ISSN:0003-2654    

DOI:10.1039/AN95378FX017

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

No. I I April, 1953 THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS BULLETIN COMMITTEES, 1953-1954 THE Couiicil of the Society has appointed the following Conimittees -- FINANCE COMMITTEE D. W. Kent-Jones (Chairman), N. L. Allport, Lewis Eynon, J. H. Hamence, H. W. Hodgson, E. B. Hughes, G. W. Monier-Williams, G. Taylor, A. M. Ward, K. A. Williams (Honorary Secretary). PUBLICATION COMMITTEE J. R. Nicholls (Chairman), N. L. Allport, A. J. Amos, A. L. Bacharach, R. C. C,hiriiside, B. S. Cooper, Lewis Eynon, D. C. Garratt, J. H. Hamence, J. Haslam, H. M. N. H. Irving, G. Roche Lynch, F. L. Okell, G. H. Osborn, J. E. Page, A. A. Smales, G. Taylor, L. S. Theobald, Eric Voelcker, C. Whalley, K. A. Williams, E. C. Wood. POLICY COMMITTEE G. Taylor (Chairman), R. C. Chirnside, J.A. Eggleston, J. H. Hamence, J. Haslam, N. Heron, H. M. N. H. Irving, D. W. Kent-Jones, J. R. Nicholls, A. M. Ward, K. A. Williams. ANALYTICAL METHODS COMMITTEE E. B. Hughes (Chairman), N. L. Allport, R. C. Chirnside, Norman Evers, J. H. Hamence, J. Haslam, D. W. Kent-Jones, R. F. Milton, J. R. Nicholls, F. L. Okell, G. H. Osborn, J. E. Page, R. W. Sutton, G. Taylor, H. C. S. de Whalley, K. A. Williams, D. W. Wilson, E. C. Wood, D. C. Garratt (Honorary Secretary). PUBLIC ANALYSTS AND OFFICIAL AGRICULTURAL AK'ALYSTS COMMITTEE G. Taylor (Chairman), C. A. Adams, F. W. F. Arnaud, H. H. Bagnall, W. Gordon Carey, H. Childs, J. F. Clark, S. Dixon, J. H. Hamence, E. S. Hawkins, N. Heron, E. T. Illing, D. W. Kent-Jones, J. King, A. Lees, J. B. McKean, T. McLachlan, C.H. Manley, S. Ernest Melling, G. W. Monier-Williams, H. E. Monk, J. R. Nicholls, C. J. Regan, J. G. Sherratt, R. W. Sutton, R. G. Thin, K. A. Williams, E. C. Wood, Eric Voelcker (Honorary Secretary). STANDARD METHODS OF ANALYSIS COMMITTEE G. Taylor (Chairman), X. L. Allport, D. C. Garratt, J. H. Hamence, D. W. Kent-Jones, J. R. Nicholls, K. ,4. Williams. LIAISON COMMITTEE G. Taylor (Chairman), J. H. Hamence, K. A. Williams. SOCIETY'S REPRESENTATIVES ON THE JOINT COMMITTEE OF THE SOCIETY AND THE ROYAL INSTITUTE OF CHEMISTRY W. Gordon Carey, S. Dixon, J. H. Hamence, T. McLachlan, J. G. Sherratt, G. Taylor, Eric Voelcker.FORTHCOMING MEETINGS Ordinary Meeting of the Society, May 6th, 1953 AN Ordinary Meeting of the Society, organised jointly by the Microchemistry Group and the Scottish Section, will be held at 7.15 p.m.on Wednesday, May 6th, 1953, in the Chemistry Department, University of Glasgow, Gilmore Hill, Glasgow. The following papers will be presented and discussed- “Geochemistry and Microchemistry,” by David T. Gibson, D.Sc. “Micro-analysis of Silicate Rocks. Part IV. The Determination of Alumina,” by Christina “Microchemical Determination of Sulphur in Organic Compounds,” by William H. The meeting will be preceded by an afternoon visit to the Clydebridge Steel Works of This meeting will take the place of the Ordinary Meeting that is normally held in London C. Miller, Ph.D., D.Sc., F.R.S.E., F.H.-W.C., and Robert A. Chalmers, BSc. Massie, B.Sc., Ph.D., A.R.I.C. Colvilles Ltd. on the first Wednesday in May.(Restricted to 30 members.) Ordinary Meeting of the Society, May 20th, 1953 , 4 ~ Ordinary Meeting of the Society will be held at 7 p.m. on Wednesday, May 20th, 1953, in the Meeting Room of the Chemical Society, Burlington House, London, W.l. The following papers will be presented and discussed- “The Determination of Ergosterol in Yeast. Parts I, 11, I11 and IV,” by W. H. C. Shaw, Ph.C., F.R.I.C., and J. P. Jefferies, B.Sc., A.R.I.C. “The Estimation of Micro Quantities of Calcium,” by G. E. Harrison, Ph.D., F.Inst.P., and W. H. A. Raymond. “The Ultra-Violet Spectrophotometric Estimation of the Quality of Mineral Oils Extracted from Bread,” by M. A. Cookson, B.Sc., A.R.I.C., J. B. M. Coppock, BSc., Ph.D., F.R.I.C., and R. Schnurmann, MSc., Dr.Rer.Nat. Meeting of the North of England Section, May 2nd, 1953 .A MEETING of the North of England Section will be held at 2 p.m.on Saturday, May 2nd, 1953, at the City Laboratories, Mount Pleasant, Liverpool, 3. At this meeting there will be a comprehensive discussion on “The Analysis of Waters, Sewages and Effluents,” introduced by W. Gordon Carey, F.R.I.C., and J. G. Sherratt, BSc., F.R.I.C. Summer Meeting of the North of England Section, June 12th to 15tl1, 1953 THE Summer Meeting of the North of England Section will be held from Friday, June 12th, to Monday, June 15th, 1953, at the Imperial Hotel, Llandudno, N. Wales. The following paper will be presented at 10.30 a.m. on Saturday, June 13th- “Random Reflections on Food Legislation,” by C. A. Adams, C.B.E., B.Sc., F.R.I.C.Meeting of the Physical Methods Group, May 8th, 1953 THE forty-first Ordinary Meeting of the Physical Methods Group will be held at 6 p.m. on Friday, May 8th, 1953, in the Oriental Cafd, Ipswich. This is a Joint Meeting with the East Anglia Section of the Royal Institute of Chemistry. The subject of the meeting will be “Spectroscopy” and the following papers will be presented and discussed- “Semi-quantitative Techniques in Spectrochemical Analysis,” by R. L. “Some Techniques of Presentation of Sample to the Spectrograph, ” “Applications of the Porous Cup Technique,” by L. G. Young. The meeting will be preceded by a visit to B.X. Plastics Laboratoiy Ph.D. , F.R.I.C. Gillieson, B.Sc., Ph.D. at 2.15 p.m. Mitchell , B.Sc., by H. C. P. at ManningtreeTHE PHYSICAL SOCIETY COLOUR GROUP THE Council of the Society of Public Analysts and Other Analytical Chemists has accepted an invitation to become a Participating Society in the work of the Physical Society Colour Group.(i) To provide an opportunity for the various groups of people concerned with colour- physicists, chemists, industrialists, etc.-to meet and become familiar with each other’s problems; (ii) To enable a representative opinion to be formed on various questions of standardisa- tion, specification, nomenclature, etc. ; (iii) Generally to encourage colorimetric investigations and to ensure that this country shall keep abreast of developments abroad. The Group will hold meetings and organise such other activities as may best achieve the above objects. Members of the Society of Public Analysts and Other Analytical Chemists are eligible to become members of the Group upon payment to the Treasurer of the Physical Society of QL special annual subscription of 5s. (Members who are also members of the Physical Society are eligible by virtue of that membership to join the Colour Group without payment of the additional subscription .) Details of the Group’s meetings and other activities can be obtained from Dr. S. T. Henderson, Acting Honorary Secretary, Physical Society Colour Group, Thorn Electrical Industries, Ltd., Great Cambridge Road, Enfield, Middlesex. The objects of the Colour Group are- PAPERS ACCEPTED FOR PUBLICATION IN THE ANALYST THE following papers have been accepted for publication in The Analyst, and are expected to appear in the near future.It is not possible to enter into correspondence about any of them. “The Determination of the Amount and Composition of Free Phenols in Phenol - Form- aldehyde and Cresol - Formaldehyde Resins and Moulding Powders,’’ by J. Haslam, S. M. A. Whettem and G. Newlands. A method has been evolved for the determination of total free phenols in phenol - formaldehyde, cresol - formaldehyde and phenol - cresol -.form- aldehyde moulding powders. In addition, tests have been devised whereby the ratio of free phenol to free cresol in the total free phenols contained in a moulding powder can be determined. It has also been shown that the ratio of m-cresol to p-cresol can be determined in the total free cresols present in a cresol - formaldehyde moulding powder prepared from m-cresol and p-cresol only.It has not been possible to extend the principles of this method to the corresponding combination of o-cresol and m-cresol or to that of 0-cresol and p-cresol. “The Colorimetric Determination of Fructose and Sorbose,” by F. J. T. Harris. When a fructose or sorbose solution is heated with the Folin- Denis reagent and tri-sodium phosphate a blue colour is produced. This colour can be used in determining the amount of sugar in the solution, with an accuracy of about 2 per cent. Sugars other than fructose or sorbose give much fainter colours. “A Research Polarograph for Photographic Recording and a Multipurpose Polarographic Cell,” by F. J. Bryant and G. F. Reynolds. The circuit lay-out and operation of a specially designed photographically- recording polarograph are described. -Incorporated in the instrument is an auxiliary potentiometer, which allows the change of applied potential per unit division of the polarogram to be varied for any required starting potential.There is also variable galvanometer damping and “forward” and “reverse” operation of the potentiometer drive, and either of two rates of change of potential can be applied to the cell. In addition, a lamp circuit is included, which may be used in marking the photographic recordat any point when recording the polarogram. Two polarograms are discussed in detail in order to illustrate the application of the instrument to polarographic problems. A multipurpose polarographic cell, which allows determination of the pH of the solution and of m and t for the electrode without removal of the solution from the cell, is also described.The dimensions of this cell allow for it to be used in a substantially unmodified Cambridge thermostat bath. NOTICES Second International Congress on Rheology A PROVISIONAL programnie for the Second International Congress on Rheology, to be held in Oxford between July 26th and 31st, 1953, has been prepared. All intending participants are asked to make their reservations as soon as possible (and in any case not later than May lst, 1953) by completing a Final Form of Application, which is attached to the provisional programme. The programme, giving full details and the form of application, can be obtained from the Hon. Organising Secretary, Dr.G. W. Scott-Blair, The University, Reading, England. The Association of Clinical Biochemists THE Inaugural General Meeting of the Association of Clinical Bi0chemist.s was held at the Postgraduate Medical School of London on March 28th, 1953. The Association will be both scientific and professional in the scope of its activities. The interim committee is acting as a provisional council, with Dr. A. L. Tgrnoky, Royal Berkshire Hospital, Reading, as Honorary Secretary, from whom further details of the Association’s future activities can be obtained. MEETINGS OF THE ROYAL SANITARY INSTITUTE Wigan Sessional Meeting, Friday, June 5th, 1953 AT this meeting the following papers will be presented: “Staphylococcal Food Poisoning in the Manchester Area,” by M. T. Parker, M.B., B.Ch., Dipl-Bact., Director, Public Health Laboratory, Manchester, and “The Changing Pattern of Refuse Disposal and its Effect on Vehicle Design,” by Clive Walker, Director, Walker Bros.Ltd., Engineers. Walsall Sessional Meeting, Thursday, July 2nd, 1953 AT this meeting the following papers will be presented: “Land Use in Walsall, with Special Reference to Slum Clearance and/Reclamation of Derelict Land,” by M. E. Habershon, O.B.E., M.Eng., M.I.C.E., M.I.Mun.E., Borough Engineer and Surveyor, Walsall, and ‘Some -4venues to a Better Environment,” by James Green, Deputy Chief Sanitary Inspector, Walsall.. London Sessional Meeting, Wednesday, July 15th, 1953 AT this meeting, to be held at 2.30 p.m. at the Royal Sanitary Institute, the following papers forming a Symposium on “Salvage and Utilisation of Food Waste for Animal Feeding” will be presented: (a) “Collection and Processing,” by John Stephen, M.Inst.P.C., Director of Public CZeansing, Luton, and (b) “Distribution and Utilisation,” by Major A. McD. Livingstone, C.I.E., M.C., M.A., B.Sc., Adviser on Agricultural Matters to the Waste Foods Branch, Ministry of Agriculture and Fisheries. Enquiries about these meetings should be addressed to the Secretary, The Royal Sanitary Institute, go, Buckingham Palace Road, London, S.W.1. - PRINTED BY W. HEFFER & SONS LTD.. CAMBRIDGE

ISSN:0003-2654    

DOI:10.1039/AN953780X019

出版商:RSC    

年代:1953

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95378BX023

出版商:RSC    

年代:1953

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95378BP039

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

APRIL, 1953 THE ANALYST Vol. 78, No. 925 PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS AN Ordinary Meeting of the Society, organised by the Physical Methods Group, was held at 6.30 p.m. on Friday, January 30th, 1953, in the Mason Theatre, The University, Edmund Street, Birmingham. The Chair was taken by the President, Dr. J. R. Nicholls, C.R.E., F.R. I.C. The subject of the meeting was “Chromatography,” and the following papers were presented and discussed: “Chromatography Past and Present,” by Trevor I. Williams, B.A., B.Sc., D.Phil. ; “Inorganic Chromatography on Cellulose. Part XIV. The Quantitative Separation of Rhodium, Palladium, Iridium and Platinum,” by D. B. Rees-Evans and R. A. Wells, RSc., A.K.I.C. ; “Chromatography of the Carbohydrates and their Derivatives,” by J.K. N. Jones, Ph.D., D.Sc. Thc meeting was preceded by an afternoon visit to the Dunlop Research Centre at the Dunlop Rubber Company Ltd. NEW MEMBERS MISS Kenate Ursula Born, 13.Sc. (Glas.) ; Dennis Green, l3.S~. (Lond.), A.R.I.C. ; Eric David Harris, M.Sc. (Lond.), A. R.I.C. ; Janies Shankland Merry, F.R.I.C., M.1nst.F. ; William Robertson, A.H-W.C,. , A.R.I.C. ; Juston O’Grady Tatton, M.Sc. (Lond.), A.R.I.C. ; Leslie George Tomlinson, M.Sc. (Lond.), F.R.I.C. ; Derick Ronald Wraige, B.Sc. (Liv.). DEATHS \c”: regret to record thc deaths of William Dickson George Eric Forstner John Myers. NORTH OF ENGLAND sEcrIoN AN Ordinary Meeting of the Section was held at 2 p.m. on Saturday, October 25th, 1952, at the City Laboratories, Mount Pleasant, Liverpool, 3.The Chairman, Mr. A. A. D. Comrie, B.Sc., F.R.I.C., presided over an attendance of fifty-seven. The following paper was presented and discussed: “Synthetic Detergents,” by C. B. Stuffins, A.R.T.C. THE Twenty-eighth Annual General Meeting of the Section was held at 2 p.m. on Saturday, January 31st, 1953, at the Engineers’ Club, Albert Square, Manchester. The Chairman, Mr. A. A. D. Comrie, B.Sc., F.R.I.C., presided over an attendance of thirty-seven. The following appointments were made for the forthcoming year :-Chairman-Mr. T. W. Lovett. Vice-Chairman-Mr. J. R. Walmsley. Hon. Secretary and Treasurer-Mr. Arnold Lees, 87, Marshside Road, Southport, Lancs. EEected Committee Members-Messrs. H. Childs, R. Crosbie-Oates, H. Dedicoat, F. Dixon, J.C. Harral and C. R. Louden. Hon. Auditors- Messrs. A. Alcock and C. J. House. The Annual General Meeting was followed by an Ordinary Meeting at which Mr. A. A. D. Comrie delivered his Chairman’s Address, the subject being “Beer Foam.” 189190 PROCEEDINGS [voi. 78 SCOTTISH SECTION THE Eighteenth Annual General Meeting of the Section was held in Glasgow on Wednesday, January 28th, 1953, at 12.30 p.m., and the following office bearers were elected for the forth- coming year :-Chairmaw-Mr. R. S. Watson. Vice-Chairman-Dr. F. J. Elliott. Hon. Secretary and Treasurer-Mr. J. A. Eggleston, Boot’s Pure Drug Co. Ltd., Motherwell Street, Airdrie, Lanarkshire. Elected Committee Members-Dr. Christina C. Miller (co-opted) and Messrs. J. K. McLellan, H. C. Moir, R. T.Potter, S. C. Sloan and A. C. Wilson. Hon. A ziditoirs-Messrs. J . Andrews and J. Gray. MICROCHEMISTRY GROUP THE Ninth Annual General Meeting of the Group was held at the Sir John Cass College, London, E.C.3, on Thursday, January 29th, 1953, at 7 pm., and the following Officers and Committee Members were elected for the forthcoming year :--Chairman-Dr. A. M. Ward. Vice-Chairman-Dr. G. F. Hodsman. Hon. Secretary-Mr. Donald F. Phillips, 101, South Promenade, St. Annes-on-Sea, Lytham St. Annes, Lancs. Hon. Treasurer-Mr. G. Ingram. Elected Committee Mewbers-Messrs. W. N. Aldridge, A. Bennett, W. Brown, G. W. C. Milner, J. T. Stock and Cecil L. Wilson. Hon. Auditors-Messrs. I.. H. N. Cooper and H. Childs. After the business meeting, the retiring Chairman, Dr. Cecil L.Wilson, F.R.I.C., gave an address on “Microchemistry : An Appraisal,” which was followed by a discussion. During the afternoon preceding the meeting, a party of members visited the new factory and laboratories of Messrs. L. Oertling Ltd. a t St. Mary Cray, Orpington, Kent. PHYSICAL METHODS GROTJP THE Eighth Annual General Meeting of the Group was held at 6 p.m. on Tuesday, November 25th, 1952, in the Meeting Room of the Chemical Society, Burlington House, London, W.l. Dr. J. Haslam, F.R.I.C., the Chairman of the Group, presided. The Group Officers and Elected Members of the Committee for the forthcoming year are as follows :-Chairman-- Dr. J. I-Iaslam. Vice-Chairman-Mr. A. A. Smales. Hon. Secretary and Treasurer-Mr. R. A. C. Isbell, Hilger & Watts Ltd., Hilger Division, 98, St.Pancras Way, London, N.W.l. Members of Committee-Messrs. W. H. Bennett, L. Brealey, J. H. Glover, A. G. Jones, R. H. Jones and F. C. J. Poulton. The Annual General Meeting was followed by the Thirty-eighth Ordinary Meeting of the Group, at which the following papers on “Forensic Science” were presented and discussed : “The Application of Some Physical Methods in Forensic Science with Particular Reference to the Examination of Materials Relating to Criminal Investigation,” by J. B. Firth, D.Sc., F.R.I.C., M.1.Chem.E. ; “The Examination of Questioned Documents,” by J. A. C. McClelland, BSc., Ph.D., A.R.I.C. POLAROGRAPHIC ISCU CUSS ION PANEL ?’HE Annual General Meeting of the Polarographic Discussion Panel was held at 6.30 p.m. on Wednesday, December 3rd, 1952, a t the Sir John Cass College, Jewry Street, London, E.C.3.The following Officers and Committee Members were elected for the forthcoming year :-Chcrirman--Dr. A. J. Lindsey. Hon. Secretary-Mr. G. W. C. Milner, Building 148, A.E.R.E., Harwell, nr. Didcot, Herks. Members of Committee-Messrs. W. Cule Davies, G. F. Reynolds and M’. Stross. After the business meeting the following papers were presented and discussed : “Modifica- tion of the Cambridge Polarograph for Derivative Polarography,” by P. R. D. Pomeroy, B.Sc., and R. A. White; “The Polarographic Determination of Cadmium in Aluminium and Some Other Alloys,” by W. Stross, M.D., F.R.I.C. Hon. Auditors-Messrs. C. A. Bassett and D. C. Garratt. BIOLOGICAL METHODS GRO‘IJP THE Eighth Annual General Meeting of the Group was held at the Chemical Society’s Rooms, Burlington House, London, W.l, on Thursday, December l l t h , 1952, a t 6.15 p.m.In the absence of the Chairman of the Group, the Chair was taken by Mr. A. L. Bacharach. The following Officers and Committee Memberswere elected for the forthcoming year :--Chairmaw--- Dr. H. 0. J. Collier. Vice-Chairman-Dr. L. J. Harris. Hon. Secretary and Treasurer- Mr. s. A. Price, Walton Oaks Experimental Station, Vitamins Ltd., Dorking Road, Tadworth, Surrey. Members of Committee-Messrs. A, L. Bacharach, E. M. Ravin, W, A. Broom,April, 19531 PROCEEDINGS 191 J. W. Lightbown, H. Pritchard and K. L. Smith. Hon.. Auditors-Messrs. D. M. Freeland and J. H. Hamence. The Annual General Meeting was followed at 6.30 p.m. by an Ordinary Meeting of the Group, at which Dr. H. 0. J. Collier was in the Chair and thirty-two other members and guests were present. The following papers were presented and discussed: “A Method of Identifying the Presence or Absence of Splenin ‘A’ and Splenin ‘B’ in Serum by the use of Guinea Pigs,” by Raymond Greene and Josephine Vaughan-Morgan ; “The Application of Large Plate Methods to Microbiological Assays of Antibiotics and Vitamin Products. Part I : Precision Assays. Part 11: Routine Assays,” by K. A. Lees and J. P. R. Tootill.

ISSN:0003-2654    

DOI:10.1039/AN9537800189

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

April, 19531 PROCEEDINGS 191 Large-Scale Chromatographic Separation of Sucrose - Raffinose Mixtures on Powdered Cellulose for the Determination of Raffinose in Raw Sugars The possibility of separating large amounts of sucrose - rafiinose mixtures on columns of powdered cellulose has been investigated. Artificial mixtures containing disproportionately small amounts of raffinose have been efficiently separated in up to 15 g of mixture per column. The liquid chromatogram technique enables complete elution of the separated sugars to be attained, with nearly quantitative recovery of the minor constituent. The method has been applied to the separation of raffinose from raw beet-sugars, 20 g of sugar being handled per run. Separation and recovery were satisfactory, and results show that agreement with raffinose analyses by the paper chromato- graphic method is good.WE have shown previously1Y2 that the quantitative determination of raffinose in raw sugars by paper chromatography can be done accurately and relatively easily. However, there is no alternative analytical method by which the results of the chromatographic determinations can be checked, as concentrations of raffinose below 0.5 per cent. in a mixture of sugars are below the sensitivity of conventional methods. If reasonable amounts of raffinose could be separated quantitatively from the bulk of the other sugars, it would appear that not only would a check be provided on the amount of raffinose present, but final proof of its identity by means of physical properties, in particular its optical rotation, would also be afforded.For a polarimetric determination, a minimum amount of 1 g of recovered raffinose was required, which, with about 0.5 per cent. of raffinose in a raw sugar, meant separating 200 g of sugar. However, by using a polarimeter micro-tube 2 dm long, but of a much smaller bore than usual, the quantity needed was reduced to about 0.1 g ; this involved the separation of 201: of sugar. The micro-tube used was a modified version of the tube described by Kacser and Ubbel~hde.~ We were confronted with three rather difficult problems with regard to the chromatographic technique : to separate quantitatively from a mixture of sugars a constituent that is present at a concentration of only 0.5 per cent. or less; to separate a sufficiently large quantity, at least 20 g, to yield enough of the constituent for its polarimetric determination; and to separate a trisaccharide from a disaccharide, sugars that have com- paratively low RF values.Quantitative separation of a minor constituent was shown to be feasible bj- means of paper chromatography. A natural developinent of this technique would be to increase the amount of sugar separated by applying in parallel a number of spots to a large sheet of paper, running the chromatogram to successful separation of the particular sugar and recovering the sugar by extraction with water, a technique that has been used satisfactorily by many workers. A simple calculation shows that with 4mg of sugar as the maximum load per spot, the number of sheets required to produce 100 rng of raffinose would be so large as to be impracticable.The only alternative seemed to be to use chromatographic columns for separating larger quantities, although it would then be difficult to attain the same accuracy as on sheets.192 GROSS AND ALBON : CHROMATOGRAPHIC SEPARATION OF [Vol. 78 By modifying the conditions, a large-scale separation with quantitative results seemed possible, even with the unfavourable proportions of sugars present. It was also considered desirable to effect the separation in one run and so avoid re-chromatographing mixed fractions. Although mixtures of sucrose and raffinose have been separated on columns before, none of the earlier workers, as far as we are aware, has dealt with similar quantities and similar unfavourable proportions in the composition of the mixture.Tiselius and Hahn4 separated raffinose from a mixture of sugars on a column of active carbon, but milligram quantities only were involved and the raffinose constituted more than one-half of the mixture ; Claesson6 designed a rather elaborate triple-filter chromatographic apparatus, with active carbon as the adsorbent, capable of dealing with large amounts of material and of separating mixtures of sucrose and raffinose. With this arrangement 1.4g of pure substance could be separated from a 50 per cent. mixture of the sugars. Lew, Wolfrom and GoeppJ6 who used a clay column, successfully separated 2 g of a mixture of sucrose and raffinose, a quarter of which was raffinose; Whistler and DursoJ7 by a modification of the Tiselius technique, resolved, on a large scale, mixtures of raffinose with other sugars on charcoal columns.Mixtures containing up to 1 g of raffinose with 2 g of disaccharides were satisfactorily separated, whilst up to 10 g of monosaccharides were separable. Powdered cellulose columns for the separation of mixtures of carbohydrates were used extensively and successfully by Hough, Jones and Wadman.8 Although the amounts of sugars separated on such columns were appreciably smaller than the quantities reported earlier, we decided that cellulose columns offered sufficient promise to be tried in preference to other adsorbents. Many data on the RF valuesof the given sugars with various solvent mixtures on paper chromatograms had already been obtained by us; they gave a good indication of the behaviour of these sugars on cellulose columns.Cellulose powder is a mild adsorbent and would not be expected to interfere with the sugars; it is commercially available in a form ready for use; the columns are comparatively easy to prepare; the rate of percolation is suitable without the use of vacuum or pressure; and the columns can be used repeatedly, which affords a great saving of time. As will be seen below, the results on cellulose columns were reproducible, with either new or old columns. Even if a column of reasonable size could cope with large amounts of sugars, and separation into single constituents in the column was clean, the possibility of recovering the individual sugars by the liquid chromatogram technique without using excessive volumes of solvent was still doubtful.With the relatively low RF values for sucrose and raffinose, the amount of solvent required to elute the zones is considerable. This means that the sugars are recovered as dilute solutions requiring concentration by evaporation. The load that can be placed on a column is proportional to the RF value of the sugar, and the time of elution is inversely proportional to its RF value. As we were interested in a quantitative recovery of the slower moving sugar, raffinose, we had first to be certain about complete elution of the sucrose. A less careful separation could easily lead to overlapping of the much wider sucrose band into the raffinose band, with subsequent failure of the separation. The task is much easier if the sucrose is to be recovered, as the loss of some of the sucrose in the “tail” of the zone will not greatly diminish the amount of pure fraction recovered.On the other hand, with a band of 100 mg of raffinose closely following a band of 20 g of sucrose, contamination by 0-5 per cent. of the sucrose will produce a 50 per cent. mixture of sucrose and raffinose. It was decided to run exploratory chromatograms with artificial mixtures of sucrose and raffinose on a gradually increasing scale and of the same unfavourable proportions as ultimately envisaged, in order to test the feasibility of the technique for our purpose. The technique was to be applied eventually to the separation of naturally occurring mixtures of these two sugars, as found in raw beet-sugars, which, owing to the presence of various impurities, might be even more difficult to separate.SEPARATION OF SUCROSE - RAFFIKOSE MIXTURES APPARATUS- Cylindrical Pyrex-glass tubes, with sintered-glass discs sealed in just above the con- striction, were used. The level of solvent above the packed cellulose was maintained constant by a simple arrangement. The flow rate of eluate was regulated to a fine degree by a needle valve. The eluate was collected by an automatic fraction collector. It was found desirable, after dry-packing the column, to fill the space below the sintered disc with solvent in order to exclude air bubbles, and to protect the top of the cellulose column by dropping anotherApril, 19531 SUCROSE - RAFFINOSE MIXTURES ON POWDERED CELLULOSE 193 sintered disc, slightly smaller than the bore of the cylinder, through the solvent on to the top layer.ADSORBENT- them through a sieve. standard grade for chromatography, was used. blr tamping and tapping to attain tight and uniform packing. with slurries of cellulose powder and solvent gave poor columns. of the column were first tested by separating a mixture of suitable dyes. columns prepared as described by Hough, Jones and Wadmans gave the best results. SOLVENT- It is well known that the movement of zones is sensitive to changes in the composition of the developer solvent. By a proper choice of solvents the rate of movement of the zones, their separability and recovery can be controlled to a certain extent. The first trial columns were run on a comparatively small scale with a solvent mixture containing pyridine, which had been found to give highly satisfactory results in the separation of raw beet-sugars on paper chromatograms.l However, it appeared that with this particular mixture it was difficult to use the same solvent repeatedly and, in particular, to maintain a constant solvent composition after re-distillation ; consequently, a simpler mixture with the same resolving power was sought.After various solvent combinations had been tested on a small scale for their capacity to separate sucrose - raffinose mixtures, two mixtures were finally found to give satisfactory results. Solvent mixture I consisted of 70 volumes of n-propanol, 20 volumes of water and 10 volumes of ethyl acetate; solvent mixture I1 consisted of 70 volumes of isopropanol, 20 volumes of water and 10 volumes of n-butanol. Solvent I gave slightly better results in paper chromatograms, whilst solvent I1 appeared to be more suitable for column work.Both mixtures are single-phase systems at ordinary temperatures, easy to re-use, provided the water content is checked, and comparatively cheap. This last point was worth considering as, with the large-scale separations in view, a considerable volume of solvent had to be used. The fairly large differences between the boiling points of the components promised good recovery of the solvent. The RF values on paper chromatograms for solvent I1 were found to be : for fructose 0.33, sucrose 0.22, and raffinose 0-08. As the sucrose moved nearly three times as fast as the raffinose and as the spots were sharp, the suitability of solvent I1 for column experiments seemed to be assured.As the chromatograms were not controlled for temperature, the figures for RF values were only approximate, although well reproducible. RECOVERY EXPERIMENTS OM ARTIFICIAL MIXTURES- To test its capacity and resolving power for sucrose - raffinose mixtures, a column 3 cm in diameter and 10 cm in height was prepared on which successively larger quantities could be separated; this gave the necessary information for future work. I t was essential to ascertain the shape of the elution curves and the sharpness of the boundaries under conditions that would give a good indication of the possibilities of the column in respect of our problem. The mixture of sugars of the desired proportions, 99.5 per cent.of sucrose and 0.5 per cent. of raffinose by weight, was dissolved in the minimum volume of solvent, the level of solvent in the column was lowered so that it just covered the top of the cellulose or upper sintered disc, and the solution of sugars was placed on the top of the column. The flow rate was then adjusted by means of the needle valve and, after all the sample had drained into the column, fresh solvent was fed from a constant-head reservoir. The eluate was collected in measured fractions, which were tested for sugars. If fractions had to be tested only for sugar generally, simple spotting on paper and spraying with a suitable sensitive reagent was sufficient. When the identity of the sugar had to be established, a paper chromatogram was run in the usual way.Fractions containing small amounts of sugars were evaporated to the necessary concentration before analysis. Hence it was possible to locate the presence of the sugars in certain fractions, establish the purity of the fractions and determine the percentage of recovery by bulking fractions containing only one sugar, evaporating to dryness under vacuum with a nitrogen leak, and weighing the residue. This ensures a flat and horizontal surface to the cellulose column. The powdered cellulose was first prepared from Whatman ashless filter tablets by rubbing For later experiments, Whatman ashless cellulose powder, the The columns were packed with dry powder Attempts to pack columns The packing and efficiency We found that The choice of solvent for any given separation must be made with great care.i ; i i 1 1 1 : I , 0.30.M c; c 00 z 020. 0- 10. 0 Fraction number Fig. 1. Powdered-cellulose column separations of sucrose - raffinose mixtures. Experiment 1 (continuous line), 0.5 g of sucrose and 2.5 mg of raffinose; experiment 2 (broken line), 1.0 g of sucrose and 5 mg of raffinose. Each fraction had a volume of 24 ml They also showed that the elution in different experiments was reproducible and, given standard conditions, such as a correctly packed column and constant composition of solvent, the breakthrough of the particular band and the volume of eluent required for it could be predicted within certain limits. This point is of interest as, with the chromatographic testing of the individual fractions, there is usually some delay before the distribution of the sugars in the fractions is known.Great variations in temperature and in the quantity of sample separated, and excessive variations of the impurities in the sample, such as the amount of salts present, may upset the reproducibility. If, however, attention is given to these points and to the water content of the solvent, a column can be used repeatedly, with the knowledge that the sugars will appear after approximately the same volumes of eluent. The fractions were tested by paper chromatography and showed the raffinose to be cleanly separated. The volume of solvent needed was 320ml and the recovery was nearly quantitative. This technique was subsequently applied to sugars when testing their purity.The degree of sensitivity attained was 0.001 per cent. of raffinose on a sucrose sample, which considerably extended the limit of 0.02 per cent. previously attained2 with the paper chromatographic method. ’ 1 : : I , 1 I . ----, 1 7 I , I I I , I I I I 1 I 1 I I I I I ! I 7 +Sucrose I $ I I I I 1 I I I Rat5 nose !----- i !----I 1 LARGE-SCALE SEPARATION OF A SUCROSE - RAFFINOSE MIXTURE- The satisfactory results with 1 g or more of the mixture prompted us to apply the technique to separations of large amounts of sugars on bigger columns. It was estimated that a column 7.5 cm in diameter and 30 cm in height might deal with up to 20 g of mixture. The column was carefully packed, thoroughly washed with solvent and the efficiency of its packing tested by running a mixture of dyestuffs of RF values approximately the same asApril, 19531 SUCROSE - RAFFINOSE MIXTURES ON POWDERED CELLULOSE 195 those of sucrose and raffinose, such as Orange G and Indigo Carmine; finally the eluate was tested for dissolved matter. The artificial mixture consisted of 15 g of sucrose and 0.1 g of raffinose, a proportion slightly more favourable than that ultimately to be analysed.The solubility of sucrose in the solvent used is 6 g per 100 ml. Consequently, the mixture was dissolved in 300 ml of solvent, placed on top of the cellulose column and allowed to pass slowly into the column. Just as the last part of the solution entered the column, pure solvent was added to develop and elute the zones of the two sugars.Even so, the experiment took several days to complete. The procedure followed was roughly that described above in connectior, with small columns. To cut down the number of operations, all fractions were tested qualitatively for purity; all pure fractions were bulked, evaporated to dryness and analysed quantitatively. This procedure effected a great economy in labour, but did not give precise information about the quantitative distribution of the individual sugars in the various fractions. However, the concentration was adequately indicated with paper chromatograms and by spot testing. Only then was the column taken into use. A flow rate of 60ml per hour was tentatively chosen. Table I shows the type of separation attained. TABLE I SEPARATION OF 15.1 g OF A SUCROSE - RAFFINOSE MIXTURE Solvent : isopropanol, n-butanol and water in the ratio 7 : 1 : 2 by volume Flow rate : 60 ml per hour Fraction of eluate, ml (r1622 Nil Type and concentration of sugar 1622-1 84 1 Sucrose increasing 1841-1 937 Sucrose maximum 1937*-2527 Sucrose decreasing 2527-2806 Sucrose traces 280633 14 Nil 3314-3749 Raffinose increasing 3749-4022 Raffinose maximum 4022-447 6 Column washed through with water Raffinose decreasing to traces Nil and tested * ,411 fractions after this were concentrated to one-hundredth of their original volume before being tested.It can be seen that the zones did not overlap; there was a definite gap between the tail of the sucrose zone and the leading edge of the raffinose zone, which makes separation highly satisfactory. The elution of the raffinose requires a rather large volume of solvent for such a small amount of sugar, but this is compensated by the purity of the fraction and the almost complete recovery of the separated sugar.DETERMINATION OF RAFFINOSE I N RAW SUGARS BY COLUMN TECHNIQUE Results of experiments with artificial mixtures were encouraging enough for an attempt to be made to separate a raw sugar with a raffinose content of about 0.5 per cent. The impurities present in raw sugar, such as inorganic salts, salts of organic acids, other sugars and organic non-sugars, were expected to make a clean separation of the two main constituents more difficult, but the previous results seemed to have clearly shown the capacity of the column to handle large quantities of mixtures, so that a slight deterioration in the quality of separation would not seriously interfere with the quantitative recovery of the raffinose.As the relevant zones on the trial chromatogram were separated by a fairly large inter-zone, it was assumed that a column of the same size would be capable of separating even 20 g of sugar, with a proportionately increased volume of eluate. EXPERIMENT 1- A cellulose column 7.5 cm in diameter and 30 cm in height was prepared and tested, as described above. Twenty grams of a British raw beet-sugar were dissolved in 400 ml of solvent and placed on top of the cellulose column. On dissolving the raw sugar, a brown relatively insoluble residue was formed, which dissolved in water and showed only a trace of sucrose when tested chromatographically. The solution was allowed to drain into the196 GROSS AND ALBON : CHROMATOGRAPHIC SEPARATION OF [Vol.78 column and the sugars were then eluted with solvent at a flow rate of approximately 60 ml per hour. The fractions were tested for sugar by spotting them on paper and spraying with a-naphthol- phosphoric acid reagent1 ; the sugars were identified by paper chromatography. After elution of the bulk of the sucrose, 10 ml of each fraction were evaporated and taken up in 0.1 ml of water before spot-testing. The fractions containing raffinose were combined and evaporated, under vacuum with a nitrogen leak, to a few millilitres, made up to 10 ml and tested chroniatographically for purity. If found free from sucrose, the raffinose in this concentrate was estimated in three ways: by spotting a certain volume on paper, spraying with a-naphthol- phosphoric acid reagent and comparing the intensity of the coloured spots with standards ; by direct polarisation; and by polarisation according to the double inversion method with invertase and melibiase, which also enables any sucrose present to be estimated.The volume of samples used for testing the fractions was taken into account in calculating the results. The results of this experiment are shown in Table 11. The sucrose concentrations were estimated approximately as before. It represented about 8 per cent. of the volume of the raffinose fractions. TABLE I1 SEPARATION OF 2Og OF RAW BEET-SUGAR Solvent : isopropanol, rt-butanol and water in the ratio 7 : 1 : 2 by volume Flow rate : 60 ml per hour Fraction Volume, Type and concentration of sugar ml 1 560 Nil 2 800 Nil 3 160 Nil 4 170 Sucrose, faint trace 5 120 )) trace 6 960 7) maximum 7 178 9 , decreasing 8 200 99 trace 9 95 99 faint trace 1 o* 127 97 strong trace 11* 134 99 strong trace 12* 127 99 faint trace 13* 120 Raffinose, trace 14' 120 7) increasing 15* 122 9 ) increasing 16* 122 99 increasing 17* 126 77 maximum 18* 123 Y ? decreasing 19" 129 99 decreasing 20" 122 9 ) decreasing 21* 126 77 strong trace 22* 132 79 trace 23* 136 99 faint trace 24* 124 Nil 25* 126 Stachyose, trace 26* 125 79 strong trace 27" 125 99 increasing 28* 125 9 ) maximum 29* 125 99 trace 30* 125 Nil * All these fractions were concentrated to one-hundredth of their original volume before being tested.The column was then washed with water and the washings were concentrated and tested for sugars. The fractions 13 to 22, inclusive, were combined, concentrated by evaporation to a few millilitres, filtered and made up to 10 ml with water, and gave an amber-coloured solution. ESTIMATION OF SEPARATED RAFFINOSE- A chromatographic test of the concentrate showed only a trace of sucrose to be present, which for all practical purposes could be disregarded. Determination of the raffinose by spot comparison gave a figure of slightly less than 1 per cent. It was found that the colour of the solution was too dark for micro-polarimetric measure- ments and decolorisation was essential for accuracy. The solution was weighed and, after No sugar could be found.April, 1953] SUCROSE - RAFFJNOSE MIXTURES ON POWDERED CELLULOSE 1 Y? making it 20 per cent.w/w in ethyl alcohol, filtered through a micro-column containing 75 mg of B.D.H. activated charcoal. The bed was washed with 10 ml of 20 per cent. alcohol; the washings, combined with the first filtrate, were evaporated to small volume to remove alcohol and finally made up to the original weight with water. I t was expected that the presence of 20 per cent. of alcohol would prevent the adsorption of raffinose on the charcoal, but not significantly interfere with the adsorption of colouring matter. Alcohol is known to displace adsorbed sugars from charcoal. To make absolutely sure that no raffinose was lost, the bed was washed with 30 per cent. alcohol, in which, after suitable concentration, no sugar could be detected. A4fter this treatment the solution, although not completelj- decolorised, was considerably paler and so suitable for polarimetric determinations.The results of the determinations of raffinose are summarised in Table 111. TABLE I11 DETEKMINA4TION OF RAFFINOSE IN EXPERIMBKT 1 Raffinose pentahydrate Method Raffinose found, in raw sugar, ing per 10 ml % Spot test . . .. ,. .. .. 108 0.54 Direct polarisation (assuming purity of raffinose to be 100 per cent.) . . .. .. .. 100 0.60 Double polarisation (2-enzyme method) . . 99 0.50 Agreement is satisfactory, and it will be shown below how the figures agree with yet .another method of estimation, the paper chromatographic routine test for raffinose ; the separation appeared to be complete and conditions for quantitative recovery good.The interesting point about this experiment was the perfect separation of the tetrasaccharide, stachyose, present in this sample of a raw beet-sugar. The amount present was not deter- mined, as this was outside the scope of our investigation. Any other sugars possibly present, such as monosaccharides, were not determined for the same reason. The comparative ease with which the tetrasaccharide has been cleanly separated illustrates the selectivity of this technique. EXPERIMENT 2- For this experiment a different raw beet-sugar with approximately the same raffinose content was selected. Twenty grams of sugar were used and the procedure followed closely that used in experiment 1. The sucrose was found to require a slightly smaller volume of solvent for elution than before, and the raffinose began to appear in the eluate earlier than anticipated. The cause of this was traced to the slightly higher water content of the solvent recovered from the previous experiment.We found that the separation was thereby some- what inferior, and a loss of 3 to 4 mg of raffinose in the recovery balance occurred according to our estimate. Another com- plication was caused by the colour of the raffinose concentrate. When the fractions containing raffinose, totalling about 1 litre, were evaporated to dryness, made up to 15 ml with 20 per cent. w/w alcohol, and filtered through 0-15g of active carbon, the solution was still too dark. 'The pH of the solution was then adjusted to a value of 7.0 and the decolorisation repeated with a similar amount of fresh carbon.There was no significant improvement in colour and it seemed as if the colour of this raw sugar was considerably less readily adsorbable than that of the previous sample. The solution was then evaporated, taken up in 10 ml of 15 per cent. alcohol and run over a column of 5 g of carbon. The result was satisfactory. No colour was eluted from the carbon on washing the column with 30 per cent. alcohol, but some sugar was detected in the washings, which were then combined with the decolorised solution. The column of carbon was then washed with 50 per cent. alcohol. No sugar could be detected in the washings. The solution, being slightly acid, was neutralised, evaporated to remove the alcohol, and made up to 10ml with water.This solution was pale amber, but could be used for polarimetric measurements. The solution was tested chromatographically for purity and it was found that apart from a trace of sucrose it contained some hydrolysis products of raffinose. The slight hydrolysis of raffinose may have occurred during the attempts to decolorise the solution. The extent of hydrolysis could not be ascer- tained quantitatively, but although it might affect the results of the direct polarisation, it would have no effect on the figures by the double-polarisation method. This point was carefully watched in later experiments.198 GROSS AND ALBON : CHROMATOGRAPHIC SEPARATION OF [Vol. 78 The amount of sucrose present was insignificant and was outside the sensitivity range of the double-polarisation method.Table IV shows the results obtained by the different methods. Allowance has been made for the use in preliminary tests of 7 per cent. of the total volume. TABLE IV DETERMINATIOS OF RAFFINOSE IK EXPERIMEST 2 Method Raffinose pentahydrate Kaffinose found, in raw sugar, mg per 10 ml % Spot test . . . . .. . . .. 86 Direct polarisation .. .. .. .. 83 Double polarisation (2-enzyme method) .. 99 0.43 0.42 0.50 EXPERIMENT 3- For this experiment we chose a raw beet-sugar of a lighter colour and lower raffinose content than before. Twenty grams of the sugar were dissolved in 300 ml of solvent, leaving only a small amount of insoluble brown residue, which on test showed no trace of sugar. The procedure was the same as in the previous experiments.The water content of the recovered solvent was checked; separation was satisfactory. Only a faint trace of sucrose could be detected in the first fraction containing raffinose (in negligible amount). For all practical purposes there was no overlapping between the sucrose and raffinose fractions. The elution of the raffinose zone required a slightly larger volume of solvent than previously, namely, 1.3 litres; no tetrasaccharide was detected. The fractions containing raffinose were evaporated to dryness and made up to 10 ml. This solution was sufficiently pale to be used €or polarisation without decolorisation. The chromatographic purity test of the separated raffinose showed it to be free from sucrose. Allowance has been made for the use in preliminary tests of 11 per cent.of the total volume. Table V shows the results obtained. TABLE V DETERMINATION OF RAFFINOSE IN EXPERIMEKT 3 Raffinose pentahydrate Method Raffinose found, in raw sugar, mg per 10 nil % Spot test .. .. .. .. . . 68 0.34 Direct polarisation .. .. .. .. 64 0.32 Double polarisation (2-enzyme method) . . 81 0.41 COMPARISON OF RESULTS OF COLUMN SEPARATIONS WITH ESTIMATIONS BY PAPER CIIROMATO- One of the objects of this investigation was to check the limits of accuracy of the quantitative routine method previously rec0mmended.l All samples were analysed for raffinose by the paper chromatographic method in the routine manner. The results bv the diflerent methods are shown in Table VI. GRAPHIC ROUTINE METHOD- TABLE VI COMPARISOX OF RESULTS EY DIFFEIZEXT METHODS Experiment No.1, Experiment No. 2, Method raffinose content, raffinose content, % % Co!utnn techniquc, spot test . . . . 0.54 0.43 77 direct polarisation . . 0-50 0.42 7 ) double polarisation . . 0.50 0.50 Mean of column determinations . . 0.51 0.45 Faper chromatography . . . . . . 0.5 1 0.5 1 Experiment Xo. 3 , raffinose content, 0.34 0.32 0.41 0.35 0.35 O / / O The paper chromatographic figures are the average values of determinations by three The agreement is good, even when the mean of the three column determinations is not On comparing the determination by any of the methods with the paper chromato- This shows that independent analysts . taken. graphic method, the differences are found to be within reasonable limits,April, 19531 SUCROSE - RAFFINOSE MIXTURES ON POWDERED CELLULOSE 199 the paper chromatographic routine method is sufficiently accurate to be considered quantita- tive and reliable.DISCUSSION On consideration of the quantities of sugar separated, it would appear that the cellulose column has a surprisingly high capacity and resolving power. I t is also worth mentioning that the eluate concentration curves are fairly symmetrical and that the emergence of each sugar is reproducible under standardised conditions. The latter feature is particularly prominent with lower concentrations and pure substances. The mechanism responsible for the high efficiency of the column is not readily obvious. The symmetry of the elution curves points to a fairly linear isotherm, irrespective of the mechanism of the separation process.The use of a solvent system that is homogeneous and so greatly undersaturated with water at room temperature that it represents a water-miscible solvent for all practical purposes would render highly improbable the functioning of the column as a partition chromatogram alone. On the other hand, the emergence of the sugars after roughly the same volumes of eluate have been collected, irrespective of whether the eluting agent is a single solute or is in admixture with others, is not entirely reconcilable with an adsorption chromatogram in which the behaviour of a given solute is often appreciably affected by other co-solutes. The low capacity usually encountered in partition chromatograms further weighs against assuming a single mechanism underlying this fractionation. For the theoretical interpretation of the separation on cellulose columns, more experi- mental data would be required to elucidate the role of adsorption and to decide whether an adsorption distribution or a liquid - liquid distribution occurs.It is safe to assume that the cellulose column possesses properties characteristic of both mechanisms. This loses its significance if all chromatography is considered as a partition process between phases, irrespec- tive of whether or not two mutually immiscible solvents are added to the column. If partition occurs between the mobile liquid phase and the solid phase of the adsorbent, instead of the stationary liquid phase, the behaviour of the solute will be governed by the adsorption isotherm instead of the partition isotherm.The substitution of the solid phase, with a more favourable isotherm, may result in a considerable increase of efficiency. Whatever the mechanism of the process may be, the fact that a clean-cut separation on a large scale has been attained is undisputable, as judged from the shape of the elution curve. With an artificial mixture of the sugars, the zones are separated by a considerable volume of pure solvent and are thus fairly certain to be homogeneous and to represent discrete substances. There is only slight diffuseness in the leading edge of the sucrose zone and not too much “tailing.” The elution curves of raw sugars are naturally affected to some extent by the impurities present, which may largely account for the deterioration of the elution curves.The resolving power of the column is still high enough to produce a satisfactory separation of the constituents, although the “tailing” of the sucrose zone tends to close the gap between the two zones. By loading the column with 20 g instead of 15 g of sugar a slight deterioration in performance was only to be expected. As long as no significant overlapping of zones took place, separation of a larger amount seemed desirable. In the experiments described there was almost no contamination of the raffinose fractions by the preceding sucrose zone; only in one instance was sucrose found in the final raffinose concentrate, and then it was less than 1 mg from nearly 20 g of sucrose. There are two obvious possibilities for increasing the capacity of the column.The first is to pre-treat the sugar sainple to eliminate most impurities, particularly the salts (ash); the second is to use a larger column. The first possibility was not tried, as it was desirable for analytical purposes to avoid anything that might lead to an inadvertent loss of raffinose from the sample. As to the second possibility, it must be realised that a liquid chromatogram requires a large volume of solvent and entails the collection and analysis of a number of fractions during several days. The proportions of the column and the volume of solvent used here were considered convenient, as a run could be completed within 1 week. The use of a column of larger diameter was intended, but met with technical difficulties in the fitting of suitable Pyrex-glass cylinders with sintered-glass discs.These difficulties have, however, been overcome, and columns 12.5 cm in diameter have been made available and are now being tested. Separations of up to 60 g of sugar mixture seemed to be feasible and results have been reported in another paper.g The effect of increased water content became apparent in those experiments in which recovered solvent was used. This risk can be avoided entirely200 GROSS ‘4ND ALBON [Vol. ‘78 by using fresh solvent for each separation or by carefully checking the water content of the recovered solvent. By comparing the results in Table VI, the agreement found between the various methods is seen to be remarkably close. As it was possible to check chromatographically the purity of the separated raffinose with a high degree of sensitivity and accuracy, the final solution used for the polarimetric tests could be considered to contain raffinose alone, or, as in one instance, a negligible amount of sucrose.Spot-testing gave a rough but fairly reliable indication of the raffinose concentration ; the final confirmation was, however, provided by the polarimetric tests. These tests not only established accurately the raffinose concentration, but proved beyond doubt that the substance recovered from the raw beet-sugars was raffinose. We were also in the favourable position of being able to follow the action of the enzymes on the raffinose by the paper chromatographic technique and so check every stage of the procedure. It was found that the formula conventionally used for the calculation of sucrose by the 2-enzyme methodlo did not hold exactly for solutions of low concentrations, such as we used, but tests with solutions of purest raffinose (Kerfoot’s) at similar concentrations showed that the small discrepancies did not affect the calculation of raffinose. We did not, therefore, attempt to apply a correction factor, as we could always determine chromato- graphically the purity of the raffinose. We felt that we had achieved our object by furnishing a method by which the results of the routine procedure could be checked independently. Our thanks are clue to Mr. H. C. S. de Whalley, Director of Research, for his advice and encouragement, and to Messrs. R. Runeckles and J. Rundell for valuable experimental assistance. We also wish to thank the Directors of Tate & Lyle Ltd. for permission to publish this paper. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Albon, N., and Gross, D., Analyst, 1950, 75, 454. de Whalley, H. C. S., Albon, N., and Gross, D., Ibid., 1951, 76, 287. Kacser, H., and Ubbelohde, A. R., J . SOC. Chem. I n d . , 1949, 68, 135. Tiselius, A., and Hahn, L., Kolloidzschr., 1943, 105, 177. Claesson, S., Ark. Kemi Min. Geol., 1947, No. 16, 9. Lew, Mi., Wolfrom, M. L., and Goepp, R. M., J . Amer. Chern. SOL, 1946, 68, 1449. Whistler, R. L., and Durso, D. F., Ibid., 1950, 72, 677. Hough, L., Jones, J. K. N., and Wadman, W. H., Nature, 1948, 162, 448; J. Chem. SOC., 1949, Albon, N., Bell, D. J., Blanchard, P. H., Gross, D., and Rundell, J . T., J. Chew?. SOC., 1253, 24. Browne, C. A., and Zerban, F. W., “Physical and Chemical Methods of Sugar Analysis,” Third 2511. Edition, John Wiley & Sons, Inc., New York, 1948, p. 464. TATE & LYLE LIMITED RESEARCH LABORATORY KESTON, KENT RAVENSBOURNE, WESTERHAM ROAD September 19th, 1962 ERRATUM: September (1852) issue, p. 463. applicable only when x = 50, as described in the procedure. should be: 500(y - titration)/x: The equation on line 4: x(y - titration)/5 is The general expression

ISSN:0003-2654    

DOI:10.1039/AN9537800191

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

April, 19531 GERRITSMA AND KOEKS 201 Determination of Theobromine in Cocoa Residues BY K. W. GERRITSMA AND MISS J. KOERS A simple, accurate method for the determination of theobromine in cocoa residues is described. The material is shaken for 5 minutes with chloroform in an ammoniacal medium ; after dehydration with anhydrous sodium sulphate, the chloroform solution is filtered and the residue and filter are washed with chloroform. The chloroform is removed from the filtrate by distillation and the residue is dissolved in water, after which silver nitrate is added and the liberated nitric acid is titrated with alkali. The results obtained by this method are compared with the values found by Wadsworth’s method, and are in agreement with them. Normally an analyst can carry out 15 to 20 determinations per day.WADSWORTH’S methodl is generally used for the determination of theobromine in cocoa residues. This method is based on repeated extraction of the material with tetrachloro- ethane after it has been triturated with magnesium oxide and water and has been brought to a certain degree of moisture by heating on a water-bath. After the extract has been evaporated to a small volume, theobromine is precipitated with ether, whilst caffeine, fat and the remaining compounds dissolve completely or almost completely in the ether. The theobromine is collected by filtration through a tared filter and is dried and weighed. A correction is applied for the solubility of theobromine in ether. There are, however, a number of objections to this method. The result of the determina- tion is influenced by the amount of water remaining in the mixture after it has been heated on the water-bath with magnesium oxide and water, and this depends on the nature of the material, the shape of the dish in which i t is heated and on the steam capacity of the water- bath.The highest result is obtained when the water content is brought to about l o g per log of cocoa residues. If less water is present after heating, then a smaller amount of theobromine is extracted; if, however, the amount of water is greater, then the theobromine separated is less pure. Even if the work is carried out in a fume-cupboard, there is a risk, owing to the high vapour pressure of tetra- chloro-ethane, of chronic poisoning being caused by the vapour penetrating the skin.Hunter2 regards tetrachloro-ethane as the most toxic of the chlorinated hydrocarbons, and Forbes3 describes some fatal cases of tetrachloro-ethane poisoning. In his article reference is made to a concentration of 1 mg per litre of air inhaled for 30 minutes or 2.3 mg per litre of air inhaled for 10 minutes being sufficient to cause headache, dizziness and signs of fatigue. Continuous exposure to tetrachloro-ethane vapours causes injury t o the body, especially to the liver. The extraction with tetrachloro- ethane is carried out by boiling the material four times with tetrachloro-ethane; after each extraction it is filtered while boiling. Because of this, the extraction is a slow process and the filtration is unpleasant, owing to the high temperature at which it takes place.When the extraction liquid is evaporated to a small volume (3 to 5ml), it is difficult to prevent charring of the crystallising theobromine. Evaporation on an asbestos ring in such a way that the flame does not touch the glass above the liquid gives the best results. Finally, filtration of theobromine precipitated with ether requires some practice, as it is difficult to bring the precipitate quantitatively on to the filter with ether. The amount of ether used must, moreover, be as small as possible, as the solubility of theobromine is 6 mg per 100 ml of ether. Various methods are described in the literature for obviating some of the objections to the Wadsworth method. McDonald4 substitutes an extraction in a Soxhlet apparatus for the boiling with tetrachloro-ethane. Although the repeated extractions and filtrations are avoided by this, there remains the objection that the purity of the theobromine depends to a great extent on the amount of water that the material contains before the extraction.The extraction method of McDonald was studied by Kay and H a y w ~ o d , ~ who paid special attention to the purity of the separated theobromine. They came to the conclusion A second objection is the poisonous nature of tetrachloro-ethane. In addition, the method involves certain difficulties.202 GERRITSMA AND KOERS: DETERMINATION OF [Vol. 78 that it was impure on separation. The purity can be established by a nitrogen determination, but, as this lengthens the method, filtration after partial distillation of the tetrachloro-ethane is recommended.This would reduce the amount of impurity to 2 per cent. of the theo- bromine content. This filtration, however, also increases the time taken for the determination, without ensuring complete purity of the theobromine. Lowes describes a special extraction apparatus by which it is possible to extract the material with moist hot tetrachloro-ethane, so maintaining the correct water content of the material. Humphries' extracts the material, after mixing it with magnesium oxide and water, for 20 hours in a Soxhlet apparatus with chloroform. The extracted fat is removed with light petroleum. The purine bases can then be weighed, and when the caffeine has been removed with benzene, the theobromine content can be determined.This method disposes of many objections, but it is too slow and laborious for routine use. Moreover, no precautions are taken to ensure the purity of the separated theobromine. As well as these modifications of Wadsworth's methods, a number of other methods are to be found in the literature.8y9~10~11~12~13 These have been critically discussed by H01mes.l~ His conclusion is that all are either inaccurate or take too much time. He therefore introduces a new method in which the cocoa, after trituration with magnesium oxide, is boiled three times with water. After clarification with basic lead acetate and filtration, the aqueous solution is evaporated to 100ml and the theobromine is extracted from this solution with chloroform. The chloroform is distilled off, and the theobromine is titrated according to Boie.15 However, this method, in our opinion, is also laborious and slow. EXPERIMENTAL In order to overcome these objections to Wadsworth's and other methods, another extraction method has been sought.It proved possible to extract theobromine quantitatively from cocoa residues by shaking with chloroform in an ammoniacal medium, a method that is used in the determination of other purine derivatives in vegetable materials, The low boiling point and low toxicity of chloroform are important advantages. The solubility of theobromine in chloroform is less than in tetrachloro-ethane. But if the material, made alkaline with ammonia, is shaken with sufficient chloroform, the theobromine is quantitatively extracted from the cocoa residues and brought into solution.SchoorP states that 1 g of theobromine dissolves in 5000 g of chloroform at 15" C. From this it follows that 400 g of chloroform is theoretically sufficient to extract the theobromine quantitatively from 2 g of cocoa residues, which contain a maximum of 4 per cent. of theo- bromine. After the extraction, the water from the dilute ammonium hydroxide added is removed with anhydrous sodium sulphate in order to bring the theobromine quantitatively into the chloroform. After removal of the solvent, the theobromine is titrated specifically according to Boie.ls This titration is based on the following reaction- The liberated nitric acid is titrated with 0.1 N alkali. theobromine + AgNO, -+ theobromine - Ag + HNO, METHOD REAGENTS- Chloroform, B.P.Ammonium hydroxide-A 10 per cent. w/w solution of ammonia in water. Sodium sulphate-The dried anhydrous salt. Silver nitrate solution, 0.1 N. Sodium hydroxide solution, 0.1 N. Phenol red indicator-A 0.1 per cent. solution in 96 per cent. ethanol. PROCEDURE- Accurately weigh 2 g of the ground cocoa residues and place the sample in a 500-ml bottle. Add successively 400 g of chloroform (about 270 ml) and 10ml of 10 per cent. ammonium hydroxide solution from measuring cylinders. Close the bottle with an ordinary cork and shake vigorously for 5 minutes. Next add 12g of anhydrous sodium sulphate, shake the bottle well and set it aside overnight. Then pass the contents quantitativelyApril, 19531 THEOBROMINE IN COCOA RESIDUES 203 through a filter into a 500-ml Erlenmeyer flask.Thoroughly wash the bottle and the filter and its contents with about 100 ml of chloroform. Distil the chloroform from a water-bath, and then heat the flask for a few minutes in an oven at 100” C in order to remove the last traces of chloroform. Boil the residue in the flask with 50 ml of water; cool it, and then add 0.5 ml of phenol red indicator. Neutralise with 0.1 N sodium hydroxide (usually 1 or 2 drops), add 20 ml on’ 0.1 N silver nitrate from a measuring cylinder, and titrate with 0.1 N sodium hydroxide solution from a micro-burette to the red colour of the indicator (pH 7.4). 1 ml of 0.1 N sodium hydroxide = 18.0 mg of theobromine. CALCULATION- t x 0.1 x 180 P Percentage of theobromine = where t = ml of 0.1 N sodium hydroxide and p = amount of cocoa residue in mg.RESULTS The method was tested on a sample of cocoa residues to ascertain the correct amounts of chloroform, ammonia and anhydrous sodium sulphate, and to investigate the time necessary for shaking and drying. The results are summarised in Table I. TABLE I INVESTIGATION OF THE PROPOSED METHOD BY APPLYING IT TO A SAMPLE OF COCOA RESIDUES THE THEOBROMINE CONTENT OF WHICH WAS 3.00 PER CENT. (CORRECTED 2.90 PER CENT.) ACCORDING TO WADSWORTH’S METHOD A 2-g portion of the sample was taken for each experiment Time of Amount Volume of Amount of drying with of ammonium Time of anhydrous anhydrous Theobroniine chloroform, hydroxide, shaking, Na,SO,, Na,SO,, found, ml minutes g hours % 300 10 5 12 400 10 5 12 500 10 5 12 600 10 5 12 18 2-85 18 3.00 18 3.00 18 3-01 400 400 400 400 400 1 5 5 18 2.71 2 5 5 18 2.81 5 6 6 18 2.83 10 5 12 18 3.01 15 5 18* 18 3.01 1 2.86 2.86 2.93 3.01 _- 400 10 5 12 12 400 10 5 12 400 10 5 12 400 10 6 12 18 i 400 400 400 400 400 400 10 3 12 18 2.99 10 4 12 18 3.00 10 5 12 18 3.01 10 15 12 18 3-01 10 120 12 18 3.01 10 300 12 18 3-0 1 400 10 5 8 18 2.99 400 10 5 10 18 3.00 400 10 5 12 18 3-00 400 10 5 14* 18 3.02 * In these experiments the residue, which was considerably greater than usual, was washed with 150 ml instead of 100 ml of chloroform. At the same time the results were compared with those found by Wadsworth’s method.For this purpose Wadsworth’s The results of this investigation are shown in Table 11.204 GERRITSMA AND KOERS DETERMINATION OF [Vol.78 method was carried out according to the original procedure whereby, after the sample was heated with magnesium oxide and water, the water content of the mass was brought to 10 g, the initial water content of the sample being taken into account.* If this was not done, the purity of the theobromine varied from 93 to 98 per cent. If the water content of the mass was brought to 50 per cent., the purity was 96 to 97 per cent. The purity of the theobromine was established by Kjeldahl determination of the nitrogen content and by titration according to Boie. The results by Wadsworth’s method, corrected for purity of the theobromine, are shown in Table 11. TABLE I1 THEOBROMINE IN COCOA RESIDUES DETERMINED BY THE PROPOSED METHOD AND BY WADSWORTH’S METHOD WITH AND WITHOUT CORRECTION Cocoa residue sample No.1 2 3 4 5 6 7 8 9 10 11 12 13 14 16 16 17 18 19 20 r by proposed method, 1-50 1-82 2-21 2.2 1 2.29 2-65 2.66 2-68 2-71 2-72 2.75 2-78 2.83 2-84 2.87 2.88 2-89 3-04 3-07 3.12 % Theobromine content A 7 - by mTadsworth’s by Wadsworth’s method, method, corrected, % % 1.59 1-50 1.82 1.76 2.23 2-14 2.25 2-17 2.29 2.18 2.68 2.59 2.63 2-54 2-77 2-68 2.77 2.63 2.7 1 2.61 2-72 2.60 2-82 2.72 2-86 2.77 2-88 2.74 2.82 2.69 2-80 2-69 2.86 2.75 3.08 2.98 3.16 3.06 3-16 3-06 It can be seen from the data in Table I that, if the theobromine content is 3 per cent., then 400 g of chloroform are necessary for the extraction. For low theobromine contents the amount of chloroform can be decreased in accordance with the solubility of theobromine in chloroform.Vigorous shaking by hand for 5 minutes is sufficient to extract all the theobromine. From this it is evident that, despite the low solubility of theobromine in chloroform a t room temperature, a rapid and complete extraction is possible. Hence any preference for tetrachloro-ethane as a better solvent for theobromine than chloroform is not justified. The amount of anhydrous sodium sulphate needed to dry the chloroform has been computed liberally, in order to be certain that all the waker is removed. Overnight drying is necessary. If the theobromine content in an aliquot part of the filtrate were determined, washing with chloroform could be eliminated. This, however, leads to inaccurate results owing to evaporation of the chloroform during filtration and alteration in weight due to loss of ammonia.The titration of theobromine is very accurate, provided direct sunlight is avoided. The reproducibility of the proposed method is good. In twenty replicate determinations t!rc results all lay between 3-00 and 3.02 per cent. The data in Table I1 show that the results by the proposed method are nearly in agree- ment with those by Wadsworth’s method. However, as the theobromine separated by il.-adsworth’s method is not absolutely pure, the content found by the proposed method is slightly greater. Additions of 0.5, 1 and 2 per cent. of theobromine to a sample of cocoa residues with a content of 1-50 per cent. of theobromine were recovered quantitatively. Ofie analyst can * We found that the extraction with tetrachloro-ethane must be finished in one day. If the mass.of cocoa residues were allowed to stand overnight with tetrachloro-ethane, results were irregular.April, 19531 THEOBROMINE I N COCOA RESIDUES ”05 carry out 15 to 20 determinations per day. Less chemicals are used than in the Wadsworth method, and the used chloroform is more easily recovered than is tetrachloro-ethane. The authors offer their sincere thanks to Professor Dr. 0. F. Uffelie, Pharmaceutical Laboratory, University of Utrecht, for the valuable advice he has given them. They haw gratefully made use of information about Wadsworth’s method provided by Mr. E. van Koolbergen, van Houten Ltd., Weesp. REFERENCES 1. 2. 3. 4. 5. 6. Lowe, E. H., Ibid., 1948, 73, 679. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Wadsworth, R. V., Apzalyst, 1921, 46, 32. Hunter, D., “Industrial Toxicology,” Oxford University Press, 1944, pp. 63-65. Forbes, G., B y i t . Med. J., 1943, i, 348. Macdonald, J . A., Ann. Rep. Cocoa Res., 1936, 6, 43. Kay, J., and Haywood, P. J. C., Analyst, 1946, 71, 162. Humphries, E. C., Ann. Rep. Cocoa Bes., 1938, 8, 36. Jalade, M., Ann. Falsif., 1929, 22, 396. Martin, F., and Clergue, H., Ann. Chim. Anal., 1942, 24, 202. Moir, D. D., and Hinks, E., Analyst, 1935, 60, 439. Moores, R. G., and Campbell, H. A., Anal. Chem., 1948, 20, 40. Parkes, A. E., and Parkes, H. A., Analyst, 1937, 62, 791. Pritzker, J., and Jungkunz, R., Mitt. Geb. Lebensm. Unters. Hyg., 1943, 34, 185. Holmes, K. l;., Analyst, 1950, 75, 457. Boie, H., Pharm. Ztg., 1930, 75, 968. Schoorl, N., “Theobrominum.” In “Conzrtzentaav op de NederZandsche Phanizacopee,” 5e Uitgave, deel IV, Utrecht, 1931, pp. 431-435. UTRECHT, THE NETHERLANDS CENTRAL INSTITUTE FOR NUTRITION RESEARCH T.N.O. July 21st, 1953

ISSN:0003-2654    

DOI:10.1039/AN9537800201

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

April, 19531 THEOBROMINE I N COCOA RESIDUES "05 The Determination of Chloromethylphenoxyacetic Acids in MCPA Formulations BY F. FREEMAN AND K. GARDNER A method is described for the chromatographic separation and determina- tion of 4 : 6-dichloro, 6-chloro, 4-chloro and unchlorinated 2-methylphenoxy- acetic acids in commercial chloromethylphenoxyacetic acid formulations. The four acids are successively eluted with a mixture of ether and chloroform from a kieselguhr column treated with phosphate buffer, and are determined by titration with dilute sodium hydroxide solution. The effect of impurities is discussed and results are shown for synthetic samples prepared from the pure acids and various commercial preparations. IJXTIL recently chloromethylphenoxyacetic acid selective weed-killer formulations containing salts or esters of 4-chloro-2-methylphenoxyacetic acid have been assayed for total acids b37 extraction with ether or chloroform and subsequent potentiometric titration of alcoholic solutions of the acids with sodium hydroxide.This method naturally does not distinguish between the 4-chloro-2-methylphenoxyacetic acid, which has a strong physiological action on plants, and the related 2-met hylphenox yace tic, 6-chloro-2-me thylphenoxyacetic and 4 :6-dichloro-2-methylphenoxyacetic acids, which are comparatively inert. Methods for the determination of the active 4-chloro acid have recently been published. Smensenl has proposed an isotope dilution method. Ultra-violet spectrophotometric methods have been suggested by Grabe2 and Hill,3 and Sjoberg4 has proposed an infra-red spectro- photometric method.The ultra-violet method determines only the 4-chloro acid and is inaccurate in the presence of appreciable amounts of the unchlorinated acid. The infra-red method stiff ers from requiring rather large mathematical corrections, owing to overlapping of the absorption spectra. In addition, the chlorocresols, which are usually present in commercial formulations, must be removed before photometric determinations can be made. By some preliminary partition experiments in separating funnels, we showed that it should be possible to separate the 4-chloro and 6-chloro acids by partition chromatography,206 FREEMAN AND GARDNER: THE DETERMINATION OF [Vol. 78 with isopropyl ether as moving phase.During these experiments a paper was presented by S t r ~ u d , ~ in which the chromatographic determination of 2 :4-dichlorophenoxyacetic acid was described. In consequence of his results, an attempt was made to adapt Stroud’s method to the determination of chloromethylphenoxyacetic acids. EXPERIMENTAL Preliminary experiments were carried out with columns similar to those used by Stroud, in which kieselguhr - phosphate buffer solutions at a pH of 5-7 to 7.0 were used for the stationary phase and ethyl ether for the moving phase. It was found that satisfactory buffer solutions could be prepared from analytical reagent grade sodium dihydrogen phosphate (NaH,P0,.2H20) and di-sodium hydrogen phosphate (Na,HPO,) more readily than they could by the somewhat protracted method previously ~uggested.~ A 0.25 M phosphate buffer solution was eventually used on the columns. The columns were packed by Martin’s A rate of flow of 1 to 1-5 ml per minute was found to be satisfactory.At a pH value of more than 6, all the acids, with the exception of the 4:6-dichloro and the 4-chloro, could be separated. By increasing the pH of the stationary phase, the bands were progressively separated, but even at a pH of 7-0 the 4:6-dichloro and the 4-chloro acids could only be partly separated. In order to devise a method for separating the 4:6-dichloro acid from the 4-chloro acid, numerous partition experiments were made in separating funnels with different mixtures of immiscible solvents and buffer solutions. From these it was predicted that chloroform would probably give a good separation of 4:6-dichloro from 4-chlor0, but a poor separation of 4-chloro from 6-chloro, and that ether would give the reverse.Columns were made up with moving phases of chloroform, ether and mixtures of chloroform and ether. I t was found that, with a (1 + 1) mixture of ether and chloroform and by adjusting the pH of the stationary phase to about 6-7, a separation was achieved by which it was possible to collect the four acids in four separate fractions. As only four fractions are taken, it is necessary to deduct one blank only for each determination; several blanks are needed when 2.5-ml cuts are taken.5 A determination by this method takes longer than one by Stroud’s method, but this dis- advantage is compensated for by the scant attention to the column that is required.The broadening of the acid bands does not affect the accuracy of the determination. The charac- teristics of the columns can be accurately reproduced after initial standardisation, and the columns can be left to run unattended in complete safety. I t has been found that the same column can be used for at least ten successive determinations of commercial MCPA. When titrating the acids in the four fractions, it is advisable to exclude carbon dioxide by passing carbon dioxide-free air or nitrogen through the titration vessel during titration. Before titration, each solvent fraction was evaporated to dryness on a warm water-bath, and the residue was dissolved in neutral alcohol through which carbon dioxide-free nitrogen was passed to displace carbon dioxide.Titrations were carried out with 0.01 or 0.004 N carbon dioxide-free sodium hydroxide solution (prepared by the method of Davies and h:ancollas7), with bromothymol blue as indicator. Evaporation of the solvent to dryness on TABLE I RECOVERY OF ACIDS FROM THE COLUMN Results are expressed in millilitres of 0.01 iV sodium hydroxide solution Theoretical Titre Acid titre, found, ml ml Theoretical Titre Acid titre, found.. ml ml 4-Chloro-2-methyl- ( a ) 2.85 8-88 4 : 6-Dichloro-2- (a) 0*96 plienoxyacetic ( b ) 3.36 3-30 methylphenoxyacetic (b) 0.70 (d) 2.68 2.72 (d) 0-65 2.98 8.98 6-Chloro-2-methq-l- (a) 0-30 2-98 2.98 plienoxyacetic ( b ) 2.06 2.86 2.84 ( d ) 2.06 4-48 4.42 2-hIethylphenoxyacetic (a) 2.50 ( d ) 1-20 (G) 2-68 2-70 (c) 2.22 0.94 0.68 2.20 0.64 0.30 2-02 2.04 2.45 1.18 XOTE-(U), (b), (c) and (d) represent the results from analyses of four separate synthetic mixtures, the remaining figures indicating recovery of pure 4-chloro acid alone from the column.April, 19531 CHLOROMETHYLPHENOXYACETIC ACIDS IN MCPA FORMULATIONS 207 a bath of boiling water resulted in the sublimation of the acids, with consequent low results.Some of the results obtained with pure chloromethylphenoxyacetic acids by the proposed method are shown in Table I. Analyses of typical commercial MCPA formulations are shown in Table 11. TABLE I1 ANALYSES OF TYPICAL COMMERCIAL FORMULATIONS Individual cresoxyacetic acid content as a percentage of total cresoxyacetic acid content r -l 4-Chloro 4 :6-Dichloro 6-Chloro Unsubstituted A 64 21 14 < 1 88 6 4 2 59.5 11 22 7.5 84 8 8 71 16 13 < 1 78 15 4 3 - METHOD REAGENTS- All reagents used should be of analytical reagent quality unless otherwise stated.Kieselguhr-Hyflo Super-Cel.* Stationary Phase solution-A mixture of 112 volumes of 0.25 M di-sodium hydrogen Ether - chloroform solution-A mixture of equal volumes of ether and chloroform DilGted hydrochloric acid (1 + 1). Sodium hydroxide solution, 0.01 N or 0.004 N-Free from carbonate. 4-Chloro-2-methyZ~henoxyacetic acid-Not less than 90 per cent. pure. 4 : 6-DichLoro-2-methyl~henoxyacetic acid-Not less than 90 per cent. pure. 6-Chloro-2-methyl~henoxyacetic acid-Not less than 90 per cent. pure. 2-Methplphenoxyacetic acid-Not less than 90 per cent. pure. (If the pure acids are not available, a commercial MCPA can be used for standardisation.) Bromothymol blue solution, 0.01 per cent.phosphate and 88 volumes of 0.25 M dihydrogen sodium phosphate. equilibrated by shaking 1 litre of the mixture with 50 ml of stationary phase solution. APPARATUS- The chromatograph tube consists of 55 cm of glass tube 1.2 cm in diameter constricted a t the lower end, and with a B19 socket a t the upper end. The eluting solvent is contained in a tap funnel provided with a ground-glass joint. A 5 or 10-ml micro-burette calibrated in 0.01 or 0-02-ml divisions is used for the titration. PREPARATION AND STANDARDISATION OF THE COLUMN- Then triturate the mixture gently with the equilibrated mixture of ether and chloroform, and pack into the glass tube by means of a perforated metal disc attached to a length of stiff wire. A thin wad of ether-extracted cotton wool will retain the packing at the bottom of the column.Prepare a solution, containing approximately 1, 5, 2 and 2 g per litre of 4:6-dichloro, 4-chloro, 6-chloro and unchlorinated 2-methylphenoxyacetic acids, respectively, in the ether - chloroform mixture. Place 1 ml of this solution on the column and force it down with nitrogen from a cylinder. Then pass through the column two l-ml portions of the ether - chloroform solution and 600 ml of ether - chloroform solvent at the appropriate gas pressure to give a flow-rate of 1.0 to 1-5 ml per minute. Collect successive 10-ml fractions, evaporate them just to dryness on a bath of warn water, dissolve each in 1 to 2 ml of neutral alcohol and dilute to 6 to 8ml with water.Titrate with 0.01 N sodium hydroxide solution under nitrogen. Standardise the column by plotting the fraction numbers against the individual titres of each fraction (see Fig. 1). Provided that the same rate of flow is maintained, the Triturate 25g of Hyflo Super-Cel with 12.5ml of stationary phase solution. * Supplied by Messrs. Johns-Manville Co., Ltd., Artillery House, London, S.W.l.208 FREEMAN AND GARDNER: THE DETERMINATION OF [Vol. 78 standardisation is reproducible, and the column can be left to run unattended. it is advisable to standardise the column periodically. PROCEDURE- Acidify a measured amount (20 to 50 ml) of commercial MCPA with diluted liydrochloric acid (1 + 1). Wash the combined ether layers with three 10-ml portions of water.Wash the combined washings with 20 ml of ether and add this to the main ether solution. Dilute the combined ether layers to 250 ml in a calibrated flask and dilute a 20-ml aliquot of this solution to 50ml. Carefully transfer, by means of a pipette, 1 ml of this final solution (which should contain about 10 mg of total acids) to the column. Wash with two 1-ml portions of ether - chloroform (as under standardisation) and elute with the solvent at a flow-rate of 1 to 1.5ml per minute. Collect four fractions of acids, taking cuts at the effluent volumes indicated from the standardisation. Evaporate each fraction on a water-bath (at about 70" C) to about 5 ml. Nevertheless, Extract with two 50-ml portions of ether. - E 0.51 W E $ *0*4- x r .- 5 0.3- Z - 8 A 0.2- f D Effluent, ml Fig.1. Standardisation of column. Peak A, 4 :8dichloro- I-methylphenoxyacetic acid; peak H , 4-chloro-2-methyl- phenoxyacetic acid ; peak C, 6-chloro-2-methylphenoxyacetic acid ; peak D, 2-methylphenoxyacetic acid Remove the residual solvent by means of a hand bulb or a stream of air. of neutral alcohol, swirl to dissolve and dilute to 15 to 20ml with water. Add 3 to 4ml Pass a stream of carbon dioxide-free nitrogen through the solution for not less than 4 minutes. Titrate the solution with 0.01 N or 0.004 N carbon dioxide-free sodium hydroxide solution, using bromothyniol blue as indicator, until the first green colouration appears. A blank value should be determined on 50ml of solvent after it has been passed through the column.This should be less than 0.01 ml of 0.01. N sodium hydroxide solution. 1 ml of 0.01 iV alkali = 2.005 mg of 4-chloro-2-methylphenoxyacetic acid or 6-chloro-2- = 2.35 mg of 4 :6-dichloro-2-methylphenoxyacetic acid. = 1-66 mg of 2-methylphenoxyacetic acid. Note-With an ester formulation it is necessary to hydrolyse with alkali before the methylphenoxyacetic acid. extraction.April, 19531 CHLOROMETHYLPHENOXYACETIC ACIDS IN MCPA FORMULATIONS 209 The results obtained for the determination of 4-chloro, 4:6-dichloro, 6-chlor0, and un- chlorinated 2-methylphenoxyacetic acids in synthetic mixtures show that accuracy is good. Uncondensed cresols and glycollic acid do not interfere in the method. With typical com- mercial l’vlCPA formulations the figure for total acids, as determined by the proposed procedure, is slightly lower (1 to 2 per cent.) than that obtained for total acids in a similar aliquot of the ether solution titrated directly.This discrepancy is under investigation, but it does not affect the accuracy of determination of the 4-chloro acid content, as outlined above. Low grade o-cresol contains an appreciable amount of phenol as its major impurity. If this material has been used in the manufacture of commercial MCPA, the final product will probably contain significant amounts of 2 :4-dichlorophenoxyacetic acid (2 :4-D). This compound has a physiological activity comparable with that of 4-chloro-2-methylphenoxy- acetic acid. Hence, when 2 :4-D is present in a sample of commercial MCPA, the method determines only 4-chloro- 2-methylphenoxyacetic acid and not the total content of “herbicidically-active acid.” The influence of wz- and 9-cresols in o-cresol on the determination of active material in the final product has not been investigated. DISCUSSION OF RESULTS In the method described, 2:4-D is eluted with the 6-chloro fraction. The authors wish to thank the Directors of Pest Control Limited for permission to publish, this paper. REFEREKCES 1. 2 . Grabe, E., lbid., 1950, 4, 806. 3. 4. 6. 8. 7. Sarensen, I)., Actu Chem. Scand., 1951, 5, 630. Hill, K., Analyst, 1952, 77, 67. Sjoberg, B., A c f u Chem. Scand., 1950, 4, 798. Stroud, S. W., Analyst, 1952, 77, 63. Martin, A. J . P., “Biochemical Society Symposium on Partition Chromatography,” Cambridge University Press, 1949, pp. 11-12. Davies, C. \Ir., and Nancollas, G. H., Nulure, 1950, 165, 237. ANALYTICAL DEPARTMENT PEST CONTROL LIMITED IIARSTON, CAMBS. August 18th, 1952

ISSN:0003-2654    

DOI:10.1039/AN9537800205

出版商:RSC    

年代:1953

数据来源: RSC

摘要:

April, 19531 CHLOROMETHYLPHENOXYACETIC ACIDS IN MCPA FORMULATIONS 209 The Reaction Between Periodic Acid and Polyhydroxy Compounds With Particular Reference to the Colorimetric Determination of Formaldehyde with Chromotropic Acid BY S. L. TOMPSETT AND D. C. SMITH The liberation of formaldehyde when a number of polyhydroxy and aminoh ydroxy compounds react with periodic acid has been studied by two procedures. The formaldehyde is determined colorimetrically with chromotropic acid. The analytical application of this reaction is discussed and applications are made to more complex substances, human urine and blood, THE determination of formaldehyde by the sensitive colour reaction with chromotropic acid was first described by Eegriwe.1 Since then various factors involved in the analytical application of chromotropic acid have been studied by MacFadyen.2 It is liberated from hexamethylenetetramine by heating with dilute mineral acids.Periodic acid reacts with various polyhydroxy compounds to produce aldehydes and organic acids, and if these com- pounds contain a terminal CH,OH group, formaldehyde is produced. In neutral medium, serine and etlianolamine react with periodic acid to give formaldehyde. A colorimetric method for the determination of serine has been described by Nicolet and Shinn.3 The determination of the formaldehyde liberated in the reaction between periodic acid and a Formaldehyde is liberated from various substances in many ways.210 TOMPSETT AND SMITH: THE REACTION BETWEEN PERIODIC [Vol. 78 polyhydroxy compound offers a sensitive means of analysis.Owing to the large number of polyhydroxy compounds, it is necessary to incorporate, in any method used, procedures that ensure some degree of selectivity. Such procedures have been devised for the deter- mination of the urinary corticoids (Lowenstein, Corcoran and Page4; Daughaday, Jaff e and Williams5). The depth of colour produced by the reaction between chromotropic acid and form- aldehyde depends on a number of factors; in particular, on the concentration of the sulphuric acid and the time of heating. It has been found that under the conditions described by Daughaday et aL5 a colour is produced that is maximal and is stable for at least 24 hours. These conditions have been used in our investigation. Before its colorimetric determination, it is desirable to separate the formaldehyde liberated by any of the above chemical reactions by distillation, or non-typical colours may be produced. In this investigation the liberation of formaldehyde by the reaction between periodic acid and polyhydroxy compounds has been studied under various conditions. METHOD Two procedures have been used in this investigation for studying the reaction between periodic acid and polyhydroxy compounds.REAGENTS- Sulphuric acid, 13 M and 9 M. Sodium sulphite-A 1 per cent. w/v solution. Periodic acid reagent-A solution of 0.01 M potassium periodate in 0.15 M sulphuric Chromotropic acid reagent-Dissolve 0.2 g of chromotropic acid in 2 ml of water and Freshly prepared Hopkins and Williams Ltd. brand of specially Stannous chloride reagent-Dissolve 6 g of stannous chloride in 100 ml of diluted hydro- acid.48 ml of 13 M sulphuric acid. purified chromotropic acid has been found suitable. chloric acid containing 10ml of concentrated acid per 100ml of solution. DISTILLATION APPARATUS- is collected in a conical tube graduated at 10 ml. PROCEDURE A- This, in general, is the procedure used by Daughaday et aL6 for the determination of corticoids in urine. Allow the substance and an excess of periodic acid to react at room temperature and then neutralise the excess of periodic acid with stannous chloride solutioii. Determine the formaldehyde colorirnetrically with chromotropic acid after its separation by distillation. It should be noted that with substances in which the CH,OH group or an adjacent CHOH group is esterified no formaldehyde is liberated.Dissolve the substance in water, transfer the solution to the distillation flask and add 3 ml of periodate reagent. Set the mixture aside at room temperature for the specified time.6 Add 2 ml of stannous chloride reagent and make up to 15 ml with water. Heat the mixture to boiling and collect the distillate in a conical tube containing 1 ml of sulphite solution. Continue to distil until a total volume of 10ml is collected. Formaldehyde is determined colorimetrically in 1 ml of distillate as described below. PROCEDURE B- Dissolve the substance in water and heat with dilute sulphuric acid and periodic acid; collect the formaldehyde in the distillate. Determine the formaldehyde colorirnetrically with chromotropic acid.A major difference between this and the previous procedure is that the reaction with periodic acid takes place at 100" C in the presence of dilute sulphuric acid. Hence hydrolysis can proceed simultaneously with oxidation and this procedure may be used for complex substances not reactive to procedure A. Dissolve the substance in water, transfer to the distillation flask and add 3 ml of periodate reagent followed by 0-5ml of concentrated sulphuric acid and a glass bead. Make up to 15ml with water and heat the mixture to boiling. Collect the distillate in a conical tube containing 1 ml of sulphite solution. Continue to distil until a total volume of 10 ml is collected. Formaldehyde is determined as described below in 1 ml of distillate.A 50-ml round-bottomed flask is attached to a water-cooled condenser. The distillateApril, 19531 ACID AND POLYIIYDROXY COMPOUNDS 211 THE COLORIMETRIC DETERMINATION OF FORMALDEHYDE- reagent. water for 30 minutes. sulphuric acid. spectrophotometer GP350 set at 565 mp. or unknown solutions against that of the blank determination. Into a test tube measure 1 ml of distillate, 2 ml of water and 5 ml of chromotropic acid Place both tubes in a bath of boiling After cooling, dilute the contents of each tube to 10 ml with 9 M Extinctions can be read in a Biochem absorptiometer with filter OY2 or in a Unicam Measure the optical densities of the standard Make a blank determination simultaneously. PURITY OF SUBSTAWES UNDER EXAMINATION- The purest substances available were used, but the purity of glyceric acid was doubtful. RESULTS AND CONCLUSIONS CALIBRATION- With British types of absorptiometers it has been found most suitable to use from 0 to 20 micrograms of formaldehyde for any one determination.For standardisation purposes, the extinctions of 5, 10, 15 and 20 micrograms of form- aldehyde were measured after treatment with the chromotropic acid reagent. The form- aldehyde solutions were prepared from AnalaR formaldehyde solution, which was standardised by an iodine method (Kolthoff and Stengere). EFFECT OF DISTILLATION- Recovery of formaldehyde by a single distillation was found to be incomplete. In procedure A, recovery was 72 to 75 per cent. of the theoretical and in procedure B, 90 to 95 per cent. In both procedures, the further addition of water and a second distillation accounted for the remainder of the formaldehyde.In both procedures, however, consistency can be attained by a single distillation, provided conditions are standardised. LIMITATIONS OF PROCEDURE A- compounds react with periodic acid has been examined. following conditions- The liberation of formaldehyde when a number of polyhydroxy and aminohydroxy Procedure A was used under the (1) After 45 minutes with periodic acid in acid medium. (2) After 24 hours with periodic acid in acid medium. (3) After 45 minutes with periodic acid in neutral medium. (4) After 24 hours with periodic acid in neutral medium. Under conditions (1) and (2), 3 ml of periodate reagent were added to the substance dissolved in 4ml of water.After setting the mixture aside at room temperature for the specified time, the reaction was stopped by adding stannous chloride. With conditions (3) and (4), experiments were carried out in a similar manner except that after the periodate reagent had been added, the mixture was adjusted to neutrality (phenol red as indicator) by adding a phosphate buffer. The time of reaction with periodic acid was occasionally extended beyond 24 hours. The results shown in Table I can be summarised as follows- (1) Serine and ethanolamine produce the theoretical amount of formaldehyde within 45 minutes, but only at neutrality. No formaldehyde was produced in the acid medium used. a(2) The sugar alcohols liberate the theoretical amount of formaldehyde within 45 minutes in both acid and neutral media..(3) The aldose pentoses and hexoses examined liberated the theoretical amount of formaldehyde within 24 hours in both acid and neutral media. The formaldehyde liberated within 45 minutes in acid media was negligible and in neutral media the reaction was often incomplete. In acid conditions, the liberation of formaldehyde from pentoses proceeds faster than from hexoses. (4) Formaldehyde is liberated rapidly from fructose, but recoveries were never 100 per cent. This is in agreement with results of Bell, Palmer and Johns,' who could only obtain a maximum of 1.7 moles of formaldehyde instead of the theoretical 2 moles.TOMPSETT AND SMITH: THE REACTION BETWEEN PERIODIC [Vol. 78 Although dihydroxyacetone has two terminal CH,OH groups, only 1 mole of form- aldehyde per mole of dihydroxyacetone could be obtained.Recoveries of formaldehyde were low from glyceric acid. This was probably due to the gross impurity of the specimen. When a fresh aqueous solution of glyceric acid was prepared, its behaviour with periodic acid was similar to that of glucose. Later, a specimen was prepared in a dilute aqueous solution of sodium hydroxide and was set aside overnight. The earlier results were probably low owing to the presence of large amounts of the lactone. With ascorbic acid and lactose, liberation of formaldehyde is slow in acid media, and even if allowed to proceed for several days the amount of formaldehyde produced is never the theoretical amount. In neutral reactions, the amounts of formaldehyde produced are nearer to the theoretical.Results similar to those in (7) have been found with glucosamine. These are in agreement with those obtained by Jeanlay and Forchielli.8 This solution behaved as shown in Table I. TABLE I LIBERATION OF FORMALDEHYDE FROM CARBOHYDRATES AND RELATED SUBSTANCES WITH PERIODIC ACID. PROCEDURE A Formaldehyde liberated Substance .. Ethanolamine Mannitol . . Erythritol . . d-Xylose . . d-Arabinose . . &Ribose . . Glucose . . Galactose . . Fructose . . .. .. .. .. .. I . .. .. .. .. .. .. .. .. .. .. .. .. .. .. d-Glucosamine hydrochloride Lactose . . - . .. Dihydroxyacetone . . .. Ascorbic acid .. .. Glyceric acid .. .. Amount taken, CLg 100 60 25 40 20 10 1000 500 250 1000 1000 1000 1000 1000 500 250 1000 1000 500 250 1000 1000 100 1000 1000 In acid reaction - for 45 minutes, tLg nil nil nil nil nil nil 330 176 76 485 20 40 39 5 5 5 6 130 50 28 5 3 31 3 230 for 24 hours, CLg nil nil nil nil nil nil 330 - - 480 204 194 190 172 83 42 130 275 120 65 93 34 31 54 230 In neutral reactio; - for 45 minutes, wg 26.5 13.0 6.9 38.7 20-8 10.9 342 - I 490 195 188 210 152 78 38 144 275 100 56 48 60 32 54 230 for 24' hours, Pg 26.2 12.9 6.8 I - - 328 - - 485 208 190 210 154 78 44 144 275 104 67 108 156 32 132 230 Theoretical amount of form- aldehyde, CLg 28-6 14.8 7.4 39.2 19.6 9-8 330 165 83 49 2 198 198 198 167 84 42 167 333 165 83 135 167 33 171 281April, 19531 ACID AND POLYHYDROXY COMPOUNDS 21 3 LIMITATIONS OF PROCEDURE B- The liberation of formaldehyde when a number of hydroxy compounds were allowed to react with periodic acid under the conditions of procedure B was examined.Amino- hydroxy compounds were not examined. TABLE I1 LIBERATION OF FORMALDEHYDE BY DISTILLATION WITH PERIODIC ACID IN PROCEDURE B PRESESCE OF SULPHURIC ACID. Substance Glucose . . Mannitol . . Erythritol . . 1-Xylose . . Arabinose . . &Ribose . . Galactose . . Fructose . . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. Glucosamine hydrochloride Maltose . . .. .. Lactose . . .. .. 1-Ascorbic acid . . .. Dihydroxyacetone. . . . Hexamethylenetetramine (periodate omitted) .. . . .. .. . . .. .. .. .. .. .. * . .. Amount taken, Pf3 1000 500 250 400 200 100 400 800 400 200 500 500 1000 500 400 200 500 400 200 100 666 333 500 667 1000 600 250 400 150 100 60 Formaldehyde determined, P.g 167 83 42 128 61 30 210 156 86 36 92 103 156 80 57 30 93 70 35 17 86 40 80 102 156 92 36 139 166 117 65 Theoretical amount of formaldehyde, Pg 167 83 42 133 67 33 196 160 80 40 100 100 167 83 66 33 83 (166) 67 (134) 33 (66) 17 (34) 92 46 88 113 170 85 43 133 171 114 -67 The results are shown in Table I1 and can be summarised as follows- (1) Theoretical amounts of formaldehyde could be obtained from glucose, mannitol, erythritol, xylose, arabinose, ribose, galactose, glucosamine, maltose, lactose, ascorbic acid and dihydroxyacetone.(2) One mole of formaldehyde is liberated from 1 mole of fructose instead of the expected 2 moles. EXAMINATION OF MISCELLANEOUS SUBSTANCES- The liberation of formaldehyde when a number of complex substances were allowed to react with periodic acid has been examined. Both procedures A and B have been used, and with some substances the effect of previous acid hydrolysis has been examined.For these, the appropriate amount of material contained in 10 ml of water was heated in a bath of boiling water with 0.5 ml of concentrated sulphuric acid for 30 minutes. In some instances the amount of formaldehyde was determined in serial distillations. Second and third distilla- tions were carried out after the addition of water to the periodic acid mixture. As the purity214 TOMPSETT AND SMITH: THE REACTION BETWEEN PERIODIC [Vol. 78 and, occasionally, the composition of some of the substances used was in doubt, the results shown in Table I11 are not strictly quantitative. TABLE I11 LIBERATION OF FORMALDEHYDE FROM COMPLEX CARBOHYDRATES AND RELATED SUBSTANCES BY PERIODIC ACID Substance Sodium /3-glycero- Starch .. .. phosphate Form- aldehyde Amount of liberated form- by Amount aldehyde procedure taken, expected, A, CLg Pg Pg 1000 139 nil 1000 174 nil Form- aldehyde liberated by procedure B (no previous treatment), CLg 140 - 37 67 18 Sodium salt of thymus gland nucleic acid (ribonucleic acid) Sodium salt of yeast nucleic acid (deoxy- ribonucleic acid) 122 (total) 3000 229* nil - 12 17 5 - 34 (total) 3000 229 nil - 83 25 5 - 113 (total) * Sodium salt of adenylic acid. Form- aldehyde liberated by procedure B after acid hydrolysis, Pg I I 106 22 6 - 133 (total) - 12 7 5 - 24 (total) 95 22 3 - 120 (total) Conditions 1st distillate 2nd distillate 3rd distillate 1st distillate 2nd distillate 3rd distillate 1st distillate 2nd distillate 3rd distillate SODIUM F-GLYCEROPHOSPHATE- a-Glycerophosphoric acid reacts with periodic acid to liberate 1 mole of formaldehyde, whereas /I-glycerophosphoric acid is unaffected.a- and P-Glycerophosphates are interconvertible under appropriate conditions. Acid favours the formation of the a-form, whereas alkali favours the formation of the p-form. Burmasterg has shown that inorganic phosphate is quantitatively liberated when ,f3-glycerophosphoric acid is heated with periodic acid in the presence of mineral acid This is due to its conversion to the a-form, which then reacts with the periodic acid. No attempt was made to determine the amount of formaldehyde so liberated. In the present investigation, it has been found that the theoretical amount of form- aldehyde is liberated in procedure B.No formaldehyde could be detected when procedure A was used. As a-glycerophosphoric acid should liberate the theoretical arnoiint of form- aldehyde in both procedures, it should now be possible to detect and measure both a- and p- forms, not only separately, but also when mixed. The a- and P-glycerophosphates are unaffected by acid hydrolysis. COMPLEX CARBOHYDRATES- Starch-No formaldehyde could be detected when starch was subjected to procedure A. Amounts of formaldehyde approaching the theoretical were obtained when starch was subjected to procedure B. The effect of previous acid hydrolysis was to speed up the liberation of formaldehyde. ATucZeic acids-The amount of formaldehyde recovered from ribonucleic acid is negligible by both procedures, and the small amount determined in practice could quite easily be due to impurities.No formaldehyde could be detected when desoxyribonucleic acid was allowed to react with periodic acid under the conditions of procedure A. Un.der the conditions of procedure B, approximately 50 per cent. of the theoretical amount of formaldehyde expected could be found. The amount of formaldehyde measured was increased slightly by previous acid hydrolysis.April, 19531 ACID AND POLYHYDROXY COMPOUNDS 215 In ribonucleic acid, the CH,OH group of the sugar is esterified with phosphoric acid. This is stable to acid hydrolysis. In desoxyribonucleic acid, the CH,OH group is unesterified. The low yield of formaldehyde may be due to the hydrolysis of purine nucleotides on heating with mineral acids ; conversely, the pyrimidine nucleotides are generally resist ant.These differences are undoubtedly due to differences in structure. APPLICATIONS- It is believed that the information reported in the previous sections, although incomplete, could have useful analytical applications. The mode of application would naturally depend upon the particular problem. We have applied the reactions to protein-free extracts of human whole blood (preserved with sodium fluoride) and human urine. Blood-Tungstic acid filtrates were used. Formaldehyde was determined after the following treatments with periodic acid- (1) In acid media after 45 minutes. (2) In acid media after 24 hours. (3) In neutral media after 24 hours.The sugar content was determined by the Hagedorn and Jensen method. Procedure A was used. From the results shown in Table IV it will be noted that glucose accounts for most of the formaldehyde liberated. TABLE IV FORMALDEHYDE LIBERATED BY THE REACTION OF WHOLE BLOOD (FOLIN-WU) Formaldehyde liberated A I -3 In acid reaction In neutral r A \ reaction for 45 minutes. for 24 hours. for 24 hours. BETWEEN PROTEIW-FREE FILTRATES AND PERIODIC ACID “Glucose” Blood sugar ea uivalent . (Haeedorn and pg per 100 ml df p g per 100 ml’of p g per 100 ml’of mg 6er 100 mi of ‘ j’ensen) , whole blood whole blood whole blood whole blood mgper 1OOml 2 4 4 3 14.0 14.0 13.2 14.2 13-8 14.0 12.8 15.0 84 84 77 00 102 87 93 89 Urine-The amount of formaldehyde liberated when urine was allowed to react with (1) In acid media after 45 minutes.(2) In acid media after 24 hours. (3) In neutral media after 24 hours. No additional information was obtained by the determination of the formaldehyde liberated in the reaction between urine and periodic acid at neutral reaction after 45 minutes, so the results of this determination have been omitted. The remainder of the results are shown in Table V. periodic acid has been measured under the following conditions- TABLE V FORMALDEHYDE LIBERATED BY THE REACTION BETWEEN URINE AND PERIODIC ACID The results are expressed in mg of formaldehyde per litre of urine Acid periodate 1 1 Urine number after 46 minutes, after 24 hours, pg per litre 1 80 155 2 92 120 3 65 74 4 100 130 6 168 412 pg per litre Neutral periodate, after 24 hours, pg per litre 200 154 98 166 692 There were appreciable differences between conditions (1) and (2).These differences can be taken as an approximate measure of the free carbohydrate content of the urine. The216 TOMPSETT AND SMITH [Vol. 78 ,difference between conditions (2) and (3) can be taken as a measure of the content of free serine and ethanolamine. A considerable amount of formaldehyde is liberated by reaction with acid periodate within 45 minutes. In view of the following experiments it is suggested that this is probably derived from sugar alcohols. These experiments included- (a) Distillation with dilute sulphuric acid, which alone produced no formaldehyde. This excludes such methylene compounds as hexamine.(b) Incubation with laked red blood cells, which resulted in no increase in the amount of formaldehyde liberated. This excludes glycerophosphates. (c) Pre-treatment of the urine with the ion-exchange resins IRA105(4) and 400(OH), which produced no appreciable change in the amount of formaldehyde liberated. Basic or acidic substances are thus eliminated. The possibility must always be taken into account that formaldehyde could be lost by the formation of a stable compound with other substances present in the reactant mixture. Although mannitol, glucose and serine added to urine could be recovered quantitatively (see Table VI), small consistent losses cannot be entirely discounted. Such stable compounds have been found to be formed with 9-aminosalicylic acid, a drug that is used in the treatment of tuberculosis. TABLE RECOVERY OF GLUCOSE, MANXITOL ASD SERINE ADDED TO URINE BY PROCEDURE A Amount of urine used-0.25 ml Initial LLg Glucose . . .. .. 62 62 45 45 Mannitol . . .. .. 50 50 50 Serine . . . . .. 76 76 76 Substance amount, Amount added, PLg 33 133 33 133 33 133 200 29 58 145 Total amount determined, LLg 101 200 83 185 81 192 272 102 121 242 Amount recovered, Pg 39 138 38 140 31 142 222 26 46 166 By the assumptions made above, the apparent glucose and serine contents of the urines that were examined have been calculated. The results are shown in Table VII. TABLE VII CA4LCULATED GLUCOSE AND SERINE CONTENTS OF URINES Urine number “Glucose” content, “Serine” content, 1 450 167 2 168 119 3 54 84 4 180 91 5 1464 980 mg per litre mg per litre REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. NORTHERN GENERAL HOSPITAL Eegriwe, C., 2. anal. Chem., 1937, 110, 22. MacFadyen, D. A., J . Biol. Chem., 1945, 158, 107. Nicolet, B. H., and Shinn, L. A., J . Anzer. Chern. SOC., 1939, 61, 1615. Lowenstein, B. E., Corcoran, A. C., and Page, I. H., Endocrinology, 1946, 39, 82. Daughaday, W. H., Jaffe, H., and Williams, R. H., J . Clin. Endocvinol., 1948, 3, 166. Kolthoff, I. M., and Stenger, V. A., “Volumetric Analyses,” Volume I, Interscience Publishers Bell, D. J., Palmer, A., and Johns, A. J., J . Chem. SOC., 1949, 1536. Jeanlay, R. W., and Forchielli, E., J . Biol. Chem., 1951, 188, 361. Burmaster, C. F., Ibid., 1946, 164, 233. Inc., New York, 1942, pp. 248 and 252. BIOCHEMICAL LABORATORY FERRY ROAD, EDINBURGH, 5 June 30t12, 1952

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DOI:10.1039/AN9537800209

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年代:1953

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April, 19531 RAY 217 Determination of Molybdenum by Ammonium Thiosulphate and Sodium Hypophosphite BY H. N. RAY Molybdenum is determined by precipitation as molybdenum sulphide by ammonium thiosulphate and sodium hypophosphite from acid solution, ignition and weighing as the oxide. Certain of the inconveniences of the direct hydrogen sulphide precipitation method are avoided, but several elements interfere. The method has been successfully applied to molybdenum steels and ferro-molybdenum. WHEN sulphurous acid or a soluble sulphite is added to an acidified solution of sodium hypophosphite, hydrogen sulphide is evolved. If sulphurous acid is replaced by sodium or ammonium thiosulphate, a mixture of gases is evolved and sulphur separates. The concentration of this gas is so low in the cold that it fails to precipitate copper or mercury as sulphide from their solutions, but on warming, the black sulphides are precipitated.This reaction has been utilised for the determination of certain elements; in particular molyb- denum. When added to the acidified solution of a molybdenum salt, ammonium thiosulphate immediately produces, even in the cold, a chocolate-coloured precipitate of molybdenum sulphide, which is sometimes contaminated with free sulphur. This precipitate becomes darker on heating. Sodium hypophosphite when present greatly enhances the reaction and facilitates complete precipitation of the molybdenum. This method has several advantages over the usual precipitation by gaseous hydrogen sulphide. In the direct determination of molybdenum by hydrogen sulphide gas, the operation is best conducted under pressure. Gassing is repeated two or three times and digestion of the solution on the steam-bath completes the precipitation of the sulphide.It is possible that the molybdenum will be reduced to its lower valency state during gassing. If this happens, complete precipitation can scarcely be expected. It is better at this stage to begin the experiment anew. Even when an alkaline solution is used, both gassing and digesting are essential in order to precipitate molybdenum sulphide completely by the decomposition of thiomolybdate with mineral acids. Both processes are objectionable in that large amounts of hydrogen sulphide are inhaled during the operations. Determination of molybdenum with ammonium thiosulphate is free from these defects.The process requires neither a pressure-bottle, nor gassing of the solution to complete the precipitation. Moreover, molybdenum is precipitated from its lower valency state. METHOD REAGENTS- containing ammonia, and dry iiz vacuo. and make up to 1 litre. sulpha t e . of water, add 2 ml of concentrated sulphuric acid and set aside. Ammonium molybdnte-Recrystallise extra pure ammonium molybdate* from water Accurately weigh 1 g of it, dissolve it in water Ammonium thiosulphate-A 10 per cent. filtered aqueous solution of ammonium thio- Sodium hypuphosphite-Dissolve 10 g of crystalline sodium hypophosphite in 100 ml Concentrated hydrochloric acid. PROCEDURE- beaker. with distilled water. to the hot solution. Accurately measure 50 ml of crystallised ammonium molybdate solution into a 100-ml Add about 8 ml of concentrated hydrochloric acid and make up to 150 to 175 ml Heat to boiling and add 20 to 25 ml of ammonium thiosulphate solution A reddish-brown precipitate immediately forms.Add 10 to 15ml of It contains 64.36 per cent. of molybdenum. * Supplied by Merck.218 RAY: DETERMINATION OF MOLYBDENUM BY AMMONIUM [Vol. 78 acidified sodium hypophosphite solution and stir vigorously for about 2 minutes. With the introduction of sodium hypophosphite solution, the reddish-brown precipitate turns dark brown and the molybdenum sulphide quickly settles. Any reddish-brown colour persisting in the supernatant liquid indicates that separation of molybdenum sulphide is incomplete. Under these circumstances add a few drops of hydrochloric acid to precipitate the molybdenum sulphide completely. The total volume of the solution should then be about 250 ml.Stir the solution again for a minute and place it on the cooler part of the hot-plate for about 10 minutes. The supernatant liquid is then clear and free from any visible colour save for some colloidal sulphur floating in the liquid. Filter through a Whatman No. 41 filter-paper and wash the precipitate thoroughly, first with dilute hydrochloric acid (1 + 99) saturated with hydrogen sulphide and then with hot water, to free it from sodium salt. If any precipitate sticks to the side of the beaker, remove it with a piece of filter-paper and add it to the main precipitate. Place the wet sulphide in a crucible and carefully ignite the sulphide in a muffle furnace provided with a pyrometer.As molybdenum oxide begins to volatilise above 600" C, the ignition is conducted at between 550" and 575" C. During heating, molybdenum sulphide is gradually transformed into molybdenum oxide, MOO,. Heat until the weight is constant and weigh the residue as MOO,. Copper, mercury, bismuth, lead, arsenic, tin, antimony are among the elements that interfere in this experiment. Results of typical experiments are shown in Table I. TABLE I RECOVERY OF MOLYBDENUM FROM AMMONIUM MOLYBDATE SOLUTION Volume of ammonium molybdate solution taken, ml 50 50 100 100 30 30 Weight of MOO, found, g 0-0400 0.0402 0*0810 0.0806 0.0223 0.0220 Weight of MOO, calculated, €! 0.04075 0,04075 0.0815 0.08 15 0.02245 0.02245 DETERMINATION OF MOLYBDENUM I N STEEL PROCEDURE- Weigh 1 to 2 g of steel drillings (depending upon the amount of molybdenum present) into a 400-ml beaker and decompose with 50 ml of diluted hydrochloric acid (1 + 1).When solution is complete, evaporate the mass to a syrupy consistency. If any black residue of molybdenum is undissolved, bring it into solution by adding a little potassium chlorate. If tungsten is present modify the procedure as follows. Dissolve the steel drillings in 50 ml of diluted hydrochloric acid (1 3. 1). When decomposition is complete, add 6 to 8 g of solid tartaric acid or, better, 30 to 40ml of a 20 per cent. solution of tartaric acid. This will keep tungsten in solution1. Heat the solution to boiling and add a little potassium chlorate from time to time to dissolve any decomposed molybdenum carbide.By this time all black particles will be decomposed and the solution will be clear. Adjust the volume of the solution to 225 to 250 ml and again bring it to the boil. Add 20 to 25 ml of ammonium thiosulphate solution and stir. Add 15 ml of acidified sodium hypophosphite solution and stir vigorously with a glass rod for about 2 minutes. During this operation the dark brown molybdenum sulphide becomes coagulated and settles at the bottom together with some of the sulphur already set free. From this point proceed exactly as described above for the determination of molybdenum. Ignite the wet sulphide at 550" to 575" C. This gives impure molybdenum oxide. It contains oxides of iron, silicon and so on, and requires purification.To purify the oxide, dissolve it by boiling with 10 to 15 ml of diluted ammonium hydroxide (1 + 1). Molybdenum oxide dissolves and oxides of iron, silicon and so on remain undissolved. Filter the solution, wash the residue with warm ammonia water. Ignite the precipitate and weigh. Deduct this weight from the weight of impure molybdenum oxide. The difference between the two weights multiplied by 66.6 and divided by the weight of steel taken gives the percentage of molybdenum.April, 19531 THIOSULPHATE AND SODIUM HYPOPHOSPHITE 219 RESULTS- In some experiments British Chemical Standard steel “W2,” No. 167, was used as a specimen of molybdenum steel. The steel contained: 0.71 per cent. of carbon; 0.14 per cent.of silicon; 0.051 per cent. of sulphur; 0.026 per cent. of phosphorus; 0.22 per cent. of manganese; 3.52 per cent. of chromium; 0.82 per cent. of vanadium; 16.12 per cent. of tungsten; 4.35 per cent. of cobalt ; 0.43 per cent. of nickel; and 0.56 per cent. of molybdenum. Results with this steel and other molybdenum steels are shown in Table 11. TABLE I1 RECOVERY OF MOLYBDENUM FROM STEELS Weight of g Sample impure MOO,, 1. B.S.S. No. 167 . . .. . . 0.0114 2. B.S.S. No. 167 . . .. . . 0.0106 3. Other steel . . .. .. . . 0.0275 4. Other steel . . .. .. . . 0.0609 5. Other steel* .. .. . . 0-0170 Weight of Molybdenum Molybdenum impurity, found, present, 0.0032 0.546 0.65 0.0026 0.539 0-55 0.0064 1-405 1-450 0.0074 3.56 3-60 0.0052 0.392 0.40 g Yo % * On 2-g samples.DETERMINATION OF MOLYBDENUM IN FERRO-MOLYBDENUM Ferro-molybdenum is an alloy of iron and molybdenum. The amount of molybdenum in it may vary from 60 to 70 per cent. PROCEDURE- Weigh 0.1 g of this finely crushed material, in a 100-ml beaker, and decompose it with 10 to 15 ml of nitric acid. Cautiously add 10 ml of diluted sulphuric acid (1 + 1) and evaporate the solution to fumes of sulphur trioxide. Remove the beaker from the hot-plate and allow it to cool. Dissolve the residue in about 40ml of water and further dilute to 200 to 250ml. Filter and heat the filtrate to boiling. Precipitate the molybdenum as sulphide from this solution with ammonium thiosulphate and sodium hypophosphite exactly as has been stated for steel. Ignite the sulphide and weigh as MOO,. Purify the ignited molybdenum oxide by dissolving the ash in ammonium hydroxide. Thoroughly wash the residue, which mainly consists of iron oxide, ignite and weigh.Deduct this weight from the weight of impure molybdenum oxide to find the true weight. RESULTS- The results found by the procedure suggested above were in good accordance with those previously found in this laboratory for samples from English suppliers, in which the molyb- denum had been determined by conversion to thiomolybdate, precipitation as sulphide and weighing as oxide. TABLE I11 Crush the alloy and sieve it through a 100-mesh sieve. Weight of MOO, x 66.6 = Percentage of molybdenum in 1 g of sample. Table I11 shows the results from three of these alloys. MOLYBDENUM IN SAMPLES OF FERRO-MOLYBDENUM Amount of molybdenum found P Weight of BY Other constituents of alloy impure Weight of By proposed thiomolybdate f A .l MOO,, impurity, procedure, procedure, C, y, Si, Mn, g g % % % % % % 0.1 147 0.0030 74.39 74.60 0.065 0.04 1-88 trace 0.1142 0.0040 73.39 73.50 0.04 0.015 0.44 trace 0.1052 0.0028 70.13 70.32 0.041 0.046 0.65 trace REFERENCE 1. ICHAPORE, BENGAL, INDIA Ray, H. N., and Ghose, S., C., Metallurgia, 1944, 30, 233. INDIAN ORDNANCE DEPARTMENT

ISSN:0003-2654    

DOI:10.1039/AN9537800217

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年代:1953

数据来源: RSC