Boronate functionalised polymer monoliths for microscale affinitychromatography

Oscar G. Potter, Michael C. Breadmore and Emily F. Hilder*

Received 26th June 2006, Accepted 15th August 2006

First published as an Advance Article on the web 29th August 2006

DOI: 10.1039/b609051f

Novel macroporous monolithic stationary phase materials

suitable for microscale boronate affinity chromatography were

developed.

The development of so-called micro-total analysis systems (mTAS)

and emerging ‘lab-on-a-chip’ technologies1 promises increased

analytical power, faster analysis speeds, decreased sample and

reagent volumes, as well as greater portability. Devices for

genomics, proteomics, and other such disciplines will most

likely contain a number of analytical processes, such as

preconcentration, chemical modification, separation and

detection, in a defined sequence to create an application-specific

device. The difficulty lies in developing and integrating each of

these processes in a simple, reproducible and effective manner.

This is particularly challenging when heterogeneous solid materi-

als, such as those used for chromatography or solid-phase

extraction, are required. One potential solution is the use of next

generation ‘monolithic’ media.

Monoliths are continuous macroporous media that can be

synthesized in situ, an approach that is much simpler than packing

particles into small-diameter capillaries or fluid channels.2 They

can be either polymeric (predominantly created through free

radical polymerisation of monomer and cross-linkers) or inorganic

(hydrolysis and condensation of alkoxy silanes) in nature.

Monoliths and in particular polymer monoliths have several

advantages over packed particle columns. First, the position of the

monolith can be controlled by a lithographic process. Second, their

high permeability allows resolution to be maintained at higher flow

rates. Third, it is possible to accurately control the surface

chemistry during polymerisation to achieve the desired surface

functionality.

Glycoconjugates have become the targets of cutting-edge

research in recent years. Glycolipids3 and glycoproteins4,5 have

been identified as biomarkers for a range of important diseases,

potentially leading to new therapeutic and diagnostic techniques. It

is hoped that the reliance on expensive mass spectroscopy or

labour-intensive gel-blotting techniques may be overcome by

mTAS. Any mTAS for glycoconjugate analysis will need to include

an extraction phase with appropriate physical properties and

selectivity. The only rigid monolithic materials developed to date

for such purposes have employed immobilised lectins, which have

been demonstrated in both capillary6 and microchip7 platforms.

Boronate affinity is another popular extraction method and it is

surprising that there has not yet been a report of rigid boronate

affinity monolithic materials.

Boronate affinity phases selectively retain molecules with 1,2-

and 1,3-cis-vicinal diol moieties, as are commonly present in

carbohydrates. This makes them an ideal general extraction

module for glycoconjugates. The primary mechanism of retention

is through the reversible formation of cyclic, anionic esters.8

Boronate ligands are an excellent alternative to lectins as they are

less carbohydrate-specific than lectins, which can be advantageous

for screening approaches and are considerably less toxic and more

stable.9 Important existing, emerging and potential applications

are described in a recent review entitled Boronic Acids as Ligands

for Affinity Chromatography.10

In this work, two approaches for the fabrication of porous

polymer monoliths with boronate affinity ligands were compared.

The performance of these materials for use as affinity supports for

chromatography and electrochromatography was evaluated and

compared using simple nucleosides as the test analytes.

Base monoliths of poly(glycidyl methacrylate-co-ethylene glycol

dimethacrylate), poly(GMA-co-EDMA) with a median pore size

of 1.19 mm and a surface area of 5.6 m2 g21 were prepared

according to the procedure described by Preinerstorfer et al.11,12

Briefly, poly(EDMA-co-GMA) polymerisation mixtures were

prepared in 2.5 g quantities with 16 wt% EDMA, 24 wt%

GMA, 30 wt% cyclohexanol and 30 wt% 1-dodecanol and 1 wt%

AIBN with respect to the total monomers. This solution was

purged of oxygen and drawn into 75 mm or 100 mm id fused-silica

capillaries (Polymicro Technologies Inc. Phoenix, AZ, USA) that

had been treated to allow bonding of polymer to the surface.13

Monoliths were formed by heating the capillaries to 60 uC in a

water bath for 20–24 h and subsequently flushed for 1 h with

MeOH at a flow rate of 30 mL h21 to remove the residual

monomers and porogens.

The first approach for attachment of a boronic acid functional

group was through nucleophilic attack of the epoxide

with p-hydroxyphenylboronic acid, as depicted in Fig. 1.

Poly(GMA-co-EDMA) monoliths were flushed with acetonitrile

before introducing a reaction solution containing 0.138 g of

p-hydroxyphenylboronic acid dissolved in 1.50 g acetonitrile

mixed with 0.324 g of triethylamine. This solution was con-

tinuously flushed through the capillary for 16–20 h at 60 uC.

Whilst m-aminophenylboronic acid has previously been employed

as a nucleophilic phenylboronate molecule,14,15 we selected

p-hydroxyphenylboronic acid as it was anticipated that the

phenoxide would be a better nucleophile and improve the

reactivity and hence surface coverage of the final boronic acid

functionality.

Australian Centre for Research on Separation Science (ACROSS),School of Chemistry, University of Tasmania, Private Bag 75, Hobart,Australia, 7001. E-mail: Emily.Hilder@utas.edu.au;Fax: +61 3 6226 2858; Tel: +61 3 6226 7670

COMMUNICATION www.rsc.org/analyst | The Analyst

1094 | Analyst, 2006, 131, 1094–1096 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

29

Aug

ust 2

006.

Dow

nloa

ded

by T

empl

e U

nive

rsity

on

22/1

0/20

14 0

6:31

:21.

View Article Online / Journal Homepage / Table of Contents for this issue

The second approach for attachment of the boronic acid

functionality was via photografting a thin layer of poly(GMA)

onto the surface of the poly(EDMA-co-GMA) monolith prior to

reaction with the p-hydroxyphenylboronic acid as discussed above.

Photografting is a term used to describe a method of attaching a

thin layer of branched polymer chains onto a surface by UV

initiated radical polymerisation, and should allow more GMA

groups to be introduced onto the surface for subsequent

modification. Photografting onto the pores of macroporous

polymer monoliths has been demonstrated by Rohr et al.13 and

is based on the method described by Ranby et al.16

Poly(GMA-co-EDMA) monoliths were formed in UV trans-

parent capillaries and a thin layer of poly(GMA) grafted onto

the surface according to the procedure of Rohr et al.17 Briefly,

the poly(GMA-co-EDMA) monolith was flushed with a photo-

grafting mixture containing 15 wt% GMA, 0.22 wt% benzo-

phenone, 63.6 wt% t-butanol and 21.1 wt% H2O that had been

purged of oxygen with nitrogen gas and exposed at an intensity of

20 mW cm22 (@ 260 nm) for 60 s. The capillary was then flushed

with acetonitrile for 1 h at a flow rate of 30 mL h21 to remove the

unreacted photografting solution and any GMA oligomers.

To evaluate the performance of the boronic acid functionalised

monolith, simple ribonucleosides were selected as model analytes.

They are readily available, relatively stable, easily observed by UV-

absorbance detection and have 2-deoxyribonucleoside counter-

parts which differ only in replacement of one hydroxyl group of

the ribose with a hydrogen. As a result of this single atom change,

2-deoxyribonucleosides are not able to form cyclic complexes with

the boronate groups and should be unretained, although they

should exhibit similar secondary interactions with the monolith

(such as hydrophobic interaction). This was verified when neither

class of molecule was observably retained on an unmodified

poly(GMA-co-EDMA) monolith. The performance of the bor-

onate affinity monoliths is shown in Fig. 2 which shows

separations of cytidine and 2-deoxycytidine using the boronic acid

functionalised monoliths in micro liquid chromatography (micro-

LC) and capillary electrochromatography (CEC) modes. It can be

seen clearly that cytidine is significantly retained in both columns

in relation to its 2-deoxy counterpart indicating successful

fabrication of a boronic acid monolith. Retention of adenosine

over 2-deoxyadenosine was also observed, while guanosine and

uridine showed significant interaction with the monolith with the

peak being too low and broad to be readily detected. These

observations are consistent with previous work that shows uridine

and guanosine to be retained more strongly than cytidine and

adenosine on some boronate affinity stationary phases.18

As expected, the p-hydroxyphenylboronate modified GMA-

photografted monolith showed considerably more retention of the

ribonucleosides than was observed for the monoliths that were

merely modified by reaction onto the pore surface. When the

separations were performed under the same conditions (except for

a different capillary diameter that would not have had a significant

effect), the retention factors of cytidine increased from 1.3 in the

surface-modified monolith to 2.6 in the photografted monolith.

This increase in affinity can be attributed directly to the

introduction of the photografted layer. The branched nature of

the photografted layer, as well as the fact that it is made up

exclusively of poly(GMA), means that there are more potential

sites on the surface of this material with which the p-hydro-

xyphenylboronic acid can react. It is important to note at this stage

that the photografting procedure used in this work was completely

unoptimised for this monomer and that it is highly probable that

the ligand density could be increased with optimisation of the

photografting process.

To confirm that the retention of the ribonucleosides was indeed

via boronate affinity, the pH of the electrolyte was varied.

Separations were performed in 50 mM HEPES at pH 6, 7, and 8,

Fig. 1 Reaction scheme for covalent attachment of p-hydroxyphenylboronic acid to a GMA monolith.

Fig. 2 Separation of 2-deoxycytidine and cytidine using p-phenylbor-

onate modified monoliths. (A) Micro-LC separation mode, 8 cm

surface modified monolith. Column: 33 cm 6 100 mm ID (8.5 cm to

the detector). BGE: 100 mM ammonium acetate, pH 9 with 100 mM

CaCl2. Sample: 200 ppm of each ribonucleoside in BGE. Conditions: 9 bar.

Injection: 18 s @ 8 bar. (B) CEC separation mode, 6.5 cm poly(GMA)

grafted monolith. Column: 33 cm 6 75 mm ID (8.5 cm to the detector).

BGE: 50 mM HEPES, pH 8.7. Sample: 100 ppm 2-deoxycytidine and

500 ppm cytidine in BGE. Conditions: 210 kV with 8 bar pressure on

both vials. Injection: 18 s @ 8 bar.

This journal is � The Royal Society of Chemistry 2006 Analyst, 2006, 131, 1094–1096 | 1095

Publ

ishe

d on

29

Aug

ust 2

006.

Dow

nloa

ded

by T

empl

e U

nive

rsity

on

22/1

0/20

14 0

6:31

:21.

View Article Online

adjusted with NaOH, with the results for adenosine and cytidine

shown in Fig. 3. As can be seen, the retention factors increased as

the pH of the buffer increased, with a 30% increase in capacity

from pH 6 to 8. This is consistent with previous results where it has

been shown that cyclic anionic ester complexes formed by the

phenylboronate groups and the ribofuranose moieties are

stabilised at higher pH.8 The results shown in this figure also

serve to demonstrate that, even at this initial stage of development,

CEC with this new material exhibits reasonable run-to-run

reproducibility and the material has a lifespan of (at least) tens

of injections.

Finally, it is necessary to note that both the surface modified

and photografted monoliths exhibited an anodic EOF ranging

from 219 to 232 6 1029 m2 V21 s21 indicating a positive charge

on the surface of the monoliths. This was not observed in an

unmodified GMA monolith suggesting the positive surface charge

was introduced during modification of the monolith with

p-hydroxyphenylboronic acid. It is proposed that triethylamine

may act as a nucleophile as well as a base in this reaction and

would generate a positively charged quaternary ammonium group.

This reaction has previously been reported as a mechanism for

functionalising the surface of resins,19 although these types of

reaction are typically performed under harsher conditions.

Verifying this, a GMA monolith was reacted with a solution

containing only triethylamine in acetonitrile and was found to

have a reversed EOF of 226 6 1029 m2 V21 s21. A similar

reaction with diisopropylethylamine, a base that is sterically

prohibited from acting as a nucleophile, did not provide a reversed

EOF supporting the notion that the positive surface charge is the

result of nucleophilic attack by triethylamine. Whilst the positive

surface charge was an unintended result, it was beneficial for

electrochromatography as the quaternary ammonium provides an

almost pH independent EOF allowing the simple pH studies

shown in Fig. 3 to be conducted without the concern of a changing

EOF. This is evidenced by the EOF times changing from

approximately 1.6 to 2.0 min as the pH was changed from 6.0 to

8.0. This will be very beneficial in a mTAS where voltages and EOF

are the most common method at the moment for controlling

analyte and fluid movement.

We have shown here the first report of boronate affinity

monoliths and their applicability to retain ribonucleosides in both

microscale liquid chromatography and capillary electrochromato-

graphy modes. These materials are part of an on-going investiga-

tion into new materials and techniques for microscale analyses of

glycoconjugates. They will be further characterised, optimised and

integrated into microfluidic devices in conjunction with other

analytical processes to create a glycoconjugate mTAS.

Acknowledgements

The authors would like to acknowledge the Australian

Research Council for funding this research and Dr Jason A.

Smith for many helpful discussions.

Notes and references

1 N. Minc and J.-L. Viovy, C. R. Phys., 2004, 5, 565–575.2 E. F. Hilder, F. Svec and J. M. J. Frechet, J. Chromatogr., A, 2004,

1044, 3–22.3 S. L. Ramsay, I. Maire, C. Bindloss, M. Fuller, P. D. Whitfield,

M. Piraud, J. J. Hopwood and P. J. Meikle, Mol. Genet. Metab., 2004,83, 231–238.

4 D. H. Dube and C. R. Bertozzi, Nat. Rev. Drug Discovery, 2005, 4,477–488.

5 G. Durand and N. Seta, Clin. Chem. (Washington, D. C.), 2000, 46,795–805.

6 F. M. Okanda and Z. El Rassi, Electrophoresis, 2006, 27, 1020–1030.7 X. Mao, Y. Luo, Z. Dai, K. Wang, Y. Du and B. Lin, Anal. Chem.,

2004, 76, 6941–6947.8 A. Bergold and W. H. Scouten, in Solid Phase Biochemistry, Wiley, New

York, 1983, vol. 66, pp. 149–187.9 W. H. Scouten, in Encyclopedia of Separation Science, ed. I. D. Wilson,

Academic Press, Sydney, 2000, pp. 273–277.10 X.-C. Liu, Sepu, 2006, 24, 73–80.11 J. P. Hutchinson, E. F. Hilder, R. A. Shellie, J. A. Smith and

P. R. Haddad, Analyst, 2006, 131, 215–221.12 B. Preinerstorfer, W. Bicker, W. Lindner and M. Lammerhofer,

J. Chromatogr., A, 2004, 1044, 187–199.13 T. Rohr, E. F. Hilder, J. J. Donovan, F. Svec and J. M. J. Frechet,

Macromolecules, 2003, 36, 1677–1684.14 M. Glad, S. Ohlson, L. Hansson, M.-O. Mansson and K. Mosbach,

J. Chromatogr., 1980, 200, 254–260.15 Z.-f. Kou, X.-d. Wang, Z.-z. Liu, W.-r. Qiu and X.-y. Wu, Huadong

Ligong Daxue Xuebao, 2001, 27, 614–617.16 B. Ranby, W. T. Yang and O. Tretinnikov, Nucl. Instrum. Methods

Phys. Res., Sect. B, 1999, 151, 301–305.17 T. Rohr, D. F. Ogletree, F. Svec and J. M. J. Frechet, Adv. Funct.

Mater., 2003, 13, 264–270.18 S. Hjerten, A. Vegvari, T. Srichaiyo, H.-X. Zhang, C. Ericson and

D. Easker, J. Capillary Electrophor., 1998, 005, 13–26.19 C.-P. Yang and C.-Y. Ting, J. Appl. Polym. Sci., 1993, 49, 1019–1029.

Fig. 3 Retention factors of cytidine and adenosine on 8 cm of surface

modified monolith. 50 mM HEPES buffers adjusted to various pH levels

with NaOH. Column: 33 cm 6 100 mm ID (8.5 cm to the detector).

Conditions: 210 kV with 8 bar pressure on both vials. Injection: 18 s @

8 bar. Samples: 200 ppm of both the appropriate nucleoside and the

corresponding 2-deoxynucleoside. All retention factors were averaged

from the results of three injections with the exception of the pH 7

adenosine value for which only two injections were performed. Error bars

show ¡ 1 standard deviation.

1096 | Analyst, 2006, 131, 1094–1096 This journal is � The Royal Society of Chemistry 2006

Publ

ishe

d on

29

Aug

ust 2

006.

Dow

nloa

ded

by T

empl

e U

nive

rsity

on

22/1

0/20

14 0

6:31

:21.

View Article Online