Easy Ion Pair question

[Mike H.]

I'm trying to get retention on a very polar basic compound. It elutes at void volume in RP so I set up ion pair conditions using 10 mmol 1-octane sulfonic acid monohydrate. The compound is retained well with this but, I'm seeing a lot of tailing ~2.8. I'm never seen any tailing in any of our other ion pair methods, so this was surprising. I really don't know how to approach this problem from an IPC standpoint. Any ideas on possible causes and/or solutions?

thx

[juddc]

I'll take a shot.

One or two things occur to me, but absent more information, they're speculative at best. First, if you're using an old style HPLC column with a significant concentration of acidic silanols and your analyte has multiple basic groups (or perhaps a secondary amine or two) I can see tailing occurring based upon interaction beween an acidic silanol and a secondary amine. Also if the molecule is small and has multiple basic groups in close proximity, it might be that the octane sulfonate is too large to effectively ion-pair with both.

What happens if you use a smaller ion-pairing agent such as TFA or heptafluorobutyric acid?

What's the pH of the MP relative to the pK or pK's of the charged groups on the molecule?

[Mike H.]

The compound I'm trying to resolve has a purine base. We have several tweaks of the molecule and have previously had no problems with tailing. The molecule in question has been phosphorylated. Now we are seeing bad tailing where as before we observed none.

We have TFA and HFBA. I've never used these before for IPC. Both are liquids so what would appropriate starting conditions be for a screening run? Volume % of each would be very helpful. Thanks.

[danko]

You can optimize the initial attempt by adjusting the pH to something like 2.5 and still use octane sulfonate.
It (the ion pair reagent concentration) can be optimized too – try higher concentration – say 20 mM

Best Regards

[Mike H.]

I'll try the 2.5 pH and post back. Current we didn't monitor pH for the base molecule. We just added 10 ml glacial acetic acid to 1 L to both mobile phase.

Incidently I did try 20mmol OSA and the molecule never eluted.

[Mike H.]

The lower pH helped. New conditions are as follows.

Mobile Phase A: 5 mmol OSA, 900 ml water, 100 ml ACN,1ml TFA

Mobile Phase B: 5 mmol OSA, 500ml water, 500 ml ACN, 1ml TFA
.
Simple gradient. 1.5ml/min, Agilent XDB c-18 column, 50mm,4.6 mm,1.8µm
0.0min 100% A. 10min 100% B
System is not fully equilibrated yet, but a preliminary injection has the tailing down to 1.8.

[danko]

I wouldn't use TFA in this case. Use phosphoric acid to adjust pH down to 2.5. pH 2.2 might be even better.
Use the sodium or potasium salt of octane sulfonic acid rather than the acid itself.

If you follow the above suggestions you might see both more symmetric and narrower peaks. It's woth trying anyway.

Best Regards