250
Carbohydrate Analysis
as optical rotation and specifc gravity.
Enzymatic methods oF analysis are becom-
ing more commonly used owing to their
specifcity and stoichiometry. Monosac-
charides derived From more complex car-
bohydrates by dilute acid hydrolysis may be
identifed and quantifed using gas–liquid
chromatography (GC), high-perFormance
liquid chromatography (HPLC), or high-
perFormance anion-exchange chromatog-
raphy with pulsed amperometric detec-
tion
(HPAEC-PAD)
by
comparison
oF
their elution positions and detector re-
sponse, respectively, with known stan-
dards (Sect. 2.2).
1.3
Oligosaccharides
Oligosaccharides containing between 2
and about 30 sugar residues may be
analyzed by means oF HPLC or, more com-
monly, HPAEC-PAD (Sect. 2.2). Known
carbohydrate oligomers are usually quan-
tifed by comparison with standards. The
structures oF unknown oligosaccharides
may be determined by means oF the
complementary techniques oF mass spec-
trometry (MS, Sect. 2.3.4) and nuclear
magnetic resonance (NMR, Sect. 2.3.5).
1.4
Polysaccharides
Polysaccharides are high molecular weight
polymers (
>
5 kDa) oF monosaccharides.
They may be linear or branched and
may contain just one (homopolysaccha-
rides) or several (heteropolysaccharides)
types oF monosaccharide. Most polysac-
charides have very high molecular weight
(
À
100 kDa) and possess repeating units
oF relatively uniForm structure. It is this
structural unit, together with the molec-
ular
weight
and/or
molecular
weight
distribution, which is generally sought,
rather than an accurate picture oF a com-
plete molecule.
The type oF polysaccharide may be con-
cluded From the component analysis ob-
tained aFter acid hydrolysis. The glycosidic
linkage positions may be determined by
means oF methylation analysis (Sect. 2.4.3)
or NMR spectroscopy (Sect.2.4.5), and the
anomeric confguration oF these links may
be Found by means oF their susceptibility
to hydrolysis using various specifc glycosi-
dases (Sect.2.4.2).
1.5
Proteoglycans
Proteoglycans consist oF a number oF
highly charged polyanionic glycosamino-
glycan chains attached to a central protein
core via serine or threonine residues.
These
glycans
are
distinguished
From
those occurring in glycoproteins in that
they are high molecular weight and es-
sentially unbranched chains oF repeat-
ing disaccharide units containing carboxyl
groups and/or substituted with sulFate
ester groups. Analysis oF proteoglycans
involves the determination oF the pro-
tein’s structure and the positions and local
confgurations at the linkage regions in
addition to the structural determination oF
the attached glycans.
Glycosaminoglycan
chain
Fragments
may be cleaved From the proteoglycans
by
β
-elimination with alkali or enzymic
hydrolysis (Sect. 2.4.2) and purifed by
high-perFormance anion-exchange chro-
matography (HPAEC). They may then
be analyzed by, For example,
13
C-NMR
spectroscopy. Although the class oF the
glycosaminoglycans can be relatively eas-
ily deduced on the basis oF a compo-
nent analysis and chemical and enzy-
matic degradation, the discovery oF their
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