HILIC peak tailing

[D.Roesch]

Greetings everyone!

I need a little bit of help with a HILIC method I am working in.

I am trying to analyse L-carnitine using a phenomenex Luna NH2 column, the method uses acetonitrile: ammonium acetate buffer pH 4.5 10mM (75:25) as mobile phase and sample diluent is water:ACN (50:50), detection wavelenght is 210 nm, and temperature is 40 °C. I get a good retention time about 12 minutes but the peak shows heavy tailing, it basically looks like a kids slide, i have tried to change pH range from 2.5 , 4.5 and 8.5 but the tailing keeps happening, tried using ammonium formate but got the same results, i also tried adding TEA to change the selectivity and perhaps help to dislocate the analyte from the column but the tailing increased.

Any advice on how to solve this? thanks for all the input.

[Andy Alpert]

I have two suggestions:

1) When the mobile phase is 75% ACN, then the sample solvent should contain at least 65% ACN, and preferably 70%. With the 50% ACN that your sample solvent contains, then at least some of the carnitine will migrate down the column faster than the rest of the carnitine until solvent exchange occurs. That produces tailing.

2) Carnitine is a zwitterion. You probably think that means that it can't interact electrostatically with the amine groups on the stationary phase. Not true. The molecule will simply migrate down the column in an oriented manner, with the carboxyl- group facing the stationary phase and the quaternary amine facing away from it. Try using more salt than your 10 mM to suppress electrostatic effects; say, 30 mM. Incidentally, you are unclear as to whether the 10 mM ammonium acetate that you're using refers to the mobile phase overall or just to the 25% of it that's aqueous. If it's the latter situation, then your ammonium acetate concentration overall is actually 2.5 mM. Again, use more than that and see if that reduces the tailing.

Just out of curiosity, why are you using a column with a charged ligand on the surface for HILIC of a charged analyte instead of using a neutral column?

[D.Roesch]

Thank you for your response Andy.

It is indeed as you say that only the 25% is 10 mM, so I will use higher concentration and in my sample diluent ill switch to a higher ACN proportion since carnitine is very soluble I don´t need a large water proportion.

Regarding your question, the amino column it´s almost the only option I have available here other than C18 and C8, I have a bare silica column aswell that it was my second option if i didn´t manage to get it done on the amino. Also I chose the amino column because I was using an alcaline pH (8.5-9) so the cuaternary amine on carnitine would be retained via electrostatic interaction but it didn´t showed the behaviour i hoped.

When you refer to a neutral column wich one would you suggest and how would it work if you could explain a little bit.

Thank you very much!

Cheers

[Andy Alpert]

Please note that an amino column consists of a column with amine groups on the surface. The amine residues will have a (+) charge at a pH below 9. Therefore, the quaternary amine group in carnitine would experience electrostatic repulsion, not attraction. The carboxyl- group in carnitine would experience attraction. That's why the molecule will migrate through the column in a highly oriented manner, with the carboxyl- group down and the quaternary amine up. That in turn will make the column sensitive to the nature of the counterion that the carboxyl- group has. I'll bet that when you used triethylamine instead of ammonium ion in the mobile phase, then retention decreased. The column will not be particularly sensitive to the counterion for the quaternary amine.

Neutral column for HILIC: This just refers to a column which has neutral ligands attached to the surface, not a ligand with amine groups (and not uncoated silica, which has a decidedly (-) charged surface above pH 4). This reduces the significance of electrostatic effects in the chromatography. Some examples include AMT's penta-HILIC material, PolyLC's own PolyHYDROXYETHYL A, and a number of others. Before you buy a new column, though, see what the effects are of using more ACN in your sample solvent and more salt in the mobile phases.

[D.Roesch]

Thank you very much for all the information! I will try it out and see what happens.

Take care,

Cheers