Anal. Chem. iga2, 5 4 , 2486-2491
2486
Multicomponent Mixture Analysis by Multidimensional Phosphorimetry Chu-Ngi Ho and lslah M. Warner”’ Department of Chemistry, Texas A&M University, College Station, Texas 77843
A technique based on the rapid scanning capability of the video fluorometer to acquire an emlssion-excitation matrix (EEM) has been developed for phosphorlmetry. This technique overcomes the problem of convoiutlon of time decay with phosphorescence excitation and emission spectra by integrating the signal on target, This method also circumvents the need for very rapid acquisition of data for samples with phosphors of very short lifetimes. A phosphorescence emission-excitation matrix (PEEM) obtained in this manner permits time resolutlan. Bets of each time-PEEM allow a ratio deconvolution aigorlthm to successfully resolve mixtures of polynuclear aromatic compounds.
The sensitivity and selectivity of luminescence spectrometry are well-known and the technique has been widely applied. The theory of phosphorescence was first described by Lewis and Kasha in 1944 (1). The applicability of phophorimetry for analysis was convincingly demonstrated by Keirs and co-workers in 1967 (2). Since then, phosphorimetry has gradually gained general acceptance as an analytical tool of merit. A number of publications in this area readily confirms this observation (3-6). The routine detection of room-temperature phosphorescence in diverse matrices (7-10) is now lending further impetus to development of a wider range of applications for phosphorimetry. In recent years, the need for multiparametric approaches to analyses of complex samples has stimulated the development of hyphenated instrumentation. Parallel technological advances in electronics, computers, and data reduction algorithms have made the acquisition of such multidimensional data possible. Wilson and Miller (11) have reported the technique of simultaneous time and component resolved phophorimetry where they obtained in essence a complete emission spectrum for each time interval on the decay curve of a phosphor. Such two-dimensional data enhance the capability of phosphorimetry for multicomponent analysis because one can now resolve components with spectral overlap by differences in their decay times and vice versa. Goeringer and Pardue (12)improved upon this technique by developing a rapid scanning phosphorimeter using a vidicon array detector. In this manner, the speed of data acquisition is increased tremendously. They also applied regression analysis to treat their data and were able to successfully analyze multicomponent mixtures whose components phosphoresce at room temperature. The use of rapid scanning spectrometers to obtain a redundant data base suitable for higher order data reduction schemes represents a major trend to effectively achieving analysis of complex multicomponent samples. A unique fluorometer, the video fluorometer, fiist developed by workers at the University of Washington (13,14) is capable of rapidly obtaining an emission-excitation matrix (EEM). The EEM is essentially a mapping of luminescence intensity Present address: Department of Chemistry, Emory University, Atlanta, GA 30322. 0003-2700/82/0354-24813$01.25/0
as a function of multiple excitation and emission wavelengths simultaneously. Another video fluorometer has also been described (15). Diverse applications of this video fluorometer have been reported (16-19). Recently, it has been extended to permit acquisition of a phosphorescence EEM (PEEM). A set of PEEM acquired along the temporal domain allow time resolution of an entire PEEM. Consequently, the applicable term multidimensional phosphorimetry is used (20). The detailed graphic representation and mathematical treatment of an EEM along with applications for qualitative (19,211 and quantitative (22,23) analysis have been published. The problems associated with energy transfer and quenching in the EEM have been discussed (17,21) and are applicable here. For ease of reference and continuity only a few equations pertinent to our discussion are presented here. An element of the EEM of a pure single component solution is given by M,, = axY.l (1) where ML,represents the luminescence intensity excited at wavelength A, and emission monitored at wavelength A,, while a is a concentration-dependent parameter. The parameters, x, and y!’ are elements of the emission and excitation spectrum, respectively. If the experimental conditions are such that synergistic effects are negligible and Beer’s law applies, then the luminescence intensity for a mixture of “r” components will be a linear sum of emission intensities contributed by each of the luminescing constituents present, i.e. r
M=
CakXkYkT k=l
(2)
where k denotes the kth component, x k and Y k are column vectors consisting of ordered values of Xkl’S and Y k J ’ S , respectively. The symbol T denotes matrix transposition. Using similar notation, one can define a standard EEM for the kth component N k
= akoXkYkT
(3)
of the mixture matrix r
Mi =
C a k / N k
k=l
(4)
where ak/ = akJ/ako (5) For ratio deconvolution (19) of an r-component mixture, one needs a set of “r” EEMs, i.e., a series of Ml for 1 = 0, 1, 2, ..., r - 1. Thus, one can obtain a set of “r” matrices, each of which contains a maximum of only “r” emitters. Then, one has a set of ‘7’’ equations in “r” unkowns, Le. M* = AN* (6) where M* and N* are a series of mixture (Ml) and standard ( N k )matrices, respectively. The matrix A is an r X r array of A solution can be found if A is invertible such that
A-1M* = N*
(7)
Experimentally, there are a number of ways to vary the to obtain a set of Ml. High-performance liquid chromatog0 1982 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, I
I
COOLED HOUSING
NO. 14, DECEMBER 1982 2487
loC
SYSTEM
hI \
n
78
II
z
L
50
25
C liB
DEWAR
.;b
t i c
TIME
Figure 2. Graphical representation of target integration process for data acquisition. Figure 1. Block diagram of video phosphorirneter. raphy (HPLC) (18)and quenching (19) have been shown to be viable approaches. For phosphorescence, time resolution of the PEEM is particularly well suited to acquisition of such a set of matrices. This i s because the decay times for many compounds differ and thus the phosphorescence intensity Qf each compound, observed at different time intervals after the excitation of the mixture has been terminated, should ideally be different. Thus, for a given concentration of each phosphor, eq 5 defines the ratio of the phosphorescence intensity at two different time delay intervals. Hence, the set of ah[ will usually form an invertible matrix, A.
EXPERIMENTAL SECTION Reagents. The polynuclear aromatic compounds used in this study, coronene, phenanthrene, and triphenylene, were direct dilutions of the solutions obtained in a polynuclear aromatic hydrocarbon kit from Chemistry Service Inc. (West Chester, PA). The solutions were used directly without further treatment. The solvent used was EPA, a 5 5 2 by volume mixture of ether, isopentane, and alcohol. The solvents were all of spectral grade quality and the EPA made from them did not have a detectable phosphorescence background. Apparatus. The vidiso fluorometer used here has been described (15). Minor modifications have been made (20). Figure 1shows the modified conifiguration of the video fluorometer for our experiments. The cuvette has been replaced by a Dewar with a cryogenic sample cell for operation at liquid nitrogen temperature. The sample cells are not the small bore cylindrical type. For reduced scattering, special rectangular cells were constructed in our glass shop. A long length of 10 mm X 10 mm square, solid quartz tube, was cut into lengths about 6.25 cm long. The bottom of each tube was sealed and the walls were maintained as parallel as possible. For the top of this cell, a round tube of 6 mm (i.d.) vycor of appropriate length was fused to the quartz. Since the tubing used for cell fabrication was solid quartz, the cells were found to withstand thermal shocks remarkably well and the samples formed a clear ,glass without significant, detrimental cracks. To prevent fogging of the Dewar due to condensation, gentle jets of dry air were blown over the window surfaces. Electronic shutters were installed at the exit of the excitation polychromator and in the path of the emission beam in front of the entrance of the emission polychromator. ‘The emission shutter was primarily used to circumvent the problem of detector lag. The shutters were opened or closed by manually activating a switch and were capable of being controlled separately. Thus, the excitation shutter may be closed to cut off the excitation beam and the emission shutter can remain closed until a desired delay period has elapsed before it was opened to allow phosphorescence to reach the detector. This two shutters may also be opened and closed alternately or simultaneously to allow alignment of optics as desired. Data Acquisition. The video fluorometer acquires a 2500 point EEM of 50 excitation wavelengths and 50 emission wavelengths in about 0.5 s. The excitation of the sample is terminated
to record only the phosphorescence signal. As soon as this occurs, the intensity of phosphorescence ideally decays exponentially according to the lifetime of the phosphor. If scanning of the camera is started as soon as the excitation of the sample is removed, the intensity of phosphorescence of the constituents would have been diminished, by the end of the scan, to an amount depending on the respective lifetimes of the phosphors. Thus, the PEEM obtained would likely be distorted, i.e. the excitation and emission spectra will have been convoluted with the readout process. Let us qualitatively examine this distortion using the first-order exponential decay equation
where 7 is the lifetime of the phosphor and Iois the initial intensity (at t = 0) and It is the intensity at any time t. We can easily calculate the lifetime of the phosphor if we assume that the change in the phosphorescence intensity is not to exceed 10% in 0.5 s (camera readout time). Thus, the lifetime should be greater than 5 s. This restriction would severely limit the scope of applications of the technique. This problem could be overcome if faster scanning were possible. However, a simpler solution which allows us to keep our present instrumental configuration is to use the integrating capability of the vidicon. Within the integratable range of the camera, we can show that the integrated PEEM preserves the integrity of the PEEM. For example, we can rewrite eq 8 in the form
I, = Ioe-kt
(9)
where k = 1 / and ~ represents the rate of decay. The integrated intensity between the time interval tl and tz ( t z> tl) for a single component with rate constant kl is given by
(10)
For an ideal r-component mixture we derive (11)
Then, the integrated PEEM is
Thus, eq 11effectivelyrepresents a multiplication factor, which is exactly what is needed for ratio deconvolution (19). Thus, it is possible to integrate the phosphorescence signal on target for any desired length of time below the saturation point of the camera. The spectral information is then scanned and recorded
2488
ANALYTICAL CHEMISTRY, VOL. 54, NO. 14. DECEMBER 1982
after an initial 15 frames of 'prep" scan. In the integration mode, the analug-to-digial(A, D)converter is disabled during the 'prep" scan and the electron beam is blanked during target integration. The electron heam scans the target and the AID is enabled when the final scan reads the accumulated signal after the prescribed integration is achieved Similarly,a blank PEEM is obtained with the emission shutter closed. This blank PEEM is taken immediately after each signal PEEM and is subtractcd from the signal PEEM. An actual experimental run would typically be as follows. The camera target is cleaned first by repeated normal scanning. Meanwhile, the sample is illuminated with the emisxion shutter closed such that nu signal is observed hy the detection system. When the sample has been excited to a steady state. the excitation shutter is closed and simultaneously the emission shutter ISo p ened. Alternatively. opening of the emission shutter may be delayed for a predetermined length of time. After a desired delay is attained. the camera is then activated to start the prep scan followed by target integration. When this process is over the PEEM is stored on the floppy disk. A hlank PEEM is then ohtained with the emission shutter closed. This hlank is subtracted from the PIXM just gathered and the resulting PEEM is stored on the disk. This background subtraction wa.. found to he satisfactory since phosphurexcence was not dekctihle in the EPA solvent. The timine of the delav has been done with a stoowatch and the shutter switch was manually operated. Untii the entire procedure is completely computerized and automated, the timing will not he exactly reproducible. We estimate our accuracy to he hetter than *lo%. However, the level of accuracy does not affect the data for application of the ratio deconvolution and to illustrate the usefulness and versatility of multidimensional phosphorimetry.
A
B
C
Flgure 3. PEEMs of standard compounds: triphenylene. (C) coronene.
(A)
phenanthrene,
(B)
at the normal rate. The length of the integration and the period of delay hefore integration actually begins, Le., the time interval between closing the excitation shutter and opening the emission shutter, can be varied to achieve time resolution. Figure 2 is a graphical representation of this concept. The parameter "6t" is the target integration time. Different time delays ( t = a, b, c, etc.) are achieved by repeating the experiment. Emrimental Procedure. Each PEEM was acquired at liquid nitrogen temperature,except as noted, with 10 integration frames A
RESULTS AND DISCUSSION To demonstrate the applicability and usefulness of multidimensional phosphorimetry, three sets of synthetic data were acquired and analyzed. The three polynuclear aromatic M), hydrocarbons used for this study were coronene phenanthrene (1W7M), and triphenylene (lo4 M). The pure PEEMs of these compounds are shown in Figure 3. From these three compounds, three samples, consisting of a single component system, a binary mixture, and a ternary mixture, were prepared. The PEEMs of phenanthrene and triphenylene overlap rather severely while coronene is quite isolated with respect to its major emission bands. I n terms B
T
*sz 333
*
C
Figure 4. Sequence of pure phenanthrene PEEMs depicting time resolutlon: (A) t = 0 s; (6) t = 5.5 s: (C) t = 12.5 s; (D) t = 17.5
S.
ANALYTICAL CHEMISTRY, VOL.
54. NO. 14, DECEMBER 1982
2489
A
C
Flgure 5. Time-resohred %EMS of binary mixture of phenanthrene and trlphenylene: (A) t = 0 8: (6) t = 5.5 s: (C) t =
12.5 s; (D)
t=
17.5
8.
0
a:!,
B ,157
,
f
Flgun 6. Ratb deconvolution of mixture shown in Figure 5 (A) phenanthrene; (B) triphenylene. of lifetime, triphenylene is the longer lived of the ternary mixture while coronene is intermediate and phenanthrene is shortest. Figure 4 very succintly demonstrates the use of our timeresolved scheme for rapid acquisition of PEEMs. The times indicated on the figures correspond to the time of delay. This figure corresponds to a sequence of time resolution PEEMs for the pure phosphor, phenanthrene. Figure 5 shows a set of time-resolved PEEMs corresponding to a binary mixture of the phasphors, triphenylene and phenanthrene. In this w e , time resolution of the mixture is not readily apparent. However, as shown in Figure 6, despite the strong spectral overlap, the results of ratio deconvolution of the binary mixture using PEEMs a t t = 0 and t = 5.5 s are extremely good. Another example (Figure 7) shows the capabilities for a ternary mixture of coronene, phenanthrene, and triphenylene. In this data set, one can clearly recognize that the phosphorescence of phenanthrene is almost completely diminished after 18 8. After 48 s, only the emission of triphenylene is discernible above the noise. The results of ratio
C
Figure 7. Time-resoivedternary mixture of coronene, phenanthrene, and triphenylene: (A) f = 0 s; (B) t = 5.5 s: (C) t = 12.5 s. deconvolution of this mixture using the set of PEEME at delay times, t = 5.5,12.5, and 17.5 s are shown in Figure 8. The three components are clearly separated. There are some residuals of triphenylene signal in the deconvoluted EEM of coronene and vice versa. Also, the noise levels of the deconvoluted EEMs are higher. This is partially due to the lower normalization factor for this plot and also partially a consequence of the ratio deconvolution procedure (19). This set of data clearly demonstrates that the combination of timeresolved PEEMs and ratio demnvolution is able to accomplish
2490
ANALYTICAL CHEMISTRY, VOL. 54, NO, 14,DECEMBER 1982 A
A
8
B
I
C
Flgure 8. Ratio deconvolution of mixture shown in Flgure 7: (A) ccfonene: (E) phenanthrene: (C) triphenylene.
highly satisfadory qualitative separation of mixtures with components which have strong spectral overlaps. The real utility of multidimensional phosphorimetry can only he demonstrated with a truly complex sample. Figure 9A is a display of the room-temperature eminsion-excitation matrix of the extract of a burned oil residue dissolved in EPA. The sample preparation procedure has been described previously (24). Figure 9B is a spectrum of the same sample obtained at liquid nitrogen temperature. The complexity of the mixture has increased since several areas of enhanced emission can he observed. In addition, the overall emission intensity has approximately doubled, largely due to decreased oxygen quenching under these cryogenic conditions. It is interesting to note that much of the phosphorescence occur8 in the m e spectral region as the emission in Figure 9 k Thus, the phosphorescence is likely contributed by components whose fluorescence is outside the accessible range of the video fluorometer, i.e., short A., The complexity of the spectrum in Figure 9B can be significantly reduced using our delayed integration procedure. Figure 9C is a PEEM obtained though our targel in-ation procedure, 9 s after closing the excitation heam. It appears that this procedure has reduced the mixture down to a single component which, unfortunately, is not in our library. However, the excitation spectrum is similar to a phenanthrene derivative previously identified in this sample (24). In addition, the ohserved phosphorescence s p e d " is consistent with substituted phenanthrenes (25, 26) when compared to the phenanthrene of Figure 3. This study has demonstrated the general utility of multidimensional phosphorimetry for multicomponent analyses. Phosphorescence information can he obtained about compcnents that do not fluoresce in the accessible range of the video fluorometer. In addition, the complexity of the sample can often he reduced down to a single component using time resolution. A particular advantage of the time-resolution
C
Figun 9. Embian-excHatbnmatricesof bum6d oil W u e : (A) man temperature: (E) IiquM nmogen temperature: (C) time delayed (t = 9 s) PEEM of Flgure 96.
approach described here is that shor&lived phosphors do not cause significant convolution problems in the acquisition of the PEEM. One can easily conceive of an algorithm which would sequentially analyze for various components after appropriate time delays. Of course, this approach would require precise computer control of the emission and excitation shutters. Such a project is currently under way.
ACKNOWLEDGMENT The authors are also grateful to Mae E. Rollie for technical assistance.
LITERATURE CITED (1) Le&. 0. N.; Kasha. M. J . Am. chsm. SOC. 1944, 66. 2100-2116. (2) Kdrs).R. J.: Bml. R. D.. Jr.: Wentwath. W. E. Anal. chem.1957,29. 202-209. (3) Parker. C. A,: Hatchard. C. G. Analysf(LMdon) 1962,8 7 , 664-676. (4) Harbaugh, G. D.: ODonndl. C. M.: Wnetwner, J. D. A d . chem. 1947,46, 12051209. (5) Boutilk. 0.0.;whletmer. J. D. Anal. chem.197% 51. 1384-1390. (6) Cortield. M. M.: Hawkins. H. L.; John. P.: Soutar. I. Am@t (LMdon) 1981. 108, 188-197. (7) ~ i i iJ.~N.. r d s AMI. m.1981, 1 . 31-34. (8) Cline Love. L. J.: Skrilec. M.: Hahrta, J. 8. Anal. Wwn. 1980. 52, 7I" I"--,-1.
Ford. C. D.; Hurtabiaae. R. J. Anal. chem.1980,50,656-662. Vo-Dlnh, T.: Wlnetordner, J. 0.A@. .%CVOSc. 1977, 13,261-294. Wilson. R. M.: Miller. T. L. Anal. Cham. 1975. 47. 256-266. meringer. 0.E.; Pardue, H. L. Anal. M r m . 1979. 51, 1054-1060. Wamer. 1. M.: Callis. J. 0.; Davklson, E. R.; Gouterman. M.; ChrisUatian. G. D. A"#. Len. 197s. 8 . 665-681. Johnson. D. W.: Gladden. J. A,: Callis. J. 8.: Chrktmn. 0. 0.Rev. Scl. Instrum. 1979. 50, 118-126. Warner. I. M.:. Fmm. ,. M. P.:. Shellv. .. D. C. Anal. Chbn. Acfa 1979.
I&. 3ii-372.
-
Shelly. D. C.: Warner, 1. M.: (IUBrlBs. J. M,~Cfkl.chem.(WmtOn-Salem, N.C.) 1980. 28. 1419-1424. Fogarty. M. P.;Wamer. 1. M. @PI. Spscbwc. 1980, 34. 438-445. Fogarty. M. P.;Shelly. D. C.; Warner, 1. M. MRC CC J . HkJh R&. Chromatog. Chromamg.. C o " . 1981. 4 . 561-568. Faearty. M. P.; Wamer. I. M. Anal. C M . 1981,53, 259-265. Ho, C.-N.; Wamer. 1. M. T d Anal. Chem. 1982. 1 , 159-103. Warner. 1. M.: Callis. J. 8.; Christian. G. D.: Davidson, E. R. Anal. Chem. 1977. 49, 564-573.
Anal. Chem. 1982, 5 4 , 2491-2495 (22) Warner, I . M.; Davldam. E. R.; Chrlstlan, (5. D. Anal. Chem. 1977, 49, 2155-2159. (23) Ho, C.-N.; Chrlstlan, G D.; Davldson. E. R. Anal. Chsm. 1980, 52, 1071-1079. (24) Shelly, D. C.; Fogarty, M. P.; Warner, I. M. HRC CC J . High ResoM. Chromafogr. Chromatogr. Commun. 1981, 4 , 616-626. (25) Cherkasov, A. S. Bull. Acad. Sci. USSR,Phys. Ser. (Engl. Trans/.) 1956, 20,436-439.
2491
(26) Cherknsov, A. S. Opt. Spekfrosk. 1959, 7 , 211-214.
RECEIVED for review June 16,1982.
Accepted September 13, 1982. This work was supported in part by grants from the Department Of and the Office of Naval Research.
Inductively [Coupled Plasma Emission Spectrometry with Internal Standardization and Subtraction of Plasma Background FIuctuat ions8 Gary J. Schmldt" anid W. Slavln The Perkin-Elmer Corporation, Norwalk, Connecticut 06856
The Improvements resulting from the use of Internal standardlzation In lnductlvely coupled plasma emission spectrometry have been experlnientaily determlned. I n addlllon, we have also slmultaneousiy measured plasma background fluctuations and corrected analyte signals for these variatlons. These technlques have been applied to the determination of several elements In various matrlx solutions and some USGS water samples and have resulted In signlflcant Improvements In both measurement preclslon and accuracy. When determining Mn in a 5 % NaCl solutlon, precision was Improved 25-fold when using internal standardization. Correlation coefficients better than 0.99 were obtained for flve elements determined In the USG!; water samples.
Since its introduction ( 1 , 2), inductively coupled plasma emission (ICP) spectrornetry has grown rapidly and has become an extremely valuable technique for trace element determination. Its usefulness has extended into many areas of interest, including the analysis of geological, environmental, biomedical, and agricultural materials. A review of many ICP applications has been given (3). A number of reports have appeared in the literature describing the use of ICP for determining trace elements in water samples including wastewaters ( 4 , 5 ) , natural waters (6, 7), and seawater (7-10). Difficulties that confronted the analyst when developing these methods included choosing the proper analyte emission line to reduce interelemeint interference and to provide adequate sensitivity and choosing the proper background correction intervals to minimize continuum or wing-overlap spectral interference. The difficulties encountered in fulfilling these requirements have been minimized by the use of modern computer-controlled instrumentation. Of equal importance to the precision and accuracy of the methodology is the choice of appropriate standardization and calibration procedures. Samples consisting of relatively pure aqueous solutions may usually be standardized with simple aqueous standards. However, samples which contain complex or variable matrix constituents may lead to falsely reduced analyte concentrations due to the effect of matrix components on the various plasma processes. In these situations, careful matrix matching of stand'ard is required to ]provideacceptable results. Unfortunately, mmples are usually highly variable
and the matrix is largely unknown. Internal standardization has been used to compensate for these changes in analyte emission intensity as well as to improve measurement precision by correcting for various plasma noise sources. Since many of these noise sources affect different analytes similarly, adding an element as an internal standard permits the emission signal from the analyte to be corrected by monitoring the internal standard signal. This results in improvements in analytical precision and accuracy. By determination of the ratio of the emission intensities of the analyte element and the internal standard, much of the noise associated with the ICP measurement can be compensated for. Barnett et al. (11,12)have studied both theoretically and experimentally the various parameters associated with selecting acceptable analyte/internal standard element pairs. Watters and Norris (13) have discussed the instrumental factors which contribute to random error in emission measurements and have noted some of the advantages of internal standardization. Several workers have used internal standardization to improve the quality of the emission measurement. Feldman (14) found a 2- to %fold improvement in precision and improved analytical accuracy when using internal standardization. Uchida et al. (15) used Y as an internal standard and found a 2- to 20-fold improvement in precision for some elements when using a microsampling technique with 5-pL sample volumes. Skogerboe and Coleman ( 1 6 ) evaluated the use of a microwave-induced plasma for multielement emission analysis and used In as an internal standard. They found a significant improvement in analytical accuracy even when large amounts of sodium were present in the sample. Salin and Horlick (17) noted that the use of internal standardization either improved or degraded precision depending on the choice of analyte/internal standard emission line pairs. The correct choice of internal standards has received considerable attention, Barnett et al. (11,1.2)proposed guidelines for choosing internal standards based on consideration of the ionization energy, excitation energy, and partition function of the elements. Recently, Myers and Tracy (18)have shown that by the proper choice of ICP operating parameters a single internal standard element improved the analytical performance independently of whether an ion or neutral atom line was ehosea fci the analyte.
0003-2700~82/0354-2491$01.25/0 0 1982 American Chemlcal Society
