Presentation on theme: "Estimation of Cholesterol Lieberman-Burchard Reaction"— Presentation transcript:
1Estimation 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.
2Estimation of Cholesterol Liberman-Burchard Reaction Blood or serum is extracted with an alcohol-acetone mixture which removes cholesterol and other lipids and precipitates protein.
3Estimation 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
4Estimation 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.
5Estimation of Cholesterol Liberman-Burchard Reaction +H2SO43,5-cholestadienepolymer containing a chromophore(blue-green color)
6until 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
7Read the Optical Density at 680 nm 2ml Acetic anhydride-sulfuricacid mixture2ml ChloroformLeave the tubes in the dark at room remperature
8Read 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