Objective:

• to detect aromatic amino acid  tyrosine or histidine

Principle:

Diazotized sulphanilic acid couples with amines, phenols and imidazole to form highly colored azo compounds. This coupling reaction must be done in cold condition since diazonium compound is formed in cold. Amino acids tyrosine or histidine coupled with diazonium salt in alkaline condition to form red coloured azo dye.

Reagents:

• 1 % tyrosine, 1 % histidine, 1%glycine
• 1 % sulphanilic acid in 10 % HCI
• 5 % sodium nitrite
• 10 % sodium carbonate
• Ice bath

** Diazonium compound=Sulfanilic acid + sodium nitrite + sodium carbonate

Procedures

1. Take 2ml test solution in dry test tube.
2. Similarly, take 2ml distilled water in another test tube as control.
3. Add 1ml of sulphanilic acid, mix well and keep in ice bath.
4. Now add 1ml sodium nitrite solution to all test tubes.
5. Leave in ice bath for 3 minutes.
6. Make the solution alkaline by adding 5ml of sodium carbonate.
7. Look for the development of red colored complex.

Result interpretation:

• Positive Pauly test: red colored azo dye ( tyrosine and histidine)
• Negative Pauly test: no red color ( glycine)

Pauly’s test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect amino acid tryptophan present in protein

Principle:

The indole group of tryptophan reacts with glyoxylic acid in the presence of conc.  H2SO4 to give a purple colored complex.  Glyoxylic acid is prepared by reducing Oxalic acid with magnesium powder or sodium amalgam. Glacial acetic acid which has been exposed to the sunlight also contains glyoxylic acid and can thus be used for this test.

Reagents:

• 1 % tryptophan, 1 % glycine, 5 % egg white (albumin)
• Adamkevich’s reagent: glyoxylic acid ( prepared by reducing oxalic acid with magnesium powder or sodium amalgam)
• H2SO4
• Dry test tubes
• Pipettes

Procedure

1. Take 2 ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 1 ml of Adamkiewicz’s reagent, mix well.
4. Now add 1ml conc. H2SO4 along the wall of the test tube.
5. A purple colored ring develops at the interface of two solutions.

Result:

Positive Hopkin’s cole test: purple color at the interface. ( tryptophan and egg albumin)

Negative Hopkin’s cole test: glycine

Adamkiewicz reaction (Hopkin’s-cole test): Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect amino acid containing phenol group (hydroxyl group attached to benzene ring) ie. Tyrosine

Principle of Millon’s test:

Compounds containing hydroxybenzene radical react with Millon’s reagent to form red complexes. The only amino acid having hydroxybenzene ring is tyrosine. Thus, this test is specific for the amino acid tyrosine and the protein containing this amino acid. Tyrosine when reacted with acidified mercuric sulphate solution gives yellow precipitate of mercury-amino acid complex. On addition of sodioum nitrate solution and heating, the yellow complex of mercury-amino acid complex converts to mercury phenolate which is in red color.

Reagents:

• test solution: 1 % arginine, 1 % tyrosine, phenol solution
• Millon’s reagent (Acidified mercuric sulphate)
• 1 % sodium nitrite

Procedure of Millon’s test:

1. Take 1ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 1ml of Millon’s reagent and mix well.
4. Boil gently for 1 minute.
5. Cool under tap water.
6. Now add 5 drops of 1 % sodium nitrite.
7. Heat the solution slightly.
8. Look for the development of brick red precipitate.

Result interpretation:

• Positive Millon’s test: Brick red color (Tyrosine and phenol solution)
• Negative Millon’s test: no red color ( arginine)

**all phenol give positive Millon’s test

Millon’s test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect amino acid gauanidium group [R-NH-C= (NH2)2+-NH2]. i.e arginine

Principle of Sakaguchi test:

This test is specific for arginine because this reaction is given by guanidinium compound. The arginine reacts with α – napththol and an oxidizing agent  such as bromine water or sodium hypochlorite/sodium hypobromite to give a red colored product. The other guanidinium containing compounds other than amino acid also give this reaction.

Reagents:

• test solution: 1 % arginine, 1 % glycine, 5 % egg white (albumin)
• 1 % α naphthol in alcohol
• Sodium hypochlorite
• 1 % urea solution
• Dry test tubes
• Pipettes

Procedure of Sakaguchi test

1. Take 1ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 2 drops of α naphthol and mix well.
4. Now add 2ml sodium hypochlorite to all test tubes.
5. Immediately add 1ml of urea solution to establish the red complex formed.

Result interpretation:

• Positive sakaguchi test: Red color ( Arginine)
• Negative sakaguchi test: no red color ( glycine, albumin)

Sakaguchi test: Objective, Principle, Reagents, Procedure and Result

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Objective:

• to detect amino acid containing sulfhydral group (-SH) ie cysteine

Principle of lead sulfide test:

When cysteine is heated with strong alkali like NaOH, some of the sulphur is converted to sodium sulphide (Na2S) which can be detected by precipitation as lead sulphide (PbS)  from alkaline solution.

Reaction:

sulfur in cystine + 40% NaOH → heating→ Na2S

Na2S + Pb (CH3COO)2 →(black precipitate) PbS

Reagents:

• test solution: 1 % cysteine, 1 % glycine, 5% egg white (albumin)
•  Foli’s reagent: Lead acetate (Pb (CH3COO)2)
• 40 % NaOH

Procedure of lead sulfide test

1. Take 1ml test solution in dry test tube.
2. Similarly, take 1 ml distilled water in another test tube as control.
3. Add 2ml of 40 % NaOH and mix well.
4. Now add 1ml Foli’s reagen ( lead acetate) t to all test tubes.
5. Heat over the flame of Business burner.
6. Look for the development of black precipitate.

Result interpretation:

• Positive lead sulfide test: Black ppt ( cysteine)
• Negative lead sulfide test: glycine, egg albumin

** Methionine does not give lead sulfide test because sulfur in methionine does not split in presence of alkali.

Lead sulfide test: Detection of amino acid containing sulfhydral group (-SH)

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General test for amino acids and proteins:

Following are the qualitative test to detect amino acids and protein in a given solution.

1. Ninhydrin test: general test for all amino acids
2. Biuret test: general test for compound having peptide bond

Specific test for specific amino acids:

1. Foli’s test ( lead sulfide test): test for cysteine
2. Pauly’s test: test for tyrosine
3. Adamkiewicz test ( Hopkin’s-Cole test): test for tryptophan
4. Sakaguchi test: test for arginine
5. Xanthoproteic test: test for tyrosine, tryptophan and phenylalanine*)
6. Million’s test: test for tyrosine

Qualitative Tests for amino acids and Proteins

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Objective:

• this test is used to differentiate aromatic amino acids which give positive result from other amino acids

Principle:

• Xanthoproteic test is used to detect amino acids containing an  aromatic nucleus (tyrosine, tryptophan and phenylalanine) in a protein solution which gives yellow color nitro derivatives on heating with conc. HNO3. The aromatic benzene ring undergoes nitration to give yellow colored product. Phenylalanine gives negative or weakly positive reaction though this amino acid contains aromatic nucleus because it is difficult to nitrate under normal condition. On adding alkali to these  nitro derivative salts, the color change fro yellow to orange.

Reactions:

Reagents:

• test solution: 1 % tyrosine, 1 % tryptophan, 1 % phenylalanine, 5 % egg white (albumin)
• Nitric acid
• 40 % NaOH

Procedures

1. Take 1ml test solution in dry test tube.
2. Similarly, take 1ml distilled water in another test tube as control.
3. Add 1ml of conc. HNO3 in all test tubes and mix well.
4. Cool the solution under tap water.
5. Now add 2ml of 40 % NaOH to all test tubes.
6. Look for the color development.

Result:

Xanthoproteic test: Objective, Principle, Reagents, Procedure and Result

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Principle of Biuret test:

• Biuret test is a general test for compounds having a peptide bond. Biuret is a compound formed by heating urea to 180° C. When biuret is treated with dilute copper sulfate in alkaline condition, a purple colored compound is formed. This is the basis of biuret test widely used for identification of proteins and amino acids.

• This test is given by compounds containing two or more peptide bond (CO-NH group). Since all proteins and peptides possessing at least two peptide linkage ie. tripeptide gives positive biuret test.
• The principle of biuret test is conveniently used to detect the presence of proteins in biological fluids.
• Alkaline CuSO4 reacts with compounds containing two or more peptide bonds to give a violet colored product which is due to formation of co-ordination complex of cupric ions with un-shared electron pairs of peptide nitrogen and O2 of water.

Requirements for Biuret test:

• 1 % alanine, 5 % egg white (albumin)
• Biuret reagent
• Water bath
• Dry test tubes
• Pipettes

Biuret reagents:

• Copper sulfate (CuSO40
• Sodium hydroxide (NaOH)
• Sodium potassium tartarate (commonly known as Rochelle salt)

Preparation Biuret reagent

• Biuret reagent is prepared by adding NaOH in CuSO4 solution, making it alkaline.
• To prepare 1000ml of  Biuret reagent
  • Take 1.5 gram of pentavalent copper sulphate (CuSO4) and 6 gram of Sodium potassium tartarate and dissolve them in 500 ml of distilled water
  • **Sodium potassium tartarate is a chelating agent and it stabilize the copper ion
  • Take 375 ml of 2 molar Sodium hydroxide
  • Mix both the solution in volumetric flask and make it final volume to 1000 ml  by adding distilled water.

Procedure for Biuret test

1. Take 1 ml of test solutions in dry test tubes and in another tube take 1 ml distilled water as control.
2. Add 1 ml of biuret reagent to all test tubes, mix well.
3. Look for the development of blue colors

Result:

Biuret test positive:

• color changes to purple
• all peptides and protein give the test positive
• Histidine is the only amino acid that give biuret test positive.

Biuret test negative:

• No color change

Precautions:

• Presence of magnesium and ammonium ions interfere in biuret test. This can be overcome by using excess alkali.

Reference:

1. laboratoryinfo.com

Biuret test-Principle, Requirements, Reagents, Preparation, Procedure and Result

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Principle:

• This test is a general test and thus given by all amino acids. This test is due to a reaction between a amino group of free amino acid and ninhydrin. Ninhydrin is a powerful oxidizing agent and its presence, amino acid undergo oxidative deamination liberating ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin ( hydrindantin). The NH3 formed from a amino group reacts with another molecule of ninhydrin and is reduced product ( hydrindatin) to give a blue substance diketohydrin ( Ruhemanns complex). However,  in case of imino acid like proline and hydroxyproline, a different product having a bright yellow color is formed. Asparagine, which has a free amide group, reacts to give a brown colored product.

Requirements

• test solution: 1 % alanine, 1 % proline , 1% asparagines
• 2 % ninhydrin in acetone
• Water bath
• Dry test tubes
• Pipettes

Procedure

i. Preparation of 2% ninhydrin solution:

• weight 0.2 gm of Ninhydrin and dissolve in 10ml of acetone or ethanol

ii. Preparation of test solution:

• prepare 1% amino acid solution in distilled water.

1. Take 1 ml test solution in dry test tube and 1 ml distilled water in another tube as a control.
2. pour few drops of 2% ninhydrin in both the test tubes
3. Keep the test tubes in water bath for 5 minutes.
4. Look for the development of blue or violet color.

**But in the case of proline and hydroxyproline, yellow color will develop instead of blue color. Similarly, asparagine will give brown color.

Result:

Ninhydrin Test: Principle, Requirements, Procedure and Result

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