Waters PIC A low UV Reagent

[agrubert]

Has anyone had any luck with Waters PIC A Low UV Reagent in a HILIC method using water/Acetonitrile as the mobile phase? I'm having trouble keeping the buffer in solution when mixed with greater than 55% ACN, which is then causing retention time trouble with my run. I was good at 65% but then the temperature dropped in the lab and it crashed out! But I LOVE the fact that the buffer gives little to no interference at 210nm and would like to use it as my mobile phase buffer. Is there anything out there that will help keep it in solution? Or should I switch to a C18 or other column with this mobile phase?

Thanks!

[carls]

I have not heard of using PIC in HILIC mode and cannot really think of a reason to do so. Can you provide some further details?

HILIC is used for the analysis of polar compounds. It is not suited for analysis of hydrophobic compounds.

PIC is typically used in RPLC to increase retention of polar compounds by giving them hydrophobic character.
PIC reagent - ion with hydrophobic tail - pairs with polar ionic analyte of opposite charge.

[tom jupille]

"PIC A" is basically tetrabutylammonium hydrogen phosphate (TBAH), so it could be used as a buffer. I'm not sure why one would want to use it that way as opposed to, say, simply preparing a phosphate buffer.

[agrubert]

I was working on a method for oxalic acid detection and HILIC was recommended. However, I tried several phosphate buffers which interfered with the analyte at 210nm. PIC A was suggested for use and provides me with no interference at 210 and good peak shape, but is not soluble in high ACN %.

[tom jupille]

What pH are you looking for? For buffer purposes, PIC A = phosphate buffer (look up the MSDS for PIC A). You might try a different salt (lithium phosphate?) for better ACN solubility or cut the concentration.

[carls]

You could try 0.1 v/v% conc H3PO4 (without PIC). It is miscible with ACN. The solubility of H3PO4 drops to near zero at 90/10 ACN/H2O with addition of any base, i.e. H2PO4- very low solubility.

You have likely permanently modifed your column by using the PIC reagent so get a new column for any further method development.

What HILIC column are you using?

Have you tried RPLC with PIC A?

[Gerhard Kratz]

I agree with Carls. Depending on the kind of HILIC you might have some RP interactions, or SEC, or IEX on the HILIC column. But with a pure HILIC phase Carls is right. Which one did you use?

[agrubert]

I'm using a Waters XBridge Amide column at this time.

My next step is now to switch to a RP column while sticking with PIC A in my mobile phase. If that doesn't work I plan on going back to HILIC and trying out another ammonium phosphate mobile phase to see if that works. I really don't want to have to purchase a new column though, but if I altered it permanently I guess I will have to do so. Any HILIC column recommendations are welcome!

Thanks.

[carls]

If you need >~85 v/v% ACN for HILIC no phosphate salt (not TEAP, not NH4) will have sufficient solubility.

Since you must use low UV (do you have another detector?) for detection your choices for buffers/additives are limited.

As I mentioned above, 0.1% conc H3PO4 is miscible with ACN and any proportion of ACN mixed with water. This is only true for H3PO4. If you add any base (i.e. make a salt) it will precipitate.

You could try 0.1 v/v% conc H3PO4 in 90/10 ACN/H2O with the amide column. If it looks promising (reduce organic to decrease retention, if needed) order a new one and hope it looks similar (i.e. the residual PIC is not responsible for the good behavior).

Bare silica is a very useful starting point for HILIC. If your compound is not retained on bare silica with 90/10 ACN/buffer its unlikely you'll retain it on another HILIC column. If the analyte is well retained on bare silica but the peak shape is poor then move to a bonded silica phase such as Luna HILIC (diol, Phenomenex) or ZIC-HILIC (zwitterion, Merck). All of these phases can be used with 0.1% H3PO4.