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Sensitive absorbance measurement method based on laser multi-wave mixing
SpecrrochrmrcaActa. Vol 496. Nos 12-14. pp 1483-1489, 1994 Copyright 0 1994 Elsewer Sctence Ltd Printed m Great Bntatn All rtghts reserved 0584-6547/94 17.00 + 00
0584-8547(94)ooo8?%3
Sensitive absorbance
measurement method based on laser multi-wave mixing*
ZHIQIANGWV, JINYINGLIU? and Department
WILLIAM
G.
TONGS
of Chemistry, San Diego State University, San Diego, CA 92182,
U.S.A.
(Received 3 May 1994; accepted 26 July 1994)
Abstract-A sensitive absorbance measurement based on nonlinear laser degenerate four-wave mixing is demonstrated for cadmium. The cadmium ions react with dithizone to form a cadium complex which is then extracted in carbon tetrachloride and analyzed. A relatively low-power argon ion laser line at 514.5 nm is used as the excitation light source. This nonlinear laser method offers many useful features including efficient and simple optical signal detection (signal is a collimated coherent beam), excellent detection sensitivity for absorbance, and efficient use of low laser power levels, small laser probe volumes and short analyte path legnths (e.g., ~0.5 mm). A detection limit of 7 fg or 0.05 ng/ml for cadmium, corresponding to an absorbance detection limit of 1.8 x 10mh AU is reported using a flowing analyte cell at room temperature.
1. INr~0nUcrt0N O~ICAL phase conjugation by degenerate four-wave mixing (D4WM) is one of the most useful and versatile nonlinear spectroscopic methods with many applications [l-4]. A phase conjugate signal beam is generated in a D4WM optical setup when a nonlinear medium (that is, an analyte) is illuminated by two exactly counter propagating pump beams (forward pump Ef and backward pump E,,), and a probe beam Ep that intersects with the pump beams at a small angle (that is, < 0.5”). Interaction of the input beams in the nonlinear medium produces a phase conjugate signal beam that retraces back the incident probe beam. Physical processes responsible for the generation of the signal beam include molecular orientation [l], change in population equilibrium [5], thermally induced refractive index change [6], and electrostriction [7]. In an absorbing liquid medium, the nonlinear effect produced by thermally induced refractive index change accompanies and often dominates other nonlinear effects. After light is absorbed by an analyte, the heat generated by analyte nonradiative relaxation results in periodic temperature and density fluctuations, and hence, in spatial modulation of the refractive index of the analyte medium. This refractive index grating diffracts one of the input beams (i.e., Eb) and produces the phase conjugate signal beam that is an exact time-reversed replica of the probe beam. Nonlinear spectroscopy based on D4WM has been demonstrated as a sensitive analytical method using different atomizers including low-pressure hollow-cathode discharge cells [g-lo], liquid flow cells [ll, 121, and analytical flame atomizers [13-161. While gas-phase atomizers yield sub-Doppler spectral resolution that is suitable for hyperfine structure analysis, liquid flow cells offer convenient detection of analytes at attomole-level detection sensitivity at room temperature [ll]. Since the D4WM signal is a collimated coherent beam and it is visible to the naked eye, optical alignment is convenient and detection sensitivity is excellent even when the excitation wavelength is more than 100 nm away from the absorption wavelength maximum [12]. In addition * This paper was published in the Special Honor Issue of Spectrochimica Acta Purr B, dedicated J. D. Winefordner. t Current address: Alliance Pharmaceutical Corp., San Diego, California, U. S. A. $ Author to whom all correspondence should be addressed. 1483
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to backward-scattering D4WM (B-D4WM) methods mentioned above, one can also use a forward-scattering D4WM (F-D4WM) setup [17], where the signal beam is generated to propagate in the same direction as that of the probe beam (i.e., forward direction), as compared to the opposite direction signal propagation as in a B-D4WM method. While a B-D4WM optical setup offers sub-Doppler spectral resolution (due to counter propagating pump beams), and less background scattering noise, a F-D4WM method yields a simpler optical setup. In this paper, we report backward D4WM as a sensitive method for measuring small absorbance values in short analyte path lengths, using dithizone as an organic chelating reagent for cadmium. Dithizone (diphenylthiocarbazone) and azo dye reagents have been widely used by many workers to increase detection sensitivity and selectivity in spectrophotometric determination of trace amounts of metals. Dithizone is one of the most important reagents, especially useful for trace-concentration detection of elements such as cadmium, lead, zinc, mercury, silver, and copper [18, 191, since these elements form intense color complexes with dithizone. Molar absorptivities of metal-dithizone complexes in Ccl, are in the range from 3 x lo4 to 9 X lo4 M-‘cm-‘. In addition, high partition coefficients of metal-dithizone complexes in organic solvents, such as CHC& and Ccl,, allow preconcentration and separation by solvent extraction, and hence offer enhanced detection sensitivity and selectivity. The backward-scattering D4WM signal intensity, I,, can be described as shown below for trace concentration analyte solutions (i.e., low absorptivity) [6, 111. 2
(1)
where I,, I,, Z,, and Z, represent beam intensities of the phase conjugate signal beam, the forward pump beam, the backward pump beam, and the probe beam, a is the analyte absorption coefficient, L is the path length of the sample cell, 8 is the angle between the forward pump beam and the probe beam, (dn/dT), is the derivative of the refractive index with respect to temperature at constant pressure, p0 is the equilibrium solvent density, C, is the specific heat at constant pressure, and X is the excitation wavelength. Equation (1) describes important characteristics of the D4WM signal including its cubic dependence on laser intensity, its quadratic dependence on absorption coefficient, and hence on analyte concentration, and its quadratic dependence on (dn/dT),. Since the D4WM signal has a quadratic dependence on analyte absorption coefficient, detection sensitivity can be significantly improved by enhancing analyte molar absorptivity using organic chelating reagents. Taking advantage of these unique nonlinear properties of the D4WM method, ultrasensitive determination of cadmium is demonstrated using the Cd-dithizone complex. In a strongly alkaline solution, cadmium ions react with dithizone to form a pinkcolored complex with a maximum absorption at 520 nm. The complex is then extracted in carbon tetrachloride and measured by D4WM detection method. Using the 514.5 nm line of an argon ion laser as the excitation source, and a laser probe volume of 0.14 ~1, a cadmium detection limit of 7 fg or 0.05 ng/ml (S/N = 2), corresponding to an absorbance-unit detection limit of 1.8 x lO-‘j AU. is determined.
2. EXPERIMENTAL Figure 1 shows a backward-geometry D4Wh4 experimental arrangement where an argon ion laser (Coherent, Palo Alto, CA, Model Innova 90-6) is used as the excitation light source. A polarizer is used to prevent the returning pump beams from entering the laser cavity. The laser beam is first split by a 70/30 beam splitter, and the transmitted beam is used as the backward pump beam I$,. The reflected beam is further split by a 50/50 beam splitter to produce the forward pump beam Ef and the probem beam E,. A half-wave plate is inserted in
Sensitive absorbance measurement
Fig. 1. Experimental arrangement for backward-geometry tion method for Cd(II)-dithizone/CCI.
1485
method
degenerate four-wave mixing detecanalyte solution.
backward pump beam path to rotate its polarization plane by 90”. The D4WM signal generated in the analyte cell retraces back the probe beam path, where a beam splitter is used to steer the signal beam into a photomultiplier tube. A mechanical chopper is used to modulate the amplitude of both the forward pump beam and the probe beam. Another polarizer, cross polarized with the polarization plane of the forward pump beam and that of the probe beam, is placed in front of the photomultiplier tube to suppress the background scattering noise caused by the forward pump beam and the probe beam. The D4WM signal from the photomultiplier tube is amplified by a lock-in amplifier after it passes through a current-to-voltage converter. The output voltage of the lock-in amplifier is recorded by a chart recorder and digitized by a persona1 computer. The absorption spectra of dithizone and cadmium-dithizone complexes are also measured by a conventional UV-visible spectrophotometer, as shown in Fig. 2. Cadmium (II) stock solution (1 ppm) is prepared by dissolving cadmium (II) chloride in a
the
0.6
04 : 5 e q0.2
0.0
I
I ,
400
b
I
600
1
600
1
700
WAVELENGTH (n “‘)
Fig. 2. Absorption spectra measured by a conventional UVNis spectrophotometer (a) extracted blank solution, (b) extracted cadmium-dithizone in CCL and (c) 0.601% dithizone in Ccl,.
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et al.
0.1 M HCl solution. Sodium hydroxide (semiconductor grade), hydrochloric acid (ACS reagent), and carbon tetrachloride (spectrophotometric grade) solutions are purchased from Aldrich. Extraction of cadmium-dithizone complex is performed using a standard procedure [18].
3. RESULTSAND DISCUSSION As shown in Eqn (l), the D4WM signal has a cubic dependence on laser intensity, a quadratic dependence on absorption coefficient, and a quadratic dependence on (dn/ dZ’),. One could take advantage of these nonlinear properties in order to enhance signal strength and detection sensitivity. For instance, due to the cubic dependence of signal on laser power, high photon density available from even a relatively weak laser beam could significantly boost the signal strength. In addition, since the background noise has only a linear dependence on intensity and the signal has a cubic dependence, signal-to-noise enhancement is very significant when the laser intensity is increased. Quadratic dependence of signal on absorption coefficient could be viewed advantageously or disadvantageously depending on whether the analyte concentration is increasing or decreasing. Nevertheless, any disadvantages due to low absorptivity or concentration could be more than compensated for by the pure strength of the “coherent” signal beam, its virtually 100% collection efficiency, and its cubic dependence on laser intensity. Taking advantage of the quadratic dependence of signal on analyte absorption coefficient, signal strength could be easily enhanced by simply improving analyte molar absorptivity using an appropriate complexing agent. One could also improve the signal strength simply by choosing a solvent with an optimum (dn/dT), value, since the signal also has a quadratic dependence on (dnldZ&. In order to enhance the absorbance detection limit for cadmium, one could use a cadmium-complex system with high molar absorptivity, a chelating agent with very low molar absorptivity at the excitation wavelength, and a solvent with a large (drill d7& value. Figure 2 shows a conventional UV/Vis spectrophotometer spectra of cadmium-dithizone complex (1 X 10e5M), dithizone in carbon tetrachloride, and a blank extraction solution. Cadmium ions react with dithizone to form an intensely colored complex with a maximum molar absorptivity of 8.8 X 10P4 M-‘cm-’ at a Amax of 520 nm [18]. Dithizone in CC& shows two maximum absorption peaks, at 620 nm and 420 nm, and a minimum absorption at 510 nm between the two absorption peaks. Carbon tetrachloride has a large (dn/dT), value, and hence, it is very suitable for use as a solvent in a D4WM detection method. A high level of blank absorption could limit detection sensitivity in a D4WM metal analysis especially at trace concentration levels, since a “blank” D4WM signal could be generated by the absorbing blank solution. To maximize the analyte D4WM signal and to minimize the background signal, one should use a laser excitation wavelength that is as close as possible to the absorption line center of the metal complex, and to the minimum absorption wavelength of the chelating reagent. For instance, the 514.5 nm line of an argon ion laser is suitable as an excitation source, since it is close to the maximium absorption peak of cadmium-dithizone complex at 520 nm and to the minimum absorption wavelength of the chelating reagent. As in other ultrasensitive spectrometric methods, impure solvents and chemicals could be a source of blank absorption, since some of the elements that might be present in the solvent could also form color complexes with the chelating reagent used. As expected, the D4WM signal amplitude, observed in a blank solution prepared by using ACS grade NaOH and Ccl4 solvents, is comparable to that of a 5 ppb cadmium solution. However, the D4WM signal amplitude, observed in blank solution prepared by using semiconductor-grade NaOH and spectrophotometric-grade Ccl, solvents, corresponds to the signal level of only 0.01 ppb cadmium solution. Hence, high-purity grade chemicals are used when trace-concentration detection sensitivity is desired. At trace-concentration detection levels, background absorption of organic chelating reagents should be also avoided. Although the 514 nm argon ion laser line is located near the maximum absorption wavelength of cadmium-dithizone complex,
Sensitive absorbance measurement
method
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a small absorption by dithizone at 514 nm could still generate some “blank” D4WM signal, and hence, it is desirable to use as small an amount of dithizone as possible. When the concentration of cadmium is less than 10 ppb, a dilute dithizone solution (0.0002%) could be used in the D4WM method. In a strong alkaline solution (e.g., 5% NaOH), cadmium ions form stable complexes with dithizone while common metal dithizone complexes such as those of lead, zinc, and bismuth do not exist. Hence, it is an effective way to improve selectivity and reduce blank absorption. As shown in Fig. 2, there is no measurable absorbance by a conventional spectrophotometer in a blank solution extracted from a strong alkaline solution using 15 ml of a 0.0002% dithizone/CCl, solution. The D4WM signal strength can be improved significantly by optimizing some D4WM experimental parameters, such as the angle between the forward pump beam and the probe beam, the path length difference of the forward pump beam and the probe beam, and the intensity distribution ratio of the input beams. Background optical scattering from the input beams can be suppressed also by using an optimum angle between the input beams (and hence, affecting the relative positions of the signal and input beams) and appropriate spatial filters to allow only the coherent collimated signal beam to pass through to the detector, and by minimizing thermal lensing or blooming as described below. In an absorbing solution, thermal gratings are formed following the absorption of light through electronic transitions of the analyte molecules. Heating via radiationless relaxation of the optically excited molecules gives rise to periodic density and temperature fluctuations that result in the spatial modulation of the refractive index of the nonlinear medium (i.e., the analyte). At the same time, heating produced by nonradiative relaxation of excited species also yields thermal lens effects, where the absorbing medium acts like a diverging lens [20]. This thermal blooming effect could interfere with the D4WM signal measurement, since thermal blooming of the backward pump beam causes its expanding beam spot to overlap with the phase conjugate signal beam spot, especially when a small angle between the forward pump beam and the probe beam is used. Optical background noise caused by thermal lensing or blooming could be effectively suppressed by the use of a mechanical chopper-based amplitude modulated detection scheme, since the chopper effectively disrupts the pump beam, and hence, does not allow sufficient time to expand and interfere with the signal beam spot. As reported previously [21], the thermal lens signal decreases rapidly at chopper frequencies higher than 2 Hz. The D4WM signal can be collected at a higher modulation frequency, at which thermal lens background signal is at a minimum. The D4WM signal for the cadmium solution is strong and stable when the modulation frequency is 30 Hz or less. As the modulation frequency is increased much higher, less time is allowed for the D4WM gratings to form, and hence, the signal decreases gradually. Nevertheless, the S/N remains reasonably high at higher modulation frequencies (up to 200 Hz studied), since the background noise is more effectively suppressed by the lock-in amplifier at a higher modulation frequency, and also because optical noise originating from E,, thermal blooming is also insignificant at a higher frequency. Hence, a reasonably wide useful range of modulation frequency is available. The optical background noise caused by thermal lens effects could also be suppressed effectively by using an analyte cell with a short path length, since the D4WM laser probe volume (and effective path length) is already short. As expected, a cell with a long path length (e.g., 10 m) yields more background thermal blooming interference than a cell with a shorter path length (e.g., 0.5 mm). Optical background noise caused by thermal lensing/blooming can be further suppressed by chopping both the forward pump beam and the probe beam, instead of just chopping the probe beam. Experimental results show that the D4WM signal is also stronger when both Ef and E, are chopped as compared to when only Ep is modulated. Signal-to-noise ratio improvement is determined to be by a factor of 2.7 when both the forward pump beam and the probe beam are chopped as compared to when only the probe beam is modulated. As expected, a cubic dependence of signal on laser power is observed with a slope
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of 3.05 in a log-log signal vs power plot. Based on this very efficient nonlinear dependency, one could take advantage of the high photon density available even from relatively weak inexpensive lasers. When relatively high laser power levels (e.g., >l W) are used, especially for the Eb beam, overheating of the analyte solution could generate more thermal blooming background noise, which could overlap with the signal spot when observed on a screen along the signal propagation path. Hence, it is desirable to distribute more laser power to the Ef and the E, beam, and less to the Eb beam. For example, a beam intensity distribution ratio of 5:3:1 for 1&l, would yield less background noise than when a ratio of 1: 1: 1 is used. As expected, a quadratic dependence of signal on analyte concentration is observed with a slope of 1.9 in a log-log signal vs concentration plot, using independent extractions of small amounts of cadmium. Quantitative measurement is still straightforward in this “nonlinear” method, since the slope is simply two instead of one as in conventional “linear” detection methods. A cadmium concentration detection limit (S/N = 2) of 0.05 ng/ml is determined in the extraction solution when a forward pump beam power of 0.35 W, a probe beam power of 0.25 W, and a backward pump beam power of 0.075 W are used. Since the laser probe volume is 0.14 ~1, the cadmium mass detection limit by D4WM is determined to be 7 fg inside the analyte probe volume. This corresponds to an absorbance-unit detection limit of 1.8 x 10e6 AU for cadmium. This detection sensitivity is still limited by blank absorption due to impurities in solvents and chemicals used, and the cadmium detection limit could be further improved by using higher purity reagents, and more effective masking agents in a cleaner laboratory environment. Since short analyte path lengths are advantageously used in a D4WM setup, the absorbance detection limit of 1.8 X 1OV AU for cadmium is especially good and compares well with other laser-based analytical methods including that of a thermallens based method [21] (i.e., 5 x lop6 AU) while using only a single laser instead of two lasers. In addition, the experimental setup is relatively inexpensive and simple in this one-color one-laser nonlinear analytical spectroscopic method, as compared to two or more lasers used in many multi-beam laser methods. The analyte cell in the D4WM detector is very simple, and the analyte solution can be conveniently introduced through the flow cell at room temperature. Some disadvantages one might consider for this nonlinear detection method include the use of two laser beams (from the same laser), and hence, slightly more complicated optical alignment as compared to the use of simply one beam in a conventional laser method, and the need to suppress and filter optical noise originating from the input beams. However, when compared to other multi-photon laser methods, the optical alignment is relatively simple and the laser requirement is minimum (i.e., one-color one-laser system as compared to twocolor two-laser systems as in other laser methods). One could use a two-input-beam forward-scattering D4WM setup to simplify the optical alignment, and use a threeinput-beam backward-scattering D4WM setup to minimize source light scattering and interference. 4. CONCLUSIONS The use of a simple nonlinear method based on laser four-wave mixing is demonstrated for enhanced absorbance measurement of metals using complexing agents. This pump-probe cross-beam laser method offers useful features including efficient and simple optical signal detection (signal is a collimated coherent beam), excellent absorbance-unit detection sensitivity, and efficient use of low laser power levels, small laser probe volumes and short analyte path lengths (e.g., < 0.5 mm). Hence, it is especially suitable for on-column absorbance detection in capillary chromatography and capillary electrophoresis systems, where the analyte path length is very short. Furthermore, since laser power requirements in a one-laser D4WM method is low, many inexpensive compact lasers could be used. Since D4WM detectability is excellent both for fluorescing and nonfluorescing analytes, this nonlinear
Sensitive absorbance measurement
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laser method offers many potential applications. Ultrasensitive detection of cadmium as demonstrated above could be obtained similarly for other metals, non-metal elements, and organic molecules, such as amino acids. Acknowledgement-We gratefully acknowledge partial support of this work from the National Institute of General Medical Sciences, National Institutes of Health under grant No. 5-ROl-GM41032, the National Science Foundation under grant No. CHE-8719843 and Beckman Instruments, Inc.
REFERENCES [l] R. A. Fisher, Ed., Optical Phase Conjugation. Academic Press, New York (1983). [2] R. W. Hellwarth, J. Opt. Sot. Am. 67, 1 (1977). [3] J. 0. White and A. Yariv, Appl. Phys. Left. 37, 5 (1980). [4] H. Fujiwara and K. Nakagawa, Opt. Commun. 55, 386 (1985). [5] D. M. Bloom, P. F. Liao and N. P. Economou, Opt. Lett. 2, 58 (1978). [6] H. J. Hoffman, IEEE J. Quantum Electron, 22, 552 (1986). [7] K. A. Nelson, R. J. D. Miller, D. R. Lutz and M. D. Fayer, Phys. Rev. 3261 (1981). [8] W. G. Tong and D. A. Chen, Appl. Spectrosc. 41, 586 (1987). [9] D. A. Chen and W. G. Tong, J. Anal. Atom. Specrrom. 3, 531, (1988). [lo] Z. Wu and W. G. Tong, Specrrochim. Acfu 47B, 419 (1992). [ll] Z. Wu and W. G. Tong, Anal. C/rem. 61, 998 (1989). [12] Z. Wu and W. G. Tong, Anal. Chem. 63, 1943 (1991). [13] W. G. Tong, J. M. Andrews, and Z. Wu, Anal. Chem. 59, 896 (1987). [14] J. M. Andrews and W. G. Tong, Spectrochim. Acta 44B, 101 (1989). [15] J. M. Andrews, K. M. Weed and W. G. Tong, Appl. Spectrosc. 45, 697 (1991). [16] Z. Wu and W. G. Tong, Anal. C/rem. 63, 899 (1991). [17] Z. Wu and W. G. Tong, Anal. Chem. 65, 112 (1993). [18] Z. Marczenko, Spectrophozometric Determination of Elementi. Ellis Horwood, Chichester (1976). [19] H. M. N. H. Irving, CRC Crit. Rev. in Anal. C/rem. 8, 321 (1980). [20] J. M. Harris and N. J. Dovichi, Anal. C/rem. 52, 695A (1980). [21] G. R. Ramos, M. C. Garcia Alvarez-Coque, B. W. Smith, N. Omenetto and J. D. Winefordner, Appl.
Spectrosc.
42, 341 (1988).
0584-8547(94)ooo8?%3
Sensitive absorbance
measurement method based on laser multi-wave mixing*
ZHIQIANGWV, JINYINGLIU? and Department
WILLIAM
G.
TONGS
of Chemistry, San Diego State University, San Diego, CA 92182,
U.S.A.
(Received 3 May 1994; accepted 26 July 1994)
Abstract-A sensitive absorbance measurement based on nonlinear laser degenerate four-wave mixing is demonstrated for cadmium. The cadmium ions react with dithizone to form a cadium complex which is then extracted in carbon tetrachloride and analyzed. A relatively low-power argon ion laser line at 514.5 nm is used as the excitation light source. This nonlinear laser method offers many useful features including efficient and simple optical signal detection (signal is a collimated coherent beam), excellent detection sensitivity for absorbance, and efficient use of low laser power levels, small laser probe volumes and short analyte path legnths (e.g., ~0.5 mm). A detection limit of 7 fg or 0.05 ng/ml for cadmium, corresponding to an absorbance detection limit of 1.8 x 10mh AU is reported using a flowing analyte cell at room temperature.
1. INr~0nUcrt0N O~ICAL phase conjugation by degenerate four-wave mixing (D4WM) is one of the most useful and versatile nonlinear spectroscopic methods with many applications [l-4]. A phase conjugate signal beam is generated in a D4WM optical setup when a nonlinear medium (that is, an analyte) is illuminated by two exactly counter propagating pump beams (forward pump Ef and backward pump E,,), and a probe beam Ep that intersects with the pump beams at a small angle (that is, < 0.5”). Interaction of the input beams in the nonlinear medium produces a phase conjugate signal beam that retraces back the incident probe beam. Physical processes responsible for the generation of the signal beam include molecular orientation [l], change in population equilibrium [5], thermally induced refractive index change [6], and electrostriction [7]. In an absorbing liquid medium, the nonlinear effect produced by thermally induced refractive index change accompanies and often dominates other nonlinear effects. After light is absorbed by an analyte, the heat generated by analyte nonradiative relaxation results in periodic temperature and density fluctuations, and hence, in spatial modulation of the refractive index of the analyte medium. This refractive index grating diffracts one of the input beams (i.e., Eb) and produces the phase conjugate signal beam that is an exact time-reversed replica of the probe beam. Nonlinear spectroscopy based on D4WM has been demonstrated as a sensitive analytical method using different atomizers including low-pressure hollow-cathode discharge cells [g-lo], liquid flow cells [ll, 121, and analytical flame atomizers [13-161. While gas-phase atomizers yield sub-Doppler spectral resolution that is suitable for hyperfine structure analysis, liquid flow cells offer convenient detection of analytes at attomole-level detection sensitivity at room temperature [ll]. Since the D4WM signal is a collimated coherent beam and it is visible to the naked eye, optical alignment is convenient and detection sensitivity is excellent even when the excitation wavelength is more than 100 nm away from the absorption wavelength maximum [12]. In addition * This paper was published in the Special Honor Issue of Spectrochimica Acta Purr B, dedicated J. D. Winefordner. t Current address: Alliance Pharmaceutical Corp., San Diego, California, U. S. A. $ Author to whom all correspondence should be addressed. 1483
to
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ZHIQLANC Wu el al.
to backward-scattering D4WM (B-D4WM) methods mentioned above, one can also use a forward-scattering D4WM (F-D4WM) setup [17], where the signal beam is generated to propagate in the same direction as that of the probe beam (i.e., forward direction), as compared to the opposite direction signal propagation as in a B-D4WM method. While a B-D4WM optical setup offers sub-Doppler spectral resolution (due to counter propagating pump beams), and less background scattering noise, a F-D4WM method yields a simpler optical setup. In this paper, we report backward D4WM as a sensitive method for measuring small absorbance values in short analyte path lengths, using dithizone as an organic chelating reagent for cadmium. Dithizone (diphenylthiocarbazone) and azo dye reagents have been widely used by many workers to increase detection sensitivity and selectivity in spectrophotometric determination of trace amounts of metals. Dithizone is one of the most important reagents, especially useful for trace-concentration detection of elements such as cadmium, lead, zinc, mercury, silver, and copper [18, 191, since these elements form intense color complexes with dithizone. Molar absorptivities of metal-dithizone complexes in Ccl, are in the range from 3 x lo4 to 9 X lo4 M-‘cm-‘. In addition, high partition coefficients of metal-dithizone complexes in organic solvents, such as CHC& and Ccl,, allow preconcentration and separation by solvent extraction, and hence offer enhanced detection sensitivity and selectivity. The backward-scattering D4WM signal intensity, I,, can be described as shown below for trace concentration analyte solutions (i.e., low absorptivity) [6, 111. 2
(1)
where I,, I,, Z,, and Z, represent beam intensities of the phase conjugate signal beam, the forward pump beam, the backward pump beam, and the probe beam, a is the analyte absorption coefficient, L is the path length of the sample cell, 8 is the angle between the forward pump beam and the probe beam, (dn/dT), is the derivative of the refractive index with respect to temperature at constant pressure, p0 is the equilibrium solvent density, C, is the specific heat at constant pressure, and X is the excitation wavelength. Equation (1) describes important characteristics of the D4WM signal including its cubic dependence on laser intensity, its quadratic dependence on absorption coefficient, and hence on analyte concentration, and its quadratic dependence on (dn/dT),. Since the D4WM signal has a quadratic dependence on analyte absorption coefficient, detection sensitivity can be significantly improved by enhancing analyte molar absorptivity using organic chelating reagents. Taking advantage of these unique nonlinear properties of the D4WM method, ultrasensitive determination of cadmium is demonstrated using the Cd-dithizone complex. In a strongly alkaline solution, cadmium ions react with dithizone to form a pinkcolored complex with a maximum absorption at 520 nm. The complex is then extracted in carbon tetrachloride and measured by D4WM detection method. Using the 514.5 nm line of an argon ion laser as the excitation source, and a laser probe volume of 0.14 ~1, a cadmium detection limit of 7 fg or 0.05 ng/ml (S/N = 2), corresponding to an absorbance-unit detection limit of 1.8 x lO-‘j AU. is determined.
2. EXPERIMENTAL Figure 1 shows a backward-geometry D4Wh4 experimental arrangement where an argon ion laser (Coherent, Palo Alto, CA, Model Innova 90-6) is used as the excitation light source. A polarizer is used to prevent the returning pump beams from entering the laser cavity. The laser beam is first split by a 70/30 beam splitter, and the transmitted beam is used as the backward pump beam I$,. The reflected beam is further split by a 50/50 beam splitter to produce the forward pump beam Ef and the probem beam E,. A half-wave plate is inserted in
Sensitive absorbance measurement
Fig. 1. Experimental arrangement for backward-geometry tion method for Cd(II)-dithizone/CCI.
1485
method
degenerate four-wave mixing detecanalyte solution.
backward pump beam path to rotate its polarization plane by 90”. The D4WM signal generated in the analyte cell retraces back the probe beam path, where a beam splitter is used to steer the signal beam into a photomultiplier tube. A mechanical chopper is used to modulate the amplitude of both the forward pump beam and the probe beam. Another polarizer, cross polarized with the polarization plane of the forward pump beam and that of the probe beam, is placed in front of the photomultiplier tube to suppress the background scattering noise caused by the forward pump beam and the probe beam. The D4WM signal from the photomultiplier tube is amplified by a lock-in amplifier after it passes through a current-to-voltage converter. The output voltage of the lock-in amplifier is recorded by a chart recorder and digitized by a persona1 computer. The absorption spectra of dithizone and cadmium-dithizone complexes are also measured by a conventional UV-visible spectrophotometer, as shown in Fig. 2. Cadmium (II) stock solution (1 ppm) is prepared by dissolving cadmium (II) chloride in a
the
0.6
04 : 5 e q0.2
0.0
I
I ,
400
b
I
600
1
600
1
700
WAVELENGTH (n “‘)
Fig. 2. Absorption spectra measured by a conventional UVNis spectrophotometer (a) extracted blank solution, (b) extracted cadmium-dithizone in CCL and (c) 0.601% dithizone in Ccl,.
1486
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0.1 M HCl solution. Sodium hydroxide (semiconductor grade), hydrochloric acid (ACS reagent), and carbon tetrachloride (spectrophotometric grade) solutions are purchased from Aldrich. Extraction of cadmium-dithizone complex is performed using a standard procedure [18].
3. RESULTSAND DISCUSSION As shown in Eqn (l), the D4WM signal has a cubic dependence on laser intensity, a quadratic dependence on absorption coefficient, and a quadratic dependence on (dn/ dZ’),. One could take advantage of these nonlinear properties in order to enhance signal strength and detection sensitivity. For instance, due to the cubic dependence of signal on laser power, high photon density available from even a relatively weak laser beam could significantly boost the signal strength. In addition, since the background noise has only a linear dependence on intensity and the signal has a cubic dependence, signal-to-noise enhancement is very significant when the laser intensity is increased. Quadratic dependence of signal on absorption coefficient could be viewed advantageously or disadvantageously depending on whether the analyte concentration is increasing or decreasing. Nevertheless, any disadvantages due to low absorptivity or concentration could be more than compensated for by the pure strength of the “coherent” signal beam, its virtually 100% collection efficiency, and its cubic dependence on laser intensity. Taking advantage of the quadratic dependence of signal on analyte absorption coefficient, signal strength could be easily enhanced by simply improving analyte molar absorptivity using an appropriate complexing agent. One could also improve the signal strength simply by choosing a solvent with an optimum (dn/dT), value, since the signal also has a quadratic dependence on (dnldZ&. In order to enhance the absorbance detection limit for cadmium, one could use a cadmium-complex system with high molar absorptivity, a chelating agent with very low molar absorptivity at the excitation wavelength, and a solvent with a large (drill d7& value. Figure 2 shows a conventional UV/Vis spectrophotometer spectra of cadmium-dithizone complex (1 X 10e5M), dithizone in carbon tetrachloride, and a blank extraction solution. Cadmium ions react with dithizone to form an intensely colored complex with a maximum molar absorptivity of 8.8 X 10P4 M-‘cm-’ at a Amax of 520 nm [18]. Dithizone in CC& shows two maximum absorption peaks, at 620 nm and 420 nm, and a minimum absorption at 510 nm between the two absorption peaks. Carbon tetrachloride has a large (dn/dT), value, and hence, it is very suitable for use as a solvent in a D4WM detection method. A high level of blank absorption could limit detection sensitivity in a D4WM metal analysis especially at trace concentration levels, since a “blank” D4WM signal could be generated by the absorbing blank solution. To maximize the analyte D4WM signal and to minimize the background signal, one should use a laser excitation wavelength that is as close as possible to the absorption line center of the metal complex, and to the minimum absorption wavelength of the chelating reagent. For instance, the 514.5 nm line of an argon ion laser is suitable as an excitation source, since it is close to the maximium absorption peak of cadmium-dithizone complex at 520 nm and to the minimum absorption wavelength of the chelating reagent. As in other ultrasensitive spectrometric methods, impure solvents and chemicals could be a source of blank absorption, since some of the elements that might be present in the solvent could also form color complexes with the chelating reagent used. As expected, the D4WM signal amplitude, observed in a blank solution prepared by using ACS grade NaOH and Ccl4 solvents, is comparable to that of a 5 ppb cadmium solution. However, the D4WM signal amplitude, observed in blank solution prepared by using semiconductor-grade NaOH and spectrophotometric-grade Ccl, solvents, corresponds to the signal level of only 0.01 ppb cadmium solution. Hence, high-purity grade chemicals are used when trace-concentration detection sensitivity is desired. At trace-concentration detection levels, background absorption of organic chelating reagents should be also avoided. Although the 514 nm argon ion laser line is located near the maximum absorption wavelength of cadmium-dithizone complex,
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a small absorption by dithizone at 514 nm could still generate some “blank” D4WM signal, and hence, it is desirable to use as small an amount of dithizone as possible. When the concentration of cadmium is less than 10 ppb, a dilute dithizone solution (0.0002%) could be used in the D4WM method. In a strong alkaline solution (e.g., 5% NaOH), cadmium ions form stable complexes with dithizone while common metal dithizone complexes such as those of lead, zinc, and bismuth do not exist. Hence, it is an effective way to improve selectivity and reduce blank absorption. As shown in Fig. 2, there is no measurable absorbance by a conventional spectrophotometer in a blank solution extracted from a strong alkaline solution using 15 ml of a 0.0002% dithizone/CCl, solution. The D4WM signal strength can be improved significantly by optimizing some D4WM experimental parameters, such as the angle between the forward pump beam and the probe beam, the path length difference of the forward pump beam and the probe beam, and the intensity distribution ratio of the input beams. Background optical scattering from the input beams can be suppressed also by using an optimum angle between the input beams (and hence, affecting the relative positions of the signal and input beams) and appropriate spatial filters to allow only the coherent collimated signal beam to pass through to the detector, and by minimizing thermal lensing or blooming as described below. In an absorbing solution, thermal gratings are formed following the absorption of light through electronic transitions of the analyte molecules. Heating via radiationless relaxation of the optically excited molecules gives rise to periodic density and temperature fluctuations that result in the spatial modulation of the refractive index of the nonlinear medium (i.e., the analyte). At the same time, heating produced by nonradiative relaxation of excited species also yields thermal lens effects, where the absorbing medium acts like a diverging lens [20]. This thermal blooming effect could interfere with the D4WM signal measurement, since thermal blooming of the backward pump beam causes its expanding beam spot to overlap with the phase conjugate signal beam spot, especially when a small angle between the forward pump beam and the probe beam is used. Optical background noise caused by thermal lensing or blooming could be effectively suppressed by the use of a mechanical chopper-based amplitude modulated detection scheme, since the chopper effectively disrupts the pump beam, and hence, does not allow sufficient time to expand and interfere with the signal beam spot. As reported previously [21], the thermal lens signal decreases rapidly at chopper frequencies higher than 2 Hz. The D4WM signal can be collected at a higher modulation frequency, at which thermal lens background signal is at a minimum. The D4WM signal for the cadmium solution is strong and stable when the modulation frequency is 30 Hz or less. As the modulation frequency is increased much higher, less time is allowed for the D4WM gratings to form, and hence, the signal decreases gradually. Nevertheless, the S/N remains reasonably high at higher modulation frequencies (up to 200 Hz studied), since the background noise is more effectively suppressed by the lock-in amplifier at a higher modulation frequency, and also because optical noise originating from E,, thermal blooming is also insignificant at a higher frequency. Hence, a reasonably wide useful range of modulation frequency is available. The optical background noise caused by thermal lens effects could also be suppressed effectively by using an analyte cell with a short path length, since the D4WM laser probe volume (and effective path length) is already short. As expected, a cell with a long path length (e.g., 10 m) yields more background thermal blooming interference than a cell with a shorter path length (e.g., 0.5 mm). Optical background noise caused by thermal lensing/blooming can be further suppressed by chopping both the forward pump beam and the probe beam, instead of just chopping the probe beam. Experimental results show that the D4WM signal is also stronger when both Ef and E, are chopped as compared to when only Ep is modulated. Signal-to-noise ratio improvement is determined to be by a factor of 2.7 when both the forward pump beam and the probe beam are chopped as compared to when only the probe beam is modulated. As expected, a cubic dependence of signal on laser power is observed with a slope
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of 3.05 in a log-log signal vs power plot. Based on this very efficient nonlinear dependency, one could take advantage of the high photon density available even from relatively weak inexpensive lasers. When relatively high laser power levels (e.g., >l W) are used, especially for the Eb beam, overheating of the analyte solution could generate more thermal blooming background noise, which could overlap with the signal spot when observed on a screen along the signal propagation path. Hence, it is desirable to distribute more laser power to the Ef and the E, beam, and less to the Eb beam. For example, a beam intensity distribution ratio of 5:3:1 for 1&l, would yield less background noise than when a ratio of 1: 1: 1 is used. As expected, a quadratic dependence of signal on analyte concentration is observed with a slope of 1.9 in a log-log signal vs concentration plot, using independent extractions of small amounts of cadmium. Quantitative measurement is still straightforward in this “nonlinear” method, since the slope is simply two instead of one as in conventional “linear” detection methods. A cadmium concentration detection limit (S/N = 2) of 0.05 ng/ml is determined in the extraction solution when a forward pump beam power of 0.35 W, a probe beam power of 0.25 W, and a backward pump beam power of 0.075 W are used. Since the laser probe volume is 0.14 ~1, the cadmium mass detection limit by D4WM is determined to be 7 fg inside the analyte probe volume. This corresponds to an absorbance-unit detection limit of 1.8 x 10e6 AU for cadmium. This detection sensitivity is still limited by blank absorption due to impurities in solvents and chemicals used, and the cadmium detection limit could be further improved by using higher purity reagents, and more effective masking agents in a cleaner laboratory environment. Since short analyte path lengths are advantageously used in a D4WM setup, the absorbance detection limit of 1.8 X 1OV AU for cadmium is especially good and compares well with other laser-based analytical methods including that of a thermallens based method [21] (i.e., 5 x lop6 AU) while using only a single laser instead of two lasers. In addition, the experimental setup is relatively inexpensive and simple in this one-color one-laser nonlinear analytical spectroscopic method, as compared to two or more lasers used in many multi-beam laser methods. The analyte cell in the D4WM detector is very simple, and the analyte solution can be conveniently introduced through the flow cell at room temperature. Some disadvantages one might consider for this nonlinear detection method include the use of two laser beams (from the same laser), and hence, slightly more complicated optical alignment as compared to the use of simply one beam in a conventional laser method, and the need to suppress and filter optical noise originating from the input beams. However, when compared to other multi-photon laser methods, the optical alignment is relatively simple and the laser requirement is minimum (i.e., one-color one-laser system as compared to twocolor two-laser systems as in other laser methods). One could use a two-input-beam forward-scattering D4WM setup to simplify the optical alignment, and use a threeinput-beam backward-scattering D4WM setup to minimize source light scattering and interference. 4. CONCLUSIONS The use of a simple nonlinear method based on laser four-wave mixing is demonstrated for enhanced absorbance measurement of metals using complexing agents. This pump-probe cross-beam laser method offers useful features including efficient and simple optical signal detection (signal is a collimated coherent beam), excellent absorbance-unit detection sensitivity, and efficient use of low laser power levels, small laser probe volumes and short analyte path lengths (e.g., < 0.5 mm). Hence, it is especially suitable for on-column absorbance detection in capillary chromatography and capillary electrophoresis systems, where the analyte path length is very short. Furthermore, since laser power requirements in a one-laser D4WM method is low, many inexpensive compact lasers could be used. Since D4WM detectability is excellent both for fluorescing and nonfluorescing analytes, this nonlinear
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laser method offers many potential applications. Ultrasensitive detection of cadmium as demonstrated above could be obtained similarly for other metals, non-metal elements, and organic molecules, such as amino acids. Acknowledgement-We gratefully acknowledge partial support of this work from the National Institute of General Medical Sciences, National Institutes of Health under grant No. 5-ROl-GM41032, the National Science Foundation under grant No. CHE-8719843 and Beckman Instruments, Inc.
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