application ce

8/7/2019 application CE

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1926 Anal. Chem. 1992, 64, 1928-1932

Capillary Electrophoresis and Sample Injection SystemsIntegrated on a Planar Glass ChipD. Jed Harrison,*l+J Andreas Manz,**s Zhonghui Fan,$ Hans Ludi) and H. Michael Widmers

DeDartment of Chemistry, Universi tyof Alberta, Edmonto n, Alberta, Canada T6G 2G2, and Forschung Ana lytik,Ciba Geig y, CH 4002 Basel, Switzerlhnd

The feadblllty of mlnlaturlzlng a chemlcal analyrls system ona planar substrate has been demonstrated for a system utHlzIngelectroklnetlc phenomena for sample upara tlon and solventpumplng. Udng mlcromachlnlng technlques, a complexmanlfold of caplllary channels has been fabrlcated In a planarglass substrate and the uparatlo n of a mixture of fluoreocelnand calceln wlthln the channels was achleved udng elec-trophoresk. The maxhnum number of theoretlcal platesobtalned was about 35 000 for calceln, wlth 5000 V applled,correrpondlng to 2100 V between the Injectlon and fluores-cence detectlon polnts In the channels. The number oftheoretical plates observed was In agreement wlth theory,lndlcatlng no lnteractlons between the analyte and the glasswalls. The electrmotlc flow rate In the glass channels was(4.5 f 0.1) X lo-' cm2/(V*s)udng a pH 8.5 50 mMborlc add,50 mM Trls buffer, comparable to (5.87 f 0.08) X lo-' cm2/(V.8) measured In fused-rlllca caplllarles. Solvent flow couldbe dlreded along a speclfled caplllary by appllcatlon ofapproprlate voltages, so that valveless swltchlng of fluld flowbetween caplllarks could be achleved. These results providea foundatlon for the dedgn of more complex sample treatmentand separatlon systems Integrated on glass or sillcon sub-strates.

Most successful analyses in the laboratory involve acomplete system of sample treatment, separation, andanalysis, designed to circum vent the com plexities of a sam pleand its matrix. These methods are often time consumingorlabor intensive. T o overcome this, the analysis process maybe a utomated , increasing its speed, precision, and reproduc-ibility. Th e use of flow injection analysis (FIA ), and itscoupling to separation methods suchas gas or liquid chro-matography, or selective chemical sensorsis one route toachieve this. High levels of autom ation have resulted in totalchemical analysis systems (TAS) that can be used to monitorchemical conc entrations continuously in industrial chemicalan d biochemical processes.1-3 T he m iniaturization of aTASonto a monolithic structure could produce a device (ap-TAS)tha t would resemble a sensor in many wa y~ .~ ,5Such a devicecould be configuredas a d ip-type probe, giving out a readingfor the analyteof interest, so th at i t behaved as a sensor fromthe perspective of the user. Sepa ration methods such as liquidchromatograph y an d capillary electrophoresis, as well as other

* Authors to whom correspondence should be addressed.+ Research performed while on leave at Ciba Geigy, Basel.

University of Alberta.$ Ciba Geigy.(1) Graber, N.; Ludi, H.; Widmer, H. M. Sens. Actuators 1990, B1,

239-243.(2) Gisin, M.; Thommen, C. Anal . Chim. Acta 1986,190 , 165-176.(3) Garn, M.; Cevey, P.; Gisin, M.; Thommen, C. Biotechnol. Bioeng.

(4) Manz, A.; Graber, N.; Widmer, H. M . Sens. Actuators 1990,B l ,

( 5 )Manz, A.;Fettinger, J. C.; Verpoorte, E. ; Ludi,H.; Widmer, H. M.;

1989, 34, 423-428.

244-248.

Harrison, D. J. Trends Anal. Chem. 1991, 10, 144-149.

0003-2700/92/0364-1926$03.00/0

bench-top analytical approaches such as FIA may also benefitfrom the p-TAS approach. It has been made clear tha t smallerdimensions result in improved performancefor these ana-lytical methods.6-9 Th e benefits of miniaturization , though ,are complicated by problems of detection an d dead volume sassociated with coupling capillaries to de tectors an d injectors.Several authors have noted t ha t th e use of microlithographictechniqu es to fabricate system s would be beneficial.*ll Th eease of fabrication of small structures should facilitatecoupling of capillary separation systems to each other for2-dimensional separa tionsor to injectors and detectors, withminim um dead volume. Increa sed speed of analysis, de-creased sample and solvent consumption, or increased de-tecto r efficiency could also be realized, as we have discussedin d etail elsewherea4v5

Microma chining of siliconor other planar materials pro-vides a path to dev elopm ent of liquid-ph ase pT A S devices.12Th e combination of microlithiography with isotropic and an -isotropic etc hing technique s, as well as controlled thin-filmdeposition, allows for the fabrication of micron-scale,3-dimensional structures.l3--'8 Terry et al.13 developed a gaschromatograph on a silicon wafer, but there have beenrelatively few extensions of th is technology to solution-phasesystems. Bergveld's group has designed micron-scale cou-lome tric titratio n systerns,lgand Shoji e t al.20have developeda dissolved02 sensor based on a microm achined device. Bothof these systems are based on the pH-sensitive field effect

transistor ( pH FE T) but, because of their integrated systemapproach, offer better performance tha n the stand-alone pHFE T does. A micromachined liquid chrom atograph has beenreported and the theoretical behavior of such a systemdiscussed, but no d ata from the system has been presented.21

Capillary electrophoresis (CE ) is a separation m ethod th atcould be coupled withFIA on a planar substrate to explore

(6 ) Small Bore Liquid Chromatography Columns: Their Properties

(7 ) Micro-ColumnHigh Performance Liquid Chromatography; Kucera,

(8) Microcolumn Separations: C olumns, Instrumentation and A ncillary

(9) van der Linden, W. E. Trends Anal . Chem. 1987,6 , 37-40.(10) Ruzicka, J.; Hansen, E. H. Anal . Chem. Acta 1984, 161, 1-10.

(11) Monnig, C. A .; Jorgenson, J. W. Anal. Chem. 1991,63,802-807.(12) Petersen, K. E. Proc. IEEE 1982, 70 , 420-457.(13) Terry, S. C.; Jermon, J. H.; Angell, J. B. IEEE Trans. Electron.

(14) Muller, R. S. Sens. Actuators 1990, A21, 1-8.(15) Esashi, M.; Shoji, S. ; Nakano, A. Sens. Actuators 1989,20,163-

and Uses; Scott, R. P. W., Ed.; Wiley: New York, 1984.

P., Ed.; Elsevier: Amsterdam, 1984.

Techniques. J. Chromatog. Libr. 1985, 30.

Deuices 1979, ED-26, 188 0-1886.

169.(16) Fan, L.-S.;Tai,Y.-C.;Muller,R.S. IEEE Trans.Electron.Deuices

(17) Sato, K.; Kawamura, Y.; Tanaka, S.; Uchida, K.; Kohida, H. Sens.

(18) Kittisland, G.; Stemme, G.; Norden, B. S e w. A c tu a to rs 1990,

(19) Olthus, W .; van der Scho ot, B. H.; Bergveld, P. S e w. A c tu a to rs

(20) Shoji, S.; Esashi, M.; Matsuo, T. Sens. Actuators 1988 ,14, 101-

1988, ED-35, 724-730.

Actuators 1990, A21, 948-953.

A21,904-907.

1989, 17 , 279-283.

1 n7*"..(21) M anz, A.; Miyahara, Y. ; Miura, J. ; Watanabe, Y .; Miyagi,H.;Sato,

K. Sens. Actuators 1990, B l, 249-255.

0 1992 American Chemical Society


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ANA1

00 0

O Sample 0

@W'SeparationChannelP

Figural. Layoutofthechannelsineplanarglasssubstrate.Channelsreferred to in me text are identifledby number (filled circles). a s ar emeinletpolnts (resewoh)to each channel(opencircles). Each channelis labeledwlth Its contentor Its function. Overall dlmensions are14.8cm X 3.9 cm X 1 cm thlck. T h e location of one pairof Pt electrodesis also shown:for clarityt h e others are not. The pointof fluorescencedetection is marked by an arrow.

the p-TAS concept, and thi s paper examines the feasibilityof doing so. There are several reasons for selecting thiscom bination of methods.s.22 FIA , of cou rse, prov idesaconvenient means of automating sample workup prior toinjection intoa separation systemor dete ctor coil. Cap illaryelectrophoresis, in which th e driving force is an elec tric field,has'proven to be a powerful separation method.23 Th ere ar etwo phenomena involved th e solvent and solutes all migratedu e toelectroosmoticmotion oft he solvent, whichisgeneratedwithin th e Helmholtzlayer near theu sually negatively chargedwalls of th e capillary, while the ions are ad ditionally drivenby migration in the electric field. Th e ions are separated d ueto the differences in their electrophoretic mobilities (migrationrates). Th e electroosmotic flow rate is usually larger th anthat ofelectrophoreticmigration, so t ha t all thesamplemovesin one direction. Because of electroosm otic flow, appliedvoltages may be used to pu mp fluid ina flow injection pre-treatm ent system,as wellas to induce separation ina coupledelectrophoresis capillary. Electroosmotic pum ping is wellsuited to the p-TAS concept, since the flow ra te of solvent iscontrolled by electrokinetic effects th at are approximatelyindepen dent of capillary dimensions. In c ontrast, methodsutilizing more conventional pum ps develop extremely highhack-pressures with small capillary dimensions and are notwell suited to delivery of suc h low volume^.',^

By m icromachininga complex manifold of flow chan nelsin a planar suhstrate, i t is possibleto fabricate a network ofcapillaries capable of sample injection, pretreatment, andseparation. We have recently described the design of sucha system.5 T o understand w hat factors playa role in theperformance of sucha device, it is useful to consider here th emodes of operation envisionedto effect a n analysis. Figure1 shows a layout of a simp le device for sam ple injection an dseparation th at has been fabricated to test th e conceptsdiscussed above. It consists of inlets, or reservoirs,at th eheads of three interconnected capillary chan nels. An inletto a fourth channel is located near the intersection of the

channels. As conceived, the application ofa voltage betweenan y two inlets should cause electroosmotic pum ping of fluidalong those channel segments hetween the inlets. Valvelessswitching of fluid flow between channels sh ould he achievedby switching the voltages applied to each channel. Forexample, voltage applied between inlets2 and 4 should dra wsample into the channel and past the intersection point.Subseq uent application ofa voltage between inlets1 and 3should then drivea small plug of sample along channel3,effecting electrophoretic separation of th e sample plug.

~~ ~

(22, Gale. R . J.: G h w w. K. In Biosensor Terhn oloyy. Fundomenlo l ian d Applications; Ruck. R . P.. Hatfield. W . E. , Umans. M , Huwder. E.F.. Eds: Mereel Dekker: New Vork. 1990: DU S S 6 2 .

' (23) Ewing, A. G.: Wallingford, R.'A.;Ol&


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1928 ANALYTICALCHEMISTRY.VOL. 64, NO. 17, SEPTEMBER 1. 1992

pedance amplifier, and th e signal was recorded with a P hillipsdual-pen strip chart recorder. Th e glass structur e was mountedon an x-y translationstage toallow fine positioningadjustme nts.The d etection point was6.5 cm from the intersec tion point alongchannel 3 (see Figure1).

A Spectra Physics electrophoresisunit equipped with an ah-sorhance detector was used for conventional capillary el ec trophoresis measurements. A 45-cm-long, 75-pm4.d. fused-silicacapillary was used with atotal app lied voltage of14.8 kV. Thedistance to the detec tor was38 cm. The nH 8.5 boric acid.Trisbuffer was used and a current of 15p A &as obtained. Samplewasinjectedoveral-speriodbyahydrodynamicmethod. Sampleconcen trations were M

The diffusion coefficient of1 mM fluorescein in pH8.5 bufferwas determined from the diffusion-limited reduction curren t2eobtained at a d ropping-H g electrode. Th e rate of flow of Hgfrom th e cap illary was measured in the sam e solution,so that thediffusion coefficient could be calculated using the Ilkovicequation.

The potential of electrodes in the channels of t he glass devicewas measured relative to ground with a Burr-Brown Model358ATM high-voltage operational amplifier. Th e amplifier wasconfiguredas a voltage follower and was supplied with+115 Vrelative to ground.

Proced ures. Solutions were introduced into the chan nels viathe 3-mm-diameterreservoirs a t the e nds of th e chann els usinga syringe. A disposable plastic pipet ti p was cutto fit the syringe

andthereservoir,taallowapplicationofmodestpressures. Somecare was taken to avoid trapping air inside. Fluorescen t dyesolutions were introduced through reservoir2 by syringe andthen driven in or out of channel 3 by application of a voltagehetweenreservoirs2 and 3. Thefluorescencedetectorwasalignedto channel 3 with dye present in the channel

Plastic pipet tips were inserted in the three reservoirs andfilled with solutions to a height of abou t1 cm above the device.Fine Pt wires were inserted in the reservoirs to sup ply the elec-trophoresis voltage.

RESULTS AND DISCUSSIONFigure 1 shows the lay out of the glass device. Separ ations

were performed in chan nel3, while sam ple was introducedthrough channel2 and m obile phase through channel1. T h e

dimensionsof the capillarychannels weredesignedto producea minimum potential drop in channe l1, which supplies th emobile phase before the sa mple injection point. T o effectthis, channels 2 and 3 were made narrow,30 jtm wide an d 10pm deep, while channel 1 was 1 mm wide and 10 jtm deep.Th e device was fabricated from an upperand lower plate,onewiththechannelsetchedinit,theotherwithPtelectrodesdeposited. ThePt electrodes prevented th e upp erand lowerglass plates of the device from contac t bonding, and hondingthem with optical cem ent still allowed waterto leak betweenthe plates. Consequently, th e plates were melted togetherunder carefully determined conditionstopre ventthe channelsfrom collapsing. Figure2 shows a photomicrograph of theintersection point of the narrow channels.It can he seen th atthe melting process used for bonding the plates did not

seriouslydistortthe channel shape. It wasalso ohse rvedthatthe glass had flowed enoughto seal the Pt electrodes an dpreven t leakage.

Unlike the other reservoirs, inlet 4 in Figure1 was notformed by a hole through the top glass plate. Instea dch anne l4 was etched out to th e edge of th e bottom plate,so tha t asmall rectangular hole allowed contact to the externalenvironment. I t was originally intended th at th e struc turewould be dipped into solution so that inlet 4 would heimmersed. Th is would have allowed rapid electrokineticinjection of sample. However, leaving this po int open causeda secondary flow of solvent due e ither t o capillary actiondrawing solvent out or the effect of hydrostatic pressure

(26) Delahay, P. Bull. Soe. Chem. 1948.15,34&350.

Figwe2. P h o t ~ ~ a p h o f m e l n t e ~ p o l n t o f m e f w c h a n ~shown in Figure 1 afler ths glasa plates have been bonded togethx.me channel wldm is 30 pm .

differences. Consequen tly, this inle t was plugged with epoxyand sample was introduce d from reservoir2.

Electrical Ch ar ac ter isti cs. Initial characterization ofth e plana r capillary electrophoresis structu re involved mea-

surem ent of t he capillary curren t versus applied voltage(I -V) characteristics, and determina tion of th e voltage range a twhich electrical failure occurred. Th e voltage was appliedbetween reservoirs1,2, or 3; these are identified in Figure1.T he I-V response was linear, with a c orrelation coefficientof 0.999, for potentials of u pto 5000 V applied between anypair of reservoirs. Th e ratio s of the resistances measuredbetween each reservoir w ere in agreem ent with th e ratios ofthe channe l lengths and cross-sectional areas. Th e reprc-ducibility of th e ch annel resistance was q uite good, varyingby *4% over 2 weeks of measurem ents. Qualitatively, theresistance is a function of th e electrolyte conductivity. Forthe channe l between reservoirs1 and 3 (channel 1-3) theresistance dro ps from7.3 GQ,when filled with th e relativelylow conductivity, p H 8.5 boric acid, Tris buffer, to0.91 GQ ,wh enf ded with am uch higher conductance, pH 7.Ophosphatebuffer. This behavior was not explored quantitatively. Th elinea rity of th eI-V curves and their dependenceon channellength an d solution conductivity indicate th e cu rrent flowwc urs through the channels. Th e l inearity further showsthat the joule heat generated in the channels at thesepote ntials is effectively dissipated.

T he poten tial distribution inside the cha nnels was mea-sured with 96.6 V applied between reservoirs l and 3 (3 a tground). A sensing electrode placed in reservoir2, whichshould be at the po tential of the intersection p oint of thechannels, gavea value of 88.3 V. On the basis of the channelcross-sectionsand lengths, a poten tial of88 V was predicted.Th is indicates tha t very little potential dro poccurs in the

wide channel segment of channel1, due to th e large cross-section over most of the path length. Th e potentialsof threePt electrodes integrated into chann el3 of the device werealso measured. Th ese electrodes, locatedat distances of 1.2,70, and 132 mm from the edge of reservoir3, had potentialsof 0.9.44, and 87.7 V, respectively. Th ese values are within3% of the predicted values, on the basis of the channelgeometries. Th e good agreement between the predicted andobserved pote ntial distribution indicates th at th e cur rent flowisthroughthechannels. Further, i t shows tha tth e impedanceof eachca pillarychannel segm ent can he carefully controlled.Isolation of th ePt electrodes from potentials other tha n thosea t the location where they contact the channel isalsoevidenced. Fu ture work using these electrodes for on-columnelectrochemical or conductivity de tectio n is planned.


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ANALYTICAL CHEMISTRY, VOL.64 , NO. 17. SEPTEMBER 1, 1992 1929

Control of the potential drop w ithin a given channel bycontrol of its cross-sectional area will prove to be a n im porta nttool in th e design of devices. It will ensure th e ma jority ofthe potential can be app lied in the active separation channel,rather than in the segment tha t connects to the externalreservoir, providing the m aximum efficiency of design. Thi sis illustrated by the device shown in Figure1. Channel1 wasmade qu ite longso th at th e reservoirs were in a convenientlocation, bu t there was no need for a large potential dro p inchann el 1, since it is located before the sam ple injection poin t.

At this sta ge of testing, when th e applied voltage increasedabove5 kV the I-Vcurves became highly nonlinear, with thecurrent increasing from 1 to 20 pA between 6 and 10 kV.Arcing began to occur in this voltage range, although theexact potential usually varied randomly between devices andover time. Th e location of th e arcing sites was not clear, bu tit appe ared to be betw een reservoirs. Arcing failure sometime soccurred a t voltages of4 kV or less, but this could be remediedby thoroughly washing the device's surface with distilled waterand the n drying it. Surface contamination with salt solutionsis th e most likely sourc e of thi s low-field failure. Very recen tlywe have shown th at linear I-V curves can be obtained a tpotentials up to 25 kV, with no arcing. Th is work will bedescribed elsewhere, but we note here th at attaining thesefields required the Pt leads contacting the reservoirs becarefully isolated from each other by placing them insideglass tubing.27 Th e minimum d istance betwee n reservoirs1and 2 is 0.20 cm. Thu s, a field of up to 25 kV/cm can besustained without care to isolate the leads, and much highervalues (>lo 0 kV/cm) can be withstood by the glass structureitself.

Electrokinetic Phenomena. T o determine whether elec-trophore tic and electroosmotic flow occurred within th e glasschann els, a mixture of fluorescein and calcein in pH8.5 boricacid, Tri s buffer was studied. Calcein is a diaminotetra ace-tic acid derivative of fluorescein th at is somew hat larger insize and has a different charge at pH 8.5. Fluorescencedetection 6.5 cm from the intersection of the channels wasused to monitor the samp le in chann el 3.24925

Sam ple was injected by syringe from reservoir2, and thenchannel 1-3 was flushed with pH8.5 buffer using a syringewith reservoir 2 blocked. A positive voltage applied betwee nreservoirs 2 and 3 (3 at ground) caused the sample solutionin channel2 to move into channel 3 and alongpas t the detector.This was evidenced by two stepwise increases of equalmagnitude in the fluorescence signal as the dyes migratedalong the channel. Th e first front corresponded to the moremobile sample component, while the second step was seenwhen the second com ponent front also reached th e detectorlocation.

A small plug of sam ple could be injected from chann el2into channel 3 a t the intersection point by applying a voltagebetween reservoirs2 an d 3 for a brief period. Application ofa positive potential between reservoirs1 and 3 then drovethis plug along channel 3 past the detector. Figure 3 showsthe resulting electropherogram for an applied potential of3000 V, which correspon ds to 1260 V between th e injectionand detection points. Th e figure demo nstrates tha t elec-trophoretic separation of th e two com ponent mixture occurs,and tha t the peaks appear nearly Gaussian in shape. Th epeak heights were proportional to th e injection vo ltageappliedbetween reservoirs2 and 3, as well as to the length of timeit was applied. A detailed study was not made, but th eprecision was approxim atelyi20%. Injection of each com-ponent separately identified the first peakas calcein and th esecond as fluorescein, on the basis of their m igration times.

(27) K. Seiler, D. J. Harrison, unpublished work.

PH a s20 pM FluoresceinI 20 pM Calcein

l 3000 V applied

I l l 1 I I I0 2 3 4 5 6

Time (min)

Flgure 3. Electropherogram of a sample plug injected from channel2 Into channel 3 wkh 250 V applied between reservoirs 2 and 3 for30 8 . A voltage of 3000 V was then applled between reservoirs 1 and3 to eff ect the separation along channe l 1-3. The sample was 20 pMfluorescein, 2 0 pM calcein, and a pH 8.5 buffer was used. Note thetime scale was expanded 2.7 mln after sample Injection.

(a ) (b )

h

Flgure 4. (a)Background fluorescence observed wkh 5000 V appliedbetween reservoirs 1 and 3 with pH8.5 buffer in channel 1-3. Initlaliy,10 pM calcein, 20 pM fluorescein sample was present in channel 2at the Intersection point. (b) The pH 8.5 buffer was then driven backInto channel 2 from the Intersection to preven t the dye from leakinginto channel 1-3 and the background fluorescence was observedwith 5000 V reapplied between reservoirs 1 and 3. A decrease Inbackground signal resulted. The fluorescence scale is expressed

relative to the signal measured for the calcein, fluorescein mixture.

These results indicate th at electrophoretic separation canbe achieved using a glass subs trate in a plana r configuration.Injection and separation of a sam ple plug from chann el2 alsodemonstrates that the concept of manipulation of flowpa tter ns selectively within the chan nel manifold is possible.Th is is the process of valveless switching discussed above,bu t its dem onstration does not indicate how exclusively theflow is restric tedto the intended channel. Consider th at oncea sample plug was injected from channel2 into channel 3,sample solution remained in channel2 a t the intersectionpoint. Th e potential at reservoir2 was the n left floating whilea field was applied betwee n reservoirs1 and 3. Co nsequently,the sample in channel2 was freeto diffuse int o the intersectionvolume or be pulled in by th e convective flow along chan nel1-3. Th is fluid leakage can be expected to limit theeffectiveness of the valveless switching concept and couldrequire active con trol of the potential of each reservoir at alltimes.

Th e leakage of fluid from chann el2 into channel 1-3 wasevaluated in three steps. In step1 a 10 pM calcein, 20 pMfluoresceinsample at p H8.5 was present throughout the entirelength of channel 2, while pH 8.5 buffer was in the otherchannels,as indicated in Figure 4a. Th en5000 V was appliedbetween reservoirs1 and 3 (3 at ground) to drive the pH8.5buffer between them, as illustrated by the flow directionmarker in Figure 4a. With th e dye solution in channel2present at and near the intersection of the channels, any


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1030 ANALYTICAL CHEMISTRY, VOL.64, NO. 17, SEPTEMBER1,

leakagefrom this channel into channel 1-3 would co ntamin atethe buffer solution moving toward the d etector. This wouldincrease the background fluorescence at the detector inchann el 3. Figure 4a shows th e background fluorescenceobserved with 5000 V applied to ch annel 1-3, after t he eq ui-librium signal in the p resence of dye at the in tersection hadbeen established. In the second step of th e experiment thedye solution in chan nel2 wa sdriven back from t he intersectionby applying 5000 V between reservoirs1 and 2 (2 at ground).As a result a large par t of channel2, including the region nearthe intersection point, was filled with pH 8.5 buffer rathertha n the d ye solution. At this stage th e 6.5-cm-long plug ofsolution in channel 3 between th e intersection point a nd t hedetector was still contaminated with dye, as a result of thefirst step of the experiment. Finally, in the third step,illustrated in Figure 4b, 5000 V was reapplied betweenreservoirs 1 and 3, to again d irect th e flow along channel 1-3.Figure 4balsoshows how th e fluorescenceintensity responded.Note that the fluorescence intensity scale in Figure 4 isexpressed as the intensity of th e observed signal, ratioed tothe inten sity tha t would be observed if instead a 10 pM cal-cein,20pM fluoresceinsolution were driven p ast th e detector.It can be seen tha t the fluorescence intensity decreased intwo steps after 5000 V was reapplied to channel 1-3. Thesesteps correspond to migration of the two contamina nts in the

6.5-cm-long plug in chan nel 3 pa st t he detector, first the cal-cein (10 pM) and then the more slowly moving fluorescein(20 pM). Th e decrease in intensity resulted from the fa cttha t any leakage from channel2 no longer delivered dye intochannel 3, since the dye was no longer present at theintersection.

Th e series of experiments described above indicate somesolution was drawn in from channel2 while solvent flowedalong channel 1-3, du e to diffusion and /or co nvective effects.However, the magnitude of the leakage was small, as th echange in fluorescence intensity caused by removal of thedye from the intersection with channel2 was about 3.5% ofthat seen for introduction of a 10pM calcein, 20pM fluoresceinsolution into channel 3. Th is level of leakage should beacceptable for many applications, and more complex flowmanifolds or control of voltages on the side ch annels couldbe used to reduce the leakage for critical applications.

Th e migration times,t,, for fluorescein and calcein weredeterm ined over an applied voltage range of 1000-5000 V,corresponding to 420-2100 V between th e injection po int an dthe detector. The overall mobility,p , is related to the appliedvoltage by eq 1,28,29 whered is the distanc e from the injection

1992

point to the detector and V,, is the total applied voltage.Since chann el 1-3 has two segm ents of differen t cross-section,the electric field in the narrow chann el is calculated from th efraction (0.91) of V,, th at drops betw een the interse ction

point and reservoir3. Th e length of tha t segment,L, is 13.9cm. P lots of l/ t, versusV,, are line ar (correlation coefficientof 0.998), giving overall mobilities of (1.90f 0.03) and (1.21f 0.06) X cm2/(V.s)for calcein and fluorescein, respec-tively. The values and errors reported here and in thefollowing text are the averages and standard deviations ofseveral replicate experiments.

Th e mobility of fluorescein in pH 8.5 buffer in a conven -tional fused-silica capillary colum n equippe d with an absor-bance detec tor was found to be (2.59f 0.05) x cm2/(V-s),and th at of the neu tral marker tryptophan was (5.87f 0.08)

(28) Jorgenson, J . W. ; Lukacs, K. D . Anal . Chem. 1981, 53 , 1298.(29) Huang, X.; Coleman, W. F.; Zare, R. N . J. Chromatog. 1989,480,

95-110.

pH 8.5 (boric acid/tris)f' 30000 1260 V in Channelv

c

0

L

0 )

n 1E3

z

0000

n-. 0 1 .1 1 1 0 10 0

Length of Injection Plug (mm)Flgure 5. Plot of the number of theoretical plates,N, as a functionof the length of sample plug injected from channel2 into channel 3.3000 V was applied between reservoirs1 and 3 (126 0 V in the actlvechannel)during the separa tions of the20 pM fluorescein,20 pM cabcein samples.

X 10-4 crnz/(V.s). Using these dat a and eq2, wherepep is th e

(2 )

electrophoretic mobility andpeOis th e electroosmoticmobility,the va lues ofpep and peO in both the fused-silica capillary andthe planar glass channel could be determ ined.It is assumedtha t pep was the same for fluorescein in the glass and thefused-silica capillaries. T he electroph oreticmob ilities of cal-cein an d fluorescein in the glass device were found t o be (-2.6f 0.1) and (-3.3 f 0.1) X 10-4 cm2/(V-s),respectively. Theelectroosmotic mobility in th e fused-silica column was (5.87f 0.08) X cm2/(V.s),while in th e glass structure it was(4.5 f 0.1) X 10-4 cm2/(V-s). These values indicate t ha t elec-troosmotic flow does occur within the glass substrate, andtha t it has a m agnitude similar to tha t in fused silica.

Separation Efficiency. Know ing the value ofp , it waspossible to estima te the length of the sample plug electroki-netically injected from channel 2 into channel 3. Th esepara tion efficiency, expressed as the num ber of theoreticalplates,N, could then be determ inedas a function of the sizeof sam ple plug injected. Figur e 5 shows a plot ofN versusthe plug length. N was calculated using eq 3,30where W, is

(3 )

P = Pe p + eo

N = 8 In 2(t,/ w,)'

the full width of the peak a t half-maximum, expressed interms of time. For both compounds,N reaches a plateauwhen th e sam ple plug length decreases below abo ut0.6-0.9mm. This is consistent with the estimated detector cell length

of abou t 0.8-1 mm . Th e fluorescence detector design was farfrom optimized and did n ot incorporate a focusing lens forthe laser excitation beam, a collection lens, or a s lit to minimizethe field of view. In light of this, it is not su rprising thedetector optics limited the separation efficiency. Th e resultsdo show th at very small sample lengths are readily injectedand d etected; the m inimum volume injected corresponds to0.8 fmol of dye. Be tter mass and conc entration detectionlimits should be readily achieved with an optimized dete ctor,allowing a f urthe r decrease in the injected sample volume.

The separation efficiency of the glass device was alsoevalua ted as a fu nction of applied voltage, as shown in Figure

(30) Karger, B. L.; Snyder, L. R.; Horvath, C. An Introduction t oSeparat ion Science; J. Wiley and Sons: New York, 1973; pp 136-137.


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ANALYTICAL CHEMISTRY, VOL. 64, NO. 17, SEPTEMBER 1, 19921831

25 I I20 FM Fluorescein,pH 8.5 I

t V channel = 0.42V applied

30000C

QEij 20000

nE 10000

L

Q)

3

z 6 - Fluorescein0 I...,

0 2 0 0 0 4 0 0 0 6000

Applied Voltage (V)Figure 6. Plot of Nversus the total applied voltage between reservolrs1 and 3 for fluorescein (0 ) and caiceln (0). The sample plug wasabout 0. 47 mm long.

6. Th e potential between the injection point an d the detectorwas 0.42 Vap,and th e injection plug length was estimate d tobe 0.47 mm. Th e efficiencies were fairly different for th e twocompone nts of the sam ple, with a maximum nu mb er of platesof about 35 OOO found for calcein.

To evalu ate the possibility of wall interactions, the heigh tequivalent to a theoretical plate, H, was evaluated experi-mentally and compared to theory. Band broadening in thecapillary arises principally from longitudinal diffusion effects,as shown by eq 4,29where H d i f f is the plate height due to

Hdiff= 2Dt,/d (4)

longitudinal diffusion andD is the diffusion coefficient. Inaddition to diffusional broadening th e injection plug lengthand th e detec tor cell volume contribute to the overall variance,u&:

(5 )Th e diffusional variance,U d i f t , is given by Dt,, and Ude? anduinj2 will be given by w2/12,29Z1where w is the length of thedetector cell or injected plug and a rectangular shapeisassumed. Zare and co-workersmhave suggested an additionalinteraction term ,uht2, should be included if the ca pillarywa lbinteract with the analyte and lead to band broadening. The yalso indicate tha t, if this term is ignored when presen t, it willlead to an effective diffusion coefficient ob tained fromU d i dtha t differs from the true value. Thu s, a comparison ofDobtained from plotting H versus t , with an independentlydeterm ined value provides insight into the ex tent of analyte-wall interactions in a capillary.

Plots of H versus t , for both fluorescein and c alcein werelinear over a range ofV,, from 1000 to 5000 V. Calcein da tashowed a slope of(7.4 f 0.8) X cm/s and a n intercep t of(1.3 f 0.2) x 10-4 cm. Th e slope corresponds to a diffusioncoefficient of (2.4 f 0.3) X lo4 cm2/s for calcein in t he pH8.5 buffer. Fluorescein dat a gave a slope of(1.02 f 0.07) X10-7 cm/s and an intercept of(1.4 f 0.3) X cm; this givesa diffusion coefficient of (3.3 f 0.2) X lo4 cm2/s. Anindepend ent measurement ofD = (3.4 f 0.3) X lo4 cm2/sforfluorescein in pH 8.5 buffer was obtained using a polaro-graphic m ethod.26 Equation4 predicts a zero intercep t fora plot of H versus t , unless there is another source ofbroadening, suchas indicated in eq5. For these experimentsthe sample plug w idth was0.047 cm and th e detector length

2 2ub: = diff2 + udet + inj + gin:

(31) Sternberg,J. C. Adu. Chromatog. 1966,2, 206-270.

iv)Q)

0c)

ii:

0 1 0 0 0 2 0 0 0 3000

V In Channel (0.42 V) (Volts)Flgure 7. Plot of the number of plates per volt between the injectionand detection points, N/(O.42Va,), for the fluorescein data in Flgure 6.The plates per volt corrected for band broadenlng introduced by theInjector and detector is also shown. The lines are theoretical curves,as discussed In the text.

was approximately0.09 cm. Th e value of the intercept in aplot of H versus t , should be given by (ain? + ude&/d. Forthe dimensions mentioned above this gives1.3 X cm, inagreement with the m easured value of(1.3-1.4) X lo4 cm.Consequently, all of th e band broadening observed in theglass structure can be accou nted for by eqs4 and 5, excludingth e uintz term. We conclude th at the system behavesessentially ideally, with no wall interactions for t he speciesstudied.

Figure 7 shows how th e numbe r of plates per volt betweenthe injection and detection points,N/(0.42VaP),varied withthe voltage between these points(O.42Va,). A range of abo ut10-15 plates/V is seen for fluorescein, decreasing a t higherapplied V. Since I.Land D were obtained experimentally forfluorescein, as was the e xtracolum n variance(ain? + Udet), itis possible to calculate N by combining eqs 1, 4, and 5 toobtain eq 6. Th is expression, divided byO.42Va,, is plotted

(6)1-l

2DdLN = d 2 + in; + det

as a solid line in Figure 7. The agreement shows that thedecrease inN/ (0.42V,,) with V,, arises from the con tributionsof extracolumn band broadening rather t ha n joule heating oranalyte-wall interactions. Th e number of plates due to thecolumn alone, corrected for the detector cell and injectionplug length contribution toH (1.4 X cm), was estimatedusing eq 7, where N,,,, is the corrected value andH is the

(7 )

d

H - 1. 4 X cmNc,,, =

observed plate height in centimeters. Th e values ofN,,,,/(O.42Va,) as a function of V,, are shown in Figure 7. Atheoretica l curve calculated according to eq6, neglecting theextracolumn contributions, is also plotted as a dashed line.These co rrected values are in th e range15-20 plates/V andare comp arable to typical results of abo ut20-25 plates/Vreported for open tubu lar, fused-silica capillaries. In fact,the calcein dye shows somewhat higher values of20-35 plates/V, after correction for ban d broadening due t o the injectorand de tector lengths. These results indicate very reasonableperformance from the prototype planar glass device, especiallygiven the limitations of the detector design used.


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1832 ANALYTICAL CHEMISTRY,VOL. 64, NO. 17, SEPTEMBER1, 1992

C O N C L U S I O N S

T o actually realize complex sample-handlingand separationsteps in an integrated, planar structure requires tha t a num berof basic principles be shown to work in such systems. Th isstudy has examined an d d emon strated th e feasibility of usingelectroosmotic pumping t o contro l flow in a manifold of flowchannels without the use of valves. Thi sis a significant aspectof the p-TAS concept, and its realization demo nstrates tha tmore complex sample-handling steps such as those used inFIA can be achieved with this approach. Th at the flow ratesare com parable to fused-silica capillaries show s th at pum pingrates will be predictable and that the glass substrate is asuitable material for this application.

Sepa ration of samples is an equally impo rtan t aspect of ap-TAS device,and the present work shows that electrophoreticseparation can be achieved on a planar substrate. Th emeasured separation efficiency is quantitatively describedby th e expected theoretical relationship, indicating the devicebehaves essentially ideally. In fac t, the efficiency expressedas the num ber of plates per volt is similar to tha t achievedwith conventionalopen tubula r, fused-silica capillaries. While5000 V applied was translated into fields in the active par tof the channel tha t did not exceed 2100 V, the data show thatredesign of th e device layout will easily increase this value.Manipulation of th e c hannel geometry to control where thebulk of an applied potential drops was shown to be easilyaccomplished. Fur ther, relatively high electric fields of 350V/cm were obtaine d with no isolation of the c ontact leads,similar to th e values typically used in conventional capillaries.Higher fields of a t least 1800 V/cm ca n be sustaine d withinthe channels when the leads are isolated, a nd by using theapp ropr iate channe l geometry virtually all of this potentialcan be applied across the active portion of the channel.Overall, glass appears to be a satisfactory substrate for

development of planar structures. It is compatible with bothmicromachining m ethods and electrophoresis.

Th e application of m icromachining techniques to prepareminiatur ized, 3-dimensional structure s for chem ical sensingand analysis is in its infancy. Th e present work suggests th atrelatively com plex system s will be realized in the fu tur e tha twill compe te with chemical sensorsand with present bench-top analysis systems. Micr ostructure s an d capillaries, inte-grated detector system s, valveless switching of sam ple flow,and electroosmotic pumping are concepts tha t can be com-

bined in a variety of ways to produce unique, miniatu rizedanalytical systems. Such systems could lead to laboratorieson a chip th at offer rapid, sophisticated analyses in a mobilepackage tha t is free to leave the laboratory. Th e possibilityof mass fabricating devices using integrated circuit and mi-cromachining technologies may lead to low-cost systems withapplications ranging from indu strial process control to clinicalanalysis. How ever, considerable effort will be required t oexplore the many possibilities of th e p-TAS conc ept and mi-cromachining technology and to establish their future impacton ap plications in chem ical analysis.

A C K N O W L E D G M E N T

We thank A. Bruno for assistance with th e fluorescence

detector and A. Pa ulus for measurements with th e commercialCE instrumentation and for valuable discussions.D.J.H.thanks Ciba Geigy for the opportunity to work in theirlaboratories and partial su ppor t during his sabbatical leave.Z.F. acknowledg es the A lberta M icroelectronic Centre for agraduate fellowship.

RECEIVEDfor review January 3, 1992. Accepted May 21,1992.

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