Analytical separations using molecular micelles in open-tubular capillary electrochromatography.

Abstract

Open-tubular capillary electrochromatography (OT-CEC) is an alternative approach to conventional CEC. The primary advantage of OT-CEC is the elimination of problems associated with frits and silica particles in conventional CEC. This report is an investigation of the utility of using a polymeric surfactant (molecular micelle) for OT-CEC. In this approach, fused-silica capillaries coated with thin films of physically adsorbed charged polymers are developed by use of a polyelectrolyte multilayer (PEM) coating procedure. The PEM coating is constructed in situ by alternating rinses with positively and negatively charged polymers, where the negatively charged polymer is a molecular micelle. This can offer a number of advantages for separation of hydrophobic analytes. In this study, poly(diallyldimethylammonium chloride) was used as the cationic polymer and poly(sodium N-undecanoyl-L-glycinate) was used as the anionic polymer for PEM coating. The performance of the modified capillaries as a separation medium is evaluated by use of seven benzodiazepines as analytes. The run-to-run, day-to-day, week-to-week, and capillary-to-capillary reproducibilities of electroosmotic flow are very good with relative standard deviation values of less than 1% in all cases. In addition, the chromatographic performance of the monomeric form of the molecular micelle is compared for the separation of these analytes. The PEM-coated capillary was remarkably robust with more than 200 runs accomplished in this study. Strong stability against extreme pH values was also observed. The general utility of this approach is discussed in detail.

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Analytic al S eparations Using Molec ular Mic elles in
Open-T ubular Capillary Elec troc hromatography
Constantina P. K apnissi,²Cevdet Akbay,²J oseph B. Schlenoff,³and Isiah M. Warner*,²
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, and Department of Chemistry and
Center for Materials Research and Technology (MARTECH), Florida State University, Tallahassee, Florida 32306-4390
Open-tubular capillary electrochromatography (OT-CEC)
is an alternative approach to conventional CEC. The
primary advantage of OT-CEC is the elimination of prob-
lems associated with frits and silica particles in conven-
tional CEC. This report is an investigation of the utility of
using a polymeric surfactant (molecular micelle) for OT-
CEC. In this approach, fused-silica capillaries coated with
thin films of physically adsorbed charged polymers are
developed by use of a polyelectrolyte multilayer (PEM)
coating procedure. The PEM coating is constructed in situ
by alternating rinses with positively and negatively charged
polymers, where the negatively charged polymer is a
molecular micelle. This can offer a number of advantages
for separation ofhydrophobic analytes. In this study, poly-
(diallyldimethylammonium chloride) was used as the
cationic polymer and poly(sodium N-undecanoyl-L-glyci-
nate) was used as the anionic polymer for PEM coating.
The performance of the modified capillaries as a separa-
tion medium is evaluated by use ofseven benzodiazepines
as analytes. The run-to-run, day-to-day, week-to-week, and
capillary-to-capillary reproducibilities of electroosmotic
flow are very good with relative standard deviation values
of less than 1% in all cases. In addition, the chromato-
graphic performance of the monomeric form of the mo-
lecular micelle is compared for the separation of these
analytes. The PEM-coated capillary was remarkably ro-
bust with more than 200 runs accomplished in this study.
Strong stability against extreme pH values was also
observed. The general utility ofthis approach is discussed
in detail.
Capillary electrochromatography (CEC) is a hybrid elec-
troseparation technique that couples the selectivity of high-
performance liquid chromatography (HPLC) and the separation
efficiency of capillary electrophoresis (CE).1-5Such studies have
demonstrated that CEC provides high resolution, short analysis
time, smaller sample and buffer consumption, and efficiencies
5-10 times higher than HPLC. The separation in CEC is based
upon the electrophoretic mobility of the solutes and their partition-
ing between the stationary and mobile phases.
In the development of CEC, both packed and open-tubular
column configurations have been reported.1-10The conventional
form of CEC uses a fused-silica capillary with a typical internal
diameter of 50-100 µm, packed with a typical HPLC stationary
phase, such as an octadecyl silica (ODS) stationary phase.2,4
However, there are several problems that need to be solved in
order for packed-CEC to be a viable alternative to either CE or
HPLC. Oneofthelimitations ofconventional CEC is thenecessity
to fabricate frits, which are required for retention of the packed
particles within the column. In addition, packed capillaries have
the tendency to form bubbles around the packing material or at
the frit. Such problems often result in an unstable baseline,
nonreproducible migration times, or even current breakdown. To
circumvent this potential problem, pressurization of both ends of
the column is required and the solvent must be thoroughly
degassed. Another common problemof conventional CEC is that
the packing procedure is more difficult than for HPLC because
of the narrow inner diameter of the capillary and the small
diameter of the particles. Finally, the separation of basic com-
pounds with packed-CEC can be difficult due to dissociation of
the silanol groups, which is needed to generate an adequate
electroosmotic flow (EOF).
Open-tubular CEC (OT-CEC) is an alternative approach to
packed-CEC.9None of the problems mentioned above are likely
to be encountered in an open-tubular format. In this CEC format,
astable coating needs to be constructed on the inner walls of the
capillary inorder toprovideefficient chromatographic separations
and reproducible EOF.10The most commonly used approaches
to wall coatings for modifying the capillary include the following:
(1) dynamic coating performed by adding the cationic or neutral
modifier to the electrolytes;11,12(2) adsorbed cationic modifier on
the capillary wall by physical adsorption;13-16and (3) fixation of
* Corresponding author. Tel: (225) 578-3945. Fax: (225) 578-3971. E-mail:
iwarner@lsu.edu.
²Louisiana State University.
³Florida State University.
(1) Liu, Z.; Zou, H.; Ye, M.; Ni, J.; Zhang, Y. Electrophoresis 1999, 20, 2891.
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10.1021/ac015733w CCC: $22.00© 2002 American Chemical Society
Published on Web 04/20/2002

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the hydrophilic layer by covalent bonding or cross-linking.17-22
Harrell et al. achieved a baseline separation of seven tricyclic
antidepressants by use of anovel nonionic micelle polymer, poly-
(n-undecyl R-D-glucopyranoside) (PUG) as a dynamic coating.23
However, dynamic coating is known to cause problems when CE
is coupled to mass spectrometry (CE/MS). In addition, the
presence of the nonvolatile buffer constituents may deteriorate
the ionization of the analytes.24,25Although physical adsorption
has asimpleandrapidcoating procedureandgoodreproducibility,
ithas beenshowntohaveashortlifetimeandlimitedpH range.24,26
In contrast, some of the covalent bonding or cross-linking have a
long lifetime, but require a more complicated coating proce-
dure.24,26Obviously, an ideal coating procedure would be one that
is both simple and stable.
(15) Preisler, J.; Yeung, E. S. Anal. Chem. 1996, 68, 2885.
(16) Cordova, E.; Gao, J.; Whitesides, G. M. Anal. Chem. 1997, 69, 1370.
(17) Towns, J. K.; Regnier, F. E. J. Chromatogr. 1990, 516, 69.
(18) Figeys, D.; Aebersold, R. J. Chromatogr., B 1997, 695, 163.
(19) Bruin, G. J. M.; Chang, J. P.; Kuhlman, R. H.; Zegers, K.; Kraak, J. C.; Poppe,
H. J. Chromatogr. 1989, 471, 429.
(20) Hjerten, S.; Johansson, M. K. J. Chromatogr. 1991, 550, 811.
(21) Cobb, K. A.; Dolnik, V.; Novotony, M. Anal. Chem. 1990, 62, 2478.
(22) Huang, X.; Horvath, C. J. Chromatogr., A 1997, 788, 155.
(23) Harrell, C. W.; Dey, J.; Shamsi, S. A.; Foley, J. P.; Warner, I. M. Electrophoresis
1998, 19, 712.
(24) Katayama, H.; Ishihama, Y.; Asakawa, N. Anal. Chem. 1998, 70, 5272.
(25) Niessen, W. M. A.; Tjaden, U. R.; Geef, J. J. Chromatogr. 1993, 636, 3.
(26) Katayama, H.; Ishihama, Y.; Asakawa, N. Anal. Chem. 1998, 70, 2254.
Figure 1. Structures of the seven benzodiazepine analytes.
Figure 2. Structures of the (a) monomeric SUG and (b) polymeric SUG.
Analytical Chemistry, Vol. 74, No. 10, May 15, 2002
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In this report, we explore an alternative to covalent linking of
a polymer to silica beads. In our approach, we use polymeric
surfactants (molecular micelles) in a simple coating procedure,
which involves layer-by-layer deposition process.27,28The coating
is thus apolyelectrolyte multilayer (PEM), constructed in situ by
alternating rinses ofpositively andnegatively chargedpolymers.29-33
Via electrostatic forces, a layer of polymer adds to the oppositely
charged surface, reversing the surface charge and priming the
filmfor theadditionofthenextlayer. Themechanismofformation
andchargebalanceinPEMs has beenexplored.33-37Such coatings
have been found to be robust and thus highly resistant to charge
and deterioration during use.24,26,32The advantages of our PEM
coating are twofold. First, since the polymeric surfactant is coated
electrostatically ontothecapillary, less consumptionofthereagent
is required. Second, with the molecular micelle coated on the
capillary, there is less detection interference with the analyte of
interest, which in turn makes the system more amenable to
coupling with mass spectrometry or other detectors where the
polymeric surfactant reagent interferes.
We describe here our PEM coating approach for fabricating
open-tubular columns for use in OT-CEC. The performance ofthe
modified capillaries as a separation medium is evaluated by use
of seven benzodiazepines as analytes. The coating was found to
be remarkably stable with excellent performance for more than
200 runs.
EX PERIMENTAL SECTION
Apparatus and Conditions. Separations were performed on
a Beckman P/ACE MDQ capillary electrophoresis system with
UV detection (Fullerton, CA). The fused-silica capillary, 57 cm
(50-cm effective length) × 50 µm i.d., was purchased from
Polymicro Technologies (Phoenix, AZ) and mounted in a Beck-
man capillary cartridge. Unless stated otherwise, the cartridge
temperature was maintained at 25 °C by use of liquid coolant.
UV detection was performed at 214 nm, and the samples were
injected by pressure (0.1 psi; 1 psi ) 6894.76 Pa) for 1 s.
Reagents and Chemicals. Flunitrazepam, temazepam, diaz-
epam, oxazepam, lorazepam, clonazepam, and nitrazepam were
purchased from Sigma Chemical Co. (St, Louis, MO). The
structures of the analytes used in this study are shown in Figure
1. Sodiumphosphate(Na2HPO4andNaH2PO4), hydrochloric acid,
and sodiumchloridewereall obtained fromFisher Scientific (Fair
Lawn, NJ). Poly(diallyldimethylammoniumchloride) (PDADMAC;
Mw)200 000-350 000) was obtained from Aldrich (Milwankee,
WI). Other chemicals, including L-glycine, undecylenic acid, and
N-hydroxysuccinimide, were also purchased from Sigma.
Sample and Buffer Preparation. Analytical standard ben-
zodiazepine stock solutions were prepared in methanol-water (1:
1) at concentrations of ∼0.15 mg/mL each. A buffer solution of
50 mM Na2HPO4 was prepared by dissolving the appropriate
amount of Na2HPO4in 10 mL of deionized water. The solution
was filtered using a polypropylene nylon filter with 0.45-µm pore
size and sonicated for 15 min before use.
Synthesis of Monomeric and Polymeric Surfactant. The
surfactant monomer of sodium N-undecylenyl-L-glycinate (mono-
(L-SUG)) was synthesized from the N-hydroxysuccinimide ester
ofundecylenic acidaccording toapreviously reportedprocedure.38
A 100 mM sodium salt solution of the monomer was then
polymerized by use of60Co γ radiation. After irradiation, the
polymer was dialyzed by use of a2000molecular mass cutoff and
then lyophilized to obtain the dry product. Structures of the
monomeric and polymeric surfactant are illustrated in Figure 2.
Procedure for Polyelectrolyte Multilayer Coating. PEM
coating was achievedby depositionofthepolymer solutions using
the rinse function on the Beckman CE system. Each polymer
deposition solution contained 0.5% (w/v) polymer in 0.2 M
aqueous NaCl solution. It was observed that the addition of NaCl
to the polymer solution resulted in enhanced thickness for each
polyelectrolyte layer.32The capillary was conditioned before
coating using a5-min rinse of water, toremove any contaminants
originating fromthecapillary manufacturing process. Thecolumn
(27) Decher, G.; Schmitt, J. Prog. Colloid Polym. Sci. 1992, 89, 160.
(28) Decher, G. Science 1997, 277, 1232.
(29) Schlenoff, J. B.; Ly, H.; Li, M. J. Am. Chem. Soc. 1998, 120, 7626.
(30) Laurent, D.; Schlenoff, J. B. Langmuir 1997, 13, 1552.
(31) Schlenoff, J. B.; Li, M. Ber. Bunsen-Ges. Phys. Chem. 1996, 100, 943.
(32) Graul, T. W.; Schlenoff, J. B. Anal. Chem. 1999, 71, 4007.
(33) Schlenoff, J. B.; Dubas, S. T.; Farhat, T. Langmuir 2000, 16, 9968.
(34) Laurent, D.; Schlenoff, J. B. Langmuir 1997, 13, 1552.
(35) Dubas, S. T.; Schlenoff, J. B. Macromolecules 1999, 32, 8153.
(36) Schlenoff, J. B.; Dubas, S. T. Macromolecules 2001, 34, 592.
(37) Dubas, S. T.; Schlenoff, J. B. Macromolecules 2001, 34, 3736.
(38) Wang, J.; Warner, I. M. Anal. Chem. 1994, 66, 3773.
Figure 3. Scheme of the PEM-coated capillary.
2330
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was then conditioned by rinsing with 1M NaOH for 60min. Pure
deionizedwater was flushedthrough thecapillary for 15minmore.
The first monolayer of polymer (PDADMAC) was deposited by
rinsing the solution of the cationic polymer through the capillary
for 20 min followed by a 5-min water rinse. All other polymer
depositions were done with 5-min rinses followed by 5-min water
rinses. All processes were performed by continuous dynamic
rinsing of the reagents through the capillary. A diagrammatic
scheme of the PEM-coated capillary is shown in Figure 3. This
diagramis not provided togiveanactual structural representation
of the bilayer, but only to represent the order of polymer
deposition. Current studies areongoing inour laboratory tobetter
understand the structure of the bilayers. The multilayer coatings
usedfor theseparationofbenzodiazepines andthereproducibility
studies consistedof10layer pairs (alayer pair is alayer ofcationic
polymer plus a layer of anionic polymer; also termed a bilayer).
The capillary was then flushed with buffer until a stable current
was achieved. The columns were conditioned with buffer for 2
min between injections.
The thickness of the coating fabricated on asilicon wafer with
arobotic platformwas determinedinoneofour laboratories using
aGaertner Scientific L116B Autogain ellipsometer. The thickness
of 10 bilayers on the wafer surface was ∼1200 Å. Typical studies
on the determination of the thickness of apoly(styrenesulfonate)
Figure 4. Stability studies of PEM coating. Conditions: 0.5% (w/v) PDADMAC and 0.5% (w/v) poly(L-SUG) with 0.2 M NaCl; pressure injection,
0.1 psi for 1 s; electrolyte, 50 mM Na2HPO4(pH 9.2); applied voltage, 20 kV; temperature, 25 °C; capillary, 57 cm (50 cm effective length) ×
50 µm i.d.; detection, 214 nm.
T able 1. Reproduc ibilities of PEM Capillary Coatinga
EOF average
(min)
RSD
(% )
run to run (n ) 50)b
day to day (n ) 5)b
week to week (n ) 3)b
capillary to capillary (n ) 25)c
9.990
9.982
9.930
9.960
0.78
0.81
0.86
0.98
an, number of runs. Conditions: same as Figure 4.bThese studies
were done in the same capillary.cThis study was done in five different
capillaries. Five consecutive runs were performed in each capillary.
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(PSS)/PDADMAC multiplayer have already been done by oth-
ers.35The thickness ofa10-bilayer filmdeposited on silicon wafer
from 0.2 M NaCl was ∼500 Å.35
RESULTS AND DISCUSSION
Column Performance. (1) Measurement of EOF. In CEC,
the transport of mobile phase through the capillary is achieved
by the EOF, which is generated due tothe electrical double layer
thatexists atthesolid-liquidinterfaceofthechargedsilicasurface
in contact with an electrolyte solution.39The EOF, µeo, is defined
as
where Ldis the distance from injector to detector, Ltis the total
capillary length, tois themigrationtimeoftheelectroosmotic flow
marker, and V is the applied voltage. The relative EOF can be
monitored by use of the values of tosince all other factors should
be constant. In the studies reported here, the values of towere
measured by use ofmethanol, which is expected tobe unretained
by the OT-CEC column. Electroosmotic mobility (µeo) and migra-
tion time of methanol (to) were also used to evaluate the stability
of the PEM coating.
The retention mechanism of benzodiazepines on this PEM-
coated phase is based on the differential partitioning of the
analytes into the coating. Their retention is determined by
hydrophobic interactions between the hydrophobic core of the
polymer and the nonpolar moiety of each analyte. Therefore, the
migration order of the benzodiazepines was tR1< tR2< tR3< tR4
< tR5< tR6< tR7(the superscripted numbers are the number
designation for the analytes in Figure 1).
(2) Endurance of PEM Coating. An important aspect of this
approach, which must be considered, is the lifetime of the
stationary phase. Thus, we examined the stability of our coating
(39) Dittmann, M. M.; Rozing, G. P. J. Chromatogr., A 1996, 744, 63.
Figure 5. Effect of applied voltage on the OT-CEC separation of benzodiazepines. Conditions: same as Figure 4, except applied voltage was
varied.
µeo) LdLt/Vto,
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