Chromatography Forum

We use a kind of column which is made by waters corporation (spherisorb○R5μm NH2 4.6mm×250mm column)to analyze sennoside A in herba medicine senna. mobile pharse is acetonitrile:PH3.5 acetate buffer(1:1). After we use the column for serval times,we found that the retention time became more and more short (0.3min per time),tail factor got more and more bigger, but back pressure did not change, we estimate that we have not clean the column corretly, please tell me how to clean and regenerate this amino column.Thak you very much!

The problem is most likely hydrolysis, and not contamination of the column. The quickest way to fix this is to inject as soon as possible about 250 microL of acetic acid onto the column.

If the change in retention has stopped, don't do anything - you are fine. But the next time you start a new column for the same assay, do the acid injection first.

Okay, Uwe, why?
(I'm not questioning the advice, I just want to understand what's happening )