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The given reagent is Zak reagent and it is a modified form of original Salkowski reagent that was developed by Salkowski E. in 1872. The Salkowski reagent or Zak reagent is an important color reagent that we often use for the colorimetric estimation of the total cholesterol in blood samples. For the preparation of the color reagent, keep reading below.
Dissolve 0.1 g of FeCl3 in 1 mL of glacial acetic acid and after complete dissolution, transfer it to a 100 mL volumetric flask. Dilute the reagent to 100 mL using concentrated sulfuric acid. Please be careful while adding the sulfuric acid, add it slowly using the 10 mL glass pipette. The reagent should be slightly yellow and transparent. If not, then filter the reagent using glass wool to remove any debris.
For the determination of the total cholesterol, add 2 mL of the Zak reagent into the 3 mL of the glacial acetic acid containing cholesterol sample. Be careful, add the reagent slowly and then mix the two layers. Initially, a light brown color appears that turns into the purple color within a minute. Let the reaction cool down to room temperature and then read the absorbance at 560 nm.
You can even scale it down so that you can perform it in a 96-well microplate. For the 96-well microplate, add 180 µL of the cholesterol standard/sample in duplicates and then add 120 µL of the Zak reagent. Mix well by pipetting up and down until the color turns violet. Incubate the plate for 5-10 minutes and read the absorbance at 560 nm.
Standard calibration curve
For the standard calibration curve, prepare the stock solution of cholesterol (0.3 mg/mL) in glacial acetic acid. Take 3.6 µL to 36 µL of cholesterol from the stock solution to microplate in duplicates and add the glacial acetic acid to make it 180 µL. These 10 standards will give the range of concentration from g ng to 60 ng. Now, add 120 µL of Zak reagent (total volume 300 µL) and mix it well by pipetting up and down. After mixing allow it to cool for 5-10 minutes and then read the absorbance at 560 nm. Draw the standard calibration curve and using the linear regression equation, calculate the total cholesterol of the serum sample.
Reference: Zlatkis, A., Zak, B., Boyle, A.J. 1953. A new method for the direct determination of serum cholesterol. Journal of Laboratory and Clinical Medicine