256
Carbohydrate Analysis
Tab. 3
HPLC, HPAEC, and GC chromatographic methods for carbohydrate analysis. There are many
commercially available columns that will perform similar functions. Choice of appropriate column for
a given separation is often due as much to personal experience, cost, and availability of materials as
choice of separation chemistry.
Column type HPLC
Mobile phase
Mechanism
Analysis
Quaternary ammonium
bonded to PS-DVB,
OH
form
Aqueous
Anion exchange
Monosaccharides,
sialic acids, uronic
acids, glycans
Anion exchange
(aminopropylsilane-
bonded silica, OH
form)
Acetonitrile/water
Hydrophobic and polar
Mono-, di-, and
oligosaccharides
Cation exchange (PS-DVB
sulfonate, H
+
form)
Acetonitrile/water
or aqueous
Ion-moderated
partition and ligand
exchange
Glycoprotein- and
proteoglycan-derived
monosaccharides
Cation exchange (PS-DVB
sulfonate, Ca
2
+
,Pb
2
+
,
Ag
+
forms)
Hot water
Ion-moderated
partition, size
exclusion, ligand
exchange
Alditols, mono-, di-,
and oligosaccharides
C
18
-bonded
reversed-phase silica
and graphitized carbon
Acetonitrile/water
Hydrophobic
interactions
Derivatized
carbohydrates and
oligosaccharides
Gel-permeation resin or
derivatized silica
Aqueous
Size exclusion
Oligosaccharides,
glycans,
glycoconjugates
Quaternary ammonium
pellicular resin
Usually alkaline
aqueous
Anion exchange
Mono-, di-, and
oligosaccharides and
glycans
Column type GC
a
Liquid phase
Derivatization
Analysis
Dimethyl
Nonpolar
Trimethylsilylated
Mono- and
disaccharides
Trifluoropropyl/methyl
Slightly polar
Trifluoroacetylated
Methyl glycosides from
glycoproteins
Cyanopropyl/phenyl/
methyl
Polar
Peracetylated
Hydrolyzed and
reduced methylated
glycans
PS-DVB, divinylbenzene/polystyrene resin.
a
Column type (silicone).
monosaccharides. Capillary electrophore-
s
is(CE
)isapowe
r
fu
l
,i
flessused
,tech
-
nique used for separating and classifying
glycans and glycoconjugates.
In GC, separation is due to the extractive
distillation of the volatile derivatives. The
normal
protocol
uses
highly
efFcient,
fused-silica,
wall-coated
open
tubular
(WCOT) columns containing a silicone
stationary liquid phase. Detection is by
means of a flame ionization detector or
by mass spectrometry. ±or low molecular
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