246
Carbohydrate Analysis
TLC
Thin-layer chromatography; a chromatographic system based on partition.
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Carbohydrates form a complex family of molecules that necessitate a variety of
analytical approaches. They are capable of forming a large number of distinct
structures using relatively few units. In addition, the more complex carbohydrates
are rarely found to exist as single structural entities; polymorphism is common, and
often a given molecule is found as an assortment of closely related structures. These
mixtures of complex structures give rise to analytical problems that are much more
difFcult to solve than those concerning protein analysis. Typical protocols involve
isolation and puriFcation, determination of the constituent carbohydrates, partial
hydrolysis of polysaccharides or glycosaminoglycans or cleavage of the intact glycans
from the protein or lipid moieties, and isolation and puriFcation of these fragments.
Chromatographic puriFcation techniques are followed by mass spectrometry and/or
NMR spectroscopy. Absolute molecular weights of larger molecules are determined
by light scattering, but uncovering their conformation often requires the assistance
of molecular dynamics. However difFcult to obtain, a solid understanding of the
structure and conformation of carbohydrates is required a our proper understanding
of their biological roles.
1
Outlines of Methodology
1.1
Introduction
Carbohydrates, which form the most di-
verse of all the major groups of biological
compounds, possess a wide variety of
structural types with very different chem-
ical and physical characteristics. They are
formed from basic structures that com-
monly have a backbone of Fve or six
carbon atoms and possess several alco-
hol groups and a hemiacetal or, more
rarely, a hemiketal group. Less commonly
found
are
carbohydrate
units
contain-
ing a nine-carbon backbone, whilst those
with seven and eight carbons are only
rarely found. In spite of the extraordi-
narily large number of isomers possible,
only relatively few basic units (
monosac-
charides
) are found as the basic building
blocks of more complex molecules (±ig. 1).
These units link in a number of ways
between the monosaccharide units utiliz-
ing different hydroxyl groups and forming
simple or complex structures. Different
monosaccharides may be joined together
in an ordered or apparently random man-
ner to form a mainly homogeneous or
heterogeneous family of structural enti-
ties, termed
glycans
.Aga
in
,on
lyal
im
i
ted
subgroup of the possible isomers is gen-
erally found. ±or example, more than
50 different D-glucose dimers and more
than 1000 trimers are possible, although
only 5 dimers and 2 trimers are com-
monly encountered. These carbohydrate
moieties may also be linked to proteins
or lipids in glycoproteins, proteoglycans,
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