ISSN:0003-2654    

DOI:10.1039/AN98611FX021

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALAO 111(6) 601-728 (1986)The AnalystJune 1986The Analytical Journal of The Royal Society of Chemistry60 160561 161 962563 163564164565 165766 166567167768168569 169570 170771 171772 1723CONTENTSLinking Low Dispersion Liquid Chromatography with Diode-array Detection for the Sensitive and SelectiveAmperometric Enzyme Electrode System for the Flow Injection Analysis of Glucose-G. J. Moody, G. S. Sanghera,Epoxy-based All-solid-state Poly(Viny1 Chloride) Matrix Membrane Calcium Ion-selective Microelectrodes-Sajeda hAnodic-stripping Voltammetry of Metal Complexes in Non-aqueous Media After Extraction: Determination of CopperVoltammetry of Hexacyanoferrates Using a Chemically Modified Carbon-paste Electrode-Ku rt KalcherDetermination of Ammonia in Wine and Milk with an Ammonia Gas-sensing Probe-Jose L.Bernal, Maria J. del Nozal,Luis Deban, Isabel TorremochaDetermination of Morphine by Flow Injection Analysis with Chemiluminescence Detection-Richard W. Abbott, AlanTownshend, Richard GillFlow Injection Spectrofluorimetric Determination of Europium(ll1) Based on Solubilising Its Ternary Complex withThenoyltrifluoroacetone and Trioctylphosphine Oxide in Micellar Solution-Makoto Ai hara, Miwako Arai,Tomitsugu TaketatsuDetermination of Ca, Mg, Na, Cd, Cu, Fe, K, Li and Zn in Acid Mine and Reference Water Samples by Inductively CoupledPlasma Atomic Fluorescence Spectrometry-Richard F. Sanzolone, Allen L. MeierDetermination of Lead in Blood by Atomic Absorption Spectrometry with Electrothermal Atomisation-Ian L.Shuttler,H. Trevor DelvesDetermination of Aluminium in Human Tissues and Body Fluids by Zeeman-corrected Atomic AbsorptionSpectrometry-Jan Rud Andersen, Susanne ReimertElimination of the Effects of lnterferents on the Quantitative Determination of Deuterium Oxide in Biological Samplesby Infrared Photometry-Ralph N. Arnold, Eric J. Hentges, Allen TrenkleBackground Correction Method for the Determination of Ascorbic Acid in Soft Drinks, Fruit Juices and Cordials UsingDirect Ultraviolet Spectrophotometry-Oi-Wah Lau, Shiu-Fai Luk, Kit-Sum WongTrace Determination of Hydroperoxides by Spectrophotometry in Organic Media-Jaroslav Petrij, SusanneZehnacker, Jifi Sedlai, Jean MarchalTernary Complexes in Solution: Complex Formation Between the 1 : 1 Thorium(lV) - Alizarin Maroon Complex andThiosalicylic Acid-Mohamed M.Seleim, Kamal A. Idriss, Magda S. Saleh, Elham Y. HashemStudy of Ternary Thorium Complexes with Some Triphenylmethane Reagents and Cationic Surfactants-MaciejJaroszDetermination of Trace Amounts of Phosphate in Water Samples by Ion-exchange Resin Thin-layer Spectropho-tometry-Kiichi Matsuhisa, Kunio OhzekiUse of a Silver - Gelatin Complex for the Microdetermination of Hydrogen Sulphide in the Atmosphere-TarasankarPal, Ashes Ganguly, Durga S. MaityRapid Automatic Gas Chromatographic Method for the Continual Measurement of Hydrogen Cyanide and Cyanogen inAir-Peter R. Fielden, Simon J. Smith, John F. AlderHigh-performance Liquid Chromatographic Analysis of Preservative-treated Timber for 2-(Thiocyanomethylthio)ben-zothiazole and Methylene BisthiocyanateMichael J.KennedyAnalytical Investigation of Some Fluorogenic Reactions of Indol-3-yl Acids with o-Phthalaldehyde. Part II. Thin-layerChromatographic Studies-Tereza C. M. Pastore, Clausius G. de LimaApplication of Computer-based Pattern Recognition Procedures in the Study of Biological Samples. Comparison of theCuticular Hydrocarbon Profiles of Different Colonies of the Black Imported Fire Ant-Jeffrey H. Brill, Tom Mar,Howard T. Mayfield, Wolfgang BertschDetermination of Vitamins A, D and E-Mary MulhollandJ. D. R. ThomasA. H. Khalil, G. J. Moody, J. D. R. Thomas, Jose L. F. C. Limawith SalicylaldoximeJose Aznarez, Juan Carlos Vidal, Jose Maria RabadanSHORT PAPERSUse of Open-circuit Pre-concentration of Sulphide Ion in Stripping Voltammetry at the Parts per Billion Level ofDirect Determination of Nickel in Human Plasma by Zeeman-corrected Atomic Absorption Spectrometry-Jan RudStudy of the Nitroprusside - Sulphydryl Test for Aromatic Thiols-James P.DanehySampleSambarnoorthy Jaya, Talasila Prasada Rao, Gollakota Prabhakara RaoAndersen, Bente Gammelgaard, Susanne Reimertcontinued inside back coverTypeset and printed by Heffers Printers Ltd, Cambridge, EnglanCOMMU NlCATlON725 Extent of the Heating of Microwave Cavities at Large Modulation Amplitudes and Its Effect on Quantitative Analysis byESR Spectrometry-D. Thorburn Burns, Barry G. Dalgarno, Brian D.Flockhart727 BOOK REVIEWS728 ERRATASimultaneous Multi-element Analysis by Continuum Source Atomic Absorption Spectrometry with Graphite ProbeElectrothermal Atomisation-John Carroll, Nancy J. Miller-lhli, James M. Harnly, David Littlejohn, John M.Ottaway, Thomas C. O’HaverDetermination of Hydroquinone in Skin-toning Creams Using High-performance Liquid Chromatography-Jane Firth,Ian RixA New Way of Organising Spectral Line Intensity Ratio Fluctuations-Bo ThelinAnnual Reports on Analytical AtomicSpectroscopy Vol. 14Edited by L. Ebdon, Plymouth Polytechnic and M. S. Cresser,University of A berdeenThis publication reports on current developments in all branchesof analytical atomic emission, absorption and fluorescencespectroscopy with reference to papers published and lecturespresented during 1984.Much of the information is presented intabular form for ease of reference.Brief Contents:ATOMIZATION AND EXCITATION:Arcs, Sparks, Lasers and Low-Pressure Discharges; Plasmas;Flames; Electrothermal Atomization; Vapour Generation.INSTRUMENTION :Light Sources; Optical Systems and Detectors; BackgroundCorrection; Automatic Sample Introduction; Instrument Controland Data Processing; Complete Instruments; CommercialInstruments.METHODOLOGY:New Methods; Detection Limits, Precision and Accuracy;Standards and Standardization.APPLICATIONS:Chemicals; Metals; Refractories and Metal Oxides, Ceramics,Slags and Cements; Minerals; Air; Water; Soils, Plants andFertilizers; Foods and Beverages; Body Tissues and Fluids.REFERENCESAUTHOR INDEXSUBJECT INDEX“. . . an essential reference work for atomic spectroscopists andfor chemists concerned with trace metal analysis.” - J . E. Page,Chemistry and Industry, reviewing Vol. 11Following publication of Annual Reports on Analytical AtomicSpectroscopy Vol. 14, this series will be discontinued. Much ofthe material covered, however, will appear in Journal ofAnalytical Atomic Spectrometry (JAAS) under the headingAtomic Spectrometry Updates.Hardcover 46Opp ISBN 085186 677 8Price S65.00 ($117.00) RSC Members M.00Ordering:RSC Members should send their orders to: MembershipManager, The Royal Society of Chemistry, 30 Russell Square,London WClB 5DT.Non-RSC members should send their orders to: The RoyalSociety of Chemistry, Distribution Centre, Blackhorse Road,Letchworth, Herts SG6 IHN, UK.ROYALSOCIETY OFCH E M ISTRYlnformat ionService

ISSN:0003-2654    

DOI:10.1039/AN98611BX023

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 601 Linking Low Dispersion Liquid Chromatography with Diode-array Detection for the Sensitive and Selective Determination of Vitamins A, D and E* Mary Mulholland Pye Unicam, York Street, Cambridge CBI ZPX, UK A low dispersion LC system using narrow-bore column technology was linked to a diode-array detector and applied to the determination of vitamins. The LC system had been evaluated for dispersion effects. The technique was robust and reliable and proved to be extremely sensitive and selective for the determination of vitamins A, D and E. Vitamin E was assayed in various vegetable oils and the percentage distribution examined. The high mass sensitivity was demonstrated with an on-column loading of 10 ng of vitamin E, which could be clearly detected and identified.Keywords: Vitamin A, D2, D3 and E determination; narrow-bore column technology; diode-arra y detection; high-performance liquid chromatography; vegetable oils analysis Reducing the size of chromatographic columns results in a decrease in the elution volume required for a given separation. Two major advantages emerge from this. Firstly, as the sample dilution is less, the sensitivity is increased. A compar- able peak size will be obtained for a 1-p1 injection of a given concentration on a 10 cm X 2.1 mm i.d. column as with a 5-1.11 injection volume on a 10 cm x 4.6 mm conventional column, both systems having equivocal flow-rates. This advantage is therefore only relevant when sample volumes are restrictive. Secondly, the amount of solvent required per analysis will be greatly reduced.Worthwhile savings can be achieved when vitamins are assayed in high sample throughput situations. In any chromatographic system the over-all efficiency achieved will be reduced owing to extra-column dispersion. This effect on the measured column effiency, N,, is shown in the following relationship: Nm = (1 - 02) where N is the true column efficiency and 0 represents the relative external contribution to band broadening. As resol- ution is proportional to the square root of the plate number, the relative loss of resolution will be W2.l Accepting, as a guideline, that the maximum acceptable loss in resolution owing to extra-column dispersion is 10%,2 a specification for equipment requirements for a given column size can be drawn UP.Conventional HPLC methods used previously were exam- ined in order to select column types and sizes that would be suitable for the determination of vitamins.3--S After making a suitable choice of column size, the instrumentation could then be optimised to fit the low dispersion requirements. A 1-p1 injection rotor, a specially designed 1-pl flow cell and minimal external tubing were therefore selected. This optimised system was evaluated for compatibility with the selected column geometry.6 The compatibility study consisted of a comparison of the column peak standard deviation with the measured external peak standard deviation to calculate the expected resolution loss? The results show that for capacity factors greater than 1 the instrumentation is fully compatible with the selected column geometry.The determination of fat-soluble vitamins presents many detection problems.8 They have differing UV absorbance maxima and are often accompanied by an excess of com- pounds of similar physical and chemical properties that can interfere with their determination. The choice of diode-array * Presented at the 30th IUPAC Congress, Manchester, UK, September &13th, 1985. detection allows each vitamin to be assayed at its wavelength of maximum adsorption (Amax.) without the loss of selectivity provided by conventional UV detection. Multi-channel UV - visible detection is based on a linear array of light-sensitive elements (diodes) etched on to a silicon chip. These diodes operate in parallel to monitor simul- taneously the absorbance over a given wavelength range.The detector thus acquires three-dimensional data of absorbance versus wavelength versus time, which, in turn, provides both qualitative and quantitative information. The peak purity can also be established by a comparison of spectra taken throughout the peak elution. The instrumen- tation employed for this work can be simply converted for use with conventional HPLC columns. Previous applications of micro-columns with diode-array detection have involved the use of specialised equipment dedicated to this column geometry .9 This paper investigates the applicability of linking narrow- bore column technology to diode-array detection for the determination of vitamins A, D and E. Experimental Materials The vitamins for use as reference materials were obtained from Sigma (St Louis, MO, USA).All solvents were of HPLC grade. (Fisons, Loughborough, UK). Instrumentation The LC system consisted of a PU4015 pump with a PU4021 multi-channel UV - visible detector equipped with a 1-p1 flow cell and a Rheodyne 7520 injection valve with a 1-p1 rotor. The detector output was connected to a PU4850 data station having two additional 128K memory boards. (All components were from Pye Unicam, Cambridge, UK.) Chromatographic Conditions Sample manipulations were performed in the absence of oxygen, direct sunlight or the light of fluorescent tubes in order to avoid degradation of the vitamins. All data were obtained as chromascans from which spectra and chromato- grams could be taken using the PU4850 data station.602 ANALYST, JUNE 1986, VOL.111 ( a ) 325.8 Ib) 110.8 0.2 a.u.f.s I 462.1 ( C) 462.1 0.2 a.u.f.s 1 Wavelength- Fig. 1. 300 nm; and ( c ) , retinol at 330 nm Chromatogram taken at the wavelength of maximum absorption of each vitamin. ( a ) , Ergocalciferol at 270 nm; ( b ) , a-tocopherol at Normal-phase Chromatography An HPLC method was developed, which resolves retinol, a-tocopherol and ergocalciferol (vitamins A, E and D, respectively) using a 3 cm x 2.1 mm i.d. guard and a 10 cm x 2.1 mm i.d. Spheri-5 silica cartridge with a mobile phase of hexane - isopropyl alcohol (99.5 + 0.5 V/V) at a flow-rate of 0.4 ml min-1. This method was altered by changing to a mobile phase of hexane - isopropyl alcohol (99.7 + 0.3 V/V) to resolve a-, p- and y-tocopherol in vegetable oils.All reference materials were dissolved in hexane and the vegetable oils were injected directly. Reversed-phase chromatography To resolve ergocaliferol and cholecalciferol (vitamins D2 and D3, respectively) a method was developed using a 3 cm X 2.1 mm i.d. guard and a 10 cm x 2.1 mm i.d. Spheri-5 RP18 cartridge with a mobile phase of methanol - water (95 + 5 V/V) at a flow-rate of 0.4 ml min-1. All reference materials were dissolved in methanol. The HPLC columns used were supplied by Pye Unicam. Results and Discussion A solution containing 0.5 mg ml-1 each of retinol, cw-toco- pherol and ergocalciferol reference materials was chromato- graphed under the conditions described. Three chromato- grams were taken at the individual wavelength of maximum absorption for each vitamin and these are shown in Fig.1. Each vitamin could therefore be assayed at its maximum sensitivity. The UV spectra illustrated in Fig. 2 were obtained at the peak apex for each vitamin. These spectra are easily distinguished , each having distinct features. The method, therefore, demonstrates good selectiv- ity. Relative standard deviations for the retention time and peak area of each vitamin obtained from ten injections were below 1%. However, care was taken to avoid degradation of the vitamins in solution by using sealed, dark glassware. Vegetable oils contain tocopherols in the unesterified form and can thus be injected on to the column directly without any pre-treatment. Fig. 3 demonstrates the resolution that was achieved between a-, p- and &tocopherol.As each tocopherol has a different degree of biological activity it is necessary to observe their percentage distribution. The results for sun- flower oil and soya oil in Table 1 show sunflower oil containing predominantly a-tocopherol whereas y-tocopherol is the largest component in soya oil. Although there was some 0.2 0.1 0 1.2 t Q) C m e a $ 0.6 0 0.1 0.05 1 I I I I 210 2 50 290 330 370 Wavelengthinm Fig. 2. Fig. 1 at each respective peak apex Spectra of the vitamins taken from the chromatograms of interference with a-tocopherol, owing to co-eluting materials, this did not greatly affect quantitation. The normalisation and subtraction of the spectrum obtained for the co-eluting material from the spectrum at the peak apex for a-tocopherol shows that the resultant spectrum can be clearly identified (Fig.4).ANALYST, JUNE 1986, VOL. 11 1 603 The features in the UV spectrum of vitamin E vary in magnitude. A graph plotting the logarithm of absorbance will therefore enable the smaller features to be examined more clearly. To examine the spectral purity of y-tocopherol in soya oil the logarithm of absorbance plots of spectra taken throughout the peak elution were compared with a plot for the Table 1. Distribution (YO) of vitamin E Total vitamin El Yo Peak 1, Peak 3, Peak 4, Sample a-tocopherol @-tocopherol y-tocopherol Sunfloweroil . . 93.1 2.2 4.6 Soyaoil . . . . 8.9 8.9 81.6 E l z != 0.02 a.u.f.s. X I I Wavelength - spectra of a-tocopherol reference material. Reference material for a-tocopherol is only available as soya oil and, as the spectrum for y-tocopherol is indistinguishable from that for a-tocopherol, it was decided to use the spectrum obtained for a-tocopherol for which the purity was assured. Examina- tion of the graphs shown in Fig.5 reveals a small shoulder at 230-250 nm. This is due to the fats present in soya oil, which leach slowly off the column. As the assay is carried out at 300 nm this will not affect quantitation. A reproducibility study of five injections of each oil gave relative standard deviations for retention behaviour and peak areas of below 2% for each isomer. Very low levels of detection and identification were achieved using this technique. This is demonstrated by Fig. 6, which shows the chromatogram and spectrum for a 10 ng on-column loading of vitamin E acetate.Ergocalciferol and cholecalciferol were resolved using a reversed-phase HPLC method. The chromascan, chromatogram and spectra for a 100-pg on-column loading of these vitamins are illustrated in 210 250 290 330 370 210 250 290 330 370 Wavelengthmm Fig. 3. Chromatograms of vitamin E in (a) sunflower oil and (b) soya oil. Peaks: 1, a-tocopherol; 2, @-tocopherol; and 3, y-tocopherol Fig. 5. Log absorbance plot of (a) a-tocopherol reference material; (6) y-tocopherol in soya oil taken on the peak slope upwards; (c) y-tocopherol in soya oil taken at the peak apex; and ( d ) y-tocopherol in soya oil taken on the peak slope downwards 210 250 290 330 370 Wavelengthlnm Fig. 4. UV spectrum of (a) a-tocopherol in sunflower oil; (6) material co-eluting with a-tocopherol in sunflower oil; and (c) after normalisation and subtraction of ( b ) from ( u ) 0.002 3.u.f.s ._.89.3 ~ 210 250 290 330 370 Wave leng t h/n m Wavelength - Fig. 6 . using a 10-ng on-column loading (a) Chromatogram and ( b ) spectrum of a-tocopherol acetate604 ANALYST, JUNE 1986, VOL. 111 1 I 2 a 9 330 360 390 300 Ti me/s Time I I I I 210 250 290 330 370 210 250 290 330 370 Wavelengthhrn Fig. 7. (a) Chromascan and ( b ) chromatogram of 1, cholecalciferol and 2, ergocalciferol reference materials. (c) Spectrum of ergocalci- ferol and ( d ) spectrum of cholecalciferol reference material Fig. 7. The method demonstrated good selectivity between the two vitamins and relative standard deviations (from five injections) for the retention time and peak area were below 1% for each vitamin.In order to demonstrate the sensitivity, Fig. 8 shows a chromatogram and spectrum obtained for a 10-ng on-column loading of ergocalciferol. Throughout this work the retention behaviour was reproducible and no column deterioration was observed, demonstrating the robustness of the columns selected. Conclusions The technique of linking narrow-bore column technology to diode-array detection was successfully applied to the determi- nation of vitamins. The use of reduced-size columns enabled exceptionally high mass sensitivities to be achieved. These columns were robust and could be used extensively without any deterioration in performance. Chromatographic peak shapes were good, demonstrating the low dispersion effects of the system.Diode-array detection proved to be extremely versatile as data, obtained from a single injection, could be extensively manipulated to give a large amount of information. The risks of misinterpreted data are minimised as chromatographic peaks can be clearly identified and their spectral purity established. These features are pertinent to the analysis of vitamins, which are often found in very complex sample matrices. The combination of narrow-bore LC and diode-array detection proved to be sensitive and selective for both the qualitative and quantitative determination of vitamins A, D and E. 339.8 I Time --t 210 250 290 330 370 Wavelengthhrn - Fig. 8. 10-ng on-column loading (a) Chromatogram and (b) spectrum of ergocalciferol using a 1. 2. 3. 4. 5. 6. 7. 8. 9. References Kucera, P., Editor, “Micro-column High Performance Liquid Chromatography,” Elsevier, Amsterdam, 1984, pp. 1-36. Bristow, P. A , , “Liquid Chromatography in Practice,” HETP, Van Neikerk, P. J., and du Plessis, L. M., J. Chromatogr., 1980, 187, 436. Abe, K., Yuguchi, Y., and Katsui, G., J . Nutr. Sci. Vitaminol., 1979, 25, 67. Van Neikerk, P. J., and Smit, S. C. C., J. Am. Oil Chem. SOC., 1980, 57, 417. Naish, P. J., Goulder, D. P., and Perkins, C. V., Chromato- graphia, 1985, 20, 335. Hupe, K. P . , Jonker, R. J., and Rozing, G., J. Chrornatogr., 1984,285, 253. Macrae, R., Editor, “HPLC in Food Analysis,” Academic Press, London, 1982, Chapter 8, pp. 187-205. Takeuchi, T., and Ishii, D., J. Chromatogr., 1984, 288, 451. UK, 1976, pp. 239-242. Paper A51400 Received November 4th, 1985 Accepted January 8th, 1986

ISSN:0003-2654    

DOI:10.1039/AN9861100601

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 605 Amperometric Enzyme Electrode System for the Flow Injection Analysis of Glucose* G. J. Moody, G. S. Sanghera and J. D. R. Thomas Department of Applied Chemistry, Redwood Building, UWIST, P.O. Box 13, Cardiff CFI 3XF, UK A flow injection system incorporating an amperometric enzyme electrode is described. Glucose oxidase immobilised on nylon mesh and held over a platinum electrode formed the basis of the electrode, which was incorporated in a three-electrode amperometric Stelte cell, modified for flow injection analysis. The flow and enzymatic reaction conditions have been optimised for maximum glucose response. The system exhibited good linearity (0.01 to 3.0 mM glucose), short response times (<45 s), good capacity (24 h with a continuous flow of 2.5 mM glucose) and long lifetimes (up to 4 months with storage at 4 "C).Data for the determination of glucose in foodstuffs with the enzyme electrode were similar t o those obtained by the Yellow Springs Instrument glucose analyser and a soluble enzyme test kit (Boehringer Mannheim method). Keywords: Amperometric glucose sensor; enzyme electrode; flow injection analysis; food analysis; glucose analysis Ever since the pioneering work of Clark and Lyons1 there has been increasing interest in the enzyme electrode field. Most immobilised enzyme probes have arisen for organic and biological substrates for which simple analyses were not available. Continuing interest in this field has largely been due to the availability of a wider range of enzymes, advances made in immobilisation technology and improvement of the asso- ciated sensor.The range of probes available today covers numerous substrates, and excellent reviews have been given by Carr and Bowers2 and by Guilbault.' Various suppport materials have been used for enzyme immobilisation,3-5 including alumina, charcoal, glass, silica, polyacrylamide gel, PVC matrix, nylon and proteins, such as bovine albumin. Attachment of enzyme directly on to the base sensor itself rather than on an adjacent support matrix has recently been reported.6 The use of enzyme probes has branched into several areas of analytical chemistry. Direct probes may employ poten- tiometry , amperometry, enthalpimetry or chemiluminescence as the base sensor and the amperometric approach is the most popular. Glucose electrodes, owing to their great scope in clinical chemistry, have been the subject of considerable interest, as can be deduced from reviews and literature s ~ r v e y s .~ ~ ~ ~ g Glucose flow injection analysis systems based on enzymes normally have a glucose oxidase reactor in a separate chamber from the signal detector.9 Present trends in clinical appli- cations are directed towards the miniaturisation and implan- tation of a glucose sensor as part of a regulated insulin infusion device.10 Active research in the area of chemically modified electrodes and direct electron transfer electrodes is of great interest. Thus, amperometric-type chemically modified elec- trodes for glucose have been reported6 and recent workll well illustrates the electron transfer electrode based on a ferrocene mediator.The determination of glucose in foodstuffs has received relatively little attention when compared with clinical glucose analysis. Among the current enzyme-based methods employed are a soluble enzyme test kit (Boehringer Mann- heim) and the Yellow Springs Instrument (YSI) glucose analyser. Foodstuffs requiring analysis may range from raw glucose products, such as simple syrups or powders, to already processed foods, such as biscuits. * Presented at the 30th IUPAC Congress, Manchester, UK, September 8-13th, 1985. This paper describes an amperometric enzyme electrode system in a modified electrochemical cell design for flow injection analysis (FIA) (Fig. 1). The excellent mechanical strength of nylon mesh with immobilised glucose 0xidase12.~~ gave a robust membrane for covering a standard platinum electrode forming the basis of the enzyme electrode.This electrode was incorporated in a three-electrode Stelte micro- cell modified for FIA, permitting the rapid determination of glucose in foods. Experimental Reagents Glucose oxidase (E.C. 1.1.3.4, 100 IU mg-1, purified from Aspergillus niger) , lysine monohydrochloride, dimethyl sul- phate, 25% glutaraldehyde solution and P-D( +)-glucose were obtained from Sigma Chemical Co. The enzyme was stored in a refrigerator. Glucose standards were prepared from a stock solution of P-D( +)-glucose in sodium dihydrogen orthophosphate (100 mM) buffer obtained from BDH Chemicals (Poole). The range of food samples was from the Laboratory of the Government Chemist (London). Nylon (6,6) mesh was obtained from Henry Simon Ltd.(Cheshire) with the following character- istics: open surfaces 38%, thread thickness 80 pm, mesh count 47.15 cm-1 and mesh aperture 132 pm. Immobilisation of Enzyme Glucose oxidase was immobilised on nylon mesh by a modification of the method devised by Hornby and Morris.12 Thus, enzyme was attached to single (1 cm2) and master (6 cm2) nylon mesh membranes, the procedure being the same in each instance. Nylon mesh was treated with dimethyl sulphate in a water-bath (75 "C) for exactly 5 min, followed by immersion in ice to stop the reaction. After cooling, the membrane was washed twice with anhydrous methanol, the first wash yielding a white precipitate. Lysine was attached to the methylated nylon mesh by immersion of the membrane in 50 cm3 of lysine (50 mM, pH 9.0) for 2 h at room temperature. After thorough washing with sodium chloride solution (100 mM), the mem- brane was placed in a 12.5% VWsolution of glutaraldehyde in saturated borate buffer (100 mM, pH 8.5) for 45 min.The enzyme was attached to the mesh by dipping the membrane in glucose oxidase (50 mg) in 5 cm3 of phosphate buffer (100 mM, pH 7.0) for 2 h at room temperature and then overnight at606 ANALYST, JUNE 1986, VOL. 111 4 “C. The enzyme attachment to nylon mesh procedure is achieved by the agency of the bifunctional glutaraldehyde with the lysine acting as a spacer between the nylon and the enzyme structure: NYLON MESH I +NH=CNHCH( COO-)CH2CH2CH2CH2N I1 CH ENZYME-N=CH The enzyme electrode was then fabricated by stretching the immobilised enzyme membrane (of specific immobilised activity 22.1 nmol cm-2 min-1) over a smooth platinum electrode of the Stelte cell. The excellent mechanical strength of the mesh permitted a taut fit over the electrode with the aid of an O-ring.Apparatus Fig. 1 shows the apparatus assembly used. Electrode poten- tials were controlled and currents monitored by means of a potentiostat (Metrohm VA-detector E611), capable of measuring current in the nanoamp range. A linear ylt chart recorder (Model 500) was used to record the FIA signals. Sample propulsion was made with a four-channel peristaltic pump (Ismatec Model IP-4) with sample injections being made with a manual (PTFE) valve (Tecator).All connecting tubing was of PTFE (nominal i.d. 1.27 mm). Pump pulsation was reduced with a suppressor situated immediately after the pump. Static noise was greatly reduced by earthing the flowing stream immediately after the injection valve. The detector was based on a three-electrode assembly incorporating a modified (Metrohm EA1102) Stelte micro-cell with a platinum - enzyme working electrode, a glassy carbon auxiliary electrode and a silver - silver chloride reference electrode [Fig. l(b)]. Electrical contact between the two chambers was achieved with a cellulose acetate membrane. The modification of the Stelte cell consisted of a Perspex block in the cell chamber in order to reduce the dead volume and to I- Linear Model 500 chart recorder C B Inject ion I d valve €@-& Waste Model IP/4 Modified Stelte cell l--tH peristaltic pump with enzyme electrode Fig.1. (a) Amperometric flow injection analysis apparatus, with ( b ) details of the modified three-electrode Stelte micro-cell and (c) a section through D to E of the micro cell. A, Reference electrode, silver - silver chloride; B, auxiliary electrode, glassy carbon; C, enzyme electrode chamber; D, sample inlet; E, sample outlet; F, reference and auxiliary electrode chamber; G, V notch on back of Perspex block; H, Perspex block; I, etched channel; and J, enzyme electrode produce a “wall jet” type of working electrode chamber [Fig. l(c)]. A channel was etched along the Perspex block in order to connect the inlet and outlet ports of the cell, enhancing the diffusion of sample to the electrode.The apparatus was used in the normal mode for flow injection analysis with standards and sample solutions of appropriate volume being introduced through the injection valve (Fig. 1). Results Optimisation for Glucose Analysis Enzymatic and flow parameters were optimised to obtain the best response to glucose. The potential of the working electrode and the pH of the carrier were optimised with a flow-rate of 2.3 cm3 min-1 and a sample volume of 850 mm3. For the optimisation of the working electrode potential, glucose (2.5 mM) was injected for 100 mV applied potential intervals over the range +300 to +1100 mV. Peak heights equivalent to the change in current (AZ, A) were plotted against potential [Fig. 2(a)]. In a similar fashion, the effect of pH on glucose response was investigated.The pH of the carrier and glucose standard (1.0 mM) was adjusted over the range 5.0-8.5 with sodium hydroxide [Fig. 2(b)]. With the potential and the pH optimised, the effect of the flow-rate of the carrier stream over the range 0-5 cm3 min-1 was determined for sample volumes of 500 and 850 mm3 (Fig. All further work used a potential of +600 mV relative to the Ag - AgCl reference electrode, a carrier stream and standard pH of 7.0, a flow-rate of 2.3 cm3 min-1 and a sample volume of 5UO mm3. 3). Electrode Calibration Prior to calibration of the enzyme electrode for glucose, the associated platinum electrode was calibrated for hydrogen peroxide. The hydrogen peroxide standardised with potas- sium permanganate solution (20 mM) was serially diluted with phosphate buffer (100 mM, pH 7.0) from an aqueous 100-volume solution.Standards were injected in duplicate and Fig. 4(a) shows a typical chart recorder output and Fig. 4(b) the corresponding calibration graph; response times to the peak and wash times are shown in parentheses. The calibration procedure was similar for the enzyme electrode. Fig. 5(a), showing the recorder output for the duplicate injection of glucose standards, and Fig. 5(b), the calibration graph, illustrate the response similarities between the peroxide - platinum and the glucose - enzyme electrodes. Repeated sampling of two glucose standards (2.5 and 5.0 mM) yielded mean currents of 116 and 192 nA, respectively, the corresponding standard deviations being 1.5 and 5.5 nA (n = 10).601 ,/ ‘I I I 1 5 6 7 8 9 I $ , 300 500 700 900 1100 Electrode potentiah\/ vs. Ag - AgCl Phosphate buffer (100 mM) pH Fig. 2. Optimisation of (a) working electrode otential (2.5 mM glucose) and (b) pH of the enzymatic reaction f1.0 mM glucose). Sample size, 850 mm3; and flow-rate, 2.3 cm3 min-IANALYST, JUNE 1986, VOL. 111 607 5 mM I20 nA 2 mM 1.5 2.5 3.5 Flow-rate/cm3 min-' Fig. 3. mm3; and B, 500 mm3. 2.5 mM glucose Optimisation of flow-rate for two sample volumes: A, 850 A 2.5 I T 300nA 200nA - Scan 1 4 3 1 I 1 -2 -1 0 Log([hydrogen peroxidelim~) Fig. 4. ( a ) Typical recorder out ut and (b) calibration gra h for hydrogen peroxide detection by tge platinum electrode. In (8) the numbers above the line indicate the response times and those below the line indicate wash times I ' I 0.5 mM 0.1 mM I 0.05 mM mM w 5 min , - Scan -2 -1 0 Log([glucosel/rn~) Fig.5. (a) Typical recorder output and (6) glucose calibration with the enzyme electrode. In (b) the numbers above the line indicate response times and those below the line indicate wash times. Determination of Glucose in Foodstuffs The selectivity of the enzyme electrode relative to seven common sugars was investigated. Each of the sugars was injected at the same concentration as the glucose standard (10 mM) and at ten times this ccrncentration (i. e., 100 mM for the Table 1. Effect of individual sugars on the glucose electrode Response normalisation value relative to glucose for the various sugars Interfering sugar lop2 M lo-' M Galactose . . .. . . 0.05 0.08 Maltose . . . . . . 0.10 0.20 Arabinose . . . . . . 0.04 0.06 Fructose . . . . . . 0.01 0.04 Sucrose . . . . . . 0.01 0.05 Lactose . . . . . . 0.05 0.12 Saccharin . . . . . . 0.02* 0. 15a * Commercially available sweetener (0.1 and 1% mlV) was used.608 ANALYST, JUNE 1986, VOL. 111 3.65 , 9.25 9.15 22.12 21.90 21.5 21.1 Table 2. Determination of glucose in foodstuffs by three different methods 9.2 22.0 21.3 Analytical data proposed by the Laboratory of the Government Chemist Sample Strawberry ice cream . . . . Vanillaicecream . . . . . . GlucosesyrupI . . . . . . Glucosepowder . . . . . . Horlicks . . . . . . . . Molasses . . . . . , . . Unrefinedglucosesyrup . . . . Glucoseinflour . . . . . . Sample pre-treatment Treat with Carrez solutions I and I1 Treat with Carrez solutions I and I1 Dissolve in phosphate buffer, gentle warming (35 "C) Dissolve in phosphate buffer, gentle warming (35 "C) Treat with Carrez solutions I and I1 Treat with Carrez solutions I and I1 Dissolve in phosphate buffer, gentle warming (35 "C) Treat with Carrez solutions I and I1 Method of analysis Soluble enzyme kit Soluble enzyme kit YSI glucose analyser Soluble enzyme kit Soluble enzyme kit Soluble enzyme kit YSI glucose analyser Soluble enzyme kit Glucose, YO by mass 4.4 4.7 14.8 88.0 4.8 9.2 21.4 22.0 interfering sugar).All standards and sugars investigated were prepared in phosphate buffer (100 mM, pH 7.0). Each peak height was normalised relative to the glucose signal (Table 1). Glucose in foodstuffs was determined with the enzyme electrode and the results obtained were compared with data obtained for other methods (Table 2).For the simplest foodstuffs, sample pre-treatment involved the leaching of glucose into phosphate buffer by gentle warming (35 "C). Protein-containing samples were treated with 5 cm3 of potassium hexacyanoferrate(II1) solution (80 mM) and 5 cm3 of zinc sulphate solution (250 mM) (Carrez solutions I and 11, respectively) in phosphate buffer and diluted to 100 cm3. After thorough shaking, the filtered solution was analysed for glucose content (Table 2). Discussion Optimisation of Glucose Analysis When optimising the potential of a platinum electrode for the anodic decomposition of hydrogen peroxide, the effect of pH must be considered. As the pH of the solution is decreased, the current - voltage oxidation wave becomes more anodicI4; this behaviour can be considered from the reaction scheme for the oxidation of peroxide: H202+02+2H+ +2e- .. . . (1) When applied to enzyme systems, this pH dependence is important for the optimum pH [Fig. 2(b)]. The potential producing the maximum glucose response was determined and the greatest response to hydrogen peroxide was attained with the electrode poised between +600 and +700 mV (vs. Ag - AgCl) [Fig. 2(a)]. Hence the working electrode potential chosen was +600 mV. With regard to the effect of pH on the electrode responses studied here from pH 5.0 to 9.0 [Fig. 2(b)], it can be seen that the optimum pH found lies between 6.8 and 7.2. Other workerslSJ6 who studied the pH dependence of solubilised glucose oxidase reactions found a broad range of pH 4-7 with a maximum around pH 5.5 The extent of such pH shifts are very much dependent on the immobilisation method.As mentioned earlier, pH 7.0 was adopted for the carrier and sample streams in this work. With regard to flow-rate (Fig. 3 ) , the plateau region is unexpected because, as the pumping rate is increased, the residence time of the substrate in contact with the electrode is reduced and, hence, a smaller peak height might be expected. This behaviour is observed between 0 and ca. 1.5 cm3 min-1, but between 1.5 and ca. 3.0 cm3 min-1 there is little change in the peak height (Fig. 3). At flow-rates greater than ca. 3.0 cm3 min-l, the peak height-determining factor again seems to be the residence time of the substrate.Initial problems with reproducibility were overcome by choosing a flow-rate in the plateau region (Fig. 3). At the adopted flow-rate of 2.3 cm3 min-1, fluctuations in the pump speed of up to k0.2 cm3 min-1 had a negligible effect on the response to glucose. Electrode Lifetime and Selectivity Important parameters when considering immobilised enzymes are the lifetime, durability and storage stability of the system. The excellent mechanical strength of nylon mesh membranes with immobilised enzyme eases handling during electrode fabrication and membranes can be assembled or detached without a decrease in enzyme performance. Membranes stored at 4 "C in buffer respond to glucose for up to 4 months. In order to determine membrane lifetime with respect to substrate, glucose (2.5 mM) was continuously pumped (2.3 cm3 min-1) over the immobilised glucose oxidase membrane electrode.At 2-h intervals, the electrode was washed and calibrated in order to measure the electrode capacity and robustness. The studies showed that membranes may with- stand 24 glucose hours before the onset of loss of enzyme activity, further demonstrating the ruggedness of this immobi- lised enzyme system. Selectivity studies of the glucose electrode relative to seven common sugars investigated (Table 1) produced a relatively small interfering signal, even when the interferent was ten times the glucose standard concentration. Determination of Glucose in Foodstuffs For the determination of glucose in foodstuffs, the electrode was pre-calibrated for glucose.Linearity was obtained over the range 0.01-3 mM [Fig. 5(b)], which was slightly greater than the range for the bare platinum electrode for peroxide standards. However, the peroxide [Fig. 4(a)] and glucose [Fig. 5(a)] response patterns are otherwise similar, although the current generated at the bare platinum electrode [Fig. 4(b)] is ten times greater than that for the enzyme electrode [Fig. 5(b)] for corresponding concentrations. This indicates that only a small fraction (about 10%) of the glucose that can be converted into peroxide is detected at the surface of theANALYST, JUNE 1986, VOL. 111 platinum electrode; hence, the upper limit of linearity for glucose is slightly increased. On this premise, the enzyme electrode might be expected to exhibit linearity up to 10 mM glucose (that is, 1 mM peroxide).However, at concentrations greater than 3 mM glucose the enzyme itself becomes saturated with substrate. Hence, increasing the concentration of glucose produces little change in the peak height at greater than 3 mM glucose. With regard to the analysis of food samples for glucose content with the enzyme electrode, there is good agreement between results obtained by the alternative methods and the glucose enzyme electrode method in this work (Table 2). Each sample was duplicated and the difference in peak heights varied between 0.7 and 2.2% for each sample with a mean variation of 1.75% for the sixteen peaks obtained for the eight samples. Conclusion Glucose oxidase immobilised on nylon mesh and fitted over platinum to form an indicator enzyme electrode in a modified Stelte micro-cell is suitable for the flow injection analysis of glucose.This is facilitated by the thin nylon mesh membrane with immobilised glucose oxidase permitting a fast response by the associated platinum electrode. The robustness and long lifetime of the system have advantages of economy of enzyme and of minimising diffusion effects by having the enzyme- catalysed reaction occurring at the sensing electrode. On-line dilution of samples could be used to increase the linear range in order to analyse clinical samples. The authors thank the Department of Trade and Industry (Laboratory of the Government Chemist) for financial sup- port and for glucose-containing samples and analytical data.Thanks are also extended to Mrs. Geraldine Alliston, Mr. 609 D. G. Porter and Mr. I. Lumley of the Laboratory of the Government Chemist for very helpful and inspiring discus- sions and interest. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. References Clark, L. C., and Lyons, C., Ann. N. Y. Acad. Sci., 1962, 102, 29. Carr, P. W., and Bowers, L. D., “Immobilized Enzymes in Analytical and Clinical Chemistry,” Wiley, New York, 1980. Guilbault, G. G., Ion-Sel. Electrode Rev., 1982, 4, 187. Silman, I. H., and Katchalski, Annu. Rev. Biochem., 1966,35, 873. Weetall, H. H., Methods Enzymol., 1976, 44, 134. Yao, T., Anal. Chim. Acta, 1983, 148, 27. Moody, G. J . , and Thomas, J. D. R., Ion-Sel. Electrode Rev., 1983, 5 , 243. Moody, G. J . , and Thomas, J. D. R., Ion-Sel. Electrode Rev., 1984, 6,209. Masoom, M., and Townshend, A,, Anal. Chim. Acta, 1984, 166, 111. Clark, L. C., and Duggan, C. A,, Diabetes Care, 1982,5,174. 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. L., and Turner, A. P. F., Anal. Chem., 1984, 56, 667. Hornby, W. E., and Morris, D. L., in Weetail, H. H., Editor, “Immobilized Enzymes, Antigens, Antibodies and Peptides,” Marcel Dekker, New York, 1975. Mascini, M., Ianello, M., and Palleschi, G . , Anal. Chim. Acta, 1983, 146, 135. Guilbault, G. G., and Lubrano, G . J., Anal. Chim. Acta, 1973, 64,439. Bright, H. J., and Appleby, M., J. Biol. Chem., 1969, 244, 3625. Weibel, M. K., and Bright, H. J., J. Biol. Chem., 1971, 246, 2734. Paper A51352 Received October 2nd, 1985 Accepted December 2nd, 1985

ISSN:0003-2654    

DOI:10.1039/AN9861100605

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 611 Epoxy-based All-solid-state Poly(Viny1 Chloride) Matrix Membrane Calcium Ion-selective Microelectrodes* Sajedah A. H. Khalil, G. J. Moody and J. D. R. Thomas Department of Applied Chemistry, Redwood Building, UWIST, P.O. Box 73, Cardiff CF7 3XF, UK and Jose L. F. C. Lima Chemistry Department, Faculty of Science, University of Oporto, 4000 Oporto, Portugal Calcium ion-selective microelectrodes have been constructed from PVC matrix membranes containing a calcium bis{di[4-( 1,1,3,3-tetramethylbutyI)phenyl]phosphate} electroactive component with dioctyl phenylphosphonate, tripentyl phosphate or trioctyl phosphate as a plasticising solvent mediator, and from the Philips IS 561/SP membrane. The electrode membranes are backed with a silver-based conductive epoxy implanted with a copper wire for electrical contact.Of the three types of electrodes designed, namely, glass capillaries with a pre-formed membrane (type A) or an applied membrane (type 6) and infrared heat-drawn Perspex capillaries with applied membrane (type C), the most functional are types B and C, which have functional lifetimes of 6 and 10 d, respectively. The electrodes have tip diameters of 1-3 pm for glass and 5-10 pm for Perspex, the bevelled tip in the latter ensuring a sharp edge. Keywords; Ion-selective microelectrodes; calcium ion-selective electrodes; intracellular and intramuscular fluid ions Ion activity measurements with ion-selective microelectrodes (micro-ISEs) are important in many areas of biophysics, physiology and other life sciences.1-4 The availability of liquid ion exchanger and neutral carrier-based liquid membrane types of ion sensors has extended the range of micro-ISEs to include lithium, potassium, calcium and chloride ions. Essen- tially, these are made by placing liquid ion exchangers in a variety of glass micropipettes57 with tip diameters as low as 0.5 vm. Conventional microelectrodes are based on an ion-selective membrane separating the test' solution from the inner refer- ence electrode, which is immersed in an inner reference solution. The small tip geometry offers advantages for the penetration of cell walls not possessed by other microelec- trodes, such as ion-selective field-effect transistors (ISFETs) and coated wires. However, they are fragile and demand patience and care during assembly and measurements.More robust versions with short, or otherwise protected tips, are needed for use in various other situations, i.e., for the measurements of ion activities in muscle fluids. It is with a view to such applications that the microelectrodes described here were developed. The studies are based on PVC mem- brane systems incorporating calcium bis{di[4-( 1,1,3,3- tetramethylbuty1)phenyllphosphate) coupled with an appro- priate plasticising solvent mediator from Orion, dioctyl phenyl- phosphonate (DOPP), tripentyl phosphate (TPP) or trioctyl phosphate (TOP). The sensor membranes heated at the tips of glass or Perspex capillaries are backed with a silver-based conductive epoxy for electrical contact to attain an all-solid- state system.Experimental Reagents and Materials All reagents, except the following, were of the best analytical grade available. Calcium bis{ di[4-( 1,1,3,3-tetramethylbutyl)phenyl]phos- phates and dioctyl phenylphosphonateg were prepared as described previously. * Presented at the 30th IUPAC Congress, Manchester, UK, September, 8-13th, 1985. Glass capillaries (2 mm external and 1.2 mm internal diameter) and Perspex tubes (5 mm external and 3 mm internal diameter) were pulled to tapered tips (of ca. 1 pm diameter) with a commercial micropipette puller at the University Hospital of Wales, Cardiff. These were baked in an oven at 150 "C for 24 h and kept over silica gel. After silanisation with trimethylchlorosilane the tubes were baked at 250 "C for 1 h and stored with the tip upwards in a metal block.Cocktails for the PVC-based sensor membranes consisted of liquid ion exchanger (0.04 g of electroactive component + 0.36 g of plasticising solvent mediator), PVC and tetrahydro- furan in the respective amounts 0.40 g, 0.17 g and 6.00 cm3. Individual Philips IS561/SP calcium ion membranes were dissolved in 1 .O cm3 of tetrahydrofuran to produce the sensor cocktail. Microelectrode Construction Preliminary experiments were made on conventional glass capillary microelectrodes in order to establish the optimum conditions for introducing the PVC-based sensor cocktail into the tapered tip glass capillaries for later back-filling with epoxy to produce all-solid-state electrodes. The vacuum-suction technique (Fig. 1) avoided the air-bubble problem mentioned by Walker,lo and the epoxy-sealed unit gave calcium micro- ISEs with good response characteristics, as illustrated in Fig.2. Three types of all-solid-state calcium micro-ISEs were constructed, namely a glass capillary tip loaded with PVC sensor cocktail followed by back-filling with silver-based conductive epoxy (type A), a glass capillary back-filled with silver-based conductive epoxy followed by application of the PVC sensor cocktail to the tip (type B), and an infrared heat-drawn Perspex capillary back-filled with silver-based conductive epoxy followed by application of the PVC sensor cocktail to the tip (type C); an air-blower heat-drawn Perspex capillary was an unsatisfactory alternative to infrared heat drawing because of threading of the Perspex owing to de-polymerisation. The full constructional details are as described below.612 +40.0 > E E LLi 2 +20.0 0.0 ANALYST, JUNE 1986, VOL.111 ' . Tip diameter -1 pm Reference solution (0.1 M CaCI2) Silanised Pyrex glass ------- - _ - - - _ - ----- ---+ _ _ _ _ _ _ _ _ _ - - - - - - - - ------ Vacuum 4- , Ag - AgCl Epoxy seal Fig. 1. tion of liquid ion-exchanger micro-ISEs Modified procedure to avoid air bubbles during the construc- 1 I I 20 40 60 Time/m in Fig. 2. D namic response of a li uid internal reference calcium micro-ISE Eased on Orion 92-20-02liquid ion exchanger, by spiked additions of 10-1 M calcium chloride to 20 cm3 of calcium chloride solution Type A electrodes The tips of the silanised glass micropipettes were broken under the microscope (Olympus Stereozoom Microscope with Research Instruments Manipulators and Dio Positioners) by gentle rubbing with a piece of tissue paper.The tip was filled by gentle vacuum suction with the appropriate PVC sensor cocktail to a depth of 100-150 pm and left to dry for 48 h. The micropipettes were back-filled with silver-based conductive epoxy using a copper wire (7/0.2 mm strands, RS, low noise cable) to push in the epoxy, the wire being finally left embedded in the epoxy to form an electrical contact. The assembly was then left for 3 d at room temperature to cure the epoxy. The tip diameters of the assembled electrodes (1-3 pm) were measured under the microscope with a calibrated eye-piece graticule. Type B electrodes The silanised glass micropipettes were first back-filled with the silver-based conductive epoxy and the copper wire contacted assembly was oven-cured at 60 "C for 4 h, whilst resting in a horizontal position in order to avoid cracking of the epoxy.The microelectrode tip was polished lightly under the micro- scope with emery paper to give a smooth flat surface, and then lightly coated with the appropriate PVC sensor cocktail. After 30-min intervals, four further PVC sensor cocktail coatings were applied. The resulting micro-ISEs had tip diameters of 1-3 pm. Type C electrodes Perspex tubes (3 mm external diameter) were carefully pulled after heating under an infrared lamp and the constriction cut at an angle with a heated metal blade to give a micropipette with a bevelled tip. The micropipettes were then assembled into micro-ISEs as detailed for type B electrodes.These electrodes had tip diameters of 5-10 pm, but with a sharp edge resulting from the bevelling. Procedures The various electrodes were normally conditioned by immer- sion in 10-1 M calcium chloride solution and calibrated at 25 k 0.1 "C in conjunction with a Corning Model 476002 reference electrode, by spiking aliquots of 10-1 M calcium chloride solution into 20 cm3 of 10-4 M calcium chloride solution. The e.m.f. measurements were made with a Corning EEL Model 112 digital millivoltmeter pH meter, used in conjunction with a Servoscribe Model RE 4541 potentiometric chart recorder. Measurements of interferences to the calcium ion responses from inorganic ions were based on selectivity coefficients , w:,t,~, measured by the separate solution approach for a 10-3 M metal ion concentration: EB - ECa mt>B = 2.303 RTl2F where Eca and EB are the e.m.f.s of calcium and interferent, respectively.Interferences to the calcium ion response from selected proteins and drugs were studied for the e.m.f. changes for 25 cm3 of 10-2 M calcium chloride solutions spiked with succes- sive 0.05-cm3 aliquots of pH 7.4 Tris buffered 0.05% mlV solutions of interferent. The solutions of proteins were aqueous, while 2.5% methanol was used to solubilise the drug-containing solutions. Results and Discussion The general approach to the study of these micro-ISEs is based on the utility of the electrically conductive epoxy for preparing ordinary all-solid-state ISEs, initially with solid sensor coatings11 and later with PVC matrix membrane trapped liquid ion-exchanger coatings.12 This design was tested in early experiments on prototypes of type A and B micro-ISEs, and the results obtained (Table 1) indicated that functional electrodes could be obtained following pre- conditioning in calcium solution before use. Inadequately pre-conditioned electrodes led to super-Nernstian slopes. (See Table 1 data on serial dilution calibration for electrode B, 2.) Table 2 summarises the characteristics of repeated calibra- tions of further typical electrodes of types A, B and C, based on the calcium bis{di-[4-1,1,3,3-tetramethylbutyl)phenyl]- phosphate} electroactive components and a dioctyl phenyl- phosphonate solvent mediator. These confirm that electrodes of good working characteristics can be obtained for all three constructional procedures.Of the two types of glass micro-ISEs, type B is clearly the more satisfactory, as illustrated by the characteristic responses shownfin Fig. 3. The responses of Type A electrodes are frequently erratic [Fig. 3(a)], the sensing membranes of these electrodes being more susceptible to damage during construc- tion. The membranes of Type B electrodes tend to peel off, which limits their functional lifetime to about 6 d. This can be caused by the hydrophilicity of glass, which causes leakage of the test solution around the PVC - glass interface with consequent peeling. There is greater compatibility between the polymer components and Perspexll,l* so that it is not surprising that type C electrodes have a longer lifetime of up to 10 d (Table 2).However, as mentioned above, these electrodes have larger tip diameters (5-10 pm) , although the bevelling gives a sharp edge, allowing their possible use for the study of ionic activities in intramuscular fluids. As the penetration of muscle flesh is to be the first application objective of these electrodes, the appraisal studies here are based on calcium ion levels in the 10-4-10-2 M range. More detailed studies have been focused on type B electrodes, although as constructional difficulties come to be solved, type C electrodes can be expected to be equally suitable in these application areas, with the advantage of longer functional lifetimes. In all of the studies described below, the electrodesANALYST, JUNE 1986, VOL.111 613 Table 1. Responses of prototype all-solid-state calcium micro-ISEs Range Linear E vs. SCE Electrode tested range at 10-3 M Type No. (-log[Ca2+]) (-log[Ca2+]) Slope/mV [CaZ+]lm\i ( a ) Runs calibrated with serially diluted CaCl, solution: A 1 3.85 - 2.1 3.25 - 2.45 33 - 180 3.85 - 2.1 As tested 28 - 155 4.00 - 2.1 As tested 24 -110 4.00 - 2.1 As tested 27 - 24 4.00 - 2.1 As tested 26 - 15 4.00 - 2.1 As tested 24 - 17 4.00 - 2.1 As tested 23 -18 B 1 4.05 - 1.5 As tested 28 - 10 2 4.05 - 1.5 As tested 42 - 192 4.05 - 1.5 As tested 59 - 183 ( b ) Runs calibrated by spiking 20 cm3 of lop4 M CaCI, with 1 M CuCl, solution: B 2 3.75 - 2.1 As tested 30 - 174 2.75 - 1.3 2.40 - 1.3 30 - 186 3.85 - 2.1 3.50 - 2.5 22 - 32 B 3 3.85 - 2.1 3.25 - 2.1 18 - 23 3.85 - 2.1 As tested 31 - 50 3.85 - 2.1 As tested 21 - 17 3.85 - 2.1 As tested 26 - 10 4.00 - 2.1 4.00 - 3.0 33 - 72 Dynamic response time at 10-3 M [Ca,+]/min Stages of electrode treatment and remarks 1 1 1 1 1 1 1 1 1 1 1 - 1 1 1 2 1 1 Dried in an oven for 18 h at 40 "C, left for 2 d in air and calibrated Conditioned for 30 rnin in 0.1 M CaCl, after previous run Conditioned for 1 h in 0.1 M CaC1, after previous run Conditioned for 18 h in 0.1 M CaC1, after previous run Conditioned for 4 h in 0.1 M CaCI, after previous run Conditioned for 45 min in 0.1 M CaCI, after previous run Conditioned for 15 rnin in 0.1 M CaCI, after previous run Air-dried for 15 h.Conditioned in 0.1 M CaCl,. PVC membrane dropped off Air-dried for 15 h. No conditioning Calibrated 15 rnin after previous run Calibrated after conditioning for 3 h in 0.1 M Calibrated immediately after previous run.Calibrated after conditioning for 2 d in 0.1 M Calibrated after air-drying for 2 d Conditioned for 1 h in 0.1 M CaCI, after previous run Conditioned for 18 h in 0.1 M CaC1, after previous run Conditioned for 2 d in 0.1 M CaC1, after previous run Conditioned for 4 d in 0.1 M CaC1, after previous run. Slope falls to near zero at -log aca = 2.1 CaCl, E (10-3 M CaCI,) extrapolated CaCI, +40.0 > +20.0 E E ui 0.0 > 'c: -20.0 10 30 50 10-4 1 10 30 50 10 30 50 Ti me/m i n Fig. 3. Dynamic response to calcium chloride of all-solid-state calcium ISEs calcium bis(di[4-(1,1,3,3-tetramethylbutyI)phenyl]- phosphate} and a DOPP plasticising solvent mediator.Electrodes: ( a ) type A; ( b ) type B; and (c) type C were pre-conditioned by soaking overnight in 10-1 M calcium chloride solution, in which they were subsequently stored. Cation Interferences Fig. 4 summarises the responses to various cations of type B electrodes based on the PVC sensor with DOPP [Fig. 4(a)], TPP [Fig. 4(b)] and TOP [Fig. 4(c)], respectively, as plasticis- ing solvent mediators, and on the sensor based on the Philips IS 561/SP membrane, while k#Yi,B data are summarised in Table 3. The superior selectivity of the Philips membrane with respect to sodium, potassium, magnesium and zinc ions is clearly demonstrated, while the electrodes made from the DOPP-based cocktail [Fig. 4(a)] is at least of equal selectivity with respect to sodium and potassium ions and only marginally less for magnesium ions.However, the DOPP-based elec- trode is far less selective for calcium over zinc ions [Fig. 4(a)] and even less so for the TOP-based electrodes. Zinc interfer- ence has always been a characteristic of the organophosphate- based calcium ISEs,l3 but this does not normally present practical problems in studies on biological fluids. The selectivity coefficient data in Table 3 are calculated forANALYST, JUNE 1986, VOL. 111 614 Table 2. Typical characteristics of calcium micro-ISEs with a DOPP plasticising solvent mediator Slope at 25 "CImV S.d. ( a ) Type A electrode* : 25.3 21.8 25.6 28.4 23.1 22.8 19.4 18.6 ( b ) Type B electrode" 40.9 32.7 28.1 25.4 26.4 26.3 26.3 22.7 22.3 1 .o 0.9 3.5 1.4 0.6 0.6 0.8 0.8 2.4 1.8 2.3 1.1 2.5 1 .o 1.2 0.8 1.1 ( c ) Type C electrode*: 29.1 1.6 31.2 1.4 27.9 1.2 27.6 1.2 27.9 1.4 28.5 0.9 26.4 0.9 28.3 1.2 28.4 1.2 28.7 1.2 30.2 1.3 EdmV +0.8 - 0.2 +8.6 +17.8 +1.4 +30.0 +18.0 +56.9 -25.7 -22.9 -26.2 -32.4 -23.4 -22.4 -1.8 + 10.6 +24.0 108.1 117.1 126.3 125.4 126.2 127.2 120.4 127.9 126.7 126.1 126.0 S.d.3.2 3.2 5.4 4.7 2.0 2.2 2.7 2.6 1.2 3.3 4.5 3.6 4.1 3.5 4.0 2.7 3.6 5.4 4.8 4.0 4.0 4.6 3.1 3.1 4.1 4.0 4.0 4.2 Remarks Conditioned for 24 h in 10-1 M CaCI, solution Conditioned for 1 h in 10-1 M CaCI, solution Conditioned for 2 h in 10-1 M CaCl, solution Conditioned for 3 h in 10-1 M CaCI, solution Conditioned for 4 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaC1, solution Conditioned for 48 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaC1, solution Air dried Conditioned for 24 h in 10-1 M CaCl, solution Conditioned for 1 h in 10-1 M CaC1, solution Conditioned for 2 h in 10-1 M CaCI, solution Conditioned for 3 h in 10-1 M CaC1, solution Conditioned for 4 h in 10-1 M CaC1, solution Conditioned for 48 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaCl, solution Conditioned for 24 h in 10-1 M CaC1, solution Air dried Conditioned for 1 h in 10-1 M CaC1, solution Conditioned for 24 h in 10-1 M CaC12 solution Conditioned for 1 h in 10-1 M CaC1, solution Conditioned for 2 h in 10-1 M CaC1, solution Conditioned for 3 h in 10-1 M CaC1, solution Conditioned for 4 h in 10-1 M CaCI, solution Conditioned for 24 h in 10-1 M CaC1, solution Conditioned for 24 h in 10-1 M CaCI, solution Conditioned for 48 h in 10-1 M CaC1, solution Conditioned for 48 h in 10-1 M CaCl, solution * The lifetimes of the electrodes were 5 , 6 and 10 d for types A, B and C, respectively. Table 3.Calcium micro-ISEs (type B) selectivity coefficients keitB (separate solution method at a cation concentration of 10-3 M) Interferent DOPP TPP TOP Philips Na+ . . . . 0.15 0.18 0.22 0.089 K+ . . . . . . 0.14 0.16 0.18 0.087 Mg2+ . . . . 0.21 0.24 0.39 0.104 Zn2+ . . . . 0.24 0.28 0.87 0.14 (B) membrane membrane membrane membrane Table 4. E.m.f. changes for type B calcium micro-ISEs for proteins added (0.001%) to calcium chloride test solution M) in Tris buffer at pH 7.40 (n = 3) AElmV DOPP electrodes TPP electrodes TOP electrodes Protein added AE S.d.Humanalbumin . . +2.1 0.07 Bovinealbumin . . -1.7 0.10 a-Globulin . . . . -1.9 0.23 fl-Globulin . . . . -0.4 0.10 y-Globulin . . . . -0.5 0.07 A E S.d. +0.2 0.20 +1.8 0.07 +0.1 0.12 -0.2 0.09 -0.9 0.11 A E S.d. -0.4 0.05 -0.6 0.17 -0.2 0.12 -0.3 0.05 -0.3 0.10 a cation concentration of 10-3 M. It is stressed that the actual w;,, data will be different at different cation concentrations, and will become smaller in magnitude for higher cation concentrations. Table 5. E.m.f. changes for type B calcium micro-ISEs for drugs added (0.001%) to calcium chloride test solution (lo-, M) in Tris buffer at pH 7.40 ( n = 3) AElmV DOPP electrodes TPP electrodes TOP electrodes Drug added AE Aminoglutethimide . . +0.7 Glutethimide . . . . -1.9 Dapsone . . . . . . - 1.7 Hydrocortisone .. -0.7 Insulin . . . . , . -0.3 Nitrofurantone. . . . -0.5 Prilocaine hydrochloride . . -3.3 Methanol . . . . . . -1.2 S.d. 0.10 0.07 0.14 0.06 0.20 0.01 0.30 0.01 AE S.d. +1.8 0.21 +5.7 0.14 +1.3 0.08 +1.0 0.13 +1.4 0.22 -2.7 0.32 -0.4 0.10 -0.9 0.07 AE S.d. +1.4 0.10 -1.7 0.08 -0.3 0.14 +1.2 0.21 -3.5 0.07 +0.2 0.17 +1.3 0.20 +0.9 0.10 Protein Interferences The TOP-based electrodes show more tolerance towards the presence of protein than either of the other type B electrodes (Fig. 5 and Table 4). This trend parallels the observations made in another study14 on the effect of biochemical components on conventional calcium ISEs. In this study the added biochemical components presented minimum interfer- ence to electrodes with an organophosphate electroactive component with a TOP solvent mediator.ANALYST, JUNE 1986, VOL. 113 61 5 50.0 c /I / Fig.4. Res onse of all-solid-state calcium micro-ISEs to various metal cations by spiking M metal chloride solutions with 10-l M metal chloriie solution. (a), ( b ) and (c) type B electrodes based on calcium bis{di 4-(1,1,3,3-tetramethylbutyl)phenyl]phosphate} and (a) DOPP, (b) TPP and (c) TOP plasticising solvent mediators; and ( d ) type B electrode fi ased on Philips IS 561/SP membrane Human albumin and bovine albumin are exceptional in giving an increase in the ISE response for the DOPP-based electrodes and the DOPP- and TPP-based electrodes [Fig. whilst impaled will be in continuous contact with the fluid under study. I . " 5(a) and (b)], respectively.Such interferences are always undesirable and can be of even greater significance in Drug Interferences intracellular fluids where protein levcls are relitively higher than in extracellular fluids. The protein interference manifests itself as a coating on the electrode tips. This can be minimised by having samples in flowing streams, but this facility is not available for micro-ISEs in intracellular and intramuscular studies. Nevertheless, towards this ideal, a single electrode Contrary to studies on the effects of other added biochemical components, in which the TOP-based electrodes showed the minimum of interference, it is the DOPP-based electrodes that generally show the least interference from added drug components (Fig. 6 and Table 5). However, the TOP electrodes showed less interference from added dapsone and616 ANALYST, JUNE 1986, VOL.111 +2.0 0.0 -2.0 +2.0 > E $ 0.0 -2.0 +2.0 I I I (b) -2.0 1 I I 1 2 6 10 [ I n t e rfe ren t 1/10 -4 O/O Fig. 5. Effect of added protein components on type B elec- trodes. Calcium bis{di[4-(1,1,3,3-tetramethylbutyl)phenyl]phos- phate} based electrodes with ( a ) DOPP, (b) TPP and (c) TOP as plasticising solvent mediators. Samples: 0, human albumin; 0, bovine albumin; A, cu-globulin; A , P-globulin; and W, y-globulin glutethimide, and differences in sign of some of the deviations. In many instances the drugs give rise to positive deviations of e.m.f., which can contribute to reducing the negative devi- ations observed for most of the protein component studies above and also for biochemical components.14 Conclusion Clearly, all-solid-state micro-ISEs based on PVC matrix membrane sensors for calcium ions and backed by a silver- based conductive epoxy are functional systems.Their selectiv- ity features resemble conventional calcium micro-ISEs. The tip diameters of 1 pm, characteristic of the electrodes studied here, were sufficiently large to permit e.m.f. measurements with a conventional millivoltmeter. This gives encouragement to applications of the devices for studies on intramuscular fluids, the choice of electrode being between types B and C. Within these types, the effect of interferences must balance selection between those based on dioctyl phenylphosphonate 0. -5A O.( > E a -5.a 0.0 -5.0 e ‘C) 2 6 10 [Interferent]/10-4°/~ Fig.6. Effect of added drug type components on type B electrodes. Calcium bis{ di[4-( 1,1,3,3-tetramethylbutyl)phenyl]phosphate} based electrodes with (a) DOPP, ( b ) TPP and ( c ) TOP as plasticising solvent mediators. Samples: 0, aminoglutethirnide; 0, dapsone; A, glutethi- rnide; A , hydrocortisone; W , insulin; V, methanol; V, nitrofuran- tone; and trioctyl phosphate plasticising solvent mediators with a calcium bis{ di-[4-( 1,1,3,3-tetramethylbutyl)phenyl]phos- phate} electroactive component and those of the neutral carrier system of the Philips IS 561/SP membrane. The authors thank the Foundation of Technical Institutes, Baghdad, Iraq for paid leave of absence and a studentship (granted to S. A. H. K.) and the North Atlantic Treaty Organisation for a travel grant (069/84).Professor A. A. S. C. Machado of the University of Oporto is thanked for helpful discussions and suggestions. References 1. LavallCe, M., Schanne, 0. F., and Herbert, N. C., Editors, “Glass Microelectrodes,” Wiley, New York, 1969. 2. Walker, J. L., and Brown, H. M., Physiol. Rev., 1977,57,729. 3 . Thomas, R. C., “Ion-selective Intracellular Microelectrodes,” Academic Press, New York, 1978. 4. Brown, H. M., and Owen, J . D., Ion-Sel. Electrode Rev., 1979, 1, 145. 5. Brown, H. M., Pemberton, J. P., and Owen, J. D., Anal. Chim. Acta, 1976, 85, 261.ANALYST, JUNE 1986, VOL. 111 617 6. Ammann, D., Morf, W. E., Anker, P., Meier, P. C., Pretsch, E., and Simon, W., Ion-Sel. Electrode Rev., 1983,5, 3. 7. Ujec, E., Keller, E. E. O., Kriz, N., Pavlik, V., and Machek, J . , Bioelectrochem. Bioenergetics, 1980, 7 , 363. 8. Craggs, A., Delduca, P. G., Keil, L., Key, B. J., Moody, G. J., and Thomas, J. D. R., J. Inorg. Nucl. Chem., 1978,40, 1483. 9. Craggs, A., Delduca, P. G., Keil, L., Moody, G. J., and Thomas, J. D. R., J. Inorg. Nucl. Chem., 1978,40, 1943. 10. Walker, J. L., Anal. Chem., 1971, 43, 89A. 11. Lima, J. L. C . , and Machado, A. A. S. C., in Albaiges, J., Editor, “Analytical Techniques in Environmental Chemistry, 2. Proceedings of the Second International Congress, Bar- celona, Spain, November, 1981 ,” Pergarnon Press, Oxford, p. 419. 12. Alegret, S., Alonso, J., Bartroli, J., Panlis, J. M., Lima, J. L. F. C., and Machado, A. A. S. C.,Anul. Chim. Actu, 1984, 164, 147. Moody, G. J., Oke, R. B., and Thomas, J. D. R., Analyst, 1970, 95, 910. Khalil, S. A. H., Moody, G. J., and Thomas, J. D. R., Analyst, 1985, 110,353. 13. 14. Paper A51359 Received October 9th, 1985 Accepted November 27th, 1985

ISSN:0003-2654    

DOI:10.1039/AN9861100611

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 619 Anodic-stripping Voltammetry of Metal Complexes in Non-aqueous Media After Extraction: Determination of Copper with Salicylaldoxime Jose Aznarez, Juan Carlos Vidal and Jose Maria Rabadan Department of Analytical Chemistry, University of Zaragoza, 50009 Zaragoza, Spain The determination of copper in biological materials and ores was carried out by anodic-stripping voltammetry in non-aqueous media after its extraction with salicylaldoxime into 1,2-dichloroethane and addition to an aliquot of the extract of 5% VNperchloric acid solution in N,N-dimethylformamide as supporting electrolyte. Different instrumental parameters were studied. The sensitivity and selectivity were considerably improved. The precision was 2.9% ( n = 8) for the determination of 2 pg ml-I of copper and the detection limit was 15 ng ml-1 of Cu(ll). The method can be applied to the determination of other elements (Sb, Bi, Cd, Sn and Pb) atthe microgram level and even the simultaneous determination of several elements after their extraction with organic reagents (8-hydroxyquinoline, benzoylphenylhydroxamic acid, sodium diethyldithiocarbamate) (Pb + Cu, Pb + Bi, Pb + Cd, Pb + Cd + Bi and Pb + Cd + Cu).Keywords: Copper determination; anodic-stripping voltammetry; extraction; polarograph y; non-aqueous media Polarography of organic substances in non-aqueous media has been used extensively and is still widely applied. In contrast, inorganic systems have hardly been studied in organic phases, except for metal complexes extracted with organic reagents.1 A preliminary separation by solvent extraction provides good selectivity for many instrumental methods of analysis that tend to suffer from matrix effects or interferences from different elements.This is particularly true of anodic-stripping voltammetry (ASV), where high concentrations of other ions, the formation of intermetallic compounds or the proximity of the stripping peaks prevent the application of this sensitive technique. Several extraction - voltammetric methods for cation analysis have been reported. Polarographic or ASV determi- nations have been made after the dilution of the extract with a polar solvent such as methanol, ethanol or acetic acid [with LiC1, NH4SCN, NaBr or tributylammonium perchlorate (TBAP) as supporting electrolyte] in order to enhance the conductivitity of the solution.Fano et aZ.2 carried out the polarographic determination of Pb, Cd and Zn, extracted into benzene or chloroform as dithizonates, by releasing the respective cation by addition of AgN03 solution in methanol. Co(II), Ni, Cd, Zn and As(II1) do not interfere in the determination of Pb, Cd and Zn as these cations are not extracted into the organic phase as dithizonates. Antimony can be extracted into toluene with tributyl phosphate (TBP) from 2 M HCl solution.3 The determination of Sb was carried out by ASV in mixtures of extracted phase with methanol that was 0.5 M in LiCl, with a detection limit of 2 X 10-8 M of Sb. Bismuth at concentrations down to 10-8 M can be determined by ASV in an organic phase (chloroform - methanol - 0.25 M NH4SCN) after extraction into chloroform with KI and methylene green.The extraction eliminates interferences from Sb, Sn and Cu in the determination of Bi.4 Pb and Sn give close peaks in ASV. However, Sn(IV) can be extracted into benzene from 0.5 M NaI - 1.5 M HC104 - 3 M NaC104 solution.5 The determination of Sn was carried out by ASV on an extract - methanol (containing 0.33 M NaBr) mixture. Pb extracted into chloroform with dithizone can be released by the addition of mercury(I1) chloride and deter- mined by ASV.6 In the determination of Cd by ASV in aqueous solution, Pb, Ni and Zn interfere. By extraction of Cd into acetonitrile [by the salting out effect with (NH4)*S04] as the iodide association complex with TBA, these interferences are eliminated.The detection limit is about 0.2 pg ml-1 of Cd in the extract.7 Molybdenum has been determined by differential-pulse polarography (DPP) after its separation by 8-hydroxyquino- line extraction into dichloromethane with 0.1 M TBAP as supporting electrolyte.8 In this work, the determination of copper was carried out by polarography and ASV after its extraction into 1,2- dichloroethane with different organic reagents. By the addi- tion of 5% V/V HC104 solution in N,N-dimethylformamide (DMF) to the extract, copper is liberated in the form of Cu2+ solvated with DMF and partially as undissociated copper perchlorate. This is due to the high solvation power and dielectric constant (36.5) of DMF. Perchloric acid in DMF also supplies sufficient electrical conductivity to organic media as a supporting electrolyte.The proposed method can be applied to the determination of other elements such as Sb, Bi, Cd, Sn and Pb, and even the simultaneous determination of several elements (such as Pb + Cu, Pb + Bi, Pb + Cd, Pb + Cd + Bi and Pb + Cd + Cu) by ASV. The method is sensitive and very selective and it has been applied to the determination of copper at the parts per million level in biological samples and ores. Experimental Apparatus Direct current polarographic and ASV measurements were made with an LKB Type 3266 Blomgren Polarolyzer, equipped with a Linear Instruments Model 252-A X - Y recorder. A dropping-mercury electrode (DME), a hanging mercury drop electrode (HMDE) (Kemula electrode), a silver - silver chloride (0.1 M KC1 in ethanol) electrode or mercury pool as reference electrodes and a platinum electrode as counter electrode were used to obtain current - potential curves.Dissolved oxygen was removed by bubbling oxygen-free nitrogen, previously saturated with solvent or solvent mixture for 10 min. The nitrogen stream was then directed over the solution surface. In polarographic determinations, the following parameters were used: DME characteristics, rTz = 2.19 mg s-1; t = 3.79 s with open circuit; scan rate, 0.2 V min-1; and sensitivity, 20 pA (full-scale).620 ANALYST, JUNE 1986, VOL. 111 In ASV determinations the instrumental conditions were as follows: sensitivity, 10 yA (full-scale); electrolysis potential (Eelect), -0.70 V; electrolysis time (telect), 6 min; rest time, 30 s; and scan rate, 0.4 V min-1. An Orion Research microprocessor was used for pH measurements with glass - calomel electrodes in the aqueous phase after extraction.Other apparatus consisted of a Haake thermostatic bath (25 "C), a Commercial Instruments Cedar Grove instrument for electrolytic conductivity measurements and Gallenkamp apparatus for mercury distillation. Reagents Analytical-reagent grade reagents were used unless stated otherwise. Doubly distilled water was used in all measure- ments. Copper stock standard solution, 1000 pg ml-1. Prepared by dissolving electrolytic copper (Merck) in HN03 (1 + l), adding 5 ml of HzS04, warming to evolution of white fumes and diluting to 1000 ml in a calibrated flask. Copper working standard solution, 10 pg ml-1.Prepared at moment of use by diluting 10 ml of the above stock standard solution to 1 1. Tartaric acid solution, 40% mlV. Perchloric acid, 70% mlV. Merck. Caution-Perchloric acid is dangerous and appropriate precautions should be taken. However, the 5% solution in DMF used in this procedure is not hazardous. N,N-Dimethylformamide (DMF). This was distilled with anhydrous sodium hydrogen carbonate, collecting the fraction between 148 and 152 "C. Bufler solution (PH 4.80). Acetic acid - 0.1 M sodium acetate . Ascorbic acid solution, 10% m1V. 1,2-Dichloroethane. Merck. Supporting electrolyte solution. A 5% VIV solution of Extraction solution. A 0.2% mlVsolution of salicylaldoxime Standard metal solutions, 1000 pg ml-1. Solutions of diverse perchloric acid in DMF.(Merck) in 1 ,Zdichloroethane. ions were used for interference studies. Procedure Sample treatment Transfer an ore sample (serpentinite type) weighing 0.2-0.5 g to a 100-ml PTFE beaker. Add 3 ml of concentrated hydrochloric acid and then 3 ml of concentrated nitric acid. Warm carefully by boiling on a hot-plate for 5 min, then add 2 ml of concentrated hydrofluoric acid (48% mlV). Evaporate the solution almost to dryness. Repeat the procedure once more. Dissolve the residue in 10 ml of 4 M nitric acid by warming and dilute to 50 ml with water in a calibrated flask. Take a biological sample weighing up to 2 g in a 250-ml beaker and decompose it with 10 ml of concentrated nitric acid on a hot-plate. Evaporate almost to dryness and repeat the procedure once more.Cool and add 10 ml of perchloric acid and heat gently at 170 "C for 15 min. Dilute to 50 ml with water in a calibrated flask. Determination Take a known volume of the sample solution (about 10 ml) in a 100-ml separating funnel, add 2 ml of ascorbic acid solution and adjust the pH by the addition of 10 ml of buffer solution (pH 4.80). Extract with 10 ml of extraction solution, shaking mechan- ically for 5 min. Allow the phases to separate. Place 9 ml of extracted organic layer in the polarographic cell and add 6 ml of perchloric acid solution in DMF. Remove the dissolved oxygen by bubbling through with oxygen-free nitrogen for 10 min. Record the polarogram or the anodic-stripping voltam- mogram at 25 "C under the specified conditions. Results and Discussion D.c.Polarographic Behaviour of Metal Complexes Preliminary polarographic studies were made for Zn( 11) and Cu(I1) complexes with trioctylamine (TOA) , a-benzoin oxime and salicylaldoxime extracted into toluene by the addition to the organic phase of a 0.15 M solution of tetrabutylammonium bromide (TBAB) in DMF as the supporting electrolyte. Table 1 lists the reagents, pH (or acidity) of extraction of the aqueous phase and half-wave potentials obtained under the experimental conditions. In the range of electroactivity available for the toluene - DMF mixture (up to -2.9 V vs. Ag - AgCl electrode) using TBAB as the supporting electrolyte, the extracted metal complexes examined gave d.c. polarograms but at too negative potentials, probably owing to the high formation constants of the complexes.The solvent used for extraction was toluene and the supporting electrolyte was a 0.15 M solution of TBAB in DMF. Generally the d.c. polarographic waves of the metal complexes appeared at fairly negative potentials, near to the discharge of the solvent or supporting electrolyte. They were often badly defined and presented a polarographic maximum and high residual currents. Additionally, the method lost selectivity because the waves of the different metal complexes accumulated in a narrow range of negative potentials. ASV df- terminations cannot be carried out under these conditions. However, the addition of a 5% VIV solution of perchloric acid in DMF to the metal complex extract releases the solvated cation of the metal complex and increases the electrical conductivity of the solutions used as the supporting electrolyte.Polarographic studies were made on 11 metal complexes with 8-hydroxyquinoline7 dithizone , diethyl dithiocarbamate (DDTC), pyrrolidine dithiocarbamate (PDTC) , benzoyl- phenylhydroxamic acid (BPHA) , tributyl phosphate (TBP) , trioctylphosphine oxide (TOPO) , cupferron, a-benzoin oxime and salicylaldoxime extracted into toluene , chloroform and 172-dichloroethane, with the addition of a solution of perchloric acid in DMF as supporting electrolyte. The pH values for these extractions have been given e1sewhere.g-11 Table 2 lists the reagents, pH (or acidity) of extraction, the solvents used for extraction and the half-wave potentials obtained under the experimental conditions. In all instances , the supporting electrolyte used was perchloric acid at a concentration in the final solution of 2% V/V, prepared by the addition to 9 ml of extract of 6 ml of 5% VIVsolution of HC104 in DMF.In the range of electroactivity available for the solvents used (up to -0.95 V vs. Ag - AgCl electrode for CHC13 and 1,2-dichloroethane - DMF or up to -1.30 V for toluene - DMF), most of the metal complexes examined gave d.c. polarograms after extraction. The half-wave potentials were nearly independent of the reagent used for extraction owing to the rupture of the Table 1. Half-wave potentials for Cu and Zn complexes with TBAB as supporting electrolyte Acidity of Cation Reagent aqueous phase EJV Cu . . . . . .TOA 1 . 0 ~ H C l -1.90 Salicylaldoxime pH 4.80 -1.60 a-Benzoin oxime pH 11.80 -2.70* Zn .. . . . .TOA 0.8 M HC1 - 1.65 * Badly defined.ANALYST, JUNE 1986, VOL. 111 621 Table 2. Polarographic data for various metal complexes Cation Reagent As(II1) . . . DDTC Bi . . . . . . 8-Hydroxyquinoline BPHA DDTC Dithizone DDTC BPHA PDTC DDTC Dithizone Cupferron Cd . . . . . . 8-Hydroxyquinoline Cu(I1) . . . . 8-Hydroxyquinoline Salicylaldoxime a-Benzoin oxime Mo(V1) . . . . 8-Hydroxyquinoline BPHA a-Benzoin oxime Ni . . . . . . BPHA Pb . . . . . . 8-Hydroxyquinoline DDTC Dithizone BPHA Sb(II1) . . . . BPHA DDTC Sn(IV) . . . . BPHA Dithizone U(V1) . . . . 8-Hydroxyquinoline BPHA TBP TOP0 Zn . . . . . . BPHA Dithizone * Polarographic maximum appears. t 1,2-DET = 1,2-dichIoroethane. pH or acidity Solvent 5 3.5 4 10 3 6.2 8.9 8.9 7 7 7 3 2 4.8 11.8 2 1 1 M HCI 9.3 8 3 8 8.9 1 M HCI 8.9 1 M HCl 8 8 3.5 0.1 M HN03 Toluene CHC13 CHC13 Toluene Toluene Toluene Toluene Toluene CHC13 CHC13 CHCI3 CHC13 CHC13 CHCl3, toluene or 1,2-DET*t CHCl3, toluene or 1,2-DET CHC13 CHC13 Toluene Toluene Toluene Toluene CHC13, toluene Toluene Toluene Toluene Toluene Toluene Toluene CHC13 CHC13 0.15 M HN03 Toluene 8.9 Toluene 4.5 Toluene EdV -0.15, -0.85* -0.15 -0.12 -0.20 No wave appears -0.95 -0.85 -0.90 -0.10, -0.32 1 Polarographic } curves badly J defined Reduction of reagent (ca.-0.1 V) -0.10, -0.30 -0.10, -0.30 -0.82, -0.43 -0.53, -0.95 -0.50, -0.95 -0.75 -0.65* No wave appears -0.65 -0.10 -0.30* -0.55 -0.25, -0.70 -0.20, -0.65 E,- -0.38, -0.80 No wave appears No wave appears 1 Badly defined waves No wave appears No wave appears metal complex by perchloric acid.The polarographic waves corresponded to slow or irreversible electrode processes and the limiting current was controlled by diffussion (id hc-& = constant). Two polarographic waves with very different diffusion currents appeared for Cu(II) (E+ -0.10 V and -0.30 V) owing to the presence of solvated Cu2+(DMF) and undissociated copper perchlorate in equilibrium (Fig. 1). In order to check this assertion, the influence of perchlorate concentration on the limiting currents (id1 and id2) was studied by the addition of lithium perchlorate to perchloric acid solution in DMF, as shown in Table 3. For the first wave the limiting current must be id1 = K1 [c~2+]solv. and for the second wave id2 = K2 [cu2+ (C104-)2]solv.but the dissociation constant for copper perchlorate should be [C~*+IsOiv. [C104-12 D2+ P O 4 -)2lsolv. Kd = Therefore, Log (f!d2/idl) = 2 log [CIO4-] -I- K The data in Table 3 yield a linear regression equation { Y = A + SX, where Y = log (id&&) and X = log [C104-1) with B = 2.24, A = -0.6460 and the correlation coefficient R = 0.993. Another confirmation of this situation was obtained by using the solubility in DMF of some copper salts, such as the perchlorate, chloride, sulphate and nitrate. The polarographic waves of copper perchlorate with HC104 in DMF were the Table 3. Influence of perchlorate concentration on the limiting currents of Cu [Clo,-]/M Log [c104-] idl/pA id2/@ Log (&/idl) 0.645 -0.1904 2.085 0.182 0.087 -1.0605 0.647 -0.1891 2.102 0.167 0.079 -1.1024 0.710 -0.1487 2.260 0.235 0.104 -0.9830 0.740 -0.1308 2.135 0.260 0.122 -0.9136 0.810 -0.0915 2.080 0.305 0.147 -0.8327 0.870 -0.0605 2.075 0.325 0.157 -0.8041 0.980 -0.0088 1.975 0.450 0.228 -0.6421 1.090 +0.0374 1.930 0.505 0.262 -0.5817 1 I 1 I 1 0 -0.10 -0.30 -0.90 Potent ia IN Fig.1. Polarographic waves of Cu(I1) after its extraction with salicylaldoxime into 1,2-dichloroethane, with addition to the extract of 5% V/V HCIO, - DMF solution. 1, Blank (without couper); 2,18 pg ml-l of Cu (idl 1.71 pA and id2 0.14 PA); and 3,21 pg ml-1 of Cu (idl 1.96 pA and id2 0.16 pA)622 ANALYST, JUNE 1986, VOL. 111 Table 4. Extraction and ASV data for various metal complexes Cation Extraction reagent Extraction solvent As(II1) . . .. . . Bi(II1) . . . . . . Cd . . , . . . c u . . . . . Mo(V1). . . . . . Sb(II1) . . . . . . Sn(IV) . . . . . . Pb . . . . . . DDTC 8-Hydrox yquinoline DDTC BPHA DDTC BPHA 8-Hydrox yquinoline Salicylaldoxime a-Benzoin oxime DDTC 8-Hydrox yquinoline PDTC BPHA Dithizone wBenzoin oxime BPHA BPHA 8-Hydroxyquinoline DDTC BPHA Toluene Toluene Toluene Toluene Toluene Toluene Toluene CHC13 or 1,2-DET CHC13 or 1,2-DET Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Eelect./V -0.75 -0.75 -0.75 -0.75 -1.20 -1.20 - 1.20 -0.70 -0.70 -0.70 -0.70 -0.70 -0.70 -0.70 -1.15 -0.70 -1.00 -0.90 -0.90 -0.90 E,IV No peak -0.26 -0.24 -0.30 -0.75 -0.80 -0.80 -0.08 -0.08 -0.08 -0.12 Badly defined -0.11 Badly defined No peak -0.28 -0.54 -0.55 -0.46 -0.58 Table 5.Extraction and ASV data for simultaneous determination of cations Cations Reagent pH or acidity Solvent Pb, Cu(I1) . . . . . . 8-Hydroxyquinoline Sn(IV),Sb(III) . . . . BPHA Pb, Sb(II1) . . . . . . BPHA Sn(IV),Cu(II) . . . . 8-Hydroxyquinoline Pb, Bi . . . . . . 8-Hydroxyquinoline Pb,Cd . . . . . . BPHA DDTC Pb,Sn(IV),Cu . . . . 8-Hydroxyquinoline Pb,Cd,Bi(III) . . . . DDTC Pb,Cd,Cu(II) . . . . DDTC * Badly defined peaks. 8 1 MHCI 1 5 6.5 8.9 8.9 6.15 8.9 9.3 Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene Toluene E,, V -0.60, -0.16 -0.55, -0.25* -0.61,* -0.11* -0.60, -0.22 -0.66, -0.84 -0.64, -0.90 -0.66, -0.84, -0.30 -0.60, -0.77, -0.10 * - * - same as those obtained by extraction. The formation of complexes or undissociated salts with chloride, sulphate or nitrate was also evident from the shift in the half-wave potential at more negative values.The effect of the water content of DMF on the polaro- graphic curves was studied. Water at levels up to 10% V/Vin DMF did not influence appreciably either the polarographic wave or the limiting currents. Solvents for Extraction Toluene (E = 2.4; b.p. 111 "C) has been used as an extraction solvent because a toluene - DMF - HC104 mixture gives a wider range of electroactivity (up to - 1.3 V) than chloroform (E = 4.8; b.p. 61.3 "C) or 1,2-dichloroethane (E = 10.2; b.p. 83.6 "C) (about -0.95 V in the presence of HC104 solution in DMF). However, copper complexes were better extracted into chloroform owing to their greater solubility in this solvent. Finally, 1,2-dichloroethane was preferred because the volatility of chloroform led to a loss of solvent during the elimination of oxygen by bubbling nitrogen, in spite of its previous saturation.The solubility of metal complexes in 1,2-dichloroethane was similar to that in chloroform and there were no difficulties in the extraction process. Anodic-stripping Voltammetry in Non-aqueous Solution From the polarographic waves obtained in the above proce- dure, it was possible to design an ASV method for the determination of copper and other ions by using a hanging mercury drop electrode (HMDE). The electrolytic potential applied in the first stage was 0.3 V more negative than the corresponding half-wave potential. However, this electrolytic Table 6. Effect of Cu concentration and teleCt.on the AS\' peak Cu(I1) concentration/ pg ml-1 telect.Jmin ASV peak i,lpA 0.405 10 1.17 1.220 6 2.31 4.560 4 5.94 6.250 2 4.04 Table 7. Tolerance limits in the determination of 40 pg of Cu(I1) Tolerance Element limithg Ion : Cu ratio (mlm) Mn(II), Al, Mg, Pb, Ca . . . . 40 1000 Zn,Sn(IV). . . . . . . . . . 30 750 Fe(III), F- . . . . . . . . . . 20 500 Ni,Cd,Co . . . . . . . . . . 10 250 V ( V ) . . . . . . , . . . . . 6 150 Bi(III), Mo(VI), Ti(IV), Sr, Ba . . 4 100 potential was verified as giving the maximum ASV peak height. The electrolysis time used was from 1 to 15 min, depending on the concentration of the extracted solution. The scan rate was 6.3 mV s-1. The results are given in Table 4. Simultaneous Determination of Different Elements by ASV The determination of different elements simultaneously extracted by the same reagent was studied.A suitable pH value of the aqueous solution must be used for quantitative extraction with the respective reagent. Table 5 lists the mixtures studied, extraction reagents, pH (or acidity) of the aqueous phase, solvents used and peak potentials ( E p ) for each element.ANALYST, JUNE 1986, VOL. 111 623 ~ ~~~~~ Table 8. Results for the determination of copper in biological samples and ore Relative standard Sample Cu present, Yo Cu found, YO * deviation, Yo * Vitis vinifera . . . . . . . . 0.0887 0.071 2.2 Pyrus malus . . . . . . . . O.OlS? 0.019 2.5 Olea europea . . . . . . . . 0.0047 0.004 2.6 Gossypium herbaceum . , . . 0.0027 0.002 3.0 Calf liver .. . . . . . . Mussels . . . . . . . . - 0.001 - 0.001 3.5 3.5 Serpentinite (IGS-22) . . . . 0.106$ 0.102 2.9 * Average of five determinations. t Approximate values according to CII (Comitk Inter-Instituts pour 1’Analyse Foliare, France). $ Certified value by Institute of Geological Sciences (London). I I I 1 -0.10 -0.60 -0.77 - 1 PotentialiV Fig. 2. ASV peaks obtained for the simultaneous determination of Cd, Pb and Cu(I1) (Ep = -0.77, -0.60 and -0.10 V, respectively) after their extraction with DDTC into toluene, by addition to the extract of 5% V/V HC104 ; DMF solution. Approximate concentra- tions in the measured solution: Cd 2.70 pg ml-1, Pb 2.40 yg ml-1 and Cu 1.60 yg ml- I PotentialiV Fig. 3. ASV peak for Cu(I1) after its extraction with salicylaldoxime into 1,Z-dichloroethane, with addition to the extract of 5% V/V HC104 - DMF solution.Concentration of Cu(I1) in the measured solution, 0.50 yg ml-1 (peak intensity, i, = 3.90 pA) The instrumental parameters for ASV determinations were as follows: telect, = 6 min; Eelect, = -1.1 V (vs. Ag - AgCl electrode); rest time, 30 s; scan rate, 6.3 mV s-1; and sensitivity, 10 pA (full-scale). Fig. 2 shows the ASV voltam- metric curves for Pb + Cd + Cu(I1). Determination of Copper by ASV Copper(I1) ions were extracted with salicylaldoxime into 1,2-dichloroethane from aqueous solution buffered at pH 4.80. After separation of the phases, 9 ml of organic phase were mixed with 6 ml of 5% V/V solution of perchloric acid in DMF. The ASV peak for copper(I1) in 172-dichloroethane - DMF - HC104 is shown in Fig.3. The instrumental parameters were as follows: Eelect, = -0.70 V; telect, = 6 min; rest time, 30 s; and sensitivity, 5 pA (full-scale). Results for different concentrations of copper(I1) and electrolysis times are shown in Table 6. The other parameters were the same as mentioned above. A caIibration graph was constructed for an electrolysis time of 6 min and the same instrumental parameters. The graph of peak height versus concentration of copper was linear from 0.1 to 4.5 pg ml-1 of Cu(I1) (peak intensity from 0.18 to 8.52 PA). The peak intensity for blank solutions due to copper contami- nation of the chemicals was less than 0.05 pA. The detection limit, as three times the standard deviation of the blanks, was 15 ng ml-1 of copper. The precision of the determination of 40 pg of copper(I1) (2.67 pg ml-1 in the final measured solution) was 2.9% (eight replicate determinations). Interference Study The effects of different ions on the determination of 40 pg of copper (2.67 pg ml-1 in the measured solution) were studied. Tolerance limits, defined as the amount of interferent that did not give an error larger than 5%, are given in Table 7. Applications The method was applied to the determination of copper in biological samples and a nickel ore (serpentinite, IGS-22) certified to contain 0.106% of copper. The results are given in Table 8. References 1. 2. 3. 4. 5. 6. Budnikov, G. K . , and Makhovich, N. A., Russ. Chem. Rev., 1980, 49, 74. Fano, V., Licci, F., and Zanotti, L., Microchem. J . , 1974, 19, 163. Nghi, T. V., and Vydra, F., Anal. Chim. Acta, 1975, 80, 267. Nghi, T. V., and Vydra, F., J . Electroanal. Chem., 1976, 71, 325. Nghi, T. V., and Vydra, F., J . Electroanal. Chem., 1976, 71, 333. Nghi, T. V., and Vydra, F., J . Electroanal. Chem., 1977, 78, 167.624 ANALYST, JUNE 1986, VOL. 111 7. Nagaosa, Y., and Yamada, T., Talanta, 1984,31, 371. 8 . Nagaosa, Y., and Kobayashi, K . , Talanta, 1984,31, 593. 9. Minczewski, J., Chwastowska, J. , and Dybczynski, R., “Sepa- ration and Preconcentration Methods in Inorganic Trace Analysis,’’ Ellis Horwood, Chichester, 1982. Elements,” Ellis Horwood, Chichester, 1976. 11. Zolotov, Yu. A . , Bodnya, V. A., and Zagrunina, A. N., CRC Crit. Rev. Anal. Chern., 1983, 14, 92. Paper A51223 10. Marczenko, Z., “Spectrophotometric Determination of Received June 21st, 1985 Accepted January 15th, 1986

ISSN:0003-2654    

DOI:10.1039/AN9861100619

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 625 Voltammetry of Hexacyanoferrates Using a Chemically Modified Carbon-paste Electrode Kurt Kalcher lnstitut fur Analytische Chemie, Karl-Franzens Universitat, Universitatsplatz 1, A-80 10 Graz, Austria A simple electroanalytical method for the quantitative determination of hexacyanoferrate(l1) and -(Ill) has been developed using a carbon-paste electrode, chemically modified with a weakly basic anion exchanger. The complexes can be pre-concentrated at the electrode surface prior to voltammetric determination. For differential-pulse voltammetric measurements with suitable methodical parameters, a linear relationship between the current and concentration was found for 0.3 to 6000 pg I-' of Fe. The method is applicable to the quantitative determination of hexacyanoferrate(l1) in wine down to 6 pg 1-1 of Fe.Factors influencing the signal response like pre-concentration time and ionic effects have also been investigated. Keywords: Chemically modified carbon-paste electrode; liquid anion exchanger; hexacyanoferrate determination; differential-pulse voltammetry; wine analysis The development of chemically modified electrodes (CME) as an electroanalytical tool has been growing rapidly during the last few years. The aim of increasing the selectivity and the sensitivity of common polarographic and voltammetric methods can be achieved by attaching functional groups to the surface of the working electrode. The feasibility of pre- concentrating electroactive species, based on chemical or physico-chemical reactions with the modifying agent, often lowers the detection limit down to the ultratrace level.Ion exchangers in particular have attracted much interest in this respect, and thus many ionic components have been analysed using electrodes chemically modified with ion exchangers for voltammetric determinations, e.g., chromate,l copper(II),2 iodide,3 gold (III)4 and iridium(IV).S Although the trapping of hexacyanoferrate(II1) in a cationic polymeric film on a platinum electrode was shown to be possible by Facci and Murray6 it has not been used for trace analytical applications. The aim of this work was to establish the optimum experimental conditions for analysing hexacyanoferrates at low concentrations. The usual methods, which include spec- trophotometry of the coloured aggregates of iron, titration of the hexacyanoferrate(I1) with permanganate or cerium(IV), determination of iodine [oxidised by hexacyanoferrate(III)], voltammetry using electrodes with working ranges at positive potentials or decomposition into iron and hydrogen cyanide ,7 are not very selective or sensitive.By adding a liquid ion exchanger to a carbon paste, voltammetric determination can be preceded by a pre- concentration period during which the ionic electroactive species accumulate on the surface of the electrode. This makes trace level analysis possible. Additionally, as this step involves no change of oxidation states, no potential needs to be applied and pre-concentration can be performed independently from the measurement. This offers another advantage; the medium exchange after the accumulation step separates off many components that interfere with the electrochemical determi- nation; these components often show little or no affinity to the ion exchanger.As it is possible to regenerate the surface to its initial state, the electrode filling can be used repeatedly, resulting in fast analyses and a low consumption of the carbon paste accompanied by greater precision. The method described above has been examined to see if it can be applied to the determination of hexacyanoferrates in wine. For the treatment of wine, K4[Fe(CN),] is often used as a fining agent and is added to the wine in order to precipitate heavy metals, including iron, and the resulting (blue) lees are filtered off. It is evident that the amount added must be evaluated on the basis of a chemical analysis to avoid too high dosages.The excess of hexacyanoferrate(I1) remaining in the wine can produce very toxic cyanocompounds and, by decomposition , potassium cyanide itself. Although many other substitutes, such as aferrine, have been introduced, hexacyanoferrate(I1) is still used owing to its low price. Because of the potential danger, it seems important to control the contents of this compound in wine even at low concentra- tions. Pure wine cannot be used directly for the accumulation phase as the high contents of organic compounds block up a large area of the surface and reduce the sensitivity drastically, so it must be diluted in a ratio of at least 1 + 9 or better still, 1 + 19. In this manner the detection limit is acceptably low with respect to the trace analysis of toxic hexacyanoferrate( 11).Of course, as hexacyanoferrate(II1) is also pre-concentrated at the electrode to almost the same degree, it cannot be distinguished from [Fe(CN)$- and is determined together with it. Experimental Apparatus For the voltammetric measurements, a PAR 264 A polaro- graph (Princeton Applied Research) was used in combination with a self-constructed electrode assembly of Plexiglass.8 The cell consisted of a titration vessel of glass (EA 880-20, 20-90 ml, from Metrohm) with a platinum wire as the counter electrode and a saturated calomel electrode (SCE) as a reference. The latter was in contact with the solution over a salt bridge with a Vycor frit, filled with 1 M KC1 solution.The curves were registered on a two-channel recorder. For determining peak heights smaller than 5 mm the data were evaluated digitally on a HP 1000 mini-computer after A - D conversion by an appropriate interface.9 The receptacle for the test solution was a 50-ml glass beaker equipped with a PTFE-coated stirring bar (30 mm, 0.d. 7 mm), which was driven by a variable speed magnetic stirrer. It was placed under a suitable holder for the electrode. Working Electrode The body of the working electrode was fabricated from a PTFE rod (0.d. 10 mm) with a 3 mm deep bore hole (diameter 7 mm) on one side for the carbon-paste filling. Contact to this was made by a platinum wire glued into a bore hole in the centre of the rod.626 ANALYST, JUNE 1986, VOL. 111 The carbon paste was prepared according to Monien et al.1" A 5-g mass of spectral carbon powder (RWB, Ringsdorff- Werke) was mixed thoroughly with 1.8 ml of liquid paraffin (Uvasol, Merck). Commercially available carbon paste (EA 267 C from Metrohm) can also be used. For each gram of this paste, 0.05 ml of liquid ion exchanger, Amberlite LA2 (Fluka), was added, and the substances were mixed thoroughly to a homogeneous consistency. This mixture was placed in the corpus of the electrode using a spatula of PTFE and smoothed off. An electrode prepared in this way can be used, if regenerated appropriately, for at least ten determina- tions without any notable change in the signal response. Reagents De-ionised and doubly distilled water that had been purified by a cartridge de-ionisation system (Nano-pure from Barn- stead) was used throughout.Hydrochloric acid and sodium hydroxide were of Supra-pure quality (Merck) and all other reagents were of analytical-reagent grade (pro analysi, Merck). Stock solutions of the hexacyanoferrates were prepared with concentrations of 1 mg ml-1 of Fe by dissolving 151.27 mg of K4[Fe(CN)6].3H20, or 117.92 mg of K,[Fe(CN),], respectively, in 20 ml of water. They were stored in the dark at 4 "C and could be used for at least one week. Solutions with lower concentrations were made freshly by dilution. For the regeneration of the electrode, 20 ml of saturated, aqueous sodium chloride solution were mixed with 0.2 ml of NaOH (30% solution). It was prepared freshly when needed. As standard wines, two Austrian types were chosen which contained no determinable amount of hexacyanoferrate( 11).The white wine was a Griiner Veltliner, a light table wine (referred to as the standard white), and the red wine (standard red) was Blaufrankisch, both originating from Burgenland. They were used directly from the bottle without any pre- treatment. Procedure Pre-concentration A 20-ml volume of the test solution containing the hexa- cyanoferrate(I1) or-(111) was acidified with hydrochloric acid to a pH of 2 (0.02 ml of 10 M HCl). When analysing wine, 1 ml of the sample was diluted with 19 ml of water and acidified with 0.02 ml of 10 M HCI. The electrode was placed in the holder exposing the carbon paste surface to the solution, which was then stirred at a rate of 300 rev.min-1. After allowing to stand for the required time, the electrode was removed, rinsed with water for about 0.5 s, placed in the voltammetric cell and connected to the polarograph. Voltammetry Water (20 ml), acidified with 0.05 ml of 10 M HCI to give a final HCl concentration of 2.5 x 10-2 M as the supporting electrolyte and de-aerated by passing through pure nitrogen (99.999%) for 3 min, was used for the voltammetric measure- ments. Quantitative analyses were performed in the differen- tial-pulse mode (DPV). The potential range was set from -0.15 to +0.4 V vs. SCE in the anodic direction regardless of the oxidation state of the iron in the cyano complex. The pulse height was 50 mV and the scan rate 10 mV s-1 with a drop time of 0.5 s. In order to settle the solution and eliminate the high background currents an equilibration phase of 15 s with an applied initial potential preceded all the voltammetric measurements. Cyclovoltammograms (CV) were recorded from -0.3 to +0.5 V vs.SCE for hexacyanoferrate(I1) and in the reverse direction for [Fe(CN),I3- The scan rate was 50 mV s-1 in both instances. The current range was selected according to the expected peak height. For accurate evaluations of the peak heights for low concentrations , the background current was synthesised and subtracted from the curve .9 Higher concen- trations yielded peaks that could be determined manually from the recorder output with sufficient precision by means of a tangent fit. Regeneration After trapping the iron complex on the surface of the electrode with a subsequent voltammetric determination, the functional groups of the ion exchanger were regenerated by a 2-min treatment with a well stirred, alkaline saturated sodium chloride solution (to remove the complex) followed by exposure for 2 min to stirred 2 M HCl (for re-formation of the ammonium groups). This procedure was also applied to the freshly prepared, virgin electrodes to yield reproducible results.Evaluation of quantitative results A quantitative determination consisted of a regeneration step, the registration of the blank of the unloaded electrode with DPV, the accumulation of the iron complex and the recording of the potential - current curve with DPV. This method was repeated twice with the appropriate addition of specified amounts of the iron complex to the test solution (standard additions method).The concentration was obtained from the peak currents by the usual mathematical methods. The peak height was calculated either by a tangent fit for higher concentrations, or synthesis and subtraction of the back- ground. The blank subtract method is also applicable. Results and Discussion In acidic aqueous solutions both hexacyanoferrate(I1) and -(III) form ion aggregates with water-immiscible, weakly basic ion exchanger molecules at the electrode - solution interface: [Fe(CN),I4- + 4R2NH,'C1- ++ (R2NHi)4[Fe(CN)6] + 4C1- (1) [Fe(CN),I3- + 3 R2NH,'C1- ++ (R2NHi)3[Fe(CN)6] + 3 C1- There is no evidence that all the negative charges are compensated by ammonium ions; protons may replace them to a certain degree.Owing to the very similar behaviour of both species, at least a one-step protonation of the hexa- cyanoferrate(I1) ion in equation (1) is probable, which would lead to an exchange reaction analogous to equation (2). However, the absolute composition of the ion pairs formed is not of great importance in the method; as these ions associate generally with ammonium compounds both complexes can be pre-concentrated at the electrode surface. As equations (1) and (2) or variations of them involve no change in the oxidation states or electron transfers, accumulation can be carried out without the application of a potential. When, after trapping the complex on the surface of the electrode, an electrical field varying with time during voltammetric measurement is imposed, oxidation of Fe(I1) or reduction of Fe(II1) can occur according to the potential and the oxidation state of the metal ion as follows: (2) (3) This electron transfer leads to a current response and can be seen clearly in the corresponding cyclovoltammograms (Figs.1 and 2). The pictures show almost reversible processes for both complex species as expressed in equation (3). The potentials of the cathodic and anodic maxima, E,, and E,,, are displayed in Table 1. For the reduction, E,, shifts much more with increasing cycle number than does E,, for the oxidation. Because of this, all differential-pulse measurements are also carried out in the anodic direction to give better reproducibil-ANALYST, JUNE 1986, VOL. 111 ity. If hexacyanoferrate(II1) is accumulated it is reduced to hexacyanoferrate(I1) during the equilibration phase and re-oxidised when recording the voltammogram. In CV, the current of the maxima decreases with growing cycle number because small amounts of the trapped-in cyanoferrate diffuse from the electrode surface leading to a signal reduction.The regeneration process makes use of this diffusion and supports it in two ways: firstly, the high concentration of chloride competes with the active sites of the exchanger and replaces cyanoferrate; and secondly, the alkaline medium reduces the protonation of the ammonium groups and thus prompts the detach reaction. Of course, the cationic state must be remade by acid treatment. As stated above , anodic differential-pulse voltammetry (ADPV) has been chosen for quantitative determinations because of its high sensitivity and accuracy.Fig. 3 shows the voltammogram of hexacyanoferrate(n) carried out in this mode. For the oxidised species the resembling curves are recorded. The peak potentials are slightly dependent on the amount of accumulated ions, being shifted in the cathodic 627 direction with increasing current. As the background shows quasi-linear functionality within the peak range, the tangent fit method can be applied fairly exactly for evaluation of peak height. Only for very small signals should more sophisticated methods be applied.9 For analysing wine, it was first checked that pre- concentration could be carried out in the alcoholic medium. The results are summarised in Fig. 4, which shows the dependence of the voltammetric peak current on the ethanol contents of an acidified aqueous solution of [Fe(CN)&-.The relationship between alcohol concentration and signal response is a linear function where increasing amounts reduce the peak height. Thus, with respect to ethanol, determinations are possible because, at low concentrations, the changes in the signal are not disastrous. Two table wines, a white and a red, with undetectable amounts of hexacyanoferrate(I1) were chosen to serve as standard media for working out the analytical conditions of the method. Practice has shown that pure, undiluted wine contains many organic compounds that produce no voltammetric signals by themselves but block the r 1 0.3 0.1 -0.1 -0.3 PotentialN vs. SCE Fig. 1. Cyclovoltammogram of hexacyanoferrate(I1) solution.[Fe(II)], 1 ng 1-1; accumulation time, 1.5 min 25 f % g o 3 0 -25 I I I I 0.5 0.3 0.1 -0.1 PotentialN vs. SCE Fig. 2. Cyclovoltammogram of hexacyanoferrate(II1) solution. [Fe(III)], 1 mg I - * ; accumulation time, 1.5 min -0.1 0.1 0.3 PotentiaW vs. SCE Fig. 3. Anodic differential pulse voltammogram of hexacyano- ferrate(I1). A, Background; B, 200 pg 1 - l of Fe(I1); and C, 400 pg 1-1 of Fe(I1). Accumulation time, 30 s 0 5.0 10.0 15.0 20.0 Ethanol content, % Fig. 4. [Fe(II)], 100 pg 1-1 as [Fe(CN),]4-; accumulation time, 1 min Relationship between peak current and ethanol content. Table 1. Maximum cathodic and anodic potentials (Ep, and Ep,) with their corresponding currents (ip, tration of Fe, 1 mg 1-1 Oxidation state Fe(I1) . .. . . . Fe(I1) . . . . . . Fe(I1) . . . . . . Fe(II1) . . . . . . Fe(II1) . . . . . . Fe(II1) . . . . . . Fe(1I) . . . . . . Fe(II1) . . . . . . Method cv cv cv cv cv cv ADPV ADPV Deposition tirnels 60 60 60 60 60 60 30 30 Cycle 1 2 3 1 2 3 - EP 1 Vvs. k E 0.030 0.033 0.035 0.029 0.030 0.031 - EP 1 Vvs. k E 0.135 0.135 0.134 0.130 0.130 0.130 0.080 0.085 34.0 32.5 31.6 28.2 27.0 26.8 - and ips). Concen- 37.0 32.8 31.5 27.0 26.8 26.6 45.0 27.6628 ANALYST, JUNE 1986, VOL. 111 surface of the electrode by adsorption and hence reduce the extent of the ion exchange drastically. Therefore, it is necessary to dilute the wine to at least 10 times (or better to 20 times) its volume prior to the accumulation step. In this way a loss of sensitivity of about 10-20% compared with aqueous solutions still permits the determination of trace levels of hexacyanoferrate.For the following investigations with respect to wine, 1-ml samples were mixed with 19 ml of water and used as described under Experimental. The effect of pre-concentration time on the peak current for an aqueous medium is displayed in Figs. 5 and 6. Similar graphs have been obtained for both types of cyano complexes. With standard white wine the dependence of the voltammetric current on the accumulation time is shown in Fig. 7. An almost identical graph has been registered for the red wine. Both are very similar to pure aqueous solutions with respect to their qualitative shape. A linear run for short periods of time soon deviates into a hyperbolic function approaching a limiting value.This behaviour can be explained by the mass action law, which governs the exchange process according to equations (1) and (2). It is self-evident that accumulation can take place to some extent only where equilibrium is reached and desorption equals sorption. Hence, the maximum amount of hexacyanoferrate on the electrode surface is controlled by the number of functional exchanger groups , the concentration of ions in the bulk solution, and the concentration distribution I I I I 1 0 1 2 3 4 5 Ti me/m i n Fig. 5. Dependence of the peak current on the accumulation time for solutions of hexacyanoferrate(I1) [Fe(II)]: A, 250 pg 1-l; and B, 5 mg 1-1 120 - 40 1 - 0 1 2 3 4 5 Time/m in Fig. 6. Relationship between the peak current and accumulation time for solutions of hexacyanoferrate(II1).[Fe(III)]: A, 250 pg I-’; and B, 5 mg 1 - 1 coefficient Dc, which expresses the affinity of an ionic species to an ion exchanger , and is defined as The subscript ex. designates the concentration of the complex as the ion aggregate with the exchanger and sol the concen- tration in the solution. The state of motion during pre- concentration also influences the absolute shape of the graphs but does not alter either the qualitative result or the equilibrium, i.e., the limiting value of the current. There may be two reasons why this maximum current is reduced in wine compared with aqueous solutions containing no alcohol: firstly, the concentration distribution coefficient [equation (4)] is decreased (as can also be seen in Fig.4); and secondly, the adsorption of organic molecules inhibits the ion exchange process notably. A transition of the ion exchanger from the surface into the alcoholic medium could not be confirmed as the reproducibility of the electrode is maintained by the regeneration step. The relationship between concentration and peak current for pure aqueous solufions at selected accumulation times, t,, is shown in Figs. 8 and 9. In spite of the different oxidation states of the metals the two types of complexes behave similarly, leading to the conclusion that their affinities to the 1.0 2.0 3.0 4.0 5.0 0 Ti me/m in Fig. 7. Dependence of the peak current on the accumulation time. [Fe(II)] [as hexacyanoferrate(II)], 250 pg I-’ after dilution of 1 ml standard white wine to 20 ml I /u f . 40 2 3 0 20 0 0.2 0.4 0.6 0.8 1 .o Fig.8. Dependence of the peak current on the concentration of hexacyanoferrate(I1) solutions. Accumulation times and concen- trationranges: A, 5 min, 0-10 pg 1-1 of Fe, current axis extended 10 times; B, 3 min, 0-100 pg 1 - 1 of Fe; C, 30 s, 0-1 mg 1 - 1 of Fe; and D, 5 s, 0-10 mg 1 - 1 of Fe Concentration rangeANALYST, JUNE 1986, VOL. 111 629 Concentration range Fig. 9. Dependence of the peak current on the concentration of hexacyanoferrate(II1) solution. Accumulation times and concen- tration ranges as in Fig. 8 I I 0 0.2 0.4 0.6 0.8 1 .o Concentration range Fig. 10. Dependence of the peak current on the concentration of hexacyanoferrate(I1) in standard white wine. Concentration ranges and accumulation times: A, 0-100 pg 1-1 of Fe, 3 min, current axis extended 5 times; B, 0-1 mg 1-1 of Fe, 2 min; C, 0-10 mg 1-I of Fe, 30 s; D , 0-100 mg 1-l of Fe, 5 s 1 1 0 0.2 0.4 0.6 0.8 1 .o Concentration range Fig.11. Dependence of the peak current on the concentration of hexacyanoferrate(I1) in standard red wine. Concentration ranges and accumulation times: A , 0-100 pg I-' of Fe, 3 min, current axis extended 10 times; B, C , and D as in Fig. 10 ion exchanger must be of the same magnitude although that for hexacyanoferrate( 11) is slightly higher. Direct propor- tionality exists over a wide range of concentration if an appropriate pre-concentration period is chosen. Hence , linear relationships can be observed for up to 6 mg 1-1 of iron for [Fe(CN)6I4- and up to 8 mg 1-1 of iron for [Fe(CN)6I3-.Therefore, the best way of determining hexacyanoferrate concentrations is the internal standard additions method. Solutions containing concentrations higher than 4 mg 1-1 of Fe must be diluted prior to analysis. In practice, the detection limit lies at 0.3 pg 1-1 of Fe, which corresponds to about 1.1 pg 1-1 of hexacyanoferrate and a molar concentration of 5.4 x M. For a series of five determinations of 50 pg 1-1 of Fe(I1) as [Fe(CN)&- (t, = 2 min) the coefficient of variation was calculated as 1.5% with two standard additions. (It is advisable to add at least two standards in order to check the linearity between the concentration and the signal response.) As shown in Figs. 10 and 11, a linear relationship exists between the concentration and voltammetric peak heights over a wide range for both white and red wine, with suitable accumulation times. Thus, reduction in the signal height by components like alcohol does not imply a breakdown in the linearity.It can be seen that both types of wine, white and red, behave similarily . With short deposition times linearity can be observed for up to 60 mg 1-1 of Fe as [Fe(CN)6]4- in the wine. In practice the detection limit lies at about 6-7 pg 1-1 of Fe in wine, corresponding to about 25 pg 1-1 of [Fe(CN)6]4-. Of course, hexacyanoferrate(II1) , [Fe(CN),I3- , which could be present in wines by oxidation processes, is co-accumulated on the electrode surface to almost the same extent as the hexacyanoferrate(I1) complex leading to a combined determi- nation of both species.From a toxicological point of view this fact is not important, as a distinction between the two types is not particularly relevant. The recovery rate was determined with spiked wines when the test solution was measured once with only one standard addition (Table 2). At low concen- trations the deviation is usually larger than at higher ones. The estimated maxima of deviations, resulting from practical experience, are also listed in Table 2. The error of the result can be decreased by performing one measurement twice, in addition to the standard additions. However, it is usually not necessary to do so because the accuracy, as indicated in Table 2, is often adequate. The fact that the recovered concen- trations of the white standard lie above (and those of the red wine below) the real contents is an artefact of this series and cannot be reproduced in any way.At this point it should be stressed that all the steps of the procedure should be carried out with the greatest care, and always in the same manner, in order to achieve the utmost homogeneity of results. Many errors arise from the non- equivalent treatment of electrodes (regenerating, rinsing, stirring, etc.) resulting in non-representative amounts of complex being accumulated by the ion exchanger. It should also be pointed out that the measurements of added standards have to be performed with the same electrode filling to avoid discrepancies on the electrode surface. For pure aqueous solutions, the influence of other ionic components on the current was investigated (Table 3).Similar behaviour was found for hexacyanoferrate(I1). Only per- chlorate and salicylate cause severe disturbances. It should be emphasised that changes in peak heights due to other ions that are larger than the standard deviations do not imply that the determination is impossible. In most instances the linear relationship between signal and concentration is maintained and only the sensitivity is altered. However, this must be checked in every single instance. Finally, the hexacyanoferrate(I1) content of ten Austrian white and red wines and two champagnes was investigated. It was shown that wines with higher sugar contents behaved analytically in the same way as the standard types used. Six of the wines had no determinable amounts of hexacyanoferrate630 ANALYST, JUNE 1986, VOL.111 ~~ Table 2. Recovery rates and maxima of deviations of hexacyanoferrate(I1) in spiked wines; evaluation with one standard addition Standard wine White . . . . White . . . . White . . . . Red . . . . Red . . . . Red . . . . Accumulation time . . 3 min . . 1 min . . 10 s . . 3 min . . 1 min . . 10 s Spiked concentration of Fe/ yg 1 - 40 400 4000 40 400 4000 Concentration of Fe foundlyg ml-1 44.7 415 4080 38.9 381 3940 Estimated maxima of deviation, '/O f 15 f 10 2 5 t 15 + 10 +5 Table 3. Interference of anionic components to the determination of hexacyanoferrate(I1) in aqueous solution. Concentrations: Fe, 100 yg 1-1; and anionic component, 10 mg I-'. Accumulation time, 60 s Change in Component Added as Concentration/M peak current, YO Br- .. * . I- . . . . * . NO2- . . . . NO,- . . . * HW4- . . . . so32- . . . . so42- . . . . CN- . . . . Acetate . . . . Oxalate . . . . Tartrate . . . . Citrate . . . . Ascorbate . . Salicylate . . Tannin . . . . c104- . . . . KBr 1.3 x 10-4 KI 7.9 x 10-5 NaN02 2.2 x 10-4 KN03 1.6 x 10-4 KH2P04 1.0 x 10-4 Na2S03 1.2 x 10-4 K 8 0 4 1.0 x 10-4 NaCIO, 1.0 x 10-4 KCN 3.8 x 10-4 G I 3 4 0 2 1.7 x 10-4 C2H204 1.1 x 10-4 C4H606 6.8 X 10-5 C ~ H S N ~ ~ O ~ 5.3 X lop5 C6H806 5.0 x 10-5 C7H603 7.2 x 10-5 C76H52046 -5.9 X lop6 +3.1 -15.0 + 10.4 -1.2 -2.6 +4.7 -7.9 -43.7 -0.2 -4.5 +7.0 + 10.2 + 10.0 -3.5 -46.5 -7.2 and four contained between 7 and 8 pg 1-1 of Fe as hexa- cyanoferrate(I1). As the wines are at least one year old it is not possible to deduce whether the original content of hexa- cyanoferrate(I1) was low or whether the complexes had vanished owing to decomposition processes. The champagnes did not show any detectable hexacyanoferrate(I1). Conclusion The basic conditions for a method to determine trace levels of hexacyanoferrate( 11) and -( 111) with a chemically modified carbon paste electrode have been described. As a linear relationship is achieved between concentration and voltam- metric signal over a wide range, the standard additions method can be applied to quantitative evaluations. The determination of hexacyanoferrate in both white and red wines is possible. The ease and simplicity of preparing the electrodes is also advantageous. 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. References Cox, J. A., Anal. Chim. Acta, 1983, 154, 71. Wang, J., Greene, B., and Morgan, C., Anal. Chim. Acta, 1984, 158, 15. Kalcher, K., Fresenius 2. Anal. Chem., 1985, 321, 666. Kalcher, K., Anal. Chim. Acta, 1985, 177, 175. Kalcher, K., Fresenius 2. Anal. Chem., in the press. Facci, J., and Murray, R. W., J. Phys. Chem., 1981,85,2870. Burger, N., Talanta, 1985, 32, 49. Kalcher, K., Fresenius 2. Anal. Chem., 1986, 323, 238. Kalcher, K., and Jorde, C., Compput. Chem., in the press. Monien, H., Specker, H., and Zinke, K., Fresenius 2. Anal. Chem., 1967,225, 342. Paper A5f398 Received November 4th, 1985 Accepted December 30th, 1985

ISSN:0003-2654    

DOI:10.1039/AN9861100625

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 63 1 Determination of Ammonia in Wine and Milk with an Ammonia Gas-sensing Probe Jose L. Bernal, Maria J. del Nozal, Luis Deban and Isabel Torremocha Department of Analytical Chemistry, Faculty of Sciences, University of Valladolid, Valladolid, Spain A method is described for the determination of ammonia in milk and wine samples using an ammonia gas- sensing probe. The procedure uses a standard additions technique without any previous treatment of the sample. The results obtained agree with those obtained using commonly recommended methods for the determination of ammonia in such samples. Keywords : A rn rn o n ia de te rrn in a tio n ; wine a na / ysis; m ilk a na / ysis; a rn rn o n ia gas-sensing p robe The development in recent years of gas-sensing probes has increased the number of compounds that can be determined using potentiometric techniques.The response of the probe is related to the partial pressure of the gas for which the probe is designed, and because of this property the presence of other ions in the sample produces little or no interfering action so that very low levels of the gas can be successfully detected. The ammonia gas-sensing probe is very useful for the determination of ammonia in water,lJ and nitrogen com- pounds in soils,3 meat, cereals, beer4>5 and wine6.7 after Kjeldahl digestion. In this work we have applied the ammonia probe to the determination of ammonia in samples of alimentary interest, such as bottled milk and wine, so that their natural or artificial contamination can be assessed.In this way we can indirectly obtain information about the hygiene standards of the milk or wine under study. Firstly, a study of the probe performance was carried out to determine the probe parameters and the optimum measure- ment conditions. Next, ammonia was determined in several milk and wine samples by using direct potentiometry and also a standard additions method. The latter technique proved to be the most efficient and can be recommended for the determination of ammonia in the samples studied. The results obtained agree with those using commonly recommended methods. Experimental Reagents All reagents were of analytical-reagent grade. Ammonia stock solution, 0.1 M. All other ammonia solu- tions were prepared by appropriate dilution of this stock solution.Total ionic strength adjustment buffer (TISA B ) solution. Sodium nitrate (85.0 g), sodium hydroxide (2.0 g) and EDTA (1.862 g) were dissolved in de-ionised water and the solution was made up to 1 1 with de-ionised water. Apparatus A digital ion-activity meter (Philips PW 9414) was used with an ammonia gas-sensing probe (Philips IS-570 NH3) and a double-junction saturated calomel reference electrode (Philips R44/2-SD/1) with its outer chamber filled with 2 M KN03 solution. For the pH measurements, a Radiometer pH-29 pH meter was used in combination with a GK 2401-C glass combined electrode. Procedures Direct potentiometry Add 50.0 ml of the TISAB to 50.0 ml of the sample in a 250-ml beaker. Place the probe and reference electrode in this solution and read the e.m.f.after 5 min of continuous stirring. The ammonia concentration is determined by comparing the e.m.f. reading with a calibration line prepared with known concentrations of ammonia under the same conditions. Recommended procedure (standard additions method) Add 50.0 ml of the TISAB solution to 10.0 ml of the sample and dilute to 100.0 ml with de-ionised water. Transfer this solution into a 250-ml beaker. Place the probe and reference electrode in it and read the e.m.f. after 5 min of continuous stirring. Next, add five 1.0-ml portions of standard ammonia solution and read the e.m.f. 5 min after each addition. Once the corresponding calculations have been made, a graph is plotted and, by extrapolation of the straight line to the ordinate, the equivalent volume and the ammonia concentra- tion of the sample are found.Results and Discussion Study of the Electrode Performance Before applying the ammonia gas-sensing probe to the study of alimentary samples, the analytical characteristics of the probe were investigated, using similar conditions to those for the samples. There are several experimental parameters that can affect the probe response, viz., ionic strength, the pH of the medium, the measurement time, temperature and stirring rate. The first three parameters have the greatest effect on the determination of ammonia8.9 and they were studied first. The optimum conditions are as follows: the pH of the medium must be between 11.0 and 12.5 to ensure that all the ammonia is in the form of gaseous dissolved ammonia; the ionic strength must be between 0.3 and 1.3 M; and the measurement time must be at least 5 min to reach a stable e.m.f.reading. The solutions must be measured according to their increasing ammonia levels, to diminish the “memory effect” of the probe and to draw a reliable calibration graph. The first two parameters can be properly regulated by the use of a TISAB solution as described under Experimental. This solution is added to the sample in the volume ratio recommended, With respect to the other experimental parameters dis- cussed, the temperature must be maintained at a constant value because the probe is affected by high temperatures. These high temperatures can produce variations in the e.m.f. reading of up to 1.6 mV “C-1. An appropriate value is 25 “C,632 ANALYST, JUNE 1986, VOL.111 regulated with the aid of a recirculation thermostat. The stirring rate of the solutions must be constant and special care must be taken to ensure that bubbles of air are not formed in the probe membrane, which would decrease the reproduci- bili ty . Under the optimum conditions a calibration graph can be drawn using solutions of a known concentration. The slope of the line obtained was 58.5 mV per decade. The Nernstian interval was between 5.0 X 10-2 and 5.0 x 10-5 M and the detection limit was 5.0 x 10-6 M. In order to apply the probe to real samples, a study of the effect produced by the presence of foreign ions was also carried out. Various concentrations of the ions under study were added to solutions of known concentrations of ammonia containing TISAB solution and, by comparing the e.m.f.reading with that obtained when the interfering ion was not present, an evaluation of the interference was obtained. Although interfering action could be expected from all the cations that give complexes with ammonia, the presence of EDTA in the TISAB is sufficient to mask all the cations, at least at the concentration levels studied (similar to those found in wines and milks). In this way all the ammonia in the solutions measured could be detected by the probe. For a sample with a higher level of interfering cations, the EDTA concentration in the added TISAB could have been increased until the interference was eliminated. However, this problem was unlikely to occur, as the wine or milk samples analysed had relatively low concentrations of ammonia complex- forming cations.Practical Applications Wine Effect of ethanol on the ammonia probe. The ethanol concentration in wine varies from about 8 to 20%, and we therefore studied the effect of ethanol on the determination of ammonia, in order to investigate its effect on the probe. Solutions were prepared, all with the same concentration of ammonia, but with varying amounts of ethanol. The e.m.f. of these solutions was measured by applying a direct potentio- metric method. The results obtained with 10-3 and 10-4 M ammonia solutions are shown in Table 1. As can be observed, the value of the e.m.f. readings increases with increasing ethanol concentration. A lower e.m.f. reading implies a lower ammonia concentration (CNH,) according to the equation E=K-plogCNfI3 a .. a . (1) where E is the potential (mV) and p is the slope (mV decade-1). There is a simple ratio between the absolute error of the e.m.f. and the relative error produced in the concentra- Table 1. Change in potential on addition of ethanol to different standard solutions of ammonia Ethanol concentration/ ml per 100 ml 0.0 1 .o 2.0 3.0 4.0 5.0 6.0 7.0 8.0 11.0 15.0 19.0 E .m . f ./mV M NH3 - 138.5 - 138.2 -137.3 - 136.2 -134.3 -132.3 - 130.0 -127.8 -125.8 -123.3 - 120.8 -118.5 10-3 M N H ~ -79.3 -78.0 -76.2 -74.0 -71.7 -69.3 -67.0 -64.8 -62.6 -59.9 -57.4 -55.0 tion of ammonia. Converting to Nernstian logarithms, the following equation is obtained: Differentiating this equation and identifying differentials and increments, we obtain p ACNH3 * * (3) AE=--.- . .. . 2.303 CNH3 and therefore 2.303 x 100 = E, = - - AE . . . . (4) ACNH~ cNH3 P where E, is the relative error in the ammonia concentration. In this instance, p is 58.5 mV per decade, so the equation becomes &,=-3.9AE . . . . * * ( 5 ) Equation ( 5 ) implies that a positive error of 1 mV in the measured e.m.f. produces a negative error in the ammonia concentration of 3.9% (when the e.m.f. error was negative, the ammonia error was positive). On applying equation ( 5 ) to our results an intolerable error was obtained. For example, taking an ethanol concentration of 6 ml per 100 ml (i.e., a hypothetical wine with a typical ethanol level of 12% V/V and diluting the solution 1 + 1 with TISAB solution) gave an error of33.2% for a 10-3 M ammonia solution, which increased to 48% for a 10-4 M solution.Therefore, because the errors obtained when the ethanol level was not taken into account were very high, the direct potentiometric method cannot be applied unless the appro- priate corrections relating to the alcoholic level of the wine are made. In this instance the ethanol concentration of the wines should have been determined previously, using, for example, a distillation procedure; this will, however, make the determi- nation of ammonia by direct potentiometry using a gas-sensing probe a complicated and time-consuming procedure. Standard additions method. Because of the problems arising from the use of the direct potentiometric method, the application of a standard additions procedure was investi- gated, as in this method the corrections for the complex matrix of the sample are not usually necessary if the volumes of the added standard are small enough to ensure that the total volume does not change significantly.Taking antilogarithms of equation (l), we obtain 10-E'P = 10-K'pCNH3 = K'CNH3 . . . . (6) From this it follows that if the ammonia concentration changes linearly, the first term of equation ( 6 ) will also do so. A volume correction must therefore be taken into account, and we finally obtain where V, is the initial volume, CoNH3 the ammonia concentra- tion of the sample and V, and C'NHi are the volume and concentration of the added ammonia standard, respectively. When the first term of equation (7) is plotted against V,, a straight line is obtained, which on extrapolation to zero gives a negative value for Veq, the volume of standard with a concentration of ammonia equivalent to that of the initial sample.The value of CoNH3 can be obtained from the following C"N I 13 veq . CONH~ = - . . . . * * (8) vo As described under Experimental, the addition of a TISAB solution in a 5 + 1 volume ratio eliminates possible interfer-ANALYST, JUNE 1986, VOL. 111 633 -8 -6 -4 -2 0 2 4 6 V,lm I Fig. 1. in wine. Samples (see Table 2): A, 1; B, 2; and C, 3 Standard additions graphs for the determination of ammonia Table 2. Results obtained for the determination of ammonia in three wine samples by different methods Ammonia found/mg I-' Proposed Wine sample method 1 2 3 139.4 103.7 79.9 Conventional method 142.8 104.5 80.9 Fig.2. in milk. Samples (see Table 3): A, 1; B, 2; and C, 3 Standard additions graphs for the determination of ammonia Table 3. Results obtained for the determination of ammonia in three milk samples by using direct determination, the Nessler method and the standard additions method Ammonia found/mg 1-l Milk Direct Nessler Standard additions method sample method method 1 14.2 12.2 12.5 2 12.4 11.0 11.3 3 9.9 8.6 8.8 ences. As the volume of the analysed solution is kept at 100 ml and the volume of standard additions is 1.0 ml, the total volume does not change significantly. In this way the composition of the analysed sample is maintained approxi- mately constant during the process, and therefore the effect of foreign species (both organic and inorganic) present in the wine is constant and their possible interfering action is obviated.Fig. 1 shows the results obtained when the recommended procedure was applied to three different commercial bottled wines. To test the validity of our method we took as a reference method the Dimotaki-Kourakau procedure,lO which is the most commonly recommended method for the determination of ammonia in wines. The results obtained by this method are given for comparison in Table 2. As can be observed, the difference between the corresponding measure- ments was no higher than 2%. Therefore, the proposed method, being much simpler and quicker to perform, can be considered as a rapid and accurate alternative for the determination of ammonia in wines.Milk We initially applied a direct potentiometric method for the determination of ammonia in milk, but the results obtained were not satisfactory because of the very poor reproducibil- ity. This effect can be attributed to the presence of fat species in the milk that interfere with the membrane probe by producing variations in the osmotic pressure and can also cause physical obstructions in the membrane pores. To avoid these complications, the fatty matter in the milk was removed by the addition of a dilute solution of acetic acid, with subsequent decantation and filtration. Next, direct potentiometry was applied to the resulting sample. Although a better reproducibility was obtained, a comparison with the standard Nesslerll method gave significantly different results.A further attempt was made to improve the accuracy and precision of the procedure by diluting the milk sample to give the same volume ratio as used for the direct potentiometric titration. The results for three different samples of commercial bottled milk are shown in Table 3. Although the values were significantly better in this instance, a comparison with those derived from the Nessler method showed differences in excess of at least 12%. These errors induced us to apply a standard additions procedure, similar to that described for the analysis of wine. The best results, also given in Table 3, were obtained when the proposed procedure (see Experimental) was applied directly without any additional treatment of the milk sample. Fig. 2 shows the results of the application of the method to milk samples. It can be seen that the differences between the results of the proposed procedure and the Nessler method are not higher than 2.7%.Therefore it can be concluded that the ammonia gas-sensing probe is a good alternative to the Nessler method for the determination of ammonia in milk samples. In conclusion, the ammonia gas-sensing probe provides a precise and accurate method for the determination of am- monia in samples of biological interest such as wine and milk. The simplicity of its use gives an additional advantage over commonly recommended procedures, which tend to be much slower and more complicated.634 ANALYST, JUNE 1986, VOL. 111 References 1. Beckett, M. J . , and Wilson, A . L., Water Res., 1974,8, 333. 2. Evans, W. H., and Partridge, B . F., Analyst, 1974, 99, 367. 3. Bremner, J. M., and Tabatabai, M. A., Comrnun. Soil Sci. Plant Anal., 1972, 3 , 159. 4. Todd, P. M., J. Sci. Food Agric., 1973, 24, 488. 5. Deschrieder, A . R . , and Meaux, R . , Analusis, 1973, 2, 442. 6. Garcia, C., Hidalgo, J. L., and Perez-Bustamante, J . A., Afinidad, 1983, 40, 41. 7. Ross, J. W., Riseman, J. H., and Krueger, J. A . , Pure Appl. Chem., 1973, 36,473. 8. McWilliam, D . J . , and Ough, C. S., Am. J . Enol. Viticult., 1974,25, No. 2,67. 9. Basica, H., J. Fish. Res. Board Can., 1973, 30, 1389. 10. Dimotaki-Kourakau, V., Ann. Falsif. Expert. Chim., 1960,53. 11. Nessler, J., Diss. Freiburg, 1856; Chem. Zentralbl., 1856, 27, 529. Paper A41410 Received November I I th, I985 Accepted December 23rd, 1985

ISSN:0003-2654    

DOI:10.1039/AN9861100631

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 635 Determination of Morphine by Flow Injection Analysis with Chemiluminescence Detection Richard W. Abbott and Alan Townshend Chemistry Department, University of Hull, Hull HU6 7RX, UK and Richard Gill Central Research Establishment, Home Office Forensic Science Service, Aldermaston, Reading, Berkshire RG74PN, UK Chemiluminescence detection has been used in combination with flow injection analysis to determine 2 1 fmol of morphine by its reaction with permanganate in an acidic tetraphosphate solution. Structurally similar narcotics can also be determined by the same procedure. A mechanism for the chemiluminescent reaction is suggested. Keywords: Morphine determination; flow injection analysis; chemiluminescence detection Until recently, reactions that produce chemiluminescence (CL) and bioluminescence have been regarded merely as interesting phenomenal and their potential in analytical methodology2 has not been fully appreciated.However, the development of extremely sensitive and reliable instrumenta- tion3 has generated much interest in such reactions. Several applications4-6 have demonstrated the remarkable sensitivity and selectivity of CL detection compared with established methods such as UV - visible and fluorescence spectroscopy. Current applications of CL to drug analysis are restricted but are expanding rapidly. The reaction of bis(2,4,6- trichlorophenyl) oxalate with hydrogen peroxide to generate an excited-state intermediate which will excite many conven- tional fluorophores has been reported by several workers.St7-8 Fluorescein and fluorescamine derivatives of thyroxine7 have been determined by this system, as have dansylated amino acids8 and fluorescamine derivatives of cat echo la mine^.^ Another area of much potential is that of immunoassay.6 The most common chemiluminescent immunoassay uses luminol- or isoluminol-labelled antigen, which competes with unlabelled antigen in the sample for the sites on the appropriate antibody.The combination of sensitive CL reagents with the specificity of immunoassay will ensure the rapid development of this technique. Recent work has involved a search for CL reactions of underivatised drugs. These reactions are conveniently studied by flow injection analysis9 (FIA). CL provides sensitivity and selectivity whereas FIA provides rapid and reproducible sample injection and mixing with the reagent. These factors, together with low cost and simplicity, make the CL - FIA combination extremely attractive, and several applications have been described.10.11 In the present system, a flow- through detector built in this laboratory is used to detect CL generated by the oxidation of morphine to the dimer, pseudomorphine, with permanganate in an acidic tetraphos- phate solution.12 Structurally related narcotic analgesics were found to give similar reactions. The system is intended to form the basis of post-column HPLC detection of such drugs. Experimental Reagents Morphine hydrochloride and the other alkaloids described were obtained from the drug collection of the Central Research Establishment, Home Office Forensic Science Service, Aldermaston.“Polyphosphoric” acid (tetraphos- phoric acid), pentasodium triphosphate, potassium dihy- drogen orthophosphate and orthophosphoric acid were of laboratory-reagent grade (BDH Chemicals); sodium tetra- meta-, trimeta- and pyrophosphate were obtained from Albright and Wilson. All other chemicals were of analytical- reagent grade and were used as received. All solutions were prepared in de-ionised water. Construction of Flow-through CL Detector Flow-through CL detectors have previously been described by Wheatley13 and Faizullah and Townshend.11 The present detector is similar, but more compact. A diagram of the detector housing is shown in Fig. 1. It contains a Perspex T-piece for the efficient mixing of the sample and oxidant, a coiled glass flow cell backed by a mirror for maximum light collection and a sensitive photomultiplier tube (PMT) (Thorn EMI, 9789QB) for measurement of the emitted light intensity.The PMT is operated at 1120 V, provided by a stable high-voltage power supply (Thorn EMI, Model 3000R). No wavelength selection is involved. The coiled glass flow cell was as used previously.llJ3 It had four turns of 0.8 mm i.d. glass tubing, enabling the flowing, emitting solution to remain in view of the detector for up to 30 s. For batch screening of drugs for CL, a Berthold Biolumat LB9500 T was used. Flow System for the Determination of Morphine by Chemi- luminescence A schematic diagram of the system used is shown in Fig. 2. Polyphosphoric acid (0.1 M, pH 1.2) was used as the carrier Housing Photocathode Stand Fig.1. Chemiluminescence detector and housing (side elevation). I, Inlet(s); W, waste636 Sample injection (25 wI) I T-Diece ANALYST, JUNE 1986, VOL. 111 Housing I Flow cell 1.3 ml min-' Acid, pH 1.2 II) (6 x 1 0 - 4 ~ ) mt 4 Waste Fig. 2. Flow injection manifold for morphine determination 1.3 ml min-' 700 > 600 E . 4- ). v) Q) .- .- E 500 C 0 v) .- .- E IJJ 400 1.0 2.0 3.0 4.0 5.0 6.0 7.0 Flow-rate/ml min-' Fig. 3. Effect of total flow-rate on CL intensity. 1 X 10-3 M morphine, 1.5 M HCl, 1 x M KMn0, stream and 6 X M potassium permanganate was used as the oxidant. Each solution was pumped at 1.3 ml min-1 by a peristaltic pump (Ismatec Mini-S-820). PTFE tubing (0.8 mm i.d.) was used to connect the reagent streams to the T-piece. Sample injection was effected with a Rheodyne Type 50 PTFE rotary valve, fitted with a 2 5 ~ 1 sample loop.The CL emission was recorded on a chart recorder (Chessell BD40 04) and the peak heights were measured. Results and Discussion Preliminary Work Several drugs were screened for CL reactions with various oxidants using a commercial luminorneter. This discrete sampling system was not ideal in that the mixing of sample and reagents was neither complete nor reproducible over the short time scale ( 4 0 s) of these CL reactions. However, it gave a qualitative selection of the drugs and oxidants that gave the most intense CL. Among these, the morphine - permanganate system was found to be very sensitive and was chosen for further study.Optimisation of Experimental Conditions A series of experiments was conducted to establish optimum analytical conditions. The parameters optimised included flow-rate, pH, permanganate concentration and buffer species and concentration. Effect of Flow-rate The effect of flow-rate is critical, as at flow-rates that are too low or too high, CL is not emitted in the flow cell and hence c '5 2.0 .- E 1.8 0, 1.6 I I I 1 -3.0 -2.0 -1.0 0.0 1 .o Log ([HCI]/M) 1.4' ' Fig. 4. Effect of acid concentration on CL intensity. 1 x 10-3 M morphine, 1 x 10-3 M KMnO,, total flow-rate 2.6 ml min-1 2.8 - 2.6 > E ). v) c . 4- .- 2.4 .- C 0 In .- .- $ 2.2 - CI) -I 2.0 I .8 Fig. 5. Effect of permanganate concentration on CL intensity. 0.2 M HCI, 1 X 10-3 M morphine, total flow-rate 2.6 ml min-1 the emitter is not detected. Fig.3 shows the effect of flow-rate on CL intensity. The greatest emission is obtained at a total flow-rate of 4.7 ml min-1 (2.35 ml min-1 €or each reagent). However, if the system was used for post-column detection in HPLC, such a high flow-rate from the column would require an unacceptably high column pressure, so a total flow-rate of 2.6 ml min-1 was chosen €or use in further work. Effect of Acid Concentration The importance of pH effects in luminescence spectroscopy has been reviewed by Schulman.14 The effect of acid concentration on the CL emission was investigated by using 0.001-2.0 M orthophosphoric acid. The results are shown in Fig. 4. The maximum response was obtained with 0.2 M acid, and this acidity was used in further studies.Effect of Permanganate Concentration The effect of permanganate concentration in the range 0.01 X 10-5-1 x 10-5 M is shown in Fig. 5. More detailed examination of the effect of 1 x 10-3-1 x 10-4 M permanganate showed that 6 x 10-4 M gave the greatest intensity.ANALYST, JUNE 1986, VOL. 111 m J 0 - 637 Table 1. Effect of 0.1 M acids on CL intensity Acid Relative CL H3P04 . . . . . . . . . . 6.6 HCI . . . . . . . . . . 8.7 H2S04 . . . . . . . . . . 10.0 H6P4OI3 . . . . . . . . . . 100.0 4.0 1 0.0 Id- I I I 1 -7.0 -6.0 -5.0 -4.0 -3.0 Log ([morphine I/M 1 Fig. 6. Effect of 0.1 M phosphates on intensity at various morphine concentrations. 1, Tetraphosphoric (“polyphosphoric”) acid; 2, tetra- metaphosphate; 3, triphosphate; 4, trimetaphosphate; 5 , pyrophos- phate; 6, orthophosphate; and 7, orthophosphoric acid.6 X M KMnO,, total flow-rate 2.6 ml min-1 / I 1 I -4.0 -1.01 ‘ -10.0 -8.0 -6.0 Log ([m orph i nelh 1 Fig. 7. four measurements) un&r the recommended conditions Calibration gra h for morphine (each point is the mean of Effect of Various Acids The effects of various acids (orthophosphoric, sulphuric, hydrochloric and “polyphosphoric”) were compared. The results are shown in Table 1. The rate of production of CL in each instance was monitored using the commercial lumin- ometer. The rates were very similar, except for “poly- phosphoric” acid, which produced a very rapid initial produc- tion of CL, unlike the other acids. The “polyphosphoric” acid used is mainly tetraphosphoric acid.As this produced the greatest intensity, the effect of other polyphosphates was investigated. Solutions (0.1 M) of ortho-, pyro-, tri-, trimeta- and tetrametaphosphate were prepared, adjusted to pH 1.2 with hydrochloric acid, and the CL intensities measured in these media and in orthophosphoric and tetraphosphoric acid at the same pH. The results are shown in Fig. 6. “Polyphosphoric” acid was again the medium that gave the greatest sensitivity. Further tests showed that 0.1 M “poly- phosphoric” acid gave the greatest CL intensity, there being less emission at lower and higher “polyphosphoric” acid concentrations. Determination of Morphine The conditions recommended for the determination of mor- phine are as follows: 0.1 M “polyphosphoric” acid, pH 1.2, as the carrier stream, 6 x 10-4 M potassium permanganate as the oxidant, a flow-rate of 2.6 ml min-1 (1.3 ml min-1 in each carrier stream) and a voltage supply to the PMT of 1120 V.An aqueous stock solution of 1 x 10-3 M morphine was prepared, from which working solutions were prepared by serial dilution with water. A linear log - log calibration graph for morphine was obtained (Fig. 7) using the flow system described in Fig. 2. The slope is 0.67, indicating an intensity a [morphine13 relationship. Four replicate injections of morphine were made per sample. The relative standard deviation of ten replicate sample injections of 1 x 10-5 M morphine was 1.8%. Fig. 8 shows some typical recorder outputs. The analysis time is short and a throughput of 150 samples per hour is possible.There was no detectable blank signal. The morphine concen- tration that gave a signal to noise ratio of 2 was 1 x 10-10 M morphine (2 fmol or 0.7 pg per injection). Interferences Narcotic analgesics Thirty-five narcotic analgesics were screened for CL using the flow injection system and recommended conditions for morphine determination. Solutions of 0.5 mg ml-1 of each of the compounds listed in Table 2 were prepared. The results for triplicate measurements are shown in Table 2. It is clear that only seven of the compounds tested emit strongly. If present they would interfere with the detection of morphine. The structural formulae of these compounds are shown in Table 3. There are close similarities in their structures so it is likely that they all react analogously.It is intended to apply the CL method for post-column HPLC of morphine in body fluids, so that related compounds would be separated and not interfere. The procedure also should be applicable to the determination of each of these compounds in , for example, pharmaceutical products. Metal ions Trace metals (100 pg ml-1) were added to the “poly- phosphoric” acid. Their effects are shown in Table 4 and compared with those obtained in orthophosphate solution. In “polyphosphoric” acid, most metal ions had a quenching effect, except silver. In orthophosphate, most metal ions enhanced the CL. The addition of manganese(I1) doubled the intensity in orthophosphate solution. However, this enhanced emission was less than that obtained in “polypho~phoric’~ acid with no added manganese(I1). In hydrochloric acid, the presence of increasing concentrations of manganese(I1) had no effect on the emission intensity from 1 X 10-4 M morphine, but in orthophosphate solution adjusted to pH 1.2 the CL638 ANALYST, JUNE 1986, VOL.111 Table 2. Relative CL intensities for narcotic analgesics and related compounds Compound Relative signal Compound Relative signal Dihydromorphine . . Buprenorphine . . Normorphine . . . . Nalorphine . . . . Morphine . . . . MorphineN-oxide . . 6-Monoacetylmorphine Naloxone . . . . Benzylmorphine . , Ethylmorphine . . Norcodeine . . . . Phenazocine . . . . Pentazocine . . . . Codeine . . . . . . Pholcodine . . . . Levallorphan . . . . Dihydrocodeine . . Morphine 3-glucuronide . . . . 100 . . . . 100 .. . . 97 . . . . 96 . . . . 96 . . , . 95 . . . . 93 . . . . 57 . . . . 7.9 . . . . 3.2 . . . . 2.6 . . . . 2.1 . . . . 2.1 . . . . 1.9 . . . . 1.8 . . . . 1.6 . . . . 1.3 . . . . 0.8 Norlevorphanol . . . . . . 0.7 Thebacon . . . . . . . . 0.7 Diamorphine . . . . . . . . 0.6 Hydrocodone . . . . . . . . 0.6 Thebaine . . . . . . . . . . 0.5 Papaverine . . . . . . . . 0.3 Oxycodone . . . . . . . . 0.2 Phenoperidine . . . . . . . . 0.1 Methadone . . . . . . . . 0.08 Normethadone . . . . . . 0.08 Dextropropoxyphene . . . . 0.03 Pethidine . . . . . . . . 0.03 Dipipanone . . . . . . . . 0.02 Norpipanone . . . . . . . . 0.02 Piritramide . . . . . . . . 0.01 Ethoheptazine . . . . . . . . 0.01 Fentanyl . . . . . . . . . . 0.00 Table 3. Structural formulae of chemiluminescent narcotics 3 4 R2 R3 Compound Morphine .. . . Normorphine . . . . Dihydromorphine . . 6-Monoace tylmorphine MorphineN-oxide . . Nalorphine . . . . Naloxone . . . . R1 CH3 . . . . CH3 CH3 . . . . O,CH3 . . . . CH2CH=CH2 . . . . CH2CH=CH2 . . . . . . . . H . . . . Buprenorphine . . . . . . R2 OH OH OH OH OH OH OH R3 OH OH OH-saturated bond at OCOCH3 OH OH =O-saturated bond at C-7-C-8 and OH at C-7-C-8 C-14 OH HO OCH3 Table 4. Effect of metal ions Relative CL intensity In “pol yphosphoric” Metal ion acid 1.00 Ca2+ - . . . . . . . . Mg2+ - Na+ . . . . . . . . . . . . . . . . - Mn2+ . . . . . . . . 0.61 cu2+ . . . . . . . . 0.49 co2+ . . . . . . . . 0.61 Zn2+ . . . . . . . . 0.93 Ni2+ . . . . . . . . 0.54 Cd2+ . . . . . . . . 0.94 Fez+ . . . . . . . . 0.47 A13+ .. . . . . . . 0.79 cr3+ . . . . . . . . 0.70 vo2+ . . . . . . . . 0.76 Mo042 . . . . . . 0.95 Ag+ . . . . . . . . 1.15 In orthophosphate solution* 0.40 0.34 0.37 0.44 0.79 0.40 0.42 0.49 - - - - - - - - * Adjusted to pH 1.2 with orthophosphoric acid. 600 500 400 > E .: 300 E . C 03 .- Lu 200 1 min E H Ill D 100 Time Fig. 8. Typical recorder outputs for permanganate - morphine CL under the recommended conditions. Morphine concentrations: A, 1 X 10-6 M; B, 4 x 10-6 M; C, 5 x M; D, 8 x 10-6 M; and E, 1 x 10-5 MANALYST, JUNE 1986, VOL. 111 639 increased as the manganese(I1) concentration increased. This effect is shown in Fig. 9. To eliminate the effects of metal ions, the use of ion-exchange columns11 is a possibility. Nature of the Chemiluminescent Reaction Oxidation of morphine [e.g., by hexacyanoferrate(II1) in alkaline solutionl5J6] produces mainly the highly fluorescent dimer pseudomorphine, and a small amount of morphine N-oxide.17 This reaction has been used for post-column fluorescence detection of morphine after HPLC separation. It is likely that pseudomorphine is also formed during the CL detection.HO n OH /N\ I CH? Pseudomorphine Structural features that influence the oxidation of morphine to pseudomorphine18 have been reported by Nelson et al. 16 and are relevant here, as some requirements for fluorescence and CL are often very similar. Darwin and Cone18 have postulated that the C-2 position of morphine is activated by the presence of the C-3 phenolic group via a quinone-type tautomer, which may undergo oxidative coupling to form the dimer.Blocking this process with an alkyl (codeine) or acyl (heroin) group at C-2 would prevent dimerisation, and such compounds do not fluoresce nor chemiluminesce. The com- pounds in Table 3, which are the CL emitters, all have a hydroxy group at C-2, which supports a mechanism for CL involving pseudomorphine formation. Another structural feature important in the production of CL is the furan oxygen bridge. Compounds without this feature (e.g., levallorphan) exhibited much less CL (Table 2). The importance of this bridge is probably in the extension of the conjugated system and increasing rigidity. t >. In C c a) C c. .- U .- 8 2 3 LL - 330 nm 800 700 600 500 400 300 Wavelength/nm Fig. 10. Fluorescence emission spectrum of Mn2+ in “poly- phosphoric” acid.R, Rayleigh scattering; kex, = 277 nm; 0.1 M polyphosphoric acid, containing 1 x 10-3 M Mn2+ t > In c c .- c .- a) In 2 7 L - 350 nm 800 700 600 500 400 300 Wavelengthhm Fig. 11. Fluorescence emission spectrum of morphine. R, Rayleigh scattering; kex. = 277 mm; 1 x 10-3 M morphine I 430 nm 700 600 500 400 300 Wavelengthhm Fig. 12. Fluorescence emission spectrum of a mixture of equal volumes of 6 x 10-4 M KMnO, in 0.1 M pol phosphoric acid and 1 x 10-3 M morphine. R, Rayleigh scattering; xex, = 277 nm640 ANALYST, JUNE 1986, VOL. 111 Other studies in this laboratory have shown that electron- donating groups in aromatic systems, such as NH2 and OH, often enhance CL, whereas electron-withdrawing moeities such as NO2 and S03H inhibit CL.This behaviour is very similar to that expected in fluorescence. 1g720 The quenching effect of electron-withdrawing species is thought to arise because of the introduction of n - n * transitions into the molecule. These are characterised by relatively low molar absorptivities and slow fluorescence emissions because there is very little overlap between the n and n* orbitals. Hence compounds with n - n* transitions rarely fluoresce efficiently. As CL and fluorescence both occur by radiative decay processes and only differ in the manner of excitation, the extension of this concept to CL would seem reasonable. The product of the reaction, pseudomorphine, is highly fluorescent and therefore a possible CL emitter. However, other oxidants such as alkaline permanganate, hydrogen peroxide, hypochlorite and hexacyanoferrate(II1) give no CL with morphine, although they can give rise to CL in other systems.21-24 This suggests that the simple formation and decay of excited pseudomorphine is an unlikely explanation for the CL.Fig. 6 shows the effect of various phosphate species on CL intensity. “Polyphosphoric” acid produces the greatest signal, yet all plots have the same log - log slope, so a common CL mechanism is likely, although the intensity cc [morphine]$ dependence indicates that it is not simple. Fig. 9 shows that manganese(I1) enhances CL in the presence of orthophos- phate, possibly by involvement of a manganese - phosphate complex in the production of CL. Manganese - polyphosphate complexes tend to be more stable as the polyphosphate chain length increases.25 Solid manganese(I1) phosphates are luminescent,26 as are various other solid compounds, e.g., those of manganese(1V) and -(V).27-29 The fluorescence emission spectrum (Fig.10) of man- ganese(I1) in polyphosphoric acid shows two peaks. One (at 330 nm) is close to the emission maximum for morphine (350 nm), which is shown in Fig. 11. Fig. 12 shows a fluorescence emission spectrum of the reaction mixture of morphine, permanganate and polyphosphoric acid. The peak at 350 nm is probably due to unoxidised morphine. The peak at 430 nm is pseudomorphine, as this emission wavelength is very close to the 436 nm obtained by Jane and Taylor15 after hexacyano- ferrate( 111) oxidation. The nature of the emitting species in this CL reaction is unknown, especially as equipment to monitor CL emission spectra was not available. However, a suggestion is proposed on the basis of the current experimental information.A manganese species, formed when permanganate is reduced by morphine, forms a complex with the polyphosphoric acid. The fluorescence spectra (Figs. 10 and 12) show that the emission spectrum of the complex overlaps the pseudomorphine emission spectrum, and energy is transferred from pseudo- morphine to the manganese complex, which is the emitter. In the presence of weaker complexing agents, such as ortho- phosphate, manganese has to be added to the system to obtain the greatest intensity. Much further work is needed to elucidate the precise mechanism, and this will be described in a later paper.The method is being applied to post-column HPLC detec- tion. The eight opiates in Table 3 can be separated and resolved by careful selection of the mobile phase. Methods for the determination of morphine in body fluids are under investigation. The authors thank the SERC and the Home Office for the provision of a studentship (to R. W. A.) under the CASE scheme. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. Harvey, E. N., “A History of Luminescence from the Earliest Times until 1900,” American Philosophical Society, Philadel- phia, 1957. Kricka, L. J., and Thorpe, G. H. G., Analyst, 1983, 108,1274. Stanley, P. E., Trends Anal. Chem., 1983, 2, 248. Burguera, J. L., and Townshend, A., Talanta, 1980, 27, 309. Kobayashi, S ., Sekino, J., Honda, K., and Imai, K., Anal. Biochem., 1981, 112, 99. Weeks, I., Woodhead, J . S . , J . Clin. Immunoassay, 1984,7,82. Mahant, V. K., Miller, J. N., and Thakrar, H., Anal. Chim. Acta, 1983, 145, 203. Kobayashi, S . , and Imai, K., Anal. Chem., 1980, 52, 424. RfiiiEka, J., and Hansen, E., “Flow Injection Analysis,” Wiley, New York, 1981. Rule, G., and Seitz, W. R., Clin. Chem., 1979, 25, 1635. Faizullah, A. T., and Townshend, A., Anal. Proc., 1985, 22, 15. Abbott, R. W., and Townshend, A., Anal. Proc., 1986,23,25. Wheatley, A., PhD Thesis, University of Hull, 1983. Schulman, S . , Rev. Anal. Chem., 1971, 1, 85. Jane, I., and Taylor, J. F., J . Chromatogr., 1975, 109, 37. Nelson, P. E., Nolan, S . , and Bedford, K. R., J . Chromatogr., 1982, 234, 407. Beaumont, I., and Deeks, T., J . Chromatogr., 1982,238,520. Dar-;in, W. D., and Cone, E. J., J. Pharm. Sci., 1980,69,253. Seitz, W. R., CRC Crit. Rev. Anal. Chem., 1980, 9, 367. Wehry, E. L., and Rogers, L. B., in Hercules, D., Editor, “Fluorescence and Phosphorescence Analysis,” Wiley, New York, 1966. Seitz, W. R., and Hercules, D. M., in Cormier, M. J., Hercules, D. M., and Lee, J., Editors, “Chemiluminescence and Bioluminescence,” Plenum, New York, 1973, p. 427. Schroeder, H. R., and Yeager, F. M., Anal. Chem., 1978,50, 1114. Burguera, J. L., Townshend, A., and Greenfield, S., Anal. Chim. Acta, 1980, 114, 209. Bostick, D. T., and Hercules, D. M., Anal. Chem., 1975,47, 447. Smith, R. M., and Martell, A. E., “Critical Stability Constants, Volume 4: Inorganic Complexes,” Plenum, New York, 1976. Kaplanova, M., Trojan, M., Brandova, D., and Navratil, J., J . Lumin., 1984, 29, 199. Flint, C. D., J . Mol. Spectrosc., 1971, 37, 414. Black, A. M., and Flint, C. D., J. Chem. SOC., Dalton Trans., 1974, 977. Chodos, S. L., Black, A. M., and Flint, C. D., J . Chem. Phys., 1976, 65, 4816. Paper A51440 Received December 2nd, 1985 Accepted January 21st, 1986

ISSN:0003-2654    

DOI:10.1039/AN9861100635

出版商:RSC    

年代:1986

数据来源: RSC

摘要:

ANALYST, JUNE 1986, VOL. 111 641 Flow Injection Spectrofluorimetric Determination of Europium(H1) Based on Solubilising Its Ternary Complex with Thenoyltrifluoroacetone and Trioctylphosphine Oxide in Micellar Solution Makoto Aihara" and Miwako Arai Faculty of Home Life Science, Fukuoka Women's University, Kasumigaoka, Higashi-ku, Fukuoka 813, Japan and Tomitsugu Taketatsu College of General Education, Kyushu University, Ropponmatsu, Chuo-ku, Fukuoka 810, Japan A flow injection spectrofluorimetric method has been developed for the determination of europium(ll1) as its ternary complex with thenoyltrifluoroacetone and trioctylphosphine oxide in a micellar solution of nonaoxyethylene dodecyl ether (BL-SEX). This procedure is satisfactory of the determination of the europium(ll1) ion in the range 1.5-150 ng mi-1 at a sampling rate of 55 per hour.The relative standard deviation was less than 1.2%. No interferences from 20-fold excesses of other rare earth ions were observed. Keywords: Flow injection; spectrofluorimetry; europium(ll1) determination; ternary complex Spectrofluorimetric determinations of europium(II1) as its ternary complex with a P-diketone and a neutral donor extracted into an organic solvent have been reported.1-3 Earlier we found that Eu(II1) - and Sm(II1) - P-diketone - trioctylphosphine oxide (TOPO) ternary complexes are readily soluble in aqueous solutions containing a non-ionic surfactant and that the strong fluorescence originating from the ternary complexes can be applied to the determination of rare earth ions.4.5 The method is simple and rapid compared with the solvent extraction method.This technique could be applied to flow injection analyses of rare earth ions. There have been few reports of applications of flow injection to rare earth ions. Burguera et aZ.6 reported the flow injection analysis of terbium(II1) as its EDTA - sulpho- salicylic acid complex. This paper describes the flow injection spectrofluorimetric determination of europium(II1) as its ternary complex with thenoyltrifluoroacetone (TTA) and TOPO in micellar solu- tion. Experimental Reagents Stock solutions of rare earth ions were prepared by dissolving the 99.9% pure oxides in dilute hydrochloric acid and standardised by titration with EDTA. Stock solutions of mixed ligands (TTA and TOPO) were prepared by dissolving the reagents (Dojin) in a 0.5% mlV aqueous solution of surfactant (BL-9EX; Nikko Chemical Co.), and the pH of the resulting solutions was adjusted to 3.5 by addition of acetate buffer.All other chemicals used were of analytical-reagent grade. Apparatus The flow injection system is shown in Fig. 1. It consisted of a reciprocating pump with two channels (plunger type, Kywaseimitu KHU-W-62), a six-way sample injection valve (Nihon Seimitsu, NV-508-6M), a spectrofluorimeter (UVIDIC-610, Japan Spectroscopic Co.) with a flow cell (32 pl), and a switching valve (Nihon Seimitsu, NV-508-3M) for * To whom correspondence should be addressed. stopped flow. The switching valve was located before the detector.7 The manifolds were made from polyethylene tubing (0.5 mm i.d.). Procedures Samples containing europium(II1) at concentrations in the range 10-8-10-6 M (1.5-150 ng ml-1) were injected into the system at a rate of 55 samples per hour.The fluorescence was measured at apparent excitation and emission wavelengths of 352 and 613 nm, respectively. For the stopped-flow method, the switching valve was switched to lead the carrier solution to waste (W) and to stop the sample zone in the flow cell for 2 min. The valve was then switched on-line to the detector again. Results and Discussion The formation of a complex of europium(II1) with TTA and TOPO in a micellar solution of non-ionic surfactant has been investigated and the following equilibrium was postulated? EU3+(b) 4- 3H'TTA(b) 4- 2TOPO,,) S E~(TTA)~(TOPO)~(I~) -k 3H+(b) where the subscripts (b) and (m) refer to mutually equilibrated bulk and micellar phases, respectively. The fluorescence intensity for the Eu(TTA)~(TOPO)~ complex was constant over the pH range 3.04.0.Changes in the concentration of surfactant in the range 0.2-1.5% m/.Y did not affect the fluorescence intensity. Therefore, detailed investigations c r f 7 n W R U Fig. 1. Flow manifold for the determination of europium(II1). C, Carrier solution (0.5% m/V BL-9EX and pH 3.5 acetate buffer solution); R, reagent solution (5 x M TTA - 5 X 10-4 M TOPO, 0.5% m/V BL-9EX and pH 3.5 acetate buffer solution); S, sample injection (100 ~ 1 ) ; M, mixing joint; RC, reaction coil (2 m); TI, stopped-flow valve; T2, three-way joint; D, detector; BC, back- pressure coil (1 m); P, pump; and W, waste642 ANALYST, JUNE 1986, VOL.111 stop A { B r Scan - 1 oc E E . c r rn 0) r al a .- g 50 1 2 3 4 5 6 7 Concentration of mixed reagents/104~ Fig. 2. A, normal FIA peak and B, stopped-flow signal obtained with the flow system. Manifold as in Fig. 1; sample concentration, 4 x 10-7 M Fig. 5. Peak height as a function of mixed reagent concentration. Manifold as in Fig. 1 except for reagent concentration. [Eu(III)]: A, 2 x 10-7; B, 4 x 10-7; C, 6 x 10-7; D, 8 x and E , 10 x M 0.6 0.8 1.0 1.2 Flow-rate/mI min-I Fig. 3. Peak height as a function of flow-rate. Manifold as in Fig. 1 except for flow-rate. [Eu(III)]: A, 2 X D, 8 X 10-7; and E, 10 x 10-7 M B, 4 X C, 6 x 200 E E -c' 'a, 100 . P, .c Y (II n I I I I I 1 2 3 4 5 Coil lengthlm Fig.4. Peak height as a function of coil length. Manifold as in Fig. 1 except for coil length. [Eu(III)]: A, 2 x 10-7; B, 4 x 10-7; C, 6 x 10-7; D, 8 x 10-7; and E, 10 x 10-7 M Q, al D F c ' 10min Scan - Fig. 6. solutions. Manifold as in Fig. 1. [Eu(III)j: A, 1 X 10-7; B, 2 X C, 4 x 10-7; D, 6 x 10-7; E, 8 x 10-7; and F, 10 x 10-7 M Peaks obtained for triplicate in'ections of standard Eu(II1) 10-7 10-6 0 1 10-8 10'-7 A Concentration of Eu(lll) solution injectedh Fig. 7. nation of europium(II1). Manifold as in Fig. 1 Typical calibration graphs for the simultaneous determi-ANALYST, JUNE 1986, VOL. 111 643 Effect of reagent concentration Table 1. Effect of other ions on determination of europium(II1). Eu(II1) taken, 76.0 ng ml-1 Sc(II1) Y(II1) La(II1) Ce(1V) Pr(II1) Nd(II1) Sm(II1) Gd(II1) Tb(II1) DY(II1) Ho(II1) Er(II1) Tm(II1) Yb(II1) Lu( 111) Eu(II1) Ion added foundhg ml-1 .. . . . . . . . . . . 80.0 . . . . . . . . . . . . 74.4 . . . . . . . . . . . . 76.9 . . . . . . . . . . . . 76.2 . . . . . . . . . . . . 77.7 . . . . . . . . . . . . 78.7 . . . . . . . . . . . . 76.0 . . . . . . . . . . . . 80.5 . . . . . . . . . . . . 77.6 . . . . . . . . . . . . 78.4 . . . . . . . . . . . . 80.3 . . . . . . . . . . . . 77.9 . . . . . . . . . . . . 75.8 . . . . . . . . . . . . 79.4 . . . . . . . . . . . . 78.9 Recovery, % 105 98 101 100 102 102 100 106 102 103 106 103 100 105 105 were made to establish the optimum conditions for the determination of europium(II1) in 0.5% mlV BL-9EX at pH 3.5. The stopped-flow pattern is shown in Fig.2. The samples were injected successively to obtain a normal flow injection signal and a stopped-flow signal. The stopped-flow signal shows a plateau, which indicates that the complex formation reaction of the europium(II1) ion with TTA and TOPO in a non-ionic surfactant proceeded to completion under the conditions described in Fig. 1. Chemical and Flow Injection Variables Effect of flow-rate The flow-rates of the reagent and the carrier solutions were studied and Fig. 3 shows the variation in peak height with flow-rate. The peak height increased as the flow-rate increased up to 1.0 ml min-1 and became almost constant above this value. For the lower europium(II1) concentration (B in Fig. 3), dispersion of the sample zone was observed at a flow-rate of 1.2 ml min-1.A flow-rate of 1.0 ml min-1 was chosen for subsequent work. Effect of reaction coil length The effect of reaction coil length was tested by the use of lengths of 1-5 m while the flow-rate was maintained at 1.0 ml min-1. The results are shown in Fig. 4. The peak heights decreased with increasing coil length, which was to be expected from the results given by the stopped-flow method. An increase in coil length caused a decrease in peak height because of increased dispersion. A 2-m coil length was chosen for subsequent work because of the reproducibility of the method and the rapid analysis achieved. The effect of reagent concentration on europium(II1) ternary complex formation with TTA and TOPO was studied, the concentrations of TTA and TOPO being varied.The results are shown in Fig. 5. The peak height increased as the mixed reagent concentration increased up to 3 x 10-4 M at each europium(II1) concentration and became almost constant above this value. A mixed reagent concentration of 5 x 10-4 M for TTA and TOPO was chosen for subsequent work. Determination of Europium(II1) Ion Samples containing 10-8-10-6 M (1.5-150 ng ml-1) eu- ropium(II1) were injected into the carrier stream under the following optimum conditions: concentration of TTA and TOPO, 5 X l o - 4 ~ ; concentration of BL-gEX, 0.5% mlV; pH, 3.5; flow-rate, 1 ml min-1; and coil length, 2 m (0.5 mm i.d.). Typical calibration peaks are shown in Fig. 6. The calibra- tion graphs obtained were linear in the range 1.5-150 ng ml-1, as shown in Fig. 7. The precision of the method was checked on ten samples containing 5 x 10-7 M europium(II1). The relative standard deviation was less than 1.2%.Interferences The influence of other rare earth ions on the determination of 76.0 ng ml-1 of europium(II1) was studied and the results are given in Table 1. It was found that 20-fold excesses of Sc, Y, La, Ce(IV), Pr, Nd, Sm, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu had no significant effect. The authors are grateful to Dr. Norimasa Yoza of Kyushu University for valuable discussions. They are also grateful to Mr. Toshihiko Imatou of Kyushu University for helpful advice. 1 2. 3. 4. 5. 6. 7 8. References Fisher, R. P., and Winefordner, J. D., Anal. Chem., 1971,43, 452. Shigematsu, T., Matsui, M., and Wake, R., Anal. Chim. Acta, 1969,46, 101. Belcher, R., Perry, R., and Stephen, W. I., Analyst, 1969,94, 26. Taketatsu, T., Talanta, 1982, 29, 397. Taketatsu, T., and Sato, A., Anal. Chim. Acta, 1979,108,429. Burguera, J. L., Burguera, M., and Gallignani, M., Acta Cient. Venez., 1982, 33, 99. Yoza, N., Kurokawa, Y . , Hirai, Y., and Ohashi, S., Anal. Chim. Acta, 1980, 121, 281. Taketatsu, T., Chem. Lett., 1981, 1057. Paper A51439 Received November 27th, 1985 Accepted January 6th, 1986

ISSN:0003-2654    

DOI:10.1039/AN9861100641

出版商:RSC    

年代:1986

数据来源: RSC