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: lihuazhang@dicp.ac.cnFax: 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.

Electrophoresis 2011, 32, 2911–29142912 Y. Liang et al.

& 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

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

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.

References

[1] Shimura, K., Electrophoresis 2002, 23, 3847–3857.

[2] Silvertand, L. H. H., Torano, J. S., Bennekom, W. P. V.,Jong, G. J. D., J. Chromatogr. A 1204, 2008, 157–170.

[3] Zhou, F., Hanson, T. E., Johnston, M. V., Anal. Chem.2007, 79, 7145–7153.

[4] Zhang, M., Rassi, Z. E., J. Proteome Res. 2006, 5,2001–2008.

[5] Dickerson, J. A., Ramsay, L. M., Dada, O. O., Cermak, N.et al., Electrophoresis 2010, 31, 2650–2654.

[6] Yu, W., Li, Y., Deng, C., Zhang, X., Electrophoresis 2006,27, 2100–2110.

[7] Huang, T., Wu, X., Pawliszyn, J., Anal. Chem. 2000, 72,4758–4761.

[8] Lochmuuller, C. H., Breiner, S. J., Ronsick, C. S.,J. Chromatogr. A 1989, 480, 293–300.

[9] Fang, X., Adams, M., Pawliszyn, J., Analyst 1999, 124,335–341.

[10] Yang, C., Zhu, G., Zhang, L., Zhang, W., Zhang, Y.,Electrophoresis 2004, 25, 1729–1734.

[11] Zhu, G., Yang, C., Zhang, L., Liang, Z., Zhang, W.,Zhang, Y., Talanta 2006, 70, 2–6.

[12] Zhu, G., Yuan, H., Zhao, P., Zhang, L., Liang, Z.,Zhang, W., Zhang, Y., Electrophoresis 2006, 27,3578–3583.

[13] Wang, T., Ma, J., Zhu, G., Shan, Y., Liang, Z., Zhang, W.,Zhang, Y., J. Sep. Sci. 2010, 33, 3194–3200.

[14] Rohr, T., Hilder, E. F., Donovan, J. J., Svec, F., Frechet,J. M. J., Rozing, G. P., Schoeamakers, P. J., Kok, W. T.,Macromolecules 2003, 36, 1677–1684.

[15] Peterson, D. S., Rohr, T., Svec, F., Frechet, J. M. J., Anal.Chem. 2003, 75, 5328–5335.

[16] Hilder, E. F., Svec, F., Frechet, J. M. J., Anal. Chem.2004, 76, 3887–3892.

[17] Stachowiak, T. B., Svec, F., Frechet, J. M. J., Chem.Mater. 2006, 18, 5950–5957.

[18] Eeltink, S., Hilder, E. F., Geiser, L., Svec, F., Frechet,J. M. J., Rozing, G. P., Schoenmakers, P. J., Kok, W. T.,J. Sep. Sci. 2007, 30, 407–413.

[19] Yang, W., Ranby, B., J. Appl. Polym. Sci. 1996, 62,545–555.

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