Biomolecles Analytical Tests

Analytical Tests for Biomolecules

Molisch’s Test

In Molisch’s test, the carbohydrate (if present) undergoes dehydration upon the introduction of concentrated hydrochloric or sulphuric acid, resulting in the formation of an aldehyde. This aldehyde undergoes condensation along with two phenol-type molecules (such as α-naphthol, resorcinol, and thymol), resulting in the formation of a purple or reddish-purple coloured complex.
The purple-coloured ring forms at the interface between the sulphuric acid and the test solution. Because the sulphuric acid is denser than the test solution, it remains above it.
3.75 gm of α-naphthol in 25 ml of Ethanol 99% is the composition of Molisch’s reagent.

Benedict’s test is a simple chemistry test used to detect reducing sugars. Reducing sugars are carbohydrates having free aldehyde or ketone functional groups in their molecular structure. These include monosaccharides like glucose and fructose and disaccharides like lactose and maltose.

Benedict’s reagent, also known as Benedict’s solution, is a chemical reagent which is made up of a complex mixture of sodium citrate, sodium carbonate, and the pentahydrate of copper(II) sulfate
It gives Red Ppt with Reducing sugars

The Barfoed’s test is used to differentiate reducing monosaccharide from a disaccharide sugar. The reaction is conducted in a slightly acidic medium.
A mixture of ethanoic (acetic) acid and copper(II) acetate, is added to the test solution and boiled.
Reducing monosaccharides react with Bedford’s reagent much faster than disaccharides and produce a copious amount of red precipitate of copper (I) oxide within three minutes. Disaccharide sugars as they are weaker reducing agents, react at a slower rate and so do not form red precipitate even for ten minutes

Seliwanoff’s test is used to distinguish aldoses from ketoses. On treatment with a concentrated Acid, ketoses are dehydrated more rapidly to give furfural derivatives and on condensation with resorcinol give cherry red complex. The test will be answered by fructose, sucrose and other keto containing carbohydrates. If the reaction is allowed for a longer time (more than 10 minutes), aldoses also may produce positive results. Seliwanoff’s test is often considered to be a test for ketohexoses in carbohydrates.

The ninhydrin test is a chemical test which is used to check whether a given analyte contains amines or α-amino acids. In this test, ninhydrin (a chemical compound with the formula C9H6O4; IUPAC name: 2,2-dihydroxyindane-1,3-dione) is added to a test solution of the analyte. The development of a deep blue colour indicates the presence of ammonia, primary/secondary amines, or amino acids in the analyte.
For ammonia, primary/secondary amines, and amino acids, deep purple colour is obtained.
For hydroxyproline and proline, a yellow colour is obtained.
For asparagine, brown colour is obtained.

The xanthoproteic reaction is a method that can be used to detect a presence of protein soluble in a solution, using concentrated nitric acid. The test gives a positive result in amino acids carrying aromatic groups, especially in the presence of tyrosine. If the test is positive the proof is neutralized with an alkali, turning dark yellow. The yellow colour is due to xanthoproteic acid which is formed due to nitration of certain amino acids, most common examples being tyrosine and tryptophan. This chemical reaction is a qualitative test, determining the presence or absence of proteins.

Millon’s test is an analytical test used for the detection of the amino acid tyrosine, which is the only amino acid containing the phenol group. Millon’s test is a specific test for tyrosine, but it is not a specific test for protein as it also detects the phenolic group present in other compounds as well. Therefore, while performing Millon’s test, it is essential that other tests like the Biuret test and Ninhydrin test also be performed. As many proteins consist of tyrosine, the test is useful in the detection of such proteins.

The biuret test is a chemical test that can be used to check for the presence of peptide bonds in a given analyte. There must be at least 2 peptide bonds Therefore, the biuret test can be also be used to gauge the amount of protein present in the analyte. In this test, the presence of peptides results in the formation of pale purple coloured (or mauve coloured) coordination compounds of the copper(II) ion (when the solution is sufficiently alkaline).
The biuret reagent is made up of hydrated copper sulfate, sodium hydroxide, and Rochelle salt (sodium-potassium tartrate). Here, the Rochelle salt acts as a chelating agent and stabilizes the copper(II) ions
Biuret is a compound also : NH2-CO-NH-CO-NH2

Bial’s test is a chemical test for the presence of pentoses. The components include orcinol, hydrochloric acid, and ferric chloride. A pentose, if present, will be dehydrated to form furfural which then reacts with the orcinol to generate a coloured substance
A blue-green colour indicates pentoses or nucleotides containing pentoses.
A yellow-green colour indicates hexoses, and disaccharides are yellow.

The picric acid test for carbohydrates is a very sensitive chemical test for the presence of reducing sugars. The reducing sugars react with Picric Acid (toxic yellow crystalline solid) also chemically known as 2,4,6-trinitrophenol (TNP) to form a red coloured Picramic Acid. All monosaccharides and disaccharides containing the potentially free aldehyde or ketone group possess reducing properties.

Anthrone test is a group test for carbohydrates that provides a rapid and convenient method for quantification of carbohydrates that are either free or bound to any lipids or proteins.
If carbohydrate is present in the form of free carbohydrate as poly- or monosaccharide or bound as in a glycoprotein or a glycolipid, the concentrated acid in the Anthrone reagent first hydrolyses it into component monosaccharide. Similarly, the concentrated acid then catalyzes the dehydration of the monosaccharides to form furfural (from pentoses) or hydroxyl furfural (from hexoses). The furfural or hydroxyl furfural formed condenses with two molecules of naphthol from the Anthrone reagent to form a blue-green complex.
Furfural