ISSN:0144-557X    

DOI:10.1039/AP98219FX008

出版商:RSC    

年代:1982

数据来源: RSC

ISSN:0144-557X    

DOI:10.1039/AP98219BX010

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

ANPRDI 19(3) 101-158 (1982) March 1982 Hon. Secretary R. Sawyer w Proceedings of the Analytical Division of The Royal Society of Chemistry AD President L. S. Bark Hon. Treasurer D. C. M. Squirrel1 Hon. Assistant Secretary D. 1. Coomber, O.B.E. Hon. Publicity Secretary Or. A. Townshend, Department of Chemistry. University of Hull, Hull, HU6 7RX Secretary Miss P. E. Hutchinson Editor, Analyst and Analytical Proceedings P. C. Weston Assistant Editors Mrs. J. Brew, Mrs. P. A. Fellows, R. A. Young Publication of Analytical Proceedings is the responsi- bility of the Analytical Editorial Board: J. M. Ottaway (Chairman) J. M. Skinner G. J. Dickes J. D. R. Thomas "G. W. Kirby A. M. Ure J. N. Miller "P. C. Weston G. E. Penketh J. Whitehead T. B. Pierce "Ex officio members All editorial matter should be addressed to: The Editor, Analytical Proceedings, The Royal Society of Chemistry, Burlington House, Piccadilly, London, W1 V OBN.Telephone 01 -734 9864. Telex 268001. Advertisements: Advertising Department, The Royal Society of Chemistry, Burlington House, Piccadilly, Analytical Proceedings (ISSN 01 44-557X) is pub- lished monthly by The Royal Society of Chemistry, Burlington House, London W1V OBN, England. All orders, accompanied by payment, should be sent t o The Royal Society of Chemistry, The Distribution Centre, Blackhorse Road, Letchworth, Herts., SG6 1 HN, England. 1982 Annual Subscription price if purchased on its own: UK f40.00, Rest of World f42.00, US $95.00, including air speeded delivery. Air freight and mailing in the USA by Publications Expediting Inc..200 Meacham Avenue, Elmont, N.Y. 11 003. USA Postmaster: Send address changes to: Analytical Proceedings, Publications Expediting Inc., 200 Meacham Avenue, Elmont, N.Y. 11 003. Second class postage paid at Jamaica, N.Y. 11431. All other despatches outside the UK by Bulk Airmail within Europe, Accelerated Surface Post outside Europe. PRINTED IN THE UK. @ The Royal Society of Chemistry 1982 London, W1 V OBN. Telephone 01 -734 9864. Editorial Signs of the Times Occasionally, changing circumstances can cause a sufficiently dramatic drop in the cost of some familiar item to force a reappraisal of its use and an a.ppreciable extension to the range of applications found for it. Computers offer a good illustration of this situation in the field of instrumentation. Not so very long ago, even small computers cost a sizeable sum and could only be considered for substantial programmes capable of supporting such an investment.Preparation of software, too, was an expensive activity. Low-level languages demanded experience from the user for efficient applica- tion and required considerable amounts of time for the preparation of programmes of any magnitude. However, the advances in elec- tronic technology that have taken place over recent years have now brought small computers almost to the stage where they rank as items of domestic electronics. They are now sold in chain stores, with the consequent exposure to the general population, and some children begin to show a computer literacy which threatens to exceed that in the more traditional academic subjects.Developments in computer technology tend to be viewed with particular interest by the analyst, as a result of the considerable use made by the analyst of instrumentation, and the analytical community has not been slow to appreciate the new opportunities open to it. However, the emergence of large-scale integra- tion has also simplified the design of digital circuits and many commercial organisations have been tempted to design and offer low-cost computers for sale. The problem for the potential user is to decide what combination of the available hardware and software best suits his purpose. This can be a formidable task, if a thorough evaluation is to be carried out, because although the cost of computers is low, computers are nonetheless complex devices.Any assessment must be matched against specific needs, and the detailed information required is not always available. Experience of existing users with similar applications is a valuable input to such deliberations, as opera- 101102 REPORTS OF MEETINGS Anal. PYOC. ting systems tends to identify advantages and limitations more clearly and it is essential that information about experience with existing systems can be passed on to potential new computer users. Meetings such as that due to be held by the Analytical Division, jointly with the Automatic Methods Group and the Micro- computers and Microprocessors Group of the RSC at the Annual Congress of the Royal Society of Chemistry this month (see the Diary, p. 155), will hopefully play their part in providing infor- mation which will extend the appreciation of the capabilities of microcomputers and will enable computer systems to be introduced more confidently into the analytical laboratory. T. B. PIERCE

ISSN:0144-557X    

DOI:10.1039/AP9821900101

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

102 REPORTS OF MEETINGS Anal. PYOC. Reports of Meetings North East Region The sixteenth Annual General Meeting of the Region was held a t 7 p.m. on Friday, November 6th, 1981, a t Scotch Corner Hotel, near Rich- mond. The Chair was taken by the Chairman of the Region, Mr. D. F. Griffiths. The follow- ing office bearers were elected for the forth- coming year : ChairmanMr. J. Vallance. Vice-Chairman and Honorary Secretary-Mr. C. L. Denton, 20 Bedford Road, Nunthorpe, Middlesbrough, Cleveland. Honorary Treas- urer-Dr. J. Newham. Honorary Assistant Secretary-Dr. C. M. Jenkins. Members of Committee-Mr. A. Atkin, Mr. P. G. W. Cobb, Mr. M. Daniel, Mr. R. Fisher, Mr. D. F. Griffiths (ex oficio), Dr. J. M. Skinner and Dr. A. Townshend. Mr. C. N Bell and Mr J. Whitehead were re-appointed as Honorary Auditors.East Anglia Region The fourteenth Annual General Meeting of the Region was held a t 2 p.m. on Wednesday, November 25th, 1981, a t the Linnean Society, Burlington House, London, W.l. The Chair was taken by the Chairman of the Region, Dr. R. J. Whiteoak. The following office bearers were elected for the forthcoming year: Chair- man-Dr. R. J. Whiteoak. Vice-chairman- Mr. G. M. Telling. HonGrary Secretary and Treasurer-Mr. A. M. C. Davies, Food Research Institute, Colney Lane, Nonvich, NR4 7UA. Members of Committee-Mr. A. Anderson, Mr. A. G. Croft (ex oficio), Mr. J. Freeman, Mr. B. Garwood, Dr. D. Simpson and Mr. B. Woodget. Mr. C. Waterhouse and Professor A. N. Worden were re-appointed as Honorary Auditors. South East Region The seventh Annual General Meeting of the Region was held at 2.15 p.m.on Wednesday, November 25th, 1981, in the Linnean Society, Burlington House, London, W.1. The Chair was taken by the Chairman of the Region, Mr. R. Sawyer. The following office bearers were elected for the forthcoming year : Chairman- Dr. S. J. Lyle. Vice-Chairman-Mr. H. I. Shalgosky. Honorary Secretary-Dr. A. H. Andrews, Beecham Pharmaceuticals, Research Division, Clarendon Road, Worthing, West Sussex, BN14 8QH. Honorary Treasurer-Mr. D. W. Houghton. Members of Committee- Mr. D. Blair, Mr. W. B. Chapman, Dr. J. G. Firth, Mr. G. F. Phillips, Mr. C. F. Simpson and Mr. D. W. Wilson. Dr. J. E. Page and Mr. D. C. M. Squirrel1 were re-appointed as Honorary Auditors. Microchemical Methods Group The thirty-eighth Annual General Meeting of the Group was held at 2 p.m.on Friday, December llth, 1981, a t the Laboratory of the Government Chemist, Cornwall House, Stam- ford Street, London, S.E.l. The Chair was taken by the Vice-chairman of the Group, Mr. A. C. Thomas. The following office bearers were elected for the forthcoming year : Chairman- Mr. G. J . Dickes. Vice-Chairman-Professor D. T. Burns. Honorary Secretary-Mr. P. R. W. Baker, Department of Physical Chemistry, Wellcome Research Laboratories, Langley Court, Beckenham, Kent, BR3 3BS. Honorary Treasurer-Mr. M. F. Cottrell. Honorary Assistant Secretary-Mr. B. T. Saunderson. Members of Committee-Mr. D. S. , Farrington, Dr. G. Ingram, Dr. E. J . Newman, Dr. B. A. Plunkett and Mr. A. C. Thomas (ex oficio). Mr. S. Bance and Mr.H. I. Shalgosky were re-appointed as Honorary Auditors. Biological Methods Group The thirty-seventh Annual General Meeting of the Group was held at 6 p.m. on Monday, December 7th, 1981, at the National Institute for Biological Standards and Control, Hamp- stead, London, N.W.3. The Chair was taken by the Chairman of the Group, Mr. V. J.March, 1982 REPORTS OF MEETINGS 103 Birkinshaw. The following office bearers were elected for the forthcoming year : Chairman- Mr. V. J. Birkinshaw. Vice-Chairman-Dr. M. Duncan. Honorary Secretary-Dr. A. H. Thomas, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London, NW3 6RB. Honorary Treasurer- Dr. L. Singleton. Honorary Assistant Secre- tary-Mr. G. A. Sabey. Members of Committee- Dr.J. A. Holgate, Mr. D. Hossack (co-opted), Miss N. Mulholland, Miss M. Rabouhans, Mr. D. Sykes, Dr. P. Turner and Dr. B. Wills. Dr. J . H. Hamence and Dr. M. Parkes were re-appointed as Honorary Auditors. Special Techniques Group The thirty-seventh Annual General Meeting of the Group was held at 2 p.m. on Wednesday, December 9th, 1981, in the Scientific Societies Lecture Theatre, 23 Savile Row, London, W. 1. The Chair was taken by the Chairman of the Group, Mr. J . T. Davies. The following office bearers were elected for the forthcoming year : Chairman-Professor D. Betteridge. Vice- Chairman-Dr. R. P. Mounce. Honorary Secre- tary and Treasurer-Mr. J. Huddleston, Building 10.2, Instrumentation and Applied Physics Division, AERE Harwell, Oxfordshire, OX11 ORA.Members of Committee-Dr. M. Adams, Dr. J. F. Alder, Dr. J. Becconsall, Mr. J. T. Davies (ex oficio), Mr. A. G. Ferrige, Dr. A. F. Taylor and Dr. J. G. Williams. Dr. D. Christopher and Professor J. N. Miller were re-appointed as Honorary Auditors. Chromatography and Electrophoresis Group The seventeenth Annual General Meeting of the Group was held at 2.15 p.m. on Thursday, December 3rd, 1981, in the Scientific Societies Lecture Theatre, 23 Savile Row, London, W. 1. The Chair was taken by the Chairman of the Group, Dr. F. K. Butcher. The following office bearers were elected for the forthcoming year: Chairman-Dr. G. H. Jolliffe. Vice-Chairman- Dr. R. M. Smith. Honorary Secretary and Treasurer-Dr. D. Simpson, Analysis For Industry, Factories 2/3, Bosworth House, High Street, Thorpe-le-Soken, Essex, C016 OEA.Members of Committee-Dr. F. K. Butcher, Mr. D. A. Elvidge, Dr. R. G. Hopkins, Dr. D. T. Plummer and Mr. V. C. Weaver. Dr. S. J. Purdy and Mr. J. S. Wragg were re-appointed as Honorary Auditors. Automatic Methods Group The sixteenth Annual General Meeting of the Group was held a t 1.20 p.m. on Thursday, December loth, 1981, at the Scientific Societies Lecture Theatre, 23 Savile Row, London, W.l. The Chair was taken by the Chairman of the Group, Mr. S. R. Hill. The following office bearers were elected for the forthcoming year: Chairman-Mr. D. G. Porter. Vice-Chairman- Mr. K. H. Wall. Honorary Secretary-Dr. C. J . Jackson, Health and Safety Executive, Occupa- tional Medicine and Hygiene Laboratory, 403 Edgware Road, London, NW2 6LN.Honorary Treasurer-Mr. J. L. Martin. Honorary Assis- tant Secretary-Mrs. E. N. Evans-Terlecki. Members of Committee-Professor D. Betteridge, Dr. A. Braithwaite, Mr. S. G. Farrow, Mr. S. R. Hill (ex oficio), Mr. R. B. Mitchell, Dr. D. Rowe and Dr. K. J. Saunders. Dr. J. E. Page and Mr. R. Sawyer were re-appointed as Honorary Auditors. Particle Size Analysis Group The sixteenth Annual General Meeting of the Group was held a t 2.15 p.m. on Wednesday, December 2nd, 1981, at the Linnean Society, Burlington House, London, W.l. The Chair was taken by the Chairman of the Group, Mr. M. W. G. Burt. The following office bearers were elected for the forthcoming year: Chair- man-Mr. M. W. G. Burt. Vice-Chairman- Dr. R. Wilson. Honorary Secretary and Treasurer-Mr.J. E. C. Harris, Materials Quality Assurance Directorate, Ministry of Defence, Puriton, Bridgwater, Somerset, TA7 8AD. Honorary Assistant Secretary-Dr. N. A. Orr. Members of Committee-Dr. T. Allen, Mr. C. P. Lenn, Mr. R. W. Lines, Mr. P. J. Lloyd, Mr. J. Spence and Dr. N. G. Stanley-Wood. Mr. W. G. King and Mr. P. W. Shallis were re-appointed as Honorary Auditors. Education and Training Group The eleventh Annual General Meeting of the Group was held at 6.10 p.m. on Friday, December 4th, 1981, at the Two Rivers Hotel, Chepstow. The Chair was taken by the Chair- man of the Group, Dr. E. J. Greenhow. The following office bearers were elected for the forthcoming year : Chairman-Dr. E. J . Green- how. Vice-ChairmanMr. A. F. Smith. Honorary Secretary-Dr. J . F. Tyson, Depart- ment of Chemistry, University of Technology, Loughborough, Leicestershire, LE11 3TU. Honorary Treasurer-Mrs. M. I. Arnold. Menz-104 UNDERGRADUATE TEACHING SYLLABUS Anal. Proc. beys of Committee-Dr. L. A. Gifford, Dr. C. Thomas and Dr. A. Townshend. Dr. N. T. Graham, Dr. E. K. Harrison, Dr. C. W. McLeod, Crosby and Dr. W. J. Williams were re- Dr. J. G. Pritchard (ex oficio), Dr. J. D. R. appointed as Honorary Auditors.

ISSN:0144-557X    

DOI:10.1039/AP9821900102

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

104 UNDERGRADUATE TEACHING SYLLABUS Anal. Proc. Undergraduate Teaching Syllabus in Analytical Chemistry Fourth Report by the Committee of the Education and Training Group of the Analytical Division of the Royal Society of Chemistry The current members of the Committee are : Dr. E. J. Greenhow (Chairman) Mr. A. F. Smith (Vice-chairman) Dr. J. F. Tyson (Honorary Secretary) Mrs. M. I. Arnold (Honorary Treasurer) Dr. E. K. Harrison Dr. C. W. McLeod Dr. J. D. R. Thomas Dr. L. A. Gifford Dr. C. Graham Dr. A. Townshend Dr. J. G. Pritchard Former members of the Committee under the Chairmanship of Dr. J. G. Pritchard were Dr. D. M. W. Anderson, Mr. H. A. Glastonbury, Mr. B. Mills, Dr. J. M. Skinner and Professor D. Thorburn Burns. Introduction This syllabus is put forward for the purpose of meeting deficiencies in the analytical chemistry content of many of the under- graduate chemistry courses in the United Kingdom.In the past few years it has been argued that, in general, undergraduate training in analyti- cal chemistry in the United Kingdom is inadequate to meet the current requirements of industry and that extensive in-service training and/or supplementary postgraduate work is necessary to remedy this situation. The first and second reports of the Committee have pointed out the generally low level of staffing in analytical chemistry as opposed to other areas of chemistry in most universities.lS2 The polytechnic sphere contains relatively more emphasis on analytical chemistry.1$2 The third report of the Committee has stressed the general opinion that exists in industry to the effect that new graduates who present them- selves for careers primarily as analysts are, in many respects, insufficiently prepared for the In addition, individual opinions to this effect have been widely publicised.* The Royal Society of Chemistry published, in July 1980, the report of a Working Party on “The Supply of and Demand for Analytical Chemists” and items from this report, including the results of a survey among employers of analytical chemists, have recently been highlighted.6s6 One of the main conclusions from the survey was that employers of analytical chemists are not, on the whole, satisfied with the standard of training of applicants for analytical chemistry posts.The main objective of the present report is to meet these criticisms with a strong recommendation for positive action.The recommendations are intended to provide graduates with a broader appreciation of chemistry and its application, and to prepare them better for available job opportunities. The Committee has taken the standpoint that Masters’ courses in analytical chemistry (under various titles) are, at present, at least in part remedial in n a t ~ r e . ~ Consequently, if the situation were remedied a t the Bachelors’ level, Masterships would inevitably increase in depth and/or in specialisation to the levels which the prior preparation of the student would then permit. Such changes would not detract from the value of specialist Bachelors’ degrees in analytical science or related subjects, for which an MSc course or PhD research course in analytical chemistry constitute a legitimate follow-up for those aspiring to an advanced degree.The Committee has confined its thought and effort to the basic material that needs to be assimilated by all students a t undergraduate level. While shorter courses below first degree level may well need improve- ment as far as analytical chemistry content is concerned, the Committee has regarded as its first priority an attempt to raise to a uniformMarch, 1982 UNDERGRADUATE TEACHING SYLLABUS 105 standard the analytical chemistry subject matter taught in undergraduate courses. The aims of the Committee in constructing the undergraduate syllabus were thus as follows. To combine and condense longer established aspects of analytical chemistry with the newer, more instrumental aspects of the subject in order to produce a balanced unit reflecting the extent to which the various analytical methods are currently utilised.To produce a course that takes a relatively small fraction of the time allocated to under- graduate training. To produce a course that integrates other branches of chemistry (and physics) and exemplifies their application. To provide a well balanced lecture scheme which, when combined with a substantial and realistic practical schedule, should allow students to develop positive practical skills and confidence in analysis, and to maximise their potential as chemists, whatever their intended career specialisation might be. General Plan The proposed course falls conveniently into two main parts.The first is a coherent basic course of approximately one conventional “course unit” in duration, a total of 173 hours, which may be taught in two sections (phases) if this is desirable. The second part is an optional, more advanced unit of 85 hours. The distribution of time to be devoted to analytical chemistry is thus: Basic unit- Phase 1-Analytical Reaction Chemistry Phase 2-General Analytical Chemistry course, and successful completion of the basic unit should enable the graduate to start useful employment in industry as an analytical chemist. However, students who also complete Phase 3 should regard themselves as well on the way to becoming analytical specialists. The Committee has recognised that the analytical chemistry course must be co- ordinated carefully with other parts of an undergraduate chemist’s normal training and education.This co-ordination should be up- dated each Session so that there are no signifi- cant gaps in the transition from “basic” chemistry to analytical chemistry. It is not the Committee’s purpose to dictate the precise manner in which this might be done, but the following two approaches readily suggest them- selves, the second of which is preferred. (i) The basic analytical chemistry unit could be placed in time after the undergraduate has completed basic units of descriptive inorganic and organic chemistry and also spectroscopy, thermodynamics, kinetics, electrochemistry, optics, electronics and the mathematics of elementary statistics. In this instance the analytical unit would appear best placed in the latter part of a three-year course.The basic 173-hour unit might be run, for example, for one six-hour working day per week for 25 weeks, the approximate length of the university undergraduate’s in-college working period per year, with one additional lecture per week on another day during the same period (or otherwise, as suits the particular Lecture Practical hours hours 18 + 49 = 67 46 + 60 = 106 - Total 173 Optional unit- Phase 3-More Advanced Topics in Analytical Chemistry The Committee has specified the lecture topics and the assigned hours in considerable detail below. The amount of time assigned to practical classes, 173 and 85 hours, are, in the Committee’s judgement, sensible amounts, appropriate to a well balanced undergraduate course.The basic unit amounts to no more than one-ninth of a typical undergraduate 30 + 55 = 85 Total 85 - - establishment). Any fragments of the proposed analytical chemistry lecture course (such as the theory of certain instru- mental methods) which have been well covered in an earlier part of the three-year period, need not be repeated unnecessarily, in which case the appropriate analytical chemistry lecture time so released might106 UNDERGRADUATE TEACHING SYLLABUS Anal. Proc. (ii) best be used for additional analytical chemistry practical work. The second, preferred, approach that suggests itself stresses how important it is for the undergraduate to have the oppor- tunity to specialise in analytical chemistry and to be committed to it early in his undergraduate life if he so desires, just as he is able to do with the other branches of chemistry.The proposed syllabus is thus presented in three phases so designed that the first phase can be taken in the first year of an undergraduate course, with phases two and three following in years two and three, respectively. This procedure should facilitate the integration of the analytical units with the other parts of an under- graduate course. While the Committee believes that a course such as the one proposed is necessary if the student is to have a thorough grounding in analytical chemistry, it is realised that some universities and polytechnics will require a shorter basic or “core” course and will be reluctant to allocate time for more than, say, 30 lectures and 60 hours of practical work.A short course of this type, intended to cover the bare essentials of analytical chemistry and to enable the institution concerned to claim that the student has reached a “level of competence” in analytical chemistry, could be constructed by judicious selection from the course recom- mended below. The content of such a course would depend to some extent on whether or not the institution had any specialisation in analytical chemistry. The Committee wish to stress that the core course must, together with topics covered in other courses, form a cohesive whole, and should not be merely a collection of unrelated sets of lectures utilising the time allocated. No attempt has been made to design a set of lectures and practical work suitable for a “service course” in analytical chemistry, intended to satisfy the requirements of non- chemists, e.g., metallurgists, geologists or biologists, as it would be extremely difficult to establish the level of background knowledge in chemistry of the students concerned. THE LECTURE COURSE Basic Unit CHEMISTRY PHASE ANALYTICAL REACTION A. Scope of analytical reactions- Lecture hours Introductory review 5 Scope and applications of analytical chemistry. Chemical equilibria, quantitative reactions and solution chemistry. Co- ordination , complex formation and chelating. Selectivity, masking. B. Titrirnetric analysis 5 Acid - base, redox, complexo- metric, non-aqueous, indicators. C. Gravimetric analysis 4 Theory of crystal formation, co-precipitation, elementary gravimetry, weighing forms, amplification reactions ; precipita- tion from homogeneous solution, titrimetric precipitation reactions.D. Colorimetry 4 Spectrophotometric reagents , elementary visible spectro- photometry, spectrophotometric titrations, turbidimetric methods. PHASE 2-GENERAL ANALYTICAL SCIENCE A. General firocedures in analytical science 5 (i) The several steps in analytical procedures, accuracy, propagation of errors, precision, limit of detection, heterogeneity of samples, sampling technique. (ii) Pre-treatment of samples to obtain homogeneous solutions, statistical treatment. ‘ (iii) Rejection of outliers, Student’s-t function, F-test. (iv) Regression lines, calibration graphs, method of standard additions. (v) Systematic errors, standard materials and procedures, standard and official methods, pharmacopoeias.B. Separatory analytical methods (i) Distillation , partition theory (ii) Principles of chromatography ; and solvent extraction. 2 theoretical plates, resolution and band spreading. Paper,March, 1982 UNDERGRADUATE TEACHING SYLLABUS 107 C. (iii) (iv) Lecture hours thin-layer, ion-exchange, gel- (i) Optical activity in analytical permeation and gas - liquid High-perf ormance liquid (ii) Nuclear magnetic resonance chromatography. 2 spectroscopy, quantitative chemistry; rotatory disper- partition methods. 5 sion; dichroism. 1 Electrophoresis. 1 applications. 3 Instrumental methods (i) General principles of (ii) Analytical application of spectrometers. 2 ultraviolet - visible absorp- tion spectroscopy. Fluorescence and (iii) Atomic spectroscopy : phosphorescence.3 absorption and emission. Flame atomisation. 4 (iv) Infrared spectrophotometry ; basic theory; solid, liquid and gas samples; group frequencies, quantitative uses. 2 (v) Potentiometry (pH and ion-selective electrodes), polarography, coulometric, potentiometric, conducti- metric, titrations. 4 (vi) Radiochemical methods. 1 (vii) Immunoassay. 1 (viii) Elementary mass spectro- metry of organic com- pounds; determination of molecular formulae and partial structure. 2 (ix) X-ray methods. 1 D. General application of analytical science Qualitative analysis of unknown samples. Schemes of wet qualitative analysis for inorganic ions, organic compounds and functional groups. Automation of quantitative analytical procedures ; applications of micro- processors ; on-line deter- mination; flow injection; continuous flow analysis; data processing.Optional Unit IN ANALYTICAL CHEMISTRY PHASE 3-MORE-ADVANCED TOPICS Lecture hours Auger electron spectrometry, ESCA. Applications to surface analysis, other surface Advanced mass spectro- metry; various analysers and sources ; inorganic and organic analysis ; isotope methods. Gas chromatography - mass Advanced electroanalytical techniques ; anodic-stripping voltammetry ; pulse polaro- Advanced immunoassay and Radiochemical methods of analysis ; neutron activation analysis; y-ray spectrometry. 3 Thermal analysis ; thermo- Kinetic methods of analysis and use of bio- and chemi- luminescence in analytical Statistical comparison of results from different laboratories ; sampling and correlation of analytical data with origin of samples, significance of correlation by “t” statistic.3 techniques. 3 spectrometry. 3 graphy. 3 electrophoretic techniques. 2 gravimetry; DTA; DSC. 2 chemistry. 3 (xi) Advanced atomic spectro- metry ; electrothermal atomisation ; arcs, sparks and plasma emission. 4 4 Schedule of Practical Work Illustrative, useful practical work is a vital part of a chemist’s training. It is intended that the following schedule be used as a suggestion pool from which the tutors concerned can select experiments best suited to their facilities and inclinations. The experiments are listed in sections which refer to the foregoing lecture syllabus. Most of these exercises and many alternatives can be found clearly described in standard texts on chemical analysis and also in 7108 UNDERGRADUATE TEACHING SYLLABUS Anal.PYOG. reference works like British Standard Specifica- tions and the British Pharmacopoeia. The following two publications on recognised methods of analysis are also excellent sources of analytical exercises for students : ‘ ‘Official, Standardised and Recommended Methods of Analysis,’’ The Society for Analytical Chemistry, London, 1973 ; “Official Methods of Analysis of the AOAC,” (Editor W. Honvitz), 13th Edition, Associ- ation of Official Analytical Chemists, Washing- ton, DC, USA, 1980. PHASE 1A Selected library exercises. PHASE 1B 1. 2. 3. 4. 5. 6. 7. 8. Use of sodium carbonate as a primary standard in acidimetry.Standardisation of sodium hydroxide solu- tion with potassium hydrogen phthalate and conventional indicator. Titrations with permanganate, dichromate and vanadium to illustrate oxidation - reduction. The cerium(IV), arsenic(II1) oxidation - reduction reaction catalysed by iodide. Saponification value for a dark-coloured specimen of fat or wax by potentiometric titration. Iodine value of unsaturated fatty acid standards and of given unknown fat or wax. Iodine - iodide coulometric titrations. Determination of iodide in iodised table salt by microtitration of iodine with thio- sulphate (back-titration with iodine). PHASE 1C 1. 2. 3. 4. 5. 6. 7. 8. 9. Gravimetric determination of sulphate as BaSO,. Determination of calcium by EDTA. Determination of phosphate in a fertiliser by gravimetry.Examination of dithizonates of various metals. Determination of aluminium and nickel. Determination of germanium in steel. Determination of molybdenum by spectro- photometric estimation of molybdenum thiocyanate extracted into 3-methylbutan- 1-01. Gravimetric determination of piperazine adipate following reaction with 2,4,6- trinitrophenol (British Pharmacopoeia Gravimetric determination of zinc in Compound Zinc Paste BP (British Pharmacopeia, 1980). 1980). PHASE 1D 1. Photometric titration [e.g., arsenic(II1) 2. Turbidimetric determination of lead ( J . with triodide ion]. Chew. Educ., 1961, 38, 358). PHASE 2A 1. Literature search problem, e.g., what methods are available for the determination of sulphur dioxide in the atmosphere? 2.Repeated use of pipette and determina- tion of statistics on the results of individual and class efforts. 3. Acid treatment of gelatin and hair to obtain clear solutions of trace cations. 4. Wet and dry combustion - oxidation to prepare a sample of foodstuff for analytical determination of trace metals. Illustration of loss of volatile metals by atomic- absorption spectrophotometry. 5. Determination of sodium chloride in selec- ted samples from a (heterogeneous) finely powdered salt - sand mixture. Deter- mination of the heterogeneity of the mixture by the 99%-point of the “t” distribution, and examination of a repre- sentative sample. PHASE 2B 1. Chromatographic identification of dyes used in coloured, boiled sweets. 2. Determination of ethanol in spirituous liquors by gas - liquid chromatography.3. Thin-layer chromatography of phosphates and phenylhydrazones. 4. Chromatographic purification and spectro- photometric determination of aspirin. 5. Ion-exchange separations of zinc and cadmium, and of sodium and copper. 6. HPLC analysis of analgesic tablets. 7. Cellulose acetate electrophoresis of the proteins of normal human blood serum. PHASE 2C 1. Determination of copper and nickel in a cupronickel alloy by controlled potential electrode position. 2. Characterisation of surfactants in com- mercial detergent preparations by infrared spectroscopy. 3. Atomic-absorption spectrophotometric determination of calcium and magnesium in tap water. Determination of sodium and potassium in plasma or urine by flame atomic emission. 4.Cold-vapour atomic-absorption spectro- photometric determination of mercury. 5. Ion-selective electrode determination of sulphide.March, 1982 ANALYTICAL METHODS COMMITTEE 109 6 . 7. 8. 9. 10. 11. 12. 1. 2. 3. 4. 5. Preparation and de-gassing of solution for polarography and determination of cad- mium. Determination of sulphur dioxide in the air through fpmation of potassium di- chlorosulphitomercurate( 11) and estima- tion of this spectrophotometrically through conversion of acid-bleached p-rosaniline to fi-rosanilinemethylsulphonic acid (West - Gaeke method). Checking an ultraviolet - visible spectro- meter. Determination of dodecanol and ethyl do- decanoate in admixture by infrared spectro- photometry of solutions. Determination of calcium in barium nitrate by flame emission photometry on an ethanol extract versus a further extract blank. Quantitative analysis of dimethylbenzene for the proportion of ortho, meta and para isomers by infrared spectrophotometry. Determination of vitamin A by ultra- violet - visible spectrophotometry. 13. Determination of quinine (and inter- ferences therewith) by spectrofluorimetry. 14. Potentiometric titration of iron(I1) with cerium (IV) , 15. Determination of iron in an ore by reaction of iron(II1) with tin(I1) and potentiometric back-titration of this with cerium(1V). PHASE 2D 1. Qualitative analysis of n-radical mixtures. 2. Qualititative analysis of a commercial detergent preparation. 3. Flow injection determinations of chloride and phosphate ( J . Cheun. Edzac., 1979, 56, 677). PHASE 3 For the practical work of Phase 3 the lecturer should demonstrate a selection of the more advanced techniques dealt with in the lectures, and the student should carry out entirely with his own hands some of the more lengthy and searching experiments in the foregoing lists for Phases 1 and 2. REFERENCES Education and Training Committee, Proc. SOC. Anal. Chem., 1972, 9, 173. Education and Training Group Committee, Proc. Anal. Div. Chem. SOG., 1977, 14, 1. Education and Training Group Committee, Proc. Anal. Div. Chem. SOC., 1979, 16, 107. Shaw, W. H. C., PYOG. Anal. Div. Chem. SOG., 1977, 14, 54. Prof. Bull. R. SOG. Chem., 1980, No. 52. 6. ThGrburn Burns, D., Anal. Proc., 1981, 18, 56.

ISSN:0144-557X    

DOI:10.1039/AP9821900104

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

March, 1982 Analytical ANALYTICAL METHODS COMMITTEE Methods Committee In order to publicise the work of this Com- mittee of the Analytical Division Analytical Proceedings will publish a summary of current projects near to the beginning of each year. The following are the current programmes of the Sub-Committees, which are expected to be completed by December 1982. Antibiotics Sub-Committee Studies on the determination of zinc baci- tracin, spiromycin and virginiamycin are almost complete. Collaborative tests of the methods for the determination of salinomycin and noracin have been organised. A method for determining nosiheptide is under development, while for monensin, salinomycin, naracin and lasalocid HPTLC identification tests are being evaluated. Medicinal Additives in Feeds Sub-Committee A The development of methods for ronidazole and ipronidazole has almost been completed, 109 and methods for clapidol, laslocid, sulphadimin and arpocox are being evaluated.Medicinal Additives in Feeds Sub-Committee B Investigation of methods for the determina- tion of halofuginone, nifursol and robenidine is almost complete, and methods for olaquinox and carbadox are under evaluation. A n i m a l Feeds Sub-committee Studies on the determination of vitamin A by HPLC are almost complete, as is a review of methods for oils and fats. Under evaluation are “fibre” methods, methods for starch and a review of methods for aflatoxin. Veterinary Residues in Fresh Meat Sub-Com- mittee A method for the determination of halo- fuginone is nearing completion,110 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSIS Anal.PYOC. Metallic Impurities in Organic Matter Sub- Committee Methods for the determination of tin and chromium are under investigation. Essential Oils Sub-Committee tic origin are being produced. Fingerprint chromatograms of oils of authen- Trace Element Speciation Sub-Committee Atomic-absorption determination of cadmium in shellfish has been studied, together with the form in which cadmium occurs in foodstuffs (i.e., ionic, chelated, metallorganic, etc.). A report on a literature survey for a number of elements is also being produced. Instrumental Criteria Sub-committee This Sub-committee is now considering atomic-absorption spectroscopy, particularly with regard to what questions a potential buyer should ask before committing himself to a particular instrument and which criterion is most important for a particular application. Persons wishing to contact any of the above Sub-Committees should write to The Secretary, Analytical Methods Committee, 8 Cadogan Court, Mulgrave Road, Sutton, Surrey.

ISSN:0144-557X    

DOI:10.1039/AP9821900109

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

110 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSIS Anal. PYOC. ANNUAL CHEMICAL CONGRESS The Annual Chemical Congress of the Royal Society of Chemistry was held at the University of Surrey, Guildford, April 7th-9th, 1981. A Symposium on Matrix and Sensitivity Problems in Analysis was organised by the Analytical Division. Matrix and Sensitivity Problems in Analysis The following are summaries of four of the papers presented at the Annual Chemical Congress on April 7th-9th, 1981. Approaches to Trace-metal Speciation in Environmental Samples L. Ebdon and R. W. Ward Department of Environmental Sciences, Plymouth Polytechnic, Drake Circus, Plymouth, Devon, PL4 8A A and D. A. Leathard Department of Chemistry, Shefield City Polytechnic, Pond Street, Shefield, S1 1 WB The interaction of metals with organic species is now being recognised as a neglected, yet vital, aspect of environmental toxicology and modelling.Atomic spectroscopy is widely applied to trace-metal determinations because of its selectivity and sensitivity. The essential simplicity of atomic-absorption spectroscopy (AAS) has led to its adoption as the technique of choice for environmental trace-metal monitoring. However, unless preceded by time con- suming sample pre-treatment atomic-spectrochemical techniques do not yield any information as to the species in which the metals are bound. Chromatography, particularly gas chroma- tography (GC) and high-performance liquid chromatography (HPLC), on the other hand, offers excellent separation of different species, but often the identification of the important organometallic moieties is difficult to achieve unambiguously when working with complex environmental samples.Hence, recently there has been a growth in hybrid chromatographic - at omic-spectroscopic inst rumen t ation for trace-met a1 speciation. Several hundred reports have now appeared concerning various metal-specific chromato- graphic detectors. Some of the more popular combinations and published environmental applications are summarised in Table I. Four requirements for a metal-specific detector for such applications can be identified: high selectivity, given the complex nature of the samples encountered; sensitivity, because of the low levels encountered and the contamination and chemical changes that may occur upon concentration; simplicity, as often the detector willMarch, 1982 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSIS 111 TABLE I EXAMPLES OF ENVIRONMENTAL APPLICATIONS OF CHROMATOGRAPHY - ATOMIC SPECTROSCOPY The meanings of the abbreviations used are as follows: G€, gas chromatography; HPLC, high- performance liquid chromatography ; FAAS, flame atomic-absorption spectroscopy ; FAES, flame atomic-emission spectroscopy ; FAFS, flame atomic-fluorescence spectroscopy ; ETA, electrothermal atomisation, AAS, atomic-absorption spectroscopy; AES, atomic-emission spectro- scopy; DCP, direct-current plasma ; MIP, microwave-induced plasma; and ICP, inductively - coupled radio-frequency plasma.Technique GC - FAAS GC - FAES GC - FAFS GC - ETA - AAS GC - DCP - AES GC - MIP - AES Application Tetraalkyllead compounds in Tetraalkyllead compounds in air Silylated alcohols Chromium chelates Chromium in orchard leaves Organotin compounds Silylated alcohols Tetraalkyllead compounds in petrol Methylcyclopentadienyl- manganesetricarbonyl in petrol Biomethylated lead Tetraalkyllead compounds in petrol petrol water Tetraalkyllead compounds in air Tetraalkyllead compounds in sea Organomercury compounds Alkylmercury compounds in fish Alklymercury compounds in Bioalkylated selenium Organotin compounds Organoarsenic compounds Alkylselenium compounds in plant transpiration Organomercury compounds in sewage sludges Me thylcyclopen tadienyl- manganesetricarbonyl in water petrol petrol Tetraalkyllead compounds in Tetraalkylead compounds Tetraalkvllead comDounds in air Methylcyclopen t adienyl- manganesetricarbonyl in petrol Alkylmercury compounds Organomercury compounds in biological and aqueous systems Organomercury compounds in fish Reference 1-6 1 7 8 9 10 7 6, 11 12 13,14 5,6,15-19 22 23 24, 25 20,21 26,27 29 29 32 32 28-31 33,34 35 36, 37 38 37 37 39, 40,41 42 Technique GC - ICP - AES GC - Arc AES HPLC - FAAS HPLC - FAFS HPLC - ETA AAS HPLC - DCP AES HPLC - ICP AES Application Reference Chromium in blood 43 Organoselenium compounds in environmental samples 46 Biomethylated selenium Elemental analysis 48 Tetraalkyllead compounds in Biomethvlated arsenic compounds 50 51 46,52 53 Silylated carboxylic acids 37,44 Alkylarsenic pesticides 45 compounds 47 petrol 49 Organosilicon compounds 49 Organochromium compounds Copper chelates Magnesium phosphate complexes Free and chelated zinc in plant Tetraalkyllead compounds in Amino acid complexes Alkyl- and arylzinc compounds Organometallic compounds in Manganese speciation Glycine metal complexes Tetraphenyllead compounds Tetraalkyllead compounds Tetraalkyllead compounds in Organotin compounds Organomercury compounds Organosilicon compounds Aluminium in serum Copper - amino acid complexes Arsenic speciation Metal chelates Cobalt in vitamin B,, Metallo-proteins Iron and molybdenum Ferrocene complexes Organoarsenic compounds Organomercury compounds Tetralkyllead compounds Copper chelates extracts petrol in oils in petroleum petrol carbonyls 54 55 56 57,58 52 58 32, 57 58, 59 60-62 63 61, 62, 64 61 65 66 67 68,69 70 71 71 72 72 72 72 72 73 need to be dismantled to allow conventional use of the chromatograph ; and compatability with existing equipment.Certain features of the couplings listed in Table I are worthy of comment in the light of the above requirements. Coupling flames that burn gases with gas chromatographs should be particularly simple. Flame AAS is probably the most selective of all the techniques listed in Table I but the attain- ment of good sensitivity with flame cells requires special precautions. Coupling GC with flame atomic-emission spectroscopy (AES) is an obvious extension of flame spectrophotometric detectors, although good selectivity is only achieved if a high-resolution monochromator is used and the flames commonly used are not hot enough to give sufficient excitation for really low level detection.Flame atomic-fluorescence spectroscopy (AFS) should, in theory, offer better sensitivity than flame AAS with equivalent selectivity together with the possibility of simple arrangements for multi-element work. The lack of readily available high-intensity, narrow-line sources has impeded the development of AFS for this application as it has for many others. A variety of devices have been proposed for the electrothermal atomisation (ETA) of GC effluents for AAS, including quartz T-shaped f~rnaces6,~~,~~,30-3~ and modified carbon The popular aim has been to increase the sensitivity of couplings with atomic-absorption detection and this is usually achieved because of the extended residence times available, rather than through any improvement in atomisation efficiency.Indeed,Anal. PYOC. electrothermal atomisers are generally designed for discrete liquid samples and can prove inappropriate and expensive for continuously presented gaseous samples. The d.c. plasma (DCP) is very sensitive to gas flows that may affect the shape and position of the inverted Y plasma continuum; perhaps it is this and its relative expense that have determined the paucity of applications for GC effluents. The microwave induced plasma (MIP) is usually regarded as the cheapest of all the analytical plasmas. It offers a high excitation temperature compared with gas temperature and is thus intolerant to solution samples but well suited to gases. Commercially available microwave plasma detectors for GC and more recently developed detectors based upon the TM,,, cavity of Beenakker,74 which is capable of sustaining an atmospheric pressure helium plasma, have been used.Careful con- trol of operating conditions is necessary to prevent extinction of the plasma by the solvent front or deterioration of the quartz walls of the atom cell. The major disadvantage of the inductively coupled plasma (ICP) as a GC detector must be its cost, The advantages of this detector lie with its multi-element detection capability and also with the compatibility of gas- chromatographic effluent flow-rates with the plasma injector flow-rates normally encountered. The suitability of various atomic-spectroscopic techniques for HPLC detection additionally involves a consideration of their ability to accept solution samples.Flames are well suited to receive liquids at typical HPLC flow-rates and most solvents are acceptable. The problems really centre around the need to achieve a high efficiency of nebulisation to obtain optimum sensitivity. This is not a problem in electrothermal atomisation, where discrete liquid drop- lets are quite acceptable, but usually at a rate of a single drop every two minutes. Hence the interest in couplings where samples are stored in an auto-sampler or the chromatograph is operated in a stop-flow mode. In both instances the analysis becomes exceedingly slow. The use of the d.c. plasma as an HPLC detector represents a growth area as this plasma is stable to quite high flow-rates and shows especial promise for reversed-phase modes. Typical eluent flows are again suitable for the inductively coupled plasma but nebulisation still presents a problem and the all-argon plasma may not be as tolerant to all of the possible solvents as a nitrogen-cooled plasma.Gas chromatography, rather than HPLC, is thus probably easier to interface to atomic spectroscopy because of the gaseous nature of the effluent. Flame AAS seems to offer the simplest detection system with best selectivity and we have attempted, by studying the be- haviour of transient signals within the flame, to improve upon reported sensitivities. Tetra- alkyl lead compounds have proved to be useful model species of some environmental signific- ance. In our work we have coupled a conventional gas chromatograph (Series 104, Pye Unicam, Cambridge), via an interface tube kept isothermal with the GC oven, to a conven- tional flame atomic-absorption spectrometer (SP 192, Pye Unicam, Cambridge), set for lead AAS.It was soon realised that to introduce the GC effluent into the burner mixing chamber caused unnecessary dilution of the sample and we proceeded to introduce effluent directly to the burner by directing it along the slot of a typical air - acetylene flame. Best results were obtained using the slower burning velocity air - propane flame as this allowed the longest residence times for the atoms within the flame. In an attempt to extend residence times further the effluent was introduced first into an air - acetylene flame and then swept into a ceramic tube (1 10 mm long) suspended above the flame. This resulted in detection limits competitive with those reported by others using coupled GC - ETA - AAS.Further improved sensitivities were obtained by separating the atomisation and tube heating functions of the flame. The nitrogen carrier gas (containing the separated analyte species) was augmented by a small flow of hydrogen via a T-piece at the end of the interface tube : a small hydrogen diffusion flame was burnt at the end of the tube and the products swept into a ceramic tube (6.25 mm internal diameter) suspended in an air - acetylene flame (see Fig. 1). The column temperature, carrier gas, hydrogen, air and acetylene flow-rates, tube-burner and interface-tube separations were optimised using the variable step-size simplex method.75 The detection limits available with this optimised system [17 pg (as lead) for tetramethyl- and tetraethyllead] are superior to any reported previously, even for GC - ETA - AAS systems, demonstrating the importance of optimising the sample residence times in the atom cell.The above systems have been applied to the determination of lead alkyl additives in petrol (unequivocal chromatograms with complete resolution can be obtained in 60 s), the analysis of mixtures of five tetraalkyl lead compounds, simulated arson samples and the separation and speciation of alkylmercury compounds. 112 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSISMarch, 1982 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSIS 113 Fig. 1. Atom cell for coupled gas chromatography - flame atomic-absorption spectroscopy : A, ceramic tube; B, air - acetylene burner; C, ceramic tube supports; D, T-piece; and E, hydrogen flame.We thank Pye Unicam Ltd. for the grant of equipment and Pye Unicam and the Science Research Council for an SRC CASE studentship (to R.W.W.), which has made this work possible. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. Chau, Y . K., Wong, P. T. S., and Saitoh, H., J . Chromatogr. Sci., 1976, 14, 162. Katou, T., and Nagawa, R., Bull. Inst. Environ. Sci. Technol., 1974, 1, 19. Kolb, B., Kemmner, G., Schleser, F. H., and Wiedeking, E., Fresenius 2. Anal. Chem., 1966, 221, Coker, D. T., Anal. Chem., 1975, 47, 386. Bye, R., Paus, P. E., Solberg, R., and Thomassen, Y., At. Absorfit. Newsl., 1978, 17, 131. Radziuk, B., Thomassen, Y ., Butler, L. R. P., Van Loon, J. C., and Chau, Y . K., Anal. Chim. Acta, Morrow, R. W., Dean, J. A., Shults, W. D., and Guerin, M. R., J . Chromatogr. Sci., 1969, 7, 572. Wolf, W. R., Anal. Chem., 1976, 48, 1717. Wolf, W. R., J . Chromatogr., 1977, 134, 159. Wright, B. W., Lee, M. L., and Booth, G. M., J . High Resolut. Chromatogr. Chromatogr. Commun., Radziuk, B., PhD Thesis, University of Toronto, 1979. Coe, M., Cruz, R., and Van Loon, J. C., Anal. Chim. Ada, 1980, 120, 171. Wong, P. T. S., and Chau, Y . K., Manag. Control Heavy Met. Environ., Int. Conf., 1979, pp. 131- Wong, P. T. S., Chau, Y. K., and Luxon, P. L., Nature (London), 1975, 253, 264. Chau, Y. K., Wong, P. T. S., and Goulden, P. D., Anal. Chim. Ada, 1976, 85, 421. Segar, D. A., Anal.Lett., 1974, 7, 89. Robinson, J. W., Vidaurreta, L. E., Wolcott, D. K., Godbread, J. P., and Kiesel, E., Sfiectrosc. Lett., De Jonge, W. R., Chakrabarti, D., and Adams, F., Anal. Chim. Acta, 1980, 115, 89. Robinson, J. W., Kiesel, E. L., Goodbread, J. P., Bliss, R., and Marshall, R., Anal. Chim. Acta, 1977, Radziuk, B., Thomassen, Y., Van Loon, J. C., and Chau, Y . K., Anal. Chim. Ada, 1979, 102, 255. De Jonghe, W. R., Chakrabarti, D., and Adams, F. C., Anal. Chem., 1980, 52, 1974. Robinson, J. W., Kiesel, E. L., and Rhodes, I. A. L., J . Environ. Sci. Health, 1979, A14, 65. Longbottom, J. E., Anal. Chem., 1972, 44, 1111. Bye, R., and Paus, P. E., Anal. Chim. Acta, 1979, 107, 169. Gonzalez, J. G., and Ross, R. T., Anal. Lett., 1972, 5, 683. 166. 1979, 108, 31.1979, 2, 189. 134. 1975, 8, 491. 92, 321.114 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. MATRIX AND SENSITIVITY PROBLEMS I N ANALYSIS Alzal. Proc. Dressman, R. C., J . Chromatogr. Sci., 1972, 10, 472. Longbottom, J . E., and Dressman, R. C., Chromatogr. Newsl., 1973, 2, 17. Chau, Y. K., Wong, P. T. S., and Goulden, P. D., Anal. Chem., 1975, 47, 2279. Parris, G. E., Blair, W. R., and Brinckman, F. E., Anal. Chem., 1977, 49, 378. Van Loon, J . C., and Radziuk, B., Can. J . Spectrosc., 1976, 21, 46. Radziuk, B., and Van Loon, J . C., Sci. Tot. Environ., 1976, 6, 251. Van Loon, J. C., Radziuk, B., Kahn, N., Lichwa, J., Fernandez, F., and Kerber, J., A t . Absorpt.Uden, P. C., Barnes, R. M., and Di Sanzo, F. P., Anal. Chem., 1978, 50, 852. Uden, P. C., Henderson, D. E., Di Sanzo, F. P., Lloyd, R. L., and Tetu, T., J . Chromatogr., 1980, Uden, P. C., Anal. Proc., 1981, 18, 189. Estes, S. A., Poirier, C. A., Uden, P. C., and Barnes, R. M., J . Chromatogr., 1980, 196, 265. Quimby, B. D., Uden, P. C., and Barnes, R. M., Anal. Chem., 1978, 50, 2112. Reamer, D. C., Zoller, W. H., and O’Haver, J. C., Anal. Chem., 1978, 50, 1449. Gohlke, R., and Schroder, U., Lab. Praxis, 1979, 3, 26. Talami, Y., Anal. Chim. Acta, 1975, 74, 107. Talami, Y., and Norvell, V. E., Anal. Chim. Acta, 1976, 85, 203. Bache, C. A., and Lisk, D. J., Anal. Chem., 1971, 43, 950. Black, M. S., and Sievers, R.E., Anal. Chem., 1976, 48, 1872. Bostick, D. T., and Talami, Y., J . Chromatogr. Sci., 1977, 15, 164. Talami, Y., and Bostick, D. T., Anal. Chem., 1975, 47, 2145. Talami, Y., and Andren, A. W., Anal. Chem., 1974, 46, 2122. Reamer, D. C., and Zoller, W. H., Science, 1980, 208, 500. Windsor, D. L., and Denton, M. B., J . Chromatogr. Sci., 1979, 17, 492. Sommer, D., and Ohls, K., Fresenius 2. Anal. Chem., 1975, 295, 337. Rraman, B. S., and Foreback, C. C., Science, 1973, 182, 1247. Jones, D. R., and Manahan, S. E., Anal. Lett., 1975, 8, 569. Jones, D. R., Tung, H. C., and Manahan, S. E., Anal. Chem., 1976, 48, 7. Yoza, N., and Kouchiyama, K., Anal. Lett., 1975, 8, 641. Umebayashi, M., and Kitagishi, K., Paper presented a t 5th International Conference on Atomic Botre, C., Cacace, F., and Cozzani, R., Anal.Lett., 1976, 9, 825. Slavin, W., and Schmidt, G. J., J . Chromatogr. Sci., 1979, 17, 610. Van Loon, J . C., Anal. Chem., 1979, 51, 1139A. Taraszewski, W. J., Paper presented a t 7th Federation of the Analytical Chemistry and Spectro- Vickrey, T. M., and Eue, W., J . Autom. Chem., 1979, 1, 198. Vickrey, T. M., Harrison, G. V., and Ramelow, G. J., A t . Spectrosc., 1980, 1, 116. Brinckman, F. E., Blair, W. R., Jewett, K. L., and Inverson, W. P., J . Chromatogr. Sci., 1977, 15, Vickrey, T. M., Howell, H. E., Harrison, G. V., and Ramelow, G. J., Anal. Chem., 1980, 52, 1743. Koizumi, H., McLaughlin, R. D., and Hadeishi, T., Anal. Chem., 1979, 51, 387. Vickrey, T. M., Harrison, G. V., Ramelow, G. J., and Carver, J . C., Anal.Lett., 1980, 13, 781. Parks, E. J., Brinckman, F. E., and Blair, W. R., J . Chromatogr., 1979, 185, 563. Van Loon, J. C., Paper presented at 7th Federation of the Analytical Chemistry and Spectroscopy Kahn, N., and Van Loon, J . C., J . Liq. Chromatogr., 1979, 2, 23. Brinckman, F. E., Jewett, F. L., Inverson, W. P., Irgolic, K. J., Ehrhardt, K. C., and Stockton, Stockton, R. A., and Irgolic, K. J . , I n t . J . Environ. Anal. Chem., 1979, 6, 313. Uden, P. C., Quimby, B. D., Barnes, R. M., and Elliott, W. G., Anal. Chim. Acta, 1978, 101, 99. Morita, M., Uehiro, T., and Fuwa, K., Anal. Chem., 1980, 52, 351. Gast, C. H., Kraak, J. C., Poppe, H., and Maessen, F. J . M. J., J . Chromatogr., 1979, 185, 549. Fraely, D. M., Yates, D., and Manahan, J. E., Anal. Chem., 1979, 51, 2225.Beenakker, C. I. M., Spectrochim. Acta, Part B, 1977, 32, 173. Ebdon, L., Cave, M. R., and Mowthorpe, D. J., Anal. Chim. Acta, 1980, 115, 179. Newsl., 1977, 16, 79. 196, 403. Spectroscopy, Melbourne, Australia, August 25-29, 1975. scopy Societies, Philadelphia, September 28-October 3, 1980. 493. Societies, Philadelphia, September 28-October 3, 1980. R. C., J . Chromatogr., 1980, 191, 31. Matrix and Sensitivity Constraints in Trace Organic Analysis J. N. Miller Department of Chemistry, Loughborough University of Technology, Loughborough, Leicestershire, LE11 3T U Organic analysis at nanogram and subnanogram levels is typified by the determination of a drug in blood plasma or of a pollutant in a filtered atmospheric sample. In each instance the analytical problems include the requirement for a sensitive detection technique, the need for extreme selectivity (the samples will contain many chemical species, including some very similar to the analyte in question) and the labile nature of the sample.Further practicalMarch, 1982 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSIS 115 problems may include the need for very rapid results, the processing of large numbers of samples and the use of cheap and straightforward techniques. Relatively few techniques fulfil these requirements : most trace organic analyses are currently carried out using chroma- tographic methods (GLC, HPLC, TLC) with appropriate detection procedures, immunoassays and sensitive spectroscopic methods (fluorescence, chemiluminescence) . In the assessment of the suitability of a method for trace analysis, much confusion remains between the related but distinct parameters sensitivity and lintit of detection. Sensitivity is determined from the slope of a calibration graph, and indicates the least detectable change in concentration that can be detected using an instrument of known signal : noise characteristics.Limit of detection (LOD), on the other hand, defines the lowest concentration or amount of analyte that can be detected at a specified degree of statistical confidence. LOD values can be determined and defined in a variety of ways, and it is essential that the method used should be adequately documented in all publications. However, published LODs should not be taken too literally, for at least two reasons : firstly, the analytical errors may not be normally distri- buted,l so the statistical significance of the LOD definition is uncertain; and secondly, in many methods, the performance characteristics of different instruments vary so widely that an LOD estimate obtained on one instrument will be only marginally relevant in other laboratories.Factors Affecting LOD and Sensitivity In any method, sensitivity and LOD will be determined by three groups of factors, those relating to the sample, those relating to the instrumentation and those relating to the influence of the sample matrix. This distinction was applied by Parker2 to photoluminescence methods, but the principle is of general application (Table I). In fluorescence spectrometry, for example, the intensity of the signal is given approximately by where I f and I , are the fluorescence and incident light intensities, respectively, K is an instru- mental (light-gathering) parameter, + f the fluorescence quantum yield and Ebc is the absorbance (assumed to be very small, less than ca.0.02). It is apparent that the sensitivity of fluorimetry, i.e., the slope of a graph of If 7)e~sus c , is dependent on (a) the properties of the sample (If cc E + f ) and (b) the instrument characteristics (If oc I,Kb). These sets of factors are entirely inde- pendent, i.e., the best fluorimeter will not yield highly sensitive results with a sample of low q 5 f , and a highly absorbing and fluorescent sample will not exhibit high analytical sensitivity on an instrument with low I,bK.In analytical practice, limits of detection in fluorimetry TABLE I EXAMPLES OF FACTORS AFFECTING SENSITIVITY AND LIMIT OF DETECTION IN TRACE ORGANIC ANALYSIS Method Analyte factors UV - visible absorption . . Molar absorptivity Fluorescence. . .. . . Molar absorptivity; quantum yield Chemiluminescence . . . . Quantum yield of CL Gas - liquid chromatography with electron-capture detection . . .. . . Electron affinity Radioimmunoassay . . . . Degree of labelling activity of labelled preparation Instrument factors Sample factors Signal to noise ratio; stability Source intensity; light-gathering power; stability; path length Light-gathering power ; photon counting sensi- tivity Background absorption Background scattering; background fluorescence ; quenching and enhance- ment effects Background CL; quenching and enhance- ment effects Pulse rate in Sample and solvent detector power interference should be supply ; contami- very small ; occasional nation of detector overlapping peaks head Counting efficiency Metabolites may cross- react; otherwise only minor effects116 MATRIX AND SENSITIVITY PROBLEMS I N ANALYSIS Anal.Proc. (especially in methods not including a separation step) are frequently dominated by the third factor, vix., the blank signal, which arises from scattered and stray light, and adventitious fluorescence from sample cells, solvents, other solutes, etc. It frequently happens that a solute that can be determined at pg ml-l levels in pure solution can only be detected at ng ml-l or pg ml-l in “real” sample matrices because of high background signals. In such instances the sample properties are often less crucial than instrumental factors: a simple filter fluori- meter will often give high sensitivities (high I&) with pure solutions of fluorescent solutes, but inferior limits of detection (compared with a spectrofluorimeter) because of larger background signals arising from poor stray light rejection, large spectral band width, etc.Conversely, if fluorimetry is used in conjunction with (for example) HPLC, background interferences will be reduced, and the efficiency of the fluorimetric detector (Io,K) will be more important. It is apparent that this approach to method evaluation is powerful if carefully applied and if LOD and sensitivity are properly distinguished.Approaches to Selectivity It is apparent that selectivity and LOD are closely connected in many methodologies and in several fields advances in selectivity have been at least as dramatic as improvements in sensi- tivity. Three general approaches to increased selectivity are a~ailable,~ vix., the use of select- ive detection systems alone, the combination of separation techniques with general or selective detectors and the use of specific biochemical assays. Sometimes, two of these approaches are employed in tandem, e g . , the use of HPLC with immunoa~says.~ In some analyses absolute selectivity may be unnecessary or undesirable. Thiis a spectroscopic evaluation of an oil spill may not require an identification or quantitation of individual hydrocarbons, merely a spectro- scopic profile that can be compared with other samples using pattern recognition methods.Again, an immunoassay for a drug molecule may use an antiserum that will cross-react with the drug’s major metabolites or with other similar drugs. Completely specific assays are only rarely accessible with methods using a simple spectro- scopic detector ; a preliminary cxtraction, or increased sophistication of the detector (derivative spectroscopy, optical multi-channel analysers) , may improve the situation, but will not eliminate all interferences. In contrast, combinations of chromatography with spectroscopy are generally highly selective. Good chromatographic technique will not only resolve similar molecules, but may also permit the spectroscopic finish to be performed in near ideal conditions, i.e., in effect with a “pure” sample in an ideal solvent.The combination of HPLC with fluorescence (and most recently chemilumine~cence~) detection procedures is a good example of this approach, which yields very low LODs. Immunoassays are of two basic types: homogeneous assays, in which no separation step is required and which are thus easily automated, and heterogeneous ones, where a separation step is necessary to separate antibody-bound from unbound materials. The separation step, while complicating attempts at automation, also serves to eliminate many background interferences when an optical detection procedure is used. Thus, while radioimmunoassays (invariably heterogeneous) and solid-phase enzyme assays ( e g ., ELISA) may be applicable at very low levels (pg ml-1 and below), homogeneous immunoassays have been largely limited to the ng ml-1 range by background effects on measurements of absorbance, fluorescence, etc. Conclusions Future developments in trace organic analysis will ensure lower LODs (more widespread use of immunoassays, chemiluminescence, etc.) and higher selectivity (GLC and HPLC combined with mass spectrometry, video-spectrometers, etc.) Equally important will be improvements in reproducibility-the latter affects LODs and is also particularly important in kinetic methods, e.g., chemiluminescence, bioluminescence. Flow-injection analysis, which is simple yet very precise, may play a major role.6 References 1. 2. 3. Curry, S. H., and Whelpton, R., in Reid, E., Editor, “Blood Drugs and Other Analytical Challenges,” Parker, C.A., “Photoluminescence of Solutions,” Elsevier, Amsterdam, 1968. Miller, J. N., Proc. Anal. Div. Chem. SOC., 1979, 16, 203. Ellis Horwood, Chichester, 1978, pp. 29-41.March, 1982 MATRIX AND SENSITIVITY PROBLEMS I N ANALYSIS 117 Teale, J. D., Clough, J. M., King, L. J., Marks, V., Williams, P. L., and Moffat, A. C., J . Forensic Kobayashi, S., and Imai, K., Anal. Chew., 1980, 52, 424. RiiZCka, J., and Hansen, E. H., “Flow Injection Analysis,” John Wiley, Chichester, 1981. 4. 5. 6. Sci. SOC., 1977, 17, 177. Problems with Food Contaminants and Residues N. T. Crosby Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SEl 9NQ Other papers at this Symposium have emphasised sensitivity problems and detection tech- niques as applied to trace analysis for both inorganic and organic analytes in a variety of sample matrices.This paper describes some specific problem areas that arise from the properties of food as a sample matrix and offers some solutions by illustration from work on the determination of polynuclear aromatic hydrocarbons (PAH), metals, mycotoxins . and synthetic colours in foods. Foods are complex mixtures of water, fats, proteins, carbohydrates, vitamins and inorganic compounds and can also include a wide range of additives and contaminants added during processing. Food composition tables1 provide no real guide to the true complexity of the substrate facing the analytical chemist. For example, Nursten and Williams2 have identified 98 volatile compounds in blackcurrants, including 23 hydrocarbons, 14 carbonyl compounds, 30 alcohols, 8 acids, 22 esters and 1 ether.Furthermore, foodstuffs, being natural products, are subject to compositional variations depending on such factors as the weather, time of harvest and nutritional status and so will vary in composition from sample to sample ostensibly of the same product. Analysis For inorganic compounds the matrix generally has to be totally destroyed, leaving the analytes in solution, whereas in the determination of organic analytes the matrix cannot be so destroyed. Methods of separation of analyte from the matrix have then to be used. Matrix problems, therefore, arise mainly during the first two stages of the analytical method.Stages 1, 3 and 4 are essentially similar for both inorganic and organic analytes. Selection of suitable techniques for the last two stages is determined principally by the chemical and physical properties of the analyte rather than by the nature of the matrix. This discussion will, therefore, be restricted to stages 1 and 2 only. Table I shows the stages in the analysis of foods for inorganic and organic analytes. TABLE I STAGES IN THE ANALYSIS OF FOODS Inorganic analytes 1. Sampling 2. Destruction of organic matter 3. Separation and concentration 4. End determination (detection, identification and confirmation) Organic analytes Separation (or isolation) from the matrix Removal of co-extractives and concentration 1. Sampling 2. 3. (clean-up) 4. End determination (detection, identification and confirmation) Sampling The purpose of sampling is to abstract from a bulk consignment a portion of suitable size for analysis.The portion taken must be representative of the bulk, otherwise the results of the analysis will be of little use however well the determination has been carried out. The mathematical and statistical treatment of this problem has been described elsewhere. I shall comment only on those sampling problems which result from the constitution of foods and their properties as a matrix. Crown Copyright.118 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSIS Anal. PYOC. As foods are such a complex mixture of inorganic and organic components, it would be surprising if they were in any way homogeneous. In practice they are not, and even liquid foods that one would expect to be homogeneous are not always so; an example is milk with cream on top or even worse when it is soured.Flowing solids, e.g., flours, are normally sampled as liquids but care must be taken to avoid stratification effects. The problems of sampling potable waters for the determination of polynuclear aromatic hydrocarbons a t the nanogram per litre level have been demonstrated by Crosby et aL3 Although water is probably the easiest substrate to sample, problems arise a t these very low levels as a result of adsorption on to the glass surface of the container and on to suspended particulate matter in the sample. This difficulty can be partially overcome by the addition of extraction solvent to the sample bottle before filling with the test water so that PAH compounds pass into the solvent before adsorption effects become important. In the analysis of foodstuffs for PAH compounds, some methods require a digestion with alcoholic potassium hydroxide solution to break up the matrix prior to extraction of the analyte in the usual way.Unfortunately, this also releases many other interfering compounds into the extraction solvent. The sampling of solid foods for trace metals has been discussed by C r ~ s b y . ~ With canned fruit and vegetables, a disproportionate concentration of metals into the solid phase by comparison with the syrup (brine) has been observed. Packetted tea is also difficult to sample, as it has been found4 that the metal content found by analysis is dependent on the relative proportions of leaf and dust in the aliquot taken for analysis, the dust containing higher concentrations of lead, iron, zinc and copper than are found in the leaf.Other problems may be encountered in the analysis of fresh fruit and vegetables where the analysis will be influenced by adhering particles of soil or washing processes that may remove surface contaminants. In migration experiments to measure the transfer of a contaminant from a packaging material into food, a concentration profile will be set up between the layer of food in contact with the container and the portion of food at the centre of the container where the concentration will be lowest. For example, in canned meats the lead content is likely to be a maximum in that portion of meat in direct contact with the soldered side seam, The problem of sampling a PVC bottle to make measurements of residual vinyl chloride has also been de~cribed.~ Stage 2 of the, Analysis (Table I) For the determination of inorganic compounds it is usually necessary to destroy the sample matrix either by dry ashing or by wet oxidation.The choice of oxidising acids in the latter process can depend on the food matrix. Carbohydrates can produce a violent reaction and need to be treated cautiously with nitric acid in the initial stages of the reaction. Fatty foods are much more difficult to oxidise and perchloric acid - sulphuric acid mixtures are often needed. Foods containing calcium (milk, cheese) after treatment with sulphuric acid form an insoluble sulphate which can occlude small amounts of lead, which are subsequently very difficult to obtain in solution.Treatment with a strontium salt can overcome this problem. When organic analytes are to be determined, the nature of the substrate is even more critical than when the object is simply total destruction of all organic matter. Fatty foods again cause many problems and are usually defatted prior to the extraction of the analyte. Alternatively, the fat may be hydrolysed using alcoholic potassium hydroxide solution but this may increase the problems of clean-up. It may also be necessary to remove moisture by grinding with anhydrous sodium sulphate. On the other hand, the presence of water enhances the extraction power of chloroform in the analysis of aflatoxins. Chloroform functions poorly on its own, as it does not “wet” the sample. Water, although a poor solvent for aflatoxins, does penetrate into the sample. The small amount of aflatoxin that then passes into the water phase is immediately removed by the chloroform so that the total extraction system then becomes much more efficient.Binding to the Sample Matrix A new analytical method is normally tested by adding a known amount of analyte to the matrix and then assessing the recovery. Obviously this procedure does not mirror the true practical situation where the analyte may be occluded within the matrix or even chemicallyMarch, 1982 MATRIX AND SENSITIVITY PROBLEMS I N ANALYSIS 119 bound to constituents of the matrix. In our work6 on the development of quantitative methods for the determination of water-soluble colours in foodstuffs, low recoveries were obtained from cooked or baked products.This problem was overcome by the use of enzymes to break down the food matrix, thus releasing additional colouring matter into the extraction solvent. The use of enzymes for digestion and breakdown of organic matrices is potentially advantageous in several other areas of analytical chemistry. References 1. 2. 3. 4. 5. 6. Paul, A. A., and Southgate, D. A, T., Editors, “McCance and Widdowson’s The Composition of Nursten, H. E., and Williams, A. A., J . Sci. Food Agric., 1969, 20, 613. Crosby, N. T., Hunt, D. C . , Philp, L. A., and Patel, I., Analyst, 1981, 106, 135. Crosby, N. T., Analyst, 1977, 102, 225. Crosby, N. T., “Food Packaging Materials,” Applied Science, London, 1981, p.52. Boley, N. P., Bunton, N. G., Crosby, N. T., Johnson, A. E., Roper, P., and Somers, L.. Analyst, Foods,” Fourth Edition, MRC Special Report No. 297, HM Stationery Office, London, 1978. 1980, 105, 589. Trace Organic Determinations in Water, Including the Influence of Some EEC Directives c. c. Scott Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SE1 9NQ Analytical work required on waters has become, in many instances, increasingly demanding in recent years as a result of increasing concern with environmental and health effects on water quality. The influence of this on trace organic determinations in waters in the UK was looked at from the viewpoint of a government chemical laboratory.In the Laboratory of the Government Chemist, work arises in this subject area both directly as a result of EEC Directive provisions and indirectly from Water Authority requirements and the desire of the government generally to behave responsibly in environmental matters. EEC Directives Agreed and draft Directives of significance to the Water Industry were described as well as the background to their development as part of the environmental programme. In particu- lar, requirements for the analysis of organic compounds were considered in some detail. These can be very demanding analytically owing to the low concentrations for which analysis is required (for example, phenols, hydrocarbons and chloroform-extractable material in the potable and raw surface water Directives).There can also be problems due to the ill-defined nature of the parameter heading. For example, “other organochlorine compounds” not covered by the pesticides parameter is of questionable meaning. The “other organochlorine compound” heading has been used to draw attention to the advisability of minimising halo- form concentrations in potable water, but the guide level figure of 1 pg 1-1 is possibly un- realistically low. However, generally in the UK a more flexible approach is favoured as different laboratories may be able to achieve their best analytical performances by different techniques. This UK viewpoint has been recognised by the inclusion of appropriate clauses in some of the Water Directives. There is, however, a need for empirical parameters, such as COD determinations, to have their analytical procedures defined in sufficient detail to allow different laboratories to obtain comparable results.Traditionally the EEC has encouraged the use of mandatory methodology. Phenol Analyses One problem considered in some detail was the analysis of phenolic compounds in water in terms of either the 4-aminoantipyrine or the p-nitroaniline spectrophotometric methods. Such Crown Copyright.120 MATRIX AND SENSITIVITY PROBLEMS IN ANALYSIS Anal. Proc. phenol index measurements fall into the category of empirical parameters, as both the specific phenolic compounds present and the exact experimental procedure followed greatly affect the value obtained. For example, the 4-aminoantipyrine procedure at pH 10 has been found to be a relatively fast, precise and accurate procedure for this purpose, but if one looks at the re- sponses of different phenols in this instance one finds huge differences, with say 2,4-dichloro- phenol responding to less than one third the extent of phenol and 4-cresol not responding at all.The position is further complicated by the normal use of a distillation clean-up stage, which, although removing many interferences, unfortunately limits the method to the determination of steam-volatile phenols. The absolute and relative responses of different phenols vary with the pH of colorimetric reaction; at pH 7.9, for example, j5ara-substituted phenols will give a stronger response than at pH 10. The arbitrariness of phenol index measurements for the control of phenolic compounds becomes apparent when one looks at threshold odour values for these compounds.Phenol itself has a threshold odour concentration at 30 "C of about 10 mg 1-1 compared with a corresponding value of 0.65 pg 1-1 for 2,4-dichlorophenol. Assum- ing that taste and odour control is a major reason for the EEC maximum admissible concentra- tion limit of 0.5pgl-1 in potable water, the relatively lower response of many substituted phenols in the 4-aminoantipyrine phenol index measurement is unfortunate. However, provided that its limitations are appreciated, such measurements can prove a useful administra- tive procedure. We have been looking at an alternative line of approach involving the gas - liquid chroma- tographic determination of specific phenols.To achieve the required sensitivity and resolution we have studied varying conditions of derivatisation and chromatography with electron- capture detection. Pentafluorobenzyl ether derivatives finally extracted into a small volume of isooctane were found to give adequate sensitivity, but interferent effects, requiring careful reagent clean-up and control of experimental conditions, become increasingly important as one analyses at concentrations lower than 1 pg 1-l. We find that the use of capillary glass columns reduces such problems. Environmental Problems Some of the analytical work undertaken by the Laboratory of the Government Chemist to assist in environmental control was discussed. Problems of interpretation of results can sometimes arise from such work. Con- centrations and composition of hydrocarbons at and near the spillage point have been moni- tored over some years in purpose-drilled test boreholes. Near the spillage point a layer of fuel was lying on top of the aquifer and it was now found to be relatively low in concentration of the lower relative molecular mass alkanes. Greater volatility of lower relative molecular mass alkanes in the fuel lying in the boreholes may well have been an important mechanismof change here. On the other hand, in a more distant test borehole slight traces of oil were detected in the water. The capillary column gas-chromatographic traces of this material showed that the composition was substantially different to that of the original aviation fuel, and this was con- firmed by mass spectrometric analysis, which showed that the oil consisted primarily of various aromatic hydrocarbons and not alkanes as in the original fuel. There are two possible explanations: (1) the water contamination is due to the original spillage, but the alkanes have been preferentially biodegraded compared with the more resistant aromatic material, and/or the aromatic hydrocarbons have been more rapidly transported through the environmental strata; or (2) the slight contamination in this area is due to a completely separate spillage of a different type of oil in a more localised unrecorded incident. Problems to be considered in carrying out meaningful biodegradation tests that will allow realistic extrapolation to behaviour in the environment were also considered briefly. The requirements of tests to be reproducible and relate effectively to environmental responses can often seem mutually antagonistic. Obviously this is a very important area of concern throughout the world, and organisations such as OECD are actively considering test criteria. One of the problems described was that arising from the spillage of an aviation fuel.

ISSN:0144-557X    

DOI:10.1039/AP9821900110

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

March, 1982 HPLC IN PHARMACEUTICAL ANALYSIS 121 H PLC in Pharmaceutical Analysis The following are summaries of the eight papers presented at a Meeting of the Joint Pharma- ceutical Analysis Group held on May 19th, 1981, at the Pharmaceutical Society of Great Britain , London. M icro-column High- perf orrnance Liquid Chromatography Steven A. Westwood, David E. Games and Michael S. Lant Department of Chemistry, University College, P.O. Box 78, Cardin, CFI 1XL and Brian J. Woodhall I C I Pharmaceutical Division, Hurdsfield Industrial Estate, Macclesfield, Cheshire, SKlO 2NA The use of packedl-4 and open-tubular4-10 capillary and packed microborell-16 columns for high-performance liquid chromatography (LC) offer a number of important advantages over conventional columns. Currently, the use of packed microbore columns (0.5-1 mm i.d.) is the easiest and most effective approach to adopt.The main advantages of microbore columns are the considerable reduction in running costs that can be achieved by the use of low solvent flow-rates and smaller amounts of column packing materials, the high efficiencies and mass sensitivities that can be achieved and the fact that the low flow-rates used make the coupling of the liquid chromatograph with a mass spectrometer (LC - MS) a much easier proposition. The technique is not without difficulties, which include the necessity of having a low dead volume injector and detector and low time constant detector, the difficulties of packing high-efficiency columns and problems encountered when too large a sample is injected on to the column, which results in column overload and loss of efficiency. Microbore LC has been used by a number of groups for LC - MS studies, mainly with systems of the direct liquid introduction t~pe,l'-~O as the use of low flow-rates enables all the eluate to be fed into the mass spectrometer ion source, resulting in considerably enhanced sensitivities.Similar advantages accrue with the interfaces of the jet21 and vacuum nebulising type.22 Although initial studies with the moving-belt interface utilised a microbore LC system,23 few other reports24 of its utilisation with this type of interface have appeared. With one exception,lg little attention was paid in these studies to optimisation of LC per- f ormance. Our interest in microbore LC originated from our studies of combined LC - MS with a moving-belt interface and we report here some of our studies with a commercial JASCO microbore LC system.Experimental A JASCO Familic-100N micro-LC equipped with a 500-pl gas-tight microsyringe pump and a variable-wavelength ultraviolet detector (UVIDEC 100-11) with a 0.3-pl quartz micro- cell was used. Microbore LC columns were made of PTFE tubing (0.5 mm i.d.) and packed using a Waters M6000 pump that had been modified with a Hoke pressure relief valve to enable a constant pressure of 1000 lb in-2 to be maintained. For combined LC - MS studies the LC column was connected directly to the moving-belt LC - MS interface of a Finnigan 4000 mass spectrometer, which was operated in the electron impact (source temperature 260 "C) or the chemical ionisation mode (source temperature 250 "C).Data were acquired and processed using an Incos 2300 data system. Results and Discussion Initial studies25 with the JASCO microbore LC system were disappointing in that efficiencies of the order of only 1000 theoretical plates were achieved with 250 x 0.5 mm i d . columns packed with 5-pm Hypersil ODs. Modification of the recommended packing technique by use of a Waters M6000 pump, which had a pressure relief valve, enabled us to maintain a constant pressure of 1000 lb in-2 during column packing and provided more efficient columns, Further improvements were obtained by modification of the coupling of the quartz microcell in the ultraviolet detector to reduce dead volumes. Typical column performances for a122 HPLC IN PHARMACEUTICAL ANALYSIS Anal.PYOC. 300 x 0.5 mm i.d. Hypersil ODS 5-pm column with a flow-rate of 10 p1 min-1 of acetonitrile - water (60 + 40), injecting 0.1 p1 of a solution containing naphthalene and biphenyl] were 6 805 and 7 999 theoretical plates, respectively, for the two components; using acetonitrile - water (40 + 60) these values were increased to 13574 and 12830, respectively, with increased capacity ratios. One advantage of the moving-belt system for LC - MS is that the microbore LC column can be connected such that it feeds its eluent directly on to the belt, thus ensuring a minimum dead volume connection; thus, we were able to obtain data of similar quality to those obtained using the ultraviolet detector, e.g., injection of 0.1 p1 of a solution of naphthalene and biphenyl on to a 300 x 0.5 mm i.d.Hypersil 5-pm column with a flow-rate of 5 p1 min-l of acetonitrile - water (70: 30) gave 7449 and 8254 theoretical plates for naphthalene and biphenyl, respectively. Studies with a variety of samples indicate that column overload begins to occur when samples of the order of 5 pg are injected on to the microbore column, resulting in loss of efficiency. This will result in problems when, e.g., a minor component is present with a large excess of other components. In order to overcome this problem we have developed a manual column switching technique. Our studies have utilised a mixture of naphthalene and biphenyl where naphthalene is present in 200-fold excess. Injection of a solution con- taining 19pg of naphthalene results in an inability to resolve the biphenyl fully from the naphthalene using an ultraviolet detector.In order to recover the resolution the flow through the LC is stopped after the bulk of naphthalene has been eluted. The column is connected to a second, longer column and the LC pump is re-started and run until the area of the chromatogram of interest has been eluted on to the second column. Pumping is then stopped again, the second column is connected to the pump and pumping is recommenced with the same or a different eluting system. This results in a chromatogram in which the two components are well resolved. Having developed this technique with a model system, we are currently studying its use for resolving minor impurities in pharmaceutical products.One major limitation of the moving-belt LC - MS interface is the difficulties experienced with aqueous solvent systems. For systems containing 10-50y0 of water only a portion of the eluate from the LC can be handled if conventional columns are used, resulting in reduced sensitivity. For larger amounts of water, beading occurs on the belt, resulting in pressure fluctuation in the ion source. A number of solutions to the problem have been described] including addition of a second water-miscible phase, e.g. propan-2-01,~~ the use of turbo- molecular pumps, together with heaters placed in each of the differentially pumped chamber^,^' and use of a segmented flow extractor.= The use of microbore LC offers an alternative solution.We have found that when the larger amounts of water present in the eluting system are used, all of the LC eluate can be handled, resulting in improved sensitivity, For higher proportions of water a miscible solvent is fed on to the belt before the LC column eluate. We have found ethanol at a flow-rate of 0.2 ml min-l to be most effective. Using this approach we have obtained excellent LC - MS data using acetonitrile - water (20 + 80) for the separation of a mixture of the phenols Ia and Ib. Similar results have been obtained by LC - MS. CHZCOR I la R = OH 8 IbR=NH2 In conclusion, we feel that microbore column LC is a useful technique, particularly when used in combination with LC - MS, where it enables considerable improvements to be obtained in terms of improved sensitivity and ability to handle aqueous solvent systems.We feel that we are unlikely to obtain much more improvement in LC efficiency with our JASCO system as it is a low-pressure system and uses flexible columns. Recent studies by Scott and c o - w o r k e r ~ ~ ~ - ~ ~ have demonstrated extremely high efficiencies with microbore columns, which will be necessary for the solution of certain types of problems and for further improve- ments in LC - MS sensitivity, and we hope to study this approach in the near future.March, 1982 HPLC I N PHARMACEUTICAL ANALYSIS 123 M.S.L. and S.A.W. thank the SRC and ARC, respectively, for financial support, and M.S.L. thanks ICI Pharmaceutical Division for additional financial support. We are indebted to the Royal Society for funds for the purchase of the JASCO Microbore LC system and to the SRC for funds for the purchase of mass spectral equipment.1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. References Tsuda, T., and Novotny, M., Anal. Chem., 1978, 50, 271. Hirata, Y., Novotny, M., Tsuda, T., and Ishii, D., Anal. Chem., 1979, 51, 1807. Hirata, Y., and Novotny, M., J . Chromatogr., 1979, 186, 521. Novotny, M., J . Chromatogr. Sci., 1980, 18, 473. Tsuda, T., Hibi, K., Nakanishi, T., Takeuchi, T., and Ishii, D., J . Chromatogr., 1978, 158, 227. Tsuda, T., and Novotny, M., Anal. Chem., 1978, 50, 632. Hibi, K., Ishii, D., Fujishima, I., Takeuchi, T., and Nakanishi, T., J . High Resolut. Chromatogr. Ishii, D., Tsuda, T., and Takeuchi, T., J .Chromatogr., 1979, 185, 73. Ishii, D., Tsuda, T., Hibi, K., Takeuchi, T., and Nakanishi, T., J . High Resolut. Chromatogr. Chro- Hibi, K., and Ishii, D., J . Chromatogr., 1980, 189, 179. Scott, R. P. W., and Kucera, P., J . Chromatogr., 1976, 125, 251. Scott, R. I-’. W., and Kucera, P., J . Chromatogr., 1979, 169, 51; 1979, 185, 27. Scott, R. P. W., Kucera, P., and Munroe, M., J . Chromatogr., 1979, 186, 475. Scott, R. P. W., J . Chromatogr. Sci., 1980, 18, 49. Reese, C. E., and Scott, R. P. W., J . Chromatogr. Sci., 1980, 18, 479. Ishii, D., Asai, K., Hibi, K., Jonkuchi, T., and Nagaya, M., J . Chrornatogr., 1977, 144, 157. Arpino, P. J., and Guiochon, G., Anal. Chem., 1979, 51, 682A. Henion, J . D., and Maylin, G. A., Biomed. Mass Spectrom., 1980, 7, 115.Brophy, J. J., Nelson, D., and Withers, M. K., I n t . J . Mass Spectrom. Ion Phys., 1980, 36, 205. Schafer, K. H., and Levsen, K., J . Chromatogr., 1981, 206, 245. Takeuchi, T., Hirata, Y., and Okumara, Y., Anal. Chem., 1978, 50, 659. Tsuge, S., Hirata, Y., and Takeuchi, T., Anal. Chem., 1979, 51, 166. Yoshida, Y., Yoshida, H., Tsuge, S., Takeuchi, T., and Mochizuki, K,, J . High Resolut. Chromatogr. McFadden, W. H., Schwartz, H. L., and Evans, S., J . Chromatogr., 1976, 122, 389. Games, D. E., Hirter, P., Kuhnz, W., Lewis, E., Weerasinghe, N. C. A., and Westwood, S. A., J . Wright, L. H., and Edgerton, T. R., “27th Annual Conference on Mass Spectrometry and Allied Dymerski, P. P., “28th Annual Conference on Mass Spectrometry and Allied Topics, New York,” Karger, B.L., Kirby, D. P., Vouros, Y., Foltz, R. L., and Hidy, B., Anal. Chem., 1979, 51, 2324. Chromatogr. Commun., 1978, 1, 21. matogr. Commun., 1979, 2, 371. Chromatogr. Commun., 1980, 3, 16. Chromatogr., 1981, 203, 131. Topics, Seattle,” American Society for Mass Spectrometry, 1979, p. 742. American Society for Mass Spectrometry, 1980, p. 624. High-performance Liquid Chromatography of Aminopyridines D. K. Scott and W. J. Irwin Pharmacy Department, East Birmingham Hospital, Bordesley Green East, Birmingham 9 Several simple monoaminopyridines and diaminopyridines are of physiological or medical interest,l-3 and some have also found uses as analytical standards4 or derivatisation reagent^.^ 4-Aminopyridine (4-AP) affects the neuromuscular junction by promoting the release of acetylcholine and was used to treat patients in the Birmingham botulism outbreak in 1978.The rapid development of a high-performance liquid chromatographic (HPLC) assay for use on serum samples collected during this treatment has been described.6 The assay was a reversed-phase ion-pair technique using an octadecylsilane column, ultraviolet detection at 268 nm and a mobile phase of 1% V/V orthophosphoric acid, 0.015 M sodium dodecylsulphate and 40% acetonitrile in water. Later, investigations were conducted into the improvement of this assay and into the behaviour of 4-AP and other aminopyridines (denoted n-AP or n,m-DAP, where TZ and m represent the substitution pattern). Much of the chromatographic behaviour was readily predictable ; methanol increased retention times compared with acetonitrile whereas higher concentrations of either decreased retention times.A decrease in the chain length of the anionic agent at equimolar concentrations (sodium pentanesulphonate, sodium hexane- sulphonate) or its concentration also reduced the retention times of the aminopyridines. In124 HPLC I N PHARMACEUTICAL ANALYSIS Anal. PIOC. contrast, the effect of acid in the mobile phase had greatly differing effects on the various aminopyridines. In non-acidic mobile phases, of varying composition but always of pH >7.0, the retention times of 3,4-DAP and 4-AP were markedly greater than those of 2-AP, 3-AP, 2,3-DAP and 2,6-DAP. In acidic mobile phases (pH 1.8-3.5, orthophosphoric or acetic acid) the retention times of the two groups were similar but the order of elution amongst the second group had changed.These results may be related to the significant differences in the basicities of the aminopyridines under investigation. Retention behaviour in this system is controlled by ion pairing between the protonated solute and the counter anion in the mobile phase. For partially ionised compounds, an increase in the extent of protonation is followed by an increase in the capacity factor.' The order of elution of the series of aminopyridines in non-acidic mobile phases may be explained by considering the extent of ionisation as indicated by the PKa values. Table I gives pK, values for these compounds and Table I1 compares the rank orders of pK, and elution. There is a positive correlation ( I = 0.89, p (0.05 by single-sided Spearman rank correlation) between pK, and retention distance, but this is not found for the acidic eluents (r = -0.83, p >0.05).In non-acidic conditions only 4-AP and 3,4-DAP are extensively ionised, hence the correlation of pK, and retention, but in acidic conditions all solutes are almost completely ionised. TABLE I PHYSICAL PROPERTIES OF AMINOPYRIDINES pKa values are taken from the literature for aqueous solutions. Values in 30% acetonitrile were found to be similar and of the same rank order. A,,,. values were measured in a typical eluent [0.5% sodium dodecylsulphate - orthophosphoric acid - acetonitrile (59 + 1 + 40)] and are the same rank order as in aqueous solution. Compound Amax./nm pKa (25 "C) 2-AP .... . . 302 6.71 3-AP .. .. .. 323 6.01 4-AP .. .. .. 268 9.11 2,3-DAP . . .. .. 322 6.73 2,6-DAP . . .. .. 335 6.00 3,4-DAP . . .. .. 293 9.19 Under acidic conditions, the formation of an ion pair, and hence the retention of the solute by any of the commonly proposed mechanisms,' will depend not on the ionisation of the compound but on the localisation of the charge into an effective binding unit that will match the sulphate or sulphonate grouping of the anion. A readily measurable physical property that may reveal this behaviour is the wavelength of maximum ultraviolet - visible absorption (Amax.), which increases as electrons (especially n-bonding electrons) become more delocalised. Table I shows the longest wavelength ultraviolet absorption maxima of these compounds in aqueous solution and Table I1 compares the rank order with that of-retention time.Again, a strong correlation is seen ( I = 0.94, p <0.01) that is not seen with non-acidic retention (r = -0.71, p >0.05). Although such phenomena may be useful only for a small series of compounds, the use of such readily available parameters as pKa and A,,,. may assist method development and may provide information on the nature of the retention processes. The optimum HPLC method for analysis of biological samples was found to involve an eluent of 0.6% sodium dodecylsulphate and 40% acetonitrile in water with extraction by TABLE I1 RANK ORDERS OF AMINOPYRIDINE PROPERTIES 2-AP 3-AP 4-AP 2,3-DAP 2,6-DAP 3,4-DAP L a x . - - .. .. .. 4 3 6 2 1 5 PK, - . .. .. .. 4 5 2 3 6 1 Acidic retention .... 4 3 5 2 1 6 Non-acidic retention . . .. 4 6 1 3 5 2March, 1982 HPLC IN PHARMACEUTICAL ANALYSIS 125 means of an equal volume of acetonitrile added to a volume of serum or urine, prior to mixing and centrifugation. This extraction method was 98.4% efficient (coefficient of variation = 0.57%) and the minimum detectable level (10-pm particle columns, 100-pl sample loop) was 5 ng per millilitre of serum. There was no interference from a wide range of serum constituents and drugs. References 1. 2. 3. 4. 5. 6. 7. Ball, A. P., Hopkinson, R. B., and Farrell, I. D., Q. J . Med., 1979, 48, 473. Bowman, W. C., Rodger, I. W., and Savage, A. O., Proc. Br. Pharmacol. Soc., 1979, 50. Baeyens, W., and De Moerloose, P., Anal. Chim. A d a , 1979, 107, 291.Bates, R. G., and Hetzer, H. B., J . Res. Nut. Bur. Stand., 1960, 64A, 427. Dombrowski, L. J., and Pratt, E. L., Anal. Chem., 1971, 43, 1042. Scott, D. K., Proc. Anal. Div. Chem. SOC., 1979, 16, 322. Knox, J . H., Editor, “High-performance Liquid Chromatography,” Edinburgh University Press, Edinburgh, 1978. Approaches to the Assay of Trientine Dihydrochloride Mrs. E. S. Zai Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SEl 9NQ Trientine dihydrochloride (BAN) is used in the treatment of Wilson’s disease, a rare condition in which patients are unable to metabolise copper because of an enzymic deficiency. The copper, a known enzymic poison, accumulates in the vital organs, leading to debilitation and death. Treatment is based on the administration of a drug to chelate with copper and facilitate its removal from the body.The drug of choice since 1956 has been D-penicillamine, but intolerance of this by a significant number of patients has led to the evaluation of trientine dihydrochloride for the same purpose. Trientine dihydrochloride, often conveniently referred to as TETA, is manufactured from ethyleneimine by polymerisation. The dihydrochloride is prepared from the base under controlled conditions to prevent variable hydrochloride and water content. Early work was carried out on a laboratory scale but the drug is now prepared commercially under a Clinical Trial certificate. Impurities are found both as products of manufacture and from degradation. These are mainly lower polymers of ethyleneimine and substituted piperazines formed by ring closure.They are known to be undesirable and the levels in the drug are controlled. The current control assay is based on spectrophotometric measurement of the blue copper complex formed with trientine. It is non-specific because the impurities also complex with copper, and the impurities are assessed by a two-stage thin-layer chromatography system. The first stage, using a silica gel plate and a four-phase solvent system, resolves all of the known impurities with the exception of diethylenetriamine, which is resolved in the second stage using a Kieselguhr plate. Clearly, a direct, specific, stability-indicating assay for trientine dihydrochloride would be preferable. Gas chromatography using an alkaline column can be used to resolve trientine from most impurities and provides a most satisfactory means of production quality control.However, the high temperatures required, over 200 “C, and the thermal lability of trientine can cause problems with respect to on-column degradation. Thus, a method was sought that would offer the resolving power of modern chromato- graphic techniques without the potential of thermal degradation. High-performance liquid chromatography (HPLC) offers these advantages but is difficult to apply directly to trientine, for two reasons. Firstly, the polyamine structure is very polar and does not elute from conventional HPLC systems, and secondly, it possesses no absorbance at accessible ultra- violet detection wavelengths. Both of these difficulties can be overcome by derivatisation.Pre-column derivatisation was chosen because it is less complex in terms of equipment than post-column derivatisation and also because the reaction conditions can be controlled more readily. This laboratory has investigated a range of derivatising agents, of which rn-toluoyl chloride offers a number of discrete advantages. This reagent was selected for further Crown Copyright126 HPLC I N PHARMACEUTICAL ANALYSIS Anal. Proc. investigation because its reaction with trientine is relatively rapid, clean, and can be stopped by the addition of ammonia. This stop mechanism offers an easy means of controlling the extent of derivatisation and yields a simple amide derivative of the m-toluoyl chloride that is easily resolved chromatographically from the trientine derivative.The derivatisation, if taken to completion, yields two derivatives of trientine but with control of the reaction conditions the reaction may be stopped after only one derivative has been formed. Sufficient derivative was prepared and purified by preparative HPLC to permit examination by NMR spectroscopy. The derivative is tetra-substituted with addition of a single m-toluoyl group at each amine centre. A linear response with respect to injection concentration has been demonstrated. Further work is planned to evaluate the system with respect to the known impurities of commercial trientine dihydrochloride. 7c - 7t Charge Transfer Interactions in High-performance Liquid - Solid Chromatography J. J. Burger and E.Tomlinson" Subfaculty of Pharmacy, University of Amsterdam, Plantage Muidergracht 24, 1018 T V Amsterdam, The Netherlands Retention in chromatography is controlled by the physico-chemistry of both the solute and the phase system. Increasingly, it is being recognised that in high-performance liquid - solid chromatography (HPLSC) descriptions of retention in terms of classical theories of adsorption and partition are somewhat naive, and that cognisance should be given to both the presence of secondary equilibria1 and to the fact that the intimate contact of both stationary and mobile phases precludes a general approach based on bulk phase properties. Although much recent attention has been given to ion-pairing2 and ion-exchange3 effects in LC, the possibilities of utilising charge-transfer phenomena have been little explored.4~5 The purpose of this contribution is to present some aspects of our recent work into this aspect of LC, and to demonstrate that this approach can be used not only for resolution purposes but also to provide a rapid method for obtaining measures of charge-transfer characteristics of organic solutes.Experimental To study charge-transfer effects we have attempted to design a very simple HPLC system by dynamically coating a strong n-electron acceptor (tetracyanoethylene, TCE) on to the surface of silica gel particles of 10 pm size (Merck SI 60). Using as eluents mixtures of hexane and 1,Z-dichloroethane of various composition (having a fixed water content of 0.004~0), the retentions of a series of n-electron-donating compounds (methyl-substituted benzenes and polyaromatics) have been determined a t various temperatures in columns loaded and non-loaded with TCE.2,2,4-Trimethylpentane was used as the non-retained marker. Detection was effected with a variable-wavelength ultraviolet - visible spectro- photometer, Results and Discussion From Fig. 1 it can be seen that (2) using a non-loaded column the donor solutes gave no signal at their specific charge-transfer complex wavelength (ACT), (ii) such a signal is given using TCE-loaded columns, (iii) retention of the charge-transfer complex peak is the same as the signal given at the wavelength of the solute (in its native form) on the loaded column, and (iv) the solute has a longer retention on a loaded column than is found in a non-loaded arrangement.To define these effects quantitatively we have attempted to study the effect of column loading on both peak height and retention times. * To whom correspondence should be addressed.127 March, 1982 HPLC IN PHARMACEUTICAL ANALYSIS Peak Height obtained using the Benesi - Hildebrand equation : Using only spectroscopy the charge-transfer complex association constant, KCT, can be where A is the absorbance, cCT the molar absorptivity of the complex and the brackets indicate molar concentrations. Thus, a plot of the ordinate term versus the reciprocal of the donor concentrations gives KCT from the ratio of the slope and intercept terms. After determining the relationship between chromatographic peak height and concentration of the charge-transfer complex, we have constructed modified Benesi - Hildebrand plots [equation (l)] based on peak-height data, and used the slope and intercept data to give KcT values.Comparison of these values with values obtained by us using only spectroscopy shows that this approach is often erroneous. For example, using spectroscopy the KcT values for pentamethylbenzene - TCE and hexamethylbenzene - TCE complexes in dichloro- methane at 22 "C are 123 and 263, respectively, although using chromatography (dichloro- ethane- hexane eluent) they are 920 and 347, respectively. Numerically, we would not expect the same values (slightly different environments and the modified Benesi - Hildebrand approach used), but the ratios of the KcT values should be similar.Examination of the residuals of the regression analysis used to obtain K,, from the peak-height 'data reveals that as the intercept term approaches zero there is a very large error in its determination and hence in obtaining K,, chromatographically. Although we are examining this effect further we present the following section on the use of peak retention data to show how the problem can be circumvented. ACT -*k- / Loaded Loaded Non-loaded 254nm t I / / f ,/ / ACT 7 / / Non-loaded / 254nm 9 Inj / / / / Retention time Fig. 1. Effect of TCE loading on the retention of hexa- methylbenzene in a straight-phase HPLSC system, using detection at 254 and 527 nm. Peak Retention Fig. 1 reveals that both donor and complex have the same retention time, and therefore using either 254nm or ACT detection we can use these altered retention times to give a measure of complexation.Introducing A C T , where .. .. . * (2) ACT = (k* - k ) .. k* and k being donor solute capacity factors in TCE-loaded and non-loaded columns, respec- tively, it can be seen that A C T is a measure of the effect of loading the column with TCE. Fig. 2 shows how there is a direct linear relationship between A C T and literature6 K C T values determined in various solvent systems. Remarkably, for similar environments the ACT ratios and K,, ratios of solutes are similar. For example, the ratio of 2.6 for hexamethyl- benzene to pentamethylbenzene obtained from the chromatographic data accords with a value of 2.1 from the literature K,, values.128 HPLC I N PHARMACEUTICAL ANALYSIS Anal.Proc. 7 6 5 z t 4 < 3 2 1 0 I- A / 5 10 15 KCT Fig. 2. Relationship between chromatographic ACT terms (equation 2) and literature KCT values6 for a series of organic n-electron donating compounds, using KCT values obtained in (A) methylene chloride, (B) chloroform and (C) carbon tetrachloride. Conclusion Dynamically coating silica with a v-electron acceptor produces changes in the retention times of r-electron donor compounds, and these changes can be related to literature charge- transfer complexation constants. Although much more work needs to be carried out on the mechanism of the effect, it does seem at this stage that parameters describing the charge- transfer complexation behaviour of, for example, drug molecules, can be obtained using HPLC and that the effect could be used in altering the selectivity of HPLC phase systems.For example, using non-loaded columns the retention order pentamethylbenzene < naph- thalene < hexamethylbenzene < biphenyl is found, which changes using TCE-loaded columns to naphthalene < pentamethylbenzene < biphenyl < hexamethylbenzene. References 1. 2. 3. 4. 5. 6. Karger, B. L., LePage, J. N., and Tanaka, N., in Cs. HorvQth, Editor, “High-Performance Liquid Chromatography,” Academic Press, New York, 1980, Volume 1, pp. 113-206. Tomlinson, E., Jefferies, T. M., and Riley, C. M., J . Chromatogr., 1978, 159, 315. Terweij-Groen, C. P., and Kraak, J. C., J . Chromatogr., 1977, 138, 245. Porath, J., J . Chromatogr., 1978, 159, 13. Nondek, L., MinArik, M., and MQlek, J., J .Chromatogr., 1979, 178, 427. Briegleb, G., “Elektronen-donator-acceptor-komplexe,” Springer-Verlag, Berlin, 196 1. A Stability Study of Aqueous Solutions of Diamorphine and Morphine Using H ig h -perf ormance Liquid Chromatography I. M. Beaumont Pharmacy Department, East Birmingham Hospital, Bordesley Green East, Birmingham 9 The aim of this work was to determine the stability of diamorphine hydrochloride and morphine hydrochloride in chloroform - water solutions, used orally in the treatment of pain in advanced ~ancer,l-~ and to compare the results obtained with the stability of more traditional mixtures containing alcohol and syrup. In addition, it was proposed to investi- gate alternative simple formulations incorporating buffering agents designed to give a preparation with the maximum shelf-life.Investigation of the stability of drugs requires an assay method that is specific and free of interference from the degradation products. The BP 1980 assay for diamorphine hydro- chloride is based on the degradation of diamorphine to morphine by heating the solution with acid, followed by spectrophotometric determination of the morphine. Thin-layer chromato-March, 1982 HPLC I N PHARMACEUTICAL ANALYSIS 129 graphy (TLC)6 has clearly indicated that breakdown by hydrolysis of diamorphine is a deacetylation process , first to 6-monoacetylmorphine and finally to morphine. The BP assay, therefore, will not detect whether this decomposition has occurred. Quantitative TLC has been attempted,’ but the accuracy of the results is questionable after elution of the separated components from the plates followed by photometric determination.Gas - liquid chromato- graphy has also been used for the quantitation of narcoticsJ6J+l0 but suffers from the limita- tions of thermal degradation and time-consuming derivatisation reactions. The method developed in our laboratory utilises an ion-pairing agent in conjunction with reversed-phase high-performance liquid chromatography (HPLC) . The method separates and quantifies the three compounds of interest : diamorphine, 6-monoacetylmorphine and morphine, with speed and accuracy, and allows analysis of the sample solutions without prior extraction or dilution being necessary. Experimental Chromatography A Pye Unicam LC3 chromatograph coupled with a Bryans 28000 chart recorder was used with the following parameters: column, Hypersil ODS, 10 cm (5-pm particle size) ; mobile phase, 0.01 M sodium pentanesulphonate - acetonitrile - orthophosphoric acid (69.5 + 30.0 + 0.5%) (pH 2.0); detector, 284 nm; sensitivity, 0.32 a.u.f.s.; flow-rate, 2.0 ml min-l; tempera- ture, ambient ; and injection volume, 20 pl.Sodium pentanesulphonate and acetonitrile were both of HPLC grade, supplied by Fisons. Orthophosphoric acid was of AnalaR grade, supplied by BDH Chemicals. The mobile phase was prepared freshly each day and degassed by vacuum for 10 min before use. Diamorphine hydrochloride and morphine hydrochloride were purchased from Macfarlane Smith, and 6-monoacetylmorphine was kindly supplied by the Laboratory of the Government Chemist.Fig. 1 shows the chromatogram produced by a mixture of the three standard materials. It can be seen that the total analytical time is rapid, the retention times being 37, 58 and 11 1 s for morphine, 6-monoacetylmorphine and diamorphine, respectively. The resolution is good, with an R, value of 2.79 between 6-monoacetylmorphine and diamorphine, and a capacity factor of 3.11. The results were calculated by measurement of peak heights and comparison with a calibra- tion graph produced daily using fresh standard solutions. No internal standard was used, as the reproducibility of the Rheodyne 7125 injection valve system was found to be excellent, giving a coefficient of variation between 20 repeated injections of the same standard of only 0.78%.Linearity of the calibration graphs was very good in the range studied. Stability Study In order to study the effect of pH and buffer strength on stability, a range of buffer solutions at pH 2.2-8.0 were prepared using the well known McIlvaine’s citric acid - sodium phosphate buffer system, in varying concentrations of buffer, and added to a diamorphine hydrochloride solution in double-strength chloroform - water. This gave a range of final solutions consisting of 1 mg ml-l diamorphine hydrochloride and single-strength chloroform - water, in a buffer of known ionic strength and pH. Each solution was divided into two parts, one being stored in a refrigerator and the other at room temperature. The solutions were assayed daily to determine the rate of hydrolysis of the diamorphine to 6-monoacetyl- morphine and ultimately morphine.Three replicate runs were performed, and all results are the means of these three results. This work was repeated using morphine hydrochloride instead of diamorphine hydro- chloride, and also using simple solutions of the analgesics in unbuffered chloroform - water (which has a pH of around 6.5) and two “traditional” mixtures, Diamorphine Elixir BPC and our own Brompton Mixture EBH (which consists of 1 mg ml-l diamorphine hydro- chloride in equal parts of brandy and syrup). Results and Discussion Fig. 2 shows the decay curve produced for 1 mg ml-l diamorphine hydrochloride in 0.5 M McIlvaine’s buffer at pH 8.0. The decay pattern can be seen to be that of a first-order breakdown process, and results for all solutions give similar curves, all giving excellent130 Anal.Proc. HPLC I N PHARMACEUTICAL ANALYSIS 4 IL 0 1 2 Time/m in Fig. 1. A typical chromatogram : A, morphine; B, 6-mono- acetylmorphine ; and C, diamorphine. 2.5 2.0 r E 2 1.5 2 0 .- + 4- 8 1.0 s 0 0.5 0 2 4 6 8 1 0 Ti meld Fig. 2. Decay curve for 1 mgml-l diamorphine in 0.5 M McIlvaine's buffer a t pH 8.0: A, morphine, B, 6-mono- acetylmorphine ; and C, diamorphine. linearity when plotted as log (concentration) versus time. The ordinate is expressed in terms of millimolar concentration in order to demonstrate clearly that all the diamorphine is breaking down to form 6-monoacetylmorphine, which is much more stable than diamorphine. Further hydrolysis to morphine was not detected in any formulation until several weeks after what would be the expiry date of the product.Using the equation for first-order kinetics, rate constants ( K ) were calculated for each solution, and from these values of K the times for 10% of the diamorphine to break down to 6-monoacetylmorphine were calculated (T,,%). T,,% is generally accepted as the basis of the shelf-life of pharmaceutical products, and so has been chosen as a useful measure of the stability of these solutions. The results for morphine solutions are not shown in Table I because all formulations were found to be very stable-no breakdown could be detected in any solution after storage for 3 months at room temperature, and so the use of the simplest formulation of morphine (k, 1 mg ml-l in chloroform - water) must be preferred to diamorphine solutions whenever possible.The effects of pH and temperature on diamorphine stability can be seen clearly from Table I. By storing the solutions in a refrigerator, shelf-lives are increased significantly, up to 10 times for certain solutions. However, this information is only of academic interest, as These results are given in Table I. TABLE I VALUES OF K AND T,,% FOR THE SOLUTIONS EXAMINED 20 "C 4 "C Solution McIlvaine's buffer (ionic strength 0.5 M) in chloroform - water- pH 2.2 . . .. .. 3.0 . . .. .. 4.0 . . .. .. 5.0 . . . . .. 6.0 . . .. .. 7.0 . . .. .. 8.0 . . .. .. Chloroform - water (pH 6.3-6.9) Diamorphine Elixir BPC . . Brompton Cocktail EBH . . r K/h-l . . 1.213 x 3.6 . . 4.886 x 8.9 . . 2.420 x 18.1 . . 2.789 x 15.7 .. 9.461 x 4.6 . . 2.834 x 10-3 1.5 . . 9.356 x 0.5 . . 1.129 x 39.0 . . 6.903 x 6.4 . . 5.177 x 10-4 8.5 1.061 x 41.4 6.430 x 68.0 5.994 x 10-5 73.3 5.994 x 10-5 73.3 1.061 x 41.4 2.556 x 17.2 6.064 x 7.2 4.145 x 10.6 - > 3 months Not testedMarch, 1982 HPLC I N PHARMACEUTICAL ANALYSIS 131 the Medicines Act prohibits storage of these drugs out of a locked safe. The room tempera- ture results are therefore more significant and show the optimum pH range for maximum stability to be 3.84.4. All of the buffer solutions were adjusted to the same ionic strength for these studies by adding various amounts of potassium chloride to the buffer s ~ l u t i o n , ~ enabling pH and temperature effects to be studied without interference from buffer ionic strength. The two well established formulations exhibit shelf-lives of around 1 week at room tempera- ture, in comparison with over 1 month for the simple solution in chloroform - water.How- ever, comparison of this chloroform - water solution with a buffered chloroform - water solution at the same pH shows a considerable difference in stability. Therefore, another factor, buffer ionic strength, also affects the drug stability. In order to study the effect of ionic strength, a range of McIlvaine’s buffers at pH 4.0 were prepared at different concentrations ranging from 0.25 to 4 times strength. For each of these solutions, the ionic strength, p, was calculated, and the shelf-life determined by performing three replicate runs as before. A plot of log Tgoy0 zleysus p gives a negative linear slope, showing that very small increases in ionic strength give very large decreases in shelf-life.The maximum shelf-life is achieved when no buffer is present, and it can be concluded that ionic strength has a far greater effect than pH on stability. Conclusion The HPLC method has proved to be rapid, sensitive and precise, and has been used successfully to analyse aqueous formulations of diamorphine and its breakdown products. The results show that morphine hydrochloride should be used in preference to diamorphine whenever possible as a simple solution in chloroform - water. It may be given a shelf-life of 3 months with 1 month from opening. Owing to the large decrease in stability caused by addition of buffer of any pH, chloroform - water should be used as a vehicle for diamorphine hydrochloride, and may be given a shelf- life of 1 month a t room temperature.This is a considerable increase from the shelf-life of 1 week of Diamorphine Elixir BPC, and has the added advantage to the hospital pharmacy department of quicker and easier preparation. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. References Twycross, R. G., BY. Med. J., 1975, 4, 212. Twycross, R. G., BY. Med. J., 1977, 1348. Twycross, R. G., Curr. Med. Res. Opin., 1978, 5, 497. Twycross, R. G., Prescribers J . , 1978, 2, 117. Twycross, R. G., Topics Therapeutics, 1978, 4, 94. Davey, E. A,, and Murray, J . B., Pharm. J . , 1969, 737. Gold, E. W., J . Hosp. Pharm., 1973, 12. Poochikian, G. K., and Cradock, J. C., J . Pharm. Sci., 1980, 69, 637.Poochikian, G. K., and Cradock, J. C., J . Chromatogr., 1979, 171, 371. Nakamura, G. R., Thornton, J. I., and Noguchi, T. T., J . Chromatogr., 1975, 110, 81. Davey, E. A., Murray, J . B., and Rogers, A. R., J . Pharm. Pharmacol., 1968, 20, Suppl., 51s. Joint Committee on Pharmaceutical Analysis, Analyst, 1978, 103, 268. Lim, H., and Chow, S., J . Forensic Sci., 1978, 23, 319. Smith, P. T., Hirst, M., and Gowday, C. W., Can. J . Physiol. Phavmacol., 1978, 56, 665. Beasley, T. H., Smith, D. W., Ziegler, H. W., and Charles, R. L., J . ASSOG. Off. Anal. Chem., 1974, 57, 85. 16. 17. 18. Knox, J. H., and Jurand, J., J . Chromatogr., 1973, 87, 95. Olieman, L., Maat, L., Waliszewski, K., and Beyerman, H. C., J . Chromatogr., 1977, 133, 382. Elving, P. J., Markowitz, J.M., and Rosenthal, I., Anal. Chew., 1956, 28, 1179. Analysis of Insulin Preparations by High-performance Liquid Chromatography L. F. Lloyd Chemistry Sectiow, National Institute for Biological Standards and Control, Holly Hill, Hampstead, London, NW3 6RB Several formulations of bovine and porcine insulins are currently used for diabetic therapy and the quality of such products is continually being improved. Analytical procedures in132 HPLC IN PHARMACEUTICAL ANALYSIS Anal. PYOC. the British and European Pharmacopoeias for characterising and assessing the purity of insulin preparations are in some instances difficult and time consuming to perform. In particular, the species of origin of insulin is determined by amino acid analysis,l which is a lengthy process.To improve the discrimination of analysis of preparations from mixed species, the insulin A chains (which alone contain the species specific amino acids) are first separated, complicating the procedure and considerably lengthening the analysis time. The simplicity, rapidity and versatility of HPLC make it an attractive technique for the develop- ment of methods for examining insulin preparations, especially as pre-treatment of samples may be avoided. We have previously developed an ion-pair reversed-phase system, which , unlike some published method^,^-^ will separate bovine and porcine insulins and their readily formed degradation products (the A,, monodesamido derivatives) , which are found to varying extents in almost all preparations. This procedure has now been applied to a range of insulin preparations and selected data are reported in this paper.Experimental An Altex 110 or LDC 720 pump was used in combination with a Cecil 212 variable-wave- length ultraviolet monitor. A flow-rate of 1 ml min-1 was used and the column effluent was monitored at either 225 or 280 nm. Columns (100 or 150 x 5 mm i.d.) were slurry packed with Hypersil ODS 5 pm (Shandon Southern), Nucleosil 5 pm C,, (Camlab) or LiChrosorb 5 pm RP-18 (BDH Chemicals). The mobile phase was made up by mixing 75 volumes of 0.1 M ammonium sulphate - 5 mM L(+)-tartaric acid (pH 3.0) with 25 volumes of aceto- nitrile and adding solid cetyltrimethylammonium bromide (cetrimide) to give an over-all concentration of 14 p ~ . Soluble acid formulations were injected directly on to the column; all other formulations were acidified with glacial acetic acid (5% V/V).Results and Discussion The effect of the cetrimide ion pair on the separation of bovine and porcine native and monodesamidoinsulins on Hypersil ODS is illustrated in Fig. 1. In the absence of cetrimide, bovine monodesamido- and native porcine insulin were inadequately resolved [Fig. 1 (a)] , whereas complete separation was achieved after addition of the ion pair to the mobile phase [Fig. l(b)]. This increased the retention times of both bovine and porcine monodesamido- insulins and reversed their elution order. Adequate separations were obtained on other C,, packings, including Nucleosil and LiChrosorb , and little variation was observed between batches of Hypersil ODs.Pre-conditioning of columns with mobile phase containing a higher cetrimide concentration (0.027 M) prior to equilibration was an important factor in achieving reproducible results. Applications Under the conditions described, formulations containing protamine or globin to prolong the effect of the insulin can be analysed without special preparation. Protamine and globin are retained on the column and the use of a pre-column is advisable if formulations containing these substances are to be routinely analysed. Preservatives commonly used in formulatiom, e.g., methyl P-hydroxybenzoate, phenol and cresol, elute before the first insulin peak and do not interfere in the separation. A representative analysis of a formulation is shown in Fig. 2. Crystalline insulin, without additives, can be analysed more rapidly using 26% V/V acetonitrile in the mobile phase.Conclusion It has several possible applications: to determine the species of origin of insulins, as an assay procedure, and to evaluate the purity of samples. With significantly degraded insulins, the deamidation products formed, ie., mono-, di-, tri- and tetradesamidoinsulins, may be separated from the native insulin. A disadvantage of this system, however, is its inability to distinguish human from porcine insulin. As formulations of human insulin (produced by semi-synthetic or DNA recombinant techniques) seem likely to be used for therapy in the near future, another HPLC system has now been developed that is capable of separating bovine, porcine and The chromatographic system described uses simple isocratic conditions.March, 1982 HPLC I N PHARMACEUTICAL ANALYSIS b) c B c a(-)= 1.06 1 15 0 30 15 0 30 Ti meim in Fig.1. Effect of cetrimide on the separation of crystalline bovine and porcine insulins. Sample : 4th International Standard for insulin. (a) 14 pm cetrimide; and (b) no cetrimide. Peaks: A, bovine native; B, bovine monodesamido ; C, porcine native ; I), porcine monodesamidoinsulin. o! is the selectivity factor. C P - 133 0.04 0.03 : 0.02 R < 0.01 30 20 10 0 Ti me/m i n Fig. 2 . Results for 10 p1 of biphasic bovine - porcine insulin formulation. Peaks A, B, C and D as in Fig. 1. P = Preservative, methyl p-hydroxybenzoate. human native insulins and their respective monodesamido derivatives.system will be described elsewhere. Details of this References 1. 2. 3. 4. “British Pharmacopoeia 1980,” Volume 2 , HM Stationery Office, London, 1980. Dinner, A., and Lorenz, L., Anal. Chem., 1979, 51, 1872. Damgaard, U., and Markusson, J., Horm. Metab. Res., 1979, 11, 533. Corran, P. H., and Calam, D. H., in Frigerio, A., Editor, “Proceedings of the 9th International Symposium on Chromatography and Electrophoresis,” Elsevier, Amsterdam, 1979, pp. 341-355. Studies of Ergot Alkaloids Using High-performance Liquid Chromatography - Mass Spectrometry and Mass Spectrometry - Mass Spectrometry Christine Eckers and David E. Games Department of Chemistry, University College, P.O. Box 78, Cardig, CF1 1XL David N. 9. Mallen and Brian P. Swann Lilly Research Centre Ltd., Erl Wood Manor, Windlesham, Surrey, GU20 6PH There has been considerable recent interest in the use of the mass spectral technique of combined high-performance liquid chromatography - mass spectrometry (LC - MS), and mass spectrometry - mass spectrometry (MS - MS), for the analysis of crude mixtures of organic compounds.As a result of recent developments, LC - MS has become a relatively routine technique for the handling of many compounds that are not directly amenable to gas134 HPLC IN PHARMACEUTICAL ANALYSIS Anal. Proc. chromatographic - mass spectrometric study. The various approaches to the technique and its applications have been extensively reviewed.1-6 MS - MS relies on utilising the mass spectrometer to effect mass separation of the compounds of the mixture and thus is followed by metastable studies of the ions of interest (usually molecular or protonated molecular ions, depending on the ionisation method used), with or without collision-activated dissociation.A wide variety of instrumentation has been developed for studies of this type and the reader is referred to various review papers7-+ for a detailed exposition of the approach. It has been suggested by some g r o u p ~ ~ y ~ ~ that MS - MS is a preferable alternative to combined chromato- graphic - mass spectrometric studies, whilst 0thers~9~ suggest that the techniques are comple- mentary and are most effective when used in combination. We have examined crude extracts of ergot fermentation broths by LC - MS, using a moving- belt interface, and a simple form of MS - MS that involves the use of B/E linked scans with and without collision-activated dissociation of the (M + 1)' ions of the components of the mixture formed under chemical ionisation conditions.In this paper we discuss the results obtained from the two approaches and their relative merits in the context of searching for new structural types in crude natural product extracts. Experimental Combined LC - MS was performed on a Finnigan 4000 mass spectrometer using a moving- belt LC - MS interface with the vaporiser at 200 "C (indicated) and the clean-up heater at 200 "C (indicated). For electron impact (EI) studies the accelerating voltage was 70 eV and a source temperature of 260 "C (indicated) was used. Isobutane was used as the chemical ionisation (CI) reagent gas with a source temperature of 230 "C (indicated).For LC studies, three types of column were used: 5-pm ODs, 250 x 5mm i.d., with methanol-water- concentrated ammonia solution (60 + 40 + 0.1) at 1 ml min-1 as eluent; Spherisorb 5W, 250 x 5 mm id., with methylene chloride - methanol - concentrated ammonia solution (95 + 5 + 0.1) at 1 ml min-l as eluent; and an N-propylethylenediamine amino phase bonded to 5-pm irregular silica gel, with isooctane - methylene chloride - methanol (5 + 4 + 1) at 1 ml min-l as eluent. A Waters M6000 pump and a Cecil CE 272 ultraviolet detector were used for the LC and LC - MS. B/E linked scad1 and collision-activated dissociation studies were performed on a VG 70-70H double-focusing mass spectrometer. Ammonia, isobutane and methane were used as CI reagent gases at a source temperature of 200 "C and source pressures between 5 x and 9 x 10-5 Torr.Helium was used as the collision gas in a collision cell in the first field- free region. B/E spectra were recorded on an ultraviolet chart recorder. Results and Discussion The ergot alkaloids can be classified into two structural types, the clavines and the peptide alkaloids.12 The fermentation extracts that we have examined to date contain only alkaloids of the clavine type and the main purpose of this study was to locate new structural types. After establishing suitable liquid chromatographic conditions, LC - MS was carried out using both EI and CI with isobutane as reagent gas. LC columns packed with amine-bonded, C,, reversed phase and silica were used in separate studies.The best results were obtained with a column packed with 5-pm Spherisorb. Examination of retention times and the mass spectral data enabled seventeen alkaloids to be located. Twelve were the known alkaloids: agroclavine (Ia) , setoclavine (IVb) , festuclavine (IIa), palliclavine (111) ,13 noragroclavine (Ib) , elymoclavine (Ic) , penniclavine (IVa) , isochanoclavine I (Vc), norchanoclavine I (Vd) and I1 (Vc) and chanoclavines I (Va) and I1 (Vb). In general, the EI spectral data were more structurally informative than the CI data. However, problems were encountered with palliclavine (111), which was present as a minor component of the mixture, as it appeared to decompose on LC - MS in the EI mode when its spectrum was compared with that obtained from the direct' insertion probe. This problem was overcome by the use of CI for LC - MS, which provided an excellent spectrum.The remaining components appear to be new alkaloids that have been isolated by preparative and semi-preparative LC for complete characterisation. Use of the reversed phase and amine-bonded phases also gave good LC - MS data with the mixture but fewer components were resolved on LC. A check was made for sample loss or decomposition during LC - MS by obtaining field desorption spectraMarch, 1982 HPLC IN PHARMACEUTICAL ANALYSIS 135 from the crude mixture. As the alkaloids exhibit only molecular ions by this technique, a relative molecular mass (Mr) profile of components present in the mixture is obtained and can be checked against the relative molecular masses obtained by LC - MS.It does not, of course, provide a complete check, as there are many isomeric components present in the mixture. R' 35 \ H P2 R' 8 H m H R1 R2 H The use of B/E scans with collision-activated dissociation for identifying components of complex mixtures has been reported.14 As a preliminary to our investigation of the fermenta- tion broth extract we carried out B/E linked scan studies of the (M + 1)+ ions of a number of known ergot alkaloids. These studies were repeated in some instances with collision- activated dissociation. Using the B/E linked scan, it was possible to differentiate between pure samples of isomeric alkaloids containing an alcohol functionality. For example, chano- clavines I (Va) and I1 (Vb) and isochanoclavine I (Vc) (Mr 256) were readily differentiated, whereas on EI and CI chanoclavines I and I1 gave very similar spectra, but isochanoclavine I can be differentiated from them.Similarly elymoclavine (Ic), setoclavine (IVb) and iso- setoclavine (IVc) (Mr 254) and N-norchanoclavines I (Vd) and I1 (Ve) (Mr 242) could be differentiated. When the alcohol functionality was absent from the molecule difficulties were encountered and festuclavine (IIa) and pyroclavine (IIb) (Mr 240) could not be differentiated. The use of B/E linked scan with collision-activated dissociation gave more complex spectra but did not lend further assistance in the differentiation of isomeric species. One feature of the B/E linked scan spectra that we found disturbing was the presence of ions not readily rationalised by conventional mass spectral fragmentation pathways.For example, strong (M + 1 - 19)+ ions were observed in the B/E linked scan spectra of the chanoclavines. We are not sure of the reason for this phenomenon, but it may be linked to the poor resolution observed for precursor ions in the B/E linked scan mode.14 Having obtained reproducible spectra from pure samples of ergot alkaloids, we proceeded to examine the crude fermentation extract. The sample was introduced into the ion source under CI conditions using the direct insertion probe and examination of mass chromatograms of the (M + 1)+ ions of interest (m/z 271, 259, 257, 255, 243, 241, 239 and 225) showed that the components of the mixture vaporised into the ion source at a similar temperature.Thus, we were certain that no components were lost or required higher probe temperatures for vaporisation into the ion source. B/E linked scans were obtained on the ions of interest in the mixture using three CI reagent gases, ammonia, isobutane and methane. B/E linked136 HPLC IN PHARMACEUTICAL ANALYSIS Anal. Proc. scan spectra were also obtained using collision-activated dissociation of the (M + 1)+ ions obtained using methane or isobutane as reagent gases. Ammonia was found to be the reagent gas of choice for the B/E linked scan studies, as there was less rapid deterioration of source conditions and there were no complications due to the formation of addition ions in the spectra of the crude mixture. As mentioned earlier, the chanoclavine isomers (Mr 256) can be differentiated on the basis of their B/E linked scan spectra.However, although examination of the B/E linked scan spectra from m/z 257 in the mixture enabled positive identification of the presence of isochanoclavine (I) (Vc), we were unable to be certain if the other isomers (Va, b), known to be present from the LC - MS, were present or not. Further problems with isomer differentiation were encountered with m/z 241. We were able to identify the norisosetoclavine (IVd) but the second component present could be festuclavine (IIa) or its isomer pyroclavine (IIb) or both, as they have identical B/E spectra. LC - MS showed only the presence of festuclavine. Difficulties were also encountered with mlz 239, the (M + 1)+ ion of agroclavine (Ia), a major component of the mixture.Whilst ions were present in the B/E spectrum corresponding to agroclavine, additional ions were present and the peak at m/z 183 was of much higher abundance than in the standard spectrum of agroclavine. Further studies showed m/z 239 to be a fragment ion from the chanoclavines and comparison of the B/E spectrum obtained from this ion in the chanoclavines showed that it accounted for the additional ions present in the spectrum and the high abundance of m/z 183. Other ions examined presented fewer problems and enabled alkaloids known to be present in the mixture to be identified. In conclusion, we found LC - MS to be preferable to MS - MS for studies aimed at the location of new compounds in complex mixtures, because it enables isomers to be readily differentiated and provides conditions that can be directly applied for the isolation of com- pounds of interest, which is necessary if they are to be fully characterised.We consider that MS - MS is a useful additional technique for studies of this type and is particularly useful when used in combination with GC - MS or LC - MS. Its major utility is for the rapid examination of mixtures for new major components (if they are not isomers) and for known compounds. Apart from our difficulties in the differentiation of isomeric species, other problems encountered with the MS - MS technique we used were rapid source contami- nation with some reagent gases, the presence of artefact peak~~~J5-1~ in some spectra and the apparently unusual fragmentation behaviour of some of the alkaloids, which we feel may be due to the lack of resolution of precursor ions in the B/E linked scan.14 Although other types of MS - MS study can overcome the latter problem, the other difficulties remain and it would appear that considerable caution should be applied in making positive identifications on the basis of MS - MS data alone.C.E. thanks the SRC for financial support. We are grateful to Dr. D. C. Honvell (Lilly Research Centre) for valuable discussions of this study and Dr. E. C. Kornfeld (Lilly Research Laboratories, Indianapolis) for provision of the crude fermentation extracts. We are indebted to the Royal Society for funds for the purchase of the linked scan and collision- activated dissociation equipment and the SRC for funds for the purchase of a mass spectral data system.1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. References Arpino, P. J., and Guiochon, G., Anal. Chem., 1979, 51, 682A. Dawkins, B. G., and McLafferty, F. W., in Tsuji, K., and Morozowich, W., Editors, “GLC and Kendler, E., and Schmid, E. R., irc Huber, J. F. K., Editor, “Instrumentation for High Performance McFadden, W. H., J . Chromatogr. Sci., 1979, 17, 2. McFadden, W. H., J . Chromatogr. Sci., 1980, 18, 97. Games, D. E., Anal. Proc., 1980, 17, 110 and 322. Kondrat, R. W., and Cooks, R. G., AmZ. Chem., 1978, 50, 81A. McLafferty, F. W., Accounts Chem. Res., 1980, 13, 33. Bente, P. F., 111, and McLafferty, F. W., in Merrit, C., Jr., and McEwen, C. N., Editors, “Practical Spectroscopy Series, Volume 3, Mass Spectrometry, Part B,” Marcel Dekker, New York, 1980, p.253. Hunt, D. F., Shabanowitz, J., and Giordani, A. B., Anal. Ckem., 1980, 52, 386. Bruins, A. P., Jennings, K. R., and Evans, S., Int. J . Muss Spectrow. Ion Pkys., 1978, 26, 395. HPLC Determination of Therapeutic Agents,” Marcel Dekker, New York, 1978, p. 259. Liquid Chromatography,” Elsevier, Amsterdam, 1978, p. 163.March, 1982 HPLC IN PHARMACEUTICAL ANALYSIS 12. 13. 14. 15. 16. 17. 18. Floss, H. G., Tetrahedron, 1976, 32, 873. Tscherter, H., and Hauth, H., Helv. Chim. Acta, 1974, 57, 113. Haddon, W. F., Anal, Chem., 1979, 51, 983. Lacey, M. J., and Macdonald, C. G., Aust. J . Chem., 1978, 31, 2161. Lacey, M. J., and Macdonald, C. G., Org. Mass Spectrom., 1979, 14, 465. Shushan, B., and Boyd, R.K., I n t . J . Mass Spectrom. Ion Phys., 1980, 34, 37. Bilton, J . N., Kyriakidis, N., and Waight, E. S., Org. Mass Spectrom., 1978, 13, 489. 137 High-performance Liquid Chromatography of Polymyxin B Sulphate and Gramicidin B. V. Fisher and R. B. Raja Central Analytical Laboratories (Chemical), Wellcome Foundation Limited, Temple Hill, Dartford, Kent, D A l 5 A H Polymyxin B and gramicidin are peptide antibiotics produced by strains of Bacillus poZymyxa and B. brevis, respectively. The polymyxins B have a general structure composed of a cyclic heptapeptide moiety and a side-chain consisting of a tripeptide with a fatty acyl residue. The major constituents of commercial polymyxin B are polymyxin B,, B, and B, (Fig. 1). As they have five unmasked 2,4-diaminobutyric acid residues, all polymyxins B are strongly basic.Both antibiotics are mixtures of at least three components. Methyloctanoic acid Polymyxin B1 I T,h r o=c \ Octanoic acid CH2-cH3 Polyrnyxin B~ \ R' = -C-CHZ-CHZ-CH~-CH2-CHZ- I 0-c I NH YAB 0 C -CH 2 - CH 2-CH 2-C H 2- C H /CH3 lsooctanoic acid Polymyxin B2 II ,HN II 'C H +CH A c NH I I \CH3 DAB I Th r DAB I I R 1 Fig. 1. Polymixins. DAB = a,y-diaminobutyric acid (all linked through the a-group except where shown) ; Leu = leucine; Phe = phenylalanine; Thr = threonine. Gramicidin is normally a complex mixture containing mainly gramicidins A, B and C. These are linear pentadecapeptide ethanolamides with their terminal amino groups blocked by a formyl group. The structures of the individual components that have been elucidated are shown in Fig.2. Gramicidins are non-polar and insoluble in water. The compendia1 methods of quantitative analysis for polymyxin B sulphate and gramicidin in the British, European and United States Pharmacopoeias are currently microbiological. These microbiological methods are capable of great sensitivity; however, if suitable chemical methods are available they are usually more reliable, reproducible and precise. Another advantage of chemical methods lies in the speed with which such analyses can be made and the possibility of automating the procedure. High-performance liquid chromatography (HPLC) is capable of determining these antibiotics rapidly, selectively and accurately and138 HPLC IN PHARMACEUTICAL ANALYSIS Anal. Proc.the method can be used to supplement results obtained by the microbiological testing of potency, or perhaps eventually to replace it. HPLC of Polymyxin B Sulphate In 1975 Tsuji and Robertson, showed that the HPLC separation of polymyxins B, and B, was possible using a pBondapak C,, column and gradient elution. In routine analysis an isocratic mode is preferable to gradient elution, particularly from the point of view of ease of automation and operation. Since Tsuji and Robertson's work, there have been studies on the separation of polymyxins by HPLC using alkyl-bonded silica packings. Terabe et aE.2 separated many of the polymyxin group of antibiotics on a Nucleosil C,, column with a mobile phase consisting of tartrate buffer, sodium butanesulphonate, sodium sulphate, water and acetonitrile.The authors' experience has been that this mobile phase causes many problems owing to crystallisation of solutes in pump heads, valves and injectors, which is unsatisfactory and occasionally expensive. At the same time, Fong and Kh03 published an HPLC separation of polymyxin B, and B, on a Hypersil C,, column using a sulphuric acid - tetramethylammonium chloride - water - acetonitrile mobile phase. In 1980 Thomas et al.4 used a Spherisorb C,, column and a mobile phase similar to that used by Terabe et aE.2 to resolve the components of commercial polymyxin B. The mobile phase suffers from the disadvantages given previously. The method used in these laboratories was devised at the same time as the method of Thomas et aZ.4 was published.The method is isocratic and without the potential problem of crystallisation of buffers from the mobile phase, making it ideal for routine use. For this work, the column packing selected was 5-pm Hypersil C,, (Shandon Southern). The support was slurry packed at 40 MPa into a stainless-steel column (25 cm x 4.2 mm i.d.). The analysis was carried out at ambient temperature using water - methanol - methanesulphonic acid (250 + 250 + 5) as the eluent. The wavelength of detection was 215 nm. The peak areas were measured with electronic integration and the results for each component expressed as a percentage of the total area. Typical chromatograms are shown in Figs. 3 and 4. The chromatographic profiles of polymyxin B sulphate are similar to those reported by Terabe et aZ.2 and Thomas et aL4 All batches of polymyxin B sulphate examined contained the two major components (polymyxins B, and B,) and more than ten minor components.The levels of minor components for all batches of polymyxin B sulphate examined were similar, but the contents of polymyxins B, and B, were different. It was possible to group samples from a common source according to the contents of polymyxins B, and B,. Manufacturer Polymyxin B,, % Polymyxin B,, yo Polymyxin B3, % A (8 batches) . . .. .. 60-80 6-1 3 2-5 B (4 batches) . . .. 50-60 20-23 2-5 USP Reference Standard . . 48 22 -3 As polymyxin B, is reported to be more active than polymyxin B,, it is necessary that in the microbiological assay the test and the standard preparations have similar component ratios, otherwise the dose - response lines of the standard and test may not be parallel.HPLC of Gramicidin In 1977, Axelsen and, Vogelsgang5 separated the components of gramicidin on a Zorbax C,, column using as the mobile phase methanol - 0.005 M ammonium sulphate solution with the column at 60 "C. The method described here has the advantage of giving satisfactory resolution when operated at ambient temperature. The chromatographic column (25 cm x 4.2 mm i.d., stainless steel) was slurry packed at 40 MPa with 5-pm Hypersil C,,. The mobile phase was methanol - tetrahydrofuran - 0.1 M sodium dihydrogen orthophosphate solution (aqueous, pH 4.5) (180 + 20 + 50). The wave- length of detection was 280 nm. The assignments of the peaks as gramicidins A, B and C were based on the observations reported by Axelsen and V~gelsgang.~ The USP ReferenceMarch, 1982 HPLC IN PHARMACEUTICAL ANALYSIS 139 HCO- y-Gly-L-Ala- D-Leu- L-Ala- D-Val 61 1 2 3 4 5 D-Leu- z- D-Leu- L-Trp-D-VaI- L-Val 12 11 10 9 8 7 L-Trp- D-Leu - L-Tr p - N H(CH&OH Y 13 14 15 c z (Val]-gramicidin A [lie]-gramicidin A [Val]-gramicidin B [Ilel-gramicidin B [Val]-gramicidin C [I lel-g ra m icidin C L-Val L-Trp L-lle L-Trp L-Val L-Phe L-lie L-Phe L-Val L-Tyr L-lle L-Tyr Fig. 2. Structures of components of gramicidin. 0 4 8 12 16 . Time/min Fig. 4. Chromatogram of commercial polymixin B sulphate. Conditions as in Fig. 3. e __ 0 4 8 12 16 Time/m i n Chromatogram of USP Refer- ence Standard polymixin B sulphate. Conditions: 5-pm Hypersil ODs; water - methanol - methanesulphonic acid (250 + 250 + 5) ; injection of 20 pl of 2 mg ml-l solution; A = 215nm. Fig. 3 Fig. 5. Chromatogram of USP Reference Standard gramicidin. Conditions : 5-pm Hyper- sil ODS; methanol - tetrahydrofuran - 0.1 M sodium dihydrogen orthophosphate (180 + 20 + 50); injection of 20 pl of 0.5 mg ml-l solution; A = 280nm. Peaks: 1, [Vall-grami- cidin C; 2, [He]-gramicidin C; 3, [Val]-grami- cidin A; 4, [Ilel-gramicidin A; and 5, [Val]- + [Ilel-gramicidin B. Standard gramicidin gave the chromatogram shown in Fig. 5. Reference Standard and samples of commercial gramicidin were estimated to be as follows: The composition of the USP140 INDUSTRIAL APPLICATIONS OF AAS Anal. Proc. Gramicidin A Gramicidin B Gramicidin C (valine + isoleucine), (valine + isoleucine) , (valine + isoleucine) , Batch % Y O Y O USP Reference . . 81.6 USNF 14432 .. 80.3 1307.. .. . . 83.9 28893 . . .. 80.8 2.9 2.6 1.8 2.2 13.2 14.1 13.0 15.8 Conclusion Because of the complex nature of these peptide antibiotics, it is more difficult to predict potency from HPLC than from microbiological assay. The disadvantages of the micro- biological assays are the lack of reproducibility of the potency determinations between laboratories, especially where the dose - response lines of the standard and test are not parallel. In combination, however, the two techniques represent a powerful means of analytical control. References 1. 2. 3. 4. 5. Tsuji, K., and Robertson, J . H., J. Chromatogy., 1975, 112, 663. Terabe, S., Konaka, R., and Shoji, J . , J. Chromatogr., 1979, 173, 313, Fong, G. W. K., and Kho, B. T., J . Liquid Chromatogr., 1979, 2, 957. Thomas, A. H., Thomas, J . M., and Holloway, I., Analyst, 1980, 105, 1068. Axelsen, K. S., and Vogelsgang, S. H., J. Chrornatogr., 1977, 140, 174.

ISSN:0144-557X    

DOI:10.1039/AP9821900121

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

140 INDUSTRIAL APPLICATIONS OF AAS Anal. Proc. Industrial Applications of Atomic-absorption Spectroscopy The following is a summary of one of the papers presented at a Joint Meeting of the North West Region and the Atomic Spectroscopy Group held on September 16th, 1980, at the University of Lancaster. Applications of Atomic-absorption Spectroscopy in Occupational and Environmental Monitoring C. J. Jackson Health and Safety NW2 6LN Executive, Occupational Medicine and Hygiene Laboratories, 403 Edgware Road, London, Aspects of the work of the Health and Safety Executive (HSE) include the assessment of potential exposure of workers to harmful contaminants in a wide variety of industrial pro- cesses and the evaluation of the effects of emissions from industrial stacks. These assess- ments necessarily result in the analysis of large numbers of samples taken on site by inspectors and scientific staff.A wide range of analytical techniques are available in HSE laboratories for this purpose and some applications of one of these techniques, namely atomic-absorption spectroscopy, are discussed in relation to the evaluation of samples, the development of sampling methods and the setting of standards. Occupational Monitoring A variety of air-quality standards are available to the occupational hygienist, of which the best known and most widely used is the listing of Threshold Limit Values (TLVs) produced annually by the American Conference of Governmental Industrial Hygienists (ACG1H)l and distributed in the UK, with certain slight modifications, as an HSE Guidance Note.2 Breathing zone concentrations are listed for a range of contaminants, which are believed to represent the limit of safe working, for the average worker, over the time limits specified.Crown Copyright.March, 1982 INDUSTRIAL APPLICATIONS OF AILS 141 The primary aim of the HSE occupational hygienist is to obtain working practices which result in workers experiencing levels of toxic hazards below (ideally, well below) these levels. In such a context the purpose of sampling is to assess the standard of process control, to measure the exposure of workers and to evaluate the background levels in the factory. Traditionally, personal monitoring for inorganic dusts and fumes has been by drawing air through a 0.8-pm 25-mm cellulose ester filter, contained in an open head, positioned on the operative’s lapel near to his breathing zone, using a portable battery-operated pump strapped to his waist and operating at 2 1 min-1.Acid and alkali mists and inorganic vapours, such as mercury, are sampled, using a similar pump operating at 1 1 min-l, into 10 ml of an appropriate absorbing solution contained in a small plastic impinger. Background (or static) samples are obtained similarly. More recently there has been a trend towards the use of enclosed head samplers, such as the UKAEA head.3 Only a 4 mm diameter orifice is avail- able for sample entry and such a head provides entry velocities closely matched to the nasal velocity as well as a degree of tamper resistance. In addition to atmospheric sampling, it would be normal to look at the various process materials, products, by-products and waste materials, general floor sweepings, ledge scrapes, etc.Environmental Monitoring Environmental monitoring by HSE is generally concerned with the evaluation and control of emissions to the atmosphere from stacks and ducts. Similar standards to those provided by the TLV list are used but much lower concentrations of toxic hazard are demanded in the general environment because exposure is on a 24-hour-day, 7-day-week basis. As far as stack emissions are concerned, total mass limits in terms of kg h-l are often specified, together with maximum contaminant concentrations in the emission. Monitoring is normally by sampling the stack emission via an inspection port, either by using a hand suction device that takes a sample on to a glass-wool pad or by the use of an isokinetic probe and control system that traps the sample on to a membrane filter.In addition, samples of the materials being handled at the works may be obtained together with material trapped by the bag filters or electrostatic precipitators. Analytical Requirements The information required by the occupational hygienist may be summarised as: (i) how do personal exposures relate to the relevant TLV?; (ii) are short-term or ceiling values exceeded even if long-term exposure appears satisfactory? ; (iii) what improvements in control measures appear necessary? ; and (iv) how do these results compare with those taken on previous occasions ? Of equal importance is speed and the ability to perform the analysis on location, thus enabling appropriate action to be taken immediately.Under most circumstances air particulate samples consist of sub-microgram or microgram amounts of toxic material. High sensitivity is therefore important, as is the ability to discriminate well between the sample and appro- priate filter and reagent blanks. Finally, as the particular contaminant being examined may be present in a large excess of a less hazardous material, reasonable freedom from interference is desirable. It is because of these requirements that techniques such as atomic-absorption spectroscopy (AAS) have found such favour for occupational and environmental analysis. Table I indicates the capability of conventional flame AAS to analyse 1-h (120 1) air samples containing as little as 0.1 TLV for a range of toxic materials.Similar questions may be posed by the environmentalist. It is clear that to answer these questions, precise and accurate analysis is necessary. Applications of Atomic-absorption Spectroscopic Analysis Lead Lead is widely distributed industrially and is of considerable toxicological significance.3-7 It is known that absorption is almost entirely through dust inhalation rather than ingestion6 and, therefore, air particulate sampling is of especial importance. The pioneering work by Williams et aZ.,s on which the ACGIH standard is based, involved the study of workers in a lead battery works using closed head samplers, but HSE have, until recently, always advocated that inorganic lead in the workplace atmosphere be sampled with an open head.In pre- paring the new Control of Lead at Work Regulations4 and Approved Code of Practice for the142 INDUSTRIAL APPLICATIONS OF AAS TABLE I TYPICAL PERFORMANCE OF FLAME AAS RELATED TO OCCUPATIONAL HYGIENE ANALYSIS Anal. Proc. Element As .. .. Be .. .. Cd .. .. Cr .. .. cu .. Ni (soluble) . Pb .. .. Sb .. .. T1 .. Z ~ C I , (as Znj ZnO (as Zn) TLV192/mg m-3 .. 0.5 .. 0.002 .. 0.05 .. 0.05 .. 1.0 .. 0.1 .. 0.15 .. 0.5 .. 0.1 .. 0.48 .. 4.0 Concentration of contaminant for 10-ml solution volume when a 120-1 air sample is obtained at 0.1 TLV levellpg ml-l 0.6 0.0024 0.06 0.06 1.2 0.12 0.18 0.6 0.12 0.58 4.8 Sensitivity of typical AA spectrometer (=0.0044 A)/ pg ml-I 0.7* 0.006 0.005 0.04 0.03 0.05 0.06 0.2 0.1 0.01 0.01 * Sensitivity can be increased by using hydride generation.Control of Lead at Work,5 a statistically valid side-by-side comparison of enclosed (UKAEA) and open head (Gelman) samplers was made in a variety of industrial situations for both personal and static monitoring. Analysis was by flame AAS, using the 217-nm lead line, after dissolution of the lead in 2 ml of 5% nitric acid, 0.2% 100-volume hydrogen peroxide and dilution to 10 ml. In general, the UKAEA head was shown to be much less susceptible to orientation of the sampler, the data obtained were considerably less statistically variable and the samples were less easily sabotaged than with an open head.g As a result of this work, the use of the UKAEA sampling head was proposed in an HSE Guidance Note.3 More recently, the EEC have proposed regulationslO that would introduce a TLV for ambient air of 100 pg m-3 of lead in air, 40-h time-weighted average (TWA), for particles of less than 15 pm aerodynamic diameter, against the current ACGIH standard1 of 150 pg m-3 of lead in air, 8-h TWA, with no particle size limit.As no data were available on the particle size distribution of industrial lead dusts and as there was some doubt that a suitable sampler to meet the EEC requirements existed, an investigation has been undertaken to look at particle size distributions and to evaluate a possible sampler. At the same time the oppor- tunity was taken to undertake a full comparison of the performance of the AAS and X-ray fluorescence (XRF) analysis techniques as applied to this work.Two sampling exercises have been undertaken, one at a lead battery works and one at a lead smelter, involving both static and personal monitoring. UKAEA and cyclone samplers (the latter modified to provide a 15-pm aerodynamic diameter cut-off) were used for sampling and standard AAS and XRF procedures were used to analyse the resultant samples. Fig. 1 shows a comparison between the results obtained by AAS and XRF for static samples from different process areas of the battery works. Clearly there is a systematic difference between the results, a linear regression analysis indicating an over-all AAS to XRF ratio of 0.786 & 0.015 over the range 0-20 pg of lead, and a more significant departure from a ratio of 1 at higher levels. Examination of the potential errors in the two procedures indicated that the discrepancy arose because the secondary X-ray responses of the spectro- meter were not uniform over the whole area of the filter samples.The sensitivity was higher for a given mass of lead in the centre of the filter than for the same mass located towards the edge. When the original calibration of the spectrometer was carried out, standards were prepared such that the lead was evenly deposited across the filter. It therefore follows that when this calibration was used to analyse real samples on which the deposit was concentrated in the centre of the filter (Le., UKAEA or cyclone sampling head), anomalously high results were found. There are two possible solutions to this problem, either to calibrate using standards with the same distribution of samples across the filter as the cyclone or UKAEA head, or to modify the geometry of the spectrometer so that there is a uniform X-ray response across the filter.It was decided that the better long-term approach was to modify the spectrometer geometry and this was done by changing from a 26-mm to a 40-mm beam limiter.March, 1982 INDUSTRIAL APPLICATIONS OF AAS 143 A comparison of analytical results from a similar sampling exercise in a lead smelter (using the 40-mm beam limiter) is shown in Fig. 2. The agreement between XRF and AAS is good, with very few XRF results differing by more than 10% from the AAS figures. A linear regression analysis gave an over-all AAS to XRF ratio of 1.012 0.011, a statistical test indicating no significant departure from theoretical slope and intercept at the 5% significance level.Lead determined by XRWpg Fig. 1. Correlation between XRF and AAS results for static samples taken at battery works. 0, Cyclone sampling device; and x , UKAEA enclosed head sampler. N = 91. Slope = 0.786 f 0.015 (tirom = 14.75). Intercept = 0.090 f 0.094 (tfrom 0 = 0.96). I? = 0.9852. t,.o,,eo 1.99. An analysis of the particle size data from both surveys showed that the particle size distribution of lead dust and fume was very dependent on both the process being carried out and also movement of workers, vehicles, etc. Clearly, the correct setting and evaluation of particle size-dependent occupational hygiene standards will be complex ; analytically, how- ever, there should be few problems and, although for highest accuracy AAS would be the preferred procedure, XRF is potentially very valuable provided that care is taken with the matching of calibration standards and high results are interpreted with caution. HSE is also considerably involved in the environmental monitoring of lead emissions from stacks and ducts at a large number of registered works in the UK.ll Monitoring stacks and ducts for compliance with emission standards is a time-consuming activity for both HSE and the occupier, and for some years HSE have been looking into the possibility of producing automatic monitors for lead (and other) fumes.One approach has been the development of an instrument for the remote analysis of representative samples collected from one or, potentially, several stacks.The monitor was constructed so that samples were taken at regular intervals using an isokinetic sampling probe and, after dilution, passed back along a heated pipe (to prevent sample drop-out), to a remotely located spectrometer consisting of a hollow-cathode source, air - propane burner, atomic-fluorescence detector and read-out device. This monitor has been described in more detail elsewhere.l2 Zinc Oxide and Zinc Chloride in Galvanising Fume Recently, a major occupational hygiene survey has been undertaken in the galvanising industry. Galvanising is a fairly complex procedure, involving degreasing, pickling, fluxing, galvanising and washing operations.13 Toxic pollutants generated in galvanising fume include zinc oxide, zinc chloride, ammonium chloride and hydrochloric acid.Sampling of the fume was on to 0.8-pm 25-mm cellulose ester filters held in open heads. Analysis involved144 INDUSTRIAL APPLICATIONS OF AAS Anal. PYOC. ion chromatography (for chloride and ammonium), potentiometric titration (for hydrogen chloride) and AAS (for zinc as zinc oxide and chloride). Of particular interest was the separation of zinc oxide and chloride, where the relevant TLVs are widely different, 1 and 5 mg m--3, respectively.lS2 Separation was based on solubility in water: zinc oxide, 0.000 16 g per 100 ml; zinc chloride, 432 g per 100 ml water.14 Sample preparation involved washing the filters in 10 ml of de-ionised water for 30 min at 23 “C to dissolve zinc chloride (and ammonium chloride), followed by digestion of the residue, after filtration, in nitric acid to dissolve zinc oxide.Under these experimental conditions a theoretical maximum of 16 pg of zinc oxide would dissolve at the water-wash stage but it was shown that, in practice, 5 p g was the maximum obtained. As air samples greater than 100 1 were taken and there is, in general, an excess of zinc chloride over zinc oxide in galvanising fume, it was considered that the maximum error in the analysis, at levels at and around 0.1 TLV, representing an over- recording of zinc chloride and an under-recording of zinc oxide, was less than 10%. This work has been discussed in more detail e1~ewhere.l~ 0 100 200 300 Lead determined by XRF/yg Fig. 2. Correlation between XRF and AAS results for static samples taken at lead smelter.0, Cyclone sampling device; and x , UKAEA enclosed head samples. N = 57. Slope = 1.012 f 0.011 (tirom = 1.07). Intercept = -3.39 f 1.93 (tiromo = -1.75). R = 0.9966. t0.06, 58 = 2.00. Conclusions The examples given have been intended to give some insight into the role of AAS in occupa- tional and environmental monitoring. It is hopefully clear that analysis cannot be divorced from sampling and sample preparation. Indeed, a full understanding of the nature and limitations of the samphg procedures is essential if the correct choice of analytical technique is to be made and the maximum information obtained from the analysis. The author thanks Dr. N. G. West for an informative discussion on the initial sources of error in the XRF analysis of lead and for permission to publish the relevant correlation data.References 1. “TLVs: Threshold Limit Values for Chemical Substances and Physical Agents in the Workroom Environment with Intended Changes for 1980,” American Conference of Governmental Industrial Hygienists, Cincinnati, 1980.March, 1982 THROWING LIGHT ON ELECTRODES 145 “Health and Safety Executive Threshold Limit Values for 1979,” Guidance Note EH15/79, HM “Control of Lead a t Work: Air Sampling Techniques and Strategies,” Guidance Note, HM Stationery “Control of Lead at Work Regulations 1980,” SI 1980 No. 1248, HM Stationery Office, London. “Approved Code of Practice for the Control of Lead a t Work,” HM Stationery Office, London, 1980. Trevethick, R. A., “Environmental and Industrial Health Hazards : A Practical Guide,” Heinemann Berman, E., “Toxic Metals and Their Analysis,” Heyden, London, 1980. Williams, M. K., King, E., and Walford, J., BY. J . Ind. Med., 1969, 26, 202. Gough, D. W., and Harvey, R. P., personal communication. “Proposal for a Council Direction on the ?otection of Workers from Harmful Exposure to Metallic Lead and its Ionic Compounds a t Work, No. C324/3, Commission of the European Communities, Brussels, 1979. “Proposals for Amendments to the Lists and Scheduled Works and Noxious or Offensive Gases,” Health and Safety Commission, London, 1979. Jackson, C. J., J . Autom. Chem., 1979, 1, 267. “General Galvanizing Practice,” Galvanizers Association, London, 1979. “Handbook of Chemistry and Physics,” Sixtieth Edition, CRC Press, West Palm Beach, FL, 1979. Jackson, C. J., Howe, A. M., and Neuberger, C., Anal. Proc., 1981, 18, 234. 2. 3. 4. 5. 6. 7. 8. 9. 10. Stationery Office, London, 1980. Office, London, 1981. Medical Books, London, 1973. 11. 12. 13. 14. 15.

ISSN:0144-557X    

DOI:10.1039/AP9821900140

出版商:RSC    

年代:1982

数据来源: RSC

摘要:

March, 1982 THROWING LIGHT ON ELECTRODES 145 Throwing Light on Electrodes The following is a summary of one of the papers presented at a Joint Meeting of the Electro- analytical Group and the Electrochemistry Group of the Faraday Division held on February 27th, 1981, at Southampton University. Analytical Applications of Spectroelectrochemistry J. F. Tyson Department of Chemistry, University of Technology, Loughborough, Leicestershire, LE11 3 T U There are many ways in which spectroscopic and electroanalytical techniques have been combined. Ellipsometry, ESCA and Auger electron spectroscopy have been used to monitor the electrode surface, UV, visible and IR absorption, NMR, ESR and luminescence spectro- metry have been used to study the layers of solution adjacent to the electrode, and Raman, specular reflection and internal reflection spectroscopy have been used to study both solution and electrode.In conjunction with these, the electrochemical techniques of chronoampero- metry (single and a double potential step), chronopotentiometry, coulometry and cyclic voltammetry have all been used. A variety of electrode - light beam configurations have been devised, of which possibly the most interesting are the optically transparent electrodes (made from a thin film on a substrate or from a minigrid), which have been used with both transmission normally through the electrode and total internal reflectance spectroscopy. These spectroelectrochemical combinations have been used to determine Eo and n values, rate constants, diffusion coefficients and reaction mechanisms and to study adsorbed species, oxide films and electrodeposits on electrode surfaces.There have been very few quantitative analytical applications reported. Analytical Possibilities The most promising candidates as analytical methods are those in which absorbance is measured whilst movement of species in solution is governed by semi-infinite linear diffusion. Starting from the Cottrell equation,lS2 a simple expression for absorbance ( A ) measured normal to the electrode surface may be deduced by a variety of methods,3y4 namely A = E ~ ~ ( D , ~ / v ) ~ C : (for a reaction 0 + ne -+ R) where is the molar absorptivity of the reduced species, t is time and Do and C: are the diffusion coefficient and bulk concentration of the oxidised species.It should be noted that the units of Ct are mol l-l, whereas in the Cottrell equation they are molml-l. The potential analytical usefulness can readily be seen from the equation’s similarity to the Beer - Lambert relationship, in which the path-length termAnal. PYOC. has been replaced by a function of time, Z(Dot/r)*. This function has, erroneously, been referred5 to as the diffusion layer thickness. However, it is not difficult to show that, by considering the expression for the variation of C, with distance (x) from the electrode surface [C, = C: erf(x/ZDo*t*j, where erf is the error function] and by finding the slope, by differenti- ation, at x = 0, the diffusion layer thickness is (nDot)*. What the equation does indicate is that, as far as the absorbance is concerned, the concentration of the absorbing reduced species may be considered constant (equal to the bulk concentration of the oxidised species) up to a boundary, which moves out into the solution as a function of the square root of the time, beyond which the concentration is zero.Thus the term Z(D,,t/r)* represents a diffusion layer thickness (but not the diffusion layer thickness6J). The problem of applying this spectroelectrochemical technique to any real system becomes immediately apparent when it is realised, for a value of Do of cm2 s-l (fairly typical of ions in solution), that after electrolysis for 10 s the path length is 0.1 mm and has increased to only 0.37 mm after 100 s. This means that for most applications a spectrometer equipped with signal-averaging facilities must be used to give meaningful signal to noise ratio charac- teristics, and makes the modification of a conventional ultraviolet - visible spectrometer for spectroelectrochemical work an unattractive proposition with any of the electrode - light beam configurations mentioned earlier.146 THROWING LIGHT ON ELECTRODES Novel Electrode - Light Beam Configuration If the configuration is changed so that a light beam of fixed height h is passed at grazing incidence over a plane electrode of length Z then the equation for the absorbance becomes8 A = (eR - E,,) x 2Z(D0t)4/h.rr*C:, again for a reaction 0 + rce + R but allowing for the possible absorbance of the oxidised species as well. It can be seen that the previous equation has been modified by term l/h.Thus, for an electrode 1 cm long and a light beam of thick- ness 0.5 mm the path-length term becomes 0.2 cm after 10 s and 0.74 cm after 100 s. This configuration has been used to study the reduction of a number of metal ions9 and both the reduction and oxidation of a number of organic molecules.8 Results consistent with the equation given above were obtained and it should be noted that the spectra observed were indeed the difference between the oxidised and reduced forms, which means that care should be taken in interpreting a spectroelectrochemical spectrum as evidence of the existence of a single absorbing species at the electrode surface. Advantages Firstly, all studies performed with optically transparent electrodes can be carried out but with freedom from the effects of the electrode transmission characteristics, and with considerably increased sensitivity, making the use of a conventional spectrometer a viable proposition.The sensitivity limit is set by the lowest value of h that can be tolerated, but the use of fibre-optic light guides or lasers (as has been reported recentlylO) may extend the system over the method in which the light beam of the conventional spectrometer is simply blanked off by the appropriate amount. The system is not only applicable to reactions of the types mentioned earlier but may be extended to more complex electrode reactions for which spectroelectrochemical equations have already been derived,ll by modification of these equations to allow for the 1 and h terms. This configuration has a number of advantages to offer. Analytical Applications The system can not only be used for any existing spectrophotometric method involving oxidation or reduction (the electrode surface substitutes for the redox reagent), but also extends the range of reactions available for analytical use as the stringent kinetic require- ments for most spectrophotometric reactions can be relaxed, highly reproducible conditions being obtained in the diffusion layer.This is well illustrated by the fact that it was shown possible to use electrogenerated hydroxide ion as a spectrophotometric reagent for a number of metal^.^ In addition, the configuration is being evaluated for use in the spectroelectro- chemical analogue of anodic stripping voltammetry, whereby metal ions are pre-concentrated and separated from the remainder of the sample matrix by electrodeposition on to the electrode surface and then anodically redissolved in a solution containing a colorimetricMarch, 1982 EQUIPMENT NEWS 147 reagent.Preliminary investigations12 have shown that there are a number of problems associated with the redox and pH behaviour of the colorimetric ligand to be overcome before the system will provide a viable analytical technique. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. References Cottrell, F. G., 2. Phys. Chem.. 1902, 42, 385. Delahay, P., “New Instrumental Methods in Electrochemistry,” Interscience, London, 1954, p. 51. Strojek, J. W., Kuwana, T., and Feldberg, S. W., J . Am. Chem. Soc., 1968, 90, 1353. Winograd, N., Blount, H. N., and Kuwana, T., J . Phys. Chem., 1969, 73, 3456. Kuwana, T., and Winograd, N., in Bard, A. J., Editor, “Electroanalytical Chemistry,” Volume 7, Marcel Dekker, New York, 1974, p. 10. Delahay, P., “New Instrumental Methods in Electrochemistry,” Interscience, London, 1954, p. 21 7. Nernst, W., 2. Phys. Chem., 1904, 52, 47. Tyson, J. F., and West, T. S., Talanta, 1980, 27, 335. Tyson, J. F., and West, T. S., Talanta, 1979, 26, 117. Pruiksma, R., and McCreery, R. L., Anal. Chem., 1979, 51, 2253. Kuwana, T., and Winograd, N., in Bard, A. J., Editor, “Electroanalytical Chemistry,” Volume 7, Monk, D., MSc Thesis, Loughborough University of Technology, 1980. Marcel Dekker, New York, 1974, pp. 9-22.

ISSN:0144-557X    

DOI:10.1039/AP9821900145

出版商:RSC    

年代:1982

数据来源: RSC