ELECTROPHORIZTIC BEHAVIOR OF SUGAR
ISOMERS INVESTIGATED BY MICROCHIP
ELECT~O~HO~SIS
AND VIDEOMICROSCOPY
F. Q. Dang’, L.H. Zhang’, M. Ishigawa’ and Y. Baba””
‘Single-Molecule
Bioanalysis Labomtov,
AIS7: Takamatsu, und 2The University oj
Tokushimu, CREST JS7: Japan.
Abstract
The imaging analysis of injection process revealed that field-amplified
stacking could
be used as an efficient method for manipulating the shape of injected sample plugs in the
absence of electroosmotic flow (EOF). However, there was a critical threshold of ionic
strength mismatch between the sample and running buffers. An increase in ionic strength
mismatch beyond this critical threshold caused surprisingly poor separation. We found
that the phosphate complexation is a pH-independent
rapid process, whereas the borate
complexation is a highly pH-dependent slow process.
Keywords:
Microchip,
sample stacking, sugar isomer, videomicroscopy
1. Introduction
Microchip electrophoresis (l&E) is a rapidly emerging analytical technology in the
past decade because it possesses substantial advantages over conventional
analytical
technologies in terms of separation speed and cost. p-CE has been used for analysis of
carbohydrate,
DNA
fragments,
peptides
and proteins.
However,
analysis
of
oligosaccharides
derived from glycoproteins
by p-CE remains challenging
because
oligosaccharides in glycoproteins are complex compounds with very subtle differences in
structure. In this work, the electrophoretic behavior of oligosaccharide isomers has been
systematically investigated by p-CE coupled with videomicroscopy,
using APTS-labeled
maltose (G?), cellobiose (G,‘), maltriose (G3) and panose (G,‘) as models. The excellent
separation of complex oligosaccharides released from glycoproteins was demonstrated in
phosphate buffer using PMMA chips with an effective separation charmel of 30 mm.
3. Experimental
Two-dimensional
images of the injection process were obtained using a Nikon Eclipse
E800 microscope with an ICCD camera (Hamamatsu Photonics, Hamamatsu, Japan).
Electropherograms
were obtained on a Hitachi SVl 100 microchip
electrophoresis
instrument with a LED detector. The PMMA microchips had a simple cross chamrel of
100 pm width and 30 pm depth. The effective separation length was 30 mm [I].
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3. Results and discussion
The dynamic formation of charged complexes between sugar units and oxy acids in
electrolyte is the basis of electrophoretic
separation of sugar isomers with the same
charge-to-mass ratio. The borate complexation is a well-studied example, and has shown
most promising results in conventional CE. However, when separation time is on a scale
of seconds, phosphate buffer allowed much good separation of APTS-labeled G2 and G2
over a wide pH range in p-CE, regardless of whether the sample was dissolved in water
or running buffer. In contrast, borate or borate-Tris
buffers gave observable poor
separations within a very narrow pH range only when the sample was dissolved in
running buffers (Fig. 1). APTS-G3 and -G3’ could not be separated in above three buffers.
In phosphate buffer, the sample in water had better separation efficiency and resolution,
as compared to those in corresponding
running buffers for APTS-sugar isomers when
buffer pH was below 7.22. When buffer pi-l rose above 7.22, the separation efficiency
and resolution decreased drastically with the sample dissolved in water, whereas kept
nearly constant with samples dissolved in corresponding rumring buffers. In addition, no
resolution of APTS-labeled
Gz and G2’ was obtained in borate and borate-Tris buffers
when the sample dissolved in water was used. It should be noted that all buffers in the
present study contained 0.5% methyl cellulose (MC) to eliminate adsorption of APTSlabeled oligosaccharides and EOF in PMMA chips.
0.8
g13wo
~,/AL--~-G-GB
$mo
*
z 4~0 +I+ 20 mM phosphate
20 mM phosphate
; VJWO
--ii--- 100 mM borate
E---O---
0.7
0.6
--+-- 20 n&f phosphate
-20
mM phosnhate
*
100 mM borate
+% 50 mM borate -Tris
Figure 1. The theoretical plates and resolution of APTS-G7 and Gi as a function of buffer pH.
Conditions: Esep = 168 V/cm; Sample, (-•-) 2.1~10’~ M APT%sugar isomers in water; (-0-),
(-A-) and (-V-) 2.1 x lo-” M APTS-sugar isomers in corresponding running buffers.
To explain the observed usual phenomena, the imaging analysis of sample injection
processes under different conditions was performed. The Still frames from a video of the
sample plug formation process without FAS (the sample dissolved in running buffer)
showed that an asymmetric sample plug with parabolic front and rear ends was formed in
the separation channel (Fig. 2A). In contrast, when the sample in water was used, FAS
due to ionic mismatch between the sample zone and buffer caused a smaller concentrated
sample plug with nearly flat front and back ends in the separation channel, as compared
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to that in running buffer, resulting in higher separation efficiency and resolution (Fig. 28).
However, there was a critical threshold of ionic strength mismatch between the sample
zone and running buffer. Once the ionic strength mismatch exceeded the critical
threshold, FAS lost its stacking effect and yielded an even larger asymmetric sample plug
than that obtained in the sample plug formation without FAS (Fig. 2C). This novel
phenomenon accounted for surprisingly poor separation performance with distorted peaks
when the pH values of phosphate buffer were higher than pH 7.22 (Fig. 3).
Figure 2. Sample injection process in the absence of FAS (A) and in the presence of FAS (B)
and (C), obtained in 20 mM phosphate buffer. (A) pH6.66,2.1 x 10m7M APTS-sugar isomers in
running buffer; (B) pH 6.66 and (C) pH 8.88,2.1x10-* M APTS-sugar isomers in water.
The present results suggest that FAS
results in smaller symmetric sample plugs
and thus higher efficiency and resolution in
phosphate buffer in the absence of EOF. In
addition, changing the sample solvent fi-om
running buffer to water led to a significant
increase of theoretical plates horn 12686 to
15226, and -20-fold enhancement of peak
intensity due to FAS effect in borate buffer.
However,
there was no resolution
of
APTS-G2
and G2’ when
the sample
dissolved in water was used (Fig. 4). These
results
strongly
indicated
that borate
complexation
is a slow process that is
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Figure 3. Separation of APTS-sugar isomers,
obtained in phosphate buffer, Sample in water.
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Analysts
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249
highly dependent on buffer pH, whereas phosphate complexation is a rapid process that is
virtually independent of buffer PH. The excellent separation of complex oligosaccharides
from al-acid glycoprotein (AGP) and IgG was shown in Fig. 5.
isolners in buffer
AGP
~
2.1x10s7Msugar
isomers in water
,011
Time (See)
Figure 4. Separation of APTS-labeled sugar
isomers, obtained in 100 mM borate buffer
(pH 8.90), E=168V/cm.
Figure 5. Separation of N-linked sugar chains
from AGP and IgG, obtained in 20 mM
phosphate buffer, pH 6.66; E=300V/cm.
5. Conclusions
We demonstrated a universal and effective method, based on FAS, for obtaining wellshaped narrow sample plugs in the absence of EOF. There was a critical threshold of
ionic strength mismatch between the sample zone and BGE. Ionic strength mismatches
exceeding the critical threshold resulted in poor separation with distorted peaks. The
present work revealed that phosphate complexation is a pH-independent
quick process,
whereas borate complexation is a highly pH-dependent slow process. I*-CE represents a
promising alternative for analysis of complex sugar chains derived from glycoproteins.
Acknowledgements
The present work is supported in part by the CXEST program of the Japan Science and
Technology Corporation (JST); a grant from the New Energy and Industrial Technology
Development Organization (NEDO) of the Ministry of Economy, Trade and Industry,
Japan; a Grant-in-Aid for Scientific Research from the Ministry of Health and Welfare,
Japan; and a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Science and Technology, Japan.
References
1. F.Q Dang, L.H., Zhang, H. Hagiwara, Y. Mishina, Y. Baba, Ultrafast Analysis I$
Oligosaccharides
by Microchip E~ect~o~ho~e~is with Light Emitting Diode Confocal
Fluorescence Detection, Electrophoresis 2003,24,7 14-72.
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