ISSN:0003-2654    

DOI:10.1039/AN95681FX029

出版商:RSC    

年代:1956

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No. 38 June, 1956 THE SOCIETY FOR ANALYTICAL CHEMISTRY BULLETIN BRITISH STANDARDS INSTITUTION DRAFT SPECIFICATION A FEW copies of the following draft specification, issued for comment only, are available to members of the Society, and can be obtained from the Secretary, The Society for Analytical Chemistry, 7-8 Idol Lane, London, E.C.3. L h f t Specification prepared by ‘Technical Sub-Cornmi ttee RUC/ 10/4-Physical Testing CW( KUC)4394-Draft B.S. Method for Determination of Abrasion Resistance of of Rubber. Vulcanized Rubber (Revision of Part 24 of B.S. 903). COMMUNICATIONS ACCEPTED FOR PUBLICATION IN THE ANALYST THE following communications have been accepted for publication in The Analyst, and are expected to appear in the near future. It is not possible for the Editor to enter into corre- spondence about any of them.“The Separation of Small Amounts of Lead from Small Amounts of Bismuth,” by V. J. Moore. (Note.) “The Determination of Small Amounts of Bismuth in‘ Copper Mattes and Concentration Products,” by V. J. Moore. “The Determination of Magnesium Oxide in Magnesium,” by H. J. Allsopp. “An Improved Method for the Quantitative Determination of Amino Acids with Indane- “Determination of Mercury in Fungicidal Preparations containing Organo-mercury “The Gravimetric Determination of Potassium in Sea Water as the Potassium Tetra- “The Polarographic Determination of Boron,” by D. T. Lewis. “The Micro-determination of Chlorine in Small Samples of Polymerised and Co- (Note.) trione Hydrate,’’ by S. Jacobs. Compounds. phenylboron Salt,” by K.F. Sporek. (Note.) , Parts I and 11,” by K. F. Sporek. polymerised Vinyl Chloride,” by J. M. Bather. “A Semi-micro Determination of Sulphur in Cystine and Methionine,” by T. T. Gorsuch. (Note.) “Determination of Volatile Oil in Effluents,” by J. G. Sherratt.LIBRARY OF THE CHEMICAL SOCIETY Summer Closing, 1956 THE Librarian of The Chemical Society has announced that from July 16th to September 30th, 1956, the Library will close at 5 p.m. instead of 7.30 p.m. The Library will be closed all day on Monday and Tuesday, August 6th and 7th. Members of the Society for Analytical Chemistry are reminded that they are entitled to use this Library. NOTICE New British Chemical Standards THE Bureau of Analysed Samples Ltd., Middlesbrough, has announced the publication of a new list of standard samples, No. 386, which may be obtained post free from the office of the Bureau. The new samples include a Permanent Magnet Alloy, Basic Slags, a Silicon Aluminium Alloy, an Alumina Firebrick, and a series of seven spectrographic standards (S.S. Nos. 11--17) of mild steels in the form of $-inch diameter rods containing increments of a large number of residual elements. In this series chemical standardisation at present extends to Ni, Cr, Mo, Cu, W, Co, Sn and V; in due course it is hoped to standardise also Ti, Al, Zr, Nb, Ta, Pb and B. Turnings are also available for use as chemical or photometric standards. PRINTED BY W. HEFFER & SONS LTD.. CAMBRIDGE

ISSN:0003-2654    

DOI:10.1039/AN956810X031

出版商:RSC    

年代:1956

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95681BX033

出版商:RSC    

年代:1956

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95681FP067

出版商:RSC    

年代:1956

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95681BP073

出版商:RSC    

年代:1956

数据来源: RSC

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JUNE, 1956 THE ANALYST Vol. 81, No. 963 MEMBERS of the Society are already aware, from the announcement made at the Annual General Meeting held at the end of February last, and from the Report of the Council for 1955 published in the May issue of The Analyst, that the subscription of Ordinary Members will be raised in 1957, and that the cost of The Analyst and Analytical Abstracts to non- members will also be increased. The subscription of Junior Members will, we are happy to say, remain unchanged. The Council has taken the step of making these increases only after the most searching enquiries into the cost of running the Society and after long deliberation of the various heads under which our expenditure falls. It will, of course, be realised that the activities of the Society have expanded very widely in the years since 1945.Not only have we extended the size and circulation of The Analyst, which now goes to 56 countries and of which we print over 6000 copies a month, but we have taken over from the Bureau of Abstracts the task of pro- viding in Analytical Abstracts a monthly journal covering the whole field of analytical chemis- try in a manner never before achieved. While this development has been going on, we have, by the formation of Subject Groups and by increasing the number of our geographical Sections, brought our scientific meetings to a very high number each year, many times that of pre-war days. We have, moreover, enabled analysts in many parts of the country to hear and discuss papers which before our expansion they could only have read.The increase in the size of The Analyst, the launching of Analytical Abstracts, and the need to provide office services for the Groups and Sections, together with the work entailed by the very greatly increased membership of the Society, have led to a big expansion of the office staff and to the need to provide proper and centralised offices. These we have found on the top floor of the new house of the Society of Chemical Industry in Belgrave Square, London. Few Societies can boast, as we can, that their annual subscription remained unchanged from 1874 to 1950, and at the modest figure of one guinea at that. We have been able to manage in the last few years by no more than doubling that amount. But the need now arises to make a further increase-of no more than another guinea, it is to be noted; and it arises not only from the development of our activities and the necessity for trying to save a little money each year against future expansion, but from those increases in prices that we see around us all today, and especially in the cost of printing.I t would have been necessary to increase the subscription of members and the cost of our publications some years ago had not the Chemical Council made us grants which enabled the Society to balance its publications accounts. These grants were made from funds subscribed by industry for the publication of chemical researches, and the continuing success of our journals prompts us to believe that we have applied them as the donors would have wished. We can never be too grateful for the aid that has thus come to us; equally it seems no more than right that we should use all our endeavours to make The Analyst and Analytical Abstracts self-supporting, or as nearly so as we can, and a t the same time to place the general finances of the Society on a footing that can cope in the foreseeable future with the demands likely to be made on them. It is with these objects in view that the Council of the Society has acted. We are a Society that has always been most reluctant to raise its subscription. K. A. WILLIAMS President 321

ISSN:0003-2654    

DOI:10.1039/AN9568100321

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年代:1956

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322 PROCEEDINGS [Vol. 81 JOINT COMMITTEE ON METHODS OF ASSAY OF CRUDE DRUGS A JOINT Committee has been formed by th.e Pharmaceutical Society and the Society for Analytical Chemistry to prepare standard rnethods of assay for crude drugs and kindred materials where such methods are required in commerce and are not included in current editions of the British Pharmacopoeia and the British Pharmaceutical Codex. The committee will receive and examine proposals for the preparation of methods and will allocate these, if approved, for detailed investigation to small working panels of experts in the subjects. Methods submitted by the panels and accepted by the committee will be published as standard methods. The constitution of the committee is as follows:-Dr. K. R. Capper (Chairman), Dr. A. J. Feuell, Dr. D. C. Garratt (ex o$cio as Chairman of the Analytical Methods Committee of The Society for Analytical Chemistry), Mr. R. Higson, Mr. C. A. Johnson, Mr. H. C. Macfarlane, Dr. W. Mitchell, Mr. W. M. Seaber, Dr. R. E. Stuckey, Dr. C. H. Tinker (Secretary) and Mr. D. Watt .

ISSN:0003-2654    

DOI:10.1039/AN956810322b

出版商:RSC    

年代:1956

数据来源: RSC

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June, 19561 HILL 323 The Determination of 4-Chlo ro-2-meth ylphenoxyacetic Acid in MCPA by a Differential Ref ractometric Method BY R. HILL (PresePzted at the meeting of the Society on Wednesday, April 4th, 1956) A differential refractometric method is described for the determination of the biologically active 4-chloro-2-methylphenoxyacetic acid in MCPA containing 80 to 100 per cent. of the active acid. The method involves the comparison of the refractive index of a saturated solution of pure 4-chloro- 2-methylphenoxyacetic acid with that of a solution of the sample prepared in such a way that all the impurities in the sample are dissolved and the solution is saturated with the main component. The difference in refractive index between the two solutions is an accurate measure of the impurity content of the sample, provided that the impurities are methyl- or chloromethyl- phenoxyacetic acids or both.The results for synthetic samples are in good agreement with the actual values and the precision of a single determination is better than & 1 per cent. expressed as 95 per cent. confidence limits. MANY selective herbicide formulations are based on MCPA, which is a mixture of chloro- methylphenoxyacetic acids containing 4-chloro-2-methylphenoxyacetic acid as the active principle together with various proportions of 6-chloro-2-methyl-, 4 : 6-dichloro-2-methyl- and 2-methyl-phenoxyacetic acids. Several methods for the determination of 4-chloro-2-methylphenoxyacetic acid in such mixtures have been published, including an isotope-dilution method by S@rensen,l,2 an infra-red spectrophotometric method by Sjoberg,3 ultra-violet spectrophotometric methods by Grabe4 and by Hill5 and a chromatographic method by Freeman and Gardner.6 The ultra-violet and chromatographic methods, which are the most suitable for routine analyses, have been used extensively in these laboratories, but because the accuracy obtainable with the ultra-violet methods is dependent to a great extent on the composition of the sample, the chromatographic method has generally been preferred.However, even this method has not proved to be entirely satisfactory, because of the considerable time required to train an unskilled operator in the art of column packing and more particularly because of the poor precision of the results, which, expressed as 95 per cent.confidence limits of a single determination, has been found to be If: 3 per cent. in a research laboratory and j-- 5 per cent. in a process-control laboratory. Attempts to improve the precision of the chromatographic method were unsuccessful and in view of the fact that a large proportion of the MCPA produced at the present time contains 80 to 90 per cent, of 4-chloro-2-methylphenoxyacetic acid attention was turned to the possibility of using a differential refractometric m e t h ~ d , ~ which it was considered would be capable of a precision of better than 5 1 per cent. This method involves a comparison of the refractive index of a saturated solution of pure standard with that of a solution of the sample prepared in such a way that all the components of the sample are dissolved except a small amount of the major component.The difference in refractive index between the two solutions is dependent upon the refractometric behaviour and amounts of the various impurities in the sample. Provided that the impurities are similar in refractometric behaviour, the difference in refractive index can be used as an accurate measure of the total impurity content. EXPERIMENTAL TECHNIQUE OF DIFFERENTIAL REFRACTOMETRY- The construction and method of use of a differential refractometer for the determination of the purity of organic compounds has been described by Hill and Jones.7 No major modi- fication was made to the apparatus described by these authors, but since brass was attacked324 HILL: THE DETESRMINATION OF [Vol.81 by phenoxyacetic acids it was found necessary to construct the three-compartment refracto- meter cell, which had a 90" central prism, from stainless steel. It was also found necessary to modify the procedure for preparing saturated solutions, since that described by Hill and Jones for gamma benzene hexachloride was not reproducible when applied to MCPA. The procedure adopted is described subsequently under " Method " (see p. 326). CHOICE OF SOLVENT- Ideally the selected solvent should be one such that a given volume will dissolve all the impurities in a relatively large sample and leave a small excess of the main component un- dissolved, For this to occur it is necessary that the ratio of the amount of main component in the sample to the amount of an individual impurity should be greater than the ratio of the solubility of the main component to that of the individual impurity, for each impurity in the sample, Le.- 100 - x Y solubility of the main component solubility of individual impurity ' > where x is the percentage of total impurity and y is the percentage of the individual impurity.If the above condition does not hold good for any one impurity, then it is necessary to add some pure main component to the sample in order that the sample solution should be saturated with this. It was known that 4 : 6-dichloro-2-methylphenoxyacetic acid, which was a likely impurity in MCPA, was in general much less soluble than the 4-chloro-2-methyl acid, and in consequence the approximate solubilities of these two acids were determined in a number of common solvents with the results given in Table I.The solubilities of 6-chloro-2-methyl- and 2-methyl- phenoxyacetic acids were not determined at this stage, since it was known that the solubility of the former acid was greater than that of 4-chloro-2-methylphenoxyacetic acid and that of the latter acid was of the same order as the solubility of 4 : 6-dichloro-Z-methylphenoxy- acetic acid. TABLE I APPROXIMATE SOLUBILITIES OF 4-CHLORO-2-METHYL- AND 4 : 6-DICHLORO-%METHYL- PHENOXYACETIC ACIDS I N !SOME COMMON SOLVENTS Solvent Benzene . . Toluene .. n-Butanol . . isoPropano1 . . Chloroform . . n-Butyl acetate Solubility of 4-chloro- 2-methylphenoxyacetic g per 100 g of solvent Temperature, acid, " C .. 21 4.2 .. 21 3.7 .. 21 56 .. 21 79 . . 21 4.2 .. 15 22 Solubility of 4 : 6-dichloro- 2-methylphenoxyacetic acid, g per 100 g of solvent 0.4 0-4 7.3 8.9 0.7 3.4 Considering a maximum impurity content of 20 per cent., all of which could be 4 : 6-di- chloro-2-methylphenoxyacetic acid, it was obvious that none of the solvents examined fulfilled the condition stated above and it would be necessary therefore to add pure 4-chloro- 2-methylphenoxyacetic acid to the sample. The solvent chosen for further work was n-'butyl acetate, since it possessed a reasonably high solubility for the acids, was not too volatile and was readily available in a sufficiently pure state. REFRACTOMETRIC BEHAVIOUR OF A NUMBER O F SUBSTITUTED PHENOXYACETIC ACIDS I N ?Z-BUTYL ACETATE SOLUTION- With rt-butyl acetate as solvent, 0.5 per cent.w/v solutions of fifteen phenoxyacetic acids were prepared and compared with pure solvent in the differential refractometer, the linear shift of the slit image, Ad, being measuredl for each solution. In addition, the relation between Ad value and concentration was investigated for solutions of 2-methyl-, 6-chloro- 2-methyl- and 4 : 6-dichloro-2-methyl-phenoxyacetic acids in n-butyl acetate. The results of these two series of experiments, which are recorded in Tables I1 and 111, showed that the differences in refractometric behaviour between mono-, di- and tri-substituted phenoxyaceticJune, 19561 4-CHLORO-2-METHYLPHENOXYACETIC ilCID I N MCPA 325 acids were relatively small and that the relation between Ad and concentration was linear over the range 0 to 2 per cent.w/v. TABLE I1 REFRACTOMETRIC BEHAVIOUR OF A NUMBER OF PHENOXYACETIC ACIDS Phenoxyacetic acid Unsubstituted . . 2-methyl . . . . 3-methyl . . .. 2-chloro . . .. 4-chloro . . .. 2 : 4-dichloro . . .. 2 : 6-dichloro . . .. 4-chloro-2-methyl . . 6-chloro-2-methyl . . 4-chloro-3-methyl . . 2-chloro-4-methyl . . 4 : 6-dichloro-2-methyl 2 : 6-dichloro-4-methyl 2 : 4 : 6-trichloro . . 2 : 4 : 5-trichloro . . Ad for 0.5 per cent. w/v solution in n-butyl acetate, cm .. 0.125 .. 0.125 .. 0.124 .. 0.125 .. 0.123 .. 0.118 .. 0.118 .. 0.120 . . 0.122 .. 0-118 .. 0.1 18 . . 0.116 .. 0-106 .. 0.109 .. 0.111 TABLE I11 RELATION OF CONCENTRATION TO Ad FOR SOLUTIONS OF %METHYL-, 6-CHLORO-%METHYL- AND 4 : 6-DICHLORO-2-METHYL-PHENOXYACETIC ACIDS Ad for 2 -methyl- Concentration phenoxy- of acid, acetic acid, % w/v cm 0-5 0.1 25 1.0 0.252 1.5 0.375 2.0 - Ad for 6-chloro-2- methylphenoxy- acetic acid, cm 0.122 0-242 0.362 0.484 Ad for 4 : 6-dichloro-2- methylphenoxy- acetic acid, cm 0.116 0.230 0.348 0.468 It was apparent therefore that for calibration purposes the nature of the impurity used was not critical.PREPARATION OF PURE 4-CHLORO-2-METHYLPHENOXYACETIC ACID- Pure 4-chloro-2-methylphenoxyacetic acid, m.p. 120.15" to 120.2" C, was prepared by the method described by SjOberg3 This method gave a consistently pure product, but was somewhat time-consuming and the possibility of preparing pure 4-chloro-2-methyl- phenoxyacetic acid from commercial MCPA containing about 90 per cent. of this acid was investigated. Although no completely satisfactory method was found, the following route usually led to material of 99.7 to 99.9 per cent.purity, m.p. 120-0" to 120.1" C, which was suitable for use as a secondary standard. About 1 kg of commercial MCPA was dissolved in about 1-5 litres of cold industrial methylated spirit. An equal volume of water was then added slowly and with stirring to precipitate most of the dissolved acids. The precipitate was removed by filtration, dried a t 105" C and crystallised successively from acetone, benzene and chloroform. The yield on starting material was about 30 per cent. DEVELOPMENT OF METHOD- It was proposed that 2.5 g of sample mixed with 2.0 g of pure 4-chloro-2-methylphenoxy- acetic acid should be extracted at 15" C with 20 ml of rt-butyl acetate and the resulting solution compared in a differential refractometer with a saturated solution of 4-chIoro-2- methylphenoxyacetic acid prepared under the same conditions.By this procedure it was considered that 0.2 per cent. of impurity should be determinable, since this would give a326 HILL: THE DETERMINATION OF [Vol. 81 Ad value of about 0.004 cm (see Table 11), which could be measured with a reasonable degree of accuracy with the instrument available. Very satisfactory results were obtained by the above procedure for artificial mixtures containing 80 to 100 per cent. of 4-chloro-2-methylphenoxyacetic acid and also for a few commercial MCPA samples. The majority of commercial samples could not be analysed, however, because of the presence in them of coloured impurities that absorbed a considerable amount of the light entering the refractometer cell and made observation of the slit image in the microscope eyepiece extremely difficult if not impossible.It was found that this difficulty could be (overcome by reducing the weight of sample taken to 1 g and increasing the weight of pure 4-chloro-2-methylphenoxyacetic acid to 3.5 g. This involved the use of large quantities of the 4-chloro acid, which was difficult to prepare in a pure state, and so the possibility of extracting the sample with n-butyl acetate mixed with some solvent of about the same boiling point in which the phenoxyacetic acids are more or less insoluble was investigated. It was found that 20ml of a (60+40 v/v) mixture of n-butyl acetate and "isooctane" (2 : 2 : 4-trimethylpentane), in which the approximate solubilities of the 4-chloro-2-methyl-, 4 : 6-dichloro-2-methyl and 2-methyl-phenoxyacetic acids at 15" C were 10.0, 1.2 and 1-0 per cent.w/w, respectively, would conveniently extract 1.0 g of sample mixed with about 1.5 g of pure 4-chloro-2-methylphenoxyacetic acid. With this system up to 19 per cent. out of a total impurity content of 20 per cent. could be tolerated for 4 : 6-dichloro-2-methyl- phenoxyacetic acid and up to 16 per cent. for the 2-methyl acid. METHOD APPARATUS- refractive index of stainless steel or some other material unaffected by phenoxyacetic acids. and able to maintain a desired temperature to within & 0.05" C. carrying stirrers. REAGEXTS- pentane). impurities. Diflerential re fractometer-A non-recording instrument, with which differences in The refractometer cell should be of A thermostatically controlled water bath capable of operating below room temperature Flat-bottomedjasks of 50 ml capacity fitted with B19 sockets and equipped with stoppers units or less can be detected.' Solvent-A (60 + 40 v/v) mixture of n-butyl acetate and "isooctane" (2 : 2 : 4-trimethyl- Commercial grade solvents may be u.sed provided that they are free from coloured 4-Chloro-2-methyl~henoxyacetic acid, pure, in.$.120.1" to 120.2" C. 6-Chloro-2-methylj1henoxyacetic acid, pure, me. 109" to 110" C. 4 : 6-Dichloro-2-methyl~henoxyacet~c acid, puae, m.9. 187" to 188" C. 2-,$fethylphenoxyacetic acid, pure, m.9. 154" to 155" C. Sulphuric acid, 10 per cent.v/v. Sodizcm bicarbonate-A half-saturated aquleous solution. Sodium sulphate, anhydrous. Sodium hydroxide, 0.1 N. Ether-Analytical-reagent grade. Chloroform-Analytical-reagent grade or the B.P. grade. Phenol~hthalein-A 0.2 per cent. solution in 50 per cent. v/v aqueous ethanol. PROCEDURE FOR COMMERCIAL MCPA- The procedure described below refers to the commercial acid as distinct from formulations containing MCPA. However, it can be adapted quite readily to formulations such as aqueous solutions of amine and alkali-metal salts of MCPA and, after preliminary hydrolysis, to esters also. Commercial MCPA, which is sold in granular or flake form, is likely to contain traces of chlorocresols and sodium chloride together with up to 10 per cent. of water, all of which interfere in the differential refractometric examination and must be removed beforehand.This means that a determination of the total chloromethylphenoxyacetic acid content of the sample is required in order to evaluate the amount of 4-chloro-2-methylphenoxyacetic acid originally present.June, 19561 4-CHLORO-2-METHYLPHENOXYACETIC ACID I N MCPA 327 DETERMINATION OF TOTAL MCPA CONTENT OF SAMPLE- Weigh out accurately sufficient sample to contain 0.5 to 1.0 g of the mixed chloromethyl- phenoxyacetic acids and dissolve it in 50 ml of chloroform in a separating funnel. Extract the chloroform solution with one 25-ml and two 10-ml portions of half-saturated sodium bicarbonate solution and combine the extracts. Acidify them carefully with dilute sulphuric acid and extract the liberated acids, free from chlorocresols, with three portions of chloroform (25, 20 and 15ml).Combine the chloroform extracts and wash the bulk with 10ml of distilled water. Transfer the chloroform layer to a 250-ml conical flask, extract the aqueous layer with 5ml of chloroform and run the latter into the flask. Boil off almost all the chloroform by heating on a steam-bath, dissolve the residue in 20ml of neutral ethanol and titrate with 0.1 AT sodium hydroxide, using phenolphthalein as indicator. Determine the equivalent weight of the extracted acids by titrating about 0.5 g of the specimen obtained for differential refractometric examination (see below) with 0.1 N sodium hydroxide as described above. Calculate the percentage of chloromethylphenoxyacetic acids from the weight of sample taken, the titre obtained and the equivalent weight.EXTRACTION OF A SUITABLE SPECIMEK OF MCPA FOR DIFFERENTIAL REFRACTOMETRIC Take about 5 g of the sample and dissolve it in 100 ml of ether in a separating funnel. Extract the ether layer with three 50-ml portions of half-saturated sodium bicarbonate solution. Acidify the combined bicarbonate extracts carefully with sulphuric acid (beware of frothing) and extract the liberated acids three times with 50 ml of ether. Dry the com- bined ether extracts over anhydrous sodium sulphate, transfer the dried ether solution to a 800-ml beaker and carefully evaporate to dryness. When dry, break up the solid into small pieces, transfer it to a smaller vessel and dry it at 80" to 100" C for + hour.Grind the product finely and store it in a stoppered bottle until required. DIFFERENTIAL REFRACTOMETRIC EXAMINATION OF THE ISOLATED SPECIMEN- Weigh out 1.Og of the extracted MCPA together with 1.3 to 1.5g of pure 4-chloro- 2-methylphenoxyacetic acid into a 50-ml flat-bottomed flask and 3-2 g of pure 4-chloro- 2-methylphenoxyacetic acid into a second similar flask. Add 20.0 ml of fi-butyl acetate - "isooctane" solvent to the first flask and 30-Om1 to the second and insert stoppers and stirrers. Support the flasks in a water bath at 15" C or any other convenient temperature between 15" C and 2" C below room temperature and stir the mixture mechanically for 2 to 3 hours. Allow the contents of the flasks to settle, withdraw the supernatant solutions through cotton-wool filters into suitable pipettes and transfer to clean dry stoppered 2-oz bottles.Ten minutes before the instrument is required switch on the mercury lamp of the differential refractometer and fill the tank surrounding the cell with water at room temperature. Clean the refractometer cell by washing it with acetone, dry it by means of a current of air and place it securely in position in the water bath. With a clean dry pipette fill each of the cell compartments with the solution of pure standard and replace the caps on the outlet tubes of the compartments, leaving the cap of the centre one quite loose. Switch on the water-bath stirrer and allow 10 minutes for the cell to reach the temperature of the water bath. Adjust the variable slit in the instrument to a convenient width and align the travelling microscope cross-wires with the image of the slit, making a fine adjustment of the focus if necessary; it is imperative that the optical distance from eye-piece to refractometer cell should not vary by more than 2 1 cm from the distance employed for the preparation of the calibration curve, Read the vernier scale to the nearest 0.001 cm to obtain the zero reading.Without removing the refractometer cell from the water bath, unscrew the cap of the centre compartment and carefully withdraw its contents with a pipette. Wash out the centre compartment with three successive small portions of the sample solution, fill with sample solution and replace the cap. Allow 10 minutes for the cell and its contents to reach temperature equilibrium with the stirred water in the bath, re-align slit image and microscope cross-wires, without making focus adjustment, and read the vernier scale to obtain the sample reading.EXAM1 NATIOK- Set the solutions aside for 1 hour to attain room temperature.328 HILL : THE DETERMINATION OF [Vol. 81 Calculate the difference between the sample and zero vernier readings to obtain Ad and read off the 4-chloro-2-methylphenoxyacetic acid content of the extracted MCPA from a calibration graph prepared as described below. From the value so obtained and the total chloromethylphenoxyacetic acid content of the sample determined above, calculate the percentage of 4-chloro-2-methylphenoxyacetic acid in the sample. PREPARATION OF CALIBRATION GRAPH- Repeat the above determination of Ad for artificial mixtures containing 4.0, 8.0, 12-0, 16.0 and 20.0 per cent.of impurity, using a mixture of equal parts of 6-chloro-%methyl- phenoxyacetic acid and 4 : 6-dichloro-2-methylplienoxyacetic acid as the impurity. Prepare the calibration graph by plotting Ad against percentage of 4-chloro-2-methylphenoxyacetic acid. This graph is suitable for the analysis of MCPA samples having equivalent weights between 200 and 205. For samples with equivalent weights between 195 and 200 a fresh series of artificial mixtures in which a mixture of equal parts of 6-chloro-2-methyl- and 2-methyl-phenoxyacetic acids are used as impurity should be examined. RESULTS AND DISCUSSION A series of artificial mixtures whose compositions were unknown to the analyst was examined, with the results shown in Table IV.TABLE IV ANALYSIS OF ARTIFICIAL MIXTURES CONTAINING 80 TO 100 PER CENT. OF 4-CHLORO-2-METHYLPHENOXYACETIC ACID Composition of substituted phenoxyacetic acid mixture 4 : 6-Dichloro- 2-methyl, % 5-0 8.3 10.1 16.0 5.1 7.5 7.0 9.7 0.0 2.5 6-Chloro- 2-methyl, % 5.0 4-5 2.0 4.0 0.0 2.5 5.1 0.8 10.0 0.0 2-Methyl, 0.0 Yo 2.1 0.5 0.0 0.0 0.0 4.2 1.4 0.7 0.0 4-Chloro- 2-me thyl, 90.0 85-1 87-4 80.0 94.9 90.0 83.7 88.1 89.3 97.5 % 4-Chloro- S-methylphenoxy- acetic acid found, 89-7 86.2 87.7 80.2 94.9 90.1 83.9 88-0 89.1 97.5 7 % All these results were within 0.3 per cent. of the actual values and this order of accuracy was considered to be satisfactory in view of the large impurity range covered, The precision or repeatability of the methold was assessed from the results of duplicate analyses of 43 MCPA samples carried out in the Research Department by experienced operators.The results were used to calculate the standard deviation, S2, of a single deter- mination by means of the following expression-- Z(x, - z1)2 + Z(x2 - g2)2 + . . . + X ( X a - zn)2 s2 = ( N , - 1) + (N2 - 1) + - + ( N n - 1) ’ Zl, z2 . . . ZB are the mean values for samples, 1, 2 . . . n, xl, x2 . . . x, are the values for individual determinations in samples 1,2 . . . n, and N,, N , . . . Na are the number of determinations made for samples 1, 2 . . . n. From this value the 95 per cent. confidence limits, i.e., the range in which 19 out of 20 results of single determinations would be expected to lie, were calculated and found to be 0.7 per cent.A further series of results obtained under routine conditions by operators with limited experience of the method is given in Table V. The 95 per cent. confidence limits for these results were 0-8 per cent. It was obvious that the results were much more precise than those obtained by any other published method, but there was some doubt about the effect on the accuracy of the method of coloured tarry materials, 1 to 2 per cent. often being present in MCPA.June, 19561 4-CHLORO-2-METHYLPHENOXYACETIC ACID I N MCPA 329 To check the effect of this tarry material, which was obtained as a residue by vacuum- sublimation of a sample of MCPA, an artificial sample was prepared containing 94.0 per cent. of 4-chloro-2-methylphenoxyacetic acid with a mixture of equal parts of the tar, 6-chloro- 2-methyl- and 4 : 6-dichloro-2-methyl-phenoxyacetic acids as impurity, and this was analysed five times.The average value obtained for the 4-chloro-2-methylphenoxyacetic acid content of the sample was 93.8 per cent., which was not significantly lower than the actual value. I t was considered therefore that any systematic error resulting from the presence of tarry impurities would be small compared with the random errors and the effect could be neglected. TABLE V REPLICATE ANALYSES OF EXTRACTED ACIDS FROM SOME COMMERCIAL MCPA SAMPLES PERFORMED UNDER ROUTINE CONDITIONS Individual values for 4-chloro-2-methylphenoxyacetic acid, % 81-9 81.3 82-2 82-2 82.6 83.0 87.3 87.1 86-7 81-9 82.2 82.6 85.2 85.5 84-9 85.3 85.7 85-0 86.5 86-0 86.3 82.7 82.8 82.8 88-1 88.4 - 81.2 81.3 - 89.0 88.9 - 89.2 89.5 - CONCLUSIONS The method described provides a precise and accurate method for the routine deter- mination of 4-chloro-2-methylphenoxyacetic acid in MCPA formulations, the extracted acids from which contain more than 80 per cent. of the 4-chloro-2-methyl acid. With suitable adjustment of the weight of extracted chloromethylphenoxyacetic acids taken and the composition of the solvent, it should be possible to apply the method to MCPA containing 60 to 80 per cent. of 4-chloro-2-methylphenoxyacetic acid. The total time required for the analysis of a single sample is 3 to 4 hours, but by examining several samples concurrently it is possible for one operator to carry out up to six determinations in a day. REFERENCES 1. 2. 3. 4. 5. 6. 7. GENERAL CHEMICALS DIVISION Sorensen, P., Actu Chem. Scand., 1961, 5, 630. - , Anal. Chem., 1954, 26, 1581. Sjoberg, R., Actu Chem. Scand., 1950, 4, 798. Grabe, E., Ibid., 1950, 4, 806. Hill, R., Analyst, 1952, 77, 67. Freeman, F., and Gardner, K., Ibid., 1953, 78, 205. Hill, R., and Jones, A. G., Ibid., 1955, 80, 339. IMPERIAL CHEMICAL INDUSTRIES LIMITED WIDNES, LANCS. Average, % 81.8 82.6 87.0 82.2 85.2 85.3 86.3 82-8 88.3 81.3 89.0 89-4 November 30th, 1955

ISSN:0003-2654    

DOI:10.1039/AN9568100323

出版商:RSC    

年代:1956

数据来源: RSC

摘要:

330 TOMPSETT THE DETERMINATION AND DISTRIBUTION [Vol. 81 The Determination and Distribution of Lead in Human Tissues and Excreta BY S. L. TOMPSETT A method was described in 1935 for the determination of lead in biological materials. Organic matter was destroyeid by ignition, and lead was separated as the diethyldithiocarbamate in ether and finally measured colorimetrically with dithizone. This paper includes descriptions of subsequent modifications and particularly the use of the reversion procedure in the final colorimetric determination. Results are given to illustrate the distribution of lead in human tissues and excreta for normal subjects and in cases of plumbism and also the effect of medication and such like in the latter condition. THIS paper is a survey of work carried out in the Biochemical Laboratory, Royal Infirmary, Glasgow, during the period 1933 to 1946, and so references are mainly restricted to work published from this laboratory.During this period, patients suspected to be suffering from lead poisoning were frequently admitted to hospital for diagnosis and treatment. Plumbism was mainly the result of industrial exposure, but in a few cases contaminated water supplies were suspected. The investigations were made for two purposes: (a) to devise a satisfactory procedure for the determination of lead in a wide range of biological materials, e.g., urine, faeces, soft tissues, bone, blood and so on, and (b) to determine satisfactory clinical methods for the assessment of plumbism and its control during treatment. THE DETERMINATION OF LEAD The method published in 1939 has in general been modified but slightly and this mainly in manipulative details.Large samples of materials were taken for analysis, since: (a) the ranges of concentrations of lead to be determined in many of these materials were uncertain, (b) to ensure that the blank represented only a small fraction of the lead to be finally deter- mined, and (c) to ensure a more accurate average value, as sampling in many instances was difficult. The procedure readily fits into the routine work of a hospital biochemical laboratory and is applicable to the biological materials, many differing greatly in composition, requiring analysis in the examination of plumbism. At certain stages, analysis may be discontinued without detriment to the final result.The procedure can be resolved into three stages, which are treated in the three sections that follow. DESTRUCTION OF ORGANIC MATTER- fume cupboard. phate was added before ashing. lead. the ashing process, could be determined quantitatively. SEPARATION AND CONCENTRATION OF THE LEAD- Before colorimetric determination of the lead, it is necessary to effect its separation. Separation as the sulphide was the usual procedure until Allport and Skrimshire2 and Lynch, Slater and Osle13 suggested the extraction of lead as the dithizonate with chloroform. I encountered difficulties with this method when it was applied to solutions of the ash of certain materials. This appeared to be due in pa.rt to the high iron content of these materials and the ease with which dithizone is oxidised to a yellow inactive compound.This aspect of the problem was not investigated further, since an alternative procedure presented itself. Sodium diethyldithiocarbamate had recently been introduced by Callan and Henderson4 for the colorimetric determination of copper. I t was found that this substance, relatively Organic matter was destroyed by ignition in a silica dish over a bunsen burner in a With materials of low ash content, e.g., blood and soft tissues, sodium phos- Sodium phosphate solutions can be readily freed from Lead added to any of these materials and subjected to the complete procedure, including No addition was made to urine, faeces or bone.June, 19561 OF LEAD I N HUMAN TISSUES AND EXCRETA 331 more stable than dithizone, formed a compound with lead that was easily soluble in ether, the extracts being colourless. Extractions were carried out at pH 7-5 to 8.0 in the presence of excess of citrate to prevent the co-extraction of iron and the precipitation of the phosphates of the alkaline earths.Under these conditions, copper is also extracted, and so the extracts are coloured yellow. Later,5 potassium cyanide was added before the formation of the heav y-met a1 diet hyldithiocarbamat es. Under these conditions, copper diet hyldit hiocar- bamate is not formed, and hence ether extracts should be colourless. This is a useful index to indicate that traces of iron are not being co-extracted. Traces of iron tend to interfere with the colorimetric determination of lead with dithizone.“Accidental” co-extraction of iron is indicated by the eth& extract assuming a dirty brown colour. In such rare cases, ether extracts must be collected, the ether removed by evapora- tion and organic matter destroyed. Extraction as lead diethyldithiocarbamate should then be repeated. Since there was little information available about the stability of sodium diethyldithio- carbamate or its heavy-metal compounds, the reagent was added after the first ether addition, and extraction and separation was carried out without delay. Extractions and separations can be made either in a separating funnel or in a glass- stoppered cylinder with the aid of a pipette. I prefer use of the latter apparatus, since contamination of the ether extract with the aqueous phase is reduced.After evaporation of the ether, organic matter in the residue was destroyed by heating it with sulphuric and perchloric acids. This acid mixture has been replaced by 100-volume hydrogen peroxide, since the use of perchloric acid in this procedure has resulted in an accident involving the loss of an eye. A modification was later introduced6 for use with materials such as bone and faeces, which contain high concentrations of the alkaline-earth phosphates. With these materials difficulties may arise because of the precipitation of insoluble phosphates in alkaline solutions, even in the presence of excess of citrate. A preliminary extraction of lead as the diethyl- dithiocarbamate with ether from an acid solution (0.5 N hydrochloric acid) was made.Ether extracts were separated, the ether was removed by evaporation and organic matter was then destroyed. Lead was then re-extracted as the diethyldithiocarbamate with ether from an alkaline solution in the presence of citrate and cyanide. Many other metals are extracted with ether as diethyldithiocarbamate when the procedure is carried out in an acid solution. This may be used as a useful index with respect to complete extraction. It was impossible to calculate whether excess of sodium diethyldithiocarbamate had been added to cope with all the reactive metals present. Additional reagent was therefore added after each ether addition, the colour of the extract being used as an index. This procedure was examined by measuring the recovery of lead added to faeces or milk.COLORIMETRIC DETERMINATION OF THE LEAD- The extracts are coloured a dirty brown owing to the predominance of iron. It had been the usual practice to determine lead in biological materials colorimetrically by the sulphide reaction. Fischer and Leopoldi’ suggested that microgram quantities of lead could be determined colorimetrically with dithizone. Anderson and the author1 success- fully applied this reaction to the colorimetric determination of lead in extracts obtained from biological materials. The colour was developed in carbon tetrachloride by using a slight excess of dithizone. The aqueous solution contained ammonium acetate and cyanide and was alkaline with ammonia. Excess of dithizone was removed by extraction of the organic phase with 1 per cent.potassium cyanide solution. The intensity of the pink colour was read against a comparable standard in a visual colorimeter. Although lacking in precision, the visual colorimeter has certain advantages over photo-electric instruments. “Off” colours caused by oxidation products of dithizone could readily be detected. Low concentrations were, however, difficult to measure. Blanks were determined by adding a known amount of lead and calculating by difference after reading against a standard. Unless precautions are taken, difficulties may be encountered when lead is determined colorimetrically with dithizone. Commercial dithizone contains an inactive yellow oxidation product, which may jnterfere. I use a freshly prepared aqueous ammoniacal extract of a carbon tetrachloride solution of commercial dithizone.Such an extract is free from the undesirable oxidation product. Precautions must Dithizone is readily oxidised to the yellow oxidation product.332 TOMPSETT : THE DETERMINATION AND DISTRIBUTION [Vol. 81 therefore be taken to prevent its formation during the colorimetric determination of lead with dithizone. The following precautions appear to be necessary- (i) iron salts must be absentthis can be ensured by suitable preliminary extraction procedures ; (ii) absence of oxidants-this can be ensured by the addition of a reducing agent, e.g., sulphurous acid; and (iii) the reaction should not be carried out in bright sunlight. It is believed that in the presence of bright sunlight the ultra-violet component liberates free chlorine produced by the decomposition of carbon tetrachloride.Before colorimetric analysis, clarification of the extracts is necessary. Clearing by centrifuging appears to be the safest procedure, Filtration through paper appears to have its difficulties. Unwashed paper contributes t.races of reactive metals, whereas the acid retained in washed papers tends to produce some reversion if the solution contains the pink lead dithizonate. Irving, Risdon and Andrew8 have introduced the principle of reversion into the use of dithizone in the measurement of micrograni quantities of metals. Irving and Butler9 have recently described a method for the determination of lead in biological materials in which this principle is incorporated. I now use a photo-electric instrument (Unicam SP350 spectrophotometer) for the assessment of the lead - dithizone reaction. Assessment is made by reversion, which is carried out with 0.1 N sulphuric acid.Absorptions are read both before and after reversion at 525mp (maximum for lead dithizonate) and 620mp (maximum for free dithizone). It has been found that the difference of absorption recorded before and after reversion followed Beer’s law within the range 0 to 20 pg of lead (10 ml of carbon tetrachloride). Much trouble can be avoided by completing the procedure with the minimum of delay. BISMUTH- Bismuth, if present, is extracted together with lead and reacts with dithizone to produce an orange-coloured solution in carbon tetrachloride. Its presence is easily detectable during the development of the dithizone reaction and is recognisable as an “off” colour by visual colorimetry.In carbon tetrachloride solution it may be removed by repeated extraction with aqueous 1 per cent. potassium cyanide solution. Lead dithizonate is more stable, but is partly removed by such treatment and as a result a comparable standard should be subjected to the same number of extractions. This property, however, is inapplicable to certain methods now in general use. Methodslo? have been published in which the presence of bismuth in the final extract is eliminated. I have described12 a method for the determination of bismuth in biological materials. Bismuth was concentrated by the sodium diethyldithiocarbamate - ether technique in the same manner as lead. It was then determined colorimetrically with thiourea.The colori- metric procedure is simple to carry out and although of low sensitivity is relatively specific. The use of the concentration procedure does peimit the determination of low concentrations of bismuth in biological materials. The procedure was devised in order to study the dis- tribution of bismuth in human tissues and excreta. It may be a component of so called “stomach powders” or given as an injection in the form of the colloidal metal or oxychloride. In the former, absorption from the alimentary tract is minute, but faeces would be heavily contaminated. In such a case the obvious procedure is to collect the sample when such therapy has been discontinued and the alimentary tract cleared of bismuth compounds. In the latter, this form of therapy is restricted to certain types of diseases.I have examined a wide range of tissues and excreta for the presence of bismuth. Bismuth was detected and measured in the urine and faeces of a young woman who had a history of treatment by injection with colloidal metallic bismuth. On the other hand, in many controls and cases suspected of plumbism, bismuth was not detectable in excreta or tissues. It does appear, therefore, that tests to show that bismuth is absent are sufficient in most analyses. (a) colorimetric determination (Unicam SP350 spectrophotometer)+orrelation between the reversion readings taken at 525 m u and 620 mp, and Bismuth is used therapeutically. I now make use of the two following criteria-June, 19561 OF LEAD IN HUMAN TISSUES AND EXCRETA 333 (b) an aliquot of the final lead extract is acidified to a concentration of 10 per cent.with sulphuric acid and 10 per cent. thiourea solution is added. Presence of bismuth is indicated by the formation of a yellow colour. In the examinations of lead in foodstuffs, Lockwood13 has also suggested the use of criterion (b). In the event of bismuth being found, an estimate would be made by the thiourea reaction and its exact significance in the dithizone reaction determined. I consider that if bismuth is present in human tissues and excreta under examination, its detection and determination should be made, since its presence could be of significance. METHOD REAGENTS- Sodium Phos9hate solutim, lead- free-A 10 per cent. w/v solution of Na2HP0,.12H20 in water.Before use, add a small quantity of sodium diethyldithiocarbamate (sufficient to cover the point of a knife blade) to about 150ml of the phosphate solution and then extract it once with 50ml of ether. Sodium citrate solution, lead-free-A 20 per cent. w/v solution in water. Store the solution over a 0.1 per cent. solution of dithizone in chloroform. Shake the solution and filter it before use. Sodium diethyldithiocarbamate solution-A 2 per cent. w/v solution in water, prepared freshly before use. Potassium cyanide, 1 eer cent. solution-Prepared freshly before use. Potassium cyanide, 10 eer cent. solution-Prepared freshly before use. Standard lead solution, 1 ml = 1 mg of lead-Dissolve 0.1831 g of lead acetate, Pb(C,H,02),.3H,0, in distilled water containing 5 ml of glacial acetic acid and dilute to 1 litre with water.Ammoniacal ammonium acetate solution-Add 1 ml of concentrated sulphuric acid, 1 ml of glacial acetic acid and 5ml of ammonia solution, sp.gr. 0.880, to water and dilute to 25ml. Dithizone reagent-Shake 5 ml of 0.1 per cent. solution of dithizone in carbon tetrachloride with 10 ml of dilute ammonia solution (0.5 ml of ammonia solution, sp.gr. 0.880, diluted to 100ml with water). Spin the mixture in a centrifuge and use the supernatant liquid. Sd~hurous acid, 5 $er cent. wlv. Hydrogen peroxide, 100-volume. Distilled water-Prepared in an all-glass still. The following reagents should all be of recognised analytical grade- Nitric acid, concelztrated. Sulphuric acid, concentrated. Hydrochloric acid, concerttrated.Acetic acid, .glacial. Ammonia solution, sp.gr. 0.880. Carbon tetrachloride. Glassware should be Pyrex whenever possible. Silica dishes (4i inches in diameter) should be cleaned with hot dilute hydrochloric acid before use. Filter-paper should be washed with dilute hydrochloric acid and then with water. PROCEDURE FOR DESTROYING ORGANIC MATTER AND EXTRACTING LEAD- Urine-Evaporate 250 ml of urine in a silica dish to dryness in a hot-air oven (100" C) or on a steam-bath. Destroy organic matter by heating the dish over a bunsen burner in a fume cupboard. Final traces of carbon may be removed by allowing the dish to cool, moistening the ash with nitric acid and re-heating. Dissolve the ash in 75ml of water containing 5 ml of concentrated hydrochloric acid, using heat to assist dissolution.Filter the solution into a 250-ml glass-stoppered measuring cylinder, using a further 25 ml of water for washing. Add 50 ml of 20 per cent. sodium citrate solution and adjust the pH to between 7-5 and 8.0 by adding ammonia solution. After cooling the solution, add 5 ml of 10 per cent. potassium cyanide. Extract the mixture three times with 50-ml quantities of ether, Prepare this solution freshly before use. APPARATUS-334 TOMPSETT THE DETERMINATION AND DISTRIBUTION [Vol. 81 adding 5 ml of 2 per cent sodium diethyldithiocar‘bamate solution after the first ether addition. Extraction involves shaking for 2 minutes. Remove the ether extract without delay with a pipette. Wash the combined ether extracts once with 10 ml of water and then evaporate to dryness in a round-bottomed digestion flask, Destroy organic matter by heating with 1 ml of concentrated sulphuric acid and 1 ml of 100-volume hydrogen peroxide.Sojt tissues (liver, brain, kidney, etc.)-Cut the tissue into small pieces with stainless-steel scissors and homogenise a 100-g portion with 1.00ml of water. Transfer the emulsion to a silica dish containing 100ml of lead-free 10 per cent. sodium phosphate solution. The procedure is then exactly as described for urine. Blood-Collect 20 ml of blood in an all-glass syringe fitted with a stainless-steel needle, and place it in a Pyrex-glass tube fitted with a glass stopper. Use of anti-coagulants should be avoided, since there is risk of contamination. Transfer the blood to a silica dish containing 100ml of lead-free 10 per cent.sodium phosphate solution. The procedure is then as described above, except that in the final digestion only 0.4ml of concentrated sulphuric acid is used. Bone-The concentration of lead depends on the type of bone and also the location from which the sample is selected. Very few laboratories have the facilities to reduce bone to a sufficiently fine state to effect accurate sampling of small quantities. I select samples as follows- Femur or tibia, a 20-g cross-section from the centre of the shaft. Rib, a “length” weighing approximately 20 g. Vertebra, a cross-section weighing approximately 20 g. Bone, selected as above and weighing approximately 20 g, should be ashed in a silica dish. With the aid of heat, dissolve the ash in sufficient N hydrochloric acid to effect solution.Dilute the solution to a convenient volume with water. Transfer 25 ml of the acid solution to a 100-ml glass-stoppered cylinder and dilute to 50x11 with water. Extract the solution three times with 25-ml quantities of ether, adding 10ml of 2 per cent. sodium diethyldithiocarbaniate solution after each ether addition. At each extraction shake the mixture for 2 minutes and separate the organic phase by means of a pipette without delay. Collect the ether extracts in a Pyrex-glass round-bottomed flask. Remove the ether by evaporation and destroy the organic matter in the residue by heating it with 1 ml of concentrated sulphuric acid and 1 ml of 100-volume hydrogen peroxide. The colour of the ether extracts may be used as an index to show that sufficient sodium .diethyldithiocarbamate has been added to combine effectively with all the reactive metals present. Heat the residue with 1 ml of concentrated hydrochloric acid and about 50 ml of water to effect solution. Repeat the diethyldithiocarbamate - ether procedure, but’ exactly as described for urine, i.e., in the presence of citrate and cyanide at pH 7-5 to 8-0. Faeces-The preliminary treatment depends upon the form of the final result. A. If the final result is to be expressed in terms of mg of lead per 100 g of dried faeces, then dry the sample on a steam-bath, grind it with pestle and mortar and ash a 10-g sample in a silica dish. If the average excretion over a period of days is to be determined, then homo- genise the complete sample with water to a convenient volume.Put 200ml of the sample in a silica dish, heat it to dryness and ash it. Then proceed as described for bone. Diets-It may be necessary to establish the ldaily intake of lead. Analysis of individual foodstuffs is not necessary. A corresponding diet received in the laboratory should be homo- genised with water to a convenient volume. The final volume depends upon the nature of the diet, but varies between 1 and 3 litres for- a 1-day diet. A suitable aliquot is then taken and examined as described under urine. PROCEDURE FOR COLORIMETRIC DETERMINATION- With the exception of blood, dilute the digests with water and add the following reagents in order : 1 ml of glacial acetic acid, 5 ml of ammonia solution, sp.gr.0.880, and water to 25 ml. Dilute the digest from blood to a volume of 10 ml containing 0.4ml of glacial acetic acid and 2 ml of ammonia solution, sp.gr. 04380. Colorimetric analysis should now be completed as quickly a s possible. B.June, 19561 OF LEAD I N HUMAN TISSUES AND EXCRETA 335 Standards-Dilute solutions containing 5, 10 and 20 pg of lead (as acetate) to 10 ml with ammoniacal acetate solution. Then add the following reagents in order: 6 drops of 5 per cent. w/v sulphurous acid, 5ml of 1 per cent. potassium cyanide solution and 10ml of carbon tetrachloride, Add dithizone reagent drop by drop Gith constant shaking until excess is present, as evidenced by the brownish colour of the aqueous phase. After shaking the mixture for 1 minute, spin it in a centrifuge.Read the absorption of the carbon tetrachloride extract against carbon tetrachloride at 525 mp and 620 mp, using a Unicam SP350 spectrophotometer with 10-mm cells. Then shake the carbon tetrachloride extract with 5 ml of 0.1 N sulphuric acid. Spin the extracts in a centrifuge and again take readings at 525mp and 620mp. The difference between the readings taken before and after reversion are indicative of the quantity of lead present. Carry out a blank at the same time, using 10ml of ammoniacal acetate reagent. Ufiknowfi-Dilute a quantity of digest containing up to 20pg of lead to 10ml with Sometimes this may necessitate a preliminary ammoniacal ammonium acetate solution. trial. BLANK DETERMINATION- Then proceed as described for the standards.A complete blank should be carried out on all procedures. TABLE I LEAD CONTENT OF “NORMAL” HUMAN TISSUES Lead content, mg per kg of fresh tissue Soft tissues- Liver . . .. .. 0.9 to 4-6 Kidney . . .. .. 0.7 to 3.7 Brain . . .. .. 0.2 to 0.7 Rib . . .. .. 5.0 to 12.9 Vertebra .. .. 2-6 to 14.7 Femur .. .. .. 18.2 to 108 Tibia . . .. .. 15.3 to 96.5 Bones- THE DISTRIBUTION OF LEAD IN HUMAN TISSUES AND EXCRETA We are indebted to Aub, Minot, Fairhall and Reznikoff14 for much of the earlier work on the metabolism of lead. It was shown that lead was preferentially deposited in the skeleton, and it was suggested that, although skeletal lead exerts no toxic action, conditions might arise to cause the transfer to the soft tissues. Such a transfer could be accompanied by the onset of the symptoms of plumbism.A high calcium diet was shown to cause a preferential deposition of lead in the skeleton, whereas the reverse was produced by a low calcium diet, acidosis and so on. Kehoe, Thamann and C h 0 1 a k ~ ~ J ~ J ~ have contributed much to the study of the excretion of lead both by normal subjects and in cases of plumbism. INGESTION OF LEAD- Under “normal” conditions a small amount of lead enters the body, mainly via the alimentary tract (in food and drinking water). This may amount to 0.2mg or more per day..1 An approximate estimate of the intake may be made from an analysis of the faeces. In industrial plumbism, considerable quantities of lead may enter the body through the respiratory tract. EXCRETION OF LEAD- The kidney is a poor excretory organ, since plasma lead together with other “heavy metals” is largely protein bound.DISTRIBUTION OF LEAD IN “NORMAL” HUMAN TISSUES- The distribution of leadl,ls is shown in Table I. Under “normal” conditions there is a retention of lead. Such retention is so small that it cannot be detected by balance experi- ment. Lynch et aL3 showed that the con- concentration of lead in such bones as femur and tibia increased with age. I have confirmed Lead is excreted mainly via the alimentary tract (bile, intestinal tract). The high concentrations in bone will be noted.336 TOMPSETT : THE DETERMINATION AND DISTRIBUTION Fol. 81 this18 and also shown that this phenomenon was not exhibited by such bones as rib and vertebra. BLOOD- It has been noted that the concentration of lead in blood is much-higher than in urine. Accurate analysis can be made with 20ml or less of blood, quantities that can easily be obtained from a patient.Normal human blood contains on an average 50pg of lead per 100 ml of whole blood. It has been suggested by Anderson and the author1 that the deter- mination of blood lead is a more reliable index in plumbism than the determination of lead in urine. Lead exists in red blood cells and plasma, and variations in distribution have been noted,19 but at present no special significance has been attached to such change. MOBILISATION OF LEAD- It is generally agreed that only lead present in the soft tissues exerts a toxic action, that in the skeleton being inert. However, conditions may exist that may produce a transfer of lead from soft tissues to skeleton or vice vewz.A high cakium diet causes lead to be deposited in the skeleton, whereas a low calcium diet produces the reverse. The hormone of the parathyroid glands20 and overdosage with vitamin D produce a transfer of lead from the skeleton to the soft tissues. Acidosis resulting from disease or by the administration of certain salts, q., ammonium chloride, also produces a transfer of lead from the skeleton to the soft tissues. It is believed that lead laid down in the skeleton under so called normal conditions is difficult to mobilise, i.e., a transfer from skeleton to soft tissues. In contrast, large amounts of lead laid down in the skeleton under abnormal conditions may readily be mobilised. Brown21 has shown that in persons with no history of abnormal exposure to lead, mobilisa- tion of skeletal lead may occur in diseases associated with decalcification. This was shown by an increase above the normal levels of the blood lead. Reference22 has also been made to a case of lead poisoning occurring in a lead worker in association with, and probably precipitated by, subacute lymphatic leukaemia.LABORATORY INVESTIGATIONS IN PLUMBISM- Haematological examination of blood and measurement of the excretion of copropor- phyrins in urine are of considerable value in the investigation of plumbism. However, only laboratory investigations concerned with the determination of lead will be considered. The function of such is to determine whether there has been abnormal ingestion of lead.TABLE I1 EFFECT OF DIET AND TREATMENT ON THE LEVEL OF THE LEAD CONTENT OF BL0O:D I N PLUMBISM Diet and treatment Lead content of blood, pgper 100ml 1. No treatment + ordinary diet . . .. .. .. .. 350 2. High calcium diet . . .. .. .. .. .. 80 180 3. Low calcium diet + treatment with ammonium chloride B 1500 3600 4. High calcium diet . . . . . . .. .. .. {': .. 70 TABLE I11 EFFECT OF DIET AND TREATMENT IN THE EXCRETION OF LEAD IN PLUMBISM The results represent the average of 3-day periods Diet and treatment 1. High calcium diet . . . . . . Lead in urine, Lead in faeces, mg per day mg per day 2. Low calcium diet + treatment with ammonium 0.3 0.2 3. High calcium diet . . . . . . .. .. 0.9 0.9 2.0 1-8 1.5 1.2 0.6June, 19561 O F LEAD I N HUMAN TISSUES AND EXCRETA 337 TABLE IV LEAD CONTENT OF TISSUES OF PERSONS WITH A HISTORY OF ABNORMAL EXPOSURE TO LEAD Lead content, mg per kg of fresh tissue r Liver Kidney Brain Rib Vertebra Femur Tibia - - Painter age41 years .. , 4.5 1.0 1.0 119.0 19.0 Painter age 60 years . . 2.4 2.9 - 22.0 9.0 - - Metal worker age 60 years 5.4 2.0 1.4 5 1.0 13.0 51.0 79.0 Condition due to contami- nation of drinking water with lead; subject age 26 years.. .. . . 7-1 4.6 3- 1 52.0 - 52-0 53.0 A measure of urinary excretion of lead is often the only practical means of investigation of persons during abnormal exposure, e.g., in factories. An excretion of more than 1OOpg of lead per day in the urine is considered to be suggestive of abnormal exposure to lead. Frequently patients are admitted to hospital when exposure to lead has ceased for a period.In such cases the urinary excretion of lead may be within normal limits and yet there may be mobilisable lead in the skeleton. The determination of the lead content of the blood under various conditions tends to produce the most satisfactory results.~+?3~2* The lead content of the blood may be normal on admission. A low calcium diet together with acidosis, which may be induced by the oral administration of ammonium chloride, may produce an increase in the level of blood lead above the limit of normality (provisional-100 pg of lead per 100 ml of whole blood) , in those persons with mobilisable lead in the skeleton. Such levels may then be brought down to within normal limits by a high calcium diet. Such changes should not occur in those persons only subject to the so called normal hazard.Some typical results are shown in Table 11. The effect of such treatment upon the excretion of lead is shown in Table 111. It will be noted that the main changes occur in faecal excretion. Some results are shown in Table IV. The markedly increased concentration of lead in rib is noteworthy. CHELATING AGENTS- The removal of excess of lead in plumbism has been an important problem. Treat- ment with a low calcium diet and ammonium chloride or other agents to increase excretion of lead by mobilisation has been considered to be dangerous, since such could potentiate symptoms of plumbism. Alternative procedures have been sought. The use of chelating agents26 appears to offer considerable promise.The use of calcium disodium ethylene- diaminetetra-acetate (EDTA) has been investigated and most promising results have been reported. I have had the opportunity to examine the lead content of the urine in a man with a history of slight abnormal exposure to lead when subjected to such treatment. Before treatment, the lead content of the urine was 50 to 80 pg per litre, whereas during treatment with EDTA the concentration had an average value of 1.46mg per litre. It may be that such substances may not only be of value in the treatment of plumbism but also in diagnosis. EXPERIMENTS ON ANIMALS- Although much useful information has been obtained from the study of plumbism in the human, more direct evidence may be obtained from experiments on animals. Using mice as the experimental subjects,23~26~27 I studied two aspects, viz., the effect of dietary composition upon the absorption of lead from the alimentary tract; and factors influencing the distribution of lead between the soft tissues and skeleton.In the first study, the quantity of lead in the whole animal was determined after various regimes. It was found that: (a) absorption of lead was least on a high calcium diet and highest on a low calcium diet, and ( b ) the addition of dilute hydrochloric acid to the diet resulted in increased absorption of lead. This could be related to the pH of the intestinal contents, a factor known to influence the absorption of “heavy metals.” When available, useful results may be obtained from the analysis of tissues.338 TOMPSETT : THE DETERMIN.ATION AND DISTRIBUTION [Vol.81 In the second study, the distribution of lead between soft tissues and skeleton was examined. It was found that: (a) the percentage of the total lead in the skeleton was highest on a high calcium diet, and (b) in contrast to controls on a high calcium diet, increased per- centages of lead were found in the soft tissues when the animals were maintained on a low calcium diet or treated with sodium bicarbonate or potassium iodide. METHODS OF ANALYSIS- Results of analyses of urine, faeces, blood and soft tissues referred to in this paper were obtained by the procedure described in 1935,l with the differences in manipulative technique referred to in this paper. Results for the lead content of bone obtained from individuals without a history of abnormal exposure to lead were obtained either by the procedure described in 1935l or by the modification described in 1939.6 No differences were noted between the ranges obtained for the four types of bone examined by the two versions of the method. Bones obtained from individuals with a history of abnormal exposure to lead were determined by the modified procedure described in 1939.6 With the exception of urines examined during therapy with EDTA, results referred to in this paper were assessed by visual colorimetry.DISCUSSION The dithizone extraction procedure described in this paper was described 20 years ago and, since the subject is of some interest, many related papers have been published. Although many modifications have been published, the determination of lead with dithizone still appears to be the most popular of the chemical methods.A notable addition has been the introduction of the reversion principleS and its use with the photo-electric colorimet er . S~ggestionslO~f3~~S for the use of a chloroform solution of diethylammonium diethyl- dithiocarbamate in place of sodium diethyldithiocarbamate and ether have been made. Such a reagent has advantages, since it may ibe readily prepared and the solvent is non- inflammable. It also appears to have a greater versatility in the separation of a wide range of trace metals, The recent publication of Gagell is of great interest. Sodium diethyl- dithiocarbamate is used and the lead compound is extracted into isoamyl alcohol - toluene. Lead is then specifically extracted from organic solution with aqueous mineral acid, thus saving the evaporation of organic solvent.It is obvious that we still have much to learn with regard to the properties of metallo-organic compounds. There has been much discussion about the respective merits of the destruction of organic matter by ignition or wet digestion.l1y2{’ The spectrograph has not been applied on a large scale, principally because of its cost and because in many laboratories it would only be put to occasional use. The polarograph30 until recently lacked the sensitivity to deal wiith the low concentrations present in many biological materials. There is general agreement with regard to the distribution of lead in human excreta and soft tissues, although there may be local differences.This could be largely related to the lead content of the local water supply or possibly to atmospheric conditions. When such material is obtainable, a knowledge of the lead content of the various bones comprising the skeleton can give very useful information with regard to abnormal exposure to lead during life. Much more investigation, halwever, appears to be required on this subject. Although numerous analyses have been published, there appears to be: (i) inadequate data to specify the normal range for any particular type of bone and also the effect of local conditions, and (ii) inadequate data about the exact relationship between age and the lead content of such bones as femur and tibia. There is also inadequate data about the type of bone that would give the most useful infonna.tion in the study of plumbism. Since the lead content of bone is relatively high in com:parison with other tissues, it would appear that analyses could be carried out by polarography, high sensitivity not being required.The introduction of chelating compounds may alter the biochemical approach to the diagnosis and control of treatment of plumbism. REFERENCES 1. 2. 3. Tompsett, S. L., and Anderson, A. B., Biochem. J., 1935, 29, 1851. Allport, N. L., and Skrimshire, G. H., Analyst, 1932, 57, 440. Lynch, G. R., Slater, R. H., and Osler, T. G., Ibid., 1934, 59, 787.June, 19561 OF LEAD I N HUMAN TISSUES AND EXCRETA 339 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. Callan, T., and Henderson, J. A. R., Ibid., 1929, 54, 650. Tompsett, S. L., and Anderson, A. B., Lancet, 1939, i, 559. Tompsett, S. L., Biochem. J., 1939, 33, 1231. Fischer, H., and Leopoldi, G., 2. angew. Chem., 1934, 47, 90. Irving, H. M., Risdon, E. J., and Andrew, G., J . Chem. SOC., 1949, 537. Irving, H. M., and Butler, E. J., Analyst, 1953, 78, 571. Lead Panel of the Society’s Metallic Impurities in Foodstuffs Sub-committee, Ibid., 1954, 79, 397. Gage, J. C., Ibid., 1955, 80, 789. Tompsett, S. L., Ibid., 1938, 63, 250. Lockwood, H. C., Ibid., 1954, 79, 143. Aub, J. C., Minot, A. S., Fairhall, L. T., and Reznikoff, P., “Lead Poisoning,” Williams and Kehoe, R. A., Thamann, F., and Cholak, J., J . Amer. Med. Ass., 1926, 87, 2081. Wilkins Co., Baltimore, 1926. 9 > , J . Ind. Hyd., 1933, 15, 257, 273, 301, 306, 320. --- , , , J . Amer. Med. Ass., 1935, 104, 90. - _ _ - Tompsett, S. L., Biochem. J., 1936, 30, 345. Tompsett, S. L., and Anderson, A. B., Ibid., 1940, 35, 48. Hunter, D., and Aub, J. C., Quart. J . Med., 1927, 20, 123. Brown, A., Ibid., 1946, 57, 77. Brown, A., and Tompsett, S. L., Brit. Med. J . , 1945, 2, 764. Chalmers, J. N. M., and Tompsett, S. L., Lancet, 1938, i, 994. Tompsett, S. L., and Chalmers, J. N. M., Brit. J . Exp. Path., 1939, 20, 408. Anon., Lancet, 1955, i, 754. Tompsett, S. L., Biochem. J . , 1939, 33, 1237. -, Brit. J. Exp. Path., 1939, 20, 512. Strafford, N., Wyatt, P. F., and Kershaw, F. G., Analyst, 1945, 70, 232. Middleton, G., and Stuckey, R. E., Ibid., 1953, 78, 532. Ferrett, D. J., Milner, G. VC’. C., and Smales, A. A., Ibid., 1954, 79, 731. BIOCHEMICAL LABORATORY NORTHERN GENERAL HOSPITAL EDINBURGH October 12th, 1955

ISSN:0003-2654    

DOI:10.1039/AN9568100330

出版商:RSC    

年代:1956

数据来源: RSC

摘要:

June, 19561 OF LEAD I N HUMAN TISSUES AND EXCRETA 339 The Photometric Determination of Silicon in Steels BY T. R. ANDREW AND C. H. R. GENTRY An improved method, based on the molybdenum-blue reaction, is proposed for the determination of silicon in steels, a mixture of ferrous ammonium sulphate and oxalic acid being used for the reduction. By making measure- ments at either of two wavelengths, a wide range of silicon contents is covered. A modification to the Spekker absorptiometer permitting it to be used at 805 mp is described. FOR the determination of the silicon content of steels, photometric methods have, by virtue of their speed and precision, largely displaced the older gravimetric methods. In this country, the so called “molybdenum-blue” method is probably more widely used than the alternative yellow silicomolybdate method.This is exemplified by the two British Standard absorptiometric methods,l ,2 one covering the “normal silicon range” of 0.05 to 2.0 per cent. and the other the “low silicon range” of 0 to 0.05 per cent. Both of these methods depend upon the determination of the silicon as molybdenum blue, but they differ, among other details, in the choice of reagent for reducing the silicomolybdate. It is not convenient to have two distinct methods for two ranges of silicon, since this necessitates keeping two different sets of reagents and may lead to confusion and error in routine practice. It would be preferable to have a single method which, by slight modification of the conditions, would permit the precise determination of silicon at all levels.It is usual in absorptiometric analysis to cover a wide range by taking either different sample weights or different aliquots ; however, this is not easily possible in molybdenum-blue procedures, since conditions for the colour formation have to be closely controlled. The main objective of the present work was to overcome this difficulty, and this aim has, in fact, been simply achieved by measuring the colour of the molybdenum blue at two wavelengths, one for low silicon contents up to 0.13 per cent. and the other for normal silicon contents up t o 3 per cent. Three reducing agents were considered, but, for reasons to be given, a mixture of ferrous The same chemical operations are used in both ranges.340 ANDREW AND GENTRY : THE PHOTOMETRIC DETERMINATION [Vol.81 ammonium sulphate and oxalic acid was eventually chosen. The method finally developed differs in two slight, but nevertheless important, points from that proposed earlier by Gentry and Sherringt~n.~ These modifications have been necessitated by the need to determine very low silicon contents. Most of the investigational work has, in fact, been concerned with finding the optimum conditions for determining: low silicon contents. These conditions have then been shown to be equally satisfactory for the normal silicon range. EXPERIMENTAL There are so many variables in the molybdenum-blue method, as applied in steel analysis, that it is no doubt possible to develop a number of distinct methods-each capable of giving results of good accuracy.For our purposes, a method of good routine-reliability was required, suitable for simultaneous determinations on six or more samples. Although speed of analysis was necessary, even more importance attached to the need to find conditions that were not highly critical, so permitting some reasonable relaxation on the part of the routine analyst. These considerations eliminated methods requiring heating of the solution to promote the rapid formation of the silicomolybdate, since it has been found that such heating is difficult to control in routine work. Some attention wats paid to the possibility of finding conditions under which the silicomolybdate formed rapidly at room temperature. However, it was subsequently realised that this was not necessary or desirable when batches of samples were being analysed.In one experimental detail we prefer the technique of Vaughan* as against that of later w ~ r k e r s . ~ ~ ~ This concerns the use of a beaker or conical flask in place of a calibrated flask for making up the final colour solution. Experience has suggested that there is little to choose between the two techniques for speed or accuracy, but has served to emphasise the fragility of the 50-ml calibrated flask. CHOICE OF REDUCING AGENT- Three reducing agents were compared : stannous ~hloride,~,~ ferrous ammonium sul- phate3~5 and l-amino-2-naphthol-4-sulphonic acid.' Yet other reducing agents could have been considered, but, since no other reagent has met with wide acceptance in steel analysis, it was obviously helpful to take advantage of the experience gained over a number of years with the reducing agents chosen.With ferrous ammonium sulphate, early experiments were carried out by the method of Gentry and Sherringt~n.~ For the 1-amino-2-napht hol-4-sulphonic acid method, a procedure previously used in these laboratories was adopted. Weigh 0.5 g of steel into a 250-ml beaker. Add 70 ml of 5 per cent. sulphuric acid and warm until the steel dissolves. Add :LOO ml of water and 2.5 g of ammonium per- sulphate. Boil for 10 minutes. Add 20-volume hydrogen peroxide dropwise until any precipitated manganese dioxide dissolves. Boil for 3 minutes, cool and dilute to 500 ml. Place by pipette two 25-ml portions (A and B) in two dry 250-ml beakers. Add to A 10 ml of 2-5 per cent.ammonium molybdate solution, mix and set aside for 5 minutes. Add 5 ml of the reducing solution and set aside for at least 10 minutes. Add to B 10ml of 4 per cent. oxalic acid, mix and add 10 ml of 2.5 per cent. ammonium molybdate, mix and then add 5 ml of reducing solution. Read the optical densities of the solutions on the Spekker absorptiometer, using Ilford No. 608 and Calorex H503 filters with 1-cm cells. Read off differences from a calibration graph. Prepare the reducing solution by grinlding 0.5 g of l-amino-2-naphthol-4-sulphonic acid to a paste with water. Add 200 ml of water, 35 g of potassium metabisulphite and 6 g of sodium sulphite. Warm gently until solution is complete, dilute to 250 ml and store in a dark bottle. As applied to plain carbon steels, there was little to choose in the precision attainable with the three methods.In each case the calibration is linear and the sensitivity not very different. Examination of the complete visible absorption spectra of the molybdenum blue produced by the three methods showed no differences. All three methods have disadvantages. The use of ferrous ammonium sulphate and oxalic acid results in a rather high background colour, which, although corrected for by the For the stannous chloride method, the ]British Standard method1 was used. It was, briefly, as follows- Add 10 ml of 4 per cent. oxalic acid, and mix.June, 19561 OF SILICON IN STEELS 341 use of a compensating solution, is not desirable when low silicon contents have to be deter- mined. The 1-amino-2-naphthol-4-sulphonic acid reagent shows a tendency to crystallise out of the final solution, as has been mentioned by Mullin and Riley.8 Although this presents no difficulty if the optical density of the solution is read fairly rapidly, it is an annoyance and possible cause of error in batch analysis.Stannous chloride as a reagent has the minor disadvantage of being rather unstable and thus requiring fresh preparation at least daily. Of more importance is the fact that the stannous chloride methods have been limited to those steels that are soluble in dilute sulphuric acid. The latter point was the main factor for preferring the ferrous ammonium sulphate- oxalic acid method to the stannous chloride method, Both have been widely favoured by steel analysts, as shown by the co-operative studies of the Glasgow Absorptiometric Panele and the Methods of Analysis Committee of B.I.S.R.A.,5 but to us the extension in scope consequent on the ability to use a range of acid solvents was an over-riding advantage.0 4 Volume of 5 per cent. sulphuric acid, ml 0.5 0.4 o . 6 ~ Volume 0 20 1 40 60 80 of 25 per cent. nitric acid, ml 0.5 ~:~~ 0 4 0 20 40 60 80 Volume of mixed acid, ml Fig. 1. Effect of concentration of solvent acid: (a) 5 per cent. sulphuric acid; (b) 25 per cent. nitric acid; (c) 20 per cent. hydrochloric acid - 6.5 per cent. nitric acid is the same within experimental error. This maximum optical density is attainable over quite wide ranges of acid concentration, the permissible variation being 80 to 120 ml of 5 per cent. sulphuric acid, 45 to 65 ml of 25 per cent.nitric acid or 55 to 75 ml of the nitric - hydro- chloric acid mixture. The pH values of the solution were measured with a glass electrode during the stage at which the silicomolybdate was forming. The limiting values of acid concentration for maximum colour formation corresponded to a pH range of 0-72 to 0-92 for the sulphuric acid solution, 0.45 to 0-70 for the nitric acid solution and 0.45 to 0.66 for the mixed-acid solution. Little physical significance probably attaches to pH measurements in solutions as complex as those studied here, but the values are given since they are considerably lower than those generally accepted for the formation of silicomolybdate. Thus Lacroix and Labaladea quote a pH range of 1.5 to 1.7, whilst Mullin and RileyS say that the rate of forma- tion of silicomolybdate is "much reduced in solutions more acidic than pH 1.0, presumably342 ANDREW AND GENTRY : THE PHOTOMETRIC DETERMINATION [Vol.81 owing to polymerisation of the silicic acid.” :Both sets of workers were, however, dealing with the formation of silicomolybdic acid in more-or-less pure solution, and Mullin and Riley indicate that a higher acidity is necessary in the presence of ferric iron. Formation of silicomolybdate-The formation of the silicomolybdate is dependent on the time of standing as well as on the acidity. This is illustrated in Fig. 2, which shows, for Time, minutes 0.9 0.8 0.7 0.6 0 5 0, 10 20 30 c 40 50 60 Time, minutes Fig, 2. Effect of time for formation of silico- molybdate : curve A, 30 ml ; curve B, 45 ml; curve C, 55 ml; curve D, 65 ml of 25 per cent.nitric acid. All curves are for steels containing 0.08 per cent. of silicon, measurements being made on a Unicam SP500 spectrophotometer in 4-cm cells Fig. 3. Effect of time for formation of silico- :molybdate as in final method : curve A, sulphuric acid; curve B, nitric acid; curve C , mixed acid; curve D, nitric acid. Curves A, B and C are for steels containing 0.1 per cent. of silicon, measure- ments being made on a Unicam SP500 spectro- photometer in 4-cm cells; curve D is for a steel containing 2 per cent. of silicon, measurements ‘being made on a Spekker absorptiometer various amounts of nitric acid, the optical density of the molybdenum blue as a function of the time of standing before reduction.At the preferred acid concentration, the maximum optical density is almost reached in 5 minutes, but at the two proposed limiting values of acid concentration more time is needed. A period of 20 minutes’ standing is sufficient to give complete colour formation under all the proposed conditions, It should be emphasised, however, that acidity and time of standing are inter-related; if desired, a shorter standing time could have been found, but the amount of acid taken would then have become more critical. That the period of 20 minutes is satisfactory for all the acid solvents has been tested at several silicon levels; this is illustrated in Fig. 3, and is confirmed by the fact that the calibration graphs for the final method are identical for all the solvents and are linear over all the silicon ranges.An additional curve, A, is given in Fig. 2 to illustrate the effect of using less acid than the optimum amount; the amount taken gave a pH of about 1.0. Under these conditions a constant optical density was reached after 15 to 20 minutes. A successful method of analysis is, therefore, still possible outside the optimum range of acid concentration , provided that rigid control is exercised over the amount of solvent acid. Under such conditions a linear working graph is also obtained. StabiZity of the molybdenum-blue complex-Bagshawe and TrumanlO have claimed that the molybdenum-blue complex produced by using a ferrous sulphate - oxalic acid mixture shows a progressive decrease in colour on standing far 1 hour.However, their conditions were markedly different from those used in steel analysis, particularly since they did not control the oxidation potential of their system by the deliberate addition of ferric iron. Under the conditions given in this paper, the colourecl complex is very stable and has shown no change when measured at intervals up to 19 hours. This is in agreement with earlier work.3 BLANKS- The determination of small amounts of silicon in steels is complicated by the need to make blank corrections. There are the fol1ow:ing three possibilities: (a) a reagent blank caused by the presence of small amounts of silicon in the reagents used and derived from the apparatus or atmosphere, (b) a colour blank caused by the presence of coloured ions or complexes in the solution, and (c) an interfering-element blank.In addition there is theJune, 19561 OF SILICON IN STEELS 343 further complication of obtaining silicon-free iron for use in the preparation of the calibration graph. For the last purpose, in the British Standard methods1P2 ferrous sulphate has been used instead of an iron or steel, since it “has been found to give less colour development due to silicon than any form of pure iron commercially available at present.” Ferrous sulphate is also used in the British Standard methods to obtain the reagent blank in actual deter- minations. Firstly, since ferrous sulphate is used in place of iron, it is necessary to take less solvent acid; thus, a true reagent blank isnot obtained. Secondly, it is implicit in the procedure that the ferrous sulphate contains a negligible amount of silicon; if any is present, the result on a steel sample will be too low by the equivalent silicon content.In our experience, these criticisms are not serious when AnalaR reagents are used. Nevertheless, when very low silicon contents are to be determined, it is preferable to use a steel of known low silicon content to obtain the reagent blank. It is also more This is open to two criticisms. Wavelength, mp Absorption spectra of molybdenum-blue complex: curve A, test solution containing 0.06 per cent. of silicon; curve B, test solution minus compensating solution ; curve C, compensating solution Fig. 4. convenient in routine practice to use in the calibration of the method a low-silicon steel to which known additions of silicon are made; it is not necessary for this purpose to know the silicon content of the steel accurately.Contrary to the statement made in Note 6 of the British Standard,2 the reagent blank, as determined in this laboratory, is not normally exceedingly small and, in fact, correction for it is essential in the lower silicon range. When AnalaR reagents are used, the blank is equivalent to about 0.005 to 0.010 per cent. of silicon. There is some evidencedhat most of this is due to traces of silicon in the ammonium molybdate, since the lowest blank is obtained when only large clear crystals of ammonium molybdate are selected. The colour blank due to the presence of coloured ions and complexes is simply corrected for by using a compensating solution, which contains all the reagents but added in an order that does not lead to the formation of silicomolybdate. Since at both wavelengths used for measurement the optical density of the compensating solution for most steels is small and constant, it is not necessary in routine practice to measure it on all samples.The interfering-element blank has not been studied in this work for the normal silicon range, since no interferences have been reported by previous workers3,* under these conditions for measuring the optical density. However, it was necessary to test the low-range method because of the different measuring conditions. For this purpose a series of experiments was conducted on two plain carbon steel samples of different silicon content to which additions were made of the several possible interfering elements in the purest available form.In this manner it was shown that the following were without effect on the method: 0.1% of phos- phorus; 0.1% of arsenic; 1% of lead; 5% of copper; 5% of vanadium; 20y0 of cobalt; 20y0 of nickel; 20y0 of chromium; and 20y0 of manganese.344 ANDREW AND GENTRY : THE PHOTOMETRIC DETERMINATION [Vol. 81 MEASURING CONDITIONS- The absorption spectrum from 500 to 1000 mp of molybdenum blue according to the present method is shown in Fig. 4. It was measured on the Unicam SP500 spectrophoto- meter, with a 4-cm cell and a slit-width of 0.017 mm. The steel contained 0.06 per cent. of silicon and measurements were made at 10-:mp intervals. As can be seen, the maximum absorption 0c:curs at a wavelength of 810 mp, in agreement with the figure found by Mullin and Riley.s T:his value should be compared with the condi- tions normally adopted when the Spekker absorptiometer is used.Vaughan4 used the tungsten-filament lamp in conjunction with an Ilford No. 608 and a heat-absorbing filter. By combining the transmission curve of the filters as measured on the Unicam SP500 spectro- photometer with the spectral response of a barrier-layer cell to a tungsten-filament lamp, the spectral response of this system has been obtained. The results, presented in Fig. 5, b 600 650 700 d Wavelength, rnp Wavelength, rnp Fig. 5. Spectral response Fig. 6. Spectral response curve for system consisting of barrier-layer cell, system consisting of “infra-red” barrier- tungsten-filament lamp, and Ilford No.608 layer cell, tungsten-filament lamp, and Ilford and Calorex H503 filters No. 608, Wratten No. 74 and Calorex H503 filters show that effectively the system acts as a filter with a peak at 665 mp and a half-band pass of 60 mp. With the limitations imposed by the barrier-layer cell this represents the best choice of conditions possible on the unmodified Spekker absorptiometer. The Ilford No. 608 and Calorex H503 filters and tungsten-filament lamp combination have therefore been used in the present work for the normal silicon range. The other commonly used conditions entail use of the mercury-vapour lamp in con- junction with Ilford No. 606 and Calorex H503 filters, which isolate the 577 and 579-mp lines. Such conditions have been favoured by steel analyst^,^^^ presumably because the mer- cury-vapour lamp is normally used in the absorptiometric analysis of steels and it is incon- venient to change over to the tungsten-filament lamp for particular determinations.As can be seen from Fig. 4, the absorption peak of the molybdenum-blue complex occurs at 810 mp. This is the preferred wavelength for measurements and the one giving the maximum sensitivity. The effect of the background colour is greatly reduced by working at 810 mp rather than at the more commonly used wavelengths, as can be seen from Fig. 4. In the present work, the low-range method has been based on measurements made at this wavelength with the Unicam SP500 spectrophotometer with a slit-width of 0-017 mm, which corresponds to a nominal band-width of 4 mp.Some consideration has also been given to modifying the Spekker absorptiometer to permit measurements to be made at this wave- length. For this purpose the barrier-layer cells have been replaced by Megatron barrier- layer cells, ‘‘ infra-red ” type. These cells, in conjunction with the tungsten-filament lamp and filter combination consisting of Wratten No. 74, Ilford No. 608 and Calorex H503, permit measurements to be made at 805 mp (as can be seen from Fig. 6). Satisfactory calibration graphs have been obtained with the Spekker absorptiometer modified in this way. The commonly used Ilford Spectrum filters show marked transmission in the near infra-red and, if the modified Spekker absorptiometer is to be used for work in the visible region, it will be necessary to verify the calibration of the instrument for the determinations in question.Alternatively, the Calorex H503 filter may be replaced by a suitable filter having a sharp cut-off at about 680 mp, thereby modifying the over-all response of the photocell - filterJune, 19561 OF SILICON IN STEELS 345 combination to approximate to that of the normal photocell. An interference filter is suitable for this purpose. Measurements made under any of the conditions discussed give straight-line calibration graphs. By using the Spekker absorptiometer, the tungsten-filament lamp and Ilford No. 608 filters, the range of the method is up to 3 per cent. of silicon with 4-cm cells. On the Unicam SP500 spectrophotometer at 810 mp, the range is up to 0-13 per cent.of silicon with 4-cm cells or up to 0.5 per cent. with l-cm cells. The calibration graphs pass through the origin if correction is made for the reagent blank. The sensitivity of our method, different measuring conditions being used, is shown in Table I, which also-gives some results obtained by other methods. It can be seen that by taking measurements at 810 mp there is a gain in sensitivity of nearly four times over measurements made at 580 mp. Differences exist between the sensitivity of the several methods when measuring under the same conditions, but this is to be attributed to differences in the conditions for the formation and reduction of the silicomolybdic acid. It is of interest, however, to note that, despite their differences, the present method and the British Standard methodl have the same sensitivity when measurements are made a t 810 mp.TABLE I COMPARISON OF SENSITIVITY WITH DIFFERENT Method Present method, Ilford No. 608 filters and tungsten- Present method, Ilford No. 606 filters and mercury-vapour British Stand&d method,a' ilford 'No. 606 fil& and Glasgow Panel method, Ilford No. 606 filters and mercury- Spekker abso@tiometev- filament lamp . . . . .. .. .. .. lamp .. mercury-vapour lamp . . .. . . .. .. vapour lamp . . . . .. .. .. .. and tungsten-filament lamp . . .. .. .. Modi$ed Sfiekker abso@tiometev- Present method, Ilford No. 608, Wratten No. 74 filters Unicam SP500 spectyophotomeier- Present method . . . . .. .. .. .. Present method . . .. .. .. . . .. Present method .. . . . . .. . . .. British Standard method' . . . . .. .. .. MEASURING CONDITIONS Wave- length, mtL 670 578 578 578 805 810 670 578 81 0 Silicon required to give an optical density of 1.00 in a 4-cm cell r \ 50 ml of Silicon in A Silicon in final solution, sample, r*g % 45 0.362 62 0.496 69 0.058 58 0.290 20 0.1 60 16 0.128 43 0.345 60 0.480 16 0.080 METHOD REAGENTS- of water, mix, cool and dilute to 1 litre. and dilute to 1 litre. of nitric acid, sp. gr. 1.42. 100ml of water. Sulphuric acid, 5 per cent.-Carefully pour 50 ml of sulphuric acid, sp. gr. 1.84, into 800 ml Nitric acid, 25 per cent.-To 500 ml of water add 250 ml of nitric acid, sp. gr. 1-42, mix Mixed acid-To 500 ml of water add 200 ml of hydrochloric acid, sp. gr. 1.18, and 65 ml Potassium permanganate solution, 2 per cent.-Dissolve 2 g of potassium permanganate in Hydrogen peroxide, 2-volume-Dilute 10 ml of 20-volume hydrogen peroxide to 100 ml.Ammonium molybdate solution, 2.5 per cent .-Dissolve 2.5 g of ammonium molybdate Oxalic acid solution, 4 per cent.-Dissolve 4 g of crystalline oxalic acid, (COOH),.2H20, in Ferrous ammonium sulphate solution, 6 per cent.-Dissolve 6 g of ferrous ammonium sul- Add 1 ml of 5 per cent. sulphuric acid, cool and dilute Mix and dilute to 1 litre. crystals in 80 ml of warm water, cool and dilute to 100 ml. 80 ml of warm water, cool and dilute to 100 ml. phate crystals in 50 ml of warm water. to 100 ml. Store in a polythene bottle.346 ANDREW AND GENTRY THE PHOTOMETRIC DETERMINATION [Vol. 81 PROCEDURE FOR SOLUTION OF SAMPLE- Use the most suitable of the following methods for dissolving the sample- Weigh 0.25 &- 0.05 g of sample (to the nearest 1 mg) into a 350-ml Erlenmeyer flask.Add 100ml of 5 per cent. sulphuric acid and simmer until solution is complete. Oxidise the hot solution by dropwise addition of 2 per cent. potassium permanganate solution until a permanent precipitate is observed. Add 2-volume hydrogen peroxide dropwise until the precipitate just dissolves. Boil gently for 5 minutes, cool and dilute to 500 ml in a calibrated flask. Weigh 0.25 -4 0.05 g of sample (to the nearest 1 mg) into a 350-ml Erlenmeyer flask. Add 100 ml of water, boil for 5 minutes, cool and dilute to 500 ml in a calibrated flask. Weigh 0.25 -4 0.05 g of sample (to the nearest 1 mg) into a 350-ml Erlenmeyer flask.Add 100 ml of water, boil for 5 minutes, cool and dilute to 500ml in a calibrated flask. (a) (b) Add 55 ml of 25 per cent. nitric acid and simmer until solution is complete. ( c ) Add 65 ml of mixed acid and simmer until solution is complete. PROCEDURE FOR COLOUR DEVELOPMENT- Place by pipette two 25-ml portions (A and B) in dry 100-ml conical flasks. Add to A from a pipette 10 ml of 2.5 per cent. ammonium molybdate, mix well and set aside for 20 minutes. Add from a pipette 10ml of 4 per cent. oxalic acid solution, mix well, add immediately from a pipette 5 ml of 6 per cent. ferrous ammonium sulphate solution and mix to give the test solution. Add from pipettes to B, in this order and mixing between additions, 10 ml of 4 per cent.oxalic acid solution, 10 ml of 2-5 per cent. ammonium molybdate solution and 5 ml of 6 per cent. ferrous ammonium sulphate solution to give the compensating solution. PROCEDURE FOR TAKING MEASUREMENTS- Normal range: read the optical densities 'of the test and compensation solutions on the Spekker absorptiometer, using the tungsten-filament lamp and Ilford No. 608 filters, together with Calorex H503 filters and the most conveniently sized cell. Use water as the reference solution. Low range: read the optical densities of the test and compensating solution on the Unicam SP500 spectrophotometer in 4-cm cells at 810 mp with a slit width of 0-017 mm, using water as the reference solution. Convert the difference between the optical densities of the test solution and the com- pensating solution to percentage of silicon by reference to the calibration graph.Correct the result for the reagent blank, which is found by treating a steel of very low and known silicon content as described in the procedure. NOTES ON PROCEDURE- For high- alloy materials the more rapid of the other solvents is used. If the solution after being diluted t o 500 ml is cloudy owing t o the presence of undissolved carbides, 100 ml should be filtered through a dry filter-paper into a dry beaker. During the colour development and subsequent optical-density measurements, the temperature of the solution should be controlled a t 20" & 3" C. Instruments other than those suggested for measuring the optical density may be used with suitable modifications of the operating details.For the normal range a wavelength of 665 m p is used and for the low range a wavelength of 810mp. Under the conditions given, the low-range method has a range of up t o 0.13 per cent. of silicon with the 4-cm cell. The normal-range method has a range of up t o 3 per cent. of silicon with a 0-5-cm cell and up t o 0.4 per cent. with a 4-cm cell. The calibration graph is prepared from a low-silicon steel (the silicon content need not be known) t o which known additions of a standard silicate solution are made. The preparation of the standard silicate solution is as described in the British Standard.l Any of the solvent acids may be used, since they all give the Same calibration graph. The series cd calibration tests is taken through all the stages of the procedure as described.A blank calibration determination is made on the steel without addition of silicate solution, t o allow correction t o be made for the silicon content of the steel plus the reagent blank. RESU L'r s A detailed assessment of the normal-range method has not been made, since it differs from the method previously described3 in only two particulars: the amount of solvent acid and the time allowed for the formation of the silicomolybdate. That these factors have 1. The 5 per cent. sulphuric acid solvent is used for all plain carbon and low-alloy steels, 2. 3. 4.June, 19561 OF SILICON I N STEELS 347 little effect on the precision has been exemplified by the results obtained in the preparation of the respective calibration graphs.The advantages of the present method over the earlier one are to be found in the less critical conditions rather than in improved precision in carefully conducted experiments. However, a study of the present low-range method has been necessary, since it is intended for types of steel outside the accurate range of the earlier method. Further, since measure- ments are made at a widely different wavelength, new or unexpected sources of error were possible. To test the precision of the low-range method a series of tests has accordingly been carried out by five analysts with three samples. The tests were made under conditions to be expected in routine practice, Hence, although the same calibration graph was used throughout, the several analysts prepared all their own reagent solutions and performed the determinations at different times of day over a period of several weeks.All but one of the operators had had no previous experience of this exact method. The results are given in Table 11. TABLE I1 PRECISION OF LOW-RANGE METHOD Steel A B C Number of determinations . . .. 30 20 20 Mean result, yo of silicon . . .. 0.0139 0.0399 0.0439 Range, % of silicon . . .. .. 0.0042 0-0039 0.0066 Standard deviation, yo of silicon . . f 0.0014 f 0-0013 f 0-0015 Number of analysts . . .. 0 . 5 5 5 It can be seen that the probable error is the same a t 0.01 per cent. as at 0.04 per cent. of silicon, and over this range there is a 0.95 probability that a single determination is correct to within & 0.003 per cent. of silicon. This precision is probably limited by the reagent blank, which includes the adventitious pick-up of silicon from glassware and the atmosphere.In the above experiments, the reagent blanks were equivalent to a silicon content in the steel of from 0.005 to 0.010 per cent. CONCLUSIONS The method that has been developed fulfils the requirements for a general photometric method of determining silicon in steels. Faster methods can be readily devised for individual types of steel, but the speed of the present method is probably adequate for most purposes. A batch of twelve steels can be analysed in 2* hours by a single operator. It is not necessary to know the approximate silicon content before a determination is started, since precise results can be obtained over a wide range of silicon contents.The conditions for carrying out a determination are fairly flexible, provided that care is taken in using pipettes to measure the solutions accurately. It is particularly useful that the volume of solvent acid is not very critical, firstly because this permits measuring cylinders to be used in routine practice and, secondly, because it means that the sample may be dissolved without the risk of error from loss of acid by evaporation. One condition that must be controlled is the temperature of the solution during the formation of the silico- molybdate and the final measurement. It is particularly important that the method should not be used at lower temperatures, since complete formation of the silicomolybdate will not then occur in the time specified. A limitation to the present method, common to most photometric methods, is that it can be applied in general only to steels that dissolve completely in one of the three solvent acids. Tungsten-bearing and similar steels that give an earth-acid precipitate can be analysed by this method only if it can be established that the precipitate absorbs a negligible amount of silica. When this is so, the photometric method offers very real advantages in speed and precision over any other chemical procedure. However, to establish that no silicon is lost requires a separate investigation of each individual type of steel and this was considered to be outside the scope of the present work. There is need for further work in this con- nection, as well as in a search for alternative solvents that will give complete solution of such steels.348 Mol POLLARD AND MARTIN : THE SPECTROPHOTOMETRIC DETERMINATION [Vol. 81 We thank Mr. R. J. Garwood for making some of the measurements and Mr. J. A. M. van and the Directors of Philips Electrical Ltd. for permission to publish this paper. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. British Standard 1121 : Part 15 : 1949. British Standard 1121 : Part 19 : 1951. Gentry, C. H. R., and Sherrington, L. G., J . SOC. Chem. Ind., 1946, 65, 90. Vaughan, E. J ., “Further Advances in the Use of the Spekker Photo-electric Absorptiometer in Metallurgical Analysis,” Institute of Chemistry, London, 1942, p. 44. Methods of Analysis Committee of B.I.S.R.A., J . Iron G. Steel Inst., 1950, 165, 430. Glasgow Absorytiometric Panel, Metallurgia, 1954, 44, 145. Olsen, A. L., Gee, E. A,, McLondon, V., and Blue, D. D., Ind. Eng. Chenz., Anal. Ed., 1944, 16, 462. Mullin, J. B., and Riley, J. P., Anal. Chim. Acta, 1955, 12, 162. Lacroix, S., and Labalade, M., Ibid., 1949, 3, 383. Bagshawe, B., and Truman, R. J., Analyst, 1954, 79, 17. PHILIPS ELECTRICAL LIMITED NEW ROAD MITCHAM JUNCTION SURREY October 25th, 1955

ISSN:0003-2654    

DOI:10.1039/AN9568100339

出版商:RSC    

年代:1956

数据来源: RSC