ISSN:1359-7337    

DOI:10.1039/AC99633FX013

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

4-40 Analytical Communications, December 1996, Vol. 33 Date Conference 20 Validative Methods in the Pharmaceutical Industry Location Cork, Ireland Contact Dr. Gerry Montgomery, The Royal Society of Chemistry, Burlington House, Piccadilly, London WlV OBN, UK Tel: +44 (0)171 437 8656. Fax: +44 (0)171 440 3 320. E-mail: montgomery@ rsc.org . 9-14 16-21 17-19 23-27 April 13-17 14-19 19-22 2 1-25 28-29 3&2/5 May 4-8 CANAS '97 Colloquium Analytische Atomspektroskopie 48th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy Fundamentals and Applications of Total-Reflection X-Ray Fluorescence Electrophoresis '97 213th American Chemical Society National Meeting Genes and Gene Families in Medical, Agricultural and Biological Research: 9th International Congress on Isozymcs Scanning 97 Seventh International Symposium on Biological and Environmental Reference Materials (BERM-7) ComDuter & Process Validation in the Freiberg/S achsen, Germany Atlanta, GA, USA Dortmund, Germany Seattle, WA, USA San Francisco, CA, USA Texas, USA Monterey, CA, USA Antwerp, Belgium Manchester, Pharmaceutical and Fine Chemical Industries UK Flavours and Fragrances Warwick, UK PBA '97, 8th International Symposium on Pharmaceutical and Biomedical Analysis USA Orlando, FL, G.Werner, Universitat Leipzig, Institut fur Analytische Chemie, Linnestrasse 3, D-04103 Leipzig, Germany Tel: +49 0341 973 6101. Fax: +49 0341 973 61 15. Linda Briggs, The Pittsburgh Conference, 300 Penn Center Blvd., Suite 332, Pittsburgh, PA 15235-5503, USA Tel: +1 412 825 3220, +1 800 825 3221.Fax: +1 412 825 3224. Mrs M. Becker, Institut fur Spektrochemie und Angewandte Spektroskopie, Bunsen-Kirchhoff-Str. 1 1,44139 Dortmund, Germany Tel: +49 231 1392 230. Fax: +49 231 1392 120. David Wiley, Electrophoresis Society, P.O. Box 1987, Lawrence, KS 66044-8897, USA Tel: +1 913 843 1221. Fax: +1 913 843 1274. E-mail: dwiley@allenpress.com.Department of Meetings, American Chemical Society, 115516th St. NW, Washington, DC 20036, USA Tel: + I 202 872 4396. Fax: +1 202 872 6128. E-mail: natlmtgs@acs.org. Mrs. Janet Cunningham, Barr Enterprises, 10 120 Kelly Road, P.O. Box 279, Walkersville, MD 21793, USA Tel: +1 301 898 3772. Fax: +1 301 898 5596. Mary K. Sullivan, FAMS Inc., SCANNING 97 Program Committee, Box 832, Mahwah, NJ Tel: + I 201 818 1010. Fax: +1 201 818 0086.E-mail: fams@holonet net; Internet: http ://w ww . scanning -fams. org . J. Pauwels, Institute for Reference Materials and Measurements, Retieseweg, B-2440 Geel, Belgium. Tel: +32 14 571 722; or Wayne Wolk, US Department of Agriculture, 10300 Baltimore Blvd, Beltsville, MD 20705, USA Tel: +1 301 504 8927. Spring Innovations Ltd,, 185A Moss Lane, Bramhall, Stockport, Cheshire, SK7 1BA Tel: +44 (0)161 440 0082.Fax: +44 (0)161 440 9 127. Elaine Wellingham, Conference Secretariat, Field End House, Bude Close, Nailsea, Bristol BS19 2FQ, UK Tel: +44 (0)1275 853311. Fax: +44 (0)1275 853311. E-mail: confsec@dial.pipex.com. 07430-0832, USA Shirley E. Schlessinger (Symposium Manager), Suite 1015, 400 East Randolph Drive, Chicago, IL, 60601, USAGordon F.Kirkbright Bursary 1996 In 1985 a fund was established as a memorial to Gordon Kirkbright and his contributions to analytical spectroscopy and to science in general. The fund is administered by the Committee of the Association of British Spectroscopists (ABS) and by the ABS Trust. The purpose of the award is to enable promising young scientists of any nation to attend a recognised scientific meeting or to visit a place of learning.Applications are invited for the 1996 Gordon Kirkbright Bursaries. The award is not restricted to spectroscopists. Full details and application forms can be obtained from Dr T L Threlfall, Department of Chemistry, University of York, Heslington, York, YO1 SDD, UK. Tel: +44 (0)1904 432576 ; Fax: +44 (0) 1904 432516 Completed application forms must be received no later than 30 April, 1996.Analytical Abstracts NOW on CDIROM! The premier source of current awareness in in analytical chemistry is now available on a Silverplatter CD-ROM. ormat ion single Analytical Abstracts on CD-ROM features: Approximately 200,000 items from 1980 onwards Easy to use Silverplatter software (WindowsTM, Quarterly updates with more than 3,000 items Unlimited searching - no additional costs DOS and MacintoshTM formats) Specia I Discount for Hardcopy Subscribers! Contact us today for further information and a FREE 30-day trial.Judith Barnsby, The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 4WF, United Kingdom Tel: +44 (0) 1223 420066.Fax: +44 (0) 1223 423429 E-mail (Internet): marketing @ rsc.org Information ServicesInternational Conference on Analytical Chemistry June 15-21, 1997 Moscow University, Moscow, Russia AIMS The objective of the conference is to highlight the most recent developments in the field of analytical science, specifically in the subject areas identified below. Presentations will be given in the form of plenary and contributed lectures as well as poster sessions.It is hoped that the poster sessions will be used to encourage scientists of different generations to exchange ideas and share experiences in their respective fields. SCOPE The following major topics will be discussed at the conference: Analytical chemistry: Philosophical aspect Preconcentration (including solid phase extraction) Chemometrics Chromatography (GC, HPLC, TLC, IC etc.) and related techniques (CE) Molecular spectroscopy (IR, Raman) Nuclear methods Kinetic methods Bioanalytical chemistry Analysis of new materials (including high-purity materials) Sampling and sample treatment Organic analytical reagents Quality assurance/quality control Atomic spectroscopy (absorption emission, Mass spectrometry Electroanalytical methods Express test methods Analysis of raw materials Analysis of food and agricultural products Clinical analysis fluorescence, XRF, lasers) ORGANISING COMMITTEE Chairperson, Yu A.Zolotov Vice-chairmen, B.F. Myasoedova, V.A. Davankov and V.G. Koloshnikov General secretary, L.N. Kolomiets Yu A. Karpov, I.N.Kiseleva, P.N. Nesterenko, G.I. Ramendik, O.A. Shpigun, S.I. Sinkov, 1.1. Smirenkina, B.Ya. Spivakov, M.M. Zaletina INTERNATIONAL SCIENTIFIC COMMITTEE Chairman, Yu A. Zolotov F. Adams, Belgium R. Barnes, USA M. Novotny, USA H. Englehardt , Germany T. Fujinaga, Japan M. Grasserbauer, Austria B. Welz, Germany A. Hulanicki, Poland B. Welz, Germany E. Mentasti, Ztaly B . F. Myasoedov, Russia V.A. Davankov, Russia H. Frieser, USA E. Pungor, Hungary I. Havesov , Bulgaria J.F.K. Huber, Austria T Yotsuyanagi, Japan M.I. Karayannis, Greece CONFERENCE SECRETARIAT For further information please contact : H. Akaiwa, Japan C. Boutron, France H. Pardue, USA K. Niemax, Germany P.G. Zambonin, Ztaly I .Kuselman, Zsruei S. Tsuge, Japan V. G. Koloshnikov, Russia G. Werner, Germany J.G.H. du Preez, South Africa J.A. Perez-Bustamente, Spain L. Sommer, Czech Republic W. Lindner, Austria F. Macasek, Slovakia M. Valiente, Spain H.M. (Skip) Kingston, USA M. W idmer , Switzeriand Yu. A. Karpov, Russia Dr L. N. Kolomiets, Scientific Council on Chromatography RAS, Leninsky Prospect 31, 117915 Moscow, Russia. E-mail : Iarionov@lmm.phyche.msk.su Tel: 7 (095) 952 0065; 7 (095) 955 4685 Fax: 7 (095) 952 0065; 7 (095) 952 5308

ISSN:1359-7337    

DOI:10.1039/AC996330X015

出版商:RSC    

年代:1996

数据来源: RSC

ISSN:1359-7337    

DOI:10.1039/AC99633BX018

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analytical Communications, April 1996, Vol33 ( I 29-1 31) 129 Determination of Liquid Accelerants in Arson Suspected Fire Debris Using Headspace Solid-phase Microextraction Alexandra Steffen and Janusz Pawliszyn" The Guelph-Waterloo Center for Graduate Work in Chemistry, University of Waterloo, Waterloo, Ontario, Canada N2L 3GI A new method for the detection of selected flammable liquid accelerants from arson-suspected fire debris has been developed using headspace solid-phase microextraction (SPME).Two different techniques were used to validate the headspace SPME method. Preliminary headspace SPME was carried out by extracting small amounts of gasoline or barbecue lighter fluid spiked onto pre-burnt carpet or wood with subsequent analysis using GC-FID. Alternatively, small amounts of accelerant were burnt with wood or carpet and headspace SPME with GC-ion-trap mass spectrometry was performed on the residue.The FID and ion current chromatograms were used to determine a visual correlation so that the presence of the components that are commonly found in petroleum-based accelerants could be confirmed. Headspace SPME was capable of detecting trace amounts of gasoline from a sample obtained from a real arson-suspected fire, while conventional extraction methods, such as static headspace, lacked adequate sensitivity for the analysis so that accelerants were not defected.Determining the presence of accelerants in debris from suspected arson fires is a challenge to many forensic scientists. In fires where arson is suspected, 35-55% of the fire-debris samples test positive for the presence of liquid accelerants, primarily petroleum-based flammable liquids such as gasoline, charcoal lighter fluids, kerosene and paint thinners.' Typically, over 60% of all fire-debris samples consist of carpet, carpet padding and wood.These samples are collected at the fire scene and brought into the laboratory, where a typical analysis might include the extraction of accelerants from the burnt matrix followed by a visual comparison of the chromatographic pattern of the burnt matrix extract to a blank.The extraction technique is vital to the success of this process, especially since these matrices are complex. The current methods for extracting accelerants from fire debris include static headspace, purge and trap and steam distillation-solvent extraction.1-3 There are limitations inherent in the current techniques, which include the lack of sensitivity and discrimination against low boiling-point analytes when using static headspace, and the use of hazardous solvents (e.g., CS2) when using adsorption methods. Other methods require dedicated instrumentation (e.g., purge and trap) and tend to be cumbersome and time-consuming.Solid-phase microextraction (SPME) has the ability to overcome difficulties with conventional extraction methods. SPME utilizes a very simple set-up and requires no additional instrumentation other than a gas chromatograph (GC) with a traditional heated injection port. In SPME, the analytes partition from the sample into the fibre coating, where they are * To whom correspondence should be addressed.subsequently transported to the injector of a GC for thermal desorption and chromatographic separation. SPME has been shown to be very sensitive towards headspace analysis and compared favourably with purge and trap.4.5 Furthermore, headspace SPME can be selective because a fibre coating can be chosen that best suits the analytes of interest and matrix interferences can be reduced.This paper describes the evaluation of SPME for the determination of accelerants in fire debris. In a similar way to current validation methods for this type of analysis, known amounts of liquid accelerants, such as gasoline or barbecue fluid, were spiked onto pre-burnt matrices, including wood or carpet, and extracted by headspace SPME.The GC-flame ionization detection (FID) or GC-ion-trap mass spectrometry (ITMS) chromatograms of the headspace SPME of the spiked sample were compared with headspace SPME of pure accel- erant to determine a visual correlation. Alternatively, gasoline or barbecue fluid were poured onto either wood or carpet and burnt simultaneously to simulate a more realistic fire situation.Finally, a real sample obtained from the interior of a burnt motor vehicle was extracted by headspace SPME to determine the presence of accelerants. Experimental Sample Preparation Gasoline and barbecue lighter fluid were the two accelerants used in this study and were purchased from a local filling (gas) station and store. Spiked samples were prepared by placing about 5 g of wood or carpet, pre-burnt without accelerant, in the extraction vessel, and pipetting between 0.1 and 2.5 yl of the fluids onto the matrices.For the fire simulation, a 2 X 4 in piece of nylon carpet was burnt together with 0.5-1 ml of gasoline and 2-4 ml of barbecue fluid inside a 1.2 1 Pyrex crystallization dish under open air. The burnt sample was placed into the extraction vessel.Headspace SPME Procedure The SPME device has been discussed in detail previously.G8 A 1 cm long x 100 vm poly(dimethylsi1oxane) coated fibre (Supelco, Canada) was used for the analysis. The fibre was conditioned in a GC injection port at 250 "C for 3 h prior to use. The sample was placed into a 100 ml round-bottomed flask, capped and mounted on a hot-plate. A modified glass stopper with a flat top and a small hole in the centre was used to allow access of the fibre to the headspace of the sample.The needle of the SPME device was put through the hole of the stopper and the device was clamped into position on top of the flask. Next, the plunger was depressed and the fibre exposed to the headspace of the sample. The sample was heated to 95 "C for 15 min.The fibre was withdrawn into the needle and transferred into the injection port of the GC, maintained at 275 "C. The fibre was130 Analytical Communications, April 1996, Vol33 exposed for 5 min to the hot injection port and the thermally desorbed analytes were focused on the head of the column on which subsequent chromatographic analysis was performed.Headspace SPME of the real fire samples was performed by piercing a small hole in the sealed jar containing the sample and plugged with a septum. The jar was placed on the hot-plate and the needle of the fibre was inserted through the hole in the lid of the jar. Headspace SPME was performed in an identical manner as described above. GC-FID-ITMS Analysis Conditions Gas-chromatographic analysis was performed using a Varian 3400 Gas Chromatograph (Palo, Alto, California, USA) equipped with a flame ionization detector and a septum programmable injector (SPT) injection port.The chromatograms were recorded and analysed using the Varian STAR system, version 4.0. The target compounds were separated using a 30 m X 0.25 mm id DB-5 capillary column with a stationary phase film thickness of 0.25 pm (J&W Scientific, Folsom, CA).Mass spectral analysis was performed using a Varian Saturn I1 GC- ion trap mass spectrometer equipped with an SPI. Analysis of the extracted components was performed by inserting the SPME device directly into the injection port of the GC. The operating conditions are described in Table 1. Results and Discussion SPME Validation Using Spiked Accelerants on Pre- burnt Matrices Validation of methods for the determination of accelerants in arson samples usually involves spiking known amounts of accelerant onto burnt fire debris and determining whether the chromatographic patterns visually match the accelerant pat- tern.9.10 Furton et al.showed that gasoline could be extracted and identified from a kimwipe, burnt plastic or wood using headspace SPME and GC-FID.9 The evaluation of headspace SPME for this type of analysis was approached in a similar manner using GC-FID, except burnt carpet or wood were used as the test matrices, and barbecue fluid and gasoline were used as accelerants because they are commonly used by arsonists.By use of GC-FID it was found that the chromatographic patterns obtained from a headspace SPME of 2 p1 of barbecue lighter fluid spiked onto about 5 g of pre-burnt carpet, and 2.5 pl onto about 5 g of pre-burnt wood, correlated well with a GC- FID chromatogram of pure barbecue fluid extracted by headspace SPME.Similarly, 1 pl of gasoline was spiked onto about 5 g of pre-burnt carpet and extracted using headspace SPME.The chromatographic pattern from this extraction was compared with one of pure gasoline and the two patterns were Table 1 GC-FID-TTMS operating conditions Operation Condition Injection port temperature Temperature programme (FID and ITMS) (FID and ITMS) FID temperature ITMS transfer line Mass range scanned (ITMS) Scan time Filament delay Peak threshold Background mass 275 "C 70 "C for 3 rnin.; ramp at 4 "C min-3 to 130 "C; ramp at 30 "C min-* to 300 "C; hold for 3 min 300 "C 260 "C 50-260 1.00 s (9 pscans) 1.00 min 1 count 49 u found to correlate visually.There were, however, some extraneous peaks in the chromatogram that could be attributed to the matrix itself which could create potential interferences. Further experiments were performed by using GC-MS. Headspace SPME for the Detection of Accelerants in Fire Simulations Previous reports have shown that headspace SPME is compar- able to conventional techniques for the extraction of spiked accelerants from fire debris using the GC-FID and has the potential to be an excellent screening technique.9 However, real arson samples contain accelerants that have been burnt along with the matrix and which could potentially interfere with the identification of the presence of accelerants in the fire debris.Attempts were made to simulate a more realistic fire situation by applying a known amount of accelerant onto wood or carpet and burning them together, followed by headspace SPME analysis to determine whether the accelerant could be recog- nized and differentiated from the burnt matrix.Since a significant portion of the accelerant would either be lost to vaporization or burnt, and matrix interferences were more likely, chromatographic separation and detection was per- formed with GC-ITMS because of its higher sensitivity and mass resolution capabilities. Studies were carried out using both accelerants; however, the gasoline burnt with carpet will be discussed at present.Fig. 1 shows an example of the selected ion current chromatograms of the m/z = 57 ion and m/z = 120 ions from the headspace SPME of gasoline burnt with carpet (top chromatograms) and pure gasoline (bottom chromatograms). A visual inspection of the two rnlz = 57 plots shown in Fig. l(a) shows that there is a good correlation between the chromatographic patterns for a head- space SPME of gasoline burnt with carpet and pure gasoline.(a) SIC of the m/z=57 ion of gasoline burned with nylon carpet SIC of the m/z=57 ion of pure gasoline i x5 c C 9 A. 5 C .- u ( b ) SIC of the m/'120 ion of gasoline burned with nylon carpet - Q) - a, cn SIC of the m/z=l20 ion of pure gasoline 0 5 10 15 Time/min Fig. 1 GC-ITMS selected ion current chromatograms of headspace SPME of gasoline simultaneously burnt with carpet and pure gasoline: (a) mlz = 57 plot for the sample (top) and pure gasoline (bottom); (h) mlz = 120 plot for the sample (top) and pure gasoline (bottom).Analytical Cornmunications, April 1996, Vol33 131 This ion corresponds to the fragments of the branched and normal alkanes that are abundant in most petroleum-derived accelerants.The earlier eluting peaks in Fig. l(a) are actually relatively small for headspace SPME of gasoline burnt with carpet, probably due to losses during combustion. Nevertheless, there is a good correlation with the peaks from the headspace SPME of pure gasoline. Fig. l ( b ) shows a similar selected ion current chromatogram for the mlz = 120, corresponding to the ion fragments derived from alkyl-substituted benzenes, which are also common in petroleum-based accelerants. By visual comparison of these selected ion current chromatographic patterns, there is a good match between the headspace SPME of gasoline burnt with carpet and pure gasoline.Additional selected ion current chromatograms representing other charac- teristic ions from abundant components in petroleum-based accelerants, including alkylated benzenes ( m l z = 9 1 , 106 and 134) and methyl naphthalenes (mlz = 142), also showed excellent chromatographic matches between the gasoline burnt with carpet and the pure gasoline.The ion selectivity of GC- ITMS was very useful because interferences from arson samples or fire debris can be distinguished from trace amounts of accelerant.Similar results were found when burning barbecue lighter fluid with the various matrices. Finally, a real fire debris sample was obtained from an arson suspected fire. The fire had been in a truck and the sample was taken from underneath the front seat within the vehicle. Fig. 2(a) shows the TIC chromatogram of a headspace SPME extraction of the real fire debris sample and Figs.2(h) and ( c ) show the selected ion current chromatograms for the mlz = 57 and 120 ions, respectively, from the same extraction. Although the TIC chromatogram in Fig. 2(a) was quite complex and visual correlation with an accelerant was nearly impossible to determine, the selected ion current chromatograms of the E (a) Real arson sample -Total ion current chromatogram ? 3 S .- ( b ) SIC of the m/z=57 ion of a real arson sample SIC of the m/z=57 ion of pure gasoline ' x 10 ( c ) SIC of the m/z=120 ion of a real arson sample 0 - x s SIC of the m/z=120 ion of pure gasoline 0 5 10 Time/min 15 Fig.2 GC-ITMS chromatograms of headspace SPME of a real fire debris sample: ( a ) total ion current chromatogram; (h) selected ion current chromatograms for mlz = 57 for the real sample (top) and pure gasoline (bottom); (c) selected ion current chromatograms for mls = 120 for the real sample (top) and pure gasoline (bottom).headspace SPME of the real sample showed good visual chromatographic pattern matches with pure gasoline when either the mlz = 57 or mlz = 120 ions were monitored. There seems to be a Iack of the earlier eluting peaks in each of the individual ion fragment plots; however, as discussed earlier, accelerants tend to evaporate during the fire or in the interval between extinguishing the fire and collection of the samples.10 Therefore, it is to be expected to see some loss of the lower boiling compounds.The same real fire sample was analysed by another laboratory using static headspace extraction coupled with a GC-FID.Surprisingly, no detectable pattern was observed that was characteristic of petroleum-based accelerants and arson was ruled out as a possible cause of the fire.12 However, as shown in Figs. 2(a-c), the real arson sample analysed by more sensitive and selective extraction methods such as headspace SPME-GC-ITMS showed that an accelerant could be present in the debris and that arson should not be ruled out as a cause of the fire.Conclusions It has been demonstrated that the headspace SPME technique can be easily applied to the extraction of liquid accelerants from fire debris. Accelerants spiked onto pre-burnt material could be extracted and detected by headspace SPME and GC-FID. This technique can be used as an excellent screening method. However, when accelerants are burnt along with sample matrices, more sensitive and selective detection techniques such as GC-ITMS may be required to discriminate between the trace amounts of accelerant and potential background interferences from the burnt matrix.A direct comparison between static headspace-GC-FID and headspace SPME-GC-ITMS showed that static headspace extraction was inadequate to determine the presence of an accelerant in a real sample from a suspected arson case.On the other hand, headspace SPME was successful in determining the presence of an accelerant from the sample. Thus, this study demonstrates that headspace SPME is a very simple and sensitive extraction method for accelerants from fire debris and simple visual correlation is often adequate to determine the presence of accelerants when selective detection methods such as GC-ITMS are used. The authors would like to thank Supelco and Varian for their financial support. References 1 2 3 4 5 6 7 8 9 10 1 1 12 Bertsch. W.. and Zhang, Q. W., Anal. Chini. Acfa, 1990, 236, 183. Nowicki, J., J . Forensic Sci., 1991, 36, 1543. Nowicki, J., J . Forensic Sci., 1990, 35, 1064. MacGillivary, B., and Pawliszyn, J., J . Chi-omarogr-. Sci., 1994, 32, 317. Zhang, Z., and Pawliszyn, J., Anal. Chem., 1993, 65, 1844. Arthur, C. L., Pratt, K., Motlagh, S . , and Pawliszyn, J., .I. High Resol. Ci?r-omatogr., 1992, 15, 74 1 . Boyd-Boland, A. A., Chai, M. Luo, Y. Z.. Zhang, Z., Yang, M., Gorecki, T., and Pawliszyn, J., Environ. Sci. Technol.. 1994, 28, Buchholz, K. D.. and Pawliszyn, J., Eni!ii-on. Sci. Tec,hnol., 1993, 27, 2844. Furton, K., Almirall, J., and Bruna, J., J . Forensic Sci., 1995, in the press. Keto, R., and Wineman, P., Anal. Chem., 1991, 63, 1964. Vella, A., J . Forensic Sci. Soc.., 1992, 32, 13 1. Martos, P., personal communication. 569-A. Paper 6100726K Received Januury 30, I996 Accepted February 20, I996

ISSN:1359-7337    

DOI:10.1039/AC9963300129

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analytical Communications, April 1996, Vol33 ( I 33-1 35) 133 Highly Selective Catalytic Spectrophotometric Determination of Nanogram Amounts of Rhenium With N,AkDimethyldithiooxamide in Alkaline Medium* ~~~ Ognyan Bozhkov" and L. V. Borisovab I I13 Sofia, Bulgaria h Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, I I7975 Moscow~, Russia Institute of General and Inorganic Chemistry, Bulgarian Academy of Sciences, The developed method is based on the catalytic action of RetV on the reduction of N,N-dimethyldithiooxamide (DMDTO) with tin(I1) in an alkaline medium, yielding a blue coloured product with maximum absorbance at 634 nm.The optimum parameters of the catalytic reaction are described. The calibration graph is plotted at fixed time intervals 10 or 33 min from the addition of tin(I1) solution.It is linear in the Re concentration range 2-15 ng ml-1. A 1000-fold excess of MoV1, WVt, Cd", Mn", Cu", FelI1, sulfate, silicate, nitrate and ammonia does not interfere with the determination. A modification of the proposed method is used as a rapid spot test. Both methods are applied to the determination of rhenium in molybdenite after alkaline fusion followed by extraction with water.No separation of the matrix is necessary. The processing of rhenium-containing materials is preferably performed in an alkaline medium, e.g., following an alkaline digestion of natural objects, alkaline anodic dissolution of rhenium-based metal wastes, separation of rhenium from the matrix in the production of potassium and ammonium per- rhenate, etc.]-9 Our previous papers reported on a number of spectroscopic methods for the determination of rhenium with various organic reagents such as hydroxylamine, thiourea and dithiooxamide.1G12 All of these methods, however, suffer from insufficient sensitivity.A catalytic spectrophotometric method has been developed for the determination of rhenium in alkaline solutions based on the catalytic action of rhenium on the reduction of azobenzeneazorhodanine with tin(1r) in an alkaline medium.]3 The method is sensitive, but not sufficiently selective (molybdenum interferes seriously).The present paper reports a highly sensitive and selective catalytic method for the determination of rhenium in alkaline medium based on its catalytic effect on the reduction of N,N- dimethyldithiooxamide (DMDTO).Experimental Reagents A stock standard solution of rhenium (1000 pg ml-l) was prepared by dissolving 0.1553 g of KRe04 in 100 ml of distilled water. Working solutions (1 pg ml-l Re) were prepared by diluting 0.01 ml aliquots of the stock solution with 10 ml water. * Presented at the 5th International Symposium on Kinetics in Analytical Chemistry, Moscow, Russia, September 25-28, 1995.The solution of DMDTO (0.04 rnol 1-1) was prepared by dissolving 0.0593 g of the reagent in 10 ml of 10 mol I-' NaOH. The use of fresh solutions is recommended. The solution of tin(I1) chloride in 10 mol I-' NaOH was prepared by dissolving 0.542 g of SnC12.2H20 in 0.3-0.8 ml of distilled water, followed by the addition of about 8 ml of 10 mol 1-1 NaOH under constant stirring until a clear, colourless solution was obtained, and finally diluting to 10 ml with 10 mol 1-1 NaOH.The solution was prepared.fresh daily. Merck sodium hydroxide solution (32%, d = 1.35, E. Merck, Darmstadt, Germany) was used throughout. Stock standard solutions of molybdenum and tungsten (1000 pg 1-1) were prepared from reagent grade Na2M004.2H20 and Na2W04.2H20 and made alkaline with sodium hydroxide (10 mol 1-I).Atomic absorption standard solutions (1000 pg ml-1) for cadmium, manganese, cobalt, iron, nickel, silicon and copper were used. Diluted solutions of sulfuric acid, nitric acid and ammonia were prepared from concentrated analytical grade acids (Merck). Apparatus A Beckman, Model DK 2A UV-VIS, near-infrared, double- beam spectrophotometer was used for the determinations (Beckman Instruments, Fullerton, CA, USA).For the digestion procedure a muffle furnace and nickel crucibles were used. Procedures Spectrophotometric determination Calibration graph. Aliquots (0, 0.02, 0.05, 0.1 and 0.15 ml) of the standard rhenium solution ([Re] = 1 pg ml-l) were pipetted into 10 ml, dry, calibrated cylinders fitted with ground-glass stoppers.Then 0.32 ml of DMDTO, followed by 0.534 ml of tin(I1) solution, were added, the volume brought up to 10 ml with 10 moll-' NaOH and the solution shaken vigorously. The reaction started on the addition of tin@) chloride reagent. The light absorbance was measured at 634 nm in a 1 cm quartz cell against distilled water at the 10th or 33rd minute.Rapid spot qualitative and semi-quantitative test. The reference colour scale was prepared as follows: 0, 2, 5 , 10 and 15 pl aliquots of the standard rhenium solution ([Re] = I pl ml-1) were placed in the wells of a PTFE spot-plate. An 8 p1 volume of the DMDTO solution and 13.33 p1 of the tin(I1) chloride solution were added and the volume brought to 250 pl with 10 moll-' NaOH.The resulting solution was stirred with a thin glass rod. The colour was developed within 6 min. To an aliquot of the unknown solution (2-20 pl) were added the134 Analytical Communications, April 1996, Vol33 volumes of the reagents described above in the cited sequence. The resulting colour was compared with the reference scale. Analysis of molybdenite.A 1 g amount of molybdenite (MoS,) was mixed with 6.5 g of Na202 and 1.5 g of Na2C03 in a nickel or iron crucible fitted with a lid and heated in a muffle furnace at 500-550 "C for 1.5 h. The melt was cooled and treated with 15-20 ml of distilled water, with heating on a water-bath (until the liberation of gas bubbles ceased) for complete dissolution of the unreacted Na202. The resulting pulp was filtered through a paper filter into a 100 ml calibrated flask by washing the crucible with small portions of distilled water (5-6 washings). The flask was made up to volume with distilled water.An aliquot of this solution was first subjected to a spot, semi-quantitative, rapid test to establish the required aliquot volume for the subsequent quantitative analysis and then analysed as described under Spectrophotometric determina- tion.Results and Discussion When a colourless, alkaline solution of DMDTO was mixed with an excess of reducing agent [tin(~r)] a blue colour formed with a maximum absorbance at 634 nm, which is an indication that a redox reaction was taking place in the system DMDTO- NaOH-Sn". The addition of perrhenate to this system does not cause any qualitative changes in the absorption spectra but only quantitative ones, viz., the light absorbance at 634 nm is rapidly increased.This is evidence that the presence of rhenium ions catalyses the reduction of DMDTO. Since the reduction of DMDTO depends on an excess of tin(rI), and rhenium is present in nanogram amounts, it could be suggested that under these conditions rhenium(vr1) is reduced to rhenium(rv), the latter being known to possess catalytic activity.This suggestion has been proved by testing the effect of both ReV1l and Re'" on the rate of reduction of DMDTO with Sn". It was established that in the presence of Re1" the initial rate of reduction was two times higher. Optim urn Reaction Parameters Reaction conditions should provide a maximum difference in absorbances of the catalytic and non-catalytic reactions at 634 nm, i.e., a maximum difference in the rates of the two reactions.In practice, this was established by measuring the light absorbance of the catalytic reaction against that of the non- catalytic reaction. Our previous studies' revealed that the formation of the blue product with A,,, at 634 nm starts at a sodium hydroxide concentration of 5 mol 1-1, reaches its maximum rate at 10 rnol I-' and that above this concentration colour formation is prevented.The course of the reaction is probably pre-determined by the state of the DMDTO. In 15 moll-' NaOH the DMDTO is very likely to be salted out, thus losing its reactivity. Below 5 mol 1-1 of NaOH DMDTO is probably not completely changed into its thiol form.The following reaction parameters have been studied: DMDTO concentration, tin(r1) concentration, duration of the interaction, rhenium concentration, temperature. The maximum difference between absorbances of catalytic and non-catalytic reactions was achieved with [DMDTO] = 1.28 X 10-3 mol 1-1 and [Sn2+] = 1.28 X 10-2 moll-'.Light absorbance increases with increase in rhenium concentration (Fig. 1). Fig. 2 shows theA634 versus time plots at various rhenium concentration. They were used to calculate the reaction order according to the initial rate equation.14 The reaction was found to be first order. The rate of the reaction was studied at 20 and 34 "C in the presence and in the absence of rhenium.The difference in the rates did not exceed 10%. The activation energies of both catalytic and non- catalytic reactions were calculated from the Arrhenius equa- tionl5 to be EAcat = 5.15 kJ mol-l and EAnon-cat = 11.9 kJ mol-l, i.e., the activation energy of the catalytic reaction is lower. Interferences The following elements usually accompany rhenium in rocks, ores and concentrates: Mo, W, Cd, Mn, Ni, Cu, Fe, S and Si.On dissolution of natural objects with alkalis a mixture of hydroxides, molybdates, tungstates, silcates, etc., is produced. Rhenium is present as soluble perrhenate. The interference of the above-cited elements with the proposed catalytic reaction was studied. It was established that a 1000-fold excess did not interfere. Calibration Graph The calibration graph is linear in the range 2-15 ng ml-1 of Re (A,,, = 4 X + 8.94 X 10-3 [Re], r = 0.9969, and A,,, = 1.46 X 10-2 + 1 S O X 10-2 [Re], Y = 0.9966 for the 10th and 33rd min from the start, respectively).The relative standard deviations for five determinations of 10 ng ml-I Re were 4.6 490 568 700 A Inm Fig. 1 Absorbance spectra of both catalytic and non-catalytic reaction with a varying rhenium concentration: A, 0; B, 2; C, 5; D, 10; E, 15 ng ml-I Re.[DMDTO] = 1.28 X mol I-'; [Sn*+J = 1.28 X rnol 1 - I ; [NaOH] = 10 mol 1-1; temperature, 20°C; absorbance measured at the 33rd minute against distilled water. 0.40 7 L 0'35 0.30 r 0.25 1 0.05 0.00 0 4 8 12 16 20 24 28 32 36 40 Umin Fig. 2 Absorbance at 634 nm versus time plot: A, 0; B, 2; C, 5 ; D, 10; E, 15 ng ml-I Re.[DMDTO] = 1.28 X rnol I-I; [Sn2+] = 1.28 X lo-* mol 1-1; [NaOH] = 10 mol 1-I. Absorbance measured against distilled water.Analytical Communications, April 1996, Vol33 135 and 2.3% at the 10th and 33rd min, respectively. The limit of detection for Re is 1 ng ml-I. Applications A sample of molybdenite was analysed by the proposed method and Re content was found to be 25.4 k 0.6 pg g-* (6 replicates).A comparison analysis was performed by ICP-AES and the Re content was found to be 25.7 f 0.4 pg g-l (3 replicates). A good agreement was achieved. Conclusions A catalytic action of rhenium(1v) on the reduction of DMDTO with tin(I1) in alkaline medium has been established. A highly selective, catalytic method for determination of nanogram amounts of rhenium has been developed.A rapid semi- quantitative spot test has been developed as well. Both methods have been applied to the analysis of molybdenite for rhenium content determination. References 1 Bozhkov, O., Thesis, Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, Moscow, 1990. 2 Linetskii, B. L., and Krunin, A.V., Plasticheskaya Deformatsiya Platinovykh Meiallov i Reniya, Metallurgiya, Moscow, Russia, 1984, Darbinyan, M. V., and Gajbikyan, D. S., in Rhenium, ed. Savitskii, E. M., Nauka, Moscow, Russia, 1964, pp. 50-54. p. 122. 3 4 5 10 I1 12 13 14 15 Zelikman, A. N., in Rhenium, ed. Savitskii, E. M., Nauka, MOSCOW, Russia, 1964, pp. 71-77. Reznichenko, B. A., Palant, A. A., and Solov’ev, V.I., Kompleksnoe Ispol’zovanie Syr’ya v Tekhnologii Tugoplmkikh Meiallov, Nauka, Moscow, Russia, 1988, pp. 201-203,214-215. Jordanov, N. P., Pavlova, M. Ch., and Bojkov, 0. D., US Pat. 4,278,641, July 14, 1981. Bozhkov, 0. D., Jordanov, N., and Borisova, L. V., and Fabelinskii, Yu. J., Fresenius’ Z. Anal. Chem., 1985, 325, 453. Krasnobaeva, N., Kaskalova, N., and Davaasuren, S., Specirochim. Acta, Part B , 1984, 35, 1323. Bozhkov, 0. D., Borisova, L. V., Jordanov, N. P., and Pavlova, M. Ch., in Khimiya i Tekhnologiya Molihdena i Vol’fr-ama, ed. Spitsyn, V. I., Izd. Kabardino-Balkarskogo Universiteta, Nal’chik, Russia, 1987, pp. 152-156. Borisova, L. V., Ermakov, A. N., and Ismagulova, A. B., Analyst, 1982, 107,495. Borisova, L. V., Ismagulova, A., and Ponomareva, E. I., Zh. Kompl. Ispol’z. Mineral’nogo Syr’ya (Alma-Ata), 1984 (12), 22. Bozhkov, 0. Jordanov, N., and Borisova, L. V., Talanta, 1988, 35, 62. Plastinina, E. I., Borisova, L. V., and Gur’eva, R. F., Zh. Anal Khim., 1992, 47, 2052. Muller, H., Otto, M., and Werner, G., Katalytische Methoden in der Spurenanalyse, Mir, Moscow, Russia, 1983, p. 56. Yatsimirskii, K. B., Kineticheskie Metody Analyza, Khimiya, Moscow, Russia, 1967, p. 51. Paper 6/00389C Received January 17,1996 Accepted February 13,1996

ISSN:1359-7337    

DOI:10.1039/AC9963300133

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analytical Communications, April 1996, Vol33 (1 37-138) 137 Solid-Liquid Extraction of Metal Ions With Thixotropic Gels Shigeki Abe, Masatoshi Endo, Misao Sat0 and Kazutoshi Awano Department of Materials Science and Engineering (Applied Chemistry Section), Yamagata University, 992 Yonezawa, Japan A solid-liquid extraction method based on thixotropy has been developed for the preconcentration of metal ions.A gelling agent, N-lauroyl-L-glutamic-a,y-dibutylamide (G-1), was used as a solid medium containing organic solvent. The G-1 gels liquefy when subjected to simple shaking and then solidify again when left standing. Thus, reversible phase separation is attained at room temperature. The gel extraction behaviour of mercury(II), copper(@ and nickel(r1) is demonstrated.Experimental Preparation of Thixotropic Gels Suitable masses of G-1 and organic solvent containing an appropriate extractant were transferred into a 10 ml glass- stoppered tube, which was heated on a water-bath (80-85 "C) until the solid G-1 completely dissolved in carbon tetrachloride or nitrobenzene. The clear solution was brought to room temperature, forming a thixotropic gel.Optimum amounts of G-1 for gelling 5 ml of carbon tetrachloride, nitrobenzene and chlorobenzene were 0.14, 0.15 and 0.15 g, respectively. Traditional solvent extraction methods for metal ion separation Extraction Procedure utilize either a liquid-liquid or a solid-liquid system. Solid- liquid extraction has gained wide acceptance over the last decade as a technique for the separation and/or preconcentration Conventionally, the majority of solid-liquid extraction systems are classified into two types according to the extractants used, liquefaction. The former is of a very different nature from the hydrophobic materials (C 8 and C,) are used as solid-phase To the solid gel in a 10 ml glass-stoppered tube, the following solutions were added: 1 ml of metal ion solution, 1 ml of buffer volume of 5 ml.The mixture was shaken mechanically for 10 min at room temperature and then centrifuged* The upper test-tube was washed with water and then dissolved in ethanol. of trace in environmenta] and biological samples. and 1 ml of extractant (if necessary) and water to make a viz, solid-phase materials and solid extractants capable of aqueous layer was removed decantation* The ''lid gel in the latter; both hydrophilic (silica, alumina and Florisil) and The metal ion concentration was determined by atomic spectrometry and spectrophotometry* .- sorbents. The latter type is subdivided according to the mode of liquefaction (or solidification) of solid (or liquid), i.e., accord- Results and Discussion ing to the mode of phase separation.Earliest studies on solid- liquid extraction were done with molten naphthalene and Selection sf Extractant Various aliphatic and aromatic hydrocarbons were assayed as media for the preparation of the thixotropic gels. The results obtained are summarized in Fig. 1. Nitrobenzene and carbon tetrachloride, with their high densities, were convenient for use because of their ease of handling in glass-stoppered tubes. The effect of shaking time on the extraction of metal complexes showed that min of mechanical shaking is sufficient to attain extraction equilibrium.benzophenone. 1 Later, these compounds were used for micro- crystalline solid formation from acetone solution; the metal chelates were quantitatively collected by them as is the case with organic precipitants.'.3 Recently, a solid-liquid system of single or mixed water-soluble polymers has been reported, in which the salt-out effect assisted phase ~eparation.~?' In principle, other physicochemical phenomena also offer un- explored opportunities that may deserve the attention of analysts.Certain gels are known to liquefy when subjected to vibratory forces. N-lauroyl-L-glutamic-a, y-dibutylamide (G- 1) solidifies large amounts of organic solvents and forms thixotropic gels; they liquefy when subjected to shaking and then solidify again when left standing.This thixotropy is observed when the gels are in contact with the aqueous phase. Thus, extraction procedures similar to those in the liquid-liquid system can be applied to the solid-liquid system.In this communication a solid-liquid extraction method for metal ions is described using thixotropic gels as solid extractants. Based on our preliminary research, good phase separation was achieved directly at room temperature. The extraction of mercury(r1) complexes with thiophene, copper(rr) chelates with dithiocarbamates and an- ionic nickel(1r) complexes with 4-(2-pyridylazo)resorcinol (PAR) has been demonstrated.To our knowledge, no practical application of thixotropy has previously been made for analytical separation purposes. Carbon tetrachloride 1,2-DichIoroethane Toluene Nitrobenzene Chlorobenzene o-Dichlorobenzene 0 0.05 0.1 0.15 G-1 content, (g per 5 ml solvent) Fig. 1 gel formation and gel suitable as solid media. Comparison of thixotropic gels used for solid-liquid extraction:138 Analytical Communications, April I996, Vol33 Extraction of Mercury(i1) Thiophene is known to bind mercury with strong preference over other heavy metals.6 This offers an analytical potential for removing trace mercury from complex environmental matrices.For the solid-liquid extraction of mercury(II), G-1 gels containing either nitrobenzene or chlorobenzene were used.The extraction curves showed that the extraction of mercury(I1) is complete in the equilibrium pH range 0.5-5.5 (acetate buffer media). The quantitative recovery of mercury(r1) from dilute aqueous samples was also verified by neutron activation analysis. Interestingly, a marked difference in the back- extractability of mercury(I1) was observed for the liquid-liquid and solid-liquid extraction systems.While the back-extraction of mercury(r1) with 6 moll-‘ hydrochloric acid was quantitative in the liquid-liquid system, the percentage back-extraction of mercury(r1) from the G-1 was low (i.e., 25-35%). This fact may reflect a unique feature of the thixotropic solid gels; presumably an additional nitrogen donor link in the G-1 gel contributed to the strong retention of mercury(I1).Extraction of Copper(i1) Two derivatives of dithiocarbamate, sodium diethyldithiocar- bamate and ammonium pyrrolidinedithiocarbamate, were used for comparison. The former was directly added to the aqueous phase, while the latter was pre-included in the thixotropic gel. In the extraction of copper(I1) chelates, the G-1 gel with carbon tetrachloride was preferable to that with nitrobenzene.Phase separation was rapid and no emulsification was observed. The pH and ionic strength of the aqueous phase showed a marked effect on the phase separation. The copper(r1) chelates were effectively extracted from citric acid media (pH 1.7). The G-1 gel and copper(I1) complexes were dissolved in ethanol for subsequent spectrophotometry .The apparent molar absorptivity of copper(r1) complexes was 1.3 X lo4 1 mol-1 cm-1 at the absorption maxima of 434 nm, which is comparable with that obtained in liquid-liquid extraction.7 Extraction of Nickel(u) The solid-liquid extraction of ion associates between the anionic nickel(1r)-PAR complexes and the tetraphenylphos- phonium (TPP) cations was investigated at pH 9.A 1000-fold excess of TPP acts also as an ionic strength modifier. Zephiramine, an effective counter cation in liquid-liquid extraction, was found to be unsuitable; it interfered with phase separation.8 For the extraction-spectrophotometry of nickel(rI), the solid G-1 gel was dissolved in ethanol. Beer’s law was obeyed up to 0.8 pg ml-1 of nickel@) at 501 nm.When the absorbance is measured at an isosbestic point (550 nm), no precise pre-adjustment of pH is required for the extraction of nickel(I1). The ternary cobalt(r1)-PAR-TPP complexes showed a similar extraction pattern. References Fujinaga, T., Kuwamoto, T., and Nakayama, E., Talanta, 1969, 16, 1225. Satake, M., Matsumura, Y., Fujinaga, T., and Takamoto, Y., Bunseki Kagaku, 1978,27,486. Bums, D. T., and Tungkananuk, N., Anal. Chim. Acta, 1988, 204, 359. Li, B. H., and Meng, R. G., Talanta, 1990, 37, 885. Sun, X. M., Li, B. H., and Fu, K. J., Mikrochim. Acta, 1990, 111, 101. Bock, R., and Puff, H. J., Fresenius’ Z. Anal. Chem., 1971, 255, 14. Bode, H., Fresenius’ Z. Anal. Chem., 1954, 143, 182. Yotsuyanagi, T., Yamashita, R., and Aomura, K., Bunseki Kagaku, 1970, 19, 98 1. Paper 6J008.576 Received February 6,1996 Accepted February 2 7, I996

ISSN:1359-7337    

DOI:10.1039/AC9963300137

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analytical Communications, April 1996, Vol33 (1 39-141) 139 Determination of Amitriptyline Using Electrogenerated Chemiluminescence Sarah J. L. Dolman and Gillian M. Greenway” School of Chemistry, University of Hull, Cottingham Road, Hull, North Humberside, UK, HU6 7RX A novel method for the selective and sensitive determination of amitriptyline, a tricyclic antidepressant, using electrogenerated chemiluminescence (ECL) is described.The ECL mechanism is based on the reaction between tris(2,2’-bipyridyl)ruthenium(11) [R~(bpy)3~+] and the tertiary amino group on arnitriptyline. After optimizing the experimental conditions, a calibration over three orders of magnitude of concentration, was obtained. A linear calibration plot was constructed in order to evaluate the limit of detection, which was determined as 3.0 x 10-7 mol 1-1 (94 ppb) with an average % s, of 2.14 for 5 replicate measurements.Antidepressants fall into two groups, the monoamine oxidase inhibitors and the thymoleptics. The thymoleptics consist of tricyclic and tetracyclic compounds, of which amitriptyline is the most therapeutically useful. 1 It is capable of alleviating a range of related symptoms, although it may have anticholiner- gic side effects.2 The chemical structure of amitriptyline is illustrated in Fig.1. Antidepressants are regularly analysed in patients receiving therapeutic doses and also in clinical and post-mortem specimens. As the dosage of a drug increases, the beneficial response increases until a point is reached where increasing the dosage further will cause toxic effects.This point is where the drug has maximum therapeutic response. The corresponding optimum plasma drug concentration can therefore be measured to indicate where the maximum response is observed. For amitriptyline, this is in the range 150-300 ppb.3 Electrogener- ated chemiluminescence (ECL) is a phenomenon where a chemiluminescent reaction is produced in the vicinity of an electrode surface when a potential is applied to it.This technique retains the advantages of conventional chemilu- minescence detection in that it is highly sensitive and selective but it also has additional advantages in that the reagents are produced in situ when required at the electrode, which allows greater control over the rate and course of the reaction by merely altering the applied potential.The emission is concentrated close to the electrode surface, which means that the optical detection system can be positioned accurately to obtain maximum sensitivity. The reactions of tris(2,2’-bipyridyl)ruthenium(11) [Ru(bpy)3*+] are one of the most promising areas of analytical Fig. 1 Chemical structure of amitriptyline.* To whom correspondence should be addressed. ECL systems since they occur at room temperature in aqueous buffered solutions and undergo reversible one-electron transfer reactions at easily attainable potentials. Ru(bpy)32+ also shows intense ECL emission, which is insensitive to the presence of dissolved oxygen and other impurities compared with other mechanisms. Ru(bpy)32+ has previously been shown to react with a variety of secondary and tertiary amines.4 Tricyclic antidepressants contain these functional groups and should therefore be detected using this mechanism.The aim of this work was to develop a novel technique for the detection of amitriptyline, based on its ECL reaction with Ru(bpy)32+, and to achieve a suitable detection limit for monitoring amitriptyline at both therapeutic and toxic concen- trations. ECL Mechanism The R~(bpy)3~+ and the tertiary amino functional group on amitriptyline are oxidized simultaneously by application of an appropriate single, positive voltage to the working electrode.The oxidation product of the amine undergoes deprotonation to form a radical, which reduces the Ru(bpy)33+ to the excited state with a subsequent emission of light.The intensity of the emission can be related back to the concentration of the amitriptyline present and hence the technique can be used to quantify this compound. The mechanism occurs as below: Ru(bpy)32+ + R~(bpy)~2+ + e- (Electro-oxidation) (C3H7)2-CH*CH2CH3--+ (C3H7)N+*-CH2CH2CH3+e- (Electro-oxidation) (Deprotonation) (C3H7)N+*-CH2CH2CH3-+ (C3H7)N-C*HCH2CH3+H+ (C~H~)N-C.HCHZCH~ + R~(bpy)33+ + H20 + R~(bpy)3~+* + (C3H7)2NH + C2HsCH20 Ru( bpy)32+*+R~( bpy)32++h~ (Chemiluminescence) Experimental Reagents Tris(2,2’-bipyridyl)ruthenium(11) hexahydrate (Pract. Grade, 90-95%) was obtained from Fluka (Gillingham, Dorset, UK).The buffer used contained sodium dihydrogenorthophosphate (AnalaR, 99-102%) obtained from Merck Ltd.(Poole, Dorset, UK). The pH was adjusted with either sodium hydroxide (analytical-reagent grade, 98%) from Rh6ne Poulenc (Man- Chester, UK) or glacial acetic acid (analytical-reagent grade, 99%) from Koch-Light (Haverhill, Suffolk, UK). Amitriptyline and a range of other tricyclic antidepressants were obtained from Sigma (Poole, Dorset, UK). Standard solutions of amitriptyline were initially prepared in AR grade methanol and subsequently diluted with buffer. All other solutions were made up in water prepared by reverse osmosis followed by ion140 Analytical Communications, April 1996, Vol33 exchange (Elgastat UHQ, PSI1 Elga Ltd., UK) and no further purification of reagents was required. Instrumentation Fig.2 illustrates the instrumentation used and this has been described previ~usly.~ A 100 pl sample loop was used and all connections in the flow injection system were constructed from 0.8 mm internal diameter PTFE tubing obtained from Anachem (Luton, Bedfordshire, UK).The flow cell was built from solid PTFE and housed in a light-tight aluminium box. Potentials were applied to the electrodes using a three-electrode potentio- stat.The electrodes consisted of a platinum-disc working electrode and a silver pseudo-reference electrode, both housed within the flow cell. A platinum-wire counter electrode was also incorporated, downstream of the flow cell. The light was detected using a photomultiplier tube (Thorn EMI, 9789QB, Ruislip, UK), which was held at 850 V. The signals were amplified and recorded using a chart recorder (Chessel, Worthing, Sussex, UK).Optimization of Factors Affecting the Reaction The amitriptyline and the R~(bpy)3~+ were pre-mixed with the buffer and were then transported directly to the flow cell where a potential was applied to induce the reaction. Five replicate measurements were carried out for each analysis. Effect of Applied Voltage and pH A voltage ramp was applied to the working electrode in the range from 0 to +1.58 V and initial concentrations of 1 mmol l-l Ru(bpy)32+, 100 pmol l-1 amitriptyline, 0.05 moll-’ phosphate buffer and a flow of 2.0 ml min-1 were used.A pH range of 4.5-8.0 was investigated. The signal response reached an optimum level at pH 7 (Fig. 3) and a voltage of 1.26 V. The Control sysiern Reagent- carrier stream I I DACIAmplifier L A ECL flow cell PMTdetector 1 potentiostat Fig.2 40 7- .......................................................................................................... Diagram of the ECL instrumentation used. 35 30 2 25 ‘b 20 5 15 5 0 cn c g 10 4 5 6 7 8 PH Fig. 3 Variation of ECL intensity with pH. optimum applied voltage did not vary significantly with varying pH.The % s, for 5 replicate measurements was less than 8.5% for the pH range investigated. An investigation of the voltage pulse rate was also carried out. A pulse of 2.5 s was found to give the optimum peak height response, as well as the most reproducible peaks. Pulses of 5 s and above gave considerable electrical spiking. Effect of Flow Rate By using concentrations of 1 mmol 1-1 R~(bpy)~2+, 100 pmol 1- amitriptyline and 0.05 moll- phosphate buffer at pH 7, the flow rate was investigated in the range 0.55-3.7 ml min-l, with a 2.5 s voltage pulse of 1.26 V.The peak responses did not change significantly as flow rate was varied, but flow rates from 2.2-3.7 ml min-1 gave electrically spiked peaks. The most reproducible results were obtained using a flow rate of 2 ml min-l and this optimum also limits reagent consumption.Effect of R~(bpy)3~+ Concentration By using the conditions described above, Ru(bpy)32+ concen- trations in the range 0.54.0 mmol 1-1 were investigated. The intensity of the signal was found to increase with increasing Ru(bpy)32+ concentration, as shown in Fig. 4. The graph depicting this relationship was sigmoidal in shape.Ru(bpy)32+ is an expensive reagent, so in order to minimize reagent consumption a concentration of 1 mmol l-1 was chosen, which produced a substantial signal. The % s, for 5 replicate measurements was less than 2.5% for the concentration range investigated. Effect of Buffer Concentration Buffer concentrations in the range 0.01-0.075 mol 1-1 were investigated by using the previously optimized conditions.Fig. 5 shows that the optimum response was gained with a buffer concentration of 0.05 mol 1-1. The % s, for 5 replicate measurements was less than 6.0% for the concentration range investigated. 0 2 4 Ru complex/mol I-’ x 10” Fig. 4 Variation of ECL intensity with Ru(bpy)3*+ concentration. 130 120 : 110 2 03 100 Y 90 .- a, c 2 80 70 60 1 I I 0.08 0.02 0.04 0.06 Buffer concentration/mol I-’ Fig.5 Variation of ECL intensity with buffer concentration.Analytical Communications, April 1996, Vol33 141 Calibration This was carried out using the optimized conditions, which are summarized in Table 1. A set of amitriptyline standard solutions in the range 1400 pmol 1-1 were analysed. The amitriptyline Table 1 Optimized conditions for amitriptyline calibration Parameter Voltage Pulse length Flow rate R~(bpy)~3+ concentration Buffer concentration PH Optimised value 1.26 V 2.5 s 2.0 ml min-' 1 mmol 1-1 0.05 mol 1-1 7 .O 3.5 , 1 x 1.5 v 0.5 I -6.5 -5.5 -4.5 -3.5 Log (amitriptyline concentration/mol I-') Fig.6 Log-log calibration graph for amitriptyline. 350 ; 300 > 250 z 6 200 .- a c Y 150 a, a 100 50 0 0 10 20 30 40 Amitriptyline concentration/mol I-' x 1 O4 Linear calilbration graph for amitriptyline.Fig. 7 was pre-mixed with the Ru(bpy)32- and injected into a buffer carrier stream. The optimized voltage was continuously applied to the working electrode. A log-log calibration (Fig. 6) was plotted by use of the resulting data. The % s, of the measurements ranged from 0.56 to 4.3, with a mean value of 2.14%.The last seven points of the calibration (0-40 pmoll-l) were used to plot a linear calibration graph (see Fig. 7). This graph was used to determine the limit of detection (LOD) as the amitriptyline concentration corresponding to a signal equal to the signal for the blank plus three times the standard deviation of the blank signal. The LOD was found to be 3.0 X moll-' (94 ppb) and the regression coefficient for the calibration was 0.9987.The sensitivity of the calibration was determined from the slope of the graph and was calculated to be 7 X lo6 mV 1 mol-1. Conclusions and Future Work Electrogenerated chemiluminescence has proved to be a suitable analytical method for the determination of amitriptyline. The method was also reproducible over a large range of amitriptyline concentrations and gave a limit of detection suitable for the detection of the drug at both therapeutic and toxic concentrations.The instrumentation could be developed as a detection system for a number of related compounds, after separation using, for example, capillary electrophoresis (CE). To enable the detection of such com- pounds in clinical samples, possible interferences from other components need to be examined. References 1 Smith, D. H., and Vernier, V. G., in New Drugs: Discovery and Development, ed. Rubin, A. A., Marcel Dekker Inc., 1978, vol. 5., p. 213. 2 Medicines: The Comprehensive Guide, ed. Morton, I., and Hall, J., Bloomsbury, 2nd edn., 1991, p.23. 3 Preskorn, S. H., Dorey, R. C., and Jerkovich, G. S., Clin. Chem., 1988, 34, 822. 4 Knight, A. W., and Greenway, G. M. Analyst, 1994, 119, 879. 5 Knight, A. W., Greenway, G. M., and Chesmore, E. D., Anal. Proc., 1995, 32, 125. Paper 61008220 Received February 5, I996 Accepted March 5,1996

ISSN:1359-7337    

DOI:10.1039/AC9963300139

出版商:RSC    

年代:1996

数据来源: RSC

摘要:

Analytical Communications, May 1996, Vol33 (143-147) 143 Analysis of Peptides by Amino Acids Com posit ion : Contribution of U It raviolet Derivative Spectrophotometry and Retention Time Prediction Emmanuel Perrin, Laurent Miclo, Alain Driou and Guy Linden Laboratoire des Biosciences de I'Aliment, Unite' associke a I'INRA Faculte' des Sciences, Universite' HP-Nancy I , BP 239,54506 Vandoeuvre-le's-Nancy CEDEX, France Amino acid composition analysis is sometimes used for the identification of peptides obtained from the hydrolysis of a protein of known sequence.Nevertheless, the interpretation of the analysis can be difficult if only one hydrochloric acid hydrolysis can be performed because of the partial destruction of some amino acids and the lack of cleavage of certain peptidic bonds.In this paper we propose combining the analysis of amino acids by two techniques (derivative UV spectrometry and retention time estimation) associated on-line with the purification step of the peptides (reversed-phase HPLC). A set of 56 peptides from the peptic and chymotryptic hydrolysis of bovine aSl-casein (asl-CN) were analysed in this manner. The difference between the theoretical and observed retention times was 1.9 min, for aromatic amino acids ratios the difference was 4.0%.The ambiguities coming from repeated residues in the sequence or the presence of Trp residues, destroyed by acidic hydrolysis, were solved and a fragment of the asl-CN sequence could be attributed to each peptide. Following the enzymic hydrolysis of a protein, the first step is to separate the peptides obtained by reversed-phase HPLC (RP- HPLC), a widely used technique.The second step is often to characterize some of the peptides or all of them. To this end, the automatic sequencing of peptides based on phenylisothiocya- nate derivatization' or the analysis of peptide mass by mass spectrometry are precise and efficient techniques.If the sequence of the protein under consideration is already known, in order to limit the cost and time of analysis, other, more simple, methods can be used. This point cannot be too strongly emphazised when the number of peptides to be identified is rather high. Among the techniques that can be used, amino acid composition analysis2 can become a powerful technique if associated with the prediction of retention time in RP-HPLC and the characterization of aromatic peptides by UV derivative spectrometry.Indeed, the identification of a peptide with an amino acid composition as the only result can be difficult. After hydrolysis in 6 mol 1-1 hydrochloric acid at 110 "C for 24 h, total destruction of Trp, a partial loss of Tyr, Ser, Thr, Cys and Met, and a resistance of peptidic bonds involving Val and Ile have been reported.3 For an accurate identification, several incuba- tion times of hydrolysis are needed, but in this case a significant amount of purified peptide must be prepared.The sequence of tryptic (E.C. 3.4.21.4) peptides often contains one arginyl or lysyl residue but, with broad specificity enzymes such as pepsin (E.C.3.4.23.1) or a-chymotrypsin (E.C. 3.4.21.1), it is not so easy to choose a reference amino acid (i.e., the amino acid, the concentration of which equals a known number of residues). Furthermore, the eventual presence of a peptidic contamination or the low quantities sometimes available after the purification steps can make interpretation difficult. In this paper, we propose a characterization of hydrolysates by using, in addition to the analysis of the composition of amino acids, the information which can be obtained on-line from the elution profile of these purified hydrolysates (retention time and UV spectrum).The solubilization of proteins and peptides in the strongly denaturing mobile phase acetonitrile-water (containing 0.1 % trifluoroacetic acid) drives them into an unfolded state.4 In the absence of residual folding, the chromatographic behaviour of peptides in RP-HPLC can then be correlated with their amino acid composition, in particular, with the sum of the relative contribution of each amino acid residue5 expressed in min (called retention coefficient, Rc).A precise estimation of the retention value of each residue is proposed by the measure of the difference between the retention time of a synthetic Ac-Gly-X- X-(Leu)3-(Lys)2-amide peptide (with substitution of X by the aminoacyl residues) and a reference substituted peptide (where X = Gln). In other work, some authors have shown that the amino acid composition is not the only parameter influencing the retention time of peptides if the chain length is greater than 15 residues.6 The predicted retention times of polymeric peptides, from 10 to 60 residues, are always overestimated with the method described above.A chain-length corrective factor must be used xC = ~ R c + t, + to - (m.ZRc.lnN + b) (1) where CRc is the sum of retention time coefficients, t, the difference between the observed retention time and the sum of the retention coefficients of a tryptic peptide chosen as standard (TTMPLW), to the retention time of an unretained compound (P-mercaptoethanol), N the number of residues of the peptide, and m and b are, respectively, the slope and the 0 intercept of the regression established (2) 'c - lobs versus ERc.lnN The recording of UV spectra during RP-HPLC analysis can be used for the confirmation of peptidic sequences containing aromatic residues.In the near-UV range, the aromatic amino acids present some characteristic absorption bands. The ratios of aromatic amino acid residues in peptides can be determined at acidic pH values by the study of derivative UV spectra between 200-300 nm.7 During RP-HPLC, the use of a 1.2 nm diode-array detector, with a mathematical resolution of 0.2 nm, allowed the transfer of the method during RP-HPLC.8 In this paper, the analysis of the composition of amino acids aided by Q-Basic software, retention time prediction in RP-HPLC and the determination of the aromatic residue ratios were considered for the identification of a set of peptides obtained from the144 Analytical Communications, May 1996, Vol33 chymotryptic and peptic hydrolysis of a sequence-knowng-11 bovine protein (a,l-CN).Materials and Methods Enzymic Hydrolysis of asl-CN Bovine a,l-CN was purified from skimmed bovine milk as described1* previously. Freeze-dried a, -CN was hydrolysed after solubilimfion, in an appropriate buffer, by insoluble a- chymotrypsin or pepsin from bovine pancreas attached to cross- linked beaded agarose (Sigma, St.Louis, MO, USA). Condi- tions for complete hydrolysis were chosen. Reversed-phase HPLC Purifications were performed using a Hitachi-Merck system with an L 6200 ternary pumping system, a Model 655A-40 automated injection and sampling system (Merck, Darmstadt, Germany), coupled with a Millipore (Malborough, MA, USA) Model 996 photodiode-array detector controlled by a 486/33i NEC (Boxborough, MA, USA) computer.Hydrolysates were run on a LichroCart C18 column (250 x 4 mm id, 5 pm particle size), obtained from Merck, and a Waters (Milford, MA, USA) Delta Pak C4 column (150 x 3.9 mm id, 5 pm particle size). Peptides were eluted with a gradient from 5 to 40% of acetonitrile (Rathbum, Walkerburn, UK) in water UHQ (Elgastat, High Wycombe, Buckinghamshire, UK) containing 0.1% (v/v) TFA (Sigma) for 70 rnin at a flow rate of 1 ml min-1.Peptides, which were partially purified after the gradient step, were re-run in isocratic conditions. The percentage of acetoni- trile used for the second isocratic step corresponded to the proportion of acetonitrile eluting the peptide in Clgor C4 gradient conditions with 2% subtracted.UV Spectral Analysis Ultraviolet spectra were recorded between 200 and 300 nm at a rate of 1 per second. The blank value was directly measured before the appearance of the peak signal. First and second derivatives were calculated by 2010 Millennium software v. 2.0.1 (Waters). Analysis of Amino Acid Composition Purified peptides were hydrolysed in 6 moll-' HCI (containing 0.5% phenol and 0.1 % 2-mercaptoethanol) at 1 10 k 2 "C during 24 h.The amino acid compositions were performed with a Biotronik LC 3000 analyser (Munich, Germany). Results Peptides Purification Fifty-six peptides were purified from the chymotryptic and peptic hydrolysate of a, l-CN after the purification steps. The individual retention times in C 18 acetonitrile gradient conditions were noted and the spectral data of the purified peptides was acquired in the range 200-300 nm.Analysis of the Amino Acids Composition The choice of the reference residue was the main difficulty for the determination of peptidic sequences from the results of the amino acid composition. A Q-BASIC software has been developed in our laboratory with the aim of resolving more easily the interpretation of amino acid analysis (unpublished results).The program compares the experimental amino acid analysis with theoretical analyses, calculated from different peptidic sequences of the native protein, and gives a matching score. The sequence of the a,l- CN, which presented the best score (i.e., the best matching with experimental amino acid composition), could be assigned to the supposed peptide.Software contribution in sequence identifica- tion was particularly helpful in the determination of the sequence of peptides with long chains. Nevertheless, some ambiguities in the identification of certain peptides remained. Confirmation of Supposed Sequences Spectrometric analysis The characteristic UV spectrum of the peptide was acquired when the peak was at the highest purity.First and second derivative spectra of the peptides were calculated. The presence of tryptophan had first to be checked and eventually added to the supposed sequence. The presence of tyrosine and phenylalanine was verified. Trp : Tyr, Trp : Phe and Tyr : Phe ratios were calculated (Tables 1 and 2) so that the Trp concentration could be calculated after analysis of amino acid composition.7,8.The difference between theoretical and experimental ratios was 4.0% on average Estimation of the retention time oj the peptides in RP-HPLC The a,] -CN tryptic peptides previously characterized by mass spectrometry (Fast Atom Bombardment and Ion-spray) and amino acid analysis were used for plotting the graph Z-tobs versus 'CRc.lnN with a view to determining the values of the slope (m) and the 0-intercept (b).However, the peptides which presented a CRc.lnN of less than 25 rnin did not seem to fit on the regression line (Dr. L. Miclo, personal communication). The case of such hydrophilic peptides is not mentioned in previous retention time stu- dies .73 Two regressions were then used for the determination of the m and b parameters.For the peptides presenting a CRc.lnN < 25 min, the regression gives m = 0.138 and b = 1.904; for the peptides with 2Rc.lnN > 25 min, m = 0.218 and h = - 11.778. The comparisons of the estimated and observed retention times are shown in Tables 1 and 2. The average difference between theoretical retention times and observed (Table 1 and 2) was 1.9 k 1.1 min Discussion The identification of the sequence of 56 peptides was performed by associating the results of the amino acid analysis with the two methods described above (i.e., retention time prediction and UV derivative spectrometry analysis).For example, the spectral determination of the ratios of the aromatic residues was particularly useful for the characterization of the Trp-containing peptides because this residue cannot be quantified by the analysis of amino acid composition following hydrolysis with hydrochloric acid.This additional characterization became essential in the determination of the peptic sequence CNasl- f(197-199) (Table 2). Indeed, the analysis of the amino acids composition yielded two residues at the same concentration, Leu and Pro, corresponding to CNaSl -f( 1 1-1 2), CNa, -f( 168- 169) or CNa,l-f( 197-198).The presence of tryptophan, a third residue, was affirmed by the zero-, first- and second-order spectral analysis. The characterization of the CNa,l -f( 197-1 99) sequence was also confirmed by the retention time study because this peptide eluted at 45.6 rnin (predicted at 43.7 min), whereas Pro-Leu or Leu-Pro were predicted at 22.2 min.For peptides containing two or three kinds of aromatic residues, the ratios allowed for the determination of Trp- concentration from the results of the analysis of amino acid composition. For peptides presenting a particular amino acid residue ratio, such as the chymotryptic fragment CNaSl -f( 174- 199) (Trp: Phe 1 : l), the spectral analysis covers the a,l-CNAnalytical Communications, May 1996, Vol33 145 region containing the peptidic sequence.Likewise, the spectral analysis, bringing to light the presence of the three aromatic residues in a high ratio, led to the identification of a long chain peptide [Trp: Tyr : Phe 1 : 3 : 1 for CNaSl-f(165-199)]. The analysis of the amino acid composition of two distinct peptides in the elution profile gave the determination of the same aSl-CN sequence. The only significant difference concer- ned the concentration of Met.We demonstrated, by induced Table 1 Experimentally detenninated sequence, retention time and aromatic residues content and ratios of a, -CN chymotryptic peptides Aromatic content Experimental Retention time/min Sequence Theoreti c a1 experimentally determined Trp Tyr Phe Trp Tyr Phe Observed Predicted Difference 1.2 2.5 2.0 -2.7 2.8 1.1 -2.0 0.0 -2.6 1.7 -3.0 -0.9 -2.2 -3.4 -2.9 -2.2 -2.0 3 .O -0.8 2.9 2.6 2.4 1.6 2.6 1 .o 0.9 -0.9 102-104 1 -9 170-173 100-104 12 1-1 27 22-23 78-9 1 15 1-154 166-173 12 1-144ox* 165-1 73 155-164 121-144 165-169 1-20 155-1 65 153-156 25-32 22-24 174-1 99 145-150 92-99 165-1990~" 165-199 143-150 24-32 128-145 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0 0 1 1 0 0 0 1 0 1 0 1 0 1 1 2 0 1 3 1 1 2 0 2 1 0 0 1 1 3 3 2 0 1 0 0 0 0 0 1 0 1 0 2 0 0 0 0 0 0 0 1 2 1 2 0 1 1 2 3 1 13.1 13.1 15.3 17.6 23.5 25.6 30.2 31.1 37.5 42.9 42.9 43.3 43.9 45.0 45.0 45.7 47.6 50.5 53.8 57.0 58.7 58.7 61.3 62.8 62.8 62.8 64.2 11.9 10.6 13.3 20.3 20.7 24.5 32.2 31.1 40.1 44.6 45.9 44.2 46.1 48.4 47.9 47.9 49.6 47.5 54.6 54.1 56.1 59.6 58.7 61.2 60.2 61.8 63.3 - - + - 1 1.07 - - + - - + + + - - - - 1 0.97 - - - + + - - - - - 1 2.02 - - 1 0.91 1 - 0.98 - 0.48 1 1 2.95 0.99 1 3.01 0.96 - 1 1.01 - 1 0.92 - - + - - + - - + * Peptide containing one oxidized methionine.Table 2 Experimentally determinated sequence, retention time and aromatic residues content and ratios of aSI -CN peptic peptides Aromatic content Sequence experimentally determined 33-39 190-196 154-156 180-189 150-153 165- I67 128-142 165-172 143-145 146-149 21-23 1-16 157-164 11 1-141 99- 109 25-3 1 90-95 170-196 197-199 143- 149 25-32 24-35 24-32 Theoretical Experimental Retention time/min Observed Predicted Difference Trp Tyr Phe Trp Phe 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 1 2 0 0 0 2 1 1 0 0 1 0 1 0 2 1 0 2 0 0 0 0 0 0 0 0 2 0 0 1 0 1 0 0 0 0 1 0 1 0 1 2 3 3 9.7 14.6 31.2 32.2 33.8 34.0 37.4 37.4 37.4 37.4 38.5 38.5 38.5 41.5 42.0 44.7 44.7 48.4 45.6 51.3 53.2 55.8 62.7 9.8 13.7 34.1 28.5 33.2 37.7 40.3 40.1 37.2 37.8 38.7 41.5 39.1 44.7 46.9 45.6 47.0 45.7 43.7 52.7 54.6 54.7 61.8 -0.1 0.9 -2.9 3.7 0.6 -3.7 -2.9 -2.7 0.2 -0.4 -0.2 -3.0 -0.6 -3.2 -4.9 -0.9 -2.3 2.7 1.9 -1.4 -1.4 -0.6 0.9 + + - + 1 + - - 0.94 - 0.92 - + - + - + - 0.89 1 - + + + + 1 2.04 - -146 Analytical Communications, May 1996, Vol33 oxidation of the hydrolysate with 1 % hydrogen peroxide,13 that Met-containing peptides decreased their hydrophobic character and, hence, their retention time.Mass spectroscopic analysis showed that the peptide molecular weight was increased by 16 following oxidation, corresponding to an oxygen atom.The identification of the oxidized, Met-containing peptides of the tryptic hydrolysate, induced by hydrogen peroxide, allowed the determination of the retention coefficient of methionine- sulfoxide (Rc = -0.5 instead of 5.5 min). The calculated Rc(Met-SO) was successfully used for the prediction of the retention times of CNa,l-f( 121-144) and CNa,l-f( 165-199) containing an oxidized methionine (Table 1).The 23-32 region of bovine a, 1 -casein (23FFVAPFPQVF32) contains four phenylalanyl residues. This region is highly hydrophobic and showed a partial hydrolysis of the potential sites of cleavage by pepsin and chymotrypsin. The interpreta- tion of the analysis of amino acid composition of the peptides Table 3 Confirmation of the sequences of the peptides from the chymotryptic and peptic hydrolysis of the 24-35 region of (x,,-CN by estimation of the retention time of the different theoretical peptides Purified peptide 0 bserved Retention time/min 55.4 44.7 62.7 55.8 Aromatic residues determined Phe Phe Phe Phe Confirmation of peptidic sequence 25-32 or 24-31 25-31 24-32 24-35 Theoretical Retention time/min 54.6 45.6 61.8 56.4 Aromatic residues content 2 Phe 1 Phe 3 Phe 3 Phe Table 4 Retention time prediction of peptides with NH2 terminal arginine residue Observed Predicted* Predicted: retention retention retention Sequence time/min time/min time/min A l/min A2/min 1-3* 5.3 10.2 3.6 -4.9 1.7 1-9 13.1 15.9 10.6 -2.8 2.5 1-20 45.7 47.9 45.3 2.2 0.4 1-16 38.5 44.6 41.5 -6.1 -3.0 90-95 44.7 51.7 47.0 -7.0 -2.3 151-154 31.1 38.1 32.6 -7.0 -1.5 Mean 5.0 1.9 * -3.0 min taken for the estimation of the contribution of the a-amino group (Guo et al.5). t -6.9 rnin taken for the estimation of the contribution of the a-amino group.I Tryptic peptide. Table 5 Estimation of the retention coefficient of phosphoseryl residues of peptic and chymotryptic peptides Observed retention Sequence time/min 41-52 15.9 105-120 41.1 109-127 38.5 64-9 1 31.1 111-141 41.5 33-91 43.3 Calculated retention coefficient Number of Number of of SerP/min SerP residues -6.1 2 12 - 18.7 I 16 -11.8 1 19 -5.8 5 28 - 16.8 1 31 -1.0 7 58 from this region had to be confirmed.The spectral analysis showed the presence of phenylalanyl residues, but could not give the number of residues.Nevertheless, the sequence of the peptides was attributed, without ambiguity, after the prediction of the retention time because of the high retention coefficient of phenylalanine5 (Rc = 8.0 min). The study did not allow for the differentiation of CNaSl-f(24-3 1) and CNaSl-f(25-32), which have the same amino acid composition, but, CNa,l-f(25-32) was chosen because this peptide corresponds to an enzymatic site-specific cleavage (Table 3).The same reasoning was applied to the tyrosine-containing peptides from the 165YYVPLGTQY 173 sequence [i.e., CNa,l-f(165-167), CNa,l-f( 165-169), CNa,l-f( 165-172), CNaSl-f( 165-173) and According to the authors,6 the charged a-amino group of an N-terminal Arg or Lys residue has a smaller effect than the a- amino group of an N-terminal residue containing an uncharged side chain6 (Rc(-NH3+) = -3.0 instead of -6.9 min).Nevertheless, we verified that the predicted retention time for the six peptides with an arginyl residue in this position was more accurate by using an Rc of -6.9 rnin for the a-amino groups as for the other residues (Table 4).The case of the post-modified amino acids is rarely mentioned in determinations of the hydrophobicity parameters of the residues.14 The six phosphoseryl residue-containing peptides were used for the calculation of the retention coefficient of the phos- phoseryl residue. Table 5 shows that no value of Rc(serP) could be estimated, but a relationship (Y = 0.81) between the calculated coefficient and the ratio number of SerP : number of residues could be established.The hydrophilic effect (expressed by the difference between the predicted retention time obtained without taking into account the phosphatidic group and the observed retention time) decreased in the presence of several SerP in the peptide. However, the hydrophilic effect measured for the SerP could be altered by the chelation of a cation.The conditions of the pH used (1.9) were approximately the value of the pK, (2.0) of the most acidic hydroxyl function of the phosphatidic group. In the presence of two repeated SerP, as in the sequence of aSl-CN, the chelation of a di-cation reduced the number of effective charges and, thus, the effect of the residues on the retention time.Two real-time methods, prediction of the retention times and determination of the aromatic residue content of peptides, were associated with the RP-HPLC purification. We have shown how this information, used with the analysis of amino acid composition, was generally sufficient to attribute a sequence to each purified peptide. CNa, 1 -f( 170-1 73)]. We thank Pamela Dubois for critical reading of the manuscript.References Edman, P., Arch. Biochem., 1949,22, 475. Moore, S. and Stein, W. H., inklethods in Enzymology, ed. Colowick, S. P., and Kaplan, N. O., Academic Press, New York, 1963, vol VI. Zumwalt, R. W., Absheer, J. S., Kaiser, F. E., and Gehrke, C. W., J . Assoc. Ofl. Anal. Chem., 1987, 70, 147. Benedek, K., Dong, S., and Karger, B. L., J . Chromatogr., 1984,317, 1227. Guo, D., Mant, C. T., Taneja, A. K., Parker, J. M. R., and Hodges, R. S., J . Chromatogr., 1986, 359, 499. Mant, C. T., Lome Burk, T. W., Black, J. A., and Hodges, R. S., J . Chromatogr., 1988, 458, 193. Miclo, L., Pemn, E., Driou, A., Mellet, M., and Linden, G., Znt. J . Pept. Protein Res.., 1995, 46, 186. Perrin, E., Miclo, L., Driou, A., and Linden, G., J . Chromatogr., Biomed. Appl., 1995, 664, 267.Analytical Communications, May 1996, Vol33 147 9 Eigel, W. N., Buttler, J. E., Ernstrom, C. A., Farrell, H. M., Jr., Harwalkar, V. R., Jennes, R., Christensen, J. A., Engelstoft, M., and Shou, H., J . Dairy Sci, 1984, 67, 1599. Nagao, M., Maki, M., Sasaki, R., and Chiba, H., Agric. Bid. Chem., 1984,48, 1663. Mercier, J. C., Grosclaude, F., and Ribadeau-Dumas, B., Eur. J . Biol. Chem., 1971,23,41. Sanogo, T., Piquet, D., Aubert, F., and Linden G., J . Dairy Sci., 1989, 72, 2242. 10 11 12 13 Cheftel, J. C., Cuq, J. L., and Lorient, D., in Prote'ines alimentaires, ed. Cheftel, J. C., Cuq, J. L., and Lorient, D., Lavoisier Paris, France, Van de Waterbeemed, H., Karajiannis, H., and El Tayar, N., Amino Acids, 1994, 7, 129. Paper 6100896H Received February 7,1996 Accepted March 7,1996 1985, pp. 255-297. 14

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DOI:10.1039/AC9963300143

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年代:1996

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Analytical Communications, April 1996, Vol33 149 ERRATUM Use of Ruthenium-(Ethylenedinitri1o)- tetraacetic Acid Monohydrate Ion Immobilized on Zirconium(iv) Oxide Coated Silica Gel Surface as an Amperometric Sensor for Oxygen in Water Carlos R. M. Peixoto, Lauro T. Kubota and Yoshitaka Gushikem Anal. Proc., 1995, 32, 503 The title of this communication should read as above. Owing to a typographical error ‘ethylenedinitrilo’ appeared as ‘ethylenedinitrito’ in the printed issue.

ISSN:1359-7337    

DOI:10.1039/AC9963300149

出版商:RSC    

年代:1996

数据来源: RSC

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Analytical Communications, April 1996, Vol33 151 Consequences of De-skilling the Analytical Laboratory Mike Sargent Laboratory of the Government Chemist, Queens Road, Teddington, Middlesex, UK TWll OLY I recently interviewed a number of senior managers of analytical laboratories, many of whom expressed concern regarding the future of the analytical sector. Their con- cern was not, as some of you might have guessed, simply their financial situation, but was, in my view, rather more fun- damental.Many interviewees noted that key aspects of their operations now depend on a single, experienced individual. This was contrasted with the situation just a few years ago when most analytical teams could offer in-depth expertise and a clear line of succession to take that expertise forward into the future.Many senior analysts and managers now believe that it will soon become increasingly difficult either to recruit experienced staff or to find others suitably experienced to train be- ginners. One does not need to look far to pinpoint the main factor that has brought about this situation. Traditionally, cornpe tition within the analytical sector has been lim- ited, with the emphasis on long-established in-house facilities complemented by a small number of specialist contract organi- zations.Even the latter have largely occu- pied their own niche and experienced only limited competition. This relatively comfortable world has changed dramat- ically in the last five to ten years. Wide- spread interest in ‘market testing’ (by government) or ‘contracting out’ (by in- dustry) has raised awareness of the possi- bilities for competition throughout the sector.This has affected the operations of not only contract laboratories but also in- house laboratories in both private sector and public organizations. Thus, margins have tightened, leading to cutbacks in staff and activities such as method develop- ment.In addition, much more time is devoted to ‘commercial’ activities, such as preparing tenders or writing project pro- posals, rather than on training or develop- ing analytical science. There is no doubt that the new commer- cial approach is here to stay. Thus, few laboratories are likely to see a return to large teams of analysts able to accumulate many years of experience in a particular application area.Extensive recruitment of school leavers or new graduates, supported by general education or in-service training programmes, also seems to be a thing of the past. We need, therefore, to examine the long-term consequences of these changes, particularly whether they will damage our capability to deliver reliable measurements. As many readers will be aware, the VAM Initiative encapsulates the way to make reliable analytical measurements in six ‘VAM principles’.One of these states that: ‘Staff making analytical measure- ments should be both qualified and compe- tent to undertake the task’. What does this mean? Essentially, that all analysts must have the knowledge required to understand the analytical problem and choose an appropriate procedure, and the skill to carry it out reliably and effectively.Refer- ring again to the wording of the VAM principles: ‘Reliable measurements can only be achieved by staff who understand what they are doing, why they are doing it and the likely effect of making changes to a method. This is true even for well documented methods, and for automated instruments’. It is widely recognized that academic qualifications, even those which result from a specialized analytical chem- istry course, only partially satisfy this need.They underpin the essential voca- tional knowledge and practical skills, which can be acquired only by working on real applications alongside experienced analysts. In the UK, this need was traditionally met through a two-pronged approach, which involved the recruitment of both school leavers and graduates. Most com- monly, these recruits would have a chem- istry qualification, usually at ‘A’ level or graduate (BSc, MSc, etc) level.Able school leavers were encouraged to seek employment as technicians in analytical laboratories by the willingness of many companies to support further education. Thus, many present-day senior analysts and managers started their careers in this way and achieved HNC or graduate qual- ifications in chemistry through part-time study. Regardless of their background, the great majority of today’s professional ana- lysts have, in effect, served an ‘apprentice- ship’, which ensured that they were indeed both qualified and competent to undertake a wide range of tasks in particular applica- tion areas of analytical chemistry.The trends mentioned above give a clear indica- I 1 VALID ANALYTICAL MEASUREMENT tion that, unless we take action now, the same will not be true for many of their successors. Of course, it may be possible to work around the problem. There are those, for example, who believe that we can use information technology, ‘expert systems’, to capture the knowledge of today’s ex- perts and make it available to future generations.Personally, I am not con- vinced. Such systems may well improve the productivity or the range of human experts but cannot replace them nor push forward the boundaries of knowledge. Others suggest that advances in instru- mentation will avoid many of today’s problems, such as sample preparation, and reduce or even remove the need for human expertise.Again, I remain sceptical. De- velopments in instrumentation over the past 30 years have solved many problems but also encouraged new demands for analysis, so that life remains as difficult as ever for the analyst. So, if technology offers no easy solutions what can be done? Recent conversations with colleagues highlighted two approaches which they believe can make a contribution.David Rudd, Head of Analytical Evalua- tion for Glaxo Wellcome Research and Development at Ware, believes that even the largest employers of analysts will be unable to return to the days of large, general training programmes. They are, therefore, looking at ways to identify specific needs for additional knowledge or expertise, with particular emphasis on asking the analysts themselves.An early result has been to highlight a lack of appropriate training in the use of the computer spreadsheets which are now so widely applied in the analytical laboratory for data reduction. Dai Bevan, Laboratory Head for Chem- ical Analysis at the European Research Division of Kodak in Harrow, is support- ing implementation of NVQs in Labo- ratory Operations through a consortium of local companies and further education colleges.The key aspect of such schemes is to provide staff, regardless of their level of academic attainment, with a clear, inde- pendent statement of their vocational achievements. This is important because Kodak, for example, are prepared to take school leavers and support their involve-152 Analytical Communications, April 1996, Vol33 ment in NVQs or to take new graduates into the same scheme for an initial period of two years.This provides Kodak with a pool of technicians and allows their staff to ‘add value’ to their record of achievement should they move on. There is no doubt that these initiatives are valuable but they do not provide a universal solution. This will require an industry-wide consensus. Employers must acknowledge that sound analytical meas- urement depends on appropriate experi- ence as well as relevant academic qual- ifications. To achieve this, colleges and employers need to work together to replace traditional ways of providing ana- lysts who are truly both qualified and competent. Your views on this topic, or information on other initiatives such as those men- tioned above, would be very welcome. Mike Sargent can be contacted at LGC, tel: 0181 943 7360, fax: 0181 943 2767. Paper 6/01 336H

ISSN:1359-7337    

DOI:10.1039/AC9963300151

出版商:RSC    

年代:1996

数据来源: RSC