml).

Chemicals and Reagents: Iodine

flask

Acetic acid Glacial (99%, m.pt 16.6ºC), Carbon tetrachloride

or / Cyclohexane or/ chlorofom, Potassium dichromate, Whatman No 1 or 4
filter paper.

1% (W/V) starch indicator solution: Make a paste of 1.0 gm soluble starch in

30.0 ml of water. Transfer the paste to 80.0 ml of boiling water and heat until a
clear solution is formed. Cool the contents and store in a tight stoppered
bottle. It can be stored for 2-3 weeks at 4ºC.

Potassium iodide solution: 10%, free from iodine and iodates.

Sodium thiosulphate solution, 0.1 N: Dissolve approximately 24.8 gm of

sodium thiosulphate crystals in previously boiled and cooled distilled water
and make the volume to 1000 ml. Store the solution in a cool place in a dark
coloured stock bottle. After storing the solution for about two weeks, filter if
necessary and standardize as follows:

22
Weigh accurately about 5.0 gm of finely ground potassium dichromate which
has been previously dried to a constant weight at 105 ± 2º in to a clean 1.0 liter
volumetric flask. Dissolve in water make up to the mark; shake thoroughly
and keep the solution in dark place. Pipette 25.0 ml of this solution into a
clean glass stoppered 250 ml conical flask. Add 5.0 ml of concentrated
hydrochloric acid and 15.0 ml of 10% potassium iodide solution. Allow to
stand in dark for 5 minutes and titrate the mixture with the solution of
sodium thiosulfate using starch solution as an indicator towards the end. The
end point is taken when blue colour changes to green. Calculate the normality
(N) of the sodium thiosulfate as follows:

25W
N= ----------------
49.03 V

W = weight in g of the potassium dichromate

V = volume in ml of sodium thiosulfate solution required for the titration.

Wijs' Reagent.: Wijs’ reagent is an iodine monochloride solution in acetic

acid. It acts to provide iodine monochloride (in excess) which reacts with the
double bonds. Prepare Wijs’ iodine monochloride solution by the following
method :

Dissolve 10.0 ml of iodine monochloride in about 1800 ml of glacial acetic acid

and shake vigorously. Pipette 5.0 ml of this, add 10 ml of potassium iodide
solution and titrate with 0.1N sodium thiosulphate solution, using starch as
indicator. Adjust the volume of the solution till it is approximately 0.2N.

Starch solution: Mix 5.0 g of soluble starch and 10.0 mg of mercuric iodide in
30 ml water. Add this mixture to 1000 ml of boiling water and continue to boil
for 3 minutes.

23
Procedure

i) Take molten ghee and filter it through Whatman No 1 or No 4 filter paper

to remove any solid debris and small amounts of water. To ensure the
complete removal of moisture from the sample, use anhydrous sodium
sulfate (Na2SO4). This can be done by placing small amount of anhydrous
sodium sulfate in the filter paper and slowly pouring the molten fat through
this.

ii) Weigh accurately 0.4 to 0.45 g of the clear ghee in a clean dried Erlenmeyer
flask.

iii) Add 20.0 ml / carbon tetrachloride/ cyclohexane/ or chloroform (Organic

solvents serves to solubilize the fat) and swirl to dissolve the sample. Add by
means of a burette exactly 25 ml of the Wijs' reagent. Close the flask with its
stopper, mix carefully and leave it standing for 1 hour in the dark.

iv) At the end of the hold, add 20.0 ml potassium iodide solution. Then
immediately add approximately 150 ml of distilled water, swirl lightly to mix,
and then promptly titrate with vigorous shaking / stirring, with 0.1 N sodium
thiosulphate solution using 50 ml burette. Continue titration until the yellow-
brown colour almost disappears, then add 1-2 ml starch indicator and
continue to titrate until the blue colour just disappears.

Addition of water forces the fat into the cyclohexane and the excess iodine
monochloride moves into the water, where it is converted to I2 and can be titrated
with the water –soluble sodium thiosulfate. Potassium iodide solution converts the
excess iodine monochloride to free iodine (blue) which can be titrated to a colorless end
point with sodium thiosulfate.

Record the volume of sodium thiosulfate used in the titration. Carry out a
blank test, using the same quantities of the reagents. Calculate the iodine
value by means of the following formula:

24
 12.69 (a – b) N
Iodine value (IV) = -------------------
W

Where:
a = Volume in ml of 0.1 N sodium thiosulphate used in the blank test.
b= Volume in ml of 0.1 N sodium thiosulphate used in the titration with
the ghee present.

N= Normality of sodium thiosulfate

W = weight of ghee taken for the analysis.

Accuracy of the method

The results of duplicate determinations should not differ by more than 0.4.

Note:
Sample size can be altered depending upon the expected iodine value e.g.
Expected iodine value Sample weight in ± 0.001 gm

<5 3.00
5 - 20 1.00
21 - 50 0.400
51 - 100 0.200
101 - 150 0.130
151 - 200 0.100

ii) Iodine values of oleic, linoleic and linolenic acids are 89.9, 181.0 and 273.5,
respectively. I.V for free acids are higher than those which are part of a triglyceride.
iii) If double bonds are conjugated, the values are not the true measures of
unsaturation.

25
Observations:

S. No Sample / Volume of 0.1N Sodium Weight of Iodine

Blank thiosulfate used the value

Initial Final sample

Vol. used
reading reading

26
Experiment 8: Estimation of unsaponifiable matter in ghee.

General: Unsaponifiable matter (USM) includes lipids of natural origin such

as sterols, higher aliphatic alcohols, pigments, vitamins and hydrocarbons as
well as any foreign organic matter, which does not form soaps with alkali and
are non volatile at 100 ºC e.g, mineral oil. Milk fat contains about 0.22-0.44%
unsaponifiable matter.
Definition: The unsaponifiable matter is defined as the substances soluble in
an oil/fat which after saponification are insoluble in water but soluble in the
solvent used for its determination.
Principle: On reacting with strong alkali, triglycerides get hydrolysed to free
fatty acids and glycerol, and form soaps with base. Matter other than
triglycerides is not hydrolysed by alkali and does not from soaps. These soaps
are water soluble and unsaponifiable matter is extracted with organic solvent.
The organic solvent containing unsaponifiable matter is washed with water to
remove traces of alkali and alkali free solvent is then dried to a constant
weight.
Reaction:

H O
| ||
H ⎯ C ⎯ O ⎯ C ⎯ R1 CH2OH
| O |
H ⎯ C ⎯ O ⎯ C ⎯ R2 + 3KOH CHOH + 3 R COOK + USM
| O |
H ⎯ C ⎯ O ⎯ C ⎯ R3 CH2OH
|
H

27
Apparatus and Glass ware:
Oven maintained at 100 ± 2°C, Boiling water bath, Flat bottom flask (250 ml
capacity), Air condenser, Separating funnels: 500ml and 250 ml capacity,
Round bottom flask.

Chemicals and Reagents:

Alcoholic potassium hydroxide: 0.5N solution of KOH in 95% (V/V) ethanol.
Dissolve 35-40gm of potassium hydroxide pellets in 20.0 ml of distilled water.
Add 1000 ml of 95% ethanol. Allow the solution to stand for 24-36 hrs and
filter the solution using Whatman No1 filter paper. Keep the filtered solution
in dark, airtight bottles.
Precaution: Strength of the solution should not be less than 0.5N in any case. Colour
of the solution should not be dark yellow (pale yellow is acceptable).
Diethyl ether: Diethyl ether (Specific Gravity at 15.5ºC in the range of 0.720 to
0.724) Non - volatile residue at 80ºC should not exceed 0.001%
Aqueous potassium hydroxide: Approximately 0.5N solution in distilled
water.
Phenolphthalein indicator: 1% solution in 95% (V/V) ethanol.

Procedure:
i) Take 5.0 gm of molten fat sample in a 250ml flat bottom flask. Add 50.0
ml of alcoholic potassium hydroxide solution and 2-3 glass beads. Mix
the contents.
ii) Attach the flask to reflux condenser properly. Now start heating the
flask on a boiling water bath. During heating, swirl the contents of the
flask frequently without detaching the condenser to ensure complete
heat transfer and reaction. Heating is continued for at least 1hr
duration from the start of boiling of the contents.
iii) After complete saponification, the contents are transferred to a 500ml
capacity separating funnel containing 150.0 ml of water. (Separating
funnel should be previously rinsed with water and diethyl ether).

28
 iv) Add 100.0 ml of diethyl ether to the separating funnel. Stopper the
funnel and shake it vigorously by intermittent removal of vapours to
release the pressure. Allow the funnel to stand until two layers are
separated.
v) Collect aqueous alcoholic soap layer in a beaker for further extraction.
vi) Collect etheral layer in a second 250ml capacity separating funnel
containing 20.0 ml of water.
vii) Extract from aqueous alcoholic soap layer (step v) twice more, each
time with 50.0ml of diethyl ether in same manner as mentioned in
(Step iv). Collect all the etheral layers in the separating funnel ( Step 6).
viii) Wash the ethereal solution twice with 20.0 ml of distilled water and
shaking vigorously on each occasion.
ix) Give successive washings with 20.0 ml of 0.5N aqueous potassium
hydroxide, 20.0 ml water and again repeat the steps. Continue the
washings with water till the wash water is free from any soap. For the
presence or absence of soap in the wash water, phenolphthalein
indicator is used. In case the soap is present in wash water, pink color
appears on adding the phenolphthalein indicator.
x) Transfer the washed ethereal solution to a previously weighed conical
flask by passing through anhydrous sodium sulfate. This will ensure
the removal of traces of moisture entrapped in the solvent during
washing.
xi) Evaporate the solvent on a boiling water bath, and finally transfer the
flask to an oven maintained at 80ºC/ 1hr.
xii) Weigh the flask and note its weight.
Calculation:
W1
% unsaponifiable matter in ghee = -------------- X 100
W
Where
W1= Weight of the residue in grams
W = Weight of the sample taken in grams.

29
Experiment 9: Colorimetric estimation of cholesterol in ghee by indirect
method using unsaponifiable matter.

General: The principal sterol of milk or milk fat is cholesterol. The cholesterol
content of milk ranges from 0.25 – 0.40% by weight of the fat. In the indirect
method of estimation of choletsrol, fat is first saponified and the
unsaponifiable matter (USM) is extracted with the help of solvents. After
washing, the solvent is evaporated and the dried USM is used for cholesterol
estimation.

Principle: Cholesterol reacts with sulphuric acid of LB- reagent and gives rise
to persulfate cholesterol, which simultaneously undergoes dehydration in the
presence of sulphuric acid. As a result of this dehydration cholesterol 3,5-
dienoid or cholesterol 2,4- dienoid is formed, which gives purple colour in
the presence of sulphuric acid. This color development is quantitatively
proportional to the amount of cholesterol present in the sample. The colour
can be estimated colorimetrically at 650nm using a red filter. This reaction is
time bound and if it is allowed to react furthers the color start fading due the
formation of polymers. Use of acetic acid can stabilize the colour for 30
minutes.
Reaction:

+ H2SO4 Æ
From LB-reagent
(C27H46O)

Dehydration action by H2SO4

Or
Purple colour

30
Apparatus and Glass ware:
Hot electric air oven, Boiling water bath, Flat bottom flask- 250 ml capacity,
Air condenser, Separating funnels: 500ml and 250 ml capacity, Round bottom
flask, Test tubes, Pipettes/ Auto pipettes of different capacities
Chemicals and Reagents:

1. Alcoholic potassium hydroxide: 0.5N solution of KOH in 95% (V/V)

ethanol. Dissolve 35-40gm of potassium hydroxide pellets in 20 ml of
distilled water. Add 1000ml of 95% ethanol. Allow the solution to
stand for 24-36 hrs and filter the solution using Whatman No1 filter
paper. Keep the filtered solution in dark, airtight bottles.
2. Diethyl ether: Diethyl ether ( Specific Gravity at 15.5ºC in the range of
0.720 to 0.724) Non - volatile residue at 80ºC should not exceed 0.001%
3. Aqueous potassium hydroxide: Approximately 0.5N solution in
distilled water.
4. Phenolphthalein indicator: 1% solution on 95% (V/V) ethanol.
5. Liebermann Burchard Reagent (LB- reagent) Take 1.0 ml of
concentrated sulphuric acid in a conical flask Add 20.0 ml of acetic
anhydride and mix the contents by keeping the flask in cold water.
Keep the reagent at 0ºC for at least 30 min. The acetic anhydride used
in the preparation of reagent acts as a diluent for the acid. Since
anhydrous conditions are required for the reaction to take place, so
aqueous diluents are not used in the preparation of the reagent.
6. Standard cholesterol,
7. Conc. Sulphuric acid – AR Grade
8. Acetic Anhydride- AR Grade.
Procedure:
A. Isolation of unsaponifiable matter:
i) Take 5.0 gm of molten fat sample in a 250ml flat bottom flask. Add 50.0
ml of alcoholic potassium hydroxide solution and 2-3 glass beads. Mix
the contents.

31
 ii) Attach the flask to reflux condenser properly. Now start heating the
flask on a boiling water bath. During heating swirl the contents of the
flask frequently without detaching the condenser to ensure complete
heat transfer and reaction. Heating is continued at least for 1hr
duration from the start of boiling of the contents.
iii) After complete saponification, the contents are transferred to a 500ml
capacity separating funnel containing 150.0 ml of water.
iv) Add 100.0 ml of the diethyl ether to the separating funnel. Stopper the
funnel and shake it vigorously by intermittent removal of vapours to
release the pressure. Allow the funnel to stand until two layers are
separated.
v) Collect aqueous alcoholic soap layer in a beaker for further extraction.
vi) Collect etheral layer in a second 250ml capacity separating funnel
containing 20.0 ml of water.
vii) Extract aqueous alcoholic soap layer ( step v) twice more, each time
with 50.0ml of diethyl ether in same manner as mentioned in ( Step iv).
Collect all the ethereal layers in the separating funnel ( Step 6).
viii) Wash the ethereal solution twice with 20.0 ml of distilled water and
shaking
vigorously on each occasion.
ix) Give successive washings with 20.0 ml of 0.5N aqueous Potassium
Hydroxide, 20.0 ml water and again repeat the steps. Continue the
washings with water till the wash water is free from any soap. For the
presence or absence of soap in the wash water, phenolphthalein
indicator is used. In case the soap is present in wash water, pink color
appears on adding the phenolphthalein indicator.
x) Transfer the washed ethereal solution to a previously weighed conical
flask by passing through anhydrous sodium sulfate. This will ensure
the removal of traces of moisture entrapped in the solvent during
washing.

32
 xi) Evaporate the solvent on a boiling water bath, and finally transfer the
flask to an oven maintained at 80ºC/ 1hr.
xii) Dissolve the unsaponifiable matter in acetic acid and transfer the
contents to a 25ml volumetric flask and make the volume to 25ml
mark. Filter the solution using whatman No1 filter paper.
xiii) Take 3.0 ml of this filtered solution in a 25.0 ml glass stoppered test
tube.
xiv) Add 4.0 ml of LB- reagent to the above tube and allow it to stand for
35min at 25ºC for colour development. Record the optical density at 650nm
against a blank consisting of 3.0ml acetic acid and 4.0 ml of LB- reagent.

B. Preparation of Standard Curve:

i) Preparation of cholesterol stock solution: Take 0.5 gm of
cholesterol standard. Dissolve it in acetic acid and transfer the
contents to a 100ml volumetric flask. Make the volume to 100ml
with acetic acid.
ii) Preparation of cholesterol working solution: Take 1.0 ml of the
stock solution in a 25.0ml volumetric flask. Make the volume to 25.0
ml with acetic acid.
iii) Take aliquots of 0.0, 0.50, 1.0, 1.5, 2.0, 2.5 and 3.0 ml of cholesterol
working solution in glass stoppered test tubes. Make the volume to
3.0 ml by adding required amount of acetic acid in respective tubes.
iv) Add 4.0 ml of LB- reagent in all the tubes and keep them in a water
bath maintained at 25ºC/ 10 min. Take absorbance at 650nm.
v) Plot absorbance readings against the corresponding cholesterol
amount ( ug) to get a standard curve.
vi) Amount of cholesterol present in the sample can be calculated from
the standard curve./

33
Observations:

Reagents (ml) Blank Tubes Number

1 2 3 4 5 6 7 S1 S2
Standard Cholesterol 0 0.50 1.00 1.5 2.0 2.5 3.0 - -
working solution
Diluted unsaponifiable - - - - - - - 3.0 3,0
matter
Acetic acid 3.0 2.50 2.00 1.5 1.0 0.5 0.0 - -
LB- Reagent 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0
Keep at 25ºC/10 min
O.D at 650 nm

Calculations:
Standard stock solution is 0.5% cholesterol in acetic acid.
Or
500mg cholesterol / 100 ml acetic acid
or
5mg / 1.0 ml
or
5000 μ g / 1.0 ml acetic acid
We have take 1.0 ml of stock solution and volume is made to 25.0 ml with
acetic acid.
Therefore,
25.0 ml of working solution contains 5000 μ g cholesterol
1.0 ml of working solution contains 200 μ g cholesterol
0.5 ml of working solution contains 100 μ g cholesterol
From the graph:
3.0 ml of diluted unsaponifiable matter contains = Y μ g cholesterol
25Y
25.0 ml of diluted unsaponifiable matter contains = --------- μ g cholesterol
3

34
 = Z μ g cholesterol
Weight of ghee sample taken = W gm.

So, W gm of ghee contains = Z

---------
W
100 g of ghee contains = Z
--------- X 100 μ g cholesterol
W

35
Experiment 10: Colorimetric estimation of total cholesterol in ghee by direct
method using whole fat.

General:
The principal sterol of milk or milk fat is cholesterol. The cholesterol content
of milk ranges from app. 0.25 – 0.40% by weight of the fat. In the direct
method of estimation of cholesterol, the whole fat as such is used after
dissolving the same in a solvent.

Principle:
Cholesterol reacts with sulphuric acid of LB- reagent and gives rise to
persulfate cholesterol, which simultaneously undergoes dehydration in the
presence of sulphuric acid. As a result of this dehydration cholesterol 3,5-
dienoid or cholesterol 2,4- dienoid is formed, which gives purple colour in
the presence of sulphuric acid. This color development is quantitatively
proportional to the amount of cholesterol present in the sample. The colour
can be estimated colorimetrically at 650 nm using a red filter. This reaction is
time bound and if it is allowed to react further the color start fading due the
formation of polymers. Use of chloroform can stabilize the colour for
15minutes.
Reaction:

+ H2SO4 Æ
From LB-reagent

Dehydration action by H2SO4

Or
Purple colour

36
Chemicals and Reagents:
i) Liebermann Burchard Reagent (LB- reagent) Take 1.0 ml of
concentrated sulphuric acid in a conical flask Add 20.0 ml of acetic anhydride
and mix the contents by keeping the flask in cold water. Keep the reagent at
0ºC for at least 30 min. The acetic anhydride used in the preparation of
reagent acts as a diluent for the acid. Since anhydrous conditions are required
for the reaction to take place, so aqueous diluents are not used in the
preparation of the reagent.
ii) Standard cholesterol, iii) Chloroform- AR Grade iv) Conc. Sulphuric
acid – AR Grade v) Acetic Anhydride- AR Grade.

Glass Ware: i) Volumetric Flask (25ml), Conical Flask (150ml), Glass

stoppered test tubes (10 ml), Pipette
Procedure: i) Take 5.0 gm of molten, clear anhydrous milk fat in a 25 ml
volumetric flask. Dissolve in chloroform and make the volume to 25ml with
chloroform.
iii) Pipette out 2.0 ml of this above solution in a glass stoppered test tube.
iv) To this add 1.0 ml of chloroform and 4.0 ml of freshly prepared cold
LB- reagent. Incubate the tubes at 25ºC /10 min in a water bath to develop the
colour.
v) Take absorbance at 650 nm using a red filter.

Preparation of Standard Curve:

vii) Preparation of cholesterol stock solution: Take 0.5 gm of cholesterol
standard. Dissolve it in chloroform and transfer the contents to a 100ml
volumetric flask. Make the volume to 100 ml with chloroform.
viii) Preparation of cholesterol working solution: Take 2.0 ml of the stock
solution in a 25ml volumetric flask. Make the volume to 25.0 ml with
chloroform.

37
ix) Take aliquots of 0.0, 0.50, 1.0, 1.5, 2.0, 2.5 and 3.0 ml of cholesterol
working solution in glass stoppered test tubes. Make the volume to 3.0 ml by
adding required amount of chloroform in respective tubes.
x) Add 4.0 ml of LB- reagent in all the tubes and keep them in a water
bath maintained at 25ºC/ 10 min. Take absorbance at 650nm.
xi) Plot absorbance readings against the corresponding cholesterol amount
( ug) to get a standard curve.
xii) Amount of cholesterol present in the sample can be calculated from the
standard curve.
Precaution: The color stability is time dependent; hence one should measure
the absorbance within 15min of LB-reagent addition.

Observations:
Reagents (ml) Blank Tubes Number
1 2 3 4 5 6 7 8 S
Standard Cholesterol 0 0.25 0.50 1.00 1.5 2.0 2.5 3.0 -
working solution
Diluted fat solution - - - - - - - - 2.0
Chloroform 3.0 2.75 2.50 2.00 1.5 1.0 0.5 0.0 1.0
LB- Reagent 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0
Keep at 25ºC/10 min
Take Absorbance at
650nm

Calculations:
0.5% cholesterol stock solution ≡ 0.5gm/100ml conc. ≡ 500mg/100ml
Therefore
100ml contains 500mg of cholesterol
1.0 ml contains = 500/100 = 5mg cholesterol or 5000 μg
Since, 25ml of working solution contains 2.0 ml of stock solution.
So,

38
25 ml of working solution ≡ 10000 μg cholesterol
0.25 ml of working solution ≡ 100 μg cholesterol

Let us say
2.0 ml of prepared sample contains “x” μg of cholesterol.
Then 25.0 ml of sample will have (25/2) x μg of cholesterol

Since this 25.0 ml contains 5.0 gm of ghee

So, 5.0 gm of ghee sample contains (25/2) x μg of cholesterol
100 gm of ghee will contain 250x μg or 0.25 x mg of cholesterol.

39
Experiment 11: Estimation of cholesterol in ghee using enzymatic
diagnostic kit

Materials and Methods

Principle:

Reagents , Glassware & Apparatus:

(i) 5% methanolic solution: prepare by dissolving the required amount of

potassium hydroxide (KOH) in methanol. (ii) Hexane (iii) Absolute methanol

(iv) Glass pipette (1.0 ml), Glass pipette (2 ml) or dropper (v) Screw capped
tubes (vi) Glass tubes

(vii) Water bath maintained at 90° C (viii) Water bath at 37°C (ix) Clinical
centrifuge (x) Spectrophotometer (xi) Enzymatic cholesterol estimation
diagnostic Kit.

Procedure:

Protocol for cholesterol estimation in milk fat after saponification using

enzymatic diagnostic kit:

Milk fat (0.1-0.15 g) in test tube with teflon lined screw cap

Add 5 ml of 5% methanolic KOH and mix thoroughly

40
 Incubate the capped tubes in water bath for 90 °C/ 20 min
with intermittent shaking after every 5 min.

Cool the contents under tap water

Add 1 ml of distilled water

Add 5 ml of hexane

Vortex the contents for 1 min

Centrifuge at 2000 rpm/ 2 min

Pipette out the upper hexane layer

Take 0.2 ml of aliquot in dry test tube

Evaporate solvent under nitrogen at 60-70°C

Add 10 µl of absolute ethanol to dissolve dried residue

Add 1.0 ml of cholesterol reagent provided in kit and incubate at 37°C/10min

Cool to room temp.

Measure colour (pink) intensity at 505 nm

41
 Calculations:

Choletserol was calculated as per the following formula:

Cholesterol (mg/100gm) = 0.02 × OD of sample × ml of hexane (5 ml) × 100

OD of standard × ml of hexane aliquot (0.2 ml) × Weight of sample (gm)

Factor 0.02 in the formula is the concentration (mg) of cholesterol in 10 µl

aliquot of standard solution used from the kit.

(Note: In some kits 20 µl of standard solution is recommended, there the factor will

change to 0.04 instead of 0.02)

Observations

Contents Blank Standard Test

Hexane Aliquot - - 0.2 ml and evaporate

Ethanol 0.0 ml - 10 µl

Cholesterol reagent 1.0 ml 1.0 ml 1.0 ml

Reagent 2: Working cholesterol - 10 µl -

standard solution (200 mg %)

Incubation at 37°C/ 15 min

Absorbance at 505 nm

42
Experiment 12: Determination of Reichert Meissl (RM) and Polenske (PV)
Values of ghee.
General:
Reichert Meissl (RM) and Polenske (PV) values are the important indices of
quality of milk fat. Reichert Meissl (RM) value is substantially a measure of
the lower chain fatty acids of ghee i.e. butyric (4:0) and caproic (6:0:). The
value of milk fat ranges from 17-35, which is well above all other fats and oils.
Butyric acid contributes about 3/4th and caproic acid 1/4th to the RM value.
PFA has also prescribed a minimum value ( 21-28, depending upon the source
of fat) for it to ensure the quality of milk fat. AGMARK also prescribes value
not less than 28 for non cotton tract areas on all India basis, and not less than
23 in winter and not less than 21 in summers for cotton tracts of Saurashtra
and Madhya Pradesh.
Polenske (PV) for milk fat ranges from 1.2-2.4. Caprylic acid (C8:0) contributes
upto 1/4th and capric acid (C10:0) contributes 3/4th to Polenske Value.
AGMARK prescribes values 1-2 for areas other than cotton tract, and 0.5-1.2
in winters and 0.5-1.0 in summers for cotton tracts of Saurashtra and Madhya
Pradesh.

The nature of fatty acids present in the RM and P V value distillates

has been computed as under:

Parameters Fatty acids

RM Value 4:0 6:0 8:0 10:0 12:0

% weight 65.3 30.1 4.6 - -

% Mole 71.8 25.1 3.1 - -

Polenske Value
% weight - 0.6 22.1 76.7 0.6
% Mole - 0.8 25.7 73.1 0.5

43
Since the presence of lower chain fatty acids is peculiar to ruminant milk fat,
the RM and PV are important characteristics of ghee. The actual figures for
both cow and buffalo ghee stand at about 28 for RM and 1.5 for the PV. Sheep
and goat ghee have RM similar to this but PV of 3 and 6, respectively. Among
common vegetable oils, only coconut and Palm Kernel contain steam volatile
acids and both exhibit RM of 7 and PV of 13.

Definitions:
Reichert Meissl Value (RM): RM value is the number milliliters of 0.1N
aqueous alkali solution required to neutralize the steam volatile and water
soluble fatty acids distilled from 5 g of ghee /fat under specified conditions.

Polenske Value: Polenske Value is the number of milliliters of 0.1N aqueous

alkali solution required to neutralize the steam volatile and water insoluble
fatty acids distilled from 5g of fat under specified conditions.

Principle:

In this method, milk fat is saponified using glycerol- alkali solution,

diluted with water and acidified by sulphuric acid to liberate free fatty acids.
Thereafter, the liberated fatty acids are steam distilled in a glass apparatus
under specified conditions and the steam volatile fatty acids are collected (as
condensate). The cooled condensate of the steam volatile fatty acids is filtered
for separation of water soluble and water insoluble fatty acids. The water
soluble fatty acids which pass through the filter paper are estimated by
titration with alkali to give RM value, while the water – insoluble fatty acids
collected on the filter paper are dissolved in alcohol and titrated to give the
polenske value.

44
Reactions:
i) Saponification

H O
| ||
H ⎯ C ⎯ O ⎯ C ⎯ R1 CH2OH
| O |
H ⎯ C ⎯ O ⎯ C ⎯ R2 + 3NaOH CHOH + 3 R COONa
| O |
H ⎯ C ⎯ O ⎯ C ⎯ R3 CH2OH
|
H

ii) Acidifcation:

2 R COONa + H2 SO4 2 RCOOH + Na2 SO4

iii) Titration:
RCOOH + NaOH RCOONa + H2O

Apparatus and Glass ware:

Graduated cylinder ( 100 and 25 ml), Pipette (50ml),
Flat bottom boiling flask (Polenske flask) 300ml, Polenske
flask
Still Head, Condenser, Receiver ( Two mark vol.
flask with graduation at 100 and 110 ml), Asbestoes
board, Gas burner, glass funnel, glass beads.

45
Chemicals and Reagents:
Glycerol (98% W/W), NaOH (50% W/W), Dil. H2 SO4 ( App. 25ml of conc.
sulphuric acid is diluted to one litre and adjusted until 40ml of it neutralizes
2.0 ml of 50% NaOH solution), Sodium Hydroxide solution ( N/10),
Phenolphthalein indicator, Ethyl alcohol (95% V/V), Whatman No 4 filter
paper 9cm dia.

Procedure

i) Weigh 5.00 + 0.1 g of ghee into a Polenske flask. Add 20 g of glycerol

and 2 ml of the 50 percent (w/w) sodium hydroxide solution. Protect the
burette containing the latter from carbon dioxide, and wipe its nozzle clear
from carbonate deposit before withdrawing solution for the tests, reject the
first few drops withdrawn from the burette. Heat the flask over a naked
flame, with continuous mixing, until ghee including any drops adhering to
the upper parts of the flask is saponified and the liquid becomes perfectly
clear. Avoid overheating during saponification. Cover the flask with a watch
glass. Carry out a blank test without ghee, but using the same quantities of
reagents and following the same procedure, again avoiding overheating
during the heating with sodium hydroxide. The over-heating would be
indicated by darkening of the solution.

ii) Measure 93 ml of boiling distilled water which has been vigorously boiled
for 15 minutes into a 100 ml graduated cylinder. When the soap is sufficiently
cool to permit addition of the water without loss, but before the soap has
solidified, add the water, draining the cylinder for 5 seconds and dissolve the
soap. If the solution is not clear (indicating incomplete saponification) or is
darker than light yellow (indicating overheating), repeat the saponfication
with a fresh sample of ghee.

46
iii) Add two glass beads, and 50 ml of the dilute sulphuric acid and connect
the flask at once with a distillation apparatus. Heat the flask without boiling
its contents, until the insoluble acids are completely melted, then increase the
flame and distill 110 ml in between 19-21 minutes. Keep the water flowing in
the condenser at a sufficient speed to maintain the temperature of the
distillate between 18 ºC and 21º C.

iv) When the distillate reaches the 110 ml mark, remove the flame and replace
the 110 ml flask with a cylinder of about 25 ml capacity to catch draining.
Close 110 ml flask with its stopper and without mixing the contents, place in
water at 15ºC for 10 minutes so as to immerse the 110 ml mark. Remove the
flask from the water, dry the outside and invert the flask carefully avoiding
wetting the stopper with insoluble acids. Mix the distillate by four or five
double inversions, without violent shaking. Filter through a dry 9-cm open
texture filter paper (Whatman No.4) which fits snugly into the funnel. Reject
the first running and collect 100 ml in a dry volumetric flask; cork the flask
and retain the filtrate for titration.

Reichert-Meissl or Soluble Volatile Acid Value

Pour 100 ml of the filtrate containing the soluble volatile acids into a
titration flask, add 0.1 ml of phenolphthalein indicator and titrate with 0.1N
sodium hydroxide solution until the liquid becomes pink. Note the burette
reading as “S” in case of sample and “B” in case of blank.

Polenske or Insoluble Volatile Acid Value

Detach the still head and wash the condenser with three successive 15 ml
portions of cold distilled water, passing each washing separately through the
cylinder, the 100 ml flask, the filter and the funnel, nearly filling the paper

47
each time and draining each washing before filtering the next. Discard the
washings.
Dissolve the insoluble acids by three similar washings of the condenser, the
cylinder and the filter, with 15 ml of neutralized ethanol each time, collecting
the solution in the 110 ml flask and draining the ethanol after each washing in
a flask. Cork the flask and retain the solution for titration.
Titrate the alcoholic solution of the insoluble volatile acids after
addition of 0.25 ml of phenolphthalein indicator with 0.1N aqueous sodium
hydroxide, until it becomes pink. Note the reading as “S1” ml in case of
sample and “B1” in case of blank.

Observations &Calculations
Reichert-Meissl value (R.M value) = 1.10 (S – B )
Polenske value (PV value) = (S1- B)

Where:
S = Volume in ml of 0.1N sodium hydroxide solution used for sample in
case of RM value.
B = Volume in ml of 0.1N sodium hydroxide solution used for blank in
case of RM value
S1 = Volume in ml of 0.1N sodium hydroxide solution used for sample in
case of PV
B1 = Volume in ml of 0.1N sodium hydroxide solution used for blank in case
of PV

Accuracy of the Method

Reichert Meissl Value: The maximum deviation between duplicate
determinations shall not exceed 0.5 units.

Polenske Value: The maximum deviation between duplicate determinations

shall not exceed 0.3 units.

48
Experiment 13 : Estimation of BHA content in ghee.

General:
The qualitative and quantitative analysis of antioxidants is necessary to
ensure adherence to regulatory requirements. Most of the synthetic
antioxidants approved for use in food products are phenolic in nature.
Among the various antioxidants available, BHA is commonly allowed in fat
rich dairy products.
The analysis of phenolic antioxidants from food products involves two
essential steps.

1. Extraction of the antioxidants from the food substrate.

2. Estimation of the extracted antioxidants

Two approaches have been suggested for the extraction of antioxidants

from foods. For high fat foods the fat and antioxidants are removed together
by solvent extraction. This is followed by extraction of the antioxidant from
fat.

For low lat foods direct solvent extraction processes may be used. Extraction
of fat from foods may be carried out by the Soxhlet procedure using
petroleum ether or similar solvents. Chloroform: methanol mixture (1:1) can
also be used.
Principle:
Determination of BHA is based upon its specific color reaction with Gibb's
reagent (2,6-dichloro- quinine - chlorimide) which permits the quantitative
estimation of BHA in the presence of other antioxidants without their
interference. The blue color of the Indophenol resulting from the reaction
exhibits maximum absorbance at 620nm and is stable for 5 hrs.

49
Reaction:

OH O OCH3
C (CH3)3 Cl ‫װ‬ Cl Cl ‫װ‬ Cl

+ Na2BO4

pH 4.4 OCH3
‫װ‬ ‫װ‬ ‫װ‬
OCH3 NCl N
(BHA) 2,6-dichloroquinone -chlorimide
C(CH3 )

OH
Blue indophhenol

Estimation of Antioxidants
Apparatus.
The list of the reagents and the precise outline of the method is as follows:
Reagents:

1. Gibb's reagent (0.01 % solution of 2,6 dichloroquinone chlorimide in 95%

methanol)

2. Borax solution - 0.5% (Prepared by dissolving disodium tetraborate in

warm D. W)

3. n-Butanol

4. Absolute methanol
5. Antioxidant (BHA)

6. Calcium carbonate (Ca Co3)

7. Whatman No.1 filter paper

8. Extraction solvent: Saturate hexane and acetonitrile by shaking them
together for 2 minutes for pre- equilibration and then separate. Use these pre-
equilibrated solvents for the extraction of BHA and other phenolic
antioxidants such as BHT, if present.

50
Procedure:

Extraction
Weigh accurately about 10 gm of the fat sample into 50 ml beaker and
quantitatively transfer to 100 ml vol. flask, rinsing beaker with hexane. Dilute
the content to 100 ml mark with hexane and mix. Take 25 ml aliquote of this
solution into a 125 ml separator and extract the antioxidants with three
successive 50 ml portions of acetonitrile. If emulsions form, break by holding
separator under hot water for 5-10 seconds. Collect all the extracts into 250 ml
separator and let the combined extracts slowly drain into a 250 ml round
bottom flask to aid removal of hexane oil droplets.

NOTE: At this point, the 150 ml acetonitrile extract may be stored overnight under
refrigeration.

Evaporate the extract completely at 40ºC, using flash evaporator equipped

with ice water cooling system completing the evaporation in less than 10 min.
However, if any traces of solvent are left remove by passing nitrogen gas.
Dissolve the dried extract containing antioxidants in 95% methanol and
transfer quantitatively to a 50 ml volumetric flask. Make up the final volume
to 50 ml with 95% methanol. Add about 1 g of CaCo3 to the flask and shake
the contents for 30 seconds. Filter through Whatman No.1 filter paper and
reject the first few ml of the filtrate before taking the clear filtrate for
colorimetric estimation of BHA.

Estimation of BHA:
Take 2 ml from the above clear filtrate containing antioxidants in a 25 ml
capacity conical flask
and add 2 ml of 95% methanol. To this add 8 ml of borax solution and 2 ml of
Gibb's reagent and mix. After 15 min., add 6 ml of n-butanol and mix the
whole contents of the flask. Measure the optical density of the blue coloured

51
solution in a colorimeter at 620 nm (red filter) against a reagent blank
prepared under identical conditions.
For the preparation of standard curve of BHA, take 0.1, 0.2, 0.3, 0.4 and 0.5 ml
of standard solution of BHA (concentration 100 µg/ml in 95% methanol ≡ 0.1
mg/ ml) in different conical flasks and make the volume to 2 ml in each case
with 95% methanol and treat with the same amounts of the reagents. From
the standard curve, the amount of BHA present in the fat sample can be
calculated and correction can be applied on the basis of recovery trials.

Observations:
Reagents Test Tubes / flasks ( 25 or 50 ml capacity)

B 1 2 3 4 5 S
Std. BHA 0 0.1 0.2 0.3 0.4 0.5 -
solution (100
μg/ml)
Filtrate - - - - - - 2.0
containing
BHA (ml)
95% methanol 4.0 3.9 3.8 3.7 3.6 3.5 2.0
(ml)
Borax solution 8.0 8.0 8.0 8.0 8.0 8.0 8.0
(ml)
Gibbs reagent 2.0 2.0 2.0 2.0 2.0 2.0 2.0
(ml)
Keep for 15 min at room temp.
n- butanol (ml) 6.0 6.0 6.0 6.0 6.0 6.0 6.0
O.D at 620 nm

52
Calculations:
From the graph:
Let the optical density for sample be “x ” and corresponding BHA conc. from
the graph be “y”.
2.0 ml of sample filtrate contains “y” μg BHA.
2.0 ml of filtrate is taken from 50.0 ml diluted extract.
Therefore,
50.0 ml diluted extract contains y X 50
----------- = 25 y μg of BHA.
2

The diluted sample is prepared from 25.0 ml aliquots

Hence 25.0 ml aliquot contains 25 y μg of BHA.

100 ml aliquot contains 25 y

--------- X 100 = 100 y μg of BHA
25
Since 100.0 ml of diluted sample contains 10.0 g ghee
So, 10.0 g ghee contains 100 y μg of BHA
100.0 g ghee contains 1000 y μg of BHA

53
Experiment 14: Determination of Vitamin D in ghee.

General:
Vitamin D is present in milk or milk products as part of the unsaponifiable
matter in the lipid phase. The vitamin D content in milk is about 23 IU/litre
( 13-33 IU/litre) and in ghee is about 10 IU/ gm fat (8-13 IU/ gm fat).

Principle:
For the determination of vitamin D, the sample of ghee or any other milk
product is saponified using alcoholic KOH and the unsaponifiable matter
containing fat soluble vitamins like vitamin D is extracted with solvent ether.
After evaporation of ether, the residue is dissolved in chloroform. The
chloroformic solution of vitamin D is treated with a mixture of antimony
trichloride-acetyl chloride dissolved in methylene chloride. The intensity of
the orange – yellow colour produced is measured at 500nm using green filter.
The concentration of vitamin D is calculated from the standard curve which is
prepared by treating different known concentrations of vitamin D with the
colour reagents.

Reaction:
Vitamin D + SbCl3 orange – yellow colour

Apparatus and Glass ware:

Saponification flask, Glass air condenser, Glass funnels, Separating funnel(
300ml), Separating funnel (250ml), Glass beads, Water bath, Oven,
Spectrophotometer.

Chemicals and Reagents:

i) 50% KOH, ii) Ethyl alcohol, iii) Diethyl ether (peroxide free), iv) Vitamin D
standard for preparation of standard curve. v) Anhydrous sodium sulfate, vi)
Whatman. No- 40 filter paper, vii) Chloroform.

54
viii) Colour reagent:
Solution A: Dissolve 20gm of Antimony Trichloride (SbCl3) in 90 ml of
methylene chloride.
SolutionB: Mix 10 ml acetyl chloride with 40 ml methylene chloride. Store in
refrigerator before use.
Mix solution A and B in the ratio (45:5), respectively to prepare color reagent.
The mixture is stable for one week at room temperature.

Working solution of vitamin D: 10 I.U / ml in chloroform

Procedure:
i) Saponification: Weigh accurately about 5 gm of ghee sample in a
saponification flask. Add 7.0 ml of 50% KOH and 50 ml of ethyl
alcohol. Reflux for 30 min in a boiling water bath using air
condenser to saponify the fat. Cool the contents and add 150ml of
distilled water. Transfer the contents to a separating funnel and
extract the unsaponifiable matter 3-times using 50 ml portion of
ethyl ether. Combine the ether extracts in another separating funnel
and wash with distilled water till it is free from alkali. Add small
quantity of anhydrous sodium sulfate to alkali free ether extract
and filter through Whatman No- 40 filter paper.
ii) Add 2-3 glass beads in the flask containing extract and evaporate
the ether solution to dryness. Dissolve the residue in 5ml of
chloroform.
iii) Place 1ml of this chloroform solution in the cell or cuvette of
spectrophotometer and add 4ml of the colour reagent. Prepare a
blank also in a similar manner. After 10 min, measure the O.D of
the orange – yellow coloured solution at 500 nm using green filter
against a reagent blank.

55
 iv) For the preparation of standard curve, prepare the standard
solution of pure vitamin D and treat with same amount of reagents.
Range of 2-10 IU can be used here.
Observations:
Reagents Test Tubes

B 1 2 3 4 5 S
Vit. D working 0 0.2 0.4 0.6 0.8 1.0 -
solution
( 10 I.U/ml)
Extract of - - - - - - 1.0
sample (ml)
Chloroform 1.0 0.8 0.6 0.4 0.2 0.0 0.0
(ml)
Reaction with color developing reagent is to be carried in
Cuvette of the spectrophotometer at room temperature
Colour reagent 4.0 4.0 4.0 4.0 4.0 4.0 4.0

O.D at 500 nm
Calculations:
Say the O.D for sample = x
Corresponding conc. from graph = y I.U
1 ml of solution has = y I.U
5 ml of solution has = 5 y I.U
i.e
5 gm of ghee has = 5 y I.U
100 gm of ghee has = 5y X 100/ 5 I.U
= 100y I.U

56
Experiment 15: Estimation of vitamin A in ghee.

General:
Vitamins A and E are a part of unsaponifiable matter present in ghee. For
their determination, ghee is saponified with alcoholic KOH and the
unsaponifiable matter containing fat soluble vitamins, sterols etc. is extracted
with solvent ether. After evaporation of ether, the dry residue is used for
estimation of vitamins like A and E. The details of estimation for vitamin A is
given below:

Principle:

The determination of vitamin A may be carried out by colorimetric method

using Carr-Price reaction. It involves the treatment of chloroformic solution of
vitamin A with the chloroformic solution of. antimony trichloride. The
resulting unstable blue colour is immediately measured at 620 nm using red
filter. The concentration of vitamin A is calculated from the standard curve
which is prepared by treating different known concentrations of vitamin A
with antimony trichloride as done for sample. Vitamin A is expressed as
International units (I.U.). One I.U. of vitamin A is equivalent to 0.3 mg of
vitamin A alcohol. Vitamin A content in ghee is about 30 I.U per gm fat.
Reaction: The belief has been ventured that the antimony trichloride becomes
attached to one of the conjugated double bonds in the carotenoid molecule.
The color reaction differs in intensity with the change in the polyene
groupings and is in all probability influenced by other portions of the
molecule.

+ SbCl3 Blue Colour

57
Apparatus and Glass ware:
i) Spectrophotometer: For reading at 620 nm., Water bath, Test tubes,
Saponification flask, air condenser, separating funnel, round bottom flask,
funnel, filter paper.

Reagents

Ethyl alcohol (95%, V/V) , Diethyl ether- peroxide free (prepared by adding
FeSO4 solvent ether and keeping overnight.) , Potassium hydroxide solution,
Aqueous (50%, W/V) Chloroform, Sodium sulphate anhydrous.
Saturated antimony trichloride solution: Dissolve 113.4 g antimony
trichloride in 300-400 ml chloroform. Add about 5.0 gm of anhydrous calcium
chloride and filter while hot. Make the volume of the filtrate to 500 ml.
Vitamin A standard: Crystalline vitamin A acetate of reference standard and
of known strength i.e 50,000 I.U
Working Vitamin A standard solution: 50 I.U / ml in chloroform.
Procedure:
Saponification of the sample: Weigh accurately a quantity of the material
containing 20-45 I.U. of vitamin A (not more than about 5.0 gm of the sample)
in a saponification flask. Add 40.0 ml of ethyl alcohol and 7.0 ml of potassium
hydroxide solution. Reflux the material for 30 to 40 minutes using air/water
condenser (rubber cork should not be used).

Extraction of unsaponifiable matter : Cool the above refluxed material, add

about 150 ml of distilled water and transfer the contents to a separating
funnel. Extract three times with 50.0 ml portions of ether and combine the
ether extracts in another separating funnel. Wash the ether extract with
distilled water (using 50-100ml each time) till the wash water is alkali free (i.e.
not alkaline to phenolphthalin). Filter the ether layer through anhydrous

58
Na2S04 and Whatman No. 40 filter paper (to absorb moisture). Rinse the
anhydrous sodium sulphate used with solvent ether to extract the vitamin A
completely, if trapped in anhydrous Na2S04. Complete extraction of vitamin A
from Na2S04 can be checked by adding a few drops of SbCl3 solution to the
residue.
Add 2-3 glass beads in the flask containing ether extract and evaporate to
dryness over a water bath. Dissolve the dried residue in 5 ml chloroform.

Determination:

Take 1.0 ml of pure CHCl3 for blank in a photometric cell tube, add 4 ml of
SbCl3 solution with cylinder and set the instrument (spectrophotometer) for
100% transmittance at 620 nm. In another cell tube, take1.0 ml chloroformic
solution of sample and 4 ml of SbCl3 solution and measure the absorbance of
blue colour within 3 seconds (the blue colour of vitamin and SbC13 complex
in stable for 3 seconds only so it should be done quickly).

Preparation of standard curve

Starting with a known quantity of vitamin A acetate, carry out the

saponification, extraction and evaporation of ether in a similar manner as
done for sample. Prepare the stock solution by dissolving the dried residue
containing vitamin A in a known quantity of chloroform. From this solution,
prepare a series of dilute solutions of vitamin A using CHCl3 so as to give a
range of 10-50 IU per ml. Take 1.0 ml each of dilute standard solutions of
vitamin A, develop the colour with 4.0 ml SbCl3 solution and measure the
absorbance as done for sample. Draw a graph by plotting the measured
absorbance against concentration of vitamin A in the standard solutions.

59
Observations:
Reagents Test Tubes

B 1 2 3 4 5 S
Vit. A working 0 0.2 0.4 0.6 0.8 1.0 -
solution
( 50 I.U/ml)
Extract of - - - - - - 1.0
sample (ml)
Chloroform 1.0 0.8 0.6 0.4 0.2 0.0 0.0
(ml)
Saturated 4.0 4.0 4.0 4.0 4.0 4.0 4.0
SbCl3
O.D at 620 nm

Calculations:

Read from the graph the concentration of vitamin A corresponding to the

absorbance (O.D.) of the sample.
Say, the O.D. for 1 ml sample is X and corresponding

concentration from graph. = Y I.U.

1.0 ml CHCl3 solution of samples has = Y.I.U.

5.0 ml CHCl3 solution of samples has = Y x 5.0 I.U.
i.e. W gm sample solution of samples has = Y x 5.0 I.U.
Y x 5.0 x 100 I.U.
100 W gm sample solution of samples has = --------------------------
W

60
Experiment 16: Determination of Vitamin E in ghee.

Principle:
Estimation of vitamin E is based on Emmerie-Engel reaction in which vitamin
E (tocopherols) reduces ferric chloride to ferrous chloride which reacts with
α,α’-dipyridyl to produce red colour whose intensity is measured at 530 nm
using green filter. For its determination in ghee, the unsaponifiable matter
obtained after saponification of the sample is extracted with ether. After
evaporation of ether, the residue is dissolved in benzene and passed through
Floridine XS earth column 'to remove interfering substances like carotenoids.
The benzene is then evaporated under vacuum. The dry residue thus
obtained is dissolved in ethyl alcohol and used for vitamin E estimation. The
concentration of vitamin E in the sample is calculated from the standard curve
which is prepared by treating different known concentrations of vitamin E
with ferric chloride α,α’-dipyridyl reagent. Vitamin E content in ghee is about
30 ug/gm.

Reaction:

+ FeCl3 FeCl2

FeCl2 + α,α’- dipyridyl Red color complex

Apparatus and Glass ware:

Spectrophotometer: for reading at 530 nm, Water bath, separating funnel,
saponification flask, Test tubes, beakers.

61
Reagents :
Ethyl alcohol-absolute, Diethyl ether- peroxide free (prepared by adding
FeSO4.7H20 to solvent ether and keeping overnight, Sodium sulphate
anhydrous, Potassium hydroxide solution aqueous (2%.W/V), Potassium
hydroxide solution Aqueous (50 % W/V) , α,α’–dipyridyl solution in absolute
ethyl alcohol. (0.5% W/V)
Ferric chloride (FeCl3.6H20) solution in absolute ethyl alcohol (0.2% W/V)
Benzene (A.R. grade), Hydrochloric acid- sp. gr. 1.16.

Floridine XS earth column: Fill a 12x30mm tube with the purified absorbent
(To purify, digest on a boiling water bath for one hour with Hydrochloric
acid. Wash with water until free of acid, then with ethyl alcohol, and with
benzene. Dry at room temperature).
Vitamin E: Reference standard.

Procedure:
a) Saponification af the sample
Weigh accurately about 5.0 gm of ghee sample in a saponification flask. Add
40 ml ethyl alcohol and 7.0 ml of KOH solution. Reflux the material for 30 to
40 minutes using air/water condenser (rubber cork should not be used).

b) Extraction of unsaponifiabIe matter

Cool the refluxed material, add about 150 mI dist-water and transfer the
contents to a separating funnel. Extract three times with 50 ml portions of
ethyl ether and combine the ether extracts in another separating funnel. Wash
the ether extract with 2% aqueous potassium hydroxide solution and then
with water till the wash water is alkali free. Filter the ether layer through
anhydrous Na2S04 and Whatman No. 40 filter paper. Rinse the anhydrous
Na2S04 with solvent ether. Add 2-3 glass beads in the flask containing ether
extract and evaporate to dryness over a water bath. Dissolve the dried residue
in about 5 ml benzene.

62
c) Carotene removal
Pass the benzene solution through the floridine XS earth column previously
wetted with benzene. Wash with benzene until the eluted volume is about 25
ml. The absorbent gets coloured a greenish blue by carotenoids and dark blue
by vitamin A. Evaporate the benzene under vacuum and dissolve the residue
in 10 ml of ethyl alcohol.

d) Determination
Take 5 ml of alcoholic solution of vitamin E in a test tube and add 1 ml of 0.2%
FeCl3 6.H20 solution and 1 ml of 0.5% α,ά - dipyridyl solution and mix each
time. Make up the final volume to 10 ml with ethyl alcohol. Prepare a blank
also in a similar manner. Allow to stand for about 15 minutes. Measure the
O.D. of the red coloured solution at 530 nm.

e) Preparation of standard curve

Prepare working standard solutions (20 μg / ml.) of pure α-tocopherol (if it is

in acetate form, then it needs to be saponified and extracted as in case of
sample) in absolute alcohol so as to get a range of 20 to 100 μg. Treat them
with the same amounts of reagents and develop the colour in a similar
manner as done for sample and measure the absorbance. Draw a graph by
plotting the measured absorbance against concentration of vitamin E in the
standard solutions.

63
Observations:

Reagents Test Tubes

B 1 2 3 4 5 S
Vit. E working 0.0 1.0 2.0 3.0 4.0 5.0 -
solution
( 20 μ g /ml)
Corresponding 0 20 40 60 80 100 -
conc.of vitamin
E ( μ g)
Vit E extract - - - - - - 5.0
from sample in
alcohol (ml)
Absolute 5.0 4.0 3.0 2.0 1.0 0.0 0.0
alcohol (ml)
FeCl3 (ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0
α,α’-dipyridyl 1.0 1.0 1.0 1.0 1.0 1.0 1.0
(ml)
Absolute 3.0 3.0 3.0 3.0 3.0 3.0 3.0
alcohol (ml)
O.D at 530 nm

Calculations

Read from the graph the concentration of vitamin E corresponding to the

absorbance of the sample.

Say, the 0.D. for sample be X and the corresponding concentration from graph
= Y μg
5 ml of alcoholic solution of sample has = Y μg

64
 Y
10 ml of alcoholic solution of sample has = ---------- X 10
5
Y
i.e. 5 gm of ghee sample has = --------------- X 10
5

Y 100
100 gm of ghee sample has = ---------- X 10 X -------- μg of vitamin E
5 5

65
Experiment 17: Estimation of phospholipids in ghee and ghee- residue.

General:
The estimation of total phospholipids contents in ghee and ghee – residue is
based on the determination of phosphorous content of the fat extracted by
organic solvents. This can be estimated as per the procedure given below.
Apparatus and Glass ware: Spectrophotometer, Heater, test tubes, volumetric
flasks (25ml), Kjeldahl flasks, pipettes, separating funnel.
Chemicals and Reagents: Conc. HNO3 (A.R), Conc. H2SO4, 0.44%
Ammonium molybedate solution in distilled water, Sulphuric acid H2SO4
(5%)

Reducing reagent: Mix 0.25 gm of 1-amino-2-napthol-4-sulfonic acid (ANSA),

15 gm of sodium bisulfite and 0.5gm of anhydrous sodium sulfite in 100ml
distilled water and filter.

Standard solution of potassium dihydrogen phosphate (KH2PO4) having a

concentration of 1 μg/ml: Dissolve 4.39 g of potassium dihydrogen
phosphate in 100 ml of 5% sulphuric acid. Take 1 ml of this and dilute with
5% sulphuric acid. To 100 ml. Take 1 ml of this diluted solution and dilute
again with 5% H2SO4 to get the final con. of 1 μg/ml.
87% ethyl alcohol ( Pre - equilibrated with petroleum ether).
Petroleum ether, Boiling point. 40 – 60ºC (pre - equilibrated with 87% ethyl
alcohol)
For equilibration mix two solvents in equal proportions in a separating funnel and
shake slowly. Keep it for some time undisturbed and then separate the two solvents.
Procedure: Estimation of phospholipids in ghee and ghee- residue involves
the extraction of phospholipids, digestion and estimation.

66
1. Estimation of phospholipids in ghee:
A) Sampling: Take the ghee sample for analysis as soon as it is made and
filtered in hot conditions.
B) Extraction: Extract the phospholipids from 10 gm of ghee by adopting
counter current distribution technique using 87% (V/V) ethanol and
petroleum ether as follows:
(i) Dissolve the ghee in 45 ml of petroleum ether (pre- equilibrated
with 87% ethanol) in a separating funnel.
(ii) Shake the above petroleum ether solution of ghee with 6
successive 15 ml portions of 87% ethanol (pre - equilibrated with
petroleum ether).
(iii) Take 45 ml of pre - equilibrated petroleum ether in a second
separating funnel.
(iv) Again shake successively the alcohol extract obtained each time
from the first separating funnel with a 45 ml portion of petroleum
ether taken in a second separating funnel.
(v) Combine all the alcohol extracts withdrawn from the second
separating funnel and evaporate to dryness in a 100 ml kjeldahl
flask.
C. Digestion: Digest the dried material obtained above as follows:
i) Add 5 ml of Conc. H2SO4 & 5 ml of conc. HNO3 in the kjeldahl flask
containing the dried material and digest it over flame (The completion of
digestion is indicated by a clear colorless or slightly yellow solution).
ii). After digestion, cool the contents and add 10 ml of distilled water. Boil
the contents till most of the water is removed. Repeat the process of
addition of 10 ml of distilled water and evaporation for a second time.
iii). Cool the digest thus obtained and dilute to 100 ml with distilled water
so that the concentration of sulphuric acid in the solution remains at about
2N level.

67
 D. Estimation: For estimation of phosphorous content in the above
solution, proceed as follows:

(i) Take 5 ml of aliquot of the solution in a test tube. To this add 4.6 ml of
0.44% ammonium molybedate solution and 0.4 ml of the reducing
agent.
(ii) Keep the test tube in the boiling water bath for 7 minutes for maximum
color development and cool the contents, measure the intensity of the
blue colour in a spectrophotometer at 720 nm.
(iii) Calibrate the colour against phosphorous contents using aliquots
drawn from a solution of potassium dihydrogen phosphate containing
1 μg of phosphorous per ml as standard.
2. Estimation of phospholipids in ghee-residue: This also involves the
extraction of fat, digestion and estimation.
Fat is extracted from ghee residue by Mojonnier fat extraction method using
sodium chloride as an additional reagent and also warming the mixture
after the addition of ethyl alcohol.

(i) Weigh accurately the appropriate quantity of ghee-residue in a

Mojonnier extraction flask.
(ii) Add 0.15gm of Sodium chloride (NaCl) and 1.5 ml of conc. ammonia
solution (Sp. Gr. 0.88) and mix.
(iii) Then add 10 ml of ethyl alcohol (95%) warm the contents and mix.
(iv) Then add 25 ml of ethyl ether and 25 ml of petroleum ether and shake
vigorously after each addition.
(v) Allow the mixture to stand for some time for clear separation of
ethereal layer.
(vi) Decant the ether layer into a 100 ml Kjeldahal flask.
(vii) Repeat the extraction procedure for second & third time using 5.0 ml of
ethyl alcohol, 15 ml ethyl ether and 15 ml of petroleum ether each time.

68
(viii) Combine all the extracts and evaporate to dryness over a boiling water
bath.
(ix) Add 0.5% sulphuric acid and make the volume to 100 ml.
(x) Subsequent Digestion and estimation steps are similar as described for
ghee at C & D above.

The quantity of ghee-residue taken should be such that the total lipids
extracted should not exceed 1.0 gm to ensure the complete digestion.

Observations:

Phospholipids in ghee:

Reagents Blank Standard Sample

Sulphuric acid 0.5% (ml) 4.5 - -
Aliquot of digested diluted solution - - 5.0
(ml)
Standard solution (ml) - 5.0 -
0.44% ammonium molybedate 4.6 4.6 4.6
solution (ml)
Reducing agent (ml) 0.4 0.4 0.4
Keep in boiling water bath for 7
min
O.D at 720 nm

Phospholipids in ghee- residue:

Reagents Blank Standard Sample

Sulphuric acid 0.5% (ml) 4.5 - -
Aliquot of digested diluted solution - - 5.0
(ml)
Standard solution (ml) - 5.0 -
0.44% ammonium molybedate 4.6 4.6 4.6
solution (ml)
Reducing agent (ml) 0.4 0.4 0.4
Keep in boiling water bath for 7
min
O.D at 720 nm

69
 Calculations:

Conversion factor of phosphorous to total phospholipids = 25.9 X P

Say
O.D of sample = x
O.D of standard = y

Y corresponds to a conc. of 1μ g
Therefore
x
x corresponds to a conc. of ---------- X 1μ g
y

x 1
= ---------- X --------- mg ( to convert μ g to mg)
y 1000
This is to be multiplied with 100 because volume was made to 100 ml after
digestion

Therefore
x 1
= ---------- X --------- X 100
y 1000

Let W be the weight in gm of ghee taken. Then for 100 g of ghee the conc.
of phosphorous will be

x 1 100
Phosphorous = ---------- X --------- X 100 X -------
y 1000 W

For conversion of phosphorus into phospholipids

x 1 100
Phospholipids (mg/100g) = ---------- X --------- X 100 X ------- X 25.9
y 1000 W

x 100
= ---------- X 0.1 X ------- X 25.9
y W
O.D of sample 100
= --------------------- X 0.1 X ------------- X 25.9
O.D of standard W

70
Experiment 18: Analysis of fatty acids of ghee by Gas liquid
Chromatography.

General: The fatty acids of milk fat can be analyzed either as the free acids or
as esters. Since the free acids are polar and interact strongly both with
themselves and with most support materials, they are very liable to produce
peaks which are badly tailed. Thus in the majority of routine analysis acids
are first converted to their respective volatile esters before chromatography.
More than 400 hundred fatty acids have been reported in bovine milk. Most of
these components could only be detected by using most advanced
chromatographic or spectroscopic techniques.

Principle: Gas – Liquid chromatography is the most elegant and useful

methods in analytical chemistry. Gas – liquid chromatography is a form of
partition chromatography in which the stationary phase is a film held in
place on a solid support and the mobile phase is a carrier gas flowing over the
surface of a liquid film in a controlled fashion. The components of a
vaporized sample are fractionated as a consequence of being partitioned
between a mobile gaseous phase and a liquid stationary phase held in a
column.

Apparatus and Glassware:

Gas liquid chromatography instrument consisting of:
Carrier Gas Supply: Helium, Argon, Nitrogen, or any inert gas. As water
may be a great nuisance, the gases must be thoroughly dried, and for this
purpose the best desiccant is a molecular sieve (Linde 5A) activated at 200 –
300 o C.
Pressure gauges: To control the flow rate of these gases.
Columns: Columns used in GLC may be of glass or metal.
Packed columns: These columns can accommodate larger samples and are
generally more convenient to use. Present day packed columns are fabricated

71
from glass or metal tubings; they are typically 2 –3 m long and have inside
diameters of 2 –4 mm. The tubes are ordinarily formed as coils with diameters
of roughly 15cm to permit convenient thermostating in an oven. The packing
or support, for a column hold the liquid stationary phase in place, so that the
surface area exposed to the mobile phase is as large as possible. The ideal
solid packing consists of small uniforms, spherical particles with good
mechanical strength and with a specific surface of at least 1m2/g. In addition
the material should be inert at elevated temperatures and be uniformly
wetted by the liquid phase. The particle size of packing for gas
chromatography typically falls in the range of 60 – 80 mesh (250 – 170μm) or
80 – 100 mesh (170 – 149 μm). The use of smaller particles is not practical
because the pressure drop with in the column becomes prohibitively high.
Open – Tubular Columns: Open tubular or capillary columns provide
unprecedented separations in terms of speed and number of theoretical
plates. Capillary columns are generally constructed of glass or fused silica.
These columns have inside diameters of 0.25 – 0.50 mm and lengths of 25 –
100 m. Their inner surfaces are coated with a thin layer of stationary phase.
Column Thermostating or oven: The column thermostating efficient to
maintain and control the temperature from 0 - 400 O C.

Flame Ionization Detector: The purpose of detector is to monitor the column

effluent, and measure variation in its composition. These devices must
respond rapidly to minute concentrations of solutes as they exit the columns.
The solute concentration in carrier gas at any instant is no more than a few
parts per thousand. Moreover, the time during which peak passes the
detector is typically one second or less, which requires device capable of
exhibiting its full response during this brief period.

Sample injection Systems: The sample may be gaseous, liquid or solid, and
the method of sample introduction may differ accordingly. Column efficiency
requires that a sample be of a suitable size and be introduced as a “plug” of

72
vapor because slow injection or oversized samples cause band spreading and
poor resolution. Sample size depends upon the sensitivity of the detector. For
ordinary packed analytical columns, sample sizes range from 2 to 20 μl.
Capillary columns require samples that are smaller by a factor of 100 or more.
Here a sample splitter is often required to deliver only a small known fraction
(1:100 to 1:500) of the injected sample, with the remainder going to waste.
Freeze drying tubes: To prepare esters of fats.
Micro syringe ( 10 or 20 μl )

Chemicals and reagents:

Sodium metal, absolute methanol.
Preparation of absolute methanol: Take one liter of analytical grade
methanol in a round bottom flask, add 5 gm of acid washed magnesium
turnings ( 5 g magnesium turnings washed with 5 – 10% HCl, till black color
of magnesium oxide is removed, dry in an oven ) and 1 gm of iodine as
catalyst. Reflux the contents of the flask for about 6 hrs, until magnesium
turnings are completely solublized. Distil the refluxed mixture at 50 – 60 OC,
using calcium carbide or calcium chloride or activated silica tube as moisture
trap and collect the distillate (absolute methanol) in an airtight bottle.

Sodium methoxide solution: For the preparation of 0.025 N Sodium

methoxide solution, required amount of dried sodium metal is dissolved in
absolute methanol. Normality is calculated by titrating this solution with
standard acid.

Procedure:
1. Take molten milk fat and filter it through Whatman No1 filter paper to
remove any debris or particles.
2. Prepare methyl esters. For this take 0.2 gm of fat sample in a freeze-drying
tube and add 0.4 ml of 0.025N sodium methoxide solution. Seal the tube on a
gas flame using glass blowing technique. Check for any leakage by inverting

73
the tube. Heat the sealed tube in an oven maintained at 80 ºC/ 1-2 hrs for
esterification of fatty acids until a uniform layer is formed.
2. Set the GLC equipment in the following manner:
i) Switch on the GLC and set Injection port temp 240ºC.

ii) FID temp (250ºC), sensitivity 10X, Polarity etc.

iii) Program oven where, initial temp 55ºC followed by a rise

@10ºC/min till 220ºC.

iv) Recorder Current: 10 mV

v) Chart speed: 10 mm / min.

vi) Switch on the carrier gas (nitrogen) supply and set the flow rate
30ml / min.

vii) Once the FID attains the set temperature, Switch on the fuel gas
(hydrogen) supply and set the flow rate 35ml / min. and ignite the
flame with electric lighter.

viii) Switch on the air supply @ 330 ml/min. to obtain a blue flame.
Take care that air supply is opened slowly so that the flame remains in
an on position.

ix) Run the column at higher oven temperature i.e 200- 240ºC for some
time with intermittent injection of absolute methanol ( 1-2 ul) so that
the column is cleaned and a stable base line is obtained.

x) Now, inject the methyl ester sample ( 1-2 ul) and press the run
button to run the set program. Simultaneously press start icon on the
computer display to obtain the digital chart.

74
3. The peaks obtained (as a result of recorder response to the detector ) are
identified by comparing their retention time with those of known standard
fatty acid esters. The area of each peak is measured by triangulation method
and expressed as relative percentage of fatty acid in the sample.
Calculation: Peak area is either calculated automatically by the software
provided by the manufacturer or manually. The manual calculation is as
follows:
Area of trapezium :
From the graph as in peak C
a = 1.0cm, b = 1.6 cm and h = 17.7 cm

Area = ½ h (a+b)
= ½ X 17.7 ( 1+1.6)
= 23.01 cm2

Area of a triangle:
From the graph as in peak B
b = 0.8cm, h = 1.9 cm
Area of a triangle = ½ bh
= ½ X 0.8 X 1.9
= 0.76 cm2

In this manner the are of all the peaks is calculated and then added to get the
total area. From this total area the per cent are of each peak can be calculated.
This represents the per cent fatty acid ester of a particular acid

75
76
Open – Tubular or Capillary Columns

Packed Column

77
 Packed column injector system

Capillary column injection system

78
Suggested Readings:

1. Hand book of food analysis, Part XI, 1981. Bureau of Indian

Standards. Manak Bhavan,9. Bahadur Shah Zafar Marg, New Delhi.

2. Ronal.E.W; Terry.E.A; Eric. A.D; Michael. H. P; David. S. R; Steven.J. S;

Charles. F.S; Denise. M.S and Peter.S (eds.) 2005. Hand book of Food
Analytical Chemistry. Wiley Interscience, John Wiley &Sons, Inc,
Hobokon, New Jersey.

3. Ramamurthy, M.K and Narayanan, K.M. 1966. Ind J Dairy Sci. 19 , 45.

4. Parrish, D.B. 1979. Determoination of vitamin D in foods: A Review.

CRC critical Reviews in Food Science and Nutrition, pp 29-57. Vol 12.

5. Douglas, A. Skooge; Donal, M. West and James Holler , F. 1994

Analytical Chemistry – An Introduction . Sunder college Publishers –
London.

6. R.Stock and C.B.F Rice. 1963. Chromatographic Methods. Chapman &

Hall.

7. Vivek Sharma, M J Sudharshana Reddy , Sumit Arora, Amit Kumar,

Darshan Lal , Raman Seth, B. K Wadhwa and G.S Sharma (2009)
Applicability of enzymatic diagnostic kit for estimation of cholesterol
in Ghee. J Food Sci Technol, 46, No 3: 24- 246

79