I'm tweaking a method (RP-HPLC) so that I can get separation of about 23 amino acids I need to quantify. As I refine the method, I am tempted to add more and more steps, to really 'tighten up' the chromatogram. Is it considered 'bad form' to have a method with too many steps?

I got an opinion on this topic from a career chromatographer; part of his reply: "putting more than 2 or 3 segments in a gradient is risky. The problem is that the conditions get harder and harder to reproduce if you change equipment, because of the washout and mixing characteristics of each instrument. If you will only use the method on a single instrument, you can get away with it. One of my colleagues did about 10 segments for a peptide separation and it was quite reproducible on a single instrument, so I know it works. So the bottom line is that it is bad form, but sometimes you gotta do what you gotta do to get the job done."