Salkowski test, 1 of the best method for total cholesterol measurement

(Last Updated On: October 15, 2023)
Salkowski reagent Zak reagent for the estimation of total cholesterol
Standard Calibration curve for the determination of the total cholesterol.

Salkowski test consists of

It consists of a coloring reagent called a Salkowski reagent (also called Zak reagent). Salkowski reagent is a modified form of the original Salkowski reagent that was developed by Salkowski E. in 1872. It is a quantitative test that is used to estimate the total cholesterol in the sample.

Materials and Reagents:

  1. Cholesterol sample (serum, plasma, or extracted from tissues)
  2. Ferric Chloride (feCl3)
  3. Sulfuric acid (concentrated)
  4. Glacial acetic acid
  5. Cholesterol standard solution (for calibration)
  6. Spectrophotometer


Sample Preparation:

  • Prepare your cholesterol sample (serum, plasma, or tissue extract) by following proper sample preparation techniques.
  • If you’re using tissues, extract cholesterol using a suitable method, such as Folch extraction.

Standard Curve Preparation:

  • Prepare a series of standard solutions with known cholesterol concentrations. These standards will be used to create a calibration curve.
  • Mix varying volumes of the cholesterol standard solution with a constant volume of glacial acetic acid in separate test tubes to create a range of standard solutions.

Salkowski reagent preparation

To carry out the Salkowski test, we first need to prepare the Salkowski reagent. The Salkowski reagent preparation involves the following steps;

  1. Dissolve 0.1 g of FeCl3 in 1 mL of glacial acetic acid
  2. After complete dissolution, transfer it to a 100 mL volumetric flask.
  3. Dilute the reagent to 100 mL using concentrated sulfuric acid (98 %). Please be careful while adding the sulfuric acid, add it slowly using the 10 mL glass pipette.
  4. The reagent should be slightly yellow and transparent. If not, then filter the reagent using glass wool to remove any debris.

Estimation of the total cholesterol by the Salkowski test

  • To perform this test for the estimation of the total cholesterol, add 2 mL of the Salkowski reagent to the 3 mL of the glacial acetic acid-containing cholesterol sample. Be careful while adding the reagent.
  • Add the reagent slowly, and then mix the two layers. Initially, a light brown color appears that turns into a 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 salkowski 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

  • During the test, you will need a standard calibration curve. To prepare a standard calibration curve, prepare the stock solution of the 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 then add the glacial acetic acid to make it 180 µL.
  • These 10 standards will give you the range of concentration from 0 ng to 60 ng.
  • Now, add 120 µL of Salkowski reagent (total reaction 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 in Excel and use the linear regression equation.
  • Now you can calculate the total cholesterol of the serum sample.

It’s important to note that the Salkowski test is a relatively simple method and may not be as accurate as more advanced techniques like enzymatic assays or high-performance liquid chromatography (HPLC). Furthermore, handling concentrated sulfuric acid requires extreme caution due to its corrosive nature, and this test should be conducted in a well-ventilated area or under a chemical fume hood.

Always follow the specific protocol provided by your laboratory or research institution to ensure accurate and safe results.

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

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