Anal. Chem. 1990, 62, 1531-1532
also affected by the time to immerse the membrane into the dodecyl-AO+solution. Thus the fluorescence intensity from the sensor depends on the preparation procedure of the plasticized PVC membrane. However, the ratio of the signal intensities for the sample and blank solutions was ascertained to be almost identical for several sensors constructed by the similar procedure. The signal intensity from the sensor gradually decreases with time, and it restricts the long time operation of the sensor. Due to a low output power of the laser, photobleaching of dodecyl-A0+ is negligible. As shown in Figure 3,dodecyl-A0+ is slightly soluble in water. Thus the signal decrease is considered to be due to leaching of dodecyl-A0+ from the plasticized PVC membrane. The more lipophilic acridine orange, 3,6-bis(dimethylamino)-l0-hexadecylacridinium bromide, was synthesized according to the procedure reported in ref 20,and this molecule was used as fluorogenic chromophore instead of dodecyl-AO+. The constructed sensor responded to the potassium ion selectively, and the signal intensity was stable at least for several hours. It implies that the lipophilic alkyl chain is essential for a long-term operation of the sensor.
Application to Other Plasticized PVC Membrane. There are many ionophores that extract only a specific cation into a membrane (21). Various ion-selective electrodes sensitive to specific cations have been developed by using the plasticized PVC membrane containing such ionophores. All these membranes might be modified to the optically sensitive membranes by immersing those into the solution containing dodecyl-A0+. The ion-selective electrodes using the plasticized PVC membranes are also useful for the measurement of anionic species (12). In a similar manner, optically sensitive membranes to specific anions might be obtained by incorporating the hydrophobic probes with a negative charge (16) in the membrane. However, such a probe currently used is highly soluble in water and leaches from the membrane. Thus it will be necessary to develop a fluorescent hydrophobic probe with a long alkyl chain. It is noted that high solubility of the chromogenic probe in the plasticized PVC membrane and presence of a charge in the probe are essential to obtain a fast and reversible response of the sensor.
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LITERATURE CITED Zhujun, 2.; Zhang. Y.; Wangbai, M.; Russell, R.; Shakhsher, A. M.; Grant, C. L.; Seitz, W. R.; Sundberg, D. C. Anal. Chem. 1888, 67, 202. Wolfbeis. 0.S.; Offenbacher, H. Sens. Actuators 1886,9 . 85. Kawabata, Y.; Tsuchida, K.; Imasaka, T.; Ishibashi, N. Anal. Sci. 1887,3 , 7. Zhujun, 2.; Seitz, W. R. Anal. Chem. 1888, 58, 220. Wolfbeis, 0.S.; Weis, L. J.; Leiner, M. J. P.; Zlegler, W. E. Anal. Chem. 1888,60, 2028. Zhujun, 2.; Seitz, W. R. Anal. Chlm. Acfa 1885, 777, 251. Kawabata, Y.; Tahara, R.; Irnasaka, T.; Ishibashi, N. Anal. Chlm. Acta l888*272, 287. Zhujun, 2.; Muliin, J. L.; Seitz, W. R. Anal. Chim. Acta 1888, 784, 251. Wolfbeis, 0.S.; Schaffar, B. P. H. Anal. Chlm. Acta 1887, 798, 1. Schaffar, B. P. H.; Wolfbeis. 0.S.; Leltner, A. Analyst 1988, 173, 693. Alder, J. F.; Ashworth, D. C.; Narayanaswamy. R.; Moss, R. E.; Sutherland, I. 0.Analyst 1887, 772, 1191. Covington, K. Ion -Selective flechode Methodology; CRC Press: Boca Raton, FL, 1979; Volume 1. Suzuki, K.; Tohda, K.; Tanda, Y.; Ohzora, H.; Nlshihama, S.; Inoue, H.; Shirai, T. Anal. Chem. 1889. 61, 382. Seiler, K.; Morf, W. E.; Rusterholz, B.; Simon, W. Anal. Sei. 1888,5 , 557. Pick, J.; Toth, K.; Pungor, E.; Vasak, M.; Simon, W. Anal. Chim. Acta 1873,64,477. Turner, D. C.; Brand, L. Blochemisby 1888, 7 , 3381. Eisenman, G. Membranes; Marcel Dekker: New York and Basel. 1975; Volume 2. Nishida, H.; Takada, N.; Yoshimura, M.; Sonoda, T.; Kobayashi. H. Bull. Chem. SOC.Jpn. 1884, 57, 2600. Waggoner. A. S. Annu. Rev. Biophys. Bioeng. 1878,8 , 47. Yamagushi, A.; Masui, T.; Watanabe, F. J . Phys. Chem. 1981, 85, 281. Dietrich, B. J . Chem. fduc. 1885, 62, 954. * Author to whom correspondence should be addressed.
Yuji Kawabata Ryuichi Tahara Toshito Kamichika Totaro Imasaka Nobuhiko Ishibashi* Faculty of Engineering Kyushu University Hakozaki, Fukuoka 812,Japan
RECEIVED for review January 17,1990.Accepted April 2,1990. This research was supported by Grant-in-Aid for Scientific Research from the Ministry of Education of Japan.
Multiway Analysis of Variance for the Interpretation of Interlaboratory Studies Sir: In a recent paper in this journal, Thompson (1) advocates the use of robust estimators to mitigate the effects of nonnormality in the statistical analysis of interlaboratory studies. To demonstrate these tools,he reanalyzes a large-scale study comparing two different catalysts for the Kjeldahl protein determination in feeds which was organized by Kane (2)in 1984. Thompson concludes that there is no difference between the two catalysts, just as Kane concluded, for most feeds, by a series of one-way analyses of variance after deleting outliers. The purpose of this communication is to point out that the same conclusion can also be reached, and presented in a concise form, by a single three-way analysis of variance (anova) before identifying outliers. It was, after all, to describe the insensitivity of the anova to mild departures from normality that the term “robust” was re-coined for circulation in the realm of statistics (3). Kane sent blind duplicates of 26 different feeds, which were grouped into 13 pairs (“Youden pairs”) of near-duplicates to 0003-2700/90/0382-1531$02.50/0
discourage data censoring, to each of 22 laboratories. He requested two Kjeldahl protein determinations on every sample, one using a mercury catalyst and one using a copper catalyst. In all, 2288 results, ranging from 10 to 90% crude protein, were reported to him. To carry out an anova on this body of data for present purposes, a linear model incorporating the main factors of the study, and all the interactions among them, was postulated Yijkl= M si + L j + Ck SLij + SCik + LCjk +
+
+
SLC,,
+ eijkl
where Yijklis the value of Ith replicate, using the kth catalyst (Ck),in the j t h laboratory (Lj),on the ith sample (Si). The term SLij is the interaction of the ith sample with the j t h laboratory, etc., eyklis the random error of measurement, and M is the mean of all values. The sample effects can be regarded as fiied and the laboratory effects, random; the catalyst effects are, of course, fixed. 0 1990 American Chemical Society
Anal. Chem. fS90, 62,1532-1536
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Table I. Three-way Anova of log-Transformed Data from Reference 2
source of variation mean samples catalysts laboratories samples X catalysts samples x laboratories catalysts x laboratories samples X catalysts x laboratories error total
sum of squares 27879.3 1054.5 0.00196 0.4747 0.0120
degrees of freedom 1
25 1 21 25
F mean square ratio 27879.3 42.2 0.00196 1.34 0.0226 43.6 0.000480 1.65
0.2996
525
0.000571
1.10
0.0307
21
0.00146
2.82
0.1526
525
0.000291
0.56
0.5931 28935.4
1144 2288
0.000518
protein determinations. The three-way anova of the logtransformed data provides no justification for doing this. The hypothesis that the adjusted treatment means for laboratories, Y..k.- Y...., constitute a sample from a normal population is easily accepted by the Shapiro-Wilk test (6). Removing laboratory treatments with the largest biases will result in serious underestimation of the variance of the laboratory effects and, hence, of the “reproducibility variance” of the analytical method (7). Thompson found that robust estimates of sample variances were noticeably greater than those computed by Kane and suggested that Kane’s outlier tests gave a significant proportion of type I errors. A more serious problem is that both Thompson and Kane sought to correct for discordance without making allowance for laboratory bias. When the linear model was collapsed to include only the main significant effects Yik= M + Si Lk eik and the AnscombeTukey test (8)for discordant residuals was applied with a 2.5% risk premium, 43 outliers were identified in the first cycle of iteration and one in the second. This compares with 117 outliers found by Kane visually from the scatter of points in a Youden plot (7). While so small a proportion of outliers should not overtax the robustness of the anova against errors in hypotheses testing, there can be a profound effect on the components of variance in terms of which the variability of an analytical method is expressed. Although the 44 outliers represent only 2% of the data, by their removal the error variance in Table I, estimated by the method of Healy and Westmacott (9), is approximately halved.
+ +
The power of the anova will be less if the data are inhomogeneous of variance, and Kane’s data are decidedly so: there is a clear dependence of the mean absolute difference between replicates on the protein level. Square-root and logarithmic transformations were tried to achieve homogeneity of variance, and the latter was found satisfactory by the Levine-median test, a robust and powerful test for homoscedasticity and itself an anova procedure ( 4 ) . Formulas for computing and comparing mean squares, with In (Y,jkJ as the random variable, were taken from Ostle and Mensing (5). The results are shown in Table I. The anova results prove that there is no overall catalyst effect; however, the F ratio for the catalyst-laboratory interactions would occur with a probability of less than 0.001, so it is likely that there are real differences between the two catalysts in particular laboratories. Also, the F ratio for the catalyst-sample interactions is marginally significant ( P = 0.025), so there is reason to doubt that both catalysts work equally well with all types of feeds studied. This is consistent with Kane’s finding of a significant method effect, at the 95% confidence level, with four of the feed samples. However, Thompson’s analysis of the data finds no matrix effects for particular materials. There is a marked laboratory effect owing to the critical nature of the Kjeldahl digestion parameters as noted by Kane who, prior to his own data analysis, discarded the results of three laboratories which were consistently high or low in their
LITERATURE CITED (1) (2) (3) (4) (5) (6) (7) (8) (9)
Thompson, Michael. Anal. Chem. 1989, 61, 1942-1945. Kane, Peter F. J. Assoc. Off. Anal. Chem. 1984, 6 7 , 869-877. Box, George E. P. Biomehka 1953, 40, 318-335. Brown, M. B.; Forsythe, A. B. J. Am. Statist. Assoc. 1974, 69, 364-367. Ostle, B.; Mensing, R. W. Statistics in Research; The Iowa State University Press: Ames, IA, 1975; Chapter 10. Shapiro, S.S.; Wilk. M. B. Biomehlka 1965, 52, 591-611. Youden, W. J.; Steiner, E. H. Statistical Manual of the AOAC; AOAC: Arlington, VA, 1975. Anscombe, F. J.; Tukey, J. W. Technometrics 1983, 5 , 141-160. Healy, M. J. R.; Westmacott, M. H. Appl. Stat. 1956, 5 , 203-206.
Neil E.Jones Michigan Department of Agriculture Laboratory Division East Lansing, Michigan 48823
RECEIVED for review November 27, 1989. Accepted March 29, 1990.
Background Emission from the Peroxyoxalate Chemiluminescence Reaction in the Absence of Fluorophors Sir: Peroxyoxalate chemiluminescence has been effectively used as a very sensitive postcolumn detector for HPLC in analyses of fluorescent analytes or fluorescent-labeled analytes including dansylated amino acids ( I ) , fluorescamine-labeled catecholamines ( 2 ) , 7-fluorobenzo-2-oxa-1,3-diazole-4sulfonate-labeled thiols (3), 4-fluoro-7-nitrobenzo-2-oxa-1,3diazole-labeled primary and secondary amines ( 4 ) ,PAHs (5), amino-substituted PAHs ( 6 ) ,and 3-aminofluoranthrene derivatives of aldehydes and ketones (7). The fluorescent species is chemically excited by a transfer of energy from an intermediate formed by the oxidation of
an oxalate derivative. Since not all fluorophors are efficient chemilumiphors, a selectivity advantage is possible in analytical applications. An additional analytical advantage is improved detection by elimination of source related problems including stray light and fluctuations in intensity. However, detection by chemical excitation is ultimately limited by background chemiluminescence observed without fluorophor present. When peroxyoxalate chemiluminescence detection is used as a postcolumn HPLC detector, the background emission is observed as a voltage offset above the photomultiplier tube
0003-2700/00/0362-1532$02,50/00 1990 American Chemical Society
