Salkowski's test

For Salkowski test's procedure one needs a sample, that is to be tested for sterols, as well as chloroform and concentrated sulfuric acid that represent Salkowski's reagent. Usually the solution of chloroform and the sample is prepared first and later treated with concentrated sulfuric (VI) acid. After that the whole solution needs to be shaken well.[2][6] It is important to use only dried glassware, as dehydration reaction occurs during the test's procedure.[8]

A solution that has tested positive on this qualitative chemical test exhibits two distinct layers in a test tube; the upper layer (chloroform) gets blueish red to violet colour, while the layer of sulfuric acid becomes yellow to green, with greenish glow being visible. If a sample does not contain any cholesterol or other sterols, colour of the tested solution remains unchanged and retains its original colour.[2][9][10]

Salkowski test can also be used to test the presence of indoles (crystalline alkaloids that are a degradation products of proteins, containg tryptophan[11]). In such cases a sample is treated with nitric acid and 2% solution of potassium nitrite, with positive reaction being shown by presence of red colour.[12]

Treating a solution of a sample, containing sterols, with chloroform and highly hygroscopic sulfuric acid, leads to a dehydration reaction (two water molecules are removed from two cholesterol molecules) and formation of new double bonds. During the chemical reaction two sterols bind together and bisterol (bisteroid) is formed; bi-cholestadien (double cholestene with two double bonds) in a case of cholesterol. Red colour of a solution is a consequence of bi-sulfonic acid of a bi-cholestadien, which is a product of sulfuric acid sulfonating bi-cholestadien.[13][14]