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Anal. Chem. 1909, $5, 2637-2042
Glass Chips for High-speed Capillary Electrophoresis Separations with Submicrometer Plate Heights Carlo S. Effenhauser,' Andreas Manz, and H. Michael Widmer Corporate Analytical Research, Ciba-Geigy Ltd., CH-4002 Basel, Switzerland
Micromachinedcapillary electrophoresissystems with integrated sample injection have been fabricated on glass chips using standard photolithographic and etching techniques. The injector permits volume-defined electrokinetic sample injection without sample biasing. Utilization of short separation capillaries and high field strengthsin combinationwith a small sample plug length results in both fast and efficient separations of fluorescein isothiocyanate- (FITC-) labeled amino acids. Analysis times range from a few seconds to a few tens of seconds with corresponding plate numbers of 5800-160 000, respectively. Plate heights down to 0.3 pm have been obtained using a separation length of 24 mm and an electric field of 1 kV/cm. As it turns out, a maximum separationefficiency has been reached, limited only by diffusion and the effects of both injection and detection. Automated repetitive sample injection and separation on a time scale of seconds is demonstrated and provides a route to quasi-continuous on-line monitoring of chemical species in a sensorlike fashion. 1. INTRODUCTION In a recent line of development, capillary electrophoresis (CE) and other separation techniques have been successfully integrated into the concept of so-called Total chemical Analysis Systems (TASI.13 The combination of all sample handling and measurement steps into a single package incorporating a high level of automation makes the TAS an ideal approach for continuous monitoring of chemical concentrations in industrial chemical and biochemical processes. As such, the TAS concept has many potential applications in biotechnology,"b process control,88and the environmental'v8 and medical sciences?Wields in which continuous monitoring has become increasinglyimportant. The TAS user is provided with chemical information in the form of electronic data at short, regularly spaced intervals. The elimination of the dependence on external laboratory analyses should have an
* Author to whom correspondence should be addressed.
(1)Widmer, H.M. Trends Anal. Chem. 1983,2,8. (2)Widmer,H.M.;Erard,J.-F.;Grase,G.Znt.J.Enuiron.Anul. Chem. 1984,18, 1. (3)Graber, N.;Lildi, H.;Widmer, H.M.Sem. Actuators 1990,Bl, 239. (4)Lildi, H.; Gam, M. B.; Bataillard, P.; Widmer, H. M. J.Biotechno2. 1990,14,71. (5) Fillipini, C.; Sonnleitner, B.; Fiechter, A.; Bradley, J.; Schmid, R. J. Biotechnol. 1991,18,153. (6) Techulena, G. Phys. Scr. 1988,2'23,293. (7)Guibault, G. G. Anal. Chem. Symp. Ser. 1983,17,637. (8) b o n d s , T. E. Trends Anal. Chem. 1985,4,220. (9)Shapiro, B.A.; Harrieon, R.A.; Cane, R.D.; Kozlowsky-Templin, R.Clinical Application of Blood Gases; Year Book Medical Publishers Inc.: chicago,1989. (10)Stinshoff,K.E.;Freytag, J. W . ; h k a , P . F.;Gill-Pazaris,L.Anal. Chem. 198S,57,114R. 0003-2700/93/0365-2637$04.00/0
enormous impact on the way chemical and biochemical processes are monitored and controlled. Among several examples of TAS that have appeared in the recent literature are a gas chromatograph-based monitor for trace analysis in air? an on-line glucose analyzer for bioprocess control,ll a supercritical fluid chromatograph-based monitor for process contro1,lZ and high-speed capillary electrophoresis as a detection method for HPLC.13 A logical extension of the TAS concept is miniaturization of these systems,to yield miniaturized TAS, or p-TAS.14 With sample handling, separation, and detection methods incorporated into a single, small probe, the p-TAS would resemble a sensor in many regards, although each function could be under the dynamic control of the user. All sample-handling steps are carried out extremely close to the location where initial sampling takes place. In order to compete with chemical sensors, the total cycle time from sample injection to the generation of an electronic signal proportional to the concentration of a certain chemical species would have to be about as long as the response time of a typical sensor, i.e., in the range from some seconds to a t most a few minutes. This would provide a means to circumvent the severe selectivity and lifetime requirements of "conventional" chemical sensors by incorporation of aseparation step in the analysisprocedure. The fact that electric field-driven separations can be very rapid and a t the same time exhibit excellent resolution performance was demonstrated by Schumacher in 1962. In these experiments, he showed that isoelectric focusing separations of CP+ and Cm3+ can be achieved in less than 90 8.15 Recently, Monnig and Jorgensode reported amino acid separations on a time scale of seconds using CE. In their experiment, small sample plugs were introduced into a short separation capillary by means of a gated laser-induced photolysis technique. Even though this method is well-suited to generating very small sample plugs, its general use is restricted by the complexityof the experiment. Furthermore, this technique leads to plugs having compositions which are not representative of the actual sample, since the individual plug lengths depend on the differingelectrokineticmobilities of these components. Pawliszyn and Wu17 have demonstrated that rapid sample separations in less than 30 scan be achieved using moving boundary CE, which also provides discrimination-free sample injection. In another study, an integrated electrokinetically driven separation system was used to separate a mixture of laser dyes in 36 8, although a t the expense of separation efficiency.18 ~~
(11)Garn,M.B.;Cevey,P.;Gisi,M.;Thommen,C.Biotechnol.Bioeng. 1989. ~ ,.34.423. - -~ ,~~-. (12)Giorgetti, A.; Periclh, N.; Widmer, H. M.;Anton, K.; DHtwyler, P. J. Chromatogr. Sci. 1989,27,318. (13)Bushey, M.M.; Jorgenson, J. W. Anal. Chem. 1990,62,978. (14)Manz, A.; Graber, N.; Widmer, H.M. Sens. Actuators 1990,Bl, 244.
(16)Schumacher, E.J. Lawrence Radiation Lab. [Rep.] UCRL 1962, UCRL-10624, 210. (16)Monnig,C. A.; Jorgenson, J. W. Anal. Chem. 1991,64802. (17)Pawliszyn, J.; Wu, J. J. Chromatogr. 1991,559,111. (18)de Bok, P. K.; Gillissen, E. E. A.; van de Weijer, P.; Bekkers, M. H. J.; van Bommel, C. H. M.; Janwn, H . 4 . J. Chromatogr. 1992,598, 115. 0 I993 Amerlcan Chemlcal Society
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Microfabrication technologies using photolithographic patterning processes were used by Terry et al. in 1975 for the integration of a gas chromatograph onto an entire silicon ~ a f e r . ’ This ~ , ~ important ~ work went unrecognized for over a decade before the fabrication by Hitachi (Japan) of a liquid chromatographic chip containing a capillary column with an electrochemical detector, in 1987 (published in 199OZ1).Since then, the integration of capillary electrophoresis channels into silicon and glass microstructures has been p r o p o ~ e d ~ ~ - ~ ~ and experimentally demonstrated.%% The photolithographic fabrication process allows for the formation of channels of almost any shape, for branched channel systems and for integration of sample preparation, injection, postcolumn Flgure 1. Layout of the glass chip with integrated sample injection; chip dimensions 80 X 70 X 3 mm. The horizontal channel was used reactions, anddetector cells (for areview, see ref 29). Recently, as separation channel. The inset showsdetailsof the channel geometry optical absorbance detector cells,30~3~ rapid CE separations,32,33 in the injection region. Reservoir numbers are indicated. and a column-switchingtechnique called “synchronized cyclic CE”34v35have been evaluated. higher electric field strengths. Optimum efficiency depends The present work reports on both fast and efficient on minimization of all unavoidable sources causing band separations of amino acids carried out on a micromachined broadening (longitudinal diffusion, finite length of injected CE device. An integrated sample injector allows electrokinetic sample plug, etc.) and the elimination of all nonideal effects injection of small sample volumes in a volume-defined like Joule heating and adsorption on capillary walls. This injection scheme. The capillary system was formed in the means that the short separation length imposes strict limits surface of a glass plate using standard photolithographic and on the contributions of injection and detection to the total glass-etching techniques, which provide an elegant and variance, if both objectives are to be obtained simultaneously. versatile route to branched capillary manifolds with virtually The use of a high field strength, although of vital importance no dead volumes.27 The first demonstration of the volumefor short analysis times, can be detrimental to efficiency due defined injection principle to prevent sample biasing was given to Joule heating effects. Fortunately, it turned out that the by Verheggen et al.36 These authors constructed a special high electric field strengths do not limit the efficiency sampling device capable of injecting sample volumes in the attainable in our experiments, due in part to the good power microliter range and plug lengths of several centimeters. In dissipation capability of rectangular-shaped capillaries of our device, this injection scheme has been miniaturized and small cross section etched in a glass matrix.22 electrokinetic pumping has been employed. This way, small sample aliquots of 100 pL with a plug length of 150 Mm can 2. EXPERIMENTAL SECTION be introduced into the separation capillary without utilization Glass Chips. The layout of the glass chips used in this work of external pumps and valves. Although electrokinetic sample is shown in Figure 1. The capillary manifold was formed into the transport is employed, sample can be injected in a nonbiased surface of a glass plate (Hoya SLW) by a standard photolithomanner, as will be described in the following section. graphic procedures7including a wet chemicaletching step. This It is clear that in order to increase the speed of analysis in was performed under contract by Baumer IMT Industrielle CE, shorter capillaries should be used in combination with Messtechnik AG, CH-8606 Greifensee, Switzerland, using their proprietary process. Briefly, this process involves deposition of (19)Terry, S.C.A gas chromatography system fabricated on a silicon separate metal and photoresist layers onto the glass substrate, wafer using integrated circuit technology. Ph.D. Dissertation, Stanford followed by illumination of the substrate through a metal mask University, 1975. containing the capillary pattern. After the photoresist is (20)Terry, S.C.;Jerman, J. H.; Angell, J. B. IEEE Trans. Electron developed, a HF-based solution is used in order to etch the Devices 1979,ED-26, 1880. (21)Manz,A.;Miyahara,Y.;Miura, J.; Watanabe,Y.;Miyagi,H.;Sato, capillarymanifold into the substrate, and the metal layer is finally K. Sens. Actuators 1990,B1, 249. removed. The dimensions of the glass plate are 80 X 70 X 3 mm. (22)Jansson, M.;Emmer,A.;Roeraade,J.J.HighResolut. Chromutogr. In order to seal the capillary system, a second glass plate of the 1989,12, 797. same dimensions was thermally bonded (4 h, 620 “C) on top of (23)Manz, A.; Fettinger, J. C.;Verpoorte, E.; Ludi, H.; Widmer, H. the plate containing the microstructures.27 Holes (diameter 2 M.; Harrison, D. J. Trends Anal. Chern. 1991,10, 144. mm) in the cover plate provide access to the capillaries; pipet (24)Harrison, D. J.; Glavina, P. G.; Manz, A. Sens. Actuators 1993, tips glued into these holes serve as reservoirs. All channels have BIO, 107. (25)Harrison, D. J.; Manz, A.; Glavina, P. G. Transducers ’91,Digest been etched to a depth of 12 pm and a width of 50 pm, except of Technical Papers; IEEE: New York, 1991,91CH2817-5,792. for the four capillaries in the left part of the glass chip (250-pm (26)Manz, A,; Harrison, D. J.;Verpoorte, E.; Fettinger, J. C.;Ludi, H.; width), as is shown in the inset in Figure 1. The smoothness of Widmer, H. M. J. Chrornatogr. 1992, 593, 253. the channel walls, the precision of the fabrication process, and (27)Harrison, D. J.; Manz, A.; Fan, Z.; Ludi, H.; Widmer, H. M. Anal. the shape of the channel cross section are clearly shown on the Chern. 1992,64, 1926. (28)Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chern., in press. electron micrograph displayed in Figure 2. (29)Manz, A.; Harrison, D. J.; Verpoorte, E.; Widmer, H. M. Adu. Sample Injection. Initially, one of the reservoirs was filled Chrornatogr., in press. with buffer solution by use of a microsyringe. The capillary (30)Verpoorte, E.; Manz, A.; Ludi, H.; Widmer, H. M. Transducers systemwas then flushedwith buffer solution by applying pressure ’91,Digest of Technical Papers; IEEE: New York, 1991,91CH2817-5, to this reservoir. In order to ensure equal hydrostatic pressure, 796. (31)Verpoorte, E.;Manz, A.; Ludi, H.; Bruno, A. E.; Maystre, F.; the remaining reservoirs, except reservoir 1,were carefully filled Krattiger, B.; Widmer, H. M.; van der Schoot, B. H.; de Rooij, N. F. Sens. with 50 p L of buffer solution using a microsyringe to prevent the Actuators 1992, B6, 66. formation of air bubbles in the reservoirs. In the same way, 50 (32)Harrison, D.J.;Fan, Z.; Seiler, K.; Manz, A.; Widmer, H. M. Anal. pL of sample solution was added into reservoir 1. Applying a Chirn. Acta, in press. voltagebetween reservoirs 1(+2 kV) and 4 (ground)causes sample (33)Manz, A.; Verpoorte, E.; Effenhauser, C.S.;Burggraf, N.; Raymond, D. E.; Harrison, D. J.; Widmer, H. M. J. High Resolut. Chromatogr., in solution to be pumped electroosmotically from 1 to 4, thereby press. fillingthe 150-pmsection (sampleloop)of the separation capillary (34)Burggraf, N.; Manz, A.; de Rooij, N. F.; Widmer, H. M. Anal. (horizontal channel in Figure 1). After a delay time of 1 I, the Methods Instmm., in press. separation voltage is applied between reservoirs 2 (positive) and (35)Burggraf, N.; Manz, A,; Effenhauser, C.S.;Verpmrte, E.; de Rooji, N. F.; Widmer, H. M. J . High Resolut. Chromatogr., in press. (36)Verheggen, T. P. E. M.; Beckers. J. L.: Everaerts, F. M. J . Chrornatogr. 1988,452, 615.
(37)See, for example: Middelhoek, S.; Audet, S. A. Silicon Sensors; Academic Press: London, 1989;Chapter 7,and literature cited therein.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 19, OCTOBER 1, 1993
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(LeCroy 9310). The whole fluorescence detector is mounted on an X-Y translational stage (Spindler & Hoyer, Gtjttingen, Germany) in order to allow variation of the location of the detection volume in the separation Capillary. Solutions and Labeling Procedure. Amino acids were labeled by adding 100 pL of a 1 mM solution of fluorescein isothiocyanate (FITC) in acetone (with a drop of pyridine added to the stock solution) to 1mL of a 1mM solution of each amino acid dissolved in buffer. After standing in the dark overnight, each solution was diluted to a formal concentration of 10 pM with respect to FITC. A pH 9.0 buffer [20 mM boric acid-100 mM tris(hydroxymethy1)aminomethane (Tris)l was used in all experiments. All solutions were filtered through 0.2-pm filters (Gelman Sciences, Ann Arbor, MI) before use. All chemicals were analytical grade and were purchased from Fluka AG, CH9470 Buchs, Switzerland. Flgue2. ElectronmkrographdlsplaylngdetaUsof the Injectionvolume. The horlzontal capillary represents the separatlon channel; the vertlcal channels in the left and right parts of the mlcrograph are connected to reservoirs 1 and 4, respectively. The distance between the outsMe walls of the lnjectlon channels defining the plug length is 150 pm.
5 (ground), inducing flow of buffer solution from 2 to 5 and carrying the sample plug into the separation channel. In this manner, geometrically well-defined sample plugs of about 100pL volume can be reproducibly injected. An additional capillary leading to reservoir 3 allows injection of two additional plug sizes (injection from 1to 3, 300 pm; from 3 to 4, 400 pm). If the injection voltage is applied for a long enough time, the injection volume will be completely filled with even the sample component possessing the lowest net mobility. The sample composition in the injection volume will thus be the same as in the sample reservoir. Although the concentration of each component in the reservoir will change slightly dependingon its mobility, this effect is negligible due to the large difference in volume between the sample reservoir and the injection capillary, provided the accumulated injection time for repetitive injections is not too long (110o0 8). This is in contrast to the conventional electrokinetic injection procedure, which produces a strongly biased sample plug due to differences in sample mobilities. For sample injection and sample separation,Models HCN 2000 (0-2 kV) and HCN 12500 (0-12.5 kV) power supplies (FUG Elektronik GmbH, D-8200 Rosenheim, Germany), respectively, were employed. Repetitivesample injection was controlled with a programmable timer clock (Alphatronic, D-7513 Stutensee, Germany). Current was monitored during separation by means of a Keithley picoammeter (Model 485). A measurement of the current as a function of voltage applied between reservoirs 2 and 5 showed a perfectly linear behavior up to a maximum voltage of 11.25 kV (corresponding current 1.0 PA). This limitation is caused by the breakthrough voltage of the high-voltage relays used for switching the potentials in our experiment. Laser-Induced Fluorescence Detection. The detection setup used in the experimenta described here was similar to the one described in ref 28. The 488-nm line of an Ar+-ion laser (Omnichrome, Model 532 AP) was coupled into a 600-pmdiameter optical fiber and used as excitation source. The unfocused output (4mW)of the fiber was positioned to illuminate a portion of the Separation capillary under an angle of about 45O. Fluorescence was collected perpendicular to the surface of the glass chip by means of a microscopeobjective (LeitzNPL Fluotar L, 25X, N.A. 0.35, working distance 17 mm) mounted on a homemade microscopebody. After passing a 514nm interference fiiter (Spindler& Hoyer GmbH, Gtjttingen, Germany, fwhm 11.5 nm) and a pinhole placed in the focal plane of the objective (diameter 1 mm), fluorescence radiation was detected with a Hamamatau R1477 photomultiplier tube (PMT). The 40-pm diameter of the viewing window of the PMT is given by the pinhole diameter divided by the magnification of the objective. In our experiment, the Corresponding detection volume amounts to about 15pL. The PMT was mounted in an integrated detection module (SMT, D-8031 Seefeld, Germany, Model NV 30-1) including HV power supply,voltage divider, and amplifier. The amplifier output was smoothed with a low-pass filter (Avens Signal Equipment Corp., Elmhurst, NY, Model AP-255-5)set at 100 Hz and finally recorded using a digital storage oscilloscope
3. RESULTS AND DISCUSSION General Remarks. Recently, several authors have investigated the influence of sample injection on resolution and quantitation in CE in great detail, pointing out the crucial role it plays for the performance of CE.@-41 Since the experiments presented here have been carried out using unusually short separation capillaries and a novel sample injection scheme, a few general remarks concerning analysis time and separation efficiency in CE might be helpful in order to simplify the discussion of the results in the following two sections. The analysis time t in CE (migration time of the slowest detectable sample constituent i) can be calculated from the relationship42
t = L/piE = L2/piV,, where L denotes the distance between the injection and detection points, pi the sum of the electroosmotic (p-) and electrophoretic (pep+)mobilitiesof component i, E the absolute value of the electric field strength in the separation capillary, and V,, the potential drop across L. Short analysis times are basically favored when short separation capillaries are employed in combination with high field strengths or high voltages, respectively. From the experimental point of view, the ultimate limit of the separation efficiency in CE is governedby three principally unavoidable sources of band broadening, namely, longitudinal diffusion and the effects of both injection and detection. If the separation efficiency is expressed in terms of the number of theoretical plates N, the ultimate upper limit N,, is given by eqs 2 and 3
(3) where Di denotes the molecular diffusion coefficient of the component i, U d d , ad2,and Udet2denote the variances due to diffusion, injection, and detection, respectively, and Bid’ denotes the s u m of the injection and detection variances. This theoretical upper limit represents the best separation efficiency that can be attained with any CE apparatus. Alternatively, the plate height H can be used as a measure of efficiency per unit separation length with an ultimate lower limit given by eq 4
H- = 2LIi/piE+ ui2/L
(4) The variances ad2and Udet2 can be written in more detail as (38)Dose, E. V.; Guiochon, G. Anal. Chem. 1992,64,123. (39) Lee, T. T.; Yeung, E. S. Anal. Chern. 1992,64,1226. (40) Delinger, S. L.; Davis, J. M. Anal. Chern. 1992,64,1947. (41) Huang, X.; Coleman, W. F.; Zare, R. N. J. Chromatogr. 1989,480, 95. (42) Jorgenson, J. W.; Lukaca, K. D. Anal. Chern. 1981,53,1298.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 19, OCTOBER 1, 1993
Table I. Experimentally Determined Diffusion Coefficients D, Electrophoretic Mobilities pep, and Effective Charges z Di(10-e cmZ/s) pep/(1G-4 cm2iVs)a tie
Arg-FITC Gln-FITC Phe-FITC Gly-FITC
*
3.9 0.4 3.0 f 0.3 3.4 f 0.3 3.7 f 0.4
-2.6 -3.0 -3.5 -3.7
f 0.2 f 0.2 f 0.2
f 0.2
-1.7 -2.5 -2.6 -2.6
f 0.2 f 0.3 f 0.3 f 0.3
0 pep= p - p w ; pw = 5.4 f 0.2 X 1Wcm2/Vs. Note that the precision of the experimentaldeterminationof the total mobilities p was better than 1%.
N
250 000
200 000
150 000 100 000
+
+
ainj2= linj2/12 2Di(tinj tdl)
(5)
50 000
0
where the injected plug length has been abbreviated to lin,, the length of the detector volume to Id&, and the variance introduced by the detection electronics to ae12. The time during which the sample plug is subject to diffusion in the separation capillary is the sum of the actual injection time tinjand the time delay tdl between the termination of sample injection and the start of the separation. In the formulation of eqs 5 and 6 it has been assumed that both the injection and detection volumes have a rectangular-shaped profile along the capillary axk43 The time constant of the detection system is denoted 7. The variances caused by the finite length of the injection and detection volumes can be calculated to lhj2/12 = 1.88 X lO-5cm2 (49%),and 1,3,?/12 = 0.13 X 10-5cm2(3%) (see also Experimental Section; the figures in brackets are fractions of aid2). A delay time t d of 1s and an injection time of 2 s are typically used in the experiments, which together with the typical diffusion coefficients listed in Table I lead to anumericalvalue of 2Di(tinj+ tdl) = 1.80 x 10-5 cm2 (47%). With the low-pass filter set a t f = 100 Hz,the time constant of the detector can be determined to be T = 1/(27rf)= 1.6 ms. For the fastest component detected in our experiment (ArgFITC), this results in UdI2 = 2.2 X lo-' cm2 (
