Hydrophilic interaction liquid chromatography (HILIC) combines a polar stationary phase (bare silica or polar bonding) and
a highly organic mobile phase (>70% acetonitrile). It is a powerful approach to retain polar compounds and is also a good
alternative to reversed-phase LC for the analysis of ionizable compounds of diverse polarity, which are often encountered
in the pharmaceutical field. In addition, HILIC presents various advantages such as a higher sensitivity when it is used with
electrospray ionization mass spectrometry (ESI-MS) and a lower back pressure than in reversed-phase LC because of the higher
volatility and weaker viscosity of the highly organic mobile phase. In a recent web seminar (
http://chromatographyonline.com/HILIC_webcast), Davy Guillarme, a senior lecturer at the School of Pharmaceutical Sciences at the University of Geneva, University of Lausanne,
in Geneva, Switzerland — and the winner of the 2013 LCGC Emerging Leader in Chromatography Award — discussed the possibilities and limitations of HILIC. Here, he answers common user
questions about HILIC.
Do you have an idea how many people use HILIC in industry?
To be honest, HILIC is not yet a widespread strategy in industry but has proved to be useful for a few specific applications
such as the determination of polar substances or inorganic ions. Today, most validated methods have been developed in reversed-phase
LC and it is often time consuming to change a method from reversed-phase LC to HILIC. However, when dealing with bioanalysis
in clinical or toxicological laboratories, I think there is a significant interest in using HILIC because of the large gain
in sensitivity that can be expected. So, I think that more and more people involved in applications such as drug metabolism
and pharmacokinetics (DMPK) will use HILIC in the future.
Based on your experience, do you think that developing a HILIC method is harder than developing a reversed-phase LC method?
People are used to working in reversed-phase LC, and it is always more difficult to develop methods with a new strategy that
has never been employed previously in a laboratory. However, if the interaction mechanism of HILIC is understood and if the
sample diluent is adequately considered, some nice separations can also be achieved under HILIC conditions. The problem with
HILIC is that it can be a kind of on–off chromatography for some compounds, whereas reversed-phase LC appears to be much more
generic. For this reason, it can be sometimes more difficult to develop HILIC methods than reversed-phase LC methods, but
the difficulty is highly dependent on the nature of the compounds.
Why is there only a limited number of chemistries for HILIC core–shell technology?
Core–shell technology is a recent development and the providers of core–shell columns have probably focused their attention
on the largest market, namely reversed-phase LC. Today, core–shell reversed-phase LC technology seems to be mature and there
are many companies that commercialize core–shell particles. All of them are now beginning to develop more exotic chemistries,
including phases dedicated to HILIC operation. For example, core–shell bare silica and amide are now available from various
HILIC–MS offers an impressive increase in sensitivity compared to reversed-phase LC–MS. However, are there some limitations
with HILIC–MS, when dealing with biological fluids?
It is true that the gain in sensitivity achieved with HILIC is impressive, on average around 10-fold. This is particularly
useful when dealing with biological fluids because the limits of detection and quantitation have to be as low as possible.
However, in the case of quantitative bioanalysis, matrix effects are also a major concern that must be considered. For example,
endogenous phospholipids are found in plasma at significant concentrations, and are typically not completely removed during
extractions. Because phospholipids are much more strongly retained in HILIC than in reversed-phase LC, it is expected that
matrix effects related to phospholipids will be more pronounced in HILIC than in reversed-phase LC. On the other hand, because
the retention mechanism of HILIC is very different from that of reversed-phase LC, the reverse situation could also occur
with other types of biological matrixes and matrix effects could possibly be reduced in HILIC.