Anal. Chem. 1983, 55, 1084-1089
1084
External Conditions and Interactions in Room-Temperature Phosphorescence of Hydroxyl Aromatics Adsorbed on Solid S urf ac es Cont aining PoIy(ac ryIic acid) R. A. Dalterlo and R. J. Hurtublse” Chemistry Department, The University of Wyoming, Laramie, Wyoming 8207 1
Poly(acryllc add)-salt mlxtures were studied as solid surfaces for Inducing room-temperature phosphorescence from hydroxyl-substltuted aromatlc compounds so surface lnteractlons and optlmal conditions for room temperature phosphorescence could be determlned. The mixtures were examined with respect to the type of salt, salt-polymer ratio, acld-base form of the polymer, and ethanol-water volume ratlo of the adsorblng solvent. A solutlon contalnlng 10% water In ethanol, whlch was used to adsorb 4-phenylphenol onto a poly(acryllc acid)-NaBr mixture, was found to Induce enhanced room-temperature phosphorescence Intenslty. Infrared data indlcated that even wlth water adsorbed on the poly( acrylic add)-NaBr matrlx room-temperature phosphorescence was observed. A comparison of room- and low-temperalure phosphorescence lntensltles was made wlth several model compounds to estimate the efflclency of room-temperature phosphorescence from the poly( acrylic add)-salt matrix. I n addltion, room-temperature phosphorescence was measured as a function of time to determlne the effects of atmospherlc humidity on the poly( acryllc acid)-salt matrlx. Fluorescence polarlratlon was also used to obtaln lnformatlon about phosphor-poly( acryllc acld) blndlng In ethanol solutions.
The sensitivity and simplicity provided by room-temperature phosphorimetry have led to a rapid development of this new technique for trace organic analysis (1-3). Optimization of conditions necessary t o induce strong room-temperature phosphorescence (RTP)can lead to lower detection limits and simpler measurement procedures and give insights into the nature of phosphor-matrix interactions responsible for RTP. Although much work on R T P has been reported in recent years, the majority of work has been in developing roomtemperature phosphorimetry as an analytical technique and less attention has been given to investigating the physical and chemical bases of the approach. Parker e t al. have recently reviewed the analytical considerations of R T P ( 1 ) and have also surveyed the aspects associated with the physical nature of R T P (2). Another recent summary of the analytical developments involving RTP and the physical and chemical interactions responsible for R T P has been provided by Hurtubise (3). Recently we found that several hydroxyl aromatics adsorbed on poly(acry1ic acid) (PAA)-salt mixtures gave strong RTP ( 4 ) . In this paper, the results of experiments for optimizing conditions for strong R T P from model hydroxyl aromatics are given. From these studies, some speculations on interactions of the PAA-salt-phosphor system are made. Also fluorescence polarization was employed t o obtain insights regarding the phosphor-polymer binding in ethanol solution and infrared spectrometry was used to measure the water content in the PAA-salt matrix.
EXPERIMENTAL SECTION Apparatus. RTP intensity measurements were obtained with
a Schoeffel SD 3000 spectrodensitometer with a modified reflection mode assembly, equipped with a SDC 300 density computer (Schoeffel Instrumenb, Westwood, NJ) and a rotating disk phosphoroscope attachment (5). The sample holder for powdered adsorbents was described elsewhere ( 4 ) . When low- and roomtemperature phosphorescence intensity measurements were compared, a Farrand MK-2 spectrofluorimeter was used that was fitted with a rotary chopper for phosphorescence data collection. Fluorescence and phosphorescence excitation and emission maxima were obtained from corrected luminescence spectra. The corrected spectra were recorded with a Farrand MK-2 spectrofluorimeter that was equipped with a corrected excitation module and an Autoprocessor-I (Farrand Optical Co., Valhalla, NY). Polarized fluorescence measurements were made with the Farrand MK-2 spectrofluorimeter fitted with dichroic film polarizers in the excitation and emission beams. Reagents. Ethanol and water were purified by distillation. Biphenyl, p-phenylphenol, p,p’-biphenol, 2-naphthol, 2,3-dihydroxynaphthalene, 9-anthracenemethanol, and a-naphthoflavone (Alrich Chemical Co., Milwaukee, WI) were recrystallized from ethanol. Poly(acry1ic acid) (secondary standard, Scientific Polymer Products, Ontario, NY) was used as received. Inorganic salts were reagent grade and used without further purification. The inorganic salts were ground to a fine powder in a ball mill and then mixed with PAA by shaking vigorously in a glass vial. Procedures. R T P Measurements. The procedure for adsorbing samples onto the PAA-salt mixtures for RTP experiments was described earlier ( 4 ) . Neutralization of Poly(acry1ic acid) with NaOH. PAA was determined to contain 13.45 mequiv of H,O’/g by titrating 100 mg of PAA with 17.56 mL of 0.07660 N NaOH to a phenolphthalein end point. Samples containing 100 mg of PAA were neutralized to 0%, 25%, 50%, 75%, and 100% by reacting PAA with 0 mL, 4.39 mL, 8.78 mL, 13.17 mL, and 17.56 mL of 0.07660 M NaOH, respectively. After the reaction, 20 g of NaCl was added to each solution and the solvent was evaporated at 90 “C in a vacuum oven for 2 h. The dried solid was then ground to a fine powder in a ball mill to yield the desired 0.5% PAA-NaC1 mixtures. Low- and Room-Temperature Phosphorescence Intensity Comparisons. For low- and room-temperature phosphorescence intensity comparisons, powdered and liquid samples of similar volume were placed in quartz tubes (3 mm i.d. X 20 cm long). A phosphor sample of 200 ng was adsorbed onto 20 mg of PAANaBr powder ( 4 ) . This amount of powder occupied about 30 pL volume. In terms of a solution,this corresponds to a concentration of 6.7 wg/mL. This concentration in ethanol (30 pL volume) was used for low-temperature phosphorescence intensity measurements so qualitative comparison of data could be made with the solid samples. The quartz sample tubes were put in a quartz Dewar assembly, with and without liquid nitrogen, and the phosphorescence was measured with a Farrand MK-2 spectrofluorimeter. Luminescence Polarization Measurements. Phosphorescence polarization measurements of compounds adsorbed on PAA-salt mixtures and filter paper were obtained with the solid adsorbent held in 3 mm i.d. quartz tubes. Room-temperature fluorescence polarization measurements from ethanol solutions were obtained with 1 cm X 1 cm quartz cells. Four luminescence intensity measurements were needed to calculate the luminescence polarization, with the excitation and emission polarizers in either the vertical or horizontal positions
0003-2700/83/0355-1084$01.50/0C 1983 American Chemical Society
ANALYTICAL. CHEMISTRY, VOL. 55, NO. 7,JUNE 1983
1085
Table 1, PAA-Salt Mixtures Examined for Inducing RTP salta NaBr LiCl
RTP re1 inten 1.0
KCl
1.5 1.9
NaCl
2.4
0.5% (w/w) PAA in salt mixture. 200 ng of 4phenylphenol adsorbed; average of four determinations; A,, = 270 nm, h e , = 4 8 5 nm. a
(6). The polarization was calculated from the various polarized luminescence intensities by the following equation:
v
0
0.10
0.20
0.30
0.40
%PAP.
where G = IHv/irHH, Iw is the luminescence intensity measured with the excitation and emission polarizers oriented vertically (parallel to the vertical lab axis), Iw is the luminescence intensity measured with the excitation polarizer vertical and the emission polarizer horizontal, and G is the correction factor for polarization of the emitted luminescence due t o the emission grating, the photomultiplier housing, and sample cell, and other optics through which the emitted luminescence passes. The value of G varied with excitation and emission wavelengths. Infrared Spectrometry with Sodium Bromide Pellets. Standard size infrared NaBr disks (13 mm) were pressed with a Perkin-Elmer evacuable die (part no. 186-0025). PAA (1.0 mg) was adsorbed onto 100 mg of NaBr from 0.5 mL volume of solution. The mixtures were dried 1-2 h at 100 "C before preparing the disks. The dlisks were then pressed at 21 000-22 000 psi for 20 min under vacuum. Infrared spect ra of PAA in sodium bromide pellets were obtained with a Perkin-Elmer 621 infrared spectrophotometer and also a Beckman IR-10 infrared spectrophotometer. The Beckman IR-10 was connected t o a Bascom-Turnier Model 4120 data handling system with floppy disk storage capabilities. The data in the infrared region between 2000 and 600 cm-' were recorded with the Bascom-Turner system with 500 data points, sampled at a rate of 1 point/532 ms. The spectra recorded were then smoothed twice Iby a five-point moving average followed by determining the derivative spectra. The derivative spectra were used to determine precisely absorption band maxima.
RESULTS AND DISCUSSION RTP w i t h Poly(acry1ic acid)-Salt Mixtures. It was previously found that sodium chloride is important for inducing R T P froim compounds adsorbed on PAA (4, 7). PAA was mixed with several different inorganic salts and the RTP intensity was measured for 4-phenylphenol adsorbed on each PAA-salt mixture. Table I lists the PAA-salt mixtures examined and the relative R T P intensity observed. The data show that the R T P intlensity decreases with the various salt mixtures in the order NaCl > KCl > LiCl > NaBr. Since NaBr mixtures yielded the lowest RTP of the salts examined, the heavy-atom effect for increasing triplet yield can be considered unirniportant relative to the other PAA-salt mixtures investigated. A previous study (4) showed that PAAsodium iodide mixtures yielded no R T P from model phenolic compounds, which is in contrast to several studies which have shown heavy atoms enhance RTP from compounds adsorbed on filter paper (4,8-12). In a previous study with benzo[flquinoline, it was also shown that a PAN-NaCl mixture induced the strongest RTP from several PAA-salt mixtures (13). It was postulated that a higher modulus and lower coefficient of thermal expansion were responsible for the greater ability of PAA-NaCl mixture to induce RTP. The same effects may be operative in the present study. The change in the RTP of model compounds was examined by varying the percent PAA in PAA-NaC1 and PAA-NaBr mixtures. The results with PAA-NaC1 for two model com-
0.50
0.60 0.70
0.80 0.90
1.00
in N o C l
Flgure 1. RTP of hydroxyl aromatics as a function of the amount of PAA in NaCI: 300 ng of a-naphthoflavone (V),300 ng of 4-phenylphenol (O),150 ng of a-naphthofhvone (W), 150 ng of 4-phenylphenol
(4. pounds are shown in Figure 1. Figure 1indicates that no RTP occurs from model compounds adsorbed on the pure salt and that a substantial rise in RTP occurs at 0.1% PAA. A gradual increase in R T P is observed from 0.1% to about 0.6 or 0.7% PAA-NaC1 after which the RTP remains constant up to 1.0% PAA. Previous studies ( 4 ) showed that above 1.0% PAANaCl the R T P intensity decreases. For the PAA-NaBr mixtures similar reuults were obtained except the sharp rise was not observed a t 0.1% PAA and the slopes of the curves were greater in the rising part of the curves. If it i s assumed that a t relatively low percent PAA (0.1-0.5%) the inorganic salt can effectively separate the PAA molecules from each other, then the carboxyl groups would not interact as readily with each other. This suggests the carboxyl groups would be available for interaction with the phosphor. However, the fact that maximum RTlP is reached beyond 0.5% PAA implies optimal matrix and/or geometric requirements are needed to obtain maximum RTP signals. The results for the PAA-NaC1 and PAA-NaBr mixtures are important analytically because they reveal the conditions for optimal and constant R T P signals with these mixtures. The percent PAA values were determined at the points where the RTP intensity vs. percent PAA graphs reached the RTP plateaus (Figure 1). A particular percent PAA value was determined by the intersection of two lines drawn along the rising portion (beyond 0.1% PAA) and the flat portion of a given graph. With the PAA percent values for the various mixtures the ratio of phosphor molecules to one PAA molecule was calculated, using 2 X lo6 as the molecular weight for PAA (14). For the PAA-Wac1 mixtures the following values were obtained: 150 ng of 4-phenylphenol, 14.4; 300 ng of 4phenylphenol, 22.0; 150 ng of a-naphthoflavone, 8.0; 300 ng of a-naphthoflavone, 17.0. For the PAA-NaBr mixtures the following values were calculated: 150 ng of 4-phenylphenol, 23.2; 300 ng of 4-phenylphenol, 22.2; 150 ng of a-naphthoflavone, 8.6; 301) ng of a-naphthoflavone, 16.9. Since there are approximately 27 778 monomer units in each PAA molecule, each PAA molecule has the potential of easily accommodating the number of phosphor molecules estimated above in the 150 ng or 300 ng samples. Also, assuming the phosphor molecules are homogeneously distributed throughout the PAA-NaC1 matrix, then interaction between the phosphor molecules would be minimized because of the distance between them (13). Neutralization of PAA. The PAA in various PAA-salt mixtures was converted partially or completely to sodium polyacrylate by treatment with NaOH solutions to study the
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
Table 11. Change in RTP of Model Compounds by Neutralization of PAA RTP re1 intensa neutralization of PAA, % 4-phenylphenolb 4,4‘-biphenolc 0
-
24
25 50 75
3.3 22
100
1.0
1.4
2-naphthold
38 5.0
no. of COOH groups per 4-phenylphenol molecule 1183 887 591 296
31
none none none none
6.0
32 21
0
a Average of four samples adsorbed on 0.5% PAA-NaC1 mixtures. 200 ng adsorbed; hex = 270 nm, hem = 485 nm. 200 ng adsorbed; h e , = 295 nm, he, = 488 nm. 300 ng adsorbed; hex = 290 nm, hem = 510 nm.
change in R T P of model compounds as a function of percent neutralization of PAA. The results with partially and totally neutralized PAA in NaCl are shown in Table 11. The large R T P intensities observed with 4-phenylphenol and 4,4’-biphenol adsorbed on 75% neutralized PAA are unexpected results since the other partially and totally neutralized PAA-NaC1 mixtures showed small RTP intensities. However, 4,4’-biphenol did show a relatively strong RTP signal at 100% neutralization. No RTP was observed from 2-naphthol on any of the neutralized surfaces. The ratio of COOH groupsl4phenylphenol molecule listed in Table I1 for the PAA-NaCl mixtures shows no obvious correlation with the RTP intensity of 4-phenylphenol. Even at 75% neutralized PAA there are an excess of carboxyl groups for interaction with 4-phenylphenol. It appears that the PAA coils achieve a conformation in the presence of NaCl which is favorable for inducing R T P from 4-phenylphenol and 4,4’-biphenol when the COO-/ COOH ratio is 3/1. This observation suggests that for the model compounds examined, the polymer chain conformation is more important for inducing R T P than the number of carboxyl groups present on the polymer chain. A similar experiment with 5,6-benzoquinoline did not show enhanced signals a t 25%, 50%, 75%, and 100% neutralization (13). From Table I1 it can be seen that the unneutralized PAANaCl mixture is the most efficient for inducing RTP from the model compounds examined. The large R T P observed from 4-phenylphenol and 4,4’-biphenol adsorbed on 75% neutralized PAA-NaC1 could possibly be used analytically for an analysis of a mixture containing these and other compounds which show little or no R T P on this surface. For example, it has been reported that benzolflquinoline adsorbed on 75% neutralized PAA-NaC1 shows a weak RTP, while very intense RTP is observed for that compound on unneutralized PAANaCl (13). This particular approach in RTP is in need of additional investigation. RTP with Ethanol-Water Solutions. Ethanol solutions with different amounts of water were used to adsorb 4phenylphenol onto PAA-NaBr mixtures for R T P measurements. It was assumed that the solubility of the salt would increase with increasing amount of water and possibly have an effect on R T P intensity (13). Figure 2 illustrates the variation in RTP intensity from samples adsorbed onto 0.8% PAA-NaBr with water-ethanol solutions. A large increase in R T P intensity is observed in going from pure ethanol to 10% water (v/v), after which the R T P decreases with increasing percent water. Infrared experiments were carried out with 1%PAA in NaBr disks in which the PAA was adsorbed onto NaBr from 100% ethanol, 90% ethanol-water, and 50% ethanol-water solutions. In the spectral region between 1800 and 1600 cm-l, the most prominent feature was hydrogen bonded C=O stretching vibration centered at 1710 cm-l (15). The PAANaBr disks which had water from the adsorbing solvent showed shoulders a t 1633 cm-l, attributed to the bending vibration of HzO. This region of the infrared spectra is illustrated in Figure 3. It can be seen in Figure 3 that the
0
20
IO
30
40
50
60
70
80
.
.
90
100
% W A T E R in ETHANOL
Flgure 2. Variation of 4-phenylphenol RTP with water content in ethanol.
I
I 2000
I800 W A V E NU M BE R
1600 ( c m- )
Figure 3. Infrared spectra from PAA-NaBr disks containing water: 100% ethanol (A), 90% ethanol-water (B), 50% ethanol-water (C).
shoulder at 1633 cm-l is most pronounced with the NaBr-PAA disk formed from 50% ethanol-water. A similar trend was noticed with the OH stretching vibration of H20 at about 3475 cm-I, which was superimposed on the broader OH stretching band of PAA. The appearance of these bands strongly suggests the presence of appreciable amounts of water retained in the PAA-NaBr samples. Leyte et al. (16) have suggested that the replacement with a more polar solvent in the environment of the carboxyl groups of PAA offers more favorable hydrogen bonding conditions for these groups. This statement implies that the addition of a more polar solvent (water) changes the conformation of
ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
Table 111. Timle Dependence of RTP Intensity time after preparation (days)
RTP re1 intensa 1.0 1.6
1.5 1.4 1.1 1.0 a 150 ng of a-naphthoflavone adsorbed on 1% PAA-Nal3r. Average of three samples; h e , = 320 nm h e , = 520 nm.
the polymer chain. Water can form stronger hydrogen bonds than ethanol (17) and because of its smaller size can form doubly H-bonded complexes (18). With a change in the conformation of the polymer, it appears that some carboxyl groups are more readily available for interaction with the phosphor. The enhanced R T P observed from model compounds with a sizable amount of water present in the PAA-salt matrix is an important result since all previously studied adsorbents showed optimal R T P with presumably little or no moisture present (1-3). Schulman (19) has shown with fiiter paper that the presence of moisture favors increased quenching of R T P by aiding the transport of O2into the sample matrix. In order for enhanced RTP to be observed with water present, the H 2 0 molecules incorporated in the PAA-salt matrix must alter the matrix structure in a way which diminishes oxygen quenching of RTP. Further studies are needed to understand the effect of water on the R T P from PAA-salt substrates. Time Dependence of RTP Intensity from PAA-Salt Matrix. Samples of a model phosphor were adsorbed onto a 1% PAA-NaBr mixture as described in the Experimental Section and the IRTP was immediately measured. The RTP was then subsequently measured after 24-h intervals for 1 week without any further heating or drying of the samples. The results of this experiment are given in Table 111. It can be seen that the IRTP intensity increased by about 60% after 24 h and then slowly diminished over the next several days. After 7 days the R T P signal had decreased to its original intensity observed right after preparation. These observations are in contrast to1 R T P signals obtained with untreated filter paper adsorbent (19, 20), where R T P intensity of certain phosphors decreased approximately to half their intensity when exposed to air after several minutes. Other R T P absorbents have been shown to be resistent to atmospheric conditions including treated filter paper (20) and sodium acetate (21,22). The increase in R T P intensity observed 24 h after adsorbing the saimple on PAA-NaBr mixture may be the result of a slow equilibrium, where the ,phosphor molecules reorient and anchor themselves in the solid matrix very slowly. Comparison of Room-Temperature and Low-Temperature Phosphorescenice Intensities for Model Compounds. Phosphorescence intensities for model compounds
1087
were compared at room- and low-temperatures for samples adsorbed on 1% PAA-NaBr and for samples at low temperature in ethanol solution (Table IV). The difference in phosphorescence intensity between the LTP powder samples and R T P samples suggests that the phosphor is not held as rigidly on the solid surface a t room temperature. Also collisional deactivation of the triplet state can occur more readily at room temperature. A quantitative comparison of the LTP powder data and R T P data with L T P solution data cannot be made because it is difficult to determine if the same number of molecules were excited with the solution sample compared to the solid surface samples. However, as discussed in the Experimental Section the sample sizes of the solution sample and solid-surfacesamples were adjusted so that approximately the same number of molecules would be excited. As shown in Table N,the LTP solution samples gave the largest signals. The previous data show that generally “maximum” phosphorescence is not achieved with the solid surface samples. It should be noted that 2-naphthol shows more intense lowtemperature phosphorescence from the solid surface than from ethanol solution. Apparently this is the first reported result of this type. 2-Naphthol, 2,3-dihydroxynaphthalene,and a-naphthoflavone, with two or more fused rings, showed a lower L T P (so1ution):LTP (powder) ratio and a lower LTP (so1ution):RTPratio than the biphenyl-type compounds. This would indicate that the more rigid molecules are held in the solid matrix more efficiently. This trend was also seen by Aaron et al. (23)in a comparative study of the RTP and LTP of several pesticides adsorbed on filter paper. Fluorescence Polarization Study of Binding of Model Compounds to PAA in Solution. The R T P of phosphors adsorbed on PAA-salt mixtures is thought to involve rigid binding of the phosphor to PAA macromolecules in the dry state. A study was undertaken to examine phosphor-PAA binding in ethanol solution because of the importance of the initial “wet” chemistry in R T P work (13). Some interactions of the phosphor with the polymer would be expected in solution because of the polar functional groups in PAA and the model compounds investigated. Fluorescence polarization measurements were made with several model compounds in PAA-ethanol solutions with different PAA concentrations. Since the Brownian rotational time of the fluorescent model compounds in ethanol solution is comparable to their fluorescent lifetimes, the fluorescence polarization measured under these conditions is virtually zero. According to Perrin’s equation (24)the rotational relaxation time is proportional to the volume of the luminescent components. If the fluorophore binds with a PAA macromolecule, the Brownian rotation might be considerably slowed and the absolute value of the polarization would be increased. The limiting fluorescence polarization values were obtained by using very dilute solutions of the model compounds at 77 K. Depolarization due to energy transfer from the originally excited molecule to a neighboring molecule is minimized in very dilute solutions and depolarization due to molecular
-
Table IV. Comparison of Relative RTP and Low-Temperature Phosphorescence Intensities of Model Compoundsa, compouhdd
i
4-phenylphenol
,,A,
nm
A,,
nm
270 26 8
48 5
4,4’-biphenol biphenyl
250
463
2-naphthol 2,3-dih ydrox ynaph thalene a-naphthoflavone
3 26 271 320
520 470 520
478
RTP 1.o 1.o 1.0 1.0 1.0 1.o
LTP
LTP
LTP (solution)/
(powder)
(solution)
LTP (powder)
2.0 14 11
8.6 2.9
22 8 93 87 4.4 11 80
114 6.4 7.9 0.51 3.6
41 2.0 a Powder samples had 200 ng of phosphor adsorbed on 1%PAA-NaBr. Ethanol solution was 6.7 kg/mL with 30 pL of ethanol. Average of two samples. Phosphorescence intensity comparisons cannot be made between different compounds due to different instrument gain settings.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 7, JUNE 1983
Table V. Low-Temperature ( 7 7 K ) Fluorescence Polarization and the Ratio of Room-Temperature Fluorescence Polarization Values for Model Compounds Uncornplexed and Complexed with PAA compound 4-phenyl henola biphenyl ! 2-naphthol 9-anthracenemethanol a
1.0 bg/mL in ethanol.
nm 271 255 277 368
hex,
4.0 pg/mL in ethanol.
nm 331 314 358 411
hem,
LTF polarizationC +0.2072 t0.2858 +0.1598 +0.2846
PcoqiplexlPeeeC (0.00538/0.00196) = 2.7 (0.00680/0.00238)= 2.8 (0.00639/0.00183)= 3.5 (0.0154/0.00764)= 2.0
Average of two samples, Table VI. Fluorescence Polarization for 4-Phenylphenol from NaCl and NaBr Saturated PAA Solutions PAA concn, salt mg/mL added
*0
24a 24 a 24 a 24b 24b 24b
,oo+
O0 0 2
o
0
4
V
10
20
CONCENTRATION
30
of PAP. In
40
50
none NaCl NaBr none NaCl NaBr
polarization
hexmax,
nm
hem
max
nm
,
+0.0056 +0.0096 +0.011 + 0.0044 t0.0075 t0.12
265 333 265 333 26 5 333 265 333 265 333 2 54 337 283 50a none +0.0051 267 333 50' NaCl 1-0.013 267 333 50a NaBr +0.032 292 340 a 4-Phenylphenol concentration, 1 pg/mL in 100% 4-Phenylphenol concentration, 1 pg/mL in 1 9 : l ethanol. ethanol-water.
E T H A N O L (rnglml)
Figure 4. Fluorescence polarization as a function of PAA concentration in ethanol: 9-anthracenemethanol, 4 pg/mL (V);biphenyl, 4 pg/mL (H); 2-naphthol, 4 pglmL (A):4-phenylpheno1, 1 pg/mL (0).
(Brownian) rotations is absent in frozen solution. Low-temperature fluorescence polarization values for some model compounds are listed in Table V. Room-temperature fluorescence polarization measurements were obtained from model compounds as a function of PAA concentration in ethanol solution. Figure 4 illustrates the change in fluorescence polarization of the model compounds with changing PAA concentration. For each model compound the fluorescence polarization reached a maximum value a t a PAA concentration between 10 and 25 mg/mL in ethanol solution. At PAA concentrations where the maximum polarization values are observed, it is assumed that the maximum number of fluorescent molecules are associated with PAA molecules. Since the polarization remains constant over a considerable range of PAA concentrations, it is evident that the increase in solution viscosity is not affecting the rotational relaxation time of the fluorophore-PAA complex in this range of concentrations. By comparison of the polarization of the fluorophore-PAA comat the plateau of the polarization) to the poplex (Pcomplex, larization of the free fluorophore (P,,,, at zero PAA concentration) a relative measure of the restriction of rotation on the fluorophore imposed by binding to PAA can be obtained. values for four model compounds Table V lists Pcomplex/Pfree examined. Larger Pcomplex/Pfree values indicate more restricted rotation of the fluorophore and implies stronger fluorophore-PAA binding. The relatively small change in polarization between the free and bound fluorophores suggests that the polymer is spread out in solution and that there is considerable flexibility in the polymer chain in ethanol solution. Nishijima et al. (25) have shown that for fluorescein covalently bound to the chain end of polyacrylamide polymers in solution, the limiting rotational relaxation time calculated from fluorescence polarization measurements is approximately four times that of free fluorescein. The increased rotational relaxation time was attributed to segmental motion of the individual monomers of the polymer chain. The rotation
relaxation time would be further increased for very large polymer chains where long-range interactions by other segments of the same polymer chain could be exerted on the fluorophore. A 4-fold increase in the mean rotational relaxation time between fluorescein covalently bound to polyacrylamide and free fluorescein corresponds to an increase in fluorescence polarization between covalently bound and free fluorescein of 3.6. Comparing Pbound/Pfree values of 3.6 for fluorescein-polyacrylamide conjugate with the values in Table V suggests that the model compounds in this work are bound quite tightly to the PAA macromolecules in ethanol solution. Additional studies were performed with NaCl and NaBr saturated solutions to study the effect of these salts on polarization. Table VI lists fluorescence polarization and XeXmax and Xemmax values for 4-phenylphenol in NaCl and NaBr saturated solutions a t several PAA concentrations. From Table VI it is seen that in each case the NaBr saturated solutions give the larger polarization values, and NaCl saturated solutions give larger polarization values than the solutions with no added salts. The solubilities of NaCl and NaBr in 100% ethanol are 0.6 and 18 mg/mL, respectively. It has been suggested (13)that halide ions in solution can interact with PAA molecules by breaking intra- and intermolecular hydrogen bonds between carboxyl groups. If this occurs, a larger number of carboxyl groups would then possibly be available to interact with the luminescent molecules and this should result in larger polarization values. Table VI shows the fluorescence emission maximum shifted to longer wavelengths for the NaBr solutions with the polarization values of +0.12 and +0.032. The solution with the +0.12 polarization value showed two excitation bands with one band shifted to a longer wavelength. The solution with the +0.032 value had one band which was shifted to a longer wavelength. The red shifts of the fluorescence excitation and emission maxima indicate relatively strong interaction of the fluorophore with PAA (26). It is important to note that PAA-NaC1 mixtures induce a larger R T P from 4-phenylphenol than PAA-NaBr mixtures (Table I). There appears to be an inverse correlation between the magnitude of fluorescence polarization with these salts in solution and magnitude of RTP. These aspects are
Anal. Chern. 1983. 55. 1089-1094
in need of additional investigations. RTP Polarization Study. The degree of polarization of R T P and LTP from compounds adsorbed on PAA-salt mixtures and filter papeir was measured. The purpose of Ibis study was to rellate the degree of phosphorescence polarization to the rigidity of the phosphor adsorbed in the solid matrix. With 4-phenylphenol adsorbed on the solid surfaces, zero polarization of the R T P and L T P was; obtained within experimental error. Thig result is not surprising since radiation scattered by a !solid surface causes loss of orientation of both exciting and emitted light (27). Scattering, therefore, causes depolarization of luminescence to occur. Teale (28)has described a method by which the polarization of luminescence emitted from turbid solutions could be measured by observing a thin layer of the luminescent volume in the plane of incidence and observation. Faucon and Lussan (29) demonstrated Teale's method of correcting for depolarization by scattering by using horizontal slits which were 1 mm high in the excitation and emission beams. We used the above method of placing 1mm horizontal slits in the exciting and emitted beams to correct for depolarization of R T P and L T P emitted from solid supports. However, within experimental error zero polarization was measured. Apparently the depolarization of RTI? from solid supports is too extensive to be corrected1 for by the method deslcribed above. Registry No. 4-Phenylphenol,92-69-3;4,4'-biphenol, 92-88-6; biphenyl, 92-52-4;2-naphthol,135-19-3;2,3-dihydroxynaphthalene, 92-44-4; a-naphthoflavone, 604-59-1; 9-anthracenemethanol, 1468-95-7;NaCI, 7647-1.4-5; NaBr, 7647-15-6;poly(acry1ic acid), 9003-01-4.
1089
(2) Parker, R. T.; Freedlander, R. S.; Dunlap, R. B. Anal. Chim. Acta 1980, 179, 189. (3) Hurtublse, R. J. "Solid Surface Lumlnescence Analysis: Theory, Instrumentation, Applications"; Marcel Dekker: New York, 1981; Chapters 5 and 7. (4) Dalterio, R A.; Hurtublse, R. J. Anal. Chem. 1982, 5 4 , 224. (5) Ford, C. D.;Hurtubise, R. J. Anal. Chem. 1979, 5 1 , 659. (6) Chen, R. F.; Bowman, R. L. Science 1985, 147, 729. (7) Ford, C. D.; Hurtubise, R. J. Anal. Chem. 1980, 52, 656. (8) Seybold, P. G.; White, W. Anal. Chem. 1975, 4 7 , 1199. (9) Jakovljevic, I. M. Anal. Chem. 1977, 49, 2048. (10) White, W.; Seybold, P. G. J . Phys. Chem. 1977, 8 1 , 2035. (11) Bower, E. L.;Winefordner, J. D. Anal. Chim. Acta 1978, 102, 1. (12) Meyers, M. L.; Seybold, P. G. Anal. Chem. 1979, 5 1 , 1069. (13) Ramasamy, S. M.; Hurtubise, R. J. Anal. Chem. 1982, 5 4 , 2477. (14) Scientific Polymer Products, Catalog 801, Ontarlo, NY; p 20. (15) Ostrowska, J.; Narebska, A. Colloid Po/ym. Sci. 1979, 257, 128. (16) Leyte, J. C ; Zuiderweg, L. H.; Vledder, H. J. Spectrochim. Acta, Part A 1987, 2 3 A , 1397. (17) Pimentel, '3. C.; McClellan, A. L. "The Hydrogen Bond"; W. H. Freeman and Co.: San Francisco and London, 1960; pp 212-213. (18) Thompson, W. K ; Hall, D. G. Trans. Faraday SOC. 1987, 6 3 , 1553. (19) Schulman, E. M.; Parker, R. T. J . Phys. Chem. 1977, 8 1 , 1932. (20) McAleese, D. L.; Freedlander, R. S.; Dunlap, R. B. Anal. Chem. 1980, 52, 2443. (21) Von Wandruszka, R. M. A.; Hurtubise, R. J. Anal. Chem. 1977, 49, 2164. (22) Von Wandruszka, R. M. A.; Hurtublse, R. J. Anal. Chim. Acta 1977, 9 3 , 331. (23) Aaron, J. J.; Kaleel, E.; Wlnefordner, J. D. J . Agric. Food Chem. 1979, 2 7 , 1233. (24) Pesce, A. J.; RosBn, C. G.; Pasby, T. L. "Fluorescence Spectroscopy"; Marcel Dekker: New York, 1971; p 96. (25) Nlshijlma, Y.; Teramoto, A.; Yamamoto, M.; Hiratsuka, S. J . Po/ym. Scl., Polym. P h p . Ed. 1987, 5 , 23. (26) Bayllss, N. S.;McRae, E. G. J . Phys. Chem. 1954, 58, 1002. (27) Reference 24, p 91. (28) Teale, F. W. J. Photochem. Photobiol. 1989, 10, 363. (29) Faucon, J. F.; Lussan, C. Biochim. Slophys. Acta 1973, 307, 459.
RECEIVED for review December 7,1982. Accepted February 10, 1983. Financial. support for this project was provided by the Department of Energy, Division of Basic Energy Sciences, Contract No. DE-AC02-80ER10624.
LITERATURE CITED (1) Parker, R. 1.; Freedlander, R. S.; Dunlap, R. B. Anal. Chim. Acta 1980, 720, 1.
Determination of Formation Constants of Calcium Complexes of Difluorornethylenediphosphonic Acid and Related Diphosphonates Tekum Fonong, Donlald J. Burton, end Donald J. Pietrzyk" Chemistry Depan'ment, The University of Iowa, Iowa City, Iowa 52242
Formation constants flor the 1:l and 2:l complexes formed between Ca2+and difluoromethylenedlptiosphonate(F,MDR), dichloromethylenediphiosphonate(CI,MDP), and i-hydroxyethane-1,l-diphosphonate(EHDP) were determlned by a potentlometric titratlon procedure where the lndlcator electrode Is a Ca Ion selective electrode and the titrant is a CaCI, solutlon. Measurements were made in the absence of lonlc strength controll and In 0.10 M NaCl and 0.10 M NH,CI solution. Logarithm formatlon constants, although similar for the 1:i complex, change In the order EHDP > CI,MDP > F,MDP which Is opposite to the1 acld strength exhlbited by the ligands. For the 2:l Ca*":llgand complex the EHDP complex formatlon constant is similar to the l:icomplex and the complex is signiflcantly more stable than either of the 2:l Ca2+:CI,MDP or Ca2+:F,MDP complexes. This Is probably the result of coordlnatlon Involving tlhe hydroxyl group In EHDP. Weak 1:i Na+:llgand complexes were also detected. 0003-2700/83/0355-1089$01 SO/O
Biological interest in pyrophosphates, compounds characterized by a P-0-P structure, increased when it was found that the presence of these substances in plasma and urine was capable of inhibiting the precipitation of calcium phosphate. Subsequent studies (1-4) have shown that diphosphonates of the type P-C-P 'we also valuable agents in the regulation of calcium metabolism in experimental laboratory animals and are potentially useful therapeutic agents for diseases involving abnormal calcification and excessive bone resorption. Success in clinical trials, for example, for osteoporosis, Paget's disease, and others, have been reported (1-6). Recent investigations have indicated high activities for the diphosphonates in inhibiting bone lysis of mice calvaria in vitro either cultivated in the presence of human tumors (7) or in tumor conditioned media (8),in rat tumor modeling studies (9),and in chemical trials for the treatment of malignant hypercalcemia (6). The two diphosphonates that have been studied extensively 0 1983 American Chemical Society
