Diode
AND PRACTICAL
Lasers
TRACE ANALYSIS Totaro Imasaka and Nobuhiko Ishibashi Faculty of Engineering Kyushu University Hakozaki, Fukuoka 812 Japan
The laser has many advantages as a light source because of its beam-focusing capability and large photon flux. It has been used in atomic and molecular spectrometries, and provides ultrahigh sensitivity. Its monochromaticity allows the recording of high-resolution spectra, providing valuable information for the assignment of chemical species. Moreover, ultrashort laser pulses are essential in the temporal discrimination of a component, which further improves selectivity. Although commercial Raman spectrometers have employed lasers for more than 25 years, the use of lasers in other commercial spectrometers has not been widespread, most likely because of the cost and difficulties in operation and maintenance. The laser is less reliable than a conventional source, and requires additional maintenance (e.g., replacing the plasma tube). In practical applications, a continuous wave (CW) laser with low output power seems to be advantageous. One possibility is an air-cooled argon ion laser (10 mW). This laser is reliable and requires no water for cooling and it costs half as much as the medium-size Ar ion laser (1 W). A He-Cd laser, which is small and generates a UV beam, might also be useful. However, the plasma tube must be replaced every 4000 h, at a cost of more than half the price of the initial equipment. 0003-2700/90/0362-363A/$02.50/0 © 1990 American Chemical Society
Another approach might be to use a small N2-laser-pumped dye laser, which contains a gas reservoir and is turnkey operated. Unfortunately, the output power is unstable and pulse-topulse variation is typically 10-50%. Moreover, rf interference noise sometimes induces a serious problem in signal measurements. A more reliable and stable laser with a lower cost is necessary in practical laser spectrometry. The diode laser Recently, a near-IR diode laser has been developed for use in telecommunications and data processing. Worldwide, more than 106 laser diodes are produced every month for use in compact disk players, bar code scanners, and laser printers. The devices are manufactured by mass production technology, and the price for one chip
and the output power can be feedbackcontrolled by an electronic circuit, providing a stability of ~10" 5 . Unfortunately, a diode laser has an inevitable disadvantage: The wavelength is restricted to the near-IR region. This results from an energy gap of the semiconductor used. Many manufacturers are working to develop a diode laser oscillating at shorter wavelengths, but the current practical limit for a commercial diode laser is 670 nm. Optical fibers We predict that in the twenty-first century all communications will be transmitted by photons through optical fibers, instead of by electrons through copper wire. In industrial operations, local area networks using optical fibers and diode lasers will connect computers, control centers, factory monitoring
INSTRUMENTATION has been reduced to about $10. Performance characteristics of the commercial diode laser are summarized in Table I. The output power of the diode laser corresponds to that of the aircooled Ar ion laser. It is tunable and has a narrow linewidth, similar to a dye laser commonly used in spectrometry. A diode laser has other advantages over conventional lasers. It is smaller and has a long life (~10 5 h). Furthermore, it has a high conversion efficiency from electricity to light and can be directly driven, even by an integrated circuit. A photodiode is usually installed in the same package for power monitoring,
instruments, and business areas. Although all analytical instruments are now designed to use electricity, we believe that in the next century all analytical data will probably be transmitted and received by optical fibers and be further processed by an optical integrated circuit and visually displayed. Thus the coupling of analytical instruments with a diode laser-optical fiber system appears likely in the future. The simplest example might be two optical fibers facing each other; this arrangement allows sensitive absorption detection because of the photothermal effect induced (1).
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Table 1.
Typical performance of a commercial diode laser
Parameter
Performance
Special features
Output power
3-40 mW 10 W 670-850 nm 420 nm 1 nm 10 MHz 20 nm 0.1-100 ns 0.006 % 10-20 %
Continuous wave Pulsed Fundamental SHG, 0.4 mW Conventional Stabilized Temperature controlled No mode locking Controlled Continuous wave
Wavelength Linewidth Tunable range Pulse width Stability Efficiency
Other electrooptical components
A variety of optical components have been developed for data communica tion and processing. For example, a package containing a laser diode and a photodiode, which might be useful for construction of a compact spectrome ter, is already commercially available. A fast detector developed for data com munication is advantageous in time-re solved fluorometry. An avalanche pho todiode has an ultrafast response time, and the transit time spread is reported to be 20 ps (2). A small package con taining an optical fiber, an avalanche diode, a Peltier electric cooler, and a discriminator is commercially available; this may be useful as a single-photon counting device in trace analysis. In this article, we will describe stateof-the-art implementation of diode la sers, optical fibers, and electrooptical components.
measurements of overtone vibration of the molecule, so that the sensitivity is limited to 2.3 mTorr · m for NH 3 . De tection of the pollutant NO2, which has an electronic absorption band in the near-IR region, is possible at pressures down to 1.8 ^Torr for a 5-cm pathlength by heterodyne spectrometry (6). Absorption spectrometry of con densed-phase samples is also reported elsewhere (7). The sensitivity is further enhanced by thermal lens spectrome try (8) and intracavity absorption spec trometry (9). Several complexes of heavy metals and organic chelates have absorption bands in the near-IR re gion. Phosphorus and iron are deter mined with 10-20X better sensitivity than with conventional absorption spectrometry. Near-IR spectrometry has an inev itable disadvantage in the application to trace analysis. Background light ab sorption in this spectral region is not
negligible in most cases, because of overtone vibrational bands of the sol vent that are appreciable for organic solvents containing hydrogen atoms (e.g., CH3CI or CH3OH). A solvent such as CCI4 or CS2 provides lower back ground, but solubility is poor with such nonpolar solvents. Thus further improvement of sensitivity is difficult in near-IR absorption spectrometry. Molecular fluorescence spectrometry
A diode laser provides better sensitiv ity when it is applied to fluorescence spectrometry. Excitation of the solvent to high vibrational levels, which pre vents more sensitive detection in ab sorption spectrometry, gives no back ground fluorescence signal. Thus ultratrace analysis is possible in fluorescence spectrometry. Further more, because most chemical species are nonfluorescent in the near-IR re gion, no impurity fluorescence inter feres with detection of the fluorescent sample molecule. In preliminary work, we could detect some polymethine dyes that fluoresce in the near-IR region down to a concen tration of 5 Χ 10~12 Μ (10). When an optical fiber and a capillary cell were used for light transmission and sample detection, the mass detection limit could be reduced to 12 fg (11). More recently, Winefordner and co workers detected 46,000 molecules of a near-IR dye [5,5'-dichloro-ll-(diphenylamino ) - 3,3'- diethyl-10,12- ethylenethiatricarbocyanine perchlorate] in a volume of 56 nL (12). These results im ply that near-IR fluorescence spec trometry using a diode laser will be use ful for ultratrace analysis.
Molecular absorption spectrometry
In 1982 we became aware of the com mercial availability of a diode laser that had an output power of 20 mW and seemed to be sufficient for analytical spectrometry. We tried to use the laser in a photoacoustic spectrometry appli cation; phosphorus was measured after color development by a molybdenum blue method (3). The configuration of the experimental apparatus is shown in Figure 1. The 780-nm diode laser is used as an exciting source, and the sound induced by light absorption is detected by a piezoelectric transducer installed in the cell. Unfortunately, this first spectrometric application of the diode laser was neither sensitive nor compact. Since then, a diode laser has been applied to various absorption spectro metries. Conventional absorption spec trometry of molecules such as NH3 (4), H2O (4), or CH 4 (5) in the gas phase has been reported. This work is based on
Figure 1. Photoacoustic spectrometer using a diode laser. (Adapted from Reference 3.)
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INSTRUMENTATION
Figure 3. Liquid chromatogram of pro tein in human serum obtained using a diode laser fluorometric detector. (Adapted from Reference 15.)
Figure 2. Chemical structures of polymethine dyes that are fluorescent in the nearIR spectral region. We have also demonstrated time-re solved fluorometry at a time resolution of 480 ps, which is useful in the evalua tion of solvent polarity (13). This work involved the use of picosecond light pulses generated by a diode laser (136 ps, 1.3 W). A potential problem in near-IR fluo rometry is the lack of fluorescent or ganic dyes in this spectral region. To our knowledge, only one group of mole cules, polymethine dyes, are fluores cent in this region. Though chromato graphic determination of polymethine dyes has been demonstrated using di ode laser fluorometry (14), practical ap plication to a real sample is quite limit ed. However, it might be possible to use polymethine dyes as probe molecules. This situation is perhaps analogous to the fact that because few substances are naturally radioactive, radioimmu noassay is extremely useful in trace —
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analysis because of the low background signal. A variety of molecular species are commercially available for polymeth ine dyes. The number of compounds synthesized and commercially avail able exceeds 3000. Developed for use as photosensitive dyes, they have been used more recently as laser dyes. The molecular structures of some poly methine dyes are shown in Figure 2. Unfortunately, they have no reactive sites in the molecule, although perhaps the ethyl side chains in the quinocarbocyanines could be modified, and the phenyl rings offer opportunities for in troducing reactive substituent®. The top three dyes are less soluble than the fourth in aqueous solution and form nonfluorescent dimers. The exception in this group is indocyanine green [2-[7-[l,3-dihydro-l,l-dimethyl-3-(4sulfobutyl)-2//-benz[e]indol-2-ylidene]-
l,3,5-heptatrienyl]-l,l-dimethyl-3-(4sulfobutyl ) - 1H - benz [ e ] indolium hy droxide inner salt sodium salt]; it has negatively charged sulfonic groups and is soluble in water. The chemical properties of indo cyanine green have recently been in vestigated. Because it has been found to be adsorbed on the surface of protein and to increase the fluorescence inten sity, this molecule can be used for la beling protein. Figure 3 shows the chro matogram recorded by labeling protein in human serum with indocyanine green and by separating the complex with a gel filtration column. The detec tion limit reported is 1.3 pmol for albu min (15). Recently, the fluorescence intensity of indocyanine green has been found to be quenched by an OH radical, proba bly because of the formation of a non fluorescent dimerlike compound. Thus an enzyme reaction producing Η2Ο2 can be monitored by using a catalyst of Fe(II), which converts H 2 0 2 to OH. By the following reaction, xanthine is de termined in the presence of xanthine oxidase (16). xanthine + 0 2 + H 2 0 xanthine oxidase > uric acid + H 2 0 2 More recently, peroxidase generating OH from H 2 0 2 has been measured in the similar reaction scheme, which is further extended to enzyme immuno assay of insulin (17). Visible semiconductor laser fluorometry Near-IR laser fluorometry has high sensitivity because of the large photon
ANALYTICAL CHEMISTRY, VOL. 62, NO. 6, MARCH 15, 1990 · 367 A
INSTRUMENTATION flux and low background signal. However, this approach suffers from a lack of suitable dyes for color development and for labeling a molecule. This situation was unchanged even when a 750nm diode laser appeared. A recently developed diode laser oscillating at 670 nm is quite versatile, however, and extends the application field because of the availability of many dyes in this spectral region. Figure 4 shows chemical structures of dyes that fluoresce in the deep-red region. Rhodamine, oxazine, and thiazine dyes are useful as chromophores for labeling biological molecules. By using a bifunctional reagent of watersoluble carbodiimide, albumin is labeled with a fluorescent tag by a covalent bond (18). The detection limit of albumin obtained by labeling it with oxazine 750 is reported to be 0.13 pmol and is determined by the limited labeling efficiency. Another dye, methylene blue, is widely used, for example, in measurements of oxidation and reduction potential, in determinations of enzymes and metabolites, and also in staining DNA by intercalation to the double helix. Therefore, visible diode laser fluorometry may have wide application in biological assay in the future. In indirect chromatography detection, the fluorescence intensity of the reagent in a carrier is measured to observe the displacement by the eluted compounds. The approach using a visi-
ble diode laser for universal detection of organic compounds has been studied elsewhere (19). Atomic absorption spectrometry The wavelength of the diode laser is determined by the cavity length, which is controlled by changing the diode current and the temperature. In atomic spectrometry the wavelength should be tuned to a resonance line within an error of 0.001 nm, so that the temperature of the laser diode is controlled within 0.01 °C. Because of mode hopping, the diode laser may skip over certain wavelength regions, so it should be carefully selected to operate at a specified wavelength. This characteristic of the commercial diode laser is reported in detail elsewhere (20). A resonance line of Rb is located at 780.2 nm and an intercombination line of Ba at 791.1 nm. A diode laser, instead of a hollow cathode lamp, has been used in atomic absorption spectrometry (21). The detection limit of Rb is reported to be 500 pg/mL. This approach not only decreases the dimension and the power consumption but also allows background subtraction by tuning the laser wavelength on and off the resonance line. This is useful when a strong molecular absorption band is superimposed. Another possible advantage of diode laser spectrometry is high sensitivity resulting from a multiple pass effect; the laser beam is well collimated and the pathlength can
be extended by reflecting the beam using a cavity filled with sample. Atomic fluorescence spectrometry Atomic fluorescence spectrometry is essentially more sensitive than atomic absorption spectrometry because the signal intensity increases linearly as the intensity of the light source increases. The application of the diode laser to atomic fluorescence spectrometry has already been studied (22). A detection limit of 2.1 ppb has been reported for Rb and was achieved by using a 200-mW diode laser. A potential problem in atomic spectrometry is the narrow tunable range of the diode laser. The resonance lines used in conventional atomic absorption spectrometry are widespread in the visible and UV regions, although some of them are in principle covered by the fundamental and frequency-doubled beams generated by the state-of-theart diode laser. An alternative method is the use of other spectral lines, although sensitivity will be substantially decreased. Other spectrometries In a refractive index (RI) detector, a light source with stable output power and good beam-pointing stability is required. The diode laser has excellent performance in these respects. In RI gradient detection, a detection limit of 4 X 10~9 RI units, corresponding to a mass detection limit of 540 pg (1.1 fmol) for polystyrene, has been reported (23). This technique has been applied to detectors for chromatography (23) and for electrophoresis (24). A diode laser has also been used to measure optical rotation and as an LC detector (25). In Raman spectrometry a near-IR diode laser is advantageously used to discriminate Raman emission from fluorescence. The low output power of the diode laser is partly compensated for by combining it with a sensitive chargecoupled device for measurements of the spectrum (26). The advantage of the diode laser has been demonstrated using a fluorescent sample in surface-enhanced Raman spectrometry (27). Frequency-doubled diode laser
Figure 4. Chemical structures of organic dyes that are fluorescent in the deep-red spectral region. (Adapted from Reference 18.)
The wavelength of the commercial diode laser is limited to 670 nm. However, the wavelength of the laser can be extended to much shorter wavelengths by second harmonic generation (SHG) or frequency mixing (FMX). Efficient SHG is achieved by passing the laser beam through a nonlinear crystal, such as potassium dihydrogen phosphate (KDP) or the more efficient potassium niobate (KNbO :) ).
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Glassy carbon electrode in solution, coated with copper, deposited and imaged at sub-nanometer resolution.
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Figure 5. Optical fiber sensor sensitive to oxygen using second harmonic emission of the diode laser, (a) Total system and (b) sensing device. (Adapted with permission from Reference 28.)
Figure 5 shows a block diagram of an optical fiber sensor sensitive to oxygen. A frequency-doubled beam is focused onto the distal end of an optical fiber, and fluorescence is measured by passing it through a different optical fiber. The conversion efficiency of SHG is 2.5 X 10~6 but it is sufficient as a light source in the optical fiber sensor system (28). The concentration of oxygen is determined at 0-15% levels. A diode laser is made of a nonlinear crystal, so that SHG occurs even in the laser diode itself. The output power obtained is at picowatt levels, but it is still sufficient for use in atomic absorption (29) and molecular fluorescence (30) spectrometries. Visible fluorometry using a frequency-doubled diode laser has wide application because visible fluorometry is already established and in use. However, some of the advantages of near-IR fluorometry (e.g., low background signal) are lost with this approach. Future developments
The technology of the diode laser is progressing rapidly. The output power doubles every year, and the cost is cut in half. A high-power, diode-laserpumped Nd:YAG laser is a hot topic in laser technology because of its high conversion efficiency (10%) from electricity to a coherent light beam. A frequency-doubled Nd:YAG laser producing 80 mW is already commercially
370 A · ANALYTICAL CHEMISTRY, VOL. 62, NO. 6, MARCH 15, 1990
available. Such a laser may be quite useful in future analytical work. On average, the wavelength of the diode laser has been shortened 10 nm every year. This rapid rate of change can be attributed to applications of the laser diode to such commercially successful products as the bar code scanner and the laser printer. We believe that a more powerful laser oscillating at shorter wavelengths will soon appear. Its availability will further extend analytical applications. For recording data to an optical disk, a laser that oscillates at much shorter wavelengths is advantageous because the focused beam diameter can be reduced to the order of the laser wavelength. Thus electronic engineers are concentrating their attention on frequency doubling of the diode laser. Their research has shown that the conversion efficiency of SHG is improved to 14% (24 mW/167 mW) (31). If this diode laser is available as an excitation source for chromatography or electrophoresis detectors, it will become a powerful tool in trace analysis. At present, no commercial diode laser spectrometer is available. We expect that a commercial instrument will appear in the next few years and will find practical analytical usefulness. Its appearance will also help stimulate the development of chemical reagents and analytical procedures for near-IR or deep-red spectrometry.
Combining a diode laser with ad vanced electrooptical components will provide the best instrument perform ance. For example, the dimension and the weight of the diode laser spectrom eter are now limited by the detector, that is, a photomultiplier and its power supply. Replacement with a sensitive photodiode is essential for its practical use. A one-chip integrated circuit con sisting of a diode laser, a transmitter, a receiver, a detector, and a data proces sor, which can be directly coupled with an optical fiber system, might be the ultimate approach for practical spec troscopic analysis.
(28) Okazaki, T.; Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1988,209, 327. (29) Sakurai, K.; Yamada, N. Opt. Lett. 1989,14, 233.
(30) Imasaka, T.; Hiraiwa, T.; Ishibashi, N. Mikrochim. Acta 1989,2, 225. (31) Goldberg, L.; Chun, M. K. Appl. Phys. Lett. 1989,55, 218.
References (1) Imasaka, T.; Nakanishi, K.; Ishibashi, N. Anal. Chem. 1987,59,1554. (2) Cova, S.; Lacaita, Α.; Ghioni, M.; Ripamonti, G.; Louis, T. A. Rev. Sci. Instrum. 1989,60,1104. (3) Kawabata, Y.; Kamikubo, T.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1983, 55, 1419. (4) Ohtsu, M.; Kotani, H.; Tagawa, H. Ja pan. J. Appl. Phys. 1983,22,1553. (5) Chan, K.; Ito, H.; Inaba, H. Appl. Opt. 1983,22,3802. (6) Lenth, W.; Gehrtz, M. Appl. Phys. Lett. 1985,47,1263. (7) Imasaka, T.; Kamikubo, T.; Kawabata, Y.; Ishibashi, N. Anal. Chim. Acta 1983, 153, 261. (8) Nakanishi, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1985,57,1219. (9) Unger, E.; Patonay, G. Anal. Chem. 1989,67,1425. (10) Imasaka, T.; Yoshitake, Α.; Ishibashi, N. Anal. Chem. 1984,56,1077. (11) Kawabata, Y.; Imasaka, T.; Ishibashi, N. Talanta 1986,33, 281. (12) Johnson, P. Α.; Barber, T.· E.; Smith, B. W.; Winefordner, J. D. Anal. Chem. 1989,67,861. (13) Imasaka, T.; Yoshitake, Α.; Hirata, K.; Kawabata, Y.; Ishibashi, N. Anal. Chem. 1985,57, 947. (14) Sauda, K; Imasaka, T.; Ishibashi, N. Anal. Chim. Acta 1986,187,353. (15) Sauda, K.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1986,58, 2649. (16) Imasaka, T.; Okazaki, T.; Ishibashi, N. Anal. Chim. Acta 1988,208,325. (17) Imasaka, T.; Nakagawa, H.; Okazaki, T.; Ishibashi, N., unpublished results. (18) Imasaka, T.; Tsukamoto, Α.; Ishibashi, N. Anal. Chem. 1989,61, 2285. (19) Folestad, S.; Ahlberg, H. International Symposium on Column Liquid Chroma tography, Stockholm, June 26-30,1989. (20) Lawrenz, J.; Niemax, K. Spectrochim. Acta 1989,44B, 155. (21) Hergenrôder, R.; Niemax, K. Spectrochim. Acta 1988,43B, 1443. (22) Johnson, P. Α.; Vera, J. Α.; Smith, B. W.; Winefordner, J. D. Spectrosc. Lett. 1988,27,607. (23) Hancock, D. O.; Synovec, R. E. Anal. Chem. 1988,60,1915. (24) Chen, C. Y.; Demana, T.; Huang, S. D.; Morris, M. D. Anal. Chem. 1989,67,1590. (25) Lloyd, D. K.; Goodall, D. M; Scriven er, H. Anal. Chem. 1989,67,1238. (26) Williamson, J. M.; Bowling, R. J.; McCreery, R. L. Appl. Spectrosc. 1989, 43,372. (27) Angel, S. M.; Myrick, M. L. Anal. Chem. 1989,67,1648.
Nobuhiko Ishibashi (left) is professor of applied analytical chemistry at Kyushu University, where he has been a member of the faculty since 1954. He is chairman of the Japanese Association of Flow Injection Analysis and past vice-president of the Japan Society for Analytical Chemistry and the Electrochemical Society of Japan. His research interests include laser fluorometry, supersonic molecular beam fluorometry, optical fiber sensors, ion-selective electrodes, ion-exchange membranes, and flow analysis. Totaro Imasaka (right) received his Ph.D. from Kyushu University, where he has been a member of the Faculty of Engineering since 1980. His research inter ests include investigations of supersonic jet spectrometry and semiconductor laser spectrometry. More recently, he has concentrated on developing a widely tunable laser system based on two-color stimulated Raman emission.
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