Separation of phenylalanine and glutamine by anion ex proble

[LCdude]

I am trying to separate phenylalanine and glutamine using a quaternary amine (on polymeric resin) anion exchange column and am having problems getting baseline resolution of these. Anyone have any suggestions or experience with this.

Thanks.

Mike Hall

[Einar Ponten]

While running on an anion exchange column both these are -1 at sufficiently high pH. In some intermediate pH range these are inner salts not having a net charge. What buffer and pH are you using?

If you instead use a cation exchanger and run at an acidic pH glutamine will be +2 and phenylalanine +1. That will probably help.

Zwitterionic compounds like these may also be nicely retained and separated on the ZIC®-HILIC column.

[SIELC_Tech]

Ion exchange columns offer only one interaction mechanism. So you best bet is to try to create the difference in the ionization state of your compounds, in some cases (some amino acids) it might be difficult. Mixed mode columns offer two type of interactions: hydrophobic and ion-exchange and you can use two mechanisms to find right conditions. Phenylalanine and glutamine are quite different in terms of hydrophobicity and have some difference in pKa. Please check the link below for the separation of 13 underivatized amino acids:

http://allsep.com/makeCmp.php?cmp=Cmp_047

I will try to run your particular mixture tomorrow to show how retention is achieved and controlled.
Your ion-exchange approach as well as HILIC might be valuable too.

Regards,

[Steve]

Ditto on the above advice. Listed is an isocratic procedure for the seperation of 20 AA. This procedure will easily seperate out phenylalanine and glutamine, as seen in the chromoatography.

https://www.macherey-nagel.ch/web/MN-WE ... =HPLC00608

[SIELC_Tech]

Steve,

How can you explain the huge fronting on almost very peak? Also if you play with conditions can you achieve base line separation, because at the conditions descibed only 6-7 acids have a base line resolution. I am just wondering if you change temperature can you achieve better separation?

regards,

[Steve]

SIELC

The chromatograph runs right to left.

Our objective here was to achieve seperation of 20 AA with isocratic conditions using a C18 column. Very high goals to achieve.

LCdude is trying to separate phenylalanine and glutamine (peaks 5 and 16). The products are different enough to achieve baseline seperation with good peak symmetry without the problems of gradient HPLC.

As you know, temperature will effect the "gram", but will not improve the seperation of these 20 AA with isocratic conditions. Depending on which AA you are working with, temperture will improve peak shape.

[LCdude]

Unfortunately, I dont have any of these other columns you all mention. I only have a strong anion (quaternary amine) and weak anion (each on polymeric resin) columns. I was trying to do this separation using 50mM buffer of boric acid/sodium borate but am not getting the desired result.

I'm trying to figure out a way to pull these two apart in an anionic environment.

Mike

[Chris Pohl]

Mike,

You don't describe your eluent systems so my suggestions will necessarily be a bit vague. One simple way to adjust the selectivity of your analyte pair is to add (or increase) solvent. This will almost certainly reduce the retention of phenylalanine more than it will reduce the retention of glutamine. Another option would be to adjust the pH of your mobile phase but to give you more specifics on your best option I would need to know your operating conditions. You should be able to reduce the retention of glutamine relative to phenylalanine by choosing a pH around 9 (assuming you are currently working at a higher pH than that).

[Einar Ponten]

Chris,

Yes, that was indeed vague... please explain the latter advice in more detail. Isn't that to walk on the edge?

[Chris Pohl]

Einar,

As you request, a bit more information behind my suggestions above:

Regarding my suggestion to add solvent in order to improve the resolution of phenylalanine and glutamine, this comes from the general fact that polymeric stationary phases nearly always contain either an aromatic backbone or an ester linkage and that both of these characteristics will result in increased retention for aromatic analytes such as phenylalanine relative to aliphatic analytes such as glutamine. In my experience, addition of solvent to the mobile phase will help minimize the interactions between phenylalanine and the polymer backbone, thus improving resolution since glutamine wouldn't be expected to interact with the polymer backbone in a similar manner. Since Steve did not give us any insight into the specific stationary phase involved, it's simply my guess based on my general knowledge of commercially available polymeric anion exchange materials.

Regarding my suggestion that adjusting the pH should help with the resolution, this is based on the general principle that you can generally achieve resolution of a pair of analytes by adjusting the ionization of one of the pair such that the ionization of one analyte is substantially greater than the ionization of the other analyte (when the retention mechanism is ion exchange). As I drafted this more detailed answer I realized that I described the effect opposite what would be expected, however, so it's just as well that you suggested providing more details. In fact, the pH at which glutamine is 50% anionic is pH 9 whereas the pH at which phenylalanine is 50% anionic is pH 9.3 so dropping the pH to 9 (assuming Steve was working at a higher pH) would have the effect of reducing the retention of phenylalanine more than glutamine (since at pH 9 phenylalanine would be less anionic than glutamine). Of course, the disadvantage of this approach is that it will reduce the retention of both analytes and conceivably result in inadequate retention for complete resolution of the pair. Alternatively, of course, Steve could increase the pH to a value high enough assure that both analytes are 100% anionic. Under these conditions, I would be surprised if the pair coelute since aromatic analytes generally always have longer retention time than aliphatic analytes on polymeric anion exchange materials.

[Einar Ponten]

Chris and others,

Thank you for the details since I was actually not understanding the advice in the first place. While going through the considerations of yours I realise that it might be an alternative. However, I am unable (maybe biased) not to recommend HILIC since, firstly, I know it will work, and secondly, when we deal with minor selectivity differences the "otherwayaround thinking" is fruitful.

Moreover, we offer both silica and polymeric alternatives.

[Chris Pohl]

Einar,

My suggestions were not meant in any way to contradict your assertions as to the capabilities of your ZIC-HILIC media. I only posted a response once it was known that Mike only had an SAX and a WAX column to work with and expressed interest in suggestions regarding available column materials.

Chris

[Kostas Petritis]

Generaly speaking, the hydrophobicity difference between these amino acids is so large that they can be easily be separated by C18. The only thing you would have to do is to retain a little bit more the glutamic acid that normally should elute in the void volume under reversed phase conditions. You can obtain this by adding a short chain ion-pair reagent (i.e. HFBA).