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Accuracy of Transmission Electron Microscopy Analysis of Asbestos on Filters: Interlaboratory Study Shirley Turner* and Eric B. Steel Center for Analytical Chemistry, National Institute of Standards and Technology, Gaithersburg, Maryland 20899
An Interlaboratory study was conducted to determine the accuracy of determlnatlons of the asbestos concentration on fllters by use of tranmlsslon electron mlcroscopy. Repllca secikrw were prepared from a single polycarbonate fllter that had chrysotlle and several types of non-asbestos partlcles deposlted on Its surface. Twenty-seven analysts from 15 laboratorles counted a mlnlmum of 3 grld squares and recorded the physical characterlstlcs of the partlcles and the methods used tor identltkatlon of the particles. One of the grid squares counted by each analyst was reanalyzed at the National Instltute of Standards and Technology (NIST) by uslng verlfled countlng methods. The mean value of TP/TNS (true posltlves/total number of structures) obtained by the laboratorbs on the verlfled grld squares Is 0.67, and the mean value for FP/TNS (false posltlves/total number of structures) Is 0.04. More than 40% of the fibers shorter than 1 pm were mlssed, whereas less than 20% of the flbers longer than 1 bm were mlssed by the analysts. Incorrect translation of the electron microscope stage Is a likely cause of many false negative values. Other possible reasons for false negatlves and false posltlves are discussed.
INTRODUCTION The determination of the concentration of asbestos in air or water is a goal common to research laboratories conducting environmental and health-related studies and to commercial laboratories involved in asbestos-monitoring and -abatement (or removal) projects. Recently, the number of asbestosabatement projects has greatly increased due to the enactment by Congress in 1986 of the Asbestos Hazard Emergency Response Act (AHERA). The determination of the presence or absence of asbestos in the nation’s schools is required by this act. It is additionally required that the quality of air in the vicinity of an abatement project be evaluated by use of electron microscopy. In response to this latter requirement, the Environmental Protection Agency (EPA) developed detailed procedures for the use of transmission electron microscopy to analyze asbestos (1). T o acquire information on the accuracy of analysis and on problems encountered by the laboratories involved in the analysis of asbestos collected onto filters, interlaboratory studies have been conducted to evaluate (1) the quality of the filter replica preparations (2) and (2) the accuracy of the operators analyzing and counting asbestos by the use of transmission electron microscopy. Results from the latter study, conducted in the fall of 1988, are reported in this paper. Membrane filters are commonly used to monitor the concentration of asbestos in air or water. Air or water is passed through a membrane filter, leaving most of the particles on or near the surface of the filter. The filter is prepared as a carbon replica and analyzed by the use of transmission electron microscopy. The accuracy of the concentration determined by the laboratories can be affected by several aspects of this multistep process, examples of which include the sample
collection efficiency, losses or contamination during sample handling, inhomogeneity in deposition of the collected particles, calibration of the instrumentation, the quality of preparation of the carbon replica, and the accuracy of the transmission electron microscope (TEM) operator. At the present time, the TEM operator performs the actual analysis, which involves scanning the filter replica, recognizing the particles of interest, and analyzing and identifying the particles. Operator precision or accuracy is typically monitored by the reanalysis of grid squares or filters by either the same or a different operator. Commonly, only the numbers obtained from the analyses are compared. A more rigorous method, “verified counting”, involves matching two analyses of the same grid square on a particle-by-particle basis ( 3 , 4 ) . This method, though the most accurate available, is time consuming and had not been implemented by most asbestos laboratories at the time of this interlaboratory study. The focus of previous interlaboratory studies on TEM analysis of asbestos on filters has been on comparing the asbestos concentrations derived from the entire laboratory procedure (5, 6). Filters with asbestos particles were distributed to laboratories, and the laboratories prepared the filters by using a variety of methods. One study showed variations in concentrations of over 4 orders of magnitude (5), and the results of a second study showed variation of a factor of 2 (6). In both studies, the numbers of asbestos particles found by each laboratory were compared. The interlaboratory study reported in this work differs from the first two because of the following: (1)the participating laboratories were sent prepared grids from a single filter, thereby minimizing variations due to the sample preparation technique, and (2) the results were evaluated by the use of verified counting rather than simple comparison of the numbers. Therefore, the accuracy of the counting by TEM operators is the focus of this study, and from this approach, the types of problems typically encountered by the analysts can be determined.
EXPERIMENTAL SECTION Filter and Grid Preparation. Five types of materials including chrysotile, gypsum, plaster of Paris, glass fibers, and a tracer material were deposited from a Freon-based suspension onto several 37-mm polycarbonate filters. The chrysotile sample consisted of the long-fiber variety obtained from the National Institutes of Environmental Health Sciences (7). The sample was ground by using a cryomill. Gypsum, plaster of Paris, and glass fibers were used because they are common materials found in buildings and, hence, at asbestos-abatement sites. One of the filters was carbon coated and divided into 80 3-mm by 3-mm sections, and the location of each section on the filter was recorded. The filter sections were placed onto 200-mesh indexed grids that contain grid openings approximately 90 jtm on a side. The filter dissolved away after approximately 2 h in a chloroform condensation washer. The quality of the filter replicas was evaluated by using criteria described in AHERA ( I ) and developed by Turner et al. (2). Replicas not satisfying the criteria given in the AHERA regulations were rejected. Preparations used in the study were not completely clear and showed some regions of ”highlighted” pores and clouded features (2).
This artlcle not subject to U.S. Copyrlght. Published 1991 by the Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 9, MAY 1, 1991
Conduct of Interlaboratory Study. Seventeen laboratories were sent grids and materials relating to the interlaboratory study. The laboratories were required to have each operator analyze at least three grid squares for the presence of countable asbestos structures as defined by the EPA specifications ( I ) . An asbestos "structure" is a general term that can refer to a single fiber or combination of fibers. A "simple" structure (or single fiber) is considered countable if it is 2 0.5pm in length and has at least a 5:l length-to-width ratio. "Complex" structures (consistingof many fibers) are considered countable if they contain fibers that fit the above criteria. To allow for subsequent verification of analyzed grid squares, the laboratoriesrecorded the orientation of the grid in the TEM by noting the orientation of one of the marker features on the index grid at the magnification chosen for analysis. The starting corner of the grid square and the initial direction of the scan were recorded. The locations of the analyzed grid squares were recorded by using a labeling system specified by NIST. An analysis consisted of scanning a grid square with parallel traverses at approximately 20000~and recording the length, width, shape, orientation (by a sketch), type of structure, presence or absence of a hollow tube (indicator of chrysotile),analytical information from energy dispersive X-ray analyses (EDXA),and selected area electron diffraction (SAED) and, finally, identifying the particle. Selected grid squares were reanalyzed at NIST. The length and width of the particles analyzed at NIST were measured with a millimeter scale on the fluorescent screen of the TEM. Measurements were made of the particles with the samples in the eucentric position of the objective lens of the electron microscope. The millimeter scale was calibrated by using a replica of an optical The accuracy of the grating containing 2160 grating lines/". reported counts could be determined by using a slight modification to the verified counting technique as described in the following section. Methods for Conduct and Analysis of Verified Counts. In previous papers, the verified counting technique was applied to internal laboratory studies, not to interlaboratory studies (3, 4). The locations of structures in a grid square were determined by using a digital voltmeter that was interfaced with the electron microscope stage, and comparisons of structures counted by two or more analysts were made on the basis of the location information in addition to length, width, and analytical information. In an interlaboratory study, it is not yet feasible to obtain and compare positional information because most laboratoriesdo not use digital voltmeters to acquire this information. In this study, matches between counts recorded by two separate analysts were made on the basis of the orientation and shape of the structure, as derived from a sketch made by the analysts, by the relative order in which the structure was counted, by the structure dimensions, and by the analytical information. Structures recorded by two or more analysts that can be matched by the criteria listed above are considered true positives (TP). Commonly, there are several other structures that cannot be matched. For identification of these unmatched structures, the grid square is rescanned to locate the reported structure. If the structure is located and is found to be a countable structure, it is also considered to be a true positive. The total number of structures (TNS) on the grid square corresponds to the number of unique true positive structures found by the analysts. For a grid square counted by two analysts, the value for the total number of structures can be derived from the following: TNS = [TP(analyst 1) + TP(ana1yst 2)] number of matched structures (1) Note that the value for TNS may increase if other analysts count the grid square-some structures on the grid square may be missed by both of the first two analysts. If an unmatched structure is determined to be a true positive for one analyst, it is a false negative structure (FN) for the other analyst. The number of true positive and false negative structures found for any analyst must equal the total number of structures on the grid square or TNS = TP(ana1yst 1) + FN(ana1yst 1) = TP(ana1yst 2) + FN(ana1yst 2) (2) If an unmatched structure is located and is found to be incorrectly identified as a countable structure, it is labeled as a false positive
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Figure 1. Distribution of the number of chrysotile structures reported by the laboratories from 82 counted grid squares.
(FP). If after repeated searches the structure cannot be found, it is termed not located (NL) and is ignored in subsequent evaluations of the grid square. For most intralaboratory verified counts, this value should be 0, because the sample need not be disturbed, i.e., by mailing, between counts. For any analyst, the sum of the number of true positive, false positive, and not located structures must equal the number of structures reported by the analyst or
reported structures = TP + FP
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Sources of Error in the Analyses. Potential sources of error in the numbers derived from verified analyses include (1)inaccuracies in the measurement of the length of fibers close to 0.5 pm and (2)unclear interpretation of the number of countable asbestos fibers in complex structure arrangements. The accuracy of length measurements on the TEM screen is affected by at least the following: the calibration of the instrumentation, the accuracy with which a TEM operator can estimate length from the TEM screen scale, the orientation of fibers in the microscope, the shape of the fibers, and the visibility of the ends of the fibers. The accuracy of measurements of fibers around 0.5 pm in length on the TEM screen is estimated to be approximately *lo%. This relative error will decrease with increasing size of fibers. A second source of error derives from unclear interpretation of countable asbestos fibers for complex fiber arrangements. The AHERA regulations specify the interpretation of the number of countable asbestos fibers for various fiber arrangements. However, the rules are ambiguous in some cases. The fiber arrangements in this study in general are fairly simple, and therefore, this problem should not be a large source of error. A detailed study of errors involved in verified count analyses is planned, and results will be presented in a subsequent paper. RESULTS AND DISCUSSION Fifteen laboratories returned count data obtained from 27 analysts. A histogram of the structure counts from 82 grid squares is shown in Figure 1. The distribution of counts has a mean value of 15.1 structures per grid square. The distribution is slightly skewed to the left as would be expected if analysts consistently undercounted the number of structures on a grid square. The accuracy of the reported counts was determined by performing a verified count analysis on one grid square counted by each analyst. Pairs of analysts from two laboratories counted the same set of grid squares; therefore, 25 unique grid squares were verified by NIST analysts. Evaluation of Laboratories. The 25 grid squares were verified by NIST analysts having average ratings on verified counts of 0.90 or above for TP/TNS and 0.05 or below for FP/TNS. Eight of the grid squares were analyzed by more
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Figure 2. Example of deposition of carbon contamination (arrowed) onto chrysotile fibers. Such contamination hinders verification of a grid square count.
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Figure 3. Histograms of the values for (a) TP/TNS and (b) FPITNS attained by the laboratories for the grid squares verified by NIST.
than one NIST analyst. If, after comparison of the analyses by the laboratory and NIST counters, more than two of the laboratory’s reported structures could not be located, the results were discarded and another grid square was verified. Only 11 structures could not be located (-2% of the total number of true positive structures). Blanks were checked periodically, and no contamination was noted. Several grid squares were more difficult to characterize than others. Most of the grid squares could be oriented in a manner similar to that used during counting by the laboratory and the square could be scanned in approximately the same direction. Therefore, the approximate relative order in which a structure was counted could be used as one of the criteria to match structures. However, several squares were counted along directions that were not parallel to the sides of the grid squares, and it was then very difficult to follow the same traverses covered by the laboratory. For these grid squares, the relative order in the count sequence could not be used as a matching criteria. In the majority of cases, the chrysotile structures that had been analyzed by the laboratory were sufficiently beam damaged that no SAED pattern could be obtained. Identification was possible, however, based on the EDXA spectra and the presence of a hollow tube and a fibrous morphology. A positive identification for chrysotile can be made based on these criteria because of our prior knowledge of the materials that had been deposited onto the filter. Identification and analysis were hindered in one case, however, by deposition of carbon contamination onto the chrysotile structures by the participating laboratory shown by the dark spots (arrowed) associated with the chrysotile fibers in Figure 2. A summary of results of verified counts for each analyst in the interlaboratory study is given in the histograms of Figure 3. The histogram of the values for TP/TNS, Figure
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Figure 4. Histograms of (a) the lengths of all true positives present in the grid squares on which verified counts were performed and (b) of the percentage of structures missed in each size class.
3a, has a range of values from 0.13 to 0.94 and a mean value of 0.67. The histogram of the values for FP/TNS, Figure 3b, has a mean value of 0.04. The mean value for TNS for the 25 verified grid squares was 20.5, compared to the mean value of 15.1 for the 82 counts reported by the laboratories. The distribution of all unique true positive structure lengths, shown in Figure 4a, has a median value of approximately 1pm and a mean value of approximately 1.3 pm. In Figure 4b, a bar chart of the percent of structures missed in each size class is given. The percentage missed decreases as the size class increases from 0.5 to 2.5 pm. There are far fewer structures in the size classes greater than 2.5 pm, and, therefore, a single miss will lead to an apparently large percentage in the histogram shown in Figure 4b. Approximately 44% of fibers less than 1pm in length were missed, whereas only 17% of the fibers longer than 1pm were missed. These values are comparable to those found by Steel and Small (3) for internal laboratory studies in which TEM operators missed more than 50% of the chrysotile structures less than 1pm in length and less than 10% of the structures longer in 1 pm. In the Steel and Small study, all structures less than 1 pm were considered countable, whereas for the present study, just those structures greater than or equal to 0.5 pm were considered countable. Only 20 structures were incorrectly identified as countable asbestos by the laboratories participating in the study. This is a relatively small number of false positive structures compared to the 553 countable structures in the grid squares analyzed. Only one non-chrysotile structure was misidentified as chrysotile. Six chrysotile structures below the 0.5-pm size limit were incorrectly reported as countable chrysotile. Thirteen structures were incorrectly reported as chrysotile due to a misunderstanding of the counting rules (1). In most of these cases, structures containing no fibers greater than 0.5 pm were incorrectly reported as countable structures. One-hundred sixty-seven structures found by NIST personnel were classified as false negatives for the participating laboratories. There are two broad categories of false negatives: (1)chrysotile structures that are observed but are incorrectly classified as noncountable asbestos and (2) those chrysotile structures that are not reported and presumably were missed entirely. Only 6 of the 167 false negative structures could be assigned to the first category. The reasons for these false negative structures include misidentification, incorrect size determination, and incorrect interpretation of countable asbestos as defined by the AHERA regulations (1). The remaining 161 structures fall into the second category of false negative structures-those that are missed entirely. This category contains by far the largest number of non-TP structures. The causes of these types of false negative structures are difficult to determine. Hypotheses are discussed in the following section.
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Flgure 5. Images of asbestos structures (arrowed) that were missed by the analysts in the interlaboratory study. Possible causes of the missed asbestos structure include (a) the presence of a sample preparation artifact that mimics a nearby chrysotile structure and (b) the presence of a large cluster of material near a chrysotile structure.
Flgwe 6. Diagrams illustrating how asbestos structures can be missed by incorrect procedures for traversing a grid square. (a) The beginning of a scan of a grid square containingasbestos structures. The dashed line denotes the path of the center of the field of the field of view on the microscope stage. (b) A correct scan of the square where the first structure is analyzed and the stage is translated back to the Original point of the traverse. (c) An incorrect traverse of a square where the first structure is analyzed, and then the stage is not translated back to the original position in the scan. (d) An incorrect traverse of a square where the first structure is analyzed, the stage is not translated back to the original position in the scan, and the operator continues the traverse in the wrong direction.
Discussion of False Negative Results. Several hypotheses can be proposed for the large number of structures that were missed by the analysts. One possibility is that features generated in the preparation of the grid replica obscure or mimic chrysotile structures. In Figure 5a, a chrysotile fiber missed by an analyst in the interlaboratory study is shown (indicated by the arrow). The fiber may have been missed due to its similarity to "highlighted pores" found on this area of the filter replica (2). A second possibility is that where a large cluster of material is present, the analyst may be distracted and may miss nearby chrysotile structures (Figure 5b). A third possibility may lie in the repeatability of translation of the microscope sample stage. Steel and Small (3)examined several microscopes and found that the stages varied widely in the degree of reproducibility of the traverses. The electron microscopes involved in this interlaboratory study were not evaluated by NIST personnel. A fourth possibility is that the grid squares are traversed too rapidly, causing operators to miss asbestos particles. A final hypothesis for the cause of structures being missed by the analysts relates to incorrect scanning or traversing procedures. For electron microscopy of most materials, TEM analysts select particles or areas of interest in a semirandom manner. For analysis of asbestos, however, systematic traverses of an entire grid square are required to determine the number of asbestos particles on a specific area of the filter replica. In Figure 6a, the beginning of a traverse of a grid square is shown. The dashed line indicates the path of the center of the field of view of the microscope screen. Correct scanning procedures are shown in Figure 6b where a structure is analyzed and the scan is continued so that the second structure will not be missed. Two types of incorrect scans are illustrated in Figure 6c where the operator does not translate back to the original position in the scan and in Figure 6d where the operator does not translate back to the original position
and, in addition, continues the scan in the wrong direction. The latter type of incorrect scan may lead to several structures in a row being missed entirely. In the interlaboratory study, it was quite common for several structures in a row to be missed. The two types of traversing problems illustrated in Figure 6c,d can be easily corrected if the analyst notes the direction of traverse and location of the structure relative to the center of the field of view prior to analyzing the structure. If the analyst then subsequently becomes confused as to the correct traverse direction, he can then backtrack to the last analyzed particle. In intralaboratory verified counts at NIST, we have found these types of scanning problems to be prevalent, especially for beginning analysts. After the problems have been recognized and corrected, the percentage of true positives found by analysts improves dramatically. Some of the problems that result in missed structures may be more likely to affect smaller structures than larger structures, thereby biasing the size distribution of the structures reported. For example, for replicas of 0.4-pm polycarbonate filters, there are many background features that are approximately 0.4-1 pm in size. These features will preferentially mimic or obscure structures that are in that size range, whereas the larger structures will be far more noticeable. Further, if the grid square is traversed rapidly, larger structures may be more noticeable than smaller structures. A final example involves incorrect traversing procedures. It is more likely that smaller structures will be entirely within the boundaries of the missed replica area and, therefore, not viewed by the TEM operator. The larger structures are more likely to occur in the area covered by two or more traverses of the grid square, and therefore, it is more likely that part of the structure would be observed by an analyst even if the replica is incorrectly scanned. These biases toward recognition of larger structures may explain the fact that structures longer than 1pm were more likely to be found than structures smaller than 1pm in
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length. A decrease in the number of false negative values is a likely result of using verified counting as a quality assurance (QA) method to check the accuracy of structure counting. Only one laboratory in this study was using verified counting as a QA method. This laboratory obtained TP/TNS values of 0.88, 0.90, and 0.92 for grid squares analyzed by three TEM operators. These values are significantly above the mean value of 0.64 obtained by the operators in the remaining laboratories. This result indicates that use of verified counting as a QA procedure will likely decrease the number of false negative values. A decrease in the number of false negative values may also result from the use of mixed cellulose ester filters rather than polycarbonate filters. The replicas of mixed cellulose filters do not have replicas of pores as a background feature and, therefore, if prepared well, provide an intrinsically cleaner background for counting than well-prepared replicas of polycarbonate filters. To check if this hypothesis is valid, we are presently conducting an interlaboratory study of structure counting by laboratories of asbestos deposited on mixed cellulose ester filters.
CONCLUSIONS Evaluation of the accuracy of the laboratory analyst is critical to evaluating the accuracy of the overall results obtained by laboratories involved in asbestos analysis. In this interlaboratory study of structure counting, analysts involved in asbestos work reported on average two-thirds of the actual countable asbestos structures on a grid square. The structures in the range of 0.5-1 pm in size were more than twice as likely to be missed than those greater than 1pm. Improved scanning and traversing techniques are likely to lead to improved values for the number of true positive/total number of structures. The number of false positives are comparatively fewer, and
they generally result from misapplication of the counting rules. The use of verified counting as a quality assurance method should decrease the number of false positive and false negative values obtained by analysts. In this study, the feasibility of interlaboratory verified counting has been demonstrated. By noting the orientation of the grid and the direction of traverses and by sketching the asbestos structures, analysts can be verified by operators from other laboratories.
ACKNOWLEDGMENT We very much appreciate the time and effort the participating laboratories voluntarily contributed to this study. We thank M. E. Beard and E. A. Dutrow of the USEPA for their support of this project. LITERATURE CITED Fed. Reg. 1987, 52(No. 210), 41826-41905. Turner, S.; Steel, E. B.; Landis, E. S. I n Specimen R e p r a t b n for TransmissionElectron Microscopy II; Anderson, R. M., Ed.; Materials Research Society: Philadelphb, PA, 1990; pp 157-166. Steel, E. 9.; Small, J. A. Anal. Chem. 1985, 57,209-213. Small, J. A.; Steel, E. B.; Sheridan, P. J. Anal. Chem. lg85, 57, 204-208. Montgomery County Asbestos Study, EPA Internal Report; US €PA: Research Triangle Park, NC, 1977. Chopra, K. S. J. Test. Eva/. 1978, 6 , 241-247. Campbell! W: J.; Huggins, S. W.; Wylie, A. G. Bureau of Mines Report of Investigations, R I 8452; US Department of the Interior: Washlngton, DC, 1980.
RECEIVED for review October 10, 1990. Accepted February 1, 1991. The information in this document has been funded wholly or in part by the United States Environmental Protection Agency under Interagency Agreement IAG No. DW13933643-01-0 to the National Institute of Standards and Technology. It has been subject to the Agency's peer and administrative review, and it has been approved for publication as an EPA document.
Direct Current Electrogenerated Chemiluminescent Microdetermination of Peroxydisulfate in Aqueous Solution Kazuo Yamashita,* Suzuko Yamazaki-Nishida, Yutaka Harima, and Akihiro Segawa Division of Material and L i f e Sciences, Faculty of Integrated Arts and Sciences, Hiroshima University, Naka-ku, Hiroshima 730, Japan Electrogenerated chemllumlnescent determination of low levels of SzOez- was studied In the aqueous [Ru( bpz),J2+/S,0,2- (bpr = 2,l'-blpyrarlne) system. Electrogenerated chemilumlnescence (ECL) was only observed when the potential applled to the worklng electrode was negative enough to cause reductlon of [Ru(bpz),r+. The ECL mechanlsm proposed was conslstent wlth the dependence of the ECL intensity on the concentrations of S,0e2- and [Ru(bpz),12+, pH, etc. Substances lnterferlng wlth the iumlnescent determlnation of S,Oez- were lnvestlgated. The ECL method based on the reductlve process of [Ru(bpr),l2+ Is hlghly specific for SzOez-, whlch can be determined In the range 10-@-10-3M when a rotating Pt dlsk electrode Is used as the worklng electrode.
INTRODUCTION Electrogenated chemiluminescence (ECL) of polypyridine transition-metal complexes such as [ R u ( b p ~ ) ~ (bpy ]~+ = 2,2'-bipyridine) has attracted interest from both theoretical 0003-2700/9 1/0363-0872$02.50/0
and practical points of view (1-10). ECL from [ R u ( b p ~ ) ~ ] ~ + has been used for the determination of oxalate or [ R u ( b p ~ ) ~ ] ~ + in the [ R ~ ( b p y ) ~ ] ~ + /and C ~the 0 ~ [~R- ~ ( b p y ) ~ ] ~ + /sysS~0~~tems (8,9). In the oxalate system, the electrolytic oxidation of [ R u ( b p ~ ) ~is] essential ~+ to produce ECL. In the peroxydisulfate system, the electrolytic reduction of [ R u ( b p ~ ) ~ ] ~ + is indispensable to it. Bard et al. have reported that ECL based on the reduction of [ R u ( b p ~ ) ~in] ~the + fully aqueous system is not observed because [ R ~ ( b p y ) ~is] +not produced or is unstable, or the excited species, [ R ~ ( b p y ) ~ ] ~is+rapidly *, quenched with Sz082-(6). For this reason, an acetonitrilewater (1:l)mixture has been used as the solvent for the luminescent determination of [ R u ( b p ~ ) ~ ]in~ +the [Ru(bpy)3]2+/Sz0,Z-system. The redox potentials for [ R u ( b p ~ ) ~are ] ~ shifted + by about 0.5 V toward more positive potentials compared with those for [ R u ( b p ~ ) ~(]2~, 11, + 12). This is an advantage in the luminescent determination in aqueous solutions because the interference due to the proton reduction that takes place a t negative potentials might be avoided. In a previous paper (13), a bright orange luminescence was observed for several hours 0 1991 American Chemical Society
