15
HILIC Phase Characterization: Understanding
the Column Chemistry
As we have seen in the previous section the stationary phases used in HILIC are quite diverse and users are often under
the misconception that ‘HILIC columns’ are interchangeable.
A rigorous testing regime, such as that proposed by Tanaka et al. [20] is essential for the characterization and
understanding of HILIC phases and should help to overcome this issue.
Understanding the possible HILIC retention mechanisms involved for a particular column-solvent-analyte system is crucial
in the process of selecting the right HILIC stationary phase leading to a successful application. HILIC stationary phases
should be characterized for:
• degree of hydrophilicity,
• selectivity for hydrophilic-hydrophobic groups,
• selectivity for positional and conformational isomers,
• evaluation of electrostatic interactions,
• evaluation of the acidic-basic nature of the stationary phases.
We performed such a study and the details of the testing conditions are described in the table on page 37.
The data from this study show structure-selectivity relationships for the various HILIC phases and are discussed
in the following sections.
HILIC Selectivity
Thermo Scientific columns for HILIC are available in both solid core and fully porous materials, with a variety of
chemistries. The data sets generated from the characterization testing regime (as developed by Tanaka [20]) and
mentioned above, highlighted important retention characteristics and differences. Stationary phase characteristics have
been visually illustrated by radar graphs [20, 21], which allow to express multi-dimensional data in a two-dimensional
format and ultimately allow to visually assess and compare columns. The separation factors obtained in the course of the
assessment for the HILIC columns were therefore arranged in radar plots. Each axis of the radar plots represents one of
the following parameters:
1.
α
(CH2) – degree of hydrophobicity
2.
α
(OH) – degree of hydrophilicity
3.
α
(V/A) – separation factor for configurational isomers
4.
α
(2dG/3dG) – separation factor for positional isomers
5.
α
(AX) – degree of anion exchange interactions
6.
α
(CX) – degree of cation exchange interactions
7.
α
(Tb/Tp) – acidic-basic nature of the stationary phase
8. k U – absolute retention
Note that a selectivity value of 1 corresponds to no selectivity (since
α
= k
2
/k
1
,
α
= 1 when k
1
= k
2
).
