8 9 6 ICGC NORTH AMERICA VOLUME 22 NUMBER 9 SEPTEMBER ZOtM www. chromatographyonline.com

Chromatography

Guest AuthorAndras Guttman

This month's "Milestones

in Chromatography"

surveys the evolution of

capillary electrophoresis

and its application in a

wide range of

biochemical applications.

The Evolution of CapillaryGel Electrophoresis: FromProteins to DNA Sequencing

Leslie S. EttreMilestones in ChromatographyEditor

E lectrophoresis (from the Greekwords elektron: electron; andphoresis: carrying) is a separation

method in which charged particles migrateunder the influence of an electric field atvarious rates, depending upon their charge-to-mass ratios. Electrophoresis as an analyt-ical method was Introduced by ArnieTiselius in the 1930s to separate humanserum into albumin, a-globulin, (3-glabu-lin, and 7-^globuIin (1). He demonstratedthe potential of electrophotesis as a researchtool In his pioneering work for which hewas awarded the Nobel Prize in 1948.Latet, antlconvective media such as srarch,agarose, and polyacrylamide gels wereintroduced to eliminate convective trans-port and reduce diffusion, which resultedin the various components of the analytemixture migrating in sharp zones duringelectrophoresis separation (2). Gels alsoacted as molecular sieves for size-basedseparations.

Getting SmallerIn the 1980s, a novel, automated, andhigh-petformance electric-field-mediateddifferential migration technique called cap-illary electrophoresis was introduced inalmost every aspect of basic and appliedbiomedical and clinical research (3). As aninstrumental approach to electrk-field-mediated separations, capillary elec-trophoresis offered full automatiog and on-line detection. Using narrow-^borefused-silica capillaries filled with the appro-priate polymer sieving media, exceptionallyhigh resolving power was achieved in theseparation of biologically important macro-molecules (4). High separation voltagesensured rapid separations with excellentefficiencies — that is, theoretical platenumbers as high as 30 million (5). Itshould be noted that the detector trace incapillary etectrophoresis is referred to as anelectropherogram, similar to the term "chro-matogram" used in chromatography. High'sensitivity detection systems such as laser-

induced fluorescence and electrochemicaldetection opened new, previously incon-ceivable detection limits as low as in thezeptomole (10"- ' mol) range. As capillarygel electrophoresis separates complex sam-ple mixtures in just minutes with excellentteproducibility, even in multicapillary for-mat, it is becoming an important separa-tion and characterization tool in thegenomics and ptoteomics era.

The Most Popular Gel:PolyacrylamideThe basic difference between traditionalslah gel elect rophoresis and capillary gelelectrophoresis is the utilization of narrow-bore, fused-silica columns containing thesieving medium. Capillaries were first filledwith cross-linked gels similar to those usedin conventional polyacryiamide gel elec-trophoresis and have proven to be particu-larly efficient in separating protein mole-cules (6). The molecular sieve created bythe three-dimensional (3-D) structure ofthe polymer chains enabled proper size sep-aration of hiopolymers. Cross-linked gelsare sensitive to temperature, pH, and saddlevoltage changes because of the so-called"phase transition" phenomena (7). For sta-bilization, the cross-linked gels are attachedcovalendy to the inner capillary surface.Samples can he introduced into the capil-laries filled with cross-linked gel only byelectrokinetic injection, a method that usu-ally results in sharp peaks due to samplepreconcentration. However, it does notaccommodate sample injection from solu-tions with high salt concentration. Cross-linked gels have been used primarily forseparating shorter single-stranded DNAmolecules and also for the separation ofproteins by capillary sodium dodecyl suifategel electro phoresis. The first report on sin-gle nucleotide resolution separation of anoligonucleorides ladder containing 40-60bases of adenylic acid (8) opened up newhorizons in DNA sequencing based onmodern capillary gel electrophoresis and

8 9 8 tCGC NORTH AMERICA VOLUMES NUMBERS SEPTEMBER 2004

20 22 24Time (min}

Figure 1: Capillary gel electrophoresis separation of single-stranded oligonucleotides ladder(poiyadenylic acid 40-60) on a cross-linked polyacrylamide gel. (Author's collection.)

actually made possible the faster-than-antic-ipated completion of the human genomeproject. Figure 1 shows an example of suchseparation.

Non-cross-linked, linear polymer matri-ces are not attached ro the inside wall ofthe capillary and feature very flexible,dynamic pore structures. Actually, the poresizes of these matrices are defined bydynamic interactions between the polymerchains and can be varied at any time bychanging such variables as capillary temper-ature, separation voltage, salt concentration,or pH. Linear polymer gels are not heatsensitive and support simple replacement ofthe separation matrix in the capillary bypressure. Such gels permit both electroki-netic and pressure injection, enabling large-scale, routine sample processing. Linearpolyacrylamide gels were introduced firstinto coated capillary columns and appliedto the separation of double-stranded DNA(dsDNA) fragments containing as many asseveral thousand base pairs (9). Figure 2

depicts the separation of a DNA restrictionfragment mixture in a linear polyacry-lamide sieving matrix. In some instances,sequence-dependent migration behavior ofdsDNA fragments was observed In capillaryelectrophoresis. Linear polyacrylamide gelswere employed successfully for the separa-tion of standard proteins and other samplesof biological origin.

Sugar-Based Sieving PolymersAgarose gel-filled capillaties were exten-sively studied for the separation of dsDNAfragments (10). In capillary electrophoresis,a low-concentration (< 0.25%) solution ofthe natural polysaccharide matrices can beapplied as a sieving additive for the separa-tion of a range of DNA fragments contain-ing as many as 1400 base pairs. The effectof temperature and the viscosity of thepolysaccharide-based sieving medium onthe elcctrophoretic behavior of sodiumdodecyl sulfate (SDS) protein complexes incapillary electrophoresis also was studied.

2

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1 : 1 1

Time mid)

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Figure 2: Separation of the DNA restriction fragments by linear polyacrylamide gel. (Author'scollection.) Peaks: 1 = 72, 2 = 118, 3 - 194, 4 = 234, 5 = 274, 6 = 281, 7 = 310, 8 = 603, 9 =872, 10 = 1078, 11 = 1353 base pairs.

www. chromatograp hyo nline.com

Low-viscosity polysaccharide matrices havebeen characterized in capillary elecrrophote-sis for the separation of DNA fragmentsand have exhibited good separation abilityfor larger DNA fragments.

In the early 1990s, Schwartz and col-leagues attempted to use derivatized cellu-loses as sieving matrices to separate DNAfragments in capillary columns (11). Stan-dard proteins ranging from 14 kDa to97 kDa were analyzed on hydroxypropyl-cellulose in less than 30 min with no appar-ent influence of various buffer composi-tions on the sieving performance (12). Themigration of dsDNA molecules was investi-gated in semidilute hydroxyethylcellulosesolution, revealing the segmental DNAmotion and allowmg quantitative descrip-tion of the changing shape of the DNA asit interacted with the sieving polymer (13).Others reported that axial difftision did notaccount for the total observed band vari-ance (14).

Recently, sugar-bearing polyacrylamidecopolymers were synthesized and adaptedas sieving matrices and capillary coatingsfbt DNA electrophoresis. Poly(A'.A -dimethylacrylamide) copoiymerized withhydrophilic monomers also was used toimprove separation performance (15). Acopolymer of acrylamide and p-D-glucopy-ranoside was used as a low-viscosity andhigh-capacity sieving matrix for the separa-tion of dsDNA molecules. The growth ofthe polymer chains was controlled by thedifferent reactivity of the two monomers.The chain length was inversely proportionalto the number of glucose residues incorpo-rated into the copolymer.

Other Sieving MediaIn addition to the most frequently usedcross-linked and linear polyacrylamides andtheir derivatives (for example, polydimethy-lacryamide), other hydrophilic polymerssuch as polyethylene oxide,polyvinylpyrrolidone, polyethylene glycol,hydroxyethylcellulose and various cellulosederivatives, polysaccharides, and agaroseproved useful for the size separation ofbiopoiymers in natrow-bore capillaries (16).

Both polyethylene oxide andpoiyvinylpytrolidone matrices providedgood separation performance in DNAanalysis based on capillary electrophoresis,also featuring self-coating properties (17).Polyvinylpyrrolidone sieving and wall-coat-ing matrix proved useful for rapid molecu-lar diagnostics capable of detecting com-mon mutations using primer extensiontechnique combined with capillary

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Figure 3: Long-read length DNA sequencingseparation by capiilary gel electrophoresis(37).

electrophoresis.Solutions of monomeric nonionic sur-

factants, «-alkyl polyoxyethylene ethersbehave as dynamic 3-D polymer structuresand rherefore can be used as sieving matri-ces for DNA fragment analysis in capillarycolumns (18). The advantages of surfactantsolutions over regularly used linear poly-mers are the ease of matrix preparation,solution homogeneity, stable structure, lowviscosity, and appropriate self-coatingproperties to suppress electroosmotic flow.Viscosity measurements revealed that inter-penetrating networks had significantlyhigher viscosity than that of the simplemixture containing the same amount ofcomponents. Transient interpenetratingnetworks were successfully attempted tothe separation of dsDNA fragments usingpolyvinyipyrrolidone with acrylamide-based polymers (19). The particularly goodsieving ability of the system was attributedto the increase in the number of entangle-ments by the more extended polymerchains. Separation of peptide-proteinmodel mixtures was attempted in surface-modified capillaries filled with liquid crys-tals acting in a secondary partition mecha-nism (20).

Breakthrough in DNA SequencingOne of the most important applications ofcapillary gel electrophoresis is DNAsequencing. During the last decade, the

lack of adequate stability of cross-linkedpolyacrylamide gels within microborecolumns initiated a rapid development tofind novel, more capillary-friendly sievingmatrices. Several reports demonstrated theusefulness of non-cross-linked polymericsolutions to attain rapid separation of sin-gle-stranded DNA molecules, also enablingthe application of high temperature duringelectrophoresis. Choosing the appropriateelectrophoresis parameters (such as separa-tion voltage and temperature) for theanalysis of DNA sequencing plays animportant role in obtaining rapid separa-tion of long-length fragments. Tempera-tures as high as 80 "C were applied success-fully to reach sequencing speeds greaterthan 1000 bases/h (21). An example isshown in Figure 1,

Novel DNA sequencing matrices weredeveloped with a thermally controllable"viscosity switch" (22). Recently intro-duced novel thermoresponsive copolymers,comprising hydrophobic and hydrophilicblocks, have exhibited promising results.They offer some practical advantages incapillary' etectrophoresis such as easier han-dling and loading of the viscous polymersolutions without the requirement of ahigh-pressure manifold. The novel blockcopolymer thermo-associating matrix forDNA sequencing (23) (comb polymers) ismade of a hydrophilic polyacrylamidebackbone, grafted with poly-A^- isopropy-lacrylamide side chains and characterizedby lower critical solution temperature.These matrices combine easy loading withhigh sieving performance due to switchingbetween a low- and high-viscosity state bytemperature change. Sequencing read

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lengths of as long as 800 bases wereattained in less than 1 h using polymerssuch as these.

Getting into BiotechCapillary SDS gel electrophoresis is a rapidautomated separation and characterizationtechnique for proteins, and it can be con-sidered as a modern instrumental approachto SDS polyacrylamide slab gel elec-trophoresis. Size separation of SDS-proteincomplexes can be attained readily in coatedcapillaries filled with cross-linked gels ornon-cross-linked polymer networks.Figure 4 depicts one of the early appiica-rions of the technique for the analysis of astandard protein test mixtures ranging insize from 14.2 kDa to 205 kDa. CapillarySDS gel electrophoresis proved to be a veryimportant separation tool for rapid molec-ular weight estimation and purity check ofrecombinant proteins in the modernbiotechnology industry. Other interesringapplicarions of capillary SDS gel elec-trophoresis have been reported for bacterialprotein characterization and human serumprotein profiling.

The usefulness of the technique wasexplored in the biotechnology industrywhere capillary SDS gel electrophoresis wasapplied to the analysis of a therapeuticrecombinant monoclonal antibody (rmAb)(24). In this instance, precolumn fluo-rophore labeling of rmAb was used toobtain low nanomolar detection limits.This parricular assay illustrated the advan-tages of enhanced precision and robustness,speed, ease-of-use, and on-line detection inmonitoring bulk manufacture of proteinpharmaceuricals. Capillary SDS gel elec-

Time (min)

Figure 4: Capillary SDS gel electrophoresis trace of a protein test mixture (38). Peaks: 1 = p-lac-talbumin, MW 14,200; 2 = carbonic anhydrase, MW 29,000; 3 = ovalbumin, MW 45.000; 4 =bovine serum albumin, MW 66,000; 5 = phosphorylase B, MW 97,400;6 = 0-galactosidase, MW 116,000; 7 = myosin, MW 205,000; OG = Orange-G tracking dye.

9 0 2 LCGC MORTH AMERICA VOLUME 22 NUMBER 9 SEPTEHeER ZOM

trophoresis of proteins in a sieving matrixusing laser-induced fluorescence detectionwas reported to attain picomolar detectionlimits with noncovalent fluorogenic label-ing of proreins using Sypro red dye (25).Rapid and efficient capillary SDS gel elec-trophoresis separation of proreins also wasreported to characterize human colon can-cer adenocarcinoma cells (26). The cellswere lysed inside a capillary, followed byprotein denaturation with SDS and fluo-rophore labeling with 3-(2-furoyl)-quino-line-2-carboxyaldehyde and separated usingan 8% sieving matrix. Typical resolutionwas found to be around 30 protein com-ponents of a single HT29 cell, similar tothe peak capacity of conventional SDSpolyacrylamide gel electrophoresis. Fluores-cent detection provided high sensitivityranging from lO"'** to 10'' ' M. Single-cell-level analysis was completed in 45 min.

In addition to DNA and proteins, otherbiopoiymers of biotechnology relevance,such as complex carbohydrates, can be ana-lyzed readily by capillary gel electrophore-sis. Separation and electrophoreric migra-tion behavior of malro-oligosaccharides,derivatized via their reducing end with 8-aminonaphthalene-l,3,6-trisulfonic acid hyreductive amination was tboroughly inves-tigated (27). Others compared capillaryelectrophoresis and micellar electrolcineticchromatography of 4-aminobenzonitriIecarbohydrate derivatives (28). Complexcarbohydrates, released from glycoproteins,were profiled by capillary gel electropbore-sis with laser-induced fluorescence-baseddetection of the sugar molecules labeledwith I-aminopyrene'3,6,8-trisulfonic acid(29). Figure 5 shows an example. Highmannose-type oligosaccharides of ribonu-clease B were derivatized by 1-aminopy-rene-3,6,8-trisulfonic acid and separated bycapillary eiectrophoresis using a polyethyl-ene oxide separation medium.

Capillary Affinity GelElectrophoresisCapillary gel electrophoresis accommodatesthe inclusion of afFinity-type ligand mole-cules in the separation matrix. These canbe either soluble in the buffer system orimmobilized by the gel matrix. An exampleof selectivity manipulation by soluble affin-ity ligand interactions was evaluated by theaddition of intercalating agents to the sepa-ration gel-buffer system in dsDNA separa-tions (30). The ligand can be immobilizedby physical entrapping into — or bychemical bonding to — the sieving poly-mer. The entrapping method was demon-

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Time (min)

Figure 5: High-performance analysis of complex carbohydrates by capillary gel electrophoresis(29). The electropherograms of aminopyrene trisulfonate labeled glycans from (middle trace)bovine fetuin and (lower trace) bovine ribonuclease B are compared with (upper trace) the elec-tropherogram of a maltooligosaccharide ladder standard. Numbers on the upper trace corre-spond to the degree of polymerization of the glucose oligomers. Peaks: F1 = tetrasialo-trianten-nary-2x(i2,6; f2 = tetrasialo-triantennary-2xa2.3; F3 = trisialo'triantennary-2xa2,6; F4 =trisialo-triantennary-2xa2,3; M5-M9 = mannose 5-mannose 9; a ,b ,c = positional isomers.

strated for the separation of chiral com-pounds by incorporating cyclodextrins intothe potyacrylamide gel-filled capillary col-umn (31). A homogenous polyacryiamide-ally I-P-cyclodextrin copolymer gel also wasattempted for the separation of drug enan-tiomers (32). The large pore size definedhy the composition of the gel ensured thenecessary electroosmotic flow for properoperation.

Further Miniaturization:Lab-on-a-ChipEarly feasibility experiments proving theusefulness of electrophoresis in microfabri-cated devices were performed more than adecade ^ o (33). Electrophoresismicrochips were developed using the tech-niques elaborated on by the semiconductorindustry, suggesting that channels andother functional elements can be fabricatedin glass substrates by photolithography.Typically, samples are loaded electrokineti-cally into the cross-section of an injector.The analyte molecules are separated hyapplying the electric field not only alongthe separation channel, but to a lesserextent, to the sample and waste reservoirsas well, preventing bleeding of the sampleinto the separation channel (34). The sepa-rated solute molecules then are visualizedmost frequently by confocal microscopywith laser-induced fluorescence or electro-chemical detection. The latter detectionsystem can be microfabricated easily intothe separation channel system. Compactinjection plugs, high electric fieldstrengths, and short effective channellengths result in separations in secondswith extremely high efficiencies because ofminimized extracolumn broadening effects.

Electrophoresis microchips using sievingmatrices were applied to the analysis ofDNA restriction fragments, polymerasechain reaction products, DNA sequencing,genotyping, and rapid polymerase chainreaction product analysis (35). A capillaryarray system reportedly analyzed 96 sam-ples simultaneously from 96 sample wellsusing a radial microchannel array elec-trophoresis microplate in high-thro ugh purDNA sizing. As a result, the 96 sampleswere analyzed in less than 90 s/microplate,showing the power of microfabricateddevices for large-scale and high-perfor-mance nucleic acid characterization (36).

InstrumentationJust like chromatography, electrophoresis isessentially an instrumental method. There-fore, we should mention briefly the state-of-art of instrumentation in electrophore-sis. Today, a number of instrumentcompanies market various systems permit-ting analytical separation of a variety ofsamples. We list them here alphabetically.

Applied Biosystems and Amersham Bio-sciences (Philadelphia, Pennsylvania) bothmarket multicapillary DNA sequencingunits (up to 384 lanes) with multispectralimaging fluorescent detection systems. Agi-lent Technologies (Wilmington, Delaware)provides a capillary electrophoresis systemwith UV and diode array detection as wellas "lab-on-a-chip" products. BeclonanCoulter (Fullerton, California) has a capil-lary electrophotesis system equipped withW, diode-array, and laser-induced fluores-cence detection systems, and also offers amulticapillary-based clinical analyzer andan eight-lane DNA sequencer. Bio-RadLaboratories (Hercules, California) offer a

9 0 4 LCGC NORTH AMERICA VOLUME 22 NUMBER 9 SEPTEMBER 2004

high-sensitivity fluorescence detector fortheir capillary clectrophoresis Instrumentline. CombiSep (Ames, Iowa) introduced ahigh throughput capillary array elec-trophoresis unit capable of doing 96 paral-lel separations with UV absorbance detec-tion. Groron Technology {Acton,Massachusetts) offers a complete separationsolution including capillary electrophoresiswith laser-induced fluorescence or diode-array detectors. Hitachi (San Jose, Califor-nia) is offering a microchip electrophoresisinstrument, while the Institute of Micro-chemical Technology (Mito, Japan) sellsintegrated glass chips and custom-madechips for environmental analysis,immunoassay, etc. MicroSolv (LongBranch, New Jersey) offers products andconsumables for capillary electrophoresisand electrochromatography. Micralyne(Edmonton, Canada) is a commercialmicrofabrication company offeringmicrochip electrophoresis equipment andstandard and custom-made glassmicrochips. Picometrics (Ramosville,France) introduced modular laser-inducedfluorescence detector units compatible withall commercial capillary electrophoresis sys-tems. Polymicro Technologies (Phoenix,Arizona) is a manufacturer of fused-silicacapillaries with polyimide, acrylate, and sil-icone coatings as well as capillary connec-tors. Shimadzu (Columbia, Maryland) hasjust entered into the lab-on-a-chip technol-ogy business. Finally, Spectrumedix (StateCollege, Pennsylvania) offers high-through-put capillary electrophoresis instruments(scalable on-site from 24 to 96 to 192 cap-illaries) for mutation discovery, forensicidentity, and DNA sequencing.

ConclusionsGels and polymer matrices greatly enhancethe productivity of capillary electrophoresisanalysis of biologically important macro-molecules including nucleic acids, pro-teins-peptides, complex carbohydrates, andsmall molecules. By automating the cur-rent manual procedures, capillary gel elec-trophoresis is reducing both the analysistime and human intervention from sampleloading to data analysis. The technique issuited ideally for handling microliter vol-umes of sample material with superiorthroughput over conventional slab gelapproaches, especially when capillary arraysare used.

At the beginning, cross-linked gels wereused in capillary electrophoresis. However,their lack of adequate stability (see phasetransition phenomena, mentioned earlier)

within the microbore columns initiated thedevelopment of novel, more capillary-friendly sieving matrices, leading to theutilization of linear polymers, the so-callednon-cross-linked gels, or polymer networksfor the analysis of biologically importantmacromolecules. With capillary gel elec-trophoresis, enormous column efficiencieswere found, as high as 30,000,000plates/m. As the advent of capillary gelelectrophoresis already has made possiblethe sequencing of the human genome, weanticipate this technique will be developedfiirther to reveal global changes at the pro'teome level, bringing about a revolutionarytransition in our views of living systems onthe molecular basis.

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Dr. Andras Guttman is affiliated with the Hor-vath Laoratory of Bioseparation Science, Uni-versity of Innsbruck. Austria. He is currentlywith with Diversa Co., San Diego, California.

Leslie S. Ettre"Milestones in Chro-matography" editorLeslie S. Ettre is aresearch affiliate ofthe Chemical Engi-neering departmentof Yale Universityand a member ofLCGC's editorialadvisory board.