Anal. Chem. 1997, 69, 4134-4142
A Transparent Flow Gating Interface for the Coupling of Microcolumn LC with CZE in a Comprehensive Two-Dimensional System Thomas F. Hooker 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 new interface for the coupling of microcolumn highperformance liquid chromatography (micro-HPLC) with capillary zone electrophoresis (CZE) is presented. The new interface is based on the original flow gated design developed in this laboratory but is now made from a clear plastic which allows for the direct observation and routine manipulation of the micro-HPLC and CZE capillaries. As with the original design, a transverse flow of CZE buffer controls analyte injection onto the CZE capillary. To evaluate the reproducibility of the interface, 400 consecutive CZE separations of a mixture of fluorescein 5-isothiocyanate (FITC)-labeled phenylalanine and glutamic acid are performed over a 2 h period. The percent relative standard deviations (% RSD) of the peak height, peak area, and migration times of 400 runs of FITC-phenylalanine are 2.5, 3, and 0.07%, respectively, and of FITCglutamic acid are 2.5, 3.5, and 0.07%, respectively. The % RSDs of the peak height, peak area, and migration time were reduced to 0.7, 0.6, and 0.02%, respectively, by normalizing the results of glutamic acid to that of phenylalanine. This interface also introduces little extra-column band broadening into the system as evidenced by plate counts of 480 000 for FITC-phenylalanine and FITCglutamic acid achieved in less than 35 s. The clarity of the interface allows the flow gating and injection processes to be directly observed using a colored dye solution. Realtime images of an injection sequence using the interface have been acquired with the aid of video instrumentation and are presented to illustrate the injection process. While separation techniques such as high-performance liquid chromatography (HPLC) and capillary zone electrophoresis (CZE) can adequately resolve the components in many samples, they lack the necessary peak capacity for more complex samples. The realization that these and other one-dimensional (1D) separation techniques are inadequate to fully resolve mixtures is growing and so is interest in two-dimensional (2D) separations. The increased peak capacity that 2D separations offer makes them attractive for the analysis of complex biological and environmental samples. Development of comprehensive 2D systems in this laboratory has focused on coupling two different modes of HPLC1-4 as well as HPLC with CZE.5-13 (1) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 161-167. (2) Holland, L. A.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3275-3283. (3) Opiteck, G. J.; Lewis, K. C.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 1518-1524.
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Giddings and Davis have set forth much of the theory upon which 2D separations are based.14-24 Their theory predicts that, as sample complexity increases, high-efficiency separations are needed to completely separate component peaks. Work by other researchers in addition to Giddings and Davis also indicates that 1D techniques lack the necessary peak capacity for separations of complex samples.25-29 The combination of microcolumn high-performance liquid chromatography with capillary zone electrophoresis (microHPLC-CZE) in a 2D system is of interest because of the high resolving power and complementary nature of the two techniques. Since the mechanism of separation in reversed phase liquid chromatography (RPLC) is based on hydrophobicity while in CZE it is a function of the analyte’s charge to size ratio, there is little redundancy in displacement mechanisms when the techniques are combined. However, the coupling of separation techniques that operate with low flow rates requires the use of an interface design capable of transferring small volumes while minimizing extra column broadening. (4) Opiteck, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 2283-2291. (5) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 978-984. (6) Bushey, M. M.; Jorgenson, J. W. J. Microcolumn Sep. 1990, 2, 293-299. (7) Lemmo, A. V.; Jorgenson, J. W. J. Chromatogr. 1993, 633, 213-220. (8) Lemmo, A. V.; Jorgenson, J. W. Anal. Chem. 1993, 65, 1576-1581. (9) Larmann, J. P., Jr.; Lemmo, A. V.; Moore, A. W., Jr.; Jorgenson, J. W. Electrophoresis 1993, 14, 439-447. (10) Moore, A. W., Jr.; Jorgenson, J. W. Anal. Chem. 1995, 67, 3448. (11) Larmann, J. P., Jr. Doctoral Dissertation, University of North Carolina, Chapel Hill, NC, 1993. (12) Lemmo, A. V. Doctoral dissertation, University of North Carolina, Chapel Hill, NC, 1994. (13) Lewis, K. C.; Opiteck, G. J.; Jorgenson, J. W.; Sheely, D. J. Am. Soc. Mass Spectrom. 1997, 8, 495-500. (14) Davis, J. M.; Giddings, J. C. Anal. Chem. 1983, 55, 418-424. (15) Giddings, J. C. Anal. Chem. 1984, 56, 1258A. (16) Davis, J. M.; Giddings, J. C. Anal. Chem. 1985, 57, 2168-2177. (17) Davis, J. M.; Giddings, J. C. Anal. Chem. 1985, 57, 2178-2182. (18) Giddings, J. C. J. High Resolut. Chromatogr. 1987, 10, 319-323. (19) Davis, J. M. J. Chromatogr. 1988, 449, 41-52. (20) Delinger, S. L.; Davis, J. M. Anal. Chem. 1990, 62, 436-443. (21) Davis, J. M. Anal. Chem. 1991, 63, 2141-2152. (22) Oros, F. J.; Davis, J. M. J. Chromatogr. 1992, 591, 1-18. (23) Shi, W.; Davis, J. M. Anal. Chem. 1993, 65, 482-492. (24) Davis, J. M. Anal. Chem. 1993, 65, 2014-2023. (25) Guiochon, G.; Gonnord, M.-F.; Zakaria, M.; Beaver, L. A.; Siouffi, A. M. Chromatographia 1983, 17, 121. (26) Guiochon, G.; Beaver, L. A.; Gonnord, M.-F.; Siouffi, A. M.; Zakaria, M. J. Chromatogr. 1983, 255, 415-437. (27) Herman, P.; Gonnord, M.-F.; Guiochon, G. Anal. Chem. 1984, 56, 995. (28) Martin, M.; Herman, D. P.; Guiochon, G. Anal. Chem. 1986, 58, 22002207. (29) Martin, M.; Guiochon, G. Anal. Chem. 1985, 57, 289. S0003-2700(97)00342-9 CCC: $14.00
© 1997 American Chemical Society
In the first automated “comprehensive” coupling of LC with CZE, Bushey and Jorgenson used conventional HPLC column and sample loop technology to interface reversed phase HPLC with CZE for the analysis of peptide samples.5,6 The use of a 1 mm internal diameter column allowed for the collection of eluted HPLC mobile phase in loops. The use of LC microcolumns in a comprehensive system is attractive because of the higher efficiencies and reduced sample dilution that are inherent in their operation. Using microcolumns makes the collection of sample in loops impractical, however, due to the small volume of eluted mobile phase and the difficulty in transferring minute quantities of material without loss, dilution, or dispersion of the sample. Therefore, a new interface was designed which allowed microHPLC and CZE to be combined in a comprehensive 2D system.8 The original, stainless steel interface used a cross flow of buffer to control injections of eluted LC mobile phase onto the CZE capillary and was called the transverse flow gating interface. Since the flow gating interface avoids the need for collection of eluted sample between the first and second dimension, small quantities of mobile phase eluted from the LC column can be transferred to the CZE capillary and a more distinct sampling of the LC dimension is obtained. The usefulness of the interface for coupling microdialysis and on-line immunoassays to CZE has also been demonstrated.30-33 The goal of the work presented here has been to design a more routine and reproducible way of interfacing micro-HPLC and CZE. The original flow gated design was the first step toward effectively coupling these two separation techniques, but further improvement was needed. Because it was constructed from steel, the old design did not allow observation of the region between the LC and CZE capillaries. Since the placement of the capillaries with respect to each other in the interface is critical for successful transfer of sample from LC to CZE, it was necessary to construct the interface from a transparent material. The new flow gating design is operated in a manner similar to that of the original, with additional modifications and improvements. The new design is constructed from a clear polycarbonate polymer, Lexan. Capillary positioning and manipulation, as well as diagnostic troubleshooting, are much more routine, as the region where the two separation dimensions are interfaced can now be clearly seen. The absence of this quality in the original design was a drawback. In addition to being transparent, the material the interface would be constructed from needed to be reasonably easy to machine and chemically inert. Lexan is machined with relative ease, and this allowed for flexibility in design and fabrication of the flow channels and the threading for the fittings. Its chemical resistance to acids, bases, and certain organic solvents also made it an attractive material. Glass would provide equal clarity and better chemical resistance to organic solvents, but it is brittle and difficult to machine. Fluoropolymers would provide better overall chemical resistance and they machine well, but they are not transparent. With the use of colored dye solutions we have been able to visually examine the flow gating and injection processes and capture them using a video camera. The design and operation of the interface are presented as well as images, captured from video, (30) Lada, M. W.; Schaller, G.; Carriger, M. H.; Vickroy, T. W.; Kennedy, R. T. Anal. Chim. Acta. 1995, 307, 217-225. (31) Lada, M. W.; Kennedy, R. T. J. Neurosci. Methods 1995, 63, 147-152. (32) Lada, M. W.; Kennedy, R. T. Anal. Chem. 1996, 68, 2790-2797. (33) Tao, L.; Kennedy, R. T. Anal. Chem. 1996, 68, 3899-3906.
of an injection sequence using the interface. Results obtained from repetitive separations of an amino acid mixture and a 2D micro-RPLC-CZE separation of fluorescently labeled urine are also shown. EXPERIMENTAL SECTION Materials. Amino acid standards, trifluoroacetic acid (TFA) and triethylamine (TEA) were purchased from Sigma Chemical Co. (St. Louis, MO). Acetonitrile (Optima grade) and sodium bicarbonate were obtained from Fischer Scientific (Fair Lawn, NJ). Boric acid and sodium phosphate were obtained from EM Science (Gibbstown, NJ). Fluorescein-5-isothiocyanate (FITC) was obtained from Molecular Probes (Eugene, OR). Dimethyl sulfoxide (DMSO) was obtained from Mallinkrodt (Paris, KY). All chemicals were used as received. Deionized water was obtained from a Barnstead Nanopure system (Boston, MA). Buffers. The CZE buffer used for the amino acid separations was 10 mM boric acid, adjusted to pH 9.25 with NaOH. The CZE buffer used for the 2D urine separation was 10 mM phosphate with 0.22% TEA (v/v) and 15% acetonitrile (v/v), pH 10.5. This buffer system was needed to prevent adsorption of certain analytes, in the urine sample, to the CZE capillary. The two previously mentioned buffers will hereafter be referred to as either the “run buffer” or “flush buffer”. The buffer used for the derivatization reaction was 0.2 M boric acid, adjusted to pH 9.25 with NaOH. This is hereafter referred to as “tagging buffer”. Buffer solutions were made with deionized water and were filtered with 0.2 µm nylon membrane filters from Alltech Associates (Deerfield, IL). FITC Derivatization of Amino Acids and Amine Samples. The FITC derivatizations were carried out in a manner similar to what was recommended by the manufacturer.34 FITC was dissolved in DMSO at a concentration of 10 mM. Aliquots (50 µL) of this solution were added to 150 µL of the tagging buffer containing the dissolved amino acid. The amino acids were in 3-fold molar excess to the FITC. Prior to tagging, the urine sample was filtered through a 10 000 molecular weight cutoff filter (Amicon, Beverly, MA) to remove any large molecular weight species that might contain multiple tagging sites. Following this, 300 µL of the urine sample was mixed with 150 µL of tagging buffer. An aliquot of 1 N NaOH (10 µL) was used to adjust the pH to 9. This solution was then mixed with 100 µL of the FITC solution. The samples were allowed to react for 4 h in the dark and stored frozen at -20 °C until diluted for use. The amino acid mixture was diluted in 80:20 water/acetonitrile prior to being run. The urine sample was diluted 100:1 in the initial RPLC mobile phase prior to being chromatographed. To remove any undissolved particulate matter, the mixtures were centrifuged at 14000g for 5 min after being diluted for use. The supernatants were drawn off and infused into the interface for repetitive run studies or injected onto the RPLC column for a 2D separation. Column Packing Procedure. The LC column used in this work was a 76 cm long, 50 µm i.d., 360 µm o.d. capillary (Polymicro Technologies, Phoenix, AZ) that was slurry packed in our laboratory with 5 µm, spherical, Zorbax C8 particles (Rockland Technologies Inc., Newport, DE). The packing procedure was described previously.35 The frit in the column used (34) Haugland, R. Molecular Probes Handbook 1992-4; Molecular Probes Inc.: Eugene, OR, 1992. (35) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 1128-1135.
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Figure 1. Schematic of instrumental setup for 2D micro-RPLC-CZE. A split injection/flow system is used to deliver a nanoliter per second flow rate to the micro-RPLC column from the gradient LC pump. The LC microcolumn is 50 µm i.d. and 76 cm long, and the electrophoresis capillary is 17 µm i.d., L ) 25 cm, and l ) 15 cm. The valve is air-actuated and controls the flow of flush buffer. See Figures 2 and 3 for more detailed illustrations of the flow gating interface and Figures 4 and 5 for information regarding operation of the interface.
to contain the packing material was made by sintering 10 µm borosilicate glass beads with a microelectric arcing device, the design and operation of which has been described fully elsewhere.36 Instrumentation. Figure 1 shows the experimental setup for 2D micro-RPLC-CZE. Reversed Phase Chromatographic System. A Beckman System Gold solvent delivery module (Beckman Instruments, Ontario, CA) was used to deliver a gradient of acetonitrile and water to the reversed phase microcolumn. The water and acetonitrile both contained 0.1% TFA. A gradient of 15-30% acetonitrile from 0 to 240 min was run. In order to generate the nanoliter per second flow rates needed for the microcolumn, a split flow system originally designed for open tubular liquid chromatography was used.37 Sample was loaded onto the microcolumn by placing the inlet of the column in a sample reservoir. Gas pressure applied to this reservoir forced sample onto the microcolumn. To start the run, the microcolumn was replaced in the tee and mobile phase flow was returned. Electrophoresis System. Capillary electrophoresis was performed in untreated fused-silica capillaries with an inner diameter of 17 µm (Polymicro Technologies). The capillary length was 25 cm with 15 cm to the point of detection. A few millimeters of the polyimide coating was removed from the inlet end of the CZE capillary to reduce adsorption of analyte and to facilitate the grinding of a pointed tip on the end, as described below. A 30 kV dc, reversible-polarity high-voltage power supply (Spellman High Voltage Electronics Corp., Plainview, NY) was used in the negative high-voltage mode. The interface was held at ground through a stainless steel tubing connection, and electrophoresis current was measured through this connection to ground. CZE injections were performed at -1 kV for 3 s, and runs were performed at -30 kV. Removal of the polyimide coating for oncapillary laser-induced fluorescence (LIF) detection was done with the use of the microelectric arc device mentioned previously.36 Capillary rinses were performed with 75% 1 N NaOH/25% methanol followed by water and then run buffer. (36) Hoyt, A. M., Jr.; Beale, S. C.; Larmann, J. P., Jr; Jorgenson, J. W. J. Microcolumn Sep. 1993, 5, 325-330. (37) Guthrie, E. J.; Jorgenson, J. W. J. Chromatogr. 1983, 255, 335-348.
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Capillary Tip Preparation. A cone was formed at the inlet end of the CZE capillary in the following manner. A few millimeters of polyimide was removed from this end of the CZE capillary. The capillary was then mounted on a jeweler’s lathe and held in a number 5 collet. The capillary was spun and stones of various coarseness were brought up against the end of the capillary at an angle of ∼45°. This process was repeated with stones of finer coarseness until the desired cone was formed. This grinding process was monitored through a microscope mounted above the spinning capillary. Upon completion, the tipped end of the capillary was placed in a solution of NaOH and sonicated for 1015 min to remove any particulates left inside the capillary from the grinding procedure. Detection. Detection was performed on-capillary using LIF. The 442 nm line from a helium-cadmium laser (Liconix, Sunnyvale, CA) passed through a laser-stabilization accessory (Liconix) and was focused on the CZE capillary using a 6.3× objective (Melles Griot, Irvine, CA). The fluorescence emission from the FITC-tagged amines was collected at 90° from excitation using a 60× objective (Edmund Scientific, Barrington, NJ), passed through a band-pass filter and a cut-on filter, and was detected with a photomultiplier tube (PMT; Model R4220, Hamamatsu, Bridgewater, NJ). The photocurrent was converted to voltage and amplified using a current preamplifier (Model SR570, Stanford Research Systems, Sunnyvale, CA). Construction of the Flow Gated Interface. Figure 2 shows a schematic of the flow gating interface. The interface was constructed in-house from clear polycarbonate (trade name Lexan, GE Plastics Group, Pittsfield, MA). The in-house built interface consists of a 1 in. diameter, 1/2 in. thick Lexan disk through which 1/ in. channels have been bored. Cones and threads for Light16 Touch nut and ferrule fittings (Upchurch Scientific, Oak Harbor, WA) were also added. The capillaries were sleeved inside 0.007 in. i.d., 1/16 in. o.d. Teflon tubing. This tubing fit snugly in the 1/ in. flow channels and was held in place with the removable 16 nut and ferrule system. The capillaries extended out from the Teflon sleeves into the flow channel where the transverse flow of run buffer was present. A Shimadzu LC 600 HPLC pump (Kyoto, Japan) was used to deliver run buffer to the interface. The buffer exited the interface through stainless steel tubing. A microam-
Figure 2. Schematic of the clear flow gating interface. The interface was constructed in-house from a 1 in. diameter, 0.5 in. thick Lexan disk. The disk is clear, which allows direct observation of the capillaries in the stream of flush buffer. The capillaries are sleeved in 1/16 in. o.d. Teflon tubing and this tubing is held in place by Lite Touch fittings (not shown). The cross flow of buffer prevents LC effluent from electromigrating onto the CZE capillary until an injection is desired.
Figure 3. Photomicrograph of the central portion of the interface. On the left is the outlet of the 50 µm i.d., 360 µm o.d. LC microcolumn that contains a frit made from sintered 10 µm borosilicate beads, which retains the 5 µm C8 modified silica particles. On the right is the coned inlet end of the 17 µm i.d., 360 µm o.d. CZE capillary. The capillary ends sit in the 1/16 in. channel through which the transverse buffer flows. In this picture, the buffer is flowing from the bottom to the top of the photo.
meter was placed between this stainless steel tubing and electrical ground to monitor electrophoresis current. Figure 3 is a photograph of the fritted outlet end of the LC microcolumn placed in the transverse flow of buffer directly across from the coned inlet of the CZE capillary. Interface Operation. Figure 4 contains a timing diagram for the injection sequence used with the flow gating interface. The transverse flow was controlled by an air-actuated valve (Valco Instruments, Houston, TX). Normally the transverse flow carried sample from the LC column or infusion capillary to waste,
preventing it from being injected onto the CZE capillary while the high voltage was being applied. When an injection was made, the following sequence occurred. The run voltage was dropped to 0 V but the transverse flow remained on. This short period, called the “slew down” time, was necessary to prevent any injection that might occur while the high voltage was dropping from the run voltage to the injection voltage. This period of zero voltage removed any fronting on the CZE peaks. After the slew down period, the transverse flow was stopped momentarily by switching the valve. This diverted the transverse buffer flow to waste and allowed sample to flow from the LC capillary across the gap to the CZE capillary. An electrokinetic injection was then performed by applying a voltage for a desired amount of time. The sample flowing out of the sample infusion capillary or the LC capillary in these studies was substantial enough to fill the region between the two capillaries in a short amount of time. It was for this reason that the injection voltage was applied at the same moment the transverse flow was stopped. If one desires, application of the injection voltage can be delayed a certain length of time after the transverse flow has been stopped. This allows the sample sufficient time to fill the region between the LC and CZE capillary. This appears to be necessary only at very low LC flow rates (
