2018
Anal. Chem. 1982, 5 4 , 2018-2022
Constant Energy Synchronous Fluorescence for Analysis of Polynuclear Aromatic Hydrocarbon Mixtures Eugene L. Inman, Jr., and James D. Winefordner" Department of Chemistty, University of Florida, Gainesville, Florida 326 1 1
Constant energy synchronous luminescence has been developed to Improve the selectivity of lumlnescence technlques. I f we scan the excltatlon and emlsslon monochromators simultaneously, synchronized so that a constant energy dlfference Is malntalned between the wavelengths, conslderable Improvements are made over conventlonal synchronous technlques. The foundational concepts of this method are descrlbed, correlatlng experlmental parameters wlth luminescence theory. Experlmental parameters are, therefore, more easily optlmlzed.
Synchronous luminescence spectrometry has been described as a method to improve the selectivity of conventional luminescence spectrometry by taking full advantage of the ability to vary both the excitation and emission wavelengths during an analysis. In this method, the excitation and emission monochromators are scanned simultaneously, synchronized so that a constant wavelength difference is maintained between the monochromators. Since its introduction by Lloyd (1-4), synchronous luminescence has been used in the analysis of crude oils (5-9), polynuclear aromatic hydrocarbons (PAHs) (10, I I ) , and pharmaceuticals (12). These applications have made use of fluorescence, low-temperature phosphorescence, and room-temperature phosphorescence. Development of the technique and the selection of optimal instrument parameters have been based on empirical results without full use of theoretical considerations. Selection of the optimal wavelength difference between monochromators, Ax, is perhaps the most important parameter in the successful application of synchronous luminescence to multicomponent solutions. In 1978, Weiner suggested the use of total luminescence to optimize the selection of A 1 in synchronous luminescence (13). Total luminescence describes the acquisition of luminescence emission spectra over a range of excitation wavelengths (14).Instrumentation and mathematical data manipulation methods have been described in some detail (15-18). The data collected by synchronous luminescence can be represented as a line in the total luminescence plane. Thus an inherent relationship exists between the two methods. While synchronous luminescence spectrometry offers reduced spectral complexity over conventional luminescence spectrometry, much of the information contained in the three-dimensional total luminescence plot is lost. An attempt was made to improve synchronous luminescence by an analysis of total luminescence plots. For this, PAHs were selected because of their analytical importance and their extensive spectral features characteristic of this class of compounds a t room temperature and reduced temperatures (77 K). Thus PAHs were selected as a model system for this analysis. The evaluation of total luminescence data and relatiosships that exist between spectral regions of PAH solutions has led to the development of the new method, constant energy synchronous luminescence (CESL). In this method, the excitation and emission monochromators are scanned simultaneously, synchronized so that a constant energy difference,
Aij, is maintained between the monochromators. Evaluation of the applicability of this method to PAH analysis has been completed and is presented herein.
EXPERIMENTAL SECTION Materials. The PAHs used were obtained from various sources as listed in previow work (19). All reference spectra were obtained from PAH solutions of 10-100 ng/mL in n-heptane. n-Heptane used was distilled-in-glass grade (Matheson, Coleman & Bell, Norwood, OH) and used as received. Instrumental Analysis. All fluorescence spectra were obtained on a laboratory-constructedfluorimeter interfaced to a PDP 11/34 minicomputer. A complete list of experimental equipment and manufacturers is given in Table I, including operating conditions. The excitation monochromator was blazed at 250 nm, with the emission monchromator blazed at 450 nm, optimum for PAH analysis. The fluorimeter was operated in a computercontrolled mode, with monochromator control via a parallel digital interface to the PDP 11/34, and data collection through the analog-to-digital converter of the LPS-11. FORTRAN callable subroutines used for control of the data collection process were obtained from Digital Equipment Corp. All programs used for instrument control, data manipulation, and two- and three-dimensional plotting were written in FORTRAN by the authors. Total luminescence spectra were obtained by using repetitive emission scans, stepping the excitation monochromator through the range of interest (20). While this is laborious (and many alternative instrument designs show substantial improvements over this design (21)),it proved sufficient for the present purposes. Data representation was achieved via isometric projections and contour plots of the total luminescence matrix on an x-y digital plotter. The contour plots use alternating contours of dark and white to represent regions of increasing intensity. While limited, this method of plotting requires few calculations and a small amount of memory because only two emission scans are required in memory at any one time. All spectra are uncorrected for instrument response and source intensity. While corrected spectra would change the relative intensities of spectral peaks, it would in no way change the conclusions drawn. The exact relative peak intensities do not affect the principles discussed throughout this work. In order to scan the monochromators and to maintain a constant energy difference between the two, we stepped the emission monochromator at a constant speed while the excitation monochromator speed was varied. A linear approximation was made for this, i.e., the spectral range to be scanned was divided into 1000 segments. The emission monochromator was scanned by a fixed increment, while the new position for the excitation monochromator was calculated and moved accordingly. Then the PMT signal was integrated over the desired time period, generally 0.5 s. The speed of the FORTRAN programs made the scan essentially continuous, with the calculation time negligible. The same procedure has been used for an assembly language microprocessor using a "look-up" table instead of real-time calculations (22). A spectral band-pass of 2 nm was used on both monochromators,unless indicated otherwise. This gave sufficient resolution for all vibrational details. Background. Vo-Dinh has presented an excellent description of the principles of conventional synchronous luminescence (23). By use of total luminescence symbolism (15),each data point for a solution containing a single emitter is defined as MLj= o,X,Y,
0003-2700/82/0354-2018$01.25/00 1982 American Chemical Society
ANALYTICAL CHEMISTRY. VOL. 54, NO. 12. OCTOBER 1982
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Table I. Experimental Equipment Used for Constant Energy Synchronous Luminescence source
model no.
item Eimac xenon arc lamp, 150 W Eimac illuminator power supply operated at 1 2 A excitation monochromator f/3.5, holographic grating 1200 grooveslmm sample housing
VL-150-2 P25oS-2
Eimac, Division of Varian, San Carlos, CA 94070 Eimac, Division of Varian, San Carlos, CA 94070
4-1OUV
American ISA, Inc., Metuchen, N J 08840
emission monochromator f/3.5, holographic grating, 1200 grooveslmm photomultiplier high-voltage power supply operated at -1000 V nanoammeter dc amplifier monochromator scan controls minicomputer
H-1OV
American Instrument Co., Silver Spring, MD 20910 American ISA, Inc., Metuchen, NJ 08840
IP928 EU-42A
Hammamatsu, Waltham, MA 02154 Heath Co., Benton Harbor, MI 49022
1020-ss PDP 11/34
laboratory constructed laboratory constructed American ISA, Inc., Metuchen, N J 08840 Digital Equipment Corp. (DEC), Maynard, M A 01754
analog-to-digital converter (A/D) digital plotter
Laboratory Peripheral System (LPS-11) HIPLOT DMP-4
Digital Equipment Corp., Maynard, MA 01754 Houston Instrument Division of Bausch & Lomh, Armtin TX 7x753
where Mi, is the measured fluorescence emission at excitation wavelength Ai and emission wavelength A,, X;is the product of the excitation wavelength dependent terms, Y;is the product of the emission wavelength dependent terms,and o( is a wavelength independent factor containing concentration information. Synchronous luminescence restricts the selection of wavelength combination to Aj - Aj = AA = comtant In CESL,a modified restriction is applied
(l/Aj
- l/Aj)
X
lo'
= AP = constant
Due to the nature of the scan process in conventional synchronous luminescence, it can he best described an constant wavelength synchronous luminescence (CWSL). The advantages of this technique over conventionalluminescence indude reduced spectral complexity, improved selectivity, reduced spectral bandwidths, and reduced Rayleigh scatter interferences, and thereforeCWSL is wfulfor qualitative and quantitative mixture analysis (23,24). While CWSL focuses on key spectral regions, the lumineecence characteristics of an unknown sample must he known beforehand to determine the proper selection of instrumental parameters. Total luminescence required minimal a priori knowledge of an unknown's luminescence Characteristicsand can be used to suggest spectral relationships useful for synchronous luminescence optimization.
RESULTS AND DISCUSSION Fluoreecence excitation and emission spectra of PAHs are generally characterized by fairly complex vibrational spectral f e a h , even at room temperature. As will become apparent, energy differences between spectral features are quite important in interpreting synchronous spectra. The 1.4 (f0.2) X 103 cm-'vibrational spacing is characteristic of many PAHs. The tatal luminescence spectrum of anthracene plotted in wavelength units is shown in Figure 1A. The high symmetry of this spectrum is clear. The lines drawn through the contour plot represent typical CWSF scan paths with Ax's selected to coincide with each peak in the tap row of emission peaks. The numbers on the top of the spectrum correspond to the Ax's used, in nanometers. Note that by optimizing each line for one peak, generally other peaks appear as weak shoulders or do not appear at all. The spectra obtained hy these scans are shown in Figure 1B. The main feature is a lack of spectral clarity and consistent peak separation. Replotted in energy units, the spectrum in Figure 2A represents the same anthracene solution. The lines drawn through the peaks illus-
EHISSILN YIYfLEffiTH LNHl
1. (A) Total fluoresance spectrum of 10 nglmL amhracene In nheptans solution. The numbers wrrespond to AA values selected to obtain lhe scan paths drawn through the spectrum. (6) Resultant CWSF spectra of 10 ng/mL anthracene In n-lmptane &n obtained from the scan paths depicted in Figure IA. AA = (A) 2 nm, (6) 23 nm. (C) 47 nm. (D) 73 nm, and (E) 90 nm. -0
trate the scan paths used in CESF. Note the coincidence of the lines with the peak maxima in each scan. The resultant spectra are shown in Figure 2B. The values selected for the difference between monochromators, Ai$ can be traced to fluorescence theory, the 0.2 X lo8 cm-' (see Figure 2A) corresponds to the approximate Stakes shift demonstrated by PAHs in n-alkane solvents, 88 used in the anthracene example. The 1.4 X 103 cm-' agrees with the vibrational e n e w dif€ersncea expected for PAHs (23).
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ANALYTICAL CHEMISTRY. VOL. 54. NO. 12. OCTOBER 1982
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EIIISSION W 1 " G l H
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Flgure 2. (A) Total R~~escmce spectrum of 10 nglmL anthracene h nheptanesoCmon. Wtted h energy mits. The m b e m carespond to As valws selected to obtain the scan paths drawn the spectrum, in kK (1 kK = IO3 cm-'). (B) Resultant CESF spectra of 10 nglmL anthracene in n-heptane solution obtained from the scan paths deplcted in FQure 2A. As = (A) 0.2 X IO3 cm-'. (B) 1.8 X IO3 cm-', (C) 3.0 x io3 cm-'. (D) 4.4 x ios cm-', (E) 5.8 x io3 cm-'.
With CWSF, these energy differenma have been approximated by AX'S of 3,520, and 34 nm (8). The selection of AA generally involves calculating the difference between the maxima in the fluorescznce excitation and emission spectra Thus a different AX must he selected for each compound of interest. For molecules demonstrating a strong 0-0 transition, the use of a 3 nm AA is a good compromk, and multicomponent analysis is possible. However, many PAHs do not exhibit a strong 0-0 transition in solutions at room temperature. Thus a AX of 20 to 30 nm is often used. Use of larger Ah's makes CWSF spectra prohibitively difficult to interpet. Figure 3A shows the total luminescence spectrum of a mixture of anthracene, perylene, and naphthacene. While this represents a simple mixture, many important mnclusiom can be drawn. CWSF lines are shown in Figure 3A and illustrate the difficulty in AA selection. The CESF paths are shown in Figure 3B on the same contour plot. The As's used for anthracene are also quite appropriate for perylene and naphthacene in this mixture. If a AA of 3 nm is used in CWSF and 0.2 X los cn-' is used in CSEF, similar spectra are obtained. However, some PAHs do not exhibit a strong 0 4 transition. Therefore, in such casea, it is necessary to increase wavelength separation by one vibrational unit. Figure 4A shows the spectra for each compound using CWSF a t a AA of 20 nm. Figure 4B shows the spectra for the same compounds using a AA of 34 nm. These can he compared to CESF using a A? of 1.6 X 10s cn-' (Figure 5A). A AA of 20 nm appears optimal for anthracene, while 34 nm appears optimal for perylene and naphthacene. It is readily apparent that 20 and 34 nm are approximations for 1.6 X 10s cm-' in each respective spectral region. Figure 5B illustrates the spectra obtained for the m e compounds using a As of 3.0 X lo3 cm-l.
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'
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600
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Flgure 3. (A) Total Rwrescence spectrum of anthracene, perylene, and naphmaoene h nheptane. The numbers carespond to AA values selected to obtain the CWSF scan paths drawn through the specbum:
A = anthracene. P = perylene. N = naphthacene.
(B) Total fluorescence s p m u m of anthracene. perylene, and naphthacene in nheptane. The numben correspond to As values selected to obtain the CESF scan paths drawn through the spectrum.
M"* E*ISSIOU D M L W E i O
IUMl
Fbun 4. (A) CWSF specha for anthracene (A), perylene (P). and naphthacene (N) using AA of 20 nm. The relative intensity scale represents the intensity relative to the maximum intensity in the total fluorescence spectrum for each compound, with the maximum equivalent to full scale. (B) CWSF spectra for anthracene. perylene. and naphthacene using a AA of 34 nm.
ANALYTICAL CHEMISTRY, VOL. 54, NO. 12, OCTOBER 1982 2021
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Flgure 5. (A) CESF spectra for anthracene, perylene, and naphthacene in n-heptane using a Ail of 1.6 X lo3 cm-'. (E) CESF spectra for anthracene, perylene, and naphthacene in n-heptane using a A8 of 3.0 X lo3 cm-'.
Comparison of CESF spectra a t 0.2 X lo3, 1.6 X lo3, and 3.0 X lo3 cm-' Ai% also demonstrates other interesting features. 2,3-Benzofluoremewill be used as an illustration. Figure 6A shows the fluorescence emission spectrum of this compound. The use of Aih3 of 0.2 X lo3, 1.6 X lo3, and 3.0 X lo3 cm-' (Figure 6B-D) show increasing spectral features with increasing Av, with spectral features matching those of the fluorescence emission spectrum. Thus the degree of complexity of the CESF spectrum can be selected as necessary. Effectively, the CESF spectrum resembles a projection of the fluorescence emission sipectrum onto the curved path through the total luminescencla plane. The relative peak intensities vary depending on the relative intensity of both the excitation and emission spectral contributions. Because synchronous luminescence data points are a product of the excitation wavelength contribution and the emission wavelength contribution, spectra obtained contain features sharper than either the excitation or emission spectra (25).
CONCLUSIONS CESF offers improved selectivity for multicomponent analysis over Conventional fluorimetry. Its use has been demonstrated for simple PAH mixtures. This work was completed as a foundational base for use in other applications. When the concepts of ICESFare applied to low-temperature (77 K) PAH analysis, even more interesting results are obtained (22). Thus the combination of band narrowing techniques with CESF is a natural extension of the fundamental concepts. Another area of potential utility includes using CESF with HPLC detection systems. Hershberger, Callis, and Christian have described the use of the video fluorometer as a liquid chromatographic detector (26). When total luminescence spectra are obtained in .this manner, one must still interpret the data after collection. CESF offers one solution. The
EMISSIM YRVELW6lH [MI1
ERISSIOH y R V a W 6 l H IMIl
Figure 6. (A) Conventional fluorescence emisslon spectrum of 2,3benzofluorene in n-heptane. A,, = 316 nm. (E) CESF spectrum of 2,3-benzofluorene in n-heptane. A8 = 0.2 X lo3 cm-'. (C) CESF spectrum of 2,3-benzofluorene In n-heptane. Aii = 1.6 X lo3 cm-'. (D) CESF spectrum of 2,3-benzofluorene In n-heptane. A8 = 3.0 X
io3 cm-'.
extension of the principles of CESF to phosphorescence spectra is also possible, using the singlet-triplet energy difference as a selectivity parameter as proposed by Vo-Dinh and Gammage (27). Thus CESF allows one to vary both the excitation and emission monochromators during an analysis. This resulta in improved spectral selectivity over conventional fluorimetry, complementing the excellent sensitivity characteristic of luminescence techniques.
LITERATURE CITED (1) Lloyd, J. B. F. Nature (London), Phys. Sci. 1071, 231, 64-65. (2) Lloyd, J. B. F. J . Forenslc Sci. SOC.1071, 2 , 83-94. (3) Lloyd, J. B. F. J . Forensic Sci. Soc. 1971, 2, 153-170.
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Anal. Chem. 1982, 5 4 , 2022-2025
(4) Lloyd, J. B. F. J . Forensic Sci. Soc. 1971, 2 , 235-253. (5) Vo-Dinh, T.; Mortinez, P. R. Anal. Chlm. Acta 1981, 125, 13-19. (6) Eastwood, D.; Fortler, S. H.; Hendrick, M. S. Am. Lab. (Fairfield, Conn.) 1978, 10, 45-51. (7) John, P.; Soutar, I., Anal. Chem. 1976, 48, 520-524. (8) Lloyd, J. B. F. Analyst (London) 1980, 105,97-109. (9) Wakeham, S. 0. Envlron. Scl. Technol. 1977, 7 1 , 272-276. (10) Vo-Dinh, T.; Gammage, R. B.; Martinez, P. R. Anal. Chem. 1981, 53, 253-258. (11) Vo-Dinh, T.; Gammage, R. B.; Hawthorne, A. R.; Thorngale, J. H., Envlron. Sci. Technol. 1978, 12, 1297-1302. (12) Andre, J. C.; Baudot, Ph.; Nlciause, M. Clin. Chlm. Acta 1977, 7 6 , 55-66. (13) Weiner, E. R. Anal. Chem. 1978, 50, 1583-1584. (14) Johnson, D. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1977, 49, 747A-757A.
(15) Warner,-I. M.; Christian, G. D.; Davidson, E. R.; Callis, J. B. Anal. Chem. 1977, 49, 564-573. (16) Ho, C.-N.; Christian, G. D.; Davidson, E. R. Anal. Chem. 1981, 53, 92-98. (17) Ho, C.-N.; Christian, G. D.; Davidson, E. R. Ana/. them. 1978, 50, 1 108-1 1 13.
(18) Ho, C.-N.; Christian, G. D.; Davidson, E. R. Anal. Chem. 1980, 52, 107 1-1 079. (19) Jursensen, A.; Inman, E. L.. Jr.; Winefordner, J. D. Anal. Chim. Acta 1961, 137, 187-194. (20) Rho, J. H.; Stuart, J. L. Anal. Chem. 1978, 50,620-625. (21) HO, C.-N.; Warner, I. M.; Fogarty, M. P. Ind. Res. Dev. 1981, 23, 118-122 . .- . (22) Inman, E. L., Jr.; Winefordner, J. D., unpublished work. (23) Vo-Dinh, T. Anal. Chem. 1978, 5 0 , 396-401. (24) Andre, J. C.; Bouchy, M.; Virlot, M. L. Anal. Chim. Acta 1979, 105, 297-310. (25) Lloyd, J. B. F.; Evett, I.W. Anal. Chem. 1977, 4 9 , 1710-1715. (26) Hershberger, L. W.; Callis, J. B.; Christian, G. D. Anal. Chem. 1981, 53, 971-975. (27) Vo-Dlnh, T.; Gammage, R. B. Anal. Chem. 1978, 50, 2054-2058.
RECEIVED for review December 28, 1981. Accepted July 6, 1982. This work Was supported by "-I-GM11373-19
and
DOE-DE-ASOJ-78EV06022AOO2.
Spectrofluorometric Determination of Calcium and Lanthanide Elements in Dilute Solution Theodore L. Miller" and Stuart I. Senkfor Department of Chemistry, Ohio Wesleyan University, Delaware, Ohio 430 15
Nonradlative energy transfer from the ligand diplcoilnic acid (DPA) to ianthanlde Ions has been used to develop a qulck, easy, and reliable method to determine calcium and lanthanide elements in dllute aqueous solution. Tb( I I I), Eu( I I I), and Dy(II1) are determined dlrectly and large useful concentratlon ranges are avallabie for quantitative analysls. When other metal ions are added to a solutlon of Tb(DPA)?-, the terbium lumlnescence Is reduced. At low metal Ion concentrations, the reductlon is linear for calclum and lanthanide Ions and can be used as an anaiytlcai callbration curve. The detection llmlt Is about 0.1 pg/L for the ions. The method Is readlly adaptable to microsampilng and caiclum can be determlned In the presence of magnesium without Interference.
Lanthanide ions have become an important tool as luminescent probes in biological systems (1-3) and for excited molecules in aqueous solution (4).As research continues and applications increase, a quick, easy, and reliable analytical method for measuring trace lanthanide ion concentrations becomes a necessity. Absorptions measurements are possible but only give satisfactory resulb for high lanthanide ion concentrations because the molar absorptivities of the ions are rather low (5). Spectrophotometric reagents (6-8) have been used to increase the sensitivity. Atomic absorption spectrometry (9) yields sensitivities not much greater than the methods based on spectrophotometric reagents since the absorptivity of the lanthanide elements is low. A promising alternative in both sensitivity and selectivity is luminescence spectrometry. In aqueous solutions high concentrations of Gd(III), Tb(III), and Eu(II1) emit with moderate strength; the emission from Sm(111)and Dy(II1) is weaker, and the other ions display little or no luminescence (10). Therefore, to develop a spectrofluorometric method for the determination of trace amounts of trivalent lanthanide ion, either the use of a high-intensity laser excitation source (analytical determinations using a
conventional spectrophotofluorometer are not possible) or excitation of the lanthanide ions by a nonradiative energy transfer process is required. Intermolecular (11) and intramolecular (12) energy transfer have both been investigated. This paper describes the use of intramolecular energy transfer from pyridine-2,6-dicarboxylicacid (dipicolinic acid-DPA) in the spectrofluorometric determination of calcium and lanthanide elements in dilute solution.
EXPERIMENTAL SECTION Apparatus. Absorption spectra were recorded at room temperature on a Cary 219 spectrophotometer by Varian using matched quartz cells with a 1-cm pathlength. All the luminescent measurements were made with an Aminco-Bowman Ratio I1 spectrofluorometer at room temperature. Either a standard 1 cm square quartz cell or a new microcell designed in our laboratory (the specific design and performance of the microcell will be discussed in a separate publication) was used with the spectrofluorometer. Reagents. The lanthanide salts were all purchased as 99.9% chlorides (except gadolinium which was prchased as the nitrate salt) from Alfa Inorganics. Calcium chloride (ultrapure) was also obtained from Alfa. All other compounds were reagent grade and used as obtained. Stock solutions of the metal ions were made about 5 mM with deionized distilled water. Stock solutions of the chelating agents were made to 0.01 M with either deionized distilled water or methanol. Procedure. Relative emission intensity measurements of the lanthanide complexes were made by adding 1 mL of the stock solution of the chelating agent and 1mL of the stock lanthanide solution to a 1 cm square quartz cell. The relative emission intensity was recorded for each chelating agent at room temperature. To evaluate the influence of pH on the Tb(II1)-DPA complex, 1mL of 9.30 mM DPA solution and 1 mL of 0.190 mM Tb(II1) solution were placed in the cell. After the initial pH and relative intensity were measured, small aliquots of 6 M NaOH or HC1 were added to the cell. The pH and relative intensity were measured after each addition. This procedure was repeated until the range of 1.1to 12.0 was covered. All subsequent studies were conducted in the optimum pH range; all solutions were made at a pH of 6.5 with 0.01 M piperazine buffer.
0003-2700/82/0354-2022$01.25/00 1982 American Chemical Society
