Chromatography Forum

Hello,

The result form this thread: http://www.sepsci.com/chromforum/viewtopic.php?t=4259

is that I am now using a different mobile phase. This mobile phase was discovered from a published paper on a validated method for the compound (Imatinib Mesylate).

The mobile phase is 30mM sodium heptane sulphonic acid in 0.01M KH2PO4 (pH=2.5):MeOH (42:58).

The RT is quite sensitive to the MeOH%. Even more concerning is that tailing varies wildly. As a result, running standards show huge varience in concentration from approx 80ppm to 180ppm (100ppm standard). Peak height changes quite a bit too. I am having a very difficult time in getting consistent results.

I am interested in doing a long equilibrium and perhaps keeping the column in the mobile phase. While I have read that storing the column in MP for ion-pairing is good, I'm not sure about the issue of the buffer. Can anyone advise on this? I should also mention that I seem to get the least amount of tailing early in the runs, and tailing increases over time. Furthermore, I'm not sure what the ion-pairing is for. The method was validated for degradants, perhaps it helps in this separation. The ion-pair agent is not needed for retention of the main compound.

How important is it that the compound is disolved/diluted in the exact mobile phase? I had made the standard solution a while ago, and it certainly is diluted in something that is different from the above mentioned MP. Would the solution used to wash the autosampler syringe have any effect?

The only other issue that I can think of is that the online mixing is not robust enough for this method, although I would think that inconsistent mixing would have a greater effect on RT and no effect on peak shape. We are currently using two pumps with a dynamic mixer, in order to avoid disportionate evaporation of the water and methanol in the MP.

I am using a Phenomenex Luna C8 150x4.6mm 3u column.

thanks

standards show huge varience in concentration from approx 80ppm to 180ppm (100ppm standard). Peak height changes quite a bit too. I am having a very difficult time in getting consistent results.

Is the amount of tailing load-dependent (i.e., do the higher standards tail more than the lower) or is the variation more random? If it is sample-size dependent, you may simply be overloading, in which case I'd suggest taking the buffer concentration up from 10 mM to mayber 25 mM.

While I have read that storing the column in MP for ion-pairing is good, I'm not sure about the issue of the buffer.

In general, you will get the longest lifetime by storing the column in organic solvent (or whatever the column manufacturere suggests ), but equilibration time is an issue with buffers and especially with ion-pair reagents. My usual recommendation is that columns are cheap, time is valuable, so go ahead and store in the mobile phase.

How important is it that the compound is disolved/diluted in the exact mobile phase?

When we teach method devlopment courses, one of the rules we present is "to do the best chromatography, disturb the equilibrium of the system as little as possible". I had one attendee critique that statement by suggesting that it implies you should never inject a sample, but the general principle is a good one. If the sample is dissolved in a solvent which is a stronger eluant than your mobile phase, you can see seemingly random variations in peak shape. I would definitely try making up at least one standard in the mobile phase and see if that improves things.

The only other issue that I can think of is that the online mixing is not robust enough for this method,

I agree, but it's easy enough to check. Pre-mix your mobile phase and put the same solvent in both A and B reservoirs.