Abstract Capillary electrophoresis (CE) enables rapid sep-arations with high separation efficiency and compatibilitywith small sample volumes. Laser-induced fluorescencedetection can result in extremely low limits of detection inCE. Single-channel fluorescence detection, however, fur-nishes little qualitative information about a species beingdetected, except for its CE migration time. Use of multi-dimensional information often enables unambiguous iden-tification of analytes. Combination of CE with informa-tion-rich wavelength-resolved fluorescence detection isanalogous with ultravioletvisible diode-array detectionand furnishes both qualitative and quantitative chemicalinformation about target species. This review discussesrecent advances in wavelength-resolved laser-induced flu-orescence detection coupled with CE, with an emphasison instrument design.

Keywords Review Capillary electrophoresis Laser-induced fluorescence Wavelength-resolvedfluorescence

List of abbreviations

CCD charge-coupled deviceCE capillary electrophoresisICCD intensified CCDIPDA intensified photodiode arrayLCW liquid core waveguideLIF laser-induced fluorescenceLINF laser-induced native fluorescenceLOD limit of detectionPDA photodiode arrayTDI time-delayed integrationTIR total internal reflectionUV ultraviolet

ChemicalsDA dopamineE epinephrineFAD flavin adenine dinucleotideFMN flavin mononucleotide5-HIAA 5-hydroxyindole-3-acetic acid5-HT or Serotonin 5-hydroxytryptamineMEL melatoninNADH -nicotinamide adenine dinucleotideNADPH -nicotinamide adenine dinucleotide

phosphateNAS n-acetyl serotoninOA octopamineTHB 5,6,7,8-tetrahydrobiopterinTrp tryptophanTrpA tryptamineTyr tyrosine

Introduction

In little over two decades capillary electrophoresis (CE)has emerged as a powerful analytical technique for deter-mining the chemical composition of mass-limited samples[1]. It enables rapid separations with high separation effi-ciency and compatibility with small sample volumes [2, 3,4, 5]. CE has been used for a variety of applications, in-cluding determination of inorganic ions [6, 7], environ-mental [8, 9], food [10], and clinical analysis [11], peptideand protein separations [12, 13], single-cell assays [14,15], and DNA sequencing [16, 17, 18]. The state-of-the-art of CE is addressed in several recent reviews [1, 19, 20].

The diminutive capillaries employed in CE, the con-comitant small peak volumes, and the temporally narrowpeaks which result from the high-efficiency separationcomplicate detection [21]. Detection in CE is most com-monly performed on-column by use of absorbance, oftendiode-array, detection [22, 23]. To achieve better perfor-mance a variety of other detection methods has been de-veloped for CE, including conductivity [24], radiochem-istry [25], refractometry [26, 27], and electrochemistry

Xin Zhang Jeffrey N. Stuart Jonathan V. Sweedler

Capillary electrophoresis with wavelength-resolved laser-inducedfluorescence detection

Anal Bioanal Chem (2002) 373 :332343DOI 10.1007/s00216-002-1288-9

Received: 29 November 2001 / Revised: 3 February 2002 / Accepted: 15 February 2002 / Published online: 19 April 2002

REVIEW

X. Zhang J.N. Stuart J.V. Sweedler ()Department of Chemistry, University of Illinois, Urbana IL 61801, USAe-mail: jsweedle@uiuc.edu

Springer-Verlag 2002

[28, 29]. Coupling CE to nuclear magnetic resonance [30,31] and mass spectrometry [32, 33, 34] enables identifica-tion of unknown compounds after a separation by provid-ing an information-rich detection scheme for structuraldetermination. Although laser-induced fluorescence (LIF),suitable for analysis of a large variety of analytes [35, 36,37], results in the highest sensitivity in CE, little structuralinformation can be obtained from single-wavelength fluo-rescence detection. Wavelength-resolved fluorescence de-tection is a means of maintaining low limits of detectionwhile furnishing valuable spectroscopic information aboutfluorescent species; this makes it an attractive mode of de-tection for trace analysis in CE. This review describes re-cent advances in instrumentation for wavelength-resolvedfluorescence detection in CE.

Laser-induced fluorescence detection in CE

In 1981 Jorgenson and Lukacs [38] described the fluores-cence detection of labeled amino acids separated by CE.In this pioneering work a high-pressure mercury arc lampwas used as the excitation source and fluorescence wasdetected by means of a standard fluorimeter. A low back-ground and a linear relationship between source and sig-nal intensity make fluorescence detection an inherentlysensitive method. Fluorescence detection for CE commonlyemploys a laser as the excitation source. Lasers emit in-tense radiation with extremely narrow bandwidths, highspatial coherence, and negligible chromatic aberration;their output is easily focused on to the small flow channelof a capillary. Since the first introduction of LIF detectionfor CE by Zare et al. [39], limits of detection (LODs) haveimproved dramatically detection limits for most research-grade instruments are in the zeptomole range for somefluorophores [2, 3, 4, 5]. Dovichi and co-workers devel-oped a more sensitive LIF method that used a sheath flowcuvette [40, 41] as the sample cell to reduce backgroundfluorescence. LOD as low as six molecules of sulforho-damine 101 [42] and ~1 molecule of -galactosidase in anenzymatic assay [43] were reported by this group. Be-cause of its low LOD, LIF is most often applied to mass-limited biological samples.

Multichannel LIF detection in CE

Combining CE with information-rich spectroscopic detec-tion, e.g. multichannel fluorescence detection, providesboth qualitative and quantitative chemical information. Thisis of interest because of possible lack of reproducibility inelectrophoretic migration times and the possibility of co-elution of analytes in a complex sample matrix. Use of atwo-dimensional system also reduces matrix interfer-ences, thus improving signal-to-noise ratios. Compoundscan therefore be identified with greater confidence by useof multi-dimensional fluorescence detection, such as wave-length-resolved, time-resolved, and polarization fluores-cence detection. Wavelength-resolved fluorescence fur-

nishes spectral information to compliment migration time.Time-resolved fluorescence [44, 45, 46] involves mea-surement of fluorescence lifetimes, to discriminate againstbackground impurities and to assist in analyte identifica-tion. Polarization fluorescence detection [47, 48] makesuse of a molecules inherent rotational correlation time.This review focuses on wavelength-resolved fluorescencedetection, one of the most widely established multichan-nel fluorescence detection schemes.

Wavelength-resolved LIF detection in CE

History

The first report of wavelength-resolved fluorescence de-tection in CE used a photodiode array (PDA) to produce a 4-nm-per-channel spectral distribution [49]. LOD forfluorescein was 60 fg, or 20 mol L1. Array detectorswith low-noise characteristics, e.g. charge-coupled devices(CCD) and intensified PDA (IPDA) enable a large im-provement in LOD [50, 51, 52]. The first use of a CCDcombined with a polychromator to achieve wavelength-resolved fluorescence in CE was reported by Cheng andcoworkers [53]. In this initial demonstration the CCD wasused in conventional camera mode with only a 3% dutycycle, and so detection limits were relatively poor ap-proximately 4 attomoles for fluorescein. In 1991 the Zaregroup modified the time-delayed integration (TDI) modeto read the CCD for their application [54]. Use of the TDImode enables synchronization of the transfer of photogen-erated charge with the movement of the image of the ana-lyte band on the array. This enabled the fluorescence to beintegrated over the entire time in the observation zone andresulted in a two-to-five-fold improvement in perfor-mance. LOD were improved dramatically to 2080 zmole(13 nL injection) for fluorescein isothiocyanate-labeledamino acids. Also in 1991 a CCD-LIF system with a 94%duty cycle, which furnished high-quality spectral informa-tion, was demonstrated [55]. The concentration LOD forJoe-A oligonucleotide primer was 26 pmol L1. Severalother research groups have since reported CCD- or IPDA-based detection with a variety of instrumental configura-tions and applications (vide infra) and CE with wave-length-resolved fluorescence detection has now been suc-cessfully commercialized. The Applied Biosystems (ABI)Prism 377 DNA sequencer is an automated instrument de-signed for analysis of fluorescently-labeled DNA frag-ments separated by capillary gel electrophoresis [56]. Aspectrograph-CCD is used to achieve wavelength resolu-tion, to enable differentiation of the different dye-labelednucleotides. Reading of up to 900 bases with 99.8% accu-racy is possible.

Instrumentation and design

Typically, fluorescence detection systems consist of threemajor subsystems excitation, collection, and detection.

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Choice of excitation laser source is based on the require-ments of a particular application. Collection optics fre-quently consist of a high numerical aperture microscopeobjective, designed to collect the maximum amount ofemitted light. Elimination of scattering and background isoften achieved by use of spectral and spatial filters, end-column sheath-flow cell detection, or, more recently, in-column detection and liquid core waveguide (LCW) end-on detection (vide infra). For multiwavelength detection awavelength selector is required; this consists either of aseries of filters for preselected wavelengths or a grating orprism for detection over a range of wavelengths. Multi-element array detectors, e.g. cryogenically-cooled CCDand IPDA, typically enable multiwavelength detection, withthe detector characteristics often dictating system perfor-mance. Cryogenically-cooled two-dimensional CCD cam-eras are integrated solid-state photometric detectors char-acterized by negligible dark current, high quantum effi-ciency, low readout noise, wide dynamic range, and theability to bin photo-generated charges from multiple pix-els [50, 51, 57]. The IPDA is a solid-state diode array cou-pled to an image intensifier via a fiber-optic bundle. Withthe image intensifier as an amplifier for photons, a gain of3000 to 9000 is typically obtained, thus the signal-to-noise ratio of the IPDA detector is significantly higherthan that of the PDA and the IPDA is thus highly sensitiveunder low-light conditions [58, 59]. In comparison, theCCD has extremely low noise, generally under 5 e andhas a dark current less than 1e per hour if properlycooled, and so is commonly used without intensificationin this application [51, 57]. Although the CCD detector isreported to have significant advantages over the IPDA interms of spectral resolution, intensity, and precision of datain the vacuum UV for laser-induced plasma spectroscopy[60], intensified detectors such as the intensified CCD(ICCD) and IPDA are suitable for time-resolved measure-ments with better temporal resolution [60]. For wave-length-resolved fluorescence detection in CE readout timeis usually not an issue. Several different instrumental con-figurations are described for wavelength-resolved fluores-cence detection in CE.

CCD-based detection

The first wavelength-resolved CE-LIF-CCD system devel-oped by us was based on previous work by Zares group,and utilized an Ar/Kr mixed gas ion laser, which providedgreat flexibility in excitation wavelength [61]. The detec-tion window (~2 mm section of the capillary) was illumi-nated axially through a cylindrical lens. The fluorescencewas collected, via a collection optic assembly at a 90 an-gle, during the entire time the analyte band was resident inthe detection window, to enhance the LIF sensitivity.Rayleigh scattering (owing to on-column detection) wasremoved by use of spatial filters. The combination of ahigh-throughput imaging spectrograph (405 grooves mm1

holographic grating) and a liquid-nitrogen-cooled CCD(1024256 pixels, 3 Hz readout, 5256 binning) enabled

use of a 500-nm spectral window adjustable in the 3001000 nm region, which is the widest fluorescence wave-length range collected in a wavelength-resolved CE-LIFsystem. LOD of 80 molecules for sulforhodamine 101(51014 mol L1) and 220 molecules for fluorescein(1.51013 mol L1) were achieved. In one applicationamino acids labeled with Bodipy 503/512 C3 and Bodipy576/589 C3 were differentiated on the basis of emissionspectrum and migration time.

Another instrumental configuration with similar spec-trograph-CCD was demonstrated in a two-lasertwo-colorflow cytometry study [62]. A sheath-flow cell (quartz cu-vette) was used as the sample chamber. Excitation of thecore stream was achieved by means of a liquid-cooled Ar+

laser and an ArKr+ laser operating at 488 nm and 568 nm,respectively. Collection optics included a microscope ob-jective (to eliminate fluorescence from the collection optics)and a series of 500-nm high-pass and 568-nm laser-line re-jection filters. The spectrograph (grating 285 grooves mm1)and CCD were oriented to enable sub-array readout andbinning (3 Hz readout, 2256 binning) while preserving1024-pixel wavelength information over the 350800 nmwavelength range. By use of single-particle fluorescenceemission spectra, submicron particles were individuallyidentified, sized, and distinguished.

Blaschke et al. recently built an on-column wavelength-resolved CE-LIF system using an Ar+ laser working at257 nm and an intensified CCD-spectrograph to achievewavelength resolution. Similar to previously describedon-column LIF detection, the capillary was illuminatedwith the laser profile for a 1.5 mm length along the detec-tion window. With an imaging spectrograph (1200 groovesmm1 grating) and an intensified CCD camera (9.9 Hzreadout, 1080 binning or 4.5 Hz, 4840 binning), a spec-tral window of 105 [63] or 160 nm [64] could be selectedover the 180400 nm wavelength range. The combinationof migration time and spectral information enabled simul-taneous detection and quantitation of etoposide and etopo-side phosphate in plasma for the first time.

An interesting and intriguing design for CE with wave-length-resolved fluorescence detection is the LCW fluo-rescence detector developed by Roeraades group [65].The principle of total internal reflection (TIR) was used inan effort to reject more stray light and for efficient illumi-nation and collection of light from large capillary arrays.TIR was achieved by coating the separation capillary ex-ternally with a polymer with a lower refractive index thanthe separation medium, thereby inducing liquid-corewaveguiding so that imaging could occur at the capillaryend. As shown in Fig.1, the laser beam from a multi-line,multi-mode Ar+ laser was expanded to ~10 mm and thenfocused by means of a cylindrical lens on to the capillaryarray positioned at the focal line (the axial extent of thebeam inside the capillary was ~25 m). The emitted fluo-rescent light was guided approximately 50 mm to the endof the capillary, which was placed in a buffer chamber.The wall of the container opposite the capillary ends wasa planar glass plate. By using glass filters to absorb theprimary light, the capillary ends were imaged via a cam-

334

era objective on to a thermoelectrically-cooled CCD cam-era. A prism was placed between the filter and the camerato allow for color dispersion. The CCD chip (1024 TKB,100 kHz read-out rate) was binned in hardware into su-perpixels (88 pixels large). A 10 superpixel-wide spec-trum covered approximately the 480620 nm range. TheCCD was operated in camera mode with an exposure timeof 0.6 s for imaging.

Some unique properties result from this instrument de-sign. The LOD for fluorescein was 62 fmol L1, which islower than for most sheath-flow systems (note that themass limits of detection cannot be specified, because theinjection volume is uncertain). The orthogonal geometryimplies that no laser light should be detected, but in prac-tice this is not so, because of dust, scratches on the capil-lary surface, and Rayleigh scattering. Yet, even this lightcan be spatially differentiated in this system, because ofthe principle of LCW. Light scattered near the peripheryof the capillary exterior should propagate down the capil-lary toward the outer surface, whereas fluorescent lightfrom the sample near the center of the capillary will result

335

Fig.1 Schematic diagram of CE with end-on wavelength-resolvedLCW fluorescence detection [65]. Figure reprinted with permis-sion from Analytical Chemistry. Copyright 2000 American Chem-ical Society

Fig.2 Top view of the two-lasertwo-window on-columnwavelength-resolved fluores-cence detection system [67].Figure reprinted with permis-sion from Analytical Chem-istry. Copyright 1993 Ameri-can Chemical Society

in a peak in the center of the CCD image. Effective straylight rejection is the most impressive accomplishment ofthe design, especially in comparison with other more labor-intensive designs in which sheath-flow cuvettes or scan-ning confocal detectors are employed. The use of interfer-ence filters and a prism for color dispersion means, how-ever, that a highly collimated excitation source is neces-sary. This high-sensitivity design is simple and robust,and has recently been adapted to four-color DNA sequenc-ing by gel electrophoresis in a ninety-one capillary array[66]. Images of the 91 microspectra (460610 nm overnine superpixel spectral elements) were demonstrated.The technique might be increasingly employed in the fu-

ture for high-throughput systems, because of its scalabil-ity and simplicity.

IPDA-based detection

Wavelength-resolved detection has also been accomplishedby use of an IPDA mounted on a spectrograph. In 1993Kargers group [67] developed a two-lasertwo-windowIPDA detection method for four-color DNA sequencingin CE. As shown in Fig.2, two different lasers (488 nmAr+ laser and 543 nm HeNe laser) were used to excitedifferent groups of fluorophores sequentially on-column.

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Fig.3 (A) Schematic diagramof the sheath-flow-based CEsystem utilizing a frequency-doubled Ar+ laser for excita-tion and a spectrograph-CCDfor wavelength-resolved fluo-rescence detection. (B) Wave-length-resolved fluorescenceelectropherogram obtainedfrom a mixture of standardswhere the y-axis shows theemission spectra wavelengthand the gray scale shows theintensity of the fluorescenceemission (with the scale bar to the right of the electrophero-gram). Excitation was at 257 nm. The two horizontallines indicated by arrows arethe major H2O Raman line at~284 nm (3654 cm1) (weaker)and the 2nd-order laser line(stronger) at 514 nm. Peak as-signments: (a) 6.2 mol L1TrpA; (b) 4.1 mol L1 5-HT;(c) 15 mol L1 OA; (d) 3.1 mol L1 NAS; (e) 4.2 mol L1 MEL; (f) 97 mol L1 DA; (g) 130 mol L1 E; (h) 3.9 mol L1 Trp; (i) 28 mol L1 Tyr; (j) 0.23 mol L1 sulforho-damine 101; (k) 2.0 mol L1THB; (l) 7.0 mol L1 5-HIAA;(m) 8.0 mol L1 FMN; (n) 11 mol L1 FAD; (o) 0.93 mol L1 fluorescein;(p) 50 mol L1 NADH; (q) 95 mol L1 NADPH. Thek, m, and n are minor impu-rity peaks for k, m, and n stan-dards, respectively.

Fluorescence spectra (510700 nm and 550700 nm, re-spectively) from these two windows were imaged on theseparate diodes in the array (1024 channels) via a spectro-graph (150 grooves mm1 grating) and band-pass filters.This design introduced use of multi-window detection tooptimize excitation wavelengths and minimize photo-bleaching. The IPDA enabled low-level detection (con-centration LOD in the pmol L1 range) and spectral reso-lution to aid dye discrimination. No further developmentor application using this system has, however, since beenreported.

Another IPDA-based wavelength-resolved fluorescencedetection system was constructed by Majidis group [68].Their detection configuration was quite different from ei-ther on-column or end-column sheath-flow-based detection.Instead, a 100 m fiber optic was inserted into a 200-mi.d. fused-silica separation capillary and extended to the endof the detection window. Electromagnetic radiation (2ndharmonic, 532 nm) from a pulsed, Q-switched Nd:YAGlaser was focused on to the fiber optic, which then sup-plied excitation energy for fluorescence measurements.Axial excitation of fluorescent molecules in-column avoidsthe common problem of light scattering (often encountered

with on-column excitation), because the excitation beamdoes not need to pass through the capillary wall [54]. Flu-orescence emission from analytes in the capillary was fo-cused through a lens to a cooled IPDA (1024 channels)mounted on a spectrograph (600 grooves mm1). Three-hundred spectra taken every 100 ms were co-added toobtain a data point. The wavelength range covered was140 nm. Separation and identification of rhodamine deriv-atives were demonstrated. Although the authors reportedunoptimized detection limits, they are currently toohigh for many applications (0.26 pmol for rhodamine 590chloride and 1.5 pmol for kiton red). In addition, the sep-aration efficiency was poor, possibly because of the lowseparation voltage (500 V) or deposition of analytes onthe surface of the fiber optic during the separation. It willbe interesting to follow further developments which usethis approach.

Native fluorescence detection

Perhaps the greatest benefit of wavelength-resolved fluo-rescence detection is laser-induced native fluorescence

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Table 1 Summary of CE with wavelength-resolved LIF systems

Detector settingsDetector

Readout(Hz)

Binning Spcg.(gr. mm1)

Detectionconfiguration

Excitationlaser (nm)

Spectralwin. (nm)

Application LOD Ref.

3 5256 405 On-column Ar/Kr+(multi-line)

500(3001000)

Bodipy-labeledamino acids

50 fmol L1

SR101,150 fmol L1

FL

[61]

3 2256 Sheath-flow Ar+ (488) andAr/Kr+ (568)

350800 Flowcytometry

6000 SR101 [62]

5 5256 Freq.-d. Kr(284)

280500 Peptides(Trp-, Tyr-)

20 nmol L1

Trp[71]

Freq.-d. Ar+

(257)[73,74]

50 K 2256

285

NeCu (248.6)

260710 Single neuron 20 nmol L1

5-HT120 nmol L1

DA[77]

4.5 4840 105(180400)

1mg L1tramadol

[63]

9.9 1080

1200 On-column Freq.-d. Ar+

(257)160(180400)

Clinicalsamples

100 mg L1etoposide

[64]

CCD

100 K 1010Imaging

Prism LCW end-on Ar+

(multi-lines)480620 DNA

sequencing62 fmol L1

FL[65]

2-laser2-win. 510700 [67]150On-column

Ar+ (488) andHe-Ne (543) 550700

DNAsequencing

Lowpmol L1

KrF-excimerdye (280 or350)

311512 Environmental [75,76]

600 On-column

Large frameAr+ (275)

410588 Isoflavones

Sub mg L1

IPDA 1024 channels

600 In-column Q-switchedNd:YAG(532)

140 0.26 pmolR590

[68]

(Abbreviations: 5-HT, 5-hydroxytryptamine; DA, dopamine; FL, fluorescein; Freq.-d., Frequency-doubled; gr., grooves; R590,rhodamine 590; Ref., Reference; Spcg., spectrograph; SR 101, sulforhodamine 101; win., window)

(LINF) studies, because different analytes have character-istic emission spectral profiles, in contrast with the con-stant fluorescent emission resulting from derivatizationwith a single fluorescent probe. Native fluorescence de-tection averts the complications associated with derivati-zation reactions, such as limited shelf life of fluorescenceprobes, lack of complete specificity for the analytes, andslow kinetics or incomplete reactions. Derivatization alsocan be problematic when dealing with small sample vol-umes (sub-microliter level) or low analyte concentrations(1010 mol L1107 mol L1), because of a decrease in thelabeling efficiency and an increase in background signal[37, 69, 70]. As an attractive alternative to labeling com-pounds with tags that absorb and fluoresce in the visibleregion, native fluorescence detection normally requires anaromatic (fluorescent) molecule and usually requires ul-traviolet (UV) laser excitation.

Taking advantage of the intrinsic fluorescence of tryp-tophan (Trp) and tyrosine (Tyr) we incorporated a fre-quency-doubled krypton laser operating at 284 nm andmodified the detection assembly to achieve the first wave-length-resolved native fluorescence detection in CE [71].Because Rayleigh scattering and capillary backgroundfluorescence are more prominent in the UV region than inthe visible, a sheath flow cell (1.0 mm1.0 mm quartz cu-vette) was employed; with the sheath flow system, no spa-

tial filters were required. A sheath flow cell can also func-tion as a post-column derivatization reactor [72], or en-able independent optimization of separation and detectionconditions [73]. In contrast with on-column detection, ob-servation time is limited by diffusion in the end-columnsheath-flow detection cell, because analytes continue todiffuse into a larger volume after leaving the capillary.The laser was therefore focused to a tight spot inside thesheath-flow cell just below the capillary outlet. The CCDwas operated under conditions similar to those in our ini-tial arrangement 5 Hz readout and 5256 binning. De-tection limits as low as 21010 mol L1 were achieved forTrp; this is among the lowest reported values for Trp byfluorescence detection in CE without derivatization.

This system was further improved for direct single-neuron analysis [74]. A schematic diagram of the instru-ment is shown in Fig.3A. A frequency-doubled Ar+ laserwas used to provide 257 nm laser excitation. Completefluorescence emission spectra (260710 nm) were recorded,and interference from endogenous impurities or co-elutingsubstances was eliminated by selection of an appropriatewavelength range for integration, thus improving the sig-nal-to-noise ratio. LOD ranged from low attomole to fem-tomole. Combination of electrophoretic migration timeand fluorescence spectral information enabled simultane-ous identification of more than 30 compounds in individ-

338

Fig.4 Three-dimensional elec-tropherogram obtained fromnaphthalenesulfonates (NS).The structures of the nine NSare shown on the y-axis. Exci-tation was at 280 nm [75]. Fig-ure reprinted with permissionfrom Journal of Chromatogra-phy. Copyright 1997 Elsevier

ual neurons. A typical wavelength-resolved electrophero-gram of selected standards is shown in Fig.3B. This fig-ure shows the separating power and the variety of analytespectral profiles that can be recorded by use of this system.

Other examples of CE with wavelength-resolved nativefluorescence detection include an IPDA-based system de-veloped by Gooijer et al., who used a pulsed KrF-excimer-dye (Rhodamine 6G) laser with a frequency-doublingcrystal to provide tunable UV output [75] or a modifiedAr+ laser to provide 275 nm excitation [76]. Fluorescencelight was collected on column via a microscope objectiveand focused on to a spectrograph-IPDA (600 grooves mm1

grating; IPDA 1024 channels). The spectral window wasadjusted to cover different wavelength ranges correspond-ing to specific applications (Table 1). Figure 4 shows athree-dimensional electropherogram (spectral window 311512 nm) of naphthalene monosulfonates, a major class of

environmental pollutant. The potential of wavelength-re-solved detection for identification purposes is apparent.

One of the largest drawbacks of UV excitation in wave-length-resolved fluorescence detection with CE has beenthe expense and the complexity of the lasers required forexcitation in the far UV wavelength range of 200300 nm.An improved turn-key metal-vapor NeCu laser operat-ing at 248.6 nm was recently demonstrated for a wave-length-resolved LINF study [77]. The performance obtainedwas similar to that from large-frame frequency-doubledAr+ lasers. The availability of inexpensive, rugged, andsmall metal-vapor lasers promises to expand substantiallythe application of wavelength-resolved UV-LINF detec-tion for CE and should enable the development of dedi-cated systems optimized for particular classes of mole-cule. Concerns about the laser lifetime might, however,prevent rapid adoption of the metal-vapor lasers.

Applications

Multi-fluorophore DNA sequencing

DNA sequencing technologies have been under rapid de-velopment, driven by the need of the Human Genome Pro-

339

Fig.5 Four-spectral-channel DNA sequencing data obtained inthe capillary electrophoretic analysis of M13mp18 with the two-lasertwo-window approach. Excitation was at 480 nm and 543nm. Asterisks mark peaks arising from false terminations. Twosites of compression (C36 and C65-G66) are labeled with arrows[67]. Figure reprinted with permission from Analytical Chemistry.Copyright 1993 American Chemical Society

ject for low-cost, high-throughput sequencing. The stan-dard method is didoxy terminator chemistry with separa-tion of tagged DNA fragments. Typically the four A-T-C-Gbases are tagged with specific fluorophores with differentfluorescence properties [78, 79]. Wavelength-resolvedlaser-induced fluorescence detection results in high sensi-tivity and enables easy discrimination and unambiguousidentification of the four bases, and CE affords a rapid andhighly efficient separation. On the basis of analysis of thespectral characteristics of the four standard dye-labeledprimers, FAM, JOE, ROX, and TAMRA, separation andidentification of DNA sequencing reaction products weredemonstrated by the Karger group using a two-lasertwo-window CE-LIF-IPDA [67] (vide supra). Figure 5 shows anexample of the four spectral channel DNA sequencing dataobtained by use of this system for analysis of M13mp18.A capillary array, such as that designed by Roeraade [66],also promises the additional characteristic of high-through-put DNA sequencing.

Cellular analyses

Simultaneous detection and quantitation of small mole-cules, metabolites, and cofactors at the level of individualcells provide fundamental information for cellular physi-ologists. By using spectral information combined with CEmigration time, the identification, purity, and amount ofmany natively fluorescent analytes in complex cell samplescan be determined. On excitation at 257 nm many classes ofbiologically important compound (e.g. aromatic amino acids,neurotransmitters, and cofactors) emit native fluorescence.Successful single-neuron analyses of diverse aromatic mono-amine neurotransmitters (indolamines and catecholamines)have been demonstrated [73, 74, 80]. LODs of 20 nmol L1

for serotonin (indolamine) [74] and 120 nmol L1 for do-

pamine (catecholamine) [73] were achieved. An exampleof a wavelength-resolved electropherogram of a seroton-ergic metacerebral neuron from Pleurobranchaea califor-nica is shown in Fig.6. The neurotransmitter, serotonin(5-HT), was detected, as were the amino acids Trp andTyr and the cofactor 5,6,7,8-tetrahydrobiopterin (THB).

Clinical assays

In clinical settings samples are characterized by samplevolumes

power of wavelength-resolved native fluorescence detec-tion, however, enables sensitive analysis of aromatic naph-thalenesulfonates in river water [75]. As shown in Fig.7,selectivity was enhanced by narrowing the emission win-dow. The reported LOD was in the low g L1 to sub-g L1range, adequate for real-world environmental monitoring.

Other biological applications

Another biological application was demonstrated by a studyof the isoflavone formononetin in red clover extract [76].Structurally related isoflavonoids and metabolites occur

together in some biological fluids. On excitation at 275 nmseveral furnish markedly different fluorescence spectraand can be separated and distinguished by means of CEcoupled to wavelength-resolved fluorescence detection.Identification and quantitation were possible at a concen-tration of 3105 mol L1, or 17 g formononetin per gramred clover.

Separation diagnostics

In addition to analytical applications, the information inthe fluorescence emission spectrum can yield diagnostic

341

Fig.7 Electropherogram ob-tained from a River Elbe sam-ple by use of on-column wave-length-resolved fluorescencedetection. Excitation was at280 nm and the emission rangewas (A) 311512 nm or (B) 349380 nm. When thesmaller wavelength window isused, only the naphthalenesul-fonates which do not have anOH or NH2 group appear in theelectropherograms [75]. Figurereprinted with permission fromJournal of Chromatography.Copyright 1997 Elsevier

information on the separation. For example, monitoring ofthe pH inside the capillary during a separation has beenachieved by use of a pH-sensitive fluorophore, carboxySNARF-1 [83]. The fluorescence emission profile ofSNARF-1 shifts at different pH, which enabled measure-ment of a counter-propagating pH gradient. Ionic strength,temperature, and ions can be measured by use of differentfluorophores. Other diagnostic possibilities include cor-rection of source drift and alignment problems, by use ofRayleigh and Raman scattering, and dynamic choice ofwavelength range with regard to background features [52].

Conclusions and outlook

Several wavelength-resolved CE-LIF systems have beendescribed. The instrumental configurations and applica-tions are summarized in Table 1. Wavelength-resolvedfluorescence detection is normally achieved by use of ei-ther a CCD or an IPDA mounted on a spectrograph. Themain design differences in the different systems are detec-tion-cell configurations, including on-column detection,in-column detection, sheath-flowed based end-column de-tection, and LCW end-on imaging. Sheath-flow detectioncells result in the lowest LOD, whereas LCW end-onimaging provides the potential of large-scale multiplexingwithout greatly sacrificing sensitivity. Wavelength-re-solved fluorescence detection, in providing informationcomplimentary to migration time, has several advantagesfor complex samples such as biological and environmen-tal material. With wavelength-resolved fluorescence ana-lyte misidentification is less likely, even under changingelectrophoretic conditions. One inherent limitation of flu-orescence detection is the inability to identify unknownpeaks without a reference compound. A combination ofmigration time and fluorescence emission spectra can,however, provide some information about analyte struc-ture [75, 82]. The high sensitivity, high electrophoreticperformance, and adequate spectral resolution of CE withwavelength-resolved laser-induced fluorescence detectionhas already been commercialized for four-color DNA se-quencing, and we expect further advances in instrumenta-tion and practical applications.

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