Interfacing commercially available capillary electrophoresis to sample preparation and/or detection systems to solve analytical problems

At-line coupled systems

At-line coupling of sample treatment device to commercial CE equipment can be performed using a robotic arm interface (Arce et al. 2000) or the modification of the replenishment system of the CE equipment (Simonet et al. 2005). The use of robotic interface requires an electronic interface and software to synchronize the movement of the robotic interface with the movement of autosampler. Figure 1 represents a scheme of the coupling of this robotic interface with CE equipment. The needles of the programmable robotic arm can be positioned in two ways with respect to the autosampler vials. While the sample treatment processing device is working, the needles are down, and the treated sample is prepared and transferred to the CE vial. When sample treatment processing device stops, the needles are lifted, and the CE analysis is started by moving the previously filled sample vial to the position where the capillary end and the electrode are located. The programmable arm can be fitted with one or two needles that can be identical or different in length – the longer one being used to fill the autosampler vials and the shorter one to drain them to maintain a preset liquid level. When a single needle is used, a constant, preset volume is delivered to each vial (e.g., by using air as the carrier). Several applications using this robotic arm interface were published applying different sample treatment devices, such as flow injection/solid phase extraction (FI/SPE), FI/gas diffusion, and SFE (see Table 2).

Figure 1

Schematic description of an at-line coupling sample treatment unit to commercial CE equipment using a robotic arm interface.

Methodologies integrating solid phase extraction (SPE) unit in continuous flow system (CFS) coupled to commercial CE using the programmable arm mode were applied to determination of chlorophenols (Mardones et al. 1999), biogenic amines (Arce et al. 1998a,b,c,d), trimethylamines (Lista et al. 2001), inositol phosphates (Simonet et al. 2003) and inorganic ions (Arce et al. 1997). The online cleanup and/or preconcentration of these analytes were accomplished by using a CFS coupled to the CE equipment. For analysis, samples were pumped through the SPE units to retain the analytes. After the samples were loaded, the SPE units were washed to remove impurities and eluted with appropriates solvents. The eluates, containing the analytes, were inserted in a minivials of the CE autosampler by actuating the programmable arm. Another example is the use of this robotic system for direct determination of trimethylamine and related amines in fish samples (Lista et al. 2001). This arrangement allowed the direct introduction and treatment of these solid samples with a high level of automation. The amine compounds were extracted from fish samples in the gas extraction sampling unit integrated in a CFS coupled to CE, and then they were conditioned in the CFS and injected into the CE vials via a programmable arm.

Supercritical fluid extraction, separation, and detection of cresols and chlorophenols in liquid samples were also achieved by a SFE-CE using this arm interface (Mardones et al. 2000). The mechanical interface used for SFE-CE coupling comprises a customized programmable arm and the autosampler of the CE equipment. This electronic interface enables automatic control of the coupled elements via the built-in microprocessor of the CE equipment. A vial that can be placed at two different positions of the autosampler establishes the link between the rinsing system and the capillary end via two programmable arms. The customized arm is furnished with two injection needles of different lengths; the longer needle is used to fill the vial and the shorter one to drain it to maintain a preset liquid level. The needles are up while the rinsing system is working and down when it is at rest. The programmable arm performs hydrodynamic injections in the conventional manner. The programmable arm was controlled by an electronic interface governed by GW Basic software. The main function of this program was to control the time for insertion of the needles into the CE vial. The determination of these analytes was carried out automatically without human intervention with good precision and throughput.

As an alternative to programmable robotic arm, sample treatment devices and CE equipment can be coupled at-line via the replenishment system, which is used to empty vials and fill them with fresh buffer in some commercial instruments. This option, proposed for the first time by Santos et al. (2004a), involves disconnecting the replenishment needles from the Teflon tube coming from the replenishment bottles and replacing them with one tube coming from the sample treatment device. This alternative was proposed to automate the monitoring of biogenic amines and sulfonamide residues in milk and wine samples, respectively (Santos et al. 2004a, 2005). For determination of sulfonamides in milk, it combines a screening unit for the total amount of sulfonamide with CE-MS equipment for processing the samples containing a detectable level of sulfonamide. The screening unit consists of CFS to precipitate the proteins connected online to the CE-MS equipment, in which a common characteristic ion of all sulfonamides was monitored with the MS detector by flushing the sample through the capillary. The confirmatory method is based on the purification and preconcentration of sulfonamides in a CFS unit and posterior analysis by CE-MS. The sample treatment unit was also online connected at-line via the replenishment system to the CE-MS equipment.

The most salient advantage of at-line coupled systems is that the CE equipment is run in its normal operation mode. This allows samples to be introduced hydrodynamically or electrokinetically into the capillary. It also facilitates other CE operations, such as capillary conditioning.

On-line coupled systems

Coupling sample treatment devices integrated in CFS on-line to commercial CE involves inserting the capillary end of the latter in a continuous steam of the former (Figure 2). Because the flow processing device and the electrophoretic system operate at a different flow rate, the two require a split-flow interface for coupling. The split-flow interface was originally developed simultaneously in a vertical configuration by Fang’s group (Fang et al. 2000) and a horizontal configuration by Karlberg (Kuban and Karlberg 1998). Figure 2 compares the two types of interface, which possess a low dead volume and are electrically grounded. Samples and electrolyte solutions are introduced in the electrophoretic capillary by effect of electroosmotic flow and electrophoretic mobility. Hydrodynamic flow should be avoided by ensuring that the liquid level at the interface (viz. the capillary inlet) coincides with that in the capillary outlet. A split-flow interface can be readily constructed from non-conducting polymer materials, such as Teflon, methacrylate, or Plexiglas. Alternatively, a piece of tygon tubing can be used to insert the electrophoretic capillary and attach it with glue. The integrated sample treatment device in CFS is made compatible with the electrophoretic system by grounding the interface. This avoids voltage differences between the interface and flow system. Electrophoretic separation is accomplished by applying a voltage difference to the capillary end. This configuration is highly recommended when an optical detector is used with the electrophoretic system, but scarcely useful with other types of detectors (e.g., mass spectrometers).

Figure 2

Schematic description of an on-line coupling sample treatment unit to commercial CE equipment using a vertical and horizontal design of split-flow interface.

In principle, the use of on-line coupled systems linked by a split-flow interface is limited to the electrokinetic introduction of samples. However, this injection mode provides biased results in some cases. This has promoted the use of hydrodynamic injection instead. Thus, Pu and Fang (1999) used a split-flow cell affording hydrodynamic injection of samples into the CE capillary by electroosmotic flow. The interface included a Nafion joint to connect the CE capillary to the tube of the flow system. An electronic time relay system was used to control switching of the high voltage. Kuban and Karlberg (1998) inserted a valve at the end of a horizontal split-flow interface. Switching the valve off caused the sample to be forced into the capillary. The greatest limitations of this approach arise from the need to control the time during which the valve is on and off and also the pressure generated in the system. One research group (Santos et al. 2006a,b) proposed controlling the pressure by using a valve loop capable of withstanding higher pressures. With commercial CE instruments, a researcher can also connect the pressure system of the equipment (Santos et al. 2006b).

It should be noted that commercial CE equipment affords injection at a low pressure (0.5 psi) with a high precision (0.01 psi). A modified version of the split-flow interface was used to accommodate a typical solid-phase microextraction (SPME) fiber precisely at the capillary end (Santos et al. 2007). Although samples were treated on the fiber and the flow system was only used to couple SPME and CE, they can also be processed at the interface connecting the fiber to the flow system. In this case detection of trace levels of tetracycline from soil samples was demonstrated. Supercritical fluid extraction (SFE) separation and detection of riboflavin vitamins in food samples were also achieved by a SFE-CE using this split-flow interface (Zougagh and Ríos 2008). This method is based on the on-line coupling of a SFE with a continuous flow-CE system with fluorimetric detection (CF-CE-FD). The whole SFE-CF-CE-FD arrangement allowed the automatic treatment of food samples (cleanup of the sample followed by the extraction of the analytes), and the direct introduction of a small volume of the extracted plug to the CE-FD system for the determination of riboflavin (RF) vitamins. Veraart et al. (1998, 1999, 2001) developed online dialysis-SPE-CE systems with a split-flow vial interface for the analysis of amphoteric solutes (Veraart et al. 1998), nonsteroidal antiinflammatory drugs (Veraart et al. 1999) and tricyclic antidepressants (Veraart and Brinkman 2001) in urine and serum samples. In these works, the dialysis-SPE unit was connected with the CE system via this interface in which the sample is delivered in a vial-like enclosure also containing an electrode and CE capillary separation. This vial interface allows the analytes to flow through to waste, while analytes are injected into the CE capillary by applying a voltage of pressure difference. In this approach, the interface has to be filled with background electrolyte (BGE) prior to starting analysis, because it also serves as the CE inlet vial. The split-flow interface can be used for the on-line coupling of both flow injection and sequential injections systems. Based on a common approach, split-flow interfaces, microsequential injection systems or integrated lab-on-a-valve systems have been coupled to CE. Sequential injection systems have also been on-line coupled via a microvalve allowing the insertion of a constant volume of sample into the capillary (Mai et al. 2012). However, the loop volume is high relative to those typically used in CE work and must be reduced by switching the valve or flushing the sample from the interface to facilitate electrokinetic insertion of a portion of the sample. Another approach for coupling SPE and CE is the use of a valve interface for introducing the eluate to CE capillary via this valve. Detailed information about this valve interface can be found in a previously published review (Tempels et al. 2008). These authors used this interface to on-line coupling of SPE microcolumn with CE-MS for peptides analysis (Tempels et al. 2007). Although the system is very robust, there are some drawbacks. The coupling via a switching valve is quite challenging, and a number of safety recommendations are essential to avoid unintended electrical discharges. In addition, the researchers concluded that it is difficult to accommodate the elution volumes and the CE injection volumes because only 4–5% of the SPE elution volume is injected. To avoid these drawbacks, Morales-Cid et al. (2009) developed an innovative way to integrate SPE, such as microextraction by packed sorbents (MEPS) into commercial CE equipment by modifying the hardware that controlled the fluid/pressure module of the CE equipment. The MEPS was integrated in the outlet region of a commercial CE equipment cartridge to provide easy manipulation and exchange. The robustness of the proposed integration was demonstrated by the design and use of a MEPS-nonaqueous capillary electrophoresis (NACE)-MS method used to determine fluoroquinolones in urine samples.

In-line coupled systems

The integration of the sample treatment devices in-line into commercial equipment can be performed in the electrophoretic capillary, the electrophoretic vial or even in an auxiliary part of the equipment, such as a replenishment system. This integration offers important advantages over coupling methodologies (described following), as there is no need for additional hardware or software. The coupled equipment described in literature includes extraction, filtering, dialysis, membrane, gas extraction and gas diffusion units; hollow fibers; and even auxiliary devices. The principal conclusion is that choosing an appropriate flow processing device can facilitate the treatment and analysis of virtually any type of sample (biomedical, pharmaceutical, environmental, food). Readers interested in a more detailed description of the types of devices coupled to CE so far are referred to the article of Marina et al. (2005). The integration of sample treatment device in the CE capillary, is probably the alternative most used to date because of coupling both solid phase extraction (SPE) and liquid phase extraction (LPE) using liquid-supported membranes (LSM) (Kuban and Bocej 2013). SPE has been integrated into the CE capillary by packing the sorbent material in the inlet region of the capillary and using it as a minicartridge (Santos et al. 2006b). For analyte determination, the sample is flushed through the capillary containing the cartridge where the analytes are retained and preconcentrated. Afterward, the sample is removed from the capillary with the electrophoretic buffer, which must avoid the elution of analytes from the minicartridge. Finally, a small plug of eluent is introduced by pressure into the capillary. This plug is moved by EOF through the capillary and, when the plug reaches the minicartridge, analytes are eluted and electrophoretic separation takes place. Some applications include the determination of heterocyclic aromatic amines in food (Viberg et al. 2004), insulin derivatives in biological fluids (Visser et al. 2003), and drugs and peptides in biological samples (Guzman 2003, Medina et al. 2014). More applications using this modality are found in the review by Guzman and Stubbs (2001). The most important shortcoming of this methodology is the need to flush the sample through the capillary, as this can result in adsorption of protein or other macromolecules from the matrix sample onto the capillary wall, disturbing the EOF. In most of these works, the solid packing material was held inside the capillary by two frit structures. However, an alternative packing material is a polymer membrane impregnated with a chromatographic stationary phase (Waterval et al. 2001). The replenishment system, which allows the electrophoretic buffer to be removed from the vial and replaced with fresh buffer, can be also used to perform SPE (see Figures 3A and 4B). For that, a typical minicolumn containing the sorbent material is located between the needles and the two bottles of the replenishment system (Santos et al. 2004a,b). One of the bottles is used as a waste and the other to provide the eluent. Once again, the minicolumn is inserted in the system and the eluent is located in the bottle, the software of the CE equipment is programmed to ensure sample aspiration, eluent elution and introduction into the vial, and subsequent CE analysis. Perhaps the most important shortcoming of this strategy is that it is impossible to control the flow rate as the replenishment system normally runs at constant pressure. This shortcoming is in part solved by adjusting the column dimensions.

Figure 3

Schematic description of in-line coupling sample treatment unit to commercial CE equipment using a (A and B) replenishment system, (C) microextraction unit in-capillary (D) microextraction unit in-vial. Reproduced with permission from John Wiley & Sons [Santos et al. 2004a,b, Nozal et al. 2006, 2007].

Figure 4

Scheme of FD designs coupled to on-capillary (A) and to end-capillary (B) modes with commercially available CE equipment. Part (B) was reprinted with permission from Timperman et al. 1995a,b. Copyright 2014 American Chemical Society.

As an alternative to SPE, liquid phase microextraction (LPME) has been coupled in commercial equipment. A simple preconcentration method was based on the formation of a small drop in the inlet region of the capillary. The drop, located inside the sample vial, acts as acceptor phase in the liquid-liquid extraction process. The main shortcoming of this strategy is that the formation of the drop strongly depends on the nature of the liquid and the capabilities of the equipment in applying controlled pressure at the outlet. LPME, based on supported liquid membranes, has also been integrated into the electrophoretic capillary by Nozal et al. (2007). This coupling was achieved by inserting a hollow polypropylene fiber in the inlet region of the capillary (see Figure 3C). The connections between the hollow fiber and the capillary were made by burning the hollow fiber on the capillary. The main difference with integrated SPE is the mode of transportation. In SPE, sample is flushed through the capillary; but, controversially, in LPME analytes migrate through the membrane, so this strategy avoids contact between the sample matrix and the internal capillary wall. The extraction system directly determines non-steroidal anti-inflammatory drugs (NSAIDs) in human urine with no sample pretreatment. This approach, which is simple and useful, can be easily implemented in routine laboratories. It affords a large number of extractions without the need to replace the unit. As with inserting the hollow fiber, Yang et al. (2006) proposed insertion of a cellulose acetate-coated porous membrane. The membrane, located at the end of the capillary, allows the passage of buffer ions but excludes larger protein molecules. The membrane is then used to retain proteins at the end of the capillary. With the retained and preconcentrated proteins, the direction of the electric field is switched to perform separation and detection.

The integration of the sample treatment devices in-line into commercial equipment can also be performed in vial (Nozal et al. 2006, Pantuckova et al. 2013) (see Figure 3D). This modality consists in the assembly of extraction unit directly in the vial containing the sample. The vial containing the microextraction unit was located in the autosampler, and the treated sample was direct analyzed by electrophoresis. The microextraction unit was designed to permit introduction of the electrode and capillary in the treated sample so that there could be both electrokinetic and hydrodynamic introduction into the capillary. The potential of an in-vial micro-SLM extraction unit with commercially available CE equipment is the direct determination of nitroimidazoles from pig-liver-tissue homogenates (Nozal et al. 2006). The same coupled system for direct determination of chlorophenols in liquid samples (Almeda et al. 2007) and ochratoxin in wines (Almeda et al. 2008) was also used.

Interfaced CE-FD systems

Several optical designs for fluorescence detection (FD) in CE have been proposed and used during the years, most of which are home-built and comprise various excitation sources and photosensitive detectors. Interested readers can find a recently published review by De Kort et al. (2013) of FD designs, coupled in the on-capillary or end-capillary modes with commercially available CE equipment. In the on-capillary mode, the system configured to make the fluorescent can be detected inside the separation capillary. A good example of a setup using the FD at-line with CE has been developed by Flux (Basel, Switzerland) that is equipped with an external detection cell mounted on the CE cassette, Xenon-mercury lamp as the radiation source, and photomultiplier tube (PMT) with a measurement range of 185–700 nm (for signal acquisition). This external detection uses total internal reflection of emitted fluorescence light to spatially separate the point of analyte excitation from the position where emission light is collected. In this way, the background noise due to scattered excitation light is seriously reduced. Excitation light is focused on the capillary by a patented light cone. After analyte excitation, part of the generated fluorescence emission light is trapped within the capillary walls due to total internal reflection, because the refractive index of fused silica (n=1.458) is higher than that of the aqueous BGE (n=1.333) and air (n=1.000). About 1 cm away from the position of analyte excitation, glycerol (n=1.473) is applied onto the surface of the detection window to match the refractive index of the capillary, so that the emission light exits the capillary and enters a light cone. The cone collimates the emission light and transfers it into an emission fiber, which guides the light toward the detector. The sensitivity of this optical application is comparable to laser-induced fluorescence detectors. Various authors have used this configuration to interface the CE instrument, for determination of B₂ and B₆ vitamins in serum samples (Priego and Luque 2005), heterocyclic amines in meat samples (De Andrés et al. 2010), and catecholamines and related compounds in urine samples (Zhu and Kok 1997).

In the end-capillary mode, the analytes migrate from the separation capillary before fluorescence detection occurs, thus preventing excitation light scatter and autofluorescence from the capillary wall. Timperman et al. (1995a), designed a fluorescence detector interfaced in CE (Figure 4B), that employs a frequency doubled krypton laser operating at 284 nm for excitation, a reflective microscope objective, an imaging spectrograph, a charge coupled detector (CCD), and a sheath-flow cell cuvette. The sheath fluid has a higher flow rate than the capillary flow, which causes elongation and focusing of the capillary flow to a small volume. The excitation light can be effectively focused onto this volume by use of a lens. The fluorescence emission is collected at 90° to the excitation with an eflective microscope objective. In this configuration, neither a Rayleigh filter nor a preexcitation laser-line filter is used, because the sheath flow cell and spectrograph eliminate and separate most of the Rayleigh scattering (Timperman et al. 1995b). Another instrumental configuration using CCD was demonstrated in a two-laser–two-color flow cytometry study (Fuller et al. 1996). A sheath-flow cell (quartz cuvette) was used as the sample chamber. Excitation of the core stream was achieved by means of a liquid-cooled Ar⁺ laser and an ArKr⁺ laser operating at 488 nm and 568 nm, respectively. Collection optics included a microscope objective (to eliminate fluorescence from the collection optics) and a series of 500-nm high-pass and 568-nm laser-line rejection filters. The spectrograph (grating 285 grooves mm^–1) and CCD were oriented to enable sub-array readout and binning (3 Hz readout, 2×256 binning) while preserving 1024-pixel wavelength information over the 350–800 nm wavelength range. By use of single-particle fluorescence emission spectra, submicron particles were individually identified, sized, and distinguished. Park et al. (1995) used the sheath flow cell to interface FD and CE for the separation and fluorescence detection of idolamine and catecholamine.

Interfaced CE-ED systems

Electrochemical detection (ED) in CE focuses on the effects of shuttling electrons, ion mobility, and membrane potentials. Modes of interfacing ED to CE included in this review are volammetry at fixed potential (amperometry), because it is by far the most implemented detection of electrochemistry owing to its excellent limit of detections (LODs). However, other ED modes such as conductivity, and potentiometry have various advantages, which are described in other published works (Timerbaev and Buchberger 1999, Huang and Fang 2000, Zemann 2001, Trojanowicz 2009).

Because, the high voltage applied to the separation capillary can lead to serious interferences with detection of the electrochemical signal, the on-capillary detection is rarely used with ED techniques (Timerbaev and Buchberger 1999). Instead, the detection electrode can be positioned at the outlet of the separation capillary (end-capillary). In this arrangement, the high electric field strength is confined to the inside of the separation capillary, whereas outside and at the position of the detection electrode the electric field strength is low and does not significantly interfere with the detection signal. Alternatively, the high electric field can be completely decoupled from the detection electrode by connecting the end of the separation capillary with a short transfer capillary by means of a porous coupling piece surrounded by a carrier electrolyte with the high voltage electrode; the current passes the porous coupling piece, whereas the electroosmotic flow (EOF) generated in the separation capillary carries the analytes through the transfer capillary to the detection electrode (Timerbaev and Buchberger 1999). Wallingford and Ewing (1987) were the first to report the interface for coupling of end-capillary amperometric detection to CE. This system couples two pieces of column together with a section of porous glass capillary, thus forming a joint that is electrically conductive. The joint was immersed in a buffer reservoir along with the ground electrode to allow the separation potential to be applied across only the first section of capillary. The strong electroosmotic flow generated in the first section of capillary serves to force solvent and analyte zones past the joint and through the second section of capillary to the detector. This configuration effectively separates the detector from the high applied voltage via the resistance of the detection capillary. The authors have obtained separation efficiencies on the order of 180,000 theoretical plates for the separation of catechol from catecholamines. Although this interface was initially used with the home-made CE, it was modified and adapted to be used in the commercial CE demonstrated by a wide variety of applications and a large volume of original research publications. In this direction, Chicharro et al. (2002) designed a good end-capillary interface to attach amperometric detector to a commercial CE. This interface, as represented in Figure 5, was mounted by assembling three pieces: two Plexiglas blocks and a PTFE gasket placed between them. Working and reference electrodes were fitted to nuts (1 and 2 in the figure), respectively, by PTFE adapters and could be simply replaced if necessary. The cell operates in an inverse wall-jet arrangement. The inlet-working electrode distance and alignment is quickly and conveniently controlled by nut (marked with 1 in the figure) and by adjusting the upper block with the screws. The electrolyte solutions can easily be changed or renewed with the help of a syringe connected to the drain outlet (marked with 7). Connection, marked with 11 in the figure, allows coupling the vacuum tube (marked with 5) to the equipment vacuum system. The vial could be easily filled and drained with the help of a syringe connected to the position marked with 12 in the figure. The body cell is fitted to this vial with a hollow screw that joins the 13 and 4 positions of the figure. The separation capillary column is inserted inside the detection cell through the hollow screw. A rubber septum fixes the capillary position and ensures the isolation between the vial and the detection cell. No modification was done to adapt the electrochemical cell on the original design of the capillary electrophoresis injection module. Various methodologies, using this interface, for coupling amperometric detection and commercial CE were applied to determination of polyphenols in white wine (Moreno et al. 2011), macrocyclic lactone mycotoxins in maize (Sanchez et al. 2009) and maleic hydrazide in potatoes (Chicharro et al. 2008).

Figure 5

Scheme of (A) on-capillary ED coupled in to commercial CE instrument, (B) detection electrochemical cell and cathodic vial for commercial capillary electrophoresis system: nut adapters for (1) working and (2) reference electrodes; (3) screw; (4) connection with vial for capillary electrophoresis system; (5) vacuum connection; (6) flow outlet; (7) drain outlet; electrode holders: (8) working; (9) auxiliary; (10) reference; (11) vacuum connection; (12) drain outlet; (13) cell connection. Reproduced with permission from Elsevier [Chicharro et al. 2002].

Interfaced CE-ELSD systems

The combination of commercially available CE with ELSD is an interesting possibility, especially for analytes no sensitive to other common detectors used in CE. ELSD can be considered to be an universal detector, as in principle the only analyte characteristic required for response is low volatility compared to the buffer solution. In the bibliography, only two CE-ELSD interfaces have been found, as can be deduced from the papers published on this topic (Szostek and Koropchak 1996, Bouri et al. 2013b). In the first interface designed by Koropchak group, the authors interfaced the ELSD to CE in two approaches. In the first system, the separation capillary was divided in two parts connected by the stainless-steel union. Nafion tubing was glued to the fused silica capillary using epoxy resin. During operation, this Nafion membrane was immersed in a vial containing sulfuric acid, along with the grounding electrode of the CE. The outlet end of the Nafion tubing was glued with epoxy to fused-silica nebulizer capillary. The nebulizer was inserted through a stainless-steel tee and the outer nebulizer capillary. The other arm of the tee was connected to the nebulizer nitrogen gas source. The nebulizer assembly was inserted through the end cap of a spray chamber, which contained a baffle assembly located ∼3 mm from the nebulizer tip. The spray chamber was connected to the ELSD using Teflon tubing. The second means of interfacing the CE with the ELSD employed either a PEEK separation capillary or a separation capillary with strong anion-exchange-coated microspheres permanently bonded to the interior walls. In either case, the capillary was directly inserted through the stainless-steel tee into the nebulizer after the last 15 cm of the exit end of the capillary was coated with copper paint containing tungsten wire to match the inner diameter of the capillary. After the paint dried, the wire was removed to leave a tip that acted as the contact for the ground with the buffer solution of the CE. This coupling is complex and requires a Nafion membrane or other material to ground CE circuit and connect the ELSD system. To solve these problems, Bouri et al. (2013b) proposed a stainless-steel triple-tube nebulizer with some similarities to those used in CE–MS interfaces by the ESI mode (Szostek et al. 1997), which was accommodated in a customized ELSD nebulizer chamber with a stainless head connected to the CE chassis with a ground cable (Figure 6B), to avoid electrical discharges. The triple tube nebulizer consists of a central tube (the CE capillary) surrounded by a second stainless-steel tube, the sheath-liquid tube (see details in Figure 6C). The sheath liquid flows between this tube and the inner CE capillary. Between the sheath-liquid tube and the third outer tube, or gas tube, flows the nebulizing gas controlled by an additional pressure regulator compatible with the CE–ELSD interface that contributes to control the nebulizing process. This arrangement is simple and does not require any special electrical connections between the CE and ELSD instruments, such as the use of Nafion membrane or nebulizer coated with a metal for the grounding of the CE circuit system (Szostek and Koropchak 1996). It is enough to connect a ground cable between the CE chassis and the head of the stainless steel nebulizer chamber (Figure 6A). Our research group recently developed a method based on CE-ELSD for direct determination of underivatized aminoacids in tea samples (Bouri et al. 2013a). The obtained results were positively compared with those obtained with a sophistical detector, such as MS (Soga and Heiger 2000). It is also expected that CE–ELSD will open new possibilities for the analytical characterization of polymers and biopolymers, macromolecules, nanoparticles, and, in general, big molecules or aggregation of molecules.

Figure 6

(A) Scheme of the interface for coupling the CE equipment with the ELSD detector via a stainless steel triple tube nebulizer; (B) picture of the nebulizer accommodated in customized nebulizer chamber and scheme of the different connections; and (C) details of the flow tubes to produce an efficient nebulization. Reprinted with permission from Bouri et al. 2013a,b. Copyright 2014 American Chemical Society.