Salkowski's test, also known simply as Salkowski test, is a qualitative

chemical test In chemistry, a chemical test is a qualitative property, qualitative or Quantitative property, quantitative procedure designed to identify, quantify, or characterise a chemical compound or substituent, chemical group. Purposes Chemical testing m ...

, that is used in chemistry and biochemistry for detecting a presence of

cholesterol Cholesterol is the principal sterol of all higher animals, distributed in body Tissue (biology), tissues, especially the brain and spinal cord, and in Animal fat, animal fats and oils. Cholesterol is biosynthesis, biosynthesized by all anima ...

and other

sterols A sterol is any organic compound with a Skeletal formula, skeleton closely related to Cholestanol, cholestan-3-ol. The simplest sterol is gonan-3-ol, which has a formula of , and is derived from that of gonane by replacement of a hydrogen atom on ...

. This biochemical method got its name after German biochemist

Ernst Leopold Salkowski Ernst Leopold Salkowski (October 11, 1844 – March 8, 1923) was a German biochemist who was a native of Königsberg. He received his education at the University of Königsberg, later working in Berlin as an assistant in the chemical laborat ...

, who is known for development of multiple new chemical tests, that are used for detection of different kinds of molecules (besides cholesterol and other sterols also for

creatinine Creatinine (; ) is a breakdown product of creatine phosphate from muscle and protein metabolism. It is released at a constant rate by the body (depending on muscle mass). Biological relevance Serum creatinine (a blood measurement) is an impor ...

carbon monoxide Carbon monoxide (chemical formula CO) is a poisonous, flammable gas that is colorless, odorless, tasteless, and slightly less dense than air. Carbon monoxide consists of one carbon atom and one oxygen atom connected by a triple bond. It is the si ...

glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecular formula , which is often abbreviated as Glc. It is overall the most abundant monosaccharide, a subcategory of carbohydrates. It is mainly made by plants and most algae d ...

and

indoles Indole is an organic compound with the formula . Indole is classified as an aromatic Heterocyclic compound, heterocycle. It has a bicyclic structure, consisting of a six-membered benzene ring fused to a five-membered pyrrole ring. Indoles are d ...

). A solution that has tested positive on the Salkowski's test becomes red and gets yellow glow.

Basic information

Procedure

For Salkowski test's procedure one needs a sample, that is to be tested for sterols, as well as

chloroform Chloroform, or trichloromethane (often abbreviated as TCM), is an organochloride with the formula and a common solvent. It is a volatile, colorless, sweet-smelling, dense liquid produced on a large scale as a precursor to refrigerants and po ...

and concentrated

sulfuric acid Sulfuric acid (American spelling and the preferred IUPAC name) or sulphuric acid (English in the Commonwealth of Nations, Commonwealth spelling), known in antiquity as oil of vitriol, is a mineral acid composed of the elements sulfur, oxygen, ...

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. It is important to use only dried glassware, as

dehydration reaction In chemistry, a dehydration reaction is a chemical reaction that involves the loss of an H2O from the reacting molecule(s) or ion(s). This reaction results in the release of the H2O as water. When the reaction involves the coupling of two molecu ...

occurs during the test's procedure. 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. Salkowski test can also be used to test the presence of

(crystalline

alkaloid Alkaloids are a broad class of natural product, naturally occurring organic compounds that contain at least one nitrogen atom. Some synthetic compounds of similar structure may also be termed alkaloids. Alkaloids are produced by a large varie ...

s that are a degradation products of proteins, containing

tryptophan Tryptophan (symbol Trp or W) is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromat ...

). In such cases a sample is treated with

nitric acid Nitric acid is an inorganic compound with the formula . It is a highly corrosive mineral acid. The compound is colorless, but samples tend to acquire a yellow cast over time due to decomposition into nitrogen oxide, oxides of nitrogen. Most com ...

and 2% solution of

potassium nitrite Potassium nitrite (distinct from potassium nitrate) is the inorganic compound with the chemical formula . It is an ionic salt of potassium ions K+ and nitrite ions NO2−, which forms a white or slightly yellow, hygroscopic crystalline powder that ...

, with positive reaction being shown by presence of red colour.

Chemistry of the test

Treating a solution of a sample, containing sterols, with chloroform and highly

hygroscopic Hygroscopy is the phenomenon of attracting and holding water molecules via either absorption (chemistry), absorption or adsorption from the surrounding Natural environment, environment, which is usually at normal or room temperature. If water mol ...

sulfuric acid, leads to a

(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 Cholestenes are chiral molecules that are derivatives of cholestanes that have a double bond. If there are two double bonds, the molecule is known as a "cholestadiene". Examples include fusidic acid, lanosterol, and stigmasterol. Cholestene is a w ...

with two double bonds) in a case of cholesterol. Red colour of a solution is a consequence of bi-

sulfonic acid In organic chemistry, sulfonic acid (or sulphonic acid) refers to a member of the class of organosulfur compounds with the general formula , where R is an organic alkyl or aryl group and the group a sulfonyl hydroxide. As a substituent, it is kn ...

of a bi-cholestadien, which is a product of sulfuric acid sulfonating bi-cholestadien.

References

{{Reflist Biochemistry detection methodsbr>Salkowski test for cholesterol – Its principle and procedure