Short Communication
Fast preparation of monolithic immobilizedpH gradient column byphotopolymerization and photograftingtechniques for isoelectric focusingseparation of proteins
A new method was developed to prepare monolithic immobilized pH gradient (M-IPG)
columns in UV-transparent fused-silica capillaries by the 5-min photopolymerization of
acrylamide and N,N0-methylenebisacrylamide, followed by the 20-min photografting of
the focused ampholine-derived glycidylmethacrylate monomer on the monolithic matrix,
by which the preparation time was reduced, and the stability of the formed pH gradient
was improved, compared with our previous methods. Using the prepared M-IPG
column, the baseline separation of proteins was achieved according to their pIs. Without
carrier ampholytes added in the running buffer, the separated components could be
detected with high sensitivity by UV at low wavelength.
Keywords:
Isoelectric focusing separation / Monolithic immobilized pH gradient column /Photografting / Proteins DOI 10.1002/elps.201100195
Capillary isoelectric focusing (CIEF), by which ampholytic
compounds could be separated according to their pIs, has
been regarded as a powerful tool for protein analysis with
the advantages of high peak capacity, high resolution and
high sensitivity [1–5]. In CIEF, carrier ampholytes (CAs), the
complex mixtures of amphoteric compounds with high
buffering capacity, are usually added in the running buffer
to establish a stable pH gradient. However, the mobile CAs
in the buffer might interfere on the detection by UV at low
wavelengths, the MS identification, and even the following
multi-dimensional separation. Therefore, to solve these
problems, besides the removal of CAs before the further
analysis and detection [6], electromigration of protons and
hydroxyl ions produced by the electrolysis of water [7], and
Joule heat-induced temperature gradient [8, 9] has been
developed to establish pH gradients without mobile CAs in
the running buffer, but few satisfactory results were
obtained since the formed pH gradients are either narrow
or unstable.
The immobilization of a pH gradient onto a support is
another strategy to avoid the interference of mobile CAs in
CIEF. In our previous work, the pH gradient was formed by
the isoelectric focusing of Ampholine (a kind of CAs) or
ampholine-derived glycidylmethacrylate (GMA) monomer,
followed by the immobilization on the monoliths by
chemical bonding or thermal polymerization [10–13]. With
the prepared monolithic immobilized pH gradient (M-IPG)
columns, proteins were separated by CIEF without CAs
added in the running buffer. However, the preparation of
such columns took a long time, including the preparation of
monoliths and the immobilization of the pH gradient. In
fact, long time required for pH gradient immobilization
(over 24 h) might be unfavorable to the stability of the pH
gradient because of the unavoidable diffusion of ampholine
or ampholine-derived GMA monomer, which could further
affect the separation performance of M-IPG columns.
In this paper, to shorten the preparation time, M-IPG
columns were prepared in UV-transparent fused-silica capil-
laries by the photopolymerization of the monolithic matrix,
followed by the photografting of the focused ampholine-
derived GMA monomer on the support. Photopolymerization
has been widely used to prepare monoliths, since the reaction
can be easily finished within a very short time even at room
temperature. The photografting of monomers on the mono-
lith has been developed by Svec et al. using a benzophenone
(BP)-initiated surface photopolymerization within the pores
of a macroporous polymer monolith, which is fast and effi-
cient [14–18]. In this work, with photopolymerization and
Yu Liang1,2
Guijie Zhu1,2
Tingting Wang1,2
Xiaodan Zhang1
Zhen Liang1
Lihua Zhang1
Yukui Zhang1
1Key Laboratory of SeparationScience for AnalyticalChemistry, NationalChromatographic R. & A. Center,Dalian Institute of ChemicalPhysics, Chinese Academy ofSciences, Dalian, China
2Graduate School of the ChineseAcademy of Sciences, Beijing,China
Received January 14, 2011Revised March 29, 2011Accepted May 6, 2011
Colour Online: See the article online to view Fig. 3 in colour.
Abbreviations: BP, benzophenone; CAs, carrier ampholytes;
GMA, glycidylmethacrylate; M-IPG, monolithic immobilizedpH gradient; poly(AAm-co-Bis), poly(acrylamide-co-N,N0-methylenebisacrylamide)
Correspondence: Professor Lihua Zhang, Key Laboratory ofSeparation Science for Analytical Chemistry, National Chroma-tographic R. & A. Center, Dalian Institute of Chemical Physics,Chinese Academy of Sciences, 457 Zhongshan Road, Dalian116023, ChinaE-mail: [email protected]: 186-411-84379560
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Electrophoresis 2011, 32, 2911–2914 2911
photografting techniques, the synthesis time of the mono-
lithic matrix and the immobilization time of ampholine-
derived GMA monomer onto the matrix were reduced to 5
and 20 min, respectively, ensuring the stability and unifor-
mity of the immobilized pH gradient.
As shown in Fig. 1A, poly(acrylamide-co-N,N0-methyl-
enebisacrylamide) (poly (AAm-co-Bis)) monolith, a kind of
neutral and hydrophilic material, was synthesized as the
matrix for M-IPG materials, by the photopolymerization of
AAm and Bis with DMSO, 1,4-butanediol and dodecanol as
porogens and AIBN as initiator. Herein, DMSO was chosen
to dissolve all components in the polymerization solution. 1,4-
Butanediol and dodecanol were used to improve the homo-
geneity and the penetrability of the monolith, respectively.
As shown in Fig. 1B and C, the ampholine-derived
GMA monomer was synthesized in the solvent, isoelec-
trically focused, and then photografted on the poly
(AAm-co-Bis) monolith. The solvent for dissolving GMA,
ampholine and BP is very important, because it can affect
the reaction of GMA and ampholine, the isoelectric focusing
of ampholine-derived GMA monomer and the photografting
efficiency. The desirable solvent must dissolve all the
components, and has low absorbance in the UV range to
exert minimum self-screening effect without hydrogen
abstraction [13]. In this study, the mixture of CH3OH and
H2O was chosen as the solvent, since CH3OH was of good
solubility to many compounds, and the addition of water
could not only facilitate the dissociation of ampholine-
derived GMA monomer for isoelectric focusing and
generation of pH gradient, but also improve the photo-
grafting efficiency because of the strong solvating ability of
water toward the hydrophilic matrix [19].
After optimization, the M-IPG columns were prepared
according to the following protocol. The internal wall surface
of UV-transparent fused-silica capillaries (75-mm id, 365-mm
od) was first vinylized to enable the covalent attachment of
Figure 1. Procedure for M-IPG columnpreparation.
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& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
the monolith, as described in our previous work [11, 12].
Then, the mixture containing 2.3 wt% AAm, 5.6 wt% Bis,
46.0 wt% DMSO, 15.3 wt% 1,4-butanediol, 30.7 wt% dode-
canol and 0.08 wt% AIBN, was purged with N2 for 30 s to
remove dissolved O2 and then filled into the capillaries. With
both ends sealed by silicon rubbers, the capillaries were
exposed to 365 nm UV light for 5 min in an XL-1500
UV-crosslinker. Finally, the prepared poly (AAm-co-Bis)
monolith was washed with methanol. According to our
previous work [13], 30 mg GMA and 50 mg ampholine were
dissolved in 75 mg CH3OH and 45 mg H2O, and vortexed for
a few minutes. After incubation at 401C for 1 h, the
temperature was dropped to 41C for 10 min to slow down the
reaction between amine and epoxy groups. After the addition
of 0.0010 g BP, the solution was degassed with N2 for 5 min
and injected into the capillary with poly (AAm-co-Bis)
monolith, followed by isoelectric focusing at the voltage of
400 V/cm, with 0.020 mol/L H3PO4 as the anolyte buffer and
0.020 mol/L NaOH as the catholyte buffer. Until the current
was decreased to stable, with both ends sealed by silicon
rubbers, the capillaries were exposed to 254 nm UV light for
20 min to photograft the focused ampholine-derived GMA
monomer. Finally, the prepared M-IPG column was washed
with CH3OH and H2O to remove the residual reagents.
To evaluate whether ampholine-drived GMA monomer
was immobilized on the monolithic matrix, a mixture of
ribonuclease B (from bovine pancreas, pI 8.8), myoglobin
(from horse heart, pI 6.8 and 7.2) and b-lactoglobulin (from
bovine milk, pI 5.1) dissolved in 10 mM Tris-HCl buffer (pH
8.0) (0.1 mg/mL for each protein) was separated under electric
field using poly (AAm-co-Bis) monolith before and after the
photografting of the focused ampholine-derived GMA mono-
mer, respectively, but no CAs were added in the running
buffer. Specifically, after the sample was filled into the
monolith with a manual pump, an electric field of 400 V/cm
was applied until the current was decreased to stable, with
20 mM H3PO4 and 20 mM NaOH as anodic buffer and
cathodic buffer respectively. The focused sample zones were
subsequently pushed through the UV detection (214 nm)
window via a 10 cm-long capillary (50 mm id, 365 mm od) from
the anode to cathode by a manual pump. As shown in Fig. 2A
and B, three kinds of proteins, including the isomers of
myoglobin, could be well separated according to their pIs only
by the photografted monolith, demonstrating that the focused
ampholine-derived GMA monomer was immobilized on the
monolithic matrix by photografting technique.
The separation performance of an M-IPG column
prepared by photopolymerization and photografting tech-
niques was also compared with that prepared by our
previous well-established method [12]. Under the same
experimental conditions, sharper peaks and higher resolu-
tion were achieved by the former column, especially for the
isomers of myoglobin, as shown in Fig. 2B and C, which
should be contributed to the fact that the shortened time
spent on the immobilization of the pH gradient by photo-
grafting technique was favorable to obtain immobilized pH
gradient with good stability and uniformity.
The reproducibility of CIEF with M-IPG columns
prepared by photopolymerization and photografting techni-
que was evaluated. As shown in Fig. 3A and B, baseline
separation of the proteins was achieved according to their pIsin both runs. Since the focused sample zones were driven by
a manual pressure pump, the migration time is slightly
different between two runs. However, the peak shape and the
resolution are repeatable. In addition, the linear relationship
between pI and the migration time of proteins was good in
both runs (shown in Fig. 3C and D), further demonstrating
the separation mechanism of CIEF with M-IPG columns.
In conclusions, a new method was developed to
prepare M-IPG columns for CIEF separation of proteins by
5-min photopolymerization of AAm and Bis and 20-min
photografting of the focused Ampholine-derived GMA
monomer on the monolithic matrix. By such column, proteins
Figure 2. CIEF separation of ribonuclease B (pI 8.8) (a), myoglobin(pI 7.2 and 6.8) (b) and b-lactoglobulin (pI 5.1) (c) without CAs inbuffer by poly(AAm-co-Bis) monolith (A), the M-IPG columnprepared by the photografting of the focused ampholine-derivedGMA monomer on poly(AAm-co-Bis) monolith (B), and the M-IPGcolumn prepared by our previous well-established method whichwas described in [12] (C), respectively. Separation conditions:column, 75-mm id, 365-mm od, 24-cm length; anolyte, 20 mMH3PO4; catholyte, 20 mM NaOH; voltage, 400 V/cm. The focusedsample zones were pushed through the UV detection (214 nm)window via a 10 cm-long capillary (50 mm id, 365 mm od) from theanode to cathode by a manual pump.
Electrophoresis 2011, 32, 2911–2914 CE and CEC 2913
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can be well separated according to their pIs without mobile
CAs added in buffer, beneficial to obtain high sensitivity with
UV detection at low wavelength. Compared with our previous
work, by this method, not only the column preparation time
could be shortened, but also the separation performance could
be improved. Further work on the preparation of M-IPG
columns on microchips and their hyphenation with MS are
undergoing in our lab, to achieve high resolution, high
sensitivity and high-throughput protein analysis.
The authors are grateful for the financial support fromNational Natural Science Foundation (20935004), NationalBasic Research Program of China (2007CB714503 and2007CB914100), Creative Research Group Project by NSFC(No.21021004), and National Key Technology R. & D. Program(2008BAK41B02 and 2009BAK59B02).
The authors have declared no conflict of interest.
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Figure 3. CIEF separation ofribonuclease B (pI 8.8) (a),myoglobin (pI 7.2 and 6.8) (b),and b-lactoglobulin (pI 5.1) (c)with the M-IPG columnprepared by the photograftingof the focused ampholine-derived GMA monomer onpoly(AAm-co-Bis) monolith induplicate runs (A) and (B) andcorresponding linearity of pIversus migration time (C) and(D). The experiment conditionswere the same as that in Fig. 2.
Electrophoresis 2011, 32, 2911–29142914 Y. Liang et al.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com