摘要:

Proceedinas - - - - - ~ -of the Analytical Division ofThe Chemical SocietyCONTENTS275 Fourth International SACConference285 Summaries of Papers285 'Original Papers in PharmaceuticalAnalysis'300 'Research and DevelopmentTopics in Analytical Chemistry'302Chemistry'306 'Analysis of IndustrialEssential Oils'309309 Sir Alan Walsh309 Biochemical Analysis Prize 1978309 CS Corday-Morgan Medal and309 CSAwards309 Conferences and Meetings31 0 Publications Received312 Analytical Division Diary'Use of Enzymes in AnalyticalNew President of the IUPACAnalytical Chemistry DivisionPrizeVolume 14 No 10 Pages 275-31 2 October 197PADSDZ 14(10)275-312(1977)'SSN 0306-1 396October 1977PROCEEDINGSANALYTICAL DIVISION OF THE CHEMICAL SOCIETYOF THEOfficers of the Analytical Divisionof The Chemical SocietyPfesidentD. W.WilsonHon. SecretaryP. G. W. CobbSecretaryMiss P. E. HutchinsonHon. Treasurer Hon. Assistant Secretaries .J. K. Foreman D. 1. Coomber, O.B.E.; D. C. M. Squirrel1Editor, ProceedingsP. C. WestonProceedings is published by The Chemical Society.Editorial: The Director of Publications, The Chemical Society, Burlington House, London, W1 V OBN.Telephone 01 -734 9864. Telex 268001.Subscriptions (non-members): The Chemical Society, Distribution Centre, Blackhorse Road,Letchworth, Herts., SG6 1 HN.Non-members can only be supplied with Proceedings as part of a combined subscription with The Analystand Anelytical Abstracts.Q The Chemical Society 1977Selected Annual Reviewsof the Analytical SciencesVolume 4Edited by Dr L.S. Bark, University of SalfordThe fourth volume continues the policy adopted in previousvolumes of presenting critical reviews of selected topics in modernanalytical science. Each of these reviews embodies the workconsidered pertinent over the four or five years up to 1974.*Advances in Voltammetric Techniques,' by B. Fleet and R. D.Jee; Advances in Instrumentation and Recent Applications ofVoltammetric Techniques in the Areas of DC Polarography, PulsePolarography, AC Polarography, Stripping Voltammetry.'Higb- frequency Electrodeless Plasma Spectrometry, ' by B. L.Sharp; Classification of Plasma Types; Emission Spectrometry-General Considerations; Nebulisers; The Inductively-coupledRadio-frequency Plasma Torch; The Microwave Plasma.73 pages Price f9.50 ISBN 0 85990 204 8Members of The Chemical Society may buy personal copies at the special price of f3.00provided they order direct and enclose remittance.Obtainable from The Chemical Society, Distribution Centre,Blackhorse Road, Letchworth, Herts., SG6 1 H

ISSN:0306-1396    

DOI:10.1039/AD97714FX032

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

October, 1977 ANALYTICAL DIVISION DIARY 31 1Analytical Division Diary, continuedNovember, continuedWednesday, 16th, 6.30 p.m. : PrestonNorth West Region, jointly with the Lancasterand District Section of the CS/RIC and thePreston Polytechnic Students ChemicalSociety.“Solvent Extraction of Metals : The CurrentPosition,” by D. S. Flett.The Polytechnic, Preston.Wednesday, 16th, 2 p.m. : FelixstoweEast A ngbia Region : Annual General Meeting,followed by a meeting on “Mineral Analysisin the Fertiliser Industry.”“The Assessment and Analysis of PhosphateRocks in the Fertiliser Industry,” by S. R.Porter.“The Determination of Quartz in AirborneDusts,” by D. R. Howlett.Followed by a tour of the laboratories ofthe Research Station.Fison’s Ltd., Levington Research Station,Felixstowe.Thursday, 17th, 10.30 a.m.: DidcotRadiochemical Methods Group : Annual Gen-eral Meeting (2.05 p.m.) and meeting on“Radioactivity and the Environment.”“Environmental Measurements of 3H and14C,” by R.L. Otlet.‘‘85Kr in Air,” by I. C. Downing, M. J. deFaubert Maunder and R. B. Pitts.“Current Practice for the EnvironmentalSurveillance of Nuclear Power Stations,”by A. R. Ware.“Radionuclides in Marine Pollution Studies,”by R. J. Pentreath.“Activation and Counting Procedures, TheirPlace in Environmental Analysis,” byL. Salmon.“Biological Tissues as Indicators of Environ-mental Health and Pollution,” by N. M.Spyrou.“Studies of the Transport of Actinides inMammals,” by G. N. Stradling.“Assessment of Radioactive ContaminationTour of selected Laboratories.B.551, AERE, Harwell, Didcot, Oxon.Biological Methods Group : Annual GeneralMeeting, followed by a lecture by SirDavid G.Evans.The Chemical Society, Burlington House,London, W. 1.Wednesday, 23rd, 6.15 p.m.: Wolverhamp-tonMidlands Region : Annual General Meeting,followed by a joint meeting with theMidlands Region of the RIC on “StudentPapers.” The Polytechnic, Wolverhampton.Electroanalytical Group on “Students’ Re-This meeting has been cancelled.Thursday, 24th, 10.30 a.m. : LondonA utonzatic Methods Group .- Annual GeneralMeeting, and a joint meeting with theSouth East Region on “Twenty Years ofAutomation. ”Speakers to include: A. C. Docherty, K.Aldous and P.B. Stockwell.Imperial College, London, S.W.7.Thursday, 24th, 4 p.m.: Glasgowin Man,” by F. Fry.Thursday, 17th, 6.30 p.m.: LondonWednesday, 23rd, 2.30 p.m. : Londonsearch.’ ’Scottish Region, jointly with the Glasgow andWest of Scotland Section of the CS/RICand the Andersonian Chemical Society.“Chemical Analysis and Water PollutionControl in the Clyde Catchment,’’ by G. A.Best.Room C133, Chemistry Department, Uni-versity of Strathclyde, Cathedral Street,Glasgow, G1 1XL.Wednesday, 30th : DidcotSpecial Techniques Group : Annual GeneralTour of selected laboratories and short pre-AERE, Harwell, Didcot, Oxon.Meeting.sentations on instruments on viewAna I yt i ca I Division DiaryOCTOBER NOVEMBERThursday, 13th, 2.30 p.m.: LondonAnalytical Division on “Topics in Environ-mental and Marine Chemistry.”“Studies on Toxic Trace Metals in theEnvironment by Advanced PolarographicMethods.” by Professor H.W. Niirnberg.“Trace Mercury and Methylmercury Com-pounds in Fish,” by H. Egan.“Some Applications of X-ray Microanalysis toBiological Problems,” by P. F. Newell.Geological Society of London Lecture Thea-tre, Burlington House, Piccadilly, London,w.l.Thursday, 13th, 4 p.m. : BelfastNorthern Ireland Sub-committee, jointly“New Analytical Techniques Based onChemistry Department, The Queen’s Univer-with the Andrews Club.Hydrogen Flames,” by A. Townshend.sity, Belfast.Friday, 14th, 7 p.m. : HarrogateNorth East Region : Social evening.Dinner, followed by “Magic and MysteryThrough the Ages,” by Professor and Mrs.E.A. Dawes.Granby Hall Hotel, Harrogate.North West Region, jointly with the SouthCheshire Branch of the PharmaceuticalSociety.“Analytical Problems in Drug Toxicology,’’by B. Scales.ICI Pharmaceuticals Division, Tenant’s HallConference Centre via Bollington Lodge,Alderly Park, Macclesfield, Cheshire.Friday, 14th, 6.30 p.m. : MacclesfieldFriday, 14th, 5 p.m. : ExeterWestern Region, jointly with the PeninsulaSection of the CS.“Polarography in Environmental and MarineChemistry,” by Professor H. W. Nurnberg.Department of Chemistry, The University,Exeter.Monday, 17th, 4.30 p.m. : EdinburghScottish Region, jointly with the Edinburghand East of Scotland Section of the CS andthe Heriot-Watt University ChemicalSociety.“Studies of Toxic Trace Metals in theEnvironment by Advanced PolarographicMethods,” by Professor H.W. Niirnberg.Mountbatten Conference Theatre, Heriot-Watt University, Grassmarket, Edinburgh.Wednesday, 2nd, 2 p.m.: SheffieldNorth East Region, jointly with the ModernMethods of Analysis Group of the SheffieldMetallurgical and Engineering Associationon “Air Pollution Monitoring.”Chairman’s opening remarks.“Analytical Techniques for the Estimation ofContaminants in Factory Atmospheres,”by A. F. Smith.“Airborne Dust Sampling in Industry,” byA. Critchlow.“Methods of Ambient Air Monitoring,” byA. J. Apling.“The Sampling and Analysis of Dust Emiss-ions,” by D. Hough.Sheffield City Polytechnic, Pond.Street,Sheffield, S1 1WB.Friday, 4th, 5 p.m.: GlasgowScottish Region : Annual General Meeting.“Potable Spirits,” by W. Dunnett.Room C129, Thomas Graham Building,University of Strathclyde, Cathedral Street,Glasgow.Monday, 7th, 6.30 p.m.: BirminghamMidlands Region.“Applications of Polarography in Environ-mental and Marine Chemistry,” by Pro-fessor H. w . Niirnberg.Lecture Theatre 201, Haworth Building,Chemistry Department, The University,Birmingham.Friday, llth, 6.30 p.m.: ChepstowWestern Region.Discussion on “Asbestos : Its Analysis andImplications,” introduced by B. Sanders.George Hotel, Chepstow.Tuesday, 15th, 6.30 p.m.: SheffieldNorth East Region and Atomic SpectroscopyGYOUP, jointly with the Modern Methods ofAnalysis Group of the Sheffield Metallur-gical and Engineering Association.Discussion on “Analytical Instrumentation-Does the Manufacturer Listen to theUser?” introduced by C. P. Cole and P. E.Kilsby.BSC Sheffield Laboratories (BISRA), HoyleStreet, Sheffield,[continued inside back coverPrinted by Heffers Printers Ltd CambridgeIEnglan

ISSN:0306-1396    

DOI:10.1039/AD97714BX034

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

Vol. 14 No. 10 October 1977 of the Analytical Division of The Chemical Society Fourth International SAC Conference Birmingham, July 1977 The Fourth SAC Conference, the largest ever -held, took place on the campus of the University of Birmingham from July 17th to 22nd. About 470 delegates and accompanying guests attend- ed, representing 33 different countries and fully justifying the description of the Conference as “International.” The Opening Ceremony was held in the Great Hall of the University on the Monday morning.Mr. H. E. Brookes, Chairman of the Local Committee, first introduced Sir Robert Hunter, Vice-Chancellor of the University, whose words of welcome were followed by short addresses from Professor J. C. Tatlow, Head of the Department of Chemistry, Professor R.Belcher, The President, Belcher. Speakers at the Opening Ceremony (L-R): MY. H. E. Brookes, Professor J . C . Tatlow, Sir Robert Hunter, M r . D. W . Wilson, Dr. G. W . C. Milner, Professor R. Belcher. representing IUPAC (and who delivered per- sonal greetings from the President of the Divi- sion of Analytical Chemistry of IUPAC, Professor N. Tanaka), Dr. G. W. C. Milner, Chairman of the Conference Steering Committee, and Mr.D. W. Wilson, President of the CS Analytical Division. Professor Belcher then delivered his Plenary Lecture on “The Resurgence of Analytical Chemistry,” and further Plenary sessions were held on the Tuesday, Thursday and Friday, with papers by Dr. R. P. W. Scott on “High- lights from Contemporary Liquid Chromato- graphy,” Professor Yu.A. ZoIotov on “Hybrid I . D. W . ilson, with Professor R. Methods of Analysis,” and Professor V. A. Fassel on “X-ray Excited Optical Lumine- scence and Phosphorescence and Their Analy- tical Apparatus.” All four Plenary Lectures will appear in full in The Analyst. Plenary Lecturer Dr. R. P. W. Scott (L) with Session Chairman Dr. J . D. R. Thomas. A very full scientific programme occupied three days, with four simultaneous streams of about 100 half-hour papers covering the whole range of analytical techniques and applications.These sessions were complemented each day by Poster Sessions, an innovation a t SAC 275276 FOURTH INTERNATIONAL SAC COKFERENCE Proc. AnaLvt. Div. Chem. Soc. Plenary Lecturer Professor Yu. A . Zolotov (L) with MY. C.Whalley. Conferences, which enable several groups to present their work in pictorial form in the same room and interested delegates to have direct contact with the presenters. About 40 papers were presented in this form. Following the main sessions each day, the specialist Subject Groups of the AD had organised Discussion Meetings on their particu- lar techniques and interests. A further new feature at SAC Conferences was the inclusion of several Workshop Sessions, devoted to newer techniques and teaching methods.Working demonstrations were given and delegates had the opportunity of having detailed discussions on the principles and applications of these novel developments. Concurrently with the scientific programme, which took place in the Lecture Theatres in the Chemistry Department, a comprehensive trade exhibition was held in the Laboratories on three floors.Over 50 companies took stands, including instrument manufacturers, consul- tants and publishers, and the serving of refresh- ments twice each day in the exhibition areas helped to promote closer contact between delegates and exhibitors. This SAC Conference at the University of Birmingham coincided with the retirement of Professor Ronald Belcher, who had been at Birmingham since 1948 and had held the Chair of Analytical Chemistry there for nearly 20 years.It was appropriate, therefore, that the Closing Ceremony on the Friday afternoon should include an appreciation of his work by one of his longest standing colleagues and friends, Dr. W. I. Stephen.In a moving eulogy, Dr. Stephen paid a personal tribute to Ron Belcher and gave his reflections about his mentor’s early days at the University of Dr. G. W. C. Milner (L) with Plenary Lecturer Professor V . A . Fassel. Activity at the Poster Sessions.October. 1977 FOURTH INTERNATIONAL SAC CONFEHENCE 277 Demonstration in progress at one of the Workshop Sessions. Aberdeen and later in the research school he founded and fostered at Birmingham. Recall- ing the authority that Belcher immediately stamped on the heterogeneous collection of students at Aberdeen, Dr.Stephen attributed the dedication that Belcher was able to inspire in them to his characteristic of fostering and encouraging young talent, and indeed it was probably this that led to the creation of one of the most productive research groups in this field in the UK.One of his most exceptional students and his first PhD student was A. J. Nutten, who had a profound influence on Belcher’s academic career and had always occupied a special place in his esteem. Nutten is now the Government Chemist in Hong Kong. In 1948, he said, Ron Belcher moved to Birmingham University, where he was joined a year later by A.J. Nutten and T. S. West from Aberdeen to form a research team that also included S. J . Clark, J. W. Robinson and R. Goulden. All of these original collaborators had since had distinguished careers in analytical chemistry. Dr. Stephen himself joined the team soon afterwards, and he recalled the enthusiasm and team spirit that existed and the high standards that were achieved.One of Ron Belcher’s greatest assets, he said, was his ability to make and keep friendships over long periods of time, and many people owed their deep respect to Belcher for the professional help they had received as a result of such friendship. He particularly mentioned Belcher’s friendship with the late Professor C. L. Wilson, with whom he resurrected the old Microchemical Club as the Microchemistry Group.Ron Belcher had received very many national and international honours throughout his career. However, one had eluded him, said Dr. Stephen, namely the Presidency of the Analytical Division, formerly the SAC. Men- tioning the well known lack of established Chairs of Analytical Chemistry in the UK, he wondered if, had Belcher been President, he might have been more successful in persuading the universities to increase the number of such Chairs, a plea first made in a Presidential Address by A.Chaston Chapman in 1915. The retirement of Ron Belcher would mark the end of an era, and nothing could be the same again at Birmingham. However, through his example and leadership, said Dr. Stephen, those who remained at Birmingham would strive to maintain the high standards and reputation of the School.On the Friday evening, the last and the most ambitious of the social events was held, namely an ox and pig roast in the grounds of High Hall. This provided a fitting climax to an extensive and most successful social programme that extended throughout the week. Lack of space prevents a complete description being given here, but mention of the Grand Buffet on the Monday, trips to Blenheim Palace and Tut- bury, an outing on the Severn Valley Railway, a cruise on the River Severn, a wine tasting and a 16th Century Banquet on the Tuesday evening, and all-day trips on the Wednesday to Harveys of Bristol, the Skol Brewery at Wrexham, Berkeley Castle and Falconry Centre, Warwick and the Stratford Memorial Theatre and the Ironbridge Gorge Museum will bring back long- to-be-retained memories to all delegates.Each278 FOURTH INTERKATIONAL SAC CONFERENCE Proc. Analyt. DiV. Clzem. SOC. of these events was complemented by excellent and appropriate food and drink, and the Social Sub-committee is to be congratulated for making this side of the Conference so enjoyable.An interesting series of industrial visits was also arranged on the Wednesday. On the Thursday evening, the Conference Banquet was held in the Hotel Metropole at Birmingham’s new National Exhibition Centre, and this was preceded by a Civic Reception. Professor Belcher’s toast to the City of Birming- ham was responded to by the Deputy Lord Mayor, Councillor Harold Powell, who also proposed the toast of the Analytical Division.The President, Mr. D. W. Wilson, replied and commented on the role of the analytical chemists in protecting the quality of the environment. Referring to the amalgamation between the SAC and the CS, about which some SAC members had had doubts, he expressed the view that the grass roots of the Society had not (L-R) Mrs.Milner and Dr. G. W . C . Milner being received by the De9uty Lord Mayor of Birmingham, Councillor Harold Powell, and Mrs. Powell. Reflections on SAC 77 Large international conferences have an import- ant and distinctive part to play in the over-all analytical scene, providing opportunities for exchanging scientific information on several levels from broad brush to fine details and from plenary lecture to personal discussion.Living on the campus and participating in the social activities also provide excellent and often unique opportunities for enhancing and en- larging those individual communication links with, for example, international workers in a similar field, which are so invaluable for the practising analyst. I have always found the SAC Conferences to be particularly good in both areas, though for me this one got off to a rather slow start.Having driven 200 miles down a wet and windy motorway I was not in the mood (L-R) Professor R. Belcher, Mrs. Belcher, Councillor Harold Powell, Mrs. Powell and MY. D . W . Wilson at the Conference Banquet. suffered, and its vigour continued within the AD, as demonstrated by this Conference. The President thanked all those who had played a part in ensuring that the Conference was a success, and proposed a toast of the guests.Professor A. W. Johnson, President of The Chemical Society, responded and congratulated the Division on its long and distinguished history. He looked forward to 1980, when it was hoped that unification between the CS and the RIC would take place, and he said that he was convinced that a majority of members favoured such a move. So ended the Fourth SAC Conference, one which again lived up to the reputation and repeated the success of earlier Conferences.In the following pages three well known and active members of the AD, Mr. G. E. Penketh, Dr. J. D. R. Thomas and Mr. J. Whitehead, give their personal views of the week’s events in the context of current developments on the analy- tical front.to receive the gloomy messages at the opening ceremony, the uncertain future of chemists, the troubles of the chemical departments in universities, “tunnelling through the hump,” the need for more chairs in analytical chemistry, etc. I happen to believe that the future of chemists in general and of analysts in particular is rather bright, especially for those who are prepared to tackle scientific and technological problems in the collaborative, interdisciplinary manner that will be required.This does not mean that we need new, trendy interdisciplinary courses that would solve the problem of vacant science places by allowing lower entrance standards; on the contrary, I think that for chemists “fewer but better’’ is the best policy.However, things began to look up when weOctober, 1977 FOURTH INTERNATIONAL SAC CONFERENCE 279 moved on to the scientific programme, even though one was faced with the usual difficulty of how to select from the 160 or so papers present- ed, in a situation where, taken overall, the signal to noise ratio was a t first sight very modest.The selection was complicated by the normal practice of organising all lectures other than the Plenary ones in three or four streams, and this year there were the additional options of poster sessions and workshops. The idea of poster sessions was imported from the Continent, where it usually works well. The author is allowed a certain area to hang his wall charts and is available for an hour or so to discuss them with interested parties.In principle, this offers much better facilities for getting to grips with the work than the more formal 20-25-min lecture with time for one or two questions at the end, but as practised a t this Conference seemed to lack cut and thrust, and certainly as a spectator sport seemed about as exciting as ladies’ cricket.Nevertheless, the potential is there and I would certainly like to see the experiment repeated. The workshop sessions were another new feature of the Conference, the idea being that working demonstrations of novel developments should be given by their inventors who would, of course, be available for extended discussions on the beauties and warts of their masterpieces.The choice of subjects seemed to me to be excellent, but again the novelty of the thing seemed to be fighting a losing battle with the reticence of the participants and, with a few honourable exceptions, I did not detect the expected enthusiasm from the delegates. The workshop seemed in a rather odd sort of way to emerge as a competitor to the exhibition rather than as an event of genuine scientific interest, but once again I would like to see the idea persevered with.To return to the difficulties of selection, I like to use a conference such as this to check out my perception of the growth points in analysis, to look out for new techniques of potential use to me and to seek information on current problems with which I am grappling. One clear growth point is the use of micro- processors ; their steadily decreasing cost taken together with the improving reliability of electronics promise to revolutionise the design of analytical instrumentation, making them both simpler to operate and capable of generating more sophisticated data.The trend was discernible in some of the equipment on display in the exhibition and in the workshop sessions on Black Boxes organised by the Special Techniques Group and on Flow Injection Analysis and Microprocessors by Rhbitka, Hansen, Betteridge and Stewart. However, the messages were less clear in the papers, with emphasis still on larger computer systems and dedicated minicomputers.An exception was an interesting paper by Gidley and Jones, who were developing microcomputer systems for handling data from a variety of relatively small instruments producing intermittent low-speed data.This would include much useful manage- ment/administration data such as costing. There were a number of papers on the really heavyweight instruments, neutron activation, nuclear microprobes, ESCA and energy- dispersive XRF, and a couple of papers from Sweden on Proton Induced X-ray Emission or PIXE.I cannot resist quoting the comment of one delegate that he hoped they would find a gnome for it! Seriously, though, I am begin- ning to have grave doubts about whether some of these instruments that are so expensive in terms of both capital and specialist personnel would stand up to a realistic cost/benefit analysis. There was, however, a most interesting series of papers on a technique that certainly would come out of such an analysis very well, namely flow injection.Papers by RfiiiCka and Hansen outlined the principles of the method, which is based on controlled dispersion of the injected sample in a laminar flow of reagent. Applica- tions described included various ‘spectrophoto- metric measurements, use of ion-selective electrodes and titrations.Karlberg described a system for redox-potential detection and Betteridge et al. one for viscosity determination. At a poster session, Stewart et al. gave further examples involving colorimetry, fluorescence and atomic absorption, and covering phosphate, trypsin, amino acids, proteins, ammonia and some heavy metals. It was inevitable, given the location, that molecular emission cavity analysis (MECA) would be prominently displayed, and the Imperial College team gave good exposure of another of the “newer” techniques, optoacoustic spectrometry.Both are now fairly well known and I regard them still as promising rather than earth-shattering novelties. There is a welcome trend towards obtaining molecular information rather than elemental composition and an example of a small step in this direction was a paper by Robinson and Keisel on a GC -‘AAS combination for speciation of metals in the environment.However, throughout the Con- ference there was no indication of any new technique to assist in the substantial problems of surface chemistry, where, for example, detailed information on the molecular events a t280 FOURTH INTERNATIONAL SAC CONFERENCE PYOC.Analyt. Div. Clzem. SOC. a catalyst surface would be invaluable. My over-all impression was that in the purely technical area analysis seems to have reached one of its periodic plateaux, although there is a lot of activity in the whole field of data handling. In discussions with a substantial number of delegates the problems of most concern seemed to be people related, managerial, administrative (with a lot of emphasis on safety) and economic, and these were not simply confined to the UK.However, the fact that the Conference provided an informal forum at which such problems could be aired was of itself beneficial and rein- forces my views on the value of international conferences in general and of the SAC Confer- ences in particular.G. E. PENKETH The Fourth SAC Conference: A Treasure Chest It cannot be claimed that every detail of the new tools and techniques of analytical chemistry was represented at the Fourth SAC Conference, but its 150 papers in lecture and poster presentation, workshops, trade exhibition, industrial visits and other activities provided a bottomless treasure chest for the more than 450 delegates present.It is an impossible task to survey the whole proceedings or to convey the impressions of all those brought together in July 1977 to Birmingham from over 30 countries spread over five continents and from academic and indus- trial walks of life. All that can be done here is to express some personal reactions. Patterns of Progress Many came for a view of analytical chemistry.This must have applied particularly to the many young people who were there. As a first conference for any one individual, the Fourth SAC Conference could not have been any less memorable than any other of this kind. How- ever, an important attraction of any conference is in what is new. The Conference did not provide anything startlingly new-few do.There are, however, patterns of progress and this Conference introduced and in some inst- ances re-introduced delegates to developments such as flow-inj ection analysis, microprocessors, minicomputers, interfacing of high-perfor- mance liquid chromatography with mass spec- trometry, foam-supported extractants, opto- acoustic spectrometry and molecular emission cavity analysis.These were enthusiastically presented, some in the workshops, which were the scene not just of white hot technology but also for illustrating some of the learning methods and concepts of modern analytical chemistry teaching in British universities and polytechnics. The workshops along with poster sessions were a new enterprise for SAC Conferences. Their warm reception is a commendation for them to be associated with future events.The hard selling line associated with the presentations in these and, indeed, with all presentations, albeit at different levels of effectiveness, has to be taken if a technique or method, however sound, is not to lie buried and forgotten. Rarely do new developments coincide with a need, as occurred with the fluoride ion-selective elec- trode, which appeared at almost exactly the moment of intense and mounting interest in the need for simple methods of fluoride analysis created by dental health and water fluoridation programmes.Tswett’s column chromato- graphy lay dormant for many years and even with a hard sell it took a few years for Walsh’s atomic-absorption spectroscopy to be properly appreciated.It can be said that chromatography, electro- chemistry and spectroscopy dominated the Conference : three quarters of the presentations were concerned in some way with one or more. Except for electrochemistry, these were reflected in the plenary lectures. In passing from one SAC Conference to another, a shift in emphasis is observed for each of these three techniques; HPLC, ion-selective electrodes and graphite furnaces were almost unheard of at the time of the First SAC Conference, held in 1965.This Conference heard much of these and an interest- ing modification for improving the graphite furnace for atomic-emission spectrometry was a nice bonus for the captive audience that was present to await the superb oration on Professor Ronald Belcher that will be remembered both for its content and for marking the end of an era at Birmingham and the beginning of another.The work horses of ultraviolet - visible and infrared spectroscopy came before the SAC Conferences commenced in 1965 and analytical chemists had already provided the sample loads for utilising the techniques and had widened their scope by such developments as those of colour-forming reagents.In these days of greater awareness of the nature and benefits to be derived from spectroscopic signals, dedicated research analysts who appreciate the kind of problems requiring solution are ready to take on variations in the harnessing of these signals. This was to be seen at the Conference for plasma sources, photoelectron and Auger spectroscopy, molecular emission cavity analysis, cando-October, 1977 FOURTH INTERNATIONAL SAC CONFERENCE 281 luminescence, optoacoustic spectroscopy, proton induced X-ray emission, etc.Laser methods were absent. Combination of Techniques The combination of techniques, or hybrid methods as used in the title of one Plenary Lecture, is tantamount to new developments and, spurred on by successes such as those of gas chromatography and mass spectrometry, there is a greater willingness to try others; not that analytical chemists have been reticent in this.Many were reported upon and generally involved a form of spectrometry. Thus, atomic-absorption spectroscopy cropped up in conjunction with gas chromatography, ion exchange and solvent extraction ; a plasma emission detector with gas chromatography ; fluorimetry with thin-layer chromatography ; and particle-induced X-ray emission analysis with gamma-ray detection.Some instances complemented each other more than others, but it is hardly possible to get more unison between vastly different techniques than in “spectro- electrochemistry,” where the intensity of a light beam passing a t grazing incidence across a platinum electrode surface is monitored during electrolysis of dilute aqueous metal ion solu- tions.The use of neutron activation with atomic-absorption spectroscopy was distinctly separate in an application concerning the extent of deposition of heavy metals in moss samples. Physical presence a t the scene was frequently needed in order to catch a glimpse of a particular item of treasure. A presentation that might from the summary have concerned simply analysis by GC - MS was almost entirely devoted to what might well be the first instance in Britain of HPLC - MS.Chemistry Remains The emphasis of the Conference did not lie purely in instrumentation and on-line continuous and automatic methods of analysis. Although the shift of analytical chemistry in this direction continues unabated, there was plenty of evidence that the chemistry still remains. There were, of course, the normal exercises in titrimetry (of metal ion containing solutions and the associated complications of hydroxide precipitations), complexometry , spectrophoto- metry, conventional potentiometry, indicator and reagent design, etc.In addition, there were derivatisation agents for GC and HPLC, design of new sensors for ion-selective electrodes and enzyme systems in further applications of the ring oven.Xanthogenate chelates and 1,lO-phenanthroline figured in a pre-concentra- tion procedure of several elements from high- purity manganese using filter-paper covered in activated carbon or silver iodide, respectively, for sorption. Although various chemical procedures provide sufficiently reproducible stoicheiometries for analytical utility, there is frequently a need for further information on mechanism.This is a realistic activity for a research analyst, an example from the Conference being a clescrip- tion of the mechanistic study of the tetrazolium method for determining corticosteroids in pharmaceuticals and biological fluids and illustrating the application of ultraviolet, infrared and mass spectrometry.Health, Safety and the Environment Various aspects of health, safety and environ- mental concern have stimulated progress in analytical chemistry. These were evident in applications described by developers of tech- niques and in presentations such as the infrared analysis of asbestos and quartz, and ultrasonic techniques in remote instrumentation in the reprocessing of corrosive, toxic and highly radioactive liquors in nuclear fuel reprocessing.Papers that scanned areas in these fields were of more general interest and although they may not have inspired specialists in techniques, they set out to give an appreciation of the problems fac- ing the analyst in areas such as natural waters and air pollution.Some of these included attention to sampling and pre-concentration. Wide-ranging presentations such as current re- search projects in air pollution control appeared to be well received. On another tack, descrip- tions of a programme concerning assessment of glass-electrode performance and alkaline errors, standard pH buffer solutions, performance of reference electrodes, etc., evoked considerable interest.The revised British Standard speci- fication on laboratory pH measurements upon which Technical Committee LBC/16 of the British Standards Institution is now engaged will provide details of the recommendations arising from this. Specialist Subject Group Sessions Although they had been introduced at the Centenary Conference in 1974, sessions by Groups of the Analytical Division to end each day’s proceedings constituted another new feature for a SAC Conference.The experience was mixed, but the themes were generally sound. A discussion on immunological assay techniques ought to have been a sell-out; was282 FOURTH IXTERNATIOKAL SAC CONFERENCE Proc. Anabi. Diu. C I Z P ~ . SOC’ this too general a conference for this theme? Much could have been gained by the presence of more delegates whose main concern was in clinical chemistry and analytical biochemistry.On the other hand, deliberations on new directions in voltammetry and ion-selective electrodes evoked a large audience and a discussion that had to be prematurely brought to an end even after I t h. How can organisers predict which discussions are likely to burn brightly ? Conclusion One may question the value of a conference of this kind, devoted as it was to the whole field of analytical chemistry.The subject is too big for all the treasure to have been at Birmingham. However, the sample was generally good and it is imperative that even specialists should from time to time look over their shoulders a t the other man’s approach.The Fourth SAC Conference provided this opportunity. The chance remark here, or the comment there, or the glimpse of something, be it in a lecture, poster session, workshop, industrial visit, exhibition or during a social event, is the gem to take away and ensure that the Conference will have been a success. Many were taken away, but some can still be shared through the Conference Handbook.* J. D. R. THOMAS Impressions of the Fourth SAC Conference 1977 “Go forth and enjoy yourselves” was the exhortation of Dr. G. W. C. Milner, Chairman of the Conference Steering Committee, a t the end of his opening remarks on the first day of the Conference, and 470 delegates did exactly that during the following week.The organisers excelled themselves in providing a very full scientific and social programme and, because of their exemplary arrangements and careful attention to detail, the programme was com- pleted without obvious mishap. All who had anything to do with the organisation and running of the Conference deserve the highest praise and must feel gratified a t its obvious success. There were occasions when the choice of what to do was bewilderingly large-one of four lecture streams, poster or workshop sessions, a visit to the exhibition or simply a * Copies of the Handbook, 4th International SAC Conference, Birmingham, 17-22 July 1977, are still available, price i4, from the Secretary of the Analytical Division, The Chemical Society, Burling- ton House, London, WlV OBN.discussion with a colleague. Inevitably, the attendance at some lectures suffered because of this and I felt sorry for some lecturers who had clearly gone to some trouble to prepare their contribution only to find that their audience did not reach double figures. It is of interest to examine the statistics of the scientific part of the Conference: 40% of the people who attended were from academic institutions, 30% from industry, 20% from public service organisations and 10% from instrument manufacturers.The number of papers amounted to nearly 150, the greater proportion of these (70%) originating from academic institutions and only 10% from industry. The position was different a t the last comparable SAC Conference at Durham in 1971 when the figures were 49% and 30%, respec- tively.Perhaps the organisers of the next Conference could bear these figures in mind and turn their attention to investigating methods for enconraging participation by analysts from industry. The Plenary Lectures will be published in full in The Analyst a t a later date and it is therefore unnecessary to comment on them in detail here. Suffice i t to say that each was excellent and the speakers fully justified the decision of the Programmes Committee to invite them.The poster sessions aroused considerable interest as this was the first time they had been used a t a major analytical conference in the UK. There were six sessions a t which 41 papers were displayed. There is no doubt that this is an attractive way of putting over scientific infor- mation.The difficulties that can occur when speakers are presenting their papers in an unfamiliar language or showing illegible slides or legible slides for too short a time are eliminated. It is also possible to have a detailed discussion with the author(s) of the paper and to discuss any points in the presentation that are not clear. Because of shortage of time and to restrict the period when the authors are present, the presentation was limited to 1 h, although in practice extra time was usually available at the beginning and end of each session.This increase in time could perhaps be recognised officially at future Conferences. All of the poster presentations were first class and there is no doubt that this method will be used at subsequent Conferences.Another innovation was the use of workshop sessions where people had the opportunity to display equipment and to demonstrate it in actual operation. There were sessions devoted to Experiments and Aids for the Teaching of Analytical Chemistry, Black Boxes, Opto-OctoSev, 1977 FOURTH INTERN-TIONAL SAC CONFERENCE 283 acoustic Spectrometry, Flow Injection Analysis and Microprocessors, and Molecular Emission Cavity Analysis.As the week progressed these increased in popularity and those on Thursday were particularly well attended. It should not be forgotten that the emphasis in these sessions should be placed firmly on the practical demon- stration and impact is lost if, for example, videotape demonstrations are used instead. A trade exhibition in which 56 organisations took part was staged in the Laboratories of the Haworth Building.This gave an excellent opportunity for delegates to have discussions with the representatives of the participating Companies and was well attended. It is difficult t o summarise the lecture programme when it is only possible to attend approximately one third of the lectures.The organisers of the programme accomplished the mammoth task of arranging the papers under the following subject headings : Chromatography and Separations, Pollution and Environmental Control, Nuclear and Radiochemistry, Newer Techniques, Particle Size Analysis, Microchemi- cal Techniques, General Analytical Methods, Computer Techniques, Spectrometric Methods, Analysis in the Life Sciences, Atomic and Molecular Spectroscopy, Electroanalytical Methods, Atomic Spectrometry and Instrumen- tation in Analysis and Molecular Spectrometry.There were instances, however, where classifica- tion proved difficult. Brief comments on the papers I attended are as follows. Miss Fujimori of the Science University, Tokyo, in a paper entitled “Round Robin Studies for the Estimation of Accuracy and Precision in Environmental Control Analysis, ” presented the results of a survey of the determin- ation of Cu, Pb, Zn, Cd and Cr in industrial waste waters and of NO, in air.Dr. Ure of the Macaulay Institute for Soil Research, in his paper “Comprehensive Quanti- tative Trace Element Analyses by Spark Source Mass Spectrometry,” showed how the technique had been used for the quantitative determination of trace elements in complex materials such as soils, rocks and plants.It was of interest that he used aluminium powder as conducting agent rather than the more conventional graphite. This paper was follow- ed by Dr. Malone of Air Pollution Control, San Bernadino, California, who described the work of his unit and, in particular, three aspects of this work.The first project was to compare two independent methods for determining nitrogen oxides, the second to determine the stability of calibration of an ozone monitor and the third the stability of air samples over 16 weeks with respect to concentrations of nitrates, sulphates, lead and iron. The candour of this lecturer in admitting he could occasionally be in error was refreshing.In a paper on ultrasonic techniques for remote instrumentation of chemical plant, Dr. Asher of Harwell described some ingenious devices for measuring concentrations, locating interfaces in liquid -liquid extraction or the depth of precipitates in systems involving hazardous chemicals. Pulsed beams of high-frequency ultrasound (0.5-10 MHz) are used and a full description of the necessary electronic techniques was given. Although primarily designed for systems containing uranium and plutonium it could be applicable to other toxic or corrosive chemicals.The last hour on Monday, Tuesday and Thurs- day was allocated to the Groups of the Division. On the first day I attended the Particle Size Group session, where Dr. Scarlett of Lough- borough University of Technology reviewed the work of the Particle Size ,4nalysis Sub-commit- tee.The results of the work of the Committee, which has been engaged in reviewing methods of particle size analysis, will be published towards the end of 1977 and for anyone interested purchase of the work is essential. Dr. Scarlett reviewed the whole field of particle size analysis and it is a pity that his excellent account had an audience of only 16.The following day I attended the first two of a series of four lectures on flow injection analysis systems. Dr. RfiiiCka of The Technical Uni- versity, Denmark, after claiming in his opening remarks that the invention of flow injection analysis was comparable to that of the pocket calculator, went on to justify his claim in his clear and concise manner.The simplicity of the equipment and the quality of the results obtained make this an attractive technique for those engaged in routine analysis of a large number of samples. Further practical examples were given by Dr. Hansen of the same University in his paper in which the uses of limited and large degrees of sample dispersion were used. In the afternoon, Dr.Wilkinson of the British Carbonisation Research Association reviewed the occurrence of trace elements in coal and coke and gave an account of the methods used in their determination. Dr. Ottendorfer of the Bundesanstalt fur Wassergiite, Kaisermuhlen, Vienna, in a humo- rous and interesting lecture discussed problems associated with the examination of natural waters and in particular the changes they undergo after sampling.His quotation of “Murphy’s Law”-if something can go wrong it284 FOURTH INTERNATIONAL SAC CONFERENCE Pvoc. Analyt. Diz. Chern. SOC. will do so, sooner or later-exemplified his grasp of the English Language. Dr. Gidley, Unilever Research Laboratory, in a paper entitled ‘Microcomputers-Evolution or Devolution in Analytical Chemistry,” des- cribed an ambitious concept for storing and processing data from a variety of analytical instruments using a Hewlett Packard 9825A calculator.A t the final lecture session on Tuesday I expected to hear a paper on metal ion separa- tion but I missed the announcement that the lecture had been cancelled. I stayed, along with an audience of 14, to hear Mr. Shaw of Glaxo Laboratories Limited give a superb presentation of his experiences with the IATROSCAN, an instrument that combines thin-layer chromato- graphy with an automated quantitative detec- tion system based on the flame-ionisation detection principle.The output from the instrument was presented graphically and audibly. At the Microchemical Methods Group meeting which ended proceedings on Tuesday, a discus- sion of Instrumental Techniques for Trace Analysis and Small Samples was led by Pro- fessor Malissa and Dr.Pantony, who considered, respectively, organic and inorganic aspects of the subject. The social side of the Conference had been well taken care of, starting with a Grand Buffet on Monday evening and the choice of five evening tours on Tuesday. The only rain of the week occurred as a torrential downpour just before the buses set out on Tuesday evening.Fortunately i t was of short duration and the evening was fine and sunny. I went on the River Severn cruise, which was most enjoyable ; the organisers even managed to select a boat with a pseudo-chemical name-the Carbolate ! Wednesday was a so-called rest day and one could choose between five tours and three industrial visits followed by a visit to the Stratford Memorial Theatre to see a perfor- mance of Henry VI, Part 3.There is no doubt that the many expressions of pleasure from delegates and their guests who went on the trips were just reward for the many hours of work put in by the organisers. On Thursday, Dr. Covington of Newcastle University discussed the work of the British Standards Institution Committee on pH mea- surement and Dr.Bogdanski of Birmingham University gave a lecture on the applications of MECA with a vapour-generating system. He presented an impressive list of results showing that the method compared very favourably with other methods of analysis. Dr. Michel described the work carried out at the University of Strathclyde on the develop- ment of an atomic-fluorescence flame spectro- photometer for the analysis of clinical materials and dealt particularly with the determination of Cd and Zn.A significant breakthrough in the construction of electrodeless discharge lamps was claimed. Discussion meetings were popular with the Groups and the Atomic Spectroscopy Group’s contribution was entitled “The Role of the Analytical Laboratory-Its Responsibilities and Obligations.” It was difficult to envisage any opposition view succeeding after the ex- cellent opening contributions by Frank Shenton, Clifford Whalley and Hugh Hughes speaking on behalf of public, industrial and state laboratories, respectively.On Friday morning I was privileged to take the chair a t the session on Instrumentation in Analysis, which included lectures by Dr.Majer of Birmingham University on “A Mass Spectro- metric Survey of Metal Chelates,” Dr. Games of University College Cardiff on “Applications of Newer Mass Spectral Techniques in the Analy- sis of Organic Compounds” and Mr. de Ceuninck of the University of Antwerp on “Digital Electrical Detection Spark-source Mass Spectro- metry.” Dr.Majer was primarily concerned with the use of mass spectrometry for eluci- dating structures of volatile metal chelates such as nickel salicylaldoximate, but I was also impressed with the sensitivity of detection of the metal, which was stated to be 10-l2 g. Dr. Games in his abstract said he hoped to present results obtained by using a moving-band HPLC - MS interface. In actual fact, although he had only been working with the technique for a few weeks, he gave a very impressive account of the results he had achieved on the analysis of drug mixtures. Mr. de Ceuninck described the work he had carried out on coupling the magnetic- scanning electrical detection output of a spark- source mass spectrometer to a multi-channel analyser. The applications he discussed were concerned with the analysis of conducting materials. In the afternoon session, Dr. Lucchesi of the North Western University, Illinois, and Con- tributing Editor of Analytical Chemistvy, gave an interesting account of the role of the analy- tical chemist in problem solving, including the important aspect of definition of the problem. Dr. Bogdain of Kontron GmbH gave a clear and concise account of the performance of an induction fusion furnace for the preparation of samples for X-ray fluorescence analysis, and finally Dr. Ottaway gave a comprehensiveOctober, 1977 ORIGINAL PAPERS IN summary of the work being carried out a t the University of Strathclyde on carbon furnace atomic-emission spectrometry. He dealt par- ticularly with modifications to the design of the graphite tube to optimise performance and concluded with an account of the many applica- tions of the technique. I was asked to write an article containing my personal views of the Conference and I must apologise for not mentioning the authors of the many papers abstracted in the Handbook, which PHARMACEUTICAL ANALYSIS 285 has now unfortunately virtually disintegrated with the amount of use it has had. My apologies also to the many other people I should have mentioned by name and have been unable to do so. In conclusion, I do not think I have come away from the Conference fired with enthusiasm to try out new techniques but what I have learnt and heard has enabled me to consolidate my views and I look forward with anticipation to the Fifth SAC Conference. J. WHITEHEAD

ISSN:0306-1396    

DOI:10.1039/AD9771400275

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

October, 1977 ORIGINAL PAPERS IN PHARMACEUTICAL ANALYSIS 285 Original Papers in Pharmaceutical Analysis The following are summaries of five of the papers presented at a Meeting of the Joint Pharma- ceutical Analysis Group held on May 17th, 1977, at the Pharmaceutical Society of Great Britain, London. Mercu rimetr ic Assay of Pen ici I I i ns A. E. Bird and Miss C. E. Redrup Beecham Pharmaceuticals Research Division, Chemotherapeutic Research Centrs, Brockham Park, Betchworth, Surrey, RH3 7AJ In this method1 the penicillin, I, is titrated with mercury(I1) ion before and after hydrolysis by /3-lactamase or alkali to the penicilloic acid, 11.The sample-blank titration of unhydrolysed penicillin gives a value for any penicilloic acid or other mercury-titratable impurities present and the purity is calculated from the titre difference, assuming a 1+1 mole reaction between mercury(I1) ion and penicilloic acid.Paal and Molnar2 investigated the chemistry of the titration and found that fission of the penicilloic acid occurs, with formation of the penillo- aldehyde and the mercaptide of dimethylcysteine. I II Results and Discussion Initial investigations (1971-72) of the method in our laboratory gave promising results, but further experience revealed an unacceptably large variation in the apparent purity with change of sample concentration, and the work was abandoned.Karlberg and Forsman3 published a modification of the method and reported excellent results for four penicillins. They used continuous addition of titrant but gave no information about the titration speed or temperature used.Application of their method (using a moderate titration speed at room temperature) to a pure benzylpenicillin (I, R = PhCH2-) sample gave results above loo%, so the effect of variation of titration conditions was investigated. Positive systematic errors can occur in titrations that use continuous addition of titrant.4 These errors can be avoided4 by maintain- ing a high value of #I, defined by #I = kct, where k is the reaction rate.between titrant and sample, c is the initial sample concentration and t is the time taken to titrate to the equivalence point. The effect of these factors on the titration of benzylpenicilloic acid (11, R = PhCH,) with 0.02 M mercury(I1) nitrate was investigated, using variation of titration temperature to change the value of k .Titrations were carried out with a Metrohm potentiograph Model E336A with 1- and 10-ml burettes for low and high sample concentrations, respectively. Platinum foil (1 x 0.5 cm) and Orion double-junction calomel electrodes were used. A total286 ORIGINAL PAPERS IN PHARMACEUTICAL ANALYSIS Proc. AnaZyt. Div.Cltem. SOC. TABLE I EFFECT OF TITRATION TIME, TEMPERATURE AND INITIAL SAMPLE CONCENTRATION ON MERCURIMETRIC TITRATION OF BENZYLPENICILLOIC ACID Concentration/mg Relative titre Approximate timelmin Temperature/"C per 35 ml (mean of 3) 5 22 6 100 10 22 6 99.2 15 22 6 98.3 20 22 6 98.1 15 15 15 15 15 15 22 35 45 22 22 22 6 6 6 6 30 60 100 98.6 98.8 100 99.8 99.2 volume of 35 ml was titrated, comprising 15 ml of aqueous sample solution and 20 ml of 0.2 M, pH 4.8, acetate buffer solution.Table I shows that all three factors significantly affect the titre, so the question arises of what titration conditions will give the correct result. Van der Linden's paper implies that the conditions should be adjusted in the direction of increasing until a constant assay result is obtained.Titrations were carried out as outlined above and the titrant was standardised with AnalaR disodium EDTA. All sample-blank titrations were carried out at room temperature in order to minimise the possibility of decomposition of the penicillin during the titration. The results in Table I1 show that the lowest value of 8, corresponding to the lowest values of titration time, concen- tration and temperature, gives an impossibly high assay.Increasing 18 by increasing the time to 15 min shows a marked drop in assay, and a further drop is observed when the temperature This is illustrated by some assays of a benzylpenicillin sample. TABLE I1 BENZYLPENICILLIN ASSAY RESULTS Approximate time] min 5 15 15 15 15 30 Variable* Concentration/ mg per 35 ml 6 6 6 60 60 60 60 Temperature/ "C 22 22 35 22 35 35 22 Assay, %f 100.9 ( 1 0) 102.8( 5) 99.6 (6) 100.1(4) 99.6 (4) 99.7 (2) 99.7 (4) * Fast to just before end-point, then very slow over end-point.Figures in parentheses are the number of assays averaged. or concentration is increased, with the increase in temperature having the greater effect. Further increase in /3 is shown to produce no additional decrease of result.The figure of 99.6 or 99.7% is probably a reasonable estimate of the purity of this sample, which gave a blank titration equivalent to 0.2% of benzylpenicilloate. Less detailed assays of several other penicillins showed similar effects of variation in titration conditions. In particular, results at low concentration at 35 "C were generally 1-274 below those obtained at 22 "C.Karlberg and Forsman3 reported satisfactory results for ampicillin (I, R = PhCH-). However, we have found (Table 111) an increase of result on increasing the titration temperature for both ampicillin and amoxycillin, (I, R = p-OHC,H,CH-) i.e., the opposite effect to that I NH2 I I NH,October, 1977 ORIGINAL PAPERS I N PHARMACEUTICAL ANALYSIS TABLE I11 287 AMPICILLIN AND AMOXYCILLIN ASSAY RESULTS WITHOUT ACETYLATION Titration time about 15 min.Assay, %* rp--- Concentration/ - mg per 35 ml Temperature/ 'C Ampicillin Amox ycillin 6 22 86.7(6) 85.8 (3) 6 35 88.2 (6) 86.5( 3) 45 22 86.0 ( 5 ) 86.1 (3) 45 35 89.0(3) 88.5(3) * Figures in parentheses are the number of assays averaged. seen with other penicillins. Further, the results obtained at 35 "C are so high that they cannot be correct, because these compounds are trihydrates with maximum purities of 86.2 and 86.7%, respectively.The titration curves of both compounds showed an additional small inflection beyond the normal second end-point (Fig. 1A) and a green - yellow coloration developed in the solution during titration. These effects were greater in titrations at 35 "C than at 22" C, although they were visible at the lower temperature, especially at the higher concentration.Thus, it is obvious that some interference occurs in mercurimetric titration of these a-aminobenzylpenicillins and it seems likely that this involves interaction with the amino group. The effect is apparently similar to that observed with 6-aminopenicillanic acid,5 which can be overcome by preliminary acetylation of the amino group.Introduction of the acetylation step for ampicillin and amoxycillin gave realistic results (Table IV) whose variation with titration temperature is consistent with that observed for other penicillins. Also, the additional inflection in the titration curve (Fig. 1B) and the coloration of the solu- tion were lost.Variation of titration temperature and time in the standardisation titration of EDTA was found to produce only small changes in titre, which were probably within experimental error, so a 15-min titration at room temperature was routinely used. It is obvious that the chemistry of this titration is very different from that of titration of a penicilloic acid and it is possible that this may introduce some bias into the results.Ideally, the standard compound should react with titrant in the same way as the samples. Preliminary studies with thiazolidine-4- carboxylic acid suggest that it may be a suitable standard if it can be prepared in a highly pure state. PotentiaVmV Fig. 1. Potentiometric titration at 35 "C with 0.02 M niercury(T1) nitrate solution added a t about 0.5 ml min-1.A, About 60 mg of ampicillin in 25 ml of water, hydrolysed with 2 ml of 8-lactamase solution (16 000 A.V.M. units) for 5 min and 20 ml of 0.2 M acetate buffer, pH 4.8, added; B, about 60 mg of ampicil- lin in 5 ml of 0.1 M borate buffer, pH 9, plus 2 ml of a 5% solution of acetic anhydride in acetonitrile for 3 min, hydrolysed with 5 ml of 2 M sodium hydroxide solution for 5 min, 5 ml of 2 M nitric acid and 20 m l of 0.2 M acetate buffer, pH 4.8, added.288 ORIGINAL PAPERS IN PHARMACEUTICAL ANALYSIS PrOC.A.naZyt. DiV. ChW. SOC. TABLE IV AMPICILLIN AND AMOXYCILLIN ASSAY RESULTS WITH ACETYLATION Titration time about 15 min. Assay, yo* Concentration/ - I A rng per 35 ml Temperature/' C Ampicillin Amoxycillin 6 22 85.9(3) 86.3 (3) 6 35 85.3(2) 85.8(3) 55 22 85.2(6) 85.6(3) 55 35 85.3(2) 85.7(3) * Figures in parentheses are the number of assays averaged. Conclusion It is clear that titration speed and temperature and initial sample concentration are inter- related parameters, the values of which can determine the validity of the result obtained.Our results suggest that titration at 35 "C for 15 min with a sample concentration of 6 mg per 35 ml or more will be satisfactory for most penicillins, but other values for these parameters could also give satisfactory results.It is important that when an analyst first uses the method, variations of conditions are investigated to ensure that a valid result is obtained. This investigation is especially important when the method is applied to a particular penicillin for the first time.It is also, of course, necessary to ensure that complete hydrolysis of the penicillin is obtained for the sample titration. References 1. 2. 3. 4. 5. Korbl, J., and Vanicek, V., Czech Pat. No. 132991, 1969. Paal, T., and Molnar, M., Gydgyszere'szet, 1976, 20, 8. Karlberg, B., and Forsman, U., Analytica Chim. Acta, 1976, 83, 309.Van Der Linden, W. E., Analytica Chim. Ada, 1975, 77, 327. Forsman, U., and Karlberg, B., Analytica Chim. Acta, 1976, 86, 87. Specific Determination of Phenoxymethylpenicillin Alan S. Hughes and Christopher R. Popplestone Dista Products Limited, Fleming Road, Speke, Liverpool, L24 9LN This summary describes our recent work on the assay of phenoxymethylpenicillin.In the first two sections are discussed the current BP method of assay and interferences arising from the presence of acid-labile penicillins, such as benzylpenicillin, or from $-hydroxy- phenoxymethylpenicillin. In the third section a new method for phenoxymethylpenicillin, in which interferences from other penicillins are eliminated or accounted for, is described. The BP assay for phenoxymethylpenicillin19z involves two absorption measurements at 268 nm, referred to as the El and E, values.A solution of the sample is prepared and the absorb- ance measured (E,) , Phenoxymethylpenicillin is precipitated, using sulphuric acid, and the absorbance of the filtrate is measured ( E J . The E, value is intended to correct for non- phenoxymethylpenicillin impurities, such as phenoxyacetic acid, which have contributed to the E, value.The E, value is subtracted from the E, value to give the effective absorbance due to phenoxymethylpenicillin. The method allows for any phenoxyacetic acid, but as this work shows, the two other major impurities in phenoxymethylpenicillin, i.e., benzylpenicillin and p-hydroxyphenoxymethylpenicillin ($-OH penicillin V) interfere with the method to an extent that it renders it unsuitable as a specific assay for phenoxymethylpenicillin. Interference by Benzylpenicillin and other Acid-sensitive Penicillins Benzylpenicillin has a very low absorbance at 268 nm and any present in samples of phen- However, benzylpenicillin is acid-unstable and under the acid conditions of the BP assay oxymethylpenicillin would not be expected to contribute to the El value.October, 1977 ORIGINAL PAPERS IK PHARMACEUTICAL ANALYSIS 289 is easily degraded to benzylpenicrllic acid, which absorbs strongly in the ultraviolet region at 268 nm, although, unlike phenoxymethylpenicillin, it does not show a maximum.The pres- ence of benzylpenicillin thus reduces the assay result in two ways.It correctly causes a low E, value but the effect of the acid treatment produces an erroneously high E, value. In fact, the combined effects of the presence of, e.g., 5% of benzylpenicillin in phenoxymethylpenicillin, causes an incorrect reduction of about 10%. This effect was demonstrated by adding benzyl- penicillin to a sample of phenoxymethylpenicillin and putting each mixture through the BP procedure to provide the results given in Table I.TABLE I BP ASSAY OF POTASSIUM PHENOXYMETHYLPENICILLIN SPIKED WITH SODIUM BENZYLPENICILLIN Penicillin V-K r A \ Found, yo Theoretical, yo .. . . 96.5 - Penicillin V-K salt control . . .. . . Penicillin V-K salt + 5% of benzylpenicillin 86.0 91.7 + 10% of benzylpenicillin . . .. . . 75.0 86.9 Interference by p-Hydroxyphenoxymethylpenicillin ( p - OH Penicillin V) Samples of phenoxymethylpenicillin have been found to contain up to 6% of @-OH penidliii V by using chromatographic methods and by measurement in the ultraviolet region. @-OH penicillin V has a relatively high absorptivity at 286 nm, showing a broad peak which has an absorbance at 268 nm that is almost the same as that of phenoxymethylpenicillin. The BP procedure provides a correction for $-OH penicillin V on the assumption that it is not precipitated, but we have shown that about 35% of the $-OH penicillin V is precipitated during the BP procedure.This precipitation means that if 3% of $-OH penicillin V is present in the sample, only 2% is allowed for in the method and the actual penicillin V content is over- estimated by 1%.This was demonstrated by adding different amounts of @-OH penicillin V to a known sample of phenoxymethylpenicillin and taking each mixture through the BP procedure to provide the results given in Table 11. TABLE I1 BP ASSAY OF POTASSIUM PHENOXYMETHYLPENICILLIN SPIKED WITH $-OH PENICILLIN V Penicillin V-K Found, 7; Theoretical, yo d r \ Penicillin V-K salt control .. .. .. .. . . 96.5 - Penicillin V-K salt control +2yo of #-OH penicillin IT . . .. .. .. . . 94.9 94.6 +4y0 of p-OH penicillin V . . .. .. .. . . 93.8 92.6 +6y0 of p-OH penicillin V . . * . .. .. . . 93.1 90.7 +8% of $-OH penicillin V . . .. .. .. .. 91.6 88.7 Specific Method for Phenoxymethylpenicillin The method consists of eliminating acid-unstable penicillins by acid degradation and determining the remaining acid-stable penicillins by using a total-penicillins method such as the imidazole assay.3 The acid-stable penicillins include phenoxymethylpenicillin and p-OH penicillin V.$-OH penicillin V absorbs at 286 nm and undergoes a “phenolic” shift in alkaline solution to give a maximum at 306 nm. The increased absorbance of a solution of the sample at 306 nm is used as a measure of the $-OH penicillin V content.Subtraction of the $-OH penicillin V content from the acid-stable penicillins gives the true phenoxymethylpenicillin content. The samples listed in Table 111, which were close to the BP specification of 94.3y0, were assayed by this method and the results compared with those obtained by the BP method.290 ORIGINAL PAPERS IN PHARMACEUTICAL ANALYSIS Proc.Az4vvt. Div. Chem. SOC. TABLE I11 ASSAY OF PHENOXYMETHYLPENICILLIN BY THE ALTERNATIVE METHOD AND BY THE BP METHOD Phenoxymethyl- Acid-stable p-OH Phenoxymethyl- penicillin by Sample penicillins, yo penicillin V, 74 penicillin, % BP method, 9; 44023 96.5 3.8 92.7 93.1 44124 98.4 3.8 94.6 95.6 39917 97.4 2.7 94.7 93.2 39898 98.3 3.1 95.2 94.2 Conclusions We think that this work has explained some of the past problems with the BP method for phenoxymethylpenicillin. It has highlighted the non-specificity of the method and also suggests an alternative, more specific, procedure that involves determination of the acid-stable penicillins as phenoxymethylpenicillin and a correction for &OH penicillin V.With minor modifications the method is also applicable to phenoxymethylpenicillin fermenter broths and should work for phenoxymethylpenicillin in the presence of the common naturally occurring penicillins.References 1. 2. 3. “British Pharmacopoeia 1973,” HM Stationery Office, London, 1973, p. 362. “European Pharmacopoeia 1969,” Maisonneuve S.,4., France, 1969, p. 341. Bundgaard, €3.. and Iliver, K., J.Pharnz. Pharmac., 1972, 24, 790. Thin-layer Bioautographical Detection of ,&Lactam Contaminants in Pharmaceuticals E. M. May and G. V. Sefton Laboratory of the Governmem! Chemist, Cornwall House, Stamford street, London, SE 1 9NQ Evidence of the contamination of pharmaceuticals with penicillin and the risk involved is given by 0’Neil.l The United States Food and Drugs Administration (FDA4) prohibits the marketing of non-penicillin oral and parenteral pharmaceuticals containing more than 0.5 I.U. and 0.05 I.U.of penicillin, respectively.2 Cephalosporins are not at present subject to this control. Checking such stringent limits requires an efficient extraction of nanogram amounts of p-lactams from gram amounts of formulated drugs, followed by a sensitive, specific method of detection.The initial problem of quantitative extraction is difficult owing to the inherent instability of the p-lactams. Each sample has to be considered individually with reference to the main drug substance. A satisfactory general procedure for penicillins is described by O’Nei1,l involving the extraction of the free “penicillin” acid from acid aqueous solution into methylene chloride, with the limitation that the zwitterionic p-lactams will remain in the aqueous layer and will require a separate extraction.Trial runs on drugs spiked with benzylpenicillin and taken through all stages of this method show acceptable recovery. A direct extraction into a low-boiling solvent may be tried if the drug formulation permits. The problem of a sensitive, specific method of detection has been more easily solved by the use of thin-layer bioautography (TLB), which is thin-layer chromatographic separation combined with bioautographic detection.The gain in sensitivity by using microbiological detection compared with chemical detection varies from 10- to 200-fold depending upon the particular /3-lactam antibiotic. Specificity is conferred by comparing the response of the micro-organism to the sample extract with and without penicillinase.Table I shows the relative mobilities (chemically detected) of nine b-lactam antibiotics together with neomycin, chloramphenicol and oxytetracycline, using three thin-layer systems that are compatible with Bacillus subtilis and A!ficrococczts Zuteus. For fl-lactams these are the organisms of choice and they are also sensitive to neomycin, chloramphenicol and oxytetracycline, but not to sulphonamides at loadings below 1 pg.October, 1977 ORIGINAL PAPERS I N PHARMACEUTICAL ANALYSIS TABLE I MOBILITY VALUES RELATIVE TO BENZYLPENICILLIN OBTAINED BY TLC (CHEMICAL DETECTIOK) Solvent system 291 Antibiotic 1* Amoxycillin .. .. . . 3.0 Ampicillin . ... .. . . 1.5 Benzylpenicillin . . . . . . 1.0 Cephalexin . . . . .. . . 1.7 Cephaloridine . . . . . . 0.25 Cephalothin . . . . . . 0.75 Cloxacillin . . . . . . . . 0.30 Phenoxymethylpenicillin . . 0.4 Propicillin . . .. .. . . 0.25 Chloramphenicol . . .. . . 1.0 Neomycin sulphate . . . . 2.05 Oxytetracycline . . .. . . 0.4s 2f 0.0 0.0 1.0 0.0 0.0 0.4 1.35 1.0 1.35 2.7 0.0 0.011 33 0.9 0.9 1.0 0.8 0.2 1.0 1.0 1.0 1 .o 1.35 0.59 0.011 * Solvent system I : sodium acetate, 0.1 M, adjusted to pH 7.4 with 5,5-diethylbarbituric acid - acetone (50 .+ I) on a Kieselgel G (Type 60) plate, 0.25 mm thick, impregnated with silicone oil (BDH MS200/200) in diethyl ether, 5% m/V.3 t Solvent system 2 : butan-%one - dichloromethane - glacial acetic acid (40 4- 20 f 1) with Kieselgel G plate, 0.25 mm thick.$ Solvent system 3 : propan-2-01 - pH 5.0 buffer (McIlvaine) (65 + 35) with Kieselgel G plate, 0.25 mni thick. § Streaks. 11 Main spot stationary with some forward streaking. Table I1 shows the loading in nanograms giving a zone of inhibition about 10 mm in diameter with both organisms, M . Zuteus being slightly more sensitive than B.subtilis. ,&Lactam antibiotics having close RF values will not give separate zones of inhibition but standard bioautograms at the loadings shown in Table I1 demonstrate resolution of amoxy- cillin, ampicillin, benzylpenicillin and cloxacillin in solvent system 1. Chloramphenicol, benzylpenicillin and cephalothin can be separated from each other and from the zwitterionic /?-lactams in solvent system 2.However, phenoxymethylpenicillin is not separated from benzylpenicillin bioautographically, nor is cephalexin from ampicillin. For samples being examined for cross-contamination, the concentrated extract should be taken up in 50-100 p1 of suitable solvent and loaded on to the thin-layer plate together with the standard p-lactam and with aliquots of both previously incubated with P-lactamase.Standards of the main drug substance and related compounds must also be included on the plate and the thin-layer system chosen must separate satisfactorily any zones of inhibition they may give from any p-lactam zones arising from potential contaminants. After development, the thin-layer plate is prepared for bioautography by drying at 50 "C for 30 min.When glacial acetic acid is present it must be completely removed, preferably by vacuum drying at 50 "C, for 1 h at 3 Torr. The plate is then placed in a levelled bioassay dish (NUNC 1015) and 150 ml of Medium A of the British Pharniacopoeia,4 inoculated a TABLE I1 LOADING XECESSA4RY TO GIVE A 10-mm ZONE DIAMETER Antibiotic Loading& Amoxycillin . . . . . . . .20 Benzylpenicillin . . . . .. . . 5 Ampicillin . . . . .. .. .. 15 Cephalexin . . . . . . . . .. 50 Cephaloridine . . .. .. .. 50 Cephalothin . . .. .. .. 50 Cloxacillin . . . . .. .. .. 200 Phenoxymethylpenicillin .. . . 5 Propicillin . . . . . . .. . . 20 Neomycin sulphate . . . . . . 1000 Chloramphenicol . . . . . . .. 400 Oxytetracycline . . .. .. .. 300292 ORIGINAL PAPERS IN PHARMACEUTICAL ANALYSIS PYOC.AnnJyt. Div. Chem. SOC. 45-50 “C with either B. sztbtiLis (NCTC 10400) or M . Zuteus (NCTC 8340), is allowed to flow over the plate, avoiding surface air bubbles. The lid is replaced, the agar allowed to set and the plate is incubated overnight at 30 “C. The outline of the zones of inhibition can be enhanced in the instance of B. subtilis by spraying the plate after incubation with a 0.5% m/V solution of 2,3,5-triphenyltetrazolium chloride and in the instance of ill.Zuteus by adding 1.5 ml of a 2 mg ml-l solution of brilliant black to the medium prior to pouring. The complete disappearance of a zone of inhibition with 13-lactamase confirms a 13-lactam, but it should be noted that in some circumstances the enzyme may cause reduced zone sizes with other antibiotics and some drug substances.This method is sufficiently sensitive to detect amounts of 13-lactams well below the FDA limits and has been used to show the presence of benzylpenicillin in chlorpropamide tablets at a level of 0.003 pg per single tablet and 0.012 5 pg g-l of a penicillin in a veterinary oxytetra- cycline syrup, and has demonstrated the absence of 13-lactams in aspirin, poldine methyl- sulphate and metaclopramide tablets. Great care must be taken against laboratory-induced contamination, and an area restricted to low levels of antibiotics is advisable.TLC should be used to supplement TLB. We thank the Government Chemist for permission to publish this summary. References 1. 2. 3. 4. O’Neil, P. J., Proc.Soc. Analyt. Chem., 1974, 11, 30. The Federal Register 1976, title 21, part 211.58. Biagi, G. L., Barbaro, A. M., Gamba, M. F., and Guerra, M. C., J . Chromat., 1969, 41, 371. “British Pharmacopoeia 1973,” HM Stationery Office, London, 1973, A97. Value of Tetraphenylborate(ll1) Complexes in the Identification of Alkaloids and Other Organic Bases in Pharmaceuticals by Infrared Spectroscopy A.F. Casy Pharmacy Department, Novfolk and Norwich HospitaI, St. Stephens Road, Norwich, NR1 3SR. The use of sodium tetraphenylborate(II1) for the quantitative determination of organic bases in pharmaceuticals was described by Johnson and King in 1962,l and the procedure of these authors has been adopted by the current British Pharmaceutical Codex for the assay of eye- drops that contain a variety of alkaloids and synthetic bases.2 The method requires addition of excess of 0.01 M sodium tetraphenylborate(II1) solution to a solution of the protonated (or quaternised) base in a buffer solution, collection of the complex on a sintered glass filter and titration of the excess of reagent in the filtrate and washings with 0.005 M cetylpyridinium chloride solution, using bromophenol blue as indicator.+ :N: + The melting-point of the complex is of some value in identifying the basel but this paper sets out to show that its infrared spectrum is of greater significance with regard to the specificity of the method. Other authors have reported the infrared spectrum of tetraphenylborate( 111) complexes of organic bases but most data relate to potassium bromide disc preparation^.^-^ In the work reported here the simpler Nujol-mull procedure was used.The washed precipitate (sucked as dry as possible) was scraped off the sinter, spread on filter-paper and left to dry, preferably overnight. The dry product was then mulled with light liquid paraffin and its infrared spectrum recorded (Perkin Elmer 157 G spectrophotometer ; mull spread between sodium chloride discs). Mulls readily formed in all instances and were prepared somewhat thicker than usual in order to compensate for the dilution factor.In spectra of such mulls the usual Nujol bands are present while bands due to the tetra- phenylborate(II1) anion are principally those near 3 050 and 1 580 cm-l (both weak but sharp), More intensive desiccation was not usually necessary.Ocfober, 1977 ORIGINAL P-4PERS IN PIIARRIACEUTICAL ANALYSIS 293 and 715 and 740 cm-1 (medium intensity), as established from spectra of potassium and sodium tetraphenylborate(II1). Provided the sample is dry, little absorption occurs in the region above 3 050 cm-1 and between 1 600 and 2 700 cm-l, i.e., regions where absorption due to common functional groups are most frequently found.Spectra of moist samples may be obscured by a broad band centred near 3 400 cm-1 and a smaller one near 1 630 cm-l. The value of the procedure lies in the fact that bands characteristic of the base as observed in spectra of mulls of conventional salts are also seen in spectra of tetraphenylborate(II1) salts, albeit in a modified form.As an example, spectra of atropine sulphate and its tetra- phenylborate(II1) salt can be compared. In the former there is a broad band centred at 3 200 cm-1 (OH) and an intense band near 1 720 cm-l (ester CO-. These bands are seen as the bands near 3 110 cm-l (sharper than that of the sulphate) and 1 700 cm-l, respectively, in the spectrum of the tetraphenylborate(II1) salt. Spectra of lignocaine hydrochloride and its tetraphenylborate(II1) complex provide a similar comparison but in this instance the three NH bands of the hydrochloride reduce to a single band for the complex while amide absorp- tions are likewise simplified (Table I).Reasons for spectral changes probably lie in the different hydrogen-bonding influences of the tetraphenylborate (111) anion and conventional inorganic anions.Note that the related compounds atropine, hyoscine and homatropine can be differentiated in tetraphenylborate(II1) spectra by the position of the carbonyl band (1 700 for atropine, 1 725 cm-1 for the other two mydri- atics) and by absorption near 850 cm-l (strong to medium band found only for hyoscine). Reserpine provides an example of a compound for which bands below 1 600 cm-l (the finger- Data for a variety of bases are summarised in Table I.TABLE I INFRARED CHARACTERISTICS OF TETRAPHENYLBORATE(III) COMPLEXES AND OTHER SALTS OF SOME COMMON PHARMACEUTICAL BASES Wavenumber* /cm-l /-___------- - Base (protonated) Lignocaine Amethocaine Cinchocaine Cocaine Pilocarpine Atropine H yoscine Homatropine Reserpine * br = broad; st-md Anion C1- Ph,B- c1- Ph,B- c1- Ph,B- c1- Ph,B- NO,- Ph,B- HSO,- Ph,R- Br- Ph,B- Br- Ph,B- Base Ph,B- Salt 3 440 (NH) 3 370 3 170 3 290 3 360 (NH) 3 380 3 500 (NH) 3 400 3 200 3 360 3 120 (ArCH) 3 060 3 260 3 120 3 200 (OH) 3 120 3 340 (OH br) 3 500 3 250 (OH) 3 120 3 420 (NH) 3 360 (br) strong to medium; md = medium intensity. Nujol mull 1 670 (amide CO) 1650 1670 1690 (CO) 1695 1660 (CO) 1 650 1635 1 725 (CO.bands well 1 710 resolved) 1 720 (resolution just 1700 apparent) 1770 (CO) 1760 (CO) 1720 (CO) 1700 1720 (CO) 850 (st-md) 1725 850 (md) 1750 (CO) 1725 1725, 1706 (CO) 1620, 1580, 1330 1230, 1 120 1715 (doublet just apparent 1 630, 1 580, 1330 1220, 1 125294 ORIGINAL PAPERS I N PHARMACE WTICAL ANALYSIS PYOC. AnUhyt. DiZT. Chew?. sot.print region) are clearly duplicated in a spectrum of the complex as well as the more readily assignable bands. To conclude, the infrared procedure described is recommended as a convenient and effective means of increasing the specificity of the tetraphenylborate(II1) assay of alkaloids and syn- thetic organic bases. The method is also of value as a facile means of concentrating and isolating a base in a form suitable for infrared examination.For example, identification of the alkaloid in atropine eye ointment can be achieved readily by shaking a sample with ether and dilute mineral acid, and precipitation of the tetraphenylborate(II1) complex from the aqueous phase followed by spectral examination as usual. In the BP 1973 identification procedure the free base must first be isolated by solvent extraction and then subjected to thin-layer chromatography.References 1. 2. 3. 4. 5. Johnson, C. A., and King, R. E., ,J. Phavm. Pharmac., 1962, 14, 77T. “British Pharmaceutical Codex 1973,” The Pharmaceutical Press, London, p. 892. Chatten, L. G., Pernarowski, M., and Levi, L., .J. Am. Pharm. Ass., Scient. Edn, 1959, 48, 276. Rich, N.W., and Chatten, L. G., J . Plaarm. Sci., 1965, 54, 995. Sinsheim, J. E., and Smith, E., J . Pharwz. Sci., 1963, 52, 1080. Characterisation of the Selectivity of Reversed Stationary Phases in H igh-performance Liquid Chromatography E. Tomlinson Shoo1 of Pharmacy and PharmaLoZogy, University of Bath, Clavcrton Down, Bath, Avon, BL4 2 7 A Y Prediction of the retention behaviour of solutes in high-performance liquid chromatographic (HPLC) systems using reversed phases is of importance in the analysis of samples obtained from a variety of sources.To ensure that such stochastic approaches will be useful, a know- ledge of the likely distribution coefficients of solutes between mobile and stationary phases is necessary. For bulk-phase systems such a requirement can be met by applying Collander’s finding1 that a linear correlation can exist between solute partition coefficients (KD) in one solvent- pair system and those in a second, i.e., Log&, = Lo@., + b ... . . . * * (1) Such a correlation is, however, inapplicable if different solvation processes take place in the non-aqueous phase. (For example, water - octane KD values are generally not correlated with water - octan-1-01 KD values, presumably owing to the existence of monomer and pent- amer forms of octan-1-01, together with the presence of a large amount of dissolved water in the octan-1-01 phase.) Similarly, to relate oil - water K D values to some function of a solute’s retention in a liquid - liquid chromatographic column is also difficult, because of the exotic nature of the chromatographic phases employed, i.e., LogKFJk # Function(so1ute retention) + c .. .. * - (2) For example, a typical reversed-phase HPLC system may, in its most simplified form, be represented as Mobile phase Stationary phase I/ I I Methanol- Water : Methanol - Chemically bonded alkyl chain on silica support Thus, a knowledge of bulk phase distribution coefficients may not be appropriate for predicting retention within any one system.The molecular interactions taking place in HPLC systems are mainly electrostatic, hydrogen bonding and dispersion. The first two are predominant in adsorption chromatography, while dispersion is generally dominant in liquid - liquid or reversed phase systems. A number ofOctober, 1977 ORIGIY-kL PAPERS I N PHARMACEUTICAL ANALYSIS 295 theoretically based approaches have been developed to assess such molecular interactions and so provide an a priori basis for estimating retention.These approaches include an extension of the regular solution theory, which employs Hildebrand's solubility parameterJ2 the specific interaction approach of Scott3 and a thermodynamic approach, in which distribution can be correlated to solute or solvent changes via effects on the excess partial molar free energy associated with the transfer of solute from one phase to a n ~ t h e r .~ The thermodynamic approach has been criticised3 on the grounds that individual interac- tions are not easily separated or identified and that there is only a limited amount of thermo- dynamic data currently available.The approach, however, is an attractive one as it should be possible to extend the information obtained from the free-energy domain to the entropic and enthalpic terms describing the chromatographic proce~s,~ which should enable a greater understanding of the nature of the molecular interactions taking place to be made. In this work the thermodynamic approach has been modified in order to estimate the contribution that each functional moiety makes in determining solute distribution between mobile and stationary phases.The column retention behaviour of each of ten homologues of ten series of monofunctional substituted benzoic acid esters and a similar series of ring unsubstituted alkylbenzenes has been studied using C, and C, chemically bonded stationary phases and water - methanol mixtures of various compositlons.Columns (3 or 10 cm) were used at pressures between 130 and 175 kgf cm-2, using loop-valve injection and refractive-index detection. The chromatographic capacity ratio K~ of a compound i can be computed from its column retention time, tRi using the equation where tg, is the retention time of a non-retained compound.related to the distribution coefficient of a solute between a mobile and stationary phase by The capacity ratio can be (4) .. .. .. K~ = VS.Vm-l. KDi . . where Vs/Vm is the volume ratio of the stationary phase to the mobile phase. The contribution of each functional group to the retention is defined by LOgrji = h g ( K j / K i ) . . .. .. .. * (5) where rji is termed the selectivity factor and is thus an indication of the effect a group will have on a measured K value.Here the selectivity factor for a methylene group is analysed in terms of the methanol conent of the mobile phase and the alkyl chain length of the bonded stationary phase. Group values for the C, phase system, which are also shown, have been calculated from data obtained elsewhere.6 Fig.1 demonstrates the effectiveness of this approach. 0.3 .- 0.2 i- -I 0" 0.1 0 1 8 18 Stationary-phase alkyl-chain length Fig. 1. Group contribution values for the methylene moiety determined a t 20 "C in a number of reversed- Methanol - water: A, 60 + 40; B, 70 + 30; phase systems. and C , 80 + 20. Closed data points calculated from ref. 6 .296 ORIGINAL PAPERS IN PHARMACEIJTICAL ANALYSIS Pf'OC.A ~ d y t . DiV. Chcm. SOG. By computing group values for other moieties in a similar fashion, it will be possible for tables to be constructed from which the retention behaviour of solutes will be able to be predicted for any one phase system. References 1. 2. 3. 4. 5. 6. Collander. R., Acta Chem. Scand., 1951, 5, 744. Tijssen. R., Billiet, H.A. H., and Schoenmakers, P. J., J . Chromat., 1976, 112, 185. Scott, R. P. W.. J . Chromat., 1976, 122, 35. Pierotti, G. J., Deal, C. H., and Derr, E. L., Ind. Engng Chem., 1959, 51, 95. Tomlinson, E., Poppe, H., and Kraak, J. C., J . Pharm. Pharmac., 1976, 28, Suppl. 43P. Tjarden, U., PhD Thesis, University of Amsterdam, 1976. Separation of the Diastereoisomers of Labetalol Hydrochloride by High-performance Liquid Chromatography D.W. Selbyand R. P. Munden A lzalytical Research Department, Allen and Hanburys Research Ltd., Priory Street, Ware, Nertfordshire Labetalol hydrochloride (I) is a new drug available for the treatment of hypertension. I As it contains two asymmetric centres, it will normally consist of two racemic mixtures, We have called the mixture of enantiomers (R) (S) + (S) (R) racemate 1 and (R) (R) + (S) (S) racemate 2.In order to maintain adequate analytical control over the product, it is necessary to measure the relative amounts of the two racemates present. This measure- ment is most easily done by using the gas-chromatographic method of Munro et aZ.1 but a procedure based on high-performance liquid chromatography (HPLC) was investigated and is reported here.Experimental Adequate separation of the two racemates could not be achieved by direct means. Forma- tion of a derivative appeared necessary, particularly one involving the amino nitrogen atom. The 1-NN-dimethylaminonaphthalene-5-sulphonyl (dansyl) derivative of labetalol hydro- chloride was prepared by treating 5 mg in 0.5 ml of water with 1.5 ml of a 2% m/V solution of dansyl chloride in acetone and adding 20mg of sodium carbonate. Reaction occurred overnight and the product was extracted into ethyl acetate and dried over anhydrous sodium sulphate.An aliquot was separated on a 20cm x 5mm column of Partisil 10 with a solvent system consisting of a 1 + 1 mixture of hexane and ethyl acetate, in which the ethyl acetate was modified by the addition of 2% V/V of methanol and 0.6% V/V of water.At a flow-rate of 1.4 ml min-1, racemate 2 had a retention time of 8.5 min and racemate 1 of 10.0 min, resolution being almost complete. Nature of the Reaction Product An amount of the dansyl derivative of labetalol hydrochloride was prepared and purified by column chromatography on neutral alumina.Elemental analysis indicated the presence of two dansyl groups and this was confirmed by means of nuclear magnetic resonance spectro- scopy. Both ultraviolet and infrared spectroscopy indicated the presence of a dansyl group on the phenolic oxygen. Mass spectroscopy gave a molecular ion at m/e 791, confirming the presence of two dansyl groups, and fragment ions at m/e 543 (M+ - dansyl group) and m / e 525 [M+ - (dansyl group + H,0)] indicated that the benzylic hydroxyl group was still intact.Other fragment ions at mle 398 and 395 placed the second dansyl group on the amino nitrogen, consistent with theOctober, 1977 ORIGINAT, PAPERS IN PHARMACEUTICAL ANALYSIS 297 fragmentation pattern described by nurden et aL2 The formation of an NO-bis(dansy1) derivative of labetalol is entirely consistent with the expected behaviour of the reagent under the conditions employed3 and with the structure of analogues reported by Durdex2 Quantification The racemate ratios were determined by a form of internal normalisation in which only the peaks of interest were considered.The areas of the two peaks as measured by electronic integration were summed and each was expressed as a percentage of the total racemate peak area.Further assumptions were: (i), that the detector responded linearly to both racemates; (ii), that no racemisation occurred during derivative formation ; and (iii), that each racemate reacted at the same rate during the dansylation procedure or that the reaction was complete. The linearity of response and absence of racemisation was checked using three mixtures of known racemate composition.The results obtained by gas - liquid chromatography (GLC) (where the response is linear and racemisation is known to be absent) were compared with those obtained by HPLC and are shown in Table I. The derivatives for HPLC were prepared by using a &fold molar excess of dansyl chloride. It was .assumed that both racemates responded identically in the detector. TABLE I COMPARISON OF THE RACEMATE RATIOS OF LABETALOL HYDROCHLORIDE OBTAINED ON STANDARD MIXTURES Racemate 1 : racemate 2 Racemate 1 : racemate 2 by GLC by HPLC 97.2: 2.8 36.5 : 63.5 97.8: 2.2 37.6 : 62.4 8.4 : 91.6 8.4 : 91.6 The ratios compared favourably with one another, indicating that detector response was linear over the range studied and racemisation did not occur.Further, reaction appeared to be complete, or both racemates reacted with dansyl chloride at the same rate. Some results obtained on standard production batches of labetalol hydrochloride are shown in Table 11. TABLE I1 RACEMATE RATIOS OF PRODUCTION BATCHES OF LABETALOL HYDROCHLORIDE OBTAINED BY GLC AND HPLC Sample No.1 2 3 4 5 6 7 Racemate 1 : racemate 2 by GLC 51.5 : 48.5 51.5 : 48.5 46.3 : 53.7 50.1 : 49.9 52.5 : 47.5 49.8 : 50.2 50.1 : 49.9 Racemate 1 : racemate 2 by HPLC 43.4 : 56.6 45.7 : 54.3 46.8 : 53.2 46.1 : 54.9 45.4 : 56.6 44.9 : 55.1 43.9 : 56.1 It will be noted that the HPLC results are consistently different from those obtained by GLC. In these studies, only a 2.5-fold molar excess of dansyl chloride was used in order to prepare the derivatives.The yield, as measured by the total area of the racemate peaks, increased as expected, reaching completion after 16 h. The racemate ratio did not remain constant throughout this time, however, as would be expected if both reacted at the same rate. The apparent percentage of racemate 1 increased rapidly at first, with racemate 2 predominant, and a constant value (coincident with that obtained by GLC) was reached after 16 h with a 5-fold excess of reagent.These results indicate that racemate 2 reacts more readily with dansyl chloride than racemate 1 and accounts for the discrepancies found in the data shown in Table 11. It appears that in order to obtain reliable data from this procedure, long reaction times and excessive amounts of reagent are necessary.It is for this reason that the GLC procedure A study of the kinetics of the dansylation reaction was made.298 ORIGINAL PAPERS IN PHARMACEUTICAL ANALYSIS Proc. Analyt. Div. Chew. SOC. of Munro et aZ.l is preferred for the determination of the racemate ratios of labetalol hydro- chloride. We thank Mr.M. Rlanchard for his technical assistance. References 1. 2. 3. Munro, G., Evans, M. B., Hunt, J . H., and Rowe, L. R., J . Pharm. Pharmac., 1976, 28, 27P. Durden, D. A., Davis, B. A., and Boulton, A. A., Biomed. Mass Sfiectrom., 1974, 1, 83. Seiler, N., J . Chromat., 1977, 143, 221. Experiences with a Semi-automated Calculation System Applied to the Technicon AutoAnalyzer P. F.G. Boon and A. W. Mace Analytical Department, CIBA Laboratories, Horsham, Sussex AutoAnalyzers produce traces with peaks whose height is a function of the concentration of the substance being determined. A typical trace consists of calibrating peaks, followed by samples, with intermediate standards to control base-line drift. There are various ways in which these traces can be evaluated: Manual A calibration graph is constructed on a transparent plastic sheet and is then used to read off the sample peaks.Fdl’ aatomatic The recorders producing the traces are interfaced directly into a minicomputer such as a PDP 12A,1 or into a terminal to a large time-sharing computer. Storage systems Using a data logger all the data from the recorders can be stored in a suitable way such as on a magnetic tape cassette.These data can then be analysed at a later time on a calculator or minicomputer. Semi-automatic systems The peak heights from the AutoAnalyzer traces can be fed into a calculator via an X - Y plotter . The scale of the problem varies considerably from one laboratory to another, depending on the nature of the work and the number of AutoAnalyzer channels involved.We have six A.A. 1 channels, which cover a variety of work including tablet-uniformity studies, release-rate monitoring and simple content determination. A.A. 1 systems often produce non-linear calibration graphs, which are more complex to analyse than A.A.2 graphs. Large numbers of peaks are produced particularly from tablet-uniformity studies. The manual method of evaluating the traces is time consuming, tedious and subject to human error.The fully automatic system is likely to be an expensive solution, unless a minicomputer or terminal is already available in the laboratory. A large amount of data-storage space is required as each channel must be scanned every few seconds in order to achieve accurate peak identification. The data-logging system avoids monopolking an expensive computer continuously and makes less demand on storage space as the results from each channel can be analysed separ- ately.However, unless each channel has its own data-logging system (which is expensive) then the tapes, or whatever is used to record the data, can only be removed when none of the channels are in operation. For us, this would only occur at the end of the day, as the channels are not related, and runs are starting and terminating in a random and overlapping fashion.This mode of operation would cause delays in the production of results, which could be serious. Both the fully automatic system and the storage systems require complex programming as all eventualities in traces must be catered for.In particular there can be difficulties in identifying the correct point on the peak for height measurement (Fig. 1). The usual strategy is to look for a certain percentage drop after the maximum has been reached. This procedure avoids some of the problems that can be encountered owing to shoulders on peaks. Noise spikes, such as may be caused by air bubbles in the flow cell, are best catered for by a curve- smoothing technique.On these fully automatic systems it is difficult to reject any particular results, add in extra or check samples or to cope with missing peaks and broken traces.October, 1977 ORIGINAL PAPERS I N PHARMACEUTICAL ANALYSIS 299 There is no doubt that given a sufficiently powerful computing system and programming expertise then all these problems could be catered for.However, this will increase the cost of the system and will only be a viable alternative for larger laboratories with perhaps a dozen or more channels. Shoulders Spikes due to on peaks\ air bubbles I ! 1 1 Break in trace + + 4 Missing 44 peak I . -Time Fig. 1. Faults on AutoAnalyzer traces causing problems in peak identification. The following advantages eventually caused us to opt for a semi-automatic system. (1) All the calculations and data handling are done automatically. (2) All chart evaluations can be done at the operator’s convenience and any urgent results can be done immediately. (3) The operator can use discretion over poor peaks and noise spikes and extra and check samples can easily be run. (4) Entry of peaks is a rapid operation and only requires a small amount of operator time. For example, a tablet-uniformity trace consisting of a total of 42 peaks can be entered in 1 min and the results produced and statistically evaluated in a further 1Q min. To read this trace manually, evaluating the statistics on a calculator takes 15-20 min. (5) Programming is relatively simple and can be done without specialist personnel. (6) The cost of the system is less than that of a fully automatic system (about fJ6000 at (7) The system can cope with expansion to the output of 10-12 channels without the addi- The system we chose consists of a PCD ZAE 5A Data Reader, which is used to measure the peak height and convert it into a digital form. This information is fed directly to a Diehl Alphatronic 160/332 calculator. The calculator programs are stored on a magnetic-tape cassette unit. Output is on the Alphatronic tally roll but could also be on a teletype or graph plotter. The mode of operation is very simple. The chart is placed on the X - Y plotter and kept flat with flexible magnetic strips. The appropriate program is called up on the calcu- lator and then the peaks are fed in by aligning a sliding cursor with the top of the peak, and pressing a button. When all the peaks have been entered the bar is moved to the extreme left of the table and the button pressed again to signal to the calculator that it has all the peak heights. The calculator works out all the individual results and performs the required st atistical analysis. The system has proved reliable since its installation 18 months ago. The accuracy of the results is better than with manual reading and considerable amounts of time have been saved. October 1975 prices). tion of further equipment. Reference 1. Overton, M. W., Alber, L. L., and Smith, n. E., J . ASS. 08. Anulyt. Chem., 1973, 56, 140.

ISSN:0306-1396    

DOI:10.1039/AD9771400285

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

330 RESEARCH AND DEVELOPMENT TOPICS R o c . Aitalyt. Div. Chem. SOC. Research and Development Topics in Analytical Chemistry The following is a summary of one of the papers presented at the Research and Development Topics in Analytical Chemistry meeting of the Analytical Division held on May 3rd and 4th, 1977, at Chelsea College, London. Summaries of eleven of the other papers presented appear- ed in the September issue of Proceedings (p.240). Comparison of the Delves Cup and Carbon Furnace Atomisation Used in Atomic-absorption Spectrometry for the Determination of Lead in Blood S. Sabet and J. M. Ottaway and G. S. Fell Department of Pure and Applied Chemistry, University of Strathclyde, Cathedral Street, Glasgow, G1 1XL Department of Pathological Biochemistry, Glasgow Royal Infirmary, Glasgow, G4 OST The determination of lead in blood is an important part of health checks for populations thought to be at risk from environmental or occupational exposure to lead.No single procedure has so far emerged as being entirely satisfactory for this determination, although the Delves cup procedure and carbon furnace atomisation, both used in atomic-absorption spectrometry, are among the most widely applied methods.In this paper the performance of these methods is compared, using a Delves cup and several different carbon furnace atomisers, with particular emphasis on rate of analysis, accuracy and precision. Investigations of the optimisation of operating conditions are also described in each instance. Atomisation Using a Delves Cup An Instrumentation Laboratory 251 spectrophotometer and Delves cup accessory were used and set up as originally described by De1ves.l The use of the continuous background correc- tion facility on the spectrophotometer enabled the determinations of lead in blood to be carried out without pre-ashing with hydrogen peroxidel or acid.2 Non-atomic-absorption signals were obtained from the blood matrix in the form of a smoke signal and molecular absorption from sodium chloride (identified by the ultraviolet spectrum).Resolution of these signals with respect to the time after which the cup is placed below the absorption tube showed that the lead atomic signal was given between the smoke signal and the molecular signal, with a maximum non-atomic overlap of 0.02 absorbance unit at the time of peak lead absorption.No spectral interference was observed when background correction was used. A loss of sensitivity was observed with time for each batch of nickel cups, which has been reported elsewhere.3 This loss could amount to well over 20% in 2 h of continuous operation and necessitated frequent recalibration with blood standards. The stability of the instru- ment and Delves cup assembly was shown to be satisfactory and a similar loss of sensitivity was also observed on integration of the signal.Replacement of the batch of five cups with a set of new cups restored the lead signal to its original value and thus ageing or changes in the cups themselves appeared to be responsible for the loss of sensitivity. Chemical analysis of new and used cups revealed that residues of the more refractory elements in the blood matrix, e.g., calcium, iron and phosphorus, were extensively retained on the surface of the cup.As the maximum temperature of the cup attained in the air - acetylene flame used was 1 040 O C , non- volatilisation of these elements could be expected. The presence of these elements, presum- ably in the form of oxides, thus appears to inhibit the rapid atomisation of lead, leading to a reduction in sensitivity. As no lead residue could be detected, the mechanism appears to be one of slowing down lead vaporisation rather than complete inhibition.Attempts made to remove the matrix elements by conversion to more volatile forms with, for example, complex- ing agents, were unsuccessful.The loss of sensitivity was found to be less serious (-lOyo in 1 h), necessitating less frequentOctober, 1977 RESEARCH AND DEVELOPMENT TOPICS 301 re-standardisation, when an alumina absorption tube with a modified hole was substituted for the normal silica tube. The sensitivity was also increased %fold owing to the improved alignment of cup and tube.Interferences on lead absorption by the inorganic constitutents of blood were investigated using aqueous solutions. Unfortunately, aqueous solutions corrode the nickel cups very rapidly but serious interference was noted from calcium, iron and phos- phorus and calibration by use of freshly prepared blood standards is essential. Large batches of blood standards were prepared by mixing aqueous lead standards with blood but were found unsatisfactory after about 2 weeks, owing to either precipitation or separation of the added lead. The physiologically bound lead was found to be stable for many weeks.Use of a microdiluter for 10 pl of blood and 30 pl of 0.01 yo Triton X-100 diluent was found to improve precision. Calibration graphs were found to be not perfectly linear and varied from day to day, with variations in alignment of the cups. Sample results must therefore be obtained from a calibration graph of blood standards, plotted manually.Carbon Furnace Atomisation Three different instruments were investigated for carbon furnace atomisation, all manu- factured by Perkin-Elmer. These instruments were an HGA-72 mounted in a 306 spectro- photometer, an HGA-74 mounted in a 360, and an HGA-76 used with an autosampling device, the AST, mounted in a 370.All instruments were fitted with background-correction devices. Optimum conditions for operating the furnaces were similar and are summarised as follows : drying stage, 100 "C for 20-30 s; ashing stage, 500 "C for 25 s ; atomisation, 2 100-2 300 "C for 10 s; tube clean-out, 2 700 "C (or maximum available) for 5 s.To obtain maximum sensitivity for lead determinations it is desirable to use the maximum volume of whole blood or a solution of it, following the minimum necessary dilution. The limits of volume and dilution are determined by (a), the ability of the deuterium background- correction system to correct for the non-atomic absorbance from the blood matrix, ( b ) , the degree of sample splutter and/or spread within the tube so that even drying conditions are achieved and (c) , the need to achieve reasonable sensitivity without excessive instrumental scale expansion at levels as low as 0.1 pg ml-l of lead in whole blood.The optimum conditions identified are shown in Table I. In all three furnaces, the gas flow can be interrupted during the atomisation stage, and in the HGA-74 and HGA-76 a mini-flow of gas can be used. The TABLE I COMPARISON OF METHODS FOR THE DETERMINATION OF LEAD IN NATIONAL QUALITY CONTROL Method Conditions Delves 10-pl, undiluted CUP sample Furnace, 20-4, 10-fold HGA-72 diluted sample : Gas-stop Furnace, 1 0-p1, 5-fold HGA-74 diluted sample: Gas-stop Mini-flow Furnace, lO-pl, &fold HGA-76 diluted sample : with AS1 Gas-stop Mini-flow BLOOD SAMPLES Sample Coefficient concentration/ of variation, 0.40 5.3 1 .oo 3.3 pg ml-1 % 0.40 4.2 1 .oo 4.0 1.00 4.6 1.00 4.1 0.25 0.65 0.25 0.65 2.3 0.9 5.5 4.5 Rate of sample analysis* / samples h-1 30 30 20 20 20 20 25 25 25 25 Comments Operator-dependent Drift must be corrected Blood calibration Blood calibration Manual operation Manual operation Blood calibration Manual operation Aqueous calibration Automatic operation Aqueous calibration Automatic operation Aqueous calibration * Each sanple analysed in duplicate.302 USE OF ENZYMES IN ANALYTICAL CHEMISTRY Proc.Analyt. Div. C h m . Soc. gas flow used has a critical effect on the ability of the instrument to give a similar response from aqueous lead standards and blood lead standards at the same concentration.Results obtained under all the configurations that gave results of acceptable accuracy and sensitivity are shown in Table I. Gas-flow during atomisation was not suitable on any of the instruments, and mini-flow in general gave a lower sensitivity and, hence, increased coefficient of variation compared to gas-stop.For routine analysis of blood samples, the HGA-72 was used under gas-stop with blood standards, the HGA-74 with mini-flow and aqueous standards and the HGA-’76 with gas-stop and aqueous standards. Although interferences were observed in aqueous solutions from calcium, potassium and sodium at the levels present in diluted whole blood on all three instruments, aqueous standard calibration graphs were parallel to blood standard calibration graphs under the conditions in which aqueous calibrations are stated to be used (Table I).A comparison of the performance of the methods described in this paper is shown in Table I. Comparative analysis of the same samples, using various combinations of the techniques shown, always gave satisfactory agreement.Results of the analysis of National Quality Control blood samples showed good agreement between all the procedures shown and the mean values quoted from many collaborating laboratories. All these methods, therefore, give results of satisfactory accuracy when optimised and operated correctly and are suitable for blood lead determinations. In terms of precision, the autosampling device shows a significant improvement, as expected, and removes the need for manual pipetting and constant operator attention. The Delves cup technique has been used for several years at the Glasgow Royal Infirmary and is still advantageous for handling large numbers of samples. It has, however, been found to be extremely operator-dependent, and the ease of aqueous calibration and automatic injection may make the furnace technique more attractive in the future, despite a slower rate of analysis. References 1. 2. 3. Delves, H. T., Analyst, 1970, 95, 431. Banthel, W. F., Smiek, A. L., Angel, G. P., Liddle, J. A., Landrigan, P. J., Gehlbach, S. H., and Joselow, M. M., and Singh, N. P., Atom. Absorption Newsl., 1973, 12, 128. Chisolm, J. J., J . Ass. 08. Analyt. Chem., 1973,56,1252.

ISSN:0306-1396    

DOI:10.1039/AD9771400300

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

302 USE OF ENZYMES IN ANALYTICAL CHEMISTRY Proc. Analyt. Div. C h m . Soc. Use of Enzymes in Analytical Chemistry The following is a summary of one of the papers presented at a Meeting of the Analytical Division, organised by the Automatic Methods and Electroanalytical Groups, held on Decem- ber 8th, 1976, in London. Summaries of three of the other papers presented at the Meeting appeared in the May issue of Proceedings (p.103). Analytical Use of Enzymes G. G. Guilbault Department of Chemistry, Louisiana State University in New Orleans, New Orleans, La. 70122, USA and M. H. Sadar Department of Basic Sciences, Faculty of Engineering, Middle East Technical University, Gaziantep, Turkey Enzymes are proteins that act as biological catalysts and enable the many complex chemical reactions, upon which depends the very existence of life as we know it, to take place a t ordinary temperatures.Because enzymes work in complex living systems, one of their outstanding properties is specificity. An enzyme is capable of catalysing a particular reaction of a parti- cular substrate, even though other isomers of that substrate or similar substrates may be present.This specificity of enzymes, and their ability to catalyse reactions of substrates at low concentrations, is of great use in chemical analysis. Enzyme-catalysed reactions have been usedOctoher, 1977 USE OF ENZYMES I N ANALYTICAL CHEMISTRY 303 for analytical purposes for a long time for the determination of substrates, activators and inhibitors, and also of enzymes themselves.Until recently, however, the disadvantages associated with the use of enzymes have seriously limited their usefulness. Frequently cited objections to the use of enzymes for analytical purposes have been their unavailability, instability, poor precision and the labour of performing the analyses. While these objections were valid earlier, numerous enzymes are now available in purified form, with high specific activity, at reasonable prices.With the advent of new techniques, fluorimetric and electrochemical as well as the successful immobilis- ation of several enzymes, many of the previous difficulties have now been resolved. The following is a Michaelis - Menten equation for an enzyme kinetics: kl k2 E + ST ES -+ E + P .. .. * * (1) . . k-1 In this mechanism, the substrate, S, combines, with the enzyme, E, to form an intermediate complex, ES, which subsequently breaks down into products, P, and liberates the enzyme.The initial rate of reaction, V,, is some function of the starting concentrations of enzyme and the substrate, [S],, as well as of activator and inhibitor concentration, if the latter two are present, and is expressed by the following relationship : where K,, the Michaelis constant, is defined as (k, + L I ) / k l .A t a fixed enzyme concentration, the initial rate increases with substrate until a non- limiting excess of substrate is reached, after which additional substrate causes no increase in rate. The concentrations of material participating in an enzyme reaction can be calculated in one of two ways: by measuring the total change that occurs by chemical, physical or enzymatic analysis of the product or unreacted starting material; or from the rate of the enzyme reaction.In the first method, large amounts of enzyme and small amounts of substrate are used to ensure a relatively complete reaction. The reaction is allowed to reach equilibrium, and the amount of substrate S in the sample can be calculated from the amount of P formed, provided that P is chemically and physically distinguishable from S.Because it is more reliable, the total change method is generally favoured over the rate method, but can only be used for substrate analysis, and not for enzyme, activator and inhibitor analysis as these are catalytic in nature and affect only the rate and not the equili- brium.In the kinetic method, the initial rate of reaction, V,, is measured in one of many conven- tional ways, by following either the production of product or the disappearance of the sub- strate. The rate is a function of the concentration of substrate (S), enzyme (E), inhibitor (I) and activator (A). Also, the rate method is faster, because the rate can be measured initially without having to wait for the reaction to go to completion.The conditions that affect the rate (pH, temperature and ionic strength) must be carefully controlled in the kinetic method in order to achieve maximum sensitivity. The temperature coefficient of the enzyme reaction rate is roughly 10% per degree,l and a 10 "C increase in temperature causes a 100 yo increase in the reaction rate.Hence, constant temperature is essential in the assay of enzyme activity. Recent work by Guilbault and co-worker~~~~ and Pardue et aL4 has indicated that with reasonable care, precisions and accuracies better than those expected within the accepted error limits can be obtained. Furthermore, some of the difficulties encountered because of side-reactions are eliminated in rate methods and greater sensitivities can be obtained in many instances.Analytical Applications Determination of the Substrates and the Enzymes At a fixed enzyme concentration, the initial rate of an enzymatic reaction increases with increasing substrate concentration until a non-limiting excess of substrate is reached, after304 Pvoc. Analyt.I>iu. ch~.znz. Soc. which additional substrate causes no increase in rate. The region in which linearity is achieved, and in which an analytical determination of substrate concentration can be made based on the rate of reaction, lies below 0.1 K,. The most important advantage of an enzymatic assay is its specificity. Frequently only one member of a homologous series is active in the enzyme- catalysed reaction; other members are totally inactive or react at much slower rates.Most enzymes are also specific for one optical isomer of the substrate. Thus, in the enzymatic assay of amino acids, bacterial amino acid decarboxylase is specific for L-amino acid 0nly.5 Another advantage in the use of enzymes for substrate analysis lies in the great sensitivity obtained.Glucose, for example, is oxidised at the rate of a few per cent. per minute, regardless of concentration. A complete compilation of enzymic methods for the assay of carbohydrates, amines, amino acids, organic acids, hydroxy compounds, esters, aldehydes and inorganic substances has been prepared by Guilbault69' and Bergmeyer.8 The recent trends in enzymic analysis have been in two main directions: to develop more sensitive procedures and to replace the long, tedious methods previously used for assay with rapid easily instrument able techniques.Common techniques such as spectrophotometry, measurements of change in pH and mano- metric measurements have been described for the assay of almost all enzymes.6JQ Because of their simplicity and susceptibility to automation, electrochemical methods have been used extensively to follow enzyme activity.10-21 USE OF EXZYMES IN ANALYlJCAL CHEMISTRY Thus, a lo-' M solution can be analysed as easily as a M solution.Determination of Activators An enzyme activator is a substance that is required for an enzyme to be a catalyst : The activity of the enzyme will increase until enough activator has been used to activate the enzyme fully. The initial rate of the enzyme reaction is proportional to the activator con- centration at low concentrations, thus providing a method for its determination. Very little work has been carried out on the analytical chemistry of activators.22-26 Determination of Inhibitors An inhibitor is a compound that causes a decrease in the rate of enzyme reaction, either by reacting with the enzyme to form an enzyme -inhibitor complex or by reacting with the enzyme - substrate intermediate to form a complex : E+I-EI E + S ~ EST P + E + I 4? E - S - I In general, the initial rate of an enzymic reaction will decrease with increasing inhibitor concentration, linearly at low inhibitor concentrations, and then will gradually approach zero.Analytical working curves for inhibitor assay are generally constructed by plotting percent- age inhibition against concentration of inhibitor. The percentage inhibition is calculated as follows : Rate NO inhibitor - Rate Inhibitor Inhibition (yo) = - x100 Rate NO inhibitor A control rate is recorded with no inhibitor present, but with the same volume of the solvent used to contain the inhibitor added.This is especially critical in studies of inhibitors added in non-aqueous solution, as most non-aqueous solvents will inhibit the enzyme at concentra- tions greater than 3%. In addition to the control (non-inhibited) rate, the rate of spontaneousOcfoher, 1977 TJSE OF ENZYMES I N ANALYTICAL CHEMISTRY 305 (non-enzymic) hydrolysis or oxidation of the substrate should be measured and all rates corrected for such non-enzymic effects, if necessary.Generally a graph of percentage inhibition against concentration is a typical exponential type of curve with a linear range extending from 0 to 60 or 70% inhibition. This linear region is the most analytically useful range. The concentration of inhibitor that causes a 50% inhibition of enzymic activity is termed the I,,, and is a measure of the strength of an inhibitor.Several analytical methods for the determination of trace amounts of pesticides, insecticides and toxic metal ions have been proposed based on the inhibition of an enzyme rea~tion.~7-~~ Inorganic Substances Although enzymic methods have been described for the assay of many inorganic substances, two very sensitive procedures that are worthy of special note are those for phosphate and ammonium ions.The enzymatic determination of ammonia in tissue body fluids was des- cribed by Faway and Dah1,37 in body fluids by Mondzac et u Z . , ~ ~ in blood and tissue by Reichelt et aZ.39 and in blood by Kirsten et Schulz et described a fluorimetric enzymic method for the measurement of inorganic phosphate.Enzymic methods have been described for many common inorganic cations and anions: Ag + ’ Al3+, Be2+, Bi3+, Ce3+, Cd2+, Co2+, Cu2+, Fe2+, Hg 2+ , Ins+, Mn2+, Ni2+, Pb2+, Zn2+, CN-, Cr20,2-, F- and S2-. A complete listing of these procedures was compiled by Guilbault.’ Conclusions Enzymes possess great potential usefulness in analytical chemistry. The specificity of enzymes can solve the primary problem of most analytical chemists, namely the analysis of one substance in the presence of many similar compounds that interfere in the analysis.The sensitivity of enzymes allows the determination of as little as 10-lo g of material. With the advent of new techniques, electrochemical and fluorimetric, many of the previous difficulties of enzymic analysis have been eliminated.The advent of the immobilized enzyme has alleviated the problem of the cost and supply of enzymes. Finally, significant progress has been made in the direction of complete automation of enzymic methods for rapid, accurate analysis. 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. References Netter, H., “Theoretische Biochemie,” Springer Verlag, Berlin, 1959, p.554. Guilbault, G. G., Brignac, P., and Zimmer, M., Analyt. Chem., 1968, 40, 190. Guilbault, G. G., Brignac, P., and Juneau, M., Analyt. Chem., 1968, 40, 1256. Pardue, H., Burke, M., and Jones, D. O., J . Chem. Educ., 1967, 44, 684. Gale, E. F., Biochem. J., 1945, 39, 46. Guilbault, G. G., Analyt. Chem., 1966,38, 537R; l968,40,459R; 1970,42, 334R.Guilbault, G. G., “Enzymatic Methods of Analysis,” Pergamon Press, Oxford, 1970. Bergmayer, H. V., “Methods of Enzymatic Analysis,” Second Edition, Verlag Chemie, Weinheim,. Blaedel, W. J., and Hicks, G. P., in Reilley, C. N., Editor, “Advances in Analytical Chemistry and Guilbault, G. G., Kramer, D. N., and Cannon, P. L., Analyt. Chem., 1962, 34, 842.Guilbault, G. G., Kramer, D. N., and Cannon, P. L., Analyt. Chem., 1962, 34, 1437. Guilbault, G. G., Tyson, B., Kramer, D. N., and Cannon, P. L., Analyt. Chem.. 1963, 35, 582. Guilbault, G. G., Kramer, D. N., and Cannon, P. L., Analyt. Chem., 1964, 36, 606. Guilbault, G. G., Analyt. Biochem., 1966, 14, 61. Malmstadt, H. V., and Pardue, H. L., Analyt. Chem., 1961, 33, 1040.Malmstadt, H. V., and Pardue, H. L., Clin. Chem., 1962, 8, 606. Pardue, H., Simon, R., and Malmstadt, H., Analyt. Chem., 1964, 36, 735. Pardue, H., Analyt. Chem., 1963, 35, 1240. Pardue, H., and Simon, R., Analyt. Riochem., 1964, 9, 204. Blaedel, W. J., and Olson, C., Analyt. Chem., 1964, 36, 343. Pardue, H., and Frings, C.. J. Electroanalyt., Chem., 1964, 7, 398. Baum, P., and Czok, R., Biochem.Z., 1959, 332, 121. Adler, E., Gunther, G., and Plass, M., Riochem. J., 1939, 33, 1028. Warburg, O., and Christian, W., Biochem. Z., 1938, 298, 150. Straub, F. B., Biochem. J., 1939, 33, 787. 1965. Instrumentation,” Volume 3, Interscience, New York, 1964, pp. 105-140.306 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. ANALYSIS OF INDUSTRIAL ESSENTIAL errs Proc.14nalyt. Div. Chem. SOC. Huennekens, F. M., and Felton, S. P., “Methods of Enzymology,” Academic Press, New York, 1957, Kitz, R. J., Acta Anaesthesiol. Scand., 1964, 8, 197. Giang, P. A., and Hall, S. A., Analyt. Chem., 1951, 23, 1830. Kramer, D. N., and Gamson, R. M., Analyt. Chem., 1957, 29 (12), 21A. Guilbault, G. G., and Sadar, M. H., Analyt. Chem., 1969, 41, 366. Guilbault, G. G., Sadar, M., and Zimmer, M., Analytica Chim. Acta, 1969, 44, 361. Abou-Donia, M. B., and Menzel, D. B., Conzp. Biochem. Physiol., 1967, 21, 99. Winteringham, F., and Fowler, K. S., Biochem. J . , 1966, 99, 6P. Guilbault, G. G., Sadar, M. H., Kuan, S., and Casey, D., Analytica Chim. Acta, 1970, 51, 83. Guilbault, G. G., Sadar, M. H., Kuan, S., and Casey, D., Analytica Chim. Acta, 1970, 52, 75. Guilbault, G. G., and Sadar, M. H., J . Agric. Fd. Chem., 1971, 19, 357. Faway, G. G., and Dahl, K., Lebanese Med. J.. 1963, 16, 169. Mondzac, A. A., Ehrlich, G., and Seegmiller, J., Lab. Clin. Med., 1965, 66, 526. Reichelt, K. L., Kjamme, E., and Tviet, B., Scand. J . Clin. Lab. Invest., 1964, 16, 433. Kirsten, E., Gerez, C., and Kirsten, R., Biochem. Z . , 1963, 337, 312. Schulz, D. W., Passonneau, J. V., and Lowry, 0. A., Analyt. Biochem., 1967, 19, 300. p- 950.

ISSN:0306-1396    

DOI:10.1039/AD9771400302

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

306 ANALYSIS OF INDUSTRIAL ESSENTIAL errs PYOC. 14na131t. Div. Chem. SOC. Analysis of Industrial Essential Oils The following is a summary of one of the papers presented at a Meeting of the South East Region and the Chromatography and Electrophoresis Group held on May Mth, 1977, a t Chelsea College, London. Some Practical Experiences of Reporting Integrators in Industrial Essential Oil Analysis D.A. Moyler Yardley of London Limited, Basildon, Essex A reporting integrator integrates a chromatographic signal in the conventional sense by giving peak-retention times and areas, etc., with the added feature of thermally plotting I 4 Fig. 1. Chromatogram of a commercial geranium bourbon oil sample. The numbers on the peaks are retention times in 1/100 min.October, 1977 ANALYSIS OF INDUSTRIiiL ESSENTIAL OILS 307 Fig.2. Chromatogram of an Italian jasmin absolute oil sample obtained on column 1. The numbers on the peaks are retention times in 1/100 min. the chromatogram. on heat-sensitive paper. integrator plus recorder are as follows. The integrals and chromatograms are tabulated as a combined report The features of reporting integrators compared to a conventional (i) Convenience: all the data and the chromatogram are on one piece of chart, making storage easier, particularly on microfilm.(ii) Ease of calculation: the chromatogram has retention times printed next to the peaks, which is easier for reference and retention index calculations, especially with complex chromat ograms. (iii) Reliability: after 20 000 reports, the only servicing done by the author has been to clean air filters.(iv) Chart cost: the cost per foot is similar to that for the accurately gridded recorder chart. Some reporting integrators can be used directly for calculating the MovAts index and the temperature-programmed retention index, including tabulating with the report. Examples were shown. A report of a commercial geranium bourbon oil sample was shown, in which an unusual peak was found at the 1% level.This was shown to be diphenyl ether by a retention index of 1374 on stationary phase SE-30 and of 2 013 on Carbowax 20M, both columns being standardised.1 The diphenyl ether does not occur naturally, but has a strong geranium- type odour and was probably added in order to enhance an inferior oil.Fig. 1 shows a chromatogram obtained by using a column of 15o/b Carbowax 2011 on Chromosorb W AW DMCS support, 2.10 m long, 4 min in diameter, of 4 000 theoretical plates, polarity 1.277, and temperature programmed from 75 to 225 "C at 2 "C min-l. Italian jasmin absolute oil, selected by odour as being the best of 30 jasmine samples in308 Proc. Analyt. Div. Cheurt. Soc.1976, was critically analysed. Two SE-30 columns were used: I , of 3 000 theoretical plates, 1.50 m by 4 mm, polarity 1.004, programmed from 60 to 300 “C at 4 “C min-l; and 2, of 200 000 theoretical plates, 50-m wall-coated open tubular capillary, programmed from 80 to 310 “C at 5 “C min-l after “splitless injection.” The chromatograms obtained by use of columns 1 and 2 are shown in Figs.2 and 3, respectively. ANALYSIS OF INDUSTRIAL ESSENTIAL OILS L I I Fig. 3. Chromatogram of an Italian jasmin absolute oil sample obtained on column 2. Column 1 gave 57 peaks, 34 of which were identified; most of the others were components specific to jasmin or components too small to identify with certainty (<0.1%). Gas chromatography - mass spectrometry was used for the identification and gas chromatography - infrared spectrometry and retention indices were used for Confirmation.Column 2 revealed 180 peaks. This sample was of interest because four of the major components occurring naturally are also available as high-purity synthetics at low cost, viz., linalol, benzyl acetate, benzyl benzoate and isophytol. Some of the more odoriferous, less abundant components were also present in higher amounts than in other samples, vix., indole, the jasmones and anthranilates. This sample also contains many components thought not to occur naturally in jasmin, e.g., diethyl phthalate and LRT 14 (2-pentyl-2-acetonylcyclopentanone). I express my appreciation for the technical help given on the jasmin sample by R. J . H. Duprey and Rarrie Frost. Reference 1. van den Dool, H., “Standardisation of Gas Chromatographic Analysis of Essential Oils,” Rijksuni- versiteit t e Groningen-Proefschrift, Rotterdam, 1974.

ISSN:0306-1396    

DOI:10.1039/AD9771400306

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

NEW PRESIDENT OF IUPAC ANALYTICAL CHEMISTRY 1)IVLSION 309 Conferences and Meet i ng s Symposium on Electrochemical Ana I ysis-Pola rog rap h y i n Act ion November 2-3, 1977, London This international Symposium is to be held at the City University and its aim is to bring the practical aspects and fields of application of polarography to the attention of practising analysts and industrial chemists.Examples will be given of how polarography is being used to solve analytical problems con- cerned with biology, dyestuffs, general analysis, heavy chemicals, marine chemistry, pharma- ceuticals, polymers, quality control and water and effluents. An exhibition of polarographic equipment is being arranged by EDT Research. Further information can be obtained from the Symposium Secretary, Scientific Symposia Ltd., 42/43, Gerrard Street, London, W1V 7LP.310 PUBLICATIONS RECEIVED Proc.AnaZyt. Div. Chew. SOC. Food Colour and Appearance Symposium April4-5, 1978, Guildford The Colour Group (Great Britian) is holding a 2-day Symposium at the University of Surrey, Guildford. It is planned to include papers dealing with assessment techniques and instru- mentation, colour specification applied to raw materials and products, quality control, food colourants and legislation, and psychophysics.Further details from : John Hutchings, Unilever Colworth/Welwyn Research Labora- tory, Colworth House, Sharnbrook, Bedford- shire. European Conference on Optical Systems and Applications (ECOSA.l) April 4-6, 1978, Brighton The Quantum Electronics Group of The Institute of Physics, in collaboration with the Institution of Electrical Engineers, the Quantum Electronics Division of the European Physical Society and, probably, with co-sponsorship of other European bodies, is arranging a Conference to be held in conjunction with the Electro- Optics -International Exhibition and Profess- ional Advancement courses, organised by Kiver Communications a t the Hotel Metropole in Brighton.Provisionally, sessions are planned on : Dye Lasers; General Applications ; Information Pro- cessing and Display ; Solid-state Lasers ; Tunable Lasers and Isotope Separation ; Spectroscopy and UV Lasers; Laser Fusion; Pollution Monitoring and Remote Sensing ; Laser Machin- ery and Materials Processing ; Gas Lasers ; Radar and Propagation ; Modulators, Para- metric and Non-linear Optical Devices ; Guided Waves and Optical Communications ; Semi- conductor and Miniature Solid-state Lasers.Further details can be obtained from The Meetings Officer, The Institute of Physics, 47 Belgrave Square, London, SWlX SQX. Fourth International Meeting on NMR Spectroscopy July 2-7, 1978, York This Meeting will take place a t the University of York and is being organised jointly by The Chemical Society and the NMR Discussion Group of The Chemical Society.The Meeting will deal with selected topics in high-resolution NMR spectroscopy and will comprise seven consecutive symposia : NMR Conformational Studies ; NMR Studies of Large Molecules ; NMR Studies of Biological Systems ; Studies of Dynamic Processes ; Relaxation Studies ; New Experimental Techniques and Applications ; and Review Lectures.The British Radiofrequency Spectroscopy Group is planning to participate, sharing a common day with the International Meeting on July 6th and arranging its own programme on July 7th. In addition to the invited lectures, there will be a number of short contributed papers in- cluded in the programme and in poster sessions. Anyone wishing to contribute a paper should submit, by February lst, 1978, a title and synopsis (ca. 250 words) to Dr. J. Feeney, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 lAA, indicating the appropriate symposium. Further information can be obtained from Dr. John F. Gibson, Assistant Secretary (Scientific & Educational), The Chemical Society, Burlington House, London, W1V OBN.

ISSN:0306-1396    

DOI:10.1039/AD977140309c

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

310 PUBLICATIONS RECEIVED Proc. Analyt. Div. Chew. SOC. Publications Received Gas Chromatography in Food Analysis. G. J. Dickes and P. V. Nicholas. Pp. xii + 393. London, Boston, Sydney, Wellington, Durban and Toronto: Rutterworths. 1976. Price fll6. Martindale. The Extra Pharmacopoeia. Twenty-Seventh Edition. Edited by L4inley Wade. Pp. xxxii + 2077. London : The Pharmaceutical Press. 1977.Price fT30. Report of the Central Drugs Laboratory, Calcutta. 1st April 1968 to 31st March 1971. Pp. iv + 57. Delhi: Government of India Ministry of Health and Family Planning. Price Rs8.70; k1.02; $3.13. British Pharmacopoeia 1973. Addendum 1977. Pp. xx + 51 + Appendices A1 to A21. Lon- don: HM Stationery Office. 1977. Price i 6 . Progress in Drug Metabolism. Volume 2. Edited by J. W.Bridges and L. F. Chasseaud. Pp. x + 348. London, New York, Sydney and Toronto: John Wiley. 1977. Price k13.50; $27. Dithizone. H .M. N. H . Irving. A nalytical Sciences Mono- grafihs, No. 5. Pp. iv + 106. London: The Chemical Society. 1977. Price k7.25 (CS members L5.50).October, 1977 ANALYTICAL DIVISION DIARY 31 1 Analytical Pyrolysis. Methods of Analysis for Herbicide Resi- Edited by C. E. Roland Jones and Carl A. dues. Second Edition. Cramers. Proceedings ofthe Third Intevnational T . H. Byast, E. G. Cotterill and R. J. Hance. Symposium o n Analytical Pyrolysis, h,eld in Technical Report Agricultural Research Council Amsterdam, September 7-9, 1976. Pp. x + 424. Weed Research Organization, No. 15. Pp. ii + Amsterdam, Oxford and New York: Elsevier. 62. Oxford: ARC Weed Research Organiza- 1977. Price $39.25; Dfl96. tion. 1977. Price k5.75.

ISSN:0306-1396    

DOI:10.1039/AD9771400310

出版商:RSC    

年代:1977

数据来源: RSC