Chromatography Forum

Does anyone know which UV transparent acids can be employed (at 200-205 nm) with a silica column in HILIC mode? I believe I saw some mention of the incompatibility of phosphoric acid but I do not understand why. What might be typical acid contents in this mode vs. reverse phase (0.05 - 0.1% v/v ok?)

Is it common to use higher concentrations of buffer in HILIC (with UV) mode than, for eg., when employing an MS detector. Again, I've read many application notes where 100 or 200 mM of formate or acetate buffer is used, and am curious how this affects the retention mechanism if silanols are fully protonated on a high purity silica at pH ≤5.

thanks.

I do not see why phosphoric acid could not be used if you are using a UV detector. You need to be careful with its solubility. This is the major problem.

Higher concentrations of MS compatible acids have been used in HILIC compared to standard RP. However, for most applications, you do not need high concentrations, and you can work with similar concentrations as in RP.

Phosphates, even phosphoric acid, absorbs much less at the low wavelengths than the carboxylates. I just used a 10+90 and 50+50 phosphoric acid(0.1M) + ethanol mobile phase without any precipitaion problems. As soon as you have Na+ in there you can get severe precipitation, though, for instance: NaPhos buffer at pH=8, 0.025M, precipitated at 20 buffer + 80 EtOH.

thanks Uwe and HW.

To be honest, we used phosphoric acid anyway (couldn't really fathom what the problem could be). It was only when nosying at Waters' Atlantis column care n' use booklet did I notice that phosphate is not recommended in HILIC mode on their silica. I therefore posed my question as a generalised one wrt any silica column.

0.1% H3PO4 gave us pretty comparable chromatography in an ACN/aqueous gradient vs TFA for a low mol weight cpd containing 1° & 2° amino and an amide functionality.