Anal. Chem. 1989, 6 1 , 955-960
955
silica phase used in this investigation. Still the observed phenomena are remarkably similar for both the polymeric stationary phases with capillary columns and the chemically bonded phases in packed columns. The consistency of the void volume measurements indicates that there is little or no problem with the determination or definition of the void volume in these systems. The basic assumptions used for the derivation of the simple equations for the tracer pulse techniques are valid. In particular, the use of the retention time of a hypothetical unretained solute (13,to,to determine the volume of the mobile phase is an accurate method to correct for the amount of COz not adsorbed on the stationary phase. The experimental results show that under normal SFC experimental conditions with C02carrier, the solid adsorbent is "coated" with at least a monolayer of adsorbed mobile phase. At temperatures and pressure close to the critical point, either sub- or supercritical, multilayer adsorption of COzis common. The exact role of the bonded phase in the adsorption process has not been determined, and even the exact nature of the "stationary phase" is unclear. In addition, the relative significance of the interactions between different solutes and the various components of the stationary phase (solid, bonded material, and adsorbed fluid) in the control of retention has not been determined for SFC systems. Thus, there is a clear need for further investigations into the fundamental processes responsible for selectivity and retention in SFC.
and 5 pm capillary restrictors (T.A.B.).
ACKNOWLEDGMENT The authors wish to express their appreciation to Dr. D. E. Martire (Georgetown University) and Dr. T. A. Berger (Hewlett-Packard, Avondale, PA) for many stimulating discussions, as well as the provision of the back pressure regulator
RECEIVED for review August 15, 1988. Accepted January 24, 1989. Acknowledgement is also made to the National Science Foundation and to the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research.
LITERATURE CITED Martire, D. M.;Boehm, R. E. J . Phys. Chem. 1987, 91, 2433-2446. Yonker, C . R.; Gale, R. W.; Smith, R. D. J . Phys. Chem. 1987, 91, 3333-3336. Yonker, C. R.; Smith, R. D. J . Phys. Chem. 1988, 92, 1664-1667. Selim, M. I.; Strubinger, J. R. fresenius' Z . Anal. Chem. 1988, 330, 246-249. Strubinger, J. R.; Selim, M. I. J . Chromatogr. Sci. 1988, 2 6 , 579-583. Selim, M. I.; Strubinger, J. R. J . Phys. Chem., in press. Springston, S. R.; David, P.; Steger, J.; Novotny, M. Anal. Chem. 1986, 58, 997-1002. Parcher, J. F. J . Chromatogr. 1982, 251, 281-288. Parcher,J. F.; Selim, M. I. Anal. Chem. 1979, 51, 2154-2156. Parcher, J. F.; Johnson, D. M. J . Chromatogr. Sci. 1980, 18, 267-272. Koch, C. S.; Koster, F.; Findenegg, G. H. J . Chromatogr. 1987, 406, 257-273. Riedo, F.; Kovats, E. sz J . Chromatogr. 1982, 239, 1-28. Krstulovic, A. M.;Colin, H.; Guiochon, G. Anal. Chem. 1982, 5 4 , 2438-2443. Knox, J. H.; Kaliszan, R. J . Chromatogr. 1985, 349, 211-234. Nagy, L. Gy.; Laszlo, K.; Foti, Gy. J . Chromatogr. 1987, 406, 311-316. Giimer, H. B.; Kobayashi, R. AIChE J . 1964, 1 0 , 797-803. Hori, Y.; Kobayashl, R. J . Chem. Phys. 1971, 5 4 , 1226-1236. Roiniak, P.; Kobayashi, R. AIChE J . 1980, 26, 616-625. Blumel, S.; Koster, F.; Findenegg, G. H. J . Chem. SOC., faraday Trans. 2 1982, 78, 1753-1764. Specovious, J.; Findenegg, 0 . H. Ber. Bunsen-Ges. Phys . Chem. 1978, 82, 174. Specovious, J.; Findenegg. G. H. Ber. Bunsen-Ges Phys. Chem. 1980, 8 4 , 690. Peng. D. Y.; Robinson, D. B. Ind. Eng. Chem., Fundam. 1978, 15, 59-64.
.
Determination of Cyclodextrin Formation Constants Using Dynamic Coupled-Column Liquid Chromatography Lisa A. Blyshak, Karen Y. Dodson,l Gabor Patonay: and Isiah M. Warner* Department of Chemistry, Emory University, Atlanta, Georgia 30322 Willie E. May
Center for Analytical Chemistry, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
Dynamic coupied-coiumn liquid chromatography Is used to determine both aqueous solubilities and cyciodextrin inciuslon complex formation constants for a series of polynuclear aromatic hydrocarbons (PAHs). This method Is based on the increase In solubility afforded by cyciodextrins upon complexation. Formation constant ( K , ) values are determined by using CY-, 8-, and y-cyciodextrins at various temperatures. The relative preclslon of the calculated K , values is best for cyclodextrln/PAH complexes with large formation constants. The calculated soluMiities are comparable to literature values, and In most cases, formation constant data are reproducible within 10%. The resulting K , values confirm that the formation of lncluslon complexes is based on the relative sizes of host and guest molecules.
* Author to whom correspondence should be addressed.
'Present address: Food & Drug Administration, 60 Eighth St., Atlanta, GA 30309. Present address: Georgia State University, Department of Chemistry, University Plz., Atlanta, GA 30303. 0003-2700/89/0361-0955$01.50/0
INTRODUCTION The environmental impact of polynuclear aromatic hydrocarbons (PAHs) has received increased attention due to the increased emission of these compounds by coal-fired power plants and automobile exhaust. The carcinogenic and mutagenic properties of PAHs are well established (1-3), and many studies have been performed to characterize PAH behavior in solution. The presence of PAHs has been observed in water, air, and soil samples ( 4 ) . Establishing the distribution properties of PAHs in the aqueous environment is of particular importance since many phenomena in water influence the accumulation and partitioning of PAHs in sediments. For example, the presence of salt in aqueous systems results in the salting-out of certain compounds onto sand or soil particles. Generally, PAH solubilities are low due to their high molecular weights and lack of polar side chains. Thus, methods for determination of aqueous solubilities of PAHs must be capable of measuring low concentrations and circumventing 0 1989 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
adsorptive losses. Several methods have been employed for this purpose including fluorescence measurements ( 5 , 6 ) ,absorption studies (7), and nephelometry (8,9).Difficulties in solution preparation, transfer, and analysis often arise when these methods are used because saturated solutions for analysis are prepared by mechanical stirring. This approach can result in adsorption onto container walls and formation of supersaturated solutions. The sensitivity of UV spectroscopy to interferents has also been a concern (7). Since many compounds are UV active, the analyte signal is often contaminated by other absorbing species. Dynamic coupled-column liquid chromatography (DCCLC) is a technique that has been developed to avoid the abovementioned problems by generating and analyzing PAH solutions within a single system (10). Analysis of solutions directly followingpreparation minimizes adsorptive losses often caused by transferring solutions from one container to another. In addition, the presence of an analytical column in the highperformance liquid chromatography (HPLC) system allows for the separation of interferent signals from the analyte signal. The DCCLC technique has been used to determine the effects of temperature and salinity on the aqueous solubility of several PAHs (11)and to study PAH sorption on sediments (12). This method may be useful for other studies in which the careful preparation of saturated solutions is important. Although several techniques have been established to obtain formation constants for different types of compounds 03-15], a concise method for compounds of low aqueous solubility, such as PAHs, has not been found. This article reports the results of a study in which formation constant (Kf)values for cyclodextrin/PAH complexes have been determined by using DCCLC. Cyclodextrins are a group of cyclic oligosaccharides that contribute to several guest-associated phenomena in solution including fluorescence enhancement (16, 17) and increased aqueous solubility of certain compounds (18). Their inclusion properties are dependent on both the size and steric arrangement of potential guests (15). In the study reported here, the complexes formed with a-,p-, and y-cyclodextrins and several PAHs are examined. Differences in the cyclodextrin cavity dimensions, based on the number of glucose residues present, allow these macrocycles to exhibit varying degrees of complexation with guests of specific sizes and shapes. The determination of formation constants for these complexes would be helpful in assessing the interactions that occur between cyclodextrins and PAHs in solution. Also, the Kf data may be valuable to researchers who wish to use cyclodextrins in extraction or chromatography for separation of PAHs. Formation constants were calculated for representative PAHs with the use of DCCLC, and the utility of this method for such determinations is evaluated.
EXPERIMENTAL SECTION Reagents. Benzo[a]pyrene (98%)was obtained from Sigma. All other PAHs were purchased from Aldrich and were reported to contain less than 1% impurities. All PAHs were used as received. Variations in solubilitieswere not observed when HPLC grade water from American Burdick and Jackson was replaced with deionized water (2.2 pmho cm-'). Thus, deionized water (ContinentalWater Systems) was used for generation of saturated solutions. Spectrograde acetonitrile from American Burdick and Jackson was used to elute PAHs from the extractor column and as a solvent for standard PAH solutions. Solvents were filtered prior to use in the chromatographic system. Each cyclodextrin was recrystallized once from boiling water. Cyclodextrin (Advanced Separation Technologies) from one lot number was used for each study.
Standard solutions were prepared by dissolving the PAH of interest, accurately weighed to 0.0001 g on a Mettler H51AR balance, in acetonitrile. The total volume of solution prepared was 500 mL in a class A volumetric flask. Cyclodextrin solutions were prepared at a concentration of M by dissolving the correct amount of solid cyclodextrin in deionized water to give a total volume of 1 L. Fresh solutions were prepared each day. Generation of Saturated Solutions. Generator columns were conditioned by flowing 1L of deionized water through the column prior to aqueous solubility determinations. Columns were prepared for cyclodextrin solubility measurements by passing 30 mL of aqueous cyclodextrin through a column that had already been conditioned with water. Solutions were prepared by flowing deionized water or cyclodextrin solution through a 60 em X 0.6 cm stainless steel column packed with glass beads coated with 1% (w/w) of the PAH (10-12). Column temperature was regulated with a constant temperature bath (Lauda RM6). Temperatures for each measurement were maintained at f O . l "C. A Perkin-Elmer Series 10 pump module set at a flow rate of 2.0 mL min-' was used to generate saturated solutions. The generator column was fitted at both ends with 2-pm frits. Extraction Procedure and Analysis of Extract. Extraction of PAH from solution was performed by flowing a measured volume of solution, typically 10 mL, through a 19 cm X 4.6 mm ODS column (Regis Chemical). The volume of water passed through the extractor column was determined by collecting the effluent in a 10-mL class A volumetric flask. After this volume of solution was collected, the generator column flow was diverted to waste while an 80-20% acetonitrile-water blend was flushed through the extractor column to elute the analyte and allow for its analysis. The aqueous solubility measuremenb made in this study were similar to those previously determined, and the efficiency of the extractor column was also similar (approximately 98%) (12). Control of the flow direction was accomplished by using a Rheodyne six-port switching valve. Solvent programming and delivery were controlled with a Perkin-Elmer Series 4 liquid chromatograph and control module keyboard. The eluted compound was passed through a 25 cm X 4.6 mm octadecyl analytical column (Regis Chemical) to separate the analyte signal from that of interferents. The eluent was analyzed with a Perkin-Elmer LC-85 dual beam spectrophotometer, and the resulting chromatographicpeaks were integrated with a 3390A Hewlett-Packard integrator. Since peak area of the analyte is proportional to the concentration, response factors for each PAH were obtained by injecting a standard solution into a calibrated injection loop (Rheodyne 7125 syringe loading sample injector). The sample loop volume was determined to be 25.7 pL by using a previously described method (12). Calculations. Detector response factors and solubilitieswere determined for each PAH as described by May et al. (12). Formation constants were determined by subetituting the aqueous solubility and cydodextrin solution solubility values into the appropriate equation. The cyclodextrin concentration used for M unless otherwise indicated. all determinations was Evaluation of Complex Stoichiometry. Since cyclodextrin complexes can form with stoichiometric ratios other than 1:l (19), complex stoichiometry was evaluated for the PAHs when solubilized in M cyclodextrin solution. Cyclodextrin/pyrene complexes have been previously characterized by several researchers, and each cyclodextrin forms a 1:l complex with pyrene at pyrene concentrations below lo4 M (20-22). The other PAHs used in this study have not been thoroughly characterized in terms of complexation, and thus, stoichiometry of these complexes was examined. Absorbance measurements for PAHs at various cyclodextrin concentrations were used to show the 1:l stoichiometry of the cyclodextrin/PAH complexes. The absorbance spectrum of each PAH was obtained in the absence of cyclodextrin and in the presence of consecutive additions of cyclodextrin. In order to solubilize sufficient PAH to obtain an absorbance spectrum, a 1%methanol solution was used in place of 100% water. Methanol was chosen as a modifier because previous studies in our laboratory have shown that small quantities of methanol do not strongly affect cyclodextrin/pyrene complexes (23). Complex formation may be indicated by a shift in the absorbance spectrum and the appearance of an isosbestic
ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
point (24). Complex stoichiometry was examined by inspection of a set of spectra for isosbestic points (24). Aqueous PAH solutions containing 1% methanol were prepared according to a previously described method (25).The concentration of PAH used for each study was 1 X lo4 M. Absorbance measurements were made with a Perkin-Elmer Lambda 3 absorbance spectrophotometer.
RESULTS AND DISCUSSION The determination of formation constants by the solubility method is simple and commonly used. This method is based on the principle that the increase in solubility of a substrate sparingly soluble in water (e.g. PAH) may be measured as a function of ligand (e.g. cyclodextrin) concentration. Although solubility measurements incorporate phenomena such as association and hydration of equilibrating components (18),the method is reproducible for characterizing the stability of inclusion complexes. Certain variations may be observed when one is comparing solubility data to data obtained from other methods; however, the data obtained in this manner are useful when compared to other values taken under the same experimental conditions. Since solubility and complexation are equilibrium phenomena, DCCLC is a useful method for determining Kf values. Although PAH solubilities increase in cyclodextrin solution, the solubility values must still remain low enough to maintain equilibration. The increases in solubility observed in the presence of cyclodextrin are not large enough to strip the coating from the generator column. This concept may be illustrated by using pyrene complexation with y-cyclodextrin, since the complex formed between these species involves the largest percentage of depletion of PAH from the generator column. At 25 OC, pyrene solubility in the presence of ycyclodextrin is 278.9 ppb, which corresponds to approximately 2.79 X lo-’ g mL-’. When generator columns are prepared, 0.20 g (1% w/w) of PAH is coated on the column packing. About 717 L of y-cyclodextrin solution would be necessary to completely strip the column of PAH. In these experiments, the volume of cyclodextrin solution passed through the column for a series of K f determinations at three temperatures is 550 mL, which is only a small fraction of the total volume necessary to deplete the column. Thus, solution generation can be accomplished in the presence of cyclodextrin without complete stripping of the generator column. The accurate determination of PAH aqueous solubilities by DCCLC depends on low PAH solubilities and column stability after large volumes of aqueous purge. Saturated solutions are formed through an equilibration process as water passes through the generator column. In fact, variations in solubility with flow rate are not observed within the range of 0.1 to 5 mL min-’ (12). Formation constant values for y-cyclodextrin with benzo[a]pyrene were not altered when flow rates were varied, indicating that complex formation is fast and an equilibration process is maintained for PAH solubility in cyclodextrin solution. Previous studies also showed that saturated solutions produced by PAH generator columns are independent of the hydrocarbon supply (12). Thus, a generator column containing coated sand generates saturated solutions much in the same way as a column packed with crystals of PAH. The use of DCCLC for formation constant determinations requires that any adsorption of cyclodextrin on the generator column is constant and that the desorption rate of cyclodextrin is fast. Adsorption of cyclodextrin on the generator column may play a role in the solubilization and complexation of PAHs. In order to understand the interaction between the cyclodextrin solution and the generator column, the effect of flowing cyclodextrin solution through a generator column for an extended period of time was determined. Approximately 550 mL of P-cyclodextrin solution were passed through a
957
~~
Table I. Formation Constants (M-*) Calculated after Different Volumes of Cyclodextrin Solution Have Been Pumped through the Generator Column total vol pumped through generator column, mL 85 110 135 160 205 240 275 310 355 400 435 480 520 av
Kf calcd at 25 “C” 3244 3262 3296 3339 3338 3294 3392 3359 3347 3379 3337 3422 3352 3336 f 51
Formation constant between 8-cyclodextrin and benzo[a]anthracene. benzo[a]anthracene generator column, and formation constants were evaluated periodically. This volume of solution required about 4.6 h to pass through the generator column and represents an average experimental period. The data are presented in Table I. Close examination of the data indicates that the Kfvalues are precise and only random fluctuations are observed for the calculated values. No specific trends of continuously increasing or decreasing Kf values were observed. Irreversible adsorption of cyclodextrin on the generator column would result in a steady decrease of the PAH solubility in the presence of cyclodextrin and, hence, a decrease in the calculated K fvalues. Since this trend is not observed, this experiment supports the assumption that the effect of cyclodextrin adsorption on the generator column is constant and does not appear to be irreversible. This study also provides further verification that a continuous flow of cyclodextrin solution does not deplete the generator column. Although 1:l complexes between cyclodextrin and guest are usually formed in aqueous solution (18),some cases of multiple complexation have been reported. Common stoichiometric ratios observed in aqueous solution include 1:2 complexes in which two guest molecules enter into a cyclodextrin cavity and 2:l complexes in which two cyclodextrin molecules “cap” a guest molecule (18). In this study, 1:2 complexes are unlikely because the cyclodextrin concentration is much larger than the PAH concentration (23). The formation of 2:l complexes is possible, however, since a large excess of cyclodextrin is present in solution. This possibility was evaluated through absorbance studies. If we assume that the absorbance of complexed PAH (1:l or 21) differs from that of the free PAH, then the presence of complexed PAH will be indicated by changes in the PAH absorbance spectrum in the presence of cyclodextrin. Under such conditions, a point of common absorption intensity called an isosbestic point will be observed. If the system possesses only two states, P and Cy:P, all the spectra will pass through this point (24). The presence of multiple complexes can sometimes be discerned by the appearance of additional isosbestic points through which all spectra do not pass. The absence of such isosbestic points does not prove the absence of multiple complexes; however, the presence of such points should prove useful for verification of some systems. Since the PAHs studied possess aqueous solubilities below 1 X lo4 M, the use of a methanol modifier was necessary to solubilize sufficient PAH for absorbance measurements. Conclusions are drawn on the basis of the assumption that
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ANALYTICAL CHEMISTRY, VOL. 61, NO.
9,MAY 1, 1989
Table 11. Aqueous Solubilities for Polynuclear Aromatic Hydrocarbons aqueous solubility, pglkg
this work compd
temp, f O . l "C
col 1
col 2
15 25 35 15 25 35 15 25 35 15 25 35
21.40 f 0.81 37.14 f 0.57 63.60 f 0.92 5.34 f 0.08 9.35 f 0.19 17.52 f 0.16 71.95 f 1.42 127.71 f 5.49 200.03 f 1.20 0.73 i 0.02 1.41 f 0.06 2.71 f 0.09
21.83 f 0.32 40.36 f 1.01 61.71 f 1.01 5.47 f 0.16 9.16 f 0.17 17.34 f 0.24 73.20 f 0.33 134.82 f 1.61 199.91 f 0.35 0.94 f 0.01 1.45 f 0.02 2.84 f 0.03
anthracene benzo[a]anthracene pyrene benzo[a]pyrene
May et aLa
Schwarz*
44.6 f 0.2
41.0 f 0.3
Wauchope and Getzen' 75.0
9.40 f 0.1
132 f 1
MacKay and Shiud
14.0 f 0.2
129 f 2
148
135 f 5
1.59 f 0.011
'Solubility determined by DCCLC method in ref 10 and 12. *Solubilitydetermined by fluorescence method and reported in mol/L in ref 'Solubility determined by UV method and reported in ppm in ref 7. dSolubility determined by fluorescence method and reported in ma/L in ref 5.
6.
The formation of the 1:lcomplex is described by eq 1. The double arrows indicate that both free and complexed PAH are present in solution at equilibrium. Equation 2 gives the cy
+ P + CyP
(1)
corresponding formation constant expression for the complex where [Cy] is the equilibrium cyclodextrin concentration, [PI is the concentration of free PAH, and [CyP] is the concentration of complexed PAH.
340
320
300
280
260
240
wavelength, nm
Figure 1. Absorbance spectra of benzo[a]pyrene in 1% methanol and (d) 4.5 X M y-cyclowith (a) 0, (b) 5 X lo4, (c)1 X dextrin.
the behavior of the complexation in the presence of methanol is similar to complexation in the absence of methanol. This assumption is based upon previous data obtained in our laboratory on the effect of alcohol modifiers on complexation behavior (23). Figure 1shows a series of absorbance spectra for benzo(a]pyrene with consecutive additions of y-cyclodextrin. As depicted in the figure, only one isosbestic point is observed for concentrations of cyclodextrin as high as 4.5 X M. When M cyclodextrin was added to some systems such as the y-cyclodextrin/anthracene system, the absorbance spectrum shifted again. This suggests that an additional change in the complexation behavior, such as the formation of 2 1 complexes, occurs at very high concentrations of cyclodextrin. Data for the other complexes studied gave similar results. Thus, a t a concentration of 1 X M cyclodextrin, multiple complex formation was not a concern. One additional piece of evidence for 1:1complexation exists for the P-cyclodextrin/benzo[a]anthracene complex. Formation constants were calculated at 25 "C for three cyclodextrin concentrations. The values obtained (M-l) were 3688 a t 1 X lo4 M, 3471 a t 5 X lo-* M, and 3525 a t 1 X M @-cyclodextrin. These formation constants show only random fluctuations with changes in cyclodextrin concentration. Variations in Kfwith changes in cyclodextrin concentration would suggest a change in the stoichiometry of the complex. The absence of such fluctuations indicates that the complex formed is not affected by increasing the cyclodextrin concentration.
The concentration of free PAH corresponds to the aqueous solubility of the PAH. In cyclodextrin solution where both free and complexed PAHs exist, the solubility value may contain contributions from free and complexed species. The solubilities of the PAH in water (So) and in cyclodextrin solution (S,) are evaluated by using DCCLC. Upon complexation, a certain amount of free cyclodextrin is used to form the complex. Even under conditions of strong complex formation and with the assumption of 2:l (Cy:P) complexation, the initial concentration of cyclodextrin is only decreased at the most by 0.1 %. Since the cyclodextrin concentration is not depleted to an appreciable extent by complexation with the PAH, the initial cyclodextrin concentration may be used in the calculation since it closely approximates the equilibrium concentration. By substitution of the solubilities and the initial cyclodextrin concentration, [Cylt, into eq 3, the formation constant can be determined.
(3) As mentioned earlier, the determination of formation constants by DCCLC requires the measurement of both aqueous solubility and cyclodextrin-enhanced solubility. Since several studies have been performed using DCCLC for aqueous solubility determinations (10, 12), aqueous solubilities for PAHs obtained in this study were compared to previous values to ensure that the solution generation method and chromatographic system were functioning properly. The aqueous solubility values determined for each PAH at three temperatures are given in Table I1 along with previously reported values obtained from DCCLC, absorbance measurements, and fluorescence measurements. It is clear that the solubilities obtained in this study are in good agreement with solubilities obtained by May et al. using DCCLC. The similarity of the resulting aqueous solubilities indicated that the chromato-
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 9, MAY 1, 1989
Table 111. Formation Constants for Cyclodextrin/PAH Complexes
temp, compd
anthracene
benzo [a]anthracene
pyrene benzo[a]pyrene
fO.l
"c
15 25 35 15 25 35 15 25 35 15 25 35
formation constants, M-' P
CY
Y
col 1
col 2
col 1
col 2
col 1
col 2
142.6 f 37.3 58.49 & 22.21 27.69 f 22.34 168.4 f 23.9 80.15 f 22.50 32.92 f 9.04 182.1 f 20.3 176.1 f 44.1 124.6 f 7.2 304.4 f 103.8 174.9 f 55.9 95.95 f 5.67
167.3 f 33.9 90.26 f 35.50 10.47 f 27.85 209.9 f 165.4 95.18 f 48.72 12.11 f 33.3 160.5 f 5.2 121.9 f 13.1 115.7 f 9.2 324.6 f 89.9 171.9 47.6 86.72 f 31.05
2753 f 109 2141 f 64 1558 f 169 3700 f 103 3324 f 105 2672 f 48 650.4 f 26.4 528.3 48.9 349.7 f 8.2 2634 f 110 2232 f 224 1036 f 86
2382 f 57 1982 f 59 1681 f 66 3906 f 386 3525 f 224 2666 f 73 620.0 f 8.8 459.0 f 14.7 234.9 f 9.5 2430 f 117 2205 f 79 1194 f 105
340.7 f 54.3 241.8 f 94.0 208.3 f 24.6 672.2 f 25.2 611.4 f 25.2 454.4 f 32.2 1855 f 45 1104 f 66 600.3 f 8.5 85350 f 2898 63180 f 4823 37940 f 1590
332.6 f 14.8 203.9 f 49.8 156.7 f 33.9 670.0 f 114.3 599.4 f 57.5 400.3 f 31.6 1846 f 13 1146 f 19 592.2 f 10.3 72550 f 2000 66310 f 1441 36150 f 1127
*
graphic system used in this experiment provides valid data. The solubility data calculated in this experiment were analyzed by using the method of error propagation so that the effect of errors in solubility measurements on formation constant determinations could be assessed. Errors in solubility and Kf data were calculated on the basis of five runs a t each temperature, and error f limits are given in terms of relative standard deviations. Formation constants, calculated from eq 3, are provided in Table 111. The only literature values for cyclodextrin/PAH complexes available were reported for pyrene by Patonay et al. (22),who studied the quenching of pyrene fluorescence by cyclodextrin. The formation constants reported (M-l) are 78 with a-cyclodextrin, 277 with (I-cyclodextrin, and 399 with y-cyclodextrin. Comparison of these values to the data obtained by use of DCCLC indicates that the same trends are observed for both types of studies, but the formation constants calculated by DCCLC are larger than those determined by quenching studies. Direct comparison of stability constants calculated from solubility measurements with formation constants obtained by other methods is often difficult because of the contributions of association and hydration inherent in solubility measurements. In addition, several basic assumptions were made in the quenching experiments that may not always be valid. Thus, it is not surprising that the values calculated by using DCCLC are different. One interesting factor is that the quenching method can only be used for PAHs that display quenching behavior upon addition of cyclodextrin. The DCCLC method, however, possesses greater general applicability. The results of the formation constant studies provide useful information concerning complexation behavior. First, all Kr values evaluated with this system show greater complex stability a t lower temperatures. For example, when one looks at the values for (I-cyclodextrin/anthracene complexes, the average Kf at 15 OC is 2568 M-l, while at 35 "C the average K fis 1620 M-l. This suggests that complex stability decreases with increases in temperature. This phenomenon has already been used in the separation of various analytes on cyclodextrin columns (26). The magnitude of this temperature effect varies for the different complexes, indicating that some complexes may be less thermally stable than others. Data obtained for a-cyclodextrin shows that the complexation behavior for all the PAHs studied was very weak. It is not unusual that the formation constants are small because the dimensions of the PAHs used in this study are larger than the cyclodextrin cavity dimensions. The complexation observed with a-cyclodextrin may result from partial entry of the PAH into the cavity. Since complexation has not been previously reported between PAHs and a-cyclodextrin (22), the increase in solubility observed with the use of this host
*
Table IV. Dimensions of the PAHs compd
anthracene benzo[a]-
dimens'
compd
dimens'
5.0 A X 9.2 8, pyrene 7.1 8, 7.1 8, X 10.4 8, benzo[a]pyrene 7.1 8,
X X
8.9 8, 10.4 8,
anthracene
'Estimates were made by using the standard bond lengths of 1.39 and 1.08 8, for the ring C=C and C-H, respectively. is probably caused by noninclusional interactions between the PAH and cyclodextrin. Complexes formed with (I- and y-cyclodextrins generally show larger Kf values. This result is expected because the 7.8and 9.5-A cavity widths of these cyclodextrins are much larger than that of the a-cyclodextrin cavity and can more adequately accommodate the size of the PAHs. The dimensions of the PAHs are provided in Table N. Benzo[a]anthracene, pyrene, and benzo[a]pyrene show larger formation constants with y-cyclodextrin than does anthracene, which is the smallest of the four PAHs. In the (I-cyclodextrin cavity, however, anthracene shows a formation constant that is comparable to those of the other PAHs. In fact, anthracene has a larger Kfwith P-cyclodextrin than pyrene does. Anthracene and benzo[a]anthracene show their largest formation constants with P-cyclodextrin, while pyrene and benzo[a]pyrene have their largest formation constants with y-cyclodextrin. Even though benzo[a]anthracene and benzo[a]pyrene have the same length and width measurements, their formation constants with y-cyclodextrin are very different. Benzo[a]pyrene has an extremely large K f with y-cyclodextrin, while benzo[a]anthracene has a K f that is closer to the values obtained for other PAHs. The differences between the interactions of benzo[a] pyrene and benzo[a]anthracene with y-cyclodextrin suggest that some other factors may be important in the complexation process. Although benzo[a]anthracene and benzo[a]pyrene have similar length and width measurements, several differences in their structure and characteristics may account for the variability in their formation constants. The surface areas of these PAHs differ because benzo[a]pyrene contains one more fused ring than benzo[a]anthracene. Thus, benzo[a]pyrene can more strongly interact with the cyclodextrin core because of its larger surface area. The manner in which the PAH orients in the cavity, in addition to the PAH surface area, may affect the complexation. An additional factor that may contribute to the variation in complexation behavior is the difference in PAH hydrophobicity. Based on the aqueous solubilities of the molecules, benzo[a]anthracene is probably slightly less hydrophobic than benzo[a]pyrene. Thus, one might expect the hydrophobic interactions between benzo-
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[alpyrene and the cyclodextrin cavity to be stronger. Such differences may contribute to the stability of cyclodextrin/ PAH complexes. Similar arguments can be made for the other complexes. The most valuable criterion for determination of complex stability still appears to be the relative size of the guest; however, some additional information about the complexation can be discerned by evaluating the contributions of the factors mentioned here. We are currently conducting an exhaustive study into understanding this phenomenon. The relative errors associated with a-CyclodextrinlPAH Kf values are much larger than those observed for the other cyclodextrins. The large errors result from the small increase in solubility in the presence of a-cyclodextrin and indicate that the determined Kf values for this cyclodextrin are not reliable. Errors are more significant in Kfdeterminations that require calculation of the difference between two solubility values (see eq 3) that are similar in magnitude. While the difference in solubilities is small, the relative error is related to the square root of the squared sums of the individual errors. This leads to large error terms even though consecutive peak area measurements are precise. The formation constants calculated for /3- and y-cyclodextrins are more precise for single column runs and show better agreement between columns than a-cyclodextrin data. Since the differences between solubility in the presence and absence of cyclodextrin are greater, the relative error is not as pronounced. Thus, determination of formation constants by DCCLC, like other solubility techniques, is most useful when reasonable complexation occurs. In systems where weak complexes are formed, the precision of the Kf data is poor, while stronger complexes result in data that are more precise.
CONCLUSIONS The DCCLC method is convenient for determining the degree of complex formation between cyclodextrin hosts and PAHs, yet relative error values of 10% are not uncommon. Since consecutive runs show reproducibility within 6%, the degree of error is a reflection of the measurement of the difference in solubilities rather than on the precision of the DCCLC technique. This method allows fairly rapid determination of formation constants with reasonable reproducibility and circumvents many of the problems associated with solution preparation and analysis in solubility measurements. No direct contact between experimenter and solutions was necessary, which is particularly important in working with hazardous substances such as PAHs. Preparation of saturated solutions of low-solubility analytes is efficient, and thus, the collection of data for aqueous and cyclodextrin solubilities can be easily performed in a few hours. Formation constant data for the cyclodextrin/PAH complexes show that the precision of the measurements is best for complexes with large formation constants. Thus, data for
a-cyclodextrin/PAH complexes are not considered reliable when obtained by this method. A useful additional application of DCCLC would be to study multiple equilibria in which cyclodextrin may complex with a variety of PAHs. An application such as this may provide useful information on competitive equilibria.
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RECEIVED for review August 18,1988. Accepted February 3, 1989. This work was supported in part by the National Science Foundation (CHE-8609372) and the Office of Naval Research. Isiah M. Warner acknowledges support from a NSF Presidential Young Investigator Award (CHE-8351675).
