Anal. Chem. 2004, 76, 4866-4874
Monolithic Column with Zwitterionic Stationary
Phase for Capillary Electrochromatography
Hongjing Fu, Chuanhui Xie, Jing Dong, Xiaodong Huang, and Hanfa Zou*
National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences,
Dalian 116011, China
A capillary electrochromatography (CEC) monolithic column with zwitterionic stationary phases was prepared by
in situ polymerization of butyl methacrylate, ethylene
dimethacrylate, methacrylic acid, and 2-(dimethyl amino)
ethyl methacrylate in the presence of porogens. The
stationary phases have zwitterionic functional groups, that
is, both tertiary amine and acrylic acid groups, so the
ionization of those groups on the zwitterionic stationary
phase was affected by the pH values of the mobile phase,
and further affects the strength and direction of the
electroosmotic flow (EOF). Separations of alkylbenzenes
and polycylic aromatic hydrocarbons based on the hydrophobic mechanism were obtained. Separation of various
types of polar compounds, including phenols, anilines,
and peptides, on the prepared column were performed
under CEC mode with anodic and cathodic EOF, and
different separation selectivities of those polar analytes
were observed on the monolithic capillary column by
using mobile phases with different pH values.
Ionizable groups on the surface of the stationary phase are
necessary to generate substantial electoosmotic flow (EOF) for
capillary electrochromatography (CEC).1 Strictly speaking, most
stationary phases used in CEC are ion-exchangers. Silica-based
packing materials, which have been most widely used in CEC and
can be regarded as weak cation exchangers, generate cathodic
EOF due to the ionization of the residual silanol groups on the
surface of packings. Recently, strong ion exchangers and so-called
mixed-mode packing materials consisting of ionic groups such
as sulfonic acids or quaternary amines and hydrocarbon chains
have attracted much attention in CEC, because these packings
ensure stable EOF over an extended pH range.2 The direction of
the EOF depends on the charges on the surface of the stationary
phases, so stationary phases with positively charged functional
groups, such as amino groups or ammonium groups, generate
an EOF from cathode to anode, whereas stationary phases
carrying negatively charged groups, such as sulfonic acid and
carboxylic acid, generate cathodic EOF. To date, the vast majority
of reports on CEC concern EOF with one direction, that is,
cathodic or anodic EOF, for a CEC column; however, zwitterionic
* To whom correspondence should be addressed. Fax: +86-411-83693407.
Phone: +86-411-83693409. E-mail: [email protected], hanfazou@
dicp.ac.cn.
(1) Rathore, A. S. Electrophoresis 2002, 23, 3827-3846.
(2) Pursch, M.; Sander, L. C. J. Chromatogr., A 2000, 887, 313-326.
4866 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
stationary phases, which can make it possible to generate EOF
with different directions for one column, are seldom investigated
in CEC. In fact, there are many reports concerning chromatographic separation with zwitterionically modified materials, although it is still one of the new liquid chromatography separation
modes studied in recent years.3-6 Hu et al.7 established the
zwitterionic functionality via dynamically adsorbing the ODS
(octadecyl silica) column with a sulfobetaine-type zwitterionic
surfactant by the interaction between the C18 groups on the surface
and the hydrophobic tail of the surfactant. To overcome the
drawback of inferior stability due to loss of functional moieties
from the dynamically attached layer on the surface of stationary
phases, covalently bonded zwitterionic separation materials for
liquid chromatography were prepared by direct surface reaction8
or surface graft polymerization.9,10 Zwitterionic monoliths were also
prepared by photoinitiated copolymerization of monomers with
zwitterionic functional groups and methacrylic cross-linkers.11 The
zwitterionic stationary phases for high performance liquid chromatography columns show the ability of simultaneous separation
of inorganic anions and cations and also acidic and basic proteins
in a single run.12 However, most zwitterionic separation materials
used in HPLC are the materials with sulfobetaine-type zwitterionic
functional groups, which carry both positively charged quaternary
ammonium groups and negatively charged sulfonic groups and
belong to the strong/strong category; i.e., both the negative and
positive groups retain their charge over the entire operational pH
range. In other words, the overall zwitterionic moiety maintains
a 0 net charge. This character of such kind of zwitterionic
materials is disadvantageous for CEC because charged groups
on the surface of stationary phases are required to promote EOF.
In this work, zwittterionic monolithic columns with in situ
polymerization of butyl methacrylate (BMA), ethylene dimethacrylate (EDMA), methacrylic acid (MAA), and 2-(dimethyl amino)
ethyl methacrylate (DAMA), which carry tertiary amine groups
and acrylic acid groups and belong to weak/weak category, were
(3) Hu, W. Z.; Haddad, P. R.; Hasebe, K.; Tanaka, K. Anal. Commun. 1999,
36, 97-100.
(4) Tramposch, W. G.; Weber, S. G. Anal. Chem. 1986, 58, 3006-3010.
(5) Yu, L. W.; Hartwick, R. A. J. Chromatogr. Sci. 1989, 27, 176-185.
(6) Nomura, A.; Yamada, J.; Tsunoda, K. Anal. Chem. 1988, 60, 2509-2512.
(7) Hu, W.; Takeuchi, T.; Haraguchi, H. Anal. Chem. 1993, 65, 2204-2208.
(8) Tramposch, W. G.; Weber, S. G. J. Chromatogr. 1990, 544, 113-123.
(9) Jiang, W.; Irgum, K. Anal. Chem. 2002, 74, 4682-4687.
(10) Jiang, W.; Awasum, J. N.; Irgum, K. Anal. Chem. 2003, 75, 2768-2774.
(11) Viklund, C.; Irgum, K. Macromolecules 2000, 33, 2539-2544.
(12) Hu, W. Z.; Tao, H.; Tominaga, M.; Miyazaki, A.; Haraguchi, H. Anal. Chim.
Acta 1994, 299, 249-256.
10.1021/ac0496695 CCC: $27.50
© 2004 American Chemical Society
Published on Web 06/26/2004
prepared. Both the direction and strength of EOF can be
controlled easily by changing the pH values of the mobile phase.
Diverse series of neutral and ionic/ionizable analytes, such as
alkylbenzenes, aromatic hydrocarbons, phenols, anilines, and
peptides, were separated on the prepared CEC columns.
EXPERIMENTAL SECTION
Materials. Butyl methacrylate and ethylene dimethacrylate
were purchased from Aldrich Chemical (Milwaukee, WI). 2-(Dimethyl amino) ethyl methacrylate, ethylene dimethacrylate and
γ-methacryloxypropyltrimethoxysilane (γ-MAPS) were obtained
from Sigma (St Louis, MO). Methacrylic acid, purchased from
Shenyang Xinxi Chemical Plant (Shenyang, China), was distilled
under a vacuum before use. 1-Propanol, 1,4-butanediol, and
azobisisobutyronitrile (AIBN) were obtained from Shanghai
Fourth Reagent Plant (Shanghai, China), and HPLC-grade methanol and acetonitrile (ACN) were supplied by the Yuwang Chemical
Plant (Zibo, Shandong Province, China). EDMA and BMA were
extracted with 5% aqueous sodium hydroxide solution and dried
over anhydrous magnesium sulfate. Thiourea and aromatic
compounds of analytical grade were purchased from Tianjin
Chemical Plant (Tianjin, China). Peptides were purchased from
Serva (Heidelberg, Germany). Double-distilled water purified by
a Milli-Q (Millipore Co., Milford, MA) system was utilized
throughout the experiments. The sample solution of aromatic
hydrocarbons was prepared by dissolving them in ACN at a
volume ratio of 1:10 and then further diluted to the appropriate
concentration range from 0.01 to 0.03 µg/µL with the mobile phase
before injection. Anilines, phenols, aromatic acids, and peptides
were dissolved in the mobile phases at a concentration range from
0.1 to 2 µg/µL directly.
Capillaries of 100-µm i.d. and 375-µm o.d. were purchased from
the Yongnian Optic Fiber Plant (Hebei, China). All of the mobile
phases were prepared by mixing phosphate buffer with ACN, and
the pH values of phosphate buffers before mixing were used as
the pH values of the mobile phases. Thiourea was chosen as the
marker molecule of EOF.
Instruments. A Hewlett-Packed 3DCE system (Hewlett-Packard, Waldbronn, Germany) was used for all CEC experiments and
a pressure of 5 bar was applied on both ends of the capillary
column to avoid bubble formation during the CEC separation. A
Waters 510 HPLC pump (Waters, Milford, MA) was utilized to
flush the columns.
Preparation of Monolithic Columns. Prior to the polymerization, the capillary was pretreated with the following procedure:
13 First, the capillary column with a length of 40 cm was rinsed
with 0.1 M NaOH for 1 h and then with water until the outflow
reached pH 7.0. After subsequent flushing with methanol for about
10 min, it was dried by passage of nitrogen gas. γ-MAPS solution
by its dilution with methanol at a volume ratio of 1:1 was injected
into the capillary with a syringe, then the capillary was sealed
with rubber at both ends and then submerged in a water bath at
50 °C for overnight. Finally, the capillary was rinsed with methanol
and water to flush out the residual reagent. Thereby, Si-O-Si-C
bonds were formed between the capillary wall and the reactive
methacryloyl groups, which are available for subsequent attachment of monolith to the wall during the polymerization reaction.
(13) Wu, R. A.; Zou, H. F.; Ye, M. L.; Lei, Z. D.; Ni, J. Y. Anal. Chem. 2001, 73,
4918-4923.
The monolithic column was prepared from a polymerization
reaction of mixtures consisting of the following monomers: 20%
(v/v) butyl methacrylate, 20% (v/v) ethylene dimethacrylate, 5%
(v/v) methacrylic acid and 5% (v/v) 2-(dimethyl amino) ethyl
methacrylate, 50% (v/v) binary porogenic solvent of 1-propanol,
and 1,4-butanediol using AIBN (0.3 wt % with respect to the
monomers) as an initiator. The polymerization mixture was
sonicated for 20 min to obtain a homogeneous solution and then
purged with nitrogen for 10 min. After the pretreated capillary
was completely filled with the mixture, it was sealed at both ends
with rubber stoppers. The sealed capillary was submerged into a
water bath and allowed to react for 2 h at 60 °C. The resultant
monolithic capillary column was washed with methanol for ∼2 h
using an HPLC pump to remove unreacted monomers and
porogens. At the end of this period, the detection window was
made by burning off 1-2 mm of both the coated polymer outside
and the monolith inside the capillary using flames.14 The ashes
of the organic monolith inside the capillary were flushed out by
methanol for about 30 min with the HPLC pump under the applied
pressure at ∼80 bar. Capillaries, without visible compression of
the monolith, were cut at both ends to a total length of 32 cm and
effective length of 8.5 cm. Finally, the column was equilibrated at
10 kV for 30 min before running.
Macroscopic materials were prepared in larger amounts of
corresponding mixtures in 4.6 mm × 10 cm HPLC columns at
various reaction temperatures for 2 h. After the polymerization
completed, the rods were washed with methanol for 6 h, flashed
out from the columns, cut into small pieces, and finally, dried in
vacuo at 50 °C for 24 h. The pore properties were determined by
mercury intrusion porosimetry, and its specific surface area was
calculated from nitrogen adsorption/desorption isotherms using
a combined BET sorptometer and mercury porosimeter (9310
Mercury Porosimeter, USA). The elemental analyses of monolithic
rods were carried out on a Eager 300 microelemental analyzer.
Scanning electronic microscopic (SEM) images were obtained
using a JEOL JSM-5600 scanning electron microscope (JEOL
company, Japan).
RESULTS AND DISCUSSION
Monolith Synthesis. Monolithic stationary phase has been
regarded as a suitable and potential separation material for CEC
because of its stability, simple synthesis procedures, and no need
for frit fabrication and packing.15-19 Moreover, flexibility of surface
chemistries of monolithic stationary phases because of a wide
variety of monomers enables easy tailoring of the stationary phases
with both chromatographic groups and charged functionalities.
To synthesize zwitterionic monolith for CEC, three functional
monovinyl monomers, namely, BMA, MAA, and DAMA, were
used for this study. BMA provides a hydrophobic C4 surface, and
MAA affords negatively charged functionalities to generate
(14) Hoegger, D.; Freitag, R. J. Chromatogr., A 2001, 914, 211-222.
(15) Zou, H. F.; Huang, X. D.; Ye, M. L.; Luo, Q. Z. J. Chromatogr., A 2002,
954, 5-32.
(16) Svec, F.; Peters, E. C.; Sýkora, D.; Yu, C.; Fréchet, J. M. J. J. High Resolut.
Chromatogr. 2000, 23, 3-18.
(17) Legido-Quigley, C.; Marlin, N. D.; Melin, V.; Manz, A.; Smith, N. W.
Elecrophoresis 2003, 24, 917-944.
(18) Gusev, I.; Huang, X.; Horváth, C. J. Chromatogr., A 1999, 855, 273-290.
(19) Zhang, S. H.; Huang, X.; Zhang, J.; Horváth, C. J. Chromatogr., A 2000,
887, 465-477.
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
4867
Figure 1. Scanning-electron micrograph with magnification of (a) 900 and (b) 3000 for the end of the poly(BMA-co-EDMA-co-MAA-co-DAMA)
monolithic stationary phase in a fused-silica capillary column with 100-µm i.d.
cathodic EOF, whereas DAMA affords positively charged functionalities to promote anodic EOF. Because pore formation
requires high levels of cross-linking, EDMA was used as the
divinyl monomer throughout this research.
Most polymerizations of monoliths concerning hydrophobic
monomers were performed in organic solvents,20,21 and the
selection of the porogenic solvents is crucial for the preparation
of the monolithic CEC columns. Peters et al.22,24 demonstrated
that the composition of the porogenic solvent has great influence
on the porous properties of the monolithic materials for CEC. The
binary porogenic system consisting of 1-propanol/1,4-butanediol
was chosen in the preparation of zwtterionic monolith for CEC
because the polymerization mixture contains both hydrophobic
monomer and monomers carrying ionizable functional groups. It
was observed that 1-propanol/1,4-butanediol binary porogenic
system is well-suited for the preparation of the zwitterionic
monoliths for their compatibility. The effect of porogenic solvent
composition on the porosity of the poly(BMA-co-EDMA-co-MAAco-DAMA) monolithic columns was investigated by changing the
ratio of 1-propanol to 1,4-butanediol (keeping the monomers/
porogens ratio at 5/5, v/v). Dark monoliths with low permeability
were observed under microscope by using porogenic mixtures
containing less than 50% 1,4-butanediol with reaction temperature
of 60 °C for 2 h. As the content of 1,4-butanediol increased to
80%, the permeability of the columns became better and the drops
of mobile phase could be seen at the end of the capillary columns
when the mobile phase was pumped into the capillary by a syringe
pump. It was concluded that a high content of 1,4-butanediol in
the 1-propanol/1,4-butanediol binary porogenic system favored
production of the zwitterionic columns with good permeability.
This is because a higher content of 1,4-butanediol resulted in more
large pores, which was also observed by Peters et al.23 in the case
of methacrylate monomers with negatively charged groups.
The scanning-electron micrograph of the end of the poly(BMAco-EDMA-co-MAA-co-DAMA) capillary column is shown in Figure
(20) Bedair, M.; El Rassi, Z. Electrophoresis 2002, 23, 2938-2948.
(21) Yu, C.; Xu, M.; Svec, F.; Fréchet, J. M. J. J. Polym. Sci., Part A: Polym.
Chem. 2002, 40, 755-769.
(22) Peters, E. C.; Petro, M.; Svec, F.; Fréchet, J. M. J. Anal. Chem. 1997, 69,
3646-3649.
(23) Peters, E. C.; Petro, M.; Svec, F.; Fréchet, J. M. J. Anal. Chem. 1998, 70,
2288-2295.
4868 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
Table 1. Results for Element Analysis of Zwitterionic
Monolithic Rodsa
DAMA (%, v/v)b
N%
C%
H%
25
50
75
0.615
1.415
2.230
60.46
60.81
61.46
8.405
8.525
8.490
a Polymerization conditions: reaction mixtures containing (DAMA
+ MAA) 10% (v/v), BMA 20% (v/v), EDMA 20% (v/v), 1-propanol 10%
(v/v), 1,4-butanediol 40% (v/v), AIBN 0.15 mg; reaction temperature
at 60 °C; polymerization reaction time for 2 h. b Volume percentage of
DAMA in the (DAMA + MAA) mixture.
1. It can be seen that the monolithic bed with macropores linked
to the pretreated capillary wall. The pore size measurements were
performed with the zwitterionic polymers reacted with a polymerization mixture containing 20% BMA (v/v), 20% EDMA (v/v),
5% DAMA (v/v), 5% MAA (v/v), 25% 1-propanol (v/v), 25% 1,4butanediol (v/v), and 0.3% AIBN (wt) with respect to the
monomers at different reaction temperatures. The surface areas
of zwitterionic material were 52.3, 55.6, and 58.0 m2/g at the
reaction temperatures of 50, 60, and 70 °C, respectively. As the
temperature increased, the surface area of zwitterionic material
increased. Figure 2 shows the pore size distribution profiles of
zwitterionic polymer at different reaction temperatures. It can be
seen that a lower reaction temperature favored zwitterionic
polymer with larger pore size and that the reaction temperature
is a convenient variable to control the pore size distribution of
the molded rods without requiring any change in the composition
of the polymerization mixture. Because only DAMA contains
nitrogen, to investigate how DAMA takes part in the polymerization, elemental analyses of the zwitterionic rods were carried
out, and the results are shown in Table 1. As can be seen, although
the nitrogen content of the rods increased with the increasing
amount of DAMA in the polymerization mixture, the linear
relationship between the nitrogen content of zwitterionic rods and
the DAMA content in the polymerization mixture was not
obtained, which may be caused by the fact that the four monomers
may not have the same reactivity during polymerization, and their
incorporation into the monoliths may not be equal.
(24) Peters, E. C.; Petro, M.; Svec, F.; Fréchet, J. M. J. Anal. Chem. 1998, 70,
2296-2302.
Figure 2. Effect of reaction temperature on the pore size distribution
of the zwitterionic monolithic rods. Experimental conditions: monolithic
rods prepared with polymerization mixture of 20% (v/v) BMA, 20%
(v/v) EDMA, 5% (v/v) MAA, 5% (v/v) DAMA, 10% (v/v) 1-propanol,
40% (v/v) 1,4-butanediol, and 0.3% (w/t) AIBN with respect to the
monomers at different reaction temperature for 2 h.
EOF of the Zwitterionic Monolithic CEC Column. In
contrast to silica-based media for CEC, the ability to easily control
the level of charged functionality for generation of the electroosmotic flow is a major advantage of directly produced monolithic
capillaries.25,26 The surface charge density in monolithic capillary
columns can be easily controlled simply by changing the percentage of charged monomers in the polymerization mixture.22,23 CEC
with monolithic columns bearing positively charged groups or
negatively charged groups generally generate the anodic EOF or
cathodic EOF. However, the poly(BMA-co-EDMA-co-MAA-coDAMA) columns with tertiary amine groups or with acrylic acid
groups resulted in a weak anion or cation exchanger and,
therefore, the generation of anodic or cathodic EOF, which can
be easily controlled via the pH value of the mobile phase. Figure
3 shows the effect of pH on EOF on the zwitterionic monolithic
column, where negative values of EOF denote cathodic EOF,
whereas positive values denote anodic EOF. Anodic EOF of 2.47
× 10-8 m2 V-1 s-1 was obtained using the mobile phase at pH 2.0
because the tertiary amine groups on the surface of the stationary
phase were ionized and the acrylic acid groups were suppressed
under such an acidic condition. With the increase of pH values of
the mobile phases, the anodic EOF decreased as the ionization
of tertiary amine groups became weaker and the ionization of
acrylic acid became stronger. As the pH reached 6.2, both the
positively charged groups and the negatively charged groups
remained equal, and the net charge of the stationary phase was
zero. Therefore, no EOF was generated. However, as the pH
increased further, the direction of EOF changed, and cathodic
EOF was obtained. The cathodic EOF increased with the increasing pH values of the mobile phases because the ionization of
acrylic acid groups became stronger and the ionization of tertiary
amine groups was suppressed. The cathodic EOF reached its
maximum of 1.99 × 10-8 m2 V-1 s-1 at pH 10. Therefore, the EOF
(25) Pursch, M.; Sander, L. C. J. Chromatogr., A 2000, 887, 313-326.
(26) Colón, L. A.; Maloney, T. D.; Fermier, A. M. J. Chromatogr., A 2000, 887,
43-53.
Figure 3. Effect of the pH values on the electroosmotic mobility of
the zwitterionic monolith. Experiment conditions: capillary column with
effective length of 8.5 cm (total length 32 cm) × 100 µm i.d. × 375
µm o.d. was prepared with a polymerization mixture containing 200
µL of BMA, 200 µL of EDMA, 50 µL of MAA, 50 µL of DAMA, 300 µL
of 1-propanol, 300 µL of 1,4-butanediol, and 0.15 mg of AIBN at 60
°C for 2 h; mobile phases, 40% (v/v) acetonitrile in 10 mM phosphate
buffer with various pHs; applied voltage, 10 kV or -10 kV; injection,
5 kV × 5 s. Thiourea was chosen as the marker molecule of EOF.
Negative values of EOF denote cathodic EOF, whereas positive
values denote anodic EOF.
of the zwitterionic stationary phase is pH-dependent and can be
controlled easily by adjusting the pH values of the mobile phases.
In addition, the monolithic capillary columns with different
amounts of charged groups were prepared according to the
polymerization mixtures listed in Table 1 at 60 °C for 2 h. The
Effect of DAMA content in the polymerization mixture on the
inflection points of the EOF was investigated by changing the ratio
of DAMA to MAA (keeping V(DAMA+MAA)% at 10% in the polymerization mixture), using the mobile phase containing 40% acetonitrile in 10 mM phosphate buffer with various pH values. The EOFs
of the zwitterionic capillary columns, reacted with volumne ratios
of DAMA to MAA at 3/1, 2/2, and 1/3, changed their directions
using the mobile phase with pH values of 5.3, 6.2, and 6.8.
Therefore, as the DAMA content in the polymerization mixture
increased, the inflection points of the zwitterionic columns
decreased. However, the inflection points of the zwitterionic
columns are hard to be predicted just by the pKs of DAMA and
MAA and the content of both ionic monomers in the polymerization mixture because the monomers may not have the same
activity in the polymerization reaction.
The effect of acetonitrile concentration on the EOF was
investigated by keeping the phosphate concentration at 10 mM
and the pH 2.0 and 9.0, respectively. As shown in Figure 4, the
EOF slightly increased with increasing acetonitrile concentration
from 30 to 60% (v/v) under both acidic and basic eluent conditions,
which was consistent with the trend reported by some authors.27,28
However, contrary results were also reported by Yamamoto et
al.29 and Jin et al.,30 which was attributed to a decrease in the
(27) Rebscher, H.; Pyell, U. Chromatographia 1994, 38, 737-743.
(28) Dittmarm, M. M.; Rozing, G. P. J. Chromatogr., A 1996, 744, 63-74.
(29) Yamamoto, H.; Baumann, J.; Erni, F. J. Chromatogr. 1992, 593, 313-319.
(30) Jin, W. H.; Fu, H. J.; Huang, X. D.; Xiao, H.; Zou, H. F. Electrophoresis 2003,
24, 3172-3180.
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
4869
Figure 4. Influence of acetonitrile concentration on the electroosmotic mobility (µeof). Experimental conditions: mobile phases, 10 mM
phosphate buffer at pH 2.0 and pH 9.0 with various concentration of
acetonitrile. Other conditions are the same as in Figure 2. Thiourea
was chosen as the marker molecule of EOF. Negative values of EOF
denote cathodic EOF, whereas positive values denote anodic EOF.
Table 2. Effect of Phosphate Concentration on
Electroosmotic Mobility (µeof)a
electroosmotic mobility
(µeof, 10-8 m-2 V-1 s-1)
phosphate concn (mM)
pH 2.0
pH 9.0
5
10
25
50
-2.47
-2.40
-2.36
-2.29
1.80
1.77
1.63
1.59
a Experimental conditions: mobile phases, 40% (v/v) acetonitrile in
various concentration of buffers. Other conditions are the same as in
Figure 2.
dielectric constant and the magnitude of the ζ-potential with
increasing acetonitrile concentration in mobile phases. Effect of
the phosphate concentration in the mobile phase on the EOF
was also investigated by keeping the acetonitrile content at 40%
(v/v), and the results are shown in Table 2. The anodic EOF
decreased slightly from 2.47 to 2.29 × 10-8 m-2 V-1 s-1 when the
phosphate concentration in the mobile phase increased from 5 to
50 mM at pH 2.0, whereas the cathodic EOF decreased from 1.80
to 1.59 × 10-8 m-2 V-1 s-1 with a phosphate concentration increase
from 5 to 50 mM at pH 9.0. The bubble formation was avoided by
applying a pressure of 5 bar at both outlet and inlet even though
the mobile phase containing 50 mM phosphate buffer was used.
Evaluation of Column Performance. A zwitterionic column
was evaluated for CEC separation performance using the mobile
phase containing 60% (v/v) acetonitrile in 10 mM phosphate at
pH 2.0 and 8.0, respectively. To test the ability of the monolithic
columns to dissipate excessive Joule heating, the current was
measured in function of the applied voltage by keeping a
phosphate concentration of 10 mM. In this work, it was observed
that the current linearly increased from 1.3 to 37 µA by increasing
the applied voltage from 1 to 30 kV with linear regression
coefficients of 0.9997 and 0.9996 at pH 2.0 and pH 8.0, respectively,
which indicated that excessive heat generation thus does not seem
4870 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
Figure 5. Plot of plate height (HEPT) of retained benzyl alcohol
versus the linear velocity. Experimental conditions: mobile phase,
10 mM phosphate buffer containing 40% (v/v) acetonitrile, pH 2.0
and 8.0; applied voltages from 5 to 25 kV. Other conditions are the
same as in Figure 2.
to be a cause of concern. Run-to-run, column-to-column, as well
as batch-to-batch reproducibility was investigated in terms of t0
and the retention factor (k*) of aromatic compounds, including
benzene, toluene, ethyl benzene, and propyl benzene, using the
mobile phase containing 40% (v/v) acetonitrile in 10 mM phosphate buffer at pH 2.0 and 8.0. It was observed that reproducibility
of EOF for the batcht-to-batch preparation of columns had a RSD
smaller than 3.9% (n ) 5) and that the reproducibility of the k*
values for different solutes based on the run-to-run injections,
column-to-column preparation, and batch-to-batch preparation of
columns was smaller than 0.56% RSD (n ) 10), 3.9% (n ) 5), 8.1%
(n ) 5), respectively. These results indicated that the reproducibility of the prepared monolithic columns is acceptable. The
column efficiencies of thiourea and alkylbenzenes varying between
79 000 and 220 000 plates/m were obtained using the mobile
phases containing 60% (v/v) acetonitrile in 10 mM phosphate at
pH 2.0 and 8.0, respectively.
The relationship between the theoretical plate height (HEPT)
and the velocity of the mobile phase for benzyl alcohol was
studied, and the obtained result is shown in Figure 5. It was found
that even when the velocity increased to 1.1 mm/s, no apparent
loss of column efficiency was observed, which would be one of
the typical behaviors for the monolithic stationary phases.
Separation of Analytes on Monolithic Column. The prepared zwitterionic monolithic columns were applied for the
separation of neutral compounds using acidic and basic electrolyte
solutions, respectively. Figure 6 shows the typical electrochromatograms for separation of alkyl benzenes on the poly(BMA-coEDMA-co-MAA-co-DAMA) monolithic columns. The analytes were
eluted in the order of thiourea < benzene < toluene < ethyl
benzene < propyl benzene < butyl benzene according to their
hydrophobicities using a mobile phase with pH values at 2.0 and
8.0, and it can be deduced that the separation of alkylbenenes on
the poly(BMA-co-EDMA-co-MAA-co-DAMA) column is mainly
based on the reversed-phase mechanism, although the stationary
phase generated EOF with different directions under different pH
eluent conditions. A mixture of polycyclic aromatic hydrocarbons
(PAHs) was separated on the column, and 5 PAHs were baseline-
Figure 6. Separation of alkyl benzenes on poly(BMA-co-EDMA-co-MAA-co-DAMA) monolithic CEC column. Experimental conditions: mobile
phases, 10 mM phosphate buffer at pH (a) 2.0 and (b) 8.0 containing 60% (v/v) acetonitrile. Other conditions are the same as in Figure 2.
Solutes: (1) thiourea, (2) benzene, (3) toluene, (4) ethyl benzene, (5) propyl benzene, and (6) butyl benzene.
Figure 7. Separation of polycyclic aromatic hydrocarbons on poly(BMA-co-EDMA-co-MAA-co-DAMA) monolithic CEC column. Experimental conditions: mobile phase, 40% (v/v) acetonitrile in 10 mM
phosphate buffer at pH 7.0; applied voltage, 20 kV. Other conditions
are the same as in Figure 2. Solutes: (1) benzene, (2) indene, (3)
naphthalene, (4) acenaphthylene, and (5) anthracene.
separated within 4 min, as the obtained electrochromatogram
shown in Figure 7. Similar to alkylbenzenes, PAHs were eluted
according to their hydrophobicities. It was confirmed that the
hydrophobic interaction is responsible for the retention of the
neutral compounds on the zwitterionic monolithic column.
Phenols compose a large category of environment pollutants.
The separation of phenols was investigated on the zwitterionic
monolith in the CEC mode, and typical electrochromatograms are
shown in Figure 8. It was observed that the elution order of
phenols on the zwitterionic column at pH 2.0 basically agree with
the hydrophobicities of the phenols with the exception of the last
eluted compound of m-nitrophenol, which has moderate hydrophobicity. The strong retention of m-nitrophenol on the zwitterionic column can be explained by the electrostatic interaction
between the analyte and the positively charged stationary phase
at pH 2.0. However, the elution order of phenols became phenol
< p-cresol < m-nitrophenol < 2,3-dimethylphenol at pH 7.0, which
is according to their hydrophobicity. Because the electrostatic
interaction between analytes and stationary phase lost importance
when the ionization of tertiary amine groups was partially
suppressed, therefore, as noted by Hilder et al.,31 in addition to
(31) Hilder, E. F.; Svec, F.; Frechet, J. M. J. Electrophoresis 2002, 23, 39343953.
the ability of ionizable surface functionalities to control the EOF,
changes in surface chemistry can also be used to control both
the nature and the strength of the chromatographic interactions.
Different selectivities of phenols can be obtained on the zwitterionic stationary phase in CEC mode using a mobile phase with
different pH values, which leads to both a different EOF and a
different surface chemistry of the stationary phase.
As mentioned above, CEC has been a useful tool for the
separation of neutral compounds. However, the separation of basic
compounds is still problematic for this technique because of the
peak tailing of basic analytes on conventional silica-based CEC
column. Many approaches, such as adding competing bases to
the mobile phase32,33 and separation in “counterdirectional mode”34
and “ion-suppressed mode”30 of CEC, have been undertaken to
overcome these problems. The separation of anilines was also
investigated in the CEC mode on the zwitterionic stationary phase.
Figure 9 shows the effect of acetonitrile concentration on the
retention factors of anilines. As can be seen, the retention factors
of anilines decreased with the increasing concentration of acetonitrile in the mobile phase at pH 2.0 and 8.0. It is induced that
the hydrophobic interaction between the anilines and the stationary phase should be one of the key factors to contribute to the
retention of anilines, no matter what pH values of the mobile
phases were applied. Figure 10 shows the typical electrochromatograms of anilines on the poly(BMA-co-EDMA-co-MAA-coDAMA) monolithic CEC column. At pH 2.0, both anilines and the
surface of zwitterionic stationary phase are positively charged, and
the separation of anilines on the column was performed under
“counterdirectional mode”, an approach suggested by Hjetérn and
co-workers,34 and the electrostatic adsorption between the analytes
and the stationary phase was avoided. However, the surface of
the stationary phase was negatively charged at pH 8.0, the anilines
were positively charged or neutral depending their pKa values,
and electrostatic interaction between the anilines and the stationary phase occurred in addition to the hydrophobic interaction.
As a result, no serious peak tailing of the anilines on the
zwitterionic stationary phase was observed with both acidic and
basic electrolyte solutions. As also can be seen in Figures 9 and
(32) Euerby, M. R.; Johnson, C. M.; Bartle, K. D. Anal. Commun. 1996, 33,
403-405.
(33) Lurie, I. S.; Meyers, R. P.; Conver, T. S. Anal. Chem. 1998, 70, 32553260.
(34) Ericson, C.; Hjertén, S. Anal. Chem. 1999, 71, 1621-1627.
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
4871
Figure 8. Separation of phenols on poly(BMA-co-EDMA-co-MAA-co-DAMA) monolithic CEC column. Experimental conditions: mobile phase,
40% (v/v) acetonitrile in 10 mM phosphate buffer at pH (a) 2.0 and (b) 7.0; applied voltage, 20 kV. Other conditions are the same as in Figure
2. Solutes: (1) hydroquinone, (2) phenol, (3) p-cresol, (4) m-nitrophenol, and (5) 2,3-dimethylphenol.
Figure 9. Effect of acetonitrile concentration on the retention factors of anilines. Experimental conditions: mobile phase, 10 mM phosphate
buffer containing various concentration of acetonitrile at pH (a) 2.0 and (b) 8.0; applied voltage, 20 kV. Other conditions are the same as in
Figure 2. Solutes: (1) 4,4′-metholenedianiline, (2) 3,3′-dimethoxybenzidine, (3) 4,4′-diaminobiphenyl, (4) 4-aminobiphenyl, (5) 2,4-dinitroaniline,
(6) 2,6-dichloro-4-nitroaniline, and (7) 1,2-diphenylhydrazine.
Figure 10. Electrochromatograms for the separation of anilines on the poly(BMA-co-EDMA-co-MAA-co-DAMA) monolithic CEC column.
Experimental conditions: mobile phase, 10 mM phoshate buffer at pH (a) 2.0 and (b) 8.0 containing 40% (v/v) acetonitrile; applied voltage, 20
kV. Other conditions are the same as in Figure 8.
4872 Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
Figure 11. Electrochromatograms for separation of peptides on the poly(BMA-co-EDMA-co-MAA-co-DAMA) monolithic CEC column.
Experimental conditions: mobile phases, 20 mM phosphate buffer at pH (a) 2.0 and (b) 8.0 containing 20% (v/v) acetonitrile; applied voltage,
10 kV. (c) Stepwise pH gradient by balancing the CEC column first with mobile phase at pH 2.8, then separating the peptides with mobile phase
at pH 8.0. Other conditions are the same as in Figure 2. Solutes: (1) Gly-Glu, (2) Ala-Ile, (3) Gly-Phe, (4) Gly-His, (5) His-Phe, (6) Lys-Ser-Try,
and (7)Gly-Asp.
10, different separation selectivities of anilines were obtained with
pH values of the mobile phases at 2.0 and 8.0, respectively.
Figure 11 shows typical electrochromatograms of peptides on
zwitterionic monolithic CEC at pH 2.0 and 8.0. Similar to anilines,
the peptides were separated on the poly(BMA-co-EDMA-co-MAAco-DAMA) column using the “counterdirectional mode” under
acidic conditions for both the peptides and stationary phase
bearing positive charges. The ionization of acrylic acid groups
on the zwitterionic stationary phase will result in a weak cation
exchanger under basic mobile phase, and the electrostatic
interaction of the basic peptides and the stationary phase is not
so strong as the basic peptides and the strong cation exchanger;
therefore, no serious peak tailing of basic peptides was observed.
Similar to anilines, different separation selectivities of peptides
were obtained on the zwitterionic stationary phase under the acidic
and basic mobile phases. The separation of peptides on the
zwitterionic columns was also carried out by using a stepwise pH
gradient by balancing the CEC column first with mobile phase at
pH 2.8, then separating the peptides with mobile phase at pH 8.0.
Peptides were eluted with high column efficiency over 800 000
N/m when they were separated individually, as shown in Figure
11c. However, separation of the peptide mixture could not be
obtained. The abnormal high column efficiency of peptides may
be caused by the stacking effect. Furthermore, we are developing
Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
4873
a method for the separation of peptides with a pH gradient from
inlet acidic buffer to outlet basic buffer.
CONCLUSION
Monolithic columns with a zwitterionic stationary phase for
capillary electrochromatography within the confined capillary
columns were prepared by the copolymerization of butyl methacrylate, ethylene dimethacrylate, methacrylic acid, and 2-(dimethyl amino) ethyl methacrylate in the presence of porogens,
and the tertiary amine and acrylic acid groups coexisted on the
surface of the zwitterionic stationary phase. The ionization of those
groups was affected by the pH values of the mobile phases;
therefore, they affect the direction and strength of the EOF. The
monolithic CEC columns generated anodic EOF under acidic
eluent and cathodic EOF under basic eluent. Neutral compounds,
such as alkylbenzenes and PAHs, retained on the stationary
phases mainly on the basis of the reversed-phase mechanism. CEC
separations of ionizable phenols, aniline, and peptides were successfully achieved on the zwitterionic monolithic column. In
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Analytical Chemistry, Vol. 76, No. 16, August 15, 2004
addition to hydrophobic interaction, the electrostatic interaction
between the ionic or ionizable analytes and the stationary phase
plays an important role on the rentention behavior of the analytes.
Different separation selectivities of phenols, anilines, and peptides
were observed on the zwitterionic stationary phase under the basic
and acidic mobile phases.
ACKNOWLEDGMENT
Financial supports from the China State Key Basic Research
Program Grants (001CB510202 and 2003CB716002), the China
State High-Tech Program Grant (2003AA233061), and the Knowledge Innovation Program of DICP to H.Z. is gratefully acknowledged.
Received for review March 2, 2004. Accepted May 20,
2004.
AC0496695