Carbohydrate Analysis
259
conditions or through complexation with
borate or similar ions. Sensitive detection
techniques such as pulsed amperomet-
ric detection and fluorescence detectors
are required owing to the tiny amounts
of material separated. Like HPLC, it is
suited to the analysis of both polar and
nonpolar carbohydrates and it is replacing
many of the HPLC techniques. CE offers
fast and efFcient separation, relatively af-
fordable and durable capillary columns,
requires small sample volumes, and has
low reagent consumption. It is also a flex-
ible technique available in a number of
modes including zone, isoelectric, and mi-
cellar electrokinetic. However, it cannot be
used for preparative-scale applications.
2.3
Determination of Size and Conformation
Historically, the molecular weight (
M
)o
f
pure macromolecules, such as glycopro-
teins, has been determined by ultracen-
trifugation, using the relationship shown
in Eq. (1)
s
D
=
M
(
1
−¯
νρ)
R
T
(
1
)
where both the sedimentation coefFcient
(
s
) and the diffusion coefFcient (
D
)a
r
e
determined under the same conditions.
T
is the temperature (K), R is the gas
constant,
¯
ν
is the partial speciFc volume
(the
effective
volume
per
unit
mass)
of
the
solute,
and
ρ
is
the
solution
density.
Today,
this
methodology
has
been largely replaced by size-exclusion
chromatography, Feld flow fractionation,
and laser light scattering methodology.
2.3.1
Light Scattering
It has been known for many years that
light
is
scattered
from
molecules
in
solution in a way that depends on the
concentration and molecular size of the
molecules present. Recently, the use of
multiangle laser light scattering (MALLS)
has enabled the accurate nondestructive
determination of the molecular weight,
molecular weight distribution, and radius
of gyration of many high molecular weight
carbohydrates without the need for known
calibrating standards.
Light
scattering
from
molecules
in
solution may be described by Eq. (2)
Kc
R
θ
=
1
M
w
µ
1
+
16
π
2
3
λ
2
h
r
2
g
i
sin
2
(θ/
2
)
+
2
A
2
c
(
2
)
where
K
is an optical parameter given in Eq. (3)
K
=
4
π
2
n
2
µ
d
n
d
c
2
λ
2
N
A
(
3
)
n
is the refractive index
d
n
d
c
is the RI increment of the solute.
c
is the sample concentration, calculated
from the differential RI response
RI
=
K
RI
c
µ
d
n
d
c
(
4
)
where
K
RI
is the instrumental calibra-
tion constant,
R
θ
is the excess intensity of scattered
l
igh
ta
tang
lethe
ta(
θ
).
M
w
is the weight
average molar mass (molecular weight,
Eq. 5)
M
w
=
X
i
n
i
M
2
i
X
i
n
i
M
i
(
5
)
where
M
i
is the mass of the
i
th component
and
n
i
is
the
number
of
component
molecules with that mass.
λ
is the wavelength of the scattered light
in vacuum.
previous page 933 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 935 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off