Chromatography Forum

Im using the bds hypersil c18 column, reversed-phase, mobile phase: 90% phosphate buffer pH= 7.5, 6% ACN, 4% MeOH retention time of analyte is 17 minutes(basic compound),

now using the same column and the same system with mobile phase: 90% phosphate buffer pH= 7.5, 6% ACN, 4% H2O, retention time of analyte is 14 minutes???

why do i get a decrease in retention when using water, shouldn't be the opposite, isnt methanol a stronger solvent??

I'm surprised you are able to get stable chromatography using only 10% organic solvent. You are on the borderline of having column collapse. Going to 6% might have pushed it over the edge.

You might consider going to a different form of chromatography than reverse phase for such a polar compound. Ion-exchange perhaps.

Otherwise i believe Agilent makes stable bond columns which are more stable at low organic solvent concentrations.

MSChemist, do you assume or do you know that this column dewets at about 94% aqu.? One can imagine that a pH shift could be responsible for the observation.

I assume he checked the pH of the mobile phase as he states it is 7.5. I just think in any case it is far from ideal to be running RP columns at less than 10% organic and is likely to be problematic/unstable. There are plenty of better options.

Methanol is a slightly weaker elutant in reverse phase. If you found it necessary to go down from 10% organic perhaps see if you can cut out the acetonitrile entirely and just use methanol at 10%.

Thank you for ur replies,,, but from my finding today i can say there is no column collapse, and also do you think that such a small % of water will shift my pH at a 20mM buffer???????? helpppppppppppppp!!!!!!

Some of us are convinced that there is no column collapse that grossly influences retention time, but some have shown that there is certainly the possibility of dewetting, which appears to happen rather suddenly and extensively (I have never observed it probably because I junked old fashioned C-18 columns early??). I therefore would expect a stronger lowering of rt than seen here.
At a pH of 7.5 the pH is quite touchy even with a good buffer, remember that pH can be stongly influenced by organic content.