Anal. Chem. 1999, 71, 5253-5257
Coupling of a Gas-Phase Chemiluminescence Nitrogen Detector and a Capillary Electrophoretic System Alex D. Sokolowski and Gyula Vigh*
Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012
A capillary electrophoretic system has been successfully connected to a gas-phase chemiluminescence nitrogen detector via a pneumatic nebulizer interface. The interface, built in-house, consists of a nebulizing gas delivery subsystem, a sheath liquid subsystem, a short spray tube, and a liquid gap. The liquid gap is formed at the point where the spray tube, the separation capillary, and the sheath liquid subsystem meet. The sheath liquid subsystem consists of a grounding connection to complete the electric circuit for the electrophoretic system, a sheath liquid delivery pump, a sheath liquid overflow outlet maintained at atmospheric pressure, and a sheath liquid conduit with a hydrodynamic resistance that is much smaller than that of the separation capillary. The interface operates at the natural self-aspiration rate of the short spray tube. The design ensures that the natural selfaspiration rate of the nebulizer is higher than the maximum electroosmotic flow rate that can be produced in the separation capillary. The flow difference is made up by the sheath liquid which, due to the hydrodynamic resistance differences, is sucked into the liquid gap preferentially from the sheath liquid conduit. Thus, the spray tube and the separation capillary are decoupled from each other hydrodynamically, and any laminar flow-induced additional band broadening in the separation capillary is avoided. Using the combined electrophoretic separation and gas-phase chemiluminescence nitrogen detector system, the mass detection limit for five nucleotide bases used as test compounds was found to be about 10 pmol nitrogen. Several recent reviews describe the use of capillary electrophoresis (CE) 1 for the separation of small molecules,2 pharmaceuticals,3 peptides,4 proteins,5 and nucleic acids.6 Though UV detectors1 are the most commonly used detectors in CE, other modes of detection were also explored because many analytes * Corresponding author: (phone) 409-845-2011; (fax) 409-845-4719; (e-mail)
[email protected]. (1) Beale, S. C. Anal. Chem. 1998, 70, 279R-300R. (2) Shihabi, Z. K.; Friedberg, M. A. Electrophoresis 1997, 18, 1724-1732 (3) Thormann W.; Caslavska, J. Electrophoresis 1998, 19, 2691-2694. (4) Messana, I.; Rossetti, D. V.; Cassiano, L.; Misiti, F.; Giardina, B.; Castagnola, M. J. Chromatogr., B. 1997, 699, 149-171. (5) Dolnik V. Electrophoresis 1997, 18, 2353-2361. (6) Righetti, P. G.; Gelfi, C. Anal. Biochem. 1997, 244, 195-207. 10.1021/ac990679t CCC: $18.00 Published on Web 10/13/1999
© 1999 American Chemical Society
lack suitable UV chromophores. There are excellent recent reviews on laser-induced fluorescence7 (LIF), mass spectrometric8 (MS), electrochemical,9 and chemiluminescence10 CE detectors. Gas-phase chemiluminescence nitrogen detectors (CLNDs)11 are attractive as selective detectors because they respond only to nitrogen, a frequent element in compounds of biological importance. Though CLNDs have been used extensively as detectors in HPLC,12-16 there are no papers on the application of CLNDs in CE, except for a patent which was issued after the work described here was completed.17 In a CLND system, nitrogen-containing organic molecules are combusted at 1050 °C in the oxygen-rich gas atmosphere of a quartz furnace:11
1 1 y CxHyNz + x + y + z O2 f zNO + xCO2 + H2O 4 2 2
(
)
(1)
The effluent gas from the furnace is passed through a cold trap and/or a membrane drier where water is removed. The dry gas then enters the ozone-rich environment of the reaction/ detection cell where NO is oxidized to excited-state NO2*:
NO + O3 f NO2* + O2
(2)
NO2* f NO2 + hv
(3)
The photons produced in the last reaction pass through a highpass optical filter and are detected by a photomultiplier against a dark background. The number of photons is proportional to the number of nitrogen atoms introduced into the reaction cell. High partial pressures in the ozone reaction cell are undesirable (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17)
MacTaylor, C. E.; Ewing, A. G. Electrophoresis 1997, 18, 2279-2290. Banks, J. F. Electrophoresis 1997, 18, 2255-2266. Vogel, P. D.; Baldwin, R. P. Electrophoresis 1997, 18, 2267-2278. Staller, T. D.; Sepaniak, M. J. Electrophoresis 1997, 18, 2291-2296. Birks, J. W. Chemiluminescence and Photochemical Reaction Detection in Chromatography; VCH Publishers Inc.: New York, NY, 1989. Fujinari, E. M.; Manes, J. D. J. Chromatogr. A, 1994, 676, 113-120. Bizanek, R.; Manes, J. D.; Fujinari, E. M. Pept. Res. 1996, 9, 40-44. Fujinari, E. M.; Manes, J. D.; Bizanek, R. J. Chromatogr., A 1996, 743, 85-89. Fitch, W. L.; Szardenings, A. K.; Fujinari, E. M. Tetrahedron Lett. 1997, 38, 1689-1692. Taylor, E. W.; Qian, M. G.; Dollinger, G. D. Anal. Chem. 1998, 70, 33393347. Abubaker, M. A.; Fujinari, E. M.; Petersen, J. R.; Bissell, M. G. US Patent. 5,888,363, March 30, 1999.
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because, as shown by eq 4, the chemiluminescent photon output decreases as a result of collisional relaxation of NO2*
NO2* + M f NO2 + M
(4)
where M is any third body in the ozone reaction cell. Therefore, the chemiluminescent reaction cell is kept at reduced pressures (10-20 Torr). When a CE system is coupled to a gas-phase detection system, the liquid-phase analytes must be converted into an aerosol. Pneumatic nebulizers (e.g., refs 18-26) or electrospray devices (e.g., ref 27) are the most commonly used converters: Olesik et al.18 reported the first use of a pneumatic nebulizer as an interface between CE and an ICP-MS detector. Their work was followed by many papers.19-26 When a separation capillary is connected directly to a pneumatic nebulizer, liquid is sucked out of the capillary. This induced laminar flow causes additional, excessive band broadening that drastically reduces the separation efficiency of the system. In these coupled CE-nebulizer systems, separation efficiencies (numbers of theoretical plates) range from a low of about 400018 through 10 00021 to a high of about 22 000.23 The high value was achieved by Olesik et al.23 who, while coupling CE to ICP-MS, reduced the effects of laminar flow by exactly matching the aspiration rate of the nebulizer to the sum of the electroosmotic flow rate and the makeup liquid flow rate. Unfortunately, when the exact match between the aspiration rate and combined liquid flow rate is lost because of slight changes in the experimental conditions, forward or backward laminar flow is generated in the CE capillary, and separation efficiency is lost. Taylor et al. used an alternative approach to obtain about 80 000 theoretical plates26 in their CEICP-MS system: they applied vacuum at the inlet of the capillary to balance the suction created by the nebulizer. A similar pressurebalancing system is disclosed in the patent that describes the coupling of CE and CLND.17 Despite the pressure-balancing system, the separations shown in the patent indicate that only about 2000 theoretical plates were obtained with that particular combined CE-CLND system.17 This paper describes the coupling of a CE system to a CLND via a pneumatic nebulizer interface which is operated at its natural self-aspiration rate. The design ensures that the self-aspiration rate of the nebulizer exceeds the highest electroosmotic flow (EOF) rate that can be generated in the separation capillary. The critical feature of the nebulizer is the liquid gap which is formed between the outlet of the separation capillary, the inlet of the spray tube, (18) Olesik, J. W.; Kinzer, J. A.; Olesik, S. V. Anal. Chem. 1995, 67, 1-12. (19) Lu, Q.; Bird, S. M.; Barnes, R. M. Anal. Chem. 1995, 67, 2949-2956. (20) Liu, Y.; Lopez-Avilla, V.; Zhu, J. J.; Weiderin, D. R.; Beckert, W. F. Anal. Chem. 1995, 67, 2020-2025. (21) Kinzer, J. A.; Olesik, J. W.; Olesik, S. V. Anal. Chem. 1996, 68, 32503257. (22) Michalke, B.; Schramel, P. J. Chromatogr., A 1996, 750, 51-62. (23) Szostek, B.; Koropchak, J. A. Anal. Chem. 1996, 68, 2744-2752. (24) Hooijschuur, E. W. J.; Kientz, C. E.; Brinkman, U. A. Th. J. High Resolut. Chromatogr. 1998, 21, 540-544. (25) Tangen, A.; Lund, W.; Josefson, B.; Borg, H. J. Chromatogr., A 1998, 826, 87-94. (26) Taylor, K. A.; Sharp, B. L.; Lewis, D. J. J. Anal. At. Spectrom. 1998, 13, 1095-1100. (27) Smith, R. D.; Barinaga, C. J.; Udeseth, H. R. Anal. Chem. 1988, 60, 19481952.
5254 Analytical Chemistry, Vol. 71, No. 22, November 15, 1999
Figure 1. Experimental setup of the CE-CLND system.
and the outlet of the sheath liquid conduit. Since the flow resistance is much smaller in the sheath liquid conduit than in the separation capillary, only sheath liquid is sucked into the gap to adjust, automatically, the combined flow rates of sheath liquid flow and EOF to match the self-aspiration rate of the nebulizer. This paper describes the nebulizer design and the first experimental results obtained with the coupled CE-CLND system. EXPERIMENTAL SECTION Chemicals. All chemicals were purchased from Aldrich (Milwaukee, WI). The background electrolyte (BE) used for the determination of separation efficiency in the CE-CLND system was 25 mM boric acid that was titrated to pH 9.2 with a saturated LiOH solution using a combination glass electrode and a precision pH meter (Corning Science Products, Corning, NY). The BE used for the separation of adenine, cytosine, guanine, and uracil was 50 mM citric acid that was titrated to pH 2.6 with a saturated LiOH solution. The sheath liquid solution was 10 mM acetic acid. All analytes were dissolved in, and injected from, the respective BE solutions. Instrumentation. The home-built CE-CLND system is shown in Figure 1. It consists of a model EH30 power supply (Glassman, Whitehouse Station, NJ), a model 200 UV detector (Linear, Reno, NV), a home-built pneumatic nebulizer interface, a gas-phase chemiluminescence nitrogen detector, CLND HPLC 7060 (ANTEK, Houston, TX), a model 2100 HPLC pump (Varian, Walnut Creek, CA), and an AD406 dual-channel data acquisition system (Beckman-Coulter, Fullerton, CA). The data acquisition system was operated under Gold version 8.1. software control (BeckmanCoulter) that was running on a 486DX2 personal computer (Computer Associates, College Station, TX). The separation capillaries were 75-µm i.d., 150-µm o.d. bare fused silica capillaries (Polymicro Technologies Inc., Phoenix, AZ). The total length of the CE capillary was 48.8 cm; the UV detection window was placed 23.9 cm away from the inlet. The inlet of the CE capillary was immersed into the BE containing inlet vial of a home-built low-high-pressure injector. The CE capillary was threaded through the UV detector, and its outlet was inserted into the nebulizer. The inlet and outlet of the separation capillary were adjusted to the same height by placing the CLND on adjustable lab jacks. The CE system was grounded via the sheath liquid at the stainless steel cross. The Pneumatic Nebulizer. The CE was interfaced to the CLND by terminating the CE capillary in the home-built pneumatic nebulizer (Figure 2). The stainless steel nebulizer was assembled
Figure 2. Schematic representation of the nebulizer interface: separation capillary (1), capillary positioning screw (2), 1/16′′ stainless steel cross (3), stainless steel needle (4), modified stainless steel tee (5), bottom part of the ANTEK HPLC nebulizer (6), 1/16′′ stainless steel tube (7), top part of the ANTEK HPLC nebulizer (8), 1/16′′ PEEK tubing (9), spray tube (10), and high-temperature epoxy (11).
from a 1/16′′ SGE cross (3) (SGE, Dallas, TX), a modified SGE tee (5) and the bottom part (6) of a 1/4′′ stainless steel HPLC nebulizer (ANTEK, Houston, TX). Oxygen was used as the nebulizing gas, and it was introduced through the SGE tee (3), coaxially, to a 5.75-cm-long 1/16′′ o.d., 0.046′′ i.d. stainless steel tube (7) (Upchurch, Seattle, WA). A 9.75-cm-long, 0.013′′ o.d., 0.007′′ i.d. stainless steel needle (4) (Small Parts, Inc., Miami Lakes, FL), which extended out of the back of the nebulizer and held the separation capillary (1), was epoxied concentrically into the front of tube 7. A 2-mm-long axial slit was machined into the top of needle 4, 2 mm away from its tip. The outlet of separation capillary 1 ended under this slit. A 1-cm-long, 75-µm i.d., 150-µm o.d. fused silica spray tube (10) was epoxied into the tip of needle 4, such that the inlet of spray tube 10 also lay under the 2-mmlong slit in needle 4. The distance between the outlet of separation capillary 1 and the inlet of spray tube 10 could be adjusted by positioning screw 2. The outlet of the separation capillary, the inlet of the spray tube, and the axial slit in needle 4 form the liquid gap in the nebulizer. The nebulizing orifice (9) was made from a 5-mm-long, 0.010′′ i.d., 1/16′′ o.d. PEEK tube (Upchurch) that was epoxied by high-temperature-resistant titanium putty (Devcon, Danvers, MA) into the tip of the adjustable upper part (8) of the 1/4′′ stainless steel HPLC nebulizer (ANTEK). By rotating upper part 8 with respect to stationary bottom part 6, spray tube 10 can be moved in and out from nebulizing orifice 9 to finely adjust the size of the gas orifice around spray tube 10. Because of the small cross-sectional area of the gas orifice, this design leads to high nebulization efficiencies at relatively low gas flow rates. The entire nebulizer is connected to the quartz furnace of the CLND 7060 through the gas-tight, original nebulizer attachment port (ANTEK) of the CLND. A needle valve between the quartz furnace and the ozone reaction cell of the CLND 7060 controls the amount of combustion products transported to the ozone reaction cell. The Varian 2100 pump supplies the sheath liquid at 0.9 mL/ min to the free overflow outlet on cross 3. Since the flow resistance of this overflow outlet is practically zero, the sheath
liquid is delivered to the liquid gap only by suction through stainless steel tubing 7. The electrophoretic circuit is closed by connecting cross 3 to the ground terminal of the power supply. Procedures. The first step in setting up the CE-CLND system is the establishment of a stable spray plume from the nebulizer. Using 60 psi oxygen as the nebulizer gas, spray plumes became stable, at least for 10-30 min long periods of time, at oxygen flow rates around 70 mL/min. Long-term plume stability (not requiring any adjustment for at least 12 h) was achieved at or above oxygen flow rates of 150 mL/min. Stable plumes could be maintained with gas flow rates as high as 350 mL/min. With an oxygen flow rate of 170 mL/min (the setting used for most of the experiments), the self-aspiration rate of the nebulizer was about 200 µL liquid/ min. The next step in the setup calls for the adjustment of the length of the liquid gap between the outlet of the separation capillary and the inlet of the spray tube to eliminate any nebulizationinduced laminar flow in the separation capillary. This adjustment can be carried out in one of two ways (or both). According to the first method, the nebulizer is connected to the CLND furnace, without the separation capillary, and the sheath flow pump is started. The CLND signal that is caused by residual nitrogen contamination in the sheath liquid is monitored to establish the reference baseline. Then, the separation capillary is slid into the interface until its outlet touches the inlet of the spray tube. The CE inlet vial is filled with a nitromethane solution, prepared in the BE, the vial is pressurized, and the solution is pushed into the separation capillary until an increase in the CLND signal is observed. Then, the injection pressure is released. A high, steady CLND signal remains because suction, created by the nebulizing action, keeps delivering the nitromethane solution through the separation capillary. Next, the separation capillary is slowly withdrawn using positioner 2, until the CLND signal level drops back to the original, reference baseline level (sheath liquid only, sans nitromethane), indicating that the nitromethane solution is no longer sucked out of the separation capillary. The second method utilizes the on-line UV detector to direct the positioning of the separation capillary in the liquid gap. First, the separation capillary is filled with the BE, and the UV absorbance at 214 nm is monitored by the UV detector to establish the BE baseline. Then, a 3-s-wide band of nitromethane is injected into the separation capillary. The nitromethane band is moved into the separation capillary by applying pressure onto the BE inlet vial until the UV absorbance signal begins to rise. As soon as the absorbance begins to decrease, pressure is released from the inlet vial. If there is no suction, siphoning, or back-flow, the UV absorbance level remains constant in time. Decreasing absorbance indicates that there is suction-induced forward flow or siphoninginduced forward flow in the separation capillary. Increasing absorbance indicates that there is backward flow in the separation capillary. Backward flow could be caused either by excessive sheath liquid delivery into the liquid gap or by backward siphoning. Thus, the length of the liquid gap is adjusted with positioner 2 until the absorbance signal becomes constant in time. Then, the point where the separation capillary enters stainless steel needle 4 is marked on the separation capillary. We monitored the UV absorbance signal for as long as 3 h and could not detect any change in the absorbance level. This indicated that the liquid Analytical Chemistry, Vol. 71, No. 22, November 15, 1999
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gap was sufficiently long to hydrodynamically decouple the sheath liquid subsystem from the separation capillary. Once all the CE measurements were completed, the nebulizer was taken apart, and the actual liquid gap distance was determined by measuring, under a microscope with a graduated ocular, the position of the marker spot on the separation capillary. After pressure-driven flow in the separation capillary was eliminated, the analytes were injected either electrokinetically, or by pressure, and the CE separations were completed as usual. RESULTS AND DISCUSSION A good pneumatic nebulizer interface should not reduce the separation efficiency of the CE system significantly. We achieved this objective by recognizing the critical importance of the following principles: (i) in a first approximation, the CLND signal is proportional to the mass flow rate of the analyte; (ii) the spray plume is very stable when the nebulizer is operated in its selfaspiration mode; (iii) the nebulizer is self-regulating when it operates in its self-aspiration mode (i.e., it does not draw mores or lesssfeed liquid than its self-aspiration rate); (iv) if the maximum EOF rate in the separation capillary is lower than the self-aspiration rate of the nebulizer, the flow rate difference must be made up either by suction-induced laminar flow through the separation capillary or by makeup liquid (sheath liquid) added to the effluent of the separation capillary; (v) the effluent of the separation capillary and the sheath liquid can be combined at the inlet of the spray tube (in a liquid gap); (vi) sheath liquid can be drawn into the spray tube from an open reservoir by suction exerted by the nebulizer; (vii) if the hydrodynamic resistance of the sheath liquid conduit is much lower than that of the separation capillary, suction will primarily draw liquid from the sheath liquid reservoir, rather than the separation capillary, to satisfy the selfaspiration rate of the nebulizer; (viii) if the volume of the liquid gap is small and the self-aspiration rate of the nebulizer is high, the liquid gap can be flushed efficiently with the sheath liquid, and back-mixing of the analyte bands at the end of the separation capillary can be minimized. Our design ensures that any change in the nebulizing gas pressure or solution viscosityswhich would alter the self-aspiration rate of the nebulizerswill only effect the sheath flow rate, but not the bulk flow rate in the separation capillary, and the adjustment will be automatic and self-regulating. The length of the spray tube is kept short to permit high self-aspiration rates and minimize the laminar-flow-induced broadening of the analyte bands as they move through the spray tube. The separation capillary and the spray tube are well-aligned with respect to each other because their o.d. is 150 µm and the i.d. of stainless steel needle 4 which holds them is 178 µm. In addition, the i.d. of the spray tube is larger than that of the separation capillary (100 vs 75 µm), and the outlet end of the separation capillary is beveled to facilitate a smooth, coaxial sheath flow pattern around the effluent of the separation capillary. Thus, with an optimum gap distance of about 100 µm (see below), the total liquid volume in the gap is less than 9 nL. Since at a 170 mL/min oxygen gas flow rate (see later) the self-aspiration rate of the nebulizer is 3.3 µLs-1, the nominal residence time in the gap (assuming plug flow) is less than 3 milliseconds. Thus, the eluted sample bands are rapidly removed from the gap. The 3.3 µLs-1 sheath flow rate can be maintained by suction alone through the low-resistance sheath 5256
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liquid conduit. Thus, the analyte is delivered into the spray tubes and the combustion furnacesby suction alone; the suction takes only as much sheath liquid as it needs, no more, no less. Consequently, the nebulization and feed flow rates are always balanced automatically. Since the separation capillary and the spray tube are hydrodynamically decoupled through the liquid gap, the CE subsystem and the nebulizer subsystem can be operated and optimized independently from each other. Some band broadening will inevitably occur when the analyte band exits the separation capillary, mixes with the liquid in the gap, is delivered to the furnace via laminar flow through the short spray tube, and passes through the CLND. The extent of this additional band broadening caused by the nebulizer and the CLND can be determined by comparing the bandwidths detected by the on-line UV detector and those detected by the CLND. The total peak variance recorded by the UV detector, σ2tot,UV, is
σ2tot,UV ) σ2inj + σ2D,UV + σ2UV
(5)
where σ2inj, σ2D,UV, and σ2UV are the variance contributions caused by the finite width of the injection band, the longitudinal diffusion of the analyte during its passage to the UV detector, and the finite length of the detector cell, respectively. Since σ2tot,UV can be measured and σ2inj and σ2UV can be calculated as28
σ2inj )
l2inj 12
(6)
σ2UV )
l2det 12
(7)
with linj and ldet as the length of the injected sample band and the aperture of the UV detector, respectively, the diffusion coefficient, D, can be calculated 28 from
σ2D,UV ) 2DtUV
(8)
where tUV is the migration time of the analyte to the UV detector. Since the analyte peak appears in the CLND at migration time tCLND, the theoretical total peak variance one would observe in a UV detector located at the end of the capillary, σ2tot,UV, theor, can be calculated with eqs 6-8, using D and tCLND, to obtain the theoretical separation efficiency of the CE system. One can then compare this efficiency with the actual separation efficiency calculated from the total peak variance recorded by the CLND, σ2tot,CLND. Comparison of these values yields the extent of efficiency loss that is due to the combined effects of the nebulizer interface and the CLND. Figure 3 shows a typical dual-channel detector trace for the electroosmotically migrated neutral analyte, nitromethane. The 1-s-long electrokinetic injection at 5 kV resulted in an initial bandwidth of 0.79 mm. The theoretical separation efficiency of the system obtained by a UV detector at the end of the capillarys calculated as outlined abovesis 109 630 plates. The actual separation efficiency calculated from the CLND trace is 97 200 plates. This represents a nebulizer-CLND related efficiency loss of only (28) Reijenga, J. C.; Kenndler, E. J. Chromatogr., A 1994, 659, 403-425.
Figure 3. UV (top) and CLND (bottom) traces obtained for an electroosmotically migrated N,N-dimethylformamide sample. Injection: electrokinetic, 1 s, 5 kV. Applied potential: 5 kV. Background electrolyte: 25 mM boric acid titrated to pH 9.2 with LiOH. For other conditions, see Experimental Section.
Figure 4. UV (top) and CLND (bottom) traces for the separation of four nucleotide bases (adenine (A), cytosine (C), guanine (G), and uracil (U)). The migration order of the bases is A, C, G, U. The amount of nitrogen represented by each peak is 10.35, 10.41, 8.37, and 7.58 pmol, respectively. Injection: pressure, 3 s, 0.039 psi nitrogen. Applied potential: 15 kV. Background electrolyte: 50 mM citric acid, titrated to pH 2.6 with LiOH. For other conditions, see Experimental Section.
11%. During the course of this investigation, the best separation efficiency we observed in the combined CE-nebulizer-CLND system for nitromethane was 113 000 theoretical plates and a peak asymmetry value (calculated at 10% of peak height) of 1.02. These values indicate that the gap is flushed quite rapidly, without extensive back-mixing, as predicted. Figure 4 shows the separation of adenine, guanine, cytosine, and uracil. Since the CLND signal is proportional to the number of moles of NO reaching the reaction chamber, the LOD is best given in terms of picomoles of nitrogen injected. In Figure 4 the adenine, guanine, cytosine, and uracil peaks represent 10.35, 10.41, 8.37, and 7.58 pmol of nitrogen, respectively. These values are close to the LODs for these analytes. The 3 s long pressure injection resulted in an initial bandwidth of 2.9 mm and lowered
Figure 5. Calibration curve for the coupled CE-CLND system: peak area vs number of moles of nitrogen injected. Analyte: N,Ndimethylformamide. Symbols: UV detector (×), CLND (+), average of triplicates at each concentration.
the separation efficiencies compared with what was observed for the 1-s electrokinetic injection of nitromethane. The calibration curves obtained for the CLND and UV detectors with N,Ndimethylformamide as analyte are shown in Figure 5. The CLND is linear (r2 ) 0.99992) over the 2 orders of magnitude range tested here, while the UV detector signal quickly falls off at this high concentration level. At all concentrations examined here, the %RSD values for peak areas on the CLND were better than 8% (five parallel runs). CONCLUSIONS The on-line coupling of CE with CLND has been accomplished by the nebulizer interface described here. Using a liquid gap and a sheath liquid subsystem with low flow-resistance, the interface hydrodynamically decouples the separation capillary from the spray tube which is operated in the self-regulating, natural selfaspiration mode. The new interface-CLND combination reduces the separation efficiency of the CE system by only about 10%. The mass detection limit observed with the current CE-CLND system is about 10 pmol of nitrogen; the linearity is good over the 2 orders of magnitude tested here. The nebulizer design described here should be usable for other mass flow sensitive gas-phase detection systems as well, provided that the gas-phase detection system can tolerate the relatively high self-aspirating liquid flow rates. ACKNOWLEDGMENT Partial financial support of this project and the loan of the CLND 7060 detector by Antek Inc. (Houston, TX) is gratefully acknowledged. We would also like to thank Stacey Collins, Mark Homan, and Randy Wreyford of Antek for their valuable input and advice. Received for review June 21, 1999. Accepted September 7, 1999. AC990679T
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