HILIC and moving between buffers

[Serpico]

Hi all,

I have a successful separation using a ZIC-pHILIC column with 60:40 ACN/25mM ammonium acetate. I happy with the chromatography (not optimized but fit-for-purpose) although detection is currently by mass spec only.

I now need to move this to a low wavelength UV compatible system. Are there any guidlines on how to do this or is it impossible to translate this type of method between buffers (say move to Potassium Phosphate)? Compounds require the method to be at 195nm wavelength.

If not possible I may look into ELSD or CAD detection but UV would be my first preference.

Thanks.

[Bintang]

Phosphate buffer is generally not recomended for HILIC separations due to its low solubillity in high acetonitrile eluents. Since you are using a high proportion of water in your eluent you may still be able to use phosphate buffer but make sure that you do not use a buffer strength too close to the solubillity limmit. It could also be wise not to mix the eluent online but use a premixed buffer to minimize the risk of percipitation in the mixer. If your analyte is ionic you may experience selectivity differences between buffers, but since there is so little work done using phosphate buffer in HILIC you are entering relatively unchartered waters.
I would advise you not to use gradients with phosphate buffers in HILIC.
We have one application note using phosphate buffer

http://www.sequant.com/files/documents/ ... phates.pdf

[Andy Alpert]

You can use an inorganic anion like phosphate provided you use an organic cation like triethylammonium ion (i.e., triethylamine phosphate). Dissolve a known amount of HPLC-grade phosphoric acid in water and then add triethylamine until you reach the pH you want. As Bintang says, though, selectivity will be different with different salts. The following link takes you to a paper that compares the selectivity in HILIC of triethylamine phosphate and sodium methylphosphonate (another salt you could use here): http://pubs.acs.org/doi/pdf/10.1021/ac070997p
You will get very different selectivity for different classes of charged compounds depending on which salt you use. The paper goes into the mechanism of this in some detail.

[carls]

What pH are you using for the MS method? The mobile phase pH changes signifcantly from the 100% aqueous pH value when mixed with the high organic concentration normally used in HILIC separations. The pH change depends primarily on the nature/charge of the components providing the buffering. Large changes in selectivty could be observed if pH change when moving from one buffer to another spans the pKa range of the analytes.

[Serpico]

Thanks guys, some really good information in this thread.

I will follow up some leads but it looks like it won't be easy to switch into a UV compatible system whilst being relatively certain of retaining a similar separation.