摘要:

of the Analytical Division ofThe Chemical SocietyCONTENTS313 CS Autumn Meeting31 3Gold MedalSociety for Analytical Chemistry314 AD Distinguished Service Award314 Council Nominations314 AD Questionnaire314 Proposed Legislation319 Summaries of Papers31 9 'Electroanalysis in BiologicalFluids'347 Equipment News350 Correspondence352 Conferences and Meetings352 Course352 Publications Received354 Analytical Division DiaryVolume 14 No 11 Pages 31 3-354 November 197PADSDZ 14(11)313-354(1977)ISSN 0306-1 396November 1977PROCEEDINGSANALYTICAL DIVISION OF THE CHEMICAL SOCIETYOF THEOfficers of the Analytical Divisionof The Chemical SocietyPresidentD. W. WilsonHon. SecretaryP . G. W. CobbSecretaryMiss P. E. HutchinsonHon. Treasurer Hon.Assistant SecretariesJ. K. Foreman D. I. 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 Analytical Abstracts.Q The Chemical Society 1977THE ANNUAL MEETINGonR AND D TOPICS IN ANALYTICAL CHEMISTRYwill be held at theUniversity of Wales Institute of Science and Technology,Card iffonJune 28th and 29th, 1978Papers are invited describing work carried out by postgraduate research studentsin Universities and Colleges and by young research workers in industrial and otherestablishments. Contributions are to be presented by the student or his industrialcounterpart during a 20-minute lecture.Those who wish to offer a paper or who have any queries about the meetingshould write to the Secretary, Analytical Division, Chemical Society, BurlingtonHouse, Piccadilly, London, W1 V OBN

ISSN:0306-1396    

DOI:10.1039/AD97714FX036

出版商:RSC    

年代:1977

数据来源: RSC

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November, 1977 ANALYTICAL DIVISION DIARY 353Analytical Division Diary, continuedDecember, continuedWednesday, 7th, 2.15 p.m.: LondonElectroanalytical Group : Annual GeneralMeeting, followed by a meeting on “TheUses of Electroanalgtical Techniques inPharmaceutical Studies.”“The Use of Polarographic Techniques inPharmacokinetic Studies,” by R. Cooper.“The -4pplication of Polarographic OxidationWaves in Pharmacy and Biochemistry,”by I. E. Davidson.“The Use of Electrochemistry in the Pharma-ceutical Industry,” by V. Kirsner.“The Application of Polarography to ForensicProblems,” by J. S. Burmicz.Wellcome Foundation Auditorium, 183Euston Road, London, N.W. 1.Wednesday, 7th, 3 p.m.: LondonAnalytical Division : Fourth SAC SilverMedal Lecture. “Gamma ActivationAnalysis-An Appraisal,’’ by J.S. Hislop.Scientific Societies Lecture Theatre, 23 SavileRow, London, W. 1.Tuesday, 13th, 9.30 a.m.: SheffieldThermal Methods Group : Annual GeneralMeeting, followed by a meeting on“-4pplications of Thermal Methods toInorganic Materials.”Speakers to include: F. W. Wilburn, F. P.Glasser, P. F. James, F. Sale and H. A.Davis.Department of Ceramics, Glasses and Poly-mers, The University, NorthumberlandRoad, Sheffield.ANALYTICAL DIVISIONTIEThe wider version of the tie bearing the SAC Coat of Arms is still available forsale to members of the CS Analytical Division. The tie, which carries a simp-lified version of the Coat of Arms woven in red, silver and gold as a singlemotif, is available in three different background colours-dark blue, dark greenand maroon. The tie is manufactured in a Crimplene - Terylene mixture.The price of the tie is Ll.60, post free.The traditional, narrower versionof the tie is also still available (with dark green or maroon background only)at Ll .lo, post free. Orders, accompanied by the appropriate remittance, shouldbe sent to The Secretary, Analytical Division, The Chemical Society, Burling-ton House, Piccadilly, London, W1V OBN.Please make cheques payable to The Chemical Society, and ensure thatthe background colour required on the tie is statedAnalytical Division DiaryNOVEMBERWednesday, 23rd, 6.15 p.m. : WolverhamptonMidlands 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.Wednesday, 23rd, 2.30 p.m.: Londonsearch.”Thursday, 24th, 10.30 a.m. : LondonAutomatic 4Wethods 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.: GlasgowScottish Region, jointly with the Glasgow andWest of Scotland Section of the CSIRICand 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, GI 1XL.Wednesday, 30th: DidcotSpecial Techniques Group : Annual GeneralTour of selected laboratories and short pre-AERE, Harwell, Didcot, Oxon.Meeting.sentations on instruments on view.DECEMBERThursday, lst, 2 p.m.: SunderlandNorth East Region and ,Microchemical MethodsGroup on “Microchemistry in the Pharma-ceutical Industry.”“Microchemistry in the Control of Pharma-ceuticals,” by C.A. Johnson.“The Identification of Trace Impurities inPharmaceutical Products by GC - MS,” byD. Catlow.“The Radioimmunoassay of Drugs in Bio-logical Fluids,” by F. J. Rowell.“Some Aspects of Automation in Pharma-ceutical Analysis,” by C . A. Young.“What of the Future? Summing-up andSchool of Pharmacy, The Polytechnic, Sun-Closing Remarks,” by C.A. Johnson.derland.Thursday, 1st: LondonEducation and Trainin<< Group : 14nnualGeneral Meeting; 2.15 p.m.Particle Size A naZysis Group : Annual GeneralMeeting; 2.30 p.m.Followed a t 2.45 p.m. by a Joint Meetingon “Education and Training of PowderTechnologists and Particle Size Analysts.”Wellcome Foundation Auditorium, 183Euston Road, London, N.W.1.Friday, 2nd, 7 p.m.: CardiffWestern Region, jointly with the South EastWales Section of the CS/RIC and theUWIST Student Chemical Society.“Electrochemical Methods in WaterAnalysis,’’ by M. J. Stiff.Department of Chemistry, UWIST, Cardiff.Tuesday, 6th: LondonAtomic Spectroscopy Group .- Annual GeneralMeeting; 2.15 p.m.Chromatography and Electrophoresis Group :Annual General Meeting; 2.30 p.m.Followed at 2.45 p.m. by a Joint Meetingon “Detectors for Chromatography.”“Selective Gas Chromatography Detectors,”by E. R. Adlard.“Performance of the Microwave InducedPlasma as an Element Selective Detectorin Gas Chromatography,” by C. I. M.Beenakker.“Directly Coupled Gas Chromatography -Atomic Spectroscopy,” by L. Ebdon andD. A. Leathard.Geological Society Lecture Theatre, Burling-ton House, Piccadilly, London, W. 1.Tuesday, 6th, 6.30 p.m. : EasthamNorth West Region, jointly with the Carlett“One Hundred Years of Analytical ChemistryCaxlett Park College of Technology, Eastham,Park Chemical Society.in Teaching,’’ by K. Jones.Wirral .[continued inside back coverPrinted by Heffers Printers Ltd Cambridge Englan

ISSN:0306-1396    

DOI:10.1039/AD97714BX038

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

Vol. 14 No. 11 November 1977 Proceedings of the Analytical Division of The Chemical Society CS Autumn Meeting University of Aberdeen, September 20th-22nd, 1977 Tlie Analytical llivision participated in the Soil Research, where they were welcomed by the recent CS Autumn Meeting, held at the Univer- Ijirector, l’rofessor T. S. West. sity of hberdeen, and organisetl a Symposium Social events during this successful meeting on “Soils, Geochemistry and the Exploitation of included a dawn visit to the Aberdeen Fish Mineral Rcsources.” A full programme of Market, a Civic Reception a t the Town House papers was arrangccl on the Rilonday ant1 o:i and a Conference Reception and Dinner at Tuesday morning, while 011 Tuesday aiternoon delegates visited the Macaulay Institute for Elpliinstone Hall.A checrful group of visitors to The Macaulay Institute for Soil Research from the C S Autumn iweeting at Aberdeen. Front row (below step, L-R): M r . R. J . Hall, M Y . D. W . Wilson (AD President), Dv. J . K . Foreman, Pvofessov T . S. West (Director, Macaulay Institute) and ( f a r right) Dv. A . ill. Uve (Chairman, AD Scottish Region). Society for Analytical Chemistry Gold Medal On the recommendation of its Honours Com- GoldMedalto mittee, the Council of the Analytical Division, at Dr.James Kenneth Foreman its meeting on September 27tl1, awarded the Deputy Director of the National Physical Lab- thirteenth Society for Analytical Chemistry oratory, formerly Deputy Government Chemist.314 AD DISTINGLJISHED SERVICE AWARD Proc. Analyt. Div. Chem. SOC. Ana I ytica I Division Distinguished Service Award On the recommendation of its Honours Coni- mittee, the Council of the Analytical Division, a t its meeting on September 27th, conferred the third Analytical Division Distinguished Service Award on Mr. Harold Edwin Brookes of The Boots Company Limited, Nottingham.

ISSN:0306-1396    

DOI:10.1039/AD9771400313

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

314 AD DISTINGLJISHED SERVICE AWARD Proc. Analyt. Div. Chem. SOC. Proposed Legislation Part II*: EEC Legislation Legislation that will be proposed as a result of EEC discussions or published Directives and Regulations is described in the second part of this article on proposed legislation for food- stuffs. A brief outline of any analytical methodology under discussion is also given. Acids, Bases and Salts Used in the Preparation of Food The EEC Commission has drawn up a working document setting out proposals for a Directive on the approximation of the laws of the Member States relating to substances used during the manufacture of foodstuffs to change * Part I of this series appeared in Proc. Analyt.Div. Chem. SOC., 1977, 14, 237. or stabilise their acidity or alkalinity. The scope of this document may now be said to include all acids, bases and salts used in the preparation of foods.The proposals have been widely circulated by the Ministry of Agri- culture, Fisheries and Food (MAFF). Antioxidants Specific criteria of purity for some of those substances which are defined by the Community as being antioxidants (and therefore have E numbers in the three hundreds) are being discussed.There is a proposal for a Council Directive laying down the specific criteria of purity for antioxidants that may be used in foodstuffs intended for human consumption,November, 1977 PROPOSED LEGISLATION 315 which has recently been submitted to the Council by the Commission and which will shortly be published in the Oficzal Journal of the Euvopean Counmztnities. (Methods of ana- lysis for criteria of purity of all food additives are considered in a later section.) There has been a proposal, dating from 1971 (i.e., prior to UK accession to the Community), within tlie Commission for a method of analysis to detect propyl, octyl and dodecyl gallstes, butylated liydroxyanisole and butylatetl hydr- oxytoluene in oils, fats and bakery products, based on separation of oils and fats by tliethyl ether - petroleum spirit, isolation o f the anti- oxidants present using acetonitrile and petro- leum spirit and a tliin-layer chromatographic identification step.Casein and Caseinates The Commission issued a revised text of thc draft proposal for a Council Directive on the approximation of the laws of tlie Member States relating to edible caseins, caseinates and co-precipitates in 1977.Methods of analysis for caseins and caseinates have also been considered and, in particular, the following determinations are under active discussion. Lactose: by the extraction of lactose from the sample into the filtrate obtained by dissolution of the sample in water, sodium hydrogen carbonate or sodium phosphate solution and precipitation with an acetic acid - sodium acetate buffer solution (pH 4.6).The lactose is then determined by its reaction with phenol in sulphuric acid medium and colorimetric measurement. Copper : by a spectrophotometric procedure using the formation of the copper salt of dicthyl- dithiocarbamic acid in neutral solution after sulphuric acid - nitric acid digestion of the sample or by an atomic-absorption spectro- photometric procedure.Iron : by an atomic-absorption spcctrophoto- metric procedure. Solubility : by centrifuging a sample solution. Moisture : by loss of mass on drying at 102 & 2 "C. Free acidity of acid casein : by titration of sample solution against sodium hydroxide so1utio:i using phenolphthalein as indicator.Fat content : by sample digestion with hydro- chloric acid followed by diethyl ether - petro- leum spirit extraction. pH determination : by measurement with a pH meter oi a sample solution. Scorched pirrticlcs in acid caseins : by visual comparison of a suitakk sample solution with standard discs. Protein : by the Kjeldahl method. Ash: by incineration at 825 -f 25 "C, using magnesium acetate as ashing aid for acid casein samples. Cocoa and Chocolate Products Tlic Council Directive, 73/241/EEC (as amended), on the approximation of the laws of the Member States relating to cocoa and chocolate products intended for human con- sumption has been translated into UK law as The Cocoa and Chocolate Products Regula- tions 1976.2 The methods of analysis that will be required to enforce tlie criteria laid down in the Directive are about to be discussed, and an initial Draft Directive laying down such methods has been proposed by the Commission and given a general UK circulation by MAFF.The Draft describes methods for tlic determination of the following : Moisture: by Office International du Cacao et clu Chocolat (OICC) method 3/1952.Total fat: by OICC method 8a/1972. Milk f a t : by OICC method 8e/1960. Total ash: by OICC method 4a/1952. .2lldinity of ash: by OICC method 4b/1952. Unsaponifiable matter : by the International IJnion of Pure and Applied Chemistry (IUPAC) oils and fats methods, II.D.5.2. Acidity: by IUPAC method II.D.l. Solvent residues : by a head-space gas-chromato- graphic technique. Coffee Extracts A Directive on coffee extracts and chicory extracts has been adopted by Member States ; this Directive will be published in the Oficial Journal o f tlze Euvopean Conamztnities.Dis- cussions on the methods of analysis required to enforce the provisions of the proposed Directive have also been proceeding. Determinations that have been considered include those for caffeine (Levine spectrophotometric procedure), loss on drying (vacuum oven drying a t 70 "C), extraction rates and insoluble matter content of coffee extracts.There has been some work on methods for the determination of de- caffeinating solvent residues but these methods will now be considered when methods of analysis for all solvents are discussed. Most of the methods are standard; it is unlikely that any procedure suitable for the determination of extraction rates of coffee extracts will be proposed.Criteria of Purity of Food Additives It is the aim of the Community to specify purity criteria for all the permitted food additives. Some such criteria are already in existence (e.g., colours, preservatives), while others are in the course of preparation (e.g., antioxidants).It is also the intention that methods of analysis will be agreed to enforce such criteria. The Commission has asked IUPAC to provide an initial series of methods that would be suitable to enforce some of the316 PROPOSED LEGISLATION Proc. Analyt. Dzv. Chem. SOC. criteria; this has been done by IUPAC. The methods provided are extensive, numbering about 80, and tend to be based on “classical” techniques.Emulsifiers and Stabilisers Specific purity criteria for emulsifiers and stabilisers are under discussion a t the Com- mission; they will shortly be sent to Council for further discussions. These criteria relate to Annex I of the Emulsifiers and Stabilisers Directive .3 Erucic Acid The Directive (number 76/62 1/EEC) relating to the levels of erucic acid in foodstuffs has now been adopted4 and UK legislation is in the course of preparation.It is intended that the level of erucic acid in the fatty acid content of oils and fats and foodstuffs to which oils and fats have been added will be limited to 10% until June 30th, 1979, and 5% thereafter. These limits will only apply to foodstuffs in which the total fat content is greater than 5yo, except that for infant foods any fat will be required to meet the limit.The analysis of erucic acid is particularly difficult as it has very similar analytical charac- teristics to those of cetoleic acid, which is a common component of fish oils. Possible analytical procedures that may be considered in order to determine erucic acid specifically are those based on capillary-column gas - liquid chromatography, thin-layer chromato- graphy using an argentation technique and high-performance liquid chromatography.Foods for Particular Nutritional Uses The Directive (number 77/94/EEC)5 on the approximation of the laws of the Member States relating to foods for particular nutritional uses has been adopted by the Council of Ministers. The Directive encompasses foods that are of a special composition and that are clearly distinguishable from foods for normal con- sumption, that are suitable for a particular nutritional purpose and are marketed in a way which indicates their suitability for a particular nutritional use.A particular nutritional use is that which provides the nutritional requirements of persons with abnormal physiological conditions or whose digestive processes, or metabolism, are dis- turbed.Certain aspects also apply to foods for healthy infants and young children. The labelling of foods claimed to be for particular nutritional uses must : (a), indicate as part of the description the particular nutri- tional characteristic of the food ; (b), indicate exactly how this characteristic has been achieved; and (c), give information on energy, carbohydrate, protein and fat contents.It is intended that the Directive will be the forerunner to a series of Directives for specific groups of products. Fruit Juices The Directive (number 75/726/EEC) relating to the approximation.of the laws of the Member States concerning fruit juices and certain similar products has now been adopted.6 U1Z legisla- tion to implement the Directive is in the course of preparation.Various methods of analysis have been considered within the Community. These are for the following : Carbon dioxide : by the International Federation of Frilit Juice Producers (IFJU) method 42. Soluble solids: by IFJU method 8. Sugar : by IF JTJ method 4. Sulphur dioxide: by IFJU method 7. Citric acid: by the Office International de la Alcohol: by IFJU method 2.Titratable acidity: by IFJU method 3. Ferrocyanide: by OIV method A.24. Ash: by IFJU method 9. pH : by I F J U method 11. Vigne et du Vin (OIV) A.29 procedure. The Directive, however, in Article 13.a.ii, “requires that the analytical characteristics of the products defined in Articles (5) to (8)” are to be specified. Such Characteristics may be a series of chemical parameters b u t will enable the analyst to make a “legislative” assessment of fruit content.Honey The Community Directive on the harmonisa- tion of the laws of Member States relating to honey’ has now been translated into UK law as The Honey Regulations 1976.8 The Regula- tions lay down certain compositional require- ments for honey as well as a method for deter- mining diastase activity.This method was adapted from that laid down in the FAO/WHO Codex, “Recommended European Regional Standard for Honey.”g The Commission has recently proposed methods t o enforce the compositional criteria laid down in the Direc- tive; included are methods for the following : Moisture : by refractometry or pycnometry.Apparent sucrose content and apparent reducing sugar content : by the Luff-Schoorl procedure. Water-insoluble solids : by filtration of a solution of honey sample through a sintered-glass crucible and weighing of the water-insoluble residue. Ash: by calcination a t 600 “C.Xovcmber, 1977 PROPOSED LEGISLATION 317 -1cidity: by plotting a pH curve and calculation of the acidity at the equivalence point. Diastase activity : by determining the number of millilitres of 1 o/o starch solution hydrolysed by the enzyme in 1.0 g of honey in 1 h a t 40 “C.Hydroxymethylfurfural : by the Winkler photo- metric procedure. The above procedures are mainly based on the Codex European Standardg methods except that a Luff-Schoorl procedure has been suggested for the sugar determinations instead of the Lane and Eynon method.Materials and Articles in Contact with Food X Council Directive on the approximation of the laws of the Member States relating to materials and articles intended to come into contact with foodstuffs (number 76/893/EEC) lo has been adopted. This Directive is, in effect, an enabling Directive permitting work on Directives relating to specific materials and articles to be undertaken.Among the materials being considered are ceramics, glass, cellulose, and plastics generally as well as, specifically, those plastics containing vinyl chloride. The proposal for a Council Directive on the approximation of the laws of the Member States relating to materials and articles containing vinyl chloride monomer and intended to come into contact with foodstuffs has now been published .l 1 This proposal was submitted by the Commission to the Council on December 21st, 1976, and should shortly be discussed in Council. The proposal, in Annex I, lays down limits for vinyl chloride in materials and articles of 1 mg kg-l (except that in the instance of materials and articles prepared with vinyl chloride copolymers and not intended for use in the containing or packaging of liquids for human consumption, the limit shall be 6 mg kg-l) and in foodstuffs of 0.050 mg kg-l.Xnnex I1 of the proposal describes a method of analysis for the determination of vinyl chloride in materials, articles and foodstuffs. The procedure is based on the gas -liquid chromatographic analysis of the head-space obtained after dissolution or suspension of the sample in dimethylacetamide.The procedure, at present, permits an unrestricted choice of gas-chromatographic columns and recommends that confirmatory analysis of the vinyl chloride peak is obtained by using a Hall microelectro- lytic conductivity detector l2 or mass spectro- metry. The proposal for a Directive13 on ceramic articles intended to come into contact with food (Idimitation of Extractable Quantities of Lead and Cadmium) has been passed by the EEC Commission to the Council.The proposal stipulates limits on the extractable concentra- tions of lead and cadmium in various classes of ceramic articles to be used in contact with food. These classes include tableware and kitchenware, cooking ware and hollow ware articles.Limits are expressed in either milli- grams of extractable lead or cadmium per square decimetre for “flat” articles or milligrams of extractable lead or cadmium per litre for “hollow” articles. The method of test is described in Annex I1 of the proposal. The test uses a 4y0 V / V acetic acid solution as the simulating solvent; the extraction tests are carried out using specified conditions over a period of 24 h.The concentration of (extracted) lead or cadmium is determined in the acetic acid solution by atomic-absorption spectrophotometry, using the guidelines stated in Annex I11 of the proposal. Natural Mineral Waters There have been discussions within the Community on a Directive relating to natural mineral waters. A text resulting from these discussions should be available soon.Various methods of analysis have been proposed for the determination of constituents of natural mineral waters. Included are methods for the follow- ing : Lithium : by atomic-absorption spectrophoto- Sodium : by atomic-absorption spectrophoto- Potassium : by atomic-absorption spectrophoto- Magnesium : by atomic-absorption spectrophoto- Calcium : by atomic-absorption spectrophoto- Strontium : by atomic-absorption spectrophoto- Boron : by spectrophotometric determination Fluoride : by an ion-selective electrode procedure.Ammonia : by a potentiometric procedure. Sulphate : by gravimetry using barium chloride Silica : by spectrophotometry using ammonium .Alkalinity : by successive titrations with standard Total residue: by oven drying of a sample a t Chemical Oxygen Demand : by a permanganate - metry.metry. metry. metry. metry. metry. using curcumin reagent. reagent. molybdate reagent. sulphuric acid. 180/260 “C. sulphuric acid procedure. Preserves The amended proposal for a Council Direc- tive14 on the approximation of the laws of the Member States relating to fruit jams, jellies and marmalades and chestnut puree was pre-318 PROPOSED LEGISLATION Proc.Analyt. Div. Chem. SOC. sented by the Commission to the Council in August, 1975. It is expected that a final Directive will be adopted by the Community in the near future. The Community has not yet commenced any discussion on methods of analysis for preserves. Preserved Milk Products The Community Directive on the approxi- mation of the laws of the Member States con- cerning certain partly or wholly dehydrated preserved milk intended for human con- sumption15 will be translated into UK law as The Condensed Milk and Dried Milk Regulations 1977.The Commission has recently proposed methods to enforce some of the compositional criteria laid down in the Directive. These methods include the following: Dry matter content of condensed milk: by dilution of the sample with water, mixing with sand and drying a t 99 Loss of mass on drying of milk powders: by drying at 102 f 1 "C.Fat content of condensed milks and milk powders : by the Rose-Gottlieb procedure. Sucrose content of sweetened condensed milks : by polarimetry before and after inversion. Lactic acid and lactates in milk powders: by colorimetric determination of lactic acid after removal of fat, protein and lactose from sample and conversion of lactic acid/lactates to acetaldehyde.Phosphatase activity of milk powders : by the Sanders and Sager procedure. All of the methods are based on the International Standards prepared by the In- ternational Dairy Federation. The Standards are FIL/IDFs-15 : 1961, 26 : 1964, 13A : 1969 and 9A: 1969 (both Rose-Gottliebj, 35 : 1966, 69: 1972 and 63: 1971.1 "C. Solvents There have been preliminary discussions on a Directive relating to extraction solvents. Provisions relating to residues of such solvents under discussion in commodity Directives have been deleted and will reappear grouped in a solvents Directive. Sugars The Community Directive on the approxi- mation of the laws of the Member States con- cerning certain sugars intended for human consumption (number 73/437/EEC) l6 has been translated into UK law as The Specified Sugar Products Regulations 1976.l7 The Commission has proposed certain methods of analysis to enforce the standards laid down in the Directive. These methods are for the determination of the following: LOSS of mass on drying in semi-white sugar or extra-white sugar: by drying at 103 & 2 "C to constant mass.Reducing sugars (expressed as invert sugar) in semi-white sugar or extra-white sugar : by the Berlin Institute procedure. Reducing sugars (expressed as invert sugar or D-glucose) in the sugar solutions and syrups, glucose syrup and dried glucose syrup and dextrose (monohydrate and anhydrous) : by the Luff -Schoorl procedure. Dry matter content in sugar solutions and syrups : by refractometry of sample a t 20 "C.Dry matter content in glucose syrup, dried glucose syrup and dextrose (monohydrate and anhydrous): by drying a t 70 "C in a vacuum oven. Sulphated ash in glucose syrup, dried glucose syrup and dextrose (monohydrate and anhydr- ous): by ashing sample a t 525 & 25 "C in the presence of sulphuric acid.Polarisation in semi-white, white or extra-white sugar: by measuring the polarisation of a 267; m/ V solution of the sample. There is a major departure from common UK foodstuff analytical practice in this series of methods as the Luff-Schoorl procedure has been proposed rather than the Lane and Eynon. It would appear that the former method is standard in Continental official enforcement laboratories.It appears likely that the Commission will be proposing further methods to enforce the remaining standards in The Certain Sugars Directive, e.g., the standard for suiphur dioxide. Thiabendazole The Preservatives in Food Regulations 1975, l8 which implement various Community Directives, require that the concentration of thiabendazole in bananas and citrus fruit shall not exceed 3 or 10 mg kg-l, respectively.The Community has been considering various methods of analysis to enforce this standard, these being based on either ultraviolet solution spectrophotometry or gas - liquid chromato- graphy on an (ethyl acetate) extracted portion of the fruit.Water in Poultry A Council Regulation (EEC) No. 2967/76, laying down common standards for the water content of frozen and deep-frozen chickens, hens and cocks, has recently been published. l9 The Regulation lays down the amount of extraneous water poultry may take up in terms of the analytical procedure employed to assess the uptake. Procedures that are permitted are (a), slaughterhouse ("in-plant") weighing of test poultry carcasses, (b), a rapid drip loss detection method (thawing of whole bird in aIVolircznber, 1977 water-bath at a collection of the chemical analysis water to protein genised bird or the ELECTROANALYSIS IN temperature of 42 “C and water released) and (c), a procedure using either the ratio of the whole homo- water to dry fat-free protein iatio of the edible portions of the b&d.It woulcl seem likely that the former chemical method will be the chemical method of choice; the protein and water contents are obtained by using established IS0 procedures. The slaughterhouse checks will apply from July lst, 1977, and the remainder of the provisions of the Regulation from December lst, 1977. The slaughterhouse weighing checks and the drip loss test are to be used as guidelines to the amount of extraneous water present in poultry. It is on the results of the chemical procedure, which should determine the excess water uptake quantitatively, that any administrative action is to be taken. The above list of topics is not exhaustive; it should be noted, however, that discussion on BIOLOGICAL FLUIDS 319 some topics, which was active, has been left in abeyance. Such topics include bread, edible ices and emulsified sauces. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. References Off. J . Eur. Communities, L228, 16/8/73. SI 1976 No. 541. Off. J . Eur. Communities, L189, 12/7/74. Off. J . Eur. Communities, L292, 28/7/76. OH. J . Eur. Communities, L26, 31/1/77. Off. J . Eur. Communities, L311, 1/12/75. Off. J . Eur. Communities, L221, 12/8/74. SI 1976 No. 1832. FAO/ WHO Codex Alimentarius Commission, “Recommended European Regional Standard for Honey,” No. CAC/RS 12-1969. Off. J . Eur. Communities, L340, 9/12/76. Off. J . Eur. Communities, C16, 21/1/77. Hall, R. C., J . Chromat. Sci., 1974, 12, 152. Off. J . Eur. Communities, C46, 27/2/75. Off. J . Eur. Communities, C202, 4/9/75. Off. J . Eur. Communities, L24, 30/1/76. Off. J . Eur. Communities, L356, 27/12/73. SI 1976 No. 509. SI 1975 No. 487. Off. J . Eur. Communities, L339, 8/12/76.

ISSN:0306-1396    

DOI:10.1039/AD977140314c

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

IVolircznber, 1977 ELECTROANALYSIS I N BIOLOGICAL FLUIDS 319 Electroanalysis in Biological Fluids The following are summaries of ten of the papers presented at a Meeting of the Electro- analytical Group held on April 13th and 14th, 1977, in London. The organising Committee and some of the lecturers (L-R) ; MY. I . E. Davidson, Mr. B . 2. Chowdhry, Mr. J . P. Hart, Mr. R. R. Rowe, Dr. B. Birch, Dr.W. Franklin Smyth, MY. J . S . Burmicz, Pvofessor P. Zuman and Dr. B. Fleet.320 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc. Analyt. Div. Chew. Soc. Polarographic Methods for Use in Biological Systems: Plenary Lecture P. Zuman Department of Chemistry, Clarksogz College of Technology, Potsdam, N . Y . 13676, USA Polarographic and related methods have recently been applied to a vast number of problems involving the analysis of biological fluids.In pharmacology, such methods have been used to follow changes in concentration of the parent compound and metabolites in blood and urine after administration of the drug, and in toxicology drugs, poisons and metal ions have been determined in the same material and in body tissues. In clinical chemistry, it is difficult to have adopted electroanalytical methods, but some methods are being used for the determina- tion of ions (such as chloride), small molecules (both inorganic, such as oxygen, and organic, such as pyruvic acid) and enzymes in blood and urine.In veterinary medicine, the fate of administered drugs has been followed. In agricultural and food chemistry, concentrations of metal ions, vitamins, preservatives, pesticides, herbicides and insecticides have been determined both in formulations and as residues.Antibiotics and other additives have been determined in feeds. In botany, analyses of plants for mineral materials, vitamins, alkaloids, dyestuffs, etc., have been carried out. Further, applications have been described in the related fields of natural product chemistry, medicinal chemistry (in particular the relation- ships between electrochemical data and biological activity) and especially in pharmacy, where hundreds of papers dealing with electroanalytical methods are published each year.It is possible to ask why the application of polarography and related techniques in the analy- sis of biological fluids is so popular.Firstly, the mentioned parallelism between electrochemi- cal activity and biological reactivity results in the fact that many biologically active compounds give polarographic waves. This is perhaps not so surprising if we realise that an electrochemical reaction is only a special type of chemical reaction in which the chemical transformation is a result of an interaction with electrons rather than reagents.When biological activity is governed by the chemical reactivity of the drug at the reaction site rather than by transport phenomena or steric conditions, the parallelism between the electrochemical and biological activity is understandable. A further reason for the applicability of polarography to the analysis of biological fluids is that macromolecules of natural origin, present in the material obtained from plants, animals or humans, do not often prevent polarographic analysis.This is often due to the fact that even when proteins and other biological polymers are adsorbed at the surface of the dropping- mercury electrode, their adsorption does not result in the formation of a rigid film (as it is with some pyridine derivatives or dyestuffs) that completely covers the electrode and prevents electrolysis from taking place.Rather, adsorbates of proteins and some other biological polymers seem to have net-like properties. Adsorption of proteins and other macromolecules may affect the transport of the electroactive species to the electrode surface and the rate of the electrode process. As a result, polarographic waves in the presence of proteins and similar polymers can be smaller and more drawn out and/or deformed in comparison with waves in aqueous solutions, but are seldom completely suppressed.The recently expressed opinion that in order to be able to determine the organic compound in the presence of proteins or other macromolecules the organic compound must be strongly adsorbed has still to be proved.The strong adsorbability might favour the reduction process, but it has to be proved whether it is a condition that must be fulfilled. Polarography thus offers, at least in some instances, the possibility of the direct determination of components of a biological sample without separ- ation. This may result in a considerable shortening of the time needed for analysis in com- parison with conventional methods.Another aspect that makes polarographic methods attractive is their selectivity and the possibility of checking how carefully the polarographic analysis was carried out. Polaro- graphic curves often indicate the presence of interfering substances by the formation of new waves or by significant changes in the wave shape. Any carelessness in the handling of the electrode o the electrolysis cell is often indicated by disturbances on the polarographic curves.hTovembey, 1977 ELECTROANALYSIS IN BIOLOGICAL FLUIDS 32 1 Similar “distress” signals would often be recognised in methods based on electronic absorption if whole spectra were recorded.The common practice in spectrophotometric and colori- metric methods, which are the most important competitors in the analysis of biological fluids, is nevertheless to carry out the measurement only over ranges of few nanometres or few tens of nanometres.The numerical value obtained in such a way can easily be affected by absorbance by a species in the sample other than that being followed. Such a numerical value, worst of all, does not indicate possible interferences, or indicate whether the analysis has been carried out carefully or not.Polarographic methods need only an average technician, but an experienced supervisor. In the proper hands, polarographic methods in some instances can offer better selectivity than the more widely used spectrophotometric methods. The sensitivity of d.c. polarographic methods, with detection limits between and mol l-l, was considered to be sufficiently sensitive in the 1950s. Increased analytical requirements for trace determinations to provide evidence of purity (in environmental, food and drug analysis), together with development of more potent drugs administered in smaller amounts, and the tendency for smaller sample sizes to be available (particularly when blood samples are involved), resulted in a quest for more sensitive methods.Increases in the numbers of samples and the need for manpower economy led to a search for rapid methods that might be automated and pressure to develop new electroanalytical methods that would fulfil at least some of these requirements. In order to understand attempts to foster the development of new electroanalytical methods, it seems useful first to recall the advantages and limitations of d.c.polarography. The essential advantages are the well controlled and defined transport of electroactive species to the electrode surface and the virtually potentiostatic conditions for electrolysis (where pro- ducts are formed at the electrode surface at potentials constant within 10-20 mV and the surface of every drop is fresh and clean and virtually unaffected by previous electrolysis).The main limitations are that the compound studied must be electroactive, i.e., give a wave on polarographic curves. As was mentioned before, many biologically active species are also electroactive and those which are not can often be converted into such species (indirect methods, derivatisation).The compounds must be soluble in a solvent that can be made conductive, but this is of limited importance as most biologically active compounds are soluble in either water or oil and the choice of solvent is not difficult. More important is the absence of interfering substances, which can be either compounds with half-wave potentials similar to that of the compound being studied or compounds that give a large wave preceding the wave of the species to be determined.Sensitivity is limited by the capacity current (“noise”), the mean value of which becomes comparable with the faradaic current (“wanted signal”) when the concentration of the electroactive species is of the order of 1 0 - 5 ~ . The difficulty of measuring wave heights due to the capacity current means that d.c.polarographic curves can rarely be used for the analysis of solutions less concentrated than about 1 x M. A further limitation is the dissolution of mercury a t +0.4 to +0.3 V, which prevents systems oxidised at more positive potentials from being followed. Next we shall discuss how recently developed techniques have dealt with some of these limitations.In hydrodynamic voltammetry, stirring of the solution prevents the depletion of the electroactive species at the electrode surface. When used with mercury-pool electrodes, either as a stirred pool or a stirred solution, the measured current and the sensitivity are increased. Current - voltage graphs obtained with such electrodes have rarely been used for the analysis of biological fluids.Solid electrodes (graphite, gold or platinum) are used as rotating or vibrating wire or disc electrodes or electrodes for which the surface is periodically renewed by gas bubbles. Such electrodes are useful for studies of oxidations at positive potentials and have been applied successfully to oxidations of phenols, amines and heterocyclic compounds. For such systems they have to be the electrodes of choice, but it is necessary for one to be prepared to solve problems arising from adsorption of macromolecular compounds and from changes of the electrode surface in the course of electrolysis. When a voltage ramp is applied to an electrode with an unchanged surface, the method is described as linear sweep polarography when the electrode is a dropping-mercury electrode (and the sweep is usually fast ; cathode-ray polarography) or linear sweep voltammetry, when Other factors that favour polarography are the sensitivity and the speed of analysis.322 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc.AnaZyt. Div. Chem. SOC. the electrode is a hanging mercury drop or a solid electrode in an unstirredsolution (and the sweep can be either fast or slow).The latter methods have found practical application in anodic and cathodic stripping. These methods, which involve pre-accumulation followed by a voltage sweep, are very sensitive (10-s-10-9 M) but are restricted to the analysis of species that form amalgams or slightly soluble compounds with electrode material. The current - voltage graphs obtained by linear sweep polarography are unsymmetrical, the rising portion corresponding to an increasing rate of the electrode process and the decreas- ing portion to the depletion of the electroactive species at the electrode surface.The method offers some increase in sensitivity in comparison with d.c. polarography and can be usedwhen the most positive reduction peak or most negative oxidation peak is measured, or if individual peaks in the investigated solution differ by about 0.4 V or more.Otherwise measurements of the second and third peaks are difficult and show poor accuracy. Attempts to eliminate such problems by comparison of two electrolytic solutions are possible in principle, but in practice are tedious. This method is recommended only when some special reasons exist against using differential pulse or integral pulse polarography.Other methods use dropping-mercury electrodes, but differ from d.c. polarography in the applied voltage. In a.c. polarography an alternating voltage of small amplitude is superimposed on a ramp of d.c. voltage. This method has been applied to the analysis of biological systems, but seems to be useful only in some special cases.The current at the summit of the recorded peaks depends not only on the concentration and nature of the substance involved and the number of electrons transferred, but also on the rate of the electrode process. Only for rapid (“reversible”) processes is the peak summit current higher than the d.c. polarographic current and an increase in sensitivity results.A.c. polarographic curves are susceptible to adsorption - desorption phenomena and change in the presence of biological macromolecules. Special attention must be paid to calibration. Some of the complicating factors can be eliminated or minimised when the second harmonic is measured. The most successful methods for application to biological fluids are based on rectangular voltage polarisation.In the two most important practical methods, in integral and differential pulse polarography, a voltage pulse of short duration is applied and the current is measured in the last quarter of this pulse. In the integral method the voltage during the rest period is equal to the initial voltage and the amplitude of the pulse gradually increases. The current - voltage graphs show waves as in d.c.polarography, but the wave height is larger by a factor of 10-50. In differential pulse polarography, the voltage pulse has a constant amplitude and is super- imposed on a gradually increasing d.c. ramp. The current is measured shortly before the application of the pulse and during the last quarter of the pulse. The difference between these two currents is plotted as a function of the ramp voltage.The current - voltage graph has peaks that are larger by a factor of 10-50 than those in the d.c. curve. Small peaks following large peaks can be measured provided that their separation is greater than about 0.25 1’. Differential pulse polarography becomes a method of choice for the analysis of biological fluids when high sensitivity is required, but even here a few words of warning should be given.The theory of differential pulse polarography has been rigorously developed only for simple systems; unexpected phenomena can occur and the role of solution composition should be checked in each instance. For the quantitative evaluation of two adjacent waves, the addi- tivity of integral pulse polarograms sometimes offers a better measurement than that of over- lapping peaks obtained by differential pulse polarography.Electroanalytical methods have adapted well to the changing requirements of sensitivity, selectivity and speed in the analysis of biological fluids. If the system is too complex, as is frequently the case in urine analyses, they can be combined with chromatographic techniques.This, together with possibility of using electrical signals for automation, seems promising for the future. Problems arise when a small wave should be measured following a large wave.SovcmbeY, 1977 ELECTKOANALYSIS I N BIOLOGICAL FLUIDS 323 Cathodic-stripping Voltammetry of Some Sulphur-containing Organic Compounds in Biological Fluids I . E. Davidson Wyeth Laboratories, Hudercombe Lane South, Taplow, Maidenhead, Berkshire, SL6 OPH and W.Franklin Smyth Che~nistvy Department, Chelsea College, University of London, Jfanresa Road, London, S . W.3 The technique of anodic-stripping voltammetry is widely used for the determination of metals in a variety of medial and it is comparable in sensitivity and accuracy with techniques such as atomic-absorption spectrophotometry.2 Metals are plated on the electrode following reduc- tion of the corresponding ions and are re-oxidised in an anodic scan to give a peak-current presentation. Anions that react with mercury are determined by cathodic-stripping voltam- metry, e.g., C1-, Br-, I-, S2-, CrO,,-, W042-, and VO,,-., Of recent environmental interest has been the determination of Se2-.I n addition, some organic species have been determined by cathodic-stripping voltammetry, e.g., oxalate, succinate, dithizonate, diethyl- dithiophosphate, cysteine, rubeanic acid, thioanilide, dimercaptothiodiazole, thiourea and 2-mercapt obenzo t hiazole .4 The theory and application of stripping voltammetry to organic species and molecules has received scant attention in the literature.There are three basic mechanisms that can be used for the analysis of organic species and molecules by stripping voltammetry : (a) pre-concentration by adsorption of the species to be determined or by a product of an electrode reaction followed by stripping of the adsorbed species ; (b) anodic pre-concentration as sparingly soluble salts or complexes with mercury ions followed by cathodic stripping; and (c) complex or compound formation with excess of a selected metal ion followed by deter- mination of the remaining metal ion by anodic-stripping voltammetry.Method (a) has been used for the determination of methylene blue,5 and (c) has been applied to the determination of CN- and the benzodiazepine tranquilliser bromazepam,6 following complexation with Cu2+.Method (b) is, to date, most commonly used in analytical stripping voltammetry of organic species and molecules and is discussed, with reference to thioamides, in this paper. Fig. 1 shows the structures of several thioamides of pharmacological importance, both primary (I-V) containing the CSNH, moiety and secondary (VI) containing the CSNHCH, group. The d.c.polarography of thioamides that contain unsaturated N-heterocycles generally yield anodic waves owing to the formation of mercury compounds with the sulphur- containing group, and one or more cathodic waves, which are usually catalytic in origin, due CSN H*- HC I I I I I l l CH3 0 CH2CSN H2. HC I I V I CSN H CH,. HCI CSN H2. HC I V V I Fig. 1. Thioamides of pharmacological importance.324 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc.Analyt. Div. Chcm. SOC. to the N- heterocycle^.^ The thioamide group is the most labile in such compounds and its anodic wave is therefore of great analytical use. For example, the chemical degradation products of compound I have been shown to be the corresponding amide and nitrile with simultaneous production of hydogen sulphide and elemental sulphur.The amide and nitrile show catalytic behaviour similar to that of compound I itself, so this is of little analytical value. Differential pulse polarography of thioamides has been reporteds and the peak due to the anodic process can be used for measuring thioamides in pharmaceutical preparations, where con- centrations of the order of However, owing to the proximity of the peak potential to the potential corresponding to the oxidation of mercury, the sensitivity of the method is of the order of M, which is insufficient for studies of biological fluids, where drug concentrations of the order of lop7 M need to be reliably determined.A method has been developed for the direct determination of drugs such as compound I in both plasma and urine.A detailed paper on the subject is currently in the press,s so only a brief summary of the technique and results will be presented here. A Princeton Applied Research 174A polarograph was used throughout. Using a hanging mercury drop electrode and electrolysing 2 ml of plasma or urine containing up to mol 1-1 of drug, mixed with 2 ml of buffer (pH 4.8), at +0.05 V V e n u s a saturated calomel electrode with subsequent cathodic stripping, a voltammogram is produced that contains a sharp peak at -0.50 V owing to removal of plated material.The height of this peak is proportional to sample concentration in the range 5 x to M and the standard addition technique is used for calculating the drug concentration. Naturally occurring sulphur compounds such as glutathione, thiamine, methionine, cystine, cysteine and hydrogen sulphide and anionic species such as chloride ion have been shown not to interfere.M level on 2-ml samples. Britton - Robinson buffer (pH 4.78) was found to be the optimum supporting electrolyte. Not only were well defined peaks produced in this medium but also the pH was high enough for protein precipitation in plasma not to occur.M can easily be achieved. The sensitivity could be improved by the use of differential pulse stripping, but the scan rates would be slower (1-2 mV s-l), leading to longer analysis times. M are involved. The results are accurate, with a precision of 2.9% at the 5 x A sensitivity of about 2 x Practical points of importance in this type of analysis are as follows: (a) Sample volume and temperature must be accurately controlled.(b) A cyclic electrolysis -stripping procedure using a reverse scan back to the plating potential after the cathodic stripping scan must be used. Instant return to the electrolysis potential after the stripping process leads to non-reproducible results, probably owing to a chemical reaction at the unpolarised electrode.(c) Accurate micropipettes must be used for standard additions. (d) Adequate sample de-gassing must be performed prior to analysis. (e) No heavy metals or elemental mercury must be allowed to enter the bulk sample solution in order to avoid sample loss by chemical reaction. For pharmacokinetic and drug metabolism studies, it is necessary to consider the methods available for analysing the compounds in blood and urine in the presence of their possible metabolites.Taking compound I as an example, gas - liquid chromatography is excluded as its lability is such that on-column degradation to the corresponding nitrile (a possible metabol- ite) occurs under a wide variety of conditions tested. Also, the compound is non-fluorescent. Thus two major techniques are immediately excluded.Radiochemically, the compound can be determined after organic solvent extraction, thin-layer chromatographic plate elution and subsequent liquid scintillation counting.1° However, it was considered necessary to have a complementary “cold” method, ideally one which would eliminate the extraction step with its possibility of producing compound degradation to the nitrile.It was for these reasons that cathodic-stripping voltammetry was investigated as an assay method and was successfully developed for the direct determination of thioamides in plasma and urine.Il‘oi.qember, 1977 ELECTROANALYSIS IX BIOLOGICAL FLUIDS 325 The cathodic-stripping technique has been compared with standard radiochemical methods on plasma samples taken from two monkeys after oral administration of therapeutic doses of compound I (radioactively labelled).Samples of plasma taken at several time intervals up to 3 h after dosing were analysed by (a) cathodic-stripping voltammetry and (b) solvent extrac- tion - thin-layer chromatography - liquid scintillation counting of the eluate containing com- pound I. Results from (a) were higher than (b), although they were in reasonable agreement and followed the same trend.The total radioactivity in the plasma samples was also measured and gave the required result of a figure higher than (a) or (b). The higher results from (a) compared with (b) possibly indicate the presence of a sulphur-containing metabolite and studies are currently in progress to elucidate these findings. Cathodic-stripping voltammetry has so far been applied to direct sample measurements. However, in the presence of sulphur-containing metabolites, solvent extraction could be applied with subsequent thin-layer chromatographic separation and stripping analysis on the eluate.The advantages of a precise, accurate, inexpensive and non-radiochemical technique would still be maintained.1 . 2. 3. ‘4. 5. 6. 7. 8. 9. 10. References Copeland, T. R., and Skogerboe, R. I<., Analyt. Chem., 1976, 46, 1257h. Cernik, A. A., PYOC. Analyt. Div. Chem. SOC., 1976, 13, 227. Rrainina, Kh. Z., “Stripping Voltammetry in Chemical Analysis,” John Wiley, New York, 1975, Vydra, F., Julakove, E., and Stulik, I<., “Electrochemical Stripping Analysis,” John Wiley, N e w Perone, S.P., and Oyster, T. J., Analyt. Chem., 1964, 36, 235. Smyth, M. R., personal communication, 1977. Brezina, M., and Zuman, P., “Polarography in Medicine, Biochemistry and Pharmacy,” Interscience, Davidson, I. E., Proc. Analyt. Div. Chem. SOC., 1976, 13, 229. Davidson, I. E., and Smyth, W. F., Analyt. Chem., 1977, in the press. Pierce, D. Rl., Wyeth 1,aboratories. personal communication, 1976.p. 211. York, 1976. New York, 1958. Use and Limitations of Polarography in Determining Plasma Drug Levels in Pre-clinical and Clinical Pharmacology J. M. Clifford G. D. Seavle 6 Co. Ltd., Lane End Road, High Wyconzbe, Buckinghamshire and W. Franklin Smyth Chemistry Department, Chelsea College, University of London, Manresa Road, London, S . W . 3 The measurement of blood and plasma levels of investigational and therapeutic chemical compounds and drugs by polarography in pre-clinical and clinical pharmacology has generally been limited to those instances where the molecules are thermally labile during gas - liquid chromatography and where spectrophotometric methods are either time consuming or lack sensitivity.In general, the molecular structure has to contain a reducible group or be made to do so by a derivatisation procedure.:C = N-containing Molecules The concentrations of several benzodiazepine sedatives have been reported in blood follow- ing administration to test animals and humans, but only with chlordiazepoxide (I), diazepam (11) and bromazepam (111) have plasma levels been reported after a single oral therapeutic dose.326 ELECTROANALYSIS IN BIOLOGICAL FLIUIDS Proc.Analyt. Div. Chem. SOC. Chlordiazepoxide (I) has three reducible groups: an N-oxide and two azomethine groups with E, values [determined by differential pulse polarography (DPP)] separated by hundreds of millivolts. Its N-desmethyl metabolite also gives a three-peak pattern whereas the lactam metabolite is reduced in two steps.Hackman et aZ.l have used DPP to determine this drug and its metabolites in serum following therapeutic administration. The assay was rapid and the selectivity of the method improved by a thin-layer chromatographic step. Levels of parent drug and metabolites were measured following a single 30-mg i.v. and oral dose and following chronic oral administration (36 days).The method compared well with an altern- ative spectrofluorimetric assay and also avoided derivatisation, which is necessary in the latter method. Gas - liquid chromatography, although sensitive to 5 ng of chlordiazepoxide per millilitre of plasma, could not determine the two major metabolites. The minimum concentration of chlordiazepoxide measured was 60 ng ml-l in plasma, that of N-desmethyl metabolite was 100 ng ml-l and that of the lactam metabolite was 50 ng ml-l.Chlordiazepoxide H ( 1 ) Diazepam (I I ) Bromazepam (I I I ) Nitrazepam (IVJ FlurazepGm (V) Clonazepam (V I ) SC-13504 (VII) Nitroimidazole (VIII) O'C H3 N H2 Phenobarbital (IX) Trimethoprim ( X INozewaber, 1977 ELECTROANALYSIS IN BIOLOGICAL FLUIDS 327 Diazepam (11) gives a well defined wave using linear-sweep voltammetry and DPP due to a two-electron reduction of the azomethine group in the pH range 0-13.It is not possible to resolve the reduction peak due to diazepam from that of its major metabolite, N-desmethyl- diazepam, which is a common metabolite of several benzodiazepines and thus a thin-layer chromatographic separation step prior to polarography is needed in order to effect complete resolution of diazepam from its metabolite(s). Plasma levels of diazepam following 10-mg doses both orally and by the intravenous route have been measured by linear-sweep voltam- metry.The minimum plasma level that could be measured was 30 ng ml-l in plasma, but less than 20% of N-desmethyldiazepam was probably present and measured as diazepam.2 A method using selective extraction into two organic solvents, DPP and a cumbersome calcula- tion failed to detect plasma diazepam levels below 100 ng ml-l in a rhesus monkey that had been given 1 mg kg-l intravenously3 while electron-capture gas - liquid chromatography measured diazepam down to 60 ng ml-l after it had been given intraperitoneally to rhesus monkeys at 3 mg kg-l.Brooks and de Silva4 have shown that DPP could measure diazepam and its desmethyl metabolite following therapeutic administration. A sensitivity of 0.05-0.10 ,ug ml-l was claimed using a 2-ml plasma sample. The method differed from other reported assays in that diethyl ether was preferred to light petroleum or benzene as the solvent. Diethyl ether had a greater extraction efficiency and was used prior to thin-layer chromatographic separation of diazepam and its desmethyl metabolite. The first peak corresponds to the reduction of the nitro group and the second to the azomethine and hydroxylamine groups.The sensitivity of cathode-ray polarography to nitrazepam in plasma from beagle dogs given 0.75 mg kg-l intravenously was found to be 80 ng per millilitre of plasma.Such sensitivity as was found in dog studies could have been applied to expected human plasma levels only in the first 2 3 h after subjects had received 10 mg of nitrazepam orally and intravenously. A spectrofluorimetric method has detected 10-20 ng of nitrazepam per millilitre of plasma, which occurred about 68 h later. It has also been found that the peak due to the nitro group reduction in clonazepam was unsuitable for measuring clonazepam extracted from the plasma of a rhesus monkey who had received 1 mg kg-l intravenously; the second wave, presumably due to the simultaneous reduction of the hydroxylamine and azo- methine groups, however, measured clonazepam and its metabolites in this study and has also been used to determine the levels of the urinary metabolites in human urine separated by t hin-layer chromatography.Flurazepam (V) is reduced in a two-electron step corresponding to saturation of the azo- methine group, and different substituents occurring in the C-1 position as a result of metabolism do not effect its reduction potential. Flurazepam and its metabolites have been measured in the plasma of rhesus monkeys given 5 mg kg-l intravenously using DPP after selective extrac- tion into solvents at different P H .~ It would appear that DPP is much less sensitive than electron-capture gas - liquid chromatography for this determination, and is thus unsaitable for studying plasma levels of flurazepam after therapeutic dosage. In a recent study, Brooks and Hackman6 determined bromazepam (111) in blood with a sensitivity of 10-20 ng ml-l using a 0.5 ml micro-cell for DPP analysis.The assay employed selective extraction of bromazepam from alkalinised blood into benzene - methylene chloride (9 + 1). The residue of this extract was dissolved in phosphate buffer (pH 7.0) prior to the determination step. The DPP and electron-capture gas - liquid chromatographic assays measured blood levels in man following chronic single daily oral administration of 3 mg of bromazepam.The two assays were similarly sensitive and yielded comparable results, although the chromatographic method was regarded as more selective. Only DDP, however, was successfully applied to the determination of metabolites in urine. SC-13504 (VII) is a diphenylpiperazine investigational compound that has shown anti- convulsant properties in various animal models of epilepsy.A DPP method was developed to determine plasma levels of SC-13504 in pre-clinical pharmacological studies. SC-13504 gives rise to five polarographic waves over the pH range 1-11. One was found to be mainly diffusion controlled, two others showed catalytic behaviour and two further waves were found to be due to kinetic processes.The first was found to be the most suitable for analytical purposes and corresponded to reduction of the >C=N-N < entity.' DPP was used to determine the plasma levels of SC-13504 in photo-sensitive baboons Nitrazepam (IV) gives two well defined four-electron reduction peaks on DPP.328 ELECTROANALYSIS IN BIOLOGICAL F L u I i i s Proc. Analyt.Div. Chem. SOC. treated with SC-13504 at 4,6 and 8 mg kg-l intravenously and a correlation was found between plasma level and the reduction in the intensity of intermittent light induced seizures in these baboons. The plasma levels of SC-13504 determined by DPP were compared with those determined by a radiochemical method in one baboon which was given 8 mg kg-l SC-13504 containing 50 ,uCi of [14C]SC-13504.Approximately similar levels of SC-13504 were found using both methods during the 6-h duration of the experiments on the photo-sensitive baboons.8 Another baboon which was given 12.5 mg kg-l intravenously had three sequential blood samples withdrawn and the plasma obtained was divided, half of one sample being analysed for SC-13504 by gas - liquid chromatography in a laboratory in the USA, the other half for SC-13504 by DPP in a laboratory in the UK; the results from these tv70 methods were in good agreement. DDP has also been used to measure SC-13504, diazepam and clonazepam (VI) simultaneous- ly in the plasma of rhesus monkeys treated with either one, both or all three of these agents intravenously.In this instance the plasma was extracted with light petroleum, which extrac- ted SC-13504, diazepam and N-desmethyldiazepam, or diethyl ether, which extracted clon- azepam, the azomethine-containing metabolites of diazepam and clonazepam and a possible azomet hine-cont aining metabolite of SC-13504 .g -N02-containing Molecules Nitroimidazoles (VIII) such as metronidazole and ornidazole have been determined in blood samples using solvent extraction and thin-layer chromatography prior to polarographic determination.lOJ1 A relatively rapid assay results if the thin-layer chromatographic step is omitted but the method is then no longer specific for the parent compound as nitro-contain- ing metabolites interfere.Brooks et aZ.12 have developed a DPP method for the determination of phenobarbital (IX) in blood to measure therapeutic and overdose levels.Phenobarbital is selectively extracted from blood buffered to pH 7.0 into an ethyl acetate - butanol (8 + 2) mixture, subjected to clean-up procedures, nitrated and subjected to polarography in pH 7.0 phosphate buffer. Quantitation was based on the measurement of the reduction peak for the nitro derivative.A sensitivity of 1 pg ml-l in blood was reported, with a recovery of 72.3 & 6.5%. A modified polarographic method for the determination of both phenobarbital and diphenylhydantoin, an anticonvulsant drug, employing thin-layer chromatography of an initial plasma extract, was also reported. 0 t her Molecules Trimet hoprim [2,4-diamino-5- (3,4,5-trimet hoxybenzyl) pyrimidine] (X) , one of the two active ingredients in Bactrim (the other ingredient is sulphamethoxazole), is reported to have in vivo antibacterial activity.Brooks et a1.l3 have reported differential pulse polarographic analyses of the trimethoprim in blood. To determine trimethoprim the drug was extracted into chloroform from blood buffered to pH 11.5. The drug was then back-extracted into 0.1 N sulphuric acid and analysed by differential pulse polarography.The polarographic blood assay was found to be twice as fast as the fluorimetric assay, primarily due to the elimination of the time-consuming conversion of trimethoprim to the trimethoxybenzoic acid. The differential pulse polarographic peak at -1.070 V vwsus S.C.E. corresponds to a reduction process in the pyrimidine ring. Conclusion In conclusion, it would appear that sensitive polarographic methods such as DPP are useful analytical tools when other analytical methods are inappropriate, e.g., thermal instability under gas - liquid chromatographic conditions, poor sensitivity with spectrophotometric methods and when the dose is high enough to give plasma levels >50-80 ng ml-l or when there is value in measurement of the total drug present such as in toxicological studies to monitor gut absorption. Generally the poor sensitivity of DPP has precluded its use in clinical pharmacology, although the method has advantages in clinical toxicology. Human therapeu- tic doses at the level of at least 200 mg are required in order to ensure that detectable plasma levels are present for a time period sufficiently long to study compound disposition.Novc 1. 3. 3. 4. 5. 6. 7 . 8. 9. 10. 1 1 . 12. 13. wzber, 1977 ELECTROANALYSIS I N BIOLOGICAL FLUIDS References 329 Hackman, hl. R., Brooks, M. A., de Silva, J. A. F., and Ma, T. S., Analyt. Chem., 1974, 46, 1075. Berry, D. J., Clin. Chim. Acta, 1971, 32, 235. Smyth, M. R., unpublished results, 1976. Brooks, M. A., and de Silva, J. A. F., Talanta, 1975, 22, 844. Clifford, J. M., Smyth, M. R., and Franklin Smyth, W., A . Analyt. Chem., 1974, 272, 19s. Brooks, M. A., and Hackman, M. R., AHalyt. Chem., 1975, 47, 2059. Smyth, M. K., Franklin Smyth, W., Palmer, R. F., and Clifford, J. RI., Analyst, 1976, 101, 469. Medrum, B. S., Smyth, M. R., Franklin Smyth, W., and Clifford, J. M., Psychopharmacology, 1976, Clifford, J. M., Methods Dev. Biochem., 1976, 5, 203. Brooks, AT. A., D’Arconte, L., and de Silva, J. A. F., J . Pharm. Sci., 1976, 65, 112. Iiane, P. O., J . Polarogr. Soc., 1961, 8, 73. Brooks, 31. A., de Silva, J . A. l?., and Hackman, 11. R., Analytica Chim. Acta, 1973, 64, 165. Brooks, M. A., de Silva, J . A. F., and D’Arconte, L., J . Pharm. Scz., 1973, 62, 1395. 51, 59.

ISSN:0306-1396    

DOI:10.1039/AD9771400319

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

N ovem bey, 19 77 ELECTROANALYSIS I N BIOLOGICAL FLUIDS 329 Non-invasive Methods of Blood Gas Analysis Irving Fatt School of Optometry, University of California, Berkeley, Calif. USA The ideal procedure for blood gas analysis (pH is included although not strictly a blood gas) would need no blood sample or a very small sample, be rapid and accurate, be continuous and be simple and inexpensive. Blood gas analysis methods used today in the operating room or intensive care unit meet few of these criteria for the ideal procedure.The switch from the older Van Slyke gasometric analysis to modern electrochemical methods has reduced the size of the blood sample needed, but withdrawal of even a small sample can be very traumatic if the sampling must be done often or if the patient is an infant.Sampling of arterial blood is particularly traumatic. Modern electrochemical methods (the Clark polarographic oxygen sensor, the Severinghaus carbon dioxide sensor and the glass electrode for pH) are reasonably accurate and rapid. Unfortunately, many hospitals do not have blood gas analysis equipment at each operating table or at each bedside. Blood samples are often taken to a central laboratory and the report is sent back by messenger or telephone. The time delay in this procedure can be dangerous to the patient.The procedure is inherently discontinuous; the attending physician waits upon the first result before requesting a second sample. Modern electrochemical blood gas analysis apparatus is not expensive when compared with other major items of hospital or clinical equipment but the space needed, the need for auxiliary standardising gases and solutions and the training of operating personnel raise the cost of each analysis.,Ilthough improvements to presently used blood gas analysis methods are continually being made, there is the widespread belief that the next step should be towards methods that require no blood sample and are continuous.Such methods can be divided into those which are invasive, that is, the sensor is placed in the blood stream via a catheter or other hypo- dermic device, and those which make the blood analysis through the skin. The latter are called non-invasive methods and are the subject of this paper. Those not familiar with blood gas analysis methods are sometimes led to believe that the use of the so-called oximeter is a non-invasive method for measuring blood oxygen tension.In a commercially available oximeter (Hewlett-Packard), the tip of a light pipe is clamped to the earlobe and the reflectance at two wavelengths from the blood in the earlobe is analysed in terms of the oxyhaemoglobin level in the blood. This quantity does not have a linear or otherwise simple relationship to blood oxygen tension, although in some instances it can be used as a measure of blood oxygen tension.The true non-invasive blood gas analysis methods make an electrochemical measurement at the surface of the skin or other exposed body tissue. Oxygen movement through the skin was detected over 125 years ago. Baumberger and Goodfriend found, 25 years ago, that330 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc.Analyt. Div. Chem. SOC. oxygen tension in a warm (45 "C) saline solution surrounding a finger would equilibrate with the arterial blood in 15-60 min. The method was slow and uncertain because the rate of equilibration depended upon the volume of solution in which the finger was immersed and because there were no reliable methods for the determination of oxygen tension in small amounts of solution.Two basic concepts or developments were needed to make possible a useful measurement of blood oxygen tension at the skin surface. Clark provided this when, in 1956, he described a solid cathode polarographic sensor that was sensi- tive to cbxygen tension but not poisoned by materials normally associated with body tissues and fluids.Secondly, a method for making the oxygen tension at the surface of the skin representative of the oxygen tension in the arterial blood underlying the skin was needed. The procedure for obtaining this result is called vascularisation of the skin and involves either application of an irritant chemical or heat to the surface of the skin.The commercial blood oxygen tension skin sensor, supplied by Hoffmann-LaRoche, uses a carefully controlled heater to bring the surface of the skin to 44 "C. The polarographic current from the skin sensor is then linearly related to arterial blood tension. For the Hoffmann-LaRoche sensor the relationship is The development of the practical skin oxygen sensor is a more recent event.Firstly, a practical oxygen sensor was needed. Skin oxygen tension (mmHg) = 8 + 0.95 [arterial blood oxygen tension (mmHg)] For 490 measurements the correlation coefficient is 0.93. The Hoff mann-LaRoche sensor, when used on newborn infants, has a response time of about 1 min, that is, there is a change in sensor current 1 min after there is a change in oxygen tension level of the inspired gas or after a change in breathing level.For adults, whose skin is thicker, the response time of the sensor is increased to several minutes. The Hoffmann-LaRoche skin oxygen tension sensor seems well suited for monitoring newborn infants in respiratory distress. It seems less applicable to adults, particularly during surgery when an instantaneous response to a decrease in blood oxygen tension is essential.Application of the skin sensor to surgical patients in the USA may be made difficult by the US Food and Drug Administration (FDA) requirement that the patient be electrically isolated from the mains. The polarographic current could be telemetered by an F M radio link or via a light pipe but it will be more difficult to isolate the heater from the mains current supply.Beran, Huxtable and Sperling have used for this purpose an antimony oxide pH sensor in the Severing- haus configuration. The pH-sensitive surface is covered with a thin layer of hydrogen carbonate buffer, which is held against the sensor by a thin, carbon dioxide permeable Teflon membrane. Changes in carbon dioxide tension at the outer surface of the membrane are reflected in the pH change of the buffer and this change is read out by the sensor as a change in carbon dioxide tension.As with the blood oxygen skin sensor, the skin must be vascularised so that the surface carbon dioxide tension reflects that of the arterial blood. For 29 simul- taneous measurements on rabbits of skin-surface carbon dioxide tension (at 42 "C) and arterial blood carbon dioxide tension the correlation was given by Skin carbon dioxide tension (mmHg) = -4.39 + arterial carbon dioxide tension (mmHg) The carbon dioxide skin on the forearm of a adult human was found to respond to breathing of a gas mixture of 4% carbon dioxide, 21% oxygen and 75% nitrogen but no quantitative data have been collected.No relationship between skin and blood pH has been demonstrated. Another form of skin blood gas sensor is the so-called eyelid sensor.A discussion of this sensor must be prefaced with a description of the eyelid's unique function and properties. The cornea is the thin, transparent tissue at the centre of the exposed portion of the eyeball through which light enters the eye. The cornea has no blood vesels because such vessels would scatter light and reduce the cornea's transparency.In the absence of blood vessels the cornea must acquire its oxygen by diffusion across its front and back surfaces. Thus thc cornea of the open eye is the only tissue of the body that obtains oxygen directly from the air. When the eye is covered by the lids during sleep the cornea must draw oxygen from the capillary bed on the underside of the eyelid.The tissue lining the underside of the eyelid is called the palpebral conjunctiva. It has a dense bed of capillaries very close to the surface, as can be verified in front of a mirror by gently pulling down the lower eyelid. The eyelid The carbon dioxide tension of the blood can also be measured through the skin.,Voiwmbey, 1977 ELECTROANALYSIS IN BIOLOGICAL FLUIDS 33 1 sensor detects the oxygen or carbon dioxide tension or the pH at the surface of the palpebral conjunctiva.As this tissue has as its function the transfer of oxygen from its capillary bed to the cornea, there can be no intervening layer of cells that would themselves take up the available oxygen. It is at this point that the eyelid sensor differs from the skin sensor.When using the skin sensor the capillaries under the skin must be enlarged by heating so that additional blood will flow. This additional flow provides sufficient oxygen so that some will reach the surface even though the highly active skin cells consume a large amount. No vascularisation is needed for the palpebral conjunctiva because nature has designed this tissue so that oxygen will reach the surface.Measuring carbon dioxide tension and pH at the surface of the palpebral conjunctiva is straightforward. Both carbon dioxide and hydrogen ions move easily from the blood vessels in the palpebral conjunctiva to the surface of that tissue where their concentration can be measured and related to their concentration in the blood. The eyelid oxygen sensor is simply a Clark polarographic oxygen sensor held against the palpebral conjunctiva by a scleral contact lens.Prototype models of this sensor have been tested on human surgical patients. In this application they have been found invaluable in giving the surgeon and anaesthetist an almost instantaneous warning of a decrease in the patient’s blood oxygen tension level.The arterial vessels of the palpebral conjunctiva come from the ophthalmic artery in the head, which in turn is closely related to the cerebral blood supply. The eyelid sensor therefore gives an early indication of the loss of oxygen supply to the brain, the one organ in the body that can stand only a very few minutes of oxygen deprivation. The relationship between arterial blood oxygen tension and palpebral conjunctiva oxygen tension, as measured on the rabbit, has been found to be Arterial oxygen tension (mmHg) = 2.3 [palpebral oxygen tension (mmHg)] -75 On seven human subjects the relationship was found to be Arterial oxygen tension (mmHg) = 1.53[palpebral oxygen tension (mmHg)] +0.36 The difference between the measured palpebral oxygen tension and the arterial oxygen tension increases as the oxygen tension increases.This result must stem from a process that regulates blood flow to the conjunctiva as the level of arterial oxygen tension increases. The response time of the eyelid oxygen sensor is about 60 s to 9574 of the new reading. This response time, in the rabbit, has been found to be a function of blood volume in the animal and suggests a means of determining the level of a patient’s blood loss in an accident or in surgery.The carbon dioxide tension at the surface of the palpebral conjunctiva can be easily measured by gently pressing a Severinghaus sensor, based on a flat-tipped pH electrode, against this tissue. Experiments on dogs have yielded the relationship Arterial carbon dioxide tension (mmHg) = l.O3[palpebral carbon dioxide tension (mmHg)] + 2.44 Arterial pH can be measured by gently pressing a bare flat-tipped glass pH electrode against In the dog the relation was found to be the palpebral conjunctiva.Arterial pH = palpebral pH + 0.07 When used under medical supervision on adults, the eyelid sensor causes no damage to the eye tissues and is well tolerated by the patient.The prototypes have used wires to connect the sensor to the auxiliary electronic and recording equipment. Design work has begun on the use of commercially available miniaturised FM transmitters to link the patient to the read- out equipment. This step is necessary to meet the FDA requirements for electrical devices attached to surgical patients but will also make possible monitoring of blood gases on ambula- tory patients.332 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc.Analyt. Div. Chenz. Soc. The Virtues and Limitations of Ion-selective Electrodes for Measurements in Biological Systems : Plenary Lecture R. P. Buck Wm. R. Kenan Laboratories of Chemistry, University of ATorth Carolina, Chapel Hill, N.C. 27514, US,4 Ion-selective electrodes (ISEs), as a sub-class of the more general category of potentio- metric (potential difference generating) sensors, are always best used under ideal experimental conditions.The ideal sample for most electrochemical sensors is a pure, homogeneous, single electrolyte solution at uniform temperature and pressure. Whenever real problems and real conditions are imposed, there is some required compromise in the experimental situation, and some inevitable loss in the quality of the electrode response: loss of slope, sensitivity, selec- tivity or reproducibility.ISEs in biological systems cannot be expected to behave at the same high level of quantitative response performance, established by calibration using ideal, interference-free electrolyte standards.Physiologists, pathologists and clinical chemists who wish to use ISEs for batch measure- ments and for continuous measurements in monitors are faced with a thorough chemical characterisation of their system followed by specific chemical or physical treatment of samples. Chemical treatment of electrode - solution interfaces or specific design and fabrication of electrodes may be needed in order to avoid interferences and to bring responses to the opti- mum level, determined by thermodynamic limitations.Take, for example, the apparently simple chemical system of dialysed blood samples for chloride measurement. If an ordinary chloride-sensitive silver chloride electrode is used, chloride responses at low levels deviate from pure calibration-based responses because of significant amount of bromide and uric acid anions, both of which form insoluble silver salts.In whole blood, even after precautions to correct for interferences, ISE results fail to agree with titrimetric results because of the high suspended protein content. The TSE gives apparently high results because true electrolyte salt concentration (activity) is measured; the titrimetric result gives the total number of moles of chloride divided by the total sample volume, which includes the electrolyte-excluding volume of the protein. Virtues of conventional ISEs in biological applications follow from (1) the frequent need for only qualitative potential measurements, and (2) the possibility that nearly ideal quanti- tative results can often be achieved after thorough characterisation and treatment of real systems.The most dramatic example of the qualitative use of ISEs is in vivo measurements within single ce1ls.l These electrodes, which may contain only potassium chloride and a silver chloride electrode, respond to the cell membrane established potential difference between inside and outside of cells. The electrodes can be large (a few microns diameter open tip) and respond only to liquid junction potentials between the test solution and the filling solution at the tip, or they can be of sufficiently narrow bore that the intersecting, inter- facial space charge provides a degree of selectivity (albeit sub-Kernstian) in response to ions of only one sign. Even moderate selectivity, established by filling tips with ion exchangers, has provided new techniques, not only for following action potentials, but also for following individual ion-activity changes.Many innovations in t?ie manufacture of micro-tip electrodes (open pipette and closed-tip types) have been introduced by physiologists and pathologists. When systems are chemically well characterized, the ultimate sensitivity and selectivity of a given ISE can usually be achieved in the biological experiment.Blood parameter analysers that are now commercially available and that monitor Na+, K+, Ca2-b, C1-, HC0,- (via COS2-), ions and carbon dioxide and oxygen are largely based on applications of ISEs. The precise tailoring of electrodes to samples, the use of surface coatings such as Cellophane films between the sample and the electrode, and the use of specially stable and water-insoluble solvents in liquid membranes are matters of developmental research that are not described in the open literature. In addition to using ISEs for conventional ionic species in biological environments, much progress has occurred in adapting conventional electrodes to the measurement of species mainly of interest in biology and medicine.2 These examples have involved use of silver sulphide electrodes to measure species that form complexes with silver ions.Among these are molecules and ions that contain amino, mercaptan or sulphide moieties, including proteins and some antibody - antigen species.Nowmzber, 1977 ELECTROAl\r’ALI-SIS IK BIOLOGICAL FLUIDS 333 The monitoring of biological species, as a rule, requires greater selectivity than is found among ordinary ISEs.There are too many biological molecules and ions of similar chemical character that will give similar responses at comparable concentrations. If one needs only the total activity of some general category of species, ISEs may be directly applicable. To obtain greater selectivity, modified electrodes with interposed chemical reactions have been developed.The opportunity to increase specificity is provided by using highly discriminating, interposed chemical reactions. This type of electrode, associated originally with Severinghaus, and more recently with Guilbault, has been applied to monitoring the activities of many compounds that can be converted selectively into a measurable ionic form using enzymes.What one gains in selectivity, however, is lost in response time. This field is also being actively pursued by Rechnitz and colleagues, whose recent achievement has been the use of living cells (to bring about selective reactions) in the interposed section of an electrode. Gas sensing electrodes also base their selectivity on the use of interposed chemical reactions.Those electrodes responsive to carbon dioxide and ammonia are interesting for biological applications. Limitations of ISEs for measurements in biological systems are a result of limitations on the potential-determining processes. All of the potentiometric ISE sensors, both the mem- brane type and conventional electrodes of the zeroth, first and second types, possess the property of rapid, reversible ion or electron exchange (high exchange current density) between a species in a bathing solution and the same species in the membrane or the metal phase.A requirement for rapid, reversible charged-species exchange arises if one insists upon electrodes with optimum thermodynamic reponses-Nernstian dependences of measured potential on activity. This requirement has two consequences : on the one hand, film-forming impurities destroy the electrode response, and on the other hand, electrodes measure reliably only material near the surface and this material may not reflect the bulk properties.For example, when in a biological sample some charged or uncharged film-forming species is present, then surface coating can create an insulating region, with a near-linear potential profile, and produce a profound decrease in the rate of ion or electron exchange.The characteristic high exchange current density no longer prevails. The electrode potential is not firmly established, but tends to drift or to become sensitive to all species that undergo ion exchange at comparable or faster rates.This means that coated electrodes become drifty, even though, in principle, slow processes should ultimately reach equilibrium, in the absence of interferences. On the other hand, ISEs with ideal, high exchange current densities tend to “tell the truth” even when the local surface information is not desired. For example, ISEs are slow to respond to ambient activity changes because the changing bulk activity requires time to carry new activity information, by diffusion, to an electrode surface. Of course stirring minimises the static Sernst diffusion layer, but never eliminates it, Another example is the response of a silver iodide electrode to ambient cyanide activities.The response is stirring-sensitive because cyanide diffuses constantly to the silver iodide surface, displaces iodide and generates a silver cyanide complex.The electrode response, which is a measure of surface silver-ion activity, is a steady-state-determined process and reaches a steady value only when diffusion flux balance occurs. As a final, related example, measurements using ISEs in systems con- taining high concentrations of polyelectrolytes, with the reference electrode separated by a salt bridge and free from polyelectrolyte, give apparently erroneous readings because of the local variations of solution potential from space charge regions near polyelectrolyte surfaces to electroneutral regions away from the surfaces.This effect, a version of the medium effect, has been found to plague pH measurements in electrolyte samples that contain high concentra- tions of polyelectrolytes, ion-exchange resin beads and charged suspensions.The main potential off set occurs a t the junctions between the suspension-filled electrolyte and the reference electrolyte. The precaution suggested is that the reference electrode be placed in the same vessel in j unctionless configuration, if possible. Interfer- ences that operate by metathesis of the underlying electrode substrate ( e g . , Br- reacting at a silver chloride electrode) continue to create surface layers of foreign material.In principle, an electrode surface should regenerate itself spontaneously by washing a contaminated electrode in the soluble salt of the ion replaced (e.g., potassium chloride). However, in practice, it may not be convenient to wait for these processes to occur, or the rates may be slow. Then the electrode must be polished with fine rouge or etched in a soluble, complex-forming medium.General interference by foreign ions in biological systems is a cumulative effect.334 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc. AnaZyt. Div. Chem. SOC. Protection of surfaces by cellulose films prolongs responses in the presence of large, hydrophilic and film-forming molecules.However, the protecting surface films must admit ions to be measured, and so must allow ionic interferences by other small size ions that can pass to the electrode surface. This problem has been partially overcome by judicious use of silanising or applying fluorocarbon oils to the electrode surfaces. Overcoating surfaces with polymer films with a variety of compositions (and with usually unknown transport characteristics for ions) is difficult and is best left to manufacturers who have time to prepare and survey many film compositions for selective rejection of ionic and molecular interferences while retaining reversible transport of species to be determined.In the long run, this option is undesirable. Sample pre-treatment requires chemical steps such as the introduction of reagents (and impurities), and physical steps such as dialysis.Certainly one of the aims of clinical analysis is simplification and speed of execution of analytical procedures because of the many thousands of analyses that must be done daily. Consequently, extensive sample pre-treatment means that the progress of the analysis is impeded, not speeded up.In addition, if bed-side analyses in hospitals are to become a reality, as well as continuous monitoring of patients’ parameters, one must look beyond discrete or continuous batch-type analysis involving sample treatment. Electrode construction must therefore receive the greatest developmental attention. Future research effort must involve new formats for old electrodes and simplified, interference-minimising formats for new electrodes as a goal for the recognition of ISEs as practical devices in biology and medicine.There are some remarkable advances on the horizon. Among these are the chemically sensitive semiconductor devices, ISFETs and CHEMFETS,~ which show some promise for in vivo applications.Although these devices depend upon the generation of interfacial potentials via ion exchange or adsorp- tion (and are therefore subject to interferences), the physical layout is capable of miniaturis- ation using modern solid-state fabrication techniques. Semiconducting devices can be built as potential-to-potential transducing devices, or in potential-to-current modes, and they have been implanted into animals with self-powered transmitters for remote monitoring of pH.The need for providing pre-treated samples for analysis has been mentioned. References 1. 2. 3. Kessler, M., Clark, L. C., Jr., Lubbers, D. W., Silver, I. A., and Simon, W., Editors, “Ion and Enzyme Buck, R. P., Analyyt. Chem., 1976, 48, 23R. Janata, J., and Rfoss, S. D., IEEE Trans.Bionzed. Engng, 1976 BME-23, 241. Electrodes in Biology and Medicine,” Verlag, Urban and Schwarzenberg, Munich, 1976. Measurement of Plasma Potassium Using lon-selective Electrodes Tom Treasure and D. M. Band St. Thomas’ Hospital, LoNdon, S.E. 1 Why Measure Potassium? Potassium is one of the two principal cations in biological systems. The human body contains about 3 mol but the majority is intracellular, with less than 1% circulating in blood plasma.Sodium is present in about the same amount but is the principal extracellular cation. It is the unequal distribution of sodium and potassium across cell membranes that is responsible for the membrane potential. The proper function of irritable tissues such as brain and nerve cells, the heart and other muscular tissues depends on the maintenance of this membrane potential.Potassium depletion can occur in the long term, that is over days and weeks, owing to vomiting, diarrhoea, fluid loss from surgical fistulae and abnormal urinary losses. I n the short term, disturbance of the normal equilibrium between intra- and extra-cellular potassium can occur and result in abnormal muscular activity and potentially dangerous abnormalities of heart rhythm due to redistribution with or without a net potassium deficit.In the care of patients, the measurement of plasma potassium is frequently undertaken. Although only such a small fraction is in the plasma, this is the commonest measurement made, while losses of potassium in urine and gastro-intestinal secretions are also measured toXonembeY, 1977 ELECTROANALTSIS I N BIOI.OGICAL FLUIDS 335 calculate day-by-day electrolyte balance.In addition, various methods have been devised to estimate total body, intracellular or tissue potassium. Although these measurements have their protagonists and may find a place in the investigation of chronic potassium depletion, they are not in routine clinical use and will not be considered further.The normal concentration range of potassium in the plasma i~3.2-5.4mrnoll-~ (95% extreme range) and survival is unusual below 2 or above 8 mmol l-l. This range is narrow in absolute terms but in proportional terms is greater than the normal range of sodium or calcium. I t represents a change of 11.35 mV for a theoretical electrode. In realistic terms, a precision of 0.2 mmol 1-1 would be adequate for clinical purposes, so a cell with a reproducibility of about 1 mV is required.Brief History of Ion-selective Electrodes (ISEs) for Potassium Measurement Eisenman et aZ.l experimented with a range of glass compositions to extend the range of sensitive glasses to include other cations apart from Hf. Sodium glasses were adequately selective but potassium glasses of various compositions2~3 did not achieve a selectivity coeffic- ient (KKNa) better than 0.1.As sodium is in a 35-fold excess over potassium in the plasma, this selectivity of only 10 : 1 for potassium over sodium is inadequate for direct measurement. The liquid ion exchangers based on the lipophilic anion tetraphenylb~rate~ were a considerable improvement (KKNa= 1.2 x but the high selectivity of cation-complexing macrocyclic antibiotics and valinomycin in particular led to the first highly selective potassium electrode^.^,^ By combining valinomycin and potassium tetraphenylborate in a liquid membrane, Band and Kratochvil' achieved a membrane of lower resistance with better anion rejection while retaining the high selectivity of the neutral carrier.The obvious advantages of incorporating ligands in a polymer matrix were described by Moody et aZ.8 and polymer-membrane electrodes are now widely used. Measurement of Blood Potassium with Ion-selective Electrodes PVC membranes of the composition given in Table I have been used in our own s t u d i e ~ . ~ Originally we mounted them on small pieces of PVC tube but more recently have cast the membranes directly on to porous ceramic.1° The ion-selective half-cell was completed by filling it with 4 mM potassium chloride solution and inserting a silver - silver chloride internal reference electrode.These electrodes are mounted in a thermostated Perspex cell so that the membrane forms one side of a small cuvette (Fig. 1).This system has been used to measure potassium in clinical samples of whole blood and the results have been compared with those obtained by flame photometry.ll TABLE I COMPOSITION OF ION-SELECTIVE MEMBRAEE Material Amount/g Valinomycin . . . . . . . . . . . . . . 0.001 5 Ris-2-ethylhexyl adipate . . . . . . . . 0.15 Nitrobenzene . . . . . . . . . . . . . . 0.05 Potassium tetraphenylborate .. . . . . 0.000 025 High relative molecular mass P\'C . . . . 0 075 The electrode was calibrated with potassium chloride standards made up in a constant background of sodium chloride. This has the effect of bringing the activity coefficient to the same value in all the standards and the blood specimens. The results of the first 50 samples are shown in Fig. 2. The first simple conclusion that can be drawn is that the agreement is sufficiently close and ISE measurements are sufficiently reproducible to permit the use of ion- selective measurements instead of the customary flame photometric measurements.On closer inspection, however, it is found that there is a systematic discrepancy with the electrode reading on average 0.2 mM higher, probably because the volume sampled by the flame photo- meter includes 5-7% of proteins by volume that are space occupying and ignored by the elec- trode, which measures potassium activity in plasma water.In most instances this is of little importance but in the occasional patient where the discrepancy is significant (for instance, in336 ELECTROANALY~IS IN BIOLOGICAL FLUIDS Proc. Annlyt.Div. Chem. soc. diabetics where there may be excessive amounts of lipid) it is the electrode which, in fact, gives the more useful information. PVC "washer" \ Screened cable "/ Water I on-selective Jacket half cell I 5cm I .- 2 5 - + W - % c 4 - 2 E 2 3 - .- I a - 2 3 4 5 6 Plasma I<'/mM (flame photometer) Fig. 1. The potassium ion-selective half-cell is mounted so that its membrane forms one side of the cuvette.The PVC "washer" is compressed between the cuvette and electrode carrier. The capillary forms a liquid junction with the saturated KC1 of a Radiometer K497 calomel reference electrode, thus completing the ion-selective cell. The e.m.f. can be displayed on any suitable high- impedence electrometer. For convenience, the CO, channel of a blood-gas analyser (e.g., Radio- meter PHM27) can be used and potassium con- centration read directly from the logarithmic scale by dividing pC0, (10-100 mmHg) by 10 to give potassium in the range 1.0-10 mmoll-l.Fig. 2 . Duplicate readings made with the electrode are compared with duplicate measurements of potassium using the flame photometer. The plasma was obtained from a series of 50 consecutive clinical blood samples.The ion-selective electrode gives the same reading with plasma or whole blood. (Data from Band, Kratochvil and Poole Wilson.) The close agreement between the two methods does, however, illustrate that there is no significant protein binding for potassium. Further, as the electrode was calibrated with potassium standards in 140 mni saline, these results confirm that the activity coefficient for potassium in blood is essentially the same as for that in saline.The Place of ISE Measurements The clinical chemistry laboratory of a general hospital measures plasma potassium in hundreds of samples a day. Automated emission flame photometry is used and sodium, urea and one or two other measurements are generally made at the same time and results reported to the clinician as a chemical profile.In considering the place of ion-selective electrodes in patient care, we must consider their advantages and limitations as compared with existing services. We must not only try to define the areas where their advantages will be most usefully exploited, but also the extent to which they can be used alongside more familiar measurements.For routine day-to-day use this service is perfectly adequate. Activity of Concent rat ion The first important consideration is that the electrode measures ionic activity, and this is the biologically important quantity. As the ionic strength of plasma is maintained within narrow limits and potassium is not protein bound, activity and concentration measurements bear a fairly simple relationship to each other, as we have seen in the experimental study described.Calcium represents a much more difficult problem for measurement. About 50% is protein bound and another smaller fraction is complexed to organic anions, and there is the further problem of a variable degree of dissociation and a less easily defined activity coefficient in biological fluids.At this point it is worth noting that this is not the case for all ions.November, 1977 ELECTROANALYSIS I N BIOLOGICAL FLUIDS 337 Again the ionic activity is the importaxt quantity and all of the evidence suggests that it is controlled with great precision in health while the customarily measured total calcium can vary independently owing to changes in the protein-bound fraction.To return to potassium, provided that the electrode is suitably calibrated, for nearly all clinical purposes activity and concentration measurements can be used interchangeably. Therefore, however theoretically attractive an activity measurement might be, it rarely gives more information to the clinician involved in the practical treatment of potassium problems.Practical Convenience The advantages of the electrode became clear during this experimental period when it was in use in the intensive-care unit. Rapid, reproducible results were obtained on small samples of whole blood, which endeared it to the medical staff. It also became popular with the anaesthetists and heart surgeons who sent samples across from the operating theatres for urgent measurements.In our view, this is the place for an electrode of this type, rather than trying to replace the well tried, established, routine laboratory service. Potassium measurements required in tlie context of coronary care, cardiac surgery and emerg- ency diabetic therapy have more in common with blood gas and arterial pH measurements. Blood gas and potassium determinations are often required quickly and are repeated more frequently than the investigations supplied in a centrally reported “chemical profile.” A potassium electrode sited alongside the blood gas apparatus in one of the emergency areas fills this clinical requirement and justifies its existence for a relatively small number of samples, provided that it is kept simple, reliable and therefore cheap to build and maintain.Continuous In Woo Measurement The ion-selective membrane will respond rapidly to small changes of potassium in whole blood without contaminating or consuming the sample. We have exploited these features in making continuous potassium measurements in patients and experimental animals.12 The use of double lumen tubing to make these catheters permits the reference liquid junction to be made near the catheter tip.The experimental trace shown in Fig. 3 was obtained using two indwelling probes of this kind. The membrane is cast on the tip of a fine PVC catheter 1.5 mm in diameter. 1 0.: rnrnol of KCI I I I I 1 rnin Fig. 3. A bolus of potassium chloride (1 ml of 200 mM solution) was injected into the right common iliac vein in a greyhound (arrow).The bolus is seen as a rapid response by a catheter in the inferior vena cava (IVC). After passing through the capillary circulation of the lung and out on the arterial side, the potassium is seen returning from the periphery as an incremental rise in the femoral vein by a second potassium-sensing catheter. For physiological research, electrodes of this type have many exciting possibilities. Elec- trodes of our design and contruction have already been applied in neurophysiological research13 and we are using them to study changes in plasma potassium in animals.Continuous monitor- ing of potassium for clinical purposes is possible, but readily available electrodes for the338 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc. Annlyt.Div. Chem. SOC. measurement of potassium in blood samples are at present the best means of providing reliable results where an urgent service is required. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. References Eisenman, G., Rudin, D. O., and Casby, J. V., Science, N.Y., 1957, 126, 831. Portnoy, H. D., Thomas, L. M., and Gurdjian, E. S., Talnnta, 1962, 9, 119. Dahms, H., Clin. Chem., 1967, 13, 437.Wise, W. M., Kurey, M. J., and Baum, G., Clin. Chem., 1970, 16, 103. Pioda, L. A. R., Stankova, V., and Simon, W., Analyt. Lett., 1969, 2, 665. Pioda, L. A. R., Simon, W., Bosshard, H.-R., and Curtius, H. Ch., Clin. Chim. Acta, 1970, 29, 389. Band, D. M., and Kratochvil, J., J . Physiol., 1974, 239, 1OP. Moody, G. J., Oke, R. B., and Thomas, J. D. R., Analyst, 1970, 95, 910.Provisional patent specification 46024/76. Provisional patent specification 49537176. Band, D. AT., Kratochvil, J., and Treasure, T., J . Physiol., 1977, 265, 5P. Band, D. M., and Treasure, T., J . Physiol., 1977, 266, 12P. Gardner-Medwin, A., Proc. Physiol. SOC., in the press. Microelectronic lon-selective Electrodes R. G. Kelly J. R. Jordan and A. E. Owen Wolfson Microelectronics Unit, University of Edinburgh, Edinburgh, EH9 3 J L Department of Electrical Engineering, University of Edinburgh, Edinburgh.EH9 3 J L The techniques of micro-circuit production may offer a new approach to the construction of ion-selective electrodes. Micro-circuit processing is concerned with the deposition of thin films (50 nm to 50 pm) in the crystalline or amorphous state.The techniques used allow excellent control of the dimensional tolerance and chemical purity of the deposited films, controllable doping levels of the order of 1 in lo7 atoms being achievable, for example. The operations involved are capital intensive and low unit costs are possibie in large-scale production. Micro-circuits can be divided into two main types, vix., monolithic silicon integrated circuits and hybrid film circuits.The former type are produced by diffusion of various components into a wafer of semiconductor material so that the entire circuit is realised on a single piece of silicon. In the hybrid circuit, the conductor, resistor and dielectric materials are deposited on t o an inert substrate, active devices (transistors) in discrete form being bonded to the substrate at a later stage.The ion-sensitive field effect transistor (ISFET) is an example of the applica- tion of silicon technology to production of ion-sensitive devices.l The alternative approach, using “thick film” hybrid circuit techniques, is the subject of current research by the authors. For compatibility with microelectronic techniques, an electrochemical sensor will need to have an essentially planar structure and to employ solid-state electrical connection to the ion- sensitive material.It is expected that several advantages may result from the micro- electronic approach. The sensor will be mechanically robust and the absence of a filling solution will allow it to be used over a wide temperature range. Its small size and the possibility of including an in situ pre-amplifier having a low impedance output will simplify the associated instrumentation and allow operation over a wide band width.By including an integral temperature sensor, it will be possible to achieve accurate temperature com- pensation. I t remains to be seen whether the precision of the deposition techniques will lead to improvements in the stability and reproducibility of the electrode.A longer term possibility is the employment of arrays of materials sensitive to different ions. By combining the responses of the imperfectly selective individual electrodes, it may be possible to achieve improvements in the over-all selectivity of the array. This will only be possible, however, if the characteristics of the individual sensors are sufficiently stable.The ISFET, originally suggested by Bergveld, is based on the conventional metal oxide- silicon field-effect transistor (MOSFET). The metal gate of the conventional transistor is not present in the ISFET and the gate dielectric, which is in direct contact with the solution, functions as an ion-sensitive material. A potential is set up between the dielectric and theNovember, 1.977 ELECTROAXALIWS I N BIOLOGICAL FLUIDS 339 solution, which modulates the drain-source current through the transistor.A qualitative description of the operation of the ISFET has been given2 in which it is shown (contrary to previous suggestions in the literature) that for stable operation the device must be used in conjunction with a conventional reference electrode. While the active area of an ISFET could be made very small (e.g., 5 x 10 pm), the size of the final device after connection has been made is likely to approach the size of a typical low-power, transistor package.As contacts have to be made to the diffused areas on the active face of the device, the problems of encapsulation against the aqueous environment are particularly formidable. For these reasons an alternative approach using “thick-film” hybrid-circuit techniques is under investig- ation.The approach currently being followed is the deposition of films approximately 20 pm thick on to a ceramic substrate on to which a metal has previously been evaporated. Contact to the evaporated metal film will then be made through the substrate material.I t is intended that the substrate will then be bonded to a tube using techniques well known in the assembly of ceramic micro-circuit packages. In this way a water-tight seal will be achieved without recourse to the use of organic adhesives, which have been shown to be a source of difficulty by those working with ISFET device^.^ This approach clearly involves a direct solid-state connection to the ion-sensitive material.Solid-state connection to various types of electrode membrane (e.g., metal - metal salt and glass electrodes) has already been s ~ g g e s t e d . ~ ~ ~ With the glass electrode, doubts have been cast on the long-term stability of the standard potential when a metal connection has been used.5 A possible solution to this problem is the use of a two-layer contact.An electrochemically active metal is employed and a layer of a halide or oxide of the metal is interposed between it and the glass.6 It is easier to envisage a reversible process and stable potential with this structure than with the simple metal contact. One of the objectives of our current research has been the investigation of the stability of solid contacts to glass electrodes For the purpose of research into the nature of the solid connection, membranes formed from bulk glass rather than deposited films have been used.Discs, typically 300 pm thick, were cut from rods (approximately 22 mm in diameter) of pH glass having the simple composition 72% SiO,, 23% Na,O and 6% CaO, kindly supplied by the Department of Ceramics, Glasses and Polymers, University of Sheffield.The discs were assembled into electrodes by attaching them to Pyrex tubes using a silicone adhesive. Contacting metal films were deposited on to the discs by vacuum evaporation prior to asembly. Both electrochemically active metals (copper and silver) and the noble metal gold were used. For comparison, membrane electrodes using conventional filling solutions were assembled by a similar technique. Well developed pH responses have been measured but a decline in the response has generally been observed over an extended period of use.A typical pattern of behaviour has been for an initial response of 55 mV pH-l, to decrease to approximately 40 mV pH-l after continuous use for 20 d. After this initial decrease, the responses have remained fairly steady for up to a further 100 d. A drift of the standard potential equivalent to several pH units has been observed during the initial period, after which stability to within 20 mV has been obtained.In addition to these long-term instabilities, a temperature coefficient of the standard potential of typically 3 mV “C-l has been noted with metal-connected electrodes.I t is suspected that the observed declining response may be due to electrical leakage effects associated with the use of silicone-rubber adhesives. Independent methods of measuring the electrical leakage resistance have, however, suggested that it is too large to explain the observed results. Further work being carried out to resolve this problem will employ an electrode structure using the ceramic-to-glass sealing technique described above. It is clear, however, that well developed electrode responses can be achieved with metal-connected elec- trodes.When the question of leakage has been resolved, it will be necessary to carry out further experiments to explain the observed drifts in both the response and standard potentials.In addition to these studies on the nature of the solid-state contact, the feasibility of produc- ing electrodes by deposition of films of the same pH glass using thick-film circuit techniques is being investigated. Visually satisfactory films have been obtained and measurements of electrode response will be made in the near future. References 1. Bergveld, P., I E E E Trans.Biorned. Engng, 1972, BME-19, 342.340 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc. A d y t . Div. Chem. SOC. 2 . 3. 4. 5 . 6. Kelly, R. G., Electrochim. Acta, 1977, 22, 1. MOSS, S. D., Janata, J., and Johnson, C. C., Analyt. Chem., 1975, 47, 2235. Ruck, R. P., and Rogers Shepard, V., Jr., Analyt. Chewz., 1974, 46, 2097. Guignard, J., and Friedman, S. M., J . Appl.Plays., 1970, 29, 254. Rd. Pat., No. 1260 065, 1972. lon-selective Electrodes for Biomedical Applications G. J. Moody and J. D. R. Thomas Chemistry Depnvtinent, U W I S T , Caidiff, C F l 3N U Most types of ion-selective electrodes have been used in studies on biological fluids, including body and tissue fluids and plant fluids.1 The main attention here is devoted to body and tissue fluids of which serum, plasma and whole blood are the most important, although interest also centres on the non-blood fluids of urine, gastric juice, saliva, sweat, cerebrospinal fluid, milk, intracellular fluids, etc.Glass Electrodes The interest in sodium was stimulated by an appreciation of the marked cation selectivity of glass electrodes with more than 1 mol-yo of aluminium oxide in the glass2 and which led to the commercial availability of cation-sensitive (mainly sodium) glass-membrane electrodes.Glass-electrode studies on serum, plasma and blood indicate a correlation with flame-photometer sodium levels amounting to no sodium b i n d i r ~ g , ~ , ~ although the possibility of weak complexes involving sodium should not be o~erlooked.~ Outside the laboratory, the use of the sodium ion-selective electrode is more convenient than flame methods for applicatiors to materials such as blood fluid, sweat and saliva.Microcapillary glass electrodes can be drawn from cation-sensitive glass with functional impaling tips of less than 1 pm for intracellular work, but there is a tendency for the cation selectivity to be poor. Glass electrodes are widely employed for pH and sodium-ion determinations.Fluoride Ion-selective Electrode Its development6 coincided with the interest and demand for determining trace amounts of fluoride in water- fluoridation and dental-health programmes. I t is now available in a completely solid-state version. Fluoride is normally present in blood serum at about the 5 x lo-' M level, but this level can be considerably higher in patients anaesthetised with certain fluorocarbons and in em- ployees of certain important industries.The problem of access to low-fluoride serum for preparing standards caused by the fluoridation of water supplies may be overcome by using fluoride-free water or by taking the blood serum of a normal young adult who has not drunk fluoridated water for 24 h.' The need to monitor fluoride has been brought into greater prominence since 1960 by the introduction of fluorinated methyl ethyl ethers and similar materials into clinical practice.In patients anaesthetised without dinitrogen oxide supplementation, peak serum values of 190.4 In this respect, renal failure occasionally follows anaesthesia with metho~yflurane,~ this being related to dose and linked to serum inorganic fluoride.Less than 50% of the methoxyflurane is exhaled and the remainder is seen as serum or urinary metabolites, for example as methoxy- fluoroacetic acid or inorganic fluoride, which is regarded as the nephrotoxin.1°711 It can be used directly for free inorganic fluoride, but total fluorine requires recourse to preliminary ashing or Schoniger flask combustion.The fluoride electrode is the most successful ion-selective electrode. 20.9 p~ have been recorded,* probably owing to high doses of methoxyflurane. The fluoride ion-selective electrode is among the easiest to use.November, 1977 ELECTROANALYSIS I N BIOLOGICAL FLUIDS Chloride Ion-selective Electrode 34 1 In order to give faster response times and smaller photoelectric potentials than simple silver chloride membranes, chloride ion-selective membranes consist of intimate mixtures of silver sulphide and silver chloride.12 Although chloride is the principal anion in blood fluids, the electrode is not used for these and the most important role for the chloride ion-selective electrode is for screening sweat chloride levels in cystic fibrosis diagnosis where it is normally used in a combination mode with a reierence e1e~trode.l~ Possible interferences from skin cleansing materials such as Hibitaine (chlorhexidine gluconate) have to be guarded against .14 There are instances of other solid-state crystal-membrane electrodes having been used, such as in the measurement of blood bromine by the bromide ion-selective electrode following treatment of patients with bromo sedatives.15 Use of the lead electrode for determining lead in urine is difficult16 ; indeed, measurements below M levels with ion-selective electrodes is generally unsatisfactory unless the user has considerable expertise.Liquid Ion Exchanger and Neutral Carrier Systems The principal electrode systems based on liquid ion-exchanger and neutral carrier sensors of interest in the biomedical field are those for calcium and potassium.These can be obtained commercially in various modes, but a convenient version is that with the liquid ion exchanger or neutral carrier sensing system trapped in a PVC membrane.17 Such membranes are easily made in the laboratory.l* Blood serum potassium levels obtained by ion-selective electrodes match those obtained by flame photometer.There is a possible role for the electrode in monitoring potassium-ion levels in blood fluids away from the laboratory. Serum calcium exists in three forms, namely non-diffusible protein bound (3045% of the total), complexed but diffusible (5-15% of the total) andionised (generally rather less than half).Although ionised calcium is recognised to be important in various physiological and biochemical processes, few routine calcium-ion determinations in blood and other fluids have been reported. However, the range of research studies concerning use of calcium ion-selective electrodes in calcium-ion investigations is extensive and has recently been reviewed.l Regarding calibration, uncertainties in ionic activity coefficients (and liquid junctions) must be overcome.The calcium electrode is normally calibrated in 0.15 M sodium chloride solution containing calcium chloride in the range 0.5 x to 2 x 1 0 - 3 ~ , but a proposal concerning reference standards for both calcium and potassium ions in blood serum should also be noted.lg Complementary Roles for Ion-selective Electrodes Ion-selective electrodes have been given complementary roles in the monitoring of enzymes and their substrates and in determining blood gases.An early example in the enzyme field was the immobilisation of urease in a layer of acrylamide gel covering the surface of an ammonium ion-sensitive glass electrode.20 The ammonium ion produced by the action of urease on urea contained in the sample solution is sensed by the electrode and is related to the amount of urea present.Various requirements have to be fulfilled in order to obtain a sensor with reproducible parameters and there is the limitation that the selectivity order of ammonium ion-sensitive electrodes can change from one batch to another. Increasing the pH to about 12 in the above instance shifts the equilibrium from the ammon- ium ion to ammonia gas, for which there are available gas-sensing probes.21T22 However, use of the gas-sensing probe requires urea - urease incubations to be completed a t the optimum pH of 7.4 in a static stage followed by removal of samples in which the pH is increased to 12 by quenching with 0.5 M sodium hydroxide solution and the ammonia gas is measured with an ammonia gas electrode.23 The air-gap e l e c t r ~ d e ~ ~ , ~ ~ can be used in the secondary quantitative stage just as conveniently as other designs of gas electrode with the added advantage that the sensing surface of the ion- selective electrode does not make contact with the fouling proteins of clinical samples.Enzyme systems need not be dependent on the availability of ion-selective electrodes that are compatible with the substrate or product. For example, neither substrate nor product can342 ELECTROANALYSIS I N BIOLOGICAL FLUIDS PYOC.I ~ a b t . DiV. Ch?f?Z. SOC. be directly detected by ion-selective electrodes in Glucos~ oxidase P-D-Glucose + H,O + 0, -___-- -+ D-Gluconicacid + H,O, Nevertheless, the consumption of iodide by the hydrogen peroxide product at pH 5.1 : Mo(V1) catalyst H202 + 2H30+ + 21- -____- + - 1, + 4H20 can be monitored with an iodide ion-selective electrode26 to give an indirect assay of that product .Conclusion There is a considerable range of ion-selective electrodes that can be used for the analysis of biological fluids. Their use demands familiarity with their properties and modes of operation in order that those which are available can be more widely exploited and so that those which are yet to appear will be better appreciated.1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 23. 23. 24. 25. 26. References Moody, G. J., and Thomas, J. D. R., Pvog. Med. C h e w , 1977, 14, 51. Eisenman, G., Rudin, D. C., and Casby, J.U., Science, N . Y . , 1957, 126, 831. Moore, E. W., Ann. N . Y . Acad. Sci., 1968, 148, 93. Wise, W. M., Kurey, M. J., and Baum, G., Clin. Chem., 1970, 16, 103. Midgley, D., Chem. SOC. Rev., 1975, 4, 549. Frant, M. S., and Ross, J. W., Science, AT.Y., 1966, 154, 1553. Fry, B. W.. and Taves, D. R., .J. Lab. Clin. Med., 1970, 75, 1020. Taves, D. R., Fry, R. W., Freeman, R.B., and Girter, A. J., J . Am. Med. Ass., 1970, 214, 91. Van Dyke, R. A., and Wood, C. Id., Anesthesiology, 1973, 39, 613. Mazze, R. I., and Cousins, M. J., Can. Anaesth. SOC. J . , 1973, 20, 64. Orion Research Incorporated, Brit. Pat., No. 1 150 698, 1969. Bray, P. T., Clark, G. C. F., Moody, G. J . , and Thomas, J. D. R., “A Perspective of Sodium and Chlor- ide Ion-Selective Electrode Sweat Tests for Screening in Cystic Fibrosis,” UWIST, Cardiff-’, 1975.Bray, P. T., Clark, G. C. F., Moody, G. J., and Thomas, J. D. R., Clin. Chim. Acta, in the press. Degenhart, H. J . , Abelin, G., Bevaart, B., and Baks, J., Clin. Chim. Acta, 1977, 77, 69. Rechnitz, G. A., and Kenny, N. C., Analyt. Lett., 1970, 3, 259. Moody, G. J., Oke, R. B., and Thomas, J. D. R., Analyst, 1970, 95, 910.Craggs, A., Moody, G. J . , and Thomas, J . D. R., J . Chem. Edacc., 1974, 51, 551. Covington, A. K., and Robinson, R. A., ANalytica Chim. Acta, 1975, 78, 219. Montalvo, J . G., and Guilbault, G. G., Analyt. Chpm., 1969, 41, 1897. Ross, J. W., Riseman, J. H., and Krueger, J. A., Pure Afipl. Chem., 1973, 36, 473. Bailey, P. L., and Riley, M., Analyst, 1977, 102, 213.Llenado, K. A., and Rechnitz, G. A., Analyt. C h e w , 1974, 46, 1109. RbiiCka, J., and Hansen, E. H., Analytica Chim. Acta, 1974, 69, 129. Hansen, E. H., and RBiiCka, J , , Analytica Chim. Actn, 1974, 72, 353. Llenado, R. A., and Rechnitz, G. A., Annlyt. Chem., 1973, 45, 826. .Mazze, R. I., Trudell, J. R., and Cousins, M. J., Anesthesiology, 1971, 35, 247. Problems of Redox Measurements in Cell Physiology J.Chayen Divisioiz of Cellular Biology, Kennedy Institute of Rheumatology, Bute Gardens, London, LV6 7 0 W Electroanalysis is becoming of increasing obvious importance in cell physiology. The purpose of this paper is to draw some selected phenomena to the attention of electrochemists in the hope that they may stimulate rigorous electrochemical analyses of such problems in cellular physiology.Role of an Oxidation - Reduction Environment in Cell Division There is a great deal of information in the older literature (reviewed by Brachetl) that mitosis and cell division are related to, or possibly dependent on, a reductive redox balance in the cytoplasm of the dividing cells. An oxidative phase, related to biosynthesis, may precede the reductive phase., In suitable cells, some workers have measured the increase in reducedXovember, 1977 ELECTROANALE-SIS I N BIOLOGICAL FLUIDS 343 glutathione that occurs just prior to cell division.Other workers have shown that substances that block sulphydryl groups inhibit mitosis and that this inhibition can be overcome by cysteine or thioglycolic acid. Similarly, Fildes3 showed that the bacteriostatic action of mercury can be reversed by cysteine or reduced glutathione.More recently, Allen and Curtis4 claimed that bacterial proliferation can be considerably altered by exposing the bacteria to a suitable electrode potential. Other evidence, quoted by Brachet,l indicated that reduced glutathione, and other sulphydryl-containing molecules, stimulate the growth of cells in vitro.The importance of a highly reductive redox state during the actual process of cell division, in contrast to the interphase period of synthesis for the next mitosis, is shown in the studies of Chayenj and of Sandritter and Krygier.6 Both of these groups of workers found the sulphur-containing proteins of the cell to be predominantly in the reduced (sulphydryl) condition at the time of chromosome division.All of these results are indicative of a redox change that may be related to, or directly involved in, the process of cell division. Yet, even though this process is fundamental to biological problems such as wound healing and malignant growth, there is too little quanti- tative and direct measurement of the -SH to -S-S- ratios, or of the redox potential operating in such cells, to allow us to know whether these factors are relevant or not.Metabolic Effects Peculiar to Carcinomata There now seem little grounds to doubt that possibly all truly malignant growths of epithelial origin (carcinomata) have elevated activity of certain enzymes of the hexose monophosphate pathway, namely glucose 6-phosphate and 6-phosphogluconate dehydrogenases. This was first shown for human bronchial cancer by Chayen et aZ.,7 then for carcinoma of the female genital tract by Bonham and Gibbsg and by Cohen and Way.s The latter results were con- firmed by Cameron and Husain.10 More recently, similar results have been found in breast cancer by Altman et aZ.11 Obviously, if cells contain a high activity of a particular dehydro- genating enzyme contained within the cell, it is difficult to demonstrate this unless the cells are removed and examined cytochemically or biochemically.However, there is evidence that malignant cells are “leaky” and that their enzymic activities “spill out” through the cell membrane into the inter-cellular spaces where they might be detectable by an electrochemical probe.Further, there is some indication12 that high levels of intra-cellular dehydrogenation can be detected electrochemically by changes that occur a t the outer surface of such cells. If this were to be true, there would be great advantages that could accrue to electrochemistrj- in detecting and delimiting the presence of such malignant cells. The practical possibility of producing such a method of detecting malignant growth may be increased by another feature that seems to be peculiar to malignant carcinoma cells.In the cytochemical demonstration of these dehydrogenase enzymes, a tetrazolium salt is used as the hydrogen acceptor (or electron receptor) ; this acceptor replaces oxygen and, on becoming reduced, yields a highly coloured, insoluble formazan.The amount of the formazan produced is a measure of the oxidative activity in respect of the particular substrate used. In all normal tissue studied up to the present, oxygen competes successfully with the tetrazolium salt so that the reaction has to be carried out in an atmosphere of nitrogen if a response is to be elicited from the cells. The peculiarity of malignant carcinoma cells is that oxygen is largely unsuccessful in competing with the tetrazolium salt for the reducing equivalents, producing perhaps only 50% inhibition of the reaction.Hence there appears to be some mechanism in the malignant cells which restricts the influence of atmospheric oxygen.13 There is some evidence from studies using electron spin resonance in support of this ~0ntention.l~ In theory, at least, it might be possible to measure, electrically, the 6-phosphogluconate dehydrogenase activity around an external surface of a particular region in the body (such as the cervix uteri) by exposing that tissue to a film of the substrate and the required coenzyme (NADI’) in the presence or absence of oxygen; the response from normal and from malignant cells should be characteristica!ly different.Response of Tissues to Hormones The original concept was that hormone molecules become attached to many receptors sited at the outer surface of the “target” cells and that, when enough were combined, they344 ELETROANALYSIS IK BIOLOGICAL FLUIDS Proc. AnaZyt. Div. Chem. SOC. stimulated the cell to respond, for example through the activation of adenyl cyclase.More recent information indicates that, for some hormones at least, this is unlikely to pertain in life; certainly this is so as regards the adrenocorticotrophic hormone, ACTH, acting on the cortex of the adrenal gland to stimulate it to produce cortisol (in man). The work of Hudson and iClcMartin15 and of Moyle et ~ 1 . l ~ indicates that the activation of adenyl cyclase and its associated systems may not be a prerequisite for the action of this hormone.The remarkable sensitivity of the cytochemical bioassay systems,17 which are 1 000 times more sensitive than the equivalent radioimmunoassays, indicates that the sensitivity of response depends on cell- to-cell interactions, probably involving co-operation between cells, as has been described by Orci et ~ ~ 1 .~ 8 In the study by Chambers and Chayenlg it was shown that a response to the hormone could be detected even when there could not be more than one molecule of the hormone to every seven “target” cells. Yet when the activation in each of the target cells was measured, it was found that they all responded equally, with as little scatter of values as was found, with considerably greater activation, when there were los more molecules capable of acting on the same number of cells.This applies only when the cells are held together in their natural histological matrix. Thus it implies that the attachment of a molecule of the hormone at one site on a cell will influence not only that cell, but many cells around the point of attach- ment.Hence it would be helpful to be able to have electrochemical measurements of the effect of binding of a hormone molecule to its “target” cell, and possibly of transmembrane potentials or of other stimuli which, initiated by the binding of a hormone, could synchron- ously stimulate many cells to a co-operative chemical activity. Effect of the Redox Balance in the Environment of Cells There is some evidence that the chemical state of the cells lining the synovium of joints differs in rheumatoid arthritis.The synovial cells of rheumatoid joints have a greater propor- tion of sulphydryl to disulphide in the sulphur-containing amino acids of their proteins20; they have a greater rate of generation of NADPH under conditions where the activity of glucose 6-phosphate dehydrogenase is tested20 and their lysosomal membranes show increased permea- bility.21 As it is suspected that the erosion of the cartilage is caused by lysosomal enzymes which escape, or are extruded from these cells,22 the possibility that the primary condition is related to a changed redox balance could be important.I t has been shown23 that the rheumatoid tissue, in vitro, requires a more acidic pH than does the non-rheumatoid synovial tissue.However, the rheumatoid tissue tolerates a higher pH if a hydrogen donor is added to the medium.24 Equally, the non-rheumatoid tissue can be cultured at a lower pH, at which it would normally die, if the culturemedium contains a suitable hydrogen acceptor. Thus it would be of interest to be able to define the redox conditions of the culture medium and to demonstrate whether there is a particular redox condition that is essential for the survival of these tissues.Moreover, as the biochemical characteristics of the rheumatoid synovial cells can be changed towards normality by the use of suitable hydrogen acceptors, it would be valuable to have rigorous electrochemical information both as regards the electrochemical state of the tissue and the influence of the oxidation - reduction state of the environment on the behaviour of the cells.References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Rrachet, J., translated by Barth, L. G., “Chemical Embryology,” Interscience, New York, 1950. Zeuthen, E., Archs Ne‘erl. Zool., 1952, 10, Suppl.1, 31. Fildes, P., Brit. J . E x p . Path., 1940, 21, 67. Allen, M. J., and Curtis, J., Abstr. Commun. Meeting Fed. Eur. Biochem. SOC. (Amsterdam), 1972, 8, Chayen, J., I n t . Rev. Cytol., 1953, 2, 77. Sandritter, W., and Krygier, A,, Z. Krebsforsch., 1959, 62, 596. Chayen, J., Bitensky, L., Aves, E. K., Jones, G. R. N., Silcox, A. A., and Cunningham, G. J., h’ature, Bonham, D.G., and Gibbs, D. F., Brit. Med. I . , 1962, ii, 823. Cohen, S., and Way, S., Brit. Med. .J., 1966, i, 88. Cameron, C. B., and Husain, 0. A. N., Brit. Med. J.. 1965, i, 1529. Altman, F. P., Bitensky, L., Chayen, J., and Daly, J. R., Proc. Ass. Clin. Biochenz., 1968, 5, 119. Fensom, D. S., Can. J . Bot., 1959, 37, 1003. Altman, F. P., Bitensky L., Butcher, R. G., and Chayen, J., in Evans, D.M. D., Editor, “Cytology 823. Lond., 1962, 195, 714. Automation,” Livingstone, Edinburgh, 1970, p. 82..November, 1.977 ELECTROANALYSIS I N BIOLOGICAL FLUIDS 345 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. Slater, T. F., and Cook, J . W. I<., in Evans, D. 31. D., Editor, “Cytology Automation,” Livingstone, Hudson, A. M., and McMartin, C., Ciochem. J . , 1975, 148, 539.Moyle, W. R., Macdonald, G. J., and Garfink, J . E., Biochem. J . , 1976, 160, 1. Chayen, J., Daly, J . R., Loveridge, N., and Bitensky, L., Rec. Prog. Hovnz. Res., 1976, 32, 33. Orci, L., Malaisse-Lagae, F., Ravazzola, M., Rouiller, D., Renold, A. E., Perrelet, A., Unger, R., and Chambers, D. J., and Chayen, J., J . Endocrinol., 1976, 68, 24P. Butcher, R. G., Bitensky, L., Cashman, R., and Chayen, J., Beitr.Path., 1973, 148, 265. Chayen, J., Bitensky, L., Butcher, R. G.. and Cashman, B., Beitr. Path., 1971, 142, 137. Chayen, J., and Bitensky, Id., Ann. Rheum. Dis., 1971, 30, 522. Poulter, L. W., Bitensky, L., Cashman, B., and Chayen, J., Virchows Arch. Path. Anat. Physiol., B, Chayen, J., Bitensky, L., Butcher, R. G., and Cashman, B., Beitr. Path., 1973, 149, 127.Edinburgh, 1970, p. 108. Unger, R. H., J . Clin. Invest., 1975, 56, 1066. 1970, 4, 303. Electrodes in Clinical Chemistry A. D. Hirst Depavtinent of Chemical Pathology, King’s College Hospital, Denmark Hill, London, S E 5 9RS Use in Routine Laboratories In spite of considerable advances in the theory of ion-selective electrodes and the development of several new electrodes in recent years, they are not in use in most routine clinical chemistry laboratories, except for blood pH, PO, and pC0, determinations. The reasons for this lack of adoption are complex, but can be split into four main categories.The first reason is that many clinical chemists tend to be cautious, and adhere to analytical methods that have known limitations. This attitude is a reflection of a general medical concept that the safety of the patient is the first priority.It is no surprise that clinical chemists are reluctant to adopt methods in which they have no experience in preference to well documented existing methods, whatever their known limitations may be, unless there is a clear advantage to be gained. The second reason is that few of the electroanalytical methods published so far offer a substantial improvement over existing methods, and any theoretical improvement is off set by the unreliability of many electrodes.Reliability is an important factor in situations where the management of a patient requires rapid production of sound analytical data. The third reason is that although clinical chemists would welcome the use of electrodes for determinations that are at present unsatisfactory, the electrodes required are not yet available.The fourth reason is that many of the blood constituents that clinical chemists wish to determine exist in a bound form, necessitating chemical pre-treatment before an electrode can be used. This invalidates one of the main reasons for changing from the existing methods, i.e., rapidity and simplicity of analysis. Advantages of Electrodes I t is recognised that despite these reasons for the lack of adoption of electrodes in routine clinical chemistry, there are advantages to be gained from their use in medicine when suitable electrodes become available. The main advantages are rapidity, direct analysis with direct readout, use with turbid solutions, use of invasive techniques and a replacement for difficult assay techniques. The demand for rapid analyses is small, but increasing, and appears to be related to currently available technology.The current demand for rapid potassium and blood gas determinations has been established for only a few years, and it is reasonable to predict that several other determinations would be requested urgently if the techniques were available, e.g., toxicological investigations. Turbid solutions present a problem in photometric analysis that could be circumvented by the use of electrodes, and are the result of either turbid reagents or turbid samples.Turbid reagents are employed in amylase and lipase determinations, and prevent the use of zero-order kinetic measurements, while turbid samples may be whole blood, lipaemic serum, urine or346 ELECTROANALYSIS IN BIOLOGICAL FLUIDS Proc.Analyt. Div. Chew. SOC. homogenised faeces, all of which require either a blank correction which may be invalid, or a time-consuming separation step. The main, and as yet largely unexplored, advantage of electrodes is their use as a replace- ment for difficult assays.The difficulty may arise from either the lability of the compound to be assayed or the lack of acceptable methodology. The former category includes blood con- stituents such as lactate and pyruvate, the determination of which requires immediate de- proteinisation of the sample to prevent their formation by glycolysis, while in the latter could be placed urine hydroxyproline, oxalate and 6-aminolaevulinic acid.Problems Associated with Routine Use While the advantages of using electrodes are marked and self-evident, there are several problems that must be solved before electroanalytical techniques are adopted by clinical chemists. Selectivity and sensitivity are problems that must be solved by electrochemists. Response time and linearisation are a problem in popular tests that require automation.Interference in response requires careful consideration when dealing with biological material of unknown composition. The major objection to the use of electrodes is, however, the fact that they respond to activity rather than molar c0ncentration.l The consequences of this property are: (a) activity coefficients are unknown and may be variable, (b) suitable calibra- tion material is difficult to define, (c) correlation with existing techniques may be poor, and (d) while it is possible that there may be direct relationships between chemical and biological activity, this has not been substantiated, and until it is clinical chemists are obliged to continue reporting molar concentration. Plasma Electrolyte Analyser In the face of all of these apparent drawbacks to the application of ion-selective electrodes, we decided that the advantages were sufficient to attempt to construct a continuous-flow plasma electrolyte analyser based on electrodes for use in the routine hospital laboratory.The advantages over existing techniques (utilising a Technicon SMA 6/60) were, in addition to those stated earlier: (1) the complexity of the instrument would be reduced by the use of simple chemical reactions, (2) the dwell time of a sample within the system would be reduced by the use of rapid chemical reactions, (3) the reduced dwell time would permit the elimination of constant re-phasing of individual channels and (4) the start-up time of the instrument would be reduced.The disadvantages of using electrodes were overcome by using several standard techniques. Selectivity and sensitivity were not problems, as all of the electrodes used (sodium, potassium, pH, ammonia and oxygen) were known to be suitable for use with human blood plasma, with the possible exception of sensitivity in the sodium determination, into which we decided to incorporate a dialysis step that reduced the sensitivity by a factor of 20.The reasons for this decision were that we found in early trials that diluted plasma appeared to clog the electrode, and Westgard et aZ.2 had reported a poor correlation with flame photometry in their evaluation of the Technicon SMAC. Protein interference, which was thought to be the cause of the poor correlation, would be eliminated by the inclusion of a dialysis step in the determination. We found linearisation of the signals difficult owing to the different millivolt per decade responses of each ele~trode.~ The use of an anti-log amplifier alone is not sufficient to linearise the signal. An additional variable-gain pre-amplifier is required in order to convert the Nernst equation into the form V , = CAga/ga, where V , is the output voltage, C is sample concent- ration, A is the variable gain of the pre-amplifier, K , is the millivolt per decade response of the electrode and K, is the anti-log amplifier constant in volts per decade. Linearisation is achieved by varying the pre-amplifier gain ( A ) to make the power of C unity. The response times of the electrodes (except potassium) were found to be too slow for the anticipated work load of the instrument, for which we required a sampling rate of 60 specimens per hour. Processing of the linearised signal with electronic curve regeneration, using the equation V , = V , + c(dV,/dt), where c is a constant related to the i+ of the response, produced an adequate improvement in electrode response. The objection to the response of electrodes to activity was overcome by pre-treatment of the samples in a continuous flow mode, e.g., by dialysis in the instance of sodium and dilution in a buffer for potassium. The pre-treatment stage enabled us to calibrate the machine,?VovmibeY, 1977 EQUIPMENT NEWS 347 accurately with aqueous standards, but in practice we find lyophilised bovine serum more convenient as a secondary calibration standard, as the same material is used for calibrating the other laboratory instruments. Caution should be used in choosing a suitable lyophilised serum, as many of the preparations that are available have an ammonium carbonate recon- stituting solution that affects the urea determination, for which an ammonia electrode is used. Five-channel Analyser : Technical Details4 The instrument, which determines plasma sodium, potassium, total carbon dioxide, urea and (1) Sodium, potassium and total carbon dioxide: the sample is diluted in 0.18 M sulphuric acid. (2) Sodium: the acidified stream is dialysed against a 0.2 M ammonia buffer, pH 8.5, which is pulled through an EIL sodium electrode. (3) Potassium: an equal volume of 0.25 M triethanolamine buffer, pH 8.0, is added, and the stream is sucked through an EDT potassium electrode. (4) Total carbon dioxide: the stream is passed through a dialyser with a silicone-rubber membrane, through which the carbon dioxide diffuses into a 2 mM sodium hydroxide solution which is sucked through an EIL pH electrode. (5) Urea: the sample is diluted in urease (0.1% in 1 M EDTA). After 60-s incubation, an equal volume of 0.5 M sodium hydroxide solution is added and the stream is sucked through an EIL ammonia electrode. (6) Glucose: the sample is diluted in Tris buffer (0.1 M, pH 7.0). Glucose oxidase is added to the stream and, after 30-s incubation, the solution is sucked through a Radiometer oxygen electrode. The data obtained from this instrument show good correlation with standard analytical methods for normal and pathological specimens. glucose, was constructed on the following principles : References 1. 2. 3. 4. Dahms, H., Rock, R., and Seligson, D., Clin. Chem., 1968, 14, 859. lliestgard, J. O., Carey, R. N., Felbruegge, D. H., and Jenkins, L. M., Clin. Chem., 1976, 22, 489. Gay, P., Med. Biol. Engng Cornput., 1977, in the press. Hirst, A. D., Gay, P., Richardson, P., and Howorth, P. J. K., Natn. Bur. Stand. Spec. Publs. 1975, No. 450.

ISSN:0306-1396    

DOI:10.1039/AD9771400329

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

EQUIPMENT NEWS 347 Equipment News pH Meters The Model 3030 is a hand-held battery-powered pH meter with a resolution of 0.1 over the pH range 0-14. It is supplied with a conventional silver chloride electrode ; a platinum resistance thermometer sensor gives automatic tempera- ture compensation over the range 0-100 "C. The Model 3040 operates from a.c. mains powcr supply a t 110 or 240V, 50Hz, and likewise gives a resolution of 0.1 over the pH range 0-14, with an accuracy of reading of 0.1 :/o.Incorporated is automatic tempera- ture compensation over the range 0-100 "C. Alarm options can also be fitted according t o requirements. The model is available with a choice of platinum resistance sensors and silver chloride combination pH electrodes. Jenway Ltd., 26 Broomhills Industrial Estate, Rsyne Road, Braintree, Essex.pH Electrodes A novel glass, coded 213, is available in the complete range of laboratory and industrial pH electrodes, which can also be made to suit individual requirements. The glass is tough, giving electrodes that can be steam sterilised and used at temperatures from 0 to 120 "C. In pH response properties mean alkaline errors in 14.1 pH solution and mean acid errors in 0.07 pH solution are not greater than 0.03 pH.Russell pH Ltd., 44 High Street, Auchter- muchty, Fife, KY14 7AP. A shatter-proof polymer-encased pH electrode with a low-resistance 10-15-M R sensing bulb, which incorporates a sealed reference gel that does not evaporate has been recently intro- duced. Graphic Controls Ltd., Clyde Vale, Forest Hill, South London.348 EQUIPMENT SEWS Proc.Analyt. Div. Chent. SOC. Low-resistance Electrode By changing the coating on the one-piece sensor from silver to silver chloride, the ECG electrode now yields a faster response time and thus improves defective signal recovery caused by potentially over-long retention of the electric charge following defibrillation. Graphic Controls Ltd., Forest Hill, South London.Micro Stirrer The Temtron Micro Stirrer is designed to stir continuously micro-amounts of aqueous com- pounds without interfering with light-sensing detectors. The stirring rate is continuously variable from 0 to 30 Hz. One-millilitre cuvettes with openings down to 3 x 10mm can be stirred. TEM Sales Ltd., Gatwick Road, Crawley, Sussex. Ultraviolet - Visible Spectrophotometer A new dual-wavelength, double-beam, ultra- violet - visible spectrophotometer, Model 556, contains two separate diffraction-grating mono- chromators, with independent selection of wave- length.Two sampling positions and the wide acceptance angle of the detector make it possible to examine all types of sample, turbid, fluorescent, suspension, solids or frozen solids.Perkin-Elmer Ltd., Post Office Lane, Beaconsfield, Bucks., HP9 1QA. Atomic-absorption Spectrophotometers Models 372 and 373 are double-beam atomic- absorption spectrophotometers controlled by microcomputer. Continuous readings with variable integration times can be taken or integrated readings held in store. By installing a printed circuit board, Model 373 can be coupled to the TR-2 Teletypewriter Readout. Model 272 is a single-beam atomic-absorption spectrophotometer, also microcomputer con- trolled, with the same electronics as Models 372 and 373.Perkin-Elmer Ltd., Post Office Lane, Beaconsfield, Bucks., HP9 1QA. Rapid-scan Spectrometer The TN-1710-21 Diode Array Rapid Scan Spectrometer systems provide simultaneous parallel high-resolution spectral measurements over the range 200-1 100 nm with coverage of 1.25 nm per element.Real-time dynamic range is 4 096: 1. A 512-element detector is scanned in 5.12 ms, with exposure times per scan adjustable from 5.16 ms to 8 192 s. Tracor Northern, Inc., 2551 West Beltline Highway, Middleton, Wisc., 53562, USA. Transient- signal Processor A new measuring system, using Lissajous figures, is in use for studying non-linear a.c. conductivity of materials a t low frequencies.The transient-signal processor automates the final analysis and extends the frequency range covered. Model 512A is a dual-channel model that can store values of current and voltage in a 2 048- word, 10-bit memory, and it has been used in place of the X - Y recorder to analyse current/ voltage loops.Bryans Southern Instruments Ltd., TYillow Lane, Mitcham, Surrey, CR4 4UL. Automatic HPLC Injector Model 725 Automatic Injector for high- performance liquid chromatography systems allows unattended injection of up to 64 con- secutive samples with negligible carryover. Only 700 p1 of sample are required. A 10-p1 sample loop and injection valves operable to 6 000 p.s.i.are fitted as standard. Fine Particle Group, Coulter Electronics Ltd., Coldharbour Lane, Harpenden, Herts. Liquid Chromatograph The 1081A liquid chromatograph is added to the HP1080 range. It incorporates a single- head diaphragm pump to give a stable flow and a valve loop injector, which allows sample introduction a t full column pressure. The single-wavelength (254 nm) ultraviolet detector can be linked directly to conventional inte- grators and strip-chart recorders.Hewlett-Packard Ltd., King Street Lane, Winnersh, Wokingham, Berks., RG11 5AR. Adsorbent for Liquid Chromatography Lichroprep comprises a range of adsorbents for liquid chromatography having close and constant particle-size classification. Available now are Lichroprep Si60 with particle sizes 5-20 (9319) 15-25 (9336) and 25-40 p (9390), while in preparation are Lichroprep RP.2, particle size 25-40 p, and Lichroprep RP.8, particle sizes 5-20 and 25-40 p.Anderman & Co. Ltd., Central Avenue, East Molesey, Surrey, KT8 OQZ. Miniature Valves A wide range of compact, top-loaded ball valves for shut-off and switching applications include 2-, 3-, 4-, 5-, 6- and 7-way patterns and vented versions.A top-loaded TFE capsule packing leaves no internal voids to trap liquid. Materials of construction are brass or 316 stain- less steel. Flow capacities (C,) range fromn’ovember, 1977 EQUIPMENT NEWS 349 0.07 to 9.8 a t temperatures of 10-65 “C under vacuum or to 3 000 p.s.i.g. A choice of forged body valves is also avail- able i n brass, 316 stainless steel, carbon steel, monel and aluminium.Orifice sizes range from 0.080-0.375 in. Ratings are up to 3 000 13.s.i.g. and 230 “C, with possible extension to 315 “C. Techmation Ltd., 58 Edgware Way, Edg- ware, Middx., HA8 8 JP. Computing Infrared Analyser Model 80 is a computing infrared analyser, which combines a single-beam infrared spectro- photometer with a programmable minicom- puter.I t can accept solid, liquid or gaseous samples without the need to vaporise or dissolve them. Measurements of up to 18 wavelengths arc possible in less than 2 min. Wilks Scientific Ltd., 64 Burners Lane, Kiln Farm, Milton Keynes, Bucks. Data Processor - Plotter Transcribe 10 is a “combination” instrument, which offers facilities for processing transient signals and producing hard copy plots in a compact package.It offers a choice of single- or tlual-channel operation. The two com- ponents of the Transcribe 10, the transient processor and the recorder, can be operated separately when required. Bryans Southern Instruments Ltd., 1 Willow Lane, Mitcham, Surrey, CIC4 4UL. Scintillation Counting Bottle Opener ,4 servo-assisted opener - sealer for scintillation counting bottles or vials is announced.The unit is wall-mounted and is operated with electrical power. It can be used also with other types of containers and caps. Koch-Light Laboratories Ltd . , Colnbrook, Uucks., SL3 OBZ. Oil Immersion Heaters Unbreakable elements, which slide into mild- steel pockets and are rated down t o 8 W in-2, have been added to the Eltron range of immersion heaters suitable for oil-heating appli- cations.Eltron (London) Ltd., Strathmore Road, Croydon, Surrey, CR9 2NA. Preparative Ultracentrifuges Prepspin 65 and 75 are new preparative ultra- centrifuges. Features include a new speed selector, a semi-hermetic refrigeration unit, brush-wear warning indicator and cushion start. Both ultracentrifuges comply fully with German safety regulations.MSE Scientific Instruments, Manor Royal Crawley, Sussex. Widefield Microscope The TIYODA Super Widefield Microscope features eyepieces that allow a field of view 90 yo larger than do conventional eyepieces. An 1 l-element apochromatic condenser is in- corporated with plan apochromatic objectives ranging from 2 to 60x. A 0 . 6 6 ~ lens in the photo tube and a low- power photo eyepiece enable an area of 14 x 22 mm to be photographed on 35-mm film when using the Plan Achromat l x objective. Hall Bros.(Optical) Ltd., Unit 4, Hill Farm Industrial Estate, Hill Farm Avenue, Leaves- den, Watford, Herts. Right-angle Prisms A range of right-angle prisms is available in sizes from 3 to 100 mm square, with either plain or aluminised hypotenuse face.Material is optical-grade BK7 with surfaces to 2 lambda overall; angular tolerances are to + 3 arc min with the more popular sizes to +30 arc s. Melles Groit B.V., Nieuwekade 10, Postbus 567, Arnhem, The Netherlands. Solvents Eleven solvents have been developed for use in analysis of pesticide residues. Each has a guaranteed maximum level of organic impurity to ensure suitability for gas-chromatographic analytical methods.J. T. Baker Chemicals B.V., P.O. Box 1, Deventer, The Netherlands. Literature The latest edition of Perkin-Elmer Analytical News, PELAN-14, is devoted to high-perfor- mance liquid chromatography, and includes papers presented at the Company’s 1976 Symposium held a t Brunel University. Perkin-Elmer Ltd., Post Office Lane, Beaconsfield, Bucks., HP9 1QA.Tables of analytical data obtained by means of infrared spectroscopy, dealing with gases for which 1977 OSHA concentration limits have been fixed, as well as some for which no such exposure limits have yet been fixed, are available. Wilks Scientific Corporation, 140 Water Street, Box 449, South Norwalk, Conn. 06856, USA.350 CORRESPONDENCE Proc. Analjvt. Div. Clzem. SOC. The May 1977 edition of The AutoAnalyst features the use of the InfraAlyzer for immedi- ate analysis of oil, protein and moisture contents of cereal crops for the feed industry. Technicon Instruments Co. Ltd., Evans House, Hamilton Close, Basingstoke, Hants., RG21 2YE. A leaflet describes the sizes and materials in which Apollo LC tubing, for use in liquid chromatography, is available. Small Tube Division, T. I. Accles & Pollock Ltd., Rounds Green Road, Oldbury, Warley, W. Midlands, B69 2DF. A shortform catalogue of recording systems is available. Bryans Southern Instruments Ltd., Willow Lane, Mitcham, Surrey, CR4 4UL.

ISSN:0306-1396    

DOI:10.1039/AD9771400347

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

350 CORRESPONDENCE Proc. Analjvt. Div. Clzem. SOC. Correspondence Correspondence is accepted on all matters of interest to analytical chemists. Letters should be addressed to the Editor, Proceedings of the Analytical Division. The Chemical Society, Burlington House, London, W1 V OBN. Basic Safety in Volumetric Ana I ys is Sir, I have been very pleased to see the series “Safety in Analytical Laboratories” and have noted with interest the sensible recommenda- tions made therein.The article on volumetric analysis1 quite rightly stressed the dangers from the production of heat when strong acids or alkalis are diluted. There are practical problems of cooling larger vessels externally, and it can be inconvenient to wait for solutions to cool before making up to volume.A method we have used with some success in this Department is internal cooling by the presence of ice. In practice, good-quality flake ice is stirred well with de-ionised water, and the concentrated acid or base added to the ice - water mixture. The heat liberated has then to overcome the enthalpy of melting of the ice as well as the thermal capacity of the solution. With a rough calculation and/or a little practice, it is possible to add just sufficient ice for the resulting solution to be a t around room temperature.For example, the making up of a 1 M solution from concentrated sulphuric acid will give a solution about 20 “C above ambient. The energy required to melt and raise 1 g of ice to room temperature is about 420 J or 100 cal, so that around 200 g should be used for 1 1 of solution.In many cases we have found that polypro- pylene vessels offer advantages over glass ones for such dilutions. They will withstand tem- peratures in excess of 100 “C without deforma- tion, and of course are essentially free of the hazard of breakage due to scratches or mechani- cal or thermal shock. Pellets of sodium hydroxide or potassium hydroxide are not prone to stick to the surface as they are with glass, and the solution is not contaminated by silicate formation.Polypropylene is however slightly attacked by fuming or strongly oxidising acids, and should not be directly exposed to these in concentrated form. Reference 1. Proc. Analyt. Div. Chern. SOC., 1977, 14, 225. Martin J. Pitt Depadment of Chemical Engineering, Aston University, Bivmingham, B4 7ET Sir, After reading the article on Basic Safety in Volumetric Analysis (Proc.Analyt. Div. Chem. Soc., 1977, 14, 225) I find myself wondering who wrote it, for whom it was intended, and whether it was designed as a means of reducing the number on the dole by manufacturing extra work. Some of the instructions seem most peculiar. Take the preparation of dilute hydrochloric acid, for example.Why use only twice the volume of water? If a large enough volume is used it will do all the cooling necessary without any help. How dilute is dilute? The method would not work for 6 M acid! Why should we not add water to pellets of caustic soda or potash? The rate of dissolution is fairly low, so if sufficient water is added and stirring is efficient, there should be no heatNovembeY, 1977 CORRESPONDENCE 351 problem except when a highly concentrated solution is prepared.For cleaning glassware my preference is benzene followed by con- centrated sulphuric acid, which is the fastest and most efficient method I know. Toluene can be used instead of benzene, if preferred. As for forbidding pipetting by mouth, I have seen much more serious accidents caused by so- called safety devices than by mishaps with the chemicals pipetted.Of course it is stupid to use suction by mouth for substances such as ammonia, or concentrated acids, but anyone who has learned to “switch off” suction or blowing (into old-fashioned “proper” wash- bottles) should not be in much danger from the usual run of reagents.In my youth it was common practice to pipette even cyanide solutions by mouth-one quickly learned to be careful. One is amused by the emphasis on “correct” methods. Anyone using a funnel and a “small container” for filling a burette must like washing up, and is quite likely to overfill the burette or leave the funnel in the top while doing the titration. Experts can fill a burette from a IVinchester.I t is not a t all obvious to this operator why i t is dangerous to sit down to do a titration, other than the risk of a practical joker taking the stool away. I agree that it is essential that proper tech- nique should be taught, and that sources of danger should be pointed out, b u t safety pre- cautions can be carried to rather absurd extremes, and one wonders whether those who would observe all those in this article would also ever have the temerity to reach the laboratory to clo so.The real answer is personal teaching and pride in the acquisition of skill and dex- terity. Legend has it that one chief chemist judged applicants for jobs purely on the speed with which they could empty a IT7inchester of water. Nowadays the job would go to the applicant doing it by remote control [provided he (she) did not do him(her)self an injury in handling the controls].R. A. Chalmers Chemistry Department, University of A berdeen, Old A berdeen, A B9 2 UE, Scotland Sir, In the article on Basic Safety in Volumetric Analysis in the August issue of Proceedings, I feel that two of the stipulations are obsessive in the context of technicians’ commonsense.In 2.2, an absolute ban on mouth pipetting is absurd when i t comes, for example, to dis- pensing distilled water. Whilst dispensing aids are rightly the mainstay of assay manipulations, their use in teaching students is unduly costly and, in any case, conveys no appreciation of manipulative skills and errors; to see students shakily dispensing even innocuous liquids with a bulb controlling the pipette is agonising to a teacher.Moreover, the ban in paragraph 3 on titrating while seated seems ridiculously sweep- ing; surely there should be some judgement as to the hazard of the liquid concerned? E. Reid TVolfsovl Bioanalytical Centre, Uniuersity of Survey, Guildford, Surrey, G U2 5XH Code of Practice for the Use of Gas Cylinders in Analytical Laboratories Sir, This Code of Practice1 should find wide use in analytical and other laboratories.I would offer comment on two points. Section 2.3 might imply that cylinders of liquefied gas in general may safely be heated to 45 “C. This is surely not so for hydrogen chloride, for example. In the case of liquid ammonia, while the integrity of the cylinder might be assured a t 45 “C so long as accidental overfilling did not occur, it could be unwise to attempt to withdraw liquid from a cylinder which had been heated in any way. Section 3.3.3 suggests “snifting,” although with qualifications, as one method of ensuring removal of dirt from cylinder valves. It may not be well enough known that hydrogen can ignite spontaneously when discharged from a cylinder in this way. Reference 1. Proc. Analyt. Div. Chem. SOC., 1977, 14, 57. W. H. Passway Technical Services Department, Glaxo Labovatories Limited, Cobden Street, Montrose, Angus, DDlO 8EB

ISSN:0306-1396    

DOI:10.1039/AD9771400350

出版商:RSC    

年代:1977

数据来源: RSC

摘要:

352 CONFERENCES AND MEETINGS Proc. AnaZyt. Div. Chem. SOC. Publications Received Annual Reports on Analytical Atomic Spectroscopy, Reviewing 2976. Volume 6. Edited by C. W. Fuller. Pp. viii + 382. London : The Chemical Society. 1977. Price i l 8 ; $36 (CS Members i13.60). Advances in X-Ray Analysis. Volume 20. Edited by Howard F. McMurdie, Charles S. Barrett, John B. Kewkirk and Clayton 0.Iiuud. Proceedings of the Twenty-Fifth ,4 nnual Confev- ence o n Applications of X - R a y Analysis held in Denver, August 4-6, 1976. Pp. xviii + 604. New York and London: Plenum Press. 1977. Price $51. Modern Physics in Chemistry. Volume 1 . Edited by E. Fluck and V. I. Goldanskii. Pp. xiv + 406. London, New York and San Francisco: Academic Press. 1976. Price i18.50; $36.10. Cane Sugar Handbook. Tenth Edition. (The late) George P. Meade and James C. 1’. Chen. Pp. xxii + 947. New York, London, Sydney and Toronto: John Wiley. 1977. Price k35; $59.

ISSN:0306-1396    

DOI:10.1039/AD977140352c

出版商:RSC    

年代:1977

数据来源: RSC