Estimation of Cholesterol Lieberman Burchard Reaction Cholesterol is
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 C 3 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 + H SO 2 - H 2 O 4 3, 5 -cholestadiene polymer containing a chromophore (blue-green color)
Unk. Boilingthe Water Bath 5 min Shaking mixture until the solvent begins boil 0. 2 ml. COOL blood to to Room Temperature 10 ml alcohol-aceton solvent Centrifuge tube
Read the Optical Density at 680 nm 2 ml Acetic anhydride-sulfuric acid mixture 2 ml Chloroform Leave the tubes in the dark at room remperature
Tube # Acetic anhydride-sulfuric Blank 2 St. Unk. 2 2 acid mixture (ml) Leave the tubes in the dark at room remperature Chloroform (ml) 2 2 2 Read the Density D. W Optical (ml) 0. 2 0 at 680 0 nm Unknown (ml) 0 0 0. 2 Standard (ml)Mix Well 0 0. 2 0
* Calculations Absorbance of Unknown Absorbance of Standard = Concentration of Unknown Concentration of Standard CUn. = A Un. × C St. A St. Samar A. Damiati
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