Introduction
3
Hydrophilic Interaction Liquid
Chromatography (HILIC) is the most
successful approach for the retention and
separation of polar compounds. HILIC has
seen a substantial increase in popularity
over the last decade, driven by the
increase in the development of polar drugs
within the pharmaceutical industry and
also the growing field of metabolomics,
which primarily involves the analysis of
polar molecules. This technique has been
described as “reversed ‘reversed phase’”
[1], since in HILIC the stationary phase is
polar and the aqueous portion of the mobile
phase acts as the stronger solvent, which is
the opposite of conventional reversed phase
chromatography. There are many stationary
phases that can be used in a HILIC mode
and many phases are generically described
as HILIC, independently of their chemistry.
There is also a variety of misinformation
regarding the use of this technique. So
what column should be used? What are
the best mobile phase starting conditions?
What are the common issues in HILIC
method development that need to be
addressed? These are the type of questions
that HILIC users face and will be addressed
within this guide. A range of applications
will also be described which will highlight
the use of this technique and how it can be
successfully implemented into a working
laboratory.
Early History
The HILIC mode of separation can be traced back to 1951, when Gregor
et al
. [2] described a water-
enriched layer on an ion-exchange resin surface. The following year, Samuelson and Sjöström analyzed
monosaccharides on an ion-exchange column [3]. Then, in 1954, Rückert and Samuelson [4] postulated
that a stagnant water layer could be responsible for the uptake of analytes. Several years later, in 1975,
the analysis of sugars was accomplished on amino columns [5, 6]. Nowadays, the existence of an semi-
immobilized water-enriched layer on the polar stationary phase, combined with a partitioning equilibrium of
analytes into the mobile phase are accepted as the dominant HILIC mechanisms [1, 7]. However, it was not
until 1990 that the acronym HILIC was first introduced by Alpert [7]. Hemström and Irgum [1] have written a
comprehensive review on the subject matter which supplies an excellent background to the technique and
how it has developed.
