Chromatography Forum

Hi all

I am working on a related compounds method using xterra column RP8 3.5 um : MP is ammonium bicarbonate buffer10mM pH10 and ACN gradient. When i condition the column i see an interference peak comes at 50:50 buffer: ACN; This interferes with my impurites. i couldn't get rid of the peak. It is present in water. i used high purity water. it is still there. If i do only 50:50 isocratic i dont see that peak. my gradient starts 95:5(buffer:ACN) stays for 5 mins. goes to 50:50 in 20 mins. i need this gradient for separation of 7 impurities in the method.

can some body help me with this ( i don't want to develop a method around an interference peak)

Wavelength used is 230; present is all wavelengths


thanks

From your post, I infer that you see this interference even when you run a "dummy" gradient (i.e, either no injection, or injection of mobile phase).

Try running two such dummy gradients following different equilibration time; for example:
- dummy gradient to flush the system
- equilibrate for 10 minutes
- dummy gradient #1
- equilibrate for 30 minutes
- dummy gradient #2.

If the interference peak is larger in gradient #2, then you have confirmed that you are looking at contamination in your A solvent. Possible sources:
- the water supply (obviously); try a different source of water.
- your buffer salts (try different lots and see if that helps)
- the pH electrode (if you are dipping the electrode in the solvent, you may have buffer leakage; try measuring pH on aliquots removed from the main vessel)
- contaminated glassware (detergent?)

When you solve the problem, let us know what you found (so we can all learn!)