DETERMINATION OF CHOLESTEROL CONTENT
Aim: To estimate the amount of cholesterol in the given sample by Zack’s method.
Introduction: This experiment aims to determine cholesterol content in biological or food
samples using Zack’s method.
Principle: In this method , proteins in serum or protein part are participated with ferric
chloride -acetic acid reagent. The protein free filtrate containing cholesterol is treated with
concentrated sulphuric acid. Cholesterol in presence of sulphuric acid undergoes dehydration
to form 3,5-cholestadiene which gives color to the solution. This color intensity is measured
at 540 nm.
Reagent Preperation:
1. Stock solution: 10mg of cholesterol in 10ml acetic acid.
2. Working solution: 1ml of stock solution made up to 5ml by 4ml acetic acid.
3. FeCl3-acetic acid reagent: 0.05% FeCl3 in 35ml acetic acid solution.
4. Concentrated H2SO4.
PROCEDURE:
1. Pipette out the working solution in series of 0.2 ,0.4, 0.6, 0.8 and 1.0ml.
2. Make up the volume to 5ml in each test tube with 0.05% FeCl3 acetic acid solution
including blank ( without working solution).
3. Similarly pipette 0.1ml sample and make up to 5.0ml using 0.05% FeCl3 acetic acid.
4. Add 2ml of concentrated H2SO4 to all the test tubes including blank and sample.
Sl.no Working 0.05% FeCl3 Concentrated Incubate at OD at
standard solution H2SO4 37 for 20 560nm
solution minutes
Blank - 5000 2000
1 200 4800 2000
2 400 4600 2000
3 600 4400 2000
4 800 4200 2000
5 10000 4000 2000
S-1 100 4900 2000
5. Incubate all the test tubes at 37 for 20min.
6. After incubation observe for the color change and record the color intensity of the solution
at 560nm.
DETERMINATION OF ACID VALUE
Aim: To determine the acid value of th given oil sample.
Introduction: The acid value is determined by titrating a known quantity of the sample (e.g.,
oil or fat) with a standard solution of potassium hydroxide (KOH) in the presence of an
indicator, such as phenolphthalein, to measure the amount of free fatty acids present. It is
expressed as the milligrams of KOH required to neutralize the free acids in 1 gram of the
sample.
Principle: The acid value is the number that expresses in milligrams the quantity of Potassium
hydroxide required to neutralize the free acids present in 1g of the substance. The acid value
is often a good measure of the break down of the triacylglycerols into free fatty acids, which
has an adverse effect on the quality of many lipids.
RCOOH + KOH KOH + KOH
Reagents required:
1. Potassium hydroxide: 0.1N KOH in 50ml distilled water.
2. Phenophthalein indicator: 1% phenolphthalein in 95% ethanol.
3. Neutral solvent: 25ml petroleum ether, 25ml of 95% ethanol and 1ml of 1%
phenolphthalein at ph-7.
Procedure:
1. Place 0.5ml of oil in dried conical flask
2. Add 5ml of neutral solvent and add 100 microliter of phenolphthalein.
3. Titrate the solution against 0.1N KOH until pink color appears (end point).
4. Repeat the titration for concordant value and calculate the acid value using the formula.
AV=ml of KOH x N of KOH x Molecular weight of KOH
Weight of sample
Tabulations
Burette readings Trial 1 Trial 2 Trial 3
Initial burette 0.0 0.3 0.5
reading(ml)
Final burette 0.3 0.5 0.7
reading(ml)
Volume of KOH 0.3 0.2 0.2
used(ml)
Calculations:
AV = ml off KOH x N of KOH x M.wt of KOH
Volume of sample
= 0.2x0.1x56.1 /0.5
= 1.122 / 0.5
= 2.244 mg
Result: The acid value of the given oil sample is found to be 2.244 mg
DETERMINATION OF IODINE VALUE
Aim: To determine the iodine value of the given oil sample.
Introduction: The iodine value measures the degree of unsaturation in fats and oils by
determining the amount of iodine (in grams) absorbed by 100 grams of the sample. It is a key
parameter for assessing the quality and stability of lipids.
Principle: The iodine value gives a measure of the average degree of unsaturation of a lipid.
The higher the iodine value the greater the number of C=C double bonds. By definition the
iodine value is expressed as the grams of iodine absorbed per 100g of lipid. One of the most
commonly used methods for detrmining the iodine value of lipids is “Hanus method”.
Reagents required:
1. Hanus solution: 18.2g of iodine in 1000ml of glacial acetic acid and then add 3ml of
bromine water.
2. 0.1N Sodium thiosulphate solution.
3. 10% Potassium iodide
4. Choloform
5. Starch indicator: 1% starch in 5ml of distilled water.
Procedure:
1. Add approximately 1ml of oil sample into conical flask.
2. Add 5ml of chloroform and shake well.
3. Add 20ml of Hanus solution, shake well and incubate in dark for 30minutes.
4. Add 5ml of 10% potassium iodide and add 50ml of distilled water to separate the oil and
the solvent.
5. Titrate the solution against 0.1N sodium thiosulfate solution till yellow color formed, then
add 2-3 drops of starch solution where blue solution formed and continue titration until blue
color disappears. Titrate for concordant values.
6. Do same above procedure but without sample which serves as blank.
7. Calculate the iodine number by using the following law:
I.V=(B-S) x N of Na2S2O3 x 12.69 x 100
Weight of sample
Where,
B= Vml of Na2S2O3 of blank
S= Vml of Na2S2O3 of sample
12.69= The conversion factor from mEq sodium thiosulfate to grams of iodine
