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Review
Micellar electrokinetic chromatography:Methodological and instrumental advancesfocused on practical aspects
This article reviews the latest methodological and instrumental improvements for
enhancing sensitivity and resolution in MEKC-based determinations. On-line sample
concentration (stacking, sweeping and combination of two protocols) and other on-
capillary approaches such as in-capillary derivatization and the coupling of flow-injection
systems with MEKC are the most relevant methodological approaches discussed for
improving sensitivity. At the same time, changes in the BGE through the use of organic
modifiers, ionic liquids, cations, CDs, non-ionic and zwitterionic surfactants, mixed
micelles, vesicles and carbon nanostructures as well as 2-D CE and chemometric tech-
niques for enhancing resolution are also considered in detail. Two instrumental
approaches such as MS and LIF are also discussed, covering the proposals for over-
coming the difficulties arising from the direct coupling of MEKC with MS and the
excellent detection sensitivity achieved by LIF using the typical argon-ion laser and recent
growth in the use of diode lasers as excitation sources. Some thoughts on potential future
directions are also expressed.
Keywords:
LIF / MEKC / MS / Stacking / Sweeping DOI 10.1002/elps.200800414
1 Introduction
Many recently published studies highlight how MEKC
systems have become viable and popular ways to analyze
samples containing a broad range of compounds. That this
is truly a reality at the present time is reflected in the
bibliography covered in this review (2006 and 2007) where
the number of routine MEKC methods considerably
outweighs the number based on methodological and
instrumental advances (which, in general, also focuses on
more practical determinations than those reported some
years ago). This situation clearly reflects just how readily this
analytical technique is being accepted by the scientific
community. Consequently, the assertion that MEKC is a
useful alternative to the liquid chromatographic technique is
becoming a more tangible reality with each passing day. As
in the previous review [1], some current relevant aspects to
do with MEKC, such as the use of polymeric pseudo-
stationary phases (PSPs), its implementation on microchip
devices and advances in MEEKC (at present, a consolidated
extension of MEKC), have been omitted in this review as
they are now the subject of dedicated reviews in this issue.
On the other hand, some papers related to theoretical
aspects of MEKC have also been published in this period,
the most relevant being those focusd on studies on the
selectivity of single, mixed and modified PSPs [2], on the
origin of peak asymmetry and isotherm non-linearity [3], on
the analysis of substances to be used as internal standards
[4] and on migration models for acidic solutes [5].
The methodological and instrumental approaches covered
by this review are focused on expanding MEKC ability to
improve sensitivity by providing LODs in the sub-mg/L region
and resolving more and more complex mixtures of neutral and
charged analytes in real samples. The use of sample stacking
and sweeping techniques in routine analysis (the most widely
used methods for on-line sample concentration in MEKC) in
order to enhance sensitivity has grown considerably in the
period covered by this review. At present, these techniques can
Manuel Silva
Department of AnalyticalChemistry, Rabanales Campus,University of Cordoba, Cordoba,Spain
Received June 30, 2008Revised August 27, 2008Accepted August 27, 2008
Abbreviations: ANNs, artificial neural networks; APFOA,
ammonium perfluorooctanoate; CD-MEKC, CD-modifiedMEKC; DTAF, 5-(4,6-dichloro-s-triazin-2-ylamino)fluorescein; EKSI, electrokinetic stacking injection; FASI,
field-amplified sample injection; FQ, 3-(2-furoyl)quinoline-2-carboxaldehyde; MCA, methyl chloroacetate; MPE,
multiphoton excitation fluorescence; NBD-F, 4-fluoro-7-nitro-2,1,3-benzoxadiozole; NDA, naphthalene-2,3-dicarboxaldehyde; PEO, poly(ethylene oxide); PF, partialfilling; PSP, pseudo-stationary phase; RMM, reversedmigrating micelles; SAMF, 6-oxy-(N-succinimidyl acetate)-9-(20-methoxy-carbonyl) fluorescein; SC, sodium cholateSC-CNT, surfactant-coated carbon nanotube; SWNT, single-walled nanotube
Correspondence: Professor Manuel Silva, Department of Analy-tical Chemistry, Marie-Curie Building (Annex), RabanalesCampus, University of Cordoba, E-14071 Cordoba, SpainE-mail: [email protected]: 134-957-218614
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Electrophoresis 2009, 30, 50–6450
really be considered useful alternatives to classical off-line pre-
concentration procedures. Regarding approaches for improv-
ing resolution, few contributions have been reported with
respect to the proposal for alternative PSPs to SDS, except for
polymeric ones (the study of which, as stated above, is outside
the scope of this review). Thus, SDS has become the most
effective pseudostationary phase (PSP), as reflected in the great
number of papers published over the last 2 years; in many
studies, conventional or modified with an organic solvent, SDS
micelles provide the required selectivity for analysis, although
in others, SDS micelles are coupled with other separation
equilibriums in the aqueous phase to enhance MEKC
separation, such as CDs and carbon nanostructures among
others, as well as those provided by other micellar systems
(mixed micelles) to enhance MEKC separation. The use of
non-aqueous MEKC, ionic liquids as alternatives to organic
modifiers, mixed micelles instead of conventional SDS and
coated capillaries has not totally succeeded in fulfilling the
expectations hoped for in the last review. On the other hand,
2-D CE and chemometric techniques have grown extensively
as tools for improving resolution in MEKC, and therefore they
have been included in the present review. Regarding detection
techniques, new contributions involving MS and LIF detection
are discussed here as in the last review. Despite improvements
in MS detection, especially those based on the use of polymeric
surfactants, this subject is still pending in MEKC, as stated by
Klampfl and Buchberger [6] in a recent article: ‘‘Whereas in
some fields CZE–MS may be accepted as a routine technique
in the near future, coupling of MEKC or even MEEKC with
MS is still in its very early stages’’. On the other hand, the use
of LIF detection in MEKC has grown dramatically over the
period covered by this review, the most relevant research topics
being: the consolidation of ‘‘classical’’ fluorescent probes with
an argon-ion laser as the excitation source, the development of
new contributions based on LEDs and the novel proposal for
fluorescence detection by using continuous wave-based
multiphoton excitation. Worthy of special note in this context
is a new contribution on the use of thermal lens microscopic
detection in MEKC. In an interesting study, Kitagawa et al. [7]
describe how this detection system in MEKC is improved
through the use of an interface chip to achieve highly sensi-
tivity detection and high reproducibility for the determination
of non-fluorescent and neutral analytes. Furthermore, nearly
million-fold sensitivity enhancement can be achieved by
coupling this detection system with sweeping as an on-line
sample concentration procedure.
2 Approaches for improving sensitivity
2.1 On-line sample concentration methods
On-line sample concentration methods are one of the
simplest ways for sample enrichment in MEKC, since the
concentration step is performed within the same capillary
used for analysis. Sample stacking based on the manipula-
tion of analyte migration velocity and sweeping on its
partitioning into a moving PSP are by far the most widely
used methods. Different trends were observed in the topics
dealt with during the period covered by this review with
respect to those included in the last one, namely: (i) few
studies have been carried out to expand the scope of
application of sweeping techniques by modifying the PSP,
resulting in SDS micelles being the most commonly
sweeping carrier used in many analytical applications; (ii)
a considerable growth in the use of sample stacking
methods; (iii) a lack of development in the expected
potential of other on-line sample concentration approaches
such as those based on ‘‘selective exhaustive injection-
sweeping’’ and ‘‘dynamic pH junction-sweeping’’, probably
due to their low reproducibility, which often prevents their
use as quantitative tools for the ultra-trace analysis of
analytes in complex matrices and (iv) some new proposals
have also been reported, mainly focused on increasing the
injected sample volume. These trends lead to the conclusion
that these techniques now offer enough reliability to be
considered useful alternatives to off-line concentration ones
to increase sensitivity in MEKC-based determinations. In
this context, the combination of both on- and off-line sample
concentration approaches is another significant trend to
achieve higher enhancement factors; SPE and solid-phase
microextraction are the most frequent off-line sample
concentration techniques, while the use of other current
alternatives such as stir bar sorptive extraction and
liquid–liquid semimicroextraction has also been reported.
2.1.1. Sample stacking
Sample stacking in MEKC is an on-line sample concentra-
tion technique based on differences between electric field
strength in the sample zone and that of BGE. The focusing
effect occurs at the boundary between the high electric field
sample zone and the low electric field BGE zone. It is a
result of a rapid change in micelle migration velocity when
passing from one region to another. There have been
remarkable reports on the use of the different stacking
modes and new alternatives in MEKC over the period
covered in this review. Ordinarily SDS is used as PSP and
the most frequent approaches are field-enhanced sample
injection [8–10], stacking with reversed migrating micelles
(RMM) [11–14] and field-enhanced sample injection
with RMM [14, 15]. The ensuing analytical methods
have been used to determine a great variety of analytes,
namely aristolochic acids in Chinese medicine preparations
[8], PAHs in airborne particulates [9], melatonin and
related indoleamines in rat pineal glands [10], melatonin
and its precursors/metabolites in human serum [11],
paraben preservatives in cosmetic products [12], pesticides
in fruits and vegetables [13], non-steroid anti-inflammatory
drugs in mineral water [14] and saponins in Panax
notoginseng [15]. Between ca. 100- and 200-fold enhance-
ment in detection sensitivity can be achieved compared with
that obtained in the simple MEKC method; which can be
increased to 300-fold by using the method proposed by
Electrophoresis 2009, 30, 50–64 CE and CEC 51
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Zhuang et al. [12] in which large volumes of sample can be
successfully injected.
In this context it is noteworthy to point out two inter-
esting stacking approaches for the analysis of large-volume
samples. Chiu and Chang [16] reported a stacking method
in the presence of poly(ethylene oxide) (PEO) for neutral or
anionic solutes, such as amino acids after their derivatiza-
tion with naphthalene-2,3-dicarboxaldehyde (NDA). In
addition to SDS, PEO is essential for the stacking and
separation of large-volume (e.g. 0.53 mL) NDA–amino acid
derivatives, which allows sensitivity enhancements to be
achieved in the 50–800-fold range. The basic principles
behind this approach are illustrated in Fig. 1. Initially, the
capillary is filled with Tris-borate buffer containing SDS in
order to reduce PEO adsorption and thus generate a high
and repeatable EOF (Fig. 1A). A mixture of NDA–amino
acid derivatives is hydrodynamically injected from the
anodic end to the capillary for a length of time, e.g. 240 s,
(Fig. 1B). When high voltage is applied, the PEO solution
enters the capillary from the anodic end. SDS micelles
migrating from the zone of Tris-borate buffer interact with
and thus sweep the negatively charged NDA–amino acid
derivatives, demonstrating less electrophoretic mobility than
that of SDS micelles in the sample zone. Both SDS micelles
and NDA–amino acid derivatives migrate against EOF
(Fig. 1C). When anionic NDA–amino acid derivatives and
the aggregates between SDS micelles and NDA–amino acid
derivatives migrate into the PEO zone, they are stacked in a
narrow band as a result of the decrease in electric field and
increase in viscosity (Fig. 1D). Finally, aggregates between
SDS micelles and NDA–amino acid derivatives are separated
according to the mechanisms of MEKC. Zeng and
co-workers [17] proposed the so-called pressure-assisted
field-amplified sample injection (FASI) with RMM for the
on-line concentration of neutral analytes in MEKC with a
low-pH BGE. Briefly, the stacking mechanism of pressure-
assisted FASI with RMM is as follows: in step 1, after the
capillary is initially conditioned with the micellar BGE, a
water plug was hydrodynamically injected by pressure. In
step 2, the injection end was switched into the sample vial
that contained the analytes, which were prepared in low-
conductivity matrices. At the same time, the outlet end was
still in the micellar BGE vial. Analyte focusing was started
by applying a negative voltage in conjunction with a pres-
sure that generates a hydrodynamic flow in the direction
opposite to the EOF, which prevents the water plug from
moving toward the inlet of the capillary too fast. Thereafter,
micelles and neutral analytes solubilized in it were injected
electrokinetically into the capillary for a period of time much
longer than usual for FASI (e.g. 8 min). Under these
conditions, 1000–3000 stacking enhancement factors in
terms of peak area can be observed for the determination of
trace steroids. The method also provides about seven times
more peak height enhancement for the analytes when
compared with those obtained by the sweeping technique.
Other ‘‘classical’’ stacking approaches have been used
for concentrating analytes from different samples. Rodrı-
guez-Delgado and co-workers [18] reported several meth-
odologies based on the reversed-electrode polarity stacking
mode for pesticide analysis in water and wine [19–21]
samples. To increase concentration factors, off-line
concentration strategies were combined with the reversed-
electrode polarity stacking mode; thus, SPE was useful for
multiresidue analysis of pesticides in water samples,
providing LODs at ng/L levels, whereas solid-phase micro-
extraction was recommended for the determination of these
compounds in wine samples, resulting in less sensitivity,
only just reaching LODs at mg/L levels. The combination of
so-called ‘‘ACN stacking’’ and ACN deproteinization with
salting-out extraction is proposed by Li and Huie [22] as a
new sample pre-treatment approach for biological samples
for additional enhancement in concentration detection
sensitivity. The approach was useful for the determination
of hydrophobic porphyrins with clinical significance at
ng/mL in urine samples. In contrast to other methods
already commented on, sodium cholate (SC) was used as the
PSP. This micellar system has also been used by Collins and
co-workers [23] for the on-line concentration of nitroaro-
matic explosives in seawater by using the high-salt stacking
mode. Although longer injection times result in a loss of
resolution due to limited interaction between the explosives
and the SC micelles, LODs below 100 mg/L can be obtained
successfully. At this point, it is worth mentioning the
interesting results also reported by Collins and co-workers
[24] to understand the role of stacked micelles in the sample
concentration. The paper is a fine study on the differences
between the mechanisms of sweeping and high-salt stack-
ing as on-line sample concentration techniques.
Detection
+
+_
_
SiO µEOF µEP PEO solution TB buffer
NDA-amino acid derivatives SDS micelle
Detection
A
B
C
D
+
+_
_
SiO µEOF µEP PEO solution TB buffer
NDA-amino acid derivatives SDS micelle
Figure 1. Evolution of stacking and separation of NDA–aminoacid derivatives by CE-LED induced fluorescence in the presenceof EOF and PEO solutions. Reprinted from [16] with permission.
Electrophoresis 2009, 30, 50–6452 M. Silva
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
Finally, there is a noteworthy new on-line sample
concentration approach based on the combination of large
volume sample stacking and the dynamic pH junction
technique for the determination of weak acidic compounds
[25]. Figure 2 shows the scheme of the method. The capil-
lary is filled with the sample solution in sodium borate pH
9.5 while sodium phosphate pH 2.5 with SDS is in the inlet
and the outlet vials (Fig. 2A). Application of a negative
electric field causes the co-electroosmotic migration of
analytes and pre-concentration due to the pH boundaries.
Analytes are immobilized in the junction of phosphate and
borate due to changes in their dissociation. This leads to a
situation where these compounds have mobility close to
zero at pH 2.5 and are stopped on reaching the phosphate
zone. Analytes are also pre-concentrated at the second pH
boundary between the inlet vial and the capillary. They
migrate with the EOF to the boundary where they are
protonated and get stacked (Fig. 2B). These two pre-
concentrated zones are joined during migration (Fig. 2C)
and then the common MEKC proceeds (Fig. 2D). The
method was tested to determine nanomolar concentration
levels of sorbic and benzoic acids and compared favorably
with another on-line sample concentration technique,
isotachophoresis, providing 10–70 times lower LODs.
2.1.2 Sweeping
Sweeping is another effective and convenient way to do on-
line sample concentration in MEKC. It is based on the
accumulation and isolation of analytes, injected by micelles
in a large sample volume, to concentrate them into a narrow
zone and enhance detection sensitivity. From an experi-
mental point of view, sweeping occurs when the sample
matrix is prepared in a buffer solution with conductivity that
is similar to or higher than BGE and without PSP. The basic
condition for sweeping is for the separation buffer to
contain a surfactant at a concentration above its CMC, while
the sample solution is free of the surfactant. When charged
micelles in BGE penetrate the sample zone during the
application of voltage, the picking or accumulating of
analytes occurs due to the partitioning or interaction of
analytes with PSP. As stated above, little work has been
conducted to improve the phenomenon of sweeping; thus,
the contributions have mainly been focused on the analytical
use of this on-line concentration technique. In most cases
the protocol for sweeping neutral and anionic analytes uses
anionic micelles like SDS and suppressed EOF in a low pH
BGE provided by phosphoric acid or phosphate buffer (pH
2–3) [26–32]. Under suppressed EOF conditions, other
anionic surfactants, di(2-ethylhexyl) sulfosuccinate and
Brij-S, and cationic ones, CTAB, octyltrimethylammonium
bromide and tetradecyltrimethylammonium bromide, have
also been used as alternatives for SDS, but with lower
enhancement factors [33]. In general, up to 100-fold
improvement in concentration sensitivity can be achieved
in the determination of different analytes, mainly in
samples of pharmaceutical or clinical interest such as
strychnine and brucine in Chinese medicinal preparations
[26, 27], phenethylamine designer drugs [28] and ketamine
and norketamine [29] in urine samples and carbamazepine
in tablet and human serum [32]. In some cases, LODs were
also enhanced by combining on-line sweeping with an off-
line concentration approach such as liquid-phase microex-
traction [27] or by using a more sensitive detection
technique such as LED-LIF detection [28, 30]. The sweeping
protocol has also been applied under basic conditions (BGE
with borate buffer at pH over 8.5–9.5), but only two
approaches have been reported. The first one, concerning
the determination of all-trans- and 13-cis- retinoic acids in
rabbit serum, was developed in order to overcome the poor
resolution obtained under suppressed EOF conditions
caused by strong interaction of the analytes with the PSP
[34]. Although under basic conditions the resolution was
increased, only ca. 19-fold improvements in sensitivity were
achieved for retinoic acids. The second one deals with a
sweeping technique using CTAB to improve sensitivity
detection in flunitrazepam and its major metabolites in
urine samples [35]. In this approach the focusing effect is as
follows: after a negative voltage was applied from inlet
(cathode), the EOF, under the influence of the cationic
CTAB surfactant, moved toward the outlet (anode). Because
the velocity of the EOF was higher than that of the CTAB
micelle, the analytes stacked at the boundary by the CTAB
micelle and moved toward the anode. Using this sweeping
protocol, sensitivity enhancement for each compound was
within the range of 110–200-fold.
_ +
_
_ +
_
detector
concentrated analytes
separated analytes
sodium phosphatepH 2.5 with SDS
sodium borate pH 9.5with analyte
+
+
_ +
_
_ +
_
detector
concentrated analytes
separated analytes
sodium phosphatepH 2.5 with SDS
sodium borate pH 9.5with analyte
A
B
C
D+
+
Figure 2. (A–D) The scheme of proposed large volume samplestacking and the dynamic pH junction technique pre-concentra-tion method. The dots represent micelles. Reprinted from [25]with permission.
Electrophoresis 2009, 30, 50–64 CE and CEC 53
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
2.1.3 Combination of stacking and sweeping
protocols
The so-called ‘‘selective exhaustive injection-sweeping’’
techniques based on the combination of stacking and
sweeping protocols are the most sensitive approaches for
on-line sample concentration; up to a million-fold sensitivity
increase for cationic analytes (cation-selective exhaustive
injection-sweeping) has been reported by Quirino and
Terabe [36]. Some contributions to these techniques have
been reported in the period covered by this review [37–41],
but fewer than expected despite the high values of
improvement factors it can involve. This behavior can be
ascribed to the poor reported run-to-run reproducibility, in
many cases over 10% for low concentrations. In an
interesting study, Chen and co-workers [37], after a
systematic optimization of the process, concluded that there
are five key factors that must be controlled to achieve highly
reproducible results, namely, the conductivity of the sample
solution, the conductivities of high- and medium-conductiv-
ity buffers, the fraction of the column filled with high-
conductivity buffer, electrokinetic injection time and the
surfactant concentration. The authors recommended prepar-
ing the sample in a solution with moderately low conductiv-
ity in order to increase the reproducibility of both the
injection process and the whole analytical procedure. Illicit
amphetamines [38, 39] and other abuse drugs [40, 41] in
urine [38–40] and hair [41] samples can be detected at a few
ng/mL or even at pg/mL levels, thanks to the higher
enrichment factors that can be obtained, which ranged from
1000- to 6000-fold. Anion-selective exhaustive injection-
sweeping is a more recent approach than cation-selective
exhaustive injection-sweeping although its principle is
basically the same. Few contributions in this respect have
been reported in this on-line sample concentration technique
for negatively charged analytes during the period covered by
this review [42], and there has even been a straightforward
and easy approach reported by Heineman and co-workers
[43] as an alternative to the anion-selective exhaustive
injection-sweeping scheme, which does not require polarity
changes during stacking and sweeping steps. The authors
developed a new electrokinetic stacking injection (EKSI)
scheme to concentrate anionic analytes by sweeping with
cationic surfactants in a dynamically coated capillary. Figure
3 shows a schematic diagram of the stacking and sweeping
processes. Under optimal experimental conditions, the
injected sample plug length for analytes under 20.1 kV for
60 min was estimated as ca. 800 cm, the effective capillary
length corresponding to ca. 25-fold. Compared with tradi-
tional pressure injection, the EKSI scheme resulted in an
increased detection factor of ca. 4.5� 103.
2.1.4 Other on-capillary approaches
In addition to the on-line sample concentration methods
reported above, other on-capillary approaches with different
aims have also been proposed in the period covered by this
review, e.g. to achieve full automation and simplification of
the analytical procedure with a further improvement in
reproducibility by using so-called ‘‘in-capillary derivatiza-
tion’’ [44–47] or by the combination of flow injection with
MEKC [48, 49], and to increase sensitivity by coupling stir
bar sorptive extraction [50] or liquid–liquid semimicroex-
traction with MEKC [51].
Despite its attractive features, little work has been
reported on the use of in-capillary derivatization in MEKC in
recent years. On the other hand, a great deal has been
devoted to studying enzymatic reactions through electro-
phoretically mediated microanalysis [44]. Only three refer-
ences to in-capillary derivatization have been reported so far
in the context of MEKC analysis; the first one evaluated the
potential of 4-fluoro-7-nitro-2,1,3-benzoxadiozole (NBD-F) as
an in-capillary derivatization reagent for the analysis of
organophosphorus pesticides [45]. A mixed in-capillary
mode combining zone-passing mixing and at-inlet reaction
was proposed, in which plugs of sample and NBD-F solu-
tions were introduced successively to the anodic end of the
capillary. The sample and reagent zones were electro-
kinetically mixed by applying a potential of 5 kV for 30 s, and
the reaction was allowed to stand for 7.5 min in the absence
of potential. Although the LODs achieved for the determi-
nation of pesticides were comparable with those obtained
+
+
+_
+_
+_
_
Steadyinjection
Vial exchange
Sweeping
Zone migration
Separation
_
Startingsituationof EKSI
+_
lSB, max
lL
Veo1Veo2
Veo1
lSB, max
lSB
V´eo2 V´eo1
lSB, max
V´eo2 V´eo1V´eo1
V´eo1 V´eo2
V´eo1
Detectionpoint
+
+
+__
+_ +__
+__
__
Steady stateinjection
exchange
Sweeping
Zone migration
Separation
__
A
B
C
D
E
F
Startingsituationof EKSI
+__
lSB, max
lL
Veo1Veo2
Veo1
lSB, max
lSB
V´eo2 V´eo1
lSB, max
V´eo2 V´eo1V´eo1
V´eo1 V´eo2
V´eo1
Detectionpoint
Figure 3. Evolution of the EKSI and sweeping. The dense-dotpart represents sample solution in phosphate buffer. The blankpart refers to the separation buffer containing dodecyltrimethyl-ammonium bromide (DTAB), phosphate and ACN. The sparse-dot denotes the phosphate sample buffer. I and L are theeffective and total capillary lengths, respectively. ISB and ISB,max
are the sample buffer plug length and its maximum, respec-tively. Veo1 (Veo1) and Veo2 (Veo2) are the local EOF velocities.Reprinted from [43] with permission.
Electrophoresis 2009, 30, 50–6454 M. Silva
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
from conventional pre-capillary derivatization, the sensitivity
could be improved remarkably by its coupling with an on-
line sample concentration technique. The other two contri-
butions rely on the in-capillary derivatization of enantiomers
of chiral amino compounds [46] and amino acid mixtures
[47] with o-phthalaldehyde and the chiral reagent N-acetyl-L-
cysteine as derivatizing reagents using DAD and LIF
detection, respectively. As with in-capillary derivatization
methods, few works have been devoted to flow injection-
MEKC approaches. Two contributions from Chen and
co-workers [48, 49], one related to the separation and
determination of alpinetin and cardamonin in Alpinia
katsumadai Hayata [48] and the other to the determination
of berberine, palmatine and jatrorrhizine in herbal medicine
[49], are the only ones that have been reported. The main
features of these methods were automation, rapidity (typical
sample throughput ranges from 10 to 24 h�1) and even high
sensitivity because low LODs can be achieved by using an
additional on-line sample concentration step, as in the
second approach, in which 64–86-fold improvement in the
detection sensitivity was obtained by using head-column
FASI and sweeping.
Finally, an interesting contribution of Wang et al. [51]
about the use of methyl chloroacetate (MCA) as an extrac-
tion solvent for coupling liquid–liquid semimicroextraction
with MEKC is worthy of note: it is based on its on-capillary
decomposition for the separation of neutral compounds
with concentration enhancement. This solvent overcomes a
significant drawback involved in the on-line coupling liqui-
d–liquid extraction with MEKC, which is the poor electrical
conductivity of the solvents that are not able to conduct a
high enough current along the capillary. Thus, after
microextraction of analytes into MCA, the organic phase was
directly subjected to separation by MEKC, where MCA
underwent in-capillary decomposition into methanol and
chloroacetic acid. Due to the features of MCA, a plug of
catalyst (e.g. NaOH) was not required as in the method
reported by Zhan et al. [52] based on the use of ethyl acetate
as the extraction solvent. Concentration enhancement for
alkylphenones such as acetophenone, butyrophenone and
valerophenone was between several multiples of 10 to more
than 100 times.
3 Approaches for improving resolution
The approaches described in this section for improving
resolution in MEKC have been classified into two main
topics: (i) incorporation of additives into the aqueous phase
to modify the distribution constant between micelles and
the surrounding medium or to alter its apparent distribution
constant by using coupled equilibriums and (ii) modifica-
tion of the micellar phase by using other surfactants as an
alternative to SDS as well as mixed micelles. This section
also considers the use of coated capillaries, 2-D CE and
chemometric techniques in MEKC as approaches for
improving resolution.
3.1 Incorporation of additives into the aqueous
phase
Organic modifiers are a widely known option for enhancing
MEKC separations because they alter the micellar structure
(solute partition coefficients) and expand the migration
window by reducing the EOF. Up to now, ACN has been the
most commonly found organic modifier used in MEKC; of
the methods reported based on the use of organic modifiers
for improving resolution in MEKC, over 75% use ACN as an
organic modifier [53–58], and to a lesser extent, methanol
[59–62], ethanol [63, 64] and mixtures [65]. Urea [66], ionic
liquids [67, 68] and cations [69] are the most significant
alternatives for organic modifiers in MEKC reported during
the period covered by this review. Urea was used as an
alternative to methanol to reduce the migration window
without detriment to the resolution of eight adrenocortico-
tropic steroid hormones with SDS micelles and acidic BGE
[66]. As stated by the authors, urea forms hydrogen bonds
with SDS micelles and weakens the hydrophobic interac-
tions of the separated components with the surfactant,
which results in an increase in separation efficiency. Few
contributions have been reported in the last 2 years on the
use of ionic liquids in MEKC as an alternative to organic
modifiers, despite their recent introduction in MEKC by
Warner and co-workers in 2003 [70]. These authors reported
a comparison of ionic liquids and organic solvents as
additives for chiral separations in MEKC using polysodium
oleyl-L-leucylvalinate as molecular micelle and 1-alkyl-3-
methylimidazolium tetrafluoroborate as ionic liquids, where
the alkyl group was ethyl, butyl, hexyl or octyl [68].
Equivalent chiral resolution and selectivity can be achieved
when using low contents of ionic liquids as an alternative to
high volumes of organic solvents; however, ionic liquids
provide faster separations without current breakdowns.
Another way to modify the EOF to improve resolution is
reported by Qu and co-workers. [69], based on the addition
of divalent and monovalent cations to the BGE as additives,
which is used to enhance the separation of amino acids as
p-acetamidobenzenesulfonyl fluoride derivatives. A compro-
mise solution can be derived from this study, because the
resolution cannot be improved easily using the buffer cation
that provides the fastest decrease for the EOF. Thus,
although the reduction of the EOF followed the order
Mg2+4K+4Na+4Li+, the authors propose Na+ as the best
additive to reach a compromise among migration time,
resolution and peak shape. Therefore, 20 mM Na+ as
electrolyte was added to the BGE consisting of 20 mM
borate at pH 9.3 containing 140 mM SDS.
The addition of CDs as additives to the aqueous phase is
another effective option for increasing resolution in MEKC,
especially in enantiomeric separations. The so-called
CD-modified MEKC (CD-MEKC) method is widely used to
achieve chiral separations [71], which is based on the
combining of chiral CDs with achiral micelles, such as SDS.
However, chiral separations in MEKC have been recently
drawn toward using chiral polymeric surfactants (also called
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molecular micelles); the description and applications of
molecular micelles is beyond the scope of this review, which
is now focused on the use of CD-MEKC and other reported
chiral alternatives. As in the previous review, b-CDs are by
far the most widely used additives for improving resolution
in CD-MEKC; SDS is commonly used as a micellar system
[9, 28, 72–76], but other micellar systems such as bile salts
[77] and mixed micelles [78] have also been employed. In
addition to b-CDs, derivatives such as hydroxypropyl-b-CD
[79–82] and 6-O-a-D-glucosyl-b-CD [83], g-CD [84, 85],
hydroxypropyl-g-CD [31] and the currently reported native
d-CD [86] have also been used as chiral selectors in MEKC to
increase enantioselectivity. Pharmaceutical [28, 74–77, 79,
84, 85] and food [31, 72, 80, 81, 83] fields are the main areas
of application of the methods reported on. In this context,
two relevant contributions are noteworthy. The first one is
devoted to the study on the competition mechanism
between b-CD and SDS reported by Liu and co-workers [74],
which led the authors to conclude that CD-MEKC improves
selectivity in hydrophobic compounds, because for hydro-
philic analytes, which have weaker hydrophobic interactions
with SDS, this approach is sometimes not efficient because
the competition between SDS and the aqueous PSP is too
strong and will lead to a negative effect on separation. The
second one introduces native d-CD as a chiral selector in
CD-MEKC [86] comparing it with conventional BGE addi-
tives a-, b- and g-CDs. Experimental results showed that,
depending on the structure of the analytes, the resolving
property of d-CD is similar to or quite different from g-CD.
Although CD-MEKC is the most widely used approach for
chiral analysis in MEKC, some interesting alternatives have
been proposed, one of which reports a new way to find novel
types of chiral derivatizing reagents for amine compounds
[87]. The method is based on the synthesis of isothiocyanate
derivatives of chiral amine reagent, like dehy-
droabietylamine, through reaction with Et3N, CS2 and
POCl3 in a dried ethyl ether medium.
3.2 Use of a different micellar phase
The choice of surfactant seems to be the most important
variable in optimizing the resolution in MEKC; out of the
numerous surfactants that have been evaluated, SDS is by
far the most widely used. However, SDS-MEKC does not
provide suitable resolution in all cases and therefore other
micellar phases from monomeric (e.g. bile salts, cationic and
non-ionic surfactants, mixed micelles, etc.) to polymeric
surfactants have been used to extend the application of
MEKC to more complex matrices. As in other sections, the
description of contributions based on the use of polymeric
surfactants is beyond the scope of this review.
Lithium dodecyl sulfate [88] and sodium octane sulfo-
nate [89] are two salient examples of the use of monomeric
surfactants as PSPs with a similar structure to SDS. When
using lithium dodecyl sulfate, selectivity can be improved
because Li+ ions will bind the surface of the micelles less
tightly than Na+ ions and therefore the repulsions with
sulfate groups are more important and a more ‘‘open’’
micelle with a higher mass transfer is obtained. In conse-
quence, the residence time of analytes in the hydrophobic
core of micelle becomes more important and narrower peak
widths can be obtained [88]. On the other hand, surfactants
with a carbon number lower than that of SDS, such as
sodium octane sulfonate, can be a useful alternative to the
use of SDS micelles in the presence of organic modifiers to
decrease the migration window without affecting separation
efficiency [89]. The number of contributions/year using
other conventional PSPs such as bile salts, cationic and non-
ionic surfactants, and mixed micelles is practically the same
as in the last review. In general, the methods reported have
been developed in order to determine analytes of clinical
and pharmaceutical interest in different samples, and only
some of them are worth pointing out. Regarding bile salts,
SC micelles have been used for the separation of bioactive
licorice compounds [77] (alkyltio)acridines [90] amino acids
after derivatization with phanquinone [91] four streoisomers
of palonosetron hydrochloride [92], whereas sodium deox-
ycholate was a useful PSP for the separation of nitrofuran
antibiotics [93] and a mixture of herbs commonly present in
herbal Chinese formulas [94]. Mixed micelles of SC with
SDS [95] or another bile salt like taurine [96] have also been
reported for the separation of ten flavonoid aglycons
belonging to four different classes, and for the determina-
tion of shikimate in raw plant extracts, respectively. As is
well known, relatively few cationic surfactants have been
used in MEKC, and consequently few contributions have
been reported in the period covered by this review [97, 98].
In an interesting paper, Palmer and co-workers [98] char-
acterize the solute distribution between water and self-
assemblies formed from ionic liquid type mono-chain
cationic surfactants containing a cyclic pyrrolidinium head
group. The kind of PSPs formed by these N-alkyl-N-
methylpyrrolidinium bromides interacts more strongly with
polar compounds and less strongly with compounds having
non-bonding or p-electrons and are more cohesive
compared with the well-studied CTAB. Regarding non-ionic
surfactants, polyoxyethylene sorbitan monolaurate (Tween
20) [49, 99], polyethylene glycol dodecyl ether (Brij-35) [85,
100] and mixed micelles of Brij-35 with SC [78] and 3-(N,N-
dimethylhexadecylammonium) propanesulfonate [101, 102]
are the useful PSPs reported for the separation of compo-
nents in herbal medicines [99], evaluation of the enantio-
meric purity of an oxazolidinone drug candidate [85],
determination of phosphoamino acids [100], profiling of
amine metabolites in human biofluids [78] and peptide
mapping [101] by using acidic [49, 85, 101, 102] or basic
[78, 99, 100] BGEs.
Between monomeric surfactants and polymeric ones
with multiple hydrophobic chains and ionic head groups are
the dimeric surfactants, which are made up of two amphi-
philic moieties connected at, or close to, the head groups by
a spacer. In an interesting paper, van Biesen and Bottaro
[103] describe the synthesis of dimeric anionic surfactants
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with a flexible hydrophobic spacer (2, 4, 6, 8, 10 and 12
methylene groups) close to the two head groups and the
influence of these spacers on the selectivity. No significant
differences were observed in the selectivity for each surfac-
tant, which means that the cohesiveness of the micelles and
their interface with the aqueous bulk phase is very similar,
regardless of the length of the hydrophobic spacer.
Compared with SDS, these dimeric surfactants do seem to
be better at separating more hydrophobic solutes at much
lower concentrations because of their lower CMCs. This
could be an advantage for applications where a low current
is required to minimize Joule heating or for direct coupling
of MEKC with MS. One other contribution of this kind of
surfactants is that of using di(2-ethylhexyl) sulfosuccinate as
the PSP for the separation of synthetic phenolic antioxidants
in edible oils [104].
Liposomes are self-assembled vesicles commonly
consisting of phospholipid bilayers enclosing an aqueous
solution, with other lipid aggregates found as well. When
using liposomes in MEKC, the lipophilic phase is suspen-
ded in the aqueous mobile phase (or vice versa) in the form
of micelles, microemulsions or other particles forming a
PSP [105–107]. These particles are normally electrically
charged and move thus with their own electrophoretic
velocity under the influence of the electric field applied.
Kenndler and co-workers [108] reviewed in detail the work
on liposomes in the context of electrically driven separation
methods in the capillary format; and concretely one topic is
related to the use of liposomes as PSP in MEKC. These
vesicular structures obtained by mixing cationic and anionic
surfactants at a concrete mass ratio, phospholipids or lipids
as well have rarely been used in MEKC before now for the
separation of hydrophobic compounds. Recently, Jiang
et al. [109] have demonstrated that the use of liposomes as
PSP in MEKC is a good and dynamic method to study
of the interaction between cell membranes and important
biomolecules.
The recent introduction of carbon nanostructures as
PSPs in MEKC is also worth commenting on [110–112]. The
use of nanoparticles as PSP in CE is a growing area of
interest because they have the advantage of introducing a
novel interaction phase for every analysis, as can be stated in
the current and excellent review by Nilsson and Nilsson
[113]. These authors discuss the use of micelle polymers,
dendrimers, silica nanoparticles, molecularly imprinted
polymer particles, polymer nanoparticles and gold nano-
particles; however, carbon nanostructures (carbon nano-
tubes and fullerenes) are not considered in this review.
Valcarcel and co-workers [110] have recently developed a
new MEKC procedure based on the use of surfactant-coated
carbon nanotubes (SC-CNTs) as PSPs, in which single-
walled nanotubes (SWNTs) are dispersed into a micellar
media by sonication to obtain SC-CNTs. The approach
allows the use of CNTs as PSPs (avoiding their insolubility
in water) and makes use of their advantages, namely, both
the high surface area and the capacity to adsorb organic
analytes; thus, additional interactions are introduced into
the micellar medium with the subsequent improvement in
the resolution. Figure 4 shows the significant effect of SC-
CNTs on the separation of a mixture of chlorophenols by
MEKC using SDS micelles. As can be seen, overlapped
peaks can be resolved by adding SC-CNTs to the BGE
without an appreciable increase in the migration window.
Other interesting potential features of SC-CNTs as PSPs in
MEKC are their use as an analytical tool in sequential
injection to improve sensitivity and resolution and as a
strategy for isomeric separation. Regarding the latter,
SWNTs and multiwalled nanotubes (MWNTs) have been
evaluated as chiral selectors for direct enantiomeric separa-
tion of ephedrines [111]. MWNTs demonstrate a better
capability to resolve enantiomeric mixtures by using partial
filling (PF) of the capillary with concentrated SC-MWNTs,
and the approach is a promising way of using carbon
nanostructures in MEKC to develop new direct enantio-
meric methods that can be a rapid and simple alternative to
A
B
Figure 4. Electropherograms of chlorophenols obtained in(A) absence and (B) presence of SC-SWNTs (3.2 mg/L).The BGE was 25 mM sodium hydrogenphosphate, 15 mMsodium tetraborate, 100 mM SDS and 6% ethanol at pH 7.95.Other operating conditions: capillary 30 cm�75 mm id, 15 kV,201C. Peaks: (1) 2,6-dichlorophenol; (2) 3,5-dichlorophenol;(3) 2,3-dichlorophenol; (4) 2,5-dichlorophenol, (5) 3,4-dichloro-phenol; (6) 2,3,5-trichlorophenol; (7) 2,4,5-trichlorophenol;(8) 2,3,6-trichlorophenol; (9) pentachlorophenol. Reprinted from[110] with permission.
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those based on the use of CDs. Surfactant-coated fullerenes
C60 have also been proposed as novel PSPs in MEKC to
improve the separation of different aromatic compounds,
such as b-lactams antibiotics, non-steroidal anti-inflamma-
tory drugs and amphenicols [112]. Compared with other
carbon nanostructures such as SWNTs and MWNTs, full-
erenes C60 have a similar potential as PSPs to improve
resolution, but for some analytes they can provide better
results; hence, additional work is required to assess the real
potential of carbon nanostructures in MEKC as PSPs.
3.3 Use of other approaches
At present, the potential of coated capillaries, 2-D CE and
chemometric techniques in MEKC as tools for improving
resolution is quite different because, as stated above, coated
capillaries have not lived up to the expectations raised in the
last review, whereas the use of 2-D CE has grown widely as
well as that of chemometric techniques during the period
covered by this review.
The most relevant contributions on coated capillaries in
MEKC reported in the last 2 years are the characterization
of the SDS-induced EOF in MEKC by using the polyelec-
trolyte multilayer coating approach [114] and a current
review of the application of physically adsorbed polymer
coatings in CE [115]. The first report constitutes a new
contribution of Pranaityte and Padarauskas [114] on the use
of bilayer coatings whose capillaries are coated with
poly(diallyldimethylammonium chloride) followed by the
physical adsorption of SDS in a second layer under MEKC
conditions. The concentration of SDS is a critical parameter
in this process: at approximately or just below the condi-
tional CMC value, a variable but highly reproducible cath-
odal EOF can be achieved, whereas at greater concentrations
the EOF is independent of the pH. The paper also revises in
detail the effect of organic solvents, urea, b-CD and non-
ionic surfactants on EOF behavior, and the results can be
helpful for separation optimization in MEKC using dyna-
mically coated capillaries. In addition, Blomberg and co-
workers [116] report on the first approach for the separation
and characterization of anacardic acids by MEKC-MS using
polydimethylacrylamide-coated capillaries. The best resolu-
tion of the analytes (nine different isomers were identified)
requires the use of a complex BGE consisting of 10 mM
phosphate buffer pH 6.5 with the addition of 1 M urea,
20% ACN, 20 mM SDS, 10 mM of b-CD and 1 mM of
heptakis-6-sulfo-b-CD.
The use of 2-D separation systems is an emerging area
in CE for achieving high-resolution capabilities, especially
for protein analysis in clinical samples. Most of the papers
on this topic in the period covered by this review are
contributed by the Dovichi group [117–122], who are an
important reference in this field. Two important contribu-
tions can be highlighted based on modifications of the first-
generation 2-D CE instrument developed by the group in
2004, both focused on decreasing analysis time. In the first,
narrow inner diameter capillaries are used to facilitate the
application of higher electric fields than in the original 2-D
CE instrument in order to reduce separation time from ca.3–5 to 1 h [117]. In this 2-D CE system, the first capillary
employs capillary sieving electrophoresis using a replaceable
sieving matrix, and the fractions are periodically transferred
across an interface into a second-dimensional capillary
where components are further resolved by MEKC. In addi-
tion to high resolution, the 2-D CE system also provides
high sensitivity because analytes are detected by LIF using
3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) as the labeling
fluorogenic reagent. Analysis of Barrett’s esophagus tissues
[117], characterization of single MCF-7 breast cancer cells
[118], separation of the subcellular fractioning of isolated
components from mouse adrenal gland cells (AtT-20) [119]
and characterization of protein expression from a single
mouse embryo [120] are interesting applications of this
approach. The second modification of the original 2-D CE
design is the substitution of the one pair of capillaries to
analyze a single sample for a multiplexed system that allows
separation of five samples in parallel [121]. Therefore,
samples are injected into the five first-dimension capillaries,
capillary sieving electrophoresis fractions are transferred
across an interface to five second-dimension capillaries
(MEKC) and finally FQ-labeling analytes are detected by LIF
in a five-capillary sheath-flow cuvette. The 2-D CE system
has been successfully used for the separation of proteins and
biogenic amines from homogenates prepared from lung
cancer (A549) and mouse AtT-20 cell lines [121, 122].
The design of the capillary–capillary interface is a
significant feature for developing a 2-D CE instrument with
an optimum transfer of the analytes from the first to the
second dimension. Recently, Sahlin [123] has developed a
novel low dead-volume capillary–capillary interface that
allows straightforward filling of the systems with different
solutions in the two different capillaries. The design of this
interface allows two channels to be tangentially in contact
with each other and connected through a small opening at
the contact area. Since the channels do not cross each other
in the same plane, the capillaries can easily be filled with
different solutions. This home-made 2-D CE system is easy
to implement in the laboratory because commercial fused-
silica capillaries can be used and the interface can be easily
made up reproducibly with simple equipment without
microfabrication technology. The approach has great
potential for use in different types of CE applications, such
as CZE at two different pH values and CZE followed by
MEKC. Finally, another noteworthy and relevant contribu-
tion is a simple methodology for converting a commercial
CE-MS instrument into an integrated 2-D CE system [124],
which only requires the construction of a modified electrode
and a two-way electrical switch. The modified electrode
(constructed from a commercial electrode) allows the
simultaneous introduction of two capillaries and the two-
way electrical switch is used to apply the voltage difference
to the first or second capillary but must be compatible with
the application of high voltage. The first-dimension capillary
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operates as a typical CE instrument with UV/visible detec-
tion; then fractions leaving the first dimension are auto-
matically collected and introduced into the second
dimension, for analysis on a CE-MS apparatus. The
approach proposed, which operates in a highly automatic
manner, is flexible and allows various combinations of CE
modes to be implemented, such as the screening of anti-
biotic families (nitroimidazoles and tetracyclines) in the first
CZE dimension and the subsequent resolution of each
family (nitroimidazoles by MEKC and tetracyclines by CZE)
in the second dimension.
The use of chemometric techniques is another current
approach for improving MEKC resolution. Thus, optimiza-
tion of separation parameters for the baseline resolution and
short migration times has been the subject of studies
reported by several authors using different approaches in
the period covered by this review [125–129]. Statistical
experimental [125] and factorial [126, 127] designs, multi-
linear regression [128] and genetic algorithms [129] are the
chemometric tools used to search for the optimum condi-
tions in MEKC separation. In general, resolution, peak
symmetry and analysis time are established as responses in
the approaches tested, which provide a good optimization of
the MEKC buffer system. The selection of the appropriate
chemometric technique is closely related to the complexity
of the mixture under study, and therefore the use of
powerful chemometric tools is not always the best choice.
However, these powerful chemometric techniques are
needed to improve resolution based on the quantification of
highly overlapping CE peaks, which is a growing area of
interest in the last years. Therefore, Li and co-workers [130]
reported the application of three different kinds of artificial
neural networks (ANNs) such as radial basis function,
generalized regression and linear neural networks in order
to resolve overlapped peaks. ANN inputs are selected by
applying principal component analysis to the 2-D array of
data generated by MEKC with DAD detection. The results
proved that the proposed ANN approach based on principal
component analysis input selection is suitable for the
quantification of the four components at their overlapped
MEKC peaks.
4 Detection techniques
4.1 MS
As the hyphenation of MEKC and MS has been a
challenging topic for several years, an array of means has
been researched to accomplish this coupling. As stated in
the previous review [1], additional work is still required to
reach a similar potential to that shown by this detection
technique in CZE, where robust CZE-MS methods are
currently available, especially in pharmaceutical analysis.
Two interesting overview articles have been published on
the period covered by this review [6, 131], mainly focused on
recent developments in interfacing and soft ionization
techniques, atmospheric-pressure photoionization versusthe typical ESI, as approaches to circumvent compatibility
problems in MEKC-MS. However, the contributions
reported in the period covered by this review are largely
focused on the use of polymeric surfactants with ESI-MS
detection to afford practical MEKC-MS determinations. This
trend can be ascribed to the fact that the use of these PSPs
with ESI interface is to date a more useful and practical
choice because the atmospheric-pressure photoionization
interface, although seeming to overcome many of the
deficiencies of MEKC-MS, does not provide the needed
sensitivity for the analysis of real samples [6].
As stated above, polymeric surfactants have been the
most frequent choice for developing practical MEKC-MS
determinations in the period covered by this review,
although the PF approach with conventional SDS micelles
has also been proposed along with the use of BGEs
containing volatile surfactants and buffers to afford the
direct coupling of MEKC to MS. The PF approach was
employed for the determination of nitroimidazoles using
ammonium acetate as the electrolyte [124] as a second
dimension of a 2-D CE system in which nitroimidazoles and
tetracyclines are separated in the first dimension by CZE at
pH 8.5. The method was really optimized with the aim of
being an application in the proposed integrated 2-D CE
system described in Section 3.3, instead of a sensitive
MEKC-MS practical methodology. Also the potential of the
hyphenation of MEKC with ESI-MS for the impurity
profiling of drugs using galantamine and ipratropium as
test samples has been evaluated using anionic SDS micelles
although in a BGE containing sodium phosphate and ACN
[132]. The authors have demonstrated that despite ionization
suppression by non-volatile buffer and surfactant, MS
detection of impurities present around 0.1% level can be
possible, demonstrating the great potential of the MEKC-
MS/MS system for the detection and structure elucidation
of minor impurities in drug substances. As in the previous
review, little work has been done toward finding suitable
volatile BGEs for the direct coupling of MEKC to MS. Biesen
and Bottaro [133] have tested the potential of ammonium
perfluorooctanoate (APFOA) for the analysis of N-methyl-
carbamate pesticides by MEKC-ESI-MS. This volatile
surfactant can be introduced into an MS without the adverse
effects of less volatile surfactants such as SDS, and its
residue can be easily removed from the ion-source after use,
as has recently been reported by Petersson et al. [134]. The
proposed MEKC system using a BGE consisting of 50 mM
APFOA at pH 9.0/isopropanol 98:2 was sensitive enough to
afford LODs between 0.01 and 0.08 mg/L in the SIM mode,
and even lower if an SPE pre-concentration step is used.
APFOA seems to be an effective alternative to SDS in
MEKC-ESI-MS because more cumbersome techniques such
as PF and reverse migrating micelles can be avoided.
Another strategy for the direct coupling of MEKC to
ESI-MS is based on the use of polymeric surfactants,
Professor Shamsi’s research group being a significant
reference in this field. In the period covered by this review,
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several contributions have been reported by the Shamsi
group, such as the determination of ephedrine alkaloids
using polysodium N-undecenoxycarbonyl-L-leucinate as
chiral polymeric surfactant [135, 136], bezodiazepines and
benzoxazocine with poly(sodium N-undecenoxy carbonyl-L,L-
leucyl-valinate) [137] and warfarin enantiomers using poly-
sodium N-undecenoyl-L,L-leucyl-valinate [138]. The sensitiv-
ity and enantioselectivity provided by these methodologies
are in general better than those achieved through UV
detection. The spray chamber parameters as well as sheath
liquid conditions are found to significantly influence the MS
S/N of the analytes, whereas enantioselectivity is closely
related to the selected polymeric surfactant. Recently, this
research group has synthesized three amino acid-derived
(L-leucinol, L-isoleucinol and L-valinol) sulfated chiral
surfactants and evaluated their potential in MEKC and
MEKC-MS [139]. Among the three polymeric sulfated
surfactants studied, polysodium N-undecenoyl-L-isoleucine
sulfate with two chiral centers on the polymer head group
provided an overall higher enantioresolution in acidic
medium for acid and/or basic analytes, such as pseudoe-
phedrine, b-blockers, phenoxypropionic acid, benzoin deri-
vatives, benzodiazepinones, etc. Regarding sensitivity, lower
LODs were also obtained at acidic pH 2.0 (325 ng/mL),
approximately 16 times better than at pH 8.0 (5.2 mg/mL).
This work opens new perspectives in MEKC-MS using
polymeric surfactants, concretely anionic chiral polymeric
surfactants, due to the superiority of chiral separation and
sensitive MS detection at low pH over conventional high-pH
chiral separation and detection.
4.2 LIF spectroscopy
MEKC with LIF detection is currently a powerful analytical
tool routinely used for the sensitive monitoring of fluorescent
analytes, either native fluorescent or dye-labeled compounds.
LIF detection is consolidated in MEKC analysis as shown in
the large number of applications that have been reported over
the period covered by this review, mainly involving the
determination of analytes showing no native fluorescence.
Despite this fact, the contributions reported on MEKC-LIF are
mainly focused on the consolidation of ‘‘classical’’ excitation
sources like the argon-ion laser and evaluation of the use of
diode lasers as alternative sources, instead of on proposals for
new labeling schemes. This is because the existing probes
provide adequate sensitivity for the determination of a great
number of compounds in many samples; the new contribu-
tions try to achieve better derivatization chemistry, especially
by decreasing the derivatization time and increasing the
stability of the derivatives.
The argon-ion laser emitting at 488 nm is the most
frequent excitation source employed in LIF detection.
Although this excitation source has been used for the
sensitive determination of native fluorescent compounds
such as doxorubicin and doxorubicinol by CD-MEKC [84],
the majority of the MEKC-LIF methods reported in the
period covered by this review are focused on the determi-
nation of amine compounds after their derivatization with
two popular amino-reactive labels: fluorescein- and nitro-
benzo-2-oxa-1,3-diazol-based reagents. Among fluorescein
analogs, classical FITC [72, 73], 5-(4,6-dichloro-s-triazin-2-
ylamino) fluorescein (DTAF) [140] and 6-oxy-(N-succinimi-
dyl acetate)-9-(20-methoxy-carbonyl) fluorescein (SAMF) [58,
100, 141] have been used for the determination of amino
acids, biogenic amines and catecholamines in foods [58, 72]
and pharmaceutical [140] and biological samples [100, 141].
SAMF is the best alternative due to its better labeling
chemistry: derivatization was carried out at room tempera-
ture for ca. 10 min whereas FITC needs hours to complete
the derivatization and DTAF 30 min at 451C. Moreover,
FITC forms several by-products and DTAF and derivatives
are relatively unstable in water. Regarding resolution, a BGE
consisting of SDS [58, 141] or Brij-35 [100] and borate buffer
at an alkaline pH provides efficient baseline separation of
labeled analytes and also from the interfering peaks of label
excess. NBD-F [45, 78, 142] and 4-chloro-7-nitro-2,1,3-
benzoxadiazole [143, 144] are the amino-reactive labels with
the nitrobenzo-2-oxa-1,3-diazol moiety that are employed
most frequently for MEKC-LIF determination of amine
compounds using the argon-ion laser as excitation source.
These reagents, and especially NBD-F, provide improved
label chemistry with respect to fluorescein analogs, except
for SAMF. The proposal of the 4-chloro-7-nitro-2,1,3-
benzoxadiazole as an alternative to NBD-F is mainly based
on the high cost of the latter for practical applications. The
improved labeling chemistry afforded by NBD-F has fores-
ted its implementation in the in-capillary format for the
determination of organophosphorus pesticides in spiked
river water samples [45]. Profiling of amine metabolites in
human biofluids [78] and determination of spermine
synthase activity [142], epinephrine and dopamine in tradi-
tional Chinese medicines [143] and catecholamines and
amino acids in human urine samples [144] are the MEKC-
LIF applications reported using these labels in the period
covered by this review. As with fluorescein derivatives, good
separations can easily be achieved between derivatives and
from the excess of the label using a simple BGE containing
SDS and borate buffer (organic modifiers are required in
some cases) with significant sensitivity: LODs at ng/mL
level are generally reported for the analytes. Another inter-
esting contribution to LIF detection in MEKC is that
reported by the Dovichi group for 2-D CE systems [117–122].
As stated in Section 3.3, MEKC analysis is carried out in the
second dimension and LIF is used for the detection of
compounds of biological and clinical interest after their
labeling with FQ. In the built in-house instrument devel-
oped by the Dovichi group, the fluorescence of the labeled
analytes is detected by using a sheath-flow cuvette, and
argon-ion [119] and solid-state diode [117, 118, 122] lasers
were used as excitation sources.
The use of diode lasers as light sources for LIF has
noticeably increased in the last 2 years as an alternative to
the argon-ion laser because comparable results in terms of
Electrophoresis 2009, 30, 50–6460 M. Silva
& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com
sensitivity can be achieved with these relatively lower cost
and longer lifetime excitation sources. The increase in
research based on the use of LEDs as excitation sources in
LIF has also contributed to this growth; although LEDs have
recently been extended to the UV region, built in-house
equipment must still be used to date. As with MEKC-LIF
methods using the argon-ion laser as the excitation source,
the reported methods are useful for the determination of
amine compounds after their labeling; as a variety of diode
lasers can be used due to their lower cost, from UV to near
infrared spectrum, different labels have been proposed as a
result. Sulfoindocyanine succinimidyl ester (Cy5-NHS) [57,
145] and NDA [88, 146] are the labels whose luminescent
properties match well with the 635 and 415 nm laser
modules, respectively, built-in commercial equipment.
These reagents have a good labeling chemistry, derivatiza-
tion time between 10 and 30 min at room temperature and
provide good sensitivity, although in some cases an SPE pre-
concentration step is necessary, such as in the determina-
tion of b-lactam antibiotics [145] and glyphosate [146] in
environmental waters, in which LODs at ng/L level can be
achieved.
The contributions toward the use of LED-induced
fluorescence detection in MEKC in the period covered by
this review are essentially those reported by Li et al. [28, 30,
147]. Thus, blue (476 nm) [28], violet (410 nm) [30] and UV
(380 nm) [147] LEDs have been used for the determination
of phenethylamine designer drugs with FITC, dopamine
and norepinephrine with NDA, and tryptophan with
phenylglyoxal, respectively. In all cases, on-line sample
concentration techniques were used to improve sensitivity.
Another interesting contribution in this field is that reported
by Aspinwall and co-workers [47] based on the utilization of
a high power UV (365 nm) LED in MEKC-LIF. In addition to
its high emission intensity, also noteworthy is its excitation
wavelength, which is particularly useful for the fluorescence
detection of common small-molecule fluorogenic labels.
This compact and inexpensive UV-LED allows the deter-
mination of native fluorescent PAHs and glutamic and
aspartic acids, proteins and peptides after their derivatiza-
tion with o-phthalaldehyde with LODs at nM levels and
without the need for on-line sample concentration.
Finally it is noteworthy to point out the introduction by
Chen et al. [148, 149] of a novel luminescence detection
method in MEKC based on continuous wave-based multi-
photon excitation fluorescence (MPE) detection using cheap
diode laser instead of high cost femto-second pulse laser.
Two major benefits can be reported on the use of MPE in
MEKC: (ii) an ultra-low detection background that allows
LODs about zeptomole level to be readily achieved and (ii)
capability of simultaneous multicolor excitation in a broad
range; hence, using only one excitation line it is possible to
detect complex components labeled with different fluor-
escent tags. Although the end-column MPE configuration
exhibits better detectability, due to less light scattering,
the LODs achieved are not as good as those provided by
the fento-second pulse laser; however, when compared with
the classical argon-ion laser excitation source, better mass
detectability and higher separation selectivity were achieved,
although the sensitivity in concentration was not so
encouraging.
5 Conclusions and prospects
From the advances reported in the different topics covered
by this review on approaches for increasing sensitivity and
resolution in MEKC-based determinations, it can be inferred
that further improvements are still needed, especially in
sensitivity, because of the extremely rapid and extensive
developments in resolution in recent years. Reasonably,
initial improvements in MEKC were focused on increasing
resolution and thus research areas such as MEEKC and
polymeric PSPs in MEKC have developed to such an extent
so far that they warrant separate discussion and thus are not
included in this review. The ‘‘classical’’ BGE containing SDS
micelles with the incorporation of different additives is a
useful and consolidated choice to afford the resolution of
complex mixtures of charged or neutral analytes. The recent
introduction of carbon nanostructures as PSPs is an
interesting topic that requires further studies to assess their
real potential. One of the biggest drawbacks of MEKC so far
is problems associated with its direct connection with MS.
The advances in this field have not progressed as expected in
the period covered by this review, and from the reported
contributions it can be inferred that the use of polymeric
surfactants seems to be the alternative with the greatest
potential for avoiding the problems associated with this
direct coupling, although the design of new approaches for
interfacing MEKC to MS is another topic of great relevance.
The consolidation of on-line sample concentration techni-
ques in routine MEKC analysis, the noticeable growth in LIF
detection using the ‘‘classical’’ argon-ion laser excitation
source also for routine analysis and the improvements in
diode lasers including LEDs as excitation sources are the
most significant approaches for improving sensitivity in
MEKC. With advances in laser technology, lower cost diode
excitation sources will be integrated in commercial instru-
ments providing excellent detection sensitivity, especially
through the use of LEDs.
The author gratefully acknowledges funding by SpainsDepartment of Research of the Ministry of Education andScience under Project CTQ2007-63962 and by FEDER.
The author has declared no conflict of interest.
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