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Anal. Chem. 1982,54, 1834-1839
Two-Photon Photoionization Detection of Polycyclic Aromatic Hydrocarbons and Drugs in a Windowless Flow Cell Edward Volgtman and James D. Wlnefordner" Chemlstry Department, Unlversity of Florida, Gainesville, Florlda 326 I 1
A laserexclted wlndowless flow cell has been developed that has simultaneous detectlon capabllltles by molecular fluorescence, photoacoustlc effect, and two-photon photolonlzatlon processes. Factors of primary Influence on the analytlcal performance of the photolonlratlon mode have been evaluated with polycycllc aromatlc hydrocarbons (PAHs) as model compounds and a variety of drugs havlng dlverse structures have been detected In the photolonlratlon mode. A llquld phase photolonlzatlon chromatogram demonstrates the analytlcal merit of the technlque.
In a recent paper (1))we have described our design and use of a windowless flow cell, intended for potential liquid chromatographic applications, which employs simultaneous laser-excited molecular fluorescence, photoacoustic effect, and two-photon photoionization detection. The significance of such a three-mode detection scheme is that it provides the opportunity to monitor the three most probable photophysical deactivation pathways and thereby provides more information about the adsorbing species than any one of the processes alone can provide. Since our windowless flow cell is an instantiation of the windowless flow cell principle employed by Diebold and Zare (2) for the extremely sensitive detection of aflatoxins and since the principles of liquid phase photoacoustic (optoacoustic) detection with pulsed laser excitation, "submersed" or external piezoelectric transducer, and gated (boxcar) averager detection have been eloquently summarized by Pate1 and Tam (3))we will not describe them further. However, the two-photon photoionization detection scheme has not previously been employed for analytical purposes, to the best of our knowledge, and, therefore, we were interested in investigating those factors which were of direct relevance to the optimization of the photoionization (PI) detection process. Interest in photoionization processes, both single photon and multiphoton, has increased greatly with the advent of high-power tunable laser excitation sources and the recognition that numerous photoionization mechanisms exist. Indeed, several PI mechanisms can operate concomitantly given the right conditions of source intensity, focusing, solution dielectric constant, and solution viscosity (4,5). Accordingly, the study of PI processes in condensed media has been actively pursued with the aim of determining the requisite factors for each such PI mechanism. See, for example, the survey by Lesclaux and Joussot-Dubien (6) and the papers by Beck and Thomas (7, 8) and Piciulo and Thomas (9) for details of several such mechanism studies. Although it is unlikely that a detailed and substantially complete understanding of photoionization processes in condensed media will soon be at hand, such an understanding is not absolutely essential to the analytical application of the PI detection technique if we proceed with a judicious appreciation of the information presently available from the many physical studies which have been published. That such a procedure is feasible has been demonstrated previously (1) in our sensitive detection of 12 PAHs by all
three modes in our windowless flow cell. It is clear that, for a laser-based analaytical technique to achieve the greatest utility, the laser must be relatively simple, inexpensive, and reliable. In addition, it must not be necessary to employ rigorously purified solvents (if possible), and extreme bandwidth and sensitivity requirements must be avoided. The first of these conditions is well satisfied by N2 laser excitation or excimer laser excitation since there is general agreement that ionization potentials in condensed phase are 2 to 3 eV below the gas-phase values due to polarization energies associated with the photogenerated ions and solvated electrons (9, 10). In addition, Siomos and Christophorou (11)and Siomos et al. (12) have employed a photoconductivity detection scheme with a N2laser-pumped tunable dye laser excitation source and computer-based signal acquisition to obtain detailed P I spectra of pyrene, fluoranthene, and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) in n-pentane. The significance of their results for detection purposes is that the ionization potentials determined directly from the PI spectra are -2.6 eV below the gas-phase values. Thus a N2 laser at 337.1 nm (2 photons = 7.35 eV) or an excimer laser (XeCl excimer) at 308 nm (2 photons = 8.05 eV) is quite capable of providing the excitation for most organic compounds of interest. Although the photoconductance techniques presently available are inadequate for subnanosecond studies of photogenerated ions and electrons, the technique is much more simple than picosecond spectroscopic schemes and is far more suitable for the analytical application of PI detection. An additional advantage of the photoconductance detection scheme is that both polar and nonpolar solvents of ordinary purity may be employed since the mobility of solvated electrons in nonpolar solvents such as n-hexane is -100 times the mobility in polar solvents such as ethanol (7). Note, however, that the initial quantum efficiency of photoionization of pyrene is essentially independent of solvent polarity, but a t later times (nanoseconds after photoionization) the apparent PI quantum efficiency in polar solvents is -100 times that in nonpolar solvents due to the efficiency of geminate ion recombination in nonpolar solvents (9). Since the P I current is proportional to the product of carrier mobility and carrier concentration, these factors approximately cancel. It is also clear from the results to be presented below that impurities must be extraordinarily active and/or must be present in high concentrations to significantly interfere with the P I detection scheme. Otherwise, they simply add a small "leakage current" to the PI photocurrent signal.
EXPERIMENTAL SECTION Since the design and operation of the windowless three-mode flow cell have been previowly described ( I ) , we will limit discussion here to additional details and changes in the construction and use of the flow cell. Briefly stated, surface tension supports an approximately cylindrical column of solution between a metal reservoir tip and a metal pedestal. The reservoir is biased to -500 V, unless otherwise indicated, through a current limiting 10 Mil resistor and removable contact to the reservoir is through a looped spring. The limiting resistor has no effect on the observed PI
0 1982 American Chemical Soclety 0003-2700/82/0354-1834$01.25/0
ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982
1835
Table I. Solvents, 'Leakage Currents, and Nominal Purities leakage currents at solvent
1
Bo"
PHOTO ACOUSTIC PREAMPLIFIER
lr,-F
PHOTOIONIZATION
3AK
[Byparsedl
L - e - g Y
I / V CONVERTERS
.....
27011
~
Eo
11071
Figure 1.
Photoacoustic and photoionization1 preamplifiers.
wave form and merely pirotects the preamplifier from inadvertent shorting of the electrode gap. The photocurrent at the pedestal is prevented from reaching the piezoelectric transducer by a quartz disk insulator glued to 2 0.267 1.720 0.268 0.014 0.141 >2 0.022 0.008 0.642 0.812
100 5600 400 1100 650 2 100 32000 70 10 1600 3400
limit of detection (LOD), ng mL-' 500 2 1000 30 6 20 9 00 600 3000 10000 4 10000
Excitation wavelength (hex)= 337.1 nm; pulse energv (Ep) c: 300
pJ.
he, =
use anticonvulsant; analgesic anticonvulsant tranquilizer diuretic topical antiinfective thyroid inhibitor diagnostic aid antibacterial an tihypertensive psychoactive agent cathartic psychomime tic
430 nm; E ,
c: 200 pJ.
hex = 290 nm;
E,, r l p J .
L-d
102 102 103 CIRBAMAZEPINI
lo4 105 CONCENTRATION
[ni/mLl
Flgure 6. Calibration curve for carbamazepine. The laser pulse energy was 340 pJ and the voltage mode preamplifier was used (Flgure 1 top).
essentially equal PI signals. A typical calibration curve for one of the drugs listed in Table I1 is shown in Figure 6. Note the slight curvature which is probably caused by a small shift in the temporal position of the PI wave form maximum with drug concentration. This problem may be easily solved by replacing the boxcar averager with a suitable peak detector signal acquisition system. In addition to drugs which could be detected by the PI detector, a number of drugs produced no detectable P I signal at any concentration in the range from -100 ng/mL to -100 pg/mL. These are listed in Table 111. Most have negligible absorbance a t 337.1 nm with the exceptions of anisindione and flurazepam. Hence these two drugs would be readily detectable by the PA mode but would be essentially "invisible" to the PI detector. It is also interesting that A9-THC is detectable while the inactive drugs cannabidiol and cannabinol produce no PI signals despite similar molar absorptivities. It is also apparent that rifampin cannot be ionized by two photons at 475. nm (5.26 eV) but is readily ionized by two photons at 337.1 nm (7.35 eV). Thus, just as Siomos and co-workers ( I I , 1 2 ) have utilized tunable dye lasers to accurately determine condensed phase ionization potentials, the same procedure might suffice to selectively excite individual drugs in a mixture by manning the laser. In terms of sensitivity, the PI technique is comparable t , condensed ~ phase PA detection with a PZ'I' transducer and the cuvette sample cell. Considerable room for improvement, exists as shown by the effects of incident laser pulse energy (Figure 4) and bias voltage (Figure 3). In addition, electrode spacing and geometry effects, which are difficult to determine with this flow cell design, have been determined with a madified, three-mode cuvette cell (22) and Yaniada et al. (23)have already improved our (1)pyrene P I detecition limit by 60-fold, a t S I N = 3, by
Table 111. Drugs Not Detected by the P I Detection Mode molar absorptivity absorptivity (E), Muse (a),cm-' drug anticoagulent 6200 anisindionea >2 anticholinergic 0.0 0.000 atropinea 0.0 CNS 0.000 caffeine" stimulent inactive cannabidiol 0.006 3 (controlled) inactive 9 cannabinola 0.007 (controlled) skeletal 2 carisoprodol a 0.003 muscle relaxant antidepressant 4 clomipramine HCla 0.012 narcotic 2 codeinea 0.001 analgesic hypnotic 150 flurazepama 0.170 antiinflam5 oxy phenbutazonea 0.016 matory primaclone'" 0.0 anti0.000 convulsant theobromine 0.000 0.0 diuretic; cardiac stimulent thepphylline a diuretic ; 0.004 2 cardiac stimulent tolazoline HCla 2 anti0.004 adrenergic rifampinb antibacterial >2 19000 a he, =
337.1 nm; E ,
= 300 pJ.
hex = 475 nm; E,
1
200 pJ.
optimization of these factors. The primary disadvantage of P I detection is that ionic solutions cannot be used so that, for example, pH effects would not be readily accessible to the technique. At this point, we are in position to realize the goal stated in the introduction of this paper and of ref 1: the application of the flow cell to HPLC detection purposes. To this end, the reservoir was replaced by an externally threaded, 30% by weight graphite-filled Teflon rod, drilled to accept a standard stainless steel HPLC tubing section in. o.d., 0.020 in. i.d.). The conductive Teflon rod insert positioned the tubing end 2 mm above the pedestal and allowed the tubing to be biased to -2 kV. The ordinary bias BNC employed previously was replaced with a high-voltage BNC connector. The chromatographic system employed (Altex Scientific, Berkeley, CA,
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 11, SEPTEMBER 1982 A I
,
,
,
,
,
,
,
12
14
16
PI a
b
c
n u)
2
0
6 S 10 Time (Min)
4
B
1
425.11171 n
“negative PI” mode is observed and the presence of a substantial concentration of “impurity” (aniline) does not interfere with the chromatographic separation or the PI detection mode. It should also be noted that naphthalene (peak b), when (nonoptimally) excited a t 308 nm and monitored by fluorescence emission at 425 f 8 nm, gives a very small fluorescence chromatographic peak so that the P I detection mode is superior in this case. Note that coabsorption is not problematic. In Figure 7B, a 1% solution of benzene in ethanol is seen to produce a “negative fluorescence” chromatographic peak when the mobile phase is spiked with 200 ng/mL of anthracene and emission is monitored a t 425 f 8 nm. The cause of the anomalous elution time shift is presently unknown. The benzene result clearly shows that the negative detection modes must be carefully optimized and the excitation light source must have rather good intensity stability as was the case in the above reference (25).
u)
a U V abs
I
I
254. nm 0
2
4
6
8
Time (Min)
Figure 7. (A) PI and conventional UV absorbance chromatograms in the presence of 20 pg/mL aniline in the 70130 (v/v) acetonitrilelwater mobile phase. The laser pulse energy at 308 nm was 1 mJ. (6)FL and UV absorbance chromatograms in the presence of 200 ng/mL anthracene in the mobile phase. Same laser pulse energy as above.
Model 312,1.5 mL/min flow rate, isocratic operation, 4.6 mm X 25 cm column with Spherisorb ODs, 10 pm packing, 20 pL sample injections) was interfaced to a commercial UV absorbance detector (Altex, Model 153, 254 nm) with chart recorder output, and the chromatographic effluent from the UV absorbance detector was then routed to the modified flow cell through 0.6 m of narrow bore Teflon tubing. Insulating tubing is necessary for the connection to the chromatographic system because of the bias voltage employed in the PI detection mode. Although full details of the chromatographic performance of the modified flow cell, including three-mode chromatograms and comparison UV absorbance chromatograms, a detailed diagram of the modified flow cell, and a table of detection limits, have been submitted elsewhere for publication (24), it should be noted that three additional modes of operation are possible. In these additional modes of operation, the HPLC mobile phase is “spiked” with a readily detectable substance so that elution of substances which are chromatographically “invisible” to the detector produce a decrease in the otherwise constant signal due to the spiking substance. This is the so-called “negative” operation mode. The demonstration by Su e t al. (25) of “negative fluorescence” in the HPLC flow cell detector with detection limits approaching 10 ppm is encouraging in this regard. Note that substances which normally produce strong signals will continue to do so if the spiking substance is properly chosen, so that selectivity is not greatly reduced. Reduction in the signal produced by the spiking agent is due to dilution and, if applicable, quenching. Shown in Figure 7A are the P I and UV absorbance chromatograms of a 20-1L injected ethanol solution of (a) acridine (20 pg/mL), (b) naphthalene (22 pg/mL), (c) 7,8-benzoflavone (20 pg/mL), (d) N-ethylcarbazole (22 hg/mL), and (e) anthracene (8.0 pg/mL). The 70/30 (v/v) acetonitrile/water mobile phase was spiked with 20 bg/mL of aniline (since it readily photoionizes in polar solvents when excited a t 308 nm) which produced the base line offset shown. Reduction of the aniline concentration to 400 ng/mL produced a 50-fold reduction in the base line offset with no change in peak heights. Elimination of the aniline eliminates the offset. Hence no
CONCLUSIONS We have demonstrated that the photoionization detection mode of our laser-excited windowless flow cell operates as a photoconductance device. Hence, the P I detection scheme may be a useful adjunct to conventional fluorescence and photoacoustic techniques, especially where both absorbance and fluorescence (or phosphorescence) are weak or otherwise difficult to determine. An additional advantage of the three-mode flow cell is that all three of the most important photophysical deactivation pathways may be monitored simultaneously. It has also been demonstrated that sensitive detection of a variety of organic compounds (drugs) is easily achieved without special problems due to concomitant impurities. A “negative fluorescence” HPLC detection mode of low sensitivity has been demonstrated and the PI detection mode for HPLC has been shown to be essentially free of impurity effects with no “negative PI” mode occurring under the circumstances employed. ACKNOWLEDGMENT The authors thank Applied Sciences, Ayerst, Ciba-Geigy, Hoffman-LaRoche, Lilly, Merck, Riker, Schering, Sigma, and the National Institute of Drug Abuse for supplying the drugs studied. LITERATURE CITED Volgtman, E.; Jurgensen, A.; Winefordner, J. D. Anal. Chem. 1981, 53, 1921-1923. Diebold, G. J.; Zare, R. N., Science 1977, 796, 1439-1441. Patel, C. K. N.; Tam, A. C. Rev. Mod. Phys. 1981, 5 3 , 517-550. Ottolenghi, M. Chem. Phys. Lett. 1971, 72, 339-343. Gary, L. P.; de Groot, K.; Jarnagin, R. C. J . Chem. Phys. 1968, 4 9 , 1577-1587. Lesclaux, R.; Joussot-Dubien, J. I n “Organic Molecular Photophysics”; Birks, J. B., Ed.; Wlley-Interscience: New York, 1973; Vol. 1, p 457. Beck, G.; Thomas, J. K. Chem. Phys. Lett. 1972, 73, 295-297. Beck, G.; Thomas, J. K. J . Chem. Phys. 1972, 5 7 , 3649-3654. Piclulo, P. L.; Thomas, J. K. J . Chem. Phys. 1978, 6 8 , 3260-3264. Kellmann, A.; Tflbel, F.; Chem. Phys. Lett. 1980, 6 9 , 61-65. Slomos, K.; Chrlstophorou, L. G. Chem. Phys. Lett. 1980, 72, 43-48. Slomos. K.; Kourouklis. G.; ChristoDhorou. L. G. Chem. Phys. Lett. 1981, 80, 504-511. Travis, J. C.; Schenck, P. K.; Turk, G. C.; Mallard, W. G. Anal. Chem. 1979. 51. 1516-1520. Turk,’G. C.;Travis, J. C.; DeVoe, J. R.; O’Haver, T. C. Anal. Chem. 1979, 57, 1690-1896. Mlnday, R. M.; Schmidt, L. D.; Davis, H. T. J . Chem. Phys. 1971, 5 4 , 31 12-3 125. Schmidt, W. F.; Allen, A. 0. J . Chem. Phys. 1970, 5 2 , 4788-4794. Sze, S.M. “Physics of Semiconductor Devices”; Wiley-Interscience: New York, 1959; pp 655-656. Von HipDel, A. R. “Dielectrics and Waves”; Wiley: New York. 1954; pp 270-271. Braun, C. L.; Scott, T. W.; Albrecht, A. C. Chem. Phys. Lett. 1981, 84. 248-252. VoigFman,ET; Jurgensen, A.; Winefordner, J. D. Anal. Chem. 1981, 53, 1442-1446. Voigtman, E.; Jurgensen, A.; Wlnefordner, J. D., Ana/yst (London) 1982, 107, 408-413. Voigtman, E.; Wlndfordner, J. D., submltted for publication in Talanta . Yamada, S.;Kano, K.; Ogawa, T., submltted for publication in Bunseki Kagaku .
Anal. Chem. 1982, 5 4 , 1839-1843 (24) Volgtman, E.; Wlnefordner, J. D. J . Liq. Chromafogr., In press. (25) SU, s. Y.; Jurgensen. A.; Bolton, D.; Winefordner, J. D., And. Lett. i o a i , 14, 1-6.
RECEIVED for review Nlovember 16,1981. Accepted June 15,
1839
1982. This work was supported by Grant No. NIH-GM11373-17and by Grant No, DOE-AS05-78-EV06022-MODAOOE. Portions of this paper were presented at FACSS VIII, Philadelphia, PA, on Sept 23,1981.
Trace Metal Determination by Metastable Transfer Emission Spectroscopy Henry C. Na' and Thomas M. Nlemczyk" Department of Chemistv, Unlvers/?yof New Mexico, Albuquerque, New Mexico 8713 1
An emlsslon technlque based on excltatlon of atomic species by an energy transfer process from an active nitrogen plasma Is dlscussed. The malin excltation pathway appears to be a colllslonal energy transfer from the N2(A3&+) species In the actlve nltrogen plasma to the atomic specles of Interest. Aqueous solutions of trace metals are eiectrothermally dried and atomlzed from a tantalum boat. The actlve nitrogen Is produced In a mlcrovvave discharge and mlxed wlth the electrothermally produiced atomlc vapor In a flow cell. Detectlon llmlts for Ag, El, Cd, Cu, Mg, Pb, and TI are reported, and a h e a r dynamic range of 4 to 5 orders of magnltude Is seen In all cases. The iupper llmit to the llnear range Is related to the maxlmum conceiitratlon of the N2(b3Zu+)specles in the actlve nltrogen plasma. The technlque shows an lmmunlty to Interferences and the potentlal for multlelement analysis.
There exist many techniques that can be used for the cletermination of trace elements. None, however, possesses all the characteristics of an ideal trace element technique. Recently, a new technique has been investigated that shows a great deal of promise in fulfilling many of the requirements of the ideal technique. The technique involves the activation of nitrogen molecules in an electric discharge and the subsequent transfer of energy from the activated, Le., excited, nitrogen molecules to the atomic species of interest. Nitrogen so activated has been termed "active nitrogen" and has been the subject of many investigations. The first report on active nitrogen was made by Warburg in 1884 ( 2 ) . He observed a peach yellow glow when air was subjected to a discharge a t reduced pressures. The glow persisted for several seconds after the discharge was turned off and has since been referred to as the "nitrogen afterglow". In 1900,Llewis made the first systematic study of the nitrogen afterglow and confirmed its long lifetime and identified its band splectrum (2-5). He also found many metallic lines corresponding to electrode material and mercury lines due to the diffusion of mercury vapor into the afterglow region from the pumping system. The chemical activity of the afterglow was first emphasized by Strutt who suggested that recombination of nitrogen atoms might be responsible for the phenomenon (6). In 1935 Rayleigh reported that the lifetime of the afterglow was very much dependent upon the nature of the walls of the reaction chamber (7). Many adPresent address: PPG Industries, Inc., P.O.Box 4026, Corpus
Christi, TX 78408.
ditional studies involving the properties of active nitrogen have appeared in the literature, and much of this work has been summarized by Wright and Winkler (8). The excitation of metallic species in an active nitrogen afterglow has been mentioned in or has been the subject of many reports. Most of these reports have focused on the nitrogen afterglow ~rori the mechanism of metal species excitation. The principal energy carriers in active nitrogen are either the metastable triplet state Nz(A32,+) molecules or the vibrationally excited ground-state Nz(X'Zg+). Many differing claims concerning the detailed excitation mechanism have appeared in the literature (e.g., ref 9-15);however, it is generally accepted that the Nz(A3Z,+) state is responsible for excitation of levels requiring more than 4.5 eV while the vibrationally excited ground state might participate in the excitation of lower energy levels. The potential for an analytically important technique is indicated in many studies where metal atom emission was discussed. For example, Meyer et al. observed emission from Hg a t the 253.7-nm line for concentrations of Hg as low as lo9 atoms cm-3 (16). Sutton and co-workers have shown a number of important applications and named the technique metastable transfer emission spectroscopy (MTES) (17-19). The applications have included the detection of metal vapors produced in a furnace (18) and the determination of P b in aqueous samples (19). The detection limit for P b in the aqueous samples was reported to be 0.2 ng. More recently Dodge and Allen have reported the detection of Hg and Zn using an active nitrogen excitation technique (20). The active nitrogen generation used in their system was a dielectric discharge, which they report produces a higher concentration of the metastable Nz(A38,+) and a lower concentration of nitrogen atoms. On the basis of the difference in the nitrogen excitation system they called the technique METAL (for metastable energy transfer for atomic luminescence). Detection limits of lo7 atoms/cm3 and los atoms/cm3 in the emission cell for Hg and Zn, respectively, as well as a linear dynamic range of 7 orders of magnitude for Zn determinations were reported. On the basis of the results of the previous work we have designed an experimental system to determine trace metal concentrations in aqueous solutions using an active nitrogen excitation system. The system employs a microwave discharge to produce the metastable Nz(A3Z,+),thus in accordance with the previous terminology we refer to the technique as MTES. EXPERIMENTAL SECTION An overall block diagram of the experimental setup is shown in Figure 1. The atomic emission from the flow tube was
0003-2700/82/0354-1839$01.25/00 1982 American Chemical Society
