Identification test for Casein: Biochemistry Practical
| S.NO. | EXPERIMENT | OBSERVATION | INFERENCE |
| 1 | XANTHOPROTEIN TEST: To 3ml of protein solution add 1ml conc.nitric acid boil for about half a minute. Cool and observe yellow colour. Now, add 2ml of conc.ammonia or 40% sodium hydroxide and observe the change in colour and after addition of alkali. | yellow colour is appears | Presence of aromatic amino acid. |
| 2 | MILLON’S TEST: To 1ml of protein solution add 1ml of 10% mercuric nitrate (prepared in 10% sulphuric acid). Boil gently for half a minute. Cool and add 3 drops of 1% solution of sodium nitrate. Mix and observe. | The red colour precipitate is obtained | Presence of tyrosine in a solution |
| 3 | SULPHUR TEST: To 3ml of protein solution add an equal volume of 40% sodium hydroxide and boil for 3 minutes. Cool and then add 1ml of lead acetate solution. | No dark grey or black precipitate is obtained. | Absence of sulphur containing aminoacid cysteine and cystine. Methionine does not answer to this test due to presence of thioether linkage which does not allow the release of sulphur in this reaction. |
| 4 | HELLER’S TEST: Take 3ml of con. Nitric | No white ring of meta proteins appears at the | Absence of protein like albumin & globulins. |
| acid and add 2ml of urine along the sides of test tube. | junction of the fluids. | ||
| 5 | PRECIPITATION BY NEUTRAL SALT: HALF SATURATION: Take 3ml of protein solution in a test tube; add an equal volume of saturated ammonium sulphate solution to it, mixed and allowed to stand. FULL SATURATION: Take 3ml of protein solution, add ammonium sulphate salt to it and keep on adding and at the same time mix till protein solution become saturated. | White precipitate is formed. No white precipitate is formed. | Presence of Protein like Globulin ,Casein. Absence of Albumin |
| 6 | NEUMANN’ TEST FOR ORGANIC PHOSPHOROUS: Take 5ml of solution add 0.5ml of 40% sodium hydroxide.Heat for one minute and cool spontaneously. Add 0.5ml of con.nitric acid and 1ml of saturated ammonium molybdate solution. | A canary yellow colour or precipitate is formed. | Presence of inorganic phosphorous. |
| 7 | PRECIPITATION AT ISOELECTRIC pH To 3ml of casein solution add a drop of bromocresol green (BCG) solution. Note the colour; add 2% acetic acid drop by drop till colour changes to light green. | Note the formation of precipitate of casein. Bromocresol green has a green colour at a pH of 4.6 which is the isoelectric point of casein | Presence of casein |
The Xanthoprotein test
The Xanthoprotein test is a chemical test used to detect the presence of proteins in a given sample. This test is based on the reaction of proteins with concentrated nitric acid, which produces a yellow color.
Here are the steps for performing the Xanthoprotein test:
- Take a small amount of the sample to be tested in a test tube.
- Add a few drops of concentrated nitric acid to the sample.
- Observe the color of the mixture. If the sample contains proteins, the color of the mixture will turn yellow.
- If necessary, heat the test tube in a water bath to enhance the color change.
- Compare the color of the test sample with a blank (control) that contains only the solvent and reagent.
It’s important to note that other compounds, such as aromatic amino acids, can also produce a yellow color with nitric acid, so additional tests may be necessary to confirm the presence of proteins. The Xanthoprotein test is a quick and easy method to detect the presence of proteins, but it is not a quantitative test and cannot provide information about the amount or type of protein present in the sample.
Millon’s test
Millon’s test is a chemical test used to detect the presence of proteins in a given sample. This test is based on the reaction of proteins with mercuric and nitric ions, which produces a red color.
Here are the steps for performing the Millon’s test:
- Take a small amount of the sample to be tested in a test tube.
- Add a few drops of Millon’s reagent, which is a solution of mercuric and nitric ions.
- Heat the test tube in a water bath or over a flame.
- Observe the color of the mixture. If the sample contains proteins, the color of the mixture will turn from white to red.
- Compare the color of the test sample with a blank (control) that contains only the solvent and reagent.
It’s important to note that other compounds, such as certain aromatic amino acids, can also produce a red color with Millon’s reagent, so additional tests may be necessary to confirm the presence of proteins. The Millon’s test is a sensitive test that can detect even small amounts of protein, but it is not a quantitative test and cannot provide information about the amount or type of protein present in the sample.
Neumann’s test is a chemical test used to detect the presence of casein, a protein commonly found in milk and milk products. This test is based on the precipitation of casein in the presence of acetic acid and copper sulfate.
Steps:
Take a small amount of the sample to be tested in a test tube.
Add a few drops of glacial acetic acid to the sample to acidify it.
Add a few drops of a 10% solution of copper sulfate to the test tube.
Mix the solution thoroughly and observe for the formation of a white or blue precipitate.
The formation of a white or blue precipitate indicates the presence of casein in the sample.
It’s important to note that other proteins may also precipitate with this test, so additional tests may be necessary to confirm the presence of casein. Also, the amount and purity of the casein in the sample may affect the result of the test. The Neumann’s test is a simple and quick method for detecting the presence of casein, but it is not a quantitative test and cannot provide information about the amount or type of casein present in the sample.
Suggested readings:
First Year B Pharm Notes, Syllabus, Books, PDF Subjectwise/Topicwise