I'm having a little trouble separating some very closely related peptides. The only difference in structure is one contains a Gln (glutamine) residue while the other contains a Glu (glutamic acid) residue at the same position. Gln has an amide side chain (CONH2) while Glu has an acid side chain (COOH). The peptides are 20 amino acids long. The Glu peptide closely follows the Gln peptide. I need to get baseline separation because I will be conducting a quantitative kinetic study.
So far I've tried C8 and C18 columns with a mobile phase of TFA in water (pH 2.6) along with either acetonitrile or methanol or a combination of both. I tried both gradient and isocratic elution. Isocratic gave better separation though run times were longer. C8 worked better than C18 (better separation and shorter run times). So far C8 with methanol works the best but I still do not have baseline separation. I am thinking of trying to increase temperature (from 25 to 45) or increasing the mobile phase pH to a higher value (maybe 3.0), so that the glutamic acid (pKa = 4.2) is more ionized and might separate better from the unionized amide in the Gln peptide.
Any other suggestions?

Do you know the respective isoelectric points of the two cpds? In order to make use of the difference in pKa's you'll probably need to do an ion exchange method (which can sometimes be done on a reverse phase column).
Call Supelco Technical Service, 800-359-3041. Dr. Barb Vogler is very good at these sorts of problems.

A pH change is likely to work best. While the traditional way of peptide separations uses straight TFA at different concentrations, you can get decent results by using s true TFA buffer and manipulating the pH.
More complicated alternatives are using different buffer components, e.g. hexafluorobutyric acid, or the old-fashioned HPLC buffers phosphoric or acetic acid.
Do you only need to separatew these tow or are they part of a more complicated mixture?

Hi Uwe Neue,
Thanks for the reply to my query in the chromforum. I am trying different TFA
concentrations. I had started with 0.024% (pH 2.5) which didn't help. I am now
trying 0.0024% (pH 3.5) with the logic that at a higher pH the COOH group in the
glutamic acid peptide would be more ionized (its pKa is 4.2) and hence it would
separate from the unionized CONH2 in the glutamine peptide. That does not seem
to be working too well either. I will try a lower pH too (maybe 0.1% TFA) but I
dont see the logic behind that working because both acid and amide groups will be
similarly unionized.
And yes, the separations I am trying are part of a very complex mixture, but I am
interested in just 3 peaks for quantitation, so they need to be separated well from
other closely eluting degradation products.
Do you think an ion pairing agent might help? I was thinking about looking into that.
Thanks for your suggestion.

Hi ABJ,
I suggest that you do not just vary the TFA concentration, but also use true buffers, i.e. TFA adjusted with a base to the appropriate pH. To get to still higher pH values, use acetic acid / ammonium acetate buffers. I would be very surprised if this does not change the selectivity of the separation. I just don't know if it will get better or worse.

Hi Uwe Neue,
I am trying the ammonium acetate/acetic acid buffer at pH 4.6 (buffer conc = 10 mM) to see if it improves selectivity. Just had another question. If I need to run a gradient with this buffer, how do I compensate for the drift in baseline. It goes down as % organic is increased. Do you know of an absorbing material I can add to the organic phase?
Thanks a lot for your suggestions.
ABJ

The common trick is to use the same buffer at the same concentration in the A and the B mobile phase. For your buffer this means that B is not 100% acetonitrile, but you should have no problem going to 80% acetonitrile.

Going away from TFA seems reasonable. We have had only trouble with TFA and higher perfluoro acids. In the future I will only use these if there is no other way. Also, since TFA is highly dissociated it is not much of a buffer. For instance, its buffer index (differential of added base or acid to conc. change of pH) is about 0.02 for a 0.1% aqu. solution, pH~2, only slightly better than that of H2SO4. Furthermore, this buffer index actually indicates only a resistence to higher acidity. Your newer buffer, NH4Ac has an even lower buffer index (though about equally efficient in both directions): ~ 0.005. At a conc. of 100mM the index would be ~ 0.06, a value which indicates reasonable buffering, or a robust method. But, robust or not, it would be interesting to find out how you finally solve your problem with what peptides. It seems that each peptide or protein has its own solution.

Another good way to obtain improved selectivity is to use a bonded phase w/ an imbedded polar group such as PRISM RP (Keystone Scientific). This also allows you to reduce the TFA concentration to 0.001% or so. Also using diffent acid modifiers such acetic acid or changing from 0.1%to 0.01% TFA (or other acid modifier) can change selectivity considerably. Generally, w/ accetic acid the concentration will be 1.0%. I have found this to be true for Antiotensins I, II, and III. Keystone Scientific does have a technical bulletin on this topic. Please feel free to contact me to request a copy or to discuss this topic further. Good Luck! aheyrman@keystonescientific.com

Hello all,
Just in case anyone is interested, I was able to separate one of my Gln peptides from its deamidated (Glu) products very nicely using a C4 column (Vydac) and a mobile phase of methanol:TFA solution (pH 2.5) with isocratic elution at 45% methanol. The retention times are more than 4 minutes apart at 1 ml/min flow rate. This particular peptide was 14 amino acids long. I am still having a little trouble with the larger peptides (20 and 29 aa long).....but I'm working on them!