20
Organic Solvent Type
Although acetonitrile is the most popular solvent used in HILIC, several other polar, water-miscible organic modifiers
can be used. The elutropic strength is generally the inverse of that observed in RPLC, with the relative solvent strength
summarized in the following table.
In HILIC, it is generally advised to use aprotic solvents (solvents which cannot exchange a proton). However, it has been
demonstrated that alcohols can be used as the weak eluent [22], although they will generally cause a decrease in the
strength of the partitioning, leading to less retentivity; this is due to their hydrogen bonding interactions with the water
molecules and hence the competition between the two to solvate the surface of the HILIC stationary phase [17, 23].
Periat et al. unequivocally proved that large amounts of alcohols caused drastic reduction in retention [15]. Methanol and
IPA – when used in mixtures with acetonitrile (80/20 ACN/MeOH or ACN/IPA) - are not able to provide significant changes
in selectivity and lead to significant loss in kinetic performance and peak broadening [17]. In some instances – where
weakly polar analytes would precipitate in water-containing mobile phases, protic solvents replaced water (technique
known as ‘non-aqueous HILIC’) [24].
As outlined in the flow chart on page 11, the starting mobile phase we suggest consists of 80/20 acetonitrile/aqueous
buffer; the elution strength is then adjusted until acceptable retention is achieved. Alternatively, a generic gradient can be
run, starting from 95% acetonitrile, holding for 2 minutes – or longer, according to column length - (to establish whether
compounds are weakly retained in HILIC mode) and gradually increasing the aqueous buffer percentage to about 40%
over 15 minutes (for a 100 mm long column). A two minute hold should follow, to establish whether compounds are
strongly retained in HILIC.
If using a gradient, it is vital to have a sufficiently long post gradient re-equilibration stage, to allow for the volume of the
water layer (as advocated by the HILIC partitioning model) to re-establish its initial conditions. We generally recommend a
post gradient re-equilibration of approximately 20 column volumes.
Increasing
solvent
elutropic
strength in HILIC
Solvent
Chemical Formula
Aprotic solvents
Tetrahydrofuran (THF)
/−CH
2
−CH
2
−O−CH
2
−CH
2
−\
Acetone
CH
3
−C(=O)−CH
3
Acetonitrile (ACN)
CH
3
−C
≡
N
Protic solvents
Iso-propanol (IPA)
CH
3
−CH (−OH)−CH
3
Ethanol (EtOH)
CH
3
−CH
2
−OH
Methanol (MeOH)
CH
3
−OH
Water
H−O−H
