Planar glass chips for capillary electrophoresis: repetitive sample

using a low-speed drill and a diamond bit, as sketched in the inset of Figure 4. Following this the active channel lengths were as follows: reservoir ...
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Anal. Chem. 1993, 65, 1481-1488

Planar Glass Chips for Capillary Electrophoresis: Repetitive Sample Injection, Quantitation, and Separation Efficiency Kurt SeilerJ D. Jed Harrison,’J and A. M a d Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2,and Central Analytical Research, Ciba-Geigy, Basel, Switzerland

A sample injection system and a capillary electrophoresis channel integrated together on a planar glass substrate are described, and the performance is evaluated. Voltages of at least 25 kV may be applied over an 11-cm-longcapillary channel with sufficient dissipation of the Joule heat generated (1.8 W/m). The glass substrate sustains at least lo6V/cm dc without dielectric breakdown. Using laser-induced fluorescence detection, mixtures of fluorescein derivatives and fluorescein isothiocyanate-labeledamino acids were injected and separated. Up to 100 000 theoretical plates and -20 plates/V were obtained under optimized conditions,comparable to results with fused-silica capillaries. Quantitative analysis showed that peak areas were proportional to the amount injected for injected plug lengths greater than 200 pm. The presence of two rectangular corners in a 0.85-cm-longseparation channel did not increase dispersion detectably, indicating a serpentine capillary channel 0.5 m in length could be fabricated in less than a 1-cm2area. INTRODUCTION Capillary zone electrophoresis (CE) introduced by Mikkers et al.1 and Jorgenson and Lukacs293 is an exciting separation technology that has attracted the attention of many analytical laboratories.4~5It has shown the ability to resolve very complex mixtures of many different species within a short time and exhibits an efficiency that can exceed 106 theoretical plates. We have demonstrated that both an electrophoresiscapillary and a sample injection system can be integrated together on a planar device, using microlithographictechnology known aa micromachining.6~7Miniaturized systems for total chemical analysis (p-TAS) of a complex sample have been previously proposeds11but only recently demonstratede~perimentally.~J In this report we present data for a planar electrophoresis

* Author to whom correspondence

should be addressed. University of Alberta. Ciba-Geigy. (1) Mikkers, F. E. P.; Everaerta, F. M.; Verheggen, Th. P. E. M. J. Chromatogr. 1979,169, 11-20. (2) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981,53,1298-1302. (3) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981,218,209-216. (4) Ewing, A. G.; Wallingford, R. A.; Olefirowicz, T. M. Anal. Chem. 1989, 61, 292A-294A. (5) Capillary Electrophoresis: Theory and Practice; Grosman, P. D., Colburn, J. C., Eds.; Academic Press: San Diego, CA, 1992. (6) Harrison, D. J.; Manz, A.; Fan, 2.;Ludi, H.; Widmer, H. M. Anal. Chem. 1992,64, 1926-1932. (7) Manz, A.; Harrison, D. J.; Verpoorte, E.; Fettinger, J. C.; Ludi, H.; Widmer, H. M. J. Chromatogr. 1992,593, 253-258. (8) Manz,A.;Fettinger, J. C.;Verpoorte, E.; Liidi,H.; Widmer, H. M.; Harrison, D. J. Trends Anal. Chem. 1991, 10, 144-149. (9) Manz, A,; Graber, N.; Widmer, H. M. Sens. Actuators 1990, B l , 244-248. (10) Pace, S.J. US. Patent, 4.908.112, 1990. +

0003-2700/93/0365-1481$04.00/0

device obtained with a more highly optimized fluorescence detector design than we described previously.6 It should allow for better detection limits and a much lower contribution to band-broadening. Other instrumentation and experimental method improvements described here have provided precise control of the injection and separation processes and allowed the application of much higher applied potentials. Several features important to the performance of p-TAS systems based on an integrated electrophoresis device could be examined due to these instrumental improvements. The ability to use higher potentials provides for more efficient separation of samples in electrophoresis,’-f-and it is important to demonstrate that high efficiencies can be obtained within the planar devices. In order to make a more compactdesign than the 15 X 4 cm device we have studied: it is important to explore the electrical breakdown characteristics of the glass substrate. Cross-talkbetween channels due to dielectric breakdown could limit how close together channels may be located on a device. Also, corners or curves will be required for any serpentine or coiled arrangement intended to increase capillary length within a small device area. Consequently,it is necessary to understand the effect of corners on band-broadening. We have shown that by use of electroosmotic pumping it is possible to control the direction of fluid flow in a manifold of intersecting capillaries withoutthe use of valves.6 However, in avalveless device there will be both diffusion and convection phenomena. These will lead to leakage and mixing between solutions of different composition when they meet at channel intersections. Recently, Dose and Guiochon12have derived a theoretical understanding of CE in this respect. They have pointed out that hydrodynamic flow and diffusion are two significant factors that need careful control to reduce quantitative errors. Consequently,we have explored the effect of diffusion of sample at an intersection of capillaries in an integrated manifold on the process of sample injection. In addition, the problem of quantitative analysis of electrokinetically injected sample at a capillary channel intersection has been examined. Since diffusion may play a significant role in performance it is important to precisely control injection, separation, and delay times, and a computercontrolled system for this purpose has been developed. Such a system makes periodic cycling of injection and separation simple and so may facilitate automation of analysis in the f~ture.~-g Our previous study examined a very simple separation of fluorescein derivatives and required 5 or 6 min. To show the ability to deal with more complex and realistic samples we present here the separation of several amino acids.13-15 Using

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(11) Gale, R. J.; Chowski, K. In Biosensor Technology, Fundarnentak and Applications; Buck, R. P., Hatfield, W. E., Umana, M., Bowder, E. F., Eds.; Marcel Dekker: New York; 19W, pp 55-62. (12) Dose, E. V.; Guiochon, G. Anal. Chem. 1992,64, 123-128. (13) Cheng, Y.-F.; Dovichi, N. J. Science 1988,242, 562-563. (14) Liu, J.; Dolnik, V.; Hsieh, Y.-Z.; Novotny, M. Anal. Chem. 1992, 64, 1328-1336. (15) Monnig, C. A.; Jorgenson, J. W. Anal. Chem. 1991,63, 802-807. 0 1993 American Chemlcai Society

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993 MOBILE PHASE

I

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SAMPLE -----o

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Flguro 1. Top view of the layout of the channels Integrated on a planar glass substrate. The open circlesshow the nwnkrlng for the reservolrs

contaotlng the channels,and the filled clrclesshow the channelnumbers.

The channel leadlng to the edge of the device was plugged wlth epoxy. The location of several of the electrodes integrated on the device is shown (Pt). The detector was placed at (I) for most of the studies and at (11) for the study on the effect of corners. The overall dimenslons are 14.8 cm X 3.9 cm X 1 cm. The inset shows a magnification of the intersectlon, where the sample was Injected.

the higher voltages and improved detector now available, we were able to attain high separation efficiencies, and these compare favorably with conventional fused-silica capillaries.

EXPERIMENTAL SECTION Reagents and Solutions. Three different buffer solutions were used for the experiments: a pH 8.0 buffer [50 mM boric acid, 50 mM tris(hydroxymethy1)aminomethane (tris)],apH 9.1 buffer (10 mM sodium borate), and a pH 9.2 buffer (30 mM sodium carbonate) adjusted with HCl. The fluoresceinsodium salt, the L-amino acids arginine (Arg),phenylalanine (Phe), and glutamine (Gln), and fluorescein 5-isothiocyanate (FITC) were obtained from Sigma (St. Louis). The fluorescein 5- (and 6-) sulfonic acid sodium salt (isomeric mixture) was obtained from Molecular Probes, Inc. (Eugene, OR). All were used as received. Labeling of Amino Acids. Labeling of the amino acids was performed as described in ref 15. To 1 mL of 6 mM FITC in acetone was added 3 mL of 3 mM solution of each amino acid in a pH 9.2 carbonate buffer (30 mM). This was allowed to react at room temperature in the dark overnight. It was further diluted with the mobile phase (pH9.2,30 mM carbonate buffer)to obtain a solution of 10 pM of each of the labeled amino acids. Before use, all solutions were passed through a 0.22-rm filter (MillexGV, Millipore, Bedford, MA) to remove particulates. Devices. The glass capillary electrophoresis-based TAS (CETAS) structures were fabricated under contract by Mettler AG (Switzerland) as discussed A bottom glass plate had channels etched in it, while a top plate had Pt electrodes defined on it. These plates were melted together under controlled conditions, so that the channel shape was not distorted.6 The layout of the capillaries and some of the electrodes is shown in Figure 1. The two narrow channels, 2 and 3, are 30 pm wide and 10rm deep, while channel 1is 1mm wide and 10pm deep. Three holes were made through the top plate to contact the channels and serve as reservoirs. The channel lengths are as follows: reservoir 1 to beginning of narrow channel, 162 mm; from the end of the wide channel to the intersection, 9.3 mm; reservoir 2 to intersection, 166.6 mm; reservoir 3 to intersection, 139 mm. Plastic pipet tips were shortened so that they fit into the holes of the top plate to form larger reservoirs for the electrolyte solutions. The Pt wires (0.3-mm diameter) inserted into these reservoirswere further isolated with a glass tube (wallthickness 1.5mm, length 2 cm) placed over the reservoirsand electrodes.

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Flguro 2. Schematlc representation of the instrumental setup. For clarlty only one power supply and one high-voltage (HV) relay are shown, aithough more were used. The computer controls the HV power supplies (I) and the HV relays, whloh are activated with a 2 4 4 power supply vla digitally controlled relays (11).

One device was prepared with no Pt electrodes by etching the Pt away in 80 OC concentrated aqua regia before bonding the plates. It was then cut with a glass saw to remove the contact holes, and new holes were drilled by hand into the bonded device, using a low-speed drill and a diamond bit, as sketched in the inset of Figure 4. Following this the active channel lengths were as follows: reservoir 1 to beginning of narrow channel, 2 mm; from the end of the wide channel to the intersection, 9.3 mm; reservoir 2 to intersection, 90 mm; reservoir 3 to intersection, 100 mm. This device was used only to study the application of high electric fields. Sample Introduction. The manifold of channels was initially flushed with the buffer by applying pressure to one of the reservoirs with a syringe. It was very important to clean the surface of the glass carefully with distilled water to remove conductivity paths from electrolyte solution on the surface. Fine Pt wires were inserted in the reservoirs to supply the electrophoresis voltage. The sample was driven from reservoir 2 to the location of the detector by applying a voltage between reservoirs 2 and 3, until a stable signal was obtained. The sample in the separation channel was flushed to waste by applying a voltage between reservoirs 1 and 3, before the cycling of the injections and separations was started. Instrumentation. A schematic representation of the instrumental setup is shown in Figure 2. FUG Elektronik Model HCN 2000 (0-2 kV) and Model HCN 12500(0-12.5 kV) power supplies (Rosenheim, Germany) were used for sample injection and for separation,respectively. The current in the respective channels was monitored by measuring the voltage drop across a 10-kS2 resistor placed between one solvent reservoir and ground and recorded with a Hewlett-Packard dual-stripchart recorder Model 7128A. Connections between the power supplies and ground to the devices were interrupted or switched between reservoirs using high-voltage relays (Model HVS 10/S3, Kilovac, Santa Barbara, CA, and Model 24HVlA-100, Douglas Randall, Pawcatuck, CT). The relay armatures were activated with a 24-V (2.4-A) power supply (GHOF2-24,GFC Power Ltd.,Ontario, Canada). Contact of this supply to the high-voltage relay armatures was controlled by the computer using a set of digitally controlled relays (Control 488/16, Iotech, Cleveland, OH). A LabVIEW (National Instruments) program running on a Macintosh IIci with a laboratory interface board (Model NBMIO-16L,National Instruments) was used to control the output of the power supplies through two digital-to-analog converters. The Iotech relays were controlled via the digital 1-0lines, and data acquisition utilized the analog-to-digitalconverters used in the double-ended input mode. A measuring cycle comprised generally the following operations: (1)activate (close) relay to sample reservoir; (2) apply injection voltage for the injection time; (3) deactivate (open) relay to sample reservoirand activate relay to buffer reservoir; (4) apply separation voltage. The rise and decay times of the power supplies when connected to the device were -0.3 s (to 99%). Optical Setup and Signal Processing. The optical system was constructed on an optical breadboard (3 f t X 3 ft, Melles Griot). A 488-nm air-cooled Ar ion laser (Cyonics/Uniphase, Model 2011-20SL,Newport Research) operated at 4 mW served

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This could be done either from analyzing peak height and area or from calculating the second statistical moment. There was good agreement except when the signal-to-noiseratio was poor.

RESULTS AND DISCUSSION

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Flguro 9. Schematic of the opticaldetectionsystem. k ion laser light was focused with a lens (1) onto the separation channel, which was held in place with a plexiglase holder (2). Fluoreecence emission (3) was collected with a rnlcrowpe objedive (4), focused onto an ak slit (5),tiltered(6),andthendetectedwithaphotom~iertube. Objective. air slit, and filter were mounted In a microscope body, which was fixed on x-y translation stages.

as the fluorescence excitation source. It was focused onto the channel of the p-TAS at -45O to the surface using a lens with a focal distance of 30 cm, as shown in Figure 3. The surfaces of the CETAS device were roughened during the bonding process, so the beam diameter focused in the channels tended to vary between 100 and 200 pm, depending on position. The laser position was shifted along the channel to study the effect of injector-to-detectordistance. The paths of the reflected beams were arranged so that they did not strike the capillary channels elsewhere, in order to avoid photobleaching. The fluorescent radiation was detected with the optical axis of the assembly shown in Figure 3 perpendicular to the plane of the device. Light was collected with a microscope objective (10: 1, NA 0.30, working distance 6 mm, Rolyn Optical) mounted on a microscope body (MellesGriot). A Hamamatsu Model R1477 photomultiplier tube (PMT) powered by a Hamamatsu HC12301 high-voltage supply was mounted in a sealed housing at the exit of the microscope. A 100-pmair slit (MellesGriot)was placed at the field stop plane of the microscope in a specially made adaptor, to restrict the channel length viewed by the detector to 10 pm. An Omega 518 DF25 interference filter (transmitting 505-538 nm) was mounted over the entrance to the PMT housing to eliminate scattered laser light. The entire assembly was covered with a large box to reduce, but not eliminate, ambient light. Both the device mounting stage and the microscope assembly were mounted on Newport Research x-y translation stages. The PMT output was connected across a l-MQresistor. The potential developed was recorded with a Linear Instruments Model 500 strip chart recorder and was also filtered through two stages of active, six-poleButterworth filters (Krohn-HiteModel 3342, Avon, MS) both set at 50 Hz, before inputting the signal to the A-D converter. The response time of the detector was 40 ms with this configuration. The sampling rate of the A-D converted varied between 10 and 20 counts per second. Once acquireddigitally,the electropherogrampeak parameters (center of gravity, peak area, variance) were obtained by statistical moment analysis. In this report, the time of migration of the center of gravity is used as the peak migration time. The number of plates was calculated after baseline correction for each peak.

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The capillary electrophoresis TAS device (CETAS) layout is illustrated in Figure 1. The channels were etched into one glassplate, and a second was then bonded on top. Thissecond piece had holes drilled into it to contact the channels. Pt electrodeswere alsofabricated on this plate, as shown in Figure 1. The reservoir and channel numbering referred to in the text is also shown. As discussed previously,6the fourth outlet was sealed with epoxy to prevent undesired hydrodynamic flow. Electrical Characteristics. When avoltage was applied between any pair of reservoirs in a device in which integrated Pt electrodes were present, the current was stable up to between 8 to 10 kV. The channel resistances evaluated from the current-voltage (1-V) response were in agreement with the channel geometry,indicating the current path was through the channels. As reported previously,6 9% of the potential was distributed between reservoir 1 and the channel intersection point when reservoir 3 was at ground. The large channel (1) is separated by 1 mm from an integrated Pt electrode that contacts channel 2. With a potential applied between reservoirs 2 and 1 (1at ground) this electrode will be floating at a potential very close to the applied voltage. Observation of a stable current a t 10kV which was consistent with the channel resistances indicates that glass can sustain at least lo5V/cm. The maximum potentials utilized in this work are about twice those achieved previously6 and were attained by insulating the Pt wires contacting the reservoirs with glass sleeves. At still higher applied potentials the current in the channel became unstable, and this was correlated with the evolution of gas in the channels at the Pt wires integrated in the device. While these wires were not connected to the power supplies they contact the channels at different locations and lead to the edge of the device, where the metal contact pads are located 1 mm from each other. Arcing between these contact pads, which are floating at the various channel potentials, is clearly the event which leads to current flow and water electrolysis at the Pt electrodes, as demonstrated below. To further evaluate the possible breakdown of the glass dielectric, a device was prepared without integrated Pt electrodes. The contact reservoirs were further separated by drilling new access holes in the bonded device, and the device was cut into two sections along a plane parallel to that running through the original reservoirs. The channels exposed at the edge by this cut were sealed with epoxy. The I-V curves for this device were stable and linear at potentials up to 25 kV (limited by the power supply), as shown in Figure 4. With 10 mM borax buffer at pH 9.1 as electrolyte the channel resistances were 5.64 and 3.16 f 0.01 GQ between reservoirs 2 and 3 and 1 and 3, respectively. The ratio of these values is in agreement with the channel geometry for this structure, indicating that the current flow is through the channels. A voltage of 25 kV corresponds to an electric field of 2.3 kV/cm when applied between reservoirs 1 and 3, which can be compared to the value of 0.3 kV1cm frequently used in CE in conventional fused-silica capillaries. The linearity of the I-V curve indicates that Joule heat is effectively dissipated a t an energy density of at least 1.8 W/cm. This is larger than the value of 1W/cm that has been suggested as the limit for CE in uncooled fused-silica capillaries.15 In addition, the potential thiough the glass between the channels contacting reservoirs 1and 2 corresponds to 7.8 X 104Vlcm, corroborating the good insulating ability of the bulk glass and the bonded joint between the top and bottom plates, as discussed above.

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Table I. Mobilities and Other Parameters. mobilitiesb (X104) (cm2/V.s) sample I (n = 8) fluorescein fluorescein 5- (and 6-) sulfonic acid sample 11 (n = 5 ) d k g Phe Gln

slope of peak calcd plug areas vs tmc lengthb (arb unita/s) (Nm)

1.932 f 0.006 1.083 f 0.004 0.975 f 0.004

44.3 f 1.6 6.4 f 0.1 24.6 f 0.5

432 231 212

2.345 f 0.007 1.875 f 0.007 1.319 f 0.006

73.3 f 2.4 70.3 f 2.4 31.7 f 2.3

1193 953 670

"Parameters obtained for a series of separations at different separation voltages (from 2 to 10 kV). The injection parameters were constant (for sample I, 110 Vi60 a; for sample 11, 250 Vi60 a). The mobilities (SDgiven) and the plug lengths were calculated with eqs 1 and 2, respectively. The listed slopes (=kjliCi,SD given) were obtained from a proportionality analysis of area vs migration time (see eq 3). Arg, arginine;Phe, phenylalanine; Gln, glutamine.

*

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POTENTIAL [kV] Figure4. Current vs the applied potential difference between channels 1 and 3 or 2 and 3 in a modified device similar to that shown in Figure 1. The integrated pt electrodes were not present, and new contact holes had been drilled (see inset and text).

, 111

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ELECTRIC FIELD DURING INJECTION [V/cm] 15

0 ELECTRIC FIELD DURING SEPARATION lV/cml

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Figure 5. Three cycles (a-c), each including injection and separation of a sample of 10 pM fluorescein (I) and 10 pM isomeric mixture of fluorescein 5- (and 6-)sulfonic acid (I1 and 111) In a pH 8.0 tris-boric acid buffer. The length between injection and detection was 6 cm. The

figure also shows the potential program applied between reservoirs 2 and 3 for sample injection and between 1 and 3 for separation.

Repetitive Injections. Sample was introduced through reservoir 2 and small sample plugs were injected through the intersection point toward reservoir 3 by applying a low potential between reservoirs 2 and 3 for a short period, as discussed previously.6 A separation was then performed by applying a positive voltage between reservoirs 1 and 3 (3 a t ground). The fluorescence detector could be located at different points between the intersection point and reservoir 3. To control and automate this injection and separation procedure, an apparatus consisting of several high-voltage power supplies, high-voltage relays, and digitally controlled relays under the control of a microcomputer was assembled, as described in the Experimental Section. This apparatus was intended to allow precise cycling and separation times so that band-broadening effects due to diffusion may be minimized and controlled (vide infra), Three repetitive cycles, each consisting of sample injection (60 s), separation (280s), and delay (10 s) steps are shown in Figure 5. The sample was a mixture of 10pM fluorescein and 10 p M fluorescein 5- (and 6-) sulfonate in 50 mM tris-boric

acid buffer, pH 8.0. The first peak was identified as fluorescein, while the second two were due to the fluorescein sulfonate sample. These two peaks may represent the two isomers or the presence of an unidentified impurity. The figure shows qualitatively that the amount injected, as indicated by the peak heights and areas, decreased as the injection voltage was decreased. The reproducibility of peak areas was estimated from the error in the slope of a plot of areas vs migration time, as discussed below and presented in Table I. The standard deviation in peak area was about i 3 % , The peak migration times were reproducible to less than 1% , and it was possible to make more than 40 repetitive injections. It can be seen in Figure 5 that the fluorescence intensity decreased during the delay and injection period. The laser intensity was sufficient to photobleach the dye essentially completely, so that when the flow rate was low or zero (Le., during the 10-s delay, or when the injection voltage was applied) the signal changed rapidly to background levels obtained in the absence of fluorescent dye. Once the separation voltage was reapplied, the higher velocity increased fluorescence to a higher background level. This level was controlled by the extent of leakage of dye from channel 2 into 3.6

Amino Acid Separation. To demonstrate the ability to deal with more complex samples, fluorescein isothiocyanatelabeled amino acids were separated. The electropherogram of three amino acids (Arg, Phe, Gln) is shown in Figure 6. This represents the first separation of amino acids in aplanar device. The separation voltage of 10 kV applied between reservoirs 1 and 3 corresponds to 6.3 kV over the injectiondetection distance of 9.6 cm or to an electric field of 655 V/cm. The three amino acids are clearly separated in only 120 s. The peaks were identified by injecting each amino acid separately. The glutamine peak exhibited a shoulder, which originated from the glutamine solution itself. The first peak elutedwas identified as an impurity in the phenylalanine solution. The migration times of each component were linearly dependent on the injection-detector distance, did, between 3.0 and 9.6 cm. This indicates the channel and electric field were spatially uniform in the longitudinal direction. The electrokinetic mobilities of the three principal peaks were evaluated from the slope of linear regression lines of the inverse of the migration time, t m Vs the electric field,E, applied during separation. This relationship is given by

where ~i is the electrokinetic mobility of component i. This mobility is the vector sum of the electroosmotic and elec-

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TIME (sec) Flguro6. Electropherogram of three 10 pM FITC-labeled amino acids (Arg, Phe,and Gln in a pH 9.2 carbonatebuffer). ElectrokineticInjection was performed from channel 2 by applying a voltage of 250 V for 80 8 (- l-mm plug length). For the separation,a voltage of 10 kV was applied between reservoirs 1 and 3 (8.3 kV between injector and detector). The separation distance from Injection to detection was 9.8

cm. trophoretic mobilities. The separation voltage was varied from 2to 10 kV (131to 655V/cm) for a specific set of injection parameters, and the distance between the points of injection and detection, did, was 9.6 cm. Excellent linearity was obtained throughout for the plots of tm-1 vs E(R2 > 0.9999), and migration times were reproducible within 1%for replicate injections at each potential. The data for both amino acid and fluorescein samples are given in Table I. The small standard deviations in pi and the linear dependence of tm-l on E confirm that Joule heat is sufficiently dissipated in the device up to at least 655 V/cm. Sample Injection and Detection. Electrokinetic injection of sample from channel 2 results in different injected plug lengths for each ion, due to their differences in mobility. The plug length of an injected component, li, is given by16

0.05

0.10

0.15

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PLUG LENGTH [cm] Flgurr 7. Peak area as a function of the calculated plug length for the FITC-labeied amino acids Arg and Phe. T a b l e 11. Calculated P l u g Length Dependence on P e a k

Areasa

sample I fluorescein (n = 4) fluorescein 5(and 6-)sulfonic acid (n = 3) sample I1 Arg (n = 4) Phe (n = 4) Gln (n = 4)

interceptb (pm)

slopeb (pmlarb units)

SEc (rm)

35.7 f 6.7 64.9 f 26.3 5.7 f 10.3

0.089 f 0.001 0.40 f 0.007 0.037 f 0.001

f7.8 f23.0 f8.2

141.2 f 12.9 99.1 f 32.2 39.7 f 15.3

0.978 f 0.012 0.694 f 0.026 0.822 f 0.020

f14.5 f35.5 f16.3

Parameters of linear regression analysis of Calculatedplug length, (pm) vs peak area, Ai (arb units) (lin,,i = slope X Ai intercept). The analysis includes plug lengths in a range from 200 to 1600 pm. The correlation coefficienta of all linear regressions were larger than 0.996. fSD. Standard error.

+

linj

*

(3) where Ci is the component's concentration and ki is a proportionality constant which depends on the detector design, the detector and device geometry, and the optical properties of the analyte. The same data used to evaluate the pi were integrated and subjected to proportionality analysis of peak area vs migration time. The slopes obtained (kiliCi) are given in Table I. The low standard deviation in the slopes indicates that the detector response is proportional to concentration in the range studied. The relationship between the calculated plug length and the experimentally measured peak area was evaluated by varying the injection parameters (injection voltage and injection time). The separation voltages were kept constant at 4 and 6 kV for the fluorescein derivatives and for the amino acid mixture, respectively. The smallest injection was made

with 9 V for 10 s, the largest with 1 kV for 60 8, whereas the time between the separations was kept constant at 70 8. Plots of the peak area vs the injected plug lengths calculated with eq 2 are shown in Figure 7 for Arg and Phe. For plug lengths greater than -200 pm a linear correlation with peak area was observed, while the relationship deviates for smaller plugs. Data in the linear region were subject to linear regression of plug length on peak areas. The slope (l/kit,Ci) and intercepts (pm) obtained for both fluorescein and the various derivatives are reported in Table 11. At this state it is unclear whether the greater indeterminant error lies in the areas or the plug lengths, and we have arbitrarily chosen to treat li as the dependent variable in the analysis. This offers the advantage that the uncertainty is expressed in units of plug length, which are easily interpreted in terms of the injection performance. The high linearity (R2 > 0.996) of the linear regressions shows that, by careful cycling of injection and separation, it is possible to inject a well-defined sample plug of length 2200 pm into the separation channel. The standard error given in Table I1 correspondsto the error in the mean of the plug lengths studied (-900 pm) and indicates the average precision was about f2 % for a 900-pm plug. Plug lengths of 200pm are about the minimum length used with electrokineticinjections into fused-silicacapillaries, whereas typical injection lengths are 5-10 times larger." For all sample components the peak areas still decrease with smaller injected plug lengths, as calculated using eq 2 and shown for Arg and Phe in Figure 7. Extrapolating from the linear range, the smallest values for the areas correspond to injection lengths in the range of 100pm, corresponding to

(16) Huang, X.; Gordon, M. J.;Zare, R.N.Anal. Chen. 1988,60,375377.

(17) Huang, X.; Coleman, W. F.; Zare, R.N. J . Chronatogr. 1989,480, 95-110.

where Einjis the electric field applied during injection and ti,j is the period it is applied for. From this expression it is possible to estimate the plug lengths, ignoring any diffusion, possible convective effects, or electric field distortions at the channel intersection. For a detector response that is proportional to analyte concentration the peak area, Ai, will be proportional to the amount of sample injected and be described by

A; = kiZitmCi

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1 pM fluorescein pH 8.0 buffer 168 V/cm separation

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( G C )

Figure 8. Peak area for 1 pM fluoresceln, pH 8.0, vs square root of Injection time, with no applied voltage during Injectlon. This tlme Is simply the period between two appllcations of the Separationpotential.

an injected volume of 30 pL and thus to 300 amol of the component. The amount injected is larger than what would be estimated from the calculated injection plug length when it is below 200 pm. This effect is likely due to the influence of diffusion during injection on the sample plug shape and concentration gradient at short times, as discussed below. The negative intercept observed in Figure 7 for extrapolation of the linear region of the data may arise from the fact the background level during sample separation includes a contribution from sample leakage from the side channel. Diffusional leakage from the sample channel during the separation step will partially deplete the concentration near the intersection within the sample channel. Consequently, sample injection would first require the sample solution move a distance along the side channel before much is introduced into the separation channel. This means the sample plug length calculated from eq 2 would be overestimated, which would result in an offset on the x-axis and a negative y-intercept. Any convective flow (leakage) from the side channel during separation, arising from the Venturi effect or from hydrostatic pressure differences between the reservoirs, would reduce the magnitude of the offset by moving'fresh sample solution toward the intersection. In fact, to obtain the positive deviation from linearity observed in Figure 7 for short sample plug lengths appears to require a convective effect. Otherwise diffusion during the 10-60-s injection would not compensate for diffusional losses from the side channel during the 70-s separation. Further detailed study of solvent flow at the intersections will be required to evaluate these effects. Dose and Guiochon12calculated that for injections into fused-silica capillaries the amount of sample diffusing into the capillary is approximately proportional to the square root of time after immersion in a sample solution. We wished to evaluate the contribution of diffusion to dispersion of the injection plug. To do this we allowed sample to diffuse out of channel 2 into the intersection for a fixed period of time without using an injection voltage. The 1 p M fluorescein sample plugs were then separated under fixed conditions (168 V/cm, did = 6 cm, constant separation time). Figure 8 confirms experimentally the theoretical prediction,12 a t least for the more complex geometry involved in valveless injection at the channel intersection of the planar device and longer delay times. However, there appear to be two regions, with the linear behavior offset from the origin a t short times. This may arise from nonlinear (i.e,, radial) diffusion effects a t short times due to the geometry of the T-shaped intersection and is certainly related to the offset observed in Figure 7. A plot of peak area vs injection time (not shown) was highly

200

300

MIGRATION TIME

400

[SI

Figure 9. Theoretlcal plate helght, Hhvs migration time for the FITClabeled amino acids Arg and Phe. The migration tlmes correspond to separation voltages In the range from 2 to 10 kV. The Injection parameters (250 V for 60 s) were kept constant.

nonlinear, indicating that some form of convectiveflow during sample injection did not lead to the distortion in the expected area vs t112dependence. Figure 8 also shows that sample is injected above background levels for injection (diffusion) times as short as 1 s. To obtain a small, well-defined plug shape the data show that it is crucial to reduce diffusion effects by keeping time delays between injection and separation and between cycles to a minimum. The delay periods should also be as precisely constant as possible. The planar, multichannel manifold construction should prove better suited for achieving this than conventional CE in which mechanical transfer of the capillary between solutions is required. Separation Efficiency. The most important on-column contribution to band-broadening in CE is longitudinal diffusion, for electric fields of 300 V/cm or less.17 Off-column contributions arise from the injected and detected volumes and the detector response time. In the planar device there is an additional contribution from dispersion of the sample during the delay time and injection period, and the variance contributed due to this term can be approximated as w i t & where Di is the diffusion coefficient of component i and td is the combined delay and injection time. Considered together these effects lead to

20. Hi = - % m , i did

w. .!I

2

in] Wdet + td) + +12did 124,

(4)

where Hi is the height equivalent to a theoretical plate of species, i, tmi is its migration time, and winj,i and Wdet are the lengths of the injected plug and the detector ~ e l l . ~ JIt~isJ ~ assumed that these are rectangular in shape in obtaining the relationship in this form. This expression predicts a linear relationship between Hi and migration time when td is kept constant. In Figure 9 a plot of Hi vs migration time is shown for the labeled amino acids Arg and Phe. The plate height does decrease linearly to -2.2 pm, but then it tends toward a constant value at applied voltages higher than -6 kV (393 V/cm). Analysis of the linear region according to eq 4 gave diffusion coefficients of (3.8f 0.5) X 10-6 cm2/s(n = 3) and (4.3 f 0.1) X 10-6 cm2/s (n = 4) for labeled Arg and Phe, respectively. The time-independent variances, obtained from the intercept of the plots were (12.7 f 0.2) X 10-4cm2for Arg and (12.4 f 0.6) X 104 cm2 for Phe. (18)Sternberg, J. C.Adu. Chromatogr. 1966, 2, 206-270.

ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1093

The off-column variance may be estimated from the appropriate terms in eq 4. The sum of the variances of the detector cell (W&t = lOpm), the calculated injected plug length (for Arg wid = 1200 pm, for Phe W i d 954 pm),and the dispersion during injection and delay is 17.2 X lo-" and 13.6 X lo-" cm2 for Arg and Phe, respectively. The calculated value for Phe compares quite well with the experimentally obtained value, whereas the one for Arg is higher by 26 % The calculated value does not include the contribution of the detector response time to H,.However, at a value of 40 me this effect contributes only0.37 X lo-" cm2to the totalvariance for the fastest eluting and sharpest peak, so it is not significant.18 For the data shown in Figure 9 a separation voltage of 4 kV between reservoirs 1and 3 correspondsto 260 V/cm, which is a value typically in use in CE, and the plate heighta in CETAS under these conditions for labeled Arg and Phe are 2.4 and 3.2 pm, respectively. These are comparable to a value of 2.5 pm reported in fused-silica capillaries for FITC-labeled amino acids.l3 We note that the delay time contribution can be easily reduced, as can the rather large injection plug length, leaving room for further increases in efficiency. For an injection time of 2 s at 37.9 V/cm (200 V/cm) the value of plate height, H, contributed from the column is not controlled just by the longitudinal diffusion term and can in fact increase with increasing velocity (increasingpotential). They have argued that this effect can be ascribed to wall interaction or adsorption leading to nonequilibrium contributions to band-broadening. This is consistent with the trend shown in Figure 9 at short migration times (E > 400 V/cm), where H tends toward a voltageindependent value, rather than following eq 4. The peaks were seen to exhibit some tailing at higher voltages. Similar observations were made for the fluorescein dye mixture as the electric field was varied from 229 to 665 V/cm. For fluorescein, the first eluted component, a minimum plate height of 5.5 pm was found at the lowest electric field. At higher electric fields the peak showed a distinct tailing,leading to larger plate heights of up to 12 pm at 655 V/cm. In contrast, the two peaks due to fluorescein sulfonate showed a linear decrease in H with t, to a minimum of 3.7 pm for the last peak, but then reached a plateau and began to climb again at fields exceeding 500 V/cm. The peak tailing and the observation that the faster eluting, less negatively charged component exhibita less ideal behavior strongly supports the existence of adsorption effecta. Notably, these effecta were observed at high fields for both the FITC-derivatized amino acid and the fluorescein dye samples we examined. The adsorption-desorption rate is apparently high enough that there ia little effect at the lower electric fields used in a previous study or at the lower fields used in the present work for all compounds except fluorescein. To verify the factors contributing to band-broadening at lower electric fields, the separation voltage was kept constant at 6 kV, while the injected amount was varied. Figure 10 shows a plot of the theoretical number of plates, N,vs the injected plug length calculated with eq 2 for Arg and Phe. The solid lines were calculated from eq 4 and N = didH, taking longitudinal diffusion during injection and separation, response time of the detector, and the length of the detection cell into account. For calculation of the contributions of longitudinal diffusion, the diffusion coefficients evaluated from linear regression analysis of Figure 9 were used. The good agreement between the experimentallyobtained values and the calculated curves indicates that contributionsby large

-

.

1487

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3E

'

60000

L

6m

32

40000 20000

0

I

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.

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' ' ""'-

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10.'

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loo

PLUQ LENGTH [cm] Flguro 10. Theoretical n u m k r of plates vs the calculated plug bngth. The separatbn voltage between r e w o k 1 and 3 was kept constant at 8 kV. The eoM lines were calculated with eq 4.

injection plugs are as anticipated. It further shows that no additional contributions to band-broadening have to be considered when small sample plugs are injected and separated below a field of r100Vlcrn. The data in Figure 7 indicate that injection plugs of less than 100 pm are not achieved in the CETAS device, so the shortest plug "lengths" in Figure 10 are not realistic. Nevertheless, plug lengths of 200 pm give plate numbera on the limiting theoretical plateau, indicating the efficiency is limited by diffusion (since the detector contribution is very small). Figure 10 indicatesthere is a maximum injection plug length that should not be exceeded if high efficiency is required. For these data, obtained with 3770 V dropped between the injection and detection points, the maximum average plate number was 63000. The resolution of Arg and Phe was estimated using eq 5.19 A value of 14 was obtained, while the

-

(5) resolution per volt was 3.7 X lO-3/V, and the number of plates per volt was 16.6/V. These values may be compared with those observed in a conventional fused-silica capillary with 25 kV applied. Cheng and Dovichi13 have reported resolution of 15 amino acids in such a system with a resolution of between 36 and 70 for Arg and Phe, correspondingto a resolution of (1.5-3) X 10-W and 16-32 plates/V. (The range depended on the capillary age.) Monnig and Jorgenson16 reported a resolution of 19 for Arg and Phe with 7000 V applied, in a system exhibiting a resolution of 2.7 X lO-s/V and 12 plates/ V. These values agree reasonably well with those obtained in CETAS and indicate that separation of complex sample mixtures will be possible, particularly at higher applied voltages. Channel Geometry. The observation of wall interaction effecta in this work and that of Novotny et al.14indicates that there may be a limit to the maximum electric field used. Increased efficiency would then require longer columns than achieved in the CETAS design. Ideally this could be accomplished by coiling the column on a chip or making it follow a serpentine path. However, the effect of such coiling on efficiency requires examination, since the presence of corners will create nonuniform electric field and solvent flow profiles. Although lithographic procedures are readily capable of forming curved shapes, many mask design programs do not allow for such geometries, so that angular shapes may have to be used. A right-angle corner is one of the worst case (19)Jorgenson,J. W.; Lukacs, K.D.Anal. Chem. 1981,53,1298-1302.

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ANALYTICAL CHEMISTRY, VOL. 65, NO. 10, MAY 15, 1993

examples in which to study this effect, and several corners were available for study in the CETAS device. The influence of rectangular corners on the separation efficiency was tested with a sample of 10 pM fluorescein in a 10mM borax buffer (pH 9.1). Again, the sample was injected from side channel 2, but the detector was now located at (ii) (see Figure 1). The distance between injection and detection corresponded to a separation length of -0.85 cm and included two corners. When a voltage of +5 kV was applied between reservoirs 1and 3 (1at GND), a retention time of 33.17 f 0.03 s (SD, n = 5) and a theoretical plate number of 1354 f 94 (corresponding to 4.9 f 0.3 plates/V) were obtained for fluorescein. The potential across the distance did in this experiment was 278 V (327 V/cm). The number of plates is comparable to the 4.8 f 0.2 plates/V obtained in the straight channel over a separation distance of 1.2 cm, when a voltage of 4 kV was applied. This potential corresponds to 314 V across did (262 V/cm). To keep the contribution by the injection as small as possible, a voltage of 10 V was applied for only 5 s, which corresponds to a -1OO-pm plug length, based on Figure 7. This sample plug size represented 1.6% of the total observed variance of the peaks, assuming a rectangular injection plug shape, although it is likely somewhat larger. Within the constraints of the CETAS geometry we did not observe a significant decrease in efficiency induced by the two corners. To determine the effect more precisely, separation channels with more corners per unit length will need to be fabricated. Our results indicate that long channels can be integrated on a small area without dramatic loss of efficiency. The integration of a 50-cm-longcolumn (30pm wide) in a serpentlike geometry with two corners per 0.85 cm would need an area of just 0.65 cm2 (dimensions of 0.84 by 0.77 cm). This calculation assumes that 30 kV is applied over the whole channel and that the electric field strength between the

channels (Le., through the glass) does not exceed a value of 105V/cm. Together with the injection channel an area of less than 1 cm2 would be needed.

CONCLUSIONS We have previously suggested that a set of capillary manifolds integrated on a planar device can act as a chemical analysis system with both flow injection analysis and sample separation capabilities. In this work we have shown that using electrokinetic phenomena as the driving force it is possible to achieve many of the required features of such a device. By increasing the accessible voltage range and improving our detector we have increased the separation efficiency in a planar device about 4-fold over our previous results and shown that more challenging samples may be separated. While the increased voltage has also shown that wall adsorption effects may be present to some extent in the glass capillary, such effects have also been reported in conventional fused-silica capillaries. The demonstration of quantitative analysis of sample composition, and the ability to reproducibly and repetitively inject and separate sample plugs, are key steps in developing the p-TASconcept. Their realization showsthat an integrated system can perform quantitatively and suggests that even more complex sample-handling steps should prove possible.

ACKNOWLEDGMENT We thank the Natural Sciences and Engineering Research Council of Canada, the University of Alberta, and Ciba-Geigy for support. RECEIVEDfor review November 2, 1992. February 10,1993.

Accepted