Amino Acid + Amino Acid + Amino Acid Peptide linkageProteins (many peptide linkages)

Proteins Amino Acids

Casein Cysteine

Gelatin Arginine

Albumin Tryptophan

Peptone Tyrosine

Arginine

Cysteine

Tryptophan

Tyrosine

Tests to be done:

Ninhydrin’s test (specific to all Proteins & Amino acids)

Biuret’s test (specific to Proteins)

Sakaguchi’s test (specific to Arginine)

Nitropruside’s test (specific to Cysteine)

Millon’s test (specific to Tyrosine)

Hopkins-Cole’s test (specific to Tryptophan)

Ninhydrin reaction

Principle:Ninhydrin is Specific for Amino Acids & Proteins – to differentiate betweenCarbohydrates (-ve) and Amino Acids & Proteins (+ve).Ninhydrin Reacts with α-amino acids (–NH2) in proteins giving a purple coloredcomplex, except Proline and hydroxy proline gives yellow color(no –NH2).

Ninhydrin is most commonly used as a forensic chemical to detect“fingerprints”, as amines left over from proteins sloughed off in

fingerprints react with ninhydrin giving a characteristic purple color.

Procedure & observation:− To 1 mL amino acid solution in a test tube, add 2-3 drops of ninhydrin reagent.- Put in a boiling water bath and observe the formation of a purple color.

Ninhydrin’s testNinhydrin’s test

Ninhydrin’stest

Ninhydrin’stest

2-3 drops of B

1ml ofsolution A

Mix Mix

C

Heat

2-3 min

ResultUnknown A Proteins or Amino Acids & Carbohydrates

Reagent B Drops of Ninhydrin reagent

Observation C Dark purple colour on heat Proteins or Amino Acids(free –NH2)

C No reaction (no change in colour) Carbohydrates

Biuret’s testBiuret’s testPrinciple:Biuret test is Specific for Proteins – To differentiate between Proteins (+ve) andAmino Acids (-ve). The biuret reagent (copper sulfate in a strong base) reacts withpeptide bonds in proteins to form a violet complex known as the “Biuretcomplex”. Two peptide bonds are at least required for the formation of thiscomplex , this is why amino acids give negative results with Biuret test.

Procedure & observation:− To 1 mL of protein solution (Albumin – Casein – Gelatin – Peptone) in a testtube, add 1 mL of 10% sodiumhydroxide solution and 2-3 drops of 1% copper sulfate solution.− Mix well; a violet color is obtained with albumin, casein & gelatin and apinkish violet color with peptone.

Biuret’s testBiuret’s test2-3 drops of CuSO4

1ml ofsolution A

Mix Mix

C

ResultUnknown A Proteins or Amino Acids

Reagent B 2 ml NaOH, then 2 drops ofCuSO4 (1%)

Observation C Violet colour Proteins (reacts with Di-peptide bonds)C No change in colour (Blue) Amino Acids (No di-peptide linkage)

1ml of NaOH

Sakaguchi’s testSakaguchi’s testPrinciple:Sakaguchi test is Specific for free Arginine or proteins containingArginine.Sakaguchi's test is positive for the amino acid containing the guanidinegroup in Arginine. Guanidine group present in the amino acid reacts withα-Naphthol and alkaline hypobromite to give red-coloured complex.

Procedure & observation:− To 1 mL of protein solution in a test tube, add 40%NaOH (2-3 drops) +Ethanolic a-Naphthol (2-3 drops) and Bromine water (2-3 drops)− Mix well; a red-colour complex will be formed with Arginine or proteincontaining Arginine.

Sakaguchi’stest

Sakaguchi’stest

B

1ml ofsolution A

Mix Mix

C

ResultUnknown A Proteins or Amino AcidsReagent B 2-3 drops 40%NaOH, then 2-3 drops alcoholic

α -Naphthol, mix then• add 2-3 drops Bromine water or• (for safety, use Chlorine water)

Observation

C Red colour Proteins containing Arginine

C Red colour Arginine (Guanidine gp)

Nitroprusside’s testNitroprusside’s test

Principle:It is specific for Amino Acids or Proteins containing sulfur , -SH (incysteine & cystine) gives a red-purple colour called “Mörner test”.

Procedure & observation:− To 1 mL of protein solution containing Cysteine/Cystine in a test tube,add 3 drops of a 5% solution of sodium nitroprusside- Mix well and add few drops of ammonia solution, a deep red-purplecolor appears; called also Mörner test.

l

Nitroprusside’stest

Nitroprusside’stest

B

1ml ofsolution A

Mix Mix

C

ResultUnknown A Cystine or Cysteine in

Proteins or Amino AcidsReagent B 3 drops Sodiun nitropruside,

Then, 3-5 drops Ammoniasoln.

Observation C Deep Red – purple colour Amino Acid: Cystine or Cysteine (-SH gp)C Deep Red – purple colour Proteins containing Cystine or Cysteine (-SH gp)

Millon’s testMillon’s test

Principle:It is specific for Tyrosine. Millon's reagent (Hg/HNO3) gives positiveresults (red – pink colour appears) with proteins containing the phenolicamino acid “tyrosine”.

Procedure & observation:− To 1 mL of protein solution in a test tube, add 1ml of Millon’s reagent[Hg(SO4) + HNO3]- Heat up for few min− A red – pink colour appears

Millon’s testMillon’s test1 mL of B

1ml ofsolution A

Mix Mix

C

Heat

2-3 min

ResultUnknown A Tyrosin in Proteins or Amino

AcidsReagent B 1 mL Millon reagent [Hg(SO4) +

HNO3] then heat

Observation C Red-Pink colour Amino Acid: Tyrosin (-Ph-OH gp)C Red-Pink colour Proteins containing Tyrosin (-Ph-OH gp)

Aldehyde testHopkin-Cole

Aldehyde testHopkin-Cole

Principle:It is Specific for Tryptophan amino acid, whether it is free or in proteins.Sulphuric acid in presence of mercuric sulphate oxidizes the indole nucleus oftryptophan. The product formed reacts with aldehydes to form violet-purplecolored complex (ring).

Procedure & observation:− To 1 ml of protein solution in a test tube, add 2 drops of [0.2 of Formalin + 1drop of 10% HgSO4] and mix well.− Incline the test tube and slowly add 2 ml of concentrated H2SO4 on the innerwall of the test tube to form violet-purple ring at the junction of the 2 layers.− A reddish violet ring is formed at the junction between the 2 layers with albuminand casein; gelatin gives negative results.

Aldehyde testHopkin-Cole

Aldehyde testHopkin-Cole

ResultUnknown A Tryptophan in Proteins or

Amino AcidsReagent B • 2 drops [Formalin + HgSO4]

• then add 2 mL conc H2SO4

Observation C Violet/Purple colour ring Amino Acid: Tryptophan (Indole ring)C Violet/Purple colour ring Proteins containing Tryptophan (Indole ring)

2 mL of H2SO4On the side wall

1ml ofsolution A

Mix

C

2 drops of [Formalin + HgSO4]

Mix

Tryptophan

H2SO4

Wait 1 min

Some additional tests for qualitativeidentification of proteinsA) Heat coagulation testPlace about 5 ml of egg-white solution (albumin solution) in atest tube and heat the top part of the solution only. Note that itgradually becomes cloudy and a flocculent precipitate ofcoagulated protein is produced.

D) Picric acid testTo 3 ml of gelatin solution in a test tube, add 2ml of saturatedpicric acid solution, a yellow gelatinous precipitate is formed.

Proteins & Amino AcidsUnknownObservationProcedure

All A.A. having –NH2except proline &

hydroxyproline

Purple colour isformed

1- Ninhydrin testSample solution (l ml) + Ninhydrin reagent (2-3 drops). Observe onCold or if not, Boil in a water bath for (5 min) and Cool

All Proteins (notA.A.) as it needs atleast 2 peptidebonds

Violet colour is formed2- Biuret testSample solution (1 ml) + Biuret reagent (1 ml)(Better to warm it)

Tyrosincontains a phenolicgroup

Red colour/precipitateis formed

3- Millon's testSample solution (1 ml) + 1 mL Millon reagent [HgS04 reagent 0.5 ml) +0.5 ml 2M HNO3]Boil in water bath for (2 min) and Cool it.

Cysteine or CystineContain –SH group

Deep dark purple redcolour called alsoMörner test

4- Nitroprusside's test (Mörner test)Sample solution (1 ml) of protein/Amino acid containing CysteineAdd (3) drops of sodium nitroprussideAdd (3-5) drops of ammonia solution

ArginineContains guanidinegroup

Red colour is formed5- Sakaguchi's testSample solution (1 ml) + 2-3 drops of Sakaguchi reagent [40%NaOH (4drop) + Ethanolic a-Naphthol]Add Bromine water (1-2) drops [for safety, use chlorine water]

TryptophanContains Indolegroup

A violet or purple-coloured ring appearsat the junction of twolayers

6- Hopkin-Cole test (Aldehyde-test)Sample solution (1 ml) + 2-3 drops of Hopkin-Cole reagent [DilutedFormalin (2 drops) + 10% Mercuric sulphate (4 drops)] – Mix wellAdd Conc. H2S04 on the side of test tube (1-2) ml

Identification of unknown protein solutionBiuret’s Test

Violet Color No Violet ColorSolution is protein Solution is not protein

(carbohydrates or amino acids)Heat coagulation test

Cloudness at top part No Cloudness at top partof the solution of the solution

(Albumin) (Casein-Gelatin-Peptone)

Reaction to litmus paper

Neutral AlkalineSoln. is (peptone or Gelatin) Soln. is (Casein)

No yellow gelatinous ppt. Yellow gelatinous ppt.Soln. is (Peptone) Soln. is (Gelatin)

(Peptone gives pink color with biuret)

Picric acid Test

Identification of unknown Amino AcidsBiuret Test

Molisch’s test

Violet ring No violet ringCarbohydrate Amino Acids

(Cysteine – Tryptophan – Tyrosin - Arginine)

Hopkins-Cole’stest

Not Violet Color Violet ColorSolution is not protein Solution is protein

(carbohydrates or amino acids)

Millon’stest

Cysteine

Sakaguchi’stest

Nitroprusside’stest

Tryptophan ArginineTyrosin