Estimation of Cholesterol

Burchard Reaction - Lieberman

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.

+
H2SO4
polymer containing a chromophore
(blue-green color)
- Estimation of Cholesterol Liberman
Burchard Reaction
- H2O
3,5-cholestadiene
Unk.
Centrifuge tube
0.2ml blood
10ml alcohol-aceton solvent
Boiling Water Bath
until the solvent begins
to boil
Shaking the mixture 5 min
COOL to Room Temperature
2ml Chloroform
2ml Acetic anhydride-sulfuric
acid mixture
Leave the tubes in the dark at room remperature
Read the Optical Density at 680 nm
Acetic anhydride-sulfuric 2 2 2
acid mixture (ml)
Tube # Blank St. Unk.
Chloroform (ml) 2 2 2
Leave the tubes in the dark at room remperature
Mix Well
Read the Optical Density at 680 nm
D.W (ml) 0.2 0 0
Standard (ml) 0 0.2 0
Unknown (ml) 0 0 0.2
* Calculations
Absorbance of Unknown Absorbance of Standard
=
Concentration of Unknown Concentration of Standard

CUn. = A Un. C St.
A St.
Samar A. Damiati