Biuret reagent is originally used to detect biuret. Because protein molecules contain many peptide bonds with similar structure to biuret, it can also occur with copper ions in alkaline solutionBiuret reaction。When the substrate contains peptide bond, the copper in the test solution coordinates with the polypeptide, and the complex is purple.The concentration can be analyzed by colorimetry, and the wavelength in the ultraviolet visible spectrum is 540nm.The sensitivity of the identification reaction is 5-160mg/mL.
What really works in biuret reagent iscopper sulphate, andPotassium hydroxideOnly to provide an alkaline environment, it can be replaced by other alkalis, such as sodium hydroxide.Add to reagentpotassium iodide, will extend the service life of the reagent.Potassium sodium tartrateIts function is to protect the complex ions generated by the reaction from being precipitated into precipitation, thus making the reagent invalid.
Chinese name
Biuret reagent
Foreign name
Biuret Reagent
Application
Detection of substances containing peptide bonds, including biuret, protein, etc
Biuret reagent A is NaOH aqueous solution with mass fraction of 0.1g/mL;
Biuret reagent B is CuSO with mass fraction of 0.01g/mLfourAqueous solution
First, add 3mL of biuret reagent A into the solution to be tested, shake it evenly (to create an alkaline environment), and then add 1-2 drops of biuret reagent B, shake it evenly.If the solution to be tested contains protein, the solution will turn purple.Compounds with two or more peptide bonds can react purple with biuret reagent.
The peptide bond of protein can interact with Cu in alkaline solution2+Complex to form purple complex.The color depth is proportional to the protein concentration.
matters needing attention
Announce
edit
When using biuret reagent, it must be noted that 0.1g/mL sodium hydroxide solution must be added first, and then 0.01g/mL copper sulfate aqueous solution must be added.If the copper sulfate solution is added first and then the sodium hydroxide solution is added, the alkaline environment cannot be fully created. At this time, CuSOfourWill occur with NaOHMetathesis reactionBlue copper hydroxide precipitate is generated, which leads to unclear phenomenon and fails to achieve the experimental purpose[1]。
Different from biuret
Announce
edit
Biuret (NHtwoCONHCONHtwo）It is the product obtained after two molecules of urea are heated at about 180 ℃ and one molecule of ammonia is released.
Biuret reagent is composed of biuret reagent A and biuret reagent B.
The composition of biuret reagent A is an aqueous solution of sodium hydroxide with a mass fraction of 0.1g/mL;
The composition of biuret reagent B is an aqueous solution with the mass fraction of copper sulfate of 0.01g/mL.
Biuret reagent can verify the existence of protein and produce purple color when meeting protein.
In brief, biuret is a chemical substance, and the two reagents used for protein detection are called biuret reagents because the detection principle is similar to the formation of biuret.
Methodological evaluation
Announce
edit
Advantages: The determination range of protein by biuret method is 1~10mg protein, and the operation is simple and fast.It is suitable for both manual operation and automatic analysis, with good repeatability and linear relationship. Biuret reagent can be stored for a long time (if there is black precipitation in the storage bottle, it needs to be re prepared).
Disadvantages: poor sensitivity, narrow measurement range, large sample demand, different proteins have similar color depth, so it is often used for fast but not very accurate protein determination.The substances that interfere with the determination include: buffer solutions that are amino acids or peptides in nature, such as TrIs buffer solutions, because they produce positive color reactions, copper ions are also easy to be reduced, and sometimes red precipitates appear[1]。
