Anal. Chem. 1986, 58,2469-2473
LITERATURE CITED (1) Mandal, K.;Hauenstein, B. L.: Demas, J. N.: DeGraff, B. A. J . Phys. Chem. 1983, 87, 328. (2) Hauenstein. B. L.; Dressick, W. J.; Gilbert, T. B.: Demas, J. N.; DeGraff. 9.A. J . Phys. Chem. 1984, 88, 1902. (3) Arnold. A. P.: Daianault. S.A.: Rabenstein. D. L. Anal. Chem. 1985. 57, 1112. (4) Busch, N.: Freyer, P.: Szameit, H. Anal. Chem. 1978, 50, 2166. (5) Seemuth, D.:Hall, J.: Robertson, K. A,: Huber. c 0.J Chern. Educ. 1979, 65, 6556. (6) Doane, L.; Stock, J.; Stuart, J. J . Chem. Educ. 1979, 56, 415. (7) Velinov, G. Tala& 1963, 30, 687. (8) Demas. J. N.; Addington, J. W. J . Am. Chem. SOC. 1976, 9 8 , 5800.
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(9) Buell, S. L.: Demas, J. N. Anal. Chem. 1982, 5 4 , 1214. (IO) Buell, S.L.: Demas, J. N. Rev. Sci. Instrum. 1962, 53, 1298. (11) Demas, J. N.: Flynn, C. M. Anal. Chem. 1976, 4 8 , 353. (12) Dressick, W. J.: Demas, J. N.: DeGraff, B. J . Photochem. 1964, 2 4 ,
45.
RECEIVED for review December 2, 1985. Resubmitted May 28,1986. Accepted May 28,1986. We gratefully acknowledge support by the donors of the Petroleum Research Fund, administered by the American Chemical Society, and the National Science Foundation (CHE 82-06279).
Multidimensional Data Formats for Phase-Resolved Fluorometric Multicomponent Determinations Using Synchronous Excitation and Emission Spectra Kasem Nithipatikom and Linda B. McGown* Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078
The use of multldlmerwlonal data formats comblnlng wavelength and fluorescence llfethe as selecthrkty parameters Is described for determlnatlons of two-, three- and four-component systems of polycycllc aromatic hydrocarbons (9phenylanthracene, 9,10-dIphenylanthracene, benro[k 1fluoranthene, and benro[a]pyrene). I n the data formats, phase-resolved fluorescence lntenslty Is measured as a functlon of elther emlsslon wavelength or synchronously scanned excltatlon and emlsslon wavelengths, as well as detector phase angle and excltatlon modulation frequency. Results were compared wlth results for determlnatlons using wavelength selectlvlty alone. Best results for the two- and threecomponent systems were obtalned wllh the steady-state measurements, but the PRFS-synchronous data format was much better for the fowtomponent system (-1.2% average relative error) and gave the best, most consistently good overall results for the multicomponent systems.
The use of phase-resolved fluorescence spectroscopy (PRFS) to incorporate fluorescence lifetime selectivity into multicomponent determinations has been explored in recent years ( I ) . Previous studies describe PRFS determinations of two-, three-, and four-component systems (2-6). All of these studies used experimental conditions chosen for the quantitation of the known components in the mixtures, although none of the conditions were rigorously optimized for those particular components. In one of these studies (3),it was demonstrated that the phase-resolved approach gave better accuracy and smaller average error magnitudes than the equivalent steady-state (non-phase-resolved) determinations based on wavelength selectivity alone. Determinations described in two of the other studies (2, 4 ) could not be accomplished with wavelength selectivity due to extensive spectral overlap of the components. One of our goals for multicomponent PRFS determinations is the development of a generalized data format for multicomponent determinations for identification and subsequent quantitation of the components. One possible data format consists of a three-dimensional representation of phase-re-
solved fluorescence intensity (PRFI) as a function of synchronously scanned wavelength on one axis and detector phase angle setting (providing the fluorescence lifetime information) on the other, independent axis ( I ) . The use of synchronous excitation spectroscopy for the determination of polynuclear aromatic hydrocarbons has been previously described (7-9). The work described here compares results obtained by using the PRFS-synchronous excitation data format with results obtained by using a similar data format in which emission spectra are used rather than synchronous spectra. The results for both PRFS formats are compared with results for steady-state determinations using only synchronous or emission spectra and no fluorescence lifetime dimension. Four polycyclic aromatic compounds were used in these studies, including 9-phenylanthracene (9PA), 9,10-diphenylanthracene (9,1ODPA),benzo[a]pyrene (BaP), and benzo[k]fluoranthene (BkF). These compounds were chosen because of the high degree of overlap between both their emission and their synchronous spectra. Their fluorescence lifetimes are within 10 ns of each other, with less than 1.5 ns difference between 9,lODPA and BkF. Four different systems were studied, including two two-component systems (9PA/9,10DPA and 9,1ODPA/BkF), a three-component system (9PA/ 9,1ODPA/BkF), and a four-component system containing all four of the compounds.
EXPERIMENTAL SECTION Materials. The BaP (98%),9PA (98%),and 9,lODPA (99%) were purchased from Aldrich and recrystallized from absolute ethanol (US.Industrial Chemical Co.). The BkF (99%) was purchased from Foxboro and used without further purification. Dimethylbis(5-phenyl-2-oxazolyl)benzene(Me,POPOP) used as a reference for fluorescence lifetime determinations was purchased from Aldrich (scintillation grade) and used without further purification. Stock solutions of each PAH were prepared in absolute ethanol. Mixtures were prepared from the stock solutions. The compositions of the solutions used for each of the two-, three-, and four-component systems (five solutions per system) are shown in Table I. Apparatus. An SLM 4800s phase-modulation spectrofluorometer (SLM Instruments, Inc., Urbana, IL) was used for all steady-state and phase-resolved fluorescence measurements.
0 1986 American Chemical Society 0003-2700/86/0358-2469$01.50/0
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12,OCTOBER 1986
Table 11. Relative Molar Fluorescence Intensities and Fluorescence Lifetimes of the PAHs Used in These Studies
Table I. Compositions of Mixtures and Standards for Multicomponent Determinations" system 2-component: 1 2
3 4 5
9PA
9,lODPA
1.50 0.588 1.20 0.148 1.80
0.332 0.679 0.499 0.859 0.169 0.351 0.175 0.516 0.0859 0.698 0.388 0.169 0.684 0.0672 0.0360 0.326 0.0824 0.659 0.0331 0.330 0.702
2-component: 1 2
3 4 5 3-component: 1 2
3 4 5 4-component: 1 2
3 4 5 standardsb
0.609 1.22 0.308 0.605 0.135 0.578 1.19 0.277 0.506 0.138 1.26
BkF
BaP
re1 molar intens"
TPb
TmC
9PA 9,lODPA BkF BaP
1.049 2.725 2.475
4.78 f 0.03 6.09 f 0.02 7.52 f 0.05 14.60 f 0.07
4.74 f 0.02 5.96 f 0.02 7.17 f 0.02 14.68 f 0.04
1.000
Relative fluorescence intensity per mole, determined at the approximate wavelength maximum of the synchronous spectrum (AA = 15) for each component. bFluorescence lifetimes (ns) calculated from phase-delay using Me,POPOP as a reference ( T , , ~= 1.45 ns), 30-MHz modulation frequency, with standard deviations shown for five determinations. Fluorescence lifetimes (ns) calculated from demodulation under same conditions as T".
0.417 0.625 0.208
0.836 0.0622 0.402 0.201 0.0813 0.798 0.428 0.388 0.392 0.0979 0.787 0.196 0.834
PAH
0.437 0.241 0.441 0.202 0.882 0.939
Standards used to determine a Concentration (FM) in cuvette. molar intensities (I). Excitation was achieved with a 450-W xenon arc source and emission detected with Hamamatsu R928 photomultiplier tubes. Three modulation frequencies, including 30,18,and 6 MHz, are achievable on the instrument. An Apple IIe microcompouter was used for on-line data acquisition. A Hewlett-Packard 9920U computer was used for analysis of the data matrices. The spectrofluorometer sample chamber was maintained at 20 0.1 "Cwith a Haake-Buchler AlOO temperature control unit. Samples were contained in disposable polystyrene cuvettes (Evergreen Scientific). The cuvette solutions were not purged to remove dissolved oxygen. Data Collection. All fluorescence measurements were made in a ratiometric mode to compensate for source output and, in the case of dynamic lifetime and PRFS measurements, source modulation fluctuations. The intensity measurements were all made in triplicate in the 10-average mode, in which each measurement is the average of 10 samplings made over a total period of approximately 10 s. Two modulation frequencies, 30 and 18 MHz, were used in these studies. The PRFS data matrices were generated by measuring the PRFI at each combination of wavelengths/detector phase angle/modulation frequency. Steady-state matrices were generated by measuring the steady-state intensity a t each wavelength combination. All steady-state and synchronous spectra were collected with slit settings of 16 nm for the excitation monochromator entrance, 0.5 nm for the excitation monochromator exit and the modulation chamber exit, and 8 nm for the emission monochromator entrance and exit. The monochromator stepping interval was 5 nm for the synchronous PRFS spectra, 10 nm for the emission PRFS spectra, and 2 nm for both the synchronous and emission steady-state spectra. The stepping intervals were chosen to provide sufficient numbers of total data points for each technique so that equal numbers of equations could be generated for the data matrices for all four techniques. The PRFS and steady-state synchronous spectra were obtained with a constant AA of 15 nm maintained between the excitation and emission monochromators. Data Analysis. Multicomponent determinations are performed in PRFS by measuring the PRFI of samples and standards at a series of detector phase angle ( 4 ~and ) wavelength conditions (A,,, Aem) at each modulation frequency (w)used, as previously described (3-6). Equations of the form
*
m
PRFI
(AeJern,htW)
=
xfi(Aei,Aemt6JD,u)Ci
(1)
1-1
express the measured sample PRFI as the sum of the individual
0
EXCITATION WAVELENGTH (nm)
Figure 1. Steady-state exchation spectra (uncorrected) of 9PA (-), 9,1ODPA (- - -), BkF (.- -), and BaP (. .). Emission was monitored at 405 nm except for BaP which was monitored at 428 nm.
-
.
PRFI contributions due to each emitting component present a t concentration Ci. The f values are the molar PRFIs for each component found by measuring a standard solution of each component under the same modulation frequency, detector phase angle, and wavelength conditions. The concentrations of the standard solutions are shown in Table I. An n X m matrix is thereby generated where n is the number of equations (corresponding to the number of different sets of measurement conditions used) and m is the number of components possible in the multicomponent sample. Steady-state data matrices are similarly generated and represented except that each equation corresponds to a unique excitation-emission wavelength condition alone. Therefore, the steady-state data matrices in these studies are two dimensional, whereas the PRFS data matrices are three dimensional, or four dimensional if more than one modulation frequency is used. The linear sets of equations were solved for the concentrations of the individual components using a nonnegative least squares (NNLS) routine (IO),since the system is generally overdetermined ( n > m). Previous studies (4-6) used a Gauss-Newton iterative procedure, which is lengthier and yields similar (but not identical) results. The NNLS solution does not allow negative concentrations in the solutions to the equations.
RESULTS AND DISCUSSION The relative fluorescence intensities and fluorescence lifetimes were determined for each of the four components and are shown in Table 11. The relative intensities were measured at the maximum of the synchronous spectrum of each component. The steady-state excitation and emission spectra and synchronous spectra, obtained by using the same instrumental settings used to generate the data for the determinations, are shown in Figures 1,2, and 3, respectively. The synchronous spectrum in Figure 3 was obtained by using a 15-nm difference between the excitation and emission wavelengths. This difference provided higher intensities, much lower scatter background and better overall resolution between the four components used in these studies than the synchronous spectrum acquired with much smaller differences such as 3
ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986 I
I
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Table 111. Summary of E r r o r s for Phase-Resolved ( P R F S ) and Steady-State (SS)Determinationsasb 9PA
9,lODPA
BkF
BaP
PRFS Synchronous'
EMISSION WAVELENGTH (nm)
Figure 2. Steady-state emission spectra (uncorrected), excitation at 375 nm. Legend is same as Figure 1.
2-component [ 370-3951 2-component [ 380-4051 3-component [370-4051 4-component [ 370-4051 average:
-1.0 (1.5)
3.3 (3.3) -2.2 (2.2)
3.6 (4.1)
-4.3 (4.3)
0.6 (2.9)
1.1 (1.1)
-2.9 (2.9)
-3.8 (6.4)
-0.1 (2.5)
1.9 (5.9)
-2.7 (2.9)
-0.5 (3.7)
1.5 (2.6)
1.9 (5.9)
PRFS Emissiond 2-component [ 390-4351 2-component [ 395-4451 3-component [390-4601 4-component [ 390-4601 average:
0.0 (1.8)
2.4 (2.8) -0.8 (1.6)
-6.2 (6.2)
5.1 (5.3)
4.2 (23.5)
6.6 (7.1)
0.4 (2.6)
e
1.8 (3.5) -2.3 (6.2)
-1.9 (3.5)
e
4.5 (5.3) -2.3 (6.2)
SS Synchronousf
EXCITATION WAVELENGTH (nm)
Figure 3. Steady-state synchronous excitation spectra (uncorrected), L A = 15 nm. Legend is same as Figure 1
2-component [364-4101 2-component [370-4161 3-component [ 360-4221 4-component [ 360-4221 average:
-1.2 (2.1)
0.7 (1.3) -0.4 (1.6)
1.8 (2.1)
-2.8 (2.8)
-3.7 (3.7)
1.7 (2.0)
-1.6 (1.6)
5.4 (5.4)
-3.0 (3.9) -8.8 (8.8)
-1.9 (2.2)
0.7 (2.8)
0.2 (2.7) -8.8 (8.8)
SS Emissionf 2-component [ 390-4361 2-component [394-4401 3-component [ 390-4521 4-component [ 390-4521 average: 450
350 EXCITATION WAVELENGTH (nm)
Figure 4. Steady-state synchronous excitation spectra (uncorrected), AA = 3 nm. Legend is same as Figure 1. PRFI
+I 0
-
5
Figure 5. Depiction of PRFS synchronous excitation data format for four-component solution 1 (see Table I), showing phase-resolved fluorescence intensity (PRFI) as a function of detector phase angle (4D) and synchronously scanned wavelength. Modulation frequency = 30 = 15 nm. MHz,
-0.5 (1.1)
-1.3 (1.3)
1.1 (1.5) -2.1 (2.3)
4.1 (4.1)
-2.2 (5.5)
-0.2 (0.8)
-2.2 (3.7) -18.2 (19.2) -2.0 (2.4) -1.0 (3.2) -1.3 (2.0)
-5.1 (6.1)
0.6 (2.4) -1.0 (3.2)
"Relative errors (%) averaged for all five solutions, with average relative error magnitudes (70)in parentheses. b A total of 24 equations was used for each of the two-component systems and 32 equations for the three- and four-component systems. For synchronous spectra, A,, = A,, + 15 nm; for emission spectra, A,, = 375 nm. The spectral wavelength range is given in brackets for each system (Aex for synchronous and A,, for emission spectra, in nm). Equations generated using wavelengths a t 5-nm intervals in spectral range, acquired a t each of four detector phase angle settings (0°/45" a t 30 MHz and 135O/18Oo at 18 MHz for the twocomponent systems; Oo/900 at 30 MHz and 18Oo/27O0a t 18 MHz for the three- and four-component systems). Equations generated using wavelengths a t 10-nm intervals, with the same detector phase angle settings and modulation frequencies used for the PRFS synchronous determinations. e Undefined relative error for solution 4 due to zero concentration found. f Equations generated usine waveleneths at 2-nm intervals.
nm (Figure 4). A depiction of the three-dimensional PRFS-synchronous excitation data array obtained for solution 1 in the four-component system (see Table I) is shown in Figure 5 . Comparison of the Determination Techniques, Results are summarized in Table 111 in terms of relative errors and error magnitudes for each component averaged for the five solutions used for each system (described in Table I). The errors averaged for all four multicomponent systems are also shown for each technique. Results were calculated from the
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 12, OCTOBER 1986
Table IV. Errors for Each Solution for the PRFS and Steady-State Analysis of the Four-Component System Using Synchronous Spectraa,b solution
9PA
PRFS 1
-5.2 -4.2 -1.1 -1.2 -2.9 -2.9 (2.9)
2 3 4 5
avd
9,lODPA -7.7 -17.5 -0.2 2.7 3.9
-3.8 (6.4)
BkF
BaP
average'
4.4 1.5 0.1 -2.2 -4.1 -0.1 (2.5)
11.0 7.9 0.5 -6.4 -3.7 1.9 (5.9)
0.6 -3.1 -0.2 -1.8 -1.7
(7.1) (7.8) (0.5) (3.1) (3.6)
-5.3 -17.0 -6.3 -10.4 -4.8 -8.8 (8.8)
-0.6 -3.0 -3.9 -0.2 -2.4
(2.4) (6.4) (5.1) (6.1) (4.5)
Overall Average? -1.2 (4.4)
ss 1 2 3 4 5 avd
-0.7 -1.7 -0.7 -0.8 -4.3 -1.6 (1.6)
2.8 5.6 2.4 11.8 4.2 5.4 (5.4)
Overall Average:'
1.0 1.3 -11.1 -1.4 -4.6
-3.0 (3.9) -2.0 (4.9)
Relative errors (70)shown for each component in each solution, corresponding to the same four-component determinations used for Table 111. bNumbers in parentheses are averages of relative error magnitudes (70).'Average error for all components in the solution, with average error magnitudes in parentheses. Average error for the component in all solutions, with average error magnitudes in parentheses. eAverageerrors for all components in all solutions. with average error magnitudes in Darentheses.
same data sets using many other combinations of evenly spaced wavelengths and detector phase angles at 30- and/or 18-MHz modulation frequencies for the PRFS techniques, or just wavelengths for the steady-state techniques, to generate anywhere from 6 to 64 equations. The results shown are typical of all of the results in terms of comparisons between the different techniques. The results indicate that the steady-state techniques are better for the two- and three-component systems. The PRFS emission technique gave the generally poorest results. Therefore, it can be concluded that resolution for these systems is adequately achieved using wavelength selectivity alone and the fluorescence lifetime dimension is not needed. However, a dramatic decrease in performance occurs using the steady-state techniques for the four-component system. Also, the PRFS synchronous technique gives better overall results than any of the other techniques. The hardest component to determine is 9,lODPA when emission spectra are used, and both 9,lODPA and BaP for the steady-state sjmchronous case. Difficulties associated with the presence of BaP in multicomponent determinations using synchronous excitation spectroscopy have been reported elsewhere (9). Results for the emission techniques were in some cases better than the results for the other techniques. However, the emission results were less consistently good and much more dependent upon the particular combinations of equations used, especially in the case of PRFS emission. Therefore, it appears that the use of synchronous spectra is preferable for a generalized data format. This may be due in part to the wider wavelength range covered by the emission spectra relative to the synchronous spectra, so that a given number of evenly spaced wavelengths in the emission range is more likely to omit key wavelengths vital to good resolution. Errors for each component in each solution are shown in Table IV for the four component system corresponding to the same PRFS and steady-state synchronous determinations summarized in Table 111. The PRFS average results are significantly better than the steady-state results for all components except the 9PA, and the overall average errors and error magnitudes are also better for the PRFS technique. It should be noted that results for the four-component system were better using only 18-MHz modulation frequency rather than the 18/30 MHz combination (discussed below, see Table V), giving the same average overall relative error of -1.2% but reducing the average error magnitude to 3.5%. However, the
18/30 MHz combination is probably better for a generalized data format, judging from the overall results for the four different multicomponent systems. Effect of Modulation Frequency. Errors for each of the multicomponent systems obtained for PRFS synchronous determinations using 30-MHz and/or 18-MHz modulation frequencies are shown in Table V. Since the optimal modulation frequency depends upon the fluorescence lifetimes of the analytes, it is not surprising that 30 MHz is best for the two two-component systems in which both analytes have short (
