Anal. Chem. 1999, 71, 1400-1407
Luminescence Detection with a Liquid Core Waveguide Purnendu K. Dasgupta,* Zhang Genfa, Jianzhong Li, C. Bradley Boring, Sivakumar Jambunathan, and Rida Al-Horr
Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-1061
A new fluoropolymer tube is proposed as the basis of a novel class of liquid core waveguide-based luminescence detectors. Both chemiluminescence and photoluminescence detectors are possible. In the latter case, illumination is transverse to the main axis of the tube. With such a geometry, it is even possible to operate without monochromators, although limits of detection do improve with the incorporation of monochromators. The nature of the design is such that it is particularly simple to fabricate detectors in a flow-through configuration and where the light from the cell is coupled to a photodetector by an optical fiber. No focusing optics are necessary. A number of applications are illustrated. Attainable limits (LODs, S/N ) 3) of detection include 150 pM fluorescein with a 254-nm excitation source, 200 amol of fluorescein in a capillary electrophoresis setup with excitation by two blue light-emitting diodes, 35 nM NH3 as the isoindole derivative in a flow injection analysis system using a photodiode detector, 50 nM methylene blue and 1 nM Rhodamine 560 using respectively red and green LED arrays and an avalanche photodiode and a PMT in a FIA configuration, 100 parts per trillion by volume gaseous formaldehyde as the Hantzsch reaction product with cyclohexanedione using a diffusion scrubber, 2.7 µM and 17 nM hypochlorite based on its chemiluminescence reaction with luminol with photodiode and PMT detectors, respectively, and 1 ppm SO42- based on nephelometric detection at 470 nm. The approach described herein leads to particularly simple and inexpensive luminescence detectors with excellent sensitivity. An optical fiber carries light with minimal loss because of a refractive index (RI) difference between the core and the cladding of the fiber. Light remains trapped in the optically denser fiber core. It has long been realized that long-path absorbance measurements for liquid samples can only be realized if the cell behaves as an optical fiber or waveguide. Otherwise, too much light is lost to the walls and excessive noise results. For the absorbance measurement cell to function as a liquid core waveguide (LCW), the cell material needs to have a lower RI than the liquid. Early work has involved systems such as carbon disulfide (RI 1.63 for Na D-line) in glass tubes (RI typically 1.52)1 or ethanol (RI 1.36) in a fluorinated ethylene-propylene (FEP) copolymer (RI 1.34) (1) Fujiwara, K.; Fuwa, K. Anal. Chem. 1985, 57, 1013.
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tube.2 However, the most important system of practical importance remains purely aqueous solutions. Therefore, the most important development in this regard has been the introduction of a new amorphous fluoropolymer, Teflon AF, which has an RI less than that of water.3 The general area of long-path absorbance measurements with LCW cells, with specific reference to Teflon AF-based cells, has been reviewed.4,5 Tubes of Teflon AF and fused-silica tubes coated externally with Teflon AF are now commercially available,4-6 as are long-path cells and detectors based on such cells.7,8 Fujiwara et al. had carried out much of the early work on absorbance measurements in waveguide cells. It is not surprising that the first use of liquid core waveguides for fluorometric measurements is also due to these authors.9,10 The general preoccupation in these efforts seems to have been the use of a laser as the excitation source. As such, the authors considered only situations where the excitation light is launched axially (or nearly so, at a critical angle that fosters the axial propagation of the light into the cell) into a tube that may be maintained in a linear, a U-shaped, or a coiled configuration. The fluorescence can be read axially from the other terminus of the tube. With this geometry, very effective means of rejecting the excitation light are required. The excitation or emitted light or both can be attenuated by the medium, and as such, the maximum fluorescence signal is reached after a finite cell length. A “side-view cell” was also investigated. A typical implementation involves a spirally shaped tubular cell; the plane of the spiral is placed next to the photosensitive window of a “end-on” type photomultiplier tube (PMT) while light is launched axially into the spiral. For this geometry, the fluorescence signal is said to be linearly dependent on the refractive index of the solution.9 In terms of continuous illumination energy, nonlaser sources still have the edge, especially when one considers the great (2) Tsunoda, K.; Nomura, A.; Yamada, J.; Nishi, S. Appl. Scpectrosc. 1990, 44, 1163. (3) DuPont Fluroproducts. Teflon AF Amorphous Fluoropolymers. H-16577-1, Wilmington, DE 19880-0711, December 1989. (4) Altkorn, R.; Koev, I,; Gottlieb, A. Appl. Spectrosc. 1997, 51, 1554. (5) Dasgupta, P. K., Zhang, G., Poruthoor, S. K.; Caldwell, S.; Dong, S.; Liu, S.-Y. Anal. Chem. 1998, 70, 4661-4669. (6) http://www.polymicro.com/tsu.htm. Information on series TSU fluorocarboncoated capillaries, Polymicro Technologies, Phoenix, AZ. (7) http://www.wpiinc.com/WPI_Web/Spectroscopy/LWCC.html. (8) http://www.thermoseparation.com/Set_02.html. (9) Fujiwara, K., Ito, S. Trends Anal. Chem. 1991, 10, 184. (10) Fujiwara, K., Ito, S.; Kojyo, R.-E., Tsubota, H.; Carter, R. L. Appl. Spectrosc. 1992, 46, 1032. 10.1021/ac981260q CCC: $18.00
© 1999 American Chemical Society Published on Web 02/18/1999
diversity of wavelengths that can be economically obtained from them. Thus, to consider only axial illumination in fluorometric measurement with a LCW cell unnecessarily limits one’s vision. Many illumination sources are available in the linear format (including linear flash lamps) that can be used to illuminate a tube transversely along its long axis. Many other sources provide a circular spot of light that can conveniently provide transverse illumination to a tube that has been coiled into a spiral. To a first approximation, light incident on the surface of an ideal fiber (transverse illumination) simply passes radially through the fiber and out. It does not propagate down the lumen. However, light can be generated within the lumen of the fiber itself. This may occur through excitation by the transversely passing radiation, due to elastic or inelastic scattering. Or light may be generated through a chemiluminescent reaction. A substantial fraction of this light, the precise extent depending on the numerical aperture of the fiber and the radial position at which the light is generated, will be emitted within the launch angle of the fiber and will propagate down the fiber. “Scintillating” optical fibers were originally developed as a simple means of detecting nuclear radiation.11,12 The cores of these fibers is typically doped with an organic scintillator, often in conjunction with a fluorescent dye such as fluorescein, serving as a “wavelength shifter”. A fiber like this can be immersed or imbedded in a medium producing nuclear radiation. Radiation that passes through the fiber has a high probability of interacting with a scinitillator molecule and producing visible radiation. Again, a substantial fraction of the light emitted propagates down the fiber and is harnessed for conventional photometric detection. Such fibers, with a polystyrene core doped variously with fluorescent dyes that emit in the visible and clad with a lower RI poly(methyl methacrylate) polymer, are readily available in round or square cross sections.13,14 In examining such a fiber, for example, one doped with fluorescein, the remarkable intense green fluorescence one observes at the termini is qualitatively the same, regardless of whether one is under daylight, fluorescent light, or tungsten light. Of course, this is because the nature of the emitter is the same. However, the results are dramatically different if the surface of the fiber is covered up and the same suite of excitation lights is launched, one at a time, through one of the termini and the resulting light, the sum of the fluorescent emission and the remnant excitation light, is viewed through the other. The striking conclusion is that when fluorescence is axially observed in a transversely illuminated optical fiber, it can be observed by itself, with little interference by the excitation light. If we translate the same situation to a liquid core optical fiber, axial fluorescence detection should be possible with a “white” light source illuminating the surface of such a fiber, without any excitation or emission monochromators. In this paper, we examine the utility of this uniquely simple geometry for fluorescence and other related luminescence detection methods. EXPERIMENTAL SECTION Materials and Equipment. Teflon AF 2400 capillaries in different sizes were obtained from Biogeneral Inc. (San Diego, (11) Binns, W. R.; Israel, M. H.; Klarmann, J. Nucl. Instrum. Methods Phys. Res. 1983, 216, 475. (12) White, T. O. Nucl. Instrum. Methods Phys. Res. 1988, A273, 820. (13) www.edsci.com. (14) http://www.bicron.com/fibers.htm.
CA). Teflon AF 1600-coated fused-silica capillaries (360-µm o.d., 100-µm i.d., 15-µm coating) were obtained from Polymicro Technologies (Phoenix, AZ). Light sources used in this work include light-emitting diodes (LEDs, Panasonic high brightness LNG992CFBW emitting at 470 nm with a half bandwidth of 40 nm, Nichia superluminescent 590S, emitting at 495 nm, Gilway E201, emitting at 660 nm with a half bandwidth of 60 nmsthese are of only modest brightness), low-pressure mercury pen lamps emitting at 254 nm and a phosphor-coated version of the same (∼0.25-in. diameter, illuminated lengths 1, 2, and 7 in., BHK Inc., Claremont, CA), miniature versions of similar 254-nm lamps in both glass and quartz envelopes (JKL Components Corp., Pacoima, CA), mercury black light 365-nm lamps in customary black glass envelopes in standard F4T5 (0.5 × 4.25 in.) and subminiature sizes (3-mm diameter, 5 mm and 20 mm long, JKL Components), linear Xe flash lamp source (model FLS 01, Chapparal Technologies, Albuquerque, NM; up to 10 J discharged/flash), and household-type white fluorescent lamps. Detectors used ranged from very inexpensive silicon photodiodes (3 × 3 mm, type S2007, Electronic Goldmine, Phoenix, AZ) and various photodiodes (Hamamatsu Corp. and others). These were typically used with simple home-built operational amplifier based current to voltage converters; in some experiments, a commercial picoammeter (Keithley model 480) was used. For higher sensitivities, we used a miniature PMT equipped with its own high-voltage (HV) power supply and current to voltage converter (H5784) and a temperature-controlled avalanche photodiode (APD) module equipped with its own HV supply (S5460, both from Hamamatsu). We should like to emphasize that with the exception of the pulsed Xe source and the commercial current amplifier (neither of which is involved in the best results presented in this work), the parts cost to build any complete luminescence detector including a flowthrough cell, light source, and all necessary power supplies and signal processing electronics, ranges from under $100 to $1000. The general design shown in Figure 1 was used for experimenting with the detection scheme. An opaque PEEK tee T for 1/ -in. tubes (Upchurch Scientific) constitutes one end of the cell. 8 In the center of the tee, an LCW tube AF butts against an 1-mm core acrylic or silica optical fiber F that is coupled to a photodetector. The liquid enters through the perpendicular arm of the tee, enters the AF tube through the gap that unavoidably remains between AF and F, and proceeds through AF to the other end where a connecting tubing is put in with a chromatographic malemale union U. A light source L illuminates the LCW as shown in the figure. For subminiature fluorescent lamp or LED excitation sources (in single or arrayed configuration), isolation from external light is provided by an opaque tubular shell S that fits within the hub of the nut in fitting T and within the inner half of U (which is drilled out to accommodate it). Electrical leads to L are brought out through the walls of S. The inside of the shell S was polished to improve excitation light throughput. Larger sources required a larger shell, put in on the exterior of the nut at T and that of U, with a spacer element, if necessary. Many experiments were conducted where light shielding was simply provided by a dark cloth draped over the experimental arrangement, or the setup was put inside a cardboard box. For optical filtering, interference filters were used in a few experiments but most commonly we used inexpensive colored Analytical Chemistry, Vol. 71, No. 7, April 1, 1999
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Figure 1. Typical arrangement for fluorescence detection. F, acrylate or silica optical fiber butted against LCW tube AF in the center of tee fitting T. The other end of AF is connected to waste tubing WT by compression fitting union U. A tubular shell S houses the tubing AF and the light source L.
plastic sheets available from Edmund Scientific (Catalog No. E60403), and filter numbers quoted in the text refer to the numbers from this vendor. Capillary electrophoresis experiments were conducted using a homemade CE instrument that has been briefly described previously.15 A 60-cm-long Teflon AF-1600-coated 100-µm-i.d. capillary (TSU100360, Polymicro Technologies) was used and the hardware for a radial path homemade capillary absorbance detector16 was used to hold a pair of blue LEDs on either side of the capillary, ∼10 cm from the grounding end. The grounding end of the capillary was put in a subminiature polypropylene tee and a 1-mm core silica fiber was butted against it. The other end of the fiber addressed the PMT to perform fluorescence detection. Fluid communication with the background electrolyte (BGE, 4 mM Na2B4O7) in the vial is maintained through the tee arm, and the ground electrode is maintained in the vial. Injection was hydrodynamic (∆h ) 10 cm, t ) 5 s, which resulted in an injected volume of 24 nL), and electrophoresis was conducted at an applied voltage of +20 kV. Nephelometric experiments were conducted with a 470-nm LED as the transverse illumination source located 1.5 cm from a 1-mm core silica fiber in a cell built with 0.79-mm-i.d. Teflon AF tubing. The detection of sulfate by the classical, albeit relatively insensitive technique BaSO4 turbidimetry was studied in a flow injection configuration. The sulfate sample (65 µL) was injected into a water carrier (160 µL/min) and merged with an equal flow rate of a reagent stream containing 5% BaCl2, 10% ethanol, and 10% ethylene glycol. The detection cell was placed after a 60-cm length of 0.7-mm-i.d. reaction coil. Phosphorimetric detection was studied with the pulsed Xe light source (arc length 13 cm, 3.5 J/pulse used for the data reported here). This linear flash lamp source was located within a reflective elliptical cavity, the lamp being located on one of the foci and a 0.79-mm-i.d. Teflon AF tube (40 cm) running through the other focal point. One side of the AF tube is connected to a waste line; the other side is connected to a tee for solution input and optical connection to a 1-mm core silica fiber that terminates in a UVsensitive photodiode (Hamamatsu HUV1000 BQ). The photodiode output was converted to a voltage signal by a rapid response i to V converter (Keithley model 427) and read by a storage oscilloscope (Tektronix, model 5103N). Except as stated, reagents were obtained from Aldrich Chemical and used without further purification. (15) Liu, S.; Dasgupta, P. K. Anal. Chim. Acta 1992, 268, 1. (16) Boring, C. B., Dasgupta, P. K. Anal. Chim. Acta 1997, 342, 123.
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RESULTS AND DISCUSSION Rejection of Excitation Light. In a conventional fluorometric setup, fluorescence is viewed at right angles to the excitation beam, typically using 10-mm path length cells. Due to reflection and scattering at the windows and at the interfaces, some light reaches the detector even with the latter placed at right angles. Using 5-mm circular apertures with a conventional cell filled with water and a divergent source, we measure that 0.06-0.1% of the source light is scattered into the detector. When the same experiment is conducted with a LCW cell, with the cell illuminated over a ∼3-mm length, ∼100 mm away from the detector, 0.0010.002% of the source light can be measured to be axially propagating through the cell. It is this degree of rejection of the excitation light that allows fluorescence measurement without any or with only rudimentary wavelength discrimination in the light propagating through the lumen of the LCW cell. It is also this characteristic that makes possible the uniquely simple design of such fluorescence detectors. Light Conduction to a Photodetector. Initial experiments were conducted with a design in which the detector end of the LCW cell ended in a thin transparent glass window made of a microscope slide cover glass. This was placed directly on top of the PMT window. Studies with the design described in the Experimental Section showed, however, that light is just as efficiently and far more conveniently conducted to the detector when a high numerical aperture fiber (a silica fiber of the same or greater core diameter as the inner diameter of the LCW cell or a larger diameter acrylic fiber) is butt-jointed to the LCW tube. This arrangement was used henceforth. When used, colored plastic filters could be directly attached to the detector end of the fiber optic by UV-cure adhesive or a suitably cut circle of the filter put on the face of the photodetector. LCW Cell Geometry. With a core RI of 1.33 (water) and a polymer RI of 1.29, a Teflon AF 2400 tube has a numerical aperture of ∼0.32 and an acceptance angle of 37°. Coiling any fiber into a small-diameter coil causes loss of light from the lumen; the exact coil diameter at which the loss becomes significant depends on the numerical aperture. However, theoretical computations alone are of limited help because this is also greatly influenced by the imperfections in a fiber. With water-filled Teflon AF 2400 tubes, we find that loss of light becomes significant only below coil diameters of 5 cm. Not only light is lost from the lumen in smalldiameter coils; the ability of transverse illumination to propagate through the axial mode increases. The signal-to-background ratio is generally unacceptably poor if, for example, the LCW cell is coiled on a conventional 3.7-cm-diameter fluorescent lamp (as
Figure 3. Reciprocal of the limit of detection plotted against the square root of illuminated volume.
Figure 2. Response of repeated injections of 10 nM fluorescein in a FIA system. LCW cell i.d. 0.79 mm, illuminated length 80 mm, 254nm Hg lamp unfiltered illumination, and PMT detector with No. 877 green plastic filter. The upper inset shows the response from a commercial HPLC detector for the same sample.
commonly used in overhead lighting applications). Most lamps are smaller than this in diameter. So coiling an LCW cell directly on a lamp source (in many cases heat dissipation would also be an additional problem) is not practical. In all further work, we therefore used the LCW cell in a linear configuration. Effect of Illumination Length. This was investigated by using a 17-cm-long Hg pen lamp emitting at 254 nm. The LCW cell (0.79mm i.d.) was placed along the length of the lamp, a few millimeters above its top. Fluorescence intensities from a 10, 30, 50, 100, and 200 nM fluorescein solution and the absolute level of the background were measured in a FIA configuration. The lamp was covered with a metal sheet. By moving the sheet, different lengths of the lamp could be exposed. The tee end with the optical fiber was 10 cm distant from the exposed terminus of the lamp. Exposed lengths of 1, 4, 8.5, and 17 cm were studied. In each case, the response was linear with concentration with an intercept that was statistically indistinguishable from zero and the linear r2 value ranged from 0.9994 to 0.9998. The response slope increased with increasing length, being in the ratio 1: 5.0:7.3:15 for the four above illumination lengths. Considering that the intensity of illumination is not identical in different portions of such a lamp and it is difficult to have a very well defined length of the lamp providing the illumination, these results do indicate that the observed fluorescence intensity increases in direct proportion to the illumination length. The background signal, on the other hand, increases in a proportion less than linearly with the illumination length, being in the ratio of 1:2.6:3.8:6.0 for the 1:4:8.5:17 cm illumination lengths. Moreover, at least under these conditions, the detector noise does not increase in proportion to the background signal, the ratio being 1:1:1.5:2 in the order of
increasing illumination lengths. Thus, the LOD improves with increasing illumination length. Figure 2 shows replicate injections of a 10 nM fluorescein solution (illuminated volume 39 µL). The LOD (S/N ) 3) is computed to be 150 pM. The inset shows a similar experiment with a commercial fluorescence detector for HPLC (cell volume 20 µL) made by a major manufacturer, set at the optimum excitation and emission wavelengths for the measurement of fluorescein. The superior performance of the present detector is readily apparent. Admittedly, the excitation source of the commercial detector is no longer in its prime, S/N measurement for quinine sulfate indicates a performance 3 times worse relative to manufacturer’s specifications. However, the performance difference between the present detector and the commercial detector is considerably greater even after accounting for this and the difference in illuminated volume (which accounts for a factor of 21/2; see below). It was also of interest to us to determine that if light intensity is increased, what type of LODs may be attainable with an inexpensive photodiode detector. The linear flash lamp source illuminated a 0.79-mm-i.d. LWC, placed in its close proximity, protected by a roll of blue plastic (No. 35,135). A silica fiber led the emitted light to a very inexpensive photodiode covered with No. 877 green plastic. The flash lamp was fired with 5-J pulses. The actual flash duration was of the order of 1 µs, but the high capacitance of the photodiode broadened the observed response to a half-width of 40 µs. The S/N was found to increase directly with the square root of the number of pulses accumulated, with 100 pulses, the LOD was 12 nM. Effect of Illuminated Volume. A more detailed study of the effect of varying both the illumination length and the diameter of the tube was conducted. From basic considerations, S/N should improve with the square root of the illuminated volume, which should therefore be linearly related to the LOD. This proves to be the case. Figure 3 illustrates this for tubes of three different diameters and different illumination lengths. A clear establishment Analytical Chemistry, Vol. 71, No. 7, April 1, 1999
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Figure 4. (a) Response from 100 nM formaldehyde, PMT detector with No. 856 (blue) plastic filter; (b) long-term response stability from repeated exposures to 5 ppbv level of gas-phase formaldehyde collected by a Nafion membrane diffusion scrubber and same detector. Table 1. Lamp Performance in Fluorometric LCW Measurement of Formaldehyde lamp type 4-cm active length Hg pen lamp 254 nm 2.5-cm active length Hg pen lamp 406 nm phosphor coated 4-cm active length F4T5 1.25-cm-diameter blacklight, 365 nm 4-cm active length 11 × 90 mm glass envelope Hg lamp, 254 nm 4-cm active length 8 × 100 mm quartz envelope Hg lamp, 254 nm 3 × 50 mm miniature Hg blacklight lamp, 365 nm
LOD, nM 34 80 150 13 3 11
of this basic behavior is very useful because it allows one to calculate a priori what changes in performance can be expected on the basis of changing cell volume or tube diameter. Illustrative Examples. It appears to us that the real merit of the proposed approach is not in general studies of luminescence spectrometry but in actual applications to design a dedicated detector that may constitute an inexpensive module for a complete measurement system. Formaldehyde. Formaldehyde is a simple compound of considerable environmental interest that can be selectively and sensitively determined by a fluorogenic reaction with cyclohexanedione and ammonium acetate; a FIA adaptation of this reaction has been published.17 The optimum excitation and emission wavelengths are 395 and 465 nm. However, both are broad and this allows considerable latitude in choosing excitation wavelengths and in the choice of detectors/filters. The signal-to-noise performance of different types of photodiodes were as follows (the relative S/N is indicated): G1115 (GaAsP) and S1226-5BQ (UV(17) Fan, Q.; Dasgupta, P. K. Anal. Chem. 1994, 66, 6, 551.
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enhaned silicon) 11, S2007 (silicon) and G3273 (large-area highperformance silicon) 17, G118 (GaAsP, diffusion type) 85, and G1742 (GaAsP, Schottky type) 100. With direct fiber coupling to a photodiode, a large active sensing area is not necessary, any sensing area beyond the fiber optic diameter merely contributes to noise. Lens ends are not desirable either. In this particular case, best results are obtained with small active area high quantum efficiency GaAsP photodiodes where the active surfaces are close to the top of the case. The two best performers cost less than $10 each. Variations in the lamp characteristics also affect the performance. Table 1 lists the attainable LODs with a G1118 photodiode, a light blue (No. 856) plastic filter, and several different types of lamps. Of course, in most applications, a choice has to be made based on the cost/performance ratio. Here the performance attainable with thin (3-mm diameter), inexpensive (less than $20, including power supply) blacklight (365 nm) lamps is particularly attractive. The detector geometry shown in Figure 1 is such that it is readily amenable to clustering several lamps around the LCW tube. Up to four lamps clustered around the LCW tube were studied. As may be intuitive, the response improves and the LOD decreases with the number of lamps. Of course, a much greater increase in the LOD is achieved with a PMT detector. Figure 4a shows the response from 100 nM HCHO with a 3 × 50 mm 365-nm lamp as the excitation source and a PMT detector. Figure 4b shows the response from repeated injections of 2.5 parts per billion by volume levels of gaseous HCHO (a key marker molecule in atmospheric photochemistry studies) in zero air, using a Nafion membrane diffusion scrubber as a collector and the same detection arrangement.17 Ammonia. Ammonia is another simple compound the determination of which is needed over a very wide range in different applications. The sensitivity of applicable methods is taxed when
Figure 5. (a) Response to 670 nM aqueous methylene blue in a FIA system, 5 mM HCl carrier, LWC illuminated by a 660-nm red LED array, illuminated volume 15 µL, avalanche photodiode detector, and No. 35136 plastic filter. (b) Response to 3 nM Rhodamine 560, LWC illuminated by dual array of green LEDs, illuminated volume 12 µL, No. 806 plastic filter, and PMT detection.
one needs, for example, to measure the concentration of ammonia over remote oceans.18 One of the more sensitive methods is the derivatization of ammonia with sulfite and o-phthalaldehyde to form 1-sulfonatoisoindole; FIA adaptations19 and application to the measurement of gaseous ammonia20 have been described. In this case, the optimum excitation wavelength is 365 nm, perfectly matched by a Hg blacklight lamp. The emission is centered at 425 nm. The detection limit with a commercial fluorescence detector with a PMT detector equipped with a long-pass excitation filter was 20 nM.19 With the same excitation arrangement as for formaldehyde above, the fluorescence in this case is sufficiently intense that a detection limit of 35 nM could be obtained with $15 detector (blue/UV-sensitized photodiode detector with integral amplifier (Burr-Brown OPT 301) with blue (No. 856) plastic filter). Methylene Blue and Rhodamine. Methylene blue (MB) is an intensely colored dye that absorbs in the deep red (absorption maximum 664 nm). It is widely used as a biological stain and also as a cationic ion-pairing agent for measuring anionic surfactants in water and wastewater. MB fluoresces with an emission maximum at 690 nm. Although the emission is not particularly strong, the fluorescence is selectively quenched by purine nucleotides; this has important applications in biological analysis.21 The dye or its ion pairs/conjugates are usually determined by absorption spectrometry; the fact that it fluoresces is less widely exploited. Spaziani et al.22 recently described a diode laser-based (18) Genfa, Z.; Uehara, T.; Dasgupta, P. K.; Clarke, T.; Winiwarter, W. Anal. Chem. 1998, 70, 3656. (19) Genfa, Z.; Dasgupta, P. K. Anal. Chem. 1989, 61, 408. (20) Genfa, Z.; Dasgupta, P. K.; Dong, S. Environ. Sci. Technol. 1989, 23, 1467. (21) Dunn, D. A.; Lin, V. H.; Kochevar, I. E. Photochem. Photobiol. 1991, 53, 47.
fluorescence detector in which sulfide is determined via the formation of MB.23 In our experiments, to excite MB, a 29-mmlong LED array consisting of 14 LEDs, connected in parallel (each with its own current limiting resistor), was constructed by removing much of the epoxy molding from each LED (both from the sides and top), cementing them together with epoxy adhesive, and polishing the top of the array to create a flat surface. The array was placed in close lateral proximity of the LCW tube. An avalanche photodiode is well suited for the detection of fluorescence in this case. While red-sensitive PMTs are expensive, silicon APDs have usable sensitivities at such wavelengths. Moreover, while the active area of a PMT tends to be a minimum of 1 cm in diameter, APDs are readily available with active area diameters of the order of 1 mm, which is perfectly adequate for coupling to a fiber optic as used in the present cells and obviates the need for more expensive APDs with larger active areas. A response to an injection of 670 nM MB in a FIA system with water carrier is show in Figure 5a, the S/N at this level is such that an S/N ) 3 LOD of 50 nM can be estimated. A similar experiment is shown for Rhodamine 560, another fluorescent dye widely used as a tag. In this case, much brighter green LEDs, two arrays (of six LEDs each) were deployed on opposite sides of the LCW tube, providing an illuminated volume of 12 µL. The results for 3 nM injections with a PMT detector are shown in Figure 6b; the baseline appears between the two sets of injections and the fluctuations are largely due to pump pulsations. Even then, the LOD is equal to at least 1 nM. With a (22) Spaziani, M. A.; Davis, J. L.; Tinani, M.; Carroll, M. Analyst 1997, 122, 1555. (23) Kuban, V.; Dasgupta, P. K.; Marx, J. N. Anal. Chem. 1992, 64, 36.
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Figure 6. Chemiluminescence signals recorded by a PMT detector for 500 nM ClO- in a luminol reaction system in a FIA configuration.
pulseless pumping arrangement, the LOD is expected to be in the high picomolar range. Capillary Electrophoresis. Detection of molecules tagged with fluorescent taggants is in extensive use in the practice of capillary electrophoretic separations, especially in biological and biomedical research. The separation and detection of amino acids as derivatized with fluorescein isothiocyanate (FITC) is in wide use, for example. The limits of detection for FITC-derivatized amino acids are typically in the low-attomole range with laserinduced fluorescence detection.24 When a Teflon AF-coated fused-silica tube filled with an aqueous liquid is illuminated, both the water core and the silica wall can act as waveguides. If such a tube is filled with water and transversely illuminated with the blue LEDs as in the present experiment, dark-field microscopic examination of the tip of the capillary shows that a small amount of blue light is transmitted through the silica wall. If on the other hand the tube is filled with a fluorescein solution, it can be clearly observed that the fluorescence emission is confined to the liquid core. To reject the stray excitation beam transmitted through the silica wall, one can either provide an opaque coating to the tip of the capillary or use a collection fiber that is matched to the core diameter of the LCW such that it collects only the light conducted by the liquid core. We chose the first alternative. The face of the capillary was made opaque by heavy silvering and the emitted fluorescent light carried through the liquid core was transmitted to a large (1-mm)diameter silica optical fiber. The results are shown in Figure 7. The injected sample amount is 2.5 fmol. We estimate an LOD of ∼200 amol under these conditions. The LEDs were very modestly driven in these experiments, if they are operated in a pulsed mode much higher illumination intensities can be obtained. We note in this context that if four different colored LEDs are staggered around the capillary to selectively excite four different colored fluorescent tags that themseleves selectively attach to four different nucleotide bases, an excitation pulse train sequence can (24) Mattusch, J.; Dittrich, K. J. Chromatogr., A 1994, 680, 279.
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Figure 7. Response from a mixture of 100 nM fluorescein (tm ∼3.5 min) and 100 nM carboxyfluorescein (tm ∼5 min) in a capillary electrophoresis system. Computed injection volume 25 nL. See text for details.
be used to perform four-color sequencing very effectively and inexpensively. Chemiluminescence (CL) Detection. Aqueous chlorine and hypochlorite are important analytes that can be measured by their reaction with luminol that results in CL. Under carefully optimized conditions, the LOD has been reported to range from 0.3 26 to 12 µg/L25 for a custom-built luminometer (S/N ) 3). In our experiments, the same detection arrangement as used for the fluorometric measurement of ammonia and/or formaldehyde was used, except the lamp was not turned on and no filter was used. Using 2 mM luminol in a 0.2 M carbonate buffer (pH 11.2) in a flow injection analysis (FIA) configuration, the LOD was 2.75 µM ClO- with a photodiode detector (as used for ammonia above) and 15 nM ClO- for the PMT detector (as used for formaldehyde). System output in the latter case is shown in Figure 6. It is known that ammonia rapidly reacts with hypochlorite to form chloramine; the latter is incapable of eliciting CL from luminol.27 Injection of ammonia into a luminol-hypochlorite system (that provides a CL background signal) thus produces negative CL signals. Using this principle and a ammoniahypochlorite reaction time of 90 s, we were able to attain LODs of 9.5 µM NH3 for the photodiode detector and 0.34 µM for the PMT detector. These results will be published in greater detail in a future paper. (25) Gonzalez-Robledo, D.; Silva, M.; Perez-Bendito, D. Anal. Chim. Acta 1990, 228, 123. (26) Marino, D. F.; Ingle, J. D., Jr. Anal. Chem. 1981, 53, 455. (27) Balciunas, R. T. An Automated Reagent Preparation System for Fast ReactionRate Analyses. Ph.D. Dissertation, Michigan State University, 1981.
Nephelometric Detection. Formation of particles in any system causes scattering. Consequently, precipitation can be detected by the present detection arrangement. The length of a nephelometric cell, specifically, the distance between the excitation source and the receiving fiber, must be relatively short, else the scattered light will be attenuated by the flowing scattering particles responsible for the nephelometric signal. Using the arrangement described in the Experimental Section, the LOD for injected sulfate was 0.9 mg/L (S/N ) 3). This is quite comparable to LODs attainable with the current practice of BaSO4 turbidimetry. Phosphorimetric Detection. An interesting room-temperature phosphorimetric detection technique, applicable to solutions, has recently been described by Segura Carreto et al.28 For several naphthalene derivatives, phosphorescence signals can be elicited by adding large concentrations of a heavy atom salt (e.g., KI) in the presence of an oxygen scavenger such as Na2SO3. Using the experimental arrangement described, the LOD was found to be limited primarily by the variations in the blank signal. In a medium of 0.25 M KI and 12.5 mM Na2SO3, we found the S/N ) 3 LOD for naphthoxyacetic acid to be ∼1.2 µM (∼150 µg/L) with the
pulsed Xe lamp excitation system. Without the KI-Na2SO3 matrix, the signal from even a 1000-fold more concentrated analyte solution was indistinguishable from the blank. Raman Scattering. It is obvious that Raman scattered light will also be guided by the LCW cell and can be detected. This is an important outcome on its own and such results will appear in a separate paper.29
(28) Segura Carretero, A.; Cruces Blanco, C.; Can ˜abate Dı´az, B.; Ferna´ndez Gutie´rrez, Anal. Chim. Acta 1998, 361, 217.
(29) Holtz, M.; Dasgupta, P. K., Genfa, Z., unpublished work, Texas Tech University, 1998.
CONCLUSIONS We have described here some very simple and inexpensive but nevertheless sensitive arrangements to perform various types of luminescence detection, made possible by LCW cells fabricated from a new fluoropolymer with a RI lower than that of water. The transverse illumination LCW fluorescence detection technique greatly reduces the need for high-throughput monochromators, and the general geometry is ideally suited for efficient fiber optic coupling permitting the use of a variety of remotely located detectors. Received for review November 17, 1998. Accepted January 20, 1999. AC981260Q
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