Bioanalysis Zone

Heroics of HILIC in tempestuous times



In this instalment of Robert MacNeill’s (Covance) column, Robert explores hydrophilic-interaction liquid chromatography (HILIC) and how the technique is often overlooked.

Robert MacNeill received his Bachelor’s degree with Honors in Chemistry from Heriot Watt University then his MSc in Analytical Chemistry from the University of Huddersfield, both in the United Kingdom. Robert is also a Chartered Chemist and a Fellow of the Royal Society of Chemistry. With 22 years of experience in all aspects of quantitative bioanalytical LC–MS/MS method development, 13 of these years heading method development activities within the Princeton site that has housed HLS/Envigo (now Covance), and a regular author and peer reviewer for the journal Bioanalysis, Robert is a recognized expert and innovator in the field.

We are all acutely aware, especially as patrons of the life sciences, of how everyday existence has recently changed so much. Cue the opportunity to make pitifully amusing remarks about toilet paper battling and the isolation of celery as the only vegetable remaining on supermarket shelves.

For myself, I am fortunate enough to take great pleasure in what I do for a living. The chance to mentally dissociate somewhat from the immediate stresses of coronavirus-related issues is presented to me by the likes of, well, chromatography. It so happens that at the moment there is a plethora of bioanalytical activity at my site and within this I have seen a couple of LC methods that curiously juxtapose in terms of anticipations and outcome. More than enough to capture a lot of interest, I would think. At the forefront is a moment of serendipity experienced when we tentatively attempted a certain chromatographic mode for one of our lipid applications and it really came up trumps!

Looking back at the content of this column over the years, it will be of little surprise that I am a big advocate of hydrophilic-interaction liquid chromatography (HILIC). Innately, this is a technique used for the analysis of compounds that are more polar than non-polar, broadly speaking. Thankfully, as with so many things, it is not quite as simple as that. To gain a solid foothold on a key chemical characteristic and use this as a basis to climb the rest of the mountain, can be priceless in the grand scheme of things. This is exactly what happened in this situation. An analyte molecular entity that, from the structure, was strongly indicative of an underlying lipophilic nature and sure enough showed a clear proclivity to dissolve readily in organic rather than aqueous. However, amidst the bulging hydrophobic parts, there is a secondary amine group, outside the reach of conjugation, that makes it a pretty strong base. It is this which makes for the HILIC possibility. It is a welcome option too, since the initial reversed-phase tests indicated difficulties synonymous with chromatographing lipids. Over-excessive retention and concomitant broadening, a high risk of matrix effect arising from the profusion of endogenous lipids affecting the signal integrity and carry-over. The HILIC avenue addresses all of these and this is the avenue we have taken for this method. A hint of buffer salts, a favorite amide chemistry in the stationary phase, an endearing 3-minute gradient optimized, oodles of sensitivity and away we go. It probably makes a clear statement about the involvement of electrostatic interactions as part of the complexity of HILIC, especially where the silica base is involved and that is of course greatly abundant. To round this off, not to assume that a great many lipid methods over years gone by could have been translated from reversed-phase even with a readily-charged moiety, but to just think of all the applications that could have benefitted in this way and the likes of HILIC was not given a proper chance, it’s almost astounding.

This article is part of Robert MacNeill’s (Covance) quarterly column for Bioanalysis Zone, which focuses on quantitative method design. Click here to read past instalments of the column!

I may dwell on HILIC a lot in my writing, but it doesn’t mean I have severely polarized vision; rather, it’s a lot to do with just trying to widen the exposure. There is certainly no aversion to reversed-phase around here. For instance, at the same time and a few instruments over, and by contrast to the above, a separate application involved a very hydrophilic small molecule, quite unique in its structure too. Suffice to say it was a contender for HILIC as a first port of call for the chromatography. However, lo and behold, screening a few columns, mobile phase pH etc. brought nothing but very broadened peaks, not feasible to be used with a practical run time and sufficiently high-sensitivity quantification as an analytical endpoint. Furthermore, a pair of isomers were not fully resolved. Then, setting up a reversed-phase gradient, beginning at very high aqueous and a mild excursion to 20% less aqueous over a couple of minutes and using a popular contemporary C18 column packed with superficially porous particles, we were able to sit back in relief. Sufficient retention, sharp, symmetrical peaks, precision of response and emphatic isomeric resolution. Rescued by reversed-phase.

I suppose this article provides a little diversion from the main subject of current day-to-day news and discussion, but in its own way remains informative, perhaps even spurring to those in similar chromatographic predicaments. If the coronavirus proves to be, with a nod to the Latin, the ‘crowning’ theme of 2020, I will not be at all surprised. Bioanalytical scientists have a weighty part to play in overcoming it, of course, and I just hope the enjoyment of the nuts, bolts and nuances of our particular specialties remains in full force or something similar during this tough time period. For the time being, in my domain, here’s a big thank you to analytical flexibility and fortune, but especially to the growing usefulness and visibility of polar-based chromatographic options!


Disclaimer: the opinions expressed are solely my own and do not express the views or opinions of my employer.


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