60
hilic
HIGHLIGHTS
Stable bonding chemistries
Unique polar phases
Handle a wide spectrum of sample polarities
Stable in 100% organic
Separate many different types of polar molecules
3 µm particle size for LC/MS analysis
Hydrophilic interaction chromatography (HILIC) is used primarily for the
separation of polar and hydrophilic compounds. HILIC has similarities
with traditional normal phase chromatography, but themobile phases for
HILIC are similar to those known from reversed phase chromatography
(RPC). They include polar organic solvents like acetonitrile. Based on
hydrogen bonds the aqueous content of the mobile phase creates a
water-rich layer on the particle surface. This allows for partitioning
of polar compounds between the more organic mobile phase and
the aqueous layer
(FIGURE 1)
. The number of polar groups, as well
as the conformation and solubility of the sample in the mobile phase
determines the elution order.
Typical mobile phases consist of acetonitrile buffer mixtures. Samples
are eluted from the column by increasing the percentage of the aqueous
component. Compared to RPC the elution order in HILICmode is inversed
for most substances.
HILIC is often used to separate hydrophilic compounds such as
peptides, carbohydrates and small polar drug candidates or metabolites.
Hydrophilic compounds are retained on the polar bonded phase column
while non-polar sample impurities elute unretained in the void volume. In
addition it is ideally suited for sensitive LC-MS analysis of water soluble
polar compounds because the high organic content in the mobile phase
provides rapid evaporation of solvent during electrospray ionization.
TSKgel HILIC columns are available in various dimensions and particle
sizes, functionalized with carbamoyl-groups (TSKgel Amide-80) or
amino-groups (TSKgel NH
2
-100).This enables the user to perfectly
match HILIC selectivity to specific application needs.
For more detailed information, please refer to our
TSKgel HILIC
brochure
on
, or request a printed copy at
.
The
TSKgel Amide-80
column offers an excellent alternative to amino-
bonded stationary phases and consists of 3, 5 or 10 μm silica particles
in a stainless steel format. Spherical silica particles are covalently
bonded with carbamoyl groups. For years TSKgel Amide-80 columns
have been the standard for the analysis of glycans. TSKgel Amide-80
columns packed with 3 μm particles are the newest addition to the
TSKgel Amide-80 series. The 3 μm HILIC columns reduce analysis time
and improve peak capacity and sensitivity for HPLC and LC-MS analysis.
TSKgel NH
2
-100
3 μm columns are the latest addition to the TSKgel HILIC
family. They expand the selectivity range of TSKgel HILIC solutions by
a new, robust amino-phase. In contrast to conventional silica-based
amino phases the new column offers expanded stability under HILIC
conditions. It is well suited for the analysis of all types of hydrophilic
compounds like carbohydrates, peptides, vitamins, polar drugs or
metabolites.
The NH
2
-100 phase is based on a silica particle with 3 μm particle and
10 nm pore size, treated with a special endcapping procedure. Amino
groups are introduced step wisely after endcapping. These columns
are unique in that the bonded phase ligand not only, as expected, has
a terminal primary amino group, but that the spacer also incorporates
secondary as well as tertiary amino groups. The amino groups act as
HILIC functional groups without any peak splits. Due to their high ligand
density and large surface area TSKgel NH
2
-100 3 μm columns show high
retention for very polar compounds. Anionic compounds are retained on
the column by ionic interaction. This allows for the use of salt gradients,
in addition to gradient elutions with acetonitrile. Since the TSKgel NH
2
-
100 has cationic sites, it can be used as mixed mode phase under some
conditions.
INTRODUCTION TO TSKgel HILIC COLUMNS
figure 1
HILIC principles
