ISSN:0003-2654    

DOI:10.1039/AN95176FX037

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176BX039

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176FP087

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

THE ANALYST ixOSEPH LUCAS LIMITED require Engineers to fill theJfollowing Vacancies in the factory which they propose. toopen in Liverpool for the manufacture of Fuel Injectionequipment.MizTALLuRGIcAL ASSISTANTS and experience in at least oneof the following:-1. Production Heat Treatment Checking.2. Physical Testing of Raw Materials.3. Pyrometry and Radiogaphy.1.2. Analysis of Raw Materials.3. Analysis of Plating Solutions.CHEMICAL LABORATORY ASSISTANTS and experience in oneProcess Control (Plating, Blacking, etc.).Selected applicants will be expected to spend a short periodat the Company’s Laboratories in Birmingham prim totaking up their positions in Liverpool. The Company willpay the expenses in connection with such visits.Application, giving full details of previous experience,should be made in writing to the Personnel Manager, JosephLucas (Gas Turbine Equipment) Limited, Shaftmoor Lane,Hall Green, Birmingham, 28.or more of the following:-UNLOP have two vacancies for assistant chemists a tDthe Research Centre, Birmingham.Applicants shouldbe aged 20-26, with a degree in chemistry.concerned with the development of new analytica%&% k the field of rubber and plastics the other with microchemicalanalysis. Previous analytical’ experience though desirableis not.essentia1. salary according to age,’ q-catiom andexpenence. Applications in writing, quoting ref. A S 100,To: Personnel Manager, Dunlop Rubber Co. Ltd., FortDunlop, Erdington, Birmingham. 24.ROCESS CONTROL CHEMISTS required for rapidlyPexpanding Chemical Works East.Anglia.The postsvacant are for men not necessanly qu&ed, but of at leastInter. BSc. standdd, with experience in Heavy Chemical andAllied Industry, and having a preference for plant control andmanagement. Age preferably 25-35. Excellent prospects.Salary €4504600 per annum according to qualifications andexpenence. Write (quoting Ad. 616), stating age and fullparticulars of experience to Box No. 3781, THE ANALYST,47, Gresham Street, London, E.C.2.CHEMISTS are invited by the MINISTRY OF SUPPLYto apply for an appointment in the grade of PRINCIPALSCIENTIFIC OFFICER at a Research and DevelopmentEstablishment near London. Candidates must be at least31 years of age and have a 1st or 2nd Class Honours degreeor equivalent in Chemistry with many years responsibleresearch experience in modern methods of analysis of in-organic materials, including microchemical methods..Salary will be assessed according to age, qualificationsand experience within the range €960 to €1,295. Rates forwomen somewhat lower. The p s t is unestablished butcarries benefits under F.S.S.U. Selected candidates may berequired to work elsewhere near London for a short while,before taking up duty at the station referred to above,where housing accommodation may be available later.Application forms obtainable from The Ministry of Labourand National Service, Technical and Scientific Register (K),York House, Kingsway, London, W.C.2, quoting F.640/51/A.Closing date: 27th October, 1951.CROWN AGENTS FOR THE COLONIESSCIENTIFIC ASSISTANT (ANALYST) required by.theEast Africa High Commission for the East AhwanAgriculture and FGrestry Research Organisation for onetour of 30 to 48 months in the first instance. Salary accordingto qualifications and experience in the scale €650 rising toL840 a year plus temporary allowance between €82 and€126 a year,’according to salary and station. Outfit allow-ance €30. Free passages and liberal leave on full salary.Gratuity of 13+ per cent. of total substantive salary onsatisfactory completion of engagement. Candidates mustbe experienced analysts. Apply a t once by letter, statingage, full names in block letters, and full particulars of quali-fications and experience and mentioning this paper to theCrown Agents for the CAlonies, 4, Millbank, London, S.W.1,quoting M.2776J.G, on both letter and envelope.TheCrown Agents cannot undertake to acknowledge all applica-tions and will communicate only with applicants selected forfurther consideration.HEMIST for work initially on electrochemical methodsc o f analysis is required by Research Department of TheCambridge Instrument Company Limited, Cambridge.Degree or equivalent essential and some experience desirable.Applications must be in writing, giving details of education,qualifications and experience, as the salary offered willdepend on these. The post offers good prospects with apension scheme.HIS MAJESTY’S COLONIAL SERVICECHEMIST (27106/35/51), TANGANYIKAAPPLICATIONS are invited for the following post:-Appointment on 2 years’ probation for permanent andpensionable employment.Salary, B ccording to qualificationsand experience, in the scale L620-€1,320 lus an emergencyallowance of 20 per cent. of salary, sub;& to a maximumof €200 per annum. Quarters are provided, if available,subject to a rental deduction of 10 per cent. of salary. Freefirst class passages are provided for officer and wife and forchildren up to the cost of one adult fare. Officers are requiredto travel by air on leave. Taxes, including Income Tax,a t low local rates. Tour of service, 2 to 3 years. Six days’leav? on full salary for each completed month of residentsemce. Candidates should be between 20 and 30 years ofage and should possess an Honours degree in Chemistry orAssociateship of the Royal Institute of Chemistry. Dutiesinclude general analytical work of a development researchnature, particularly in connection with Agriculture ; generalall round interest in Industrial Chemistry, Food and DrugsToxicology; supervision of African Assistant Chemists andChemical Assistants.Intending candidates should apply in writing to theDirector of Recruitment (Colonial Service) Colonial OfficeSanctuary Buildings, Great Smith Streei, S.W.1, givinibrief details of their age, qualifications and experience.They should mention this paper and quote the referencenumber (27106/35/61).CHEMISTS REQUIRED for (a) Research Work (b)Analytical Work connected with the developmen( andproduction of jet engines.Midland area. State agequalifications and salary required. Write Box 3787, T H ~ANALYST, 47, Gresham Street, London, E.C.2.SOUTHERN RHODESIA GOVERNMENTVACANCY: CHEMIST: GOVERNMENTMETALLURGICAL LABORATORYPPLICANTS should have as a minimum qualification aAB.Sc. degree in Chemistry of a recognised University andat least four years’ experience in the analysis of all typks ofores and their products.Salary Scale: €468 x A66 to €600 x €34 to A668 x A33 to€800 x Lroo to €900 x L4o to A1140 per annum. Thecommencing salary may be higher than the minimum, butnot exceeding €668 per annum, in recognition of approvedpost qualification experience. In addition to salary, a costof living allowance of approximately 25% of salary is payableand, when applicable, marriage and children’s allowances.Application forms and further details may be obtainedfrom the secretary, Rhodesia House, 429, Strand, London,W.C.2, to whom completed forms should be returned notlater than 31st October.NALYTICAL CHEMISTS.Three required with BSc.,AA.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 35 years of age. State age, qualificationsand experience to Personnel Manager, Henry Wiggin &Co., Ltd., Wiggin Street, Birmingham, 16.LD-ESTABLISHED LONDON ANALYSTS requireOCHEMIST with experience of analysis under Fertiliserand Feeding Stuff Acts.Salary according to experienceand qualifications. Write Box 3783, THE ANALYST, 47,Gresham Street, London, E.C.2.HEMIST, recently graduated, wanted for work in con- C nection with the control and development of worksprocesses by manufacturers of Aluminium Sheet, Extrusionsand Paste. The chemist’s work would involve both organicand inorganic chemistry. Write Box 3784, THE ANALYST,47, Gresham Street, London, E.C.2.LARGE and progressive engineering company in WestALondon have a vacancy for a CHEMIST to undertakedevelopment work on corrosion prevention and metalfinishing. Candidates should hold a degree or HigherNational Certificate in Chemistry with a good knowledgeof organic chemistry and industrlal experience of rust pre-’ventives. 6-day week. Pension fund. Good prospects fora keen worker. Write Box 3786, TILE ANALYST, 47, GreshamStreet, London, E.C.2.CHEMIST requires ap intment; six years industrialthree years researchexpenence. Familiar with ph *od-chem. methods, analysesof ferrous and non-ferrous x!, foundry materials, etc.Trace analyses. Box 3786, THE ANALYST, 47, GreshamStreet, London, E.C.2.( w y t i c a l and devegment

ISSN:0003-2654    

DOI:10.1039/AN95176BP093

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

OCTOBER, I 95 I Obituary Vol. 76 No. 907 WALTER COLLINGWOOD WILLIAMS WALTER COLLINGWOOD WILLIAMS died on April 5th, 1951, in his 89th year; by his death the Society has lost probably the last of the original pioneers in “blazing the trail” of the public analyst. He was apprenticed to one of the London Guilds and, after serving his full term, he studied at London University, obtaining his BSc. degree with honours in chemistry and physics. He then attended Mason College, Birmingham, and was elected Associate of the College in 1884. On leaving Birmingham he became an assistant to Dr. J. Campbell Brown and shortly afterwards was appointed, jointly with Dr. Brown, Public Analyst for Lancashire, Liverpool and several boroughs. On the death of his chief in 1910 he became whole time Public Analyst for Lancashire, a post which he held until his retirement in 1926.He became a Fellow of the Institute of Chemistry in 1893; he joined the Society in the same year and served for two periods on the Council. 561562 OBITUARY [Vol. 76 In the early days of his career the post of public analyst had only recently been established. Very little attention had been given to the composition of foods or to the detection of adultera- tion. Hassall had published his classical researches on microscopy applied to foodstuffs, but no corresponding work had appeared relating to analytical investigations. Consequently, in the Lancashire County Laboratory, as in other laboratories, research was carried out to obtain information regarding the composition of foods, the detection of adulteration and to establish limits and standards.Williams threw himself whole-heartedly into this work ; his enthusiasm was such that it stimulated the deep and permanent interest of one of the writers, who served under him. A vast amount of information was accumulated as the years passed, but little or none of it was published, with the exception, it is believed, of a paper on Jamaica rum. Had his policy been to make known the results of his work he would have occupied a much more prominent position than he did in the profession. The mind of Collingwood Williams might aptly be described as a mathematical one. He faced certain types of investigation armed with algebraic equations, much, it would seem, as at a later date and on a grander scale, Einstein did when striving to elucidate the problem of the universe.The analytical results were expected to agree with those derived from the equations or he must know the reason for the divergence. His attitude had something in common with that of Martin Arrowsmith when he said in his prayer, “. . . give me a restless- ness whereby I may neither slumber nor accept praise until my observed results equal my calculated results or, in pious glee, I discover . . . my error.” He manifested the spirit of the true scientist in that he imposed on himself and on his assistants the most severe standards for laboratory work; no pains were spared to arrive at the truth, time being a secondary consideration. He made a thorough study of mistakes, how, why and when they were made and he elaborated strict rules of procedure to guard against errors and detect them.Working under such stringent conditions, with a minimum in the way of encouragement, was, in the case of young assistants, a “chastening” that could scarcely be described as “joyous,” but later on it was much appreciated, particularly by the assistant who, in course of time, became the head of the laboratory. Williams had a very reserved temperament ; and a sense of humour, though very seldom did evidence of it emerge in working hours. In this connection it should, in fairness, be mentioned that, at the period usually regarded as the prime of life, his eyesight began to fail, and it is quite likely that this misfortune emphasised a disposition that had always been retiring. The chief recreation of his active life was mountaineering and he scaled many peaks on the Continent and in Britain. He was very much attracted to a11 mechanical contrivances; microscopy claimed much of his spare time and he attained a wide and thorough knowledge of the subject. In 1949 he presented a badge to the North of England Section for the use of the Chairman -a gift that was gratefully accepted. One of the writers made his acquaintance only a few years before his death, but in that short time derived great pleasure from listening to his reminiscences which appeared to become clearer as sight and hearing failed. N. Heron represented the Society at the funeral service at Anfield Crematorium. He was a devoted member of the Liverpool Microscopical Society. J. R. STUBBS N. HERON

ISSN:0003-2654    

DOI:10.1039/AN951760561b

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Oct., 19511 HAMENCE 563 The Determination of the Availability of Nitrogen in Nitrogenous Fertilisers. Part 11* BY J. HUBERT HAMENCE (Presented at the meeting of the Society on Wednesday, April 4th, 1951) I n 1949 the author described a method for the determination of the available nitrogen in fertilisers based upon a determination of the nitrate produced when the fertiliser is mixed with soil under conditions that give maximum nitrification. The nitrate produced is compared with the nitrate produced from dried blood containing an equivalent amount of nitrogen and treated under similar conditions. The availability of the fertiliser is calculated by taking that of dried blood as 100. This test has now been applied to the majority of the common nitrogenous fertilisers and the results are given in this paper.It is shown that additional valuable information concerning the availability of the nitrogen may be gained by determining the nitrate produced a t the end of three or more different intervals of time and plotting and studying the time - nitrate curve. With very slow-acting fertilisers it is necessary to extend the period of test beyond the usual 21 days. The initial absorption of nitrate nitrogen from the soil, which takes place during the decomposition of some fertilisers containing substantial amounts of carbohydrates, can be demonstrated by this test. IN an earlier paper1 the author described a method for the determination of the relative availability of the nitrogen in nitrogenous fertilisers. This method is based upon a deter- mination of the nitrate nitrogen produced from the fertiliser when it is mixed with soil under such conditions as to give the maximum rate of nitrification. The nitrate nitrogen produced from the fertiliser in 18 days is compared with the quantity of nitrate nitrogen produced from dried blood containing an equivalent amount of nitrogen and treated under identical conditions.The availability of the fertiliser is calculated by taking that of dried blood as 100. The results for the availability, as determined by this method, of some of the more common nitrogenous fertilisers were given in that paper (Zoc. cit.). From the results the opinion was expressed that this test gave a better measure of availability of the nitrogen in nitrogenous fertilisers than the Street permanganate test,2 which has been widely used for this purpose.The same conclusion was arrived at by Clark and G a d d ~ , ~ who compared the behaviour of the water-insoluble nitrogen in a wide range of mixed fertilisers, which are available on the American market, in the neutral and alkaline permanganate tests with the corresponding nitrification values. As the result of this work they report that the lack of correlation between alkaline and neutral permanganate activities and observed nitrification values indicates that the official methods of the A.O.A.C. are inadequate for proper characterisation of the insoluble nitrogen content of many organic materials. A similar conclusion was arrived at by Owen, Rogers and Win~or,~ who were carrying out nitrification studies of nitrogenous fertilisers at the same tifie as the author.They used larger quantities of soil than did the author, although in some of the tests the ratio of fertiliser to soil was substantially the same. The tests differ in that the nitrification was studied over a longer period than the 21 days that was suggested as the standard time in the new technique. Where the results of the two laboratories impinge they are in general agreement. Time - “nitrate produced” graphs are given by Owen, Rogers and Winsol“‘ for a number of the commoner nitrogenous fertilisers. The new test has been applied also to new materials of unknown value and the results of laboratory tests with this technique have been confirmed subsequently by field trials. * Part I appeared in J .Sci. Food Agric., 1950, 1, 92.564 HAMENCE : THE DETERMINATION OF THE AVAILABILITY OF SURVEY OF AVAILABILITY OF NITROGENOUS FERTILISERS [Vol. 76 Since its publication this method has been applied in our laboratory to nearly all common nitrogenous fertilisers, and it is the purpose of t'his communication to describe and discuss the results that have been obtained. In the course of this work the practice of determining the nitrate nitrogen at the end of three fixed intervals of time has been maintained and experience has shown that this technique is valuable in that it gives a more complete picture of the decomposition taking Nitrate Nitrogen, mg Fig. 1 Nitrate Nitrogen, mg Fig. 2 place in the soil than could possibly be obtained by making only one determination of the nitrate nitrogen a t the end of 21 days.This supports the findings of Owen, Rogers and Winsor4; moreover, the curve obtained by plotting the production of nitrate nitrogen against time yields valuable information and the different types of nitrogenous fertilisers have each their own characteristic type of curve. For some slow-acting fertilisers the tests have been continued beyond the normal period of 21 days and the results of these extended tests are given. For the purpose of reporting the results, the fertilisers have been divided into classes; the classification is based partly on composition and partly on the origin of the materials, as follows- 1. Protein by-products. 2. Protein and bone by-products. 3. Treated protein materials. 4. Seed and plant residues.5. Excreta. 6. Synthetic compounds. 7. Other fertilisers. Protein by-products are taken as the first grsup because dried blood, the material used for comparison purposes, falls within it. In the results that foUow each group is treated separately. Besides the results of the availability tests, typical curves for the different materials are given and the results are discussed from the point of view of the composition of the fertiliser. PROTEIN BY-PRODUCTS- divided state, as in this test (see Table I). nature, and Fig. 1 shows the curves for dried blood and shoddy. Protein by-products all have a,similar availability in the soil when in the same finely The nitrate - time curves are also all similar in They indicate a steadyOct., 19511 NITROGEN IN NITROGEKOUS FERTILISERS 565 decomposition of the protein material with the rate of nitrification slowing as decomposition in the soil proceeds and reaches completion.Moreover, although the rate of nitrification slows, the curve does not become parallel to the time axis, as occurs with some fertilisers, TABLE I 1. PROTEIN BY-PRODUCTS Fertiliser Nitrogen, Availability per cent. Dried blood . . .. .. .. 14.00 100 (by definition) Hoof meal . . . . .. .. 15.10 118 Hoof meal . . .. .. . . 14.85 80 Hoof and horn meal . . .. . . 14.30 100 Shoddy . . .. . . .. . . 10.00 91 Gluten* . . .. .. .. .. 14.70 114 * Not a usual fertilising material, but included for comparison purposes. and if the time of the test is extended to 60 days nearly the whole of the organic nitrogen is recovered in the form of nitrate.It is interesting to note that gluten, a protein material, behaves in a similar manner. TABLE I1 2. PROTEIN AND BONE BY-PRODUCTS Fertiliser Nitrogen, Availability per cent. Meat and bone meal . . .. . . 8.57 Fish meal .. . . .. . . 10.67 Bone meal . . . . .. . . 4.19 Bone meal . . . . . . . . 3.75 Steamed bone flour . . .. . . 1-64 104 131 18 14 80 PROTEIN AND BONE BY-PRODUCTS- Fig. 2 shows typical curves for bone meal and for meat and bone meal. These results indicate that those fertilisers of this group in which the predominating material is protein have an availability similar to that of dried blood, and the nitrate - time curves are also similar to the dried blood curve. Bone meal, in contrast, presents a different picture, showing only a comparatively small availability and a correspondingly different nitrate - time curve.In other words the test shows bone meal to be a slow-acting nitrogenous fertiliser compared with dried blood and with meat and bone meal-thus confirming the accepted view of its slow-acting nature. TABLE I11 Table I1 shows the availability of the nitrogen in protein and bone by-products. RESULTS OF EXTENDED TESTS Nitrogen converted to nitrate in A \ Fertiliser 21 days, 39 days, 77 days. % % % Hoof meal . . .. .. .. .. 59 Bone meal .. .. .. . . 11 8 Plastic by-product, 22.2% of nitrogen . . Sewage sludge . . .. .. .. 9 77 19 10 10 In the studies of the availability of a slow-acting material like bone meal, much valuable information is gained by prolonging the tests beyond the normal 18 to 21 days.By extending the test for a further 21 days it is found that an increase in the rate of nitrification and con- sequently of the availability takes place, and this differentiates bone meal from very slow- acting materials, such as certain plastic by-products, which do not exhibit this increased rate of nitrification during the second period of 21 days (see Table 111). The extended period of time immediately shows a difference between bone meal and the other slow-acting materials.566 HAMENCE : THE DETERMINATION OF THE AVAILABILITY OF [Vol. 76 A t the present time the reason for the slow-acting nature of the nitrogen in bone meal is not by any means clear, but two possibilities present themselves; either (i) the protein, being embedded in the phosphate matrix, is protected by the calcium phosphate from the attack of micro-organisms, or (ii) the protein, being in the form of collagen, needs to be hydrolysed to gelatin before it is capable of being attacked by bacteria.Steamed bone meal presents an entirely different picture from bone meal in that it has a high availability, presumably owing to the steaming process rendering the protein more available for decomposition. The high nitrogen availability would appear to support the second possibility as being the more correct, as the steaming process would bring about hydrolysis of the collagen to gelatin; again, if the first hypothesis were the more probable, it would be expected that the nitrogen in steamed bone meal would be less available, as the nitrogen remaining in steamed bone meal is the residue after the more easily extractable protein has been removed.Some interesting results on the availability of the nitrogen in bone meal have been obtained by Long, Owen and Winsor,6 who showed that availability depends upon the age of the bone meal and the extent to which decomposition has occurred. # TABLE I’V 3. TREATED PROTEIN MATERIAL Fertiliser Bark tanned leather Chrome leather . . Treated leather meal Treated leather meal Casein waste . . Comb dust . . Keronikon . . Keronikon . . Nitrogen, Availability per cent. .. .. . . 5.07 . . . . .. 8.38 .. .. .. 6.67 .. .. . . 6-7 1 .. . . .. 13.38 .. .. .. 12.05 .. .. .. 15.03 .. .. .. 13.30 nil 8 46 47 30 53 53 46 TREATED PROTEIN MATERIAL- The results for treated and untreated leather waste have already been discussed in the original paper and are repeated here in Table IV for the sake of completeness.Typical curves for these materials are shown in Fig. 3. The considerable increase in availability as the result of treatment of leather wastes is immediately apparent from Table IV and Fig. 3. The treated products yield a substantial amount of readily available nitrogen in the first 7 days-more than the majority of other fertilisers-but the rate of breakdown soon slows up very considerably. They may thus be regarded as consisting of two parts, one a quick-acting fertiliser and the other slow-acting. TABLE V 4. SEED AND PLANT RESIDUES Fertiliser Nitrogen, Availability per cent. Castor meal . . .. .. .. 4.75 Castor meal . ... .. .. 5-47 Cotton-seed meal . . .. . . 5.62 Cotton-seed meal . . .. . . 5.74 Rape-seed meal .. .. .. 5.16 Cocoa shell . . .. .. .. 2.68 Dried grass meal . . .. .. 1.95 96 108 36 39 69 nil nil SEED AND PLANT RESIDUES- The materials in this group are mainly the residues remaining after the extraction of the oil from oil-bearing seeds. It will be seen from the results in Table V that castor meal has by far the highest avail- ability of the materials in this group. The availability is similar to that of dried blood and this fact probably accounts for the extensive use of castor meal as an organic nitrogenous fertiliser in past years.Oct., 19511 NITROGEN I N NITROGENOUS FERTILISERS 567 Curves for castor meal and cotton-seed meal are shown in Fig.4. This group of materials is interesting in that it shows a phenomenon that has not been referred to before in this work, namely, the non-availability of the nitrogen after 21 days in cocoa-shell meal and the dried-grass materials, which should break down easily in the soil. Nitrate Nitrogen, mg Fig. 3 Nitrate Nitrogen, mg Fig. 4 This result is merely a laboratory demonstration of the well-known phenomenon that often occurs when attempts are made to increase the organic matter in soils by the incorporation of straw or of a green crop. This practice often leads to the utilisation of the natural soil nitrate for the decomposition of the carbohydrate portion of the organic material. The substantial withdrawal of nitrate nitrogen from the soil by cocoa shell and dried grass is readily seen from Fig.5. \ \ \ \ I Cocoa Shel,l - ', Dried Grass - - - - \ I -I Ir 5 -10 Nitrate Nitrogen, mg Fig. 5. Nitrate absorbed from the soil Nitrate Nitrogen, mg Fig. 6568 HAMENCE THE DETERMINATION OF THE AVAILABILITY OF [Vol. 76 Fertiliser TABLE VI 5. EXCRETA Nitrogen, Availability per cent. Urea . . .. .. . . .. 46.60 170 . . 16.8 163 1.46 nil Bird guano . . .. . . Fresh horse-manure . . . . .. Sewage sludge . . .. . . . . 3.04 32’ Sewage sludge . . .. . . .. 2.74 34 Sewage sludge . . . . .. .. 2.14 14 EXCRETA- It will be seen from Table VI that there is a very wide difference in the availability of these different products, which represent excreta i n different forms. The nitrogen in urea is very rapidly broken down, and the figures show urea to be the most readily available of all nitrogenous fertilisers apart from the alkali nitrates.Bird guano similarly breaks down with extreme rapidity and behaves in many respects similarly to urea in that decomposition is so rapid that it appears to stimulate the natural formation TABLE VII 6. SYNTHETIC COMPOUNDS Fertiliser Nitrogen, Availability per cent. Hexamine . . .. .. .. 40.00 Cyanamide . . .. . . .. 16.04 Ammonium nitrate . . .. .. 35.00 Ammonium sulphate . . . . .. 21.00 163 84 120 164 of nitrate from the soil; moreover that this stimulation is only temporary and subsequently loss of nitrate takes place. These observations are in accord with the fact that the nitrogen in bird guano is mainly in the form of urea or ureides. They also afford an explanation of the great value that was placed on guano by the horticulturalists at the beginning of this century. Fresh horse-manure shows a strong initial absorption of soil nitrate by the residual carbohydrate material, so demonstrating one reason for the age-old custom of not using this type of fertiliser in its fresh condition.The curves for some of these materials are shown in Fig. 6. The availability of the nitrogen is very much less than that of urea-containing substances in this group, and reference to the curves in Fig. 6 shows that, although there is a small, fairly rapid, initial formation of nitrate, this in some instances diminishes by the absorption of nitrate presumably required for the TABLE VIII Sewage sludge presents a contrasting picture.7. MI SCELLANE ou s Fertiliser Nitrogen, Availability per cent. Hop manure . . .. .. .. 3.46 128 Peat . . . . .. .. .. 0.90 16 Humic acid* . . .. .. .. 2.00 12 * Contaminated with sand. decomposition of the other organic matter present in the sludge. Experiments in which the time has been extended beyond the normal 21 days show that the subsequent breakdown of the sewage sludge is extremely slow and therefore only a relatively small part of the nitrogen in the sludge is available for immediate plant growth. It seems possible that the very slow- acting nature of a substantial part.of the nitrogen in sewage sludge may account for its unpopularity in some quarters as a nitrogenous fertiliser. Clearly, in the process of sewage purification the readily decomposable and water- soluble nitrogenous compounds are washed out and.removed, leaving behind material having a humic and somewhat inert nature.Oct., 19511 KITROGEX IN NITROGENOUS FERTJLISERS 569 SYNTHETIC COMPOUNDS- ability, and follow the normal type of curve as shown in Fig. 7. employed as a fertiliser, behaves in the soil in a manner similar to urea. from the other members of the group in that it is slow-acting in the initial stages. The ammonium salts in Table VII show, as would be expected, high figures for avail- Hexamine, a compound not Cyanamide is different 0 5 10 15 Nitrate Nitrogen, mg Fig. 7 MISCELLANEOUS FERTILISERS- Most of the materials in this group call for some special attention. Hop manure-Hop manure frequently consists of spent hops suitably reinforced with concentrated fertilisers such as ammonium sulphate. The figures in Table VIII show that the nitrogen in hop manure is very readily available.It may be argued that this would be expected, as the main bulk of the nitrogen is in the form of ammonium salts; on the other hand, the fertiliser contains a substantial amount of organic matter and therefore an initial loss of the readily available nitrogen by decomposition of the organic matter might be expected. Peat and hzmic acid-Peat yields a small amount of available nitrogen and in that respect is similar to humic acid. Clearly, the accumulation of these substances in the soil is only possible if they are relatively inert in nature and decompose only slowly in the soil. ABSORPTION OF SOIL NITRATE DURING DECOMPOSITION This phenomenon is readily shown in this test by studying the rate of nitrate formation from protein-enriched bread and from the major constituents of this bread, namely, wheat starch and gluten.With wheat starch the nitrate nitrogen of the soil is completely removed or utilised by carbo- hydrate decomposition and even after 21 days the soil contains only insignificant traces of nitrates. With the protein gluten, decomposition is slow in starting, but having once started it proceeds rapidly. With bread itself the carbohydrate a t first removes soil nitrate, but once gluten decomposition gains pace the absorption of nitrate by the carbohydrate is more than compensated by the production of nitrate from the gluten, and free nitrate again appears in the soil.If we consider the nitrate balances at the end of 13 and 21 days, from the curves shown in Fig. 8, we obtain the results shown in Table IX. These results show that the calculated balance of nitrate from the individual gluten and starch curves agrees well with the balance of nitrate as determined from the bread. It follows therefore that the two reactions, viz., The nitrate formation with these materials is shown in Fig. 8.570 HAMENCE : THE DETERMINATION OF THE AVAILABILITY OF [Vol. 76 the decomposition of carbohydrate involving the absorption of nitrate and the decomposition of protein with accompanying nitrification proceed simultaneously in the soil. I Nitrate Nitrogen, mg Fig. S TABLE IX NITRATE BALANCES FOR BREAD AND ITS CONSTITUENTS After 13 days, After 21 days, g 0.0078 0.0072 0.0006 0*0005 Nitrate produced from gluten .. .. Nitrate absorbed by starch . . . . .. Free nitrate on balance . . .. . . Free nitrate found from decomposition of .. .. .. .. bread . . g 0.0089 0.0077 0.0012 0.0014 RAPID ROUTINE TEST The principal disadvantage of the method is the time it takes; as the method requires three weeks for completion, it is hardly justifiable suggest its adoption as a routine test in, say, the regulations of the Fertilisers and Feeding Stuffs Act. Clearly there is still room for a more rapid method, and search for such a method :has been made in our laboratory for some RESULTS OF PEPSIN DIGESTIBILITY TEST Fertiliser Dried blood . . .. .. .. Castor meal . . .. .. .. Casein waste . . .. .. .. Sewage ..Sewage .. .. Urea plastic . . .. .. .. Leather meal . . .. .. . . .. . . . . .. . . Total nitrogen, per cent. 14.0 5.47 12.75 1.72 2.74 19-46 6.13 Availability (by soil test) 100 10s 35 30 34 12 54 Nitrogen insoluble in pepsin, per cent. 0.44 0.60 6.00 1-33 2.20 3.00 3.15 Availability in pepsin 97 89 56 23 20 84 49 time, all possible sorting methods being compared with the results of the longer test that has just been described. So far, the most promising rapid test has been the one that is commonlyOct., 19511 NITROGEN IN NITROGENOUS FERTILISERS 571 used for the determination of available protein in feeding stuffs. This is a pepsin digestibility test. Procedwe-Dissolve 1 g of pepsin in 480 ml of water and 10 ml of 126 per cent. v/v hydrochloric acid. Add 1.6g of the finely ground fertiliser, mix thoroughly and incubate at blood heat for 24 hours.At the end of 24 hours add 10 ml of 12.5 per cent. v/v hydro- chloric acid and incubate for a further 24 hours. Filter off the insoluble matter, wash well with water and determine the nitrogen in the insoluble matter remaining on the filter-paper. As will be seen from these results for a number of fertilisers, the pepsin solubility test is a useful guide to the availability of the nitrogen, but unfortunately it breaks down seriously with the urea- formaldehyde type of plastic waste material that is now becoming available for fertilising purposes. It is conducted as follows. Resalts-By this test the results shown in Table X have been obtained. REFERENCES 1. 2. 3. 4. 5 .Hamence, J. H., J . Sci. Food Agric., 1950, 1, 92. “Official Methods of Analysis of the Association of Official Agricultural Chemists,” Association of Clark, K. G., and Gaddy, V. L., J . Ass. Off. Agric. Chew., 1950, 33, 480. Owen, O., Rogers, D. W., and Winsor, G. W., J . Agric. Sci., 1951, 40, 185. Long, M. I. E., Owen, O., and Winsor, G. W., J . Sci. Food Agric., 1951, 2, 125. Official Agricultural Chemists, Washington, 1950. DR. BERNARD DYER AND PARTNERS LONDON, E.C.3 DISCUSSION THE PRESIDENT congratulated Dr. Hamence on producing a most important paper on the availability of fertilisers. DR. 0. OWEN said he considered the technique was valuable in two ways. First it indicated the extent of conversion of the source of nitrogen into nitrate. Secondly, it indicated the rates a t which nitrogenous compounds decomposed, provided that periodic determinations were carried out. For this second purpose the work a t Cheshunt had shown that both nitrate and ammonia nitrogen should be determined, as their sum represented the decomposition in the early stages and ammonia was known to be a precursor of nitrate. Dr.Hamence’s results for cocoa shell suggested that the use of this material as a filler for quick-acting top dressings might defeat the object of such fertilisers in that the correct timing of a nitrogenous fertiliser for a rapidly growing crop was often critical. The results shown for horse manure were unexpected, as this material was considered to be so valuable in intensive horticulture. Similar work had been carried out on a range of urea - formaldehyde condensation products a t Cheshunt and some of the American conclusions had been confirmed. There still remained the problem of the fate of that part of the nitrogen that had not been converted into nitrate a t the end of an experiment.MR. D. D. MOIR enquired whether the author could give any information as to the degree of fineness of the various fertilisers, or whether the commercial articles had been used without any additional grinding. Since the availability would be dependent on the fineness of grinding this would appear to be important. DR. HAMENCE expressed his pleasure that Dr. Owen had been able to come to the meeting, as they had both been working independently on the problem of nitrogen availability for a number of years.In reply to Dr. Owen’s remarks, he said that, under the conditions in which the decomposition of the fertiliser was carried out with his test, it was his experience that no appreciable quantities of free ammonia were produced, and it was for this reason that he had confined himself almost entirely to nitrate determina- tions. Moreover, he had expressed the view in the earlier paper that, if ammonia accumulated in the system, the mechanism of nitrification was not functioning properly. He agreed with Dr. Owen that the results of this work had shown that great care must be taken when selecting an organic filler for a compound fertiliser. If the filler contemplated had the power of absorbing substantial quantities of nitrate from the soil, it was important to ensure that adequate supplies of readily available nitrogen were present to counteract this tendency.In connection with the speaker’s remarks on the subject of horse manure it was important to remember that the sample examined was fresh and that i t was unusual to use the material in this fresh condition in practice. Presumably this was one of the reasons for avoiding the use of the fresh material, and that one of the functions of the preliminary “aging” before use was to enable the readily available carbohydrate fraction to expend its nitrogen absorbing power before it was applied to the crop. In reply to Mr. Moir, Dr. Hamence said that all materials used in the test were in the same finely divided state. The sample, after having been ground as finely as possible in the mill, was weighed and then thoroughly ground with about 10 g of air-dried soil, together with 0.1 g of chalk.In this way i t had been found possible to reduce even very coarse and fibrous materials Indeed, this was part of the technique.572 HAMENCE [Vol. 76 that were otherwise difficult to grind in a laboratory mill to the same very finely divided state. This intimate mixture of soil and fertiliser was then mixed with the bulk of the soil that was used for the test. MR. HASLAM said he gathered that Dr. Hamence was surprised that, on treatment of urea - formaldehyde plastic waste with dilute acid and pepsin, relatively large amounts of nitrogenous substances passed into solution. It would occur in the absence of pepsin with most urea - formaldehyde preparations from the original urea - formaldehyde syrup to the moulding powder used in the preparation of the final moulded article.Simple treatment of most urea -formaldehyde preparations with weak acid would yield a comparatively large amount of soluble nitrogenous matter. DR. HAMENCE thanked Mr. Haslam for these very helpful observations, which provided an immediate answer to a problem that had perplexed him for some time, as he had always regarded urea - formaldehyde plastic as being an inert material and certainly not one that would have a water solubility. DR. K. A. WILLIAMS asked if the theobromine in cocoa shell had any effect on the bacteria of thesoil. DR. HAMENCE said that he doubted it very much, as nitrogenous compounds of the nature of theo- bromine were usually broken down rapidly in the soil.DR. E. C. WOOD asked if it were possible to draw up a balance sheet for soil nitrogen in all forms during the period of denitrification that preceded nitrification when fertilisers containing carbohydrate were added to the soil. Was the nitrogen withdrawn only temporarily, to re-appear a t a later stage as available nitrogen, or was there a net loss of nitrogen in the early stages-as ammonia, for instance? DR. HAMENCE replied that it appeared from the work they had carried out that the course of the decomposition would depend largely on the ratio of nitrogen to carbohydrate in the fertiliser and also .on the form in which the nitrogen was present. It was evident also that both the process of nitrification and of absorption of nitrate by the carbohydrate portion of the fertiliser went on simultaneously and that the proportion of free nitrate available for plant growth depended on the resultant of the two processes. As regards losses from the soil in the form of nitrogen, there was now fairly substantial evidence of such a phenomenon taking place in certain conditions and he thokght that, now a labelled isotope of nitrogen was available, more information on the point should soon be forthcoming. MR. R. F. MILTON asked if the apparent slow availability of certain organic fertilisers was not due to the “locking-up” in micro-organisms of nitrogen that became available later. DR. HAMENCE in reply said that he thought this a re,asonable explanation; a t the present time in his laboratory they were trying out a method that he thought might provide a measure of the nitrogen present in this form. In his view it was the only reasonable explanation of strong residual nitrate action observed some three or four years after the application of unusually heavy dressings of readily available organic nitrogenous fertilisers, and he hoped that the test now being developed would give a definite answer to this problem. That did not surprise him in the least.

ISSN:0003-2654    

DOI:10.1039/AN9517600563

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Oct., 19511 BAIN 573 The Evaluation of Drugs in Man, with Special Reference to Antihistaminics BY W. A. BAIN (Presented at the meeting of the Biological Methods Groufi on Monday, March 12th, 1951) The advantages of graded or quantitative responses over “all or none” or quantal responses in determining dose - response relationships in human subjects are discussed. There are special difficulties and limitations in the evaluation of drugs in man. As an example of the application of a graded response method, the evaluation in man of various antihistaminic drugs, by measurement of the reduction of the response to intracutaneous histamine, is described. In earlier published work, which is briefly reviewed, comparisons of relative potencies, durations of action and therapeutic efficacies were made in separate sets of experiments.More recently, estimates of relative potencies and durations of action have been derived simultaneously from the same subjects; some comparisons made in this way are presented, including hitherto un- published data for chlorcyclizine, chlorprophenpyridamine and compound Antihistaminics will only reduce, and not abolish, the effects of the 3-pg dose of intracutaneous histamine used in these experiments. They may, however, prevent or abolish the symptoms of hay fever and chronic urticaria in doses that produce only a small percentage reduction in the effect of the 3-pg test dose of histamine. From a consideration of the dose - response curve to intracu taneous histaminics, an explanation for this seeming discrepancy between pharmacological and the therapeutic results is suggested, and a clearer picture of how the drugs produce their therapeutic effects is obtained..B.W. 405-C-49. THE evaluation of drugs in man involves essentially the application to man of those principles of quantitative pharmacological investigation that have hitherto been applied chiefly to biological assay and st andardisation procedures on animals and isolated animal tissues. I t was, indeed, largely for the solution of the problems of biological standardisation that these methods were first developed. In general they are methods that permit, among other things, the establishment of dosage - response relationships. The pharmacologist has two different types of data that can be used for the determination of such relationships.The first is got by measuring the degree of effect produced by a drug (graded or quantitative response), the second by noting the presence or absence of a drug effect (“all or none” or quantal response). The quantitative method is capable of yielding much more information than the other and is preferable when it can be applied. It has the further advantage that, if the same subjects are used for all the comparisons, only small numbers need be used. The quantal method, besides yielding less information, involves larger numbers of subjects. In man, however, because of subjective or other effects that can be arbitrarily graded, responses that would be quantal in animals can often be made to yield data that are essentially quantitative and can be dealt with as such.Further, there is usually more than one effect or manifestation of a particular drug that can be recognised and may be of importance in man: consequently, even when the different effects are strictly “all or none,” there is more information to be extracted from the quantal type of response in man than from the corresponding type in animals. I t seems that the future advancement both of human pharmacology itself and of rational pharmacotherapeutics must depend largely on how far such methods can be developed and applied to the investigation of drug actions in man. The extent to which they can be developed will itself depend largely on the success achieved in recognising or establishing measurable criteria of the effects of the drug under test.Within a class of drugs the short-term evaluation in man should involve not only the determination of relative weight for weight potencies when the drugs are administered by their usual routes, but also the determination of relative rates of onset and duration of action and the relative immediate toxicities. This pharmacological evaluation can sometimes be Both are important.574 BAIN: THE EVALUATION OF DRUGS Il\i MAN, WITH [Vol. 76 carried out on normal people, but at other times only on patients suffering from some particular condition. In either event, since the object of obtaining the pharmacological information is to make possible its rational application in pharrnacotherapeutics, a therapeutic evaluation of the most promising drugs of the class should !succeed the pharmacological one.Often, however, the pharmacological evaluation on patients will constitute, by its nature, the short-term therapeutic evaluation as well. Quantitative comparisons of drugs in man are subject to the same variables as are comparisons in animals and animal tissues, but additional difficulties and limiting factors immediately present themselves. Thus, in obtaining dosage - response data in animal experiments, an objective record of the whole effect of a given dose can often be obtained, leaving no doubt about the maximum effect. Even where no objective record is possible, the number of observations yielding the required data for a given dose can be made sufficiently great to give a smooth curve of response. Such curves are clearly necessary for dosage - response data in man and to determine difference:; in duration of action of different drugs, but continuous and objective records of the drug effect can seldom be obtained, and the number of observations that can be made to determine the rise and fall of the effect is limited.Further, the dose-range used in animals can be as great as desired, but is necessarily limited in man. The element of personal danger is never entirely absent in human experiments, and particular caution has to be exercised in investigating new or hitherto untried compounds. Finally, since only small numbers of subjects are usually available for such tests, all com- parisons have to be made on the same people, who, if the experiments are numerous and of long duration, begin, sooner or later, to find them exceedingly tedious.The illustrations in this paper are simple examples of the application of a graded response method to the comparison of antihistaminics in man. The antihistaminics are drugs of varied structure, whose most important action is to antagonise, presumably by receptor competition, certain of the actions of histamine. Most antihistaminks have other actions, such as the hyoscine-like effects (both central and peripheral) seen on systemic administration and the anaesthetic effects produced by local application to suitable sites and lesions. The hyoscine-like effects, while sometimes useful therapeutically, more often constitute the most undesirable side-action of the drugs. Since histamine itself, injected into the skin, gives characteristic effects-the wheal and the flare-that can be measured, the quantitative modification of either or both of these effects by antihistaminics can be used as the basis for an evaluation and comparison of different antihistaminic drugs in man.The techniques used in our original experiments have been published1*2 and need not be detailed here. Three drugs, promethazine, mepyramine and antazoline, were compared for relative potencies and durations of action, and the first two for their therapeutic efficacies. Comparisons of potency were made by determining, for each drug, under standard conditions, the maximum reduction of the intradermal histarnine response in each of a group of six subjects to three different oral doses of the drug.The average maximum response to each dose of each drug was expressed as the average percentage reduction of wheal area. As the dose-response curves for the three drugs were parallel, a simple ratio expressed the potency differences. Promethazine was about seven times more potent, weight for weight, than mepyramine and about fifteen times more potent than antazoline. Relative durations of action were determined by following the reduction of the effect of an intradermal dose of histamine, usually 3 pg, to and beyond its maximum after adminis- tering approximately equi-effective doses of the different antihistamine drugs to another group of subjects. Relative durations of action were expressed as the average times taken for the full effect of the drug to be reduced to half.For promethazine, mepyramine and antazoline these times were 194, 5Q and 34 hours respectively. A therapeutic comparison of promethazine and mepyramine was then carried out on 20 patients with chronic urticaria. All these had first been treated with mepyramine and the daily dose to control the urticaria was determined. They were then taken off all therapy and, when the reactions to intradermal histamine had returned to normal, treated with promethazine. This drug, in view of its long duration of action, was given in a single dose at night, the dose being adjusted to give as nearly as possible the same effect as the mepyramine in the earlier period of treatment. The results showed that promethazine was, on the average, about fourteen times more potent therapeutically than mepyramine.In the pharmacological tests it was about seven times more potent. It is clearly to the prolonged duration of action of promethazine that thisOct., 19511 SPECIAL REFERENCE TO ANTIHISTAMINICS 575 difference between the pharmacological and therapeutic potencies was due. A further point brought out by this comparison was that the effective dose of promethazine produced fewer side reactions than the corresponding dose of mepyramine, so that 14 of the 20 patients preferred the promethazine treatment. These first comparisons of potency and duration of action were made in separate experi- ments on different groups of people. The necessity for reducing the total number of observations to a minimum has led us t o modify this proced~re,~ and now all the necessary I I 1 1 I I t .1 L I I I I I I 1 1 I I I I I I I I 100 3 0 0 1000 3000 I0000 10 30 TIME, MINUTES Fig. 1. Graphical representation of time - response data for compound “405” and chlor- Left-hand family of curves, effects of 12.5. 25 and 50 mg of “405”; right-hand Points of maximum action and of half-action prophenpyridamine. curves, 18.5, 25 and 50 mg of chlorprophenpyridamine. are indicated on the graphs information is obtained simultaneously from the same subjects. Each subject receives duplicate or triplicate intracutaneous injections of 3-0 pg of histamine, estimated as base, in 0.05m1, and the resulting wheal areas are recorded 5 to 7 minutes later. The anti- histaminic is then taken on an empty stomach. The test dose of histamine is injected half- hourly until the maximum effect has been reached, and then hourly or at longer intervals, until the effect has obviously passed its half maximum. By plotting the percentage reduction of wheal area against time for each subject, an approximate curve of action for the dose of the drug in each subject is determined.From each such curve is derived an estimate of the maximum effect, the time for the establishment of this and the time for it to be reduced to half. The individual results in the group are averaged. Three different doses of each drug are usually investigated. Table I gives the main data abstracted from nine experiments of this kind on three different drugs. A graphic representation of the results with two of these, chlorprophen- pyridamine, and a compound prepared by Dr.Adamson of the Wellcome Laboratories and known as “405,” is shown in Fig. 1. The comparison of these drugs is of particular interest, as they both have the same potency but differ in the rate at which the maximum effect is reached and, more important, in the rate at which it falls to half. Compound “405” thus seems an exception to the general rule that the more potent an antihistaminic the longer its duration of action. The averages of the individual maximum percentage reductions of wheal area for each dose of an antihistaminic (see Table I) constitute the data from which dose -response relationships are obtained. Fig. 2 shows such relationships for some drugs, derived in this576 BAIN: THE EVALUATION OF DRUGS I N MAN, WITH TABLE I SUMMARY OF DATA ON RELATIVE POTENCIES AND DURATIONS OF ACTION FOR THREE ANTIHISTAMINICS [Vol. 76 Drug Dose, mg Chlorcyclizine 25.0 100.0 250.0 Chlorprophenpyri- 12.5 damine 25-0 50.0 “405” 12.5 25.0 50.0 Average of individual maximum percentage reductions & S.E.23.2 -k 2-4 44.3 5 2-7 57.9 * 2.3 36.0 & 4.5 48.3 i 2.1 56.7 & 1.7 39.1 2 1-75 46.6 & 2.66 55.8 & 2.36 Times to maximum and to half-maximum action 7 A 7 Average of individual half-action times, Average time i.e., time to half- for all doses Average half- action less time to maximum action time to maximum action, action for all doses 932 - 348 = 584 294 minutes 1024 minutes minutes 1082 - 278 = 804 (5 hours) (17 hours) 1940 - 255 = 1685 1600 - 190 = 1410 187 minutes 2050 minutes 2640 - 188 = 2452 (3 hours) (34 hours) 2470 - 183 = 2287 760 - 120 = 640 122 minutes 679 minutes 863 - 128 = 735 (2 hours) ( l l g hours) 779 - 117 = 662 way from a group of six people. The slopes of the lines are less than in our original experi- ments on other subjects and are such that if the dose to produce a 30 per cent.reduction in the response is taken as unity, then for a 65 per cent. reduction it is ten, and, assuming the lines to maintain their courses indefinitely, the dose to abolish the effect of 3 ,ug of histamine is approximately 100, i.e., about 1-5 g of promethazine or 10.0 g of mepyramine! (The doses of the different drugs required to produce a 30 per cent. response are indicated below the abscissae in Fig. 2 and give a measure of the relative potencies.) U W 4 J U W a $ 5 0 a a 2 t- U ’> w UI 0 4 t- z W U w a a 70 60 5 0 40 30 2 0 10 0 00 ORAL DOSE, m9 Fig.2. Dose - response relationships for different antihistaminics. The dose of each pro- ducing-a 30 per cent. ieduction in whLal areais shown below the abscissa; these indicate relitive potencies. Thus, taking promethazine as 1, the corresponding doses required to produce the same maximum effect are : chlorprophenpyridamine (and “405”) +, chlorcyclizine 24, mepyramine 6*. antazoline 144, thonzylamine 20Oct., 19511 SPECIAL REFERENCE TO ANTIHISTAMINICS 577 I t is clearly impossible to abolish the effects of a 3-pg intracutaneous test dose of histamine with any of the antihistaminics at present available. Equally clearly, it is possible to prevent or abolish many of the effects of histamine liberated in the body by doses of these drugs that produce only a moderate reduction in the response to the 3-pg intracutaneous test dose.Clinical experience, indeed, especially with the longer acting drugs, indicates that the symptoms of some allergic conditions, e.g., hay fever, can be controlled by a level of anti- histaminic effect represented by no more than a 10 or 15 per cent. reduction of the 3-pg test wheal. How, then, can these percentage reductions of wheal area be interpreted so as to give a picture of how the antihistaminics produce their therapeutic effects? 0.001 : 0.602 0 :012 0.1 : 0.2 5 1.0 i 1.5 10.0 100.0 INTRADERMAL HISTAMINE, I*g Fig. 3. Shift of dose - response curve for intracutaneous histamine by an antihistaminic.Experiment on five subjects. Assuming the regressions are parallel, the shift shown, representing a 25 per cent. reduction of the 3-pg test wheal, involves a six-fold increase in the dose required to produce any given effect. For further explanation see text Upper line before and lower line after dosage of antihistaminic. As the percentage reduction of wheal area that we measure is an expression of the shift of the dose - response curve to intracutaneous histamine, the following considerations provide a possible answer. The upper graph in Fig. 3 shows the log-dose - response regression for intracutaneous histamine in five of the subjects used in the foregoing experiments. The lower graph shows the shift of this 3 hours after ingestion of an antihistaminic.If it is assumed that the two regressions are parallel, the shift is such that a six-fold increase in dose is now required to produce the same effect as originally. Now the percentage reduction of wheal area to the 3-pg test dose in this experiment is about 25. Thus a 25 per cent. reduction of wheal area involves a six-fold increase in the dose of intracutaneous histamine required to produce any given original effect within the dose range represented on the graphs. By drawing parallel dose - response regressions through points representing different percentage reductions of the effect at the 3-pg dose level, the relationship between the percentage reduction of wheal area to this test dose, and the extent to which any dose of intracutaneous histamine must be raised to produce its original effect, is immediately established.This relationship, calculated from Fig. 3, is shown by the lower line in Fig. 4. If the slope of the dose - response curve to intracutaneous histamine is shifted slightly, so as to meet the abscissa at 040.2 instead of 0*0015pg, the upper line of Fig. 3 results. Thus, though the exact relationship will depend on the slope of the dose -response regression, it is obvious that a relatively small percentage reduction in the intracutaneous wheal response represents a considerable578 BAIN: THE EVALUATION OF DRUGS I N MAN, WITH [Vol. 76 increase in the dose of histamine required to produce any given response, including a threshold response. Thus it is not difficult to see how the effects of extrinsic histamine liberated in the body may be prevented or abolished by doses of antihistaminics that produce only a moderate reduction in the effect of an intracutaneous test dose, for this relatively small reduction may involve increasing many-fold the threshold for the production of extrinsic histaminic effects.Fig. 4. Relationship between percentage reduction of wheal area from a 3-pg test dose of histamine by an orally administered antihistaminic, and the extent t o which the intracutaneous dose of histamine must then be increased to produce the same effect as originally. Abscissa: reduction of wheal area from 3-pg intracutaneous test dose. Lower graph from data based on normal (upper) dose - response line in Fig. 3. Upper graph shows effect of modifying slope of normal dose -response line so that it cuts abscissa in Fig. 3 a t 0.0015 instead of 0.002.Note that 10 per cent. reduction of wheal area more than doubles dose required to produce a given effect. Similarly, a 30 per cent. reduction involves a 9 to 10-fold increase, a 60 per cent. reduction an 80 to 100-fold increase and so on. Ordinate : multiple of dose of intracutaneous histamine (log scale). For further explanation see text This affords one way of resolving what many clinicians regard as an impossible discrepancy between pharmacological experiments and clinical experience. In conclusion it should perhaps be pointed out that the methods very briefly outlined here, while probably capable of development to the same degree of precision as other quantita- tive biological methods, have been used simply as a better means than any other available of obtaining comparative information about antihistaminics in man.But no undue stress should be placed on the numerical results. Nor must it ever be forgotten how wide is the individual variation in response to these drugs4 and how necessary it is, in their clinical use, to take account of this. REFEREKCES 1. 2. 3. 4. SCHOOL OF MEDICINE Bain, W. A., Broadbent, J. L., and Warin, R. P., Lancet, 1949, 3, 47. Bain, W. A., PYOC. Roy. SOC. Med., 1949, 42, 615. Bain, W. A., Batty, J. E., and Broadbent, J. L., 1951, Communication to British Pharmacological Bain, W. A., Hellier, F. F., and Warin, R. P., Luncet, 1948, ii, 964. Society, Jan., 1951. DEPARTMENT OF PHARMACOLOGY UNIVERSITY OF LEEDS DISCUSSION MR.N. T. GRIDGEMAN asked if it would not be useful to take, as a measure, response (reduction of PROFESSOR BAIN replied that he thought i t better to deal with these separately. wheal area) multiplied by time from maximum to half-maximum effects.Oct., 19511 SPECIAL REFERENCE TO ANTIHISTAMINICS 579 DR. H. 0. J. COLLIER commented on the difficulties encountered in the experimental development of new paralysants because their relative potencies in man did not agree with their relative potencies in experimental animals. Did the relative potencies of antihistaminics assessed by Professor Bain’s method agree with those obtained by any method using animals ? He thought it was clear, however, that drugs that were very weak antihistaminics by animal tests proved weak in man, but drugs that were powerful in man often did not seem to be so in animal tests.But i t was obviously the results on man that were the more important. DR. L. J. HARRIS enquired whether tests had been made in which two drugs were administered simultaneously-for example, one producing a rapid, short-lived amelioration together with another producing a more prolonged action. Might the resultant time curve be expected to be a summation of the two individual curves? PROFESSOR BAIN said there were differences of opinion on this point. PROFESSOR BAIN replied that this would be expected, but had not been tried. DR. J. V. SMART asked if the magnitude of the initial response to intradermal histamine had any PROFESSOR BAIN replied that there was no apparent correlation, i.e., individual variation in the response DR. J. RAVENTOS wondered what was the explanation of the increased effect of histamine immediately PROFESSOR BAIN said he had originally attributed this to an inhibition of diamine oxidase, subsequently DR. F. J. DYER asked whether there was any difference in response of different age groups. PROFESSOR BAIN said that no clear differences in responses of different age groups had been detected. effect on the amount of an antihistaminic necessary to produce a given response. to an antihistaminic did not seem to depend on the magnitude of the intradermal histamine reaction. after the administration of promethazine that was apparent in one of Professor Bain’s graphs. masked by the antihistaminic action of the drug, but this was by no means certain. The difference between individuals was much greater.

ISSN:0003-2654    

DOI:10.1039/AN9517600573

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Oct., 19511 Inorganic SPECIAL REFERENCE TO ANTIHISTAMINICS Chromatography on 579 Cellulose Part VI The Extraction and Determination of Gold* BY N. F. KEMBER AND R. A. WELLS Two chromatographic methods are described for the quantitative separa- For micro quantities a paper strip In both procedures the Special attention has been given to the separation of gold from the six tion of gold from many other metals. is used and for higher ranges a column of cellulose. solvent is a mixture of wet ethyl acetate and nitric acid. platinum metals. ALTHOUGH it is well known that gold can be separated from the platinum metals by liquid - liquid extraction, the procedure is not recommended for quantitative separations as small amounts of platinum are also extracted. The extraction of gold by paper chromatography with ethyl ether containing dry hydrogen chloride and methyl alcohol as solvent has already been described,l and reference has been made to a method for dealing with larger quantities of gold with the aid of a cellulose column.2 This paper describes improved methods by which gold has been separated and estimated both with the aid of strips of absorbent paper and with columns of cellulose.In both procedures separation from a large number of metals in addition to those of the platinum group has been attained. The extreme volatility of the ethereal solvent previously used for the separation of gold from the platinum metals on strips of filter-paper led to difficulty in maintaining a suitably saturated atmosphere in the separation vessel.Accordingly some other solvents were examined, and ethyl acetate containing 5 per cent. v/v of water and 10 per cent. v/v of nitric acid, sp.gr. 1.42, was found to extract gold very efficiently from a large number of other metals. The test solution, containing up to 15 per cent. w/v of metals, was prepared in moderately strong hydrochloric acid. A measured volume was then placed in a spot on a * Presented at the XIIth International Congress of Pure and Applied Chemistry, New York, September 10th to 13th, 1951.580 KEMBER AKD WELLS: IKORGANIC CHROMATOGRAPHY ON CELLULOSE [Vol. 76 strip of Whatman No. 1 filter-paper, dried for 1 hour and extracted in the manner and with the apparatus described in Part 1I.l The gold was removed from the paper by tapering the strip and allowing the solvent to run off the end; gold was thus isolated in a few drops of organic solvent.Attempts to recover the gold by ashing part of the paper or by washing it from the paper did not give quantitative results. After removal of the organic solvent and solution of the residue in 0.1 N hydrochloric acid, the gold was estimated over the range 1 to 10 pg by the method of Sandell,3 but using $-dimethylbenzylidene-rhodanine, instead of the p-diethyl derivative. Earlier separations of gold from the platinum metals on columns of cellulose were carried out with ethereal solvents (see experimental section) but the solvent finally adopted was ethyl acetate containing 2 per cent. v/v of nitric acid, :;p.gr. 1-42, and 3 per cent. v/v of water. By the use of cellulose columns 2 cm in diameter and 7.5 cm long, quantities of gold in the range 0.0005 to 0-2 g were extracted quantitatively from chloride solutions of mixtures of the platinum metals and a number of other metals.The gold was recovered from the ethyl acetate solvent, prepared in nitrate-free chloride solution and estimated by electrolytic deposition on platinum. Provided that the solution was diluted to contain less than 0-2g of gold per 100m1, no difficulty was experienced in attaining quantitative recovery of gold direct from the chloride solution. PAPER STRIP SEPARATIONS QUALITATIVE EXPERIMENTS- A solution containing 50 pg of gold per 0.05 ml in 2 N hydrochloric acid was used in preliminary tests. As already reported,l use of more diluted acid resulted in partial reduction of the gold during drying of the strip.A spot containing 0-05 ml of the test solution was placed on the paper, which was then allowed to air-dry for at least 1 hour. It will be shown later that a shorter drying time caused iron to move with gold in the solvent front. After running the solvent, the positions of the gold and platinum metals were located by spraying with stannous chloride solution containing a few crystals of potassium iodide, prepared by dissolving 5.6 g of SnC1,.2H20 in 10 ml of concentrated hydrochloric acid, diluting to 100 ml with water and then adding 0.2 g of potassium iodide. This was found to give a more intense black colour with gold than the "Purple of Cassius" formed with stannous chloride alone. After extraction with the recommended solvent no difficulty was experienced in detecting 0.1 pg of gold on a 2.5-cm wide strip.Various mixtures of ethyl ether and methyl alcohol together with concentrated hydro- chloric acid were first examined, but bad trailing of gold invariably occurred. Ethyl acetate with added ethyl alcohol containing concentrated hydrochloric acid was then used in place of the ethereal solvent. The alcohol content was varied over the range 0 to 30 per cent. v/v, and the proportion of the acid was varied between 2 and 40 per cent. v/v of hydrochloric acid, sp.gr. 1.18. Increase of acid and alcohol content improved the extractibn of gold but at the cost of increased movement of platinum metals. The compromise concentrations found most satisfactory were 15 per cent.v/v of ethyl alcohol and 2 per cent. v/v of hydro- chloric acid, sp.gr. 1.18. The ethyl alcohol in the ethyl acetate mixtures was replaced by methyl alcohol, but this led to increased movement of the platinum metals with no improvement in the extraction of gold. This was even more pronounced when methyl acetate was used in place of ethyl acetate. The nitric acid concentration was varied from 1 to 10 per cent. v/v, above which the interaction between acid and solvent became too vigorous. Gold was extracted in the solvent front in a band, which decreased in width as the acid strength increased. The addition of water still further improved the extraction of gold and also resulted in a decrease in the movement of iron. Ethyl acetate containing 10 per cent. v/v of nitric acid, sp.gr.1-42, and 5 per cent. v/v of water (i.e., saturated with water) gave an extremely efficient extraction of gold, as shown in Fig. 1, and was finally adopted as the most favourable solvent. The behaviour of a number of metals under the conditions for gold extraction was examined, with the following results- (1) Pd", Pt"", Rh"', Ni", Co", Cu", Al"., Mn", Cr"', Ca", Sr", Ba", TeO,", Ti"', Na', K', Pb", Ag', VO,' and the rare earths all remained in the original spot. Ethyl acetate containing nitric acid was then examined as a solvent.I25 pg of plat i nu m , etc. 2 5 of gold Fold- Test - Solution ' Fig. 1. Normal strip v A I I I 4cm I 0.15 g of platinum. I etc. F 5cm I x I I I I I I I 1 I I Ocm 1 1 I I I I I I I 1 dr Fig. 3. Quantitative strip 0*0075 g of platinum, etc.2 t-4 of gold (4 ( b ) Fig. 2. Heavily loaded strips ( a ) Developer : Stannous (b) Developer: SO, chloride Fig. 4. Twelve inch wide stripOCt., 19511 PART VI. THE EXTRACTION AXD DETERMINATION OF GOLD 581 (2) Ir””, Os””, Ru”’, Hg”, Cd”, Bi”’, Se03”, Li’, ZrO” and Mg” moved only slightly from the original spot. (3) Zn”, As04’” and MOO,” moved and had the following approximate RF values: Zn = 0-32, As = 0.48, Mo = 0.50. (4) Sn”, Sn””, UO,” and Sb”’ moved with gold in the solvent front. (5) The movement of iron was very sensitive to changes in water and acid concentra- tion. With dry ethyl acetate containing 10 per cent. v/v of nitric acid, sp.gr. 1.42, as solvent, iron moved with gold in the solvent front. The addition of 5 per cent.v/v of water to the solvent resulted in a decrease in the movement of iron. The precise extent of this decrease was dependent on the time of drying of the original spot. If the original solution was prepared in 2 N hydrochloric acid and the strip dried for 1 hour, the RF value for the iron was about 0-5; on drying for 2 hours little or no movement of iron was observed. Very strong solutions of the platinum metals can be dealt with and solutions containing a total of 15 per cent. by weight of metals were used. At this concentration movement of some of the platinum metals from the original spot occurred (see Fig. 2), but not to an extent that would affect the subsequent detection and estimation of gold. QUANTITATIVE EXPERIMENTS- For experiments on quantitative separations and estimations, three solutions were prepared to contain 1, 5 and 10 pg of gold per 0.05 ml.In addition, each contained 12.5 g of platinum metals in the ratio of 4 parts of platinum to 3 of palladium, 1 of rhodium, 1 of iridium and 1 of rubidium, together with 2 g of nickel and 0.5 g of iron per 100 ml. The solutions were prepared in 2 N hydrochloric acid and, to overcome the tendency for gold to precipitate from the strong platinum metal solutions, a few crystals of sodium chlorate were added. The use of the sodium salts of the metals, the presence of an excess of sodium chloride or a small amount of nitric acid was not found to affect the extraction. With an “Agla” pipette, 0.05 ml of the test solution was placed on the strip and allowed to dry for 1 hour.In initial experiments strips 2.5 cm wide and 30 cm long were used and, after running the solvent for 1 hour, the gold was recovered from the small area of cellulose near the solvent front, in which .it was concentrated. Various methods were examined for the recovery of gold from the cellulose, as follows- (a) The paper strip was cut 03cm below and 16cm above the solvent front. The small section of paper was ignited and the ash dissolved in aqua regia. The acid was allowed to evaporate under atmospheric conditions and the gold was estimated in the residue. (b) The strip was cut as in (a) and the paper was destroyed with a mixture of fuming nitric and perchloric acids. The solution was evaporated to dryness and the residue redissolved in 0.1 N hydrochloric acid saturated with bromine.The bromine was removed by bubbling air through the solution and the gold estimated in the dilute acid. Results were low and erratic. A section of the strip was removed as in (a) and extracted with successive small quantities of 0.1 N hydrochloric acid. Results were very low, and the experiments were repeated with 0.1 N hydrochloric acid saturated with bromine. After removal of bromine by bubbling air through the solution the gold was estimated, but the recovery was usually only 90 per cent. As these methods failed to give satisfactory results, the strips were tapered and the solvent allowed to drip from the pointed end of the paper into a small beaker. Conditions were standardised as regards dimensions of the strip and time of running.With strips of the dimensions shown in Fig. 3, the solvent reached the bottom of the strip in 40 minutes and was allowed to run for a further 40 minutes. The collected solvent was evaporated just to dryness on a water-bath and the residue dissolved with slight warming in 03ml of 0.1 N hydrochloric acid saturated with bromine. The bromine solution was washed into a 5-ml graduated flask with 3.5 ml of 0.1 N hydrochloric acid and, after aeration to remove bromine, the gold in solution was estimated colorimetrically with p-dimethylben- zylidene-rhodanine. The volume of rhodanine reagent used was less than that recommended by Sandell. With the dimethyl derivative it was found that the yellow colour of the reagent interfered with the estimation of quantities of gold less than 2.5 pg if the quantity of reagent Results were very erratic.(c)582 KEMBER AND WELLS: INORGANIC CHROMATOGRAPHY ON CELLULOSE [VOl. 76 was not reduced. and the following method was adopted. Satisfactory recovery and estimation of gold was achieved by this technique METHOD FOR THE SEPARATION. AND ESTIMATION OF 1 TO 10 pg OF GOLD REAGENTS- of AnalaR ethyl acetate. Ethyl acetate solvent-Add 5 ml of water and 10 ml of nitric acid, sp.gr. 1.42, to 85 ml Hydrochloric acid, 0.1 N . Hydrochloric acid, 0.1 N , saturated with bromim-Make from a portion of the reagent Sodium juoride solution, 1 per cent. w/v-Dissolve 1 g of sodium fluoride in 100 ml of p-Dimethylbenzylidene-rh.odanine sohtion-Dissolve 0.05 g of solid reagent in 100 ml of 0.1 N hydrochloric acid by shaking with sufficient bromine.water. absolute alcohol. PROCEDURE- To the test solution, prepared in 2 N hydrochloric acid, add a few crystals of sodium chlorate. With an “Agla” pipette place 0.05 ml of test solution in a band near the top of the strip of Whatman No. 1 filter-paper (see Fig. 3) and allow to dry for 1 hour in the air. Place the strip with the wide end in the solvent container and allow the tapered end to hang in a 25-ml beaker. Any of the conventional forms of capillary descent apparatus is suitable. Add the ethyl acetate solvent and allow it to flow clown the strip for 80 minutes. Evaporate the solvent that collects in the beaker to dryness on a steam-bath. Add 0.5 ml of 0.1 N hydrochloric acid saturated with bromine to the residue and warm gently.Wash the solution into a 5-ml graduated flask with 3.5 ml of 0-1 N hydrochloric acid and remove the excess of bromine by drawing a brisk stream of air through the solution for 10 minutes. Add 0.25 ml of 1 per cent. w/v sodium fluoride solution to complex any traces of iron impurity from paper and reagents, and then accurately add 0.2 ml of a 0.05 per cent. solution of p-dimethylbenzylidene-rhodanine solution, preferably from an “Agla” pipette. Make up to the 5-ml mark with 0.1 N hydrochloric acid, shake the flask and allow it to stand for 10 minutes. The same hydrochloric acid solution should be used throughout the estimations as the colour reaction is sensitive to acidity. Determine the transmission of the solution on a Beckmann spectrophotometer after 10 minutes, accurately measured from time of addition of the rhodanine reagent.IJse a wavelength of 480 mp and slit-width of 0.1 mm. Read off the gold concentration from a previously prepared graph. Prepare the calibration graph with the aid of standard gold solutions in 0.1 N hydrochloric acid, check it frequently and construct a new graph with each new batch of reagents. RESULTS- estimations was made, as shown in Table I. With chloride solutions containing 15 per cent. w/v of metals a number of satisfactory TABLE I ESTIMATION OF GOLD I N PRESENCE OF LARGE AMOUNTS O F OTHER METALS Weight of metal present A I 7 Weight of gold Weight of gold 6250 1000 250 1.0 1.0, 1.0, 1.0 6250 1000 250 10.0 9.9, 10.0, 10.0 Iron, taken, found, Platinum metals, Nickel, pg/O.O5 ml pg/0.05 ml pg/0*05 ml pgl0.05 ml p.g/0-05 ml 6250 1000 250 5.0 5.2, 4.9 The time taken to complete one separation and estimation was 3 hours, although the time for which the operator was actively employed was considerably less.By hanging the strip in a suitably designed tank, several dozen separations have been made concurrently. Estimations have been made over a range limited by the colorimetric method, but there appears no reason to doubt that the separation is quantitative for both larger and smaller quantities of gold. The sensitivity of the method could be further increased by the use ofOCt., 19511 PART VI. THE EXTRACTION AND DETERMINATION OF GOLD 583 thicker paper, e.g., Whatman No. 3, on which a larger volume of test solution could be placed, or by using wider strips of paper.Strips up to 12 inches wide, on which it is possible to place 1 ml of solution containing 0.15 g of metal, have been used; such a strip is shown in Fig. 4. The method provides a speedy and accurate means for the detection and estimation of small quantities of gold, and requires a minimum of manipulative skill. COLUMN SEPARATION The use of cellulose columns in the separation of metals has been recorded in a number of previous reports. The procedure for the separation of gold described in the present paper is essentially the same as that for the separation of uranium described in a previous paper4 where the extraction tube, its treatment with a water repellant and the packing of the column are also described, together with the method of pulp preparation.THE SOLVENT- The solvent recommended is ethyl acetate containing 2 per cent. v/v of nitric acid, sp.gr. 1-42, and 3 per cent. v/v of water. Earlier experiments were made with ethereal solvents containing hydrogen chloride. The acid was added both as the dry gas and in aqueous solution. The effect of variation in the concentration of the dry gas, in the con- centration of aqueous acid used and in the amount of aqueous solution added was studied. In all experiments the majority of the gold was readily extracted but a small part of it trailed badly. This effect was particularly noticeable at low concentrations of acid, when a tendency for gold to reduce to the metal became apparent. Addition of methanol to the ether enabled a larger volume of aqueous acid to be added and this resulted in a gradual improvement in the extraction of gold as the methanol content of the solvent was increased, but a greater tendency for the movement of the platinum metals was observed. Similar results were attained with acetone in place of methyl alcohol.The results were good for some determinations with solvents containing as much as 10 per cent. v/v of methyl alcohol, but tended to be erratic owing to streaking of platinum metals down the column. In view of the favourable results for paper-strip separations with ethyl acetate as solvent, the ethereal solvent was abandoned in favour of the ester. The movement of gold and the platinum metals was investigated using as solvent ethyl acetate with additions of from 1 to 5 per cent.v/v of concentrated nitric acid. With 1 per cent. v/v of acid most of the gold was extracted by 60 ml of solvent, but excessive trailing of traces was observed. This was avoided by increasing the acid concentration to 2 per cent. v/v. The movement of the platinum metals increased with increase of acidity so that ethyl acetate with 2 per cent. v/v of nitric acid, sp.gr. 1.42, was used in further experiments. In these experiments a solution of the chlorides of gold and the platinum metals in 2 N hydrochloric acid was used, but, as will be explained in the next section, a solution of the sodium chloro-salts is preferred. With these salts a slower rate of extraction was observed and traces of gold still remained in the column after the passage of 150ml of solvent. Saturation of the solvent with water prevented this effect and gold was completely extracted by 100 ml of ethyl acetate containing 2 per cent.v/v of nitric acid, sp.gr. 1-42, and 3 per cent. v/v water. The solvent was freshly prepared from AnalaR ethyl acetate before each extraction. Ethyl alcohol present in com- mercial ethyl acetate gave rise to excessive movement of the platinum metals, particularly iridium. Only slight improvement was possible by further increasing the acidity. PREPARATION OF THE SOLUTION- In preliminary experiments with ethyl acetate containing nitric acid as solvent, traces of platinum were found in the eluate together with the gold, although little movement of the platinum metals was apparent in the column. Different methods of preparation of the solution were therefore investigated.In the first, chloroplatinic acid was used; in the second a solution of chloroplatinic acid in dilute hydrochloric acid was evaporated to dryness with an excess of sodium chloride; in the third, chloroplatinic acid was fused with an excess of sodium chloride in a stream of chlorine at a dull red heat. Each of these products was dissolved in 5 ml of 2 N hydrochloric acid and extracted with 100 ml of solvent through a 5-cm column. Platinum was found in the eluate from the first but not in the second or third. In further584 KEMBER AND WELLS : INORGANIC CHROMATOGRAPHY ON CELLULOSE {Vol. 76 experiments the sodium salts were therefore used. A large excess of sodium chloride was avoided because it crystallised out of solution, occluding gold and causing trailing in the column.A tendency for small amounts of gold to precipitate from solution, particularly in the presence of a large excess of platinum metals, was overcome by addition of nitric acid to the test solution. This addition, however, tended to inhibit the extraction of gold, and in further experiments the test solution was prepared by dissolving the sodium salts in a mixture of 1 ml of concentrated hydrochloric acid saturated with chlorine gas and 4 ml of water. This permitted the complete extraction of gold with but little trailing. The presence of an excess of chlorine, e.g., by use of saturated chlorine water, accelerated the movement of iridium. THE EXTRACTION TUBE AND CELLULOSE PULP- The glass tubes used for extraction were similar to those described in Part V,4 but the method of treating the inner surface with a water repellent silicone derivative was modified.Instead of the obnoxious dichlorodimethylsilane previously used, silicone Fluid 200, supplied by the Dow Corning Co. of America, was used. This, properly a ~ p l i e d , ~ resulted in a surface that prevented "wall" effects. The cleaned and dried tube was filled with a 2 per cent. solution of silicone fluid in carbon tetrachloride and allowed to stand for half an hour. After draining, the tube was heated to 260" C and maintained at this temperature for 3 hours. The process was repeated if necessary. The pulp used was prepared by the action of dilute nitric acid on Whatman Ashless Tablets. A fin& commercially-available pulp was tried but proved too retentive of gold.In qddition it contained a large proportion of extractable organic matter, which interfered with subsequent analysis. THE EXTRACTION PROCEDURE- A column of cellulose 7.5 cm long and 2 cm in diameter was prepared by packing it sufficiently tightly in ethyl acetate- nitric acid solvent to allow 100 ml of solvent to be collected in 45 minutes. The test solution was absorbed on about 2 g of cellulose pulp, transferred to the extraction tube, beaten up with a plunger and packed down to form a continuous part of the column (see Part V4). By this method of addition of the sample, which was found preferable to adding the solution directly to the top of the tube, the aqueous phase was evenly distributed throughout a comparatively large volume of cellulose.Direct addition resulted in the top part of the cellulose column becoming completely saturated with the viscous aqueous phase, which tended to act as a barrier to the organic solvent. Solvent was then run through the tube until 100ml had been collected. This volume was found sufficient to extract gold completely and without the platinum metals. Water was added and the solvent removed by evaporation. Gold was estimated in the residue as described later. BEHAVIOUR OF OTHER METALS- The movement of a number of metals other than gold under the same conditions of extraction was investigated and the results are recorded in Table 11. TABLE XI BEHAVIOUR OF METALS OTHER THAN GOLD ON THE CELLULOSE COLUMN Metal Movement down column, cm Detected in eluate Fe"' .... .. .. . . See notes below UO" .. . . .. .. . . Extracted in solvent front but trailed badly. Zn" . . . . .. .. .. . . Extracted in solvent front CU" . . .. . . .. .. .. 2 nil pt ."., pd", Ir*--*, Rh**., RU"* .. 1 nil Ni" . . .. .. .. .. .. nil nil CO" .. . . .. .. .. nil nil Mn" .. .. .. .. .. nil nil *Pb" . . .. .. .. .. .. nil nil *Age . . . . .. .. .. .. nil nil Incomplete extraction * A paste of the chlorides was used in these experiments but to avoid possible occlusion of gold, insoluble chlorides were normally removed before extraction.OCt., 19511 PART VI. THE EXTRACTION AND DETERMINATION OF GOLD 585 When no other metal was present, ferric iron was extracted because it moved in the solvent front, although as it trailed considerably there was only partial extraction. In the presence of gold, iron moved behind the gold in a well defined front and started to emerge from the column after the passage of about 80ml of solvent. The addition of phosphoric acid to the original solution did not affect the movement of iron.RECOVERY AND ESTIMATION OF GOLD- After removal of the organic solvent from the column eluate, a solution of gold in dilute aqua regia was left. This was evaporated with concentrated hydrochloric acid to remove nitric acid, and the gold was then estimated in the chloric solution by a suitable method. Al R e Al R Fig. 5. Macro electrolytic cell Fig. 6. Semi-micro electrolytic cell. (Scale = 2/3 actual size) When the amount of gold in the eluate was less than 20mg, the organic matter present (paper, dust, etc.) brought about partial reduction of the gold solution.To prevent this the eluate was evaporated to dryness in a silica crucible and incinerated a t 600" C; the gold was redissolved in aqua regia. In the initial experiments the gold was precipitated with sulphur dioxide, but the electro- lytic method developed later was much preferred. The electrolytic estimation of gold in cyanide solution has been described,6 and we have achieved some success with this medium, particularly for very small amounts of gold. A method for the electrolysis of the chloride solution has, however, been developed and found satisfactory over the range 0.005 to 0.200 g of gold. The apparatus used for the recovery of quantities between 0.010 and 0.200 g is shown in Fig.5. It consisted of a 100-ml beaker in which was arranged an 80-mesh platinum- gauze cathode of 3.5 cm diameter and 3 cm height surrounding a platinum-wire spiral anode.586 KEMBER AND WELLS : INORGANIC CHROMATOGRAPHY ON CELLULOSE [F'ol. 76 The solution was agitated by hydrogen or air bubbled through a tube between the anode and cathode and the whole was heated by placing the beaker on a hot plate; to prevent loss as spray the beaker was covered by a suitably designed polythene top. Full-wave rectified A.C. was used with a variable resistance to adjust the current. The following factors were found to be important for quantitative yields. Cortcentration-Complete recovery of gold was iound possible only from a solution containing less than 0.2 g of gold per 100 ml of 2 N hydrochloric acid.At higher concentra- tions of gold chlorine was liberated at the anode. Voltage-A voltage of 2.5 to 3.5 v. was used. Higher voltages up to 12 v. gave low results owing to the formation of oxidising agents, such as chlorine, at the anode. Current-A current density of 1.5 to 3-0mA per sq. cm was used. Higher currents gave low results for the same reason as did high voltages. A darkening of the electrolyte adjacent to the anode or evolution of gas at the cathode were signs of oxidation taking place. Temperature of electrolyte and agitation-The rate of electrolysis was found to increase very rapidly with increase of temperature and electrolysis was carried out at 50" to 70" C. Rapid agitation of the solution was found to be of the utmost importance and a fast stream of air or hydrogen was used.Under these conditions the electrolysis was complete in 30 minutes. For quantities of gold between 1 and lOmg, a semi-micro cell as shown in Fig. 6, of total capacity 35 ml, and designed to give greater agitation, was used. The conditions of voltage and current were the same. In each type of cell the gold chloride solution was reduced to a suitable volume and made 2 N with respect to hydrochloric acid. For quantities less than 1 mg a micro cell of similar design to the macro cell but of 15 ml total capacity was used together with a cyanide base solution. METHOD FOR THE SEPARATION AND ESTIMATION OF 0.0005 TO 0.20g OF GOLD REAGENTS- Hydrochloric acid containing chlorine-Pass dry chlorine into concentrated hydrochloric acid until a pale yellow solution is obtained. Ethyl acetate solvent-Add 4 ml of nitric acid, sp.gr.1.42, and 6 ml of water to 190 ml of AnalaR ethyl acetate. Cellulose @l+-To 4 litres of boiling 5 per cent. v/v nitric acid add 100 Whatman Ashless Tablets. Wash free from acid with water and then remove water by washing first with alcohol and then with ether. PROCEDURE- Prepare a solution of the sample containing up to 0-25 g of platinum metals and gold in aqua regia and evaporate to a syrupy mass on a steam-bath. Add 5 ml of hydrochloric acid, sp.gr. 1.18, and repeat the evaporation. To the residue add 10 ml of 2 N hydrochloric acid and 0-5 g of sodium chloride and take nearly to dryness. Dissolve the sodium salts in a mixture of 1 ml of concentrated hydrochloric acid containing chlorine and 4 ml of water.Prepare 200 ml of ethyl acetate solvent and pack an extraction tube with cellulose pulp to a depth of 7.5 cm in the organic solvent. Add 2 g of pulp to the test solution, stir to form a friable mass and transfer it to the top of the column. Beat up the added cellulose and pack it to form a continuous column. Extract with 100ml of solvent, using 10-ml fractions of solvent, each of which is used to wash the sample beaker. Collect the eluate, which should run through in 45 minutes, in a 500-1-111 flask without allowing the column to run dry at any stage. Add 5ml of hydrochloric acid, sp.gr. 1.18, to the residue and evaporate to a small volume. Wash the gold solution into the appropriate electrolytic cell (see Figs.5 and 6) with 2 N hydrochloric acid and heat to 50" C. Agitate the solution with a stream of hydrogen or air. Place the tared cathode in the electrolyte and electrolyse a t a current density of 1-5 to 3 mA per sq. cm and at an e.m.f. of 2-5 to 3.5 volts. After 30 minutes, remove the cathode, wash it with distilled water, then with acetone and dry it at 110" C. Weigh the cathode, replace it in the cell and electrolyse for it further 15 minutes. Wash, dry and reweigh the cathode, repeating the procedure until a constant weight is reached. Several electrolyses may be carried out on the same cathode without removal of the gold. To remove the gold, immerse the cathode in potassium cyanide solution containing a few drops of hydrogen peroxide.Boil for 2 minutes, dilute with cold water and filter rapidly. Add 50ml of water to the eluate and distil off the solvent.Oct., 19511 PART VI. THE EXTRACTION AND DETERMINATION OF GOLD 587 RE su LT s- The results of direct electrolyses of solutions of known strength in Table I11 indicated that recovery of gold by electrolysis was satisfactory and further determinations were made on gold solutions prepared from column separations (see Table IV). TABLE I11 DIRECT ELECTROLYSIS OF SOLUTIONS OF GOLD OF KNOWN STRENGTH Metals present Weight of gold, Gold . . .. .. ,. 7, . . .. .. .. 5) . . .. .. .. 3) . . .. .. .. Gold + 1 g of FeCl, . . 0.5 g of UO, in HCl . . .. .. RESULTS OF ELECTROLYSIS OF Weight of platinum metals” Weight of present, gold present, g g nil 0.1857 0.1250 0.00050 0.0500 0.1571 0.0500 0.2013 0.1250 0.0845 0.1250 0.1260 0-1250 0.1810 0.2500 0.0526 0.2500 0-0045 0.2500 0.0130 0.0500t 0- 1107 g 0*00050 0.0050 0.1055 0.2046 0.1055 nil TABLE SOLUTIONS OF Weight of gold found, g 0.1855 0.00049 0.1570 0-2009 0.0844 0.1258 0.1808 0.0526 0-0046 0.0130 0.1 107 Type of Increase in weight solution Cell used of cathode, g cyanide micro 0.000496 chloride semi-micro 0.00489 Y Y macro 0.1053 Y 3 9, 0.2047 Y Y 73 0.1054 Y Y Y, 0~0001 GOLD PREPARED FROM COLUMN ELUATES Method of estimation Cell used SO, precipitate - cyanide electrolysis micro chloride electrolysis macro >> Y Y Y > YY 19 Y, Y t semi-micro macro YY * Platinum metals were present in the ratio-Pt, 4 : Pd, 3 : Ir, 1 : Rh, 1 : Ru, 1.t In this experiment the following metals were also present to a total weight of 0.25 g-Fe, Cu, Ni, Co, Mn. These determinations were considered to be satisfactory. The maximum error was 2 per cent. for 5mg or less of gold; for larger quantities the maximum error decreased to 0.2 per cent. The method is speedy and, like the paper-strip separation, gives good results when the gold is present with those metals giving maximum interference in normal analysis. The authors are indebted to Miss’ P. Swain for experimental assistance. This work was carried out on behalf of the Ministry of Supply, by whose permission it is published. REFERENCES 1. 2. 3. 4. 5. 6. Burstall, F. H., Davies, G. R., Linstead, R. P., and Wells, R. A., J . Chem. Soc., 1950, 516. Burstall, F. H., Davies, G. R., and Wells, R. A., Disc. Farad. Sac., 1949, No. 7, 179. Sandell, E. B., Anal. Chem., 1948, 20, 253. Burstall, F. H., and Wells, R. A., Analyst, 1951, 76, 396. Johannson, 0. K., and Torok, J. J., Proc. Inst. Radio Eng., N.Y., 1946, 34, 296. Scott, W. W., and Furman, N. H., “Standard Methods of Chemical Analysis,” Fifth Edition, D. Van Nostrand Co. Inc., New York, and The Technical Press Ltd., London, 1939, Volume I, p. 435. CHEMICAL RESEARCH LABORATORY TEDDINGTON, MIDDLESEX April, 1951

ISSN:0003-2654    

DOI:10.1039/AN9517600579

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

588 JONES: A PLATE ASSAY TECHNIQUE; FOR INOSITOL IN YEAST [Vol. 76 A Plate Assay Technique for Iaositol in Yeast BY A. JON.ES A Petri-plate technique is proposed for the assay of inositol in yeast by means of Saccharonzyces carlsbergeizsis; and, for one yeast, the results are compared with those of the h7eurosPora method. INOSITOL has been assayed in various products with the yeasts Saccharomyces cereviseae172 and Saccharomyces ca~lsbergensis~ and also with the inositol-free mutant 37401 of NeurosPora c~assa.~ Plate assays have been used previously for assaying several of the vitamins of the B complex5s6s7s8 and the possibility was therefore investigated of using a similar technique for inositol to that used for the assay of vitamin B6.9 In these laboratories the Neurospora assay for inositol was found to be unsatisfactory and a more reliable method, such as a plate assay with its simple apparatus, was desirable.EXTRACTION OF INOSITOL- Beadle4 and WoolleylJo used hydrolysis with either 3 per cent. sulphuric acid or 16 per cent. hydrochloric acid. The latter appeared to give a better extraction of inositol from dry brewer's yeast.4 Several methods of extraction were investigated in the plate assay, i.e., hydrolysis with N , 2 N , 3 N and 5 N hydrochloric acid and N , 3 N and 5 N sulphuric acid, and also with takadiastase plus papain, in which method 0.3 g of takadiastase and TABLE I METHODS OF EXTRACTION OF INOSITOL FROM YEAST Inositol content, mg/g A f \ Extraction method HCl N .. 2 N .. 3 N .. 5 N .. %SO, N .. 3 N .. 5 N .. Takadiastase + papain .... .. .. .. .. .. .. Yeast 129 r-A-, Mean 2.75, 2.75 2.75 2.94, 3.06, 2-76, 2.76, 2-96, 2.61, 2.50, 2-53, 2.72 2.48, 2.70, 2-66, 2.67, 2-70, 2.75 1.95, 2.40, 2.22 2.33, 2.19 2-36, 1-97, 2.06, 2.18, 2-16, 1.70, 1.95 1.78, 1-83, 1-70, 1.76 0.99, 1.08 1.04 Yeast 885 Yeast Y r+ Mean Mean 1.44 1 4 4 1.44 1.44 1.99, 2-04, 2.75, 3.33, 2-04, 1.83 1.98 3.08, 3-44, 3-37, 3.36, 3.27, 3.39, 3.14 2-79, 2.70, 3.00, 3.12, 3.19, 3.12, 3.23 0-98, 1.10 1.04 0.90 0.90 0.3 g of papain were added to 2.0 g of yeast in 15 ml of acetate buffer at pH 4.5 and incubated overnight at 37" C. When either hydrochloric or sulphuric acid was used, the yeast samples were hydrolysed with 10 times their weight of the appropriate acid. After hydrolysis by autoclaving at 15 lb pressure for 2 hours with sulphuric acid, or for 1 or 2 hours with hydro- chloric acid, the pH value of the sample was adjusted to 4.6 with sodium hydroxide.When hydrochloric acid was used, heating in an autoclave for 2 hours appeared to extract no more inositol than heating for 1 hour.OCt., 19511 JONES: A PLATE ASSAY TECHNIQUE FOR INOSITOL I N YEAST 589 The high concentrations of sodium chloride in the solutions after neutralisation of the yeast samples hydrolysed by 5 N hydrochloric acid did not appear to have any effect on the growth of S. carlsbergensis on the plates, but when 5 N sulphuric acid was used the sodium sulphate formed on neutralisation crystallised out readily and led to error in assessing the volume of the samples. No more inositol appeared to be released by 5 N than by 3 N hydrochloric acid, but the definition of the growth-zones after extraction with 5 N acid was generally better than with 3 N .Takadiastase plus papain appeared to release little inositol from yeast; this is in agreement with the findings of Beadle.4 Hydrolysis with hydrochloric acid gave higher results for yeast than did sulphuric acid. The results of these different methods of hydrolysis are shown in Table I. BASAL MEDIUM- The basal medium was essentially that of Atkin, Schultz, Williams and Frey,ll with the addition of 2.5 per cent. of agar. The higher concentration of agar was found necessary during the summer months, when growth zones tended to be less sharp with 2 per cent. of agar, as was shown with the basal mediumg used for vitamin B,.METHOD Distribute the medium (Table 11) in volumes of 20 ml in boiling-tubes and sterilise by steam for 20 minutes. TABLE I1 BASAL MEDIUM Glucose . . .. .. .. 50g Aneurine hydrochloride . . 250 pg Potassium dihydrogen phosphate 0.55 g Calcium pantothenate . . 1000 pg Citric acid .. .. .. 1.0 g Pyridoxine hydrochloride . . 1000 pg Casein hydrolysate . . .. 4.0 g Salt solution*. . .. .. 25ml Volume made up to . . . . 900ml pH value adjusted to . . .. 4-6 Agar .. .. .. . . 22.5 g .. .. 8 CCg Potassium citrate .. .. 5.0 g Biotin . . .. Nicotinic acid . . .. .. 1000 pg * The salt solution contains 1.7 g of KC1; 0.5 g of CaC4.6KO ; 0-5 g of MgS0,.7H20; 0.01 g of FeC1,.6H20 and 0.01 g of MnS0,.4H20 per 100 ml. PREPARATION OF THE INOCULUM- Maintain S.carlsbergensis by fortnightly sub-cultures on 4 per cent. malt agar, at a pH value of 5 to 5-6, in 1-ounce screw-capped bottles, with incubation for 18 to 24 hours at 28" to 30°C. Wash off the growth of yeast from each bottle with 10 ml of sterile saline, centrifuge, wash once with saline and resuspend the yeast growth in sufficient saline solution to make the opacity of the suspension correspond to tube No. 9 or 10 of the Wellcome standard opacity tubes. PREPARATION OF THE PETRI PLATES- Take five tubes for the standard and five for each test and hold at a temperature of 48" to 50" C. Add 1.0 ml of inoculum suspension to each tube, mix thoroughly before pouring and allow the plates to cool on a flat surface. Cut four holes in each plate by means of a 10 mm cork-borer and remove the agar discs.Just before use, heat the tubes in a bath of boiling water. PREPARATION OF THE YEAST SAMPLES- To 2.0 g of yeast add 20 ml of 5 N hydrochloric acid and sterilise at 15 lb pressure in the autoclave for 1 hour. Cool and adjust the pH value to 4.6 by the careful addition of approximately 50 per cent. sodium hydroxide solution. Adjust the volume to give an estimated concentration of approximately 400pg of inositol per ml, filter and dilute the filtrate 1 to 2, 1 to 4 and 1 to 8.590 JONES: A PLATE ASSAY TECHNIQUE FOR INOSITOL 1s YEAST [Vol. 76 Prepare standard plates to cover the rapge 50, 100, 200 and 400 pg of inositol per ml. Place 0-1 ml of each dilution of the standard and of the test solutions in the appropriate holes of the Petri plates, and incubate a t 28" to 30" C overnight. Yeast 12.5 25 50 100 mg per mi Standard 50 100 200 4 0 0 ~ per mi Fig. 1.Dose Dose - response curves for standard and for yeast Measure the diameters of the growth-zones in millimetres. For the standards, plot the mean diameters against the concentrations in pg per ml. For the samples, plot the mean diameters against the concentrations in mg per ml. As in the vitamin B, assay,n the effect of doubling the concentration of the substance being estimated is to increase the zone diameters by 2.5 mm. TABLE 11" ZONE DIAMETER READINGS FOR STANDARD INOSITOL AND A YEAST SAMPLE Inositol standard Concentration, pg/ml r 50 mm 25.0 25.5 25.5 25-0 25-5 100 mm 27.5 28.0 28-0 28.0 28.0 200 mm 30.0 30-5 30.5 30.5 30.5 3 400 mm 32.5 33.0 33-0 33.0 33.0 25.3 27.9 30.4 32.9 Yeast sample Concentration, mg/ml 12.5 mm 23.0 22-5 23.0 22-5 23.0 25 mm 25.5 25.0 25.5 25.0 25.5 50 mm 28.5 27.5 28.0 27.5 28.0 100 mm 31.0 30.0 30.5 30.0 30.5 22.8 25.3 27.9 30-4Oct., 19511 JONES: A PLATE ASSAY TECHKIQUE FOR INCSITOL IN YEAST RESULTS 591 Table I11 and Fig.1, in which the zone diameters are given to the nearest 0.5 mm, show typical results for a standard and for a yeast sample a t various dose levels, From these can be seen the spread of the readings for any given dose throughout the five plates, as also the linearity of the dose response to the standard and to the yeast sample. In a recovery experiment a yeast sample solution was taken, containing 200 ,ug of inositol per ml (mean value of four levels of assay), and to 10 ml of this solution were added (a) 10 ml of a solution containing 200 pg of inositol per ml and (6) 10 ml of a solution containing 400 pug of inositol per ml.They gave mean assay values of (a) 200 pg of inositol per ml and (b) 303 pg of inositol per ml. These mixtures were assayed at four levels. COMPARISON WITH THE Neuros$ora ASSAY- the same hydrolysed samples being used in both assays. of N . crassa was found to be essential for a reasonably satisfactory assay. in two separate assays are shown in Table IV. For yeast 129 a comparison was made between the plate and Neurospora methods, A heavy inoculum of the spores The values obtained TABLE IV A COMPARISON OF INOSITOL ASSAY VALUES BY S carlsbergensis AND Newospora Yeast 129 S. carlsbergensis Autoclaved 15 lb pressure for Xeuvospova assay plate assay 1 hour with 5 N HC1 Inositol, Inositol, mg/g mg/g First assay 1 .. .. .. 3.21 2-04 2 . . .. .. 3.33 3-06 Second assay 1 . . .. .. 3.83 2 .. .. .. 3-92 3 .. .. .. 3-89 4 .. .. .. 3.79 5 .. a . .. 4-01 2- 70 2-75 2.70 2.66 2.67 The agreement shown by the first assay is good, but in the second, the Neurospora method shows considerably higher values. These may be accounted for by the fact that the growth responses to the standard inositol in the Neurospora assay were almost identical in both assays, but the growth responses to the test samples were very much greater in the TABLE V RESULTS OBTAINED IN A (2 + 2) ASSAY OF INOSITOL I N YEAST Zone diameters in millimeters 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Totals Means Standards lob pg/ml 31.5 29.5 32-0 31.5 31.5 31.0 31.0 31-5 31.0 31-5 31.5 31-0 31.0 31.0 31.0 31.0 ..498.5 1 400 pg/ml 36-5 35.0 37.0 36.5 36.5 36.0 36.5 36-5 36-0 36.5 36.5 36.0 36.0 36.0 36.0 36.0 579.5 7 1 to 60 30.0 29.0 30.0 29.0 29.0 29.0 30.0 29.5 29.0 29.0 29.0 28.5 28.5 29.0 29- 0 29.0 466.5 31.156 36.219 29,156 .. Yeast -7 A- 1 to 15 35.0 34.0 35.0 34.0 34.0 34.0 35.0 34.5 34.5 34.0 34.0 33.5 33.5 34.0 34.0 34.0 547.0 34.187592 [Vol. 76 second. Other Neurospora assays on yeast 129 failed through lack of growth in two-thirds of the flasks. Table V shows the results obtained in a (2 + 2) assay using 16 plates, with a dose-ratio for standard and yeast Y of 4 to 1. When these results were submitted to variance analysis, no significant difference was found between the slopes for standard and test sample; this confirmed the validity of the assay technique.The estimated potency from the 64 readings was 3.29 mg of inositol per g with limits of 3.05 to 3.54 mg per g at a probability level of 5 per cent. From the 20 readings of the first 5 plates, which showed the greatest variation in response, the estimated potency was 3-60 mg per g with limits of 3.12 to 4-20 mg per g (Table VI). JONES: A PLATE ASSAY TECHNIQUE FOR INOSITOL I N YEAST TABLE V I VARIANCE ANALYSIS OF (2 + 2) ASSAY AS IN TABLE v Treatment Degrees of freedom Variance Difference between samples . . 1 Linear regression .. .. 1 Unparallelism , . .. 4 . 1 Error . . .. .. .. 60 65-00 407.59 0.0039 0.27 Difference between samples . .1 17.112 127-51 { Error . . . . .. .. 16 0.28 Linear regression . . .. .. 1 5 Plates Unparallelism . . .. .. 1 0.0125 The average value, 3.14 mg per g, for yeast Y (Table I) obtained from a total of 76 plates, assayed on different days, is in good agreement with the value of 3.29 mg per g from the 64 plates in the (2 + 2) assay. SENSITIVITY OF METHOD- By this method amounts of inositol of 20pg per ml can be detected, but for greater precision the extracts should contain at least 60 pg per ml, which is equivalent to 0.9 to 1-0 mg of inositol per g of original sample. The sensiti.vity and range of the assay method are, possibly, not as great as could be desired, but, against that, it is rapid and simple. The Neurospora method has the advantage of a lower assay level, but the disadvantage of a long incubation period and irregularity in the mould growth in the liquid medium.This irregularity appears to be an inherent defect in all Neurospora assays, and it has been all too frequently encountered in these laboratories with the N . crassa mutants in the assays of inositol, choline and biotin, and with N . sitophila in the vitamin B, assay. The plate assay has also been used to determine inositol in molasses (2.80 mg per g). Thanks are due to the Directors of Beecham Research Laboratories, Limited, for permission to publish,^ to Dr. S. Morris for his interest and encouragement in this work, and to Mr. V. Thorne for assistance with the statistical analysis. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. REFERENCES Woolley, D. W., J . Biol. Chem., 1941, 140, 453. Williams, R. J., Stout, A. K., Mitchell, H. K., and McMahan, J. R., University of Texas Some, S., and Sobotka, H., Arch. Biochem., 1947, 14, 93. Beadle, G. W., J . Biol. Chem., 1944, 156, 683. Bacharach, A. L., Nature, 1947, 160, 640. Jones, A., and Morris, S., Analyst, 1949, 74, 333. Bacharach, A. L., and Cuthbertson, W. F. J., Ibid., 1948, 73, 334. Genghof, D. S., Partridge, C. W. H., and Carpenter, F. H., Arch. Biochern., 1948, 17, 413. Jones, A., and Morris, S., Analyst, 1950, 75, 613. Woolley, D. W., J . Biol. Cheun., 1943, 147, 481. Atkin, L., Schultz, A. S., Williams, W. L., and Frey, C. N., Ind. Eng. Chern., Anal. Ed., 1943, Publication No. 4137, 1941, p. 27. 15, 141. BEECHAM RESEARCH LABORATORIES LIMITED BROCKHAM PARK, BETCHWORTH, SURREY February, 195 1

ISSN:0003-2654    

DOI:10.1039/AN9517600588

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Oct., 19511 BELCHER AND TATLOW 593 The Determination of Fluorine in Organic Compounds BY R. BELCHER AND J. C. TATLOW A method is described for the determination of fluorine in organic compounds. Decomposition is effected with sodium in a nickel bomb of special design and then the fluoride ion is determined gravimetrically as lead chlorofluoride, THE accurate determination of fluorine in organic compounds is complicated both by the difficulty of decomposing the sample effectively and by the lack of convenient or reliable methods for determining the fluoride ion. Whilst certain fluorine-containing compounds can be decomposed readily by simple hydrolysis, the method is restricted mainly to acyl fluorides and compounds in which fluorine atoms are activated by other groupings present in the molecule; where the fluorine is bound more firmly much more drastic treatment is necessary.Fluorocarbons or mixed halofluorocarbons are particularly resistant to decom- position. Elving and Ligett,l who have very comprehensively reviewed the methods of n 1‘ Fig. 1. Bomb for combustion A, nickel cup ; B, nickel lid ; C, hole to take bar for opening; D, steel nut; E, copper gasket decomposition and determination, recommend decomposition with sodium or potassium in a sealed tube or nickel bomb and determination of fluoride ion by titration with thorium nitrate or precipitation as lead chlorofluoride. From our own experience the most convenient and effective method of decomposition is with sodium in a nickel bomb. Decom- position with sodium peroxide in a Parr bomb2 is effective for certain compounds but does not completely decompose some of the fluorocarbons we have encountered.The design of the bomb we use is shown in Fig. 1. It is easy to manipulate and sufficiently compact to enable several bombs to be placed in a small muffle furnace at the same time. After destruction of the organic matter and conversion of the fluorine to sodium fluoride, there are several possible methods for carrying the determination to completion. Because of its rapidity, considerable attention was devoted to the thorium nitrate method, but for our purpose it did not prove satisfactory. The many modifications of this method, including that described in the Analytical Methods Committee’s “Determination of Fluorine in food^,"^ We have not found it necessary to use potassium.594 BELCHER AND TATLOW: THE DETERMINATION OF [Vol.76 to which we propose to return at a later date, have not been examined. As results were urgently required, a modification of the well-known lead chlorofluoride method was used, the working details of which were supplied to us in a private communication by Mr. H. S. Stretch of the Ministry of Supply. This procedure required about 80 mg of fluorine to be present, which necessitated more sample than could normally be afforded. Accordingly, the method was further modified to make it effective for samples of limited size that contained from 30 to 60mg of fluorine. When the amounts of sample were very limited, sometimes only 20 mg or even 10 mg of fluorine have been determined, but the error is then slightly larger.In general, with 20mg of fluorine the results are about 0.5 per cent. lower than theoretical, and with 10mg about 1 per cent. lower. The final gravimetric estimation of the fluoride ion is preferred because of the favoura.ble conversion factor. By this procedure, satisfactory results, some of which are shown in Table I, have been obtained, for a wide variety of compounds. TABLE I RESULTS OF A TYPICAL SERIES OF ROUTINE DETERMINATIONS BY THE RE COMMENDED METHOD (NOT SPECIALLY SELECTED) Fluorine content Compound m-Trifluoromethylbenzoic acid . . .. .. 2-Nitro-5-acetaminobenzotrifluoride . . .. m-Acetaminobenzotrifluoride . . . . . . 3 : 3'-Bis(trifluoromethyl)azobenzene . . . . N-Trifluoroacetyl-2 : 5-dichloroaniline .. .. Y-Trifluoroacetylbenzylamine . . .. .. Perfluorodimethyldecalin . . . . .. .. Perfluorodicyclohexylethane . . .. . . N-Trifluoroacetylaniline . . .. . I * . Heptafluorobutyric acid . . .. .. . . Perfluorodicyclohexylpentane . . .. .. Benzotrifluoride . . .. .. . . .. 1 : l-Bis(~%fluorophenyl)-2 : 2 : 2-trichloroethane Trifluoroacetamide . . .. .. . . 4 : 6-Benzylidene trifluoroacetyl-a-methylglucoside .. .. .. .. .. .. .. .. .. .. .. .. .. . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Found, 30.0 22.8 27.7 35.6 22-1 28-2 39.1 11.4 74.0 74.9 49.9 15.0 30.0 61.8 74.7 % Calculated, 30.0 23.0 28.1 35.8 22-1 28.1 39.0 11.8 74.4 74.6 50.4 15.1 30.1 62.1 74.9 % So far compounds containing only carbon, hydrogen, oxygen, chlorine and nitrogen as well as fluorine have been encountered.The method might require some modification for other types of compound and we hope to address ourselves to this problem in the near future. We have found that m-trifluoromethylbenzoic acid is a suitable standard substance, for it is readily purified and is non-hygroscopic. METHOD THE BOMB- The bomb, shown in Fig. 1, has already been described in detail.4 The cup, A, is made of nickel, the upper portion being threaded to take the nut, D, and the lower portion being hexagonal to fit a spanner. The thread on the body of the bomb should be relieved to a very slight taper to avoid binding during the unscrewing process. The lid of the bomb, B, is also of nickel, a hole, C, being drilled in the top to enable a bar to be inserted to open the bomb.As the nut, D, is unscrewed, the bar impinges on the top of this nut and the cup and lid of the bomb are separated. The nut, D, is made of stainless steel"; brass has also been used successfully but is not as satisfactory as steel, particularly at high temperatures. The sealing ring, E, is made of copper, and is a hollow rolled gasket similar to a sparking-plug washer (there is no asbestos packing). Before use each gasket is cleaned and is then softened by being heated in a flame to redness and cooled in ethanol. A new gasket is necessary for each determination. It is convenient to construct a bench jig by welding or brazing the hexagonal section of a 5/16-inch box spanner to a steel plate that can be mounted on the bench. The cup, A, can rest on this while the bomb is being assembled or taken down.* Firth Vickers H.R. Crown Max.Oct., 19511 FLUORINE I N ORGANIC COMPOUKDS 595 After each determination, the flanges of the bomb and lid should be cleaned by means The bomb components may be mounted in a lathe chuck for rotation of fine emery paper. during the cleaning procedure. REAGENTS- Nitric acid-5 N. Acetic acid-A 30 per cent. v/v solution. Lead chloride-A saturated solution. Lead chlorojuoride-A saturated solution. *4 bsolute alcohol. A cetone. PROCEDWRE- Weigh sufficient sample from a weighing tube to yield between 30 and 60 mg of fluorine and transfer to the cup of the bomb. Add sodium in the form of small pellets, taking about 2 to 3 times the weight of sample, place the treated copper washer in position and screw the nut down tightly with a spanner.Place the sealed bomb for at least 1 hour in a furnace maintained at 550" to 600" C. Some fluoro- carbons may require a temperature of 650" to 700" C. After heating for the required time, remove the bomb and allow it to cool. Remove the lid of the bomb and wash any material adhering to the underside into a 100-ml beaker with a fine jet of water, not exceeding a total of 10 ml. Place the cup in the beaker and very carefully add 1 ml of absolute alcohol to destroy the residual sodium. Allow to stand for 10 minutes, then stir with a nickel rod and leave for a further 3 minutes. Add 2 or 3 drops of water at intervals to complete the destruction of the sodium. Empty the contents of the bomb into the beaker, wash the bomb thoroughly inside and out and remove it from the beaker.Filter through a Pregl sintered-glass filter of No. 1 porosity. Transfer the filtrate to a 100-ml beaker and wash the filter tube with water. Add 8 drops of a 0.1 per cent. solution of methyl red and add 5 N nitric acid dropwise until the indicator just turns pink. Add 0.25 ml of the acetic acid solution and heat to 60" to 70" C. Meanwhile, heat 200 ml of saturated lead chloride solution, containing 0-5 ml of the acetic acid solution, nearly to boiling in a 400-ml beaker. Add the hot fluoride solution to the lead chloride solution and wash the 100-ml beaker twice with 15-ml portions of saturated lead chloride solution. Place a clock glass over the 400-ml beaker and heat gently to boiling. Allow to stand for at least 4 hours or, preferably, overnight.Wash the beaker twice with 15-ml portions of lead chloride solution taking care to remove all traces of precipitate. Finally, wash the beaker with a jet from the wash-bottle containing the lead chloride solution. Wash the precipitate thrice with 15-ml portions of lead chlorofluoride solution, then thrice with 15-ml portions of acetone. Suck the crucible dry, wipe the outside and then place in an oven at 110" C for 20 to 30 minutes. Cool in a desiccator for 20 minutes, place in the balance case and weigh after a further 10 minutes. Weigh liquids in a gelatin capsule, size No. l.* Filter the precipitate through a clean, dry, weighed No. 4 sintered-glass crucible. Weight of fluorine = weight of precipitate x 0.07263 The authors express their gratitude to the Department of Scientific and Industrial Research for a grant in support of this work; to Professor M. Stacey, F.R.S., for his interest and help; to Mr. W. Massingham for the construction of the bombs; and to Mr. B. S. Noyes, who has been responsible for the majority of the routine determinations. REFERENCES 1. 2. 3. 4. Eking, P. J., and Ligett, W. B., Ind. Eng. Chem., Anal. Ed., 1942, 14, 449. Nicholls, M. L., and Olsen, J. S., Ibid., 1943, 15, 342. Analytical Methods Committee, "Determination of Fluorine in Foods," A nalysl, 1944, 69, 243. Stacey, M., Tatlow, J. C., and Massirlgham, W. E., British Patent Application 3631/51, dated February 14th, 1951. DEPARTMENT OF CHEMISTRY THE UNIVERSITY BIRMINGHAM, 15 March, 1951 * Manufactured by Eli Lilly and Co., Indianapolis, U.S.A.

ISSN:0003-2654    

DOI:10.1039/AN9517600593

出版商:RSC    

年代:1951

数据来源: RSC