754
Anal. Chem. 1980,
52, 754-759
Analysis by Micelle-Stabilized Room Temperature Phosphorescence in Solution L. J. Cline Love,” Marie Skrilec, and J. G. Habarta Department of Chemistry, Seton Hall University, South Orange, New Jersey 07079
The analytical usefulness of a new method based on room temperature phosphorescence (RTP) in aqueous micellar solution was examined. The analytical figures of merit are given for naphthalene, pyrene, and biphenyl in TVNa and Ag/Na mlxed counterion lauryl sulfate micelles. The average precision of the measurements is - 6 % , and the sensitivities are competitive with other phosphorescence techniques. Sample temperature was found to have a moderate effect on RTP intensities. The ratio of heavy metal/sodium in the micelle had a dramatic affect on relative luminescence intensities up to -10-20% heavy atom. The fluorescence was greatly diminished and the RTP enhanced. No RTP was observed in the absence of heavy atoms. Luminescence spectra for naphthalene in Na micelles, in TVNa micelles, in Ag/Na micelles, and in EPA solution at 77 K are compared. Some reasons for the observed curvature in the analytlcal curves of these micellar-stabilized probes are discussed. A prospective is also given on the general applicability of this method in analytical chemistry.
Although weak emission from the triplet state in fluid solution has been observed (1-31, recent reports indicate that dramatic enhancement of room temperature phosphorescence (RTP) in solution can be achieved when the species is incorporated into a micellar assembly (4-7). The heterogeneous nature of these surfactant aggregates greatly affects the photophysics of aromatic probe molecules by mechanisms such as altered microenvironment and orientational constraints. These properties make them of special interest for use as model membrane systems in biochemistry. Additionally, micelles can increase the solubility of hydrophobic species in water, effectively screen t h e excited triplet state from quenchers present in solution, and increase the proximity of interacting species. T h e 2nd two properties have profound implications for the development of a practical analysis method of aqueous solutions of aromatic species based on their phosphorescence intensities. Although phosphorimetry has been shown to be a sensitive method for the analysis of numerous species, many of biological interest (8,9), its widespread use has been hampered by the inconvenient and often time-consuming sample conditions required. Generally, it has been necessary to immobilize the sample in low-temperature glasses or adsorb the sample on an inert substrate such as filter paper (IO,11). The latter technique does allow observation of phosphorescence at room temperature, but has the disadvantages of rather cumbersome sample preparation, critical drying requirements, and high phosphorescent background intensity from the filter paper substrate. This paper demonstrates the first analytical application of micelle-stabilized room temperature phosphorescence in aqueous solution. Criteria regarding critical micelle concentration, reagent purity, oxygen quenching, sample temperature, and heavy atom enhancement will be presented. The analytical figures of merit, specifically limit of detection, linear 0003-2700/80/0352-0754$01 O O i O
Table I. Spectrofluorimeter/RT Phosphorimeter Components
component lamp power supply xenon lamp lamp housing excitation monochromator emission monochromator sample compartment photomultiplier tube (PMT) PMT housing PMT power SUP.PfY
amplifier/recorder
model and manufacturer Sorensen, Model X L S l A Canrad Hanovia, Inc., No. 901C1100, 150 W (Newark, N.J.) Schoeffel Instruments, Rlodel LH 1 6 0 (Westwood, N.J.) Heath C o . , Model EU 700-58 (Benton Harbor, Mich. ) GCA/iVcPherson, Model E L - 7 0 0 (Acton, Mass.) laboratory constructed Hamamatsu R 4 4 6 (Middlesex, N.J.) GCAiMcPherson, Model E U 701-93 (Acton, Mass.) Hewlett-Packard, custom design (Palo Alto, Calif.) Heath Co., LogiLinear Current Module E U 20-28 (Benton Harbor, Mich.)
dynamic range, and precision are given for some aromatic hydrocarbon probe molecules. Spectral comparisons between R T P and 77 K phosphorescence are also examined, as well as the effects of heavy atoms on the spectra. Several significant advantages of this analytical technique are illustrated. For example, the ability to work in fluid solution a t room temperature greatly eases sample handling, sample preparation, and increases speed of analysis. Further, it will be shown that interferences from scattered light and fluorescence are greatly diminished, even for species exhibiting small Franck-Condon spectral shifts, thus allowing the use of a conventional fluorimeter for measurement of the phosphorescence of many species of interest. Comparable or improved sensitivity compared to 77 K data was achieved for naphthalene, pyrene, and biphenyl.
EXPERIMENTAL Apparatus. Uncorrected room temperature phosphorescence and fluorescence solution spectra were obtained using the laboratory-constructed spectrofluorimeter described elsewhere (12, 13). The individual components are summarized in Table I. To obtain uncorrected 77 K phosphorescence spectra, a rotating can (modified, American Instrument Co., Silver Springs, Md., phosphorescope attachment) was added to the spectrofluorimeter and the excitation monochromator was replaced by a 250-380 nm transmittance filter (Corning ;7-54 with >65% transmittance). Samples were rotated at a constant speed with a specially constructed sample tube rotator (Hacker Machine Shop, East Lansing, Mich.). Sample cells used for room temperature phosphorescence and fluorescence spectra were conventional 10 X 10 X 45 mm quartz cells (S-18-260, Pyrocell, Westwood, N.J.). The liquid-nitrogen temperature spectra were obtained using a phosphorescope Dewar flask (American Instrument Co.) and rotated 5 X 178 mm sample tubes (Wilmad Quartz). Absorbance spectra were obtained using a Cary 15 spectrophotometer. E‘ 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL 52, NO. 4, APRIL 1980
Temperature control of fluid samples used in the room temperature phosphorescence spectra studies was achieved by thermostating the sample compartment with a laboratory-constructed constant-temperature water bath having a circulating pump (Fisher Scientific Co.). CMC Flow System. The critical micelle concentration was established by monitoring the onset of fluorescence using a laboratory-constructed fluorescence flow system. A constant-feed delivery system was constructed from a 2-L bottom-feed reservoir containing the surfactant solution with a 0.5-cm-bore Tygon tube and ending in a B-D Yale z23 hypodermic syringe needle. By movement of the gravity-feed reservoir, siphoned flow into the reaction mixing chamber was maintained constant in the 1-2 mL/min range. The solutions were magnetically stirred in a standard laboratory beaker used as the "reaction" chamber prior to the sample flow cell. A 1 cm2,3-mL capacity conventional quartz flow cell was used in a modified laboratory-constructed sample compartment attached to the spectrofluorimeter. Recirculated flow from the mixing chamber was controlled at 100, 150, or 200 mL/min by a laboratory micropump. Reagents. Methanol and acetone solvents (Fisher Spectroscopic Grades) were further purified by distillation. USP ethanol (absolute) was used without further purification. Water used for purification of reagents and to prepare each micellar solution was purified in the following order: distillation, filtration through a Millipore filtration unit, (Milli. Q Water Purification System), followed by double distillation. Ultra-high purity nitrogen (AGL Welding Supply Co., Clifton, N. J.),used to deoxygenate the samples, was passed through an oxygen trap (Indicating Oxy-Trap, Alltech Associates, Arlington, Ill.). Pyrene (Matheson) was double recrystallized from ethanol and found to be >99.5% pure by an HPLC method. Biphenyl (Matheson, lot 18F19) and naphthalene (J.T. Baker, lot 334161) were recrystallized once from ethanol. The heavy metal salt, thallous nitrate (Fisher, lot 7924381, was used without further purification. Silver nitrate (J & S Scientific, Inc.) was recrystallized once from water. The surfactant, sodium lauryl sulfate (NaLS) (BDH Biochemicals, Poole, England) specially purified for biochemical work, was used without further purification. NaLS used to prepare the mixed salts was of lesser purity. To prepare thallium lauryl sulfate (TlLS) and silver lauryl sulfate (AgLS), approximately stoichiometric amounts of each reagent (NaLS, AgN03 or T1NO3) were dissolved in hot water and the two solutions were then mixed. The precipitate was allowed to form overnight. TlLS was recrystalized once from water and then dried in a vacuum oven at 40 "C. AgLS was recrystalized once from water and once from methanol, and then dried in a vacuum oven at 40 "C. Most of the volumetric flasks used were baked in a lo00 K oven prior to being used for the first time for room temperature phosphorescence work. The same volumetric flasks and pipets were used throughout the study. Micellar solvent blanks were checked for fluorescence and/or phosphorescence interferences prior to use. Procedures. For the CMC determinations, an aliquot of the stock probe solution, dissolved in either ethanol or methanol, was transferred to the beaker reaction chamber and the solvent was gently evaporated. After all the solvent was removed, 20-25 mL of water was added and the chamber was connected to the buret input and flow cell input/output. It was allowed to equilibrate to constant fluorescence intensity. A solution of the surfactant under study was metered in at a constant rate, the start of which was coordinated with the start of the stripchart recorder. The fluorescence output of the sample probe in the flowcell was monitored at or near the fluorescence wavelength maximum of the particular probe. The concentration of the surfactant solution used was approximately lox greater than the molarity at the CMC for each particular surfactant. After each determination, lasting about 45 min, the CMC was calculated from extrapolation of the two intersecting lines of the plot representing the fluorescence intensity before and after the CMC. The CMC was corrected for volume changes due to addition of titrant. The values obtained for the NaLS CMC were in good agreement, with those obtained using a tensiometer (Fisher Surface Tensiomat, Model 21, Fisher
755
Scientific Co.) to measure changes in surface tension with increasing surfactant concentration. For the solutions used for the standard calibration curves, a known amount of probe was dissolved in a known amount of acetone and an aliquot of the acetonic solution was transferred to a volumetric flask. Acetone was then evaporated under nitrogen and the residue was redissolved in an aqueous mixed micellar solution using an ultrasonic bath. An aliquot of the sample solution was transferred to a sample cell and deoxygenated for 30 min using purified nitrogen and a vacuum pump, while the sample was kept in an approximately 35 "C water bath. Samples of lower concentration were obtained by dilution from the more concentrated stock solutions. The typical time for analysis of a single sample, including sample preparation, deoxygenation, and measurement, is 1 h. However, simultaneous deoxygenation of several samples considerably reduces the total time of analysis. The surfactant ratio study was carried out by first dissolving the probes in an aqueous NaLS solution, which was further diluted. Different ratios of surfactants were obtained by adding the stock solutions of NaLS to individual weighings of TlLS or AgLS. Prior to recording the room temperature phosphorescence spectra, the samples were illuminated for 15 min or more until a constant phosphorescence signal was obtained. The temperature study was performed as follows. A sample giving a relative intensity on the linear portion of the standard curve as selected. Readings were taken at 20, 25,30, and 35 "C, and again at 25 "C to check for any drifts in the intensity other than changes related to temperature effects. Prior to the first temperature analysis, actual temperature in the sample compartment was calibrated. The temperatures of the samples were equilibrated 10 min before readings were taken.
RESULTS A N D DISCUSSION Micelles are aggregates of amphiphilic molecules formed in solution due t o hydrophobic repulsive interactions of the nonpolar portions of the molecules. T h e monomer has two regions of widely different polarity. T h e nonpolar, hydrophobic "tail" is usually a hydrocarbon chain, and the polar, hydrophilic "head group" is either ionic or neutral. At concentrations just above the CMC in aqueous solution, spherical or ellipsoidal micelles have the head groups oriented outward and the nonpolar tails oriented inward where they are more protected from the polar water molecules. The critical micelle concentration (CMC) is the minimum concentration of monomer necessary for micellization to occur. CMC Estimation. Appreciable stabilization of the triplet state does not occur a t concentrations below the CMC, and no room temperature phosphorescence was observable. Thus, it is imperative t o adjust the monomer concentration t o be well above the CMC of the surfactant under study. T h e surfactant used in this study, sodium lauryl sulfate (NaLS) was found to have a CMC of -9.0 x M by tensiometry and fluorescence titration (13). However, the lauryl sulfate anion was used with mixed cations, Na+ and TI+ or Na+ and Ag', which would not necessarily be expected to have the same CMC as NaLS. T h e hydrophobicity, bulk, and charge of the head groups interact differently with different counterions. T h e latters' hydration requirements and size affect the CMC by disrupting the solvent structure and electrostatic repulsion (14 - 1 6 ) . Generally, surfactant micelles with larger counterions have been found to have lower CMCs, and t o form more ordered micellar structures with increased micellar size and aggregation number, because of their effects on head group electrostatic repulsion (14-16). CMCs of 4.7 x 10-'-8.4 x M have been reported for AgLS (17). Though no data are available for TlLS or the mixed systems, AgLS/NaLS and TlLS/NaLS, it would be reasonable to predict that CMCs for these systems would have lower values than for NaLS alone. Following the same logic, increased ordering, micellar size, and aggregation number could be assumed. In the present study, surfactant concentrations of the mixed-counterion micelles were adjusted to be
756
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
i I
I
I
0
1
[NAPTH;LEYiI
(X
los
M ) O
Figure 1. Log-log plot of naphthalene concentration in WmollL vs. RTP intensity in PA: (-0-)TILS/NaLS = 30 %; total detergent concentration = 0.15 M; temperature = 23 O C ; he, = 476 nm; (-0-)AgLS/NaLS = 5 0 % ; total detergent concentration = 0.08 M; temperature = 20 OC;,A , = 500 nm: ,A, = 276 nm for both curves: coefficients of determination = 0.998 (TI' system) and 0.999 (Ag' system)
r
I
I
I
0
l
2
0
10
[PYREYE]
[X 10'MI
Flgure 2. Log-log plot of pyrene concentration in pmol/L vs. RTP intensity in PA: see Figure 1 caption for conditions; ( - 0 - )TILS/NaLS system, ,A, = 596 nm; (-0-)AgLS/NaLS system, ,A, = 600 nm: A, = 336 nm for both curves: coefficients of determinination = 0.974 7 (TI system) and 0.986 (Ag' system) approximately ten times that of the CMC for NaLS alone. A n a l y t i c a l W o r k i n g Curves. Analytical curves of room temperature phosphorescence vs. probe concentration were typical for some aromatic hydrocarbons in mixed-counterion micelles. Figure 1 shows the working curves for naphthalene in NaLS/TlLS and NaLS/AgLS micelles. The linear dynamic ranges (LDR) are both over two decades, and the limit of detections (LOD) are 7 x and 9 X M , respectively. M for These compare favorably with the value of 5.5 x LOD found in EPA solution (EtOH:isopentane:ether, 2:5:5) at 77 K (18). The overall precision found in the measurements was -6%. Using the same instrument, the intensity was even reproducible in this range from day to day. Initially degassed standard solutions gave reproducible intensities over a period of days without further degassing, indicating relatively slow diffusion of oxygen under the experimental conditions used. AgLS/NaLS solutions had to be protected from light during this extended time. T h e analytical curves for pyrene in NaLS/TlLS and NaLS/AgLS shown in Figure 2 gave LDRs of one decade, and M. Those for biestimated LODs of 1 X lo4 and 3 X
:slPHEavl]
I X 1O6.M
Figure 3. Log-log plot for biphenyl concentration in pmol/L vs. RTP intensity in PA: see Figure 1 caption for conditions; (-0-)TILS/NaLS system, A,, = 470 nm; (-0-)AgLS/NaLS system, ,A, = 490 nm: he, = 266 nm for both curves: coefficients of determination = 0,999 for both TI' and Ag' curves phenyl, in Figure 3, have an LDR and LOD of two decades and 1 x lo-' M in NaLS/TlLS, respectively, and an LDR and M, respectively, LOD of one and a half decades and 2 X in NaLS/AgLS micelles. Again, these compare favorably with values obtained a t 77 K (18). Thallium counterions gave consistently higher signals and increased sensitivity, compared to silver counterions. No R T P was observed for probes in NaLS micelles without some heavy metal counterions. C a u s e s of C u r v a t u r e . T h e possible reasons for the curvature in the analytical working curves a t higher concentrations must be reconsidered because of the presence of the micellar assemblies. The three most probable concentration-dependent mechanisms will be considered in turn. First, triplet-triplet annihilation (TTA) or excimer formation has been commonly observed for aromatic hydrocarbons, particularly for naphthalene where TTA is the principal deactivation mechanism at room temperature in solution (15, 19). This could be an operative mechanism in micellar aggregates only if two probes were closely positioned within the same micelle. It has been shown that the probe molecules are distributed among the micelles according to Poisson-Boltzmann statistics (5, 1 5 ) , and the probability of finding one, two or more probes in a given micelle can be calculated by
P, = mnem/n!
(1)
where P, is the probability of finding IZ probe molecules and m is the average number of probes per micelle. The micelle concentration a t different monomer levels can be calculated from the CMC and mean aggregation number by the following relationship: [micelle] = [surfactant - CMC] /aggregation no. (2) Thus, for a n accurate estimate of the distribution of the number of probes per micelle, it is necessary to know the CMC and mean aggregation number accurately. Table I1 shows the probability of finding two or more probes in a micelle at a constant CMC level and different aggregation numbers. The latter values bracket the number of 63 found experimentally for NaLS (16). The concentration of probe used in the calculations was taken from Figure 1 as the concentration of naphthalene where observable curvature ocM. For a mean aggregation number of curred a t 1.5 X 100, approximately 4 % of the probes share the same micelle,
ANALYTICAL CHEMISTRY, VOL. 52, NO. 4, APRIL 1980
Table 11. Percentage of Occupied Micelles Containing Two o r More Probesa aggregation no. 40
60 80 100 100
500 a
Tl/NaLS,b c/ IG
2.6 3.4 3.9 4.2
757
Table 111. Decrease in Luminescence Intensity Predicted from Inner Filter Effects‘l
Ag/NaLS,b %
2.0 3.9 4.2 6.2
8.3‘
22.0
See reference 1 5 ; no CMC subtracted (-3%).
Naphthalene concentration used where curvature was M at 2.65 X 10.’ A ; total Tl/NaLS -= 0.15 M ; total Agih’aLS = 0.08 M. Naphthalene concentration used = 2.5 X 1 0 at 2.40 X lo-* A.
97
1.0x 10-5
90 82 75
0.5 x 1 0 - 4 1.0 Y 1 0 - 4 1.5 x 10-4
61
2.5 x
“ See reference 22 for method of calculation. I*,, intensity expected if inner filter effect is present, and I f h e o r is intensity if not present.
is
first observed = 1 . 5 x
which would result in an 8% decrease in intensity due to TTA. This certainly could account for some of the observed decrease in intensity. Note that 99% of the micelles are empty, containing no probe molecule. However, if the aggregation number were 500, which may be possible with the high concentration of heavy atoms, a far larger probability would result with a correspondingly larger decrease in phosphorescence. No excimer emission was observed and it is not considered a viable mechanism. Unfortunately experimental CMCs and mean aggregation numbers are not available for the mixed heavy atom colinterion micelles used in this study, so these calculations are only suggestive of possible effects. As mentioned earlier, larger counterions would be expected to slightly decrease the CMC and increase the number of monomers associated. It does seem likely that some of the observed decrease in intensity at higher concentrations does occur via TTA. A second possible reason for curvature is collisional quenching. T h e actual chemical environment is a complex mixture of micelles in dynamic equilibrium with monomers, dimers, trimers, etc., with hydrated counterions, and with the aqueous solvent sheath. The probes enter and exit the micelles, as well as migrate internally on a constant, dynamic basis. The probe-containing micelle can undergo either micelle-micelle collisions on a macro level, or micelle-subunit collisions. T h e overall effect of these collisional quenching pathways would be measurable only a t higher probe concentrations, and might account for the observed curvature in the analytical working curve (20, 21). A third and very likely cause of most of the curvature is due to the inner filter effect (20). If the phosphorescence intensity from the part of the cell nearest the excitation light source is not measured, a bending off in the analytical curve is seen a t higher concentrations of analyte. In the present experiments, only a portion of the 1 cm2 sample cell surface was observed by the acceptance angle of the monochromator, thus giving rise to the inner filter effect. Alternatively, one can estimate if concentrations employed would give rise to curvature by calculating the product ccb (molar absorptivity x concentration (M) X path length (cm)). Linearity is usually observed in simple systems if this product is
