摘要:

Classical wet ashing versus microwave-assisted attacks for the determination of chromium in plants A. Sahuquillo, R. Rubio* and G. Rauret Departament de Química Analítica, Universitat de Barcelona, Avd. Diagonal 647, E-08028, Barcelona, Spain Received 5th November 1998, Accepted 5th November 1998 Microwave-assisted pre-treatments for the determination of Cr in plants are compared with classical wet-ashing procedures using open attacks in sand-baths. Four certified plant reference materials were analysed: pine needles (NIST SRM 1575), rye grass (BCR CRM 281), beech leaves (BCR CRM 100) and an aquatic plant (Trapa natans) (BCR CRM 596).The use of acidic procedures with HClO4 or H2SO4 yielded different Cr results for these materials when classical wet-ashing procedures were used, as the use of HClO4 caused losses of volatile chromium compounds. The shorter time of analysis required (60 min) in open-focused microwave-assisted attack allows the use of HClO4 for obtaining results very close to the certified values for CRM plant materials.This type of microwave digestion also led to good reproducibility values with relative standard deviations between 5 and 10%. Introduction Chromium is determined routinely to monitor pollution levels in both environmental and biological matrices. For the latter, Cr is usually determined in plants since its concentration level provides information about plant uptake from polluted soils and entry into the trophic chain.The chromium content in these matrices is generally lower than 5 mg kg21, which requires the use of sensitive analytical techniques.1 The technique most widely used for chromium determination is atomic absorption spectroscopy with electrothermal atomisation (ETAAS).2,3 Nevertheless, it is widely accepted that the measurement of chromium in complex matrices by AAS involves serious difficulties.2,4 Other powerful spectroscopic techniques, such as inductively coupled plasma mass spectrometry (ICP-MS), are subject to spectral and non-spectral interferences5,6 and consequently this technique is unsuitable for the certification of chromium in reference materials.4,7 Recent developments in ETAAS include different sample introduction procedures such as the preparation of slurries.However, this system presents homogeneity problems for sample intake at the milligram level, especially for field samples, and therefore sample pre-treatment is mandatory to obtain good reproducibility.8,9 With regard to the attack of sample materials for chromium determination, the use of classical open attacks can presumably lead to losses of volatile chromyl chloride or organic chromium compounds, due to the reduction of perchlorate anion to chloride and due to the oxidation of organic substances.10,11 Sample digestion is usually the most time-consuming step of the analysis.With the aim of shortening the time and the total volume of reagents, and to avoid contamination in the digestion process, the use of microwave sample pre-treatment is increasing, as shown by the large number of reviews dealing with the principles.6,10,12–14 The main microwave-assisted digestions described for chromium determination in plants consist of microwave PTFE closed-vessel attacks using different acidic mixtures for short periods of time.15–17 In this work, different digestion procedures for the determination of Cr in plants were studied with the aim of establishing a suitable method that ensures no losses of volatile chromium compounds, has the ability to use as little reagent as possible and is rapid.For this purpose, classical open wet ashing procedures with sand-baths were compared with open focused microwave-assisted pre-treatments optimised for the determination of chromium in plants. In both cases, HClO4 and H2SO4 were tested for sample mineralisation. The instrumental conditions for final determination by ETAAS with Zeeman effect background correction (ZETAAS) were optimised for the different extracts obtained after sample pre-treatment.Experimental Apparatus A Pselecta Recisplac sand-bath (Afora, Barcelona, Spain) was used for sample pre-treatment when heating by conduction. A Microdigest A301 open-focused microwave digestor (Prolabo, Paris, France) was used. The magnetron worked at a maximum power of 200 W (100%) and it could be regulated from 10 to 100% in steps of 5%.A Perkin-Elmer (Norwalk, CT, USA) Model 4100ZL atomic absorption spectrometer with longitudinal Zeeman effect background correction and transversal heating, equipped with an automated autosampler able to perform standard additions, was used for Cr determination. Pyrolytic graphite-coated tubes with a pre-built L’vov platform were used. Reagents All solutions were prepared using doubly de-ionized water (Culligan Ultrapure GS, 18.3 M½ cm21) in a class 100 work bench with vertical air flow, in accordance with the USA Federal Standard 209b/d norm.18 The class of the laboratory was checked annually.19 All the concentrated acids used for the attacks were of Suprapur quality (Merck, Darmstadt, Germany).Chromium(vi) standard solutions were prepared from certified National Institute of Standards and Technology (NIST) potassium dichromate of 99.984 ± 0.010% purity. The working calibrant solutions were prepared in 0.3 mol l21 HNO3. Analyst, 1999, 124, 1–4 1Otherwise, Cr(iii) commercially available calibrant solution was used and checked against K2Cr2O7 standard solutions.As chemical modifiers, 0.02 mol l21 Mg(NO3)2 and 0.01 mol l21 Pd of Suprapur quality were used (Merck). Samples Four certified plant reference materials were used: pine needles (NIST SRM 1575), rye grass [Bureau Community of Reference (BCR) CRM 281], beech leaves (BCR CRM 100) and an aquatic plant (Trapa natans) (BCR CRM 596). The certified chromium contents in these materials were 2.6 ± 0.2, 2.14 ± 0.12, 8.0 ± 0.6 and 36.3 ± 1.7 mg kg21, respectively, on dry mass.Further information on these materials is available in the certification reports.4,7,20 Sample preparation HClO4 digestions by conduction. A 5 ml volume of 14 mol l21 HNO3 was added to 0.5 g of sample in a PTFE beaker. The beakers were then placed in a sand-bath and heated to between 120 and 150 °C. Subsequent aliquots of HF–HClO4 (2 + 1) were added until the remaining residue did not have a siliceous aspect.A total volume of 33 ml of the mixture was added in four steps. A total volume of 6 ml of 8.8 mol l21 H2O2 was added to the sample in three steps for complete mineralisation. Finally, 2 ml of HClO4–H2O2 (1 + 2) were added and the sample was evaporated to dryness. The residue was dissolved in dilute HNO3 and diluted to 50 ml with doubly deionized water. The whole sample pre-treatment process lasted between 40 and 56 h.H2SO4 digestions by conduction. The above procedure was also used but HClO4 was replaced with H2SO4. At the end of the digestion, a residue of insoluble sulfates was obtained. The suspension was filtered through an ashless Whatman (Maidstone, Kent, UK) No. 42 filter-paper. The residue was washed carefully with doubly de-ionized water and the filtrate was diluted to 50 ml. When using H2SO4, the total time of attack increased to 90 h. Microwave-assisted digestion procedures.Table 1 gives the optimised programmes for the microwave digester used. As for the open attacks in sand-bath, the final solution was filtered through an ashless Whatman No. 42 filter-paper to ensure the separation of any fine particles, and the filtrate was diluted to 50 ml with doubly de-ionized water. Measuring conditions ZETAAS determination was performed at 357.9 nm with a slitwidth of 0.2 nm. Peak areas were considered for signal treatment. The standard additions method was used for calibration. A 5 ml volume of sample, diluted to a final volume of 20 ml with blank and Cr(vi) standard solution, was injected.The working linear concentration range was from 2 to 30 mg l21. Table 2 gives the optimised programme of temperatures and times, which was established for each matrix studied. All measurements were performed in duplicate. Results and discussion Establishment of graphite furnace heating programmes The acidic solutions obtained from each plant material were used for the optimisation of the final ZETAAS instrumental conditions. Each programme of temperature and times was established from the construction of pyrolysis and atomisation curves.The pyrolysis curve was obtained at a fixed drying and atomisation temperature by increasing the temperature of pyrolysis from 900 to 1800 °C in steps of 100 °C. For the atomisation curve, the drying and pyrolysis temperatures were fixed and the atomisation temperature was increased from 1900 to 2600 °C in steps of 100 °C.At each temperature, 20 ml of plant extracts and 20 mg l21 Cr(vi) calibrant solution were injected in duplicate. Fig. 1 shows the curves obtained for each of the materials studied. The criterion for choosing the working temperatures was a maximum peak area with a good peak shape, that is, non-tailing and reproducible peaks. Table 2 gives the final optimised programmes obtained from the pyrolysis and atomisation curves.No significant differences were observed between the final temperatures obtained for standard solutions in 0.3 mol l21 HNO3 and those established for plant extracts. Table 1 Microwave digestion procedures for plants Programme A (HClO4) Programme B (H2SO4) 5 ml HNO3; 30 W (10 min); 40 W (5 min) 5 ml HNO3; 30 W (10 min); 40 W (5 min) 5 ml HF–HClO4 (2 + 1); 30 W (10 min); 50 W (5 min); 60 W (15 min)a 5 ml HF–H2SO4 (2 + 1); 30 W (10 min); 50 W (5 min); 80 W (15 min)a 10 ml HNO3 (1 + 10); 20 W (5 min) 5 ml HNO3 (1 + 10); 20 W (5 min); 40 W (5 min)a 2 ml H2O2; 20 W (5 min); 40 W (10 min)a 10 ml HNO3 (1 + 10); 20 W (5 min) Total time: 50 min Total time: 75 min a Evaporation to dryness.Table 2 Graphite furnace programme for plant materials Step T/°C tramp/s thold/s Drying 1 110 10 20 Drying 2 130 15 30 Ashing 1400 1300a 10 20 Atomisationb 2200 0 4 Cleaning 2400 1 2 a Ashing temperature for beech leaves. b Stopped flow during atomisation. Fig. 1 Pyrolysis and atomisation curves for plant materials and Cr(vi) calibrant solution. 2 Analyst, 1999, 124, 1–4The ashing temperature was set to 1300 °C for beech leaves since slightly higher peak area values were obtained. The extracts resulting from HClO4 and H2SO4 digestions yielded the same pyrolysis and atomisation conditions. Atomisation profiles and analytical performance Two calibration methods were used: linear calibration graph and standard additions. Chromium(vi) and (iii) solutions were tested as calibrants.The results obtained using both types of calibrant solution and calibration method agreed closely, as also observed in previous studies with different matrices.3 Since slightly better reproducibility was obtained with the standard additions method, the final Cr determination was carried out with this method and using Cr(vi) as calibrant solution. Atomisation profiles and background correction signals were studied by adding some of the more commonly used chemical modifiers mentioned in the literature for chromium, such as Mg(NO3)2 21,22 and Pd.22 The characteristic masses obtained for rye grass with and without addition of Mg(NO3)2 were 7.10 ± 0.05 and 7.53 ± 0.18 pg Cr, respectively, with similar atomisation peak profiles.On the other hand, the addition of 0.01 mol l21 Pd increased the background signal significantly without improving either the atomic signal or the peak profile. Therefore, no chemical modifier was used for Cr quantification in these materials.Classical digestion procedures Two series of open attacks in a sand-bath examined, one using concentrated HClO4 as oxidant acid and the other using H2SO4 and maintaining the same acid volume-to-sample mass ratio. Addition of HF was necessary to remove the siliceous structural component of the plants. The initial volumes tested were 5 ml of each of the acids and the final volumes were established according to the aspect of the residue obtained.Complete mineralisation was obtained only after the addition of H2O2. Five replicates for each plant material were digested in different working sessions. Some differences were observed between extracts resulting from HClO4 and H2SO4 attacks. Whereas when HClO4 was used clear, colourless solutions were obtained, the use of H2SO4 led to a black, insoluble residue which required a filtration step and the final solutions were slightly coloured. Owing to the difficulty in removing H2SO4, the final time of attack was increased to between 30 and 50 h.Table 3 gives the results obtained by applying the two digestion procedures. The mean value and the standard deviation for five determinations are compared with the certified values. For Cr determination in pine needles, both attacks yielded concentration values that agreed closely with the certified value. However, a negative bias was observed for the other three materials when HClO4 was used.The losses of analyte detected were about 35%, with reproducibility ranging between 11 and 19% (RSD), showing different behaviour depending on the type of matrix. When samples were digested with conduction heating only H2SO4 gave good results. The main disadvantage of this acidic digestion was that a different procedure would have to be followed if other heavy metals such as Pb are to be determined. Microwave-assisted attacks With the aim of shortening the time of analysis and studying the behaviour of chromium when using other heating systems such as microwaves, two different programmes were optimised for sample pre-treatment by using HClO4 or H2SO4, both combined with HF.The same acidic mixtures as used for sand-bath procedures were tested in different steps. Before the addition of a new acidic mixture the aspect of the remaining residue was monitored until a crystalline residue was obtained. The microwave power was increased in subsequent heating processes. Microwave powers up to 80 W were only necessary in drying steps with H2SO4 owing to its high boiling-point.As can be seen in Table 1, 50 min were required when using the HClO4 and 75 min when using the H2SO4 procedure. In the same way as for the open attacks, the extracts were filtered and diluted to 50 ml with doubly de-ionised water. Although the microwave digester allowed the possibility of evaporating the sample to dryness, several drops of condensed HF remained on the walls of the PTFE tubes.Therefore, 10 ml of saturated H3BO3 were added to the digestion vessels in order to eliminate any remaining HF. The addition of H3BO3 did not have any effect on the heating temperature programmes in the graphite furnace. Table 4 gives the results obtained for pine needles, rye grass and beech leaves. The results obtained using the HClO4 and H2SO4 procedures were consistent with the certified values (recoveries ranged from 92 to 108%) for all matrices except rye grass when HClO4 was used (86% recovery). Since the digestion time when using microwaves was reduced by a factor of about 60, no significant losses of chromium occurred.The RSD obtained for HClO4 were lower than 5% for triplicate analyses and slightly higher values were obtained for H2SO4 attack (4–13%). Conclusions The use of HClO4 during the sample pre-treatment step in classical open attacks with conduction heating (sand-bath) led to losses of volatile chromium compounds for some of the plant Table 3 Comparison of open attacks with HClO4 and H2SO4 (conduction) (mg kg21 Cr, mean ± s, n = 5) Sample Certified value RSD% HClO4 attack RSD% H2SO4 attack RSD% Pine needles 2.6 ± 0.2 7.7 2.15 ± 0.26 12 2.45 ± 0.14 5.7 Rye grass 2.14 ± 0.12 5.6 1.48 ± 0.28 19 2.33 ± 0.31 13 Beech leaves 8.0 ± 0.6 7.5 5.12 ± 0.80 16 7.27 ± 0.44 6.1 Aquatic plant 36.3 ± 1.7 4.7 23.08 ± 2.63 11 32.01 ± 4.66 14 Table 4 Comparison of open attacks with HClO4 and H2SO4 (microwaves) (mg kg21 Cr, mean ± s, n = 3) Sample Certified value RSD% HClO4 attack RSD% H2SO4 attack RSD% Pine needles 2.6 ± 0.2 7.7 2.40 ± 0.07 2.9 2.45 ± 0.14 5.7 Rye grass 2.14 ± 0.12 5.6 1.84 ± 0.05 2.7 2.33 ± 0.31 13 Beech leaves 8.0 ± 0.6 7.5 8.12 ± 0.80 9.8 8.07 ± 0.80 9.9 Analyst, 1999, 124, 1–4 3materials studied. From this it can be concluded that losses of chromium are matrix dependent and therefore validation of the method is required for samples of different origins. When classical open attack is the only possibility, the use of H2SO4 is mandatory in spite of the longer time of analysis required.The open-focused microwave heating system is suitable for Cr determination in plants, even when HClO4 is used in the acidic mixtures. Moreover, the time of analysis and the volume of reagents can be reduced by factors of 60 and 6, respectively. Hence it can be considered a cleaner method than the open attack method.The proposed method provides good reproducibility. References 1 J. W. Moore, in Inorganic Contaminants of Surface Water. Research and Monitoring Priorities, ed. R. S. De Santo, Springer, New York, 1991, ch. 9, pp. 82–97. 2 R. Rubio, A. Sahuquillo, G. Rauret and Ph. Quevauviller, Int. J. Environ. Anal. Chem., 1992, 47, 99. 3 A. Sahuquillo, J. F. López-Sánchez, R. Rubio and G. Rauret, Mikrochim. Acta, 1995, 119, 251. 4 Ph. Quevauviller, J. G. Van Raaphorst and H. Muntau, Trends Anal.Chem., 1996, 15, 259. 5 S. Wu, X. Feng and A. Wittmeier, J. Anal. At. Spectrom., 1997, 12, 797. 6 F. E. Smith, Talanta, 1996, 43, 1207. 7 J. G. Van Raaphorst, Ph. Quevauviller and H. Muntau., The Certification of the Mass Fraction of Chromium in Rye Grass (CRM 281), Beech Leaves (CRM 100), Aquatic Plant (Trapa Natans) (CRM 596), Fly Ash (CRM 038) and Sewage Sludge (CRM 597), EUR 16840 EN Report, European Commission BCR Information. Reference Materials, Brussels, 1996. 8 R. E. Sturgeon, Spectrochim. Acta, Part B, 1997, 52, 1451. 9 A. Carlosena, M. Gallego and M. Valcárcel, J. Anal. At. Spectrom., 1997, 12, 479. 10 I. Kubrakova, Spectrochim. Acta, Part B, 1997, 52, 1469. 11 M. J. T. Carrondo, R. Perry and J. N. Lester, Anal. Chim. Acta, 1979, 106, 309. 12 M. Burguera and J. L. Burguera, Quim. Anal., 1996, 15, 112. 13 R. Chakraborty, A. K. Das, M. L. Cervera and M. de la Guardia, Fresenius’ J. Anal. Chem., 1996, 355, 99. 14 A. Sinquin, T. Görner and E. Dellacherie, Analusis, 1993, 21, 1. 15 D. H. Sun, J. K. Waters and T. P. Mawhinney, J. AOAC Int., 1997, 80, 647. 16 I. V. Kubrakova, T. F. Kudinova, E. B. Stavnivenko and N. M. Kuz’min, J. Anal.Chem., 1997, 52, 522. 17 R. Chakraborty, A. K. Das, M. L. Cervera and M. de la Guardia, J. Anal. At. Spectrom., 1995, 10, 353. 18 J. R. Moody, Anal. Chem., 1982, 54, 1358A. 19 Report of Verification and Control of Clean Laboratories, Sociedad de Validación de Sistemas (SVS), Sant Cugat, Barcelona, 1998. 20 Certificate of Analysis of SRM 1575 (Pine Needles), National Institute of Standards and Technology, Gaithersburg, MD, 1993. 21 M. Hoenig and A. M. de Kersabiec, L’Atomisation � Electrothermique en Spectrométrie d’Absorption Atomique, Masson, Paris, 1989, pp. 137–165. 22 A. Sahuquillo, R. Rubio and G. Rauret, in Quality Assurance for Environmental Analysis, ed. Ph. Quevauviller, E. A. Maier and B. Griepink, Elsevier, Amsterdam, 1995, pp. 39–62. Paper 8/08659A 4 Analyst, 1999, 124, 1&ndash

ISSN:0003-2654    

DOI:10.1039/a808659a

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Influence of sediment grain size on the efficiency of focused microwave extraction of polycyclic aromatic hydrocarbons M. Letellier and H. Budzinski* LPTC, UPRESA 5472 CNRS, 351 cours de la libération, 33405 Talence, France Received 25th September 1998, Accepted 6th November 1998 The efficiency of focused microwave (FMW)-assisted extraction of polycyclic aromatic hydrocarbons (PAHs) at atmospheric pressure was investigated for sediments with different grain size distributions. The PAH contents and distribution profiles obtained by FMW extraction for a dry matrix and a remoistened dry matrix were compared with those obtained by Soxhlet extraction for a bulk matrix and six fractions.The effect of moisture depended on the composition of the matrix and the grain size: an improvement in PAH recovery with the addition of water was noted for coarse fractions, but not for fine fractions. Application to other matrices of different grain sizes and contamination levels showed that FMW-assisted extraction is a good alternative to Soxhlet extraction.FMW extraction efficiency was tested on a naturally moist sediment. PAH concentrations were compared with those obtained by extraction of dry and remoistened dry matrices by FMW extraction and with those obtained by extraction of a dry matrix by Soxhlet extraction. PAH recoveries, compared with those obtained by Soxhlet extraction, were satisfactory. Therefore, it is possible to avoid the drying step with the FMW method.The FMW technique might be suitable for field studies, for example, on a boat during an oceanographic cruise. The developed procedure cosists of an extraction step of 10 min with a few millilitres of solvent, reconcentration steps and micro-column purification. The treatment of the sample can be performed immediately after sampling. The method affords good recovery. The reproducibilities are comparable to, or better than, those obtained by conventional extraction. Introduction Contaminants of anthropogenic or natural origin such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment.1–3 Such persistent compounds damage the entire ecosystem and especially the aquatic environment. Sediment, because of its accumulation capacity, is a huge sink for contaminants arising from atmospheric contaminated particles, gas transfer and effluents. Because of the bioavailability of contaminants, present in the sedimentary matter, there is a toxicological risk to fauna and flora.In this respect, monitoring of contamination levels and research into the association of contaminants with sediment have been conducted. In environmental studies, the improvement in analytical methods has allowed more accurate quantification of individual compounds of different toxicity levels, which allows an estimation of the exposure of organisms to contaminants. However, the limiting factor in the analysis of numerous samples is the treatment of the sample.The conventional procedure consists in drying the sample followed by Soxhlet extraction, which involves the percolation of the sample for 8–72 h by a solvent. This method is time and solvent consuming and is not easy to automate. The development of new extraction methods, based on microwave irradiation, showed that such methods might be a good alternative to Soxhlet extraction.4–12 A focused microwave (FMW)-assisted extraction method at atmospheric pressure gives satisfactory results with a reduction of time (10 min), a reduction of solvent (30 mL) and with safety.11 The procedure of extraction of PAHs has been optimised on a certified matrix (NIST SRM 1941a).12 This preliminary study12 has shown the influence of moisture on the extraction efficiency.Indeed, the specific interaction of microwaves with polar compounds allows local heating of the moist matrix and an overall improvement in extraction recovery.10,12 The optimum amount of moisture in sediment has been determined (30%) and allows a significant improvement in recovery.12 However, the effect of moisture varies depending on the matrix. The preliminary study12 could not show a relationship between the nature of the matrix and water content.One interesting characteristic of a matrix appears to be the grain size. The efficiency of microwave procedures according to grain size was studied in this paper. Firstly, it is important to study the selectivity of the FMW extraction towards compounds of different origin and associated with different particles.Some studies showed a difference in the distribution profile with the grain size of the sediment for different classes of contaminants, such as PAHs and PCBs.13,14 Secondly, it is interesting to study the influence of moisture content on the microwave extraction efficiency. In this study, efficiencies obtained by Soxhlet extraction and FMW extraction were compared for a bulk muddy sediment and for six sub-fractions of this matrix. The effect of moisture for each sub-fraction and for different compounds was studied. Two other sediments with different levels of contamination and different grain sizes were also studied.Finally, the extraction of a naturally moist sediment was compared with the extraction of the freeze-dried matrix to determine whether a drying step is necessary for the FMW method. Experimental Standards, solvents and reagents The PAHs studied ranged from three-ring aromatics (phenanthrene) to six-ring aromatics (benzo[ghi]perylene).The Standard Reference Material, SRM 2260, Aromatic Hydrocarbons in Toluene (Nominal Concentration 60 mg mL21), a standard solution of 24 aromatic hydrocarbons (23 are certified), was provided by the National Institute of Standards and Technology (NIST, Gaithersburg, MD, USA) and used for calibration. Analyst, 1999, 124, 5–14 5The compounds used as internal standards were perdeuterated PAHs.Phenanthrene-d10, benzo[a]pyrene-d12 and benzo[ ghi]perylene-d12 were purchased from Cambridge Isotope Laboratories (CIL, Andover, MD, USA), and fluoranthene-d10, pyrene-d10 and chrysene-d12 from MSD Isotopes (Division of Merck Frost Canada, Montreal, Canada). Pestinorm dichloromethane was purchased from Prolabo (Fontenay-sous-Bois, France). HPLC-grade isooctane and extra-pure pentane (Scharlau) were purchased from ICS (St Médard en Jalles, France).Pentane was distilled. Copper (40 mesh, 99.5% purity) (Aldrich, Saint Quentin Fallavier, France) was activated with hydrochloric acid (7 M), then washed with water, acetone and dichloromethane. Alumina (150 basic, Type T, 0.063-0.2 mm) and silica (silica gel, 0.063–0.2 mm) (Merck, Darmstadt, Germany) were washed with dichloromethane, deactivated at 150 °C overnight and then stored at 150 °C. Sediment sampling For the study of grain size fraction and of the influence of the matrix state (dry or moist), a sediment was sampled in the superficial layer (0–5 cm) in the harbour of Arcachon (south west Atlantic coast of France).It was homogenised and frozen (220 °C) until treated. For grain size study (sand or silt), two sediments (‘OPCB 5b’ and ‘OPCB 9b’) were sampled in the Gironde estuary (south west of France) by Flusha or Shipeck grab. They were chosen for their different grain sizes and contamination levels.Sediment fractionation For the study of grain size fraction, bulk sediment was wetsieved: sediment samples were gently shaken by hand in sieves with water (2000, 500, 300, 125, 63, 40, 15 mm). Six grain-size sub-fractions were defined as follows: 500–300 mm (coarse sands), 300–125 mm (medium sands), 125–63 mm (fine sands), 63–40 mm (silts), 40–15 mm (medium silts), 15–0 mm (fine silts and clays). Wet-sieving and manual stirring were employed to avoid possible carry-over of finer particles, which might reach the coarser fractions if dry-sieving was performed.Each subfraction was centrifuged to remove water and freeze-dried. The weight of each sub-fraction of sediment was carefully recorded to estimate the relative weight contribution. During the sieving, attempts were made to minimise sediment weight loss. For the study of the influence of the matrix state (dry or moist), bulk sediment was wet-sieved in sieves of 300 mm to remove large debris in order to improve homogeneity.The 0–300 mm sub-fraction was centrifuged to remove as much water as possible and to keep about 30% of water as in the natural bulk sediment. Half of the material was stored at 220 °C until treatment. The other half was freeze-dried. For the study of grain size (sand or silt), bulk sediments were freeze-dried and then sieved at 500 mm. Soxhlet extraction procedure Soxhlet extractions with dichloromethane (2 3 250 mL) were performed using 0.3–10 g of sediment (Table 1) spiked with perdeuterated internal standards for quantification. The extraction lasted 48 h.Blank experiments were performed. The extract was reduced to a small volume (a few millilitres) using a rotary evaporator. The organic extract was desulfurized on activated copper and then purified as described below (Fig. 1). Microwave extraction procedure Apparatus. FMW-assisted extractions in open cells were performed at a frequency of 2450 MHz using a SOXWAVE 100 Table 1 Extracted amount of sediment by Soxhlet and FMW-assisted methods and weight and number percentage, organic carbon and mineral carbon content (weight-%) for each sub-fraction of Arcachon sediment.Sediment Extracted amount/g Weight distribution (%) Number distribution (%) Organic carbon content (weight-%) Mineral carbon content (weight-%) Arcachon sediment: < 500 mm 1 Arcachon sediment: 500–300 mm 1 25 24 0.27 0.01 Arcachon sediment: 300–125 mm 1 45 32 0.30 0.00 Arcachon sediment: 125–63 mm 1 14 11 0.97 0.10 Arcachon sediment: 63–40 mm 0.5 5 5 2.45 0.25 Arcachon sediment: 40–15 mm 0.5 9 17 7.60 0.35 Arcachon sediment: 15–mm 0.3 2 11 7.90 0.25 Silty sediment: OPCB 5b 10 Sandy sediment: OPCB 9b 10 Naturally moist Arcachon sediment 2 Fig. 1 Schematic diagram of the sample preparation procedure. 6 Analyst, 1999, 124, 5–14apparatus (Prolabo) with a programmable heating power (from 30 to 300 W). The use of focused microwaves allowed homogeneous and reproducible treatment of samples.The SOXWAVE 100 operates at atmospheric pressure. Procedure. The procedure has been optimised on a certified matrix, NIST SRM 1941a.12 The FMW extractions were performed using conditions as close as possible to those of Soxhlet extractions using 0.3–10 g of sediment (Table 1). A solution containing the perdeuterated PAHs used for quantification was added to the matrix prior to the extraction. A level of 30% of moisture (g of water per g of sediment) was added to the freeze-dried sediment in the vessel, after which 30 mL of dichloromethane were added.The power (30 W) and time (10 min) were programmed. Blank experiments were performed. The extract was filtered and reduced to a small volume (a few millilitres) using a rotary evaporator. The organic extract was desulfurized on activated copper and then purified (Fig. 1). Purification The extract was purified on a micro-column containing alumina by eluting with dichloromethane. The purified extract was then fractionated on a micro-column containing silica in order to collect separately saturated and aromatic compounds eluted with, respectively, pentane and pentane–dichloromethane (65 + 35, v/v).15 The purified aromatic fraction was finally reconcentrated to a few microlitres in isooctane and analysed by gas chromatography-mass spectrometry (GC-MS). Gas chromatography-mass spectrometry conditions The analyses were performed on an HP 5980 Series II gas chromatograph (Hewlett-Packard, Palo Alto, CA, USA) equipped with a splitless injector (purge delay: 1 min; purge flow: 60 mL min21). The injector was maintained at 270 °C.The temperature program was: 50 °C (2 min) to 290 °C (20 min) at 5 °C min21. The carrier gas was helium at a constant flow rate of 1 mL min21. The capillary column used was a PTE-5 (Supelco, Bellefonte, PA, USA), 60 m 3 0.25 mm id (0.25 mm film thickness). The gas chromatograph was coupled to an HP 5972 Mass Selective Detector (MSD) (electron impact: 70 eV, voltage: 2000 V) operated in the single ion monitoring (SIM) mode using the molecular ion of each compound at 1.23 scans s21.The interface temperature was maintained at 290 °C. The PAHs were quantified relative to perdeuterated PAHs. The response factors of the different compounds were measured by injecting a solution of SRM 2260, containing 23 PAHs at certified concentrations and spiked with perdeuterated compounds used as internal standards for the extraction of the samples.Some co-elutions were noted between structural isomers: (i) chrysene co-eluted with triphenylene; (ii) benzo[b]fluoranthene co-eluted with benzo[j]fluoranthene and benzo[k]fluoranthene; (iii) dibenz[a,h]anthracene co-eluted with dibenz[a,c]anthracene. The concentrations given for the compounds suffering from these co-elutions take this factor into account: Chry* = Chry + Trip, BF = BbF + BjF + BkF, DaA = DahA + DacA.Carbon analyses Each fraction was analysed with a LECO CS 125 carbon analyser (LECO, MI, USA), according to Cauwet,16 to evaluate the total carbon and organic carbon content. Results and discussion Extraction efficiency of PAHs from a muddy sediment and from sub-fractions Characteristics of sediment. Arcachon sediment is a silt. The grain size distribution was determined by sieving (% in Table 2 PAH concentrations (ng g21) of the different fractions of Arcachon sediment obtained by Soxhlet extraction.Conc. = concentrationa < 500 mm 500–300 mm 300–125 mm 125–63 mm 63–45 mm 45–15 mm 15–0 mm Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) P 182 ± 52 28 279 ± 257 92 101± 21 20 224 ± 11 5 246 ± 9 4 283 ± 16 6 234 ± 45 19 Fluo 423 ± 100 24 637 ± 477 75 244 ± 72 30 566 ± 45 8 627 ± 45 7 500 ± 45 9 280 ± 90 32 Pyr 641 ± 88 14 550 ± 398 72 243 ± 64 27 595 ± 77 13 944 ± 125 13 959 ± 126 13 598 ± 220 37 BaA 179 ± 16 9 296 ± 223 75 128 ± 32 25 309 ± 12 4 322 ± 29 9 239 ± 48 20 136 ± 58 43 Chry* 443 ± 16 4 353 ± 241 68 179 ± 40 22 466 ± 16 3 656 ± 40 6 792 ± 16 2 555 ± 77 14 BF 569 ± 129 23 678 ± 477 70 307 ± 71 23 946 ± 39 4 1305 ± 10 1 1641 ± 123 7 1226 ± 137 11 BeP 254 ± 16 6 261 ± 183 70 120 ± 29 24 377 ± 19 5 520 ± 10 2 680 ± 30 4 530 ± 70 13 BaP 230 ± 24 10 337 ± 250 74 160 ± 44 27 399 ± 30 8 457 ± 87 19 339 ± 98 29 220 ± 96 44 Per 86 ± 4 4 107 ± 80 75 51 ± 13 26 125 ± 7 6 152 ± 18 12 151 ± 17 12 157 ± 33 21 IP 324 ± 25 8 306 ± 235 77 141 ± 36 25 401 ± 6 2 599 ± 53 9 850 ± 111 13 736 ± 54 7 BP 225 ± 5 2 253 ± 185 73 120 ± 33 28 347 ± 8 2 470 ± 19 4 602 ± 3 1 521 ± 70 13 DaA 64 ± 2 4 57 ± 39 69 27 ± 5 18 88 ± 2 2 129 ± 12 9 159 ± 17 10 135 ± 35 26 SPAHs 3619 ± 413 11 4115 ± 297372 1822 ± 449 25 4843 ± 214 4 6427 ± 414 6 7196 ± 254 4 5329 ± 939 18 a PAH identification: P = phenanthrene; Fluo = fluoranthene; Pyr = pyrene; BaA = benzo[a]anthracene; Chry* = chrysene (Chry) + triphenylene (Trip); BF = benzo[b]fluoranthene (BpF) + benzo[j]fluoranthene (BjF) + benzo[k]fluoranthene (BkF); BeP = benzo[e]pyrene; BaP = benzo[a]pyrene; Per = perylene; IP = Indeno[1,2,3-cd]pyrene; BP = benzo[ghi]perylene; DaA = dibenz[a,h]anthracene (DahA) + dibenz[a,c]anthracene (DacA).Fig. 2 Correlation between organic carbon content and the concentration of the sum of PAHs in sub-fractions of Arcachon sediment.Analyst, 1999, 124, 5–14 7weight) and by laser diffraction (% in number) (Table 1). In both cases, the major fraction is medium sands (300–125 mm). If the weight of bulk matrix which has been sieved, corrected for the percentage of moisture, is compared with the sum of the weight of each freeze-dried sub-fraction, the difference is 7.5%. This error is due to the loss of the finer particles during the removal of water by centrifugation.Organic and mineral carbon contents of each fraction were determined (Table 1). Fractions of the finest particles (40–15 and 15–0 mm) have a significant organic carbon content. PAH content in bulk sediment. The study of the bulk sediment (sieved at 2 mm to remove parts of shell or stone) was not possible because of problems of homogeneity. RSDs of PAH concentrations were above 50%. The sample amount of a few grams was not representative owing to the presence of vegetal fragments.Hence, the studied sediment was not strictly bulk but was sieved at 500 mm for this study. PAH concentrations obtained by Soxhlet extraction are given in Table 2. The RSD is under 28% for individual compounds and 11% for the sum of PAHs. The level of contamination is a few mg g21 for the sum of the PAHs studied. The profile of contamination shows that the contamination is both pyrolytic and petrogenic. There is a wide range of the parent PAHs in similar abundance which is characteristic of pyrolytic input and the presence of alkylated compounds shows an additional input of petrogenic contaminants. 17 Indeed, this area is fairly urbanised and the sample location is a yachting harbour. Grain-size distribution of PAHs. Concentrations of PAHs for each sub-fraction obtained by Soxhlet extraction are given in Table 2. If the contamination of the bulk sediment (0–500 mm) is calculated according to the weight percentage and the concentration of each sub-fraction, the result (3602 ng g21) is in agreement with the experimental value (3619 ± 413 ng g21) for the sum of PAHs.This shows that the fractionation is accurate and that the extractions performed on bulk sediment or subfractions are correct. The RSDs of concentrations obtained by Soxhlet extraction for the different sub-fractions are very different and depend on Fig. 3 Relative distribution of PAHs in sub-fractions of Arcachon sediment. PAH identification as in Table 2.Table 3 PAH concentrations (ng g21) of the < 500 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 145 ± 39 27 202 ± 44 22 80 ± 21 111 ± 24 Fluo 385 ± 106 27 610 ± 364 60 91 ± 25 144 ± 86 Pyr 581 ± 134 23 778 ± 287 37 91 ± 21 121 ± 45 BaA 187 ± 45 24 333 ± 201 60 104 ± 25 186 ± 112 Chry* 334 ± 60 18 532 ± 200 38 75 ± 14 120 ± 45 BF 600 ± 129 21 960 ± 413 43 105 ± 23 169 ± 73 BeP 241 ± 50 21 382 ± 157 41 95 ± 20 150 ± 62 BaP 233 ± 60 26 404 ± 232 57 101 ± 26 176 ± 101 Per 75 ± 18 24 145 ± 86 59 87 ± 21 169 ± 100 IP 293 ± 73 25 413 ± 126 30 90 ± 23 127 ± 39 BP 219 ± 51 23 339 ± 140 41 97 ± 23 151 ± 62 DaA 58 ± 21 36 83 ± 41 50 91 ± 33 130 ± 64 · PAHs 3351 ± 778 23 5180 ± 2275 44 93 ± 21 143 ± 63 a PAH identification as in Table 2.Table 4 PAH concentrations (ng g21) of the 500–300 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture.Conc. = Concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 85 ± 18 22 557 ± 476 86 30 ± 6 200 ± 171 Fluo 278 ± 69 25 1170 ± 878 75 44 ± 11 184 ± 138 Pyr 251 ± 52 21 977 ± 727 74 46 ± 9 178 ± 132 BaA 137 ± 31 23 572 ± 381 67 46 ± 10 193 ± 129 Chry* 174 ± 45 26 592 ± 367 62 49 ± 13 168 ± 104 BF 322 ± 87 27 1106 ± 705 64 47 ± 13 163 ± 104 BeP 118 ± 30 25 426 ± 276 65 45 ± 11 163 ± 106 BaP 165 ± 45 27 658 ± 454 69 49 ± 13 195 ± 135 Per 51 ± 12 24 222 ± 174 79 48 ± 11 207 ± 163 IP 137 ± 42 30 506 ± 350 69 45 ± 14 165 ± 114 BP 120 ± 35 29 460 ± 325 71 47 ± 14 182 ± 128 DaA 30 ± 9 31 93 ± 55 59 53 ± 16 163 ± 96 · PAHs 1866 ± 467 25 7340 ± 5166 70 45 ± 11 178 ± 126 a PAH identification as in Table 2. 8 Analyst, 1999, 124, 5–14the composition of the fraction.The RSD for the 500–300 mm fraction for the sum of PAHs is 72%. This might be explained, as stated previously, by the non-representative nature of the small amount (a few grams) of sediment owing to the presence of vegetal fragments. The RSD for the 300–125 mm fraction is lower but remains significant (25%). The concentrations obtained for the extracts of the 125–63, 63–40 and 15–40 mm fractions are very reproducible ( < 6%). The RSD obtained for the 0–15 mm fraction is more significant (18%) and might be explained by the very small sample amount (0.3 g) which is limited by the total amount of this fraction (3.2 g).Each sub-fraction has a different contamination level. Concentrations in the 500–300 mm fraction are significant and can be explained by the presence of vegetal fragments which Table 5 PAH concentrations (ng g21) of the 300–125 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture.Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 152 ± 3 2 135 ± 24 18 150 ± 3 134 ± 24 Fluo 324 ± 10 3 351 ± 33 10 133 ± 4 144 ± 14 Pyr 282 ± 1 0 321 ± 26 8 116 ± 0 132 ± 11 BaA 151 ± 16 10 177 ± 30 17 118 ± 13 138 ± 23 Chry* 214 ± 10 5 235 ± 36 15 120 ± 6 131 ± 20 BF 355 ± 59 17 451 ± 92 20 116 ± 19 147 ± 30 BeP 134 ± 20 15 174 ± 36 21 112 ± 17 145 ± 30 BaP 173 ± 26 15 229 ± 51 22 108 ± 16 143 ± 32 Per 55 ± 6 12 73 ± 21 28 108 ± 12 143 ± 41 IP 145 ± 14 9 208 ± 55 27 103 ± 10 148 ± 39 BP 127 ± 17 13 169 ± 43 26 106 ± 14 141 ± 36 DaA 32 ± 7 20 35 ± 1 3 119 ± 26 130 ± 4 · PAHs 2145 ± 180 8 2559 ± 387 15 118 ± 10 140 ± 21 a PAH identification as in Table 2.Table 6 PAH concentrations (ng g21) of the 125–63 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 204 ± 13 7 233 ± 70 31 91 ± 6 104 ± 31 Fluo 516 ± 24 5 581 ± 115 20 91 ± 4 103 ± 20 Pyr 567 ± 44 8 651 ± 86 13 95 ± 7 109 ± 14 BaA 283 ± 13 5 302 ± 51 17 92 ± 4 98 ± 17 Chry* 449 ± 28 6 475 ± 58 12 96 ± 6 102 ± 12 BF 845 ± 45 5 940 ± 159 17 89 ± 5 99 ± 17 BeP 335 ± 26 8 373 ± 61 16 89 ± 7 99 ± 16 BaP 368 ± 15 4 428 ± 71 17 92 ± 4 107 ± 18 Per 126 ± 6 5 142 ± 31 22 101 ± 5 114 ± 25 IP 375 ± 32 8 431 ± 62 14 94 ± 8 107 ± 15 BP 324 ± 21 6 370 ± 60 16 93 ± 6 107 ± 17 DaA 75 ± 11 15 85 ± 2 2 85 ± 13 97 ± 2 · PAHs 4467 ± 234 5 5002 ± 774 15 92 ± 5 103 ± 16 a PAH identification as in Table 2.Table 7 PAH concentrations (ng g21) of the 63–40 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 186 ± 24 13 220 ± 12 5 76 ± 10 89 ± 5 Fluo 556 ± 41 7 649 ± 33 5 89 ± 7 104 ± 5 Pyr 823 ± 22 3 927 ± 106 11 87 ± 2 98 ± 11 BaA 288 ± 23 8 371 ± 32 9 89 ± 7 115 ± 10 Chry* 623 ± 35 6 718 ± 42 6 95 ± 5 109 ± 6 BF 1166 ± 99 8 1346 ± 108 8 89 ± 8 103 ± 8 BeP 481 ± 57 12 563 ± 30 5 93 ± 11 108 ± 6 BaP 391 ± 8 2 494 ± 54 11 86 ± 2 108 ± 12 Per 153 ± 13 8 169 ± 15 9 101 ± 9 111 ± 10 IP 520 ± 33 6 671 ± 92 14 87 ± 6 112 ± 15 BP 439 ± 44 10 526 ± 29 6 93 ± 9 112 ± 6 DaA 97 ± 14 15 127 ± 15 12 75 ± 11 98 ± 12 · PAHs 5724 ± 359 6 6780 ± 218 3 89 ± 6 105 ± 3 a PAH identification as in Table 2.Analyst, 1999, 124, 5–14 9greatly adsorb lipophilic PAHs. Raoux and Garrigues13 showed that concentrations in vegetal debris are 10–25 times higher than those in the sediment. Fractions less than 63 mm are also highly contaminated. The PAH level can be compared with the organic carbon content (Fig. 2). The two are not strictly correlated but a tendency can be observed.The relationship between organic carbon content and the contamination level based on hydrophobicity is generally accepted.18 Karickhoff et al.19 and Rao et al.20 showed the association of compounds with the finest and richest organic matter particles. However, such relationships are not always strictly observed. Raoux and Garrigues13 observed an enrichment of fine particles for sediments with low contamination but an enrichment in coarse fractions for highly contaminated sediments. The association of PAHs in sediment is not simply a result of their hydrophobicity but also the result of the characteristics of the different sedimentation processes which affect each specific location.The adsorption of contaminants on to particles rich in organic carbon content may be followed by internal diffusion as is the case for most lipophilic compounds. Concerning individual compounds, the fingerprints are different. Fig. 3 represents the weight percentage of each PAH in each fraction. Concentrations of phenanthrene, fluoranthene, benzo[a]anthracene and benzo[a]pyrene are more significant in coarse fractions. Concentrations of benzo[b+j+k]fluoranthene, benzo[e]pyrene, indeno[1,2,3-cd]pyrene, benzo[ghi]perylene and dibenz[ah+ac]anthracene are more significant in fine fractions. This might be explained by the atmospheric source of pyrolytic high molecular weight PAHs associated with fine particles.21,22 Such preferential relative enrichment of high molecular weight compounds has been observed in the smallest particles ( < 10 mm) and might also be explained by preferential adsorption of the more hydrophobic higher molecular weight compounds.23 Table 8 PAH concentrations (ng g21) of the 40–15 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture.Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 257 ± 4 2 272 ± 42 16 91 ± 1 96 ± 15 Fluo 474 ± 33 7 441 ± 15 3 95 ± 7 88 ± 3 Pyr 976 ± 42 4 941 ± 67 7 102 ± 4 98 ± 7 BaA 258 ± 26 10 251 ± 33 13 108 ± 11 105 ± 14 Chry* 743 ± 32 4 767 ± 82 11 94 ± 4 97 ± 10 BF 1562 ± 68 4 1490 ± 83 6 95 ± 4 91 ± 5 BeP 675 ± 29 4 629 ± 41 7 99 ± 4 93 ± 6 BaP 370 ± 60 16 289 ± 97 33 109 ± 18 85 ± 29 Per 154 ± 7 5 138 ± 27 20 102 ± 5 91 ± 18 IP 760 ± 30 4 684 ± 35 5 89 ± 4 80 ± 4 BP 628 ± 22 4 597 ± 40 7 104 ± 4 99 ± 7 DaA 156 ± 6 4 180 ± 12 7 98 ± 4 113 ± 8 · PAHs 6929 ± 162 2 6680 ± 320 5 96 ± 2 93 ± 4 a PAH identification as in Table 2.Table 9 PAH concentrations (ng g21) of the 15–0 mm fraction of Arcachon sediment obtained by FMW extraction without and with 30% of moisture. Conc. = concentration; SOX = Soxhleta FMW FMW (30% of moisture) FMW (30% of moisture): Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) FMW: SOX (%) SOX (%) P 122 ± 26 21 160 ± 16 10 52 ± 11 68 ± 7 Fluo 249 ± 39 15 240 ± 26 11 89 ± 14 86 ± 9 Pyr 569 ± 95 17 530 ± 63 12 95 ± 16 89 ± 11 BaA 160 ± 7 4 132 ± 11 8 118 ± 5 97 ± 8 Chry* 487 ± 4 1 483 ± 13 1 88 ± 1 87 ± 2 BF 1098 ± 47 4 1197 ± 98 8 90 ± 4 98 ± 8 BeP 454 ± 17 4 518 ± 40 8 86 ± 3 98 ± 8 BaP 258 ± 37 14 188 ± 46 24 117 ± 17 85 ± 21 Per 121 ± 12 10 136 ± 3 2 77 ± 8 87 ± 2 IP 521 ± 44 8 606 ± 38 6 71 ± 6 82 ± 5 BP 482 ± 24 5 518 ± 52 10 93 ± 5 99 ± 10 DaA 95 ± 6 6 151 ± 32 21 70 ± 4 112 ± 24 · PAHs 4615 ± 333 7 4697 ± 125 3 87 ± 6 88 ± 2 a PAH identification as in Table 2.Fig. 4 Comparison of recoveries of the sum of PAHs for FMW extraction without and with 30% of moisture compared with Soxhlet extraction for each sub-fraction of Arcachon sediment. 10 Analyst, 1999, 124, 5–14Hence, there is a difference in the association of compounds. An extraction method must be efficient for all compounds whatever their origin and the grain size of the particles. Comparison of Soxhlet extraction and FMW-assisted extraction.Concentrations of PAHs for each sub-fraction obtained by FMW-assisted extraction for a dry matrix or a remoistened dry matrix (30% of moisture) are given in Tables 3–9. The extraction recovery for the 0–500 mm fraction obtained by FMW is good compared with Soxhlet extraction being 93% for the sum of PAHs. With 30% of moisture, the recovery is higher, but the RSD is very high (44%). Hence, in this case, the results are not significant.For the 500–300 mm fraction, the results are not interpretable because of the heterogeneity of this fraction which is rich in vegetal matter, which was also the case with Soxhlet extraction. For the fractions from 300–125 to 15–0 mm, RSDs for FMW extraction with or without water are equal to, or lower than, those obtained by Soxhlet extraction (except for FMW extraction with 30% of moisture for the 125–63 mm fraction). Data for the sum of PAHs are summarized in Fig. 4, which shows the recovery for FMW compared with Soxhlet extraction.FMW extraction affords better recoveries than Soxhlet extraction for the 300–125 mm fraction (recoveries of 118 and 140%, respectively, for FMW extraction and FMW extraction with 30% of moisture). For the 125–63 and 63–40 mm fractions, recoveries obtained by FMW extraction are acceptable (92 and 89%, respectively), but can be improved by adding moisture to the matrix, giving recoveries of 103 and 105%, respectively.For the 40–15 and 0–15 mm fractions, moisture content does not seem to affect extraction recoveries. Maximum recoveries are 96% for the 40–15 fraction and 88% for the 15–0 mm fraction. All the recoveries are above 80%, which is acceptable when considering the benefits in terms of time and solvent. Fine fractions seem to be more difficult to extract by FMW extraction whereas coarse fractions are better extracted by FMW than Soxhlet extraction. FMW recoveries of the sum of PAHs vary between 140 and 88%.However, this selectivity linked to grain size is not very important, especially if one considers the difference in the RSDs obtained for each fraction. The effect of moisture is not the same for each fraction. For fine and high organic carbon content particles, containing in particular clays (40–15; 15–0 mm), the effect of water on the recovery is not significant. Improvements of up to 22% are observed for coarse and low organic carbon content particles (300–125; 125–63; 63–40 mm).This dismisses the hypothesis that an improvement in the extraction efficiency is related to a specific interaction of clays with water under microwave irradiation. The dependence of the extraction efficiency on the analyte was studied. Fig. 5 shows the recoveries of a tricyclic (phenanthrene), a tetracyclic (chrysene), a pentacyclic (benzo[ a]pyrene) and a hexacyclic compound (benzo[ghi]perylene) for the different fractions. The compounds can be associated with different particles in different ways according to their origin.Paschke et al.24 showed the influence of the nature of a sample on the extraction efficiency by supercritical fluid extraction for nitro-PAHs from diesel exhaust particles and diesel soot. Diesel exhaust particles, where PAHs are formed at the same time as the growth of the particles and are adsorbed in the internal structure, are more difficult to extract than diesel soot, where PAHs are adsorbed on the surface.The pyrolytic compounds (associated with finer particles) may thus be more difficult to extract than petrogenic compounds, which are adsorbed on the surface. For phenanthrene, recoveries vary from 52 to 152% depending on the fraction, whereas for the other compounds, recoveries only vary between 86 and 120%. This difference can be explained by the different origin of these compounds (pyrolytic, petrogenic and diagenetic). Benzo[ghi]perylene, the heaviest compound, is well extracted whatever the fraction.This contradicts the perceived idea that high molecular weight compounds are more difficult to extract than low molecular weight compounds. The recoveries of phenanthrene are influenced by moisture whatever the fraction but the reproducibility is lower. The extraction of chrysene in the 300–125, 125–63 and 63–40 mm fractions is improved with moisture, but the improvement in recovery for the 40–15 and 0–15 mm fractions is not significant.For benzo[ghi]perylene, the same tendency is apparent, except for the 0–15 mm fraction for which there is a slight improve- Fig. 5 Comparison of recoveries for FMW extraction without and with 30% of moisture compared with Soxhlet extraction for each sub-fraction of Arcachon sediment for (a) phenanthrene, (b) chrysene, (c) benzo[a]pyrene and (d) benzo[ghi]perylene. Analyst, 1999, 124, 5–14 11ment. For benzo[a]pyrene, the extraction of the 40–15 and 0–15 mm fractions is better without moisture, but the RSD is significant.Other pentacyclic compounds (benzo[e]pyrene, perylene) do not show the same behaviour. Application to other sediments The efficiency and selectivity of FMW extraction were tested for other matrices with different contamination levels and different grain sizes. The method can be validated if the treatment of the sample does not influence the contamination level determination and does not change the biomarker parameters useful for the determination of contaminant origin.Table 10 shows the results for a muddy sediment, ‘OPCB 5b’ for which 94% of particles are under 63 mm. Three Soxhlet and three FMW extractions of the dry matrix and the remoistened dry matrix (30% of moisture) were performed. The RSDs are comparable. Recoveries of the sum of PAHs obtained for FMW extraction are 84% without water and 101% with 30% of water. Individual recoveries for FMW extraction without water are comparable and close to 90–100%, except for phenanthrene, anthracene and perylene which are, respectively, 64, 53 and 73%.With 30% of moisture, recoveries are better and vary between 87 and 120%. In this case, there is no extraction selectivity. This is confirmed by the biomarker parameters (concentration ratios of isomers) calculated for each extraction technique, which are comparable and generate the same interpretation of contamination origin. Table 10 shows the results for a sandy sediment, ‘OPCB 9b’ in which 15% of particles are under 63 mm.Whatever the extraction technique, the reproducibility is very poor, owing to the heterogeneity of the matrix, the relatively small sampling amount and the presence of vegetal debris. The ratio of phenanthrene to anthracene is influenced the most by the Table 10 Comparison of PAH concentrations (ng g21) of ‘OPCB 5b’ (silty sediment) and ‘OPCB 9b’ (sandy sediment) obtained by FMW extraction without and with 30% of moisture and by Soxhlet extraction (SOX).Conc. = concentrationa OPCB 5b FMW FMW (30% of moisture) SOX FMW (30%) FMW: of moisture: Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) SOX (%) SOX (%) P 68 ± 2 3 127 ± 7 6 106 ± 14 14 64 ± 2 120 ± 7 A 11 ± 1 5 23 ± 4 16 21 ± 6 28 53 ± 3 112 ± 18 Fluo 102 ± 3 3 96 ± 7 7 102 ± 3 2 100 ± 3 95 ± 7 pyr 82 ± 5 6 83 ± 5 6 84 ± 2 2 98 ± 6 99 ± 6 BaA 58 ± 5 8 55 ± 5 10 60 ± 1 2 97 ± 8 92 ± 9 Chry 68 ± 6 9 66 ± 6 10 71 ± 3 4 97 ± 9 93 ± 9 BF 117 ± 6 5 107 ± 8 7 116 ± 9 8 101 ± 5 92 ± 7 BeP 49 ± 3 5 49 ± 3 5 53 ± 1 2 92 ± 5 93 ± 5 BaP 61 ± 3 5 57 ± 4 7 65 ± 4 6 95 ± 5 87 ± 6 Per 105 ± 1 1 153 ± 8 5 143 ± 2 1 73 ± 1 107 ± 6 IP 42 ± 2 5 56 ± 3 4 58 ± 4 7 72 ± 5 97 ± 6 BP 49 ± 2 4 50 ± 3 6 55 ± 2 4 90 ± 4 91 ± 6 DaA 12 ± 1 6 12 ± 1 8 14 ± 2 12 86 ± 5 88 ± 7 SPAHs 824 ± 37 4 933 ± 53 6 927 ± 51 6 89 ± 4 101 ± 6 ratio ratio ratio P :A 6.2 ± 0.3 5 5.6 ± 0.6 11 5.2 ± 0.7 13 Fluo : Pyr 1.2 ± 0.0 0 1.2 ± 0.0 0 1.2 ± 0.0 0 Chry : BaP 1.2 ± 0.0 0 1.2 ± 0.0 0 1.2 ± 0.0 0 BeP : BaP 0.8 ± 0.0 0 0.9 ± 0.0 0 0.8 ± 0.1 13 OPCB 9b FMW FMW (30% of moisture) SOX FMW (30%) FMW: of moisture: Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) Conc./ng g21 RSD (%) SOX (%) SOX (%) P 28 ± 11 39 36 ± 6 17 48 ± 17 35 58 ± 23 75 ± 12 A 4 ± 2 50 6 ± 3 50 10 ± 6 60 40 ± 22 60 ± 29 Fluo 53 ± 36 68 24 ± 8 33 66 ± 50 76 80 ± 54 36 ± 12 pyr 41 ± 30 73 18 ± 7 39 46 ± 37 80 89 ± 66 39 ± 15 BaA 26 ± 25 96 16 ± 16 100 34 ± 29 85 76 ± 75 47 ± 49 Chry 29 ± 24 83 19 ± 18 95 35 ± 23 66 83 ± 69 54 ± 52 BF 48 ± 45 94 26 ± 24 92 44 ± 25 57 109 ± 101 59 ± 54 BeP 19 ± 17 89 11 ± 9 82 18 ± 10 56 106 ± 90 61 ± 48 BaP 28 ± 28 100 15 ± 14 93 24 ± 17 71 117 ± 116 63 ± 57 Per 17 ± 10 59 10 ± 5 50 20 ± 6 30 85 ± 52 50 ± 23 IP 16 ± 15 94 12 ± 8 67 15 ± 7 47 107 ± 98 80 ± 55 BP 18 ± 16 89 9 ± 6 67 14 ± 7 50 129 ± 108 64 ± 42 DaA 5 ± 4 80 2 ± 2 100 4 ± 2 50 125 ± 102 50 ± 52 SPAHs 332 ± 260 78 204 ± 126 62 378 ± 235 62 88 ± 69 54 ± 33 ratio ratio ratio P :A 9.4 ± 4.1 44 6.7 ± 1.9 28 4.9 ± 1.2 24 Fluo : Pyr 1.4 ± 0.3 21 1.4 ± 0.1 7 1.5 ± 0.1 7 Chry : BaP 1.2 ± 0.2 17 1.3 ± 0.1 8 1.2 ± 0.3 25 BeP : BaP 0.8 ± 0.2 25 0.8 ± 0.1 13 0.8 ± 0.2 25 a PAH identification as in Table 2. 12 Analyst, 1999, 124, 5–14vegetal debris, but the difference is not significant. The biomarker parameters generate the same interpretation. Therefore, it is impossible to test the precision of the method with this matrix. However, the order of magnitude, between 100 and 600 ng g21, is in agreement with that obtained by Soxhlet extraction.Extraction of a naturally moist matrix The extraction of a naturally moist matrix (30% of moisture) by FMW extraction was compared with the extraction of a freezedried matrix by the same technique and by the conventional method of Soxhlet extraction and with the extraction of a freezedried matrix to which 30% of water had been added before the extraction (remoistened dry matrix).Drying of the matrix is generally used to improve the extraction efficiency of Soxhlet extraction. Moreover, a dry matrix is easier to store than a frozen matrix and avoids alteration of the matrix by bacterial degradation, for example. However, this step is very long and can lead to a loss of volatile compounds. Table 11 shows the concentrations obtained for a dry matrix, a remoistened dry matrix and a naturally moist matrix by FMW extraction and for a dry matrix by Soxhlet extraction (n = 3).Compared with Soxhlet extraction, the recovery obtained for the dry matrix by FMW extraction is 91% for the sum of PAHs. The recovery is improved by the addition of 30% of moisture before extraction. Concentrations are comparable to those obtained by Soxhlet extraction with a recovery of 104% for the sum of PAHs. The extraction of the naturally moist matrix is less efficient than for the remoistened dry matrix with a recovery of 84% for the sum of PAHs.This might be because some of the water is trapped in the pores and prevents access of the solvent (dichloromethane). The use of other solvent mixtures, richer in ethanol to dissolve the water better or having a higher boiling-point, such as heptane–ethanol (80 + 20, v/v), does not improve the recovery. The concentration obtained with heptane–ethanol (80 + 20, v/v) for the sum of PAHs is 3.7 ± 0.6 mg g21 compared with 3.6 ± 0.3 mg g21 obtained with dichloromethane.The use of a drying agent, such as anhydrous sodium sulfate, was also tested but the concentration obtained (3.8 ± 0.4 mg g21) was not significantly better. However, the extraction efficiency is acceptable for environmental matrices. The RSDs are comparable to those obtained for a dry matrix (under 20%, except for anthracene) and lower than those obtained by Soxhlet extraction. Table 11 also shows the biomarker parameters calculated in each case from the concentrations of isomers. The FMW treatment of the sample has no influence on the parameters, whatever the state of the matrix.There is no selectivity for the compounds studied. The ratios of the concentrations of phenanthrene to anthracene are all less than 10. The ratios of the concentrations of benzo[e]pyrene to benzo[a]pyrene are all less than 2. This suggests a pyrolytic contamination. The ratios of the concentrations of fluoranthene to pyrene are all less than 1, whereas the ratios of the concentrations of chrysene to benzo[a]anthracene are all greater than 1.This suggests a petrogenic contamination. In conclusion, the analysis of a naturally moist matrix can be performed rapidly by FMW extraction without a drying step with good precision and reproducibility in terms of individual concentrations and for biomarker studies. The FMW apparatus is not bulky and might be used for field work during an oceanographic cruise on a boat for example. The only requirement is refrigeration with water circulation.No gas supply is necessary. It should be possible to perform homogenisation of the matrix (removal of stones and shells), extraction, reconcentration under microwave irradiation, and purification on micro-columns as the sampling is performed. Results could then be obtained rapidly after sampling. Conclusions FMW extraction at atmospheric pressure is a good alternative to Soxhlet extraction for the analysis of PAHs in sediments with various grain size distributions.There is no extraction selectivity concerning contaminants or particles. Good recoveries and good reproducibility are obtained. The procedure allows a Table 11 Comparison of PAH concentrations (ng g21) obtained by FMW extraction without and with 30% of moisture for the freeze-dried Arcachon sediment, by FMW extraction for the naturally moist sediment and by Soxhlet extraction (SOX) for the freeze-dried sediment. Conc. = concentrationa FMW (dry matrix) FMW (remoistened dry matrix) FMW (naturally moist matrix) SOX (dry matrix) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) Conc./ ng g21 RSD (%) FMW (dry matrix): SOX (%) FMW (remoistened dry matrrix): SOX (%) FMW (naturally moist matrix): SOX (%) P 457 ± 78 17 515 ± 8 2 327 ± 65 20 357 ± 231 65 128 ± 22 144 ± 2 92 ± 18 A 84 ± 11 13 79 ± 9 11 66 ± 18 28 72 ± 18 25 116 ± 15 109 ± 13 91 ± 25 Fluo 357 ± 68 19 447 ± 23 5 354 ± 57 16 388 ± 59 15 92 ± 18 115 ± 6 91 ± 15 pyr 587 ± 46 8 666 ± 15 2 579 ± 33 6 646 ± 68 11 91 ± 7 1`03 ± 2 90 ± 5 BaA 213 ± 38 18 229 ± 16 7 212 ± 28 13 243 ± 46 19 88 ± 16 95 ± 7 88 ± 12 Chry 393 ± 51 13 425 ± 6 1 340 ± 31 9 442 ± 58 13 89 ± 12 96 ± 1 77 ± 7 BF 626 ± 76 12 718 ± 3 0 556 ± 40 7 722 ± 118 16 87 ± 10 99 ± 0 77 ± 6 BeP 260 ± 29 11 290 ± 2 1 224 ± 13 6 293 ± 25 9 89 ± 10 99 ± 1 76 ± 5 BaP 238 ± 33 14 282 ± 6 2 238 ± 20 8 290 ± 55 19 82 ± 11 97 ± 2 82 ± 7 Per 77 ± 10 13 79 ± 4 5 64 ± 5 7 99 ± 11 11 78 ± 10 80 ± 4 64 ± 5 IP 297 ± 28 9 416 ± 38 9 337 ± 5 1 311 ± 17 5 95 ± 9 134 ± 12 108 ± 1 BP 237 ± 34 14 282 ± 8 3 203 ± 14 7 264 ± 40 15 90 ± 13 107 ± 3 77 ± 5 DaA 56 ± 7 13 76 ± 13 17 65 ± 4 6 77 ± 14 18 73 ± 9 99 ± 17 84 ± 5 SPAHs 3880 ± 484 12 4421 ± 81 2 3565 ± 269 8 4243 ± 625 15 91 ± 11 104 ± 2 84 ± 6 ratio ratio ratio P :A 5.4 ± 0.4 8 6.6 ± 0.8 13 5.1 ± 0.4 8 4.3 ± 5.4 125 Fluo : Pyr 0.6 ± 0.1 11 0.7 ± 0.0 7 0.6 ± 0.1 11 0.4 ± 0.1 14 Chry : BaP 1.9 ± 0.1 5 1.9 ± 0.1 6 1.6 ± 0.1 6 1.2 ± 0.2 20 BeP : BaP 1.1 ± 0.0 4 1.0 ± 0.0 2 0.9 ± 0.0 5 0.6 ± 0.1 13 a PAH identification as in Table 2.Analyst, 1999, 124, 5–14 13reduction of time and solvent amount. Numerous samples can be rapidly analysed at less cost. The method is simple and can be used in field work. Acknowledgements Prolabo is acknowledged for financial support and the loan of the microwave system. Cécile Campoy is acknowledged for technical assistance. References 1 K. L. White, Environ. Carcin. Rev., 1986, C4, 163. 2 J. M. Neff, in Polycyclic Aromatic Hydrocarbons in the Aquatic Environment. Sources, Fates, and Biological Effects, Applied Science, Barking, 1979, pp. 7–43. 3 A. E. McElroy, J. W. Farrington and J. M. Teal, in Metabolism of Polycyclic Aromatic Hydrocarbons in the Aquatic Environment, ed. U. Varanisi, CRC Press, Boca Raton, FL, 1989, pp. 1–40. 4 V. Lopez-Avila, R. Young and W. F. Beckert, Anal. Chem., 1994, 66, 1097. 5 F. I. Onuska and K. A. Terry, Chromatographia, 1993, 36, 191. 6 J. R. J. Paré and J. M. R. Belanger, Trends Anal. Chem., 1994, 13, 176. 7 K. Ganzler, A. Salgo and K. Valco, J. Chromatogr., 1986, 371, 299. 8 I. J. Barnabas, J. R. Dean, I. A. Fowlis and S. P. Owen, Analyst, 1995, 120, 189. 9 H. Budzinski, A. Papineau, P. Baumard and P. Garrigues, C. R. Acad. Sci. Paris, 1995, t.321, Series II b, 69. 10 H. Budzinski, P. Baumard, A. Papineau, S. Wise and P. Garrigues, PAC J., 1996, 9, 225. 11 M. Letellier, H. Budzinski, P. Garrigues and S. Wise, Spectroscopy, 1997, 13, 71. 12 H. Budzinski, M. Letellier, P. Garrigues and K. LeMenach, J. Chromatogr., submitted. 13 C. Raoux and P. Garrigues, in Polycyclic Aromatic Compounds, ed. P. Garrigues and M. Lamotte, Gordon and Breach, Amsterdam, 1993, pp. 443–450. 14 C. Piérard, H. Budzinski and P. Garrigues, Environ. Sci. Technol., 1996, 30, 2776. 15 F. Behar, C. Leblond and C. Saint-Paul, Rev. Inst. Fr. Pét., 1989, 44, 387. 16 G. Cauwet, Chem. Geol., 1975, 16, 59. 17 P. Baumard, H. Budzinski and P. Garrigues, Mar. Pollut. Bull., 1998, 36, 577. 18 J. P. Knezovich, F. L. Harrison and R. G. Wilhelm, Water, Air Soil Pollut., 1987, 32, 232. 19 S. W. Karickhoff, D. S. Brown and T. A. Scott, Water Res., 1979, 13, 241. 20 P. S. C. Rao, L. S. Lee and R. Pinal, Environ. Sci. Technol., 1990, 24, 647. 21 M. A. Sicre, J. C. Marty, A. Saliot, X. Aparicio, J. Grimalt and J. Albaiges, Atmos. Environ., 1987, 21, 2247. 22 J. Grimalt, J. Albaiges, M. A. Sicre, J. C. Marty and A. Saliot, Naturwissenschaften, 1988, 75, 39. 23 J. W. Readman, R. F. C. Mantoura and M. M. Rhead, Fresenius’ Z. Anal. Chem. 1984, 319, 126. 24 T. Paschke, S. B. Hawthorne, D. J. Miller and B. Wenclawiack, J. Chromatogr., 1992, 609, 333. Paper 8/07482H 14 Analyst, 1999, 124, 5–14

ISSN:0003-2654    

DOI:10.1039/a807482h

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Determination of trace metal impurities in high purity silver by two step selective precipitation separation followed by neutron activation analysis M. Y. Shiue,a Y. C. Sun,b J. J. Yeh,a J. Y. Yangb and M. H. Yang*a a Department of Nuclear Science, National Tsing-Hua University, 30043 Hsinchu, Taiwan b Nuclear Science and Technology Development Center, National Tsing-Hua University, 30043 Hsinchu, Taiwan Received 14th September 1998, Accepted 20th November 1998 A neutron activation analysis method for the determination of Au, Co, Cu, Fe, Hg and Zn in high purity silver materials with prior isolation of the analytes by a two-step selective precipitation separation from the silver matrix was developed. In the first step, the silver matrix was separated from the trace impurities of interest through the addition of hydrochloric acid to form a silver chloride precipitate.The principle of this separation is based on the extreme difference in the solubilities of the chlorides of silver and the trace elements of interest.In the second step, the trace elements remaining in the solution were subsequently coprecipitated with the Pb salt of pyrrolidine dithiocarbamate, Pb(PDC)2. The concentrations of six elements (Au, Co, Cu, Fe, Hg and Zn ) collected in the precipitate were determined by neutron activation analysis. Limits of detection of 0.001, 0.1, 0.08, 10, 0.1 and 1 mg g21 for Au, Co, Cu, Fe, Hg and Zn, respectively, were obtained.The proposed method was validated by the analysis of NIST SRM 8171 Fine Silver and applied to the determination of metal impurities in two high-purity silver samples (EM9465 and EM9343). Introduction The determination of trace impurities in high purity materials is essential in order to control and to improve manufacturing technology. An additional reason for the determination of trace element impurities in high purity silver is the effect of impurities on the freezing and melting properties when silver is used for primary and secondary temperature calibration.1,2 For the determination of trace impurities in high purity materials, a combination of chemical pre-treatment processes with instrumental analysis can generally achieve the best analytical performance.Atomic spectrometric methods, e.g., ETAAS and ICP-OES,3–10 and electrochemical methods,11–13 combined with preconcentration and/or separation procedures, have been reported for the determination of trace impurities in various materials.With the advent of ICP-MS, many attempts10,14 at the determination of trace element impurities in high purity materials both with or without preconcentration processes have been made. Even with this powerful analytical technique, the interference effect caused by the parent matrix may restrict its direct applicability to the determination of trace impurities in the samples. Among sample preconcentration methods, solid–liquid extraction, 10,14 ion exchange,12 coprecipitation5,7 and electrodeposition4,15 –19 are most commonly used for the separation of trace impurities from the sample matrix.However, in these multi-stage combined procedures there is a risk of increasing contamination and consequent worsening of the detection limits. To attain high sensitivity and reliability, the analytical blank and systematic error inherent in extreme trace analysis should be critically controlled.20 Instrumental neutron activation analysis (INAA) is a unique analytical technique which can sometimes be used for the direct determination of trace impurities in high purity metals.21 However, owing to the unavailability of suitable standards and difficulties connected with matrix interference, quantitative applications of this physical method may be severely restricted. 22 It is impossible to achieve the direct determination of trace impurities in high purity silver by NAA without pre- or post-irradiation separation, because the very high levels of gamma activity produced by the silver matrix may cause serious spectral interference in the determination of trace analytes.A method has recently been developed in our laboratory for the analysis of high purity silver by isolating analytes from the silver matrix with selective precipitation followed by ICP-MS determination.23 The elements including Al, Au, Cu, Cd, Co, Fe, Mg, Mn, Ni, Pb and Sn can be quantitatively separated from the precipitate of silver chloride.However, the determination of Hg was difficult because of the serious memory effect in the measurement by ICP-MS. In this study, the feasibility of applying a coprecipitation method for preconcenting Au, Cu, Fe, Hg and Zn from high purity silver followed by NAA was investigated. The effect of hydrochloric acid concentration on the recovery of trace elements from the Ag matrix and the effect of pH on the coprecipitation of the analytes by Pb(PDC)2 were investigated.Experimental Reagents, containers and samples All reagents were of analytical-reagent grade, unless stated otherwise. High-purity water was obtained by purification through de-ionization and double distillation. The purification of nitric acid and hydrochloric acid was carried out by subboiling distillation of the analytical-reagent grade acids. PTFE and glass containers were used throughout and were cleaned by immersion in HNO3 (1 + 1) for at least 24 h.Prior to use, they were rinsed with doubly distilled, de-ionized water and air-dried in a class 100 clean bench. Ammonium pyrrolidinedithiocarbamate (APDC) was used as a coprecipitant together with Pb(NO3)2. pH measurements were Analyst, 1999, 124, 15–18 15made with a conventional pH meter and the pH was adjusted with HCl and NH3 solution after adding originally 1% of 1 m acetate buffer (1 + 1). Samples of high purity silver EM9343 (6A9 grade silver shot) and EM9465 (5A9 grade silver shot) were obtained from Johnson Matthey Electronics (Royston, Hertfordshire, UK).Silver SRM 8171 (Fine Silver FS 14 Block) was obtained from NIST (Gaithersburg, MD, USA). Sample cleaning A 2 g sample of high purity silver was weighed into a 30 mL PTFE beaker and 25 mL of cold 0.1 m nitric acid were added with approximately 10 min of agitation, followed by thorough rinsing in doubly distilled, de-ionized water and air drying in a class 100 clean bench.A similar procedure was also applied to silver standard samples. Sample pre-treatment A flow chart of the proposed separation procedure is given in Fig. 1. A 0.1–0.3 g silver sample was weighed into a 20 mL PTFE beaker and 1 mL of water and 1 mL of concentrated nitric acid were added. The sample was heated below the boilingpoint of nitric acid until complete dissolution of silver was achieved, then 4 mL of 3 m hydrochloric acid were added progressively to form a fine precipitate of silver chloride.The solution was filtered with a 0.45 mm membrane filter and the filtrate collected was heated to near dryness. To the residue, 0.5 mL of 3 m hydrochloric acid was added to form a fine precipitate of silver chloride and the solution was filtered again. The filtrate collected was adjusted to pH 4 with 1% of 1 m acetate buffer (1 + 1), then 50 mg of Pb(NO3)2 and 5 mL of 1% APDC were added to the solution. The solution was allowed to stand for 0.5 h and the precipitate was filtered off.The filterpaper was inserted into quartz ampoules and was heat-sealed. Neutron activation analysis studies Several precipitated samples from silver were irradiated either with multi-element standards or using the monostandard method. Neutron irradiation was performed at fluxes of 1 3 1012–5 3 1013 cm22 s21 in THOR for 1 min (for the determination of Cu) and 30 h (for the determination of Au, Co, Fe, Hg and Zn). The counting system consists of a 38 cm3 Ge(Li) detector coupled with a TN-1710 4096-channel pulseheight analyser (Tracor Northern) and high voltage supplier (Canberra).The energy resolution of the system was 2.4 keV for 1332 keV. The irradiated samples were counted for 10 and 30 min for the measurement of short- (64Cu) and long-lived nuclides (203Hg, 198Au, 60Co, 59Fe and 65Zn), respectively. Results and discussion Precipitation separation procedure To achieve high sensitivity and accuracy for the determination of trace impurities in silver, a method based on the separation of the matrix element prior to the determination of isolated trace elements was developed. Coprecipitation of trace analytes should be avoided during the precipitation of silver chloride in order to achieve effective separation of impurities from the sample matrix.Furthermore, as Ag ions will be simultaneously collected with the trace analytes in the APDC coprecipitation step, the Ag matrix remained in the supernatant should be minimized to prevent the occurrence of interferences during the analysis by NAA.The effect of hydrochloric acid concentration on the recovery of trace elements and Ag matrix remaining in the supernatant after precipitation of silver chloride is shown in Table 1. The experiments were carried out in high purity silver spiked with 25 mg of the elements of interest followed by the proposed procedure and determination by ICP-OES. It can be seen from Table 1 that quantitative recoveries of most of the spiked elements, except Hg, from the silver chloride precipitate are obtained for concentrations of hydrochloric acid from 2.4 to 4 m.In contrast, the Ag matrix remaining in the supernatant was only 0.03–0.12% over the same acid range. It is interesting that the recoveries of Hg and Au show a significant dependence on the concentration of HCl. As Table 1 shows, quantitative recovery of Au can be achieved as the acid concentration increases to about 2.0 m, and that of Hg can only be achieved by further increasing the acid concentration to about 4.0 m. This observation can probably be explained on the basis of the relative tendency for the formation of soluble complexes (chloroauric and chloromercuric complexes) between Hg and Au with chloride ion in the HCl medium.Basically, the complexation behavior of Hg and Au towards chloride ion can be reflected in their respective stability constants. As indicated in the literature, the stepwise stability constants of Au3+ and Hg2+ with chloride ion are 8.5/6.7(log k1), 8.1/6.5(log k2), 7.0/6.9(log k3) and 6.1/1.0 (log k4), respectively. 24 Obviously, the higher stability constants of Au3+, compared with those of Hg2+ will result in a stronger interaction Fig. 1 Flow chart of the separation procedure for the determination of trace metal impurities in high purity silver by NAA. Table 1 Recovery of (%) of trace impurities and Ag matrix remaining in the supernatant after precipitation of silver chloride at different concentrations of HCl HCl concentration/m Element 1 2 2.4 3 4 Au 74 94 101 104 101 Co 93 87 97 94 98 Cu 96 93 100 104 104 Fe 94 87 99 96 98 Hg 40 55 75 81 105 Zn 101 96 105 104 104 Ag 2 0.04 0.03 0.04 0.12 16 Analyst, 1999, 124, 15–18between Au3+ and Cl2 and thus result in a quantitative recovery of Au at lower chloride concentrations than that of Hg.The differences in recovery between Hg and Au at different HCl concentrations can be explained on this basis.Coprecipitation of analytes with Pb(PDC)2 Following the separation of the Ag matrix, the trace analytes that remain in the supernatant should be further concentrated to a minimum sample size in order to be used effectively for NAA. For NAA, solid samples are preferred as they can be irradiated for longer times at higher fluxes of neutrons without the difficulties arising from the considerable gas generation in the radiolysis of water.Many processes can be used for the enrichment of trace elements in solid matrices,15,25 such as ion exchange, sorption on activated carbon, sorption on chelating agents immobilized on silica gel or a polymer chain and coprecipitation. Each of the different methods has its own advantages and the choice between them depends on the elements to be determined, the exact matrix and the method used for the detection of these elements. Coprecipitation is one of the most appropriate methods for our purpose because it provides a convenient way to collect trace elements from a large volume of sample solution on a small solid precipitate ( < 100 mg).The trace elements collected in this small sample can be used as a whole for NAA and can therefore result in a large enhancement of the analytical sensitivity. Concerning the coprecipitating agent, Pb(PDC)2 is considered to be one of the best choices for collecting trace elements in water for NAA,26 because APDC is a well recognized chelating agent which can react with over 30 elements,27 and the major constituent elements of the chelating agent (C, H, O, N) and also Pb do not form radioisotopes or form a beta emitter(209Pb) or have a very short half-life (207Pb, t1/2 = 0.8 s).28 The effect of pH on the coprecipitation of trace impurities in aqueous solution by Pb(PDC)2 was studied and the results are given in Table 2.The experiments were carried out by adding 25 mg of the respective elements to the supernant which was obtained by following the sample pre-treatment process shown in Fig. 1. Quantitative recoveries of most of the spiked elements with Pb(PDC)2 coprecipitation can be obtained from pH 3 to 6. A relatively lower recovery of Fe (90%) is observed however. This may be due to the formation of soluble complexes of iron hydroxide at higher pH.15 On the basis of the above experimental results, a pH in the range 3–5 is suitable for the coprecipitation of trace impurities by Pb(PDC)2 from aqueous solution.A pH of 4.0 was chosen for subsequent studies. Neutron activation analysis of high purity silver The determination of trace impurities in a silver sample is performed first by separation of the silver matrix in a medium of 3 m HCl followed by coprecipitation of the analytes of interest with Pb(PDC)2 at pH 4.0, and finally NAA of the coprecipitate sample. Six elements, Au, Co, Cu, Fe, Hg and Zn, were to be determined in this study.Among the radionuclides produced by the (n, g) reaction, only 64Cu has a short half-life of 12.7 h; the others (198Au, 60Co, 59Fe, 203Hg and 65Zn) have much longer half-lives from days to years. To achieve the quantitative determination of all the elements in the sample, two analytical schemes with a prescribed program of irradiation, cooling and counting were designed. For the determination of 64Cu (t1/2 = 12.7 h), a short irradiation time of 1 min followed by 1 h cooling and 10 min counting was applied, whereas for the longer lived nuclides a longer irradiation time of 30 h followed by 1 week cooling (to allow the decay of shorter interfering nuclides) and 30 min counting was applied.Fig. 2 shows the g-ray spectrum of a silver sample (NIST SRM 8171) which was treated by a two step precipitation separation followed by neutron irradiation. The g-spectrum was obtained for the sample subjected to 30 h irradiation, 1 week cooling and 30 min counting.The gamma peaks of the nuclides of interest including 198Au, 60Co, 59Fe, 203Hg and 65Zn are all clearly identified, but the g-spectrum of 110mAg, resulting from silver matrix remaining in the coprecipitate, also appeared. Since the majority of silver matrix was removed in the separation process, as evidenced in Table 1, the presence of minor activity arising form 110mAg would not constitute a perceivable interference effect on the accurate determination of the analyte nuclides.For the determination of 64Cu, the measurement of the 511 keV g-ray which resulted from the annihilation of b+-emission from 64Cu was performed. Basically, the 511 keV g-ray is not an appropriate choice for the characteristic identification of a specific nuclide because it can originate from both b+-emission and any g-decay with energy higher than 1022 keV. In this study, however, the applicability of determining 64Cu via measurement of 511 keV was tested by the selection of a suitable neutron irradiation time.The assumption underlying this study is that for a short irradiation time (1 min), 64Cu can be produced at a certain level owing to its relatively short half-life, whereas the long-lived nuclides would not be produced with such a short irradiation time at a perceivable level to cause interference in the measurement of 64Cu at 511 keV. In order to prove the correctness of this assumption, a neutron irradiated sample obtained by following the procedure in Fig. 1 was analyzed by tracing the half-life of the 511 keV g-peak. The result indicated that the half-life of this peak is very close to the 12.7 h of 64Cu . From the good coincidence of the half-life of the 511 keV g-peak with that of 64Cu, it may be concluded that the radioactivity measured at the 511 keV g-peak should come Table 2 Effect of pH on the recovery (%) of trace elements by coprecipitation with Pb(PDC)2 pH Element 3 4 5 6 Au 95 99 99 98 Co 97 99 99 99 Cu 98 100 99 100 Fe 97 99 99 90 Hg 94 100 100 100 Zn 95 97 99 99 Fig. 2 g-Ray spectra of a silver sample (NIST SRM 8171) treated by precipitation separation followed by neutron irradiation. Irradiation time, 30 h; cooling time, 1 week; counting time, 30 min. Analyst, 1999, 124, 15–18 17solely from the decay of 64Cu and can therefore be used for the quantification of this nuclide in high purity silver samples. The reliability of the proposed method was evaluated by the analysis of a certified silver standard.Table 3 gives the method detection limit and the analytical results for trace impurities in the sample of NIST SRM 8171 (Fine Silver FS14). Since no certified values are given for Co and Hg in the sample, the analytical reliability was tested with the method of standard additions. From Table 3, it can be seen that the analytical data obtained are in reasonably good agreement with the certified values and the spike recoveries are also within the reasonable range obtained in the previous study.The limits of detection were determined experimentally based on the lowest concentration of the analytes which produced the observed analytical signal on the addition of the analyte standard solution, and were 0.001, 0.1, 0.1, 10, 0.1 and 1 mg g21 for Au, Co, Cu, Fe, Hg and Zn, respectively. Further increase in the detection sensitivity would be possible if an increased neutron flux of the irradiation site can be applied. The developed method was applied to the analysis of the high purity silver samples EM9343 and EM9465 (Johnson Matthey) and the results are given in Table 4. The concentration levels of Co, Cu, Fe and Zn are found to be close to or below the limits of detection, and therefore quantification of these elements in these two samples is not possible under the present experimental conditions. However, the determination of Au and Hg was possible and the results were 0.10 ± 0.02 and 2.2 ± 0.4 mg g21 for EM9343 and 1.3 ± 0.3 and 4.3 ± 0.5 mg g21 for EM9465, respectively.An intercomparison study with the use of ICP-MS for the analysis of the EM9343 sample was also performed, and concentrations of 0.09 ± 0.01 and 2.0 ± 0.3 mg g21, for Au and Hg respectively, were found, which are in good agreement with those obtained by the proposed method and the values provided by Johnson Matthey. Conclusion A method for the determination of trace metal impurities in high purity silver which consists of selective separation and coprecipitation followed by NAA determination is established.Silver in the dissolved sample solution can be nearly quantitatively separated from the trace impurities by the formation of a silver chloride precipitate and the trace impurities remained in the aqueous solution can be subsequently coprecipitated by Pb(PDC)2 under the established conditions. The concentrations of Au, Co, Cu, Fe, Hg and Zn in the solid precipitate are finally determined by neutron activation analysis.The data with reasonably good accuracy and precision can be achieved by the proposed method. Acknowledgements The authors gratefully acknowledge the financial support of the National Science Council of Taiwan, (NSC 87-2212-E- 007-057). References 1 E. H. MacLaren, Can. J. Phys., 1957, 35, 1086. 2 B. W. Mangun, E. R. Pfeiffer, G. F. Strouse, J. Valencia-Rodriguez, J. H. Lin, T. I. Yeh, P. Marcarino, R.Dematteis, Y. Liu, Q. Zhao, A. T. Ince, F. Cakiroglu, H. G. Nubbemeyer and H. J. Jung, Metrologia, 1996, 33, 215. 3 J. D. Mullen, Talanta, 1976, 23, 846. 4 T. Tanaka, Y. Maki, Y. Kobayashi and A. Mizuike, Anal. Chim. Acta, 1991, 252, 211. 5 W. Reichel and B. G. Bleakley, Anal. Chem., 1974, 46, 59. 6 W. Lund, B. V. Larsen and N. Gundersen, Anal. Chim. Acta, 1976, 81, 319. 7 M. W. Hinds, J. Anal. At. Spectrom., 1992, 7, 685. 8 M. Hiraide, Y. Mikuni and H. Kawaguchi, Fresenius’ J.Anal. Chem., 1990, 354, 212. 9 I. G. Yudelevich, B. I. Zakda, V. P. Shabarova and A. S. Chereko, At. Spectrosc., 1992, 13, 108. 10 G. Kudermann, Fresenius’ J. Anal. Chem., 1988, 331, 697. 11 R. Naumann, W. Schmidt and G. Höh, Fresenius’ J. Anal. Chem., 1990, 347, 133. 12 S. R. Kayasth, A. K. Basu, N. Chattopadhyay and H. B. Desai, Anal. Chim. Acta, 1990, 231, 133. 13 T. Matsuda and T. Nagai, Anal. Sci., 1990, 7, 75. 14 R. J. Stummeye and G. Wünsch, Fresenius’ J. Anal.Chem., 1991, 340, 269. 15 A. Mizuike, Enrichment Techniques for Inorganic Trace Analysis, Springer, Berlin, 1983. 16 R. C. Chirnside, H. J. Chuly and P. M. C. Proffit, Analyst, 1957, 82, 18. 17 A. Mizuike, N. Mirsuya and K. Yammgai Bull. Chem. Soc. Jpn., 1969, 42, 253. 18 B. H. Vassos, R. F. Hirsch and H. Letlterman, Anal. Chem., 1973, 45, 792. 19 H. Malissa and I. L. Man, Mikrochim. Acta, 1971, 2, 241. 20 P. Tschöpel and G. Tölg, J. Trace Microprobe Tech., 1982, 1, 1. 21 G.Kudermenn, K. H. Blanfuss, C. Lührs, W. Vielhaber and V. Collisi, Fresenius’ J. Anal. Chem., 1992, 343, 734. 22 G. Tölg, Pure Appl. Chem., 1978, 50, 1075. 23 Y. C. Sun, J. Mierzwa, C. F. Lin, T. I. Yeh and M. H. Yang, Analyst, 1997, 122, 437. 24 J. Bjerrum, G. Schwarzenbach, and L. G. Sillen, Stability Constants, Special Publication No. 7, Chemical Society, London, 1958. 25 J. Minczewski, J. Chwastowska and R. Dybczynski, Separation and Preconcentration Methods in Inorganic Trace Analysis, Ellis Horwood, Chichester 1982. 26 C. R. Lan, Y. C. Sun, J. H. Chao, M. H. Yang, N. Lavi and Z. B. Alfassi, Mikrochim. Acta, 1990, 50, 225. 27 D. K. John and V. L. Jon, Anal. Chem., 1974, 46, 1894. 28 G. Erdtmann, Neutron Activation Tables, Verlag Chemie, Weinheim, 1976. Paper 8/07137C Table 3 Analytical results for trace impurities in high purity silver (NIST SRM 8171) determined by precipitation separation followed by NAA MDL Impurity concentration/mg g21 Detection limitb/ Element Certified Measureda Recovery (%) mg g21 Au 26.7 ± 6.4 23.5 ± 2.4 88 0.001 Co 2 (117 ± 6)c 88 0.1 Cu 65.2 ± 3.7 65.7 ± 9.4 100 0.1 Fe 48.9 ± 2.6 50.9 ± 2.0 104 10 Hg 2 (105 ± 5)c 79 0.1 Zn 7.2 ± 0.8 7.7 ± 1.4 107 1 a Mean ± s (n = 3). b Calculated based on the lowest concentration of the analyte which produced the observed analytical signal in the spectra on addition of the analyte standard solution. c 133 ppm of standard solution was added to the sample prior to sample pre-treatment. Table 4 Analytical results for trace elements (mg g21) in high purity silver samples (from Johnson Matthey Company) EM9465 EM9343 Reference Reference Element This worka value This worka ICP-MSb value Au 1.3 ± 0.3 1.0 0.10 ± 0.02 0.09 ± 0.01 2 Co N.D.c 2 N.D.c 2 2 Cu N.D.d 0.1 N.D.d 2 0.1 Fe N.D.e 2.0 N.D.e 2 0.3 Hg 4.3 ± 0.5 2 2.2 ± 0.4 2.0 ± 0.3 2 Zn N.D.f 2 N.D.f 2 2 a n = 5. b n = 3. c < 0.1 mg g21. d < 0.1 mg g21. e < 10 mg g21. f < 1 mg g21. 18 Analyst, 1999, 124, 15–18

ISSN:0003-2654    

DOI:10.1039/a807137c

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Rapid preconcentration method for the determination of azadirachtin-A and -B, nimbin and salannin in neem oil samples by using graphitised carbon solid phase extraction Atmakuru Ramesh* and Muthukrishnan Balasubramanian Department of Pesticide Chemistry, Fredrick Institute of Plant Protection and Toxicology, Padappai, Kancheepuram District, Chennai-601301, India. E-mail: fippat@giasmd01.vsnl.net.in Received 18th August 1998, Accepted 9th November 1998 A simple and rapid method involving solid phase extraction and liquid chromatography for the determination of azadirachtin-A and -B, nimbin and salannin at nanogram levels in neem oil samples is presented.The neem oil samples are defatted and the compounds of interest extracted by mixing the sample with hexane and passing the hexane solution through a graphitised carbon black column. After washing the column with 2 ml of hexane, azadirachtin-A and -B, nimbin and salannin are eluted with 5 ml of acetonitrile and quantified using HPLC with UV detection. The recoveries of azadirachtin-A and -B, nimbin and salannin in fortified oil samples were 97.4–104.7%.The upper limit of quantification is up to 100 mg ml21 without any additional clean-up and with little interference from lipids during the analysis by HPLC. The method was successfully applied to various neem oil samples collected from different locations in India. Introduction Major attention has been paid in recent years to the use of neem based formulations (Azadirachtica indica, A.Juss) in the control of pest infestation.1 The main active ingredient, azadirachtin, has been found to exhibit a variety of properties such as antifeedant and anti-ovicidal effects, disrupting the life cycle of different insects.2–4 During the past few years it has been found that neem oil/extract based products are highly active against 200 insect species belonging to different orders.Also, azadirachtin has low mammalian toxicity and does not affect most beneficial organisms.5,6 Unlike synthetic chemical insecticides, which are mostly contact neurotoxins, azadirachtin is a selective compound affecting the endocrine system of insects in addition to being an anti-feedant.7 Because of this selectivity and its rapid degradation,2,8 azadirachtin is considered to be less damaging than synthetic insecticides to the environment and to pose a much smaller threat to non-target organisms, including humans via food residues, surface and ground water contamination or accidental exposure.9–11. Further, it is readily biodegradable and hence is perceived to be environmentally safe and ecologically acceptable.12–14 In general, neem oil contains azadirachtin at a concentration of approximately 0.3% (but this may vary depending on the locality), along with other components such as nimbin, salannin and meliantriol.Most of the formulations currently used are based on the content of azadirachtin,15–17 although other active ingredients of neem oil have also been found to exhibit potent insecticidal activity.There is currently an increasing demand for the ability to monitor azadirachtin at lower and lower concentrations because standardisation and commercialisation of all the neem products are solely based on azadirachtin content. In recent years, the use of graphitised carbon black (GCB) solid phase extraction (SPE) methods have been described for the analysis of environment samples for a variety of pesticides and herbicides.18,19 In continuation of our experiments20 with graphitised carbon black as a solid phase extraction material for the concentration of pyrethroids in oil samples, we report a rapid and simple method for the determination of active ingredients azadirachtin-A and -B, nimbin and salannin in neem oil samples.The method involves a simple clean-up procedure for the removal of interferences. Experimental Reference standards of azadirachtin-A and -B, nimbin and salannin were obtained from Trifolio-M, (Lahnau 2, Germany).Trace analysis grade acetonitrile was supplied by Merck, (Darmstadt, Germany). All other chemicals were of analytical reagent grade. The high performance liquid chromatographic (HPLC) system, supplied by Shimadzu (Kyoto, Japan) consisted of a model LC-10 AT pump and SPD-10A UV–VIS detector interfaced to a Winacds data station supplied by Aimil (Bangalore, India).A Supelcosil LC-18 stainless steel reversed phase column (15.0 cm 3 4.6 mm id) was used for HPLC separation. An isocratic solvent system consisting of methanol, acetonitrile and water (35 + 15 + 50) was prepared and used as the mobile phase at a flow rate 1.0 ml min21. Standard solutions Individual stock standard solutions of azadirachtin-A and -B, nimbin and salannin were prepared in trace analysis grade acetonitrile by dissolving 1 mg of each compound in 10 ml of acetonitrile and storing at 24 °C. Working standard solutions were prepared by diluting the stock standard solutions to obtain final concentrations of 10 mg ml21 of each compound.These standard solutions were used for the preparation of calibration solutions and for the preparation of fortified samples. Solid phase extraction Graphitised carbon black has been shown to be a valuable sorbent material for SPE for a variety of pollutants in water 21–24. Graphitised carbon black (500 mg) from Indo-National, (Chennai, India), was placed in a 1 cm stainless steel–glass cartridge between two Teflon frits.The cartridge was attached Analyst, 1999, 124, 19–21 19to a solvent recovery flask connected to a vacuum pump and was conditioned by rinsing with 10 ml of hexane. Preconcentration of azadirachtin-A and -B, nimbin and salannin on solid phase extraction cartridges A 1.0 ml volume of oil sample was taken in a test-tube and 0.2–0.5 ml of 10.0, 20.0, 30.0, 50.0 and 100 mg ml21 standard solutions of azadirachtin-A and -B, nimbin and salannin were added, mixed thoroughly and allowed to stand for 5 min.A 10 ml volume of hexane was added and each tube was shaken vigorously for 3 min. The sample was transferred to the SPE column reservoir and allowed to percolate for 5 min, then a vacuum was applied to drain the oil out completely. After ensuring that the oil had drained completely, the column was slowly washed with 2 ml of hexane. Azadirachtin and other active ingredients were slowly eluted with 5 ml of acetonitrile.The acetonitrile layer was collected, filtered and analyzed by HPLC. Recoveries of azadirachtin in fortified neem oil samples Neem oil samples purified after passage through the GCB cartridge column were fortified with known standards of azadirachtin-A and -B, nimbin and salannin and processed as described earlier. The recovery details are presented in Table 1. Azadirachtin-A and -B showed 99.2–104.7% recoveries at fortification levels of 10–100 mg ml21 with relative standard deviations (RSDs) of 1.61–3.18%, whereas nimbin and salannin showed 97.4–102.0% recoveries with RSDs in the range 1.57–3.18%. Results and discussion The proposed procedure consists in the SPE extraction of azadirachtin in place of the usual liquid–liquid partition step followed by column clean-up.Relatively large volumes of oil sample can be passed through the cartridge and pesticides in small amounts can be concentrated on the surface of the sorbent.No major interference from lipids was observed during the process. In addition, SPE columns can be used repeatedly at least four times by simply washing with 10 ml of acetone followed by 10 ml of water each time. The elution of the oil takes place efficiently and also more quickly after mixing the oil with 5 ml of hexane. Absolute recoveries were determined by using external calibrations. Additionally there were no interferences during the analysis of oil samples by HPLC for azadirachtin-A and -B, nimbin and salannin. Use of a graphitised carbon black SPE method facilitated the preconcentration of azadirachtin-A and -B, nimbin and salannin and the removal of all impurities associated with oil samples to a major extent, because there are no silanol group interactions with graphitised carbon black, and the adsorption of the compound is solely on carbon.As a result of the factors noted above, this method is an improvement on other techniques reported for these compounds. 13,17,27 The chromatogram presented in Fig. 1 shows the clear, sequential separation of azadirachtin-B and -A, nimbin and salannin at retention times of 6.0, 7.0, 14.2 and 16.4 min, respectively. Effect of storage All the cartridges containing azadirachtin and other active ingredients were stored at three different temperatures, 4, 20 and 30 °C, for 72 h to determine the effect of storage conditions on stability.Cartridge samples were stored in a temperature controlled oven/refrigerator. After 72 h, the cartridges were removed from the oven/refrigerator and allowed to come to room temperature. Azadirachtin and other active ingredients were eluted using 5 ml of acetonitrile and analysed by HPLC (Table 2). The results show that samples stored at 4 °C for 72 h retain the initial recovery levels. At 20 °C, on average 10–15% lower recoveries were observed and at 30 °C the recoveries fell by 18–27%.Table 1 Mean recoveries (n = 6) of azadirachtin-A and -B, nimbin and salannin Amount Azadirachtin-A Azadirachtin-B Nimbin Salannin fortified/ mg ml21 Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) Recovery (%) RSD (%) 100 104.7 2.14 102.0 1.95 101.3 1.97 102.0 1.86 50 103.8 3.13 100.5 3.18 101.9 3.18 99.1 2.19 30 100.8 2.27 99.2 2.42 100.4 2.64 100.5 1.34 20 102.4 2.41 99.3 1.61 99.2 1.97 99.3 1.47 10 99.6 2.04 99.4 1.77 98.2 1.57 97.4 1.70 Fig. 1 HPLC of (a) standards and (b) sample. Peaks: 1, azadirachtin-B; 2, azadirachtin-A; 3, nimbin; and 4, salamin. Table 2 Effect of temperature on the stability of azadirachtin-A and -B, nimbin and salannin (30 mg g21 fortification level) when stored in a graphitised carbon black cartridge column at different temperatures Recovery (%) At 6 h At 72 h Active ingredient 4 °C 20 °C 30 °C 4 °C 20 °C 30 °C Azadirachtin-A 102.6 101.8 99.8 102.4 91.2 81.2 Azadirachtin-B 100.8 100.4 98.8 100.8 88.3 78.4 Nimbin 99.6 99.2 98.7 99.6 89.9 74.1 Salannin 99.4 98.9 98.3 99.6 86.2 75.2 20 Analyst, 1999, 124, 19–21Applications to real samples The method was successfully applied to real samples.Neem seeds collected from three locations in India (Padappai, Chennai; Rajendra Nagar, Hyderabad; and Bangalore City) were crushed and the oil extracted. A 1–2 g amount of oil sample was weighed and processed as described earlier. Analysis of the results showed the maximum concentration of nimbin (3.24–3.53%) in the neem kernels collected from Hyderabad, Andhrapradesh, whereas kernels from Karnataka (Bangalore) and Tamilnadu (Chennai) showed almost equal concentrations (0.70–0.80%) of nimbin.Small amounts of salannin (0.06–0.07%) were also observed in the sample collected from Andhrapradesh, whereas the samples from Karnataka and Tamilnadu did not contain detectable levels of salannin. Samples from Andhrapradesh contained the highest levels (0.24–0.31%) of azadirachtin-A and -B whereas the Karnataka and Tamilnadu samples contained less azadirachtin (0.17–0.21% and 0.26-0.33%, respectively).In general, azadirachtin is very labile when exposed to air, moisture and sunlight. This may be due to the presence of C–C p-bonds.25,26 The strained molecular structure consisting of epoxide rings and ester groups may make azadirachtin prone to undergo addition, ring cleavage, etc. Further, its instability to UV radiation may also affect the percentage of azadirachtin present in neem seed kernels.Conclusions A sensitive and rapid method for the sequential determination of azadirachtin-A and -B, nimbin and salannin in neem oil samples has been developed. The method shows no interferences during the HPLC analysis of the neem oil samples after preconcentration using graphitised carbon black. Further, the recoveries of azadirachtin-A and -B, nimbin and salannin were higher in comparison with other methods.Major interferences associated with fats/lipids of neem oil samples were simply removed without affecting the active components. As there is no other simple and rapid method for the determination of azadirachtin- A and -B, nimbin and salannin in neem oil samples, the proposed method should be of value. Further, the utility of graphitised carbon black was established once again in the SPE and preconcentration of pesticides. Similar results were observed for some organophosphorus and organochlorine pesticides when tested under the same conditions.Further studies are in progress and the results will be published elsewhere. The authors thank the management and Director of the Institute and colleagues of FIPPAT for their cooperation in conducting this study. References 1 H. Schmutterer, J. Insect Physiol., 1988, 34, 713. 2 M. A. Barnby, R. B. Yamasaki and J. A. Klocke, J. Econ. Entomol., 1988, 82, 58. 3 K. N. Singh, Pestology., 1996, 20(3), 29. 4 C. M. Ketkar and M. S. Ketkar, Int. Proc. IPM in Tropical and Subtropical Cropping Systems, Frankfurt, Germany, Deutsche Landwioitschafts- Gesell-Schaft, 1990, (3), 689. 5 M. Jacobson, Focus on Phytochemical Pesticides. Vol. I. The Neem Tree. CRC Press. Boca Raton, FL, 1988, p. 178. 6 R. C. Saxena, in ACS Symp. Ser., 1989, 387, 110. 7 A. J. Mordue and A. Blackwell, J. Insect. Physiol., 1993, 39, 903. 8 S. V. Ley, A. A. Denholm and A. Wood, Nat. Prod. Rep., 1993, 10, 109. 9 O. Koul, M.B. Isma, C. M. Ketkar, Can. J. Bot., 1989, 68, 1. 10 M. B. Isman, Pestic. Sci., 1993, 38, 57. 11 W. Quarles, IPM Practitioner, 1994, 10, 1. 12 J. D. Stark and J. F. Walter, J. Environ. Sci. Health Part B, 1995, 30, 685. 13 K. M. S. Sundaram, J. Environ. Sci. Health Part B, 1996 31, 913. 14 K. M. S. Sundaram and J. Curry, J. Environ. Sci. Health Part B, 1996, 31, 1041. 15 T. R. Govindachari, G. Sandhya and S. P. Ganeshraj, Chromatographia, 1994, 31, 303. 16 M. Ganeshkumar, R. Jayakumar, A. Regupathy and B. Rajasekaran, Pestology, 1994, 18(11), 26. 17 M. E. Azam, S. Rangasamy and B. S. Parmer, JOAC Int., 1995, 78, 893. 18 A. Dicorcia and M. Marchetti, Anal. Chem., 1991, 63, 580. 19 A. Dicorcia and M. Marchetti, Environ. Sci. Technol., 1992, 26, 66. 20 A. Ramesh and M. Balasubramanian, Analyst, 1998, 123, 1799. 21 C. Crescenzi, A. Dicorcia, G. M. Passariello, R. Samperi and M. I. Turnes Carou, J. Chromatogr., 1996, 733, 41. 22 A. Dicorcia, A. Marcomini and R. Samperi, Environ. Sci. Technol., 1994, 28, 850. 23 A. Dicorcia, C. Crescenzi, R. Samperi and L. Scappaticcio, Anal. Chem., 1997, 69, 2819. 24 T. D. Bucheli, F. C. Gruebler, S. R. Muller and R. P. Schwarzenbach, Anal. Chem., 1997, 69, 1569. 25 C. J. Hull, W. R. Dutton and B. S. Switzer, J. Chromatogr, 1993, 633, 300. 26 J. B. Stokes and J. E. Redfern, J. Environ. Sci. Health Part A, 1982, 17, 57. 27 M. A. Barnby, R. B. Yamasaki and J. A. Klocke, J. Econ. Entomol., 1989, 85, 53. Paper 8/06527F Analyst, 1999, 124, 19–21 21

ISSN:0003-2654    

DOI:10.1039/a806527f

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Membrane solubilization with tetramethylammonium hydroxide for the preconcentration and electrothermal atomic absorption spectrometric determination of trace amounts of arsenic in water Noriko Hata,* Hiromi Yamada, Issei Kasahara and Shigeru Taguchi Faculty of Science, Toyama University, Toyama 930-8555, Japan. E-mail: noriko@sci.toyama-u.ac.jp Received 2nd September 1998, Accepted 11th November 1998 Solubilization of a mixed cellulose ester membrane filter (MF) with tetramethylammonium hydroxide (TMAH) is proposed for the preconcentration and electrothermal atomic absorption spectrometric (ETAAS) determination of trace amounts of arsenic in water.Arsenic at not more than 0.4 mg in 100 ml of sample solution was retained on the MF by filtration as an ion associate of arsenomolybdate and tetraphenylphosphonium ions.The ion associate was dissolved in a small volume of TMAH together with the MF. After being made up to 2 ml with water, the arsenic in the concentrate was determined by ETAAS in the presence of zirconyl nitrate as a chemical modifier.This method is very simple and rapid. The detection limit, defined as three times the standard deviation of the blank, was 0.04 mg l21. Inorganic matrix components in river waters, Na+, K+, Ca2+, Mg2+, SO4 22, NO3 2 and silicate at high concentrations did not interfere with the determination. Trace components, phosphate at 0.3 mg l21, dodecyl sulfate at 14 mg l21 and aluminium at 5 mg l21, also did not interfere with the determination.The proposed method was applied to the analysis of river water samples. Electrothermal atomic absorption spectrometry (ETAAS) is widely applied for trace analysis because of its high sensitivity and the requirement for only small amounts of samples. In environmental trace analysis, however, sometimes its sensitivity is insufficient and interference from matrix components also causes serious problems. In most such cases preconcentration techniques such as solid-phase extraction and solvent extraction are employed to concentrate and separate the analyte from the matrices before instrumental analyses. We proposed a soluble membrane filter (MF) technique for solid-phase extraction of trace elements in water.1 In this technique, the analyte was converted into hydrophobic species and the species was retained on an MF by filtration, then the collected material was dissolved in a small volume of organic solvent or sulfuric acid together with the MF.This technique is simple, rapid and versatile and has been applied to the spectrophotometric determination of trace amounts of phosphate,1,2 ETAAS determination of cadmium,3 copper4 and chromium5 and inductively coupled plasma atomic emission spectrometric (ICP-AES) determination of arsenic.6 Most of the MF solubilization methods are associated with sulfuric acid3,5,6 or organic solvents such as N,N-dimethylformamide (DMF),4 dimethyl sulfoxide (Me2SO)1 and 2-methoxyethanol.2 In our preliminary experiments on the application of the soluble MF technique to the ETAAS determination of arsenic, solubilizers such as DMF, Me2SO and 2-methoxyethanol, which have been successfully applied in previous studies, were not suitable for the ETAAS determination of arsenic, because white curds of the material of the mixed cellulose ester MF were precipitated when a chemical modifier aqueous solution was added to the concentrate.Probably in these solvents the material of the MF is dissolved as the original polymer and is not decomposed to small molecules. Therefore, the addition of an aqueous solution of a modifier to the polymer solution decreased the solubility of the material of the MF and the material was precipitated. Concentrated sulfuric acid, which dissolves the mixed cellulose ester MF, can be diluted with an aqueous solution to prevent the precipitation, but its viscosity is extremely high.In our preliminary experiments it was found that a small volume of tetramethylammonium hydroxide (TMAH) dissolves a mixed cellulose ester MF quickly and does not give viscous solution. TMAH has been used as a ‘tissue solubilizer’ for various zoological samples prior to analysis for minor inorganic elements by ETAAS.7 In this work, TMAH was studied as a solubilizer for MF and the solubilization technique was applied to preconcentration and ETAAS determination of trace amounts of arsenic in water.Experimental Apparatus A Hitachi (Tokyo, Japan) Model Z-8000 flame and graphite furnace atomic absorption spectrometer equipped with a Zeeman-effect background corrector and an optical temperature control system (Hitachi Model 180-0342) was used. Sample solution (20 ml) was injected by an autosampler. The analytical wavelength and slit width were 193.7 and 1.3 nm, respectively. The electric current of the hollow cathode lamp was 17.5 mA. The lamp current was set according to the manufacturer’s instrumental manual.A graphite tube cuvette was used as the furnace. The argon gas flow rate was 200 ml min21 except for atomization, where it was 30 ml min21. The optimum furnace operating conditions for the determination of arsenic used in this study are given in Table 1. Reagents All the chemicals were analytical-reagent grade or of the highest purity available. They were used as received. Analyst, 1999, 124, 23–26 23Molybdate reagent solution.Dissolve 18 g of sodium molybdate dihydrate (Na2MoO4·2H2O) (Wako, Osaka, Japan) in water, add 100 ml of concentrated sulfuric acid, then dilute to 400 ml with water. Tetraphenylphosphonium bromide solution, 0.02 mol l21. Dissolve 0.84 g of tetraphenylphosphonium bromide [(C6H5)4PBr] (Tokyo Chemical Industry, Tokyo, Japan) in 100 ml of water. TMAH solution (25%, TAMAPURE AA grade). This was purchased from Tama Chemicals (Tokyo, Japan) and was diluted to 12.5% with water before use.Zirconyl nitrate solution, 0.02 mol l21. Dissolve 0.53 g of zirconyl nitrate dihydrate [ZrO(NO3)2·2H2O] (Wako, extra pure grade) in 100 ml of water. Membrane filter An Advantec Toyo (Tokyo, Japan) A045A025A MF (25 mm in diameter, 0.45 mm pore size, mixed cellulose ester) was used. Mixed cellulose ester MFs of different brands may also be used. An Advantec Toyo KG-25 filter support (effective filtration area 2.0 cm2) was used as a filter support.Recommended procedure 1. Determination of inorganic AsV (arsenate) (Procedure A). Place 100 ml of a sample solution containing not more than 0.4 mg of AsV in an Erlenmeyer flask. Poor linearity for ETAAS determination of arsenic was obtained with more than 0.4 mg of arsenic. Add 8 ml of the molybdate reagent solution and set aside for 5 min, then add 2 ml of tetraphenylphosphonium bromide solution, set aside for at least 2 min and pass the solution through an MF by filtration under suction, collecting the arsenomolybdate as its ion associate with a tetraphenylphosphonium cation.Wash the MF twice with about 5 ml portions of water. Remove the MF from the holder and place it in a 10 ml beaker containing 0.4 ml of 12.5% TMAH solution. Heat the beaker at about 100 °C for 1 min on an electric heating plate with swirling to dissolve the MF completely. Alternatively, without heating, allow the beaker to stand for at least 6 h to dissolve the MF in 0.4 ml of 12.5% TMAH solution with swirling at intervals.Add 1.2 ml of 0.02 mol l21 zirconyl nitrate solution and dilute to 2 ml with water. Inject 20 ml of the solution into the cuvette with an autosampler and measure the absorption at 193.7 nm. 2. Determination of total inorganic arsenic [arsenite (AsIII) plus arsenate (AsV)] (Procedure B). Place 100 ml of a sample solution containing not more than 0.4 mg of arsenic in an Erlenmeyer flask. Before addition of 8 ml of molybdate solution as in Procedure A, add and dissolve 1 g of potassium peroxodisulfate to oxidize arsenite to arsenate. Arsenite is oxidized to arsenate immediately after dissolving potassium peroxodisulfate.Heating should be avoided in this procedure. Follow Procedure A from ‘Add 8 ml of the molybdate reagent solution . . .’. 3. Determination of total arsenic (Procedure C). Place 100 ml of a sample solution containing not more than 0.4 mg of arsenic in an Erlenmeyer flask. Prior oxidation and decomposition of organoarsenic species to arsenate can be accomplished by heating for 20 min after addition of 1 g of potassium peroxodisulfate before addition of 8 ml of molybdate solution as in Procedure A.Follow Procedure A from ‘Add 8 ml of the molybdate reagent solution . . .’. Results and discussion Formation and collection of arsenomolybdate The conditions for the formation of arsenomolybdate6 are similar to those reported by Wadelin and Mellon,8 except that sulfuric acid is used in place of hydrochloric acid.The choice of a counter ion is very important for the quantitative collection of the ionic species. In our previous study we found that both tetrapentylammonium and tetraphenylphosphonium are effective and successful for the collection of arsenomolybdate.6 In this work, tetraphenylphosphonium cation was applied. Material of MF and solubilizer A solubilizer applicable to the MF solubilization and ETAAS method for the determination of trace amounts of arsenic was investigated.Organic solvents, in which the materials of MF are dissolved as an original polymer, can hardly be diluted with water. Both hydrochloric acid and nitric acid, which dissolve polyamide MF, can also hardly be diluted with water for a similar reason. As stated above, concentrated sulfuric acid was not suitable because of its extremely high viscosity. Although both TMAH solution and sodium hydroxide solution in which mixed cellulose ester MF dissolves can be diluted with water, the ETAAS signal for arsenic in TMAH solution was sharper and more reproducible than that in sodium hydroxide solution.Eventually, among the materials of MF and the solubilizer tested, the combination of mixed cellulose ester and TMAH solution was selected as the best. A piece of mixed cellulose ester MF (25 mm in diameter) was dissolved and decomposed in 0.3 ml of 12.5% or 0.2 ml of 25% TMAH solution by heating for about 1 min.In the range 2.5–6.3% of TMAH in the concentrate, a constant absorbance in ETAAS determination was obtained. Chemical modifier Chemical modifiers have been recommended to improve the AAS sensitivity.9 Twenty metal salts were investigated as chemical modifiers for the preconcentration and ETAAS determination of arsenic. Cobalt nitrate, nickel nitrate, zinc nitrate and potassium permanganate were not suitable because they gave precipitates when their solutions were added to the TMAH solution for dissolving the mixed cellulose ester MF.Zirconyl nitrate, chromium(iii) nitrate, iron(iii) nitrate, palladium chloride, ammonium tungstate, ammonium vanadate(v), copper(ii) sulfate and lead nitrate solutions did not give precipitates in the TMAH solution for dissolving the MF and Table 1 Temperature–time programme optimized for the determination of arsenic Temperature/°C Stage No. Stage Start End Ramp or hold time/s 1 Drying 80 120 30.0 2 Ashing 400 800 10.0 800 800 20.0 3 Atomization 2800 2800 10.0 4 Cleaning 2900 2900 3.0 24 Analyst, 1999, 124, 23–26were effective in enhancing the sensitivity.Although some metals such as iron(iii) produced precipitates with pure TMAH solution they did not produce precipitates in the TMAH solution for dissolving the MF. Among the metal salts tested, zirconyl nitrate, chromium(iii) nitrate and iron(iii) nitrate gave comparable and excellent results. The effects of their concentrations on the arsenic signal are shown in Fig. 1. All of these three nitrates could be used successfully and their effects were almost the same. Zirconyl nitrate was adopted in this study as a chemical modifier. Ashing Temperature The influence of the ashing temperature on the signal was studied in the range 400–1200 °C. When zirconyl nitrate was used as a chemical modifier the maximum absorbance was independent of ashing temperature over the range 400–900 °C. The ashing temperature adopted in the ETAAS determination was 800 °C.Effect of foreign substances Interference from aluminium, sodium, potassium and sulfate in the direct ETAAS determination of arsenic has been reported.10 However, as previously reported in the ICP-AES determination of arsenic,6 most of spectrally interfering ions are eliminated by the MF retention procedure. Table 2 shows the effects of foreign substances on the determination of arsenic. Concentrations of 5 mg l21 of aluminium, 2.0 mol l21 of sodium, 0.1 mol l21 of potassium and 1.0 mol l21 of sulfate did not interfere.Interference from phosphate in the determination of arsenic without separation has been reported.11,12 In this study, phosphate up to 0.3 mg PO4 l21 did not interfere. In our experience, in most river waters not strongly polluted, the concentrations of the phosphate were below this limit. At phosphate levels higher than the limit, this method was not suitable unless the sample solution was diluted.Phosphate also formed a heteropolymolybdate and was collected on the mixed cellulose ester MF under the same conditions as arsenate. A sample solution containing 1 mg l21 of phosphate took longer than 8 min to be filtered and the mixed cellulose ester MF with collected heteropolymolybdates did not dissolve completely in 0.4 ml of 12.5% TMAH solution. Reproducibility The correlation of the calibration graphs based on peak height (correlation coefficient = 0.999) was better than that based on peak area (correlation coefficient = 0.994), and the peak height was more reproducible than the peak area.Peak height was therefore adopted. At a concentration of 0.5 mg l21 As, the relative standard deviation (RSD) was 8.8%, at 1.0 mg l21 the RSD was 6.3%, at 2.0 mg l21 the RSD was 4.9% and at 4.0 mg l21 the RSD was 2.9% (n = 6). These are mainly due to the instrumental errors in measurement. The blank was 0.016 absorbance units (peak height) and the RSD was 12.4%.The detection limit, defined as three times the standard deviation of the blank, was 0.04 mg l21 (n = 7). Determination of arsenic in natural water samples The acute toxicity of arsenic species decreases in the order arsenite (AsIII) > arsenate (AsV) > > dimethylarsinic acid (DMAA) > monomethylarsonic acid (MAA).13 Trimethylarsine oxide (TMAO) and arsenobetaine (AB) are considered to be relatively non-toxic. The acute toxicities of organoarsenic species are several orders of magnitude less than those of inorganic arsenic (arsenite and arsenate).With respect to the acute toxicity, at least arsenite and arsenate should be determined. Table 3 gives the analytical results of arsenic in natural water samples. Using Procedure A, only arsenate (AsV) formed arsenomolybdate and was collected on the MF and was determined by ETAAS. Therefore, arsenate is identical with the value obtained from Procedure A. Arsenite (AsIII) was oxidized to arsenate by addition of peroxodisulfate.AsIII is given by subtraction of the value obtained by Procedure A from that obtained by Procedure B. The decomposition of methylated arsenic, DMAA, with peroxodisulfate was investigated. Although the addition of 1 g of potassium peroxodisulfate did not decompose DMAA to arsenate, heating for 10 min after addition of the reagent gave arsenate. In natural water, arsenic species are mainly present as inorganic arsenic and most of the rest is as methylated arsenic.The total As is given by the value obtained by Procedure C. However, in sea-water samples the value from Procedure C was slightly lower than that from Procedure B, perhaps because some of the chloride may be oxidized to hypochlorite and be converted into chlorine by acidification. When hypochlorite solution was added to tetraphenylphosphonium after acidification, yellowish white curds precipitated and were collected Fig. 1 Effect of metal salts as chemical modifiers on arsenic signal intensity (arbitrary units) at 193.7 nm.Arsenic, 2 mg l21; sample volume, 100 ml; concentration factor, 50. Metal salts: 5, ZrO(NO3)2·2H2O; :, Fe(NO3)3·9H2O; 8, Cr(NO3)3·9H2O. Table 2 Effect of foreign substances on the determination of arsenic in water Substance added Added as Concentration/ mg l21 As founda (%) Sodium Na2SO4 46 000 102 Magnesium MgCl2·6H2O 2 400 88 1 200 96 Potassium KCl 3 900 99 Calcium Ca(NO3)2·4H2O 4 000 99 Aluminium Al(NO3)3·9H2O 5 95 Chloride NaCl 35 000 100 Sulfate Na2SO4 96 000 102 Phosphate KH2PO4 0.3 (as PO4) 96 0.5 (as PO4) 90 Silicate SiO2 + Na2CO3 200 (as SiO2) 102 Dodecyl sulfate SDSb 28.8 85 14.4 96 a Arsenic concentration, 2 mg l21; sample volume, 100 ml; concentration factor, 50.b Sodium dodecyl sulfate. Analyst, 1999, 124, 23–26 25on the MF. Arsenic is known to form gaseous molecules with chlorine.14,15 Therefore, this pre-treatment was not adopted for higher salinity samples. Table 3 gives the results of recovery tests at different arsenic levels. The recoveries of the added arsenic were quantitative, which indicates that the major salts in the river water did not interfere.Conclusion TMAH solution was successfully applied as the solubilizer in MF solubilization methods for the ETAAS determination of trace amounts of arsenic. TMAH solution dissolves the mixed cellulose ester MF to give a solution of low viscosity. The method will be applied to other trace elements and/or other instrumental analyses, especially atomic spectroscopic determinations.Acknowledgement The authors thank Dr. K. Goto, Professor Emeritus at Toyama University, for his valuable suggestions. References 1 S. Taguchi, E. Ito-oka and K. Goto, Bunseki Kagaku, 1984, 33, 453. 2 C. Matsubara, M. Takahashi and K. Takamura, Yakugaku Zasshi, 1985, 105, 1155. 3 S. Taguchi, S. Yamazaki, A. Yamamoto, Y. Urayama, N. Hata, I. Kasahara and K. Goto, Analyst, 1988, 113, 1695. 4 M. Kan, T. Nasu and M. Taga, Anal. Sci., 1991, 7(Suppl.), 1115. 5 Z. Q. Li, Y. Z. Shi, P. Y. Gao, X. X. Gu and T. Z. Zhou, Fresenius’ J. Anal. Chem., 1997, 358, 519. 6 N. Hata, I. Kasahara, S. Taguchi and K. Goto, Analyst, 1989, 114, 1255. 7 S. B. Gross and E. S. Parkinson, At. Absorpt. Newsl., 1974, 13, 107. 8 C. Wadelin and M. G. Mellon, Analyst, 1952, 77, 708. 9 D. L. Tsalev, V. I. Slaveykova and P. B. Mandjukov, Spectrochim. Acta Rev., 1990, 13, 225. 10 D. Chakraborti, W. D. Jonghe and F. Adams, Anal. Chim. Acta, 1980, 119, 331. 11 K. Saeed and Y. Thomassen, Anal. Chim. Acta, 1981, 130, 281. 12 Z-M. Ni, Z. Rao and M. Li, Anal. Chim. Acta , 1996, 334, 177. 13 M. Andou and Y. Magara, Shigen Kankyo Taisaku, 1997, 33, 113. 14 K. Fujiwara, J. N. Bower, J. D. Bradshow and J. D. Winefordner, Anal. Chim. Acta, 1979, 109, 229. 15 J. Koreckov, W. Frech, E. Lundberg, J. A. Persson and A. Cedergren, Anal. Chim. Acta, 1981, 130, 267. Paper 8/06856I Table 3 Analyses of natural water samples and recovery of arsenic added Inorganic Recovery of added As Samplea AsV added/mg l21 AsV found/mg l21 AsIII foundb/mg l21 Total As foundc/mg l21 mg l21 % River water A 0 0.97 ± 0.14(5)d 0.29 1.95 ± 0.07(4)d — — River water B 0 0.62 — — — — 1.00e 1.67 — — 1.05 105 2.00e 2.67 — — 2.05 103 River water C 0 0.09 ± 0.03(5)d NDf 0.32 ± 0.15(4)d — — Sea-water 0 0.50 ± 0.27(3)d 0.19 NAg — — a River waters A and B were taken from Jinzu River on different days. Concentration factor, 50. River water C was taken from Oyabe River. Concentration factor, 50. Sea-water was taken from the seashore at Toyama Bay on the Japan Sea. Concentration factor, 50. b AsIII is given by subtraction of the value obtained by Procedure A from that obtained by Procedure B. c Total As is given by the value obtained by Procedure C. d Mean ± confidence limits (No. of runs). e Arsenic added as arsenic(v). f Not detected. g Not available (see text). 26 Analyst, 1999, 124, 23–26

ISSN:0003-2654    

DOI:10.1039/a806856i

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Indirect determination of trace amounts of fluoride in natural waters by ion chromatography: a comparison of on-line post-column fluorimetry and ICP-MS detectors María Montes Bayón, Ana Rodríguez Garcia,† J. Ignacio Garc�ýa Alonso* and Alfredo Sanz-Medel Department of Physical and Analytical Chemistry, University of Oviedo, c/Julián Clavería 8, 33006 Oviedo, Spain Received 10th September 1998, Accepted 20th November 1998 An alternative method for the determination of trace levels of fluoride in drinking and sea-water samples is presented. It is based on the formation of the aluminium monofluoride complex in excess of Al3+ and separation of the two species formed (AlF2+ and Al3+) in a small (5 cm long, CG2) ion exchange guard column.The final determination is accomplished by both ICP-MS specific detection and post column derivatisation with fluorimetric detection. Fundamental studies on the formation kinetics of the complex, ion chromatographic separation and optimum aluminium concentration were carried out using spectrofluorimetric detection by post-column reaction of the species with 8-hydroxyquinoline-5-sulfonic acid in a micellar medium of cetyltrimethylammonium bromide.Fluorimetric detection showed good detection limits, but interferences from cations such as Mg2+ and Zn2+ required the use of the longer CS2 ion exchange column. Iron interfered in relatively large amounts but adding EDTA to the sample solution eliminated the interference.A similar separation methodology was applied using ICP-MS detection for the indirect determination of fluoride, by monitoring aluminium at mass 27. In this case, a detection limit of 0.1 ng ml21 was obtained using 0.45 m HNO3 as eluent and no interference caused by high concentrations of iron was observed. The proposed method was applied to the determination of very low levels of fluoride in natural waters. Introduction During the last decade, the majority of fluoride determinations have been performed using techniques such as potentiometry with fluoride ion selective electrodes (ISE),1,2 ion-exchange chromatography with conductivity detection,3–4 spectrophotometry5 and most recently capillary electrophoresis.6,7 The use of ISEs has been the preferred technique for this determination, but their sensitivity is insufficient to measure fluoride at ng ml21 levels. A spectrophotometric method using SPADNS has been applied to drinking waters.5 In the case of ion chromatography, the weak binding affinity of fluoride to the ion exchangers used to perform the separation process causes its early elution from the column, too close to the so-called injection peak, containing non-retained compounds and also the sample solvent.Most interferences in fluoride determination come from the presence of high levels of iron or aluminium in the sample. In these cases distillation of fluoride as HF can be performed.8 Atomic spectrometric techniques have not been used so far for direct fluoride determinations.The high excitation and ionisation potentials presented by this halogen resulted in poor sensitivity for atomic emission spectrometric (AES) detection even using powerful spectrochemical sources such as heliumbased plasmas (e.g., He MIP or He ICP). In this respect, good detection limits have been achieved for other halogens such as chloride or bromide,9,10 but no results have been reported so far on fluoride determinations by AES.One interesting and recently developed alternative involves fluoride determination by electrospray mass spectrometry, with promising results.11 Several groups have investigated indirect fluoride determination. Marco et al.12 determined fluoride by measuring the molecular absorption of the AlF2+ complex in a graphite tube using a Pt hollow cathode lamp. The chromatographic separation of Al–fluoride species was first described by Bertsch and Anderson,13 who determined the stability constants of the several possible AlFx species.Later, Jones14 determined fluoride as AlF2+ after chromatographic separation from the excess of Al3+ using indirect fluorimetric detection with 8-hydroxyquinoline-5-sulfonic acid, obtaining detection limits in the low ppb range. Previous work in our laboratory15 showed that aluminium could be better detected in the presence of cationic micelles of cetyltrimethylammonium bromide (CTAB) and it was also observed that the aluminium monofluoride complex could be detected by this fluorimetric reaction after ion chromatographic separation from Al3+.16, 17 Here, the optimum conditions for the formation of the AlF2+ complex were studied using ion chromatography and post-column fluorimetric detection.Two types of elution conditions were evaluated, using K2SO4 14,16 and HNO3 as eluents. The analytical characteristics using fluorimetric detection were compared with ICP-MS as a specific detection method monitoring aluminium at m/z 27.Examples of the application of the proposed method are presented for the determination of low fluoride levels in drinking and sea-water samples. Experimental Instrumentation The chromatographic system used consisted of a Pharmacia (Uppsala, Sweden) Model P-500 medium pressure pump and an Model 5M PA inert valve from Pharmacia fitted with a 100 ml sample loop and a 5 cm long Dionex (Camberley, UK) Ion Pac † Present address: Ingenieros Asesores SA, Polígono Silvota, Llanera, Asturias, Spain.Analyst, 1999, 124, 27–31 27HPIC-CG2 ion exchange column. For the experiments using the K2SO4 as eluent, the column was immersed in a water-bath at 50 °C. The spectrofluorimetric detector was a Shimadzu (Kyoto, Japan) R-F5000 equipped with a 12 ml flow cell. The excitation and emission wavelengths were 390 and 500 nm, respectively, and the chromatograms obtained were recorded using a Shimadzu Chromatopac C-R3A integrator.The post-column reagent was pumped using a Scharlau (Barcelona, Spain) HP4 peristaltic pump. Al-specific ICP-MS detection was carried out using an HP 4500 instrument (Hewlett-Packard, Yokogawa Analytical Systems, Tokyo, Japan) fitted with a concentric nebuliser and a Peltier cooled (2 °C) spray chamber. All chromatograms were obtained monitoring aluminium at m/z 27 using time resolved analysis. Fig. 1 shows the instrumental set-up of the system using alternatively fluorimetric or ICP-MS detection.Neither detection system could be used on-line because of incompatibility of the eluents and post-column reaction. For fluorimetric detection, the eluent from the column was mixed with the postcolumn reagent using a T-piece and a 2 m 3 0.5 mm id PTFE reaction coil. For ICP-MS detection, the eluent leaving the column was connected directly to the nebuliser. Reagents All chemicals were of analytical-reagent grade unless stated otherwise and water obtained from a Milli-Q system (Millipore, Molsheim, France) was used to prepare stock standard solutions of all the reagents.Aluminium standard solution (1000 mg ml21) was obtained from Merck (Darmstadt, Germany). Stock standard solutions of F2 (1000 and 1 mg ml21) were prepared by dissolving solid NaF (Merck) in water. For fluorimetric detection, K2SO4 (Merck) was used to prepare the mobile phase. The post-column reagents, 8-hydroxyquinoline- 5-sulfonic acid (HQS) and CTAB, were obtained from Sigma Aldrich, (St.Louis, MO, USA). The pH of the postcolumn reagent was adjusted using acetic acid–sodium acetate buffer (Merck). To prevent interferences from other metals present in the sample such as Fe, a standard solution of EDTA (Sigma Aldrich) was also used. For ICP-MS determination, no post-column reagent was necessary and the mobile phase used was prepared from 65% HNO3 (Suprapur, Merck) and diluted with Milli-Q water.Preparation of AlF2+ complex Samples and standard solutions were adjusted to pH 3 with nitric acid and spiked with Al3+, at least a five-fold mass excess of Al to fluoride being required to ensure that only AlF2+ was formed. The samples were diluted by volume (fluorimetric detection) or mass (ICP-MS detection), transferred into 10 ml polypropylene test-tubes and immersed in a water-bath at 50 °C for 60 min. Th ensured quantitative formation of the AlF2+ complex. For natural water samples and fluorimetric detection, EDTA was added at 1.6 3 1025 m.Chromatographic separation For fluorimetric detection, the mobile phase was 0.1 m K2SO4 in water adjusted to pH 3 with nitric acid. A flow rate of 1 ml min21 was used. The CG2 HPLC column was immersed in a water-bath at 50 °C following the recommendations of Jones et al.18 For ICP-MS detection, the mobile phase was 0.45 m HNO3 at a flow rate of 0.5 ml min21 and the column was kept at room temperature. Spectrofluorimetric detection The post-column reagent contained HQS and CTAB at optimum concentrations of 1 3 1023 and 2 3 1023 m, respectively and the optimum pH of 6 was adjusted with 0.25 m acetic acid–sodium acetate buffer.The optimum flow rate was 0.43 ml min21. The excitation and emission wavelengths selected were 390 and 500 nm respectively, providing the highest analytical signals for AlF2+ determination. The excitation and emission slits were both 5 nm. ICP-MS detection The ICP-MS operating conditions are summarised in Table 1. The output from the column was fed directly to the inlet of the concentric nebuliser and the m/z value monitored was 27 using the time resolved analysis mode, a 0.5 s integration time and 1 point per mass unit.Results and discussion Kinetic studies The initial aim of this work was to obtain chromatographic conditions that allowed the separation and detection of the AlF2+ complex from the excess Al3+, to provide a sensitive method for indirect fluoride determination. First, some studies on the formation kinetics were performed and the determination Fig. 1 Instrumental set-up of the system using (a) fluorimetric and (b) ICP-MS detection. Table 1 Typical operating conditions Instrument Rf power Nebuliser Spray chamber Sampling depth Gas flow rates— External Intermediate Carrier Ion lens settings— Extract 1 Extract 2 Einzel 1, 3 Einzel 2 Omega bias Omega (+) Omega (2) QP focus Ion deflector Oxide level (CeO+/Ce+) Double charged level (Ce2+/Ce+) HP 4500 1300 W Meinhard Scott type, double pass, cooled at 2 °C 5.7 mm 15 l min21 1 l min21 1.17 l min21 2221 V 2106 V 2144V 39.3V 248V 6 V 27 V 8 V 39 V < 0.5% < 1% 28 Analyst, 1999, 124, 27–31of the complex was carried out using spectrofluorimetric detection.In aqueous acidic solution, aluminium ions are present as [Al(H2O)6]3+, which can react with F2 to form the AlF2+ complex. It has been demonstrated1 that in a highly acidic medium, F2 reacts with H+ to form HF, leading to a decrease in the rate of complexation of Al3+ with F2.At pH > 3.0, the hydrolysis reaction of Al3+ can take place with the formation of Al(OH)i (32i)+, which reduces free aluminium and therefore the concentration of the complex. The optimum pH for the complex formation seems to be between 2 and 4,1,14 and therefore in this work the pH selected was 2.6–3, where the complex AlF2+ proved to be stable. Under these conditions, several parameters were evaluated in order to determine the formation kinetics of the complex, such as temperature and excess of aluminium necessary to obtain the quantitative formation of the AlF2+.Fig. 2 shows the fluorimetric peak heights obtained for the AlF2+ complex as a function of solution temperature (15, 22 and 50 °C with solutions containing 100 ng ml21 fluoride and 1 mg ml21 aluminium) and complexation time. As can be appreciated, on heating the solution containing fluoride and aluminium at 50 °C, the formation of the complex can be considered quantitative after 50 min.At room temperature, more than 5 h are necessary to obtain stable signals for the complex. The slow reaction kinetics of the AlF2+ complex formed at room temperature could allow its separation from the excess of Al3+ without any decomposition or formation of alternative species during passage through the chromatographic column. In order to optimise the aluminium concentration to be added for complete formation of the complex, solutions containing 200 ng ml21 fluoride were tested.Increasing amounts of aluminium were added to each sample and the solutions were heated at 50 °C for 1 h. The results obtained are shown in Fig. 3 and were measured fluorimetrically as peak height. As can be observed, a plateau was reached when using an aluminium concentration of 1 mg ml21 or higher. In order to increase the linear range as much as possible, a concentration of 10 mg g21 of aluminium was used in subsequent studies using fluorimetric detection.Ion exchange separation: study of the mobile phase Fig. 4 shows the typical chromatograms obtained for the separation of AlF2+ and Al3+ using 0.1 m K2SO4 as eluent and increasing amounts of fluoride from 0 to 400 ng ml21. As can be observed, using 0.1 m K2SO4 the AlF2+ peak increases with increase in fluoride concentration and the excess Al3+ elutes after 2.5 min.In order to optimise the concentration of K2SO4 in the eluent, several concentrations were evaluated and Fig. 5 shows the standard representation of the logarithm of the capacity factor (log kA) versus the negative logarithm of potassium concentration (2log [K+]).19 As can be observed, the slopes of the lines are 1.90 and 3.12 for AlF2+ and Al3+, respectively, and therefore it seems clear that the charge of the compounds is +2 and +3, respectively, and the structure of the complex is the one proposed. Fig. 2 Fluorimetric peaks heights obtained for the AlF2+ complex as a function of solution temperature (D, 15; -, 22; and 2, 50 °C) containing 100 ng ml21 fluoride and 1 mg ml21 aluminium. Fig. 3 Optimisation of the aluminium concentration required for complete formation of the AlF2+ complex in a solution containing 200 ng ml21 fluoride. Fig. 4 Typical chromatograms obtained for the separation of AlF2+ and Al3+ using 0.1 m K2SO4 as eluent and increasing amounts of fluoride from 0 to 400 ng ml21.Fig. 5 Representation of the logarithm of the capacity factor (log kA) obtained both for AlF2+ and Al3+ using different K2SO4 eluents. The slope of the log–log plot provides the charge of the species.19 Analyst, 1999, 124, 27–31 29Because saline solutions, such as K2SO4, are not recommended in ICP-MS (possible clogging of the central channel of the torch and deposits on the cones), alternative mobile phases were tested. HNO3 was found to be a good eluent for the separation of AlF2+ and Al3+ and detection by ICP-MS.Different concentrations of nitric acid, from 0.15 to 0.75 m, were tested for the above mentioned separation. The conditions chosen for subsequent studies were 0.45 m nitric acid at a flow rate of 0.5 ml min21 and detection at m/z = 27. The chromatogram obtained under these conditions for 20 ng g21 F2 in the presence of 100 ng g21 of Al is shown in Fig. 6. As can be observed, two aluminium containing peaks are detected.The first peak could be ascribed to the AlF2+ complex as its peak height/area was found to be proportional to the concentration of fluoride in the sample. Analytical performance characteristics Analytical performance characteristics for both detection modes are summarised in Table 2. The linear dynamic range for fluoride determination depends on the excess of aluminium added to the sample. It was observed with both detection modes that, for a given aluminium concentration, the upper linear limit for fluoride determinations was about one fifth of the total aluminium concentration.For ICP-MS, aluminium concentrations higher than 500 ng g21 were not tested. Using fluorimetric detection, linear upper limits up to 2000 ng ml F2 were obtained (using 10 mg ml21 excess Al3+). The detection limits obtained were 0.6 ng ml21and 0.1 ng g21 for fluorimetry and ICP-MS, respectively, calculated as three times the standard deviation of the blank divided by the slope of the linear calibration graph between 0 and 5 ng g21.The detection limit using ICP-MS detection is one of the lowest ever reported for the determination of fluoride. Interference studies An exhaustive study of possible interferences from other anions and cations was carried out first using spectrofluorimetric detection. The results are summarised in Table 3. As can be observed, typical anions present in natural waters such as HCO32 and Cl2 do not interfere at the maximum concentration tested (200 mg ml21).Other anions such as H2PO42 and BO3 32 can be present at 100 and 200 mg ml21, respectively, without causing interference. The main interferences were observed from trace metals, which can be present in natural waters. Fe3+ at concentrations higher than 0.5 mg ml21 decreased considerably the peak height from the AlF2+ peak owing to competition with Al3+ for fluoride or quenching of the fluorimetric reaction. However, in the presence of 1.6 3 1025 m EDTA this interference was eliminated.Ca2+ and Sr2+ up to 100 and 50 mg ml21, respectively, did not show any interference effect. Cu2+ and Pb2+ at concentrations higher than those in Table 3 produced a small decrease in the fluorimetric signal of AlF2+, which can be ascribed to co-elution with AlF2+ and the formation of competing chelates with the post-column reagent. On the other hand, Zn2+ and Mg2+ formed fluorescent chelates with the postcolumn reagent and co-eluted with AlF2+ using the 5 cm CG2 column. The use of a 25 cm CS2 column and a lower eluent concentration of 0.05 m K2SO4 resulted in the separation of the AlF2+ peak from Mg2+ and Zn2+.However, in this case the retention time for Al3+ increased to 30 min, as shown in Fig. 7 for a real water sample. Using ICP-MS aluminium specific detection, no effect on detection from co-eluting divalent cations should be expected. The only possible interference could be with the formation and retention of AlF2+ complex. No effect of Fe3+ on the height or area of the AlF2+ peak was obtained for Fe3+ concentrations up to 100 ng g21 at the same aluminium concentration and 20 ng g21 of fluoride.Also, when monitoring at m/z 57, Fig. 6 Chromatogram obtained for 20 ng g21 of fluoride in the presence of 100 ng g21 of aluminium using ICP-MS detection. Eluent, 0.45 m nitric acid. Table 2 Comparative analytical performance characteristics of spectrofluorimetric and ICP-MS detection Spectrofluorimetric ICP-MS Analytical characteristics detection detection Detection limit Precision Linear range Regression coefficient (r) (n = 7 points) 0.6 ng ml21 2.3%a Up to 2000c ng ml21 0.9995 0.1 ng g21 4%b Up to 100d ng g21 0.9993 a For five injections of 20 ng ml21 fluoride.b For five injections of 20 ng g21 fluoride. c Using 10 mg g21 aluminium. d Using 500 ng g21 aluminium. Table 3 Effect of foreign ions on the determination of fluoride with spectrofluorimetric detection (200 ppb F2, 10 ppm Al3+) Maximum concentration allowed/mg ml21 Interference (recovery 100 ± 5%) Fe3+ Zn2+ Mg2+ Cu2+ Ca2+ Sr2+ Pb2+ HCO2 (as NaHCO3) Cl2 (as KCl) H2PO42 (as NH4H2PO4) BO3 32 (as Na3BO3) 0.5 (10, in the presence of 1.6 3 1025 m EDTA) Interferes 10 1.6 100a 50a 2 200a 200a 100a 200a a Maximum concentration tested.Fig. 7 Chromatogram obtained for a real water sample using the CS2 column (25 cm long) and 0.05 m K2SO4 as eluent with fluorimetric detection. 30 Analyst, 1999, 124, 27–31representing a minor iron isotope, no elution of Fe from the column could be detected owing to insufficient sensitivity. Wang et al.1 have reported that the formation of the Fe–fluoride complex is strongly pH dependent. The optimum pH for iron– fluoride complex formation is 1.5 but at pH 2.5 complex formation decreased significantly.1 Our results on pH effects agreed well with such observations. Determination of fluoride in fresh and sea-water samples Fluorimetric detection.Natural water samples may contain a large range of fluoride concentration from a few ng ml21 to several mg ml21. The concentrations of interfering compounds should be well below those indicated in Table 3 except, perhaps, for Fe, Mg and Zn in some samples. The chromatogram of a mineral water sample diluted 1 + 9 with Milli-Q water and containing 3.1 mg ml21 of fluoride determined by ISE is shown in Fig. 7; 0.05 m H2SO4 as eluent and the CS2 column were used.As can be observed, the AlF2+ peak is well separated from Mg2+ and Zn2+. The concentration found in this sample by reference to a calibration graph was 3.1 ± 0.1 mg ml21 (n = 5) and the recovery for spiked fluoride was 97%. Unfortunately, under these conditions the retention time for Al3+ was > 30 min, increasing dramatically the time required for each measurement as Al3+ must be eluted from the column before the next injection.ICP-MS detection. Under the optimum separation conditions using HNO3 for elution, the retention time for Al3+ is < 8 min, allowing a sampling rate of 6–7 h21, and the detection is much more selective. Therefore, the proposed ICP-MS method was applied to the determination of fluoride in natural and drinking waters from a variety of sources and with different saline concentrations. As no stable aqueous fluoride reference material was available, it was decided to compare the proposed methodology with the fluoride ion selective electrode (FISE) and spiking the samples with fluoride to calculate the recoveries.In order to minimise aluminium addition to the samples and contamination of the ICP-MS system, up to 200-fold dilution of some drinking and sea-water samples was necessary. The results obtained are summarised in Table 4. As some of the concentrations found were around 150 ng g21, and sometimes lower, the determination using FISE was adequate in only a few cases. As can be observed, the results obtained were in good agreement with the values found by FISE, when this determination was possible.In the other cases tested, recoveries of 100 ± 10% were obtained, showing the applicability of the proposed methodology to perform fluoride determinations at extremely low levels. Conclusions The formation of the AlF2+ complex in excess of Al3+ can be considered quantitative after thermal treatment of the sample for 1 h at 50 °C.Once formed, the complex is stable and can be separated from the excess of aluminium by cation exchange chromatography without decomposition even using highly acidic eluents (0.45 m HNO3). The detection of aluminium can be accomplished either by a post-column fluorimetric reaction, with interferences from other cations such as Fe3+, Zn2+ and Mg2+, or by ICP-MS. The latter detection method proved to be extremely sensitive, with a detection limit of 0.1 ng g21 of F2, and free from interferences from other cations and anions in natural water samples. In comparison with the fluoride ISE, the proposed indirect ICP-MS method is at least two orders of magnitude more sensitive, it is not affected by interferences from aluminium and iron (in fact, the formation of the AlF2+ complex is the basis of the method) and it can be applied without modification to a large range of water samples of different salinity.Acknowledgements We thank Hewlett-Packard for the loan of the HP 4500 instrument and the DGCYT (Madrid) for financial support through Project DG-94-PB-1331.References 1 H. Wang, Z. Zhang, A. Sun, D. Liu and R. Liu, Talanta, 1996, 43, 2067. 2 R. W. Kahama, J. J. M. Damen and J. M. ten Cate, Analyst, 1997, 122, 855. 3 T. A. Biemer, N. Asral and A. Sippy, J. Chromatogr. A, 1997, 771, 355. 4 J. M. Talmage and T. A. Biemer, J. Chromatogr. A, 1987, 410, 494. 5 S. A. Sen, K. Kesava Rao, M. A. Frizzell and G. Rao, Field Anal.Chem. Technol., 1998, 2(1), 51. 6 P. Wang, S. F. Y. Li and H. K. Lee, J. Chromatogr. A., 1997, 765, 353. 7 S. A. Shamsi and N. D. Danielson, Anal. Chem., 1995, 37, 1845. 8 Standard Methods for the Examination of Water and Waste Water, American Public Health Association, New York, 15th edn., 1980, p. 337. 9 F. Camuña, J. E. Sánchez-Uría and A. Sanz-Medel, Spectrochim. Acta, Part B, 1993, 48, 1115. 10 A. H. Mohammed, J. T. Creed, T. M. Davidson and J. A. Caruso, Appl. Spectrosc., 1989, 43, 1127. 11 D. A. Barnett and G. Horlick, J. Anal. At. Spectrom., 1997, 12, 497. 12 V. Marco, F. Carrillo, C. Pérez-Conde and C. Cámara, Anal. Chim. Acta, 1993, 283, 489. 13 P. M. Bertsch and M. A. Anderson, Anal. Chem., 1989, 339, 535. 14 P. Jones, Anal. Chim. Acta., 1992, 258, 123. 15 J. I. Garcia Alonso, A. López García, E. Blanco González and A. Sanz-Medel, Anal. Chim. Acta, 1989, 225, 339. 16 J. I. Garcia Alonso, A. Rodriguez Garcia and A. Sanz-Medel, paper presented at Euroanalysis VII, Vienna, 1990. 17 A. Rodriguez Garcia, Master’s Degree, Faculty of Chemistry, University of Oviedo (1991). 18 P. Jones, L. Ebdon and T. Williams, Analyst, 1988, 113, 641. 19 R. Rosset, H. Conde and A. Jordy, Manuel Pratique de Chromatographie en Phase Liquide, Masson, Paris, 2nd edn., 1982. Paper 8/07079B Table 4 Results obtained for the determination of fluoride in water samples using ICP-MS after dilution of the samples Water sample (dilution factor) Concentration found (n = 3) by ICP-MS/ ng g21 Concentration found by FISE/ng g21 Spiked amount/ ng g21 Recovery (%) Fontecelta (200) Font-Vella (10) Tap water (10) Sea-watera (100) 8050 ± 80 182 ± 2 161 ± 1 1030 ± 60 7700 — — 1080 4300 205 210 1080 104 97.8 90 97.5 a Collected at Gijon, Asturias, Spain. Analyst, 1999, 124, 27–31 31

ISSN:0003-2654    

DOI:10.1039/a807079b

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Measurement of the cumulative particle size distribution of microcrystalline cellulose using near infrared reflectance spectroscopy Andrew J. O’Neil,* Roger D. Jee and Anthony C. Moffat Centre for Pharmaceutical Analysis, The School of Pharmacy, University of London, 29–39 Brunswick Square, London, UK WC1N 1AX Received 14th September 1998, Accepted 9th November 1998 The cumulative particle size distribution of microcrystalline cellulose, a widely used pharmaceutical excipient, was determined using near infrared (NIR) reflectance spectroscopy. Forward angle laser light scattering measurements were used to provide reference particle size values corresponding to different quantiles and then used to calibrate the NIR data.Two different chemometric methods, three wavelength multiple linear regression and principal components regression (three components), were compared. For each method, calibration equations were produced at each of eleven quantiles (5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95%).NIR predicted cumulative frequency particle-size distributions were calculated for each of the calibration samples (n = 34) and for an independent test set (n = 23). The NIR procedure was able to predict those obtained via forward angle laser light scattering. Measurement of the particle size distribution of powdered pharmaceutical raw materials is an important task that must be performed prior to manufacturing processes. This is because the distribution determines physical properties such as powder flow (Hausner ratio), dissolution rate and compressibility.1 With microcrystalline cellulose, a range of different grades are commercially available, each with different physico-chemical properties.2 These grades are classified by their median particle size and by their cumulative particle size distribution.2 Each grade should have a nominal median particle size and a cumulative particle size distribution which agrees with the range set by a pharmacopoeial monograph.3 Typically, particle size analysis of this material is by forward angle laser light scattering (FALLS) or sieve analysis.4 However, a drawback with these methods is that the analysis is time consuming and sample destructive.5 Recently, a near infrared (NIR) spectroscopic method of analysis has been described which is capable of measuring the median particle size using a two wavelength multiple linear regression of NIR reflectance, R, and FALLS data.6 The best calibration results were obtained using the logarithm of the FALLS median particle size versus reflectance data.6 Taking this method a step further, it should be possible to produce calibrations for particle size quantiles other than just 50% (median size), for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 95%.These calibrations would be produced in the same manner as described in our previous paper,6 and thus permit the measurement of a sample’s cumulative frequency particle-size distribution from its NIR spectrum.A manufacturer should then have sufficient information to assess the likely physicochemical properties of the sample.2 The aim of this work was to measure the cumulative percentage frequency particle-size distribution of microcrystalline cellulose. Two chemometric methods of calibration were compared: three wavelength multiple linear regression (MLR)7 and principal components regression (PCR)7 using three principal components, each model using log (FALLS particle size) values and NIR reflectance data.The robustness of the calibrations was assessed using an independent validation set. Experimental Instrumentation NIR measurements were made using an FT-NIR NIRVIS spectrometer (Model 100.1, Buhler, Uzwil, Switzerland) fitted with a Buhler fibre-optic probe (Model 110.2). Reflectance spectra were recorded over the range 4008–9996 cm-1 (500 data points), each spectrum being the average of six scans.Particle size data were acquired by forward angle laser light scattering using a Malvern 2600C particle sizer (Malvern Instruments, Malvern, UK). Sieve fractions were produced using a machine sieve (Endecotts, London, UK). Materials The grades of microcrystalline cellulose used were Avicel PH101 (16 batches), Avicel PH102 (19 batches) and Avicel PH200 (single batch), all from FMC International (Wallingstown, Little Island, Co. Cork, Ireland). Sample preparation and presentation Sieve fractions from single batches of Avicel PH101, Avicel PH102 and Avicel PH200 were produced by machine sieving using a nest of progressively finer stainless steel wire mesh sieves (150, 90, 63, 45, 38 and 32 mm).In addition, material falling through the 32 mm sieve was collected. The sieved fractions and samples of all the original Avicel batches were particle sized by FALLS. Each sample was suspended in cold, distilled water with surfactant (dilute household detergent) prior to particle sizing and was gently shaken using a vortex mixer to prevent the formation of agglomerates.NIR diffuse reflectance measurements were made on the samples of sieved and bulk materials in narrow disposable glass vials to permit a consistent compaction pressure. Sieved and bulk materials were scanned on different days over the course of several weeks. Analyst, 1999, 124, 33–36 33Data analysis Data were processed using in-house computer programs written in C and in Matlab 5 Scientific and Technical Programming language (Mathworks, Natick, MA, USA).The MLR program was based on the routine svdfit, available in the literature.8 Programs were run on an Acer Pentium II 333 MHz machine. Results and discussion Preliminary investigation The results of previous work6 have shown that useful calibrations for median particle size can be obtained by using NIR reflectance data with a logarithmic transform of the FALLS particle-size data, hence these data were used in this work.With MLR calibrations, preliminary work showed that a three wavelength linear regression at any of the FALLS quantiles produced calibrations more robust than a two wavelength fit. It was therefore decided that three wavelength MLR calibrations would be employed subsequently. With PCR models, three principal components were required to obtain satisfactory calibrations and this was used for all subsequent calibrations.Spectral characteristics Each powdered sample exhibited an NIR reflectance spectrum with a curved baseline resulting from multiple scattering (Fig. 1). Across the spectrum of each sample, the apparent offset appears to increase and this has previously been attributed to variations in pathlength,9 which in turn is dependent on particle size and sample porosity. Model generation The FALLS instrument gives values of the cumulative percentage frequency particle-size distribution at 64 particle sizes (range 564–5.8 mm) at intervals which follow a geometric progression.For each sample, linear interpolation of the measured FALLS values was used to calculate the particle size values corresponding to the 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 95% quantiles. The samples exhibited a wide range of particle sizes at each quantile (Table 1) and a wide variety of distributional shapes. Of the 57 samples available, 34 were chosen at random for the calibration set; the remaining 23 samples were used as an independent validation set.To aid comparison of the two calibration methods, the same calibration and validation data were used for each method. Multiple linear regression. Data from the calibration samples were used to generate calibration equations for each quantile by fitting the logdx values to the NIR reflectance values according to the equation logdx = b0 + b1Rl1 + b2Rl2 + b3Rl3 (1) where d is the FALLS interpolated particle size at quantile x, R is the reflectance at wavelength l and b are the MLR coefficients. The selection of wavelengths was performed on a reduced data set of every other wavelength to reduce the computation time required.A full three wavelength search for each particle size quantile calibrated therefore used 250 of the 500 available wavelengths. This reduced the total computation time for all 11 calibrations to about 10 h, compared with an estimated 80 h if all 500 wavelengths had been searched.For each calibration equation, the three chosen wavenumbers (Table 2) were those which gave the smallest standard error of calibration (SEC).9 The optimum wavenumbers were similar for the 30–60% quantiles, but varied for the extreme quantiles. The calibration equations were then used to predict the validation set (n = 23) to give an indication of the robustness of the method (Table 3). Principal components regression. This calibration method required the generation of a principal components analysis (PCA) model.This consists of a set of new variables which are uncorrelated and represent linear combinations of the original NIR reflectance data. Fig. 1 NIR spectra of microcrystalline cellulose samples with different particle-size distributions and median particle sizes, d50: (a) 24, (b) 45.8, (c) 93.4, (d) 261 and (e) 406 mm. Table 1 Particle size ranges at each quantile for the calibration and validation sets as determined by FALLS Particle size/mm Calibration set (n = 34) Validation set (n = 23) Quantile (%) Minimum Median Maximum Minimum Median Maximum 5 6.45 25.72 216.52 7.21 23.06 167.11 10 9.92 37.14 268.91 11.44 32.34 187.67 20 14.48 52.92 311.96 18.05 45.40 219.33 30 18.39 67.10 345.62 22.55 56.36 251.13 40 21.40 81.41 376.44 26.27 67.35 283.66 50 23.99 96.59 406.07 29.82 78.98 319.67 60 26.47 112.82 436.21 33.71 91.57 359.67 70 29.25 131.29 466.55 38.30 105.95 402.81 80 33.11 154.78 497.51 44.94 124.03 451.54 90 40.62 197.11 529.54 57.16 152.54 504.66 95 48.47 240.07 546.76 70.34 184.74 533.21 Table 2 MLR wavelengths and PCs selected for each percentage quantile calibration Percentage MLR wavenumber/cm21 PCs 5 4008 9300 9528 28 22 17 10 4008 9300 9528 29 27 14 20 5640 5676 6216 29 27 14 30 4464 9852 9864 29 28 27 40 5736 9852 9864 27 15 1 50 5736 9852 9864 20 15 1 60 5496 9852 9864 20 15 1 70 6024 6948 9168 15 14 1 80 5664 5796 9432 23 9 3 90 5952 6996 8280 28 18 6 95 7632 8532 8664 28 18 6 34 Analyst, 1999, 124, 33–36The PCA model, X, was obtained as the product of a score matrix, T, with a loadings matrix, U, plus a residuals matrix, E: X = T U + E (2) where X represents the original spectral data.Principal components (PCs) are arranged such that the first represents the variable describing the largest amount of variance in the data set, the next represents the largest residual variance, and so on until all PCs are extracted.Regression of FALLS data was as described above for MLR, except that PC scores were used in place of reflectance values. For each calibration, the three PCs selected were those which gave the highest correlations with the FALLS data (Table 2). The total time required to compute PCs and PCR calibration equations was much faster than MLR, requiring only about 20 min. Calibration and validation precision With both methods, individual calibrations were the most precise at the 40% and 50% quantiles (Table 3).This is clearly seen from the plot of SEC versus percentage quantile (Fig. 2). The decrease in the precision of individual calibrations at the extreme quantiles probably reflects the shape of the distribution curves for the particle sizes in the calibration sets. The shapes of the distributions become more skewed at the extreme quantiles. With both MLR and PCR, excellent calibration results were obtained, with low SECs (Table 3). The SECs at each quantile are smaller with MLR; however, the standard errors of prediction (SEPs)9 for the independent validation set are smaller with the PCR model (Table 3).This suggests that the PCR model is more robust. Table 3 also gives the slopes and intercepts for the plots of NIR predicted logdx versus FALLS measured logdx values at each quantile. The slopes and intercepts were not significantly (5% probability level) different from 1 and 0, respectively, apart from a few values (indicated with superscript b) which occurred at some of the extreme quantiles.Cumulative particle size distributions The percentage quantile value was plotted against the NIR predicted logdx of each sample in the calibration and validation sets to give cumulative particle-size distribution curves for both the MLR and PCR methods. The MLR and PCR results for the first four validation samples are shown in Fig. 3, which also shows the FALLS measured cumulative percentage frequency distributions overlaid.Predicted distributions for both calibration methods closely follow those obtained by FALLS, although PCR predicted distributions match the FALLS measured distributions more closely than with MLR. In this work, the number of quantiles at which calibration equations were set up was restricted to 11. In principle, more or Table 3 MLR and PCR calibration and validation results at various percentage quantiles Percentage Parametera 5 10 20 30 40 50 60 70 80 90 95 MLR calibration set (n = 34)— R 0.977 0.980 0.984 0.987 0.989 0.988 0.984 0.979 0.972 0.932 0.889 m 0.954 0.960 0.968 0.974 0.978 0.975 0.968 0.958 0.945 0.869b 0.791b c 0.065 0.063 0.055 0.048 0.042 0.049 0.065 0.088 0.121 0.301b 0.497b SEC [log(dx/mm)] 0.084 0.071 0.055 0.046 0.039 0.039 0.042 0.046 0.052 0.080 0.104 RSD (%) 19.3 16.3 12.7 10.6 9.0 9.0 9.7 10.6 12.0 18.4 23.9 Validation set (n = 23)— R 0.951 0.951 0.965 0.971 0.959 0.955 0.950 0.959 0.964 0.897 0.822 m 0.876 0.950 0.977 0.978 0.984 0.973 0.943 0.986 0.980 0.921 0.734b c 0.140 0.064 0.046 0.021 0.013 0.040 0.108 0.050 0.057 0.216 0.657b SEP [log(dx/mm)] 0.131 0.109 0.074 0.066 0.074 0.073 0.073 0.070 0.061 0.106 0.132 RSD (%) 30.1 25.1 17.0 15.2 17.0 16.8 16.8 16.1 14.0 24.4 30.4 PCR calibration set (n = 34)— R 0.969 0.973 0.978 0.980 0.981 0.981 0.976 0.968 0.959 0.898 0.858 m 0.939 0.946 0.956 0.960 0.963 0.961 0.953 0.937 0.921 0.806 0.737 c 0.086 0.084 0.076 0.074 0.070 0.077 0.096 0.133 0.174 0.445b 0.627b SEC [log(dx/mm)] 0.096 0.082 0.065 0.057 0.051 0.049 0.051 0.057 0.062 0.097 0.116 RSD (%) 22.1 18.9 15.0 13.1 11.7 11.3 11.7 13.1 14.3 22.3 36.8 Validation set (n = 23)— R 0.980 0.981 0.981 0.978 0.984 0.981 0.969 0.965 0.953 0.924 0.842 m 1.128b 1.045 0.998 0.969 0.967 0.970 0.977 0.975 0.946 0.932 0.861 c 20.16b 20.071 0.005 0.053 0.051 0.042 0.024 0.034 0.079 0.092 0.258 SEP [log(dx/mm)] 0.085 0.062 0.051 0.050 0.041 0.045 0.056 0.057 0.071 0.094 0.124 RSD (%) 19.6 14.3 11.7 11.5 9.4 10.4 12.9 13.1 16.3 21.6 28.5 a R is multiple correlation coefficient, m and c are slope and intercept of plots of NIR predicted logdx versus FALLS measured logdx; n is the number of samples in each data set.b m significantly different from 1, or c significantly different from 0. Fig. 2 Standard errors of calibration (SEC) versus cumulative percentage quantile: (A) MLR; and (B) PCR. Analyst, 1999, 124, 33–36 35less could be used.With the present data sets the errors do not justify the need for smaller intervals (Table 3). Conclusion NIR spectroscopy may be used to measure the cumulative percentage frequency particle-size distribution of powdered microcrystalline cellulose. This represents a development over previous studies which have focused on measurement of only the median or mean particle size.5,10–12 Although setting up the calibration equations is time consuming, once generated they allow the rapid determination of the cumulative frequency distribution of subsequent samples.Both MLR and PCR provide excellent results; however, the PCR method is computationally faster and slightly more robust. The method should be applicable to other powdered pharmaceutical materials. The authors are grateful to Buhler for the loan of the NIR instrument and to Mathworks for providing Matlab 5 software. They thank P. A. Hailey, Pfizer, Sandwich, UK and FMC International for advice and providing samples of pharmaceutical excipients and A.J. O’Neil thanks Pfizer for a research grant. Kevin Taylor and Keith Barnes, The School of Pharmacy, University of London, are thanked for assistance with forward angle laser light scattering and sieving. References 1 C. Washington, Particle Size Analysis in Pharmaceutics and Other Industries, Ellis Horwood, New York, 1992. 2 Handbook of Pharmaceutical Excipients, ed. A. Wade and P. J. Weller, American Pharmaceutical Association, Washington, DC and Pharmaceutical Press, London, 2nd edn., 1994. 3 British Pharmacopoeia 1993, H.M. Stationery Office, London, 1993, vol. 1. 4 M. E. Aulton, Pharmaceutics: the Science of Dosage Form Design, Churchill Livingstone, Edinburgh, 1988. 5 P. A. Hailey, P. Doherty, P. Tapsell, T. Oliver and P. K. Aldridge, J. Pharm. Biomed. Anal., 1996, 14, 551. 6 A. J. O’Neil, R. D. Jee and A. C. Moffat, Analyst, submitted for publication. 7 B. G. Osborne, T. Fearn and P. H. Hindle, Practical NIR Spectroscopy with Applications in Food and Beverage Analysis, Longman, Harlow, 2nd edn., 1993. 8 W. H. Press, S. A. Teukolsky, W. T. Vetterling and B. P. Flannery, Numerical Recipes in C. The Art of Scientific Computing, Cambridge University Press, Cambridge, 2nd edn., 1992. 9 H. Mark, Principles and Practice of Spectroscopic Calibration, J. Wiley, New York, 1991. 10 J. L. Ilari, H. Martens and T. Isaksson, Appl. Spectrosc., 1988, 42, 722. 11 E. Ciurczak, P. Torlini and P. Demkowicz, Spectroscopy, 1986, 1, 36. 12 P. Frake, C. N. Luscombe, D. R. Rudd, J. Waterhouse and U. A. Jayasooriya, Anal. Commun., 1998, 35, 133. Paper 8/07134I Fig. 3 Cumulative percentage frequency particle-size distributions for the first four validation samples with FALLS measured values overlaid. Sample 1: (A) MLR and (B) PCR. Sample 2: (C) MLR and (D) PCR. Sample 3: (E) MLR and (F) PCR. Sample 4: (G) MLR and (H) PCR. 36 Analyst, 1999, 124, 33–36

ISSN:0003-2654    

DOI:10.1039/a807134i

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Perspective Direct qualitative and quantitative characterization of a radiosensitizer, 5-iodo-2A-deoxyuridine within biodegradable polymeric microspheres by FT-Raman spectroscopy Annabelle Geze,a Igor Chourpa,*b Franck Boury,a Jean-Pierre Benoita and Pierre Duboisb a UPRES EA 2169, Facult�e de Pharmacie, Universit�e d’Angers, 16 Boulevard Daviers, 49100 Angers, France b Laboratoire de Chimie Analytique, Facult�e de Pharmacie ‘Philippe Maupas’, Universit�e de Tours, 31 Avenue Monge, 37200 Tours, France Received 25th August 1998, Accepted 29th October 1998 Non-destructive qualitative and quantitative characterization of a radiosensitizer, 5-iodo-2A-deoxyuridine (IdUrd), incorporated within injectable microspheres of a biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA), was performed using Fourier transform (FT) Raman spectroscopy.Raman spectra of IdUrd, free and entrapped in microspheres, were recorded under fluorescence-free conditions, described and assigned.For the Raman bands of the PLGA microspheres, assignments with preferential localization of the corresponding vibrations at lactic or glycolic units were proposed. No evidence for drug–polymer interactions in microspheres was found. This allowed the FT-Raman spectra to be used for the quantification of the IdUrd content in the samples. For the microspheres with IdUrd loadings varying from 2 to 27% of the total weight, the methodology used provided good reproducibility and precision (1%).Within the sensitivity of the technique, samples exposed to sterilization doses (27 kGy) of g-radiation did not exhibit marked changes in the drug structure. Nowadays, Raman spectroscopy has become an easily used technique with a very wide range of applications. For the resolution of numerous analytical problems, the molecularspecific information obtained in a non-destructive way seems to be irreplaceable. Moreover, Raman spectroscopic data can be successfully used for quantitative measurements.Owing to some particular advantages, such as fluorescence-free conditions, Fourier transform (FT) Raman spectroscopy with excitation in the infrared region is especially useful in analytical studies of raw samples. In this study, we aimed to apply FT-Raman spectroscopy to the non-destructive qualitative and quantitative characterization of a drug incorporated within a polymeric matrix. The polymers concerned are bioresorbable aliphatic polyesters based on copolymers of lactic (LA) and glycolic acid (GA).These biocompatible polymers degrade hydrolytically in the body with the formation of non-toxic products.1 Poly(d,l-lactide-coglycolide) (PLGA) polymers have been studied as materials for osteosynthesis, sutures and prosthetic devices.1 They are widely used for therapeutic purposes especially to make sustained drug release delivery systems.2 We focused on PLGA-based delivery systems as used for the sustained release of 5-iodo-2A-deoxyuridine (IdUrd).This molecule is a thymidine analog and is a powerful radiosensitizer. 3 This halogenated pyrimidine competes with thymidine in the biosynthesis of DNA. The treatment of malignant brain tumors based on conventional radiotherapy is far from satisfactory. Therapeutic IdUrd concentrations within the tumor during the time course of radiotherapy might improve treatment results, by increasing the lethal effects of radiation on the tumor cells having incorporated the radiosensitizer.The intracranial implantation of IdUrd-loaded microparticles in the vicinity of the cancer cells can meet these requirements.4 The study involved the preparation of PLGA microspheres loaded with different amounts of IdUrd, by using a phase separation technique. Intact IdUrd-loaded microspheres were characterized qualitatively and quantitatively by FT-Raman spectroscopy. Since the microspheres are intended to be administered in vivo into the brain, they need to be sterile. The sterilization of biodegradable drug delivery systems is often carried out by g-irradiation.The stability of the drug entrapped in the microspheres5,6 after exposure to g-radiation was determined from the respective Raman spectra. Experimental Chemicals IdUrd (99% pure, odorless, white, crystalline powder, slightly soluble in water: 2 mg ml21) was obtained from Sigma-Aldrich Chimie (St. Quentin Fallavier, France). PLGA 50/50 was purchased from Boehringer Ingelheim (RG 506, B.I.Chimie, Paris, France). The composition of the chains includes 25% llactic units, 25% d-lactic units and 50% glycolic units. The mass- and number-average molecular masses were 75 000 and 48 000, respectively. These values were determined in tetrahydrofuran by size exclusion chromatography (SEC Waters, St. Quentin en Yvelines, France), referred to polystyrene standards. Methylene chloride, silicone oil (Rhodorsil, viscosity 300 cSt, relative density 0.97) was obtained from Prolabo (Paris, France).Heptane was purchased from Verbi`ese (Wasquehal, France) and dimethyl sulfoxide (DMSO) from Carlo Erba (Val de Reuil, France). IdUrd crystal milling Grinding of IdUrd crystals was performed with a Pulverisette 7 planetary micro-mill (Fritsch, Idar-Oberstein, Germany). A 800 mg amount of IdUrd was milled for 10 min at a rotation speed of 2500 rpm. Analyst, 1999, 124, 37–42 37Microsphere preparation The coating polymer PLGA (250 mg) was dissolved in methylene chloride to reach a concentration of 1.3% m/m.Various amounts of milled IdUrd crystals (18 ± 3 mm, SD of different mean size values) were then dispersed in the organic phase with sonication for 3 min. A separation phase inducer (silicone oil, 8 g) was added to the mixture and stirred magnetically at room temperature for 2 min in order to precipitate the polymer around the drug particles. The resulting dispersion (semi-formed microparticles or coacervates) was poured into 400 ml of heptane (hardening agent), stirred at 600 rpm (Heidolph RGH500, Prolabo, Paris, France).After 30 min of agitation, the solidified microparticles were filtered on a 0.45 mm filter (HV type, Millipore, Maurepas, France) and washed with heptane (50 ml). The resulting microspheres were dried under reduced pressure for 60 h at 35 °C. They were stored at 6 °C shielded from light. Microsphere size distribution analysis The average size of the microparticles was determined using a Coulter Multisizer (Coultronics, Margency, France) after dispersion of the microparticles in a conducting liquid (Isoton II, Coultronics).Crystal size distribution The average size of the crystals was determined using a Mastersizer S (Malvern Instruments, Malvern, Orsay, France). Size measurements were performed in the liquid phase, using a 300 RF lens, in the size range 0.05–880 mm and an MSI module as a manual liquid sampler.A 30 mg amount of milled powder was suspended in 3 ml of cyclohexane with sonication and immediately analyzed. g-Irradiation of microspheres IdUrd-loaded microspheres were accurately weighed into 100 mg samples, transferred to 2.5 ml glass vials and sealed. The vials were irradiated at a dose of 26.7 kGy 60Co source, (Ionisos, Dagneux, France). This was done in triplicate. IdUrd content determination This was achieved using two methods, spectrophotometry and Raman spectroscopy.The former method provides direct access to the drug concentration via molar absorptivity (7.9 3 103 l mol21 cm21 at 287 nm) but was destructive, since the microspheres (6–8 mg) needed to be dissolved in DMSO. The latter method, allowing non-destructive quantification of IdUrd, was developed using the Raman spectra obtained from intact microspheres. These spectra contained a contribution of the polymer vibrations which could be useful as an internal standard.The IdUrd/PLGA peak area ratio of the Raman bands allowed the calculation of the corresponding ratio of the concentrations of these molecules. This is discussed below. Spectroscopic instrumentation The absorption of IdUrd was measured with a Uvikon 922 UV spectrophotometer (Kontron Instruments, St. Quentin en Yvelines, France). The IdUrd solutions were shielded from light. The Raman spectra were excited with 1.06 mm radiation from an ADLAS Nd:YAG laser and recorded with a Bruker (Wissembourg, France) RFS100/D418-S FT-Raman spectrometer.The laser power at the sample was about 140 mW. No laser-induced sample degradation was noted during the experiments. All the measurements were repeated at least three times. The data appeared to be well reproducible. The data obtained were analyzed with Labspec software (DILOR, Lille, France), which permitted the very easy and rapid treatment of the spectra (baseline correction, peak area analysis, normalization, etc.) and statistical analysis of any spectral parameters.In addition, this software package allowed us to treat simultaneously and in exactly the same manner all the sets of the recorded spectra. Results and discussion In order to analyze the Raman spectra of the drug-containing microspheres, the model Raman spectra of the blank PLGA 50/50 microspheres and of IdUrd crystals (Fig. 2) were recorded. FT-Raman spectra of IdUrd crystals In the FT-Raman spectra of IdUrd crystals (Fig. 2 and Table 1), both the bands of iodo-substituted aromatic nucleus (5-iodouracil) and those of the deoxyribose moiety (Fig. 1) can be observed. As a result, the spectra appeared very rich in vibrations, especially within the 1400–1800 cm21 region. Since the Raman spectra of IdUrd have not been reported previously, we describe them in detail. Our discussion is limited to only the more intense bands between 1800 and 700 cm21. The CH region bands above 2800 cm21 and the lower wavenumber weak deformational bands were less interesting with respect to the main task of the present study. Fig. 1 Structural formulae of IdUrd and PLGA. Fig. 2 FT-Raman spectra of IdUrd crystals (bottom) and the blank PLGA microspheres (top). 38 Analyst, 1999, 124, 37–42As expected for the IdUrd molecule, the very characteristic stretching bands of the non-conjugated and conjugated C =O groups were clearly observed at 1696 and 1676 cm21, respectively (Fig. 2 and Table 1).In their neighborhood, at 1611 cm21, one could observe another strong band, that of the n(C = C) vibration. The region of the CH2 deformation motions was represented by a weak doublet at 1460/1444 cm21 encircled with two even weaker bands which are not discussed here (see Table 1). A strong band at 1350 cm21 had the characteristic wavenumber of H–N–C = O stretching (amide III). A group of weak bands located at lower wavenumbers, down to 1230 cm21, was due to CH deformation and CH2 twisting.The bands within the 1200–1140 and 1105–1020 cm21 regions were assigned to asymmetric and symmetric COC stretching, respectively. The vibrations observed between 1010 and 850 cm21 were attributed to the C–C stretching and CH2 rocking. The very strong band at 779 cm21 was assigned to a ring breathing mode of the 5-iodouracil moiety. The Raman bands of IdUrd located near 750 cm21 (dC = O) and lower were those of the various deformational motions of the C = O, CCO, etc., groups.The FT-Raman spectra of IdUrd after mechanical milling (see Experimental) were devoid of any detectable changes (data not shown). Based on this and on the data from X-ray diffraction (not shown), it was concluded that no polymorphic forms were present in detectable amounts in these samples. Therefore, the crystallinity of IdUrd was preserved after milling. FT-Raman spectra of the blank PLGA 50/50 microspheres FT-Raman spectra of blank microspheres (Fig. 2 and Table 2) were analyzed in comparison with previously reported Raman spectra (in the visible region) of poly(d,l-lactide) (PLA)7 and poly(glycolide) (PGA)8 polymers. The band positions and shapes in the spectra of PLGA microspheres indicated an amorphous form of polymer.9,10 In general, the PLGA spectra contained both the PLA and PGA Raman bands, the former being more pronounced (see Table 2). With respect to this comparison, we proposed a temptative attribution of the observed PLGA bands to vibrational modes of lactic (LA) or/and glycolic (GA) units (Table 2).Whereas both PLA and PGA spectra have already been well documented,7-10 to our knowledge, there has been no report on the Raman spectra of PLGA. For this reason, only some particular features in the Raman patterns which differentiate the PLGA samples from PLA and PGA (Table 2), are discussed. Table 1 Major Raman wavenumers (cm21) and their tentative assignments for IdUrd in pure crystals and when in PLGA microspheresa Microspheres with IdUrd Blank microspheres Assignment Crystals of IdUrd Assignment 3080 w 3080 m nasCH 3010 w 3010 m nsCH 3002 3002 s nasCH3 2965 sh 2968 vs nasCH2 2953 s 2956 s nasCH2 2947 vs 2947 vs nsCH3 + nasCH2 2936 m 2936 m nasCH2 2908 sh 2908 m nCH 2876 s 2876 s nCH 1769 s 1769 s nC =O ~ 1760 sh ~ 1760 sh nC =O 1696 m 1696 m nC = O, non conj. 1676 s 1676 s nC = O, conj. 1611 s 1611 s nC = C 1458 s 1452 s dasCH3 + dCH2 1460 mw dCH2 1442 sh 1445 w dCH2 1427 m 1427 m dCH2 1390 w 1384 w dsCH3 1395 w dCH2 1350 s 1345 vw d1CH + dsCH3 1350 s Amide III + dCH 1300 w 1303 w d2CH 1296 w dCH 1274 vw twCH2 1268 w 1267 mw twCH2 1252 vw twCH2 1239 w 1239 mw twCH2 1199 mw 1198 m nasCOC 1145 w 1146 w nasCOC 1130 m 1130 m rasCH3 1102 vw nsCOC 1093 w 1093 w nsCOC 1096 w nsCOC 1075 vw nsCOC 1055 vw nsCOC ~ 1048 sh nC–CH3 1032 w 1032 w nsCOC 1028 w nsCOC 990 w 989 w nC–C + rCH2 961 w 961 w nC–C + rCH2 953 vw nC–C + rCH2 + rCH3 920 vw nC–C + rCH2 913 w nC–C + rCH2 892 m nC–C + rCH2 885 m 886 m nC–C + rCH2 874 s 874 s nC–COO 878 m nC–C + rCH2 849 sh 847 mw rCH2 855 mw rCH2 779 vs 779 vs Ring breathing 746 w 750 vw dC =O 756, 749 w dC =O 706 vw 706 vw gC = O + dC =O a Abbreviations: v = very; s = strong; m = medium; w = weak; sh = shoulder; subscript s = symmetric; subscript as = asymmetric.Analyst, 1999, 124, 37–42 39The wavenumbers that appeared significantly shifted in PLGA are given in italics in Table 2.This particularly concerned the stretching (3002 and 2947 cm21) modes of the CH3 (LA) and CH2 (GA) groups (Table 2). The interesting observation was that the copolymer formation seemed to affect the dCH2 (1427 cm21) more than the dCH3 (1384 cm21) wavenumbers and the d1CH (1345 cm21) more than the d2CH (1303 cm21) wavenumbers. The perturbations of the nC–C and rCH2 modes (both 920 and 892 cm21) were less predictable than those for dC = O (750 and 706 cm21). FT-Raman spectra of the PLGA microspheres loaded with IdUrd These logically contained both drug and polymer bands (Fig. 3 and Table 1). We subtracted from these spectra the model spectra of the pure drug and blank microspheres (Fig. 4). The difference spectra, even obtained from those with a significant contribution of the polymer or drug to the Raman bands, did not reveal any noticeable changes as compared with their respective model Raman patterns. Therefore, no indication either of structural modification of IdUrd and/or PLGA matrix or of their interactions could be discerned.It was also noted that unchanged spectra (in wavenumbers and relative intensities) could be considered as the sum of the model spectra. This allowed the quantitative considerations discussed in the following section. Determination of Raman IdUrd content in the IdUrd-loaded PLGA microspheres The methodology used with incorporation of a phase separation technique yielded PLGA microspheres of nearly regular size in the range 40 ± 5 mm (SD on mean size of different microsphere batches) as established by particle sizing.The first step in the current work was to establish the calibration curve, i.e. the function describing the Raman spectral parameters as a function of the drug/polymer relative concentrations. For this purpose, microspheres with different IdUrd loadings, ranging from 2 to 27% [IdUrd (mg)/microspheres (mg) 3 100: incorporation ratio, determined using spectrophotometry] were analysed.Comparison of the Raman spectra of the drug-loaded microspheres with the spectra of the pure IdUrd crystals and blank PLGA microspheres (Fig. 3) revealed several regions devoid of superposed bands of these molecules. In particular, in the region between 1600 and 1820 cm21, the drug and polymer vibrations were in a close neighborhood but well separated. This made these vibrations usable as an internal standard. Three strong Raman bands of IdUrd, at 1696, 1676 and 1611 cm21, and a very large band of PLGA with a maximum at about Table 2 Major Raman wavenumbers (cm21) and their tentative assignments for PLGA 50/50 microspheres compared with PLA and PGA polymersa Raman (lex 514.5 nm)b,c FT-Raman (lex 1064 nm): PLA amorphousb PGA amorphousc PLGA50/50 microspheres Localizationd Assignment 2997 s 3002 s LA nasCH3 2942 vs 2947 vs LA + GA nsCH3 + nasCH2 2954 vs 2877 m 2876 s LA nCH 1769 s 1769 s LA nC =O 1760 s ~ 1760 sh GA nC =O 1455 s 1450 sh 1452 s LA + GA dasCH3 + dCH2 1423 s 1427 GA dCH2 1400 sh — wCH2 1386 m 1384 w LA dsCH3 1365, 1355 m 1345 w LA d1CH + dsCH3 1296, 1300 s 1303 w LA d2CH 1274 m 1274 w GA twCH2 1128 s 1130 m LA rasCH3 1092 s 1090 w 1093 w LA + GA nsCOC 1042 s ~ 1048 LA nC–CH3 1029 m 1032 GA nsCOC 953 sh 950 m 953, 920 LA + GA rCH3 + nC–C + nC–C + rCH2 885 s 892 m GA nC–C + rCH2 873 vs 874 s LA nC–COO 845 s 847 w GA rCH2 740 m 750 vw LA dC =O 700 vw 720 w 706 vw LA + GA gC = O + dC =O a Abbreviations: b As referred in ref. 7. c As referred in ref. 8. d With respect to the d, l lactic (LA) or glycolic (GA) units. v = very strong; s = strong; m = medium; w = weak; sh = shoulder; subscript s = symmetric; subscript as = asymmetric. Values in italics are wavenumbers shifted for PLGA compared with those of PLA and/or PGA. Fig. 3 Low-wavenumber region of the FT-Raman spectra: (a) IdUrd crystals; (d) blank PLGA microspheres; (b) and (c) PLGA microspheres containing different amounts of the drug. To emphasize the comparison, the spectra have been normalized using nC = O bands (see Table 1) of the drug [(a) and (b)] or of the polymer [(c) and (d)]. 40 Analyst, 1999, 124, 37–421769 cm21 were observed (for assignments, see Tables 2 and 3).Therefore, this spectral region was selected for quantitative measurements. Hereafter, the peak area ratio of the 1586–1723/1723–1815 cm21 spectral regions, representing the drug/polymer Raman spectral ratio, Rd/Rp, was considered.Rd/Rp plotted as a function of the respective incorporation ratio (%) is presented in Fig. 5. These experimental points fitted well a second-order polynomic curve (Table 3). In the range of incorporation ratios used for clinical applications (16–23%) the calibration curve was quasi-linear. This could be used to simplify the quantitative calculations for the commonly used incorporation ratios. The largest deviations of the observed Rd/Rp value from the fitted curve gave higher deviations of IdUrd incorporation within ±1% (see the shaded area in Fig. 5). Therefore, the reproducibility of the data was good and the approach allowed the determination of the IdUrd content in ‘non-calibrated’ microspheres from their Raman spectra with a precision of at least 1%. Effects of exposure to g-rays on IdUrd-loaded PLGA microspheres Two sets of microspheres, loaded with 20 and 27% of IdUrd, were exposed to 27 kGy of ionizing radiation. A dose of 25 kGy was considered to be the minimum necessary for sterilization specified by the European Pharmacopoeia.Strong ionizing radiation is known to be destructive for the polymeric matrix. Several reports have commented on g-ray irradiation-induced chain scission and cross-linking11-13 in polylactide and polyglycolide. However, concerning the active agent, the influence of irradiation on the drug structure remained to be defined. This point was essential since microspheres must be radiosterilized before implantation. In the present study, we focused on the irradiation effect on the sole IdUrd structure.As followed from both spectrophotometric and Raman data, the irradiated PLGA microspheres exhibited nearly the same (±1%) IdUrd content as before the irradiation process. The FTRaman bands of the irradiated IdUrd crystals, free or when incorporated within the microspheres, also remained unchanged, both in wavenumers and intensities (Fig. 6). This was also supported by the infrared absorption spectra (data not shown) of the same samples.Therefore, no drug degradation was found. This was a promising observation with respect to clinical application. Conclusion We interpreted each of the FT-Raman spectra of IdUrd-loaded PLGA microspheres in both qualitative and quantitative ways. Fig. 4 (a) Model FT-Raman spectrum of IdUrd crystals; (b) and (c) difference spectra obtained by subtraction of the polymer model spectrum from those of the microspheres loaded with (b) 27% and (c) 4% of IdUrd; (d) model FT-Raman spectrum of the blank PLGA microspheres; (e) and (f) difference spectra obtained by subtraction of the drug model spectrum from those of the microspheres loaded with (e) 4% and (f) 27% of IdUrd.The difference spectra have been normalized for comparison with corresponding model spectra. Table 3 Results of fitting of the Rd/Rp data to a second-order curve: y = a + bx + cx2 Parameter Value Standard error 95% confidence interval A (fixed constant) 0.0 B 0.028141 0.003231 0.0202343– 0.0360485 C 0.001818 0.000141 0.00147298– 0.00216377 Degrees of freedom 6 r2 0.998181 Absolute sum of squares 0.007489 Syx 0.0353 No.of x values 10 No. of y replicates (mean analyzed) 5 Total no. of values 8 No. of missing values 42 Fig. 5 Drug/polymer Raman peak area ratio, Rd/Rp, versus IdUrd incorporation ratio (%). Fig. 6 Comparison of the FT-Raman pattern of IdUrd in PLGA microspheres (a) before and (b) after irradiation with 27 kGy of g-rays.These are difference spectra obtained by subtraction of the model spectra of the blank microspheres from the spectra of the microspheres loaded with 20% of IdUrd. Analyst, 1999, 124, 37–42 41The simultaneous access to both qualitative and quantitative information illustrated the major advantages of the proposed analytical approach. The qualitative information included the detailed analysis of the spectral shape, i.e., band position and relative intensity.This allowed the molecular characterization of the samples and therefore detection of probable structural changes of the drug and/or polymer matrix induced by interaction, degradation, etc. The quantitative analysis included the peak area ratio calculation for the Raman bands. The analytical method presented several important advantages. First, the evaluation of the drug content was nondestructive and the assayed microspheres could be used for further in vitro investigations. Second, this information can be obtained rapidly: about 1 min is necessary to record a Raman spectrum and the computer-assisted mathematical treatment can be almost instantaneous.Compared with more conventional methods, including spectrophotometric measurements, the Raman approach brought an additional facility and economy of time due to elimination of extended sample preparation (source of errors): for instance, no accurate weighing operation was required. Compared with certain reported quantitative Raman measurements of drugs within polymeric implants,14 the advantage of the proposed method was the use of the band peak area instead of intensity and of relative instead of absolute values of the spectral parameters, which eliminated numerous experimental errors and complicated data manipulations.The use of the Raman band of the polymer as an internal intensity standard made the measurements completely independent of the overall intensity of the Raman spectra.Hence control of the laser power, focusing and other instrumental conditions was unnecessary. The methodology therefore had a more general analytical character, i.e., not related to particular experimental conditions (instrumental parameters, polymer content, etc.). Finally, the proposed approach provided reproducible results. The precision of 1% obtained in determining the IdUrd incorporation ratio makes it possible to study the in vitro drug release kinetics by assaying, by Raman spectroscopy, the remaining drug amounts in the microspheres as a function of time. References 1 S. Li and M. Vert, in Degradable Polymers, ed. G. Scott and D. Gilead, Chapman and Hall, London, 1995, ch. 4, pp. 43–87. 2 J. P. Benoit, H. Marchais, H. Rolland and V. Vande Velde, in Microencapsulation. Methods and Applications, ed. S. Benita, Marcel Dekker, New York, 1996, ch. 3, pp. 35-71. 3 B. Djordjevic and W. Szylbalski, J. Exp. Med., 1960, 112, 509. 4 P. Menei, J. P. Benoit, M. Boisdron-Celle, D. Fournier, P. Mercier and G. Guy, Neurosurgery, 1994, 34, 1058. 5 A. G. Hausberger, R. A. Kenley and P. Deluca, Pharm. Res., 1995, 12, 851. 6 G. Spenlehauer, M. Vert, J. P. Benoit, F. Chabot and M. Veillard, J. Controlled Release, 1988, 7, 217. 7 G. Kiester, G. Cassanas, M. Vert, B. Pauvert and A. T�erol, J. Raman Spectrosc., 1995, 26, 307. 8 G. Kiester, G. Cassanas and M. Vert, Spectrochim. Acta, Part A, 1997, 53, 1399. 9 G. Cassanas, M. Morssli, F. Fabr`egue and L. Bardet, J. Raman Spectrosc., 1991, 22, 11. 10 G. Kiester, G. Cassanas, F. Fabr`egue and L. Bardet, Eur. Polym. J., 1992, 28, 1273. 11 K. M�ader, A. Domb and H. M. Swartz, Appl. Radiat. Isot., 1996, 47, 1669. 12 A. G. Hausberger, R. A. Kenley and P. P. DeLuca, Pharm. Res., 1995, 12, 851. 13 M. C. Gupta and V. G. Deshmukh, Polymer, 1983, 24, 827. 14 P. Milne, S. Gautier, J. M. Parel and V. Jallet, Proc. SPIE, 1997, 2971, 137. Paper 8/06678G 42 Analyst, 1999, 124, 37&ndas

ISSN:0003-2654    

DOI:10.1039/a806678g

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Comparison of four fluorescence Edman reagents with benzofurazan structure for the detection of thiazolinone amino acid derivatives Akira Toriba, Tomofumi Santa, Takayuki Iida and Kazuhiro Imai* Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan Received 28th September 1998, Accepted 12th November 1998 Two newly synthesized fluorescence Edman reagents with the benzofurazan structure, 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (PSBD-NCS) and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS), were compared with 7-aminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (ABD-NCS) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) for peptide and protein sequence analysis by the generation of fluorescent 2,1,3-benzoxadiazolylthiazolinone (TZ)-amino acids.The effects of the substituent group at the para position to the isothiocyanate moiety of these reagents on the rate of the cyclization/cleavage reaction, the repetitive yield and the fluorescence quantum yield and stability of TZ amino acids were investigated.MSBD-TZ-amino acids were most sensitively detected and the detection limit for MSBD-TZ-Pro was 7 fmol (S/N = 3). ABD-NCS afforded the highest repetitive yield in the sequencing analysis. Fewer interfering peaks were observed in the chromatogram with DBD-NCS. Introduction Amino acid sequence analysis of peptides and proteins using phenyl isothiocyanate (PITC) was first reported by Edman;1 Nterminal amino acids are derivatized with aryl isothiocyanate, cleaved and cyclized to thiazolinone (TZ)-amino acids with anhydrous acid, and then the TZ-amino acids are recyclized to aryl thiohydantoin (TH)-amino acids and identified by HPLC.Since then, various techniques have been introduced to improve the detection sensitivity.2–4 Although the introduction of the gas-phase sequencers substantially reduced the amounts of sample peptides and proteins required, the automated sequencers are still unable to analyze peptides and proteins at the sub-picomole level. On the other hand, various fluorescence Edman reagents have been reported to enhance the detection sensitivity of the final product, TH-amino acid.These include fluorescein isothiocyanate (FITC),5, 6 4-{[(5-(dimethylamino)-1-naphthyl)sulfonyl]- amino}phenyl isothiocyanate (dansylamino-PITC)7 and 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)- 2,1,3-benzoxadiazole (DBD-PyNCS).8 However, these reagents are not yet routinely utilized as compared with PITC, since (1) the bulkiness of the fluorophore lowers the derivatization and cleavage reaction yield, (2) the hydrophobic fluorophore interferes with the simple removal of excess reagents without loss of the sample peptides, (3) only about 50% of the generated TZ-amino acids are converted to TH-amino acids and (4) the strong fluorescence of the reagents themselves sometimes interferes with the detection of the generated TH-amino acids.To overcome these disadvantages, we have recently reported a new Edman procedure using the fluorescence Edman reagent, 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS),9–11 in which DBD-TZ-amino acids generated by the cleavage/cyclization reaction were detected fluorimetrically and the step of the conversion reaction to THamino acid was eliminated to simplify the sequencing process.Furthermore, DBD-NCS itself did not fluoresce and thus the interfering peak derived from the excess reagents did not interfere with the detection of the TZ-amino acids. In a previous paper, we reported that the fluorescence of the generated 2,1,3-benzoxadiazolyl-TZ-amino acids was more intense when a stronger electron-withdrawing group is present at the para position to the isothiocyanate moiety.10 DBD-TZamino acid, which had the strongest electron-withdrawing para substituent at that time, gave the strongest fluorescence intensity. Therefore, in this study, two new benzofurazan fluorescence Edman reagents with stronger electron-withdrawing substituents, which have larger Hammett substituent constants (sp)12 than that of DBD-NCS, namely 7-phenylsulfonyl- 4-(2,1,3-benzoxadiazolyl) isothiocyanate (PSBDNCS) and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS), were synthesized and applied to sequencing analysis.Furthermore, we examined the effects of the para-substituent group on the fluorescence quantum yield and stability of TZ-amino acids, the rate of the cyclization/ cleavage reaction and the repetitive yield of the sequencing analysis utilizing these fluorescence Edman reagents, PSBDNCS, MSBD-NCS, DBD-NCS and 7-aminosulfonyl- 4-(2,1,3-benzoxadiazolyl) isothiocyanate (ABD-NCS)10,13 (Table 1). Experimental Materials The following materials were employed: b-casomorphin-7 (bovine), Gly–Leu and Leu–Gly (Peptide Institute, Osaka, Japan); Pro–His–Leu (Backem, Bubenford, Switzerland); Tyr– Val, Pro–Leu, Phe–Gly and leucine (Leu) (Sigma, St.Louis, MO, USA); sequencer-grade trifluoroacetic acid (TFA) (Wako, Osaka, Japan); sequencer-grade pyridine (Tokyo Chemical Industry, Tokyo, Japan); and HPLC-grade acetonitrile (Kanto Chemical, Tokyo, Japan). Water was purified using a Milli-Q Analyst, 1999, 124, 43–48 43system (Millipore, Bedford, MA, USA).All other reagents were of analytical-reagent grade and used without further purification. Apparatus The following apparatus was used for the identification of synthesized compounds. Melting-points were measured on a micro melting-point apparatus (Yanagimoto, Tokyo, Japan) and are uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra were obtained on a GSX-400 spectrometer (JEOL, Tokyo, Japan) with tetramethylsilane as the internal standard (abbreviations used: s, singlet; d, doublet; t, triplet; m, multiplet).Mass spectra were measured on an M-1200H mass spectrometer (Hitachi, Tokyo, Japan) with an atmospheric pressure chemical ionization system (APCI-MS). HPLC was carried out using an L-7100 intelligent pump an L-4000H UV detector, an L-7480 fluorescence detector and a D-7500 integrator (all from Hitachi). 2,1,3-Benzoxadiazolyl isothiocyanates PSBD-NCS and MSBD-NCS were synthesized as described below and ABD-NCS and DBD-NCS were synthesized as described previously.13 4-Amino-7-phenylsulfonyl-2,1,3-benzoxadiazole 4-Amino-7-phenylthio-2,1,3-benzoxadiazole (180 mg), synthesized as described previously,14 and 400 mg of m-chloroperoxybenzoic acid were dissolved in 5 ml of dichloromethane and the mixture was stirred at room temperature for 5 h.The reaction mixture was poured into 100 ml of 1 mol dm23 Na2CO3 solution and extracted twice with 100 ml of dichloromethane.The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was chromatographed on silica gel (dichloromethane) to afford 155 mg of the corresponding amine as a yellow powder, mp 216–217 °C. 1H NMR (CDCl3): d 8.16 (2H, t), 8.11 (1H, d, J = 8.0 Hz), 7.49–7.56 (3H, m), 6.40 (1H, d, J = 8.0 Hz), 5.28 (2H, s, br). APCI-MS: m/z 276 ([M + H]+). 7-Phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (PSBD-NCS) 4-Amino-7-phenylsulfonyl-2,1,3-benzoxadiazole (150 mg) was dissolved in 15 ml of acetonitrile and 1.5 ml of 25% v/v thiophosgene in benzene was added slowly. The mixture was refluxed for 5 h.The reaction mixture was evaporated to dryness in vacuo and the residue was chromatographed on silica gel (ethyl acetate–hexane) to afford 50 mg of the corresponding isothiocyanate as a yellow powder, mp 149–150 °C. 1H NMR (CDCl3): d 8.19–8.25 (3H, m), 7.56–7.64 (3H, m), 7.20 (1H, d, J = 8.0 Hz). APCI-MS: m/z 317 (M2). 4-Amino-7-methylthio-2,1,3-benzoxadiazole 4-Methylthio-7-nitro-2,1,3-benzoxadiazole (180 mg), synthesized as described previously,14 was dissolved in 5 ml of dichloromethane.After addition of 1 ml of concentrated hydrochloric acid, methanol was added to the mixture until it became homogeneous. After addition of 120 mg of iron powder, the mixture was stirred vigorously for 30 min. After the iron had been removed by filtration, the reaction mixture was poured into 100 ml of a 1 mol dm23 NaOH solution and extracted twice with 100 ml of dichloromethane.The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was chromatographed on silica gel (dichloromethane–hexane) to afford of 50 mg of the corresponding amine as a red powder, mp 104–105 °C. 1H NMR (CDCl3): d 7.15 (1H, d, J = 8.0 Hz), 6.32 (1H, d, J = 8.0 Hz), 4.54 (2H, s, br), 2.57 (3H, s). APCIMS: m/z 180 ([M 2 H]2) 4-Amino-7-methylsulfonyl-2,1,3-benzoxadiazole 4-Amino-7-methylthio-2,1,3-benzoxadiazole (46 mg) and 150 mg of m-chloroperoxybenzoic acid were dissolved in 5 ml of dichloromethane and the mixture was stirred at room temperature for 5 h.The reaction mixture was poured into 100 ml of 1 mol dm23 Na2CO3 solution and extracted twice with 100 ml of dichloromethane. The organic layer was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was chromatographed on silica gel (dichloromethane) to afford of 39 mg of the corresponding amine as a deep orange powder, mp 238–239 °C. 1H NMR [CDCl3–(CD3)2SO (9 + 1, v/v)]: d 7.95 (1H, d, J = 8.0 Hz), 6.42 (1H, d, J = 8.0 Hz), 4.76 (2H, s, br), 3.26 (3H, s). APCI-MS: m/z 212 ([M 2 H]2). 7-Methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MSBD-NCS) 4-Amino-7-methylsulfonyl-2,1,3-benzoxadiazole (35 mg) was dissolved in 15 ml of acetonitrile and 1.5 ml of 25% v/v thiophosgene in benzene was added slowly. The mixture was refluxed for 5 h. The reaction mixture was evaporated to dryness in vacuo and the residue was chromatographed on silica gel (ethyl acetate–hexane) to afford 10 mg of the corresponding isothiocyanate as a yellow powder, mp 148–149 °C. 1H NMR (CDCl3): d 8.14 (1H, d, J = 8.0 Hz), 7.24 (1H, d, J = 8.0 Hz), 3.83 (3H, s). APCI-MS: m/z 255 (M2). HPLC conditions 2,1,3-Benzoxadiazolyl-TZ-amino acids were separated using two columns in tandem, i.e., an ODS column (TSK gel ODS- 80Ts, 250 3 4.6 mm id, 5 mm; Tosoh, Tokyo, Japan) and a phenyl function bonded porous silica gel column (YMC-Pack Ph, 250 3 4.6 mm id, 5 mm; YMC, Kyoto, Japan) for sequencing analysis.Isocratic elution of TZ-amino acids was employed with acetonitrile–water (50 + 50, v/v for ABD-TZamino acids, 55 + 45 v/v for MSBD-TZ-amino acids; 60 + 40 v/v for DBD-TZ-amino acids and 65 + 35 v/v for PSBD-TZamino acids) containing 10 mmol dm23 formic acid at a flow Table 1 Structures of para-substituted 2,1,3-benzoxadiazolyl isothiocyanates Reagenta R Hammett constant (sp) ABD-NCS SO2NH2 0.60 DBD-NCS SO2N(CH3)2 0.65 PSBD-NCS SO2C6H5 0.68 MSBD-NCS SO2CH3 0.72 a ABD-NCS = 7-aminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate; DBD-NCS = 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate; PSBD-NCS = 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate; MSBD-NCS = 7-methylsulfonyl-4- (2,1,3-benzoxadiazolyl) isothiocyanate. 44 Analyst, 1999, 124, 43–48rate of 0.5 ml min21. Fluorescence detection was performed at the maximum excitation and emission wavelength for the respective TZ-Leu (see Table 2).TZ-amino acids and 2,1,3-benzoxadiazolylthiocarbamoylated (TC)-amino acids were also determined under the following conditions (RP-HPLC): eluents A and B containing 10 mmol dm23 formic acid were 30 + 70 v/v and 70 + 30 v/v acetonitrile–water, respectively, and the analytical column was ODS-80Ts using gradient elution from 0 to 55 min (eluent B composition, 0–100 %) at a flow rate of 1.0 ml min21.Preparation of standard 2,1,3-benzoxadiazolyl-TZ-amino acids Dipeptides were dissolved in pyridine–water (1 + 1 v/v) (0.2–2.0 mmol dm23). A 100 nmol amount of a benzoxadiazolyl isothiocyanate (ABD-NCS, DBD-NCS, PSBD-NCS or MSBD-NCS) was dissolved in 20 ml of the solution and the mixture was vortex mixed and heated at 50 °C for 20 min. After the coupling reaction, the excess reagent and by-products were removed by washing three times with 200 ml of heptane– dichloromethane (4 + 1 v/v).The aqueous phase was evaporated to dryness using a centrifugal evaporator (SPE-200; Shimadzu, Kyoto, Japan) at 50 °C for 15 min and 40 ml of trifluoroacetic acid (TFA) were added to the residue. The mixture was heated at 50 °C for 10 min and dried under a stream of nitrogen. After the cleavage/cyclization reaction, 20 ml of water were added to the residue and the solution was extracted twice with 100 ml of ethyl acetate. The combined organic phase was dried under a stream of nitrogen. The resulting residue was dissolved in the HPLC eluent and immediately analyzed. Characteristics of 2,1,3-benzoxadiazolyl-TZ-amino acids TZ-Leu prepared as described above was collected by RPHPLC and dried in a centrifugal evaporator at 50 °C.The residue was dissolved in acetonitrile and the solution was subjected to LC-MS. The fluorescence intensity and UV absorption of the solution were measured with a spectrofluorimeter (F-4010; Hitachi) and a spectrophotometer (Ubest 50; JASCO, Tokyo, Japan), respectively.10 Further, TZ-Leu fractions were subjected to conversion reaction and hydrolysis to obtain 2,1,3-benzoxadiazolyl-TH-Leu and -TC-Leu, respectively.The molecular masses and UV spectra of the THand TC-Leu fractions were measured.9 Stability of 2,1,3-benzoxadiazolyl-TZ-amino acids The residue of TZ-Leu prepared as described above was dissolved in acetonitrile–water (1 + 1 v/v) and kept at room temperature. Aliquots of the solution were subjected to HPLC at appropriate time intervals.The observed rate constant (kobs) for the disappearance of TZ-Leu was calculated as described previously.9 Coupling reaction with benzoxadiazolyl isothiocyanate Leu was dissolved in pyridine–water (1 + 1 v/v) (0.1 mmol dm23). A 100 nmol amount of a benzoxadiazolyl isothiocyanate (ABD-NCS, DBD-NCS, PSBD-NCS or MSBDNCS) was dissolved in 20 ml of the solution and the mixture was vortex mixed and heated at 50 °C, samples being withdrawn after appropriate time intervals.After the coupling reaction, 50 ml of 1 mol dm23 hydrochloric acid solution were added to the mixture and the solution was extracted twice with 100 ml of ethyl acetate. The combined organic phase was dried under a stream of nitrogen and dissolved in the HPLC eluent. The generated TC-Leu was subjected to RP-HPLC and the UV detection was carried out at 385 nm. Cyclization/cleavage reaction by TFA Pro–His–Leu was dissolved in pyridine–water (1 + 1 v/v) (0.1 mmol dm23).The 2,1,3-benzoxadiazolyl-TC derivatives of Pro–His–Leu were prepared by reaction with ABD-NCS, DBDNCS, PSBD-NCS and MSBD-NCS in a manner similar to that described above. TFA was added to the residue and the mixture was heated at 50 °C, samples being withdrawn after appropriate time intervals. The resulting TZ-Pro was subjected to RPHPLC. Liquid phase sequencing of b-casomorphin-7 with 2,1,3-benzoxadiazolyl isothiocyanates The peptide was dissolved in pyridine–water (1 + 1 v/v) (0.1 mmol dm23).A 100 nmol amount of a benzoxadiazolyl isothiocyanate (ABD-NCS, DBD-NCS, PSBD-NCS or MSBDNCS) was dissolved in 20 ml of the solution and the mixture was vortex mixed and heated at 50 °C for 20 min. After the coupling reaction, the excess reagent and by-products were removed by washing three times with 200 ml of heptane–dichloromethane. As the sequencing cycle proceeded, the dichloromethane concentration was decreased, i.e., those in cycles 1, 2, 3, 4, 5 and 6 were 20, 10, 5, 0, 0 and 0% v/v, respectively.The aqueous phase was evaporated to dryness using a centrifugal evaporator at 50 °C for 15 min and 40 ml of TFA were added to the residue. The mixture was heated at 50 °C for 10 min and dried under a stream of nitrogen. After the cleavage/cyclization reaction, 20 ml of water were added to the residue and the solution was extracted twice with 100 ml of ethyl acetate. The aqueous phase was dried in a centrifugal evaporator and subjected to the next cycle.The combined organic phase was dried under a stream of nitrogen. The resulting residue was dissolved in 1 ml of the HPLC eluent and 0.5% of the solution was subjected to HPLC. Results and discussion Characteristics of 2,1,3-benzoxadiazolyl-TZ-amino acids The molecular masses, fluorescence spectra and UV spectra of four TZ-Leu fractions isolated are summarized in Table 2. The identification of TZ-Leu was carried out not only by the molecular mass but also by the absence of a UV absorption maximum at 266 nm that can be assigned to the TH ring since TH derivatives have the same molecular mass as TZ derivatives.To compare the fluorescence quantum yields of TZ-Leu derivatives, we used the ratio of fluorescence intensity (at lem) to absorbance (at lmax)10 as shown in Table 2. The ratios for PSBD-TZ-Leu and MSBD-TZ-Leu were about six and ten times higher than that of DBD-TZ-Leu, respectively, and it was Table 2 Characteristics of 2,1,3-benzoxadiazolyl-TZ-Leu Derivative m/za lmax/nm lem/nm Ratiob ABD-TZ-Leu 370 384 522 1880 DBD-TZ-Leu 398 386 524 380 PSBD-TZ-Leu 431 483 525 2100 MSBD-TZ-Leu 369 386 519 3630 a Measured by APCI-MS.b Ratio of fluorescence intensity (lem) to UV absorption (lmax). Analyst, 1999, 124, 43–48 45noted that the fluorescence quantum yield of TZ-amino acid increases with increasing electron-withdrawing activity, as was expected.With all the reagents, TH-Leu and TC-Leu derivatives gave no fluorescence. Stability of 2,1,3-benzoxadiazolyl-TZ-amino acids In an aprotic solvent such as acetonitrile, DBD-TZ-amino acids were extremely stable in comparison with a protic solvent such as water or methanol.9 Hence TZ-amino acids were stored in acetonitrile. Next, the stability of TZ-amino acids in the HPLC eluent, acetonitrile–water (1 + 1 v/v), at room temperature was examined. Fig. 1 shows the relationship between sp of the parasubstituent group of the isothiocyanate moiety and the observed rate constant (kobs) for the disappearance of TZ-Leu.A linear relationship between sp and kobs for the disappearance of TZLeu was obtained. An electron-withdrawing group is considered to promote the hydrolysis of TZ ring to the corresponding TCamino acids. However, even the MSBD-TZ-amino acid containing the largest sp para-substituent group did not cause any problems in the sequencing analysis, since the t1/2 of MSBDTZ- Leu was 3.7 h, which was sufficient for HPLC detection.Coupling reaction with 2,1,3-benzoxadiazolyl isothiocyanate Four benzoxadiazolyl isothiocyanates were reacted with Leu and the reaction mixtures were subjected to RP-HPLC at intervals to observe the effects of a substituent group on the coupling reaction. The reaction was nearly completed in about 20 min and no difference in the reactivity of the benzoxadiazolyl isothiocyanates examined was observed under these conditions (Fig. 2). Cyclization/cleavage reaction by TFA We have reported that the use of an aprotic acid, boron trifluoride (BF3), facilitated the cyclization/cleavage reaction and provided higher yields of TZ-amino acids than TFA without racemization of the chiral center of amino acid.11,16–18 We used TFA since it has usually been used as the cyclization/cleavage reagent and the racemization of TZ-amino acids was not examined in this study. In order to observe the effect of the substituent group on the reaction, the time course of the yield of the TZ-Pro generated from TC-Pro–His–Leu was examined, since the N-terminal Pro–His linkage of a peptide is known to be particularly resistant to cleavage.2 The production of MSBD-TZ-Pro and PSBD-TZ-Pro required a longer reaction time, since MSBD and PSBD bear a strong electron-withdrawing substituent group and the electron density at the sulfur atom seems to be small15 (Fig. 3). The yield of TZ-Pro decreased after attainment of the maximum yield. This was presumably due to the conversion to TH-Pro by trace amounts of contaminant water and/or decomposition of the generated TZ-Pro in TFA.The yield of other TZ-amino acids decreased significantly with the reaction time, completely cleaving the respective Pro–His linkage. Therefore, a reaction time of 10 min, which did not cause carryover and a decrease in the repetitive yield in sequencing analysis, was selected in subsequent work.Detection of 2,1,3-benzoxadiazolyl-TZ-amino acids In general, the fluorescence intensity of compounds containing a benzofurazan moiety increases with increasing the content of organic solvent in the HPLC eluent. Furthermore, TZ-amino acids were extremely stable in an aprotic solvent such as acetonitrile. Therefore, two analytical columns, i.e., an ODS column and a phenyl bonded silica gel column, were used in tandem as in previous work.9 PSBD-TZ-amino acids were the most hydrophobic and were strongly retained on the columns with the eluent of the highest acetonitrile content.It is worth Fig. 1 Relationship between the Hammett para-substituent constants (sp) of benzoxadiazolyl isothiocyanates and the observed rate constants (kobs) for the disappearance of TZ-Leu. The sp values of the para-substituent groups are taken from ref. 12. Fig. 2 Time course for the coupling reaction of Leu (2 nmol) with benzoxadiazolyl isothiocyanates at 50 °C.The yield of the coupling reaction at 50 °C after 30 min was taken as 100%. (5) ABD-NCS; (8) DBD-NCS; (-) PSBD-NCS and (2) MSBD-NCS. Fig. 3 Time course for the cyclization/cleavage reaction of 2,1,3-benzoxadiazolyl- TC-Pro–His–Leu at 50 °C. (5) ABD-NCS; (8) DBD-NCS; (-) PSBD-NCS; and (2) MSBD-NCS. The 2,1,3-benzoxadiazolyl-TC derivatives of Pro–His–Leu were prepared by the reaction of Pro–His–Leu (2 nmol) with benzoxadiazolyl isothiocyanates. 46 Analyst, 1999, 124, 43–48noting that TZ-Pro, particularly MSBD-TZ-Pro, was most sensitively detected among the TZ-amino acids.The detection limits for MSBD-TZ-Pro, PSBD-TZ-Pro, DBD-TZ-Pro and ABD-TZ-Pro were 7, 10, 15 and 30 fmol (S/N = 3), respectively, and those of the other TZ-amino acids were from 0.1 pmol to the sub-picomole level. The suggestion that TZamino acids containing an electron-withdrawing group would be more sensitively detected was confirmed. Since the most sensitive reagents previously reported were FITC (10 fmol)6 and DBD-PyNCS (20 fmol),8 it appears that the sensitivity of the TZ derivatives is slightly better than or comparable to them.Liquid phase sequencing of b-casomorphin-7 with 2,1,3-benzoxadiazolyl isothiocyanate b-Casomorphin-7 was adopted as a model peptide in this study. b-Casomorphin-7 is a short peptide composed of hydrophobic amino acid residues and would be easily lost in the washing step, hence being suitable for the comparison of the repetitive yields of the sequencing analysis with these reagents.Fig. 4 shows the results of the sequence analysis using ABD-NCS, DBD-NCS and MSBD-NCS. From the chromatograms obtained in the respective cycles, the peptide sequence was identified as Tyr–Pro–Phe–Pro–Gly–Pro, whereas the C-terminal amino acid, Ile, was not detected, presumably owing to the production of TC- or TH-Ile. As shown in Table 3, ABD-NCS gave the highest repetitive yield, which was calculated by comparison of the fluorescence intensities of Pro2 and Pro6, among these three reagents.These results suggest that the Fig. 4 Chromatograms obtained from sequencing analysis of b-casomorphin-7 (2 nmol) with (A) ABD-NCS, (B) DBD-NCS and (C) MSBD-NCS. Table 3 Analysis of peptides by the manual sequencing method with 2,1,3-benzoxadiazolyl isothiocyanates Reagent Repetitive yield (%)a ABD-NCS 83 DBD-NCS 80 MSBD-NCS 76 a Mean values from 2 or 3 experiments. Analyst, 1999, 124, 43–48 47repetitive yields might be lowered with increase in the electronwithdrawing activity of the substituent group, although the differences in the repetitive yields were not large. When the Pro residue was determined (cycles 2, 4, and 6) in sequencing with MSBD-NCS [Fig. 4(C)], an unknown peak (tR = 24.3 min) derived from MSBD-TZ-Pro increased during the cyclization/cleavage reaction. This suggests that MSBD-TZPro was significantly unstable in TFA. MSBD-NCS, which gave the most fluorescent TZ-amino acids but gave a slightly low repetitive yield, is considered to be applicable to the identification of the trace amounts of a short peptide.The Gly (cycle 5) and Pro (cycle 6) in the sequence analysis using PSBD-NCS afforded a small amount of the corresponding TZamino acids, and the repetitive yield calculated from the fluorescence intensities of Pro2 and Pro4 was 40% (data not shown). It is considered that the loss of the peptide in the washing step was significant owing to the hydrophobicity of the PSBD-TC-peptide.There seem to be a few disadvantages for DBD-NCS with regard to the stability and sensitivity of TZamino acids, the rate of the cyclization/cleavage reaction and the repetitive yield. Furthermore, fewer interfering peaks were observed in the chromatograms compared with other reagents. In conclusion, the para-substituent group of benzoxadiazolyl isothiocyanate was demonstrated to affect the fluorescence quantum yield, stability and retention in the reversed-phase HPLC of TZ-amino acids, the rate of the cyclization/cleavage reaction and the hydrophobicity of the reagent.DBD-NCS was the most appropriate reagent, and the development of a double coupling method with PITC and an automated sequencing method is in progress. We thank Drs H. Homma and T. Fukushima, University of Tokyo, for the valuable suggestions and discussions. We gratefully acknowledge Dr C. K. Lim, University of Leicester, for revision of manuscript and useful comments. We also thank YMC and Tosoh for the gift of a YMC-Pack Ph column and a TSK gel ODS-80Ts column, respectively.This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (Grant 10557212). References 1 P. Edman, Acta Chem. Scand., 1950, 4, 283. 2 G. E. Tarr, Methods Enzymol., 1977, 47, 335. 3 J. M. Bailey, J. Chromatogr., 1995, 705, 47. 4 G. A. Grant, M. W. Crankshaw and J. Gorka, Methods Enzymol., 1997, 289, 395. 5 K. Muramoto, H. Kamiya and H. Kawauchi, Anal. Biochem., 1984, 141, 446. 6 K. Muramoto, K. Nokihara, A. Ueda and H. Kamiya, Biosci. Biotechnol. Biochem., 1994, 58, 300. 7 H. Hirano and B. Wittmann-Liebold, Biol. Chem. Hoppe-Seyler, 1986, 367, 1259. 8 T. Toyo’oka, T. Suzuki, T. Watanabe and Y. M. Liu, Anal. Sci., 1996, 12, 779. 9 H. Matsunaga, T. Santa, K. Hagiwara, H. Homma, K. Imai, S. Uzu, K. Nakashima and S. Akiyama, Anal. Chem., 1995, 67, 4276. 10 H. Matunaga, T. Santa, T. Iida, T. Fukushima, H. Homma and K. Imai, Analyst, 1997, 122, 931. 11 H. Matsunaga, T. Santa, T. Iida, T. Fukushima, H. Homma and K. Imai, Anal. Chem., 1996, 68, 2850. 12 C. Hansch, A. Leo and R. W. Taft, Chem. Rev., 1991, 91, 165. 13 K. Imai. S. Uzu, K. Nakashima and S. Akiyama, Biomed. Chromatogr., 1993, 7, 56. 14 S. Uchiyama, T. Santa, T. Fukushima, H. Homma and K. Imai, J. Chem. Soc., Perkin Trans. 2, 1998, 2165. 15 G. E. Tarr, in Methods of Protein Microcharacterization, ed. J. E. Shively, Humana Press, Clifton, NJ, 1986, p. 155. 16 T. Iida, H. Matsunaga, T. Fukushima, T. Santa, H. Homma and K. Imai, Anal. Chem., 1997, 69, 4463. 17 T. Iida, H. Matsunaga, T. Santa, T. Fukushima, H. Homma and K. Imai, J. Chromatogr. A, 1998, 813, 267. 18 T. Iida, T. Santa, A. Toriba and K. Imai, Analyst, 1998, in the press. Paper 8/07520D 48 Analyst, 1999, 124, 43–48

ISSN:0003-2654    

DOI:10.1039/a807520d

出版商:RSC    

年代:1999

数据来源: RSC

摘要:

Application of partial least squares multivariate calibration for the determination of mixtures of carbaryl and thiabendazole in waters by transmitted solid phase spectrophosphorimetry L. F. Capitán-Vallvey, Mahmoud Kalil A. Deheidel, I. de Orbe and R. Avidad* Department of Analytical Chemistry, University of Granada, Granada 18071, Spain. E-mail: lcapitan@goliat.ugr.es Received 7th October 1998, Accepted 5th November 1998 Mixtures of the pesticides carbaryl and thiabendazole were studied using solid phase spectrophosphorimetry at room temperature with Whatman No. 4 filter-paper as solid support and Pb(ii) as enhancer of the phosphorescence. The transmitted phosphorescence was used as an analytical signal and its measurement was performed by placing the paper containing the sample between two quartz plates. In this way, the use of a dry inert gas flow to avoid the quenching of atmospheric agents (oxygen and moisture) is not needed. The influence of several experimental parameters (e.g., pH, nature of the enhancers and solid supports, gate and delay times) on the phosphorescent emission of both chemicals was also studied.As a result, a new method for the simultaneous determination of these pesticides is proposed, using partial least squares (PLS-1) multivariate calibration. The statistical parameters of the optimised matrix, are included as a table. The applicable concentration ranges were from 0.16 to 1.20 mg l21 for thiabendazole and from 0.50 to 4.00 mg l21 for carbaryl.The method was applied to the determination of both pesticides in water, with recoveries between 93.4 and 105.6% for carbaryl and between 90.0 and 105.0% for thiabendazole. HPLC was used as a reference method. Introduction One of the current requirements in analytical chemistry is to detect and quantify smaller amounts of chemical contaminants in samples of diverse natures, with low cost and time consumption. Hence selective, sensitive, rapid and inexpensive methods of analysis, using readily available, conventional instrumentation, must be developed.Room temperature phosphorimetry is a technique that allows the development of analytical methods that combine these qualities because, together with the natural sensitivity and selectivity of luminescent methods, it can reduce costs through the use of inexpensive materials and reagents, and provides short analysis times. The methodology employed in room temperature phosphorescence (RTP) is based on the measurement of the phosphorescence intensity in a solid phase,1–5 or in solution if the analyte forms an inclusion complex (cyclodextrins),6,7 or micellar-stabilised media with non-polar molecules containing a polar group.8–10 Obviously, the use of paper as a solid support diminishes the cost of the analysis, but it has the disadvantage of showing a background signal produced by the substrate, which can limit its application in the detection of trace analytes.There are several ways to diminish the background signal of the paper used as a solid support, the best known being chemical pre-treatment, irradiation with ultraviolet light or extraction with water in a Soxhlet apparatus.11–15 With these treatments of the paper used as support, the background signal can be 6% of its initial value, but the time of analysis increases and the precision of the measurements can decrease. Additionally, a factor influencing strongly the phosphorescence of the sample and decreasing the intensity of the emitted signal, both in solution and in the solid phase, is the quenching effect produced by environmental moisture and oxygen. This effect has been avoided in the past by the addition of sulfite, in the case of measurements in solution,16 or by using a flow of a dry, inert gas (usually N2 or He) in the compartment of the spectrometer when the measurement is performed in the solid phase.17 However, it is clear that the use of a flow of inert gas and/or pre-treatment of the solid support to avoid background signals increases the time and cost of the analysis.With the aim of improving the experimental procedures, using paper as a solid support, we studied the phosphorescent behaviour of phosphors, modifying the experimental procedure that is usually used in phosphorimetry. This modification consists of the measurement of the diffuse transmitted phosphorescence emitted from the analyte, placing the paper containing the sample between two quartz plates.In this way, a flow of inert gas and pre-treatment of the paper used as a solid support are not needed and the analysis is consequently faster. A second objective was to test the suitability of multivariate calibration methods for the determination of mixtures of analytes in the solid phase whose phosphorescence spectra overlap substantially. The phosphors studied here were two pesticides widely used in agriculture, carbaryl (CBL) and thiabendazole (TBZ).The former has been described as a phosphorescent chemical,1,2,9,15,18 but we did not find any references to the phosphorescence emission of TBZ. These chemicals show similar spectra, and to resolve the considerable spectral overlap we used the partial least squares (PLS) multivariate calibration method, developed and applied by Wold et al.19–21 The main advantages of this statistical method are the speed of the handling of results and the determination of the analytes without previous separation. PLS methods have been used in several studies for the determination of different analytes (e.g., pesticides, sulfonamides and flavour enhancers) 22–29 in solution, although the main application of the PLS methods has been for calibration in near-infrared and nuclear magnetic resonance spectroscopy in the solid phase.30 In this work, in addition to the study mentioned above of the phosphorescent behaviour of CBL and TBZ, we applied the PLS method in solid phase spectrophosphorimetry to test its suitability for this methodology.As a result of this study, a new method for the determination of CBL and TBZ in mixtures is proposed. The method combines the advantages of solid phase Analyst, 1999, 124, 49–53 49spectrophosphorimetry (selectivity and sensitivity) and of multivariate calibration (speed and no previous separation of components). It is also inexpensive, because only common reagents and materials and conventional instrumentation are needed.Experimental Apparatus The measurements of phosphorescence were performed with a Perkin-Elmer (Norwalk, CT, USA) LS-50 luminescence spectrometer, working in the phosphorescence mode, and equipped with a Hammamatsu R289 photomultiplier, two Monk– Gillieson F/3 monochromators and a xenon discharge lamp (with a power equivalent to 20 kW during 8 ms), and interfaced to a PS/230-386 microcomputer.The spectrometer was checked daily using a P1 standard (12.5 3 12.5 3 45 mm) containing europium(iii) thenoyltrifluoroacetonate dissolved in a transparent matrix of poly(methyl methacrylate), supplied by Perkin- Elmer. Other apparatus and laboratory materials were a Crison (Barcelona, Spain) digital pH meter with a combined glass– saturated calomel electrode, a micropipette Biohit Proline (Helsinki, Finaland) microtip 10 ml, a Selecta (Barcelona, Spain) ultrasonic bath, a Braun Silencio 1600 hair-dryer (maximum power 2000 W) and a 250 W infrared heat lamp.Two rectangular (45 3 12 3 1 mm) Hellma Suprasil (M�ullheim, Baden, Germany) quartz plates were also used to perform the measurements of the transmitted RTP. Software programs used for the treatment of the spectral data were Grams/286 Software Package Version 1.0, Add Application PLS Plus Version 2.1 (Galactic Industries, Salem, MA, USA) and Data Leader Software Package (Beckman, Fullerton, CA, USA; 1987). Reagents Stock standard solutions of TBZ (Dr.Ehrenstorfer, Ausburg, Germany) and CBL (Riedel-de Haën, Hannover, Germany) were prepared in ethanol at a concentration of 100.0 mg l21. Working standard solutions were prepared by appropriate dilution with reverse osmosis quality water (obtained using a Milli-Ro 12 plus Milli-Q Station from Millipore, Bedford, MA, USA). Stock standard solutions of saturated Pb(ii), 1 m Tl(, 1 m Ag(i) and 1 m KI were prepared from lead acetate, AgNO3, TlNO3 and KI solid salts (Merck, Darmstadt, Germany), respectively.A buffer solution of the required pH (4.0) was prepared from 1 m sodium acetate and 1 m acetic acid solutions (Merck). All reagents were of analytical reagent grade unless stated otherwise. Phosphorescence measurements Diffuse transmitted phosphorescence spectra were recorded between 400 and 570 nm, the plane of the sample forming two angles of 45° with the excitation and emission beams (Fig. 1).31 These spectra were obtained with a delay time td = 0.1 ms and a gate time tg = 12 ms. The excitation and emission slits were 2.5 and 5.0 nm, respectively, and the scan speed was 240 nm min21. The solid support containing the sample was placed in the holder of the spectrometer shown in Fig. 1. Procedure On a 50 312 mm strip of Whatman (Maidstone, Kent, UK) No. 4 filter-paper soaked in acetic acid–sodium acetate buffer solution (pH 4.0) and then dried, 3 ml of saturated Pb(CH3COO)2 solution and 3 ml of sample solution containing between 0.5 and 3.6 ng of TBZ and between 1.5 and 12.0 ng of CBL were spotted with the aid of a micropipette.The paper containing the reagents was dried for 3 min by means of a hot air stream from the hair-dryer placed 7 cm above the sample at 600 W power. Next, the solid support with the sample was placed between the quartz plates and the assembly was inserted in the sample compartment of the instrument. The phosphorescence spectra were obtained as described in the previous section.Results and discussion Spectral characteristics CBL and TBZ in the solid phase gave the emission spectra shown in Fig. 2. It can be seen that the spectra of both analytes Fig. 1 Placing of the sample and quartz plates in the holder of the spectrometer. Fig. 2 Phosphorescence spectra of TBZ and CBL fixed in Whatman No. 4 filter-paper. (a) Blank; (b) CBL at 3.8 mg ml21; (c) TBZ at 1.0 mg ml21; (d) CBL (2.8 mg ml21) and TBZ (0.8 mg ml21) mixed. 50 Analyst, 1999, 124, 49–53overlap greatly and they are clearly different from the blank spectrum. The peak wavelength in the emission spectrum of the TBZ is 472 nm (lex = 303 nm) and for CBL there are two peaks in the emission spectrum at 486 and 520 nm (lex = 282 nm). This overlap hinders the simultaneous determination of the two chemicals by conventional spectrophosphorimetric methods, but the problem can be resolved by using multivariate calibration after the optimisation of the experimental parameters that influence the phosphorescence intensity emitted by the phosphors.The experimental parameters, individually optimised for each pesticide, were as follows: pH, volume of analyte, nature and volumes of buffer and heavy ion solutions, delay and gate times, nature of the solid support and drying time of the sample. Whatman No. 4 and Albet No. 1305 filter-papers and Whatman P-81 and Whatman DE-81 ionic exchange papers were checked as solid supports.It was found that the greatest difference between the signals produced by the sample and the respective blank were obtained when Whatman No. 4 filterpaper was used as the solid support. Next, in order to minimize the background signal produced by the solid support, several strips of Whatman No. 4 paper were subjected to extraction with reverse osmosis quality water in a Soxhlet column for 24 h, after which they were dried and irradiated with ultraviolet light for 12 h.The blanks prepared with these pre-treated supports presented a background signal three times lower than the blanks prepared with the paper without pre-treatment; however, the net phosphorescence intensity (sample signal 2 blank signal) obtained by using pre-treated papers was around 14% lower with papers without pre-treatment. As a consequence, Whatman No. 4 filter-paper without pre-treatment was used as the solid support in subsequent experiments, giving a better analytical signal than with pre-treated paper, and in a shorter time.Second, the influence of the heavy ion usually used as an enhancer of phosphorescence intensity was tested, using Pb(ii) as the acetate salt, Tl(i) and Ag(i) as the nitrate salts and KI at different concentration levels. The best net phosphorescence intensity was obtained using saturated Pb(ii) solution as the enhancer for TBZ and 1 m Tl(i) or saturated Pb(ii) solution for CBL. As a consequence, Pb(ii) was selected as the enhancer of phosphorescence intensity for both analytes.Next, different volumes of saturated Pb(ii) solution, ranging between 1.0 and 5.0 ml, were tested in order to find the optimum volume. It was found that 3.0 ml produced the best net phosphorescence intensity (NPI). Hydrochloric acid and sodium hydroxide solutions of different concentrations were used to test the influence of pH on the phosphorescence emitted by the analytes. As can be seen in Fig. 3, the phosphorescence intensity emitted by CBL remains constant at pH values between 2.0 and 8.0, decreasing rapidly at lower or higher pH values. The decrease at pH > 8.0 is due to the hydrolysis of CBL, producing the derivative 1-naphthol, which does not show phosphorescence under the present experimental conditions. For TBZ, pH has no influence on the phosphorescence intensity in the range 1.0–10.0, and only in a very basic medium (pH � 12.0) is a decrease noticeable. As a consequence of these results, although the pH did not appreciably influence the phosphorescence intensity of either chemical, standards and samples were measured at pH 4.0 to ensure that the CBL was not hydrolysed during the preparation of the sample.A 1 m acetic acid–sodium acetate buffer solution (pH 4.0) was used for this purpose. The influence of the drying time of the samples on the phosphorescence intensity was studied using a 250 W infrared heat lamp and a hair-dryer operated at 600 W.Different samples, all prepared in the same way, were placed at different distances and for different time intervals. The best results were obtained when the hair-dryer was placed 7 cm above the sample for 3 min. The sample volume spotted on the solid support also influenced the phosphorescence intensity emitted by the analytes. To test this influence, different volumes of standard solution, ranging between 0.5 and 5.0 ml, and containing 1.0 mg ml21 of each analyte were tested.It was observed that the phosphorescence intensity increased when the sample volume was increased from 0.5 to 3.0 ml, and decreased very slowly when the sample volume was higher than 4.0 ml. As a consequence, 3.0 ml was adopted as the working volume in subsequent experiments. The influence of instrumental parameters such as delay and gate times, size of the excitation and emission slits and scan speed was also studied. It was found that the last parameter did not influence appreciably the phosphorescence intensity emitted by the phosphors but, as expected, td and tg exerted a notable influence.To optimise these parameters, different values of td (between 0.1 and 0.5 ms) and tg (between 4.0 and 14.0 ms) were checked, and it was found that 0.1 and 12.0 ms, respectively, produced the maximum phosphorescence intensity for both analytes. The optimum sizes of the excitation and emission slits were 2.5 and 5.0 nm, respectively.Finally, the quenching effect produced by the presence of atmospheric moisture and oxygen was studied using four samples, each containing 1.0 mg ml21 of each analyte. The phosphorescence intensities were measured at timed intervals, between 1 and 30 min, counted from the preparation of the samples. These samples were measured with and without quartz plates and with and without a dry N2 flow. The samples measured using quartz plates (with or without an N2 flow) showed phosphorescence intensities higher than those without quartz plates (Fig. 4). Hence if the phosphorescence intensity of the sample is measured before 8 min after its preparation, an N2 flow is not needed, thus simplifying the measurement process. Application of the PLS-1 model Optimising the data. A training set of 12 samples, randomly selected, was prepared to obtain the calibration matrix using the experimental data obtained fm single and binary mixtures of both pesticides at low, medium and high concentration levels.Table 1 gives the concentrations of each pesticide in each sample of the set. These concentrations were chosen avoiding correlation between the different samples, because this can produce underfitting in the PLS models. The spectra of these samples, obtained under the previously optimised experimental conditions described above, were recorded from 400 to 570 nm, with an interval of 0.5 nm between consecutive points, resulting in 341 experimental points per spectrum.PLS algorithms consist of two steps, calibration and prediction. In the calibration step we assumed that the Fig. 3 Influence of pH on the phosphorescence intensities of (a) CBL and (b) TBZ. Analyst, 1999, 124, 49–53 51concentration of the different analytes is related to the experimental measurements by the equation ck = R bk + e where ck is the vector of concentrations of analyte k in the N samples of the calibration set, R is the matrix of the instrumental measurements of the N samples in the different channels, e is the vector of the residuals of the concentrations that not are fitted in the model and the coefficients vector bk is obtained in the calibration step from the expression bk = R+ ck where R+ is the pseudo-inverse matrix of the matrix R.In PLS models, R+ is obtained from the decomposition of the original matrix R taking into account the information contained in the concentration vector ck of the calibration set.In the prediction step the concentration of the analytes (k = 1 for CBL, k = 2 for TBZ) are obtained from the matrix expression ck = rT bk + ek where rT is the vector of the instrumental measurements of the sample when this is measured in the j wavelength channels. Here, b coefficients were estimated by PLS-1 and the decomposition and regression of the matrix were performed separately for each component,28 taking into account the collinearity between the different wavelength channels. In our case, to select the optimum number of factors, the crossvalidation method32 was used and, as the training set was formed with 12 spectra, the calibration was performed on 11 of them, thus predicting the concentration of the excluded standard.The process was repeated 12 times (one for each standard) and the predicted and known concentrations were compared. The fitness of the PLS model was calculated by the prediction error sum of squares (PRESS), applied each time that a new factor was added to the model, using the F-statistic as a significance test.Applying the Haaland and Thomas criterion,33 seven factors (half of the standards + 1) were accepted as the maximum number of initial factors and the optimum number of factors was calculated for the first value of PRESS whose Fratio probability fell below 0.75. In this way, three factors were selected as optimum. These three factors should correspond to two analytes plus the background variation of the paper used as the solid support.Table 2 gives the estimated values of residual mean standard deviation absolute (RMSD): RMSD c c N i i i N = - = Â ( � )2 1 as an indicative value of the average error in the determination of each component and the values of R2: R c c c c i i i N i i i N 2 2 1 2 1 = - - = = Â Â ( � ) ( ) indicating that the data are fitted to a straight line. In order to determine the potential interferences produced for organic species and foreign ions usually present in waters, a systematic study of the effects produced by these chemicals was carried out.Standard solutions containing 1.0 mg l21 of CBL and 0.8 mg l21 of TBZ were spotted with the potentially Fig. 4 Influence of time on the phosphorescence intensity emitted by the analytes under different conditions. (A) Carbaryl and (B) thiabendazole. In both cases: (a) with quartz plates and N2 flow; (b) with quartz plates and without N2 flow; (c) with N2 flow and without quartz plates; (d) without quartz plates or N2.Table 1 Composition of the training set Standard CBL/mg ml21 TBZ/mg ml21 1 0.0 0.5 2 0.0 1.1 3 3.5 0.0 4 1.0 0.0 5 2.2 0.7 6 3.8 0.3 7 0.8 1.0 8 2.8 0.8 9 1.4 0.2 10 1.9 0.6 11 3.1 0.4 12 0.4 0.5 Table 2 Statistical parameters of the optimised matrix using the PLS-1 model Pesticide No. of factors RMSDa R2 b CBL 3 0.22713 0.96912 TBZ 3 0.028233 0.99315 a Residual mean standard deviation. b Square of the correlation coefficient.Table 3 Effects of foreign ions or organic species on the determination of CBL (1.0 mg l21) and TBZ (mg l21) Ions or species Tolerance level/ mg l21 Humic acids, dichlone, captan, atrazine, morestan, hexametazone, folpet, carbendazime 10.0 SO4 22, Cl2 , Ca(ii), Mg(ii), Cu(ii) 10.0 Al(iii), Be(ii), NO32 5.0 Fe(iii), PO4 32, warfarin 2.0 52 Analyst, 1999, 124, 49–53interfering species at a 10.0 mg l21 concentration and the concentrations of the analytes (CBL and TBZ) were determined using the PLS-1 method under the conditions established above. If interference occurred the concentration level of the foreign species was reduced until the error produced did not exceed ±5.0% in the determination of either of the two analytes.As can be seen in Table 3, the greatest interference was produced by warfarin and Fe(iii). Application of the model to real samples. In order to test the accuracy and applicability of the method, the optimised matrix obtained by the PLS-1 model was applied to the analysis of real samples of different kinds of water.Because the samples of waters did not contain pesticides (or the concentration levels were lower than the detection limit of the method), a recovery study was carried out. These samples (which were different from the 12 samples used to obtain the calibration matrix) were also analysed by using HPLC as a reference method.34 Table 4 gives the results obtained with the two methods.These data are the average values from three measurements of each sample by PLS-1 and HPLC. The results obtained by the two methods were compared statistically and the values of P are included in Table 4. Conclusions The use of the quartz plates during the measurement of the phosphorescence of the analytes improves the analytical process because a flow of inert gas is not needed. The PLS-1 method can be applied for the statistical treatment of the experimental spectrophosphorimetric data obtained from mixtures of phosphors in the solid phase.The use of a readily available filter-paper as a solid support allows for an inexpensive method, the main advantages of which are the sensitivity and selectivity that derive from the phosphorescence technique and the simplicity and speed that derive from the application of PLS-1. In all cases examined the results obtained by PLS-1 and HPLC were similar, as proved by the applied test. However, we are conscious of the need to carry out further work to demonstrate the applicability of the method to other samples under different conditions, and this is our intention.References 1 J. J. Vannelli and E. M. Schulman, Anal. Chem.,1984, 56, 1030. 2 S. Y. Su, E. Asafu-Adjaye and S. Ocak, Analyst, 1984, 109, 1019. 3 R. Q. Aucélio and A. D. Campiglia, Anal. Chim. Acta, 1995, 309, 345. 4 T. Vo-Dinh, E. Lue Yen and J. D. Winefordner, Anal. Chem., 1976, 48, 1186. 5 M. C.García-Alvarez Coque, G. Ramis Ramos, A. M. O’Reilly and J. D. Winefordner, Anal. Chim. Acta, 1988, 204, 247. 6 S. Scypinski and L. J. Cline Love, Anal. Chem., 1984, 56, 322. 7 S. Scypinski and L. J. Cline Love, Anal. Chem., 1984, 56, 331. 8 W. J. Jin and C. S. Liu, Anal. Chem. 1993, 65, 863. 9 Y. S. Wei, W. J. Jin, R. H. Zhu, C. S. Liu and S. S. Zhang, Talanta, 1994, 41, 1617. 10 S. Panadero, A. Gómez-Henz and D. Pérez-Bendito, Anal. Chem., 1994, 66, 919. 11 R. A. Paynter, S.L. Wellons and J. D. Winefordner, Anal. Chem., 1974, 46, 736. 12 R. P. Bateh and J. D. Winefordner, Talanta, 1982, 29, 713. 13 S. Su, D. L. Bolton and J. D. Winefordner, Chem. Biomed. Environ. Instrum., 1983, 12, 55. 14 D. L. McAleese and R. B. Dunlap, Anal. Chem., 1984, 56, 600. 15 A. D. Campiglia and C. G. de Lima, Anal. Chem., 1987, 59, 2822. 16 M. E. Díaz-García and A. Sanz-Medel, AnChem., 1986, 58, 1436. 17 M. C. Simeone, A. Gioia and A. D. Campiglia, Anal. Chim. Acta., 1994, 287, 89. 18 C. G. De Lima, M. M. Andino and J. D. Winefordner, Anal. Chem., 1986, 58, 2867. 19 H. Wold, in Research Papers in Statistics, ed. F. David, Wiley, New York, 1966, pp. 411–444. 20 S. Wold, in Food Research and Data Analysis, ed. H. Martens and H. Russwurm, Applied Science, London 1983. 21 S. Wold, M. Martens and H. Wold, in The Multivariate Calibration Problem in Chemistry by PLS, ed. A. Ruhe and B. Kagstrom, Springer, Heidelberg, 1983, pp. 286–289. 22 I. Durán-Merás, A.Muñoz de la Peña, A. Espinosa-Mansilla and F. Salinas, Analyst, 1993, 118, 807. 23 A. Espinosa-Mansilla, F. Salinas and I. de Orbe, Fresenius’ J. Anal. Chem., 1996, 354, 245. 24 A. Espinosa-Mansilla, F. Salinas and A. Zamoro, Analyst, 1994, 119, 1183. 25 A. Espinosa-Mansilla, A. Muñoz de la Peña and F. Salinas, Anal. Chim. Acta, 1993, 276, 141. 26 R. G. Brereton, Analyst, 1997, 122, 1521. 27 P. W. Araujo, D. A. Cirovic and R. G. Brereton, Analyst, 1996, 121, 581. 28 F.R. Burden, R. G. Brereton and P. T. Walsh, Analyst, 1997, 122, 1015. 29 C. Demir and R. G. Brereton, Analyst, 1998, 123, 181. 30 H. Martens and T. Næs, Multivariate Calibration, Wiley, Chichester, 1994. 31 L. F. Capitán-Vallvey, F. Ojeda, M. del Olmo, R. Avidad, A. Navalón and T. Vo-Dinh, Appl. Spectrosc., 1998, 52, 101. 32 M. Stone, J. R. Stat. Soc. B, 1974, 36, 111. 33 D. M. Haaland and E. V. Thomas, Anal. Chem., 1988, 60, 1193. 34 M. V. Dilna, R. E. Hall, J. D. Shamis and S. A. Whitlock, J. Chromatogr., 1984, 283, 383. Paper 8/07825D Table 4 Recovery study of mixtures of TBZ and CBL in tap water samples spotted with one or two components CBLb TBZb Sample No.a Taken/ mg l21 PLS-1/ mg l21 RSD (%) HPLC/ mg l21 RSD (%) P (%)c Taken/ mg l21 PLS-1/ mg l21 RSD (%) HPLC/ mg l21 RSD (%) P (%)c 1 0.00 (0.01) — — — — 1.00 0.93 3.1 0.96 4.5 18.4 2 3.00 3.15 2.0 3.10 3.0 27.4 0.50 0.45 4.7 0.47 3.2 8.0 3 2.50 2.64 2.5 2.59 3.4 21.1 0.60 0.63 2.9 0.60 4.9 8.7 4 3.50 3.41 2.3 3.48 3.4 24.2 0.40 0.40 2.9 0.41 3.9 19.0 5 1.00 1.09 3.2 1.08 4.6 64.0 0.60 0.59 2.5 0.61 5.3 29.5 6 0.00 (0.02) — — — — 0.40 0.39 4.1 0.40 3.1 16.1 7 1.00 1.01 3.5 0.98 5.1 20.8 0.40 0.39 4.8 0.41 2.7 32.0 8 3.80 3.55 6.0 3.60 4.1 60.2 0.00 (0.02) — — — — 9 2.00 1.97 7.1 2.00 4.5 64.8 0.50 0.48 5.5 0.49 3.7 41.8 10 1.50 1.41 3.4 1.44 4.1 20.4 0.30 0.30 3.1 0.32 5.2 6.6 11 1.80 1.72 4.6 1.75 5.2 31.2 0.00 (0.04) — — — — 12 2.50 2.41 1.9 2.44 2.4 23.2 0.85 0.87 2.0 0.86 3.9 32.0 a Samples 1–3, tap water, Granada City; samples 4–6, Genil river water (Granada); samples 7–9, mineral water from Lanjarón (Granada); samples 10–12, mineral water from Fontvella (Gerona). b The values in parenthesis are those measured by the method when this compound is not present. c P value of the comparison test. Analyst, 1999, 124, 49–53 53

ISSN:0003-2654    

DOI:10.1039/a807825d

出版商:RSC    

年代:1999

数据来源: RSC