Carbohydrate Analysis
261
Cross-flow outlet
Carrier inlet
Sample inlet
Sample outlet
0.2 mm
Fig. 4
Outline of asymmetric flow Feld flow chromatography. Sample (about
100
µ
L containing 100
µ
g) is injected and concentrated onto the crossflow
membrane. Eluant then carries it along the surface of the crossflow membrane
to the sample outlet. Eluant but not sample leaves through the crossflow outlet
at a rate determined by the flow rate and the chamber’s asymmetry, thus
maintaining the sample close to this surface. Molecules possessing greater
diffusivity are able to enter the faster flowing stream above the surface and are
carried out of the chamber Frst.
coefFcients with those that have higher
values (i.e. smaller molecules) exiting Frst
and the largest molecules exiting last. The
concentrations of the chromatographed
material on exit are usually determined
from the RI changes and their absolute
molecular weights are determined using
MALLS. As the separation only depends
on the diffusion coefFcient and there is
effectively no absorption, this method is ca-
pable of giving the absolute hydrodynamic
size of molecules plus useful information
concerning the molecules’ conformations
and structures (e.g. the degree of chain
branching).
2.3.4
Mass Spectrometry (MS)
Mass spectrometry (MS) is a sensitive
and powerful technique for the analysis
of glycans. It involves the production of
ionized carbohydrate moieties that are in
the gas phase. The charged ions move in a
magnetic Feld along curved paths, which
are determined by the imposed magnetic
Feld and by the ratio of their mass to
their charge (
m
/
z
). Usually, singly charged
cations are produced most abundantly
and may be detected with masses up
to several kilodaltons with an accuracy
approaching 1 ppm. The key stage of the
technique is the ionization step. Sugars
are not normally volatile and must be
derivatized or otherwise persuaded to enter
the gas phase.
In electrospray ionization mass spec-
trometry (ES-MS), a strong electric Feld
is applied between the end of a capil-
lary tube containing the sample solution
and a counter electrode, to disperse the
emerging sample into an aerosol of highly
charged droplets. As the droplets evapo-
rate, assisted by the flow of warm gas,
multiply charged ions emerge free from
solvent (±ig. 5). This rapid and sensitive
method produces intact molecular ions of
even very large molecules and allows the
determination of the molecular weight of
glycoproteins with an accuracy of 0.01%
us
ingasl
i
t
t
leas1pmo
lofsamp
le
.Thus
,
glycosylation heterogeneity can be accu-
rately quantiFed. As an example, the ES-
MS of the carboxy-terminal half of hen ovo-
transferrin gives 3 major peaks centered on
molecular masses of 40 220, 40 056, and
39 854. This indicates microheterogeneity
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