10 min readConcept sheetExam experiment on a protein solution; 'which amino acid does it detect?' is the viva question

Colour reactions of proteins

Biuret shows peptide bonds, ninhydrin shows free amino groups, and the other tests pick out particular amino acids: tyrosine, tryptophan, arginine, cysteine, histidine.

BiuretAlkaline CuSO4 + two or more peptide bonds → violet

NinhydrinFree α-amino group → Ruhemann's purple (proline gives yellow)

Tyrosine testsMillon's (brick red) and xanthoproteic (yellow, orange in alkali)

Specific testsHopkins-Cole: tryptophan; Sakaguchi: arginine; lead acetate: cysteine and cystine; Pauly's: histidine, tyrosine

Aim and principle

Aim: To perform the colour reactions on the given protein solution (egg albumin; gelatin or casein for comparison) and find which groups and amino acids are present. Principle: A protein gives colours through its backbone and its side chains. Biuret: in alkali, Cu2+ binds the nitrogen atoms of two or more neighbouring peptide bonds and forms a violet complex; free amino acids and dipeptides do not answer. Ninhydrin: on boiling, ninhydrin oxidatively deaminates and decarboxylates free α-amino acids; the ammonia released joins with ninhydrin and its reduced form to give Ruhemann's purple (proline and hydroxyproline give yellow). Xanthoproteic: hot nitric acid nitrates the rings of tyrosine and tryptophan to yellow nitro compounds, which turn orange in alkali. Millon's: mercury salts in nitric acid react with the phenol group of tyrosine to give a red colour or precipitate. Hopkins-Cole: the indole ring of tryptophan condenses with glyoxylic acid in strong sulphuric acid to give a violet ring. Sakaguchi: the guanidino group of arginine gives a red colour with α-naphthol and sodium hypobromite. Lead acetate (sulphur) test: boiling with strong alkali frees the sulphur of cysteine and cystine as sulphide, which forms black lead sulphide. Pauly's: diazotised sulphanilic acid couples with the imidazole of histidine and the phenol of tyrosine in alkali to give red azo dyes.

Requirements

Reagent or item	Made of	What it does
Protein solution	1-2% egg albumin (egg white in water or saline); gelatin and casein for comparison	The sample
Biuret test	10% NaOH and 1% copper sulphate	Alkali and Cu2+ form the violet peptide complex
Ninhydrin	0.1-0.2% ninhydrin in ethanol or acetone	Reacts with free α-amino groups
Xanthoproteic test	Conc. HNO3; 40% NaOH or ammonia	Acid nitrates aromatic rings; alkali deepens yellow to orange
Millon's reagent	Mercury dissolved in conc. HNO3 and diluted (mercuric and mercurous nitrates)	Reacts with the phenol of tyrosine; poisonous
Hopkins-Cole reagent	Glyoxylic acid (oxalic acid reduced with magnesium); used with conc. H2SO4	Condenses with the indole of tryptophan
Sakaguchi reagents	10% NaOH, 1% α-naphthol in ethanol, bromine water or sodium hypobromite, 40% urea	Hypobromite and naphthol react with arginine; urea removes excess hypobromite so the red stays
Lead acetate test	40% NaOH and 10% lead acetate	Alkali frees sulphide; lead forms black PbS
Pauly's reagents	1% sulphanilic acid in dilute HCl, 5% sodium nitrite (ice-cold), 10% sodium carbonate	Form a diazonium salt that couples with histidine and tyrosine
Apparatus and safety	Test tubes, boiling water bath, ice bath, droppers, pipettes with a bulb; gloves, goggles, fume hood	Concentrated acids and mercury need care

Procedure

Use 2 mL protein solution for each test. Typical volumes are given. Follow the exact volumes in your lab manual.

1
Biuret: 2 mL protein + 2 mL 10% NaOH; add 1% CuSO4 drop by drop (2-3 drops), mixing.
Too much CuSO4 hides the violet with blue
2
Ninhydrin: 2 mL protein + 0.5 mL ninhydrin; boil for 1-2 minutes.
Blue-violet colour
3
Xanthoproteic: 2 mL protein + 1 mL conc. HNO3 (fume hood); heat in a boiling water bath; cool; add 40% NaOH drop by drop until alkaline.
Yellow, then orange
4
Millon's: 2 mL protein + 3-4 drops Millon's reagent; heat gently.
White precipitate turns brick red
5
Hopkins-Cole: 2 mL protein + 2 mL Hopkins-Cole reagent; mix; tilt the tube and run 2 mL conc. H2SO4 slowly down the side.
Violet ring at the junction
6
Sakaguchi: 2 mL protein + 2 mL 10% NaOH + 2 drops α-naphthol; mix; add 4-5 drops bromine water (or hypobromite), then 2 drops 40% urea.
Red colour
7
Lead acetate: 2 mL protein + 2 mL 40% NaOH; boil for 2 minutes; cool; add 2-3 drops 10% lead acetate.
Black or brownish-black precipitate
8
Pauly's: 1 mL sulphanilic acid + 1 mL sodium nitrite, keep in ice for 3 minutes; add 1 mL protein, then 2 mL 10% sodium carbonate.
Red to orange-red colour

Test | Observation | Inference

Test	Observation	Inference
Biuret test	Violet (purple) colour	Two or more peptide bonds: protein or peptide present
Ninhydrin test	Blue-violet (purple) colour on boiling	Free α-amino groups (amino acids, peptides, proteins)
Xanthoproteic test	White precipitate turns yellow on heating and orange on adding alkali	Aromatic amino acids: tyrosine, tryptophan
Millon's test	White precipitate turns brick red on heating	Phenol group: tyrosine
Hopkins-Cole test	Violet ring at the junction of the two layers	Indole group: tryptophan
Sakaguchi test	Red colour	Guanidino group: arginine
Lead acetate (sulphur) test	Black or brownish-black precipitate	Sulphur of cysteine and cystine
Pauly's test	Red (histidine) or orange-red (tyrosine) colour	Imidazole (histidine) and phenol (tyrosine) groups

Egg albumin vs gelatin

Test	Egg albumin	Gelatin	Why
Biuret	+	+	Both are proteins with peptide bonds
Hopkins-Cole	+	−	Gelatin has no tryptophan
Millon's	+	− or faint	Gelatin has very little tyrosine
Sakaguchi	+	+	Gelatin is rich in arginine
Lead acetate	+	− or faint	Gelatin has very little cysteine or cystine

Precautions and result

Wear gloves and goggles; use conc. HNO3 and H2SO4 in a fume hood and run acid slowly down the side of the tube.
Millon's reagent contains mercury: use 3-4 drops only, avoid skin contact and pour waste into the marked container. Excess reagent can give a yellow colour that hides the red.
Add copper sulphate drop by drop in the biuret test; excess blue Cu(OH)2 masks the violet.
Ammonium salts also give a ninhydrin colour, so use clean, ammonia-free tubes and reagents.
Never pipette by mouth; use a bulb.
Result: The given solution is a protein (biuret positive) containing tyrosine, tryptophan, arginine, cysteine or cystine, and histidine, as shown by the positive tests above.

Nutrition link

Colour tests show why proteins differ in quality. Gelatin gives no Hopkins-Cole colour because it lacks tryptophan, an essential amino acid, so it is an incomplete protein; egg albumin answers every test. Tryptophan also matters because the body makes some niacin from it (60 mg tryptophan ≈ 1 mg niacin), which links to pellagra in maize-based diets. The biuret reaction is the basis of routine serum total protein tests.

Exam angle

Short answer: “Why do free amino acids give a negative biuret test?” The violet complex needs Cu2+ bound to at least two peptide bonds (a tripeptide or larger). Free amino acids and dipeptides do not have enough.

Short answer: “Why does gelatin give a negative Hopkins-Cole test?” Hopkins-Cole detects the indole ring of tryptophan, and gelatin contains no tryptophan.

Structured question: “Explain the colour reactions of proteins with the amino acids they detect.” Give each test with reagent, colour and group: biuret (peptide bonds, violet), ninhydrin (α-amino group, purple), xanthoproteic (tyrosine and tryptophan, yellow to orange), Millon's (tyrosine, brick red), Hopkins-Cole (tryptophan, violet ring), Sakaguchi (arginine, red), lead acetate (cysteine and cystine, black PbS), Pauly's (histidine and tyrosine, red).

Remember it

My Tyre, Hop's Trip, Saki's Argument, Lead's Sulphur: Millon's = tyrosine, Hopkins-Cole = tryptophan, Sakaguchi = arginine, lead acetate = sulphur amino acids.