Carbohydrate Analysis
257
weight sugars, the choice between HPLC
and GC is not often clear-cut, but in gen-
eral, HPLC has been preferred for the
separation and analysis of oligosaccharide
mixtures, while GC has been used for more
complex mixtures of monosaccharides and
disaccharides.
A wide variety of columns has been used
for the HPLC of carbohydrates. Sulfonated
cross-linked
styrene-divinylbenzene col-
umns containing metal-loaded cation ex-
changers (H
+
,Ca
2
+
,Pb
2
+
,orAg
+
)used
at moderately high temperatures (
85
C)
or amino-bonded silica columns operat-
ing at around ambient temperature are
both popular. The cation exchangers act
by
ion-moderated
partition,
ligand
ex-
change, and size exclusion, elution being
roughly in order of decreasing molecu-
lar weight. Amino-bonded silica columns,
which use an acetonitrile–water mobile
phase, separate by hydrophobic and po-
lar interactions and partition between the
acetonitrile-rich mobile phase and the
water-enriched stationary phase. Under-
ivatized carbohydrate molecules generally
do not absorb light of useful wavelengths,
so mass detectors, changes in refractive
index (RI), or post-column chromophore-
producing reactions are used for detection.
Columns are short (generally 5–25 cm)
and contain 5
µ
m, or 3
µ
m usually spher-
ical particles. Silica-based packing mate-
rials have a high rigidity and do not
swell in any solvent. Their major weak-
ness is their limited stability in aqueous
mobile phases at high pH. Polymeric-
based packing materials are more com-
pressible, but they are compatible with
all mobile phases including the entire
aqueous pH range. Both the inorganic-
and
the
polymeric-based
materials
of-
fer a wide range of separation modes
including reversed-phase, ion-exchange,
hydrophilic-interaction,
hydrophobic-int-
eraction, and size-exclusion chromatogra-
phy, which is discussed in Sect. 2.3.2.
UV detectors are not commonly used
for carbohydrate analysis, though it is
possible to monitor sugars directly at
wavelengths below 200 nm. However, at
such short wavelengths, low sensitivity
and background interference levels of-
ten make their use impractical. Refractive
index (RI) detectors work on the prin-
ciple
of
differential
refractometry
and
have, until recently, been the mainstay
of HPLC carbohydrate detection. How-
ever, RI detectors have low sensitivity,
are temperature-dependent, and cannot be
used with a gradient, as they are extremely
sensitive to changes in mobile-phase con-
centration. Pulsed amperometric detectors
offer much more versatility and sensitivity
and are commonly used in tandem with
anion-exchange chromatography.
High-performance liquid chromatogra-
phy, on strongly basic anion-exchange
resins (HPAEC), is generally the method
of choice for the analytical separation of
disaccharide, oligosaccharide, and glycan
mixtures. Coupled with pulsed amper-
ometric detection (PAD), HPAEC-PAD
offers high resolution at picomole sen-
sitivity. Polyhydroxyl compounds, such as
the carbohydrates, are negatively charged
at high pH and are separated by anion
exchange with the degree of ionization,
composition, size, linkage position, and
anomeric conFguration all affecting reten-
tion. Retention times can be manipulated
by altering the strength of the sodium hy-
droxide eluant or by addition of the more
strongly eluting acetate anion (±ig. 3).
Pulsed amperometric detection is a very
sensitive technique (
1ng glucose) that
makes
use
of
the
electrochemical ox-
idation, at a gold electrode poised at
positive potential, of a tiny proportion of
th
ea
l
k
a
l
in
ean
i
on
i
cc
a
r
b
oh
y
d
r
a
t
e
s
.Th
e
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