Stage Procedure Conditions
1 precipitation with methanol v/v ( 4:6-8:2) followed by centrifugation at 12 g, 7 min
2 SepPak C18 in presence of methanol >70%
3 HiTrap SP HP Methanol<20%,  pH 2.95
4 HiTrap Q HP Methanol<20%,sample application pH 7.0, elution with 0.4% of formic acid (pH~2)
Table 1: C-peptide purification scheme. Methanol precipitation was followed by centrifugation; the supernatant was then loaded onto C18 SepPak cartridge. The effluent was then applied to the ion exchanger after dilution of the methanol concentration and pH adjustment.