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Transverse Flow Gating Interface for the Coupling of Microcolumn LC with CZE in a Comprehensive Two-Dimensional System Anthony V. Lemmo and James W. Jorgenson* Department of Chemistry, University of North Carolina at Chapel Hill, Venable Hall, CB 3290, Chapel Hill, North Carolina 27599-3290
A comprehensivetwo-dimensional(2D) separation system for the study of proteins based on size exclusion chromatography (SEC)and capillary zone electrophoresis (CZE)is presented. A new interface design is used to couple SEC,carried out in packed capillary columns of 250- and 100-Fm inner diameters, together with CZE. This interface design uses a transverse flow of CZE buffer to prevent SEC column effluent from being electroinjected onto the CZE capillary until an injection is desired. This interface provides reproducible CZE injections while introducing minimal extracolumn band broadeninginto the system.The 2D system is completely automated under computer control. The relative merits of this interface design are presented through a comparison between the separations of protein standards using this interface and separations using a valve/loop approach.
INTRODUCT1ON The analysis of complex mixtures often requires more than one separation process in order to resolve all the components present in a sample. This realization has generated a considerable interest in the area of two dimensional (2D) separations techniques. Giddings and Davis are largely responsible for laying the conceptual foundation upon which 2D separation systems are built.l-4 Bushey and Jorgenson introduced the first automated “comprehensive” 2D system that coupled column liquid chromatography (LC) with capillary zone electrophoresis (CZE).5,6 Capillary zone electrophoresis is particularly well-suited as a second dimension separation because high-speed separations can be achieved without significantly sacrificing efficiency and resolution. The system of Bushey and Jorgenson coupled reversed-phase (RP) HPLC with CZE for the analysis of peptide samples. The system operated in a comprehensive manner, and as such, all sample components separated in the RPLC dimension were subjected to separation by CZE (although the entire RPLC elution volume was not reinjected). The coupling of the two dimensions was facilitated by the use of conventional column and sample loop technology. Part of the success of this 2D system was due to this feature. In addition to this system of Bushey and Jorgenson, 2D systems comprised of LC/LC
* Author to whom correspondence should be directed.
(1) Giddings, J. C. Anal. Chem. 1984,56, 1258A. (2) Davis, J. M.; Giddings, J. C. Anal. Chem. 1985, 57, 2168. (3) Davis, J. M.; Giddings, J. C. Anal. Chem. 1985, 57, 2178. (4) Giddings, J. C. HRC CC, J.High Resolut. Chromatogr. Commun. 1987, 10, 319. (5)Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990,62,978. ( 6 ) Bushey, M. M.; Jorgenson, J. W. J. Microcolumn Sep. 1990,2,293.
0003-2700/93/0365-1576$04.00/0
techniques7 and GC/GC techniques8 have also been devised. Work in our group has emphasized designing new 2D systems that couple other forms of LC together with CZE. This paper presents a 2D system that couples microcolumn size exclusion chromatography (SEC) with CZE for the separation of proteins. Our interest in microcolumnsis aimed a t exploiting the increased separation efficiency (theoretical plates) achieved through their use. The relative merita of using microcolumns rather than conventional columns have been previously discussed.9 Microcolumns, and their inherently small column volumes and operating flow rates, pose a considerable engineering problem in terms of designing a suitable 2D interface for use with CZE. We present two different approaches to couplingmicrocolumnSEC with CZE. The first approach is an extension of the valve/loop design used previously by Bushey and Jorgenson. With this approach we achieved limited succesa at coupling a 250-pm inner diameter microcolumn with CZE. As will be shown, currently available valving schemes for sample collection are essentially impractical for use with packed capillary columns. To address this problem, we have designed and utilized a new interface scheme that circumvents collection of sample in a loop and therefore overcomes the shortcomingsassociated with sample collection. The design, operation, and advantages achieved over the valve/loop system using the so-called “transverse flow gating” interface approach are presented.
EXPERIMENTAL SECTION Reagents. Protein standards and buffer reagents were purchasedfromSigmaChemical Co. (St. Louis, MO). Formamide was obtainedfromFisherScientific (Raleigh,NC). Allchemicals were used as received. The buffer used for both separations was 10 mM tricine, 25 mM Na2S04, 0.005% sodium azide (w/v) adjusted to pH 8.23 with NaOH. Buffer solutions were made with deionized water purified with a Barnstead Nanopure system (Boston, MA) and were filtered with 0.2-pm nylon membrane filters from Alltech Associates (Deerfield, IL). Sample Preparation. A mixture of protein standards was made from thyroglobulin (THYRO), bovine serum albumin (BSA),chicken egg albumin (OVA),and horse heart myoglobin (MYO). This mixture also contained formamide (FA). The relative percent (w/v) of these Components varied depending on the interface used to couple the SEC microcolumn to the electrophoresis capillary. For the valve/loop interface experiments, each protein was present at 2% (w/v) with 5% (w/v) formamide. For the flow gating interface experiments, each protein was present at 0.5% (w/v) with 3% formamide. Rather than filtering to remove any undissolved particulate matter, the mixtures were centrifuged at l6000g for 2 min. After centrifugation the supernatants were drawn off and stored at 4 O C . Column Packing Procedure. Two size exclusion microcolumns were used. The first was a 250-pm inner diameter (i.d.) ~
~~
(7) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990,62, 161. (8)Liu, 2.;Phillips, J. B. J. Chromatogr. Sci. 1991,29, 227. (9) Kennedy, R. T.;Jorgenson, J. W. J.Microcolumn Sep. 1990,2,120.
0 1993 American Chemical Society
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Figure 1. Schematlc of InstrumBntBI setup fw 2D SECCZE. See Figures 2-4 for specific lnformatlon regarding the interface designs. A descrlptbn of the components Is given In the text. Deliin Tee
1 ~
50 vm ID Fused Dlaa Eledrophoreris Caplllani
Frit
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1116'OD Teflon Tubing
Flgur. 2. Schematic 01 me vahe/bp interface. The valve is a six-porlelectrically actuated valva fitted with a 300-nL-volume semple Imp made hom fused silica. The SEC column is a 250-pm4.d.. 105-cmbng minocolumn. The Inset shows the union between the SEC microcolumn and the connecting tubing used to lnhoduce the mlaOcOlumn to #a sixpod valva.
fused-silicacapillary of length 105cm (PolymicroTechnologies, Phoenix,AZ). Thesecond was8 100-pn4.d. fused-silicacapillary of length 110 cm. The capillaries were slurry packed in our laboratory with 6-pm, spherical, Zorbax GF450 particles (Rockland TechnologiesInc., Newport, DE). The packing procedure has been described previouslyg and was followed with slight modification to the fritting process.1° Instrumentation. Figure 1 is a schematic of the entire instrumental setup. Chromatographic System. The chromatographic injection system has been modified from a static split injection system which had beendevelopedfor open tubular LC inour laboratory." Injections were madefromapressurereservoircontainingasmall vial of sample. The microcolumn was removed from the static split injection tee and inserted into the pressure reservoir where head pressure of 7 bar (1M) psi) was applied for a predetermined time to inject the desired volume of sample. After the injection was complete, the microcolumn was returned to the static split injectiontee where thedesired head pressurewasapplied to begin chromatography. Thisapproach wasusedratherthanperforming astatic split injection,where severalhundred microlitersof sample are required per injection. Electrophoresis System. Capillary electrophoresis was performed in untreated fused-silica capillaries with inner diameter 50pm (PolymicroTechnologies). Capillarylengtbs and distance (10) Hop.A. M..Jr.; Beale. S.C.; Larmann. J. P., Jr.; Jorgenoon, J. W. J. Microcolumn Sep.. in press. 111) Cuthrie, E..I.; .lorgemon. J. W. J . Chromotog. 1983.255.335.
M the detection window varied and are reported in the figure captions. A *30-kV high-voltagepower supply (Spellman High Voltage Electronics Corp., Plainview, NY) was used in the negative high-voltagemode. Details concerning CZE operating parameters are provided in the figure captiuns. SEC-CZEInterface. Twodifferent approaches were taken tu interface the SEC microcolumn to the CZE system. Figure 2 shows a schematic of a valve sample loop design. A six.port electrically actuated valve (Valco Instruments, Houston. TXJ was fitted with a 300-nL collection loop. This cullection loop was made in-house from a 15-cm-longpiece of 50-pm4.d.. 350urn-o.d. fused-silica capillary. Each end of the fused-silica loop was held in place in the valve by sleeving through PEEK tubing 'liners". The liners were made from 2.cm lengths of 0.020-in: i.d.,' ,An.-o.d. PEEK tubing (UprhurchScientifir.Oak Harbor, WA). Eachofthese liners hasa-Light-Touch"removableferrule system (Upchurch Scientific) and a standard ' ,win. stainless steel nut for fittings. Rather than directly exposing the relatkely fagile fritted end of the microcolumn to the fittings necessary to bold the capillary in place within the valve, the microcolumn wascoupled ma O-rmlong section ofS0-pm-i.d..350-urn-0.d.fused-silicarapillary. The union between these capillaries was made by deevinp the capillaries with a I-cm piece of 0.007-in.-i.d.,' li-in.-o.d. Teflon tuhing. When heated, the Teflon tubing expanded and allowed insertionofthecapillaries. Uponconling.asnug fit wasubulinrd between the capillaries and the tubing. The 3-cm fused-silica connecting tube was also held in place within the valve by a
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 11. JUNE 1, 1993 Bvnsr CzE
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Figure 3. Schematlcof the flow gating Interface. The interface was buiit In-house from two 3-in Aiameter plates of stainless steel. Sandwlchedbetweentheseplates is a Teflongasket (shownhere with a thickness of 127 pm) wnh a channel cut in it to allow liquid Mw. Cetails of the operetlon of the device are glven in the text.
PEEK tubing liner. Protecting the delicate frit in this manner increases column lifetime and allows easy insertion and removal of the SEC microcolumn from the valve. The electrophoresiscapillarywascoupledto thevalve through a Delrin tee (Alltech Associates) which was connected to the valve hya 5-cm section of 0.005-in.-i.d., 'Ils-in.-o.d. PEEK tubing (Upchurch Scientific). The capillary extended into the tee and was held in place near the center of the tee with a PEEK tubing liner. A 20-cm section of 0.040-in-i.d. stainless steel tubing was also inserted into the Delrin tee. This tubing serves as both a waste line and the ground electrode (anode) for the CZE system. A microammeter was placed between the tubing and the connectiontogroundtoallow for current monitoring. Theoutlet of the stainless steel waste line was kept at the same height as the liquid level of buffer in the cathodic buffer reservoir to minimize hydrodynamic flow within the CZE capillary. The general operation and timing sequence of the valve have been previously described? A flush rate of 100 pLlmin was provided by a Hewlett-Packard 1050 pump (Hewlett-Packard. Palo Alto, CAI. Figure 3 shows a schematic of the second interface design used to couple the SEC microcolumn with the CZE system. The inhouse built flow gating interface consisted of two stainless steel plates, 3 in. in diameter and ' I 2in. thick, separated hy a Teflon gasket. The apparatus was assembled with six holta to create a liquid-tight seal. The outlet of the SEC microcolumn was positioned directly across from the inlet of the electrophoresis capillary, separated only hy the thickness of the Teflon gasket (127 pm). The gasket had a 1-mm channel cut in it to allow liquid flow between the twoplates. Normally,atransverse "flush" flow of buffer (controlled by an electrically activated valve) entered through the top port of the interface, swept through the channel, and exited through the bottom port. This flow of CZE buffer carried SEC effluent away to waste, preventing transfer of sample to the CZE capillary. When an injection was desired, thisflushflowwasinterrupted,allowingtheflowofSECeffluent to carry sample into the narrow gap separating the SEC microcolumn and CZE capillary. Electromigration from this volume injected sample onto the CZE capillary. After the desired injection time the transverse flow was resumed, terminating the injection process. All SEC effluent was again carried to waste until the next injection was to he made. Figure 4 shows a timing sequence for the entire injection process. Figure 5 is an expanded view of the central region of the flow gating interface. The SEC microcolumn was interfaced via a section of fused-silicaconnecting tubing and a Teflon union, as described previously for the valvelloop interface. For the 250pm-i.d. microcolumn the connectingtube was 3 em of50-pm-i.d., 375-pm-0.d. fused silica. For the 100-pm-i.d. microcolumn, 3.5 cm of 15-pm-i.d., 375-pm-0.d. fused silica was used. The end of the connecting tubing terminated at the inner face of the flow cell and was mounted flush with a liner made from 0.007-in.-i.d., 'il6-in.-o.d. Teflon tubing and a Light-Touch removable ferrule
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Fbure 1. Timing sequence used to wntrol injections wllh the Row gatlng Interface. The transverse flow of flush buffer is normaliy on so that all SEC effluent is carrkd to waste. When an injection is deslred. the high voltage is slewed from the running voltage to 0 V. while the flush flow through the interface channel is maintained. The flush flow Is maintained for a 2-s period after the high-voltage slew and then is terminated to allow effluent to fill the gap within the interface. During this fill time or '"injection"time, there is no applied potential. At the end of the fill time. the high voltage slews to the run voltage while the valve controlling the flush flow simultaneously actuates to begin the flushing process. See Figures 7-9 for specificvalues of run voltage and times for CZE injection and run duration. Fused - Silica Cmnening TubicQ
Fbure 5. Expanded view of the central region of liw flow gating interface. The SEC mlcrocoiumn is elther a 250 @m1.d.. 105cmlong column a a lOO-wn-.d., t lkmiong column. The microwlumn is coupled to the interfacevia a section 01 fused-silicaconnectlngtubing. Both the SEC Connecting tubing and the CZE capillary are mounted flush w~ the inner wails of the f ow cell so they are semrald onlv by the thickness of the Teflon gasket system. The electrophoresis capillary was mounted within the interface in the same manner as the SEC connecting tuhing. Approximately I mm of polyimide coating was removed from the end of the CZEcapillary oriRinatingat the interface to reduce adsorption of a n a l w at the interface. The flow of flush buffer was controlled hy an electrically actuated sir-port valve (Valco Instruments). Theinlet tuhingronnertingthevalveandinterface was40cmof0.020-in.-i.d.,I ,,-in.-o.d.PEEK tuhing. Theoutlet line was 30 cm of 0.040-in.-i.d., ,.-in.i.d. Teflon tubing (Alltech Associates). The flow of flush buffer was provided hy helium head pressure. For the 250-pm-i.d. micromlumn, 3 har (50 pxij head pressure was applied. This pressure provided a flushing flow rate of approximately fiOO pL min. For the 100-pm-i.d. microcolumn, 0.5 har (7 psi) was npplied to generate a flushing flow rate of approximately 100 pL min. In general, the flush flow rate required to effectively sweep out analyte from the interfacescales directly with the volumetric flow rate oft he SKC column. Although the flush flow rates used here were eaperimentnlly determined. the ratio of flush flow rates was approximately equal to the ratio of the cross-sectional areas of the two SEC columns. This relationship was used as a general guideline for determining flushing flow rates. The entire interface served as the ground electrode (anode) for the CZE system. The microammeter was placed hetween the interfare and electrical ground to monitor current. The height of the outlet of the flush buffer waste line was kept lev4 with the height of huffer in the cathodic reservoir to minimize hydrodynamic flow within the C%Krapillary. Defection. A Linear Model 200 variahle-wavelength UV visible detector outfitted with an on-column capillary flow cell
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J
THYRO
0
10
20
30
40
50
60
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CZE (seconds) Figure 7. Gray-scale image of 20 repeat Injections of 1% (wlv) formamide using the flow gating interface. The formamide soiutbn was eluting in a constant stream from the 250-pmi.d. microcolumn under 55 bar (800 psi) head pressure. The electrophoresis capillary was 35 cm overall, 10 cm lo the detection window, CZE condtions were 5-8 electromigrathn injectlon at 0 hV and 2-min runs at -7 kV. Head pressure at 3 bar (50 psi) generated a flush flow rate of 800 pLlmin. The Teflon gasket thickness was 127 pin.
the actual CZE run time. The actual run time in Figure 6was 9 min. To overlap the CZE runs, an injection was made every 4.5 min. In general, overlapping of CZE runs is only possible when detection of the first sample component does not begin until after half of the CZE run time has passed. In addition, the time in which all components migrate must be less than half of the total CZE run time. For samples that satisfy these criteria, CZE overlap helps to maximize use of the separation time and space available. The peak identifications indicated were verified by running each protein separately, by CZE. The use of formamide serves several important roles in the 2D separation. Because it is a neutral species it serves as an electroosmotic flow marker in the CZE separation. Variations in migration time for formamide can be attributed to changing electroosmotic flow. Also, because neutral species do not tend to adsorb to fused-silica capillary surfaces, evidence of peak asymmetry in CZE for formamide indicates an injection problem or poor interface washout characteristics. As far as the SEC separation is concerned, formamide serves as a low molecular weight marker. Any peak elution later in time than formamide indicates that retention has occurred by mechanisms in addition to size exclusion. Figure 7 is a gray-scale image of 20 individual injections of 1%formamide using the flow gating interface. The 1% formamide solution eluted in a constant stream from the 105cm-long, 250-pm4.d. microcolumn under 55 bar (800psi) head pressure. CZE injections were done a t 0 ' kV" for 5 s (see Figure 4). The 5-8 "injection" was really a filling period t o allow sufficientflow of SEC effluent into the region between the microcolumn and the CZE capillary so that electromigration injection could occur as the high voltage slewed from 0 kV to the run voltage (-7 kV). Coincident with the highvoltage slew, the valve controlling the flush flow was actuated to assume the flow of buffer and terminate the injection. This injection differed from more conventional CZE electromigration in that the injection voltage was completely afunction of the run voltage. As shown by the well-defined black band at71 sinFigure7,veryrepeatabletransferofsampleoccurred with this interface. This image was obtained by "stacking" each successive CZE run of formamide on top of the previous run and interpolating the data between each run to generate a continuous band. The reproducibility of the 20 injections
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Flgurb 8. Separation of protein standards by 2D SEClCZE with b flow gating interface and a 250-pm-1.d. microcolumn. Each protein waspresentat 0.5% (wlv) with 2.5% (w/v)formamlde. SEC injection was 10 min at 7 bar (100 psi). Head pressure of 34 bar (500 psi) was applied to generate a flow rate of 235 nllmin for the SEC separation. Theeieclrophoresisceplllarywas45cmiong.21.5cmtothedetection window. CZE condkions were 5-s elechomigration injection at 0 kV and 2-min overlapped runs at -10 kV. The actual CZE run time was 4 min. Head pressure of 3 bar (50 psi) generated a flush flow rate of 600 pL1min. The Teflon gasket thickness was 127 pm. Data collection was 3 pointsls.
in terms of percent relative standard deviation (% RSD) was determined to he 0.18% RSD in migration time (first statistical moment), 1.9% RSD in peak area, and 3.1 % RSD in peak height. The concentrationofsample transferredusingthis interface was maintained a t a much higher level than with the valve/ loop interface. This is demonstrated in Figure 8 hy the grayscale image of the 2D separation of protein standards with a 250-pm4.d. microcolumn using the flow cell interface. Each proteinwaspresentatO.5% (wiv) with2.5% (w/v)fommide. Asshown inFigure6,CZEruns wereoverlapped. The relative concentration of protein used for this separation was 4-fold less than used in the separation with the valve/loop interface in Figure 6,yet the intensities of most of the peaks have also increased by -2-fold. On average, an 8-fold improvement in sensitivity over the valve/loop scheme was achieved with the flow gatingdesign. Also, because there wasnot afixedvolume of effluent to he collected during a given sampling period, lower operational SEC flow rates were available. For the separation shown here, the flow rate was -235 nL/min. A more dramatic example of the ability to use lower SEC flow rates is seen in Figure 9. The gray-scale image seen in Figure 9 is the result of the 2D separation of the protein standard mixture used in the separation shown in Figure 8 using the flow gating interface and a 100-pm-i.d. SEC microcolumn operated a t -20 nL/ min. The improvement in SEC resolution over that seen in Figures 6and 8 was due to both operating a t a flow rate closer to the optimum value and an increase in separation efficiency due to a decrease in column inner diameter. This trend of enhanced performance has been demonstrated previously.~
DISCUSSION The work presented shows two different approaches to coupling capillary LC columns with CZE in a 2D system. Although both approaches allow the effective coupling of the two microscaleseparation techniques, the flow gating interface designis theonlytrulyviahleapproachiffreedominoperating parameters is desired. The necessity to collect a fixed volume
200
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CZE (minutes) Fwre #. Separation of protein standards by 2D SEClCZE with b
flow gating interface and a 100-pm1.d. mlcrocolumn. Each protein waspresentatOS% (w/v)with2.5% (w/v)formamMe. SECinjecUon was 8 min at 7 bar (100 psi). Head pressure of 14 bar (200 psi) was applied to generate a flow rate of 20 nLlmin for the SEC separation. The electrophoresis capillary was 53 cm long. 33 cm to the detection window. CZE conditionswere 30-9 elactromigration injection at 0 kV and Cmin overlapped runs at -1 1 kV. The actual CZE run time was E min. Head pressure of 0.5 bar (7 psi) generated a flush flow rate of 100pUmin. TheTeRwlgasketthldme%swas76pm. DatacolMhw was 2 pointsls. of effluent is the major shortcoming of the valvelloop approach. The minimum amount of effluent that can he collected in any size sample loop is determined by the inherent extra (nonloop) volumes contained within the valve itself. For the six-port valve used here, eachport contributea 16OnLof extra volume. The valve rotor, used to make the internal connections hetweenporta, has threeengravings that eachmntrihute an additional 220 nL of volume. These volumes are in addition to the volume of the sample loop. For the sample loop itself, theminimumdistancetoconnectanytwoports (in the proper configuration) and allow for fittings is 15 cm. The smallest i.d. tubing (of 15-cm length) that could he used without generating excessively high backpressure upon flushing was determined to he 50 pm. A 15-cm-longsection of 50-pm4.d. fused-silica capillary provides 300 nL of volume. With the extra volume of the valve taken into account, the valve/loop assembly shown in Figure 2 requires approximately 900 nL of sample effluent to enter the valve in order to just fill the 300-nL loop with sample. This fixed volume of 900 nL must he generated within the time of one CZE run. Although it is possible to 'underfill" the sample loop with less effluent and thereby allow a slower SEC flow rate or smaller column inner diameter, adequate signal would not be seen a t the detector due to dilution and dispersion effects within the valve. In fact, thevalve/loop providesenoughadded dilution and dispersion that in order to obtain an appreciable signal upon detection, each protein had to he present a t 2% (wiv). Protein levels of this magnitude are already restrictive and impractical. Increasing the necessity for greater sample concentrations by underfilling, which reduces the volume of sample collected, is clearly not an option. Because the flow gating interface design is not based on sample collection and storage, the added dilution and dispersion associated with loop systems is not present. The flow gating design has a major advantage over a valve1 loop system because the sample is not stored in any sort of collection tubing that needs to he flushed out. Elimination of collecting tubing helps to reduce the extracolumn dilution which occurs in the transfer of sample from the SEC
ANALYTICAL CHEMISTRY, VOL. 85, NO. 11, JUNE 1, 1993
microcolumn to the CZE capillary. Also, peak elution volumes from the SEC microcolumn are typically 2 orders of magnitude greater than the geometric volume of liquid contained in the cylindrical cross section between the two capillaries. For example, assuming a cylindrical cross section, the gap within the interface provides a “volume for injection” with respect to the 50-pm-id. CZE capillary of -0.25 nL when the 127pm-thick gasket is used and -0.15 nL when the 76-pm-thick gasket is used. The flow rate from the 250-pm4.d. SEC microcolumn used in Figure 8 is 235 nL/min or -4 nL/s. The flow rate from the 100-pm4.d. SEC microcolumn used in Figure 9 is 20 nL/min or -0.3 nL/s. A typical peak elution volume from the 250-pm4.d. microcolumn operating a t 4 nL/s is -1900 nL [based on a peak width (4u) of 8 minl. For the 100-pm4.d. microcolumn operating at 0.3 nL/s, a typical peak elution volume is -200 nL [based on a peak width (4u) of 10 minl. In the case of the valve/loop approach, where 900 nL of effluent must be collected, a peak eluting in 200 nL would undergo significant dilution prior to injection into the CZE capillary. In contrast, because the “collection volume” with the flow gating interface is typically 2 orders of magnitude lower than peak elution volumes, sample is presented to the CZE capillarya t essentiallythe same concentration as it eluted from the SEC microcolumn. Because sample concentration is well preserved through the transfer from the SEC dimension to the CZE dimension, lower sample concentrations yield adequate signal upon detection. The combined effects of lower required sample concentrations and more efficient transfer between separation steps results in an 8-fold improvement in sensitivity over the valve/loop approach. This can be seen when the separations shown in Figures 6 and 8 are compared. This flow gating interface design also loosens the restrictions on usable SEC flow rates and column inner diameter that the valve/loop system imposed. By changing the thickness of the gasket, the volume of effluent “collected” in the gap between the two capillaries can be varied. This provides flexibility for using different size microcolumns operated over a wide range of flow rates. This is particularly important when microcolumn SEC is employed for protein separation, where long isocratic runs are necessary to maximize separation efficiency.9 Changing the Teflon gasket is a simple task because the two capillaries are held firmly in place within the plates of the flow gating interface. The last unique characteristic of the flow gating design is that sample injections onto the CZE capillary are represen-
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tative of discrete points along the SEC peak profile. In this way, the CZE capillary analyzes ”snapshots” of the SEC effluent. This is in contrast to the valve/loop approach, which averages sample composition over each sampling period. This feature of the flow gating design is not a disadvantage as long as several transfers between SEC and CZE are carried out along the width of each SEC peak. This will help ensure that the SEC column run is not ”undersampled” and misrepresented. It is of interest to note that the SEC separation in Figure 6, which used the valve/loop interface, approached minimum sampling by CZE. By minimum sampling it is meant that for two of the components (myoglobin and formamide) only one CZE run occurred over the width of the SEC peak. Sampling a t a greater frequency was restricted due to the sample volume/collectionconsideration previously mentioned. By comparison, using the flow gating interface design, the separation shown in Figure 8 has at least three CZE runs occurring across each eluting SEC peak. The results shown here are promising for the future use of the flow gating design as an interface for 2D microscale separation techniques. Further scaledown in SEC column inner diameter and flow rate is currently limited in this system by the use of UV detection for CZE. The short path length for detection provided by the electrophoresis capillary, in conjunction with the inherently low sensitivity of UV detection, precludes the 2D separation of protein speciespresent at less than -0.3% (w/v). The full utility of the flow cell design as an interface for microscale separation techniques would be best shown with a system that employs a more sensitive detection system, such as laser-inducedfluorescence. Future work will be directed toward investigating the use of this flow cell interface to couple other microcolumn LC techniques together with CZE.
ACKNOWLEDGMENT We thank Hewlett-Packard for the donation of the pump and computer used in this work, and Rockland Technologies Inc., for the donation of the Zorbax GF450 packing material. Support for this work was provided by the National Science Foundation under Grant CHE-9215320. RECEIVEDfor review December 10, 1992. Accepted February 16, 1993.
