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Anal. Chem. 1989, 61,272-275
the electrode at a sweep rate of 0.5 V/s. The current recorded at the anodic switching potential of each potential cycle during consecutive exposures of the electrode to the electroactive mixture is shown in Figure 3B. It is clear that the potential scanning scheme greatly improves the stability of the response. The remaining instability may be further decreased by the inclusion of a delay time between anodic scans to more thoroughly reduce the ferricinium. CONCLUSION The results presented in Figures 2 and 3 demonstrate that microelectrodes coated with ionically conductive polymer membranes provide practical devices for electrochemical investigations in flowing supercritical COz without added supporting electrolyte. The requirement for a polar modifier does not seem to present a major drawback as modifiers are widely used in chromatographic systems for which these detectors are of interest. Furthermore, as mentioned above, other available ionically conducting membranes do not require such modifiers. Particular attention has to be paid to the potential waveform used for detection to circumvent deleterious effects of electrochemical products in the film. Electrochemical detection in supercritical fluids is anticipated to be quite sensitive because of the gaslike diffusion coefficients of solutes. Diffusion through membranes, however, is often very slow. If the membrane is sufficiently thin compared to the dimension of the diffusion layer, as in Figure 2, the voltammetry becomes characteristic of the fluid diffusion coefficient (20). Thus, the use of polymer films is not expected to significantly counteract this attractive aspect of supercritical fluids. In liquid flow systems, microelectrodes coated with very thin Nafion films display response times that are very similar to uncoated electrodes (21). These latter two points suggest that membrane-coated microelectrodes have the requisite properties for an electrochemical detector of supercritical fluid chromatography that may match or surpass the performance of electrochemical detectors currently available for liquid chromatography (22). Registry No. Pt, 7440-06-4;COz, 124-38-9;ferrocene, 102-54-5; Nafion, 39464-59-0.
LITERATURE CITED Crooks, R. M.; Bard, A. J. J. Electroanal. Chem. 1988, 243, 117-131. Philips, M. E.; Deakin, M. R.; Novotny, M. V.; Wightman, R. M. J. fhys. Chem. 1987, 97,3934-3936. Novotny. M. V.; Springston, S. R.; Peaden, P. A,; Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1981, 53,407A-414A. Wightman, R. M. Science 1988, 240, 415-420. Pons, S.; Fleischmann, M. Anal. Chem. 1987, 59, 1391A-1399A. Beran, P.; Bruckenstein, S. Anal. Chem. 1980, 52, 1183-1186. Kaaret, T. W.; Evans, D. H. Anal. Chem. 1988, 60,857-862. DeWulf, D. W.; Bard. A. J. J. Electrochem. SOC. 1988, 735, 1977-1985. Reed, R. A.; Geng, L.; Murray, R. W. J. Electroanal. Chem. 1988, 208, 185-193. Geng, L.; Reed, R. A.; Longmire, M.; Murray, R. W. J. Phys. Chem. 1987, 97,2908-2914. Wang, S.S.J. Electrochem. SOC. 1988, 135, 15312, Abstract #461. Brina, R.; Pons. S.; Fleischmann, M. J. Electroanal. Chem. 1988, 244,81-90, Moore, R. E., 111; Martin, C. R. Macromolecules 1988, 27, 1334-1339. Aoki, K.; Akimoto, K.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. 1984, 177, 219-230. Wightman, R . M.; Wipf, D. 0. I n Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1988; Vol. 15. Martin, C. R.; Dollard, K. A. J. Electroanal. Chem. 1983, 759, 127- 135. Parcher, J. F.; Barbour, C. J.; Murray, R. W. Anal. Chem., submitted. Buttry, D. A.; Anson, F. C. J. Am. Chem. SOC. 1983, 705,685-689. Moran, K. D.; Majda, M. J. Electroanal. Chem. 1986, 207, 73-86. Peerce, P. J.; Bard, A. J. J. Electroanal. Chem. 1980, 172, 97-115. Kristensen, E. W.; Kuhr, W. G.; Wightman, R. M. Anal. Chem. 1987, 59, 1752-1757. Weber, S. G.; Long, J. T. Anal. Chem. 1988, 60,903A-913A. Author to whom correspondence should be addressed.
Adrian C. Michael
R. Mark Wightman* Department of Chemistry Indiana University Bloomington, Indiana 47405
RECLWED for review September 23,1988. Accepted November 1,1988. This research was supported by the Chemical Analysis Section of the National Science Foundation.
Enhanced Detection of Sulfite by Inductively Coupled Plasma Atomic Emission Spectroscopy with High-Performance Liquid Chromatography Sir: Inductively coupled plasma atomic emission spectroscopy (ICP-AES) has become a standard analytical technique for sulfur determination in soil ( I ) , plant tissue @), and coal (3). In most cases, the sample is digested or extracted and aspirated directly from solution and the concentration determined. Speciation information is, however, unavailable. Concentrations of particular species of sulfur in solution have been determined with emission spectroscopy by separation prior to quantification. High-performance liquid chromatography (HPLC) with ICP-AES detection has been used to quantify various sulfur-bearing surfactants (4). Sulfur-containing biological compounds from rat organs have been determined by ICP-AES after extraction and separation on a TSK Gel G3000 S W column (5). The conversion of an analyte from a solution species to a gaseous product is the basis of cold vapor and hydride generation techniques. In both of these techniques, sensitivity and detection limits are significantly improved over more conventional nebulization modes (6). Gaseous analyte is also produced and quantified by using a heated graphite atomizer
(7). ICP-AES detection has been used to determine hydrogen sulfide from solution as a gaseous product (8). Samples of groundwater, preserved with 0.1% KOH, were acidified to contain 0.5% HCl to liberate the hydrogen sulfide. Argon was used to flush the hydrogen sulfide from the top of a gas-liquid separator into the torch. The sensitivity of determination of osmium was enhanced by a factor of approximately 100 over conventional nebulization by converting the analyte to osmium tetraoxide vapor (9). The improvement was attributed to avoiding the inefficiency of the nebulization process as well as the cooling of the plasma by the solvent. The plasma served as an ion source for a mass spectrometer used to determine osmium isotopic ratios in nanogram quantities. HPLC with ICP-AES detection is significantly less sensitive than other detection methods used in liquid chromatography because of the loss of from 80% to 99% of the analyte upon nebulization to an aerosol (IO). Results from the investigation herein reported indicate, however, that the detection of sulfite by this method is far more sensitive than detection of sulfite under other conditions. It is proposed that a second mech-
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ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1, 1989
anism, which converts sulfite under acidic conditions t o gaseous sulfur dioxide, accounts for an enhanced transport of analyte into the plasma of the torch.
EXPERIMENTAL SECTION Reagents. Weighed amounts of ACS grade sodium bisulfite and anhydrous sodium sulfate from Fisher Scientific (Pittsburgh, PA) were used to prepare standard solutions. ASTM Type I distilled deionized water was degassed by vacuum filtration and used to prepare standard solutions as well as mobile phases. The standardization of the sulfite solutions was as follows: The sulfite-S standards were injected into a liquid ion chromatograph and the amount of sulfate4 present quantified by using sulfate-S standards. The sulfite standard was then oxidized with hydrogen peroxide and the resulting sulfate3 quantified by again using sulfate-S standards. The concentration of the sulfite4 standard was calculated by subtracting the sulfate-S in the sulfite-S standard before oxidation from the total sulfate-S determined after oxidation. Apparatus. An LDC ConstaMetric I1 G Pump (LDC, Riviera Beach, FL) was used with a Rheodyne injector (Rheodyne, Cotati, CA) equipped with a 175-pL sample loop. A sulfite ion exclusion column, 100 X 7.8 mm, of polystyrene-divinylbenzene, 9 pm, with 10% cross-linking (Benson Polymeric, Inc., Reno, NV) was used. The ICP-AES was an Applied Research Laboratories 3520 sequential spectrophotometer equipped with a vacuum monochrometer (ARL, Valencia, CA) operated at 1220 incident watts and less than 5 reflected watts. The plasma flow rate and the coolant flow rate were 1.0 and 15.0 L/min, respectively. A Dionex 2010i liquid ion chromatograph with a conductivity detector (Dionex,Inc., Sunnyvale, CA) was used to determine sulfate and sulfite concentrations. The integrator was a Hewlett-Packard Model 3398A (HP, Santa Clara, CA). The interface from the HPLC to the ICP-AES was a direct connection of the HPLC eluent to the concentric nebulizer in the ICP-AES 50-mL spray chamber regularly used. HPLC flow rates used in these experiments ranged from 1.0 to 2.7 mL/min, typical of sample introduction rates for ICP-AES. Initial experiments to characterize the nebulization process did not include the use of HPLC instrumentation. The detection limits for both sulfite-s and sulfate-S were determined in continuous sample presentation of separate standards. The pH dependences of the sulfite43 and sulfate-S signals were investigated with the addition of an HPLC pump and the injector. Final experiments to determine the detection limit, linear working range, and retention times included a full chromatographic setup with an analytical column. During operation of the ARL 3520 ICP-AES, the signal from the photomultiplier tube (PMT) is summed by a capacitor over a programmed integration time and the resulting voltage measured as optical signal intensity. This method is inadequate for the continuous output needed in a chromatographic application. The ARL 3520 ICP-AES with “A” electronics has a 12-bit analog to digital (A/D) conversion line from the PMT which is normally used in the positioning routine of the instrument, as well as in diagnostics. Under “test” conditions it is possible to position the PMT on one particular element channel, set the attenuation of the PMT, and in the profile mode obtain a continuous output from the voltage line going to the A/D converter. The voltage can be output to a strip chart recorder, an integrator, or any other signal recording device. Procedure. Four 50-mL solutions were prepared containing 1000 ppm sulfite-S, two acidified to 1%HC1 (v/v), and two left unacidified. Each solution was aspirated into the nebulizer and the sample that flowed down the drain was collected in a 200-mL volumetric flask. The drain solutions were oxidized with 1 mL of 30% hydrogen peroxide, made to volume, and analyzed by ion chromatography to determine the sulfur content. The percentage of the sample that reached the torch was calculated as the difference between the analyte aspirated and the analyte detected in the drain solution. The sample introduction system for the plasma was modified in order to detect the evolution of a gaseous analyte. The carrier argon gas was directed across the surface of a solution in a 300-mL Erlenmeyer flask and directly into the torch. The monochromator was set at the 182.037-nmemission line in the third order of sulfur, the PMT set on an attenuation of 120, and the output monitored.
273
Solutions containing lo00 ppm sulfate-S, lo00 ppm sulfite-S, and 10 ppm sulfite-s were prepared. Each solution in turn was placed in the flask, acidified to contain 0.1% HCl, mixed, and ultrasonicated. Any sulfur-containing gas evolved in the flask would be detected. The mobile phase flow rate and the ICP-AES carrier flow rate were optimized. A 10 ppm sulfite solution containing 0.1% HCl (v/v) was pumped into the nebulizer. The mobile phase flow rate was set a t 2.0 mL/min and the ICP-AES carrier gas flow rate varied. The sulfur signal was continuously recorded on a strip chart recorder. With the argon gas carrier rate then optimized, the rate of pumping was then optimized. In order to determine the detection limits of pure standards, separate solutions of 10 ppm sulfite-s and sulfate3 were prepared in 0.1% HC1 (v/v) and aspirated in a normal ICP fashion. The detection limits were calculated as 3u. The HPLC without an analytical column was then connected to the ICP-AES. The pH dependences of the sulfite-S and sulfate-S signals were determined by using mobile phases prepared from concentrated HC1 with pH values ranging from 0.86 to 5.72. By use of each mobile phase in turn, triplicate 50-pL injections were made of 10 ppm sulfite4 solution and the peak heights plotted as a function of pH. The procedure was repeated with injections of 1000 ppm sulfate-S. To determine the difference in sensitivity between the sulfite and sulfate signal under identical chromatographic conditions, an ion exclusion column was added. A mixed standard was prepared of 10 ppm sulfite4 and 10 ppm sulfate4 as well as a second standard of 10 ppm sulfite-S and 1000 ppm sulfate-S. These standards were injected with a mobile phase of pH 1.84 (0.1% HC1 (v/v)), at a flow rate of 2.0 mL/min. Peak areas and heights were compared. The same chromatographic system was used to determine the detection limit of sulfite-s. Triplicate 50-pL injections of a 1ppm solution containing 50 ng of sulfur were analyzed and the peak height and area determined. The detection limit was determined at a signal to noise ratio of 3u. The wide dynamic linear range of plasma emission in analysis is widely known. Emission is typically linear over 4-5 orders of magnitude. The linearity of response of the chromatographic system used in this study was determined at an attenuation of 95 (the maximum attenuation is 128). Triplicate 50-pL injections of 1.0,5.0,10.0,50.0, and 100.0 ppm of sulfite-s were injected and regression analysis was applied to the data obtained.
RESULTS AND DISCUSSION The first set of solutions were aspirated to determine the amount of acidified sulfite-S and unacidified sulfite-S which reached the plasma. The drain solutions collected from the acidified sulfite solutions (1% HCl (v/v)) were 50 f 2 ppm in total sulfur indicating that 81 f 3% of the analyte reached the torch. For the unacidified sulfite solutions, the concentration of sulfur in the diluted drain solutions was 248 f 6 ppm, indicating that less than 2% of the sulfur in these solutions reached the torch for detection. With argon carrier gas purging the headspace over the lo00 ppm sulfate-S solution in the Erlenmeyer flask, no observable sulfur signal was observed when the solution was introduced to the flask, acidified t o contain 0.1% HC1, shaken, or ultrasonicated. The 1000 ppm sulfite-S solution contributed a considerable background signal for sulfur even before acidification. After acidification of the solution the P M T became saturated because of the evolution of large amounts of sulfur dioxide and the solution was discarded. Amounts of sulfur gases which evolved from the 10 ppm sulfite-S standard were a maximum from the acidified solution when swirled and not as large when untrasonicated. The presence of a large sulfur signal when sulfite solutions a t concentrations as low as 10 ppm are acidified and swirled confirms the production of gaseous sulfur dioxide. The large signal from the swirling suggests that this enhancement is maximized by having a large surface to volume ratio, a ratio augmented by nebulization to an aerosol and spraying the sample onto the
ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1, 1989
274
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Dependence of the signal of a 10 pprn acidified sulfite-S solution on the argon carrier flow rate. Flgure 1.
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pH dependence of ICP as a detector for separate injections 50-pL of a 10 ppm sulflte-S solution and injections of a 1000 ppm sulfate4 solution. Instrumental conditions are listed in the text (n = Figure 2.
of
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inside surface of the spray chamber. When pumping an acidified 10 ppm sulfite-s standard into the nebulizer, the optimum sulfite signal was determined to be at an argon carrier flow rate of 0.4 L/min (Figure 1). After the carrier flow was optimized, it was determined that increasing the flow rate from 1.0 to 2.6 mL/min increased the signal. The pumping rate of 2.0 mL/min was chosen as a comparison between maximizing sensitivity and minimizing column back pressure. When separate 10 ppm sulfite-S and 10 ppm sulfate-S standards acidified to 0.1% (v/v) were aspirated with continuous nebulization without HPLC sample introduction, the detection limit of sulfate-S using a detection limit of 3u was 0.062 ppm and the detection limit of sulfte-S was O.OOO8 ppm, an improvement of 78 times. The detection limit of sulfite4 is comparable to the literature value for sulfide of 0.0002 ppm detected as hydrogen sulfide gas (9). After an HPLC unit with an injector was added, the signal response to 50-pL injections of 1000 ppm sulfate3 was not observed to be pH dependent (Figure 2). Sulfite peak height graphed as a function of p H yielded a typical sigmoidal p H titration curve. The maximum sulfite-S emission signal was achieved by using mobile phases of pH 2.0 or below. Because of the corrosive nature of HCl solutions on the stainless steel of the LC pump, an eluent of pH 1.84 in HC1 (0.1%HCl) was
2
4
TIME ( m i d
TIME (minl
C A R R I E R ARGON F L O W ( L / m l n )
Sulfate-S
0
Flgure 3. (A) 50-pL injection of a
solution 10 ppm in sulfite-S and 1000 in sulfate-S. (B) 50-pL injection of a 1 ppm sulfite4 standard. Instrumental conditions are listed in the text. selected for the remainder of the investigation. The standard composed of 10 ppm sulfate-S and 10 ppm sulfite-S was injected in triplicate. At a flow rate of 2.0 mL/min and a mobile phase pH of 1.84, the retention time for sulfite was 1.4 min. Sulfate under the same conditioins was not retained and eluted after only 0.8 min. The ratio of peak area for equal concentrations of sulfite-s to sulfate-S was 70:l and for peak height 601. These results demonstrate the increased sensitivity of ICP-AES detection of sulfite under these conditions. A second mixed standard of 10 ppm sulfite-s and 1OOO ppm sulfate-S was used in order to obtain peaks of comparable height and area (Figure 3A). Peak area analysis of this mixed standard indicates that ICP-AES detection is 85 times more sensitive for sulfite3 than for sulfate-S. Analysis of triplicate 50-mL injections of a 1 ppm solution of sulfite-S (Figure 3B) yielded a percent relative standard deviation of 4.4%for peak area and 6.2% for peak height. The signal to noise ratio based on peak height was 16 and on peak area 23, or about 40% higher. By assuming a detection limit of 3u, 0.08 ppm sulfite-s could be quantified by peak area with 50-pL injections. From the calibration curve of standards from 1 to 100 ppm, the correlation coefficient from regression analysis was calculated to be 0.996 for both peak area and peak height. Under acidic conditions the sulfite forms sulfurous acid which is in equilibrium with a gaseous product, sulfur dioxide
HSOB- + H+
H2SO3 + H2O + SO2t
(1)
Typically, the equilibrium is shifted to the left, accounting for the considerable solubility of sulfur dioxide in aqueous solutions. However, in the process of nebulization and subsequent impact against the spoiler bead in the spray chamber, the acidity of the eluent, the increased surface area, and the rigorous conditions cause the evolution of gaseous sulfur dioxide to be enhanced. Because one product of the equilibrium, sulfur dioxide, is removed, the equilibrium shifts further to the right, evolving even more sulfur dioxide.
CONCLUSIONS HPLC/ICP-AES is a species specific analysis technique with a special sensitivity for sulfite in acidic media. Often considered to be the weakest link in plasma spectroscopy, the nebulization process in this application has been used to considerable advantage to extract the analyte from the eluent and produce a gas-aerosol mixture in which the analyte is concentrated by almost 2 orders of magnitude. The use of
Anal. Chem. 1989, 6 1 , 275-279
chromatography with ICP-AES allows for the separation of the sulfite from the oxidative product of sulfite, sulfate, as well as from any other sulfur-bearing species in more complex matrices. The wide linear response range as well as the low detection limit indicates that this method meets the requirements for sulfite determination in beverages and foods, as well as for other types of sulfite analysis. ACKNOWLEDGMENT The author thanks James Harley and Harry W. Wonders of Applied Research Laboratories (Dearborn, MI), James Benson of Benson Polymeric, Inc. (Reno, NV), and my colleague, Vipin Agarwal, for assistance in this investigation. Registry No. Sulfite, 14265-45-3;sulfate, 14808-79-8. LITERATURE C I T E D (1) Maynard, D. 0.; Kaira, Y. P.; Radford, F. G. SoilSci. SOC.Am. 1987, 57, 801-806.
275
(2) Novozamsky, I.; Van Eck, R.; Van der Lee, J. J.; Houba, V. J. G.; Temminghoff, E. Commun. Soil Sci. Plant Anal. 1986, 1 7 , 1147-1157. (3) Caroll, S.; Farina Mazzeo, A.; Laurenzi, A.; Senofonter, 0.; Violante, N. J . Anal. At. Spectrom. 1988, 3 , 245-248. (4) Irgolic, K. J.; Hobill, J. E. Spectrochim. Acta, Part 8 1987. 428, 1-2. (5) Sunga, H.; Kobayashi, E.; Shimojo, N.; Suzuki, K. T. Anal. Biochem. 1987, 160. 160-168. (6) Gardiner, P. E. J . Anal. At. Spectrom. 1988, 3 , 163-168. (7) Casetta, 6.; DiPasquale, G.; Sofflentinl, A. A t . Spectrosc. 1985, 6 , 62-64. (8) Lewin. K.; Walsh, J. N.; Miles, D. L. J. J. Anal. At. Spectrom. 1987, 2 . 249-250. (9) Russ, G. P., 111; Bazan, J. M.; Date, A. R. Anal. Chem. 1987, 59, 984-989. (10) LaFreniere, K. E.; Fassel, V. A.; Eckels, D. E. Anal. Chem. 1987, 59, 879-887.
Dennis R. Migneault The Connecticut Agricultural Experiment Station New Haven, Connecticut 06504 RECEIVED for review July 25,1988. Accepted October 24,1988.
TECHNICAL NOTES Solubilization of Cyclodextrins for Analytical Applications Daniel Y.P h a r r , ’ Zheng Sheng FU? T h u y K. Smith, and Willie L. Hinze* Department of Chemistry, Laboratory for Analytical Micellar Chemistry, Wake Forest University, P.O.Box 7486, Winston-Salem, North Carolina 27109 INTRODUCTION During the past decade, there have been increasing accounts of the utilization of cyclodextrins (CDs) in chemical analysis (1-26). Cyclodextrins (also referred to as cycloamyloses or Schardinger dextrins) are cyclic, homologous oligosaccharides containing rings of a-(1,4)-linked D-glucose moieties (27,28). The three most commonly employed CDs contain six, seven, and eight such glucose residues and are referred to as CY-,0-, and y-cyclodextrin, respectively. Applications include their use as mobile phase additives in liquid chromatography (1-9), fluorescence and chemiluminescence enhancement agents (1&18,26), a medium for the observation of room temperature liquid phosphorescence (19-21 ), and reagents in isotachophoresis (22,23),spectrophotometric assays (241, and NMR optical purity measurements (25) among others. In many of these applications, the CD concentration is very important and dictates the maximum analytical response obtainable. For instance, chromatographic retention (k’and R, values) of solutes as well as the magnitude of luminescence intensity enhancement9 observed for analytes can strongly depend upon the CD concentration (1-23, 26). While the aqueous solubilities of a-and y C D at 25 “C are 0.121 and 0.168 M, respectively, that of O-CD is roughly a factor of 10 less, Le. 0.0163 (29,30). Consequently, this can create problems in those applications involving 0-CD and limita the beneficial effects possible. Previously, mixed solvent systems (3) or water-soluble /3-CD derivatives (i.e. hydroxyethyl or methylated CDs (31-34) and polymeric CDs (28,35, 36)) have been employed in attempts to overcome such solubility problems. However, such soluble @-CDderivatives are Present address: Department of Chemistry, Virginia Military Institute, Lexington, VA 24450. *Presentaddress: Chemistry Department, Northwestern Teachers’ College, Lanzhou City, Gansu Province, People’s Republic of China. 0003-2700/89/0361-0275$01.50/0
much more expensive than 0-CD itself (37)and their use (as well as use of mixed solvent systems) can alter the CD-solute binding interaction (1,31,38). In addition, we and others had previously mentioned that base and/or urea can be used to increase the water solubility of cyclodextrins, particularly p-CD, for use in some analytical procedures (13,15,39-41). Aside from two preliminary reports concerning p-CD in urea (13,41),no detailed quantitative investigation of the ability of such media to solubilize the different CDs is reported in the literature. In this note, we detail the results of our quantitative solubility study and demonstrate that either base or urea can be utilized to easily solubilize appreciable quantities of p-CD in aqueous solution. Solubility data for a-and y C D in these media are also presented. Lastly, preliminary results that illustrate the viability of using such solubilized 0-CD in selected analytical applications are summarized. EXPERIMENTAL SECTION Apparatus. A Model 7637 “Roto Torque” heavy duty rotator (Cole-Parmer Instrument Co., Chicago, IL) was used to agitate and mix the solutions in the cyclodextrin solubility studies. Thermal analysis studies on selected samples were made by using a Mettler system that consisted of a TG-50 thermogravimetric unit, M3 microbalance, and a TC-10 controller T A processor equipped with TA 3000 version 3-1 software attached to an IBM PC. The chemiluminescence measurements were made on a Turner Designs 20-000 luminometer. This latter system is described in more detail elsewhere (13). Materials. CY-, p-, and y-Cyclodextrins (CD) were obtained from Advanced Separation Technologies,Inc. (Whippany, NJ), Sigma Chemical Co. (St. Louis, MO), or Aldrich Chemical Co. (Milwaukee,WI). Certified ACS or A grade urea was obtained from Fisher Scientific Co. (Raleigh, NC) and CalBiochem (San Diego, CA). Basic solutions were prepared from certified standard 0.20, 1.00, 2.50, 5.00, or 10.0 M solutions of sodium hydroxide (Fisher). Lucigenin (lO,lO’-dimethyl-9,9’-biacridinium dinitrate) 0 1989 American Chemical Society
