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buffer strength, the retention is much higher at pH 6.5.
These experiments were undertaken using acids on a zwitterionic stationary phase exhibiting a weak anionic character. All electrostatic interactions were thus repulsive, but when separating bases electrostatic attraction would be dominant on this type of phase. By ionising a base its retention will however always increase (on the ZIC-HILIC column), both from increased hydrophilicity and through ionic attraction. Shielding the attractive forces will not reduce the retention below that which comes from polar partitioning.
Conclusion
Figure 5: Relative impact of selectivity (α), efficiency (N) and retention (k) on chromatographic resolution (Rs). Graphs were obtained by plotting the resolution equation against one parameter while keeping the others fixed at the intersection point
values (α = 1.05, N = 5000, k = 5).
molecule is allowed to influence its retention so that it is longer retained than glutamine.
The glutamic acid elutes last, maintaining its
relative elution order to α-ketoglutaric acid since it is a more polar molecule.
Discussion
Chromatographic resolution in HILIC depends on the same factors as in reversed phase LC i.e. retention (k), column efficiency (N) and selectivity (α) (Figure 4).
As already indicated, manipulating selectivity is highly effective for resolving critical pairs in a chromatogram. Changing pH has long been used as a means of altering selectivity in reversed phase chromatography. It has even been shown that RP-HPLC at pH 2.6 and pH 10 is orthogonal enough to use for 2D chromatography of peptides4
.
Changing pH in HILIC has the same effect on the ionisation state of weak acids and bases as in reversed phase chromatography. Retention will also be affected by the increased polarity of an ionised analyte but with opposite impact to reversed phase chromatography, retention will increase rather than decrease for ionised analytes. Additionally the profound effect that coulombic interactions can have in HILIC separations (a.k.a. eHILIC, ERLIC6 normal phase7
, ion-pair ) has been illustrated here.
Coulombic interactions are 10 times more powerful than hydrophilic partitioning forces
(hydrogen bonding, dipole-dipole or -). In order to manage these interactions, a buffer is required in the mobile phase. When trying
to understand elution profiles, it also worth considering that the ZIC-HILIC column is unaffected by changes in pH which can greatly simplify the interpretation of the results. This separation depends both on the ionic nature of the analytes and their coulombic interactions with the stationary phase, which differs with both eluent pH and ionic strength changes.
Resolving the complexity of the discussed separation depends on observing that the retention time for glutamine was the same under all three mobile phase conditions. Glutamine has no net charge and is thus not affected by changes in the buffer conditions. The only way to change glutamine’s retention time would be to change the acetonitrile content in the eluent.
The effect of pH changes on the carboxylic acids are two-fold, but opposing. By deprotonating the acids they become much more hydrophilic, but at low buffer strength they are also strongly repelled from the distal negative charge on the stationary phase. In order to be retained by hydrophilic partitioning the analyte needs to enter the stagnant water layer at the stationary phase surface. If electrostatic repulsion prevents this, retention will be low even though the acid is more hydrophilic. Depending on the ionic strength, deprotonating an acid could give any outcome in terms of retention; shorter, longer or unchanged. This is
exemplified with the behaviour of the α- ketoglutaric acid which at low buffer strength has a slightly lower retention at pH 6.5 compared to at pH 3.0, but when the electrostatic repulsion is shielded by a high
It has been shown that by controlling the ionisation state of weak acids selectivity can be altered by changing the mobile phase ionic strength. Depending on how much salt is shielding the electrostatic repulsion between stationary phase and analyte the outcome of ionising an acid can be manipulated at will. Retention can be reduced by promoting electrosatatic repulsion or increased by shielding the repulsion and allowing hydrophilic partitioning of the more polar ionised form of the acid. By balancing these two forces, retention may also remain unchanged.
References
1. N.P. Dinh, T. Jonsson, K. Irgum, J. Chromatogr. A, 1218 (2011) 5880-91.
2. D. McCalley, "Hydrophilic Interaction Chromatography: Is it a Viable
Complimentary Method to Reversed-phase for the Separation of Polar or Ionisable Compounds?" Presentation T01 at HPLC 2011, June 2011.
3. Y. Guo, et al., Chromatographia, 66, August (No. 3/4) (2007) 223-229.
4. Y. Takegawa, et al., J. Sep. Sci. 29 (2006) 2533 – 2540.
5. M. Gilar, P. Olivova,A.E. Daly, J.C. Gebler, Anal. Chem. (2005), 77, 6426–6434.
6. A. J. Alpert, Anal. Chem. 80, (2008) 62-76.
7. W. Ding, et al., Mol. Cell. Proteomics, 8, September 1, (2009) 2170-2185.
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