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:
- Cholesterol sample (serum, plasma, or extracted from tissues)
- Ferric Chloride (feCl3)
- Sulfuric acid (concentrated)
- Glacial acetic acid
- Cholesterol standard solution (for calibration)
- 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;
- Dissolve 0.1 g of FeCl3 in 1 mL of glacial acetic acid
- After complete dissolution, transfer it to a 100 mL volumetric flask.
- 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.
- 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