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.
 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.
 Estimation of Cholesterol Liberman-
Burchard Reaction
• 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
 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.
Estimation of Cholesterol Liberman-
Burchard Reaction

- H2O
+ H SO
2 4

3,5-cholestadiene

polymer containing a chromophore

(blue-green color)
 Shaking the mixture 5 min
Unk.
Boiling Water Bath
until the solvent begins
to boil
COOL to Room Temperature
0.2ml blood

10ml alcohol-aceton solvent

Centrifuge tube
Read the Optical Density at 680 nm

2ml Acetic anhydride-sulfuric

acid mixture
2ml Chloroform

Leave the tubes in the dark at room remperature

 Tube # Blank St. Unk.

Acetic anhydride-sulfuric 2 2 2
acid mixture (ml)

Chloroform (ml) 2 2 2
D.W (ml) 0.2 0 0
Unknown (ml) 0 0 0.2

Standard (ml)
Mix Well
0 0.2 0

Leave the tubes in the dark at room remperature

Read the Optical Density at 680 nm
* Calculations
Absorbance of Unknown Absorbance of Standard
=
Concentration of Unknown Concentration of Standard

CUn. = A Un. × C St.

A St.

Samar A. Damiati