28
May/June 2013
Controlling Selectivity on Zwitterionic HILIC Columns by Adjusting pH and Buffer Strength
by Petrus Hemstrom1 , Amos Heckendorf2 , Wen Jiang1 , Tobias Jonsson1 , Patrik Appelblad1 1) Merck SeQuant AB, Box 7956, 90719 Umeå, Sweden, 2) The Nest Group, Inc. 45 Valley Road, Southborough, MA, USA
Ionic interactions are important in determining selectivity in HILIC separations. Knowing how to utilise these interactions in order to selectively change retention of analytes or interfering peaks in the chromatogram is a valuable tool when developing HILIC methods. By changing the ionisation state of the analytes and buffer strength of the mobile phase, retention of electrostatically repelled analytes can be increased or decreased depending on the extent to which the stationary phase surface repulsion is ‘shielded’ by the buffer salt.
Introduction Hydrophilic Interaction Chromatography (HILIC) is a powerful technique for the separation of polar compounds. HILIC has emerged as the second most used HPLC technique after reversed phase chromatography. This growth has been all the more remarkable since the retention mechanism is still being debated and it is only just starting to be incorporated in University curricula.
There are numerous HILIC stationary phases with different bonded phase chemistries, most of which carry ionic charges, either deliberately incorporated during synthesis or from residual silanol groups. There is a general consensus that in HILIC a buffer salt needs to be used in order to control the ionic interactions between the analytes and the stationary phase. This is true also for nominally neutral stationary phases. Polar partitioning is the main retention mechanism in HILIC but the presence of ionic interactions can have a tremendous impact on retention. It is this mixed mode separation aspect which was explored in the study described in this paper.
Numerous papers have been published examining the differences between HILIC stationary phases and the effects on retention from altering chromatographic conditions. There are four distinct classes of HILIC stationary phases, neutral, anionic (silica), cationic (amine containing phases) and zwitterionic1,2
.
A charged functional group on a column's surface has an order of magnitude greater free energy of interaction with charged analytes than that of an uncharged stationary phase (Table 1). These electrostatic interactions provide the possibility of changing retention time and controlling selectivity by altering pH and/or buffer strength. The strength of ionic interactions require, however, the addition of high concentrations of salt in order to overcome the electrostatic interactions between charged (anionic or cationic) stationary phases and charged analytes to promote reasonable retention.
Truly zwitterionic HILIC stationary phases also provide sites for such electrostatic interactions, but at a much lower magnitude, due to close proximity of ion and counter ion
Interaction Covalent Interaction Ionic Interaction Polar Interactions (hydrogen bonding, dipole-dipole, π-π) Non polar interactions (van der Vaals or dispersion) Table 1. Free energy of interaction in different bond types.
in balanced proportions within their functional groups. In the ZIC-HILIC column the distal charge, the sulphonate, will dominate the interaction and the phase will behave as a net cation exchanger1,3-4
at
buffer concentrations low enough to expose analytes to this charge. The possibility of changing column selectivity is a very effective means of improving resolution and in HILIC (especially with zwitterionic phases) it is more easily utilised than in reversed phase chromatography. By changing the buffer strength or pH similar dramatic effects on selectivity can be achieved as when adding ion-pairing agents in reversed phase. This is of course quite complicated unless the charge state of the stationary phase is pH independent. It was therefore sought to demonstrate how compounds can be selectively moved to avoid co-elution with other similar compounds by simply adjusting pH and buffer strength.
Experimental
Preparation of a stock solution of buffer at two different pH simplified solvent preparation. Listed buffer concentrations in
Energy (kcal/mole) 100-300 50-75
3-7 1-2
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