摘要:

Communication The preparation of a sol–gel glass oxygen sensor incorporating a covalently bound fluorescent dye Chris Malins,a Stefano Fanni,b Herveline G. Glever,a Johannes G. Vosb and Brian D. MacCraith*a a Optical Sensors Group, School of Physical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland b School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland Received 9th November 1998, Accepted 10th December 1998 The synthesis of a novel ruthenium(ii) polypyridyl complex incorporating silane pendant moieties is described.This fluorescent material was covalently bound to an organically modified silica glass precursor to give thin films suitable for optical oxygen sensing. These films showed good oxygen sensitivity characteristics, and possess the distinct advantage over physically entrapped dyes of being chemically attached to the support membrane. This approach to the preparation of luminescent micro-porous glass films provides sensitive membranes suitable for applications where zero dye leaching is a requirement.The sol–gel route to silica glass thin films is commonly used to immobilise dyes for application in optical chemical sensors.1 However, when only physical entrapment is employed, leaching of the dopant into the analyte solution is usually encountered. This phenomenon arises when the average pore diameter of the encapsulating membrane is larger than the size of the dopant molecule, and represents a serious problem in terms of signal stability and membrane longevity.2 One approach to solving this problem has been to covalently bind the dye using 3-aminopropyltriethoxysilane. Silanisation of a glass surface can be achieved by extended reflux in a solution of this reagent.3 The amino group is then used to covalently bind to a suitable dyestuff, such as pyrene4 for oxygen sensitivity, for fluorescein isothiocyanate5 for fluorimetric pH measurements.Recently, amino fluorescein has been immobilised using the sol–gel process, by mixing with 3-(trimethoxysilyl)propylisocyanate,6 although this was shown to be an inefficient encapsulation strategy, as the combination of the silane and dye was incomplete.Surprisingly, a similar approach has never been reported for ruthenium(ii) polypyridyl complexes, generally accepted to be the most useful dyes for optical oxygen sensing applications.7 In this communication we wish to report our novel route to a glass in which a ruthenium complex is covalently bound, for use in optical oxygen sensors.We have successfully functionalised a suitable ruthenium(ii) polypyridyl moiety with triethoxysilane groups, and utilised the resulting functionalised complex to prepare a glass using a standard sol–gel method. Silanisation of the dye, rather than the glass substrate, provides the advantage of a more homogeneous and well-defined distribution of the dye in the glass film.To the best of our knowledge, this is the first example of a ruthenium(ii) polypyridyl dye covalently incorporated onto a silica glass film. Experimental Molecular structures for the product (2) and precursors in the synthetic scheme are given in Fig. 1. [4,4A-Bis(chlocarbonyl)- 2,2A-bipyridine]bis(2,2A-bypiridine)ruthenium(ii) dichloride (1) was synthesised as described in the literature.8 [4,4A-Bis[(3- triethoxysilyl)propylamide]-2,2A-bipyridine]bis(2,2A-bypiridine) ruthenium(ii) dichloride (2) was obtained by reacting 1 with 3-aminopropyltriethoxysilane (2.2 equivalents) in dry acetonitrile, in the presence of an excess of triethylamine.After removal of the solvent, the highly reactive, moisture sensitive bis-amide 2 was used without further purification to prepare a glass using the sol–gel process. A 4 : 1 molar ratio of water to glass precursor was employed in this instance. To a stirred solution of the bis-amide 2 (60 mg) in ethanol (3.0 g) and aqueous HCl (pH 1, 1.6 g) was added methyltriethoxysilane (4.0 g).Stirring was then continued for a further hour, after which thin films (of thickness 400 nm) were deposited onto soda glass slides by a dip-coating procedure.9 The coated glass slides were then dried overnight (70 °C), and left to stabilise at room temperature in the laboratory for one week, prior to examination. Characterisation of the sensitivity towards oxygen of the fluorescent glass films was undertaken using a planar waveguide configuration described elsewhere,10 and the details of the gas and aqueous flow systems can be found in the literature.11 A pulsed blue light emitting diode (lmax = 450 nm) was used to directly illuminate a coated glass slide.The resulting fluorescence is guided along the substrate and collected at the end face by a photodiode controlled by dedicated lock-in circuitry. Results and discussion The covalently immobilised dye material displays an emission band centred at 610 nm upon excitation with a blue light Fig. 1 Molecular structure of the product (2) and intermediates for the synthetic route. Anal. Commun., 1999, 36, 3–4 3emitting diode. This fluorescence is dynamically quenched by molecular oxygen, as described by the Stern–Volmer relation: I0/I = 1 + KSV pO2 (1) where I0 and I are the emission intensities in the absence and presence of oxygen respectively, KSV is the Stern–Volmer constant and pO2 is the partial pressure of oxygen.The emission was monitored upon exposure to both gaseous and dissolved oxygen. The fluorescence emission upon repeated exposure to oxygen/nitrogen cycles in the gas phase is shown in Fig. 2. We can see that the sensor response is stable and very rapid, being of the same order as is found for physically entrapped ruthenium(ii) polypyridyl dye complexes.11 Linear regression of the Stern–Volmer plots obtained for gas phase and dissolved oxygen sensitivity shown in Fig. 3 reveals a limit of detection12 of below 1% oxygen. The sensitivity towards oxygen of these films is reflected in Stern–Volmer constants of 0.005 and 0.008 O2%21 for gas and aqueous phases, respectively. These values are similar to those previously reported for physically immobilised tris(2,2A-bipyridine)ruthenium(ii) thin films.13 We can conclude, therefore, that the oxygen sensing characteristics of the dye are not impaired by the presence of the silane pendant groups. Conversely, when the 4,4A-dicarboxy derivative 3 was used as the dopant in sol–gel films prepared by the same procedure, the resulting glass showed only a very weak fluorescence emission centred at 640 nm, with negligible sensitivity to oxygen.We can therefore state that the acid chloride (1) has been successfully converted to the silane in good yield. Preliminary dye-leaching investigations in a range of solvents have revealed enhanced performance for sol–gel glass thin films incorporating the covalently bound dye, in comparison with similar films incorporating physically entrapped tris(2,2-bipyridine)ruthenium(ii) chloride.Therefore, this material is suitable for the preparation of sol–gel derived glass films of increased porosity. If the molar ratio of water to glass precursor is reduced below 4 : 1 the extent of leaching for physically immobilised dye materials is greatly increased.2 However, such conditions are desirable due to the greater sensitivity encountered with such films, due to the increased surface area of the membrane and the dependency of the Stern– Volmer constant upon diffusion of the analyte through the membrane.Work is currently underway to fully characterise these, and other oxygen sensitive films incorporating covalently bound ruthenium(ii) complexes with longer luminescence lifetimes, which should further improve sensitivity for a novel class of oxygen sensitive membranes that shows a very high degree of chemical stability.Conclusions This material offers the attractiveness of ruthenium(ii) polypyridyl complexes for oxygen sensing applications, the stability, robustness and ease of preparation of sol–gel derived glass, alongside the resistance to leaching of a covalently bonded dyestuff. This, therefore, represents a significant advance in the field of optical sensor chemistry, and opens the way to use in in vivo biomedical applications, such as the fluorescence-based monitoring of oxygen uptake during anti-cancer photodynamic therapy,14 and more widespread utilisation for oxygen monitoring tasks in the food and beverages industry.References 1 O. Lev, M. Tsionsky, L. Rabinovich, V. Glezer, S. Sampath, I. Pankratov and J. Gun, Anal. Chem., 1994, 66, 1120A. 2 T. M. Butler, B. D. MacCraith and C. McDonagh, J. Non-Cryst. Solids, 1998, 224, 249. 3 A. Bromberg, J. Zilberstein, S. Reisemberg, E. Benori, E. Silberstein, J. Zimnavoda, G. Frishman and A.Kritzman, Sens. Actuators B, 1996, 31, 181. 4 J. Zilberstein, A. Bromberg and G. Berkovic, J. Photochem. Photobiol. A: Photochem., 1994, 77, 69. 5 (a) G. E. Badini, K. T. V. Grattan and A. C. C. C. Tseung, Rev. Sci. Instrum., 1995, 6, 4034; (b) G. E. Badini, K. T. V. Grattan and A. C. C. C. Tseung, Analyst, 1995, 120, 1025. 6 A. Lobnik, I. Oehme, I. Murkovic and O. S. Wolfbeis, Anal. Chim. Acta, 1998, 367, 159. 7 J. N. Demas and B. A. DeGraff, J. Chem. Edu., 1997, 74, 690. 8 P. D. Beer, F. Szemes, V. Balzani, C. M. Sala, M. G. B. Drew, S. W. Dent and M. Maestri, J. Am. Chem. Soc.,1997, 119, 11864. 9 C. J. Brinker, A. J. Hurd, P. R. Schmuck, G. C. Frye and C. S. Ashley, J. Non-Cryst. Solids, 1992, 147, 424. 10 J.-F. Gouin, A. Doyle and B. D. MacCraith, Electronics Lett., 1998, 34, 1685. 11 (a) A. K. McEvoy, C. M. McDonagh and B. D. MacCraith, Analyst, 1996, 121, 785; (b) C. McDonagh, B. D. MacCraith and A. K. McEvoy, Anal. Chem., 1998, 70, 45. 12 J. N. Miller and J. C. Miller, Statistics for Analytical Chemistry, Ellis Horwood, Chichester, 1988, pp. 115. 13 A. Mills and F. C. Williams, Thin Solid Films, 1997, 306, 163. 14 J. Zilberstein, A. Bromberg, A. Frantz, V. Rosenbach-Belkin, A. Kritzmann, R. Pfefermann, Y. Salomon and A. Scherz, Photochem. Photobiol., 1997, 65, 1012. Paper 8/08731H Fig. 2 Fluorescence emission on cyclic exposure to oxygen and nitrogen in the gas phase for a [4,4A-bis[(3-triethoxysilyl)propylamide]-2,2A-bipyridine] bis(2,2A-bypiridine)ruthenium(ii) dichloride/methyltriethoxysilane film. Fig. 3 Stern–Volmer plots for a [4,4A-bis[(3-triethoxysilyl)propylamide]- 2,2A-bipyridine]bis(2,2A-bypiridine)ruthenium(ii) dichloride/methyltriethoxysilane film in the gaseous and aqueous phases. 4 Anal. Commun., 1999, 36, 3–4

ISSN:1359-7337    

DOI:10.1039/a808731h

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Preliminary study on fluorimetric detection of aflatoxins Q1, P1 and B1 using heptakis-di-O-methyl-b-cyclodextrin as post-column HPLC reagent B. I. Vázquez,*a C. A. Fente,a C. M. Franco,a A. Cepeda,a G. Mahuzierb and P. Prognonb a Laboratorio de Higiene e Inspección de los Alimentos, Dpto. de Química Analítica Nutrición y Bromatología, Facultad de Veterinaria, 2700 Lugo, Spain b Laboratoire de Chimie Analytique II—Bioanalyse, Faculté de Pharmacie, Université Paris-Sud, 5, rue J.B. Clément, 92290 Châtenay-Malabry, France Received 7th July 1998, Accepted 23rd November 1998 Post-column fluorimetric detection for the determination of aflatoxins Q1, P1 and B1 was carried out by using HPLC with a 2.0 mm id column. The post-column reagent consisted of a 1022 M aqueous solution of heptakis-di-O-methyl-b-cyclodextrin. The fluorescence enhancement achieved was 37- and 27-fold for AFQ1 and AFB1, respectively, whereas the AFP1 signal was increased just about 2-fold.With the proposed method, aflatoxins Q1, P1 and B1 can be simultaneously determined in human urine. The detection limits (S/N = 3) were as follows: 0.7 ng ml21 for AFQ1, 0.5 ng ml21 for AFP1 and 0.3 ng ml21 for AFB1. Introduction Aflatoxin B1 (AFB1) is a toxin produced by the widespread moulds Aspergillus flavus and Aspergillus parasiticus.1,2 Following consumption of food and feed contaminated with this mycotoxin, it is known that AFB1 is hydroxylated in the liver by several mammalian species, including humans, to yield aflatoxins Q1 (AFQ1) and P1 (AFP1), the two major metabolites produced (Fig. 1).3–6 Aflatoxins are powerful carcinogenic and mutagenic agents,7 and some of these metabolites have been explored as urinary biomarkers for hepatocellular carcinoma.8,9 All these reasons have focused the efforts of analytical chemists on studying highly sensitive methods to detect AFQ1, AFP1, AFB1 and AFM1. The use of pre-column TFA derivatization has been reported for their determination,10,11 as well as halogenation using bromine12,13 or iodine,14 in order to improve the fluorimetric detection.Nevertheless a major common drawback of these procedures is the instability of the derivatives and the fact that the use of bromine with AFP1 produces a damping of the native fluorescence of the compound.12 The use of cyclodextrins in a post-column reaction system has proved successful in improving the detection limit of the important fungal toxin B1, due to the high fluorescence enhancement obtained with b-CD derivatives.15,16 Our laboratory has determined, in previous work, some spectroscopic data (i.e., Stokes shifts, E(0-0) energy levels, relative quantum fluorescence yields, etc.) for the interaction between the hydroxylated aflatoxins Q1, M1 and P1, and different cyclodextrins. 17 AFM1 was deliberately excluded from the present study due to the demonstrated absence of fluorescence enhancement upon cyclodextrin addition.17 This absence of effect was observed with all cyclodextrins tested i.e., a-, b-, g-, hydroxypropyl-band heptakis-di-O-methyl-b-cyclodextrin. This feature was attributed to a lack of convenient fit between host and guest compound owing to the position of the furanic hydroxyl, which distinguishes AFM1 from AFB1.The aim of the present work was to develop a reversed phase liquid chromatographic system using post-column cyclodextrin derivatization with heptakis-di-O-methyl-b-cyclodextrin in order to enhance the signals of AFQ1, AFP1 and AFB1.A practical example of the feasibility of the proposed method was demonstrated by using human urine. Experimental Chemicals All reagents were of analytical grade. The purity of the organic solvents and ultra-pure water (MilliQ®-quality, Millipore, Molsheim, France) were checked via fluorescence prior to use. Aflatoxins Q1, P1 and B1 were obtained from Sigma (St. Louis, MO, USA), AFQ1 being supplied as a mixture of epimers. Heptakis-di-O-methyl-b-cyclodextrin (DIMEB) was purchased from Wacker (Munich, Germany).Solutions Individual aflatoxins, in crystalline form, were dissolved in acetonitrile as this solvent was recommended to avoid rapid aflatoxin degradation.10 Each solution, protected from light by aluminium foil, was kept at 4 °C for no longer than 1 month. Standards were prepared from these solutions, containing 9.8 3 104 ng ml21 for AFQ1, 4.5 3 103 ng ml21 for AFP1 and 9.4 3 105 ng ml21 for AFB1, and these were stored for one week at 4 °C.Successive dilutions were made with the mobile phase in order to achieve working concentrations and stored at 4 °C for no longer than 24 h. Fig 1 Chemical formulae of the three aflatoxins studied. Aflatoxins Q1 and P1 are hydroxylated derivatives resulting from aflatoxin B1 metabolism. Anal. Commun., 1999, 36, 5–7 5Stock aqueous solutions (1022 M) of the cyclodextrin cited above were prepared daily and maintained at room temperature before use.Urine samples Human urine samples were randomly collected and chromatographically tested for the absence of any of the aflatoxins assayed. Urine samples were prepared and extracted as follows: 50 ml of urine were spiked, by means of a syringe, with 100 ml (calibration limit of syringe) of appropriate amounts of mixtures of AFQ1, AFP1 and AFB1. After homogenization by vigorous shaking, chloroform (5 ml) was added, and then the tube was shaken mechanically for 3 min.After centrifugation for 5 min, the upper aqueous layer was discarded and the organic layer transferred into an amber screw-capped tube and evaporated to dryness under a stream of nitrogen. The residue was reconstituted in 0.5 ml of the mobile phase, and 20 ml injected. Chromatographic conditions The chromatographic system consisted of two LC6A metering pumps (Shimadzu, Kyoto, Japan), one for the mobile phase, a methanol–water mixture (40 + 60 v/v) at a flow rate of 0.2 ml min21, equipped with a Rheodyne® Model 7125, 20 ml loop injector (Cotati, CA, USA), and the second for introducing the post-column reagent, a 1022 M aqueous cyclodextrin solution pumped at a flow rate of 0.3 ml min21.The mixing of the mobile phase and the post-column reagent was performed with a Tee mixer (Supelco, Bellefonte, PA, USA). For chromatographic separation a C18 Nucleosil column (5 mm particle size, 120 Å pore size, 150 3 2.0 mm id) from Tecknochroma (Barcelona, Spain), was used.All measurements were made at room temperature (22 ± 2 °C). A Perkin-Elmer LC240 fluorescence detector (Norwalk, CT, USA) was programmed with the following excitation and emission wavelengths: lex = 365 nm, lem = 466 nm for AFQ1; lex = 365 nm, lem = 504 nm for AFP1; and lex = 360 nm, lem = 435 nm for AFB1, according to ref. 14. The chromatograms were recorded on a Shimadzu CR5A Chromatopac integrator (Kyoto, Japan).When cyclodextrins were added in the mobile phase, they were first dissolved in water and then methanol was added to yield the final desired concentration. Results and discussion Reversed phase HPLC methods (RP-HPLC) are the most widely used for aflatoxins. So, from our own experience with aflatoxins B and G, an isocratic mobile phase of methanol– water (40 + 60 v/v) was envisaged. Methanol was preferred over acetonitrile because of its lower association constant with cyclodextrins.18 A common C18 Nucleosil column (5 mm particle size, 120 Å pore size, 150 3 2.0 mm id) was employed.These toxins are simultaneously determined, in less than 30 min, and the elution order was Q1 < P1 < B1, in accordance with their polarities. Adding cyclodextrins in the mobile phase and post-column optimization In our previous work,17 it was spectroscopically observed that DIMEB, a b-cyclodextrin derivative, was the most suitable for the enhancement of the fluorescence emission of these three mycotoxins.Hence, DIMEB at a concentration of 1022 M, was directly added to the methanol–water mobile phase. Despite the increase in the signal, the analytical problems encountered with the direct introduction of cyclodextrin in the eluent are first, the decrease of the resolution, and second, the back pressure increase due to a higher viscosity of the eluent. Consequently, a post-column addition was preferentially used. For a mobile phase flow rate of 0.2 ml min21, and a fixed concentration of AFQ1, AFP1 and AFB1 (150 ng ml21 each), the post-column dilution effect was studied, first from 0.1 ml min21 to 0.5 ml min21 using deionized water as the reagent.A progressive decrease in the fluorescence signal from about 50 to 10% of the initial signal was observed for these aflatoxins. The decrease ratio (flow rate mobile phase : flow rate post-column solution) does not exactly correspond to that expected theoretically.This could be due to the addition of the water leading to quenching of the fluorescence signal, as already reported.18 Then, in the same experimental conditions, an aqueous 1022 M DIMEB solution (the optimal concentration, yielding the highest signal) was used as post-column reagent and the highest enhancement of the fluorescence signal was achieved at a postcolumn flow rate of 0.3 ml min21 (37-, 2- and 27-fold for AFQ1, AFP1 and AFB1, respectively, referred to the fluorescence signal obtained with post-column water).Although this postcolumn flow rate is larger than that of the mobile phase, the cyclodextrin concentration achieved seems to form inclusion complexes in an environment suitable for an optimum fluorescence signal. AFQ1 was the more highly promoted, even more than AFB1 (37- and 27-fold, respectively); in contrast, AFP1 remained nearly constant (about 2-fold), which may be due to its phenolic structure, which leads to some different spectroscopic behaviour.It should be noted that, although disappointing in comparison with the other aflatoxins tested, this finding allows us to determine AFP1, which is not possible by the destructive post-column halogenation derivatization.12 From a spectroscopic point of view, neither spectral shifts of the emission nor of the excitation have been noticed upon cyclodextrin addition. This indicates that the change in polarity of the eluent after post-column reagent addition has no effect on the environment of the aflatoxin included.Analytical figures of merit. Analysis of real examples The method was tested in real conditions by spiking human urine samples with standards containing a mixture of AFQ1, AFP1 and AFB1. Data from the extraction recovery and F-linearity test of the different aflatoxins, as well as repeatability and reproducibility of the method, are summarized in Table 1. The detection limits (S/N = 3) were as follows: 0.7 ng ml21 for AFQ1, 0.5 ng ml21 for AFP1 and 0.3 ng ml21 for AFB1, with 20 ml injections of the standard samples analysed by HPLC.Table 1 Analytical figures of merit Aflatoxin Extraction recovery (%)a RSD (n = 5) (%) F-linearity (r)a (p < 0.001) (n = 4) (r = ) Repeatabilityb RSD (n = 8) (%) Reproducibilityb RSD (n = 8) (%) LOD (S/N = 3) /ng ml21 Q1 96 9 0.998 9 11 0.7 P1 87 17 0.992 4 7 0.5 B1 94 6 0.996 5 5 0.3 a Extraction recovery and F-linearity test were demonstrated for: AFQ1 from 3 ng ml21 to 66 ng ml21; AFP1 from 2 ng ml21 to 60 ng ml21; and AFB1 from 2 ng ml21 to 31 ng ml21.b Repeatability and reproducibility were demonstrated for c = 15 ng ml21 for AFQ1, AFP1 and AFB1. 6 Anal. Commun., 1999, 36, 5–7These limits certainly do not reach the ng l21 levels for AFB1 and AFQ1 reported in other procedures in the literature (6.8 ng l21 and 18 ng l21, respectively).12,13 Nevertheless, taking into account the aflatoxins levels reported in human urine (0.5–16 ng ml21 for AFP1,3 0.1–4.8 ng ml21 for AFB1 19–21), the method should be suitable.It can be envisaged that with a higher volume of urine sample extracted and with a fluorescence detector of better performance, the limited accuracy of the proposed method will be significantly increased and thus will lie with the best limit of detection reported in the literature. Finally, Fig. 2 shows the typical chromatograms of a human urine blank (A) and a spiked urine (26 ng ml21 AFQ1, 3 ng ml21 AFP1 and 19 ng ml21 AFB1) (B). For both (A) and (B) deionized water was added post-column in order to get the times to match and to obtain the same quenching-water effect as in (C).(C) is the same spiked urine sample analysed by the proposed method with 1022 M DIMEB post-column reagent, showing the improvement of the fluorescence signal for these aflatoxins. References 1 P. M. Scott, J. Assoc. Off. Anal. Chem., 1987, 70, 276. 2 I. Dvorackova, in Aflatoxins and Human Health, CRC Press, Boca Raton, FL, USA, 1990, 458. 3 G. S. Qian, R. K. Ross, M .C. Yu, J. M. Yuan, Y. T. Gao, B. E. Henderson, G. N. Wogan and J. D. Groopman, Cancer. Epidemiol. Biomarkers. Prev., 1994, 3, 3. 4 B. D. Roebuck and G. N. Wogan, Cancer Res., 1977, 37, 1649. 5 G. H. Büchi, P. M. Müller, B. D. Roebuck and G. N. Wogan, Res. Commun. Chem. Pathol. Pharmacol., 1974, 8, 585. 6 G. E. Neal and P. J. Colley, Biochem. J., 1978, 174, 839. 7 W. F. Busby and G. N. Wogan, in Aflatoxins, ed.G. N. Searleed, American Chemical Society, Washington, DC, USA, 1985, p. 1. 8 R. K. Ross, J. M. Yuan, M. C. Yu, G. N. Wogan, G. S. Qian, J. T. Tu, J. D. Groopman, Y. T. Gao and B. E. Henderson, Lancet, 1992, 339, 943. 9 J. D. Groopman, J. Q. Zhu, P. R. Donahue, A. Pikul, L. S. Zhang, J. S. Chen and G. N. Wogan, Cancer Res., 1992, 52, 45. 10 D. L. Orti, J. Grainger, D. L. Ashley and R. H. Hill, Jr., J. Chromatogr., 1989, 462, 269. 11 H. Joshua, J. Chromatogr.A, 1993, 654, 247. 12 A. Kussak, B. Andersson and K. Andersson, J. Chromatogr. B, 1994, 656, 329. 13 A. Kussak, B. Andersson and K. Andersson, J. Chromatogr. B, 1995, 672, 253. 14 H. Jansen, R. Jansen, U. A. Th. Brinkman and R. W. Frei, Chromatographia, 1987, 24, 555. 15 A. Cepeda, C. M. Franco, C. A. Fente, B. I. Vázquez, J. L. Rodríguez, P. Prognon and G. Mahuzier, J. Chromatogr. A, 1996, 721, 69. 16 O. J. Francis, G. P. Kircheneuter, J. R. G. M. Ware, A. S. Carman and S. S. Kuan, J. Assoc. Off. Anal. Chem., 1988, 71, 725. 17 C. M. Franco, C. A. Fente, B. I. Vázquez, A. Cepeda, G. Mahuzier and P. Prognon, J. Chromatogr. A, 1998, 815, 21. 18 Y. Matsui and K. Mochida, Bull. Chem. Soc. Jpn., 1979, 52, 2808. 19 G. Niedwetzki, G. Lach and K. Geschwill, J. Chromatogr. A, 1994, 661, 175. 20 R. Guan, C. J. Oon, C. Wild and R. Motesano, Ann. Acad. Med. Singapore, 1986, 15(2), 201. 21 J. D. Groopman, A. J. Hall, H. Whittle, G. J. Hudson, G. N. Wogan, R. Motesano and C. P. Wild, Cancer Epidemiol., Biomarkers Prev., 1992, 1, 221. 8/09150A Fig 2 Chromatograms of human urine extracts: urine blank (A); spiked urine (26 ng ml21 AFQ1, 3 ng ml21 AFP1 and 19 ng ml21 AFB1) (B); the same spiked urine sample, detected with the proposed method, with DIMEB (1022 M) as post-column reagent (C). For detailed chromatographic conditions, see text. Anal. Commun., 1999, 36, 5–7 7

ISSN:1359-7337    

DOI:10.1039/a809150a

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Anion-modulated switching of retention properties of a zwitterionic stationary phase Jayakumar M. Patil† and Tetsuo Okada* Department of Chemistry, Tokyo Institute of Technology, Meguro-ku, Tokyo 152, Japan. E-mail: tokada@chem.titech.ac.jp Received 2nd November 1998, Accepted 1st December 1998 Stationary phases modified by zwitterionic molecules act both as an anion- and a cation-exchanger. This characteristic property of zwitterionic stationary phases can be modulated only by changing the nature of anions added in mobile phases. Anion-exchange properties emerge when small and well hydrated anions are added in mobile phases, while cation-exchange properties appear when mobile phases contain large and poorly hydrated anions.The nature of cations is much less important than that of anions. These unusual retention properties come from the structure of a zwitterionic molecule used as a stationary phase modifier, which has an inner cationic and an outer anionic group.The surface adsorption of functional molecules has largely enhanced the versatility of reversed-phase chromatography, and allowed the separation of various compounds that are not retained on such hydrophobic stationary phases.1–11 Amphoteric compounds, such as surfactants, have been most extensively used for modifying chromatographic stationary phase surfaces, and are often called ion-pair or ion-interaction reagents.1–9 Ion-pair reagents, for example, make the surfaces of hydrophobic stationary phases ionic.The resulting stationary phases can be used as ion-exchange resins; this mode has significantly contributed to the development of chromatography of ions.12 Unstable retention and poor reproducibility are disadvantages of these so-called ‘dynamically-coated’ stationary phases. However, these have important advantages over chemically bonded ion-exchange resins. The surface densities and the structures of active molecules are two principal factors controlling the retention in this chromatographic mode; these factors can be easily changed in dynamically-coated stationary phases.The former mainly affect the retention ability of the stationary phases, while the latter modify separation selectivity. Recently, Hu, Haraguchi and their coworkers13–19 developed a method named electrostatic chromatography, in which the stationary phases are coated with zwitterionic surfactant and are thus capable of simultaneous retention of cationic and anionic species.The main purpose of their work was the development of a method permitting the use of pure water as a mobile phase for the separation of ions because of the advantage of this highly resistive mobile phase in the conductivity detection of ions. Since the properties of zwitterionic surfaces (as well as those of zwitterionic surfactant micelles) are affected by mobile phase compositions, such as the concentration and the nature of an added salt, detailed studies on salt effects are important for further methodological developments of electrostatic chromatography and for the understanding of its separation mechanism.In the present paper, we demonstrate that the ion uptake properties of stationary phases coated with 3-(N-dodecyl-N,Ndimethyl- ammonio)-propane-1-sulfonate (DDAPS) can be modulated by the nature of the anions added to the mobile phase, i.e., they act as a cation-exchanger under certain conditions, but as an anion-exchanger under different conditions. The characteristic properties of DDAPS-coated stationary phases can be explained by the calculation of electrostatic potential.Experimental section The chromatographic system used was composed of a Tosoh (Tokyo, Japan) computer-controlled pump Model CCPD, a Rheodyne injection valve equipped with a 100 mL sample loop, a JASCO (Tokyo, Japan) UV-Vis detector JASCO Model 875-UV, and a chart recorder. The separation column was immersed in water thermostatted at 25.0 °C.PAR [4-(2-pyridylazo) resorcinol] solution was delivered by another singleplunger pump (Nihonseimitsu Co.) for the postcolumn reaction of transition metal ions. The separation column was a 4.6 3150 mm stainless steel column packed with Wakosil 5C8 (Wako Pure Chemicals, Osaka, Japan) (particle size = 5 mm, specific surface area = 300 m2 g21, mean pore size = 12 nm). The critical micelle concentrations (c.m.c.) of DDAPS were determined by a dye solubilization method with Coomassie Brilliant Blue G-250 under various conditions.20 The adsorption amounts of DDAPS were determined by a breakthrough method; the elution of DDAPS was monitored with a Tosoh refractive index detector Model RI-8010. DDAPS was purchased from Tokyo Kasei (Tokyo, Japan), and recrystallized twice from acetone containing a small amount of ethanol.Other reagents were of analytical grade. Distilled deionized water was used for solution preparation. Results and discussion It is known that anions are partitioned into DDAPS micelles better than cations.This has been confirmed by self-diffusion measurements,21 fluorescence quenching,22 and chromatography. 16–17 This anion-dominated partitioning has been explained in our recent work on chromatographic modeling based on electrostatic theories.23 The polarity of the DDAPS molecule is the most important factor in determining anion-dominated partitioning; i.e., it has an inner cationic and an outer anionic group.According to the developed model, the electrostatic potential at the interface between solution and the DDAPSmodified surface is illustrated in Fig. 1, where 10 mM NaCl or NaClO4 mobile phases are assumed. The transfer free energies of ions are included in the developed model to represent the partition selectivity. The large crystalline ionic radius (thus low charge density) and large polarizability of ClO42 allow its enhanced invasion to the DDAPS layer and the developments of the negative electrostatic potential region as a result.In contrast, the low penetrable nature of Cl2 keeps the potential of the inside DDAPS layer positive, allowing the partition of anions. These calculations predict that DDAPS-modified stationary phase acts as a cation-exchanger in the former case, but as an anion-exchanger in the latter case. The electrostatic potential is † Permanent address: Department of Chemistry, Textile and Engineering Institute, Ichalkaranji-416115, India.Anal. Commun., 1999, 36, 9–11 9not, in contrast, affected by the nature of cations; in most cases, cation effects are negligible. This result agrees well with the experimental facts reported so far.16,17,21,22 The above calculation results were confirmed by chromatographic experiments. Small amounts of DDAPS were added to mobile phases to prevent the desorption of DDAPS from the equilibrated stationary phase surface during a series of measurements. The adsorption equilibrium is usually established for monomer surfactant molecules, suggesting that the c.m.c.of DDAPS is an important factor to optimize the DDAPS concentration in the mobile phases. Salt effects on the c.m.c. of DDAPS were therefore studied. The c.m.c. of DDAPS is 3.0 mM in water (the same as literature values),24 but that the addition of salts reduces the c.m.c. the value of which varies depending upon the nature and the concentration of the salts added (see Table 1).The preliminary experiments showed that 2 mM was high enough to prevent the desorption of DDAPS. Since micellar partition effects are also avoidable for 2 mM DDAPS mobile phases, this concentration was adopted for further chromatographic experiments. It is also predictable that the addition of salts affects the adsorption of DDAPS. However, changes in the adsorption amount were very small; the adsorption amount of DDAPS was 7.1 3 1024 mol per column from 5 mM aqueous solution (without added salts), 6.9 3 1024 mol from 0.1 M NaClO4 solution, and 6.8 3 1024 mol from 0.1 M NaCl solution.Increasing salt concentration affects adsorption in (at least) two ways; (1) enhancing the adsorption by salting-out and (2) lowering the adsorption due to a decrease in the monomer surfactant concentration (due to low c.m.c.). A constant adsorption amount must imply that these opposite effects happen to cancel each other.Thus, we can discuss chromatographic results on the basis of constant surface density of DDAPS molecules and no micellar partitioning. The chromatographic retention of I2 and Cu(ii) on the DDAPS-modified stationary phases was studied as a function of salt concentrations. Fig. 2 clearly indicates the anion-modulated switching of the ionic partition properties of the DDAPS layer. As predicted from the calculation of electrostatic potential (Fig. 1), an anion and a cation show the opposite retention dependence on the nature of anions; the largest retention is seen for Cu(ii) with ClO42 mobile phases, but for I2 with Cl2 mobile phases.It is also an important feature that the plots for anion retention show maxima at particular salt concentrations. This maximum formation was explained by the thickening of the DDAPS layer with increasing salt concentrations.23 The peak broadening was so marked with low concentration mobile phases that maximum appearance was not confirmed for Cu(ii).It can be explained in the following way that DDAPSmodified stationary phase distinguishes anions better than cations. The interaction energy between an ion and the dipolar layer of the DDAPS phase might be substituted by the free energy of transfer from water to a less polar medium of weaker solvation ability [DG°tr (W ?S)] if electrostatic interaction can be ignored. Although electrostatic interaction should be a major source of total interaction energy, it must be negligible if we discuss the selectivity (or relative interaction) of identically charged ions.Although we do not know DG°tr (W ? S) for S = DDAPS (or the dipolar layer of DDAPS), considering typical organic solvents instead of DDAPS is significant to infer the origin of the anion selectivity of the DDAPS-modified phase. DG°tr (W ? S) values of cations do not vary as much as those of anions; e.g., differences in DG°tr (W ? S) between Na+ and Cs+ are 1.3 kJ mol21 for S = methanol, 0.4 kJ mol21 for S = DMF, 28.8 kJ mol21 for S = acetonitrile, and 2.1 kJ mol21 for S = DMSO, while those between Cl2 and I2 are 25.9 kJ mol21, 227.3 kJ mol21, 223.5 kJ mol21, and 229.4 kJ mol21.25 This might be explained by the fact that anions are predominantly solvated through hydrogen bonds in water but mainly by dispersion energy in other solvents.Though we do not know the proper circumstances in the dipolar layer of the DDAPS phase, ions should be solvated in a different way from solvation in bulk water.Since the solvation of anions in the DDAPS layer must be weaker than that in water (similar to that in organic solvents), it can be reasonably understood that the Fig. 1 Profiles of electrostatic potential at the interface of solution and the DDAPS-modified surface. Table 1 The c.m.c. of DDAPS in various solutions Solution c.m.c./mM Water 3.0 0.01 M NaClO4 2.4 0.05 M NaClO4 2.0 0.1 M NaClO4 2.0 0.1 M NaCl 2.6 0.2 M NaCl 2.2 0.4 M NaCl 2.0 1.0 M NaCl 2.0 Fig. 2 Changes in the retention of (a) I2 and (b) Cu(ii) on the DDAPSmodified stationary phase with the salt concentration. 10 Anal. Commun., 1999, 36, 9–11DDAPS phase shows anion-selectivity rather than cationselectivity. The above results clearly suggest that the properties of the DDAPS-modified stationary phases can be adjusted to a particular separation in a very simple way, i.e., by changing electrolyte compositions. Fig. 3A shows the separation of some UV-absorbing anions with Cl2 eluents. It should be noted that the selectivity is identical with that in usual anion-exchange chromatography. In contrast, a less solvated anion should be added in mobile phases for the separation of cations. Fig. 3B is an example of separation of transition metal ions with the mixed mobile phase of tartaric acid and NaClO4; the former is a complexing agent, while the latter is necessary to make the stationary surface negative.Thus, switching the nature of the stationary phase in ionic separation is possible only by varying anions added in mobile phases. This is an important characteristics of zwitterionic stationary phases. Thus, although uses of zwitterionic micelles or zwitterionic surfaces in separation have not been common in developing analytical methods, we believe that this approach is of potential importance and versatile applicability. Acknowledgement This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports, Japan (Monbusho).J.M.P. thanks the UNESCO and the Monbusho for providing a fellowship. References 1 H. Miwa and M. Yamamoto, J. Chromatogr. A, 1996, 721, 261. 2 M. Piotte, F. Boss�anyi, F. Perreault and C. Jolicoeur, J. Chromatogr. A, 1995, 704, 377. 3 S. Fichtner, F. Th. Lange, W. Schmidt and H.-J. Brauch, Fresenius’ J. Anal. Chem., 1995, 353, 57. 4 J.-F. Jen and C.-S.Chen, Anal. Chim. Acta, 1992, 270, 55. 5 Y. Michigami, K. Fujii and K. Ueda, J. Chromatogr. A, 1994, 664, 117. 6 M. Adachi, K. Oguma and R. Kuroda, Chromatographia, 1990, 29, 579. 7 C. Sarzanini, M. C. Bruzzoniti, G. Sacchero and E. Mentasti, Anal. Chem., 1996, 68, 4494. 8 Y. Inoue, K. Kawabata and Y. Suzuki, J. Anal. At. Spectrom., 1995, 10, 363. 9 S. Zappoli and C. Bottura, Anal. Chem., 1994, 66, 3492. 10 J. D. Lamb and R. G. Smith, J. Chromatogr., 1993, 640, 33. 11 S.H. Hansen and J. Tjørnelund, J. Chromatogr., 1991, 556, 353. 12 M. C. Gennaro, Adv. Chromatogr., 1995, 35, 343. 13 W. Hu, T. Takeuchi and H. Haraguchi, Anal. Chim. Acta, 1992, 267, 141. 14 W. Hu and H. Haraguchi, Anal. Chim. Acta, 1994, 289, 231. 15 W. Hu and H. Haraguchi, Anal. Chim. Acta, 1994, 285, 335. 16 W. Hu, T. Takeuchi and H. Haraguchi, Anal. Chem., 1993, 65, 2204. 17 W. Hu, H. Tao and H. Haraguchi, Anal. Chem., 1994, 66, 2514. 18 W. Hu, A. Miyazaki, H. Tao, A. Itoh, T. Umemura and H. Haraguchi, Anal. Chem., 1995, 67, 3713. 19 W. Hu and H. Haraguchi, Bull. Chem. Soc. Jpn., 1993, 66, 1420. 20 C. Samsonoff, J. Daily, R. Almog and D. S. Berns, J. Colloid Interface Sci., 1986, 109, 325. 21 N. Kamenka, M. Chorro, Y. Chevalier, H. Levy and R. Zana, Langmuir, 1995, 11, 4243. 22 S. Brochsztain, P. B. Filho, V. G. Toscano, H. Chaimovich and M. J. Politi, J. Phys. Chem., 1990, 94, 6781. 23 T. Okada and J. M. Patil, Langmuir, 1998, 14, 6241. 24 J. P. Berry and S. G. Weber, J. Chromatogr. Sci., 1987, 25, 307. 25 I. Sakamoto and S. Okazaki, Yobai in Ion (Solvents and Ions), Taniguchi Insatsu, Matsue, 1990. Paper 8/08477G Fig. 3 Typical separation of selected anions (A) and transition metal ions (B). Mobile phase: (A) 2 mM DDAPPS + 10 mM NaCl; (B) 2 mM DDAPS + 20 mM tartaric acid + 0.1 M NaClO4 (pH 4.0). Detection: (A) UV at 220 nm; (B) PAR postcolumn reaction, detection at 540 nm. Anal. Commun., 1999, 36, 9–11

ISSN:1359-7337    

DOI:10.1039/a808477g

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Evaluation of internal standardisation in electrothermal atomic absorption spectrometry Bernard Radziuk,a Natalya P. Romanovab and Yngvar Thomassenc a Bodenseewerk Perkin-Elmer GmbH, Postfach 101761, D-88647 � Uberlingen, Germany b Department of Analytical Chemistry, St. Petersburg State Technical University, St. Petersburg 195251, Russia c National Institute of Occupational Health, P.O. Box 8149 DEP, N-0033 Oslo, Norway Received 20th November 1998, Accepted 10th December 1998 Significantly improved performance in electrothermal atomic absorption spectrometry is possible using an internal standardisation technique.A Perkin-Elmer SIMAA 6000 simultaneous multielement spectrometer was used to study the correlation between two integrated absorbance signals. The behaviour of Pb (analyte) in different urine, blood and placenta samples was compared to that of Bi or Tl used as the internal standards. All samples were spiked with known amounts of Pb and Bi or Tl.A satisfactory signal correlation (r = 0.94) between the integrated absorbances for spikes of the analyte and internal standard was observed with Bi as the internal standard. After signal correction, the relative standard deviation of the integrated absorbance for Pb spikes reduced from 29 to 7% for urine, from 19 to 2% for blood and from 22 to 4% for placenta. The mean difference between Pb concentration found in analysed samples by the method of additions and using an internal standard was 10%.Introduction Although classical internal standardisation is a well established technique in multielement emission spectroscopy, very few studies have been carried out for electrothermal atomic absorption spectrometry (ETAAS). Internal standardisation in atomic absorption spectrometry was introduced in 1965 by Massmann1 when he reported on the use of an internal standard to reduce the variability of sample introduction. In those experiments, he used a laboratory-made multi-channel spectrophotometer.The scatter among results arising from inaccurate introduction of samples was slightly reduced for certain combinations of elements and increased for other combinations. Later, the concept of internal standardisation was used by Katskov and L’vov2,3 for determining trace elements in powdered samples, in particular, in zirconium dioxide and powdered graphite. When zirconium dioxide was being analysed, zinc was used as the internal standard, and when graphite was analysed, silver was used.These elements were introduced into the samples in the form of solutions. The use of internal standards made it possible to allow reliably for the weight of the powder samples and to obtain a standard deviation of 6.2% in the determination of cadmium. The recent development of commercially available simultaneous multielement atomic absorption spectrometers has made it practicable to apply internal standardisation techniques in order to improve analytical performance in ETAAS. In this work, we have tested Tl and Bi as internal standards when measuring Pb in blood, placenta and urine samples.Many authors use aqueous lead standards for calibration while analysing lead in biological materials. However, these matrices sometimes cause a significant decrease in the absorption signal of Pb. In this work we investigated the effectiveness of internal standardisation under these circumstances.We have studied the causes of the variation in the lead signal and have made an attempt to improve both precision and accuracy by internal standardisation. The criteria used for the selection of the internal standard were similarity of chemical/ physical properties to those of the analyte and that the internal standard concentration was negligible in the sample population. Table 1 summarises some chemical/physical parameters of Bi, Pb and Tl. Since these three elements have very similar characteristics we could expect both Tl and Bi to be appropriate internal standards for Pb.Experimental Instrumentation A Perkin-Elmer SIMAA 6000 ( � Uberlingen, Germany) simultaneous multielement atomic absorption spectrometer equipped with a Perkin-Elmer AS-71 autosampler and transversely heated graphite atomiser (THGA) was used for all measurements. A hollow cathode lamp was used for Pb and electrodeless discharge lamps for Bi and Tl. The measurement wavelengths were 283.3, 223.1 and 276.8 nm, respectively.End-capped graphite tubes with integrated L’vov platform supplied by Bodenseewerk Perkin-Elmer ( � Uberlingen, Germany) were used (Part No. B-300-0655). For the measurement of major constituents in the urine samples (Ca, K, Mg, Zn, P, S) a Perkin-Elmer Optima Model 3000 inductively coupled plasma atomic emission spectrometer was used (Perkin-Elmer, Norwalk, CT, USA). Reagents All standard and modifier solutions were prepared by dilution of 1 mg ml21 Pb, Bi, Tl and Pd Stock Standards (Spectrascan CertifiedTM, Teknolab AS, Drøbak, Norway).For dilution and Table 1 Some physical and chemical parameters for elements under study and their oxides4,5 Parameter Tl Pb Bi Atomic number 81 82 83 Molar mass/kg m23 0.204 0.207 0.209 Melting point/K 577 601 545 Boiling point/K 1748 2018 1825 Heat of vaporization/kJ mol21 180 196 199 Tl2O3(s) PbO(s) Bi2O3(s) Activation energy for oxide atomisation/ kJ mol21 225 268 257 Dissociation energy of MO(g)/kJ mol21 230 372 339 Dissociation energy of MCl(g)/kJ mol21 368 297 301 Anal.Commun., 1999, 36, 13–16 13digestion of urine, heparinised whole blood and placenta samples, water purified by reverse osmosis and deionisation and ultrapure 65% nitric acid (Scan Pure, Chemscan, Elverum, Norway) were used. Samples Whole blood and urine specimens were obtained from male workers exposed to lead; placenta samples were obtained from healthy mothers using protocols which conformed to the ethical guidelines of the Declaration of Helsinki.We tested the sampling equipment by leaching with 0.5% nitric acid; no detectable lead contamination occurred ( < 1 mg l21). All samples were also checked for possible contamination of bismuth and thallium using ETAAS and the content of these two candidate internal standards was below the detection limits ( < 1 mg l21). This is in good agreement with the results reported by e.g.Schramel et al. who documented very low physiological levels of bismuth ( < 0.01 mg l21) and thallium (0.25 mg l21) in urine.6 Analytical procedure The placenta tissue samples were after homogenization, freezedried in a standard laboratory system. Two and a half ml HNO3 were added to 0.3 g placenta weighed in a 13.3 ml polypropylene tube. After the 1.5–2 h necessary for the completion of active reaction, tubes containing the samples were heated at 95 °C for 90 min in a laboratory oven.After cooling, samples were diluted with H2O to final volume (13.30 ml ± 0.04). Two ml of a heparinised blood sample were transferred using a positive displacement micropipette to a 13.3 ml polypropylene tube and 2.5 ml HNO3 were added. The rest of the procedure (heating and dilution) for the preparation of blood samples was the same as for placenta tissue. Urine samples were diluted 1 : 1 with 0.5% HNO3. In all experiments, the sample and modifier aliquots were 10 ml.Modifier solution (0.1% Pd for urine and 0.05% Pd for other samples) was taken first, followed by sample. To check the accuracy of internal standardisation the method of standard additions was used routinely for determination of Pb in all the materials mentioned above. The final concentrations of spiked elements in the sample solutions to be analysed were 25 mg l21 for Bi and Pb, and 10 mg l21 for Tl. The experimental conditions for the graphite atomiser are given in Table 2.In order to better demonstrate the effectiveness of the internal standardisation, we intentionally selected the higher than recommended (850 °C) pyrolysis temperature for Pb. It led to increased scatter of results (see below). Results and discussion Possible causes of interference on Pb and selection of internal standard The integrated absorbance signal for Pb is suppressed in all three matrices under study here. In order to shed light on the ori this interference, the signals were correlated with the concentrations of major components in a series of urine samples with both low, medium and high matrix salt content.The best correlation (r = 0.81) is obtained with the concentration of phosphorus, representing the phosphate content of the urine sample (Fig. 1a). Thus, an appropriate internal standard should be affected in the same way as lead is, especially by phosphorus. The depression of the signal in the presence of other major constituents such as creatinine and sulfur may not be component specific, but rather the result of physical expulsion due to the rapid formation of molecules during the atomisation phase.This indicates a second important characteristic of the internal standard, i.e., the appearance temperature should be the same as that of the analyte. Based on this consideration, and on the general properties listed in Table 1, Bi and Tl were selected as the most likely internal standard candidates.Initially, qualitative studies on the effectiveness of these two elements were carried out. The correlation between Pb and Tl signals in different urine samples is shown in Fig. 2. It illustrates the variation of the signals for Pb and Tl spikes in different samples during the simultaneous detection of Pb and Tl. It is evident from this figure that these elements differ significantly in their behaviour and for this reason Tl cannot be used as internal standard for Pb.Bismuth gives much better correlation with Pb for urine samples (Fig. 3) and placenta and blood samples (Fig. 4). Calculation scheme When internal standardisation is used, all calculations are based on the supposition that sensitivity of analyte (SPb) and internal standard (SBi) depend identically on sample matrix and Fig. 1 Correlation between Pb-spike signal and concentration of P in urine samples: a, uncorrected Pb-spike signal; b, Pb-spike signals after correction by Bi.Concentrations of spike in samples are 25 mg l21 of both Pb and Bi. Table 2 THGA heating program used for the SIMAA 6000 spectrometer Step Temperature/°C Ramp/s Hold/s Dry 110 1 30 Dry 130 15 30 Pyrolysis 1100Bi/600Tl 10 20 Cool-down 20 1 5 Atomisation 1600Bi/1800Tl 0 3Bi(7aqua)/5Tl Clean 2450 1 3 14 Anal. Commun., 1999, 36, 13–16uncontrolled variations of conditions during pyrolysis and atomisation steps. Therefore, the ratio of sensitivity of aqueous solutions (Sa) must be related to that of real samples (Ss) as S S S S Pb a Bi a Pb s Bi s = (1) Assuming that the integrated absorbance, Q = º A dt, is proportional to mass, m, of element in the tube, we may express the sensitivity as S = Q/m (2) In this case, we can use for the calculation of the mass of Pb in the sample the expression m Q Q Q Q m Q k Q m Pb s Bi a Bi s Pb s Pb a Pb a Pb s Bi s Pb a = ¥ ¥ = ¥ ¥ (3) which follows from eqn.(1), (2) and mBi a = mBi s . Here k Q Q = Pb a Bi a (4) If the masses of Pb and Bi, spiked in samples and aqueous solutions, are equal, eqn. (1), (2) and (4) may be rewritten as: (QPb s )cor = QPb s + k (QBi a 2 QBi s ) (5) where QPb s and (QPb s )cor are the original and the corrected signals for Pb, respectively.Eqn. (5) will be used for comparison of corrected signals of Pb in different samples. Evaluation of results The cross-correlation function calculated by the SPSS statistical software package (SPSS Inc., Chicago, IL, USA) revealed reasonable correlation of Bi and Pb spike signals for all the materials investigated.Correlation coefficient equals 0.94. This led to significant improvement in the variation of Pb signals corrected with eqn. (5). Fig. 1b shows that the dependence of Pb sensitivity on phosphorus concentration was much reduced when Bi was used as internal standard. However, the slight upward trend in the corrected results indicates that the effect of phosphorus on Bi is somewhat stronger than that on Pb under the pyrolysis conditions used in this study.The improvement in the results for the determination of Pb in real samples is demonstrated in Table 3. In general, the use of the internal standard technique provides a 4- to 9-fold improvement in precision for different samples. Comparison with the integrated absorbance for the spikes in aqueous solution shows that recovery, i.e. accuracy of the determination, is also improved dramatically. The signals for Pb spikes are comparable after correction, irrespective of sample matrix (urine, placenta and blood).In addition to the above calculations of integrated absorbance signals for Pb in spikes, the concentrations in the samples were determined and compared with those obtained using the method of standard additions (Table 4). The sample numbers in Table 4 correspond to those in Figs. 3 and 4. Concentrations of Pb in several samples of urine (4, 8, 9, 12, 15, 17, 18, 24, 25, 29 and 30) were close to or below the detection limit and hence were Fig. 2 Correlation between Pb (—) and Tl (- - -) spike signals in urine samples. Concentrations of spike in samples are 25 mg l21 of Pb and 10 mg l21 of Tl. Fig. 3 Correlation between Pb (—) and Bi (- - -) spike signals in urine samples. Concentrations of spike in samples are 25 mg l21 of both Pb and Bi. Fig. 4 Correlation between Pb (—) and Bi (- - -) spike signals in blood and placenta samples. Concentrations of spike in samples are 25 mg l21 of both Pb and Bi.Table 3 Statistical evaluation of integrated absorbance signals (s) for 25 mg l21 of Pb corrected by Bi as internal standard Urine (n = 36) Placenta (n = 5) Blood (n = 5) All samples (n = 46) Parameter Original Corrected Original Corrected Original Corrected Original Corrected Mean signal 0.0171 0.0308 0.0158 0.0301 0.0146 0.0291 0.0167 0.0305 Min signal 0.0047 0.0272 0.0107 0.0292 0.0116 0.0282 0.0047 0.0272 Max signal 0.0230 0.0348 0.0205 0.0322 0.0180 0.0299 0.0230 0.0348 s 0.0049 0.0021 0.0035 0.0012 0.0028 0.00064 0.0046 0.0020 RSD (%) 29 7 22 4 19 2.2 28 6 Recovery (%) 52 94 48 92 45 89 51 93 Anal.Commun., 1999, 36, 13–16 15omitted in Table 4. It can be seen from the comparison of these data that the difference between concentration values includes both random and systematic errors. The last error is probably dominated by the uncertainty in the determination of the k value. In our experiments, the k value (1.4 ± 0.1, n = 3) was estimated as the ratio between the integrated absorbances of Pb (QPb a ) and Bi (QBi a ) in aqueous solution. At k = 1.3, chosen as an empirical example, the systematic discrepancy practically disappears and the mean difference value reduces from 10 to 5%.Conclusions Thallium and bismuth have been tested as internal standards when measuring lead in blood, placenta and urine samples. Bismuth has been found to be the best match in thermal volatility and atomisation behaviour to Pb in different matrixes.It was used to compensate for changes in Pb signals resulting from the matrix effects. This made it possible to obtain good recoveries for Pb in various matrices without the use of the method of standard additions. Nevertheless, it will be necessary to continue this study and to investigate the effectiveness of the internal standard technique under optimum sample pretreatment conditions and to improve the reliability of the determination of the sensitivity ratio (QPb a /QBi a ) during the analysis process.It should be stressed, however, that the selection of the internal standard in ETAAS is not a straightforward task. There must be a close coincidence in chemical and physical properties between analyte and internal standard. An inappropriate selection of internal standard can even be detrimental to the quality of an analysis. Acknowledgement We gratefully acknowledge Professor Boris L’vov, St.Petersburg State Technical University, for his active participation in the discussion of the results, which was essential to the success of this work. References 1 H. Massmann, in Second International Symposium Reinststoffe in Wissenschaft und Technik, ed. G. Ehrlich, Academie-Verlag, Berlin, 1966, pp. 297–308. 2 D. A. Katskov and B. V. L’vov, Zh. Prikl. Spektrosk. 1969, 10, 382 (in Russian). 3 B. V. L’vov, Atomic Absorption Spectrochemical Analaysis, Adam Hilger, London, 1970, p. 247. 4 Tables of Physical Quantities. Handbook, ed. I. K. Kikoin, Atomizdat, Moscow, 1976 (in Russian). 5 Disruption Energies of Chemical Bonds. Ionization Potentials and Electron Affinity Handbook, ed. V. N. Kondratiev, Nauka, Moscow, 1974 (in Russian). 6 P. Schramel, I. Wendler and J. Angerer, Int. Arch. Occup. Environ. Health, 1997, 69, 219. Paper 8/09096C Table 4 Lead concentrations in urine measured by method of addition and internal standardisation Concentration/mg l21 Sample Method of addition Internal standard Difference (%) Urine 1 5.0 4.4 11 2 4.2 3.8 11 3 4.7 4.2 12 5 5.2 5.4 23 6 3.3 3.2 4 7 5.3 5.1 4 10 5.5 5.1 7 11 7.4 6.8 9 13 10.4 10.1 3 14 6.4 5.9 7 16 8.8 7.2 18 19 5.3 4.6 14 20 6.4 5.7 12 21 3.2 2.8 11 22 5.1 4.5 11 23 4.8 4.2 12 26 3.6 3.0 16 27 5.8 6.1 25 28 4.6 4.3 7 31 4.5 4.4 4 32 5.9 5.4 8 33 4.4 3.9 9 34 3.7 3.4 8 35 4.5 3.9 13 36 4.5 4.6 22 Concentration/ng g21 Placenta 1 0.21 0.21 23 2 0.38 0.33 13 3 0.50 0.43 14 4 0.26 0.24 8 5 0.27 0.23 13 Concentration/mg l21 Blood 6 63.1 52.8 16 7 51.2 43.8 14 8 54.1 47.5 12 9 48.3 40.9 15 10 53.6 45.0 16 mean 10 16 Anal. Commun., 1999, 36, 13–16

ISSN:1359-7337    

DOI:10.1039/a809096c

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication Investigation of imaging X-ray photoelectron spectroscopy for surface analysis of atmospheric particulates Bernie M. Hutton* and David E. Williams University College London, Chemistry Department, Christopher Ingold Laboratories, 20 Gordon Street, London, UK WC1H 0AJ. E-mail: b.hutton@ucl.ac.uk Received 19th November 1998, Accepted 10th December 1998 X-Ray photoelectron spectroscopy (XPS) is used to investigate surface species on atmospheric particulates as part of an investigation into the sources and health effects of such particulates. Oxygen and carbon species dominate the surface with trace quantities of Na+, NO32, Cl2 and SO4 22.Multiple species for carbon and oxygen were evident, with major species identified as oxidised carbon, graphitic/ aliphatic carbon and ruthenium oxide or a carbide. The potential of imaging XPS to show localised variation of surface species across atmospheric particulates is demonstrated. Introduction Elevations in airborne particulates, especially those of aerodynamic diameter less than 10 mm (PM10) increase the incidence of human cardiac and respiratory diseases1,2 but the mechanisms involved are poorly understood. Also, the formation mechanisms of particulates need to be investigated to establish a link between the source and effect of such particles.The chemical characterisation of the surface of atmospheric particulates is critical in identifying particulate sources because new species produced from chemical reactions in the atmosphere occur on the particle surface.Also the species present on the surface are critical for effects on health because it is the particulate surface which is directly accessible to biological fluids after inhalation. Particle size determines the penetration depth in human airways, with coarse particles (greater than 2.5 mm) depositing in the upper tract and finer particles penetrating further into the lungs.3 Because cardiac and respiratory diseases occur in specific regions in the airways associations between particle size, surface chemistry and resultant disease can be inferred given appropriate characterisation of the particles.Techniques currently used for particle characterisation include light microscopy4 and electron microscopy with energy dispersive spectroscopy (EM-EDS).5,6 The techniques do not, however, deliver information on the surface composition, which may differ from the bulk.In this present study X-ray photoelectron spectroscopy (XPS) is used to identify and quantify major chemical species on the surface of London airborne particulates. XPS offers the advantage over other surface-sensitive techniques of relatively low beam damage and suitability of insulating materials for analysis. The inherent weakness in XPS has been the lack of spatial resolution but the recent development of parallel imaging, which obtains positional information from dispersion characteristics of the hemispherical analyser, has produced photoelectron images with spatial resolution better than 5 mm.7–9 The introduction of a magnetic objective lens has improved the sensitivity for a given spatial resolution,9,10 enabling the new technique of imaging XPS (IXPS) to be realized. The present work assesses the capability of IXPS to obtain spatially resolved chemical maps across the surface of atmospheric particulates.Nonuniform variation of major species across the surface is demonstrated.The sensitivity was insufficient to produce images for trace species. Experimental Cu foils (10 mm 3 6 mm), used as passive samplers were cleaned in UHV to remove residual carbon by sputtering with argon ions at 4 kV with an ion beam current of 1.5 mA for 15 min. This was verified by comparing spectra before and after cleaning. The cleaned foils were positioned 2 m from a roadside location in Bloomsbury, London for collection of atmospheric particulates over 1 week.A VG ESCALAB 220i XL with a magnetic objective lens was used with monochromatic Al radiation (1486.6 eV) at 10 kV and a 600 mm spot size throughout this work. Large area spectra were collected with the analysis area defined by the monochromator spot size. Survey spectra of the particulates were collected over a 1100 eV range at a resolution of 0.8 eV step21, 100 ms step21 and pass a energy of 80 eV. High resolution spectra were collected for species of interest at a pass energy of 20 eV.Charge compensation was achieved by using low energy electrons (4 eV) to flood the sample and referencing to the C 1s binding energy for graphitic carbon at 285 eV. Binding energies were taken at peak maxima for all species. Peak and background images (800 mm 3 600 mm) were obtained for all relevant species detected from the survey scan. A retard ratio of 4 was an adequate energy resolution for imaging species across a wide energy range.Background images were recorded at slightly lower binding energy than each relevant peak to remove imaging artefacts caused by non-uniform secondary electron generation across the image. Accumulation times of 240 min were used for each image to obtain good S/N ratios. Images were stored in 256 3 256 pixel format with an 8-bit grey value resolution and the resultant images (peak 2 background) were examined. Fig. 1 XPS survey spectrum of atmospheric particulates collected on copper foil.Anal. Commun., 1999, 36, 17–18 17Results and discussion The wide area survey of particulates deposited on the Cu sampler is shown in Fig. 1. Peaks denoted * were caused by photoelectron emission from the sampler and the remainder were identified as carbon and oxygen as the dominant species with traces of Na+, NO32, Cl2 and SO4 22. Species such as Mg2+, Ca2+ and NH4 + were not detected due to the inefficient sampling process. Three major bands in the carbon region in Fig. 2 were identified as oxidised carbon (288.8 eV), graphitic/ aliphatic carbon (285.0 eV) and puzzlingly ruthenium oxide (280.9 eV). RuO2 is used as a catalyst in fuel cells and in the production of transportation fuels. Another possibility is copper carbide, a byproduct of Cu used as a catalyst in diesel vehicles. In either case this warrants further investigation. The resultant XPS images for the carbon regions are shown in Fig. 3(a)–(c) and indicate the non-uniform distribution of species across the clusters of particulates in the carbon region, with the lighter shades of grey representing increased pixel intensity.The segregation of surface species is due to the heterogeneity of particulates derived from numerous sources and interactions in the atmosphere. Residual carbon in the analysis chamber was not significant during the imaging process as Fig. 3(b) indicated a non-uniform distribution of carbon species. Images obtained for all species had a pixel resolution of 3 mm, reducing to 10 mm after smoothing, which is sufficient for identifying small clusters of atmospheric particulates.The low intensity region in the centre is thought to be caused by side-on X-ray illumination casting a shadow from the cluster of particles at the bottom of the image. The oxygen and oxidised carbon images showed similar spatial variation across the particle surface, so the oxygen image is not presented. No clear features were observed on images obtained for trace species due to poor S/N ratios.The image shows three spatially distinct, chemically different regions: one in which oxidised carbon dominates; one which is a mixture of oxidised and graphitic/aliphatic carbon; and one which is a mixture of ruthenium oxide and graphitic/aliphatic carbon. This work has shown evidence of localized chemical variation across the particulate surface. The next step will be to correlate such variation with the particle sources. Long acquisition times and lack of sensitivity has limited the study to major species at present.Improvement of spatial resolution to enable identification of individual particles is currently being investigated, but is far from routine. More efficient sampling methods such as the Burkard Spore trap4 are being explored. Acknowledgements The authors would like to thank the Jackson Environment Institute, UCL for the funding of this project. Special thanks to Tim Carney, VG Scientific for his advice on the intricacies of imaging XPS.References 1 D. W. Dockery, F. E. Speizer, D. O. Stram, J. H. Ware, J. D. Spengler and B. G. Ferris, Am. Rev. Respir. Dis., 1989, 139, 587. 2 C. A. Pope III, Arch. Environ. Health, 1991, 46, 90. 3 D. W. Dockery and C. A. Pope III, Annu. Rev. Public Health, 1994, 15, 107. 4 J. L. Battarbee, N. L. Rose and X. Long, Atmos. Environ., 1997, 31, 2, 171. 5 S. Weinbruch, M. Wentzel, M. Kluckner, P. Hoffmann and H. M. Ortner, Mikrochim. Acta, 1997, 25, 1–4, 137. 6 N. L. Rose, S. Juggins and J. Watt, Proc. R. Soc. London, Ser. A, 1996, 452, 881. 7 E. Adem, R. Champaneria and P. Coxon, Vacuum, 1990, 41, 7–9, 1695. 8 N. M. Forsyth and P. Coxon, Surf. Interface Anal., 1994, 21, 6–7, 430. 9 N. M. Forsyth and P. Coxon, Fresensius’ J. Anal. Chem., 1993, 346, 218. 10 I. W. Drummond, Philos. Trans. R. Soc. London, Ser. A, 1996, 354, 2667. Paper 8/09067J Fig. 2 High resolution XPS spectrum in the carbon region for atmospheric particulates. Fig. 3 Resultant XPS images for (a) C–O, (b) C–C/C–H and (c) RuO2 in the carbon region. 18 Anal. Commun., 1999, 36, 17–18

ISSN:1359-7337    

DOI:10.1039/a809067j

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Communication A new long-wavelength fluorigenic substrate for alkaline phosphatase: synthesis and characterisation G. Hussain Sarpara,a Si Jung Hu,a Derek A. Palmer,b Martin T. French,b Mark Evansb and James N. Millera a Department of Chemistry, Loughborough University, Loughborough, Leicestershire, UK LE11 3TU b Kalibrant Ltd., Department of Chemistry, Loughborough University, Loughborough, Leicestershire, UK LE11 3TU Received 18th November 1998, Accepted 9th December 1998 Naphthofluorescein diphosphate has been synthesised from the parent dye, and shown to be an attractive longwavelength alternative to other fluorigenic substrates for the determination of alkaline phosphatase.Its application to the determination of theophylline, an inhibitor of this enzyme, has been demonstrated. The optimum excitation wavelength of the hydrolysis product naphthofluorescein has been found to depend on the presence of additives such as cyclodextrins and (3-[3-cholamidopropyl]-dimethylamino)-1-propane sulfonate (CHAPS): such effects can be used to raise the excitation wavelength to match the output of a 635 nm diode laser in a simple and sensitive fluorescence detector. Amongst many recent developments in fluorescence spectrometry, the increased use of long wavelength probes and labels is one of the most important.1,2 The major benefits include the much reduced ‘autofluorescence’ background obtained from many biological samples if the excitation wavelength exceeds ca. 550 nm and the emission wavelength ca. 600 nm, and the ease with which long wavelength fluorescence is detected by using diode laser sources, simple fibre optics and solid state detectors in small, low-cost and robust instruments. The application of fluorigenic substrates, which are non- or weakly fluorescent but are converted by an appropriate enzyme to a highly fluorescent product, is widespread in biochemical analysis.3 Such methods combine the amplification effects of the catalytic enzyme action with the high sensitivity of fluorescence spectrometry. Some applications are straightforward enzyme assays, while in others the enzyme is a label through which other reactions such as immunoassays are monitored.4 Alkaline phosphatase, which hydrolyses phosphate esters in alkaline solution, is one of the most widely used labels in such indirect assays.The most commonly used fluorigenic substrates are those based on a number of umbelliferyl(7- hydroxycoumarin) derivatives,5 but the reaction products fluoresce at ca. 460 nm, a wavelength at which there is much background fluorescence from biosamples. Assays based on fluorescein (emission at ca. 520 nm)6 and resorufin (emission at ca. 590 nm)7 conjugates have also been described. Here we describe the synthesis and properties of naphthofluorescein diphosphate (I), a new long-wavelength fluorigenic substrate for alkaline phosphatase, and show how the fluorescence properties of its hydrolysis product can be red-shifted by appropriate choice of the solvent environment, facilitating analysis using diode laser light sources.Previous studies have demonstrated the applicability of carboxynaphthofluorescein as a solution pH probe,8 and a naphthofluorescein calcein derivative as an intracellular pH probe.9 Naphthofluorescein has also been investigated as a possible acceptor for use in fluorescence energy transfer assays.10 Experimental Naphthofluorescein diphosphate (NFDP) was synthesised by reacting naphthofluorescein (NF, Molecular Probes, Oregon; 0.25 mmol) with 10.7 mmol POCl3 in 4 ml dry pyridine under nitrogen gas at 0 °C.11 The reaction was monitored by thin layer chromatography on silica gel, using ethyl acetate : methanol : water : acetic acid (7 : 1 : 1 : 1 v/v/v/v) to develop the chromatogram: NF and NFDP had Rf values of 0.8 and 0.2 respectively.This method showed that 30 min reaction was sufficient, after which the reaction mixture was quenched by pouring into 40 ml of cold water and neutralising to pH 7.0 with ammonia.The pyridine was extracted with excess chloroform and the aqueous layer lyophilised. The resulting pink solid was characterised using IR spectrometry (phosphate absorption band at 1402 cm21) and mass spectrometry (NFDP molecular ion at m/z = 593). Fluorescence spectra were measured at room temperature using a Perkin-Elmer LS-50B spectrometer fitted with an R928 red-sensitive photomultiplier tube and a spectral bandwidth of 10 nm.Alkaline phosphatase (from calf intestine, 4.3 units mg21) was obtained from Sigma (Poole, Dorset) and was immobilised on 200–400 mesh amino-propyl controlled pore glass beads using glutaraldehyde prior to incorporation in a flow injection solid phase reactor of volume ca. 100 ml.12 Cyclodextrins and (3-[3-cholamidopropyl]-dimethylamino)- 1-propane sulfonate (CHAPS) were also products from Sigma. Fluorescence detection in flow injection analysis experiments was performed using a small laboratory constructed spectrometer incorporating a 635 nm diode laser light source (Power Technology Inc.), a 640 nm cut-off filter in the emission beam, and a silicon photodiode.Flow tubing of 0.8 mm diameter and a Hellma flow cell of 45 ml volume were used. The flow injection manifold used in the estimation of theophylline is shown in Fig. 1: the buffer flow rate was 1.5 ml min21, the injected sample and substrate volumes were 25 ml in each case, and the substrate concentration was 10 mg ml21.Results Fig. 2 shows that NFDP is effectively non-fluorescent, but is hydrolysed by alkaline phosphatase to yield the fluorescent naphthofluorescein, with excitation and emission wavelengths Anal. Commun., 1999, 36, 19–20 19of ca. 595 and 660 nm respectively in 0.1 M NaOH. Similarly, UV–visible absorption spectrometry shows that NF has a strong absorption band near 600 nm, while NFDP has only a weak band at ca. 500 nm. The effects of a range of shift reagents on the spectroscopic properties of NF were studied (Table 1). Addition of a-, b- and g-cyclodextrins (5% w/v) had relatively little effect on the absorption wavelength, while methyl-bcyclodextrin and 2-hydroxy-b-cyclodextrin at the same concentration produced shifts to 609 and 615 nm respectively. The most dramatic wavelength shifts were obtained with CHAPS, which produced shifts to ca. 630 nm (excitation) and 680 nm (fluorescence) when present at the 5% level.Lower levels of this reagent produced only slightly less significant shifts while 10% CHAPS produced a small further shift in the excitation wavelength, but had little effect on the fluorescence emission wavelength. Modest but useful increases in fluorescence intensity (ca. 2-fold) were also obtained by the use of CHAPS (Table 1). The combined effects of the wavelength shift and intensity enhancement facilitated the use of NFDP as a fluorigenic substrate.This was demonstrated by the determination of the anti-asthmatic drug theophylline by using its inhibitory effect (Fig. 3) on alkaline phosphatase immobilised in the flow injection microreactor system. The solid state fluorescence instrument could detect NF at levels of ca. 1029 M: although the inhibition of the enzyme by theophylline in the flow injection system was modest, it sufficed to determine this drug at therapeutic (mg ml21) levels. Conclusions The results presented here demonstrate the development of a new fluorigenic substrate for alkaline phosphatase, and show that the hydrolysis product of this and other NF based substrates can be used for trace analyses using diode laser based fluorescence detection.The novel use of CHAPS to shift the fluorescence, and especially the excitation wavelength of NF is crucial in matching the characteristics of this fluorophore to those of the lowest wavelength diode lasers currently available at low cost.Further new NF-based substrates for the determination of esterase and other enzymes are currently being developed in our laboratory, and are expected to be of importance in high throughput screening applications. Preliminary studies also suggest that naphthofluorescein monophosphate might be an even better substrate, with a lower Michaelis constant, than NFDP; and that the wavelength shift effect of CHAPS might be extended to other long-wavelength and near-IR fluorophores.The origin of this interesting phenomenon is also under further study. Acknowledgements G.H.S. thanks the Government of Pakistan for financial support. References 1 J. N. Miller, M. B. Brown, N. J. Seare and S. Summerfield, in Fluorescence Spectroscopy, New Methods and Applications, ed. O. S. Wolfbeis, Springer-Verlag, Berlin, 1993. 2 M. B. Brown, T. E. Edmonds, J. N. Miller, D. P. Riley and N. J. Seare, Analyst, 1992, 118, 407. 3 D. Robinson, Biochem. J., 1956, 62, 39 4 J. F. Bard, R. J. Carrico, M. C. Fetter, R. T. Buckle, R. D. Johnson, R. C. Boguslaski and J. E. Christner, Anal. Biochem., 1977, 77, 56. 5 D. H. Leaback, Biochem. J., 1961, 78, 22P. 6 A. Plovins, A. M. Alvarez, M. Ibanez, M. Molina and C. Nombela, Appl. Environ. Microbiol., 1994, 60, 4638. 7 J. Hofmann and M. Sernetz, Anal. Chim. Acta, 1984, 67, 163. 8 A. Song, S. Parus and R. Kopelman, Anal. Chem., 1997, 69, 863. 9 Y. Zhou, E. M. Marcus, R.P. Haugland and M. Opas, J. Cell Physiol., 1995, 164, 9. 10 A. P. Weir, J. N. Heron and D. A. Christensen, ACS Symposium Series, 1992, 511, 105. 11 H. D. Hill, G. K. Summer and M. D. Water, Anal. Biochem., 1968, 24, 9. 12 M. Masoom and A. Townshend, Anal. Chim. Acta, 1984, 166, 111. Paper 8/09018A Fig. 1 Flow injection manifold for the determination of theophylline. S = sample, A = buffer, R = substrate injection, P = peristaltic pump, DIV = dual injection valve, IMER = immobilised enzyme reactor; D = detector, W = waste. Fig. 2 Excitation and emission spectra in 0.1 M NaOH of (1) naphthofluorescein diphosphate (A and B, respectively) and (2) naphthofluorescein (C and D, respectively). Fig. 3 Inhibition of immobilised alkaline phosphatase by theophylline, determined with the use of naphthofluorescein diphosphate using the solid state fluorescence spectrometer. Table 1 Effect of additives on the fluorescence properties of naphthofluorescein Additive Excitation maximum/nm Emission maximum/nm Intensity enhancement factor (%) None 595 660 — a-Cyclodextrin (5% w/v) 596 660 11 b-Cyclodextrin (5% w/v) 603 660 43 g-Cyclodextrin (5% w/v) 599 660 37 Methyl b-cyclodextrin (5% w/v) 609 660 — 2-Hydroxypropyl-bcyclodextrin (5% w/v) 616 660 — CHAPS (5% w/v) 630 680 47 20 Anal. Commun., 1999, 36, 19–20

ISSN:1359-7337    

DOI:10.1039/a809018a

出版商:RSC    

年代:1999

数据来源: RSC