Please help-
My method uses a mobile phase of 60% 25mM heptanesulfonic acid/25mM sodium phosphate, pH 2.5, 40% methanol. My column is metachem inertsil ODS3, 3.6 x 150 mm, 3 micron. My problem is that my peak of interest shifts by up to 4 min from column to column and mobile phase to mobile phase. Could this be that sensitive that 1/2% change in mobile phase would shift the RT that much? Or could it be the column / heptanesulfonic combination? Thanks in advance...
-Anonymous

The answer depends on the actual rethetion time of your analyte, on whether or not it is basic, and on whether you are running at "ambient" temperature.

1. If the "correct retention time is 6 minutes and you're seeing a 4-minute shift, then something is *definitely* wrong. If your retention time is 40 minutes, and your're seeing a 4-minute shift, on the other hand, things are not *that* bad.

2. If your compound is basic, then it is likely being retained by a mixture of hydrophobic interaction ("reversed-phase") with the ODS phase and ionic interaction with heptane sulfonate stuck on the surface. A small shift in %B can cause a relatively large swing in the equilibrium surface concentration of of heptane sulfonate, so the answer to your question is "yes, a 1/2% change in %B *can* make a 10-15% difference in retention.

3. Along the same lines, a change in *temperature* can also change the equilibrium surface concentration of of heptane sulfonate.

Bottom line is that ion-pair separations are typically more fussy about mobile phase composition and operating temperature than we might like.

-- Tom Jupille / LC Resources

1. 25 mM of the ion-pair reagent is fairly high, standard concentrations are 2x lower.

2. Do you allow the column to equilibrate with the reagent? It takes a long time for a column to equilibrate with ion-pair reagents. If this is the problem, dedicate the columns to the mobile phase with the reagent and store the column in this mobile phase with the reagent as well.

Dear Anonymus:

I have validated many Ion-Pair LC methods using conditions similar to those you describe, mostly with gradient conditions. When I study the riuggedness of the separation is my experience that three of four factors always make the most difference in retention;

1.- The Ion-Pair reagent concentration is always critical, changes in suppliers or errors in mobile preparation can shift retention very much. In most cases I use 0.01 M but is not unusual to use 0.02 or slightly higher.

2.- The second most frequent problem is mobile phase pH. I hope you control this carefully. I have seen cases where 0.1 pH units shift retention of some compounds by 10-20%.

3.- % organic content is always important. Make sure your pump is working well if you are pump-mixing your eluent. If you are premixing the mobile phase then this is not likely to be a problem.

4.-Buffer salt molarity. This can also have a marked effect on retention. I have seen significant changes when the molarity changes by as little as 0.005M. is it possible your buffering capacity is being overwhelmed by the sample concentration?. Try using 0.05M instead.

The column equilibration time can also be rather long. In most of my methods I recommend to run a couple of gradient blanks before any samples. Or if isocratic elution is employed, then I recommend to inject the sample 2-3 times until constant retention is obtained.

Another factor that can be giving you problems is the sample solvent. In this mode the column is always in some state of delicate equilibrium with the mobile phase. If your samples are not dissolved and injected in mobile phase, that could lead to retention shifts and abnormal peak shapes.

Temperature has an effect. But in my experience it has been always minor between 20-30C.

Good Luck;

Benjamin

All-
Thanks for the advice. We'll look into all that was mentioned. I appreciate your help.
-Anon