ISSN:0003-2654    

DOI:10.1039/AN95176FX045

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176BX047

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176FP105

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

THE ANALYST xiIMPERIAL COLLEGE OF SCIENCEAND TECHNOLOGYSOUTH KENSINGTON,Lomom-, s.w.7PHYSICS DEPARTMENT(Technical Optics Section)A COURSE OF EIGHT LECTURESbYonM I C R O S C O P Ywill be given onTuesdays and Thursdays at 4 p.m., commencing onTuesday, 15th January, 1952The lectures, which will be accompanied by practicaldemonstrations will include modem advanced methods inmicroscopy. ?hey will be suitable for those having to usethe microscope in technical practice.B. K. JOHNSON, D.I.C.,Application for admission should be made to the Registrarof the Imperial College, prince Consort Road, S.W.7. Thefee is €2 2s. Od. for the Lectures. Students of the College andInter-Collegiate students will be admitted free (on productionof an Inter-Collegiate ticket).ANALYTICAL CHEMISTS.Three required with BSc.,A.R.I.C. or equivalent qualifications for MetallurgicalAnalysis in Laboratories to be erected in Birmingham.The positions are permanent and pensionable, applicantsshould not be over 36 years of age. State age, qualificationsand experience to Personnel Manager, Henry Wiggin &Co., Ltd., Wiggin Street, Birmingham, 16.VACANCY exists for a male chemist for analysis of castAiron and related materials. Age preferably 20 to 30.Previous analytical experience of ferrous alloys an advantage.Applications should be sent to British Cast Iron ResearchAssociation, Bordesley Hall, Alvechurch, Birmingham.ANALYTICAL CHEMIST required to control AnalyticalLaboratory of Industrial Research Organisation in NorthKent, BSc.or A.R.I.C., age about 30. Candidate should beinterested in analytical work, conversant with modemtechniques and able to train and supervise junior staff. Postis permanent and pensionable. Basic commencing salary€500 to €600 per annum, depending on qualifications. WriteRox No. 3796, THE ANALYST, 47, Gresham Street, London,E.C.2.N EXPERIENCED ANALYTICAL CHEMIST with adegree will accept a post with a public analyst, whereWrite Box No. 3795, THE future partnership is possible.ANALYST, 47, Gresham Street, London, E.C.2.XPERIENCED ANALYST required to take charge ofEAnalytical Department in research and control unitApplicant to sess qualifications in chemistry of B.Sc. 0;A.R.I.C. s t a n a , with pharmaceutical qualifications and/orexperience in the analysis of pharmaceuticals and finechemicals. A five-day week and a pension scheme are inoperation.Apply stating full details of age, qualificationsand experience to the Pharmaceutical Development Director,Evans Medical Supplies Ltd., Speke, Liverpool, 19.NALYST May & Raker Ltd Dagenham Essex have aAvacancy h their Analytical ‘bntrol Ladorat06 for anassistant holding either the BSc., A.R.I.C., B.Pharm., orPh.C. qualification. Salary according to age, qualificationsand experience. Contributory Pension Scheme. Five-dayweek. Apply initially in writing to the Personnel Officer,quoting referen6e No. 8616.SSISTANT CHEMIST required for the anal sis of copperAand nickel base alloys, stainless steel.$nowledge ofspectrographic analysis an advantage. Salary according toage, quaIifications and experience. Applications, PersonnelDepartment, Langley Alloys Ltd., Burks.UALIFIED ANALYST required for new laboratory at 0 Loughton Essex. General analytical experience necessaryand acquaintance with organic intermediates. An adequatesalarywill be paid to a suitable Applicant. Details to WorksController, Ozalid Company Limited, 19, Queensway, Enfield,Middlesex.NATIONAL COAL BOARD-NORTH-WESTERNDIVISIONPPLICATIONS are invited from Graduates with a F k tSecond Class Honours Degree in Chemistry for the postof Scientist, Grade 111, at the Central Laboratory, ShadeHouse, Swinton. The salary will be in the scale L445 x L25to 5845, and the commencing salary will be commensuratewith qualifications and experience.The successtul applicantwill be expected to undergo a medical examination forentrance into the Board’s Superannuation Scheme. Applica-tions, giving full details of age, experience and qualifications,should be submitted to the Divisional Establishment Officer,National Coal Board North-Western Division 40 PortlandStreet, Manchester, 1: within fourteen days of h i s advertise-ment.NATIONAL COAL BOARD-NORTH-WESTERNDIVISIONPPLICATIONS are invited for the post of Scientist,AGrade I1 in the No 4 (Burnlev) Area Laboratory atAltham Cokk Works, Ahham, Acc;ington. This appoint-ment will be within the scale 6645 x &40-&995, and thecommencing salary will be according to qualifications andexpenence. Candidates should possess an Honours Degreein Science or the equivalent, and have had several years’industrial experience. The successful applicant will beexpected to undergo a medical examination for entrance intothe Board’s Superannuation Scheme. Applications givingfull particulars of age, education, qualifications and experience(with dates) should be sent to the Divisional EstablishmentOfficer, National Coal Board, North-Western Division, 40Portland Street, Manchester, 1, within fourteen days of thedate of this advertisement.THERE are thirty - sixtypes of JOHNSONtest books and indicatorpapers available to chem-ists, analysts and worksmanagers for testing thepH value of products inthe course of manufacture.LITMUS (PURE GRAN)can also besupplied inany quantityJOHNSONS of HENDON LTDLONDON, N.W.4

ISSN:0003-2654    

DOI:10.1039/AN95176BP113

出版商:RSC    

年代:1951

数据来源: RSC

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DECEMBER, 1951 THE ANALYST Vol. 76, No. 909 PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS AN Ordinary Meeting of the Society was held at 7 p.m. on Wednesday, October 3rd, 1951, in the Meeting Room of the Chemical Society, Burlington House, London, W.l. The Chair was taken by the President, Dr. J. R. Nicholls, C.B.E., F.R.I.C. The following papers were presented and discussed : “A Critical Investigation of the Use of the Silver Reductor in the Micro-Volumetric Determination of Iron, especially in Silicate Rocks,” by Christina C. Miller, Ph.D., DSc., F.R.S.E., F.H.-W.C., and Robert A. Chalmers, B.Sc.; “A Technique to Improve the Efficiency of Desiccators,” by J. King, O.B.E., F.R.I.C. ; “Controlled Potential Electrolysis in the Analysis of Copper-Base Alloys,” by G.W. C. Milner, B.Sc., F.R.I.C., A.Inst.P., and R. N. Whittem, B.Sc., A.R.I.C. NEW MEMBERS Houston Adam Anderson, A.R.I.C., A.I.M. ; John Banks, A.H.-W.C., A.R.I.C., M.1nst.F. ; Arthur Bilsborough; Andrew Carey, Ph.C., M.P.S. ; Alan John Durre, M.Sc. (Melbourne) ; Leslie Norman Harper; Cyril David Hough, B.Sc. (Lond.), A.R.I.C.; Alan Macey, B.Sc., Ph.D. (Birm.), A.R.I.C. ; George Edward Maxwell, A.R.I.C. ; Dennis Francis Pinnington; Geoffrey Irving Smales, B.Sc. (Lond.), A.R.I.C. DEATHS WE regret to record the deaths of Frederick Charles John Bird Richard Victor Briggs Henry Edward Cox Fred Beresford Richardson Charles Reginald Wilkins. A MEETING Of the Grow MICROCHEMISTRY GROUP was held iointlv with the LiverDool and North-Western Section of the Royal Institute 0; Chemistry ’on Thursday, OctoberL18th, 1951, in Liverpool. Some mem’tters of the North of England Section also participated.During the afternoon visits were made to the works and laboratories of Lever Brothers {Port Sunlight) Ltd., and J. Bibby & Sons Ltd., Liverpool. In the evening a meeting was held in the Chemistry Lecture Theatre of the University of Liverpool. The following papers on Chemical Microscopy were presented and discussed : “Some of the Principles of Quantitative Microscopical Analysis,” by ,J. G. A. Griffiths, B.A., Ph.D., F.R.I.C. ; “Some New and Simple Techniques for the Application of Fluorescence Microscopy,” by J. King, O.B.E., F.R.I.C. ; “Applications of Polarisation Microscopy in Chemical Practice,’’ by N. H. Hartshorne, M.C., M.Sc., Ph.D., F.R.I.C.About 200 members and guests were present. PHYSICAL METHODS GROUP THE Thirty-third Ordinary Meeting of the Group was held at 7 p.m. on Friday, October 19th, 1951, at the William Newton School, Norton. This was a joint meeting with the Tees-side Section of the Royal Institute of Chemistry and was preceded by a visit to the new Research Lahoratory of British Titan Products Co. Ltd., at Billingham. The meeting was opened by Mr. Newton Wilson, who invited Mr. B. S. Cooper to take the Chair. Fifty-two members and visitors were present. 679680 OBITUARY [Vol. 76 The following papers on Physical Methods in the Pigment Industry were presented and discussed: “Physical Methods in the Titanium Pigment Industry,” by F. R. Williams, Ph.D., F.R.I.C.; “Rapid Methods of Quantitative Analysis of Rutile - Anatase Mixtures,” by W. Hughes, Ph.D., and H. Smith, M.Sc.; “Surface Area Measurements by the High Vacuum Nitrogen Adsorption Method,” by C. T. Morley Smith, B.Sc., F.R.I.C. BIOLOGICAL METHODS GROUP A MEETING of the Group was held at the Medical Society of London, Chandos Street, London, W.l, on Friday and Saturday, October 26th and 27th, 1951. The meeting was in the form of “A Symposium on the Evaluation of Chemotherapeutic Substances,” and the Chair was occupied by Dr. G. M. Findlay. ?he first part of the meeting was held in the afternoon and evening of Friday, October 26th, and began a t 2.30 p.m., with a break for tea. After the Chairman’s Introduction the meeting was devoted to “Antibiotics, Antituberculous and Antiviral Substances,’’ and the following papers were presented and discussed : “The Design of Antibiotic Assays,” by Dr.W. L. M. Perry; “Evaluation of the Biological Properties of Newly-Isolated Anti- biotics,” by Dr. J. Ungar; “Factors Determining the Character of Inhibition Zones,” by Dr. J. H. Humphrey; “Evaluation of Antituberculous Compounds in vivo,” by Dr. J. M. Robson ; “Practical Aspects of the Routine ’Testing of Antituberculous Compounds,” by Dr. A. R. Martin; “Evaluation of Antiviral Compounds,” by Dr. L. Dickinson. The second part of the meeting was held. on the morning of October 27th and began at 10 a.m. The meeting was devoted to “Antiprotozoal Substances,’’ and the following papers were presented and discussed : “The Evaluation of Amoebicidal Substances in vivo,” followed by a film, “The Chemotherapy of Experimental Amoebiasis,” by Dr. L. G. Goodwin; “Routine Testing of Amoebicidal and Leishmanicidal Agents,” by Dr. J. D. Fulton ; “The Evaluation of Chemotherapeutic Agents directed against Trypanosome Infections,” by Dr. E. M. Lourie; “The Evaluation of Antimalarial Substances,’’ by Dr. D. G. Davey; “Problems of Drug Resistance, with Special Reference to Malaria,” by Dr. Ann Bishop. The Chairman’s Summary followed. About 75 members and guests were present,

ISSN:0003-2654    

DOI:10.1039/AN9517600679

出版商:RSC    

年代:1951

数据来源: RSC

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680 OBITUARY [Vol. 76 Obituary THOMAS HAROLD FAIRBROTHER THOMAS HAROLD FAIRBROTHER, who held the position of Joint Managing Director of McDougalls Ltd., died on August 4th, 1951, the day before his 58th birthday. He had been in ill-health for some months, but had continued to perform actively his responsible work until shortly before his death. Fairbrother was educated at Wigan Grammar School and Manchester University, where he studied under Rutherford and Weizmann. He had a brilliant career at college and gained the Leblanc Medal. During the 1914-18 war he obtained a commission in the Army, but relinquished it to work on explosives under Professor H. B. Dixon, and later became manager of H.M. factory at Lytham, making picric acid and T.N.T. In 1918 he joined Levinsteins and, when that firm became incorporated in British Dyestuffs Corporation, he was made head of the Fine Chemical Department.In March, 1927, he joined the well-known milling firm, McDougalls, as the Chief Chemist, and for his excellent work in this new field in manu- facturing self-raising flour he was promoted to the Board in 1937 and became Joint Managing Director in 1946. He thus joined the ranks of the relatively few chemists who rose from the position of chemist to that of one of the principals of the firm, and of a firm that was not primarily a chemical firm. His rise to this position was not only due to his outstanding grasp of the technicalities of his work, but to his judgment, wise counsel and personality. To chemists his name will always be associated with his work on evaporative loss from flour in storage, to which his paper on “The Influence of Environment on the Moisture Content of Flour and Wheat” (Cereal Chem., 1929, 6 , 379) was an important contribution; but his published work shows that his interests were wide, covering the relationship between antiseptic actions and chemical constitutions in synthetic aniline dyestuffs ( J .Path. Bact., 1922, 145), the etiology and treatment of diabetes (Brit. Med. J., April 29th, 1922), and the preparations of certain substituted ureas and their use in the treatment of trypanosomiasis ( J . Path.Dec., 19511 j PICKFORD 681 B a d , 1925, 515). He was a prolific and forcible writer on many cereal and nutritional problems in publications dealing with food. Fairbrother was an outstanding member of the Chemical Club, occupying the positions of President, Chairman of the Executive Committee and Honorary Treasurer. He nearly always attended meetings of our Society when papers were read on cereal problems and when he spoke he always had something constructive and interesting to say. He married in 1920 a cousin of Ernest Melling, Muriel Peirpoint Melling, who survives him. He had no children. To his fellow cereal chemists his passing is a blow, and he will be remembered for long by his wide circle of friends in the profession. Although he attained considerable advance- ment, he remained unaffected, was always cheery and was always affectionately known as Tommy Fairbrother. D. W. KENT- JONES

ISSN:0003-2654    

DOI:10.1039/AN9517600680

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Dec., 19511 j PICKFORD 681 Hypophysectomy in the Preparation of Mammals for the Bio-Assay of Adrenocorticotrophic Hormone Activity BY M. PICKFORD This paper formed the basis of an introductory address to four papers* o n the Biological Assay of Adrenocovticotrophic and Cortical Hormones @resented at the meetings of the Biological Methods Group on Tuesday, October 24th and Tuesday, December 19th, 1950. HYPOPHYSECTOMY is now frequently performed on a wide variety of vertebrate species as a preliminary to investigating the activities of other endocrines, and it is particularly needed before the bio-assay of adrenocorticotrophic hormones (ACTH). Removal of the pituitary is necessary because, in this instance, the endogenous production of ACTH interferes with the action of the ACTH injected for assay.For reasons of convenience, rats are frequently the animal of choice in large-scale assay work. They can be obtained easily, they are not large and a number can be kept in a small space, and they are remarkably resistant to infections, so that, provided reasonable cleanliness prevails at the operation, it is certain that no sepsis will result. This saves all the trouble involved in aseptic techniques. When performed by an expert the operation of hypophysectomy of rats appears easy. In fact it is not so, both innate skill and experience being necessary for its successful performance. The method generally used for hypophysectomising rats is in essence that described by Smith in 1927 and by Richter and Wislocki in 1930 and uses the parapharyngeal approach.The anaesthetic of choice is ether, despite the resulting free formation of fluid and mucus in the respiratory tract. Rats need large doses of atropine for even some control of this secretion and it is necessary to use suction as well t o keep the airways clear. Suction is also used for removal of the gland. It is possible to choose a suction tube whose tip is of such a size that the gland blocks the end, is held there and can be picked out and laid aside for examination later. There is little danger of injury to the hypothalamus because the rat, like man, has a diaphragm of dura across the sella between the pituitary and the base of the brain. It is advisable to keep the rats in a cage warmed to 28" C for some hours post-operatively, and the animals should never again be allowed to get cold.In expert hands mortality from hypophysectomy is nil and haemorrhage minimal. It takes 4 to 15 minutes to complete the operation without assistance. Hypophysectomised rats survive for long periods, although on the whole they do not live as long as normal ones. The two chief precautions needed to ensure survival are warmth, as they are unable to adapt well to low temperatures, and an always-available supply of food to reduce the risk of hypoglycaemia. A number of species may be hypophysectomised by a similar parapharyngeal route, including mice, ferrets, hedgehogs, guinea pigs and cats. With cats some workers prefer * Analyst, 1951, 76, 461-481.682 PICKFORD [Vol. 76 to use the buccal route and, in any event, the technique must be accompanied by thorough aseptic precautions, as cats tend to develop pneumonia. In certain species the buccal route is safer and easier than the parapharyngeal, for example, rabbits, fowls and dogs.From the first two the gland can be removed by suction, but for the last dissection is necessary. For total hypophysectomy in the dog it is not enough to remove all visible fragments of the gland, as the pars tuberalis runs up the stalk and spreads fanwise below the hypothalamus. The only certain way of destroying this part is by careful use of the thermocautery. Some injury to the hypothalamus cannot be avoided, but unless the pars tuberalis is destroyed the results of the operation are uncertain. A small viable portion of any part of the anterior lobe is enough to maintain the adrenals and the dog in almost normal condition.This must mean that the remaining fragment of gland is making an appreciable amount of ACTH. There is no diaphragm across the sella in the dog, so that some escape of cerebrospinal fluid is inevitable, but this seems to be of no moment. At the end of the operation plugging is unnecessary nor need the soft palate be stitched.. Infection is never seen unless the operator has been careless about asepsis. It is pleasing and surprising how little shock follows the buccal removal of the gland. The post-operative requirements are warmth, ample food and glucose added to the milk. The dog is generally well for the first one or two days, then less well and quieter. Later its condition again improves, but it is never as vigorous as before operation.The advantage of this method is that the stalk of the pituitary and the base of the hypothalamus can be seen. The disadvantages are that part of the zygorna has to be removed, the temporal muscle reflected, a large aperture made in the skull and the temporal lobes of the cerebrum retracted. This method, therefore, is followed by considerable operative shock and discomfort for the animal and needs far greater care as to asepsis. Before attempting hypophysectomy on any species it is advisable, and certainly helpful, to watch the performance of the operation by an expert. In dogs and monkeys the transtemporal route can be used. BIBLIOGRAPHY Allan, H., and Wiles, P., (Cats) J . Physiol., 1932, 75, 23. Dott, N. M., (Dogs) Quart. J . Exp. Physiol., 1923, 13, 241. Fisher, C., Ingram, W. R., and Ranson, S. W., (Cats, Monkeys) “Diabetes Insipidus,” Edwards Hill, M., and Parkes, A. S., (Ferrets, Hedgehogs, Guinea Pigs and Fowls) Proc. Roy. Soc. B, 1932, McPhail, M. K., (Cats) Ibid., 1935, 117, 45. Newton, W. H., (Rabbits) Endocrinology, 1939, 24, 468. Richter, C. P., and Wislocki, G. B., (Rats) A n w . J . Physiol., 1930, 95, 481. Smith, P., (Rats) J . Amer. Med. Ass., 1927, 58, 168. Sweet, J. E., (Dogs) Ann. Surgery, l.935, 102, 1069. Thompson, K. W. , (Rats) Endocrinology, 1932, 16, 257. White, E. W., (Rabbits) Proc. Roy. Soc. B, 1933, 114, 64. Bros. Inc., Ann Arbor, 1938. 112, 138. PHYSIOLOGY DEPARTMENT EDINBURGH UNIVERSITY

ISSN:0003-2654    

DOI:10.1039/AN9517600681

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Dec., 19511 BUSH AND HIGGS 683 The Estimation of Boron in Boronised Metals BY G. H. BUSH AKD D. G. HIGGS (Presented at the meeting of the Society on Wednesday, May 2nd, 1951) The more general methods for the separation of boron from associated elements, and for its estimation, are briefly considered. Work carried out in adapting Tschischewski’s method to the estimation of boron in boronised iron, nickel, cobalt and copper is described, in which these metals are removed by mercury cathode electrolysis. In addition, a rapid method for the estimation of boron in boronised molybdenum is described in which the metal is separated as the sulphide. The boron after conversion to mannitoboric acid is finally determined by potentiometric titration with sodium hydroxide, which obviates the difficulties inherent in the use of colour-change indicators.The methods as described have an accuracy of about 1.4 per cent. of the amount of boron present over the range 0.27 to 4.30 per cent., which may be extended with slight modification. IN an investigation into the coating of iron, nickel and molybdenum with boron it was found that no approved method for the estimation of boron was available. The boronised samples were believed to contain amounts of boron varying between 1 and 10 per cent. Of the published methods for the separation of boron from associated elements that proposed by Kollock and Schmidt, referred to by Tschischewskil but without a detailed reference, appeared to be suitable to the problem in hand. Tschischewski separated boron from iron and nickel by mercury cathode electrolysis and this principle has now been extended to cover also the separation from cobalt and copper by the modification described below.In addition, an independent method has been devised for the separation of boron from molybdenum, for which the mercury cathode electrolysis is ineffective; in this the molybdenum is separated as sulphide. The object of this paper is not to discuss at length the various methods for the separation and estimation of boron, but it is appropriate to consider briefly the possible methods. Separation of boron by distillation as methyl borate-The distillation as methyl borate is perhaps the best known method for the separation of the element and it has been suggested as a method of separation from iron in It is however a tedious procedure and unsuited to the work under consideration. Separation by precipitatiotn of the associated elements--Wherry3 separated boron from iron by precipitation of the latter with calcium carbonate, and.other methods4 are described whereby iron and aluminium are precipitated with sodium hydroxide between pH 6 and pH 7.5. The large volume of the precipitates involved, with the danger of occlusion of the boron, renders these methods of doubtful value. Separation by means of mercury cathode electrolysis-The method of Kollock and Schmidt referred to by Tschischewski for the separation of boron from iron and nickel has the advantage that precipitation is avoided, the volume of solution required is small and the number of manipulative operations is reduced to a minimum.Estimations of boron-The hydrolysis of the distilled methyl borate with calcium oxide and the determination of the increase in weight due to the formation of calcium borate has been described by Gooch5 and later modified by Gooch and Jonesg The method, although capable of giving good results, is tedious. Colorimetric methods making use of curcumin, h ydroxyquinones, p-nitrobenzene-azo- chromotropic acid,7 carminic acid, etc., are mainly of use in the detection and estimation of boron in very small quantities, whilst methods proposed by Klinger,8 by Evans,g who proposed the estimation of boron in a boron alloy by the iodine liberated from potassium iodate in sulphuric acid solution, and by Glaze and Finn10 are considered less suited to the present investigation than the method to be described.684 BUSH AND HIGGS: THE ESTIMATION OF BORON PRELIMINARY EXPERIMENTAL WORK [Vol.76 ESTIMATIOK OF BOKOX IN BOKONISED IRON, NICKEL, COPPER AND COBALT- Electrolysis-An illustration of the cell used by Tschischewski is to be found in his published work, but the cell illustrated in Fig. 1 was considered to be more satisfactory. This piece of apparatus is a water-jacketed variety of the Cain cell containing an internal syphon that is closed, when the cell is held vertically, by the mercury level inside. By tilting the cell the liquid may be syphoned away through the tap in the stem. This avoids the use of an external syphon to remove the contents of the cell and facilitates washing. The cell is fitted with a piece of nickel - iron wire fused into the glass to make contact with the 50 ml of mercury used as the negative electrode.The anode is a cylinder of platinum foil capable of being rotated within the cell at about 100r.p.m. It was found that, with the exception of iron and molybdenum, all the metals deposit easily over a mercury cathode in a relatively wide range of acidity with a current of 4 amperes from a 12-volt supply. Iron, however, must be in nearly neutral solution before complete removal can be effected, and Fig. 2. Typical curve showing end-point of titration of boric acid with sodium hydroxide in presence of mannitol it is necessary to add small quantities of alkali, at frequent intervals, to neutralise the acid liberated during electrolysis.The temperature of the solution during electrolysis in an air- cooled cell may reach 65" C, but it has been found that this does not result in any appreciable loss of boron. Molybdenum cannot be deposited quantitatively at the mercury cathode. An alternative method of separation is described later in this paper. Titration of boric acid-Early titrations were carried out with p-nitrophenol as the indicator for the first stage of the titration and phenolphthalein as end-point indicator for the alkali titration of the monohydric acid formed by the addition of mannitol. It is assumed that the titration is stoicheiometric and that- The titration of boric acid with these indicators presented difficulty. The colour change was not always sharp and this could lead to appreciable personal error.The operation could be carried out with greater ease and accuracy by titrating the boric acid solution by means of a calomel - glass electrode system and a Cambridge pH meter. A boron solution was prepared by dissolving pure recrystallised borax in distilled water, free from carbon dioxide, and diluting to 1 litre in a calibrated flask to make a solution containing 0-001082 g of boron per ml. Aliquot portions of this solution were titrated with 0.10 N sulphuric acid by means of a calomel - glass electrode system and a Cambridge pH 1 ml of 0.1 N sodium hydroxide = 0.001082 g of boron.Fig. 1. Electrolytic cellDec. , 19511 I N BORONISED METALS 685 meter, Readings of pH against acid additions were recorded throughout the titration and the end-point was determined by plotting the differential and obtaining the point of inflection of the curve.This titration was used to standardist: the sulphuric acid. The pH value of the solution was then adjusted to that of the end-point, 5 g of mannitol were added and the solution titrated with 0.1 N sodium hydroxide, free from carbon dioxide, and readings of pH against alkali additions were recorded as before. The end-point was determined by noting the point of inflection of the curve. The amount of sodium hydroxide used is that required to neutralise the mannito-boric acid. A curve obtained for the end-point of this titration is shown in Fig. 2. A titration of the standardised sulphuric acid against sodium hydroxide solution was carried out by the potentiometric method and the concentration of the alkali found. The - r\ ' \ # \ I \ d \ l b - - f \ Titre equivalent to boron - I I I content of metal - '\ , -- \ .- - - , , I \ - 'I /- .d I' f ; i v0 -0 /* /* i /* i ,,I' &. ---- 1.0 0.8 0.6 0.4 D I 0.2 - - u - 2 amount of sodium hydroxide of this concentration required for the titration of the mannito- boric acid corresponded to a boron content of 0.02708 g, compared with the amount added, 0.02705 g . To apply the principle to the titration of the boric acid in the solution after removal of the associated metals by electrolysis, the pH at the beginning of the titration should be that at which the free mineral acid has been neutralised, which is determined by adjusting the solution to about pH 4.0, then titrating with sodium hydroxide and recording pH values against alkali additions.. The point at which the free mineral acid is neutralised is obtained by noting the point of inflection of the curve. It is unnecessary to adjust the pH of the solution to that of the end-point before adding mannitol and continuing the titration as described earlier. The standard sodium hydroxide corresponding to the boron present is that required to titrate the solution between the points of inflection of the two curves. A typical titration is shown in Fig. 3. Efect of glassuare-In the course of preliminary work a Pyrex beaker was inadvertently used for reducing the volume of the solution under mildly alkaline conditions and a serious error was found in the subsequent results. Boron added = 0.02040 g., Boron found = 0.02275 g.Boron added = 0.02754 g., Boron found = 0.02894 g. It is recommended that only boron-free glassware should be used. On the other hand, Pyrex and other boro-silicate glassware is reasonably resistant to acid attack and as none686 BUSH AND HIGGS: THE ESTIMATION OF BORON [Vol. 76 of the operations to be described requires the use of hot alkaline solutions, there is much less serious objection to its use. Under the conditions to be described no appreciable extrac- tion of boron takes place. Efwt of residual elements remaining after electrolysis-The presence of metals incompletely removed by electrolysis, e.g., iron, copper, nickel, cobalt, chromium and manganese, or not removed at all, e g . , aluminium, molybdenum, titanium and vanadium, may have serious effects on the titration of boric acid with sodium hydroxide; this depends on the pH range at which the corresponding metallic hydroxide - borate is formed.Residual manganese not deposited at the anode or cathode, but remaining in the solution as permanganic acid, can be removed by discharging the pink colour with sulphurous acid and boiling the slightly acid solution with potassium persulphate under a reflux condenser, when manganese will be precipitated as the dioxide. If the amount of manganese is small it is unnecessary to remove it from the solution, otherwise it may be removed by passing the cooled solution through a pulp filter. In alloys containing large amounts of aluminium the method described is inapplicable and an alternative separation is now being worked out.Blank due to reagents-The titration of the borax solution described earlier was conducted without dilution and when the same titration was carried out in a volume of 400m1, the sodium hydroxide required for neutralisation was 0.30 ml more. This amount represents the excess alkali required to change the pH of the increased volume of solution from one end of the titration range to the other. It is therefore necessary to perform a blank determination if the final volume of the solution is as much as 400ml. The blank appears to be due to (a) the volume of solution and @)-the reagents used, with possible boron pick-up from glassware. That due to (a) appears to be far the greater. The blank is determined by taking an amount of boron-free metal equal to that of the sample and treating it as follows.The acidity of the final solution is adjusted to about pH 4-0 and the solution titrated with 0.1 N sodium hydroxide to a pH slightly higher than the point of neutralisation of free acid in the sample, recording pH against titre throughout. Five grams of mannitol are then added and the titration continued to a pH just higher than that obtained for the end-point of the sample, recording pH against titre as before. In the absence of boron in the blank no depression of pH will occur on the addition of mannitol and the curve will be continuous. The amount of alkali required to titrate the blank solution over the pH range of the sample titration can be interpolated from a graphical representation of the results. The curve obtained before the addition of mannitol is used for the commencement of the titration and the curve after the mannitol addition for the end-point of the titration.In the present work the value of the blank was found to be 0.25 to 0.35 ml of 0-1 N sodium hydroxide and appeared to be independent of the amount and nature of the metal sample used. REAGENTS- METHOD All the reagents used complied with recognised analytical standards. Sulphuric acid (1 + 3)-Add 1 part of sulphuric acid to 3 parts of cold distilled water. Sulphuric acid, 0.1 N-Dilute 2.7 ml of concentrated sulphuric acid to 1 litre with distilled water, free from carbon dioxide. Standardise the solution against pure Na,B,O,. 10H,O. Sodium hydroxide, 0.1 N-Dissolve 4.00 g of sodium hydroxide in 1 litre of distilled water, free from carbon dioxide.Add 10ml of 10 per cent. barium hydroxide solution to the solution before making up to volume, and fit the bottle with a carbon dioxide trap. Standardise the solution against the standardised 0.1 N sulphuric acid reagent (above) or directly against standard borax solution as described earlier. Sodium hydroxide-A 20 per cent. solution. Sodium hydroxide-A 1 per cent. w/v solution in carbon dioxide-free water, to which add 10 ml of 10 per cent. barium hydroxide solution before making up to volume. Fit the container with a carbon dioxide trap. Sulphuric acid, 1 per cent .-Dissolve approximately 5.5 ml of concentrated sulphuric acid in 1 litre of distilled water, free from carbon dioxide.Dec., 19511 IN BORONISED METALS Hydrogen peroxide, 100-volume.Mercury-Pure redistilled metal. Mannitol-Pure reagent. 687 Distilled uater, free from carbon dioxide-Boil distilled water vigorously for 30 minutes and cool. Fit a carbon dioxide trap to the container. APPARATUS- Rejux unit-A 400-ml Erlenmeyer flask fitted to a straight tube water-cooled condenser was used in this work. Electrolytic cell-The modified Cain cell described earlier and illustrated in Fig. 1 was used. Titration unit-This consisted of a Cambridge pH meter, used in conjunction \yith a calomel positive electrode and a glass negative electrode suitable for use with solutions of pH 8 to 9. DETERMINATION OF BORON IN BORONISED IRON, NICKEL, COPPER AND COBALT- Weigh an appropriate amount of sample (not exceeding 2 g for iron samples) preferably in a fine state of division, into a 400-ml conical flask, attach the flask to the lower end of the water-cooled condenser and carefully add down the condenser column, 20 ml of diluted sulphuric acid (1 + 3) followed by 10 ml of 100-volume hydrogen peroxide, a little at a time.As the reaction may become violent, care must be taken in attacking boronised iron samples by this method. When the initial reaction has subsided, boil gently to complete the solution of the alloy and decompose the residual peroxide. The initial reaction with other alloys is not violent and heat may be applied immediately after the addition of the solvent. More peroxide may be added from time to time should the reaction become sluggish.When the sample is completely dissolved, should any residue remain, remove it by passing the cooled solution through a small filter, wash with distilled water and gently ignite the paper and contents in a platinum dish at a temperature just sufficient to destroy the organic matter. Fuse the residue with the minimum quantity of fusion mixture and dissolve out the melt in the original sulphuric acid solution. Cool the solution thoroughly and transfer it to any convenient electrolytic apparatus containing 50ml of mercury, or to the special cell described above and shown in Fig. 1. Reduce the acidity by adding 20 per cent. sodium hydroxide solution until the precipitate formed just re-dissolves. Electrolyse the solution with a current of 4 amperes from a 12-volt supply, until all trace of initial colour has disappeared.Frequent additions of sodium hydroxide will be necessary to neutralise the acid formed, more particularly when dealing with iron, otherwise complete deposition will not be effected. Continue to pass the current for 30 minutes after the solution has become colourless, or until a drop removed from the solution gives no reaction with the appropriate reagents. When electrolysis is complete, syphon off the electrolyte into a 1-litre conical flask and wash out the cell thoroughly with about 150 ml of distilled water, added in 30-mi quantities, so that the resultant volume is approximately 350ml. Add 1 per cent. sodium hydroxide solution until the electrolyte shows only a slight acid reaction to litmus paper, reconnect the flask to the reflux unit previously used and boil gently for 10 to 15 minutes to expel .all carbon dioxide, and then cool thoroughly. Transfer the liquid to a 600-ml squat-form beaker marked at a volume of 400 ml, adjust the volume up to the mark with distilled water, free from carbon dioxide, and measure the pH of the solution.Adjust the acidity to about pH 4-0 and then titrate with 0.1 N sodium hydroxide solution, recording the pH value against the titre throughout. Determine the point of neutralisation of the free mineral acid by plotting the differential and noting the point of inflection of the curve. Add 5 g of mannitol and continue the titration, recording pH against sodium hydroxide addition as previously. The end-point is at the point of inflection of the curve.The amount of 0.1 N sodium hydroxide solution required to titrate the test solution between the point of inflection of the two curves is equivalent to the amount of boron present. A blank on a sample of boron-free metal should be run under the conditions described.688 BUSH AND HIGGS: THE ESTIMATION OF BORON [Vol. 76 CALCULATION- [(mi of std. NaOH x factor of 0.1 N soln.) - ml of blank] x 0.001082 x 100 Weight of sample Boron, yo = REsurrs By the method described, the figures for recovery of known amounts of boron added as boric acid to iron, nickel, copper and cobalt were found and are shown in Tables I to IV. TABLE I RECOVERY OF ADDED .BORON FROM IRON Weight of iron taken, g 1.0 1.0 1.0 2-0 2.0 2.0 Weight of nickel taken, g 1.0 2.0 2.0 2.0 2.0 2-0 Weight of copper taken, g 1.0 1-0 1.0 2.0 2.0 2.0 Weight of cobalt taken, g 1.0 1.0 1.0 2-0 2.0 2.0 Boron added, g 0.00541 0.02705 0.03787 0.00541 0-01082 0.01623 Titration minus Blank 0-10N NaOH, ml 5.10 25.13 34.79 4.96 10.18 14.75 Boron Found, Added, 0.00552 0.541 0.02719 2.705 0.03764 3.787 0.00537 0-27 1 0.01 101 0.541 0.01596 0.812 A g % TABLE I1 RECOVERY OF ADDED BORON FROM NICKEL Boron added, g 0.03246 0.00541 0.01082 0.01623 0-02164 0.03030 Titration minus Blank.0.10 N NaOH, ml 29-70 5.08 10.36 15.64 19-84 27.87 Boron r Found, g 0.03214 0.00550 0*01121 0.01 692 0.02147 0.03016 Added, 3.246 0-27 1 0.541 0-812 1-082 1.515 % TABLE I11 RECOVERY OF ADDED BORON FROM COPPER 7 Found, Yo 0.552 2.719 3.764 0.269 0.551 0.798 7 Found, 3.214 0.275 0.561 0-846 1.074 1.608 Y O Boron added, g 0.01082 0.02164 0.03246 0.00541 0.0081 2 0-0 1623 fitration minus Blank.0.10N NaOH, ml 10.11 20.18 29.73 5.06 7.60 15.00 7 Found, g 0.0 1094 0.02 183 0.03217 0.00547 0.00822 0.01 623 Boron Added, Found, 1.082 1 -094 2.1 64 2.183 3.246 3-21 7 0.27 1 0.274 0.406 0.41 1 0.811 0.81 1 - % Yo TABLE I:V RECOVERY OF ADDED BORON FROM COBALT Boron added, g 0,01623 0.02 164 0.03030 0-00541 0.01 082 0.01623 Titration minus 0.10 N NaOH, ml 14-90 20.30 27.80 4.95 10.14 14.79 . Blank. f Boron Found, g 0.01612 0.02196 0.03008 0.00536 0.01097 0.01600 Added, 1.623 2-164 3-030 0.27 1 0.54 1 0.812 YO -b Found, Yo 1.612 2-1 96 3.008 0.268 0.549 0.800Dec., 19511 IN BORONISED METALS 689 Tables I to IV show that good agreement exists between the amount of boron added and that found, a mean value for the error over all the results being about 1-3 per cent.of the amount of boron present. The method described presents a fairly rapid and accurate method for the evaluation of the boron content of metals containing this element in amount between 0.27 and 3-76 per cent. There would, however, appear to be no reason why, with slight modifications, it should not be satisfactory for boron contents well outside this range. DETERMINATION OF BORON IN BORONISED MOLYBDENUM As molybdenum cannot be completely deposited on a mercury cathode, it was necessary to examine the precipitation methods for its separation. In addition to the occlusion of boron in bulky metal precipitates, the objection to this type of separation is the possibility of co-precipitation of metal borate taking place when the precipitation is made in alkaline solution.Precipitation in acid solution is not subject to this objection. The possibility was examined of precipitating molybdenum as sulphide and determining the boron, after elimination of the hydrogen sulphide, by titration with standard sodium hydroxide, as described earlier in this paper. The main problems investigated were solution of the sample, completeness of precipitation of molybdenum as sulphide and the blank due to the reagents used. Sohtion of the sample-The samples of boronised molybdenum received could be dissolved practically completely in aqua regia, any slight residue could be filtered, the paper and contents gently ashed at a temperature just sufficient to destroy the organic matter, the residue fused with a little fusion mixture and the melt leached out with dilute sulphuric acid.The solution so obtained could then be added to the original solution before proceeding with the separation. PRECIPITATION OF MOLYBDENUM AS SULPHIDE- Molybdenum can only with difficulty be completely precipitated by hydrogen sulphide in acid solution and it is necessary, after saturation with the gas, that the solution should be warmed under pressure. If, however, a solution of molybdenum in sodium hydroxide solution is saturated with hydrogen sulphide and then acidified with dilute sulphuric acid and heated, molybdenum trisulphide is completely precipitated in a flocculent form that settles readily. The red colour developed by the hydrogen sulpbide in alkaline solution is due to the formation of the thiomolybdate.The clear supernatant liquid from this separation was tested and found to be free from molybdenum. The thiomolybdate decomposes with sulphuric acid according to the reaction- Na2MoS4 + H2S04 = h’a,S04 + MoS, + H2S THE REAGENT BLANK- The remarks on this subject in the earlier part of this paper apply equally to this procedure and it is necessary to make a blank determination under the conditions to be described, carrying out the final titration as described earlier. METHOD REAGENTS- The following reagents are required in addition to those listed on p. 686. Bromine water-A saturated aqueous solution. Hydrogen sulphide-This should be washed by passing the gas successively through 10 per cent.hydrochloric acid and water. PROCEDURE FOR BORONISED MOLYBDENUM- Weigh 2 g of sample into a 400-ml conical flask, attach the flask to the lower end of a water-cooled condenser and carefully add 20 ml of aqua regia down the condenser column, a little at a time. When the initial reaction subsides, heat the flask and contents to effect complete solution, adding a little extra nitric acid from time to time if necessary. Should a small residue be left after the acid attack, cool the flask, dilute with 100 ml of distilled water and filter off the residue through a small pulp filter. Wash with warm distilled[Vol. 76 water and ignite gently in a platinum dish at a temperature just sufficient to destroy the organic matter. Fuse the residue with a small quantity of fusion mixture, dissolve the melt in a little dilute sulphuric acid and add it to the solution from the original acid attack.Neutralise the solution (or the combined solutions, when a fusion has been necessary) with 20 per cent. sodium hydroxide solution, adding 5 ml in excess and then saturate, in the cold, with hydrogen sulphide. When the solution has absorbed sufficient hydrogen sulphide it assumes a reddish colour owing to the formation of thiomolybdate. Neutralise the solution (in a fume cupboard) by the careful addition of diluted sulphuric acid (1 + 3). If the neutralisation is effected too rapidly, loss of sample may result from the rapid evolution of hydrogen sulphide. Add 5 ml of diluted sulphuric acid (1 + 3) in excess, fit the flask to the reflux condenser (see p.687) and bring the contents to boiling-point. Cool thoroughly, make up the contents of the flask to 500ml in a calibrated flask, return the contents to a 500-ml conical flask and leave the precipitate to settle. Decant 250ml of the supernatant liquid through a No. 41 Whatman filter-paper into a 250-ml calibrated flask. Transfer to a l-litre conical flask and add bromine water until a faint yellow colour persists. Connect to the reflux condenser and boil until the solution is colourless, to destroy any residual hydrogen sulphide and to expel bromine and carbon dioxide. Cool and transfer the solution to a 600-ml squat beaker marked at 400m1, measure the pH of the solution and adjust the acidity to about pH 4.0 by the addition of alkali, free from carbon dioxide.Dilute to 400ml with distilled water, free from carbon dioxide, and carry out the titration as described in the method for boron in iron, nickel, copper and cobalt. A blank on a sample of boron-free metal should be run under the conditions described. 690 BUSH AND HIGGS: THE ESTIMATION OF BORON CALCULATION- [(ml of std. NaOH x factor of 0.1 N soln.) - blank] x 2 x 0.001082 x 100 Weight of sample Boron, yo = RESULTS The results obtained using molybdenum with varying additions of boron as boric acid to the solution are shown in Table V. TABLE V RECOVERY OF ADDED BORON FROM MOLYBDENUM Weight of molybdenum taken, g 1.0 1.0 1.0 2.0 2.0 2.0 Boron added, g 0-00541 0.02164 0-04328 0.0054 1 0.00757 0.01623 Titration minus 0-10 N NaOH, ml 2.55 10.00 19.89 2.54 3.49 7.23 Blank.r ~~ Found, g 0.00552 0.02 164 0.04304 0.00550 0.00755 0.01564 Boron Added, 0.541 2- 164 4.328 0.27 1 0.379 0.812 - Yo v Found, % 0.551 2- 164 4.304 0.275 0.378 0*782* * The precipitation of molybdenum was incomplete in this experiment and a double treatment was necessary, hence slight loss of boron. The figures in Table V show good agreement between the amounts of boron added and those found and indicate that the analytical process described provides a satisfactory method for the determination of boron in boronised molybdenum. The method can be used without modification in the presence of small amounts of group I1 A metals, but in the presence of iron, nickel, cobalt and manganese it is necessary to remove these metals by mercury cathode electrolysis after elimination of the molybdenum and before titration.Thanks are due to the Director General of Scientific Research for permission to publish this paper.Dec., 19511 IN BORONISED METALS 691 1. 2. 3. 4. 5. 6. 7 8. 9. 10. REFERENCES Tschischewski, X., Ind. Eng. Chem., 1926, 18, 607. Lundell, G. E. F., Hoffman, J. I., and Bright, H. A., “Chemical Analysis of Iron and Steel,” Wherry, E. T., and Chapin, W. H., J . Amer. Chem. SOC., 1908, 30, 1687. Scott, W. W., and Furman, N. H., “Standard Methods of Chemical Analysis,” Fifth Edition, Volume 1, D. Van Nostrand Co., New York, and The Technical Press Ltd., London, 1939, p. 177. John Wiley & Sons Inc., New York, 1946, p. 390. Gooch, F. A., Anzer. Chem. J., 1887, 9, 23. Gooch, F. A., and Jones, L.C., “Methods in Chemical Analysis,” First Edition, John Wiley & Komarowski, A S , and Poluekstoff, N. S., MzRrochem., 1933-34, 14, 317. Klinger, P., quoted by Lundell, G. E. F., Hoffman, D. I., and Bright, H. A., “Chemical Analysis Evans, B. S., unpublished work. Glaze, F. W., and Finn, A. N., J . Res. Vat. Bur. Stand., 1941, 27, 33. Sons Inc., New York, 1912, p. 204. of Iron and Steel,” John Wiley & Sons Inc., New York, 1946, p. 396. MINISTRY OF SUPPLY ARMAMENT RESEARCH ESTABLISHMENT FORT HALSTEAD, SEVENOAKS, KENT DISCUSSION MR. G. R. BALL asked what was the useful working range of the method. He pointed out that the constant pH method of titration, in which the same pH was used for both end-points, could probably be conveniently applied to this method.This technique automatically eliminated interference from most radicals except phosphate and germanium. MR. BUSH replied that the method was not suited to the estimation of small amounts of boron in steel, but amounts of the order of 0.1 per cent. of boron could be determined readily with a titration figure about five times that of the blank. The higher range was limited by the initial weight of sample and possibly by the necessity for slight modification of the solution technique. He did not see any reason. why individual analysts should not use the method of fixed end-points if they wished. The procedure described was more fundamental and was preferred for that reason. MR. C. WHALLEY said that during the war he had had to make many analyses of boron, both in steels and in de-oxidising alloys of high silicon content.The boron content of the alloys was of the order of 2 to 3 per cent. and of the steels of the order of 0.001 per cent. To determine the lower concentrations of boron he had used the mercury-cathode separation followed by an absorptiometric measurement making use of quinalizarin. He had found no evidence of the loss of boron during the mercury-cathode separation, which conclusion the authors confirmed. He had checked his procedure from amorphous boron as distinct from solutions of boric acid and had found satisfactory results. For the de-oxidising alloys a sodium peroxide fusion had been followed by distillation of the boron as methyl borate and subsequent hydrolysis and titration. He felt that for these alloys the distillation pro- cedure was superior to the mercury-cathode separation owing to the complex nature of the materials.He asked the authors if they had had experience of the procedure of adding the mannitol in small increments during the titration rather than all a t once a t the beginning and he wondered if the results obtained by the two procedures were comparable. MR. BUSH agreed that the method used by Mr. Whalley for determining low boron contents in steel was probably one of the best for the purpose and was glad to hear that his results confirmed the authors’ experience that no appreciable loss of boron took place during mercury-cathode electrolysis. Before the introduction of the water-cooled cell, the temperature of the electrolyte often reached 60” to 70” C, but the results obtained were within the limits of experimental error. MR. HIGGS stated that during initial experiments he added mannitol in small portions, but later found that results were the same whether additions were made gradually or all at one time, providing the total addition was sufficient for the complete titration of the boric acid. Good quality mannitol was not found to give any appreciable acid or alkaline reaction. Were the boron content of the sample to exceed the limits quoted in the paper, i t might be necessary to increase the amount of mannitol used. MR. E. C. MILLS asked how far the results obtained would be representative of the whole consignment from which the sample was taken, considering the heterogeneous nature of the wire samples analysed, and also what degree of accuracy could be expected. MH. HIGGS replied that the question of a representative sample did not arise in connection with the work undertaken, since the whole sample, which weighed between 1 and 2 g, was used. If large supplies were available, the question of heterogeneity would have to be considered and would entail analyses of samples a t intervals along the specimen.

ISSN:0003-2654    

DOI:10.1039/AN9517600683

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

692 HART: THE LEAD CONTENT OF WHEATEN FLOUR [Vol. 76 The Lead Content of Wheaten Flour and its Determination BY H. V. HART A method for the estimation of lead in flour and other wheat products is described. The method involves dry ashing of the sample, preliminary separation of lead as the diethyldithiocarbainate complex and its determination absorptiometrically by the mixed colour technique. Destruction of organic matter by dry ashing under controlled conditions has been investigated and shown not to result in any significant loss of lead from flour. Quantities of lead of less than 1 p.p.m. were estimated with good duplica- tion of results. In the samples of flour examined, the normal range of lead was from 0.03 to 0.20p.p.m. (average 0.10). TRACES of lead in wheat and wheaten products hiave been reported by a number of investi- gators;lJJ the work described in this paper was undertaken to obtain figures for the lead content of commercially milled National flour.The amount of lead was not expected to exceed 1 p.p.m., and in the choice of a method of estimation dithizone offered much greater sensitivity than the sulphide procedure. The accurate application of the former method, however, is subject to certain precautions (Clifford and Wichmann4) and the method used in the final estimation of the lead was the mixed colour method of the Association of Official Agricultural Chemists’ “Methods of Analysis,” Sixth Edition, 1945, with certain adjustments of volume to give maximum sensitivity. A Committee of the Society of Public Analysts and Other Analytical Chemists has also been considering the same problem and details of a method they are examining (not yet published) were made available to us.As shown later, the two methods, which in fact follow similar lines, agree well. DESTRUCTION OF OKGANIC MATTER- Wet oxidation involves less risk of loss of lead, but it was found that the process resulted in the extraction of small but variable amounts of lead from the glass, which was a hard borosilicate type. Further, the blank on the reagents formed a large proportion of the total lead because of the limitation of the amount of sample taken. Dry ashing is generally considered to involve some loss of lead, and it was therefore necessary to carry out determinations of lead added to flour (as acetate or nitrate) in order to examine this point.Certain of the results are shown in Table I. Ashing was carried out in silica dishes, overnight, in a muffle a t a temperature not exceeding 500” C. For this range of lead, the recoveries were satisfactory and dry ashing was therefore adopted. Organic matter can be destroyed either by wet oxidation or by dry ashing. TABLE I EFFECT OF DRY ASHING ON LEAD CONTENT Initial lead content of flour, Added lead, p.p.m. p.p.m. 0.01 0.20 0.01 0.40 0.16 0.20 0.16 0.40 0.16 0.60 0.16 0.80 0.07 0.60 0.07 1.00 Total lead found, p.p.m. 0.19 0.44 0.35 0.53 0.74 0.95 0.67 1.06 Recovery, p.p.m. 0-18 0.43 0.19 0.37 0.58 0.79 0.60 0.99 Recovery, 90 108 95 93 97 99 100 99 Yo SEPARATION OF DIETHYLDITHIOCARBAMATE COMPLEX- In order to avoid precipitation due to the presence of calcium and magnesium phos- phates when the ash solution was made alkaline, a preliminary separation of lead and otherDec., 19511 AND ITS DETERMINATION 693 metals as the diethyldithiocarbamates was made from a slightly acid solution (Tompsetts).A chloroform solution of diethylammonium diethyldithiocarbamate was used (Strafford, Wyatt and Kershaw6) and the lead was readily extracted at a pH between 3 and 4. The subsequent wet oxidation of the carbamate residue was rapid and did not involve any measurable contamination from the glass. Filtration-Filtration at any stage of the estimation was avoided when it was found that the last traces of metals were not removed from filter-paper by dilute nitric acid or by a chloroform solution of dithizone.Silica-Solution of the ash in dilute hydrochloric acid usually resulted in a slightly opalescent liquid, probably because of the presence of traces of silica. By taking to dryness with hydrochloric acid and dehydrating at 100" C, the insoluble material became aggregated and the supernatant liquid cleared. Such treatment, however, always involved loss of lead, no doubt by adsorption on the silica. Recovery of this lead would involve treatment of the insoluble material with hydroffuoric acid. As the recovery of added lead was satisfactory, however, it was decided to ignore the slight opalescence of the ash solution. Bismuth-The method does not distinguish lead from bismuth, which forms an orange- coloured dithizonate, but the latter may be separated with dithizone at pH 3 (Sandell'). Several separations were carried out on the samples, but no evidence of interference by bismuth was found.An alternative method of separation is to extract the bismuth from a hydrochloric acid solution, which must be at least 2 N , with diethylammonium diethyldithiocarbamate solution (Strafford et al).6 METHOD REAGENTS- Nitric acid-AnalaR grade acid redistilled in an all-glass Pyrex still. Hydrochloric acid, diluted (1 + 1)-Mix equal volumes of AnalaR grade acid, sp.gr. 1.18, Kedistil in an all-glass Pyrex still and adjust the distillate to 5 iV. Sulphuric acid-AnalaR grade acid selected for its low lead content. Ammonium hydroxide, sp.gr. 0-880-We have found the AnalaR grade satisfactory. Ammonium citrate solution, 50 per cePzt.-Dissolve 500 g of citric acid in 300 ml of distilled water and carefully add about 450 ml of ammonium hydroxide, sp.gr.0-880, until the solution is neutral to thymol blue. Extract with successive quantities of dithizone in chloro- form until all traces of metal complexes are removed and the last extract is green. Finally decolorise by extraction with pure chloroform (Blaxter and Allcrofts) . Chloroform-Redistil from calcium hydroxide. Diethylammonium diethyldithiocarbamate solution-Prepared as described by Strafford Stock solzttion-Dilute 3.0 ml of redistilled diethylamine to 10.0 ml with chloroform and add slowly, with stirring, to 1.0 ml of redistilled carbon disulphide, previously diluted to 10.0 ml with chloroform. Cool and preserve in a dark-coloured glass-stoppered bottle.Extraction reagent-Dilute 5.0 ml of stock solution to 100 ml with chloroform. Prepare the stock solution once a week and the dilute solution daily. Dithizone-The commercial reagent was purified by the following method. and water. Filter. et aL6- Dissolve about 1 g in 100 ml of chloroform and filter off any insoluble material. Extract with four 100-ml portions of dilute ammonium hydroxide (1 ml of ammonium hydroxide, spgr. 0.880, added to 99 ml of water). Combine these extracts and precipitate the dithizone by adding a slight excess of sulphurous acid, containing about 5 per cent. of sulphur dioxide, as recom- mended by B a r n e ~ . ~ Extract the dithizone with two or three 20-ml portions of chloroform. Wash the combined extracts twice with water, and evaporate the chloroform gently.Dry the residue in a desiccator. Prepare a 0.1 per cent. stock solution in chloroform, and store away from light. Standard dithizone solzttion-Dilute the 0.1 per cent. stock solution to give a solution containing 4 mg per litre in chloroform. Nitric acid, 1 per cent.-As A.O.A.C. Methods of Analysis.lo Dilute 10 ml of fresh water- white nitric acid, sp.gr. 1.40, to 1 litre with distilled water. Boil off nitrous fumes from the re-distilled acid before making the dilution. To 100 ml of a 10 per cent. solution of recrystallised phosphate-free potassium cyanide in a 500-ml volumetric Store in a cool place away from light. Ammonia - cyanide mixture-As A.O.A.C. Methods of Analysis.1°694 HART: THE LEAD CONTENT OF WHEATEN FLOUR [Vol.76 flask add sufficient ammonium hydroxide to introduce 19.1 g of NH, and make to volume with distilled water. Standard lead solution-Prepare from AnalaR lead nitrate a solution containing 0.1 mg of lead per ml in 1 per cent. nitric acid. Potassium cyanide solution, 10 per cent.-Prepare a 50 per cent. w/v solution in water and extract lead with small portions of dilute dithizone in chloroform. Finally extract the excess of dithizone with chloroform and dilute the solution to 10 per cent. (Sandell,' p. 395). APPARATUS- Wherever possible apparatus should be made of Pyrex glass. Apparatus must be carefully cleaned with nitric acid; particular attention should be paid Reagents should be stored in Pyrex bottles. All apparatus should be kept for lead The first few results should be viewed with caution, in case lead has not been removed to ground-in stoppers and taps.estimation only. completely from the apparatus. PROCEDURE- Ash 25 g of flour in a 3-inch silica basin overnight (16 hours) in a muffle at a temperature of 450" to 500" C. If the residue is still black, :moisten it with the minimum of nitric acid, dry it at the entrance to the muffle and then complete the ashing in the interior for a few minutes. Add 5 ml of 5 N hydrochloric acid and 26 ml of water, and allow to simmer gently for 30 minutes. Cool the solution and transfer it to a separating funnel with water to bring the total volume to about 40ml. Add ammonium citrate solution to bring the pH between 3 and 4 (full yellow colour to thymol blue, olive to bromophenol blue).Extract with three 5-ml portions of diethylammonium diethyldithiocarbamate solution in chloroform. Run the extracts into a Pyrex micro-Kjeldahl flask, add 2 ml of diluted sulphuric acid (1 + 1) and evaporate the chloroform on a water-bath. Continue heating over a small flame and swirl the tube to dissolve all the residue until sulphur trioxide fumes are evolved. Continue heating to oxidise organic matter and drive off the excess of nitric acid. Cool slightly, add approximately 1 ml of water and evaporate to fuming again. Cool, dissolve the residue in a little water and then dilute to 15 ml. Add 2 ml of ammonium citrate solution and make alkaline to the blue colour of thymol blue with ammonium hydroxide, sp.gr. 0.880. Cool and add 1 ml of potassium cyanide solution.Transfer to a separating funnel and wash in with 5 to 10ml of water. Extract with 10-ml portions of a 10 mg per litre solution of dithizone in chloroform, shaking for about 1 minute. Two portions are sufficient if an excess of dithizone is present in the first extract. Run the chloroform layers carefully into a separating funnel. Shake the combined chloroform extracts with 15 ml of 1 per cent. nitric acid for 1 minute. Transfer the aqueous layer without any of the chloroform into a graduated flask (25 ml for each 10 pg of lead). Extract again with 10 ml of nitric acid. The first extract removes practically all the lead, the second acts as a washing. Measure 25 ml into a separating funnel, add 5 ml of ammonia - cyanide solution and 12.5 ml of standard dithizone solution from a burette.Dry the stem of the separator with filter-paper and run the chloroform layer carefully into a dry &inch test tube. Allow to stand for a few minutes, when any traces of water will collect on the side of the tube. Slight haziness of the solution may be removed by gentle warrning. The chloroform should show a mixed colour indicating a slight excess of dithizone. Measure the absorption with a Spekker absorptiometer at 510 mp in a 2-cm cell, using Ilford 604 filters and comparing with pure chloroform. The volume of standard dithizone used must be sufficient to fill the 2-cm cell and may be different from the 12.5 ml used above. Should the final colour be red only, indicating insufficient dithizone, then the nitric acid solution should be diluted and a fresh 25 ml treated as above.If all the solution has been used, then all the lead must be extracted again with the 10 mg per litre solution of dithizone in chloroform and the analysis repeated from that stage. Cool slightly and add 2 drops of nitric acid. Dilute to the mark with 1 per cent. nitric acid. Shake for 1 minute. This volume should be kept to a minimum.Dec., 19511 AND ITS DETERMINATION 696 Blank determinations must be made at the same time as each set of determinations, exactly the same procedure and quantities of reagents being used. The blanks under our conditions were normally from 0-3 to 0.5 pg of lead. Prepare a standard curve for the range 0 to 10 pg of lead by diluting appropriate quanti- ties of standard lead solution to 25 ml with 1 per cent.nitric acid, saturated with chloroform. Add 5 ml of ammonia - cyanide solution, 12.5 ml of standard dithizone solution and shake for 1 minute. The standard dithizone solution retains its strength fairly satisfactorily if stored in a Pyrex bottle in a dark place, but the standard curve should be checked periodically at a few points. RESULTS Measure the absorption of the solution as in the estimation. Results of estimations of lead on 24 commercially milled National flours of 81 per cent. extraction by this method and by the Society’s method are shown in Table 11. Sample C58 60 61 63 64 65 66 68 69 70 71 73 75 76 78 79 81 83 84 85 87 88 90 92 TABLE I1 ESTIMATION OF LEAD IN FLOUR Estimate by method described, p.p.m. 0.03, 0.04, 0.06 0.09, 0.10, 0.04, 0.06. 0.07 0.16, 0-20 0.14, 0.13 0.18, 0-24, 0.33, 0.32 0.12, 0.06, 0.08 0.07, 0.08 0-07, 0.06 0.14, 0.14 0.06, 0.08 0.10, 0.06 0.08, 0.19, 0.14 0.20, 0.20 0-06, 0.08 0.18, 0.18 0.10, 0.12 0.18, 0.15 0.08, 0.06 0.04, 0.02 0.08, 0.07 0.09, 0.08 0.08, 0-05 0.03, 0.06 Average 0.04 0.08 0.07 0.1 8 0.14 0.27 0.09 0.08 0.07 0.14 0.07 0.08 0.14 0.20 0.07 0.18 0.11 0.17 0.07 0.03 0.08 0.09 0.07 0.05 Estimate by Society’s method, p.p.m.0-04, 0.06 0-04, 0.07 0-06, 0.04 0-16, 0.20 0-22, 0.13 0.21, 0-30 0-06, 0.09 0-05, 0.06 0.08, 0.08 0-15, 0.14 0.08, 0.08 0.08, 0.07 0.13, 0.13 0-23 0-06, 0.12 0-16 0.10, 0.16 0-17, 0-20 0.03, 0.05 0.03, 0.04 0-04, 0-07 - - 0.04, 0.03 Average 0.05 0.06 0.05 0.18 0.18 0.26 0.08 0.06 0.08 0.15 0-08 0.08 0.13 0.23 0.09 0.16 0.13 0.19 0.04 0.04 0.06 - - 0.04 By the method described, 23 of the 24 samples are estimated to contain lead in the range 0.03 to 0-20 p.p.m., with an average of 0.10 p.p.m.The other sample (C 65), averaging 0.27 p.p.m., appeared to be unusual. Twenty-two of the samples were examined by the Society’s method and, again excluding sample C 65, the range of results is 0.04 to 0.23 p.p.m., the average being 0.10 p.p.m. Destruc- tion of organic material in this method was by ashing in the presence of magnesium nitrate, and the method was modified slightly for greater sensitivity. A t present calcium carbonate (Creta praeparata) is added to flour at the rate of 14 02. per 280 lb. of flour. Determinations on 8 different samples of commercial Creta praeparata indicated a lead content of 1 to 4 p.p.m.The B.P. limit for this substance is, however, 20 p.p.m., at which level it would account for 0-06 p.p.m. of lead in the flour. My thanks are due to Mr. C. L. Hinton (British Food Manufacturers’ Research Associa- tion) and Dr. H. H. Green (Ministry of Agriculture and Fisheries) for their advice in the early stages of this work. I am also indebted to Mr. G. Taylor, lately President of the Society of Public Analysts and Other Analytical Chemists, for kindly allowing me to see details of the method developed by the Society.696 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. HARRISON, LEES AND WOOD: THE ASSAY REFERENCES [Vol. 76 Kent, N. L., J . SOC. Chenz. Ind., 1942, 61, 183. Kehoe, R. A., Cholak, J., and Story, R. V., J . Nutr., 1940, 19, 588; 590. Monier-Williams, G. W., “Lead in Food,” Reports on Public Health and Medical Subjects, No. 88, Clifford, P. A., and Wichmann, H. J., J . Ass. 08. Agric. C h e w , 1936, 19, 130. Tompsett, S. L., Biochem. J., 1939, 33, 1231. Strafford, N., Wyatt, P. F., and Kershaw, F. G., Analyst, 1945, 70, 232. Sandell, E. B., “Colorimetric Determination of Traces of Metals,” Interscience Publishers Inc., Blaxter, I<. L., and Allcroft, R., Veterinary Laboratory, Ministry of Agriculture, Weybridge. Barnes, H., Analyst, 1947, 72, 469. Association of Official Agricultural Chemists, “Methods of Analysis,” Sixth Edition, 1945, p. 456. 1938. New York, 1950, p. 398. Private communication. RESEARCH ASSOCIATION OF BRITISH FLOUR-MILLERS CEREALS RESEARCH STATION ST. ALBANS, HERTS. June, 1961

ISSN:0003-2654    

DOI:10.1039/AN9517600692

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

696 HARRISON, LEES AND WOOD: THE ASSAY [Vol. 76 The Assay of Vitamin B12 Part VI Microbiological Estimation with a Mutant of Escherichia coZi by the Plate Method BY ELEANOR HARRISON, K. A. LEES AND F. WOOD A method for the microbiological cup-plate assay of vitamin B,, with a mutant of Escherichia coli as test organism is presented. The assay is an advance on other methods previously published as Parts I11 and IV of this series because (a) a simple chemically defined medium is used, (b) changes in the EH of the test medium have little effect on the response, (c) the zones of exhibition are reproducible and well defined in character and (d) the degree of specificity and general freedom from interfering effects and inexplicable variations is compatible with a large output of assays on a routine basis.The inoculum consists of a small volume of a culture grown overnight in peptone water, and the assay plates ma-y be incubated at any temperature between 27" and 37°C. Zones of exhibition can be obtained with solutions containing one milli- microgram of vitamin B,, per ml. The dose response line is rectilinear over the range 0.005 to 5.0 pg per ml and hence a (2 + 2) assay design can normally be used. The effects of times of incubation and of standing before incubation have been investigated. A streptomycin-resistant strain of the test organism has been developed for the purpose of assaying directly the vitamin B,, content of Streptomyces griseus fermentation samples. Thymidine does not interfere with the assay. Standard errors of 0.14 to 0.23mm per zone are obtained, 0.18mm being the normal error encountered in assays on Petri dishes or large glass plates.METHODS for the microbiological assay of vitamin B,, in tubes or on plates with strains of These micro-aerophilic bacteria respond uniformly to graded doses of vitamin B!, within a narrow EH range only, so that variations in oxygen tension of test media profoundly influence the growth of the test organism in the presence of sub-maximal quantities of the vitamin. In view of these difficulties, a plate assay with L. Zeichmannii 313 as test organism was developed (unpublished), but it suffered from the disadvantage that certain of the vitamin B,, group of factors proved difficult to assay; in particular vitamin B12c containing the -NO, grouping gave poorly defined zones.Lactobacillus Zeichmannii or L. Zactis Dorner as test organisms have been published. 1 7 2 9 3 9 4,6,6,7Dec., 19511 OF VITAMIN BIZ, PART VI 697 Normally it is not practicable in this country to use wholly synthetic media and the consequent need for some complex constituent, such as casein hydrolysate, adds to the difficulties of preparing reproducible standard media. The receipt of a mutant strain of Escherichia coZi responsive to vitamin B,, and methionine encouraged us to determine its nutritional requirements and the conditions under which it would yield zones of exhibition in an agar medium. E. coZi strains are not as a rule nutrition- ally exigent and are classed as aerobic in their oxygen requirements; this suggested that it should be possible to devise for the assay a simple medium in which the organism would not be sensitive to small changes of oxidation - reduction potential.Preliminary work indicated that inocula grown in peptone water yielded better zones than those grown in synthetic media and that inocula grown under conditions of aerobiosis, i.e., shaken, were more vigorous than those grown in static culture. Initial work was per- formed with a washed inoculum, but later it was found that an unwashed inoculum gave excellent assay plates with no perceptible growth over the background of the plate. Zones of exhibition were obtained on assay plates by using a previously described synthetic medium* enriched with a trace element solution first described by Beadle and T a t ~ m .~ Preliminary details of the assay method have already been published.lO The test media used in these lactobacillus assays are complex and expensive. ASSAY METHOD TEST ORGANISM- A mutant of Escherichia coli, kindly supplied by Dr. Bernard Davis of New York, was used. This organism appeared as Gram negative rods measuring about 2 p by 0.5 p when grown on the synthetic agar slant medium detailed below. A culture of the mutant strain has been deposited with the National Collection of Industrial Bacteria (Chemical Research Laboratory, Teddington, Middlesex), whence sub-cultures can be obtained. For the purpose of determining the vitamin B,, content of StqXomyces gviseus fermenta- tion liquors containing both streptomycin and vitamin B,,, we have developed a streptomycin- resistant strain of the test organism.Our first attempt to induce resistance in E . coZi was not successful. It happened that a vitamin B,, “non-requiring” variant was produced. This proved difficult to separate from the parent culture. Further attempts were made to induce resistance by steps of increasing dosage, the organism being plated out after each transfer. On checking the purity of an isolate by using the colony as a parent inoculum for a few assay plates, increased resistance was recorded, until finally a strain resistant to 20,000 units of streptomycin per ml was obtained. Consequently, growth in the inoculum medium and within the zones on the assay plates is slightly less dense than that of the parent strain, which we prefer to use for the assay of samples that do not contain streptomycin. This resistant variant grew rather less vigorously than the parent mutant. MAINTENANCE OF CULTURE- the composition shown in Table I.Stock cultures of the mutant strain are maintained on a synthetic agar slant medium of TABLE I SYNTHETIC AGAR SLAKT MEDIUM Acid hydrolysed casein (Allen and Hanbury) . . .. 6.0 g Di-potassium hydrogen phosphate . . .. .. . . 0.2 g . . .. . . 5mg Ferrous sulphate (7H,O) . . .. Magnesium sulphate (7H,O) . . . . . . .. .. 0.2 g L-Asparagine . . . . . . . . . . .. . . 0.15 g Distilled water to . . .. .. .. . . 700ml Dissolve the constituents with gentle heating in the order shown in Table I, with a few drops of hydrochloric acid to dissolve the asparagine, and adjust the pH of the solution to 7.2 After cooling add 2.0 g of glycerol and 20.0 g of agar (Davis Gelatine Ltd.).Make up the volume to 1 litre with distilled water and steam to dissolve the 0.1; boil and filter.698 HARRISON, LEES AND WOOD: THE ASSAY [Vol. 76 agar, re-adjust the pH to 7.2 0.1 and add 400 pg of crystalline vitamin B1, (or its equiv- alent). After thorough mixing, dispense the medium in 10-ml amounts in 6-inch by $-inch resistance glass tubes, then plug and autoclave them for 15 minutes at 15 lb pressure. After sterilisation, “slope” the tubes and store them subsequently at 4” C. The master culture is stored at 4” C; from this, sub-master cultures are prepared weekly. Transfers are made daily into peptone water inoculum medium (see below) and incubated a t 37” C for twenty-four hours.INOCULUM MEDIUM- Dissolve 20 g of peptone (Evans Medical Supplies Ltd.) and 5 g of sodium chloride in distilled water and make up to 2 litres. Adjust the pH to 7.2 & 0.1 and dispense the medium into flasks of such dimensions that 100 ml in each occupy a depth of between three and five millimetres.* Many samples of peptone have proved unsuita.ble, but Evans and Difco brands both give satisfactory growth. As SAY MEDI u M- Autoclave the medium for 20 minutes at 15 lb pressure. Prepare the stock solutions shown in Table 11. TABLE I:[ ASSAY MEDIUM Ammonium chloride . .) . . .. .. Ammonium nitrate . . .. .. .. Distilled water to . . .. .. .. Stock solution A- Stock solution B- Potassium dihydrogen phosphate . . . . Di-potassium hydrogen phosphate .. . . Distilled water to . . .. . . . . Stock solution C- Sodium borate . . .. .. . . . . Copper sulphate (5HzO) . . . . . . Ferrous sulphate (7HzO) . . . . .. Manganese chloride (4H20) . . .. .. Zinc sulphate (7H,O) . . .. . . . . Ammonium molybdate .. I . . . Distilled water to . . .. .. . . .. .. .. . . . . . . . . . . . . . . . . . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. 200 g 80 g 2 litres 40 g 120 g 2 litres 0-094 g 0.250 g 0.540 g 0.460 g 4.900 g 1 litre 0.020 g Add 36 per cent. WIW hydrochloric acid dropwise until the solution is clear (approximately 0.5 ml is required) From these stock solutions, which can be stored in a refrigerator, the agar plate medium is prepared in the following manner. Mix in the order given- Stock solution A .. .. .. .. . ... Stock solution R .. . . . . .. .. I . Stock solution C .. .. .. .. .. . . 5 per cent. w/v magnesium sulphate (7HzO) . . * . Asparagine . . . . .. . . .. . . .. 0.1 per cent. calcium chloride . . . . . . .. Adjust the pH to . . .. . . .. . . . . Distilled water to . . .. .. .. . . .. 250 ml 250 ml 0.5 ml 10.0 ml 5.0 ml 4.6 litres 7.2 2 0.1 7.5 g Dispense 120-ml volumes into 250-ml conical flasks ;%rid add 2 g of agar (Davis) to each. Plug the flasks and autoclave them at 15 lb pressure for 15 minutes. The sterile medium is stored at 4” C and has a storage life of four to six weeks. USE OF ASSAY MEDIUM- When required for use, the flasks are steamed for thirty to forty minutes to melt the The glucose is sterilised separately agar, and then 10 ml of glucose solution are added to each.* Penicillin flasks (“Glaxo” type) are eminently suitable and can be got from Messrs. Townson and Mercer.Dec., 19511 OF VITAMIN Biz. PART VI 699 as a 13 per cent. w/v solution in an autoclave for 15 minutes at 15 lb pressure. medium is allowed to stand in a water-bath a t a temperature of 46" to 48" C. DETERMINATIOS OF INOCULUM VOLUME- The turbidity of the 24-hour inoculum culture in peptone water is determined with a Spekker photo-electric absorptiometer. An inoculum reference graph has been prepared relating the density of inoculum to the volume required to yield zones of the best character and a dose - response slope giving the greatest difference in response for standard dose increments. It is assumed that the variation from day to day in the ratio of viable to total bacterial count is insignificant.The molten PREPARATION OF ASSAY PLATES- The flasks of molten plate medium are inoculated at a temperature not exceeding 48" C and poured into plates immediately. Into each 9-cm Petri dish 12.5 ml of inoculated medium is poured. High temperatures or delay in pouring the plates a t this stage will materially reduce the initial seeding rate, reduce the density of the growth in the zone of exhibition and thus inflate the mean zone size. With the aid of a guide frame and a stainless steel borer of about 8 mm diameter, six cups are punched into each agar plate, the discs of agar being removed by means of a vacuum attachment. REFERENCE STANDARD- We use sealed vials each containing approximately 50 pg of vitamin B,, freeze-dried on about 40 mg of sodium chloride; the exact vitamin-B,, content is stated on the label in terms of anhydrous vitamin B,,, having been determined by ultra-violet spectrophotometric measurement of E:k at 207 mp.The contents of the vial of standard are diluted appropriately with distilled water and stored in the refrigerator away from direct light. The solutions are not kept for more than four days. The storage life of these prepared plates is about three days. PREPARATION OF SAMPLE DILUTIOW- In attempting to evaluate the potency of pure crystalline vitamin B12a, B,,, and BIZd in terms of vitamin B,,, we experienced difficulty through lack of consistency in the assay from day to day and through lack of parallelism between the curves for the crystalline factors on certain days.Consequently accurate evaluation of the potency of the crystalline vitamin B,, factors was not possible, although the results indicated an order of activity very similar to that of vitamin B,, referred to an anhydrous weight basis or the colour of the solution determined on the Spekker photo-electric absorptiometer. Having been informed by Cuthbertsonll that lack of parallelism and inconsistencies in the assay could be eliminated by treating the assay samples with potassium cyanide solu- tion, we now use the following dilution technique. The assay samples are accurately diluted with distilled water to about 20 pg per ml in graduated flasks and to 1 ml of this solution in a graduated flask one drop of 1 per cent. potassium cyanide solution is added, the solution being allowed to stand for fifteen minutes and then further diluted with distilled water as required.Three drops of test or standard solution are now added to the appropriate cup from a pipette in which the dropper has a platinum tube, of 0.0365-inch external and 0.0295-inch internal diameter and about 10 mm long, fused into the tip at an angle of about 130" to take up a position normal to the Petri dish during delivery. The diameter of the zones of exhibi- tion can be measured after 18 hours' incubation, but this time is not critical and little change in zone size or appearance is detectable on further incubation. For the purpose of determining the amount of vitamin B,, in crude sources of the vitamin such as feeding materials, liberation of the combined vitamin can be achieved by steaming the preparation in solution for 30 minutes a t a pH value between 5.0 and 6.0.The liberation is facilitated by the presence of potassium cyanide, as recommended by Wijmenga, Veer and Lens.,, In view of the recent reports13 of factors active in this assay and also for EzgZena gracilis, but not active for L. Zeichmanii 313, it is advisable to include in any preliminary investigations of crude sources of vitamin B,, a qualitative control by paper chromatography to demonstrate the absence of factors other than vitamin B12. It is advisable to use for this700 HARRISON, LEES AND WOOD: THE ASSAY [Vol. 76 chromatography a developing solvent containing a trace of potassium cyanide to ensure that the vitamin B,, is not converted to slower moving factors on the strip, as reported by Woodruff and Foster.14 ASSAY DESIGN- The dose - response curve is rectilinear for doses between 5.0 and 0.005 pg of vitamin B,, per ml applied to the plate.Any of the conventional microbiological plate assay designs can be used and results computed from the mean slopes of standard and sample dilutions by reference to suitably We normally use a (2 + 2) assay with a dilution ratio of 1 to 10. 12 x 12 LATIN SQUARE DESIGN Totals . . H E 13.9 18.7 F I 18.7 19.2 L K 13-7 13.7 B C 19.8 14.0 19.0 13.5 13.8 19.3 E H 19.3 13-7 A F 19-3 19.3 D G 13.4 13.6 I D 18-4 13.7 G A 13.8 18.8 K B 12.9 18.0 76.0 75-5 J L C J G B D 13.2 18.8 14.0 E D G 19.2 13.8 13.8 13.8 19.8 20.0 D I F 13.8 19.7 19.7 K C R 13.7 13.5 10.5 B A L 19.2 19.2 14-0 A K B 19.5 13.9 19.0 19.3 13.4 13.4 H E K 13-6 18.8 13.5 L F J 13.4 19.0 19.0 I L C 18.8 13-3 13-4 F H A 18.0 12.9 18.3 C J I J G H 75.5 76.1 77.6 I L F 19.6 13.7 18.7 H C L 13.7 13.7 13.7 G A D 14.0 19.5 13.6 L G A 13.8 13.7 19.0 A B H 19-5 19.4 13.7 D H K 13.9 13.9 13.9 13.5 19.0 19.0 E K C 19.3 13.6 13.7 19.1 18.8 19.1 K E G 13-4 18-9 14.0 B F E 18.7 18.9 19.1 18.4 13.2 18.4 C I J F J B J B I 76.9 76.3 75.9 C J A K 13.8 19.0 18.7 13.4 A K J B 18.7 13.5 18.7 18.7 B F H E 19.3 19.3 13.7 19.3 K H E J 13.6 13.7 19.0 19.0 F G D I 19.0 13.8 13.7 19.2 E I F G 19.3 19.3 19.5 13.7 D L G F 13.0 13-4 13.5 18.9 I B L D 19.2 19.2 13.7 13-4 L C I A 13.7 13.0 18.9 18-9 H A B C 13.3 18.9 18.8 13.3 J D K H 18.7 13.2 13.0 13.0 G E C L 12.9 18.0 13.0 12.9 74-5 74.3 74.2 73.7 Totals 75.5 75.4 79.7 78.8 77.5 79.0 75.7 76.8 74.4 74.1 72.7 66.9 906.5 1.which prepared tables or nomograms. gives the maximum likelihood solution of any assay. To use the nomogram, abstract from a (2 + 2) assay the values of (RT, - RS,) and (RS, - RT,), where RS,, RS,, RT, and RT, refer to the mean zone sizes of the responses of low and high standard and low and high doses of sample dilution respectively. By joining these two values on the ordinates of the nomo- gram with a perspex ruler, the potency of the sample referred to the highest dose is obtained. This value is then multiplied by the dilution that has been made on the sample. In view of the slope of the dose - response curve (1.2 to 1.7 mm for 100 per cent. increase in dose) we consider it necessary to employ replication on ten plates if the zone diameters are being measured only to the nearest 0.5 mm.We prefer to read the zone diameters to the nearest 0.1 mm with replication on five plates, For this measurement we use slide calipers modified by the addition of needle tips by which zones are measured on the surface of the agar, as shown in Fig. 2. This method of reading avoids errors due to parallax. For the accurate evaluation of special samples or the critical examination of interfering agents on the assay we prefer to use larger assay plates to obtain more precise comparisons. These large assay plates are made from plate glass on the lines previously reported by Brownlee, Loraine and Stephens16 for penicillin and streptomycin assays.The use of these large plates for microbiological assay can only be justified if a statistical design is used to obviate effects similar to those reported by Brownlee et al. We prefer to use the nomogram shown in Fig.RT,- RS , RS,-RT, Fig. I . Nomogram for (2 + 2) assay, 10 to 1 dilutionsRT,- RS , RS,-RT, Fig. I . Nomogram for (2 + 2) assay, 10 to 1 dilutionsDec., 19511 OF VITAMIN BIZ. PART VI 701 The precision of the assay is demonstrated in Tables I11 to V. A 12 x 12 Latin square assay was performed to investigate the effect of the pH and of the cyanide content of sample dilutions on the zone size. Table I11 shows the plan of the assay; the letters indicate the experimental treatment being assayed. The letters were randomised against the treatments as shown in Table IV.The solutions were introduced into the cups in horizontal row order beginning H, E, G, . . . and finishing E, C , L. The use of the design has improved the TABLE IV TREATMENT TABLE All dilutions performed in 0-05 M phosphate buffer Treatment and dose, p g per ml r > A Vitamin B,, KCN pH 5-0 0.1 0.0 0.01 0.0 pH 5.0 0.1 0.3 0-0 1 0.03 pH 5.0 0.1 15.0 0.01 1.5 0.0 1 0.0 pH 8-0 0.1 0.3 0.01 0.03 pH 8.0 0.1 15.0 0.01 1.5 pH 8.0 0.1 0.0 Randomised plating code E C J L B D A G I H Total of twelve responses 228.1 162.5 228.0 162.8 228.0 162.7 228.3 163.4 229.7 162.5 F 228-1 K 162.1 TABLE V ANALYSIS OF VARIANCE Source D.F. Mean square .. 11 0.9960 Rows .. .. .. Columns . . .. .. .. 11 0.1 146 .. .. .. 1 .. .. 1 Level of CN .... .. 1 Effect of CN . . .. .. 1 .. .. 1 Zone size Effect of pH . . .. .. 1 Slope Effect of pH . . 1 Effe'c't of 6N 1 Zone size pH interaction with Slope pH interaction with 1 1 1 Error .. 110 Level of CN 1086-7656 0-0059 0.0460 0-0153 0.0126 0.0201 0.0201 0.00 17 0,0376 0.0767 0.0176 0.0548 Significance highly significant significant highly significant not significant not significant not significant not significant not significant not significant not significant not significant not significant not significant not significant accuracy of the experiment by eliminating significant extraneous variation in the rows and the columns. Hence it can be concluded that the only effect that is significant is the slope of the dose - response line, which demonstrates that neither pH nor cyanide within the ranges examined have any interfering effects in the E .coli assay. The analysis recorded in Table V showed that- The variation in responses within the rows and columns was highly significant. This effect has been eliminated in the analysis. There was no observable difference in zone size or slope of the dose - response line between pH 6.0 and pH 8.0. Potassium cyanide, at either level, had no observable effect on zone size or slope. There were no interactions between cyanide and pH treatments. The standard error of the response zones was 0.23 mm.702 HARRISON, LEES AND WOOD: THE ASSAY [Vol. 76 It must be noted that the tolerance with respect to pH has been demonstrated in the presence of 0-05 M phosphate buffer and no information was obtained as to the pH of the solutions after diffusion into the agar medium.REPRODUCIBILITY OF ASSAYS- The results for several samples, each assayed on two days, are shown in Table VI. Six cups were punched on each plate and the equivalent of two (2 + 2) assays were performed on five plates, i.e., two samples are referred to one set of dilutions of standard. It follows, therefore, that a total of one hundred and thirty plates has been used to assay the twenty-six samples. TABLE 'VI REPRODUCIBILITY OF ASSAYS Sample 1 2 3 4 5 6 7 8 9 10 11 12 13 Day 1 5.6 2-8 20.0 15.5 19.5 17.5 19.5 0.22 2.2 2.3 2-2 1-9 7.8 Day 2 5.6 3.1 2 1.0 17.0 21.0 17.6 18.5 0.18 2.0 2.0 2.2 1.7 7.0 Reported value 5.5 3.0 20.5 16.5 20.0 17.5 19.0 0.20 2.1 2.1 2.2 1.8 7.5 Sample 14 15 16 17 18 19 20 21 22 23 24 25 26 Day 1 6.0 5.2 27.0 27-0 27.0 23.6 21.6 22.6 23.5 4.6 0.25 19.5 1.60 Day 2 5.0 4-7 25.0 26-0 26-5 21.0 18.0 19.5 26.0 4-8 0-2 1 20.8 1.42 Reported value 6.5 5.0 26.0 26.5 27.0 22.0 19.5 21-0 24.0 4.7 0.23 20.0 1-46 THE EFFECT OF PRE-COOLING ON THE DIFFUSION OF VITAMIN BIZ THROUGH THE AGAR- It was thought that, if diffusion of vitamin B,, could be allowed to take place before growth of the test organism, not only would the zone sizes be larger, but that the differences between zone sizes given by graded doses of vitamin B,, might also be increased.In this manner the gradient of the response line would be increased and the assay made more precise. Accordingly an experiment was performed in which the growth of the organism was delayed by refrigeration for various times up to three hours, while graded doses of vitamin B,, were diffusing through the agar.Following this refrigeration the plates were incubated in the normal manner and the resultant zones measured to the nearest 0.1 mm. Five equally spaced times of pre-refrigeration, 0, 45, 90, 135 and 180 minutes, were combined in all possible ways with five logarithmically spaced doses of vitamin B,,, 0.2, 0.1, 0.05, 0.025 and 0.0125 pg per ml. The resulting twenty-five treatments were replicated six times by using in all thirty five-hole plates. In order that the results should be as precise as possible the twenty-five treatments were arranged in an incomplete block design. Table VII shows the analysis of variance of this experiment ; the estimated and calculated regression values are shown in Table VIII.TABLE 'VII ANALYSIS OF VARIANCE Source D.F. Mean square Linear effect of time . . .. .. 1 119.347 Linear effect of dose . . .. .. 1 541.417 Interaction of time and dose.. .. 1 1.980 Deviation . . .. .. .. 21 0.082 Error .. .. .. . . .. 96 0.060 Significance significant highly significant not significant not significant Intra-plate error : + 0-229 Inter-plate error : 5 0.42 1 Error of weighted results : + 0-244 iO.100 Error of estimates (mean of 6) :Dec., 19511 OF VITAMIN Biz. PART VI 703 TABLE VIII ESTIMATED AND CALCULATED REGRESSION VALUES Dose Time 0 45 90 135 0-2 Estimate 25.1 1 24-27 23-33 22-80 Regression 25.09 24.34 23-60 22.85 0.1 Estimate 23.69 22.95 22.28 21-65 Regression 23.63 22.94 22.25 21-57 0.05 Estimate 22.35 21.64 21.00 20.22 Regression 22.17 21.54 20.91 20.28 0.025 Estimate 20.75 19.99 19-50 19.11 Regression 20.71 19.57 19.57 18.99 0.0125 Estimate 19.27 18.70 18.16 17.54 Regression 19.26 18.74 18-22 17.71 Standard error of estimate : & 0.1 mm.This experiment showed that- 180 22-18 22.10 21.02 20-88 19.52 19.65 18.48 18-42 17.32 17.19 (i) For any given dose, the zone size increased linearly with increasing diffusion time in the refrigerator at a mean rate of 0.84 mm per hour. (G) For any given pre-refrigeration time the zone size is linearly related to the logarith- mic dose and therefore suitable for assay. The mean slope over all pre-refrigeration times is 1.34 mm per doubling dilution. (iii) The slope of the linear response of (ii) increases with increasing diffusion time in the refrigerator at a mean rate of 0.0766 mm per doubling dilution per hour. Thus the slope without any pre-diffusion, corresponding to existing methods of assay, is 1-23 mm per doubling dilution, while after 3 hours pre-diffusion this is improved to 1.46 mm per doubling dilution.This corresponds to a reduction of a 10 per cent. assay error to about 7 per cent. (iv) In view of the low experimental error of 20.1 mm per estimate, the above effects are known to be linear with considerable accuracy. A further experiment on similar lines to that described above, making use of refrigeration times greater than seven hours, was performed. In this the slope of the dose - response line increased at a mean rate of 0.09 mm per doubling dilution per hour, resulting in a final slope of 2.0 mm for a doubling dilution after the full refrigeration time.The zones of exhibition resulting from the vitamin B,, solutions that have been permitted to diffuse for seven hours before incubation are indistinguishable in character from zones resulting from normal treatment. For routine assay we do not find it convenient to employ a pre-diffusion period, but when critical comparisons or determination of effects are required we use such a pre-diffusion treat- ment (up to seven hours if possible) to improve the precision of the determinations. The effect of incubating the assay plates before adding the samples to the cups was examined in a similar manner. The results, together with those from the above refrigeration experiment, are shown in Fig.3, where it can be seen that the slope of the dose - response curve decreases at a mean rate of 0.11 mm per hour per doubling dilution. DISCUSSION OF RESULTS The optimal conditions for the growth of the inoculum, together with other unpublished results from the use of the E. coli mutant in tube assays, indicated that a highly aerated medium favours rapid dense growth of this strain. The thin agar layer in the assay plates therefore results in sharply defined dense zones of exhibition. The character of these zones may be seen in Fig. 2. It is important that the growth on the assay plates should be preceded by growth of an inoculum in shallow layers of medium in order to obtain zones of the best character superimposed on a clear transparent background.As previously reported,1° zones of exhibition can be obtained by using ammonium dihydrogen phosphate, potassium chloride, magnesium sulphate and dextrose as the only constituents of the medium, but the growth within the zones is not so dense as with the recommended complete medium, and we prefer this for routine assay.704 HARRISON, LEES AND WOOD: THE ASSAY [Vol. 76 We have found that incubation of the assay plates may be performed either at 27" C or 37" C, a small increase in the slope of the dose - response line sometimes being observed at 27" C. With the Lactobacillus Zaclis Dorner plate assay,, vitamin BlZc often gives assay vdues three to four times as high as those obtained by tube methods or physico-chemical procedures. We have also found the same effect with L.Zeichmannii 313 plate assays, in which the zones are characterised by a certain fuzziness, but are entirely different in appearance from the zones given by desoxyribosides. With the E . col% mutant plate assay, however, activity approximately equivalent on a weight basis to that of the vitamin B,, reference standard is obtained for vitamin B1*, and there is no unusual appearance of the zones. 30.0 - 25.0 - E & L s 5 2 20.0 - 4 c 15.0 - Normal assay treatment it.. plating out of dilutions I I I I 1 7 6 5 4 3 2 I 0 I 2 3 12.0 Time, hours Fig. 3. The effects of refrigeration and incubation of the assay plates on the assay of vitamin B,, by the Escherichia coli mutant The standard error of the (12 x 12) Latin square assay quoted above is 0.23 mm per zone, rather higher than we normally experience. With (11 x 5) or (13 x 4) cyclic incomplete block designs it is usual to find experimental errors of 0.14 to 0.20 mm per zone.This high error of 0.23 mm we attribute to the unusually large design employed, for no evaluation of the effect of time of plating out on the slope of the dose - response line could be made and any consequent error is inflating the assay error. This effect of time of plating out on the slope of the dose - response line is not very great when smaller designs are being used. The time taken to plate out the (12 x 12) design was forty-four minutes. Methionine, as noted by Davis,16 gives zones of exhibition with the E. coZi mutant. A solution of 1 mg per ml gave a zone of approximately 35 mm, and dilutions of 100 and 10 pg per ml also stimulated growth, but the zones were indistinct and not measurable.A full account of the interfering effects of various substances in this assay method has been published by Cuthbertson, Pegler, Quadling and Herbert.,' Consideration of Fig. 3 shows the effect of deviation from normal assay treatment in plating out and incubation of the assay plates. The graphs serve to emphasize that the assay plates must be retained in the refrigerator until required for plating out and must during this operation be as cold as possible. For accurate assay comparisons an obvious gain in precision will be obtained if pre- refrigeration of the assay plate between plating out and incubation be performed. This mayDec., 19511 OF VITAMIN BIZ.PART VI 705 conveniently be done when the large assay plates are used, since they are easily handled as individual units. The analysis of the (12 x 12) Latin square experiment in Table V demonstrates the wide tolerance of the assay method to differences in pH and cyanide content of the test samples. Neither alteration in the pH values of the dilutions of the vitamin B,, reference standard from 5.0 to 8.0 nor an alteration in the cyanide content from three times to one hundred and fifty times the vitamin B,, concentration significantly affected the response. We are much indebted to Dr. Bernard Davis of the Public Health Laboratory, Cornell University Medical College, New Uork City, not only for having originally called Dr. E. Lester Smith’s attention to the mutant, but also for supplying the culture that enabled us to carry out the work reported here. We wish to acknowledge the help given by our colleagues Miss A. Gibson and Mr. J. P. R. Tootill for developing the streptomycin resistant strain and for the statistical design and analysis, respectively. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES Caswell, M. C., Koditschek, L. K., and Hendlin, D., J . Bid. Chem., 1949, 180, 125. Cuthbertson, W. F. J., Pegler, H. F., and Lloyd, J. T., Analyst, 1951, 76, 133. Emery, W. B., Lees, K. A., and Tootill, J . P. R., Ibid., 1951, 76, 141. Foster, J. C., Lally, J. A., and Woodruff, H. B., Science, 1949, 110, 507. Larkin, F. E., and Stuckey, R. E., Analyst, 1951, 76, 150. Skeggs, H. R., Hepple, €3. M., Valentik, K. -\., Huff, J. W., and Wright, L. D., J . Biol. Chem., Thompson, H. T., Dietrich, L. S., and Elvehjem, C. A., Ibid., 1950, 184, 175. Tatum, E. L., and Lederberg, J., J . Bact., 1947, 53, 672. Beadle, G. W., and Tatum, E. L., Apner. J . Rot., 1945, 32, 678. Bessell, C. J., Harrison, E., and Lees, K. A., Chem. & Ind., 1950, 561. Cuthbertson, W. F. J. (Personal communication). Wijmenga, H. G., Veer, W. L. C., and Lens, J., Biochim. Biophys. Acta, 1950, 6, 229. Coates, M. E., Ford, J. E., Harrison, G. F., Kon, S. I<., Porter, J. 117. G., Cuthbertson, W. F. J., and Pegler, H. F., Biochem. J . , 1951, 49, lxvii. Woodruff, H. B., and Foster, J. C., J . Biol. Chem., 1950, 183, 569. Brownlee, K. A., Loraine, P. K., and Stephens, J., J . Gen. Microbiol., 1949, 3, 347. Davis, B. D. (Personal communication). Cuthbertson, W. F. J., Pegler, H. F., Quadling, C., and Herbert, V., Analyst, 1951, 76, 540. 1950, 184, 211. NOTE-References 2, 3 and 17 are to Parts 111, V and IV of this series, respectively. GLAXO LABORATORIES LIMITED SEFTON PARK STOKE POGES, BUCKS. June, 1951

ISSN:0003-2654    

DOI:10.1039/AN9517600696

出版商:RSC    

年代:1951

数据来源: RSC