controlling retention time and pH

[sluggo]

Hello,

I recently started analyzing a new compound (to us) and found some developed methods in literature. After testing a few of the methods I decided on one that seemed to give good peaks and a workable time scale (ie the retention time was neither too soon nor too long).

The mobile phase consists of water + 1% TEA, adjusted to pH 2.4 by phosphoric acid. I then mix the water with methanol 65/35 v/v (but measured gravimetrically). The pH is then re-adjusted to 2.4.

Having made three batches of mobile phase, it seems that the retention time is extremely sensitive to pH. It appears that even a very small change in pH (difference of 0.05) can result in the retention time shifting from 7.5min to 6.5min.

Is it common to experience this type of behaviour? Should the mobile phase be controlled within such a tight window? Or perhaps a standard can be used to adjust the retention time at the start of each day or whenever new mobile phase is added to the system.

I had also tried using a phosphate buffer with water and methanol, but the peak shape (tailing) wasn't as good as the above mentioned mobile phase.

Regards

[Consumer Products Guy]

Slug - I suspect your pH variability is due to measuring apparent pH of a water-methanol solution. I think I'd decide if I wanted the 6.5 or 7.5 minute retention time (maybe you don't care, as long as it's more consistent). I'd devise a more-robust way of making the mobile phase, such as adding known amounts of your TEA and phosphoric acid, or adjusting pH of the aqueous-only phase that gives you a "desired" pH once the methanol is added. Also, as methanol selectively evaporates in your solvent reservoir, your mobile phase concentration will change somewhat. Or you could just adjust for the retention time drift over time, or make sure if your mobile phase volume is low to add new MP first thing that day, mix it well, purge the lines, so all your results that day will be with same MP.

[sluggo]

Slug - I suspect your pH variability is due to measuring apparent pH of a water-methanol solution.

I bet that's definitely part of the problem. The first indication I that pH had a great effect on RT is when I made my second batch of mobile phase. Because of the difficulty in measuring pH of water-methanol, in my second try I decided not to re-adjust. I had hoped that the results wouldn't change very much. In fact, the RT went from 7min to over 15min. I had stopped the run at 15m and decided that that was not anything I was interested in...

I like your idea with changing the method of the mobile phase mixing. I think I'll try to decide on an appropriate pH prior to methanol as you've suggested. I'll then move towards a fixed mixture of water (mass), methanol (mass), TEA (pipette), phosphoric acid (pipette). Does that seem reasonable?

[Mark Tracy]

Yes. pH 2.40 is nicely in the buffer range for phosphate, and mixing by accurate measurement is the best way to go in most situations. In your case, however, it is possible that the pKa (or worse yet the pI) of your analyte is too close to the pH of the buffer (all measured in the methanol/water solvent system). In that case, the retention time will be excessively sensitive to pH changes. You may need to pick a different target pH.

[Consumer Products Guy]

Yes, that seems reasonable. I've seen published procedures that are vague in the language concerning mobile phase preparation. Of course, I've also seen procedures from our suppliers where the analyte standard did NOT dissolve in the specified dissolution solvent (not good !). You may even want to consider purchasing pre-made mobile phase from a vendor such as Chata.

[sluggo]

The MSDS for the analyte says the pH is 5.5 (1%) and I don't know the pKa. However, several published papers (using validated methods and optimized methods found through DOE) use pH 2.4 to 2.6 for the mobile phase.

I'm not deviating from the papers too much, but I am optimizing RT with peak shape. The published methods either had relatively fast runs but less desirable asymmetry, or long run times with good symmetry. So I've come up with a "best" method for us, and now I just need to improve my mobile phase mixing technique as suggested.

If there are any other thoughts or suggestions, I'd love to hear them. Learning is always a good thing!

thanks

[HW Mueller]

If the work of your references was done by measuring the pH of the aqueous solution only, then it´s likely that you have the wrong pH (since you readjusted it after adding MeOH). Mark already mentioned that: your pH is possibly at a value which is non-robust. Also, we had many examples, mentioned in this forum, of validated (or whatever) methods which certainly had far from optimum pH. Furthermore, there have been reports of TFA instability and other TFA problems.

[Peps]

I assume TEA is short for triethylamine? I'm not really sure about these things but my guess is that TEA does not buffer your mobile phase very well at pH 2.4. Therefore I would suggest that you used some kind of salt e.g. sodium or potassium phosphate or acetate in order to actually buffer your mobile phase at your desired pH.

/Peps

[HW Mueller]

Darn, just noticed that I read TFA instead of TEA, the latter has its problems also, though.

[Alfred88]

Dear sluggo:

TEA has pKa ~8, and you are working with pH ~3; therefore TEA here is added to suppress tailing only. We avoid adding TEA to mobile phase for this purpose.

How about using a different buffer system: Potassium Phosphate Dibasic & Monobasic instead? (Phosphate has pKa1 ~2, pKa2 ~7). If tailing is unacceptable, try using column from a different vendor.

Alfred

[Albany-12303]

At such a low pH, there should not be very much retention variability with most basic compounds (I will assume that your analyte is basic, since TEA is in the MP - to prevent tailing, as the above post mentions).

My suggestion is to not readjust the pH after mixing with methanol, (maybe you can adjust to a lower pH ie 2.0 to begin with).

It may be that using such an acidic pH with such a highly buffered MP (probably over 20g/L TEA.PO4) may be destroying your column.

[sluggo]

Hello All,

As suggested, I've moved to a phosphate buffer where I lower the pH of the buffer to 2.4 and then do not re-adjust after mixing with methanol. I also switched to a different column to help reduce tailing. I haven't yet determined how stable the retention time will be from one day to the next, but I feel that I've taken the best steps to avoid the problem. I think methanol evaporation may become an issue, so I may put the methanol on a different pump and do on-line mixing. However, this has it's own problems.

Am I to understand that TEA reduces tailing only on basic compounds?

thanks

[Albany-12303]

Many of the older columns (lichrosorb, Novapak, ubondapak) have silanol groups that are somewhat exposed. The positive charge on amine groups is attracted to these free silanols - this gives tailing. Adding a large amount of an amine such as TEA covers up these groups (the ethyl groups cover the silanols a bit like an umbrella).

Newer types of colums dont have this problem very much.

[HW Mueller]

Reading the original post again and recalling that at a pH of 2.4 there should not be many, if any, SiO-, I wonder why TEA improved peak shape. Is the stationary phase a very old type with high metal ion contamination? Was your phosphate concentration or the pH quite different for the aqu. phos/MeOH? Did you, sluggo, compare peak shape at the same retention time for the two mobile phases? How does the 1% TEA influence retention time?

[sluggo]

Hello,

The best peak shape I had was using Luna C18 column with the 1%TEA, and a low amount of MeOH. However, this resulted in a long run time. Using the phosphate buffer with approx 50:50 MeOH, there was a lot of tailing. However, by switching to a different C8 column the peak shape improved considerably. This is what I am now using, and I am no longer using TEA. The retention time is still very sensitive, but my mixing procedure is more robust now.

I never tried varying the %TEA. However, the paper I gathered information from did a four-factorial design of experiment whereby they judged according to peak separations. They optimized there method by adjusting the %MeOH and %TEA.