No repeatability on HPLC c8 column

[guyka]

I try to purify 4kDa peptide on semiprep Kromasil column, and recently I get different separations on repeating injections of the same material.

What could be the reason?

[tom jupille]

That's like going to a mechanic and saying "my car is making a funny noise; what could be causing it?"

What do you mean, exactly, by "different separations"? Extra peaks? Missing peaks? tailing? Excessively wide peaks? Retention times different?

[KM-USA]

That's like going to a mechanic and saying "my car is making a funny noise; what could be causing it?"

Hey - just had this situation this weekend, daughter drove down from college this weekend. But I'm the mechanic (but don't have any cGMP documentation for this). It took me 3 minutes to pull the belt and spin the pulley, had bad bearing. New $30 pulley, installed in another 3 minutes, noise gone.

Had this been under cGMP, only a trained mechanic with a certificate on file, and documentation of expertise on that very same vehicle could've done this....and even with the noise being gone: still would've needed performance qualification.