Anal. Chem. 2001, 73, 4545-4549
A Miniaturized Liquid Core Waveguide-Capillary Electrophoresis System with Flow Injection Sample Introduction and Fluorometric Detection Using Light-Emitting Diodes Shi-Li Wang, Xiao-Jing Huang, and Zhao-Lun Fang*
Research Center for Analytical Sciences, Northeastern University, Chemistry Building, Box 332, Shenyang 110006, P. R. China Purnendu K. Dasgupta
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061
A novel miniaturized capillary electrophoresis (CE) system is described where a Teflon AF-coated silica capillary serves both as the separation channel and as a transversely illuminated liquid core waveguide. This device uniquely uses flow injection (FI)-based split-flow sample introduction through a falling-drop interface. An H-channel structure fixed on a microscope glass slide utilizes a horizontal separation capillary with tubular sidearms on each end that serve as inlet and outlet flow-through electrode reservoirs. The inlet reservoir also functions as a falling-drop interface for coupling to the FI system. A blue LED is used as excitation source. A large-core optical fiber takes the emitted fluorescence to an inexpensive PMT with two layers of green plastic used for optical filtering. No focusing arrangement is needed. Continuous FI introduction of a series of 30-µL samples containing a mixture of of fluorescein isothiocyanate (FITC)-labeled amino acids allowed a throughput rate up to 144 samples/ h, with ∼2% carryover and good precision (3.2% RSD). Baseline separation was achieved for FITC-labeled arginine, phenylalanine, glycine, and FITC in sodium tetraborate buffer (pH 9.5) with plate heights of 5.4-5.5 µm and plate numbers of 2.34 × 104-2.37 × 104 under electrical field strengths of 214 V/cm for injection and 500 V/cm for separation (14-cm capillary, 48-µm i.d.). Detection limits (S/N ) 3) were 1.3 µM for arginine and 1.9 µM for phenylalanine and glycine. The combination of flow injection (FI) sample introduction and pretreatment techniques with capillary electrophoresis (CE) separation is demonstrably advantageous over other CE sample introduction techniques.1-3 The continuous sample introduction * Corresponding author: (e-mail)
[email protected]; (fax) +86-2423968628. (1) Fang, Z.-L.; Chen, H.-W.; Fang, Q.; Pu, Q.-S. Anal. Sci. 2000, 16, 197-203. (2) Fang, Z.-L.; Liu, Z.-S.; Shen, Q. Anal. Chim. Acta 1997, 346, 135-143 (3) Kuban, P., Engstrom, A.; Olsson, J. C.; Thorsen, G.; Tryzell, R.; Karlberg, B. Anal. Chim. Acta 1997, 337, 117-124. 10.1021/ac010341a CCC: $20.00 Published on Web 08/14/2001
© 2001 American Chemical Society
ability of a FI-CE system enhances reproducibility and throughput and allows on-line sample pretreatment. Recently, the entire FI-CE system was miniaturized using a 15 × 70 mm microscope slide as the base.4-7 This device contains an H-shaped channel with a horizontal several centimeter-long separation capillary connecting to two vertical sidearm tubes, one on each end of the slide. Either FI-4,5 or sequential injection (SI)6based sampling was demonstrated with laser-induced fluorescence (LIF),6 amperometric,4 and chemiluminescence (CL)5 detection. Conceptually, the approach is midway between conventional CE and microfabricated CE on a chip. It approaches the high separation speed and efficiency of the latter but is more readily coupled to FI- or SI-based sampling. The device is ideal for basic studies of microfluidic systems and can be inexpensively produced without microfabrication equipment. If desired, it can be mass produced in molded polymeric versions with a user-replaceable separation capillary. LIF is presently the workhorse for chip-scale CE systems. Detection sensitivity comes at a cost of complicated optical setup, need for critical adjustments by experts, and significant investment. They are elephantine in size compared to the separation systems they serve. The H-channel FI-CE device can be exploited for producing a more compact and robust fluorometric CE system by using a Teflon AF-coated capillary functioning as a liquid core waveguide (LCW). The light emission is guided through the LCW and collected by an optical fiber positioned adjacent to the capillary outlet. Teflon AF- and Teflon AF-coated tubes have thus been successfully used for FI and CE determinations this way.8-11 Light (4) Fu, C.-G.; Fang, Z.-L. Anal. Chim. Acta 2000, 422, 71-79. (5) Huang, X.-J.; Pu, Q.-S.; Fang, Z.-L. Analyst 2001, 126, 281-284. (6) Fang, Q.; Wang, F.-R.; Wang, S.-L.; Liu, S.-S.; Xu, S.-K.; Fang, Z.-L. Anal. Chim. Acta 1999, 390, 27-37. (7) Fang, Z.-L.; Fang, Q. Fresenius’ J. Anal. Chem., in press. (8) Li, J.; Dasgupta, P. K.; Genfa, Z.; Hutterli, M. A. Field Anal. Chem. Technol. 2001, 5, 2-11. (9) Li, J.; Dasgupta, P. K. Anal. Chem. 2000, 72, 5338-5347 (10) Li, J.; Dasgupta P. K.; Zhang, G. Talanta 1999, 50, 617-623 (11) Dasgupta, P. K.; Zhang, G.; Li, J.; Boring, C. B.; Jambunathan, S.; AI-Horr, R. Anal. Chem. 1999, 71, 1400-1407
Analytical Chemistry, Vol. 73, No. 18, September 15, 2001 4545
Figure 1. Schematic structure of the micro-CE device (not to scale) with falling-drop interface. CP, Teflon AF-coated fused-silica capillary, 50-µm i.d., 360 -µm o.d.; P, planar glass base plate; G,: epoxy; Pt, platinum electrode; OF, optical fiber; W, waste; X, glass plug.
from the excitation source impinges perpendicularly on the capillary axis and passes through, without being axially transmitted through the waveguide, the fluorescence emission need only be minimal filtered for stray excitation light. Previously fluorometric LCW-CE was demonstrated only for separating two model fluorescent dyes without any optimization. Here we introduce a novel, very inexpensive LED/LCW-based fluorometric FI-CE system in which continuous automated FI-based sampling is coupled to the LCW-based CE system on a chip-scale interface. EXPERIMENTAL SECTION Reagents. All reagents used were of reagent grade, and deionized (DI) water was used throughout. The carrier solution for the FI system and the working electrolyte for CE separation were 10 mM Na2B4O7 buffer, adjusted to pH 9.5 using 1 M NaOH solution. HCl (0.1 M) and NaOH (0.1 M) were used as capillary wash liquids. Stock solutions of 10 mM L-arginine, L-phenylalanine, and L-glycine (Kangda Amino Acid Works, Shanghai, China) were prepared in DI water. FITC (Sigma), 40 mM, in acetone (containing ∼1% v/v pyridine) was used as labeling reagent and prepared fresh before use. Stock FITC-labeled amino acid solutions containing 3.5 mM of each amino acid were prepared by mixing 0.35-mL stock solutions of each amino acid with 0.55 mL of the borate buffer and 0.1 mL of labeling reagent, and the resultant mixture was left in the dark overnight at room temperature. Working solutions were prepared by diluting the stock with the borate buffer. Apparatus. A model FI-2100 flow injection analysis system (Vital Instruments, Beijing, China) with two variable-speed peristaltic pumps and a 16-port valve was used for sample introduction. PTFE tubing (0.7-mm i.d.) was used for connecting all components of the FI system upstream of the sample loop, while a 14-cm length of 0.5-mm-i.d. PTFE tubing was used to connect to the split-flow interface. An electronic timer (JS14S, Taihua Electronics Co., Shanghai, China) switched the 0-30-kV high-voltage power supply (model 9323-HVPS, Institute of Applied New Technology, Beijing, China) between two different voltage settings. The miniaturized CE device (Figure 1) was constructed using a 15 × 70 × 1 mm microscope glass slide as a base plate, on which was integrated a falling-drop interface, a separation capillary, 4546
Analytical Chemistry, Vol. 73, No. 18, September 15, 2001
and an end-column reservoir. The falling-drop interface (left inlet terminal in Figure 1) and the end-column reservoir (right outlet terminal in Figure 1) were fabricated from 10 × 15 × 10 mm Plexiglas blocks that were first connected with the separation capillary and then fixed as shown on the slide with epoxy. A 0.5mm-diameter, 5-mm-long vertical hole, terminating in a conical top and bottom (4-mm diameter at the openings) formed the inlet terminal. A 0.5-mm hole, drilled at the upper end of the vertical hole, accommodated the separation capillary. On the opposite side, 1.5 mm from the upper end of the channel, a 1-cm length of 0.5mm-diameter platinum wire, inserted through another hole (and fixed in place with epoxy), served as the anode. In the right terminal, a 2-mm-diameter vertical hole serves as the cathode reservoir. A 0.5-mm-diameter horizontal through hole, intersecting the vertical aperture, held the capillary outlet at the inner end. The optical fiber (quartz, 300-µm diameter) was coaxially butted against the capillary outlet (that protruded to the center of the well) from the outer end. The free end of the optical fiber addressed the PMT (Hamamatsu 1P28) with two layers of green plastic film, acting as a broadband filter inserted between them. The PMT electronics (model GD-1, Ruike Electronics, Xi′an, China) provided an output signal recorded on a strip chart recorder (BD41, Kipp & Zonen). A Teflon AF 1600-coated fused-silica capillary (TSU 050375, 48-µm i.d., 362-µm o.d., 140 mm long, Polymicro Technologies) served as the LCW separation capillary; the central section, coiled into a loop, is fixed on the base plate with adhesive tape. A blue light-emitting diode (LED) (LNG992CFBW, 478 nm, Panasonic) was used as the excitation source, located 12 mm from the capillary outlet in contact with the outer wall, transversely illuminating the lumen. Excess plastic was first carefully removed from the top of the LED by grinding with sandpaper, leaving
