PRACTICAL 6 LIPIDS

6.1 Introduction
6.2 Lipids-Basic Concepts
6.2.1 Neutral Fats- Characterization and Assessment
6.2.2 Cholesterol- The Derived Lipid
Experiment 1:Determination of the Iodine Number of Lipids using Hanus Method
Experiment 2: Determination of the Saponification Number of Fats
Experiment 3: Estimation of Cholesterol using Zlatkis Method
-
6.1 INTRODUCTION
You have studied that lipids together with carbohydrates and proteins are important
as food for many animals. Lipids include neutral fats, phospholipids, cholesterol etc.
The lipids are also of great biochemical importance because of their role as chief
storage form of energy and in cellular structure. In addition, they are commercially
important in their role in detergents, soaps and in the chemical and pharmaceutical
industry. Fats, because of high degree of concern over health implications and also its
extensive use in preparation of food products, have become the area o f interest in

Various parameters are used for the assessment of the quality of fats and oils. Some
of the parameters including determination of moisture content, colour, impurities, acid
value, peroxide value and tests for the presence of adulterants in fats and oils, we
have already covered in the Principles of Food Science Practical Course - MFNL-
008. In this practical, we shall learn about other tests which will help us in
characterization of fats and help us assess the purity of fats and oils. Bureau of
Indian Standards (BIS) has standardized procedures available for these tests with
recommended values and limits. We will be doing some of these tests to assess the
purity of fats and oils in this practical. Using the combination of these tests, you will
realize, you can determine the identity, the structure, the functional groups, physical
properties etc. of the lipid components. Further, the practical shall also focus on
methods used for estimation of cholesterol in a given solution.

After studying this practical and undertaking the experiments given herewith you will

perform tests for partial characterization of fats,

differentiate between adulterated and non-adulterated fats and oils,
determine the purity of fats and oils based on physical and chemical tests,
interpret the results obtained, and
estimate the amount ofcholesterol in a given solution.

6.2 LIPIDS - BASIC CONCEPTS

You have studied the classification of lipids in your theory lesson. Lipids, you learnt, are
classified as simple, compound or derived lipids on the basis of their chemical structure.
Simple lipids are esters of fatty acids with various alcohols. They include neutral fats
(which are esters of fatty acid with glycerol. Compound lipids are esters of fatty acids
containing groups in addition to an alcohol and a fatty acid. Examples include
phospholipids, glycolipids etc. Derived liplds are substances derived from above groups
by hydrolysis. This group includes fatty acids, glycerol, steroids, sterols, fatty aldehydes
and ketone bodies, vitamin A, D, E and K etc. Cholesterol belongs to the family of
steroids. Let us learn about neutral fats and the tests involved in the assessment oftheir
qual~tyin the next sub-section
157
Nutritional 6.2.1 Neutral Fats -.Characterization and Assessment
Biochemistry

and fatty acids.

triglyceride glycerol fatty acid

Figure 6.1: Triglyceride

When fats are in liquid form they are called oils.

Fatty acids are grouped into two classes - saturated and unsaturhted fatty acids. When
a fatty acid contains one or more double bonds (4= C-), it is said to be unsaturated,
otherwise it is a saturated fatty acid. They are esterified to glycerol in several permutations
and combinations resulting in varying composition of fats and oils. If all the three fatty
acids present in a triglyceride are the same, then the fat is referred to as simple glyceride
and if they vary then it is referred to as mixed glyceride.
A list of fatty acids present in natural fats in given in Table 6.1. You will find the
food sources ofthese fatty acids also included in this table for your information.

Table 6.1: Fatty acids in natural fats

No. of Structure Name Source
carbon
atoms
*
4 CH,CH$H,COOH Butyric acid Butter
6 CH,(CH,),COOH Caproic acid Butter
8 CH,(CHJ,COOH Caprylic acid Coconut oil
10 CH,(CY)FOOH Capric acid Coconut oil
12 CH,(CH,),,COOH Lauric acid Palm
kernel oil
14 CH, (CH, ),, COOH Myristicscid Oil of nutmeg
16 CH, (CH2),4COOH Palmitic acid Palm oil
18 CH,(CH,),,COOH Stearic acid Beef tallow
18 CH,(CH,),CH= CH(CH,),COOH Oleic acid Olive oil
18 CH,(CH,),CH= CHCH,CH= CH(CH2),COOH Linolenic acid Soyabean oil
18 CH,CH,(CH= CHCH2)3(CH2)6COOH Linolenic acid Fish oil
20 CH,(CH,),(CH= CHCH,),CH,CH,COOH Arachidonic Liver
acid

The properties of fats areto a large extent dependent on the properties of the constituent
fatty acids. The solubility, melting and boiling points vary with the varying chain length
and the degree of unsaturation. You may recall studying about the chemical properties
of fatty acids in the Nutritional Biochemistry Theory Course (MFN-002) in Unit 2,
section 2.4, which include esterification, hydrogenation, halogenation etc. Recognition
of these properties, you will realize, is ~mpottantfor a better understanding of fatty
acids. Along with the chemical properties of fatty acid, we have also looked at
saponification, hydrogenation, hydrolysis which are chemical properties specific to neutral
To determine the purity of fats and oils, several physical, chemical and puri9 tests are Lipids
performed. Some of them we have already covered in the MFNL-008 course. Bureau of
Indian Standards (BIS) has standardized procedures available for these tests with
recommended values and limits. Two important tests are: Iodine Number Estimation and
determining the saponification number of fats. We will be doing these tests to assess the
purity of fats and oils. These estimations will help in characterization of fats.
Let us get to know about these tests. We begin with the Iodine Number Estimation,

A. Iodine Number Estimation

As you may recall studying in the theory course, in Unit 2, fatty acids accept chlorine,
bromine and iodine at the double bond when treated with reagents such as iodine
monochloride and a fatty halide is formed. This is the halogenation property of
unsaturated fats and is used in iodine number estimation.
Let us get to know the principle behind this estimation.

Iodine number is defined as the number of grams of iodine absorbed by 100 grams
of fat. .,lgives an idea of the relative degree of unsaturation of fats or the amount of-
douMe bonds present in the fats. Iodine number is directly proportional to the degree of

In actual practice, Iodine monobromide (IBr) (Hanus method) or Iodine monochloride

(ICI) (Wij's method) is used in preference to iodine solution, as these are more reactive
and give more reliable and reproducible results. The method is based on the absorption
of halogen by the unsaturated fatty acids present in fats.
The method employs dissolution of fat in chloroform (CHCI,) and treatment with a
known excess of halogen solution. After allowing for absorption of the halogen, the
excess halogen is liberated as I, and is back titrated against standard sodium thiosulphate
solution using starch as indicator. By using an excess of sodium thiosulphite to react
with each double bond in the fatty acid chains and measuring the amount used by back
titrating, we can determine the amount of sodium thiosulphate used and thus the relative
nuniber of double bonds.
We will conduct this experiment later in this practical. Next, let us also learn about the

B. h ~ n i f i c a t i o nNumber of Fats
F k t let us begin by understanding what is meant by saponification. Fats when boiled
with alcoholic solution of NaOH or KOH undergo hydrolysis into glycerol and fatty,
acids and the latter form soaps with Na or K. The reaction is known as saponification.
The sapdnification value is the number of milligram ofpotassium hydroxide required
tosaponifi 1 goffat under the conditions specifled. In other terms, the saponification
number of fat or oil is the number of milligrams of KOH required to neutralize free or
combined fatty acids in one gram of oil or fat. ~ a ~ i n i f i c a t i onumber
n indicates the
molecular weight of the fatty acids present in the oil or fat and is inversely
proportional to the molecular weight of fat. Since natural fats and oils are mixtures
of fatty acids, it becomes an academic exercise to calculate the saponification number
for the component fatty acids. The saponification number 6f a fat is determined by
hydrolysis of the fat in an excess standard alcoholic alkali and determination of the
excess alkali titrimetrically. Let us get to know the principle behind saponification.

159
Yutritional the number of fatty acid chains in the solution. In this experiment, the hydrolyzing agent
Biochemistry is KOH. By using an excess of KOH and back titrating the unused excess with
hydrochloric acid, we will know the number of moles of KOH used.
Like iodine number, this method is also used for estimation of purity of fat. A given
amount of fat or oil is refluxed with a known excess of alcoholic KOH and then the
excess is back titrated against standard HCI using phenolphthalein as an indicator.
This concept will become more clearer as we carry out this technique in Experiment 2
included in this practical.
Next, we shall learn about cholestrol and study the methods we can use in the lsooratory
for its estimation.

6.2.2 Cholesterol
Cholesterol is an acyclic compound whose structure includes a cyclopentano
perhydrophenanthrene nucleus with four fused rings, a single hydroxyl group at carbon
3, double bonds between carbons 5 and six. a hydrocarbon chain with 8 carbon atoms
attached to carbon 17 and 2 methyl groups at carbon 10 and 13.

Can you draw the structure of cholesterol with this information? Try completing the
structure given herewith where the cyclopentano perhydrophenanthrene nucleus with
four fused rings is already illustrated.

Now refer to the cholesterol structure given in Figure 6.2, and judge your understanding
of the structure.

Figure 6.2: Cholesterol

Cholesterol, we already know, belongs to the family of steroids. It is poorly soluble in

water but soluble in fat solvents. It has an alcoholic group and thus forms esters with
fatty'acids. In the body cholesterol is esterified with oleic, linoleic and palmitoleic acid.
Thus, 70% of the cholesterol present in the blood is esterified whereas only 30% of
cholesterol occurs as free cholesterol.
The two sources of cholesterol in the body are:
1. Dietary cholesterol - Dietary cholesterol is mainly obtained from foods of animal
origin.
2. De novo synthesis -Cholesterol is synthesized in every living cell. The major sites
of synthesis are liver, adrenal cortex, aorta, intestine, brain etc.The biosynthesis
involves a complex pathway consisting of 24 steps, about which you may recall
studying in the theory Course, Unit 7. If not, we suggest you get back to the theory
course. Look up Unit 7, sub-section 7.3.3, page 213 for studying the biosynthesis
process of cholesterol. The starting material for cholesterol synthesis, you would
160 have noticed. is acetyl CoA.
Cholesterol is vital for the body to function properly i.e. for hormones and the bile acid Lipids
production. Elevated cholesterol levels, on the other hand, have been associated with an
increased risk of atherosclerosis (narrowing of the arteries because of fatty deposits).
Hence, the importance of estimating cholesterol. Let us then move on to the methods
we can use for estimation of cholesterol.

Estimation of cholesterol
Many methods for estimation of cholesterol are available, and have been used, which
are both chemical and enzymatic. Most of the chemical methods used until recently
were based on the ability of cholesterol to be converted to highly coloured substances in
strong acid solvents that possess dehydrating, oxidizing and sulphonating properties.
For many years, the Liberman Burchard Reaction was used for estimation of cholesterol
which gave a green colour resulting from a reaction of cholesterol with acetic anhydride
and sulphuric acid. At present, in chemical methods, the method of Zlatkis et al, 195.8
is most popular. This is a colorimetric method. Colorimetric methods are based on the
ability of cholesterol to be converted to highly coloured substances in solvents which
have dehydrating, oxidizing and sulphonating properties. Let us study this method of
estimation of cholesterol. We begin with the principle behind this estimation.

Zlatkis and associates have evolved a method of quantitative determination of cholesterol

based on a reagent containingferric chloride, glacial acetic acid and concentrated sulphuric
acid. Cholesterol gives a purple colour with this reagent. The colour development is due
to a dehydration reaction in the cholesterol molecule to form 3,5 cholestadiene which is
oxidized by H,S04 and polymerises to form a diamer or trimer. The cholastadiene and
its polymers react with H,S04 to form mono and disulfonic acids which are highly
coloured. In the presence of added metal ions like ferric, the disulphonic acids are
preferentially formed.

The detailed step-by-stepprocedure for cholesterol estimation is included in Experiment 3

later in this practical. Study the procedure carefully before conducting the experiment.

With this basic understandingabout iodine number, saponification technique and methods
for cholesterol estimation, we are well equipped to carry out the experiments given in
this practical. There are three experiments in this practical. Let us get started with

161
 DETERMINATION OF THE IODINE NUMBER OF

Date: .......................... Aim: To determine the Iodine Number of mustard, sesame or groundnut oil by Hanus
methqd.

Reagents
The following reagents are required to conduct this experiment.
1. Hanus solution (Iodine monobromide solution).
2. Chloroform
3. 15% potassium iodide solution
4. Standard sodium thiosulfate solution - 0.1 N
5. 1 % starch solution

Principle
(We have already described the principle in section 6.2 above. Read and understand
the principle and write the same in the space provided herewith).

Procedure
The technique is conducted in two steps. Follow the procedure given herewith and
carry out the experiment.

1. Sample titration
With a clean dry pipette, accurately measure 0.3 mL of oil into a clew and dry idination
flask (DuplicateJask required). AlternativeIy, weigh and transfer 0.3 g of the oil into
the flask for better accuracy. Add 10,ml CHCI, with a measuring cylinder and mix.
Using an automatic pipette, add exactly 25 ml Hanus iodine solution. Mix well. Stopper
the flask. Add distilled water in the cup around the stopper to prevent loss of iodine.
Keep the flask in the dark for 30-40 minutes. Remove the stopper and let the distilled
water in the cup fall into the flask. Add 10 ml of 15% KI solution and 100 ml of freshly
boiled and cooled distilled water. Shake. Titrate the iodine liberated in the process with
standard sodium thiosulfate solution (0.1 N) stopperingthe flask and shaking vigorously
from time to time while titrating until a pale yellow colour is obtained. Now add 2 ml of
0.5% starch solution. Continue the titration until the blue colour disappears. After the
blue colour disappears from the aqueous phase, note the chloroform layer in the bottom
of the flask which may contain untitrated iodine as indicated by a pink or violet colour.
By continuous shaking bring this iodine into the aqueous layer. The end pojnt of the
titration is reached when the aqueous layer, as well as, the lower chloroform layer are
completely colourless.
162 Titrate the duplicate iodine flask in a similar manner.
Using an autopipette, measure 25 ml of Hanus iodine reagent accurately and transfer
into a 250 ml iodine flask. Add 10 ml CHCI,with a measuring cylinder. Mix. Stopper
the flask and add distilled water in the cup around the stopper to prevent loss of iodine.
Keep the flask in the dark for 30-40 minutes. Titrate the contents of the flask according
to the method described for sample titration.
Since there is no'oil in the blank flask and no reaction will take place between the oil and
Hanus solution, the blank titration could be performed without keeping the flask in dark
for 30-40 minutes to reduce the time for analysis. It could also be done with reduced
volumes of the reagents as follows: measure accurately 5 ml of Hanus iodine reagent
into a 250 ml conical flask, add 2 ml of CHCI, and immediately titrate using 115th
volume of all the reagents used in the sample titration except the oil.

Precautisnr
1. If using the pipette for transferring the oil, allow the pipette to drain slowly
and compietely, as oil takes time to get transferred.
2. Do not pipette Hanus iodine reagent, since the glacial acetic acid vapours are
corrosive and can be dangerous.
3. Allow iodination to take place in the dark.,
4. Keep the flask stoppered to avoid loss of iodine by sublimation.
5. Always use freshly boiled and cooled distilled water.

Method of Calculation
1. Blank Titration:
i) Volume of Hanus Iodine = 5 ml
u i Volume of 0.1 N Na,S,O, required
Buret reading (ml)
S.No.
Initial Final Difference
Pilot
1
2
3

Titer value = (a). ......ml

iii) 5 ml of Hanus Iodine = (a) ....................ml of 0.1 N Na2S20,

:. 25 ml of Hanus Iodine = a x 25
,.......... = (b) .............ml = .................ml of 0.1 N Na2S20,

2. Sample Titration:
\
Volume of oil = 0.3 ml
Specific gravity of oil = (c) ........ (You may use the value 0.91 since this 1s
approximately the specific gravity of mustard and peanul 3il).

iv) Weight of oil = specific gravity x volume

:.Weight of our oil sample = 0.3 x c = 0.3 x c
= (a) ............... gm = ..............
v) Volume of Hanus Iodine = 25 ml
163
 vi) Volume of 0.I N Na,S,O, required
Buret reading (ml)
S.No.
Initial Final Difference
Pilot
1
2
3

Titer value = (e) ml = ................ml

vii) Volume of Hanus Iodine = Volume of 0. IN Na$i,O, added = (b) ........ ml
viii) Volume of Hanus Iodine = Volume of 0.1 N Na,S jO, in excess = (e) .......ml
ix) Volume of Hanus Iodine = Volume of 0.1 N N%S,O, absorbed =. b - e ml = (f) ml
- ..............ml

x) 1000 ml of IN Na,S,O, = 1000 ml of IN lodine (I,)

:. (f) ..........ml of 0.1N N~,s,O, = (f).........ml of 0.1 N 1,
xi) 1000 ml of IN I, = 127 g I, (based on molecular weight)
:. 1000 ml of O.lN I, = 12.7 g 1,
:. (f) .......ml of 0.1 N I, = 12.7 x (f) = 12.7 x ....a* gm = (g) ............
1000 1000
xii) (d) ............gm of oil absorbs (g)............gm of 1,
:. 100 g of oil absorbs E x 100 = ..........x 100 = hgm= ............
d ............
Result
The iodine number of the given oil is................................ J

To help you consolidate your understanding about Iodine Number, we have included
some review questions herewith. Answer these questions and see where you stand.

Review Questions
1 . Define iodine number.

........................................................................................................................
........................................................................................................................
........................................................................................................................
2. What is the significance of determination of iodine number.
........................................................................................................................
........................................................................................................................
........................................................................................................................
........................................................................................................................
3. What is the indicator used in iodine number titration?
........................................................................................................................
.......................................................................................................................
........................................................................................................................
164
.................................................................. ................................ .
Now submit the experiment and the review question answers for evaluation.

..........,,..........,..........,
Counsellor Signature
 DETERMINATION OFTHE SAPONIFICATION

Date: .......................... Aim: To determine the saponification number of a given sample of oil.

Apparatus
Burettes
Pipettes
Conical flasks 250 ml, I00 ml
Volumetric flask I00 ml
Reflux condenser
Tripod stand
Water bath (portable)

Reagents
0.5 N alcoholic KOH
0.5 N HCI
Solid sodium carbonate
1% alcoholic solution of methyl orange
1% alcoholic solution of phenolphthalein

Principle
(We have already described the principle in section 6.2 above. Read and understand the
principle and write the same in the space provided herewith).
.-

Procedure
Follow the steps enumerated herewith and conduct the experiment.
1. Standardization of HCl - Precisely weigh 2.65 g of anhydrous sodium carbonate
and transfer it to 100 ml volumetric flask. Make the volume to the mark with
distilled water. This is a solution of 0.5 N sodium carbonate. Pipette 10 ml of this
solution into a 100 ml conical flask and add a drop of methyl orange as an
indicator. Titrate with standard HCI till the colour changes to red. Calculate the
normality of the HCI.
2. Sample titration - Measure 2 ml of oil with the help of a pipette and transfer this oil
to a 250 ml conical flask. Add 40 ml of alcoholic KOH to the flask. Fit a reflux
water condenser on the flask. Put a small water bath on a tripod stand and place
the flask fitted with the condenser in the water bath. Heat the water bath for 30-
35 minutes until the fat globulesin the flask are not visible. Cool. Add a few drops of
phenolphtalein as an indicator and titrate against the standardized HCI.
3. Blank titration - Pipette 5 ml of alcoholic KOH in a 250 ml conical flask. Titrate
against standardized HCI using phenolphtalein as an indicator. The values can later
be multiplied by 8.

Calculations
Now carry out the calculations following the lead given herewith:
1. ~tand&dizationof HCL:
Weight of Na,CO, = 2.65 g
2.65 g of Na,CO, diluted to 100 ml
iii) Normality of Na,CO, solution = 0.5 N
iiii Volume of Na,CO, solution = 10 ml
iv) Volume of HCI required
Buret reading (ml)
S;No.
Initial Final Difference
+

Pilot
1
2
-
3
Titre value = (a) ..........'ml
v) Now using the formula given below calculate the normality of HCI.
N,V, = N,V,
where N, = Normality of NqCO, solution
'V, = Volume of Na,CO, solution
N, = Normality of HCl
V, = Volume of HCl = (a)
:. N, = N,V,= 0.5 x 10 = 0.5 x 10 = b N = ..................N

:. Normality of HCI = .................... N

2. Blank titration:
vi) Volume of alcoholic KOH = 5 ml
vii) Volume of (b).......................N HCI required

viii) 5 ml of alcoholic KOH = (c)..................ml of (b).................N HCI

...............
3. Sample tit~ation:

x) Weight of oil = volume x specific gravity

Volume of oil = 2 ml
Specific gravity of oil = (e) ................(You may use the value 0.91 since this is .
approximately the specific gravity of mustard and peanut oil).
:. Weight ofoil = 2 x e = 2 x ................. = (f) ........ &m
xi) Volume of alcoholic KOH = 40ml
xiii Volume of b ........... N HCI required
 Titer value = ................. mI = g ml
xiii) Volume of alcoholic KOH =Volume of (b) ...........N HCI added = (d) ........... ml
xiv)Volume of alooholic KOH =Volume of (b) ...........N HCl in excess = (g) ....... ml
xv) Volume of alcoholic KOH =Volume of(b) .......N HCI used = d - g = ... m l = h mi'
xvi)1000mlof 1 N H C l = l 0 0 0 m l o f 1 N K O H
:. (h)..............ml of (b) .......N HCI = (h) ...........ml of (b) ................... N KOH
xvii) 1000 ml o f 1 N KOH = 28 g of KOH (based on molecular weight)
:. 1000 ml of (b)........N KOH = 28 x b = 28 x ........ = ( i ) g o f K O H Z .........

:( h ) . . m l of (b)........N KOH= g x h x 1000 .......mg of KOH

= ( ) mg =
i ooo
(we have multiplied the above value with 1000 to convert g into mg)
xviii) (0........ g of oil is saponified by 6)........ mg of KOH
:. 1 g of oil is saponified by j& = ............. mg
f
Result
The saponification number of the oil is .............................................
To help you consolidate your understanding about saponification number, we have included
some review questions herewith. Answer these questions and see where you stand.
Review Questions

1 . What is saponification?

.......................................................................................................................
.......................................................................................................................
2. Define saponification number.

.......................................................................................................................
3. In what way do saponification number help us characterize fats?
.......................................................................................................................
........................................................................................................................
.......................................................................................................................
4. How will you assess the purity of a fat using saponification number?
.......................................................................................................................
.......................................................................................................................
Now submit the experiment and the review question answers for evaluation.

..............................
Counsellor signature
ESTIMATION OF CHOLESTEROLUSING ZLATKIS

~ i m To: draw a standard curve for cholesterol and estimate the amount of cholesterol Date: ..........................
in the given solution.

Apparatus & Instruments

Test tubes Borosil(6x314 inch)
Test tube stand
Pipettes 1 ml, 5 ml, 10 ml
Beakers
Measuring cylinders
Volumetric flasks I00 ml, 50 ml
Glass marker & labels
Spectrophotorneterlcolorimeter
Single pan balance
Cuvettes
Tissue roll

Reagents
2. Colour development for standard solution:
a) Take different concentrations ofthis standard solution (1 .O, 2.0,3.0,4.0,5.0 and
6.0 ml) in 6 test tubes (S 1 4 6 ) and make the volume in each test tube upto 6 ml
with glacial acetic acid. *
b) Add 4 ml of colour reagent to each tube carefully from the side of the tube. A
ring will be formed.
c) Shake well and let it stand for 30 minutes for colour development.
3. Preparation of the unknown solution: Dilute the given stock solution to 50 ml with
glacial acetic acid. Mix well.
4. Colour development for unknown solution: Pipette 3.0 rnl o f the diluted
unknpwn solution in duplicate test tubes Sal and Sa2. Similarly pipette 4.0 ml of
the diluted unknown solution in duplicate in tubes Sa 3 and Sa 4. Follow the same
procedure as you did for standard solution for colour development.
5. Preparation of blank: Pipette 2.0 ml of glacial acetic acid into a clean dry test
tube. The blank will contain no standard cholesterol. Follow the same procedure as
you did for standard solution for colour development.
6. Measurement of 0.D:Read the absorbency of all solutions in a colorimeter
or spectrophotometer at 540 nm after setting the colorimeter at 100% transmittance
using the blank solution.
7 . Standard Curve: Plot a standard curve of concentration of cholesterol in standard
vs. OD.
8. Determination of cholesterol content: Calculate the amount of cholesterol present
in the given solution from standard curve as well as from 0.D for standard solution.

Precautions
1. Use aldehyde free glacial acetic acid.
2. Use only glacial acetic for making up and adjusting the volume. Do not use
distilled water.
3. Allow 30 minutes for colour development.
4. Before taking the readings, if a coloured layer is formed in the test tube, make
sure that the contents in the,test tube are shaked and mixed well once again.
5. Make sure there are no air bubbles when taking the readings.
6. Be very careful while working with glacial acetic acid, since it is very toxic.

Observations and Calculations

Now record your observations and do the calculations as suggested in the section below.
i) Preparation of Standard Cholesterol
Weight of cholesterol taken = I00 mg
100 mg cholesterol was dissolved in glacial acetic acid and solution was made up to
100 ml Out of this we took 10 ml & diluted it to 100 ml with glacial acetic acid.
:. Concentration of cholesterol solution = 0.1 mg/ml.
Now we took different concentrations of cholesterol solution, so calculate for the different
concentrations taken (i.e. 2.0 ml, 3.0,4.0, 5.0,6.0) in the space provided herewith. We
have calculated for one, now you proceed with the others:
Since 1 .O ml of cholesterol solution = 0.1 mg cholesterol
:. 2.0 ml of standardvcholesterol solution = 0.1 x 2.0 = 0.2 mg cholesterol

:. 3.0 ml
:. 4.0 ml
:. 5.0ml

.: 6.0ml
 I ll m IV V M
Volume of Conc. of Glacial Acetic Colour Optical Optical Density
Std. Sol. Cholesterol Acid (ml) Reagent Density at for 0.4 mg
(in ml) (mg) (mi) 540 nm Cholesterol

1 .o

2.0

3O
.

4.0

5 .o

I 'n m IV
Volume of ~ l a c i aAcetic
l Coloufieagent Optical
Std. Sol. Acid (ml) (ml) Density at
(in ml) 540 nm
3.0 ml
Sal
Sa2

4.0 ml w

Sal
Sa2
 C1. ........ mg of cholesterol is present in 3.0 ml of unknown

:. I00 ml of unknown contains = C1 x l o o = ........ mg of unknown solution = Dl

3.0
Observed Value = (D, Dl). .............
*
Expected Value = (F) ..............
Now calculate the % error in the space provided using the formula given
,, herewith:
F - D x 100
:. % error (based on optical density) = -
F

v) Determination of Cholestrol Content from Standard Curve

On the standard curve prepared earlier, plot the OD of the unknown solution on the y-
axis. Now check the corresponding concentration of cholesterol (in the two unknown
samples taken) for this OD on the x-axis. Say the value obtained is E and E l .

So 3 ml of unknown solution conthins E ml of cholesterol

:.I00 ml of unknown'solution will contain = E x 100 = F = .............
3
Observed Value = F = .............
Expected value = D =............(Take it from the counsellor)

Similarly, using this calculation now you calculate the cholesterol content in the second
unknown samples (4.0 ml of which we had taken). Write your calculations here in the
space provided.

Observed Value = F1 = .............

Expected value = D =............(Take it from the counsellor)

Now calculate the % error in the space provided using the formula given herewith:

.: % error (based on o ~ t i c a density)

l D --
=- F x 100
D
Result
The concentration of cholesterol in the given solution is.. ......... 1 100 ml

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To consolidate your understanding on the topic do attempt the questions included in
the review questions included herewith.

Review Questions
I . Why is it important to estimate cholesterol in blood? What does a high
blood cholesterol level predict?
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2. What is the role of sulphuric acid in cholesterol estimation?

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3. In what form is cholesterol present in blood?
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Now submit the experiment along with the answers to the review questions for
evaluation.

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