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RP-HPLC of reduced Antibody- Peak tailing and incorrect %
Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.
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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??
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