Chromatography Forum

Our compound shows a degradation peak at 1.99min in NaOH forced degradation. Mobile A is 20mM phosphate buffer pH 6.8 and B is AcN/H2O 80/20. 100%A is used for the first 4 minute in the gradent. The pKa of the compound is 5. And it is not stable at low pH, so pH 6.8 was chosen for the buffer. The column used is Phenomenex Gemni 150mm, 4.6mm and 3.5um.

My question is that if there is a way to push back that early eluting peak? I tried different columns Xterra, Aquasil, symmetry, Zircrom. Only Aqusil can solve this early eluting peak(the peak elutes at 3.0min), but it by no means can base-line separate our main peak with two impurities.

Any suggestions?

This would be a classic situation for eitehr the use of an ion-pair reagent (e.g., tetrabutylammonium) or a bimodal columns that has mixed reversed-phase/anion exchange functionality.

If you go the ion-pair route, you can start by adding tetrabutylammium phosphate to the A solvent at a fairly low level (e.g., 1 mM). Keep doubling the concentration on successive runs until you see some effect, then adjust as necessary.

Be aware that you may be in for long equilibration times between gradient runs.

My recommendation would be to use the Atlantis dC18. We have shown that it gets you more retention in 100% water than other approaches. Of course, we make this stuff too, but I do think that it is worth giving it a shot.

Here are a few examples of retention of highly hydrophilic acidic compounds on our Primesep mixed mode columns. The hydrophilic ionizable compounds are retained mostly by ion-exchange mechanism. Everything else is retained by combination of reverse phase and ion-exchange. With the right mobile phase you can achieve K’ of much more then 1 or 2.

http://www.sielc.com/compound_157.html
http://www.sielc.com/compound_178.html
http://www.sielc.com/compound_127.html

If you would like you can send us a sample and we can develop method for your free of charge –we just did it for another “help seekerâ€

Question to Tom: How do we know that a compound with a pKa of 5 is anionic at pH 6.8? Is it possible it could be a protonated nitrogen base, for example something like ArNH3+Cl- or some other strong acid salt, and so running neutral under the current conditions.

Or am I off base here and something about the forced degradation inferred the compound is a carboxylic acid.

JA, you've got a good point: I have no proof that the degradant is acidic.

Basically, I was "playing the odds":
1. there aren't that many bases with a pKa as low as 5
2. A free base would have to be quite hydrophilic to be unretained in 100% water on a reversed-phase column.
3. ergo, it's probably an acid.

If it's a base, I stand corrected, and suggest something like HFBA as an ion-pairing reagent. The reason for HFBA (which is not a very effective ion-pairing reagent) is the very slow equilibration times characteristic of the more commonly used alkly sulfonates.

Thanks for your reply, Tom. As usual I was reluctant to post incase I would be following it up with a:

1. I looked at a chart and figured there might not be that many acids with a pKa as high as 5.
2. This however, feels like the strongest point to me

I apologise to the OP for my digression and not really having anything to add. Especially when I could of just asked what the nature of the compound is.

I tried TBA in the mobile phase. At 10mM TBA, that early eluting peak eluted at 1.88min instead of 1.99min. It seems to be the rare case of what Tom mentioned.

In this case, should I try HFBA? Is there any other reagent I can try since Tom said HFBA is not effective and needs more equilibration time?

Thanks!

Apple

That's what I get for jumping to conclusions!

Actually, I didn't mean to imply that HFBA is ineffective, merely that it's not as effective an ion pair reagent as some of the more "classical" things like the sulfonates. If you do a search of the forum using HFBA as the keyword, you'll find a number of threads dealing with various aspects.

Are you sure that the peak in question is a degradation product? If your detection wavelength is low enough you could be seeing junk from the sodium hydroxide. I usually inject solutions that mimic the degradation conditions but without the sample (e.g. neutralized base).

We injected the blank(neutralized base) as you did. That peak doesn't show in the blank. Then we concluded it is a deg peak.

Thanks Tom anyway. You showed me a direction already. I feel I am approaching a solution now

Besides, I tried the Atlantis dC18, unfortunately it didn't work for our case.
Thanks all for your input!

There a lot of “basesâ€

Apple,

I can provide you with free Primesep column (to try for a month) if you agree to share results (retained vs. not retained) and comparison to whatever you already tried

Contact me if would like to use this opportunity vlad.orlovsky@sielc.com

Regards,

Vlad

Is your parent compound an acid or base? Do you have any idea whether your degradation product is an acid or base? Anyway, here are some other suggestions. You may want to tried a stronger ion-pair reagent such as SDS with phosphate buffer e.g. 20 mM SDS & phosphate buffer pH 6.8. Another option is to use a much higher pH (e.g. 10) with a Waters Xterra or Agilent Extend column that are stable at high pH. Maybe, the degradation product have a higher pKa and therefore, the higher pH would unprotonate the degradation product. If these and other options presented by others do not work, you may want to try normal phase chromatography.