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Light at the end of the tunnel: recent analytical applications of liquid-core waveguides
Trends in Analytical Chemistry, Vol. 23, No. 5, 2004
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Light at the end of the tunnel: recent analytical applications of liquid-core waveguides Tim Dallas, Purnendu K. Dasgupta Optical chemical analysis systems are the most important tools to analytical chemists in need of sensitive measurement techniques. Liquid-core waveguides (Fig. 1) have proved to be an important innovation that has led to improvements in detection limits when incorporated into many types of optical analysis systems, especially UV–Vis absorbance, fluorescence and Raman measurements. ª 2004 Published by Elsevier B.V.
1. Introduction Tim Dallas, Purnendu K. Dasgupta* Texas Tech University, Lubbock, TX, USA
*Corresponding author. Tel.: +1-806-742-3064; Fax: +1-806-742-1289; E-mail: [email protected]
It has long been recognized that photoluminescence and related (chemiluminescence, electroluminescence, etc.) spectroscopies are excellent ways to detect and to characterize materials. Although specific electron-excitation mechanisms differ, the radiation interactions that produce electron transitions in atoms and molecules provide an unique optical signature of the sample under study. It has become increasingly important to have field-portable tools for measurement of trace amounts of chemicals, especially those that pose environmental and human health hazards. Analytical chemistry has benefited from innovations in optics, light sources and detectors, resulting in improved sensitivity and speed, while greatly reducing size and cost. Readily available light-emitting diodes (LEDs) [1], across a wide wavelength range, even deep into the ultraviolet, now permit precise tailoring of instrumentation to applications. Many diode lasers have even become inexpensive commodity items. For any photometric analysis technique, it is important to capture and to detect as much of the optical signal as possible, while minimizing background and noise. There are many excellent bench-top spectroscopy systems that can accomplish these goals. However, cost and size issues
0165-9936/$ - see front matter ª 2004 Published by Elsevier B.V. doi:10.1016/S0165-9936(04)00522-9
can often pre-empt them from a sensing application. One approach, increasingly used to enhance sensitivity without special optics, involves a liquid-core waveguide (LCW). Analogous to an optical fiber, a typical LCW is a tube (cladding) with a refractive index (RI) lower than the RI of the fluid (core) inside it. Light propagates through the fluid core by total internal reflection (TIR) if the RI conditions are satisfied. The LCW serves as the ideal flow cell, as it is both the optical component and the fluid conduit. LCWs have been used for fluorescence [2–22] and Raman [23–33] measurements, as well as long-path-length UV–Vis absorption/transmission [10,20, 34–62] and IR [63,64] spectrometry. Water has an (RI) of 1.33 (Na D-line). Until the late 1980s, there were no coating or tubing materials available with a lower RI. The use of glass or polymer tubing as an LCW meant that either non-aqueous liquids with even higher RI have to be used as the core or one must rely on TIR at the cladding-air interface. Air, with an index of refraction of 1.0, is the ultimate low-RI material. However, to achieve efficient TIR at the cladding-air interface, the surface of the tubing must remain scrupulously clean to prevent light loss through absorption or scattering. Light propagating through the LCW passes through the cladding region, effectively shortening the interaction distance within the core [45,65]. Absorption, fluorescence, and scattering in the cladding is possible as well. These problems were eliminated when DuPont introduced a fluoropolymer that had a RI less than 1.33 [66]. The Teflon AF family of fluoropolymers (2,2-bistrifluoromethyl-4,5-difluoro-1,3,-dioxole) have RI in the range 385
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throughput with inexpensive LED sources for lengths up to 15 cm. Thus, they provide better performance than AF tubes as gas sensors because gas transfer through microporous membranes is much more efficient [76]. In yet other applications, permeability of AF tubing to various gases and vapors is a hindrance. The tubing can be altered by gases and chemicals absorbed into it, thereby degrading the optical performance. Multiple polyelectrolyte layers have been used to greatly reduce the permeability of AF tubes [78].
2. Liquid-core waveguide optics
Figure 1. Blue LED transversely illuminating a Teflon AF tube filled with dilute fluorescein. The guided fluorescence is visible at the tube end.
1.31–1.29. The first implementation of Teflon AF in a LCW was in solution form. It was applied to the inner surfaces of glass capillaries through dip-coating techniques [35,67,68]. As AF-polymer tubing became commercially available [69,70], it has been the mainstay of many LCW aficionados [23]. AF-clad, low-loss silica tubes have also become available [71]. In the latter case, TIR occurs at the silica-AF interface (Fig. 1). Following one manufacturer’s terminology [72], an LCW made of an AF tube is often called Type I and an AF-clad silica tube is called a Type II waveguide. The relative merits of each type have been discussed energetically in the literature [50,51]. In an extreme case, to make extremely thin-walled LCWs, Dasgupta et al. [37] made a device from the Type II waveguide and then dissolved the silica with HF to make a waveguide with wall thickness of 18 lm. In addition to a low-RI, AF has other useful material properties. AF tubing is flexible and can be readily coiled for applications (portable detectors) that demand long path-length without increasing system footprint. A molecularly porous structure makes AF very gas permeable and its hydrophobic nature leads to selective permeation, making possible the utilization of Type I waveguides as gas sensors [37,53,73–75]. Microporous hydrophobic membrane tubes are not nearly as good conductors of light as AF tubes (which in turn show much greater light losses than traditional optical fibers) but, in many applications, they provide acceptable light 386
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While an AF tube or an AF-coated tube is a humble optical device, its unique properties have led to many applications. Aside from the sub-1.33 RI, it is transparent over 200–2000 nm, according to the manufacturer [66]; and, low UV transparency in commercially available AF tubing varies [75]. In either Type I or II waveguides, the AF surface can be smooth enough to reduce losses from scattering and other optical irregularities. The tubular geometry allows easy integration with other components, especially an optical fiber. A vast array of optical fibers is available for matching with a particular LCW. Light undergoes TIR when, going from one medium (RI ¼ n1 ), it is incident on a second medium (RI ¼ n2 , where n1 > n2 ) with an angle (hi ) is less than the critical angle (hC ) given by 1 n2 hC ¼ cos : ð1Þ n1 For a LCW, n2 is the index of the cladding material and n1 is the index of the fluid-core. hC is measured with respect to the axial direction in the core. For Teflon AF 2400 (n ¼ 1:29) and water, hC is 14.1. LCWs have been used quite successfully to increase the signal-to-noise (S/N) ratio in absorption systems by greatly increasing the path-length compared to standard cells, while maintaining very low sample volumes. A 1 m long LCW with 250 lm i.d. has a volume of only 50 lL. It is quite convenient to launch light axially into an LCW using a size-and-numerical-aperture-matched optical fiber. Beer’s Law states that IðxÞ ¼ I0 eðaþbÞx ;
ð2Þ
where I0 is the initial light intensity, IðxÞ is the light intensity at a distance x away from the origin, a is the absorption coefficient of the solution being analyzed, and b is the intrinsic loss coefficient of the LCW. An increase in the path-length, x, increases the light attenuation both via absorption by the solution as well as guiding losses. Given a particular light source and detector, the optimum length is dictated by the quality of the lumen and its diameter. The smaller the diameter, the more reflections and more intrinsic losses per unit length
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occur. Miller et al. [55] reported a 1.6 dB/m loss for 600 lm i.d. internally AF-coated glass capillaries. In contrast, the data presented by Toda et al. [76] for a 1.1 mm i.d. AF tube indicated a loss >35 dB/m. Obviously, there is considerable disparity in these reports; whether the difference arises from poor surface quality in the latter case or is from intrinsic differences in the test conditions is not known to us. Nevertheless, at some distance, the gain realized by the increased pathlength is offset by decreased light throughput. Inya-Agha et al. [77] summarized optimum LCW lengths for Raman measurement as a function of laser wavelength, optical loss and waveguide geometry. A fluorescing molecule or a chemiluminescent reaction occurring within the core of an LCW radiates light isotropically. Because water-core AF fibers have a rather low critical angle, the fraction of light collected in each direction is also low. In photoluminescence systems, the analyte in the fluid-core can be illuminated axially or radially (transversely). In absorption measurement systems, excitation light is coupled axially and transmitted light is also measured axially at the other terminus. In Raman or fluorescence experiments, axial illumination/ backscattered detection of the same axial location can be attractive, if the analyte fills most or all of the core volume, because a greater number of molecules are interrogated, thus increasing the signal. However, a monochromator/ filter is needed to prevent the excitation light from reaching the detector. This arrangement is commonly used for Raman spectrometry because the Raman signal is typically weak and backscattered detection is advantageous. Small volume fluorescence detection is advantageously carried out with transverse illumination. Fig. 2 illustrates such an arrangement originally used by Dasgupta et al. [3]. One or more transversely located sources can be used to simultaneously [3] or serially excite the analyte with different wavelength sources [20]. The excitation light not absorbed by the solution mostly passes through the cladding. For a really good optical fiber, <1 in 106 excitation photons make it to the detector in the
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transverse illumination geometry. For practical AF-based waveguides, 1 in 105 is more realistic, but even this degree of exclusion of the excitation light can vastly relax the requirement for optical filtering used in signal detection. One of the early practical demonstrations of a transversely illuminated LCW fluorescence system was in the detection of ammonia using a small, inexpensive 365 nm source (3 50 mm black light) to illuminate an 11.5 cm LCW. The limit of detection (LOD) with a $15 photodiode detector was 35 nM, with a S/N of three [5]. This was comparable to the 20-nM LOD attainable with a commercial photomultiplier-based filter fluorometer.
3. Experimental systems Long-path absorption measurements are made using long waveguides, which provide good LODs for trace analysis. Yao et al. [39] used a 0.56-mm i.d., 4500-mm long LCW as a 10 cm coil for trace nitrate/nitrite measurement. Fig. 3 shows a typical LCW-based fluorescence detection system. The AF-based LCW is used in lengths typically 1.5–50 cm, with an i.d. typically 0.1–1 mm, and is confined within an opaque tube/container to prevent external light from getting into the system. An optical fiber with an equal or higher core diameter and high numerical aperture is used to collect light from the LCW and guide it to a detector. This allows excellent coupling without focusing optics. At the tee, the gap between the
Outlet
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LEDs
Fluid Inlet
Connection Tee
Optical Fiber Figure 2. Cross-sectional view of a LCW. Photoluminescence generated in the lumen is guided if the photons are emitted within the critical angle. Illumination light not absorbed by the solution passes through the cladding and is not guided to the detector.
Detector Figure 3. LCW fluorescence detector.
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end of the LCW and the optical fiber is maintained as small as possible to minimize light losses. In most systems, the light from only one end of the LCW is detected; however, in at least one commercial chemiluminometer, tees are used at both ends, and light is collected at one or both ends [79]. The flexibility of illumination allows the use of single/multiple LEDs at single/multiple wavelengths for continuous/time-resolved/multiplexed measurements, the latter by pulsing the LEDs. Compact mercury pen lamps and miniature cold cathode lamps have also been used. Detector options depend on the sensitivity, specificity and the wavelength range needed. Applicable detectors range from the simplest photodiodes [80,81], to interference filter-equipped photodiodes [82], to low-cost photomultiplier tubes [83] and to fiber input CCD-[84] and photodiode array-[85] spectrometers. Formation of microscopic gas bubbles that adhere to the LCW wall (more of a problem in hydrophobic AF tubes than AF-coated silica tubes) can create problems by scattering the excitation light down the lumen. Backpressure can be applied to the system through the outlet tubing, or the tube can be mounted vertically. To remove bubbles, a low surface-tension solvent (e.g., methanol) can be used to wash it through.
4. Applications 4.1. Long-path absorption LCW-based long-path absorption spectroscopy has found a real niche in performing trace analysis in natural water and marine chemistry. Measurements have been made for hydrogen sulfide [10], iron [36,56], nitrate and nitrite [39,47,48,54], phosphate [57], chromium and molybdenum [43], copper [49,59], total inorganic carbon [52], colored dissolved organic matter [55] and phytoplanktons [46]. The long path-length attainable with an AF LCW and its inert nature have for the first time permitted direct, convenient spectroscopic studies of hydroxyl radical reactions [50]. Of course, many other general applications of long-path absorption spectrometry have also been described. 4.2. Sensing dissolved organics and gases In addition to serving as fluid and light conduits, AF LCWs can uniquely be used as an analyte pre-concentrator, a gas-permeable membrane or a conduit for electrophoresis. Many gases permeate readily through AF, and filling the lumen with a selective chromogenic liquid can be the basis of a selective, sensitive gas sensor, as demonstrated for H2 S [37], Cl2 [37], NO2 [37], and CO2 [37,53]. Because two gases that react identically with the same chromogenic agent can have different permeabilities through AF, it may still be possible to make a differential measurement by using AF tubes of different thicknesses or lengths, as demonstrated for 388
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HONO and NO2 with a reagent that cannot discriminate between these gases [74]. Non-polar analytes can adsorb on AF. Tanikkul et al. [33] developed a Raman sensor with a two-stage AF tube system. The first tube was used for the preconcentration of analytes (e.g., benzene, toluene, and p-xylene), which were then eluted and the Raman spectra of the 14·-concentrated analytes were then measured. The LOD for benzene was 730 lg/l. Larsson et al. [75] reported on the possibility of measuring similar compounds based on long-path UV absorption spectrometry. The long-path AF-based absorbance cell capitalized on the gas-permeable properties of the AF tubing. Large-core, silica-fiber-coupled light from a pulsed xenon flash lamp (200 Hz) was input into 20–100 cm long LCW cells and a photodiode array was used for detection in the 200–280 nm range. The LOD was 10 lg/l for benzene in a 1 m LCW for a 10 min sample. 4.3. Chemiluminescence In many chemiluminescence (CL) and bioluminescence experiments, light is emitted as soon as solutions are mixed and reactions occur. Some CL reactions take place in ms time-scales, requiring that solutions be mixed in front of a photomultiplier tube window that must therefore be of significant area and hence expensive (e.g., the hypobromite-urea reaction reaches a peak CL intensity 20 ms after mixing and the signal decays to nearly zero in 120 ms). Li et al. [4] reported on the luminol-hypochlorite reaction that reaches a maximum intensity 800 ms after mixing. This system can be used to detect ammonia by measuring the drop in background CL as ammonia consumes hypochlorite. They compared the performance of the system (a) with the solutions mixing with each other for the first time within the LCW, and (b) coming to a confluence point within a tee and then going into the LCW. Configuration (a) was found to provide superior performance. Li et al. also reported on the use of a scrubber system for collecting atmospheric gaseous hydrogen peroxide (H2 O2 ) and then generating CL by reacting with luminol within a LCW. The maximum CL intensity occurred within 2 s after mixing, necessitating reaction and detection in the same vessel. A PMT (photomultiplier tube) was used for detection with a 83 ll cell; the S/N was 3 and LOD was 25 pptv (parts per trillion by volume) [14]. 4.4. Electrophoresis The applicability of AF-coated silica capillaries for conducting transversely excited fluorescence detection in capillary electrophoresis (CE) was demonstrated early on [3], when these authors demonstrated 200 amol LODs for fluorescein derivatives using excitation by a pair of LEDs and detection by an inexpensive PMT.
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Belz et al. [19] performed LCW capillary gel electrophoresis in a multi-capillary format with fluorescence detection establishing the potential for very high throughputs with a large array of capillaries, four-color detection and high-speed separation. Liu and Pawliszyn [22] used a type I axial laser-illuminated LCW-based capillary isoelectric focusing system with whole-column image detection. The unique properties of AF, beyond its RI, relevant to this application, namely minimal protein adsorption and low electroosmotic flow, eliminate the need for polymeric additives. The detection sensitivity of the system was enhanced by three to five orders of magnitude compared to commercial UV absorbance-based detection systems.
Wang et al. [12] subsequently coupled a flow-injection sample-introduction system to a similar AF-based short CE capillary for the separation of fluorescein isothiocyanate-labeled amino acids. The S/N was 3 and LODs were 1.3 lM for arginine and 1.9 lM for phenylananine and glycine for this LED-excited PMT detected system with a throughput of 144 samples/h. Ball lens coupling of the excitation light to the LCW resulted in a four fold improvement in S/N. Hanning et al. [11] described a similar transverse illumination laser-excited, CCD-based fluorescencedetection system with a CCD detector, achieving a 0.55-amol LOD for fluorescein in the illuminated volume. They also applied it to DNA sequencing, including four-color detection. A similar scheme was then applied to a two-dimensional array of 91 capillaries. Olivares et al. [17] showed whole column imaging for both DNA electrophoresis separations and isoelectric focusing of proteins. The utility of the system is demonstrated for DNA-fragment sizing and protein separations. Scanning the excitation laser along the length of the electrophoresis capillary excites individually separated analyte bands, while the fluorescence is collected end-on by an optical fiber coupled to a PMT, thus creating an image of the separation along the length of the capillary with an ability to detect target molecules in the zeptomolar range.
2.5
5. LCWs in field-deployable instruments LCWs uniquely allow small compact fluorescence detectors particularly suitable for field use. This has been exploited in field instruments to measure formaldehyde [15] and hydrogen peroxide [9]. Gas–liquid scrubbers and appropriate reaction chemistries are used to allow sensing of specific analytes. In many LCW systems that require high sensitivity, the most expensive component is the detector. PMTs offer high sensitivity but can be relatively expensive.
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Time, s Figure 4. Instrument response: (a) photomultiplier tube (PMT) response as seen by the PC, consisting of the composite signal from two channels. The sample contained 2.0 ppbv hydrogen peroxide (H2 O2 ) and 3.4 ppbv methyl hydroperoxide (MHP). The lower and upper envelopes of the composite trace constitute the individual signals for H2 O2 and MHP, respectively; (b) software-isolated signal for the two channels.
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Li et al. [21] took advantage of the ability to switch LEDs readily to construct a multiplexed LCW-based detector for measuring two analytes sequentially. Using different scrubbers and different enzyme catalysts, one of these detectors measured H2 O2 and the other methyl hydroperoxide (MHP). Both LCWs were optically coupled through fiber to the PMT. Each tube was separately illuminated in staggered 5-s intervals, allowing the detector to read out each LCW separately (Fig. 4). Using this system, LODs of 25 pptv (H2 O2 ) and 15 pptv (MHP) were attained (see Fig. 5). A rather versatile portable, hybrid fluorescence/ absorption system has also been described recently [20]. It incorporated a 50-cm long AF tube in a horseshoe path embedded in a black plastic housing to reject ambient light, and it contained a miniature multichannel pump and an injection valve (Fig. 5). Both transverse and axial illumination schemes were possible. A 12-LED array that covered the wavelength range 375–620 nm was mounted transversely to the LCW. The LEDs was controlled by a microcontroller. Light from a white LED was used for long path-length absorption measurements. An optical fiber coupled the light into the LCW. An Ocean Optics spectrometer with a fiber input was used as the detector permitting a LOD of 51 lM for aqueous H2 O2 with an integration time of 2 s. When using a miniature PMT, the LOD was 16 nM. In this system, the S/N was found to increase linearly with increasing integration time since the noise was nearly independent of the integration time. The 50 cm cell had
50x the sensitivity of a standard 1 cm cell (not LCW), although this performance was not realized for situations with high background absorbance.
6. Raman spectroscopy While Raman spectroscopy (RS) can provide detailed vibrational information from aqueous solutions, including biological samples, it suffers from poor sensitivity. Typically, a laser spot impinges on a sample and the backscattered light is collected using high aperture optics. The vast majority of the light collected is still at the excitation frequency (Rayleigh scattering), but a small fraction is inelastically scattered, the decrease (increase) in frequency corresponding to energy loss (gain) to characteristic vibrational modes in the material. LCWs have been used to dramatically improve the sensitivity of RS by axially illuminating a LCW. As the laser propagates through the lumen, Raman scattered light from within the entire illuminated volume of the lumen, within the capture angle of the LCW is backscattered to the detector (alongside the excitation light), effectively greatly increasing the interrogated sample volume [86]. A suitable monochromator system is needed to discriminate against the excitation light. Altkorn et al. [23] showed that, to generate the same Raman signal intensity for acetonitrile in a conventional cell compared to an acetonitrile-filled long LCW, nearly 300 times the laser power was required! The relative Raman intensity from the 2253 cm1 CN band increased as a function of the LCW length in accordance with the numerical model of Walrafen and Stone [87] IR ¼ Kxeax ;
Figure 5. Photograph of a complete flow injection analyzer that has a multi-wavelength source for LED excitation, a PMT for fluorescence or chemiluminescence measurements and a CCD spectrometer for multi-purpose measurements. The system uses a 50 cm Type I LCW with an axial white LED source for absorbance measurements.
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ð3Þ
where, IR is the Raman intensity; K is proportional to the product of intensity of the exciting radiation, the Raman-scattering cross-section of the observed bands in the liquid-core and the solid angle of light guided in the core; x is the length of the LCW; and, a is the attenuation coefficient, assumed to be the same for both the excitation and the emission light. In this particular case, the calculated attenuation coefficient was 4.39 dB/m. Subsequently, this group developed a figure of merit to describe the enhancement given by (adÞ1 , where a is the loss coefficient and d is the diameter of the LCW, and they established parameters for maximizing performance for six different excitation and detection schemes [30]. Other important work has been directed towards using RS as a detection technique in liquid chromatography (LC); given the notoriously poor sensitivity of conventional RS, this would have been considered impossible only a few years ago. A novel, real-time, liquid-core, Raman waveguide detector designed for LC applications was first described by Marquardt et al. [26]
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with the detection volume compatible with microbore and minibore LC; LOD enhancements of over 1000-fold were achieved for the measurement of alcohols in aqueous solution. The LOD of 2-propanol was established to be 2 ppm. Dijkstra et al. [32] has described optimum LCW lengths and choice of laser wavelength in this connection. Much of the effort in LCW-based RS relies on axial illumination. Typically, a notch filter that very effectively removes the laser wavelength is placed before further monochromators and the detector. Holtz et al. [25] used transverse illumination to allow low-energy Raman bands from CCl4 in solution to be measured using a multi-channel CCD detector without a notch filter. As noted earlier, a transverse illumination geometry does not cause many photons to be scattered into the lumen. It is usually very difficult, if not impossible, to obtain a spectrum of CCl4 with a single grating monochromator; typically, a double grating system is needed to prevent the Rayleigh tail from the laser from saturating the detector. Holtz et al. used the 488 nm line of argon laser, a 0.5 m monochromator, and a liquid nitrogen-cooled CCD detector to measure down to 200 cm1 without a notch filter, using 1–60-s integration times. The laser spot created a 6-nL probe volume, usually 3–5 cm from the end of LCW. In order to increase the amount of signal in another series of experiments, the laser light was recycled using a multi-pass configuration. With two and three passes, the signal increased 1.9 and 2.3 times, respectively. Smaller diameter LCW tubing leads to higher power density per unit cross section if the same laser is focused to illuminate the lumen. This can be fortuitous, as it can often photobleach unwanted fluorescent impurities very effectively, thus further improving the ability to look at the Raman signal. Pelletier and Altkorn [29] showed that, in addition to a two order of magnitude improvement in the Raman signal using a long path-length cell, the photobleaching of fluorescence resulted in a two orders of magnitude reduction in the background fluorescence. However, this approach is not universally applicable, as many analytes will themselves be photosensitive.
7. Conclusions The availability of water-core waveguides has allowed a significant increase in performance of a number of analytical systems that rely on solution phase spectrometry. In addition to its low index of refraction, the gas permeability and hydrophobic properties of Teflon AF permit innovative sensing techniques. The relatively low cost, integration, simplicity and durability of the optics will allow expansion in its use in field-deployable sensor systems.
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Acknowledgements This work was made possible by the J.F. Maddox Endowment at Texas Tech University and the US National Science Foundation. References [1] P.K. Dasgupta, I.-Y. Eom, K.J. Morris, J. Li, Anal. Chim. Acta 500 (2003) 337. [2] K. Fujiwara, S. Ito, Trends Anal. Chem. 10 (1991) 184. [3] P.K. Dasgupta, Z. Genfa, J. Li, B. Boring, S. Jambunathan, R. Al-Horr, Anal. Chem. 71 (1999) 1400. [4] J. Li, P.K. Dasgupta, Anal. Chim. Acta 398 (1999) 33. [5] J. Li, P.K. Dasgupta, Z. Genfa, Talanta 50 (1999) 617. [6] P. Lacki, A. Nowakowski, P. Dress, H. Franke, Proc. SPIE-Int. Soc. Opt. Eng. 3730 (1999) 112. [7] E.J. D’Sa, R.G. Steward, A. Vodacek, N.V. Blough, D. Phinney, Limnol. Oceanogr. 44 (1999) 1142. [8] A. Hanning, J. Westberg, J. Roeraade, Electrophoresis 21 (2000) 3290. [9] J. Li, P.K. Dasgupta, Anal. Chem. 72 (2000) 5338. [10] R.H. Byrne, W.S. Yao, E. Kaltenbacher, R.D. Waterbury, Talanta 50 (2000) 1307. [11] A. Hanning, P. Lindberg, J. Westberg, J. Roeraade, Anal. Chem. 72 (2000) 3423. [12] S. Wang, X. Huang, Z. Fang, P.K. Dasgupta, Anal. Chem. 73 (2001) 4545. [13] P.K. Dasgupta, Luminescence detector with liquid core, US Patent 6,332,049, 2001. [14] J. Li, P.K. Dasgupta, Anal. Chim. Acta 442 (2001) 63. [15] J. Li, P.K. Dasgupta, Z. Genfa, M. Hutterli, Field Anal. Chem. Technol. 51 (2001) 2. [16] K. Toda, P.K. Dasgupta, J. Li, G.A. Tarver, G.M. Zarus, Anal. Chem. 73 (2001) 5716. [17] J.A. Olivares, P.C. Stark, P. Jackson, Anal. Chem. 74 (2002) 2008. [18] P. Langer, R. M€ uller, S. Drost, T. Werner, Sens. Actuators B 82 (2002) 1. [19] M. Belz, P. Dress, A. Sukhitskiy, S. Liu, M. Curcio, P. Stalhandske, P. Lindberg, J. Roeraade, Electrophoresis 23 (2002) 1467. [20] Q. Li, K.A. Morris, P.K. Dasgupta, I.M. Raimundo Jr., H. Temkin, Anal. Chim. Acta 479 (2003) 151. [21] J. Li, P.K. Dasgupta, G.A. Tarver, Anal. Chem. 75 (2003) 1203. [22] Z. Liu, J. Pawliszyn, Anal. Chem. 75 (2003) 4887. [23] R. Altkorn, I. Koev, R. Van Duyne, M. Litorja, Appl. Opt. 36 (1997) 8992. [24] L. Song, S. Liu, V. Zhelyaskov, M.A. El-Sayed, Appl. Spectrosc. 52 (1998) 1364. [25] M. Holtz, P.K. Dasgupta, Z. Genfa, Anal. Chem. 71 (1999) 2934. [26] B.J. Marquardt, P.G. Vahey, R.E. Synovec, L.W. Burgess, Anal. Chem. 71 (1999) 4808. [27] R. Altkorn, I. Koev, M.J. Pelletier, Appl. Spectrosc. 53 (1999) 1169. [28] R.J. Dijkstra, A.N. Bader, G.P. Hoornweg, U.A.Th. Brinkman, C. Gooijer, Anal. Chem. 71 (1999) 4575. [29] M.J. Pelletier, R. Altkorn, Appl. Spectrosc. 54 (2000) 1837. [30] R. Altkorn, M.D. Malinsky, R.P. Van Duyne, I. Koev, Appl. Spectrosc. 55 (2001) 373. [31] M.J. Pelletier, R. Altkorn, Anal. Chem. 73 (2001) 1393. [32] R.J. Dijkstra, C.J. Slooten, A. Stortelder, J.B. Buijs, F. Ariese, U.A.Th. Brinkman, C. Gooijer, J. Chromatogr. A 918 (2001) 25. [33] S. Tanikkul, J. Jakmunee, M. Rayanakorn, K. Grudpan, B. Marquardt, G.M. Gross, B.J. Prazen, L.W. Burgess, G.D. Christian, R.E. Synovec, Talanta 59 (2003) 809.
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[34] P. Dress, H. Franke, Proc. SPIE-Int. Soc. Opt. Eng. 2686 (1996) 157. [35] P. Dress, H. Franke, Rev. Sci. Instrum. 68 (1997) 2167. [36] R.D. Waterbury, W. Yao, R.H. Byrne, Anal. Chim. Acta 357 (1997) 99. [37] P.K. Dasgupta, Z. Genfa, S.K. Poruthoor, S. Caldwell, S. Dong, Anal. Chem. 70 (1998) 4661. [38] P. Dress, M. Belz, K.F. Klein, K.T.V. Grattan, H. Franke, Appl. Opt. 37 (1998) 4991. [39] W.S. Yao, R.H. Byrne, R.D. Waterbury, Environ. Sci. Technol. 32 (1998) 2646. [40] M. Belz, P. Dress, K.F. Klein, W.J.O. Boyle, H. Franke, K.T.V. Grattan, Water Sci. Technol. 37 (1998) 279. [41] C. Gooijer, G.P. Hoornweg, T. de Beer, A. Bader, D.J. van Iperen, U.A.Th. Brinkman, J. Chromatogr. A 824 (1998) 1. [42] B. Schelle, P. Dress, H. Franke, K.F. Klein, J. Slupek, J. Phys. D: Appl. Phys. 32 (1999) 3157. [43] W.S. Yao, R.H. Byrne, Talanta 48 (1999) 277. [44] M. Belz, P. Dress, A. Sukhitskiy, S. Liu, Proc. SPIE-Int. Soc. Opt. Eng. 3856 (1999) 271. [45] S. Jambunathan, P.K. Dasgupta, J. Soc. Leather Technol. Chem. 84 (2000) 63. [46] G.J. Kirkpatrick, D.F. Millie, M.A. Moline, O. Schofield, Limnol. Oceanogr. 45 (2000) 467. [47] J.Z. Zhang, Deep Sea Res. 47 (2000) 1157. [48] J.Z. Zhang, R. Wanninkhof, K. Lee, Geophysical Res. Lett. 28 (2001) 1579. [49] H.W. Chen, Y.B. Cao, S.B. Han, H.Q. Zhang, A.M. Yu, Q.H. Jin, Chin. J. Anal. Chem. 29 (2001) 478. [50] R.H. Byrne, E. Kaltenbacher, Limnol. Oceanogr. 46 (2001) 740. [51] E.J. D’Sa, R.G. Steward, Limnol. Oceanogr 46 (2001) 742. [52] R.H. Byrne, X.W. Liu, E.A. Kaltenbacher, K. Sell, Anal. Chim. Acta 451 (2002) 221. [53] Z.H. Wang, Y.H. Wang, W.J. Cai, S.Y. Liu, Talanta 57 (2002) 69. [54] E.T. Steimle, E.A. Kaltenbacher, R.H. Byrne, Mar. Chem. 77 (2002) 255. [55] R.L. Miller, M. Belz, C. Del Castillo, R. Trzaska, Cont. Shelf Res. 22 (2002) 1301. [56] J.Z. Zhang, C. Kelbe, F.J. Millero, Anal. Chim. Acta 438 (2001) 49. [57] J.Z. Zhang, J. Chi, Environ. Sci. Technol. 36 (2002) 1048. [58] A.C. Pappas, C.D. Stalikas, Y.C. Fiamegos, M.I. Karayannis, Anal. Chim. Acta 455 (2002) 305. [59] M.R. Callahan, J.B. Rose, R.H. Byrne, Talanta 58 (2002) 891. [60] C. George, D. Rousse, E. Perraudin, R. Strekowski, Phys. Chem. 5 (2003) 1562. [61] K. Tsunoda, T. Umemura, T. Watanabe, H. Takiguchi, H. Asano, H. Itabashi, T. Ishibashi, S. Sato, Tetsu To Hagane 89 (2003) 979. [62] G. Deng, L. Wei, G.E. Collins, Talanta 60 (2003) 9. [63] S.J. Saggese, J.A. Harrington, G.H. Sigel, R. Altkorn, R. Haidle, Appl. Spectrosc. 46 (1992) 1194. [64] J. Meister, S. Diemer, R. Jung, S. Klein, W. Fuss, P. Hering, Proc. SPIE-Int. Soc. Opt. Eng. 2977 (1997) 58. [65] K. Tsunoda, A. Nomura, J. Yamadam, S. Nishi, Appl. Spectrosc. 43 (1989) 49.
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[66] DuPont Fluoroproducts, Teflon AF Amorphous Fluoropolymers, H-16577-1, DuPont, Wilmington, DE 19880-0711, USA, December 1989. [67] P. Dress, H. Franke, Appl. Phys. B 63 (1996) 12. [68] P. Dress, M. Belz, K. Klein, K. Grattan, H. Franke, Sens. Actuators B 51 (1998) 278. [69] Available from. [70] Available from . [71] TSU series. Available from . [72] Available from . [73] P.K. Dasgupta, S.-Y. Liu, Chemical sensing techniques employing liquid core optical fibers, US Patent 6,011,882, 2000. [74] M.R. Milani, P.K. Dasgupta, Anal. Chim. Acta 431 (2001) 169. [75] H. Larsson, P.K. Dasgupta, Anal. Chim. Acta 485 (2003) 155. [76] K. Toda, K.I. Yoshioka, S.I. Ohira, J. Li, P.K. Dasgupta, Anal. Chem. 75 (2003) 4050. [77] O. Inya-Agha, S. Stewart, T. Veriotti, M.L. Bruening, M.D. Morris, Appl. Spectrosc. 56 (2002) 574. [78] G.E. Walfren, Phys. Blatter 30 (1974) 540. [79] Available from . [80] Available from . [81] Available from . [82] Available from . [83] Available from . [84] Available from . [85] Available from . [86] D. Che, S.Y. Liu, Long capillary waveguide Raman cell, US Patent 5,604,587, 1997. [87] G.E. Walrafen, J. Stone, Appl. Spectrosc. 26 (1972) 585. Tim Dallas received the B.A. degree in Physics from the University of Chicago in 1991. He received his M.S. and Ph.D. degrees in Physics from Texas Tech University (TTU) in 1993 and 1996. There followed a year in the semiconductor equipment industry and a year as a postdoctoral fellow at The University of Texas – Austin, in the Department of Chemical Engineering. He has been an Assistant Professor of Electrical and Computer Engineering at TTU since 1999. He is the Associate Director of the Nano Tech Center at TTU. His research interests include microfabrication, microanalysis systems, and MEMS. Available from
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Light at the end of the tunnel: recent analytical applications of liquid-core waveguides Tim Dallas, Purnendu K. Dasgupta Optical chemical analysis systems are the most important tools to analytical chemists in need of sensitive measurement techniques. Liquid-core waveguides (Fig. 1) have proved to be an important innovation that has led to improvements in detection limits when incorporated into many types of optical analysis systems, especially UV–Vis absorbance, fluorescence and Raman measurements. ª 2004 Published by Elsevier B.V.
1. Introduction Tim Dallas, Purnendu K. Dasgupta* Texas Tech University, Lubbock, TX, USA
*Corresponding author. Tel.: +1-806-742-3064; Fax: +1-806-742-1289; E-mail: [email protected]
It has long been recognized that photoluminescence and related (chemiluminescence, electroluminescence, etc.) spectroscopies are excellent ways to detect and to characterize materials. Although specific electron-excitation mechanisms differ, the radiation interactions that produce electron transitions in atoms and molecules provide an unique optical signature of the sample under study. It has become increasingly important to have field-portable tools for measurement of trace amounts of chemicals, especially those that pose environmental and human health hazards. Analytical chemistry has benefited from innovations in optics, light sources and detectors, resulting in improved sensitivity and speed, while greatly reducing size and cost. Readily available light-emitting diodes (LEDs) [1], across a wide wavelength range, even deep into the ultraviolet, now permit precise tailoring of instrumentation to applications. Many diode lasers have even become inexpensive commodity items. For any photometric analysis technique, it is important to capture and to detect as much of the optical signal as possible, while minimizing background and noise. There are many excellent bench-top spectroscopy systems that can accomplish these goals. However, cost and size issues
0165-9936/$ - see front matter ª 2004 Published by Elsevier B.V. doi:10.1016/S0165-9936(04)00522-9
can often pre-empt them from a sensing application. One approach, increasingly used to enhance sensitivity without special optics, involves a liquid-core waveguide (LCW). Analogous to an optical fiber, a typical LCW is a tube (cladding) with a refractive index (RI) lower than the RI of the fluid (core) inside it. Light propagates through the fluid core by total internal reflection (TIR) if the RI conditions are satisfied. The LCW serves as the ideal flow cell, as it is both the optical component and the fluid conduit. LCWs have been used for fluorescence [2–22] and Raman [23–33] measurements, as well as long-path-length UV–Vis absorption/transmission [10,20, 34–62] and IR [63,64] spectrometry. Water has an (RI) of 1.33 (Na D-line). Until the late 1980s, there were no coating or tubing materials available with a lower RI. The use of glass or polymer tubing as an LCW meant that either non-aqueous liquids with even higher RI have to be used as the core or one must rely on TIR at the cladding-air interface. Air, with an index of refraction of 1.0, is the ultimate low-RI material. However, to achieve efficient TIR at the cladding-air interface, the surface of the tubing must remain scrupulously clean to prevent light loss through absorption or scattering. Light propagating through the LCW passes through the cladding region, effectively shortening the interaction distance within the core [45,65]. Absorption, fluorescence, and scattering in the cladding is possible as well. These problems were eliminated when DuPont introduced a fluoropolymer that had a RI less than 1.33 [66]. The Teflon AF family of fluoropolymers (2,2-bistrifluoromethyl-4,5-difluoro-1,3,-dioxole) have RI in the range 385
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throughput with inexpensive LED sources for lengths up to 15 cm. Thus, they provide better performance than AF tubes as gas sensors because gas transfer through microporous membranes is much more efficient [76]. In yet other applications, permeability of AF tubing to various gases and vapors is a hindrance. The tubing can be altered by gases and chemicals absorbed into it, thereby degrading the optical performance. Multiple polyelectrolyte layers have been used to greatly reduce the permeability of AF tubes [78].
2. Liquid-core waveguide optics
Figure 1. Blue LED transversely illuminating a Teflon AF tube filled with dilute fluorescein. The guided fluorescence is visible at the tube end.
1.31–1.29. The first implementation of Teflon AF in a LCW was in solution form. It was applied to the inner surfaces of glass capillaries through dip-coating techniques [35,67,68]. As AF-polymer tubing became commercially available [69,70], it has been the mainstay of many LCW aficionados [23]. AF-clad, low-loss silica tubes have also become available [71]. In the latter case, TIR occurs at the silica-AF interface (Fig. 1). Following one manufacturer’s terminology [72], an LCW made of an AF tube is often called Type I and an AF-clad silica tube is called a Type II waveguide. The relative merits of each type have been discussed energetically in the literature [50,51]. In an extreme case, to make extremely thin-walled LCWs, Dasgupta et al. [37] made a device from the Type II waveguide and then dissolved the silica with HF to make a waveguide with wall thickness of 18 lm. In addition to a low-RI, AF has other useful material properties. AF tubing is flexible and can be readily coiled for applications (portable detectors) that demand long path-length without increasing system footprint. A molecularly porous structure makes AF very gas permeable and its hydrophobic nature leads to selective permeation, making possible the utilization of Type I waveguides as gas sensors [37,53,73–75]. Microporous hydrophobic membrane tubes are not nearly as good conductors of light as AF tubes (which in turn show much greater light losses than traditional optical fibers) but, in many applications, they provide acceptable light 386
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While an AF tube or an AF-coated tube is a humble optical device, its unique properties have led to many applications. Aside from the sub-1.33 RI, it is transparent over 200–2000 nm, according to the manufacturer [66]; and, low UV transparency in commercially available AF tubing varies [75]. In either Type I or II waveguides, the AF surface can be smooth enough to reduce losses from scattering and other optical irregularities. The tubular geometry allows easy integration with other components, especially an optical fiber. A vast array of optical fibers is available for matching with a particular LCW. Light undergoes TIR when, going from one medium (RI ¼ n1 ), it is incident on a second medium (RI ¼ n2 , where n1 > n2 ) with an angle (hi ) is less than the critical angle (hC ) given by 1 n2 hC ¼ cos : ð1Þ n1 For a LCW, n2 is the index of the cladding material and n1 is the index of the fluid-core. hC is measured with respect to the axial direction in the core. For Teflon AF 2400 (n ¼ 1:29) and water, hC is 14.1. LCWs have been used quite successfully to increase the signal-to-noise (S/N) ratio in absorption systems by greatly increasing the path-length compared to standard cells, while maintaining very low sample volumes. A 1 m long LCW with 250 lm i.d. has a volume of only 50 lL. It is quite convenient to launch light axially into an LCW using a size-and-numerical-aperture-matched optical fiber. Beer’s Law states that IðxÞ ¼ I0 eðaþbÞx ;
ð2Þ
where I0 is the initial light intensity, IðxÞ is the light intensity at a distance x away from the origin, a is the absorption coefficient of the solution being analyzed, and b is the intrinsic loss coefficient of the LCW. An increase in the path-length, x, increases the light attenuation both via absorption by the solution as well as guiding losses. Given a particular light source and detector, the optimum length is dictated by the quality of the lumen and its diameter. The smaller the diameter, the more reflections and more intrinsic losses per unit length
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occur. Miller et al. [55] reported a 1.6 dB/m loss for 600 lm i.d. internally AF-coated glass capillaries. In contrast, the data presented by Toda et al. [76] for a 1.1 mm i.d. AF tube indicated a loss >35 dB/m. Obviously, there is considerable disparity in these reports; whether the difference arises from poor surface quality in the latter case or is from intrinsic differences in the test conditions is not known to us. Nevertheless, at some distance, the gain realized by the increased pathlength is offset by decreased light throughput. Inya-Agha et al. [77] summarized optimum LCW lengths for Raman measurement as a function of laser wavelength, optical loss and waveguide geometry. A fluorescing molecule or a chemiluminescent reaction occurring within the core of an LCW radiates light isotropically. Because water-core AF fibers have a rather low critical angle, the fraction of light collected in each direction is also low. In photoluminescence systems, the analyte in the fluid-core can be illuminated axially or radially (transversely). In absorption measurement systems, excitation light is coupled axially and transmitted light is also measured axially at the other terminus. In Raman or fluorescence experiments, axial illumination/ backscattered detection of the same axial location can be attractive, if the analyte fills most or all of the core volume, because a greater number of molecules are interrogated, thus increasing the signal. However, a monochromator/ filter is needed to prevent the excitation light from reaching the detector. This arrangement is commonly used for Raman spectrometry because the Raman signal is typically weak and backscattered detection is advantageous. Small volume fluorescence detection is advantageously carried out with transverse illumination. Fig. 2 illustrates such an arrangement originally used by Dasgupta et al. [3]. One or more transversely located sources can be used to simultaneously [3] or serially excite the analyte with different wavelength sources [20]. The excitation light not absorbed by the solution mostly passes through the cladding. For a really good optical fiber, <1 in 106 excitation photons make it to the detector in the
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transverse illumination geometry. For practical AF-based waveguides, 1 in 105 is more realistic, but even this degree of exclusion of the excitation light can vastly relax the requirement for optical filtering used in signal detection. One of the early practical demonstrations of a transversely illuminated LCW fluorescence system was in the detection of ammonia using a small, inexpensive 365 nm source (3 50 mm black light) to illuminate an 11.5 cm LCW. The limit of detection (LOD) with a $15 photodiode detector was 35 nM, with a S/N of three [5]. This was comparable to the 20-nM LOD attainable with a commercial photomultiplier-based filter fluorometer.
3. Experimental systems Long-path absorption measurements are made using long waveguides, which provide good LODs for trace analysis. Yao et al. [39] used a 0.56-mm i.d., 4500-mm long LCW as a 10 cm coil for trace nitrate/nitrite measurement. Fig. 3 shows a typical LCW-based fluorescence detection system. The AF-based LCW is used in lengths typically 1.5–50 cm, with an i.d. typically 0.1–1 mm, and is confined within an opaque tube/container to prevent external light from getting into the system. An optical fiber with an equal or higher core diameter and high numerical aperture is used to collect light from the LCW and guide it to a detector. This allows excellent coupling without focusing optics. At the tee, the gap between the
Outlet
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Connection Tee
Optical Fiber Figure 2. Cross-sectional view of a LCW. Photoluminescence generated in the lumen is guided if the photons are emitted within the critical angle. Illumination light not absorbed by the solution passes through the cladding and is not guided to the detector.
Detector Figure 3. LCW fluorescence detector.
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end of the LCW and the optical fiber is maintained as small as possible to minimize light losses. In most systems, the light from only one end of the LCW is detected; however, in at least one commercial chemiluminometer, tees are used at both ends, and light is collected at one or both ends [79]. The flexibility of illumination allows the use of single/multiple LEDs at single/multiple wavelengths for continuous/time-resolved/multiplexed measurements, the latter by pulsing the LEDs. Compact mercury pen lamps and miniature cold cathode lamps have also been used. Detector options depend on the sensitivity, specificity and the wavelength range needed. Applicable detectors range from the simplest photodiodes [80,81], to interference filter-equipped photodiodes [82], to low-cost photomultiplier tubes [83] and to fiber input CCD-[84] and photodiode array-[85] spectrometers. Formation of microscopic gas bubbles that adhere to the LCW wall (more of a problem in hydrophobic AF tubes than AF-coated silica tubes) can create problems by scattering the excitation light down the lumen. Backpressure can be applied to the system through the outlet tubing, or the tube can be mounted vertically. To remove bubbles, a low surface-tension solvent (e.g., methanol) can be used to wash it through.
4. Applications 4.1. Long-path absorption LCW-based long-path absorption spectroscopy has found a real niche in performing trace analysis in natural water and marine chemistry. Measurements have been made for hydrogen sulfide [10], iron [36,56], nitrate and nitrite [39,47,48,54], phosphate [57], chromium and molybdenum [43], copper [49,59], total inorganic carbon [52], colored dissolved organic matter [55] and phytoplanktons [46]. The long path-length attainable with an AF LCW and its inert nature have for the first time permitted direct, convenient spectroscopic studies of hydroxyl radical reactions [50]. Of course, many other general applications of long-path absorption spectrometry have also been described. 4.2. Sensing dissolved organics and gases In addition to serving as fluid and light conduits, AF LCWs can uniquely be used as an analyte pre-concentrator, a gas-permeable membrane or a conduit for electrophoresis. Many gases permeate readily through AF, and filling the lumen with a selective chromogenic liquid can be the basis of a selective, sensitive gas sensor, as demonstrated for H2 S [37], Cl2 [37], NO2 [37], and CO2 [37,53]. Because two gases that react identically with the same chromogenic agent can have different permeabilities through AF, it may still be possible to make a differential measurement by using AF tubes of different thicknesses or lengths, as demonstrated for 388
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HONO and NO2 with a reagent that cannot discriminate between these gases [74]. Non-polar analytes can adsorb on AF. Tanikkul et al. [33] developed a Raman sensor with a two-stage AF tube system. The first tube was used for the preconcentration of analytes (e.g., benzene, toluene, and p-xylene), which were then eluted and the Raman spectra of the 14·-concentrated analytes were then measured. The LOD for benzene was 730 lg/l. Larsson et al. [75] reported on the possibility of measuring similar compounds based on long-path UV absorption spectrometry. The long-path AF-based absorbance cell capitalized on the gas-permeable properties of the AF tubing. Large-core, silica-fiber-coupled light from a pulsed xenon flash lamp (200 Hz) was input into 20–100 cm long LCW cells and a photodiode array was used for detection in the 200–280 nm range. The LOD was 10 lg/l for benzene in a 1 m LCW for a 10 min sample. 4.3. Chemiluminescence In many chemiluminescence (CL) and bioluminescence experiments, light is emitted as soon as solutions are mixed and reactions occur. Some CL reactions take place in ms time-scales, requiring that solutions be mixed in front of a photomultiplier tube window that must therefore be of significant area and hence expensive (e.g., the hypobromite-urea reaction reaches a peak CL intensity 20 ms after mixing and the signal decays to nearly zero in 120 ms). Li et al. [4] reported on the luminol-hypochlorite reaction that reaches a maximum intensity 800 ms after mixing. This system can be used to detect ammonia by measuring the drop in background CL as ammonia consumes hypochlorite. They compared the performance of the system (a) with the solutions mixing with each other for the first time within the LCW, and (b) coming to a confluence point within a tee and then going into the LCW. Configuration (a) was found to provide superior performance. Li et al. also reported on the use of a scrubber system for collecting atmospheric gaseous hydrogen peroxide (H2 O2 ) and then generating CL by reacting with luminol within a LCW. The maximum CL intensity occurred within 2 s after mixing, necessitating reaction and detection in the same vessel. A PMT (photomultiplier tube) was used for detection with a 83 ll cell; the S/N was 3 and LOD was 25 pptv (parts per trillion by volume) [14]. 4.4. Electrophoresis The applicability of AF-coated silica capillaries for conducting transversely excited fluorescence detection in capillary electrophoresis (CE) was demonstrated early on [3], when these authors demonstrated 200 amol LODs for fluorescein derivatives using excitation by a pair of LEDs and detection by an inexpensive PMT.
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Belz et al. [19] performed LCW capillary gel electrophoresis in a multi-capillary format with fluorescence detection establishing the potential for very high throughputs with a large array of capillaries, four-color detection and high-speed separation. Liu and Pawliszyn [22] used a type I axial laser-illuminated LCW-based capillary isoelectric focusing system with whole-column image detection. The unique properties of AF, beyond its RI, relevant to this application, namely minimal protein adsorption and low electroosmotic flow, eliminate the need for polymeric additives. The detection sensitivity of the system was enhanced by three to five orders of magnitude compared to commercial UV absorbance-based detection systems.
Wang et al. [12] subsequently coupled a flow-injection sample-introduction system to a similar AF-based short CE capillary for the separation of fluorescein isothiocyanate-labeled amino acids. The S/N was 3 and LODs were 1.3 lM for arginine and 1.9 lM for phenylananine and glycine for this LED-excited PMT detected system with a throughput of 144 samples/h. Ball lens coupling of the excitation light to the LCW resulted in a four fold improvement in S/N. Hanning et al. [11] described a similar transverse illumination laser-excited, CCD-based fluorescencedetection system with a CCD detector, achieving a 0.55-amol LOD for fluorescein in the illuminated volume. They also applied it to DNA sequencing, including four-color detection. A similar scheme was then applied to a two-dimensional array of 91 capillaries. Olivares et al. [17] showed whole column imaging for both DNA electrophoresis separations and isoelectric focusing of proteins. The utility of the system is demonstrated for DNA-fragment sizing and protein separations. Scanning the excitation laser along the length of the electrophoresis capillary excites individually separated analyte bands, while the fluorescence is collected end-on by an optical fiber coupled to a PMT, thus creating an image of the separation along the length of the capillary with an ability to detect target molecules in the zeptomolar range.
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5. LCWs in field-deployable instruments LCWs uniquely allow small compact fluorescence detectors particularly suitable for field use. This has been exploited in field instruments to measure formaldehyde [15] and hydrogen peroxide [9]. Gas–liquid scrubbers and appropriate reaction chemistries are used to allow sensing of specific analytes. In many LCW systems that require high sensitivity, the most expensive component is the detector. PMTs offer high sensitivity but can be relatively expensive.
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Time, s Figure 4. Instrument response: (a) photomultiplier tube (PMT) response as seen by the PC, consisting of the composite signal from two channels. The sample contained 2.0 ppbv hydrogen peroxide (H2 O2 ) and 3.4 ppbv methyl hydroperoxide (MHP). The lower and upper envelopes of the composite trace constitute the individual signals for H2 O2 and MHP, respectively; (b) software-isolated signal for the two channels.
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Li et al. [21] took advantage of the ability to switch LEDs readily to construct a multiplexed LCW-based detector for measuring two analytes sequentially. Using different scrubbers and different enzyme catalysts, one of these detectors measured H2 O2 and the other methyl hydroperoxide (MHP). Both LCWs were optically coupled through fiber to the PMT. Each tube was separately illuminated in staggered 5-s intervals, allowing the detector to read out each LCW separately (Fig. 4). Using this system, LODs of 25 pptv (H2 O2 ) and 15 pptv (MHP) were attained (see Fig. 5). A rather versatile portable, hybrid fluorescence/ absorption system has also been described recently [20]. It incorporated a 50-cm long AF tube in a horseshoe path embedded in a black plastic housing to reject ambient light, and it contained a miniature multichannel pump and an injection valve (Fig. 5). Both transverse and axial illumination schemes were possible. A 12-LED array that covered the wavelength range 375–620 nm was mounted transversely to the LCW. The LEDs was controlled by a microcontroller. Light from a white LED was used for long path-length absorption measurements. An optical fiber coupled the light into the LCW. An Ocean Optics spectrometer with a fiber input was used as the detector permitting a LOD of 51 lM for aqueous H2 O2 with an integration time of 2 s. When using a miniature PMT, the LOD was 16 nM. In this system, the S/N was found to increase linearly with increasing integration time since the noise was nearly independent of the integration time. The 50 cm cell had
50x the sensitivity of a standard 1 cm cell (not LCW), although this performance was not realized for situations with high background absorbance.
6. Raman spectroscopy While Raman spectroscopy (RS) can provide detailed vibrational information from aqueous solutions, including biological samples, it suffers from poor sensitivity. Typically, a laser spot impinges on a sample and the backscattered light is collected using high aperture optics. The vast majority of the light collected is still at the excitation frequency (Rayleigh scattering), but a small fraction is inelastically scattered, the decrease (increase) in frequency corresponding to energy loss (gain) to characteristic vibrational modes in the material. LCWs have been used to dramatically improve the sensitivity of RS by axially illuminating a LCW. As the laser propagates through the lumen, Raman scattered light from within the entire illuminated volume of the lumen, within the capture angle of the LCW is backscattered to the detector (alongside the excitation light), effectively greatly increasing the interrogated sample volume [86]. A suitable monochromator system is needed to discriminate against the excitation light. Altkorn et al. [23] showed that, to generate the same Raman signal intensity for acetonitrile in a conventional cell compared to an acetonitrile-filled long LCW, nearly 300 times the laser power was required! The relative Raman intensity from the 2253 cm1 CN band increased as a function of the LCW length in accordance with the numerical model of Walrafen and Stone [87] IR ¼ Kxeax ;
Figure 5. Photograph of a complete flow injection analyzer that has a multi-wavelength source for LED excitation, a PMT for fluorescence or chemiluminescence measurements and a CCD spectrometer for multi-purpose measurements. The system uses a 50 cm Type I LCW with an axial white LED source for absorbance measurements.
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ð3Þ
where, IR is the Raman intensity; K is proportional to the product of intensity of the exciting radiation, the Raman-scattering cross-section of the observed bands in the liquid-core and the solid angle of light guided in the core; x is the length of the LCW; and, a is the attenuation coefficient, assumed to be the same for both the excitation and the emission light. In this particular case, the calculated attenuation coefficient was 4.39 dB/m. Subsequently, this group developed a figure of merit to describe the enhancement given by (adÞ1 , where a is the loss coefficient and d is the diameter of the LCW, and they established parameters for maximizing performance for six different excitation and detection schemes [30]. Other important work has been directed towards using RS as a detection technique in liquid chromatography (LC); given the notoriously poor sensitivity of conventional RS, this would have been considered impossible only a few years ago. A novel, real-time, liquid-core, Raman waveguide detector designed for LC applications was first described by Marquardt et al. [26]
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with the detection volume compatible with microbore and minibore LC; LOD enhancements of over 1000-fold were achieved for the measurement of alcohols in aqueous solution. The LOD of 2-propanol was established to be 2 ppm. Dijkstra et al. [32] has described optimum LCW lengths and choice of laser wavelength in this connection. Much of the effort in LCW-based RS relies on axial illumination. Typically, a notch filter that very effectively removes the laser wavelength is placed before further monochromators and the detector. Holtz et al. [25] used transverse illumination to allow low-energy Raman bands from CCl4 in solution to be measured using a multi-channel CCD detector without a notch filter. As noted earlier, a transverse illumination geometry does not cause many photons to be scattered into the lumen. It is usually very difficult, if not impossible, to obtain a spectrum of CCl4 with a single grating monochromator; typically, a double grating system is needed to prevent the Rayleigh tail from the laser from saturating the detector. Holtz et al. used the 488 nm line of argon laser, a 0.5 m monochromator, and a liquid nitrogen-cooled CCD detector to measure down to 200 cm1 without a notch filter, using 1–60-s integration times. The laser spot created a 6-nL probe volume, usually 3–5 cm from the end of LCW. In order to increase the amount of signal in another series of experiments, the laser light was recycled using a multi-pass configuration. With two and three passes, the signal increased 1.9 and 2.3 times, respectively. Smaller diameter LCW tubing leads to higher power density per unit cross section if the same laser is focused to illuminate the lumen. This can be fortuitous, as it can often photobleach unwanted fluorescent impurities very effectively, thus further improving the ability to look at the Raman signal. Pelletier and Altkorn [29] showed that, in addition to a two order of magnitude improvement in the Raman signal using a long path-length cell, the photobleaching of fluorescence resulted in a two orders of magnitude reduction in the background fluorescence. However, this approach is not universally applicable, as many analytes will themselves be photosensitive.
7. Conclusions The availability of water-core waveguides has allowed a significant increase in performance of a number of analytical systems that rely on solution phase spectrometry. In addition to its low index of refraction, the gas permeability and hydrophobic properties of Teflon AF permit innovative sensing techniques. The relatively low cost, integration, simplicity and durability of the optics will allow expansion in its use in field-deployable sensor systems.
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