258
Carbohydrate Analysis
01
0
3
0
4
0
Retention time (min)
15
20
25
30
35
40
45
50
10
20
Fig. 3
HPAEC elution profle oF the components oF debranched wheat
amylopectin on a HPIC-AS6 (250
×
4 mm i.d.) column using a gradient oF
200 mM sodium acetate at the start, 250 mM at 2 min, 300 mM at 10 min, and
400 mM at 40 min in 150 mM sodium hydroxide solution (1 mL min
1
;
ambient temperature) and PAD detection. The number on each peak indicates
the number oF glucose residues. Redrawn From Koizumi et al. (1991) with
permission From Elsevier Science.
electrode surface is kept clean by using
short pulses of higher positive and neg-
ative potentials, thus removing unwanted
oxidation products. The advantage of de-
tection with PAD for carbohydrates is that
no pre- or post-column derivatization is
necessary. Sample preparation is also sim-
pliFed because only oxidizable analytes
will be detected by PAD, and the sensitivity
for carbohydrates is orders of magnitude
greater than that for the usual contami-
nant species. HPAEC-PAD has been used,
for example, to estimate the chain length
of different amylopectins after enzymatic
digestion by isoamylase (±ig. 3).
Capillary electrophoresis (CE) is a rela-
tively new technique that provides rapid,
highly efFcient separations for carbohy-
drates and possesses several advantages
over HPLC owing to its ease of operation,
low cost per analysis, and the use of
nontoxic chemicals. It provides great res-
olution by high-voltage (10–20 kV) elec-
trophoresis of mixtures within a polymer-
coated fused-silica capillary tube (generally
50 or 70
µ
m i.d., 375
µ
m o.d., and between
30 to 100 cm in length). The separation
process involves electroendosmotic flow
owing to interactions between the charged
capillary surface and oppositely charged
ions in the buffer. Charged species are sep-
arated on the basis of their electrophoretic
mobility (charge:mass ratio) and the elec-
troosmotic flow
(migration of
positive
buffer ions to the cathode under the in-
fluence of the applied Feld). Separation
can be manipulated by changing either
buffer composition or pH. The technique
is also applicable to neutral polysaccha-
rides, which can be charged under alkaline
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