Anal. Chem. 1991, 63, 2685-2688 (12) Schuster, G. B. Acc. Chem. Res. 1979, 72, 366-373. (13) Catherell. C. L. R.; Palmer, T. F.; Cundall, R. B. J . 0”.Soc., Faradey Trans. 1984, 80, 837-849. (14) Alvarez, F. J.; Parekh, N. J.; Matuszewski, B.; Givens, R. S.; Higuchi, T.; Schowen, R. L. J . Am. Chem. SOC. 1988, 108, 6435-6437. (15) Hanaoka, N.;Givens, R . S.; Schowen, R. L.; Kuwana, T. Anal. Chem. 1988, 60, 2193-2197. (16) Hanadta, N. J . Chrometog. 1990, 503, 155-165. (17) Mann, 8.: Grayeskl, M. L. Anal. Chem. 1990, 62, 1532-1536.
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(18) Instructlon Manual ot HPLC column. STR ODS-M; Shlmadzu Techne Research: Kyoto, Japan. (19) Bayer, E.; Grom, E.; Kaltenegger, 8.; Uhmann, I?.Anal. Chem. 1976, 48, 1106-1109.
RECEIVED for review April 24,1991. Accepted September 12, 1991.
Direct Comparison of Two-Photon and One-Photon Excited Fluorescence Detection in Liquid Chromatography Using an Excimer-Pumped Dye Laser Ronald J. van de Nesse, Arjan J. G. Mank, Gerard Ph. Hoornweg, Cees Gooijer,* Udo A. Th. Brinkman, and Ne1 H. Velthorst
Department of General and Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
An excimer-pumped (XeCi) dye laser has been used to study two-photon excited (TPE) fluorescence detection in conventional-sire column liquid chromatography (LC) with aromatic compounds as test solutes. Excitation was performed at three different wavelengths, 1.e. 514, 586, and 650 nm, while emlssion light was collected around 410 nm. The results obtained with TPE at 586-nm excitation have been compared with onephoton excitation (OPE) at 293 nm; since frequency doubling was utilized, these measurements were performed under exactly the same experimental conditions. The relative peak heights observed for the various analytes with both modes of detection are distinctly different, demonstrating a noticeable difference In selectivity between TPE and OPE. Detection with TPE will in general be less sensitive than with OPE because of the Inherently less efficient excitation of the former technique; stili a detection limit as low as 1.0 nM was obtained in LC-TPE for the dye 4,4’-diphenylstilbene.
INTRODUCTION In two-photon excited (TPE) fluorescence, visible laser light is generally used for excitation of UV-absorbing analytes and the fluorescence spectrum is on the short-wavelength side of the excitation light. This implies that problems due to Rayleigh and Raman scatter and to reflection and refraction of laser radiation, as encountered in conventional laser-induced fluorescence (LIF) detection (based on one-photon excitation, OPE), are more easily solved in the TPE fluorescence detection mode. At first sight the analytical potential of T P E fluorescence detection seems to be limited, since the excitation efficiency of T P E as compared to OPE is extremely low. However, interesting detection limits can be obtained by invoking high-power pulsed lasers because the TPE efficiency is proportional to the square of the excitation power. As early as 1977, Sepaniak and Yeung published the first paper on laser T P E fluorescence detection for column liquid chromatography (LC) (I). Utilizing a 4-W continuous-wave argon-ion laser operating at 514 nm, chromatograms of oxadiazoles at concentration levels of 10-5-104 M were shown.
* To whom correspondence should be addressed. 0003-2700/91/0363-2685$02.50/0
The selectivity of T P E fluorescence detection was obvious: the highly fluorescent compounds phenol, fluorene, anthracene, and chrysene, also present in concentrations of M, did not show up. Unfortunately, the selectivity of the system could not be fully exploited because of the limited availability of laser output frequencies. The lowest limit of detection (LOD) of 3 x IO-’ M (SIN = 3;time constant, 0.5 s), obtained for 2-phenyl-5-(4-biphenylyl)-1,3,4-oxadiazole (PBD), obviously was not low enough for on-line coupling to LC. Considerable improvement has been achieved since then by applying lasers which provide high peak powers. The most recent achievement in combination with LC is in a paper by Pfeffer and Yeung (2) who used a copper-vapor laser (510/578 nm) with an average power of 3 W and a peak power of 20 kW as an excitation source for T P E fluorescence detection in a micro-LC system. For the same test solute as used before, the oxadiazole PBD, now an LOD of 9 X M ( S I N = 3; time constant, 3 s) was recorded. Recently, Wirth and Fatunmbi (3)reported the in-batch detection of 2.3 X M bis(methylstyry1benzene) ( S I N = 1; time constant, 1s) in a 1-cm cuvette using a Nd:YAG synchronously pumped dye laser tuned at 600 nm with an average power of 245 mW and a peak power of 600 W. Until now a systematic study of the analytical potential of laser-induced T P E fluorescence detection in LC has not been available. In this context the difference in wavelength dependences of T P E and OPE will be especially interesting. In the present paper an excimer-pumped (XeC1) dye laser was used, which provides an average power of about 300 mW with 1-MW pulses for efficient excitation. Since the absorbed power and, hence, the fluorescence signal are linear with the product of the peak power and average power (31,these characteristics imply that the system should allow for sensitive T P E fluorescence detection. Furthermore, the presence of a dye laser enables wavelength tuning. In our setup the dye-laser output also can be frequency-doubled, so that LC detection can be performed using either OPE (conventional LIF detection) or T P E fluorescence with exactly the same experimental assembly. That is, a direct comparison can be made between the two modes. Mixtures of polycyclic aromatic hydrocarbons (PAHs) and some other model compounds were studied in conventional-size LC at three excitation wavelengths, 514, 586, and 650 nm. 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63,NO. 23, DECEMBER 1, 1991
EXPERIMENTAL SECTION Chromatography. The LC system consisted of a Philips PU 4100 LC pump (Pye Unicam, Cambridge, Great Britain) and a laboratory-made injection valve with an external loop of 50 pL. All separations were performed by isocratic elution at a flow rate of 1 mL/min on a 200 X 3.0 mm i.d. column packed with 5-pm RoSil C18(Research Separations Laboratories, Eke, Belgium) with methanol-water (95/5, v/v) as the eluent. TPE Fluorescence Measurements. The excitation light was provided by a Questec 5200B dye laser (Spectra Physics, San Jose, CA) which was pumped by a Questec Q2440 excimer (XeCl) laser (Spectra Physics) operating at a repetition rate of 30 Hz. Depending on the energy of the pump pulse and the efficiency of the laser dye, the peak power of the 15-11s pulse from the dye laser was about 1 MW. For the OPE experiments, the light was frequency-doubled through an ammonium dihydro arsenate (ADA) crystal. A 5 mm path length cuvette filled with an aqueous 0.5% (w/v) K2Cr207solution was placed after the dye laser to reject 308-nm pump light. The laser beam was focused on the flow cell with a 24 cm focal length quartz lens. To increase the excitation efficiency, lenses with shorter focal points were also applied ( 4 , 5); although the fluorescence signals were higher, proper optical alignment was more difficult and damage to the quartz flow cell occurred. With the current optical setup and used powers, the flow cell remained undamaged during the whole period of the investigation. A laboratory-made quartz flow cell (Suprasil I quality) with external dimensions of 4 X 4 X 10 mm was used; the internal bore was 1.1mm. For the collection of the fluorescence light, a 0.6 mm i.d. quartz fiber was put into the bore of the flow cell, as shown in the design in Figure 3 of ref 6. The light from the fiber was converted into a parallel beam by a 5 cm focal length quartz lens. To select the appropriate spectral window at the emission side, a 2-cm cuvette filled with 5% (w/v) CuS04.5H20in 1.2 M ammonia solution was used. The filter was transparent for wavelengths of between 360 and 480 nm with a maximum of 65% transmittance at 410 nm. For TPE fluorescence measurements at 514-nm excitation, a larger filter function was required to reject all laser stray light; therefore a 4-mm Schott BG3 filter was added to the filter system. A cooled XP 20206 photomultiplier tube (Philips) operating at 2200 V was employed for detection. The photomultiplier signal was processed by a Stanford Research (Palo Alto, CA) SR 250 boxcar integrator. The necessary trigger pulse for the boxcar was generated by a photodiode detecting a small portion of the excimer laser light. As the fluorescencelifetimes of moat compounds under study were too short to discern them from the 15-ns laser pulse, no net delay was used; the gate width was 90 118. The signal pulses were averaged over 30 samples, which corresponds to a time constant of 1 s. A Stanford Research SR 235 analog processor unit was used for the conversion to voltage read-out on a Kipp & Zonen (Delft, The Netherlands) BD 8 strip-chart recorder. Materials. Anthracene, benzo[b]chrysene (both from Fluka, Buchs, Switzerland), chrysene (Materials Ltd.,Englewood Cliffs, NJ) benzo[blfluoranthene, benzo[k]fluoranthene, benzo[a]pyrene (all three from the Joint Research Centre of the European Economic Communities, Petten, The Netherlands), 2-phenyl-5-(4biphenylyl)-1,3,4-oxadiazole(Exciton, Dayton, OH), and 4,4'diphenylstilbene (Lambda Physik, Gottingen, Germany) were used as received. The eluent was composed of HPLC grade methanol from Baker (Deventer, The Netherlands) and demineralized water. The various excitation wavelengthswere acquired by three laser dyes: Coumarin 521, Rhodamine 6G (both from Exciton), and DCM (Radiant Dyes, Wermelskirchen, Germany). R E S U L T S A N D DISCUSSION TPE Fluorescence Detection. As stated in the Introduction, the excimer-dye laser provides wavelength tunability, so that the potential of TPE fluorescence detection in LC can be studied as a function of the excitation wavelength. It should be realized, however, that the tuning range per dye is limited, i.e. typically 50 nm, and that the laser efficiency is dye- and wavelength-dependent. In our experiments three excitation wavelengths were chosen, rather far apart from each
zol A
TPE
5
0
-
10 I (min)
15
TPE
0
5
10 ___)
"1
hex= 650 nm
15
1 (min)
c
I
0
Lex-586 nm
5
-
I
I
10
15
t (min)
Flgure 1. Lc chromatograms of a mixture of 2-phenyld-(4-blphenylyl)-l,3,4-oxadiazole (PBD),chrysene (CHR), benzo[k] fluoranthene (B[k]F), benZO[8 1pyrene (B[8 ]P), benzo[b Ichrysene (B[bIC), and 4,4'diphenylstilbene (DPS), excRed with (A) 586 nm (TPE), (B) 650 nm (TPE), and (C) 293 nm (OPE). The fluorescence detection window was between 360 and 480 nm. The concentrations in Figure 1A,B were 5 X lo-' M CHR and 5 X 10" M for the other analytes; in Flgure 1C the concentrations for all analytes were 2.5-fold lower. Note the different scales on the y axes; zero intensity corresponds to the dark
current.
other, i.e. 514,586, and 650 nm. T o generate these linea, thee different laser dyes were applied; the difference in excitation power at the three wavelengths considered was less than 50%. As a test mixture, five PAHs and two other aromatic compounds were studied using conventional-size reversed-phase LC. In Figure 1two chromatograms are shown measured with (A) 586-nm and (B) 650-nm excitation. The concentrations were 5 X lo-' M for chrysene and 5 X M for the other analytes. Anthracene is not present in the chromatograms because it coelutes with PBD and was injected separately. The
ANALYTICAL CHEMISTRY, VOL. 63,NO. 23,DECEMBER 1, 1991
Table I. Limits of Detection (LOD) of Selected Analytes in LC with TPE Fluorescence for Three Excitation Wavelengths (514,686, and 660 nm) and Molar Absorption Coefficients (e) at 293 and 325 rima
analyte PBD anthracene chrysene benzo[k]fluoranthrene benzo[a]pyrene benzo[b]chrysene DPS
k'
lO@LOD,Mb 514 586 650 nmc nmd nmd
10-4~, L mol-' cm-' 293 325 nm nm
2.3 4.3 6.8
3.5 65 250 17
20 16 16 5
6 110 350 7
3.5
1.9 0.23 0.26 0.63
7.9 12.3
21 11
4 3
11
9
4.5 6.5
0.39 0.71
16.0
12
1
11
1.1
1.8
2.3
4.3 0.044 1.1
For LC system, see text; injection volume, 50 pL. S I N = 3, n = 3-5. cAem = 360-460 nm, 30% at 410 nm. dA,, = 360-480 nm, 65% at 410 nm. LODs are reported in Table I ( S I N = 3) together with the chromatographic capacity factors. No correction was made for differences in laser excitation power. It is obvious from Table I that the LODs for the analytes under study are strongly dependent on the excitation wavelength. Except for PBD, the lowest values are found at 586-nm excitation; they range from 1to 20 nM. It is interesting to note that, despite its larger retention time (k' = 16), the lowest LOD is obtained for DPS. The data in Table I on 514-nm excitation may well be compared with the early results of Sepaniak and Yeung (I) who applied the same excitation wavelength, provided by a continuous-wave argon ion laser. A chromatogram of a test mixture containing 1.5 X lo4 M PBD and 10" M of anthracene and chrysene only showed the PBD peak, while the PAH signals obviously were not strong enough to discern them from the base-line noise. This is in line with our results for the same excitation wavelength the LODs of anthracene and chrysene are about 20-fold and 70-fold higher, respectively, than the LOD of PBD. The results obtained with the copper-vapor laser (2) are more difficult to compare, because this laser provides 510-nm as well as 578-nm light; these lines were not separated by the authors, and the relative intensities were not reported. The LODs of PBD at 514 nm (3.5 nM) and 578 nm (7 nM; not shown in the table) in our study are in the same range as the LOD reported for the copper-vapor laser. This indicates that the general performance of our experimental assembly is similar to that of the system used by Pfeffer and Yeung, despite the differences in laser-pulse power, pulse width, and repetition rate as well as chromatographic instrumentation. Comparison of the LODs reported for 586- and 650-nm excitation (the LODs at 514 nm cannot easily be compared because the spectral emission window is slightly different) reveals that the T P E efficiency cannot simply be related to the absorptivity responsible for conventional OPE. This is obvious from Table I wherein the molar absorption coefficients of all test solutes at 293 and 325 nm are included; they were measured in methanol-water (9515, v/v). Whereas the LODs of anthracene and DPS at 650-nm excitation are about 10 times higher than at 586 nm, for anthracene the absorptivity at 65012 nm is &fold higher than at 58612 nm, and for DPS it is %fold higher. For the larger PAHs, benzo[k]fluoranthene, benzo[alpyrene, and benzo[b]chrysene, the absorptivities at 65012 nm are 6-11-fold lower than at 586/2 nm, but the loss in detectability upon going from 586- to 650-nm excitation is far less pronounced. For PBD, however, the reverse is true.
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Table 11. OPE (Excitation Wavelength 293 nm) Fluorescence Limits of Detection (LOD) Measured for Two Emission Windows: Comparison with TPE (Excitation Wavelength 586 nm) Fluorescenceo
analyte PBD anthracene chrysene benzo[k] fluoranthrene benzo[a]pyrene benzo[blchrysene DPS
~ O ~ L O~bD , 360-480 403 nmc nmd 0.5 55 15 0.3
LODpEC/ LODOPE'
nd'
50 0.3 1.0 0.15 17 1.0 0.35 4 0.75 0.40 4 2.5 nde 0.4 a For LC system, see text; injection volume, 50 pL. S I N = 3, n = 3. A, = 360-480 nm, 65% at 410 nm. A, = 394-412 nm, 9% at 403 nm. 'Not determined. 10 3.0
Its absorptivity decreases about 2-fold on going from 58612 to 650/2 nm; the LOD, however, improves from 20 to 6 nM. The above results can be summarized as follows: under the present experimental conditions, the LODs attainable with an excimer-pumped dye laser are in the low nanomolar range (1-20 nM, corresponding to 10-200 pg injected). As expected, the LODs differ strongly from analyte to analyte and, additionally, there is a strong dependence on the excitation wavelength used which cannot be predicted on the basis of conventional (single-photon) absorption spectra. As expected, due to the less efficient excitation, the signal intensities in T P E are lower than in OPE (see next section). The background signal, however, is also low, showing little noise; therefore low detection limits can be obtained in TPE. The background is attributed to fluorescence from impurities in the eluent and/or flow cell induced by TPE. The Rayleigh and Raman scatter are fully rejected: upon adding a 515-nm cutoff filter to the 360-480-nm band filter the background was completely removed. Most likely the noise on the low background is dominated by shot noise-which is proportional to the square root of the background signal-while laser fluctuations are of minor importance. This implies that in principle the signal-to-noise ratio can be improved by increasing the laser excitation power until the noise due to laser fluctuations becomes predominant. From a practical point of view, damage to the detector flow cell limits the allowed spectral irradiance; this parameter was not examined in the present study. OPE (LIFB3)Detection. For the present test solutes W E fluorescence detection at an excitation wavelength of 586 nm is a suitable means of detection (see Table I). Evidently, 586-nm two-photon excitation provides the same energy tu 293-nm one-photon excitation. The excitation efficiencies of T P E and OPE are, however, fundamentally different. It is therefore interesting to study OPE-that is, LIF-detection under the same experimental conditions (excitation/collection geometry, spectral window, and detection performance) as utilized for T P E fluorescence detection. To this end the dye-laser output at 586 nm was frequency-doubled to generate 293-nm laser light. The initial output from the frequency doubler was attenuated by a pinhole aperture to prevent saturation of the detector system. The laser power used in OPE was not exactly known. In Figure 1C an LC-OPE chromatogram is shown, using analyte concentrations which are 2.5-fold lower than in Figure 1A,B, i.e. 2 X lo-' M for chrysene and 2 X 10-e M for the other analytes. The LODs measured with OPE (LIFm3)detection are reported in Table 11. With the same spectral emission window as was used in the TPE experiments, the LODs range from 0.3 to 10 nM. Upon installation of a 403-nm interference
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 23, DECEMBER 1, 1991 DPS
10
TPE
A
hex -586nm
m
53 7.6 m
-5
:5:
5
W
z I
2.5
0
5
-
10
15
((min)
0
5
-
10
15
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Figure 2. LC chromatograms of a sediment sample containing various PAHs, among which were benzo[k]fluoranthene (B[k]F) and benro[elpyrene (B[a]P), p e n t in concentrations of about 1 X lo-’ M, spiked with 5 X 10- M 4,4’diphenyistilbene (DPS), excited with (A) 586 nm (TPE) and (B) 293 nm (OPE). The fiuorescence detection window was between 360 and 480 nm. Note the different scales on the y axes: zero intensity corresponds to the dark current.
filter in addition to the copper-ammonia filter, the LODs of some analytes considerably improved. The gain depends on the overlap of the emission spectrum of the analyte with the spectral window (Aem = 393-412 nm, 9% at 403 nm). The above results may be considered disappointing in view of the high efficiency of the excitation process. As can be seen from Figure 1C and from comparing it with Figure lA, this is due to the relatively high background signal. Consequently, noise contributions proportional to the pulse-to-pulse variations of the excitation source overshadow the shot noise. In other words, both the fluorescence signals and the noise on the background now are linearly proportional to the laser intensity. This implies that the use of higher excitation powers will not result in improved LODs. For this reason the use of an interference filter which offers more spectral selectivity, although it admittedly reduces the application range of the detection system, yields better detection limits, as shown in Table 11;the effect was studied for the PAHs only. Since the
light level reaching the detector was now considerably lower, the pinhole aperture was removed and the full UV output was exploited. The several subnanomolar detection limits demonstrate that the excimer-dye laser combination equipped with a frequency doubler is suitable for conventional OPE detection of analytes at trace levels. Comparison of TPE and OPE Using an ExcimerPumped Dye Laser. The results discussed above reveal that, with the same emission window, for five out of the seven analytes tested, the LODs achieved by TPE fluorescence detection with 586-nm excitation are higher than those obtained by OPE (LIFZg3).The difference in detectability between the two modes is not the same for each analyte. For instance, for PBD and DPS the data in Table I1 indicate that their relative signal intensities differ by a factor of 125 in going from one mode to another. In Figure 1 it can be seen that with OPE (L1Fzg3)detection (Figure IC) the DPS peak is about 5-fold lower, whereas with TPE using 586-nm excitation (Figure 1A) it is 20-fold higher. The differences in excitation properties (wavelength dependence and excitation efficiency) between the two modes are not unexpected since different selection rules are involved (7, 8). Finally, it is interesting to compare the background signals observed in OPE and T P E fluorescence detection. Such information is virtually nonexistent although it has been suggested (9) that for urine and plasma samples the background is negligible even at high-sensitivity settings. For this reason, OPE and TPE chromatograms were recorded for a sediment extract known to contain various PAHs and, for this particular case, spiked with DPS (exhibiting a high T P E fluorescence response). Obviously, also in Figure 2A a substantial background is observed: after the column dead time of about 1min it increases about a factor of 3-5, an increment similar to that observed in the OPE mode. Thus, in this example the background problem is not reduced by utilizing TPE instead of OPE. In the present study only a limited number of analytes was studied. General conclusions can therefore not easily be drawn. This is even more true because in the literature hardly any information is available on the wavelength dependence of two-photon cross sections (IO). LITERATURE CITED (1) (2) (3) (4)
(5)
(6) (7) (8) (9)
(IO)
Sepaniak, M. J.; Yeung, E. S. Anal. Chem. 1977, 49, 1554-1556. Pfeffer, W. D.; Yeung, E. S. Anal. Chem. 1986, 56, 2103-2105. Wirth, M. J.; Fatunmbi, H. 0. Anal. Chem. 1990, 62, 973-976. Huff, P. E.; Tromberg, 6. J.; Sepaniak, M. J. Anal. Chem. 1982, 54, 946-950. Swofford, R. L.; McClain, W. M. Chem. Phys. Lett. 1975, 3 4 , 455-459. Sepaniak, M. J.; Yeung, E. S. J . Chromatogr. 1980, 790, 377. Friedrich, D. M. J . Chem. Educ. 1982, 59, 472-481. McClain, W. M. Acc. Chem. Res. 1974, 7, 129-138. Yeung. E. S.; Sepaniak, M. J. Anal. Chem. 1980, 52, 1465A-1481A. Chen, C. H.; McCann, M. P. Optics Commun. 1987, 63, 335-338.
RECEIVED for review May 29,1991. Accepted September 12, 1991. The investigation was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Scientific Research (NWO), Grant No. 700-344-006.
