Chromatography Forum

Dear all,
I am using an Acquity BEH C18 column at 40C

If I use a mobile phase A of 90%phosphate buffer (pH 7), 10% AcN, 2mM ammonium acetate and 0.1%f.a. and a mobile phase B of 10%phosphate buffer (pH 7), 90% AcN, 2mM ammonium acetate and 0.1%f.a., will I have buffer precipitation??

Regards,
T

You didn't give the concentration of phosphate in the buffer.

That said, the easy (and smart) thing to do is to mix your A and B solvents in different ratios in test tubes and see what happens.

You are so right!! Your tip is highly appreciated.

(FYI: phosphate concentration is 25 mM)

Phosphate buffer (i.e. any phosphate salt) is not soluble in 90% ACN. I tried to get as little as 1mM into solutuion for use in HILIC but it precipitated. Phosphoric acid, without ANY salt e.g. ammonium formate, is miscible with ACN.

Pete Carr published an article on buffer solubility back in 2004 (http://tinyurl.com/4lrr6r4). His data are for 25 degrees. About 10 years ago we looked at the issue (albeit much less rigorously) when we were working on an automated method development system. We used 10 mM buffers and ran gradients only up to 85% ACN because we saw evidence of precipitation at 90% (again, this was ambient temperature). That system operated at temperatures ranging from 35 to 55 degrees (if memory serves!), so that provided a bit of margin.

When all is said and done, though, I'm an empiricist: when in doubt, try it and see what happens!

dear all, thank you so much for your replies!