Construction of a simple, economic, and versatile

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In this article we describe in detail the construction and use of a simple, ... including sample provision, capillary conditioning, buffer replacement, and injection procedures. ..... Besides, rise of air humidity in the protection case and consequently the ... been the use of a peltier thermostated cooler but for laboratory ambience, ...

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Construction of a simple, economic, and versatile capillary electrophoresis system for coupling with syringe based analytical flow techniques Burkhard Horstkottea, Carlos M. Duartea, Olaf Elsholzc, Víctor Cerdàb,* a

Department of Global Change Research. IMEDEA (CSIC-UIB), Institut Mediterráni d'Estudis Avançats, Miquel Marques 21, 07190 Esporles, Spain b University of the Balearic Islands, Department of Chemistry, Carreterra de Valldemossa km 7,5, 07011 Palma de Mallorca, Spain c Hamburg University of Applied Sciences, Faculty Life Science, Lohbrügger Kirchstraße 65, 21033 Hamburg, Germany *Author for correspondence: Víctor Cerdà, e-mail: [email protected] Received 27 Jul 2010; Accepted 8 Nov 2010; Available Online 12 Nov 2010

Abstract In this article we describe in detail the construction and use of a simple, economic, and versatile capillary electrophoresis system. The system is coupled to a sequential injection analysis system for operation control including sample provision, capillary conditioning, buffer replacement, and injection procedures. Considerations about design and operation modes are discussed and coupling with a preconcentration flow system is further explained. Keywords: Capillary electrophoresis; Sequential injection analysis; Description of Construction; Operation modes and versatility

1. Introduction At its beginning, capillary electrophoresis (CE) has been considered to become a major separation technique comparable to liquid chromatography and gas chromatography. Karger (1989) [1] stated "High performance capillary electrophoresis is expected to be the fastest-growing analytical technique since HPLC". However, most likely due to the high instrument purchase costs and the wellknown limitations originated from the typical dimensions of the capillary and injected sample being a moderate sensitivity, CE is still not used as wide-spread as it has been predicted. On the other side, CE has gained importance and the analyst's interest in areas where high analyte concentrations are estimated or where the nanoliter sample volumes are an important benefit such as pharmaceutical, clinical, forensic, and biomolecular analysis and research [2]. Single cell analysis has been reported further [3]. CE separations yield generally high efficiencies with plate numbers beyond 105 and often within minutes. An outstanding characteristic of CE and related techniques is the variety of operation and detection modes developed, enabling their application for a wide range of analytical tasks, among these the separation of uncharged analytes by micelle interactions, chiral separations, or implication of solid structures such as gels, monoliths, or most recently, carbon nanotubes [4,5]. Several on-capillary concentration and stacking techniques have further been described [6], which can enhance the detection sensitivity by orders of magnitude. This offers the potential for CE application to environmental analysis. The coupling with mass spectrometry or chemiluminescence as highly sensitive detection methods with CE has further become the state-of-the-art. A frequently posed drawback of CE and related techniques is the reproducibility of injection and separation. While for hydrodynamic injection, the injection volume depends on the possibility to exhibit a controlled and reproducible – general positive – pressure on one side of the capillary and the samples viscosity, for electrokinetic injection, differences in samples conductivity are the main factor of error. The reproducibility of the separation is mainly influenced by the adsorption of matrix components on the capillary inner walls, favored by the high surface to volume ratio in this technique, Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010

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GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY which affects the electro-osmotic flow and by this the separation time and resolution. Sample clean up and filtration and frequent conditioning of the capillary are therefore required. Analytical flow techniques (FT) are characterized by their potential of automation of laboratory preparative and analytical procedures with high reproducibility and low consumption of time and reagents while on the other side they show a low capacity of analyte discrimination. Coupling of FT and CE has therefore been proposed as a powerful marriage combining high efficient separations and automation of analyte preconcentration, sample treatment, provision of reagents for pre- or postcapillary reactions, or to enable interfacing CE analysis with process monitoring. The different modes and interfaces used for coupling of FT and CE including detailed advantages and analytical applications of FT-CE combined analytical systems have been reviewed by the authors recently [7]. Coupling of FT with commercial CE instruments has been successfully carried out in the past using either flow-through vials [8] or batch-wise vial flushing [9] by taking benefit of the autosampler of the commercial CE instrument. However, this task is in general problematic since the safety measures and appliances considered by the producer of the commercial CE instrument might have to be violated or even mechanical modifications of the instrument have to be done. Data connection and software control of both the FT and the CE instrumentation can present a further bottleneck. Finally, the possibilities of flow interfacing of the CE capillary and the FT manifold are limited, which can result in a lack of versatility due to higher dead volumes or lower sample frequency. Therefore, in this paper we describe in detail the construction of a simple, economic, and versatile CE system for coupling with FT to facilitate this task for other researchers and to favor the development and improvement of FT-CE systems and their application. Analytical applications of the presented system have been published elsewhere [10, 11] . We would like to stress out that the high separation voltage presents a risk for electronic instrumentation and health and therefore carefulness in the system design considerations and manipulation is required. 2. Construction of the home-made FT-CE system 2.1. Preliminary considerations 2.1.1. Versatility and reproducibility In order to achieve high reproducibility even for the analysis of samples with complex matrices, a home-made CE system should enable capillary flushing in-situ (without dismounting) by pressure application and conditioning as well as the automated replacement of the separation buffer on both capillary ends (both buffer reservoirs). Both features are further obligatory for the accomplishment of on-capillary concentration techniques, where a major part of the capillary has to be filled with sample or further solutions required to perform analyte sweeping or stacking. Finally, capillary flushing with water for storage should be possible. For electrokinetic injection and on-capillary stacking, the change of the separation voltage level and polarity should further be possible without manual modifications but by remote computer control. To accomplish hydrodynamic injection, the reproducible application of a variable pressure on one side of the capillary should be further enabled. Finally, the injection of sample should be possible on both ends of the capillary, either on the high voltage side if end-of-capillary detection should be performed (e.g. coupling with mass spectrometry) or – generally – on the grounded side performing contact-less on-capillary detection. 2.1.2. Safety Since harmfully high voltage is applied for CE separation, the entire CE system should be enclosed and the galvanic separation of all instrumentation from the high voltage must be guaranteed throughout. Short-circuits by possible leakages of separation buffer or sparks by accumulating humidity must further be prevented. Finally, the automated shut-down of the separation voltage whenever not required or during manual interventions has to be guaranteed. 2.2. Interfacing to flow system manifold The objective of the system autonomy and applicability for monitoring purposes demanded for the control of the entire system including the buffer reservoirs at both sides of the capillary. A second requirement was the possibility to perform hydrodynamic sample injection and capillary flushing. This required the ability of pressure application at least at one side of the capillary. Interfaces between the flow system manifold and both sides of the capillary acting also as buffer reservoirs were developed. The interfaces are shown with photos in Figure 1.

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Figure 1. Grounded split-flow (A) and high voltage falling drop (B) interfaces with both photo and technical drawing: electrodes (E), capillary (C), flow entrance (IN) and flow outlet (OUT).

Following the common principle of former works, the flow system manifold was galvanically connected to the grounded buffer reservoir, which was also used for sample injection. The corresponding grounded interface followed a tubular split-flow design as shown in Figure 1A. Pressure application was enabled by placing a 3-way solenoid valve (V7) at the outlet of the interface. The position OFF was permanently closed with a blind connector, while the position ON was used as the atmospherically open interface outlet. The intermediate closure of the interface outlet allowed forcing liquid through the capillary (see section 2.9). A similar functionality was achieved using pinch valves in former works [12,13]. Permanently open or membrane interface designs were rejected as less versatile since they do not enable pressure application at the capillary entrance. For connection of the HV-side to the flow manifold, the formerly reported principle of a falling-drop interface was applied (see Figure 1B) [14, 15]. Thus, separation buffer renewal was possible by dropping the solution from about 3 cm above into a miniature funnel of the interface. By this, galvanic separation of the flow manifold and simultaneously the exchange of the contact liquid between the capillary were guaranteed. The hydrostatic pressure of the introduced buffer forced interface flushing and gravimetric flow-out at the sideward opening. This flow was sufficient to remove stacked gas bubbles originated from water electrolysis during HV application. Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010

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Figure 2. Detailed view of the solenoid valve and silicone tube compliance used as pressure regulator and reservoir. A: not inflated, ambient pressure, B: inflated with 300 µL (solenoid valve in position of the closed port) corresponding to the pressure used for capillary flush.

The flow channel of the grounded interface was made small with 0.7 mm i.d. in order to reduce its dead volume and sample dispersion. On the other hand, it still allowed the visual positioning of the capillary tip in the center of the flow channel. The flow channel inner diameter of the HV interface was 2 mm in order to facilitate the gravimetric flow-out during the cleaning procedure. The electrode containing sections of the interfaces were made with diameters in order to guarantee the galvanic, i.e., liquid contact between the capillary and the respective electrode. The application of pressure at the grounded interface for capillary flushing required tight sealing between the capillary and the interfaces. So, the capillary ends were inserted into short pieces of PTFE tube (ca. 2 cm, 0.5 mm i.d.), whose ends were squeezed into the conical capillary inlets of the interfaces by screwing-in the respective tube fittings. The electrodes were prepared from pieces of platinum wire (ca. 1.5 cm, 0.5 mm o.d.), soldered to copper contact cables, cemented into commercial fittings of polyphenylene sulfide with commercial adhesive, and sealed into the interface using slices of silicon tube of 1 mm thickness as washers. 2.3. Pressure reservoir The objective of construction of an autonomously operating and long-term stable CE system implied the need of regular and in-situ rinsing of the capillary for re-conditioning of the capillary inner walls. However, the minimal performable flow rate with the used syringe pump would have corresponded to a pressure far beyond the nominal pressure stability of the solenoid valves required for the manifold set-up ruling out direct capillary flushing. Therefore, pressure build-up was done by inflation of a silicone rubber tube (3.5 cm length, 5.5 mm o.d., 1.5 mm i.d.) acting as compliance or pressure reservoir, respectively. It was fixed with nylon tie wraps to commercial tube fittings and placed between the outlet of the grounded interface and V7. Propelling liquid towards the grounded interface with V7 in position OFF (interface outlet closed) led to the expansion of the silicone tube and pressure increase as consequence of the tube wall tension (see Figure 2). With the interface outlet kept closed, the pressure caused by the inflation with 300 µL resulted in a pressure of about 3 bar. This pressure enabled capillary flushing over about 10 minutes. Activation of V7 led to instantaneous pressure release by opening of the interface outlet. By this, external pressure sources as well as continuous operation of the syringe pump affecting the operational versatility and the integrity and functionality of manifold components were avoided. Since pumping was required only for pressure build-up, other operations were practicable during capillary flushing (background operation). The pressure increased with increasing inflation Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010 www.simplex-academic-publishers.com © 2010 Simplex Academic Publishers. All rights reserved.

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Figure 3. Mounted (A) and demounted (B) spectrophotometric detection cell. The cell was made of aluminum, both parts with optical fiber connections. The capillary was fixed with black nail polish. The required small optical aperture was achieved by adjusting aluminum foil strips, which where fixed black nail polish and adhesive tape afterwards

volume and tube bursting became probable above 400 µL. An inflation volume of 5 µL was applied to generate low, reproducible pressure for hydrodynamic injection. 2.4. Spectrophotometric detection cell The construction of a detection cell for on-capillary photometry implicates three main challenges: Achieving an aperture smaller than the capillary inner diameter since the maximal i. sensitivity is obtained when the detector visual field is limited to the liquid filled part of the capillary. Alignment of the aperture, optical waveguide fiber, illuminating light source, and the ii. capillary and maximization of the illumination intensity to minimize the baseline noise. Stress-free fixation of the capillary in the detection cell since the capillary fragility is iii. increased by the required removal of the polyimide protection coating in order to obtain a transparent window and to avoid displacement and affection of the detector cell's performance. The proposed detection cell design is shown in Figure 3. The detection consisted of two metal parts (aluminum or brass). Both parts were turned in one cycle to achieve highest manufacturing precision and center-alignment and to assure the perpendicularity of light path and the contact areas. On the outer face, both parts showed UNS ¼'' 36/'' threaded holes for screwing-in short segments of threaded pipes as SMA 905 junctions for the connection of an optical light fiber (detection part) or LED support (illumination part), respectively. The center opening on the illumination cell part was done with a 0.4 mm drill. A trapezoid, centered groove was milled afterwards on the inner face of the illumination cell part (0.4 mm depth and width) to align and hold the capillary. The transparent window on the capillary was prepared by removing the polyimide coating from about 5 mm by burning and cleaning the zone with methanol afterwards. For stress-free fixation, the capillary was placed into the groove, aligned with the prior produced transparent window to the opening, fixed with glue and adhered with black nail polish (both removable with acetone). To reduce the aperture to the inner diameter of the capillary, two pieces of commercial aluminum foil were adjusted to cover the illuminated part of the capillary afterwards by the visual aid of a binocular microscope (20-fold magnification) leaving a slit of about 50 µm and adhered with black Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010

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Figure 4. Front-view of the safety case (open) of the capillary electrophoresis instrument.

nail polish. Afterwards, the aluminum foil pieces were fixed and protected against mechanical stress with strips of commercial adhesive film. Finally, the cell was assembled with the LED support and the optical fiber connected to the spectrometer. Both cell parts were carefully aligned monitoring the obtained intensity gain of the spectrometer and securely fixed (screwed). Fabrication of the detection cell and mounting of the capillary in the detection cell were the most time-consuming tasks for the setup of the FT-CE system and required calm and precision. 2.5. Protection case For reasons of convenient working, labor safety, minimization of external influences, transportability, and robustness, the components of the CE system (interfaces, detector, and capillary) were assembled in a protection case. It was made of polymethylmethacrylate (PMMA) providing both visual control and a high dielectric strength (electrical resistance > 1015 Ωm). The base and several smaller components were made of PVC, showing an even high electrical resistance than PMMA. The case was made as two parts as shown with photo in Figure 4 and 5. On removing the front part, an integrated push-button opened and disabled the activation circuit of the analog control of the high voltage source, leading to its immediate deactivation. The back panel was mounted securely onto a support panel. The back part of the protection case held an eight-pin coil support for the separation capillary, adaptors for both interfaces, which allowed an easy installation or removal of each one, and apertures for the outlet tube of the grounded interface, the falling-drop tube outlet above the HV interface, the montage of the detection cell, the connected optical fiber, the electrical supply cable of the LED used as light source, and sockets for the both-way removable electrical connections to the capillary interfaces (male, inner side) and the HV source (female, outer side). On the opposite side of the back panel, the solenoid valve V7 was mounted and connected with the common port to the outlet of the grounded interface by a short PTFE tube (10 cm, 0.8 mm id.) and the silicon rubber tube acting as pressure reservoir (see section 2.9). The valve position OFF was closed with a blind connector, in position ON, a rigid curved PEEK tube (15 cm, 0.8 mm id.) was used to adjust the atmospheric outlet of the interface to the same level as the outlet of the high-voltage interface in order to avoid laminar flow in the separation capillary by siphoning. Two drain-groves were milled into the support panel to avoid the unhindered spread-out of eventually leaking liquid from the interfaces and by this to reduce the risk of short circuits. By four plastic screws at each corner of the support panel, declination of the surface could be balanced by height adjustment. Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010 www.simplex-academic-publishers.com © 2010 Simplex Academic Publishers. All rights reserved.

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Figure 5. Back-view of the safety case (closed) of the capillary electrophoresis instrument

The side, front, and top panels of the protection case were made-up as a single unit, which could be removed completely. The only component included on the front part was a common computer fan located oppositely to the capillary coil support. This allowed the efficient dissipation of joule heat from the capillary generated by HV application and by this the reduction of thermal diffusion and current increase as explained. Besides, rise of air humidity in the protection case and consequently the risk of sparking were avoided by the continuous air exchange. A possible improvement would have been the use of a peltier thermostated cooler but for laboratory ambience, the used assembly proved to be fully adequate. The clearances remaining between front and the back part allowed the passage for the inlet tube of the grounded interface. 2.6. Power source and remote safety control A HCP 35-35000 high voltage source from F.u.G. Elektronik GmbH (Rosenheim, Germany) was used and remote controlled via the analog interface of the source as shown in Figure 6. Using one auxiliary supply of the multi-syringe pump, a safety circuit was controlled via a relay for initiation and

Figure 6. Connection scheme for control of the high voltage power source HCP35-35000 by the use of the analogous interface and external supplies of the multisyringe device, enabling electrokinetic on-capillary counter-voltage pre-concentration. A second relay for is used for remote polarity reversal switching. It either connects or disconnects pin 6 and 7 (negative or positive polarity, respectively) and simultaneously pin 8 (external voltage control) to the sliders of one of two potentiometers between pin 9 (analog ground) and 10 (+10 V reference voltage). By this, different voltage levels are pre-adjustable for both polarity states. Remote high voltage application is initiated by the safety circuit of pin 6 and pin 12. Connection of pins 5 and 15 enabled the adjustment of the maximal current by the source's front panel. Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010

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Figure 7. Manifold of the proposed MSFIA-CE system. SV: selection valve (Pos1: Waste, Pos2: IV, Pos3: Eluent, Pos4: Water, Pos5: HVI, Pos6: HCl 10 mmol L-1, Pos7: NaOH 10 mmol L-1, Pos8: Sample), V1 – V7 solenoid valves with normally open position (OFF, deactivated) dotted (V3 and V7 of enhanced pressure stability), IV: Injection valve with SPE column, D: detector, HC: holding coils (1: 500 cm, 1.5 mm i.d., 2: 150 cm, 0.8 mm i.d., 3: 175 cm, 0.8 mm i.d., tube a: 40 cm, 0.5 mm i.d., tube b: 50 cm, 0.5 mm i.d., tube c: 40 cm, 0.8 mm i.d., tube d: 20 cm, 0.8 mm i.d., e: 20 cm, 0.8 mm i.d., knitted, tubes f & g: 10 cm, 0.8 mm i.d., tubes h & i: rigid PVC tubes 4 cm, 0.8 mm i.d., X: confluence, BGE: background electrolyte, S: sample, W: waste, Aux1: Auxiliary solution for column conditioning and loading, Aux2: Auxiliary solution for column cleaning. The different instruments are circles by dashed lines, the pre-concentration part is circled by pointed line.

turn-off of the high voltage. The formerly described safety push-button of the protection case was integrated into this safety circuit. A second auxiliary supply of the multi-syringe pump was used for the optional change of the voltage polarity and voltage height using a second relay and two potentiometers, enabled by this electrokinetic injection mode. 2.7. Flow instrumentation The flow analyzer used for the operation and control of the CE system is shown in Figure 7. It consisted of a valve module VA 1 + 1 equipped with an 8-port rotary selection valve and a 6-port injection valve and a multi-syringe module Bu 4 S equipped with three glass syringes purchased from Hamilton Bonaduz AG. Syringe 3 (2.5 mL total dispense) was used for the control of the CE instrument via the selection valve. Syringe 1 (10 mL) and syringe 2 (2.5 mL) were used for optional sample cleanup and analyte pre-concentration (see below). The original 3-way solenoid head valve of the syringe 3 was replaced by a single-line syringe holder (Sciware SL). Instead, a 3-way solenoid valve of enhanced pressure stability (V3) of nominal 600 kPa (type MTV-3-1/4UKGH) from Takasago Electric Inc. (Nagoya, Japan) was used. A second solenoid valve (V7) of 600 kPa nominal pressure stability was used for the intermediate closure of the grounded capillary flow interface outlet and powered by the supplying port 7 of the syringe module. Excessive heating of the external solenoid valves was prevented by using protection circuits from Sciware S.L. Position ON of V3 was connected to the central port of the selection valve via holding coil 3 (PTFE, 175 cm, 0.8 mm i.d.), while position OFF was connected to the reservoir of the applied background electrolyte or separation buffer, respectively. The lateral selection valve ports of the selection valve were used for waste disposal (Pos 1), injection valve and split-flow interface (Pos 2), water (Pos 3), sample (Pos 4), falling-drop interfaces (Pos 5), air (Pos 6), 10 mmol L-1 HCl (Pos 7), and

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GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY 10 mmol L-1 NaOH (Pos 8). Connection to the interfaces was done via PTFE tubes of reduced diameter (0.5 mm id) aiming low sample dispersion and dead volumes. The pre-concentration part of the system is indicated in Figure 7 by a pointed line, interfaced to the CE part by the 6-port injection valve. A miniature solid phase extraction (SPE) column was mounted on the valve instead of the normal injection loop. In position LOAD, column cleaning and analyte preconcentration was carried out, in position INJECT, elution was done towards the split-flow interface. Three further 3-way solenoid valves (V4, V5, V6) were used in the preconcentration part. V4 enabled the aspiration of a segmentation air bubble prior to sample aspiration from V6. By this, sample dispersion was inhibited and time efficiency of the preconcentration procedure was improved. S2 provided an auxiliary solution (AUX1) for conditioning of the SPE column and – by inline addition to the sample flow – to improve analyte retention during sample loading. Another auxiliary solution (AUX2) was accessible via V5, used for column cleaning after analyte elution. Dead volumes of all connection lines were minimized; between the confluence X, V6, and V4, two-sided tube fittings were used as shortest possible connectors. A miniature USB 2000 spectrophotometer from Ocean Optics Inc. (Dunedin, USA) was connected to the detection cell via an optical fiber of enhanced UV transparency (Premium Grade Xtreme XSR solarization-resistant Optical Fiber, Ocean Optics Inc.) with a core diameter of 455 µm. This fiber features considerably lower attenuation for UV in comparison with laboratory-grade optical fibers. Use of a spectrometer without the typical slit metal membrane is highly recommended when working with monochromatic LED as light sources since a considerable gain of sensitivity and in consequence much lower baseline noise is achievable. 2.8. Software control For the described system, the software AutoAnalysis 5.0 from Sciware SL was used [16]. One important advantage of this program is its simultaneous applicability for instrumental control as well as for data acquisition and data evaluation and the possibility to define procedures and variables, which facilitate the creation of the analytical method by module-selection and automation of the instrument's optimization. Performance of the electrophoretic separation in parallel to the analyte preconcentration has formerly been demonstrated [11]. 2.9. Operation The operation scheme is schematically described in Figure 8. First, a segmentation air bubble of 100 µL is aspirated by S1 from V4 followed the aspiration of sample (up to 6 mL). Then, the sample is forced through the SPE column while simultaneously, AUX2 is added from S2, adjusting the sample pH to an optimal value for analyte retention of the respective solid phase resin. The segmentation air bubble is discharged to waste with the injection valve in position INJECT and after switching the valve back to position LOAD, remaining, not retained sample components are flushed out by the carrier of S1 being water. Afterwards, the eluent is aspirated by S3 from the respective port of the selection valve and forced through the column in position INJECT. It is of highest importance to use an eluent, that allows uniform elution profiles for all analytes of interest and to adjust the volume in a way that the maximum concentration of the eluted analytes are positioned at the entrance of the capillary in the split-flow interface. In case of hydrodynamic injection, V7 is deactivated (interface outlet closed) and the compliance tube is inflated by a minimal volume (few µL) to apply low pressure during a time of about 1 s. Afterwards, V7 is deactivated (interface opened) and the split-flow interface is flushed with the background electrolyte being the carrier of S3 and the separation voltage is turned on. The interface outlet has to be maintained open to avoid hydrodynamic flow in the capillary due to EOF suction. The following steps were possible to perform optionally in parallel to the running separation: 1st: aspiration of AUX2 from V4 and flushing the SPE column with AUX2 for cleaning using S2, 2nd: passing an appropriate volume of S2 carrier (AUX1) through the column for conditioning, and 3rd: analyte pre-concentration as formerly described. An important feature of the present system is the possibility to replace the BGE of the HV buffer reservoir. For this, 500 µL of BGE were dropped from above into the interface for flushing out stacked bubbles and used BGE by gravimetric flow and the connecting flow line

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Figure 8. Scheme of the sampling, pre-concentration, elution, and injection procedure on the CE-MSFIA analyzer. 1: Aspiration of segmentation air bubble, 2: Aspiration of sample, 3: In-line addition of solution Aux1 and loading of SPE column, 4: Discharge of segmentation air bubble, 5: Washing of column with water, 6: Aspiration of eluent, 7: Elution from SPE column, 8: Positioning of sample, 9: Hydrodynamic injection, 10: Flushing of interface with separation buffer.

was emptied afterwards by aspiration of the containing BGE. By this, the probability of electrical shortcircuit over this flow line by sparking is further decreased. Capillary flushing for cleaning with acid, base, and water and conditioning with BGE was performed in-system by aspiration of the required solution, propelling it into the split-flow interface, and inflation of the compliance tube by 300 µL (shown in Figure 2) and holding for the required time. Since the pressure reservoir itself acted as liquid driver, during the pressure holding time, other operations such as formerly described cleaning of the HV interface were possible to perform in parallel. 3. Analytical example The following application of separation of mono-nitrophenols (NP) including study of the influencing parameters has been described in detail by the authors elsewhere [10, 11]. It is used here as an analytical example and operational parameters and analytical characteristics are summarized in Table 1. An example of peak separation and calibration is given in Figure 9. Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010 www.simplex-academic-publishers.com © 2010 Simplex Academic Publishers. All rights reserved.

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Table 1. Summarization of analytical parameters for the separation of mono-nitrophenols

Capillary Light source SPE Column SPE Resin Background electrolyte Sample volume Time of analysis Auxiliary solution 1 (sample modifier) Auxiliary solution 2 (column cleaning) Eluent Hydrodynamic injection Separation conditions Plate number Concentration factor Repeatability of retention times Mean recovery of analytes Limits of detection Repeatability of peak height

Fused silica 65 cm length, 55 cm effective, 75 µm id Polymicro technologies LLC (Phoenix, USA) Ultra-bright blue LED Cu6SMA1 (401 nm) Roithner Lasertechnik (Vienna, Austria) PMMA 7.5 mm in length, 4.5-mm id Polymeric reversed phase resin strata-X 33 µm Phenomenex (Aschaffenburg, Germany) 40 mmol L−1 sodium borate, pH 9.7, 10% v/v methanol 6 mL of sample for SPE preconcentration 19 min including preconcentration and conditioning Hydrochloric acid 100 mmol L-1 Sodium hydroxide 10 mmol L-1, 40 % v/v methanol Sodium hydroxide 10 mmol L-1, 10 % v/v acetonitrile, 300 µL with pressure volume of 5 µL during 800 ms - 25 kV at detection side (falling drop interface), < 10 min ca. 12.000 ca. 120 including in-capillary sample stacking < 2.5 % (comparing different samples and standards) 98 - 102 % 0.11 µmol L−1 2-NP, 0.35 µmol L−1 3-NP, 0.03 µmol L−1 4-NP < 3 % 2-NP, 4-NP, < 6 % 3-NP

As can be seen, the system featured high repeatability and analyte recovery near to 100 %, indicating low influence on the sample matrix. These satisfying characteristics were the result for one of the possibility to carry out hydrodynamic injection with high reproducibility and the integration of the analyte pre-concentration part. In a former study omitting any sample cleanup but filtration, the CE system was susceptible to matrix components, leading to prolonged migration times and broader, lower peaks. Sample cleanup enables also a better performance of electrokinetic injection since the influence of the ionic strength of the sample is mostly eliminated.

Figure 9. Electrophoretic separations in triplicate of aqueous standards, pre-concentrated according the example application. Concentrations of o-NP, m-NP and p-NP in µmol L−1: (a): 0.43, 0.87, 0.22; (b): 2.17, 4.35, 1.09. The time scale refers to time of data acquisition. Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010

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Figure 10.Schematic representation of operation order of the different analytical procedures carried out three times on the described system

Further important advantages of the described system are the possibility to perform the preconcentration procedure including SPE column conditioning in parallel to the electrophoretic separation as shown schematically in Figure 10. By this, the required time of analysis, i.e. the time between two injections was reduced from about 27 min to 19 min. In contrast, in most formerly presented analytical systems combining CE and FT, neither control of both buffer reservoirs, in-line flushing of the capillary, versatile combination of SE preconcentration and CE separation, nor operation in parallel to capillary flushing or separation was enabled. The system's characteristics would not have been achievable without using the multisyringe flow injection analysis. Main advantages of this technique: pressure robustness required for both the pre-concentration as well as on the CE part, and parallel driving of solutions in a multichannel manifold were key requirements for the obtained operational versatility. Changing the SPE resin, the system can easily be applied to other analytical problems. Simplification of the system by omitting S2 and V5 and addition of the sample modified manually is further possible, however, the applicability to fully automated purposes such as process monitoring would be lost. 4. Conclusions In the present work, an analytical system was described, being a hyphenation of the multisyringe flow injection technique and capillary electrophoresis. Operation schemes and construction of components such as the detection flow cell and the flow interfaces are detailed and documented by engineering drawings and photos to give a through documentation and help the other researchers. References 1. Karger, B.L., High-performance capillary electrophoresis, Nature, 339 (1989), 641-642. 2. Altria K.D., Overview of capillary electrophoresis and capillary electrochromatography, Journal of Chromatography A, 856 (1999), 443-463. 3. Olsson J., Nordström O., Nordström A.-C, Karlberg B. (1998) Determination of ascorbic acid in isolatedpea plant cells by capillary electrophoresis and amperometric detection, Journal of Chromatography A, 826, 227-233. 4. Gerald Gübitz, Martin G. Schmid, Recent advances in chiral separation principles in capillary electrophoresis and capillary electrochromatography, Electrophoresis, 23 (2004), 3981–3996. 5. M. Valcarcel, S. Cardenas, B. M. Simonet., Analytical Chemistry, 79 (2007), 4788-4797. 6. Shihabi Z.K., Stacking in capillary zone electrophoresis, Journal of Chromatography A, 902 (2000), 107-117. Global Journal of Analytical Chemistry | Volume 1 | Issue 3 | November 2010 www.simplex-academic-publishers.com © 2010 Simplex Academic Publishers. All rights reserved.

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GLOBAL JOURNAL OF ANALYTICAL CHEMISTRY 7. Horstkotte B. Cerdà V., Journal of Chromatographic Science (ISSN 0021-9665), 47 (2009), 636-647. 8. Dantan, N., Frenzel, W., Küppers. S., Flow injection analysis coupled to HPLC and CE for monitoring chemical production processes, Chromatographia, 54 (2001), 187-190. 9. Hinsmann, P., Arce, L., Rios, A., Valcárcel, M., Determination of pesticides in waters by automatic on-line solid-phase extraction–capillary electrophoresis, Journal of Chromatography A, 866 (2000), 137-146. 10. Horstkotte, B., Elsholz, O., Cerdà, V., Development of a capillary electrophoresis system coupled to sequential injection analysis and evaluation by the analysis of nitrophenols, International Journal of Environmental Analytical Chemistry, 87 (2007), 797-811. 11. Horstkotte, B., Elsholz; O., Cerdà. V., Multisyringe Flow Injection Analysis coupled to Capillary Electrophoresis (MSFIA-CE) as a novel analytical tool applied to the preconcentration, separation, and determination of nitrophenols, Talanta, 76 (2008) , 72-79. 12. Kubáň, P., Pirmohammadi, R., Karlberg, B., Flow injection analysis - capillary electrophoresis system with hydrodynamic injection, Analytica Chimica Acta, 378 (1999), 55-62. 13. Wang J., Cai P., Mo J., A sample introduction method based on negative pressure in flow injection-capillary electrophoresis system and its application to the alkaline-earth metal cation separation, Analytical Letters, 38 (2005), 857–867. 14. Fu, C.-G.; Fang, Z.-L., Combination of flow injection with capillary electrophoresis, Part 7, Microchip capillary electrophoresis system with flow injection sample introduction and amperometric detection, Analytica Chimica Acta, 422 (2000), 71-79. 15. Wang, S.-L., Huang, X.J., Fang, Z.-L., A miniaturized liquid core waveguide-capillary electrophoresis system with flow injection sample introduction and fluorometric detection using light-emitting diodes, Analytical Chemistry, 73 (2001), 4545-4549. 16. Becerra E., Cladera A., Cerdà V., Design of a very versatile software program for automating analytical methods, Laboratory Robotics and Automation, 58 (1999), 131-140.

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