ISSN:0003-2654    

DOI:10.1039/AN99318BP015

出版商:RSC    

年代:1993

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN99318FX017

出版商:RSC    

年代:1993

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN99318BX019

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, MAY 1993, VOL. 118 53N Book Reviews Journal of Bioluminescence and C hemi luminescence : Studies and Applications in Biology and Medicine Vol- ume 4. Number 1. Proceedings of the Fifth International Symposium on Bioluminescence and Chemilumines- cence, September 1988 Edited by M. Pazzagli, E. Cadenas, L. J. Kricka, A. Roda and P. E. Stanley. Pp. vi + 646. Wiley. 1989. Price f92.00. ISSN 0-884-3996. This immense volume (a special issue of the journal concer- ned) contains almost 80 papers, many by well-known authors, covering every major aspect of this rapidly growing field. The exceptional sensitivity of these methods, coupled with the relatively simple instrumentation used, makes them extremely attractive in many areas of biochemical analysis, and the growing diversity of luminescent reactions is another striking feature of the area, which is well covered in this volume.The currently fashionable dioxetane systems were only just appearing at the time of this conference, so this is one of the few areas that may now seem to be less than fully covered. The papers are sensibly divided into sections, the longest inevitably and rightly being that devoted to immunoassay applications (16 papers). This section also contains a paper on time-resolved fluorescence studies: interesting, but out of place in this volume. Much later in the volume there are two separate sections devoted entirely to particular commercial systems (11 papers in all). These sections also deal with immunoassays, but any possible confusion is alleviated by the provision of a good index, a rare and welcome feature in a volume of this kind, and one on which the editors are much to be congratulated.The growing application of CL and BL methods to the study of nucleic acids is also well featured (10 papers), and other sections to catch the eye are those dealing with HPLC detection (4 papers) and with instrumental developments including biosensors (8 papers). Contributions to a conference proceedings volume of this kind are inevitably a mixed bag. Their length varies con- siderably, and reviews and general interest articles are well mixed with specific research papers. However, the over-all level is very good indeed, and matched by high production standards with very few misprints. Whether to refer to or to dip into, this is an excellent volume, and well worth having as a companion to one of the most dynamic areas of modern analytical science.James N . Miller Radiochemistry and Nuclear Methods of Analysis By William D. Ehmann and Diane E. Vance. Volume 7 76in Chemical Analysis: A Series of Monographs on Analytical Chemistry and its Applications. Pp. xviii + 532. Wiley. 1991. Price f75.00. ISBN 0-471-600-76-8. This book is volume 116 in a series of well-respected books on chemical analysis and is a welcome addition to the rather sparse literature concerning radiochemistry. The target groups for this book are defined as those at undergraduate or beginning graduate level and, in the main, the book will satisfy most of the requirements of these groups. The authors' style and clarity of writing together with the excellent presentation of the book make for easy reading and understanding although the large number of topics covered in the book results in some of them being treated rather trivially.This criticism is somewhat overcome by the inclusion of a further reading list at the end of each chapter. Chapters 1-8 cover a general introduction to radio- and nuclear chemistry and Chapters 9-14 explain radioanalytical methods and applications of the use of radionuclides. Chapter 1 starts with a very readable account of the discovery of radioactivity and chronicles the discoveries of early workers. The chapter is enhanced by the inclusion of a number of interesting photographs. Chapters 2-6 explain the fundamentals of radio- and nuclear chemistry in a straightforward, clear and concise style.Derivatations of equations are fully explained and useful example calculations are included throughout the chapters. One criticism is the continual use of the curie, the'old unit of activity, instead of the SI unit, the becquerel. Chapter 7 describes aspects of health physics, an important topic, which clearly deserves attention. However, again the examples make use of old units (rad and rem) instead of the SI units, the gray and the sievert. Chapter 8 provides a useful explanation of instrumentation used in radiochemistry although measurement techniques such as liquid scintillation counting deserve a fuller explana- tion. Nuclear activation analysis is covered well in Chapter 9 as are radiotracer methods (Chapter lo), ion beam analysis, chemical applications of radioactivity (Chapter 11) and nuclear dating methods (Chapter 12).The book concludes with chapters on the origin of the chemical elements and on particle generators. The book is clearly a compilation of lectures and is written by experienced teachers. A valuable addition to any library but I fear that the cost of the book will prohibit its purchase by students. P. Warwick Multidimensional Chromatography: Techniques and Applications Edited by Hernan J. Cortes. Chromatographic Science Series. Volume 50. Pp. viii + 378. Marcel Dekker. 1990. Price US $99.75 (USA and Canada), US $1 19.50 (export). ISBN 0-8247-81 36-8. This volume, consisting of ten chapters, two of which are by the editor, covers the theoretical and practical aspects of performing multidimensional separations, using a variety of different chromatographic techniques. These are, in brief, gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), super- critical fluid chromatography (SFC) , supercritical fluid extrac- tion (SFE) on-line with SFC, and HPLC on-line with GC.Although covering a wide variety of topics, many of which warrant and indeed have constituted complete texts in their own right, the book succeeds in illustrating the power afforded by multidimensional means and the limitations of the single dimensional approach. The theoretical case for multidimensional separation as a whole is presented appropriately in the first chapter by Giddings whose work has been instrumental in the under- standing of the process involved.Whether they are performed off-line or on-line, this chapter presents the case that multidimensional strategies are essential to achieve the required analytical resolution, when dealing with many complex matrices. Three of the remaining chapters are devoted to GC. The first by Bertsch is a contemporary evaluation of multidimen- sional GC, focusing on recent advances using capillary columns. It concentrates on the practical issue of two-dimen- sional GC and is extensively referenced. The capillary theme is maintained in the next chapter on GC, which explains the considerations to be made when tuning a GC system for54N ANALYST, MAY 1993, VOL. 118 selectivity, illustrating the logic of phase selection, phase mixing and the importance of pressure drop , compressibility effects etc.Practical examples are described principally for mixed phase and tandem coupled columns, with true multi- dimensional systems addressed towards the end. The final chapter on GC concerns its use in process control and the practical considerations to be made , including valveless column switching using both the ‘Deans-type- and ‘live-type’ systems. A single, extensively referenced chapter provides an overview of what constitutes modern-day TLC, and indicates the recent revolution within the field including overpressure TLC and circular/anticircular TLC. Theoretical considera- tions of unidimensional development are presented, with explanations and examples of continuous and multiple devel- opment, as well as two-dimensional development and bimodal chromatography.Multidimensional HPLC and HPLC-GC are covered in two chapters by the editor. Both address the practical problems, solutions and instrumental requirements of the techniques , which are illustrated by a number of selected applications. The newer techniques of multidimensional SFC and SFE- SFC are covered in two separate chapters, although the SFC-SFC chapter also briefly includes SFC-GC, LC-SFC and SFE-chromatography . Examples are restricted mainly to fossil fuel analysis, as the associated industries have initiated the early developments of the technique. The SFE-SFC chapter concentrates on microscale SFE and its application to PAH analyses. Hardware considerations for the techniques presented throughout the text, are addressed in the final chapter. This book will be useful to chromatographers who have considered attempting multidimensional separation, but have been dissuaded by the apparent complexity of the techniques, as well as those familiar with multidimensional methods. The theoretical case, convincingly presented, along with numerous practical examples, demonstrate the advantages to be gained, at the cost of an increased, yet readily achieved complexity.Colin Chappell Surfactants in Solution. Volumes 7,8,9 and 10 Edited by K. L. Mittal. Plenum Press. 1989. Price US $1 15.00 ISBN 0-306-43332-X. (Vol 7); 0-306-43333-8 (Vol 8 ) ; 0-306-43334-6 (Vol 9); 0-306-43335-4 (VOI 10). These 4 volumes contain the proceedings of the 6th Inter- national Symposium on Surfactants in Solution (SIS) held in 1986 in New Delhi and published in 1989.The 6th Symposium represents the 10th anniversary of the start of the meetings in 1976. The previous 2 meetings were held in Lund (1982), producing volumes 1, 2 and 3 in the series, and in Bordeaux (1984) resulting in volumes 4, 5 and 6. Three other meetings have been held since the one in New Delhi, in 1988 in Ottawa, 1990 in Gainsville and 1992 in Varna (Bulgaria). Surfactant science, which is the essence of the SIS meetings, is an area of physical science not well-represented in the UK although worldwide it is very much alive and well, particularly in a number of other European countries as well as in the USA. Certainly surfactants are used very widely in many commercial processes and products and so industrial interest is predictably strong.Now, the old view that colloids, of which surfactant solutions are an important example, are ‘things indefinite in shape, indefinite in chemical composition and physical properties, fickle in chemical deportment, things unfilterable and unmanageable’ is no longer tenable. Indeed, the shape (and other properties) of surfactant aggregates is the subject of much of the material of the volumes under review. Physical properties of surfactant solutions are now being widely studied, as is the way in which ‘composition’ affects the properties of surfactant systems. It does have to be admitted that surfactants can be a little unmanageable at times. Nonetheless, the detergent industry for example, does an excellent job in surfactant management in the production of a range of products with reproducible properties.The contents of these 4 volumes are wide ranging (148 papers are included) and are organized into seven sections. Part I covers aggregation of surfactants and the structure, dynamics and characterization of micelles, and Part I1 deals with biological amphiphiles. The aggregation of surfactants in apolar media is the subject of Part 111, and Part IV contains papers on micellar catalysis and reactions in surfactant solutions. Monolayers and surfactant adsorption are discussed in Part V. Microemulsions and reactions in microemulsions are dealt with in Part VI and the final part (VIT) consists of a collection of general papers that do not readily fall into any of the above categories.Volume 7 contains some of Part I, the remainder appearing in Volume 8 together with Part 11. Volume 9 contains Parts 111, IV and V, and Parts VI and VII make up Volume 10. As can be expected, the quality, nature and size of the contributions vary, although the general standard is good; all the contributions have been refereed. It will have been noticed that there is an inordinately long gap between the conference and appearance of these proceedings. Nonetheless, the authors were all given the opportunity to revise and update their contributions in 1988. There are several useful review- type articles, including NMR self-diffusion and relaxation techniques for the study of structure and dynamics of surfactant solutions (Lindman et al. , Volume 7); interfacial chemistry of bilayer lipid membranes (Tien, Volume 8); developments in the study of insoluble monolayers (Voll- hardt , Volume 9) ; preparation of monodisperse colloidal metal particles (Nagy, Volume 10); and spectroscopic tools in the study of micelles and membranes, (Zachariasse, Volume 7). Otherwise the papers are mainly concerned with experimental work with relatively few theoretical contribu- tions. Each volume contains an index and some short notes about the contributors. Also, at the front of each volume there is a list of the contents of all 4 volumes, which is useful. The books are well produced from camera-ready copy. It is a pity that one of the contributions in Volume 9 contains a very large number of unsightly hand-written equations. Because these books report work presented in 1986 (and was possibly updated in 1988), they will now be of limited use for those seeking up-to-date information on their own research areas. Nonetheless, they can be recommended for browsing and for picking up references; they will also be of interest to those wishing to familiarize themselves with areas of surfactant science peripheral to their own. R. Aveyard

ISSN:0003-2654    

DOI:10.1039/AN993180053N

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, MAY 1993, VOL. 118 55N Conference Diary Date June 2 4 2-4 3 3-4 7-9 8-1 1 13-17 13-17 14-16 14-16 14-18 14-20 16 16-18 17-18 Conference Location ICES-ELPHO ’93, Meeting of the International Council of Electrophoresis Norway Societies Sandefjord , International Symposium on Analysis of Peptides Sweden Stockholm, NMR Symposium Turku , Finland European Conference on Analytical Chemistry, Chromatography and Czechoslovakia Spectroscopy and Thermal Analysis Brno , ESIS ’93: European Seminar on Infrared Spectroscopy France Lyon, The Seventh International LIMS Conference Egham, Surrey , UK 6th European Congress on Biotechnology Firenze , Italy 3rd Scandinavian Symposium on Chemometrics Arhus , Denmark 23rd Annual Symposium on Environmental Analytical Chemistry USA Jekyll Island, GA, PREP-93, 10th International Symposium on Preparative Chromatography USA Arlington, VA, EOQ ’93 World Quality Congress: Information, Communication, Knowledge and Finland Quality European Organization for Quality Control Helsinki, Helsinki, Suomi-Finland Substances and Processes Dangerous to the Environment Lancashire, Preston, UK 5th International Symposium on Chemically Modified Surfaces USA Malvern , PA, International Conference on Analytical Toronto, Chemistry and Applied Chromatography/ Canada Spectroscopy Contact Professor Nils Olav Solum, Research Institute for Internal Medicine, Rikshopitalet, Pilestredet 32, N-0027 Oslo, Norway Tel: +47 2 868 226.Fax: +47 2 868 303 The Swedish Academy of Pharmaceutical Sciences Symposium on ‘Analysis of Peptides’, P.O.Box 1136, S-111 81 Stockholm, Sweden Tel: +46 8 24 50 85. Fax: +46 8 20 55 11 Professor J. Mattinen, Abo Akademi, Institution for Organisk Kemi, Akademig 1, SF-20500 Abo 50, Finland Dr. V. M. Bhatnagar, Alena Chemicals of Canada, P.O. Box 1779, Cornwall, Ontario, Canada K6H 5v7 Tel: +l 613 932 7702. G. Lachenal, Laboratoire d’Etudes des MatCriaux Plastiques et des BiomatCriaux, UniversitC Claude Bernard, Lyon 1, 43 Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France Tel: +33 72 43 12 11. Fax: +33 78 89 25 83 The Conference Registrar, 18 Portway Drive, West Wycombe, Buckinghamshire, UK HP12 4AU Tel: +44 494 448048. Fax: +44 494 448154 Congress Secretariat, c/o Professor Laura Frontali, Department of Cell and Developmental Biology, University of Rome ‘La Sapienze’, P.le Aldo Moro 5 , 00185 Rome, Italy Tel: +39 6 445 3950. Fax: +39 6 499 12351 SSC3 Secretariat, Department of Chemical Technology, Danish Technological Institute , Teknologiparken, DK-8000 Arhus C, Denmark Tel: +45 86 14 24 00. Fax: +45 86 14 74 45 Dr. Wayne Garrison, Athens Environmental Research Laboratory, US EPA, Athens, GA 30613, USA (for the USA) or M. Frei-Hausler, Postfach 46, CH-4123 Allschwil 2, Switzerland (for Europe) Ms. Janet Cunningham, Barr Enterprises, P.O. Box 279, Walkersville, MD 21793, USA Tel: + 1 301 898 3772. Fax: + 1.301 898 5596 Ann-Kristin Bergh, EOQ ’93 Congress Secretariat, P.O. Box 1, SF-02731 Espoo, Finland Tel: +358 (9)800 08254. Fax: +358 0 509 1807 Mrs. Tarja Jalasto, Finnish Society for Quality Control, Laaksolahdentie 41, P.O.Box 1, SF-02730 Espoo , Suomi-Finland Tel: +358 800 08254. Fax: +358 0 509 1807 Dr. Paul Illing, Health and Safety Executive, R425 Magdalen House , Stanley Precinct , Bootle , UK L20 3QZ Tel: +44 51 951 3420. Fax: +44 51 922 7918 Dr. Ivan E. Leigh, CertainTeed Corp., 1400 Union Meeting Road, P.O. Box 1100, Blue Bell, PA Tel: + 1 215 341-6622. Fax: + 1 215 341-6291 Dr. V. M. Bhatnagar, Alena Chemicals of Canada, P.O. Box 1779, Cornwall, Ontario, Canada K6H 5v7 Tel: +1 613 932 7702. 19422-0761, USA56N ANALYST, MAY 1993, VOL. 118 Date 27-117 28-29 29 29 29-417 July 4-6 4-6 4-7 4-7 4-8 4-9 4-9 7 11-14 11-15 11-15 Conference Fullerenes '93, 1st International Interdisciplinary Colloquium on the Science and Technology of the Fullerenes XXVIII CSI Pre-Symposium: Analytical Spectroscopy in the Earth Sciences XXVIII CSI Pre-Symposium: Introductory Chemometrics XXVIII CSI Pre-Symposium: Vapour Generation Techniques XXVIII Colloquium Spectroscopicum Internationale XXVIII CSI Post-Symposium: Spectroscopic Data-Handling XXVIII CSI Post-Symposium: 5th Surrey Conference on Plasma Source Mass Spectrometry XXVIII CSI Post-Symposium: Graphite Atomizer Techniques in Analytical Spectroscopy XXVIII CSI Post-Symposium: Analytical Location Santa Barbara, CA, USA Kingston, UK York, UK York, UK York, UK York, UK Durham, UK Durham, UK York, Applications of Glow Discharges in Optical and UK Mass Spectrometry 6th International Conference on Indoor Air Quality and Climate, Indoor Air'93 11th International Meeting on NMR Spectroscopy UK Helsinki, Suomi-Finland Swansea, 22nd Meeting of the Federation of European Biochemical Societies Sweden Stockholm, XXVIII CSI Post-Symposium: Trace Elements Durham, in Clinical Chemistry UK 6th International Symposium on Polymer Analysis and Characterization Greece Crete, 25th Conference of the European Group for Atomic Spectroscopy France Caen, Chemometrics 111, 3rd Czechoslovak Brno, Chemometrics Conference Czechoslovakia Contact Gill Spear, Pergamon Seminars, c/o Elsevier Advanced Technology, Mayfield House, 256 Banbury Road, Oxford, UK OX2 7DH; Tel: +44 865 512242.Fax: +44 865 310981 or for North America, Kim Cavellero, Pergamon Seminars, 660 White Plains Rd., Tarrytown, NY 10591-5153, USA XXVIII CSI Secretariat, Department of chemistry, Loughborough University of Technology, Leicestershire, UK L E l l 3TU Tel: +44 509 222575.Fax: +44 509 233163 XXVIII CSI Secretariat, Department of Chemistry, Loughborough University of Technology, Leicestershire, UK L E l l 3TU Tel: +44 509 222575. Fax: +44 509 233163 XXVIII CSI Secretariat, Department of Chemistry, Loughborough University of Technology, Leicestershire, UK LE11 3TU Tel: +44 509 222575. Fax: +44 509 233163 Dr. B. L. Sharp, Loughborough University of Technology, Department of Chemistry, Loughborough, Leicestershire, UK LE11 3TU XXVIII CSI Secretariat, Department of Chemistry, Loughborough University of Technology, Leicestershire, UK L E l l 3TU Tel: +44 509 222575. Fax: +44 509 233163 Kym E. Jarvis or John G. Williams, NERC ICP-MS Facility, Department of Geology, Royal Holloway College, Egham, Surrey, UK TW20 OEX Tel: +44 784 44383514.Fax: +44 784 443836 XXVIII CSI Post-Symposium, Department of Chemistry, (CSI Secretariat), Loughborough University of Technology, Loughborough, Leicestershire, UK L E l l 3TU Tel: +44 509 22575. Fax: +44 509 233163 XXVIII CSI Secretariat, , Department of Chemistry, Loughborough University of Technology, Leicestershire, UK L E l l 3TU Tel: +44 509 222575. Fax: +44 509 233163 Professor Olli Seppanen, SF-02150 Espoo , Finland Dr. J. F. Gibson, Secretary (Scientific), The Royal Society of Chemistry, Burlington House, Piccadilly , London, UK W1V OBN Tel: +44 71 437 8656. Fax: +44 71 437 8883 Dr. Stefan Nordlund, FEBS '93, Department of Biochemistry, Arrhenius Laboratories, Stockholm University, S-10691 Stockholm, Sweden XXVIII CSI Secretariat, Department of Chemistry, Loughborough University of Technology, Leicestershire, UK L E l l 3TU Tel: +44 509 222575.Fax: +44 509 233163 Judith A. Sjoberg, Professional Association Management, 815 Don Gaspar, Sante Fe, NM 87501, USA Tel: +l 505 989 4735. Fax: +1 505 989 1073 25th EGAS Secretariat, IS-MRA, Labo. Spectro. Atom., Boulevard MarCchal Juin, F-14050 Caen, France Dr. Josef Havel, Department of Analytical Chemistry, Masaryk University, Kotlarska 2, CS- 61137 Brno, Czechoslovakia Tel: +42 5 712984. Fax: +42 5 740108ANALYST, MAY 1993, VOL. 118 57N Date 12-14 19-21 19-23 26-29 Conference R & D Topics Meeting 1993 6th Symposium on Handling of Environmental and Biological Samples in Chromatography 12th International Symposium on Nuclear Quadrupole Interactions August 107th AOAC International Annual Meeting and Exposition 9-1 1 9-13 9-13 22-25 22-27 22-27 22-27 23-27 23-27 23-27 23-27 26-119 3rd Soil and Sediment Residue Analysis Workshop Asianalysis 11: Second Asian Conference on Analytical Chemistry ILC '93: International Conference on Luminescence and Optical Spectroscopy on Condensed Matter EUROTOX'93 (32nd Congress of Toxicology) Gordon Research Conference on Reactive Polymers, Ion-exchangers and Adsorbents 206th ACS National Meeting (with Sessions of the Divisions of Analytical Chemistry, Environmental Chemistry, Chemical Health and Safety, etc.) Third International Symposium on Separation Technology 9th Meeting of EURO CVD 9th International Conference on Fourier Transform Spectroscopy 6th Hungaro-Italian Symposium on Spectrochemistry , Advances in Environmental Sciences 9th Danube Symposium on Chromatography 5th International Conference on Electron Spectroscopy Location Bradford, W.Yorkshire, UK Guildford, Surrey, UK Zurich, Switzerland Washington, DC, USA Winnipeg, Manitoba, Canada Changchun, China Storrs, CT, USA Uppsala, Sweden Newport, RI, USA Chicago, IL, USA Antwerp, Belgium Tampere, Finland Calgary, Alberta, Canada Lillafiired, Hungary Budapest, Hungary Kiev, Ukraine Contact Miss P. Hutchinson, Analytical Division, The Royal Society of Chemistry, Burlington House, Piccadilly , London, UK W1V OBN Tel: +44 71 437 8656. Fax: +44 71 734 1227 M. Frei-Hausler, IAEAC Secretariat, Postfach 46, CH-4123 Allschwil2, Switzerland Tel: +41 61 632789.Fax: +41 61 4820805 Professor D. Brinkmann, Physik-Institut , University of Zurich, Schonberggasse 9, CH-8001 Zurich, Switzerland Margaret Ridgell, AOAC, 2200 Wilson Boulevard, Suite 400, Arlington, VA 22201-3301, USA Dr. G. R. Barrie Webster, Pesticide Research Laboratory, Department of Soil Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2. Tel: + 1 204 474 6039. Fax: + 1 204 275 6019; or Professor Dr. Joseph Tarradellas, IGE, Federal Technical Institute EPF-L, CH-1015 Lausanne Ecublens, Switzerland Professor Erkang Wang, Asianalysis 11, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, P.O. Box 1022, Changchun, Jilin 130022, China Tel: +86 431 682 801 (ext. 562). Fax: +86 431 685 653 Professor Douglas Hamilton, Physics Department, 2152 Hillside Road, University of Connecticut, Storrs, CT 06269-3046, USA Dr.R. A. Ettlin, EUROTOX Secretary General, Sandoz Pharma Ltd., Toxicology, Building 881, P.O. Box, CH-4002 Basle, Switzerland Professor Cs. HorvBth, Department of Chemical Engineering, Yale University, P.O. Box 2159, Yale Station, New Haven, CT 06520, USA Tel: +1203 432 2217. Fax: +1 203 432 4360 Mr. B. R. Hodson, American Chemical Society, 115516th Street N.W., Washington, DC 20036, USA Tel: + 1 202 872 4396. Mrs. M. Stalmans, University of Antwerp (UIA), Department of Chemistry, Universiteitsplein 1, B-2610 Antwerp- Wilrij k, Belgium Tel: +32 3 820 23 75. Fax: +32 3 820 23 74 Ms. Raili Siekkinen, Tampere University of Technology, P.O. Box 527, SF-33101, Tampere, Finland Lois Kokoski, Conference Office, The University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4 Tel: +1 403 220 5051.Fax: +1 403 284 5696 Dr. Gy. Zhray, Institute of Inorganic and Organic Chemistry, Eotvos University, P.O. Box 32, H-1518 Budapest 112, Hungary Professor L. Szepesy, Hungarian Chemical Society, Budapest, Hungary Tel: +36 1 186 9000. Fax: +36 1 181 2755 J. J. Pireaux, LISE, rue de Bruxelles, 61, B-5000 Namur, Belgium58N ANALYST, MAY 1993, VOL. 118 Date 29-319 30-119 30-219 30419 31-119 Conference Location 9th International Symposium: Advances and Application of Chromatography in Industry Bratislava, Czechoslovakia 15th International Symposium on Safety in Interaction with Quality, Productivity and Economy Second European Symposium on Near Infrared Spectroscopy Denmark Lugano, Switzerland Kolding , 13th European Conference on Surface Science Warwick, UK 106th Annual International Meeting and Exposition of AOAC International Czechoslovakia Prague, September 2-3 5-10 5-10 5-10 5-1 1 5-1 1 6-10 6-10 6-10 6-10 7-8 7-10 2nd UK International Meeting on Biological and Biomedical Applications of Scanning Probe Microscopy Nottingham, UK Ninth International Biodeterioration and Leeds, Biodegradation Symposium UK 5th European Conference on the Spectroscopy Lontraki, of Biological Molecules Greece Second International Conference on the Biogeochemistry of Trace Elements Taipei, Taiwan, Republic of China Euroanalysis VIII: European Conference on Analytical Chemistry UK Edinburgh, Pharmacy World Congress '93 Tokyo, Japan 18th International Conference on Infrared and Colchester , Millimetre Waves UK Defect Recognition and Image Processing in Santander, Semiconductors and Devices Spain 11th Specialised Colloque Ampere on Magnetic Menton, Resonance in Homogeneous and France Heterogeneous Catalysis Second International Conference on Magnetic Heidelberg, Resonance Microscopy Germany Environmental Fate of Chemicals Lancaster, 12th International Symposium on Biomedical Applications of Chromatography and Electrophoresis and 2nd International Symposium on the Applications of HPLC in Enzyme Chemistry UK Verona and Soave, Italy Contact Assoc.Prof. Jozef Polonsky, Department of Analytical Chemistry, Slovak Technical University, Radlinskkho 9, 812 37 Bratislava, Czechoslovakia Tel: +42 7 560 43.Fax: +42 7 49 31 98 Secretariate ISSA, Section Chemistry, c/o BG Chemie, P.O. Box 10 14 80, D-W-6900Heidelberg 1, Germany Lone Vejgaard, Biotechnological Institute, Holbergsvej 10, P.O. Box 818, DK-6000 Kolding, Denmark Tel: +45 75520433. Fax: +45 75529989 Dr. C. F. McConville, ECOSS-13, Department of Physics, University of Warwick, Coventry, UK CV4 7AL Tel: +44 203 523353. Fax: +44 203 692016 J. Barek, Department of Analytical Chemistry, Charles University, Albertov 2030, 12840 Prague 2, Czechoslovakia Tel: +42 2 292051, +42 2 297541. Fax: +42 2 291958 The SPM Laboratory, Department of Pharmaceutical Sciences, University of Nottingham, Nottingham, UK NG7 2RD Tel: +44 602 515101. Fax: +44 602 515102 The Conference Secretary (RE), Department of Chemical Engineering, The University of Leeds, Leeds, UK LS2 9JT Professor Th.Theophanides, National Technical University of Athens, Department of Chemical Engineering, Zogratou 15780, Athens, Greece Dr. Shang-Shyng Yang, Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 106, Republic of China; or Dr. Domy C. Adriano, University of Georgia, Savannah River Ecology Laboratory, Drawer E, Aiken, SC 29802, USA Miss P. E. Hutchinson, Analytical Division, The Royal Society of Chemistry, Burlington House, Piccadilly, London, UK W1V OBN Tel: +44 71 437 8656. Fax: +44 71 734 1227 Professor D. J. A. Crommelin, FIP Congress Department, The Hague, The Netherlands Tel: +3170 363 1925.Fax: +3170 363 3914 Professor T. J. Parker, Department of Physics, University of Essex, Wivenhoe Park, Colchester, UK C04 3SQ Dr. Juan JimCnez, Drip 5, Universidad de Valladolid, 47011 Valladolid, Spain Professor J. Fraissard, Laboratoire de Chimie des Surfaces, Universitk P. et M. Curie, 4, Place Jussieu (Boite 196), 75252 Paris Cedex 05, France Dr. Bernhard Blumich, c/o Max Planck-Institute fur Polymerforschung, Postfach 3148, D-6500 Mainz, Germany Dr. D. Osborn, Institute of Terrestrial Ecology, Monks, Abbots Ripton, Huntingdon, UK PE17 2LS Dr. Franco Tagliaro, Scientific Secretariat, c/o Istituto di Medicina Legale, Policlinico Borgo Roma, 1-37134 Verona, Italy Tel: +39 45 8074 618. Fax: +39 45 505 259ANALYST, MAY 1993, VOL. 118 59N Date 7-12 8-10 12-17 13-17 13-17 19-22 19-22 20-24 20-26 21-22 21-23 22-24 26-1/10 30 Conference 12th International Symposium on Microchemical Techniques 4th Workshop on Chemistry and Fate of Modern Pesticides and Related Pollutants 9th International Conference on Heavy Metals in the Environment International Conference on Nuclear Analytical Methods in the Life Sciences Workshop in Liquid Scintillation Counting 4th International Symposium on Chiral Discrimination 2nd National Symposium on Planar Chromatography: Modern Thin-Layer Chromatography Dioxin 93: 13th International Symposium on Chlorinated Dioxins and Related Compounds 173rd Annual Meeting of the Swiss Academy of Natural Sciences (including Symposia of the Swiss Society for Analytical and Applied Chemistry, the Swiss Society for Microchemistry and Instrumental Analysis, the Swiss Association on Environmental Research, and other Societies, in German and French) 4th German Symposium on Near Infrared Spectroscopy The Royal Society of Chemistry 1993 Autumn Meeting XIIth Conference of Analytical Chemistry of Romania 12th Australian Symposium on Analytical Chemistry incorporating 3rd Environmental Chemistry Conference Duration of Repeated Dose Toxicity Studies- A Commonsense Approach? October 4-8 ECASIA 93, 5th Conference on Application of Surface and Interface Analysis 5-7 Laboratory Exhibition and Conference Locat ion Cordoba, Spain Prague, Czechoslovakia Toronto, Canada Prague, Czechoslovakia Loughborough, Leicestershire, UK Montreal, Quebec, Canada Research Triangle Park, NC, USA Vienna, Austria CH-1936 Bagnes- Verbier , Switzerland Essen, Germany Warwick, UK Constanta, Romania Perth, Australia Bath, UK Catania, Italy London, UK Contact Professor M.Valchrcel, Quimica Analitica, Facultad de Siencias, 14004 Cordoba, Spain Tel: +34 57 234453. Fax: +34 57 452285 M. Frei-Hausler, IAEAC, P.O. Box 46, CH-4123 Allschwil 2, Switzerland Tel: +4161 632789. Fax: +41 61 482 08 05 Heavy Metals Secretariat, CEP Consultants Ltd., 26-28 Albany Street, Edinburgh, UK EH1 3QH Tel: +44 31 557 2478. Fax: +44 31 557 5749 Jan Kucera, Nuclear Research Institute, CS-250 68 Rez near Prague, Czechoslovakia Tel: +42 2 685 7831 ext. 2268. Fax: +42 2 685 7567 Dr. Peter Warwick, Nuclear Chemistry Laboratories, Loughborough University of Technology, Loughborough, Leicestershire, UK LEll 3TU Tel: +44 509 222585.Fax: +44 509 233163 Chiral Secretariat, Conference Office, McGill University, 550 Sherbrooke St. West, West Tower, Suite 490, Montreal, Quebec, Canada H3A 1R9 Tel: +1 514 398 3770. Fax: +1 514 398 4854 Ms. Janet E. Cunningham, Barr Enterprises, P.O. Box 279, Walkersville, MD 21793, USA Tel: +1301898 3772. Fax: +1 301 898 5596 Symposium Secretariat, Dioxin '93, Gesellschaft Osterreichischer Chemiker , Nibelungengasse 11, A-1010 Vienna, Austria Tel: +43 222 587 3980/4249. Fax: +43 222 587 8966 General Secretary, Swiss Academy of Sciences, Barenplatz 2, P.O. Box 2535, CH-3001 Berne, Switzerland Professor Dr. H. W. Siesler, University of Essen, Schutzenbahn 70, P.O. Box 103764, D-4300 Essen 1, Germany Miss P. E. Hutchinson, Analytical Division, The Royal Society of Chemistry, Burlington House, Piccadilly, London, UK W1V OBN Tel: +44 71 437 8656.Fax: +44 71 734 1227 Dr. Gabriel-Lucian Radu, The Romanian Society of Analytical Chemistry, 13 Bul.Caro1 I, Sector 3, 70346 Bucharest, Romania Valerie Landgrebe, Symposium Secretariat, 12ACl 3EC, Conference and Seminar Management, UWA Extension, The University of Western Australia, Nedlands, Perth, Western Australia 6009, Australia Tel: +619 380 3181/2433. Fax: +619 380 1088/1066 Dr. Paul Illing, Health and Safety Executive, R425 Magdalen House, Stanley Precinct, Bootle, UK L20 3QZ Tel: +44 51 951 3420. Fax: +44 51 922 7918 G. Marletta, Consorzio Catania Ricerche, V. Le Andrea Doria, 6, 1-95125 Catania, Italy Tel: +39 95 221635. Fax: +39 95 339734 Evan Steadman Communications Group Ltd., 90 Calverley Road, Tunbridge Wells, Kent, UK TN12UN60N ANALYST, MAY 1993, VOL.118 Date 5-8 10-15 11-13 13 17-21 17-22 18-22 19-23 20-22 21-22 Conference Location Contact 5th Meeting of the Nuclear Magnetism and Professor M. Malet-Martino, Laboratoire IMRCP, Biology Group France Universite Paul Sabatier, 118, route de Narbonne, F-31062 Toulouse Cedex, France Electrochemical Society Meeting New Orleans, LA, Electrochemical Society Inc, 10 South Main Street, Toulouse, I USA VIth National Symposium on Mass Dehradun, Spectometry India FT Microscopy-10 Years On: 4th European Seminar on FT-IR Microscopy UK Manchester, Eighth Symposium on Separation Science and Oak Ridge, TN, Technology for Energy Application USA FACSS XX, 20th Annual Meeting of the Detroit, MI, Federation of Analytical Chemistry and USA Spectroscopy Societies Modern Electrochemistry in Industry and for Krakow , the Protection of the Environment Poland EXPOQUIMIA '93: Applied Chemistry Barcelona, Technical Fair Spain Hygiene and Health Management in the Working Environment Belgium Ghent, International Conference on Analytical Casablanca, Chemistry, Biochemistry and Pharmaceutical Morocco Sciences November 1-3 2 7-10 7-1 1 7-12 11-12 Chernyaev Conference on Chemistry, Moscow, Analysis, Technology and Application of Platinum Metals Electro-Membrane Processes London, Russia UK Electrophoresis '93 Charleston, SC, USA 7th International Forum-Electrolysis in Lake Buena Chemical Manufacture Vista, FL, USA Symposium on Supercritical Fluid Phenomena St.Louis , MO , (1993 Annual Meeting of the AIChE) USA International Conferences on Analytical Chemistry, Biochemistry, Pharmaceutical India Sciences, and Water QualitylEnvironmental Pollution New Delhi, Pennington, NJ 08534i2896, USA Dr. Pradeep Kumar, Indian Institute of Petroleum, Dehradun-248 005, India, and Dr. S. K. Aggarwal, Honorary Secretary-ISMAS, c/o Fuel Chemistry Division, Bhabha Atomic Research Centre, Bombay-400 085, Maharastra, India Michelle Barker, Conference Co-Ordinator, Spectra-Tech Europe Limited, Genesis Centre , Science Park South, Birchwood, Warrington, UK WA3 7BH Tel: +44 (0) 925 830 250. Fax: +44 (0) 925 830 252 J. T. Bell, Oak Ridge National Laboratory, Post Office 2008, Oak Ridge, TN 37381, USA FACSS, 198 Thomas Johnson Drive, Suite S-2, Fredericks, MD 21702, USA Tel: +1 301 846 4789.Fax: +1 301 694 6860 Dr. Andrzej Kowal, Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, ul. Niezapiminajek 1 , 30-239 Krakow, Poland Fira de Barcelona, Avda. Reina Ma Cristina, 08004 Barcelona, Spain 3rd International Symposium, 'Hygiene and Health Management in the Working Environment', c/o TI-K VIV, Attn. Ms. Rita Peys, Desguinlei 214, B-2018 Antwerp, Belgium Dr. V. M. Bhatnagar, Alena Chemicals of Canada, P.O. Box 1779, Cornwall, Ontario, Canada K6H 5v7 Tel: +1 613 932 7702. Dr. I. B. Baranovsky, Kurnakov Institute of General and Inorganic Chemistry, 31 Lenin Avenue, Moscow 117907, Russia Dr. T. R. Ralph, Johnson Matthey Technology Centre, Blounts Court, Sonning Common, Reading, Berkshire, UK RG4 9NJJ Tel: +44 734 722811 ext. 2257. Fax: +44 734 723236 Mrs. Janet Cunningham, Electrophoresis '93, c/o The Electrophoresis Society, P. 0. Box 279, Walkersville, MD 21793, USA Tel: +1 301 898 3772. Fax: +1 301 898 5596 Dr. N. Weinberg, 72 Ward Road, Lancaster, NY Tel: +1 716 684 0513. Fax: +1716 684 0511 Michael A. Matthews, Chemical Engineering Department, University of Wyoming, Box 3295 , University Station, Laramie, WY 82071-32, USA Tel: + 1 307 766 5769 Fax: + 1 307 766 4444. Or: Ted W. Randolph, Chemical Engineering Department, Yale University, 9 Hillhouse Avenue, New Haven, Tel: + 1 203 432 4375. Fax: + 1 203 432 7232 Dr. V. M. Bhatnagar, Alena Chemicals of Canada, P.O. Box 1779, Cornwall, Ontario, Canada K6H 5v7 Tel: +1 613 932 7702. 14086-9779, USA CT 06520-2159, USA Entries in the above listing are at the discretion of the Editor and are free of charge. If you wish to publicize a forthcoming meeting please send full details to: The Analyst Editorial Office, Thomas Graham House, Science Park, Milton Road, Cambridge, UK CB4 4WF. Tel: +44 (0)223 420066. Fax: +44 (0)223 420247.

ISSN:0003-2654    

DOI:10.1039/AN993180055N

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, MAY 1993, VOL. 118 61N Conference Report Huayi International Symposium on Analytical Chemistry: October 20-24, 1992, Wuhan, People‘s Republic of China The Huayi International Symposium on Analytical Chemistry brought together over 200 participants from different countries. Most of the delegates were Chinese-speaking (‘huayi’) analytical chemists so the official languages for the symposium were Chinese and English. The programme was extensive and included 10 Plenary Lectures, a number of invited lectures and oral presentations that covered various aspects, fundamental as well as applied, of analytical chemistry. After a ‘welcome’ by the organizer, Yun’e Zeng (Wuhan University, Wuhan, P.R. China) and the opening by Hong Gao (Nanjing University, Nanjing, P.R. China), the Plenary Lecture by K.L. Cheng (University of Missouri, Kansas City, MO, USA) discussed some common and significant miscon- ceptions in analytical chemistry present in the literature and textbooks. One example of these misconceptions is the mis-use of the Nernstian equation in non-faradaic poten- tiometry. According to Cheng, this is the second Nernstian hiatus in electroanalytical chemistry after the first as pointed out by John Bockris (Modern Electrochemistry, Plenum Press, New York, 1977, vol. 1, p. 17). Cheng pointed out that the important role of OH- in the development of a potential by a glass electrode was ignored, and talking about the measure- ment of hydrogen ion concentration in a solution of, say, 18 moll-’ KOH was a nonsense, as 1 1 of such a solution did not contain a single hydrogen ion! Chromatography Chromatography was the main topic in the section on separation science.Peichang Lu (Dalian Institute of Chemical Physics, Academia Sinica, Dalian, P.R. China) gave an upbeat presentation of the state-of-the-art of expert systems in chromatography. He reviewed the systematic research work of his group in the field of high-performance liquid chromato- graphy (HPLC) expert systems. The recommendation of column systems, intelligent optimization of the chromato- graphic process and qualitative identification of unknown samples were important parts of the designed expert system. Jieke Cheng (Wuhan University), the General Secretary of this symposium, reviewed the recent works of his group in the field of HPLC of metal complexes, the synthesis of tetra- phenylporphyrin derivatives with fluorine, chlorine or bro- mine substituents and their retention characteristics.A micro-volume detector for HPLC and high-performance capillary electrophoresis (HPCE) was presented by Yanzhuo Deng (Wuhan University). The He-Ne laser beam focused by a lens passes through the sample cell and produces an interference pattern. The change of light intensity in the interference ring centre, which is proportional to the change of refractive index, is monitored using a photodetector. The laser interferometric detection was also used by Deng for capillary zone electrophoresis (CZE). Yung-Lin Chen (J & W Scien- tific, Folsom, CA, USA) discussed tailor made stationary phases and other recent works in the field of HPLC.The synthesis of nitrogen-containing crown ether bounded station- ary phase, BCN18-C6, for HPLC via a solid-phase reaction pathway was reported by Shilu Da (Wuhan University). Caiying Wu (Wuhan University) studied the selectivity of a crown ether polysiloxane stationary phase. Jianxiong Feng (Jiangxi Academy of Agricultural Science, Nanchang, P.R. China) used HPLC to study the correlation between the content of free monosaccharides and disaccharides in stalks, roots of tomato varieties and their antiviral ability. The protein folding on the hydrophobic surface of stationary phases for high-performance hydrophobic interaction chro- matography (HPHIC) was studied by Xindu Geng (Northwest University, Xi’an, P.R. China). Boli Zuo (Chemical College, Beijing, P.R. China) designed a stainless-steel flow cell with ZnSe as the window material for construction of a CO2 laser induced photoacoustic detector for gas chromatography (GC).Two-dimensional GC was used for the analysis of selenomethionine in complex mixtures of the hydrolytic products of proteins in selenium-enriched mushrooms by Bingjiu Xu (Beijing Medical University, Beijing, P.R. China). Qiang Gu (Hua Feng Nutrition Factory, Wuxi, P.R. China) used GC for determining trans-10-hydroxy-2-decenoic acid, which was claimed to have an anticancer effect, in a popular Chinese tonic Royal Jelly. Weixi Yao (Dionex Corporation, Sunnyvale, CA, USA) reviewed the recent development of capillary electrophoresis (CE) and the important criteria for comparison of different CE apparatus. David T.Mao (J & W Scientific) discussed ways of solving problems associated with the use of capillary gel electrophoresis (CGE) . One approach is the development of replaceable polymerhetworks as the sieving media to avoid contamination carried from previous analysis. Weixi Yao (Research Centre for Eco-environmental Science, Beijing, P.R. China) and Eugene Lo Wei (Dionex Corporation) reviewed supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE) techniques. It is possible to use SFE to separate carcinogenic benzo[a]anthran- cene (BaA) and benzo[j]fluoranthene (BjF) from their isom- ers chrysene and benzo[k]fluoranthene (BkF) , respectively, the last two being of low toxicity. Fullerenes Several speakers presented work on fullerenes and their fluorinated derivatives.Wenkuan Zhao (Wuhan University) reported the preparation of a dark red-brown liquid consisting of Cm-C, by extraction of a black graphitic soot, which was produced by arcing in a vacuum using boiling benzene in a Soxhlet extractor. Chromatographic separation and mass spectrometry (MS) , 13C nuclear magnetic resonance spectros- copy (NMR) and Fourier transform infrared (FTIR) spec- trometric identification were reported. Yiliang Sun (Peking University, Beijing, P.R. China) reported the chromato- graphic analysis of fullerenes in the toluene extract of the soot produced by contact-arc vaporization of a graphite rod. Besides Cm and C7,,, a small amount of some higher fullerenes probably also existed.Rongsheng Sheng (Wuhan University) used surface-enhanced Raman spectra (SERS) for studying the symmetry of C6,, prepared in their laboratory. They claimed that the symmetry of Cm adsorbed onto the silver mirror was changed and adopted a more ellipsoidal shape and Cm was unstable under laser radiation. Yi Wu (Institute of Microchemistry, Beijing, P.R. China) reported the field desorption (FD) mass spectra of Cm, C70 and fluorinated fullerene C60F60.62N ANALYST, MAY 1993, VOL. 118 Spectroscopy A number of new spectroscopic techniques along with some traditional ones attracted the attention of delegates. Xihui Luo (Fushun Research Institute of Petroleum & Petrochem- icals, Fushun, P.R. China) reported the use of positron annihilation spectroscopy for chemical analysis (PASCA) for studying the catalysts used in petrochemical industries.As PASCA is based on the chemical probes, positrons and ortho-positroniums, which can react chemically with the active sites on a catalyst surface it should be a powerful complement to electron spectroscopy for chemical analysis (ESCA) and extended X-ray absorption fine structure spec- troscopy (EXAFS), which are based on physical phenomena. By using PASCA, a method for explaining the essential features of the surface of alumina was formulated. The interaction between metal and its supported material and formation of metal support interaction complex were found. Meisheng Zhou (National University of Singapore, Singa- pore) described construction and calibration of her interesting apparatus for photopyroelectric spectroscopy (PPES) .When a metal is coated with a polymer layer, and the temperature of metal-polymer interface increases due to non-radiative relax- ation processes following optical absorption, the polarized film aligns its polar constituent crystallites along the temperat- ure gradient field, thus producing a net dipole moment, which results in a pyroelectric voltage. The PPES based on this phenomena was used for identification and characterization of the electrode surface coating. Yongfa Zhu (Tsinghua Univer- sity, Beijing, P.R. China) reported the method for calculation of chemical shift in Auger electron spectroscopy by XPS (X-ray photoelectron spectroscopy) binding energy. He used this technique to study surface reaction of oxygen on zinc, Ti/Si interface reaction and the structure of lubrication films.H. C. Kang (National University of Singapore) studied the reconstruction of platinum and gold films on 11oPd using Auger electron spectroscopy and low-energy electron diffrac- tion (LEED). In a Plenary Lecture, Guo-Qin Xu (National University of Singapore) reported his studies on alkali overlayers and their oxides supported on 1olRu using high- resolution photoemission spectroscopy and thermal desorp- tion. By using these techniques, it was observed that upon the exposure of oxygen to potassium overlayer, the initial oxidation occurred in the ‘bulk’ layer, not the surface layer as expected. Tsutomu Fukasawa (Yamanashi University, Japan) reported the use of an X-ray diffraction technique for speciation and quantification of inorganic compounds, in particular the characterization of airborne particulates from Tokyo, Japan, and Sichuan, P.R.China. He claimed that some new compounds with a high-lead content, which were isostructural with potassium lead chromates, were found. Yang Lu (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, P.R. China) showed a special software program NOMCSDP for routine analysis of organic molecules, especially natural organic compounds, by using X-ray diffraction methods. Guangzhong Tu (Beijing Institute of Microchemistry) used NMR for analysis of liquorice, a well known traditional drug in China. The structure of squasapoge- no1 extracted from liquorice was identified as olean-11,13( 18)- diene-3,22-diol.Proton NMR was used by Yong-Cheng Hing (Tsinghua University) for identification of the viper venom components, which were used for treating thrombosis and atrophic gastritis. Xiao-Yuan Li (Hong Kong University of Science and Technology, Hong Kong) gave an informative Plenary Lecture on the use of resonance Raman spectroscopy (RRS) and related Raman techniques for studying the structure and dynamics of metalloporphyrins, which are the prosthetic group of haemproteins. Rongsheng Sheng (Wuhan University) reported the in situ SERS of 5,10,15,2O-tetra-(4- trimethylammoniumpheny1)porphyrin at a silver electrode. The use of IR photoacoustic spectroscopy for analysis of fibrous samples such as acrylic, wool and ramie was reported by Xueguang Shao (Chinese University of Science and Technology, Hefei, P.R.China). Fluorescence Fluorescence and related methods were the subject of a number of presentations. Yaoqun Li (Xiamen University, Xiamen, P.R. China) derived an equation for calculating the wavelength position of solvent Raman scatter appearing on constant-wavelength synchronous fluorescence spectra. Longdi Li (Tsinghua University) studied the room tempera- ture phosphorescence of 6-coumarinsulfonyl chloride as a labelling reagent for amino acids. Zhujun Zhang (Shaanxi Normal University, Xi’an, P.R. China) discussed the new approaches to solid surface chemiluminescence (SSCL). By using foaming plastic as the solid support, a method for determining 0.1-10 pg of gold was developed for ore and steel analysis. Zhang also reported a fibre-optic biosensor for 0.1-10 pg ml-1 of lactic acid by covalently coupling lactate oxidase and horseradish peroxidase to 3-aminopropyl porosity silica via glutaraldehyde and attaching the silica to the end of fibre-optic handle. Yuanbao Zhu (Hunan University, Chang- sha, P.R.China) reported the design of two fibre-optic sensors for ammonia. The membranes contained a neutral carrier for ammonium ion and a lipophilic pH indicator. Kemin Wang (Hunan University) reported an optical sensing membrane containing dinaphthyl-20-crown-6 and ETH5294 as the neutral carrier and lipophilic pH indicator, respectively. The membrane was sensitive to primary amine drugs. Micellization Micelle-sensitized spectrophotometry and fluorimetry was studied by a number of investigators. Yong-Xi Zheng (Tsing- hua University) proposed a new mechanism for the sensitizing effect of cetyltrimethylammonium on the absorbance and fluorescence of the Zr-morin complex.A sensitive method for determination of Ru was reported by Zuting Pan (Wuhan University) using 01, p, y ,&te trakis(4- trime thylammonium- pheny1)porphyrin and dodecylbenzenesulfonate. The micelli- zation behaviour of the steroidal surfactant sodium deoxy- cholate was studied using fluorescent probe, optical rotation and conductivity measurements by Yunbao Jiang (Xiamen University). New Reagents A number of new analytical reagents were reported: 2,4- dimethoxyphenylfluorone (Jinduan Zhao, China University of Geosciences, Wuhan, P. R. China), 4-formacylbenzene- diazoaminoazobenzene (Yinglu He, China University of Geosciences) , rneso-tetra(4-methoxy-3-sulfonatophenyl)por- phine (Yushou Chen, Fuzhou University, Fuzhou, P.R.China), 5-(6-bromobenzothiazole-2-azo)-8-aminoquinoline, diantipyryl-o-chlorophenylmethane (Qiheng Xu, Yunnan University, Kunming, P.R. China), 2,3,7-trihydroxy-9-lyral- 6-fluorone (Xianchun Li, Jiangxi Normal University, Nan- chang, P.R. China), 9-(3,5-dibromo-4-amino)phenylfluorone and other derivatives of phenylfluorone (Huashan Zhang, Wuhan University), 1-(4-antipyriny1)-3-(4-nitrophenyl)- triazene (Bin Xu, Hubei Normal University, Huangshi, P.R. China). Atomic Spectrometry Some new techniques for atomic spectrometry were presented at the symposium. Benli Huang (Xiamen University) des- cribed in his Plenary Lecture a simple and compact nebulizer- hydride generator system for simultaneous determination of hydride forming elements and monohydride forming ones.The sensitivity for the hydride forming elements was substan-ANALYST, MAY 1993, VOL. 118 63N tially improved. Hydride generation was also studied by Li Zhang (Perkin-Elmer, Norwalk, CT, USA). The matrix effect in inductively coupled plasma atomic emission spectrometry (ICP-AES) was studied by Zhanxia Zhan (Zhongshan Uni- versity, Guangzhou, P.R. China) using an optical-fibre probe to sample the spatial position of plasma and constructing the three-dimensional spatial distribution of electron densities in the presence of different elements. Li-Ching Tian (Nanjing University) combined a movable sampling device with a pulsed laser to eliminate the effect of inhomogeneity of powdered solid geological samples in ICP-AES.Zucheng Jiang (Wuhan University) studied the use of a poly(tetra- fluoroethylene) slurry fluorinating reagent in electrothermal vaporization (ETV) in ICP-AES to avoid the formation of refractory carbides by converting the analytes into their corresponding halides. The detection limit was improved by 1-2 orders of magnitude compared with the conventional ETV procedure. The advantage of an end-on viewing ICP for AES has been recalled and re-evaluated for its suitability to ICP-AES coupled with a charge-injection device as the mass detector. Pengyuan Yang (Xiamen University) reported his latest work using an end-on viewing ICP-AES instrument with D.Nygaard (Baird Corporation, Bedford, MA, USA). Jianshi Ren (Institute of Metal Research, Shenyang, P.R. China) studied the cathode sputtering of alloys in glow discharge lamp and showed the hyperbolic relationship between the sputtering rate and concentration of constituents of these alloys. Xiaobin Zeng (Wuhan University) studied the high-temperature reaction of A1 compounds on a graphite substrate for elucidation of their interference on the determi- nation of precious metals by electrothermal atomic absorption spectrometry (ETAAS). Gongke Li (Zhongshan University) studied the characteristic mass for many elements under standardized conditions for absolute analysis by ETAAS. A correction factor was defined to account for the effect of experimental parameters.Electroanalysis The electroanalytical chemistry section was one of the largest sections of the symposium. In his Plenary Lecture, Hong Gao (Nanjing University) reviewed the systematic study of oscillo- graphite analysis undertaken by his group and gave a comprehensive classification of different methods. The improved oscillographic chronopotentiometry, for instance, changes the incision of the dEldt versus E curve into a very sharp peak with a height directly proportional to the concen- tration of sought-for species. Ultramicroelectrodes have been the subject of many investigations for their ability to offer dramatic improvement in the quality of electrochemical data. The theory of these ultramicroelectrodes was studied by Zuxun Zhan and Hongyuan Chen (Nanjing University).Chen derived the current equations of single sweep and first-, second- and third-order differential voltammetry at ultramic- rodisc electrodes. Li Jiang (University of Oxford, Oxford, UK) reported the fabrication of a microelectrode array on silicon substrates with individual elements of from 0.3 to 5 pm dimensions by photolithography and electron beam litho- graphy. The arrays retained the advantages of single ultra- microelectrodes while substantially increasing the current response. Jinyuan Mo (Zhongshan University) reported a series of advances in cyclic staircase pulse voltammetry (CSPV), which improved the symmetry of the waveform of square-wave voltammetry. In the middle of each staircase step of cyclic scan a pulse is superimposed. The currents are measured at both the advancing and following edge of each pulse. After addition the forward and reverse charging currents are eliminated.The theoretical equations of CSPV for different types of electrode reactions were derived by Mo. In his Plenary Lecture, Ying-Sing Fung (University of Hong Kong, Hong Kong) described the theoretical equation derived and verified by him for the transient current response at planar electrodes for a reversible electrode reaction in steady-flow system. Jem-Mau Lo (Tsinghua University, Hsinchu, Taiwan) combined differential-pulse anodic stripping voltammetry with a preconcentration extraction procedure for determina- tion of 0.1 pg 1-1 of metal in sea-water. Shaojun Dong (Changchun Institute of Applied Chemistry, Changchun, P.R. China) reviewed her recent research in designing chemically modified electrodes. The enzyme sensors for glucose, alcohol, lactate and malate with mediators and improved sensitivity and selectivity were reported.Some porphyrin derivatives were synthesized as a kind of artificial enzyme for biosensor design. The ion transfer at the liquid liquid interfaces of membranes containing some newly syn- thesized bis-crown ethers were studied by Jinyao Yin (Beijing Research Institute of Chemical Engineering & Metallurgy, Beijing, P.R. China) for design and characterization of K+ and pH sensors. Shouzhuo Yao (Hunan University) discussed the design and theory of piezoelectric sensors in the liquid phase. An immunosensor was reported by Guoli Shen (Hunan University) for a-fetoprotein, which is an important index for clinical diagnosis of liver cancer. C hemome trics Chemometrics were the subject of a series of presentations.The author of this report gave a Plenary Lecture entitled ‘Analytical chemometrics: theory and methodology of chem- ical measurement’. The development of analytical chemo- metrics including analytical information theory, theories of sampling, analytical detection, calibration, etc., stimulated formulation of a sound mathematical theory of chemical measurement, which should play an important role in the development of modern analytical chemistry. Zhenpu Wang (Nanjing Institute of Chemical Technology, Nanjing, P.R. China) made a comparison of different chemometric methods for multivariate calibration in FTIR analysis using Monte Carlo simulation.The Monte Carlo simulation was also used for studying the nebulization and evaporation process in ICP-AES by Jianguo Zheng (Zhongshan University). Bo Deng (Tsinghua University, Beijing) used the fuzzy orthogo- nal design for the optimization of ETAAS. Yaoguang Wang (Fuzhou University) combined Kalman filtering with fast Fourier transform smoothing and a standard additions method to resolve overlapping neopolarographic peaks. The Kalman filtering method was also used for photodiode-ICP-AES for the determination of trace ytterbium in vanadium by Xiaoguo Ma (Zhongshan University). Jian Fan (Zhongnan University of Technology, Changsha, P.R. China) reported the simul- taneous indirect determination of anions using their interfer- ence effect on the atomic absorption of calcium.The data were analysed by the partial least squares (PLS) method. Spline smoothing was used by Jinyuan Mo (Zhongshan University) for step voltammetry data treatment. Tianming Yang (Zhongnan College for Minority Nationalities, Wuhan, P.R. China) reported the experiment of using pattern recognition for carcinoma diagnosis by using the trace element content of patient’s hair as feature variables. Zheng Li (Shanghai Institute of Organic Chemistry, Shanghai, P.R. China) used neural networks for prediction of furnace lining durability in some industrial plants. Applications The problem-oriented applications of various analytical methods focused on environmental chemistry and life science. Jiunn-Guang Lo (Tsinghua University, Hsinchu, Taiwan) gave an informative Plenary Lecture on trace gas analysis in connection with the CATS programme (Climate and Air- quality Taiwan Station) for studying long-term air quality change and their impact on the climate of Taiwan.Xiaobai Xu64N ANALYST, MAY 1993, VOL. 118 (Research Centre for Eco-environmental Sciences) discussed the analytical aspects of a series of papers on polycyclic aromatic hydrocarbons and some recently discovered environ- mental carcinogens and mutagens. The variation of iron and molybdenum concentration in the sea-water during the red tide (RT) was studied by Xiuhuan Yang (Zhongshan Univer- sity) by using Zeeman-effect background corrected ETAAS. The iron concentration within the RT area was relatively high, decreasing rapidly in the course of RT and rising again after the disappearance of RT.On the contrary, the molybdenum concentration first decreased, then rose gradually and sus- tained for 1-2 d after the disappearance of RT. Tonghui Zhou (Institute of Materia Medica, Beijing) gave a Plenary Lecture on the use of rat liver microsomes for in vitro metabolic studies of drugs. Erik Lund (Huddinge University Hospital, Hud- dinge, Sweden) studied the mechanism of peroxidation of cholesterol into monooxygenated products by lipoxygenase system. He also reported the analysis of the ginseng G155 extract. The optimized procedure with alkaline hydrolysis was found to produce 20-s-protopanaxadiol and 20-s-protopanax- atriol from ginsenosides with about 80% yield. Te-Hsien Lin (School of Technology for Medical Sciences, Kaohsiung Medical College, Kaohsiung, Taiwan) measured lipid peroxi- dation in tissues and body fluid of mercury intoxicated rats to study the molecular mechanism of cell injury in acute HgC12 poisoning. Conclusion Space does not permit the description of many excellent presentations. The symposium was well organized and managed, the absence of a language barrier and the friendly atmosphere throughout prompted optimum interaction between the delegates. An interdisciplinary symposium of this kind has the advantage of the cross-fertilization that occurs when people dealing with various aspects of analytical chemistry come together and discuss diverse ways of looking at similar problems. The diversity and depth of study reported was truly impressive. The next Huayi Symposium on Analy- tical Chemistry will take place in Guangzhou, China, in 1995. Ru-Qin Yu Department of Chemistry and Chemical Engineering, Hunan University, Changsha 41 0082, Peoples Republic of China

ISSN:0003-2654    

DOI:10.1039/AN993180061N

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

64N ANALYST, MAY 1993, VOL. 118 Future Issues will lnclude- Preliminary Characterization of Inductively Coupled Plasma Mass Spectrometry With Flow Injection into a Gaseous (Air) Carrier-Diane Beauchemin Determination of Anatoxin-a and Homoanatoxin in Blue- Green Algal Extracts by High-performance Liquid Chroma- tography and Gas Chromatography-Mass Spectrometry- Anastasia Zotou, Terry M. Jefferies, Paul A. Brough and Timothy Gallagher From Flow Injection to Sequential Injection: Comparison of Methodologies and Selection of Liquid Drives-Ari Ivaska and Jaromir RiZi2ka Calculation of the Confidence Range in Order to Obtain a Linear Calibration Graph in Stable Isotope Dilution Mass Spectrometry: Application to Reference Methods and Phar- macological Studies-Jean-Frangois Sabot and Henri Pinatel Microcomputer-aided Titrimetric Determination of Bromine- containing Active Ingredients in Some Drug Formulations- Biljana F.' Abramovic, KornCIia S.Horvath and Ferenc F. Gaal Spectrophotometric Determination of Lead in Tap Water With 5,10,15,20-Tetra( 4-N-sulfoe thy1pyridinium)porphyrin Using Merging Zones Flow Injection-Jeffery A. Schneider and James F. Hornig Bioluminescent Flow-sensing Device for the Determination of Magnesium(I1)-Stefan0 Girotti, Elida Ferri, Severino Ghini, Pave1 Rauch, Giacomo Carrea, Roberto Bovara, Aldo Roda, Maria Antonietta Giosui?, Pier0 Masotti and Gianni Gangemi Recent Strategies in Automated Reaction Rate Based Deter- minations-Manuel Silva Analysis of Mixtures of Polycyclic Aromatic Hydrocarbons in Sea-water by Synchronous Fluorescence Spectrometry in Organized Media- J.J. Santana Rodriguez, J. Hernandez Garcia, M. M. Bernal Suarez and A. Bermejo Martin-Lazaro Multicomponent Determination of Flavour Enhancers in Food Preparations by Partial Least Squares and Principal Component Regression Modelling of Spectrophotometric Data-Isabel Duran-Meras, Arsenio Munoz de la Pena, Anunciacih Espinosa-Mansilla and Francisco Salinas Partial Least Squares Resolution of Multianalyte Flow Injec- tion Data-Paul J. Worsfold, Paul MacLaurin, Philip Norman and Michael Crane Determination of Carboxylic Acids by High-performance Liquid Chromatography With 2-( 2,3-Anthracenedicarbox- imido)ethyl Trifluoromethanesulfonate as a Highly Sensitive Fluorescent Labelling Reagent-Hiroshi Meguro, Kazuaki Akasaka and Hiroshi Ohrui Development of a Highly Sensitive Fluorescence Reaction Detection System for Liquid Chromatographic Analysis of Reducing Carbohydrates-Shuji Yamauchi, Chie Nakai, Noriyuki Nimura, Toshio Kinoshita and Toshihiko Hanai Photobleaching of Methylene Blue in Continuous Wave Thermal Lens Spectrometry-R. D. Snook and R. D. Lowe Attomole Detection of Nitroaromatic Vapours Using Res- onance Enhanced Multiphoton Ionization Mass Spec- trometry-Alastair Clark, Kenneth W. D. Ledingham, Archibald Marshall, Joseph Sander and Ravi P. Singhal Typification of Alcoholic Distillates by Multivariate Tech- niques Using Data From Chromatographic Analysis-M. C. Ortiz, J. A. Saez and J. L6pez Palacios

ISSN:0003-2654    

DOI:10.1039/AN993180064N

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, MAY 1993, VOL. 118 455 Tutorial Review Outliers in Experimental Data and Their Treatment James N. Miller Department of Chemistry, Loughborough University of Technology, Loughborough, Leicestershire, UK LEI1 3TU This review summarizes critically the approaches available to the treatment of suspect outlying results in sets of experimental measurements. It covers the use of parametric methods such as the Dixon test (with comments on the problems of multiple outliers); the application of non-parametric statistics based on the median to by-pass outlier problems; and the application of robust statistical methods, which down-weig ht the importance of outliers. The extension of these approaches to outliers occurring in regression problems is also surveyed. Keywords: Outliers; exploratory data analysis; significance tests; non-parametric and robust statistics; regression methods Introduction The quantitative measurements which dominate modern analytical chemistry are inevitably subject to random varia- tion.As a result such measurements are almost invariably replicated. The number of replicates performed may be limited by factors such as the availability of the test material, reagents or time, but in every case the aims are the same. Replicates allow the magnitudes of the random variations to be estimated, for example by calculating standard deviations or confidence limits, and the means of the replicates are expected (if systematic errors are absent or have been corrected) to be closer to the true value of the measurand under study than the individual readings.In practice, when a series of replicate measurements is obtained, it is often found that one or more of the values seem to be substantially different from the others. The practical question then posed is clear: should such outlying results be rejected or not before the mean, standard deviation, etc., of the data are calculated? The dilemma involved is equally clear. The replicate measurements are regarded as a small statistical sample from a potentially infinite population: the latter is generally assumed to have a Gaussian or ‘normal’ distribution (see below for further comments on this assump- tion), in which readings near the mean value are much more likely than readings distant from the mean. Nonetheless, there is some probability of obtaining a value well removed from the mean, and such a reading may appear in the small sample of measurements made in practice.If this is so, what justification can there be for rejecting the suspect data point? These issues have caused concern and controversy among experimental scientists (not just analytical scientists) for many years, and indeed continue to generate new research, and further controversy! Three separate statistical approaches to the problem can be identified: (1) use of statistical significance tests that assume a Gaussian (or some other defined) error distribution for the population; (2) use of non-parametric statistical methods, which make no such assumptions; and (3) use of robust statistical methods, the underlying principles of which are fully discussed below.Each of these approaches can be criticized, and rather than recommend any one of them, this review seeks to summarize their principles, strengths and weaknesses. What is certain is that all analytical scientists must make a considered decision as to how they are going to treat potential outliers: equally, that decision must be a clearly stated part of reports, published papers, etc. One further obvious approach to the problem of outliers is to seek more data! If we have four replicates, one of which is suspect, our treatment of the latter may have a crucial effect on our final results. If we could make four further measure- ments (and assuming that none of these is suspect) our concerns are much reduced, as any statistical test applied will have more power, and in any case the decision as to whether to eliminate the outlier or not will have a smaller effect on the calculated mean, standard deviation, etc. Of course, it may not be possible to obtain more data if the material under study has been exhausted or irreversibly changed by previous measure- ments, but if more material is available then the first expedient the analytical scientist should consider when outliers arise is to obtain further readings.Before considering statistical approaches to outliers in more detail, the established methods of exploratory data analysis are often valuable. The simplest statistical packages available for personal computers will rapidly present (for example) dot plots (in which the data are effectively plotted as one- dimensional graphs) or box and whisker plots (which show the range, interquartile range, mean, confidence intervals, etc., and often highlight outliers immediately).These elementary visual data displays readily show up suspect values, and also give an immediate overview of the data distribution, thus helping a judgement on whether (for example) the assumption of normality is valid. It is worth noting that, in some cases, the suspect measurement can readily be explained. A check on the instrumentation used may, for example, reveal an intermittent fault. In other cases such as in the data set: 12.11,12.27,12.19, 21.21, 12.18, we can be fairly certain that a transcription error has occurred in the fourth result, which should really be 12.21. In such cases outliers can be omitted or corrected with a relatively easy conscience.The statistical methods now to be summarized need only to be used in those all too frequent cases where outliers occur without obvious explanation. Parametric Tests for Outliers The methods described in this section are probably still the most commonly used solutions for outlier detection.’-5 They assume that the data are a sample taken from a population with a Gaussian error distribution, and apply conventional significance test approaches to decisions on (in the simplest456 ANALYST, MAY 1993, VOL. 118 cases) the rejection of single outliers. The probability level used in the significance test determines, as usual, the likelihood of a type I error, i.e. , the probability that a suspect value will be rejected when in fact it should be retained.As in other significance tests applied in analytical science, the p = 0.05 level is most frequently used. The principal points of interest in such tests are the test statistics to be used. Their selection is not as easy as it seems. It would clearly be illogical to take the complete data set, i.e. , including the suspect results, use it to calculate, for example, 95% confidence limits, and then reject any measurements falling outside such limits. Even less defensible would be outlier rejection on the basis of confidence limits determined with the suspect values excluded! In practice, the test statistics usually applied are those described by Dixon over 40 years ago, and often described as ‘Dixon’s Q’.They utilize comparisons of the difference between the suspect value(s) and the values close to them and the over-all range of the results. For n = 3-7 (as always, n is used to describe the total number of measurements) with the ordered sample values labelled xl, x2, etc., the test statistic for a single outlier is: Qio = (x, - xn-i)/(x, - xi) or (x2 -xiY(xn - XI) (la) according to whether the suspect value is at the high or low end, respectively, of the data set. These equations can be replaced by: Qlo = I(suspect value - nearest value)I/range (lb) A simple example of this approach is instructive. Suppose that we obtain the results: 9.97, 10.02, 10.05, 10.07 and 10.27 cm3 in a titrimetric analysis. Given the precision with which titrimetry can be performed in expert hands, the last result, 10.27 cm3, must be regarded as suspect.The test statistic, Qlo, is given in this case by (10.27 - 10.07)/(10.27 - 9.97) = 0.667. The critical value for n = 5 andp = 0.05 is 0.710 (tables of such values are given in several of the books listed at the end of this review). In our example, the test statistic is less than the critical value, so the null hypothesis underlying the test (i.e., that all the results could come from the same population) has to be accepted: the doubtful result 10.27 cannot be rejected. This example is a good illustration of an important principle, viz., that when n is small, as it so often is in analytical work, one result has to be very different from the others before it can be rejected by criteria such as the Dixon test.In this case the suspect titre would have to be as high as 10.32 cm3 (when Qlo is 0.2Y0.35 = 0.714) before it could (just) be rejected at the p = 0.05 level. Such findings emphasize the need to obtain extra data wherever possible. The same set of results can be used to emphasize a point made earlier. When n is small, acceptance or rejection of an outlier makes a large difference to the results of mean and standard deviation calculations. For the five results 9.97, 10.02, 10.05, 10.07 and 10.32cm3, the mean and standard deviation if the last result is retained are 10.086 and 0.136 cm3, respectively. If the figure of 10.32 cm3 is rejected then the mean and standard deviation fall to 10.0275 and 0.043 cm3, respectively. Note that the estimated standard deviation, s, is reduced by more than two-thirds as a result of rejecting the outlier.As s is routinely used to estimate confidence limits, and in significance tests which compare means, variances, etc. , the importance of making a sensible judgement about the suspect result is very clear. It is at this point, however, that we have to confront some of the difficulties of the Dixon method and other parametric significance tests. One immediate problem is that, as n increases, there is a growing likelihood of obtaining two or more suspect outliers in the same data sample. The identifica- tion of multiple outliers is a serious problem (to which we shall return briefly below), but here we are interested only in the effects of increasing n on the identification of a single outlier.Most authorities recommend that modified forms of eqn. (1) are used. Hence for n = 8-12, we use: and for n 2 13, we use: Q22 = ( ~ 3 - XI)/(X, - 2 - xi) or (xn - xn - 2)/(~, - ~ 3 ) (3) according to whether x1 or x,, respectively, is the suspect value. Somewhat surprisingly, there is considerable controversy about the ranges of n values over which eqns. (1)-(3) should be used, and even over the critical values associated with these statistics. (This review follows the recommendations, although not the nomenclature, of the book by Wernimont and Spendley.5) Further difficulties in using this approach to outliers become apparent when we are suspicious about two (or more) of the measurements. Consider the situation where there are two values which (for example) are similar but suspiciously higher than the rest.In that case we clearly cannot simply take the highest result and determine Qlo from eqn. (l), becausc the numerator in the equation will be small, being given by the difference between the two suspicious values. This effect is known as masking. Two other approaches suggest themselves. One (the so-called block method) considers the two results as a pair, the test statistic being (for example) their mean divided by the mean of the whole set of measurements. The clear danger here is that we necessarily discard or retain both values, even in the situation where it might be right to retain one and reject the other. A consecutive procedure, on the other hand, takes the outliers one at a time: the one that is the nearer to the remainder of the data is tested first, and if it can be rejected, the more pronounced outlier is rejected together with it.If the nearer outlier is retained, the further outlier is tested separately. The relative merits of these block and consecutive procedures are still causing controversy. Still more complications arise when there are two suspect results at opposite ends of the data sample. Tests for all these situations are thoroughly summarized in the book by Barnett and Lewis’ but there is a growing feeling that such methods should, if possible, be by-passed by using the alternative approaches discussed in later sections. Before leaving the realm of parametric tests, however, we must re-emphasize that they do assume the presence of (in most cases) a Gaussian distribution of error.If this is not present, the tests are completely invalid. Take, for example, the following set of numbers: 1.26,1.58,2.29,2.51,3.02,3.98, 7.94. The value 7.94 certainly seems suspicious, and the Dixon test shows that, at p = 0.05 it could be rejected, if a Gaussian error distribution is assumed. However, suppose that, in reality, these numbers were a sample from a log-normal distribution, i.e. , one in which the logarithms of the numbers are normally distributed. Their logarithms (base 10, but any base would give the same result) are: 0.100, 0.199, 0.360, 0.400,0.480,0.600,0.900. Now there is no suggestion that the last result is suspicious: the appropriate Qlo value is 0.3/0.8 = 0.375, well below the critical value (p = 0.05) of 0.569.Hence a result that seems suspicious on the assumption of a particular error distribution may not be at all suspicious when the correct distribution is used. In summary, parametric significance tests are widely used, no doubt because of their superficial simplicity. However, even in their most elementary forms, they are controversial and need to be used with care. In the presence of multiple outliers still further complexities arise, and it is not surprising that the present trend is for such tests to be replaced by other approaches. Non-parametric Statistical Methods In the last section, it was noted that the use of significance tests which assume a particular distribution of errors is fraught withANALYST, MAY 1993, VOL. 118 457 danger.The obvious question therefore arises: why not use methods which utilize no such assumptions? Such tests have been widely available for many years, although much of their use has in practice been in the social sciences. These distribution-free or non-parametric techniques68 are charac- terized by their use of the median rather than the arithmetic mean as the ‘measure of central tendency’. The benefits of this approach are immediately apparent if we reconsider the data given in the previous section. In numerical order, the five results examined were 9.97,10.02,10.05,10.07 and 10.27. The median of these results, i.e., the middle value when the numbers are ordered, is 10.05. (If the number of results is even, the median is the average of the two middle values.) It is clear that the median is entirely unaffected by the suspect value, and will remain 10.05 as long as the highest value is >10.07.This valuable behaviour contrasts with that of the mean, as already noted, and extends to the situation in which there are two outliers: if the first value had been mistakenly recorded as 9.77, or the fourth value as 10.70, the median of the five numbers would remain as 10.05. These advantages extend to other methods which utilize medians directly, for example the Theil methods used in regression (see below). The use of the median therefore circumvents rather than confronts the problems of outlier identification, and is valuable when n is small. The most common non-parametric measure of dispersion ( i e . , the ‘spread’ of the results) is the inter-quartile range (IQR).If we imagine a set of ordered numbers to be divided into two groups, one above and one below the median, each of these groups can itself be divided into two by the quartiles: the difference between the two latter numbers is the IQR. As it does not involve the highest and lowest values this statistic also has the valuable property of being unaffected by outliers. It may also be shown that, for a Gaussian distribution of error, the IQR is about 1.350, where 0 is the population standard deviation: this relationship allows us to estimate the standard deviation of a set of data in a manner which is independent of outliers. Unfortunately, the IQR is a rather unrealistic concept for small samples, and there are a number of different conventions for calculating it, which give significantly different results when n is small.It has, therefore, seen relatively little use in analytical chemistry calculations. The number of non-parametric significance tests is very large indeed, and space only permits a summary of their strengths and weaknesses. Their value in dealing with suspicious results is well illustrated by the following example. Suppose that the levels of metal ion (in ng cm-3) in the water from 32 rivers in two areas is as follows. Area A: 29,42,60,80, 83, 110, 130, 168, 194,230, 260, 270, 275, 280, 350, 780; and area B: 122, 140, 160, 220, 245, 250, 260, 268, 348, 390, 420, 430, 445, 454, 482, 498. Is there any evidence that the metal ion levels in the rivers in the two areas differ? The first point to note about these measurements is that they are not replicates: the metal ion concentration has been measured only once for each water sample from each area.There are, therefore, almost no circumstances in which the apparently anomalous result of 780 ng cm-3 from area A could even be considered as an outlier in the usual sense. It may be very different from all the other area A data, but there will normally be no reason for rejecting it. It is also important to note that there is no reason to think that these samples are necessarily drawn from populations with Gaussian error distributions, so conventional parametric tests such as the t-test may not be justifiable. The best-known non-parametric approach to problems of this type is the Mann-Whitney U-test, sometimes known now as the Wilcoxon-Mann-Whitney test as it is closely related to the Wilcoxon Rank Sum test.The idea underlying the Mann-Whitney formulation is very simple. Inspection of the data shows that, in general, the metal ion levels from area A are lower (median = 181 ng cm-3) than those from area B (median = 308 ng cm-3). Hence the number of occasions on which individual area B results are exceeded by individual area A results should be fairly small. Performing the test simply involves counting the number of occasions on which this occurs. Hence the result 122 from area B is exceeded by 130 . . . 780 from area A, i.e., by ten area A values; the result 140 from area B is exceeded by nine area A values, and so on. Allowing for one ‘tie’ (i.e., when the two equal results of 260 ng cm-3 are compared), which counts 0.5 in our tally, the total number of cases in which area B data are exceeded by area A data is 66.5.Reference to statistical tables shows that, for n1 = n2 = 16, this test statistic must be less than or equal to 75 (p = 0.05) if the null hypothesis (z.e., that the two sets of measurements have equal medians) is to be rejected. This is clearly the case in this example, so the Mann-Whitney method suggests that the metal ion levels in the two areas are probably different. It is of interest that, at the same probability level, the t-test (just) fails to detect this difference. As already noted, the t-test may not be appropriate anyway, not, at least, until we have checked for the normality of the two sets of data, but it is clear that the Mann-Whitney method has the advantage that it has effectively down-weighted the significance of the anomalous area A result of 780 ng cm-3.The same Mann- Whitney result would have been obtained if this reading had taken any value 3499, i.e. , any value higher than the highest area B value. By contrast, the reading of 780 ng cm-3 has greatly inflated both the mean and the standard deviation of the results for area A. This property of the Mann-Whitney method (i.e. , of being little affected by anomalous results) is known as robustness, and is more fully explored in the next section. It arises because the test really considers not the absolute values of the measurements but their ranks, i.e., their numerical positions if the data are arranged in order.This is not immediately apparent from the way the test has been described here, but is clarified in the next example. Two different methods were utilized to determine the concentration of nitrate ion in a single sample of tap water. Each method was used six times, with the following results (in pg cm-3). Ion chromatography: 0.57, 0.60, 0.62, 0.66, 0.67, 0.68; and ion-selective electrode: 0.46, 0.50, 0.59, 0.69, 0.83, 0.85. In this case the question to be addressed is whether the two methods differ significantly in their precisions. (The median concentration is the same in each case, 0.64 yg ~ m - ~ . ) If we can assume a Gaussian error distribution for each data set, and if we set aside worries about the two possible outliers (0.83, 0.85) in the ion-selective electrode data, we could use the well-known F-test to make this comparison.The alterna- tive is the Siegel-Tukey method, in which the results are first written down as one continuous and ordered list with, for example, the ion chromatography results underlined: 0.46, 0.50,-=, 0.59,0.60,0.62,0.66,0.67,0.68,0.69,0.83,0.85. A little consideration suggests that, if the spread of the two sets of results was roughly the same, the underlined and non-underlined data would appear roughly at random across the list. In practice, the underlined results tend to be concentrated in the middle of the list. This is expressed numerically by the use of paired alternate ranking. The lowest result is ranked 1, the highest result is ranked 2, the second highest 3, the second lowest 4, the third lowest 5 , etc.These rankings are written down with the underlining retained: 1,4, 5, 8, 9, 12. 11. 10, 7. 6. 3. 2. / I , I , _ , I I - - - - - The sum of the underlined ranks is 5 + 9 + 12 + 11 + 10 + 7 = 54, and the sum of the remaining ranks is 1 + 4 + 8 + 6 + 3 + 2 = 24. (It is worth verifying that the two sums total 78, the sum of the first 12 natural numbers.) We must now subtract from each of these sums n(n + 1)/2 and rn(m + 1)/2, where n and rn are the numbers of measurements in the two data sets. This may seem unnecessary in this case, as n = rn = 6, but the test is formulated in this way to allow for cases where n # m. Hence we subtract 21 from each sum giving us 33 and 3, respectively. We now take the lower of these two results, i.e., 3 as our test statistic.The critical value (which is derived from458 ANALYST, MAY 1993, VOL. 118 the same set of tables as are used in the Wilcoxon-Mann- Whitney test described above) for n = rn = 6 at p = 0.05 is 5, i.e., our test statistic must be ~5 for the null hypothesis of equal spreads to be rejected. (Note that this test and the closely related Wilcoxon-Mann-Whitney method are unusual in that the critical region, i.e. , the region leading to rejection of the null hypothesis, occurs when the test statistic is less than or equal to the critical value in the tables.) In this example we have clearly demonstrated that the null hypothesis can be rejected. The Siegel-Tukey method is also robust: the ion-selective electrode value of 0.46 could have been replaced by any value <0.50, and the values 0.69, 0.83 and 0.85 could have been replaced by any values B0.68 without disturbing the rank order calculated above.However, this test is open to criticism on the grounds that it lacks power, even when, as in our example, the medians of the two sets of results are equal or close to each other. The power of a test is its ability to reject correctly a false null hypothesis. Generally speaking, signifi- cance tests are most powerful if they use the greatest amount of available information. In the Wilcoxon-Mann-Whitney and Siegel-Tukey tests we gain both simplicity (examples with small n, rn, can often be calculated mentally) and robustness by replacing the actual measured concentrations etc.by ranks, but the inevitable price paid is some loss of power. Many non-parametric methods are robust (although the opposite is not true), but some non-parametric methods even lack robustness. This is true of, for example, Tukey’s quick test, which is often used as a rule-of-thumb for comparing two sets of results such as the river water data given above. Tukey’s test declares that, for two statistical samples to be determined to come from different populations, the one with the higher median must have some data that are higher than all the measurements in the other sample, and the one with the lower median must have some results which are lower than all the data in the ‘higher’ sample. This is clearly not the case in our river water example, because of the anomalous result of 780ngcm-3 for area A, so the Tukey test would suggest, because of its lack of robustness, that the two samples were not significantly different.By contrast, some non-parametric methods have been specifically designed to identify outliers. These techniques, summarized in the classic text by Barnett and Lewis,l seem to have found little practical application. In the context of analytical measurements, the greatest objection to the use of non-parametric methods is that they discard too much information. The common use of ranking methods, and the associated loss of power, has already been mentioned. More fundamental is the belief that in many analytical situations the data do come, at least in part, from populations with Gaussian error distributions, but with contamination by outliers arising from gross errors. There is also increasing evidence that, in practice, many data sets come from heavy-tailed distributions, i.e., with an excess of measurements distant from the mean. Such a situation might arise from the superposition of two or more normal distribu- tions with similar means but with different standard devia- tions, an outcome that might occur if, for example, two or more individuals, pieces of equipment or sets of experimental conditions had been used in making the measurements. In these circumstances, the ideal way to treat suspect or outlying results would be to use methods which down-weight , without entirely rejecting, measurements which are far distant from the mean. Methods using this approach are summarized in the next section.Robust Statistics Robust statistical methodsg-11 have been studied extensively over the last two decades or more, but have only recently become widely available to practising scientists. This is because, despite the underlying simplicity of their principles, they are frequently iterative methods, which cannot usually be undertaken without a personal computer. It is, therefore, the widespread availability of ample computing power that has brought these methods into sharper focus (although many otherwise excellent software packages still omit robust statis- tics), and they may in future become the methods of choice for dealing with data sets containing suspicious results. Numerous robust tests have been developed; again, only a summary can be attempted here.Some very simple robust approaches do not require iterative computations. One fairly obvious calculation is that of the trimmed mean. This involves the deliberate omission of a certain percentage of the measurements (in practice 10-25% trimming is common: a ‘10% trimmed mean’ is the mean determined after the top 10% and the bottom 10% of the results have been omitted). This procedure has the obvious drawback of being arbitrary. How do we decide the extent of the trimming, why trim at both ends of the data sample when suspicious results may only occur at one end and why remove such results altogether when, as already noted, it might be better simply to reduce their weights? Moreover, trimming is of little value in many cases where the number of measure- ments is small.With a sample of five measurements, the least trimming that can be carried out is to eliminate the top and bottom measurements (i.e., 20% trimming) leaving only three data points: this is clearly unacceptable. A variant on the trimming approach is Winsorization, in which an outlying result is not removed, but ‘moved’ so that its residual (i.e. , the difference between the single result and the sample mean) is reduced so that it becomes the same as the second (or perhaps third) largest or smallest result. Although Winsorization is still sometimes used in regression problems (see below), these relatively crude methods have been largely supplanted by the more sophisticated iterative approaches. At this stage, it is important to introduce a robust dispersion estimate known as the median absolute deviation (MAD!).The MAD is given by: MAD = median[lxi - median(xi)I] (4) If we apply this equation to the data given at the beginning of this review (9.97, 10.02, 10.05, 10.07, 10.27; median 10.05), the individual absolute deviations from the median are 0.08, 0.03, 0, 0.02 and 0.22, respectively. The MAD is, therefore, the median of the five latter numbers, i . e . , 0.03. The MAD has several useful attributes. For example, it provides the basis of a crude outlier test, based on the ratio [Ixo - median(xi)l]/ MAD. If this ratio exceeds 5 for any single outlier, XO, then that outlier can be rejected. Note that this test suggests that, in the above data, the result 10.27 could be rejected, the ratio being 0.22/0.03: this conclusion disagrees with that of the Dixon method, rightly making us suspicious of both methods! A more important property of the MAD is that MAD/0.6745 can be shown to give a useful and robust estimate (6) of the population standard deviation, 0.In our example, MAD/ 0.6745 = 0.03/0.6745 = 0.0448. We can now make a robust estimate (p) of the population mean, p, using a distance function different from that usually used. In conventional statistics our estimate of the mean is obtained by minimizing a sum of squares (SS), Z( Ixi - p1)2. In this case the distance function is (xi - p)2: as we have already seen, it is the use of the sum of such squared terms which makes this estimate of the mean so sensitive to large errors. We now use an alternative distance function, simply Ixi - PI.Any measurement for which this function exceeds 1.50 is down-weighted. It should be noted that the value of the constant 1.5 is not obligatory, but is fairly general. In our example 1.50 = 1.5 x 0.0448 = 0.0672. The iterative process of calculating p needs an initial estimate, which we can conveniently take as the median of the measurements, 10.05. Deviations from this value exceeding 0.0672 therefore need down-weighting, a process achieved by replacing such resultsANALYST, MAY 1993, VOL. 118 459 by F k 0.0672 as appropriate. This process clearly moves the value 9.97 to a new value of 10.05 - 0.0672 = 9.9828, and the value 10.27 is reduced to 10.05 + 0.0672 = 10.1172. We therefore have a new set of five values, usually called pseudo-values; three remain unchanged (10.02, 10.05 and 10.07) but the other two are altered from their original values to those just calculated.A new mean can, therefore, be determined as a second estimate of p: note that although the initial ji estimate may be the median, all subsequent estimates of p are means. This second estimate is 10.048: further down-weighting may now be required if any of the pseudo- values lie outside the range 10.048 & 0.0672, i.e., 9.9808- 10.1152. The highest pseudo-value calculated in the first iteration (10.1172) lies just outside this range, and is, therefore, altered to 10.1152. The new set of pseudo-values (now 9.9828, 10.02, 10.05, 10.07 and 10.1152) gives us a third estimate of p, i.e., 10.0476. This value is so close to the previous estimate that we can safely take a rounded value of 10.048 as a robust estimate for $.Clearly, this calculation will be more complex with a larger set of measurements, and the p estimates may converge more slowly, hence the need for a personal computer to expedite the calculations. The principles underlying this method ('Huber's rn-estimator') are clear and simple, but other methods are available. In this case the use of MAD/0.0645 as a robust standard deviation estimate survived through each iteration, i.e., the assumed value of 6 was constant: in other methods a robust estimate of the mean is the starting point for iterative calculations of the standard deviation, and in still others both mean and standard deviation are calculated iteratively and simultaneously.In summary, robust statistics provide a sound and logical approach to the problem of suspect results, by down-weight- ing them to an extent that depends on their degree of departure from the remaining data. Although the computa- tions may be tedious to explain, they are simple to perform with the aid of suitable software. It must be added that several of the books on the theory of robust methods are forbidding in the extreme: preferable are two excellent summaries provided by the Analytical Methods Committee.9.'" Outliers in Regression The methods discussed in the previous sections are applicable to replicate measurements of the same signal, titre, intensity, etc. However, perhaps the commonest approach .to the handling of analytical data is to use regression techniques.12.13 These find application in two areas in particular: in the plotting of calibration graphs in quantitative analysis, and in method comparison studies.In calibration graphs a series of specimens of known concentration is examined, the instrumental signals resulting are recorded, and the data plotted with the signals on the y-axis and the standard concentrations on the x-axis. An appropriate calibration line is drawn, and then used to estimate the concentrations of test specimens by interpolation. The calculations in this applica- tion usually assume that errors occur only in the y-direction, i.e., that the standard concentrations are error-free: this assumption may not always be valid. In method comparison studies, a number of specimens are examined by each of the two methods under study, and the two sets of results obtained are plotted on the x- and y-axes. Each point on the graph therefore represents a single specimen examined by the two methods.If the methods gave identical results in every case, the graph would be a straight line with zero intercept, unit slope and a correlation coefficient of 1: of course such results are never obtained in practice, but the closeness of these statistics to their ideal values is used as a measure of the agreement between the two methods. In this instance it is obvious that measurement errors must be expected in both the x- and y-directions. In either of these applications of regression methods, it is 70 1 I I *LA 60 40 I 0 10 20 30 40 50 60 70 X Fig.1 A, Straight line and B, quadratic least-squares fits to a set of data points (for data see text). The straight line fit gives an adjusted R2 value of 99.4%, with the point (70,64) marked by MINITAB as a possible outlier. The quadratic fit gives an adjusted R2 value of 99.6%, with no outliers identified clear that outliers or suspicious results may occur, especially because in many cases restrictions of time or material mean that only single measurements or very small numbers of measurements are made on each standard or test specimen. The identification and/or treatment of such results is, there- fore, as important in regression calculations as it is in replicate measurements. Moreover, it is apparent that in regression calculations outlier problems are rather more complex.There are several reasons for this. One is that, as already noted, outliers may occur in the x- andor the y-directions. (It must be added that in multiple regression, e.g. , with the form y = a + blxl + b2x2 + bg3 + ... outliers are even harder to identify, but this problem will not be treated here.) A second problem is that, to an even greater extent than in replicate measure- ments, the status of possible outliers depends crucially on the model assumed. This is exemplified in Fig. 1, which shows a fairly simple calibration graph. If the analyst was absolutely certain on the basis of experience , underlying physico-chem- cia1 principles, etc. , that the plot should be linear, then the last point on the graph might be regarded as an outlier. However, as Fig.1 also shows, the points are excellently fitted by a quadratic model, in which case no question of outlier identification arises. Lastly, we must observe that some apparently obvious approaches to outlier identification are not really admissible. It might be supposed, for example, that in an unweighted regression calculation (i. e. , one in which the y-direction error is assumed to be the same at all values of x ) , outliers could simply be identified by treating the y-residuals as a set of replicate results, and applying the Dixon or other test methods. (The y-residuals are the - 91 values, i.e. , the y-direction distances between the experimental points and the fitted line at any x-value.) This method is unsound, however, as the y-residuals are not independent measurements, as they total zero.Despite these concerns, it is found in practice that the approaches to outlier diagnosis in regression mirror those used in replicate measurements. There are outlier tests, some fairly simple and some less so; non-parametric methods are avail- able; and robust regression methods have also found recent use in analytical work. However, an additional concept of particular importance to regression problems is worth intro- ducing at this point, viz., the breakdown point (BDP). Put simply (there is, of course, a mathematical definition also) the breakdown point is the percentage of outlier points on the graph that can be tolerated before the regression line determined is significantly altered. Common sense indicates that the maximum value of the BDP is 50% (beyond that level, who is to say which points are the genuine ones, and which are460 ANALYST, MAY 1993, VOL.118 the outliers?). It is easy to show that, in conventional least-squares regression, even a single outlier can greatly change the estimates of the regression coefficients: the BDP of this approach is, therefore, 0%. In analytical science, we would hope that in most cases the number of outliers will not be large, so any method with a BDP of, say, 220% will be of importance. The use of relatively simple test methods, based as so often in regression statistics on residual diagnostics, is exemplified in many elementary statistical packages. (As already noted, residuals are [y - 91, i.e., [experimental - fitted] values.) For example, MINITAB,14 widely used in both teaching and research, will list the residuals obtained in a least-squares regression calculation, and convert them into standardized residuals ( i e ., with mean = 0 and standard deviation = 1). Points whose standardized residuals are >2 or <-2 are highlighted in the printout of the results. Note that, as with ordinary residuals, standardized residuals are not indepen- dent, so this method of highlighting outliers has to be treated with reserve, especially when n, the number of points on the graph, is small. However, two additional ways of manipulating residuals do not suffer from this disadvantage. Studentized residuals, ri, are given by: ( 5 ) In this equation, ei stands for the original residuals; sylx is the residual standard deviation, [Eei2/(n - k - 1)]4, where k is the number of terms in x, x2, etc., in the regression equation, and hi is the leverage value of the ith point, given by: hi = (l/n) + (xi - %)2/(n - l)s,* where sx2 = X(xi - x)z/(n - 1).In simple linear regression, therefore, the leverage of a point is a measure of its distance from the mean of the xi values. It may be shown that, for a graph which is not forced through the origin, i.e., has a constant term, a, hi lies between lln and 1. MINITAB and other statistics packages highlight xi-values with high leverages, a facility of obvious value in identifying x-direction outliers when a regression line is used in method comparisons. Note that a point with a high leverage is not necessarily a y-direction outlier, and vice versa.A further modified form of residual is the jackknife residual, ri, given by: r-i = ei/[si(l - hi)$] (7) where si is analogous to s,/,, except that the ith point is omitted from the calculation. Both the Studentized and jackknife residuals have the property that they approximately follow a t-distribution, with (n - k - 1) and (n - k - 2) degrees of freedom, respectively. Moreover, if n is greater than about 30, the distributions of these residuals are approximately standard normal distributions. These properties make the identification of outliers relatively simple at any chosen p-value. Yet another and distinct approach is to use Cook’s distance, di, a statistic that measures the extent to which the regression coefficients (a, b, etc.) are influenced by the omission of individual points.The equation is: It can be shown that di is always 30, and should normally be less than 1; values >1 are regarded as worthy of further investigation as possible outliers. These and related methods are fully discussed by Kleinbaum et al. 13 A previous section of this review showed that non-para- metric methods based on the median were of value in by-passing, as it were, the problems associated with possible outliers. This advantage extends to non-parametric regression methods, of which the best known is that of Theil. His original (‘complete’) method involved taking all the possible pairs of points from the n points on the graph, and estimating the slopes of the lines joining them. The median of these n(n - 70 , I 9 / A 40 ~- 1 / I I I 1.I I 0 10 20 30 40 50 60 70 X Fig. 2 A, Least-squares and B, Theil incomplete methods applied to fit a straight line to the data plotted in Fig. 1 (for data see text) 1)/2 estimates was taken as the best estimate of the slope, b, of the regression line. Use of this estimate together with the coordinates of the n points and the equation a = y - bx provided n values of the slope, a, of the regression line, and again the median was chosen as the best estimate of a. This procedure is open to at least two objections, of which the simpler is the practical one, that the number of slope estimates is very large: even a graph with only eight points would provide 28 separate estimates of b. The second and more serious objection is that the method gives equal weight to the slope estimates from pairs of points that are close together and well separated on the graph; surely the latter estimates should carry more weight? Both problems are overcome by the simpler procedure used in Theil’s ‘incomplete’ method, in which n/2 slope estimates are given by using (xl, yl) and the point immediately above the median value of xi, ( x 2 , y2) and the second point above the median, and so on. If, as is usually the case in calibration graphs, the xi values are equally spaced, then the slope estimates obtained using this method will rightly be accorded equal weight.(If n is odd, then the point with the median value of xi is omitted from the calculation.) We can apply this method to the data used in Fig. 1, which were: x = 0,10,20,30,40,50,60,70; y = 1,9,21,30,41,49, 59, 64.The four slope estimates obtained from these measurements are clearly 40/40,40/40,38/40, and 34/40, so the median slope estimate is 39/40, 0.975. The eight individual intercept estimates, determined as described above, are then 1, -0.75, 1.5, 0.75, 2, 0.25, 0.5, and -4.25. The median of these values is 0.625. Hence the Theil method gives a straight line equation of y = 0.625 + 0.975~. The conventional least-squares method applied to the same data gives a result of y = 1.50 + 0.936~. Both these lines are plotted in Fig. 2: it is clear that the Theil method has effectively ignored the point (70, 64) which indeed does not figure directly in either the median estimate of b, or that of a. (This is not simply because it is the last point of the eight: the advantages of the Theil method apply to any of the points.) By contrast the least- squares line has given the point (70, 64) a weight equal to any other point, so the least-squares line passes closer to it than the non-parametric line.Again, therefore, the non-parametric method has effectively circumvented the outlier problem. [Note that the MINITAB output for this set of data highlights the point (70, 64) as a possible outlier, with a standardized residual of -2.14, and that, as already shown, a straight line plot may not be the most appropriate for this data set.] The BDP of the Theil method is about 30%, more than adequate for most purposes, and it can be improved by using iterative methods to estimate the median slope and intercept.It can be freely used in cases where there are both x- and y-direction errors. Moreover, the principles underlying theANALYST, MAY 1993, VOL. 118 46 1 method are fairly general, and can be extended to polynomial and multiple regression problems. Lastly, we consider briefly the use of robust regression methods. By analogy with the robust techniques discussed earlier, we expect these to modify suspect or outlying results to an extent that depends on their departure from normal behaviour. One approach that has been shown to be powerful, leading in some circumstances to methods with BDPs of 50% , is that of trimming. The least trimmed squares and least Winsorized squares methods both utilize the least-squares concept, but the residuals concerned are modified by trim- ming or Winsorization, respectively.In this case, and possibly in contrast to repeated measurements, there have been claims that trimming is the superior approach. Several other robust methods have been developed. Over a century ago, the least absolute values method was proposed, i.e., the sum of the residuals without regard to their signs is minimized. This fairly simple method has a BDP of 50% with respect to y-direction outliers, but its BDP in the x-direction is zero. A much more recent and widely applied method is the least median of squares technique, in which an iterative process is used to minimize the median value of e,2. The main disadvantage with this method in practice is that the con- vergence of values can be fairly slow, but this is not a major problem for the small data sets encountered in analytical science. These and other robust regression methods are summarized in another established text, by Rousseeuw and Leroy.11 The importance of these methods to analysts is underlined by an increasing number of research papers in which robust regression is used, or the various robust approaches compared. This, in turn, should encourage the wider availability of appropriate software. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 References Barnett, V., and Lewis, T., Outliers in Statistical Data, Wiley, New York, 2nd edn., 1984. Hawkins, D. M., Identification of Outliers, Chapman and Hall, London, 1980. Miller, J. C., and Miller, J. N . , Statistics fur Analytical Chemistry, Ellis Horwood, Chichester, 3rd edn., 1993. Anderson, R. L. , Practical Statistics for Analytical Chemistry, Van Nostrand Reinhold, New York, 1987. Use of Statistics to Develop and Evaluate Analytical Methods, eds. Wernimont, G . T . , and Spendley, W., Association of Official Analytical Chemists, Arlington, VA, 1985. Sprcnt, P., Quick Statistics, Penguin, Harmondsworth, 1981. Sprent, P., Applied Non-Parametric Statistical Methods, Chap- man and Hall, London, 1989. Conover, W. J . , Practical Non-Parametric Statistics, Wiley, New York, 2nd edn., 1980. Analytical Methods Committee, Analyst, 1989, 114, 1489. Analytical Methods Committee, Analyst, 1989, 114, 1497. Rousseeuw, P. J., and Leroy, A. M., Robust Regression and Outlier Detection, Wiley, New York, 1987. Draper, N. R., and Smith, H . , Applied Regression Analysis, Wiley. New York, 2nd edn., 1981. Kleinbaum, D. G., Kupper, L. L., and Muller, K. E., Applied Regression Analysis and Other Multivariable Methods, PWS- Kent, Boston, MA, 1988. MINITAB (Minitab Inc.), User's Manual, Addison Wesley, London. Paper 3100356F Received January 20, 1993 Accepted February 24, 1993

ISSN:0003-2654    

DOI:10.1039/AN9931800455

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, MAY 1993, VOL. 118 463 Surface Modification of the Biomedical Polymer Poly(ethy1ene terep h t h a late) Ldn N. Bui and Michael Thompson* Department of Chemistry, University of Toronto, 80 Saint George Street, Toronto, Ontario, Canada M5S ?A I Neil B. McKeown,+ Alex D. Romaschin and Peter G. Kalman Vascular Surgery Division, Toronto General Hospital, 200 Elizabeth Street, Toronto, Ontario, Canada M5G 2C4 X-ray photoelectron spectroscopy was used to characterize modified surfaces of a biomedically important polymer, poly(ethy1ene terephthalate). Several modification schemes were investigated and direct silanization with 3-aminopropyltriethoxysilane was found to be the optimum procedure, resulting in an aminated surface. Surface coverage of up to 100% was achieved with retention of the polymeric structural integrity.Further activation of the silanized surface was accomplished with two cross-linkers, glutaraldehyde and sebacoyl chloride. A simple biomolecule, L-cysteine, was successfully immobilized onto a surface pre-treated with 3-aminopropyltriethoxysilane and glutaraldehyde, with a coverage of 42%. Keywords: Biomaterial: biocompatibility; poly(eth ylene terephthalate); silanization; X-ray photoelectron spectroscopy An important aim in the field of biomaterial technology is the improvement of the biocompatibility of existing synthetic vascular grafts. Currently, the most common arterial substi- tutes are Dacron, composed of woven or knitted fibres of poly(ethy1ene terephthalate), (PET), and Gortex, produced from expanded poly(tetrafluoroethy1ene) (PTFE).These graft materials possess the required mechanical properties and excellent chemical stability in vivo. However, both types of graft are susceptible to occlusion as a result of thrombus formation,’ particularly under low flow, high resistance conditions present in small diameter arterial by-passes. Poly(ethy1ene terephthalate) is inherently more thrombogenic than PTFE;2?3 therefore, its primary application is restricted to larger diameter by-passes in the coronary area. The high flow rate and low resistance in this region tend to minimize thrombus formation. Poly(tetrafluoroethy1ene) is the conduit of choice for smaller diameter arterial by-passes. The cumula- tive five years patency for one such type of operation, a lower extremity by-pass, is only 17~25% .I The usual consequence of a graft failure is the amputation of the diseased appendage; therefore, substantial improvements in the biocompatibility of both graft materials are clearly required.Despite its greater thrombogenicity , PET is still favoured for large diameter arterial reconstructions because of its excellent handling characteristics during an operation. Its superior elasticity and pliancy make PET the preferred material, provided that its thrombogenicity can be improved to or beyond that of PTFE. It has been demonstrated that the surface properties of any graft material are of prime importance in the determination of its biocompatibility;4.5 hence a decrease in thrombogenicity may be achieved through modification of the surface of the polymer.There have been numerous attempts to attain this objective with the common approach being the immobiliza- tion of anti-thrombogenic moieties onto the surface of the polymer.6 Numerous compounds have been investigated, among them heparin,’ various prostaglandins8 and albumin.9 The nature of these attachments has largely been by physical adsorption. This has resulted in only limited success as the adsorbed compounds can be slowly removed by the blood flow. The stability of the modification will be enhanced if the inclusion of chemical covalency in all aspects of the modifica- * To whom correspondence should be addressed. + Present address: Department of Chemistry, University of Man- Chester, Manchester, UK. tion process can be effected.A different approach is the modification of the existing material so that it ‘mimics’ that of a biological membrane, a biomimetic surface.10 A natural cell membrane consists of a bilayer of phospholipids, in which proteins and glycoproteins are supported. Model films of phospholipids have been prepared using the Langmuir- Blodgett technique and such films have been shown to be generally thrombo-resistant .I1 These films are, however, too unstable to be transferred effectively onto any prostheses; hence more stable lipids and lipid-like surfaces must be developed. These can then be immobilized onto the polymer to provide non-thrombogenic surfaces. Other constituents of a membrane, such as those found in endothelial cells, including cholesterol and polymeric saccharides, can also be incorpor- ated.8 The inclusion of chemical covalency is still of prime importance because, unless a covalent attachment of the lipid layer can be achieved, any gain in biocompatibility would be short-lived.The lack of indigenous reactive functional groups on the PET surface precludes any covalent attachment unless the introduction of free, reactive chemical species onto the surface can be achieved. The functionalization of the polymer surface will allow further modifications of a more permanent, covalent nature. The target functional groups are hydroxyl or amino groups as both can facilitate further chemistry. Poly- (ethylene terephthalate) is a polyester; hence it is susceptible to a variety of chemical reactions at the carbonyl sites. Dave eta1.12 reported the saponification of PET, giving rise to a surface comprised of carboxylic and hydroxyl functional groups. Aminolysis using multifunctional amines to create free NH2 moieties was investigated by Avny and Reuben- feld.13 Multifunctional reagents were employed to reduce the reactivity and control the degree of polymeric degradation.The derivatization of PET with poly(ethy1ene glycol), using a diamine as a cross-linker, was reported by Kim and K0.14 Poly(ethy1ene terephthalate) can also be reduced using a strong reducing agent such as a metal hydride. The reduction of PET with LiAlH4, yielding a hydroxylated surface, was patented by Collins.15 In this investigation, all of these procedures were attempted and the resultant surfaces were analysed to determine which method provides the optimum modification. As these reactions involve the cleavage of the ester bonds, there exists a possibility of structural degrada- tion.This aspect was investigated for each experiment through the implementation of a degradation reaction, i.e., modifica-464 ANALYST, MAY 1993, VOL. 118 tion using an analogous but more concentrated solution or by simply adopting a longer reaction time. After functionalization has been achieved, the polymer can be further activated using various cross-linkers. This would facilitate the eventual immobilization of phospholipids or any other non-thrombogenic entities. An important class of coupling reagents are multifunctional silanes. The utility of these silanes can be attributed to the fact that they can form strong covalent bonds to a substrate and yet still retain their chemical reactivity.These silanes have been used to improve the bonding of polymer resins to metal surfaces and glass fibre. 16717 Several procedures have also been developed employing silanes and other cross-linkers to promote the covalent bonding of organic complexes to a functionalized surface. These include the development of a surface acoustic wave chemical sensor,18 the attachment of antibodies to a substrate for affinity chrornatography,'g the construction of a polymer-modified electrode20 and the immobilization of enzymes on solid supports for enzyme assay techniques .2l,Z2 Recently, Kallury et al.23 reported the covalent linkage of modified phospholipids to activated gold and glass surfaces, utilizing reactive silanes such as 3-aminopropyltriethoxysilane (APTES) and dichlorodimethylsilane (DCDMS) and also other cross-linkers such as sebacoyl chloride (SB) and monomethyl azelate.These procedures can be readily adap- ted for an activated PET surface as the initial active moieties would be the same, hydroxyl or amino groups. The immediate aim of this work was the adaptation of these methodologies to immobilize a simple biomolecule, L-cysteine, onto an acti- vated PET surface. X-ray photoelectron spectroscopy (XPS) was chosen as the primary means of analysis of the surfaces studied. Advancing water contact angle (WCA) measurement and scanning electron microscopy (SEM) were used to complement the XPS results. Experimental Materials and Chemicals The modifications were performed on thin films (0.25 mm) of PET (Mylar, DuPont, Toronto, Canada) (hereafter this will be referred to as surface 1) and on the manufactured woven Dacron graft (Meadox Medical, Oakland, NJ, USA) (surface 2).Owing to the prohibitive cost of the Dacron graft, most of the experiments were performed solely on the PET polymer. However, certain important experiments were performed concurrently on Dacron to ensure the applicability of the procedure to the actual graft material. All surfaces were cleaned by Soxhlet extraction with tetrahydrofuran (THF) for 4-6 h prior to and after all modifications to ensure the removal of most physically adsorbed contaminants. This was followed by rinsing with ethanol and chloroform. Toluene and THF were refluxed, prior to collection, over sodium and benzophe- none. Dichloromethane was distilled over phosphorus pento ide, also for several hours. All three solvents were distilled under a nitrogen atmosphere.Water was doubly distilled and de-ionized prior to use as a solvent and rinsing agent. Hydrazine, LiAlH4, HCI, 1,3-diaminopropane (DAPr), 1,5- diaminopentane (DAPe), tetraethylenepentaamine (TEPA), DCDMS, trifluoroacetic anhydride (TFAA), SB, 50% v/v glutaraldehyde (GA) in water, L-cysteine and all solvents were purchased from Aldrich (Milwaukee, WI, USA). Tetra- ethoxysilane (TES) and APTES were obtained from BDH (Toronto, Ontario, Canada). 3-Aminopropyldiethoxymethyl- silane ( APDEMS) and 3-aminopropylethoxydime thylsilane (APEDMS) were acquired from Fluka (Ronkonkoma, NY, USA).All silanes were vacuum-distilled prior to use and all reactions, except for base saponification, hydrolysis and cysteine immobilization, were carried out under a nitrogen atmosphere. Degradation experiments were performed with neat solutions of the specified reagents where possible. Modification Procedures Virgin PET (1) was incubated in a 10% v/v aqueous solution of hydrazine at 60 "C for 0.5 and 1.5 h to provide surfaces 3A and 3B, respectively. The degradation of each surface was achieved through an incubation period of 24 h in the same solution. Poly(ethy1ene terephthalate) samples (1) were incu- bated in 5% v/v toluene solutions of TEPA, DAPe and DAPr for 6 h under nitrogen. The results were the aminated surfaces 4,5 and 6, respectively. 1,3-Diaminopropane was also reacted with Dacron (2) for a period of 12 h to give surface 7.Degradation experiments were carried out using neat reagents. Virgin PET (1) was reduced using a solution of 0.5 g of LiAIH4 in 25 ml of dried THF for 20 min under nitrogen. The resulting sample, 8, was then acidified with 0.01 mol 1-1 HCI. The degradation experiment was carried out by incubating a sample in a similar solution for 1 h. All reactions with silanes were performed using 2% v/v toluene solutions under nitrogen atmospheres for periods of 24 h. The only exception was the silanization involving DCDMS, for which dichloro- methane was employed as the solvent. All silanized samples were cured for at least 48 h at ambient temperature and pressure prior to any subsequent modification.Samples previously reduced by LiAIH4 (8) were silanized with APTES and DCDMS to give surfaces 9 and 10, respectively. Both virgin PET (1) and virgin Dacron (2) were also silanized with APTES to give surfaces 11A and 12, respectively. An incubation time of 48 h was also used for PET to produce surface 11B. Silanized sample 11A was immersed in a buffer solution (pH 9) for 24 h, yielding surface 11C. Poly(ethy1ene terephthalate) (1) was also silanized with TES, APEDMS and APDEMS to give samples 13, 14 and 15, respectively. The degradation of PET was attempted by leaving samples in neat reagents for periods of up to 5 d. Poly(ethy1ene terephthalate) (1) and Dacron (2) were incubated in a solution of 2 ml of TES in 100 ml of dried toluene with 0.2 ml of DAPr added as an initiator.The reaction was carried out under a nitrogen atmosphere for a period of 24 h, resulting in surfaces 16 and 17. An unmodified PET sample (1) and silanized PET samples ( l l A , 14, 15 and 16) were suspended over neat TFAA in a vapour phase reaction for 24 h. The resulting surfaces were designated 18-22, respectively. A control PET (1), and an APTES-modified surface (11) were incubated in 5% v/v aqueous GA solution. The reaction was performed at ambient pressure and temperature for 24 h. The resulting surfaces were designated 23 and 24, respectively. A control PET sample (1) and an APTES-modified sample (11) were incubated in 3% v/v solutions of a second type of cross-linker, SB, to give specimens 25 and 26. Toluene was used as the solvent and 0.1 ml of pyridine was also added as a proton scavenger.The incubation time was 24 h and a nitrogen atmosphere was employed. Surfaces modified with APTES and APTES-GA (11 and 24) were incubated in a solution consisting of 1.0 g of L-cysteine dissolved in 10 ml of doubly distilled, de-ionized water for a period of 3 h. The resulting samples were designated 27 and 28, respectively. Instrumentation Some XPS spectra were acquired using an SSX-100 (Moun- tainview, CA, USA) electron spectroscopy for chemical analysis instrument at the University of Western Ontario (UWO) Surface Science Laboratory. The spectrometer was equipped with an unmonochromated aluminium K a source and the sampling area was 1 mm in diameter. A flood gun setting of 1.0 eV was required to compensate for charging effects.Scofield constants were used to derive sensitivity factors for the aluminium source [C(ls) = l.OO,O(ls) = 2.49, N(1s) = 1.68 and Si(2p) = 0.901.' Peak deconvolution wasANALYST, MAY 1993, VOL. 118 465 performed on a Hewlett-Packard (Palo Alto, CA, USA) 9836 computer using software provided by the manufacturer. All other spectra were taken using a Leybold MAX-200 surface analysis system (Leybold, Cologne, Germany) employing an unmonochromated magnesium Ka source with an excitation energy of 1253.6 eV. No flood gun was employed but the resulting spectra were calibrated by positioning the aliphatic carbon peak at 285.0eV. The observed charging effect is usually of the order of 3 4 eV. The sampling area utilized for all spectra was 4 x 7 mm.Survey and high- resolution spectra were obtained using pass energies of 192 and 48 eV, respectively. The elemental composition was calculated from satellite-subtracted spectra, normalized for constant transmission using sensitivity factors previously calculated for the Leybold MAX-200 system [F(ls) = 1.00, O(1s) = 0.78, C(1s) = 0.34, Si(2p) = 0.40 and N(1s) = 0.541. Peak deconvolution was performed using a program supplied by the manufacturer. Most spectra were obtained with the detector at 90" to the sample except during the angular dependency study, for which detector angles of 60,45 and 30" were employed. Consecutive area analyses of the carbon 1s peak of the control PET surface (surface 1) were performed to determine the extent of the damage to a sample caused by the X-ray source. The measured decrease in the intensity of the carbon peak was on average approximately 2% of the original peak, well within experimental error; hence, damage can be concluded to be minimal.The decrease may in fact be due only to the sputtering of adsorbed hydrocarbon contaminants by the X-ray source. Scanning electron microscopy was performed using a Hitachi (Tokyo, Japan) S-570 system with an accelerating voltage of 18 kV and a working distance of approximately 15 mm. The samples were gold-coated in a Polaron (Watford, Hertfordshire, UK) vapour deposition unit at 20 mA and 2.4 kV for a period of 120 s. Advancing WCAs were measured on a Remy-Hart (Mountain Lakes, NJ, USA) goniometer at 20°C. Measurements were taken for both sides of the advancing drop and the average was calculated.This was repeated three times for each sample to arrive at a more reliable mean. Results and Discussion Control Polymer The XPS survey spectrum of control PET (Fig. l), obtained using the XPS spectrometer at the UWO Surface Science Laboratory, yields the expected carbon (285 eV) and oxygen (532 eV) peaks (1 of Table 1). High-resolution analysis of the C(1s) peak reveals the three carbon bond types expected from an ester: carbonyl(289.0 eV), ether (286.5 eV) and hydrocar- bon (285.0 eV). The scanning electron micrograph shows a smooth, clean surface with only several white, circular artefacts [Fig. 2(a)]. These are either adsorbed dust particles or gold clusters, deposited during the coating process.The solid line near the top left-hand corner of the scanning electron micrograph is a photographic defect and not an inherent feature of the surface. The advancing WCA measure- ment of 82" (1 of Table 2) corresponds well with the reported literature range of 79-83.5".1*313 The survey and high-resolu- tion C( 1s) spectra of Dacron are almost identical with those of PET with the differences in percentage compositions being statistically insignificant (2 of Table 1). Scanning electron micrographs of the Dacron surface show the characteristic weaveknit pattern of the graft, the individual strands being smooth and even [Fig. 2(6) and ( c ) ] . The few white specks on the strands are again simply adsorbed dust particles or gold clusters. In view of the morphology of the surface, WCA measurements of Dacron could not be obtained for compari- son.The same PET and Dacron samples, analysed using the Leybold MAX-200 spectrometer, yield significantly different numerical values (1A and 2A of Table 1). This is probably 240 - (a) 220 - 200 180 - 160 - - - 60 - E 20 C 3 8 $ 280 *c 240 -r 260 2 220 5 200 - != 180 1 60 140 120 100 80 60 40 20 = 900 800 700 600 500 400 300 200 100 292 290 288 286 284 282 280 Binding energyleV Fig. 1 ( a ) Survey and (b) high-resolution C(1s) spectra of control PET Table 1 Elemental and high-resolution XPS of nucleophilic modifica- tions* Oxygen Nitrogen Surface Carbon (285.0 eV) (532.1 eV) (400.0 eV) 1. Control PET (UWO) Yo c 79.5 Yo 020.5 YO C-C 67.2 (285.0 eV) % C-0 17.2 (286.5 eV) YO C=O 15.8 (289.0 eV) 1A.Control PET (MAX-200) Yo C 71.2 Yo 0 28.8 Yo C-C 61.9 Yo C-0 20.7 Yo C=O 17.4 2. Control Dacron (UWO) Yo C 79.2 % 020.8 % c-c 67.4 Yo C-0 17.1 Yo C=O 15.6 2A. Control Dacron (MAX-200) %C 72.0 Yo 0 28.0 Yo C-C 62.1 Yo C-020.9 Yo C=O 17.0 3A. PET-hydrazine 3B. PET-hydrazine (0.5 h) YOC 75.3 Yo 023.4 Yo N 1.3 (1.5 h) % c 75.8 Yo 021.5 % N2.7 Yo c-c 59.9 YO C-0 22.8 Yo C=O 17.3 4. PET-TEPA Yo C 76.6 Yo 023.0 % N0.5 5. PET-DAPe YoC 75.1 Yo 023.9 Yo N 1.0 6. PET-DAPr % C 74.8 Yo 023.1 Yo N2.1 7. Dacron-DAPr % C 71.3 Yo 021.5 % N7.2 % C-C 65.0 Yo C-0 14.5 YO C=O 7.1 (288.9 eV) % C-N 5.9 (287.8 eV) * Binding energies for all high-resolution C(1s) data are similar to those of 1 except for figures accompanied by binding energies in parentheses.466 ANALYST, MAY 1993, VOL.118 Fig. 2 Scanning electron micrographs of control surfaces: (a) PET at magnification of X3000; (b) Dacron at a magnification of X 100; and (c) Dacron at a magnification of X3000 0 II Table 2 Advancing WCA measurements of selected surfaces Surface WCA 1. Control PET 82.0" I NH2 NHI 8. PET reduced using LiAIH4 63 .0" NH7 9. Reduced PET modified with APTES 67.5" NH2 NHz I NH2 NH2 NH2 I 10. Reduced PET modified with DCDMS 11. PET modified with APTES 13. PET modified with TES 14. PET modified with APEDMS 15. PET modified with APDEMS 76.8" 69.0" 77.0" 78.9" 71.2' I I 1 16. PET modified with TES/DAPr 27.0" NH 26. PET modified with APTES then SB 87.8" NH2 NH2 NH2 NH2 because different sensitivity factors were utilized for the elemental composition calculation. Of the two sets of values, the set obtained using the Leybold MAX-200 spectrometer (C = 71.2%, 0 = 28.8%) seems to be more consistent with the actual elemental composition of the monomeric structure (C = 71.4%, 0 = 28.6-/0).For meaningful comparison, each set of results will be referenced against its respective control data. Spectra were acquired at the UWO laboratory for surfaces 1, 2, 3A, 3B, 8, 9 and 10. Saponification With Hydrazine Saponification with hydrazine did not result in any significant degree of modification. A nitrogen peak at 400 eV is seen, but the amount detected is minimal, constituting only 1.3% of the over-all atomic composition (3A of Table 1). An increase in the reaction time produced a small increase in the elemental nitrogen, but again, not to any appreciable extent (3B of Table 1).A decrease of the carbon peak and a relative increase of the oxygen peak, compared with unmodified PET, can be explained by the fact that XPS is a surface-sensitive technique. Some of the carbon atoms on the surface may have been masked by the hydrazine, while the more mobile hydroxyl ends are closer to the surface and hence can be detected more easily. High-resolution analysis of the C( 1s) peak does not reveal an amide bond, but the -NCO peak may be too small and may be submerged beneath the carbonyl peak, which has a similar binding energy. Another possible explanation is that most of the observed hydrazine is not covalently bound but instead is physically adsorbed in the numerous pits and cracks of the modified polymer.Fig. 3 Mechanism of polymeric degradation It was observed that the original shiny, smooth PET was dulled and some scoring of the surface can be seen. This observation combined with the limited amount of modifica- tion led to the hypothesis that the observed phenomenon is the result of concurrent nucleophilic attacks at adjacent or neighbouring carbonyl sites. The subsequent multiple cleavages of the ester linkages can result in the loss of modified organic fragments (Fig. 3). This would lead to a peeling effect, i.e., any modified layer would be stripped off, leaving a fresh, unmodified polymer surface. This peeling phenomenon would effectively negate any modification generated by the saponifi- cation process. The degradation of the polymeric structure was confirmed by the observation that exposure of a PET sample to the hydrazine solution for 24 h resulted in complete destruction.The PET sample was reduced to tiny, barely visible fragments. It is clear that base saponification is not an applicable process of modification owing to this deleterious consequence. Aminolysis Avny and Reubenfeldl3 and Kim and K014 reported the introduction of free amino groups onto the surface of PET via reactions with organic diamines or higher amines. Aminolysis using 1 ,2-diaminoethane was also reported by Desai and Hubbe1124 as a method for introducing amino functional groups onto a PET surface, prior to the covalent binding of poly(ethy1ene oxide) (PEO). The aminolysis mechanism is simply a nucleophilic attack at the carbonyl site of the polymer by the amine. Modification of PET by this process was attempted, using DAPr, DAPe and TEPA, but the resultsANALYST, MAY 1993, VOL.118 467 were similar to those of hydrazine: minimal modification accompanied by extensive degradation of the polymer (4-6 of Table 1). All three aminated surfaces exhibit an increase in the percentage composition of carbon and a corresponding decrease of oxygen. This can be attributed to the masking effect of the carbon-containing amines. In the modification of PET, DAPr produced the largest nitrogen peak among the three amines, a still insignificant 2.1%. It is postulated that owing to its length, it is entropically favourable for DAPr to form a ring structure with the polymer through reactions at both of its NH2 ends.This would result in a greater amount of modification but the required primary amine functional group may now be in a less reactive secondary form. The failure of TEPA (0.5% N) seems to be in contradiction with results published by Avny and Reuben- feld. 13 It is possible that the nitrogen-containing entities detected by Avny and Reubenfeld were degraded PET fragments (Fig. 3) that were not properly rinsed off the surface, whereas our surfaces were more thoroughly extracted with THF after every modification. Extensive degradation of the sample was observed within 1 h, if neat solutions were employed, and within 48 h for 5% solutions. Scanning electron microscopy of the DAPr-modified PET surface reveals that the amine has degraded the polymeric structures considerably, stripping large fragments off the surface [Fig.4(a)]. Under higher magnifications, large craters within the polymeric structure can clearly be seen alongside the PET fragments [Fig. 4(b)]. The film became brittle and was easily torn. As it produces the most significant modification, DAPr was used to treat a Dacron surface. Initially, the result was encouraging as the amount of nitrogen observed increases to 7.2% (7 of Table 1). This indicates that a large number of amino groups had been introduced onto the surface. However, the scanning electron micrograph of this surface shows that the degree of degradation has also increased substantially [Fig. 4(c)]. A large number of the strands that make up the weaveknit pattern are damaged or completely severed, as a result of the modification.Under higher magnification, it can be seen that the polymer has 'melted' into a film, covering the surface [Fig. 4(d)]. This degree of degradation, undoubtedly, would greatly affect the tensile strength, and hence, the structural stability of the graft. The SEM results help to provide an explanation for the increase in the amount of amine. The Dacron graft is much thicker than the PET film; therefore, it can withstand more degradation, and hence, additional modification. The weaveknit structure of the graft also allows for the retention of some adsorbed amine, despite the intensive extraction process. It is clear that the increase in the amount of modification cannot compensate for the magnitude of degradation. Although the damage to the surface could probably be controlled by varying the experimental conditions such as concentration and incubation time, this method of modification is obviously unsuitable for the functionalization of the delicate Dacron graft material.Reduction using LiAlH4 This reaction should yield two alcohol functional groups per PET monomer. A significant decrease in hydrophobicity is observed for this surface (8), with the WCA measurement dropping from 82 to 63" (Table 2). This indicates the presence of polar groups on the surface. However, high-resolution C(1s) analysis discloses that the percentages of carbonyl and ether carbons of this surface (8 of Table 3) are not significantly different from those values obtained for the unmodified PET (1 of Table 1).In fact, the percentage of carbonyl carbons actually increases by 1.2%. A possible explanation is that the LiAlH4 may have cleaved the ester bond but did not completely reduce the resultant carboxylic acid. The alkoxide functionalities, created through ester cleavages, may have destroyed the metal hydride and interrupted the reduction process. The increase in the amount of oxygen and the corresponding decrease of carbon support the cleavage theory, as the oxygen-containing termini are more surface- oriented and hence can be detected more easily than the carbon backbone. Another possible explanation can be derived from the observation that the reduction process also affects the integrity of the polymeric structure. Extensive degradation of the sample was observed when the reduction time was extended for a few hours, indicating that the reduction may have only served to strip away the top layer of the PET surface, exposing a fresh, unmodified layer of PET.The resultant degradation, again, places limits on the utility of this procedure as a modification technique. Subsequent Reactions With DCDMS and APTES Reasonable amounts of APTES and DCDMS were found to have reacted with the reduced PET surfaces (9 and 10 of Table 3). The fact that more substantial silanizations are not observed can be linked to the limited success of the reduction process. The WCA measurements for the APTES- and DCDMS-modified samples are 67.5 and 76.8", respectively. These figures are higher than that of the reduced surface, and can be explained by the fact that the silanes on the surface are not as hydrophilic as the hydroxyl groups of the reduced sample.The APTES-modified sample contains polar amino groups, whereas the DCDMS-modified surface is comprised of highly non-polar methyl termini. Significantly more APTES is observed to be immobilized on the surface than DCDMS, in spite of the fact that DCDMS is considerably more reactive. It was also found that whereas a reasonable magnification of x 1000 amount of APTES and DCDMS reacts with the reduced PET, Fig. 4 Scanning electron micrographs of DAPr-modified surfaces: (a) PET at a magnifiLation of ~3000; (b) PET at a ma nification of x22 OOO; (c) Dacron at a magnification of ~ 2 2 2 ; and (&Dacron at a468 ANALYST, MAY 1993, VOL. 118 Table 3 XPS data for LiAlH4 reduction and subsequent silanization* 10.PET-LiAlH4-DCDMS 11A. PET-APTES (24 h) 11B. PET-APTES (48 h) 11C. PET-APTES (PH 9) 12. Dacron-APTES Oxygen Surface Carbon (285.0eV) (532.1 eV) 8. PET-LiAlH4 Yo C 75.2 Yo 0 23.8 YO C-C 64.3 % C-0 18.7 Yo C=O 17.0 Yo C-C 66.2 Yo C-0 20.7 Yo C=O 13.1 Yo C 68.8 Yo 0 26.2 Yo c-c 64.1 % C-0 19.5 Yo C=O 16.4 Yo C 65.8 Yo 0 22.3 Yo C-C 65.6 YO C-0 23.2 9. PET-LiAlH4-APTES % C 68.0 Yo 0 23.3 YO C=O 3.4 (289.3 eV) % C-N 7.7 (288.1 eV) Yo C 56.4 Yo 022.7 Yo C 68.5 Yo 023.2 Yo C 65.7 Yo 0 21.9 Yo C-C 69.0 Yo c-0 18.8 Yo C=O 6.9 YO C-N 5.3 (288.1 eV) % Si 5.1 Silicon Nitrogen (400.0 eV) (102.5 eV) YON 3.3 Yo N 0.7 Yo Si 4.3 % N 5.6 YO Si 6.3 YO NH2 44.8 (399.3 eV) % NH3+ 55.2 (401.1 eV) Yo N 10.2 Yo Si 10.8 Yo N 3.9 YO Si 4.5 YON 6.9 Yo SI 6.1 * Binding energies for all high-resolution C(1s) data are similar to those of Table 1 except for figures accompanied by binding energies in parentheses. an even larger amount of APTES is detected on the control, unmodified PET, silanized in the same reaction vessel (11A of Table 3).It is then apparent that more APTES is immobilized on the reduced surface because it can react with both the reduced and the unmodified areas on the surface, whereas DCDMS can only undergo a reaction with the hydroxylated portion. This unpredicted reactivity between APTES and virgin PET was investigated as it may provide a direct way to silanize a PET sample. Reaction With APTES The silanization of PET with APTES, without any prior reduction, results in the immobilization of a large amount of the silane (11A of Table 3).Surface coverage is calculated to be 79% for an incubation time of 24 h. A similar modification was obtained for the Dacron graft, with the amount of silane detected being of the same magnitude as that for PET (12 of Table 3). When the reaction time was increased to 48 h (11B of Table 3), surface coverage of 100% was achieved. Coverages of APTES on PET samples were calculated using observed intensities of the nitrogen peaks and the equation below:' where F is the percentage coverage, Ip is the intensity of the nitrogen peak, I, is the intensity of the nitrogen signal expected for a pure APTES layer of infinite thickness (7.1%), i . e . , the ratio of nitrogen atoms in an APTES molecule, d is the depth of the APTES layer, h is the escape depth of N(1s) electrons (25 A) and 8 is the angle of the detector with respect to the sample (90").As APTES tends to cross-link to form a multilayer siloxane network with a thickness well above the sampling depth of the XPS instrument (200 A), d can be set at It has been demonstrated that the deposition of APTES from an organic solvent usually results in the formation of a two- (monolayer) or three-dimensional (multilayer) poly- meric silane structure.25-27 Polymerization occurs via reac- tions between silyl ends of different APTES molecules to form 200 A. Si-0-Si bonds. Monolayer formation is caused by siloxane bond formation between surface-bound APTES molecules (horizontal polymerization). Multilayer generation is the result of reactions between surface-bound APTES molecules and solution-phase APTES molecules (vertical poly- merization). The thickness and stability of the silane poly- meric network depends greatly on the experimental con- ditions.The anhydrous, organic conditions employed in this work often result in thick multilayers with irregular mor- phology. It has also been determined that the curing process of an APTES-modified surface plays a very important role in the determination of its stability. Curing increases the number of siloxane bonds in the APTES film up to the maximum of three per monomer and can be carried out at ambient or elevated temperatures.28 For this investigation, curing was carried out under ambient conditions for at least 48 h prior to any subsequent modification.The curing process conferred a stability to the APTES layer immobilized on PET as Soxhlet extraction with hot THF and chemical rinsing with ethanol and chloroform failed to remove any significant amount of the silane. The modification is also fairly stable under aqueous conditions, although a loss of 30% of the silane is observed after immersion in a basic solution for 24 h (11C of Table 3). This figure is in agreement with previously published results.29 The fact that no further loss occurs suggests that only the loosely bound outer layers, which are either held together by weak forces (hydrogen bonds or hydrophobic interactions) or are incompletely polymerized (less than three siloxane bonds per monomer), are affected by an aqueous medium.The completely cross-linked inner layer is inert to nucleophilic attacks by the basic solution. In previous investigations, the mechanism for the attach- ment of the 'APTES film to the substrate has always been considered t o be a simple nucleophilic substitution, with the surface hydroxyl groups attacking the silyl group, displacing the ethoxide moiety and forming an 0-Si bond. This is also true for the reaction between APTES and the partially reduced PET. However, the reaction between APTES and the unreduced PET must proceed via a different pathway, as there is not a sufficient amount of nucleophile present on the unreduced PET, apart from a limited number of hydroxylANALYST, MAY 1993, VOL. 118 469 termini from the polymer chain ends. These termini are not present in any significant amount as indicated by the lack of reaction between unreduced PET and a highly reactive chlorosilane, DCDMS.It is proposed that the initial reaction occurs via nucleophilic attack by the amino group of the APTES molecule on the ester linkage of the PET in a manner analogous to the aminolysis reaction. The aminopropyl group of APTES can be considered to be comparable to that of DAPr. However, unlike all other aminolysis reactions, no degradation is observed even on prolonged exposure (5 d) to pure APTES. A secondary phenomenon must be responsible for the preservation of the PET polymeric structure, It is believed that the alkoxide group created by the ester cleavage may play a role in the preservation of the polymeric structure. A reaction between this anion and the silyl end of APTES, in a substitution reaction similar to the well-documented reaction between a hydroxyl group and APTES,18-23 would have the effect of holding both ends of the ester together. As the negatively charged alkoxide end of the cleaved ester is a much stronger nucleophile than a hydroxyl group, the second reaction is favourable.The proximity of the alkoxide moiety to the silyl group of the attacking APTES molecule favours a secondary reaction between these two moieties. This inserted APTES molecule would then cross-link horizontally and vertically with other APTES molecules near the surface to form the APTES flm. Not all silane molecules will be covalently attached to the PET surface but those that are act as anchors to bind the APTES overlayer to the substrate.This two-step mechanism is summarized in Fig. 5 . There are several other pathways by which the APTES coating can be anchored to the PET surface. The cleaved alkoxide end could react with a second APTES molecule, which may already be attached to the PET surface via its amine end, or is already part of the APTES polymeric network [Fig. 6(a)]. Another pathway may have the initial APTES molecule cross-linking with the APTES overlayer without undergoing the second step of the proposed two-step mechanism [Fig. 6(b)]. In either scenario, the integrity of the PET molecule would still be preserved as both ends of the cleaved ester are now bound to the APTES film. Two other pathways, not related to the proposed mechanism, may also exist.The APTES layer may be bound to the substrate via siloxane bonds with any existing hydroxyl groups on the surface, i.e., polymeric chain ends [Fig. 6(c)] or through hydrogen bonds between the amino groups and the ester EtO-S;, EtO' '*OEt I 0 II C- / 0 4 *+ EtO OEt oxygen [Fig. 6 ( d ) ] . All of the above schemes were examined in greater detail through reactions with several close derivatives of APTES and the results are discussed in a later section. High-resolution analysis of the C( 1s) peak for surface 11A by XPS (Fig. 7) reveals the presence of a new carbon bond type with a binding energy between those of the carbonyl and ether bonds (288.1 eV). This peak can be assigned to the carbons attached to the nitrogen atoms within the APTES layer (C-N).A marked decrease in the amount of carbonyl carbon (C=O) and a corresponding increase in the amount of ether carbon (C-0) are also observed. Although the mor- phology of the APTES layer is uneven, ranging from several to hundreds of monolayers thick,*5-27 its average thickness is significantly greater than that of the escape depth of electrons APTES multilayer 111111111111u111u1111*111111111111111111111111111111*1 (a) I Si I A PET substrate PET substrate APTES multilayer NH I c=o PET substrate APTES multilayer IIIUIIIIIIIIIIII1I111111111111111111111111IB111IIIIIIBIIIIWI H HN\ I 0 g PET substrate Fig. 6 Schemes of interaction between the APTES overlayer and PET; (a) linkage via the alkoxy group; (b) linkage via the amino group; (c) linkage via surface hydroxyl group; and ( d ) linkage via surface hydroxyl group; and ( d ) linkage via hydrogen bonding 200 180 160 140 120 100 80 60 1, 40 2? 20 8 1000 900 800 700 600 500 400 300 200 100 r. 3 N 0 7 200 > 'u, 180 $ 160 5 140 4- 4- 120 100 80 60 40 20 294 292 290 288 286 284 282 280 Bind i ng ene rgyIeV (a) Survey and (6) high-resolution C(1s) spectra of APTES- Fig.7 modified PET Fig. 5 Mechanism of APTES insertion into PET470 ANALYST, MAY 1993, VOL. 118 ~~~ ~~ Table 4 Angle-resolved XPS data of APTES-modified PET sample Detector angle c (Yo) 0 (Yo) N (Yo) Si (YO) 90" 65.8 22.3 5.6 6.3 60" 67.8 21.6 4.8 5.8 45" 66.6 20.8 6.0 6.8 30" 68.0 19.9 4.7 7.3 Fig. 8 Scanning electron micrographs of APTES-modified surfaces: (a) PET at a magnification of ~ 7 0 0 ; (b) PET at a magnification of x 14 800; (c) Dacron (2Y0, 24 h) at a magnification of X 10 0oO; and (d) Dacron (5%, 48 h) at a magnification of ~ 5 0 0 0 originating from the PET bulk.The decrease in the amount of carbonyl carbons can, therefore, be attributed to the shielding effect of the APTES layer. In fact, as the APTES layer does not contain any carbonyl bonds, the peak at 289.3 eV can be attributed to amide carbons, which have a similar binding energy. This statement is correct only if the coverage of the multilayer APTES formation is 100% , i.e., there is no exposed PET. Previous mathematical calculation places the coverage at approximately 79% ; therefore, some contribution from bulk PET is probable, with the balance being contributed by the amide bond at the PET/AFTES interface.The presence of the APTES layer also accounts for the increase in the ether carbon percentage as the APTES layer contains numerous ether-like carbons (C-0-Si). As the amino groups of the APTES layer are not involved in the cross-linking process, they are available for any subse- quent reaction. However, a certain proportion will be in the unreactive, protonated or H-bonded form.29 Protonation occurs via proton transfer from uncondensed silanols or ambient moisture while hydrogen bonding may exist between the amino groups and such silanols. High-resolution analysis of the nitrogen peak reveals that there are indeed two types of nitrogen species present on the surface, with binding energies of 399.3 and 401.1 eV, respectively. The peak at 399.3 eV can be attributed to free amine nitrogens while the 401.1 eV peak belongs to protonated amine nitrogens.As only the unproto- nated amines are able to participate in any future nucleophilic attack, it is clear that less than half of the amines present are in an active form. Pyridine was added in an attempt to scavenge free protons, but the concentration was kept low to prevent degradation of the polymer. An assessment of the over-all reactivity of the silanized surface through a reaction with TFAA was conducted prior to any subsequent immobiliza- tion. An angular dependence study was executed to determine the profile of the immobilized APTES layer but the results (Table 4) reveal that no angular dependency exists. The scanning electron micrographs are consistent with these results in showing the uneven topography of the siloxane polymer on the PET [Fig.S(a)]. Prominent on the APTES- treated surfaces are hemispherical 'islands' with diameters of less than 1 ym. These hemispheres are present singly or in linked aggregates. On closer inspection, it can be seen that the surface seems to be covered with a thin film of silane, out of which grow the macroscopic islands [Fig. 8(b)]. The SEM results are in agreement with a previous study of the polymerization structure of APTES .29 The origin of these macroscopic structures has not been conclusively determined but it is believed that they form in the reaction solution, prior to attachment to the APTES layer.29 Scanning electron micrographs of the Dacron surface treated with APTES show a similar pattern but the silane layer also exhibits a 'scab-like' appearance in addition to the hemispherical clumps [Fig.8(c)]. An interesting phenomenon is observed on a Dacron surface modified using a higher silane concentration and a longer reaction time. A coating of material can be seen covering most of the surface. This layer seems to serve as the anchor for a complex network of linked spheres [Fig. S(d)]. It is hypothesized that the outer network is the loosely bound portion of the APTES multilayer, some of which can be displaced by a basic aqueous medium. The basal portion is more strongly bound, rendering it stable in most media. Reactions With Other Silanes In an effort to elucidate further the mechanism of the PET-APTES interaction, the reactions between PET and several silane derivatives, structurally related to APTES, were examined.The first compound examined was TES, a silane in which the aminopropyl moiety has been replaced by another ethoxy group. The failure of the reaction between TES and PET (13 of Table 5 ) strongly suggests that the amino moiety does indeed participate in the reaction with PET. The minimal amount of TES detected can be attributed to the small amount of hydroxyl chain ends present on the surface of the polymer. The lack of reactivity suggests that the reaction between the silyl end of the silane and the hydroxyl termini does not play an important role in the anchoring of the APTES layer to the substrate [Fig. 6(c)]. Physical adsorption of the silane onto the substrate is also a possibility.This minimal amount of TES reduces the WCA measurement to 77" (Table 2). As TES polymerizes to a greater extent than APTES, the lack of reactivity between TES and PET also suggests that the initial attack by the amino group is the determining factor in the immobilization process. The adsorptiodpolymerization effect exerted by the silyl end of the molecule is a secondary process and by itself cannot account for the reaction between APTES and PET. The next silane examined was APEDMS. This molecule is similar to APTES, except that two of the three ethoxy groups have been replaced by inert methyl groups. As it has only oneANALYST, MAY 1993, VOL. 118 47 1 ethoxy entity that can be displaced, extensive polymerization is not possible with this compound. There are two possible routes for the reaction to proceed following the initial attack by the amino group: dimerization with another silane mol- ecule, which would result in degradation of the surface, or displacement of the ethoxy group by the alkoxy anion, which would preserve the polymeric structure. No degradation of the PET structure is observed, which eliminates the dimerization pathway and lends credence.to the hypothesized two-step mechanism. X-ray photoelectron spectroscopy data (14 of Table 5) reveal that only a small amount of APEDMS is actually bound to the surface. A reduction in WCA measure- ment to 78.9" seems to corroborate this observation. The reduced reactivity of the silane can be attributed to the steric hindrance caused by the substitution of the two ethoxy moieties with inert methyl groups.The lack of extensive polymerization also contributes to this reduction as the degree of immobilization is now primarily dependent on the reactivity of the amino group. The result shows that the amino functionality is reactive only to a moderate degree. This can be attributed to the fact that APTES is known to form an internal zwitterion, through the co-ordination of the amino group with the silyl skeleton, in the presence of trace amounts of water.30 The nucleophilicity of the amino group would be diminished as the lone pair is involved in the co-ordination. High- resolution analysis of the C(1s) peak shows slight decreases in the amount of ether and carbonyVamide carbons. This corresponds to a thin, incomplete coverage of the silane.As the third APTES derivative, APDEMS, has one less ethoxy group than APTES, the degree of polymerization should be reduced; hence a thinner and more even coverage can be achieved. This is indeed the observed result as demonstrated by XPS data (15 of Table 5). High-resolution analysis of the C(1s) peak reveals a trend similar to that of APTES, but with a smaller magnitude. This agrees with the result of the survey spectrum and confirms the presence of a thinner layer of silane. The reduced polymerization is not detrimental to any great extent as the top layer is loosely bound and can be easily solubilized chemically. The WCA measurement of 71.2" is close to that of the APTES-modified sample; hence it may be postulated that the number of NH2 groups present at the surface may be close to that for APTES.The success of this reaction provides a cleaner method of silanizing a PET surface. However, as high-resolution analysis of the nitrogen peak shows that only 34.6% of the amines are in the active unprotonated form, this surface may not be as reactive as that modified with APTES. The reactivity of this silanized surface was determined through a subsequent reaction with TFAA and the results are discussed in a later section. Reaction With TES, Initiated by DAPr As the APTES molecule may be thought to possess two distinct reaction sites, the aminopropyl end and the silylethoxy end, the combination of a molecule containing an amino- propyl group, DAPr, and TES should provide a reaction medium analogous to that of APTES, with the important difference being that the two functional groups are not connected. Such a reaction was attempted and the XPS results (Fig.9 and 16 of Table 5) reveal that a considerable amount of TES is immobilized on the surface of the polymer. This layer of TES was hydrolysed by atmospheric water vapour to yield a surface comprised of silanol groups. The presence of these silanol functions is substantiated by an extremely low WCA measurement of 27". Another observation acquired from this experiment is the fact that the surface of the PET shows obvious signs of degradation. Recalling that TES does not react with unmodified PET in the absence of an initiator, it seems that the immobilization of TES occurs only because the initial aminolysis by the diamine creates a reactive alkoxy anion, which can then displace the ethoxy group on the TES molecule. This initiation process appears to play a critical role in the success of the reaction, even though only a minimal amount of the amine was required.The degradation of the PET can be explained by the fact that the two reactive components are not connected together as with APTES. The integrity of the polymeric structure is compromised because, of the three pathways that could have preserved the polymeric structure, two have been eliminated [Figs. 5 and 6(b)], leaving only one [Fig. 6(a)]. The degree of degradation is minimal, which may be attributed to the action of the third pathway and the low concentration of the amine. High-resolution analysis of the C(1s) peak shows a trend similar to that of APTES, a reduction in carbonyl carbons and an increase of ether carbons, consistent with the presence of the silane.However, no C-N peak is detected as the amount of amine is minimal and any initial amide formation would be rapidly masked by the layer of TES. The large amount of silane detected suggests the presence of a thick multilayer, consistent with the greater polymerization capability of TES. Nevertheless, the fact that silanol groups are present on the surface has important ramifications as these provide an alternative route for further chemistry. However, Table 5 XPS data for silanizations involving APTES derivatives* Surface Carbon (285 .O eV) 13. PET-TES Yo C 71.2 14. PET-APEDMS Yo C 73.0 Yo C-C 67.6 Yo c-0 18.1 Yo C=O 14.3 15.PET-APDEMS Yo C 70.2 Yo C-C 70.2 % c-0 18.1 % C=O 8.3 % C-N 3.4 16. PET-TES/DAPr Yo C 36.9 Yo C-C 64.6 Yo C-0 24.1 Yo C=O 11.3 17. Dacron-TES/DAPr % C 26.4 % C-C 68.2 % C-0 26.6 Yo C=O 5.2 Oxygen (532.1 eV) Nitrogen (400.0 eV) Silicon (102.5 eV) Yo 0 26.6 Yo N 5.6 'YO Si 0.5 Yo 024.2 YON 1.4 YO Si 1.4 Yo 0 18.2 YON 5.1 % Si 6.2 YoNHZ 34.6 YoNH,+65.4 Yo 045.7 %N 2.5 YO Si 14.9 % 049.0 % N 2.2 % Si22.4 * Binding energies for all high-resolution C(1s) data are similar to those of Table 1.472 ANALYST, MAY 1993, VOL. 118 tertiary silanols may not be very reactive, owing to steric constraints; hence a reaction with TFAA is required to determine the extent of their reactivity. A similar modification was carried out on a Dacron surface. The result is even more significant as the atomic composition of Si reaches the extremely high figure of 22.4% (17 of Table 5 ) .This reflects a high degree of polymerization, which may or may not be suitable for a modification process, but it can be controlled through variation of experimental condi- tions. High-resolution analysis of the C( 1s) peak displays the same trends as those of the PET modification, but to a greater degree. Assessment of Reactivity Using TFAA A subsequent reaction between the silanized surfaces and TFAA was performed to assess the reactivity of the amino nucleophiles on these surfaces. A significant amount of the trifluoroacetate moiety is detected on the surface pre-treated with APTES (19 of Table 6), which attests to the high chemical reactivity of the NH2 groups.The apparently anomalous increases in elemental nitrogen and silicon are due to the use of a different pre-modified sample. High-resolution analysis of the C(1s) peak shows a large, distinct C-F peak at 500 450 400 350 300 250 200 150 2 50 8 900 800 700 600 500 400 300 200 100 1, 100 C 3 900 800 700 600 500 400 300 200 100 Binding energyleV Fig. 9 uninitiated and (b7 initiated with DAPr Survey s ectra of PET surfaces modified with TES, (u) 293.1 eV. The carbonyl peak also increases in size, which corresponds well with the presence of the trifluoracetate groups on the surface. X-ray photoelectron spectroscopy data for the reaction between the APEDMS-modified PET sample and TFAA reveal that TFAA is immobilized on this surface (20 of Table 6) to the same minimal degree as that of the control (18 of Table 6).This indicates that the surface-bound amino groups may be in an inactive conformation. One such conformation is the amide bond, formed in the initial reaction. A subsequent dimerization through the silyl end would have regenerated the reactive amine group. The lack of reactivity seems to exclude the dimerization pathway and supports the hypothesis that the ethoxy group present may have reacted with the cleaved alkoxide end of the PET monomer. Analysis by XPS of the APDEMS-modified surface reacted with TFAA (21 of Table 6) confirms that the surface-bound amino groups are chemically reactive although to a lesser degree than for the APTES-treated surface. This can be explained by the smaller number of amino groups and the higher proportion of the unreactive protonated form present on the former surface (15 of Table 5).Virtually no trifluoroacetate is detected on the surface modified with TES and TFAA (22 of Table 6), suggesting the failure of the tertiary silanols to act as nucleophiles. This supports the contention that TFAA is covalently bound to the aminated surfaces as opposed to being physically adsorbed onto the polymeric silane network. If physical adsorption is the mechanism of attachment, a significant amount of TFAA would have adsorbed onto the extensive siloxane network on the TES/DAPr sample. Reactions With Cross-linkers After the reactivity of the APTES-modified surfaces had been verified, a common cross-linker, GA, was reacted with this surface in an effort to facilitate further linkages.This di-aldehyde molecule, often used for protein coupling, reacts directly with the amino groups on the silanized surface to form a Schiff's base. The other terminal aldehyde can form a second Schiff's base with the amino moiety on any amino acid or protein. An APTES-modified sample, subsequently treated with GA, shows decreases in the nitrogen and silicon signals and an increase in the oxygen composition (24 of Table 7). This corresponds to the immobilization of a layer of the GA on top of the silane surface. More importantly, the control PET surface shows no such deposition, substantiating the covalent nature of the reaction. Surface coverage cannot be determined owing to the lack of a label element in the GA molecule but a sub-monolayer coverage is a probable result as it has been determined that only half of the amino groups in the APTES Table 6 XPS data for reactivity assessments with TFAA Carbon Oxygen Nitrogen Silicon Fluorine Surface (285.0eV) (532.1 eV) (400.0eV) (102.5eV) (688.4eV) 18.PET-TFAA Yo C 69.9 Yo 028.3 - 19. PET-APTES-TFAA % c 40.7 yo 020.5 % N6.5 % Si 8.8 % F23.4 - Yo F 1.6 Yo c-c 44.6 Yo C-0 17.0 Yo C=O 16.0 Yo C-N 6.7 % C-F 14.1 (293.1 eV) 20. PET-APEDMS-TFAA Yo C 70.8 % 024.5 21. PET-APDEMS-TFAA Yo C 61.9 Yo 023.0 YO C-C 61.1 Yo C-0 19.2 Yo C=O 16.5 % C-F 3.2 22. PET-TES/DAPr-TFAA YO C 31.0 YO 0 47.4 Yo N 2.3 YO Si 19.3 Yo F 0.0 % N 1.4 %Si 1.1 Yo F 1.3 Yo N 2.7 YO Si 2.4 Yo F 7.4ANALYST, MAY 1993, VOL. 118 473 Table 7 XPS data for reaction with cross-linkers and L-cysteine* Carbon Oxygen Nitrogen Silicon Chlorine Sulfur Surface (285.0 eV) (532.1 eV) (400.0 eV) (102.5 eV) (199.6 eV) (163.6 eV) 23.PET-GA Yo C 71.0 % 029.0 - - - - 24. PET-APTES-GA Yo C 65.7 % 024.6 % N4.5 Yo Si 5. I - - YO C-C 68.8 Yo C-0 19.2 YO C=O 10.9 (288.0 eV) YO C-N 3.7 (290.9 eV) 25. PET-SB Yo C 71.8 Yo 0 28.2 26. PET-APTES-SB Yo C 58.8 Yo 0 17.4 Yo C-C 72.3 Yo c-0 18.0 Yo c = o 5.5 YO C-F 4.2 Yo c-c 60.8 Yo C-0 26.0 Yo C=O 13.2 27. PET-APTES-cysteine YO C 63.9 Yo 0 28.8 28. PET-APTES-GA-cysteine YO C 60.1 Yo 022.1 Yo N 8.9 YO Si 2.0 - Yo s 7.0 Yo C-C 53.2 Yo C-0 29.6 Yo C=O 17.3 (288.3 eV) - - % c10.0 Yo N6.4 % Si9.9 Yo CI 7.5 - YO Si 1.8 Yo N4.1 % S1.4 * Binding energies for all high-resolution C(1s) data are similar to those of Table 1 except for those followed by figures in parentheses.200 150 7 100 v) c 3 0 v) 50 4 4 900 800 700 600 500 400 300 200 100 m 2 320 .z 280 2 240 200 160 120 80 40 . > S C - 292 290 288 286 284 282 280 Binding energylev Fig. 10 (a) Survey and (6) high-resolution C( 1s) spectra of immobi- lized 1.-cysteine on an APTES-modified PET surface, cross-linked with GA layer are in an active conformation. High-resolution analysis of the C(1s) peak reveals a reduction in the ether peak and a large increase in the carbonyl signal, again consistent with the presence of the aldehyde (24 of Table 7). The carbonyl signal was shifted to a lower binding energy of 288.0 eV, characteris- tic of an aldehyde functionality. A small peak is seen at 290.9 eV, which can be assigned to the imine linkage of the Schiff's base.This signal is smaller than that of the free aldehyde owing to the masking effect of the GA layer. A second type of cross-linker, SB, was also employed in the conversion of the amino groups of the APTES surface into more reactive sites. The amino group can form an amide bond with one end of the di-acid chloride, leaving the distal acid chloride free for subsequent nucleophilic attack. The success of this experiment is confirmed by the XPS results. Survey data (26 of Table 7) establish the presence of the acid chloride on the APTES-treated surface and confirm the inertness of the same molecule to the control PET sample (25 of Table 7). High-resolution analysis shows an increase in the carbonyl peak, consistent with the presence of the distal acid chloride moiety and the amide group.Reductions in the C-0 and C-N contributions also support this observation. A WCA measure- ment of 87.8" reflects an increase in hydrophobicity caused by the introduction of the non-polar Cx chains onto the surface. Reactions With L-Cysteine A PET surface, initially modified with APTES, does not couple to L-cysteine to any significant extent (27 of Table 7). The low reactivity between the amino group on the silane and the carboxylic function of the amino acid clearly demonstrates the need for a more reactive cross-linker. A successful coupling is achieved with a similar surface, after treatment with GA. A considerable amount of L-cysteine is bound to the surface as demonstrated by a 7% sulfur peak (28 of Table 7).An approximation, using eqn. (1), places the surface coverage of the amino acid at 42%. An increase in the nitrogen signal and a decrease in the Si peak substantiate the presence of a surface-bound cysteine layer. High-resolutim analysis o f the C(1s) peak reveals a further increase in the carbonyl bond type (Fig. lo), compared with surface 24, which agrees well with the introduction of the carboxylic acid-containing L-cysteine. The calculated cysteine coverage Of 42% indicates that the loss of any previous modification is minimal (assuming an initial APTES coverage of loo%, 45% of which are in an active conformation, and a 1 : 1 : 1 reaction ratio of APTES, GA and L-cysteine). This suggests that the APTES + G A surface is sufficiently stable in an aqueous medium to ensure the success of any further immobilization.This inertness to an aqueous environment is a requirement for any modification to a biological implant . The immobilization of I>-cysteine was not attempted on the surface pre-modified with APTES and SB, as an aqueous reaction medium, necessary for the solvation of the highly polar cysteine molecule, would result in the hydrolysis of the acid chloride. The SB surface can be employed for the immobilization of molecules that are soluble in a non-polar solvent, such as various phospholipids, as outlined by Kallury et a1.23474 ANALYST, MAY 1993, VOL. 118 Conclusions The stated aim of creating reactive surfaces on which the covalent immobilization of possible non-thrombogenic enti- ties can take place has been achieved.Aminated surfaces were generated via direct silanization with APTES and APDEMS, with surface coverages of up to 100%. A surface consisting of silanols has also been created via reaction with TES, initiated by DAPr. These modifications have been shown to be stable in most chemical environments; hence application to clinical uses is feasible. Reaction mechanisms between PET and the silanes have also been postulated. It is hypothesized that the silane films are anchored to the PET surface via several interactions. Pathways that involve a nucleophilic amino functionality play a more important role than those that make use of just the siloxy end of the molecule. Glutaraldehyde and SB were employed to activate the aminated surface further.A simple biomolecule, L-cysteine, was successfully immobilized onto a surface pre-treated with APTES and GA. The surface coverage of L-cysteine was calculated to be 42%. The surface modified with SB is unstable in an aqueous medium owing to the high reactivity of the acid chloride moiety to water; hence this surface is reserved for future immobilization of non-thrombogenic species that are soluble in a non-polar solvent. This work was supported by the Medical Research Council of Canada and the Physicians’ Services Incorporated Founda- tion.The authors also thank Drs. K. M. R. Kallury, D. C . Stone, S. Vigmond and M. Yang of the University of Toronto for helpful discussions during the preparation of this manu- script. Thanks are also due to Dr. R. Sodhi for assistance with the XPS experiments. References Anderson, J. M., and Kottre-Marchant, K., CRC Crit. Rev. Biocompatibility, 1988, 1, 111. Lord, R. S. A., Nash, P. A., Raj, B. T., Stary, D. L., Graham, A. R., Hill, D. A., Tracy, G. D., and Goh, K. H., Ann. Vascular Surg., 1988, 2, 248. Kottre-Marchant, K., Anderson, J. M., Miller, K. M., Mar- chant, R. E., and Lazarus, H., J. Biomed. Muter. Res., 1987, 21, 379. Andrade, J. D., in Surfaces and Interfacial Aspects of Bio- medical Polymers, ed. Andrade, J. D., Plenum, New York, Ratner, B. D., Johnston, A. B., and Lenk, T. J., J. Biomed. Muter. Res., 1987, 21, 59. 1985, ch. 5, pp. 105-191. 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Jozefowicz, M., and Jozefowicz, J., Pure Appl. Chem., 1984, 56, 1335. Barbucci, R., Castellani, C. B., Delfini, M., and Ferruti, P., Inorg. Chim. Acta, 1984, 93,47. Llonos, G., and Sefton, M. V., Biomaterials, 1988, 9, 429. Kottre-Marchant, K., Anderson, J. M., Umemura, Y., and Marchant, R. E., Biomaterials, 1989, 10, 147. Durrani, A. A., and Chapman, D., in Polymer Surfaces and Interfaces, eds. Feast, W. J., and Muno, H. S., Wiley, New York, 1987, p. 189. Hayward, J. A,, and Chapman, D., Biomaterials, 1984, 5 , 135. Dave, J., Kumar, R., and Srivastava, H. C., J. Appl. Polym. Sci., 1987, 33, 155. Avny, Y., and Reubenfeld, L., J. Appl. Polym. Sci., 1986,32, 4009. Kim, K., and Kop, S., J. Appl. Polym. Sci., 1986, 32,6019. Collins, R. J., US Pat. 2 955 954, 1964. Schmidt, R. G., and Bell, J. P., in Advances in Polymer Science, ed. DuSek, K., Springer-Verlag, Berlin, 1986, vol. 75, pp. 33- 71. Rosen, M. J., J. Coatings Technol., 1978, 50, 70. Heckl, W. M., Marassi, F. M., Kallury, K. M. R., Stone, D. C., and Thompson, M., Anal. Chem., 1990, 62, 32. Sportsman, J. R., and Wilson, G. S.. Anal. Chem., 1980, 52, 2013. Kannuck, R. M., Bellam, J. M., and Durst, R. A., Anal. Chem., 1988,60, 142. Loscascio-Brown, L., Plant, A. L., and Durst, R. A., Anal. Chim. Acta, 1990, 228, 102. Schifreen, R. S., Hanna, D. A., Boners, L. D., and Carr, P. W., Anal. Chem., 1977,49, 1929. Kallury, K. M. R., Ghaemmaghami, V., Krull, U., Davies, M. C., andThompson, M., Anal. Chim. Acta, 1989, 225, 369. Desai, N. P., and Hubbell, J. A., J. Biomater. Res., 1991, 25, 829. Kallury, K. M. R., Krull, U. J., and Thompson, M., Anal. Chem., 1988,60, 169. Pluddeman, E. D., in Silylated Surfaces, eds. Leyden, D. E., and Collins, W. T., Gordon & Breach, New York, 1980, p. 31. Murray, R. W., in Silylated Surfaces, eds. Leyden, D. E., and Collins, W. T., Gordon & Breach, New York, 1980, p. 125. Boerio, F. J., Schoelein, L. H., and Greivenkamp, J. E., J. Appl. Polym. Sci., 1978. 22, 203. Vandenberg, E. T., Bertilsson, L., Liedberg, B., Uvdal, K., Erlandsson, R., Elwing, H., and Lundstrom, I., J. Colloid Interface Sci. , 1991, 149, 1. Chiang, C. H., Lui, N. I., and Koenig, J. L., J. Colloid Interface Sci., 1982, 86, 26. Paper 2106339E Received November 26, 1992 Accepted January 13, 1993

ISSN:0003-2654    

DOI:10.1039/AN9931800463

出版商:RSC    

年代:1993

数据来源: RSC

摘要:

ANALYST, MAY 1993, VOL. 118 475 Intercomparison of Methods for the Determination of Vitamins in Foods Part 1 Fat-soluble Vitamins Peter C. H. Hollman and Jean H. Slangen DLO-State Institute for Quality Control of Agricultural Products (RIKIL T-DLO), Bornsesteeg 45, NL-6708 PD Wageningen, The Netherlands Peter J. Wagstaffe and Uta Faure Commission of the European Communities, Community Bureau of Reference (BCR), Rue de la Loi 200, B- 1049 Brussels, Belgium David A. T. Southgate and Paul M. Finglas AFRC Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colne y Lane, Norwich, UK NR4 7UA An intercomparison of methods involving 18 European laboratories was organized to assess the state-of-the-art of vitamin determination in foods. Each laboratory received identical samples of dry food reference material (homogeneous powders, milk powder, pork muscle and haricot vert beans), which were recently certified for major dietary components and elements.Each laboratory was requested to perform the analyses by its own methods. Results for fat-soluble vitamins are reported. All participants isolated the fat-soluble vitamins by alkaline saponification. For retinol, only high-performance liquid chromatography (HPLC), reversed- or normal-phase, was applied, with both ultraviolet (UV) and fluorescence detection. Results in milk powder showed a relative standard deviation of reproducibility (RSDReprod) of only 10%. Carotene was determined by HPLC (reversed- and normal-phase) and with open-column chromatography at atmospheric pressure. For p-carotene results in milk powder agreed very well; the RSDReprod was 14%. The values reported for haricot vert beans showed poor agreement; the RSDReprod was 52%.A major part of this variability was due to differences in methodological principles. The results for a-tocopherol in milk powder and haricot vert beans agreed very well, with RSDSReprod of 16 and 15%, respectively. Only HPLC (reversed- and normal-phase) with UV and fluorescence detection was applied. Keywords: Intercomparison; food: retinol; /3-carotene; a-tocopherol Vitamins are a large group of compounds, which differ in their chemical composition, physiological action and nutritional importance. From a nutritional point of view a sufficient intake of the fat-soluble (pro)vitamins is of great importance. This is also reflected in the legislation and labelling guidelines for retinol and vitamin D3 (both dangerous in excess), and tocopherols.Therefore, accurate methods of analysis for these vitamins are needed. So far, no data are available in the open literature on the between-laboratory reproducibility for the determination of fat-soluble vitamins in foods using routine methods. Recently, the Community Bureau of Reference of the Commission of the European Communities undertook a programme to improve the quality of vitamin determination in food. The following aspects will be covered: improvements in method- ology, intercomparison of methods and preparation of refer- ence materials. As a first step in this programme, an intercomparison of methods for fat- and water-soluble vit- amins in foods was planned.The purpose of this intercompari- son was to assess the state-of-the-art of vitamin determination and to identify problem areas. The results of this intercomparison of methods for the determination of the fat-soluble (pro)vitamins retinol, car- otene and a-tocopherol in food are reported in this paper. Seventeen laboratories with experience of vitamin determina- tion in food participated in this study. Participants were encouraged t o apply the methods of analysis routinely used in their respective laboratories. A subsequent paper deals with water-soluble vitamins (see Part 2).13* * A report with detailed data can be obtained from Peter C. H. Hollman, DLO-State Institute for Quality Control of Agri- cultural Products (RIKILT-DLO), Bornsesteeg 45, NL-6708 PD, Wageningen, The Netherlands.Experimental Protocol The participating laboratories were invited to use their routine methods for the analysis of each food sample and vitamin. The laboratories had to carry out at least three separate determinations on three separately weighed sub-samples taken from at least two of the sachets provided. Results were expressed on a dry-matter basis as determined by drying under prescribed conditions. The vitamin standards used as cali- brants can be an important source of variation. To be able to assess these errors, a multivitamin reference mixture of known composition was sent with the samples. Determinations on separately weighed sub-samples taken from two units had to be made.Guidelines for the preparation of stock solutions of this multivitamin mixture were given. Materials Three dry food certified reference materials (CRMs) in the form of homogeneous powders [whole milk powder (not enriched, CRM 380), freeze-dried pork muscle (CRM 384) and dried haricot vert beans (CRM 383)] were selected for this intercomparison. Only pork muscle was used for the deter- mination of the water-soluble vitamins. The samples were packed into heat-sealed laminated-foil sachets, flushed with nitrogen. These foods have been developed as CRMs for major nutrients and elements, and proved to be homogeneous with respect to more than 10 elements and major nutrients.2 The homogeneity with respect to retinol, a-tocopherol, vitamin B1 and B2 was also studied in the milk powder and pork muscle (only vitamin B1 and B2) and was found to be adequate.Additional evaluation of the stability study3 showed476 ANALYST, MAY 1993, VOL. 118 that the between-sachet RSD of a-tocopherol and vitamin C content for haricot vert beans was smaller than 4%. Long-term stability at 4 "C was demonstrated for: retinol, a-tocopherol, vitamin B1 and B2 in milk powder; vitamin B1 in pork muscle; and a-tocopherol and vitamin C in haricot vert beans. In addition, short-term stability at 30°C of the most labile vitamins, retinol and a-tocopherol in milk powder and of vitamin C in haricot vert beans was known to be acccptable,3 hence the stability of the vitamins was sufficient to allow normal postal shipment. However, data on the multivitamin mixture and of other vitamins were lacking.Therefore, a linear regression analysis was carried out for all vitamins and samples to study a possible relationship between the concentration (logarithmic concentration) and the date of analysis in the laboratory. Correlation coefficients calculated did not indicate a significant negative correlation (t-distribu- tion according to Fisher, one sided, P >0.05). The multivitamin reference mixture was composed of lactose with vitamins A and E added in the form of beadlets (made of gelatine, starch and saccharose), the other vitamins were added as the pure substances. In order to assess between-sample variation, five bottles were randomly chosen and analysed for a number of fat-soluble vitamins in one laboratory. The variation between the samples for fat-soluble vitamins was rather small (RSD = 2.3-2.7%0) compared with the analytical variation (RSD,,,,,,) of the laboratories (Table 1) and thus the multivitamin mixture can be regarded as homogeneous.Technical and Statistical Evaluation Statistical evaluation followed the principles of the Interna- tional Organization for Standardization (ISO) norm, I S 0 5725,4 to calculate the RSDReprod and the relative standard deviation of repeatability (RSD,,,,,,). Strictly speaking, I S 0 5725 was designed to evaluate collaborative studies with one, well-defined method and, therefore, RSD,,,,,, represents the average repeatability of all methods as applied in this study. This statistical evaluation guided the technical evaluation of the study at a meeting with the participating laboratories.In addition, the Youden rank sum test was applied.5 For vitamin B1 this test identified a laboratory with possible systematic bias. Outlying laboratories identified by the statistical tests were only rejected from the calculations if supported by technical considerations, for instance, inadequate methods or poor laboratory performance. Suppressed results will be discussed subsequently. The aim of this intercomparison was to investigate the influence of different procedures routinely used by different laboratories. Results Laboratories carried out the analyses within a period of 3 months, with instruction to store the samples at 4 "C until use. Table 1 Summary of the variation in the results for fat-soluble vitamins in the multivitamin mixture, and the effect of the correction on the reproducibility Multivitamin mixture Milk powder Haricot vert beans RSD,* RSDR+ RSD,* RSDR RSDR(~~,.~)§ RSDR RSI?,R(~<,~~) Vitamin ( Y o ) (Yo) (Yo) (Yo) ( Y ) (Yo) (/A) - - Retinol 9.1 24 4.7 10 21 Carotene 7.3 35 5.1 14 16 52 46 a-Tocopherol 4.1 19 3.2 16 21 1s 26 * RSD, = RSD,,,,,,.* RSD, = predicted RSDRcprod according to the equation of Horwitz.6 + RSDR = RSDncprod. RSDR(,,,,, = RSDReprod of corrected results. Multivitamin Reference Mixture Table 1 summarizes the precision achieved by the different laboratories in analysing the multivitamin mixture. An indica- tion of the RSDKeprod expected for the determinations in the multivitamin mixture was obtained from the empirical equa- tion of Horwitz:6 RSDReprod = 2 exp(1 - 0.5 logc).This equation related the reproducibility observed in collaborative studies, when the same rigidly defined standardized methods were applied, to analyte concentration, c , expressed as a decimal fraction. When comparing the actual RSDReprod in this study with this estimated RSDReprotl, the results for fat-soluble vitamins are very poor. Inhomogeneity of the multivitamin mixture can be ruled out (see under Materials). Segregation of the beadlets during shipping could have caused inhomogeneity; however, this could not have resulted in the large variation observed, because the sample sizes prescribed (31 g) would have ensured a representative sample. This large RSDRcprod could possibly indicate differences in calibration procedures of the laboratories.To study this effect a correction factor for each vitamin was separately calculated for each laboratory. Division of the theoretical level of the specified vitamin in the multivitamin reference mixture by the mean value determined in the multivitamin mixture by the laboratory concerned, yielded the correction factor for this laboratory. Next, results of the laboratory for each sample and the specified vitamin were multiplied by the correction factor. The corrected values were calculated for all laboratories, food samples and vitamins. Finally, RSDReprod of the corrected results was calculated. By comparing RSDKeprod of the food samples with the RSDReprod of corrected results [RSDR(cor,) in Table 11, no decrease in the variation is noticeable. If differences in the calibration of vitamin standards are an important source of variation between laboratories, RSDR(corr) would be expected to be smaller than the uncorrec- ted RSDReprod; however, correction only increased the variability.Most likely, this was caused by inadequate execution of the extraction procedures in the standard mixture. These procedures were prescribed in the protocol, and participants were not familiar with them. Consequently, this intercomparison was not able to reveal the possible effects on the precision of differences in the calibration procedures of vitamin standards. In order to avoid the type of extraction problems with the powdered standards of the present intercomparison, liquid solutions of vitamin standards should be used, and the laboratories should try out the prescribed procedures before starting the trial.Retinol Results for milk powder show an RSDReprod of 10% (Table 2), and compare well with an interlaboratory study using enriched skimmed milk powder containing >0.3 mg of retinol per 100 8.7 In this International Dairy Federation study, with partici- pants applying a uniform high-performance liquid chromato- graphic method, the following precision data were found: All participants isolated vitamin A by alkaline saponifica- tion, followed by extraction of the retinol. Conditions for extraction varied (Table 3). Laboratories 4 and 17 used solid-phase extraction instead of liquid-liquid extractions used by the other participants. Only high-performance liquid chromatography (HPLC) with reversed- and normal-phase (laboratories 1, 6 , 7, 10 and 17) was used (Tables 4 and 5).Both ultraviolet (UV) and fluorescence detection (labora- tories 1, 4, 15 and 17) were used. Results of one laboratory (not shown) were judged to be unreliable because of insuffi- cient extraction of retinol after saponification and inadequate chromatographic resolution. The material used in this intercomparison was purchased from a commercial supplier, so the cis-isomers (13-cis, 9,13-di-cis and 9 4 s ) can be expected to be present in the RSDReprod = 15%, RSD,,,,,, = 5%.477 ANALYST, MAY 1993, VOL. 118 Table 2 Summary of the results of the intercomparison (expressed as mg per 100 g of dry mass) Number of Mean* RSDrepeat RSDReprod RSDpt Vitamin laboratories (range) (Yo) ("/I (Yo) Ratio$ Milk powder 12 0.267 6.7 10 14 0.7 Retinol- (0.2224293) Carotene- Milk powder 7 0.119 7.3 14 16 0.9 Haricot vcrt beans 9 0.222 7.6 52 14 3.7 (0.098-0.146) (0.063-0.398) a- Tocopherol- Milk powder 11 0.603 7.0 16 12 1.3 Haricot vert beans 10 0.335 8.1 15 12 1.2 (0.503-0.771) (0.25 14.406) * Mean = mean of means of the laboratories.t RSD, = predicted RSDReprod according to the equation of Honvitz.6 Ratio = RSDReProd/predicted RSDReprod. Table 3 Extraction methods used for the determination of fat-soluble (pro) vitamins. All laboratories applied alkaline saponification Extraction Laboratory Rctinol Carotene 1 Diisopropyl ether Diisopropyl ether 2 - Acetone-hexane (2 + 3), acetone, 3 Light petroleum (3x) Diethyl ether, (3x) 4 Extrelut, hexane - 6 Light petroleum-diethyl ether - hexane* (1 + I), (3x1 7 Diethyl ether, (3x) Diethyl ether 8 Diethyl ether Light petroleumt 9 - - 10 Diethyl ether - 11 Dichloroet hane Diisopropyl ethcr 13 - Diethyl ether+ 14 Hexane, (2x) Hexane, (2x) 15 Hexane, (5x) Hexane, ( 5 x ) 16 Light petroleum-diethyl ether Light petroleum-diethyl ether 17 Extrelut, hexane - (1 + I), (2x1 (1 + 11, (2x) * Only haricot beans were analysed, saponification was not applied.t Only haricot beans were analysed. a-Tocopherol Diisopropyl ether Light petroleum (3x) Extrelut, hexane Light petroleum-diethyl ether Diethyl ether Diethyl ether Hexane ( 2 x Dichloroethane Diethyl ether, (4x) Hexane, (2x) Hexane, (5x) Light petroleum-diethyl ether (1 + 1) (1 + I), (3x1 - Extrelut, hexane Table 4 Normal-phase HPLC conditions used for the determination of retinol and Laboratory HPLC 1 Column Eluent Detection Eluent Detection Eluent Detection Eluent Detection Eluent Detection Eluent Detection Eluent Detection Eluent Detection Eluent Detection 3 Column 6 Column 7 Column 10 Column 14 Column 15 Column 16 Column 17 Column Retinol Polygosil Si-60,5 pm, 250 x 4.6 mm Hexane-CH2CI2-propan-2-ol (90 + 9 + 1) Fluorescence, 333/470 nm - - - p-Porasil, 10 pm, 300 x 4.0 mm Isooctane-propan-2-01(985 + 15) UV, 340 nm Kieselgel, 5 pm, 250 x 4.6 mm Propan-2-ol-heptane (gradient) UV, 325 nm LiChrosorb Si-60,5 pm, 250 x 4.6 mm Isooctane-propan-2-01(98.5 + 15) UV, 312 nm - - - - - - - - - APS Hypersil, 3 pm, 100 x 4.6mm Isooctane-butan-2-ol(96 + 4) Fluorescence, 328/477 nm a-tocopherol a-Tocopherol Polygosil Si-60,5 pm, 250 X 4.6 mm Hexane-diisopropyl ether (90 + 1) Fluorescence, 296/320 nm LiChrosorb Si-60,5 pm, 250 x 4.6 mm Hexane-propan-2-01(99 + 1) Fluorescence, 290/325 nm p-Porasil, 10 pm, 300 X 4.0 mm Isooctane-propan-2-01(996 + 4) Fluorescence, 290/325 nm Kieselgel, 5 pm, 250 x 4.6 mm Propan-2-ol-heptane (gradient) Fluorescence, 290/327 nm - Spherisorb SSW, 5 pm, 250 X 4.6 mm Hexane-propan-2-01(985 + 15) UV, 295 nm LiChrospher Si-60,s pm, 125 x 4 mm Hexane-1.4-dioxane (97 + 3) Fluorescence, 293/326 nm LiChrosorb Si-60,5 pm, 250 x 4.6 mm Hexane-propan-2-ol(99.5 + 5 ) Fluorescence, 290/330 nm APS Hypersil, 3 pm, 100 X 4.6 mm Isooctane-butan-2-01(96 + 4) Fluorescence, 29Y327 nm478 ANALYST, MAY 1993, VOL.118 Table 5 Reversed-phase HPLC conditions used for the determination of retinol and a-tocopherol Laboratory HPLC 3 4 8 9 11 13 14 15 16 Column Eluent Detection Column Eluent Detection Column Eluent Detection Column Eluent Detection Column Eluent Detection Column Eluent Detection Column Eluent Detection Column Eluent Detection Column Eluent Detection Retinol p-Bondapak CI8, 10 pm, 250 x 4.6 mm Methanol-water (90 + 10) UV, 325 nm Hypersil ODs, 10 pm, 100 x 4.6 mm Methanol Fluorcscence, 32Y4.50 nm LiChrosorb RPls, 10 pm, 2.50 X 4.6 mm Methanol-water (93 + 7) UV 328 nm - - - Hypersil ODs, 5 pm, 250 X 4.6 mm Methanol-water (97 + 3) UV, 325 nm - - - Spherisorb ODs, 3 pm, 150 X 4.0 mm Methanol-water (95 + 5 ) UV, 325 nm LiChrospher 100 RP18, 5 pm Methanol-water (98 + 2) Fluorescence, 325/470 nm Partisil ODs-2,lO pm, 250 X 4.6 mm Methanol-water (90 + 10) UV, 325 nm a-Tocopherol - - - Hypersil ODs, 3 pm, 100 x 4.6 mm Methanol Fluorescence, 290/330 nm LiChrosorb C18, 10 pm, 250 X 4.6 mm Methanol-water (93 + 7) UV 292 nm LiChrosorb Cls, 5 pm, 250 x 4.6 mm Methanol-water (97 + 3) Fhorescence, 293/326 nm Hypersil ODs, 5 pm, 250 x 4.6 mm Methanol Fluorescence, 290/330 nm C18, 5 pm, 220 x 4.6 mm Acetonitrile-CH2CI2-MeOH (70 + 20 + 10) UV, 294 nm - - - - - - - - - 0.35 - A l l - t r a n s b A l l - t r a n s + 13-cis- T L U 0 o1 0.30 7 c Q 7 0.25 .I ................................................................ b 1 1 i 1 4 i ............. I I . - n I - .- ; I s T - tT I 0.20 1 1 1 1 1 1 1 1 1 1 1 1 6 16 10 7 1 15 17 3 8 14 4 11 Labcode Fig.1 Results of individual laboratories for retinol in milk powder (mg per 100 g dry mass). Data represent the mean k standard deviation of at least three separate determinations for each laboratory samples. Woollard and Indyk8 determined cis-isomers in different commercial samples of milk powder and found 13-cis to be the most predominant isomer in all samples. Levels ranged from 9 to 20% of the all-trans-retinol. Laboratories 1,6,7,10 and 16 reported all-trans values, which were used for the statistical calculations and are shown in Fig. 1. The majority of the laboratories did not separate 134s and all-trans-retinol, so 13-cis is included in their results. However, the contribution of 13-cis to the total value for retinol depends on the type of detection chosen. With UV detection at 325 nm the absorbance of 13-cis is 8% smaller than the absorbance of all-trans, whereas with fluorescence detection at 314/485 nm, the intensity of 13-cis is only one third of the intensity of all-trans.Laboratories 7 and 16 determined 13-cis, and found values of 10 and 13% of the all-trans-retinol content, respectively. This is in agreement with the data of Woollard and Indyk.8 However, it is documented that during the analytical procedure isomerization can occur depending on the type of sample and conditions of saponification and extraction.9~10 Laboratories 3,8,11 and 14 using UV detection reported an average retinol content of 0.28 mg per 100 g, about 10% higher than the average value of 0.26 reported by laboratories 1, 6, 7, 10 and 16 only reporting all-trans.This compares well with the estimated content of 13-cis. It is concluded that the results for retinol in milk powder agreed very well between laboratories. Precision can be improved by taking into account the two isomers of retinol, viz . , 134s and all-trans. p-Carotene Reproducibility of the determination of carotene in haricot vert beans is very poor; results range from 0.063 to 0.398 mg per 100 g of dry mass (Table 2). Collaborative studies, using uniform methods, show an analyte at this level would be expected to give an RSDReprod of about 15% according to the equation of Honvitz,e as opposed to the RSD of 52% obtained. On the other hand, results for milk powder agree very well; the RSDReprod of 14% calculated for milk powder is identical with the predicted value.All laboratories, except No. 2, extracted carotenes after alkaline saponification (Table 3). As is shown in Table 6, most of the participants subsequently used HPLC, both in normal- phase (laboratories 7 and 14), and in reversed-phase mode (laboratories 1, 11, 13, 15 and 16). Laboratories 2, 3 and 8 used methods based on open-column chromatography at atmospheric pressure, and consequently determined the total of all-trans carotenes (a, B, y and 6) and their stereoisomers, calculated as (3-carotene .I1 Of these laboratories, only laboratory 3 carried out analyses in the milk powder. Indyk12 determined carotenoids in milk powder using HPLC and found that B-carotene is the principal carotenoid; no a-carotene was present. Several participants using HPLC confirmed that no &-carotene or other carotenes were present.Consequently, the results of carotene in milk powder obtained with open-column chromatography and HPLC should theoretically agree. In this intercomparison, the results of laboratory 3 agreed with the HPLC results. Laboratory 16 determined the a-carotene content of haricot vert beans and found 0.083 mg per 100 g (21% of the (3-carotene content); probably no y- and &carotene were present. So the results of laboratories 2 , 3 and 8 are expectedANALYST, MAY 1993, VOL. 118 479 0.5 I 1 Table 6 Methods and conditions used for the determination of carotene (for extraction methods see Table 3) Laboratory HPLC Conditions Normal-phase HPLC- 7 Column Kieselgel, 5 pm, 250 X 4.6 mm Eluent Propan-2-01-heptane (gradient) Detection Visible, 4.50 nm 14 Column Partisil, 5 pm, 250 X 4.6 mm Eluent Hexane-ethanol(9999 + 1) Detection Visible, 450 nm Reversed-phase HPLC- 1 Column Hypersil ODs, 5 pm, 250 x 4.6 mm Eluent Acetonitrile-CHCI3-acetone-water Detection Visible, 445 nm Eluent Acetonitrile-CHCI3-methanol-water Detection Visible, 445 nm 13 Column CI8, 5 pm, 220 x 4.6 mm Eluent Acetonitrile-CH2CI2-methanol (70 + 20 + 10) Detection Visible, 450 nm Column Eluent Acetonitrile-methanol-CHZC12 (36 + 40 + 24) Detection Visible, 450 nm Eluent Acetonitrile-CH2C12-methanol (70 + 20 + 10) Detection Visible, 450 nm (750 + 150 + 100 + 20) 11 Column Hypersil ODs, 5 pm, 250 X 4.6 mm (85 + 8 + 5 + 2) 15 LiChrospher 100 RP18, 5 pm, 125 X 4.6 mm 16 Column Zorbax ODs, 5 pm, 250 X 4.6 mm Open-column chromatography- 2 Column Activated magnesia-diatomaceous earth (1 + 1) Eluent Hexane-acetone (9 + 1) Detection Visible, 436 nm Eluent Hexane Detection Eluent Detection Visible, 452 nm 3 Column De-activated aluminium oxide 90 Visible, 4.50 nm, E(1%, 1 cm) = 2590 Diethyl ether-light petroleum (1 + 3) 8 Column Aluminium oxide neutral activity to be higher than the results of HPLC methods (Fig.2). However, in this intercomparison these laboratories using open-column chromatography at atmospheric pressure, found values lower than the trial mean. Laboratory 14 did not succeed in separating a- and @-carotene and probably re- ported a- + p-carotene. Chromatograms for haricot vert beans showing adequate resolution of a- and @-carotene were given by laboratories 11 and 16, reporting the highest p-carotene values, but also by laboratory 13 giving one of the lowest values.So poor resolution of the carotene isomers seems not to be the only problem in the analysis of @-carotene. Summarizing, the results for @-carotene in milk powder agreed very well between laboratories. On the other hand, the reproducibility of the carotene determination in haricot vert beans was very poor. Poor resolution between a- and @-carotene in haricot vert beans as determined by HPLC does not explain the variability found. Methods based on open- column chromatography at atmospheric pressure tended to give the lowest results, even in the haricot vert beans where both a- and @-carotene were present. a-Tocopherol Results for milk powder, with an RSD,,,,,, of 7.0% and an RSDReprod of 16% (Table 2), compare favourably with the results of a collaborative study of tocopherols in vegetable oils and fats organized by the International Union of Pure and Applied Chemistry (IUPAC).13 In this IUPAC study an RSDrepeat of 5% and an RSDReprod of 31% were found for a sample with a tocopherol content of 1.7 mg per 100 g.Values +Open column- HPLC _____( wi 8 0.3 ....... \ S s 0.1 2 u € . . . . . . . . . . . . € m . . . . . . . . . . . . . . . . . . . . . . . . . . 01 I I I I 1 I I I I I 3 2 8 13 14 15 1 11 16 Labcode * Fig. 2 Results of individual laboratories for carotene in haricot vert beans (mg per 100 g dry mass). Data represent the mean k standard deviation of at least three separate determinations for each laboratory v) 1.0 v) T 17 3 7 6 1 5 9 11 8 1 6 1 4 1 4 2 0 .4 l ' I ' ' ' ' I ' ' ' Labcode Fig. 3 Results of individual laboratories for a-tocopherol in milk powder (mg per 100 g dry mass). Data represent the mean k standard deviation of at least three separate determinations for each laboratory reported for haricot vert beans agreed very well. For both products the RSDReprod is comparable to the predicted RSDReprod when uniform methods are used (Table 2).6 All participants used alkaline saponification, mostly iden- tical with the procedure used for retinol and carotene. The tocopherols were extracted with hexane or light petroleum, mixtures of light petroleum and diethyl ether, diisopropyl ether and dichloroethane (Table 3). Laboratories 4 and 17 used solid-phase extraction, and again laboratory 4 (see under Retinol) showed a high intralaboratory variation.All used reversed-phase HPLC (laboratories 4, 8, 9, 11 and 13) or normal-phase HPLC (Tables 4 and 5). Most of the labora- tories used fluorescence detection; only laboratories 8,13 and 14 used UV detection. No effect of this choice of chromato- graphy and detection on the results was apparent (Fig. 3). Results of one laboratory (not shown) applying a continuous- flow method with fluorescence detection were rejected, because for the determination of a-tocopherol in milk powder and haricot vert beans, its method reached the detection limit. High results from laboratory 4 were not included in the statistical evaluation, because of the calibration procedure.This laboratory used a-tocopherol as a standard, without checking the content. Because the tocopherol supplied never has 100% purity, this leads to erroneous results. Laboratory 14 reported difficulties during the extraction of the haricot vert beans caused by formation of emulsions. The results given by this laboratory are very high (2.48 mg per 100 g) and show a very large variation (RSD = 50%) and have not been included. In conclusion, the results for a-tocopherol in milk powder and haricot vert beans obtained with different types of HPLC, agreed well between laboratories.480 ANALYST, MAY 1993, VOL. 118 Discussion A summary of the results of this intercomparison on methods for the determination of fat-soluble vitamins in foods is given in Table 2.Although homogeneous materials were involved, it can be argued that the homogeneity of carotene in these materials was not demonstrated beforehand. As participants carried out separate determinations in at least two different sachets, non-homogeneity will be reflected in the analytical variation (RSD,,,,,,) of the laboratories. Comparing RSD,,,,,, and RSDReprod for carotene (Table 2), it can be concluded that possible non-homogeneity was not an impor- tant factor. Experienced food laboratories participated; in this inter- comparison the choice of method was left to the participants and was only subject to the requirements of achieving the best level of accuracy. The reproducibility ( RSDReprod) shown gives an impression of the state-of-the-art of fat-soluble vitamin determination.In order to evaluate the results the approach of Horwitz et aZ.14 was followed. In this, the ratio (Table 2) was calculated, with as the numerator RSDReprod found in this intercomparison, and as denominator the RSDReprod achievable. From collaborative studies with uni- form methods Honvitz6 had concluded that the KSDReprod achievable is mainly a function of concentration, largely independent of analyte, matrix and method. It represents the analytical variation caused by different laboratories with different operators using different equipment, but using the same well-defined method. In the present intercomparison laboratories used different methods. Therefore, if different procedures used by different laboratories do not have a strong influence on the results, the ratio will be close to 1.This proved to be true for the analysis of retinol and 6-carotene in milk powder and a-tocopherol in milk powder and haricot vert beans. The results agreed well; sometimes it was necessary to exclude one or two laboratories using inadequate methods. This good agreement allowed indicative values for retinol and a-tocopherol to be issued in these food RMs, which have already been certified for major dietary components and elements.2 High-performance liquid chromatography was the method of choice of most of the participants for the determination of fat-soluble vitamins. Future intercomparisons for retinol need to take into account all-trans and 13-czs-retinol, because of the different response depending on the type of detection.As was evident from the results of p-carotene in haricot vert beans, the traditional methods based on open-column chro- matography proved to be biased. In the evaluation of results of a-tocopherol, calibration procedures as a source of variation was emphasized. The present intercomparison failed to identify the role of different calibration procedures, because of the inadequacy of the extraction procedures prescribed for the multivitamin mixture. The skilful participation of the following laboratories is gratefully acknowledged: Tnstituto del Frio, Instituto del Fermentaciones Industriales CSIC, Madrid, Spain; Labora- tory of the Government Chemist, Teddington, UK; The National Food Agency of Denmark, SQborg, Denmark; Schweizerisches Vitaminin-Institut , Basel, Switzerland; State Institute for Quality Control of Agricultural Products (RIKILT) , Wageningen, The Netherlands; Unilever Research, Bedford, UK; VTT Food Research Laboratory, Espoo, Finland; TNO-CIVO Institutes, Zeist, The Nether- lands; Bundesforschungsanstalt fur Ernahrung, Stuttgart, Germany; Servicio de Nutricion, Madrid, Spain; Leatherhead Food R.A., Leatherhead, UK; University College Cork, Cork, Ireland; Federal Dairy Research Institute, Liebefeld- Bern, Switzerland; Swedish National Food Administration, Uppsala, Sweden; Produits Roche, Fontenay sous Bois, France; Food Inspection Service, Maastricht, The Nether- lands; and AFRC Institute of Food Research, Norwich, UK.1 2 3 4 5 6 7 8 9 10 11 12 13 14 References Hollman, P. C. H., Slangen, J. H., Wagstaffe, P. J . , Faure, U . , Southgate, D. A. T., and Finglas, P. M., Analyst, 1993, 118, 469. Hollman, P. C. H., Boenke, A., and Wagstaffe, P. J., Fresenius’ J. Anal. Chem., 1993,345, 174. Hollman, P. C. H., Slangen, J. H., Finglas, P. M., Wagstaffe, P. J., and Faure, U., Fresenius’ J. Anal. Chem., 1993,345,236. International Standard I S 0 5725-1986, International Organiza- tion for Standardization, Geneva, 1986. Youden, W. J., and Steiner, E. H., Statistical Manual of the Association of Official Analytical Chemists, The Association of Official Analytical Chemists, Washington, DC, 1975. Horwitz, W., Anal. Chem., 1982, 54, 67A. IDF Questionnaire 1188/E, International Dairy Federation, Brussels, 1988. Woollard, D. C., and Indyk, H . . J. Micronutr. Anal., 1986, 2, 125. Landers, G. M., and Olson, J. A., J. Assoc. Of5 Anal. Chem., 1986, 69, 50. Steuerle, H., 2. Lebensm. Unters. Forsch., 1985, 181, 400. Brubacher, G. Miiller-Mulot, W., and Southgate, D. A. T., Methods for the Determination of Vitamins in Food, Elsevier, LondonRVew York, 1985. Indyk, H., J . Micronutr. Anal., 1987, 3, 169. Pocklington, W. D., and Dieffenbacher, A., Pure Appl. Chem., 1988, 60, 877. Horwitz, W., Albert, R., and Deutsch, M., J. Assoc. Off. Anal. Chem., 1990, 73, 661. Paper 21061 67H Received November 19, 1992 Accepted January 18, 1993

ISSN:0003-2654    

DOI:10.1039/AN9931800475

出版商:RSC    

年代:1993

数据来源: RSC