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Electrophoresis 2014, 00, 1–9
Wojciech Grochocki1 Michał J. Markuszewski1 ∗ Joselito P. Quirino2
Review
Multidimensional capillary electrophoresis
1 Department
of Biopharmaceutics and Pharmacodynamics, Medical University of Gdansk, Gdansk, Poland 2 Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Hobart, Australia
Received August 29, 2014 Revised September 11, 2014 Accepted September 12, 2014
Multidimensional separation where two or more orthogonal displacement mechanisms are combined is a promising approach to increase peak capacity in CE. The combinations allow dramatic improvement of analytical performance since the total peak capacity is given by a product of the peak capacities of all methods. The initial reports were concentrated on the construction of effective connections between capillaries for 2D analysis. Today, 2D and 3D CE systems are now able to separate real complex biological or environmental mixtures with good repeatability, improved resolution with minimal loss of sample. This review will present the developments in the field of multidimensional CE during the last 15 years. The endeavors in this specific field were on the development of interfaces, interface-free techniques including integrated separations, microdevices, and on-line sample concentration techniques to improve detection sensitivity. Keywords: Capillary electrophoresis / Comprehensive / Heartcutting / Interfaces / Multidimensional separations DOI 10.1002/elps.201400416
1 Introduction Multidimensional (MD) separations are used in order to increase the peak capacity and thus allow the separation of very complex mixtures. There are two criteria introduced by Giddings for multidimensional separation. First, the separations must be orthogonal. The orthogonal separations must be based on different chemical or physical properties of the molecule, such as charge, molecular mass, hydrophobicity, or chirality. The second criterion defines that the resolution obtained in the first dimension must be preserved in the subsequent dimension. When on-line separation is performed, the second dimension running must be completed faster than the first one [1,2]. In ideal comprehensive 2D separations, the peak capacity is given by the product of the individual separation peak capacities. For example, if two techniques with a peak capacities of 1000 each were coupled, the obtained 2D system would have maximum peak capacities of 1 000 000. In practices, it is much lower. This is on account of the incomplete orthogonality between two dimensions as well as the result of first-dimension undersampling. The history of MD separations is summarised in Table 1. The first use MD/2D separation was reported by Martin and coworkers in 1944 when two chromatographic steps were used in paper analyses [3]. In 1975, O’Farrell reported the
Correspondence: Dr. Joselito P. Quirino, Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Hobart 7001, Australia E-mail: [email protected]
Abbreviations: AFMC, analyte focusing by micelle collapse; CNGSE, capillary nongel sieving electrophoresis; CSE, capillary sieving electrophoresis; MD, multidimensional C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
separation of a complex protein mixture using combination of isoelectric focusing and SDS-PAGE as the first and second dimension, respectively. The separation produced a large number of spots that could be correlated with pI and molecular weight of the proteins within the sample [4]. This opened the age of 2D PAGE that is widely used for the separation of complex mixtures of proteins and peptides in biological samples [5, 6]. Despite the fact that 2D PAGE has high resolution and orthogonal nature, it also has some essential limitations. These are: (i) only proteins or peptides can be separated; (ii) it is time-consuming where analysis can take even two days; (iii) on-line coupling with MS requires sophisticated systems or manual intervention; (iv) quantitative analysis is not possible; and (v) efficiency may be insufficient to separate all components present in the sample, e.g. proteins with extreme value of pI, mass or hydrophobicity cannot enter the gel. 2D separations based on chromatography and/or electrophoresis in columns or capillaries may overcome some of the limitations of 2D PAGE. These separations are amenable to almost all molecules and are also easy to couple with wide range of detection methods including UV absorbance, LIF and MS. The interest of 2D column separations flowered in the 80s when Guiochon and coworkers described the 2D LC system [7]. A significant development was the direct coupledcolumn LC with MS detection for protein identification described by Yates et al. in 2001 [8]. This method employs one micro-capillary column packed with two different stationary phases instead of two regular columns. The first dimension is strong cation exchange chromatography, while the second is RP-LC. Another milestone was the on-line coupling of LC with CE (2D LC × CE) [9, 10]. The near orthogonality between LC ∗
Additional corresponding author: Dr. Michał J. Markuszewski, E-mail: [email protected]
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Table 1. History of 2D separations
Separations techniques
Connection between dimension
Analyte
Application content
Ref.
Double-way paper chromatography IEF/SDS-PAGE SCX/RP-LC
Amino acids Proteins Proteins
Analysis of peptide hydrolysate Separation of compounds from E. coli Proteome of the S. cerevisiae strain BJ5460
[3] [4] [8]
RP-LC/CZE
— Manual Micro-capillary column packed with two different stationary phases Six-port valve
Proteins
[9]
IEC/SEC
Eight-port valve
Proteins
Submicellar CE/MEKC CZE/CSE
Flow-gated interface Single capillary interface-free
[16] [43]
MEKC/CZE
Microchip device
Proteins Polystyrene sulfonates Peptides
Analysis of peptide standards and fluorescently labeled peptide products from a tryptic digest of ovalbumin Separation of protein standards and serum proteins Separation of proteins from single cancer cell Separation of mixture of standards Analysis of mixture containing tryptic digests
[57]
and CE modes allows increasing peak capacity. Furthermore, CE is much quicker than LC and provides high resolution of the sample. However, the issues in 2D LC × CE were (i) dead volume caused by the interface connecting column with the capillary and (ii) the large volume difference of effluent from LC and the sample requirement in CE. A flow valve was developed by Larmann and coworkers to address these issues [11]. Other notable developments were (i) 2D RP-HPLC × CE platform coupled to ESI-TOF-MS to characterize the phenolic fraction in olive oil [12]; (ii) 2D RP-HPLC × MEKC for separation of phenolic acids and flavonoids in green tea sample [13, 14]. CE is an effective, powerful separation technique and has been used in many areas of chemistry, pharmacy, biology, etc. Nevertheless, there are some limitations of CE in analyses of complex mixtures. The peak capacity and sensitivity can be too low for handling such samples. The development of multidimensional CE was then pursued by several research groups, most notably by Dovichi and Cottet groups, to improve the peak capacity. The plurality of CE techniques such as CZE, MEKC, capillary sieving electrophoresis (CSE), ITP, CIEF, CEC, CGE provides a great number of possible 2D systems, many of which have already been applied [15]. Compared to HPLC, CE allows higher resolution expressed as a number of theoretical plates. In spite of the fact that sensitivity of electromigration techniques is lower than chromatography, on-line sample concentration and stacking methods have been developed and improved the LOD of CE. Moreover, CE is a rapid process, e.g. a single run takes usually a few minutes In addition, very little amounts of chemicals and sample are needed which make CE relatively cheap and environmentally friendly. A combination of two or more CE techniques into MD systems should provide a powerful, quick, and cheap tool in an increasingly demanding analysis. The aim of this review is to introduce and describe the evolution of MD-CE systems that have been reported over the last 15 years. The reports were mainly for the improvement of resolution power and peak capacity of extremely complex samples, as well as detection sensitivity. The very first 2D-CE systems, especially employing interfaces between C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[10]
capillaries struggled with many problems. The Dovichi team faced issues such as dead volume, long time of analysis, and loss of sample during transfer from the first to the second dimension [16]. However, many of these adversities have been eliminated. Recently described systems use improved leakage-free interfaces that allow for high efficiency, reproducible, and accurate analysis of the sample. Similarly, in the case of systems employing a single capillary, such as the so-called heart-cutting techniques, the problems encountered such as peak broadening during the transfer to dimensions have been overcome [17–19]. In the case of sensitivity, stacking steps were popularly implemented [20–22]. On the applications side, the analysis of biological and bioactive analytes such proteins and peptides, amino acids, plant metabolites, and drugs and of environmental pollutants were reported. In addition, the possible ways of MD-CE development in the future are also included. There were 64 articles regarding MD-CE published since 2000, including seven reviews. For the first five years, when MD-CE began to enter labs, researchers reported 16 scientific papers. The next five years resulted in 20 research articles and the first summary. A total of six review papers were also printed. The last five-year period provided a similar number of research papers. It must be noticed that our review is prepared in the middle of the year and a few more articles may appear until December. A steady number of papers were observed during the entire review period, but we hope in the future that other scientists in the field will discover the elegance of MD-CE.
2 Types of two-dimensional capillary electrophoresis The 2D systems can be divided according to the strategy of transport of a sample from the first to the second dimension. The first one, which involved the transfer of the entire sample from the first dimension to the second one, is called “comprehensive 2D separation.” The advancements here were mainly on interface development. The second one, www.electrophoresis-journal.com
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so-called “heart-cutting 2D separation,” involved only one or a couple of fractions from the sample. Therefore, only an isolated cut of mixture is analysed, while the rest of compounds were eluted from the capillary. Heart-cutting 2D separations can be performed through on-line, at-line, or off-line modes. The on-line mode was used when the sample was transferred directly from the first to the second dimension. Other modes were the so-called diagonal (at-line) and integrated (off-line) 2D-CE where some modification of molecules (e.g. derivatization or digestion) was necessary before analysis in the second dimension [23].
2.1 Comprehensive 2D-CE 2.1.1 Initial interfaces and their variations The comprehensive 2D-CE involved using two separation capillaries, which must be tightly connected to avoid leakage. The development of interfaces to link the capillaries in MD-CE is therefore an important area of research. Table 2 provides a summary of the interfaces described in literature. The first fully automated 2D-CE was reported by Dovichi et al. [16]. Dovichi developed a four-crossed interface that connected separation capillaries and two waste capillaries. LIF was used as an ultrasensitive detector of the separated proteins. The samples were analyzed by submicellar capillary electrophoresis in the first dimension. Then, fractions were transferred through the interface to the second capillary for CZE in the second dimension. Zeptomoles of proteins were separated and detected. The evolution of the above method was using CSE and MEKC in the first and second dimension, respectively. However, the obtained peak capacity was much lower than theoretical. The possible reason could be diffusion during the long separation time in the CSE dimension. The peak capacity in the MEKC dimension on one hand was dominated by the transfer time and the relatively short separation time. The researchers noticed that the separation efficiency must be enhanced by sealing the joint between dimensions and optimizing the parameters such a separation time, buffer composition, or length of the capillary [24]. Despite the resolution difficulties, the technology was able to separate and detect the labeled proteins from single-mammalian cell [25]. Further modifications included the use of narrow inner diameter capillaries as well as higher electric fields that allowed significant decrease of analysis times. Moreover, the use of dynamic coating and temperature control stabilised the electric current and EOF. The control of EOF then resulted to improved repeatabilty of separation performance and component mobility. However, the analysis of Barrett’s esophagus homogenates was still unsatisfactory even with a 1 h separation time [26,27]. A considerable improvement of resolution was achieved by the use of CD as buffer additives in the MEKC dimension. The Dovichi’s team suggested that resolution enhancement was achieved by two factors. First, the CDs formed new complexes with similar compounds, which had C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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different electrophoretic mobility. Second, the decreased EOF caused by the rise of conductivity of the buffer after addition of CDs [28]. Other variations (interface for two capillaries) and applications of the cross-interface 2D-CE were also described by Dovichi’s group [29–31]. The new fluorogenic reagent, the Chromeo dyes that has been compatible with CIEF, allowed the coupling of CIEF and CZE with LIF detection for protein analysis. The two separation capillaries were aligned using a buffer-filled interface. However, the 2D system resulted in poor resolution when compared to the 1D separation. This was explained by the relatively large volume of sample transferred to the second dimension [32]. More recently, Dovichi and coworkers designed a nicked-sleeve, leak-free interface (see Fig. 1). This new interface enhanced the transfer of molecules from the first to the second capillary as well as improved the precision with respect to peak area. For example, more than 90% and less than 70% transfer efficiency was observed from the new and original/traditional interface, respectively [33]. A 2D-CE using tangentially connected capillaries was reported by Sahlin [34]. The low dead-volume capillary–capillary interface that allowed easy filling of the two capillaries with different BGEs was prepared without microfabrication technology. The system was able to resolve the tryptic digest of a protein with acceptable repeatability. The RSD of migration time in the second dimension was from 1.7 to 4.0%. Whereas most of the reported 2D systems were concentrated on the separation proteins or its digest, Zhang and coworkers proposed an on-line sample concentration with 2D separation of low-molecular-weight cationic compounds via hyphenation of CZE with cyclodextrin-modified MEKC [35]. In the first dimension CZE, cation-selective exhaustive injection and transient ITP were employed for preconcentration of the analytes. Then, the concentrated and separated fractions were transferred by a newly designed interface into the second capillary. The approach showed distinct positive characteristics. First, the full automation of the system with one high voltage and four switches that were controlled by a computer. Second, the resolution was excellent and the detection limit was lowered more than 10 000-fold. Furthermore, because of the difficulty in designing a tight interface, the focusing step was a promising solution that avoided the effluent fraction from the first capillary to diffuse at the interface. The efficiency of the system was demonstrated by the analysis of complex samples of wastewater and urine that contained enantiomeric drugs [35, 36]. In another report, Zhang’s group proposed a 2D separation combining CZE and MEKC that were connected by a microhole interface, while detection was performed electrochemically. A dual on-line sample concentration strategy, pH junction, and sweeping were employed to stack the analytes in the first capillary. Cardiovascular drugs were selected as a model compounds to evaluate effectiveness and reliability of the described system. The acceptable performance was due to a few factors. First, the microhole interface improved the transfer of analytes from the first to the second capillary. Second, the pH junction sweeping produced narrow analyte www.electrophoresis-journal.com
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Table 2. Interfaces described in MD-CE
Specific name of interface
CE modes
Analyte
Application content
Ref.
Flow-gated interface
Proteins
Separation of proteins from single cancer cell
[16]
Microhole interface
Submicellar CE/MEKC CZE/MEKC CSE/MEKC
Interface between five pair of capillaries Ten-port valve interface with two conditioning loops Microdialysis junction
CSE/MEKC
Proteins
Determination of cardiovascular drugs in mouse blood; analysis of drugs in urine sample Analysis of Deinococcus radiodurans protein homogenate; protein fingerprinting of single mammalian cell; Barrett’s esophagus tissues; analysis of proteins in the mouse tumor cell homogenate; analysis of single mouse embryo lysate Analysis of lung cancer cell homogenate
[17, 36]
Cross-interface
Cardiovascular drugs Proteins
MEKC/CIEF
Proteins
[38]
CIEF/CZE
Proteins
Dialysis interface Buffer-filled interface Partially etched porous interface
CIEF/CNGSE CIEF/CZE CIEF/CZE
Proteins Proteins Proteins
Nicked-sleeve interface Hollow-fiber membrane interface
CZE/CZE CIEF/CNGSE
TAMRA Proteins
Etched porous interface
CIEF/CZE
Proteins
Analysis of tryptic digests of trypsinogen and cytochrome c Separation of tryptic digests of cytochrome c, ribocnuclease A, carbonic anhydrase II Separation of ribonuclease Analysis of proteins standards Separation of proteins standards and proteins extracted from milk Transfer efficiency evaluation Separation of Hb and proteins excreting from lung cancer cells of rat Separation of proteins standards
Figure 1. Nicked-sleeve capillary with separation capillary. The influence of human factors was eliminated by use the computerprogrammed microdicing saw to nick the sleeve capillary (adapted from ref. [33]).
bands and prevented sample zone diffusion at the interface. Finally, the system was fully controlled by a computer [17]. Wei and coworkers developed a microinjection valve that was used in a potential-free interface that connected CIEF and pressurized CEC [37]. CEC is an excellent analytical tool that combined the benefit of high selectivity of HPLC and high efficiency of CE. These features make it very promising method in proteomic study. Although good results were obtained, there were future modifications proposed, e.g. more sensitive detectors such as ESI-MS or MALDI-MS and use of a monolithic column to improve the flow of the BGE. The majority of comprehensive 2D-CE systems employed interfaces based on cross-linked methodology whereas only few different types such a tangentially connected capillaries or microhole interfaces were reported. The development of new interfaces is extremely difficult for a few reasons. First, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[24–30]
[31]
[39] [40] [32] [18] [33] [40] [41, 42]
capillaries must be precisely connected to avoid leakage and loss of analytes. Second, the interface must provide the possibility to fill the capillaries with various electrolytes. This includes the ability to alter the matrix of the fractions from the first dimension for sample compatibility for the next dimension or MS detection. Third, commercial CE instruments were not suitable for the developed interfaces. Self-made CE systems were normally used. Lastly, dead-volume that resulted from the interface significantly decreased 2D-CE separation efficiency as well as caused peak broadening. Innovative interfaces to address some of these issues are discussed below.
2.1.2 Dialysis interfaces An interesting approach to connect the capillaries between two dimensions is the use of dialysis interfaces. Compared to other types of connections, they allow, e.g. desalting the sample that may have importance in coupling the 2D system with MS; and performing field enhanced stacking in the second dimension. Sheng and Pawliszyn developed an interface using a 10-port valve with two conditioning loops to connect CIEF and MEKC. The interface enabled performing both comprehensive collection as well as dialysis desalting of the first dimensional effluent. Tryptic digests of trypsinogen and cytochrome c were resolved [38]. Microdialysis interfaces were developed by Lee’s and Zhang’s group for the analysis of proteins or digests. Mohan’s group connected CIEF with CZE using a microdialysis junction. Increased sample www.electrophoresis-journal.com
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Figure 2. Schematic diagrams of (A) the etched fused-silica porous junction interface and (B) on-line coupling of the CIEF with CZE systems using an etched porous junction interface. (a) and (c) are grounded, and (b) is connected to a high-voltage power supply (adapted from ref. [41]).
load and analyte concentration in CIEF and further sample concentration via transient ITP prior to CZE separation was shown. Their system could be easily coupled with ESI-MS for protein or peptide detection in proteomic studies [39]. Zhang and coworkers also combined CIEF and CZE but using a hollow-fiber membrane interface [40]. The system enabled better resolution than 1D mode but it was still not able to perform a satisfactory separation. Zhang’s group then combined CIEF with capillary nongel sieving electrophoresis (CNGSE) using the same hollow-fiber membrane interface. CNGSE as the second dimension eliminated the incompatibility of CIEF with cross-linked polyacrylamide gel. The system had a higher resolving power and peak capacity than 1D separation. However, the connection with MS was not possible due to the use of SDS in the running buffer. This limited the usage of the system in proteomic research.
2.1.3 Etched porous interface One of the greatest improvements in 2D-CE systems was the removal of the dead-volume by using etched porous interfaces. It is noted that the dead-volume dramatically decreased the efficiency of 2D-CE. Liu and coworkers described an oncolumn etched fused-silica porous junction for the on-line coupling of CIEF with CZE. They prepared the interface with hydrofluoric acid to etch part of the wall of a fused-silica capillary. The interface acted like a porous glass membrane that allowed only small molecules to pass through. This included ions during electrophoretic separation. Figure 2 presents the scheme of reported system where a mixture of proteins was successful separated [41, 42]. Afterwards, the robustness of interface was improved by modification of the etching technique where only a partially etched porous interface was made. The partially etched interface was easier to fabricate and allowed the separation of a mixture of protein standards and proteins extracted from milk using monolithic immobilized pH gradient-based IEF and CZE [18]. All reported interfaces are summarised in Table 2. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.2 Heart-cutting 2D-CE 2.2.1 Interface-free heart-cutting 2D-CE Heart-cutting 2D-CE in a single capillary was reported for the first time by Cottet et al. [43]. The usage of a single capillary excluded the possibility of loss of material between the two dimensions. In addition, the approach did not require any special equipment, thus a conventional apparatus was employed. The technique is referred to here as interface-free heart-cutting 2D-CE since the 2D separation did not require any interface. The fractions of interest were mobilized via pressure or voltage in order to facilitate analysis in the second dimension. The selectivity of the second dimension was also achieved by pressure or voltage application to introduce the BGE or pseudostationary phase. In the initial report, a mixture of polymers were analysed using CZE and CSE in the first and second dimension, respectively. To isolate the fraction of interest at the end of the first dimension, three various strategies were proposed. The first one required switching the voltage polarity; the second used pressure or hydrodynamic flow; and the third needed polarity switching and EOF manipulation. It was required that the second separation medium must enter the capillary with a higher apparent velocity than the isolated fraction. To achieve this, Cottet’s group proposed to perform the second dimension in counter-EOF mode [43]. The potential of interface-free heart-cutting 2D-CE was then investigated for the separation of sample containing ten polyelectrolytes of different charge densities and different molecular masses [44]. Interface-free heart-cutting 2D-CE was then applied for the on-line purification and separation of derivatized amino acids. The by-products coming from the sample derivatization step was cleaned or removed by CZE. Then, only an isolated fraction was transferred into the second dimension (i.e. MEKC) by a specific pressure and voltage program. The hydrodynamic mobilization of the fraction, however, reduced the efficiency of the second dimension because of peak broadening due to Taylor dispersion. The possible designs
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Figure 3. Schematic representation of interface-free heart-cutting 2D-CE procedure. The schema illustrates two different approaches. (A) is possibly when the second dimension of the separation can enter in the capillary by EOF pumping, whereas (B) involves hydrodynamic mobilization of isolated fractions. Steps includes: (1) first-dimension separation (injection by the inlet end of the capillary); (2) isolation and return of the fraction of interest (fraction B) to the inlet end of the capillary; (3) starting of the second-dimension separation; (4) separation of the fraction of interest in the second dimension (adapted from ref. [45]).
of interface-free heart-cutting 2D-CE were discussed and a schematic representation of their procedure is illustrated in Fig. 3 [45]. The use of multiple detection points was then implemented in interface-free heart-cutting 2D-CE. Cottet’s group used two contactless conductivity detectors to monitor the purification/isolation steps. This was critical in the selection of the pressure and voltage used in the first dimension. They analyzed a mixture of 22 underivatized amino acids using achiral and chiral running buffers in the first and second dimension, respectively. To prevent peak broadening that was due to hydrodynamic mobilization, a 10 m id capillary was used. The narrower capillary also allowed the use of high pressures that reduced the analysis time. The manipulation of parameters using commercial CE instrumentation was however, a challenge due to software constraints. The connection of extra equipment to the CE system could also be problematic [46]. The sensitivity of the 2D-CE method was later improved using transient moving chemical reaction boundary as the stacking technique [47]. Zhang and coworkers then implemented an interfacefree heart-cutting 2D-CE with electrochemical detection for the analysis of cardiovascular drugs in blood samples. CZE as the first dimension was used to purify a mixture of -blocker
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drugs and to eliminate unwanted compounds. To overcome the peak broadening that resulted from the applied pressure during the transfer to the second dimension MEKC, a dual on-line sample concentration or stacking method using field amplified/enhanced sample stacking and sweeping was proposed. The stacking method also improved the detection sensitivity 3.8-fold compared to the separation without stacking [48]. The method was improved via stacking and was applied to the analysis of the components of Herba Leonuri sample and yielded 1000-fold sensitivity enhancements [49, 50]. The stacking was via sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) [51]. More recently, an interface-free heart-cutting CE was reported by Kukusamude et al. [19]. They stacked and separated a mixture of neutral and cationic analytes by using CZE and MEKC in the first and second dimension, respectively. The control of EOF was critical in the stacking and the implementation of the 2D separation. Figure 4 describes the steps in the 2D-CE procedure. Stacking of both species was with one or with a combination of two on-line sample concentration mechanisms. Two complex samples were tested. First, contained eight cationic drugs and five neutral steroids, all of them were soluble in water. The stacking was via two steps, namely sweeping and AFMC with organic solvent [52]. The
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Figure 4. Stacking and separation of two different classes of compounds (cationic (+) and neutral (n) analytes) in interfacefree 2D heart-cutting CE (CZE × MEKC). (A) The fused silica capillary was filled with a low pH CZE electrolyte. The sample was injected as a long plug. The CZE electrolyte was placed at both ends of the capillary. (B) A voltage was applied at positive polarity (cathode at the detector end). The analytes were focused by stacking. (C) Continued application of voltage caused the migration of the concentrated cationic analytes to the detector. (D) The cationic analytes migrated out of the capillary and the concentrated neutral analytes were purified and remained inside the capillary. The 1st dimension analysis ended. (E) The start of the 2nd dimension analysis was the replacement of the CZE electrolye at both ends of the capillary by the low pH MEKC electrolyte with SDS micelles. The presence of micelles is depicted by the square patterned zones. Application of voltage at negative polarity (anode at detector end) caused the electrophoretic migration of the SDS micelles into the capillary. The micelles eventually penetrated or swept the neutral analyte fraction. (F) Continued application of voltage caused the micelle bound analytes to separate and migrate to the detector. The 2nd dimension and analysis ends when all the analytes were detected (adapted from ref. [19]).
Table 3. Interface-free heart-cutting 2D-CE
CE mode
Analyte
Application content
Ref.
CZE/CSE CZE/MEKC Achiralcze/chiralCZE CZE/MEKC MEKC/CZE CZE/MEKC
Polystyrene sulfonates Amino acids Amino acids -Antagonists Flavonoids Cationic and neutral analytes
Separation of mixture of standards Separation of mixture containing 12 standards Chiral separation of mixture of 22 native amino acids Determination of drugs in rat blood Determination of flavonoids in Leonura cardiac and in mouse blood Analysis of artificial mixture and real water sample
[43, 44] [45] [46, 47] [48] [49, 50] [19]
second, consisted of three herbicides and three poorly water soluble neutral pesticides. The solubilization of the analytes was facilitated by the addition of micelles in the sample and stacking was via AFMC with organic solvent. It was noted that the stacking mechanisms of sweeping and AFMC employed the presence of electrolytes in the sample diluents, thus the 2D-CE methods should be amenable to samples with salt. Stacking provided one to two orders of magnitude improvement in concentration sensitivity. The increase in peak capacity by the multidimensional separation approach and improved concentration sensitivity by stacking may further expand the applicability of CE for the analysis of complex and dilute samples. Table 3 summarizes the reports on interfacefree heart-cutting 2D-CE for easy reference.
2.2.2 Diagonal and integrated 2D-CE Two interesting forms of 2D-CE called diagonal CE [53–56] and integrated 2D-CE [23] were reported. In both approaches, the analytes migrated outside the first dimension and were processed before analysis in the second dimension. Dovichi’s group developed diagonal CE that employs two identical C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
separation modes in both dimensions. In the initial demonstration, the first dimension was used to separate proteins. At the end of the capillary, a microreactor containing enzyme that modified some part of the analytes was placed. Thereafter, fractions were transferred into the second capillary and analyzed. Diagonal CE is a promising technique whereby stoichiometry of enzymatic processes, such as protein phosphorylation, could be determined [53–56]. An integrated 2D-CE using a modified commercial CEMS system was proposed by Santos et al. [23]. The separated zones in the first dimension were collected into a vial and then were reinjected into the second dimension using the same CE-MS instrumentation. The vial where the fractions were collected may be considered as a "pseudo-interface." They coupled MEKC as the first dimension and CZE as the second dimension. The flexibility, efficiency, and robustness were satisfactory, however, the sensitivity of the system was limited by the fraction collection (i.e. the method requires about 1 L of buffer in the vial to collect molecules). The sensitivity issue was solved by modifying the arrangement of electrodes or by using on-line sample concentration in the second dimension. The system could be used in a combination of various electrophoretic modes.
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3 Microchip devices Ramsey and coworkers reported a number of combination of electromigration modes placed in a microchip, including MEKC and high-speed CZE, electrochromatography and CZE, and IEF and CZE [57–59]. Microfabricated fluidic devices were potentially useful for MD separation because highefficiency separations could be achieved and small sample volumes could be manipulated with minimal dead volumes between interconnecting channels. The microchip platform was improved by optimizing the turns in the serpentine channel [60]. This minimized the geometrical contributions to band broadening and provided enough channel length for high-efficiency separations. Finally, the use of higher applied voltages for microfluidic 2D separations offered the potential for extremely fast, high resolution, and high peak capacity analyses of peptides and protein digests.
4 Three-dimensional capillary electrophoresis In spite of the fact that there is still a lot of problems to improve the 2D-CE systems, some research groups have started to construct more advanced instruments for MD separations. The first 3D-CE apparatus was reported by Hanna et al. in 2000 [61]. Their system coupled cITP with CZE and MEKC but only applied to standard samples. There was renewed interest to 3D-CE in 2013 when Mikus and coworkers coupled ITP-ITP-CZE modes for the determination of phthalic acids in urine matrix [62]. Their instrument employed three capillaries that were wider than usually used in CE (300– 800 m id). The first ITP mode was performed as a preseparation step, followed by separation using ITP and then CZE. The ITP steps were run under different pH values ITP1 (pH 3.1) and ITP2 (pH 4.5), while the pH of the BGE in CZE step was close to ITP2. The efficiency, repeatability, and good concentration detection limits of the proposed approach were attributed to the hydrodynamically closed separation system implemented. The advantages over 2D-CE claimed were better selection of the zone of interest for transfer to the analytical step, more effective sample cleanup, and improvement of separation selectivity.
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ity in both comprehensive and heart-cutting modes and thus stacking in MD-CE research will continue. The use of very sensitive and selective detectors such as MS is attractive to decrease the LOD and to identify components in the analysis real complex and diluted mixtures. The demonstrated use of diagonal and integrated MD-CE should find more applications such as in biological screening and food analysis. One of the most promising research areas is the miniaturization of the MD-CE systems that may reduce the costs and make MD-CE separations a portable and common tool in many fields in medicine, pharmacy, or environment sciences. Last, but not least, connecting more dimension in 3D and more dimensional systems, which have already started, gives a wide range of possibilities in demanding research works.
6 Conclusion Many diverse methods for performing 2D-CE have been presented in this review. However, all of them have the same purpose that is to improve the resolution and peak capacity. In spite of the fact that 2D-CE is not common strategy performed in many area of science, it has a great chance to become a widely used method. More recent systems not only enhance the resolution, but also lower the LOD. Furthermore, the reported 3D system heralds the new chapter in MD-CE that may significantly facilitate more complex analysis. Employing more sensitive detectors as well as using the on-line sample concentration strategies allowed the analysis of real complex samples such as those found in the biological, medical, or environmental fields. The growing importance of proteomics and metabolomics requires more efficient techniques that permit saving time, reagents, and money. The multidimensional capillary electrophoresis seems to be a high-efficiency, relatively cheap, and rapid analytical method that may be applied in many research areas in the future. The authors have declared no conflict of interest.
7 References [1] Giddings, J. C., Anal. Chem. 1984, 56, 1258–1270. [2] Giddings, J. C., J. High Resolut. Chromatogr. 1987, 10, 319–323. [3] Consden, R., Gordon, A. H., Martin, A. J. P., Biochem. J. 1944, 23, 244.
5 Future of the multidimensional capillary electrophoresis The progress made in recent years offered a glimpse of the future of MD-CE. The improved methods of interface fabrication that can be fully controlled and automated will eliminate the human factor and thus provide the reproducibility of obtained devices. For the heart-cutting mode, a single capillary is used which allowed the use of commercial available CE systems without the necessity of preparing interfaces between dimensions. On-line sample concentration was an excellent way to improve the results especially the detection sensitiv C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Electrophoresis 2014, 00, 1–9
Wojciech Grochocki1 Michał J. Markuszewski1 ∗ Joselito P. Quirino2
Review
Multidimensional capillary electrophoresis
1 Department
of Biopharmaceutics and Pharmacodynamics, Medical University of Gdansk, Gdansk, Poland 2 Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Hobart, Australia
Received August 29, 2014 Revised September 11, 2014 Accepted September 12, 2014
Multidimensional separation where two or more orthogonal displacement mechanisms are combined is a promising approach to increase peak capacity in CE. The combinations allow dramatic improvement of analytical performance since the total peak capacity is given by a product of the peak capacities of all methods. The initial reports were concentrated on the construction of effective connections between capillaries for 2D analysis. Today, 2D and 3D CE systems are now able to separate real complex biological or environmental mixtures with good repeatability, improved resolution with minimal loss of sample. This review will present the developments in the field of multidimensional CE during the last 15 years. The endeavors in this specific field were on the development of interfaces, interface-free techniques including integrated separations, microdevices, and on-line sample concentration techniques to improve detection sensitivity. Keywords: Capillary electrophoresis / Comprehensive / Heartcutting / Interfaces / Multidimensional separations DOI 10.1002/elps.201400416
1 Introduction Multidimensional (MD) separations are used in order to increase the peak capacity and thus allow the separation of very complex mixtures. There are two criteria introduced by Giddings for multidimensional separation. First, the separations must be orthogonal. The orthogonal separations must be based on different chemical or physical properties of the molecule, such as charge, molecular mass, hydrophobicity, or chirality. The second criterion defines that the resolution obtained in the first dimension must be preserved in the subsequent dimension. When on-line separation is performed, the second dimension running must be completed faster than the first one [1,2]. In ideal comprehensive 2D separations, the peak capacity is given by the product of the individual separation peak capacities. For example, if two techniques with a peak capacities of 1000 each were coupled, the obtained 2D system would have maximum peak capacities of 1 000 000. In practices, it is much lower. This is on account of the incomplete orthogonality between two dimensions as well as the result of first-dimension undersampling. The history of MD separations is summarised in Table 1. The first use MD/2D separation was reported by Martin and coworkers in 1944 when two chromatographic steps were used in paper analyses [3]. In 1975, O’Farrell reported the
Correspondence: Dr. Joselito P. Quirino, Australian Centre for Research on Separation Science (ACROSS), School of Physical Sciences-Chemistry, University of Tasmania, Hobart 7001, Australia E-mail: [email protected]
Abbreviations: AFMC, analyte focusing by micelle collapse; CNGSE, capillary nongel sieving electrophoresis; CSE, capillary sieving electrophoresis; MD, multidimensional C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
separation of a complex protein mixture using combination of isoelectric focusing and SDS-PAGE as the first and second dimension, respectively. The separation produced a large number of spots that could be correlated with pI and molecular weight of the proteins within the sample [4]. This opened the age of 2D PAGE that is widely used for the separation of complex mixtures of proteins and peptides in biological samples [5, 6]. Despite the fact that 2D PAGE has high resolution and orthogonal nature, it also has some essential limitations. These are: (i) only proteins or peptides can be separated; (ii) it is time-consuming where analysis can take even two days; (iii) on-line coupling with MS requires sophisticated systems or manual intervention; (iv) quantitative analysis is not possible; and (v) efficiency may be insufficient to separate all components present in the sample, e.g. proteins with extreme value of pI, mass or hydrophobicity cannot enter the gel. 2D separations based on chromatography and/or electrophoresis in columns or capillaries may overcome some of the limitations of 2D PAGE. These separations are amenable to almost all molecules and are also easy to couple with wide range of detection methods including UV absorbance, LIF and MS. The interest of 2D column separations flowered in the 80s when Guiochon and coworkers described the 2D LC system [7]. A significant development was the direct coupledcolumn LC with MS detection for protein identification described by Yates et al. in 2001 [8]. This method employs one micro-capillary column packed with two different stationary phases instead of two regular columns. The first dimension is strong cation exchange chromatography, while the second is RP-LC. Another milestone was the on-line coupling of LC with CE (2D LC × CE) [9, 10]. The near orthogonality between LC ∗
Additional corresponding author: Dr. Michał J. Markuszewski, E-mail: [email protected]
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Table 1. History of 2D separations
Separations techniques
Connection between dimension
Analyte
Application content
Ref.
Double-way paper chromatography IEF/SDS-PAGE SCX/RP-LC
Amino acids Proteins Proteins
Analysis of peptide hydrolysate Separation of compounds from E. coli Proteome of the S. cerevisiae strain BJ5460
[3] [4] [8]
RP-LC/CZE
— Manual Micro-capillary column packed with two different stationary phases Six-port valve
Proteins
[9]
IEC/SEC
Eight-port valve
Proteins
Submicellar CE/MEKC CZE/CSE
Flow-gated interface Single capillary interface-free
[16] [43]
MEKC/CZE
Microchip device
Proteins Polystyrene sulfonates Peptides
Analysis of peptide standards and fluorescently labeled peptide products from a tryptic digest of ovalbumin Separation of protein standards and serum proteins Separation of proteins from single cancer cell Separation of mixture of standards Analysis of mixture containing tryptic digests
[57]
and CE modes allows increasing peak capacity. Furthermore, CE is much quicker than LC and provides high resolution of the sample. However, the issues in 2D LC × CE were (i) dead volume caused by the interface connecting column with the capillary and (ii) the large volume difference of effluent from LC and the sample requirement in CE. A flow valve was developed by Larmann and coworkers to address these issues [11]. Other notable developments were (i) 2D RP-HPLC × CE platform coupled to ESI-TOF-MS to characterize the phenolic fraction in olive oil [12]; (ii) 2D RP-HPLC × MEKC for separation of phenolic acids and flavonoids in green tea sample [13, 14]. CE is an effective, powerful separation technique and has been used in many areas of chemistry, pharmacy, biology, etc. Nevertheless, there are some limitations of CE in analyses of complex mixtures. The peak capacity and sensitivity can be too low for handling such samples. The development of multidimensional CE was then pursued by several research groups, most notably by Dovichi and Cottet groups, to improve the peak capacity. The plurality of CE techniques such as CZE, MEKC, capillary sieving electrophoresis (CSE), ITP, CIEF, CEC, CGE provides a great number of possible 2D systems, many of which have already been applied [15]. Compared to HPLC, CE allows higher resolution expressed as a number of theoretical plates. In spite of the fact that sensitivity of electromigration techniques is lower than chromatography, on-line sample concentration and stacking methods have been developed and improved the LOD of CE. Moreover, CE is a rapid process, e.g. a single run takes usually a few minutes In addition, very little amounts of chemicals and sample are needed which make CE relatively cheap and environmentally friendly. A combination of two or more CE techniques into MD systems should provide a powerful, quick, and cheap tool in an increasingly demanding analysis. The aim of this review is to introduce and describe the evolution of MD-CE systems that have been reported over the last 15 years. The reports were mainly for the improvement of resolution power and peak capacity of extremely complex samples, as well as detection sensitivity. The very first 2D-CE systems, especially employing interfaces between C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[10]
capillaries struggled with many problems. The Dovichi team faced issues such as dead volume, long time of analysis, and loss of sample during transfer from the first to the second dimension [16]. However, many of these adversities have been eliminated. Recently described systems use improved leakage-free interfaces that allow for high efficiency, reproducible, and accurate analysis of the sample. Similarly, in the case of systems employing a single capillary, such as the so-called heart-cutting techniques, the problems encountered such as peak broadening during the transfer to dimensions have been overcome [17–19]. In the case of sensitivity, stacking steps were popularly implemented [20–22]. On the applications side, the analysis of biological and bioactive analytes such proteins and peptides, amino acids, plant metabolites, and drugs and of environmental pollutants were reported. In addition, the possible ways of MD-CE development in the future are also included. There were 64 articles regarding MD-CE published since 2000, including seven reviews. For the first five years, when MD-CE began to enter labs, researchers reported 16 scientific papers. The next five years resulted in 20 research articles and the first summary. A total of six review papers were also printed. The last five-year period provided a similar number of research papers. It must be noticed that our review is prepared in the middle of the year and a few more articles may appear until December. A steady number of papers were observed during the entire review period, but we hope in the future that other scientists in the field will discover the elegance of MD-CE.
2 Types of two-dimensional capillary electrophoresis The 2D systems can be divided according to the strategy of transport of a sample from the first to the second dimension. The first one, which involved the transfer of the entire sample from the first dimension to the second one, is called “comprehensive 2D separation.” The advancements here were mainly on interface development. The second one, www.electrophoresis-journal.com
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so-called “heart-cutting 2D separation,” involved only one or a couple of fractions from the sample. Therefore, only an isolated cut of mixture is analysed, while the rest of compounds were eluted from the capillary. Heart-cutting 2D separations can be performed through on-line, at-line, or off-line modes. The on-line mode was used when the sample was transferred directly from the first to the second dimension. Other modes were the so-called diagonal (at-line) and integrated (off-line) 2D-CE where some modification of molecules (e.g. derivatization or digestion) was necessary before analysis in the second dimension [23].
2.1 Comprehensive 2D-CE 2.1.1 Initial interfaces and their variations The comprehensive 2D-CE involved using two separation capillaries, which must be tightly connected to avoid leakage. The development of interfaces to link the capillaries in MD-CE is therefore an important area of research. Table 2 provides a summary of the interfaces described in literature. The first fully automated 2D-CE was reported by Dovichi et al. [16]. Dovichi developed a four-crossed interface that connected separation capillaries and two waste capillaries. LIF was used as an ultrasensitive detector of the separated proteins. The samples were analyzed by submicellar capillary electrophoresis in the first dimension. Then, fractions were transferred through the interface to the second capillary for CZE in the second dimension. Zeptomoles of proteins were separated and detected. The evolution of the above method was using CSE and MEKC in the first and second dimension, respectively. However, the obtained peak capacity was much lower than theoretical. The possible reason could be diffusion during the long separation time in the CSE dimension. The peak capacity in the MEKC dimension on one hand was dominated by the transfer time and the relatively short separation time. The researchers noticed that the separation efficiency must be enhanced by sealing the joint between dimensions and optimizing the parameters such a separation time, buffer composition, or length of the capillary [24]. Despite the resolution difficulties, the technology was able to separate and detect the labeled proteins from single-mammalian cell [25]. Further modifications included the use of narrow inner diameter capillaries as well as higher electric fields that allowed significant decrease of analysis times. Moreover, the use of dynamic coating and temperature control stabilised the electric current and EOF. The control of EOF then resulted to improved repeatabilty of separation performance and component mobility. However, the analysis of Barrett’s esophagus homogenates was still unsatisfactory even with a 1 h separation time [26,27]. A considerable improvement of resolution was achieved by the use of CD as buffer additives in the MEKC dimension. The Dovichi’s team suggested that resolution enhancement was achieved by two factors. First, the CDs formed new complexes with similar compounds, which had C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
CE and CEC
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different electrophoretic mobility. Second, the decreased EOF caused by the rise of conductivity of the buffer after addition of CDs [28]. Other variations (interface for two capillaries) and applications of the cross-interface 2D-CE were also described by Dovichi’s group [29–31]. The new fluorogenic reagent, the Chromeo dyes that has been compatible with CIEF, allowed the coupling of CIEF and CZE with LIF detection for protein analysis. The two separation capillaries were aligned using a buffer-filled interface. However, the 2D system resulted in poor resolution when compared to the 1D separation. This was explained by the relatively large volume of sample transferred to the second dimension [32]. More recently, Dovichi and coworkers designed a nicked-sleeve, leak-free interface (see Fig. 1). This new interface enhanced the transfer of molecules from the first to the second capillary as well as improved the precision with respect to peak area. For example, more than 90% and less than 70% transfer efficiency was observed from the new and original/traditional interface, respectively [33]. A 2D-CE using tangentially connected capillaries was reported by Sahlin [34]. The low dead-volume capillary–capillary interface that allowed easy filling of the two capillaries with different BGEs was prepared without microfabrication technology. The system was able to resolve the tryptic digest of a protein with acceptable repeatability. The RSD of migration time in the second dimension was from 1.7 to 4.0%. Whereas most of the reported 2D systems were concentrated on the separation proteins or its digest, Zhang and coworkers proposed an on-line sample concentration with 2D separation of low-molecular-weight cationic compounds via hyphenation of CZE with cyclodextrin-modified MEKC [35]. In the first dimension CZE, cation-selective exhaustive injection and transient ITP were employed for preconcentration of the analytes. Then, the concentrated and separated fractions were transferred by a newly designed interface into the second capillary. The approach showed distinct positive characteristics. First, the full automation of the system with one high voltage and four switches that were controlled by a computer. Second, the resolution was excellent and the detection limit was lowered more than 10 000-fold. Furthermore, because of the difficulty in designing a tight interface, the focusing step was a promising solution that avoided the effluent fraction from the first capillary to diffuse at the interface. The efficiency of the system was demonstrated by the analysis of complex samples of wastewater and urine that contained enantiomeric drugs [35, 36]. In another report, Zhang’s group proposed a 2D separation combining CZE and MEKC that were connected by a microhole interface, while detection was performed electrochemically. A dual on-line sample concentration strategy, pH junction, and sweeping were employed to stack the analytes in the first capillary. Cardiovascular drugs were selected as a model compounds to evaluate effectiveness and reliability of the described system. The acceptable performance was due to a few factors. First, the microhole interface improved the transfer of analytes from the first to the second capillary. Second, the pH junction sweeping produced narrow analyte www.electrophoresis-journal.com
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Table 2. Interfaces described in MD-CE
Specific name of interface
CE modes
Analyte
Application content
Ref.
Flow-gated interface
Proteins
Separation of proteins from single cancer cell
[16]
Microhole interface
Submicellar CE/MEKC CZE/MEKC CSE/MEKC
Interface between five pair of capillaries Ten-port valve interface with two conditioning loops Microdialysis junction
CSE/MEKC
Proteins
Determination of cardiovascular drugs in mouse blood; analysis of drugs in urine sample Analysis of Deinococcus radiodurans protein homogenate; protein fingerprinting of single mammalian cell; Barrett’s esophagus tissues; analysis of proteins in the mouse tumor cell homogenate; analysis of single mouse embryo lysate Analysis of lung cancer cell homogenate
[17, 36]
Cross-interface
Cardiovascular drugs Proteins
MEKC/CIEF
Proteins
[38]
CIEF/CZE
Proteins
Dialysis interface Buffer-filled interface Partially etched porous interface
CIEF/CNGSE CIEF/CZE CIEF/CZE
Proteins Proteins Proteins
Nicked-sleeve interface Hollow-fiber membrane interface
CZE/CZE CIEF/CNGSE
TAMRA Proteins
Etched porous interface
CIEF/CZE
Proteins
Analysis of tryptic digests of trypsinogen and cytochrome c Separation of tryptic digests of cytochrome c, ribocnuclease A, carbonic anhydrase II Separation of ribonuclease Analysis of proteins standards Separation of proteins standards and proteins extracted from milk Transfer efficiency evaluation Separation of Hb and proteins excreting from lung cancer cells of rat Separation of proteins standards
Figure 1. Nicked-sleeve capillary with separation capillary. The influence of human factors was eliminated by use the computerprogrammed microdicing saw to nick the sleeve capillary (adapted from ref. [33]).
bands and prevented sample zone diffusion at the interface. Finally, the system was fully controlled by a computer [17]. Wei and coworkers developed a microinjection valve that was used in a potential-free interface that connected CIEF and pressurized CEC [37]. CEC is an excellent analytical tool that combined the benefit of high selectivity of HPLC and high efficiency of CE. These features make it very promising method in proteomic study. Although good results were obtained, there were future modifications proposed, e.g. more sensitive detectors such as ESI-MS or MALDI-MS and use of a monolithic column to improve the flow of the BGE. The majority of comprehensive 2D-CE systems employed interfaces based on cross-linked methodology whereas only few different types such a tangentially connected capillaries or microhole interfaces were reported. The development of new interfaces is extremely difficult for a few reasons. First, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
[24–30]
[31]
[39] [40] [32] [18] [33] [40] [41, 42]
capillaries must be precisely connected to avoid leakage and loss of analytes. Second, the interface must provide the possibility to fill the capillaries with various electrolytes. This includes the ability to alter the matrix of the fractions from the first dimension for sample compatibility for the next dimension or MS detection. Third, commercial CE instruments were not suitable for the developed interfaces. Self-made CE systems were normally used. Lastly, dead-volume that resulted from the interface significantly decreased 2D-CE separation efficiency as well as caused peak broadening. Innovative interfaces to address some of these issues are discussed below.
2.1.2 Dialysis interfaces An interesting approach to connect the capillaries between two dimensions is the use of dialysis interfaces. Compared to other types of connections, they allow, e.g. desalting the sample that may have importance in coupling the 2D system with MS; and performing field enhanced stacking in the second dimension. Sheng and Pawliszyn developed an interface using a 10-port valve with two conditioning loops to connect CIEF and MEKC. The interface enabled performing both comprehensive collection as well as dialysis desalting of the first dimensional effluent. Tryptic digests of trypsinogen and cytochrome c were resolved [38]. Microdialysis interfaces were developed by Lee’s and Zhang’s group for the analysis of proteins or digests. Mohan’s group connected CIEF with CZE using a microdialysis junction. Increased sample www.electrophoresis-journal.com
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Figure 2. Schematic diagrams of (A) the etched fused-silica porous junction interface and (B) on-line coupling of the CIEF with CZE systems using an etched porous junction interface. (a) and (c) are grounded, and (b) is connected to a high-voltage power supply (adapted from ref. [41]).
load and analyte concentration in CIEF and further sample concentration via transient ITP prior to CZE separation was shown. Their system could be easily coupled with ESI-MS for protein or peptide detection in proteomic studies [39]. Zhang and coworkers also combined CIEF and CZE but using a hollow-fiber membrane interface [40]. The system enabled better resolution than 1D mode but it was still not able to perform a satisfactory separation. Zhang’s group then combined CIEF with capillary nongel sieving electrophoresis (CNGSE) using the same hollow-fiber membrane interface. CNGSE as the second dimension eliminated the incompatibility of CIEF with cross-linked polyacrylamide gel. The system had a higher resolving power and peak capacity than 1D separation. However, the connection with MS was not possible due to the use of SDS in the running buffer. This limited the usage of the system in proteomic research.
2.1.3 Etched porous interface One of the greatest improvements in 2D-CE systems was the removal of the dead-volume by using etched porous interfaces. It is noted that the dead-volume dramatically decreased the efficiency of 2D-CE. Liu and coworkers described an oncolumn etched fused-silica porous junction for the on-line coupling of CIEF with CZE. They prepared the interface with hydrofluoric acid to etch part of the wall of a fused-silica capillary. The interface acted like a porous glass membrane that allowed only small molecules to pass through. This included ions during electrophoretic separation. Figure 2 presents the scheme of reported system where a mixture of proteins was successful separated [41, 42]. Afterwards, the robustness of interface was improved by modification of the etching technique where only a partially etched porous interface was made. The partially etched interface was easier to fabricate and allowed the separation of a mixture of protein standards and proteins extracted from milk using monolithic immobilized pH gradient-based IEF and CZE [18]. All reported interfaces are summarised in Table 2. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2.2 Heart-cutting 2D-CE 2.2.1 Interface-free heart-cutting 2D-CE Heart-cutting 2D-CE in a single capillary was reported for the first time by Cottet et al. [43]. The usage of a single capillary excluded the possibility of loss of material between the two dimensions. In addition, the approach did not require any special equipment, thus a conventional apparatus was employed. The technique is referred to here as interface-free heart-cutting 2D-CE since the 2D separation did not require any interface. The fractions of interest were mobilized via pressure or voltage in order to facilitate analysis in the second dimension. The selectivity of the second dimension was also achieved by pressure or voltage application to introduce the BGE or pseudostationary phase. In the initial report, a mixture of polymers were analysed using CZE and CSE in the first and second dimension, respectively. To isolate the fraction of interest at the end of the first dimension, three various strategies were proposed. The first one required switching the voltage polarity; the second used pressure or hydrodynamic flow; and the third needed polarity switching and EOF manipulation. It was required that the second separation medium must enter the capillary with a higher apparent velocity than the isolated fraction. To achieve this, Cottet’s group proposed to perform the second dimension in counter-EOF mode [43]. The potential of interface-free heart-cutting 2D-CE was then investigated for the separation of sample containing ten polyelectrolytes of different charge densities and different molecular masses [44]. Interface-free heart-cutting 2D-CE was then applied for the on-line purification and separation of derivatized amino acids. The by-products coming from the sample derivatization step was cleaned or removed by CZE. Then, only an isolated fraction was transferred into the second dimension (i.e. MEKC) by a specific pressure and voltage program. The hydrodynamic mobilization of the fraction, however, reduced the efficiency of the second dimension because of peak broadening due to Taylor dispersion. The possible designs
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Figure 3. Schematic representation of interface-free heart-cutting 2D-CE procedure. The schema illustrates two different approaches. (A) is possibly when the second dimension of the separation can enter in the capillary by EOF pumping, whereas (B) involves hydrodynamic mobilization of isolated fractions. Steps includes: (1) first-dimension separation (injection by the inlet end of the capillary); (2) isolation and return of the fraction of interest (fraction B) to the inlet end of the capillary; (3) starting of the second-dimension separation; (4) separation of the fraction of interest in the second dimension (adapted from ref. [45]).
of interface-free heart-cutting 2D-CE were discussed and a schematic representation of their procedure is illustrated in Fig. 3 [45]. The use of multiple detection points was then implemented in interface-free heart-cutting 2D-CE. Cottet’s group used two contactless conductivity detectors to monitor the purification/isolation steps. This was critical in the selection of the pressure and voltage used in the first dimension. They analyzed a mixture of 22 underivatized amino acids using achiral and chiral running buffers in the first and second dimension, respectively. To prevent peak broadening that was due to hydrodynamic mobilization, a 10 m id capillary was used. The narrower capillary also allowed the use of high pressures that reduced the analysis time. The manipulation of parameters using commercial CE instrumentation was however, a challenge due to software constraints. The connection of extra equipment to the CE system could also be problematic [46]. The sensitivity of the 2D-CE method was later improved using transient moving chemical reaction boundary as the stacking technique [47]. Zhang and coworkers then implemented an interfacefree heart-cutting 2D-CE with electrochemical detection for the analysis of cardiovascular drugs in blood samples. CZE as the first dimension was used to purify a mixture of -blocker
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drugs and to eliminate unwanted compounds. To overcome the peak broadening that resulted from the applied pressure during the transfer to the second dimension MEKC, a dual on-line sample concentration or stacking method using field amplified/enhanced sample stacking and sweeping was proposed. The stacking method also improved the detection sensitivity 3.8-fold compared to the separation without stacking [48]. The method was improved via stacking and was applied to the analysis of the components of Herba Leonuri sample and yielded 1000-fold sensitivity enhancements [49, 50]. The stacking was via sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) [51]. More recently, an interface-free heart-cutting CE was reported by Kukusamude et al. [19]. They stacked and separated a mixture of neutral and cationic analytes by using CZE and MEKC in the first and second dimension, respectively. The control of EOF was critical in the stacking and the implementation of the 2D separation. Figure 4 describes the steps in the 2D-CE procedure. Stacking of both species was with one or with a combination of two on-line sample concentration mechanisms. Two complex samples were tested. First, contained eight cationic drugs and five neutral steroids, all of them were soluble in water. The stacking was via two steps, namely sweeping and AFMC with organic solvent [52]. The
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Figure 4. Stacking and separation of two different classes of compounds (cationic (+) and neutral (n) analytes) in interfacefree 2D heart-cutting CE (CZE × MEKC). (A) The fused silica capillary was filled with a low pH CZE electrolyte. The sample was injected as a long plug. The CZE electrolyte was placed at both ends of the capillary. (B) A voltage was applied at positive polarity (cathode at the detector end). The analytes were focused by stacking. (C) Continued application of voltage caused the migration of the concentrated cationic analytes to the detector. (D) The cationic analytes migrated out of the capillary and the concentrated neutral analytes were purified and remained inside the capillary. The 1st dimension analysis ended. (E) The start of the 2nd dimension analysis was the replacement of the CZE electrolye at both ends of the capillary by the low pH MEKC electrolyte with SDS micelles. The presence of micelles is depicted by the square patterned zones. Application of voltage at negative polarity (anode at detector end) caused the electrophoretic migration of the SDS micelles into the capillary. The micelles eventually penetrated or swept the neutral analyte fraction. (F) Continued application of voltage caused the micelle bound analytes to separate and migrate to the detector. The 2nd dimension and analysis ends when all the analytes were detected (adapted from ref. [19]).
Table 3. Interface-free heart-cutting 2D-CE
CE mode
Analyte
Application content
Ref.
CZE/CSE CZE/MEKC Achiralcze/chiralCZE CZE/MEKC MEKC/CZE CZE/MEKC
Polystyrene sulfonates Amino acids Amino acids -Antagonists Flavonoids Cationic and neutral analytes
Separation of mixture of standards Separation of mixture containing 12 standards Chiral separation of mixture of 22 native amino acids Determination of drugs in rat blood Determination of flavonoids in Leonura cardiac and in mouse blood Analysis of artificial mixture and real water sample
[43, 44] [45] [46, 47] [48] [49, 50] [19]
second, consisted of three herbicides and three poorly water soluble neutral pesticides. The solubilization of the analytes was facilitated by the addition of micelles in the sample and stacking was via AFMC with organic solvent. It was noted that the stacking mechanisms of sweeping and AFMC employed the presence of electrolytes in the sample diluents, thus the 2D-CE methods should be amenable to samples with salt. Stacking provided one to two orders of magnitude improvement in concentration sensitivity. The increase in peak capacity by the multidimensional separation approach and improved concentration sensitivity by stacking may further expand the applicability of CE for the analysis of complex and dilute samples. Table 3 summarizes the reports on interfacefree heart-cutting 2D-CE for easy reference.
2.2.2 Diagonal and integrated 2D-CE Two interesting forms of 2D-CE called diagonal CE [53–56] and integrated 2D-CE [23] were reported. In both approaches, the analytes migrated outside the first dimension and were processed before analysis in the second dimension. Dovichi’s group developed diagonal CE that employs two identical C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
separation modes in both dimensions. In the initial demonstration, the first dimension was used to separate proteins. At the end of the capillary, a microreactor containing enzyme that modified some part of the analytes was placed. Thereafter, fractions were transferred into the second capillary and analyzed. Diagonal CE is a promising technique whereby stoichiometry of enzymatic processes, such as protein phosphorylation, could be determined [53–56]. An integrated 2D-CE using a modified commercial CEMS system was proposed by Santos et al. [23]. The separated zones in the first dimension were collected into a vial and then were reinjected into the second dimension using the same CE-MS instrumentation. The vial where the fractions were collected may be considered as a "pseudo-interface." They coupled MEKC as the first dimension and CZE as the second dimension. The flexibility, efficiency, and robustness were satisfactory, however, the sensitivity of the system was limited by the fraction collection (i.e. the method requires about 1 L of buffer in the vial to collect molecules). The sensitivity issue was solved by modifying the arrangement of electrodes or by using on-line sample concentration in the second dimension. The system could be used in a combination of various electrophoretic modes.
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3 Microchip devices Ramsey and coworkers reported a number of combination of electromigration modes placed in a microchip, including MEKC and high-speed CZE, electrochromatography and CZE, and IEF and CZE [57–59]. Microfabricated fluidic devices were potentially useful for MD separation because highefficiency separations could be achieved and small sample volumes could be manipulated with minimal dead volumes between interconnecting channels. The microchip platform was improved by optimizing the turns in the serpentine channel [60]. This minimized the geometrical contributions to band broadening and provided enough channel length for high-efficiency separations. Finally, the use of higher applied voltages for microfluidic 2D separations offered the potential for extremely fast, high resolution, and high peak capacity analyses of peptides and protein digests.
4 Three-dimensional capillary electrophoresis In spite of the fact that there is still a lot of problems to improve the 2D-CE systems, some research groups have started to construct more advanced instruments for MD separations. The first 3D-CE apparatus was reported by Hanna et al. in 2000 [61]. Their system coupled cITP with CZE and MEKC but only applied to standard samples. There was renewed interest to 3D-CE in 2013 when Mikus and coworkers coupled ITP-ITP-CZE modes for the determination of phthalic acids in urine matrix [62]. Their instrument employed three capillaries that were wider than usually used in CE (300– 800 m id). The first ITP mode was performed as a preseparation step, followed by separation using ITP and then CZE. The ITP steps were run under different pH values ITP1 (pH 3.1) and ITP2 (pH 4.5), while the pH of the BGE in CZE step was close to ITP2. The efficiency, repeatability, and good concentration detection limits of the proposed approach were attributed to the hydrodynamically closed separation system implemented. The advantages over 2D-CE claimed were better selection of the zone of interest for transfer to the analytical step, more effective sample cleanup, and improvement of separation selectivity.
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ity in both comprehensive and heart-cutting modes and thus stacking in MD-CE research will continue. The use of very sensitive and selective detectors such as MS is attractive to decrease the LOD and to identify components in the analysis real complex and diluted mixtures. The demonstrated use of diagonal and integrated MD-CE should find more applications such as in biological screening and food analysis. One of the most promising research areas is the miniaturization of the MD-CE systems that may reduce the costs and make MD-CE separations a portable and common tool in many fields in medicine, pharmacy, or environment sciences. Last, but not least, connecting more dimension in 3D and more dimensional systems, which have already started, gives a wide range of possibilities in demanding research works.
6 Conclusion Many diverse methods for performing 2D-CE have been presented in this review. However, all of them have the same purpose that is to improve the resolution and peak capacity. In spite of the fact that 2D-CE is not common strategy performed in many area of science, it has a great chance to become a widely used method. More recent systems not only enhance the resolution, but also lower the LOD. Furthermore, the reported 3D system heralds the new chapter in MD-CE that may significantly facilitate more complex analysis. Employing more sensitive detectors as well as using the on-line sample concentration strategies allowed the analysis of real complex samples such as those found in the biological, medical, or environmental fields. The growing importance of proteomics and metabolomics requires more efficient techniques that permit saving time, reagents, and money. The multidimensional capillary electrophoresis seems to be a high-efficiency, relatively cheap, and rapid analytical method that may be applied in many research areas in the future. The authors have declared no conflict of interest.
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