Analysis of underivatized amino acids by IP-HPLC

[fuelquest]

Does anyone have experience with an IP-HPLC method, reported by P. Chaimbault, et al, for seperation and quantification of underivatized amino acids? The method uses Hypersil's Hpercarb column (porous graphitic carbon) and evaporative light scattering detection. The ion-pair reagent is nonafluoropentanoic acid.

I would like to evaluate this method which reports baseline resolution for most of the 20 amino acids without derivatizaton, but I would like some input from some IP-HPLC pro's who have tried this method before proceeding.

[tom jupille]

Looks like you're the pioneer. Let us know how it works out!

[Kostas Petritis]

Fuelquest,

As a co-author of the paper, I can tell you that the method was working but there are two amino acids that were tricky to resolve and these were Lys-Leu and Met-Ile. Maybe smaller particle of the same packing can do a better job. Also remember that you will need to cool your column at 10 C.

[SIELC_Tech]

Fuelquest,

We have a few methods for analysis of underivatized amino acid, our Primesep approach involves stationary phase with ion-pairing reagent attached to silica. One of the methods below shows separation of 12 amino acids. We never tried to separate all 20 amino acids. There is no problem to separate different combinations of amino acids on Primesep columns. You can use different buffers and may be you can find conditions to separate all of them (ammonium formate, ammonium acetate, TFA, sulfuric acid, phosphate and sulfate buffers).
You also have to consider effect of you sample and if you have a lot of "junk" this might effect your separation.

Here are a few methods:

http://hplcmethods.com/compound_002.php
http://hplcmethods.com/compound_096.php

One of the methods separates 12 amino acids, it is a gradient method and may be you can optimize it for 20 amino acids.
The column will retain other basic molecules too (amines, amidines, quaternary amines, etc.)


Regards,

Vlad

[HW Mueller]

Kostas, just curious, in view of your references on amino acid analysis (in the other chain) which are probably not done with the Hypercarb (?), did you use the very expensive Hypercarb just to demonstrate what can be done?
It would be nice to get the ref for that paper.
A side remark: I just tried to use the positive surface charge of this column to analyze a single amino acid (a cis/trans proline derivative) and gave up, because of weird peak shapes (no ion pairing agents used). A very old TLC method (from E. Stahl´s classic) modified and transferred to a silica column (the method would now be called HILIC) gave nicely shaped peaks in a short and straightforward development.

[Kostas Petritis]

Hans,

Some years ago, the quest for us was to be able to separate all the 20 proteogenic amino acids with means other than ion-exchange and only Hypercarb could do it. Now in addition to everything else, Hypercarb gave different amino acid selectivities over the C18 under the same chromatographic conditions.

Once we started using mass spectrometry we saw that we do not have to separate all the amino acids just a certain among them, so we speed up our separations. We also did some more work to evaluate possible ion-suppression etc...

Concerning your side remark, I have used the negative charge of this column as well for amino acid separation and was succesful (unpublished results). However it was not as powerful as when using ion pairing reagents (but I had not problem with weird peak shapes).

[HW Mueller]

Very interesting, Kostas.

Well, I guess I just lost patience. This was mentioned before: We did oxalic on Hypercarb with TFA, nice peaks were obtained within a fairly narrow TFA concentration. Outside of that, peak shapes also became very strange. On C-18 columns a more familiar looking peak spreading usually results when ion interactions are not under control (actually, this could be more of a feeling than a real difference??).

.

[Kostas Petritis]

Hans,

I agree that Hypercarb is more unpredictable than the silica C18 columns but it can be rewarding (if you have the time to mess with it, which I guess in an academic environment you can afford to do so). However, you may end up with results difficult to interpret not to mention publishing.

For example when we reported that you can separate inorganic anions with Hypercarb (which have/had the reputation of the most hydrophobic stationary phase) one of the referees more or less said that this is not possible and that we do not know what we are doing...

In the case of amino acids, in silica C18, Ile is eluted before Leu, in Hypercarb the selectivity is inversed. Furthermore, under the same ion-pairing conditions, Asp and Asn are the amino acids that eluted first in silica C18, in Hypercarb it is Gly followed by Ser... go figure...Of course you can always push it with amino acid molecular volumes and specific surfaces but you are safer if you just describe your observations without getting into possible explanations...

[HW Mueller]

Well I am not throwing the columns away, anyway, it can be nice to jolt the referees a bit.

[Kostas Petritis]

Hans,

Just to clarify, when I said "if you have the time to mess with it, which I guess in an academic environment you can afford to do so" I was not referring to you but to me as when I did the work I was at the university... so I was not trying to imply anything...

[HW Mueller]

Kostas, that applies to me as well, there usually is time to play a bit, but right now there is an upheaval at some university medical schools in Hessia, time is getting a bit tight.