ISSN:0003-2654    

DOI:10.1039/AN95277FX005

出版商:RSC    

年代:1952

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95277BX007

出版商:RSC    

年代:1952

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95277FP009

出版商:RSC    

年代:1952

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95277BP019

出版商:RSC    

年代:1952

数据来源: RSC

摘要:

KSRUARY, I952 Vol. 77, No. 911 Some Observations on the Determination of the Activity of Rennet BY N. J. BERRIDGE (Presented at the meethg of the Society on Wednesday, November 7th, 1951) Experiments on the measurement of the clotting time of rennet in a substrate made by reconstituting skimmed milk powder in 0.01 M calcium chloride are described in detail, as is the method of observing the clotting- point in a thermostatically controlled enclosure. The reproducibilities of activities of rennet so determined are tabulated and it is shown that differences of 2 per cent. between samples should be recognisable. Comparisons have been made between the clotting times in calcified milk and in bulk milk to show the suitability of the artificial substrate for the assay of rennet for cheesemaking.It is suggested that either dry powdered rennet or a special batch of milk powder could be used as a standard. LARGE quantities of rennet are used in the manufacture of cheese and it.is the only enzyme to be sold widely by retail. The determination of rennet strength and the standardisation of rennet are, therefore, important. The technique of measurement described below was used in work that culminated in the crystallisation of rennin1 and it is hoped that the method n i l 1 assist in the solution of the problems of standardisation. The enzymic activity of a rennet 5758 BERRIDGE : SOME OBSERVATIONS ON THE [Vol. 77 solution may be determined by measuring the time taken by a given volume to clot a given volume of milk; but in order to obtain reproducible values each of the many variables in the process of clotting must be adequately controlled and certain precautions are necessary on account of the nature of both enzyme and substrate.The precautions to be observed with rennet differ but little from those required bj- enzymes in general. For example, its destruction must be avoided during dilution and assay and the activation of any pro-rennin that may be present must be prevented. This activation may be rapid in dilute, slightly acid, solution and if a rising activity is observed in a series of replicates it is necessary to make fresh dilutions at a controlled pH value of, say, 6 to 7. Although the rennet itself introduces no unusual difficulties, the substrate requires particular care and attention because of the complex and variable nature of milk, the clotting of which is not a direct result of the action of the enzyme but of a secondary reaction that appears to follow the enzymic reaction after a time lag.The variations in the reactivitj- of milk may be partly obviated by making special casein preparations, but this introduces further difficulties and it is better, in general, to use a good quality milk powder, prepared from skim milk with the minimum of heating, from which a more reproducible substrate for rennet testing can be prepared; although the readiness with which the reconstituted milk clots is liable to vary with the age of the powder and with the conditions of storage. This variation is largely due to changes of state in the calcium phosphate between the ionised and the colloidal condition.2 These changes can to some extent be prevented by adding an excess of calcium ions, and milk prepared by reconstituting a powder in 0.01 M calcium chloride was stable enough for use as a substrate.The time lag between the enzymic change and the clotting can be a serious source of emor because it may be unnoticed and can vary with the history of the milk and with the conditions of the test. The existence of the time-lag is made evident by lack of linearit>- between the clotting time and the concentration of rennet, and also by a point being reached beyond which further increases in the concentration of rennet fail to reduce the clotting time. The time-lag, or period of time between the action of the rennet and the non-enzymic change that causes clotting, is influenced by temperature and the concentration of electrolytes in the milk; it can be considerably shortened by reconstituting the skimmed-milk powder with 0.01 M calcium chloride solution instead of distilled water.The non-linearity mentioned above is diminished, but not entirely removed, by recon- stituting in calcium chloride; and, as other factors in the milk probably have some effect, it is necessary with a new substrate to plot a graph of enzyme concentrations against reciprocal clotting times in order to determine the range of clotting times over which linearity may be assumed to hold good without significant error. OBSERVATION OF THE CLOTTING-POINT The precise observation of the moment of clotting is a little difficult.Various kinds of apparatus have been devised to facilitate it.3,435 The visual method described here h a the advantage of needing only simple apparatus that is readily available and easily cleaned. It gives, moreover, a particularly sharp end-point ; the little skill required is readily acquired with practice. METHOD APPARATUS- A stop-watch. Volumetric apparatus for diluting the rennei. Test tubes-These should hold approximately 20 ml to the brim. Glass rods-These should be 3 to 4 inches longer than the test tubes. A "barrel-aizd-plunger" uhisk for reconstituting the milk. A ra$id measuring device-This is required for dispensing 10-ml quantities of milk, and may be, e.g., an ordinary 10-ml pipette with a wide tip. The pipette used in this work had a delivery time of about 4 seconds and an accuracy of 50.1 per cent.with water and of 5 0 . 2 per cent. with milk. A water-bath controlled at 30" 5 0.2" C-This should be made of glass, or fitted with ;t transparent front, and provided with a rack suitable for holding at least 9 test tubes in batches of 3 and a convenient clamp for holding one tube at a time near the glass front for observation of the clot t ing-poin t .Feb., 19521 DETERMINATION OF THE ACTIVITY OF RENNET 69 The tube should be illuminated by a 100-watt electric lamp placed in front of the water- bath, just above and slightly to the left of the operator. The lamp should be shielded so that the test tube is brilliantly lit, but neither direct light nor reflection from the glass should dazzle the operator.The background behind the tube should be as dark as possible. These conditions are necessary for accurate observation and to prevent fatigue, which leads to inaccuracies. Like many other proteins, rennin tends to be adsorbed on glass surfaces. Cleanliness of all glassware is therefore particularly important and soaking in strong sulphuric acid - bichromate mixture followed by thorough rinsing, first with tap water and then with distilled water, is essential. RE-4GENTS- Calcium chloride solution-This should be accurately adjusted to 0.01 M. Spray-dried skim milk powder-This should be prepared without pre-heating. (The powder used in this work was supplied by Messrs. Aplin and Barrett Ltd., Yeovil, Somerset.) PROCEDURE- All weighings and volume measurements should be made with errors not greater than &0.3 per cent.This wide latitude is permissible because of the, so far, unavoidable variations in the clotting times. Weigh 12.0 g of the milk powder and reconstitute with 100 ml of the 0.01 M calcium chloride solution. Pipette 10.0 ml of the “milk” into a number of test tubes and place them, in batches of three, in a suitable rack in the water-bath at intervals of 15 minutes between .each batch. Since the clotting time with a constant rennet concentration changes slightly with the time during which the milk is warmed, this time is kept approximately constant at 30 to 35 minutes. Thus, after the first half-hour there will be a batch of tubes ready for use every 15 minutes as long as the supply is maintained.If the approximate activity of the rennet is not known, make a preliminary dilution to give, on the basis of an assumed activity, a clotting time of 1 to 2 minutes and use the first batch of tubes to obtain an approximate figure for the clotting time. From this figure, calculate by simple proportion the dilution necessary to give a clotting time of about 5 minutes. (The actual range of clotting time permissible will depend on the length of the linear portion of the graph, as mentioned in the introduction.) Make this new dilution with distilled water not more than .? minutes before the milk is ready for use. Now clean the thumb of the right hand thoroughly, dry it on a clean cloth and take care not to contaminate it with rennet during the test. When the milk is ready, add to one of the tubes 1.00 ml of the rennet dilution in spch a way that it runs down the side of the tube to form a layer on the surface of the milk, close the tube with the cleaned thumb, and mix by inversion, starting the stop-watch simultaneously.Repeat the inversion and reversion twice more to wash all the rennet from the side of the test tube. Repeat the whole procedure with the second tube of the set, timing the first inversion to coincide with a reading of 60 seconds on the stop-watch, and do so similarly with the third, mixing it a t 120 seconds. About 1 minute before the expected end-point in the first tube, insert a clean stirring rod and clamp the tube firmly so that the level of the milk is well below that of the water in the bath and the tube wall above the milk is clearly visible.After stirring thoroughly for a few seconds, raise the rod until its lower end touches the side of the test tube 1 or 2 cm above the surface of the milk and hold it in that position to allow a thin film of milk to run from it over the inner surface of the tube. Give the rod a vertical jerk at intervals of 1 or 2 seconds to assist the flow of the milk film from the reserve held by surface tension between the end of the rod and the wall of the test tube. Just before this supply .of milk is exhausted, spread the remaining milk towards the side of the tube and repeat the operation on a fresh but easily visible section of the test tube. Watch the old film until the new one is properly established. Keep the film flowing continuously but as thinly as possible so as to be only just visible; clotting is then easily observed as the sudden appearance of heterogeneity, the faint grey film breaking up into white particles on a black background.During this observation place or hold the stopwatch only just out of sight so that the instant clotting takes place a slight movement of the eyes enables the time to be observed within a second. Immediately transfer the rod to the second tube and repeat the above procedure. Similarly determine the third clotting time and stop the watch. In this way clotting times are obtained in triplicate.60 BERRIDGE: SOME OBSERVATIONS ON THE [Vol. 77 With routine samples it is convenient for one worker to assay 4 samples per hour. Larger quantities of “milk” may then be made a t one time, providing its properties do not change before use.The “milk” used in this work was stable for several hours at room temperature. The strength of the rennet may be expressed in arbitrary units per given volume by dividing the dilution used by the mean clotting time in seconds, and when a. sufficient number uf samples have been measured the replicate clotting times can be used to estimate the standard error by the analysis of variance in the usual way. RESULTS The reproducibility of activities determined by the method described is shown in Table I. I‘wenty-three rennet samples were diluted equally and assayed in triplicate. The mean dotting time for the least active was 419 seconds, that for the most active, 311 seconds. The average value of all the means was 358 seconds and the standard error of each mean was :bH seconds.Similar sets were assayed 7 and 9 months later. The three sets of results are TABLE I STANDARD ERRORS OF RENKET ASSAYS Average of the mean (average of 23 means) (means of 3 observations) times Range of the mean Standard errors oi clotting times clotting times the mean clotting [seconds) Set 1 .. .. .. 358 Set 2 .. .. .. 147 S e t 3 .. .* .. 191 (seconds) 311-419 131-1 67 16 1-242 (seconds) 3.80 1.35 1.29 collected in Table I, in which the first two columns indicate the spread of values due to the differences between the rennet samples, but the standard error in the last column reflects only the unavoidable variations in the clotting times when the rennet is kept constant. The h i n u t i o n in the standard errors compared with the actual values of the means reflects the increasing skill of an assistant who was new to the test when the first batch of assays was done.Taking the last value of 1.29 for the standard error and a probability limit oi 4t.05 as an example, the minimum significant difference between 2 means, each of 3 clotting times, becomes 3.7 seconds. Comparing this with clotting times of the order of 200 seconds, it is clear that differences of 2 per cent. between samples will usually be recognisable. In order to get some idea of the suitability of this substrate for the assay of rennet for- cheesemaking, the activities of a number of enzyme preparations in bulk milks before and after pasteurising and after ripening in the cheese vat were compared with their activities in the calcified milk.The results are shown in Table 11, in which the figures in the main section are ratios. Each figure was obtained by dividing the caiculated clotting time of the particular enzyme in calcified milk by the calculated clotting time of the same enzyme at the same concentration in the bulk milk indicated. All the rennets were diluted to give clotting times of 6 to 8 minutes whatever the milk in use; corresponding times at a rennet concentration of 0.1 per cent. were then obtained by simple proportion to facilitate the calculation of the ratios. I t is rather remarkable that the calcified milk also behaved so much like bulk milk towards pepsin and papain. An alternative substrate consisting of milk powder reconstituted with phosphate buffer was tried at the same time.This clotted with rennin more slowly than bulk milk, so that most of the ratios were between 5 and 10, and even slower with pepsin, for which the ratios rose to 11.6, but it clotted more quickly with papain, giving ratios from 0.5 to 0.8. It can be seen from the table that there were some samples that responded differentl!. to the different rennets; for example, milks Nos. 1 and 2 behaved similarly to all four rennets. but for milk No. 3 the ratio for the first rennet does not agree with those for the other three rennets. For other samples, smaller differences will be noticed and it is clear that these discrepancies are not of practical importance. The four rennet preparations differed in the method of manufacture.DISCUSSION OF RESULTS Although the rate of clotting of milk is sensitive to the pH value there is no point in imfTering the milk, for its buffering power is already very high compared with the minuteFeb., 19521 DETERMINATION OF THE ACTIVITY OF RENNET 61 quantities of acid that may be added with normal rennet. Cheesemaking rennet, for example, needs diluting several hundred times before testing. However, if very dilute rennet preparations are to be assayed, the role of acidity must be remembered. The variations between different milk powders are also partly due to differences in acidity, but it is a simple Rennet No. Milk No. Acidity * 1 2 3 4 Pepsin Milk No. Acidity" 1 e 3 4 Papain Pepsin Milk No. Acidity* 1 e 3 4 TABLE I1 CLOTTING TIME I N CALCIFIED MILK CLOTTING TIME IN BULK MILK 'rHE RATIOS RAW MILK 1 2 3 4 5 0.23 0-21 0.21 0.20 0.20 1-43 1.17 1.40 1-13 0-93 1-46 1.21 1.08 1-12 0.9 7 1.46 1.23 1.08 1.08 0.93 1-46 1.22 1.07 1.10 0.90 1.29 0.98 - - PASTEURISED BULK MILK, H.T.S.T.8 9 10 11 12 0.21 0.19 0.22 0.20 0.22 1.22 1.07 1.42 1.13 1.67 1.21 1.08 1.40 1-09 1.57 1.21 1-08 1-38 1.10 1-03 1-20 1.12 1.46 1.07 1.64 - 1.16 1.09 1.07 1-32 - - DIRECT FROM CHEESE VAT JUST BEFORE RENNETING 14 16 16 17 18 0.19 0-18 0.19 0.19 0.19 1-05 0.73 0-84 0.86 0.83 0.95 0-72 0.83 0.86 0.82 0.98 0.73 0.82 0.84 0.82 0.97 0.73 0.88 0.86 0.85 - - CLOTTING TIMES FOR CALCIFIED MILK 6 7 0.20 0.20 1.09 1.21 1.02 1.23 1.14 1-38 0.99 1.18 - - 13 0.21 1.20 1.20 1.17 0.70 - I 19 0.75 0-74 0.73 0- 74 - Calculated to 0.1 per cent.concentration of the enzyme preparation Rennet No. .. .. 1 2 3 4 Papain Pepsin Time, seconds . . . . 220 168 81 78 7600 88 * Titratable acidity as percentage of lactic acid determined by the creamery. matter to compare a fresh batch of standard milk powder with the old and calculate a correction factor. Frost6 has succeeded in diminishing this change in sensitivity of the substrate with a new batch of milk powder by including acetate buffer in the liquid for reconstitution. This suggests that a further improvement might be effected by buffering the calcium ion activity with a partly ionised calcium salt. The work of Pyne7 shows that calcium citrate would probably be suitable. For example, when 1 per cent. of sodium chloride was added to a diluted rennet and 1 ml was added to 10 ml of milk, the clotting time was increased by 8 per cent.These errors will arise when rennet solutions of low activity are being tested. A further step towards standardisation can be made by using dry powdered rennet as a standard. In fact, two master standards could be maintained, a special batch of milk powder m d a special batch of rennet powder. Over a limited period a rennet powder has been used as a standard. No variation in the sensitivity of the substrate during this time was noticed. It appears to be the general opinion that dried rennet is particularly stable. See, for example, Havenhill. 8 The other ionic constituents also play their part. The author wishes to thank the Directors of Benger's Limited for the award of a Fellow- ship, during the tenure of which this work was carried out.Thanks are due also to Miss M. Haskins for technical assistance and to Miss Rothwell for hospitality at the M.M.B. Creamery at Sturminster Newton.62 BERRIDGE [Vol. 77 1. 2. 3. 4. 5. 6. 7. 8. REFERENCES Berridge, N. J., Biochem J., 1945, 39, 179. Pyne, G. T., Ibid., 1945, 39, 385. Mattick, E. C. V., and Hallett, H. S., J . Agric. Sci., 1929, 19, 452. Kunitz, M., J. Gen. Physiol., 1935, 18, 459. King, C. W,, and Melville, E. M., J . Dairy Res., 1939, 10, 340. Frost, H. F., Private cgrnmunication. Pyne, G. T., Nature, 2948, 162, 925. Havenhill, L. D., J. Amer. Pitarm. Assoc., 1930, 19, 720. THE NATIONAL INSTITUTE FOR RESEARCH IN DAIRYING SHINFIELD, NR. READING DISCUSSION THE PRESIDENT congratulated the author on the skill with which he had tackled the essentially biological problem of the activity of rennet.DR. J. G. DAVIS said that he was particularly interested in the apparent lack of systematic behaviour of the four samples of rennet with ordinary raw and pasteurised milks, in contrast to which the relative behaviour of all four rennets with cheese-making milk seemed remarkably consistent. He asked whether the bulking of the ordinary raw and pasteurised milks was as great as that of the cheese-making milk; presumably the bulks of the latter would have been a t least 750 or 1000 gallons. Otherwise the only difference appeared to be that, if the cheese-making milks were tested a t rennetting time, the pH would be slightly lower a t about 0.4. DR. BERRIDGE said that the raw milk consisted of drip samples taken from a junction in the pipeline supplying the pasteuriser.Pasteurised samples were taken in a similar way out of milk flowing away from the pasteuriser. This seemed the nearest possible approach to truly representative bulk samples, and it seemed likely that the lower pH value of the ripened milks made them behave more uniformly towards rennet. MR. P. J. GOODE asked whether the author could advise on the storage of dried milk powder. His experience had been that, after a few weeks, dried milk kept in large tins would not set when reconstituted and treated with rennet. Keeping the milk in small, brim-full, airtight cans had no apparent effect on the storage life. He also asked if the author could give him some information about variations in clotting time with different types of milk, e.g., pasteurised, unpasteurised and homogenised, and with large variations of fat content up to about 20 per cent., such as a housewife might use when taking the “top off the milk” for her junket.Indeed, it was partly because its stability seemed too good to be true that dried rennet was used as a confirmatory standard. The quality of the dried milk appeared to be the controlling factor; if a batch seemed to be unstable, it should be discarded. It was well known that pasteurising increased the clotting time of milk, but homogenising had no effect, and variations in fat content had only the small effect that results from the corresponding change in the proportion of aqueous phase present.MR. A. L. BACHARACH suggested that the use of a freeze-dried “standard preparation” of rennet, which could probably be prepared in sufficient quantities to last a large number of people for a long time, might compensate for variations in behaviour of different samples of milk powder, especially under the standardised conditions Dr. Berridge had laid down. It would not then be necessary, and would indeed probably involve practical difficulties because of the amounts of substrate required, to prepare large quantities of “standard” milk powder, for each fresh batch of this could be checked against the standard rennet, which could also be used for comparison with the “test samples” of rennet coming up for assay. The procedure would be strictly analogous to that of using a standard vitamin preparation and a variable rat colony. DR. BERRIDGE replied that no trouble had been experienced with the storage of milk powder. DR. BERRIDGE thanked Mr. Bacharach for his valuable suggestion. DR. J. H. HAMENCE congratulated the author on the method, and said that he himself had used calcium ions in the form of calcium chloride to assist in clotting reconstituted dried milk and had found the technique to work very well. In view of the difficulty of obtaining sufficient quantities of dried milk powder he asked the author if he could give him any idea of the maximum range of variation that ‘was likely to occur between different batches of dried milk. DR. BERRIDGE replied that the milk powders used for some of the illustrative data showed that varia- tions could be very great. On the one hand there was milk, dried without pre-heating, that clotted readily without additional calcium ions, and with calcium gave a straight-line response over a wide range of rennet concentrations; on the other hand there was milk that hardly clotted at all unless calcium ions were added, and even then the straight-line response was limited to a narrow range of rennet concentrations. These variations could doubtless be reduced by selecting the milks.

ISSN:0003-2654    

DOI:10.1039/AN952770057b

出版商:RSC    

年代:1952

数据来源: RSC

摘要:

Feb., 19521 STROUD 63 The Determination of 2 : 4-Dichlorophenoxyacetic Acid BY S. W. STROUD (Presented at the meeting of the Society on Wednesday, November 7th, 1951) A rapid and accurate method is described for the separation and deter- mination of 2 :4-dichlorophenoxyacetic acid in a mixture of chlorinated phenoxyacetic acids. It is based on the separation of the acids by partition chromatography between ether and strong phosphate buffers on a kieselguhr column and their determination by titration of the carboxylic ac'd groups. In this way an accurate determination of the individual acids in a mixture of chlorinated phenoxyacetic acids can be made on 10 to 20 mg of a sample. THE use of chlorinated phenoxyacetic acids as plant-growth regulating substances and as selective herbicides has become well established, the most widely used of the group being 2 : 4-dichlorophenoxyacetic acid (2 : 4-D).Several reviews on the relative activities of this group of compounds have been published and it has been shown that, although the 2-chloro- and 4-chlorophenoxyacetic acids are active, they are less so than 2 : 4-D and 2 : 4 : 5-trichlorophenoxyacetic acid (2 : 4 : 5-T) ; 2 : 4 : 6-trichlorophenoxyacetic acid (2 : 4: 6-T) is inactive. Despite the extensive use of 2:4-D, no satisfactory simple chemical method has been described for its determination in a complex mixture. Bandurskil described a method based on the spectrophotometric determination of 2:4-D in soil extracts, but the method was not specific for 2 :4-D. Freed2 used a sensitive colorimetric method, chromotropic acid being used for the detection of 2:4-D, but again the method was not specific and proved unsuitable for the determination of 2 : 4-D.Other methods used for the determination of 2: 4-D involve the estimation of the total chlorine content followed by titration of the carboxylic acid group. Details of such methods have been given by Rooney3 and Heagy.* These authors point out, however, that the methods described are not entirely satisfactory, as estimations involving total chlorine content will give erroneous results if other chlorine compounds are present; likewise the estimation by titration of the acid group by the methods stated will also include other organic acids that may be present. I t can be seen that the presence of other chlorine-substituted phenoxyacetic acids would falsify the results ; for example, an equimolecular mixture of 4-chlorophenoxyacetic acid and 2 : 4 : 6-trichlorophenoxyacetic acid would analyse by these methods as 2:4-D.For the accurate estimation of 2:4-D it is necessary, therefore, to ensure a complete separation of the 2:4-D from the related compounds that may be present, by virtue of the method of preparation, in the product used for horticultural purposes. A method of separa- tion and estimation of 2:4-D has been described by Warshowsky and Schant~,~ but the method involves the use of the Craig counter-current extraction machine followed by the spectrophotometric determination of the 2 : 4-D in the selected tubes. It has been found that the separation of 2 : 4 D from the other chlorinated phenoxyacetic acids can be rapidly and effectively accomplished by means of partition chromatography between ethyl ether and strong phosphate buffer, advantage being taken of the differences in the pK values and partition coefficients of the various acids.By this means it has proved possible to separate 2-chloro-, 4-chloro-, 2 : 4-dichloro- and 2 : 4 : 6-trichlorophenoxyacetic acids from a mixture of them all and to estimate them separately by titration. Whilst 2 : 4 : 6-trichlorophenoxyacetic acid and 2 : 4-D can be individually separated and estimated in one procedure with a buffer at pH 6-35, under these conditions 4-chloro- and 2-chloro- phenoxyacetic acids remain on the column and are not eluted. For a complete analysis, therefore, a further separation is required, at pH 5-80, to separate 2-chlorophenoxyacetic acid and 4-chlorophenoxyacetic acid and to allow them to be determined separately.The method given below can be used for the determination of the salts, amides, esters, and so on, of 2 : 4-D and for their determination in horticultural preparations after the libera- tion of the free acid by the usual methods.64 STROUD THE DETERMINATION OF METHOD APPARATUS- - E u’ 5- E e W c .- 5 4,- Q 3 - U v) 2 0 2- 1.- jyol. 7‘1 A chromatographic tube, which is a glass tube of length 40 cm and diameter 1.2 cm, A separating funnel, fitted with a cork to fit the open end of the column, to act as a closed at one end by a perforated silver disc covered with a filter-paper disc. reservoir for the eluting solvent.MATERIALS AND SOLUTIONS- KieseZguhr, “Hy~o-SuperceZ”-This material* is suitable for use without pre-treatment. Sodium phosphate bufler A-A 25 per cent. v/v solution of sodium phosphate buffer adjusted to pH 6.35 and prepared as follows. Add sufficient of a 30 per cent. w/v aqueous :j 6 1 30 Fig. 1. The separation of 2 : 4 : 6- trichlorophenoxyacetic acid and 2 : 4- dichlorophenoxyacetic acid by means of phosphate buffer, pH 6-36 6 1 Combined 2 : 4 : 6-T and 2:4-D 1 \ A 2-Chioro- Fig. 2. The separation of khloro- and 2-chlorophenoxyacetic acids from the combined 2 : 4 : 6-trichloro- and 2 : 4-dichlorophenoxyacetic acids by means of phosphate buffer, pH 5.80 solution of sodium hydroxide to a saturated aqueous solution of sodium dihydrogen phosphate to bring the solution to approximately pH 6.35.Cool and allow the solution to stand over- night to avoid supersaturation. Then add just sufficient water to redissolve any precipitate and dilute the resulting solution with three times its volume of distilled water. Adjust this solution accurately to pH 6.35 by adding either 10 per cent. w/v aqueous phosphoric acid solution or 10 per cent. w/v aqueous sodium hydroxide solution. Sodium phosphate bufer B-A 25 per cent. v/v solution of sodium phosphate buffer prepared as for buffer A, but adjusted at all stages to pH 5.80. Ether-Sp.gr. 0.720, equilibrated with the appropriate buffer solution. Sodium hydroxide solution-0*002 N . Rromothymol blue solution-4-01 per cent. w/v. * Supplied by Messrs. Johns-Manville Co., Ltd., Artillery House, Artillery Road, London, S.W.1.Feb., 19521 2 : 4-DICHLOROPHENOXYACETIC ACID 65 PROCEDURE FOR THE SEPARATION AND DETERMIMATION OF 2:4:6-T AND 2:4-D- Mix intimately 15 g of kieselguhr and 7-5 ml of sodium phosphate buffer A, pH 6.35, and pack the mixture dry into the chromatographic tube. Displace the air in the column by running through a quantity of ether, previously equilibrated with the buffer solution. Mixture A B C D E F G TABLE I ANALYSIS OF MIXTURES OF CHLORINATED PHENOXYACETIC ACIDS Compounds Weights Titration Weights present taken, sum found, Recovery, mg ml mg % 2: 4: 6-T 2.00 3-85 1-967 98.4 2 4-D 5.00 11-30 4.995 99.9 2 4 : 6-T 1-00 1-95 0.996 99.6 2 : 4-D 7-50 16.85 7.448 99.3 2 : 4 6-T 2-00 3-90 1.992 99.6 2 : 4-D 5.00 11-20 4.950 99.0 4-chloro- 2.00 5-30 1.977 98.9 2 : 4-D 5.00 4-chloro- 5.00 2-chloro- 5.00 2 : 4-D 5-00 4-chloro- 2-00 2-chloro- 3.00 2: 4: 6-T 2-16 2 : 4-D 6.95 4-chloro- 2-13 2-chloro- 3-34 2 : 4 : 6-T 1.40 2 4-D 5.85 4-chloro- 2.40 2-chloro- 1.30 11.20 4.950 13-35 4.980 13.90 5.185 11.25 5.30 8-10 4.972 1.977 3.02 1 4.15 2.121 15.65 6.917 5.75 2.145 8.90 3.320 99.0 99.6 103.7 99.4 98.9 100-7 98.2 99.5 100.7 99.4 2.75 1.405 100.4 13-20 5.834 99.7 6-50 2.425 101.0 3.40 1.268 97.6 Place on the column 2 ml of an ether solution of the material under test, which should contain approximately 10 mg of 2 : 4-D, and apply gentle air pressure to the top of the column to force the solution into the kieselguhr mixture.Apply two further l-ml portions of ether to the column in a similar manner to ensure that all of the material is on the column before the elution is begun.Then fix the separating funnel, filled with the ether, to the open end of the tube and perform the elution, using gentle air pressure to speed the flow through the column. Take successive 2.5-ml fractions of the eluate, add 2 ml of distilled water and 2 drops of bromothymol blue indicator solution to each fraction and titrate with 0.002 N sodium hydroxide solution to the blue-green shade of the indicator. Deduct the blank value, which is obtained by titrating 2 6 m l of the ether solution used for the elution under the same conditions as above, and plot the corrected titration figures against the number of the fraction and draw the graph. To ensure the complete elution of the 2:4-D from the column, a total of thirty fractions is required.A typical graph obtained is shown in Fig. 1, two peaks corresponding to the 2 : 4 : 6-T and 2 : 4-D contents being obtained. Under the above conditions the 4-chloro- and 2-chlorophenoxyacetic acids remain on the column. To determine the weight of the acid represented by an individual peak, the corrected titration figures corresponding to all the points determined on that peak are summed and the sum is multiplied by the appropriate factor to give the weight of the acid in mg; e.g., if V , and V2 are the respective sums of the titration figures corresponding to the points on the 2:4:6-T and 2:4-D peaks in ml of 0.002 N sodium hydroxide, then- the weight of 2:4:6-T = V , x 0.511 mg, and the weight of 2:4-D = V 2 x 0.442 mg.This separation is sufficient for the determination of 2:4-D in a mixture.66 STROUD [Vol. 77 PROCEDURE FOR THE SEPARATION AND DETERMINATION OF 4-CHLORO- AND 2-CHLOROPHENOXY- ACETIC ACIDS- Pack the column dry as before with a mixture of 15 g of kieselguhr and 7.5 ml of sodium phosphate buffer B, pH 5.80. Apply the same load to the column as for the separation of 2:4:6-T and 2:4-D and take forty fractions; titrate as before and draw the graph (see Fig.2). Under these conditions the 2:4:6-T and 2:4-D are eluted together as one peak, followed by the peaks corresponding to the 4-chloro- and 2-chloro- compounds. If V , and V , are the respective sums of the titration figures corresponding to the points on the latter peaks in ml of 0-002 N sodium hydroxide, then- the weight of 4-chlorophenoxyacetic acid = V , x 0.373 n g , and the weight of 2-chlorophenoxyacetic acid = V , x 0.373 mg.RESULTS The results shown in the table below have been obtained from the analysis of a series of mixtures of the various chlorinated phenoxyacetic acids. Figs. 1 and 2 show the graphs obtained from mixture G. From the results obtained it can be seen that the method is capable of the separation and determination of 2:4-D and the other constituents with a high degree of accuracy. By decreasing the cross-section of the tube, maintaining the same length, and by using a more dilute solution of sodium hydroxide and micro-burettes, it is possible to carry out the analysis on smaller quantities of materials, which should render the method suitable for estimations on soil extracts.I wish to thank Sir Jack Drummond, F.R.S., Director of Research, Boots Pure Drug Co. Ltd., for his encouragement and permission to publish this work; Dr. H. A. S. Stevenson, for supplying the compounds used; and Messrs. R. Abrahams and D. Shooter for technical assistance. REFERENCES 1. 2. 3. 4. 5. BOOTS PURE DRUG Co. LTD. Bandurski, R. S., Bot. Gaz., 1947, 108, 446. Freed, V. H., Science, 1948, 107, 98. Rooney, 33. A., Anal. Chem., 1947, 19, 475. Heagy, A. B., J . Ass. 08. Agric. Chem., 1950, 33, 764. Warshowsky, B., and Schantz, E. J., Anal. Chem., 1950, 22, 460. RESEARCH DEPARTMENT NOTTINGHAM DISCUSSION THE PRESIDENT remarked on the neatness of this technique, which doubtless would find many other applications. MR.N. HERON suggested that an indicator could be adsorbed on the column, and so allow the flow of the acids to be followed readily. MR. STROUD said that it was doubtful whether there was any practical advantage in using an indicator adsorbed on the column to make the various bands visible. His colleague, Mr. P. B. Baker, had established a rapid paper-chromatographic method for the qualitative analysis of chlorinated phenoxyacetic acid mixtures with the same buffer - ethyl ether systems, and he used a methyl red spray for the detection of the bands. MR. A. L. BACHARACH asked whether any advantage had been found in using a system of automatic or semi-automatic “cut-takers.” MR. STROUD replied that the use of an automatic or semi-automatic fraction cutter, where available, was an advantage provided that sufficient care was taken in the design of the syphon system in the apparatus to ensure satisfactory working when ethyl ether was the eluting solvent.As the total number of cuts and the time taken for the estimation were both small, manual manipulation was easy. MR. K. GARDNER asked whether phenol and its chlorinated derivatives preceded the phenoxyacetic acids through the column and whether unchlorinated phenoxyacetic acid appeared after the chlorinated acids. He also asked how long a complete determination of isomers took, and whether ethers other than ethyl ether, for example, isopropyl ether, had been used in this type of chromatographic estimation.Feb., 19621 HILL 67 MR. STROUD said that, although no systematic study of the behaviour of phenol and chlorinated phenols had been made, i t was possible to predict from the respective dissociation constants and partition coefficients that phenol and its chlorinated derivatives would run ahead of the chlorinated phenoxyacetic acids. However, these compounds did not interfere with the determinations as they were not titrated under the conditions used. Unchlorinated phenoxyacetic acid appeared after the 2-chlorophenoxyacetic acid and therefore did not interfere. The complete determination of the isomers could be carried out in half-an-hour to an hour, though the time per determination could be reduced if several determinations were carried out simultaneously. Other ethers and also other immiscible solvents could be used, although ethyl ether was preferred as i t gave a good rate of flow through the column and was readily available. A further advantage was its easy removal by distillation when the acid in 8 fraction is to be isolated.

ISSN:0003-2654    

DOI:10.1039/AN9527700063

出版商:RSC    

年代:1952

数据来源: RSC

摘要:

Feb., 19621 HILL 67 Determination of 2-Me t h yl-4- C hlor ophenox yace tic in Presence of 2-Methyl-6-Chloro- and Acid 2-Me t hyl=4:6=Dichlor ophenox y acetic Acids Ultra-Violet Spectrophotometry BY R. HILL A method is described for the determination of 2-methyl-4-chloro- phenoxyacetic acid in presence of 2-methyl-6-chloro- and 2-methyl-4 : 6- dichlorophenoxyacetic acids by measurement of their ultra-violet absorption. The method is based on that given by Morton and Stubbs for the analysis of vitamin-A oils and depends upon the fact that the absorption due to 2-methyl-6-chloro- and 2-methyl-4:6-dichlorophenoxyacetic acids is approxi- mately linear over the wavelength range 275 to 285 mp, while 2-methyl-4- chlorophendxyacetic acid exhibits a peak absorption at 279 mp. It involves measurements of the extinctions of a solution of the acids in methanol at 275, 279 and 285 mp, from which the corrected extinction at 279 mp is obtained and thence the 2-methyl-4-chlorophenoxyacetic acid content of the mixture. For synthetic mixtures, the accuracy has been found to be 5 per cent.or better. SINCE the discovery in 1941 by Slade, Templeman and Sexton1 of the selective weed-killing activity of 2-methyl-4-chlorophenoxyacetic acid, extensive use has been made of preparations containing this acid. Such preparations are usually aqueous solutions of the sodium salt of 2-methyl-4-chkorophenoxyacetic acid, which, as a consequence of the method of manu- facture, contain in addition the sodium salts of 2-methyl-6-chloro- and 2-methyl-4 :6-dichloro- phenoxyacetic acids, together with sodium chlorocresylate, sodium glycollate and sodium chloride in small amounts.Extraction of the mixed methyl-chlorophenoxyacetic acids from the remainder of the material can be readily achieved, but there is as yet no published chemical method for the analysis of the extracted mixture of acids. Sjoberg2 has published an infra-red spectro- photometric method for the analysis of such mixtures and Grabe3 a method for the deter- mination of 2-methyl-4-chlorophenoxyacetic acid in mixtures with other methyl-chloro- phenoxyacetic acids by ultra-violet spectrophotometry. The latter method takes advantage of the fact that, of the methyl-chlorophenoxyacetic acids likely to be present, only 2-methyl- Pchlorophenoxyacetic acid absorbs strongly at 287 mp.In this laboratory, however, it has been found that this method is not altogether suitable for application to commercial products owing to the presence of impurities that absorb in the region of 287mp. An alternative ultra- violet method, based on that used by Morton and Stubbs4 for the determination of vitamin A in oils, has been found to be applicable to a large number of commercial samples containing 2-methyl-4-chlorophenoxyacetic acid as the active constituent. The Morton - Stubbs method requires the accurate measurement of the intensities of the total absorption of a mixture at three suitably chosen wavelengths, sufficiently close together to68 HILL : DETERMINATI0:N OF [Vol. 77 permit the assumption that the irrelevant absorption, Le., that part of the total absorption due to unwanted components, is linear over the wavelength range. If this assumption can be made, then from a consideration of the geometry of the problem it can be shown that- where El, E2 and E3 are the observed extinctions (expressed as log I0,4) at wavelengths hl, A, and A3.respectively and k, and k2 are defined by the ratios EAJEA, and EAJEA, for the pure component that it is desired to estimate.When the wavelengths can be so chosen that kl = k2 = k, i.e., when EA, = EA,, equation (1) reduces to the form-- EXPERIMENTAL DETERMINATION OF ABSORPTION SPECTRA- The ultra-violet absorption spectra of 2-methyl-4-chloro-, 2-methyl-6-chloro- and 2-methyl-4:6-dichlorophenoxyacetic acids were determined for 0.01 per cent. w/v solutions of the acids in AnalaR methanol with quartz cells of path length 1 em and a Hilger “Uvispek” spectrophotometer (Fig.1). Wavelength, mv Fig. 1. The ultra-violet absorption spectra of (A) 2-methyl-4-chlorophenoxy- acetic acid, m.p. 120” C, (B) 2-methyl-6- chloro-phenoxyacetic acid, m.p. 110’ C, and (C) 2-methyl-4 : 6-dichlorophenoxyacetic acid, m.p. 184” C From a study of these spectra it was considered that by using the wavelengths 275, 279 and 285 mp, the Morton - Stubbs method could be applied and equation (2) used, since (a) the extinction coefficients of both 2-methyl-6-chloro- and 2-methyl-4 :6-dichlorophenoxy- acetic acids are small at these wavelengths, compared with the 2-methyl-4-chloro-isomer andFeb., 19521 2-METHYL-4-CHLOROPHENOXYACETIC ACID 69 (b) the extinction values for 2-met hyl-6-chloro- and 2-methyl-4 : 6-dichlorophenoxyacetic acids at 275, 279 and 285 mp lie approximately on straight lines.By substituting A, = 279 mp, A, = 275 mp and A3 = 285 mp in equation (2), the following expression was obtained- where k = E2,,/Ezs5 = E,79/E275 for pure 2-methyl-4-chlorophenoxyacetic acid. VALIDITY OF BEER’S LAW AND EVALUATION OF k- The extinctions of a number of solutions of 2-methyl-4-chlorophenoxyacetic acid, of various concentrations from 0.004 to 0.02 per cent., were measured at 275, 279 and 285 mp with l-cm cells (Table I) and plots of extinction against concentration were made from which it was deduced that Beer’s law holds up to a concentration of 0.016 per cent., but for higher concentrations there was a slight falling off from linearity. The value of the constant k was determined by taking the average of the values for E27g/E,,, for concentrations of 2-methyl-4-chlorophenoxyacetic acid of 0.004, 0.008, 0-010 and 0.016 per cent.(Table I). Substitution for k (= 1.1914) in equation (3) gave the following expression- E2,9 (corrected) = 6.224 [E279 (obs.) - 0.6E2,, (obs.) - 0.4E2,5 (obs.)] . . * * (4) TABLE I VALIDITY OF BEER’S LAW AND THE EVALUATION OF k Conc. of s o h , E276my E279mp E285mp k = E279/E285 % w/v 0.004 0.265 0.319 0.267 1.1963 0.008 0.543 0.648 0.544 1.1912 0.010 0.676 0.802 0.674 1.1900 1.1872 0.016 1.063 1.267 1.057 0.020 1-334 1.580 1.322 - Average value of k = 1.1914. APPLICATION OF EQUATION (4) TO SYNTHETIC MIXTURES- Six mixtures containing known amounts of 2-methyl-4-chloro-, 2-methyl-6-chloro- and 2-methyl-4 :6-dichlorophenoxyacetic acids were examined with the results shown in Table 11.These results were obtained by taking 0.01 per cent. w/v solutions of each mixture, measuring the extinctions in l-cm cells at 275, 279 and 285 mp and calculating the corrected extinctions at 279 mp, from which the 2-methyl-4-chlorophenoxyacetic acid contents were computed. Replicate determinations on each sample gave results which were satisfactorily reproducible. TABLE I1 APPLICATION OF EQUATION (4) TO SYNTHETIC MIXTURES Composition, % L I 3 Mixture 2Me-4C1 2Me-6C1 2Me-4:6-C1 1 95 2 3 2 85 3 12 3 80 15 5 4 71 26 3 5 62 30 8 6 49 36 15 2-Methyl-4-chloro-acid found, yo Individual values Average 92, 94, 95 94 84, 86, 85 85 80.79, 79 79 72, 71, 73 72 63, 62, 63 63 48, 48, 47 48 r A \ METHOD The following procedure was adopted for commercial preparations containing sodium 2-methyl-4-chlorophenoxyacetate in aqueous solution.70 HILL [Vol. 77 REAGENTS- Hydrochloric acid, 5 N . Chloro form-A.R. or B.P. Sodium bicarbonate-A half-saturated aqueous solution. Methyl alcohol-AnalaR. PROCEDURE- Take sufficient of the sample to give 0.5 to 1.0 g of mixed methyl-chlorophenoxyacetic acids, dilute to 50 ml in a separating funnel and make distinctly acid with hydrochloric acid. Extract three times with portions of chloroform and combine the chloroform extracts, Extract the chloroform layer three times with half-saturated sodium bicarbonate solution to obtain the acids free from chlorocresols and combine the extracts.Acidify with hydro- chloric acid and extract the liberated acids three times with chloroform. Run the chloroform extracts into a small flask and dry by addition of 1 to 2 g of anhydrous sodium sulphate. After a few minutes pour off the chloroform into a beaker, washing the sodium sulphate with small amounts of chloroform. Evaporate the chloroform and dry the residue of methyl- chlorophenoxyacetic acids on a steam-bath for exactly 10 minutes after removal of the solvent. Weigh out accurately 100 4 06mg of the extracted acids and dissolve in methanol, making up the volume of solution to .100ml in a standard flask. Pipette 10ml of this solution into a second 100-ml standard flask and dilute to the mark with methanol to give a 0.01 per cent.w/v solution of the acids. With a photo-electric spectrophotometer, measure the extinctions of this solution at 275, 279 and 285 mp in a 1-cm cell, using methanol from the same batch as is used to prepare the solutions in the “blank” cell. With the extinction values so obtained, use equation (4) to calculate- E,,, (corrected) = 6.224 [E279 (obs.) - 0.6E2,5 (obs.) - 0.4E2,, (obs.)] and read the 2-methyl-4-chlorophenoxyacetic acid content from a previously prepared calibration curve. CONCLUSIONS The results of tests on synthetic mixtures indicate that the method is reliable for samples of mixed acids containing 50 per, cent. or more of 2-methyl-4-chlorophenoxyacetic with 2-methyl-6-chloro- and 2-methyl-4 :6-dichlorophenoxyacetic acids as the only other major constituents. The chief advantage of this method is speed, it being possible to analyse in duplicate three samples of extracted acids in less than 2 hours. The disadvantages of the method are (a) the relatively large values of the constants in equation (4), which means that great care must be taken in the determination of extinction values, and (b) probability of interference from 2-methyl-phenoxyacetic acid, which has an absorption maximum at 277 mp. However, it does not appear that, in general, the proportion of this acid will exceed 5 per cent. of the total acids in the sample, in which circumstances the accuracy of the result will not be appreciably affected. REFERENCES 1. 2. 3. 4. GENERAL CHEMICALS DIVISION WIDNES LABORATORY Slade, R. E., Templeman, W. G., and Sexton, W. A., Nature, 1945, 155, 497. Sjoberg, B., Acta Chem. Scand., 1950, 4, 798. Grabe, E., Ibid., 1950, 4, 806. Morton, R. A., and Stubbs, A. L., Analyst, 1946, 71, 348. IMPERIAL CHEMICAL INDUSTRIES LTD. RESEARCH DEPARTMENT WIDNES August, 1961

ISSN:0003-2654    

DOI:10.1039/AN9527700067

出版商:RSC    

年代:1952

数据来源: RSC

摘要:

Feb., 19521 HASLAM AND SQUIRRELL 71 The Determination of o-Tolyl Ester in Tritolyl Phosphate BY J. HASLAM AND D. C. M. SQUIRRELL A method has been devised for the determination of the o-tolyl ester in commercial tritolyl phosphate. The test, which is based on previous work carried out by Wurzschmitt, involves the preliminary isolation of the mixed cresols from the ester, followed by condensation of these cresols with benzaldehyde in a sulphuric acid medium. On being made alkaline the product obtained from o-cresol under these conditions yields a coloured solution, which is examined absorptiometrically. The method is suitable for the determination of up to 6 per cent. of o-tolyl ester in commercial tritolyl phosphate, without modification, and of a corresponding amount of o-cresd in mixed cresols.The influence of phenol and various xylenols on the test has been investigated. CONSIDERABLE interest has been taken recently in this country in the proportion of o-tolyl esters in commercial samples of tritolyl phosphate. The reason for this is that there appears to be no doubt that tri-o-tolyl phosphate is a dangerously toxic material. There is a voluminous literature on the subject and Blomqvistl has summarised the position as follows- Of the esters with phosphoric acid the m- and $-isomers are innocuous, but tri-o-cresyl phosphate is a dangerously toxic material. I t attacks the central nervous system, causing paralysis of the extremities. As little as 0-15 to 0-3 g produces the characteristic symptoms. Poisoning has mainly occurred by consumption of the substance, but some German cases are reported where paralysis has been caused by vinyl sheet plasticised with tricresyl phosphate containing a high proportion of the o-isomer.Tri-o-cresyl phosphate in liquid form being absorbed through skin, it is advisable to specify not more than 3 per cent. of o-isomer in tricresyl phosphate for use in vinyl plastics. Experience has shown no hazards need be anticipated then.’’ Certain authorities have taken steps to restrict the proportion of o-tolyl ester in tritolyl phosphate. For example, in the Eastern Zone of Germany a limit of 6 per cent. has been set2 and in Sweden a limit of 3 per cent. has been advised by Blomqvist.1 It is understood that a limit of 3 per cent. is being contemplated in America.Apparently most of the work on toxicity has been concerned with the dangerous character of the tri-o-tolyl ester, so although it may be possible at a later date to obtain more precise information about the relative toxicities of tritolyl phosphates of mixed 0-, m- and $-esters, it seems desirable at the present time to limit the proportion of o-cresol in the mixed cresols used in the preparation of the tritolyl ester, as this will a t least limit the proportion of tri-o-tolyl ester that can possibly be present in a commercial product. We do not doubt that if the demand arises, it will be possible, at a later date, possibly by infra-red methods, to determine the relative proportions of mixed tolyl esters in the commercial products. The purpose of this paper, therefore, is to describe the methods that we have found to be most useful in the determination of (a) o-cresol in mixed cresols and (b) the o-tolyl radicle in commercial samples of tritolyl phosphate.This work is based on previous work of Wurz~chmitt,~ who showed that o-cresol, when heated with freshly distilled benzaldehyde in the presence of 75 per cent. sulphuric acid, produced a benzein dyestuff of the formula- “Cresol is a mixture of three isomers. OH 072 HASLAM AND SQUIRRELL : THE DETERMINATION [Vol. 77 This dyestuff is red in acid solution and blue - violet in alkaline solution. Under corre- sponding conditions m- and p-cresol do not give coloured products. In our hands Wurzschmitt’s method failed in several respects and we have had to modify his test in at least five important particulars, as follows.Method of extraction and purification of the mixed cresols from the hydrolysis products of iritoZyZ$hosphate-After the hydrolysis of the tritolyl phosphate we find it necessary to extract with ether, first on the alkaline side in order to remove interfering substances, then on the acid side in order to extract the resulting cresols. The residue of cresols obtained on removal of the ether solvent is not a satisfactory product for benzaldehyde condensation and requires purification by distillation. Conditions of temperature, time, acid concentration and so on used in the Condensation with benxaldehyde-These conditions must be well defined and strictly adhered to in order to obtain a resinous condensation product that is completely soluble in methanol. Extraction and purification of the condensation product and its subsequent solution-A fter careful washing, the condensation product contains a small amount of sulphuric acid that in its turn produces a small amount of sodium sulphate when made alkaline in the colorimetric test.I t is necessary to pay strict attention to the ratio of methyl alcohol to water used at this stage of the procedure in order that any traces of sodium sulphate produced do not interfere in the colorimetry. Method of preparatzon of standards-In Wurzschmitt’s paper but little attention was paid to the preparation of standards. In our work, the colour standards have been prepared from known amounts of o-cresol mixed with a known amount of m- and p-cresols, free from o-cresol.Use of the Spekker absorptiometer and Lovibond colour disc in the &a1 colour comparison- Wurzschmitt gives no detailed information about the method used to effect colour comparison of the o-cresol - benzaldehyde condensation product. In the method that we normally use the final colour comparison is made with a Spekker absorptiometer, the range covered being from 0 to 12mg of o-cresol per 50ml of coloured solution. With the co-operation of The Tintometer Ltd., two colour discs have been prepared to cover the range 0 to 12 mg of o-cresol per 50ml of coloured solution in 13 steps, and it is suggested that these discs may be of value in the day-to-day control of the quality of mixed cresols used for the preparation of tritolyl phosphate.The observations of Mr. G. J. Chamberlin of The Tintometer Ltd. on the colours obtained may be of interest. In his view, with low concentrations of o-cresol the colour obtained is predominantly yellow. With increasing amounts of o-cresol the colour then changes through a greenish grey to a dirty mauve. This appears to be a dichroic solution with two absorption bands that change in relative importance as the concentration increases. The method that we have evolved for the determination of the proportion of o-tolyl esters in tritolyl phosphate is given below. It should be remembered that it is the o-cresol content of the mixed cresols recovered from the tritolyl phosphate that is determined, and it is inferred that this figure is precisely the same as the calculated proportion of the tri-o-tolyl ester in the original tritolyl phosphate under test.METHOD REAGENTS- Cellosolve potash, 50 per cent. w/v-Dissolve 12.5 g of potassium hydroxide pellets in 25 ml of ethylene glycol mono-ethyl ether by heating under reflux for 5 minutes in the hydrolysis flask. Sulphuric acid-Diluted (1 + 1) and 75 per cent. v/v. Ether. Sodium sulphate-Anhydrous. Benzaldehyde-Redistil AnalaR grade. Ammonium hydroxide, 0.5 per cent. w/v-Dilute 20.5 ml of ammonium hydroxide, Methyl alcohol. Sodium hydroxide, 10 per cent. w/v. sp.gr. 0-880, with water to 1 litre.Feb., 19521 OF O-TOLYL ESTER IN TRITOLYL PHOSPHATE 73 PROCEDURE FOR THE HYDROLYSIS OF THE TRITOLYL PHOSPHATE AND EXTRACTION OF THE Cool the solution and dilute with 150 ml of distilled water, and then extract successively with two 25-ml portions of ether; discard the extracts.Acidify the aqueous layer by the dropwise addition of diluted sulphuric acid (1 + 1) with cooling and extract the acid solution successively with two 25-ml portions of ether; wash the combined ether extracts twice with 50-ml portions of distilled water and dry over anhydrous sodium sulphate before evaporation of the bulk of the ether on a water-bath. Transfer the residual cresols to a small (10-ml) distillation flask and distil. After the preliminary removal of the remaining ether, collect the fraction distilling to the dry point. PROCEDURE FOR CONDENSATION WITH BENZALDEHYDE- Transfer 14 drops (about 0.3 g) of the mixed cresols to the bottom of a 6-inch by &-inch hard-glass test tube.Add 14 drops of redistilled benzaldehyde and mix thoroughly with the weighed amount of mixed cresols. Immerse the tube and contents to a depth of 2 inches in an oil-bath maintained at 130" 5 1" C. After half a minute, add 5 ml of 75 per cent. v/v sulphuric acid dropwise over a period of 1 minute, with constant stirring by means of a glass rod. Allow the test tube and contents to remain in the oil-bath for a further minute; i.e., the total time of immersion should be 2; minutes. After cooling for 20 minutes dilute the mixture with 10ml of distilled water and filter through a compressed cotton-wool pad in a small porcelain Gooch crucible, 3.5 cm by 2 cm, suction being applied by means of a water pump. With a glass rod,.thoroughly break up the insoluble residue on the pad and wash it carefully with at least 100 ml of cold water, This operation is very important, as it is desirable to remove occluded sulphuric acid at this stage.Wash the resin with 40 ml of water at 50" to 60" C, then with 2 ml of 0.5 per cent. w/v ammonium hydroxide and finally with cold water. Transfer the crucible and contents to a flat-bottomed flask, of 100-ml capacity and with a 3-cm neck, and heat under reflux for 10 minutes with 15ml of methanol to dissolve the resin. Filter the solution of the resin through a 3-cm Whatman No. 1 filter-paper supported by a filter disc contained in a funnel over a side-arm boiling-tube graduated at 30 ml. Wash the flask and crucible with methanol until the volume of the filtrate is 30 ml. RESULTING CRESOLS- Heat 4 g of the sample with 25 ml of Cellosolve potash under reflux for 2 i hours.Weigh the test tube before and after the addition of the cresols. COLORIMETRY- Transfer 20ml of this methanol solution, by means of a pipette, to a 50-ml standard flask. Add 0.5 ml of 10 per cent. w/v sodium hydroxide solution and 12 ml of distilled water. Filter this solution through a Whatman No, 42 filter-paper into a l-cm cell and measure the colour in a Spekker absorptio- meter 15 minutes after making alkaline and diluting to the mark. Use spectrum yellow filters, No. 606, and Calorex heat absorbers. For the blank use a 75 per cent. v/v solution of methanol in water. From mixtures made by adding known weights of o-cresol to a 50 per cent. w/w mixture of m- and 9-cresol, prepare by the above procedure a calibration curve to cover the range 0 to 12 mg of o-cresol per 50 ml of final coloured solution, Le., from 0 to 6 per cent.of o-cresol on a 0.3 g sample. From this curve or from the appropriate Lovibond disc deduce the o-cresol content of the unknown cresol mixture. Should the cresols contain more than 6 per cent. of o-cresol, a proportionately smaller sample should be taken and diluted to approximately 0.3 g with the 50 per cent. w/w mixture of m- and 9-cresol before the condensa- tion with benzaldehyde. The percentage of o-cresol found is assumed to be equivalent to the tri-o-tolyl phosphate content of the tritolyl phosphate originally submitted to test. Mix and dilute the solution to the mark with methanol. A typical calibration curve is a straight line passing through the following points- Indicator drum reading .. . . 0.002 0.058 0.115 0.170 0.226 0.284 0.340 o-Cresol corresponding to 50 ml of coloured solution, mg . . .. 0 2 4 6 8 10 12 NOTES In the course of the work certain other conclusions have been reached and they may be of interest.74 HODGSON AND GLOVER : THE DETERMINATION [Vol. 77 Eficiency of hydrolysis-It has been shown that the hydrolysis of the tritolyl phosphate with Cellosolve potash is very efficient. In given experiments less than 0.2 per cent. of the tritolyl phosphate remained unhydrolysed at the conclusion of the operation. Interference of phenol and xylenols-The interference of phenol and 2 :3-, 2 :4-, 2 :5-, 2 :6-, 3 :4- and 3 :5-xylenols in the test has been investigated.We are indebted to Mr. P. J. C. Haywood for the supply of authentic samples of these xylenols. There is no interference from 2:3-, 2:4-, 2 5 , 3:4- and 3:5-xylenols, but 2:6-xylenol and phenol do interfere in the test. The magnitude of the interference is demonstrated by the fact that 3 per cent. of phenol would be reported as 1-8 per cent. of o-cresol and 3 per cent. of 2:6-xylenol would be reported as 3.6 per cent. of o-cresol if examined as unknown samples. In the examination of commercial material it is unlikely that this interference would prove to be serious, as we understand that the maximum 2:6-xylenol content of commercial cresylic acid is of the order of 0.3 per cent. and the maximum phenol content is of the order of 1 per cent. FZasks-The hydrolysis with Cellosolve potash proceeds smoothly in soda-glass flasks and the life of these flasks is satisfactory. In contrast, Pyrex flasks do not withstand the conditions of the test and should riot be used. Stability of colour-The condensation product is not stable if allowed to stand for a long time in methanol solution, so the colorimetry should be completed within 3 hours of dissolving the resin in methanol. Throughout the test the same time intervals should be used for the samples as are used in the preparation of the standard colour. REFERENCES 1. 2. 3. Wurzschmitt, B., 2. anal. Chem., 1949, 129, 233. Blomqvist, O., Svenska Plast Foreningen Tekniska Meddelanden, Vol. 5, No. 1, January, 1950. Chem. Tvade J., 1951, 128, 218. IMPERIAL CHEMICAL INDUSTRIES LIMITED WELWYN GARDEN CITY, HERTS. PLASTICS DIVISION August, 1951

ISSN:0003-2654    

DOI:10.1039/AN9527700071

出版商:RSC    

年代:1952

数据来源: RSC

摘要:

74 The HODGSON AND GLOVER : THE DETERMINATION Determination of Water in Liquid [Vol. 77 Ammonia BY H. W. HODGSON AND J. H. GLOVER A method is described for the determination of water in liquid ammonia. The ammonia is removed by evaporation after addition of ethylene glycol to retain the water. The water is titrated with Fischer reagent after neutralisa- tion of traces of residual ammonia with acetic acid. Results are given to show the advantage of the method over that of direct evaporation with no water retaining agent; the accuracy and reproducibility are also shown. A METHOD for the determination of water in liquid ammonia was described by Iljin and his co-workers.1 Liquid ammonia was allowed to evaporate from a Dewar flask through weighed absorption tubes containing sodium hydroxide pellets ; the water content was calculated from the increase in weight of the absorption tubes and the weight of ammonia lost from the Dewar flask.There is no evidence to support the assumption that water is carried over quantitatively with the ammonia in this method. Pleskov2 described a method that depended on the blue colour of a solution of sodium in liquid ammonia. He added a weighed amount of sodium to a known volume of liquid ammonia and titrated the solution with more liquid ammonia until the blue colour was discharged. Experiments were carried out by Pleskov’s method, but it was found that the amount of sodium required to produce a permanent blue colour was much more than could be accounted for by the known water content of the sample.According to Mitchell and Smith3 the water in liquid ammonia can be determined by Fischer reagent after the addition of methanol and the subsequent removal of ammonia by vacuum distillation. The method is not described in detail and the result quoted is cited from unpublished work.Feb., 19521 OF WATER IN LIQUID AMMONIA 75 SAMPLING OF LIQUID AMMONIA METHOD- The low boiling-point and the hygroscopic nature of liquid ammonia necessitated the development of a special technique for sampling and storing the material. The sample is taken in a small steel cylinder, of about 2 litres capacity, fitted with a needle valve at each end. The cylinder is dried by rinsing with acetone and blowing through with compressed air. The dry sampling cylinder is connected by an adaptor and flexible metal tube to the source of liquid ammonia, and, with both valves open, ammonia is blown through until the temperature has dropped sufficiently for liquid ammonia to issue from the bottom valve.This valve is then closed, the sampling cylinder is left in position for five minutes, the other valve is closed and the cylinder disconnected. APPARATUS- The apparatus is illustrated in Fig. 1; the tube, A, is graduated with one mark a t 12 ml and constructed from a 6 x 1-inch Pyrex boiling tube fitted with a standard ground joint To Pump Fig. 1. Evaporation assembly A, titration tube; B and D, traps; C, mercury gauge ; E, stopcock,to give controlled variable leak Plunger Stirrer T Y Pe Rubber stop I A 'I Fig. 2. Titration assembly for and carrying a side-arm that joins the main tube just above the graduation mark.The r 1 A . I % ? I % % 1 1 1 1 n 1 1 1 1 7 1 1 1 tuDe, A, is connectea ~y a stanaara cone to me rrap, D, ana rnence rnrougn me mercury gauge, C, to the trap, D, and finally to the water pump. A controlled variable leak is provided by the stop-cock, E. PROCEDURE- Attach the sampling cylinder to the side-arm of the tube, A, by a short length of pressure tubing. Immerse the trap, B, in a bath containing acetone and solid carbon dioxide at -78" C, and reduce the pressure in the system to about 40 mm of mercury for ten minutes so that traces of water in the tube, A, and its connections are transferred to the trap, B. Restore to atmospheric pressure by opening the stop-cock, E. Place 2 ml of ethylene glycol,76 HODGSON AND GLOVER: THE DETERMINATION [Vol.77 containing less than 0.1 per cent. of water, in the tube, A, by means of a pipette, replace the connecting cone, and immerse the tube and its contents in another bath containing acetone and solid carbon dioxide at -78" C. Cautiously open the valve on the sampling cylinder, so that liquid ammonia is run into the tube, A, via the side-arm. The side-arm acts as a short condenser and prevents loss of ammonia as gas. Close the valve as soon as the liquid level reaches the graduation mark. Remove the cooling bath from around the tube A, wipe the outside of the tube and immerse the bottom part of the tube in the ether vapour boiler. The boiler, a 100-ml beaker containing ether, is heated by a small electric hot-plate.The condensing ether supplies enough heat to the ammonia to maintain steady boiling. Reduce the pressure to about 650mm, so that the ammonia gas not condensed in the trap, B, is removed by the water pump. When the bulk of the ammonia has evaporated, further reduce the pressure to 150mm, continue to heat with the ether vapour bath and introduce a single-coil copper condenser around the tube, A, to prevent loss of ether. After 15 minutes, restore to atmospheric pressure and disconnect the tube, A, from the trap, B. Take 10 ml of a 10 per cent. solution of acetic acid in methanol,. and titrate with Fischer reagent. Add about 10 ml of the titrated solution to the residue in the tube, A, immediately after disconnecting, and attach the tube to the titration apparatus, Fig.2. Titrate the water in the sample and glycol (T,) with Fischer reagent, then add 2ml of the glycol with the original pipette and titrate to determine the glycol blank (T,). Water, yo w/v = (TI - T,) x 10 x W.E. where W.E. is the water equivalent of the Fischer reagent. DISCUSSION Preliminary experiments indicated that ammonia interferes seriously with titrations with Fischer reagent, and early work was directed towards neutralising the ammonia before titration. Various methanolic solutions of acids were tried, but it was found that no satis- factory method could be developed with this procedure; the large amounts of reagents needed to neutralise 10 ml of liquid ammonia gave rise to manipulative difficulties, and heavy precipitates were formed. Spontaneous evaporation of 50 ml of liquid ammonia and titration of the residual water with Fischer reagent gave low and erratic results, which were only useful as an indication of the water content.Experiments were made to determine the effectiveness of separation of liquid ammonia from water by evaporation. Known weights of water were added to liquid ammonia in the tube, A, the ammonia was evaporated under the conditions of the method and the residual water determined by titration with Fischer reagent. Results are shown in Table I. TABLE I SEPARATION OF LIQUID AMMONIA FROM WATER BY DIRECT EVAPORATION Liquid ammonia taken, ml 10 10 10 10 10 10 10 Water taken, g 0.050 0.100 0.100 0.200 0.250 0.300 0.500 Water found, g 0.049 0.1 01 0.098 0.180 0.213 0.260 0-276 Recovery of water, 98 101 98 90 85 87 55 % These results showed that loss of water is significant if more than 1 per cent.is present in the sample. Although most samples of liquid ammonia would be expected to contain less than 1 per cent. of water, it was necessary to extend the method to samples containing water in excess of this amount. Loss of water was prevented by the use of a high-boiling hygroscopic liquid as a retaining agent. The effect of using ethylene glycol was demonstrated by a series of tests in which mixtures of glycol and water were subjected to conditions similar to those of the determination. Table I1 shows results obtained in this way. The accuracyFeb., 19521 Liquid ammonia taken, ml nil nil nil 10 10 OF WATER I N LIQUID AMMONIA TABLE I1 ETHYLENE GLYCOL AS A WATER RETAINING AGENT Ethylene glycol taken, Water added, ml g nil 0.047 2 0.705 2 0.191 2 0.300 2 0-086 Recover of Water found, water, g % 0.03 1 65 0.702 99 0-190 99 0.291 97 0.087 101 77 of the method has been examined by analysing a series of synthetic mixtures prepared by adding anhydrous ammonia to weighed amounts of water in glycol.The results for the synthetic mixtures are shown in the first part of Table 111; the second part shows results of replicate analyses of samples derived from commercial sources. The trap shown in TABLE I11 ACCURACY AND REPRODUCIBILITY Synthetic mixtures- Water added, 0-86 0.64 0-71 0-88 0.48 0.41 0-41 0.34 % Commercial samgles- Water found, 0.87 0.64 0.68 0.84 0-49 0.44 0-42 0-30 % Water found, % 0.04, 0-04 0.07, 0.08 0.01, 0.01 Water added, 0.23 0.21 0.09 0.08 0.06 0-05 0.01 % Water found, 0.23 0.19 0.12 0.09 0-07 0-03 0.02 % Water found, % 0.07, 0.08 0.09, 0.09 0.11, 0.12, 0.09 Fig. 1 has proved to be the most efficient type for preventing water vapour from entering the apparatus from the pump. As the determination proceeds, ammonia condenses in the trap, and this, together with the low temperature, prevents any passage of moisture to the sample. The authors express their thanks to the Directors, British Oxygen Co. Ltd., for permission to publish this paper. REFERENCES 1. 2. 3. Iljin, N. V., Libschitz, G. L., and Tichvinskaja, E. I., J . Chem. Ind. RUSS., 1935, 12, 1, 54. Pleskov, V. A., Zavod. Lab., 1937, 6, 177. Mitchell, J., and Smith, D. M., “Aquametry,” Interscience Publishers Inc., New York, 1948. ANALYTICAL LABORATORY &SEARCH AND DEVELOPMENT DEPARTMENT THE BRITISH OXYGEN COMPANY LIMITED LONDON, S. W. 19 July, 1951

ISSN:0003-2654    

DOI:10.1039/AN9527700074

出版商:RSC    

年代:1952

数据来源: RSC

摘要:

78 Inorganic KEMBER : INORGANIC CHROMATOGRAPHY Chromatography on [Vol. 77 Cellulose Part VII The Determination of Thorium in Monazite and of Thorium and Uranium in Uranothorianite BY N. F. KEMBER A method is described for the quantitative separation of thorium from other metals; it is based on the extraction of thorium nitrate with ether containing nitric acid on a column of cellulose. The method has been applied to the determination of thorium in monazite sands and, by means of a double extraction technique, uranium and thorium have been determined on the same sample of uranothorianite. The behaviour of a number of other anions and cations has been investigated. ALTHOUGH the solubility of thorium nitrate in organic solvents has been widely observed,l this factor has not been used previously for the determination of thorium.The successful use of organic solvents in conjunction with columns of cellulose absorbent for the determina- tion of various metals suggested that a similar procedure could be developed for the determination of thorium. The separation of thorium nitrate from a number of other metals on a paper strip with tetrahydrosylvan containing nitric acid has already been recorded.2 It was observed during work with cellulose columns on the analysis of uranium products3 and in preliminary work by T. V. Arden of this laboratory, that thorium nitrate was partly extracted by solvents consisting of ethyl ether containing small amounts of nitric acid. By increasing the concentration of nitric acid, sp.gr. 1.42, to 12.5 per cent.v/v it was possible to extract thorium nitrate quantitatively, and this observation has been made the basis of a method for the determination of thorium in minerals and ores. The values obtained by this procedure show greater consistency than those by established procedures, particularly for very crude ores. The procedure consisted in preparing a nitrate solution of the sample of monazite sand, free from phosphate, and allowing it to percolate through a cellulose column previously prepared in ether containing 12.5 per cent. v/v of nitric acid, sp.gr. 1-42. Thorium nitrate passed quantitatively into the solvent and was extracted, whereas most of the other metals present remained in the column. The thorium nitrate was recovered from the eluate after removal of the solvent by distillation and the thorium precipitated as oxalate, ignited and finally weighed as Tho,.The method was also adopted for the analysis of uranothorianite for both uranium and thorium on the same sample. The uranium was extracted with ether containing 3 per cent. v/v of nitric acid, sp.gr. 1.42, which left thorium at the top of the column. When extraction of uranium was complete, the solvent was changed to ether containing 12.5 per cent. v/v of nitric acid, sp.gr. 1.42, and the thorium was extracted. Under the conditions used for the extraction of thorium, scandium and zirconium were extracted, although more slowly and less completely than thorium. The movement of these metals was prevented by addition of tartaric acid to the original nitrate solution.The theoretical considerations controlling the efficiency of the separation are similar to those already recorded for ~ r a n i u m . ~ EXPERIMENTAL EXTRACTION OF THORIUM NITRATE- Preliminary experiments were carried out in order to ascertain the lowest concentration of nitric acid in the ether and the volume of solvent needed to extract the thorium nitrate quantitatively. The original solution contained 0.4706 g of thorium dioxide in each 5 ml of 6 N nitric acid and the extractions were carried out in 25-cm columns of cellulose, theFeb., 19521 ON CELLULOSE. PART VII 79 eluate being collected in three fractions of 150ml each. The results in Table I show that the best acidity was 12-5 per cent., no advantage being gained a t higher acidity. TABLE I EFFECT OF ACIDITY OF THE SOLVENT ON THE EXTRACTION OF THORIUM NITRATE Nitric acid in solvent, 3 5 7-5 10 12.5 15 % Thorium nitrate (expressed as Tho,) in r 1 First 150 ml, Second 150 ml, Third 150 ml, mg % mg % mg % <0*05 <0.01 <0*05 <0*01 20.54 4-37 0-25 0.05 0.30 0.06 39.30 8.36 5-95 1.26 11.48 2.44 240.2 51.06 227.3 48.3 160.0 34.0 17.85 3-80 398.0 84.5 34.06 7.2 6.67 1-41 400.1 85.0 34.19 7.3 5.12 1.09 A Total, 4.37 8.47 54-76 86-1 93.1 93-3 % In order to ascertain the volume of solvent needed to give complete extraction at this acidity a further experiment was carried out in which a larger volume of solvent was used.Only 0.05 mg of thorium dioxide was detected between 700 and 800 ml and none after that, 99-5 per cent. of the original weight of thorium dioxide being recovered from the eluates.In an attempt to reduce the volume of solvent needed, the effect of shortening the column was tested, but gave no advantage. It was apparent that the large volume of solvent needed was not caused by adsorption on the cellulose, but by the low solubility or partition of the thorium nitrate in the solvent. A number of extractions were carried out with various quantities of thorium nitrate and 400ml of solvent. The results in Table I1 show that complete extraction of 0-25 g of thorium dioxide was achieved, but larger amounts were not completely extracted. TABLE I1 EFFECT OF INCREASE IN AMOUNT OF THORIUM NITRATE IN ORIGINAL SOLUTION Tho, extracted Tho, present, in 400 ml, Tho, extracted, g g % 0.2481 0.4706 0.9412 1.8824 0.2481 0.4516 0-866 1 1.6720 100.0 94-8 92-0 88.8 In the analyses carried out at a later stage, 600 ml of solvent were used.This increase was necessary to compensate for the increase in the volume of the original solution, as it was found that the rate of extraction of thorium nitrate decreased with an increase in the volume of water in the original solution. Since monazite sands contain a large proportion of phosphate, the effect of phosphate ions on the extraction of thorium nitrate is important. A number of experiments were carried out to investigate the effect of addition of phosphoric acid when ether containing 12.5 per cent. of nitric acid was used as solvent. After the addition of one equivalent of phosphoric acid to a known amount of thorium nitrate, 48 per cent.of the thorium was recovered after the passage of 300ml of solvent, and after the addition of 0.1 equivalent, 94 per cent. was recovered; this demonstrated the necessity of removing phosphates. The addition of sulphate or oxalate to the solution also led to low results. MOVEMENT OF METALS OTHER THAN THORIUM As the final estimation of thorium was carried out by precipitation with oxalic acid, a study was made of those metals that precipitated or tended to co-precipitate under these conditions. The nitrates of Ca, Sn, Pb, Fe, Co, Ni, Cu and Ti did not move more than 2 cm down the column. THE RARE EARTHS- Quadrivalent cerium moved with thorium as a yellow band, but in the cerous form it moved no more than 2 cm; hence it was necessary to reduce all the cerium to its lower valency The remaining nitrates of interest moved as described below.80 KEMBER : INORGANIC CHROMATOGRAPHY [Vol. 77 form before extraction.With samples containing small amounts of cerium, the reducing powers of the cellulose and solvent were sufficient, but in most samples the cerium content was too high for the reaction to go to completion. Ferrous sulphate was effective as a reducer, but a cleaner and more efficient method was to add a few drops of 20-volume hydrogen peroxide to the sample solution and boil to remove excess of peroxide. With non-activated cellulose pulp, i.e., pulp made by mechanical disintegration of cellulose in water, some difference in the rate of movement of the rare-earth nitrates was detected, those of higher atomic weight moving faster.Fig. 1 shows the distribution of a I In wad I In column - n n eluate Fig. 1. Movement of rare earths in a column of non-activated cellulose pulp number of rare earths and pseudo rare earths (scandium and yttrium) in such a column after the passage of 500 ml of solvent. With activated pulp, Le., pulp made by boiling cellulose with dilute nitric acid, the movement of all the rare earths is reduced, and after the passage of 600 ml of solvent none was detected as having moved more than 3 cm. YTTRIUM- The movement of yttrium was shown to be largely dependent on the type of pulp used. No difficulty was found in holding back the yttrium nitrate when activated pulp, i.e., pulp treated with dilute nitric acid, was used, and although yttrium moved down the column more rapidly than any of the true rare earths, none was detected as having moved more than 3 cm after the passage of 600 ml of solvent. With non-activated pulp, however, partial extraction of yttrium nitrate was observed and a purification was carried out on a sample of commercial quality “yttria,” which contained some cerium and a trace of gadolinium.After solution of 0.75 g of the oxide in nitric acid the sample was transferred to a column and extracted with 800ml of solvent. From the eluate 0.18 g of oxide was recovered and was shown spectrographically to be free from cerium and gadolinium. SCANDIUM- It was shown that the extraction of scandium nitrate was almost as rapid as that of thorium nitrate, so that increasing the length of the column gave no advantage.As ammonium tartrate has been used for the partial separation of scandium and thorium, its effect on the chromatographic separation was investigated. In one column it was shown that the addition of up to 3 g of tartaric acid to the nitrate solution did not affect the extraction of thorium; in a second column 0.1 g of Sc,O, as nitrate was added to a known weight ofFeb., 19521 ON CELLULOSE. PART VII 81 thorium nitrate and 3 g of tartaric acid. After extraction with 600 ml of solvent, a quantita- tive yield of oxide (Tho,) was recovered from the eluate, whilst the scandium was found to be entirely within the top 10 cm of the column. On spectrographic analysis the scandium was shown to be completely free from thorium. Scandium can normally be disregarded when dealing with monazite sands, in which it rarely occurs.ZIRCONIUM- Preliminary experiments showed that zirconyl nitrate moved almost as rapidly as thorium nitrate in the column. As in uranium e~traction,~ the addition of tartaric acid to the original solution was also found to inhibit the movement of zirconium. Most monazite sands, however, contain not more than 1 per cent. of ZrO, and the final oxalate precipitation was sufficient to purify the thorium from zirconium, as zirconium oxalate is soluble in an excess of reagent. To test the efficiency of the separation of thorium nitrate from other nitrates, a number of extractions of synthetic mixtures was carried out. The results shown in Table I11 indicate complete separation in all experiments. The oxides obtained on ignition of the oxalates were always pure white.TABLE I11 ANALYSIS OF SYNTHETIC MIXTURES CONTAINING THORIUM NITRATE Tho, present, Other metals added Tho, found, Yield, 0.4706 1 g of La, 0.1 g of Ce* 0.4737 100.7 0.1882 0.1 g of sct 0.1893 100.6 0.2481 0.1 g each of La, Ce, Fe, Co, 0.2489 100.3 g g % 0.2481 1 g of La, 0.1 g of Ce 0.2485 100.2 Ni, Cu, Ti, Sn, Y , Ca, Yb * 800 ml of solvent used. t Tartaric acid added to original solution. It was shown in Table I that no thorium was extracted by up to 300ml of a solvent consisting of ether containing 3 per cent. v/v of nitric acid. Reasonable quantities of uranium could be quantitatively extracted in 150 ml of this ~olvent.~ These were analysed by the double extraction technique with the results shown in Table IV. TABLE IV ANALYSIS OF SYNTHETIC MIXTURES CONTAINING URANYL AND THORIUM NITRATES Uranium Thorium r A \ I 7 Sample Present, Found, Difference, Present, Found, Difference, A 0.1613 0.1617 +0.0004 0.2824 0.2834 +0-0010 B 0.0865 0.0863 - 0*0002 0.3764 0.3748 -0.0016 g g g g g g METHOD SOLVENT PREPARATION AND RECOVERY- The solvent for thorium nitrate extraction was prepared freshly by the slow addition The ether of 12-5 ml of nitric acid, sp.gr.1.42, to each 87.5 ml of peroxide-free ethyl ether. was prepared and recovered by the methods described in Part V of this series3 PREPARATION OF THE CELLULOSE COLUMN- The extraction tube and method of packing were the same as those described in Part V.3 For a normal thorium extraction the column was packed in the solvent containing 12.5 per cent.v/v of nitric acid, but when a double extraction of both uranium and thorium was desired, the column was packed in ether containing 3 per cent. v/v of nitric acid. The minimum length of column depended on the type of cellulose pulp used. A column length of 5 cm was quite sufficient with pulp prepared from ashless tablets, although longer columns were used for most of the analyses quoted. The controlling factor for the length of column was82 KEMBER : INORGANIC CHROMATOGRAPHY [Vol. 77 found to lie in the retention of other metals rather than in the quantity of thorium to be extracted. The movement of yttrium nitrate was a useful guide to this hold-back, as it was found to be the most rapidly moving of the interfering metals usually associated with thorium in minerals; thus the movement of yttrium can be used to indicate the minimum length of column for any particular type of pulp.PREPARATION AND TRANSFER OF SAMPLE- As the original solution for extraction had to be phosphate-free, several methods of phosphate removal were investigated. The methods finally used were as follows- After solution of the sample, thorium, the rare earths, calcium, etc., were pre- cipitated with oxalic acid, the precipitate treated with fuming nitric acid to destroy the oxalate radical and then dissolved in water. This procedure was particularly useful for low grade samples, where the oxalate precipitation also served as an enrichment stage. The monazite sand was treated by the method of Seelye and Rafter4 with con- centrated hydrofluoric acid and taken nearly to dryness on a steam-bath.Chemical decomposition of the sand took place, the thorium and rare-earth phosphates being converted to insoluble fluorides with the release of phosphoric acid. The physical appearance of the sand did not change as a result of this treatment, so that after addition of water the supernatant liquor was easily removed from the heavy sand particles by means of a polythene filter stick (see Fig. 2). Three treat- ments with water were sufficient to remove all traces of phosphate; the resulting product was then fused with potassium hydroxide and the melt leached with hot water. The residual hydroxides were filtered and dissolved in nitric acid. Uranothorianites were usually soluble in hot nitric acid and did not contain phosphate; hence digestion of the sample with concentrated nitric acid was sufficient preliminary treatment.Star-shaped filter paper support cut from co-axial cable r paper disc 0 I Polythene 2 1 I I inches Fig. 2. Polythene filter-stick The strength of nitric acid in the original solution and its total volume were important factors in the efficiency of extraction. AS observed in uranium extractions,3 the movement of a number of impurities, particularly iron, was accelerated in higher acid concentrations. The volume of solution used had considerable effect on the volume of organic solvent needed. For the transfer of the sample to the column the “wad” technique, as described in Part V,3 was used for monazite sands. Uranothorianites gave a less viscous solution that could be poured directly on the top of the column.THE EXTRACTION OF THORIUM- The procedure for the extraction of thorium from monazites was the same as that already described for uranium in Part V,3 except that the solvent used was ether containing 12.5 per cent. v/v of nitric acid. After the passage of 600 ml of solvent, ammonium hydroxide was added to the eluate to neutralise part of the acid, and the excess of solvent was distilled for recovery. Ammonium hydroxide was added to neutralise the remaining acid, and the solution was then re-acidified with hydrochloric acid. Oxalic acid was added to the boiling solution, the precipitated thorium oxalate filtered, ignited to Tho, and weighed.Feb., 19521 ON CELLULOSE. PART VII 83 For the analysis of uranothorianites, ether containing 3 per cent.v/v of nitric acid, sp.gr. 1.42, was first used until 150 ml of eluate had been collected; this extracted the uranyl nitrate. The receiving flask was changed, and 600 ml of the solvent of higher acidity was then passed through the column to extract the thorium nitrate. The uranium was estimated in the first fraction by ceric sulphate titration5 after removal of the excess of solvent by distilla- tion and conversion of the uranyl nitrate to sulphate by boiling with sulphuric acid in a Kjeldahl flask. The thorium in the second fraction was estimated by oxalic acid precipitation. ANALYTICAL PROCEDURES I. MONAZITE SANDS : FIRST PROCEDURE- Weigh into a platinum dish sufficient sample to yield not more than 0.25g of Tho,, add 10ml of hydrofluoric acid and evaporate nearly to dryness on a steam-bath.Add 20ml of 5 per cent. hydrofluoric acid, warm, and remove the supernatant liquor with a polythene filter stick, Fig. 2, provided with a disc of Whatman No. 540 or 541 filter-paper. Repeat the complete hydrofluoric acid treatment twice, then wash the sample into a nickel crucible and remove the supernatant liquor as before. Add the paper disc from the filter- stick to the crucible and evaporate to dryness on a steam-bath. Add potassium hydroxide pellets corresponding to 5 times the weight of sample and remove excess of water from the alkali by placing the crucible under an infra-red lamp for 15 minutes; or, failing this, heat very gently over a low gas flame.Then fuse strongly for 45 minutes, although red heat is not necessary as the phosphate has been removed and the sand is less refractory than previously. Cool and transfer the crucible and lid to a 250-ml beaker and extract the hydroxides by boiling with 200 ml of water. Remove and wash the crucible and lid. Add a few grains of soluble starch to the suspension of hydroxides and boil. Cool and allow to stand for 5 minutes; the hydroxides should settle and leave a clear supernatant liquor. Remove the supernatant liquor to about 0.5 crn above the hydroxides with the aid of the polythene filter-stick and a disc of Whatman No. 541 paper. Add 200 ml of water, boil and allow to settle, removing the supernatant liquor as before. Should the filter-stick clog at any stage, replace the filter-paper disc and keep all discs used.Transfer all the discs to a 50-ml beaker, destroy the paper by digestion with 1 ml of fuming nitric acid and transfer to the main beaker. Add 20 ml of concentrated nitric acid to the main beaker and boil gently. The solution should clear; if not, add concentrated nitric acid until it does and then evaporate to dryness. Caution should be observed at this stage as occasionally a little zirconium phosphate settles out and causes bumping. Add 5 ml of water, 0.2 ml of con- centrated nitric acid and 0.5 ml of 20-volume hydrogen peroxide. Warm on a steam-bath until all the red colour is discharged. Prepare a cellulose column 7.5 cm long, as previously described in Part V,3 packing it in a solvent consisting of ethyl ether containing 12.5 per cent.v/v of nitric acid, sp.gr. 1.42. Add sufficient dry cellulose pulp to the sample solution, with stirring, to give a semi-dry mass; an addition of about 6 g is usually sufficient. Transfer this to the top of the column and beat up the “wad” so formed with a glass rod to form as continuous a part of the column as possible, Begin extraction with the solvent, transferring it at first via the sample beaker to wash it out. The column should never be allowed to run dry and the supernatant liquor should not exceed 10 ml. Collect the eluate in a 1-litre flask to a total of 600 ml. After the extraction, add slowly to the receiver 100ml of water containing 30ml of ammonium hydroxide, sp.gr. 0.880. Remove the excess of ether by distillation and transfer the acid solution to a 500-ml beaker and cool.Just neutralise with ammonium hydroxide, sp.gr. 0.880, add 25 ml of concentrated hydrochloric acid, make up the volume to about 400 ml with water and boil. Add 20 g of oxalic acid crystals slowly with stirring and allow to cool. Filter the precipitated thorium oxalate through a Whatman No. 42 filter-paper or Gooch crucible and wash the precipitate with 100ml of a wash liquor consisting of 2 per cent. of oxalic acid in 0.1 N hydrochloric acid. Ignite the precipitate at 850” C and weigh as Tho,. Cool and add 2 ml of concentrated nitric acid. 11. MONAZITE SANDS: SECOND PROCEDURE- Take a sample of the material containing not more than 0.25 g of Tho, into a sulphate or chloride solution. A pure monazite will dissolve readily after digestion in hot concentrated sulphuric acid; cruder samples containing highly refractory minerals will require fusion with potassium hydroxide as described in the first procedure, above.Adjust the acidity of the84 KEMBER : INORGANIC CHROMATOGRAPHY fVol. 77 solution to 0-5 N with hydrochloric or sulphuric acid, at the same time adjusting its volume to 200 ml, and then boil. Add 100 ml of boiling 25 per cent. oxalic acid solution with stirring. Allow to cool and filter through a Whatman No. 542 filter-paper, washing the precipitate with 2 per cent. of oxalic acid in 0.1 N hydrochloric acid. Wash the precipitate into a 100-ml beaker with water and evaporate to dryness. Add 20 ml of fuming nitric acid andevaporate to dryness; repeat the nitric acid treatment and evaporation. It is essential to destroy all traces of oxalate, as its presence in the column retards the extraction of thorium.Dissolve the dry nitrates in water, treat with hydrogen peroxide, add 2ml of concentrated nitric acid and continue with the extraction and recovery as described in the first procedure. 111. URANOTHORIANITES- Weigh 0.4 to 0.5 g of the finely ground sample into a 100-ml beaker, add 10 ml of con- centrated nitric acid, cover with a watch glass, heat gently under reflux for 2 hours and then add 5 ml of water. This treatment usually gives a clear solution containing a little silica in suspension. If any heavy precipitate remains, remove it by filtration and fuse it with a few pellets of potassium hydroxide, extract the melt with hot water and transfer the hydroxides to the original solution.Evaporate the solution to dryness and dissolve the residue in 5 ml of water. Treat with hydrogen peroxide and add 2 ml of concentrated nitric acid as described above for monazite sands. Prepare a cellulose column 15 cm long in ether containing 3 per cent. v/v of nitric acid, sp.gr. 1.42. Run out the solvent until there is no supernatant layer, then pour on the prepared nitrate solution and wash it into the column with more solvent from a wash bottle. Extract with 150ml of the solvent, taking the same precautions as before, and collect the eluate in a 500-ml flask. Replace this receiver by a l-litre flask and change the solvent to ether containing 12.5 per cent. v/v of nitric acid, sp.gr.1.42, and extract with 600ml. To the first flask, which contains all the uranium and no thorium, add 100 ml of water and remove the ether by distillation. Determine the uranium by any suitable method, e.g., by ceric sulphate titration.6 Determine the thorium in the second flask by oxalate precipita- tion as described in the first procedure, above. RESULTS Tables V and VI show a number of results by the methods described and by standard methods, vix., iodate - oxalate precipitation. TABLE V MONAZITE ANALYSES BY EXTRACTION COLUMN PROCESS Sample and method Procedure I- 1 1 2 2 2 2* ? 3 4 5 Procedure II- 6 4 4 Sample weight, g 2.1241 2.0387 2.5734 2.8578 2-5499 2.1247 3.6834 3.5546 3.0861 2.3061 7.3 105 2.1225 2.2677 2.6095 Weight of Tho, extracted, g 0.2046 0.1960 0.2398 0.2660 0.2392 0-1993 0-2322 0-2215 0.1956 0-1639 0.0520 0-2086 0.1616 0.1860 Tho, found, % 9.63 9.62 9.32 9-31 9-38 9-38 6-3 1 6.23 6.34 7-12 0.7 1 9.83 7-10 7.13 Results by standard methods , % } 9-65 } 9.48 } 6.0 7-17 0.71 9.80 } 7.17 * To this sample was added 0.1 g each of Y , Sc, Zr, Ti, Fe, Co, Ni, Cu, Pb and Sn before analysis.t The sample was of crude monazite that gave erratic results by the normal analytical procedure (i.e., Tartaric acid, 3 g, was added to the original solution. the iodate - oxalate method).Feb., 19521 ON CELLULOSE. PART VII 85 TABLE VI ANALYSIS OF URANOTHORIANITE BY THE DOUBLE EXTRACTION PROCEDURE Sample 7 7t 8 8 9 9 10 Weight of sample, g 0.4589 0.47 1 1 0.4828 0.4938 0.4997 0.4676 0.4924 Weight of extracted , g 0.1761 0.1798 0.1703 0.1749 0.0682 0.0650 0.1301 ‘3’8 Uranium* P standard u3°8 by U,O,, methods, 38.4 38.4 % % 38.2 99 35.3 38-5 35.4 99 13-7 13.3 13.6 99 26.4 26.4 .Weight of Tho, extracted , g 0.2491 0.2558 0.2335 0.2387 0.3777 0-3535 0-2881 Thorium 54.2 48-4 48.4 75.9 75-6 58.5 Tho, by standard methods, 53.7 % 97 48.7 74-1 57.5 99 99 * Uranium was determined by titration with ceric sulphate in both the extraction and standard t To this sample 0.1 g each of Fe, Mo, V, Ca, La, Nd, Sm, Ti, Zr, Ce, Co, Ni, Sn and Pb were added. procedures. CONCLUSION The work carried out, besides being the basis of a number of quantitative analytical techniques, has shown the possibilities of development on parallel lines of the extraction of a number of other metallic nitrates, such as scandium, zirconium and yttrium. Further, although previously limited to comparatively small quantities, indications are that the scale of the extractions can be increased, making the procedure useful for the preparation of extremely pure samples for research purposes. This work has been carried out on behalf of the Ministry of Supply, and is published with the permission of the Ministry and the Director of the laboratory. REFERENCES 1. 2. 3. 4. 5. NOTE-ReferenCeS 2 and 3 are to parts I1 and V of this series. Rothschild, B. F., Templeton, C. C., and Hall, N. F., J . Phys. Coll. Chem., 1948, 52, 1006. Burstall, F. H., Davies, C. R., Linstead, R. P., and Wells, R. A., J . Chem. SOC., 1950, 516. Burstall, F. H., and Wells, R. A., Analyst, 1951, 76, 396. Seelye, R. T., and Rafter, T. A., “Report of the Dominion Laboratory, Wellington, N.Z.” (un- “Handbook of Chemical Methods for the Determination of Uranium in Minerals and Ores,” H.M. published). Stationery Office, London, 1950. RADIOCHEMICAL GROUP CHEMICAL RESEARCH LABORATORY TEDDINGTON. MIDDLESEX Augwst, 1951

ISSN:0003-2654    

DOI:10.1039/AN9527700078

出版商:RSC    

年代:1952

数据来源: RSC