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Zak’s Method for Cholesterol Estimation

    Zaks method for cholesterol estimation based on the red-violet colour which is proportional to the amount of cholesterol and formed after the reaction of cholesterol melted in acetic acid,FeCl3 and sulfuric acid. Zak’s test for cholesterol employing ferric chloride-acetic and sulphuric acid reagents. Nowadays clinical laboratories use cholesterol oxidase-peroxidase enzymatic method instead of Zaks method for cholesterol estimation.

    Zak's method principle

    Cholesterol estimation by zak’s method based on the  red-violet colour which is proportional to the amount of cholesterol and formed after the reaction of cholesterol melted in acetic acid,FeCl3 and sulfuric acid. Cholesterol in glacial acetic acid gives a red colour with ferric chloride and a polar sulphuric acid. This reaction has been employed by ZAK’S to estimate the cholesterol in an unknown serum sample.

    Zak's method Requirements

    Materials

    1. Pipette

    2. Spectrophotometer

    3. Test Tube Stand

    4. Test Tubes

    Chemicals

    1. Glacial acetic acid

    2. Ferric chloride

    3. A polar sulphuric acid

    4. Cholesterol

    Reagents

    Stock Standard: About 100 mg of cholesterol was dissolved and made up to 100 ml with glacial acetic acid (concentration 1 mg / ml).

    Working Standard: About 4 ml of stock solution was made up to 100 ml with ferric chloride acetic acid reagent (concentration in 40 mg / ml).

    Standard sample: 20 ml of sample and 40 ml of chloroform was added and centrifuged. The supernatant was used for estimation.

    Zak's test for Cholesterol

    1. 0.5 ml to 2.5 ml of working standard were Pipetted out into a clean test tubes. About of 0.5 ml and 1 ml of unknown sample supernatant was taken in a test tubes.

    2. The volume was made up to 5.0 ml with ferric chloride and 3.0 ml of concentrated sulphuric acid were added.

    3. The test tubes were kept at room temperature for 15 minutes. The pinkish red colour formed was measured at 540 nm.

    4. Standard graph was drawn for the values obtained. From the standard graph the amount of cholesterol present in the serum sample can be calculated.

    Zak's test Calculations

    Zak’s Method for Cholesterol Estimation

    0.5 ml of standard corresponds to O.D 0.03 O.D corresponds to ———- mg.  0.5 ml of unknown corresponds 100 ml of unknown corresponds to ——– 100x ———.

    Zak's test Result

    The amount of cholesterol present in unknown sample was found to be —–.