ISSN:0003-2654    

DOI:10.1039/AN96287FX009

出版商:RSC    

年代:1962

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN96287BX011

出版商:RSC    

年代:1962

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN96287FP053

出版商:RSC    

年代:1962

数据来源: RSC

摘要:

ConfidentiaCompany NamConfidentiaCompany NamConfidentiaConfidentia

ISSN:0003-2654    

DOI:10.1039/AN96287BP067

出版商:RSC    

年代:1962

数据来源: RSC

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MARCH, 1962 THE ANALYST Vol. 87, No. 1032 PROCEEDINGS OF THE SOCIETY FOR ANALYTICAL CHEMISTRY NEW MEMBERS ORDINARY MEMBERS Derek Charles Abbott, B.Sc., Ph.D. (Lond.), A.R.I.C.; Lionel Stuart Bark, B.Sc. (Lond.), F.R.I.C. ; Michael Raymond Barker; Humphry John Moule Bowen, M.A., D.Phi1, (Oxon.) ; James Francis Chissell, MSc. (Lond.) ; William Edward Driver, B.Sc. (Lond.) ; Jaroslav Franc, B.Eng. ; Luigi Guiducci; John Haydock; Francis John Kelland, B.Sc. (Wales) ; David John Lewis, A.R.I.C. ; Denis Birkby Lisle, B.Sc., Ph.D. (Leeds) ; John William McMillan, B.Sc. (Binn.), A.R.I.C. ; Thomas Bryan Pierce, B.Sc., M.A., D.Phi1. (Oxon) ; Harry Ian Shalgosky, B.Sc. (Lond.), A.R.I.C. ; Jawahir Lekhraj Sipahimalani, B.Sc. (Bombay), B.Phann. (Lond.), F.P.S. ; John Clarence Bonshill Smith, A.R.I.C.; William Bland Smith, B.Sc. (Lond.) ; Keith Harvey Squire, A.R.I.C. ; John Malcolm Steele, A.R.I.C. ; Mark Noel Strachan, B.Sc. , Ph.D. (Lond.), A.R.C.S., D.I.C., A.R.I.C.; Yerzy Bozeslaw Szymanski, B.Sc. (Lond.). JUNIOR MEMBERS Michael Joseph Hadley ; Alan Hedley ; John Patrick Hollins ; Geoffrey Plant. DEATH Arthur Alcock. WE record with regret the death of NORTH OF ENGLAND SECTION THE thirty-seventh Annual General Meeting of the Section was held at 2.15 p.m. on Saturday, January 27th, 1962, at the City Laboratories, Mount Pleasant, Liverpool, 3. The Chairman of the Section, Mr. J. Markland, B.Sc., F.R.I.C., presided. The following appointments were made for the ensuing year: Chairman-Mr. J. Markland. Vice-Chairman-Mr. C. J. House, Hon. Secretary and Treasurer-Mr.B. Hulme, Ch. Goldrei, Foucard & Son Ltd., Brookfield Drive, Liverpool, 9. Members of Committee-Mr. J. F. Clark, Mr. G. B. Crump, Mr. A. 0. Jones, Mr. A. N. Leather, Mr. G. F. Longman and Mr. R. Sinar. Mr. A. A. D. Comrie and Mr. F. Dixon were re-appointed as Hon. Auditors. The Annual General Meeting was followed by an Ordinary Meeting of the Section at which the following paper was presented and discussed: “The Work of the Laboratory of the Government Chemist,” by D. T. Lewis, Ph.D., D.Sc. , M.R.S.H., F.R.I.C. SCOTTISH SECTION THE twenty-seventh Annual General Meeting of the Section was held at 1.45 p.m. on Friday, January 26th, 1962, at the Grosvenor Restaurant, 72 Gordon Street, Glasgow, C.1. The Chair was taken by the Chairman of the Section, Mr. A.F. Williams, B.Sc., F.R.I.C. The following office bearers were elected for the forthcoming year: Chairman-Mr. A. F. Williams. Vice-Chairman-Dr. R. A. Chalmers. HOW Secretary and Treasurer-Mr. J. Brooks, Re- search and Development Department, Imperial Chemical Industries Ltd., Nobel Division, Stevenston, Ayrshire. Members of Committee-Mr. J. K. McLellan, Mr. H. C. Moir, Mr. W. J. 161162 PROCEEDINGS [vol. 87 Murray, Dr. J. Sandilands, Mr. S. C. Sloan and Miss D. A. Thomson. Mr. J. S. Foster and Mr. R. A. Sutter were appointed Hon. Auditors. The Annual General Meeting was followed by an Ordinary Meeting of the Section at which the following paper was presented and discussed : “The Application of Analysis to Research Problems in the Gas Industry,” by G.R. Boreham, B.Sc., A.R.I.C. WESTERN SECTION THE seventh Annual General Meeting of the Section was held at 6 p.m. on Friday, January 12th, 1962, in the University College, Cathays Park, Cardiff. The Chair was taken by the Chairman of the Section, Dr. G. V. James, M.B.E., F.R.I.C. The following appointments were made for the forthcoming year : Chairman-Dr. F. H. Pollard. Vice-Chairman-Mr, E. A. Hontoir. Hon. ’ Secretary and Treasztrer-Dr. T. G. Morris, BrocMeigh, Clevedon Avenue, Sully, Glamorgan. Members of Committee-Dr. L. E. Coles, Dr. G. V. James, Mr. G. M. Telling, Mr. J. D. R. Thomas and Dr. W. J. Williams. Mr. C. H. Manley and Mr. S. Dixon were appointed Hon. Auditors. The Annual General Meeting was followed by an Ordinary Meeting of the Section at which the Chair was taken by the new Chairman, Dr.F. H. Pollard. The following paper was presented and discussed: “Radio Activity Measurements in Monmouthshire,” by G. V. James, M.B.E., M.Sc., Ph.D., F.R.I.C. MIDLANDS SECTION AN Ordinary Meeting of the Section was held at 2.30 p.m. on Wednesday, January loth, 1962, at the Hills Lecture Theatre, The University, Edgbaston, Birmingham, 15. The Chair was taken by the Chairman of the Section, Dr. H. C. Smith, bip.Ed., F.R.I.C. The subject of the meeting was “Developments in Gas Chromatography, as Applied to Polymers,” and the following papers were presented and discussed: “Recent Advances in Technique,” by D. H. Desty; “Gas Chromatography and its Use in Polymer Chemistry,” by C . A. Finch; “The Separation of the Degradation Products of Polymers,” by R. S. Lehrle. MICROCHEMISTRY GROUP THE thirty-third London Discussion Meeting of the Coup was held at 6.30 p.m. on Wednesday, January 24th, 1962, at “The Feathers,” Tudor Street, London, E.C.4. The Chair was taken by the Chairman of the Group, Mr. C. Whalley, B.Sc., F.R.I.C. A discussion on “Determination of Traces of Copper” was opened by E. I. Johnson, M.Sc., F.R.I.C., and D. B. Adams, B.A., B.Sc.

ISSN:0003-2654    

DOI:10.1039/AN9628700161

出版商:RSC    

年代:1962

数据来源: RSC

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March, 19621 CARTWRIGHT 163 Studies in Precipitation from Homogeneous Solution by Cation Release at Constant pH Part IIT.* The Influence of the Solubility of the Precipitates? BY P. F. S. CARTWRIGHT (Department of Chemistry, S i r John Cuss College, London, E. C. 3) The scope of precipitation from homogeneous solution by cation release at constant pH is limited by the fact that some substances are immediately precipitated at room temperature when suitable anions are added to solutions containing metal - EDTA complexes. This behaviour has been related to the solubilities of the precipitates and the apparent stability constants of the complexes. Under these conditions, EDTA may be of limited use in the absence of oxidising agents as a masking agent; an example is given in the separation of barium and calcium.DURING experiments with several different cations and anions it became evident that the solubilities of the precipitates had a pronounced effect on the ease with which precipitation occurred. In some instances, the combination of low stability of the complex with low solubility was sufficient to render the cation-release technique unsuitable for precipitation from homogeneous solution ; immediate precipitation occurred a t room temperature when the precipitating anion was added to the solution of the metal - EDTA complex. The ability of certain anions to precipitate metals from their complexes with EDTA has been described by Flaschka,l and more recently by Cheng2 in studies of masking action. Such behaviour imposed a limitation on the method, and experiments were carried out to determine the importance of this aspect in the field of precipitation from homogeneous solution.EXPERIMENTAL REAGENTS AND APPARATUS- As described in Part I of this series.3 Stock solutions of the cations studied were prepared to contain approximately 10 g of metal per litre. PROCEDURE- Aliquot portions of the stock solutions containing about 250 mg of metal were transferred to beakers, sufficient EDTA solution just to complex the metal was added to each, and the solutions were diluted to about 250 ml. The pH values were adjusted to greater than 9.0 by adding sodium hydroxide solution, and, after an excess of the appropriate anion had been added, they were checked and if necessary adjusted. The solutions were set aside at room temperature for 10 minutes to determine whether or not any precipitates were formed.If no precipitation occurred, the pH values of the solutions were slowly decreased by adding dilute nitric acid to 8-0, 6.0, 4.0 and 2.0, and the appearance of precipitates was noted. DISCUSSION OF RESULTS- The results are plotted in Fig. 1, in which the behaviour of the solution is referred to the values of apparent stability constant and solubility product of the precipitate; the key to the diagram is shown in Table I. The apparent stability constants of the metal-EDTA complexes at the pH values studied were calculated as described by Flaschka and Barnard.4 Solubility products should ideally be derived for each precipitate under the conditions of its formation.This was impracticable, as a method was required by which the likelihood of precipitation occurring * For details of Part I of this series, see reference list, p. 167. f The substance of this paper and other parts of the series was presented a t the meeting of the Society on Wednesday, November lst, 1961.164 CARTWRIGHT : STUDIES I N PRECIPITATION FROM HOMOGENEOUS [Vol. 87 at any pH could be predicted from readily available constants. For this reason, published values of solubility products were used, although the limitations of this practice, as discussed by L e ~ i n , ~ were fully realised. The values shown in Fig. 1 are only approximations and are denoted by large circles indicating regions rather than fixed points. The boundaries of the circles do not indicate the limits of uncertainty. Number on figure 1 2 3 4 5 6 8 9 10 11 12 F TABLE I KEY TO FIG.1 Compound BaSO, or BaCrO, CaC,O, MgNH,PO, AgI W O H ) 2 ZnS CdS PbS PbCrO, c u s NiS Cr ( OH ) 3 Number on figure 13 14 15 16 17 18 19 20 2’1 22 23 Compound Fe(OH)3 A1 (OH 13 AgCl CaSO, CaF, MnS BiOCl FePO, BaHPO, SrSO, Bi(OH)3 I t is possible to distinguish those compounds that, at the concentrations studied, formed precipitates immediately the solutions were mixed at room temperature from those with which no precipitation occurred; the two classes are separated by a shaded region in Fig. 1 (a) and by broken lines in Figs. 1 ( b ) , 1 (c) and 1 (d). The graphs can be used to give a first indication of those compounds that may be precipitated from homogeneous solution by cation release at constant pH and of the range of pH values over which the method may be applied.The results in Fig. 1 are based on the first appearance of a precipitate and do not indicate that precipitation is complete. From the point of view of precipitation from homogeneous solution, formation of even a trace of precipitate when the reagents are first mixed is sufficient to render the method unsuitable, as the solutions are then no longer initially unsaturated. The results reported by Flaschka,l who studied the masking action of EDTA in the deter- mination of certain metals as sulphides, and by Cheng2 who studied the masking of metals in the presence of various anions, appear to agree with these results. Somewhat different behaviour might be expected at other concentrations or in the presence of excess of EDTA, and an increase in the temperature of the solutions will increase the likelihood of precipitation occurring.APPLICATION TO GRAVIMETRIC ANALYSIS Although the immediate formation of precipitates can impose limitations on the method, it may be advantageous in certain circumstances. When precipitation is brought about by osidation of metal - EDTA complexes, selectivity is poor, since all the cations are released into solution. The fact that, at a given pH, some metal complexes break down immediately the precipitating anion is added, whereas others remain stable, offers the possibility of selective precipitation without the use of an oxidising agent. The success of such a method will depend on the relative stabilities of the metal - EDTA complexes at the pH values selected and can be demonstrated by the behaviour of calcium, strontium and barium.It can be seen from Fig. 1 that, in the presence of sulphate ions, precipitation of barium began at about pH 8.0, and strontium behaved similarly between pH 4 and 6, whereas calcium remained in solution below pH 4. Experiments were therefore made to determine whether or not the differences between the stabilities of the complexes were sufficient to permit a separation of the metals. Precipitation of alkaline-earth sulphates Preliminary experiments were carried out to determine the pH at which the barium - EDTA complex was completely dissociated in the presence of sulphate ions. In order that precipitation shouId take place from homogeneous solution, the sulphate ions were generated by hydrolysis of sulphamic acid in accordance with Wagner and Wuellner’s method.6March, 19621 SOLUTION BY CATION RELEASE AT CONSTANT pH.PART 111 24 I 01 1 i I I t I I I I 0- 8 16 24 32 4 Solubility product Fig, 1. Graphs showing behaviour of solutions a t pH values of (a) 2.0, (b) 4-0, (G) 6-0 and (d) 8.0: 0, no preci- pitation ; c.:::, precipitate formed ; 0, uncertain behaviour. (For key to numbers, see Table I) 165166 CARTWRIGHT : STUDIES I N PRECIPITATION FROM HOMOGENEOUS p o l . 87 PROCEDURE- An approximately 0.1 M solution of barium was prepared, and its barium content was checked by precipitation as barium sulphate in the conventional manner. Aliquot portions of this solution were transferred to separate beakers, and to each was added sufficient EDTA solution to complex the metal.After the addition of 5 g of sulphamic acid, the pH of each solution was adjusted to the required value, and the solutions were gently boiled for 30 minutes on a hot-plate. The precipitates were separated by filtration, washed, and then dried to constant weight at red heat. DISCUSSION OF RESULTS- For aliquots of solution each containing 113.8mg of barium, the results were- pH of solution . . .. .. . . 2.0 3.0 4.0 5.6 Barium precipitated, mg . . . . 113.8 113.6 111.1 109.4 Recovery of barium, yo . . . . 100.0 99.8 97.6 96.1 These results show that precipitation was complete at pH 2.0 and complete to within 0.2 per cent. at pH 3.0. At higher pH values, however, results were low, owing to incomplete dissociation of the barium - EDTA complex.It was evident that the method was not suitable for separating barium from stronium, as precipitation of the latter begins between pH 4 and 6. It was thought, however, that separation of barium from calcium might be possible, since, when solutions containing the calcium - ED’TA complex were boiled in the presence of sul- phate, no precipitation occurred until the pH was less than about 2 to 2.5. Precipitation of barium sulphate in presence of calcium PROCEDURE- Aliquot portions of barium solution were transferred to separate beakers, together with the required amounts of calcium, and to each was added sufficient EDTA solution to complex the metals. After the addition of 5 g of sulphamic acid, the pH values of the solutions were adjusted to between 2-5 and 3.0 by adding nitric acid.The solutions were warmed on a hot-plate for 30 minutes and then filtered through weighed, sintered-porcelain filter crucibles. The precipitates were washed with distilled water and were dried to constant weight at red heat. The residues were subsequently examined for the presence of calcium by the spot-test method involving use of alkaline sodium rhodizonate solution as described by Feigl.7 DISCUSSION OF RESULTS- The results are shown in Table 11, from which it can be seen that the method afforded good recovery of barium with little or no contamination by calcium. The quality of the precipitates, however, was not noticeably better than that of those formed by direct addition of sulphate ions to hot solutions of barium.TABLE I1 BARIUM RECOVERED IN PRESENCE OF CALCIUM Each solution contained 113.8 mg of barium Calcium present, Barium found, Recovery of barium, Result of spot test for calcium mg mg % 113-8 100.0 Trace present None detected 11 3.4 113-5 11 3.4 99-6 250.0 500.0 Trace present In this separation, the role of the EDTA was that of a masking agent to retain calcium The subsequent release of calcium by oxidation of its complex with EDTA by in solution. hydrogen peroxide was not studied.March, 19621 SOLUTION BY CATION RELEASE AT CONSTANT pH. PART I11 167 Thus, use can be made of the relative stabilities of metal - EDTA complexes to effect separations of certain cations. The stabilities must be sufficiently different to allow clean separation to be made, and the method is also limited by the degree of dissociation of the metal - EDTA complex under the conditions of the reaction. In Part IV of this series, the qualities of precipitates obtained by cation release at constant pH will be discussed and the potentialities of the method will be critically surveyed.REFERENCES 1. 2. 3. 4. Flaschka, H., Chemist-Analyst, 1955, 44, 2 . Cheng, K. L., Anal. Chem., 1961, 33, 783. Cartwright, P. F. S., Analyst, 1961, 86, 688. Flaschka, H., and Barnard, A. J., jun., in Wilson, C. L., and Wilson, D. W., Editors, “Compre- Volume lB, Classical Analysis,” Elsevier Publishing Co. Ltd., Lewin, S., “The Solubility Product Principle,” Sir Isaac Pitman & Sons Ltd., London, 1960. Wagner, W. F., and Wuellner, J.A., Anal. Chem., 1952, 24, 1031. Feigl, F., “Spot Tests in Inorganic Analysis,” Fifth Edition, Elsevier Publishing Co. Ltd., Am- hensive Analytical Chemistry. Amsterdam and London, 1960, p. 301. 5. 6. 7. sterdam and London, 1958, p. 223. NOTE-Reference 3 is to Part I of this series. Received July 28th, 1961 D I s c u s s I o N MR. F. CLERMONT SCOTT asked if use could be made of the method of precipitation from homogeneous solution in the separation of calcium and magnesium as oxalate. MR. CARTWRIGHT replied that a method of precipitation from homogeneous solution had been used for the separation of calcium and magnesium, as oxalate, by Gordon and Wroczynski (AnaE. Chem., 1962, 24, 896). However, the method under consideration was not suitable, since the hydrogen peroxide, which was added to break up the metal - EDTA complex, would also destroy any oxalate ions present.MR. C. WHALLEY congratulated the author on a most interesting paper and commented on how the oxidation rate - pH curve followed the hydrogen peroxide decomposition curve. He thought it a pity that the author had started off with elements such as bismuth and lead, which were active decomposition catalysts for hydrogen peroxide. If a start had been made with barium, calcium or magnesium, the author might have been successful with precipitating agents other than phosphate. Mr. Whalley added that he thought that peroxide decomposition was probably an essential step in the oxidation and he asked if other per-compounds had been tried for the oxidation.Bismuth was purposely chosen, since i t afforded an extreme instance owing to the stability of its EDTA complexin acid solution. Other metals were subsequently selected to cover a range of complex stabilities. The use of ammonium persulphate had been reported in the precipitation of barium sulphate by cation release of barium from its EDTA complex (Heyn and Schupak, Anal. Chem., 1954, 26, 1243). Mr. Cartwright added that he had tried to use percarbonates and perborates, but had found that both classes of compounds behaved as hydrogen peroxide and their use offered no advantage. MR. R. C. CHIRNSIDE said he assumed that the principal objective in homogeneous precipitation was to bring about nucleation and crystal growth so that precipitates would not only settle and filter well, but so that adsorption of foreign ions would be kept to a minimum.MR. CARTWRIGHT replied that he had not made quantitative comparisons, but studies by other workers had clearly shown that substantially purer precipitates had been obtained in a wide variety of precipitations from homogeneous solution. DR. J. A. W. DALZIEL asked what were the relative merits of the method described compared with the one in which bismuth was precipitated from homogeneous solution by hydrolysis of metaphosphoric acid in the presence of nitric acid (Ross and Hahn, Anal. Chew., 1960, 32, 1690). MR. CARTWRIGHT agreed with the comments on barium, calcium and magnesium.168 CARTWRIGHT [Vol. 87 MR. CARTWRIGHT said that he believed both methods worked equally well, and that neither method DR.J. HASLAM asked how accurate were the tests for sulphate. He also asked if all the EDTA were oxidised by the hydrogen peroxide and what was i t oxidised to. MR. CARTWRIGHT replied that the accuracy of all determinations was that afforded by normal tech- niques to the limit of the analytical balance. The products of the oxidation of EDTA were not known, but Welcher had said that strong oxidising agents slowly oxidise EDTA to form cyclic ureides. During the reaction with hydrogen peroxide some carbon dioxide was liberated, which suggested that decarboxylation occurred, There was evidence that ammonia was released during the final stages of oxidation, but further investigation could lead to explosive reaction. Mr. Cartwright said that he thought i t was unlikly that all the EDTA was necessarily destroyed during the reaction and that the release of the cation was clue, to some extent, to the presence of a precipitating anion. Beck (Chemist-A?zaZyst, 1961, 50, 14) had shown that, in the absence of metal ions, EDTA could be oxidised by acid permanganate solution a t 60" (3, but a bismuth - EDTA complex was not attacked under these conditions. DR. G. W. C. MILNER said that, since some elements formed peroxy-EDTA complexes, would the author care to comment on the behaviour of his method for the determination of this type of element. MR. CARTWRIGHT said that if the peroxy complex were sufficiently stable to resist boiling in the presence of a precipitating anion the method would clearly be unsuitable for the determination of the element. It might, however, be possible to make use of the formation of peroxy complexes to improve the separation of pairs of metals. Thus Babko and Shtokalo (Zuvod. Lab., 1958, 24, 674) had separated zirconium from titanium by precipitation of zirconium phosphate by cation release from a zirconium - EDTA complex in strongly acid solution, to which hydrogen peroxide had been added to convert the titanium to the peroxy complex. afforded a separation of bismuth from lead in a single precipitation.

ISSN:0003-2654    

DOI:10.1039/AN9628700163

出版商:RSC    

年代:1962

数据来源: RSC

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March, 19621 GOODW’IN, GOULDEN BND REYNOLDS 169 Rapid Identification and Determination of Residues of Chlorinated Pesticides in Crops by Gas - Liquid Chromatography BY E. S. GOODWIN, R. GOULDEN AKD J. G. REYNOLTIS (Il’oodstock .-3g~~icziltzwal Rpsenvch Crutre, “S12ell” Heseaifclz Ltd., Sittingbouriir, I<ciifl THE paper published under this title in the Xovember issue of The Afzalyst, 1961, 86, 697, was presented and discussed at an Ordinary Meeting of the Society on November lst, 1961, as first reported on p. 685 of the November issue. DISCUSSION NR. E. D. CHIL~VELL expressed interest in the authors’ use of “clectron capture” for determining malathion, and enquired if it was known which group in malathion was responsible for its electron capture properties. MR. REYNOLDS, in reply, indicated that in methyl parathion the nitro-group probably played a major part in the electron capture phenomenon, bnt he did not know which group in malathion was responsible.MR. J. V. 3IoRnmx said that, although he was familiar with the principles involved, he had had no practical experience with the electron capture detector. He asked if the speaker would elaborate on three points: what features in the I,ovelock design made i t so much more sensitive than the Shandon detector; how linear the detector response was : what degree of ionisation voltage stability was necessary to ensure satisfactory quantitatiTe performance. MR. GOODWIN replied that the geometry of the detector was the main factor accounting for the improved sensitivity. I n the Lovelock design the gas from the column was fed through the anode, thus the electron field was a t its greatest at the point where the gas was emerging from the column, and also the negative molecular ions produced by electron capture were swept out of the detector by the flow of gas before they could be attracted t o the anode.He thought these points could best be appreciated by inspect- ing the detectors on display. The linear range was not large, but adequate calibration ensured the accuracy of quantitative work. Ordinary grid-bias and high-tension batteries gave a sufficiently stable voltage for quantitative performance. No trouble with insulation or capacitance effects had been encountered. M R . C. A. JOHNSOX asked the author if they had applied this procedure to the determination of residues in animal tissues.If the method were applicable to animal tissues, he would like t o know if the possible metabolites of the insecticides would be determinable. He also asked if the author had experienced any clifficulty owing to the break-down of chlorinated insecticides on the column. Finally, he enquired if the recovery of aldrin from an alumina column, such as the speaker, Mr. Reynolds, had described for preliminary “clean-up” of certain extracts, was quantitative. MR. REYXOLDS replied that the procedure had been successfully applied wifhout clean-up to extract? of pigs’ blood, fat, brain, liver and meat. For the f a t the amount of tissue material actually injected into the column in the same volume of solution was a hundred times greater than in the other extracts, b u t the background trace was quite satisfactory, probably owing t o the involatility of the fatty material.He felt that the presence of metabolites would be observed if they possessed sufficient electron affinity and if their retention times were different from those of the parent compound. They must also, of course, be volatile under the conditions used. Break-down of chlorinated insecticides on columns was well known and was troublesome because it was not reproducible. It could be avoided by the use of Epikote resin in admixture with the silicone elastomer. The amount of alumina used on the preliminary “clean-up” was very small, but sufficient to remove interfering peaks in the particular background. Recovery of the aldrin was quantitative. THE YRESIDEKT, DR. Ilvos, said that the results presented showed that the background curve obtained from oats in the absence of a special “clean-up” prxedurc exhibited a peak that coincided with a peak indicative of the presence of a pesticidal residue. He asked if any other cereal crops behaved similarly or if oats were anomalous in this respect. MR. GOOD\VIX replied that whole wheat, barley and oats gax‘e a peak a t the retention time of approxi- mately the same amplitude. Polished rice and maize also showed it, but at a lower response

ISSN:0003-2654    

DOI:10.1039/AN9628700169

出版商:RSC    

年代:1962

数据来源: RSC

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170 CLULEY : SUSPENSION SCINTILLATION COUNTING [Vol. 87 Suspension Scintilla tion Counting of Carbon44 Barium Carbonate* BY H. J. CLULEY (The General EZectric Company Limited, Centval Research Laboratories, Hirst Research Centre, Wembley, Englaud) A study has been made of the suspension scintillation counting of carbon-14 barium carbonate with a very finely divided silica as the suspending agent, as proposed by Ott, Richmond, Trujillo and Foreman. A 4 per cent. w/v concentration of the silica in the scintillator solution (0.3 per cent. w/v of 2,5-diphenyloxazole in toluene) gives an almost transparent gel capable of suspending up to 1 g of barium carbonate in 10 ml of solution. Counting efficiency was found to decrease somewhat with increasing concentration of barium carbonate; 0.1 and 0.5 g of barium carbonate gave counting efficiencies of 60 and 49 per cent., respectively, compared with 71 per cent.for solution scintillation counting of carbon-14 under the same instrumental conditions. The high efficiencies with which substantial amounts of barium carbonate can be counted by the suspension technique represent a considerable gain over the efficiency of about 1 per cent. obtained by Geiger counting of barium carbonate under “infinite thickness” conditions. IN connection with carbon-14 tracer studies of the system graphite - carbon monoxide - carbon dioxide under irradiation conditions, it has been required to determine small amounts of carbon-14 in samples of graphite weighing up to several grams. In such an instance, i.e., when the sample is of appreciable weight and is chemically identical to the isotope to be determined, two factors affect the over-all sensitivity of detection: (i) the counting efficiency achieved ; (ii) the proportion of the original sample, after suitable chemical processing, that can be presented to the counter consistent with reasonable counting efficiency.Originally, the determination of tracer carbon-14 in graphite samples was effected by combustion in oxygen, absorption of the carbon dioxide in sodium hydroxide solution, pre- cipitation of barium carbonate from an aliquot of the solution and Geiger counting of the precipitate. To permit large precipitates to be handled without undue loss of counting efficiency, thereby enhancing the over-all sensitivity, a 2-inch diameter de-mountable filteE was used to collect the precipitates, which were counted with a 2-inch diameter end-window counter (G.E.C.2B2 counter). With the 2-inch diameter (approximately 20 sq. cm) sources prepared in this manner, “infinite thickness” was achieved with amounts of barium carbonate greater than 0-5 g, i.e., > 25 mg per sq. cm. Amounts of 0-5 to 1 g of barium carbonate were counted with 1 to 0.5 per cent. counting efficiency; the over-all efficiency of the process was appreciably lower in that only a fraction of the absorption solution (usually one twentieth for l-g samples of graphite) was taken for the precipitation. This procedure, which required only simple counting equipment, proved generally satisfactory, but it became clear that for certain aspects of the work greater sensitivity would be required.It would obviously be convenient to use the same chemical processing irrespec- tive of the level of carbon-14 activity to be determined in the graphite specimens. An alternative method of counting substantial weights of barium carbonate, affording much greater counting efficiency than that given by Geiger counting, was therefore sought. Available informationl229314Jj suggested that the technique of suspension scintillation counting might provide an answer. Liquid scintillation counting, which has been widely used for soft 13-emitters, such as carbon-14, normally involves dissolving the material to be counted in the solution of the organic scintillator. The suspension method permits the extension of this technique to the counting of solids that are insoluble in the usual scintillator solvents.For this purpose a gelling additive is incorporated in the scintillator solution, so that the resulting gel can support a suspension of the solid material to be counted. Little has been published on the suspension * Presented at the meeting of the Society on Thursday and Friday, October 5th and Gth, 1961.arch, 19621 OF CARBON-14 BARIUM CARBONATE 171 chnique, but it has been claimed that several grams of solid can be suspended in the normal lunting volumes and that the counting efficiencies obtained approach the high efficiencies :hieved by scintillation counting in solution. The application of a suspension technique to the counting of carbon-14 barium carbonate described in this paper.EXPERIMENTAL KOICE OF GELLING ADDITIVE- In previous work on suspension scintillation counting three types of gelling agent have :en used. These are aluminium stearate1j2 and two proprietary materials of American -igin, T h i ~ c i n ~ 9 ~ (a castor oil derivative) and Cab-0-Si15 (a very finely divided silica). Of iese, aluminium stearate has the obvious disadvantage that heating is necessary for formation the gel and for the work described here additives of the other two types were tried. With Thixcin, and with a similar material of British origin, difficulties were found in lending it with a toluene-based scintillator solution to form a homogeneous gel, possibly ecause suitable high-speed blending equipment was not available. Experiments with a ery finely divided silica as the gelling additive were much more promising; almost transparent sls could be prepared by separately putting the silica and the scintillator solution into a counting mtainer and then shaking the container.This ability to form the gel in situ in the counting mtainer was clearly advantageous; the problem of introducing into the container a precise .eight or volume of a viscous liquid or gel was thereby avoided. A silica additive was ierefore adopted for subsequent work. The silica used was Degussa Aerosil (obtainable in this country from Bush Beach and egner Bayley Ltd., Marlow House, Lloyds Avenue, London, E.C.3). This material is a ery finely divided silicz, having the low bulk density of 0-04g per C.C. and apparently imilar in properties to the American material Cab-0-Sil. 'REPARATION AND COUNTING OF SUSPENSIONS- The use of finely divided silica as a gelling additive was advocated by Ott, Richmond, 'rujillo and F ~ r e m a n .~ These authors recommended a silica concentration of 3 to 5 per cent. not stated whether w/w or w/v) in the toluene-based scintillator solution and claimed that ounting efficiency and suspending capacity were relatively independent of silica concen- ration in this range. It was stated that 2 g of a heavy solid (lead chloride or barium carbonate) ould be suspended in 20 ml of scintillator solution. The brief paper by Ott ef al. contained ittle experimental information and gave no results. In the present work, experiments were first made to ascertain a suitable concentration if Aerosil.Suspensions were prepared by putting into glass counting containers weighed .mounts of Aerosil and of inactive barium carbonate, adding 10 ml of toluene-based scintillator olution and then shaking mechanically to form the gel and to disperse the barium carbonate. With a 3 per cent. w/v concentration of Aerosil, appreciable settling of the barium carbonate vas detectable after 1 hour; 5 per cent. w/v of Aerosil gave an unnecessarily viscous, almost ,olid gel and the intermediate concentration of 4 per cent. w/v was provisionally adopted for #ubsequent work. At this concentration the barium carbonate dispersed readily and stable uspensions of up to 1 g of barium carbonate could be obtained. Because of the extremely light fluffy nature of Aerosil, it was found most convenient to lispense it from a thin-walled polythene bottle.Into the cap of the bottle was inserted a ihort length of polythene tubing, which was a sliding fit into the necks of the glass counting :ontainers. Aerosil could thus be introduced into the containers by tapping or squeezing :he bottle. For this purpose active Darium carbonate precipitates were ground and mixed to form a stock of known activity. Suspensions containing various weights of this material were prepared as described above, LO ml of 0.3 per cent. w/v 2,5-diphenyloxazole in toluene being used as the scintillator solution Eor each. Immediately after preparation each suspension was stored in - the scintillation zounter for 10 minutes to permit decay of any short-lived fluorescence acquired by the counting container, and then counted. The scintillation counter, operated a t room tem- perature, was of the single photomultiplier type (Ekco type N664A, used with an Ekco automatic scaler type N530F).The colourless-glass counting containers used were those Attention was then directed to the counting of suspensions.172 CLULEY : SUSPENSION SCINTILLATION COUNTING [Vol. 8 supplied by the manufacturers (Ekco type N671B, capacity 16 ml) ; the polythene stopper supplied with the containers showed a tendency to crack on continued use with toluen solutions and were later replaced by stoppers turned from polytetrafluoroethylene rod. The results of the preliminary experiments on counting of suspensions are shown ii Table I. At constant Aerosil concentration, the ratio of count rate to weight of bariun carbonate suspended decreased somewhat with increasing weight of barium carbonate Similar variations in counting efficiency with concentration have been observed by othe workers.The results at constant concentration of barium carbonate showed that som decrease in counting efficiency occurred with increasing concentration of Aerosil. I t therefor appeared dcsirable to use a precise and constant concentration of Aerosil. ’TABLE 1 COUNTING OF BARIUM CARBONATE SUSPENSIOSS PRELIMINARY EXPERIMENTS Weight of barium carbonate in 10 ml of gel, g 0.02 0.1 0.2 0.3 0.5 Counts per second pcr 0.1 g of barium carbonate I I 3.5% w / v of 4-00/, w/v of 4.5% w;v of Aerosil .\erosil ,\erosil - 175 - _ - - 17.5 - - 168 - 169 165 1.59 - 148 ___ Calculation showed that the count rates given in Table I corresponded to countini efficiencies in the range 50 to 60 per cent., a promising improvement on the efficiency o about 1 per cent.obtained by Geiger counting. However, one possible difficulty became apparent at this stage. It was noticeable that the active barium carbonate dispersed rathe less efficiently than the inactive (AnalaR) barium carbonate that had been used in the earlies experiments on preparation of suspensions. With suspensions of the active material somf aggregates of barium carbonate always remained undispersed, this behaviour presumablj arising from some feature in the method used for precipitating the active material. On the assumption that aggregates were undesirable and a possible source of variatior from suspension to suspension, attempts were made to improve the dispersion of the active barium carbonate.Longer and more vigorous mechanical shaking and the use of wetting agents were tried ; neither expedient gave any visibly detectable improvement in the dispersior of the barium carbonate nor, encouragingly, did they have any significant effect on the counl rates obtained. Finally, the effect was tried of removing the aggregates at source by sifting the active barium carbonate through an 86-mesh silk screen. Comparison of the couni rates obtained for suspensions of the sifted and unsifted barium carbonate is shown in Table IT TABLE I1 EFFECT OF SIFTING THE BARIUM CARBONATE ON THE COUXT RATE OF SUSPENSIONS Observed count rate with- 1 A_--- ~ - _ _ 0.1 g of 0.3 g of 0.5 g of barium Carbonate, barium carbonate, barium carbonate, c.p.s.C.P.S. C.P.S. Unsifted barium carbonate 124,123 342 533 Sifted barium carbonate (through 86 mesh) 122, 121 336 48; It will be seen that the sifted barium carbonate consistently gave slightly lower count rates than the unsifted material. (This observation is in agreement with that of White and Helf,3 who found almost constant counting efficiency over a range of mesh sizes, the finest particles being counted least efficiently.) It was thus apparent from the sifting experiments and from the attempts to improve dispersion of the barium carbonate that the aggregates were inimical neither to consistent count rates nor to high counting efficiencies.I t was concluded that sifting of the barium carbonate was unnecessary and that the presence of some aggregates in the suspensions could be tolerated, possibly because the aggregates were permeable by scintillator solution and for other reasons (see “Discussion of method,” p. 175).:arch, 1962: OF CARBON-14 BARIUM CARBONATE METHOD ROCEDURE- 173 From the preliminary experiments described above, the procedure detailed below was volved for preparation and counting of suspensions. A dry, weighed barium carbonate precipitate was gently ground in a glass mortar for bout 30 seconds, and then the required amount (0.1, 0.3 or 0.5 g) was weighed. A clean, ry counting container was weighed, and Aerosil was dispensed into the container until .4 g (accurate to the nearest 2 or 3 mg) had been added. The weighed portion of barium xrbonate and then, by pipette, 10 ml of 0.3 per cent.w/v 2,5-diphenyloxazole in toluene 'ere added. The stoppered container was vigorously shaken for 2 minutes on a mechanical haker, stored in the scintillation counter for 10 minutes, and then counted. A similar uspension of an inactive barium carbonate precipitate was used to determine the back- round count rate. If absolute activities were required, the activity of the suspended barium carbonate 'as calculated from the known counting efficiency (determined as described below) correspond- ig to the weight of barium carbonate suspended. The activity in the whole original pre- ipitate, of known weight, could then be calculated. After it had been counted the suspension was washed out of the container with a jet f acetone from a wash-bottle.Dilute hydrochloric acid and a few pieces of cellulose floc Jere then added, and the container was shaken to promote solution of any residual traces If barium carbonate and to assist mechanical removal of any adhering Aerosil. After copious vashing with water, the container was washed with acetone and air-dried. .4LIBRATION- The suspension procedure was calibrated with barium carbonate precipitates derived rom two sodium carbonate solutions of certified carbon-14 activity. The results obtained, xpressed as percentage counting efficiency observed, are given in Table 111. These figures hould be compared with a counting efficiency for solution scintillation counting of 71 per .ent., obtained under the same conditions with n-hexadecane of certified activity.The certi- ied sodium carbonate solutions and hexadecane were obtained from the Radiochemical kntre, Amersham. TABLE 111 COUNTING EFFICIEKCIES OF BARIUM CARBONATE SUSPENSIONS IVeight of Counting efficiency, yo 7 - barium carbonate 7- suspended, g A B Mean 0.1 60, 60 59, 61, 60 60 0.3 54, 53.5 56, 54, 55 54.5 0.5 49,49 49, 48.5, 49.5 49 A = Barium carbonate precipitates from sodium carbonate reference solution batch 3A, B = Barium carbonate precipitates from sodium carbonate reference solution batch 23, 0.78 pC per g. 0.62 pC per g. The results in Table I11 show good agreement between the two series derived from the :wo certified sodium carbonate solutions ; the mean counting efficiencies used for calibration we given in the final column of the Table.These figures substantiate the levels of counting :fficiency expected from the preliminary experiments and again show the reduction in counting :fficiency that occurs with increasing weight of barium carbonate. If required, a curve -eIating counting efficiency to weight of barium carbonate suspended could be plotted. It Nas found simpler in practice to adhere to the weights of barium carbonate used for calibration, Le., 0.1,0.3 or 0.5 g, and to employ the corresponding directly determined counting efficiencies if calculation to absolute activity were required. STABILITY OF SUSPENSIONS As the counting of suspensions can begin 10 minutes after their preparation, long-term It was, however, observed that suspensions stability of the suspensions is not required.174 CLULEY : SUSPENSION SCINTILLATION COUNTING [Vol.8' stored in the dark could be re-counted with little error one day after their preparation, a: shown by the results in Table IV. Some settling of the suspensions was usually apparen after several days. TABLE IV RE-COUNTING OF SUSPENSIONS ONE DAY AFTER PREPARATION Counts per second observed for duplicate suspensions r -3 0.1 g of 0.3 g of 0.5 g of barium carbonate barium carbonate barium carbonate After preparation . . 116,116 312, 309 471,479 One day later . . .. 116, 118 311, 312 469, 479 EFFECT OF VARYING AEROSIL CONCENTRATION The preliminary experiments had shown that variations in the concentration of Aerosi could affect counting efficiency somewhat (see Table I) ; this effect was subsequently investi. gated in more detail.From a stock of active precipitated barium carbonate, suspension: were prepared from 0-1 , 0.3 and 0.5 g of the carbonate and the usual concentration of Aerosil i.e., 4 per cent. w/v. Parallel series of suspensions were prepared with 3.5 and 4.5 per cent w/v of Aerosil. The count rates observed for these two series, expressed as percentages of the corresponding count rates observed with 4 per cent. w/v of Aerosil, are shown ir, Table V. TABLE V EFFECT OF AEROSIL CONCENTRATION ON COUNT RATE Count rate, expressed as percentage of corresponding count rate with 4 per cent. w/v of Aerosil Concentration 7 per cent. w/v barium Carbonate barium carbonate barium carbonate 3.5 107-5 103 105.5 4.5 99 99.5 95 A > of Aerosil, 0.1 g of 0.3 g of 0.5 g of These results confirmed the earlier observation that counting efficiency decreased some- what with increasing concentration of Aerosil.It was, however, apparent that the practice of weighing the normal amount of Aerosil (0.4 g) to the nearest 2 or 3 mg was adequate to reduce the errors from this source to negligible proportions. The results in Table V did not support the claim of Ott et aL5 (who used Cab-0-51) that the counting efficiency is relatively independent of the silica concentration over the range 3 to 5 per cent. RESULTS The suspension technique has proved to be a valuable adjunct to the conventional process of precipitating and Geiger counting cart o 1-14 in the form of barium carbonate. If a precipitate shows too feeble an activity for reliable assessment by Geiger counting, it is simple and quick to suspend and count a portion of the precipitate; no chemical processing of the precipitate is required and preparation of a suspension takes only 10 to 15 minutes.In addition, if required, the suspension method provides a means of checking activity figures deduced from Geiger counting. Table VI shows the results obtained from a cross-check of the two methods of counting applied to duplicate (in one instance triplicate) precipitations in a series of experiments. These results show good agreement between suspension counting on different weights of the same precipitate and between suspension and Geiger counting over a range of activities. SENSITIVITY * In practice, the gain in sensitivity achieved by suspension counting, relative to Geiger counting, will depend on a variety of factors.These include the weight of the precipitate, the proportion taken for suspension counting and also the instrumental conditions used for the suspension counting, these last affecting both the efficiency of the suspension countingMarch, 19621 OF CARBON-14 BARIUM CARBONATE 175 TABLE VI COMPARISON OF RESULTS BY GEIGER AND SUSPENSION COUNTING OF BARIUM CARBONATE number 105 106 107 108 109 110 111 112 113 114 115 Activity of precipitate found by- A I 1 suspension scintillation counting on- 0.1 g of 0.3 g of 0.5 g of A f > Geiger Precipitate counting, barium carbonate, barium carbonate, barium carbonate, CLC CLC CLC CLC 0.0030 0.0028 0.0061 0.006 1 0.0080 0.0080 0-0309 0.0316 0.1 14 0.112 0.111 0*0030 0.0030 0.0060 0*0060 0.008 1 0.0080 0.0303 0.0302 - - - 0.0031 0.0030 0-0061 0.0062 0.0081 0*0080 0.0312 0.0308 - - - and the magnitude of the background count.The example in Table VII shows the kind of gain in sensitivity realised in this work. In this example, Geiger counting over a long period would be required to achieve any sort of accurate assessment of the activity present, whereas if only half the precipitate is suspension counted, counting for 15 to 30 minutes would be adequate to reduce the counting error to a few per cent. , TABLE VII COMPARISON OF SENSITIVITIES OF GEIGER AND SUSPENSION COUNTING Counting 1 g of barium carbonate containing 0.0005 pC of carbon-14 Method of counting Gross, Background, Net, Net count rate c.p.m.c.p.m. c.p.m. background Barium carbonate as 2-inch diameter source Suspension counting of 0.5 g of the barium counted with 2-inch Geiger counter . , 33 28 5 0.2 carbonate . . . . .. .. . . 350 90 260 2-9 W’ith a background for suspension counting (i.e., the count rate observed for an inactive suspension) of abmt 1-5 c.P.s., an increase of 1 C.P.S. above this level has been taken as significant. This corresponds to the detection of 5 x pC of carbon-14 in 0.5 g of barium carbonate. This level of sensitivity has proved adequate for this work, but further gains in sensitivity would appear to be practicable, A few experiments showed that 0.7 and 1.Og of barium carbonate could be counted with efficiencies of about 45 and 39 per cent., respectively; hence, if large precipitates are available sensitivity can be increased by increasing the weight of precipitate suspended.Precipitation in the form of calcium carbonate, containing about twice as much carbon as barium carbonate, would presumably double the sensitivity, Finally, use of other instrumental conditions, e g . , coincidence counting, could effectively increase sensitivity by reducing the background count rate. DISCUSSION OF METHOD Following on the work of Hayes, Rogers and Langham6 (who counted suspensions in the absence of a suspending or gelling agent) and of White and Helf,3 an attempt was made to determine the extent to which self-absorption of the carbon-14 beta-activity occurs in the particles of the suspended barium carbonate.For this purpose suspensions were prepared from inactive barium carbonate precipitates by using scintillator solution containing a known concentration of n-hexadecane of certified carbon-14 activity. Counting of these suspensions176 CLULEY SUSPENSION SCINTILLATION COUNTING [Vol. 87 permitted salculation of the efficiencies of solution scintillation counting in the presence of inactive r ispensions Compared with these conditions, the onljr additional factor affecting the efficiency with which active suspensions are counted is self-absorption within the particles of the suspension, and comparison of the two series of counting efficiencies should show the extent to which the self-absorption occurs. TABLE WIT EFFICIENCIES OF SUSPENSION COI~STIKG ASI) OF SOLUTIOS c ou S n x G I l i THE PKESEXCE 01; INACTIVE SUSPENSIOSS \\'eight of Suspension counting Solution counting cAfficienc-\- in prcscnce liatio L H barium carbonate, efficiency (from g Table 111) (A), y,, of suspensions ( R J , ;{) 0.1 0.3 0.5 60 49 66 66*5 6fi The results obtained (see Table VIII) show a constant ratio between the two counting efficiencies of a little less than unity.This confirms the conclusions of Hayes et d . , 6 whose corresponding mean ratio for barium carbonate was 0.94 0.02, that the loss of counting efficiency of suspensions due to self-absorption amounts to only a few per cent. and is independent of the weight of barium carbonate suspended. It is clear therefore that some additional factor must be adduced to account for the appreciable reduction in counting efficiency of suspensions that occurs as the concentration of barium carbonate is increased. White and Helf3 have suggested that this effect is attributable to the increasing opacity of the suspension to the light emitted by the scintillator. Taking this factor into account, it would appear that at constant barium carbonate concentration, variation of the particle size of the suspension will produce two opposing effects on the counting efficiency.A reduction in particle size will reduce the loss of activity by self-absorption, but will increase the loss of light by scattering, because of the larger total surface area of the particles. These two effects would appear to be largely self-cancelling in that White and Helf3 (though not using barium carbonate) found only small differences in counting efficiency for suspensions of 60 to 120 and 325 and 400 mesh particles; in the work described here only slight differences in counting efficiency were observed for suspensions of sifted and unsifted barium carbonate (see Table 11).This comparative insensitivity of the counting efficiency of suspensions to variation in particle size is of obvious practical advantage. It is clearly unnecessary to take elaborate precautions in sample preparation to ensure perfectly consistent particle size distribution. In this work, a short period of grinding is all that has proved necessary in the way of sample preparation, although care has been taken to use a consistent procedure for the precipitation of the barium carbonate.As referred to earlier, even the presence of some aggregates in the suspensions is no bar to the attainment of consistent counting efficiencies, REFERENCES 1. 2. 3. 4. 5. 6. Funt, B. I,., NucZeonics, 1956, 14, (8), 83. Funt, B. L., and Hetherington, A., Science, 1957, 125, 986. White, C. G., and Helf, S., Nucleonics, 1956, 14, (lo), 46. Helf, S., White, C. G., and Shelley, K. N., Anal. Chem., 1960, 32, 238. Ott, I>. G., Richmond, C. R., Trujillo, T. T., and Foreman, H., Nucleonics, 1969, 17, (9), 106. Hayes, I;. N., Rogers, B. S., and Langham, W. H., Ibid., 1956, 14, (3), 48. Received December 6th, 1961 DISCUSSION DR. T. T. GORSUCH asked if Dr. Cluley had any experience of direct beta-scintillation counting of DR. CLULEY said that, in connection with another aspect of his work with carbon-14, he had counted He had had no experience aqueous solutions of carbonate.aqueous carbonate solutions directly using a plastic phosphor as the scintillator.March, 19621 OF C-4RBON-14 BARIUM CARBONATE 177 of the technique by which an organic scintillator solution was modified, tag., by addition of dioxan, to permit miscibility with small amounts of aqueous solution. MR. M. R. HAYES asked if Dr. Cluley would care to compare the suspension scintillation counting of barium carbonate with the direct gasometric method, which appeared to be applicable to the determination of carbon-14 in graphite with considerable simplification. He also asked if the Van Duran type of beta counter had been tried. From the comparison given of a beta counter with a suspension scintillation counter it appeared that the insertion of the 1 to 2 counts per minute background counting rate of the Van Duran counter would in fact have made beta counting more attractive than suspension scintillation counting.DR. CLULEY said, with regard to gas counting, that this had been considered, but rejected for more than one reason. The characteristics of carbon dioxide were not favourable for Geiger counting, and for such a method addition of other gases to the carbon dioxide would be desirable. In addition, there was the problem of storage; this was difficult with gas samples, whereas, when the carbon dioxide was absorbed in sodium hydroxide solution, the solution was easily stored for subsequent re-examination if required.Although a low background count would be advantageous, the efficiency of solid counting would still be low because of the large losses by self-absorption. The higher efficiency of suspension counting would permit a shorter period of counting to attain the number of counts required to achieve a given accuracy. MR. D. I?. WOOD asked if the author could give some details of the reason for the differences in ratios of total count to background count obtained by Geiger counting and scintillation counting. DR. CLULEY said that the much more favourable ratio obtained with suspension scintillation counting of small amounts of carbon-14 arose from the much higher efficiency of this method of counting. DR. R. G. MONK said that, in view of the low energy of carbon-14 beta particles, it seemed to him that the use of barium carbonate as a counting source made unnecessary difficulties. The high atomic number of barium resulted in greater self-absorption losses than would occur with the same thickness of calcium or strontium. The use of calcium or strontium carbonate would present no difficulties, as both compounds were slightly less soluble than barium carbonate. He added that these remarks applied only t o the counting of solid thick sources and not to the author’s suspension scintillation method. DR. CLULEY agreed that precipitation in the form of calcium or strontium carbonate would give more efficient solid counting, although the resulting gain in efficiency would still be small compared with that achieved by suspension scintillation counting. In the suspension method, as pointed out in the paper, the loss of counting efficiency due t o self-absorption by the barium carbonate particles amounted to only a few per cent. MRS. M. P. TAYLOR asked whether a coincidence circuit was used in the apparatus for counting the samples. DR. CLULEY replied that a coincidence circuit was not used; the scintillation counter was of the single photomultiplier type. MR. J. W. OGELBY asked whether the suspension method of counting was restricted to the measurement of white compounds or compounds that would not absorb the light emitted by the scintillator. DR. CLULEY said that, as far as he knew, the suspension technique had been applied only to white or colourless compounds. The Van Duran type of counter had not been tried.

ISSN:0003-2654    

DOI:10.1039/AN9628700170

出版商:RSC    

年代:1962

数据来源: RSC

摘要:

178 GIBBONS: THE DETERMINATION OF GLYCOLLOYL [Vol, 87 The Determination of Glycolloyl in Substances containing Neuraminic Acid BY R. A. GIBBONS (,National Institute f o r Research in Dairying, Shinjield, Reading, Berks.) A procedure is described for determining the glycolloyl residue in substances containing neuraminic acid. The glycollic acid in acid hydroly- sates is freed from interfering substances by adsorption on an ion-exchange resin and subsequent elution, the glycollic acid in the effluent being deter- mined colorirnetrically with a solution of 2,7-dihydroxynaphthalene in sulphuric acid. The glycolloyl contents of several substances containing neuraminic acid are reported. NEURAMINIC acid (5-am~no-~,~-d~deoxy-~-g~cero-~-~~~o-nonulos-l-onic acid), I , occurs natu- rally both as its N-acetyl and N-glycolloyl derivatives ; several naturally occurring 0-acetyl derivatives have also been described.These acylated derivatives of neuraminic acid are referred to collectively as sialic acids. A method of determining the glycolloyl group in materials containing sialic acid was required, and the only procedure available was that described by Eegriwe,l which necessitated isolation of the sialic acids in a fairly pure form before hydrolysis.2 This procedure is wasteful both in time and material. The N-glycolloyl group is readily liberated by acidic hydrolysis, but other substances present in materials of biological origin, notably hexoses, hexosamines and some amino acids, react to a con- siderable extent with Eegriwe’s reagent (2,7-dihydroxynaphthalene in concentrated sulphuric acid).H H H OH OH I I I I L HOOLCO-CH2-C--C--C-C- -CH,OH I I I I I OH NH, OH H H (1) Because of the ease with which neuraminic acid can be quantitatively separated from interfering substances by passage through a column of strongly basic anion-exchange resin in the acetate form,3 procedures along these lines were investigated. Acetate was found to interfere with the determination of glycollic acid if present in large amounts ; the carbonate and free-base forms of the resin retained significant amounts of hexose and hexosamine, but the benzoate form appeared to be satisfactory. Two hydrolytic procedures were investigated; one involved use of 0.5 N sulphuric acid, the other of a strongly acidic cation-exchange resin.The latter was satisfactory only when the specimen for analysis was salt-free and readily soluble in water. With both procedures, other neutral and basic products of hydrolysis, e g . , hexoses and hexosamines, can conveniently be determined in the effluents from the column. METHOD REAGENTS- Strongly basic anion-exchange resin-2QQ to 400 mesh. Strongly acidic cation-exchange resin-200 to 400 mesh. Hydrochloric acid, 2 N. Sodium hydroxide, 2 N. Sodium benzoate, I M. Potassium sulphate, 1 M. Eegriwe’s reagent-A 0.1 per cent. solution of 2,7-dihydroxynaphthalene in sulphuric acid, sp.gr. 1-84; prepare this solution freshly each day. Standard glycollic acid sol.ution--Dissolve 213 mg of zinc glycollate dihydrate , previously stored over calcium chloride, in 100 ml of water to give a solution 0.1 per cent.with respect to the glycolloyl (CH,.OH-CO-) group. Store this solution frozen, and dilute suitably before use.March, 19621 I N SUBSTANCES CONTAINING NEURAMINIC ACID 179 PREPARATION OF ION-EXCHANGE COLUMN- Set the anion-exchange resin aside at 80" C with 4 to 5 volumes of 2 N hydrochloric acid for 30 minutes, decant the "fines," pour the resin into a tube, and wash with water until the effluent is neutral. Slowly wash with 20 volumes of 2 N sodium hydroxide and then with water until the effluent is neutral; then wash with 10 volumes of 1 M sodium benzoate and finally with 10 volumes of water. Use a small tube (internal diameter 0-7 cm) tapered at the base and plugged with a little glass-wool, and fill with resin to a height of 6 to 7 cm (this quantity is somewhat critical), HYDROLYSIS AND ION-EXCHANGE PROCEDURES- Hydrolysis by sul~huric acid-Dissolve the sample in 0.5 N sulphuric acid, seal the solution in a Pyrex-glass test-tube, and heat at 100" C for at least 10 hours (see Fig.1, p. 180). Withdraw not more than 3 ml of solution containing 10 to 100 pg of glycolloyl (equivalent to 50 to 500 pg of N-glycolloylneuraminic acid). Place this solution on the column, and wash with 20 ml of water; the effluent can be reserved for determining hexoses and hexosamines. (As benzoic acid is relatively insoluble, the effluent is only slightly acid and requires no further neutralisation before analysis.) Elute the glycollic acid with 1 M potassium sulphate to a volume of 10 ml; collect the effluent in a calibrated vessel. Hydrolysis by cation-exchange resin-Seal in a Pyrex-glass test-tube an aqueous salt-free solution of the sample (10 to 100 pg of glycolloyl) and an equal volume of moist cation- exchange resin, prepared as described by D i x ~ n , ~ and heat at 100" C for 24 hours.Quantita- tively transfer the contents and rinsings of the tube to a small column containing an approxi- TABLE I Wash the column with 10 ml of water before use, OPTICAL DENSITIES WITH AND WITHOUT REMOVAL OF INTERFERING SUBSTANCES Optical density (corrected for blank value) of- Sample I 3 effluent from sample diluted (1 + 9) Weight taken column and measured directly Bovine submaxillary mucoid . . . . 10mg 0-440 Bovine cervical mucoid . . . . . . 0.44 1 Galactose . .. . . . . . .. Fucose . . . . . . . . . . . . 0.1 mg of each Serine, threonine, aspartic acid, glutamic acid, glycine, alanine, valine, arginine, lysine, proline and leucine Glycollic acid . . . . . . N-Acetylglucosamine . . . . . . 0.261 0.533 0.267 0-131 . . Glycollic acid . . . . . . .. 100 pg* 50 P.g* 25 pgY * As glycolloyl. 0.744 0.637 0.805 0.540 0-27 1 0.140 mately l-cm layer of the cation-exchange resin, and elute with water directly into the column of anion-exchange resin in the benzoate form ; with this procedure, a column containing an approximately 5-cm layer of the latter resin is sufficient. The aqueous effluent contains only the neutral compounds, as hexosamines are quantitatively retained on the cation- exchange resin, from which they can be eluted with 2 N hydrochloric acid.5 Elute the glycollic acid from the anion-exchange resin as described above.REGENERATION OF ANION-EXCHANGE COLUMN- benzoate and water (in that order). Treat the column with 15-ml portions of 2 N sodium hydroxide, water, 1 M sodium DEVELOPMENT OF COLOUR- Place 1 ml of the potassium sulphate effluent in a stoppered Pyrex-glass test-tube, cool in ice-water mixture, and add 10ml of Eegriwe's reagent. Mix well, heat in a bath of boiling water for 20 minutes, cool in tap-water, and measure the optical density (2-cm cells) at 535 mp with a Unicam SP600 spectrophotometer; the colour is stable for many hours.180 GIBBONS : THE DETERMINATION OF GLYCOLLOYL [Vol. 87 A blank solution and standards should be subjected to the ion-exchange chromatographic procedure, as there is usually a small column blank value.This value depends on the resin used; if many determinations are to be made, examination of several resins to find one giving a low or negligible blank value is worth-while. A batch of De-Acidite FF resin was used for most of the work described here. A C I.. I L---_I_ 2 4 8 16 24 +-h Time of hydrolysis, hours Fig. 1. Rates of hydrolysis at 100" C of glycolloyl residue in: curve A, N-glycolloylneuraminic acid ; curve B, bovine cervical mucoid ; curve C, bovine submaxillary mucoid. Hydrolysis by 0-5 N sulphuric acid (curves A and C) or Zeo-Karb 225 resin (curve B) 1 Wavelength, m p Absorption spectra of colours produced by heating various compounds with Eegriwe's reagent for 20 minutes: curve A, 4 pg of glycollic acid; curve B, 2 pg of formaldehyde; curve C , 70 bg of glucuronic acid; curve D, 100 p g of tartaric acid; curve E, 100 p g of lactic acid; curve F, 100 pg of gluconic acid Fig.2. RESULTS Table I shows results found when the proposed procedure was applied to standard glycoIIic acid, an artificial mucoid hydrolysate and bovine cervicals and submaxillary7 mucoids ; the optical densities obtained without removal of interfering substances are also recorded. Fig. 1 shows the rates of hydrolysis of the glycolloyl group present in N-glycolloylneuraminic acid and the same two mucoids.March, 19621 IN SUBSTANCES CONTAINING NEURAMINIC ACID 181 INTERFERING SUBSTANCES- There is no interference from any of the usual constituents of mucoids, although the hexoses and hexosamines cause serious disturbance if not removed as described above.A number of hydroxy acids other than glycollic caused negligible interference unless present in large amounts; the absorption spectra of some of these substances are shown in Fig. 2. Uronic acids, however, interfere with the column procedure and must be removed or allowed for: the optical density produced by 22 pg of glucuronic acid is equivalent to that developed by 1 pg of glycolloyl. Fortunately, uronic and neuraminic acids have not so far been found to co-exist in the same molecule.8 Synthetic N-acetylneuraminic acid has a negligible glycolloyl content, ie., the acid break-down products of neuraminic acid do not interfere. MECHANISM OF COLOUR REACTION- The colours produced by reaction of formaldehyde and glycollic acid with Eegriwe's reagent have almost identical absorption spectra.Glycollic acid also gives the same colour as does formaldehyde with a solution of chromotropic acid in 90 per cent. sulphuric acid, but does not do so under MacFadyen's less strongly acid condition^.^ Probably, therefore, the reaction proceeds via the oxidative decarboxylation of glycollic acid. There appears to be no advantage in replacing 2,7-dihydroxynaphthalene by chromotropic acid, as the latter is slightly less sensitive and has no greater specificity. TABLE II GLYCOLLOYL AND NEURAMINIC ACID CONTENTS OF VARIOUS SAMPLES Calculated N-Acetyl- N-glycolloyl- Neuraminic acid neuraminic acid Glycolloyl neuraminic acid content determined content calculated content con tent directly as N-acetyl from A and B Sample found (A), (5.51 x A), compound (B), (see text) % % % 4.99 20.5" 15-8 3.42 13.3* 10-15 Bovine submaxillary mucoid Bovine submaxillary mucoid after neuraminidase treat- ment .. .. , . Bovine submaxillary mucoid after hydrolysis (0.1 N sul- phuric acid; 1 hour; 80" C) Bovine cervical mucoid . . Bovine cervical mucoid after neuraminidase treatment Pig submaxillary mucoid . . Goat submaxillary mucoid . . Pig cervical mucoid . . Human bronchial mucoid . . Colominic acid . . .. K-Casein . . . . .. Human serum glycoprotein and dialysis . . . . % 0.90 0.62 0.23 4.01 2.07 2.20 0.18 0.11 t 0 . 0 2 <0*01 (0.01 0.27 1.27 22.1) 3*5* 14.8* 2.3 - 11.4) 7-O* -_ 12.1 9 . l t - 0.99 18-37 17.3 0.61 1.5t 1.03 - 8.2t 8.2 - 88.6* 88.6 1-49 1.9t 0.76 - 0.8t 0.80 * Determined by the Ehrlich reaction.t Determined by the thiobarbituric acid reaction. DISCUSSION OF RESULTS In the second column of Table I1 are shown the glycolloyl values for twelve preparations. This figure multiplied by 5.51 appears in the third column and represents the percentage of N-glycolloylneuraminic acid present if all the glycolloyl in the sample is originally present as N-glycolloylneuraminic acid. This assumption is apparently not justifiable for at least one type of sample, since the results show that bovine cervical mucoid contains more glycol- loyl residue than would be required for the N-acylation of all the neuraminic acid present. However, it can be seen from Fig. 1 that the rates of hydrolysis of the glycolloyl present in N-glycolloylneuraminic acid and in bovine submaxillary mucoid are similar and follow first- order kinetics reasonably well.Further, the relative amounts of N-glycolloyl- and N-acetyl- neuraminic acids found in this work agree well with those reported for the sialic acids crystallised from a similar source.2 For bovine submaxillary mucoid and many other samples,182 GIBBONS [Vol. 8‘7 therefore, the assumption is probably justified. The total neuraminic acid content, deter- mined directly as N-acetylneuraminic acid, is shown in the fourth column of Table 11; from this figure and the glycolloyl value, the content of N-acetylneuraminic acid present can be calculated (see fifth column of Table 11). This is valid if the preparation contains no N-acylated neuraminic acid other than the acetyl or glycolloyl derivative.In making this calculation it is necessary to allow for the difference between the molar extinction co- efficients of N-acetyl- and N-glycolloylneuraminic acids, and this difference depends on the method of determination used.1° When N-acetylneuraminic acid is used as standard, the correction to be subtracted from the amount of N-acetylneuraminic acid found in order to give the true value is 5-24 GR, where G is the percentage of glycolloyl found and X is the ratio between the molar extinction coefficients of N-glycolloyl- and N-acetylneuraminic acids appropriate to the method of determining the N-acetylneuraminic acid. (The values of R for the direct Ehrlich reaction, the resorcinol reaction and the thiobarbituric acid reaction were foundlo to be, respectively, 1-00, 1.17 arid 0.81.) Neuraminic acid values, referred to N-acetylneuraminic acid as standard, may therefore be misleading if the value of R differs significantly from unity when the sample contains much N-glycolloylneuraminic acid.Gottschalks states that the relative amounts of N-acetyl- and N-glycolloylneuraminic acids in a sample vary with the species and also with the site from which the specimen originates. This is borne out by the results reported here, which also show that, for bovine submaxillary mucoid (containing both derivatives), hydrolysis by neuraminidase or 0.1 N acid at 80” C fails to remove either derivative preferentially. I t may be remarked in passing that, after hydrolysis by 0.1 N sulphuric acid at 80” C for 1 hour, bovine submaxillary mucoid still retains an appreciable content of bound neuraminic acid, as do bovine cervical mucoid and colominic acid. For these substances the thiobarbituric acid assay for neuraminic acid is inapplicable. The “additional” glycolloyl residue in bovine cervical mucoid appears to be associated with the neuraminic acid, as removal of part of the latter with neuraminidase results in removal of the same proportion of the total glycolloyl residue.The presence of di-ON-glycolloylneuraminic acid is a possible explanation. The work described forms part of a study of cervical mucus supported by the Population Council, Rockefeller Institute, New York. I gratefully acknowledge gifts of N-glycolloyl- neuraminic acid and pig submaxillary mucoid from Professor E. Klenk, synthetic N-acetyl- neuraminic acid from Dr. Patricia Carrol and colominic acid from Dr. D. A. L. Davies. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Eegriwe, E., 2. anal. Chern., 1932, 89, 121. Klenk, E., and Uhlenbruck, G., 2. physiol. Chern., 1957, 307, 266. Svennerholm, L., Acta Chern. Scand., 1958, 12, 547. Dixon, A. S., Biochern. J., 1955, 60, 165. Boas, N. I?., J . Biol. Chem., 1953, 204, 553. Gibbons, K. A., Biochem. J . , 1959, 73, 209. Heimer, R., and Meyer, K., PYOC. Nut. Acad. Sci., Washington, 1956, 42, 728. Gottschalk, A., “The Chemistry and Biology of the Sialic ,4cids,” Cambridge tTniversity Press, MacFadyen, D. A., J . Biol. Chem., 1945, 158, 107. U7hitehouse, M. H., and Zilliken, F., in Glick, D., Editor, “Methods of Biochemical Analysis,” London, 1960. Interscience Publishers Inc., New York and London, 1960, Volume VIII, p. 199. Received Septenzber 6th, 1961

ISSN:0003-2654    

DOI:10.1039/AN9628700178

出版商:RSC    

年代:1962

数据来源: RSC

摘要:

March, 19621 LESTER SMITH, MARTIN, GREGORY AND SHAW 183 Standardisation of Hydroxocobalamin BY E. LESTER SMITH, J. L. MARTIN, R. J. GREGORY AND W. H. C. SHAW (Glaxo Laboratories Ltd., Greenford, Middlesex) Hydroxocobalamin can be standardised spectroscopically. The value of Ei& a t 351 mp may be taken as 190 for the pure anhydrous substance. Acidic and neutral cobalamins present as impurities may be separated from hydroxocobalamin (in its basic aquocobalamin form) with DEAE and CM ion-exchange celluloses for spectroscopic determination. SEVERAL publications have appeared recently1 9 , p3j4 suggesting that hydroxocobalamin offers some advantages over cyanocobalamin for patients requiring treatment with vitamin B12. The main superiority is greater retention in the body; with cyanocobalamin the major part of a large dose (e.g., 1 mg) is quickly excreted, but the proportion so lost is much smaller with hydroxocobalamin.In addition, it is becoming clear that cyanocobalamin is not the functional form of the vitamin and that it is probably inactive until converted via hydroxo- cobalamin to a coenzyme form, The structure of a B,, coenzyme, which is claimed to be the major component of the vitamin B,, reserves in human liver5 has recently been revealed.6 It contains adenosine linked via C, of the ribose to the B,, cobalt atom in place of cyanide. Accordingly, hydroxocobalamin is becoming available, as a bulk chemical and as a solution in ampoules, and it seems appropriate to suggest analytical methods and standards for the material, especially since some of the published constants appear to be incorrect.Cyanide can be removed from cyanocobalaniin in various ways; for example, by photoly- sis7 or with reducing a g e n t ~ ~ 9 ~ that yield vitamin B12, containing bivalent cobalt (or a more highly reduced product), which readily passes to hydroxocobalamin on aerial re-oxidation. In acid solutions the neutral hydroxocobalamin takes up a hydrogen ion, which converts the hydroxyl to a co-ordinated water molecule, as shown below.1° In this form the molecule (aquocobalamin) is basic and forms salts with acids. Thus in solution it exists as an equili- brium mixture with hydroxocobalamin, and, since it is more stable at acid pH values, commercial products are likely to be mainly in the aquocobalamin form.Therefore “aquoco- balamin” would be a more appropriate designation and it is indeed the only one sanctioned for this substance in the Nomenclature Rule+ ; however, the term “hydroxocobalamin” has become established in the medical literature (hydroxycobalamin is incorrect). “Vitamin B1,,” and “vitamin Blzb” are also synonyms for the same substance. Cyanocobalamin Hydroxocobalamin Aquocobalamin chloride Hydroxocobalamin occurs as very dark red (nearly black) crystals or crystalline powder, either as the free base or as a salt such as the chloride or sulphate. Its solubility characteris- tics and other general properties are similar to those of cyanocobalamin. The absorption spectrum is also similar, but the peaks occur at different wavelengths and they are displaced somewhat by pH changes1, The value of E:c2 at 351 mp appears to be much higher than that given in the original publications.8.12 This does not necessarily mean that the early product was less pure than the 98 per cent.claimed; the measurement was made on material dehydrated by heating at 100” C for 2 hours under reduced pressure, and it now appears that this treatment causes some decomposition (although cyanocobalamin is unaffected). It is therefore recommended that absorption measurements be made on the hydrated material as received. A separate portion should be taken for determination of moisture. Solubility analysis does not commend itself for this somewhat unstable substance existing as an equilibrium mixture of two forms, probably having different solubilities.In acid solution the main peaks are at 351 and 525 mp. It is difficult to find independent criteria for the purity of a reference sample.184 LESTER SMITH, MARTIN, GREGORY AND SHAW: [Vol. 87 Three samples of highly purified aquocobalamin chloride were examined ; the values for E:Z at 351 mp were 190, 194 and 191, calculated for anhydrous material. This suggests that all the preparations were substantially pure and that the rounded-off value of E:cz at 351 m,u = 190 may be accepted for pure anhydrous aquocobalamin chloride. Two of these preparations were also assessed after quantitative conversion to cyanocobalamin. This was done by adding a trace of cyanide to a solution containing a known weight of sample, allowing 2 hours for complete conversion to dicyanocobalamin, then acidifying with acetic acid and allowing a further 2 hours for conversion to cyanocobalamin by removal of the second cyanide group.The values (corrected for the small change in molecular weight) were 209 and 210, compared with the accepted value of 207 for cyanocobalamin. This provides further assurance that the hydroxocobalamin preparations were pure. In the preparation of hydroxocobalamin from cyanocobalamin some hydrolysis of the most labile amide group to a carboxylic acid is liable to occur13; also, unchanged cyanoco- balamin and possibly other neutral and acidic cobalamins may be present in the product. There may also be cobalamin analogues (in their hydroxo-forms), derived from impurities in the cyanocobalamin used, which are not detected by the tests prescribed in the British Pharmacopoeia, 1958.In principle, chromatography on paper could be used to separate all impurities, but difficulties would arise from their very slow movement in the usual solvent systems. Electrophoresis on paper at various pH values has been enlisted to separate the red acidic and neutral impurities from the basic aquocobalamin, but in order to have measur- able amounts of the minor constituents it is necessary to apply an inconveniently heavy load to the paper, and complete separation is not easy to achieve. By contrast, small columns of ion-exchange celluloses provide dependable, rapid and quantitative fractionation, provided certain simple precautions are taken. Analogues having bases other than 6,B-dimethylbenziminazole in the “nucleotide” part of the molecule can be detected by paper chromatography with wet secondary butanol con- taining traces of acetic and hydrocyanic acids.13 In this system all other cobalamins are converted into cyanocobalamin, but any carboxylic acids present would separate and cause confusion.Accordingly, chromatography woiild preferably be done after removal of “red acids’’ with DEAE cellulose and concentrating the effluent by evaporation or phenol extrac- tion.13 Experiments are in hand to determine how best to make this procedure quantitative. The solution was then diluted suitably for measurement of EtCE at 361 mp. METHOD PROCEDURE FOR DETERMINING MOISTURE- Dry about 50mg, accurately weighed, at 105°C under reduced pressure to constant Do not use this dried material for the other analytical weight (3 hours is normally sufficient).tests. PROCEDURE FOR DETERMINING HYDROXOCOBALAMIN CONTENT (plus COLOURED IMPURITIES), Weigh accurately about 60 mg of the sample into a 25-ml calibrated flask, dissolve, and dilute to the mark with water. By pipette, place 1.00ml of this solution (reserving the remainder) in a 100-ml flask, and dilute to volume with acetate buffer solution, pH 4.5 (pre- pared by mixing 600 ml of 0.2 N acetic acid with 400 ml of 0.2 N sodium acetate). Determine the absorption at the maximum near 351 mp in a 1-cm cell against a buffer solution blank. Express the result as a percentage of hydrosocobalamin (calculated on the dry sample) assuming E:cZ = 190 for the pure dry substance.Determine the absorption spectrum over the range 250 to 600 mp (conveniently with a recording spectrophotometer) on the same solution in a 1- or a 2-cm cell against an acetate buffer solution blank. For identification, compare with the curve for a reference sample. AND IDENTIFICATION- PROCEDURE FOR DETERMINING “RED ACIDS” AND OTHER COBALAMINS- Apparatus and results- (a) Two 9-inch x *-inch glass chromatographic tubes fitted with taps. ( b ) DEAE cellulose (Whatman DE50 powder). (c) Carboxymethylcellulose (Whatman CM70 floc.)March, 19621 STANDARDISATION OF HYDROXOCOBALAMIN 185 Make slurries of the DEAE cellulose with 0.5 N sodium hydroxide and the CMC with 0.5 N hydrochloric acid. Dilute each slurry with distilled water, allow to settle, and then remove most of the supernatant liquid by decantation.Stir each sediment with distilled water, transfer to Buchner funnels, and wash thoroughly, pressing out surplus water after each wash. Transfer portions of the suspensions to the empty tubes (one for each cellulose), after constrictions have been plugged with glass-wool. The DEAE column (A) should be allowed to fill with solid under gravity to about 8 inches. The CMC column (B) should be tamped down firmly, as it is filled, to a final depth of 4 inches. Wash each column with water until the pH of the wash-liquor remains constant. When free from alkali, the DEAE cellulose column should be gently tamped with a glass rod to a depth of about 6 inches. I t is important that this should not be overdone, as this column gives a slow rate of flow when compacted.Add to each column a further plug of glass-wool, and drain until only a small amount of water remains above the packing. Place the DEAE cellulose column (A) above the other (B) so that the eluate from (A) flows into it. Check the pH of the undiluted solution prepared for spectroscopy, and, if it is not already acid, add a trace of hydrochloric acid to bring it below pH 4, to ensure complete conversion to the aquo-form. By pipette, place 20 ml of this solution in the top column (A), and allow to run through both columns, rejecting the first colourless eluate. Continue adding water to column (A), collecting the coloured eluate from (B) in a 50-ml calibrated flask until 50 ml have been collected. At this stage the eluate from both columns should be nearly colourless.Record the absorption of the eluate from (B) at the 360 to 361 mp maximum, with water as blank, and calculate the percentage of other cobalamins as dry B,, equivalent (E:cE = 207). In (A) there will be one or more pink bands due to “red acids.”” In (B) the aquocobalamin is strongly held as a dark red band at the top; a slight red staining further down this column is due to weakly held hydroxocobalamin. The eluate contains any cyanocobalamin and other neutral cobalamins. Elute the pink bands from column (A) directly into a 50-ml calibrated flask with 1 per cent. aqueous sodium chloride, and dilute to the mark. Mix, and measure the absorption at the maximum (355 to 360 mp) in a 1-cm cell against a 1 per cent. solution of sodium chloride as blank.Calculate the red-acid content as the hydroxocobalamin equivalent on the dry substance (Le., use an E:cZ value of 190). If desired, the aquocobalamin may be eluted from column (B) with 1 per cent. aqueous sodium chloride into a 250-ml calibrated flask until the washings are colourless. The absorp- tion at the 350 to 351 mp maximum is then recorded against a 1 per cent. aqueous sodium chloride blank in a 1-cm cell on an appropriate dilution in 1 per cent. aqueous sodium chloride, and the aquocobalarnin chloride content of the dried sample is calculated by using Ei& = 190 for the pure substance. If only a limited amount of sample is available or if ampouled material has to be tested, then a moderately accurate assessment can be made on a few milligrams with scaled-down columns and solutions made up to a volume only just large enough to fill the spectrophotometer cell.The CMC and DEAE packings may be used again after the appropriate treatment (acid or alkali, respectively). Standards for hydroxocobalamin may be suggested as follows. Since it appears hazardous to dehydrate hydroxocobalamin, air-dried hydrated material containing 10 to 20 per cent. of moisture should be acceptable. The purity, calculated on a moisture-free basis and taking E:cZ at 351 mp = 190 for the pure anhydrous substance, should be not less than 95 per cent. in conformity with the B.P. standard for cyanocobalamin. This value determined by direct spectroscopy will include any other cobalamins and analogues. Not more than 3 per cent.of red acidic impurities and (in addition) not more than 3 per cent. of red neutral impurities should be present, calculated on the total red material as measured by direct Suspensions of these prepared celluloses may be stored ready for use. At this point the appearance of the columns should be as described below. * The monocarboxylic acid in its aquo-form has zero net charge, but nevertheless it is held strongly enough by the basic cellulose to permit separation, which can be considered satisfactory provided colour does not extend to the bottom of the column. Confirmation may be obtained if necessary by treating another sample with cyanide before running on DEAE; in its cyano-form the acid is strongly held by DEAE.186 LESTER SMITH, MARTIN, GREGORY AND SHAW [Vol.87 spectroscopy. Tentatively, a limit of 3 per cent. may also be set on analogues not derived from cyanocobalamin. The results of some typical analyses are set out in Table I. TABLE I TYPICAL ANALYSES OF COMMERCIAL HYDROXOCOBALAMIN Sample No. . . .. .. 1* 2 3 4 5 Loss a t 105” C in vacuo, yo . . 17.8 16-9 16.4 17.5 17-4 “Red acids,” % .. . . 0.5 2.8 2.4 1.8 1-8 Total absorption at 351 mp (calculated as percentage of hydroxocobalamin in dry material): . . .. . . 100.5 99-1 96.3 98-9 99.2 Other cobalamins, yo . . . . 0.1 1-6 5.4t 3.1 1.9 * Sample specially purified. t Residual cyanocobalamin. : Includes “red acids” and other cyanocobalamins. 6 Ti 17.0 16-7 1.2 0.3 3.0 0-3 97.5 97.1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. REFERENCES Skeggs, H. R., Hanus, E. J., McCauley, A. B., and Rizzo, V. J., Proc. SOC. Exp. Biol. Med., 1960, Killander, A., and Schilling, R. F., J . Lab. Clin. Med., 1960, 56, 917. Glass, G. B. J., Skeggs, H. R., Lee, D. H., Jones, E. L., and Hardy, W. W., Nature, 1961, 189, 138. Samson, G. D., Yeh, S. D. J., and Chow, 13. F., Fed. Proc., 1961, 20, 451. Toohey, J. I., and Barker, H. A., J . Biol. Chem., 1961, 236, 560. Hodgkin, Dorothy C., and Lenhert, P. G., paper presented a t the 2nd European Symposium on Vitamin R,, and Intrinsic Factor, Hamburg, August, 1961. Veer, W. L. C., Edelhausen, J. H., Wijmenga, H. G., and Lens, J., Biochim. Biophys. Acta, 1950, 6, 225. Kaczka, E. A., Wolf, D. E., and Folkers, K., J . Amer. Chem. SOC., 1949, 71, 1514. Beaven, G. H., and Johnson, E. A., Natum, 1955, 176, 1264. Cooley, G., Ellis, B., Petrow, V., Beaven, G. H., Holiday, E. R., and Johnson, E. A., J . Phavm. I.U.P.A.C., J . Amer. Chem. SOC., 1960, 82, 5582. Kaczka, E. A., Denkewalter, R. G., Holland, A., and Folkers, K., J . Amer. Chem. SOC., 1951, .4rmitage, J. B., Cannon, J. R., Johnson, .A. W., Parker, L. F. J., Smith, E. Lester, Stafford, Received Septembey 2272d, 1961 105, 518. Pharmacol., 1951, 3, 271. 73,335. W. H., and Todd, A, R,, J . Chem. SOC., 1953, 3849.

ISSN:0003-2654    

DOI:10.1039/AN9628700183

出版商:RSC    

年代:1962

数据来源: RSC