Chromatography Forum

Hello,

I have recently started to use Tetrabutylammonium hydrogen sulfate to separate nucleotides/nucleosides on a C8 column. The separation works quite well but unfortunately I got a ghost peak at the end of my run-allthough the ion pairing agent should be HPLC and gradient grade. After I had to order a new batch (same lot and filling code!) the ghost peak moved closer to my last sample compound, allthough I did not change anything. I have tried changing the ion pairing agent concentration, bufffer concentration, organic modifier, pH.....but I could not eliminate this peak, however, I found out that its retention time decreases with increasing % of methanol or acetonitrile. I never had problems with water, buffer or methanol/acetonitrile (all HPLC grade) in runs without this IP agent.

The buffers I am using are:

Buffer A: 50 mM potassiumphosphate buffer, pH 6, 4 mM TBAHS
Buffer B: 50 mM potassiumphosphate buffer, pH6, 4 mM TBAHS, 25%
MetOH
Gradient profile: 0 to 100% Buffer B in 10 minutes
100% Buffer B from 10-15 minutes

the ghost peak comes off at ca. 12/13 minutes and its UV absorbance maximum is around 215 nm...

I am still waiting for the supplier to get back to me, but I was wondering if anyone has any good suggestions what else I could try....

Many thanks,

Steffi

Some things to try:

1. Higher detector wavelength (if you can sacrifice sensitivity)
2. Try C18 instead of C8. Chances are the unknown will "hang around"
longer and won't be as much of a threat to interfere with your last peak.
3. Go on a hunt to find the culprit:
In- house water supply
Buffer (less likely)
MeOH
TBAHS

You can do UV scan of each solution. Or try a gradient without the IP, if there's no peak - the IP is the culprit.
If the uknown peak is still there then it's mostly likely from the water or MeOH.