Chromatography Forum

I am trying to separate L-arginine, L-citrulline, L-ornithine using HILIC conditions
0.1 % TFA or weaker in ACN(A) and Water(B)
Luna Silica or LiChrospher Silica
230 nm detection
scout gradient 0% B to 100 % B.

Unfortunately, I have not been successful. No apparent retention. Thought maybe too much retention, but have eluted w/ hi water % to check this possibility with no observed peaks.

I have previously separated an arginine-like compound and its process impurities using similar conditions. In addition, I have searched the literature and found reports of successful retention of L-arginine - all be it with 0.1 % formic acid.

Am I missing something in my old age? Comments and suggestions are appreciated (about the LC... not my age).

Kind regards

Here is a much better alternative to HILIC (same mobile phase ACN/water/TFA). The link below decribes separation of arginine and other underivatized amino acids on Primesep 100 column (ASP, GLU, ALA, VAL, MET, LYS, HIS, CYS, PHA, ILE):

http://allsep.com/makeCmp.php?cmp=Cmp_006

Contact us if you have more questions (we are in Prospect Heights, IL)

Hi Robotjock,

Normally you should have retention with HILIC (I had been sucessful to retain these compounds with the Polyethylhydroxy aspartamide column). Mayb the problem is that you start with very high organic conditions so you might have solubility problems and/or the solvent that you dissolve your amino acids is not compatible.

These amino acids are so polar that you do not need to go up to 100% ACN. I would start from 80 to 70% ACN and try to dissolve my amino acids in the highest % of ACN they do not precipitate. Then a gradient with water. In my application I have use 10 mM of ammonium formate as I have seen that I obtained better peak shapes.

Finally, I wouldn't use any ion-pairing reagents (even as weak as TFA) with HILIC as you may destroy your separation (I had read this somewhere but I can not remember where).

Kostas

Thanks for the input. I am able to dissolve the compounds in 80% ACN/20% water. I am, however, confused about your middle paragraph. Do you mean 100 % water? Please restate it. Also what pH did you use / the NH4 formate?
regards, Robotjock

Hi Robotjock,

Normally you should have retention with HILIC (I had been sucessful to retain these compounds with the Polyethylhydroxy aspartamide column). Mayb the problem is that you start with very high organic conditions so you might have solubility problems and/or the solvent that you dissolve your amino acids is not compatible.

These amino acids are so polar that you do not need to go up to 100% ACN. I would start from 80 to 70% ACN and try to dissolve my amino acids in the highest % of ACN they do not precipitate. Then a gradient with water. In my application I have use 10 mM of ammonium formate as I have seen that I obtained better peak shapes.

Finally, I wouldn't use any ion-pairing reagents (even as weak as TFA) with HILIC as you may destroy your separation (I had read this somewhere but I can not remember where).

Kostas

I have not run your application, but in your case you can use two mechanisms to manipulate retention. The first one is the real HILIC, which is an extraction of a very polar compound from the high-acetonitrile mobile phase to water that is adsorbed on the surface of the packing. In order to do that, you need water on the surface. Thus I usually recommend not to start with 100% acetonitrile in the gradient, but with 95%. The second mechanism is ion-exchange with the silanols. Since silanols are very weak acids, you will suppress the silanol activity with your TFA. As you have seen, a lower pH is more useful.

Sorry ... a HIGHER pH than provided by TFA is more useful.

Robotjock,

Glad to hear that you can resolve your compounds in 80:20 ACN:H2O.

I think that my statement is correct. I really wanted to say that you do not need to go as high as 100% ACN due to the high polarity of your compounds. Uwe explained already the kind of mechanisms that can have in the HILIC separation (and I agree that 95% ACN should be the maximum due to the desired water layer on the surface of the packing).

So... the more your compound is polar the less ACN you need to retain it... (in general though you need more than 50-60% ACN in order to say that you operate in the HILIC mode).

I have used pH 4 for my application.

We have retained arginine using the following conditions:
ZIC®-HILIC 250x4.6 mm, isocratic ACN/H2O 70:30, with 55 mM ammonium acetate (k'~ 4).

The two others should be retained as well (L-citrulline and L-ornithine) and I am quite sure that you will have selectivity.

Einar, Kostas and Uwe;

From your comments and additional thought on my part, I agree that a higher pH is necessary, and increased % water. (Although I am now wondering how I was successful before?!!) Will experiment first thing Monday morning. You all have been very helpful.

Kind regards to all,
Robotjock

[I might have completed graduate school sooner if this forum had been available then.]