Qualitative Analysis of Amino Acids

chemical formula of amino acid

The qualitative analysis of amino acids is a qualitative measure,  where a chemical change brings some changes like the colour change, precipitation, ring formation etc. on the basis of which the amino acids can be detected and classified. The colour change is due to the change in the moiety or the structural configuration where the functional groups of amino acid react with the specific reagent to give specific results.

Amino acids are the basic building blocks of protein. The structure of amino acid consisting of amine group (-NH2), carboxylic group (-COOH) and R-group or side chain.

The side chain differs among all 20 different naturally occurring amino acids. These 20 naturally occurring amino acids are the essential amino acids, which broadly classified into three types:

  • Polar amino acids
  • Non-polar amino acids
  • Aromatic amino acids

The melting point of an amino acid is about 200 degrees Celsius. The net charge of all amino acids becomes zero at neutral pH or at isoelectric point (pI), where they occur as “Zwitter ions”.

At isoelectric point, the amino acid carries both positive and negative charge or it will neither move to cathode nor anode, even under the influence of the electric field. Isoelectric point or pI is different for different amino acids.

Accept glycine, all the amino acids are having an asymmetric C-atom (Carbon linked to 4 different groups), which shows optical activity to rotate a polarized light either to left or right.

Content: Qualitative Analysis of Amino Acids

  1. Definition of Qualitative Analysis of Amino Acids
  2. Methods for Qualitative analysis of amino acids

Definition of Qualitative Analysis of Amino Acids

Qualitative analysis of amino acids can define as the analytical method which detects the presence or absence of amino acids in a solution on the basis of colour change. precipitation etc.

We can see the change that will produce as a result of the reaction between the functional groups of amino acids and the chemical reagents which gives characteristic colour change.

Methods for Qualitative analysis of amino acids

Several methods are used for the qualitative analysis of amino acids, which includes the following tests that are given below:

Ninhydrin Test

This test is used for the detection of all α-L-amino acids.

Principle: Ninhydrin test is based upon the principle which makes the use of reagent “Ninhydrin”. The amino acid reacts with the chemical reagent Ninhydrin to form an intermediate “Hydrindantin”. Hydrindantin further reacts with Ninhydrin and ammonia to form a blue-purple pigment or Ruhemann’s purple compound refers as “Diketohydrin”. Therefore, the amino acid undergoes degradation through the series of chemical reaction to give specific results.

Other than amino acids, imino acids like proline, hydroxyproline also reacts with the Ninhydrin and gives a yellow coloured compound. Amines also react positively by reacting with the Ninhydrin reagent and gives a blue colour.

Method:

  1. Prepare a 1ml solution of the given sample.
  2. Then, add a few drops of Ninhydrin solution.
  3. Boil the solution for 2 minutes and then cool the content.

Observation: Observe the tubes for the appearance of any colour change.

ninhydrin test

Inference: The appearance of purple colour indicates the presence of α -amino acids and yellow colour indicate the presence of imino acids.

Xanthoproteic Test

This test is specific for the detection of aromatic amino acids containing active benzene ring or aromatic nucleus.

Principle: The Xanthoproteic test is based upon the principle of “Nitrification” reaction. In this test, the active aromatic amino acids undergo nitrification in the presence of concentrated nitric acid which forms a yellow coloured nitro-derivatives. The yellow colour turns into orange by the ionization of the phenolic group at alkaline pH.

Method:

  1. Prepare a 1ml solution of the given sample.
  2. Then, add a few drops of nitric acid.
  3. Boil the solution for 2minutes and then cool the content.

Observation: Observe the tubes for the appearance of yellow colour.

xanthoproteic test

Inference: If a solution gives a yellow colour then it indicates the presence of aromatic amino acids.

Pauly’s Diazo Test

This test is specific for the detection of amino acids like histidine and tyrosine.

Principle: The principle of Pauly’s diazo test is based upon the “Diazotation” reaction. Pauly’s diazo test makes the use of chemical reagent (Sulphanilic acid) which undergoes Diazotation reaction to form a “Diazonium salt” in the presence of sodium nitrite and hydrochloric acid. The diazonium salt then couples with the amino acids (either tyrosine or histidine) by giving a red colour to the solution in the alkaline medium.

Method:

  1. Take Sulphanilic acid reagent in a test tube and place in an ice bucket to cool.
  2. Then add prechilled sodium nitrite solution and few drops of chilled amino acid solution.
  3. At last, add sodium carbonate until the colour appears.

Observation: Observe the test tube for the appearance of red colour by the addition of sodium bicarbonate.

pauly's diazo test

Inference: The appearance of the red colour will indicate the presence of tyrosine and histidine.

Millon’s Test

This is the specific test for the detection of phenolic amino acids like tyrosine.

Principle: The principle of Millon’s test is based upon the “Nitration” reaction. Millon’s test makes the use of nitrifying agent i.e. concentrated nitric acid, by which the phenolic amino acids are converted into nitrated amino acid by giving a red colour to the solution. The nitrated compound further forms a deeper yellow coloured salt of amino acids.

Method:

  1. First, take the sample of amino acid.
  2. Then, add Millon’s reagent to the above and mix all the contents.
  3. After that, boil the solution in a water bath for 5 minutes and then cool for a while.
  4. At last, add sodium nitrite solution until the colour appears.

Observation: Observe the test tube for the appearance of a red colour.

millon's test

Inference: The appearance of red colour indicates the presence of tyrosine.

Histidine Test

As from the name, it is clear that this test is specifically used to detect the presence of Histidine. This test was discovered by Knoop.

Principle: The principle of Histidine test is based upon the principle of “Bromination” reaction. In Histidine test, Bromination of amino acid occurs to give a yellow coloured compound in the presence of bromine in acid solution.

Method:

  1. Prepare 1 ml sample of given amino acid.
  2. Then add an acid solution containing 5% bromine in 33% acetic acid.
  3. After 10 minutes, add 2 ml of 5% sodium carbonate solution.
  4. Boil the solution in a water bath for 10 minutes.

Observation: Observe the test tube for the appearance of blue colour in a solution.

histidine tset

Inference: The appearance of a blue colour indicates the presence of histidine.

Hopkin’s Cole Test

This test is specific for the detection of tryptophan.

Principle: The principle of Hopkin’s Cole test is based upon the “Dehydration” reaction. In Hopkin’s Cole test, the amino acid reacts with the reagent glyoxylic acid in the presence of concentrated sulphuric acid. This reaction causes the dehydration of tryptophan which forms a purple coloured ring between the junctions of two solutions.

Method:

  1. Prepare 1 ml sample of given amino acid.
  2. Then add 1 ml of glyoxylic acid.
  3. Mix the contents and add concentrated sulphuric acid from the side of the tube in an inclined position.

Observation: Observe the test tube for the appearance of a violet ring in between the solution.

hopkin's test

Inference: Appearance of a violet coloured ring will indicate the presence of tryptophan.

Sakaguchi Test

This test is specific for the detection of arginine.

Principle: The Sakaguchi test is based on the principle of “Oxidation” reaction. Sakaguchi test makes the use of oxidising agent i.e. sodium hydroxide and α- naphthol reagent. Both sodium hydroxide and α- naphthol reacts with the arginine to give a characteristic red colour on the treatment with hypochlorite.

Method:

  1. Take 1 ml of given amino acid sample.
  2. Then, add 2 drops of sodium hydroxide.
  3. After that, add 2 drops of α- naphthol and mix all the contents.
  4. At last, add a few drops of hypochlorite solution.

Observation: Observe the test tube for the appearance of the red colour in the solution.

sakaguchi test

Inference: The appearance of the red colour indicates the presence of arginine.

Lead Sulphide Test

This test is useful for the detection of amino acids like cysteine which contains the –SH or Sulfhydryl group.

Principle: The principle of Lead sulphide test is based upon the “Precipitation” reaction. In Lead sulphide test, the cysteine reacts with sodium hydroxide and after boiling, it gets converted into sodium sulphide. Then, sodium sulphide reacts with the lead acetate and undergo precipitation reaction by forming black precipitates as lead sulphide.

Method:

  1. Prepare 1 ml solution of given amino acid.
  2. Then add a few drops of 40% sodium hydroxide.
  3. Boil the solution in a water bath for about 5-10 minutes and then cool.
  4. At last, add 10% of lead acetate solution.

Observation: Observe the test tube for the formation of black precipitate in a solution.

lead sulphide test

Inference: Formation of a black precipitate indicates the presence of cysteine.

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