Early eluting peaks splitting on Agilent LC

[DrathsRob]

I'm running an Agilent 1100 with an Agilent SB-Aq column, 150 x 4.6, 5 mcm, and using it to separate dehydroshikimic acid, fumaric acid, and various isomers of muconic acid. I first noticed the peak splitting with trans, trans muconic acid, but it seems to be splitting all my early eluting peaks, including the shikimic acid, fumaric acid, and t,t muconic acid, but not c,t or c,c muconic acid. I replaced the tubing and fittings going into the column, as well as the needle seat, and of course made new mobile phases and diluent, as well as tried a new column. Mobile phases are 25 mM KH2PO4 at pH 2.0 and ACN, running isocratic at 84:16. Sample diluent is 0.2M KH2PO4 at pH 7, with the fumaric acid spiked in as an internal standard with a 2 g/L concentration. Previous analyses showed no peak splitting at all, with generally good chromatography.

[Uwe Neue]

The cause of your problem is the different pH between your mobile phase and the sample. Adjust the pH of the sample to acidic, and the problem will go away. If there is a good reason why you do not want to do that (such as sample stability), consider lower injection volumes or a giant tube between injector and column to mix the sample with the mobile phase.

[DrathsRob]

Thanks for the reply, yes in fact I do want to keep that pH 7 buffer to provide sample stability. I did install a larger diameter peek tubing with a little more length at the head of the column, but that did not affect the problem. My injection volume is currently 5 mcL, and I hesitate to go much lower, as the signal is not terribly large as it is. Unfortunately, this problem is new, and this method has been run quite a few times previously without the peak splitting problem, so I'm doubting it's a problem with the pH differential. I did check the pH of both my mobile phase and diluent to be sure they haven't changed, and they have not. Agilent suggested I bypass the column switching valve to see if the rotor seal was problematic, which is the next solution I'm going to try, will report on the results as soon as I have them.

[rhaefe]

What's the retention time of the first and the last eluting compound and at what point does the splitting stop?
I have seen columns with cracked column beds and/or voids that produced split peaks but only the early eluting compunds were affected. The late eluters didn't show splitting.
I would still investigate pH as Uwe suggested.

[DrathsRob]

. . . and got bupkis Peaks are splitting on both systems with different columns, which also leads me to think that the valve seals aren't the problem. Someone here suggested I drop the mobile phase pH to 1.7 to further protonate the analytes, but the pka of the fumaric acid is above 3, so I don't think that's the problem (it would exacerbate the pH problem everyone here is describing anyway). I'm going to try and lower the pH of the diluent on one system and make a new solution, and raise the pH of the mobile phase on another system and see what shakes out, will report when I have something. Thanks again to everyone for the help!

[HW Mueller]

How would you "exacerbate" the mobile phase-sample solvent incombatibility by going to the pH of the mobile phase?
Regarding earlier success of this method: You wouldn´t be the first one to measure a different pH than what you actually have.

[DrathsRob]

When I referred to the exacerbation of the pH problem, I meant that lowering the pH of the mobile phase to 1.7 would be creating an even bigger difference between the mobile phase and the diluent.

it turns out that both raising the pH of the mobile phase or lowering the pH of the diluent works perfectly!!! Sigh, wish I hadn't assumed that previous analyses that I didn't do were done properly, especially given the quality of the documentation that went with it, but when you're really new at a company you don't want to start stepping on toes. Good thing the person who developed this is on her way out then, because I'm going to readjust this method to make more sense. Hopefully by adjusting the nonpolar content downwards a bit I can make up for the increased polarity of my analytes at pH 7, since the various forms of muconic acid will convert to the t,t form in a low pH environment, and I need to keep things in their original form.

Thanks so much everyone, I'm quite relieved that our systems are actually working fine, and it was just an iffy method that needed modifying. Cheers, Rob.

[Rdsxbaseball]

Rob,
Was the peaks splitting after a certain number of injections? Or was it noticeable right away on a new column?