Chromatography Forum

if having no information(only solubility/moleculer structure) on analyte than how should i start my HPLC method development

In principle, run a gradient from 5% acetonitrile to 90% acetonitrile. If you suspect that you have ionizable compounds in your sample, add an acid to the mobile phases: about 5 mM phosphoric acid (check the miscibility/solubility in the 90% acetonitrile/10% 5 mM phosphoric acid) for UV detection or 5 mM formic acid for MS detection. With UV, monitor at a low wavelength (~215 nm) to see everything that elutes from the column.

See where the stuff is coming out and narrow the gradient or go isocratic.

first look at the detection possibilities -UV is easiest, if it has a chromophore.

then at solubility- if its water/methanol/ACN soluble try Uwe's way,
if its only soluble in apolar compounds, go for normal phase

Alex

I am aware of a lot of labs where the start to inject the compound on a HILIC column.

If that doesn't work, then they try RP columns.



The reason for the strategy is that HILIC typically will give better sensitivity in MS or ELSD, faster and better sample preparation, and retention also for impurities. So the gain is considerable.