Anal. Chem. 1993, 65, 3313-3319
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This Research Contribution is in Commemoration of the Life and Science of I. M. Kolthoff (1894-1993).
Continuous Electrophoretic Separations in Narrow Channels Coupled to Small-Bore Capillaries J. M. MesarosJ G. Luo> J. Roeraadef and A. G. Ewing*J 152 Davey Laboratory, Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, and Department of Analytical Chemistry, The Royal Institute of Technology, S-10044 Stockholm, Sweden
Continuous zone electrophoretic separations in channels have been demonstrated. This new technique has the potential to continuously sample and separate analytes from volume-limited microenvironments. A small-bore capillary is used to electrophoretically sample, but not separate, a mixture of dansylated amino acids. The capillary is coupled to a quartz channel structure in a manner which allows continuous injection of the sampled material into the channel. The channel functions to continuously separate the sampled material via electrophoresis. A laser-induced fluorescencedetection scheme,which involves two fiber optic arrays situated at the channel exit, monitors eluting analytes. A continuous separation of dansylated amino acids on the time scale of a few minutes demonstrates the utility of the technique. Sampling has been performed continuously up to 400 s, and initial detection limits are -30 pM. INTRODUCTION The development of analytical techniques which sample and separate material from microenvironments has become a priority in many laboratories. In particular, techniques which can manage nanoliter to picoliter quantities are needed in the investigation of processes at the single-cell level. Capillary electrophoresisl-6 and microcolumn liquid chromatography'ss are existing techniques which can be used in the analysis of single cells. Although these techniques have the ability to analyze microenvironments, they cannot continuously sample material over long time periods. A continuous technique to sample and separate materials from microenvironments would offer several advantages over techniques capable of only incremental analysis. With a continuoustechnique, processes which occur in or near a single cell could be monitored over time periods of several minutes to hours. The long-term response of cells to external stimuli
* Author to whom correspondenceshould be addressed. t The
Pennsylvania State University. t The Royal Institute of Technology. (1) Chein,J.B.;Wallingford,R.A.;Ewing,A.G.J.Neurochem. 1990,54, 633-638. (2) Olefirowicz, T. M.; Ewing, A. G. Anal. Chem. 1990,62,1872-1876. (3) Olefirowicz, T. M.; Ewing, A. G. Chimia 1991,45, 106-108. (4) Jorgenson, J. W., Lukacs, K. D. Science 1983,222,266272. (5) Ewing, A. G.; Wallingford, R. A.; Olefirowicz,T. M. Anal. Chem. 1989,61, 292A-303A. (6) Lee, T. T.; Yeung, E. S. Anal. Chem. 1992,64, 3045-3051. (7) Oates, M. D.; Cooper, B. R.; Jorgenson, J. W. Anal. Chem. 1990, 62,1573-1577. (8) Cooper, B. R.; Jankowski, J. A.; Leszczyszyn, D. J.;Wightman,R. M.; Jorgenson, J. W. Anal. Chem. 1992,64,691-694.
A.
quartz plates
\ 4810 110 T
cm channel)--( spacers
B.
2.5 cm
1 L
2 - 7.8 cm
channel spacers
+ flow
I
.
A %
2 - 7.8 cm
>
Flgure 1. (A) Perspective view of the channel showing plate length, plate width, and placement of channel spacers. Plates were 5 mm thick. (B) Top view of the channel showing flow axis in relation to the placement of the channel spacers.
or pharmacological agents could then be examined. In addition to the investigationof biological microenvironments, a continuous technique could be applied to analyses where low sample volumes are desirable or necessary. These may include the monitoring of chemical concentrations in process streams or from toxic environments. In the research described in this paper, a new analytical technique is outlined which has the potential to continuously monitor the chemical composition of microenvironments. The scheme involves electrophoresis in narrow channels with rectangular cross sections to affect a continuous separation. Electrophoretic separations in structures with rectangular cross sections were first described by Tiselius in 1937.9 In 1989, Jansson et a1.lO published the results of a theoretical examination which compared structures with circular and rectangular cross sections as applied to zone electrophoretic separations. Their calculations indicated that more efficient heat dissipation would occur with rectangular structures. Tsuda et al.11 used rectangular capillaries in the zone electrophoretic separation of dansylated amino acids and pyridoxine. Efficient heat dissipation in the rectangular structures allowed larger sample volumes to be employed. The technique developed in our research has taken advantage of the large sample capacity of channel structures to achieve a two-dimensional, continuous electrophoretic separation.
EXPERIMENTAL SECTION Chemicals. a-Dansyl-L-arginine hydrochloride, Ne-dansylL-lysine, dansyl-L-alanine cyclohexylammonium salt, and 3-(cy(9) Tiselius, A. Trans. Faraday SOC.1937,33, 524-530. (10) Jansson, M.; Emmer, A.; Roeraarde, J. HRC CC,J. High Resolut. Chromatogr. Chromatogr. Commun. 1989,12, 797-801. (11) Tsuda, T;Sweedler,J. V.; Zare, R. N. Anal. Chem. 1990,62,21492152.
0 1993 American Chemical Society QQQ3-27QQ/93/Q3653313$Q4.QQ/ Q
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 22, NOVEMBER 15, 1993 collection fiber opticarray
J-,
excitation fiber optic array -325 nm
Flgure 2. Fiber optic array detection scheme. The excitation fiber optic array was secured to the bottom channel plate using a Hoffmann screw clamp (not shown). The collection fiber optic array was secured onto the side of the top channel plate.
clohexy1amino)-1-propanesulfonicacid (CAPS) were obtained from Sigma (St. Louis, MO). Buffer was prepared by dilution of proper amounts of CAPS in doubly distilled water. The pH was adjusted with 1M NaOH. All standards were prepared in CAPS buffer. Buffers and standards were refrigerated prior to use. Sample Injection. Fused-silicacapillaries,38-41-pm i.d., 144pm o.d., 60-cm length (Polymicro Technologies, Phoenix, AZ), were used for continuous injection of analytes into the channel. Prior to each experiment, a capillarywas filled with CAPSbuffer via a solventreservoir pressurized to 60-80 psi with helium. With the anodic electrode placed in the sample reservoir, a Spellman (Plainview, NY) power supply was used to apply high voltage acrossthe capillaryfor continuoussample injection. The operator was protected from the high-voltage end of the capillary via a Plexiglas box interlock system. Capillaries were filled with analyte via electromigration before each experiment. The capillary was attached to a sliding platform (Newport Research Corp., Fountain Valley, CA) which was coupled to a stepper motor (Hurst, Princeton, IN). Approximately 1mm of the end of the capillary was placed into the entrance of the channel. The motor controlled movement of the capillary across the entrance of the channel in 25.4-pm steps. The motor driver (built in-house) was computer controlled via a 24-bit parallel digital input/output interface (National Instruments Corp., Austin, TX) installed in an IBM PS/2 computer (Armonk, NY). The software to control the motor movement was written in-house. Channels and Channel Reservoirs. Ground and polished quartz plates, 2-7.8 cm long, 2.5 cm wide, and 5 mm thick, were used to construct the channels (Figure 1). Plates were obtained from Quartz ScientificInc. (Fairport Harbor, OH) in either 6.8or 7.8-cm lengths and were cut to appropriate lengths in-house. All edges of the quartz channel plates were beveled, except for the channel top plate edge supporting the fiber optic collection array (discussed under Fiber Optic Arrays and PDA). The beveled edges allowed up to 1mm of the end of the capillary to be inserted and held in the channel entrance. Channel top plates were cut at least 1.7 cm shorter than bottom plates to allow placement of the fiber optic excitation array. The thickness of the separation space between the channel plates was controlled using glass microspheres (Duke Scientific Corp., Palo Alto, CA) mixed in UV cure adhesive (Pacer Technology, Rancho Cucamonga, CA). By use of different diameter microspheres, the channel spacer thickness could be varied from 48 to 110 pm. The mixture was applied in -1mm-wide strips along the length of the channel plates parallel to the intended flow axis. The channel plates were then held together with Hoffman screw clamps and cured under a 50-W mercury lamp (Carl Zeiss Inc., Oberkochen, Germany) for -20 min. Adhesive (Miller Stephenson, Danbury, CN) was used to attach 0.5-cm-wide plastic strips, cut from Nalgene bottles, onto the channel sides and bottom. The result was a plastic enclosed region on the outside of the channel. When a channel was fitted into the reservoirs (see below), hot glue was applied to the plastic region and the channel was pressed into the reservoirs. The use of the plastic was necessary to avoid leaks across the channel reservoirs,as the hot gluedid not bond well to quartz. Separations were carried out in the center 1-1.5-cm portion of the channel
A.
HIGH VOLTAGE
6.
flow
-3. (A)Experimentalse~forcontinuouselectrophoresis.Sample was placed in reservoir 1. The capillary was attached to a stepper motor which moved it across the entrance of the channel. The channel was suspendedacross buffer reservoirs 2 and 3. (B) Imageof sequential stages of a separation process in the channel for three analytes with different electrophoretic mobilities. The image on the left shows the analytes first beingdepositedinto the channel. The middle image shows the migration paths after the capillary has been moved to the middle of the channel. The image on the right shows the capillary at the end of the channel entrance.
to avoid effects of the spacers on the electroosmotic flow. After each day of experimentation, the channel was immersed in water for -24 h until the spacers weakened and the channel could be easily disassembled. The channel was then reassembled for the next day of experimentation. This disassembling and reconstruction of the channel served two purposes. First, the glass microspheres could be easily changed between experiments to alter channel internal height. Second, problems associated with weakened or poorly made spacers would not be propagated to the next experiment. The reservoirs were constructed from the bottom halves of two rectangular Nalgene bottles. A rectangular section was cut from the sides of the bottle halves, and the halves were hot glued together with the rectangular openings matched. The channel was glued into the rectangular opening. Prior to each experiment, the channel/buffer reservoir assembly was placed in an ice bath to aid in heat dissipation. A Bertan (Hicksville,NY) high-voltage power supply was used to apply the separation potential across the channel. The ground electrode was placed in the buffer reservoir, which supported the sample introduction side of the channel. No safety interlock system was used with the channel high-voltage power supply; therefore, care was taken during experimentation to ensure that both high-voltagepower supplies were shut off before the channel was handled. Fiber Optic Arrays and PDA. Fiber optics (100-pm core, 110-pmcladding, 125-pmbuffer) were obtained from Polymicro Technologies (Phoenix, AZ). Excitation fiber optic arrays were constructed in house from 80 fibers, each 50-60 cm long. Individual fibers were placed side by side in a linear array and covered with epoxy (5 Minute; Devcon Corp., Danvers, MA). After the epoxy cured, the ends of the fibers were covered with adhesive (Norland Optical Adhesive No. 68; Norland Products, Inc., New Brunswick, NJ). The opposite end of the excitation arraywas gathered into a bundle, secured with epoxy,and covered with adhesive as above. Both the array and the bundle end were polished with 600-gritCarbimet paper strips (Buehler,Lake Bluff, IL) followed by fine polishing with 1-63-pm A1203 or Sic Unilap lapping film (Photonics Inc., Hicksville NY). Polymicro Technologies supplied one fiber optic excitation array which was constructed from 160 fibers. A Hoffman screw clamp was used to attach the excitation array to the bottom plate of the channel. The excitation light source was the 325-nm line (10 mW) of a HeCd laser (Liconix,Sunnyvale, CA). The laser beam was either
ANALYTICAL CHEMISTRY, VOL. 65, NO. 22, NOVEMBER 15, 1993
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472 s
0.16 V
time
0.00v
0.0s 1
diode number
512
Flgure 4. Continuous migration of 20 mM dansyk-alanine in 24 mM CAPS buffer, pH 9.64. Channel top and bottom plate lengths were 2 and - 5 cm, respectively. Spacers were created using 109-pmdiameter glass microspheres in UV-cure adhesive. The capillary was 3&pm i.d., 144-pm o.d., and 60-cm length. Channel and capillary separation potentials were -400 V and + I 5 kV, respectively. The capillary was moved across the channel entrance for 400 steps. The collection and excitation fiber optic arrays were both constructed from 160 fibers. The excltation array was supplied by Polymicro Technologies Inc. and was slightly modified. The excitation array bundle was completely illuminated by placing a plano convex lens in the laser beam path and positioning the bundle past the focal point of the converged beam. A total of 2500 scans were collected with every 5 scans averaged for the above topographic representation. The first scan of the averaged data was backgroundsubtracted. The channel current was not recorded for this experiment.
coupled directly into the excitation fiber optic bundle or a plano convex lens was used to diverge the laser beam before coupling to the bundle. To avoid UV damage to the eyes, UV-grade safety goggles were worn when the laser was in operation. Collection fiber optic arrays were constructed in-house from 150-160 fibers each 40-50 cm long. Epoxy (5 Minute) was used to attach the collection array directly onto the channel top quartz plate as shown in Figure 2. The edge of the plate, which faced the inside of the channel, was not beveled. The opposite end of the collection array was also arranged in a linear array configuration and was placed directlyagainsta photodiode array(EG&G Reticon, Sunnyvale, CA). Care was taken when the collection arraywas constructed to ensure that each fiber optic at the photodiode array (PDA) end corresponded to its same position at the channel end of the array. Both ends of the array were covered with adhesive and polished as discussed above for the excitation array. The PDA was received outfitted with a fiber optic face plate, which allowed direct coupling of the fluorescent signal from the collection fiber optic array to the diodes. The PDA had a total of 512 photodiodes. Each diode was 2.5 mm high with 50-pm center-to-center spacing between diodes. Data Acquisition and Treatment. The PDA data acquisition rate was 73 ps/diode and was controlled via mother and satellite evaluation boards (EG&G Reticon, Sunnyvale, CA). A 16-bitMC-DAS interface (ScientificSolutions,SolonOH) coupled to an IBM PS/2 computer was used to collect the PDA signal. The software to control data acquisition and treatment was
written in-house. Software-controlledoptions for data treatment included background subtractionand signal averaging. For each experiment, several scans of the PDA were collected before the migrating analytesreached the end of the channel. These initial scansrecorded any background signaldue to the buffer. In cases where no signal averaging was used, one of the initial scans was considered the background for the entire experiment and subtracted diode by diode from all the scans collected for the experiment. Data averaging was accomplished by taking the average of a designated number of scans and was used primarily to condense large data sets into manageable sizes for graphing and storage. Background subtraction from averaged data seta was done by subtracting one of the first averaged scans.
RESULTS AND DISCUSSION The key component in our continuous separation technique is the channel structure. The internal height of the channel can be varied from 48 to 110 pm, which makes it suitable to manage small volumes of analyte. The usable internal width of the channel is 1-1.5 cm, allowing multiple injections of material to be made across the channel entrance. These two aspects of the channel make it extremely useful asa continuous separation structure for microvolume analysis. The experimental setup for continuous electrophoresis in narrow channels is shown in Figure 3A. The setup involves three
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ANALYTICAL CMMISTRY, VOL. 85, NO. 22, NOVEMBER 15, 1993
0.20 v
V
:
signal
a-dansyl-I-arginine
NE dansyl-I-lysine
signal
0.00 v
os
time
420 s
Flgwe 5. Signal vmus time response for dbde 280 from the data represented In Figure 4. The data were not signal averaged. Scan 1 was background subtracted. 0.IOV
, 261
j
144 s time 285 s Figure 7. Slgml versus time response for dbde 279 from tho data represented In F W e 6. No data averam was used. Scan 1 was background subtracted. Signal scale dlffers from Figure 6 becaw no -1 avemglng was used.
Table I. Theoretical and Experimental Efficiencier for a Linear SepPration of Three Amino Acid" a-damyl-barginiie N,-damyl-L-lysine dannyl-talanine
theor efficiency exptl efficiency
signal
owv 1
diode number
512
Flgum 6. Linear separatbn of a 1-8 Injection plug of 5 mM each adansyk-arghlne(eluted Mt), Npansyk-lyslne(middle peak), and dansyk4anlne (duted last) In 24 mM CAPS, pH 10.18. Channel top andbottomplate~were4.9and8.8cm,respectlvely.~channd separatkm space was created using 47.4-pmdiameter glass mlcre spheres In UVcue adhesive. The capillary was 4%" Ld., 14Cm o.d., and 80cm length. Channel and capRlary separatlon potentlab were -550 V and +15 kV, respecthrely. No motor " e n t was used. The cdlectknand excltatkm Rber optk arrays were consttucted from 18Oand80flberoptics, respahrely. The laser beamwas coupled directly Into the exdtatbn amy bundle. A total of 700 scam were colectsd.Everyfhm~weresigralaveraged.Scan3oftheavera~ data was backgound sublracted. The channel cunent was 338 pA.
essential components: the injection capillary, the channel, and the detection fiber optic arrays. The injection capillary continuously samples material and introduces it into the channel. This is accomplished when a high-potential field is applied across the capiuaryand analyte continuouslyelectromigrates though it. In the experiments to be discussed, the analyte is f i t allowed to completelyf i i the capillary via electromigration before injection into the channel. The capillary is attached to a sliding platform which is controlled by a stepper motor. The end of the capillary is placed into the entrance of the channel. The capillary is moved across the width of the channel entrance in 25.4-pm steps, which allows continuous injection of analyte into the channel. The channel is suspended across two buffer reservoirs as shown in Figure 3A. A high-potential field applied acroas buffer reservoirs 2 and 3 results in a net flow of all species toward thecathodedue to e1ectroosmosis.l2 Chargedanalytes ~
~~
(12) Rice, C. L.,Whitehead, R. J. Phys. Chem. 1966,69,4017-4024.
15 100-75 600
12 300-61 600
9 700-48 700
26 OOO
14 600
9500
Theoretical efficiencies, N,were calculated wing the following , + pJV/2D, where k ia the coefficient for equation: N = & electrmotic flow, ia the electrophoreticmobility,Via thechannel separationvoltage, and D ia the diffusion coefficient. Analwere assumed to have a diffusion coefficient lying W e e m 1 X 1V end 6 X lVcm*/s. Experimentalefficiencieean8k++wwerecalculated from the data in Figure 6.
n.2nv
r a-dansyI-I-arginine
signal
-
1 noov
I
1
*' .+-fl--wdL~'.-A4**--..-
, ..?. *',&"A+~&,&**,L*A
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diode number
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Fbure 8. Sbnal v b l s u ~dbde ~ number for a-dansyl++ginlne from the data rep"W In FWe 8. No dcJMI averaging was used. Scan 1 of tho averaged data was backsubtracted. Scale d",from Figure 8 because no signal averaging was used.
separate in the channel due to their electrophoretic mobility.
An image of the continuous injection and separation process involving three species with different mobilities is shown in Figure 3B. As analytmemerge fromthe channel, they must be detected in a manner which retains their spatial integrity across the width of the channel exit. Fluorescence detection utilizing
ANALYTICAL CHEMISTRY, VOL. 65, NO. 22, NOVEMBER 15, 1993
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552 s
I
0.18 V
€7a
time
...
?@
....... ?:%{? n9i’ ...
...I.. ._I.-”
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Flgure 9. Dynamic separation of three dansylated amino acids. A continuous injection of 0.5 mM samples each of cudansykdrginine (eluted first), N&nsyk-lysine (middle band), and dansyk-alanine (eluted last) in 25 mM CAPS buffer, pH 10.20, was made over 38 s. Channel top and bottom plate lengths were 4.9 and 6.8 cm, respectively. The channel separation space was created using 47.4-pm glass microspheresIn UV-cure adhesive. The Capillary was 41-pm i.d., 144-pm o.d., and 60-cm length. Channel and capillary separation potentials were -550 V and 4-12 kV, respectively. The capillary was stepped once every 0.38 s for 350 steps. The collection and excitation fiber optic arrays were constructed from 160 and 80 fibers, respectively. A total of 1440 scans were collected. Every three scans were averaged. Scan 1 of the averaged data was background subtracted. The channel current was 341 pA.
two fiber optic arrays is employed in our system. The placement of the fiber optic arrays is shown in Figure 2. An excitationfiber optic array illuminatesthe exit of the channel with 325-nm light. As fluorescent species emerge from the channel they absorb excitation light and fluoresce. The fluorescent signal is collected via a collectionfiber optic array and is delivered to a photodiode array where the signal is quantified. ContinuousMigrationin Channels. Experiments have been performed to determine whether continuous injection of one analyte species into the channel results in straight and equal migration. In these experiments, dansyl-L-alanine continuously flows into the channel over a time period of 400 s as the capillary is moved 1 cm across the channel entrance. A typical migration path of dansyl-L-alaninein CAPS buffer is shown in Figure 4 as a topographic view of time versus diode number. Figure 5 shows the signalversus time response of a single diode in the central region of the channel. In addition to information related to migration in the channel, this experiment also demonstratesthe abilityof this technique to continuously sample material over relatively long time periods. A closer examination of the topographic representation of the data reveals several features typical of data acquired with this system. The diagonal line represents the fluorescent response of the analyte as it flows under the collection array.
The vertical lines which flank the analyte signal and increase in intensity with time are not due to analyte but are apparently caused by particles, such as dust, becoming lodged at the channel exit and scattering light into the collection fiber optic array. Changes in signal intensity are most likely due to flaws in either excitation or collection array construction. A careful examination of the analyte signal reveals a slightly bowed migration path. This might be due to nonuniformity of the channel quartz plates caused by minute scratches or curved surfaces. Despite these plate irregularities,the flow did not vary greatly throughout the channel structure and the migration paths appear to be straight. The use of higher grade quartz plates in channel construction, with special attention to flatness and other minor surface imperfections, should result in more equal electroosmotic flow throughout the channel structure. The signal versus time graph for diode 280 shows that band broadening, but not band distortion, occurs. The band broadening appears to be due to the high concentration of analyte used. Linear Channel Separations. Small plugs of analyte can be injected into the channel by first positioning the capillary at the center of the channel with the capillary separation voltage off and the channel separation voltage on. Application of the capillary separation voltage with the capillaryheld stationaryresults in a plug of analyteintroduced
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 22, NOVEMBER 15, 1993
into the channel. A 1-8 injection of a-dansyl-L-arginine,N,dansyl-L-lysine, and dansyl-L-alanine into the channel has been made employing this technique to examine the quality of a linear separation. A three-dimensional representation of the separation is shown in Figure 6. The signal versus time response of the diode centered on the peaks, shown in Figure 7, reveals information concerning the amount of diffusionin the channel parallel to the flow axis. This longitudinal diffusion can be examined by a comparison of theoretical and esperimental efficiencies, as shown in Table I. The theoretical efficiencies are determined by use of a range of diffusion coefficients typical of molecules with sizessimilar to the dansylated amines used in the experiment. In addition, the diffusion coefficient for &alanine in water at 25 "C is 9.33 X 10-8 cm2/s;thus the range of D used (1 X 10-8 to 5 X 10-8 cm2/s) in determining theoretical efficienciesis conservative when compared to this value. The experimental efficiencies obtained fall within or near the range of those predicted by theory for molecular diffusion alone. Sources of band broadening other than molecular diffusion couldbe attributedto"atched volume flow rates in the capillary versus the channel, which are 2.30 nL/s and 610 pL/s, re~pective1y.l~This could have lead to channel overloading or irregular flow paths a t the channel entrance. The placement of the excitation fiber optic array may also have caused irregular flow paths by blocking the channel exit. Another source of band broadening could have been due to convection currents caused by joule heating in the channel. Figure 8 shows the signal versus diode response of the scan centered on the a-dansyl-L-arginine peak. The plot reveals information related to diffusion in the channel perpendicular to the flow axis, or lateral diffusion. The amount of lateral broadening due only to molecular diffusion can be estimated by calculatingthe theoretical standard deviation of the peak, u, where u = (2Dt)1/2; (D is the diffusion coefficient, and t is time). Using the retention time of the peak and assuming the diffusion coefficient of a-dansyl-L-arginine lies between 1 X l0-8and 5 X10-8 cm2/s,the expected standard deviation of the peak would be approximatelyO.l&0.40 mm. The peak shown in Figure 6 spans 30 diodes at its base, which corresponds to 1.5 mm. This distance can be used as an approximate value of effective zone width, which equals 40. The experimental standard deviation of the peak therefore equals 0.38 mm, which falls in the range predicted by theory for molecular diffusion alone. Possible sources of band broadening mentioned previously,such as mismatched volume flow rates, blockage of the channel exit by the excitation fiber optic array, and joule heating, may have also been factors contributing to lateral diffusion. Apparent sources of band broadening also need to be considered. These apparent sources are not extraneous band broadening processes occurring in the channel, but are inherent in the signal transduction mechanism used. A sample zone's shape may not be conveyed perfectly by the collection fiber optic array. For example, consider an analyte band migrating under the fiber optic collection array. The fluorescence is collected by the 100-pm core of each fiber optic. Signal may be collected at the edges of the analyte band even if the analyta edge does not completely overlap the fiber optic core area. Therefore, a 125-pm-widepeak may be perceived as a 200-pm-wide peak by the collection fiber optic array. Other sources of apparent (13) Volume flow rates in the capillarywere calculatedby multiplying the velocity of a-dansyl-L-arginine in the capillary by ita crow-sectional area. Volume flow rates in the channelwere dculated by multiplyingthe velocity of a-dansyl-carginine in the channel by the cross-sectionalarea of the channel in which an injection wae made. This cross-sectionalarea wae approximated by multiplying the internal height of the channel by the intarnal diameterof the injection capillary.The velocity of a-dansylL-arginine wae used becam it wm neutral at the pHs ueed.
0.20 v ;.
a-dansyl-l-arginine
Ne dansyl-l-lysine ..i dansyl-l-alanine
..
signal
0.00 v 171 s
time
545 s
FIQW 10. Signal versus time response for dlode 250 from the data represented In Figure 10. The data were not signal averaged. Scan 1 was background subtracted. Assumlng a detectkn llml of 2X the peak-t-k noise, the detection llmlt for N&nsyk-lysine was estlmated to be 30 pM.
band broadening may be due to minor construction flaws in the collection array or slight misalignment of the collection array at the PDA. Dynamic Channel Separations. Continuous injection of 0.5 mM cu-dansyl-Larginine,N~-dansyl-clysine, and dansylL-alanine into the channel has been carried out while the capillary is moved across the channel entrance. The volume flow rates are 1.85 n L / s in the capillary and 530 pL/s in the channel. Figure 9 shows the results in a topographic representation. Figure 10 shows the signal versus time response for a diode corresponding to the center portion of the channel. The unidentified peak that eluted after dansylL-alanine is also seen in capillary electrophoreticseparations of the same three dansylated amino acids and is assumed to be an impurity. The results shown in Figure 9 verify that highly resolved, continuous separations of several species can be performed successfully in the channel system over a time scale of min. The total injection time is 38 s and can be increased by moving the capillary back and forth across the channel entrance. Aa in the case with the continuous injection of daneyl-L-alanine (Figure 4), a slight bowing of the analyte bands can be seen. Again, this could be due to defecta in the quartz channel plates used. The plates used in this experiment contained several visible scratches from repeated use. Other evidence to the cause of the bowed bands is the observation that the effect is most pronounced for the dansyl-calanine band, which elutes last. This indicates that the phenomenon may be caused in part by insufficient removal of joule heat in the channel as the analysis time increases. The signal versus time response for diode 250 shows baseline resolution of the analytes with high signal intensities (Figure
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VOL. 65, NO. 22, NOVEMBER 15,1993
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10). A comparison of the signal for dansyl-L-alanine in this experiment and in the continuous migration experiment (Figure 5) reveals similar signal intensities, despite the large difference in analyte concentrations. This is the result of improvements in the construction of the collection and excitationfiber optic arrays. Also,the laser beam was directly coupled into the excitation array bundle for the continuous separation of the three amino acids, allowing more excitation light to enter the channel. These improvements resulted in an enormous increase in signal generated and collected, and it is expected that further experimentation will reveal other possibilities for refinement. As of these experiments, the concentration detection limit is estimated at 30 pM as calculated from theN,-dansyl-L-lysinepeak in Figure 10. The detection limits are expected to decrease as the system is improved further.
system which utilizes electrochemical detection is under construction. Other modifications, such as the development of a native fluorescence detection scheme involving the use of deep UV lasers to analyze biological samples,@can be envisioned. The separation scheme presents an interestingnew concept for continuous analysis, and much information concerning flow characteristics in the channel can be gathered using fluorescence detection. In particular, different volume flow rates in the capillary versus the channel are being tested to determine whether a focusing effect occurs at the capillary/ channel interface. These factors, and others, are currently under investigation to determine their affect on peak shape and efficiency. Methods to control Joule heating, such as thinner channel plates and salt/ice baths, are ale0 being considered and tested.
CONCLUSIONS
ACKNOWLEDGMENT
Capillary electrophoresis coupled to electrophoresis in narrow channels allows continuous separations of analytes to be carried out. A continuous separation of three dansylated amino acids has been demonstrated. In these separations, complete and continuous resolution of the solutes has been achievedwith concentration detection limits of 30pM. The preliminary results presented in this paper are very promising; however, refinement of the detection scheme is needed. Future optimization in the construction of the fiber optic arrays w i l l be necessary to provide increased consistency in signal transfer by the collection array and equal illumination across the channel exit by the excitation array. In addition to optimization of the fluorescent detection scheme,a channel
The funding for this research was provided by the National Institutes of Health and the National Science Foundation. A.G.E. is a Camille and Henry Dreyfus Teacher-Scholar. Scientific Parentage of the Authors. Jody M.Mesarm, Ph.D. expected under A. G. Ewing, Ph.D. under R. M. Wightman, Ph.D. under R. W. Murray, Ph.D. under R. C. Bowers, Ph.D. under I. M. Kolthoff.
-
RECEIVED for review May 24, 1993. Accepted August 25, 1993.' ~
~~~
Abstract publiihed in Advance ACS Abstracts, October 1,1993.
