Quantify of amino acid using ninhydrin

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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