NTD - Lab Manual of Analysis of Milk Lipids (Ghee) - 2007 - India

A LABORATORY MANUAL
On
ANALYSIS OF
MILK LIPIDS (GHEE)
Vivek Sharma
Sumit Arora
Darshan Lal
B. KWadhwa
{(~\zt ~ 31iJIitrrat l:imrcr

~
(JII'Il tlnilGiQll'IQ)
(1{l.~.3{.q.) iM<t11'l-132001 IIRi[
NATIONAL DAIRY RESEARCH INSTITUTE

11111111 ~ . (Deemed University)
Call No. 637.25 P7 (I CAR) Karnal-132001 India
.-
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/B Techl MScl 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-200? Dr. Vivek Sharma

Dr. Sumit Arora
Dr. Darshan Lal
Dr. B.K Wadhwa
Acknowledgement
Support and encouragement from Director, National Dairy Research
Institute, Karnal is thankfully acknowledged. The authors are also

thankful to Jo int Director (Al and Registrar , Dairy Science College, NDRI
for providing funds under the scheme on strengthening the post graduate
educational programme at reAR institutes .
Dr. Vivek Sharma

Dr. Sum it Arora
Dr. Darshan Lal
Dr. B. K Wadhwa
No. part of this publication can be reproduced without the prior
pennission of Director, National Dairy Research Institute.
Cover Design: Vlvek Shanna
First Edition: March, 2007

N.D.R.I. Publication No. 2912007
Published by: Director,

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

# 51-A, Model Town, Karnal_132001
Ph : 0184-2267451, 3292951.
Email: vivek.intech@gmail.com
CONTENTS
S.No Name of Experiment Page

No
-3
4-5
Determination of peroxide value in ghee.

~~c::-==----j--:;;;-.......,--1
method.
estimation of cholesterol in ghee by i i

method.
method.
11 Determination of Reichert Meissl (RM) and Polenske (PV) 40 - 45
Values of ghee.
Estimation of vitamin A in ghee.
residue .
18
Experiment 1: Determination of butyro refractometer (B.R) reading of
ghee.
General:
Ghee exhibits a B.R reading about 43 at 40C, corresponding to a refractive
index of 1.4545. Most vegetable oils have a B.R value of about 75 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.
Temperature is indirectly proportional to B.R. reading i.e
Temp <>0 1
--------
B.R.
Therefore, if observations are taken at temperature other than 40C the

correction has tc? be applied , as follows:
Correction = 0.54 B. R. I '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 (B.R.) reading) measures the index of refraction
between air and liquid fat, and varies with the nature of fat. The instrument is
calibrated in butyro- refractometer degrees instead of the usual absolute
refractive indices, the two values being interconvertible as follows:
B.R. in ghee = 42 + Factor (observed refraclive index - 1.4538)
Refraclive index = 1.4538 + Factor (observed B.R. - 42)

Apparatus and Glass ware:
Butyro refractometer, Circulatory water bath maintained at 40C , Glass rod,

Beaker, Tissue paper.
Chemicals and Reagents:

Ethyl alcohol I 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 + 1' C by adjusting the
thermostat of circulatory water bath.
iii ) Clean the prism of the Instrument with cotton plug! tissue paper moistened
with Ethyl alcohol I toluene or petroleum ether and allow it to dry. Repeat
cleaning step between readings.
iv) Place 1-2 drops of sample on the lower prism. Close prisms and 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, an
optical contact with the prism being made by a drop of.(bromonapthalene) or
by using distilled water which has refractive index of 1.3330 at 20.0 ' C and
1.3306 at 40.0 ' C, the usual temperature of taking readings.
Light Source:
Normal day light or if the refractometer is equipped with a compensator, a
tungsten lamp.
2
Observations:
S.No Type of fat Observed B.R at Corrected B.R- at
40' C 40'C
._-
--
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 -40C to 40C. 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.

Melting point tubes ( Capillary tubes .- thin walled, uniformly bored , open at
both 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 I capillary 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 I capillary tube in a horizontal position in
a freezer for at least 1 hr.
iv) Fill water at 10C 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 25C @ 2CI min. After attaining 25C further increase the
temperature @ 0.5C/min . The melting point tube I capillary tube is observed
4
-
Butyro refractometer
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 OS".
vii) Note down the temperature at which ghee sample starts melting as "T1 "
and "T2" when ghee melts completely.
Observations:
S.No Sample/BI Observed temperature C
ank
Initial Final Slip temperature

Range
temp. temp.
Experiment 3: Determination of Free Fatty Acid (FFA) value in ghee.
General:
Free fatty acids (FFA) are an indication of hydrolytic rancidity. but other lipid
oxidation processes can also produce acids. Free fatty acids (FFA) 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 (!.nd/or lypolytic enzyme lipase. Free fatty "acids are significant for
the quality of the milk fat because they increase the fa!"s susceptibility to
oxidation andean contribute to bitter or soapy flavours.
Fresh ghee has an acidity in the vicinity of 0.2% oleic acid. As per PFA
standards the maximum permitted FFA level in ghee is 3% oleic acid.
Definition: The FFA is defined as the percentage by weight of free acid

groups in the oil or fat.
Principle of the method
The lipids are extracted from the food samplel milk product and then
dissolved in neutralized alcohol containing an indicator. This solution is then
titrated with an alkali until a pinkish color appears.
Reactions:
o o
"
R - C - OH + NaOH - - - - .
(FFA)
"
R-C-ONa + H20
6
Water balh (boiling), Beaker 250 ml capacity , Conical Flask (250ml).
Ethanol (Ethyl alcohol) :- 95% ethanol or rectified spirit.

Phenolphthalein indicator solution: - Weigh 19m phenolphthalein and
place tihe powder in a 100ml volumetric flask containing about 50ml of 95%
etihanol. 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
100.0 mi.
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.
"recetlure:
i) Mix tihe melted fat thoroughly before weighing. Filter it using Whatrnan 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 neutralised ethanol and about one ml of
phenolphtihalein indicator solution.
iii) Boil tihe mixture for about five minutes on a boiling water bath and titrate
while hot against standard alkali solution shaking vigorously during the
titration.
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 mi.
7
Calculation:
ml NaOH used X Normalily of NaOH X 2.82

FFA (% oleic acid) = ---------------------------------
Weight of sample
Derivation:
1L of 1.0N sodium hydroxide" 1L of 1.0N Oleic acid.
Mol. weight of Oleic acid = 282.

Hence
1 N oleic acid contains 282gm of oleic acid IL 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)1W gm of oleic acid.
100 gm of sample contains = [(0.0282T)IW]X100
% FFA = 2.82TI W
8
Observations :
S.No Sample Volume of O.1N NaOH used Weight

IBlank of %FFA
sample
Initial Final
Vol.Used ( gm)
reading temp.
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 expressed in many ways (i) as mmol active oxygen (Le
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 0(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 oXygenlKg 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 fonned . This is done by titration with sodium thiosulfate and a starch
10
indicator. The amount of sodium thiosulfate required for titration in the
reaction is related to the concentration of peroxides in the original sample.
Reaction:
ROOH + Kl excess ROH + KOH + 12

(blue colour) (colourless)
Glass stopperec Erlenmeyer flask (250 ml), Glass pipette (1-2 ml) or
micropipettor, Graduated class-A glass burette.(l 0 or 25 ml), Oven 100-110'C
Sodium thiosulfate pentahydrate ( Na 2S20,.5H 20 , M.wt; 248.17) AR grade.
Sodium Carbonate (Na2CO,),
Potassium Iodate (KIO, ) as primary standard. Before use heat the primary
standard in an oven maintained at 11 COC 11 hr and then cool in a desiccators.
Potassium iodide (KI), iodate free
Hydrochloric acid (6.0 M)
0.1 N Sodium thiosulfate solution: Add 25 gm of sodium thiosulfate

pentahydrate into previously boiled & cooled distilled water. Stir the contents
until ali crystals are dissolved. Make up the volume to 1000 mi. 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:
II
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
gm of iodate free potassium iodide. Then add 2 ml of 6.0 M HCI 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 dropwise until the blueiviolet colour disappears. Record the
total volume of added sodium thiosulfate solution. Repeat the titration
twice/thrice to record the exact volume of (0.1 N) 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 KIO,
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 to cool. To a portion, add enough potassium iodide to ensure a
saturated solution (10 gm KI in 6 ml water). Store it overnight to ensure that
undissolved crystals remain there. To test stability, dilute 0.5 ml saturated KI
solution in 30 ml of 3:2 01N) 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 O.1N sodium thiosulfate solution to discharge, discard the
iodine solution and prepare a fresh one.
12
1% (WN) starch indicator solution: Make a paste of 1 gm soluble starch in
30 ml of water. Transfer the paste to 80 ml of boiling water and heat until a
clear solution is formed . Cool the contents and stofe in a tight stoppered
bottle. It can be stored for 2-3 weeks at 4'C.
Mixed Solvent: 2:1 01N) glacial acetic acid! chloroform solution
Procedure:
i) Accurately weigh 1 .!.-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 mi.
ii) Add 20 ml of mixed solvent and swirl flask until fat is dissolved.
iii) Add 0.5 ml of saturated KI solution to flask and allow solution to stand
exactly for 1 min with occasional shaking and then add 25 - 30 ml of distilled
water.
iv) Gradually add 0.002N standardized sodium thiosulfate solution from a

graduated burette and titrate until y~lIow colour has almost disappeared.
Shake the contents constantly and vigorously.
v) Add 0.5 ml of starch indicator solution to the above flask . Continue titration ,
shaking flask vigorously near the end point to liberate all iodine from the
chloroform layer. Add sodium thiosulfate solution dropwise until the violet
colour disappears. Record the total added sodium thiosulfate volume.
--
Conduct a simultaneous blank, without fat.
13
Calculations: Peroxide value (PV) as miliequivalents of peroxide oxygen per
Kg of sample is :
(S-B)X N X 1000 X 8
PV=
W
Expressed as meq active oxygen I 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 mllll 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,
From peroxide value , the quality of ghee can be judged as follows:
Peroxide value in terms of volume Quality of ghee

inml 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
14
the use of peroxide value as an indicatior of lipid oxidation. Firstly, peroxides
are primary products that are broken down in the latter stages of lipid
oxidation. Thus, a low value of PV 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/SI Volume of O.002N Sodium mUll Weight of

ank thiosulfate used equivalents the sample
of oxygen
Initial Final
Vol. used per Kg of
reading temp.
fat
15
Experiment No 5. Determination of thlobarbiturlc acid (TBA) value in
ghee.
General:
TBA is the mosl widely used tesl 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 r~QiJlment which is quantified
at 532nm. 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 detenmined by using a standard of acetal
tetraethoxy propane, which hydrolyzes under acidic conditions to generate a
source of unstable mal on aldehyde.
Chemical Reaction:
malonaldehyde TBA TBA chrom.,en

Water bath, Glass stoppered test tubes, volumetric flask (100ml), Glass rod,
Funnel.
16
i) Carbon tetrachloride (CCI,) density 1.:.
ii) Glacial Acetic acid.
/
ii) TBA reagent: Take O.76gm 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 gm of molten fat in a glass stoppered test tube.
ii) To this add 10 ml of carbon tetrachloride solvent and 10 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 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
spectrophoto"meter.
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 ~mol/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
hydroperoxides.
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 pr~sent 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:
/
NHNH,
@NO:
/
"- C=O
~~~" + H,O
NO, NO,
2,4- Dinitrophenyl Carbonyl 2,4- Dinitrophenyl Water
Hydrazine Hydrazone

Pesije and Mortar, Spectrophotometer, Glass column, Glass rod having
flattened end . Flasks 25ml capacity.
18
Procedure:
A. Preparation of carbonyl free hexane: Take 1 It of hexaneibenzene in
a round bottom flask. Add 5 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 ml of 87% orthophosphoric acid in a pesUe & 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 Cellite545 by heating at 120'C for 12hrs.
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 tree 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 tree 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.
3. Take reading at 340nm. If the absorbance is more than 0.9, dilute the
contents by mixing 1 ml of solution to 9 ml of carbonyl free hexane.
Absorbance should be in the range of 0.2-0.8.
4. Prepare a corresponding blank.
19
Calculations:
Extinction coefficient ( E) = DI CXd
Where
o =Absorbance
C = Conc. of absorbing species in moles/It
d = length of light path in solution.( e.g 1.0cm)
Then
E= D/C or C= DIE moles/lt
For milk fat E= 22,500
So
DX1X10"
C= ~ moles/ml
22500 X 1000
= D/22.5 ~ moles/ml
If, W gm of fat was taken and diluted to V ml.

Then, Conc. of total carbonyl = (D/22.5) X rylW ) ~ moles/gm of fat.
Observations:
Say
Weight of sample (W) =
Absorbance(D) =
20
Experiment 7: Oetenminatlon of Iodine Value of ghee by the Wijs '
method.
General:
The iodine value (IV) gives a measure of the degree of unsaturatioll 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 01 oils ,
and to follow processes such as hydrogenation and oxidation that inl,olv
changes in the degree of unsaturation. One of the Illost 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 1009 of
lipid.
Principle:
The lipid to be analyzed is weighed and dissolved in a suitable o rgon i ~
solvent, to which a known excess of iodine mono chloride (lei) is added.

Some of the lei reacts with the double bonds in the unsaturated lipids, while
the rest remain~ un reacted. The amount of lei that has reacted is determined
by measuring the amount of lei remaining after the reaction has gone to
completion (lei reacted ;ICI excess - lei remaining) . The amount of lei remaining is
determined by adding excess potassium iOdide in the solution to liberate
iodine, ::nd then titrating with a sodium thiosulfate (Na2S203) 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 goes a
dark blue. The solution is then titrated with a sodium thiosulfate solution of
known molarity. While there is any b remaining in the solution it stays blue,
but once all of the 12 has been converted to 1 - it turns colorless. Thus, a
change in appearance of solution from blue to colorless can be used as the
end-point of the titration.
21
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.
Reaction:
R-CH=CH-R + ICl exce,"",----+

R-CHI-CHCI-R + IClremalning
IClremalnlng + 2KI - - --... KCI + KI + 12
b + starch + 2Na2S203.-----. 2Nal + starch + Na2S406

( Blue Colo pur) (Colourless)
Apparatus and glassware:
Analytical balance (sensitivity 0.1 mg.),
Erlenmeyer flask with ground glass stopper (300-500 ml), Graduated burettes
(50 ml).
Acetic acid Glacial (99%, m.pt 16.6'C), Cyclohexane, Potassium dichromate,

Whatman no 1 or 4 filter paper.
1% (WN) starch indicator solution: Make a paste of 1 gm soluble starch in

30 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 4'C.
Pota~sium 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
22
and make the volume to 1000 ml. Store the solution in a cool place in a dark
coloured stock bottle. After staring the solution for about two weeks, filter if
necessary and standardize as follows:
Weigh accurately about 5 gm of finely ground potassium dichromate which

has been previously dried to a constant weight at 105 ~ 2 in to a clean 1.0 It
volumetric flask. Dissolve in water make up to the mark; shake thoroughly and
keep the solution in dark place. Pipette 25 ml of this solution into a clean glass
stoppered 250m I conical flask. Add 5 ml of concentrated hydrochloric acid
and 15 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 ml of iodine monochloride in about 1800 ml of glacial acetic acid

and shake vigorously. Pipette 5 ml of this, add 10 ml of potassium iodide
so lution ana titrate with 0.1N sodium thiosulphate solution , using starch as
indicator. Adjust the volume of the solution till it is approxi mately 0.2N.
Starch solution: Mix 5 g of soluble starch and 10 mg of mercuric iodide in 30

ml water. Add th is mixture to 1 000 ml of boiling waler and continue to boil for
3 minutes.
23
Procedure
i) Take molten ghee and filter it through Whatmall 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
(Na2S04). 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 cleaf ghee in a clean dried
Erlenmeyer flask.
iii) Add 20 ml cyclohexane (Cyclohexane sen'es 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 if! the dark.
iv) At the end of {he hold, add 20 ml potassium iodide solution. Then
immediately add approximately 150 ml of distilled water, swirl lightly to mix,
and then promptly titrate with vigorous shaking I stirring, with 0.1 N sodium
thiosulphate solution using 50 ml burette. Continue titration until the yeJlow-
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 12 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:
12.69 (a - b) N
Iodine value (IV) =
W
24
Observations:
S.No Sample! Volume of O.1N Sodium Weight Iodine

Blank thiosulfate used olthe value
sample
Initial Final
Vol. used
reading temp.
26
Experiment 8: Estimation of unsaponifiable matter In ghee.
General :
Unsaponifiable matter 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 0 C 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 un saponifiable matter is washed with water to remove
traces of alkali and alkali free solvent is then dried to a constant weight.
Reaction:
H 0
II
H- C- O- C - Rl
o
N
H- C- O- C - R2 +3KOH ---+~ CHOH +3R COOK+ usm
o
N
H- C- O- C- R3
27
Oven mainlained al 100 2C, Boiling waler bath, Flat bottom flask (250 ml
capacity), Air condenser, Separating funnels : 500ml and 250 ml capacity,
Round bottom flask.

Alcoholic potassium hydroxide: 0.5N solution of KOH in 95% ryN) 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 N01 filter pater. Keep the filtered solution in
dark, airtight bottles.
Precaution:Strength of the solution should not be less than O.SN in any case.
Colour of the solution should not be dark yellow (pale yellow is acceptable).
Diethyl ether: Diethyl ether (Specific Gravity at 15.5'C in the range of 0.720
---- ~
to 0.724) Non - volatile residue at eo'c should not exceed 0.001%
Aqueous potassium hydroxide: Approximately 0.5N solution in distilled
water.
Phenolphthalein Indicator: 1% solution in 95% ryN) ethanol.
Procedure:
i) Take 5 gm of molten fat sample in a 250ml flat bottom flask. Add 50 ml
of alcoholic potassium hydroxide solution and 2-3 glass beads, Mix the
contents.
ii) Attach the flask to reflux condenser prope~y. 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 SOOml
capacity separating funnel containing 150 ml of water. (Separating
funnel should be previously rinsed with water and diethyl ether).
iv) Add 100 ml of diethyl ether to the separating funnel . Stopper the
funnel and shake it vigorously by intenmittent removal of vapours to
28
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 250 ml capacity separating funnel
containing 20 ml of water.
\ vii) Extract from aqueous alcoholic soap layer (step v) twice more, each
time with 50 ml of diethyl ether in same manner as mentioned in (Step
Iv). Collect all the etherallayers in the separating funnel ( Step 6).
viii) Wash the ethereal solution twice with 20 ml of distilled water and
shaking vigorously on each occasion.
i.J.) Give successive washings with 20 ml of O.5N aqueous potassium
hydroxide, 20 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 80C/1 hr.
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
Hot electric air oven, Boiling water bath, Flal bottom flask- 2S0 ml capacily,
Nr condenser, Separating funnels: SOOml and 2S0 ml capacily, Round bottom
ftask, Test lubes, Pipettes! Auto pipettes of different capacities
1. Alcoholic potassium hydroxide: O.SN solution of KOH in 9S% 0JN)

elhanol. Dissolve 3S-4Ogm of potassium hydroxide pellets in 20 ml of
distilled waler. Add 1000mi of 9S% elhanol. Nlow the solulion 10 sland
for 24-36 hrs and filler the solution using Whalman N01 filter paper.
Keep the fillered solulion in dark, airtight bottles.
2. Dlethyl ether: Diethyl ether ( Specific Gravily at 1S.S'C in the range
of 0.72010 0.724) Non - volalile residue at SO'C should not exceed
0.001%
3. Aqueous potassium hydroxide: Approximalely O.SN solution in
distilled waler.
4. Phenolphthalein Indicator: 1% solution on 9S% 0JN) ethanol.
S. Liebermann Burchard Reagent (LB- reagent) Take 1.0 ml of
concentraled 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 O'C for at leasl30 min. The acetic anhydride used
in the preparation of reagenl 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
S. Acelic Anhydride- AR Grade.
31
Procedure:
A. Isolation of unsaponifiable matter:
i) Take 5 gm of molten fat sample in a 250 ml flat bottom flask. Add 50 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 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 ml of water.
iv) Add 100 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 tv.Io layers are
separated.
v) Coliect aqueous alcoholic soap layer in a beaker for further extraction.
vi) Coliect etheral layer in a second 250 ml capacity separating funnel
containing 20 ml of water.
vii) Extract aqueous alcoholic soap layer (step v) tv.Iice more, each time
with 50.0ml of diethyl ether in same manner as mentioned in ( Step iv) .
Coliect all the ethereal layers in the separating funnel ( Step 6).
viii) Wash the ethereal solution tv.Iice with 20 ml of distilled water and
shaking vigorously on each occasion.
ix) Give successive washings with 20 ml of 0.5N aqueous Potassium
Hydroxide, 20 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) Tran .rer the washed ethereal solution to a previously weighed conical
flask l; passing through anhydrous sodium sulfate. This will ensure the
remove; l of traces of moisture entrapped in the solvent during washing.
32
xl) Evaporate the solvent on a boiling water bath, and finally transfer tho
flask to an oven maintained at eO'C/1 hr.
xii) Dissolve the un saponifiable matter in acetic acid and transfer the
contents to a 25 ml volumetric flask and make the volume to 25ml
mark. Filter the solution using whatman N01 filter paper.
xll) Take 3.0 ml of this filtered solution in a 25 ml glass stoppered tesl
tube.
xiv) Add 4 ml of LB- reagent to the above tube and allow it to stand for
35min at 25'C for colour development. Record the optical density al
650 nm against a blank consisting of 3.0ml acetic acid and 4 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 ml of the
stock solution in a 25 ml volumetric flask. Make the volume to 25 ml
with acetic acid.
ill) 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 ml of LB- reagent in all the tubes and keep them in a water
bath maintained at 25'C/10 min. Take absorbance at 650nm.
v) Plot absorbance readings against the corresponding cholesterOl
amount (~g) to get a standard curve.
vi) Amount of cholesterol present in the sample can be calculated from
the standard curve.
33
Observation.:
Reagents (mil Blank Tubes Number

2 3 4 5 6 7 S1 S2
Standard Cholesterol 0 O.SO 1.00 1.S 2.0 2.S 3.0 - -
working solution
Diluted unsaponifiable - - - - - - - 3.0 3,0
matter (mil
Acetic acid 3.0 2.S0 2.00 1.S 1.0 O.S 0.0 - -
LB- Reagent 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0
Keep at 2S'C/10 min
0 .0 at 6S0 nm
Calculations:
Standard stock solution is 0.5% cholesterol in acetic acid .
Or
SOOmg cholesterol 1 100 ml acetic acid
or
Smg 1 1.0 ml
or
SOOO ~ 9 11.0 ml acetic acid
We have take 1.0 ml of stock solution and volume is made to 2S .0 ml with
acetic acid.
Therefore,
2S.0 ml of working solution contains SOOO ~ 9 cholesterol
1.0 ml of working solution contains 200 ~ 9 cholesterol
O.S ml of working solution contains 100 ~ 9 cholesterol
From the graph :
3.0 ml of diluted unsaponifiable matter contains; Y ~ 9 cholesterol
2SY
25.0 ml of diluted unsaponifiable matter contains;. -------- ~ 9 cholesterol
3
; Z ~ 9 cholesterol
Weight of ghee sample taken; W gm.
34
So, W gm of ghee contains = Z
W
100 9 of ghee contains = Z
-------- X 100 ~ 9 cholesterol
W
35
Experiment 10: Colorimetric estimation of total cholesterol In ghee by
direct method.
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.
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 calorimetrically at 650nm 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:
+ H2SO, -+
From LBreagent
cftJlesleroi
-'--'._-
Dehydration action by H2S04
1
or
Purple colour
36
i) Liebermann Burchard Reagent (LB. reagent) Take 1 ml of
concentrated sulphuric acid in a conical flask Add 20 ml of acetic anhydride
and mix the contents by keeping the flask in cold water. Keep the reagent at
DOC 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 Anh ydride AR Grade.
Glass Ware: i) Volumetric Flask (25ml), Conical Flask (150ml), Glass

stoppered test tubes(1 Oml), Pipette
Procedure:
(I) Take 5 gm of molten, clear anhydrous milk fat in a 25 ml volumetric
flask. Dissolve in chloroform and make the volume to 25 ml with
chloroform.
(ii) Pipette out 2 ml of this above solution in a glass stoppered test
tube.
(iii) To this add 1 ml of chloroform and 4 ml of freshly prepared cold LB
reagent. Incubate the tubes at 25'C 110 min in a water bath to
develop the colour.
(iv) Take absorbance at 650nm using a red filter.
Preparation of Standard Curve:

i) PreparcULon of cholesterol stock solution: Take 0.5 gm of
,
cholesterols tandard . Dissolve it in chloroform and transfer the
contents to a 100ml volumetric flask. Make the volume to 100ml
with chloroform.
Ii) Preparation of cholesterol working solution: Take 2 ml of the
stock solution in a 25 ml volumetric flask. Make the volume to 25 ml
with chloroform.
37
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 chloroform in
respective tubes.
iv) Add 4 ml of LB- reagent in all the tubes and keep them in a water
bath maintained at 25'C/l0 min. Take absorbance at 650nm.
v) Plot absorbance readings against the corresponding cholesterol
amount (~g) to get a standard curve.
vi) 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 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 25' C/l 0 min
Take Absorbance at
650nm
Calculations:
0.5% cholesterol stock solution "0.5gmil OOml conc. " 500mg/l OOml
Therefore
100ml contains 500mg of cholesterol
38
1.0ml contains = 5001100 = 5mg cholesterol or 5000 ~g
Since, 25ml of working solution contains 2.0 ml of stock solution.

So,
25 ml of working solution" 1000 ~g cholesterol
0.25 ml of working solution" 100 ~g cholesterol
Let us say
2.0ml of prepared sample contains x ~g of cholesterol.
Then 25.0 ml of sample will have (2512) x ~g of cholesterol
Since this 25.0 ml contains 5.0 gm of ghee

So, 5.0 gm of ghee sample contains (25/2)x ~g of cholesterol
100 gm of ghee will contain 250x ~g or 0.25x mg of cholesterol.
39
Since the presence of lower chain fatty acids is peculiar to ruminant milk fat,
tile RM and PV are important characteristics of ghee. The actual figures for
botll 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: ;I (
Reichert Mels.1 Value (RM): RM value is the number milliliters of 0.1N

aqueous alkali solution required to neutralize the steam volatile and "Yater
soluble fatty acids distilled from 5 9 of ghee Ifat under specified conditions .
.1- ~t O
~olen.ke 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 s01uble 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.
41
Reactions:
Q' Saponmeatlon
H 0
II
H-C-0-C-R1
N
o
H-C- O - C-R2 +3NaOH ._ _ CHOH + 3 R COONa
,",' 0
H- C- 0 - C - R3 CH,OH
II} Aeldlfeatlon:
2 R COONa + H, SO, - - - 0 ' 2 RCOOH + Na, SO,
IiQ Titration:
RCOOH + NaOH -,---0' RCOONa + H,O
Apparalu. and Glass ware:

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

Glycerol (98% WIW), NaOH (50% WIW), Oil. H, SO, (App. 25ml of conc.
o'
sulphuric acid is diluted to one litre and adjusted until 40ml of it neutralizes 2.0
ml of 50% NaOH solution), Sodium Hydroxide solution ( N110),
Phenolphthalein indicator, Ethyl alcohol (95% VN), Whalmsn No 4 filter paper
9cm dia.
42
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 ove r a naked flame ,
with continuous mixi ng , until ghee in cluding any drops adhering to the upper
pa!lJ.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 overwheating 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.
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 11 0 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 ' C and 21 ' 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 ,
43
place in water at 15'C 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 (Wnatman 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.
Relchert-Melssl or Soluble VolaUie Acid Value
Pour 100 ml of the filtrate containing the soluble volatile adds into a
titration flask, add 0.1 ml of phenolphthalein indicator and titrate with 0.1 N
sodium hydroxide solution until the liquid becomes pink. Note the burette
reading as ' 5' 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, nea~y filling the paper 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 "S1
in case of bl ank.
44
Observations &Calculations
Reichert-Meissl value (R.M value) ; 1.10 (S - B )

Polenske value (PV value) ; (S,- B)
IMlere :
S = Volume in ml of 0.1N sodium hydroxide solution used for sample of

RM .
B ; Volume in ml of 0.1N sodium hydroxide solution used for blank of RM
81 = Volume in ml of 0.1 N sodium hydroxide solution used for sample of
PV
II, ; Volume in ml of 0.1N sodium hydroxide solution used for blank 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.
45
Experiment 12 : 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
AO 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
ethcr 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- quinonc-chlorimide) which permits the quantitative
estimation of BHA in the presence of other antioxidants wit-lout their
interference. The blue color of the Indophenol resulting from the reaction
exhibits maximum absorbance at 620nm and is stable for 5 hrs.
46
ReacHon:
H o
C (CH,h CI II CI
~I I
V
+ Na,BO
pH 4.4 OCH,
I I
OCH3 NCI N
(BHA) 2,6-dichIQrochlorimide o C(CH, )
OH
Blue indophhenol
Spectrophotometer, Separating funnel , test tubes, beakers, pipiettes.
1. Gibb's reagent (0.01 % solution of -,6 dichloroquinone chlorimide in 95%

methanol) 2. Borax solution - 0.5% (Prepared by dissolving disodium
tetraborate in "farm D. W)
3. n-Butanol
4. Absolute methanol
5. Antioxidant (BHA)
6. Ca Co)
7. Whatman No.1 filter paper

8. Extraction solvent: Saturate hexane and acetonitrile by shaking them
together for 2 minutes and thl:n separate. Use these saturated solvent for the
extraction of BHA and BHT.
47
I
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 rnl 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 ovemight
under refrigeration.
Evaporate the extract completely at 40C, 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 CaCO, 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 :
(i) 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 thc blue coloured solution in a
colorimeter at 620 nm (red filter) against a reagent blank prepared
under identical conditions.
(Ii) For the preparation of standard curve of BHA, take 0.1, 0.2, 0.3, 0.4
48
Calculations:
From the graph:
n
Let the optical density for sample be "x and corresponding BHA cone. from
the graph be "yo.
2.0 ml of sample filtrate contains"y" ~g 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 ~g of BHA.
2
The diluted sample is prepared from 25.0 ml aliquots

Hence 25.0 ml aliquot contains 25 y ~g of BHA.
100.0 n1 aliquot contains 25 y

--------- X 100 = 100 Y ~g of BHA
25
Since 100.0 ml of diluted sample contains 10.0 9 ghee

So, 10.0 9 ghee contains 100 y ~g of BHA
100.0 9 ghee contains 1000 y ~g of BHA
50
Experiment 13: Determination of Vitamin D in ghee.
General:
Vitamin 0 is present in milk or milk products as part of the unsaponifiable
matter in the lipid phase. The vitamin 0 content in milk is about 23 IUllitre (
13-33IU/lilre) and in ghee is about 10 lUI gm fat (8-13 lUI gm fat).
Principle:
For the determination of vitamin 0 , the milk product is saponified using
alcoholic KOH and the unsaponifiable matter containing fat soluble vitamins
like vitamin 0 is extracted with solvent ether. After evaporation of ether, the
residue is dissolved in chloroform . The chloroformic solution of vitamin 0 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 0 is
calculated from the standard curve which is prepared by treating different
known concentrations of vitamin D with the colour reagents .
Reaction:
Vitamin D + SbCl, - - - - - orange - yellow colour

Saponification flask, Glass air condenser, Glass funnels , Separating funnel (
300ml). Separating funnel (250ml), Glass beads. Water bath. Oven.
Spectrophotometer.
Chemicdls and Reagents:
i) 50% KOH . ii) Ethyl alcohol. iii) Oiethyl ether ( peroxide free) . iv) Vitamin D
standard for preparation of standard curve. v) Anhydrous sodium sulfate, vi)
Whatman. No- 40 filter paper, vii) Chloroform.
viii ) Colour reagent:
Solution A: Dissolve 20gm of SbCt, in 90 ml of methylene chloride .
SolutionS: Mix 10 ml acetyl chloride with 40 ml methylene chloride. Store in
~ 'I
" - II
refrigerator before use. r
51
Mix solution A and B in the ratio (45:5), respectively. The mixture is stable for
one week at room temperature.
Working solution of vitamin D: 10 I.U I ml in chloroform
Procedure:
i) Saponification: Weigh accurately about 5 gm of ghee sample in a
saponification fiask. 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 th is chloroform solution in the cellar cuvette of
spectrophotometer and add 4ml of the colour reagent. Prepare a
blank also in a similar manner. After 10 min, measure the 0.0 of
the orange - yellow coloured solution at 500 nm using green filter
against a reagent blank.
iv) For the preparation of standard curve , prepare the standard solution
of pure vitamin 0 and treat with same amount of reagents . Range of
2-10 IU can be used here.
52
Observations:
Reagents Test Tubes
B 1 2 3 4 5 S
ViI. 0 working 0 0.2 0.4 0.6 0.8 1.0 -
solution
( 10 ioU/mil
Extract of - - - - - - 1.0
sample (mil
Chloroform 1.0 0.8 0.6 0.4 0.2 0.0 0.0
(mil
Colour reagent 4.0 4.0 4.0 4.0 4.0 4.0 4.0
Keep at room temp. for 10 min

0 .0 at 500 nm
Calculations:
Say the 0 .0 for sample; x
Corresponding cone. 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 ofghee has; 5 y I.U
100 gm ofghee has; 5y X 100/ 51.U
;100yt.U
53
Experiment 14: 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 A and E. The details of estimation 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.
+ SbCI, ---+. Blue Colour

yitamin A
54
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%, VN) , Oiethyl ether- peroxide free (prepared by adding
FeSO.. solvent ether and keeping overnight.) , Potassium hydroxide solution,
Aqueous (50%, WN) 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 I 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 airlwater
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 phenol phthalin). Filter the ether layer through anhydrous
Na2SO. 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
55
from Na2S0. can be checked by adding a few drops of SbCl, 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 ml of pure CHCI, for blank in a photometric cell tube, add 4 ml of

SbCI, solution with cylinder and set the instrument (spectrophotometer) for
100% transmittance at 620 nm. In another cell tube , take1 .0 ml chioroformic
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 SbCh 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 CHCI3 so as to give a
range of 10-50 IU per ml. Take 1 ml each of dilute standard solutions of
vitamin A, develop the colour with 4 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.
56
Observations:
Reagents Test Tubes
B 1 2 3 4 5 S
Vi!. A working 0 0.2 0.4 0.6 0.8 1.0 -
solution
( 50 LU/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
SbCI3
0 .0 at 620 nm
Calculations:
Read from the graph the concentration of vitamin A corresponding to the

absorbance (0.0.) of the sample.
Say, the 0 .0 . for 1 ml sample is X and corresponding
concentration from graph. ;;: Y I.u.
1.0 ml CHCI 3 solution of samples has = Y.LU.

5.0 ml CHCI3solution of samples has = Yx5.0 LU.
i.e. W gm sample solution of samples has;;: Yx 5.0 I.U.
Yx 5.0xl00LU .
100 W gm sample solution of samples has =
W
57
Experiment 15: Detennination of Vitamin E in ghee.
Principle:
Estimation of vitamin E is based on EmmerieEngel reaction in which vitamin
E (tocopherols) reduces ferric chloride to ferrous chloride which reacts with
a ,6dipyridyl to produce red colour whose intensity is measured at 530 nm
using green filter. For its determination in ghee, the un saponifiable 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 carotenOlds.
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 a,a -dipyridyl reagent. Vitamin E content in ghee is about 30
ug/gm.
Reaction:
HO + FeCI, ---+. FeCI,

Cf; Cf;
H,C
Vitamin E
Toco pherol
FeCI2 + a,a -dipyridyl Red color complex

Spectrophotometer: for reading at 530 nm, Water bath. , separating funnel ,
saponification flask, Test tubes, beakers.
Reagents :
Ethyl alcohol-absolute. Diethyl ether- peroxide free (prepared by adding
FeSO.7H 20 to solvent ether and keeping overnight. , Sodium sulphate
58
anhydrous, Potassium hydroxide solution aqueous (2%. WN), Potassium
hydroxide solution Aqueous (50 % WN) , 0 ,6 --<lipyridyl solution in absolute
ethyl alcohol. (0.5% WN)
Ferric chloride (FeCI,.6H, O) solution in absolute ethyl alcohol. (0.2% WN)
Benzene (A.R. grade), Hydrochloric acid-sp. gr. 1.16.
Florldine XS earth column: Fill a 12x30mm tube with the purified absorbent
(To purify, digest on a boiling water bath for one hour with HCI. Wash with
water until free of acid, then with ethyl alcohol, and with benzene. Dry at room
temperature).
Vitamin E: Reference standard.
Procedure:
aJ Saponification af the sample
Weigh accurately about 5 gm of ghee sample in a saponification flask . Add 40

ml ethyl alcohol and 7 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 unsaponifiable matter
Cool the refluxed material, add about 150 ml 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 ethe;- extract with 2% aqueous potassium hydroxide solution and then with
water till the wash water is alkali free. Filter the ether layer through anhydrous
Na,SO, and Whatman .No. 40 filter paper. Rinse the anhydrous Na, SO, with
solvent ether. Add 2-3 glass beads in the flask containing ether extract and
evaporate to dryness over a water bath. Dissol ve the dried residue in about 5
ml benzene.
59
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% FeCI, 6.H, O solution and 1 ml of 0.5% a,6 - dfpyridyl 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 0 .0 . of the red coloured solution at 530 nm.
e) Preparation of standard curve
Prepare working standard solutions ( 20 ~g I mI.) of pure a-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 ~g . 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.
60
Observations:
Reagents Test Tubes
B 1 2 3 4 5 5
ViI. E working 0. 0 1.0 2.0 3. 0 4.0 5.0 -
solution
(20~g /ml)
Corresponding 0 20 40 60 80 100 -
conc.of vitamin
E (~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)
FeCI, (ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0
a ,a-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)
0 .0 at 530 nm
Calculations
Read from the graph the concentration of vitamin E corresponding to the

absorbance of the sample.
Say, the 0.0 . for sample be X and the corresponding concentration from
graph ~ Y ~g
5 ml of alcoholic solution of sample has ~ Y ~g
61
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 ~g of vitamin E
5 5
62
Experiment 16: Estimation of phospholipids in ghee and ghee- residue.
General:
The total phospholipids contents of 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 proced ure given below.
Apparatus and Glass ware : Spectrophotometer, Heater, test tubes,
volumetric flasks ( 25ml), Kjeldahl flasks, pipettes, separating funnel.
Chemicals and Reagents : Conc. HN03 (A.R), Conc. H2S04 , 0.44%
Ammonium molybedate solution in distilled water, Sulphuric acid H2S04 (5% )
Reducing reagent: Mix 0.25 gm of 1-amino-2-napthol-4-sulfonic acid

(ANSA), 15 gm of sodium bisulfite and O.5gm of anhydrous sodium sulfite in
100ml distilled water and filter.
Standard solution of potassium dihydrogen phosphate (KH,PO,) having

a concentration of 1 pglml: 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% H2S04 to get the final con . of 1 ~g /ml.
87% ethyl alcohol ( Pre - equilibrated with petroleum ether).

Petroleum ether BPt. 40 - 60'C (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.
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.
63
B) Extraction: Extract the phospholipids from 10 gm of ghee by adopting
counter current distribution technique using 87% 01N) 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
(vi) 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. H2 SO, & 5 ml of conc. HN03 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.
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.
64
(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 ~g of phosphorous per ml as standard .
2. Estimation of phosphorous 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 NaCI 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 mi x.
(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 .
(viii) Combine all the extracts and evaporate to dryness over a boiling
water bath.
Add 0.5% sulphuric acid and make the volume to 100 mL
Subsequent Digestion and estimation steps are similar as described
for ghee at C & D above.
65
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% (mil 4.5 - -
Aliquot of digested diluted solution - - 5.0
(mil
Standard solution (ml) - 5.0 -
0.44% ammonium molybedate 4.6 4.6 4.6
solution (mil
Reducina aaent [ml) 0.4 0.4 0.4
Keep in boiling water bath for 7
min
O.D at 720 nm I I
Phospholipids in ghee- residue:
Reagents Blank Standard Sample

Sulphuric acid 0.5% (ml) 4.5 - -
Aliquot of digested diluted solution - - 5.0
(mil
Standard solution (mil - 5.0 -
0.44% ammonium molybedate 4.6 4.6 4.6
solution (mil
Reducing agent (mil 0.4 0.4 0.4
Keep in boiling waler bath for 7
min
O.D at 720 nm I I
66
Calculations:
Conversion factor of phosphorous to total phospholipids = 25.9 X P
Say
0 .0 of sample = x
0.0 of standard = y
Y corresponds to a conc. of 11-1 9

Therefore
x
x corresponds to a con . of ---------- X 11-/9
Y
x 1
= ---------- X --------- mg ( to convert to mgW 9
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 9 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
0.0 of sample 100
= -------------------- X 0.1 X ----------- X 25.9
0.0 of standard W
67
Experiment 17: 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 CQuid 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 SA) activated at 200 -
300 ' 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 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
68
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 - 170~m)
or 80 -100 mesh (170 -149 ~m). 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 0 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 n of
vapor because slow injection or oversized samples cause band spreading and
poor resolution . Sample size depends upon the sensitivity of the detector. For
Jrdinary packed analytical columns, sample sizes range from 2 to 20 ~I.
::;apillary columns require samples that are smaller by a factor of 100 or more.
69
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 ~I )
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
tumings ( 5 g magnesium turnings washed with 5 - 10% HCI, 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 tumings are
completely solublized. Dis~iI the refluxed mixture at 50 - 60 C, 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 N01 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
the tube. Heat the sealed tube in an oven maintained at 80 CI 1-2 hrs for
esterification of fatty acids until a uniform-layer is formed .
2. Set the GLC equipment in the following manner:
;) Switch on the GLC and set Injection port temp 240'C.
70
ii) FlO temp (250'C), sensitivity 10X, Polarity etc.
iii) Program oven where , initial temp 55C followed by a rise

@10'C!mintiI1220'C.
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 FlO 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- 240C 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.
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.
71
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 =1.0em, b =1.6 em and h =17.7 em
Area = Y, h (a+b)
= y, X 17.7 ( 1+1 .6)
= 23.01 em'
Area of a triangle:
From the graph as in peak B
b = 0.8em, h = 1.9 em
Area of a triangle = Y:z bh
= Y, X 0.8 X 1.9
= 0.76 em'
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
72
Suggested Readings:
1. Hand book of food analysis. Part XI . 1981 . BUreau 01 Indian Standards.

Manak Bhavan.9. Bahadur Shah Zalar Marg. New Delhi.
2. RonaI.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 &Son5 , Inc, Hobokon,
New Jersey.
3. Ramamurthy. M.K and Narayanan. K.M. 1966. Ind J Dairy Sci. 19 . 45.
4. Parrish, 0.8. 1979, Determoination of vitamin 0 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.
73
" ,
Open - Tubular or Capillary Columns
Packed Column
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IDisuHol\\;11,l's Gu.idf'
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Lin t . - 0, 1' 0 Top
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SupportUl!!. l\1l'tal
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Canil'1 Silicon' ' O\'en Top
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;,

NTD - Lab Manual of Analysis of Milk Lipids (Ghee) - 2007 - India

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A LABORATORY MANUAL

{(~\zt ~ 31iJIitrrat l:imrcr

NATIONAL DAIRY RESEARCH INSTITUTE

phospholipids and cholesterol. Today most undergraduates and

The manual consists of detailed information on several

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

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

YEAR-200? Dr. Vivek Sharma

Support and encouragement from Director, National Dairy Research

Institute, Karnal is thankfully acknowledged. The authors are also

educational programme at reAR institutes .

Dr. Vivek Sharma

Cover Design: Vlvek Shanna

First Edition: March, 2007

Published by: Director,

Printed at: Intech Graphics

S.No Name of Experiment Page

Determination of peroxide value in ghee.

estimation of cholesterol in ghee by i i

Estimation of vitamin A in ghee.

Therefore, if observations are taken at temperature other than 40C the

Refraclive index = 1.4538 + Factor (observed B.R. - 42)

Butyro refractometer, Circulatory water bath maintained at 40C , Glass rod,

Chemicals and Reagents:

Apparatus and Glass ware:

Initial Final Slip temperature

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

Definition: The FFA is defined as the percentage by weight of free acid

Principle of the method

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

Chemicals and Reagents:

Ethanol (Ethyl alcohol) :- 95% ethanol or rectified spirit.

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

Standard aqueous potassium hydroxide or sodium hydroxide solution

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

ml NaOH used X Normalily of NaOH X 2.82

Mol. weight of Oleic acid = 282.

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

S.No Sample Volume of O.1N NaOH used Weight

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

ii) 0.5 X mmol active oxygen! Kg

iii) 8 X milligram-equivalents of oxygen

Principle of the method:

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

ROOH + Kl excess ROH + KOH + 12

Apparatus and Glass ware:

Chemicals and Reagents:

Sodium thiosulfate pentahydrate ( Na 2S20,.5H 20 , M.wt; 248.17) AR grade.

Sodium Carbonate (Na2CO,),

Potassium iodide (KI), iodate free

Hydrochloric acid (6.0 M)

0.1 N Sodium thiosulfate solution: Add 25 gm of sodium thiosulfate

Calculate the normality of the sodium thiosulfate solution as follows:

W= weight in grams of KIO,

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

Mixed Solvent: 2:1 01N) glacial acetic acid! chloroform solution

i) Accurately weigh 1 .!.-0.05 gm of molten fat into a 250 ml glass stoppered

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

Expressed as meq active oxygen I Kg sample.

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.