Anal. Chem. 1987, 5 9 , 1830-1834
1830
7440-02-0;Cu, 7440-50-8; As, 7440-38-2;Se, 7782-49-2; Zr, 744067-7; Nb, 7440-03-1; Mo, 7439-98-7; Ag, 7440-22-4. LITERATURE C I T E D Beske. H. E.; Welter, J. M.; Frerichs, G.; Melchers, F. G. Fresenius’ Z . Anal. Chem. 1981, 309, 269. Gray, A. L.; Date, A. R. Analyst(London) 1983, 108, 103. Muller, K. H.; Oechsner, H. Mikrochim. Acta, Suppl. 1983, 70, 51. Beckrnann, P.;Kopnarski, M.; Oechsner, H. Mikrochim. Acta, Suppl. 1985, 11, 79. Heinen, H. J.; Meier, S . ; Vogt, H.; Wechsung, R. Int. J . Mass Spectrom. Ion Phys. 1983, 4 7 , 19. Coburn, J. W.; Harrison, W. W. Appi. Specfrom. Rev. 1981, 17,95. Bentz. B. L.; Bruhn, C. G.; Harrison, W. W. Int. J . Mass Spectrom. Ion Phys. 1970, 28, 409. Colby, B. N.; Evans, C. A. Anal. Chem. 1974, 4 6 , 1236. Loving, T. J.; Harrison, W. W. Anal. Chem. 1983, 5 5 , 1526. Jakubowski, N.; Stuewer, D.; Toelg, G. Int. J . Mass Spectrom. Ion Proc. 1986, 71, 183.
(11) Caroli. S.; Alimonti, A.; Delle Fernmine, P. Spectrosc. Lett. 1979, 12, 871. (12) Caroli, S.; Senofonte, 0.; Alirnonti, A,; Zirnrner, K. Spectrosc. Lett. 1981, 14, 575. (13) Radmacher. H. W.; de Swardt, M. C. Spectrochim.Acta, Part B 1975, 3 0 8 , 353. (14) Kruger, R. A.; Bornbelka, R. M. ; Laqua. K Spectrochim. Acta, Part B 1980, 356. 589. (15) Dogan, M.; Laqua, K.; Massrnann, H. Spectrochim. Acta, Part B 1971, 268, 631. (16) Bubert, H.; Klockenkarnper, R. Fresenius’ Z . Anal. Chem. 1983, 316, 186.
R E C E ~ Efor D review jmuary 5 , 1987. Accepted ~
~6, 1987. ~ i This work has been supported financially by the Ministerium fur Wissenschaft und Forschung des Landes Nordrheinand by the Bundesministerium fur Forschung und Technologie.
Measurement of Fluorescence Lifetimes during Liquid Chromatography David J. Desilets, Peter T. Kissinger, a n d F r e d E. Lytle*
Department of Chemistry, Purdue Uaiversity, W e s t Lafayette, Indiana 47907
A method is presented whereby the fluorescence lifetime of an eluting compound can be determined “on the fly” during liquid chromatography. The measurement is made by using puised-laser excitation with sknuitaneous detemdnation of two values on the fluorescence decay. A ratio of these two decay values is used to determine the lifetime. Since chromatographic retention time and fluorescence lifetime are unrelated, complementary information about the identity of the analyte is obtained. Fifteen polycyclic aromatic hydrocarbons(PAH) are identified in a combustion sample by ushrg this technique. Effects of poor chromatographic resolution on the measured lifetimes are discussed, and optimization of instrument parameters is addressed.
Fluorescence detection is generally acknowledged as an appropriate tool for confirming or recognizing the presence of anticipated trace-level constituents separated by liquid chromatography (LC). Many organic compounds either exhibit native fluorescence or may be induced t o fluoresce by some derivatization scheme ( I ) . Frequently, peak identities are assigned on the basis of retention time alone even though, in principle, spectral information is available. This situation occurs with samples so dilute that the sacrifice of sensitivity for the bandwidth required to obtain a fully resolved spectrum becomes impractical. Often, the analyst using broad band detection must resort to replicate injections with different spectral parameters (excitation wavelength, emission filter) in an effort to confirm peak identities. Some of the loss in selectivity could be recouped by scanning a low-resolution monochromator during elution in order to optimize the wavelength match with the retention time of anticipated sample constituents. Alternatively, additional selectivity can be obtained by resorting to time-resolved fluorometric detection. Time resolution has already been used successfully to improve the signal-to-background ratio in fluorescence LC (2,3). However, these methods relied upon the instrumentation to reject only scatter and short-lived fluorescence from 0003-2700/87/0359-1830$01 S O / O
the signal. No new identifying parameter was measured. Clearly, the largest selectivity advantage in the time domain is realized by obtaining information about the lifetime of the eluting compound. As with spectral resolution this will also involve some degree of sensitivity loss as the temporal precision of the measurement is improved. Recently, we described a concentration-independent method for measuring fluorescence lifetimes in flowing solutions ( 4 ) . A natural extension of this technology would be to use it as a detection method in liquid chromatography. In this paper, we show that fluorescence lifetimes can be measured “on the fly” in liquid chromatography. In turn, the lifetimes provide the additional selectivity needed t~ confirm peak assignments based on retention time. I t should be emphasized that a combination of coarse measurments of the lifetime and retention time can yield more information about the identity of an unknown than extremely fine measurement of either one alone. Results of experiments with polycyclic aromatic hydrocarbon (PAH) standards of known identity are presented as a demonstration. In addition, 15 PAH are positively identified in combustion products, illustrating the applicability of this new method to real samples. EXPERIMENTAL SECTION Instrumentation. The instrument used to measure fluorescence lifetimes on the fly has already been described in detail ( 4 ) . Briefly, it consists of a pulsed nitrogen laser (Princeton Applied Research Model 2100) operated at 10 Hz, with 1.5-11s pulses fwhm. The 337.1-nm excitation radiation was focused into a custom-built, 20-wL flow cell (Hellma cells). Emission from the chromatographic effluent was collected by a J-Y H-20 monochromator, and an 8-nm bandwidth was selected and focused onto a photomultiplier tube (RCA 931A). Photomultiplier anode current was divided evenly with a power divider (General Radio), with both halves of the split signal used as input to a two-channel sampling oscilloscope (Tektronix 5S14N). To one channel of input, a 10-ns delay was added by inserting an appropriate length of coaxial cable (Figure 1). The second channel received the signal with no delay. A fraction of the excitation beam was diverted by a quartz plate to a fast photodiode (Texas Instruments TIED 56) whose output was used to trigger the oscilloscope. The amplified signals from 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987 Anthracene, Fluoranthene. Pyrene "
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Position of Oscilloscope Apertures for Ratio Measurement
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Flgure 2. Channel 1 and channel 2 chromatograms with ratiogram for three PAH standards. From left to right: anthracene, fluoranthene, and
pyrene.
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T h e (nanoseconds) Simultaneous Measurement of Two Points on Fluorescence Decay I
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Figure 1. (a) Simplified diagram illustrating how the signal from the delay. (b) Signals photomuttiplier is d i v i with half being given a 10-n~
seen by the two-channel sampling oscilloscope. (c) Effective monitoring of the fluorescence decay at two points in time by poising the sampling apertures at the point shown in b. the oscilloscope (both channels) were digitized with a Keithley Series 500 interface and stored by an IBM PC-XT. Liquid Chromatography. The liquid chromatograph (Bioanalytical Systems Model 152) was equipped with a 0.46 X 25-cm, reverse-phase, C18analytical column. Elution was isocratic with a mobile phase consisting of 80/20 acetonitrile/water (v/v before mixing). Chromatographic determinations were carried out at ambient temperature, with a flow rate of 1.4 mL/min. Samples were dissolved in acetonitrile, and a 20-pL injection volume was used. Fluorescence detection wavelengths varied depending on the experiment being performed. For absorbance detection at 254 nm, an Altex Model 153fixed-wavelength detector was used. Solvents. Water was distilled in glass (Corning Mega-Pure). Acetonitrile was spectroscopic grade (Baker Resianalyzed) and was fractionally distilled in glass prior to use. Both solvents were checked periodically for residual fluorescence and discarded if some was observed. PAH Standards. Standard compounds were obtained from a variety of commercial sources. Purity was assured by fluorescence and UV-vis absorption spectroscopy and by chromatography. Measurement of Fluorescence.Lifetimes. The method used to measure fluorescencelifetimes in flowing systems with changing concentrations has been described previously ( 4 ) . The photomultiplier anode current is divided evenly, and half of the signal is delayed by 10 ns before it is allowed to reach its channel of the sampling oscilloscope (Figure la). When the two-channel oscilloscope is made to scan the inputs, two identical fluorescence decays are observed 10 ns out of phase with each other (Figure
lb). If scanning is stopped, and the two sampling apertures are positioned at the location shown by the arrow in Figure Ib, two points on the fluorescence decay (produced from a single excitation pulse) may be observed simultaneously (Figure IC).It has been shown that a ratio of these two points ( F ( t 2 ) / F ( t lin ) Figure IC) is concentration independent, greatly decreases the contribution from excitation intensity fluctuations to the total noise, and is related to the fluorescence lifetime ( 4 ) . For chromatographic applications, the sampling apertures are positioned as described above, and two channels of data are continuously digitized and stored. Two chromatograms result, one of which is lower at all points to a greater or lesser extent than the other, with the degree of difference depending on the excited-state lifetimes of the fluorophores. A "ratiogram" is created by dividing the lower chromatogram by the upper at all points above a user-selected threshold. The threshold is used to prevent division by zero for meaningless points falling on the base line. In order to avoid a mathematical error, it is essential to subtract the base line offset from each peak before a ratio is obtained. This is especially true for peaks in the center of a complex chromatogram that appear to be resting on a shallow "bulge" in the base line. Failure to subtract the base line with result in ratios that are base line dependent and/or ratios with artificial curvature. The ratiogram yields values related to the fluorescence lifetime of each analyte. If the technique is to be used for peak identification only, it is sufficient to match the ratio of the analyte to that of one or more standard compounds having the same chromatographic retention time. If actual lifetimes are to be measured, a calibration curve of ratio vs. lifetime must be constructed. This can be done by collecting a ratiogram for compounds of known lifetime and plotting the measured ratios vs. the known lifetimes. One may also construct a calibration curve by measuring the instrument impulse response, generating by computer a family of convolutes spanning the lifetime range of interest, and plotting the ratio of two selected points on each convolute vs. the "lifetime" (exponential time constant) used to create the convulute ( 4 ) .
RESULTS AND DISCUSSION Standard Compounds. The results of two-channel, time-resolved monitoring of the chromatographic effluent are shown in figure 2. Notice that the ratios are relatively flat across the width of each peak despite large changes in analyte concentration. The average height of the ratio peak is proportional to the fluorescence lifetime. Figure 3 shows the chromatographically determined ratios of several PAH plotted as a function of the fluorescence lifetime measured in the same solvent by a different convolute and compare) method ( 5 ) . Note that the ratio technique, using the computer-generated calibration curve, agrees well with lifetime values obtained by the independent method (Table I). Quantitation. Analyte quantitation is performed in the usual fashion by using numerical integration of chromatographic peaks. The earlier of the two channels of raw data
1832 1.88
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987 Fluorsnthene
Conparison of Hearumd PAH Data to Calibration Curve
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Figure 3. Chromatographically obtained ratios for some PAH vs. independently determined Ilfetimes. The solid Hne is a cabration curve produced from synthetic convolutes.
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Figure 4. Expanded view of the two channels of raw data, and the ratio for a PAH standard (fluoranthene). Here, neutraldensity filters were placed in the collection optics (denoted by arrows) to illustrate the insensitivlty of the ratio to fluctuations in signal strength.
Table I
Benzo(e)pyFsne and Psrylene
compound
lifetime, ns
lifetime estd from ratiogram, ns
anthracene dibenzo[a,h]pyrene benzo[a] fluoranthene
4.3 6.0 6.6 15.4 16.8 17.8 18.8 19.2 23.2 23.9 24.3 28.0 30.0 41.6
4.4 5.5 5.8 15.8 16.7 17.4 19.7 18.5 26.5 23.7 24.3 26.9 28.7 43.7
measd
benzo[a]pyrene
chrysene dibenz[a,h]anthracene benz [a]anthracene dibenzo[a,e]pyrene dibenzo[a,i]pyrene benzo [e]pyrene benzo[ghi]perylene pyrene
fluoranthene coronene
is used for this purpose since the signal-to-noise is greater near the maximum of the fluorescence decay. Limits of detection vary with the compound but usually fall in the range of l e 5 0 ng/mL for PAH without taking any special precautions to set the emission wavelength at a band maximum. This corresponds to 0.2-1.0-ng injected, while still yielding a usable ratio. Other related studies of the same PAH samples yielded limits of detection of 2-48-ng injected with a Hewlett-Packard diode array absorbance detector, 0.4-4.8-ng injected for an Altex Scientific 254-nm absorbance detector, and 0.04-0.2-ng injected for a Kratos Analytical Instruments filter fluorometer detector. Sensitivity could be improved substantially by using a cutoff filter instead of a monochromator to increase the emission bandwidth. However, spectral selectivity would be lost, and the limit of detection might not improve. Also, 337 nm is not a particularly efficient excitation wavelength for most PAH containing three to five rings. A source which emits a t shorter wavelengths in the ultraviolet, such as a XeCl laser (Aex = 308 nm), would be expected to improve detection limits, provided that the average power of the excitation remained the same. Insensitivity to Source Noise. The ratio chromatograms are, within limits, quite insensitive to changes in excitation intensity. The intensity of the excitation was made to fluctuate by attenuating the laser with neutral-density filters or by stopping down the beam diameter with an iris. Since both channels of the two-channel detector are reduced by the same attenuation factor, no change is observed in the ratio (Figure 4). The same result is obtained with a drifting or misfiring laser as the source. Resolving Chromatographically Overlapped Peaks. Like absorption or emission wavelength ratios, two-point decay ratios are very useful for finding pairs of compounds which
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Figure 5. Two-component peak showing how the ratiogram can resolve overlapping species. are only slightly resolved by the chromatograph. Figure 5 shows a mixed peak consisting of the isomers perylene and benzo[e]pyrene. These compounds are partially resolved as shown by the ratio. Even when a peak looks perfectly symmetric, if there is a small amount of chromatographic resolution, it can often be picked up by the ratio. Unlike wavelength ratios from sequential injections, the time-resolution system allows two channels of raw data to be acquired simultaneously (much like a diode-array system monitoring at two wavelengths). The simultaneous data acquisition reduces the guesswork involved in ascertaining peak purity. Peak wandering in a second injection, even by only a few seconds, can ruin the shape of chromatographic ratios. Since the second injection is eliminated with decay ratios, slight changes in the flatness of a ratio are significant and can no longer be attributed to data files which do not overlap perfectly in time. For those few stubborn pairs of compounds that show virtually no chromatographic resolution under isocratic conditions, certain additional steps must be taken to ensure correct compound identification. One such step is to change to a new emission wavelength. Such a change varies the relative contributions of the individual decays to the measured sum of component intensities. Thus, a peak whose ratio changes when measured at a different wavelength is not pure and consists of at least two coeluting species with measurably different lifetimes and different emission spectra. On the other hand, a peak whose ratio is invariant with wavelength is not necessarily pure. If the lifetimes of the coeluting species are indistinguishable to within the precision of the measurement, the observed ratio will be unchanged regardless of grossly different spectral pattern or partial chromatographic resolution. Similarly, an invariant ratio could
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987 Flu Ash Extract
Table I1 compound
rt
anthracene fluoranthene pyrene benzo[b]fluorene chrysened benz [a]anthracened
benzoli]fluoranthene benzo[b]fluoranthene perylened Ninuter
Flgure 8. Chromatogram of combustionderived PAH. Peak identities are given in Table 11.
arise from compounds whose chromatographic peaks overlap "perfectly", and whose emission spectra are sufficiently alike so that the observed ratio does not change with wavelength even though the lifetimes are quite different from each other. Of course, if one of two overlapping components is in much lower concentration than the other or gives a smaller signal for some other reason, it will be difficult to resolve the pair just as with other types of detection. The possibility for determinate error exists if there is a fixed amount of quencher in the mobile phase, or if a peak coelutes with a nofluorescing quencher. However, the quencher would, in most cases, have to be in considerably higher concentration than the fluorophore for any quenching effects to be observed. When working with unknowns, ratios for peaks of mixed composition are obtained a t a different wavelength chosen to optimize the signal for each suspected compound in the presence of the interfering compound(s). Typically, a compromise wavelength (420 nm) giving measurable signal for most suspected compounds is selected, so that only one injection need be made. If we know from standard chromatograms that a sample peak could be composed of a pair of compounds, the ratio a t 420 nm is carfully examined. If it is not "flat", and all values of the ratio for that peak are between extremes dictated by the standards, then the presence of both compounds is indicated. If the ratio peak is flat, but remains somewhere within the bounds specified by the standards, then both compounds may still be assigned to that peak, especially if work with standards indicates that the compounds coelute "perfectly". In general, wavelength selectivity is not necessary, a t least for PAH, as we have found only two sets of compounds (benzo [b] fluoranthene/ benzo [e]pyrene/ perylene and chrysene/benz[a]anthracene) out of a total of approximately 25 compounds, that coelute so well that additional selectivity is needed. Those in the first group have quite different emission spectra, and changing the emission wavelength usually resolves them. The second pair poses a more serious problem because their lifetimes are nearly identical and they have similar emission spectra and thus are not easily resolved by measuring the ratio a t a different wavelength. For real samples, it is possible to determine that both compounds are present, but only because the analyst is forearmed with a priori knowledge that for this pair, a special resolution/lifetime/spectral problem exists, and that the two compounds nearly always occur together. Individual quantitation of chrysene and benz[a]anthracene is not possible with the lifetime instrument. It is expected that the two groups of compounds mentioned above are a t least partially resolvable with suitable packing material and gradient elution (6, 7). Analysis of a Real Sample. Figure 6 shows a chromatogram of the PAH fraction of a dichloromethane extract of
1833
benzo[k]fluoranthene benzo(a1pyrene dibenz[a,c]anthracene dibenz[a,h]anthracene benzo[ghi]perylene anthanthrened
peak PAH standards fly ash sample no." Rb f uc R i a 1 2 3 4 5 6 7
8 9 10 11 12 13 14 15
0.134 f 0.016 0.771 f 0.046 0.671 f 0.055 0.274 f 0.036 0.589 f 0.031 0.561 f 0.074 0.283 f 0.023 0.721 f 0.084 0.139 f 0.021 0.357 f 0.030 0.609 f 0.017 0.599 f 0.029 0.567 f 0.017 0.753 f 0.054 0.120 f 0.012
0.135 f 0.060 0.782 f 0.039 0.668 f 0.049 0.271 f 0.048 0.575 f 0.147 0.575 f 0.147 0.264 f 0.060 0.749 f 0.032 0.267 f 0.040 0.351 f 0.043 0.581 f 0.060 0.583 f 0.036 0.574 f 0.038 0.748 f 0.043 0.222 f 0.039
"Peak number in Figure 6. *Average value of the ratio across the width of the peak or part of the peak. Standard deviation of the ratio measurement for one injection. Corresponds to the "noise" in the ratiogram across a given peak. See text for explanation. combustion-derived fly ash (8). This sample had been characterized previously by GC-MS and LC-diode array spectrophotometry. Eighteen parent PAH are known to be present. Although the chromatogram is complex, we were able to identify 15 PAH in this sample based on measured ratios and retention times (Table 11). Since fluorescence lifetime and chromatographic retention time are unrelated, low-precision estimates yielded complementary information sufficient for the unambiguous identification of many of the components. Criteria for positive identification were as follows: (1) Retention times for standards and samples had to be equal to within a tolerance of less than 2% (Le., less than 1 2 s in 10 min). (2) Ratios (lifetimes) were required to be equal to within a tolerance of less than 1standard deviation (SD) of the ratio. For example, for fluoranthene (Table 11),the retention times were 376 and 382 s for the standard and sample, respectively. Peak identity was confirmed because the ratios were 0.771 f 0.046 and 0.782 f 0.039, that is, equal to within less than 0.039, the smaller standard deviation. Entries in Table I1 labeled with an asterisk have poorly matching ratios because these compounds were part of a coeluting pair. For example, the perylene ratio was made artificially high by the presence of an unknown compound (probably benzo[e]pyrene, but not confirmable). Similarly, the anthanthrene ratio was too high due to the presence of a t least one long-lived, coeluting species. Because the ratios for these peaks approached their true value as the emission wavelength was optimized to select for these compounds, their presence was confirmed. Some major peaks remained unidentified because none of our standards matched in retention time. Also, it is interesting to note that although we knew the isomers triphenylene and benzo[c]phenanthrene were present in this sample from a previous study [8], and even though appropriate standards were available, we were unable to confirm their presence with the ratio method because the lifetimes for the suspected peaks differed from those of the standards by more than one standard deviation. It is apparent that the presence of many interfering compounds in a complex real sample can impose some limits on the utility of this two-channel technique, especially in the absence of gradient elution. It could also be true that this flat ratio is due to a pure compound not contained in the standard set. Previously, using liquid chromatography with diode-array spectroscopy, we were able to identify 18 PAH in this sample,
Anal. Chem. 1987, 59, 1834-1841
1834
which were accessible with the nitrogen laser (8). We still possess standards for 16 of those compounds, and of those 16, 1 2 were conclusively identified by the two-point lifetime technique. With the aid of new standards, three additional PAH were found. It is interesting to note that there were many other compounds in our standard set that were not identified in the sample by either method. That is, no compound listed as clearly absent by the diode-array technique was subsequently found by using the ratio method. The agreement between the two different techniques is extremely encouraging. Work is now in progress to expand the application of the new method to other systems.
LITERATURE CITED (1) Seitz, W. R.; Frei, R. W . CRC Crit. Rev. Anal. Chem. 1880, 18(14), 367-405.
(2) Richardson, J. H.; Larson, K. M.; Haugen, G. R.; Johnson, D. C.; Clarkson, J. E. Anal. Chim. Acta 1880, 116, 407-411. (3) Imasaka. T.; Ishibashi, K.; Ishibashi, N. Anal. Chim. Acta 1982, 142, 1-12. (4) Desiiets, D. J.; Coburn. J. T.; Lantrip. D. A.; Kissinger, P. T.; Lytie, F. E. Anal. Chem. 1988, 58, 1123-1128. (5) Lytle, F. E. Photochem. Photobiol. 1973, 17, 75-78. (6) Sander, L. C.; Wise, S. A. In Advances in Chromatography; Giddings, J. C., Grushka. E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1986; Voi. 25, pp 139-218. (7) Wise, S. A.; Bonnet, W. J.; May, W. E. I n Polynuclear Aromatic Hydrocarbons: Chemistry and Siological Effects; Bjorseth, A,, Dennis, A. J.. Eds.; Battelle Press: Columbus, OH, 1980; pp 791-806. (8)Desiiets, D. J.; Kissinger. P. T.; Lytle, F. E.; Horne, M. A,; Ludwiczak, M. S.; Jacko, R. B. Environ. Sci. Techno/. 1984, 18, 386-391.
RECEIVED for review August 7 , 1986. Accepted April 6, 1987. This work was supported in part by the American Cancer Society, the Indiana and the National Science Foundation, Grant CHE-8320158.
Photochemical Amplifier for Liquid Chromatography Based on Singlet Oxygen Sensitization Curtis L. Shellum and John W. Birks*
Department of Chemistry and Biochemistry and Cooperative Institute for Research i n Environmental Sciences ( C I R E S ) , Campus Box 449, University of Colorado, Boulder, Colorado 80309
A postcolumn photochemical reaction scheme designed to enhance the detectability of UV-absorbing compounds has been coupled to high-performance liquid chromatography (HPLC). Specifically, the method detects members of the large class of organic compounds termed “singlet oxygen sensitizers”. These compounds transfer excitation energy to ground-state oxygen, forming the excited singlet species, O,( -let oxygen in turn reacts with a substituted furan such as 2,54lmethylfuran (DMF) or 2,l-diphenylfuran (DPF), and UV absorption or fluorescence is used to detect either the loss of reactant (DMF or DPF) or appearance of a product. The reaction sequence is photocatalytic In nature, resulting in a large chemical ampllflcation of the signal. Detection h i t s are Improved by 1 to 2 orders of magnitude for a wide variety of UV-absorbing compounds. Discussed in this report are the theory and characterization of the detectlon system as well as its application to several classes of compounds including polycyclic aromatic hydrocarbons, substituted anthracenes, anthraquinones, and polychlorinated biphenyls (PCBs).
‘4).
Sufficient detection sensitivity is not currently achievable for many types of molecules separated and analyzed by HPLC. Particularly significant are UV-absorbing organic molecules having low or negligible fluorescence quantum yields as a result of high quantum efficiencies for singlet-to-triplet intersystem crossing. At low temperatures or in organized media such compounds are often phosphorescent. The excited triplet to ground singlet state transition is spin forbidden, however, yielding an inherently long triplet state lifetime of typically to 10 s (1). More rapid deactivation processes prevent phosphorescence under the usual conditions in HPLC of room temperature and the presence of dissolved oxygen. Oxygen quenches the excited triplet state at a diffusion-controlled rate
-
and is present at a level of M in most solvents ( 2 , 3 ) . Phosphorescence has been used for detection in HPLC, however, under conditions of virtually complete oxygen removal. The approaches include micelle-stabilized room-temperature phosphorescence ( 4 ) ,sensitized room temperature phosphorescence (5),and phosphorescence quenching (6). The necessity of rigorous oxygen removal is a disadvantage of all of these detection methods. Furthermore, these approaches have not resulted in substantially improved detection limits as compared to conventional UV absorption. This paper describes an approach that uses to advantage the triplet quenching ability of molecular oxygen. Groundis promoted to an excited singlet state, state oxygen, 02(3C.J, either Oz(l&+) or O#$) (2). O#.&,+) decays to the metastable 02(lAg) state within s (7). The OZ(lAg)species is involved in a variety of photochemical oxidations, some of the most rapid of which occur with substituted furans such as 2,5-dimethylfuran (DMF) and 2,E~diphenylfuran(DPF). Figure 1presents absorption spectra of four furans including DMF and DPF. These compounds may serve as “singletoxygen traps”. The analytical signal may be based on a decrease in concentration of the furan or an increase in concentration of the oxidation product, as measured by a spectroscopic property such as UV absorption or fluorescence. A large sensitivity advantage results in that the analyte molecule may absorb light many times and produce large numbers of singlet oxygen molecules during its residence time in a postcolumn photochemical reactor. As a result, detection limits for a wide variety of UV-absorbing compounds may be improved by between 1and 2 orders of magnitude by using this photochemical reaction scheme. The only modification to a standard HPLC apparatus is the insertion of a photochemical reactor between the analytical column and the detector. The compound serving as a singlet oxygen trap (substituted furan) is spiked into the mobile phase M. The at a concentration typically in the range to
0003-2700/87/0359-1834$01.50/0 0 1987 American Chemical Society
