Presentation on theme: "Estimation of Cholesterol Lieberman-Burchard Reaction"— 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 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 +H2SO43,5-cholestadienepolymer containing a chromophore(blue-green color)
6 until the solvent begins COOL to Room Temperature Unk.Shaking the mixture 5 minBoiling Water Bathuntil the solvent beginsto boil0.2ml bloodCOOL to Room Temperature10ml alcohol-aceton solventCentrifuge tube
7 Read the Optical Density at 680 nm 2ml Acetic anhydride-sulfuricacid mixture2ml ChloroformLeave the tubes in the dark at room remperature
8 Read the Optical Density at 680 nm Mix Well Tube # Blank St Unk.Acetic anhydride-sulfuricacid mixture (ml)Leave the tubes in the dark at room remperatureChloroform (ml)Read the Optical Density at 680 nmD.W (ml)Unknown (ml)Mix WellStandard (ml)
9 * Calculations CUn. = A Un. × C St. Absorbance of Unknown Absorbance of Standard=Concentration of Unknown Concentration of StandardCUn. = A Un. × C St.A St.Samar A. Damiati