Anal. Chem. 1087, 59, 419-422
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High-Performance Liquid Chromatography with Supersonic Jet/Laser Fluorometric Detection Totaro Imasaka, Noriyuki Yamaga, and Nobuhiko Ishibashi* Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812,Japan
Anthracene derivatives are separated by conventlonal hlghperformance liquid chromatography and Introduced Into a contlnuowflow supersonlc jet nozzle. A carrler of methand Is malntalned at 200 O C and 40 atm, and It Is expanded from a plnched restrlctor Into a dlluent gas of argon. The mixed gas Is succeedhgly expanded from a plnhole Into a vacuum. The molecular specks are detected by a laser fluorometrlc system. Anthracene derlvatlves are selectively determlned by adjmtlng the excltatlon wavelengths to the pure electronk transitkns. The detecHon Hmn Is 40 ng for anthracene, whkh Is shnllar to those reported for naphthalene derlvatlves uslng a gas chromatograph equlpped wlth a supersonic jet/fluorometrlc detector.
In supersonic jet spectrometrythe sample molecule is cooled down to several kelvin, and clearly resolved features are observed in the spectrum. Therefore, selective determination of chemical species can readily be achieved from their sharp fluorescence and multiphoton ionization spectra (1,2).When the sample contains many chemical species, a separation procedure is necessary for identification of the compounds. A gas chromatograph equipped with a supersonic jet/fluorometric detector has already been reported by Small et al. (3). It is advantageousespecially for determination of samples with similar chemical structures such as isomers. We have also reported a gas chromatography system with a supersonic jet/multiphoton ionization detector (4),which has been applied to capillary gas chromatography (5). More recently, we have studied capillary gas chromatography/supersonic jet/ multiphoton ionization/mass spectrometry (6). These methods are useful for sensitive and selective determination of volatile chemical species. Most of the analytical samples are nonvolatile or thermally labile at a high temperature and should be measured in a solution phase. High-performance liquid chromatography (HPLC) has been used for separation of the sample species. Absorption or fluorescence detectors are frequently used with HPLC because of their high sensitivity. Unfortunately, these spectrometric techniques are not so selective especially for samples with similar spectral properties. A mass spectrometric detector has greater performance with respect to selectivity, but it still suffers from poor selectivity in assignment of isomers. The HPLC system equipped with a supersonic jet spectrometric detector may provide a new analytical tool because of its high selectivity. But such a study has not been reported previously since supersonic jet spectrometry is essentially an investigation for a gas-phase molecule. Many spectroscopists as well as analytical chemists are interested in application of supersonic jet spectrometry to nonvolatile samples. In 1984 Levy et al. tried to demonstrate supersonic jet spectroscopy for a nonvolatile molecule that ordinarily exists in a solution phase (7). They measured a multiphoton ionization spectrum for tryptophan by using a thermospray technique (8). However, they could only observe a nonresonant two-photon ionization (broad) spectrum. In 0003-2700/87/0359-0419$01.50/0
the later publication they reported observation of sharp, clearly resolved spectral features whose widths reflected their rotational contour (9). The technique they used involved forcing tryptophan in methanol through a heated capillary and spraying it into the channel, depositing tryptophan on the cylinder wall. The solution flow was then stopped, and a free jet was produced by pulsing argon or helium over the tryptophan on the hot (230 “C) wall. By their experiment they verified that the sample had not been introduced as a supersonic jet in their previous study and that tryptophan is not a nonvolatile molecule. However, we would like to emphasize that it is the first published report of trying to introduce a liquid sample to form a supersonic jet. Since 1983 we have also studied the application of supersonic jet spectrometry to nonvolatile samples. However, we were unsuccessful in observing a cold spectrum; the fluorescence spectrum was sometimes broad and sometimes structured but irreproducible. Later we found that the sample can be sufficiently cooled when it was expanded from a supercritical fluid into a vacuum. In 1985 we first reported this new sample introduction technique (10). We used perylene as a sample and used n-hexane, n-pentane, benzene, methanol, and C02 as a carrier, which were maintained at around supercritical conditionsbefore jet expansion. Lubman et al. also succeeded in the experimental work of pulsed supersonic jet expansion from supercritical C02. They observed a cold multiphoton ionization spectrum for acenaphthene, phenanthrene, and carbazole (11).Later they also reported the mass spectrum for tyramine, benzimidazole,and tryptophan using COzand N20 as superctitical fluids (12).Nishi et al. reported a similar sample introduction technique, that is, “liquid expansion method”, in which they expanded liquid acetonitrile from the continuous-flow nozzle (13). They also performed the spectroscopic works for formic acid (14)and l-methoxynaphthalene using this technique (15).However, the carriers are maintained a t a high pressure and temperature, and therefore their method is basically identical with our previous method. Unfortunately, they have used electron impact ionization/mass spectrometry,so that it is, at present, difficult to assert whether the sample molecule is cooled or not. In these investigations including our previous work, the researchers presented the advantage of their methods using volatile compounds by demonstrating their work at a sufficiently low temperature, where the vapor pressure can be assumed to be negligible at an atmospheric pressure. Supersonic jet spectrometry using a supercritical fluid for sample introduction may directly be coupled with supercritical fluid chromatography (SFC). For example, the components in the sample can be separated by a SFC column using a supercritical fluid as an eluent and be detected by supersonic jet spectrometry. This method is advantageous especially for selective determination of the thermally labile molecule. We tried to use C02as a carrier and SFC for sample separation. But, we had some difficulties in handling of C 0 2 and could not find a commercial column for this purpose. Most of the nonvolatile samples are determined by HPLC, and it is a more universal method than SFC. Many chemical 0 1987 American Chemical Society
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HPLC Column Nozzle
lniecter
I
to Vaccum Pumps
II
(PulseGeneratorH Transmitt-
Figure 1.
Supersonic jet spectrometer combined with HPLC.
species are already separated by a commercial column, and their applications are already published in scientific journals. Furthermore, careful adjustments of the pressure and temperature, which are essential in SFC, are unnecessary in HPLC. It is pointed out that methanol is frequently used as a carrier for HPLC and it is already used as a superctitical fluid for sample introduction in supersonic jet spectrometry (9). Therefore, HPLC might be directly coupled with supersonic jet spectrometry. In this study we report supersonic jet spectrometry of a thermally labile molecule. By careful optimization of the experimental conditions, the sample molecule is sufficiently cooled even when methanol is used as a carrier. We demonstrate selective determination of anthracene derivatives by combination of HPLC and supersonic jet spectrometry. We also presented the advantage of the supersonic jet detector in comparison with a conventional fluorometric detector for HPLC.
EXPERIMENTAL SECTION Apparatus. The experimental apparatus for HPLC combined with supersonicjet/laser fluorometry is shown in Figure 1. The instrument for supersonic jet spectrometry is reported in detail elsewhere (9) and briefly describedhere. The sample is introduced by a loop injector (Reodyne, Model 7125, 20 fiL) into a carrier supplied by a high-pressure pump (Shimadzu, LC-5A). The sample components are separated by a commercialcolumn (Toyo Soda, TSK-GEL,ODS-EWAQ and detected either by a supersonic jet system or by a conventional fluorescence spectrometer equipped with a xenon arc lamp (Hitachi, 204). In the former case, the stream is introduced into a nozzle through a narrow stainless steel tube restricted at the end. The carrier stream is maintained typically at 40 atm and 200 "C in the nozzle. The expanded sample gas from the capillary is mixed with argon at the nozzle port and succeedingly expanded from a pinhole into a vacuum. The exhaust gas is evacuated by a vacuum system consisting of a 6-in. diffusion pump equipped with a cold trap, which is followed by mechanical booster and rotary pumps. The pressure of the vacuum chamber is monitored by a pirani gauge (Ulvac,GP-2T). The exciting source is an excimer-laser-pumped dye laser, whose output power and line width are 3 mJ and 0.007 nm, respectively. The dye laser excites the sample molecule in the supersonic jet 10 mm away from the nozzle. Fluorescence is collected onto a slit of a monochromator equipped with a photomultiplier. The signal is measured by a boxcar integrator. Reagents. Anthracene was obtained from Tokyo Kasei Kogyo. Other polycyclic aromatic hydrocarbons such as 9-methylanthracene, 9-chloroanthracene, 1-chloroanthracene, 2-chloroanthracene, and perylene were purchased from Aldrich Chemical Co. A laser dye of phenylbiphenyl-1,3,4-oxadiazole(PBD) was supplied from Nakarai Chemical Co. RESULTS AND DISCUSSION Thermal Lability. Thermal stability of the sample was investigated by heating chemicals on a hot plate. Anthracene
was directly sublimated from the solid phase by increasing the temperature, and it was confirmed to be a volatile compound. Other anthracene derivatives (9-methylanthracene, 9-chloroanthracene, and 1-chloroanthracene) were melted on the hot plate and later vaporized at a higher temperature; they are also volatile compounds. On the other hand, 2-chloroanthracene decomposed and formed a black polymerized compound. It was found to be a thermally labile molecule. Therefore, advantage of the present method may be demonstrated by applying supersonic jet spectrometry to thermally labile 2-chloroanthracene. Optimization. For sample introduction, the top of the stainless steel tube should be properly restricted to maintain a sufficient pressure. When it was too restricted, the nozzle was easily clogged with small particles remaining in the stream, despite installation of a filter in the pump system. In our experiment the minimum flow rate was practically limited to 0.05-0.1 mL/min. The argon gas should be mixed as much as possible at the nozzle port for sufficient sample cooling, as described later. A large pumping system is required to increase the total flow rate, but the capacity of the pump is practically limited. The flow rates of methanol and argon should be carefully optimized to cool the sample molecule efficiently. We connected a pirani gauge to the vacuum chamber and monitored the pressure. We observed the change of the fluorescence spectrum for perylene by increasing the flow rate of methanol from 0.05 mL/min at the fiied flow rate of argon (50-100 mL/min). When the stagnation pressure increased from 300 to 500 torr, the pressure in the vacuum chamber increased from 0.006 to 0.5 torr. The spectrum consisted of clearly resolved spectral features a t 0.003 torr, and the structure remained at 0.02 torr. However, it was completely broadened a t 0.5 torr. The signal intensity decreased more than 10 times by increasing the stagnation pressure. The optimum stagnation pressure was 350 torr in our experiment. The flow rate of methanol was adjusted to 0.1 mL/min, and the flow rate of argon was changed. The signal intensity gradually increased with increasing the flow rate of argon up to 150 mL/min and decreased at 200 mL/min, where the spectral line shape was slightly broadened. For the present nozzle (0.3 mm i.d.), the distance between the nozzle and a Mach disk (X,) can be calculated to 6 cm Patagnation pressurelpvacuum pressure = 300 torr/O*W3 torr), 2.5 cm (400/0.02), and 0.5 cm (600/0.5). Thus,the insufficient cooling at the higher pressure might be ascribed to formation of the Mach disk behind the nozzle. In this study we typically adjusted the flow rate of methanol to 0.05-0.2 mL/min. The argon gas was mixed as much as possible, by monitoring the pressure of the vacuum chamber not allowing it to exceed 0.005 torr. Optimization of the conditions was further carried out case by case. The present optimization is tedious and time-consuming, and efficient cooling was achieved only when a cold trap was filled with a sufficient amount of liquid nitrogen. We believe that a vacuum system with a larger pumping capacity or a new type of nozzle reliably operated at a lower flow rate should be developed for routine work. The nozzle pressure and temperature were typically adjusted to 40 atm and 200 OC, which are close to the critical pressure and temperature for methanol (P, = 79.9 atm, T,= 239.4 "C). At lower pressure and temperature the cooled spectrum had not been observed. Thus it is confirmed that the carrier should be maintained close to the supercritical fluid before sample expansion. Cooling Effect. Figure 2 shows fluorescence spectra for anthracene measured after optimization of the experimental conditions. These spectra show that the sample is sufficiently
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
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9-chloroanthracene
.-h 4-
v)
C
Q
c
-C
0,
9-rnethylanthracene
0 C Q
0 v)
2
0 3
i
356
360
385
3 5
370
Wavelength (nm)
360
Figure 4. Excitation spectra for anthracene, 9-methylanthracene, and 9-chloroanthracene measured by using methanol for sample introduction.
1
380 400 Wavelength (nm)
F@tm 2. Fluorescence spectra for anthracene: (A) the sample diluted with argon is expanded from the gaseous phase; (8)the sample is introduced by dissolving in methand and expanded into a vacuum after sample mixing with argon.
12-chloro-
l-chloro/anthracene
Q-chloro-
355
360
365
370
375
Wavelength (nm)
360.5
361.5
Wavelength(nm)
360.5
361.5
Waveiength(nm)
360.5
361.5
Waveiength(nm)
Figure 3. Line proflles of pure electronic transition for anthracene: (A)
the sample diluted wlth argon is expanded from the gas phase; (B) the sample is introduced by dlssoiving in methanol and expanded into a vacuum after sample mixing wlth argon; (C) the sample is introduced by dissolving in methanol and expanded into a vacuum without mixing with argon.
cooled, and the present method is useful for selective determination of the molecular species. However, the spectral line width is slightly increased, when methanol is expanded without using argon as a diluent gas. A similar result was observed for excitation spectra. Figure 3 is the peak profile at around the pure electronic transition (0 transition). When the sample is expanded from the gas phase, the line width is 3 cm-l. When only methanol is used for sample introduction, the line width is increased to 11cm-I and a long tail appears. The line broadening might be due to increase of the rotational temperature and formation of van der Waals complex of anthracene with methanol. With argon, the line width decreased to 8 cm-' and the long tail substantially disappears. 9-Position-Substituted Anthracenes. Figure 4 shows excitation spectra for anthracene, 9-methylanthracene, and 9-chloroanthracene measured by using methanol for sample introduction. The results show that these molecules are sufficiently cooled. The mixture of these compounds was measured by use of a chromatograph with a conventional
Flgure 5. Excitation spectra for a mixture of chloroanthracene derivatives. The expanded spectrum in the region 367-368 nm is shown in the figure.
fluorometric detector. All the components are resolved by the separation column, and they are simultaneously and selectively determined. The chromatogram was also measured by use of a supersonicjet/fluorometric detector. Every component is selectively determined by adjusting the excitation and fluorescence wavelengths to the specified values: anthracene, A,, 361.03 nm A,, 380.3 nm; 9-methylanthracene, A,, 371.16 nm, A,, 390.6 nm; 9-chloroanthracene, A,, 373.25 nm, A, 394.0 nm. In this case the sample should be repetitively injected for determinations of these components. It is apparent that the conventional nonselective detector is preferable when all the components can be separated by the column. Chloroanthracene Derivatives. Figure 5 shows excitation spectra for the mixture of 1-chloroanthracene, 2-chloroanthracene, and 9-chloroanthracene. It is emphasized that thermally labile 2-chloroanthracene can be measured by supersonic jet spectrometry and gives clearly resolved spectral features. This is a great advantage of the present sample introductiontechnique using a supercritical fluid. The spectral properties for 1-chloroanthraceneand 2-chloroanthraceneare very close, but the wavelengths of the pure electronic transitions are slightly different. Selective excitation of these compounds is readily achieved by supersonicjet spectrometry. The chromatograms for chloroanthracene derivatives and for their mixture shown in Figure 6 were obtained with a con-
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
n
9-chloroanthracene
I\
fI 2-chloroanthracene
A em-
14
111 I
U I
l./3..y.y*....*I.,
0
La ( A ) 2-chloroanthracene
0
5 10 15 20 Retention Time ( m i d
Figure 6. Chromatograms for chloroanthracene derivatlves and their mixhres measured by conventionalfluorometric detector. The samples were dlssolved in methanol until the solution was saturated. The concentration for anthracene was 1 X M. The excitation and fluorescence wavelengths were adjusted to 365 nm and 400 nm, respectively.
ventional fluorometric spectrometer. The retention time for 9-chloroanthracene is longer than that for other compounds, so 9-chloroanthracene can be selectively determined. On the other hand the retention times for 1-chloroanthracene and 2-chloroanthracene are almost identical, so their selective determination with the conventional chromatograph is difficult. Figure 7 shows the chromatogram obtained with the supersonic jet detector. By use of the excitation and fluorescence wavelengths, these compounds are selectively determined by the chromatograph equipped with the supersonic jet detector. Detection Limit. The analytical curve was constructed for anthracene. It was straight from 0 to 800 ng, the detection limit (SIN = 3) being 40 ng. The sample determination at yet higher concentrations might be achieved until overloading to the column, but it was not experimentallyconfirmed in this study. The background signal was negligible, and the detection limit was determined by the dark current noise from the photomultiplier. No signal saturation was observed at present power levels, 80 that the sensitivity might be improved by using the laser with a larger output power. The achieved detection limit was similar to those reported for naphthalene derivatives by a gas chromatography system combined with a supersonic jet/fluorometric detector (3). Figure of Merit. In supersonic jet spectrometry, the compounds with similar chemical structures can be selectively determined by specifying the excitation and fluorescence wavelengths. This provides a distinct advantage with respect to selectivity over conventional spectrometry. It is noted that a real sample frequently contains more than 100 chemical species. Therefore, it is quite difficult to resolve and detect all the components by conventional HPLC. In this case a selective detector such as a supersonic jet spectrometer is essential for their determination. The present method using a supercritical fluid for sample introduction can be applied to a thermally labile molecule.
10
15
X e x = 387.09 nm
20
h e m = 387.5 nm
Retention Time (mid
1
(B) I -ch I oraan thracene
5
387.5 nm
Flew0 7. Chromatograms for mixture of chloroanthracene derivatives by supersonic jet/fluorometrlc detector. The excitation and fluorescence wavelengths are specified in the figure. Other condltions are identical with those for Figure 6.
As described, 2-chloroanthracene decomposes at a high temperature. However, the residence time of the sample at the nozzle port maintained at 200 OC is only 1s, so that no degradation occurs in this period. In the present application supersonic jet spectrometry was so selective that all the components could be determined without using a separation column. Then, one may consider that combination with HPLC is unnecessary. However, we would like to point out that optically active molecules give completely identical spectra even by supersonic jet spectrometry. In this case HPLC is essential for selective determinations. It is noted that many separation columns packed with optically active materials are currently used for the determination of biochemical species. The present approach based on HPLC/supersonic jet/laser fluorometry may be advantageous in such applications. We expect that the present technique will be very useful for trace analysis of medicines, whose biochemical activities change with slight modification of the chemical structure. Registry No. Anthracene, 120-12-7; 1-chloroanthracene, 4985-70-0; 9-chloroanthracene, 716-53-0; 9-methylanthracene, 779-02-2; 2-chloroanthracene, 17135-78-3.
LITERATURE CITED (1) (2) (3) (4)
(5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)
Hayes, J. M.; Small, G. J. Anal. Chem. 1983, 55, 565A. Johnston, M. V. Trends Anal. Chem. 1984, 3 , 58. Hayes, J. M.; Small, G. J. Anal. Chsm. 1982, 5 4 , 1204. Imasaka, T.; Shgezumi, T.; Ishibashi, N. Analyst (London) 1984, 109, 277. Imasaka, T.; Okamura, T.; Ishibashi, N. Anal. Chem. 1988, 58, 2152. Imasaka, T.; Tashiro, K.; Ishibashi, N. Anal. Chem. 1988, 58, 3242. Rizzo, T. R.; Park, Y. D.; Levy, D. H. J . A m . Chem. SOC.1985, 107, 277. Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983, 55, 750. Rizzo, T. R.; Park, Y. D.: Peteanu, L.; Levy, D. H. J . Chem. Phys. 1985, 83, 4819. Fukuoka, H.; Imasaka, T.; Ishibashi, N. Anal. Chem. 1988, 58, 375. Sin, C. H.; Pang, H. M.; Lubman, D. M.; Zorn, J. Anal. Chem. 1988, 58, 490. Pang, H. M.; Sin, C. H.; Lubman, D. M.; Zorn, J. Anal. Chem. 1986, 58, 1581. Nishi, N.; Yamamoto, K.; Shinohara, H.; Nagashima, U.; Okuyama, T. Chem. PhYS. L 8 f f . 1985, 122, 599. Yamamoto. K.; Nishi, N. J . Spectrosc. SOC. Jpn. 1988, 35, 163. Nishi, N.; Okuyama, T. The Chemlcal Society of Japan, Annual Meeting, 1985, Proceedings, p 168.
REfor review June 30,1986. Accepted October 2,1986. This research is supported by Grant-in-Aid for Scientific Research from the Ministry of Education of Japan and by Nissan Foundation.
