Capillary electrophoresis in two-dimensional separation systems: Techniques and applications.

144 Felix J. Kohl ´ ´ Laura Sanchez-Hern andez ¨ Christian Neusuß Department of Chemistry, Aalen University, Aalen, Germany

Received July 25, 2014 Revised September 16, 2014 Accepted September 17, 2014

Electrophoresis 2015, 36, 144–158

Review

Capillary electrophoresis in two-dimensional separation systems: Techniques and applications The analysis of complex samples requires powerful separation techniques. Here, 2D chromatographic separation techniques (e.g. LC-LC, GC-GC) are increasingly applied in many fields. Electrophoretic separation techniques show a different selectivity in comparison to LC and GC and very high separation efficiency. Thus, 2D separation systems containing at least one CE-based separation technique are an interesting alternative featuring potentially a high degree of orthogonality. However, the generally small volumes and strong electrical fields in CE require special coupling techniques. These technical developments are reviewed in this work, discussing benefits and drawbacks of offline and online systems. Emphasis is placed on the design of the systems, their coupling, and the detector used. Moreover, the employment of strategies to improve peak capacity, resolution, or sensitivity is highlighted. Various applications of 2D separations with CE are summarized. Keywords: Comprehensive / Coupling / Electromigrative techniques / Heart-cut / Orthogonality DOI 10.1002/elps.201400368

1 Introduction Two-dimensional separations combine two different separation techniques with different selectivity. Accordingly, a sample is first subjected to separation by one technique (named as first dimension), and subsequently separated by an additional technique (second dimension). This combination offers an improved resolving power providing enhanced peak capacity compared to a 1D separation system and, thus, improving the separation of complex samples. In an ideal 2D analysis, the two separation mechanisms have to be orthogonal in order to obtain the maximum peak capacity [1]. Orthogonality is defined as the lowest degree of correlation between the selectivity of the two dimensions. When orthogonality is given, the peak capacity of the 2D system (nc,2D ) would be the product of the peak capacities of the components of each dimension (nc,2D = 1 nc × 2 nc ). This shows the high analytical potential of these systems even when one dimension contributes only a limited peak capacity. Two-dimensional separation, where only the portion of interest is transferred from the first dimension effluent to

¨ Correspondence: Professor Christian Neusuß, Department of Chemistry, Aalen University, Beethovenstra␤e 1, D-73430 Aalen, Germany E-mail: [email protected]

Abbreviations: CRPLC, capillary RPLC; CSE, capillary sieving electrophoresis; FTICR, Fourier transform ion cyclotron resonance; UV, ultraviolet

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

the second dimension, is referred to as “heart-cutting.” By contrast, a system where sequentially the entirety of the firstdimension effluent is transferred to the second dimension is known as “comprehensive” 2D [2]. In comprehensive approaches, the second dimension must be capable of completing fast separation in order to not diminish the resolution achieved in the first dimension [1]. Additionally, both dimensions may include an online preconcentration step and/or a cleanup step, e.g. to remove a high salt concentration from the analytes. A wide variety of combinations for 2D separations are present employing two identical techniques with different selectivity, introduced by, e.g., using different stationary phases, such as TLC-TLC [3], LC-LC [2,4], GC-GC [5], or a combination of two techniques (e.g. LC-GC). Within this range of options, the employment of different CE techniques (such as CZE, MEKC, CIEF) in 2D systems allows relative fast separations with high efficiency and resolution compared to LC, which makes it well suited as the second dimension. Nevertheless, CE has also been used as first dimension. Some general reviews on 2D systems include CE separations [6–9]. Several aspects of 2D CE separations have been reviewed by Valcárcel et al. in 2001 [10] and Mikuˇs et al. [11]. The present review provides to the readers an actual and general overview on techniques and applications of 2D systems where CE is involved. This review is divided into two sections to describe offline systems, where fractions from the first-dimension effluent are collected and transferred individually to the second dimension, and online systems, where the two separation techniques are hyphenated. Furthermore, the online techniques have been classified into 2D separation

www.electrophoresis-journal.com


systems using (i) a single capillary, (ii) an interface, or (iii) a valve. In the past years, 2D or multidimensional electrophoretic separation in the microfluidic chip format also became very important. Microfluidic devices are not covered here as special coupling aspects and their material has to be considered; this subject has been reviewed recently [12–15].

2 Offline 2D systems with electrophoretic separation techniques Offline 2D methodologies principally consist of three steps: (i) separation of the sample compounds in the first dimension, (ii) collection of different fractions for subsequent sample treatment, and (iii) injection of each of the fractions in the second dimension to be subject to analysis. This concept is preferred when the samples leaving the first dimension must be treated (e.g. sample washing, digestion, derivatization, etc.) before introduction to the second dimension. This principle is simple and standard commercially available instrumentation with any detection system can be applied. Furthermore, any size or type of column or capillary can be used and compatibility of different chemical (e.g. solvents, additives) and/or physical (e.g. volume of sample, flow rate, pressure, temperature) conditions can often be achieved. Therefore, it is quite easy to reach a high degree of orthogonality in offline 2D systems. However, their automation is often not possible and a lot of analysis time is consumed because each fraction is injected individually. Furthermore, the analytes are often diluted, especially when CE is used as first dimension, because of the low CE effluent. Several research groups have been focusing on the development of offline 2D systems. There have been described methodologies based on HPLC/CE [16–30], capillary LC/CE [31–35], and SEC/CE [36]. However, approaches by CE/HPLC [37–39] or even CE/CE [40] have also been developed. As can be seen, in order to obtain the maximum orthogonality, combinations of chromatographic and electrophoretic techniques are widely used. The majority of these systems were designed for proteome and/or peptide analysis, although metabolites, antibiotics, phenols, and flavonoids have been also analyzed. Table 1 presents the offline 2D works in which at least one dimension is an electrophoretic separation technique. This table is certainly not a complete overview since many articles are applying offline 2D techniques without highlighting this approach. The table includes important technical aspects as well as the given applications. The main issues of offline couplings are discussed in the following. Issaq’s research group used offline 2D systems with chromatographic separation techniques in the first dimension and CZE as second dimension for the analysis of proteins [16, 17, 23, 27]. The charge-to-size selectivity of CZE provides the necessary high degree of orthogonality in conjunction with short analysis time and high peak capacity. In an early RPLC/CZE approach with ultraviolet (UV) detection, a mixture of digested cytochrome c and myoglobin C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CE and CEC

145

was analyzed [16]. After vacuum drying and dissolution in water, the 1-min fractions were injected in the CE capillary where the separation of the protein mixture was achieved in 14 min/fraction. In order to speed up the total analysis and increase the throughput of the method, a 96-array CZE system with LIF detection was proposed for the second dimension [23]. For this, 96 capillaries of the same length were sealed directly in the holes of a standard 96-well microtiter tray. Thus, the 96 fractions collected from HPLC were analyzed simultaneously by CZE-LIF after derivatization with FITC. Using this technique, the analysis of all collected fractions was achieved within less than 10 min. The same setup using a 12-capillary array was used for protein mapping of cancer cell extracts [17]. UV detection was employed in this case in order to avoid the derivatization step needed for LIF detection. However, due to the short optical path length and the typical small injection volumes in CE, large volume sample stacking was necessary for in-capillary sample preconcentration in order to improve the sensitivity. Another possibility to gain sensitivity is the use of enrichment sample treatment techniques, such as SPE. Significant advancements have been described for the assembly of online SPE-CE devices in order to combine the benefits of sample concentration and selectivity of a preparative technique with the resolving power of CE in a single analysis [20, 21]. An HPLC/SPE-CZE methodology was proposed for the analysis of tryptic BSA peptides [20]. Using in-line SPE-CE with a concentrator tip, containing a bed of reverse phase material, a sensitivity enhancement of about 100-fold was obtained compared to a standard CE approach. The combination of HPLC and CZE was also used with intermediate offline SPE for the analysis of impurities of octreotide [21]. Further, HPLC/CZE applications with UV detection have been carried out for the analysis of collagen peptides [18, 19], and the determination of apolipoproteins A–I in human blood serum [22]. The employment of MS detection in 2D systems is a very promising approach in order to achieve high sensitivity and the possibility of identification and characterization of the analytes. In this way, a mass dimension is added to the two dimensions of separation. Issaq’s group developed a 2D offline methodology using RPLC and CZE-MS with a sheathless ESI interface for the identification of human serum proteins [27]. MS/MS experiments carried out by an IT MS allowed the identification of 130 proteins. Offline RPLC/CZEESI-MS was also applied to the characterization of phenolic fractions from extra-virgin olive oils [24] and for the analysis of proteins isolated from breast cancer cell lines [25]. Besides, the combination of HPLC, CE, and MALDI-MS has been used as well [28, 29]. After optimization of the orthogonality, this hyphenation of three techniques allowed the identification of more than 30 glycoforms found in a heterogeneous glycoprotein [28] or the fingerprinting of tarantula venom peptides [29]. SEC or IEC was also combined offline with different CE modes. Cesla et al. developed an offline RPLC/MEKC approach for the analysis of phenolic compounds and flavonoids [30]. In this system, the outlet of the LC column www.electrophoresis-journal.com

UV (214 nm) UV (214 nm) UV (214 nm) UV (214 nm) UV (215 nm) UV (210 nm) UV (214 nm) UV (214 nm) UV (240 and 280 nm) UV (214 nm) MS (Q) UV (214 nm) UV (190–640 nm) UV (215 and 280 nm) UV (220, 254, 280 and 350 nm) UV (210 nm) UV (254 nm) UV UV UV (215 nm) UV (280 nm) UV (214 nm) UV (260 nm)

250 mm × 4.6 mm id

250 mm × 4.6 mm id 150 mm × 2.1 mm id 250 mm × 2.0 mm id 220 mm × 2.1 mm id 250 mm × 4 mm id 125 mm × 4 mm id

250 mm × 4.6 mm id 250 mm × 10 mm id

330 mm × 4.6 mm id 250 mm × 4.6 mm id 250 mm × 4.6 mm id 250 mm × 3.0 mm id

125 mm × 4.6 mm id

50 mm × 2.1 mm id

250 mm × 0.2 mm id

500 mm × 0.2 mm id 300 mm × 0.25 mm id

300 mm × 0.25 mm id

300 mm × 0.25 mm id

300 mm × 10 mm id -

100 cm × 200 ␮m id 80 cm × 100 ␮m id 60 cm × 50 ␮m id

RPLC

RPLC RPLC RPLC RPLC RPLC RPLC

RPLC RPLC

RPLC RPLC RPLC RPLC

RPLC


RPLC

CRPLC

CRPLC CRPLC

CRPLC

CRPLC

SEC CIEF

CIEF CITP/CZE CZE

CRPLC: Capillary RPLC; FTICR: Fourier transform ion cyclotron resonance; Q: quadrupole. a) Total length × id. b) Analysis time for all the fractions.

10 cm × 0.2 mm id 15 cm × 50 ␮m id 60 cm × 50 ␮m id

60 cm × 50 ␮m id 14 mm × 4.6 mm id

20 cm × 100 ␮m id

20 cm × 75 ␮m id

60.5 cm × 75 ␮m id 35 cm × 75 ␮m id

60.5 cm × 75 ␮m id

48 cm × 25 ␮m id

70 cm × 100 ␮m id

50 cm × 40 ␮m id 57 cm × 75 ␮m id 60 cm × 30 ␮m id 64.5 cm × 75 ␮m id

52 cm × 50 ␮m id 85 cm × 50 ␮m id

55 cm × 75 ␮m id 37 cm × 75 ␮m id 57 cm × 75 ␮m id 54 cm × 100 ␮m id 50 cm × 50 ␮m id 25 cm × 50 ␮m id

31 cm × 50 ␮m id

LIF (␭ex 532 nm, ␭em 550 nm) MALDI-MS (TOF/TOF) UV (280 nm) UV (214 nm) MALDI-MS (TOF) ESI-MS (FTICR) ESI-MS/MS (IT) ESI-MS (Q)

UV (254 nm) LIF (␭ex 473 nm, ␭em 520 nm)

UV(210, 230 nm)

LIF (␭em 510 nm) ESI-MS (TOF and QTOF) ESI-MS (TOF) UV (190–230 nm) nanoESI-MS (IT) UV (190–640 nm), MALDI-MS (TOF) UV (210 nm), MALDI-MS (TOF) UV (280 nm)

UV (214 nm) UV (214 nm) UV (214 nm) UV (200 nm) UV (200–365 nm) UV (200–214 nm)

UV (214 nm)

Detection

35 120 4

35 11

30

10

15 10

30

15

20

10 17 20 45

10 15

30 40 75 30 70 7

14

b)

b)

b)

Separation time per fraction (min)

Proteins Proteins Antibiotics

Proteins Proteins

Proteins

Proteins

Metabolites Proteins Peptides

Phenols and flavonoids Metabolites

Peptides

Proteins Peptides Proteins Proteins

Proteins Phenols

Proteins Peptides Peptides Peptides Peptides Proteins

Proteins

Application

[38] [39] [40]

[36] [37]

[35]

[34]

[32] [33]

[31]

[30]

[29]

[25] [26] [27] [28]

[23] [24]

[17] [18] [19] [20] [21] [22]

[16]

Reference F. J. Kohl et al.

CRPLC CRPLC MEKC/CZE

CIEF RPLC

CIEF

CIEF

CZE and MEKC CZE CIEF

MEKC

CZE

CZE CZE CZE CZE

CZE CZE

CZE CZE CZE CZE CZE CZE

CZE

Technique

Detection

Capillary or column a) dimensions

Technique

Capillary or column a) dimensions

Second dimension

First dimension

Table 1. Offline 2D separation systems with CE technique

146 Electrophoresis 2015, 36, 144–158



was connected to the CE autosampler by a switching valve in order to automate the collection of the fractions from the first dimension. This design decreased time limitations in the second dimension and avoided the possible sample loss during the manipulation of the collected fractions. Moreover, this system achieved a double peak capacity compared to a previous LC/LC system from the same group for the same application (i.e. the analysis of phenolic compounds and flavonoids). An RPLC/CE-UV system was described for the analysis of metabolites of a cell culture [31]. CZE with dynamic pH junction was used to analyze the early-eluting fractions containing hydrophilic metabolites. Sweeping MEKC was used to analyze the late-eluting fractions containing strongly hydrophobic metabolites and the middle fractions were separated by both strategies. LODs from 0.2 to 30 ng/mL were achieved for 54 standard metabolites. However, in spite of the sensitivity improvement, a smaller number of signals could be detected in the cell extracts using these techniques. Zhang’s group demonstrated the combination of capillary RPLC (CRPLC)-UV and CIEF using LIF [33, 34] or MALDI-MS [35] detector. In a first work, the collected fractions from CRPLC were derivatized by FITC and/or borondipyrromethene and were injected in the CIEF as second dimension [33]. This 2D system provided high sensitivity by LIF detection with LODs of femtomols of peptides from a BSA digest and analysis time of 10 min/fraction in the second dimension. Further, an array of up to 60 capillaries was assembled to this system in order to increase the speed of the total analysis time [34]. Figure 1A shows this setup where 6 cm polyimide coating was removed from the capillaries and the electrical connections for each of them were made using a porous polymer. Figure 1B illustrates the protein expression profile of a liver cancer tissue after 2D separation. Concurrently, a CRPLC/CIEF approach was performed using MALDI-MS detection [35]. Deposition of the CIEF fractions was carried out on a MALDI 192-well plate using a sheath liquid flow to maintain the electric field. After washing and digestion of the sample, the MALDI-MS analysis was carried out. Using this system and MS/MS experiments by a TOF– TOF analyzer, 11 proteins from a single RPLC fraction of a rat liver tissue were identified. CIEF with UV detection has been also reported as first dimension for the separation of proteins, using RP-HPLC [37] or CRPLC [38] with MS detection in the second dimension. As example, Gao et al. [38] collected protein fractions from CIEF and digested with trypsin. The peptides were analyzed by RPLC-Fourier transform ion cyclotron resonance (FTICR)-MS. Finally, some offline systems have been also reported with use of two different electrophoretic modes (such as MEKC or CZE) with MS detection [40]. A commercial CEMS system was modified by incorporation of a modified electrode that allowed the simultaneous introduction of two capillaries and a two-way electrical switch to apply the voltage in the first or second capillary. In the first dimension, two antibiotic families (tetracyclines and nitroimidazoles) were separated by CZE and collected. Subsequently, C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

CE and CEC

147

Figure 1. Offline combination of CRPLC and 60-capillary array CIEF for protein analysis. (A) Schematic diagram of the LIF system for capillary array with scanning. (B) Protein expression profile of the liver cancer tissue after 2D separation. Reprinted from [34]. Copyright (2006), with permission from WILEY-VCH Verlag GmbH & Co. KGaA.

tetracyclines and nitroimidazoles were analyzed by CZE-MS and MEKC-MS.

3 Online 2D systems with electrophoretic separation techniques Online coupling of two separation techniques, containing at least one electrophoretic driven technique, can be divided into three groups: first, 2D systems using two different electrophoretic modes in a single capillary; second, hyphenation of two different separation techniques by an interface; third, coupling of two different separation techniques by a mechanic device such as a multiport valve. Most variants of these three groups can be operated as a heart-cut as well as a comprehensive system. The various systems are discussed in the following sections.

3.1 Two-dimensional separations in a single capillary When a single capillary is used as a 2D system, the separation of both dimensions is performed in the same capillary, and therefore, no coupling device is required. A first separation occurs by the initial CE mode. When the matrix is out of the capillary and the analytes of interest are stored www.electrophoresis-journal.com

148

F. J. Kohl et al.


Table 2. Two-dimensional CE separations in a single capillary a)

First dimension

Second dimension

Capillary dimensions

Detection

Separation time (min)

Application

Reference

CZE CZE CZE CZE CZE MEKC MEKC CZE RPLC RPLC

CSE CSE MEKC EKC EKC CZE CZE MEKC CZE CZE

30 cm × 50, 25, 10 and 5 ␮m id 30 cm × 75 ␮m id 30 cm × 25 and 50 ␮m id 60 cm × 10 ␮m id 60 cm × 10 ␮m id 60 cm × 50 ␮m id 60 cm × 50 ␮m id 50 cm × 50 ␮m id 25 ␮m id -

UV (214 nm) UV (214 nm) UV (214 nm) C4 D, UV (200 nm) C4 D EC EC UV (200/254 nm) MS (IT) MS (IT)

50 25 45 45 60 35 100 30 20 20

Polymers Polymers Amino acids Amino acids Amino acids Flavonoids Flavonoids Small molecules Proteins Proteins

[41] [42] [43] [44] [45] [46] [47] [48] [53] [54]

CSE, capillary sieving electrophoresis. a) Total length × id.

inside the capillary, the buffer is exchanged. By using a different buffer containing, e.g., enantioselective compounds, a second separation step with a selectivity different from the first step can be performed. Table 2 summarizes the reported studies. Cottet’s group used CZE as first dimension and different techniques such as capillary sieving electrophoresis (CSE) [41, 42], MEKC [43], or EKC [44, 45] as the second dimension with C4 D and UV detection. In a first work, three strategies to separate and isolate the fraction of interest (a standard polymer mixture of two polystyrene sulfonates) were proposed [41]. As can be seen in Fig. 2, the anionic analytes are separated by CZE toward the cathodic capillary outlet by a strong EOF. In the first strategy, the analytes are then mobilized in the capillary inlet by switching the polarity. After changing the inlet vial for a hydroxyethyl cellulose buffer, the analytes were separated in CSE mode in cathodic outlet direction and with a strong EOF (see Fig. 2A). In the second strategy, mobilization of the analytes was performed by a reversed hydrodynamic flow (Fig. 2B), while in the last strategy the exchange of the buffer vial at the outlet is accomplished before polarity switching (Fig. 2C). Using the first isolation strategy, this technique was also applied for the separation of three poly-styrene sulfonates [42]. In a further work, Anouti et al. developed a heartcutting 2D CE approach in a single capillary for purification and separation of 12 amino acids derivatized with benzyl 4-(3-(2-chloroethyl)-3-nitrosoureido)butylcarbamte [43]. In this application, the first CZE dimension stands for a clean-up step in order to separate the amino acids from derivatization products, while in the second MEKC separation step the derivatized amino acids were separated. A similar strategy was used for the chiral analysis of the amino acids D,L-tyrosine, D,L-tryptophan, and, D,L-threonine in a mixture of 22 underivatized amino acids with C4 D [44]. Separation in first dimension was achiral in order to separate the different amino acid fractions, while in the second dimension the buffer contained (+)-(18-crown-6)-2,3,11,12tetracarboxylic acid as chiral selector. This technique was also used for the chiral analysis of D,L-phenylalanine and D,L-threonine in a mixture of 22 amino acids with C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

transient moving chemical reaction boundary for online preconcentration in order to increase the sensitivity [45]. Zhang’s research group developed a 2D system in a single capillary using MEKC as first dimension and CZE as second dimension with electrochemical detection for the analysis of flavonoids [46, 47]. When using MEKC as the first dimension, the analytes have to be released from the micelle interior. By a micelle collapse technique, namely “analyte focusing by micelle collapse,” the micelles can be destroyed and the analytes are released. Thus, the selected zone of a Leonurus cardiac sample from the first dimension was transferred into the second dimension by pressure and, after the analyte focusing by micelle collapse strategy, six flavonoids were separated by CZE [46]. The same approach involving an online combination of sweeping and electrokinetic injection with MEKC separation was used [47]. More than 6000fold improvement relative to the traditional pressure injection method was achieved and the separation of eight flavonoids in Herba Leonuri and postdosing mouse blood samples was obtained. Two-dimensional separation in a single capillary was used by Kukusamude et al. for the simultaneous analysis of cationic and neutral compounds by CZE followed by MEKC [48]. In the first step, the cationic analytes were separated by CZE using a low pH electrolyte, while the neutral compounds remained at the inlet tip of the capillary due to the suppressed EOF. In the second step, both buffer vials were exchanged by an SDS-containing MEKC electrolyte. By the application of negative potential, the SDS migrates into the capillary and sweeps the neutral analyte fraction forming micelles for second-dimension MEKC separation. In this approach, an additional mobilization step is not required, which helps to avoid diffusion. All these methodologies comprise a technically simple way for the analysis of target analytes in complex samples. However, the one-capillary approach is only useful if the first dimension separates classes of molecules into discrete bands and if a higher resolution separation is available for the second dimension. Furthermore, coupling to MS or other external detectors is not possible because of the need of a (pressurized) outlet vial.



CE and CEC

149

Figure 2. Heart-cutting 2D-CE separations in a single capillary. Schematic representation of three different strategies (A–C). Each strategy is based on four key steps. Return of fraction B to the inlet end of the capillary by the application of a switched polarity (A) or a reverse hydrodynamic flow (B). Both fractions B and C are submitted to the second dimension of the separation (C). The electrolyte used for the second dimension of the separation is depicted in gray. Reprinted from [35]. Copyright (2003), with permission from Elsevier publications.

In-line SPE-CE is one of the most effective in-line preconcentration techniques in CE [49–51]. When applying multistep elution, a 2D RPLC-CE approach is possible, similar to the multidimensional protein identification technology approach [52]. In contrast to the prior discussed strategies of 2D separation in a single capillary, MS coupling is possible here because no outlet vial is needed. On the other hand, only comprehensive separation can be carried out. The feasibility of this system for protein analysis was demonstrated by Tong et al. [53] as well as Lee et al. [54] whereby the extracted analytes were eluted in 10 and 11 steps, respectively.


3.2. Hyphenated 2D capillary electrophoretic techniques Besides 2D separation in a single capillary, online coupling of two separation techniques can be carried out by an interface. In contrast to the single-capillary approach, these systems are more flexible and allow the combination of chromatographic and electrophoretic separation techniques. Besides LC, different modes of CE have been used for the first dimension. As second dimension, CE has been the preferred technique. Several different interface systems have been employed: dialysis [55–59], porous [60, 61], tee-union [62–64],



CSE

CSE

CSE CSE

Flow gating

Flow gating

Flow gating Flow gating

MEKC MEKC MEKC MEKC

20 cm × 31 ␮m id 40 cm × 31 ␮m id 20 cm × 30 ␮m id

50 cm × 31 ␮m id 20 cm × 30 ␮m id

20 cm × 31 ␮m id

25 cm × 31 ␮m id

LIF (␭ex 473 nm, ␭em 580 nm) LIF (␭ex 473 nm, ␭em 580 nm) LIF (␭ex 488 nm) LIF (␭ex 473 nm, ␭em 580 nm)

LIF (␭ex 488 nm) LIF (␭ex 442 nm, ␭em 520 nm) FTICR MS MS (TOF) MS (Q) UV UV (210 nm) LIF LIF (␭ex 488 nm) LIF (␭ex 473 nm)

8 cm × 10 ␮m id 25 cm × 17 ␮m id 30 cm × 50 ␮m id 20–25 cm × 50 ␮m id 15 cm × 29 ␮m id 50 cm × 75 ␮m id 36 cm × 75 ␮m id 40 cm × 50 ␮m id 30/40 cm × 50 ␮m id 20 cm × 30 ␮m id

UV (280 nm) UV (214/280 nm) UV (214/280 nm) FTICR-MS MS (QTOF) UV (280 nm) UV (214 nm) UV (214 nm) UV (214 nm) MS (QTOF) UV (214 nm)

25 cm × 50 ␮m id 21 cm × 50 ␮m id 18 cm × 50 ␮m id 20 cm × 50 ␮m id 5 cm × 300 ␮m id 17 cm × 50 ␮m id 30 cm × 50 ␮m id 40 cm × 50 ␮m id 40 cm × 50 ␮m id 30 cm × 50 ␮m id 35 cm × 50 ␮m id

Detection

18 s

20 s

3 min

1 min 30 s 4 min 1.5 min 2.6 min 2 min 12 s

2.5 s 1 min

b)

22 min 5 min 6 min 15 min 10 min 4 min 10 min 6 min 6 min 8 min 4 min

Separation time per fraction

Proteins Single-cell proteins

Proteins

Peptides Peptides Peptides Acids Alcohols and ketones Proteins Proteins Proteins and biogenic amines Proteins

Peptides Amino acids

Proteins Proteins Peptides/proteins Proteins Proteins Proteins Proteins ␤-Blocking drugs ␤-Blocking drugs Peptides Proteins

Application

[76] [77]

[75]

[74]

[68] [82] [83] [69] [70] [71] [72] [73]

[66] [67]

[55] [56] [57] [58] [59] [60] [61] [62] [63] [64] [65]

Reference

F. J. Kohl et al.

25 cm × 31 ␮m id

CZE CZE CZE CZE MEKC MEKC MEKC MEKC

10–12 cm × 200 ␮m id 10–12 cm × 200 ␮m id 33 mm 15 cm × 320 ␮m id 80 cm × 250 ␮m id 40 cm × 50 ␮m id 30 cm × 50 ␮m id 20 cm × 30 ␮m id

CRPLC CRPLC RPLC CRPLC CRPLC CSE CSE CSE

Flow gating Flow gating Flow gating Flow gating Flow gating Flow gating Flow gating Flow gating

CZE CZE

RPLC CRPLC

Optical gating Flow gating

CSE CSE tcITP-CZE tcITP-CZE CRPLC CZE CZE MEKC MEKC CZE CZE

30 cm × 50 ␮m id 32 cm × 50 ␮m id 33 cm × 50 ␮m id 40 cm × 50 ␮m id 55 cm × 50 ␮m id 33 cm × 50 ␮m id 20 cm × 50 ␮m id 48 cm × 50 ␮m id 48 cm × 50 ␮m id 20 cm × 100 ␮m id 105 cm × 250 ␮m id 110 cm × 100 ␮m id 15cm × 2.1 mm id 76 cm × 50 ␮m id

CIEF CIEF CIEF CIEF CIEF CIEF CIEF CZE CZE ITP ␮SEC

Technique

Capillary/column a) dimensions

Technique


Second dimension

First dimension

Dialysis Dialysis Dialysis Dialysis Dialysis Porous Porous Tee-union Tee-union Tee-union Flow gating

Interface

Table 3. Hyphenated 2D capillary electrophoretic techniques

150 Electrophoresis 2015, 36, 144–158


CE and CEC

[86] Proteins CZE 30 cm × 250 ␮m id CRPLC

a) Total length × id. b) Analysis time for all the fractions.

[85] Proteins/peptides MS (IT) CZE 36.5 cm × 50 ␮m id CZE

30 cm × 50 ␮m id

2 min

[84] Proteins/peptides 3 min

Zhang’s research group used a dialysis interface for online coupling of CIEF and CGE in order to carry out 2D electrophoretic separation of hemoglobin variants [55]. This system transfers the principle of 2D-PAGE separation to the capillary format. Thus, proteins were focused by capillary ITP and then transferred through the dialysis interface to the second CGE dimension. The interface consisted of a bolster reservoir containing methacrylate resin plate (see Fig. 3A). On each side of the reservoir a Teflon tubing section is mounted with an additional hollow fiber partly inserted into each of the two Teflon tubes. Figure 3B shows the 2D-CIEF-CSE electropherogram of the separation of the hemoglobin variants (A, F, S, and C) using a polyacrylamide gel buffer in the second dimension [55]. After the CIEF separation, the hollow fiber was rinsed with the SDS-containing CGE buffer. The proteins were subsequently complexed with SDS and injected into the gel-filled capillary for further separation. Using this setup, 2D separation required total analysis time of almost 1 h (about 22 min for the second dimension). The same group used a similar interface for the hyphenation of CIEF and nongel CSE [56]. In this approach, a replaceable dextran-containing electrolyte was used in the second dimension instead of a cross-linked polyacrylamide gel. In contrast to the CGE approach [55], even shorter analysis time of 5 min per fraction

MALDI-MS (TOF–TOF)

[79] [81] 6 min 3 min

MEKC CZE CZE CZE 40 cm × 50 ␮m id 20 cm × 50 ␮m id 40 cm × 50 ␮m id 31.2 cm × 48 ␮m id

Flow gating Flow gating Nicked sleeve Flow gating/ microreactor Flow gating/ microreactor Hydrodynamic

CSE CIEF CZE CZE

40 cm × 50 ␮m id 20 cm × 50 ␮m id 40 cm × 50 ␮m id 31 cm × 48 ␮m id

flow gating [65–83], flow gating microreactor [84, 85], and hydrodynamic [86]. Table 3 summarizes the different interface systems including technical information for the first and second dimensions as well as the application.

30 cm × 75 ␮m id

[78]

Proteins and primary amines Proteins Proteins 18 s

LIF (␭ex 473 nm, ␭em 580 nm) LIF (␭ex 488 nm) LIF (␭ex 473 nm) LIF (␭ex 532 nm) MS (QqQ) MEKC 20 cm × 30 ␮m id CSE Flow gating

Technique Capillary/column a) dimensions Technique

20 cm × 30 ␮m id

Detection Second dimension


151

3.2.1 Dialysis interface

First dimension Interface

Table 3. Continued

1 min

Reference Separation time per fraction

Application



Figure 3. (A) Construction of 2D CIEF-CGE framework and the dialysis interface that consists in (1) methacrylate plate, (2) capillaries, (3) Teflon tubes, (4) hollow fiber, and (5) buffer reservoir. (B) 2D CIEF-CGE electropherogram of hemoglobin. Reprinted from [43]. Copyright (2003), with permission from ACS publications.


152

F. J. Kohl et al.

in the second dimension was achieved for the separation of hemoglobin [56]. In comparison to 2D separation in a single capillary, the flushing steps and the hydrodynamic diffusion disappear. However, only comprehensive coupling is possible because no fraction can be cut by the interface. Alternatively, Mohan et al. developed a microdialysis interface in order to set up a CIEF-CZE system for separation of cytochrome c, ribonuclease A, and carbonic anhydrase II and the corresponding proteolytic peptides [57]. The interface consisted of a polysulfone dialysis tube that was placed in an electrolyte reservoir. The capillaries were inserted into the tubing after coating with hydroxypropyl cellulose. Transient ITP focused the fractions prior to CZE separation enhancing the dynamic range and the sensitivity of the system. This 2D approach achieved a total peak capacity of 1600 (90 for each of the 18 fractions) and was further coupled to ESI-FTICR-MS for the proteome analysis of Shewanella oneidensis [58]. A microdialysis membrane cathodic cell interface was used for the hyphenation of CIEF and RPLC with MS detection for protein characterization [59]. In this case, CIEF was used as the first dimension and not as the second dimension in order to overcome the limitation of the direct CIEF-MS coupling caused by interferences and signal quenching by the ampholytes in ESI-MS. The dialysis membrane was placed between the grounding electrode and a tee union, which separated the ampholyte from the protein molecules. Focused proteins were transferred then hydrodynamically to the injection loop of a six-port valve. Remaining ampholytes were removed by an RP trap column installed in a second six-port valve serving as an injection system for RPLC.

3.2.2 Porous junction interface Zhang’s group used an etched porous junction interface as an advancement to the dialysis interface [60]. The junction was prepared by etching a small part of a fused silica capillary with hydrofluoric acid in order to achieve a porous glass membrane. Using this technique, high voltage can be applied separately to the first as well as to the second part of the single capillary. This type of interface has been applied to the 2D separation of protein mixtures by CIEF in the first dimension and CZE in the second dimension. Due to problems with the fragileness of the whole etched porous interface, an additional partially etched porous interface was developed [61]. A small part (about 0.5–2 mm in length and 1/6–1/2 of the capillary diameter) of the polyimide coating of a fused silica capillary was removed. Hydrofluoric acid was then used to etch the exposed fused silica section according to the procedure used for the interface in [60]. The partially etched porous interface provided effective electrical contact and improved robustness in comparison to the whole etched porous interface. The system was used for the separation of a mixture of six proteins by monolithic IPG CIEF and subsequent CZE-UV. The monolithic IPG capillary was prepared separately and connected to the interface by a PTFE tubing. The complete 2D-CE C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


separation was attained within 116 min, yielding to a total peak capacity of about 200.

3.2.3 Tee-union interface An additional third connection provided by a tee-union interface enables some advantages in comparison with the dialysis and porous interfaces, such as the possibility to flush both dimensions separately. This kind of interface was used for the hyphenation of CZE-UV and CD-modified MEKC-UV for the determination of cationic ␤-blocking drugs in wastewater [62]. It consists of a PTFE tube with 350 ␮m id, where the two capillaries were placed coaxially and equipped with a microhole at this gap. In addition, “cation-selective exhaustive injection” and transient ITP were used for online preconcentration. Excellent resolution between the ionic, neutral, and structural isomers of the standard drugs was reached and more than a 10 000-fold improvement in sensitivity was achieved with LODs between 0.03 and 0.1 ␮g/L [62]. The same research group employed this interface again in a CZE-MEKC approach for the determination of the same drugs in human urine [63]. Kler et al. worked with a polyether ether ketone tee-union with 150 ␮m thru-holes resulting in a swept volume of 20–81 nL for the hyphenation of capillary ITP and CE with mass spectrometric detection [64]. Based on the analysis of angiotensin peptides, the sample transfer characteristics of the tee-union interface were compared to the characteristics of a microfluidic interface. It was found that the microfluidic interface provides much better transfer characteristics due to distortion of the electric and fluid flow fields in a commercial polyether ether ketone tee-union. Analysis was carried out with C4 D in the first dimension and mass spectrometric detection in the second dimension.

3.2.4 Gating interfaces The most widely employed interface up to now has been referred to as flow gating interfaces. Jorgensen’s research group designed first a transverse flow gating interface that circumvents sample collection in a loop to connect ␮SEC and CZE for the separation of standard proteins [65]. It consisted of two stainless steel plates separated by a Teflon gasket with a thickness of 127 ␮m equipped with a 1 mm channel that allowed liquid flow between the plates. The SEC column was placed toward the CZE capillary orthogonal to the flow channel inside the interface. By applying a liquid flow to the channel inside the interface, effluent coming from the SEC dimension was drawn to waste. For injection of a sample from the first dimension into the CZE capillary, the transverse flow was stopped. This allowed the upcoming effluent to enter in the CZE capillary for further separation. An improved transparent flow gating interface for the hyphenation of CRPLC and CZE-LIF was constructed by a polycarbonate disk with cross-shaped channels (Fig. 4A) and used for the analysis of amino acids or human urine [67]. www.electrophoresis-journal.com


CE and CEC

153

Figure 4. Schematic of the clear flow gating interface (A). Frame-grabbed video images of the injection process using the flow gating interface (B). Reprinted from [67]. Copyright (2003), with permission from ACS publications.

The two dimensions were connected via two opposite connections similar to the transverse flow gating interface [65]. The transparency of this interface allowed the flow gating and injection process to be directly observed using a colored dye solution. Figure 4B shows frame-grabbed video images of the injection process of this device. This interface was employed in CRPLC-CE systems for the analysis of peptides with quadrupole-MS [83], TOF-MS [82], and FTICR-MS [68], for separation of the two acids, 4-hydroxybenzoic acid and 3,4,5 trimethoxycinnamic acid, with UV detection [69], and for the analysis of alcohols and ketones from traditional Chinese medicines with UV detection [70]. Furthermore, this flow gating interface was used in order to hyphenate two capillary electrophoretic techniques. In this system, the entire fraction of the first dimension was transferred to the second dimension to avoid the loss of sample. Dovichi’s research group used this interface for the hyphenation of CSE and MEKC for the analysis of protein homogenate of Deinococcus radiodurans (see Fig. 5) [71], protein fingerprinting of single mammalian cells [72], analysis of Barrett’s esophagus tissues [73, 74], analysis of proteins in mouse tumor cell homogenate [75,76], single-cell protein analysis in a mouse embryo [77], characterization of single breast cancer cells [78], and separation of proteins from human adenocarcinoma cells [79]. Moreover, a CIEF-CZE system was used for the analysis of a protein mixture [81]. In all applications, the analyses were carried out with fluorogenic marked proteins in combination with LIF detection to reach detection limits down to zeptomoles [79]. As shown in Fig. 5, proteins can be effectively separated by 2D CE. RPLC was hyphenated to CZE via an optical gating interface [66]. A combination of short capillaries and relative high voltage levels was used in order to perform rapid CZE analysis. Thus, a constant high voltage was applied to the capillary and the sample migrates through the capillary. An argon ion laser was focused near the injection end of the capillary. The majority of the sample, previously treated with FITC, is photodegraded by the intense light of the gating beam. Therefore,


Figure 5. Hyphenation of CSE and MEKC. Two-dimensional separation of protein homogenate from D. radiodurans. Image processed to resemble a silver-stained gel. The intensity of each spot is proportional to the logarithm of the fluorescence intensity (A). Image processed to resemble a landscape. Peak height is proportional to fluorescence intensity (B). Reprinted from [71]. Copyright (2004), with permission from WILEY-VCH Verlag GmbH & Co. KGaA.


154

F. J. Kohl et al.

in contrast to the previously described interfaces, upcoming analytes are not stopped or drawn to waste but destroyed by the intense laser. In order to carry out an injection, the gating beam was momentarily blocked allowing a small slug of unbleached material to pass through. The sample is then subsequent separated by fast CZE-LIF.


Effluent coming from the LC dimension was collected in a droplet at the LC column tip and injected hydrodynamically by the siphoning effect into the CZE. The efficiency of this system was demonstrated by analysis of proteins in liver cancer tissue. 3.3 Two-dimensional systems using mechanical valve

3.2.5 Microreactor interface Schönherr et al. implemented a pepsin microreactor into a CZE-CZE-MS system with flow gating interface [84]. A mixture of intact proteins was first separated and the fractions were then online digested by a monolithic microreactor. This 5-cm-long reactor was carrying immobilized pepsin and was placed at the end of the first-dimension capillary. The proteolytic peptides were then transferred to the second dimension via the interface. This system combines protein separation, digestion, and fast peptide separation (3 min per fraction) in one fully automated step with MS/MS detection and identification of the peptides. In a further CZE-CZE-MS approach, trypsin was immobilized on magnetic beads, which allows to replace the microreactor [85]. The performance of this methodology was demonstrated by the analysis of insulin chain b and beta-casein requiring roughly 30 min to digest them and 1 min to separate each of the 30 fractions with subsequent MS detection.

3.2.6 Nicked-sleeve interface A nicked-sleeve interface was developed providing improved transfer efficiency [80]. This redesigned interface uses only one sleeve capillary that was cut approximately halfway through the inner diameter by a microdicing saw, resulting in a nick that exposed the inner diameter of the sleeve capillary. For this reason, alignment and therefore performance of the new interface is more reproducible. Transfer efficiencies were compared to those achieved by traditional 2D separations by reference to carboxytetramethylrhodamine-labeled proteins as a model analyte using LIF detection where the nicked-sleeve interface showed a transfer efficiency of 94% while the traditional 2D interface only showed 68% with respect to a 1D approach.

3.2.7 Hanging droplet interface Zhang et al. proposed a comprehensive CRPLC-CZE system with MALDI-MS detection [86]. In order to carry out 2D separations as well as coupling to the MALDI detection, a gravitation interface for CRPLC-CZE separation and an interface for CE-MALDI coupling were employed. The gravitation interface consisted of a slide bar that could be moved in two positions by a step motor. The CZE capillary inlet was fixed on that slide bar that enables positioning in a buffer vial for analysis as well as at the tip of the LC column for injection. C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The main challenges for using a mechanical valve in 2D systems containing electromigrative separation techniques are the electrical isolation of the valve parts and avoiding high dead volumes in the system. Most of the commercial available valves are equipped with an electrical control unit and, therefore, can be fully automated and integrated with the separation apparatus. Furthermore, both heart-cut and comprehensive systems can be realized. Mechanical valves have been mainly employed for the coupling of chromatographic and electrophoretic techniques such as LC-CE [87–89], SEC-CE [90,91], or CE-LC [92–94], but also for the coupling of CE-CE [95,96].The works published for 2D systems regarding this coupling are summarized in Table 4. Jorgenson’s group developed a 2D RPLC-CZE-LIF system, using a mechanical six-port valve, for the separation of fluorogenic labeled peptides [87]. Because first dimension is HPLC, it is possible to ground the CE capillary at the connection at the valve. Therefore, an electrically isolated valve is not necessary and hence, commercially available HPLC valves can be used. In run position, the upcoming HPLC effluent filled the 10 ␮L sample loop of the six-port valve. An additional syringe pump provided, via the valve, a BGE sheath flow surrounding the CE separation capillary coaxially. CE injection was carried out electrokinetically direct from the sheath flow. Therefore, CE sample composition does not depend on the HPLC solvent gradient. Because only a less amount of HPLC effluent is drawn to waste, this system can be considered as comprehensive. In a further work of the same group, a sixport valve setup for coupling of ␮SEC to CZE was studied for the analysis of thyroglobulin, BSA, chicken egg albumin, and horse heart myoglobin [89]. In this case, the six-port valve was equipped with a 300 nL sample loop in order to collect the proteins previously separated by SEC, which then were hydrodynamically transported through a tee interface, providing enclosure of the CZE electric circuit. Figure 6 shows the scheme of this setup. Working with the sample loop, this system turned out as a heart-cut approach. A similar system was further used for the analysis of peptides [90] and enkephalins in cerebrospinal fluid [91] by SEC in the first dimension and CZE in the second dimension. The homemade interface consists of 20 mm of PTFE tubing. At the bottom of the PTFE tubing, a stainless steel tubing is inserted supplying the SEC effluent as well as the CZE buffer and also works as the grounding electrode. On the top, a second PTFE tubing is inserted supporting the CZE capillary and resulting in an interface compartment with a volume of 11 ␮L, which can be flushed with either SEC effluent for injection or buffer for second-dimension CZE separation. www.electrophoresis-journal.com

CE and CEC


155

Table 4. Two-dimensional CE techniques using mechanical valve

First dimension

Second dimension

Application

Reference

Technique


Technique


Detection

Separation time (min)

RPLC

250 mm × 1 mm

CZE

38 cm × 41 and 50 ␮m id

RPLC

250 mm × 4.6 mm

40/15 cm × 1000 ␮m id

␮SEC SEC SEC CIEF CIEF CIEF CIEF CIEF ␮-SCXLC

105 × 250 ␮m id 30 mm × 4.6 mm id 30 mm × 4.6 mm id 30 cm × 100 ␮m id 60 cm × 100 ␮m id 84 cm × 100 ␮m id 50 cm × 100 ␮m id 44 cm × 50 ␮m id 10 cm × 0.3 mm id

Wide-bore electrophoresis CZE CZE CZE CRPLC CRPLC nRPLC CEC CEC RP-pCEC

Fluorescence (␭ex 365 nm, ␭em 470 nm) UV (214/230 nm)

1

Peptides

[87]

5–12

Small molecules

[88]

38/58 cm × 50 ␮m id 90 cm × 50 ␮m id 90 cm × 50 ␮m id 10 cm × 150 ␮m id 15 cm × 180 ␮m id 15 cm × 50 ␮m id 20 cm × 150 ␮m id 41 cm × 100 ␮m id 55 cm × 150 ␮m id

UV (214 nm) UV (215 nm) UV (215 nm) MS MS (IT) MS (QTOF) UV (214/280 nm) UV (280 nm) UV (230 nm)

4–9 25 25 30 90 90 30 20 60

Proteins Peptides Peptides Peptides Peptides Peptides Peptides/proteins Peptides/proteins Proteins

[89] [90] [91] [92] [93] [94] [95] [96] [97]

SCXLC, strong cation-exchange liquid chromatography. a) Total length × id.

Figure 6. Scheme of instrumental setup for 2D SEC-CZE using a valve. Reprinted from [89]. Copyright (1993), with permission from Elsevier.

Samples were first separated by SEC in the first dimension. Further, the fraction of interest is cut by a first six-port valve and introduced to a C18 trap column. The sample is then washed, eluted by a solvent plug, and introduced to the interface by a second six-port valve. Electrokinetic injection is carried out directly from the interface and separated by CZE after exchanging the sample by CZE buffer. Wu et al. used RP CEC hyphenated to strong cation exchange chromatography [97]. A six port-valve was used to inject the sample fractions into the second dimension. The valve was isolated from electric circuit of the CEC instrument by a grounded cross-union. This system combines the strong cation exchange chromatography with RP chromatography and electrophoresis mechanisms, which provided high selectivity, high resolution, and a total peak capacity of 1200 for the 2D system. However, analysis time of around 1 h for


each fraction was necessary to attain the separation profile of medicine Cortex Phellodendri, a digest of BSA, or human serum. Li et al. used a six-port injection valve with a 400 nL sample loop for coupling of HPLC and wide-bore electrophoresis (1000 ␮m capillary id) [88]. The performance of this system was studied by the analysis of the two model substances, benzoic acid and phenylacetic acid, with UV detection. Lee’s group used a four-port nano-injector valve with an internal 200 nL sample loop for the hyphenation of CIEF and CRPLC for protein and peptide separation [92]. The CIEF zones were introduced to the sample loop by gravity mobilization via raising the inlet reservoir vial about 5 cm above the outlet reservoir. Analytes were further separated by CRPLC and detected by ESI-MS. Further, CIEF was coupled to


156

F. J. Kohl et al.

CRPLC-ESI-MS for proteome analysis but with an additional multitrap column [93]. Focused sample zones from the CIEF dimension were hydrodynamically loaded into a 400 nL sample loop in a six-port valve and further injected into one of the six reverse-phase columns. Remaining ampholyte was eluted from the trap columns prior to the CRPLC separation. Lee’s research group used this CIEF-CRPLC platform with ESI-MS detection successfully also for proteome analysis of tumor tissues [94]. Alternatively, a six-port valve was used for the hyphenation of CIEF and CEC for peptide and protein mapping with UV detection [95]. The valve was isolated from the electric circuit in both dimensions. A homemade electrical decoupler was introduced to the first CIEF dimension, while the second CEC dimension was isolated via a grounded crossunion. Moreover, the cross-union worked as a flow splitter for pressurized CEC. The focused sample zones from the first dimension were transported to the 500 nL sample loop by a moderate EOF. After filling of the loop, the sample was injected into the second pressure-driven CEC dimension. Instead of isolating the valve from the electric circuits, a six-port microinjector valve with a full ceramic internal pathway and a 25 nL port-to-port volume was used for the coupling of CIEF and CEC, for the analysis of proteins, peptides, and human serum [96]. Because of the nonconductive valve material, an additional isolation of the two dimensions is not required. Both the outlet tip and inlet tip of the first dimension CIEF capillary were connected to the nano-injector valve. Electrical connections as well as connections to the anolyt/catholyt reservoir were realized by two additional short capillaries. Thus, the CIEF capillary could be connected directly to the inlet tip of the second-dimension CEC capillary by switching the valve. The focused sample zones were further mobilized hydrodynamically by a microsyringe pump and introduced to the CEC dimension.

4 Conclusions Two-dimensional separation systems containing at least one capillary eletrophoretic dimension have been summarized in this review. Offline approaches benefit from flexibility, possible sample preparation (including, e.g., digestion of proteins), and ease of use as well as the possibility to use standard instruments. Most recent developments have been focused on online methodologies offering a more precise control of fraction collection, reducing sample loss and dilution in between the two separation steps, as well as shorter analysis time. Applying subsequently different electrolytes in a single capillary is the technically simplest way to perform online 2D separation omitting any dead volumes, sample loss, sample dilution, or flow problems by the absence of an interface providing a heart-cut separation of target analytes. A gain in flexibility is achieved when two separation techniques are interfaced, enabling also the coupling of chromatographic to electrophoretic separation techniques. Besides dialysis and porous junction interfaces, especially open tee, cross- or C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


nicked-sleeve junctions have been used modulating the injection by flow gating. In this way, comprehensive analysis can be performed. Furthermore, a mechanical valve as an interface is a promising option since the highest degree of flexibility in the combination of the two separation techniques is achieved due to the complete partition of the two dimensions. Mainly UV and LIF detections have been used for both dimensions. However, the coupling to MS detection in the second dimension is a promising trend in 2D systems because it allows the implementation of analyte identification. However, several approaches, such as 2D separation in a single capillary or the implementation of a nonisolated valve with a subsequent tee for high-voltage supply, cannot be used with MS detection. Several applications have been performed with electrophoretic separation interfaced with another separation technique. Peptide and protein analysis is particularly the focus of most studies. In this way, it is possible to overcome some drawbacks from classic 2D electrophoresis such as limited detection sensitivity or the tedious manipulation when large-scale protein analysis projects are performed. Moreover, it is possible to design in-capillary strategies or online sample treatments to improve the sensitivity depending on the sample to be analyzed. Furthermore, online approaches in capillaries allow automation and strongly decrease analysis time. In this regard, complex (biological) samples have been analyzed also for small molecules such as metabolites and drugs, separating acids, alcohols, ketones, or amino acids. In conclusion, 2D techniques with CE involved provide a powerful tool for separation and will further develop due to achievements in micromechanics and microfluidics driven by the need for better analytical tools for the analysis of complex (biological) samples. The authors gratefully acknowledge the assistance of Dr. Cristina Montealegre (Aalen University, Aalen, Germany) and Priv.-Doz. Dr. Philippe Schmitt-Kopplin (Helmholtz Zentrum München, Munich, Germany), and the financial support (FKZ 17N1110) of the Federal Ministry of Education and Research (BMBF). The authors have declared no conflict of interest.

5 References [1] Giddings, J. C., Anal. Chem. 1984, 56, 1258A–1270A. [2] Jandera, P., Cent. Eur. J. Chem. 2012, 10, 844–875. [3] Tscherch, K., Biller, J., Lehmann, M., Truschb, M., Rohn, S., Phytochem. Anal. 2013, 24, 436–445. [4] Bedani, F., Schoenmakers, P. J., Janssen, H.-G., J. Sep. Sci. 2012, 35, 1697–1711. [5] Seeley, J. V., J. Chromatogr. A 2012, 1255, 24–37. [6] Evans, C. R., Jorgenson, J. W., Anal. Bioanal. Chem. 2004, 378, 1952–1961.



[7] Stroink, T., Ortiz, M. C., Bult, A., Lingeman, H., Jong, G. J. d., Underberg, W. J. M., J. Chromatogr. B 2005, 817, 49–66. [8] Huang, Y.-F., Huang, C.-C., Hu, C.-C., Chang, H.-T., Electrophoresis 2006, 27, 3503–3522. [9] Xu, X., Liu, K., Fan, Z. H., Expert Rev. Proteomics 2012, 9, 135–147. ´ [10] Valcarcel, M., Arce, L., R´ıos, A., J. Chromatogr. A 2001, 924, 3–30. ´ ´ K., Biomedical Engineering—From [11] Mikus, P., Marakov a, Theory to Application, Intech, Rijeka 2011. [12] Gao, D., Liu, H., Jiang, Y., Lin, J.-M., Lap Chip 2013, 13, 3309–3322. [13] Tia, S., Herr, A. E., Lap Chip 2009, 9, 2524–2536. [14] Kasicka, V., Electrophoresis 2014, 35, 69–95. [15] Guihen, E., Electrophoresis 2014, 35, 138–146. [16] Issaq, H. J., Chan, K. C., Janini, G. M., Muschnik, G. M., Electrophoresis 1999, 20, 1533–1537. [17] He, Y., Yeung, E. S., Chan, K. C., Issaq, H. J., J. Chromatogr. A 2002, 979, 81–89. [18] Eckhardt, A., Miksik, I., Charvatova, J., Deyl, Z., J. Liq. Chromatogr. Relat. Technol. 2005, 28, 1437–1451. ´ K., Eckhardt, A., Miksik, I., J. Sep. Sci. 2006, [19] Mikulikova, 29, 1126–1131. [20] Strausbauch, M. A., Madden, B. J., Wettstein, P. J., Landers, J. P., Electrophoresis 1995, 16, 541–548. [21] Jaworska, M., Klaczkow, G., Wilk, M., Anuszewska, E., Acta Pol. Pharm. 2012, 69, 799–808. ¨ [22] Voelter, W., Schutz, J., Tsitsiloni, O. E., Weiler, A., ¨ Grubler, G., Paulus, G., Stoeva, S., Lehmann, R., J. Chromatogr. A 1998, 807, 135–149.

CE and CEC

157

[35] Yu, W., Li, Y., Deng, C., Zhang, X., Electrophoresis 2006, 27, 2100–2110. [36] Shen, Y., Berger, S. J., Smith, R. D., J. Chromatogr. A 2001, 914, 257–264. [37] Wall, D. B., Kachman, M. T., Gong, S., Hinderer, R., Parus, S., Misek, D. E., Hanash, S. M., Lubman, D. M., Anal. Chem. 2000, 72, 1099–1111. [38] Gao, H., Shen, Y., Veenstra, T. D., Harkewicz, R., Anderson, G. A., Bruce, J. E., Pasa-Tolic, L., Smith, R. D., J. Microcolumn Sep. 2000, 12, 383–390. [39] Fang, X., Yang, L., Wang, W., Song, T., Lee, C. S., DeVoe, D. L., Balgley, B. M., Anal. Chem. 2007, 79, 5785–5792. ´ [40] Santos, B., Simonet, B. M., R´ıos, A., Valcarcel, M., Electrophoresis 2007, 28, 1345–1351. [41] Cottet, H., Biron, J. P., Taillades, J., J. Chromatogr. A 2004, 1051, 25–32. [42] Cottet, H., Biron, J. P., Macromol. Chem. Phys. 2005, 206, 628–634. [43] Anouti, S., Vandenabeele-Trambouze, O., Koval, D., Cottet, H., Anal. Chem. 2008, 80, 1730–1736. [44] Anouti, S., Vandenabeele-Trambouze, O., Koval, D., Cottet, H., Electrophoresis 2009, 30, 2–10. [45] Anouti, S., Vandenabeele-Trambouze, O., Cottet, H., Electrophoresis 2010, 31, 1029–1035. [46] Zhang, X., Zhang, Z., J. Chromatogr. B 2011, 879, 1641–1646. [47] Zhang, Z., Du, X., Li, X., Anal. Chem. 2011, 83, 1291–1299. [48] Kukusamude, C., Srijaranai, S., Quirino, J. P., Anal. Chem. 2014, 86, 3159–3166. [49] Ramautar, R., Somsen, G. W., de Jong, G. J., Electrophoresis 2010, 31, 44–54.

[23] Issaq, H. J., Chan, K. C., Liu, C. S., Li, Q., Electrophoresis 2001, 22, 1133–1135.

[50] Ramataur, R., De Jong, G. J., Somson, G. W., Electrophoresis 2012, 33, 243–250.

´ [24] Garc´ıa-Villalba, R., Carrasco-Pancorbo, A., Vazquez´ Mart´ın, A., Oliveras-Ferraros, C., Menendez, J. A., ´ Segura-Carretero, A., Fernandez-Gutierrez, A., Electrophoresis 2009, 30, 2688–2701.

[51] Puig, P., Borrull, F., Calull, M., Carme, A., Trends Anal. Chem. 2007, 26, 664–678.

[25] Zhu, K., Kim, J., Yoo, C., Miller, F. R., Lubman, D. M., Anal. Chem. 2003, 75, 6209–6217.

[53] Tong, W., Link, A., Eng, J. K., Yates, J. R., Anal. Chem. 1999, 71, 2270–2278.

[26] Sanz-Nebot, V., Benavente, F., Barbosa, J., J. Chromatogr. A 2002, 950, 99–111.

[54] Lee, W.-H., Wang, C.-W., Her, G.-R., Rapid Commun. Mass Spectrom. 2011, 25, 2124–2130.

[27] Janini, G. M., Chan, K. C., Conrads, T. P., Issaq, H. J., Veenstra, T. D., Electrophoresis 2004, 25, 1973–1980.

[55] Yang, C., Liu, H., Yang, Q., Zhang, L., Zhang, W., Zhang, Y., Anal. Chem. 2003, 75, 215–218.

[28] Udiavar, S., Apffel, A., Chakel, J., Swedberg, S., Hancock, W. S., Pungor, E., J. Anal. Chem. 1998, 70, 3572–3578. ´ erier, ´ [29] Escoubas, P., Whiteley, B. J., Kristensen, C. P., Cel M.-L., Corzo, G., Nakajima, T., Rapid Commun. Mass Spectrom. 1998, 12, 1075–1084. [30] Cesla, P., Fischer, J., Jandera, P., Electrophoresis 2010, 31, 2200–2210. [31] Jia, L., Liu, B.-F., Terabe, S., Nishioka, T., Anal. Chem. 2004, 76, 1419–1428. [32] Jia, L., Tanaka, N., Terabe, S., Electrophoresis 2005, 26, 3468–3478. [33] Mao, Y., Zhang, X., Electrophoresis 2003, 24, 3289–3295. [34] Mao, Y., Li, Y., Zhang, X., Proteomics 2006, 6, 420–426.


[52] Delahunty, C. M., Yates, J. R., Biotechniques 2007, 43, 563–569.

[56] Liu, H., Yang, C., Yang, Q., Zhang, W., Zhang, Y., J. Chromatogr. B 2005, 817, 119–126. [57] Mohan, D., Lee, C. S., Electrophoresis 2002, 23, 3160–3167. [58] Mohan, D., Pasa-Tolic, L., Masselon, C. D., Tolic, N., Bogdanov, B., Hixson, K. K., Smith, R. D., Lee, C. S., Anal. Chem. 2003, 75, 4432–4440. [59] Zhou, F., Johnston, M. V., Anal. Chem. 2004, 76, 2734–2740. [60] Liu, H., Zhang, L., Zhu, G., Zhang, W., Zhang, Y., Anal. Chem. 2004, 76, 6506–6512. [61] Wang, T., Ma, J., Wu, S., Sun, L., Yuan, H., Zhang, L., Liang, Z., Zhang, Y., J. Chromatogr. B 2011, 879, 804–810.


158

F. J. Kohl et al.


[62] Zhang, Z.-X., Zhang, M.-Z., Zhang, S.-S., Electrophoresis 2009, 30, 1958–1966.

M. J., Dovichi, N. J., Mol. Cell. Proteomics 2002, 1, 69– 74.

[63] Zhang, Z.-X., Zhang, X.-W., Li, F., Sci. China 2010, 53, 1183–1189.

[80] Flaherty, R. J., Huge, B. J., Bruce, S. M., Dada, O. O., Dovichi, N. J., Analyst 2013, 138, 3621–3625.

[64] Kler, P. A., Posch, T. N., Pattky, M., Tiggelaar, R. M., Huhn, C., J. Chromatogr. A 2013, 1297, 204–212.

[81] Dickerson, J. A., Ramsay, L. M., Dada, O. O., Cermak, N., Dovichi, N. J., Electrophoresis 2010, 31, 2650–2654.

[65] Lemmo, A. V., Jorgenson, J. W., Anal. Chem. 1993, 65, 1576–1581.

¨ [82] Bergstrom, S. K., Samskog, S., Markides, K. E., Anal. Chem. 2003, 75, 5461–5467.

[66] Moore, A. W., Jorgenson, J. W., Anal. Chem. 1995, 67, 3448–3455.

[83] Lewis, K. C., Opiteck, G. J., Jorgenson, J. W., Am. Soc. Mass Spectrom. 1997, 8, 495–500.

[67] Hooker, T. F., Jorgenson, J. W., Anal. Chem. 1997, 69, 4134–4142.

[84] Schoenherr, R. M., Ye, M., Vannatta, M., Dovichi, N. J., Anal. Chem. 2007, 79, 2230–2238.

¨ ¨ [68] Bergstrom, S. K., Dahlin, A. P., Ramstrom, M., Andersson, M., Markides, K. E., Bergquist, J., Analyst 2006, 131, 791–798.

[85] Li, Y., Wojcik, R., Dovichi, N. J., J. Chromatogr. A 2011, 1218, 2007–2011.

[69] Cabaleiro, O., Lores, M., Cela, R., Analusis 1999, 27, 468–471. [70] Zhang, X., Hu, H., Xu, S., Yang, X., Zhang, J., J. Sep. Sci. 2001, 24, 385–391. [71] Michels, D. A., Hu, S., Dambrowitz, K. A., Eggertson, M. J., Lauterbach, K., Dovichi, N. J., Electrophoresis 2004, 25, 3098–3105. [72] Hu, S., Michels, D. A., Fazal, M. A., Ratisoontorn, C., Cunningham, M. L., Dovichi, N. J., Anal. Chem. 2004, 76, 4044–4049. [73] Kraly, J. R., Jones, M. R., Gomez, D. G., Dickerson, J. A., Harwood, M. M., Eggertson, M. J., Paulson, T. G., Sanchez, C. A., Odze, R., Feng, Z., Reid, B. J., Dovichi, N. J., Anal. Chem. 2006, 78, 5977–5986. [74] Gonzalez-Gomez, D., Cohen, C., Dickerson, J. A., Chen, X., Canada-Canada, F., Dovichi, N. J., Talanta 2009, 78, 193–198. [75] Chen, X., Fazal, M. A., Dovichi, N. J., Talanta 2007, 71, 1981–1985.

[86] Zhang, J., Hu, H., Gao, M., Yang, P., Zhang, X., Electrophoresis 2004, 25, 2374–2383. [87] Bushey, M. M., Jorgenson, J. W., Anal. Chem. 1990, 62, 978–984. [88] Li, Y., Fang, X., Zhao, S., Zhai, T., Sun, X., Bao, J. J., Chem. Lett. 2010, 39, 983–985. [89] Lemmo, A. V., Jorgenson, J. W., J. Chromatogr. A 1993, 633, 213–220. [90] Stroink, T., Schravendijk, P., Wiese, G., Teeuwsen, J., Lingeman, H., Waterval, J. C. M., Bult, A., de Jong, G. J., Underberg, W. J. M., Electrophoresis 2003, 24, 1126–1134. [91] Stroink, T., Wiese, G., Teeuwsen, J., Lingeman, H., Waterval, J. C. M., Bult, A., de Jong, G. J., Underberg, W. J. M., Electrophoresis 2003, 24, 897–903. [92] Chen, J., Lee, C. S., Shen, Y., Smith, R. D., Baehrecke, E. H., Electrophoresis 2002, 23, 3143–3148. [93] Chen, J., Balgley, B. M., DeVoe, D. L., Lee, C. S., Anal. Chem. 2003, 75, 3145–3152.

[76] Fazal, M. A., Palmer, V. R., Dovichi, N. J., J. Chromatogr. A 2006, 1130, 182–189.

[94] Wang, Y., Rudnick, P. A., Evans, E. L., Li, J., Zhuang, Z., DeVoe, D. L., Lee, C. S., Balgley, B. M., Anal. Chem. 2005, 77, 6549–6556.

[77] Harwood, M. M., Christians, E. S., Fazal, M. A., Dovichi, N. J., J. Chromatogr. A 2006, 1130, 190–194.

[95] Wei, J., Gu, X., Wang, Y., Wu, Y., Yan, C., Electrophoresis 2011, 26, 230–237.

[78] Harwood, M. M., Bleecker, J. V., Rabinovitch, P. S., Dovichi, N. J., Electrophoresis 2007, 28, 932– 937.

[96] Zhang, M., Rassi, Z. E., J. Proteome Res. 2006, 5, 2001–2008.

[79] Michels, D. A., Hu, S., Schoenherr, R. M., Eggertson,


[97] Wu, Y., Wang, Y., Gu, X., Zhang, L., Yan, C., J. Sep. Sci. 2011, 34, 1027–1034.


Recent advances in coupling capillary electrophoresis-based separation techniques to ESI and MALDI-MS.

Chiral separation by capillary electrophoresis with oligosaccharides.

Analytical and micropreparative separation of peptides by capillary zone electrophoresis using discontinuous buffer systems.

Recent applications of on-line sample preconcentration techniques in capillary electrophoresis.

Applications of capillary electrophoresis for industrial analysis.

Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual cyclodextrin-crown ether systems.

Separation and characterisation of detonation nanodiamond by capillary zone electrophoresis.

Enhancing separation in short-capillary electrophoresis via pressure-driven backflow.

Separation of cationic proteins via charge reversal in capillary electrophoresis.

The application of capillary electrophoresis techniques in toxicological analysis.

Separation by capillary electrophoresis followed by dynamic elution.

Isotopic separation of lithium ions by capillary zone electrophoresis.

Separation of globins using free zone capillary electrophoresis.

Optimizing separation conditions of 19 polycyclic aromatic hydrocarbons by cyclodextrin-modified capillary electrophoresis and applications to edible oils.

Capillary electrophoresis.

Capillary electrophoresis.

Separation of dideoxyribonucleosides in trace amounts by automated liquid chromatography and capillary electrophoresis.

Efficient capillary electrophoresis separation and determination of free amino acids in beer samples.

Experimental evaluation of the separation efficiency in capillary electrophoresis using open tubular and gel-filled columns.

High-speed separation and detection of amino acids in laver using a short capillary electrophoresis system.

Low electric field DNA separation and in-channel amperometric detection by microchip capillary electrophoresis.

Characterization and separation of semiconductor quantum dots and their conjugates by capillary electrophoresis.

The separation and detection of PET tracers via capillary electrophoresis for chemical identity and purity analysis.

The use of capillary electrophoresis in a micropreparative mode: methods and applications.