摘要:

ANALYST, JANUARY 1992, VOL. 117 39 Digestion of Soil Samples for the Determination of Trace Amounts of Lead by Differential-pulse Anodic Stripping Voltammetry Angelo Ransirimal Fernando and James Alan Plambeck" Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 An HN03-HC104 dissolution procedure followed by evaporation, re-dissolution and dilution with a CH3C02H-KN03 electrolyte is shown to be effective for the treatment of soil samples prior to the determination of trace amounts of lead by differential-pulse anodic stripping voltammetry. Keywords: Digestion procedure; lead determination; differential-pulse anodic stripping voltammetry; soil sample Differential-pulse anodic stripping voltammetry (DPASV) is now a commonly used technique for determining heavy metals such as lead at trace levels.However, recalcitrant real samples such as soils usually require spectroscopic methods for their analysis. In a previous paper,' some of the possible adverse effects of the reagents used for dissolution were examined. The present work applies the results of that study'.' to the determination of lead in actual soil samples. Experimental A local standard (LS) soil sample was collected from an urban backyard in Edmonton, Alberta, in an area known to have grown only grass and to have been unfertilized and undis- turbed for at least 10 years. Topsoil (exclusive of grass, vegetation and roots) was collected to the extent of 5 kg in polyethylene pails pre-cleaned with a 10% HN03 soak; soil that came into contact with the shovel was discarded.The black soil was oven-dried overnight at 30°C on aluminium foil. Initial sieving to remove stones and other foreign matter with a No. 10 stainless-steel sieve removed very little (<20 g) of the sample. The entire sample was then re-sieved, with grinding (CRC Micro Mill, The Chemical Rubber Co., Cleveland, OH, USA), to 25 mesh and stored in sealed glass jars. The results of the sieving (Canadian Standard Sieves, W. S . Tyler Co. of Canada, St. Catherines, Ontario, Canada) of the LS soil sample are given in Table 1. After mixing, sub-samples were withdrawn and re-sieved with grinding to 100 mesh and then to 200 mesh, then mixed by rotation. Sub-(sub-samples) (-20 g) were taken using a weighing bottle. After drying overnight at 100 "C and cooling in a desiccator, samples ( = I g) were removed for digestion and analysis. The closed weighing bottle prevented absorption of moisture (about 0.17% observed increase in mass if open bottles were used).In addition to the LS soil sample, soil certified reference materials (CRMs) SO-1 Regosolic Clay Soil, SO-2 Podzolic B Horizon Soil, SO-3 Calcareous C Horizon Soil and SO-4 Chernozemic A Horizon Soil [Canada Centre for Energy and Mineral Technology (CANMET). Canada] were used after drying according to the instructions.3.4 Open Beaker Digestions As HC104 digestion with a final evaporation step has been applied' to biological samples prior to analysis by DPASV, and as such a digestion procedure will result in only small amounts of organic residue, this procedure was considered to be the 'normal' method.The exact procedure used was that of the Canadian Society of Soil Science,h which has found widespread use.7.8 In this procedure, 1 g of soil ground to 300 mesh or below is digested successively in 20 ml of HN03, 20 ml of HC104 and 20 ml of HF followed by heating to near dryness, re-dissolution in 25 ml of 1 mol dm-3 HN03 and dilution to 50 ml. The procedure is designed for use with atomic absorption analysis, and was used with the 100 and 200 mesh soil samples without any modifications. Teflon Bomb Digestions The HN03 procedure for dissolution in Teflon-lined steel bombs (Parr 4745, Parr Instrument Co., Moline, IL, USA) described by Reddy et u1.9 was modified on the basis of other workI0.l1 to give the following procedure: weigh 0.3 g of sample into the Teflon cup, add 3 ml of concentrated HN03 and heat the mixture in an oven at 150 "C for 1.5-3 h.Cool with air (or in ice), transfer the solution into a 50 ml poly(propy1ene) calibrated flask and make up to volume with distilled water. This procedure did not dissolve the sample completely. Modification of the procedure by heating 0.2 g of sample at 150 "C for 3 h with 4 ml each of concentrated HN03 and HF produced much more complete dissolution. Although this procedure is a modification of that of Hsu and Locke,l' who used a mixture of HN03, HCI04 and HF, the use of HCI04 contrary to the manufacturer's recommendations'? was not considered safe. Perfluoroalkoxy( PFA)-Teflon bombs (Digestion Vessel 561, Savillex Corporation, Minnetonka, MN, USA) were also used for some microwave-assisted dissolutions.The procedure was the same as that used for the HN03-HF dissolution described above except that the 3 h of heating at 150 "C was replaced by 7 min of heating at 50% power (400 W) for eight of the bombs. The oven used was a standard household oven (Kenmore 88760, Sears Canada, Toronto, Canada) using a Table 1 Rcwlts of the sieving of SO g samples of LS soil Retention as a % of sample massY Sievc size (meshNo.) Run 1 Run2 Run3 Mean 40 18 17 17 17 60 19 19 I Y I9 80 11 I 1 1 1 1 1 100 S 5 5 s I40 7 7 8 7 170 5 S 5 5 200 4 4 4 4 325 10 10 9 1 0 (-325) 21 23 22 22 Bottom plate Total: 100 100 101 100.33 *: Rounded off to the nearest whole number. * To whom correspondence should be addressed.40 ANALYST, JANUARY 1992, VOL. 117 Table 2 DPASV data for the 100 mesh open-beaker digcsted LS soil samples Sample No.1 2 4 5 6 7 9 10 11 13 14 15 17 19 Lead found 29.7 28.0 38.0 33.5 38.0 29.8 35.5 37.8 32.9 23.7 23.1 28.7 30.0 30.1 (PPm) Mean: 31.4 Standard deviation: 4.9 Standard Relative deviation standard 1.8 6 1.1 4 3.3 9 2.6 8 4.4 11 0.5 2 3.3 9 3.2 8 3.2 10 2.0 8 1.5 7 1.9 7 0.92 3 0.96 3 (ppm) deviation (%) Table 3 DPASV data for the open-beaker digested 200 mesh LS soil samples Lead Standard Relative found deviation standard Sample No. (ppm) (ppm) deviation (YO) 1 31 .5 1.7 5 2 27.3 0.67 2 4 25.3 0.61 2 5 28.0 2.5 9 6 27.0 2.3 9 Mean: 27.8 Standard deviation: 2.3 Table 4 DPASV data for open-beaker digested samples of soil CRMs SO-1 and SO-2 Lead Standard Relative Soil Sample found deviation standard CRM No.(ppm) (ppm) deviation (%) SO- 1 * 1 19.7 0.73 4 2 18.9 0.91 5 3 22.9 2.06 9 so-2* 1 23.1 1.6 7 2 24.1 1.6 7 3 21.0 0.65 3 * Certified lead content for SO-1 and SO-2 = 21 k 4 ppm. standard household microwave turntable. In order to prevent leakage of acid fumes, the bombs were placed in a plastic container with a snap-fit lid, which prevented loss of fumes without the danger of explosion inherent in the use of a tightly sealed small container. Reagents, Apparatus and Other Procedures The reagents and the procedures used for their purification were as described previously.'.' All of the reagents were of analytical-reagent grade and were used as received, except as follows. Water was re-distilled from alkaline permanganate at very slow rates (0.5 1 h-1) by using an insulated vertical open column.Aqueous stock solutions of 2 mol dm-3 KN03 and of acetate buffer (1 mol dm-3 in both CH3COONa and CH3COOH) were further purified by using bulk electrolysis in a large-volume cell with a mercury cathode and a large-volume calomel anode separated from the reduction compartment by glass frits. The potential of the mercury pool was held at - 1.4 V against the calomel electrode for one month while the solution was slowly stirred for bubbling with de-oxygenated Table 5 Dissolution data for HN03 digestions in Teflon-lined steel bombs Lead Standard Sample Residue Dissolved found deviation Sample mass/g mass/g (YO) (ppm) (ppm) M200-01 0.3041 0.2109 30.7 27.5 2.3 M200-02 0.3448 0.2429 29.6 28.9 2.9 Average values: 30.1 28.19 Table 6 DPASV data for the 200 mesh microwave-digested soil samples Sample No.1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 Lead found 22.2 20.7 22.0 18.2 20.1 15.8 23.3 19.5 14.6 20.3 18.6 15.4 15.1 14.9 18.5 (PPm) Mean: 18.6 Standard deviation: 2.9 Standard deviation 2.1 1 .o 1.1 1.4 0.9 1.8 1.9 1 .0 1.3 1.8 1.5 1 .0 0.5 4.9 1.4 (PPm) Re 1 at i ve standard deviation (YO) 10 5 5 8 4 11 8 5 9 9 10 6 4 33 7 and water-saturated nitrogen. High-purity nitrogen was further de-oxygenated using acidified vanadium(i1) chloride solution and zinc amalgam bubblers followed by aqueous washing towers. Potassium chloride was further purified by recrystallization and crystal adsorption. Triply distilled mercury was purified by anodic oxidation at +0.3 V against a saturated calomel electrode under a continuous stream of filtered laboratory air obtained by applying a mild vacuum to the cell.Positioning of the air inlet below the mercury surface also allowed the air to agitate the mercury. Acetic acid was further purified by isopiestic distillation. The apparatus used for analysis was as described previ- ously.1 The DPASV parameters1 (Model 174A Polarographic Analyzer, Princeton Applied Research, Princeton, NJ, USA) were: deposition potential, -0.9 V; modulation, 25 mV; clock, 1 s; scan rate, 5 mV s-1; deposition time, 90 s; and equilibration time, 30 s. Results Complete dissolution of the samples was achieved in the normal open-beaker digestion method, as expected, if the samples were heated to near dryness and then re-dissolved.This procedure also eliminates the residual HF and HCI04. The results of the analyses of the LS soil sample and of soil CRMs SO-1 and SO-2, obtained using this dissolution method, are given in Tables 2-4. The use of Teflon pressure bombs with HN03 alone dissolved only about 30% of the sample mass for 200 mesh LS soil samples, but apparently all of the lead (Table 5). Quantitative sample dissolution could be achieved by using HF together with HNO,; this resulted in a more yellowish solution . The same HF-HN03 mixture was also used with microwave heating. Dissolution of the sample was again essentially complete (>98.5%). However, a lead blank of 3.5 ppb together with significantly lower observed lead levels ('TableANALYST, JANUARY 1992, VOL.117 i7j 41 (c) 6) casts doubt on the validity of this protocol. Moreover, both of the HF-HN03 protocols produced solutions which, even when diluted, were reactive towards mercury, resulting in film formation on the mercury surface and plugging of the hanging mercury drop electrode capillary. These effects, which were presumably caused by HF, could be eliminated by conven- tional open-beaker heating of the microwave-digested solu- tions, but only with heating times comparable to those for open-beaker digestions. (Attempts to eliminate these effects by the addition of analytical-reagent grade H3B03 gave significantly higher lead blanks.) Discussion All of the acid digestion methods used required re-dissolution in a highly acidic medium after heating to near dryness.Attempts to re-dissolve the residue quantitatively in 0.1 rnol dm-3 HN03 resulted only in incomplete dissolution even after boiling for 1 h, whereas re-dissolution in 1.0 rnol dm-3 HN03 was rapid and apparently complete. Although the high acidity might not be a problem in atomic spectrometric analysis,lI.l~-1s it reduces the potential window available in DPASV to the point where analysis might become impossible (amplification does not help because the noise caused by the onset of hydrogen evolution is also amplified). Storage of the solutions in a concentrated and acidified form is better, a) J I I J -1.0 -0.5 0.0 +0.5 -1.0 -0.5 0.0 +0.5 PotentialN Fig. 1 Effect of dilution. The interfering signal observed at about -0.7 V in solutions prepared by diluting 1 ml of digested solution with 5 ml of water and sufficient 1 rnol dm-3 NaOH to adjust the pH to 2 [ ( u ) and ( h ) ] disappears completely on reduction of the volume of digested solution added to 0.1 ml [(c) and ( 4 1 .Deposition time was I min for ( a ) and ( b ) and 2 min for (c) and (d). For (c) and (d). current scnsitivity was also increased and no additional pH adjustment was made however, because adsorption on the container walls is then less of a problem. Three possible solutions to the problem of the analysis of very acidic solutions by DPASV are: partial neutralization (e.g., with NaOH); dilution with water; and dilution with a constant weak acid electrolyte. Partial neutralization was not successful. Attempts to dilute 1 ml of the digested solution to 5 ml followed by adjustment of the pH to 2 with NaOH required different amounts of NaOH for repeated samples.As a consequence, an aliquot was taken and titrated with NaOH to determine the amount required and this amount was added to a second aliquot. The volume of NaOH solution required was 600-1000 pl of 1 mol dm-3 NaOH. In the course of the titrations it became clear that a yellow coloration developed at a pH value above 2.7 followed by precipitation of Fe(OH)3 as the pH increased further (the LS soil sample contained 1.25 mmol of Fe per gram of soil as measured by atomic absorption spectrometry). Even when the pH was not allowed to rise above 2 and the solution was apparently clear, DPASV of solutions to which NaOH had been added showed, in addition to the clear and well-defined lead peak, an abnormal and ill-defined peak near -0.7 V (Fig.1). This peak prevented accurate measurement of the baseline of the lead peak. It is probable that the interference is caused by iron, as it disappears on precipitation of Fe(OH)3 at higher pH, but the lead is then removed also. Addition of ascorbic acid or hydroxylamine hydrochloride was not effective, Dilution of the solution, either with water or constant weak acid electrolyte, did remove the interfering peak, as would be expected if this peak were caused by interference from another matrix component, which was not reduced to a form that was soluble in mercury. Therefore, the dilution of the stripping solution with a comparable increase in deposition time results in a satisfactory voltammogram.Dilution of the dissolved sample has been suggested'.9 and is indeed effective in reducing matrix effects. Excellent baseline stability was obtained in repetitive runs on a single solution produced by diluting 100 pl of the digested sample to 5 ml with water. However, the pH of the solution (about 2) depended markedly on the dissolution method and on the extent to which the sample was evaporated prior to re- dissolution. The difference in the results of the analysis of the LS soil sample after different dissolution procedures had been applied was so large as to be unacceptable even though the precision of repetitive runs on a single dissolution was excellent. Dilution of the dissolved sample with a constant weak acid electrolyte makes the procedure less dependent on the amount of acid remaining from the dissolution stcp and is therefore preferable. Chloroacetic acid buffers produced unacceptably high background currents, viz., more negative than -0.8 V; however, solutions of CH3C02H at pH 3 were found to give excellent results if sufficient supporting elec- trolyte (KNOT) was present. Accordingly, the procedure chosen was dilution of 100 pl of the highly acidic dissolved sample with 5.00 ml of CH3C02H + K N 0 3 diluent (0.10 + 0.10 rnol dm-3).De-aeration of the diluent overnight in situ (a 500 ml repipette bottle) reduced the de-aeration time in each analysis from 5 to 1 min with no adverse effects. The dispenser was precise to 0.1%. (Acetic acid alone is not a buffer at pH 3. However, 0.1 mol dm-3 CH3C02H provides considerably greater protection against loss of protons during DPASV than does approximately 0.001 rnol dm-3 HN03.Acetic acid can also be easily purified.') The final procedure involved dilution with CH3C02H and standard additions of two 10 ~1 aliquots of a 5 ppm standard lead solution for each analysis. Four voltammograms were recorded for each of the three solutions. The mean values and standard deviations were calculated by computer programs.? The baseline was taken as tangential to the curve before and after the lead peak. Hsu and Lockel? have suggested that the colour of the42 ANALYST, JANUARY 1992, VOL. 117 digested solution might be caused by incomplete oxidation of organic matter. I n the present work it was found that the colour of the final solution was dependent on the extent of evaporation to dryness.Evaporation of the solution until the first signs of a solid were observed give a brown solid that on dissolution yielded a yellow solution. Further evaporation left a light-yellow solid residue which produced a nearly colourless solution on dissolution. The ultraviolet-visible spectrum of the yellow solution described above showed peaks near 300 and 260 nm, similar to those reported1619 for aqueous solutions of iron(il1) salts. The spectrum is very similar to that of 1.0 mmol dm-3 Fe2(S04)3 in 0.025 rnol dm-3 KCl, 0.02 mol dm-3 K2C2O4 and 0.1 rnol dm-3 ethylenediaminetetraacetic acid (EDTA) or KCI recorded in this work. The colourless digested solution turned yellow on addition of KCI, K2C204 or EDTA, whereas the yellow colour of the coloured solution was extracted into isobutyl methyl ketone, suggesting that chloro complexes of iron are the source of the yellow colour.Chloride might be present in the original sample or from HC104, whereas oxalate might be produced by the oxidation of organic matter by HCI04,Z0 as with the oxidation of soils by peroxide .2 * .22 The lead content of the LS soil sample and of the soil CRMs SO-1 and SO-2 is given in Tables 2-4. The over-all results for the LS soil sample are 31.4 +_ 4.9 pprn of lead (100 mesh sample) and 27.8 k 2.3 pprn of lead (200 mesh sample), whereas the values for SO-1 and SO-2 are well within the range given for their certified lead content, 21 5 4 ppm. Conclusion The DPASV method developed previously' for the analysis of synthetic samples can be used successfully for the determina- tion of total lead in soil samples following dissolution of the soil samples in HN03-HC104, evaporation, re-dissolution in 1.0 rnol dm-3 HN03 and dilution with 0.1 rnol dm-3 CH3COZH-KNO3.The authors are grateful to Dr. Byron Kratochvil and Dr. Gary Horlick for the loan of the different types of Teflon bomb. This research was supported by the Department of Chemistry of the University of Alberta. Taken in part from the thesis of A. R. F. submitted to the Faculty of Graduate Studies and Research in partial fulfilmcnt of the requirements for the degree of Ph.D. 1 2 3 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 References Fcrnando, A. R.. and Plambeck. J . A., Anal.Chem.. 1989.61, 2609. Fernando, A. R.. Ph.D. Thesis. University of Alberta, 1988. Bowman. W. S . , Faye, G. H . , Sutarno, R.. McKcaguc. J . A.. and Kodama. H., CANMET Report 79-3, Soil Sumples SO-I, SO-2, SO-3 und SO-4-Certified Reference Materials, Energy Mines and Rcsourccs Canada, Ottawa, 1979. Steger, H. F., CANMET Report 80-6E, Certified Reference Materials, Energy Mines and Resources Canada, Ottawa, 1980. Oehmc. M., and Lund, W., Fresenius Z. Anal. Cliem., 1979, 298, 260. Munuul on Soil Sumpling unti Methods of Anulysis. cd. McKcague, J. A , , Canadian Society of Soil Science, 2nd cdn., Ottawa, 1978. McKeague, J . A.. Desjardins. J . G., and Wolynctz, M. S . , Minor Elements in Cunudiun Soils. Agriculture Canada. Research Branch, Ottawa, 1979. Dudas. M. J . , and Pawluk, S . . Can. J . Soil Sci.. 1980, 60, 763. Reddy, S. J . , Valcnta, P . , and Nurnbcrg, H. W.. FreseniuA Z . Anal. Chem., 1982, 313. 390. Stoeppler, M.. and Backhaus, F., Fresenius Z. Anal. Cliem.. 1978, 291, 116. Stoepplcr. M., Muller. K . P., and Backhaus. F., Fresenius Z. Anal. Cliern., 1979, 297, 107. Hsu, C.-G., and Lockc, D. C., Anal. Chim. A m , 1983, 153, 313. Microwave Acid Digestion Bombs, Bulletin 4780, Parr Instru- ment Co., Moline, IL, 1986. McLarcn, J . W., Berman, S. S . , Boyko. V. J.. and Russcll, D. S . , Anal. Chem., 1981, 53, 1802. McQuaker. N . R.. Brown, D. F.. and Kluckncr. P. D.. Anal. Cliem., 1979, 51, 1082. Bastian. R., Wcbcrling, R., and Palilla, F., Anal. Cliem.. 1953, 25, 284. Ferguson, R. C., and Banks. C. V.. Anal. Chem., 1951.23,448. Medalia, A . I . . and Byrne. B. J.. Anal. Chem., 1951, 23. 453. Buck. R. P.. Singhadeja, S.. and Rogers. L. B., Anal. Ciiem., 1954. 26, 120. Smith, G. F.. The Wet Chemical Oxidution of Orgunic Composi- tions Employing Perchloric Acid, The G. F. Smith Chcmical Co., Columbus, OH, 1965. Martin. R. T., SoilSci., 1954. 77, 143. Farmcr, V. C., and Mitchcll. B. D., Soil Sci., 1963, 96. 221. Paper 1/01 9921 Receiiied April 29, 1991 Accepted August 8, 1991

ISSN:0003-2654    

DOI:10.1039/AN9921700039

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST. JANUARY 1992, VOL. 117 43 Determination of Selenium(iv) by Anodic Stripping Voltammetry Using Extraction With Pentyl Alcohol Hasan Aydin Department of Chemistry, Gazi Universitesi, Fen-Edebiyat Fakultesi, 06500 Ankara, Turkey Abdul Hamid Yahaya Department of Chemistry, University of Malaya, 59100 Kuala Lumpur, Malaysia A method is described for the specific and accurate determination of Se in samples by anodic stripping voltammetry following separation of the Se with pentyl alcohol. The separation procedure removes Cull ions with a single extraction. After the extraction, the acidic solution can be used for the determination of Cull because most of these ions remain in the solution. The height of the anodic stripping peak and the standard additions method were used as a quantitative measurement.Following the extraction, 1 x 10-9 mot dm-3 Se can be determined; the relative standard deviation is 1 % ( n = 4). Keywords : Selenium determination; pent yl alcoh o I; anodic stripping voltam m etr y Selenium can be determined by atomic absorption spec- trometry,' polarography,' cathodic stripping voltammetry,3 anodic stripping voltammetry,J-h titrimetry7 and spec- trometry.8 Although there are many methods for the determi- nation of Se, its determination by anodic stripping voltam- metry is still of interest because of the sensitivity and simplicity of the procedures. Vadja" stripped Se from a mercury electrode in acidic solution and was able to determine down to 8 ppb of Se'". Andrews and Johnson l o significantly extended the sensitivity of the method by electrodeposition of Se'" and stripping from a rotating gold disc electrode.In both of these methods, metal ions such as Cu" and Cd" can interfere. Hamilton et af.h reported a procedure for the determination of Se and Te in electrolytic Cu using anodic stripping voltammetry at a gold film electrode. In this method, Se and Te were separated from Cu by passing an ammoniacal solution of the sample through Chelex-100 resin. Adeloju et al." developed a method for the stripping voltammetric determination of Se in biological materials by direct calibration. They used the stripping voltammetric technique following ion-exchange separation of Se from the bulk of the acid digested matrix. Aydin and SomerS studied the anodic stripping voltam- metric determination of Se in the presence of Cu" ions and demonstrated that 2 x 10-8 mol dm--i Se could be determined in the presence of Cull at concentrations of Cu" up to 10% of that of Se with a relative standard deviation of 3.7% ( n = 6).I t was shown that when the amount of Se lay in the range 1.8 < Y < 11, where Y is the concentration ratio of Cu to Se, Se could be determined without elimination of Cu from the solution. When the concentration ratio was less than 1.8, the peak of the intermetallic Cu-Se compound did not increase with the addition of Se because of the absence of free Cu. At a ratio of Cu to Se greater than 11, the Cu peak overlapped the peak of the intermetallic compound. This paper describes a simple voltammetric method for the determination of Se in the presence of Cu" ions based on the use of a combination of voltammetric techniques and extrac- tion with pentyl alcohol.The proposed method permits the determination of Se in the presence of Cull ions at low and trace levels. Experimental Apparatus The apparatus and plating of the graphite wax test electrode with mercury have been described previously. l 2 Reagents All reagents were of analytical-reagent grade. Triply distilled water was used for preparation of all solutions and at all other stages of analysis. Stock solutions of 0.16 mol dm-3 HgCI?, 1 X lo-? mol dm-3 Se03'- and 1 x 10-1 mol dm-3 Cull were prepared by dissolving the appropriate amounts of the salts in water. These solutions were further diluted to the appropriate concentrations.Procedure The sample solution was prepared by mixing Sel" and Cull solutions containing various concentrations of HN03. It was then mixed with pentyl alcohol and the mixture allowed to stand so that the two phases could separate. Solutions of NaOH of various concentrations were added to the organic phase. After shaking, the mixture was set aside for 1 d to allow the two phases to separate. In order to determine the amount of Se and Cu, 70 PI of concentrated HC104 were added to 100 PI of the aqueous phase. The solution was heated to boiling for about 1-2 min and 5 ml of triply distilled water were added. Nitrogen was passed through the solution to effect de-oxygen- ation prior to plating of Sel" and Cu" ions at potentials between -0.10 and -0.30 V. After a short rest period (30-40 s) a potential sweep of 60 mV s-I was applied in the positive direction and the current was recorded.The standard addi- tions were made by using 1 x 10-5 or 1 x 10-6 mol dm-3 Se03'- or Cu" solutions. The same procedure was used for preparation of the blank solutions, without Se and Cu. Results and Discussion Optimization of Experimental Conditions Several experiments were performed in order to determine the optimum conditions following extraction with pentyl alcohol. The best results were obtained at pH 1-2. Therefore, the pH of the plating solution was adjusted to the appropriate value by using HC104. The sensitivity of the method was affected by the deposition potential, Ed, the deposition time, the scan rate and the thickness of the mercury film.The peak height of Se increased with an increase in the negative deposition potential up to a limiting value of -0.350 V. Hence, a potential between -0.10 and -0.30 V was used and the electrode was again plated to the appropriate thickness as described under Procedure.44 ANALYST, JANUARY 1992, VOL. 117 Interference of Cu The specific and accurate determination of Se in various samples by stripping voltammetry requires the separation of this element from interferents.11.13-16 Copper is the ion that interferes most seriously among the metal ions because its deposition potential is close to that of Se'". The procedure developed in this work is designed to prevent the interference of Cu. The procedure can also be used to eliminate other interferents, for example Pb and Cd, and also those having deposition potentials close to that of Se or fairly small solubility products with Se'" ions.As shown in Fig. 1, a broad peak was observed at about 0 V. After the application of the procedure, a peak was only observed at about 0.06 V. The height of this peak increased on addition of S e 0 3 2 - or Cu" ions. After the addition of a solution of Cu", the peak potential was shifted towards a more n Ix -0.2 0.0 0.2 EdN versus SCE Fig. 1 Interference of Cu on the determination of Se. A. Sample solution (100 pl) (23 O00 ppb of Cu and 707 ppb of Se) + HCIOl (70 pl) + triply distilled water (5 ml); B. sample solution (100 pl), taken after extraction with pentyl aleohol, + HCIOl (70 pl) + triply distilled water (5 ml); and C, as for B + 707 ppb of H2Se03 (100 PI).Measurement of the peak height is indicated by x Ix -0.2 0.0 0.2 EdN versus SCE Fig. 2 Effect of Cu as an interferent on the determination of Se. A, Sample solution (100 pl) (2300 pb of Cu and 707 ppb of Se) + HCI04 (70 pl) + triply distilled water 8 ml); and B, sam le solution (100 pl), taken after extraction with pentyl alcohol, + H&O4 (70 pl) + triply distilled water ( 5 ml). Measurement of the peak height is indicated by x positive value. This showed that the ratio of the concentration of Cu to Se in the medium was less than 1.8. When synthetic sample solutions (3-6 in Table 4) were used directly, either two peaks or a single peak were observed for Cu and Cu-Se, respectively. However, after the extraction of Sel", the peaks for Cu and Cu-Se were not observed (Fig.2). Extraction of Se The results in Tables 1-3 show the appropriate concentrations of HN03 and NaOH that are necessary for the successful determination of Se in samples containing Cull ions. When the NaOH concentration is less than 1 rnol dm-3, the amount of Se extracted decreases and the Cu peak is not observed. It was Table 1 Determination of Se in NaOH solution (90% confidence interval was used) Se found in NaOH Synthetic HN03/ NaOH/ Se added to solution* 1 1 3 707 7 538k 10 1 1 3 707 6 543+2 2 1 1 707 7 706+ 1 7 706+ 1 2 1 1 707 3 0.1 1 707 5 698k5 3 0.1 1 707 7 707k 1 4 0.1 0.1 707 6 21 k 6 4 0.1 0.1 707 6 21 + 2 sample mol dm-3 mol dm-3 acid (ppb) n (PPb) * k standard deviation. Table 2 Determination of Cu in NaOH solution (9070 confidence interval was used) Cu found Cu added in NaOH solution* sample mol dm-3 mol dm-3 (ppm) n (10-3ppm) 1 1 3 23 5 115f20 2 1 1 23 6 13 _+ 3 3 0.1 1 23 7 1 3 f 1 4 0.1 0.1 23 5 Nopeak Synthetic HN03/ NaOW to acid * k standard deviation.Table 3 Determination of Se in acidic solution after extraction with pentyl alcohol (90% confidence interval was used) Synthetic HN03/ NaOH/ Se added to Se found in sample mol dm-3 mol dm-3 acid (ppb) n acid* (ppb) 1 1 3 707 8 114 k 5 1 1 3 707 8 1 1 2 f 3 2 1 1 707 6 38 k 4 2 1 1 707 7 3 3 5 8 3 0.1 1 707 5 66 k 7 3 0.1 1 707 5 66 k 2 4 0.1 0.1 707 6 92 k 12 4 0.1 0.1 707 5 87 f 6 * k standard deviation. Table 4 Application of the method to synthetic samples containing Se'" and Cu" ions (90% Confidence interval was used) Concentration of ions in 0.1 mol dm-3 HN03 Synthetic sample Cu (ppb) Se (ppb) 1 23 000 707 2 23 000 707 3 2 300 707 4 2 300 707 S 690 707 6 690 707 * f standard deviation.Se extracted" n (PPb) 6 699 k 7 6 693 k 4 6 693 k 5 6 699 k 3 6 693 k 3 6 701 kSANALYST, JANUARY 1992, VOL. 117 4s found that the NaOH concentration had to be equal to or greater than 1 mol dm-3 for the best results. Pentyl alcohol was selected for the extraction because it extracted Se selectively under the given conditions and was immiscible with water. The results in Table 3 show that the extraction procedure permits almost complete recovery of Se in the presence of Cu. The results in Tables 1 and 2 show that HN03 and NaOH concentrations of 0.1 and 1 mol dm-3, respectively, are necessary for the success of the method.Above 5.5 mol dm-3 HN03, pentyl alcohol did not separate from water. In order to determine the sensitivity of the method, 23 ppm of Cu were used and the results are shown in Table 2. The results in Tables 2 and 4 demonstrate that the extraction procedure was successful in preventing the interfer- ence of Cu. As shown in Tables 1 and 3, Se could be extracted from acidic solution. The reaction occurring in acidic media is: SeO? H3SeO3 or + 2ROH $ (RO)?SeO + H20 (R = alkyl) I t was found that this reaction was reversible and depended on the pH of the medium. For this reason, the concentration of NaOH must be greater than 0.1 mol dm-3 for the quantitative extraction of Se. Conclusion Anodic stripping voltammetry has been shown to be a sensitive and accurate method for the determination of Se.The minimum recovery of Se is 76%. The method is reproducible with a relative standard deviation of 1% for the determination of Se. 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 1s 16 References Sturgeon. R. E., Willie. S. N . , and Berman. S. S.. Anal. Chcm.. 1985.57.6. Hasdcmir. E., and Somcr. G . , Analyst, 1990. 115. 297. Dennis, B . L . , Moyers. J . L . , and Wilson. G . S . . Anal. Cliem.. 1976.48, 161 1. Poscy, R . S . , and Andrews, R. W., Anal. Chim. Actu, 1981, 124. 107. Aydin, H., and Somcr, G.. Anul. Sci., 1989, 5 , 89. Hamilton, T. W.. Ellis. J . , and Florence, T. M.. Anul. Clrim. Acru, 1979. 110. 87. Aydin. H.. and Somcr, G.. Tulunra, 1989, 36, 723. Bodini. M. E . , Pardo, J., and Arancibia. V . . Tuluntu, 1990,37, 439. Vadja, F., Actu Chim. Acud. Sci. Hung., 1970, 63, 257. Andrcws, R. W.. and Johnson. D. C.. And. Cliem.. 1975, 47. 294. Adeloju, S. B., Bond, A. M., Briggs, M. H.. and Hughes, H. C., Anul. Cliem.. 1983, 55. 2076. Somer, G . , and Aydin, H., Analyst. 1985. 110. 631. Howard, A. G., Gray, M. R., Waters, A. J . . and Orcmichic. A. R.. Anal. Chim. Actu. 1980, 118, 87. Forbes, S . , Bound, G. P., and West. T., Tuluntu, 1979,26.473. Andrews, R . W., and Johnson, D. C., Anal. Chem., 1976,48, 1056. Rasmussenh. L., And. Clzirn. Acru, 1981, 125, 117. Paper 1l01519B Received April 2, 1991 Accepted August 12, 1991

ISSN:0003-2654    

DOI:10.1039/AN9921700043

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST, JANUARY 1992, VOL. 117 47 Flow Injection Amperometric Determination of Cyanide on a Modified Silver Electrode SneZana D. Nikolic and Emil 6. Milosavljevic Faculty of Chemistry, University of Belgrade, P.O. Box 550, I1001 Belgrade, Yugoslavia James L. Hendrix and John H. Nelson Departments of Chemistry and Chemical and Metallurgical Engineering, Macka y School of Mines, University of Nevada, Reno, NV 89557, USA A composite coating based on a mixture of phosphatidylcholine, cholesterol and stearic acid was used for fabricating a silver-based amperometric sensor. The sensor was used in a flow injection (FI) configuration for the selective and sensitive determination of cyanide. The method is based on the permselectivity of the lipid membrane, which rejects from the surface of the working silver electrode the undesired, potentially interfering species, while allowing the transport of the analyte as hydrogen cyanide. Logarithmic calibration graphs were linear up t o the maximum concentration of CN- investigated (0.100 mmol dm-3).The precision of the technique was better than a relative standard deviation of 3.5% at the 0.5 Vmol dm-3 level and better than 2% at the 0.1 mmol dm-3 level, with a throughput of 30 samples h-1. Under optimum conditions, the detection limit was 0.1 pmol dm-3 (0.26 ng CN-). The effects of working potential, type of electrode coating, acidity of the reagent stream and interferents on the FI signals were studied. Keywords: Flow injection; amperometric detection; modified silver electrode; cyanide determination Amperometric detection for flowing streams based on a silver working electrode is inherently sensitive.However, it is also non-selective, as any species that forms either an insoluble silver salt or stable complex with Ag+ would necessarily interfere. Hence, in order to solve a particular analytical problem, such detection has to be coupled with a suitable separation step. Rocklin and Johnson' developed a method for the determination of cyanide, sulfide, iodide and bromide utilizing ion chromatography and electrochemical detection via a silver working electrode. More recently, the same group2 developed an automated system for the determination of cyanide in water samples. Two in-series separation steps were employed. The analyte (as HCN) is separated from most interferents using gas diffusion and then from the remaining interferents, such as sulfide, by ion-exchange chromatography and is detected by an amperometric detector with a silver working electrode.Flow injection (FI) methods based on the same detection principle have also been successfully used for cyanide3-5 and sulfide determination.6 However, either continuous distillation3 or gas diffusion44 pre-separation steps had to be employed. On the other hand, lipid membranes, based on stearic or palmitic acid or phospholipids, have previously been used for modifying carbon electrode~7-~~) to be employed under static conditions. As was pointed out recently by Wang and Lu," a major obstacle to the development of lipid-based flow detectors had been the poor mechanical stability of the lipid layer.However, they developed a highly stable composite phospholipid-cholesterol electrode coating for the glassy carbon electrode for amperometric monitoring of hydrophobic substances in flowing streams. Under the FI regime, they employed the developed amperometric sensor for the determination of hydrophobic drugs (e.g., promethazine and trimipramine) while nearly eliminating interferences from ascorbic acid and tyrosine. The same group" described an interesting and innovative approach for simultaneous analyses using FI. They used a sensor array of several glassy carbon amperometric electrodes, each coated with a different permselective film, one of which was a phospholipid. Successful multicomponent analyses of neurologically significant catechol compounds were performed using pattern recognition.The technique is based on the partial but different selectivity of each sensor. This paper describes a novel approach for the selective and sensitive FI determination of CN-. An in situ separation step is incorporated in a relatively simple FI manifold by modifying the silver electrode with a phosphatidylcholine-cholesterol- stearic acid coating. The lipid composite membrane acts as a barrier for ionic species while retaining permittivity for the hydrophobic HCN. This work also demonstrates the possibil- ity of modifying electrodes that participate directly in the amperometric detection (anodic dissolution of silver in the presence of the analyte), as opposed to those which are only a medium for the electron transfer (glassy carbon or platinum electrodes).Experimental Reagents and Materials All chemicals were of analytical-reagent grade. The aqueous reagent and standard solutions were stored in glass containers. De-ionized water was used throughout. A stock solution of 0.1 mol dm-3 CN- was made from potassium cyanide (Merck, Darmstadt, Germany) and checked using the Liebig method. 13 Standard cyanide solutions, which in most experi- ments were made to be 1 mmol dm-3 in NaOH, were prepared by diluting aliquots of the stock solution to the appropriate volume. i2-cx-Phosphatidylcholine type XI-E (Sigma, St. Louis, MO, USA) and stearic _acid (Merck) were used as received. Cholesterol (Zorka, Sabac, Yugoslavia) was re- crystallized from chloroform. Instrumentation and Apparatus The FI manifold is illustrated in Fig.1. Two peristaltic pumps were used, Mini S-840 (Ismatec, Zurich, Switzerland) and a Model HPB 5400 (Iskra, Kranj, Yugoslavia). The injection valve was a Model 5020 (Rheodyne, Cotati, CA, USA) equipped with a 100 1-11 sample loop. Interconnecting poly- (tetrafluoroethylene)(PTFE) tubing had an i.d. of 0.5 mm. [Caution: to prevent HCN evolution, the waste lines should be connected to the NaOH solution.] A thin-layer flow-through amperometric cell was part of an LC-17A package (BAS, West Lafayette, IN, USA) and was equipped with a Model MF-1008 silver working electrode (BAS) and a Model MW-2021 Ag-AgCI reference electrode (BAS). A 0.13 mm48 ANALYST, JANUARY 1992, VOL. 117 I RE I II P I I W Fig. 1 FI manifold used for amperometric determination of cyanide: C, carrier (water); R, reagent (pH 8 borate buffer); P, peristaltic pump; I .injection valve; MC, mixing coil (30 cm X 0.5 mm i.d.); EC, electrochemical flow-through cell; PO, potentiostat; RE, recorder; and W, waste. Flow rates are given in ml min-l thick Model MF-1047 PTFE gasket (BAS) was used to separate the working electrode from the cell body. Before coating the electrode, the silver surface was polished with polishing alumina (BAS) and thoroughly rinsed with de-ionized water. The composite coating was prepared by adding the desired amounts of cholesterol (usually 12 mg) and stearic acid (12 mg) to 1 ml of chloroform solution containing 10 mg of L-a-phosphatidylcholine. Each silver disc of the dual-disc electrode block was covered with 5 1.11 (1 drop) of the chloroform solution.A 10-20 PI Wiretrol I1 micropipette (Drummond Scientific, Broomall, PA, USA) was used for the delivery of chloroform solution. The potential was applied to the flow-through amperometric cell and currents were measured with a Model MA 5450 polarograph (Iskra); the resulting FI signals were recorded on a Servograph Model 61 strip-chart recorder (Radiometer, Copenhagen, Denmark) equipped with a REA 110 unit. Results and Discussion The FI amperometric determination of cyanide on a modified silver electrode was performed with the manifold illustrated in Fig. 1. The alkaline (pH 11) CN- standard or sample, after injection (I), is washed by the water carrier (C) to a mixing point with a reagent (R) (pH 8 borate buffer).The mixing coil (MC), positioned downstream, ensures thorough trans- formation of CN- to HCN (pKHCN = 9.21).14 The hydro- phobic hydrogen cyanide formed on-line in the FI manifold diffuses through the lipid membrane and is detected at a silver working electrode. The anodic current measured is propor- tional to the CN- concentration in the injected standard or sample. The effects of several parameters on the performance of the FI system were studied. The effect of the applied potential at the working silver electrode was investigated in the range -0.20 to +0.30 V versus the Ag-AgC1 reference electrode. Taking into account the signal-to-noise ratio achieved, and the interference effects, the optimum potential was found to be 0.10 V.It had been established earlier' that the optimum potential for CN- determination at the bare silver electrode is 0.0 V. Of the four acceptor solutions tested (0.1 mol dm-3 HCl, pH 4.5 acetate buffer, 0.1 mol dm-3 KN03 and pH 8 borate buffer), the best results were obtained with the borate buffer. Also, the highest FI signals were achieved with the shortest mixing coil utilized. Hence, for all subsequent experiments, a potential of 0.10 V versus Ag-AgCI reference electrode, a borate reagent stream and a 30 cm X 0.5 mm i.d. mixing coil were used. As noted earlier, Wang and Lull developed a highly stable phosphatidylcholine-cholesterol coating for the glassy carbon electrode. These experiments indicated that better selectivity towards CN- is achieved when stearic acid is added as a third component to the lipid membrane.The addition of stearic acid Scan --.-c Fig. 2 Response of the amperometric detector with ( a ) bare and ( 6 ) lipid-modified silver electrodes t o three repetitive injections of: A, cyanide (0.100 mmol dm-'); B, thiosulfate (0.100 mmol dm-3)); and C, thiosulfate (1 .OO mmol dm-3) increases the concentration of negatively charged sites at the surface of the coating, which then, in turn, cause better discrimination against potentially interfering anions. The permselectivity of the three-component membrane is illus- trated in Fig. 2, which depicts typical FI peaks for CN- and S2032- at the bare and lipid-modified silver electrodes. As can be seen, unlike the complete elimination of the thiosulfate signals at 0.1 mmol dm-3 levels, large peaks are still observed for CN- at the coated electrode. It was suggested" that the ratio between the currents at the film-coated electrode over that at the bare electrode, im/ih, could be used as a measure of the permeability and thus selectivity.These values for S2032- and CN- at 0.1 mmol dm-3 concentrations were found to be 0 and 0.10, respectively. Even for a thiosulfate concentration ten times higher this ratio was only 0.004. Hence the membrane exhibits effective discrimination against S2032- and tolerable attenuation of the signal for CN- ion. The corresponding im/ib values for a two-component (phos- phatidylcholine-cholesterol) membrane at 0.1 mmol dm-3 concentrations of CN- and S2032- were found to be 0.11 and 0.07, respectively.A plausible rationalization that clearly explains the ob- served experimental selectivity relationship could be based on the Donnan equilibrium effects. A composite membrane based on a mixture of phosphatidylcholine, cholesterol and stearic acid can be envisioned as a cation exchanger. The membrane has fixed carboxylic acid sites. At pH values greater than about 6.8 these sites are completely ionized and therefore it is possible that the membrane would exclude co-ions (in this instance anions) to an extent determined by the Donnan equilibrium. This phenomenon could be responsible for hindering the passage of the ionized species (S2032-, for example) to the lipid-modified silver electrode, while allowing the transport of the non-ionized analyte (HCN) through the membrane to the working electrode.The ability of the FI manifold developed here to determine CN- selectively in the presence of other anions will be illustrated later. Wang and Lull demonstrated that composite coatings fabricated from cholesterol and phosphatidylcholine were stable when applied to the glassy carbon electrode. In order to show that addition of the stearic acid to the membrane and its application to the silver working electrode also produce a stable coating under dynamic FI conditions, the following experiment was performed. A 0.1 mmol dm-3 CN- standard was sequentially injected for a 2 h period. No change in the FI signals was observed, which indicates the integrity of the lipid membrane under dynamic flow conditions. The same experi- ment has an additional significance: it shows that it is possibleANALYST, JANUARY 1992, VOL.117 I I I I 49 Table 1 Comparison of FI results for the determination of CN- in the presence of other anions (all samples contained 50.0 pmol dm-3 CN- and 1 .00 mmol dm-3 of the anion investigated) t l 1 al d.-0.15 pA 1 rnin scan - Fig. 3 Ra id scan response to two repetitive injections of a 0.100 mmol dm-?cyanide standard at ( a ) bare and ( b ) lipid-modified silver electrodes. Valve switchings are indicated by the arrows 2.8 2.4 h p 2.0 2 2 1.6 1.2 L 2 0 -I to modify, with lipid coatings, electrodes that participate directly in the amperometric detection process (dissolution of silver electrode) and not only those which function solely as a medium for electron transfer (glassy carbon or platinum electrodes). A comparison of the responses of the bare and modified silver electrodes to the dynamic changes in the concentration of the analyte is shown in Fig. 3.As can be seen, the time elapsed from the injection of the CN- standard to the appearance of the FI signal is almost the same with both the bare and the lipid-modified silver electrodes [compare the traces in Fig. 3(a) and (b)]. This indicates that the kinetics of the diffusion of HCN across the lipid layer are fast on the FI time-scale. Also, it can be seen that there is no significant change in the peak profiles. This indicates an effective wash out of the analyte from the lipid membrane, which is a prerequisite for successful FI operation. The linearity studies were conducted by injecting in triplicate eleven CN- standards with concentrations between 0.500 pmol dm-3 and 0.100 mmol dm-3. A linear relationship was found between the logarithm of the anodic peak current (nA) and logarithm of the concentration (pmol dm-3).A typical calibration graph, illustrated in Fig. 4, had a slope of 0.55 k 0.02 with a correlation coefficient of 0.9978 (all the statistics were calculated for a 95% confidence level). The detection limit, calculated according to the recommended procedure,lS was found to be 0.1 pmol dm-3, which corre- sponds to 0.26 ng of CN- (the sample loop volume was 100 yl). Under these conditions, the relative standard deviations for the determination of cyanide were 1.7 and 3.1 % (n = 9) for 0.1 mmol dm-3 and 0.5 ymol dm-3, respectively.As can be seen from Fig. 4, a similar linear relationship was found also for a bare silver electrode. Hence it can be inferred that the nature Ion added Acetate B ro mat e Bromide Chloride Carbonate Citrate Fluoride Iodide Nitrate Nitrite Oxalate Phosphate Sulfate Sulfide Sulfite Thiocyanate Thiosulfate Added as CH3COONa KBr03 KBr NaCl Na2C03 Sodium citrate KF KI KNO3 KNO? K?C?OJ KZHPOJ Na7S04 Na2S Na2S03 KSCN Na2S203 Difference (%)* 0 0 0 0 0 -1.8 1.9 121-1 0 0 0 0 0 --$ 0 0 0 * Compared with a pure CN- (50.0 vmol dm-3) standard; as -t Does not interferc at a 2-fold cxccss. -$ Out of scale; interferes significantly at all concentrations. determined by a t-test at a 95% confidence level. Table 2 Analysis of a synthetic water sample Concentration/pmol dm-3 Taken Found* 1 .00 1 .oo 5 0.01 5.00 4.96 k 0.01 10.0 10.03 k 0.02 25 .o 25.22 * 0.04 50.0 51.6 k0.l * Mean k standard deviation ( n = 5 ) .~ ~ Table 3 Comparison of CN- determination by FI and argentimctric titrationI3 in samples of industrial salt wastes obtained after thermic treatment of steel Concentration ( % ) 4: Sample No. I t 20.1 k 0 . 1 20.3 1 2 0.04 2 3 A rge n t i me t ri c F1 (4.7 * 0.1) x 10-4 (1.4 k 0.6) x 10-4 (5.05 k 0.04) x lo-' ( 1 .30 -t 0.01) x 1 0 - 4 * Mean t standard deviation ( n = 5 ) . t Before thermic treatment. of the calibration relationship is not influenced by the lipid coating . The effects of possible interferents on the cyanide determi- nation are summarized in Table 1. As can be seen, the presence of the following ions in a 20-fold excess relative to CN- do not interfere: acetate, bromate, bromide, chloride, carbonate, citrate, fluoride, nitrate, nitrite, oxalate, phos- phate, sulfate, sulfite, thiocyanate and thiosulfate.I t is noteworthy that Br-, C1-, SCN- and S203z- do not cause a positive error in CN- determination at the lipid-modified silver electrode as they would at a bare electrode. Iodide ion interferes at a 20-fold excess, more than doubling the anodic peak current. However, it does not cause a change in the CN- peak when present in a 2-fold excess. The Donnan equilibrium effects could also be used to explain why higher levels of I- (1.00 mmol dm-3) interfered whereas the same levels of Br-, for example, did not. The Donnan equilibrium-type exclusion is species dependent, so it is plausible that at higher I - concentrations some of the anion is transported across the composite membrane and is detected at a silver working electrode.However, the selectivity of the membrane is still operational as the i,,,/i,, value for this species is less than 0.03.50 ANALYST, JANUARY 1992, VOL. 117 Sulfide ion was found to be the only serious interferent. This is not surprising, as H2S formed on-line in the manifold would be expected to diffuse through the lipid coating. However, if SZ- is present in the sample, it can be removed by precipita- tion with lead(i1) acetate; lead(i1) does not form strong complexes with the CN- ligand. I n order to illustrate the potential of the proposed FI method, synthetic water samples (Table 2) and industrial salt wastes obtained after thermic treatment of steel (Table 3) were analysed.This work demonstrates that composite permselective lipid coatings, by rejecting the potentially interfering species from the surface of the electrode while allowing the transport of the analyte, offer a substantial enhancement of amperometric FI methods. The authors acknowledge the financial support of the US Bureau of Mines under the Mining and Mineral Resources Institute Generic Center programme (Grant number G1125132-3205, Mineral Industry Waste Treatment and Recovery Generic Center) and the Serbian Republic Research Fund. References 1 Rocklin, R. D., and Johnson. E. L.. Anal. Chem.. 1983,55.4. 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Liu, Y . , Rocklin. R. D., Joyce. R. J.. and Doyle, M. J., Anal. Cliem., 1990, 62. 766. Pihlar, B., and Kosta, L., Anal. Chim. Acta, 1980, 114, 275. Utley, D., Analyst, 1990, 115, 1239. Pollema, C. H., Hendrix, J. L., Milosavljevid, E. B . , Solujid, L., and Nelson, J. H.. J. Photochem. Pliotobiol. A: Cliem., submitted for publication. Milosavljevid, E. B., SolujiC, L., Hendrix, J . L., and Nelson, J. H., Anal. Chem., 1988.60, 2791. Garcia. 0. J.. Quintela, P. A., and Kaifer. A. E., Anal. Chem.. 1989, 61, 979. Chastel, O., Kauffmann, J. M., Patriarche, G. J., and Christian, G. D., Anal. Chem.. 1989. 61. 170. Tanaka. K., and Tamamushi. R., J . Electrounal. Cliem., 1987. 236, 305. Uchida, I . , Ishiho. A., Matsue, T., and Itaya, K.. J . Electyo- anal. Cliem.. 1989, 266. 455. Wang. J., and Lu, Z . , Anal. Cliem.. 1990, 62. 826. Wang. J.. Rayson. G. D.. Lu, Z . , and Wu, H . , Anal. Chem.. 1990, 62, 1924. Vogel. A. I . , A Text Book of Quantitative Inorganic Analysis, Longman, London. 1961, 3rd edn.. pp. 271-272. Izatt. R. M., Christenscn, J. J., Pack, R. T., and Bench, R., Inorg. Cliem.. 1962. 1, 828. Analytical Methods Committee, Analyst, 1987. 112, 199. Paper I l02344F Received May 20, 1991 Accepted August 29, I991

ISSN:0003-2654    

DOI:10.1039/AN9921700047

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST, JANUARY 1992, VOL. 117 51 Comparison of the Influence and Contribution of the Response Times of Coated Open-tubular Solid-state Bromide- and Chloride-selective Electrodes on the Analytical Throughput (Dispersion) in Flow Injection Systems Jacobus F. van Staden Department of Chemistry, University of Pretoria, Pretoria 0002, South Africa An evaluation of the influence and contribution of the response times of coated open-tubular solid-state bromide- and chloride-selective electrodes on the analytical throughput (dispersion) in flow injection (FI) systems is presented using concentration ranges of 10-5000 mg dm-3 for bromide and chloride, respectively, in the study. For both electrodes the adsorption rate is independent of concentration and does not contribute significantly to any decrease in the sampling rate, although the adsorption rate of the chloride-selective electrode in the FI system seems to be slightly faster than the bromide system.For both electrodes the desorption mechanism process is mainly responsible for the sampling rate obtained in an optimized FI-ion-selective electrode system. An electrode memory of the bromide-selective electrode is the main reason for the sampling rate of this system being lower than that obtained for a similar FI system with a chloride-selective electrode. Keywords: Flow injection; tubular solid-state bromide- and chloride-selective electrodes; flo w-through system; dispersion; response times It was shown in a previous paper' that the shape and quality of the analytical signal obtained from a coated open-tubular inorganic-based solid-state silver-silver chloride ion-selective electrode incorporated into a flow injection (FI) system are directly influenced by both the electrode and the FI manifold.It was also shown that the electrode characteristics and response of a chloride-selective electrode in an FI flow- through system are basically controlled by two mutually dependent components: ( i ) a contribution from the type of electrode used, which depends on the chemical properties of the specific electrode used at the moment in time, e.g., silver-silver chloride, i.e., the electrode aspect; and (ii) a contribution from the partial dispersion originating from flow dynamics resulting in a concentration-time profile obtained from both convection and diffusion transport, i.e., the FI manifold aspect.The study was extended in this paper to compare the influence and contribution of the response times of coated open-tubular inorganic-based solid-state bromide- and chloride-selective electrodes on the analytical throughput (dispersion) in FI systems using a slightly different approach in connection with the data acquisition system. Experimental Reagents and Solutions Analytical-reagent grade chemicals were used throughout unless specified otherwise. Doubly distilled, de-ionized water was used to prepare all solutions. The water was tested beforehand for traces of chloride. All solutions were de- gassed before measurements were made by using a vacuum pump system. The main solutions were prepared as follows.Ionic strength adjustment reagent Dissolve 202.22g of potassium nitrate in 2dm3 of distilled water in a calibrated flask to obtain a 1 mol dm-3 solution of potassium nitrate. Standard bromide solutions Dissolve 29.7860 g of dried potassium bromide in 2 dm3 of distilled water to give a stock solution with a bromide concentration of 10 000 mg dm-3. Prepare working standard bromide solutions by dilution of appropriate aliquots of the stock solution to cover the range 5-5000 mg dm-3. Standard chloride solutions Dissolve 32.9680g of dried sodium chloride in 2dm3 of distilled water to give a stock solution with a chloride concentration of 10 000 mg dm-3. Prepare working standard chloride solutions by dilution of appropriate aliquots of the stock solution to cover the range 5-5000 mg dm-3.Instrumentation Construction of the coated tubular flo w-through electrodes The basic design, preparation and conditioning of the coated tubular flow-through solid-state bromide-selective' and chloride-selective3 membrane electrodes were similar to those described previously.2.3 Five different coated tubular flow- through solid-state bromide- and chloride-selective electrodes were prepared using the same procedures as previously described2.3 in order to validate the data. Flow injection system, data acquisition and assimilation A manifold system with a sampler unit, sampling valve and peristaltic pump similar to the system described previously~-3 was used in this study. The tubular flow-through solid-state bromide- and chloride-selective electrodes were incorporated separately, as needed, into the conduits of the FI system for the specific studies.Experiments were performed on five different coated tubular flow-through solid-state bromide- and chloride-selective membrane electrodes, respectively, in order to validate the data properly. Bromide and chloride standard solutions were injected into the corresponding FI-ion-selective electrode (ISE) systems in order to obtain representative runs. The experiments were also conducted in a series of five consecutive runs for each electrode type to ensure that the results obtained fell within the required accuracy and precision. A very good reproducibility between the different prepared ISEs was obtained and also between the series of five consecutive runs.The potentials were measured at room temperature with an Orion Model 901 microprocessor Ionalyzer. A binary coded decimal (BCD) output signal, equivalent to the displayed value in front of the Orion microprocessor Ionalyzer, was directed via a 40-pin 8-4-2-1 BCD connector at the rear of the instrument with appropriate interfacing to an XT IBM-com- patible microcomputer, 640 kbyte RAM, equipped with an 80287 mathematics co-processor, EGA colour graphics card with 256 kbyte graphics memory and high-resolution monitor.52 -40.0 0) ANALYST, JANUARY 1992, VOL. 117 I 'Oo0 .- 5000, I I I The acquired data were manipulated using ASYST software (ASYST Software Technologies, Version 1.04, Macmillan Software, New York). The background noise received with the raw data from the electrodes was removed by applying the DS (data set) Cat and Smooth unary operation of the file processor of the ASYST software.With the DS Cat and Smooth operation, consecutive data sets in long data files are catenated and then smoothed by convolution with a filter computed from a low-pass Blackman window frequency response. Selection of a cut-off frequency (cycles per point) is available in a prompt list to allow flexibility in the amount of smoothing. These background-corrected data were plotted on a Hewlett-Packard Model 7475A-compatible serial plotter. The constructed flow-through tubular indicator electrodes were used in conjunction with an Orion Model 90-02 double-junction reference electrode with 10% m/v potassium nitrate as the outer chamber filling solution.Results and Discussion The relevant parameters that are normally selected in the quantitative evaluation of the shape and quality of the analytical signal obtained from coated open-tubular inorganic- based solid-state ion-selective electrodes interfaced to an FI system are peak intensity and residence time. These two parameters resulted in a concentration-time profile and the observed peak dimensions revealed valuable information on the electrode characteristics of a specific type of electrode used. This ultimately formed a very important part in the selection of optimum conditions as far as precision, accuracy and sample throughput are concerned in analytical measure- ments for a specific application. The electrode characteristics and response, in a flow-through system, are controlled by the chemical properties of the specific electrode used and the partial dispersion arising from the flow dynamics of the system.The basic background related to the contribution of the electrode and FI manifold to the analytical signal has been described previously. 1 The BCD acquired raw data for representative runs, transferred as raw data to the ASYST system, contained less background noise than the detector output as previously directed via an analogue-to-digital ( N D ) converter to the ASYST system.' The main reason for the noisier background previously experienced' was probably the accumulation of noise in the detector output and the channelling of the analogue data from the detector output (normally used for a recorder) via a screened cable to the daughter board of the LabMaster card. The integration time function in the A/D converter of the Orion model circuit consists of a conversion cycle of the integrator output which contains four phases.The first phase (zero period) is initiated by the line synchronization and is terminated by the counter exactly 100ms later. The second phase (input integration) is initiated by the counter 100 ms later, i.e, 200 ms after synchronization. The third phase (reference integration) is initiated by the counters at 300 ms after line synchronization and is terminated by a zero crossing, 0 to approximately 260 ms later, depending on the value of the integrator voltage at the beginning of phase three. The fourth phase (idle) is initiated at the end of the update pulse.The counters are then reset to zero, turning on the zero switch to hold the integrator at zero while it waits for line synchronization and the start of a new conversion cycle. The purpose of the idle period is to provide synchronization with the a.c. line frequency. Two features combine to provide rejection of noise at the a.c. line frequency. Firstly, line synchronization ensures that each conversion begins at the same point in the line waveform. Secondly, the input integration period is chosen to be an exact multiple of both the 50 and 60Hz line frequencies, so that in either situation the integrator receives a whole number of line cycles of any power frequency ripple superimposed on the input from the pre- amplifier. This study, however, still showed some amount of background noise with the raw data.By applying the DS Cat and Smooth unary operation of the file processor of the ASYST software, the background noise was removed. The background-corrected graphs, as they appeared on the moni- tor screen, are represented in this paper. A typical representative DS Cat and Smooth operated background-corrected graph for the determination of bromide with the FI system and coated tubular bromide-selective electrode is presented in Fig. l ( a ) . Standard bromide solutions in the range 10-5000 mg dm--7 were injected, each standard in duplicate. The total run was performed in 2300 s and the signal range varied between -60 and 140 relative mV. An experimental calibration plot of analytical signal versus pBr = -log[Br] of a typical representative DS Cat and Smooth operated background-corrected run, illustrating the dynamic linear response of the bromide-selective electrode, is pre- sented in Fig.l(b). The linear range depends on the pre- treatment of the electrode and care should be taken during the preparation of each new coated tubular electrode. When incorporating an electrode into the conduits of an FI system, the sample volume is not only crucial to the design of efficient FI systems but also plays a major part in the sensitivity and linearity of a calibration graph. The DS Cat and Smooth unary operation of the file processor of the ASYST software might also have a very small effect on the represented background- corrected graph. The calculated Nernstian response of the tested electrodes at steady state, using the same procedure as described previously,z is 57.7 k- 1 mV per decade with a correlation coefficient of 0.9996, which confirmed previous results.' The dynamic linear response range in Fig.l(b) was less than those at steady state, but was consistent with those previously obtained.' The results from Fig. l ( a ) revealed two 120.0 80.0 40.0 0.0 250 750 1250 1750 2250 .- - 60 - 40 - 20 0 20 40 60 80 100 I I I I I 0 - 1 -2 -3 -4 PBr Fig. 1 ( a ) Typical representative DS Cat and Smooth operated background-corrected graph of raw data for a run with different standard bromide solutions from the tubular bromide-selective electrode-FI system as directed via a BCD output and as transferred to thc ASYST system and as it appeared on the monitor screen.From left to right, 5000-10 mg dm-3 standard bromide solutions; each standard injccted in duplicate. Total run performed in 2300 s and the signal range varied between -60 and 140 relative mV. ( 6 ) Experimen- tal calibration plot of analytical signal versus pBr of a typical representative DS Cat and Smooth operated background-corrected runANALYST, JANUARY 1992, VOL. 117 important points: the tendency of a slower return to the baseline of the higher bromide standards compared with the lower standards and a slight shift in the baseline over the entire run. When these results are compared with a typical represen- tative DS Cat and Smooth operated background-corrected graph for the determination of chloride with a similar FI system and a coated tubular chloride-selective electrode as outlined in Fig.2(a) (chloride solutions in the range 10- 5000 mg dm-?, running time 2300 s, variation of the signal range between SO and 205 relative mV), the first observation is that the return to baseline of the higher chloride standards seemed faster and the baseline seemed to be constant over the entire run. An experimental calibration plot of analytical signal i'emfs pC1 = -log [Cll of a typical representative DS Cat and Smooth operated background-corrected run, illustrating the dynamic linear response of the chloride-selective electrode, is presented in Fig. 2(h). The calculated Nernstian response of the tested electrodes at steady state, using the same procedure as previously described,3 is 57.7 k 1 mV per decade with a correlation coefficient of 0.9998, which confirmed previous results.-3 Again the dynamic linear response range in Fig.2(6) was less than that at steady state, but was consistent with those previously obtained.3 The typical respresentative DS Cat and Smooth operated background-corrected graph for the deter- mination of chloride obtained riia the BCD output signal [Fig. 2(u)] is a better representation of the chloride-selective electrode-FI system than that described previously. 1 The main reason for this conclusion is that the amount of background noise received previously' together with the raw data was of such a nature that it definitely influenced the DS Cat and Smooth unary operation of the file processor of the ASYST software. 195.0 165.0 135.0 105.0 75.0 li I loo0 [15000, I 1 I 250 750 1250 1750 2250 .- 4- a Time/s - L .- m o zn v) .- 50 100 150 200 I I I I 0 -1 -2 -3 -4 PCI Fig. 2 ( L I ) Typical representative DS Cat and Smooth operated background-coi-rcctcd graph of raw data for a run with different standard chloride solutions from the tubular chloride-sclcctivc clcc- trodc-FI systcm as directed viu a BCD output and as transferred to the ASYST system and as it appeared on the monitor screen. From lcft to right, 5000-10 mg dm-.3 standard chloride solutions: each standard injcctcd in duplicatc. Total run pcrformcd in 2300s and thc signal range varied between 50 and 205 relative rnV. ( h ) Experimental calibration plot of analytical signal wI:su.s pCI of a typical rcprcscnta- tivc DS Cat and Smooth operated background-corrected run 53 The two typical representative graphs [Figs.l(a) and 2(a)J did not give a detailed observation of the individual peaks. In both graphs all the peaks did not, for example, reach the baseline before the start of the next peak, a condition that is more prominent within the higher concentration range. In order to compare the different peaks over the whole concen- tration range a more in-depth study of the performance of individual peaks of the different bromide concentrations was necessary. Separate representative runs of the different bromide concentrations were therefore conducted in order t o obtain a baseline-to-baseline performance of each individual peak. This was carried out as follows. Each bromide standard solution was injected in duplicate into the ISE-FI system allowing sufficient time for both peaks in a run to reach the baseline.This was also conducted in a series of five consecu- tive runs to ensure that the results obtained fall within thc required accuracy and precision. By accumulating data in such a way, it was possible to evaluate the results in a more logical way, which led to better conclusions. The beauty of the ASYST system is the manipulation of individual peaks from which valuable information can be revealed. Figs. 3 and 4 show a selection of individual pcaks from the above-named runs of individual bromide (Fig. 3) and chloride (Fig. 4) standards where the position of the individual peaks was manipulated in such a way that overlapping of peaks was possible.The main aim was to try to obtain an overlapping of individual peaks from different concentrations at a fixed point, thereby enlarging the selective individual bromide and chloride peaks to the same graphical intensity on the y-axis and to study the response behaviour of the bromide- and Time - Fig. 3 Enlargements of thrcc sclcctcd individual bromide pcaks to the samc graphical intensity on the y-axis: 5000 mg dm- 3 of Br- bctwccn -SO and 130 relative mV, 500 mg dm-3 of Br- bctwccn 0 and 130 relative mV and SO rng dm-3 of Br- bctwccn 59 and 135 relative mV. A cut-off frequency of 1000 s was used on the x-axis 5000, 50 5b0 500C ,-500 50 Time - Fig. 4 Enlargements o f thrcc sclcctcd individual chloridc peaks to the samc graphical intensity on the y-axis: SO00 mg dm-3 of CI- between 58 and 203 rclativc mV.500 mg dm-3 of Cl- bctwccn 1 1 0 and 205 relative mV and 50 mg dm- 3 of CI- bctwccn 164 and 204 rclativc mV. A cut-off frequency of 1000 s was used on the x-axis54 ANALYST, JANUARY 1992, VOL. 117 chloride-selective electrodes for different bromide and chloride concentrations, respectively, from this common point. The rising part of each curve was used for simultaneous overlapping. Three selected individual bromide peaks were enlarged to the following graphical intensity on the y-axis: 5000mgdm-3 Br- between -50 and 130 relative mV, 500mgdm-3 Br- between 0 and 130 relative mV and 50 mg dm-3 Br- between 59 and 135 relative mV (Fig. 3). The three selected individual chloride peaks were enlarged to the following graphical intensity on the y-axis: 5000 mg dm-3 Cl- between 58 and 203 relative mV, 500 mg dm-3 CI- between 110 and 205 relative mV and 50 mg dm-3 CI- between 164 and 204 relative mV (Fig.4). In order to comply with the main goal of baseline-to-baseline performance, no enlargement on the 124.0 93.2 62.0 30.8 -0.400 620 628 636 644 652 122.0 1 I E . - 126.0 0, m .- 107.0 87.5 68.5 620 628 636 644 652 128.0 113.0 97.0 81.4 65.8 624 632 640 648 656 Time/s Fig. 5 Enlargements of the leading edges of four selected individual bromide peaks to the same graphical intensity on the y-axis and the same cut-off frequency of 40 s on the x-axis. ( a ) 1000 mg dm-3 of Br- between -16 and 140 relative mV; ( 6 ) 500 mg dm-3 of Br- between 0 and 135 relative mV; (c) 100mgdm-3 of Br- between 40 and 135 relative mV; and (d) 50 mg dm-2 of Br- between 58 and 136 relative mV x-axis was made and a relatively large cut-off frequency of 1000 s was used.It is clear from the above information that the influence of the ‘wash-in’ and ‘wash-out’ of a bromide analyte sample plug in the flow detection conduits of the coated tubular bromide- selective electrode can be divided into four sections: two for the wash-in part and two for the wash-out part. As previously outlined,’ the output form for a certain part of a peak is controlled by the electrode mechanism, whereas the flow dynamics of the manifold system dominate another output region of the peak. Also, the distortion of the Gaussian peak shape at the lower front and rear part of the peak was attributed to dominance of the electrode mechanism, which is in agreement with the adsorption-desorption mechanism that occurs at low concentrations.1 198 174 1 50 126 1 02 647 655 663 671 679 200 180 160 140 > E 120 Q, .- 644 652 660 668 676 *.’ - Q, 191 179 167 154 620 628 636 644 652 1681 , . , , , ,c 660 668 676 684 692 Time/s Fig. 6 Enlargements of the leading edges of four selected individual chloride peaks to the same graphical intensity on the y-axis and the same cut-off frequency of 40 s on the x-axis. ( a ) 1000 mg dm-2 of CI- between 90 and 210 relative mV; ( b ) 500mgdm-3 of C1- between 109.8 and 209.8 relative mV; (c) 100 mg dm-3 ofC1- between 148 and 210 relative mV; and ( d ) 50 mg dm-3 of CI- between 164 and 205 relative mVANALYST. JANUARY 1992.VOL. 117 55 Although the plots in Figs. 3 and 4 clearly differentiate between the leading (wash-in) and tailing (wash-out) edges of the peaks, the large cut-off frequency of 1000 s on the x-axis only gave a global, but still valuable, insight into these two parts. A closer examination of the baseline-to-baseline performance of the ‘wash-in’ part of the bromide and chloride samples was necessary, however, in order to analyse the leading edges of the peaks. In Figs. 5 and 6, enlargements of the x-axis were made by using a cut-off frequency of 40 s. The results in Fig. 5 clearly divide the performance of the bromide-selective electrode-FI system of the leading edge (wash-in part) of the bromide peaks into two sections. A time delay was observed in the first section near the baseline, where the front part of the sample plug starts to move into the conduit of the tubular bromide-selective electrode.As the bromide concentration in a very small fraction of the front edge of the sample plug is very low, the time delay was probably mainly due to the chemical properties of the electrode at the moment in time and more specifically to the adsorption mechanism process at the initial lower portion of the peaks. The flow dynamics of the manifold system dominate the second section of the leading edge, where the bulk of the sampling zone starts to move through. This became more obvious (Fig. 5 ) when the front part of the sampling zone (where the bromide is concentrated) in the FI conduits took over for a period of time, after which the peak jumped to a maximum output. The adsorption rate seems to be indepen- dent of the concentration evaluated. I t seems that the increase in the dominance of the adsorption of the electrode mechan- ism becomes more obvious with a decrease in bromide concentration as observed in Fig.l ( a ) , where the peak intensities of the range of peaks are different. This is, however, not the situation as seen from Fig. 3, where the three peaks reflecting different concentrations of 50, 500 and 5000mgdm-3 of Br- were enlarged to the same peak intensity, a fact which is also true for the concentration range 10-5001) mg dm-3 of Br-. This is confirmed by the enlarged plots given in Fig. 5. It is, therefore, clear that the leading edge of the bromide peaks with different bromide concentrations in this FI-ISE study did not influence the analytical throughput of a specific optimized FI-system. The influence of the flow dynamics and adsorption rate of the bromide-selective elec- trode-FI system (Figs.3 and 5 ) was also compared with the flow dynamics and adsorption rate of a similar chloride- selective electrode-FI system (Figs. 4 and 6). The contribu- tion from the flow dynamics of the manifold system was identical for both electrode systems, but the adsorption rate of the chloride-selective electrode-FI system seems to be slightly faster than the bromide system. The results in Figs. l(a) and 3 clearly illustrate that for the top part of the peak, where the centre portion of the sample plug with the higher bromide concentration is moving through the electrode conduit, the influence of dispersion flow dynamics becomes predominant.I t also follows (Fig. 3) that the top portion near the peak maximum of the peak shape is due to the bulk of bromide analyte moving through the electrode conduit. This is confirmed by the results in Fig. 7, where enlargements of both the x- and y-axis were made. This is followed by peak tailing until the baseline is reached. The results in Figs. 3 and 7 also clearly distinguish between two sections in the performance of the bromide-selective elec- trode-F1 system of the wash-out part of the bromide peaks. The first portion of the rear part of the peak tailing (Figs. 3 and 7) is attributed to the tendency of the rear of the sample zone to move to the tube centre.This output region of the peak tailing nearest to the peak maximum is mainly due to the wash-out effect originating from the flow dynamics of the manifold system, where the rear part of the sample zone moves through the electrode conduits. Confirmation of this is obvious from Fig. 7. Although this tailing of the peaks has an effect on sampling rate, the contribution is reduced for bromide concentrations between 20 and SO0 mg dm-3. The influence, however, increases for bromide concentrations above 500mgdm-3 as observed from Figs. 3 and 7. In the second section, as the peaks approach the baseline, the effect of the rear part of the sample zone decreases owing to the flow dynamics of the manifold system and the desorption mechan- ism starts to dominate. The major contribution to any delay in analytical sampling rate of a specific optimized FI-ISE system for different bromide concentrations comes, however, from the part nearest to the baseline, where the peaks of 10- 250 mg dm-3 of Br- approach baseline conditions.From 250 to 5000mgdm-3 of Br-, the influence of the desorption process on sampling rate becomes more marked (Fig. 7). Figs. 3 and 7 clearly illustrate that the desorption mechanism process is mainly responsible for thc sampling rate obtained in an optimized FI-ISE system with a coated tubular bromide- selective electrode. When the rear part of the peaks of the coated open-tubular 124.0 93.2 62.0 30.8 -0.400 658 694 730 766 802 122.0 94.5 67.5 40.5 > 5 13.5 F > .- +d - \ - m & 126.0 v) .- 107.0 87.5 68.5 49.5 688 724 760 796 832 658 694 730 766 802 735 771 807 663 699 Time/s Fig.7 Enlargements of the tailing cdges of four sclcctcd individual bromide pcaks to the same graphical intensity o n the y-axis as in Fig. 5 . but with a cut-off freqiicncy of 180 s on thex-axis. ( a ) 1000; ( h ) 500; (c.) 100; and ( d ) 50 mg dm--’ of Br-56 ANALYST, JANUARY 1992, VOL. 117 198 174 150 126 102 688 724 760 796 832 200 180 1 60 140 > 5 120 > .- .I- - 683 719 755 791 827 2 . - m & 204 v) 191 .- 1 79 167 154 663 699 735 771 807 I V I I I I I 703 739 775 811 847 Time/s Fig. 8 Enlargcmcnts of the tailing cdges of four selected individual chloride peaks to the same graphical intcnsity on the y-axis as in Fig. 6 , but with a cut-off frequency of 180 son thcx-axis. ( a ) 1000; (6) S00; (c) 100; and (d) 50 mg dm-3 of Cl- solid-state bromide-selective electrode-FI system (Figs.3 and 7) was compared with the corresponding results obtained for the chloride-selective electrode-FI system (Figs. 4 and 8), the following observations were made. In the first section of the rear part of the output region of the peak tailing for both the bromide- (Figs. 3 and 7) and chloride-selective (Figs. 4 and 8) electrodes, where the sample zones in both instances moved to the tube centre, the contributions to peak shapes were mainly due to the wash-out effect originating from the flow dynamics of the manifold system. It seems from Figs. 3 and 4 that the flow dynamics of the manifold systems contribute more to the first section of the tailing of both the bromide and chloride peaks, which is actually not the whole explanation.Although there is a differentiation between the performance of the wash-out part of the bromide and chloride peaks, when compared in Figs. 3 and 4, the large cut-off frequency of 1000 s only gives a global view of the situation. When an enlargement I ; Time - Fig. 9 Enlargcmcnts of pairs of bromide and chloride pcaks to the same graphical intensity on thc y-axis as in Figs. 5-8. The pairs of pcaks wcrc manipulated in such a way that overlapping of twin pcaks was possible. A cut-off frcqucncy of 1000 s was used on thc x-axis. A , 1000 mg dm-3 of Br-; B, 1000 mg dm-3 of CI-; C, S O 0 mg dm-3 of Br-; D, 50Omgdm--i of C1-; E, 100mgdm-2 of Br-; and F, 100 mg dm-3 of CI- (Figs. 7 and 8) of the x-axis was achieved by using a cut-off frequency of 180 s, a better view of the situation was obtained.I t was clear from these results (Figs. 7 and 8), and more obviously from the bromide peaks (Fig. 7), that the desorption rate is already starting to contribute in the first section of the tailing edge. The contribution of the desorption rate increases as the peaks move nearer to the baseline and eventually dominates in the region of the baseline. A further conclusion to be drawn is that the desorption rate of the bromide- selective electrode system is slower than that of the chloride- selective electrode system and that this contribution also comes into effect in this rear part of the peak shape. This conclusion is confirmed from the representations in Figs. 4 and 8. I t is clear from the results that the chloride-FI system responds faster than the bromide-FI system for the tailing edge of the peak shape. The results, however, show that the major contribution to the analytical throughput of both electrode systems in the tailing edge comes from the desorp- tion process. The desorption rate of the chloride-selective electrode is, however, faster than that of the bromide- selective electrode. This is confirmed in Fig. 9, where three peaks reflecting different concentrations of 100, 500 and 1000 mg dm-3 of Br- and CI- were enlarged to the same peak intensity and curves were drawn wherc the corresponding peaks overlapped. I t can therefore be concluded that the electrode memory of the bromide-selective electrode is mainly responsible for the sampling rate of the bromide-selective-FI system being lower than that obtained for a similar FI system with a chloride- selective electrode. The Foundation for Research Development (FRD) , Pretoria, and the University of Pretoria are thanked for financial support. J. C. Lindeque is also thanked for assistance in performing some of the experiments. References 1 2 3 van Stadcn, J . F.. Anulysr, 1990, 115. 581. van Staden, J . F., Analysr. 1987. 112, 595. van Staden, J . F., Anal. Cliim. A m . 1986, 179, 407 Paper O105092.1 Receiried Nmyernber 13, I990 Accepted June 18. I991

ISSN:0003-2654    

DOI:10.1039/AN9921700051

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST, JANUARY 1992, VOL. 117 57 Characterization Lipophilized Ana of an Optode Membrane for Zinc(ii) Incorporating a logue of the Dye 4-(2-Pyridylazo)resorcinol Kemin Wang," Kurt Seiler,t Bruno Rusterholz and Wilhelm Simon Swiss Federal institute of Technology (ETHJ, Department of Organic Chemistry, Universitatstrasse 16, CH-8092 Zii ric h, Switzerland A chemical optical sensing system (optode) for Zn" based on a lipophilized metal ion indicator dye [ 1 -octadecyloxy-4-(2-pyridylazo)resorcinol; ETH 24641 dissolved in a plasticized poly(viny1 chloride) membrane has been developed. In the measuring range from 1 x 10-6to 3 x 10-3 mol dm-3Zn2+ (pH 4.8), the absorbance response shows a good correlation with the theoretically derived formulae. The optode membranes were found to reach 95% of the final signal within 5 min (&).When exposed to a continuous sample flow (1 ml min-I), the optode membrane did not lose a perceptible amount of the indicator over a period of 10 h. The selectivity of the optode membrane for the response towards Zn2+ ions in pH-buffered solutions is described. Keywords: Optode membrane; indicator; plasticized poly(vin yl chloride) membrane; spectrophotometry; zinc(//) ion Organic indicator dyes, which are normally used in the spectrophotometric determination of various metal ions, play a central role in the design of chemical optical sensors (optodes). I-8 Many optical sensing layers with immobilized classical indicators," which exhibit a marked change in their absorption spectrum on complexation of the metal ion, have been described.Most frequently, the immobilization of the indicator is achieved by covalent binding or by electrostatic attraction to a resin. Unfortunately, the group responsible for the complexation is often inaccessible to the metal ion after the immobilization process. In addition, very strong binding of the metal ion by the indicator often prohibits the reversible response of the sensor. Saari and SeitzJ.i have developed a fluorescence sensor for and Be" by binding the ligand morin with cyanuric chloride on a cellulose membrane. Zhujun and Seitz6 have described a fluorescence sensor for the detection of Allt1, Mg", Zn" and Cd", which is based on 8-hydroxyquinoline-5-sulfonic acid attached electrostatically to an ion-exchange resin. Chau and Porter7 have utilized a similar procedure for the immobil- ization of calcichrome on an anionic polymer film.An attempt to construct a sensor for transition metal ions with the fluorogenic indicator calcein was not successful, because of its lack of reversibi1ity.x The performance of these sensors is usually given in terms of dynamic measuring range. response time and selcctivity, whereas data on lifetime and signal recovery are often absent. A different approach has recently been proposed with the use of homogeneous plasticized poly(viny1 chloride) (PVC) membranes, incorporating lipophilized components as the chemically active species.lo In order to reduce the possible influence of surface effects on the response signal, a sensing layer with a thickness of the order of 3-4 pm was proposed. As a consequence, these layers can be treated as bulk phases with uniform properties.Highly lipophilic ionophores are the selective components of the widely used ion-selective elec- trodes and have been incorporated in such optode membranes together with specially designed chromo-ionophores. The free mobility of the complexing agent allows its active participation in the extraction of the substrate. This type of system appears to be well-suited for the detection of many analytes.10-1' In this paper it is shown that not only ionophores, but also lipophilized conventional dye indicators" are useful as cam- + O n leave from the Hunan Univcrsity. Changsha. China. .i_ To whom correspondence should be addressed. plexing agents in such layers.An optode membrane incorpor- ating a lipophilized analogue of the well-known indicator 4-(2-pyridylazo)resorcinol (PAR)'"-" and its response towards Zn'+ ions in a sodium acetate pH buffer are described. Experimental Reagents Aqueous solutions were prepared with water that had been doubly distilled from a quartz still and with salts of the highest purity available. For the preparation of the membrane, PVC (high relative molecular mass), tris(2-ethylhexyl) phosphate (TEHP) and tetrahydrofuran (THF) were used and were obtained from Fluka (Buchs, Switzerland). The buffer solutions used for the characterization of the optode membrane were acetate buffers of pH 4.8 and 5.6 (see Table 10.18 in ref. 16). Synthesis of 1-Octadecyloxy-4-(2-pyridylazo)resorcinol (ETH 2464) A solution of 2.0 g (9.3 mmol) of PAR (Fluka, p.a.), 3.03 g (9.3 mmol) of caesium carbonate (Fluka, puriss., p.a.) and 3.1 g of 1-bromooctadecane (Fluka, purum) in 100 ml of N,N-dimethylformamide (Fluka puriss., p.a.) was stirred for 1 h at 140 "C.The solvent was evaporated and the residue dissolved in 200 ml of CH2C17. This solution was washed with two 200 ml portions of distilled water and the solvent was evaporated again. The residue was purified by flash chroma- tography on silica gel 60 (Fluka) using ethyl acetate as eluent, and on neutral alumina (Woelm; activity grade 1) using hexane, and subsequently ethyl acetate, as eluent. The product was recrystallized twice from hexane to yield 51 mg (0.1 mmol, 1.2%) of the pure product (m.p. 69.5-70.5 "C). The structure of ETH 2464 (see Fig.1) was confirmed by proton nuclear magnetic resonance spectroscopy (300 MHz, CDC13) and fast atom bombardment mass spectrometry. The purity was determined by elemental analysis [Found: C , 74.4; H, 10.0; N, 8.7. Calc. forC2c,H45N307 (467.7): C, 74.5; H, 9.7; N , 9.0%]. Membrane Preparation The optode membranes were prepared from a mixture of 79 mg of PVC, 157 mg of plasticizer (TEHP) and 3.0 mg of ETH58 ANALYST, JANUARY 1992, VOL. 117 ETH 2464 Fig. 1 Structures of the indicator PAR and its lipophilized analogue (ETH 2464) Sample I 1 To detector - 1 To waste Fig. 2 Schematic reprcsentation of the flow-through cell: 1. poly- (propylenc) support with sample inlet and outlet; 2. O-ring seal; 3, quartz glass support; 4, ion-sensing optodc membrane; 5 , Plexiglas cell wall; and 6 , screw for fixation 2464.The membrane components were dissolved in 2 ml of freshly distilled THF. By means of a spin-on device, two identical membranes of approximately 4 vm thickness were cast onto two quartz glass plates, which were subsequently mounted in the specially designed flow-through measuring cell (see Fig. 2).".17 Apparatus Two quartz glass plates without membranes were mounted in the reference cell. Both measuring and reference cells were then placed in a conventional double-beam ultraviolet-visible spectrophotometer (Perkin-Elmer, Model Lambda 2, Kiis- nacht, Switzerland) to measure the absorbances. A specially designed base for the cells was flushed with thermostated water to keep the temperature constant at 25 "C.Procedure Except for the response behaviour, which was studied under batch conditions, all other measurements were carried out in the flow-through mode. The sample solutions were pumped (SJ-1211 Perista Mini-Pump, ATTO, Japan) through the measuring and reference cells at a constant flow rate of 1 ml min-1. The absorbance values A l and A. were determined with the optode membrane in contact with 0.1 rnol dm-3 HCI and 0.1 rnol dm-3 ZnS04, respectively. Table 1 Effect of diffcrcnt metal ions on the absorbance values of the optodc mcmbranc. in contact with a fixed concentration of 3 X rnol dm-3 ZnSO, in an acctatc buffer at pH 4.8 Metal ion Zn2+ K+ Na + Caz+ Mg2+ ~ 1 3 + Fe3+ Ni*+ Mn2+ Pb2+ Cd? + co2+ Cu2+ Concentration/ rnol dm-3 0.1 0.1 3 x 10-2 3 x 10-2 3 x 10-5 3 x 10-3 3 x 10-3 3 x 10-3 3 x 10-3 3 x 10-3 3 x 10-3 3 x 10-3 3 x 10-4 3 x 10-4 3 x 10-3 3 x 10-4 Absorbance 0.2178 0.2178 0.2178 0.1809 0.2125 0.1812 0.2129 0.1743 0.2061 0.2188 0.2000 0.2179 0.2154 0.2238 0.2603 0.2809 Absorbance change (%) 0 0 -16.9 -2.4 -16.8 -2.2 -20.0 -5.4 +0.5 -8.2 +0.05 -1.1 +2.7 +19.5 +29.0 - For the selectivity measurements, the membrane was equilibrated with a 3 X 10-5 rnol dm-3 Zn'+ sample solution (pH 4.8), before the solutions with the respective metal ion as background were injected (see Table 1).The absorbance readings for the selectivity studies were taken 15 min after the sample had been changed. Owing to the strong binding, Cu and Co were removed from the membrane by pumping 1 x 10-2 rnol dm-3 HCI through the measuring cell.In this way, Cu could be removed within 30 min, whereas more than 6 h were required to remove Co. Results and Discussion Principle of Operation The optode membrane described here belongs to the class of ion-exchange systems described previously. 1 0 The extraction of Zn'+ from the aqueous sample solution into the membrane phase and its complexation by the lipophilized metallochromic indicator ETH 2464 (HL) proceed with the loss of two protons from the hydroxy groups of two indicator molecules. This ion-exchange process is determined by the electroneutrality in the organic membrane phase. Fig. 3 shows the absorption spectra of the optode membrane incorporating ETH 2464, as obtained after equilibration with pH-buffered solutions (acet- ate buffer, pH 4.8) containing different ZnS04 concentra- tions.Obviously, the formation of the Zn complex induces a bathochromic shift of the absorption band from 380 nm (protonated form) to 523 nm. The over-all equilibrium between the organic membrane phase (org) and the aqueous sample solution (aq) is described as follows: K,,CI, ZnL?(org) + 2H+(aq) e 2 H L ( o r g ) + Zn2+(aq) (1)ANALYST, JANUARY 1992. VOL. 117 59 0.4 0.3 0) C m + 0.2 s a I] 0.1 0 I 1 I I 1 1 260 340 420 500 580 660 Unm Fig. 3 Absorption spectra of two 4 pm thick optode membranes after equilibration with different ZnSO, concentrations in an acetate buffer at pH 4.8. The ligand shows an absorbance maximum in its protonated form at 380 nm and the Zn complex an absorbance maximum at 523 nm. A, Buffer [ZnSO,]; B, 1 X 10-6; C, 3 x D, 1 X 10-5; E, 3 x 10-5; F, 1 x 10-4; G, 3 x lo-,; H, 1 x 10-3; and I, 3 x 10-3 mol dm-3 The corresponding equilibrium constant Kexch depends on the complex formation constant and on the distribution coef- ficients of the H+ and Zn?+ ions between the aqueous sample solution and the organic membrane phase.It can be assumed that a 1 : 2 Zn2+-ligand complex is formed in the optode membrane, as this stoichiometry was found in aqueous solutions for both indicator dyes, 1-(2-pyridylaz0)-2-naphthol (PAN) and PAR,13 and also in chloroform for PAN.18 Substituting the activities of the species in the membrane phase by their concentrations,I0.l I and with the introduction of a [the ratio of the concentration of the protonated ligand relative to the total amount of ligand (LT) present in the membrane phase] the response of the optode membrane can be given by: ffL” = (UH)’Kcycl,( 1 - CY)/(2CY’Lr) (2) where aL,, and aH denote the activities of the Zn’+ and the H+ ion, respectively.The high concentration of the acetate buffer in the sample solution provides a nearly constant ionic strength and hence the activity coefficient of Zn?-+ can be assumed to be constant. About 60% of the total Zn in the sample solution is complexed by the acetate anion.19 However, in the concentration range considered the relative amount of free Zn’+ does not change significantly, and hence it is approximately proportional to its total concentration c::. If LT and Kexcl, are assumed to be constant over the whole dynamic range, and if all the constant values are summarized in K ’ , then eqn.(3) can be derived from eqn. (2): (3) t o t CZ” = K’(1 - a)/& The measured absorbance A is directly related to a, if the optode membrane obeys Beer’s law: CY = (A0 - A)/(Ao - A , ) (4) where A I and A,) are the limiting absorbance values for CY = 1 (fully protonated ligand L) and CY = 0 (fully complexed ligand L), respectively. Response Behaviour In Fig. 4 the relative absorbance values, a, are given as a function of log ~2: for two different pH values. The curve fittings for the experimental points were calculated from eqn. (3) with log K’ = -4.92 (pH 4.8) and -6.40 (pH 5.6), respectively. The good correlation of the measured data with the theory confirms the validity of the assumptions made in 1 .o 0.8 0.6 0.4 0.2 0 8 I I I -8 -6 -4 -2 Log c g Fig.4 Relative absorbance values at 523 nm as a function of log c:’,: at A, pH 4.8; and B , pH 5.6. The curves fitting the experimental points were calculated from eqn. (3). Membrane: ETH 2464 + TEHP + PVC t 0 B 4 rnin - 10.02 I 0.3038 t 0.2686 4 rnin - Io.02 1 x 10-3 rnol drn-3 ZnS04 t t t 0.2687 0.2681 0.2677 3 x 10-4 rnol dm-3 ZnS04 Time - Fig. 5 membranes at 523 nm after several concentration step changes Absorbance response versus time for two 4 pm thick optode eqn. (3). In order to avoid depletion of the analyte close to the optode membrane, which would result in a large concentra- tion gradient in the sample solution, a sufficient amount of the analyte must be ensured. For this reason, the steady state was reached significantly faster for a total Zn concentration of < I X 10-5 rnol dm-3, when the sample solution was pumped through the measuring cell.At low Zn concentrations a reduction of the total amount of the indicator dye would of course also lead to shorter response times, but unfortunately at the expense of sensitivity. Short-term Reproducibility The reproducibility of the optical signals was evaluated by repetitively changing between two samples of 1 x 10-’ and 3 x 10-4 rnol dm-3 ZnSOj in an acetate buffer at pH 4.8. Thc absorbance response at 523 nm versus time is shown in Fig. 5 . The mean absorbance values and their standard deviations were determined after 5 min and were found to be repro- ducible to 0.3037 * 0.0005 (1 x 10-3 rnol dm-3 ZnS04) and to 0.2683 k 0.0005 (3 x rnol dm-3 ZnS04). Response Time The tgS values are reached within 5 min after concentration step changes from 1 x 10-3 to 3 x 10-4 rnol dm-3 ZnS04 and vice versa, as illustrated in Fig.5. The optode membranes described here exhibit a longer response time than the membranes that incorporate electrically neutral ionophores60 ANALYST, JANUARY 1992, VOL. 117 and which reach the final steady state within 3-4 s.IO-I1 The response time of such optodes is mainly governed by diffusion processes in the bulk of the membrane. For the optode membranes with ETH 2464, the rate of complex formation and particularly the rate of complex dissociation (see Fig. 5 ) might play an important role. Lifetime The absorbance signal at 523 nm for the optode membrane in contact with a 3 x 10-4 mol dm--? ZnS04-acetate buffer solution (pH 4.8; flow rate, 1 ml min-*) was recorded over a period of 10 h.From the absorbance values taken every 30 min ( n = 21) a mean absorbance of 0.2682 and a standard deviation of k0.0004 were obtained. No significant loss of the ligand was observed during this period of time. Selectivity The selectivity of optode membranes incorporating lipophilic dye indicators is expected not to deviate significantly from the selectivity behaviour of the less lipophilic analogues used in conventional extraction systems. The influence of other metal ions on the absorbance value at 523 nm, at a level of 3 x 10-5 mol dm-3 Zn2+, was investigated (see Table 1). As the rate of complex dissociation was slow for Cu, and complex dissocia- tion almost irreversible for Co, the selectivity towards these metal ions was determined at the end of the selectivity measurements.An exact description of the selectivity of the optode membrane described here can only be given for ions of the same charge and for chelates with the same stoichiometry and the same spectral properties. For most of the interfering ions considered, these prerequisites are unfortunately not fulfilled.ls Further, the activities of the interfering ions in the sample solution should be known, which requires certain assumptions and extensive calculations. The indicator acts as a non-selective chelating agent towards many metal ions. However, by analogy with extraction systems, the inter- ference from metal ions can be reduced by prior extraction, by masking with certain ligands or by an appropriate choice of the pH.'" Conclusions It has been shown that a lipophilized analogue of the classical indicator dye PAR is well-suited for the construction of an optode membrane based on plasticized PVC.The system described here offers significant advantages over other optodes reported to date, which make use of indicator dyes. These membranes might not only be used as reversibly functioning sensing layers, but also as simple extraction devices for the preconcentration of certain metal ions. Preconcentration by conventional solvent extraction provides a useful, but often sophisticated, means of improving the sensitivity of a determination, e.g., in atomic absorption spectrometry." The extraction of metal ions from a largc volume of the aqueous phase into a small volume of the organic liquid can be a necessary step in trace determinations or can enable the separation of the metal ions from an interfering matrix.An efficient phase separation, e.g., no solvent entrainment phenomena after the extraction, is an important criterion in the design of an extraction system, which is fulfilled in the system described here. There are many possibilities for introducing such optode membranes into other fields of application, e.g., in the titration of trace amounts of metals. This work was partly supported by the Swiss National Science Foundation. The authors thank S. Tan for careful reading of the manuscript. 1 2 3 4 5 6 7 8 9 10 I 1 12 13 14 15 16 17 18 19 20 21 References Seitz.W. R.. Anul. Chem., 1984. 56, 16A. Wolthcis, 0. S . . Fresrnius 2. Anul. Cliem.. 1986, 325. 387. Scitz. W. R., CRC Crit. Rev. Anul. Cliem.. 1988, 7. 173. Saari, L. A., and Scitz, W. R., Anal. Cliem., 1983. 55. 667. Saari, L. A., and Scitz, W. R., Analyst. 1984, 109, 655. Zhujun, Z . , and Seitz, W. R.. Anal. Cliim. A m . 1985, 171,251. Chau, L. K., and Porter. M. D., Anal. Cliem., 1990, 62, 1964. Saari, L. A., and Seitz. W. R.. Anul. Cliern.. 1984, 56. 810. Marczenko, Z., Separution and Si~ectroi,liotomt.rsic Determina- tion of Elemcwts. Wiley, New York, Chichester, Brisbane and Toronto. 1986. Morf, W. E.. Sciler, K., Sorcnscn. P., and Simon. W.. in /on-selc.ctiw Electrodes, cd. Pungor, E., Akadkmiai Kiad6. Budapest, 1989, vol. 5. p. 141. Seilcr. K., Morf. W. E . . Rusterholz. B., and Simon. W.. Anal. Sci.. 1989, 5 , 557. Seilcr, K . . Wang. K.. Kuratli, M.. and Simon. W., Anul. Chim. Actu, 1991. 244, 151. Corsini, A . , Yih, I . M.-L., Fernando. Q.. and Freiser, H., Anal. Chem., 1962. 34. 1090. Wehbcr, P., Fresenius 2. Anul. Chem.. 1957, 158. 10. Cheng, K . L . , and Bray, R. H., Anul. Cliem., 1955. 27, 782. Perrin, D. D.. and Dempsey, B., BuffersforpH and Metal /on Control, Chapman and Hall, London and New York. 1983. Sciler. K.. Dissertation, ETH Zurich. ETH No. 9221, 1990. Bctteridge, D.. Fernando, Q.. and Freiser, H.. Anal. Cliem., 1963, 35. 294. Perrin. D. D., Urgunic Liguntls, Pergamon Press, Oxford, New York. Toronto, Sydney, Paris and Frankfurt. 1979. Shibata. S . . Anul. Cliim. A m . 1960, 23. 367. Agrawal, Y. K.. and Mehd. G. D., Rev. Anul. Cliem., 1982. 6. 185. Paper 1 l03682C Receiiyed July 19, 1991 Accepted September 4, I 991

ISSN:0003-2654    

DOI:10.1039/AN9921700057

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST, JANUARY 1992, VOL. 117 61 Poly(ani1ine): A Conducting Polymer as a Novel Anion-exchange Resin Akheel A. Syed and Maravattickal K. Dinesan Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India Poly(ani1ine) resins were prepared by a one-step redox polymerization of aniline with persulfate in various molar ratios. The resin samples were characterized by elemental analysis, solid-state conductivity and solid-state cross-polarization magic angle spinning 13C nuclear magnetic resonance studies. The resins were used for binary and tertiary separations. Keywords: Poly(ani1ine); conducting polymer; anion-exchange resin; binary and tertiary separations Organic polymers, with conjugated n-electron backbones, display electronic conductivity upon doping.I This novel property, not associated with other commonly used polymers, has attracted considerable attention in recent years.' Polymers of this type are known as conducting polymers. Of late, there has been a marked resurgence of interest in the conducting polymer, poly(ani1ine) (PANI), as indicated by extensive studies.3-33 Unlike other conducting polymers, such as poly- (acetylene), poly(pyrro1e) and poly( thiophene), the conduc- tivity of PAN1 exhibits a strong dependence on solution PH,?-~ oxidation state"-s and water content.".')-I I Furthermore, PANI exists in a number of forms, characterized by the extent of oxidation, i.e., the ratio of amine to imine nitrogen, and the extent of protonation. These different forms can be intercon- verted by acid/base treatment, and/or by a redox These features make PAN1 a novel conducting polymer.1' Poly(ani1ine) can be synthesized by electrochemical methodsI?-ls or by chemical oxidative polymerization of aniline.The hitherto reported applications of PANI synthesized by chemical methods are: (i) as an electrode material in rechargeable batt~ries;l~-"-'-i'5-'~ and (ii) as a chemical indicator.28 I n previous publications's--"' the ion- exchange properties of chemically synthesized PANIs have been described. In the present paper, the synthesis, charac- terization and the potential application of PANI as an anion-exchange polymer are reported. Experimental Reagents All reagents were of analytical-reagent grade and were purchased from Glaxo Laboratories (India).Aniline was doubly distilled before use. Ammonium persulfate was standardized by an iodimetric method before each operation. Silver nitrate solution was standardized by titration with standard sodium chloride solution, using potassium chromate as indicator. All other chemicals were used as purchased. Apparatus A Bruker CXP 300 nuclear magnetic resonance (NMR) spectrometer was used. Conductivity measurements were carried out o n a GR impedence bridge/l608A and a GR capacitance bridge/l615A. Synthesis Poly(ani1ine) was prepared by the oxidative action of ammo- nium persulfate on aniline. Ammonium persulfate (and not any other persulfate salt) was used in order to avoid the presence of unwanted cations. Purified aniline (0.4 mol) was dissolved in 1000 ml of a sulfuric acid solution containing 0.5 mol dm-3 sodium sulfate (pH = 1 ) and the required amount of ammonium persulfate (solid) was added while stirring.The reaction was allowed to continue for 1 h at room temperature (-27 "C). The insoluble precipitate formed after each synthesis was filtered through a sintered glass crucible (porosity 2) and washed with sulfuric acid solution (pH = I ) . The black precipitate was air dried, transferred into a Soxhlet apparatus and washed with acetonit- rile (in order to remove the soluble species) until the extract was colourless. The resultant material was dried in a vacuum desiccator for about 24 h. The black amorphous powder was poly(ani1ine) sulfate. The syntheses were carried out at different persulfate : aniline molar ratios, Z = 0.5, 1 .O, 1.5, 2.0, 3.0, 4.0 or 5.0.Poly(ani1ine) sulfate (1 g) was transferred into a 250 ml beaker containing 100 ml of 1 mol dm--3 HCI and the mixture was stirred for about 12 h (equilibration) at room temperature. The mixture was filtered through a sintered glass crucible (porosity 4). The powder retained in the sintered glass crucible was rinsed with acetonitrile and dried under vacuum. The resultant powder was weighed as poly(ani1ine) chloride. For comparison, the production of poly(ani1inc) chloride was also achieved by direct synthesis, as for poly(ani1ine) sulfate, using a hydrochloric acid solution containing 0.50 mol dm-3 sodium chloride (pH = 1). A 1 g amount of poly(ani1ine) sulfate, poly(aniline) chloride [derived from poly(ani1ine) sulfate] or poly(ani1ine) chloride prepared in HCl medium (pH = 1 ) was transferred into a 250 ml beaker containing 100 ml of a 1 + 1 ammonia solution and the mixture was stirred for about 12 h .The mixture was then filtered and the material retained was dried as described above. The resultant powder was PANI base (PANI-R). The PANIs prepared are represented by the general formula PANI(Z)-X, where Z is the persulfate : aniline molar ratio, and has the value 0 . 5 , 1.0, 1 . S , 2.0, 3.0, 4.0 or S . 0 : and X is SO4'-, C1k0, CI-, or B, where SOJ?- represents counter ions incorporated in the polymer matrix during its formation in sulfuric acid medium; Cl-,, refers to chloride ions imposed on the molecular matrix (replacing sulfate ions) by cquilibra- tion with 1 mol dm-3 HCl; C1-, corresponds to chloride ions incorporated in the molecular matrix during its formation in HCI medium (pH = 1 ) ; and B indicates the base form of the polymer.Ion-exchange Studies The ion-exchange studies with all the PANI samples (50 mesh) were carried out using column techniques. lon-t.xc.hange cupcity Sodium nitrate solution ( 1 mol dm-3) was passed through a glass column (20 x 1.2 cm i.d.) containing 1 g of dry PANI(Z)-CI-(, or PANI(2)-CI-, (dried in an oven for 6 h at SO "C). The elution was continued until the eluted volume wasANALYST, JANUARY 1992, VOL. 117 free of chloride (AgN03 test). The elution was carried out at a flow rate of 0.5 ml min-1. The eluate was collected quantita- tively and made up to the mark in a 100 ml calibrated flask.A 10 ml volume of this solution was pipetted into a conical flask and titrated with standard AgN03 solution (0.02 rnol dm-3) in order to measure the chloride ions eluted from the polymer. From the titre values and mass of the polymer taken, the ion-exchange capacity (in mequiv g- 1) was calculated as, Exchange capacity = No. of equivalents of C1- Mass of polymer taken x 1000 The results are presented in Table 1 A n ion -exchange chroma tograp h y Synthetic mixtures of halide ions, CI-, Br- and I-, were used in this study. Aliquots (2ml) (in the concentration range 0.08-0.25 moldm-3) were passed through a column (20 x 1.2 cm i.d.) packed with approximately 4 g of resin [PANI(Z)- SO4’-, SO mesh size] and preconditioned with 0.25 rnol dm-3 sodium nitrate solution at a flow rate of 0.25 ml min-1.The eluate was collected in sequential 2 ml fractions. Each fraction was transferred into a conical flask and was analysed for the halide ion by titrating with standard silver nitrate solution (0.02 rnol dm-3) using 1% m/v potassium chromate as indica- tor. Separation of CI- and Br-, Br- and I-, or CI- and I- mixtures was also performed on the column containing the PANI resin, and the results are presented in Table 2. Solid-state Studies Conductivity measurements The conductivity experiments were carried out on the pellets pressed on a 1 cm die at a pressure of 2942 N m-2 to a thickness of =3 mm, employing an impedence and a capaci- tance bridge in a dry nitrogen atmosphere. Both sides of the -3 mm pellet were coated with silver in order to render them conducting.The d.c. conductivity of PAN1 compressed pellets (area 7.86 x 10-5 m’) was determined using the logfversus o plots, where f i s the frequency of the a.c. supply and o is the observed conductivity. The frequency-independent conduc- tivity is taken as the d.c. conductivity. The results are presented in Table 3. Cross-polarization magic angle spinning (CPMAS) 13C N M R Carbon- 13 nuclear magnetic resonance spectra were recorded on a Bruker CXP-300 solid state, high resolution spec- trometer, operating at 75.47 MHz (magnetic field 7.05 T). The spe’ctral width was SO kHz and the number of scans was typically of the order of 5 x 103. Results and Discussion In order to select better PANI resin(s) from the samples synthesized in this study, several preliminary ion-exchange studies were carried out on all the PANIs synthesized.The results are presented in Table 1. It was found that PANI- ( 1.0)-S04’- and PANI( 1.5)-S04’- are more suitable as anion-exchange resins compared with the other polymers synthesized here. Although PANI(0.S)-S04’- has the highest ion-exchange capacity, it has two serious limitations: first, its yield is only 4.5% and second, the separation of the halide mixture is poor. Effect of Eluent Concentration Different concentrations of sodium nitrate (eluent) were employed to study the elution behaviour of 2 ml aliquots of C1- ion (0.3-0.4 rnol dm-3) from the exchange columns loaded with different resins. The results for a few selected resins are presented in Fig. 1. From the results it can be seen that with higher concentrations of sodium nitrate, the elution curves shift to the left, as is observed for Dowex 1 or Amberlite.Amongst the PANI resins, good symmetrical curves are seen for PANI( l.0)-S04’- and PANI( 1 .5)-S04’- with 0.25 rnol dm-3 NaN03. The quantitative analysis of a mixture of chloride, bromide and iodide (0.08-0.25 rnol dm-3 of each component) using 0.25 rnol dm-3 sodium nitrate as eluent is not satisfactory, because the iodide is removed too slowly from the column. By applying a more concentrated solution of sodium nitrate as eluent, as soon as the chloride and bromide are removed from the column, the removal of iodide is greatly enhanced. The effect of the concentration of sodium nitrate on the elution behaviour of iodide ion on PANI(l.S)-S04’- is shown in Fig.2. Similar results were obtained with PANI( 1 .0)-S04’-. Analysis of Halide Mixtures The elution graphs of halide mixtures (Cl-, Br- and I-) using PANI(0.S)-S04’-, PANI( 1 .0)-S04z-, PANI( 1 .5)-S04’- and PANI(l.5)-CI-i resins are displayed in Fig. 3. The results Table 1 Ion-exchange studies on PANI resins” Ion-exchange capacity of the Volume capacity Persulfate : aniline Yield of insoluble resin, CI- of the molar ratio ( Z ) Resin polymert (%) form/mequiv g-1 rcsin/mequiv cm-3 Sulfuric acid + 0.5 rnol dm-3 Na2S0, (‘pH = 1)- 0.5 PAN I(0. 5)-SOj’- 45.2 3.58 0.85 1 .0 PANI( 1 .O)-SOj’- 90.2 3.03 0.75 1 .s PANI( 1 .5)-SOj’- 89.2 2.46 0.67 2.0 PANI(2.0)-SOj’- 78.4 1.75 0.44 3.0 PANI(3.0)-SOA2- 60.5 1.03 0.27 5.0 PANI(S.O)-SOj’- 48.5 0.98 0.29 4.0 PANI(4.0)-SOj2- 50.2 1.18 0.36 1 .0 PANI( 1 .0)-CIkl 83.5 I .98 0.50 1.5 PANI(1 .S)-Clkl 82.7 1.38 0.34 4.0 PAN I (4.0) -Cl - I 45.0 0.71 0.22 Hydrochloric acid + 0.5 rnol dm--3 NaCl ( p H = 1)- Separation of 2 ml of C1-. Br- and I- mixture (0.08- 0.25 rnol dm-3 of each component) Poor Good Good N o separation of C1- and Br- No separation No separation No separation Poor Poor No separation * Column = 13.0 x I .2 cm i.d., eluent = 0.25 rnol dm--3 NaN03 for C1-, 0.50 mol dm--3 NaN03 for Br- and 2.OU rnol dm-3 NaN03 for 7’ The anion (monovalent) content of 0.5 per ring is taken into consideration.The cxtrancous water present in the powder is not taken into I-. account. Mere drying in a vacuum desiccator is not sufficient to eliminate water completely.ANALYST, JANUARY 1992, VOL.117 63 Table 2 Quantitative separation of halide anions achieved on a PANI(1 .5)-S04’ - column” Sample No. Cl- - Br - mixtures- 1 2 3 4 5 Br--I - mixtures- 1 2 3 4 5 CI --I- mixtures- 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 Cl- -Br- -1- mixtures 1 2 3 Separation achieved CI - Br- CI - Br- CI - Br- CI - Br- CI - Br- Br- I - Br- I - Br- I - Br- I - Br- I - CI-~ I - CI - 1- CI - I - c1- I - CI - I - CI - I - CI - I -- CI - I - C1- I - CI - I - CI - I - CI - I - CI - I- c1 - I - CI - I - Cl- Br- I - CI - Br- I - CI - Br- I - Molarity of the eluent Volume of (NaN03)/mol dm-3 eluatei/ml 0.25 0 . 50 0.25 0.50 0.2s 0.50 0.25 0.50 0.25 0.50 0.25 I .OO 0.25 1 .00 0.25 1 .00 0.25 1 .00 0.25 1 .oo 0.25 0.50 0.25 0.50 0.25 0.50 0.25 0 . 50 0.25 0.50 0.25 1 .oo 0.25 1 .00 0.25 1 .oo 0.25 1 .00 0.25 1 .OO 0.25 2.00 0.25 2.00 0.25 2.00 0.25 2.00 0.25 2 .00 0.25 0. 50 2 . 00 0.25 0.50 2.00 0.25 0.50 2.00 Similar results were obtained with a PANI( 1 .0)-S04,’- column. -t Volume required to elute all the ions (Cl-. Br- or I - ) . 3 Average of five replicates. 10 20 10 20 10 20 6 20 10 20 14 24 14 24 14 24 12 24 14 22 12 28 14 28 14 28 12 28 14 28 14 22 14 22 14 22 14 22 14 22 14 16 14 16 14 16 12 16 14 16 12 22 18 12 22 18 12 22 18 Amount loaded/mg 9.4 21.9 17.4 40.1 28.5 64.1 5 .0 37.4 17.1 11.4 12.2 19.4 30. I 50.5 48.1 75.9 6.9 50.2 31.6 10.5 4.6 16.5 12.6 44.6 21.3 76.3 4.6 50. 8 14.2 15.8 4.6 16.5 12.6 44.6 21.6 76.3 4.6 50.8 14.2 15.8 4.6 16.5 12.6 44.6 21.3 76.3 4.6 50.8 14.2 15.8 5.8 12.8 20.3 10.7 22.5 38.3 17.6 39.9 63.4 Amount reeoveredS/mg 9.2 21.6 17.2 40.1 28.4 64.2 5 .o 37.4 17.1 11.3 12.2 19.3 29.9 50.6 47.8 75.7 6.6 49.5 31.1 10.6 4.9 15.9 12.4 44.5 21.3 75.2 4.6 51.1 14.2 16.1 4.9 16.5 12.4 44.5 21.3 74.9 4.6 51.2 14.2 16.2 5.0 16.5 12.5 44.5 21.2 75.0 4.6 50.9 14.2 16.1 5.6 12.9 20.1 10.6 22.5 38.6 17.5 39.6 63.6 Relative standard deviation 0.023 0.012 0.013 0.005 0.009 0.005 0.039 0.008 0.012 0.024 0.015 0.013 0.009 0.004 0.006 0.0 14 0.037 0.007 0.01 I 0.027 0.048 0.022 0.014 0.012 0.0 I6 0.006 0.044 0.01 1 0.017 0.017 0.041 0.022 0.106 0.013 0.015 0.0 1 0 0.046 0.008 0.021 0.019 0.047 0.0 I7 0.01 1 0.013 0.018 0.010 0.040 0.01 I 0.014 0.022 0.044 0.020 0.016 0.015 0.008 0.010 0.0 1 0 0.013 0.004 demonstrate that the resolution of halide mixtures with PAN1 synthesized in sulfuric acid medium is better than that of the corresponding resins synthesized in a hydrochloric acid teristic ion-exchange properties (Table 1).medium. Another interesting facet of this study is that, as the 2 value of the polymer increases, the resin loses its charac-64 ANALYST, JANUARY 1992. VOL. 117 140 120 - - 100 80 - - 60 - 40 20 - - 0 - ' - ," 160 0 C .O 140 E 4- 4- : 120 u" 100 0 C 80 60 40 20 0 140 120 100 80 60 40 20 L 0 10 20 30 40 50 60 Volume of eluate/ml Fig. 1 Effect of clucnt conccntration o n the elution of 2 ml aliquots of chloride ion (0.35 rnol dm-3) for ( u ) PANI( 1 .O)-SO,'-, ( h ) PANI( 1 .S)-SO,'-, ( c ) PAN1(3.0)-S04'- and (d) PANI(S.O)-SO,'-: A, 1 .OOO mol dm' NaN03; B , 0.500 rnol dm-3 NaN03; C, 0.250 rnol dm-3 NaN0,; and D, 0.125 rnol dm-3 NaN03.Column dimensions, 13.0 X 1.2 cm i.d. Table 3 D.c. conductivity of a number of PANls Resin Conductivity"'/B-1 cm-1 255 x I O k - 3 413 x 1Ok-1 PANI(l.0)-CI- 230 x 10-6 PANI( 1 .S)-CIk 195 x lo-' PANI( 1 .0)-B 125 x lo-" PANI( 1 .5)-B 203 x lo-" PANI(l.O)-SOJ'- PANI( 1 .S)-SO,'- * Measured on pellets of =3 mm in thickness and 1.86 x lo--' m' in area, using an a x . impcdencc bridge and a capacitance bridge. in a dry nitrogen atmospherc. m 80 m 0 - E 60 m I z -. 40 .c C 0 .- - 20 2 *-' Q) C 0 0 0 A 20 40 60 80 100 Volume of eluate/ml Fig. 2 Effect of eluent concentration on the clution of 2 ml aliquots of iodide ion (0.35 rnol dm-3) for PANI( I .S)-SO,?-: A, 2.00 rnol dm-3 NaNO,; B , 1 .OO rnol dmp3 NaNO,; C, 0.50 rnol dm-3 NaNO,; and D.0.25 rnol dm-3 NaNO,. Column dimensions. 13.0 x 1.2 cm i.d. Characterization As PANIs are insoluble in most of the common organic solvents, only limited information relating to their characteri- zation is supplied by chemical analyses. Raman studies of PANI films and powders have identified the presence ofpara- disubstituted benzene and quinone diimine moieties, and, when taken together with infrared spectra, they have provided evidence for a head-to-tail polymerization of aniline with no ortho incorporation of any groups.30.31 Chemical analysis The elemental analyses were carried out in the Microanaly- tical Laboratory, Calcutta University, Calcutta, India. The elemental compositions for ( i ) PANI( l.0)-S04'- or PANI( 1.5)-S042-, (ii) PANI(l.O)-CI- or PANI(l.5)-CI- and (iii) PANI(l.0)-B or PANI(l.5)-B are consistent with the relative atomic structures: (C6H5.IN)(S04)0,2h( H20)o,h5; (S04)0 01(H20)o.33, respectively. These results clearly show that almost all the sulfate anions initially present (39%) in the polymer have been replaced by chloride as a result of the equilibration of the sulfate forms of PANIs with 1 mol dm-3 HCI. The polymers described above, PANI( 1 .0)-S04'-, PANI(1.S)-S04'-, PANI(l.O)-CI- and PANI(l.5)-CI-, upon equilibration with NH3/NaOH, form neutral species. Thus, the polymers exhibit high resistance, and hence behave as insulators (Table 3). I t has been observed, under a wide range of experimental conditions, that equilibration of an insulating form of PANI with an acid solution incorporates anions in the polymer (thereby making the polymer a conductor), whereas the equilibration of a conducting form of PANI with a basic solution eliminates the counter ions (thereby making the polymer an insulator).Moreover, these reactions are revers- ible. An anion content of 0.5 per ring was observed in most of the syntheses carried out here. Hence, the structures shown in Fig. 4 are proposed for PANIs. (ChH5. "C~)0.5I(SO4)0. I(H2O)0.87 and (ChHS.IN)(C90.0,ANALYST, JANUARY 1992, VOL. 117 60 65 0.25 mol dm-3 /-0.50 rnol d w 3 -1 2.00 mol dm-3 -1 (b) -- NaN03 I NaN03 I- NaN03 I I 2.00 rnol dm-3 -I 0.25 mol dm-3 1-0.50 rnol dm-3 .-+I I- 60 NaN03 +j NaN03 I NaNOs I 60 2.00 rnol dm-3 ~ (d I- 0.25 mol dm-3 L; -0.50 rnol dm-3-’ - - NaN03 I NaN03 I NaN03 I I I I CI-A - - I I I I I I I I t “10 20 30 40 50 60 70 80 90 Volume of eluate/ml Fig.3 Elution graphs for 2 ml aliquots of halide mixtures; column dimensions, 13.0 X 1.2 cm i.d. Eluent: 0.25 rnol dm-? NaNOz for CI-, 0.50 mol dm-i NaNOi for Br- and 2.00 mol dm-? NaN03 for I-. ( n ) , PANI(0.S)-SO12-; ( h ) , PANI( l.0)-SO12-; (c). PANI( 1 .5)SO12-: and ( d ) . PANI(1 .S)-Cl-, The gravimetric ratios of 0.9457 (-t2%) and 0.9562 (&2%) for the dried PANI( 1 .0)-S04’- : PANI( 1.0)-Cl and PANI( l.0)-S04’- : PANI( 1 .O)-B, respectively, are in agree- ment with the results of elemental composition data. Support- ing evidence for the proposed structure is provided by conductivity data and NMR (CPMAS). Conductiivity Electrical charges, delocalized on the polymer matrix, are responsible for the d.c.conductivity of the resin. The conductivity data are presented in Table 3. The concomitant increase in conductivity of PANI(2)-B upon treatment with H2S04 or HCI is due to the conjugation of the n-electron system in the polymer chain. From the data it can be observed that equilibration of poly(ani1ine) sulfate with HCI reduces the conductivity of the resultant poly( aniline) chloride. N M K studies The CPMAS I T NMR spectra resolve the bcnzenoid and quinonoid ring structure of the PAN1 base only.32-33 The typical 13C NMR spectra are shown in Fig. 5. The PANI( 1 .O)- B or PANI( 1 .S)-B shows a well defined structure with three peaks, while PANI( 1 .0)-S042-/CI- or PANI( 1 .S)-S04’-/C1- gives a broad structureless spectrum.Although these peaks are broader than those in Fig. 5 , curve B, their position, as far as it can be determined, remains the same (curve A). This suggests that the transition from the conducting to the insulating form involves the removal of protons and anions, with no further alteration of the polymer structure. Selective polarization experiments prove that the high-field peak [ 122.2 ppm from tetramethylsilane (TMS)] originates from the proton-bonded carbons (2,3,5,6) of the benzenoid rings, whereas the low-field peak is from carbons 1 and 4 of the66 ANALYST, JANUARY 1992, VOL. 117 c c n (3 1 Fig. 4 form and (3) base form Proposed structures for PANI: (1) sulphate form, (2) chloride 250 200 150 100 50 0 6 (ppm) Fig. 5 13C NMR spectra of PANI: A. PANI(l.5)-CI- and B, PANI( 1 S)-B quinonoid rings (156.7 ppm from TMS).The signal at about 137.5 pprn is from carbons 2,3,5 and 6 of the quinonoid rings and carbons 1 and 4 of the benzenoid rings. After curve resolution and integration, the observed peak intensities are in the ratio 6.00: 4.87 : 1.01 for the peaks at 122.2, 137.5 and 156.7 ppm, respectively. This matches the expected peak intensity ratio of 6 : 5 : 1 in the PANI-B structure (3) shown in Fig. 4. Conclusions The studies on the ion-exchange properties of different PANIs have shown that PANI( 1 .0)-S042- and PANI( 1 .5)-S04'- are suitable as anion-exchange resins. The advantages include the inexpensive starting materials and the simple method of synthesis. It is suggested that the electronic conductivity of PANI, combined with its ion-exchange properties, will open up new technological applications.This phenomenon is also likely to be useful in understanding the chemistry and physics of PANIs. The authors are grateful to K. M. Dastur, formerly at the Central Food Technological Research Institute, Mysore, and Professor D. S. Mahadevappa, University of Mysore, Mysore, for valuable discussions in connection with this work. M. K. D. gratefully acknowledges the award of a Senior Research Fellowship by the University Grants Commission, New Delhi. 1 2 3 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 References Frommer, J. E., and Chance, R. R., in Encyclopediac?fPolymer Science and Engineering. eds. Grayson. M., and Kroschwitz, J., Wiley. New York.2nd edn.. 1986, vol. 5 . pp. 462-507. Handbook of Conducting Polymers, ed. Skotheim. T. A , , Marcel Dekker, New York, 1986, vols. I and 2. MacDiarmid, A. G., Chiang. J . C., Huang, W. S . , Humphrey. B. D.. and Somasiri, N. L. D., Mol. Cryst. Liq. Cryst.. 1985, 125, 309. Chiang, J . C., and MacDiarmid, A. G.. Syntlz. Met.. 1986, 13. 193. MacDiarmid. A. G.. Chiang. J . C., Richter, A. F.. and Epstein. A. J.. Synrli. Met., 1987, 18. 285. Fockc. W. W.. Wnek, G. E., and Wei, Y., J . Phys. Chem.. 1987, 91. 5813. Travers, J . P., Chroboczek, J., Devreux. F.. Genoud, F., Nechtschein, M.. Syed, A. A., Genies, E. M.. and Tsintavis, C., Mol. Cryst. Liq. Cryst.. 1985, 121, 195. McManus, P. M., Yang. S . C., and Cushman, R. J . , J . Chem. Soc., Chem. Commun.. 1985, 1556.Nechtschein, M., Santier. C., Travers. J. P., Chroboczek. J., Ailx, A., and Ripert, M., Synth. Met.. 1987, 18, 311. Angelopoulos, M., Ray, A., MacDiarmid, A. G.. and Epstein, A. J . , Synrh. Met.. 1987, 21, 21. Travers, J . P., and Nechtschein, M.. Synth. Met.. 1987.21, 135. Huang, W. S.. Humphrey, B. D., and MacDiarmid, A. G., J. Chem. SOC., Faraday Trans. I , 1986, 82, 2385. Genies, E. M., Syed, A. A., and Tsintavis. C., Mol. Cry~t. Liq. Cryst.. 1985. 121, 181. Genies. E. M.. and Tsintavis, C . , J . Electroanal. Chem., 1985. 195, 109. Genies, E. M., and Tsintavis, C.. J . Elecrroanal. Chem., 1986. 200. 127. Diaz. A. F.. and Logan, J . A., J . Electroanal. Chem., 1980,111, 111. Noufi. R.. Nozik, A. J . , White, J., and Warren, L. F . , J. Electrochem. Soc., 1982.129, 2261. Kitani, A., Izumi, J . , Yano, J., Hiromoto. Y.. and Sasaki, K . , Bull. Chem. Soc. Jpn., 1984, 57, 2254. Willstatter. R., and Dorogi. S . , Ber. Drsch. Chem. Ces., 1909. 42, 2147. Green, A. G., and Woodhcad, A. E., J . Chem. Soc., 1910,97. 2388. Green. A. G., and Woodhead, A. E., J . Chem. SOC.. 1912.101, 1117. DeSurville, R.. Jozefowicz. M.. Yu, L. T.. Perichon, J., and Buvet, R., Electrochim. Acfa, 1968, 13, 1451. Syed, A. A., Genies, E.. and Santier, C., in Materials forSolid State Batteries, eds. Chowdari, B. V. R., and Radhakrishna, S., World Scientific, Singapore, 1986. pp. 435439. Syed, A. A.. Dinesan, M. K., and Genies, E. M., Bull. Elecrrochem. (India), 1988. 4. 737. MacDiarmid, A. G., Mu, S. L., Somasiri, N . L. D.. and Wu, W.. Mol. Cryst. Liq. Cryst., 1985, 121, 187. MacDiarmid, A. G., Yang. L. S . . Huang, W. S . , and Humphrey, B. D.. Synth. Met.. 1987, 18, 393. Mermilliod, M.. Tanguy, J . , Hoclet, M., and Syed. A. A., Synth. Met., 1987, 18, 359. Syed, A. A., and Dinesan, M. K., Synth. Met., 1990.36. 209. Syed, A. A., and Dinesan, M. K., in Solid State Ionic Devices. eds. Chowdari, B. V. R., and Radhakrishna, S . , World Scientific, Singapore, 1988. pp. 481-487. Kuzmany. H., Genies, E. M.. and Syed, A. A., Springer Series in Solid State Sciences, Vol. 63, Springer-Verlag, Berlin, 1985. Furukawa, Y.. Hara, T.. Hyodo. Y., and Harada, I . , Synth. Met., 1986, 16, 189. Devreux. F.. Bidan, G., Syed, A. A., andTsintavis, C., J . f h y s . (Paris), 1985,46, 1595. Kaplan, S . . Conwell, E. M.. Richter, A. F., and MacDiarmid. A. G.. J . Am. Clzem. Soc., 1988, 110, 7647. pp. 223-226. Paper 01038135 Received August 21, I990 Accepted July 24, 1991

ISSN:0003-2654    

DOI:10.1039/AN9921700061

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST, JANUARY 1992, VOL. 117 67 Development and Evaluation of Analytical Methodology for the Determination of Aflatoxins in Palm Kernels Sadat Nawaz School of Biological and Chemical Sciences, Thames Polytechnic, Wellington Street, London SE 18 6PF, UK Raymond D. Coker" Mycotoxins Section, Natural Resources Institute, Chatham Maritime, Central Avenue, Chatham, Kent ME4 4TB, UK Stephen J. Haswell School of Chemistry, The University of Hull, Hull HU6 7RX, UK A rapid, simple and reproducible method for the simultaneous estimation of aflatoxins AFB1, AFB2, AFG1 and AFG2 in palm kernel samples has been developed by optimizing the sample preparation, solvent extraction, sample clean-up and quantification procedures. The aflatoxins are extracted from a slurried palm kernel sample with an acetone-water (80 + 20, v/v) mixture and the crude extract is cleaned up by solid-phase extraction using a phenyl bonded phase cartridge.The extract is passed through the cartridge with a water-methanol (93 + 7) mixture. Subsequent elution of the aflatoxins retained on the cartridge is achieved with a 3 m l aliquot of chloroform. The aflatoxin content of the eluate is quantified using a bi-directional high-performance thin-layer chromatography procedure. A critical evaluation of the proposed method was carried out by statistical comparison with the British Standard Method. The proposed procedure was shown t o be more efficient and precise. Consistent recoveries of over 90% were achieved from spiked palm kernel extracts and detection limits were found t o be 3.7, 2.5, 3.0 and 1.3 pg kg-1 for AFB1, AFB2, AFG, and AFG2 aflatoxins, respectively. Keywords: A flatoxin; palm kernel; sample preparation; solid-phase extraction; high-performance thin-layer chromatography Aflatoxins are highly toxic secondary metabolites of the moulds Aspergiffus ffavus and A.parusiticus. The moulds thrive on substrates rich in carbohydrates and lipids under the conditions of high temperature and relative humidity' that occur in tropical countries. The oil palm Efueis guineensis Jucquin3 exists in wild, semi- wild and cultivated states in the equatorial land areas of Africa, South East Asia and America. The main product of the palm is the fruit, which is rich in palm oil. The fruit also contains a nut, which is cracked in order to obtain the palm kernel.Palm kernels, which on average form 20% by mass of the whole fruit, are typically composed of at least 50% oil. Palm kernel oil is used mainly in the food and detergent industries. Palm kernel cake and meal, by-products of the palm kernel oil extraction process, are used as protein supplements in compound animal feeds.4 These tropical areas provide ideal conditions for mould growth and the production of aflatoxins on palm kernels5 both at the pre- and post-harvest stages. Damage caused to the kernels due to the mechanical cracking and insect infestation in addition to poor storage conditions can aid mould infection. The introduction of legislation to limit the levels of aflatoxins in foods and feedstuffs in over 56 countries6 necessitates the development of efficient analytical methods for quality control purposes.The British Standard Method (BS 5766) for the determina- tion of aflatoxins in palm kernels7 is a slow and costly procedure involving a chromatographic clean-up stage using a silica gel based column with quantification by thin-layer chromatography (TLC). A method using a combination of an immunoaffinity clean-up stage and quantification by high- performance liquid chromatography (HPLC) has also report- edly been applied to the analysis of palm kernels.8 However, no data describing the efficiency of the method have been *: To whom correspondence should be addressed. published. No other methods have been reported to date for the determination of aflatoxins in palm kernels apart from the methods described above.Consequently, a demand has arisen for a simple, rapid, accurate and reproducible assay method for the determination of aflatoxins in palm kernels. Solid-phase extraction procedures, which are ideally suited to automation, have greatly simplified the sample clean-up stage.Y The use of non-polar phenyl (PH) bonded phase cartridges for the clean-up of maize10 and peanut butter" extracts with subsequent quantification using high-perfor- mance thin-layer chromatography (HPTLC)l* has been re- ported. In this paper, the development and validationl3-15 of a method suitable for the determination of aflatoxins in palm kernel samples is described. It should be noted that, owing to the highly toxic nature of aflatoxins, extreme care16 must be taken when carrying out the procedures described in this paper.Experiment a1 Apparatus An Apex knife mill (3 mm screen) was used for grinding the kernels and a rotary cascade divider (Pascall Engineering, Crawley, UK) was employed for sub-sampling the ground samples. Waring blenders (Dynamics Corporation of America, New Hartford, C?', USA) were used for slurry preparation (4 I) and for the acetone-water extraction (1 1). Whatman No. 1 filter-papers were used for the filtration of blended mixtures. The clean-up apparatus, supplied by Jones Chromato- graphy (Hengoed, Mid-Glamorgan, UK), consisted of a vacuum manifold (Vacelut, A 1600) used in conjunction with a disposable 500 mg PH bonded phase cartridge (Cat. No. PH; 608303) selectively coupled with 4 ml (Cat. No. 600400), 25 ml (Cat.No. 602500) or 75 ml (Cat. No. 607500) reservoirs using68 ANALYST, JANUARY 1992, VOL. 117 1 2 75 ml reservoir - Phenyl cartridge 25 ml reservoir Phenyl cartridge Anhydrous sodium sulfate column Fig. 1 Palm kernel extract clean-up using bonded phase cartridges prior to HPTLC quantification. 1. Cartridge activation: pass 1 ml of methanol followed by 10 ml of water through the cartridge. 2, Extract clean-up: (i) 5 ml of aqueous acetone treated with 1 ml of lead acetate solution. (Addition of 1 g of Celite also aids the clean-up process.) (ii) Pass 5 ml of the treated aqueous acetone sample extract through the cartridge together with 5 ml of methanol and about 63 ml of water. under vacuum, at a rate of 1 0 ml min-1. 3 , Washing the cartridge: wash with 1 0 ml of water and dry the cartridge (2 min).4. Aflatoxin elution: with 3 ml of chloroform adapters (Cat. No. 636001) and Luer stopcock (Cat. No. A 16078) (Fig. 1). The glass vials (8 ml) used for eluate collection and work-up were supplied by Merck (Cat. No. 215/0073/05). Additional equipment used for the British Standard Method included a wrist action shaker (Voss Instruments, Maldon, Essex, UK), glass columns (22 x 300 mm), and a Buchi rotary evaporator (Laboratorium-Technik AG CH- 9230, Flawil/Schweiz, Switzerland). A sample concentrator (Tecam, UK, DM-Block DB-3) was used for drying chloroform extracts prior to quantification. A Perkin-Elmer Lamda 3 ultraviolet-visible (UV/VIS) spectro- photometer was used to determine the concentrations of the aflatoxin standard and spiking solutions.The aluminium backed HPTLC plates were supplied by Merck (Cat. No. 5547). The plates were spotted using an automated TLC sampler (Camag Cat. No. 27200). A conventional TLC tank and a continuous linear TLC tank were employed during the chromatographic development of the plates. 1' The TLC scanner I1 (Cat. No. 76610) and TLC integrator SP 4270 (Cat. No. 76650) (Camag, Switzerland) controlled by a personal computer with link-up software (Quadrant Scientific, UK) were employed for the densitometry measurements. 17 Reagents Lead acetate (20%) solution was prepared as described by Stoloff.18 All chemicals and solvents were AnalaR or HPLC grade (Merck) and distilled water was used throughout. Aflatoxin Standards Crystalline aflatoxin standards purchased from Sigma were diluted to concentrations of about 10 pg ml-1 in a mixture of benzene-acetonitrile (93 + 2) and the exact concentrations of the solutions were determined by UV absorbance measure- ments.l y Standard solutions, prepared by diluting the above solutions to give aflatoxin concentrations of 1.0 pg ml-1 (AFB,, AFGl) and 0.5 pgml-1 (AFB?, AFG?) in benzene- acetonitrile (98 + 2), were stored at -20 "C. [Caution: Aflotoxins are carcinogenic to humans. Gloves and other protective clothing must be worn for all operations involving the handling of these compounds or their solutions. All work should be carried out in a well ventilated area.161 Palm kernel samples collected from West Africa during the autumn of 1988 were used during this investigation.Procedure Sample preparation Riffle division. A finely ground palm kerncl sample (1 kg) was divided into six sub-samples, using a rotary cascade sample divider (spinning riffle). The aflatoxin content of each sub-sample was determined in replicate ( n = 4) using the proposed method. Comparison of methods used in sample dillision. A finely ground palm kernel sample (1.8 kg) was divided into two parts using a rotary cascade divider. One of the 0.9 kg sub-samples was slurried with 1.35 1 of water (1 : 1.5 m/v) in a 4 1 blender at high speed for 3 min. Ten aliquots (100s) of the resultant slurry were individually blended at high speed in a 1 1 blender with 240 ml of acetone for a further 3 min. Each of the ten mixtures was then filtered through a Whatman No.1 filter- paper. The aflatoxin content of each filtrate was determined using the proposed method. The other 0.9 kg sub-sample was divided into eighteen 50 g sub-samples using a rotary cascade divider. Ten of the sub-samples were individually blended with 300 ml of acetone and 75 ml of water in a 1 I blender and the resultant mixtures were filtered and analysed by solid-phase extraction and bi-directional HPTLC. Optimization of the solvent extraction procedure Choice of solvent. Sets of five aliquots (100 g) of palm kernel slurry (1 : 1.5 m/v) were blended in a 1 1 Waring blender, with 240 ml portions of five different acetone-methanol mixtures (Table 3). The ratio of water to organic phase in the solvent mixture remained constant at 20 + 80. The resultant mixtures were analysed by solid-phase extraction and bi-directional HPTLC.Optimization o f the soliient : sample ratio. Sets of five aliquots of palm kernel slurry were extracted with mixtures of acetone-water with solvent : sample ratios which ranged from 7.5 : 1 to 25 : 1 (Table 4). Effectirieness of the extraction procedure. Replicate 100 g palm kernel slurries (60 ml of water : 40 g of sample) were each blended with 240ml of acetone and filtered through a Whatman No. 1 filter-paper. The first 230ml of the filtrate were collected in a measuring cylinder and taken through the clean-up and quantification procedures. The residue (which contained 70 ml of solvent mixture) and the filter-paper were transferred into a Biichner funnel and washed with excess (approximately 480 ml) of acetone-water (80 + 20), the wash was made up to a final volume of 500 ml and quantified for aflatoxins.The residue in the Buchner flask was then blended with 250ml of acetone-water (80 + 20), filtered, cleaned-up and again quantified for aflatoxins. Use of lead acetate for the clean-rip The minimum amount of lead acetate required to achieve a sufficient precipitation of the colloidal components present in the crude acetone-water (80 + 20) extracts was investigated. Crude sample extracts were mixed with various volumes (0-5 ml) of a 20% m/v lead acetate solution, followed by the proposed clean-up and quantification steps. The process was duplicated for each volume of lead acetate. Composition of the mobile phase Use of acetic acid. Aliquots ( 5 ml) of palm kernel extracts were applied to pH bonded phase cartridges along withANALYST, JANUARY 1992, VOL.117 69 aliquots (63 ml) of acetic acid solutions (0-3% v/v) and quantified using the proposed method. The process was duplicated for each acetic acid concentration. Volume of methanol in the mobile phase. Various volumes of methanol and the crude acetone-water extract were applied to the PH cartridge along with various volumes of distilled water, and the cleaned-up extracts were quantified using bi-directional HPTLC. Elution and work-up stage Two experiments were conducted in order to optimize the elution and work-up stages. Firstly, 4.8 ml of a mixed aflatoxin solution in benzene-acetonitrile (98 + 2), was evaporated under nitrogen at 45 "C on a sample concentrator.The residue was dissolved in 120 ml of an acetone-water mixture (80 + 20) and homogenized. Twenty four aliquots ( 5 ml) of the above solution were each treated by the PH bonded phase clean-up procedure. Replicate elutions ( n = 4) of the aflatoxins were carried out with varying amounts of chloroform (1, 2, 3, 4, 5 and 7 ml). The chloroform extracts were dried and quantified, for the aflatoxins, using bi-directional HPTLC. In the second experiment, 42 equal volumes (200~1) of a mixed aflatoxin standard solution in benzene-acetonitrile (98 + 2) were evaporated to dryness. The residues were reconsti- tuted in seven different volumes of chloroform (1-7 ml) to give six replicates at each volume. Solvent evaporation was performed under a gentle stream of nitrogen at 45°C on a sample concentrator.The times required to evaporate increas- ing volumes of chloroform are summarized in Table 5. Aflatoxin quantification was carried out using bi-directional HPTLC. Comparison of Analytical Methods Two 1 .0 kg samples of naturally contaminated (approximately 100 and 600 pg kg-* of AFB!) ground palm kernels were halved using a rotatory cascade divider, to produce two representative sub-samples (500 g), [IA and IB (100 v g kg-1 AFBI) and IIA and IIB (600pgkg-1 AFB1)] at each contamination level. British Standard Method7 Sub-samples IA and IIA were each divided into ten equal 50 g portions. Each portion was shaken with 25 ml of water, 250 ml of chloroform and 25g of diatomaceous earth in a 500ml stoppered flask, on a wrist action shaker, for 30min.The mixture was passed through a Whatman No. 1 filter-paper and the filtrates were combined to give bulked extracts, one at each level of contamination. The following procedure was repeated ten times for each of the bulked extracts. A chromatographic column was prepared by two thirds filling a glass column (22 x 300mm) with chloroform, to which anhydrous granular sodium sulphate ( 5 g) was added, followed by the addition of silica gel (log) as a chloroform slurry. After the silica gel had settled, a further 10 g of sodium sulfate were added. The chloroform was allowed to drain until it just reached the upper surface of the sodium sulfate layer. A 50 ml portion of the bulked extract was mixed with 100 ml of hexane in a 250 ml measuring cylinder and the solution was quantitatively transferred into the chromatographic column.The solution was allowed to pass through the column at a rate of about 10 ml min-1 until it was level with the upper surface of the sodium sulfate layer. The column tap was then closed and 100ml of anhydrous diethyl ether were added to the column. The tap was then reopened and the eluate allowed to flow until it was again level with the upper surface of the sodium sulfate layer. The aflatoxins were eluted with 150 ml of a chloroform-methanol mixture (50 + 50), the eluate was collected in a 500 ml round-bottomed flask and evaporated to dryness on a rotary evaporator. The residue was quantitatively transferred into an 8 ml glass vial with a small amount ( 5 ml) of chloroform. Proposed bonded phase clean-up method The sub-samples IB and IIB (500g) were each slurried by blending at high speed €or 3 min, with 750 ml of water in a 4 I Waring blender.A 100 g portion, from each of the two slurries, was extracted by blending with 240 ml of acetone in a 1 1 Waring blender and filtered through a Whatman No. 1 filter-paper. An aliquot ( 5 ml) of the crude extract was mixed with methanol ( 5 ml), 20% m/v lead acetate solution (1 ml), Celite (1 g) and water (63ml). The mixture was then applied to a solvated PH bonded phase cartridge at a rate of 10ml min-I. (The solvation step involved the passage of 10ml of methanol followed by 10ml of water through the cartridge.) After washing the cartridge with 10 ml of water, the aflatoxins were eluted using 3ml of chloroform.The eluate was passed through a 4 ml reservoir containing anhydrous sodium sulfate, to eliminate any water, and collected in an 8 ml glass vial. The above procedure was carried out under reduced pressure on a Vacelut vacuum manifold. Work-up. Each chloroform solution was evaporated under a steady stream of nitrogen at 45 "C on a sample concentrator in order to give clean, dry sample extracts. Quantification using bi-directional HPTLC. High-perfor- mance TLC plates (20 x 20 cm) were cut in half and immersed in methanol for 1 h in order to remove atmospheric contami- nants deposited on the plate during storage. The plates were dried for 5 min in an oven at 100°C, situated in a fume cupboard, and stored in a desiccator until required. The following procedures were performed under darkened conditions.The clean, dried sample extracts were dissolved in 300 pI of a benzene-acetonitrile mixture (98 + 2). Aliquots of the above extracts ( 5 PI) along with external standards (1 p1 aliquots of a mixed aflatoxin solution) were applied, using an autosampler, as a row of spots at 5 mm intervals 3 cm from the top edge of an HPTLC plate. One 10 x 20 cm HPTLC plate accommodated up to 30 sample spots together with three standards along the 20 cm edge. A strip of silica gel (approximately 3mm wide) was removed from the edges of the plate, parallel to the direction of development, in order to eliminate edge effects. A further sample clean-up was performed by developing the plate in 20 ml of diethyl ether for 17 min in a continuous horizontal tank.[NB. The eluent reached the top of the plate within 3-4 min. Continuous development was then allowed to proceed for a further 13-14 min, hence there is no solvent front.] The plate was dried for 3min in a darkened chamber under a stream of nitrogen and the top portion (2cm) of the plate, containing sample interferences, was cut and removed using a sharp knife. The plate was rotated through 180" and developed for 20min in a conventional TLC tank, using 20ml of a chloroform-xylene-acetone (CXA) (6 + 3 + 1) mixture for 20 min. After drying ( 5 min) the bottom portion (1 cm) of the plate was removed, as before, in order to decrease the development time and improve the resolution of the aflatoxin spots. This was followed by another CXA development (16 min).These times for the CXA development were found to be optimum for the conditions existing during the method development. Factors such as temperature variations may have a slight effect on the method and should be modified accordingly until the solvent front is approximately 1 cm from the top of the plate. The plate was finally dried (1 min) in a fan assisted oven at 100 "C before quantification of the aflatoxins by UV fluorescence in the reflectance mode using the TLC I1 scanner supported by appropriate software. 17 Statistical Evaluation of the Proposed Method Nine 30ml aliquots of aflatoxin-free crude palm kernel extracts and nine 30 ml aliquots of an acetone-water (80 + 20)70 ANALYST, JANUARY 1992, VOL. 117 mixture were each accurately spiked with an acetone-water (80 + 20) solution of aflatoxins, in order to produce two sets of nine concentrations in the range 0-300 pg kg-I for AFBl and AFG1, and 0-150pgkg-1 for AFB2 and AFG?.Aliquots (5m1, in replicates of six) from each of the spiked crude extracts and solvent mixtures were processed using the proposed clean-up method followed by quantification using HPTLC. Nine solutions of aflatoxins in benzene-acetonitrile (98 + 2) within the same concentration range of 0-300 pg kg-1 for AFRl and AFG, and 0-150 pg kg-l for AFB? and AFG2 were also quantified by bi-directional HPTLC. Calculation Effective weight The effective weight of the extracts from the proposed clean-up procedure were calculated using the following equation: Vextract X Msample (Mslurry - Msampie) + Vsolvent) Effective weight = where Vextract = volume of extract = 5 ml; Msample = mass of sample in the slurry; Mslurry = mass of slurry = 1OOg; and Vsolvent = volume of the organic solvent = 240 ml.The mass of sample in the slurry is given by mass of sample slurried mass of the total slurry X Mslurry Msampie = If the mass of sample slurried = lOOOg, and the mass of the total slurry = 2500g then Msampie = 100 x 1000/2500 = 40g Hence, 5 x 40 Effective weight = = 0.667 g (100 - 40) + 240 Spiking volume The volumes of the spiking solution required to produce an artificially contaminated palm kernel extract at a specified level was calculated using the following equation D x effective weight V = C where V = volume of the spiking solution required (PI); D = desired contamination level (pg kg-I); and c = concentration of the spiking solution (pg ml-1). Results and Discussion Method Development The results discussed below refer to AFBl only (unless otherwise stated), as AFBl was the major toxin found in naturally contaminated samples in this study.Sub-sampling and sample preparation It was important to establish early in this work whether the sub-sampling of ground palm kernels would introduce bias into the analytical procedures. The sub-sampling, using a rotary cascade divider, and the analysis of a 1 kg sample of ground palm kernels facilitated the separation of variances due to both the sub-sampling and analytical procedures. From the analysis of variance (ANOVA) calculations, these variances were found to be 4.67 and 81.728, respectively (Table 1).The calculated value of F was found to be 1.229, compared with the critical value for Fs,18 (p = 0.05) of 2.773, thus confirming that the variance due to sub-sampling did not significantly differ from zero. Table 1 Analysis of variance for AFBl to estimate the variances associated with spinning riffle sub-division. Sum Degrees Variance of of Mean com- Between sub-samples 502.04 5 100.41 4.67 Within sub-samples 1471.10 18 81.73 81.73 Source of variation squares freedom squares ponent - - Total 1973.14 23 Table 2 Comparison of sample preparation techniques Dry sampling Slurry Parameter method method Mean recovery of AFB ,/pg kg- 1 120.5 207.2 Variance 105.4 340.0 Standard deviatiodpg kg- 1 10.3 18.4 Relative standard deviation (Yo) 8.9 8.5 No.of replicates, n 10 10 Computed t statistic = 13.0 Ratio of variances = 3.2 F9,Y 0, = 0.95) = 4.03 t 0, = 0.05) = 2.1 Table 3 Comparison of solvent systems for the extraction of aflatoxins Composition of acetone- methanol- water (%) a, 80+00+20 b, 70 + 10 + 20 c, 60 + 20 + 20 d, 40 + 40 + 20 e. 00 + 80 + 20 * n = 5 . Average AFB I extracted*/ - Yg kg-I 475.2 441 .5 433.8 409.2 360.7 95% confidence interval*/yg kg-1 Upper Lower 492.6 457.4 465.5 417.5 456.1 41 1.5 420.1 398.3 394.7 326.7 Standard deviation/ 14.0 19.3 18.0 8.8 27.4 P8 kg- I The production of sub-samples using a rotary cascade divider (spinning riffle) was compared with the slurry method”) by preparing two equivalent 0.9 kg samples of ground palm kernels according to the two sample preparation methods.Analytical results obtained using each of the sample preparation methods showed good repeatability, with relative standard deviations of 8.9 and 8.5% for the spinning riffle and slurry techniques, respectively. Statistical analysis of the results (Table 2) showed that there was no significant difference at the 5% significance level between variances associated with the methods. However, the slurry technique was found to extract significantly more aflatoxins than the dry extraction method. Optimization of extraction procedure f o r aflatoxins Choice of solvent. Solvent systems composed of acetone- methanol-water in various ratios were assessed for their abilities to extract aflatoxins from palm kernel samples. Inspection of the confidence intervals (Table 3) for the mean recoveries of AFB, indicated that solvent mixture ‘a’ ex- tracted significantly more AFBl than the other solvent systems with the exception of solvent system ‘b’.However the t-test used to compare the mean extracted value of AFB using the solvent systems a and b ( t = 3.16 and critical value for t (p = 0.05) for 8 degrees of freedom = 2.31), showed that the solvent system a extracted significantly more of AFBl than system b at the 95% level of confidence. An F-test showed that the variance associated with the solvent system a did not differ significantly from the variances for the other extraction mixtures. Optimization of tfie extraction solvent volume. Slurried palm kernel samples were extracted with varying volumes of the acetone-water mixture in order to optimize the sample : sol- vent ratio.The recovery results for AFB, (Table 4) indicated that changing the ratios did not lead to a pronounced change inANALYST, JANUARY 1992, VOL. 117 71 Table 4 Volume of acetone-water (80 + 20) mixture required for the efficient recovery of aflatoxin Amount of slurry/g 100 75 60 50 35 25 * n = 5 . Volume of water added/ml 0 9 21.6 30 35 35 Volume of acetone added/ml 240 2 16 230.4 240 224 200 Total solvent volume/ml 300 270 288 300 280 250 Solvent: sample ratio 7.5: 1 9 : 1 12: 1 15: 1 20: 1 25 : 1 Effective weight/g 0.67 0.56 0.42 0.33 0.25 0.20 95% confidence interval for AFB, extracted*/yg kg-I Upper Lower 320.3 277.6 326.5 275.6 332.9 268.8 308.2 286.3 291.2 254.4 297.4 250.9 the quantity of aflatoxins extracted from the palm kernel samples.The lowest possible solvent : sample ratio of 7.5 : 1 v/m was adopted as it afforded the highest effective weight and, hence, lower detection limits. Consequently, the extraction of a 100 g slurry containing 40 g of sample and 60 ml of water, with 240 ml of acetone, was adopted as the optimum extraction procedure. Effectiveness of the extraction procedure. The investigation showed that the first 230 mi of filtrate collected contained 2.03 pg of AFB1. The residue and the filter-paper were washed with an excess of the solvent and contained 0.61 pg of AFB1. Quantification of the third extract failed to show the presence of any aflatoxin. From these results, it would appear that the initial extraction with 300 ml of acetone-water (80 + 20) solvent mixture removed all the aflatoxins from the matrix.However, owing to the difficulty in recovering all of the solvent approximately 70 ml (23.33%) remained unfil- tered. The concentrations of aflatoxins (8.83 and 8.71 pgml-1) in these two fractions were not found to be different, using a t-test, at the 95% level of significance, indicating that no gradient was present in the two portions of the solvent mixture analysed. Precipitation of interfering components prior to column clean-up Lead acetate18 solution has previously been used to eliminate, by precipitation, proteins, lipids and other colloidal com- ponents from cottonseed extracts. However, because of the toxicity of lead acetate, studies were carried out in order to minimize its usage.Visual inspection of the extracts obtained from the bonded-phase clean-up, showed that the use of low levels of lead acetate afforded oily residues. This was particularly apparent when no lead acetate was used. The presence of the oily residues artificially increased the volume of the cleaned-up benzene-acetontrile extract, causing a dilution effect and an increase in interferences, which led to lower recoveries. As the volume of lead acetate was increased above 1 mi, no differences were observed in the aflatoxin recoveries or the cleanliness of the extracts. Hence, it was concluded that 1 ml of lead acetate solution would be sufficient for the satisfactory precipitation of interfering colloidal components from palm kernel extracts. Celite washed with methanol was also used as a filter aid.composition of the mobile phase In a previous method reported for the analysis of maize,") the mobile phase was composed of a methanol extract in a 1% acetic acid solution. In these studies, the presence of acetic acid in the mobile phase appeared to have no effect on the aflatoxin retaining properties of the PH bonded phase cartridges. More importantly, however, a visual comparison of the resultant extracts showed that the acid-free mobile phase led to the cleanest extract. The presence of methanol has been reported to have a significant effect on the retention of aflatoxins o n the PH I 100 90 - 8 H 8o > 8 70 a, U 60 50 0 2 4 6 Volume of chloroform/ml Fig. 2 Effect of changing the volume of chloroform as eluting solvent on the percentage recoveries of aflatoxins.A, AFB,; B, AFB2; C, AFG,; and D, AFG2 Table 5 Effect of drying times on aflatoxin recoveries Volume of chloro- form/ml 1 2 3 4 5 6 7 * n = 6 . Average drying ti me/m i n 8 13 18 23 27 32 37 Average recovery* 99.6 93.5 93.8 81.7 82.6 82.2 73.2 ( Y o ) 95% confidence interval* Upper 102.2 96.5 96.9 83.5 86.0 84.1 75.2 Lower 97.0 90.5 90.7 79.9 79.2 80.3 71.2 cartridges.?' This was confirmed in the present study as a better retention of the aflatoxins contained in the acetone- water extracts was achieved when methanol was added to the extracts. The volume of methanol required was found to be equal to the volume of acetone in the mobile phase. However, as the proportion of the organic phase (acetone and methanol) was increased to above 20%, the recoveries of aflatoxin decreased.It was concluded, therefore, that the mobile phase should be composed of 5ml of acetone-water extract, 5ml of methanol, 1 ml of lead acetate solution (20%) and 63 mi of distilled water. Elution and work-up stage Two experiments were performed to optimize the elution and work-up stages. The fifst experiment, conducted to optimize the volumes of chloroform required for the maximum elution, showed that the recoveries of aflatoxin initially increased with an increase in the elution volume (Fig. 2). However, as the volume of the chloroform approached the 2ml level, the recoveries levelled off and gradually dropped as the volume of72 100 90 8 80 ' 70 h v B 8 a 60 chloroform was increased above 4 ml. This apparent loss of aflatoxins was investigated further in the second experiment.The analyses of variance [Fratio = 84.3, F6,35 (p = 0.05) = 2.41 of the results from the second experiment (Table 5) showed that as the exposure to heat increased during evaporation the recovery of aflatoxin was affected signifi- cantly. Fig. 3 also shows that the recovery of aflatoxin decreased as the increasing volumes of chloroform required a longer exposure to heat during the evaporation step. It is evident from Figs. 2 and 3 that the 'B' aflatoxins were less susceptible to losses during the elution and the work-up stage than the 'G' aflatoxins. Furthermore, AFB2 showed a higher recovery than AFBl and, similarly, AFG2 showed a better recovery than AFG1, giving recoveries in the order AFB2 > AFBl > AFG2 > AFGl .The same order of recovery from spiked acetone-water mixtures was found to exist in a later evaluation study (Table 6). It may also be postulated that an increase in the volume (and surface area) of the chloroform solution will lead to a greater irreversible adsorption of aflatoxin onto the wall of the glass vial. - - - - - Quantification The bi-directional HPTLC method was found to be both precise and accurate. The TLC scanner was calibrated for each plate using external standards to minimize inter-plate variabil- ity. Typical RF values, when using the bi-directional HPTLC 40 50 I 0 2 4 6 Volume of chloroform/ml Fig. 3 Effect of exposure to heat on aflatoxin recovery. A, AFB,; B, AFB,; C, AFG,; and D, AFGz ANALYST, JANUARY 1992, VOL.117 described above, were found to be 0.28, 0.3, 0.4 and 0.45 for AFBI, AFB2, AFGl and AFG2, respectively. Detection limits of 18.9, 6.4, 15.1 and 8.4pg were calculated (Table 6) for AFB,, AFB2, AFGl and AFG2, respectively, using this method. Method Validation Statistical evaluation of the proposed method The recovery data from the three evaluation procedures were used to construct calibration lines for aflatoxins using a weighted regression method.14 These statistical results were used to calculate the detection limits15 and percentage recoveries and to detect the existence of any inherent relative and/or systematic errors for the AFB1, AFB2, AFGl and AFG2. Calibration data, summarized in Table 6, for the four aflatoxins were obtained using recovery results from: (1) HPTLC quantification of the aflatoxins after a spiked palm kernel extract had been subjected to the proposed clean-up procedure; (2) HPTLC quantification of the aflatoxins after spiked acetone-water (80 + 20) solutions had been subjected to the proposed clean-up procedure; and (3) HPTLC quantifi- cation of various concentrations of the aflatoxins in a benzene-acetonitrile (98 + 2) solution.The presence of a systematic error was indicated when the 95% confidence limits for the regression intercept did not include the value of zero. Systematic errors were found to be present only for AFB2 and AFG2 standards (Table 6). Even for these, the lower limits of the lines passed very close to the origin (0.172 and 0.102). Relative errors were estimated by the percentage deviation of the confidence intervals of the regression slope, from an expected slope of unity.Such errors were found to exist for all the calibration lines (Table 6). The decreased aflatoxin recoveries, shown by the relative errors, can be attributed to the losses incurred and were discussed under Elution and work-up (Figs. 1 and 2). The overlap of the 95% confidence intervals of the regression lines, for the individual aflatoxins, indicated that the accuracy of the proposed method was not affected by the presence of sample interferences or by the solid-phase extraction step. This overlap existed for AFBI, AFB2 and AFG?. However, the regression lines for AFGl did not completely overlap as the recoveries from the spiked acetone- water (80 + 20) mixture were significantly lower than from the spiked extracts. This effect was caused by the presence of interferences around the AFGl spot on the HPTLC plate, Table 6 Summary of the calibration data for aflatoxins 95% confidence limits y-Interceptlpg kg- Slope of the line Detection limit Correlation Relative error Sample Lower Upper Lower Upper ELg kg- ' Pi? coefficient (% range) - 1.595 -0.734 -0.425 -0.068 -0.025 0.172 - 1.931 -0.295 -0.328 -0.504 -0.275 0.102 0.813 1.274 0.190 1.004 1.154 0.440 0.615 1.482 0.824 0.488 0.327 0.529 0.893 0.943 0.922 0.92 0.92 0.962 0.975 0.847 0.946 0.887 0.92 1 0.948 0.980 1.002 0.991 0.981 0.971 1 .oo4 1.073 0.911 1.031 0.964 0.985 0.997 3.74 2.38 1.70 2.48 1.04 0.58 3.04 0.98 1.36 1.26 0.73 0.76 41.6 26.5 18.9 27.5 11.6 6.4 33.8 10.9 15.1 14.1 8.2 8.4 0.9997 0.9996 0.9998 0.9982 0.9996 0.9999 0.9985 0.9998 0.9997 0.9968 0.9981 0.9998 - 10.7, -2.0 -5.7, 0.2 -7.8, -0.9 -8.0, - 1.9 -8.0, -2.9 -3.8, 0.4 -2.5.7.3 - 15.3. -8.9 -3.4, 3.1 -11.3. -3.6 -7.9, - 1.5 -5.2. -0.3 * Spiked extracts subjected to the proposed methodology. t Spiked solvent mixture (80 + 20, acetone-water) subjected to the proposed methodology. $ Standard aflatoxin solutions (98 + 2, benzene-acetonitrile), of equivalent concentration to other samples, quantified by bi-directional HPTLC.ANALYST, JANUARY 1992, VOL. 117 73 Table 7 Comparison of the precisions of the proposed method and the British Standard Method PH bonded phase method British Standard Method Degrees of Degrees of Degrees of Degrees of freedom X S2 freedom s2 freedom x S2 Sample freedom S' 1 9 13.6 122.8 9 83.6 752.7 2 9 866.7 7800.4 9 3065.8 27 592.5 - 28 345.2 Total 18 - 7923.2 18 s'p" 7923.2118 = 440.2 Spt = 21.0 Variance ratio (FObh) = 1774.7/440.2 = 3.58 * Pooled variance. -t Pooled standard deviation.28 345.2118 = 1474.7 = 39.7 Table 8 Amount of AFB, extracted using the proposed method and the British Standard Method 95% confidence Mean intervalslpg kg-' recoveries/ Sample Method pgkg-1 Lower Upper 1 PH-bonded 91.4 88.2 94.6 1 British Standard 63.4 60.4 66.6 2 PH-bonded 694.6 691.4 697.8 2 British Standard 512.0 478.3 515.2 which enhanced the fluorescence of this component. The presence of these interferences was confirmed by spraying the plate with a 50% v/v sulphuric acid solution.19 After spraying, the plate was dried in the fume cupboard and viewed under UV (365nm) light; the aflatoxin was seen to have changed fluorescence colour from green to yellow. However, the interfering compounds still displayed greenish fluorescence.This particular interference problem was only encountered during the validation studies, as the aflatoxin-free sample had been in store for a long period, leading to the development of interfering compounds not normally associated with palm kernels. However, the apparent loss of AFGl at the work-up stage (Figs. 1 and 2) was thought to be the main cause of the low over-all recovery of AFG experienced during this validation exercise. Comparison of methods The aflatoxin contents of two naturally contaminated palm kernel samples were quantified using the proposed method and the British Standard Method.' The variances associated with the two methods were compared using the pooled standard deviations procedure13 and the results are given in Table 7.The observed figure for F ratios (3.58) was found to be higher than the critical value [Fls,lx (p = 0.975) = 2.61 and thus it was concluded that the two methods had significantly different variances and that the PH bonded phase method was more precise than the British Standard Method. The abilities of the two methods to extract aflatoxins from the palm kernel samples were compared by setting up a table of 95% confidence intervalsl3 for the mean recoveries (Table 8). Table 8 clearly shows that there is no overlap in the confidence intervals for the two methods, indicating that the proposed method extracts significantly more of AFBl than the British Standard Method.Conclusion The proposed methodology for the extraction and determina- tion of aflatoxins in palm kernels has been found to be more effective than the British Standard Method in reducing time and solvent costs, affording improvements in reproducibility (average relative standard deviation of 4.1% compared with 12.6% for the British Standard Method), efficiency and accuracy (extracted 40% more of AFB,). These improve- ments were attributed to the semi-automation of the clean-up stage (using a twelve-place vacuum manifold), the commercial availability of the pre-packed PH cartridges and the use of automated HPTLC procedures (capable of 30 quantifications in a single process), The bi-directional HPTLC procedure facilitated the removal of persist en t interfering com pounds.The extracts were suited to the HPTLC quantification which utilizes the natural fluorescent properties of the aflatoxins. These factors contributed to the low detection limits for the proposed method, which were well below the current legis- lative levels.6 The proposed method gave excellent results over the ranges of aflatoxin concentrations investigated. The authors are grateful to the Natural Resources and Environmental Department of the Overseas Development Administration for funding this work. APPENDIX Summary of the Proposed Methodology The following method for the estimation of aflatoxins in palm kernel samples is proposed. ( a ) Grind the sample to afford a free-flowing product.(b) Sub-divide, using a spinning riffle, to afford a 1 kg (c) Slurry the 1 kg sub-sample with 1.5 I of water. ( d ) Blend 100g of the slurry with 240ml of acetone and filter the mixture. ( e ) Solvate a PH cartridge by passing 10ml of methanol followed by 10 ml of water through it under reduced pressure. v) Mix 5 ml of the filtrate with 5 ml of methanol, 1 ml of lead acetate solution (20% m/v), 1 g of Celite and 63 ml of water and pass the mixture through the solvated PH cartridge under reduced pressure at a rate of 10 ml min-1 (Fig. 1). (8) Wash the cartridge with 10ml of water and dry the cartridge for 2 min. ( h ) Attach the cartridge to a 3 ml reservoir containing 1 g of anhydrous sodium sulfate, and elute with 3 ml of chloroform.Collect the eluate in an 8 ml glass vial. (i) Dry the elute at 45 "C under a gentle stream of nitrogen and dissolve the residue in 300 pl of benzene-acetonitrile (98 (j) Apply 5 1-11 of the benzene-acetonitrile solution to an ( k ) Quantify using bi-directional HPTLC. sub-sample. + 2). aluminium-backed HPTLC plate. References I Hsieh, D. P. H.. in Food Toxicology. A Perspective on the Relative Risks, eds. Taylor, S . L . . and Seanlan, R. A.. Marcel Dekkcr, Ncw York, 1989. p. 1 1 .74 2 Cuero, R. G., Smith. J. E.. and Lacey, J.. Trans. Br. Mycol. Soc., 1987, 89, 221. 3 Hartley, C. W. S . . The Oil Palm, Longman Group. Harlow, 3rd edn., 1988. 4 Shibata. M., and Osman. A. H., JARQ, 1988, 22, 77. 5 Moore, A., in Fifth Meeting on Mycotoxins in Animal and Human Health, eds. Moss, M. O., and Frank, M., University of Surrey, 1984, 117. 6 Van Egmond, H. P., Food Addit. Contam.. 1989. 6, 139. 7 British Standard Method for Analysis Feeding Stuff. (IS0 6651-1987). BS 5766. Part 7. 1988. 8 Martin. C. N., Mulholland, F., and Garner, R. C., Feed Compounder, 1987. 7. 10. 9 Sorbent Extraction Technol. ed. Vanhorne, K. C., Analyti- chem. Harbor City, CA, 1985. 10 Tomlins, K . I . , Jewers, K., and Coker. R. D., Chromato- graphia, 1989. 27, 67. 11 Dell, M. P. K . , Haswell, S. J.. Roch, 0. G., Coker. R. D., Medlock, V. F. P., and Tomlins. K.. Analyst. 1990, 115, 1435. 12 Tornlins. K. I . , Jewers, K., Coker, R. D.. and Nagler. M. J., Chromatographia, 1989, 27. 49. 13 Wernimont. G. T.. in Use of Statistics to Develop and Evaluate Analytical Methods, ed. Spendlcy. W., Association of Official Analytical Chemists, Arlington, VA. 1985. 14 Miller, J. C.. and Miller, J. N.. Statistics for Analytical Chemistry, Ellis Horwood, Chichester. ANALYST, JANUARY 1992, VOL. 117 15 16 Analytical Methods Committee. Analyst. 1987, 112, 199. World Health Organization/International Agency for Rcscarch into Cancer eds. Castegnaro. M., Hunt. D. C., Sansone. E. B., Schuller, P. L . . . Siriwardana. M. E., Telling, G. M.. Van Egmond. H. P., and Walker. E. A., ]ARC Scientific publication No. 37, Lyon, France. 1980. Coker, R. D.. Jewers, K.. Tomlins. K. I . . and Blunden. G., Chr-omatographia, 1988. 25, 875. Stoloff, L., and Scott, P. M., in Official Methods of Analysis of the Association of Official Analytical Chemists. ed. Williams, S . , Association of Official Analytical Chemists. Arlington, VA, 1984. pp. 380-484. Cokcr, R. D., Jones, B. D., Nagler. M. J . , Gilman. G. A., Wallbridge, A. J., and Panigrahi, S . , in Mycotoxin Truining Manual, ODNRI, London, 1984, Section B:2. Velasco. J.. and Morris, S. L.. J . Agric. Food Chem.. 1976.24. 86. Bradburn, N., Coker, R. D., and Jewers. K.. Clrromatogr-aphia, 1990, 29, 177. 17 18 19 20 21 Paper 0104949B Received November 5, 1990 Accepted July 2, 1991

ISSN:0003-2654    

DOI:10.1039/AN9921700067

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST, JANUARY 1992, VOL. 117 75 Thin-layer Chromatographic Detection of Pyrethroid Insecticides Containing a Nitrile Group Vitthal 6. Patil, Murlidhar T. Sevalkar and Sudhakar V. Padalikar Regional Forensic Science Laboratory, State of Maharashtra, Cantonment, Aurangabad-43 I 002, India A spray reagent for the detection of pyrethroid insecticides containing a nitrile group by thin-layer chromatography is described. These insecticides on alkaline hydrolysis yield cyanide ion, which in turn reacts with copper(ii) acetate and o-tolidine in an acetic acid medium t o give a blue colour. Organochlorine, organophosphorus and carbamate insecticides do not interfere. The limit of detection is about 1 yg. Keywords: Thin-layer chromatography; copper&) acetate; o -tolidine; pyrethroid insecticide Pyrethroid insecticides are used to control a number of insect species on economic crops.These pyrethroids are effective pest control chemicals and have low mammalian toxicity. 1.2 Three major pyrethroid insecticides containing a nitrile group, Liz., fenvalerate, cypermethrin and deltamethrin, have been identified as highly effective contact insecticides. Their use is increasing for the control of insects. Owing to their availabil- ity, insecticides are misused in homicidal and suicidal poison- ing cases. Consequently, characterization of these insecticides is necessary in forensic toxicology. A large number of gas-liquid chromatographic methods"-" for residue analysis of synthetic pyrethroids have been reported, as has autoradiographic thin-layer chromatography (TLC), using l-'C-labelled compounds, particularly in meta- bolic studies where the unlabelled compounds were detected by visualization on silica gel 60 F2s4 chromatographic plates under ultraviolet (UV) light.ks Chromogenic reagents have also been reported, for example, phosphomolybdic acid (20% m/v in ethanol) for the detection of permethrin, cypermethrin and deltamethrin," palladium chloride (0.5% m/v in 12 mol dm-3 HCI) for the detection of deltamethrin10 and silver nitrate impregnated alumina G and irradiation with UV light11 for pyrethroid insecticides in general.In this paper the use of a chromogenic reagent, viz., 20% sodium hydroxide solution, 5% copper(i1) acetate solution and 1% phosphomolybdic acid solution followed by 0.1% o-tolidine in acetic acid, is described for the detection of pyrethroid insecticides containing a nitrile group by TLC.This reagent gives intense blue spots and is selective for pyrethroid insecticides containing a nitrile group. Experimental Reagents All reagents were of analytical-reagent grade. Distilled water was uscd throughout. Sodium hydroxide solution, 20%. Dissolve 20 g of sodium hydroxide in distilled water and dilute the solution to 100 ml. Copper(1i) acetaie solution, 1%. Dissolve 1 g of copper(i1) acetate in distilled water and dilute the solution to 100 ml. Phosphomolyhdic acid solution, 1%. Dissolve 1 g of phosphomolybdic acid in distilled water and dilute the solution to 100 mi. o-Tolidine reagent. Dissolve 0.1 g of o-tolidine in 100 ml of 10% v/v acetic acid.[Caution: o-Tolidine is a known carcinogen .] Procedure A standard glass TLC plate was coated with a slurry of silica gel G in water (1 + 2) to a thickness of 0.25 mm. The plate was activated by heating it at 110 "C for about 1 h. Volumes of 10 yl of standard solutions of fenvalerate, cypermethrin and delta- - Table 1 RF values of three pyrethroid insecticides containing a nitrile group. Solvent system: cyclohcxane-toluene (6 + 4) Insecticide RF value Fen val era t c Cypermethrin Deltarnethrin 0.42 0.38 0.31 methrin in ethanol (each 1 mg ml-l) were spotted on to the plate, which was then developed in a previously saturated TLC chamber using cyclohexane-toluene (6 + 4) as the solvent. After the solvent had eluted a distance of 10 cm up the plate, the latter was removed from the chamber, dried in air and sprayed successively with 20% sodium hydroxide solu- tion, 1% copper(i1) acetate solution and 1% phosphomolybdic acid solution; after 5 min it was sprayed with 0.1% o-tolidine reagent.Intense blue spots of the quinoidal oxidation product of o-tolidine developed. The RF values of three pyrethroid insecticides containing a nitrile group are given in Table 1. Discussion Pyrethroid insecticides containing hydrolysable nitrile moi- eties yield cyanide ion on alkaline hydrolysis. The cyanide ion in turn reacts with copper(ri) acetate and o-tolidine to give a blue colour.12 Normally, the blue colour thus formed fades immediately; however, it is stabilized (>1 h) by the use of phosphomolybdic acid prior to application of the o-tolidine reagent.Other pyrethroids not containing a hydrolysable nitrile group (permethrin, resmethrin, allenthrin, etc.) should not interfere; moreover, organochlorine, organophosphorus and carbamate insecticides do not give a coloured spot. The limit of detection of the method is about 1 pg. The reagent described here is sensitive and selective for pyrethroid insecticides containing a nitrile group, and might find application in the detection of these insecticides in biological materials in forensic toxicology. The authors are grateful to Dr. B. N. Mattoo, Director, Forensic Science Laboratories, State of Maharashtra, Bom- bay, for his valuable advice and encouragement during this work. We also extend our gratitude to Searle (India) Ltd., Bombay, for the supply of technical-grade fenvalerate, and to Nocil Agrochemicals Ltd., Bombay, and Hoechst (India) Ltd., Bombay, for supplying the cypermethrin and deltameth- rin samples, respectively.References 1 Elliott, M., Janes. N. F., and Potter, C., Annu. Rev. Entomof., 1978, 23. 443.76 ANALYST, JANUARY 1992, VOL. 117 2 Elliott, M., Pestic. Sci., 1980, 11, 119. Ruzo. L. 0.. Engel, J. L., and Casida. J. E., J. Agric. Food 3 Chapman. R. A., and Simmons, H. S., J. Assoc. Off. Anal. Chem.. 1979, 27. 725. Chem., 1977.60, 977. 11 Sundararajan, R., and Chawla, R. P., J. Assoc. Of& Anal. 4 Talekar, N. S., J. Assoc. Of5 Anal. Chem., 1977, 60, 908. Chem., 1983, 66. 1009. 5 Lee. Y. W.. Westcott, N. D., and Reichle, R. A..J. Assoc. Off. Feigl, F . , Spot Tests in Inorganic Analysis, Elsevier, Amster- Anal. Chem.. 1978, 61, 869. dam, 1972, p. 209. 6 Roberts. T. R.. and Standen, M. E.. festic. Sci., 1977, 8. 30.5. 7 Ruzo, L. 0.. Holmstead. R. L., and Casida, J . E., J . Agric. Food Chem.. 1977, 25, 1385. 8 Gaughan, L. C., Ackerman, M. E., Unai, T., and Casida, J . E., J. Agric. Food Chem., 1978, 26, 613. 9 Shono, T., Ohsawa, K., and Casida, J. €3.. J. Agric. Food Chem.. 1979. 27, 316. 10 12 Paper I I00988 E Received March 4 , 1991 Accepted July 31, I991

ISSN:0003-2654    

DOI:10.1039/AN9921700075

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST, JANUARY 1992, VOL. 117 77 Relationship Between the Structures of Dihydrofuro- and Di hydropyrano[2,3-b]quinolinium Alkaloids, Their Spectral Properties (Ultraviolet Absorption and Fluorescence) and Their Chromatographic Behaviour Monique Montagu, Genevieve Petit-Paly, Pierre Levillain, Jean-Claude Chenieux and Marc Rideau UFR Pharmacie, 37042 Tours Cedex, France Chromophores of dihydrofuro- and dihydropyrano[2,3-b]quinolinium alkaloids differ from each other only by the position of the electron-donating substituents attached to the aromatic ring. It is shown that there is a connection between the electronic absorption and the fluorescence properties (taking advantage of solvent effects) and the chromophore structure of the alkaloids. The non-chromophore part of these structures also has an influence on the chromatographic behaviour of the molecules.It is shown that the spectral and chromatographic properties not only permit the identification of known structures but are also useful for predicting the structure of new quinolinium alkaloids. An example is given for two alkaloids isolated from Ptelea trifoliata (Rutaceae). Keywords : Electronic absorption; flu o rescence-structu re relationship; 4 ua te rna r y alkaloid; dih ydro fu ro - and dih ydrop yrano[2,3-b] quinolinium The electronic absorption spectra of some dihydrofuro- and dihydropyrano[2,3-b]quinolinium compounds (extracted from a few species of Rutaceael) have been reported previously,'-h but their fluorescence properties have hitherto received little attention.For both absorption and fluor- escence, no relationship was established between spectral properties and the molecular structure in terms of various media. In a previous paper,7 we studied the electronic absorption and fluorescence of two dihydrofuro[2,3-h]quinolinium alka- loids, namely, balfourodinium and platydesminium; in par- ticular, attention was directed to the influence of various solvents and anions, and to the effect of pH on the spectra. It was shown that, although their electronic absorption spectra are, in practice, independent of the medium, their fluor- escence is generally strongly dependent on the medium.7 This investigation led to the development of spectrofluorimetric and spectrofluoridensitometric assays for these two alka- 1 o i ds .7-9 This work was extended to other quinolinium alkaloids that were extracted from Ptelea trifoliata L.and Ruta graveolens L. (Rutaceae). I n this paper it is shown how, with the aid of these spectral properties, it is possible, at an early stage of the isolation and with only a few picomoles of substance, to predict the structure of new dihydrofuro[2,3-b]quinolinium alkaloids. Experimental Equipment Fluorescence measurements were made with a Jobin-Yvon Model JY3CS spectrofluorimeter, equipped with a xenon arc source, with two monochromators, with an R-212 photomulti- plier, and with a 1 X 1 cm quartz cell. Spectra were recorded with a 4 nm bandwidth and a scan speed of 40 nm min-1: the fluorescence data are given without spectral correction. Reference Alkaloids Seven alkaloids were investigated: platydesminium perchlor- ate, balfourodinium chloride, ptelefolonium perchlorate, isoptelefolonium perchlorate, hydroxyluninium chloride, ribalinium perchlorate and rutalinium perchlorate.They had been isolated previously and analysed by proton nuclear magnetic resonance ( * H NMR) spectroscopy and mass spec- trometry (MS). 1 0 Apart from rutalinium, which has a di- hydropyrano[2,3-b]quinolinium structure [Fig. l(b)], these alkaloids have a dihydrofuro[2,3-b]quinolinium structure [Fig. l(a)]. All of these compounds have a methoxy group in position 4 of the quinolinium structure, but differ either in the nature and position of the substituents in positions 6 , 7 and 8, or in the nature of the R group, i.e., R = l-hydroxy-l- methylethyl or 1-methylethenyl (Fig. 1).CH3 OCH? (b) R Z = YCH2 CH3 Fig. 1 ( u ) Structure of dihydrofuro[2.3-b]quinolinium alkaloids: (h) structure of rutalinium; and (c) conjugation effect induced by a rnethoxy group in position 7 of the dihydrofuro[2.3-h]quinolinium struct urc78 ANALYST. JANUARY 1992, VOL. 117 Table 1 Absorption spectra of dihydrofuro- and dihydropyrano- [2.3-b]quinolinium alkaloids. Solvent: methanol Absorption maximumlnm (log E-I) substituent" 'Ch 'Bb ' L', I Lt, Alkaloid and Platydesminium perchlorate (A) R' 2 16(4.49) 236(4.52) 294( 4.04) 3 17( sh)t Balfourodinium chloride (B) R' ; -0CHj (8)s 2 14(4.40) 2S3(4.S 1 ) 30O(3.88) 32O( s h) perchlorate (C) Isoptelefolonium perchlorate (D) H ydrox y 1 unini um chloride (E) Ri balinium perchlorate (F) R1; -OH (6)s 220( 4.52) 246( 4.48) 294( 4.05) 336( 3.89) perchlorate (G) -OH (6)s 2 13(4.46) 248(4.48) 304( 3.97) 347( 3.85) * R1 = l-Hydroxy-l-methylethyl; R' = l-methylethenyl.t E = Molar absorptivity (dm' mol-1 cm-1). .$ sh = Shoulder. 9 Numbers in parentheses indicate the position of the substituent in 220(sh) Ptelefolonium R';-OCH3 (6.8)s 216(4.40) 259(4.56) 299(3.70) 345(3.48) R'; -0CH3 (7.8)s 226(4.38) ZSO(4.80) 322(4.23) R'; -0-CHz-O- (7.8)s 2 H(4.28) ZSs(4.45) 335(3.63) Rutalinium 294(sh) the alkaloid ring system. Results and Discussion Relationship Between the Chemical Structure and the Electronic Absorption of Alkaloids The spectral data (using methanol as solvent) are shown in Table 1. They are similar to those obtained in a 0.1 mol dm-3 HCI04 solution in methanol: only a weak hyperchromic effect is observed in the spectra of balfourodinium and isoptele- folonium (results not shown).Platt's nomenclature11 applied to quinoline and the quinolinium ion" was used to denote the absorptivity bands. From the electronic absorption properties, it is possible to dinstinguish the alkaloids in terms of the substituent in positions 6, 7 and 8 from the non-substituted platydesminium. Monosubstitution on the quinolinium structure in position 6 or 8 induces a bathochromic shift of the IB,,, lLZl and ILb bands. As observed by Zanker,I3 the shift of the longitudinally polarized 'Lb band is much more significant when a group is substituted in position 6 than when a group is substituted in position 8, which affects the transversally polarized IL;, band (Table 1 ; compare B with F and G).However, the influence of the nature of the substituent, all the substituents are electron- donating groups, is position-dependent: this influence is observed on comparing the spectra of balfourodinium (-OCH3 in position 8) and pteleatinium (-OH in position 8), the bathochromic effect being more significant in the latter, but it is not observed on comparing the spectra of ribalinium (-OH in position 6) and methylribalinium (-OCH3 in position 6).14 The bathochromic effect increases with the number of substituents; however, the effect is modified by the relative positions of the various groups. In particular, substitution in positions 7 and 8 induces a coalescence of the *L:, and 'Lb bands (Table 1; D and E).This is not observed on substituting in positions 6 and 8 (Table 1; C). Possible conjugation of the electrons of the methoxy group in position 7 might be responsible for this effect [Fig. l(c)]. The conjugated form can give an nn" transition, but this transition undergoes a hypsochromic shift caused by the methoxy group in position 4; this induces the coalescence of the 1L and nn* bands. Table 2 Fluorescence spectra of dihydrofuro- and dihydropyrano- [2,3-b]quinolinium alkaloids. Medium: methanol, unless indicated otherwise Exci t at ionlemission Alkaloid Substituent* wavelengthlnm (It) Platydcsminium perchlorate (A) RI 238,3041346 (20. 100) Balfourodinium chloride (B) RI 264,312,3361433 (8)s (79, 96. 100) Ptelefolonium perchlorate (C) R' 263,307,348l454 -0CH3 (6.8)s (48,56.100) 263,307,348l4061 (48,56, lOO)'11 Isoptelefolonium perchlorate (D) R' 256.3361478 -0CH3 (7,8)6 (44, 100) 256.33614787 (42. 1OO)y H ydrox y 1 un ini um chloride (E) R' 262,3401368 -0-CH?-O- (7.8) (54, 100) Ribalinium perchlorate (F) R1 250.3O2.3461405 -OH (6)s 250,302,3461520 (30.46, 100) 250,302.3461406~ (34.53, l0o)fl 250. 302,3461500, 5201 (12,19.36)1 (20,44,100) (10.22, SO) Rutalinium perchlorate (G) -OH (6)s 253,307,3561418 253,307,3561526 253,307,3561418~ (20.42, lOO)1 253.3O7.3561525~ (7.14.33)1 * RI and R' as in Table 1 . .i- I = Intensity ratio of the fluorescence bands in %. bSlnm 42 97 106 587 142 1421 128 59 174 608 741 62 70 62'11 169'11 $ 6 = Differcnce between the emission and excitation wave- $ Numbers in parentheses indicate the position of the substituent in 7 Medium: 0.1 mol dm-3 HCI04 in methanol.lengths. the alkaloid ring system. In contrast, the dihydrofuranoid and dihydropyranoid rings, cf. the auxochromic oxygen function, are not part of the chromophore itself. They can behave in the same way as auxochromic groups, and, in this instance, have some effect on the spectrum. As observed previously,15 a bathochromic shift of the ILb band occurs when the dihydrofuranoid group is replaced by the dihydropyranoid group (Table 1; F and G). Relationship Between the Chemical Structure and the Fluorescence of Alkaloids Alkaloids with two fused benzene rings (which give the molecule a certain rigidity) show a fairly high fluorescence, allowing the assay of these alkaloids to within a picomole.Moreover, with these alkaloids, the nitrogen lone pair is localized by methylation, thus preventing internal coupling between a singlet and a triplet state. Hence, phosphorescence, which usually develops at the expense of fluorescence, is inhibited. 16 The influence of four different media on the fluorescence spectra was studied. Table 2 shows that the excitation maxima in methanol, with or without HC104, are identical. (The same data were also obtained in water, with or without HCI04.) Investigation of the excitation spectra allows one to dis- tinguish between three groups of alkaloids, as follows: (1) twoANALYST, JANUARY 1992, VOL. 117 79 i; r”\ I t I \ \ ;\ ’ \ 200 400 600 I I I \ / \ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . , . . . a . . . . . . . . . . . . . . . . . . . * . * . . . . . . . . . . . . . . . . . . :: * . . . . . . . . . . . . . . .-._ a : : .. * : . : . : * . * . . . . * . : : : : *: . *..a .. r.. 200 400 600 200 400 AJnm 600 Fig. 2 Fluorescence spectra of dihydrofuro- and dihydropyrano[2.3-h]quinolinium alkaloids: ( a ) platydesminium; (b) balfourodinium; (c) ptclefolonium; ( d ) isoptelefolonium; (e) hydroxyluninium; (f) ribalinium; (8) rutalinium; and ( A ) and (i), new alkaloids. Solid line shows spectra in methanol medium; broken line shows spectra in 0.1 mol dm-3 HCIOl in methanol medium; and dotted line shows similar spectra in both media narrow well-separated bands are obtained for alkaloids substituted in positions 7 and 8 [Fig. 2 ( d ) and ( e ) ] ; (2) spectra with three bands, with the IL bands very close to each other or almost overlapping, are obtained for non-substituted alkaloids and alkaloids substituted in position 8 [Fig.2(a) and ( b ) ] ; and (3) spectra with three well-separated bands are obtained for alkaloids substituted in position 6 [Fig. 2 0 and (g)] and in positions 6 and 8 [Fig. 2(c)]. The emission maxima of the alkaloid spectra can be very different when using different media (Table 2). In methanol, the molecule with no substituent attached to the aromatic ring (platydesminium) re-emits the quasi-totality of the absorbed energy (see Table 2: the 6 value corresponds to the difference between the absorbed and re-emitted energies; when this value is low, most of the absorbed energy is re-emitted).On the other hand, as soon as the aromatic ring is substituted, the emission band undergoes a marked bathochromic shift. This effect is due to the existence of a very polar excited state, arising from a perturbation in the location of charges in these molecules. The bathochromic shift increases with the number of substituents. However, the non-radiative dissipated energy ( i . e . , the difference between the excitation and emission wavelengths) before re-emission is almost identical for molecules substituted in position 8 as for those substituted in positions 6 and 8 (Table 2; B and C). It increases noticeably for molecules substituted in positions 7 and 8 (Table 2; compare B and C with D and E). A hydroxy group in position 6 (Table 2; F and G) yields the most significant bathochromic shift (emission greater than 500 nm).It might be that the exceptional fluorescence is caused by the creation of a zwitterionic structure, which induces a consider- able lowering of the energy between the absorption and re-emission levels. 17 The energy reduction might be explained by an increase in the solvation effect of the molecule, which is more polar when in an excited state, which lowers the energy of the excited level and leads to a lower frequency re-emission. Derivatives with a hydroxy group in position 6 also have two emission bands, hence the existence of two possible energy levels in an excited state. By using 0.1 mol dm-3 HCI04 in methanol in place of methanol, it is possible to distinguish between two groups of dihydrofuro[2,3-b]quinolinium alkaloids according to their emission spectra: (1) non-substituted alkaloids or alkaloids substituted with a methoxy group in position 8 or in positions 7 and 8: at most a slight hyperchromic effect is observed [Fig.2(a), ( b ) , (d) and ( e ) ] ; and (2) alkaloids substituted in positions 6 and 8 or in position 6: the fluorescence spectra are modified [Fig. 2(c) and 01. However, for ptelefolonium [Fig. 2(c)], this modification is essentially a hypsochromic shift together with the hyperchromic effect of the emission band, while for ribalinium [Fig. 2 0 3 , with an emission spectrum having two maxima, it is the hyperchromic effect of the higher energy band. These exceptional effects can be compared with those observed for indolic compounds with a hydroxy group in the para position with respect to the nitrogen atom.18 These phenomena are strongly linked to the pK,, of the alkaloids in80 ANALYST, JANUARY 1992, VOL.117 the excited state. On the other hand, it is possible to distinguish dihydrofuro[2,3-b]quinolinium alkaloids from dihydropyrano[2,3-b]quinolinium alkaloids. Rutalinium [Fig. 2(g)], a dihydropyrano[2,3-b]quinolinium alkaloid, has a spectrum which is altered in the same way as that of ribalinium [Fig. 2 0 1 in HC104, but to a lesser extent. Indeed, for the spectra of ribalinium and rutalinium, the intensity ratios of the two emission maxima ( 1 4 0 ~ Z s 2 0 ) are increased by a factor of 2.8 and 1.2, respectively. The rigidity of the dihydrofuranoid ring makes the molecule planar, which would allow conjugation of the free electrons on the oxygen atom with the N-methyl- quinolinium structure.This conjugation is less likely to occur with the dihydropyranoid ring, which is not as rigid, whence the less marked difference between the spectra obtained in the two media. It should be noted that the variations of the observed spectra (in the HCI04-methanol medium) are due to the acidity of the medium and not to the C104- anion, as the spectra obtained in a methanolic solution of NaC10, are identical with those obtained in methanol. l y It should also be noted that the fluorescence spectra of all the alkaloids are identical in both water and 0.1 mol dm-3 HCIO, (results not shown). Relationship Between the Structure and the Chromatographic Behaviour of Alkaloids Dihydrofuro- and dihydropyrano[2,3-b]quinolinium alkaloids can be separated by reversed-phase liquid chromatography”) and thin-layer chromatography.8 In the latter instance the behaviour of these compounds on silica with the solvent system ethyl acetate-formic acid-water (10 + 1 + 1) makes it possible to discriminate between two alkaloid groups which separate in different RF zones according to the group present on the dihydrofuranic ring (RI = l-hydroxy-l-methylethyl and R’ = l-methylethenyl). The Rl-substituted alkaloids have lower RF values (RF = 0.20 k 0.05) than the RZ-substituted alkaloids (RF = 0.35 k 0.07).Application to the Determination of the Structures of Two Dihydrofuro[2,3-b]quinolinium Alkaloids Concurrently with this work, two new dihydrofuro[2,3-b]- quinolinium alkaloids were isolated, one from in vitro cultures and the other from leaf stems of Relea trifoliata (Rutaceae).It was possible to determine the structures of these two alkaloids from their absorption and fluorescence spectra without isolating them completely. An examination of the fluorescence spectra (Fig. 2 and Table 3), obtained in methanol, shows, for the alkaloid (H), isolated from cultured cells, an excitation spectrum with three bands; two IL bands are near to each other, and the Table 3 Absorption and fluorescence spectra of the dihydrofuro- [2,3-b]quinolinium alkaloids (H) and (I). Medium: methanol. unless indicated otherwise Absorption maximumlnm Alkaloid* (log E t ) (H) 2 16(4.30) 256(4.33) 299(3.55) 2 19(4.22) 261 (4.43) 336(3.52) 320w)ii (1) Excitationlemission wavelengthlnm ( I $ ) h$lnm 260,308,3331437 104 (71.90, 100) 260,308,33314398 1068 (71-93, l00)T 263,3441468 124 (44, loo) * Both alkaloids were in the form of their chlorides. I E = Molar absorptivity (dm’ mol-’ cm-I).$ 1 = Intensity ratio of the fluorescence bands in %. 9 6 = Difference between the emission and excitation wavelengths. f i Medium: 0.1 mol dm-3 HCIO., in methanol. 11 sh = Shoulder. wavelengths of the excitation and emission maxima indicate monosubstitution. Hence it can be deduced that this molecule has a substituent in position 8. The stability of the emission spectrum with respect to the medium reflects the similarity between the chromophoric groups of the molecule isolated from the cultured cells and balfourodinium [Fig.2(b) and ( h ) ] . For the alkaloid ( I ) , isolated from leaf stems, the excitation spectrum has two narrow bands, which are not affected by HCIO,; this indicates substitution in positions 7 and 8. However, the difference between the wavelengths of the excitation and emission maxima is less than that expected for two substituents, and this suggests substitution with a dioxolo group which is the auxochromic group giving an almost identical wavelength difference for hydroxyluninium [Fig. 2(e) and (i)]. The RF values of these two alkaloids in the solvent system ethyl acetate-formic acid-water (10 + 1 + 1) are 0.39 for (H) and 0.41 for (1). These higher RF values indicate that these alkaloids have a methyl-l-ethenyl group. Therefore, compound (H) is thought to be 2,3-dihydro-4,8- dimethoxy-9-methyl-2-( l-methylethenyl)furo[2,3-b]quino- linium and compound (I) to be 7,8-dihydro-8-( l-methyl- ethenyl)-6-methoxy- 10-methyl-1,3-dioxolo[4,.5-h]furo[2,3-b]- quinolinium. After isolation, these two assignments were confirmed by *H NMR spectroscopy and MS.”.” The alkaloids were designated as ptelecultinium (H) and ptelefolidonium ( I ) .Conclusion The results presented here show that it is possible to elucidate the structures of dihydrofuro[2,3-b]quinolinium alkaloids by studying their fluorescence spectra and chromatographic behaviour. The specificity and sensitivity of the fluorescence allows the identification of a molecule contained in the eluate from a chromatographic layer, with only a few picomoles of the alkaloid.At this level, purification is not sufficient to identify a molecule from its electronic absorption spectrum and by MS, and the available amounts preclude any possibility of determining the structure by NMR spectroscopy. In a few instances, thin-layer chromatography cannot separate alkaloids sufficiently. The use of overpressured thin-layer chromatography might lead to some improve- ment,z3 but it should also be noted that it is possible to differentiate the fluorescence spectrum of each component in a mixture by synchronous fluorescence spectrometry. A recent example was provided by a study of the alkaloidic composition of in vitro cultures of Ruta graveolens.’4 In conclusion, the method described here provides a simple means of characterization, and is faster than classical methods of identification.1 2 3 4 5 6 7 8 9 10 References Mester. I . . in Chemistry and Chemical Taxonomy of the Rutales. eds. Waterman. P. G., and Grundon. M. F., Academic Press, New York, 1983. pp. 31-96. Sangster. A. W., and Stuart, K. L., Chem. Rev., 1965, 65. 69. Rapoport. H.. and Holden, K. G., J . Am. Clrem. Soc., 1959.81. 3738. Szendrei. K., Minker. E.. Koltai, M., Reisch. J . . Novak. I . . and Buzas. G., Pliarmazie. 1968, 23, 519. Boyd, D. R., and Grundon. M. F., J . Clrem. SOC. C , 1970,556. Reisch, J . , Mirhom, Y. W,, Korosi. J., Szendrei. K., and Novak, I . , Phytochemistry, 1973, 12, 2552. Montagu. M., Levillain, P., Ridcau, M., and Chenicux. J . C.. Talunra, 1981, 28, 709. Montagu, M., Levillain, P . , ChCnieux, J .C., and Ridcau. M., J . Chromatogr.. 1986, 351, 144. Tr6mouilloux-Guiller, J., Kodja. H., Andreu. F., Creche, J.. Chknieux. J . C.. and Rideau. M., Plant Cell Rep.. 1988, 7,456. Rideau, M.. Verchere, C., Hibon, P., ChCnieux, J. C., Maupas, P., and Viel. C., Pliytocliemisrry, 1979, 18, 155.ANALYST, JANUARY 1992, VOL. 117 81 1 1 12 13 14 1s 16 17 18 I9 Platt. J . R., J . Cliem. Phys., 1949, 17, 484. Jaffc. H . H., and Orchin, M.. Theory and Applications of Ultraviolet Spectroscopy. Wiley, New York, 5th edn., 1970. Zanker, V.. Z . Phys. Chem.. 1954, 2, 52. Mitschcr, L. A.. Bathala, M. S.. Clark, G. W.. and Bcal, J . L., Lloydia. 1975.38, 109. Szcndrci, K . , Rcisch, J . . Novak, I . , Simon, L.. Rozsa, Zs.. Minkcr. E . , and Koltai, M., Herhu Hung.. 1971, 10. 131. Bccker, R . S . . Theory and Interpretation of Fluorescence und PI1 osphorcwen ce . W i 1 c y- I n t c rscic ncc . New Y ork . 1 969, Bourdon. R., in Mises au Point de Chimie Analytique, Organ- ique, Pliurmuceutique et Bromatologique. eds. Gautier, J . A.. and Malangcau. P., 15rh Series. Masson, Paris, 1967, pp. 1-41. Udcnfricnd. S . , Fluorescence Assay in Biology and Medicine, Acadcmic Press. New York. 3rd edn., 1962, pp. 125-190. Montagu. M., Thesis. Univcrsite dc Tours, 1983. pp. 1-193. pp. 345-383. pp. 155-189. 20 21 22 23 24 Montagu, M.. Lcvillain, P., ChCnieux. J. C.. and Rideau, M., J. Chromatogr.. 1985, 331, 437. Petit-Paly, G . . Montagu, M., Viel, C., Rideau, M., and Chknieux, J . C., Plant Cell Rep., 1987, 6, 309. Petit-Paly, G., Montagu. M., Merienne, C . , Ambrosc. J . D., Viel, C., Rideau, M., and Chknieux, J . C.. Planta Med., 1989. 55. 209. Pothier, J . . Pctit-Paly. G., Montagu, M.. Galand, N.. ChCni- eux, J. C., Rideau, M.. and Viel, C., J . Planar Ckromatogr.. 1990, 3, 356. Montagu, M., Pctit-Paly, G.. Levillain, P., Baumcrt, A.. Groger, D., ChCnicux, J. C., and Rideau, M., Pharmazie, 1989, 44.342. Puper 11024786 Received May 28, 1991 Accepted August 7. 1991

ISSN:0003-2654    

DOI:10.1039/AN9921700077

出版商:RSC    

年代:1992

数据来源: RSC

摘要:

ANALYST. JANUARY 1992, VOL. 117 83 Quenching of Fluorescence of Polynuclear Aromatic Hydrocarbons by Chlorine Saschi A. Momin and Ramaier Narayanaswamy" Department of Instrumentation and Analytical Science, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 IQD, UK A number of polynuclear aromatic hydrocarbon molecules, dissolved in methanol, have been investigated for their fluorescence quenching by chlorine. Extremely efficient quenching is observed between the fluorophore and the quencher, which leads to enhanced non-radiative decay of the fluorescent state of the molecule with increasing chlorine concentration. The findings demonstrate the possibility of developing an analytical method for the determination of chlorine. Keywords: Fluorescence quenching; polynuclear aromatic hydrocarbon; chlorine The use of fluorescence-based analysis is becoming increas- ingly popular in many branches of the chemical and biological sciences.The principal advantages of this technique, which encourage its use, are its high sensitivity which allows the measurement of low analyte concentrations, its selectivity which is, in part, due to the two characteristic wavelengths (excitation and emission) of each fluorescent species, and the variety of sampling methods available. In addition, the phenomenon of fluorescence quenching provides a kinetic method for the detection and determination of quencher molecules. I Fluorescence quenching is the process by which the fluorescence intensity or quantum yield of luminescent species is decreased, or even eliminated, by interaction with other chemical species.The quenching effect of oxygen on various luminescent species has been observed since the early 1930s;' however, analytical applications for the determination of oxygen were not reported until much Although there is a growing interest in the development of analytical methods based on dynamic quenching of fluorescence, which arises from the full and rapid reversibility of the process, quenching data for chlorine are not available. The need for chlorine sensing arises from its extensive use in the chemical industry and in the sterilization of drinking water. Owing to its toxic nature,' there is a risk of over exposure to chlorine, both in its manufacture and use. Hence the danger of chlorine pollution demands a sensitive and reliable method for its determination. This paper reports the extremely efficient quenching of the fluorescence of certain polynuclear aromatic hydrocarbon (PAH) molecules by chlorine and demonstrates the possibility of developing an analytical method based on this principle for the quantitative determination of chlorine.Measurement of Spectra Fluorescence spectra were recorded on a Perkin-Elmer Model LS-5 luminescence spectrometer equipped with an 8.3 W xenon discharge lamp pulsed at line frequency (50 Hz). All experiments were conducted using 1 x 1 cm rectangular quartz cells. Excitation and emission slit-widths were fixed at 2.5 nm for all the experiments. Absorbance measurements were recorded on a Perkin- Elmer Lambda 5 ultraviolet/visible spectrophotometer.Procedure The PAHs chosen were based on a high quantum yield of fluorcscence, possibility of near visible excitation and avail- ability in pure form. Before investigating the effect of chlorine on the fluores- cence of the PAH dyes it was first necessary to determine whether the process of static quenching was taking place. This was carried out by recording the absorption spectra of the PAHs in the absence and presence of chlorine in methanol. Perturbations in the absorption spectra of PAH molecules, due to chlorine, were taken as evidence of ground state quenching. Polynuclear aromatic hydrocarbons that showed static quenching were eliminated from further investigation. The effect of chlorine dissolved in methanol on the fluorescence of those dyes that did not show signs of static quenching was investigated at room temperature (22 "C).The concentration of the PAH solutions was kept constant at about 1 x 10-7 mol dm-3 in order to avoid self-quenching or inner filter effects. Experimental Reagents Polynuclear aromatic hydrocarbons of analytical-reagent grade were used as obtained. Their spectral properties were found to be in accordance with published data.8 Methanol of spectroscopic grade was dried over molecular sieves (Merck, Type 3A) and used as the common solvent. Chlorine solutions were prepared by passing gaseous chlorine through methanol. The concentration of chlorine dissolved in methanol was determined by standard iodimetric titration. *: To whom corrcspondcncc should be addressed.Table 1 Quenching of fluoresccncc of PAH moleculcs in the presence o f 3 x 10-5 mol dm-3 chlorine Polynuclear Wavelcngth of Decrcasc in aromatic cxcitationl fluorescence due hydrocarbon emissionlnm to chlorine (%) Anthraccne 9.10-Dichloroanthracene 9.10-Diphenylanthracene 9-Methylanthraccne 9-Phcnylanthracene 9-Vinylanthracenc Pcrylene Bcnzo[ghi]pcrylene Pyrene Benz[e]pyrcne Fluoranthcne 3551397 3781426 3701426 36614 10 36414 10 386141 0 3801437 3801427 3341392 3291387 3501440 41 31 26 32 30 23 39 28 22 20 984 ANALYST, JANUARY 1992, VOL. 117 Results The per cent. decrease in fluorescence of PAH molecules that did not show any static quenching, in the presence of 3 x 10-5 mol dm-3 chlorine, is shown in Table 1. All the PAH molecules show a decrease in fluorescence due to chlorine.Anthracene shows the greatest sensitivity to chlorine but has its excitation maxima too far in the ultraviolet region to be of any use in the development of inexpensive analytical techniques. Similarly, pyrene, benzo[e]pyrene and fluoranthene are not suitable owing to their short wavelength excitation. By taking 9,10-diphenylanthracene as an example, a typical fluorescence spectrum of a PAH molecule in the presence of various chlorine concentrations is shown in Fig. 1. The fluorescence intensity, I , and quencher concentration, [ Q], are related by the Stern-Volmer equation where I,) is the fluorescence intensity in the absence of quencher and Ksv is the Stern-Volmer constant. The Stern-Volmer plot of the quenching of the fluorescence of 9,lO-diphenylanthracene by chlorine is shown in Fig.2. Within the concentration range investigated, the fluorescence quenching by chlorine does not show a linear dependence as predicted by the Stern-Volmer equation. However, the Ksv value calculated using the linear part of the plot was found to be 11 120 dm3 mol-I. Discussion The results show that the fluorescence of the PAHs investi- gated is sensitive to the presence of chlorine. Fig. 2 shows a positive curvature at higher chlorine concentrations, indicat- ing the presence of more than one quenching mechanism. The diffusion-controlled bimolecular quenching constant, ko, is related to the Stern-Volmer constant by Ksv = koYo where yo is the fluorescence decay lifetime. By taking the fluorescence decay lifetime of 9,lO-diphenyl- anthracene to be 8.41 ns,8 a bimolecular quenching constant of 1.32 X 1012 dm3 mol-1 s-* was obtained.This value is larger than that predicted by purely diffusion-controlled processes. Fluorescence quenching data, obtained by intensity measure- ments alone, cannot distinguish between dynamic and static quenching. The lifetime, temperature, viscosity dependence o r careful examination of the absorption spectrum can be used to discriminate between the two processes. Observation of static quenching implies a ground state chemical reaction and might not result in the reversibility that is inherent in dynamic quenching. For this reason PAHs that showed static quench- ing, as observed by perturbations in the absorption spectra, were eliminated from further investigation.The process of dynamic quenching is a diffusion-controlled process that only takes place during the lifetime of the excited state of the molecule. As perturbations in the absorption spectra of the dyes investigated were not observed in the presence of chlorine, it has to be assumed that there is a combination of dynamic quenching with other non-radiative processes. Further, removal of chlorine from the PAH solution by a non-interfering chlorine scavenger such as sodium thiosul- phate returned the fluorescence of the dye to its original intensity. Reaction between chlorine and the PAH molecules was not observed within the concentration range investigated (<0.03 mol dm-3) and the excitation wavelengths used. Oxygen was observed to reduce the fluorescence intensity of the PAH molecules. This does not affect the quenching data provided the concentration of oxygen, or any additional quenchers, remains constant.This was taken to be the case A 380 400 420 440 460 480 500 Wavelengthlnm Fig. 1 Emission spectra of 9,lO-diphenylanthracene in the presence of various chlorine concentrations: A, 0; B. 9.2; C, 18.3; D, 27.5; E, 36.7; F, 45.8; G. 55.0; and H, 64.2 pmol dm-3 chlorine 2.0 1 I 1.8 i I .6 I Y 1.4 1.2 1 X X X X X X I .o I I I I I I 0 10 20 30 40 50 60 70 Chlorine concentration/pmol dm-3 Fig. 2 9.10-diphenylanthracene by chlorine Stern-Volmer plot of the quenching of the fluorescence of with ambient oxygen in these experiments. Where one fluorescent species is quenched by more than one quencher, the Stern-Volmer equation can be extended to take into account the contribution of the second quencher.9 With the extremely efficient quenching of the PAH molecules by chlorine, interference caused by oxygen or any other diffu- sion-controlled quencher is not thought to be significant.Conclusion The fluorescence of certain PAHs has been found to be quenched extremely efficiently by chlorine. The findings demonstrate that the PAH molecules can be applied in measurements of chlorine concentration. This might be adopted in the development of optical fibre based sensors for chlorine. Removal of the chlorine from the dye with a suitable agent will facilitate the development of a reusable sensor in which the reagent is continuously replenished. One of us (S. A. M.) acknowledges the financial support of the Science and Engineering Research Council (UK).ANALYST, JANUARY 1992, VOL. 117 85 References 1 Guilbault. G. G., Practical Fluorescence-Tlteory, Methods, and Techniques, Marcel Dekker. New York, 1973. 2 Kautsky, H.. and de Brujin, H . , Nuturwissenschufren. 1931,19, 1043. 3 Bergman, I.. Nurure (London), 1968, 218, 396. 4 Kroneis, H. W., and Marsoner, H. J., Sens. Actuators, 1983.4. 587. 5 Lubbers. D. W., and Opitz, N.. Sens. Actuators, 1983, 4, 641. 6 Li, P. Y. F., and Narayanaswamy, R., Analyst. 1989, 114,663. 7 Turner, R. M., and Fairhurst, S . , Toxicology of Substances in Relation to Mujor- Hazards. Chlorine, Health and Safety Executive, HM Stationery Office, London. 1990. 8 Berlman, I. B., Handbook of Fluorescence Spectra of Aromatic Molecules, Academic Press, New York, 1971. 9 Wolfbeis, 0. S.. Posch, H . E . , and Kroneis, H. W.. Anal. Chem., 1985.57, 2556. Paper 1 I001 31 K Received January 11, 1991 Accepted August 5, 1991

ISSN:0003-2654    

DOI:10.1039/AN9921700083

出版商:RSC    

年代:1992

数据来源: RSC