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Capillary Electrophoresis:Finding a Niche

Where HPLC struggles, CE thrives.Laura DeFrancesco

Interest in capillary electrophoresis(CE) has waxed and waned in the

decade since the first commercial instru-ment was introduced. Now, CE seemsto be establishing itself as the techniqueof choice for certain applications.

For example, according to companyrepresentatives and experts, CE pro-vides chiral analysis at a lower cost andis better than HPLC at separating high-ly charged and polar species. CE’s facili-ty for small ion analysis has made it amainstay in the food and beverage in-dustries. And biotech companies havestarted to incorporate CE their researchas well as into QA and QC. DNA se-quencers using CE also played a majorrole in sequencing the human genome.

When last reviewed in these pages in1996, the commercial CE field had nar-rowed to a handful of companies. (Dedi-cated DNA sequencing instruments wererecently reviewed and are not includedin this discussion [1].) Since that time,the field has narrowed even further tojust a few companies, with two giants,Beckman Coulter and Agilent Techno-logies, becoming the dominant players.

Table 1 lists representative featuresof CE instruments from five companies.Readers interested in these productsshould contact the companies for acomplete list of features.

AA sshhoorrtt hhiissttoorryyAs with most new technologies, CE wasinitially fraught with technical difficul-ties. Barry Karger, director of the Bar-nett Institute at Northeastern Universi-

ty, likens it to the early days of LC. Inthe beginning, he says, “[LC] was hor-rible. Every column was different. Itwas many years before the techniquebecame robust.”

However, some of the problem mayhave been the users who first boughtthe instruments. “Chromatographerstried to [use CE] and did not fully rec-ognize that it was electrophoresis. AndCE was governed by those rules, notchromatographic ones,” says DavidHeiger of Agilent. “For those who

know electrophoresis, you have a mucheasier entrance.”

Although the chromatography prob-lem still exists, Jeff Chapman of Beck-man Coulter says that the adoption of“CE thinking” has been key to thetechnique’s success. Instead of migra-tion or retention times, which are chro-matographic descriptors, CE focuses onmobility. “The use and development ofmobility has changed the face of howwe operate,” claims Chapman. “It tookCE from what was perceived as not re-

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producible to [a technique] which ishighly reproducible.” Chapman addsthat coefficients of variation of <0.3%

have been demonstrated across CE in-struments, capillaries, and buffers usingmobility as the “index of identification”.

Another problem was how CE wasbeing applied, says Jim Jorgenson fromthe University of North Carolina. “In

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Agilent Technologies2850 Centerville Rd.Wilmington, DE 19808 800-227-9770www.agilent.com/chem

$49,000 with ChemStation,PC, and printer

Pressure, vacuum, or electrokinetic

8.5 cm (from injection pointto detector); 33 cm total;25-, 50-, 75-, or 100-µmstandard i.d.

25-, 50-, 75-µm extendedpathlength; high-sensitivitycell (75 µm)

±30 kV0–300 µA0–6 W

Thermostatted 48-positioncarousel; 100-µL to 2-mLvials

UV–vis diode array

High-sensitivity pathlengthcapillaries; self-aligningcapillary cartridges; highpressure at both ends forCEC; ChemStation soft-ware; automated fractioncollection; integratedCE/MS (quadrupole and iontrap mass spectrometers)

Interfaces directly with Agi-lent 1100 Series mass spec-trometer; couples with Pi-cometrics' LIF; couples withLeap Technology's tunablefluorescence detector

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Beckman Coulter, Inc.4300 N. Harbor Blvd. P.O. Box 3100Fullerton, CA 92834-3100714-871-4848www.beckmancoulter.com/

$60,000

Pressure, vacuum, or elec-trokinetic

7 cm (from injection to detector); 27 cm total; maximum length is variable

All commercially availablecapillary dimensions

±30 kV0–300 µA0–9 W

Thermostatted autosam-pler; 2-mL and PCR vials;0.5-mL tubes and 100-µLtubes; 96-well plates

Modular UV–vis diodearray, selectable wave-length UV–vis

Automated fraction collec-tion; high pressure at bothends for CEC; recirculatingliquid temperature control;independent buffer and sam-ple temperature control; mo-bility plots; Ceaser algorithmfor quanititation; 32 K soft-ware (combined platform forLC/CE); large array of chem-istry kits and reagents

Single- or dual-channelLIF; external detectoradapter; ion-trap MS/MS;integrated CE/MS/MS andMSn single-point controland analysis

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Prince TechnologiesP.O. Box 21947801 CD Emmen The Netherlands31 (0)591 629184www.princetechnologies.com

$23,000–32,500 (includingUV–vis and software) de-pending on exchange rate

Pressure (between –180 and+250 mbar), electrokinetic

Flexible in length and diameter

±30 kv–200 to 200 µA0–6 W

4-position or 30/48 positionautosampler; variable vialsizes

Allows use of any CE compa -tible detector, including MS,fluorescence, LIF, conductivi-ty, UV –vis, photodiode array

Fully modular; high pressureat inlet, outlet or both endsfor CEC; easy-to-couple toa mass spectrometer; flexi-ble; diverse models; CEdedicated software withmobility calculations; cur-rent-controlled end ofmethod step

Single lift with optionalchoice of temperature con-trol or high pressure; dou-ble lift with optional tem-perature control; double liftwith standard pressure; in-tegrated CE and CEC

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CombiSep1915 Scholl RoadApplied Science Building IIAmes, Iowa 50011515-294-2837www.combisep.com

Price based on options

Vacuum and electrokinetic

40 cm total; 20 cm effectivelength; 75-µm i.d.

Standard; 96 capillaries inlinear array format

±20 kv0–4.0 mA total current

96-well format PCR plates,0.2 µL to 1.0 mL volumewell plates

UV at 214 nm standard;254, 280, and 305 nm alsoavailable

96 simultaneous separa-tions; software processes96 samples in <15 min

Test kits for peptide map-ping pH/buffer tests; pre-concentration; log P ; chiral screening; pKadetermination

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Micro-Tech Scientific140 South Wolfe Rd.Sunnyvale, CA 94086408-730-8324www.microlc.com

$9500–65,000

High pressure, time con-trolled injection valve, hydro-dynamic, and electrokinetic

5–50 cm

50, 75, 100, 250, and 320 µmi.d.; open or packed capillary

±60 kV1.2 mATime programmable voltage control

96 and 384 well plates, 2mL vials

UV–vis, MS

Peltier sample tray cooler;integrated capillary gradientcapillary LC, CE, and CECsystem for pH, salt concen-tration, and organic solventgradients; binary capillarygradient pumping system; 8mm � 20 nL flow cell; up to10,000 psi operation; time-programmable voltage andsolvent gradients

Could be configured to amultidimensional LC, CE, orCEC; run under Windows98, NT 4.0, Finnigan X’Cal-iburMS software; could befully automated

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the process of evaluating CE over thepast decade, people tried applying it toeverything,” recalls Jorgenson. “To thosepeople working on problems that LC isgood at handling, CE has often been adisappointment. To those working infields where LC is inadequate, CE hasbeen a godsend. It is now becomingclearer what its strengths and weaknessesare relative to LC.”

GGooiinngg wwiitthh ssttrreennggtthhssAlthough CE was initially heralded forits speed and low sample volume, thetechnique is valuable because it is quan-titative, can be automated, and will sep-arate compounds that have been tradi-tionally difficult to handle by HPLC, sayexperts. Jorgenson calls CE the “best-kept secret in chemical analysis,” capa-ble of analyzing complex carbohydratesand protein–protein interactions. Headds that CE is the basis for virtually allmicrofluidics for lab-on-a-chip devices.

Ed Burton of Procter and Gambleadds that CE can also separate polarsubstances, which are notoriously diffi-cult to analyze with HPLC. For exam-ple, Burton and his colleagues were ableto study multicharged and water-solublebisphosphonates by CE. And they havebeen able to run analyses in the presenceof surfactants, which can be found inpractically every product Procter andGamble produces.

Chiral separations is another area inwhich CE use has expanded. Chapmanreports that he and his colleagues haveseparated 90% of the compounds thatcustomers have thrown at them with asingle capillary and a proprietary line ofhighly sulfonated cyclodextrins.

WWhhaatt’’ss nneeww??The small sample volumes required forCE can be an advantage with limitedsamples; however, with its short path-lengths, the technique can suffer fromlow sensitivity. One solution, introducedby Agilent, is the “bubble cell” capillary,which increases the capillary internal dia -meter at the point of detection, increas-ing sensitivity by a factor of 3 to 5. Agi-lent has introduced a newer cell, whichthe company claims increases sensitivityby a factor of 10 over conventional capil-

laries. The proprietary cell does this byincreasing the pathlength from 75 µmto 1.2 mm while eliminating stray light.

UV is the predominant detectionmeth od used with CE; however, laser-induced fluorescence (LIF) and MS havebeen added to the detection arsenal. Agi-lent, as well as Beckman Coulter (in con-junction with Thermo Finnigan), offerCE/MS options. According to Heiger,sensitivity with CE/MS is still a problem.But, he adds, CE/MS is now as easy touse as LC/MS. Thus, for problems thatcan’t be solved by LC/MS, CE/MS isthe right choice, says Heiger.

TThhee ggooooddssIn addition to its CE system, Agilent sellsthe 2100 BioAnalyzers, lab-on-a-chip de-vices that use electrophoretic separations.The BioAnalyzer is more of an appliancethan an analytical instrument, with eachchip doing one thing such as RNA, DNA,or protein analysis. With these chips, nomethod development occurs. If it fits theapplication and the required sensitivity,you insert your sample and run it.

Beckman Coulter has eight automat-ed, application-based capillary array sys-tems, marketed under the name P/ACEMDQ. The application-based systems—chiral, carbohydrate, molecular character-ization, DNA, glycoprotein, education,and QC—differ in the way they are con-figured and the types of detectors andchemistries coupled to them. The carbo-hydrate system, for example, comes onlywith a LIF, whereas the molecular char-acterization system comes with a UV de-tector and a two-color LIF unit for de-tecting nucleic acids and proteins.

CombiSep, a start-up companyfounded in December 1999, offers theonly multiplexed absorbance-based CEinstrument. (The other multiplexed in-struments are principally for DNA se-quencing and are equipped with LIFdetectors.) On the basis of technologydeveloped by Edward Yeung’s group atIowa State University, CombiSep’s MEC2000 combines massively parallel process-ing with UV detection and software thatcan analyze 96 samples in a few minutes.

Prince Techno logies offers a com-pletely modular system, which sports apatented dynamic compression injection

unit, giving <1% relative standard devia-tion for migration times and peak areas.

OOnn tthhee hhoorriizzoonnAn area closely related to CE that hasyet to reach its stride is capillary electro -chromatography (CEC), which combinesfeatures of CE with LC by using capil-laries packed with chromatographic ma-terials. Although no products have yethit the CEC market, some of the prob-lems have been solved, so they may startappearing over the next few years.

The term “high throughput” took ona new meaning when Richard Mathiesat the University of California–Berkeleybuilt a prototype CE instrument with1000 capillaries. Mathies has designed aCE–DNA sequencer for Molecular Dyna -mics; however, he sees chips as the futureof CE. “Performing CE on microfabri-cated channels gives better performance,higher speed, and a more robust and re-liable package,” Mathies says. He recentlyran a 384-lane genotyping microchipsystem and found it to be much fasterand more reliable than the earlier 384-lane discrete capillary device.

TThhee nniicchheeWill CE ever overtake HPLC? Some saythat’s not the appropriate question any-more. Ian Mutton of GlaxoSmithKlineexplains, “CE comes into its own forlarge molecules and when sample size is limited. This tends to give its best ap-plications a biological flavor, and it canpowerfully address problems whereHPLC has little chance of success—theHuman Genome Project being the ob-vious example.”

Now that CE has been officially rec-ognized by several regulatory agencies—the Food and Drug Administration andthe Center for Drug Evaluation and Re-search among them—it is finding a nichein QA and QC labs as well. And withCE’s hugely successful foray into geno -mics—it being the undisputed reasonthe Human Genome Project finishedwell ahead of schedule—the question iswhether it can do the same for proteo -mics, the next phase of genomic research.

Laura DeFrancesco is a freelance writerbased in Pasadena, CA.