Chromatography Forum

A commonly faced problem for the ion-pair chromatography is the RT drift for the ionized species. Some peaple do not suggest a postwash of the column as the ion-pair equilibrium takes a long time in some cases. Some people insist a through postwash after each experimental run to avoid an irreversible status of the column after using a length of time without washing.

I have tried an Epic Polar column using 10 mM sodium hexanesulfonate in water from 70% to 10% in gradient with ACN, the column can be easily equilibated within 1 hour. However, the column only worked for 2 weeks. From the third week, two weak basic compounds suddenly started to shift dramatically from one injection to another, resulting a failed system suitability. I tried to wash the column with water, ACN, MeOH and combinations at 40 C, it helps, but unable to recover its status before the start of the significant drift. Does anyone have suggestions for

1) should we do postwash immediately after each run?
2) is there a way to wash out the hexanesulfonate ?
3) any comments???

Thanks,

John

With ion-pair chromatography, there is only one way in which you can get guaranteed reproducibility. This is isocratic conditions, and full equilibration with the ion-pair reagent. In the moment you run a solvent gradient in the presence of an ion-pair reagent, you are always out of equilibrium, and you method is likely to work ONLY if you keep all operating parameters 100% constant and reproducible.

Therefore, I do NOT recommend any additional washes in such an already complicated system. If you can't recover the separation, it is possible that your analytical column has changed just enough to throw you off. To test for this, use a brandnew column and see if the separation is OK on the new column.

Since such a system is so sensitive, it is also possible that other minor disturbances in your protocoll are the cause of the problem. You did not talk about the pH. How reproducible is the concentration of your ion-pair reagent? Is there a difference between the column preequilibration in your older runs and the newer runs? Any change in the column reequilibration? Many possibilities, unfortunately...

John,

You might want to consider our Primesep approach, when ion-pairing reagent is attached to the surface of silica gel. Equilibration time after gradient is extremely short (2-3 column volumes). You can use ACN-water with different additives (TFA, formic acid, sulfuric acid, phosphoric acid, ammonium acetate, ammonium formate, phosphate buffers, etc.). See the link for Method Development Guide:

http://www.hplcmethoddevelopment.com/

Depending on your detection technique you can use different additives. TFA, ammonium acetate, formic acid if you need ELSD,LC/MS or prep work; sulfuric or phosphoric acod (or buffers) if you need low UV.
Click on the link below for "Retention of Polar Compounds without Ion-Pairing Reagents"
http://allsep.com/Newsletters_Home.php

Contacty us if you need more information

regards,

Vlad