Chromatography Forum

Dear forum members,

I am wondering about the workings of zwitterionic chromatrography under hydrophilic interation chromatographic conditions (ZIC-HILIC) to analyse organic acids, such as pyruvic acid, succinic acid, citric acid, etc. with LC-MS.

In literature you will find plenty of examples where people use this, typically with an acetonitrile / ammonium acetate gradient.

I would imagine that deprotonated carboxylic acids can have ionic interactions with the permanently charged choline in the ZIC solid phase, but better separation is achieved on ZIC compared to e.g. aminopropyl columns, which are also positively charged at most LC-MS compatible pH's. A quaternary ammonium is also not a hydrogen donor, so hydrogen bridges can not be formed.

Any ideas on the precise mechanism are welcome!

DP

The ZIC-HILIC material exhibits electrostatic charge on a conditional basis. The functional group is a zwitterion and therefore is supposed to be "neutral". For whatever reason, though, the quaternary amine moiety is titrated by electrolytes in the mobile phase at a lower concentration than is the case with the sulfonic acid moiety. Consequently, at salt concentrations in the mobile phase lower than 20 mM, the material acts like a cation-exchange material, with (-) charge. If the salt concentration is 5 mM or less, then the material acts like a cation-exchanger of quite high capacity. If you don't want to have electrostatic effects (attractive or repulsive) superimposed upon your hydrophilic interactions, then include at least 20 mm salt in the mobile phases. That's 20 mM overall, not just in the aqueous portion of the mobile phase.

There's plenty of data in the literature bearing on this electrostatic effect, which would lead to electrostatic repulsion of organic acids (let me know if anyone wants references); no need to invoke hydrogen bonding or other less well-substantiated effects.

Our zwitter-ionic phase allows to have ion-exchange interaction since we don't have sulfate or Quaternary amine on the surface and two charges in Obelisc N column are "miles apart". For analysis of organic acids you can use either HILIC or mixed-mode ion exchange. The key for HILIC is to have acidic analytes be ionized.

Here are few examples of acids in mixed-mode and acids in HILIC:
http://www.sielc.com/Application-HPLC-A ... raphy.html
http://www.sielc.com/Application-Effect ... Acids.html
http://www.sielc.com/Application-HILIC- ... -EDTA.html

Contact me if you have questions