Chromatography Forum

Have anybody had experience with retaining this kind of compound on a C18 column? It is a short peptide consisting of arginines and lysines. I have tried 0.1%TFA and up to 30mM SDS but it would not retain on the column, even in 100% aqueous. I need to stick with reversed phase for this method.

small chains of arginines and lysines have for quite a while been very good at doing good drug delivery.
are you trying to develop a method where you look at the different species of the peptide and drug together or something like that?
hence you feel the need to stick to the RP method?

First, I advice to look at the pKas of the amino acids that form your peptide. Try to match pH in a way that your peptides stay unionized - in that neutral form you have a chance that it will be retained. I assume that in 0.1% TFA (pH=~2) peptides are totally positive (+1 or even +2). It is very difficult to stick ionic compounds onto C18.
I tested tri-peptide: Leu-Leu-Leu. I have used C8 column (special for small peptides), and I have used 52/48/0.1 H2O/MeOH/TFA. It was nicely retained.
The other option is to use heptafluorobutyric acid (HFBA) as ion pairing agent. I can assume that SDS as surfactant is very hard to remove from your column. HFBA is a strong acid - it will give you low pH (0.1% pH=~2) which cause your compound to be positive and HFBAcetate negative to pair.
Pay attention if your column is aqueous compatible. I would advice to use one of those for only aqueous mobile phases.
If that doesn't work go for normal phase.

These are precisely the sort of compounds that HILIC was developed for. The high polarity that prevents their retention in RPC makes them well-retained in HILIC. For an example of HILIC of the basic peptides GHK and KGHK, using a volatile mobile phase with ammonium formate and acetonitrile, look up T.F. Lane et al., J. Cell Biol. 125 (1994) 929-43. The column used there was neutral. Now, if you perform HILIC on an anion-exchange column, then you have the combination called ERLIC, cf. A.J. Alpert, Anal. Chem. 80 (2008) 62-76. ERLIC works nicely with very basic peptides; the electrostatic repulsion of the + charged groups tunes down their domination of the chromatography compared with the selectivity in regular HILIC.

Thanks Andy, I will check it out, maybe I'll get someone to demo me a HILIC column. Will HILIC provide the same resolving power as RP? The reason I wanted to stay with RP was I would like to be able to separate a number of derivatives of this peptide (with different aliphatic and aromatic groups) and I want to be able to separate them. Obviously, if I go more ion exchange mode, I would lose that opportunity, as these compounds will all be the same charge.

You could try a different ion-pairing reagent. Try a hydrophobic cation like hexylamine acetate instead of your hydrophobic anion, dodecyl sulfate.

Due to the lower viscosity of HILIC solvents, you will get better separations in HILIC than in RP. However, familiarize yourself with HILIC techniques first, since they are quite different from RP. Waters just published a beautiful HILIC brochure a few months ago, which will help you get rolling.

Alera, the 1994 reference demonstrated baseline separation of GHK and KGHK on a PolyHYDROXYETHYL A column in the HILIC mode. Now, if you want to scrutinize the nonbasic parts of these peptides, per aliphatic and aromatic sidechains, then you need to tune down the interaction with the basic parts of the peptides. That's because basic groups are the most polar ones of all. You do this by one of two alternative methods:
1) Form a hydrophobic ion pair with the basic residues using trifluoroacetate ion (Wen Ding's method);
2) Use an anion-exchange column in the HILIC mode (my method; the term ERLIC is used for this combination).

Below are basic amino acids separated on Unison UK-Amino:
http://www.imtaktusa.com/site_media/fil ... TI329E.pdf

I agree with Andy Alpert (when it comes to HILIC most sane people do). I would also like to add my two cents worth.
I would not use a neat silica column for this type of separation due to the electrostatic attraction between your basic peptides and the silica.

The ZIC-HILIC columns also have this Sio-/R+ interaction under very similar conditions where it shows up in bare silica.

I wanted to thank everybody for their advice. Unfortunately, as it often happens in a fast paced CRO environment, I did not have time to explore "new" venues (HILIC), plus I really wanted to get RP to work for this particular application. My original mistake was that I chose a completely BDS'd column, which did not help retention of basic compounds. Sometimes we forget that we can almost never have a "pure" RP or any other interaction in any given separation.
I was able to achieve beautiful peak shapes and excellent retention for the main compound and all its derivatives.

Thanks again, someday I will hopefully have a chance to explore all advantages of a HILIC separation.

If it´s not broke dont fix it (even if it is an old Skoda).
When utilizing secondary interactions for retention batch to batch variabillity may be a problem, if it not just a one off analysis.

I'd go for HIC (HIC alike anyway) if I were to find a solution in this case.

Best Regards