Basic structural unit: amino acid

The sample is heated and digested with concentrated sulfuric acid and a catalyst to decompose the protein, in which carbon and hydrogen are oxidized into carbon dioxide and water escape, while the organic nitrogen in the sample is converted into ammonia and combined with sulfuric acid to form ammonium sulfate. Then add alkali for distillation to evaporate the ammonia, absorb it with boric acid, and then titrate it with a standard acid. The protein content in the sample can be calculated based on the consumption of the standard acid.

Digestion: After sample pretreatment, add concentrated sulfuric acid, potassium sulfate (increase boiling point), copper sulfate (catalyst, end point indicator)

Distillation and absorption: Add alkali (NaOH, 400g/L), steam out ammonia, and absorb it with boric acid (20g/L)

Titration: 0.05mol/L standard acid or sulfuric acid (the solution changes from blue to reddish)

Data processing (crude protein)

The result is crude protein

Suitable for F value

Excess sulfuric acid should be used to prevent excess ammonia

Two parts of urea are condensed into biuret at 180°C, and react with copper sulfate in an alkaline environment to form a purple-red complex.

Standard protein solution

Coomassie G-520 combines with proteins to form a colored complex with a maximum absorption peak at 595nm.

Standard solution: bovine serum albumin standard solution

Amino acids contain acidic -COOH and basic -NH2, which interact to make the amino acid a neutral internal salt. When formaldehyde is added, -NH2 combines with formaldehyde, and its alkalinity disappears, making the -COOH group show acidity. NaOH can be used Titration of standard solutions.

According to the amphoteric effect of amino acids, formaldehyde is added to fix the alkalinity of the amino group and make the carboxyl group show acidity. Insert the glass electrode and calomel electrode of the acidometer into the liquid to be measured at the same time to form a battery. Use NaOH standard solution to titrate according to the instructions of the acidometer. The pH value determines the end point.

Ninhydrin→Ninhydrin hydrate→Ninhydrin hydrate→Ninhydrin hydrate→Blue-purple compound