Product
Review
HIGH-VOLTAGE AREA Capillary Electrophoresis Instruments
electrophoresis (CE), which sepIn a shrinking group Capillary arates species based on their movements under an applied electric field, is increasof competitors, CE ingly being used for biological and pharmaceutical analysis. A typical CE instrument consists of a high-voltage power supinstrument ply, two electrode assemblies, two buffer reservoirs connected by a fused silica capmanufacturers illary, a sample injection system, and a introduce new systems detector. At pHs higher than 3, the interior surface of the silica capillary is negatively and make incremental charged because of the ionization of silanol groups, and cations in the buffer are changes attracted to the surface. When the electric field is applied, the positively charged cations in the solution direct the electroosmotic flow (EOF) toward the cathode. All species—cationic, anionic, and neutral— flow toward the cathode when the EOF exceeds their migration velocity under the electric field. Cations usually elute first but EOF modifiers are sometimes added to the electrolyte solution to reverse the direction of the EOF Various CE modes including capillary zone electrophoresis (CZE) micellar electrokinetic chromatography (MEKC) caoillary gel electrophoresis (CGE) capillarv isoelectric focusing (OIEF) and isotachophoresis (ITP) can he used analytes CZE, also called free-solution CE, is the most common CE mode and is used to
separate ionic species. In CZE, the user defines the analysis environment, opening the technique to numerous applications, including the study of receptor-ligand interactions (often called affinity CE), multianalyte immunodetection, and chiral separations. In MEKC, which uses surfactants above the critical micelle concentration, the analytes partition between the buffer and the hydrophobic interior of the micelles. Because the micelles migrate, neutral species, in addition to ionic species, can be separated. Whereas CZE and MEKC are uniquely capillary techniques, CIEF, CGE, and ITP are capillary analogues of slab gel electrophoresis techniques. In CGE, the capillary is filled with a gel that separates the analytes by size. In CIEF, which uses an ampholytic solution to separate polyelectrolytes, each analyte migrates to the point at which the pH equals its isoelectric point. In ITP, two different buffers—a "leading" electrolyte and a "trailing" electrolyte sandwich the analytes, which reach a steady state and migrate in order of decreasing mobility. Because the analytes adopt the concentration of the leading electrolyte ITP is often used to separate dilute solutions When Analytical Chemistry ran its first CE product review (Anal. Chem. 1994,
Analytical Chemistry News & Features, December 1, 1996 747 A
Product Review
Table 1. Summary of representative products
Model
P/ACE Series
BioFocus 2000 and 3000
Company
Beckman Instruments 2500 Harbor Blvd. P.O. Box 3100 Fullerton, CA 92634 800-742-2345 www.beckman.com $35,100-$65,900
Bio-Rad 2000 Alfred Nobel Dr. Hercules, CA 94547 800-424-6723
INA
INA
38.1 x 51.4x63.5
56 x 49 x 58
57.5 x 42.5 x 52
45.4
59 (2000); 61 (3000)
52
1—±30 kV 1-±250/iA 0-7.5 W Constant or gradient voltage, current, and power
0—±30 kV 0-±300,uA INA Constant voltage or current
0-±30 kV 0-±300/JA 0-6 W Constant voltage, current, or power
Polarity switchable Sampling
Yes 34-position random-access autosampler and buffer exchanger
Injection
Pressure (with feedback) and electrokinetic
Yes, software programmable Two randomly accessible 32-position carousels; uses standard 500-^iL or 1.5-mL microcentrifuge tubes Pressure (time-pressure integrated) and electrokinetic
Ytes 48-position carousel randomly accessible from both cathode and anode; thermostated 10 °-40 °C Controlled pressure and electromigration from both inlet and outlet ends
Capillaries
Housed in self-aligning cartridges with selection of aperture sizes; 21cm minimum length, 7 cm to detector minimum; cartridges compatible with most commercially available capillaries, including extended lightpath bubble cells Recirculating liquid; 10 °-50 °C
Self-aligning cartridges; 24-100 cm
Self-aligning cartridges; 33-cm minimum length, 8.5 cm to detector minimum; extended light-path capillaries with bubble at detection point for three- to fivefold S/N increase
Peltier liquid cooling for capillary; Peltier air cooling for autosampler Programmable single-wavelength UV (190-365 nm) (2000); fast-scanning UV-vis (190-800 nm)
Forced air with Peltier element; 10 ° below ambient to 60 °C UV-vis diode array (190-600 nm); prealigned deuterium lamp
Windows-based software controls instrument operation, data collection, and data analysis; requires 486 or higher PC-compatible system with 8 MB RAM HV interlocks on all doors (cartridge and sample)
Windows 95 or 3.1, Pentium processor, and 24 MB RAM recommended
Price Dimensions H x W x D (cm) Weight (kg) Power supply Voltage range Current Power Modes
Temperature control
3D Capillary Electrophoresis System Hewlett Packard 3495 Deer Creek Road Palo Alto, CA 94304 800-227-9770 www.hp.com
Detectors
UV-vis diode array (190-380 nm); selectable wavelength UV (seven positions); laser-induced fluorescence; MS adapter
System control
Front panel control with LED readout; System Gold LC/CE Workstation; P/ACE Station Workstation (Windows 95)
Safety features
HV interlock system; HV protection circuitry invoking shutdown with voltage arc detection
Options
Validation services; detector modules; sample temperature control; choice of system controller; upgrades for LIF systems Integrated "all-in-one" systems in which all modules interchange with any existing P/ACE system; general instrument design compatible with all modes of CE, including CEC and endcolumn detectors
Autosampler temperature control; CE/MS interface kit for Finnigan API electrospray head
INA
High-pressure capillary purging allows use of viscous polymer solution for sieving separations
401
402
Adapter interface allows use of modular detectors and acquisition of data via HP ChemStation software; pressurization of inlet and outlet for capillary electrochromatography; automated, time-independent fraction collection 403
Special features
Reader service number
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News & Features, December
1, 1996
HV interlock system; leak sensor system
Crystal 300 Series Thermo Capillary Electrophoresis (formerly ATI Unicam) 8 Forge Parkway Franklin, MA 02038 800-824-6204
INA
Spectraphoresis Ultra Thermo Separation Products 355 River Oaks Parkway P.O. Box 49031 San Jose, CA 95161 800-532-4752 pubs.acs.org/pin/thermo2/ tsp.html INA
I Capillary Ion Analyzer Waters 34 Maple St. Milford, MA 01757 800-252-4752 www.waters.com
INA
49 x 49 x 40 (300) 4 9 x 4 9 x 50 3310) 40 (300); 62 (310)
44 x 92 x 51 (complete system)
66 x 56 x 55
62
73
0 - ± 3 0 kV 0-±200 jiiA INA Constant voltage or current
0-±30 kV 0-±300 //A INA Constant voltage, current, or power; gradient voltage or current
Yes Four vial positions (300); 30-48 vials depending on configuration (310) Electrokinetic and/or "dynamic compression injection" with
Ytes 110-position carousel
0-±30 kV INA INA Constant voltage or current; single voltage or current change during program run Yes 20-position carousel
simultaneous pressure and voltage ramping INA
Electrokinetic and hydrodynamic Hydrostatic and electromigration
22.5-63.5 cm; cassettes barcoded to track history
Self-aligning, 50-to 75-^/m i.d.; precut to 60- to 100-cm lengths; user can customize length from capillary spool
Peltier air cooling
Peltier air cooling
Peltier air cooling
UV-vis (single wavelength, dual wavelength, diode array, rapid scan, "Z-cell" technology), fluorescence, and conductivity
Programmable single-wavelength, multiple wavelength, or scanning UV-vis; 190-800 nm; prealigned deuterium and tungsten lamps PC1000 software can provide simultaneous CE and LC data handling and system control
Discrete wavelength UV absorbance; 185 and 254 nm standard; 214, 229, 280, 313, 365, 405, 436, and 546 nm optional Pentium-based PC workstation running Windows-driven Millenium software for CE
Menu-driven using 11 keys and 4 x 40 character LCD; PC1000 software with full
control of dual wavelength and scanning UV detectors HV interlock; power is off when HV interlock on CE module front door is open, capillary oven is open, or inlet or outlet vial is not present INA Sample tray temperature control
Modular system allows easy and rapid variation of detector; floating ground for use with endcolumn detectors; negativepressure capability enhances ITP experiments and pressure balances ICPMS 404
INA
Premixed electrolytes for inorganic analyses; enantioselective chiral reagents
Sample and buffer preparation with a single vial, air-driven vortex mixer/heater settable in 1 °C increments 30°-100 °C
Isomigration technology; capillary ion analysis software
405
406
66,1137 A-41 A)) ,he manufacturer population had exploded, with at least 10 companies producing instruments. Since then, the industry has experienced a "shakeout", in which Dionex, Isco, and Perkin Elmer have withdrawn from the general CE instrument market—although Perkin Elmer still offers a CE DNA sequencer {Anall Chem. 1996, 68,493 A-97 A). ATA Unicam, after being purchased by Thermo Instrument Systems, is now known as Thermo Capillary Electrophoresis (Thermo CE). The remaining companies have primarily made incremental improvements to their systems although Thermo Separations has introduced a instrument the Spectraphoresis Ultra and Beckman has introduced new members to its P/ACE family that are optimized for particular applications Most of the companies have focused on the life sciences market and such applications as DNA, carbohydrate, and protein analyses. Waters, however, has chosen to focus on the inorganic ions and organic acids analysis market. Table 1 lists representative instruments from the current manufacturers. Detectors
One of the big pushes has been in the interfacing of CE and MS. In order to mount the CE instrument on the interface designed by the MS manufacturer, the CE system must have an internal electrical circuit feedback loop so that the second electrode and outlet buffer reservoir can be removed. With this modification, the CE capillary can be inserted into the MS interface. The Thermo Separations system can be interfaced with the Finnigan ESI, and the BioFocus series from BioRad can be interfaced with Finnigan and Micromass electrospray systems. Hewlett Packard's system is designed to be interfaced with the HP 8959 ESI-MS but also be used with mass spectrometers from other manufacturers John Lake of Thermo CE says that because the Crystal 300 series was intentionally designed with a feedback loop and an extended capillary the instruments can be used with any ex-' isting ESI source without an additional module According to Jeff Chapman North American CE product manager Beckman was the first companv to offer an "MS-ready" svstem which ran he inter faced to Micro Fi i H
Hewlett Packard electrospray interfaces. Although a CE/MS combination makes demands on both methods, the major challenges of interfacing the two Analytical
Chemistry
News & Features, December
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have involved MS. CE peaks are so sharp and the absolute amount of material is so small that MS has lacked the sensitivity for easy detection. In addition, the surfactants used in CE (such as sodium dodecyl sulfate), high salt concentrations, and nonvolatile buffers are incompatible with MS detection. Those problems, however, require method development solutions rather than instrument changes. David Heiger of Hewlett Packard says that "there are no problems interfacing CE and MS if you understand the limitations." Several types of detectors are available in addition to MS. The most common of these is UV-vis, which is available from every manufacturer. However, UV-vis detection has relatively low concentration sensitivity because of the short pathlength, typically 50-75 um, associated with oncolumn detection windows. This shortcoming has been addressed in two ways. Hewlett Packard offers a capillary with a bubble at the detector window, called an extended pathlength capillary, that increases the signal without increasing detector noise or band broadening. Another important improvement is the "Z-cell" a capillary constructed with two right-angle bends so that the optical path passes along the long axis of the capillary for 3 mm According to James Jorgenson of the University of North Carolina-Chanel Hill UV-vis is most popular because manv applications involve nonfluorescent molecules "More people use UV-vis because they don't have the luxury of usingfluorescenceor laser-induced fluorescence (I W) " he says Heiser however believes that a fluorescence detector will evenhrallybe as useful with CF
concentrations, where the derivatization step is clearly the rate-limiting step. Those detectors are primarily for organic analysis. For inorganic analysis, the choices for detection are U v—vis and conductivity detectors. i nermo v/tj uses a conductivity detector that Lake claims offers higher sensitivity than do UV detectors. The mercury lamp that Waters uses permits detection at 185 nm. In response to the realization that many users are developing their own detection systems or interfacing with other detectors, the manufacturers are designing instruments that can be interfaced with third-party detectors, which require a design in which the capillary can be extended beyond the instrument housing. Susan Lunte of the University of Kansas says that the "ability to get the capillary out of the box" has been a welcome improvement in CE systems.
Manufacturers are making systems that can be interfaced with third-party detectors for the users who design their own.
ment, rather than your capillary thermoregulatory system." He says that Beckman's experience has been that complex sample matrices such as protein solutions requiring higher salt concentrations and isolation of analytes requiring larger bore capillaries increase the need for effective heat dissipation. Although a wider range of thermoregulated temperatures is achievable with air cooling, Heiger concedes that liquid cooling, which requires adding gaskets and sealed cassettes, may be necessary for applications at high currents. "I would never say that air cooling is superior to liquid cooling at high currents." In most circumstances, however, the type of heating system will not be an issue. "I haven't heard anyone say there was a real problem with a certain type of cooling system," says Jorgenson. Robert Rush of Amgen says that heat dissipation is not an issue as long as the operating parameters are within the specified range of the instrument. User friendly
CE systems have become more user friendly in terms of software, application kits, and system modularity. "Computers have made things easier," says Rush. "The software still has bugs but it's a lot better than it used to be. There are more options for how the data is handled." Hewlett Packard, for example, uses a common format for both LC, CE, and GC in its ChemStation software. Thermo Separations also offers simultaneous LC and CE control. On the other hand, Waters offers a separate CE software application that asitiswithHPTT runs with its Millennium results management software, which compensates for the Because of patent issues, Beckman is Keeping your cool the only company listed in Table 1 that ofThe high voltages used in CE (up to 30 kV shifting migration times and asymmetric peaks that make accurate and reliable fers LIF. However, Thermo CE offers a pro- for most commercial instruments) can quantitation and identification of inorganic grammablefluorescencedetector, and Per- generate large amounts of heat, which kin Elmer's Applied Biosystems Division must be dissipated with either air or liquid ions difficult manufactures the Prism 310 Genetic Anacooling. Despite the potential for high According to Chapman, "Beckman has lyzer, which also uses LIF. (Fluorescence temperatures, most applications do not improved its software in recognition [of with excitation sources other than lasers generate much heat. "For 99% of all appli- the fact] that CE software must have capahas generally been less sensitive.) LIF has cations, air cooling is all you need," says bilities unique from those of other analytibeen particularly useful for DNA analysis Heiger. "If you don't generate too much cal techniques. For example, the software with intercalating dyes, which removes the heat, why do you need to remove it?" must be capable of programming CE need for derivatization of the DNA For Heiger says that air cooling is ademethodology, calculating analyte mobility, some applications such as carbohydrate quate for applications at < 150 uA, but correcting peak areas for eletrophoretic analysis, the derivatization step is necessary Chapman disagrees, saying that the heatvelocity, and estimating molecular qualianyway to provide a charge for the separa- ing system is a real issue for certain appli- ties like isoelectric point and molecular tion step. In those circumstances LIF pro- cations: "The more you improve the abilweight." Bio-Rad has also made incremenvides sensitivity and the physical character- ity to dissipate joule heating the further tal improvements to its software, says istics necessary for the separation Chapyou can push the limits of the technology. Michael Kurokawa of Bio-Rad. man says that "too often a parallel ts drawn I cannot emphasize enough the imporBeckman is changing its approach to between LIF and having to derivatize tance of using the sample matrix to drive the CE market Rather than selling a single minute quantities of sample at subattomolar your approach to CE methods developgeneric instrument, it is modifying the in750 A
Analytical Chemistry News & Features, December 1, 1996
Just published! struments to more effectively apply CE technology to specific solutions, says Chapman. For example, the DNA analysis system is configured with all the chemistries and detection systems required to separate and quantitate PCR fragments and uses LIF because UV detection is not practical for quantitative PCR applications. In addition to the P/ACE series listed in the table—which includes the 5000 Series and DNA, chiral, and carbohydrate systems Beckman manufactures a multicapillary instrument, the Paragon CZE serum protein analyzer. Thermo Separations has just introduced a modular system that includes automated precapillary derivatization. The system is linked by fiber-optic cable to the detector, which makes it easier to interface the system with other detectors. Whither CE? Although, as Heiger points out, CE is not chromatography, an important part of CE's future will be the union of electrophoresis and HPLC in a method known as capillary electrochromatography (CEC). In CEC, the capillary is packed just as it is in HPLC, and the separation of analytes depends on both electrophoretic migration and partitioning. Unlike in HPLC, however, the EOF that propels neutral molecules through the capillary in CEC has a uniform flow-velocity profile so that there is less band broadening than in HPLC. CE users are happy with the current state of separation equipment. "The [instrument manufacturers] have done a remarkably good job providing instruments," says Rush. Although the future should bring advances in detector technology, most progress will be in the separation chemistry itself. Even more than they had been previously, manufacturers are turning to the development of CE application kits, which include the buffer solutions for specialized separations such as peptides and proteins PCR products and ion analysis; in most cases the capillary cassette is also included The kits bring "plug-and-play" ease and versatility to CE for users who may not want to spend a lons' time with method development Heiger and Eric Fotheringham of Waters say that for the CE market to really take off, industry will need to see methods that have been developed and approved by the various regulatory agencies, including the Environmental Protection Agency and the Food and Drug Administration. Celia Henry
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