expt2 schematic
TRANSCRIPT
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