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Anal. Chem. 1088. 6 0 , 1043-1045
Effects of Metal Cations on the Fluorescence Intensity of Polycyclic Aromatic Hydrocarbons in Sodium Taurocholate Micellar Solutions Kasem Nithipatikom and Linda B. McGown* Department of Chemistry, P. M.Gross Chemical Laboratory, Duke University, Durham, North Carolina 27706
The effects of Tb3+, Eu", and AI3+ on the fluorescence Intemtly of polycyclic aromatic hydrocarbon (PAH) compounds in sodlum taurocholate (NaTC) micellar solutions were determlned. Aqueous solublllty of the PAH appears to play an Important role In the effects of the catlons, whkh ranged from fluorescence quenctdng of relathrely soluble PAHs to as much as 18-fold enhancement for the more hydrophobic PAHs. Sensltlvltles and detectlon llmlts were determined for five PAHs In sodlum dodecyl sulfate (SDS) micelles, NaTC mlceiles, and NaTC mlcelles with Tb". Although poorest results were obtained with NaTC micelles, the addltlon of Tb3+ increased sensltlvltles and decreased detectlon llmlts In most cases to levels at least as good as those obtalned with SDS. Detectlon limits for benro[k#iuoranthene were an order of magnltude lower in NaTC-Tba+ than In SDS.
Table I. Effects of Metal Cations on PAH Intensity in NaTC Micellar Solutionsn compounds phenanthrene anthracene 9-methylanthracene fluoranthene pyrene 9,lO-dimethylanthracene 2-methylanthracene benzo[a]pyrene perplene benzo[ghi]perylene
Sodium taurocholate (2-[ [ (3a,5/3,7a,12a)-3,7,12-trihydroxy-24-oxocholan-24-yl]amino]ethanesulfonate sodium salt), NaTC, is a micelle-forming bile salt. The three hydroxy groups on the cholic acid part of the molecule play an important role in the micellar characteristics of NaTC and, along with the taurine group, serve to solubilize NaTC in aqueous solutions (1). The most commonly observed micellar form has an aggregation number of four and can bind other molecules in the relatively hydrophobic interior, usually with a 1:l stoichiometry (2). The formation of secondary micelles has not been observed. Insoluble biological lipids can be solubilized by comicellization with NaTC (3)and NaTC can also form mixed micelles with detergent molecules (2). The micellar properties of NaTC are relatively insensitive to experimental conditions. The aggregation number and critical micelle concentration (cmc) show very little dependence on pH in the range of 1.6-10 (I), on counterion concentration, and on temperature in the range of 10-60 "C (3). A cmc of 3 mM was found from the shift in the absorption wavelength maximum of Rhodamine 6G (3). In a recent study, fluorescent probes were used to study the properties of the NaTC micellar binding site (4). We describe here the effects of the trivalent metal cations Tb3+,Eu3+, and A13+ on the fluorescence intensities of polycyclic aromatic hydrocarbons (PAHs) in NaTC micellar solutions. Sensitivities and detection limits for five PAHs were determined in NaTC micelles alone and in the presence of each of the metals, and in sodium dodecyl sulfate (SDS) micelles.
EXPERIMENTAL SECTION The NaTC (Mallinkrodt),SDS (Fluka),carbazole and perylene (Aldrich), phenanthrene, benzo[ blfluoranthene (BbF), benzo[klfluoranthene (BkF), benzo[ghi]perylene (BgP), 9-methylanthracene (9MA), phenanthrene, and fluoranthene (Foxboro), and 9,10-dichloroanthracene (DCA, from Alfa) were all used without further purification. Pyrene, anthracene, benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), 9,lO-dimethylanthracene (DMA), 0003-2700/88/0360-1043$01.50/0
benzo[e]pyrene 9-phenylanthracene benzo[b]fluoranthene benzo[k]fluoranthene 9,lO-dichloroanthracene carbazole
solubility,b intensity ratioc W M Tb Eu A1 7.2 4.1 1.4
1.3 0.67 0.27 0.20
0.015 0.0016 0.00094
0.61 1.3 2.2 0.76
0.49 1.2 1.7 0.99
2.0 3.9
0.61 3.3
3.8 5.7 10.3
7.8 1.1
4.6 3.0 17.9 3.6 0.65
0.67
1.2
3.4
4.8 6.2 6.5
12.7
1.0 4.2
3.0 17.8 3.7
11.4
0.45
'PAHs 2.0 p M , NaTC 10 mM, metal cations 10 mM, measurements made 1 h after mixing, 25 "C. bSolubilitiesfrom ref 6. Ratio of PAH intensity in NaTC-metal to intensity in NaTC alone.
2-methylanthracene (2MA),and 9-phenylanthracene (9PA) were obtained from Aldrich and recrystallized from ethanol. The metal solutions were prepared from the nitrate salts (>99.99% purity) of the metals. Distilled demineralized water was used for solution preparations. Stock solutions of the PAHs were prepared in ethanol. Aqueous stock solutions of the micelles were prepared fresh daily to avoid errors due to aging (5). Micellar solutions of the PAHs were prepared by gently evaporating the ethanol from the appropriate volume of the PAH stock solution, followed by dilution with the NaTC micelle stock solution in a volumetric flask and sonication for at least 1 h. None of the solutions was deoxygenated. All fluorescence measurements were made with an SLM 4800s spectrofluorometer (SLM Instruments, Inc., Urbana, IL) with a 450-W xenon arc lamp source, excitation and emission monochromators for wavelength selection, and photomultiplier tube detectors. The sample chamber was maintained at 25.0 & 0.1 "C with a Haake A81 temperature controller. Fluorescence intensities were measured in triplicate in the "100-average" mode, in which each measurement is the average of 100 samplings over a 30-s period, performed internally by the spectrofluorometer electronics. The monochromator entrance and exit slits were set at 4 nm. Absorption spectra were collected on a Perkin-Elmer Lambda Array spectrophotometer.
RESULTS AND DISCUSSION Effects of Metal Cations on Fluorescence Intensity. The effects of Eu3+, Tb3+, and A13+on the fluorescence intensities of PAHs in aqueous NaTC solutions are shown in Table I. The intensity ratios are expressed as the ratio of the intensity in the presence of the metal to the intensity in the absence of the metal, measured at the emission and excitation wavelength maxima of the PAH in NaTC without the
0 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 10, MAY 15, 1988
1044
1
20.00
I
14.00 12.00
B z
10.00
6.00 4.00 2.00 0.00 0.00 2.00
1.00
Figure 1. Fluorescence intensity of PAHs (2 pM) in NaTC (10 mM) as a function of Tb3+ concentratlon: (m) BkF; (A)perylene; (e)anthracene; (X) carbazole.
4
I
1
I
,
1
I
I
I
-9.0 -8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 LOG MOLAR CONCENlRAllON
4.00 6.00 8.00 10.00 12.00 14.00 16.00 lb3CCONCEMR4110N. rnM
-8
5
Figure 3. log plots of calibration curves for pyrene: (m) SDS, 10 mM; (A)NaTC, 10 mM; (e)NaTC, 10 mM with 10 mM T ~ J ~ + .
20.00
5.50 18.00
5.00
16.00
c r
14.00 12.00 10.00
W O
3 gz
8.00
4.50 /
1f
4.00
z
3.50
' W
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3.00 2.50
4.00
ieL----
2.00
2.00 1S O-8.5 -8.0 -7.5 -7.0 -6.5 -6.0 -5.5 -5.0 -4.5
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C 3 2.00
k
4.00
-1
LOG MOLAR CONCENlRAllON
6.60 8.00 1 0 . 0 0 1 2 . 0 0 1 4 . 0 0 1 6 . ~ 3
Flgure 4. log plots of calibration curves for BgP, legend as in Figure Figwe 2. Fluorescence intensity of PAHs (2 pM) In NaTC (10 mM) as a function of Eu3+ concentratlon, legend as in Figure 1.
metal. For most of the PAHs, Eu3+and Tb3+had very similar effects. One exception to this was pyrene fluorescence, which was enhanced by Tb3+but quenched by Eu3+. This may be due to different effects of the cations on the vibronic structure of pyrene, which is very sensitive to its microenvironment. Precipitation occurred in most of the NaTC-PAH solutions upon the addition of A13+,and intensity ratios were obtained for only four of the PAHs. These results agreed roughly with those obtained for the two heavy metals. Aqueous solubilities have been reported in the literature for some of the PAHs (6) and are shown in Table I. There is a definite trend toward increased enhancement as the aqueous solubility of the PAH decreases. For all of the metals, BkF showed the most enhancement, followed by perylene and BgP which are the least soluble of those PAHs for which solubilities were reported. Carbazole, fluoranthene, and phenanthrene, all of which are relatively soluble, exhibited quenching in the presence of the metals. Fluorescence intensity of four of the PAHs as a function of metal ion concentration is shown for Tb3+ in Figure 1 and for Eu3+ in Figure 2. The curves for each PAH are very similar for both metals, except for perylene which is more strongly enhanced by Tb3+. Absorption spectra were collected from NaTC solutions of DMA and BkF in the presence and absence of Tb3+ and for DMA and phenanthrene in the presence and absence of Eu3+. Absorption bands a t 300 nm were identical with those observed in the absorption spectra
3.
of the metals in NaTC, and the bands were eliminated from the PAH-metal spectra upon subtraction of the metal spectra. No other absorption bands were observed in any of the spectra in the 250-400 nm range (the absorption bands of the PAHs were too weak to be observed), and the solutions of the metals in NaTC did not have any fluorescence emission in the region of the PAH fluorescence. Therefore, it appears that the fluorescence enhancements were due primarily to increased quantum efficiency rather than increased molar absorptivity. Further studies are needed to determine, first, the mechanisms of the quenching and enhancement caused by the metal cations and, second, their relative contributions to the observed intensity changes and their dependence on PAH structure and solubility. Collisional quenching of the excited state of pyrene in NaTC by Cu2+has been reported (3, and similar collisional quenching mechanisms may be involved in the systems discussed here. It is also possible that energy transfer from the PAH to the metal contributes to the quenching, although we did not observe any emission from the metals. Enhancements may result from increased structural rigidity of the NaTC-bound PAH in the presence of the metal cations, as well as from reduced access of the aqueous solution to the PAH molecule. These and other possible explanations for the observed intensity effects are currently under investigation. Sensitivity and Detection Limits. Calibration curves of log intensity vs log PAH concentration are shown for pyrene in Figure 3 and for BgP in Figure 4, for SDS micellar solutions,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 10, MAY 15, 1988
Table 11. Detection Limits for PAH Compounds, in nM
anthracene BgP
pyrene BkF perylene
NaTC
NaTC-Tb3+
SDS
4.4 4.4 0.76 0.49 0.36
0.68
0.46
0.69 0.090 0.021
2.0
0.059
0.15 0.20 0.12
NaTC micellar solutions, and NaTC with Tb3+. For both PAHs, highest sensitivity was obtained with NaTC-Tb3+. It is also interesting that the linear range extends to higher concentrations for SDS and NaTC-Tb3+ than for NaTC alone, especially in the case of BgP. This may be due to poorer solubilization of the PAHs in the NaTC solution, which would indicate that the addition of the metal to the NaTC solution increases either the binding equilibrium or the stoichiometry of the PAH-NaTC association. Detection limits were found for five PAHs, including anthracene, BgP, pyrene, BkF, and perylene, in SDS, NaTC, and NaTC-Tb3+ solutions. The results are shown in Table 11. The detection limit was calculated as the concentration of PAH, found from a linear calibration curve of five points run in triplicate, required to produce a signal equal to the blank signal (averaged for 15 measurements) plus three times the standard deviation of the blank signal. For all five of the PAHs, highest detection limits were found for NaTC. For all but anthracene, detection limits were significantly lower for NaTC-Tb3+ than for SDS, most notably for BkF for which the detection limits differed by an order of magnitude.
CONCLUSIONS Several conclusions may be drawn from the intensity ratio experiments. The effects of the heavy metals Eu3+and Tb3+ are similar to each other and to the effects of A13+,indicating that the ionic charge is probably more important than ionic size in determining the extent of enhancement. Ionic size is probably more critical to precipitate formation which occurred for most of the PAHs with NaTC-A13+ but not with the heavy metal ions in NaTC. It is likely that the metal cation is coordinated to the negatively charged taurine groups of the NaTC micelle. The resulting increase in structural rigidity and decrease in accessability of the interior of the micelle (including the hydrophobic binding site) to the surrounding aqueous solution should serve to decrease nonradiative deexcitation pathways of the bound PAH, thereby increasing the fluorescence quantum yield. Such an effect has been
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previously reported for Mg2+in NaTC micelles (2). It is also possible that the metal ion changes the properties of the NaTC micelle in terms of aggregation number, binding stoichiometry, and cmc. The role of PAH solubility may simply be to determine whether the PAH is located in the hydrophobic binding site, on an exterior portion of the micelle, or in the aqueous solution. Other properties of the PAHs may also need to be considered in order to explain the effects of the metal cations. These areas are currently under investigation. Studies of NaTC micelles are clearly applicable to the analytical chemistry of PAHs. Micelles are often used to solubilize PAHs in aqueous solution and, although the intensities of PAHs in NaTC micelles are generally not as high as in detergent micelles, the work described here demonstrates that the addition of metal cations to the NaTC solutions can be used to increase the PAH intensities to levels a t least as high as those observed in detergents. Aside from intensity considerations, NaTC micelles are in many ways preferable to detergent micelles. They are much easier to work with because they exhibit less sudsing and much lower light scattering levels. They are more versatile in terms of their tolerances of pH, ionic strength, temperature, and counterion concentrations and are less likely to precipitate. Therefore, the use of NaTC with metal enhancement may simplify experimental procedures without sacrificing sensitivity and detection limits, and in some cases can even improve detection limits by as much as an order of magnitude. Registry No. NaTC, 145-42-6;SDS, 151-21-3;BbF, 205-99-2; BkF, 207-08-9; BgP, 191-24-2;9MA, 779-02-2;DCA, 605-48-1;BaP, 50-32-8; BeP, 192-97-2; DMA, 781-43-1; 2MA,613-12-7; 9PA, 602-55-1; Tb, 7440-27-9; Eu, 7440-53-1; Al, 7429-90-5; phenanthrene, 85-01-8; anthracene, 120-12-7; fluoranthene, 206-44-0; pyrene, 129-00-0; perylene, 198-55-0;carbazole, 86-74-8.
LITERATURE CITED
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(1) Small, D. M. I n Molecular Association in Biological and Related Sys tems; Advances in Chemistry Series No. 84; Gould, R. F., Ed.; American Chemical Society: Washington, DC, 1966; pp 31-52. (2) Chen, M.; Gratzel, M.; Thomas, J. K. J . A m . Chem. Soc. 1975, 9 7 , 2052. (3) Carey, M. C.; Small, D. M. J . Colloid Interface Sci. 1989, 37, 382. (4) Nithipatikom, K.; McGown, L. 8 . Photochem. Photobiol., in press. ( 5 ) Gratzel, M.; Thomas, J. K. J . Am. Chem. SOC. 1973, 95, 6885. (6) McKay, D.; Shiu, W. Y. J . Chem. Eng. Data 1977, 22, 399. (7) Hashimoto, S.; Thomas, J. K. J . Colloid Interface Sci. 1984, 702, 152.
RECEIVED for review October 14,1987. Accepted January 26, 1988. This work was supported by the United States Army Research Office.
