Pharmaceutical Chemistry 127.1Group 5 – Sierra, Tolentino, Zuñiga

Experiment 2COLOR REACTIONS OF PROTEINS AND AMINO ACIDS

Ninhydrin Reaction Biuret Test Xanthoproteic Test Milton - Nasse Reaction

Test ALL samples.

a) 0.5mL sample + 1-2gtt ninhydrin reagent

b) Heat to boiling for 1-2minsc) Allow to cool

intense blue or purple solution (SR: Ruhemann’s Blue or Purple) effervescence (due to CO2 liberation)

proteins: ALL samples amino acids: ALL samples

non-halogenated waste container

Test ALL samples.

0.5mL sample + 0.5mL 10% NaOH + 1gtt 0.5% CuSO4

pink to violet blue solution (color deepens as the # of peptide bonds increases)

proteinscasein violetalbumin violetgelatin blue

amino acids

metal waste container

a) 0.5mL sample + 0.25mL conc. HNO3

b) ∆ under the hood. Observe if the white ppt turns yellow and finally dissolves. Color of the ppt &/ the sol’n?

c) Cool. + enough (4-5mL) 10% NaOH to make the solution alkaline. Color?

yellow solution (SR: nitro derivatives) upon heating with conc. HNO3

orange solution when made alkaline w/ NaOH

amino acids: tyrosine, tryptophan

acid waste container

a) 2.5mL sample + 2-3gtt HgSO4

b) Heat in a boiling H2O bath for 10mins. Cool in H2O.

c) + 0.5mL 1% NaNO2 sol’nColor of the ppt &/ sol’n?

old rose solution (SR: Hg+2 salt of the phenolic compound)

amino acids: tyrosine

metal waste container

Hopkins - Cole Reaction Bromine Water Test Pauly Reaction Lead Acetate Reaction

0.5mL sample + 0.3mL glyoxylic acid sol’n

+ 1mL conc. H2SO4

violet ring at the junction of the 2 fluids (violet complex)

amino acids: tryptophan

acid waste container

a) 0.5mL sample + freshly prepared bromine water

b) Shake w/ 0.5mL n-amyl alcohol. Color of alcohol layer?

pinkish lavender or violet alcohol layer

amino acids: tryptophan

halogenated waste container

a) 0.5mL cold sat’d sulfanilic acid sol’n + 0.25mL cold 1.0% NaNO2

b) Cool in ice w/ constant shaking for 3mins.

c) + 0.5mL sample + 10% Na2CO3 (to alkalinify)

red or purple condensation products

amino acids: tyrosine, histidine

dilute w/ H2O sink!

a) 0.5mL sample + 1mL 10% NaOH + 1gtt Pb(CH3COO)2

b) Cover w/ marble & boil in H2O bath for a few mins

black precipitate (SR: PbS)

amino acids: cysteine, cystine

sulfides waste container

Nitroprusside Reaction Sakaguchi Reaction

(Samples) proteins: [1.0%] casein, albumin, gelatin, peptone amino acids: [0.1%] glycine, alanine, tryptophan, tyrosine, phenylalanine, histidine, cysteine, cystine

Pharmaceutical Chemistry 127.1Group 5 – Sierra, Tolentino, Zuñiga

0.5mL sample + 0.25mL sat’d Na2CO3 sol’n + 1gtt freshly prep’d sat’d sol’n of sodium nitroprusside purple color?

if negative, + 1gtt 1.0% NaCN

purple color (free sulfhydryl groups are present) purple color only after the addition of NaCN (disulfide bonds are present)

amino acids: cysteine, cystine

cyanide waste container

a) 1mL 10% NaOH + 0.5mL sample + 1mL -naphthol reagent

b) + sodium hyprobromite sol’n dropwise ( 5gtt)

red or orange solution

amino acids: arginine, aminoguanidine

halogenated waste container