ISSN:0003-2654    

DOI:10.1039/AN99116BP015

出版商:RSC    

年代:1991

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN99116FX017

出版商:RSC    

年代:1991

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN99116BX019

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

ANALYST, MAY 1991, VOL. 116 437 Certification of a Reference Material for Aromatic Hydrocarbons in Tenax Samplers Stefaan Vandendriessche and Bernardus Griepink Commission of the European Communities, Community Bureau of Reference (BCR), Brussels, Belgium Jacobus C. Th. Hollander, Johannes W. J. Gielen and Fred G. G. M. Langelaan TNO, Division of Technology for Society, Dele, The Netherlands Kevin J. Saunders BP Research Centre, Sunbury-on-Thames, Middlesex TW16 7LN, UK Richard H. Brown OMHL3, Health and Safety Executive, 403 Edgware Road, London NW2 6LN, UK A homogeneous and stable reference material consisting of aromatic hydrocarbons sorbed on Tenax in stainless-steel sample tubes has been prepared and certified. An initial feasibility trial established that a homogeneous and stable batch could be prepared.Part of this batch was used in an intercomparison, which allowed the identification of various sources of error. A second test batch was used in a second intercomparison in an attempt to improve the analytical performance of the participating laboratories. A third batch (of 1000 tubes) was then prepared and certified on the basis of analyses carried out in ten laboratories. The certified values for benzene, toluene and m-xylene are, respectively, 1.053 k 0.014, 1.125 k 0.015 and 1.043 k 0.015 pg per tube. This Community Bureau of Reference (BCR) Certified Reference Material 112 is recommended for quality control and for calibration purposes. Keywords: Tenax; occupational hygiene; reference material; intercomparison; aromatic hydrocarbons National and international legislation prescribes that the exposure of individual workers to certain potentially harmful substances (defined as agents according to Directive 80/1107EEC)l shall be assessed.If the assessment indicates that exposure is likely to be in the region of the relevant exposure limit, regular monitoring must be carried out. Of the various techniques that are suitable for regular monitoring of personal exposure to a wide variety of organic vapours, a device commonly used is a sorption tube. In this technique a known volume of workplace air (usually from the worker's breathing zone) is drawn through the tube by means of a sampling pump, or a mass of analyte is collected by diffusion. The vapours collected are recovered by solvent or thermal desorption and determined, usually by gas chroma- tography (GC).The certified reference material (CRM) described [Community Bureau of Reference (BCR)*] is based on Tenax, a sorbent that is usually desorbed thermally. This is the first sorbent for which appropriate long-term stability has been demonstrated. A CRM to be used for quality control or calibration should ideally closely match the material being analysed. However, it would be impractical to have CRMs to cover all possible combinations of organic solvents. A choice of representative analytes must therefore be made, and for the first certifica- tion, benzene, toluene and rn-xylene have been chosen on the basis of their toxicity and wide usage throughout the world. Experimental Preparation of Homogeneously Charged Batches The tubes used for the preparation of the test batches and for the production of CRM 112 were commercially available stainless-steel tubes which were cleaned and filled with Tenax as shown in Fig.1. The tube dimensions were: length, 89 mm; o.d., 6.34 mm; and i.d., 5.0 mm. Proprietary brass caps with polytetrafluoroethylene (PTFE) ferrules were used to close the tubes. (Preliminary experiments had shown that the aluminium caps supplied with the tubes were inadequate for the purpose of long-term storage.) The bed of Tenax (100 mg, 60-80 mesh) was 32 mm long and was retained at one end by a metal gauze and at the other end by a plug of quartz wool, a metal gauze and a spring. All tubes were pre-conditioned for 16 h at 300 "C and with a helium flow of 30 ml min-1. Each tube contained, before charging, less than 1 ng (the limit of detection of the method is 1 ng) of benzene, toluene and m-xylene.Diffusion cells were used to blend known levels of benzene, toluene and rn-xylene vapours into a stream of clean air. A known volume of this air was drawn through each tube. The apparatus used is represented schematically in Fig. 2. The amounts of benzene, toluene and rn-xylene (approxi- mately 1 yg of each), the flow-rates and the volumes of air (approximately 1 1 per tube) were such that saturation or breakthrough did not occur. The following precautions were taken to ensure that all the tubes received the same mass of each vapour: (i) the temperature of diffusion cells was controlled to within k 0.02 "C; (ii) the mass flows of air were controlled to within k 0.2%; (iii) all tubes were charged in an uninterrupted period of time during which the atmospheric pressure (which influenced the rates of diffusion) varied only slightly ( e .g . , the finally certified batch was charged in a period of 44 h in which the lowest and highest atmospheric pressures were 100.1 and 102.0 kPa); ( i v ) the total vapour content of the air was continuously monitored by use of a photoionization detector; and ( v ) the amount of air drawn through each tube was fixed by an electronic timer which operated the valves. G H Fig. 1 Schematic representation of a sample of CRM 112. A, Stainless-steel tube; B, metal gauze; C, s ring; D, quartz wool plug; E, grooves for O-rings; F, bed of Tenax 800 mg); G, Swagelok caps (brass); and H, PTFE ferrules438 ANALYST, MAY 1991, VOL.116 The volume flow-rates were measured at regular intervals using calibrated mercury sealed piston flow meters. The diffusion rates of the cells were known accurately from gravimetric determinations which were made weekly over a period of more than 3 months. The purity of the liquids in the diffusion cells was greater than 99.9%. On the basis of the measured flow-rates, rates of diffusion and times taken to charge the tubes, the amounts of vapour received by each tube could be calculated with an estimated uncertainty [a combina- tion of precision (95% confidence limits) and bias] of 0.015 pg of each compound for individual tubes and 0.010 pg for the means of a batch.Assuming quantitative sorption on the Tenax, the amounts charged to each sample of CRM 112 were calculated to be: 1.054 pg of benzene; 1.123 pg of toluene; and 1.039 pg of rn-xylene. Homogeneity and Stability Tests The homogeneity and stability of each batch studied were tested in the following manner: (a) a number of tubes (13 for the test batches, 40 for the reference material) were selected systematically so as to include all sampling units (see Fig. 2) and to include at least one in every four of each series of 12 tubes that were loaded simultaneously. These tubes were analysed in one laboratory on the same day. ( 6 ) Tubes stored under different conditions, i.e., in a refrigerator (04 "C), at room temperature (19-23 "C) and at approximately 40 "C, were analysed at regular time intervals (in the same labora- tory, the tubes stored under different conditions all being analysed on the same day).All these tests were carried out at the laboratory which prepared the batches. Intercomparisons For each round of analysis (two preliminary intercomparisons and the certification exercise), each participating laboratory analysed from four to ten tubes. The participating laboratories were as follows: Akzo Research (Arnhem, The Netherlands); BP Research (Sunbury-on-Thames, UK); Directoraat-Gene- raal Van de Arbeid (Voorburg, The Netherlands); Dow Chemical (Nederland) (Terneuzen, The Netherlands); Eolas (Dublin, Ireland); Health and Safety Executive, Occupational Medicine and Hygiene Laboratory (London, UK); ICI Chemicals and Polymers plc.(Runcorn, UK); Koninklijke- Shell Laboratorium (Amsterdam, The Netherlands); Labora- tory of the Government Chemist (London, UK); Arbejdsmil- joinstituttet (Hellerup, Denmark); Rhone-Poulenc Indus- trialization(Decines-Charpieu, France); State Laboratory (Dublin, Ireland); and Universita Degli Studi di Urbino (Urbino, Italy). In the following text, numerical codes (not related to the above alphabetical order) are used to refer to the analytical laboratories. M FC Mixing Critical orifice Sampling I unit x 12 Fig. 2 Schematic representation of the vapour generating and tube charging apparatus; up to 12 tubes were charged in parallel. (MFC = mass flow controller) All the laboratories but one used a procedure based on thermal desorption and gas chromatographic separation and detection (represented schematically in Fig.3) as follows. (a) A carrier gas stream was passed through the tube to be analysed, which was held at approximately 250 "C (laboratory 9: 275 "C; laboratory 6: 280 "C) for 5-10 min (laboratory 10: 3 min; laboratory 6: 4 min; laboratory 8: 25 min). The gas stream was then passed into a cold trap which contained a small amount of Tenax (or similar sorbent) and which was held at approximately -30 "C (laboratory 8: -25 "C). (6) For the second step of the procedure (injection into the gas chromatograph), a valve was switched so that the gas flow was directed into the GC column and the cold trap was heated to 250-300 "C. (c) In the third step of the procedure (gas chromatographic separation and detection) the valve was switched to the original position and (unless the isothermal mode was used) the temperature programme of the chromato- graph was started. Most of the laboratories used a capillary column (length, 25-60 m; i.d., 0.20-0.32 mm) coated with a non-polar stationary phase [ e .g . , poly(dimethylsiloxane)]. Laboratory 6 used a packed column (2 m x 2 mm i.d.). A wide range of temperature programmes were used (chosen so as to complete the chromatogram in 5-20 min). A flame ionization detector was used in all instances. The procedures used in laboratories 6, 11 and 12 differed significantly from that outlined above. Solvent desorption and mass spectrometric detection were used in laboratory 11. The Tenax powder was removed from the tube and transferred with 2 ml of hexane into a 5 nil flask which was occasionally shaken.After 30-60 min at room temperature, samples from the solution were injected into the gas chromatograph with a syringe. No cold-trap was used in laboratory 6; the vapours were sorbed directly on the GC column which was held at room temperature. Laboratory 12 transferred the Tenax from the tubes supplied into 18 cm tubes before desorption. Certification of CRM 112 In the third round of analysis, the certification round, the participants had all re-evaluated their method of calibration. An example re-evaluation is given by Wright.3 All syringes and volumetric glassware used were calibrated gravirnetrically and the analytical balances were checked with certified weights. The results were calculated from the GC detector response using calibration graphs based on three or more calibration solutions. These solutions were prepared by mixing accurately known volumes (laboratories 3 and 7) or masses (other laboratories) of pure benzene, toluene and rn-xylene with an accurately known amount of solvent (methanol, cyclohexane or carbon disulphide), in systems designed so as to minimize losses through evaporation. Successive dilution was avoided.The purity of the products used was verified. In laboratories 6 and 12, microlitre amounts of standard solutions were injected directly into the gas chromatograph for calibration. In other laboratories, similar amounts of standard solutions were added to clean Tenax tubes by injection in a flow of clean gas (injection of liquid on the Tenax powder was avoided) and these tubes were analysed in the same way as the samples. Thermal desorber C Fig.3 Schematic representation of thermal desorption-gas chromatography equipmentANALYST, MAY 1991, VOL. 116 439 I I I 1 /- + C Q) c. C 1.05 8 .? 1 .oo - (0 C .- w- 0 C 0 tj 0.95 2 .- LL 1.05 1 .oo 0.95 I I 1 0 3 6 14 Time/months Fig. 4 Stability of a batch of charged tubes similar to CRM 112. Mean k one standard deviation of a set of 5-13 analyses. ( a ) Benzene; (b) toluene; and (c) rn-xylene. 0, storage at 0 4 OC; 0, storage at ambient temperature; and A, storage at 40 "C Results and Discussion Homogeneity and Stability Tests The homogeneity test yielded in all instances relative standard deviations (RSDs) of <1.5%; this corresponds with the RSD typical for the thermal desorption-gas chromatography pro- cedure used by Brown et al.4 It was concluded that no inhomogeneity could be detected.The stability test outlined above was applied only to the first test batch. The results are presented graphically in Fig. 4. Significant systematic differences which correlated to the volatility of the compound or to the storage temperature were not observed; the long-term variation in the results is thus of analytical origin and not caused by losses through evapora- tion, chemical reactions or irreversible sorption. The stability of CRM 112 has been monitored further by the periodic analysis of samples stored at room temperature (as the entire batch); no instability was detected after 25 months. Intercomparisons Figs.S(a) and (b), 6(a) and (b), and 7(a) and ( 6 ) present the intercomparison results for individual laboratories in graph- ical form where the variable plotted is the ratio of the value found to the known value, and the means and standard deviations are shown. In each instance, the mean values were close to the amounts with which the tubes were charged, but the confidence intervals were unsatisfactory as they were much larger than expected on the basis of the within-laboratory repeatability of the analysis. 1.50 1.25 1 .oo 0.75 0.50 * € 1 1 1 1 1 1 1 1 1 1 1 1 1 i i , a 3 ; f 0.50 (Illllllllllllj 1.50 1.25 1 .oo 0.75 0.50 ! 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 Laboratory n urn ber Fig. 5 Results of three successive intercomparisons for benzene on Tenax (mean value and standard deviation for each laboratory).'Known value' = value calculated from charging data. (a) First round; (b) second round; and ( c ) third round After each of the preliminary intercomparisons, the results were discussed with all the participating laboratories. They explained in great detail how their analyses had been carried out. Consequently, various potential sources of error were identified and eliminated. Considerable progress was made between the second and the third round of analysis. This improvement resulted from measures being taken, from which some laboratories expected only a minor improvement (e.g., calibration of volumetric glassware, preparation of calibration solutions in sealed systems in order to avoid losses through evaporation, gravimetric dilution and dispensing, weighing of the syringe before and after injection, and calibration and maintenance of the analytical balance).As all participating laboratories were considered to be highly competent analytical laboratories, other laboratories are expected to experience similar difficulties and therefore some typical sources of error are listed below. Errors due to the handling of the tubes Errors arising from the handling of the tubes included: loss of sorbent when applying force to remove the cap from the grooved end of the tubes; and losses of vapour (or contamina- tion risk) upon contact with the atmosphere when the tubes were opened in order to transfer the sorbent into another container for solvent desorption, or similar risks when caps440 ANALYST, MAY 1991. VOL.116 1.50 I (a) I 1.25 1 R 0.75 t 1.50 I(bl 4 1 > 1.25 0.50 \ 0.50 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 Laboratory number Fig. 6 Results of three successive intercomparisons for toluene on Tenax (mean value and standard deviation for each laboratory). 'Known value' = value calculated from charging data. (a) First round; (b) second round; and (c) third round were removed a long time before the determinations were carried out. Errors in the calibration Errors in calibration resulted from: partial evaporation of volatile compounds of interest, or of the volatile solvent, between preparation and final use of the standard solutions (especially when syringes were being manipulated); relying on uncalibrated syringes or other glassware (errors of the order of 10% can be expected); and ignoring the water content of the solvents used.Errors due to malfunctioning instruments Errors caused by malfunctioning instruments were due to leaks in the connections of the tube in the thermal desorber; the use of a malfunctioning analytical balance (if the calibra- tion is not checked periodically, an error may remain undetected as short-term repeatability may remain normal); and the use of an ionization detector that was not optimized (checking is required whenever the response factors for benzene, toluene and m-xylene differ by more than 2%). 1.50 1.25 1 .oo 0.75 0.50 1 1 I I I I I I I 1 1 I I 1 1.50 1 P) 3 m - 1.25 ; Y u 1.00 C 3 0 .c al - 3 0.75 >" 0.75 0.501 I I I I I I I I ' I 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 Laboratory number Fig.7 Results of three successive intercomparisons for rn-xylene on Tenax (mean value and standard deviation for each laboratory). 'Known value' = value calculated from charging data. (a) First round; (b) second round; and (c) third round Certification The results of the certification (effectively a third intercom- parison) are presented in Figs. 5(c), 6(c) and 7 ( c ) . For this intercomparison, the ten laboratories applying thermal desorption achieved excellent agreement. Only one result (laboratory 3, benzene) was identified as a straggler (Dixon test). As this result was associated with a calibration error, the result was rejected. The data obtained from laboratory 11 (which used solvent desorption) were also rejected, on the grounds either that solvent desorption had been used or that losses through volatilization had occurred when the Tenax was being transferred from the tube into the flask, or that desorption had been incomplete. The mean of laboratory means and the halfwidth of its 95% confidence interval for each component are as follows: benzene, (1.053 k 0.014) pgper tube; toluene, (1.125 k 0.015) pg per tube; and rn-xylene, (1.043 k 0.015) pg per tube. If the interlaboratory standard deviation is significantly larger (as calculated using the F-test) than the intralaboratory standard deviation, the presence of remaining systematic errors in the participating laboratories is indicated. It is generally assumed that these errors are randomized, i.e., they do not cause aANALYST, MAY 1991, VOL.116 44 1 systematic error in the mean of means, if the interlaboratory standard deviation does not exceed the intralaboratory standard deviation by more than a factor of 2-3; in the present instance, the factor is <1.0 for each analyte. In addition, the values measured correspond closely with the masses with which the tubes were charged (the difference being 0.1, 0.2 and 0.4% for benzene, toluene and m-xylene, respectively). These results were therefore considered to provide sufficient basis for certification. Conclusions The data presented in this paper demonstrate that a batch of Tenax tubes charged with benzene, toluene and m-xylene can be prepared and analysed to an uncertainty of better than 2%, provided the analytical laboratories apply due care and attention to detail.Initial intercomparisons gave results with much larger errors and some of the potential sources of such errors have been identified. The absence of a detectable systematic error is a sufficient reason to declare the batch certified to BCR specifications and the batch is offered for sale for calibration and quality control. The uncertainty obtained renders the material useful for these purposes. The certification of this reference material is the first major achievement in the BCR efforts to provide means of quality assurance in occupational health monitoring. Work similar to that described here is in various stages of progress for: (i) aromatic hydrocarbons on active charcoal (for solvent desorption); (ii) chlorinated C2 hydrocarbons on Tenax; and (iii) esters and ketones on Tenax. Several other possible projects are in the discussion stage; feasibility studies on amines (including triethylamine), alde- hydes (including formaldehyde) and isocyanates are planned for the near future. References Council of the European Communities, Directive 88/1107/EEC, Official Journal of the European Communities, 1980, No. L327/8. Vandendriessche, S., and Griepink, B., The Certification of Benzene, Toluene and rn-Xylene Sorbed on Tenax in Tubes, Report EUR 1 2 3 0 8 ~ ~ , Commission of the European Communi- ties, Luxembourg (Office for Official Publications of the European Communities, 1989). Wright, M. D., BCR Certification of Reference Materials- Organics on Tenax-a Critical Examination of Sources of Calibration Error, Health and Safety Executive (HSE) Internal Report IR/L/IA/90/3, HSE, London, 1990. Brown, R. H., Cox, P. C., Purnell, C. J., West, N. G., and Wright, M. D., in Identification and Analysis of Organic Pollutants in Air, ed. Keith, L. H., Ann Arbor Science Publishers/Buttenvorth, Woburn, MS, 1984. Paper 010.53936 Received November 29th, 1990 Accepted December 12th, 1990

ISSN:0003-2654    

DOI:10.1039/AN9911600437

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

ANALYST, MAY 1991, VOL. 116 443 Investigation of the Quenching of Peroxyoxalate Chemiluminescence by Amine Substituted Compounds Joseph K. DeVasto and Mary Lynn Grayeski" Chemistry Department, Seton Hall University, South Orange, NJ 07079, USA The role of amine compounds in quenched peroxyoxalate chemiluminescence was investigated. The mechanistic steps examined included (1) fluorescence quenching; (2) base hydrolysis of the oxalate; and (3) a competitive interaction between the quencher and fluorophore for the peroxyoxalate reaction inter- mediate(s). The results showed no evidence of amines causing fluorescence quenching. Base hydrolysis of the oxalate is significant only at high concentrations of amines. When the concentration of amines is greater than or equal t o the level of oxalate, the amines can compete with the fluorophore for reaction with the intermediate(s).Because competitive effects were demonstrated, one analytical implication is that caution must be exercised in applications where more than one fluorophore is present. At low concentrations of fluorophores relative to the oxalate, chemiluminescence emission of both fluorophores will be observed. At higher levels, the fluorophores can compete for reaction with the intermediate(s). Finally, an important analytical implication of this study is that the quenching response can be used t o quantify amines without the need for derivatization. The limitation is that the linear response is approximately one order of magnitude. Keywords: Peroxyoxalate chemiluminescence; aliphatic/aromatic amines; quenched chemiluminescence Rauhut et al.1 first reported significant differences in quantum yields and chemiluminescence (CL) lifetimes when adding basic compounds to a peroxyoxalate reaction containing bis(2,4-dinitrophenyI) oxalate (DNPO) and 9,10-diphenyl- anthracene. Quenched peroxyoxalate CL was also observed in the reaction of hydrogen peroxide with bis(pentach1oro- phenyl) oxalate (PCPO) using sodium salicylate as a base catalyst .? A number of substituted anilines, and organosulphur and ionic compounds have been analysed by high-performance liquid chromatography and flow injection with quenched peroxyoxalate CL detection.3.-' A competitive quenching mechanism between easily oxidizable analytes and the peroxy- oxalate reaction intermediate was initially proposed.A later report4 suggested that the quencher is involved in radiation- less deactivation of the fluorophore-charge-transfer complex. The purpose of this study was to examine the role of aliphatic and aromatic amines in quenched peroxyoxalate CL, and to develop an analytical technique for measuring amine compounds by means of this quenching phenomenon. The advantage of this approach is that a derivatization step is not required prior to detection of the aliphatic amines. 'This is particularly important for tertiary aliphatic amines for which derivatization reactions are limited. The mechanistic pathways that a quencher (Q) can follow in the reaction with bis(2,4,6-trichlorophenyl) oxalate (TCPO) and hydrogen peroxide are: TCPO + H202 + intermediate(s) (I) + 2[2,4,6-trichlorophenol (TCP)] (1) Q TCPO + OH- + 2TCP ( 1 4 I + fluorophore (FL) + [I'-FL'+] (2) I + Q + non-CL ( 2 4 [I'-FL'+] + FL" ( 3 ) FL" + FL + hv (4) FL" + Q -+ FL ( 4 4 The asterisk signifies the excited state of the fluorophore.Equations (1)-(4) [excluding (la), (2a) and (4a)l are the proposed light producing pathways. Hydrogen peroxide and * To whom correspondence should be addressed. TCPO react to form a high energy intermediate(s) that undergoes an electron charge transfer with a fluorophore. The charge-transfer process leads to radical ion annihilation and formation of an excited state fluorophore that fluoresces. The addition of an amine (or other quencher) can cause the reaction to proceed along dark pathways such as (i) base hydrolysis of the oxalate [equation (la)], (ii) competitive interaction of the quencher and fluorophore for the inter- mediate [equation (2a)l and (iii) fluorescence quenching of the fluorophore [equation (4a)l.Experimental Chemicals Triethylamine (TEA), N-propylamine (N-PA), N-isopropylcyclohexylamine (N-IPCA), 2-ethylaniline (2- EA), 4-toluidine (4-TOL), N,N-dimethylaniline (N,N-DMA) and 30% hydrogen peroxide were obtained from Aldrich. Analytical-reagent grade dibasic sodium phosphate was received from J. T. Baker and TCPO was obtained from A. Mohan (New Jersey Department of Health, Trenton, NJ, USA). Spectrophotometric grade acetonitrile (Aldrich) and analytical-reagent grade anhydrous sodium perchlorate (GFS Chemicals) were used for determining the oxidation potentials of the fluorophores. The fluorophores used in the fluorescence and CL studies, viz., 1-aminoanthracene, 1-aminopyrene, anthracene, pyrene, perylene, rubrene and 1-aminonaphthalene, were purchased from Aldrich.2,4,6-TrichlorophenoI (TCP) was obtained from Eastman Chemicals, and HPLC grade acetonitrile (Fisher) was used throughout. All chemicals were used as received without additional purification. Static Chemiluminescence: Competitive and Quenching Experiments A Turner Instruments Model TD-20e luminometer was used for CL measurements. In the competitive interaction experi- ments, the reagents were added to a 1.6ml polypropylene cuvette in the following order: (i) 1OOpl of H202 (490 mmol dm-3); (ii) 100 pl of Na2HP04 buffer ( 5 mmol dm-3) (pH 6.2); and either (iiia) 40 pl of acetonitrile for a blank run, (iiib) 20 p1 of solution for a single fluorophore444 1.5 1.0 -0 \ 9 ANALYST, MAY 1991, VOL.116 . run plus 20 pl of acetonitrile or (ziic) 20 pl of each fluorophore solution with no acetonitrile added for runs with mixtures of two fluorophores. For the quenched CL experiments, steps (i) and (ii) were repeated and either (a) 20 pl of 1-aminopyrene (1 pmoldm-3) plus 20pl of quencher solution, ( b ) 201-11 of fluorophore solution plus 20p1 of acetonitrile, (c) 201-11 of quencher solution plus 20pl of acetonitrile or (d) 40p1 of acetonitrile were added to the cuvette. The TCPO (2mmoldm-3) was injected last (50 pl) for either set of experiments into the reaction cuvette with the Turner apparatus.With the final addition of TCPO the total volume was 290 pl for each CL measurement. The CL signal was monitored for 120 s with a Fisher Series 5000 strip-chart recorder, and the CL intensity areas were determined by electronic integration (0-120 s) with the Turner luminometer. The CL reagents were prepared in acetonitrile except for the aqueous phosphate buffer. Relative Fluorescence Quenching Experiments Fluorescence quenching was measured by using a Fluorolog 2 + 2 spectrofluorimeter with a 450W Xe continuous source (Spex Industries). The fluorescence intensity of l-amino- pyrene (1 pmol dm-3) was determined by adding 0.2 ml of the fluorophore to a cuvette containing 1.0 ml of sodium phos- phate buffer (5 mmol dm-3) (pH 6.2) and 1.7 ml of acetonit- rile. Relative fluorescence quenching was measured by adding 0.2 ml of the quencher (at several concentrations examined in the quenched CL experiments) plus 0.2 ml of 1-aminopyrene (1 pmol dm-3) to 1.0 ml of phosphate buffer ( 5 mmol dm-3) (pH 6.2) and 1.5 ml of acetonitrile.Spectral peak areas were determined at an excitation wavelength of 360nm and an emission wavelength range of 390-500nm with a 1 nm bandpass. Base Hydrolysis and Apparent pH The formation of TCP from TCPO was measured at 298 nm by ultraviolet (UV) absorbance5 using a Varian 2200 UV spec- trophotometer. Absorbance readings were recorded 2 min after injecting the last reagent under two sets of run conditions. The injection volumes for each set of runs were: 0.5 ml of TCPO (2 mmol dm-3) + 1.0 ml of Na2HP04 buffer (5 mmol dm-3) (pH 6.2) + 1.0 ml of H202 (490 mmol dm-3) + (0.2 ml of 1-aminopyrene (1 pmol dm-3) + 0.2 ml of acetonitrile; and the same sequence as for the first run but with 0.2 ml of quencher solution substituted for acetonitrile.The background absorbance readings of the fluorophore, H202, buffer and quencher solution components were subtracted from the values obtained from the two sets of runs. All reagents were prepared in acetonitrile (except for the aqueous buffer). The quartz cuvette was placed in the instrument prior to injecting the final reagent (TCPO) in order to improve the experimental precision. Measurements of apparent pH were made on CL solutions that contained quenches at concentrations ranging from 0.0069 to 6.9 mmol dm-3. The injection order and concentra- tions of CL reagents were: 10 ml of H202 (490 mmol dm-3); 10ml of Na2HP04 buffer (5mmoldm-3) (pH6.2); 2ml of 1-aminopyrene (1 pmol dm-3); 2 ml of quencher solution; and 5 ml of TCPO (2 mmol dm-3).Acetonitrile (2 ml) was substi- tuted for the quencher solution when the apparent pH of the CL reaction was measured. The apparent pH was determined with an Orion Research Model 611 pH meter 2min after adding the last reagent. Oxidation Potentials Oxidation potentials of 1-aminoanthracene and l-amino- pyrene were determined by cyclic voltammetry with an IBM EC/225 voltammetric analyser. Half-wave potentials were determined by using a platinum disc working electrode, a platinum wire auxiliary electrode and an Ag-Ag+ reference electrode. The supporting electrolyte was 0.5 mol dm-3 sodium perchlorate in acetonitrile, and the sweep rate was 10 mV s-1.Results Quenched peroxyoxalate CL in a buffered solution was investigated by measuring the CL response while varying the concentration of amines over several orders of magnitude (Figs. 1 and 2). The CL signal was quantified by determining the quenching ratio IQ : 10, where IQ is the CL intensity with the quencher and Z0 is the intensity without a quencher in the CL reaction. Figs. 1 and 2 show that the aliphatic amines have a more significant quenching effect on the CL reaction in the 0.035-0.69 mmol dm-3 concentration range. Also, an increase in CL intensity is observed for 2-EA and 4-TOL at a concentration of 0.035 mmol dm-3. 1.0 . . 0.6 s 0.2 0.4 t nn 0.5 t ir I € T I I, 0 I I 1 I 1 10 100 1 000 10 000 Concentration/lO-7 mol dm-3 Fig.2 Quenched eroxyoxalate CL ratios for the aromatic amines 2-EA (a), 4-TOL 6) and N,N-DMA (A). Point representation and concentration of the fluorophore as in Fig. 1ANALYST, MAY 1991, VOL. 116 445 Figs. 3 and 4 show typical quenched CL profiles of TEA and 2-EA, respectively. Two peaks are observed when a 0.69 mmol dm-3 TEA solution is added to the CL reaction without 1-aminopyrene (Fig. 3, B). No double peak is observed, however, when the fluorophore is included in the CL reaction. Also, double peaks are not observed when aromatic amines are added to the CL reaction. A decrease in the CL rate of decay occurs at relatively low concentrations (less than 0.069mmoldm-3) of aromatic amines, and an increase in CL emission is observed.The quenched CL response is linear [analysis of variance (ANOVA) regression; F = 0.9751 over a relatively narrow concentration range, viz., 0.0174.17 mmol dm-3 for TEA, and 0.045-0.17 mmol dm-3 for N-IPCA or N-PA. The CL response curve was calculated by taking the reciprocal value of the quenched CL signal versus concentration. The lowest levels of aliphatic amines measured (n = 4) by quenched CL were 0.017 k 0.001mmoldm-3 TEA, 0.045 k 0.002 mmol dm-3 N-IPCA and 0.045 k 0.003 mmol dm-3 N-PA. Fluorescence Quenching A relative comparison of fluorescence spectral peak areas of 1-aminopyrene was made with and without an amine in solution in order to determine fluorescence quenching (Table 1). No fluorescence quenching is observed when the amines are added to 1-aminopyrene in an acetonitrile- phosphate buffer solution. Also, there is no evidence of a shift in the spectral peaks of 1-aminopyrene at any concentration of the quenchers studied.D 3 6 9 Time/m i n Fig. 3 Quenched peroxyoxalate CL with 0.69 mmol dm-3 TEA. Static CL curves: A, acetonitrile blank (CL solution without fluorophore or TEA); B, CL solution with TEA and no fluorophore; C, CL solution with 1-aminopyrene; and D, CL solution with 1-aminopyrene plus TEA. The sensitivity of the luminometer was increased 100-fold when recording CL curves A and B relative to curves C and D C ~~~ 3 6 9 Fig. 4 Quenched peroxyoxalate CL with 0.69 mmol dm-3 2-EA. Static CL curves A-D are as given for Fig. 3 with curves A and B measured at a 100-fold increase in sensitivity relative to curves C and D Timeimin Hydrolysis The apparent pH of the quenched peroxyoxalate CL reaction was measured and compared with the CL reaction without a quencher.There is no significant increase in apparent pH of the quenched CL reaction, except for aliphatic amines added at concentrations greater than or equal to 0.069 mmol dm-3. A similar experiment was conducted using UV absorbance to measure the change in the concentration of TCP in the CL reaction when adding TEA at several concentrations (Table 2). As the concentration of the aliphatic amine increases, an increase in the level of TCP relative to the CL reaction without TEA is observed. When absorbance measurements were made on the CL reaction with and without the addition of aromatic amines, there was no increase observed in the concentration of TCP.Competitive Interaction The competitive role of the quencher and fluorophore for the CL reaction intermediate(s) was examined by comparing the CL response of the reaction with one fluorophore with the CL response of the reaction with a mixture of two fluorophores. If the sum of the signals for the individual fluorophores is equivalent to the CL response of the mixture, then there is no competition between the fluorophores for interaction with the intermediate. If the CL signal of the mixture is not equivalent, the interaction with the peroxyoxalate intermediate(s) would be favoured for one fluorophore relative to the other. Two sets of conditions were evaluated in the competitive interaction experiments: (i) the concentrations of the fluoro- phores were lower than the concentration of TCPO by approximately 2-5 orders of magnitude (except for pyrene) ; and (ii) the total concentration of amino substituted fluoro- phores was within one order of magnitude of the concentra- tion of TCPO.These conditions include the entire range of concentrations of amines used in the quenching experiment. If the two fluorophores react competitively with the intermediate(s), the reactivity of one fluorophore might be favoured based on its oxidation potential or concentration. Both of these parameters were evaluated for various fluoro- phores (Table 3). Sums of the CL signals were determined for pairs of fluorophores by using a central composite experi- mental design.The results showed that the CL signal ( n = 3 injections) for a mixture of two fluorophores is equivalent Table 1 Relative fluorescence quenching. The values are relative to the fluorescence intensity of 1-aminopyrene, which is equal to 1 .00 in the absence of a quencher; relative standard deviation = 6.1% (n = 7) Quencher 6.9 mmol dm-3 0.69 mmol dm-3 0.34 mmol dm-3 TEA 1.06 1.08 1 .oo N-PA 1.04 1.03 1.01 N-IPCA 0.95 - - 2-EA 1.02 0.98 1.03 4-TOL 1.06 0.96 - N, N-DMA 0.97 0.98 - ~ ~~~~~ Table 2 TCP product formation during the peroxyoxalate CL reaction plus quencher TEA concentra- tion/mmol dm-3 TCP/mmol dm-3 0 0.54* 0.0069 0.55 0.034 0.56 0.069 0.57 0.34 0.60 0.69 0.60 6.9 0.66 * Average of duplicate injections; relative standard deviation = 1.3%.446 ANALYST, MAY 1991, VOL.116 Table 3 Oxidation potentials (Etox) and concentrations of fluoro- phores in the CL reaction. Final CL reagent concentrations in the static cell: TCPO, 0.34 mmol dm-3; H?O2, 170 mmol dm-3; and phosphate buffer, 1.7 mmol dm-3 Concentration/ E40d pmol dm-37 Fluorophore V versus Ag-Ag+ * Anthracene +0.79 7.4 3.7 0.7 Pyrene +0.86 69.7 34.8 7.0 1-Aminoanthracene +0.10 0.08 0.04 0.008 1- Aminop yrene t-0.19 0.07 0.04 0.007 Perylene +0.55 0.01 0.005 0.001 Rubrene +0.52 0.02 0.01 0.002 1- Aminonaphthalene +0.24 1.4 0.7 0.14 * References 6 and 7. t Three concentration levels were studied with pairs of fluorophores mixed in solution. (within a 95% confidence interval) to the sum of the CL signals for the individual fluorophores when measured at concentrations much lower than that of TCPO.The second set of conditions was examined by increasing the total concentra- tion of the fluorophores and decreasing the level of TCPO by one order of magnitude. 1-Aminonaphthalene (13 vmol dm-3) and 1-aminopyrene (0.71 pmol dm-3) were measured in the experiment with TCPO (0.034 mmol dm-3), H202 (170 mmol dm-3) and phosphate buffer (1.7 mmol dm-3) (pH 6.2). The CL signal of the mixed fluorophores (n = 3) is not equivalent to the sum of the CL responses of each fluorophore when compared within 95% confidence intervals. Discussion Role of the Quencher The role of amines in a buffered peroxyoxalate reaction was examined through a number of possible quenching mechan- isms. The non-chemiluminescent reaction pathways investi- gated included: fluorescence quenching of the fluorophore [equation (4a)l; base hydrolysis of the oxalate [equation (la)]; and competitive interaction of the amine and fluorophore for the peroxyoxalate reaction intermediate(s) [equation (2a)l.The results in Table 1 show that there is no significant change in the fluorescence intensity of 1-aminopyrene at the higher concentrations of quenchers used in this study. Therefore, the amines are not causing fluorescence quenching [equation (4a)l of the fluorophore in the peroxyoxalate CL reaction. The effect of TEA on the base hydrolysis of TCPO was determined at several concentrations of TEA in the CL reaction (Table 2). The formation of TCP is increased relative to the CL reaction without TEA at levels greater than or equal to 0.069 mmol dm-3.Aliphatic amines also affect the appar- ent pH of the CL reaction at concentrations greater than 0.069 mmol dm-3. These results indicate that aliphatic amines have sufficient basicity to exceed the buffer capacity of the CL reaction and cause the hydrolysis of TCPO. It has been speculated that the efficiency of the peroxyoxalate CL reaction decreases above pH 8 because of base hydrolysis of the oxalate.8 In this study, base hydrolysis also appears to be part of the quenching mechanism when high concentrations of aliphatic amines are added. There is no evidence of base hydrolysis when aromatic amines are added to the CL reaction. There is also no significant change in the apparent pH of the CL reaction when 2-EA, 4-TOL or N,N-DMA are added at a high concentration (6.9 mmol dm-3).These aromatic amines are relatively weak bases compared with the aliphatic species studied, and do not exceed the buffer capacity of the CL reaction. The possibility of a competitive quenching mechanism was investigated by measuring the CL response of pairs of fluorophores both as a mixture and separately (Table 3). If a competitive mechanism exists, then a decrease in CL intensity should be observed relative to the sum of the intensities for each species measured separately. There is no competitive interaction for the peroxyoxalate reaction intermediate(s) when the concentration of TCPO is several orders of magnitude greater than that of the fluorophores. Also, differences in the oxidation potentials of the two fluorophores do not contribute to a competitive interaction for the reaction intermediate(s) .There is evidence of a competitive mechanism when the total concentration of 1-aminopyrene or 1-aminonaphthalene is within one order of magnitude of the level of TCPO. As the concentration of the peroxyoxalate reaction intermediate(s) is limited relative to that of the fluorophores, one fluorophore acts as a competitive quencher of the CL reaction. The non-fluorescent amines used in the quenching study (Figs. 1 and 2) were added to the peroxyoxalate reaction at concentra- tions that were greater than or equal to the level of TCPO (0.34 mmol dm-3). This implies that a competitive mechanism can occur between the quencher and fluorophore for the peroxyoxalate reaction intermediate(s) if the concentration ratio of quencher:TCPO is greater than or equal to unity. This is based on most of the TCPO reagent being converted into the high energy intermediate(s) in the peroxyoxalate reaction.It is not clear which quenching mechanism operates when the concentration of the amine is significantly less than that of TCPO. An earlier report4 on the role of a quencher in the peroxyoxalate reaction postulated that the fluorophore- charge-transfer complex undergoes radiationless deactiva- tion. This suggests that a trimolecular interaction of the quencher, fluorophore and intermediate is favoured over a bimolecular light-producing fluorophore-intermediate re- action pathway. It should be noted that fluorophores with broad emission bands and some spectral overlap were included in this study.Theoretically, it should be possible to measure the emission from one fluorophore in the presence of another if the two fluorophores are sufficiently spectrally resolved and exhibit efficient CL. This would require the use of a very long wavelength emitter relative to the fluorophores usually measured with this reaction.9 For the fluorophores examined here, the emission levels were very low at all the concentra- tions studied and the total intensity was integrated over all wavelengths in order to collect an adequate signal. Analytical Implications Aliphatic and aromatic amines can be quantified by using quenched peroxyoxalate CL detection. An advantage of this technique is that aliphatic amines can be detected without derivatization.This is important in quantifying tertiary aliphatic amines where the choice of a derivatizing reagent is very limited. There are limitations that must be considered in quantifying amines by quenched CL detection. Fig. 1 clearly shows a non-linear quenching response when aliphatic amines are measured over three orders of magnitude in concentration. This is the result of the quencher causing complex kinetics that change the CL peak shape and rate of decay. A probable cause of this phenomenon (Fig. 3, C and D) is that as the concentration of the aliphatic amine increases in the buffered CL reaction, it exceeds the buffer capacity and acts as a base catalyst thus increasing the CL rate of decay.'() A linear response can only be observed over a range less than or equal to one order of magnitude in concentration.The quenched CL response of the aromatic amines (Fig. 2) is also non-linear and shows an increase in CL emission at relatively low concentrations. The cause of the increase in CLANALYST, MAY 1991, VOL. 116 emission was not investigated, but a decrease in the CL rate of decay was observed. It is possible that the aromatic amines are increasing the formation of a second peroxyoxalate reaction intermediate. 1 1 Possible evidence for this mechanism was obtained qualitatively (Fig. 3, B). The CL intensity-time curve has two peaks, which indicates that two intermediates are formed when aliphatic amines are added to the CL reaction without a fluorophore. A burst of light generates the initial peak which might correspond to a reaction pathway in which an arylperoxyoxalic acid intermediate is formed rapidly together with a dioxetane dione or dioxetanone intermediate species.Although double peaks are not observed for the aromatic amines (Fig. 4), it is still possible that a second reaction intermediate is formed but cannot be observed. The addition of aromatic amines might generate a burst of light which is too rapid to measure, o r a second low-level emission peak which cannot be detected above the main CL signal. As competition occurs, the question about mixtures of fluorophores is raised. Is one fluorophore preferentially being measured when several fluorophores are present in a solution? This information was obtained by investigating the competi- tive role of a quencher in the peroxyoxalate CL reaction. A quenched CL signal could occur when determining the concentration of more than one fluorophore in a solution.One fluorophore can act as a quencher if the total concentration is within one order of magnitude of the level of the oxalate in the CL reaction. A quantitative CL signal should result from a mixture of fluorophores when the total concentration of the fluorophores is significantly less than that of the oxalate. This research was supported through Corporation. 447 a grant from Research 1 2 3 4 5 6 7 8 9 10 11 References Rauhut, M. M., Bollyky, L. J . , Roberts, G. B., Loy, M . , Whitman, R. H., Iannotta, A. V.. Semsel, A. M., and Clarke, R. A., J. Am. Chem. Soc., 1967, 89, 6515. Catherall, C. L. R., Palmer, T. F., andcundall, R. B.,J. Chem. Soc., Faraday Trans. 2 , 1984, 80, 823. van Zoonen, P., Kamminga, D. A., Gooijer, C., Velthorst, N. H., and Frei, R. W., Anal. Chem., 1986, 58, 1245. van Zoonen, P., Bock, H., Gooijer, C., Velthorst, N. H., and Frei, R. W., Anal. Chim. Acta, 1987, 200, 131. Givens, R. S . , Schowen, R. L., Matuszewski, B., Alvarez, F., Parekh, N., and Nakashima, K., paper presented at the Federation of Analytical Chemistry and Spectroscopy Societies Symposium. St. Louis. MO, September 1986. Pysh, E. S., and Yang, N. C.. J. Am. Chem. Soc., 1963, 85, 2124. Siegerman, H . , Techniques of Electroorganic Synthesis, Part It, Wiley, New York, 1975, vol. 5. Wcinberger, R., J. Chromatogr., 1984, 314, 155. Mann, B., and Grayeski, M. L., Anal. Chem.. 1990, 62, 1532. Nozaki. 0.. Ohba, Y . , and Imai, K . , Anal. Chim. Acfa, 1988, 205, 255. Alvarez. F. J., Parekh, N. J . , Matuszewski, B., Givens, R. S . , Higuchi, T., and Schowen, R. L., J . A m . Chem. Soc., 1986,108, 6435, Paper 0103 784 B Received August 17th, I990 Accepted December 17th, 1990

ISSN:0003-2654    

DOI:10.1039/AN9911600443

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

ANALYST, MAY 1991, VOL. 116 449 Rapid Method for the Determination of the Major Components of Magnesite, Dolomite and Related Materials by X-ray Spectrometry Michael H. Jones and 6. William Wilson" CSIRO Division of Mineral Products, P.O. Box 124, Port Melbourne, Victoria 3207, Australia A new flux, MAG 5743, has been developed, which contains 57% m/m lithium tetraborate and 43% m/m lithium metaborate for use in the analysis of raw magnesite, caustic and dead burnt magnesia, (i.e., magnesite heated to 700-1100 "C and ~ 1 4 0 0 "C, respectively) and dolomite. The flux can be used for the determination of loss on fusion followed by X-ray fluorescence analysis of the glass disc for its major component elements, as their oxides MgO, CaO, SiOz, A1203 and Fe203, on the one sample. As the flux is both fluid and reactive at 1000 "C, fusion times are short (10 min) regardless of the reactivity of the sample.The results obtained from the analysis of three certified reference materials using the new flux were statistically indistinguishable at the 95% confidence level from the certificate values. Keywords: X-ray fluorescence; magnesia, magnesite and dolomite analysis; loss on fusion The rapid and accurate determination of the principal components of magnesite, magnesia, dolomite and calcite (as the oxides MgO, CaO, SO2, A1203, Fe203) and the loss on ignition (LOI) is necessary for quality control in mining and processing. Current X-ray fluorescence (XRF) techniques require the sample to be calcined before fusion,lJ determine only one component3.4 and/or require the use of high melting-point fluxes with relatively long fusion times.Overall, the tech- niques currently used, although accurate, are time consuming and labour intensive. In addition, making a separate measure- ment of the LO1 has several disadvantages, these being: the time taken for measurement; the temperature of ignition being sometimes insufficient to break down any refractory constituents; and the possibility of re-adsorption of H20 and/or C02 on cooling. These last two disadvantages can contribute to a low LO1 measurement. Furthermore, the LO1 temperatures in recommended methods range from 1000- 1100 "(25-9 The flux used to prepare discs for XRF analysis in most methods is lithium tetrab0rate.1~2~~~~0 An 'acidic' flux such as lithium tetraborate or sodium metaphosphatelo is necessary for the dissolution of samples and production of stable discs.Lithium tetraborate at the usual fluxing temperature of 1100-1200 "C has such a high viscosity that even modern fluxing machines require dissolution times of 30 min or longer. Mixed lithium tetraborate-metaborate fluxes with melting- points of t l O O O "C and a high fluidity are widely available but they usually contain a high proportion of lithium metaborate, which produces unstable discs when used with samples that have high concentrations of Mg or Ca. This paper describes the development of a new flux, designated MAG 5743, with a composition of 57% m/m lithium tetraborate and 43% m/m lithium metaborate, which not only produces stable discs but is also sufficiently reactive so as to obtain the complete dissolution of magnesites, calcined magnesites and dolomites in 10 min at 1000 "C.Two methods are described. The magnesite method, in which the glass disc composition has a flux to sample (FTS) ratio of 5 : 1, is used with samples for which the LO1 is >40% (i, e., uncalcined raw materials). This method provides oxide analysis and a loss on fusion (LOF) value simultaneously. The general method, in which the glass disc composition has an FTS ratio of 10 : 1, is essentially for samples where the LO1 is <40%. ( i . e . , calcined products). Certified reference materials (CRMs)" and four magnesite samples are analysed using the new flux. * To whom correspondence should be addressed. Experimental Sample Preparation The British Chemical Standard (BCS) CRMs magnesites 319 and 389, which are magnesias, were re-ignited at 1025 "C and the dolomite (BCS CRM 368) was oven dried at 110 "C before use.Three discs were prepared from each CRM for analysis and the results obtained were compared with the certificate values. Four samples of magnesite (labelled magnesite A, B, C and D) from the Kunwarara deposit (Queensland, Australia) with a range of MgO, CaO and Si02 contents, were ground to a particle size of t 1 5 0 pm (100%) for analysis by both the magnesite and general methods. Three glass discs were prepared from each of the magnesite samples using both methods, i.e., the LOFs were determined simultaneously. Each disc was analysed separately over a period of 4 d. The data were compared with those obtained from the same magnesites after calcination at 1300 "C; the LO1 was deter- mined separately.Preparation of MAG 5743 Flux The flux was a mixture of 57% m/m lithium tetraborate and 43% m/m lithium metaborate. This flux can be obtained custom blended (by special order from suppliers such as Sigma or Johnson Matthey) or can be blended in the laboratory by mixing the constituents, which are then dried at 500 "C in a muffle furnace for at least 4 h. After removal from the furnace, the flux was cooled in a desiccator charged with a suitable desiccant. The flux should be stored in a capped jar in a desiccator. In order to test whether the flux was in a proper condition for use, a 3 g portion was fused at 1000 "C for 10 min to determine the LOF.(If the LOF exceeds 0.2% the flux must be re-dried at 500 "C.) Preparation of Glass Discs A 0.3000 k 0.0005 g sample (general method) or, a 0.6000 k 0.0010 g sample (magnesite method) was transferred into a 95% Pt-5% Au alloy crucible which had been previously heated to 1000 "C and cooled to constant mass. The sample masses were chosen in order to prepare 30-32 mm diameter discs. Larger diameter discs required larger masses of both sample and flux. The sample was then intimately mixed (by stirring thoroughly with a clean platinum rod) with 3.000 k 0.001 g of dry flux and the total mass of the crucible and contents recorded. The crucible was placed in a muffle furnace450 ANALYST, MAY 1991, VOL. 116 (containing an agitator mechanism) and the sample fused at lo00 "C for 10 min.Alternatively, any comercially available fusion device fitted with an agitator could be used under equivalent conditions. The crucible was removed from the furnace, cooled rapidly on an aluminium block heat sink contained in a desiccator and then re-weighed. The crucible and glass were returned to the furnace at 1000 "C, allowed to melt, and then poured into a casting mould (95% Pt-5% Au alloy, 30 mm i.d.), which had been pre-heated to at least lo00 "C over an oxygen-liquified petroleum gas (LPG) flame. A muffle furnace of appropriate size (set at lo00 "C) is a suitable alternative for casting the disc. The casting mould containing the molten glass was removed from the heat source and cooled rapidly on a graphite block or an air-cooled aluminium block.The disc was then available for analysis in a suitable X-ray spectrometer. [N.B. When a very large number of fusions are made daily, it might be desirable to give the aluminium block a ceramic coating to minimize the possibility of any aluminium adsorption.] Measurement The instrument conditions for the determination of the major components MgO, CaO, SO2, A1203 and Fe2O3 are listed in Table 1. A Philips PW 1404 wavelength dispersive, sequential X-ray spectrometer equipped with a scandium-molybdenum dual anode side-window X-ray tube (operated at 40 kV), with a flow-proportional counter, was operated under vacuum to measure the Ka lines of each element. A combination of flow-proportional and scintillation counters in tandem was used to measure the intensity of the iron Ka line.Pulse-height selection or line-overlap corrections can be used to reduce interference from the fifth-order calcium Ka line on the first-order magnesium Ka line for samples with a high-calcium and low-magnesium content. Calibration measurements were carried out using high- purity oxides or carbonates. The calibration was not carried out utilizing CRMs because only three CRMs for this rock type were available, vit., BCS CRMs 319, 368 and 389. A series of fifteen calibrations on discs containing only single elements was used to calibrate the spectrometer. Multiple regression analysis was used to calibrate the concen- tration of the ignited oxide (the temperature of ignition was determined by the metal constituent) against intensity (count rate).The matrix interference correction for the element on itself was also calculated simultaneously utilizing the de Jongh equation,l2 which is provided in the Philips X40 software. A further ten discs were prepared with the elements paired as follows: MgO-CaO, MgO-Si02, MgO-Al203, MgO-Fe203, CaGSi02, Ca0-AI2O3, CaO-Fe203, SiO2-AI2O3, Si02- Fe2O3, and A1203-Fe203. The inter-element matrix correc- tion for the paired elements was calculated by regression analysis. 12 The same series of discs was used for calibration using both the magnesite and general methods. The discs were originally used for the general method but, by using the following equations, the data were corrected for use with the magnesite method. Equation (1) is a general equation that, in this Table 1 Instrumental parameters for Philips PW 1404 Element Time/s Crystal Collimator 29/" Fe 40 LiF 200 Fine 57.57 Ca 40 LiF 200 Fine 113.19 Si 40 PET* Coarse 109.17 A1 40 PET* Coarse 145.08 Mg 200 PXlt Coarse 23.16 * PET = Pentaerythritol.t PX1 = Synthetic layered crystal, 2d = 4.9800 nm. instance, becomes eqn. (2), which in turn, simplifies to eqn. (3) CMAG = CGEN x 0.3000 - x - 3.600 0.6000 3.300 (3) where, CMAG is the calibration concentration for the mag- nesite method; CGEN is the calibration concentration for the general method; SGEN is the sample mass for the general method; SMAG is the sample mass for the magnesite method; TMAG is the sample plus flux mass for the magnesite method; and TGEN is the sample plus flux mass for the general method. Repeatability The repeatability of the results using the two XRF methods was tested over a period of 2-3 d by analysing each of the three individual discs from magnesite sample C in sequence.This measurement sequence was repeated twice at daily intervals and finally one disc was analysed in triplicate. The result for an individual disc reading was compared with that from the group. Effect of Initial Temperature on Loss on Fusion Raw magnesite sample B was analysed using the magnesite method. The crucible and contents were placed in the fusion furnace, operating over a range of initial fusion temperatures (500-1000 "C), and the LOF was determined. Results and Discussion Development of Flux Bennett and Oliver13 have detailed the relative merits and disadvantages of existing fluxes, such as lithium tetraborate (100%) and mixtures with lithium metaborate, for forming fused glass discs.The MAG 5743 flux was developed from Norrish 1222 flux (a mixture of 35% m/m lithium tetraborate and 65% m/m lithium metaborate). Norrish 1222 is an alkaline flux with a low melting-point and a low solubility for oxides such as MgO, CaO and A1203 (the so-called basic oxides). Stable discs could not be produced with samples of a high magnesium or calcium content with Norrish 1222. The solubility of the basic oxides in Norrish 1222 flux can be increased by the addition of another oxide such as SO2, Ti02 or B203 in order to decrease the basicity of the flux. Boron(II1) oxide is a non-interfering oxide in XRF spectrometry and, when added as a modifier, increases the acidity and also the viscosity of the flux.A mixture of 100 g of Norrish 1222 flux plus 12 g of boron(m) oxide was considered to be necessary in order to obtain a suitable compromise of viscosity, high oxide solubility and low temperature of fusion. The use of this composite flux, however, was limited to pre-ignited samples because the mass of flux lost on fusion is approximately 15 mg g-1 which precludes its use for accurate LOF determinations. The variable water content of boron(i1i) oxide is the cause of the mass loss and makes the flux difficult to stabilize by heating or drying. The Norrish 1222 flux has a LizO : B2O3 ratio of 20 : 80 and calculations showed that the same ratio could be achieved with a lithium tetraborate-lithium metaborate mixture of 57% m/m lithium tetraborate and 43% m/m lithium metaborate.This flux, which can be obtained custom mixed from a supplier or produced by the user, is stable long-term when dried by heating at 500 "C and stored in a desiccator. The MAG 5743 is a reactive flux, which is fluid at 10oO "C, in contrast to the lithium tetraborate flux usually used for fusionANALYST, MAY 1991, VOL. 116 45 1 of carbonate rocks. The fusion of raw magnesite samples with the MAG 5743 at 1000 "C requires fluxing times (10 min) similar to those required when lithium tetraborate is used at 1100-1200 "C. This is a result of mixing due to the loss of volatiles on fusion. In contrast, caustic calcined and fused magnesia samples require 20-30 min for complete reaction with lithium tetraborate, but only 10 min with the MAG 5743 flux.The MAG 5743 is a specialized flux, hence, matrix corrections were not possible using commercial calculation programs. Therefore, the corrections obtained for the ele- ments were determined empirically and compared with those calculated using the modified NRL-XRF program14 for lithium tetraborate and the Norrish 1222 flux, because the composition of the MAG 5743 flux is between these two fluxes. The values obtained empirically were within the range of the values of the program-calculated correction coefficients for Fe203, CaO, Si02, A1203 and MgO with the lithium tetraborate and Norrish 1222 flux. Table 2 Repeatability ( n = 4) of measurements on three glass discs prepared from sample C Relative Conccntration Standard standard Determinand (Yo 1 deviation (YO) deviation (YO) 44.83 0.20 0.5 2.10 0.04 1.9 MgO Si02 1.12 0.06 5.4 Fed& 0.05 0.01 20 A1203 0.04 0.01 25 CaO LOF 51.80 0.13 0.3 Less than 1% of the beads produced from MAG 5743 cracked; these beads were, nevertheless, still usable.Repeatability Table 2 gives details of the results obtained from three separate discs prepared from the magnesite sample C. The results obtained from the individual discs showed that reproducible, stable discs giving repeatable results could be prepared, for analysis by XRF using the rapid fusion method. The discs slowly absorb moisture (at a rate determined by the relative humidity), which decreases the intensity of the signal. Therefore, long-term storage of the discs in a desiccator is necessary.Determination of the five major components together with the LOF can be obtained within 35-40 min. Analysis of CRMs The values obtained using the two methods ( i e . , magnesite and general methods) are compared with the certificate values of a series of BCS CRMs in Table 3. As both magnesite standards had already been calcined only the general method could be used. The dolomite (BCS CRM 368) had not been calcined during preparation so it could be used as a basis for comparison of the two methods. Comparison of the results for pre-ignited dolomite (BCS CRM 368) by the general method was not considered valid because the recommended tempera- ture of ignition (1025 "C) does not decompose the sample completely. The measured LOF of the pre-ignited sample was 1.1 -t 0.1%, which together with the measured LO1 at 1025 "C of 46.5 k 0.1% gave a total loss of 47.6 k 0.2%.This value compares favourably with the LOF at 1030 "C of 47.5 rt 0.3% Table 3 Comparison of XRF results ( n = 12) with the certificate values for the CRMs (all values expressed as percentages) Certified reference material BCS 319* BCS 3681- General method General method Magnesite method Deter- Certificate Concen- Standard Certificate Concen- Standard Concen- Standard minand value tration deviation value tration deviation tration deviation MgO 90.46 90.37 0.05 20.9 20.57 0.09 20.47 0.12 CaO 2.28 2.28 0.03 30.8 30.67 0.33 30.58 0.13 Si02 1.55 1.58 0.05 0.92 0.92 0.05 0.89 0.01 A1203 0.97 0.94 0.02 0.17 0.12 0.02 0.14 0.01 Fe203 4.63 4.58 0.05 0.23 0.24 0.02 0.20 0.02 LOF NA$ 0.16 0.05 ND$ 47.27 0.15 47.50 0.28 LO1 ND ND ND 46.7 ND ND ND ND * Magnesite method not applicable to these samples; pre-treatment temperature, 1025 "C.1- Pre-treatment temperature. 110 "C. $ NA = not applicable, ND = not determined. BCS 389* General method Certificate value 96.7 1.66 0.89 0.23 0.29 NA ND Concen- tration 96.65 1.66 0.90 0.21 0.32 0.21 ND Standard deviation 0.11 0.02 0.05 0.02 0.03 0.10 ND Table 4 Comparison of XRF results (n = 3) for fused-disc methods (all values expressed as percentages) Sample A B C Deter- minand 1* 2-t 3$ 1* 21 33 1" 2t 3$ MgO 46.21 46.16 46.30 45.12 45.19 45.35 45.12 45.15 45.08 CaO 1.28 1.25 1.27 2.32 2.31 2.35 2.08 2.07 2.11 Si02 0.20 0.20 0.18 0.27 0.25 0.23 1.24 1.17 1.16 A1203 0.04 0.03 0.03 0.08 0.04 0.05 0.06 0.04 0.05 Fe203 0.04 0.05 0.05 0.04 0.07 0.04 0.05 0.06 0.05 LO13 52.23 NAfi NA 52.03 NA NA 51.55 NA NA LOFll NA 52.42 52.30 NA 52.07 52.05 NA 51.83 51.66 * Results for ignited sample (0.3 g, 1300 "C) with values adjusted for LOI.t Results for sample (0.3 g) with no adjustment, and LOF determined using the general method. $ Results for sample (0.6 g) with no adjustment, and LOF determined using the magnesite method. 3 LO1 determined by igniting sample at 1300 "C before fusion. 7 NA = not applicable. (1 LOF determined after fusion at 1000 "C. D 1" 46.69 1 .oo 0.12 0.02 0.03 52.15 NA 21- 46.63 0.98 0.12 0.03 0.04 NA 52.43 3$ 46.53 1 .oo 0.10 0.01 0.03 NA 52.44452 ANALYST, MAY 1991, VOL. 116 (Table 3). Loss on ignition results are always lower because of incomplete decomposition.The LO1 results are only compar- able to LOF results if the magnesite dolomite has been ignited at 1300 "C to constant mass. Certified reference materials (BCS CRMs 319, 368 and 389) can only be used for comparison (Table 3) because CRMs 319 and 389 are burnt magnesites and 368 is a dolomite. Analysis of Magnesites Originally, the flux was developed for the rapid analysis of calcined magnesia but, because the flux was so stable, it was decided that the method could be modified and applied to unignited samples, thus including LOF in the analysis. The LOF value was therefore included as a correction factor in the de Jongh equation.15 With unignited samples, the LOF can be as much as 52% for magnesite and 42% for calcite which means that, if a 10 : 1 FTS ratio is used, the line intensities are reduced to about half those produced using a disc prepared from ignited material. In order to compensate for this loss in signal, the mass of sample was doubled thus halving the FT'S ratio.The alternative method of maintaining the signal, i.e., by doubling the count time, was rejected because of the concomitant increase in analysis time. A further benefit was achieved by increased accuracy in weighing. Table 4 shows a comparison of the results obtained for the four magnesite samples, using the conventional, general and magnesite methods. As with the results for the CRMs, agreement was achieved between the conventional analysis and the two proposed methods. Effect of Initial Fusion Temperature on LOF Value Loss on fusion values for magnesite sample B of 52.03,52.07, 52.10, 52.16 and 52.11% were obtained for initial fusion temperatures of 500,600,700,800 and lo00 "C, respectively.The LOF value obtained using the magnesite method was The results show that there was virtually no sample loss during the fusion. If any loss of mass had occurred owing to decrepitation of the sample, as opposed to loss due to CO2 and H20 evolution, the LOF value would have been greater at higher initial fusion temperatures. 52.09 k 0.13%. Conclusion A rapid fusion technique has been developed for the determination by XRF of the major oxides in magnesites, magnesias and dolomites. The total analysis time is as short as 40 min. The flux used in the fusion (MAG 5743), if prepared properly, has no LO1 and produces stable discs. Thus it can be used for the accurate and rapid simultaneous determination of the major oxides and the LOF. The validity of the method was checked by comparison of the results with the certificate values of three CRMs. The work reported here was sponsored by Queensland Magnesia Pty. Ltd. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 King, B.-S., and Vivit, D., X-Ray Spectrom., 1988, 17, 85. King, B.-S., and Vivit, D., X-Ray Spectrom., 1988, 17, 145. Simonov, K. V., Zos'ka, A. V., Polovinkina, R. S., and Dremina, V. A., Znd. Lab. (Engl. Transl.), 1979, 44, 1026. Prager, M. F., and Graves, D., J. Am. Oil Chem. Soc., 1977,60, 1386. British Chemical Standard, Certificate of Analysis. Standards Association of Australia, AS2503.4, 1987. International Standards OrganizatiodDraught International Standard, 10058. American Society for Testing and Materials, C574, 1982. Deutsche Industrie Norm, 273, 1981. Pert], A., Lehmann, H., and Grubitsch, H., Radex Rundsch., 1976, 1, 639. Govindaraju, K., Geostand. Newsl., 1989, XI11 (Special Issue), Appendix 1, p. 27. de Jongh, W. K., X-Ray Spectrom., 1973,2, 151. Bennett, H., and Oliver, G. J., Analyst, 1976, 101, 803. Norrish, K., Commonwealth Scientific and Industrial Research Organization (CSIRO), Adelaide, South Australia, Australia, personal communication, 1990. de Jongh, W. K., X-Ray Spectrom., 1979, 8, 52. Paper 0104412A Received October lst, 1990 Accepted December 19th, 1990

ISSN:0003-2654    

DOI:10.1039/AN9911600449

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

ANALYST, MAY 1991, VOL. 116 453 Kinetic Model of pH-based Potentiometric Enzymic Sensors Part 1 Theoretical Considerations Stanistaw Gtab, Robert Koncki and Adam Hulanicki Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland A theoretical, kinetic model for a pH-based potentiometric enzymic sensor has been developed. It has been shown that the response of these sensors is governed by the pH, the buffering capacity of solutions analysed, and also the stirring rate. This model takes into account the variability of the kinetics of the enzymic reaction. The final equation is presented in an algebraic form and can be used in both fitting and optimization procedures. Keywords: Potentiometric enzymic pH sensor; kinetic model; steady-state response Enzymic sensors are important analytical devices used for the determination of substances that often cannot be determined by conventional methods of analysis.' In these devices a substrate-active enzyme layer is placed directly on the surface of a classical sensor that measures the concentration of the products formed in the enzymic reaction.In a large number of enzymic reactions, substances having protolytic properties are either formed or consumed. This explains why the pH-based potentiometric enzymic sensors are probably among the most versatile of the potentiometric enzymic sensors. Development of this group of biosensors has stimulated studies on the formulation of a mathematical model , describing the physico- chemical phenomena responsible for the formation of the analytical signal.Until now, mainly diffusion models for the enzymic electrodes have been presented.2" These models lead to equations that can only be solved by assuming that the kinetics of the enzymic reaction are of first or zero order.2 Owing to the form of the Michaelis-Menten equation, in general non-linear parabolic differential equations are obtained, which cannot be solved simply. The solution of these equations can be presented either as a definite integral in a non-elementary form or as an expansion of a power series.3>4 Therefore, sometimes, instead of these equations extrapola- tion methods are used.5 Diffusion models can be applied in order to describe the response of electrodes with a thick layer of enzyme and with defined geometry. These models do not take into account the influence of the rate of stirring on the analytical signal, commonly observed in practical measurements with enzymic electrodes. In order to explain this effect, the diffusion coefficients inside the enzyme sensing layer are assumed to vary with the rate of stirring.3.4 This assumption is also made by other authors7 for electrodes with liquid-state membranes.Another treatment of this problem is based on the consider- ation of the changes of concentration in a hypothetical interfacial zone between the enzyme layer and the bulk solution.6 From a mathematical point of view this model is very complicated and special numerical procedures have to be used. The papers mentioned above deal with models of the enzymic electrode response when the potentiometric sensor is sensitive to the product of an enzymic reaction.Modifications of these diffusion models for pH-metric sensors lead to more complicated equations. Combination of the equations de- scribing the diffusion of the respective species, with equations describing the kinetics of the protolytic reactions8.9 or acid-base equilibria1@-15 leads to a set of non-linear partial differential equations of the second order. These equations can be transformed, in special instances, into a rather complicated algebraic form."-" For example, when the kinetics of a protolytic reaction are taken into consideration, the enzymic reaction is assumed to be first order and any acid product assumed to be fully disso~iated.82~ Combination of the kinetic constants of the enzymic and protolytic reactions in the same equation also seems to be unjustified, because the magnitudes of these parameters are not comparable.16 Combi- nation of equations describing diffusion with equations describing dissociation leads to algebraic equations only when it is assumed that the substrate is totally transformed into protolytic products. 10,l1 This drastic assumption is equivalent to a situation, considered by a classical equation, describing the pH of a mixture of weak acids and bases.17 Varanasi and co-workers10,*1 have taken into account the influence of stirring with an assumption that the concentrations of all of the species at the surface of the electrode and in the bulk solution are different. The diffusion model in the general form without the assumptions mentioned above and, in addition, considering the modification of the kinetics of the enzymic reaction by inhibitors or pH, leads to a very complicated expression. Therefore, special numerical procedures have to be used.12-15 The mathematical complexity of the models presented above, and the difficulties with the interpretation of the influence of the rate of stirring on measurements, are caused by an assumption that the transport rate is proportional to the gradient of the concentration.This inconvenience can be ignored if, as assumed by Morf,lS the transport rate is proportional to the concentration of the species. In this instance, the derived equations are analogous to the kinetic equations. These equations take into account the influence of stirring, which causes the change in the rate constant.The model by Morf describes an enzymic layer on the potentio- metric electrode, which is sensitive to the product of the enzymic reaction. The aim of this paper is to present a simple model of the response of a pH-based potentiometric enzymic sensor. The proposed model is a modification of the substrate-enzyme electrode model of Morf.18 Description of the Model The layer on the surface of a pH sensor contains an enzyme that catalyses the reaction of the substrate, S, leading to the formation of an acid, HA, a base, B, and a non-protoIyic product, Z : Enzyme S + nxX - nAHA + nBB + nZZ where, n denotes the respective stoichiometric coefficients and X refers to other reaction substrates present.454 ANALYST, MAY 1991, VOL.116 The rate, V, of the over-all enzymic reaction is given by the equation: where V and [S] denote the actual rate and concentration of the substrate in the enzymic layer, K , is the Michaelis- Menten constant and Vmax the maximum reaction rate. It is possible to take into account the variability of the kinetic parameters Vmax and K , [in eqn. (2)] as a function, for example, of pH, as discussed later. The protolytic equilibria in the sensing layer for the reaction products, HA and B , and for the buffer system HW-W, are described by the corresponding acid dissociation constants, Kax, these constants are assumed to be equal to those in the bulk of the solution: The mass balances in the enzymic layer and in the bulk solution, marked with the superscript B , are given by eqns.(4a)-(4e) : c b = [W]B + [HWIB ( 4 4 cw = [W] + [HW] CA = [A] + [HA] CB = [B] + [HB) CH = [HI + [HW] + [HA] + [HB] ( 4 4 ( 4 4 In these equations the symbols cx correspond to total concentration in the sensing layer, or c$, the concentration, in the bulk solution. All of the protolytic species can diffuse in either direction across the hypothetical semi-permeable mem- brane which separates the bulk of the analyte solution from the enzyme-containing sensing layer. The transport of the respective species is described by the transport rate constants, kw, k ~ w , etc. It$ assumed that there is no preconcentration of substances in the enzymic layer. This means that the transport rate constants in both directions are equal.The scheme presented in Fig. 1 illustrates the proposed model for the response of pH-based potentiometric enzymic sensors. The substrate having concentration [SIB in the bulk of the analysed solution diffuses into the enzymic layer with a transport rate constant, ks, and can pass out again with the same rate constant. A decrease in the substrate concentration of the enzymic layer is a result of the enzymic reaction occurring with the rate described by the Michaelis-Menten equation [in eqn. (2)]. The rate of changes of the total concentration of substances [eqns. (4a)-(4e)] in the enzymic layer are represented by a set of equations, which take into account the rate of transport of respective species into and out of the sensing layer, and also the rate of the following enzymic reactions: - kHW[HW] - kHW[HW] - kHA[HA] - kHB[HB] ( 5 3 At steady state, the conversion of substrate and the formation of product is compensated for by the interfacial mass transfer, and consequently the concentrations of all of the substances are constant.Therefore, the derivatives are equal to zero and eqns. (5a)-(5e) can be re-written in the forms given by eqns. (6a)-(6e): The eqns. (6a)-(6e) are based on the assumption of a steady state, which means that the rate of increase of the concen- tration in the enzymic layer (the left-hand sides of the equations) is the same as the rate of decrease (the right-hand sides of the equations). The sets of eqns. (3a)-(3c), (4a)-(4c) and (5a)-(5e) give the solution in an algebraic form [eqn. (7)] which shows the dependence of the hydrogen ion concentra- tion, inside the enzymic layer, on the substrate concentration, [SIB, in the bulk solution.In the derivation of the final equation the transport rate for the species is assumed not to depend on protonation, i. e., kA = Bulk I Sensing layer I pHsensor W I I kw w j,. c w 1Lr , HW I knw I HWd ~~~ Fig. 1 Scheme of the pH-based potentiometric enzymic sensor. Where: k and K , , with corresponding subscripts, are transport rate constants and acid dissociation constants. respectively, for species indicated by the subscripts; and HW-W is a pH buffer system. Other symbols as in eqns. (1) and (2)ANALYST. MAY 1991, VOL. 116 455 kHA etc. This is, most probably, a sufficiently good approxi- mation, but the differences can also be taken into account, thus making the final equation more complicated, but still solvable.With the assumptions mentioned above it is possible to transform eqn. (7) into: - 1 kH([HIB - + kwck 1 + Kaw/[H]B 1 + Kaw/[H] where [S], obtained by solving eqn. (6a), is given by: m I - From eqns. (8) and (9), using normalized rate constants defined by eqns. (lOa)-(lOc), a general algebraic equation describing the proposed model is obtained: (11) nB 1 -t K,B/[H] ] = o - n A 1 + [H]/K,A This equation can be transformed into simple linear relation- ships versus analyte concentration andor normalized rate constants for the following situations: firstly, for [SIB >> [S], secondly for, Km << [S] and thirdly for, Km >> [S] Because of the simplicity of the form of eqns.(11) and (12a)-(12c) it is not difficult to take into account the influence of inhibitors.1,I9 For this purpose the Michaelis-Menten equation [eqn. (2)] should only be modified to include the concentrations of the competitive, [Ic], and non-competitive, [Inc], inhibitors [eqn. (13a)].1.19 (13a) Vmax[Sl Km(1 + [L]/KI~) + [S](1+ [Incl/K~,,) I/= where KIc and, KInc are the respective inhibition constants. products, inhibitors can also be taken into account: In a similar way to the influence of the substrate and the K:, = Km(l + [S]*/KI,) (134 The kinetic parameters of the enzymic reaction, K& and V;, strongly depend on pH. The influence of pH on these parameters can be described by a simple protolytic model proposed by Waley.l71932* This model (Fig.2) assumes that only one protolytic form of the enzyme can form an activated complex with the substrate, and only one protolytic form of this complex is irreversibly decomposed upon the formation of the enzyme and products. On the basis of this model the following relationships can be obtained: where pH,,, denotes the pH at which the enzyme activity is at a maximum. Kal, Ka2, Kbl and Kb2 are acidic and basic dissociation constants, respectively (see Fig. 2). These re- lationships, can easily be introduced into the general equation [eqn. ( l l ) ] or into the approximate forms of the equation [eqns. (12a)-( 12c)l. These modifications allow the variability of the enzymic reaction kinetics, influenced by local changes of pH inside the enzymic layer, to be taken into account.Discussion The model presented in this work for the response of the pH-based potentiometric enzymic sensors assumes that the enzymic layer is separate from the bulk solution. All of the components of the solution and the enzyme layer, except for the enzyme molecules are able to diffuse through the membrane in both directions. Realistic examples which can be described by the proposed model are enzymic sensors with pseudo-immobilized enzymes connected to the surface of the pH sensor by means of a dialysis membrane. However, the application of the model is not limited to this instance only. In several situations (including that for the hydrogen ion because of the enzymic reaction), such a membrane does not exist at all, and there is only a hypothetical border between the bulk solution and the zone where the local concentration changes, which alter the analytical signal, occur.The thickness of the enzymic layer is not defined in the proposed model. Contrary to the previously described diffusion modeIs,l2-15 this means that this model is not applicable for use in describing the concentration profiles inside the sensing layer. On the other hand, the geometry of the sensing layer does not have to be known, and this is a major advantage of the proposed model. Diffusion models cannot be used for describing the response of enzymic sensors with a monomolecular sensing layer of enzyme. Because the geometry of the enzymic layer is not E L E H . 2 Kbl E H . &I + S EH + P Fig. 2 Scheme of the protolytic model by Waley to describe the enzymic reaction.Where S = substrate; P = product; and E = enzyme456 ANALYST, MAY 1991, VOL. 116 known it is not important to know the exact concentration of the enzyme in this layer. The enzyme concentration in the sensing layer, however, is required for a diffusion model. This becomes impossible when the enzyme is covalently bound to the surface of the sensor. Therefore, electrodes with thin enzymic layers can also be described by the proposed model. Diffusion models adequately describe the pH response of sensors with thick enzymic layers, whereas for electrodes with thin sensing layers the use of the model proposed by Morf is more plausible ,*8 after the appropriate modification for pH-based sensors. This is confirmed by the observation that the response of thin-layer sensors depends on the stirring rate, whereas with thick enzyme film electrodes this effect is not seen.The proposed model takes the stirring effect into account, as stirring modifies the respective transport rate constants. Apart from the fundamental differences in the treatment of the transport phenomena, the diffusion and kinetic models lead to the same conclusions. However, the model proposed in this paper appears to be more practicable because of the mathematical simplicity. The general equation [eqn. ( l l ) ] can be modified without increasing the mathematical complexity. This model can also be used when a polyprotic acid is used as a component of the buffer system and/or polyprotic products are formed in the enzymic reaction.This requires only small changes in eqns. (3a)-(3c), (4a)-(4c) and (5b)-(5c). The proposed model also allows for differences between the transport rate constants, which depends both on the type of substance and the type of protolytic form [eqn. (7)]. Similarly an assumption that the transport rates both to and from the sensing layer are different does not exclude the use of the equation. In this instance, only the distribution coefficients will quantitatively describe the process of species concen- tration. These modifications complicate the general equation [eqn. ( l l ) ] but do not cause difficulties in its use. The same modifications, when introduced to the diffusion model, markedly increase the mathematical complexity. On the other hand, some simplifications of the general equation [eqn.( l l ) ] are possible when proper approximations [eqns. (12a)-(12c)] are accepted. If the concentration of the substrate in the sensing layer is assumed to be much smaller than that in the bulk solution, i.e., [S] << [SIB, hence, only the transport phenomena govern the response, then an equation [eqn. (12a)l is obtained, which is an explicit algebraic function [SIB = f[H]. This relationship is linear versus the transport parameters and the buffer concentration, but owing to the assumption that the substrate is totally transformed into 7.00 5.0 4.0 3.0 2.0 1 .o 0 Fig. 3 Theoretical response of pH-based sensor. As an example the urea sensor is used. Phosphate buffer, pH 7.0, 0.01 mol dm-3. The curves were calculated frqm the general equation describing the proposed model: 1 and 1 , without any approximations (general equation); 2 and 2’, with the assumption that K, << [S] (zero order); 3 and 3’, with the assumption that K, >> [S] (first order); and 4, with the assumption that [S] << [SIB (substrate concentration in the sensing layer negligible).Lines l’, 2’ and 3’, the influence of pH on enzyme kinetics was taken into account -Log[SIB products in the enzymic reaction, the kinetic parameter does not appear in this equation. This equation is identical with one of the equations for the diffusion model, derived with the same assumptions10211 when the kinetic parameters, kH and kw, are replaced by the partition coefficients (defined as the concentration ratio of the species at the sensing layer and in the bulk solution).An additional assumption that th_e trans- port rate constants for all species are the same (kH = kw = 1) leads to the transformation af eqn. (12a) into the classicial equation describing the pH value of a mixture of acids and bases. 17 Equation (12a) can be used for describing the response of pH-based sensors when the concentration of the substrate, [SIB, is low. The response calculated by using the equation agrees with the experimental response10311 for urea21 and penicillin14 sensors at low concentrations of substrate. The differences at higher concentrations of the substrate (the lack of the upper limit of determination) appear because the kinetic parameters of the enzymic reaction are not taken into account. The proposed model allows the pH at the upper determination limit to be calculated, on the basis of eqn.(12b), which was obtained with the assumption that the kinetics of the enzymic reaction are of zero order. For zero order kinetics ([S] >> Km), eqn. (12b) is obviously indepen- dent of the substrate concentration and describes only the maximum value of the analytical signal, i.e., the pH which corresponds to the concentration at the upper limit of determination. The derivation of eqn. (12c) is based on the approximation of the Michaelis-Menten non-linear equation by a kinetic equation, for the first-order reaction, ([S] << K,). It should be noted that for large values of kv, i.e. , for a high level of enzyme activity, the simplified equation [eqn.(12c)l approaches that for the diffusion model, where [S] << [SIB. In all the instances mentioned, as for the general equation, the bisection method can be applied. The equations are linear versus the various parameters, which makes the use of linear algebra possible, and in consequence? simple numerical optimization and fitting procedures. The calibration graphs calculated for any given experimental conditions, approxi- mate the general equation relationships in particular regions (Fig. 3). As mentioned earlier it is not difficult to take into account the influence of inhibitors on the kinetics parameter [eqns. ( l l ) , (12b), (12c) and (13a)l. Because inhibitors are neither consumed nor formed it can be assumed that their concentra- tions in the bulk solution are equal to that in the enzyme layer.When the solutions to be analysed contain inhibitors at equal concentrations, the use of eqn. (2) without modification except for the apparent kinetic parameters K , and V,,, (i.e., z 8 . 2 5 7.00 5.0 4.0 3.0 2.0 1 .o 0 -Log[SlB Fig. 4 Influence of the value of the Michaelis-Menten constant, K,, on the response of the pH-bas_ed urea sensor. Phosphate buffer, pH 7.0, 0.01 moldm-3. Where kv = 0.01; 1 and l’, K , = O.OOO1 mol dm-3; 2 and 2’, K , = 0.001 mol dm-3; and 3 and 3’, K , = 0.01 mol dm-3. Lines l’, 2‘ and 3‘, the influence of pH on enzyme kinetics was taken into accountANALYST, MAY 1991, VOL. 116 457 9.50 I 9.50 1 7.00 5.0 4.0 3.0 2.0 1 .o 0 -Log[SIB Fig. 5 Influence of normalized rate constant of enzymic reaction, k,, on the response of the pH-based sensor (urea sensor).Phosphate buffer, pH 7.0,O.Ol rnol dm-3; K , = 0.001 rnol dm-3. 1 and l', kv = 0.1; 2 and 2', kv = 0.01; and 3 and 3 ' , isv = 0.001. l', 2' and 3 ' , the influence of pH on enzyme kinetics was taken into account zv), is possible. The response of the pH-based potentiometric enzymic sensor is affected by local changes of pH within the sensing layer, because of the change of enzymic reaction kinetics. This is taken into account in eqns. (14a) and (14b) (Fig. 3). The introduction of all of the modifications discussed above causes an increase in the complexity of the final relationships, but as previously stated there are simple algebraic equations in either non-explicit [eqn. ( l l ) ] or explicit [eqns.(12a)-(12c)] forms. The introduction of these modifications to the equations describing the diffusion models requires the use of complicated numerical methods which can give only approximate solutions. By using eqns. ( l l ) , (12b) and (12c) it is possible to anticipate the influence of kinetic parameters on the shape of the calibration graphs. The value of the Michaelis-Menten constant affects mainly the upper limit of determination and only slightly changes the analytical signal. The larger the value of K,, the further the upper limit of determination is extended, but the sensitivity decreases (Figs. 4 and 5 ) . The parameter kv indicates the influence of the enzyme activity on the calibration graph (Fig. 5 ) because it has the same function 5s the 'loading factor' in the diffusion models. An increase of kv causes an increase in the sensitivity of the sensor over a range of concentrations.For all of the substances, the transport rate constants [eqns. (lOa)-(lOc)] depend on the stirring rate to the same extent, therefore, only kv takes into account the effect of stirrkg. An increase in the stirring rate decreases the value of kv and consequently, also the sensitivity of the detector (Fig. 5 ) . The proposed model allows a prediction to be made regarding the influence of the concentration and pH of the buffer used, on the shape of the calibration graph for the pH-based potentiometric enzymic sensor (Figs. 6 and 7). An increase in the concentration of the buffer, &, shifts the calibration graph towards the higher concentration range and decreases the sensitivity (Fig.6). The sensitivity of the sensor is mainly dependent on the pH in the bulk solution, pHB. When, owing to the enzymic reaction, the pH increases, a decrease in the sensitivity of the sensor is observed for a pH-based potentiometric enzymic electrode for urea (Fig. 7). A small influence on the detection limit is primarily connected with the changes in buffering capacity. Therefore, at a pH close to the pK, of the buffer (i.e., for maximum buffering capacity) the sensor shows the worst detection limit. All of the effects mentioned above were experimentally investigated in detail and will be submitted for publication at a later date. The considerations presented in this paper refer to the steady state. When a non-steady state is considered the 5.0 4.0 3.0 2.0 1 .o 0 Fig.6 Influence of buffer concentration, & on the response of the pH-based urea sensor. Phosphate buffer, pH 7.0; K,, 0.001 rnol dm-3; and Ev, 0.01. 1 and l', c% = 1 x 10-4 rnol dm-3; 2 and 2', cyv = 1 X rnol dm-3; and 3 and 3 ' , c"w = 1 x 10-2 rnol dm-3. l', 2' and 3'. the influence of pH on enzyme kinetics was taken into account -Log[S]B 1 1' 9.00 1 3 0 -- 2' r 7.50 a 6.00 5.0 4.0 3.0 2.0 1 .o 0 -Log[S]B Fig. 7 Influence of pH of the analysed solution, pHB, on the response of the pH-based urea seasor. Phosphate buffer, 0.01 rnol dm-3; K , = 0.001 rnol dm-3; and kv = 0.01.1 and l ' , pH = 8.0; 2 and 2', pH = 7.0; and 3 and 3', pH = 6.0. l ' , 2' and 3 ' , the influence of pH on enzyme kinetics was taken into account differential equations [eqns.(5a)-(5e)] have to be solved. Because the magnitude of the transport rates, for all of the substances, and the rate of the enzymic reaction are of the same order, and because the rates of protolytic reactions are much higher,l6 the consideration may be limited to a discussion of eqn. (5a) as carried out by Morf.18 Conclusion The proposed kinetic model for the response of the pH-based potentiometric enzymic sensor has the following advantages in comparison to earlier published models. (i) The model is mathematically simple, and in order to describe it only algebraic equations are required. (ii) The geometry of the enzymic sensing layer need not be defined. (iii) Stirring effects are taken into account. (iv) Modifications of the model are possible, without further complications, and which take into account: the differences in the transport rates of the respective species; the process of concentrating the species in the enzymic layer; the decrease in enzyme activity caused by local changes in pH or by the presence of inhibitors; and all protolytic equilibria.The proposed model also leads to conclusions similar to those of the previously described diffusion models. This model can be applied not only to potentiometric sensors but generally to systems where hydrogen ions are monitored such as ISFETs (ion selective field effect transistor) and optodes.458 ANALYST, MAY 1991, VOL. 116 References 1 Carr, P. W., and Bowers, L. D., Immobilized Enzymes in Analytical and Clinical Chemistry. Fundamentals and Appli- cations, Wiley, New York, 1980. 2 Blaedel, W. J., Kissel, T. R., and Boguslaski, R. C., Anal. Chem., 1972,44,2030. 3 Hameka, H. F., and Rechnitz, G. A., Anal. Chem., 1981, 53, 1586. 4 Hameka, H. F., and Rechnitz, G. A., J. Phys. Chem., 1983,87, 1235. 5 Brady, J. E., and Carr, P. W., Anal. Chem., 1980, 52, 977. 6 Jochum, P., and Kowalski, B. B., Anal. Chim. Acta, 1982,144, 25. 7 Morf, W. E., Lindner, E., and Simon, W., Anal. Chem., 1975, 47, 1596. 8 Eddowes, M. J., Sens. Actuators, 1985, 7, 97. 9 Eddowes, M. J., Pedley, D. G., and Webb, B. C., Sens. Actuators, 1985, 7, 233. 10 Varanasi, S., Stevens, R. L., and Ruckenstein, E., AZChE J., 1987,33,558. 11 Varanasi, S., Ogundiran, S. O., and Ruckenstein, E., Bio- sensors, 1988,3, 269. 12 13 14 15 16 17 18 19 20 21 Caras, S. D., Janata, J., Saupe, D., and Schmitt, K., Anal. Chem., 1985,57, 1917. Caras, S. D., Petelenz, D., and Janata, J., Anal. Chem., 1985, 57, 1920. Caras, S. D., and Janata, J., Anal. Chem., 1985,57, 1924. Moynihan, H. J., and Wang, N.-h. L., Biotechnol. Prop., 1987, 3, 90. Bell, P. R., Acids and Bases: Their Quantitative Behaviour, Methuen, London, 1969. Hulanicki, A., Reactions of Acids and Bases in Analytical Chemistry, Ellis Honvood, Chichester, 1989. Morf, W. E., Mikrochim. Acta, 1980, 2, 317. The Enzymes, ed. Boyer, P. D., Academic Press, New York, vol. 1, 1970. Waley, S. G . , Biochim. Biophys. Acta, 1953, 10, 27. Nilsson, H., Akerlund, A. C., and Mosbach, K., Biochim. Biophys. Acta, 1975,320, 529. Paper 01031360 Received July 12th, 1990 Accepted January 14th, 1991

ISSN:0003-2654    

DOI:10.1039/AN9911600453

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

ANALYST, MAY 1991, VOL. 116 459 Studies on Enzyme Electrodes With Ferrocene and Carbon Paste Bound With Cellulose Triacetate S. K. Beh, G. J. Moody and J. D. R. Thomas* School of Chemistry and Applied Chemistry, University of Wales College of Cardiff, P.O. Box 912, Cardiff CFI 3TB, UK A ferrocene-based chemically modified electrode has been prepared from a mixture of carbon paste and ferrocene, bound with cellulose triacetate. Glucose oxidase immobilized onto nylon net placed over the chemically modified indicator electrode completed the assembly of a robust ferrocene-mediated enzyme electrode. This was housed in a three-electrode Stelte micro-cell modified for flow injection according t o previous studies, and further modified by introducing a viscose acetate exclusion membrane between the outermost nylon-enzyme mesh and the ferrocene-carbon paste layer.Glucose was determined ampero- metrically by monitoring the product of hydrogen peroxide enzymolysis at +I60 mV versus a silver-silver chloride reference electrode. The enzyme electrodes showed a detection range of 0.01-70 mmol dm-3 glucose and the lifetime of the chemically modified electrode exceeded 24 months with intermittent use. Interference from ascorbic acid was minimal, while the maximum useful range was extended t o 100 mmol dm-3 glucose by simply covering the electrode surface with an exclusion membrane. A simplex optimization procedure was employed in evaluating electrodes without the use of an exclusion membrane. Keywords: Chemically modified glucose enzyme electrode; ferrocene; flow injection; simplex optimization The use of electron-transfer mediators has significantly improved the scope and performance of amperometric probes. These mediators are redox couple agents of low relative molecular mass, which shuttle electrons from the redox centre of the enzyme catalyst to the surface of the indicator electrode.During the catalytic cycle, the mediator, M,,, reacts with the reduced enzyme, and then undergoes rapid charge transfer at the electrode surface as illustrated for adenine flavin dinucleotide (FAD): Glucose + FAD + gluconic acid + FADH2 (1) FADH2 + M,, -+ FAD2+ + Mred + 2H+ Mred -+ M,, + ne- (2) (3) Provided M,, does not react with oxygen, it substitutes for oxygen in the classical enzymic reaction [eqns. (1)-(3)], and the rate at which the reduced mediator, Mred, is produced can be measured amperometrically at a suitable electrode.A practical mediator needs to be of low relative molecular mass, easily adsorbed onto an electrode surface, reversible, fast reacting, regenerated at low potential, pH independent, stable in both oxidized and reduced forms, unreactive with oxygen and non-toxic. Among the most successful mediators are those based on ferrocene [bis(n-cyclopentadienyl)iron] and its derivatives,' all of which fulfil the stated criteria, and those with a standard electrode potential (Eo) ~ 1 6 0 mV versus a saturated calomel electrode. The first successful mediated enzyme electrode was based on 1,l-dimethylferrocene which was adsorbed onto a graphite electrode with the enzyme having been chemically immobi- lized using the carbodiimide route.' The upper linear detec- tion limit was 30 mmol dm-3 glucose, and response times were 60-90 s.A variety of oxidoreductases2 have since been used in association with the ferrocene-modified electrode. The method seems to be generally applicable3 and mediators other than ferrocene have been used but generally they do not have the versatility of ferrocene. Dimethylferrocene-mediated electrodes are the most developed and form the basis of a commercial glucose monitor.' Traditionally, graphite with immobilized glucose oxidase and coated with a ferrocene redox mediator has been used as a * To whom correspondence should be addressed. dip-type glucose sensor,' but for flow injection (FI), the modified electrode needs to be more robust.Covalent binding onto polymer film has been studied, but the system is relatively unstable.4 Another approach is the use of a carbon paste electrode, where a quinones-6 or dimethylferrocene7~~ mediator is mixed with the carbon-binder matrix to form the working electrode. In the present study the carbon powder and mediator are bound together with cellulose triacetate, and the enzyme is chemically immobilized with the use of a nylon mesh matrix. Experimental Reagents and Materials Glucose oxidase (E.C. 1.1.3.4,1.667 pkat mg-1, purified from Aspergillus niger) , p-benzoquinone, lysine, 25% glutaral- dehyde solution and P-D-( +)-glucose were all obtained from Sigma (Poole, Dorset, UK). Nylon net was obtained from Henry Simon (Stockport, Cheshire, UK), viscose acetate (Visking tubing, 0.32 mm thickness and of relative molecular mass > 150000) was obtained from Gallenkamp (Loughbor- ough, Leicestershire, UK), cellulose triacetate from Kodak (London, UK) and carbon powder from Goodfellow Metals (Cambridge, UK).The enzyme was stored desiccated in a freezer (-5 "C). All other reagents used were of the best analytical grade available and were used without further pre-treatment. Sodium dihydrogen orthophosphate buffer (0.1 mol dm-3, pH 4.5 when freshly prepared) was of pH 4 when used. This was adjusted to the appropriate higher pH values by spiking with 4 mol dm-3 sodium hydroxide. Glucose standards were prepared from fresh P-D-( + )-glu- cose (0.1 mol dm-3) in sodium dihydrogen orthophosphate buffer (0.1 mol dm-3, pH 7) which was also used in the FI carrier stream.Immobilization of Enzyme The chemical immobilization of glucose oxidase onto nylon net was carried out as previously described.9 Nylon net (95-150 pm mesh size, 1 x 1 cm) was treated with dimethyl sulphate (30 cm3) in a boiling-tube, and placed in a water-bath at 75 k 3 "C for exactly 5 min with constant swirling. The460 ANALYST, MAY 1991. VOL. 116 boiling-tube was immersed in ice to stop the reaction. After cooling, the membrane was washed twice (or more if necessary) with methanol (30 cm3) until the methanol washings became clear. The lysine spacer molecule was attached by immersing the membrane in 30 cm3 of 0.5 mol dm-3 lysine for 2 h at ambient temperature.9 After rinsing with 0.1 mol dm-3 sodium chloride the membrane was placed in a saturated solution of p-benzoquinone for 2 h at ambient temperature. Finally, in order to attach the enzyme, the membrane was dipped into a solution of glucose oxidase (50 mg) in 5 cm3 of phosphate buffer (100 mmol dm-3, pH 7) for 2 h at ambient temperature, or overnight at 4 "C.Electrode Fabrication The chemically modified electrode material was prepared by thoroughly mixing carbon powder and ferrocene, and was bound with 20% cellulose triacetate (1 + 2 + 1 m/m) in 1,2-dichIoroethane. The ferrocene, carbon powder and poly- mer mixture was then packed into the well of an electrode holder and smoothed over with a clean flat spatula. A small drop of the cellulose triacetate solution was then placed on the electrode surface to form a protective covering.The electrode was oven-dried for 24 h at SO "C and smoothed using very fine emery paper. Flow Injection Apparatus The FI system described previously9 was used to evaluate the ferrocene-type glucose oxidase electrode using glucose stan- dards. For this, the mediated glucose oxidase electrode was completed by placing the nylon net with immobilized glucose oxidase over the chemically modified carbon paste indicator electrode. This was then set up in a modified three-electrode Stelte micro-cell (Metrohm E A 1102) assembly.'* The elec- trode potential was controlled and the current was monitored by using a Metrohm (Herisau, Switzerland) VA-detector E611 potentiostat in conjunction with a Linear Model 500 y-t chart recorder.The carrier stream and sample propulsion were driven by a four-channel Watson-Marlow (Falmouth, Cornwall, UK) peristaltic pump, and an Omnifit (Atlantic Reach, NY, USA) sample injection valve was used. All connecting tubes were of either silicone rubber or poly- tetrafluoroethylene with a nominal i.d. of 1.27 mm. A pulse suppressor was fitted between the pump and the injection valve. The indicator electrode was set at +160 mV versus a silver-silver chloride reference electrode. The following scheme illustrates the reaction sequence:' Glucose + GOD,, + gluconolactone + GODred (4) GODred + 2Fecp2R+ + GOD,, + 2Fecp2R + 2H+ ( 5 ) 2Fecp2R - 2e- + 2Fecp2R+ ( 6 ) where GOD,, and GODred are the oxidized and reduced forms of glucose oxidase, respectively, and R represents substituents in the Fecp2 ring system for Fecp2R+ and Fecp2R. Results and Discussion Optimization of the FI System for Glucose Determination Optimization of reaction p H Each enzyme electrode was optimized by varying the pH between 5 and 9 through increments of 0.5 pH unit.The resulting peak height versus pH plots reached a plateau at pH 6.8-7.2. All further work was therefore carried out at pH 7. immobilization technique and the nature of the support material, and for immobilized glucose oxidase an optimum pH of 7.0 is normal. Effect of temperature The effect of temperature on glucose sensing was studied by slowly raising the temperature from 5 to 75 "C over a period of 2 h. The sample solution and carrier stream were kept at the same temperature in a water-bath for each run.The glucose signal rose from 154 nA at S "C to a maximum of 280 nA at 38-42 "C; thereafter the signal decreased (to 230 nA at 75 "C), presumably due to denaturation of the enzyme. A repeat run on the same electrode gave a similar profile, but with a reduced glucose signal for temperatures ranging from 5 to 75 "C; the signal was reduced by 27 nA at 5 "C, 52 nA at 35- 42 "C and 41 nA at 75 "C. Optimization of flow conditions The sample volume and carrier solution flow-rate were optimized for electrodes without the use of viscose acetate membranes, and on 100 mmol dm-3 glucose standards, using an additional modified simplex optimization procedure,l3 this being an adaptation of the modified simplex optimization algorithm.14+15 For this, the control parameters of flow-rate and sample volume were examined with respect to the response criteria of peak height and run time, i.e., the time taken from sample injection to the attainment of the maximum current signal and a return to the baseline.The bias of the optimization in previous studies13-15 was maximization of the peak height with minimization of the run time. Different degrees of importance can be placed on the various response criteria according to the requirement expected of the enzyme electrode. The criteria of convergence for the simplex occur when the standard deviation (SD) of the signal responses of its vertices is less than five times the signal fluctuation (noise) of the system. The signal fluctuation can be determined by taking the SD of the signals of ten runs; this is necessary to prevent degeneracy of the simplex caused by the signal fluctuation.It is important to note that with the search method of optimiza- tion the data need to be verified, as the computation of each search point, and its direction, is dependent on the previous point. Therefore, a minimum of three runs was carried out for each subsequent cycle, the data being accepted only when there were three points with a percentage SD:signal ratio within the corresponding ratio of the original ten runs. If large signals are required, regardless of the run time, then greater weighting is placed on signal size rather than on run time. At the other extreme a system may be required that can 'O 60 L L z F Other workers have reported a broad pH range of 4.0-7.0 with a maximum response at about pH 5.5 for solubilized glucose oxidase.11J2 However, the optimum pH range is a direct result of the micro-environment of the enzyme and is related to the Fig.1 Simplex optimization of :nzyme electrode without viscose acetate membrane. Open squareF represent the experimental condi- tions; closed squares represent the normalized response of peak height and experimental run time, ie., the TRFANALYST, MAY 1991, VOL. 116 461 Table 1 Study of the interference of organic acids on the glucose sensor with and without a viscose acetate exclusion membrane Separate injection Mixed injection AilnA AilnA + 160mV +600 mV + 160 mV +600 mV Organic Without With Without Without With Without acid membrane membrane membrane membrane membrane membrane Ascorbic 0.05 0.03 3.00 1.05 1.03 4.00 Gluconic 0.00 0.00 0.00 1 .OO 1.00 1 .oo Lactic 0.00 0.00 0.00 1 .oo 1 .oo 1.00 Citric 0.00 0.00 0.00 1.00 1 .oo 1.00 Acetic 0.00 0.00 0.00 1.00 1 .00 1.00 4000 3000 P 2 2000 1000 0 4 3 P a 2 a 0, 1 - 1 0 0.1 0.2 [ G lucose]/mol dm -3 -4.0 -3.0 -2.0 -1.0 Log([glucosel/mol dm-3) Fig.2 ( a ) Glucose calibration plots for electrodes with (0) and without (A) viscose acetate exclusion membranes. ( b ) Log-lo glucose calibration plots for electrodes with (0) and without (A7 viscose acetate exclusion membranes Table 2 Response parameters of the two enzyme electrodes at + 160 mV versus silver-silver chloride reference electrodes. F-factor obtained when comparing the two electrode systems = 26.67 at p = 0.01, denominator = 8, numerator = 1 with critical value = 8.29, shows that the two electrode systems gave significantly different signals Linear Aifor detection 1 mmol range/ dm-3 mmol glucose1 Response Washout Electrode dm--3 nA SD*/nA timels time/s Without 0.10-70 22.0 0.791 15 30 With 0.10-100 20.0 0.354 25 45 * n = 5 .handle a large throughput of sample, thus placing greater emphasis on minimizing the run time. Operating conditions of sample volume and flow-rate for the systems in this study were optimized with equal emphasis being placed on the two response criteria, namely peak height and run time, which together make up the total response function (TRF) (Fig. 1). The open squares represent the experimental conditions and the closed squares represent the normalized response of peak height and experimental run time, i.e., the TRF.The initial search starts from iterations 1 , 2 and 3, and the optimum range occurs at iterations 16, 17 and 18 as shown in Fig. 1. The optimum range was the same for different search starting points. As a result of the optimization, the carrier stream flow-rate adopted was 2.3 cm3 min-1 and the corresponding sample size was 0.5 cm3, based on 100 mmol dm-3 glucose standards so as to realise large currents (= 2000 nA) without recourse to the use of instrumental noise adjustments. Sensor Selectivity The possible interference of ascorbic, gluconic, lactic, citric and acetic acids, particularly important in the analysis of food, was assessed by using two different techniques. In the first, 1 mmol dm-3 of interferent was injected directly into the system and in the other a mixture of substrate and interferent, both at a concentration of 1 mmol dm-3, was injected into the sensing system.The signal was normalized with respect to the signal obtained for 1 mmol dm-3 glucose which was taken as zero (Table 1). Ascorbic acid interfered very slightly but this interference was further reduced by covering the indicator electrode with a viscose acetate membrane. However, this acid causes severe interference even at +300 mV with the analogous glucose electrode, described by Wang et al. ,8 where the enzyme is physically, rather than chemically, immobilized. Electrode Calibration The electrodes were calibrated by FI with glucose standards over the range 0.01-200 mmol dm-3 using the optimized conditions.The lowest detectable concentration of glucose was 0.1 mmol dm-3, while the calibration was linear to 70 mmol dm-3 glucose (Fig. 2). Sensitivity analysis was carried out to determine the linear portion of the calibration plot because at higher concentra- tions the tailing-off effect of the signal from the system was caused by a physical phenomenon rather than by random error. The non-linear portion of the calibration plot cannot therefore be rejected on the basis that the points on this part of the graph are simply outliers. The sensitivity analysis was performed by determining the coefficient of regression (6) of the calibration plot, wherein each point is the mean signal for three replicate determina- tions. The point representing the highest concentration is rejected and the value of 1-2 obtained from the remaining points is compared with the previous value. If the value of r2 does not approach unity, the remaining final point of the calibration plot is again rejected and the rz of the calibration determined rejecting the point of highest concentration each time until r2 approaches unity. This procedure is carried out because the ‘outliers’ are not caused by noise (or random462 ANALYST, MAY 1991, VOL.116 error), but are the result of phenomena concerning the electrode itself. After optimization, the inclusion of the viscose acetate membrane over the electrode surface extended the linear detection range to between 0.01 and 100 mmol dm-3 (Fig. 2). The inclusion of the membrane over the electrode surface probably promotes this by limiting mass transfer to the electrode surface.This effect can be seen by comparing the time of response of the electrode system without the exclusion membrane ( ~ 1 5 s) and that with the exclusion membrane in place (=20 s); respective washout times were -30 and =45 s (Table 2). These times compare favourably with those of the analogous glucose electrode described by Wang et al. ,8 whose linear range, however, is inferior, namely, 0.5-8.0 mmol dm-3. Membrane Lifetimes Membrane lifetimes and storage stability are significant factors with regard to a wider practical role for biosensors. In order to determine membrane lifetime with respect to substrate, a 1 mmol dm-3 glucose solution was continuously pumped (2.3 cm3 min-1) over the immobilized glucose oxidase electrode.At daily intervals the electrode was washed and re-calibrated. The study showed that the membrane could withstand at least 24 h of glucose flow before any loss of enzyme activity was detected. After 7 d the signal had fallen by It was also noted that membranes stored at 4 "C in buffer gave electrodes that responded well to glucose; after frequent intermittent use (1 h per week) over 4 months the signal was 70% of that for a new electrode. 25%. Electrode Lifetimes The carbon-ferrocene chemically modified electrodes are highly stable with lifetimes of >2 years with intermittent use. The immobilized enzyme membrane can be changed when there is a signal loss due to enzyme deterioration. To achieve this long lifetime the electrodes are stored in a cool, dry and dark place when not in use.This indicates that the electrode lifetime is independent of the immobilized enzyme, the long life being attributed to the carbon paste being bound with a porous polymer matrix rather than the Nujol used by other workers. 16 Conclusion Cellulose triacetate is sufficiently porous to permit electrical contact between the reaction substrate and the electrode material, and the pores are small enough to prevent the electrode modifying material from leaching away. This is indicated by the long lifetimes of the electrodes used under tortuous FI conditions. The maximum linear limit can be extended, and interference from ascorbic acid minimized, by placing a viscose acetate membrane over the electrode surface, as the mass transfer to the electrode surface is thus reduced. The authors thank the Trustees of the Analytical Chemistry Trust Fund of the Royal Society of Chemistry for the award of an SAC Research Studentship and the Committee of Vice Chancellors and Principals for a concurrent Overseas Research Scheme Studentship (both to S.K. B.). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 References Cass, A . E. G . , Davis, G . , Francis, G. D . , Hill, H. A . O., Aston. W. J . , Higgins, I. J., Plotkin, E. V . , Scott, L. D . I . , and Turner, A . P. F., Anal. Chem., 1984,56, 667. D'Costa, E. J . , Higgins, 1. J . , andTurner, A. P. F., Biosensors, 1986, 2, 71. Mattews, D. R . , Holman, R. R., Brown, E., Steemson, J., Watson, A., Hughes, S . , and Scott, D., Lancet, 1987. 1, 778. Foulds, N. C., and Lowe, C. R., Anal. Chem.. 1988,60, 2473. Ikeda, T., Hamada, H., Miki, K . , and Senda, M., Agric. Biol. Chem., 1985.49, 541. Senda, M., Ikeda, T., Miki, K., and Hasa, H . , Anal. Sci., 1986, 2, 501. Dicks, J. M., Aston, W. J.. Davis, G . , and Turner, A . P. F., Anal. Chim. Acta, 1986, 182, 103. Wang, J . , Wu, L.-H., Lu. Z . , Li. R.. and Sanchez, J . , Anal. Chim. Acta, 1990, 228, 257. Beh, S. K . , Moody, G. J . , andThomas, J. D. R., Analyst, 1989, 114, 1421. Moody, G. J., Sanghera, G. S., and Thomas, J . D. R . , Analyst, 1986, 111, 605. Bright, H. J . , and Appleby, M. J . , J . Biol. Chem., 1969, 244. 3625. Weibel, M. K . , and Bright, H. J., J . Bid. Chem., 1971, 246, 2734. Beh, S. K . , Moody, G. J., and Thomas, J. D . R . , Anal. Proc., 1990, 27,82. Beh, S. K . , Moody, G. J . , and Thomas, J . D. R., Anal. Proc., 1989, 26, 290. Beh, S. K., Moody, G. J . , and Thomas, J . D . R . , Analyst, 1989, 114, 29. Gunasingham, H., and Tan, C.-H.. Analysr, 1990, 115, 35. Paper 0102581 J Received June 11 th, 1990 Accepted January Ist, 1991

ISSN:0003-2654    

DOI:10.1039/AN9911600459

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

ANALYST, MAY 1991, VOL. 116 463 pH Dependence of Hydrochloric Acid Diffusion Through Gastric Mucus: Correlation With Diffusion Through a Water Layer Using a Membrane-mounted Glass pH Electrode C. V. Nicholas, M. Desai and P. Vadgama Department of Medicine (Clinical Biochemistry), University of Manchester, Clinical Sciences Building, Hope Hospital, Salford M6 8HD, UK M. 6. McDonnell Department of Chemistry, University of Southampton, Southampton SO9 5HH, UK S. Lucas Co m pu ta tio na I Group, Computing Department, University of Man c h este r, Stop fo rd Building, Man c h este r M73 9PT, UK Solute diffusion coefficients (D) can indicate a dependence upon actual solute concentrations. Here a single compartment has been utilized, in which effective HCI diffusion t o a membrane-mounted glass pH electrode can be measured across the pH spectrum.The study has investigated HCI diffusion through both mucus and water layers as a function of HCI concentration. The observed dynamic responses of a liquid-film and mucus- coated electrodes over a range of HCI concentrations suggest that the speed at which equilibrium is attained is pH dependent; equilibrium was reached rapidly under more acidic and alkaline conditions. Estimated values of DHCl also indicate a strong pH dependence for both liquid film and mucus. In both instances, a >lo-fold reduction in DHcl at pH 7.5 as compared with that at pH 3.5 has been demonstrated. Furthermore, estimated values of DHCl are approximately 4-fold smaller through the mucus gel, as compared with a water layer. The findings indicate that the most powerful influence on diffusional resistance is pH itself, whereby a marked drop in H+ diffusion is likely t o occur towards neutral pH irrespective of the composition of the gel barrier.Possible implications of the findings are discussed in relation t o mucosal protection from acid. Keywords: Hydrochloric acid diffusion; gastric mucus; pH electrode Mucus forms a continuous, adherent visco-elastic gel layer over the gastrointestinal mucosa, which in man has an estimated depth of 50450 pm (Kress et a f . I ) . In the stomach and duodenum, mucus has been considered to play an important role in protecting the mucosa from damage by luminal acid (Allen and Garner’). Studies of its diffusional resistance to HCI have revealed a resistance that is 4-5-fold greater than that of an equivalent unstirred layer of water (Williams and Turnberg,3 Pfeiffer4 and Turner et a f .5 ) . Furthermore, steep pH gradients have been observed within mucus, across intact gastric mucosa, by means of implanted micro-pH electrodes (Williams and Turnberg,6 Bahari et a f . ,’ Takeuchi et a f . * and Flemstrom and Kivilaakso’). These have been explained on the basis of an intra-gel neutralization of HCI with hydrogen carbonate secreted by the surface epi- thelium (Allen and Garner2 and Flemstrom and Kivilaakso’). In this concept of the mucus-hydrogen carbonate barrier, the neutralizing action of HC03- is considered to be potentiated by neutralization occurring in the restricted volume of the mucus gel phase.A mathematical model developed by Engel et a f . 1 0 for intra-mucus neutralization, however, suggested that a relatively minor (5 mmol dm-3) drop in H+ concentra- tion was likely to be generated across the mucus layer based on existing, reported values for gel layer thickness, hydrogen carbonate flux and HCI diffusion. Solute diffusion coefficients can show a dependence upon actual solute concentrations (Crank”) and in the present study HCI diffusion through both mucus and water layers as a function of HCI concentration has been examined. A signifi- cant change from earlier reported values for HCI diffusion could provide further information concerning the resistive contribution made by mucus in its protective role over the gastroduodenal mucosa. A more detailed profiling of hydrogen ion diffusion is warranted, as its diffusion is unique in involving passage from one water molecule to the next, through the formation of a sequence of hydrogen bonds (Robinson and Stokes”).In a previous study, HCI diffusion through water was observed to be retarded by a factor of 100, as compared with earlier reported values, when conditions approached neutrality (Nicholas et af. 13)- The correlation between diffusion through water and mucus layers is reported here, and possible implications of these findings for mucosal protection from acid are discussed. Theory The two-compartment diffusion chamber is appropriate for the study of most solute species (IUPAC14). However, for H+ diffusion, measurement requires highly acidic (pH <2) con- ditions (Williams and Turnberg,6 Robinson and Stokes12 and Slomiany et af.15) if buffering is to be avoided.A one- compartment system based on the glass pH electrode, which allows measurements under acidic through to neutral and alkaline conditions, was used in the present study. The dynamic response of a pH electrode and its approach to an equilibrium may be modelled in terms of diffusion through a stagnant, unstirred layer over the sensor surface (Morf and Simonl6). Provided that the diffusion layer, and not the intrinsic electrode response, is rate limiting, the change in the electrode e.m.f. showed the following dynamic time depen- dence in its approach to an equilibrium response: Here, E, is the electrode e.m.f. at any given time t , E,, is the electrode equilibrium response, S is the slope of the pH calibration graph (mV per decade), [H+Io is the H+ concentra- tion at time zero, [H+],, is the H+ concentration at the final equilibrium response and ‘I: is the time constant for the system.The value of T is governed by both the thickness of the464 ANALYST, MAY 1991, VOL. 116 unstirred layer, d , and by the H+ diffusion coefficient, D , within that layer: The measurement of T permits the calculation of D provided d is known. Alternatively, mucus and liquid films have been created over the glass surface of a pH electrode (Nicholas et aZ.13), which provided a well-defined boundary layer in a stirred solution and which, furthermore? were of sufficient depth both to define the dynamic response of the pH electrode according to eqns.(1) and (2) and eliminate the effects of an external Nernst diffusion layer. Experimental The measuring glass pH electrode (Type CETL; Russell, Fife, UK) was used in conjunction with a saturated calomel reference electrode (Microelectrodes, Londonderry , NH, USA). Electrode e.m.f. was measured using a pH meter (PCMKI, Newcastle upon Tyne, UK) and output recorded at a strip-chart recorder (Linseis, Selb, Germany). A combi- nation pH electrode served as a follower electrode to monitor bulk solution pH during the addition of HCl in the pH jump experiments. All standard reagents were of AnalaR grade and purchased from BDH (Poole, Dorset, UK); bovine serum albumin (BSA) was obtained from Sigma (Poole, Dorset, UK). Native pig gastric mucus was removed as described previously (Williams and Turnberg3), from the stomachs of abattoir animals that had been killed recently.Mucus was applied to the tip of the measuring glass pH electrode which had a pre-mounted 135 pm nylon netting that acted as a spacer. A uniform gel or mucin layer was then created by stretching an external 10 pm Cuprophan dialysis membrane layer, using a Cuprophan from a haemodialysis cartridge (Gambro, Lund, Sweden). For measurements through aqueous films the nylon spacer and dialysis mem- brane were used alone. Measuring and follower electrodes were immersed in a chamber containing 175 ml of solution that was stirred rapidly (Vadgama and Albertil’), and 1 mol dm-3 HCI was injected via an automatic pipette over a period of 1-2 s in order to create a change in the pH of the bulk solution of about 1 pH unit; the temperature of the solution was 21 k 2 “C.Results The dynamic response of the uncovered glass pH electrode in stirred solution, as monitored at the strip-chart recorder, was complete within 2 s of the addition of HCI over the entire pH range used in these studies. The magnitude and dynamic response were unaffected by either previous contact with mucus gel or with the bulk solutions used. Dynamic response profiles were reproducible to within 5% with respect to e.m.f. The observed dynamic responses of a liquid-film and mucus- coated electrodes over a range of pH values in the presence of albumin, as a non-diffusible buffer, are shown in Fig. l(a) and ( b ) , respectively. These suggest that the speed at which equilibrium is attained depends upon pH, with equilibrium being reached more rapidly under more acidic and alkaline conditions; this would not be expected on the basis of eqn.(1) (Morf and Simonl6) , which predicts similar dynamic responses across the pH spectrum, provided the magnitude of the pH jump is uniform. However, when eqn. (1) was used to calculate the dynamic electrode response, it showed good agreement with observed e.m.f changes, both for liquid-film electrodes (Fig. 2) and the mucus-coated electrodes (Fig. 3) over a range of pH changes. This indicates a change in effective diffusion coefficients for HCI (DHCI) over a range of pH values. The value of DHCl was estimated using BSA as a non-diffusible buffer to assist in the pH stabilization of the t + E ui H-r G- EF- D- C / b’T Gf F/ E A 1 min Time -c Fig.1 Dynamic responses of a pH electrode mounted with a 135 pm nylon spacer and dialysis membrane. (a) Liquid film; pH change: A , 5.59-4.98; G, 4.98-3.07; and H, 3.07-2.51. (b) Mucus layer; pH change: A, 10.02-9.08; B, 9.08-7.80; C, 7.80-6.73; D , 6.73-5.62; E, 5.62-4.72; F, 4.724.00; and G, 4.00-2.88. Albumin (20 g 1-1) was used as a non-diffusible buffer 8.70-7.42; B, 7.42-7.07; C, 6.83-6.64; D, 6.46-4.29; E, 5.89-5.59; F, bulk solution. The results are shown in Fig. 4; these indicate a strong pH dependence for DHCl for both a liquid film and mucus. In both situations there is a >lO-fold reduction in DHCl at pH 7.5, as compared with pH 3.5. In addition, estimated diffusion coefficients are approximately 4-fold smaller through the mucus gel, as compared with a water layer.Interestingly, as conditions are made more alkaline, DHCl is seen to increase again. A reliable estimation of DHCl at pH <2 was precluded by the very rapid responses of the film-coated electrode. For the citrate buffer examined here, background ionic strength had no apparent effect on the trend in DHCl values observed over the pH range investigated, as shown in Fig. 5. However, results obtained using different concen- trations of glucosamine as a diffusible buffer in the presence of albumin (Fig. 6) suggest that there may be an effective increase in DHCI, particularly at more acidic and alkaline pH values when a high concentration of such a diffusible buffer is used. This possibility receives some support from the finding of a higher DHCl with a mucus-coated electrode at pH 10.5 in the presence of 30 mmol dm-3 salicylate and albumin (Fig.7) as compared with albumin alone. Discussion The concept of the mucus-hydrogen carbonate barrier (Allen and Garner2 and Flemstrom and Kivilaaksog) has received important supportive evidence (Williams and Turnberg6 and Flemstrom and Kivilaaksog), and continues to attract interest (Munster et al. 19). The effectiveness of this barrier would be critically affected by the rate at which protons approach the surface epithelium from the lumen. A mucus gel layer with a high diffusional resistance would appear to be ideal for such a system; however, the retardation of the diffusion of H+ in mucus would appear to be insufficient to explain the type of pH gradients observed (Engel et al.lo). Part of the explanation for the retardation of the diffusion of the H+ ion in mucus is the net negative charge of the constituent glycoprotein. This may operate by means of a Donnan exclusion mechanism, although comparison with uncharged gel suggest that the effect is minor (Lee and Nicholls20). Any specific ordering of water molecules around the glycoprotein structure is likely to be minimal (Soggett21) and, therefore, unlikely to result in significant additional diffusional resistance, particularly in view of the low ( ( 5 % ) concentration of the glycoprotein in the gel (Allen and Garner2). For native mucus, particulateANALYST, MAY 1991. VOL. 116 465 20 (a) 25 20 - 15 - 10 - - 5 - 0 - 1 1 I I 5 0 20 40 60 80 100 0 100 200 300 400 500 2 0 + 30 E d 25 20 15 10 5 0 14 12 10 8 6 4 2 0 I I I I I I 0 100 200 300 400 500 0 20 40 60 80 100 Time/s Spacer and dialysis membrane mounted pH electrode response profiles tor a liquid film at various pH jumps.(a) pH 10.99-9.99; (b) Fig. 2 9.00-7.4; (c) 6.97-5.91; and (d) 4.03-2.96. Calculated (0) and measured (a) e.m.f. values are compared 0) I I I I I I I 2 0 30 60 90 120 150 180 210 .;. 40 E d 30 20 10 200 400 600 800 1000 0 0 200 400 600 800 1000 0 60 120 180 240 300 360 420 Time/s Fig. 3 7.98-7.03; (c) 6.1 1-5.04; and (d) 3.98-2.93. Calculated (0) and measured (0) e.m.f. values are compared. Spacer and dialysis membrane mounted pH electrode response profiles for a mucus layer at various pH jumps.(a) pH 11.5-9.98; (b) c 80 70 7 60 50 .O 40 = 30 N z 0, .- g 20 2 10 .- v) i 0 2 4 6 8 1 0 1 2 Mid-point pH jump Fig. 4 Effective DHCl calculated from dynamic responses of a spacer and dialysis membrane mounted pH electrode in albumin buffer: 0, liquid film only; and 0, liquid film with mucus. pH values are the mid-points of pH jumps of magnitude about 1 material, protein and lipid, do appear to confer additional diffusional resistance (Slomiany et a1.22). The summation of all the above effects is undoubtedly important in reducing DHCl relative to that of a liquid film. The effect observed in this study is consistent with that reported previously by Williams and Turnberg,3 who used a classical two-compartment diffusion chamber. However, the present work would appear to indicate that the most powerful influence on diffusional resistance is pH itself, whereby a marked drop in H+ diffusibn is likely to occur towards neutral pH irrespective of the composition of a gel barrier (Fig.4). The DHCl has not previously been measured directly, largely because of the problems of achieving a stable pH under near neutral conditions without buffering. As a result, all previous studies of DHC] in mucus have been limited to using HCI at a pH of about 1. The technique reported here permits deter- mination of &-] in mucus over most of the pH spectrum. In the present study, the small unbuffered compartment, i.e., the466 F 2 60 a 0 5 40 .- 8 .- s 20- s In b- 0 - ANALYST, MAY 1991, VOL. 116 - - c I ," 30 E 2 % 20 5 s s 10 z n r- .- 0 .- In 0 2 4 6 8 Mid-point pH jump Fig.5 Effective DHCl calculated from dynamic responses of a liquid film mounted pH electrode for solutions containing 10 mmol dm-3 citrate in 0 , 1 0 mmol dm-3 NaCl; and @, 300 mmol dm-3 NaCl (the latter data are redrawn from reference 18 for comparison) 0 0 2 4 6 8 Mid-point pH jump Fig. 6 Effective DHCl calculated for a liquid film electrode from dynamic responses in glucosamine solutions at various concentra- tions: 0, 10 mmol dm-3 (redrawn from reference 18 for comparison); A , 30 mmol dm-3; and @, 60 mmol dm-3 in albumin buffer 0 .- In 0- 4 6 8 10 Mid-point pH jump Fig. 7 Effective DHCl for a mucus-coated electrode calculated from dynamic responses for 30 mmol dm-3 salicylate in albumin buffer for a range of pH changes liquid or mucus layer over the pH electrode, was more readily controlled with regard to pH by incorporation of a non- diffusible buffer in the much.larger external compartment, i.e., the bulk solution. The effective DHC] values at about neutral pH would appear to have major implications for H+ diffusion in biological systems generally. The mechanism for such pH dependence remains to be elucidated; however, one possibility is the unique mechanism for proton transfer through water, involving multiple hydrogen bonding. The effect of a diffusible buffer (B) in solution is to augment proton transfer by means of a buffer shuttle (Engasser and Horvath23) : HB * HB H+ i I I 6- 4 B- This system would be expected to operate in a concentration- dependent manner and also to have maximal effect at a pH close to the pK, of the buffer (Vadgama and Alberti24).Both phenomena have been observed previously to affect the dynamic response of a pH glass electrode mounted with immobilized protein (Deem et al.25), and also native gastric mucus (Vadgama and Albertil7). Over the buffer concentra- tions used in the present study, any possible effect was minor, except when a high concentration of buffer was used (Figs. 6 and 7). The over-all reduction of at low glucosamine concentration may have been the result of buffer shuttling, although this could have been affected by the binding of ghcosamine to albumin. Estimated DHC] values obtained in different low ionic strength buffers of low relative molecular mass18 demonstrated some differences between citrate, ascor- bate and glucosamine, but these were relatively minor as compared with the steep drop in as neutral pH was approached.Values of DHCl were also lower for the diffusible buffers as compared with the albumin system. This raises the possibility that in the total absence of a buffer effect, values of may be even lower than reported here, as neutrality is approached. The results augment considerably the postulated resistive property of the surface mucus layer; for a relatively small rise in pH, H+ diffusion may be reduced by a factor of =lo. A possible implication of the present findings for the mucus-hydrogen carbonate barrier is that HC03- secretion into mucus may be designed to adjust the pH of the mucus to a range were DHCl is reduced, rather than to effect complete neutralization.Indeed, at the equivalence point HC03- cannot neutralize HCI; for 100 mmol dm-3 HCI the final pH would be 3-5 (Vadgama and Alberti24). It is even conceivable that a high concentration of buffer within the mucus layer, including the HC03-/C02 buffer system, would actually accelerate proton fluxes to the surface epithelium by the operation of a buffer shuttle. Thus, while the high urease activity of Heliobacter pylori generates ammonia which can neutralize H+ within mucus (Thompson et a1.26), there may be significant associated shuttling of H+ along the pH gradient in mucus which might actually contribute to mucosal damage associated with this organism. The present studies reconcile the idea of mucus as a resistive barrier (Williams and Turnberg6) with that of mucus as simply an unstirred water layer (Morris27). In conclusion, the results obtained should be regarded as effective DHCl values, but ones which nevertheless reflect the diffusion behaviour of the physiological system.Further comparative studies are in progress for other ion and solute species using the coated electrode technique described. References 1 Kress, S., Allen, A., and Garner, A., Clin. Sci., 1982, 63, 187. 2 Allen, A., and Garner, A., Gut, 1980, 21, 249.ANALYST, MAY 1991, VOL. 116 467 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Williams. S. E . , and Turnberg, L. A . , Gastroenterology, 1980, 79, 299. Pfeiffer, C. J., Am. J. Physiol., 1981, 240, B176. Turner, N . C., Martin, G. P., and Marriott, C., J. Pharm. Pharmacol., 1985, 37, 776.Williams. S. E., and Turnberg, L. A., Gut, 1981, 22,94. Bahari, H. M. M.. Ross, I . N., andTurnberg, L. A., Gut, 1982, 23, 513. Takeuchi. K.. Magee, D., Critchlow, J., Matthew, J., and Gilen, W., Gastroenterology, 1982,84, 331. Flemstrom, G., and Kivilaakso, E., Gastroenterology. 1983,84, 787. Engel, E., Peskoff, A., Kauffman. G. L., and Grossman, M. I., Am. J. Physiol., 1984, 247, G321. Crank, J., The Mathematics of Diffusion, Oxford University Press, Oxford, 1959. Robinson. R. A., and Stokes, R. H., Electrolyte Solutions. Butterworth. London, 1955. Nicholas. C. V., McDonnell, M. B., and Vadgama, P., 1. Chem. SOC., Chern. Cornmun., 1990, 4. 320. IUPAC, Conditional Diffusion Coefficients of Ions and Mol- ecules in Solution. An Appraisal of the Conditions and Methods of Measurements, Pure Appl. Chem.. 1979.51, 1575. Slomiany, B. L., Laszewicz, W., and Slomiany. A., Digestion. 1986, 33. 146. Morf, W. F., and Simon, W., in Ion-Selective Electrodes in Analytical Chemistry. ed. Freiser, H . . Plenum, New York, 1978, vol. 1, p. 211. 17 18 19 20 21 22 23 24 25 26 27 Vadgama, P., and Alberti, K. G. M. M., Experientia, 1983,39, 573. Vadgama, P., Nicholas, C. V., McDonnell, M. B., Lucas, S., and Desai, M., J. Chem. SOC., Faraday Trans., 1991,87,293. Munster, D . J., Robertson, A. M., and Bagshaw, P. F., N. 2. Med. J . , 1989, 102, 607. Lee, S. P., and Nicholls, J. F., Biotechnology, 1987, 24, 565. Soggett, A., in Water: A Comprehensive Treatise, ed. Franks, F.. Plenum, New York. 1980, vol. 4, p. 519. Slomiany, B. L., Piasek, A., Sarasick, J., and Slomiany, A., Scand. J. Gastroenterol., 1985, 20, 1191. Engasser, J. M., and Horvath, C., Biochim. Biophys. Acta, 1974, 358, 178. Vadgama, P., and Alberti, K. G. M. M., Digestion, 1983, 27, 203. Deem, G. S., Zabusky, N. J., and Sternlicht, H., J. Mernbr. Sci., 1978, 4, 61. Thompson, L., Tasman-Jones, C., Morris, A., Wiggins, P., Lee, S., and Furlong, L., Scand. J. Gastroenterol., 1989, 24, 761. Morris, G . P., Gastroenterol. Clin. Biol., 1985, 9, 106. Paper 1/001321 Received January I 1 th, 1991 Accepted January 23rd, 1991

ISSN:0003-2654    

DOI:10.1039/AN9911600463

出版商:RSC    

年代:1991

数据来源: RSC

摘要:

ANALYST, MAY 1991, VOL. 116 469 Comparative Barium Ion Sensing Qualities of Planar and Tetrahedral Tripodal Receptor Molecules Y. P. Feng, G. Goodlet, N. K. Harris, M. M. Islam", G. J. Moody and J. D. R. Thomas School of Chemistry and Applied Chemistry, University of Wales College of Cardiff, P.O. Box 912, Cardiff CF7 3TB, UK Nine acyclic polyethers, representing examples of planar and tripodal 'scorpion-like' molecules, each with oligoether 'tails' and a pair of anionic 'pincers', were evaluated as possible barium ion-selective electrodes (ISEs) when incorporated into a poly(viny1 chloride) matrix membrane with 2-nitrophenyl phenyl ether as the solvent mediator. The general performance was inferior to a traditional ISE based on the tetraphenylborate salt of the barium complex with a-( nonylpheny1)-a-hydroxy-catena-poly(oxyethy1ene) (Antarox CO-880 with 30 oxyethylene units).However, a general barium ion response seems to be favoured by a tetrahedral tripodal structure (sensor C, electrode 3)' with its design promoting good ion-dipole interactions, as seen in another study on the association constants of these acyclic polyethers with barium ions. A more extensive study of the effect of methoxylated benzyl groups on similar type groups in the pincer positions for the tetrahedral tripodal structures is indicated. Keywords: Receptor molecule; polyether; ion-selective electrode; barium; planar and tetrahedral tripodal structures Examples of strong stoichiometric complexes between alkali and alkaline earth metal cations and neutral carriers are well established.1-6 In ion-selective electrode (ISE) terms, an early example is based on the naturally occurring ionophore, valinomycin, which forms the basis of the highly selective potassium ISE.7,X However, such a rigid cyclic arrangement is not necessary for complexation, and ionophores with a more open structure have selective ion sensing capabilities.9 Systems based on complexes between polyalkoxylate systems and alkaline earth metal cations, especially barium, have been exploited for their potentiometric response in ISE mem- branes.10-15 The tetraphenylborate salt of a barium complex with a-(nonylphenyl)-o-hydroxy-catena-poly(oxyethylene) (with 30 oxyethylene units) [Ba2+ (Antarox CO-880)l [BPh4-]2, when incorporated with 2-nitrophenyl phenyl ether as the plasticizing solvent mediator in a poly(viny1 chloride)(PVC) support matrix, yields electrodes with a near Nernstian response, giving a slope of 28 mV decade-', for concentrations of barium ions between 1 x 10-1 and 1 x 10-4 mol dm-3.13 As a result of an investigation on the complexation of dibenzo-30-crown- 10,1G19 a regio-selective synthesis of acyclic polyether (based on the oxyethylene units) intermediates was devised.16 In due course, this led to the synthesis of a series of 'scorpion-like' ligands, each with oligoether 'tails' and a pair of anionic 'pincers'.*0 These were shown to be capable of metal encapsulation with the association of some members of the alkali and alkaline earth metals, with the affinity for barium being relatively strong compared with the other alkali and alkaline earth metals.'" Some possible technological benefits were indicated, such as the use of these materials to overcome clogging, e.g., by barium sulphate scale formation during oil production from oil wells.20 Additionally, there was the prospect of the application of these scorpion-like ligands as potentiometric ion sensors.Thus, some of these ligands (sensors A-I in Fig. 1 and Table 1) were studied for their suitability as possible selective sensors in PVC matrix mem- branes incorporating 2-nitrophenyl phenyl ether as the solvent mediator, and their performance was compared with that of a traditional barium sensor using [Ba2+ (Antarox CO-880)][BPh4-I2 (sensor J, Table 1). * Present address: Chemistry Department, Bangladesh University of Engineering and Technology, Dhaka.Bangladesh. Experimental Reagents Sensors A-I (Fig. 1, Table 1) were donated and synthesized by Stoddart and co-workers at the University of Sheffield, Sheffield, UK,20 and Antarox CO-880 was donated by GAF Chemicals, Manchester, UK. The [Ba2+ (Antarox c-l \o 0-x J = [ C S H ~ ~ C ~ H ~ ( ~ C H ~ C H ~ ) ~ O O H I B ~ ~ . ~ [ B P ~ ~ - Fig. 1 Structural details of sensors. A and B are planar tripodal structures, whereas C-I are tetrahedral tripodal structures. For key see Table 1470 ANALYST, MAY 1991, VOL. 116 CO-SSO)][SP~-], was prepared as described previously,l3 2-nitrophenyl phenyl ether was supplied by Eastman Kodak, Rochester, NJ, USA and PVC Breon Resin I1 EP by BP Chemicals, Barry, UK. Otherwise, analytical-reagent grade reagents were used, including the chlorides of barium, calcium, magnesium, lithium, sodium, potassium, caesium and rubidium (BDH, Poole, Dorset, UK).PVC Membrane Fabrication and E.m.f. Measurements The PVC matrix membrane ISEs were fabricated from membranes containing a sensor (2.5 mg), 2-nitrophenyl phenyl ether (360 mg) and PVC (170 mg), and assembled according to established procedures.21.22 The internal filling solution was barium chloride (0.1 rnol dm-3), and all the electrodes were conditioned in barium chloride (0.1 rnol dm-3) prior to use. The e.m.f measurements were made with a Radiometer PHM64 pH-millivoltmeter (Radiometer NS, Copenhagen, Denmark) in conjunction with a saturated calomel reference electrode (EIL Model 1370-710).A Corning pH meter and a glass electrode (EIL Model 740748) were used for pH measurements. Electrode calibrations were carried out by spiking with successive aliquots of known concentrations of the sample into doubly de-ionized water thermostated at 25 k 0.1 "C. When not in use, the electrode membranes were stored in barium chloride (0.1 rnol dm-3). Selectivity Coefficient Determination Potentiometric selectivity coefficients, (k$tt,B) were deter- mined using the separate solution method: where El and E2 are the electrode responses to the barium and interferent ion, respectively, each at a barium concentration of 1 x 10-2 rnol dm-3, S is the calibration slope and zBa and zB are the charges of the barium and interferent ions, respect- ively. For divalent interferent ions, eqn.(1) simplifies to For determination of the pH interference-free ranges, e.m.f. measurements were made on solutions of barium chloride in a 0.01 rnol dm-3 tris(hydroxymethy1)amino- methane (Trizma) buffer (obtained from Sigma, Poole, Dorset, UK), whose pH values were adjusted with 0.1 rnol dm-3 hydrochloric acid. Table 1 Structural characteristics of the planar and tetrahedral molecules used and electrode numbers (PVC matrix membrane type based on 2-nitrophenyl phenyl ether plus sensor) No. of oxyethylene Nature of X ISE Sensor units in Fig. 1 No. A 3 p-OMe-benzyl 1 B 3 Benzyl 2 C 3 Benzyl 3 D 2 H 4 E 3 H 5 F 4 H 6 G 5 H 7 H 3 CH2COOH 8 I 4 CH2COOH 9 J 30 - 10* * This electrode is based on a sensor of the tetraphenylborate salt of the barium complex with Antarox CO-880.Results and Discussion A number of points are relevant for discussion with regard to both the performance characteristics (calibration slope and kbtt,B values) of these materials as potentiometric barium ion sensors, and their selectivity characteristics, namely: (i) a general comparison of the barium ion sensing qualities of the sensors formed from compounds A-I with the traditional systems of J; (ii) a comparison of the planar tripodal (B) and the corresponding tetrahedral tripodal structure (C); (iii) the dependency of the number of oxygen atoms in the oxyethylene chain of the diphenol structures (D-G), and of the carboxyl- ates (H and I); (iv) the effect of substituting the phenolic groups of E and F by carboxylic groups (H and I), and for E by benzyl (C); and (v) a comparison of the methoxylated benzyl derivative (A) with the benzyl derivative (B) for the planar tripodal structure.Barium Ion Sensing Qualities The calibrations for barium of the various ISEs were evaluated and compared with the [Ba2+(Antarox CO-880)][BPh4-I2 mode1,13 ISE 10 (Fig. 2). All of the electrodes responded to barium ions, but to different degrees (Fig. 2); each of the new planar and tetrahedral molecule types (Fig. 1 and Table 1) were inferior to the previously established ISE 10 (Fig. 2). 8o 60 -1 UJ 40 0 v) v) 20 -20 'c: -40 -60 - 80 -100 -= E *O 60 c" 8 40 v) v) 20 > -20 E 'c: -40 LU -60 \ Ei - 80 -100 I w 60 0 40 3 2 20 E > o E 2 -20 € uj -40 -601 I ' I ' 1 ' 1 7 6 5 4 3 2 1 -Log([Ba2+l/mol dm-3) Fig. 2 Barium ion responses of various PVC matrix membrane electrodes.(a) 1, ISE 10; 2, ISE 3; 3, ISE 1; and4, ISE2: (b) 1, ISE 10; 2, ISE 7; 3, ISE 4; 4, ISE 6; and 5, ISE 5: and (c) 1, ISE 10; 2, ISE 8; and 3, ISE 9. For key see Table 1ANALYST, MAY 1991, VOL. 116 47 1 In general terms, of the electrodes based on the planar and tetrahedral scorpion-like polyethers under investigation, it is ISE 3 (sensor C) that most closely resembles the calibration characteristics of TSE 10. This is because it has a higher (near-Nernstian) e.m.f. response than any of others. The next, in order of response, are ISEs 8 (sensor H), 9 (sensor I) and 7 (sensor G). Ion-selective electrode 5 showed the best slope, and has a reasonable linear range (2.6 x 10-4-4.3 x 10-2 mol dm-3), but its overall response characteristics are greatly inferior to that of ISE 10.Sensor B (ISE 2) is inferior to C (ISE 3) and points to the tetrahedral tripodal structure being a favourable system for forming a pseudo-cavity around the metal ion in order to maximize the number of stable ion-dipole interactions as previously intimated from data for the association constants of these acyclic polyethers with barium ions.20 Association constants ( K , ) in tetrachloromethane of sensors B and C with alkali and alkaline earth metal cations as measured by the picrate extraction technique are as follows (data from refer- ence 20). For sensor B: Li+ , 160; Na+ , 1300; K+, 1600; Rb+, 610; Cs+, 220; Mg'+, 140; Ca2+, 500; Sr2+, 900; and Ba2+, 1500. For sensor C: Li+, 670; Na+, 1100; K+, 3800; Rb-+, 1100; Cs+, 800; Mg*+, 180; Ca2+, 700; Sr2+, 1700; and Ba2+ , 24 000.Regarding the effect of the dependency of the number of oxygen atoms in the oxyethylene chain, there is little difference between ISEs 4, 5 and 6 (sensors D, E and F), but ISE 7, although of short calibration (Fig. 2), indicates the extra effect of ion-dipole interaction promoted by the oxyethylene units. This is not so when ISEs 8 and 9 (sensors H and I) are compared (Fig. 2), but here the carboxylate group may have a modifying steric influence impressed on the extended oxyethylene chain (from 3 to 4 units). Appropriate groups in the 'pincer' positions certainly seem to promote barium ion-sensing (ISEs 3 and 8). After comparing the effect of the methoxylated benzyl group in the pincer positions of the planar tripodal structure (ISE 1) with just the benzyl group for ISE 2, it would seem [Fig.2(a)] that there is some virtue in a future study of the methoxylated benzyl derivative with the tetrahedral tripodal structure. Selectivity Characteristics The selectivity features of ISEs 1-10 for barium with respect to selected alkali and alkaline earth metal cations are sum- marized in Fig. 3. Again, the superiority of ISE 10 (sensor J) is demonstrated. However, it is less easy to distinguish the trends between the various other electrodes. It is of interest to note that ISE 5 (sensor E) is more selective [Fig. 3(b)] than ISE 4 (sensor D), 6 (sensor F) and 7 (sensor G). The structural reason for this is unclear. More obvious is that ISE 1 (sensor A) is more selective than ISE 2 (sensor B) [Fig.3(a)], and ISE 3 (sensor C ) is marginally more selective than ISE 2 (sensor B). For the alkali metal cations, the interferences are not as significant as indicated by the positive values for log kk?& in Fig. 3, as even when kk:t.B > 1 there can be a selectivity towards barium if B is a univalent cation.23 Nevertheless, a value of log kEt,K = 6 for JSE 3 is considerably greater than the threshold value of "2, which is necessary for the complete loss of selectivity towards barium over potassium for sensor C, and raises the question of whether the system yields a credible potassium ISE. Experiments indicated that potassium ISEs do result from the system, but they are of inferior quality to the well established ISE based on valinomycin. However, there was insufficient sensor material to permit a full definitive study, and the matter merits further investigation.With regard to barium ion selectivity, ISE 10 is still the best. 8 6 4 m + - 9 2 0) 3 0 - 2 -4 Mg Ca Li Na K 3 2 1 -m g o 0" - 1 J -2 -3 - 4 -5 Mg Ca Li Na K Rb Cs 2 1 0 g j - 1 0" -2 J -3 -4 t Mg Ca Li Na K Rb CS Element Fig. 3 Summary of selectivity coefficient data for various PVC matrix membrane electrodes. (a) 1, ISE 2; 2, ISE 3; 3 , ISE 1; and 4, ISE 10: ( b ) 1, ISE 4; 2, ISE7; 3, ISE 6; 4, ISE 5 ; and 5 , ISE 10: and (c) 1, ISE 9; 2, ISE 8; and 3 . ISE 10. For key see Table 1 Conclusion Although the general performance of ISEs, based on sensors of the planar and tetrahedral receptor molecules studied here, is inferior to that of barium ISEs based on an a-( nonylpheny1)- o3-hydroxy-catena-poly(oxyethy1ene) system, an extended study of the effect of methoxylated benzyl or similar type groups in the 'pincer' positions of the tetrahedral tripodal structures is indicated. Hunan University, China, and the British Council are thanked for supporting visiting research associateships for Y .P.F.and M.M.I., respectively. The Science and Engineering Research Council is thanked for generous financial support under their Chemical Sensors research initiative which has made possible the support of N.K.H. and of the provision of the new type receptor molecule sensors from Dr. J. F. Stoddart (University of Sheffield). Discussions with Dr. Stoddart and Dr. J . F. Costello (University of Sheffield), and with Dr. D.J . Williams (Imperial College, London) are much appreciated. References 1 Moore, C., and Pressman, B. C., Biochem. Biophys. Res. 2 3 Commun.. 1964, 15, 562. Pedersen, C. J . , J . Am. Chem. SOC., 1967,89,7017. Eisenman, G., Ciani, S., and Szabo, G.. J . Membrane Biol.. 1969, 1, 294.472 ANALYST, MAY 1991, VOL. 116 4 5 6 7 8 9 10 11 12 13 14 15 16 Truter, M. R., Struct. Bonding, 1973, 16, 72. Simon, W., Morf. W. E., and Meier, P. Ch., Struct. Bonding, 1973, 16, 113. Midgley, D., Chem. Soc. Rev., 1975, 4, 549. Pioda, L., Stankova, B., and Simon. W., Anal. Lett., 1969, 2, 665. Frant, M. S., and Ross, J. W., Science NY, 1970,167, 987. Ammann, D., Morf, W. F., Anker, P., Meier, P. C., Pretsch, E., and Simon, W., Ion-Sel. Electrode Rev., 1983, 5 , 3. Levins, R. J., Anal. Chem., 1971. 43, 1045. Levins, R. J., Anal. Chem., 1972, 44, 1544. Bauman, E. W., Anal. Chem., 1975,47, 959. Jaber, A. M. Y., Moody, G. J., andThomas, J. D. R., Analyst, 1976, 101, 179. Jaber, A. M. Y., Moody, G. J.. and Thomas, J. D. R.. J. Inorg. Nucl. Chem., 1977, 39, 1973. Levins, R. J . , Ger. Offen., 2264721, 1973. Allwood, B. L., Kohnke, F. H., Slawin, A. M. Z., Stoddart, J. F., and Williams, D. J.. J. Chem. Soc., Chem. Comm., 1985, 311. 17 Kohnke, F. H., and Stoddart, J. F., J. Chem. SOC., Chem. Comm., 1985, 314. 18 Allwood, B. L.. Kohnke, F. H.. Stoddart, J. F., and Williams. D. J., Angew. Chem., Int. Ed. Engl., 1985, 24, 581. 19 Colquhoun, H. M., Goodings, E. P., Maud, J. M., Stoddart, J. F., Wolstenholme, J. B.. and Williams, D. J., J. Chem. Soc., Perkin Trans. 2, 1985, 2, 607. 20 Bartlett, J. S., Costello, J. F., Mehani, S., Ramdas, S., Slawin, A. M. Z., Stoddart, J. F., and Williams, D. J., Angew. Chem., int. Ed. Engl., 1990. 29, 1404. 21 Moody, G. J., Oke, R. B.. and Thomas, J. D. R., Analyst, 1970, 95, 910. 22 Craggs, A., Moody, G. J., and Thomas, J. D. R., .I. Chem. Educ., 1974, 51, 541. 23 Craggs, A., Kiel, L., Moody, G. J. and Thomas, J. D. R., Talanta, 1975, 22, 907. Paper 01049340 Received November 2nd, 1990 Accepted Januury 1 Oth, I991

ISSN:0003-2654    

DOI:10.1039/AN9911600469

出版商:RSC    

年代:1991

数据来源: RSC