7
retention mechanism on diol chemistries are influenced by direct hydrogen donor bonding between the stationary phase and the analyte, a feature which could give these columns a different selectivity compared to columns more relying on partitioning and ionic interactions for retention.
Columns based on amide and zwitterionic functionality seems to be the most promising bonded phase chemistries for HILIC although they do differ in several aspects. Amide columns are supposedly neutral with very limited electrostatic interactions [39]
whereas
zwitterionic columns also rely on weak ionic attraction and repulsion interactions [40] Recent data [41]
also show that zwitterionic
sulfobetaine type phases are remarkably good in establishing the water-enriched layer necessary for HILIC partitioning.
There have been some initial attempts to classify columns and to make generalizations regarding differences in column retention and suitability for various applications. Chirita
et.al. [42]
tested 12 different columns and came to the conclusion:
i) For anionic compounds, a cationic or zwitterionic phase will provide good retention
ii) For cationic compounds, a neutral, anionic or zwitterionic phase will provide good retention
iii) For neutral and zwitterionic compounds, any HILIC phase is likely to provide good retention
However, since selectivity is the main contributor to separation, what really is needed to leave the trial and error approach to column selection are studies of selectivity differences between phases. Some attempts are in preparation [43]
, meaning that within a few years
scientific evidence will allow a systematic approach to column selection in HILIC.
Trends in HILIC Selectivity Tuning Changing the ionization stage of a molecule has a significant impact on its chromatographic properties. That is the reason why pH always has been such an important tool for changing selectivity in HPLC. Ionized and charged solutes are less hydrophobic and have lower retention in RP separations and pH has thus been used to suppress ionization. When ion suppression has not been possible, ion-pairing techniques have been the route to overcome the fundamental mismatch and tweak RP into separating charged species, but the technique has always struggled with drawbacks in terms of poor MS detection sensitivity and inherently reduced selectivity due to the complexes becoming more similar than the original ions.
With HILIC separations the opposite is true; charged compounds have more retention due to their increased hydrophilicity. Changing pH to deliberately induce ionization is thus one of the most powerful ways of altering the selectivity in HILIC mode separations, and the incentive to go to pH-extremes will thus be greater in HILIC than in RP.
An additional feature of HIILIC is that the use of charged stationary phases easily adds another dimension of selectivity while the main HILIC separation mechanism still is present. Naturally the ionic interaction properties and the response to buffer salt type and concentrations differ whether the HILIC phases are anionic, cationic or zwitterionic, but the principle is similar. Opposite net charges on the material and the analyte will induce attraction and same net charges will cause repulsion.
By combining pH tuning and charged stationary phases it is possible to utilize controlled molecular orientation to enhance selectivity [44]
precipitation by salting out, followed by centrifugation, tryptic cleavage, enrichment on a RP SPE cartridge, and direct injection of the eluate on a HILIC separation column. Conversely, removal of phospholipids was accomplished by Lindegardh et al. [46]
by using
protein precipitation in acetonitrile, followed by centrifugation, enrichment on HILIC 96-well plates, and direct injection of the eluate on a RP separation column.
In summary, combining RP and HILIC in sample preparation and HPLC separation either way can drastically simplify sample preparation schemes of biological samples where protein precipitation is needed before analysis of small molecules. The ease of automation of this combination is likely to attract a lot of interest in the near future.
. The concept is called ERLIC (or
sometimes eHILIC) and is based on repulsion of commonly occurring charged functional groups of strongly retained species having the same charge as the stationary phase at the same time as the counteracting HILIC mechanism increase retention.
The increased benefit of going to extreme pH combined with the water layer wetting a HILIC phase increases its exposure to hydrolysis. These factors will together increase the need for pH-stable HILIC columns, for example with polymeric base particles. A pH stable column with a hydrophilicity and surface charge independent of pH makes it possible to run pH gradients with predictable results. The FDA method for simultaneous quantification of melamine and cyanuric acid [21]
is a striking example of the usefulness of this approach.
Sample Preparation by HILIC The need for sample preparation is increasing with the move towards smaller particle sizes (UPLC), and to match sample preparation throughput to these fast separations, online coupled techniques will be needed.
minimized the number of sample handling steps by applying protein
One aspect that largely has been overlooked when considering the applications of HILIC is the opposite elution strengths of solvents compared to RP. For medium polarity solutes that have retention in both HILIC and RP one can couple sample preparation by SPE in one dimension with chromatographic separation in the other while benefiting from automatic band sharpening. To reduce the variability in proteomic analysis of urine proteins Loftheim et al. [45]
Conclusions There is no doubt HILIC has come to stay and that tweaks like ion-pairing RP to separate polar molecules will decline as a consequence. Our prediction is that HILIC very soon will be second to RP in terms of practitioners and eventually also in HPLC column sales. We believe that the initial excitement period over HILIC already have passed and now come the test how this separation mode will perform practically in competition with other techniques.
There are many separation technology trends that have risen rapidly in popularity among scientists only to fall short when it comes to practical use, followed by a more sober establishment after a few years. For HILIC we foresee a development without the decline phase for the following reasons; the hype is lower, the application pool is very large, and no instrument investments are needed. To summarize the present status of HILIC development we borrowed the words from a rather famous Britain [47]
:
“Now this is not the end. It is not even the beginning of the end. But it is, perhaps, the end of the beginning.”
References 1. R.E. Majors LC-GC North America, 27 (2009) 956–966 2. P. Hemström, K. Irgum, J. Sep. Sci., 29 (2006) 1784–1821 3. A.J. Alpert, J. Chromatogr. 499 (1990) 177–196 4. C.F. Poole, The Essence of Chromatography, Elsevier Science B.V; ISBN: 0 444 50199 1.
5. A. Nordström, R. Lewensohn, J. Neuroimmune Pharmacol. 5 (2010) 4–17
6. P. Wang, Hydrophilic Interaction Liquid Chromatography (HILIC) and Advanced Applications; H. Weixuan (Editor), CRC Press; Print ISBN: 978-1-4398-0753-8.
7. H.A. Lakso, P. Appelblad, J. Schneede, Clin. Chem. 54 (2008) 2028–2035.
8. P. Wang, L. Huang, J.L. Davis, Clin. Chim. Acta 396 (2008) 86–88.
9. J. Martens-Lobenhoffer, S. Postel, U. Troger, S.M. Bode-Boger, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 855 (2007) 271–275.
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