Hi Bobby12,
Just general comments:
Seems to me that the pH where you're operating (7.0) for the phosphate with the Ion-Pairing agent octanesulfonate is really kind of high...better protonation of that second nitrogen on the imidazole ring would occur at pH values of 5 or less (that's why I suggested 2.5 above). Maybe Tom Jupille will see this thread and lend a hand with embedding chromatograms...he once was kind enough to tutor me on embedding quotes, but I didn't use the knowledge he gave and misplaced his e-mail note to me.
Anyhow, if the retention time for the imidazole-octanesulfonate "ion-pair" in this case is able to be repeated consistently run-after-run...well, that surprises me, and you know what is happening better than I do.
Undoubtedly part of the shift in the baseline is due to the "proportioning-in" of organic...you've got a ternary gradient going on, and the buffer proportion decreases fron 95 to 20%. Some shift in the baseline would be expected...however, the change in percent organic will also have a possibly ill-effect on the concentration of the octanesulfonate that becomes "attached" to the stationary phase. Generally that is why gradients are not desired for use with ion-pairing agents as the ion-pairing agent can be slightly removed from the stationary phase during the course of a gradient, possibly affecting retention times for analytes in subsequent runs, and certainly contributuing to a shift in the baseline of a given chromatogram. Remember also...please allow some time for the HPLC system to recover from 20 Buffer:20 MeOH:60 ACN back to 5 Buffer:95 MeOH--didn't see this in the gradient table you typed in, and I know that the "guideline" of 10 column-volumes of initial eluent composition isn't written in stone, but one minute may not really be enough...particularly with octanesulfonate in there.
Again, you see what happens run-to-run using the separation you're working on...just something to consider. One thing, though...if your analytes are "spread-out" as much as you're describing, with that column and those conditions, you wouldn't need a column anywhere near as long as the one you're using. (As HPLCAddict wisely notes, it's not about the retention time of the analyte, it's about the retention factor of the analyte, the analyte retention time divided by the "retention time of an unretained analyte," sometimes called tm...for the imidazole-ion-pair in your case, the value is ca. 3.5, which is adequate for quantitative work.) Probably though, if this column is what you've got and you can't change...well, it's a situation I've seen in my career. Just because I think things may have been unwise didn't mean I could do a whole lot to change them...a more polar phase may help retain imidazole even without an ion-pairing agent, and not hold quite so hard onto you're other analyte of interest that contains an imidazole functional group.
My hope is that somehow you'll get to change the stationary phase to one that is more polar (for this set of analytes)...well-covered phases with excellent endcapping such as the one you're using are great for bases like imidazole, but in my experience only if I used acidic eluents and ion-pairing or basic eluents with pH greater than the pKa values of my analytes of interest. I read that Phalanx is supposed to be resistant to high pH...don't have any experience myself with that Higgins product, but running an eluent at pH 9 or so can be a scary proposition that requires the use of a sacrificial column...silica begins dissolving at pH 8 or so.
Anyhow, what I'd try is to run your separation again, but at a more acidic pH...perhaps the imidazole-ion-pair may be better-retained than it is now and the other analyte less-retained...this may work to your advantage in a couple of ways in the long term.
I wish you well, don't know what the others will say, and these are only opinions.
. It's nonsense to aim for a certain retention time, the retention FACTOR is the value which will show you if you have enough retention.
