This document describes a procedure for quantifying amino acids using ninhydrin. A series of glycine standards of increasing concentration from 0.2mM to 1mM are prepared using serial dilution calculations. The standards and unknown samples are mixed with ninhydrin solution and heated to develop a color. Absorbance is measured at 570nm and a linear relationship between concentration and absorbance is observed, verifying Beer's law.

1. Quantify of amino acid using ninhydrin .
Procedure:
Prepare 1mM solution of known amino acid in isopropane and 0.2% ninhydrin in acetone.
Added 2ml of the total vol.and 2ml of ninhydrin solu. to each tube and heat for 15 min in boiling
water. Then added 50 % 3ml of ethanol and 10 ml of water. Set the Spectrophotometer to 0
absorb at 570nm.
Procedure to prepare Ninhydrin solution:Take 0.5 g of Ninhydrin in 30ml of Acetone. Shake well so
that ninhydrin could completely solve in water. Add 20ml of acetate buffer which has 5.5 pH.
Stock solution of 1mM amino acid(Glycine): 1M =75g/1 liter ; 1mM = 75/1000=0.075g/l
Prepare a series of test tubes with amino acid.
C1V1=C2V2 (c1 stock solu. ; v1 volume stock solu. use ; c2 conc. need ;v2 vol. need)
For 0.2mM . 0.1* V1=0.2*100 ; V1=0.2*100/1 ; V1 = 20
No of
obs.
Stock solution
(ml)
0 .2mM 20
0 .4mM V1= 0.4*100/1 = 40
0 .6mM V1= 0.6*100/1 = 60
0 .8mM V1= 0.8*100/1 = 80
1 mM V1= 1*100/1 = 100
Observation:
Conc. 0 .2mM 0.4mM 0.6mM 0.8mM 1mM
absorbance 0.655 1.09 1.361 1.781 1.954 2.20
Discussion: Result proof the beer lamberd’s law as we increase the concentration absorbance
also increase.
Reference:Lab manual in biochemistry by Arti Nigam.
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