RP-HPLC of reduced Antibody- Peak tailing and incorrect %
Posted: Thu Oct 24, 2013 7:33 pm
Trying to run RP using a Zorbax 300SB-C3 column. 0.1% TFA in water and 0.1% TFA in 80/20 ACN/IPA. Antibody is denatured with guanidine and reduced with DTT. Light chain peak looks funny..assymetric peak with some tailing, but heavy chain peak looks ok. However, the % LC to HC is not right. I'm getting close to 50/50% for peak areas. It is quite reproducible. I do notice some heavy chain carryover to next blank injection, but not enough to account for % area problem. Anyone see this or have any suggestions??