Separation of Tryptic Digested IgG with HPLC

Abstract: The antibody immunoglobulin G (IgG) can be tryptically digested into smaller peptides. This study attempted to develop a separation method for those peptides using RP-HPLC with a C18 column at room temperature. Optimizing separation of trypsin cleaved cytochrome C was used as a guideline before analyzing IgG. The optimized analysis of Cytochrome C was performed at wavelength 280nm (UV) and methanol was used as an organic solvent in mobile phase (B). A fast gradient to 100% mobile phase B with low flow rate gave favorable results for cytochrome C. A slow gradient to 100% mobile phase B was suited for IgG separation. The optimized gradient elution of cytochrome C and IgG was performed at 0.3 and 0.8 ml/min, respectively.