Test Reagents Test for Result Reaction/PrincipleBiuret Test CuSO4 sol'n, diluted

NaOHIntact proteins and protein hydrlysates (at least tripeptide, 2 peptide bonds

Pink to violet blue color

ComplexationForm. Of coordination complex Cu+2 and 4 N atoms (2 from each 2 polypeptide chain)

Ninhyrin Test 1,2,3-indanetrione monohydrate or triketohydintene hydrate, ethanol

Alpha- amino group (usually a general test for amino acids)

Blue to blue-violet color (proline:hydroxyproline gives a yellow color)

Oxidative decarboxylation & deamination followed by condensation

Xanthoproteic Test

Conc. HNO3, conc. NaOH

For W,F,Y (aromatic except for H)

Yellow sol'n/ppt. with conc. HNO3,

orange with excess NaOH

Nitration via SEAr

Millon's Test Millon's rgt.(Hg2+ & Hg2

2+nitrates& nitriles/Hg(NO3)2 in conc. HNO3, w/ trace of HNO2/HgSO4,H2SO4,NaNO2)

Phenolic group of Y Old rose/flesh to red ppt.

Complexation(mercuration & nitration or nitrosation/complexation of nitrohydroxyphenyl derivatives with Hg2+)

Hopkins-Cole Test Glyoxylic acid (Mg powder, oxalic acid, HOAc), conc. H2SO4

Indole group in W Pink to violet interface

Reduction of oxalic acid to glyoxolic acid and acid-catalyzed condensation of two tryptphans w/glyoxolic acid

Sakaguchi Test Alpha-naphthol, NaOBr, NaOH, urea(to stabilize color & destroy excess OBr ions)

Un-or monosubstituted guanidines in R

Red to red-orange color

Complexation (base-catalyzed condensation of alpaha-naphthol w/ the guanidine group of R)

Fohl's Test Pb(CH3COO)2 , NaOH

Sulfur-containing (C, C-C)

Brown to black ppt. Degradation & substitution rxn to form PbS

Nitroprusside Test Na2Fe(CN)3 in dil. NH3

For cys Red coloration Complexation rxn

Test for Amide NaOH 10,20,30 amides and nitriles ; peptide bonds

Red litmus to blue Basic hydrolysis

Pauly's Test Diazo rgt. (1% sulfosalicylic acid with 5% NaNO2), 10% Na2CO3

Detects tyr and his Red coloration Formation of azo dyes