The Column - May 2009 - (Page 14)

Incognito The Column May 2009 ‘ILICs’ Everywhere!! It seems that wherever I turn recently I’m being told that the new best thing in HPLC is HILIC (or Hydrophilic Interaction Chromatography). Apparently, it’s going to retain all of my polar analytes, improve my MS sensitivity, save me lots of acetonitrile, negate the need for me to blow down and re-constitute my sample extracts and turn gradient HPLC analyses into isocratic ones at a single stroke! With the current resurgence of the technique (it’s by no means ‘new’) and the usual attendant marketing claims, this month’s column comes from the perspective of the laboratory chemist who has used HILIC and its derivatives sporadically over the past few years and who has done a little further research of late to see what all the hype is about! The name hydrophilic interaction chromatography (HILIC) was first coined by Alpert1 in 1990 to describe a variation of normal phase chromatography in which a polar stationary phase (typically bare silica or a bonded diol type phase) is used with an organic mobile phase containing small amounts of water (typically in the order of 20%), for the analysis of polar and ionic compounds. The HILIC technique, however, was first reported by Samuelsson in 1952 for the separation of monosaccharides on Amberlite IRA-400 resins.2 In simple HILIC mode, water is used as the strong solvent and, hence, the technique has also been called ‘aqueous normal phase’ or ‘reverse reversed-phase’ chromatography. The most fundamental form of HILIC chromatography works as a form of partition chromatography, where the analyte distributes between the eluent and a layer of water, which becomes absorbed by the polar stationary phase surface (Figure 1) (this is a fairly gross oversimplification but a good analogy for descriptive purposes). The eluotropic strength of the eluent is raised by increasing the amount of water in the mobile phase. Knowing analyte Log P values allows one to attempt a rudimentary prediction of the relative retention order of analytes. On this subject, I discovered a very nice free web applet for calculating Log P and other analyte physical chemical properties from simple JME structural inputs, which is really helpful when using HILIC Contact author: E-mail: Incognito chromatography — you can find the tool at So, what advantages does the HILIC mode of separation really bring to daily laboratory work? Well, in the simple form described above, silica columns might be used to gain retention for highly polar analyte molecules that would otherwise be poorly retained in the reversed-phase mode, while offering a more user friendly solution than normal phase chromatography, in which highly polar analytes may only be sparingly soluble in the eluent systems used. Of course, when using bare silica the eluent pH may be manipulated by causing the silica surface to become ionized. This offers a whole other set of advantages and at around pH 2.5 the silica will be able to interact electrostatically with analyte cationic moieties, to give improved retention. This is very useful for the analysis of basic species in electrospray LC–MS as the highest analyte sensitivities are usually achieved when the analyte is ionized in solution. This sometimes gives rise to poor analyte retention in reversed-phase mode and the ability to obtain improved retention through electrostatic as well as hydrogen bonding interactions is significant. Of course amino (cationic) and similar stationary phases may be used for the analysis of acidic species in a very similar fashion. The HILIC mode also offers an orthogonal selectivity to the reversed-phase mode for the separation of polar and ionizable compounds. Further, as the technique uses eluent systems that are highly organic, this favours the rapid desolvation of the sprayed droplets and can lead to an increase in LC–MS sensitivity. One word of caution — as the desolvation process is improved your API source conditions (drying gas temperature and flow-rate) and/or the position of the sprayer relative to the sampling orifice may need to be adjusted to realize the full potential of this effect. One potential downside is the requirement to use buffers to elute the analyte species, by disrupting the electrostatic interactions between the analyte and sorbent bonded ligand (or sorbent surface when bare silica is used). As the buffer is dissolved in the aqueous component the buffer strength will 14

Table of Contents for the Digital Edition of The Column - May 2009

The Column - May 2009
Q&A: Is Green the New Black?
Market Trends and Analysis
Quantification of Pharmaceuticals from Diminishing Small Volumes of Blood Using the UHC Small Molecule Chip Coupled to Triple Quadrupole MS
An Investigation of the Impact of Common Experimental Parameters on Signal Intensity in SFC–ESI-MS
Multicolumn Preparative SFC: An Advanced Solution to Scale-up Difficulties
Introduction to HPLC 2009
HPLC 2009 Guide

The Column - May 2009