A Charge Coupled Device Array Detector forSingle-Wavelength and Multiwavelength UltravioletAbsorbance in Capillary ElectrophoresisEdmund T. Bergstro1m and David M. Goodall*

Department of Chemistry, University of York, York YO10 5DD, UK

Boris Pokric and Nigel M. Allinson

Department of Electrical Engineering and Electronics, UMIST, P.O. Box 88, Manchester, M60 1QD, UK

A fundamental limitation to the use of single-point absor-bance detection for capillary electrophoresis is irradiance,since it is not possible to create an image at the detectionpoint on capillary that is brighter than the light source.This limitation may be overcome by illuminating a lengthof the capillary using a fiber-optic bundle and using acharge coupled device (CCD) camera that can image thefull length of the illuminated zone. The present paperdescribes design and development of a CCD detector forUV absorbance that can be used in both multiwavelengthand single-wavelength modes. The CCD camera imagesanalyte peaks in the capillary dimension, together withwavelength-resolved absorbance in the dimension per-pendicular to the capillary. Successive snapshots of thepeaks are added together, after appropriate correction fortime-dependent peak displacement, without sacrificingspatial resolution. Measured baseline rms noise valuesat 200 nm are 34 µAU using a holographic grating inmultiwavelength mode and 8 µAU with the addition of aband-pass filter. Both values are in excellent agreementwith calculations of limiting shot noise. Performance inmultiwavelength mode is constrained by the 470-msreadout time of the CCD used, which sets a maximumduty cycle of 2.3%. Noise contributions from sourceintensity fluctuations are reduced by using a portion ofthe CCD image to provide a baseline reference signal.With 4-hydroxybenzoate as test analyte, the linear dy-namic range in multiwavelength mode is shown to bebetween 3 and 4 orders of magnitude. High-qualityspectra of 2-, 3-, and 4-methylbenzoates are obtained oncapillary and used in deconvolution of closely migratingpeaks of the 2- and 3-isomers.

Capillary electrophoresis (CE) is one of the leading currentseparation techniques for analysis of water-soluble analytes.Benefits include high resolution, small sample volume, speed ofanalysis, automation, and ease of method development.

In CE, analytes are normally detected at a single point nearthe output end of the capillary. Detection occurs across thecapillary internal diameter. The small path length, typically 25-

100 µm, provides the principal reason concentration limits ofdetection (LODs) are 1-2 orders of magnitude worse than in high-performance liquid chromatography (HPLC), where the flow-through cell normally has a 1-cm path length. The factor of >102

difference in path length is to an extent offset by lower peakdispersion in CE.

While bubble1 and Z cells2,3 are available to increase the pathlength in some commercial CE instrumentation, there can be anaccompanying penalty in terms of loss of resolution. To takemaximum advantage of the superb efficiency in CE (105-106

theoretical plates), improvements in UV detection limits arerequired without compromising spatial resolution. Typical LODswith current commercial instruments are in the micromolar range.To be useful for bioanalysis, nanomolar detection limits arerequired, ideally without any preconcentration of the analytes.While laser-induced fluorescence provides this for some com-pounds, the method typically requires derivatization with fluores-cent labeling reagents and lacks the universality of UV lamp-baseddetection.

In recent work, Culbertson and Jorgenson have shown thebenefits of using a photodiode array in which each of the diodesin the array is treated as an independent detector.4 Analytes areimaged onto the linear detector array which is aligned parallel tothe capillary. Averaging the electropherogram data from each ofthe 1500 diodes, after appropriate time shifts for successivereadouts, resulted in a signal-to-noise ratio 85 times that of anelectropherogram generated from any one diode in the array. Thelight source in this work was a single-wavelength 254-nm mercurypen ray lamp.

The key challenge addressed in our project is to increase thesensitivity of UV detection with conventional deuterium lightsources. Our objective is to establish the performance character-istics of an array detector for both single-wavelength and multi-wavelength UV absorbance, using a deuterium lamp as in mostHPLC and CE instruments. Current multiwavelength UV detectorsfor CE measure absorbance at a single spatial point, with spectral

(1) Xue, Y.; Yeung, E. S. Anal. Chem. 1994, 66, 3575-3580.(2) Moring, S. E.; Reel, R. T. Anal. Chem. 1993, 65, 3454-3459.(3) Djordjevic, N. M.; Stegehuis, D.; Liu, G.; Erni, F. J. Chromatogr. 1993, 619,

135-141.(4) Culbertson, C. T.; Jorgenson, J. W. Anal. Chem. 1998, 70, 2629-2638.

Anal. Chem. 1999, 71, 4376-4384

4376 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999 10.1021/ac990035l CCC: $18.00 © 1999 American Chemical SocietyPublished on Web 08/31/1999

information obtained by using a diffraction grating and eitherimaging onto a photodiode array5 or rapidly scanning across asingle photodiode.

Most absorbance detectors for CE are shot noise limited.6

Converting from radiometric to photon quantities, the photon flux(photons per second) in a light beam of radiant power I (watts)is given by Iλ/hc, where λ is the wavelength, h Planck’s constant,and c the speed of light. Photon emission is described well bythe Poisson process, where the probability of observing a numberof photons in a given time interval is given by the Poissondistribution. In this distribution the variance, σ2, is equal to themean, µ. The photon flux is then just µ. A perfectly stable lightsource will have a fundamental rms noise level, σ, that is equal tothe square root of the photon flux, µ; this is shot noise. If adetection system incorporates a light sensor with a quantumefficiency η, such that the mean signal current (photoelectronsper second) is ηµ, then the shot noise is given by σ ) (ηµ)1/2. Ofmore interest than the absolute noise level is the signal-to-noise ratio (SNR); the signal is proportional to µ; therefore theSNR is proportional to µ/σ ) µ1/2. The noise level in terms ofabsorbance ) -log(1 - 1/σ) AU, and when 1/σ is small, ∼0.43/σAU. For a shot noise limited absorbance detector with a givenpath length, the only way to improve the sensitivity is either touse a sensor with higher quantum efficiency or to increase thelight flux.

While standard HPLC UV detectors can be used for CE,significant improvements in terms of the efficiency of couplingthe light output from a discharge lamp through a capillary can bemade by using carefully aligned fast microscope objectives.6 Themost widely used designs incorporate a sapphire ball lens.2,7

However, there is still the fundamental limit that it is impossibleto produce an image that is brighter than the source.8 In the caseof a conventional lamp-based CE detection system, the source(usually a 30-W deuterium lamp discharge) has a diameter of ∼0.5mm with an area that is much larger than the target area (thecapillary detection zone), typically 0.075 × 0.2 mm. This meansthat most of the light from the lamp cannot be focused throughthe capillary detection zone. Increasing the power of the lamptends to increase the size of the discharge, not its brightness, sothis will not increase the light flux through the capillary.8 Theapproach adopted in our work is to increase the target area,enabling more of the lamp output to be used and thereforeincreasing the total light flux. A fiber-optic bundle is used with adeuterium lamp to illuminate a 2-cm length of capillary, which ismuch greater (∼100 times) that in a conventional system. Singlefibers have been used in CE systems to produce compact andflexible UV absorbance detectors.9,10 A round to line fiber-opticbundle has been used to increase light throughput for on-columnUV absorbance detection in high-temperature open-tubular liquid

chromatography;3 an improvement in SNR was observed but atthe cost of lost spatial resolution for this single-point detector. Tomaintain the high spatial resolution necessary for the high-efficiency separations that are characteristic of CE, this longdetection zone cannot be treated as a single point but has to beimaged. For a multiwavelength detector, a 2D imaging systemhas to used, with spatial resolution parallel to the capillary andspectral resolution perpendicular to the capillary. This requirementlimits the choice of detector to charge coupled devices (CCDs).

A CCD is essentially a silicon chip that is very sensitive tolight. When a photon is absorbed, a single electron is releasedthat is free to move about the silicon crystal lattice structure.Electrodes covering the CCD surface hold these electrons in placein an array of wells, or pixels, so that during exposure of the chipto light a pattern of charge builds up that corresponds to thepattern of light. Charge on the CCD is read out by shifting inparallel the charge held in all the columns of pixels up one row,so that the charge in the first row is transferred into a serialreadout register. The charge in the serial output register is shiftedout one pixel at a time into an output node for amplification anddigitization. This process is repeated until the whole CCD is readout to give a very accurate digital representation of the light image,leaving the CCD ready for the next exposure. There is consider-able flexibility in how the CCD readout is performed. The readouttakes less time if only the area of interest rather than the entireCCD image is digitized. A process called on-chip charge binningcan also speed up readout and improve the signal-to-noise ratio.Parallel binning is where the charges from adjacent pixels in acolumn are added together in the readout register; serial binningis where the charges from adjacent readout register elements areadded together in the output node. Binning reduces the spatialresolution of the CCD but also reduces the number of digitizations,making readout quicker and reducing the contribution of readoutnoise in the output. Readout noise levels are typically <10electrons, and dark currents are measured in terms of electronsper pixel per hour for cooled CCDs. These figures have enabledCCDs to be used to provide both spatial and spectral resolutionin LIF detection for CE and capillary isoelectric focusing.11-16

CCDs have not generally been considered as suitable forabsorbance detection due to the high light levels involved andthe low saturation levels of the devices.4,17 Typically a front-illuminated, UV-sensitive CCD with large pixels (e.g., 22 × 22 µm)will have a full well capacity of ∼5 × 105 electrons compared to∼5 × 107 electrons for a 25 × 2500 µm element of a photodiodearray.4 This means that with a quantum efficiency of 10% in theUV, an exposure is limited to ∼5 × 106 photons/pixel to avoidsaturation of the CCD. However, although the signal charge perreadout is low, the signal current (photoelectrons per second) islimited by the readout rate, and if fast enough will ultimately belimited by the light flux. For a given CCD camera, the highest

(5) Kobayashi, S.; Ueda, T.; Kikumoto, M. J. Chromatogr. 1989, 480, 179-184.

(6) Flint, C. D.; Grochowicz, P. R.; Simpson, C. F. Anal. Proc. 1994, 31, 117-121.

(7) Bruin, G. J. M.; Stegeman, G.; Van Asten, A. C.; Xu, X.; Kraak, J. C.; Poppe,H. J. Chromatogr. 1991, 559, 163-181.

(8) Arriaga, E.; Chen, D. Y.; Cheng, X. L.; Dovichi, N. J. J. Chromatogr., A 1993,652, 347-353.

(9) Foret, F.; Kirby, D. P.; Vouros, P.; Karger, B. L. Electrophoresis 1996, 17,1829-1832.

(10) Lindberg, P.; Hanning, A.; Lindberg, T.; Roeraade, J. J. Chromatogr., A 1998,809, 181-189.

(11) Sweedler, J. V.; Shear, J. B.; Fishman, H. A.; Zare, R. N. Anal. Chem. 1991,63, 496-502.

(12) Lu X. D.; Yeung, E. S. Appl. Spectrosc. 1995, 49, 605-609.(13) Timperman A. T.; Oldenburg, K. E.; Sweedler, J. V. Anal. Chem. 1995, 67,

3421-3426.(14) Wu, X. Z.; Wu, J. Q.; Pawliszyn, J. Electrophoresis 1995, 16, 1474-1478.(15) Nilsson, S.; Johansson, J.; Mecklenburg, M.; Birnbaum, S.; Svanberg, S.;

Wahlund, K. G.; Mosbach, K.; Miyabashi, A.; Larsson, P. O. J. CapillaryElectrophor. 1995, 2, 46-52.

(16) Quesada, M. A.; Zhang, S. P. Electrophoresis 1996, 17, 1841-1851.(17) Spectrum, 14th ed.; L.O.T. Oriel: Leatherhead, UK, 1998.

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possible signal current will be obtained if the full well capacity isachieved just before each readout. This means that typically theshot noise on each output will be (5 × 105)1/2 ≈ 700 electrons,which dwarfs any contributions from readout noise or darkcurrent, and 12-bit digitization will be more than adequate. Insummary, the ideal CCD camera for absorbance detection shouldhave a large full well capacity and be as fast as possible. A highreadout rate will enable the high sensitivity and dynamic rangerequired for a good detector to be acquired from the summationof many individual outputs.

The CCD-based detector can be considered as a series ofsingle-point multiwavelength detectors which provides improvedsensitivity in the final output by averaging of all the individualsignals. In recent work using a linear photodiode array detectorto provide a long detection zone, it was shown that obtaining thetotal signal over a long period of time has the advantage ofeffectively filtering out low-frequency noise which contains mostof the “excess noise”, for example, the lamp flicker noise.4 Anotherway to view the signal averaging is just as a means to increasethe total useful light flux, with each detector element adding tothe total number of photoelectrons collected and thereforeincreasing the shot noise limited SNR. The ultimate noiseperformance for any absorbance detector is always going to belimited by the total light throughput.

This paper outlines the design of a CCD-based absorbancedetector and the strategies for its use in CE, including on- andoff-line data processing and signal referencing to compensate forfluctuations in illumination. Comparisons of performance are madewith shot noise limits, in both single-wavelength and multiwave-length modes, and the benefits of the latter for peak deconvolutionare illustrated.

EXPERIMENTAL SECTIONCE Apparatus. The CE apparatus used was a homemade,

unthermostated system constructed from Perspex and usedplatinum wire electrodes. The inlet vial holder has a shield andinterlock to ensure safety; it also has a syringe and connectingtube that enables the pressure in the vial to be reduced (to ∼0.3bar) for capillary rinsing. The outlet vial holder has a groundedelectrode and is contained along with the detector hardware insidea light-tight enclosure. An electronic circuit was designed and builtto program a 30-kV high-voltage power supply (Glassman PS/MJ30P0400-22; Whitehouse Station, NJ.) that allows selectablevoltage with adjustable current limiting; when turned on itproduces a linear voltage ramp from 0 V to the set voltage (10-sramp time) and then holds the set voltage for the duration of theexperiment. Sample introduction was achieved hydrodynamicallyby having the liquid level in the sample vial higher than that inthe outlet vial, allowing the sample to siphon into the capillary.For example a 5-nL injection is achieved by having a the samplelevel 11 mm higher than the output level for 30 s. The capillariesused are polyimide-coated, fused silica 75 µm i.d., 363 µm o.d.(Composite Metals, Hallow, UK), 500 mm long and 400 mm tothe end of the detection zone. The detection zone is formed byremoving ∼20 mm of polyimide from the capillary using a flameand wiping with methanol.

Reagents. The Tris-borate buffer used for all separations wasmade by adding solid boric acid (Fisons, Loughborough, UK) andTrizma base (Sigma, Gillingham, UK) to water to give concentra-

tions of 43.9 and 51.7 mM, respectively. Other reagents used were2-, 3-, and 4-methylbenzoic acids and mesityl oxide (Aldrich,Gillingham, UK), methanol and sodium hydroxide (Fisher Sci-entific, Loughborough, UK), and 4-hydroxybenzoic acid (Sigma).The sample solutions of the methylbenzoates were made up byfirst dissolving the acid in ethanol at 10 mM and then dilutinginto sodium hydroxide solution of the appropriate concentrationto give the final methylbenzoate solution. The hydroxybenzoatesample solutions were made up by dissolving the solid acid directlyinto the appropriate sodium hydroxide solution.

CCD-Based Absorbance Detector. Figure 1 is a schematicdiagram of the CCD-based UV absorbance detector. Software,Zeemax Optical Design Program (Focus Software Inc., Tucson,AZ), aided in the design of the detector. The UV source consistsof a high-output deuterium lamp, power supply, and lamp housingwith F/1.8 imaging condensers (model 60060 with 63163 lamp,L.O.T.-Oriel Ltd., Leatherhead, UK) and is operated at 300 mA.The condensers (two fused-silica elements) couple the output fromthe lamp, through a light chopper (model 9479, EG&G, Wellesley,MA.) into the round end of a round to line fiber-optic bundle(CeramOptec GmbH, Bonn, Germany). The bundle is a collectionof ∼200 Optran UV 100/110 P fibers, 500 mm long; the roundend is an SMA 905 connector with a 1.96-mm drilling; the lineoutput has dimensions 25 × 0.1 mm. The fibers that make up thebundle have a numerical aperture of 0.22; the core diameter is0.1 mm, and the overall diameter is 0.125 mm, which means 58%of the round end area is active. A 0.6 mm diameter × 25 mmlength sapphire rod (Crystan Ltd., Poole, UK.) is positionedparallel to the output line at a distance of 0.4 mm using a brassshim and epoxy glue. For single-wavelength detection an interfer-ence band-pass filter (Beckman P/ACE part 359921 (200 nm) or359926 (260 nm), Beckman, High Wycombe, UK) is placedbetween the light source and the fiber bundle.

The capillary is mounted by first hanging a length verticallywith a weight attached to the end so that the detection zone (theportion with the polyimide removed) is exposed and straight. Thecapillary is then glued at either end of the detection zone to aframe; this frame can then be mounted on optical mounts to enableprecise positioning of the capillary parallel to and almost touchingthe sapphire rod. The light that emerges from the fiber bundle isfocused by the sapphire rod, the linear analogue of a sapphireball lens used in single-point detectors, through the capillarycenter as shown in Figure 2: then folded by a flat mirror (part340093, Spindler & Hoyer, Milton Keynes, UK) onto a holographicimaging grating (Jobin Yvon, part 523-01-030, Instruments SA,

Figure 1. Diagram showing the layout of the multiwavelengthdetector. For single-wavelength detection, the light chopper is ef-fectively replaced by a 10-nm band-pass interference filter.

4378 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

Stanmore, UK) designed for use in an imaging spectrograph. Thegrating is 66 mm in diameter, disk shaped with spherical curvatureof 192 mm radius (F/2.9). The layout of the grooves, spaced at∼200/mm, has been computer optimized to produce a 1:1 imageof an entrance slit (in our case the capillary detection zone) ontoa plane surface (the CCD) and to include a high degree ofcorrection for spherical aberration. The dispersion is ∼25 nm/mm and the grating has a wavelength range of 190-800 nm. TheCCD camera (Antares Duo, LSR AstroCam, Cambridge, UK) isfitted with a front-illuminated chip (EEV 05-20 CCD) that has770 × 1152 active pixels, each 22.5 µm square; there are 24columns of inactive pixels at each edge of the CCD, making thewidth 818 pixels in total. The chip has a UV phosphor (Astro-chrome 90); the quantum efficiency is ∼10% in the range <100-480 nm, which covers the useful range of the lamp of 180-400nm. The CCD has long columns and short rows with the serialoutput register on the short edge on the chip and aligned parallelto the capillary. The camera head cools the chip thermoelectricallyto -40 °C and has a shutter. The camera controller is interfacedto a PC (Pentium 150 MHz, 64 MB RAM, Dan TechnologyComputers, London, UK) via a fast serial link. Maximum pixelreadout rate is 165 kHz at 16 bit. The controller also allowsconsiderable flexibility over on-chip charge binning procedures.The PC is used to control the camera readouts and to collect,store, and analyze the data. The software was written in house inC to run under DOS, using Microsoft Visual C++, includingroutines that were supplied with the camera system to controlthe camera and routines from Numerical Recipes18 to analyze thedata.

RESULTS AND DISCUSSIONIrradiance. The illumination of the CCD is shown in terms

of peak irradiance (the brightest part of the detection zone) as afunction of wavelength in Figure 3. The number of photoelectronsof charge was measured after a single 8.3-ms exposure of the CCD;the irradiance was then calculated by assuming the manufacturers’stated 10% quantum efficiency. The variation of irradiance withwavelength may be accounted for by the spectral distribution ofthe lamp and the wavelength dependence of transmission of thevarious optical elements and chromatic aberration of the condens-ers and the sapphire rod. The last factor allows the shape of thecurve in Figure 3 to be adjusted to some extent by changing thefocus of the condensers and the distance between the sapphirerod and the capillary.

Strategy for Using the CCD. It was found for this CCDcamera system that the full well capacity of the individual pixels,readout register elements, and output node were all very similar(5 × 105-6 × 105 e-). This means that there is no advantage interms of increased signal current (photoelectrons per second) byperforming on-chip charge binning. The size of the CCD meansthat 17.3 mm of the capillary is imaged. However, only the center11.4 mm (508 pixel columns) was used on account of its higherimage quality. The wavelength range chosen was ∼200-300 nm,which corresponds to ∼4 mm (180 pixel rows) on the CCD; thismeans that the area of interest is 508 × 180 pixels. Although onlythe area of interest needs to be digitized, most of the CCD isexposed to light to some extent during an exposure in multiwave-length mode, requiring the charge from the whole CCD to beshifted out after each exposure (Figure 4A). This could be avoidedif a mask could be laid onto the CCD surface so that only thearea of interest was exposed. However, the camera used here hasthe CCD chip mounted ∼13 mm below a fused-silica window,which prevents the efficient use of a mask. To minimize thereadout time, it is advantageous to minimize the number of shiftsof the serial readout register. This is achieved by maximizing thenumber of parallel shifts per serial discard when the charge isshifted from the portion of the CCD that is not digitized; the limitwas found to be 20, above which the charge accumulating in thereadout register overflows onto the rest of the CCD. Approximatetimings for reading out the CCD are 100 µs for a parallel shift, 3µs for a serial shift without digitization, and 6 µs for a serial shiftwith a digitization. These timing figures were used to work out asuitable binning strategy to give a suitable exposure rate, as shownin Table 1. Although it would have been possible to have anexposure time of 11 ms, it was convenient to use one slot of a30-slotted light chopper to give a 1.7% duty cycle (8.3-ms exposuresat 2 Hz). During an experiment, the camera shutter is left openand the light chopper is interfaced to the computer so that areadout is requested from the camera immediately after eachexposure.

For single-wavelength detection when an optical band-passfilter is used, the grating produces an image of the detection zonewith a narrow dispersion. Only a few pixel rows are illuminatedand the rest of the CCD remains unexposed. This means that foreach snapshot only the first few pixel rows need to be read out,

(18) Press: W. H.; Flannery, B. P.; Teukolsky, S. A.; Vetterling, W. T. NumericalRecipes in C; Cambridge University Press: Cambridge, UK, 1988.

Figure 2. Ray tracing diagram showing how the light is focusedthrough the capillary center by the sapphire rod. Paths of rays forlight at 250 nm emerging from two points of the fiber are shown.

Figure 3. Irradiance of the CCD plotted in both photon andradiometric quantities as a function of wavelength. A 20-mm portionof capillary is illuminated, and the dispersion is ∼25 nm mm-1.

Analytical Chemistry, Vol. 71, No. 19, October 1, 1999 4379

resulting in all the charge on the chip, including that accumulatedin the exposed portion, being shifted a few pixels in the directionof the readout register. In this way, the area of the CCD betweenthe exposed region and the readout register acts as temporarystorage for the snapshot information until it finally reaches thereadout register (Figure 4B). The readout time is very muchreduced, partly because the area of interest is much smaller (fewerdigitizations) and partly because it is not necessary to shift outthe charge from the whole CCD (Table 1). The total lightthroughput for the 200-nm filter is 24% of the light measured withno filter at 200 nm in a 5-nm bandwidth; similarly it is 43% for the260-nm filter. The reduced irradiance when the filters are used,coupled with the shorter readout time, allows the light chopperto be left open; an exposure rate of 10 Hz was used in a 100%duty cycle.

Data Processing. The procedure for carrying out a separationis to initiate the program that controls the camera, so that theexposure/readout cycles are in progress but with the camerashutter closed. The separation voltage is turned on and thecomputer sets migration time equal to zero halfway through theinitial voltage ramp; the camera shutter is opened 15 s into theseparation. In multiwavelength mode with 3 × parallel on-chipcharge binning, each snapshot produces an array of 508 × 60unsigned integers (2 bytes); further binning is carried out insoftware, as the data are generated, to produce a 127 × 20 arrayof effective pixels (each the sum of 36 physical pixels) representingspatial and spectral resolution of 100 µm and 5 nm, respectively.This array is stored in RAM as 3 bytes per effective pixel plus 3bytes for the current time; this gives a data rate of ∼1 MB min-1

and allows for >1 h of continuous data collection. Similarly forsingle-wavelength detection, a 508 × 14 array is reduced by thesoftware in real time to a 127 × 1 array, and the data rate is ∼230kbytes min-1. On completion of the separation the data are savedfrom RAM to the hard disk.

The rest of the data processing to produce an electrophero-gram is carried out postrun. The raw data are first corrected forfixed pattern noise (time invariant), uneven illumination, pixelresponse, and dark signal values. The most significant of these isthe uneven illumination. Figure 5 shows the number of photo-electrons for each effective pixel for one 8.3-ms exposure. Theunevenness in the spatial dimension is due to the fiber ends beingunsatisfactorily polished. Dark signal values for each effective pixelare obtained from the first 15-s (shutter closed) portion of thedata, and the pixel response and illumination pattern are takenfrom a portion of the data with the shutter open but with noanalytes in the detection zone. All data have the dark signal valuessubtracted and are normalized for uneven illumination and pixelresponse. An electropherogram is then constructed from thenormalized snapshots to produce the result that would have beenobtained if a single-point detector had been used, placed at thefurthest end of the detection zone and with a user-defined samplerate.

The normalized snapshot data comprises an array containingthe experiment time for each snapshot, T[j] for j ) [0, td × er -1], where td is the experiment duration and er is the exposurerate (2 or 10 Hz), and, for each wavelength, a two-dimensionalarray of signal data, S[i][j] for i ) [0, n - 1], where n is thenumber of effective pixels imaging the capillary detection zone.The resulting electropherogram is an array, E[k] for k ) [0, td ×sr - 1], where sr is the simulated data sampling rate for theelectropherogram. For each snapshot, the value of the effectivepixel, S[0][j], is added to E[T[j] × sr] (this effective pixel simulatesthe single-point detector at the end of the detection zone). Thetime taken for an analyte imaged by the effective pixel, S[i][j], toreach the end of the detection zone is given by ilpT[j]/(l - ilp),where l is the capillary length to the end of the detection zoneand lp is the length on capillary that is imaged by one effec-tive pixel. Therefore, S[i][j] is added to E[(T[j] + [ilpT[j]/(l - ilp)])sr]. The process is carried out for all S[i][j] and repeatedfor each wavelength. A record is made to keep track of the numberof contributions that have been made to each E[k] so that thefinal array can be normalized.19 The data can be expressed interms of absorbance, log(E[b]/E[k]), where b is chosen so thatE[b] represents part of the electropherogram where there are noanalyte peaks. Savitsky-Golay smoothing20 is used on the datapoints produced to simulate the desired detector speed of response(rise time). The size of the finished electropherogram files is nolarger than those from a conventional single-point detector andwill be determined by the number of wavelengths and thesampling rate.

Baseline Noise Levels. Figure 6 is the baseline at 250 nmobtained using the detector in multiwavelength mode with a water-filled capillary. Without exposure normalization, trace a, the noiseon the baseline, ∼0.2 mAU rms, is much higher than thecontribution from shot noise. Other noise sources include flickernoise in the lamp output and instabilities in the exposure timing.The frequency dependence of the lamp output noise was measuredusing a single UV-enhanced photodiode (Centronic OSD5.8-7Q,RS Components, Corby, UK) and a lock-in-amplifier (Stanford

(19) Pokric, B.; Allinson, N. M.; Bergstrom, E. T.; Goodall, D. M. J. Chromatogr.,A 1999, 833, 231-244.

(20) Savitzky, A.; Golay, M. J. E. Anal. Chem. 1964, 36, 1627-1639.

Figure 4. Schematic showing charge accumulation and readoutprocedure for one snapshot for (A) multiwavelength and (B) single-wavelength modes. (i) Charge accumulation during exposure. (ii) Areaof interest is read out and digitized (charge from a previous exposurein the single-wavelength case). (iii) The remaining charge is clearedready for the next exposure; this is only necessary in the multiwave-length case.

4380 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

Research Systems SR850, Speirs Robertson Ltd., Bedford, UK)in the absence of the light chopper. Figure 7 shows noise plottedas a function of inverse frequency. The 1/f dependence of thenoise evident at frequencies below ∼0.5 Hz is characteristic offlicker noise. The noise spectrum was also measured with thelight chopper present and fitted with a 90 slotted blade. The resulthad large components at the blade rotation frequency and itshigher harmonics, suggesting that the chopper was also contribut-ing to the total noise observed in Figure 6.

Compensation for Illumination Fluctuations. Variations ofirradiance with time due to instability in lamp intensity and lightchopper speed (multiwavelength) or readout timing (single-wavelength mode) are not corrected for in the data processingdescribed above. For a multiwavelength electropherogram, it may

be possible to select a wavelength at which none of the analytesabsorb and use this as a reference for all the other wavelengths.This will totally correct for chopper instability and correct for anywavelength-independent lamp instability. Trace b in Figure 6 wasobtained by referencing the data displayed as (a) to the output at295 nm.

Figure 8 shows the UV absorbance spectrum obtained usingthe detector described here by filling the capillary with a 5 mM4-hydroxybenzoate solution. In this case, the software binningstrategy was altered to give a higher spectral resolution of ∼1.7nm (3 pixels), which is the limit governed by the inner diameterof the capillary. Figure 9A is the electropherogram obtained froma 5-nL injection of 10 µM 4-hydroxybenzoate, constructed byreferencing the absorbance to that at 295 nm.

Table 1. Strategy for Using the EEV 05-20 CCD Chip When Used in Multiwavelength and Single-Wavelength Modes

total area 818 (770 active) columns × 1152 rows of 22.5 × 22.5 µm pixelsserial register full well capacity >5 × 105 e-

detection mode multiwavelength single wavelength (260 nm)area of interest 508 × 180 pixels 508 × 28 pixelsbinning 3 × parallel 2 × paralleltime to readout area of interest 200 ms 45 mstotal readout time 470 ms 58 mspeak irradiance 1.5 × 108 photons pixel-1 s-1 2.5 × 107 photons pixel-1 s-1

saturation time 11 ms 100 msbest possible duty cycle 2.3% 100%duty cycle used 1.7% (8.3-ms exposures at 2 Hz) 100% (100-ms exposures at 10 Hz)

Figure 5. Signal charge accumulated in each effective pixel (36physical pixels) during one 8.3-ms exposure.

Figure 6. Baseline measured using the multiwavelength detectorwith water in the capillary. Output at 250 nm is shown. Data havebeen processed (a) without any exposure normalization and (b) usingthe output at 295 nm as a reference.

Figure 7. Deuterium lamp noise measured using a single UV-enhanced photodiode and plotted as a function of 1/frequency. Thestraight line highlights the 1/f dependence, at low frequency, due toflicker noise.

Figure 8. UV absorbance spectrum of 4-hydroxybenzoate mea-sured by filling the capillary with a 5 mM solution and using themultiwavelength detector. The molar absorption coefficient measuredusing a 1 cm square cell and spectrophotometer has been used tocalculate the apparent path length of the capillary detection zone.

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The disadvantage of this method of exposure normalization,using a second wavelength as a reference, is that there needs tobe a wavelength at which the analyte or analytes do not absorb.In addition, it does not correct for lamp instability that is notcorrelated across the wavelength range used. To allow forexposure normalization for each wavelength individually, the datafrom each snapshot can be split into two sets, each having thefull spectral range but representing spatially distinct portions ofthe detection zone. One portion is used to produce the electro-pherogram and the other is a reference. For every snapshot thesignal charge in the reference portion is totaled for eachwavelength and then used to normalize all the pixels of thecorresponding wavelength. This procedure is carried out aftercorrection for fixed pattern noise and before the integration ofthe snapshots to produce the final electropherogram. For thismethod to work, the position on the CCD from which thereference portion is taken must be selectable for each snapshotso that it always avoids the position of any analyte peaks. This isachieved by first producing an electropherogram using the methodof having a reference wavelength so that the position of anyanalytes can be precisely measured. As long as all the peaks aresufficiently narrow and well spaced, the same raw data can thenbe reprocessed as described above so that the reference avoidsall peaks. Figure 9B uses the same data set as Figure 9A, butthis time 12 of the 127 pixels, for each wavelength, have beenused as a reference. The improvement in baseline quality compar-ing (B) with (A) is evident, with a reduction in baseline noise of

a factor of ∼2 by using spatial referencing rather than wavelengthreferencing.

The linear dynamic range of the detector in multiwavelengthmode has been investigated by filling the capillary with solutionsof 4-hydroxybenzoate at concentrations of 0.1, 0.3, 1, 3, 10, 30,and 100 mM. The range 200-300 nm was investigated in 5-nmsteps. The absorbance measured was found to scale linearly upto 0.3 AU with concentration giving correlation coefficients of>0.999 at all wavelengths. The presence of stray light means theresponse to increasing concentration is reduced above 0.3 AU,limiting the linear range of the detector at high absorbances. Thelinear range extends from 10-4 (3 × rms baseline noise) to ∼0.3AU. The lower noise levels obtained in single-wavelength modeextend the linear dynamic range to 4 orders of magnitude.

Figure 10 is the single-wavelength electropherogram of 5 nL,1 µM 4-hydroxybenzoate using a 200-nm filter. The increasedphotoelectron throughput achieved in comparison to the multi-wavelength mode has reduced the shot noise relative to the signal,which is reflected in the greater sensitivity of this mode ofoperation. With an rms noise of 8 µAU, and absorbance at thepeak maximum of 90 µAU for the 1 µM analyte, it is evident thatsubmicromolar LODs are possible using the criterion of LOD of3 × SNR. This is obtained without compromising the spatialresolution. The measured peak width gives an efficiency of 2.5 ×105 theoretical plates. A simulation using the program HPCESIM21,22

with the experimental conditions used here predicts 2.2 × 105

theoretical plates, which agrees well with the measured value.Comparison with Shot Noise Limits. The theoretical shot

noise limit of this detector was calculated numerically. A syntheticraw data set was produced in which the number of photoelectronsfor each effective pixel for each snapshot was obtained from arandom number generator. For each effective pixel, a normaldistribution of numbers was produced with mean, µ, equal to themeasured value and variance equal to the mean. For multiwave-length mode, the measured values for the means were taken fromFigure 5. The data set produced is a good simulation for the casewhere the only noise source is photon shot noise, since the normaldistribution with σ2 ) µ is a good approximation of the Poissondistribution when µ is large. The simulated data sets were then

(21) Reijenga, J. C.; Kenndler, E. J. Chromatogr., A 1994, 659, 403-415.(22) Reijenga, J. C.; Kenndler, E. J. Chromatogr., A 1994, 659, 417-426.

Figure 9. Spectrally resolved electropherogram of 10 µM 4-hy-droxybenzoate. Injection, 5 nL of sample in water. Buffer, Tris-borate,pH 8.2, ionic strength 10 mM. Separation voltage, 15 kV. (A) Exposurenormalization carried out using the output at 295 nm as a reference.(B) Data reprocessed using 12 of the 127 spatially resolved pixelsfor each wavelength as a reference for exposure normalization.

Figure 10. Electropherogram of 5 nL of 1 µM 4-hydroxybenzoate(separation conditions as for Figure 9) using the detector in single-wavelength mode with a 200-nm filter. Peak width at half-height is2.0 s, which gives an efficiency of 2.5 × 105 theoretical plates.

4382 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

processed identically to the experimentally obtained data and therms noise measured from the baseline.

Table 2 lists noise levels obtained for the various experimentalmodes of operation and compares them to the shot noise limit.When exposure normalization is carried out, the signal obtainedis the result of taking the ratio of two unreferenced signals,resulting in the attenuation of noise that is correlated betweenthe two signals (e.g., lamp flicker noise). The uncorrelated noise(e.g., shot noise) associated with these two signals, σ1 and σ2,propagates to give (σ1

2 + σ22)1/2. This increase is apparent when

the shot noise limited figures listed in Table 2 are compared withand without referencing. Comparison of shot noise limits at 200

and 260 nm shows that, while the shot noise limit is better at 260nm than at 200 nm in both multiwavelength and single-wavelengthmodes, the difference is more pronounced in the case of the single-wavelength mode. This is accounted for by greater lamp outputand higher transmittance at 260 nm. With spatial referencing,calculated shot noise values at 200 and 260 nm are 35 and 28 µAU(multiwavelength) and 7 and 3 µAU (single-wavelength), respec-tively. The experimentally obtained noise levels for the multi-wavelength electropherograms are limited by chopper speedinstabilities if no referencing is carried out using exposurenormalization. Using one wavelength to reference all the othersgives a much better noise performance but is still not shot noiselimited. However, shot noise limits are achieved in multiwave-length mode when each wavelength has its own reference (34and 29 mAU at 200 and 260 nm, respectively). In the single-wavelength mode, a performance limit of ∼6 µAU rms noise isreached, making detection at 200 nm shot noise limited but afactor of 2 higher than the theoretical value at 260 nm. Baselinenoise levels attained using commercial instruments under similarconditions are 15 µAU rms for the Beckman P/ACE 2050 single-wavelength detector with a 200-nm filter and 10 µAU rms for theHewlett-Packard HP3DCE using the UV diode array detectorcollecting data at 260 nm. The limit of 6 µAU found with the CCDsystem could not be improved on by removing the capillary, whichsuggests that the source of this residual noise is either the lightsource or the camera. One possible cause is fluctuations in the

Figure 11. (A) Electropherogram of a mixture of 100 µM each of 2-, 3-, and 4-methylbenzoate in water. Injection, 5 nL (separation conditionsas for Figure 9). Output at 230 nm from the multiwavelength detector. (B) UV absorbance spectra of 2-, 3-, and 4-methylbenzoate measured byfilling the capillary with 0.5 mM solutions for each of the compounds. (C) UV absorbance spectra taken from the maximums of each of the threepeaks numbered in (A). Assignment of these spectra and the peaks in (A) made by reference to the spectra obtained for (B). (D) Result of aleast-squares fit of a linear combination of the spectra obtained for the three compounds to the full wavelength-resolved electropherogram toestimate the concentration of each of the compounds at each point along the electropherogram. The output obtained for each compound hasbeen scaled to give the predicted absorbance at 230 nm for comparison with (A).

Table 2. Noise Performance of Detector, Measured AsIf for an Analyte Migrating at 1 mm s-1 and Using a 1-sRise Time

detection mode

multiwavelength single-wavelength

200 nm 260 nm 200 nm 260 nm

rms Shot Noise Limit Measured from Simulated Data Set/µAUno referencing 27 20 5 2wavelength reference 36 30spatial reference 35 28 7 3

rms Noise Measured from Electropherogram/µAUno referencing 210 163 120 115wavelength reference 52 61spatial reference 34 29 8 6

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spatial distribution of the deuterium discharge,23 which would bereproduced in its image on the fiber bundle. Placing an opticalscrambler on the input to the fiber bundle should reduce suchnoise.

Using the Spectral Information. A particular advantage ofmultiwavelength rather than single-wavelength detection is in peakidentification and resolving overlapped or hidden peaks. Figure11A is an electropherogram showing the separation of 2-, 3-, and4-methylbenzoates; the output at 230 nm is displayed from themultiwavelength detector. Figure 11B shows the spectra obtainedby filling the capillary with 0.5 mM solutions of these compounds.The spectra obtained from the electropherogram (Figure 11A) atthe peak maximums are shown in Figure 11C; this makes thepeak identification very easy. It is possible to carry out a least-squares fit to a linear combination of the spectra obtained for thethree compounds to the full wavelength-resolved electrophero-gram in order to estimate the concentration of each of thecompounds at each point along the electropherogram. The resultof carrying out this procedure using the method of singular valuedecomposition18 is shown in Figure 11D, with scaling to give thepredicted absorbance at 230 nm for comparison with Figure 11A.

CONCLUSIONSThis paper has demonstrated the practicability of use of a CCD

array for UV absorbance detection, in both single-wavelength andmultiwavelength modes. A two-dimensional imaging system isused, with spatial and spectral resolution parallel and perpendicu-lar to the capillary, respectively. Use of a round-to-linear opticalfiber bundle enables more of the deuterium light flux to be used

than would be possible with a conventional single-point detectorin CE. Strategies for optimal use of the CCD, data processing,and baseline noise reduction are outlined. The electropherogramsare built up by addition of successive snapshots of the analytebands progressing across the central 11 mm of the field of viewof the grating, without sacrificing any image quality and retaininghigh peak efficiency (>2 × 105 theoretical plates). Measured rmsnoise values at 200 nm of 8 and 34 µAU in single-wavelength andmultiwavelength modes accord with shot noise limits.

The present constraint of relatively low readout rate inmultiwavelength mode should be overcome in future work byusing the new generation of fast (MHz) cameras and someimprovements to the optics, and we envisage a shot noise limit inmultiwavelength mode of 1-2 µAU rms using a 1-s rise time foran analyte migrating at 1 mm s-1. This would represent asignificant improvement on the current generation of multiwave-length UV absorbance detectors and would make nanomolarLODs for many UV-absorbing analytes feasible without precon-centration. Such imaging UV detectors would also be eminentlysuitable for application in microbore HPLC and in hyphenationwith electrospray ionization mass spectrometry could provide apowerful resource for characterization and quantification ofanalytes in complex mixtures.

ACKNOWLEDGMENTThis work has been supported by EPSRC through their

Analytical Science Program, Grant GR/L01657. We also thank Dr.Craig Mackay at LSR AstroCam for helpful discussions.

Received for review January 14, 1999. Accepted June 21,1999.

AC990035L(23) Grochowicz, P. R. Ph.D. Thesis, Birkbeck College, University of London,

UK, 1997.

4384 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999