Solvent A consists of 0.1% (v/v) Trifluoro Acetic acid (TFA) in
deionized water and solvent B has 0.1% (v/v) TFA in 50% acetonitrile
in water. Solvent gradient used was as follows: 100% solvent A for
0e5 min which was reduced to 90% from 5 to 60 min, 30% from 60
to 75 min, and 0% A from 75 to 80 min. Absorbance was monitored
at 215 nmand the peak areas were used to prepare a standard curve
using the three difference concentrations of the peptide fraction in
water. The equation to calculate concentration based on the standard
curve was: