Ans. Biological molecules contain unique chemical structures called functional groups, distinguished by biochemical tests including Benedict's iodine and Sudan III/IV. Benedict's test detects the reduction of sugars in functional groups of free ketone and aldehyde. The categories reduce CuSO4, a portion of Benedict, which results in a shift in color, from turquoise to rusty-brown. Starch, a polysaccharide, can be identified in another class of carbohydrates by the iodine examination. The helical structure of the starches, creating blue-black precipitation, is accompanied by a high molecular weight, iodine. The hydrocarbon chains of the lipids are measured by the Sudan III/IV test. Sudan is a red, non-polar colored color that interacts hydrophobically with hydrocarbon lipid chains. The Brown Bag test can also be used to classify lipids since hydrocarbon chains are oily in their existence.
One such biochemical test is the biuret test. The biuret test is a chemical test for the identification of peptide bonds, also called a Piotrowski test. A copper[II] ion produces mauve-colored coordination complexes in the presence of peptides in an alkaline solution. The biuret reaction can be used to determine protein concentration since peptide bonds are contained by amino acid in the peptide at the same frequency. According to Beer-Lambert's law, the strength of the color is directly proportional to the protein concentration, and hence the absorption at 540 nm gives the amount of protein present in the sample.
Reagent for Biuret
The biuret Reagent consists of sodium hydroxide, Rochelle salt (sodium potassium tartrate), and hydrated copper sulfate. In this case, the salt of Rochelle serves as an agent for chelating and stabilizes ions from copper(II).
The Principle
The copper(II) in the biuret reagent binds to the nitrogen atoms in protein peptides. Now copper(II) is reduced and turned into copper (I). Since the presence of amino acids in the sample does not cause major disruption to this examination, the protein content in whole tissue specimens can be measured. Samples of proteins purified through precipitation by ammonium sulfate ((NH4)2SO4) are not, however, suitable for this test as ammonia in the buffer interferes with it. The copper(II) ions and the nitrogens of the peptide bonds cause peptide hydrogen to be displaced (as long as the environment is sufficiently alkaline). Now four atoms of nitrogen donate lonely pairs to form covalent relations to the cupric ion, which results in a chelate complex being formed. This chelate complex is capable of absorbing light with a 540nm wavelength that gives it a purple color. The development of a purple complex therefore reveals the existence of proteins in the sample. The peptide bond concentration in the analyte contributes to the strength of the purple color. In the biuret test, short-chain peptides mostly give blue or pink color.
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