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Anal. Chem. 19S2, 64, 2920-2928
Organized Media for Fluorescence Analysis of Complex Samples: Comparison of Bile Salt and Conventional Detergent Micelles in Coal Liquids Patricia M. Ritenour Hertz and Linda B. McGown’ Department of Chemistry, P. M . Gross Chemical Laboratory, Duke University, Durham, North Carolina 27706
Accurate quantttathredetennlnatlonrare often drmcult to obtaln from fluorescenceanalyris of complex samples due to sample mat& effectsand Intermolecular lnteractlonsbetweensolutes. Organized medla can be used to mlnknlze these unwanted processes wlthout phydcal separation or extractlon of the analytes from the sample matrlx by blatlng the analyte molecules In a unlform mlcroenvlronment within the sample. The advantages d bile salt mlcellar medla over conventlonal detergentmlcelh are demonstratedfor analydeof coal Ilqukk. The bile salt medla Is shown to Increase the sensitlvlty and dynamic range of fluorescence measurements relatlve to simple ethandlc solutions, wlthout promotlnggound-state and exclted-state Interactionsthat occur In the detergent mlcellar medla.
INTRODUCTION Sensitivity to matrix effects, heterogeneous binding microenvironments, and dynamic intermolecular interactions, which makes fluorescence excellently suited to sample fingerprinting and characterization, is a serious handicap in the quantitative determination of individual analytes in a complex sample. Accurate calibration is often not possible if the fluorescence characteristics of the analyte are highly sample dependent. Most quantitative techniques rely on extraction and/or chromatographyto remove the analyte from the samplematrix. Matrix effects may still cause inaccuracies, however, by varying extraction or separation efficiencies as well as through carryover of concomitants and interferences. We present here a different approach to this problem, in which organized micellar media is added directly to the sample in order to provide a uniform microenvironment for the analyte within the sample, thereby accomplishing an “in situ” extraction of the analyte. Incorporation of a given analyte into a uniform microenvironmentthroughout the sample and calibration standards will minimize matrix effects and increase the accuracy of calibration without necessitating a physical separation or extraction of the bulk sample. Moreover, the dynamic range and sensitivity will be increased in a micellar medium that is able to individually isolate the analyte molecules and thereby protect them from intermolecular interactions such as energy transfer, dimerization, complexation, and quenching. Micellar media, primarly those formed by synthetic detergent molecules, have been used in luminescence analysis to solubilize hydrophobic molecules in aqueous solution1J and to enhance or otherwise modify the luminescence (1) Elworthy, P. H.; Florence, A. T.;Macfarlane, C. B. Solubilization by Surface Active Agents and its Application to Chemistry and the Biological Sciences; Chapman and.Hak London, 1968. (2) Mittal, K. L., Ed. Micellization, Solubilization and Microemubions; Plenum Press; New York, 1977.
properties of molecule^.^^ However, conventional micelles formed by detergents often bind more than one molecule per micelle in a relatively fluid interior, which may actually promote energy transfer and other nonradiative processes that interfere with fluorescence determinations. Cyclodextrins have also been explored as solubilization reagents, but their use is limited by size limitations for the guest molecule and relatively low binding constants for all but a few guest compounds. Thus, cyclodextrins are useful as highly selective reagents but are limited to a relatively small number of analytes. Recent studies have explored a different class of amphiphilic compound, the bile salts, as an alternative to Synthetic detergents or cyclodextrins for luminescence analysis.l@-12 These studies have shown that bile salt aggregates are effective at individually solubilizing fluorescent molecules, thereby minimizing their interactions with each other and with sample matrix constituents. The work presented here compares two micellar media, the conventional detergent sodium dodecyl sulfate (SDS)and the bile salt sodium taurocholate (NaTC),for the fluorescence analysis of coal liquid samples. The purpose of these studies is to determine the effects of bile salt media on the sensitivity and dynamic range of fluorescence analysis of complex samples, in comparison to simple solvents and conventional micellar media.
BACKGROUND Coal Liquids and Fluorescence Analysis. Coal liquids are highly complex mixtures which contain a significant number of aromatic compounds, including a variety of polycyclic aromatic hydrocarbons (PAHs). It is important for processing as well as for environmentaland health reasons to develop methods to determine the PAHs in coal liquid ~amp1es.l~ Techniques for analysis of PAHs in coal liquids have generally involved separation step^.'^-'^ Various flu(3) Hinze, W. L. In Solution Chemistry of Surfactants; Mittal, K. L., Ed.;Plenum Press: New York, 1979; Vol. 1, p 79. (4) Singh, H. N.; Hinze, W. L. Anal. Lett. 1982, 15, 221.
(5) Singh, H. N.; Hinze, W. L. Analyst 1982, 107, 1073. (6)Taketatau, T.; Sato, A. A d . Chim. Acta 1979, 108, 429. (7) Taketatau, T. Talanta 1982, 29, 397. (8) Jing-He, Y.; Gui-Yun, Z.; Bo, W. Anal. Chim. Acta 1987,198,287. (9)Tran, C. D. Anal. Chem. 1988,60, 182. (10) Nithipatikom, K.; McGown, L. B. Anal. Chem. 1989, 61, 1405. (11) Meyerhoffer, S.M.; McGown, L. B. A d . Chem. 1991,63,2082. (12) McGown, L. B.; Kreiss, D. S.In SPIE Proceedings; Fluorescence Detection II; Menzel, E. R., Ed.; S P I E Bellingham, WA, 1988, Vol. 910, pp 73-80. (13) Kershaw, J. R. Fuel 1978,57,299. (14) Katoh, T.; Yokoyama, S.; Sanada, Y. Fuel 1980,59, 845. (15) von der Dick, H.; Kalkreuth, W. Fuel 1984,63, 1640. (16) Aigbehinmua, H. B.; Darwent, J. R.; Gaines, A. F. Energy Fueb 1987,1, 386. (17) Satou, M.; Yokoyama, S.; Sanada, Y. Fuel 1989,68, 1048. (18)Rusin, A.; Pazdziorek, T. Fuel 1989, 68, 1290.
0003-2700/92/0364-2920$03.00/0 @ 1992 Americen Chemical Sociely
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
H
1
I
Flgure 1. Sodium taurocholate (NaTC) monomer (solid circles In inset represent hydroxy groups on steroid backbone).
orescence techniques which require cryogenicconditions have also been u t i l i ~ e d . ~ ~ ~ ~ ~ It is preferable, if possible, to use methods which require no preseparation steps and which can be performed at room temperature. One such technique is synchronousfluorescence spectroscopy,21~22 which was used for coal liquid analysis with some Synchronous spectra are colleded by simultaneously scanning the excitation and emission monochromators. Most commonly, a constant wavelength difference, AA, is maintained between the two monochromators,although variable-angle methods have also been described. The synchronous excitation technique improves selectivity by combining the properties of both the excitation and emission spectra and also provides a bandwith narrowing effect as a result of the multiplication of the two spectra. Additionally, there is a significant simplification of the spectral features and a subsequent reduction of spectral overlap which make this technique very useful for the analysis of complex, multicomponent systems. This approach is limited, however, by its susceptibility to matrix effects and intermolecular interactions between solutes that could cause inaccurate determination^.^^-^^ This is particularly true for complex samples such as coal liquids. In addition to synchronous spectra, this work also uses the totalluminescence spectral format, in which the fluorescence spectral data are represented as a two-way array, or excitationemission matrix (EEM). In the EEM, fluorescence intensity is plotted as a function of excitation wavelength and emission wavelength to generate a three-dimensional surface.27 This format is particularly useful for multicomponent analysis28 in systems of independent fluorophores that have no matrix effects or interactions between solutes. For complex samples, this is generally not the case. In this work, the EEM is useful for comparing spectral features of different samplesor spectral features of the same sample in different solvents. Organized Bile Salt Media. Bile salts are biological detergents that are synthesized from cholesterol in the liver. They are typically composed of a steroidal backbone with one or more a-oriented hydroxy groups, conjugated to an anionic side chain, or “tail” (Figure 1). The a oriententation of the hydroxy groups places them on the concave side of the steroid skeleton, with the methyl groups positioned on the opposite, convex side; the polar or charged group on the (19)Yang, Y.; DSilva, A. P.; Faesel, V. A.;Iles, M. Anal. Chem. 1980, 52,1350. (20)Perry, M. B.; Wehry, E. L.; Mamantov. G. Anal. Chem. 1983,55, 1893. (21)Lloyd, J. B. F. Nature 1971,231,64. (22)Vo-Dinh, T. Anal. Chem. 1978,50,396. (23)Vo-Dinh, T. In Modern Fluorescence Spectroscopy; Wehry, E. L., Ed.; Plenum Press: New York, 1981;Vol. 4, Chapter 5. (24)Latz,H. W.; Ullman, A.H.; Winefordner, J. D. Anal. Chem. 1978, 50,2148. (25)Latz, H. W.; Ullman, A. H.; Winefordner,.J. D. Anal. Chem. 1980, 52, 191. (26)Lloyd, J. B. F. Anal. Chem. 1980,52, 189. (27)Weber, G. Nature 1961,190,27-29. (28)Warner, I.M. In Contemporary Topics in Analyticaland Clinical Chemistry; Hercules, D., Heiftje, G. M., Snyder, L. R., Evenson, M. A,, Eds.; Plenum Press: New York, 1982;Vol. 4,p 75.
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aliphatic tail will therefore interact with the hydroxy groups on the concave surface.29The resulting bile salt conformation provides a hydrophobic (methyl-containing) surface on one side and a hydrophilic (hydroxy-containing) surface on the other side. This structure is distinct from that of a conventional detergent monomer,which has a hydrophilichead group and a long, hydrophobic tail. The bile salts also exhibit unique behavior with respect to self-association and molecular solubilization.*33 In conventional detergents, solubilization sites include the micellar surface, the palisade layer at the interface between the hydrophobic taile and the hydrophilic head groups, and the hydrophobic inner core.% Analogous binding sites are not present in the smaller, more rigid, bile salt micelles. Instead, solubilization of hydrophobic compounds is accomplished through favorable interactions with the hydrophobic surfaces of the bile salt micelle^.^^^^^^ The resulting solubilization microenvironments in bile salt micelles are often highly apolar.” The bile salt used in this work, sodium taurocholate (NaTC), is a trihydroxy salt. Trihydroxy bile salts have significantly lower aggregation numbers than conventional detergent micelles; estimates range from 2 to 32,compared to 30-200 for typical detergents. It is also very interesting that bile salt micelles have unusually high internal microviscosity (2100CPfor bile salts vs 15-30 CPfor conventional detergent micelles3’). Micellar bile salt solutions have been reported to exhibit significant polydispersity with respect to aggregate size and structure.a
EXPERIMENTAL SECTION Four different coal liquid samples were used in these studies: Solvent Refiied Coal Heavy Density (SRC-11, Ft. Lewis, WA, pilot plant), V-1072(Wilsonville ACLTF, Run 258,second stage heavy distillate, derived from Black Thunder subbituminous coal), V-1074(Wilsonville ACLTF, Run 257,second stage heavy distillate,derivedfrom IllinoisNo. 6 coal),and V-178(Wilsonville ACLTF, first stage heavy distillate, derived from Illinois No. 6 coal). Sodiumtaurocholate(NaTC,purity>99%)waspurchased from Calbiochem, and sodium dodecylsulfate(SDS,purity99%) was purchased from Aldrich; both were used as received. Solutions of the coal liquids were prepared in absolute ethanol (Aaper Alcohol and Chemical Co., Shelbyville,KY)and in aqueous micellar solutions of NaTC and SDS (both at a concentration of 30 mM, calculated as total monomer, unless otherwise noted). This is well above the critical micellar concentration (cmc) of both amphiphiles: SDS has a cmc of 8 mMS and an aggregation number of approximately 6Ra NaTC has a cmc in the range 8-12 mM41and a much smalleraggregation number (the actual value is uncertain;@polydisperse solutions of dimer, tetramers and higher order aggregates are likely). AU fluorescencemeasurementswere made with an SLM48ooos spectrofluorometer (SLM Instruments, Inc., Urbana, E), using (29)Small, D. M. In The Bile Acids; Nair, P. P., Kritchevsky, D., Eds.; Plenum Prees: New York, 1971;Vol. 1, p 249. (30)Zana, R.;Guveli, G. J. Phys. Chem. 1986,89,1687. (31)Fisher, L.;Oakenfull, D. Aust. J. Chem. 1979,32,31. (32)Chen, M.; Gr&tzel,M.; Thomas, J. K. J. Am. Chem. SOC.1975,97, 2052. (33)Sugihara, G.; Yamakawa, K.; Murata, Y.; Tanaka, M. J. PhyS. Chem. 1982,86,2784. (34)h e n , M. J. Surfactants and Interfacia! Phenomena, 2nd ed.; John Wiley & Sons: New York,1988. (35)Conte, G.; Di Blasi, R.; Giglio, E.; Parretta, A,;Pavel, N. V. J. Phys. Chem. 1984,88,5720. (36)Kolehmainen, E.J. Colloid Interface Sci. 1986,105, 273. (37)Chen, M.; Gratzel, M.; Thomas, J. K. Chem. Phys. Lett. 1974,24, 65. (38)Kratohvil, J. P.; Hsu, W. P.; Jacobs, M. A.; Aminabhavi, T. M.; Mukunoki, Y.Coloid Polym. Sci. 1983,261,781. (39)Cline Love, L. J.; Dorsey, J. G.; Habarata, J. G. Anal. Chem. 1984, 56,1132A. (40)McIntire, G. L. Anal. Chem. 1990,21,257. (41)Meyerhoffer, S. M.; McGown, L. B. Langmuir 1990,6, 187.
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ANALYTICAL CHEMISTRY, VOL. 64, NO.23, DECEMBER 1. 1992
Table I. Uncorrected Matrix Correlation (UMC), Calculated between the EEM at Each Concentration*band the Mean EEM for All Concentrations for That Solvent0
SRC-I1concn (fiL/mL) solvent ethanol NaTC SDS
0.001 0.9502 0.9797 0.9730
0.005 0.01 0.05 0.1 mean f 1 sd 0.9789 0.9895 0.9580 0.8180 0.9389 0.0694 0.9851 0.9900 0.9904 0.9338 0.9758 0.0239 0.9833 0.9897 0.9910 0.9313 0.9737 0.0247
* *
a Estimated standard deviation of each EEM,based on triplicate meaaurements,is *0.001. * EEMs are shown in Figure 2. The righthand column is the mean UMC one standard deviation for each solvent.
*
of similarity between two matrices. The UMC between two m X n matrices, A and B, is defined by2
U
Lu X
Emission Wavelength (nm) Flgure 2. EEMs (Lx = 255-395 nm Increasing along y-axis, h, = 300-440 nm Increasing along x-axis) for the S R C I I coal llquld In ethanol (rlght-handcolumn),30 mM NaTC (center column),and 30 mM SDS (left-handcolumn). Concentrationsare (from top to bottom) 0.1, 0.05, 0.01. 0.005, and 0.001 pL/mL.
a 450-W xenon arc lamp for excitation and PMTs for detection. A reference PMT was used to monitor the source intensity for ratiometric intensity measurements. The spectrofluorometer sample compartment was maintained at 25.0 0.1 OC with a Haake A81 temperature control unit. Solutions were contained in quartz cuvettes and were not deoxygenated. For all measurements the entrance and exit slits were set at 16 and 2 nm, respectively, for both the excitation and emission monochromators. An IBM PC-XT was used for on-line data acquisition. Off-linedata analysis was performed on various microcomputers includingan HP 9!320U,an IBM PC-AT, a Zeos 486, a Macintosh SE, and a Macintosh LC. Blank fluorescence of the solvents (ethanol, NaTC and SDS) was not significant, and no blank correction or subtraction was performed. Excitation-emission matrices (EEMs)were collected as a series of emission spectra with 4-nm scanning intervals for both excitation and emission wavelengths. Each intensity measurement was the average of five samplings, performed internally by the instrument over a period of a few seconds. The contour plots of the EEM data were generated by Surfer software (Golden Software, Inc., Golden, CO). Reference is made in the following sections to “mean EEMs” and ‘uncorrected matrix correlations” (UMCs). A mean EEM is simplythe s u m of n EEMs divided by n. The UMC is a measure
*
Each measurement in the synchronous spectra is the internal average of 20 samplings, performed by the instrument over a period of severalseconds. Thespectra were smoothedthree timea using the smoothing algorithm provided with the instrument software. The algorithm uses a fixed bandwidth, sharp cutoff, three-point, symmetrical low-pass digital filter that causes no phase shift. Synchronous spectra were collected with a AA of either 5 or 38nm, using 1-nmscanning intervals for the excitation and emission monochromators. Plots of the synchronousspectra were generated using Sigmaplot software (Jandel Scientific). Measurementsof fluorescenceintensity at individualexcitation and emission wavelengths are reported as the averageof triplicate measurements,where eachmeasurement was the internal average of 50 samplings, performed by the instrument over a period of approximately 15 s. Absorbance measurements were made at room temperature using a Hewlett Packard 8451A diode array spectrophotometer. Absorbance at individual wavelengths is reported as the average of duplicate or triplicate measurements. All measurements and spectra were collected using the corresponding solvent (ethanol, NaTC or SDS) as the blank. Standard addition of PAHs was performed by addition of the appropriate volume of a standard solution of pyrene (Accustandard), fluorene (Accustandard), and/or carbazole (Aldrich) in ethanol to a volumetric flask, evaporation of the ethanol (for the micellar solutions)by passing N&) through the flask and dilution with the SRC-IIsolutionin ethanol, SDS, or NaTC. Thesolutions were sonicated for 1h and used immediately.
RESULTS AND DISCUSSION
Dynamic Range. Studies of dynamic range, absorbance, energy transfer, standard addition, and sensitivity were performed with the Solvent Refiied Coal Heavy Density (SRC-11) sample. Excitation-emission matrices (EEMs), shown in Figure 2, were collected in the spectral region of & = 255-395 nm, bm = 300-440 nm for five different concentrations of SRC-I1 (0.001, 0.005, 0.01,0.05, and 0.1 rcL/mL) in ethanol, NaTC, and SDS. Higher concentrations of the coal liquid were not studied due to formation of a precipitate in the NaTC and SDSsolutions. The EEMsfor a given solvent in Figure 2 were collected at the same applied voltages to the PMT so that the intensities are on the same scale. A significant shift in the fluorescence to shorter wavelengths with decreasing coal liquid concentration is observed in the ethanol solutione. The shift occurs to a much smaller degree in the micellar solutions, which are dominated by the longer wavelength fluorescence at all concentrations. Relative enhancement of fluorescence at longer wavelengths in the organized media may be due to increased contributions from more hydrophobic components with longer lifetimes. Solubilization of these components within a micelle will afford (42) Tu,X.M.; Burdick, D.
S.J. Chemom. 1989, 3, 431.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 7 . I
I
2928
i1.04 3
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6
4
Addea Pyrene ConcentraTion (pM) Figure 3. Relative fluorescence intensity vs added pyrene for SRC-I1 coal liquid (0.005 pL/mL)in ethand (squares)and30 mM NaTC (circles), measured at Lx= 339 nm, X, = 376 nm, where pyrene is the main contributer to the intensity.
e o.on
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SRC-il Concentration (pi/mi) Flgure 4. Absorbanceat 334 nm vs concentration of SRC-I I in ethanol (squares),30 mM NaTC (circles),and 30 mM SDS (triangles).
some protection from collisional quenchers such as dissolved oxygen in the bulk solution; components with longer-lived fluorescence are particularly susceptible to collisional interactions and would therefore experiencegreater enhancement upon protection from such interactions. Additional explanations for the spectral shift to longer wavelengths are considered in a later section. Similarity of the spectral features across the concentration range was quantitatively compared for the SRC-I1 in each solvent (ethanol, NaTC, and SDS) by calculating the uncorrected matrix correlation (UMC, eq 1)between each EEM for a particular concentration and the mean EEM for the five concentrations in that solvent (Table I). The average of the five UMCs and their standard deviations for each solvent are also shown in Table I. The UMCs show that the EEMs for the five concentrations are much more similar in the organized media solutions than in the ethanol solution. Moreover, the average UMCs are higher for the micellar solutions, and the standard deviations are lower. These results indicate that the similarity among spectra across the concentrationrange is greater in the micellar media, which corresponds to an increased dynamic range. The UMCs for all three solvents decrease significantly at 0.1 pL/mL due to concentration effects, but the UMCs are still much higher for the two organized media relative to ethanol. The UMCs for the NaTC solutions are slightly higher, and
0.02
0.04
0.06
0.12
SRC-I I Concentration (pi/ml) F l g w . 6. Absorbance at 550 nm vs concentratlon of SRCII In ethenol (top),30 mM NaTC (middle), and 30 mM SDS (bottom).
the standard deviation slightly lower, than for the SDS solutions. This suggests that NaTC is marginally more effectivethan SDSat extendingthe dynamic range. However, it should be noted that the concentration of micelles for 30 mM total monomer concentration is approximately 10-fold greater for NaTC than for SDS;on the other hand, it is unlikely that the small NaTC micelle can accommodate more than one solute molecule per micelle, whereas the SDS micelle can bind multiple molecules. The relative capacities of the two media are therefore probably similar, as are their effects on dynamic range. In order to further investigate dynamicrange, the standard addition technique was used to incorporate additional pyrene into the solutions of SRC-I1 in ethanol and NaTC. The fluorescence intensity of the pyrene peak increases with increasing pyrene concentration in both solvents (Figure 3), but the increase is much greater in NaTC. The EEMs (not shown) of the solutions in both solvents show an increase in the fluorescence peak around Lx= 355 nm, hem = 380 nm, which is attributed primarily to ppene with some contribution from benzoblpyrene. In ethanol, the EEMs also showed changes in spectral features of other peaks throughout the matrix. In contrast, in the NaTC solutions, the pyrene peak increases but relatively little change is observed in the other spectral features until the highest concentration of added pyrene (7.5 pM) is achieved. This absence of indirect effects of added pyrene on other peaks in NaTC again demonstrates
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
Table 11. Ratio of Sensitivity. in Organized Media Relative to Sensitivity in Ethanol
0.16
r2
peak
0.15
(AexheIJ (Im)
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t
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2951356 3111368 3231396 3351392 3371342 3501396 3751400 391t404
0 0.14
0
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2 0
0.13
3
possible fluorescent componentb carbazole Pyrene pyrene, BeP pyrene, BeP, anthracene 2,3-benzofluorene BeP, anthracene BaP, anthracene BaP
solvent NaTC SDS 1.7 1.5 2.0 1.7 3.9 2.7 5.0 3.2 1.3 1.2 2.4 1.8 1.4 1.6 2.0 3.1
E
? 0 e
a Sensitivities for each solvent are the slope of the plot of relative fluorescenceintensity w SRC-I1concentration, taken over the linear range which includesthe three lowest concentrationaof SRC-II.bFrom ref 43. BeP is benzo[elpyrene, BaP is benzo[alpyrene.
0.12
".,I
,
0
1.
2
1
6
Added Pyrene Concentration
8
P
(pM)
Figure 6. Relative fluorescence lntenslty (Lx= 303 nm, X, = 308 nm)vsadded pyreneconcentratbnfor SRCII (0.005 pL/mL)ln ethanol (squares) and 30 mM NaTC (circles).
the greater dynamic range that is afforded by this medium relative to ethanol. Absorbance Measurements. Plots of absorbance at 334 nm vs SRC-I1concentration are shown in Figure 4. The 334nm wavelength is notable because it is the primary fluorescence excitation peak in the EEMs. It is interesting that the absorbance at 334 nm is largest for ethanol over the entire concentration range, which suggests that the molar absorptivities of the sample components (both fluorescent and nonfluorescent) are generally higher in ethanol than in the organized media. Absorbances in the two organized media are similar at the three lowest SRC-I1 concentrations, above which absorbances for SDS are more similar to those for ethanol. Although none of the curves are perfectly linear, the SDS curve shows the most deviation from linearity. At longer wavelengths, deviations from linearity are observed for both ethanol and SDS but not for NaTC. As shown in Figure 5 for 550 nm, the absorbance for ethanol is relatively constant at the three lowest concentrations and then increases to a higher level at the two highest concentrations. In SDS, the absorbance remains relatively constant or even decreases slightly at the three lowest SRC-I1 concentrations and then dramatically increases. The curve for NaTC is the most linear of the three. These results suggest the formation of ground-state dimers or complexes at high SRC-I1 concentrations in ethanol and SDS, but not in NaTC, illustrating the superior ability of NaTC to isolate solute molecules from each other and from the sample matrix. Furthermore, the dramatically high absorbanceof the most concentrated solution in SDS relative to ethanol suggests that the SDS is actually promoting the dimerization/complexationby trapping two or more solute molecule in a single micelle. Standard Addition ofPAHs. The possibility that energy transfer is involved in the shift of fluorescence to longer wavelengths with increasing coal liquid concentration was studied by making standard additions of PAHs to the coal liquids in the three solvents. On the basis of the spectral overlap of the PAHs present in the SRCII coal liquid sample,43 three were considered as donors for possible energy-transfer processes: (1) fluorene (bx = 255-275 nm and bm = 305-330 nm in cyclohexane); (2) carbazole (bx = 300-340 nm and bm (43)Vo-Dinh, T.; Martinez, P.R. Anal. Chim. Acta 1981,125, 13.
12
SRC-II Concentration (pl/ml)
SRC-II Concentration (pliml)
Fburo 7. Relatlve fluorescence lntenslty versus SRCII concentratlon In ethanol(squares), 30 mM NaTC (circles),and 30 mM SDS (triangles). Intenslty measured at Lx= 375 nm, X, = 400, where the probable fluorophoresare anthracene and benro[a]pyrene (left), and at Lx= 391 nm, X, = 404 nm, where the probable fluorophore is benzo[a1pyrene 0%"
= 340-380 nm in ethanol); (3) pyrene (Lx = 300-340 nm and = 375-400 nm in cyclohexane). Standard addition of fluorene and carbazole individually to SRC-II(O.06 pL/mL) solutions in ethanol, NaTC, and SDS showed no evidence of energy transfer. Standard addition of fluorene did not affect the synchronous peak intensities of fluorene, 2,3-benzofluorene, anthracene, benzo[alpyrene, or perylene. The increasing carbazole fluorescence at X , = 334 nm, )bm = 356 nm showed a more linear range in NaTC (20 mM) than in SDS (20 mM) or ethanol. As was shown in Figure 3 (see above), the intensity of the pyrene peak upon standard addition of pyrene to SRC-I1 (0.005 pL/mL) was found to increase with increasing pyrene concentration, to a much greater extent in NaTC than ethanol. Perhaps more interesting is the significant decrease in the fluorescence at the fluorene peak (bX = 303 nm, bm = 308 nm) as pyrene is added (Figure 6), in both NaTC and ethanol. While the decrease occurs over the entire range of added pyrene concentration in ethanol, the decrease follows an increase at lower added pyrene concentrations in NaTC. These resulta again demonstrate an increased dynamic range in NaTC relative to ethanol. Although the decrease in fluorene intensity in NaTC at higher pyrene concentrations may be due to energy transfer, another possibility is that pyrene molecules may be replacing fluorene molecules in the NaTC micelles. This would also explain the absence of energy transfer upon standard addition
bm
ANALYTICAL CHEMISlRY, VOL. 64, NO. 23, DECEMBER 1, 1002 lm
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171
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lm
-
,-
-I
Emission Wavelength (nm) Firpa 8. EEMs (L = 255-395 nm lncreaslng along y-axis, b,,, = 300-440 nm increasing along x-axis) for dlfferent coal llqulds (0.005 pUmL) In ethanol: SRCII (upper left); V-178 (upper rlght); V-1072 (lower left); V-1074 (lower right).
of fluorene. The solubilities of fluorene and pyrene in water at 25 "C are reported to be 11.2 and 0.68 p M , respectively.a Since fluorene is 16 times more soluble than pyrene, it is reasonable to expect that pyrene would be preferentially solubilized in the NaTC micelles as its concentration is increased, displacing the more soluble fluorene into the aqueous environment. Thus, as the pyrene concentration is increased, fewer fluorene molecules would be protected from intermolecular interactions and matrix effects, and the fluorene fluorescence would subsequently decrease. Finally, it is also possible that pyrene, which also absorbs at the fluorene absorption peak, reduces the excitation intensity available to fluorene a t high pyrene concentrations;however, this effect should be approximately equal in ethanol and NaTC. The concentration studies and standard addition experiments suggest several possible causes for the shift of fluorescenceto longer wavelengthsas coal liquid concentration is increased. The absorbance measurements clearly indicate a concentration-dependent, ground-state dimerization or complexation in the SDS solutions, most likely facilitated by trapping several solutes in a single micelle. Energy transfer is also possible, as indicated by the decrease in the fluorene peak upon standard addition of pyrene, although our results do not support this possibility and suggest that the effect (44)Pearlman, R. S.;Yakowsky, S.H.;Banerjee, S.J. Phys. Chem. Ref. Data 1989, 13, 1984.
may be due to preferential solubilization of pyrene. Other possible causes include deviations from Beer's law, inner filtering, and self-absorption effects, which would be more likely at shorter Wavelengths due to the greater total concentration of absorbers in this region relative to longer wavelengths. These effects have been considered elsewhere to explain the decrease in fluorescence intensity for PAHs in the shorter wavelength regions for concentrated PAH solutions and for concentrated Synthoil coal liquid solutions;" other investigatorshave debated between energy transferu*u and inner filter effects26as the cause for decreased fluorene emission around 303nm in the presence of pyrene in synthetic PAH mixtures. Relative Sensitivity. Fluorescenceintensity waa meaured at eight (b,,, bm)points in spectral regions corresponding to different fluorophores for the five concentrations of SRC-I1 in ethanol, NaTC and SDS. At most wavelengths, the sensitivity (taken as the slope of the linear portion of plots of intensity vs concentration)in the organized media solutions is approximately twice that in the ethanol solutions, and the sensitivity for the NaTC solution is greater than for the SDS solution (Table 11). Since the absorbances in this excitation wavelength region were actually lowest in NaTC and highest in ethanol (see above),the increased fluorescencerepresents a true enhancement effect, i.e., an increase in quantum yield. These results substantiate the capability of the micellar media (45) Vo-Dinh, T.Appl. Spectrosc. 1982, 36, 576.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992 m
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Emission Wavelength (nm) Flgure 9. EEMs (bx = 255-395 nm Increasing along y-axls, h.m = 300-440 nm lncreaslng along x-axls) for dlfferent coal liquids (0.005 &/mL) In 30 mM NaTC: SRCII (upper left); V-178 (upper right); V-1072 (lower left); V-1074 (lower rlght).
to protect the fluorophoresfrom intermolecular interactions and supports the superiority of NaTC micelles for this purpose. An interesting feature shown in Table I1 is an apparent enhancement of the sensitivity in the SDS solutions as compared to the NaTC solutions at the two longest wavelengths. Plots of fluorescence intensity vs SRC-I1 concentration at longer wavelengths (Figure 7) show that the fluorescence levels off at high concentrations in NaTC but keeps increasing in both ethanol and SDS. The formation of ground-state dimers or complexes or energy-transfer processes could contribute additional fluorescence intensity in these longer wavelength regions, which is consistent with the results of the absorbance and standard addition experiments discussed above. Characterization of Four Different Coal Liquids. EEMs were collected for the four different coal liquids (SRC11, V-1072,V-1074and V-178)in ethanol, NaTC, and SDS (Figures 8-10),over the same wavelength region used in the above studies of SRC-11. The coal liquids were at a concentration of 0.005pL/mL,which is the next-to-lowest of the five concentrations used in the SRC-I1 studies and is below the concentrationat which significant concentration effects were observed. Spectral differences between the coal liquids are apparent. The origin of these distinguishing features, whether due primarily to intermolecular interactions and matrix effects or to differences in fluorophore composition, can be studied
by comparing the EEMs for the organized media solutions to those for the ethanol solutions. A visualcomparison of the coal liquid spectra for the ethanol and the organized media solutions was inconclusive;therefore, for each solvent,the UMCs were calculated between the EEM of each coal liquid and the mean EEM for the four coal liquids (Table 111). The UMCs show that the EEMs of the four coal liquids are most similar in ethanol. Since the micellar media are likely to enhance the contributions from the more hydrophobic components and those with longer lifetimes, which are most susceptible to quenching effects and excitedstate interactions, it is likely that the different coal liquids have significant variations in composition with respect to these components. Thus, the organized media may actually provide increased discrimination between samples at low concentrations;for this purpose, NaTC and SDS serve equally well. It is interesting to compare these results with an earlier study of crude oils, which showed that the EEMs for three different crude oils were more similar in NaTC micellar media than in a simple solvent, cyclohexane.12 The results of that study suggested that the differences between the spectra were due to dynamic interactions and matrix effects in the crude oils. Synchronousspectra (AA = 5 nm) of each coal liquid (again at 0.005 pL/mL) in the three solvents are shown in Figure 11. For all of the coal liquids, the synchronousspectral intensity is greatly enhanced in the organized media relative to ethanol.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992
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Flgurr 10. EEMs (bx = 255-395 nm increasing along y-axis, X, = 300-440 nm Increasing along x-axis) for different coal llqulds (0.005 pL/ml) In 30 mM SDS: S R C I I (upper left); V-178 (upper right); V-1072 (lower left); V-1074 (lower right).
Table 111. Uncorrected Matrix Correlation (UMC), Calculated between the EEM of Each Coal Liquideb and the Mean EEM for All Four Coal Liquids in That Solvent” coal liquid solvent V-1072 V-1074 V-178 SRC-I1 mean f 1 sd ethanol 0.9991 0.9902 0.9876 0.9986 0.9914f 0.0053 NaTC 0.9994 0.9842 0.9814 0.9763 0.9853 0.0099
SDS
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a Estimated standard deviation of each EEM, based on triplicate measurements, is f0.0001.* EEMs are shown in Figures 8-10. The right-hand column is the mean UMC f one standard deviation for each solvent. 0 300
However,a relative reduction of the peak a t 375nm (attribued to anthracene) and enhancement of the peak a t 400 nm (attributed to benzo[alpyrene) is observed in the SDS solutions, compared to both ethanol and NaTC. This is again suggestive of intermolecular interactions in SDS, especially for the SRC-11, V-178,and V-1072coal liquids.
CONCLUSIONS These studies demonstrate the benefita of NaTC micellar media for analysis of complex samples. NaTC is shown to be superior to SDS for protecting solutes from ground-state and excited-state processes that limit the dynamic measurement range and make accurate calibration difficult. In fact,
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Synchronous Emission Wavelength (nm) Flguro 11. Synchronousexcitatlonfluorescence spectra (AA = 5 nm) for different coal llqulds (0.005 pL/mL) In ethanol (dotted line), 30 mM NaTC (dashed line), and 30 mM SDS (solid Ilne): S R C I I (upper left); V-178 (upper right); V-1072 (lower left); V-1074 (lower rlght).
the SDS micelles are shownto facilitate interactions between solutes, increasing ground-state dimerization/complexation and excited-state interactions. Fluorescence sensitivity is
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approximately %fold greater in NaTC than in ethanol, while absorbance is lowest in NaTC, indicating true fluorescence enhancement in the micellar media. Comparison of the spectra of four different coal liquids at low concentrations indicates that both NaTC and SDS micellar media improve the ability to discriminate between the coal liquids, relative to ethanol.
Research, United States Department of Energy (Grant Number DE-FG0588ER13931). P.M.R.H. was supported by an Analytical Division Summer Fellowship from Dow Chemical U.S.A. The coal liquids were provided by the Pithburgh Energy Technology Center of the United States Department of Energy.
ACKNOWLEDGMENT
RECEIVED for review June 3, 1992. Accepted August 24,
This work was supported by the Division of Chemical Sciences, Office of Basic Energy Sciences, Office of Energy
1992. Registry No. NaTC, 145-42-6;SDS, 151-21-3.
