Anal. Chem. 1980, 52,
Consideration must be given to the instrument's portability if it is to be used for field studies utilizing a mobile laboratory. In this laboratory, Perkin-Elmer has proven reasonably portable. The Tracor is physically the smallest of the three but exhibits some operating difficulties in the best of laboratory conditions. The Varian is designed primarily as a laboratory research instrument but presumably is rugged enough to be used for field studies. Conclusions a n d Recommendations. It is evident from the data presented that actual experimental parameters may vary from the manufacturer's specifications as a result of laboratory conditions or differences in individual instruments. Therefore, it should be standard practice to characterize each F P D before placing any confidence in resulting data. The choice of an FPD system should be based upon careful consideration of individual laboratory needs and budgetary constraints. To select the instrument with the lowest detection limit would be unwise if the linear range does not extend to the concentration levels pertinent to a particular application. Additionally, instrumental options, such as other detectors, injection systems, and capillary accessories should be considered before purchasing.
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(3) Pearson, C. D.; Hines, W. J. Anal. Chem. 1977, 4 9 . 123-126. (4) Desouza, T. L. C.; Lane, D. C.; Bhatia, S. P. Anal. Chem. 1975, 47, 543-545. (5) Baumgardner, R. E.; Clark, T. A.; Stevens, R. K. Anal. Chem. 1975, 47, 563-566. ... .... (6) Bruner, F.; Liberti, A.; Possanzini, M.; Allegrini, I. Anal. Chern. 1972, 4 4 . 2070-2074. (7) Stevens, R . K.; Mullk, J. K.; O'Keefe, Krost, K. J. Anal. Chem. 1971, 4 3 , 827-831. (8) Stevens, R. K.: O'Keefe, A. E.; Ortman. G. C. Envlron. Scl. rechnol. 1969, 3, 652-655. (9) Gangwal. S . K.; Wagoner, D. E. J. Chromatogr. Scl. 1979, 77, 196-201. (10) Farwell, S . 0.;Rasmussen, R. A. J. Chromatogr. Scl. 1976, 74, 224-234. (11) Mizany, A. E. J . Chromatogr. Scl. 1970, 8 , 151-154. (12) Burnett. C. H.; Adams, D. F.; Farwell, S. 0. J . Chromatogr. Sci. 1978, 76, 68-73. (13) Sugiyama, T.; Suzuki, Y.; Takeuchi. T. J. Chromatogr. Scl. 1973, 7 1 , 639-641. (14) Shiftman, S.; Frank, C. W. Anal. Chem. 1974, 4 6 , 1804-1887. (15) Patterson, P. L.; Howe, R. L.; Abu-Shumays, A. Anal. Chem. 1978, 50, 339-344. (16) Patterson, P. L. Anal. Chem. 1978, 50, 345-348. (17) Patterson, P. L., private communication, Varian Associates, Walnut Creek, CA, Oct 1978. (18) Sugiyama, T.; Suzuki, Y.; Takeuchi, T. J . Chromatogr. 1973, 77, 309-316. (19) Burnett, C.H.; Adams, D. F.; Farwell, S. 0. J . Chromatogr. Sci. 1977, 75, 230-232.
ACKNOWLEDGMENT T h e authors wish to thank Mr. Raymond Michie for his technical assistance.
RECEIVED for review April 22, 1980. Accepted July 28, 1980. This work was presented at the 178th National Meeting of the American Chemical Society, Washington, DC, Sept 1979, and was supported in part by the Fuel Process Branch, U.S. Environmental Protection Agency, Research, Triangle Park, NC, under Grant No. R8040979.
LITERATURE CITED (1) Brody, S. S.; Chaney, J. E . J . Gas. Chromatogr. 1986, 4 , 42-46. (2) Pescar, R. E.; Hartman, C. H. J. Chromatogr. Sci. 1973, 11, 492-502.
High Repetition Rate Atmospheric Pressure Nitrogen Laser for Lifetime Measurements Totaro Imasaka and Nobuhlko Ishibashi" Faculty
of Engineering, Kyushu University, Hakozaki,
Fukuoka 8 12, Japan
organic molecules in the condensed phase (7 N 0.1-10 ns). Though a sophisticated design of a dye laser cavity enables the generation of short dye laser pulses (5-8),the nitrogen laser with subnanosecond pulses might be more useful for the generation of the shorter dye laser pulses and for the use of the nitrogen laser itself as an exciting source. The direct generation of the subnanosecond pulses (0.05-1 ns) from the nitrogen laser has been reported by several authors on the basis of transversely excited atmospheric (TEA) discharge excitation (9-18). However, very few applications of this TEA nitrogen laser to fluorometry have been reported. A sensitive photodetection system such as a photon counter combined with a repetitive pulse laser is useful for ultratrace analysis (19). However, low repetition rate operation of the nitrogen laser (-20 Hz) requires a long time for recording a spectrum or a fluorescence decay curve, and it limits the convenient use of the apparatus. A higher repetition rate nitrogen laser is commercially available (- 100 Hz); however, it requires a large amount of nitrogen gas and a high-flow-rate vacuum pump to supply fresh ion-free gas to the laser for each pulse. Through the use of a closed-cycle transverse gas flow system, operation of a nitrogen laser has been demonstrated at the repetition rate up to 1200 Hz (20). If the system could be simplified, this type of the laser might be useful for practical use. A transverse gas flow, TEA nitrogen laser might be very
An output of 24 mW of average power at 337.1 nm is obtained from an atmospheric pressure nitrogen laser Operating at a repetltlon rate of 600 Hz through the use of a transverse gas flow system to supply fresh ion-free nitrogen gas. A laser fluorometrlc system for the lifetime measurement of nanosecond fluorescence decay is constructed by using this nitrogen laser as an exctting source and comblning wlth a fast response photomultipller. The increase of the fluorescence quantum yleld of the zinc 5-sulfo-8-quinolinolate complex by the surfactant of Zephiramine is concluded to originate partlally from lengthening the fluorescence llfetime (3.6 5.0 ns). The use of the organic solvent of 1,2-dichloroethane for the measurement of ANS-Zephiramlne Increases the fluorescence intenslty and lengthens the fluorescence lifetime.
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Nitrogen lasers and nitrogen laser pumped dye lasers have found wide-spread appications as short intense light sources for an ultratrace analysis of organic molecules (1-4). This short pulse is essential for the application of lasers for time-resolved fluorometry. The usual nitrogen laser, which is operated a t the reduced nitrogen pressure (-50 torr), has a pulse width of several nanoseconds (3-10 ns), and it is not too short for the study of time-resolved fluorometry of the 0003-2700/80/0352-2083$01 .OO/O
C
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
'I
t'
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Figure 1. (A) Schematic diagram of the TEA nitrogen laser. (B) Schematlc diagram of the discharge channel and the hydrogen pressurized trigger gap switch.
promising for time-resolved fluorometry because of its short pulse width and its high-repetition-rate operation. Furthermore, the laser is compact and can be operated without a vacuum pump. The construction of such a laser has not, t o the best of our knowledge, been reported. This paper describes the construction and performance characteristics of a compact, transverse gas flow, TEA nitrogen laser and presents a few applications to lifetime measurements studies. In the fluorometric determination of inorganic ions by a chelate reaction, fluorescence enhancement effects can be used for the trace analysis. The convenient use of a surfactant, which makes micelle in water, provides a useful analytical method, since the micelle reduces the interaction between the solute and solvent and induces strong fluorescence. Most of the fluorescence enhancement studies have been performed by measuring the spectral shift and the enhancement of the fluorescence intensity of the complex with the surfactant. Meanwhile, many successful investigations of the interaction between dyes and surfactants have been carried out by measuring the change of the lifetime of the dyes with surfactants. I t implies that information concerned with the mechanism of the fluorescence enhancement can be obtained by measuring the change of the lifetime of the complex with the surfactant. Alternatively, solvent extraction is also used in order to increase the sensitivity of the fluorometric analysis, since the fluorescence intensity of the sample in the organic phase is usually stronger than that in the aqueous phase. This paper shows preliminary studies of the fluorescence enhancement effect of the surfactant and solvent extraction by the lifetime measurement.
EXPERIMENTAL SECTION Nitrogen Laser. A basic element of the TEA nitrogen laser is a parallel plate transmission line of the Blumlein type as shown in Figure 1. Primary ceramic capacitors (30 kV, 5 nF X 2) are charged to the supply voltage, usually 13 kV, and then discharged by a hydrogen pressurized (2 kg/cm2) spark gap switch (A). This allows resonant charging of the secondary Blumlein capacitors (B) and (C) (0.25 mm thick Mylar sheet: 1.8 nF), which can
instantly reach a voltage up to twice the primary voltage. Therefore, this resonant charging system not only requires half voltage for the power supply but also reduces the possibility of the breakdown of the thin Mylar sheet, which provides a low characteristic impedance of the transmission line to achieve very fast discharge excitation. To meet the required conditions for a very fast population inversion, a surface of the laser has a parabolic configuration (21). A small (14 mm i.d.) hydrogen pressurized (2 kg/cm2) spark gap switch (B),which locates at the focus of the parabola, is used to reduce impedance of the system. The discharge electrodes, 30 cm long, are made from aluminum of 10 mm thickness and 13 mm width with a semicircular profile ( R = 6.5 mm) and are screwed onto the Blumlein capacitors of aluminum plate of 10 mm thickness. The electrode separation of 4 mm is used. The electric discharge in atmospheric conditions gives rise to arc formation. In order to reduce this undesirable arc formation, a dc-glow discharge stabilization technique is introduced for the preionization (12).The sharp points of the resistor wires shown in Figure 1 lead to high electric field strengths, and they extract free carriers from the electrode. Therefore, this preionization acts as pilots for the formation of the main discharge glow. A rather uniform discharge along the laser channel is obtained by a row of 70 resistors (1MQ) in parallel. High-repetition rate operation is made possible through the use of transverse nitrogen gas flow between the discharge electrodes. A brass tube with a row of 150 holes (1mm id.) is placed below the electrodes and supplies fresh ion-free nitrogen gas to the laser channel for each pulse. The nitrogen gas can be recycled by using an air pump (34 L/min). No laser cavity resonator is used in order to reduce the pulse width of the laser. A larger laser transmission line would store more electrical energy but would add very little extra energy to the laser pulse. Therefore, the laser is extremely compact excluding a power supply; the entire laser has a physical dimension 51 cm long by 33 cm wide by 9 cm high. The nitrogen laser is shielded against a large quantity of the radio frequency interference (RFI) by a steel box. The output power of the laser was measured by a Molectron 53-05 DW pyroelectirc joulemeter. Lifetime Measurement. Fluorescencefrom a sample solution irradiated by the nitrogen laser was focused onto an entrance slit of a CT-40D double monochromator equipped with an HTV R905 photomultiplier. The fifth dynode of the photomultiplier was employed as the anode to improve the impulse response time (22). A power supply voltage of 1750 V was typically used. A fluorescencedecay curve was directly displayed on a synchroscope (IWATSU SS 5321; 250 MHz) and photographed. An exposure time of 15 s was typically used and then lo3 shots of fluorescence decay superimposed were observed in an oscillogram. Sample Preparation. A zinc 5-sulfo-8-quinolinolatesolution was prepared according to ref 23; the following solutions were added to a 100-mL volumetric flask in turn: 40 pg of zinc ion solution, 4 mL of 2 X lo4 M 5-sulfo-8-quinolinolsolution, 10 mL of 1 M ammonium acetate buffer solution, 10 mL of M tetradecyldimethylbenzylammoniumchloride (Zephiramine), and distilled water. The pH of the solution was adjusted to 6.9. The solution of the ion pair of 8-anilino-1-naphthalenesulfonic acid (ANSI-Zephiramine was prepared as follows. The aqueous solution consists of 5 X lo4 M of ANS and Zephiramine. The ANS-Zephiramine was extracted to 150 mL of 1,2-dichloroethane from the 150 mL aqueous solution consisting of 6 mL of 2.5 X M ANS, 10 mL of 2 M MgC12,0.75 mL of M Zephiramine, and distilled water.
RESULTS Nitrogen Laser. The operating characteristics of the transverse gas flow, TEA nitrogen laser are indicated in Figure 2. For economical use, the nitrogen gas should be recycled. A transverse gas flow by the air pump makes stable laser oscillation possible u p to 140 Hz. An output power of 0.1 mJ/pulse is achieved at these operating conditions. If the pulse width of the laser is assumed to be 0.5 ns (13,the peak power of the nitrogen laser can be estimated to be 200 kW. In spite of the use of a free-running spark gap switch, the output power of the laser was stable and observed variation of the output power was 10%. When the present laser was
ANALYTICAL CHEMISTRY, VOL. 52, NO. 13, NOVEMBER 1980
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Figure 3. Oscillogram of the TEA nitrogen laser.
Figure 2. Output power and repetition rate of the TEA nitrogen laser (20 pJ/div): (A) nitrogen gas is recycled by an air pump (34 L/min); (B) nitrogen gas is not flowed: (C) fresh nitrogen gas is flowed at the high flow rate (several hundreds L/min).
operated without the air pump, the decrease and instability of the output power were observed as shown in Figure 2B. By use of a higher flow rate of the nitrogen gas directly from a cylinder (several hundred L/min), operation of the nitrogen laser up to 600 Hz was achieved. A longitudinal flow, TEA nitrogen laser has been reported to be operated at the repetition rate of 125 Hz (IO). The present results indicate the distinct advantage of the transverse gas flow system for highly repetitive operation of the TEA nitrogen laser. In this study, the repetition rate of the nitrogen laser was limited by the flow rate of the nitrogen gas. If high repetition rate operation more than 140 Hz is required for a long time, nitrogen gas should be recycled by using a high-flow-rate air pump for the economical use of this transverse flow laser. The laser can be operated for several hours without any adjustment at the repetition rate of 100 Hz. After 3 h of operation, a small amount of powder deposited inside of the spark gap switch (A) made the laser oscillation unstable. After the spark gap switch was cleaned, the laser could be operated stably again as before. Because the lifetime of the spark gap switch (A) depends on total conducted charge, the repetition rate was adjusted to 50-140 Hz for long life operation as occasion demands. T h e use of a thyratron instead of the spark gap switch (A) might improve the reliability of the nitrogen laser; though the rise time of the thyratron switch is slower than that of the spark gap switch, the rise time of high voltage charging to Blumlein capacitors is limited by the resonance frequency of the Blumlein capacitors and coils to 1 MHz and not limited by the rise time of the switch. Later, we noticed that many spots developed on the edges of the electrodes after -50 h of operation, and this deterioration made the laser oscillation unstable (24). A pair of saw blade electrodes, made of steel, was also tested; however, a preliminary result showed that the output power of the laser was smaller than that with the round shape electrodes, because of the ununiformity of the main discharge. Though the smooth aluminum electrode can be easily recovered by cleaning the edge of the electrodes, a heavy-duty material might be desirable for the maintenance-free operation of the nitrogen laser. The steel box reduced the RFI noise to the synchroscope from the nitrogen laser down to several millivolts. This noise level allows even a single photoelectron detection by using a photomultiplier with a high gain for current amplification.
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Dye Laser. Laser oscillations of dye lasers when pumped by the present TEA nitrogen laser were investigated. T h e cavity of the dye laser was 4 cm long, which consists of a 3 cm dye cell, a quartz plate, and an aluminum mirror. The intense stimulated emissions were observed for three laser materials: 2-phenyl-5-(4-biphenylyl)- 1,3,4-0xadiazole(PBD), 2-(l-naphthyl)-5-phenyloxazole(a-NPO), and sodium fluorescein. By replacement of the aluminum mirror by a grating, a tuning capability of the present dye laser was investigated. As a result, the laser emission of a coumarin 500 dye was continuously tunable under a nearly grazing incidence configuration. This preliminary result shows that the TEA nitrogen laser pumped dye lasers have a potential to be used as subnanosecond light sources with very high wavelength resolution from the ultraviolet to visible region. Lifetime Measurement Apparatus. A typical oscillogram of the nitrogen laser pulses recorded by the detection system for the fluorescence lifetime measurement is shown in Figure 3. The profile of the nitrogen laser output can be clearly observed, and a time resolution of 1.8 ns is achieved. The pulse width of the exciting source of the nitrogen laser and the time response of the photomultiplier are estimated to be 0.5 and 0.7 ns (22),respectively. Then, the total time resolution of the fluorescence detection system is mainly limited by the bandwidth of the synchroscope (rise time, 1.4 ns). By use of faster signal processing equipment, a time resolution less than 1 ns might be achieved. The practical limit of the lifetime, which can be determined by using the present detection system, is considered to be 2 ns. For the samples with a shorter lifetime, deconvolution of the fluorescence decay curve might be useful; however, ringings observed in the oscillogram make it difficult to determine the precise lifetime of fluorescence decay. Though pulse-to-pulse instability of the exciting source is 1070,many shots are superimposed and averaged on the synchroscope during an exposure time of the photograph. Then, this averaging increases the reproducibility of the measurement, and the oscillograms of the fluorescence decay curves measured under same conditions are completely superimposed. The obsel-ved variation of the lifetime values for the samples seems to be originating mainly from the procedure in the sample preparation and partially from the errors caused by the ringings. The sensitivity of the fluorometer is estimated by the product of the sample absorbance ( A ) and fluorescence quantum yield (4) under the conditions of the detection limit (4). The present apparatus enables the lifetime measurement of the sample of Ad = 4 X lo4 under the fluorescence spectral resolution of 5 nm. Lifetime of Zinc 5-Sulfo-8-quinolinolate. A preliminary investigation of a fluorescence enhancement effect of a surfactant in a determination of a metal ion by a chelate reagent
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(B) Figure 4. Fluorescence decay curve of zinc 5-sulfd-quinolinohte (X, = 525 nm): (A) in the absence of the surfactant; (8) in the presence
of Zephiramine.
has been performed on the basis of the lifetime measurement of sample fluorescence. Figure 4 shows fluorescence decay of the zinc 5-sulfo-8-quinolinolate complex in the absence or presence of Zephiramine. The observed lifetime of 3.6 f 0.2 ns in the absence of Zephiramine was similar to the reported value (4.0 f 0.3 ns) (25). The discrepancy might be coming from the difference of the experimental conditions used (excitation wavelength, sample preparation, etc.). The longer lifetime of 5.0 ns was obtained for the sample solution including the surfactant. This result is consistent to the fact that the fluorescence quantum yield increases by a factor of 1.9 by the formation of the micelle of Zephiramine in the solution (23). Further systematic information such as the composition and stability constants of the complex may provide detailed information for the mechanism of the fluorescence enhancement effect by the surfactant. Lifetime of ANS-Zephiramine. A general characteristic of ANS has been known to have a very low fluorescence quantum yield in an aqueous solution and strong fluorescence in an organic phase. Fluorescence decay of the ion-pair solution of ANS-Zephiramine is indicated in Figure 5 . In the aqueous phase, the fluorescence intensity is very weak and has a short lifetime. Alternatively, the ANS-Zephiramine ion-pair extract to 1,2-dichloroethanefluoresces strongly and shows long fluorescence decay (11ns). This result indicates that the strong fluorescence enhancement by solvent extraction is partially coming from the decrease of the fluorescence quenching.
DISCUSSION Many designs of a TEA nitrogen laser have been reported to have very short pulses. However, few publications indicate its performance with respect to stability and reliability of the laser, which is considered to be important for the analytical application of the laser. The present transverse gas flow, TEA nitrogen laser has several advantages over previously reported instruments: (I) Transverse flow of the nitrogen gas increases stability of the ouput power a t a high repetition rate and enables the laser oscillation up to 600 Hz. (2) The resonance charging system reduces the possibility of the breakdown of the dielectric material in the capacitor, and it improves the reliability of the nitrogen laser. ( 3 ) Every element of the nitrogen laser can be taken apart, so that it is easy to repair and reconstruct. T h e combination of the TEA nitrogen laser with the high-speed photodetector of a modified photomultiplier provides a simple lifetime measurement system with a good
Figure 5. Fluorescence decay curve of ANS-Zephiramine (X,= 470 nm): (A) in water, (B) in 1,2dichloroethane.
time resolution. The stable and highly repetitive nitrogen laser enables the convenient use as the exciting source for the lifetime measurement and gives clear photographs even at a high writing speed of a synchroscope. In a conventional instrument for the lifetime measurement of nanosecond fluorescence decay, a flashlamp with a short pulse width has been used as an exciting source. By using a time correlation single photon detection system including a time-tuamplitude converter (TAC), we can achieve the precise determination of the fluorescence lifetime, since the repetition rate of the flashlamp is very high (20-200 kHz). However, the pulse width of the flashlamp is typically 2-6 ns, and it is not always short enough for the lifetime measurement of nanosecond decay. Though operation of a flashlamp with a very short pulse width (-0.8 ns) has been achieved by the careful adjustment of the conditions, it sacrifices the photon flux and the repetition rate (26).
A variety of lifetime measurement systems can be considered by using a short pulse, TEA nitrogen laser and its pumped dye laser. A streak camera has a very good time resolution and has been used for the measurement of the temporal profile of the TEA nitrogen laser pumped dye laser pulse (27). Such a system seems to be quite useful for the measurement of the samples with very short lifetimes. For the best use of the repetitive TEA nitrogen laser, it could be combined with a repetitive detection system, such as a sampling socilloscope or a boxcar integrator with a sampling head, rather than a single-shot detection system, which was carried out in this study. T h e application of the present transverse gas flow, TEA nitrogen laser to the time correlation singlephoton detection system is considered to be very promising because it has high sensitivity and good time resolution and because the TAC system requires a highly repetitive exciting source. The repetition rate of the nitrogen laser is limited to 600 Hz under the present operating conditions, and this repetition rate operation requires a long time for recording a fluorescence decay curve (1&100 min). However, it should be noted that such a lifetime measurement system is extremely sensitive. The combination of the flashlamp and the TAC has been used for the lifetime measurement under the sample M, that is A4 while laser concentrations of excitation will make it possible to measure the fluorescence or below. A lifetime under the conditions of A+ mode-locked, cavity-dumped argon ion laser and ita pumped dye laser are very useful for the lifetime measurement and have several advantages as a highly repetitive exciting source of the time correlation single photon detection system (28,
Anal. Chem. 1980, 52, 2087-2092
29). Such lasers exceed the present laser in performance of the pulse repetition rate and in average power. However, the transverse gas flow, TEA nitrogen laser and its pumped dye laser have distinct advantages with respect to the pulse energy per pulse, simplicity and a low cost for the construction. We should stress that a tunable wavelength region of the present system extends over from the ultraviolet to visible region while the mode-locked, cavity-dumped system is limited to rhodamine 6G dye region practically. In the ultratrace analysis, lasers are very useful as the exciting sources for the fluorometric analysis, since the fluorescence intensity is proportional to the intensity of the exciting source. However, laser fluorometry is so sensitive that the detection limit is not always determined by the sensitivity of the apparatus but determined by the background signal from contaminant fluorescence ( 4 ) . In order to reduce this solution blank, the temporal separation of the components has been achieved for the samples with a long fluorescence lifetime such as pyrene (30) and polycyclic aromatic hydrocarbons ( 3 I ) ,since the emission lifetime of the background signal is relatively short. The TEA nitrogen laser developed in this study s e e m to be promising for the temporal separation of the components with nanosecond fluorescence lifetimes.
ACKNOWLEDGMENT The authors wish to thank M. Ichishima for his assistance in the preparation of the sample solution. The authors also wish to thank F. E. Lytle for his helpful suggestions about the high-speed photomultiplier. LITERATURE CITED (1) Bradley, A. 6 . ; Zare, R. N. J . Am. Chem. SOC.1976, 98,620-621. (2) Geel, T. F. V.; Winefordner, J. D. Anal. Chem. 1976, 48,335-338. (3) Richardson, J. H.; George, S. M . Anal. Chem. 1978, 50, 616-620.
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(4) Ishibashi, N.; Ogawa, T.; Imasaka, T.; Kunitake, M. Anal. Chem. 1979, 57,2096-2099. (5) Lin, C.; Shank, C. V. Appl. f h y s . Lett. 1975,26, 389-391. (6) Cubeddu, R.; Polloni, R.; Sacchi, C. A. Appl. Phys. 1977, 73, 109-110. (7) Wyatt, R. Opt. Commun. 1978,26, 429-431. (8) Bor, Zs.;Opt. Commun. 1979, 29, 103-108. (9) Strohwald, H.; Salzman, H. Appl. Phys. Lett. 1976, 28, 272-274. (10) Bauer, R. K.; Kowalczyk, K. Opt. Commun. 1977,23, 169-170. (11) Bergmann, E. E. Appl. fhys. Lett. 1977,37, 661-663. (12) Hugenschmidt, M.; Vollrath, K. Opt. Commun. 1978, 26, 415-418. (13) Patel. B. S. Rev. Sci. Instrum. 1976, 49, 1361-1363. (14) Maeda, M.; Yamashita, T.; Miyazoe, Y. Jpn. J . Appl. Phys. 1976, 17, 239-240. (15) Hasson, V.; von Bergmann, H. M. Rev. Scl. Instrum. 1979,5 0 , 59-63. (16) Hugenschmidt, M.; Wey, J. Opt. Commun. 1979, 29, 191-196. (17) Cubeddu, R.; De Slkestri, S. Opt. Quantum Electron. 1979, 7 7 , 276-277. (18) Kobayashi, T.; Sugimura, K.; Inaba, H. Opt. Quantum Electron. 1979, 1 7 , 373-376. (19) Imasaka, T.; Ogawa, T.; Ishibashi, N. Anal. Chem. I97B,5 1 , 502-504. (20) Targ, R. IEEE J . Quantum Electron. 1972, QE-8, 726-728. (21) Gcdard, 0. IEEE J . Quantum Electron. 1974, QE- 70, 147-153. (22) Beck, G. Rev. Sci. Instrum. 1976, 4 7 , 537-541. (23) Kina, K.; Tamura, K.; Ishibashi, N. Bunseki Kagaku 1974, 23, 1404- 1406. (24) Wright, J. C., University of Wisconsin, personal communication. (25) Hiraki, K.;Morishige, K.; Nlshikawa, Y. Anal. Chim. Acta 1978, 97, 121-128. (26) Leskovar, 6.; Lo, C. C.; Hartig, P. R.; Sauer, K. Rev. Sci. Insfrum. 1978, 4 7 , 1113-1121. (27) Aussenegg, F.; Leitner, A,; Opt. Commun. 1980, 32, 121-122. (28) Spears, K. G.; Cramer. L. E.; Hoffland, L. D.Rev. Sc!. Insfrum. 1978, 49,255-262. (29) Koester, V. J.; Dowben, R. M. Rev. Sci. Instrum. 1978,49,1186-1191. (30) Kunitake, M.; Imasaka, T.; Ishibashi, N., submitted for publication in Nippon Kagaku Kaishi. (31) Dickinson, R. B., Jr.; Wehry, E. L. Anal. Chem. 1979, 5 7 , 778-780.
RECEIVED for review April 8, 1980. Accepted July 25, 1980. This research is supported by a Grant-in-Aid for Scientific Research (Grant No. 347054,575501) and for Environmental Science (Grant No 303046) from the Ministry of Education of Japan.
Fluorometric Determination of Secondary Amines Based on Their Reaction with Fluorescamine Hiroshi Nakamura * and Zenzo Tamura Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, 7-3- 1, Hongo, Bunkyo-ku,
A spectrophotofluorometric method has been developed for the determination of secondary amines by using fluorescamlne. When compounds having a secondary amino group were reacted with fluorescamlne at pH 12 and then heated at 70 OC for 10 min with a primary amine (L-Leu-L-Ala), they gave a bluish green fluorescence. By measurement of the fluorescence, most secondary amines could be determined in 0.5 nmol quantities. Relative standard deviations of 4.4 and 5.2 YO are observed for the analyses of 5 nmol of N-methylanlllne and sarcosine, respectively.
Scheme I
", 1
FI
11
0
Fluorescamine is known to react almost instantaneously with primary and secondary amines to give fluorescent pyrrolinones (FI) ( I , 2) and nonfluorescent aminoenones (FII) ( 3 , 4 ) respectively , (Scheme I). Fluorescamine itself is rapidly hydrolyzed to nonfluorescent products under reaction conditions ( I ) . We have previously reported both general (5-7) and selective (8-12) methods for the analysis of primary amino 0003-2700/80/0352-2087$01 .OO/O
Tokyo 1 13, Japan
Fluorescami n e
R'>" R2
aq?J COOH
FI I
groups with fluorescamine. In this paper, we plan to utilize fluorescamine for the fluorometric determination of secondary amines. We found that FII is readily converted by the reaction with primary amines to FI (Scheme 11). The reaction of 0 1980
American Chemical Society
