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Estimation of Cholesterol Lieberman-Burchard Reaction Cholesterol is a lipidic, waxy alcohol found in the cell membranes and transported in the blood plasma.

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Presentation on theme: "Estimation of Cholesterol Lieberman-Burchard Reaction Cholesterol is a lipidic, waxy alcohol found in the cell membranes and transported in the blood plasma."— Presentation transcript:

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 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 + H 2 SO 4 polymer containing a chromophore (blue-green color) Estimation of Cholesterol Liberman- Burchard Reaction - H2O 3,5-cholestadiene

6 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

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

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

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


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