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Journal of Separation Science
Communication
A cationic -cyclodextrin as a dynamic coating for the separation of proteins in
capillary electrophoresis
Joselito P. Quirino
Australian Centre for Research on Separation Science (ACROSS), School of Physical
Sciences-Chemistry, University of Tasmania, Australia 7001
jquirino@utas.edu.au
running title:
Cationic cyclodextrin as a dynamic coating in CE
Abbreviations: CD, cyclodextrin; CTAB, cetyltrimethylammonium bromide; Q--CD,
quaternary -cyclodextrin; TMA--CD, 2-hydroxypropyltrimethylammonium -cyclodextrin
keywords:
capillary electrophoresis, cationic cyclodextrins, dynamic coating, neutral surfaces, proteins
Abstract
A cationic cyclodextrin was used as dynamic coating for the capillary electrophoresis
of a model mixture of proteins (i.e., ubiquitin, -lactoglobulin, cytochrome-c, and
myoglobin) as positively charged species in a fused silica capillary. An interesting feature of
the coating is that by simple adjustment of the concentration of cyclodextrin added into the
background electrolyte, a neutral or positively charged surface, which was beneficial in
Received: 06 03, 2017; Revised: 10 10, 2017; Accepted: 10 11, 2017
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1002/jssc.201700610 .
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Journal of Separation Science
preventing protein adsorption at the inner capillary wall surface, was obtained. This is the
first demonstration of a dynamic coating that yielded a neutral surface for protein separations
in capillary electrophoresis. Based on electro-osmotic flow measurements, addition of 0.05 to
0.10 mg/mL quaternary -cyclodextrin in a low pH electrolyte resulted to a neutral or
positive surface (undetectable to very slow anodic electro-osmotic flow). The coating
approach afforded the electrophoretic separation of the mixture of proteins at positive polarity
with good repeatability and separation performance.
1. Introduction
In the CE of proteins, choosing the proper capillary wall coating is an essential part of
method development [1–8]. The coatings reduce the notorious protein adsorption at the walls
that lead to severe analyte band broadening and fouling of the fused-silica capillary, as noted
by recent reviews on the use of CE for protein separations [1, 3, 9–10]. They are also used to
regulate the EOF that can be tuned to impart separations of targeted protein mixtures.
Coatings are often classified as permanent or dynamic coatings [1], and there are also the socalled semi-permanent coatings. In permanent coatings, materials are attached to the inner
walls of the capillary, normally by covalent bonding. Semi-permanent coatings can be
obtained from polyelectrolytes or double-chain ionic surfactants [11, 12]. No additive in the
CE BGE is required to sustain permanent or semi-permanent coatings. In dynamic coatings,
BGE additives are used to continuously coat the walls during the CE separation. Common
additives are long chain cationic surfactants such as cetyltrimethylammonium bromide
(CTAB) [13, 14] that attach to the capillary walls by electrostatic interaction between the
cationic head group of the surfactant and the negatively charged silanol groups of the
capillary. These surfactants aggregate to form a bilayer at the liquid–capillary surface
interface above a critical surface aggregation concentration, leading to a positive capillary
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Journal of Separation Science
wall surface that reverses the EOF direction (to an anodic EOF). Indeed, positively charged
long chain ionic surfactants have been employed to modify the charge of the capillary wall
for control of EOF and for reduction of protein sticking during CE separation.
Cyclodextrins (CDs) are popularly added into the BGE in CE for enantiomeric separations,
where the CDs act as chiral pseudostationary phases [15–17]. There has been significant
work on the use of cationic CDs as chiral phases in CE [18–22]. Cationic CDs have also been
shown to affect the magnitude and direction of the EOF in a fused-silica capillary. For
example, addition of 2-hydroxypropyltrimethylammonium -CD (TMA--CD) has been
shown to significantly suppress the EOF, where no EOF was observed in a BGE with 1
mg/mL of TMA--CD [18]. This was perhaps due to the formation of a monolayer of TMA-CD, with the neutral side of the TMA--CD in contact with the BGE inside the capillary.
The cationic -CDs were anchored to the capillary wall by electrostatic interaction between
the cationic group of the -CD and the silanol groups at the capillary walls. Interestingly,
similar to the effect of long chain cationic surfactants, the EOF reversed and increased in
magnitude as the concentration of additive was increased. This suggests a positively charged
capillary surface due to the aggregation of cationic CDs [23] at the liquid–capillary wall
interface. In this work, the molecular organization of a cationic -CD (instead of charged
long chain ionic surfactants or polymers) at the liquid–capillary wall interface to form a
positive or neutral capillary wall surface was studied for the first time to allow the CE
separation of model proteins (i.e., ubiquitin, -lactoglobulin, cytochrome-c, and myoglobin)
in a fused-silica capillary.
2. Materials and Methods
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Quaternary -CD (Q--CD) (product number 33805) was obtained from Supelco
(USA). All other reagents (HPLC grade methanol, sodium hydroxide, acetone, ammonium
acetate, and phosphoric acid) were obtained from Sigma–Aldrich (Australia). Ubiquitin, lactoglobulin, cytochrome-c, and myoglobin were also obtained from Sigma–Aldrich
(Australia). Water was purified using a Milli-Q system (USA).
CE experiments were performed with a Beckman MDQ system (USA). The capillary
(50 m id x 365 m od, 60 cm total and 50 cm effective length from Polymicro (USA)) was
conditioned by flushing (1 bar) with purified water (1 min), 0.1 M NaOH (1 min), purified
water (1 min), 50% v/v methanol (1 min) and BGE (5 min) before each injection. New
capillary was conditioned by flushing with 0.1 M NaOH (15 min) and purified water (10
min). The BGEs contained 50 mM ammonium acetate at pH 5 or 75 mM sodium phosphate
at pH 2.5 with different concentrations of Q--CD (0 to 1 mg/mL). 10% v/v acetone in the
BGE was used as EOF marker that was injected at 25 mbar for 6s. The protein mixture in the
BGE was also injected at 25 mbar for 6 s. The applied voltage was +/–20 kV and UV
detection was at 254 and 210 nm for EOF measurements and protein separations,
respectively.
The slow EOF was measured by applying voltage (+ and – 20 kV) for 30 min after
sample injection, followed by mobilization at 1000 mbar to shorten the detection of the EOF
marker. The EOF (if the EOF marker was detected) was calculated based on the distance
travelled by the marker after voltage application, which was obtained by comparison with the
mobilization of the EOF marker but no voltage application. EOF was considered undetected
when the detection time of the EOF marker with or without voltage application was the same.
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3. Results and Discussion
The molecular organization of Q--CD at the interface between the BGE and fusedsilica capillary wall was assessed using EOF measurements. The slow EOF was cathodic
without the Q--CD (1.9 x 10–9 m2/V.s, RSD (n = 3) = 1.0%), and was undetected (at both
voltage polarities) when 0.05 mg/mL Q--CD was added into the BGE. The latter indicated a
somewhat neutral inner wall surface due to the adsorption of a layer of Q--CD, with the
neutral side of the adsorbed Q--CD molecules in contact with the BGE inside the capillary
(see Figure 1). A very slow anodic EOF was then detected when 0.1 mg/mL Q--CD was
added (–2.1 x 10–9 m2/V.s, RSD = 5.2%), and this anodic EOF increased with the addition of
higher concentrations of Q--CD into the BGE. The EOF mobility was –7.5 x 10–9 (RSD =
5.2%), –9.9 x 10–9 (RSD = 4.5%), and –1.2 x 10–8 (RSD = 4.9%) m2/V.s when 0.25, 0.5, and
1.0 mg/mL of Q--CD, respectively was added into the BGE. The results suggested a
positive surface at the capillary walls with BGEs that contained concentrations of Q--CD >
0.1 mg/mL, which was probably due to the aggregation of Q--CD at the interface. The
magnitude of the anodic EOF due to Q--CD was however slower than those typically
obtained with cationic polymers, and this can be easily attributed to the lesser positive charge
at the interface after the molecular organization of the bulky cationic CD.
The dynamic coating of Q--CD was then tested at concentrations from 0.05 to 0.1
mg/mL. Capillary conditioning and sample injection was the same as in the EOF study. There
were no separations obtained with the ammonium acetate at pH 5 buffer. However, as shown
in Figure 2, 75 mM sodium phosphate at pH 2.5 afforded the CE separation of the model
proteins at positive polarity (25 kV). Similar to the results from the pH 5 buffer, there was a
very slow cathodic EOF at Q--CD concentrations below 0.05 mg/mL. The EOF was
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undetected, suggesting the surface of the capillary was neutral at concentrations from 0.05 to
0.075 mg/mL of Q--CD. A slow anodic EOF was detected with 0.1 mg/mL of Q--CD. In
Figure 2, the migration time of each protein was similar when 0.05 and 0.075 mg/mL of Q-CD was added to the acidic phosphate buffer (see Figures 2A and 2B, respectively). The
migration time was longer when the higher concentration of 0.1 mg/mL Q--CD was added
into the buffer (see Figure 2C). This was because the EOF had a velocity directed to the
anode while the electrophoretic velocity of each protein was to the cathode. The separation
was counter-EOF but with a faster electrophoretic velocity of the solutes compared to the
EOF velocity in Figure 2C. The peaks were also sharper with the no-EOF conditions (see
Figures 2A and 2B) compared to the slow-EOF condition (see Figure 2C). This was because
the apparent velocities of the proteins were faster in the no-EOF condition, thus each protein
band spent less time at the detector window.
The addition of 1 and 2 mM CTAB to the phosphate buffer was unsuccessful in the
counter-EOF CE analysis of the tested proteins at positive or negative polarity, contrary to
that reported previously but with another set of proteins [12]. Also, without addition of Q-CD into the BGE and after a few injections, the capillary had to be replaced as the separations
were very irreproducible and there were injections where no peaks were observed.
The repeatability of the coating with 0.05 (no-EOF) and 0.1 (slow EOF) mg/mL Q-CD in the BGE using the acidic phosphate buffer was evaluated. The RSD (n = 10) for
migration time and corrected peak area with the no-EOF condition was 0.7–1.0 and 1.8–
3.3%, respectively. The RSD (n = 10) with the slow EOF condition was 1.4–2.7 and 13.3–
14.7%, correspondingly. The results indicated that elimination of EOF seems to be a better
approach to provide repeatable results.
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The long-term repeatability of results from the coating using 0.05 mg/mL Q--CD in
the acidic phosphate buffer was therefore investigated. There were 81 injections which were
conducted for 3 days (27 injections per day). The RSD (n = 81) for migration time and
corrected peak area (peak area/migration time) was 0.6–0.7 and 5.7–9.3%, respectively. The
very small variation in migration time is quite notable. The RSDs of > 5% in terms of
corrected peak area was not only due to the variation of the CE method but also due to the
variations caused by the sample. A 100 L volume of sample solution was prepared each day
in the 3-day repeatability experiments. Nevertheless, the dynamic coating with Q--CD can
provide reasonably repeatable results under no-EOF conditions. It is also noted that the
capillary after >100 injections can still be used.
4. Concluding remarks
A new dynamic coating for the CE separation of proteins was described. Addition of
very small amounts of Q--CD into the separation electrolyte can eliminate (neutral surface)
or significantly suppress (slightly positive surface) the EOF in a fused-silica capillary. CE
separation of the model positively charged proteins at positive polarity was obtained with
sharp peaks. The coating was found to provide repeatable results and there was no fouling of
the capillary after more than 100 injections.
Acknowledgement
This work is supported by the Australian Research Council Discovery Project Scheme
(DP13010406).
The author has declared no conflict of interest.
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5. References
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[3] Štěpánová, S., Kašička, V., Recent applications of capillary electromigration methods to
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Figure captions
Figure 1. Proposed adsorption of a cationic CD at the inner wall of a fused-silica capillary,
forming a monolayer of CD with the neutral side of the CD in contact with the BGE inside
the capillary. This provides a neutral surface and an undetectable EOF. The CDs were
anchored to the capillary wall by electrostatic interaction between the cationic group of the
CD and the anionic silanol groups at the capillary walls.
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Figure 2. CE separation of model proteins using 75 mM sodium phosphate at pH 2.5 and
different concentrations of a cationic cyclodextrin. 0.05 (A), 0.075 (B), and 0.1 (C) mg/mL of
Q--CD was added to the BGE. Concentrations of proteins were 0.05–0.15 mg/mL.
Separation voltage was 25 kV and detection was at 210 nm. Capillary (50 m id) was 60 cm
long with 50 cm effective length. Peak assignment: -lactoglobulin (-Lac), ubiquitin (UBI),
cytochrome-c (Cyt-c), and myoglobin (MB). The proteins were separated as positively
charged species. More explanation in the text.
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