1. Estimation of Cholesterol Lieberman-Burchard Reaction
Cholesterol is a lipidic, waxy alcohol found in the cell membranes and transported in the blood plasma of all animals.
Cholesterol is readily soluble in acetone, while most complex lipids are insoluble in this solvent.

2. Estimation of Cholesterol Liberman-Burchard Reaction
Blood or serum is extracted with an alcohol-acetone mixture which removes cholesterol and other lipids and precipitates protein.

3. Estimation of Cholesterol Liberman-Burchard Reaction
Acetic anhydride reacts with cholesterol in a chloroform solution to produce a characteristic blue-green color.
Liberman-Burchard Test:
A test for unsaturated steroids

4. Estimation of Cholesterol Liberman-Burchard Reaction
When conc. sulfuric acid is added to cholesterol, a water molecule is removed from C3 of cholesterol molecule, and it is oxidised to form 3,5-cholestadiene.This product is converted to a polymer containing a chromophore which results in the green colouration indicating a positive test.

5. Estimation of Cholesterol Liberman-Burchard Reaction+
H2SO4
3,5-cholestadiene
polymer containing a chromophore
(blue-green color)

6. until the solvent begins COOL to Room Temperature
Unk.
Shaking the mixture 5 min
Boiling Water Bath
until the solvent begins
to boil
0.2ml blood
COOL to Room Temperature
10ml alcohol-aceton solvent
Centrifuge tube

7. Read the Optical Density at 680 nm
2ml Acetic anhydride-sulfuric
acid mixture
2ml Chloroform
Leave the tubes in the dark at room remperature

8. Read the Optical Density at 680 nm Mix Well
Tube # Blank St Unk.
Acetic anhydride-sulfuric
acid mixture (ml)
Leave the tubes in the dark at room remperature
Chloroform (ml)
Read the Optical Density at 680 nm
D.W (ml)
Unknown (ml)
Mix Well
Standard (ml)

9. * Calculations CUn. = A Un. × C St.
Absorbance of Unknown Absorbance of Standard =
Concentration of Unknown Concentration of Standard
CUn. = A Un. × C St.
A St.
Samar A. Damiati