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HILIC separation of Polar and Hydrophilic Compounds for Routine and Emerging Application Areas

presented by

Patrik Appelblad
Application Manager - Chromatography
Merck SeQuant AB

February 11, 2010

The Scripps Research Institute, W.M. Keck Foundation Amphitheater


Background:

Dr. Appelblad received his PhD in Analytical Chemistry from Umea University in December 2001 focusing in Obstetrics and Gynecology. He has worked for Merck SeQuant since 2002 as a Product and Applications Manager on application and product development. His main interest is in chromatography and its applications to real-world problems.

Abstract:

"Hydrophilic interaction liquid chromatography (HILIC) has during the last 5-7 years developed from being an unknown technique to a cornerstone in many laboratories. This rapid increase in popularity has been driven by several factors and of most importance has been the wide range of application areas. Now there is almost a successful application presented every day where HILIC offers a new and mostly better way to retain and detect polar and hydrophilic compounds.


Obviously, the availability of dedicated and robust HILIC columns has contributed to the increased use by facilitating method development and making the analysis of real like samples successful. In its most straightforward configuration polar compounds are separated and detected by a UV-detector. There has, however, been a significant interest in using HILIC also for compounds that might be separated by other techniques. Since the eluent typically contain 60-80% of organic solvent it is rather volatile and this properly normally enhances the sensitivity in detectors relying on vaporazation of the eluent. Therefore, HILIC is an excellent technique in combination with electrospray mass spectrometry (ESI-MS) and evaporative light scattering detectors (ELSD) or evaporative charged aerosol detectors (CAD). The desire to both get better retention and sensitivity have made HILIC the first choice in many labs, even if the compounds may be separated by other techniques.


Another but less obvious factor for the increased use is the possibility to increase the overall speed of analysis and sample through-put. Firstly, the low eluent viscosity allow for very high flow rates without extreme back-pressures, using conventional equipment. Secondly, and probably most important is the possiblity to speed-up sample preparation. Protein precipitation protocols commonly employed in the analysis of biological samples can be used. After a quick centrifugation the solvent rich sample can be directly injected on the HILIC column relying on peak-compression of the analytes since these are dissolved in the weak solvent. An interesting option may also be to use HILIC for a polar analyte that is retained, but weakly, on a reversed phase column. In particular, peptides may show retention on both reversed phase and HILIC columns, enabling orthogonal and two-dimensional separations.


This presentation will demonstrate the palette of HILIC applications that have evolved with respect to compound characteristics and application areas. Ranging from validated methods to an outlook for future perspectives the hydrophilic stationary phase behavior, mobile phase composition, ion-strength, and pH value need to be considered to ensure a proper retention mechanism and robust results.


In conclusion, when the pieces of the puzzle is put together we see a new map guiding labs to simultaneously get faster sample preparation, resolution of tricky compounds, and increased sensitivity. Still using the same equipment, by by thinking HILIC!"

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