Help me understand HPLC gradients
Hi all, I have a chemistry degree but I’m new to industry and new to method development.
So I work in an analytical lab and I’m involved in looking at all of the methods and trying to decrease the run times. These are assay methods usually for a single analyte, so we’re only interested in one peak (or 2 or 3 depending on isomers). Most of these are pretty simple fixes: decrease the purge time, decrease the requilibriation time, decrease the time it takes to get to and from the purge %B, etc. Most methods are longer than needed on all of these points.
Where I’m struggling is with the region of the gradient where the peaks are eluting. We have quite long gradients (up to 17 minutes), and the gradient ranges are quite a bit before and after the %B where the analytes are eluting. My understanding is that we should set the gradient range so that it’s just before and just after the %B of elution. But I’ve been told that this is the wrong way to think about it.
I suppose what I’m asking is, how would you go about attempting to tweak these regions of the gradient? I appreciate that this is all very general but I’m after general advice! How does all of this work?