J. Sep. Sci.

2011, 34, 2079–2087 2079

Anthony Bruchet1,2 Research Article

Vincent Dugas2
Clarisse Mariet1
Florence Goutelard1
Jérôme Randon2
1
Commissariat à l’Energie
Atomique, Saclay, DEN/DPC/
SECR/LANIE, Gif-sur-Yvette,
France
2
Laboratoire des Sciences
Analytiques (UMR CNRS 5180),
Université de Lyon,
Villeurbanne, France
Received March 1, 2011
Revised April 11, 2011
Accepted April 11, 2011
Improved chromatographic performances
of glycidyl methacrylate anion-exchange
monolith for fast nano-ion exchange
chromatography
An efficient and reproducible photopolymerized poly(glycidyl methacrylate-co-ethylene
dimethacrylate) was synthesized in Teflon-coated fused-silica capillaries (100 mm id) and
functionalized by reaction of triethylamine with reactive epoxy groups. We report here the
successful transfer of a standard polymerization mixture optimized for the thermally
initiated synthesis of glycidyl-based monolith to photo-induced polymerization. The
monolith obtained after optimization of the photo-initiation conditions was characterized
in reverse-phase chromatography evaluating its suitability in terms of efficiency, retention
and hydrodynamic permeability. Reproducibility of the photo-induced procedure was
satisfactory with RSD below 6% for retention and efficiency and slightly higher for
hydrodynamic permeability (12%). The functionalized generic support was then used in
nano-ion-exchange chromatography. Efficiencies up to 75 000 plates/m, ion-exchange
capacity of 8 nano-equivalents/cm of monolithic column, with a combination of a satis-
factory hydrodynamic permeability allowed to perform fast separations of five inorganic
anions in o3 min maintaining baseline resolution. The efficiency of the monolith was not
retention-dependent, demonstrating its wide range of possible applications for highly
retained anions.

Keywords: Chemical functionalization / Fast nano-IEC / Monolith / Photopoly-

merization / Strong anion exchanger
DOI 10.1002/jssc.201100180

1 Introduction inorganic anions or cations [10, 11], carbohydrates [12],

Since their introduction in the early 1990s [1], polymeth-
acrylate monoliths have emerged as a powerful alternative
tool in chromatographic column technology. The combina-
tion of their singular porous properties, the various
chemistries available and their relatively simple implemen-
tation in columns with small internal diameters make them
particularly attractive for the new chromatographic chal-
lenges of complex matrices analysis [2], fast chromatography
[3] or on-chip separations [4]. Besides the use of methacry-
late monolith in many chromatographic modes [5–9], ion
exchange is of growing interest. It allows the separation of
Correspondence: Dr. Vincent Dugas, Laboratoire des Sciences
Analytiques, Université de Lyon, Université Claude Bernard
Lyon 1, Bâtiment Curien, 43 Boulevard du 11 Novembre 1918,
69622 Villeurbanne Cedex, France
E-mail: Vincent.dugas@univ-lyon1.fr
Fax: 133-472-431-078
Abbreviation: AIBN, 2,20 -azoisobutyronitrile; EDMA,
ethylene dimethacrylate; GMA, glycidyl methacrylate; IEC,
ion-exchange capacity; SEM, scanning electron microscopy;
TEA, triethylamine
small organic charged compounds [13], peptides or proteins
[14, 15]. Although high performances have been obtained
for ion-exchange separations of large biomolecules [16, 17],
monoliths suffer from a lack of ion-exchange capacity (IEC)
for small ions due to their highly porous structure. From
this point of view, the preparation of methacrylate monolith
for ion-exchange chromatography is still challenging and
two strategies can be implemented for the enhancement of
IEC: tuning the porous structure in order to increase the
surface area or maximizing the surface density of ion-
exchangeable groups.
During the last ten years, research mainly focussed on
the enhancement of the surface-density of ion-exchangeable
groups in order to obtain high capacity ion-exchange
monoliths. Such surface functionalization of methacrylate
monolith was divided into two different strategies as referred
recently by Hilder: co-polymerization and post-synthesis
functionalizations [18]. Co-polymerization has been
successfully used by Lee et al. with direct incorporation of
sulfonate, phosphonate or ammonium functions into the
monolithic structure for biomolecule separations [15, 19, 20].
Additional correspondence: Anthony Bruchet
E-mail: Anthony.bruchet@cea.fr

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
2080 A. Bruchet et al.
However, this approach suffers from some drawbacks such
as the need for mixture optimization for each type of func-
tionalization [21], poor mechanical stability and high swel-
ling [11]. Therefore, post-polymerization modifications have
emerged as versatile tools in order to easily tune the surface
properties of monolith as mentioned in numerous reviews
[18, 22, 23]. These modifications can be performed in three
different ways: electrostatic coupling (the use of surfactant or
latex particles), surface grafting on reactive groups and
surface radical grafting. Latex-coated monolith, introduced
by Hutchinson et al. [8, 24], showed improved IEC but
involved the incorporation of a negatively charged monomer
into the bulk polymer. Reaction with reactive epoxy groups
was first demonstrated by Svec and Frechet as early as 1992
[1] and is still widely employed to obtain cation or anion
exchangers [11, 20, 25–28], but the surface availability of the
reactive epoxy rings limits the capacity. Alternative approa-
ches have been developed with surface-initiated poly-
merizations. First attempt was carried out with a chemical
initiation using CeIV as the initiator [29], but photo-induced
functionalizations have been intensively developed during
the last few years with the successful grafting of 2-acryl-
amido-2-mehtyl-1-propanesulfonic acid (AMPS), methacrylic
acid (MAA) and [2(methacryloyloxy)ethyl] trimethylammo-
nium chloride (META) [10, 30, 31]. Such a technique intro-
duced by Ranby et al. involved the use of benzophenone as
free radical initiator allowing free radical processes by
hydrogen abstraction from surface available alkyl groups
[32, 33]. This led to the creation of polymer brushes from
only one reactive site, thus increasing the density of ion-
exchangeable groups compared with the modification of
surface reactive groups [34, 35].
As stated above, the enhancement of IEC can be
obtained also by tuning porous structure in order to increase
the surface area. The preparation of monoliths via photo-
initiated polymerization is well and widely used from
sample treatment to classical RP separation [36–38]. In
particular, it was already successfully applied to miniatur-
ized capillary columns [39] and microfluidics [40]. In order
to prepare the column with ion-exchange capability, most of
the previous works have used glycidyl methacrylate (GMA)
polymers as the backbone support either for surface coating
or surface grafting since they have proved efficient ion-
exchange separations. However, these polymeric monoliths
provide only small surface areas [41]. The optimized
mixtures are generally composed of GMA, ethylene di-
methacrylate (EDMA), ternary (1,4-butanediol/1-propanol/
water) or binary (dodecanol/cyclohexanol) porogens and
2,20 -azoisobutyronitrile (AIBN) as the initiator for thermally
induced free radical polymerizations. Recently, attempts
have been made in order to increase the specific surface area
by means of terminated polymerization [41] or post-poly-
merization hypercrosslinking [42] for RP-mode chromato-
graphy. As described earlier, photo-initiation process on
poly(butyl methacrylate-co-ethylene dimethacrylate) mono-
liths led to the obtention of a smaller structure and can
improve the specific surface area of the porous materials
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2011, 34, 2079–2087
[43]. Here, we showed that the transfer of well-established
thermally induced polymerization to photo-induced poly-
merization can be a relevant way to enhance the IEC of
glycidyl-based monolith. Using the efficient mixture defined
by Ueki et al. [11] and our previously published functiona-
lization procedure [44], the present study demonstrates the
potential of this strategy to obtain monoliths with unmat-
ched chromatographic performances.
2 Materials and methods
2.1 Reagents and materials
GMA, EDMA, 1,4-butanediol and AIBN were obtained from
Acros Organics (Noisy-Le-Grand, France). All HPLC-grade
solvents (1-propanol, acetonitrile, ethanol), 3-(trimethoxysi-
lyl)-propyl methacrylate and triethylamine (TEA) were from
Sigma-Aldrich (Isle-d’Abeau, France). All anions used for
the preparation of mobile phases and samples (iodate,
bromate, nitrite, bromide, nitrate, benzoate and perchlorate)
were purchased as their sodium salts from Sigma-Aldrich.
The aqueous solutions were prepared using 418 MO DI
water (Millipore, Molsheim, France). UV-transparent
Teflon-coated capillaries with an inner diameter of 100 mm
were obtained from CIL-Cluzeau Info Labo SA (Sainte-Foy-
La-Grande, France). Concentrations of individual anions
were chosen according to their UV absorbance at 210 nm as
follows: 200 mg/L iodate, 300 mg/L bromate, 100 mg/L
nitrite, 300 mg/L bromide and 100 mg/L nitrate.
2.2 Instrumentation
Vinylization of capillaries was done by flushing reagents
through capillaries by applying a 1-bar nitrogen pressure
with the Nanobaume system (CIL-Cluzeau Info Labo SA).
The illumination system used for photopolymerization
reactions was a Bio-link cross-linker (VWR International,
Strasbourg, France) equipped with five 8 W UV tubes
emitting at 365 nm. A UVX-radiometer (Fisher Scientific,
Lyon, France) equipped with a 365-nm sensor (1 cm2) was
used to record the total UV-light energy supplied during
each polymerization. An HPLC pump (Merck, Lyon,
France) and an HPLC oven were used for functionalization
steps.
Evaluation of chromatographic performances of GMA-
based monolith in reverse-phase chromatography (RP
mode) was performed using the Agilent3D CE system
(Waldbronn, Germany) operating in pressure-driven mode.
Ion chromatography experiments were performed with
a MicroPro syringe pump from SunChrom (Friedrichsdorf,
Germany) and external injection and detection devices.
Temporized manual injection was realized using a Vici
C72NH nanovolume valve (Interchim, Montluc- on, France)
in order to obtain injection volume of approximately 10 nL.
The detection was carried out at 210 nm using an Ultimate
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J. Sep. Sci. 2011, 34, 2079–2087
UV detector from LC-Packings (Amsterdam, The Nether-
lands) equipped with a detection cell of 3 nL. Strict evalua-
tion of flow rate was obtained using the liquid flow sensor
Sensirion SLG-1430 (Sensirion AG, Germany).
The study of monomer conversion was carried out with
a Waters HPLC system equipped with a W-600 pump, a
W-717 plus autosampler, a Waters 996 DAD detector and
Millennium 32 software (Waters, Guyancourt, France).
Hypercarb column (3  100 mm, packed with 5 mm parti-
cles) and a mobile phase of 50:50 v/v ACN/water were used
for reverse-phase separation of the two monomers. The
injection volume of samples diluted in acetonitrile was
20 mL. Linear response of the UV detector, set at 220 nm,
allowed the quantitative determination of non-reacted
monomers collected after the in-capillary monolith synth-
esis as previously described by Nischang et al. [41].
Scanning electron microscopy (SEM) images were
acquired using a JCM-5000 Neoscope Scanning Electron
Microscope (Jeol, Croissy-sur-Seine, France) after metalli-
zation of monolithic capillary columns.
2.3 Preparation of monolithic columns
UV-transparent Teflon-coated fused-silica capillaries were
first pretreated with 3-(trimethoxysilyl)-propyl methacrylate
according to a previously published procedure [44]. There-
after, polymerization mixture was introduced under the gas
pressure of 500 mbar for 1–2 min and the capillary ends
were sealed with silicon.
Polymerization mixture was prepared following the
procedure of Ueki et al. [11]: 900 mL of GMA and 300 mL of
EDMA were dissolved in a ternary porogen of 1050 mL of
1,4-butanediol, 600 mL of 1-propanol and 150 mL of water.
Twelve milligrams of AIBN was added to the mixture as a
polymerization photo-initiator (1 wt% relative to the
monomers). Prior to irradiation, dissolved oxygen in solu-
tion was degassed by sonication for 15 min. Photo-
polymerization was performed in room temperature at
365 nm (1.4 mW/cm2) for different irradiation times.
During the irradiation step, the temperature increased
slightly in the UV-oven (few degrees), without a impact on
the final monolith properties compared with temperature-
controlled photopolymerization. After polymerization, the
monolithic columns were rinsed with acetonitrile at 1 mL/
min for 100 min either to remove the remaining reactant or
to collect 100 mL for the monomer conversion study. A UV
detection window was then created at 8.5 cm from the inlet
of the column by depolymerization of the monolith under
exposure to a deuterium lamp, inside the CE instrument,
before reverse-phase chromatographic experiments.
Functionalization of monolith was performed according
to the optimized procedure previously described [39].
Briefly, monolithic columns were filled with diluted solution
of TEA diluted in 50:50 v/v ethanol/water mixture. The
functionalization was carried out under dynamic conditions
with a flow rate of 1 mL/min at a temperature of 851C for 4 h.
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Liquid Chromatography 2081
The resulting 3-triethylammonium-2-hydroxy-propyl func-
tionalized monolith was then rinsed at 1 mL/min succes-
sively with ethanol and water for 3 h before equilibration in
10 mM sodium perchlorate for 6 h.
2.4 Evaluation of the GMA-based monolithic column
performances in RP chromatography
As extra-column dispersion effects are a critical in nano-
liquid chromatography, the evaluation of monoliths was
performed using the CE system equipped with an in situ
hydrodynamic injection (by applying a constant pressure for
several seconds), and with in situ UV detection (214 nm)
through the capillary. This system also allowed to move the
mobile phases through the monolithic column from the
inlet to the outlet with an internal/external pressure device
(2–12 bar). Mobile phase used was 50:50 v/v ACN/water and
samples of benzene, toluene, ethylbenzene and acetone
(non-retained marker) were prepared in ACN. The injection
was performed by applying 3 bar at the inlet for 6 s.
Determination of the minimum plate height (HETPmin)
was performed by varying the pressure applied at the inlet
of the column, hence by varying the flow rate (measured
with the flow sensor). Hydrodynamic permeability was
measured by
L
K0 ¼ Z:us ð1Þ
DP
where L is the length of the column, DP is the pressure
drop, Z is the ACN viscosity and us is the superficial velocity
of the mobile phase.
2.5 Determination of the IEC
The IEC of the monolith was determined using the
breakthrough curve of perchlorate with a UV detection at
210 nm. The column was first equilibrated using low
retained benzoate anions at a concentration of 2 mM until
obtaining a stable baseline of the UV signal. After a washing
step with water in order to remove the interstitial benzoate
ions, a solution of 10 mM perchlorate was passed through
the column at 1 mL/min until breakthrough detection.
3 Results and discussion
3.1 Transfer of polymerization process from thermal
to photo-induced initiation
The elaboration of an efficient anion-exchange monolith
first relies on the synthesis of a suitable generic support
before any further modifications. We carefully designed the
experimental protocol in order to produce a monolith with
suitable chromatographic characteristics evaluated in
reverse-phase mode. Since glycidyl-based monolith
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2082 A. Bruchet et al.
described by Ueki et al. [11] is well-established, no changes
have been made as regards to initiator, monomers and
porogenic solvent, but we paid a special attention to the
photo-initiation step. In thermal initiation, the number of
free radicals produced during the overall polymerization
process is governed by temperature and time of reaction
determining the size of the polymeric structure and the rate
of incorporation of monomers. By analogy for UV-induced
polymerizations, the key parameters are the radiant power
of UV light and also the time of reaction. As our
experimental setup did not allowed to adjust the radiant
power (fixed at 1.4 mW/cm2), optimization of the monolith
structure has been carried out varying energy doses supplied
from 0.9 to 3.4 J/cm2. As recently described, the kinetic
study of the monomers incorporation is a relevant way to
improve chromatographic performances of methacrylate
monolith [41]. Conversion yield of monomers during the
photopolymerization process obtained with HPLC measure-
ments is reported in Table 1. As photopolymerization is
known to be much faster compared with thermal polymer-
ization, 50% of monomers were already incorporated in only
10–15 min and more than 90% in 40 min. The constant
ratio of 1.2 between the quantities of crosslinker and
functional monomer during the first 20 min indicated a
higher conversion of the cross-linker in the bulk polymer.
The decrease in this ratio to a value of 1.05 at 40 min reflects
a higher incorporation of GMA in surface layers, which
should be advantageous with regard to loading capacity. As
the crosslinker conversion occurs during the overall process,
even surface layers are reticulated too and thus are less
subject to the phenomenon of swelling. It should be noticed
that the ratio obtained for the photopolymerization process
differs significantly from that observed for thermally
initiated polymerization of butyl methacrylate and EDMA
[41].
The characterization of the monoliths was also
performed on the basis of chromatographic parameters:
hydrodynamic permeability K0, retentions and efficiencies
(measured for model solutes in reverse-phase chromato-
graphy). Evolution of resulting K0 was recorded as a func-
tion of irradiation time as shown in Fig. 1. Low UV dose
(o0.9 J/cm2) results in non-uniform monolithic material,
unsuitable for liquid chromatography. An increase in the
polymerization time leads to a decrease in the hydro-
Table 1. Monomer-specific conversion during photopolymerization of poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith
in Teflon-coated fused-silica capillaries (id 100 mm)
Irradiation time (min) Energy supplied (J/cm2)
10 0.9
15 1.4
20 1.8
25 2.2
30 2.6
40 3.4
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2011, 34, 2079–2087
dynamic permeability by a factor of 2 within approximately
40 min. This was correlated with SEM photographs showing
a decrease in macroporosity for monolith obtained after
15–40 min of irradiation.
Retention and minimum plate height (HETPmin) for the
three alkylbenzene are plotted in Fig. 2 as a function of the
irradiation time. The minimum of plate height is reached at
a reaction time of 30 min (HETPmin 5 10 mm for an opti-
mum velocity of 0.5 mm/s). Conversely, it corresponds to a
maximum of retention for each alkylbenzene with k 5 1.2,
1.5 and 1.8, respectively. These relatively high retention
factors, despite the poor hydrophobic character of the
monolith, illustrate the relative high surface area of the
monolith. The best compromise between acceptable
permeability (Fig. 1) and lower HEPT (Fig. 2) was chosen to
be 30 min of photopolymerization, corresponding to an
irradiation dose of 2.6 J/cm2.
SEM images of monolith obtained for this optimized
conditions are given in Fig. 3. Fine structure of monolith
clearly shows closely aggregated small globules with a
diameter of 1 mm (compared with 2–5 mm for thermally
initiated monolith [11]) and macropores of about 1 mm.
Surface roughness highlighted the relatively high specific
area developed on the monolith. The photopolymerized
monolith exhibited a permeability of 5  1014 m2 with a
total porosity eT of 0.86 (porosity was estimated from flow
rate and measurement of the dead volume). The mechanical
strength of these monolithic columns was also assessed by
recording the backpressure for different flow rates. High
rigidity of the monolithic structure was demonstrated by the
linear correlation between flow rate up to 10 mL/min (with
ACN) and backpressure for a 10 cm length column (corre-
sponding to 160 bar).
Photopolymerized monoliths are considered as repro-
ducible as thermally initiated polymers [43, 45] but because
photopolymerization process is faster than thermal initia-
tion (30 min compared with 24 h for this monolith), small
deviations of the experimental setup during the initiation
step could lead to high variations of the monolithic structure
and poor reproducibility. Robustness of the monolith
synthesis was evaluated through measurement of repeat-
ability (capillary-to-capillary RSD) and reproducibility
(batch-to-batch RSD) with three synthesis batches
composed up to three monolithic capillaries. Hydrodynamic
EDMA (% conversion) GMA (% conversion) Ratio (EDMA/GMA)
43.5 36.1 1.20
59.6 48.6 1.22
72.8 60.2 1.21
82.3 70.8 1.16
91.3 82.6 1.10
95.2 90.1 1.05
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J. Sep. Sci. 2011, 34, 2079–2087 Liquid Chromatography 2083

10 .0

9.0
A
K0 (x 10-14 m²)

8.0

7.0

6.0
B
5.0
C
4.0
10 15 20 25 30 35 40 45
Irradiation time (min)

Figure 1. Evolution of hydro-

dynamic permeability K0 of
GMA-based monolith synthe-
sized in Teflon-coated
capillaries with increased irra-
diation time and SEM images
of polymer structure corre-
sponding to polymerization
time of (A) 15 min (top right
corner), (B) 25 min (bottom left
corner) and (C) 40 min (bottom
right corner). Column:
100 mm  8.5 cm. Mobile phase:
100% ACN.

A B
2.5 60

Figure 2. Impact of irradiation

2 50
time at 365 nm on (A) reten-
Retnetion factor, k

40 tion factor, k and (B) mini-

HETPmin (µm)

1.5 mum plate height for benzene

30 (}), toluene (&) and ethyl-
1 benzene (D) using GMA-
20 based monolith. Column:
0.5 100 mm  8.5 cm. Hydrody-
10
namic injection: 3 bar during
0 0 6 s. Mobile phase: 50:50 v/v
5 10 15 20 25 30 35 40 45 5 10 15 20 25 30 35 40 45 ACN/water. UV detection at
Irradiation time (min) Irradiation time (min) 214 nm.

Figure 3. SEM photographs of the column

cross-section and surface roughness of the
optimized GMA-based monolith.

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2084 A. Bruchet et al.
Table 2. Capillary-to-capillary repeatability and batch-to-batch reproducibility relative standard deviation for permeability (K0), minimum
plate height (HETPmin) and retention factor (k)
Capillary-to-capillary repeatability
Mean
K0 (m2)a) 5.1  1014
HETPmin (mm)b) 11
kb) 1.2
a) Effective length of column: 8.5 cm. Mobile phase: 100 % CAN.
b) Effective length of column: 8.5 cm. Mobile phase: 50:50 v/v ACN/water. Hydrodynamic injections: 3 bar during 6 s. Samples: acetone
(dead-volume marker) and benzene. Linear velocity: 0.5 mm/s. UV detection at 214 nm.
permeability, efficiencies and retentions measured for the
model compounds were used to qualify the robustness of
photopolymerization. The results obtained using this stan-
dardized test are given in Table 2. Minimum plate heights
and retention factors were found to be in the range of
9–11 mm and 1.1–1.2, respectively, for benzene. Intra-batch
RSD values were below 6%. These variations were
comparable to those obtained by Ueki et al. [11] and indi-
cated a suitable monolith manufactory process. Reproduci-
bility of synthesis, evaluated through the inter-batch RSD,
was satisfactory. These results corroborated that the photo-
initiation process is as reproducible as the thermal initiation
one.
The characteristics of such optimized monolith reported
in Table 2 have been taken as a reference in order to eval-
uate the suitability of each synthesized monolith for further
functionalization and use in nano-ion-exchange chromato-
graphy (nano-IEC).
3.2 Performances of functionalized monolith for
nano-IEC
3.2.1 Evaluation of IEC
Photopolymerized monolithic capillary columns (7.5 cm
length) were obtained according to optimized conditions
previously mentioned (i.e. 30 min irradiation) in 100 mm id
Teflon-coated capillaries. Single-step preparation of
quaternary ammonium functionalized monolith has
been carried out according to a previously published
procedure [44]. Briefly, monolithic capillary columns
were filled with a solution of TEA in 50:50 v/v EtOH/water.
Total conversion of epoxy groups was then performed
under dynamic conditions through pumping the reactive
solution at 1 mL/min for 4 h at 851C leading to an
average maximized IEC of 8 nequiv/cm (measurements
realized in triplicates for each column). Such IEC compares
very favourably with results reported for methacrylate
monolith functionalized by means of latex coating [8] or
photografting [10]. Despite the relative poor surface area of
methacrylate monolith, the quantity of available ammonium
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2011, 34, 2079–2087
Batch-to-batch reproducibility
RSD (%) Mean RSD (%)
6 4.9  1014 12
4 10 6
1 1.2 5
groups allowed to develop a high IEC particularly suited for
use in nano-IEC.
3.2.2 Kinetic properties
Evaluation of the quaternary ammonium functionalized
monoliths was carried out with nano-IEC experiments using
sodium perchlorate as mobile phase and UV detection at
210 nm. Ion-exchange methacrylate monoliths are widely
used for fast separations of large biomolecules, thanks to the
convective flow of mobile phase allowed by their intrinsic
macroporous properties [46]. However, these monoliths
typically show poor performances for the separation of small
molecules due to the swelling of the surface layer mainly
constituted of non-reticulated polymer brushes. Highly
retained molecules can diffuse through this non-uniform gel
layer generating significant peak broadening [41]. However,
this phenomenon has not been investigated yet for meth-
acrylate ion exchangers. Evaluation of kinetic properties has
been carried out with the separation of three model anions to
probe a wide range of retention factors spanning from 1.1 to
8.1 in the 10 mM sodium perchlorate mobile phase. Column
efficiency was recorded at different flow rates and different
ionic strengths of mobile phase. Van Deemter curves plotted
for bromate, nitrite and nitrate in a 10-mM sodium
perchlorate mobile phase are shown in Fig. 4. Efficiencies
up to 75 000 plate/m were measured for the three anions at an
optimum linear velocity of 0.5 mm/s corresponding to a flow
rate of 100 nL/min. These high efficiencies obtained with the
optimized photopolymerized monolith are almost two to three
times higher than in previous works [8, 10, 11]. Van Deemter
plots for each anion were similar with no significative
deviation of efficiencies until 3.5 mm/s corresponding to an
average value of 18 000 N/m. This trend is conserved in all the
experimental domain explored (see Fig. 4) when retention
factors were increased by using mobile phases with lower
ionic strengths (8 and 5 mM). The photo-polymerized ion-
exchange monolith did not show any retention dependence of
kinetic performances unlike thermally initiated methacrylate
used in reverse-phase mode. This can be attributed to a less
pronounced phenomenon of swelling of the monolith surface
layer as indicated in Section 3.1.
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J. Sep. Sci. 2011, 34, 2079–2087 Liquid Chromatography 2085

3.2.3 Nano-IC separations of inorganic anions 10

9
(3)
The same set of three anions was first used to evaluate the 8
performances of the monolith in fast nano-IEC using a 10- 7

Absorbance (mAu)
mM sodium perchlorate mobile phase. An example of fast 6
(5)

separations of bromate, nitrite and nitrate is presented in 5

Fig. 5. Increasing flow rate from 0.1 to 1.7 mL/min
4
(corresponding to linear velocities of 0.5 and 4.3 mm/s, (1)
3
respectively) allowed to reduce the time of analysis from
2
15 min to o1 min maintaining baseline resolution for all (2) (4)
1
peaks.
Separations of five inorganic anions were also 0
0 0.5 1 1.5 2 2.5 3 3.5
performed at flow rates up to 1400 nL/min as illustrated in Retention time (min)
Fig. 6. Particularly interesting are the high retention
Figure 6. Fast separation of five inorganic anions using the
obtained with retention factors ranging from 0.5 to 4.3 GMA-based photopolymerized column at 1400 nm/min (corre-
(iodate, bromate, nitrite, bromide and nitrate) and the sponding to a linear velocity of 3.5 mm/s). Peaks: (1) 200 mg/L
maintainance of baseline resolution for all peaks. The iodate, (2) 300 mg/L bromate, (3) 100 mg/L nitrite, (4) 300 mg/L
analysis time was also reduced from more than 25 min for a bromide, (5) 100 mg/L nitrate. Other chromatographic conditions
are as in Fig. 4A.
flow rate of 100 nL/min (corresponding to an optimal linear

Figure 4. Plate height curves

A B as a function of linear velo-
60 60
city for (A) bromate (&),
50 50 nitrite (J) and nitrate (D) in
10 mM NaClO4 mobile phase
Plate Height (µm)
Plate Height (µm)

40 40 and for (B) nitrates in 10 mM

(D), 8 mM (m) and 5 mM (m)
30 30 NaClO4 mobile phases for
the GMA-based anion-
20 20
exchange monolith. Column:
10 10
100 mm  7.5 cm. Injection:
10 nL of anions prepared in
0 0 water. Mobile phases
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 prepared in water. UV detec-
Linear Velocity (mm.s-1) Linear Velocity (mm.s-1) tion at 210 nm.

3
1.6 2

1.4

1.2 1
A
Absorbance (mAu)

1
0.35

0.8 B 0.3
Absorbance (mAu)

0.25

0.6 C 0.2
Figure 5. Evolution of the
0.15 separation of bromate (1),
0.4 0.1
nitrite (2) and nitrate (3) for
increased flow rates of (A)
D 0.05 100 nL/min, (B) 400 nL/min, (C)
0.2 800 nL/min, (D) 1400 nL/min,
0
0 0.2 0.4 0.6 0.8 1 1.2 (E) 1700 nL/min and a zoom
E Retention time (min) for the separation at 1700 nL/
0 min (bottom right corner).
0 5 10 15 20 25
Chromatographic conditions
Retention time (min) are as in Fig. 4A.

& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com
2086 A. Bruchet et al.
Table 3. Capillary-to-capillary repeatability and batch-to-batch reproducibility RSD for retention factor of bromate, nitrite and nitrate and
for ion-exchange capacity
Capillary-to-capillary repeatability
Mean
kBromatea) 1.1
kNitritea) 2.3
kNitratea) 4.1
IECb) (nequiv/cm) 8.0
a) Effective length of column: 7.5 cm. Mobile phase: 10 mM sodium perchlorate. Injection: 10 nL. Samples : bromate 100 mg/L, nitrite
100 mg/L and nitrate 100 mg/L in water. Linear velocity: 0.5 mm/s. UV detection at 210 nm.
b) Effective length of column: 7.5 cm. Loading step with 2 mM sodium benzoate, wash step with water and breakthrough step with 10 mM
sodium perchlorate. UV detection at 254 nm.
velocity of 0.5 mm/s), to o3 min. Moreover, it should be
noticed that no overloading effect was observed when using
relatively high sample concentrations due to low UV
sensibility (ranging from 100 to 300 mg/L).
Pure anion exchange process was also confirmed by
plotting the retention factors versus the inverse of sodium
perchlorate concentration for all anions. Linear correlation
was obtained in the range of 5–10 mM of sodium perchlo-
rate (data not shown).
3.2.4 Reproducibility and robustness of functiona-
lized monolith
Each column mentioned in Section 3.1.2 was functionalized
in order to evaluate the reproducibility of the functionaliza-
tion step. The results presented in Table 3 shows capillary-
to-capillary RSD values below 2% and slightly higher batch-
to-batch RSD especially for IEC (the value reported by Ueki
was 8.2%). Injections and separations were also carried out
during 1 wk with RSD values for retentions and efficiencies
lower than 8% highlighting the reproducibility and robust-
ness of all monoliths.
4 Concluding remarks
Efficient GMA-based monoliths were successfully synthe-
sized according to the transfer from a well-established
thermally initiated procedure to photo-induced polymeriza-
tion. Optimization of the UV-irradiation dose was investi-
gated through a systematic test allowing the evaluation of
the reverse-phase performances of monolithic columns
before functionalization. IEC obtained with a single-step
functionalization procedure was 8 nequiv/cm (approxi-
mately four times higher than previous reported data),
and kinetic performances evaluation showed the potential of
these columns for high-speed analysis. These results
demonstrate the promising improvement of performances
obtained with the use of transferred procedures from
thermal to UV-induced polymerizations. In agreement with
previous works dealing with the use of GMA monoliths for
ion-exchange chromatography, this study clearly demon-
& 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
J. Sep. Sci. 2011, 34, 2079–2087
Batch-to-batch reproducibility
RSD (%) Mean RSD (%)
0.5 1.1 5
1 2.4 3
2 4.1 3
2 8.1 6
strates their great potential of separation not only for large
biomolecules but also for the separation of small ions.
Moreover, the use of the photopolymerization process is
valuable for the localized synthesis of monolithic stationary
phase in micro-Total Analysis System.
The authors have declared no conflict of interest.
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