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Appendix a Commercially Available Instrumentation for Capillary Electrophoresis

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Appendix a Commercially Available Instrumentation for Capillary Electrophoresis Appendix A Commercially available instrumentation for capillary electrophoresis Appendix At Overview of commercial instruments T. WEHR AND M. ZHU AI.I Introduction Commercial capillary electrophoresis (CE) instruments were first intro­ duced in 1988 and at the time of writing there are at least 14 vendors offering CE systems or components. During the preceding decade many researchers had contructed experimental manual systems consisting of a power supply, capillary, detector and injection device. In contrast to these simple homemade systems, many of the first commercial units were auto­ mated instruments with integrated detectors, autosamplers with multiple injection modes and provision for temperature control. In the intervening period, additional systems have been introduced onto the market and ear­ lier systems have evolved with the addition of new or more sophisticated detectors, more advance sample injection and liquid handling features and improved control and data processing software. This chapter summarizes the features of currently available CE instrumentation with a focus on automated systems (Table A1.1). AI.2 Power supply Power supplies capable of delivering constant voltage at high precision up to 30kV are standard throughout the industry. Most systems offer, in addi­ tion, constant current operation at up to 300IlA; constant current operation may be desirable in systems without adequate temperature control. Some instruments are also capable of operation in constant power mode up to 6 W; use of constant power operation permits separation time to be mini­ mized without excess heat generation. Early in the evolution of CE it was considered that efficiency in a CE separation was primarily diffusion­ limited and that operation at very high voltages (up to 60kV) could yield higher theoretical plate numbers and improved resolution. In practice, thermal effects at high voltage compromise efficiency gains, and today satisfactory separations are achieved below 30kV (typically 10-2SkV). 674 OVERVIEW OF COMMERCIAL INSTRUMENTS Table AI.I Capillary electrophoresis commercial instruments Manufacturer Advanced Molecular Applied BioSystems- ATIIUnicam Systems Perkin Elmer Product Model 2000 270-HT/HS Crystal CE 300 High voltage supply Operation models Constant voltage or Constant voltage Constant voltage or current current Voltage range 0-30kV 0-30kV 0-30kV Current range 0-330 /-L A 0-330 /-L A 0-2oo/-LA Power range Up to 9W Polarity reversal Manual Through software Through software Injection modes Electrokinetic, Electrokinetic, Electrokinetic, vacuum vacuum pressure Capillary temperature control Mode Liquid Forced air Forced air Range 2-75°C Ambient-60°C 10°C below ambient- 60°C Liquid handling Capillary purge Up to 800mbar in 20 Vacuum Vacuum mbar increments Buffer replenishment Yes, inlet and outlet Yes Yes automatically Autosampler Inlet positions 4, 8, 96-well tray Fifty 0.5 ml positions 4 (Model CE 300), 30 and eight 4.0ml (Models CE 310 positions and 31OC) Outlet positions One One 15ml, Up to 8 replenish able Temperature control Liquid Optional external Optional external bath bath Range 2-75°C 5-60°C 4-40°C Detectors Absorbance Photodiode array, Variable "A, 190- Selectable "A (filter) 190-288nm 700nm or diode array Fluorescence 190-288 nm excitation, Dual monochromator, 450-628 nm emission UV-vis excitation and emission Mass spectrometer Available for ESI coupling Other Conductivity Fraction collection Automatic Manual * 48 positions total, each position can be stationed at capillary inlet or outlet. POWER SUPPLY 675 Table At.t Continued Beckman Bio-Rad Dionex GTIISpectro­ Instruments Laboratories Vision PlACE 5000 Series BioFocus Series CES 1 Modular Constant or Constant or Constant or Constant voltage programmable programmable programmable or current voltage, constant voltage, constant voltage, current current or power current or power 1-30kV 0-30kV 0-30kV 0-30kV 1-2S01lA 1-3001lA 0--400 1lA 0-2S0llA 0-6W O-{iW Manual Through software Manual Manual Electrokinetic, Electrokinetic, Electrokinetic, Electrokinetic pressure, or both pressure pressure, gravity Liquid Liquid Forced air 5°C below ambient 1S-6SoC, to 50°C programmable Pressure,20psi Pressure,S or 100 Pressure, 20 psi Manual psi No No Yes 24 32 40 10 32 1 with automatic purging Optional external Optional integrated bath peltier 4-40°C Selectable A (filter) or Variable A (190-800nm) Variable A (190-800nm) diode array or fast -scanning (190-{i00 nm) (190-800nm) Argon ion laser Deuterium and source tungsten sources Available for ESI Available for ESI Enhanced fluorescence kit Automatic Automatic 676 OVERVIEW OF COMMERCIAL INSTRUMENTS Table AI.I Continued Manufacturer Hewlett Packard Kontron Otsuka Product HP3D Eureka 2100 CAP 1-3100 High voltage supply Constant or Constant or Constant or Operation models programmable programmable programmable voltage, current voltage or voltage or or power; CEC current current Voltage range 0-30kV 0-30kV 0-30kV Current range 0-300)lA 0-300)lA 1-300)lA Power range 0-6W Polarity reversal Through software Yes Automatic Injection modes Electrokinetic, Electrokinetic, Electrokinetic, pressure, pressure, gravity vacuum gravity Capillary temperature Forced air Forced air Forced air control 10°C below 5-50°C 5-45°C Mode ambient-60°C Range Liquid handling Pressure, up 12 20 psi 20psi Capillary purge bar Buffer replenishment Yes No No Autosampler 48* 20 45 sample (0.5 ml) Inlet positions 6 buffer (4ml) Outlet positions 10 7 buffer (15 ml) 6 fraction Temperature control Optional external Optional external Optional external bath bath bath Range 1O-40°C 4-45°C 4-45°C Detectors Diode array Diode array Diode array Absorbance (190-600nm) (190-800nm) (190--600 nm) Fluorescence Mass spectrometer Adapter kit for all coupling MS platforms Other Fraction collection Automatic No Yes POWER SUPPLY 677 Table At.t Continued Thermo Separation Products Waters Associates Zeta Technology SpectraPhoresis Series Quanta 4000E modular Constant or programmable Constant voltage, constant Constant voltage or current (optional) voltage, constant current, or both current ('programmable isomigration') 0-30kV 0-30kV 0-30kV 0-300JlA 0-2S0JlA 0-300JlA Through software Manual Manual Electrokinetic, vacuum Electrokinetic, hydrostatic Electrokinetic, vacuum Forced air Peltier IS-60°C, programmable with 1O-4SoC optional software Vacuum Vacuum Yes Yes 12 (Model 100), 82 (Models SOO, 20 1000,2(00) S Integrated peltier Selectable Ie (Model 100), Selectable Ie Argon ion laser source variable Ie, (Model SOO), fast (lamp, filter) scanning 190-800 nm (Models 1000, 2000) Automatic 678 OVERVIEW OF COMMERCIAL INSTRUMENTS High voltage is applied at the capillary inlet, with the outlet (detector) at ground potential. In many applications, capillary zone electrophoresis (CZE) is performed in uncoated fused-silica capillaries and electro osmotic flow (EOF) carries all analytes, regardless of charge, towards the cathode. In these cases, the inlet (high voltage) electrode is the anode (,normal' polarity). In an increasing variety of applications, however, 'reversed' polarity is used with the inlet electrode as cathode. These cases include analysis of anionic analytes in the absence of EOF (e.g. capillary gel electrophoresis and CZE using neutral coated capillaries) and analysis with reversed EOF (e.g. CZE using capillaries with positively charged coatings or with osmotic flow modifying additives in the background electrolyte). All commerical instruments have reversible polarity, although for frequent switching among different applications, polarity reversal through software is more convenient than manual reversal. Six commercial instruments (Beckman, Bio-Rad Dionex, Hewlett­ Packard, Kontron and TSP) offer time-programmable high voltage; this feature may be useful in preventing artifacts during application of high voltage, in reducing analysis time for samples containing low mobility com­ ponents and in modulating migration rates in special applications (e.g. fraction collection and resolution of temperature-sensitive conformers of biopolymers ). Al.3 Injection Sample injection in CE requires the introduction of very small amounts of analyte at the capillary inlet with high precision. All commercial instru­ ments offer electromigration injection and at least one type of displacement injection. Electromigration is the simplest injection method in CE; the capillary inlet is immersed in the sample solution and high voltage is applied for a brief period (typically a few seconds). If no EOF is present, sample ions enter the capillary by electophoretic mobility alone. If EOF is present, sample ions will be introduced by a combination of electrophoretic mobility and EOF; this mode is generally termed electrokinetic injection. Electrophoretic injection offers two advantages. First, since only species of like charge will enter the capillary, it enables discrimination against compounds of opposite charge, simplifying the separation problem. Sec­ ond, by varying the ionic strength of the sample relative to the background electrolyte, a focusing or stacking effect is created which allows the forma­ tion of very sharp sample zones with high analyte concentration. Unfortu­ nately, these advantages are countered by two major limitations. Since sample ions enter the capillary based on mobility, low-mobility ions will be present in lower concentrations, decreasing detector response. More impor­ tantly, presence of non-analyte ions in the sample will reduce injection CAPILLARY TEMPERATURE CONTROL 679 efficiency,
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