ISSN:0003-2654    

DOI:10.1039/AN95075FX037

出版商:RSC    

年代:1950

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95075BX039

出版商:RSC    

年代:1950

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95075FP071

出版商:RSC    

年代:1950

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95075BP077

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

OCTOBER, 1950 Vol. 75, No. 895 INTERNATIONAL UNION OF PURE AND APPLIED CHEMISTRY SECTIOX V-ANALYTICAL CHEMISTRY THE Section of Analytical Chemistry of the International Union of Pure and Applied Chemistry, organised at the meeting of the Union, September, 1949, has announced its complete member- ship, which is as follows- Honorary President-Sir Ian Heilbron, F.R.S. Presidertt-Prof. Dr. C. J. van Nieuwenburg, Technische Hoogeschool, Delft, Netherlands. Vice-President-Prof. I. M. Kolthoff, University of Minnesota, Minneapolis, Minn. Ewopea.tz Secretary-Prof. Dr. Paul E. ’illenger, L’Ecole de Chimie de l’Universit6, Geneva, Switzerland. A mericnn Secretary-hilr. Samuel E. Q. Ashley, General Electric Company, Pittsfield, Massachusetts. Other Members-Prof. Dr. Fritz Feigl, Laboratorio da Producao Mineral, Rio de Janeiro, Brazil.Dr. Jan Gillis, University of Ghent, Belgium. Prof. Sir Shanti S. Bhatnagar, Dept. of Scientific Research, New Delhi, India. Mr. R. C. Chirnside, Research Laboratories of The General Electric Company, Ltd., Wembley, England. Prof. F. E. Beamish, University of Toronto, Canada. Prof. G. Charlot, L’Ecole de Physique et de Chimie, Paris, France. ‘This membership, which is considered to be temporary, will function until the meeting in New York in September, 1951. At present the Section membership is concerned with the establishment of the two new Commissions on Physico-chemical Constants and on the Expression of Analytical Results. The Commission on New Analytical Reagents and Reactions, which has been functioning for some time, will continue under the sponsorship of this Section. 509 Dr. Edward Wichers, Sational Bureau of Standards, Washington, D.C. Mr. N. Strafford, Imperial Chemical Industries Ltd., Manchester, England.

ISSN:0003-2654    

DOI:10.1039/AN950750509b

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

ti10 WILLIAMS : DETERMIKATIOX OF FLUORIDE BY ETCHISG [Vol. 75 PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS Determination of Fluoride by Etching BY H. AMPHLE‘CT T31‘ILLIAMS (Read at the meeting of the Society on Wednesday, May 3rd, 1950) SuNoPsIs-The reactions involved in the etching of glass hat-e been studied with a view- to the development of a specific quantitative method for the determination of small quantities of fluoride. Optimum conditions for high sensitivity have been found by trial, and a method capable of detecting 0.1 pg. of fluoride ion is described. The reactions which take place under these conditions have been found to comprise the dissolution of silica from the glass by the hydrogen fluoride, and the deposition of hydrated silica through hydrolysis of the silicon tetrafluoride formed, with the production of a matt “etching” and the regeneration of hydrogen fluoride.It is concluded that a process in which the fluoride acts as a catalyst provides no sound basis for quantitative analysis. THE etching test, in which the fluoride ion is identified by its property of attacking glass, is the oldest known test for fluorides; it is still one of the most widely used. The reason for its popularity lies in its specificity, for the acids of fluorine are believed to be the only acids that will dissolve glass rapidly and in dilute solutions. Numerous attempts have been made to derive a reliable quantitative method for the determination of fluoride ion by iitilising this property, a few with some measure of success.Thirty of the more promising methods published during this century have recently been studied by the author,* but only brief reference can be made here to the literature. The majority of published methods afford only an approximate estimate of quantity, since they depend upon visual comparison of the depth of an etching produced on 3 piece of glass that has been exposed to the vapours resulting from treatment of the fluoride with concentrated sulphuric acid with that produced under similar conditions by a known weight of fluoride ion. The minimum quantity of F’ which will give a visible etching has been variously reported as between 10 and 0.1 mg., the average being about 0.5 mg.; by using modified techniques higher sensitivities have been claimed by some analysts, notably Woodman and Talbotl and Gautier and Clausmann,2 who found that by heating the reaction mixture to 220” C .in suitable apparatus a few micrograms of F’ could be detected. With quantities of that order, however, these workers were unable to obtain consistent etchings. Several workers have attempted to estimate fluoride ion from the loss of weight observed when a piece of glass is exposed to the hydrogen fluoride evolved by treating the sample, or a purified concentrate obtained from the sample, with sulphuric acid. In Brauns’ m e t h ~ d , ~ for instance, for the determination of F’ in organic compounds, the material is digested in a small Pyrex flask with sulphuric acid and potassium nitrate until decomposition is complete, when the fluorine content is calculated from the loss of weight of the flask.But, unfor- tunately, almost all minerals containing fluoride ion also contain silica, the presence of which might vitiate an etching test through combining with the fluoride ion as silicon tetrafluoride; and the complete separation of the F’ from the Si”” is a lengthy and unsatisfactory operation. With skilful manipulation, however, Mayrhofer and Wasitzky4 obtained good results with known weights of sodium fluoride; the average loss in weight of a small, inverted Jena glass funnel, fitted into the top of a crucible in which the purified fluoride residue was heated with concentrated sulphuric acid, being 0.96 mg. of glass per mg. of F’. In another ingenious method,2 the fluoride residue after removal of silica is treated with sulphuric acid in a closed platinum crucible containing a small basket of fragments of * A full review of the literature of etching and other tests for fluoride appears in the author’s Doctorate Thesis entitled “A Study of Existing Methods of Fluorine Analysis and the Development of an Improved Method of General Application” (University of London, 1948), in which is also included the greater part of the experimental work upon which this paper is based.October, 19501 WILLIAMS : DETERMINATION OF FLUORIDE BY ETCHING 51 1 optical glass (containing 42 per cent.of lead) suspended above the reaction mixture; after treatment for 5 hours at 140” C. the lead fluoride, which is formed on the glass in proportion to the hydrogen fluoride liberated, is dissolved in potassium chlorate solution and the lead determined colorimetrically.Quantitative etching methods of this type have been used where the quantity of fluoride ion to be determined exceeds 0.1 mg.; where the F’ is less than this, they have been found generally unreliable owing to errors inherent in the procedures available for concentration of the F‘ and its separation from Si““. The object of the present investigation was to ascertain whether the attractive, if elusive, possibilities suggested by the solvent action of hydrogen fluoride on glass could be utilised as a basis of a specific quantitative method for the determination of very small quantities of fluoride in diverse materials. Although no such object has been attained, it is hoped that the conclusions and the sensitive etching test which follow may be of interest to other analysts seeking the same goal.Owing to the practical drawbacks of weighing microgram quantities, gravimetric methods were regarded as undesirable and consideration was first given to the principle of producing a permanent etched mark, the extent of which could be determined by optical methods. But before the development of any such method is justified two questions must be answered: whether sufficient sensitivity could be obtained, and whether proportionate and comparable manifestations of the extent of attack on glass could be obtained with known amounts of fluoride. Since the only way in which these questions could be answered was by the method of trial and error, a very large number of etching tests have been performed, under various conditions.The results summarised below show the effect of varying the experimental conditions. EXPERIMENTaL The method usually given in the textbooks of analysis was first tried, a known weight of fluoride being heated with sulphuric acid in a platinum crucible covered with a waxed watch glass engraved with a distinctive mark. In the majority of these trials, the fluoride ion was introduced in the form of weighed quantities of intimately ground mixtures of purified calcium fluoride and potassium sulphate, free from silica, containing 1, 0.1 or 0.01 per cent. of fluorine ; measured volumes of standard sodium fluoride solution, gently evaporated in the reaction vessel, were substituted occasionally as a check.In the general textbook method the reaction mixture cannot be strongly heated owing to the wax melting, and further trials were carried out with an unwaxed watch glass at a higher temperature. Sensitivity appeared to be slightly higher, as shown in Table I. TABLE I ETCHINGS OBTAINED BY “TEXTBOOK” METHOD -1. Apparatus-Platinum crucible with waxed and marked watch glass. F’ introduced, Time of heating, Etching produced mg. min . 10 20 deep 1 20 fairly deep 0.5 30 slight 0-3 40 very faint 0.1 40 nil (bis) I3. Apparatus-Platinum crucible with unwaxed watch glass ; higher temperature. 0.5 0-3 0.2 0-2 0.1 0.1 30 definite but irregular 30 slight but irregular 15 faint 60 faint 30 nil 60 nil Etchings were patchy and the methods insufficiently sensitive, probably owing to the imperfect fitting of the watch glass on the crucible rim and the large area of glass exposed.To ensure better fitting and exactly comparable conditions, a rectangular lead block was cast with two parallel cylindrical cavities. The top of the block was ground flat and the cavities,512 WILLIAMS DETERMISATION OF FLUORIDE BY ETCHING [Vol. 75 after introduction of the fluoride and acid, were covered with a microscope slide held in position by a lead cover over a felt pad. The tests were carried out by heating the lead block in an oil-bath and varying the temperature, time of heating and quantity of acid. TABLE I1 EFFECT OF VARIATION OF TIME AND TEMPERATURE OF HE.4TING AND QUANTITY OF -4CJ.D Apparatus-Lead block with two caxTities, covered with unwaxed microscope slide.First four trials with cavities 7/16 inch in diameter, remainder with new lead block having smaller cavities 5/16 inch in diameter. Time of F' taken, H,SO,, heating, Teniperatu re, Etching mg. ml. min . O c. 0.3 2 30 "0 very distinct 0.2 2 30 2000 definite 0.1 3 30 800 faint 0.05 2 30 800 nil 0.2 1.5 30 300 well defined 0.1 1.5 30 200 slight but definite 0.05 1.5 30 200 very faint (bis) 0.03 1-5 30 300 nil 0.05 1.0 30 200 very faint 0-05 2.0 30 200 very faint 0.05 1.5 60 150 very faint 0.03 1.5 60 200 nil 0.03 1.5 9 0 200 nil Sensitivity was increased in this way to about 0-05 mg. of fluoride ion, but could not be further increased by variations of time of heating, temperature, volume of acid, nor by B Cooling Water c A Lead Cover Fig.1 G I yce ri n-Li n t Pad / ,GI ass Cove r-si i p ,Lead Block ,Medicinal Parafi n insertion of lead washers having holes of 1/16, 118 and 3/16 inch diameter over the cavities and under the glass slide, which was tried with a view to reducing the area of glass exposed to attack. The temperature could not be raised appreciably above 200" C. owing to the risk of acid creeping up and covering the glass, but 150" C. appeared to be sufficient for the etching.October, 19501 WILLIAMS : DETERMINATION OF FLUORIDE BY ETCHISG 5 13 This is in general agreement with Caley and Ferrer's conclusions,j using a similar technique in 1937. For the next series of trials, on the recommendation of certain of the earlier workers, direct cooling of the glass was introduced.A simple cooling apparatus consisting of flat- bottomed test tubes through which cold water circulated was employed, as shown in Fig. 1 ; the bottoms of the test tubes resting upon the glass, with pads of lint soaked in glycerin inserted between the glass surfaces to assist conduction. Microscope cover-slips were sub- stituted for the thicker slides, to avoid cracking through the wide difference of temperature on the two sides of the glass. TA4BLE 111 EFFECT OF COOLING THE GLASS -4pparatzts-Lead block : cooled, unwaxed , cover-slips. F' taken Time of heating, min. 30 30 3 0 30 30 60 90 30 30 30 30 45 60 90 45 45 45 Temperature, c. 150 150 150 150 150 150 150 165 180 180 180 180 180 180 180 180 200 Etching very deep deep definite slight nil nil nil faint definite faint nil just perceptible very faint very faint nil (gave smear when breathed upon) nil 2 spoiled, 2 faint but very irregular The results, given in Table 111, show that a remarkable increase in sensitivity can be achieved by keeping the glass cool throughout the test.Since it is difficult to conceive that the rate of attack of hydrogen fluoride on glass would be greater at low temperatures, the increased action is ascribed to concentration of the hydrogen fluoride in the droplets of condensation which form on the glass, and to a prolonged opportunity for attack through avoiding the evaporation from these droplets that probably occurs when the glass becomes heated. The trials show that by heating for 45 minutes at 180" C. a perceptible etching can be obtained from as little as 1 pg.of fluoride ion, and they suggest that still less might be detect- able if the temperature could be raised above 180" C. Difficulties were encountered when this was attempted, the causes being (i) flooding of the cover-slip by creeping or distillation of sulphuric acid, or both, and (ii) deposition of sulphur and water on the cover-slip owing to increased action of the sulphuric acid on the lead. Even at 180" C., etchings were very irregular, the attack producing deep pits in some parts of the glass and a rough, matt surface in other parts, and critical visual comparison of one etching with another was impossible. Moreover, in the course of further trials, a third source of error was encountered, viz., (iii) it was found that after an etching test had been carried out with a relatively large quantity of fluoride ion (20 to 100 pg.), subsequent etchings from very small quantities tended to be erratic and often high, although the cavities were washed out thoroughly between tests.This was ascribed to adsorption of hydrogen fluoride or formation of lead fluoride in the upper part of the cavity, and it was found that distinct etchings sometimes appeared with no added fluoride. Satisfactory "blanks" were only obtained in such circumstances after the lead block had been strongly heated in sulphuric acid, a procedure which caused further corrosion. These three difficulties render the estimation of microgram quantities of fluoride by etching with lead apparatus impossible, and an alternative material was sought.Eventually a new apparatus was constructed in the following manner.514 WILLIAMS : DETERMINATION OF FLUORIDE BY ETCHING [Vol. 75 To contain the reaction mixtures and to provide satisfactory seating for the cover-slips, three platinum tubes, 1& inches long by $ inch internal diameter, with flat 1-inch diameter flanges at the open end, were made. The oil-bath, which had proved troublesome owing to the creeping of the fluid (whether medicinal paraffin, wax, or glycerin), was replaced by a solid block of Duralumin, 3 x 24 x 2& inches, with three equidistant holes, 5/16 inch in diameter and 12 inches deep, drilled to take the platinum tubes, and a fourth hole drilled obliquely to the centre of the block to take a thermometer. A few drops of medicinal paraffin, sufficient to cover the bulb of the thermometer, were inserted in the oblique hole, and a small coiled wire spring in each of the other holes; the platinum tubes when inserted fitted snugly into the holes and no conducting liquid was found to be necessary or desirable.Heating was effected by means of an adjustable electric hot plate upon which the block was placed, and cooling by means of a conical flask condenser, the bottom of which was ground flat. The fluoride and acid were placed in the tubes, covered with No. 3, $-inch circular cover-slips, and the flask, supported by a retort stand, was gently lowered into position. Contact with the cover-slips was effected by the use of glycerin-soaked lint washers, # inch in diameter. By means of this apparatus, shown in Fig.2, three tests may be carried out simultaneously under identical conditions and interference from extraneous reactions and the creeping of oil is obviated. U Platinum Tube C 0 0 0 0 Plan of Block Fig. 2 The most suitable amount of acid, to leave only a small air space but to avoid risk of creeping up to the cover-slip, was found to be 0.7 ml., and this volume of pure A.R. sulphuric acid was used in each tube. The first 30 trials, with quantities of fluoride ion ranging from 0-1 t o lopg., produced no etchings whatever, no matter what the temperature, time of heating, type of glass or surface condition; even the possibility of lead acting as a catalyst was considered and some lead turnings were added, but without effect. Results with the platinum apparatus proved disappointing and puzzling.October, 19501 WILLIAMS DETERMISATION OF FLUORIDE BY ETCHING 515 Eventually another supply of sulphuric acid was tried; this gave a slight etching with 2pg. of fluoride ion.Four other supplies were then tried, one of which, from the same bottle of ordinary “pure” acid that had been used in the trials with the lead block, gave much better results. Since the principal variant in the different supplies of sulphuric acid was probably the moisture content, it seemed that this might be the essential factor. On determining the strength of various acids by titration and on trying dilutions prepared from the AnalaR acid which had failed to produce any etchings, this proved to be so. The results are shown in Table IV, from which it will be seen that the optimum concentration is apparently 96 to 96.5 per cent.w/w of sulphuric acid (H,SO,). TABLE IV EFFECT OF CONCENTRATION OF SULPHURIC ACID ,-1PPnl.n2us-Platinurn tubes ; cooled cover-slips ; heating for 1 hour at 200” C . F’ taken, /*g- 5 5 5 5 > Grade of sulphuric acid =Inalali (B.D.H.) “Sitrogcn-free” (B.U.H.1 ‘‘Technical” (B.D. H.) “Pure” (T. & 31.) “Pure” (B.D.H.), as used with lead block -4nalaR (B.D.H.), diluted ditto ditto ditto ditto ditto H,SO,, per cent. w/w 97.6 96.8 92.3 98.8 96.3 Etching nil slight nil slight definite very faint definite (ter) definite (bis) faint doubtful nil ’The trials with different supplies of acid were conducted at 200” C. for 1 hour. At this temperature droplets of moisture condense on the cooled cover-slip and the evolved hydrogen fluoride dissolves in them.The character of these droplets is critical, since if they do not form rapidly enough the hydrogen fluoride may be evolved first and escape under the cover- slip without being dissolved, and if they are too large the solution of hydrofluoric acid formed will be too dilute to attack the glass. According to Olivier,6 solutions of hydrofluoric acid containing less than 0.00033 per cent. of fluorine do not visibly corrode glass. Moreover, whilst a very fine mist produces an even, matt etching of a type suitable for comparison, larger droplets produce irregular craters in the glass. This seems to be the explanation of the vital importance of the moisture content of the sulphuric acid. \Wh the exclusive use of 96.5 per cent.sulphuric acid, further trials were carried out with the platinum apparatus to ascertain the optimal conditions, firstly as to temperature and time of heating. Results were not entirely consistent, and even when simultaneous triplicate tests were performed differences in the area or depth of the etchings would occur; but from a large number of trials the following inferences appeared to be justified: (i) that the hydrogen fluoride is not completely evolved at 180” C.; (ii) that a temperature of 220” t o 240” C. is necessary; (iii) that the action is not completed by the time the tubes reach 220” or 240” C,., a further period at that temperature being necessary; (iv) that no further action occurs at higher temperatures ; (v) that gradual heating increases the sensitivity, the best method apparently being to raise the temperature to 120” C.in 45 minutes, to 220” C. in the next 45 minutes, and to maintain at 220” to 230” C. for 45 minutes more; (vi) that no appreciable increase in the sensitivity can be achieved by raising the temperature of the cover- slip during the test; and (vii) that effective contact between the cover-slip and the cooling surface is important. To secure this, the glycerin-soaked lint pad, which tended to enclose air bubbles, was replaced by a small drop (0.02 ml.) of glycerin containing about 2 per cent. of agar, and a fine wisp of cotton wool, placed in the centre of the upper side of the cover-slip. The experimental requirements of thinness and flatness practically restricted the choice to microscope cover- slips, but careful selection of the grade of cover-slip was found necessary, as some supplies showed a finely pitted surface when examined microscopically with oblique illumination, and variations in sensitivity to attack were also found.Regarding the preparation of the glass surface for etching, it was thought possible that destruction of the molecular symmetry of the surface layer, for example bj- grinding or by pi-eliixiiiar>- attack, might render the The type and condition of the glass to be etched was next considered.516 WILLIAMS : DETERMISATIOS OF FLUORIDE BY ETCHISG [Vol. 75 substance of the glass more vulnerable. Superior etchings of greater depth and clarity were in fact obtained with the ground and polished glass cover-slips with optically plane surfaces which are specially made for use with haemocytometer cells ; the sensitivity, however, was not greatly enhanced.Pre-treatment of cover-slips by immersion in hydrofluoric acid baths of a strength just too low to cause pitting (> 1 per cent.) did not appear to render the glass any more susceptible. Further trials indicated that the state of hydration of the glass surface exerted a marked influence on sensitivity. The best results were obtained when the glass was in a highly hydrated state, and the cover-slips, selected after careful examination from a single packet, were cleaned in chromic acid mixture, soaked in distilled water, and air-dried immediately before use. On applying the technique and precautions outlined above, a marked increase in sensitivity was obtained with the platinum apparatus, as little as 0-2 pg.of fluoride ion giving a visible etching. The platinum tube overcame the difficulties encountered with the lead block at the higher temperatures, and produced, in the centre of the exposed area of the cover-slip, round matt etchings of a type which would readily admit of comparison, but replicate etchings from similar quantities of fluoride were not always consistent. Before examining further means of increasing the sensitivity, the question was considered whether the maximum degree of attack theoretically possible had not already been attained. Could it be obtained, a measure of the quantity of silicon separated from the glass in the process of etching or of the amount of combined fluoride ion in the end-products might show whether the whole of the F' taken had been induced to attack the glass.Attempts to extract and determine the silicon in the deposit of condensed moisture and end-products that formed on the cover-slip during the etching, by solution in alkalies, etc., were unsuccessful owing to high blanks from unetched cover-slips, and determinations of the residual combined F' by distillation of the deposit on the cover-slip from perchloric acid, followed by thorium nitrate titration, yielded only from one-fifth to one-quarter of the F' originally taken. I t seemed possible, however, that a proportion of both Si"" and F' might have escaped in the form of silicon tetrafluoride. In these circumstances the only practicable way to ascertain how much glass had been attacked appeared to be by direct weighing of the cover-slip before and after attack.A further series of etching tests, with larger amounts of fluoride, was therefore carried out, the cover-slips being weighed on a microgram balance after standardised cleaning and pre-weighing technique. T-~BLE V LOSS OF WEIGHT OF GLASS COVER-SLIPS Apparalus-Platinum tubes; cooled cover-slips; heated to 120" C. in 45 minutes, to &-O b'') ' C. in next 45 minutes, and a t 220" to 230" C. for 45 minutes more. Weight of cover-slip before F' taken, etching, mg. mg. nil* 148.535 nil* 145.824 nil* 138-867 nil* 129.086 nil* 139.005 Weight after etching and cleaning, mg. 148.533 145-8 19 138.856 129*080 139.001 Loss corrected for Glass to Loss, ' 'blank' ' F' ratio mg. 0.002 0.005 0.01 1 0.006 0.004 Mean cleaning blank., .. .. 0-006 1.000 139.900 138.862 1 *038 1.000 200.760 199.747 1.013 1.000 11 1.808 110.822 0.986 0.500 128.804 128.2 84 0.520 0.200 109.432 109.2 16 0.216 * Cover-slip cleaned only. 1.032 1.032 1.007 1.007 0.980 0.980 0.514 1.028 0.210 1.050 From the results shown in Table V it can be seen that the average loss of weight by etching was equal to 1.02 mg. per 1 mg. of fluoride ion. This ratio of loss, it may be observed, is higher than any found in the literature (0.5 to 0.96 rng, per 1 mg. of F'). Enquiries elicited the information that the glass used approximated in composition to the "formula,"October, 19501 U'ILLIAMS : DETERMISATIOS OF FLUORIDE BY ETCHISG 517 Na,O.CaO.SSiO,; assuming this to be true, the theoretical loss of weight, on the basis of the "equation"- should be 0-916 mg. per 1 mg.of F'. Even allowing for considerable variation in the com- position of the glass, it would seem evident that the attack effected by the F' had been not less than 100 per cent. efficient. Since no appreciable increase of sensitivity could therefore be expected, attention was directed to the effect caused by the presence of traces of silica in the fluoride tested, a question Sa2O.Cs0.5SiO, +- 24HF -+ Sa,SiF, + CaSiF, - 3SiF, -+ lPH,O, TABLE VI ETCHING IN THE PRESEXCE OF SILIC.~ ~ppnl.atzfs-Platinnin tubes; cooled cover-slips; heated to 120" C. in 43 minutes, to 220" C. in next 45 minutes, and at 220" to 230" C. for 46 minutes more.F' taken 1 CLg. 1 CLg- 1 Ph". 1 ELg. 1 P.9. 1 LLg. 1 pg. 1 tLg. 2 pg. 5 pg. 0.4 pg. 1 pg. 1 nil nil 1 mg. 1 mg. 0.2 mg. 1 CLg. 0.1 pg. 1.5 pg. 0.4 pg. 0.2 pg. 0.1 pg. 0.1 pg. 0.1 pg. 0.1 pg. 0.2 pg. 0.06 pg. 0.6 pg. 0.3 pg. 0.7 pg. 0.5 pg. 0.5 pg. 0.8 pg. 0.8 pg. 0.8 pg. 0.4 pg. 0.3 pg. 0.26 pg. 0.13 pg. Source of F' CaF, CaF, CaF, CaF, NaF S a F CaF, CaF, CaF, CaF, CaF, CaF, CaF2 Ca 17, - - c a F, CaF, Sa,SiF, Xa,SiF, Sa,SiF, Sa,SiF, Sa,SiF, Sa,SiF, Sa,SiF, Sa,SiF, Sa,SiF, CaF, CaF, CaF, CaF, CaF, XaF XaF well water well water, H,SiF, distillate ditto well water, ditto ditto ditto S O , added 2 rug. 4 pg. 3 pg. 20 pg. 100 pg. 2 pg. 4 PLg. 20 pg. 1 ing. 1 mg. 2 mg. I mg. 10 mg. 10 mg. 25 mg. 1 mg. r 1 ing. { Img. I 1 mg. 1 Illg. - - - - - - 3 1ng. -- 2 nig. -- 2 mg.L 1 ing. t 1 mg. 0.2 pg. 0.5 pg. 2 pg. 10 ing. 10 ing. lmg. 26 mg. - - -- 10 ins. - 10 ing. - - - Source of SiO, Sa,SiO, soh. ditto ditto ditto ditto dltt0 ditto pure SiO, pure SiO, purified sand ditto ditto ditto ditto ditto ditto powdered glass SiO, 7 glass } sand ,I _- suid >ant1 sand hand >and Sa$iO, S O h l . cl i t to ditto sand sand sand sand _- sand sand - - - Etching very strong very strong none strong weak weak normal very strong normal very strong none strong normal weak slightly strong normal strong weak normal slightly weak strong just visible strong strong strong normal none strong definite definite normal none normal normal normal normal normal normal normal strong strong of fundamental importance to the general application of an etching process to the determina- tion of fluoride.If sufficient silica is present, the F' must be evolved, partly at least, in the form of silicon tetrafluoride, and it has been shown by Germanii and Booth' that pure silicon tetrafluoride does not attack glass, though Roses (in 1850) observed that a slow attack occurred in the possible presence of moisture, and Reichg and Olivier6 also noticed some attack on moistened glass.518 WILLIAMS : DETERMINATION OF FLUORIDE BY ETCHING pa. 75 To ascertain the degree of separation from silica that would be necessary before microgram quantities of fluoride could be estimated by etching, a number of trials were conducted in which different amounts of silica, in various forms, were introduced into the platinum reaction tubes before adding the sulphuric acid.Representative results are shown in Table VI, the etchings being designated by comparison with the average result produced by a similar weight of F’ without silica. As the first trials with added silicate recorded in Table T I showed no reduction in the depth or area of the etching, the quantities of silica were increased, ultimately to proportions far in excess of those likely to be encountered in ordinary analytical work. Sext, a standard silicofluoride solution was tested, the measured volumes being gently evaporated to dryness in the platinum tubes before addition of sulphuric acid; finally, a sample of a London well water containing 1.3 parts of fluorine per million was tested, with and without additional silica. In many of these tests, some with proportions of Si”” to I?’ exceeding 1000 times the theoretical equivalent, there can be little doubt that much, if not all, of the F’ must have been evolved as silicon tetrafluoride ; yet, contrary to expectation, definite etchings were obtained.Indeed, although somewhat erratic, the etchings tended to be more marked than those without silica; in a few instances remarkably so and of a rather superior character. Since silicon tetrafluoride could, presumably, only attack glass after conversion to H,SiF,, it seems probable that the larger etchings observed with sub-microgram quantities of fluoride ion when silica was present are connected with the humidity, which, as shown earlier, was found to be necessary for high sensitivity; for silicon tetrafluoride, being more hydrophilic than hydrogen fluoride, as indicated by its vigorous reaction with water, would probably be more quickly and completely absorbed in the moisture condensing on the cover- slip than would hydrogen fluoride, and the H2SiF, formed would then be favourably placed for the production of hydrogen fluoride by dissociation in direct contact with the glass.The proportion of F’ converted to H2F2 might thus be from a third to the whole of the F’ absorbed, according to either or both of the reactions- H,SiF, -+ H2F, -+ SiF, H,SiF, -y- 4H,O --+ 3H2F2 -+ Si(OH),, the course of which would depend upon local conditions of temperature, moisture, etc. In this connection the importance of cooling the cover-slip, at least in the early stages, may also be explained, for JacobsoiilO has shown that the liberated SiF, and H,O do not react together if the temperature exceeds 30” C., although they immediately combine when cooled to form H2SiF,, which subsequentl? dissociates into H,F, and SiF, when the temperature rises.The possibility of increasing the sensitivity by placing a very small drop of water on the cover-slip before the test, to accelerate and localise the hydrolysis, was suggested by the above hypotheses. It was found, however, that small drops of water, or a thin film, on the under- side of the cover-slip quickly disappeared, owing to the desiccating action of the sulphuric acid. A 10 per cent. solution of glycerin was therefore employed instead, a droplet of 0.5mm. diameter being placed in the centre of the cover-slip.\Vith this solution, the droplet was somewhat reduced in volume at the commencement of the test and did not materially interfere with subsequent comparison of the etchings. The use of the glycerin droplet was found to stimulate attack in its immediate vicinity, 0.2 and 0.1 pg. quantities of fluoride ion giving distinct etchings round the droplet that were several times as great as without the droplet, with or without the addition of 2 mg. of sand, but the sensitivity was not reduced appreciably below 0.1 pg. of F’. This quantity of F’ is probably approaching that required to produce the minimum concentration of free H,F, in the condensed water necessary to attack glass. The discovery that silicon tetrafluoride produces an attack at least as vigorous as that of hydrogen fluoride under the conditions of the test, however welcome from some points of view, raises questions of fundamental importance to the quantitative application of the etching test, So far as is known to the author, SiF, can only attack glass by hydrolysis, 3SiF, -+ 4H,O --f 2H2SiF6 + Si(OH), .. .. . . . . (i) thus- \1 2H,F, -+ 2SiF,October, 19501 WILLIAMS : DETERMISATIOS OF FLUORIDE BY ETCHISG 519 The residual SiF, shown in the equation may be volatilised and gradually lost, or it may continue to undergo hydrolysis until the reaction becomes ultimately- 3SiF, + 12H,O --f 6H2Fz + 3Si(OH), . . . . . . . . (ii) The next stage comprises the attack by the liberated H,F, on the glass; this reaction cannot be precisely specified, but the production of more SiF, is to be expected, as in the simplified equation usually quoted- 2H,F, + SiO, -+ SiF, + 2H,O .. . . . . . . . . (iii) Hydrolysis of the secondary quantity of SiF, thus formed would lead to a chain reaction, with the F' assuming the role of catalyst. It cannot be assumed, however, that the liberated H2F, would confine its attack to the glass, and leave the precipitated Si(OH), untouched. To some degree, therefore, it may reverse the reaction shown in equation (ii). In any case there can be no prospect of finality. For these reasons any hydrolysis of silicon tetrafluoride would have disastrous consequences to quantitative analysis ; consequences, moreover, which could not be avoided by complete elimination of silica from the fluoride residue to be tested.In view of the evident importance of establishing more clearly what reactions take place during the etching, the end-products remaining on the cover-slip were micro-analysed. For this purpose, 1-mg. etchings were made, with and without added sand, and the moist white deposits found on the exposed areas carefully removed by wiping with fragments of wet filter-paper. Both deposits proved to consist almost entirely of sodium and calcium silico- fluorides and water, the weight of the dry silicofluorides in each test being approximately 400pg. A slight scum was observed on the surface of the sulphuric acid in the platinum tubes after the tests, both with and without sand; this consisted of silicic acid. Except for this small quantity (about 30 pg.of SO,), no significant amount of silicic acid or silicate was found in the deposits analysed. But having regard to the loss of weight of the glass, and the loss of fluoride ion unaccounted for, it is evident that a relatively large proportion of dissolved silica must have escaped under the cover-slip in the form of silicon tetrafluoride. To reduce such losses, further trials were carried out with the cover-slips sealed to the platinum tubes. The under-sides of the cover-slips were moistened with fodr O.5-mm. diameter droplets of the glycerin - agar solution, near to the periphery, forming nearly complete seals by capillary attraction when the cover-slips were placed upon the platinum flanges; also the time of heating was increased to 34 hours, the temperature being very slowly raised to 260" C.during this time. The resultant etchings exhibited a remarkable form, being apparently raised above the surface of the glass and covered with a powdery micro-crystalline deposit. Repeated cleaning treatment with boiling chromic and sulphuric acid mixture failed to remove this deposit, though silicofluoride would have been dissolved. It was found, however, that much of the deposit could be removed by scraping and that, operating under a dissection microscope with a scalpel, some of the matt surface of the etching could also be removed. On re-examina- tion of some of the previous etchings, obtained from microgram quantities of fluoride ion, it was found that much of the apparent etching could also be removed from these cover-slips in the same way, although this did not apply to the larger quantities employed before the platinum apparatus was devised, where the cover-slips were deeply pitted.Micro-analysis of the substance removed showed-that it consisted entirely of hydrated silica. The loss on ignition was 11.3 per cent., suggesting the hemihydrate, 2Si0,.H20 (13.0 per cent. of H,O), but the degree of hydration in the silica when deposited may not have been uniform, and may have differed from that existing after the cleaning treatment. The formation of the matt surfaces on the cover-slip by the deposition of hydrated silica is analogous to commercial matt etching processes, which have been shown to depend upon the growth of fine crystals of silicofluorides or of insoluble double fluorides on the surface of the glass, so protecting the glass underneath from further attack; the pitted appearance is in fact due to minute elevations, the size of which is determined by the degree of saturation of reacting ions and undissociated salts in the etching r n i x t ~ r e .l l ~ ~ ~ The evidence of a deposit of hydrated silica confirms the supposition that hydrolysis of silicon tetrafluoride takes place, and a means of ascertaining the extent of the hydrolysis was sought. Since in the previous experiments the silica remained firmly attached to the glass, it is clear that the loss of weight shown in Table V does not represent the full extent of the attack. Direct determination of the weight of silica deposited could not readily be effected520 WILLIAMS DETERMIKATIOK OF FLUORIDE BY ETCHING [Vol.i 5 as the silica could not be quantitatively removed by scraping, and alkali extraction proved unsatisfactory owing to dissolution of the glass, but it was thought that, with the knowledge of the water content of the silicic acid remaining after the cleaning and drying treatment, a sufficiently close approximation might be obtained by finding the loss on ignition of the cover-slips. Corrected for a small “ignition blank,” some representative results are shown in Table 1711, together with the estimated ratio of glass attacked per 1 mg. of fluoride ion. With the larger etchings, obtained from 0.5 to 1.0 mg. quantities of fluoride ion, a further, cloud-like, area of attack was noticeable round the periphery of the first sharp etching in trials where the heating was prolonged; this appeared to be due to secondary attack caused by hydrolysis, with further liberation of hydrogen fluoride, of the deposit of silicofluorides first formed; its area extended as the heating was continued.TABLE 1711 TOTAL WEIGHT OF GLASS ATTACKED Afiparatus-Platinum tubes, with liquid-sealed cover-slips ; heated gradually to 260” C. F’ taken, mg. . . . . . . Silica added . . . . . . Duration of test . . . . Loss of weight of cover-slip, mg. Weight of silicic acid, mg. (further loss on ignition, x 100/’88*7) . . . . . . Total glass attacked, mg. . . Glass to F’ ratio . . . . 1.000 0.200 0.100 1.000 none none none 10 mg. glass 31)hr. Z$hr. 34hr. 39 hr. 1.001 0.210 0.194 0.118 0.772 0.193 0.174 0.489 1.773 0.403 0.368 0.607 1-8 2.0 3.7 0.6 0.200 1 mg.each sand, glass and silica 29 hr. 0.069 I 0.100 0.169 0.8 0*100 10 mg. sand 39 hr. 0.163 It is noteworthy that the glass attacked per unit weight of fluoride ion was greater with the smaller quantities of F’; this is ascribed to the greater protection afforded by the thicker coating of deposit which forms with larger quantities of F’, and it is probable that with microgram quantities of F’ the ratio of glass attacked would be higher still. It is now apparent that the losses of weight shown in Table V are understated by the weight of water held by the silicic acid, since the cover-slips were not ignited before being weighed. Moreover, the strange circumstance that, whilst sub-microgram etchings showed inconsistencies of as much as 100 per cent. on visual comparison, the losses of weight from milligram etchings showed fairly close agreement, becomes explicable ; the visible etching represents the amount of glass changed into silicic acid, but the loss of weight represents the amount of glass converted into sodium and calcium silicofluorides and silicon tetrafluoride (plus any silicic acid dissolved or dislodged by the cleaning treatment).If the whole of the fluoride ion were evolved in the form of SiF,, and the “reaction”- In the presence of added silica the losses of weight were extremely variable. proceeded to completion, no appreciable loss of weight would be anticipated. But part of the F’ in the substance under test may be evolved as H2F2, although an excess of silica is present, as postulated by Schneider13; part of the SiF,, and part of the deposited silica, may ’ be lost in various ways, and since, moreover, the reactions taking place appear to depend upon fortuitous local concentrations of SiF, and H,O at the glass surface, the variations of weight are not surprising.To summarise the results of the investigations, an extremely sensitive etching test for fluoride, reacting to 0.1 pg. F’, even in the presence of silica, has been developed; but it has also been shown that the etching of glass, under the conditions necessary for high fluoride sensitivity, comprises a catalytic structural transformation of the silica, which, being capable of proceeding indefinitely, appears to provide no reliable basis for the determination of fluoride.REFERENCES 1. 2. 3. 4. 5. Woodman, A. G., and Talbot, H. P., J . Amer. Chew Soc., 1906, 28, 1437; 1907, 29, 1362. Gautier, A., and Clausmann, P., Bull. Soc. Chirm., 1912, 11, 872. Rrauns, D. H., J . Res. Nut. Bur. Stand., 1941, 27, 105. Mayrhofer, A., Schneider, C., and Wasitzky, A,, Biochew. Z . , 1929, 204, 62. Caley, E. R., and Ferrer, J. M., jun., Mikrochem. Actu, 1937, 1, 160.October, 19501 COX: OBSERVATIONS ON VITAMIN D 6. Olivier, E., Rev. Universelle Mines, 1921, 14, 25. 7. Germann, A. F. O., and Booth, 14. S., J . Phys. Cheni., 1917, 21, 81. 8. Rose, H., Pogg. Ann., 1850, 80, 406. 9. Reich, J. A., Z h e m Zeit., 1896, 20, 985. 10. Jacobson, C. A,, J . Phys. Chem., 1923, 27, 517-80 am1 761-70, 11. Spencer, C. D., and Ott, L., J . Amev. Ceram.Soc., 1927, 10, 403. 12. Schiilz, H., Glushutte, 1938, 68, 828-30. 13. Schneider, L., Uesterr. Z. Berg. u. Hiittenwesen, 1913, 61, 365; Anulyst, Xbst., 1913, 38, 475. THE LABORATORY, 1, SOUTHWARK STREET, LONDON, S.E.1. 521 DISCUSSION DR. J . R. NICHOLLS said that this test had always been applied to the etching of glass and it appeared that secondary reactions caused complications. I t might, however, be possible to use material other than glass. Suppose the sample were mixed with silica and sulphuric acid and the cover-slip were made of plastic. The liberated silicon tetrafluoride would react with the film of moisture on the cover-slip leaving a deposit of silica and secondary reactions should not take place. The difference in the amount of light passing through the cover-slip before and after the test might be proportional to the deposited silica. There seemed no reason why a cover-slip of clear fused silica should not be used since that material is very unreactive to hydrofluoric acid. DR. J. G. A. GRIFFITHS said that, bearing in mind that a t least part of fluorine present as hydrofluoric acid or silicon fluoride appears to become fixed on the glass microscope cover-slip as sodium and calcium silicofluorides, and therefore no longer available to “catalyse” the etching process, it would be interesting to know what happens if a silica cover-slip is used in place of a glass slip, and whether the possibility of an apparently interminable cycle of reactions involving hydrofluoric acid would lead to an even smaller quantity of fluorine ion than the quite remarkable minute figure of 0.1 pg. being detected if the glass slip is replaced with a silica slip, DR. WILLIAMS said, in reply to Dr. Nicholls, that the deposition of silicic acid when silicon tetrafluoride reacted with water provided a very sensitive test for fluorine, and a large number of modifications of this method had been published. In the quantitative variations the silicic acid formed was usually determined colorimetrically by development of the molybdenum blue complex, but he did not know of any procedure by which microgram quantities or less of fluorine could be estimated. Dr. Griffiths’ suggestion was an interesting one, but he did not think the sensitivity could be increased much further because a certain minimum strength of hydrofluoric acid, said by Olivier to be 0*0003 per cent., was necessary for the solution to attack glass a t all.

ISSN:0003-2654    

DOI:10.1039/AN9507500510

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

October, 19501 COX: OBSERVATIONS ON VITAMIN D 521 Observations on the Spectrophotometric Estimation of Vitamin D BY H. E. COX (Read at the meeting of the Society on Wednesday, May 3rd, 1950) SYNoPsIs-The International Unit of vitamin D has recently been changed by the World Health Organisation, but the new unit has absorption constants similar to those of the old unit and gives a similar reaction with the acetyl chloride - antimony chloride reagent first proposed by Nield, Russell and Zimmerli. A study of this method shows that it works well for control purposes provided that (a) the amount of vitamin D present is sufficient, (b) the amount of vitamin A is not more than about five times that of the vitamin D and (c) suitable correction is made for the absorption due to certain other sterols.Data are given on interfering substances and on commonly occurring fats that are used in the preparation of commercial vitamin products. When there is too high a concentration of vitamin A it may be separated by chromatography. THERE is at present no recognised chemical, physico-chemical or even microbiological method applicable to the estimation of vitamin D in foodstuffs. The biological method is so costly and time-consuming that there is great need for a simpler or cheaper process and every contribution to this end is to be welcomed. To appreciate the problem it is necessary first t o define what has to be determined and then to consider possible methods and probable522 COX: OBSERVATIONS ON THE [vol. 75 interfering substances. Until quite recently the International Unit of vitamin D was defined in terms of a solution of irradiated ergosterol that: was in fact mainly a solution of vitamin D, and was equivalent to 0.025 pg.of calciferol. Calciferol is now available as a synthetic chemical of a high degree of purity, has the empirical formula C,,H,OH, and is the subject of a specification in the British Pharmacopoeia. More recently the World Health Organisation, which is the body charged with the duty of defining units and issuing appropriate international standard preparations of certain of the vitamins, has decided that the solution of irradiated ergosterol, being mainly of vitamin D2, has not proved to be sufficiently representative of the D vitamins. Also a suggestion made at an earlier conference that the standard should be replaced by pure crystalline calciferol is now unacceptable. So the standard has been re-defined in terms of the alcohol C2,HBOH, formerly known as vitamin D,.This compound differs from calciferol in the nature of its side-chain; the methyl group at C, is absent and there is one double bond less. It is available as a pure substance in crystalline form and has a melting- point of 87" to 89" C., [a]: + 110" and E:&. at 265 mp. = 490; 0.025 pg. of this substance now becomes the International Unit of vitamin D. It will be noted that this substance is defined as having a high extinction coefficient; in 1 per cent. solution in alcohol it is as high as 480, and E, the molecular extinction coefficient, is 18,800. So it is evident that it would be quite easy to determine the amount of this sub- stance in a pure solution or in the absence of interfering substances.Besides natural products containing vitamin D, there are many proprietary preparations that contain and claim it, and the Labelling of Food Order requires that certain specified proportions of the normal daily requirement (500 I.U. of vitamin D) shall be present. This brings an insistent demand for its estimation, and it might be said that it is the duty of the Public Analyst to determine it. In general, he cannot do so. The vitamin is always associated with the fatty portion of the food in which, of course, it is soluble. Even if the fat is extracted, and saponified and the unsaponifiable matter dissolved in pure solvent, such as cyclohexane, there is almost always enough impurity to mask the specific absorption curve of the vitamin. Nearly all the sterols associated with the unsaponifiable portion of fatty oils have absorptions in the region of 260 mp., so that a direct method such as is used for vitamin A is inapplicable.Moreover, if vitamin A is also present in quantity substantially greater than vitamin D, its absorption will overlap the 260 mp. region. The well known Can- - Price reaction for vitamin A was, of course, very attractive and as long ago as 1936 it was observed that vitamin D or calciferol gave a pink or orange colour with the same reagent, but it was not sufficiently stable and reproducible for a quantitative method, and many substances interfere. In 1940, however, Nield, Russell and Zimmerlil~2 introduced the use of acetyl chloride into the reagent and since then various improvements have been made or proposed.The reagent is a solution containing 20 per cent. w/v of antimony chloride to which is added 4 per cent. of pure acetyl chloride. The reagent must be fresh and must be quite free from alcohol. When 9 volumes of this reagent are added to 1 volume of a solution of vitamin D in cyclohexane or in chloroform, an orange colour is rapidly developed; it reaches a maximum in about 4 minutes and retains it for about 12 minutes. The optimum conditions of time and temperature have been studied by various authors. The colour is fairly specific and has a characteristic absorption curve with a maximum a t 600 mp. At this wavelength the value of EiZ!.is 1880, a figure that makes the test four times as sensitive as the absorption at 265 mp. Vitamin A with this reagent gives the well known blue colour; this fades quickly and is, indeed, somewhat suppressed by the acetyl chloride. The blue colour has Em,,. at about 620 mp., which is well clear of the 500 mp. line. However, when there is much vitamin A relative to the vitamin D, the blue colour does seriously interfere. In my experience the blue colour begins to cause difficulty when the quantity of vitamin A is more than about five times that of the vitamin D. If calciferol is the only sterol present in the mixture the problem is easy and the determina- tion can be made with a good degree of accuracy down to small quantities. Unfortunately the orange or pink colour is given in greater or less degree by various sterols and some polyenes.The literature is somewhat confusing and, as one does not know just what sterols may be present in an unknown material, it is difficult to apply any general correction. Those sterols other than calciferol that do give a colour with acetyl chloride - antimony chloride all give a less intense one, so there is a practical possibility of establishing blanks applicable toOctober, 19501 SPECTKOPHOTOMETRIC ESTIMATION OF VITAMIN D 523 particular types of material. Measurement of the absorption of some of the sterols gave the following extinctions- E;&. at 500 mp. Calciferol . . .. .. .. .. 1800 Sitosterol . . .. .. .. .. nil Phytosterol (B.D.H.) . . . . .. nil Cholesterol .. .. .. . . .. 80 Ergosterol . . .. .. .. .. 3 00 It will be seen that each is small compared with that of calciferol, and for such a quantity as 10 pg., which is convenient for vitamin D, or D,, in a total volume of 2 ml., the extinction will be- E;& a t 500 mp. Calciferol . . .. .. .. .. 0.90 Cholesterol . . .. .. .. f . 0.04 Ergosterol . . .. .. .. .. 0.15 Phytosterol .. .. .. .. 0.00 As the composition of a sample in terms of its sterols is not always known, it becomes necessary to establish data on the basis of the common fats. A few such results may be given; they refer to the unsaponifiable matter calculated on the original sample, i.e., 1 per cent. of the oil in the mixture after addition of the antimony chloride reagent. Ei& at 500 mp.Arachis oil . . 1 . .. .. .. 0-06 Butter fat . . .. .. .. .. 0-05 Castor oil . . .. .. .. .. 0.06 Cocoa butter . . .. .I .. 0.025 Cod-liver oil .. .. .. .. 0.057 Olive oil . . .. .. .. .. 0.033 Tea-seed oil .. .. .. .. 0.025 It will be noticed that margarine is not included; this is because its composition is variable and generally unknown and is likely to include large proportions of fish oils which themselves vary greatly in their content of unsaponifiable matter and its constituents. It has been shown by Henry and Kon3 that cows' butter maintains a constant level of about 0.07 to 0.1 I.U. per gram in the winter and 0.55 to 0.97 I.U. during the summer. These quantities would account for an extinction of only about 0.002 on 1 gram, which is much too small for satisfactory measurement and would be overshadowed by irrelevant absorption. It appears at present that the spectrophotometric method can only be applied when the composition is known or may be assumed.The method has distinct usefulness as a control on manufacture, as once the necessary blanks have been set up it is only necessary to observe the extinction under standardised conditions and ensure that the total E value does not fall below a pre-determined figure. The Public Analyst is not always in the happy position of knowing what is present or of having a good blank. He can, however, establish a maximum. Clearly the vitamin D cannot be more than is indicated by the value E:,%, = 1800 and is almost certainly less, unless the mixture under examination contains calciferol but no animal oil.If the oils or fats present are known or can be determined, then the extinction due to them can be estimated within a margin of error and allowance made. This method has been applied to the control of certain vitamin products now enjoying a considerable sale and it has been found to work. The amounts found have generally agreed reasonably well with the amounts of vitamin added by the manufacturer. For example, the unsaponifiable matter extracted from 3 g. of a sample of a well-known product containing about 33 per cent. of cocoa butter. The extraction of the unsaponifiable matter was carried out as for a cod-liver oil with all due precautions against atmospheric oxidation. The un- saponifiable matter was dissolved in 10 ml. of cyclohexane. To 0.2 ml.of the solution was added 1.8 ml. of the acetyl chloride - antimony chloride reagent a t a temperature of 20" C. The colour was a t first bright blue, but the blue soon faded and a pale orange tint remained; it is necessary to use a quantity of the sample such that the blue colour disappears within 4 or 5 minutes. The mixture was transferred to the l-cm. cell of a Spekker spectrophoto- From the observed value is deducted the appropriate blank, which is usually about 0.03. It is not practicable to measure less than about 50 or 100 I.U. per gram. meter and the extinction measured after 6 minutes, using the stages 0.02,0-04,0.06 . . . 0.30.524 COX: OBSERVATIONS ON VITAMIN D [Vol. 75 If 3-0 grams were taken, the final concentration is 3 per cent., so the amount of vitamin D is: (observed E - blank) x 600 I.U.per gram. It sometimes happens that the amount of vitamin A is inconveniently large in relation to the vitamin D. This is shown by the blue colour being so intense that it does not fade sufficiently within 5 minutes for a clear reading to be obtained, while if a smaller quantity be taken the colour due to vitamin D is too faint for satisfactory measurement. In these circumstances I have applied the chromatographic method devised for whale liver oils by Gridgeman, Gibson and S a ~ a g e . ~ All the vitamin A can be removed and the cholesterol - calciferol fraction afterwards washed out with petroleum spirit and ether. This enables a satisfactory reading at 500mp. to be obtained if it is known that there is no substantial amount of cholesterol present.It remains to be mentioned that certain other reactions have been proposed, e.g., with glycerol dichlorhydrin containing acetyl chloride (by Sobel, Mayer and Kramers) , which are said to be more specific for vitamin D and to be unaffected by cholesterol. However, the extinction curves have a less well-designated peak and the method has not been extensively studied. REFERENCES 1. 2. -,- , Ibid., 1943, 148, 245. 3. 4. 6. 10 BILLITER SQUARE Nield, C. H., Russell, W. C., and Zimmerli, A, J . Biol. Chem., 1940, 146, 73. Henry, K. M., and Kon, S. K., Biochem. J., 1942, 36, 456. Gridgeman, N. T., Gibson, G. P., and Savage, J. P., Analyst, 1948, 73, 662. Sobel, A. E., Mayer, A. M., and Kramer, B., Ind. Eng. Chem., Anal. Ed., 1945, 17, 160. THE LABORATORY LONDON, E.C.3 DISCUSSION DR. J. GREEN said that the reagent must be meticulously prepared and be quite pure, or very erratic results would be obtained. With oils containing usual amounts of vitamin A or products containing irradiation intermediate sterols or 7-dehydrocholesterol, direct estimation fails and separation techniques must be used. DR. Cox, in reply, agreed with Dr. Green that the method was not applicable to oils containing relatively large proportions of vitamin A, or to whale oils and products containing unknown sterols. The method he was putting forward should only be applied to known products or mixtures when the necessary blanks and controls could be established. He agreed as to the need of preparing pure alcohol-free reagents.

ISSN:0003-2654    

DOI:10.1039/AN9507500521

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

October, 19501 CLARK 525 The Testing of Atmospheric Conditions in Theatres and Cinemas BY J. F. CLARK (Read at the meeting of the Society on Wednesday, May 3rd, 1950) SVNoPsIs-The terms on which theatre and cinema licences are granted usually include some requirements regarding the ventilation, and an investiga- tion has been carried out to test the adequacy of the conditions laid down in the licences. Temperatures, humidities and carbon dioxide concentration were determined at several points in each. It was found that temperature and humidity varied more with weather conditions than with the internal atmosphere, but the carbon dioxide content of the air was a reliable indication of the ventilation. In particular it was noticed that “stufiness” became apparent to the observer a t a carbon dioxide level of 15 to 20 parts per 10,000.A simple method of sampling and testing for carbon dioxide concentra- tion, accurate to 0.2 part per lu,uOO, was developed. Rapidity of sampling was essential because not infrequently adjustments of ventilation were made while sampling was in progress. With most systems of ventilation it was considered that the usual require- ments of a maximum of 10 parts per 10,000 was too stringent, since to maintain this level near draught screens and in high balconies an air supply that produced pronounced draughts in the more open parts of the auditorium was required. Ventilation giving a maximum of 15 parts of carbon dioxide in the least ventilated areas resulted in an average concentration of under 12 parts throughout the auditorium (corresponding to a rate of 700 to 800 cu.f t . of fresh air per head per hour) and this could be maintained by any efficient ventilating system without producing noticeable draughts. Theatres and cinemas were inspected at peak periods. CINEMAS and theatres are subject to licensing and control by local Justices of the Peace and in laying down the conditions under which a licence is granted an attempt is usually made to ensure that an adequate standard of ventilation is maintained. During a review of these conditions of licence it was decided to investigate the practicability of the Justices’ requirements. The standard of comfort and hygiene of an auditorium atmosphere depends on many factors, including temperature, humidity, outside weather conditions, arrangement of seating, supply of fresh air and its velocity, and suspended impurities; for control purposes it is desirable to have some index of an easily determinable nature to show the state of the air.It is not proposed to enter into a discussion of the whole question of ventilation here. That was very ably done some time ago by R. C. Frederick at an Institute of Chemistry lecture, and the monograph1 is available to anyone wishing to follow up the subject. We might briefly define an undesirable atmosphere in the circumstances we are now considering as one that has been too much used. It will be hot, humid, hazy (if smoking is permitted) and will contain too high a proportion of the products of respiration and, not infrequently, perspiration also.Such an atmospheric state is the result of an insufficient supply of fresh air, but an excessive supply, although more hygienic, may be almost as unpleasant if it results in a cold draughty hall. The object of adequate ventilation is to strike the mean between draughts and “ stuffiness.” The optimum temperature is a matter of personal preference and is relative to outside weather conditions and other factors. It is a matter of comfort more than health, and so may be regarded as something to be arranged by the manager to suit his patrons in com- petition with similar establishments. It should, however, be borne in mind that it is cheaper to use the bodily heat of an audience to warm the atmosphere than to supply warm air. This will be referred to later. Humidity is influenced both by the audience and by outside conditions, and also by526 CLARK: THE TESTING OF ATMOSPHERIC [Vol.75 the fact that in many modern buildings the air drawn in is washed by passing through a water spray. The katathermometer, by measuring rates of heat loss, gives a very useful indication of the combined effect of temperature, humidity and air flow, but unfortunately it is not a suitable instrument for operating in crowded cinemas in the dark, so that it was decided not to use it in the investigation. An interesting possibility in this direction would be the use of one of the “drinking birds” which appeared as toys a year or two ago. If such a device could be standardised or calibrated, it would provide a very simple wet kata, and it would only be necessary to take the rate of dipping.A large number of tests were made with a whirling hygrometer, from which it was found that the relative humidity was lower inside a building than outside, owing to the higher temperatures, but there was little consistency i,n the results. Calculated as the percentage of water vapour in the air, the humidity ran parallel with the atmospheric conditions on any particular occasion, but no correlation was found possible in the results from different theatres on different days. The method was discarded with regret, as it seemed a very suitable one by which managements could have checked the efficiency of ventilation for themselves. Respired air, of course, contains more carbon dioxide and water vapour than fresh air, and also contains in suspension moisture droplets which can carry infection.The hygienic reason for ventilation is the need for the removal of these droplets and the associated bacteria, and also the avoidance of the effect of high temperature and humidity on respiratory surfaces, which are rendered less resistant to infection. Since the attempt to assess ventilation conditions by humidity measurements proved unsuccessful, owing to the many contributory factors, the accepted method of using the carbon dioxide concentration as an index of the extent to which the air had been used was investigated. It cannot be regarded as more than an indicator, as in the worst ventilation conditions the amounts found are of no toxic significance. One of the first symptoms of an excessive carbon dioxide concentration is a diminution of the critical faculties and a tendency to hilarity, which might not be disadvantageous at times in theatres and cinemas, The normal carbon dioxide output of a man at complete rest is just over 0.5 cu.ft. per hour, which can increase to 2 or even 3 cu. ft. with heavy exertion. For women and children the amounts are less. The average output of an audience under normal conditions was taken as 0.6 cu. ft. per person per hour, and on this basis, knowing the carbon dioxide contents of the fresh air supply and the air in the auditorium, it is possible to calculate the rate of supply of fresh air in terms of cubic feet per hea.d per hour. Working on these lines, the testing of ventilation resolves itself into problems of sampling and estimation of carbon dioxide.A reasonable size of sample is desirable, to cover a small area rather than a single point, as the latter might consist of almost undiluted exhaled air. It should be taken at about the height of the faces of the audience, and with as little noise and fuss as possible, since the most useful time of sampling is when the house is full and settling down, and distracting circumstances are then unpopular. It must be taken fairly rapidly, in view of the fact that some economically-minded managers stop the fans for a while after the house has refilled, to allow the theatre to warm up from the body heat of the audience. In such instances, the immediate reaction to the knowledge that tests are being made is generally a speeding up of the ventilation fans.EXPERIMENTAL First tests were carried out with absorption trains using various absorbents, but the amount of air to be passed to give a weighable amount of carbon dioxide necessitated too rapid a flow for efficient absorption. A small calibrated electric pump was useless, in view of the varying resistance to gas flow in the tubes. More success followed the use of a standard hand pump drawing 125 ml. per stroke, but this, had to be used very slowly. The apparatus was cumbersome and attracted undue attention. With too slow a stroke, the full 125 ml. was not drawn, and too rapid a stroke meant incomplete absorption. In the latter event, with solid absorbents, a loss in weight was recorded in some instances, possibly because of the removal of fine particles of absorbent from the tubes by the pumping.In view of these difficulties, the attempt to combine sampling and estimation in one operation was discontinued, and sampling alone was done on the site. This was accomplished most successfully by blowing up football bladders, which, when fitted with well-greased glass stopcocks, proved most useful gas containers. They can be filled quickly and, if in good condition, will take 4 to 5 litres of air and hold it without determinable change for severalOctober, 19501 CONDITIONS I N THEATRES AND CINEMAS 527 days. Some apprehension was felt about the possible condensation of moisture inside the bladders and at first they were pumped up through a tube containing anhydrone, but this introduced complications, as allowances had to be made for loss in volume of the gas on drying. It was found that con- densation did not take place in the bladders if extreme changes in temperature were avoided.When exhausted they contain only 3 or 4ml. of residual air. -APPARATUS- A simple apparatus, shown in Fig. 1, was devised for the estimation of the carbon dioxide, and consisted of an aspirator and a bubbler. The aspirator was an ordinary 5-litre bottle with a pressure tube so that the internal gas pressure could be observed and adjusted to atmospheric. The bubbler consisted of a large test tube with a centre tube, blown into two bulbs, fitting fairly closely inside it. The absorbent was barium hydroxide, roughly 0.1 N , containing about 5 per cent. of barium chloride. The effect of this type of bubbler is that the gas is held below and between the bulbs in contact with the liquid and released in a stream of bubbles through two layers of absorbent.With the carbon dioxide concentrations experienced and rates of flow not exceeding 2 litres per hour, it was found that one bubbler was quite sufficient and no carbon dioxide was absorbed in a second tube connected in series. 5 LlTRE ASPlRATOR BUBBLER Fig. I FOOTBALL BLADDER METHOD- Ten millilitres of baryta were used in the tube and in a “blank” tube of the same type, kept closed during the passage of the air; the same pipette was used to measure into both tubes. After passing the air, the contents of the tube and the blank were washed into flasks with carbon dioxide-free water and titrated with oxalic acid adjusted to such a strength (approximately 0.084 N ) that 1 ml.was equivalent to 1 ml. of carbon dioxide gas under room conditions of temperature and pressure. Phenol thymol phthalein was found to be the most suitable indicator, with a good colour change and sensitive to 0-02ml. The titration gave a direct reading of the volume of carbon dioxide in the volume of air passed. The procedure was to connect the bubbler to the aspirator and the bladder to the bubbler. The bladder stopcock was partially and cautiously opened, and when the aspirator was in equilibrium with the bladder (at a pressure of about 30 cm. of water) the water was drawn from the aspirator into a measuring cylinder at a slow trickle. The control of pressure from the bladder enabled a very steady gas flow to be maintained.When about 2 litres had been drawn off (or less, if barium carbonate was seen to be forming quickly) the bladder stopcock was closed and the water st.ream continued until the pressure in the aspirator was atmospheric. The volume of water collected was taken as the volume of gas passed at atmospheric pressure.628 CLARK: THE TESTING OF ATMOSPHERIC p o l . 75 It was actually very slightly higher, owing to the amount of water vapour picked up by the gas on passing through the bubbler, but the difference is well within the experimental error. At all the theatres and cinemas, samples of street air were taken and, although on the outskirts of the town, or in the town itself on breezy days, the carbon dioxide concentration was steady at 3.2 to 3.6 parts per 10,000, on still days in the town it sometimes rose as high as 6 per 10,000.These figures were taken into’ account in assessing ventilation efficiency. RESULTS AND CONCLUSIONS- It was concluded from the investigation that the usual limit of 10 parts per 10,000 in any part of the auditorium (necessitating an air supply of 1000 cu. ft. per head per hour in the most sheltered areas) was too stringent to be effectively operated, as it involved violent draughts and an average carbon dioxide concentration of 7 to 8 parts per 10,000 throughout the theatre. With a concentration of 5 to 6 parts per 10,000 in the outside air, this level is impracticable. It was decided that a practical limit which could be reasonably enforced was 15 parts per 10,000 in any part of the auditorium.In a modern cinema, when this level is reached in the least ventilated areas, the average concentration is found to be below 12 parts per 10,000, corresponding with an air supply of 800 c x . ft. per head per hour. It was found that 15 parts per 10,000 was the approximate point where “stuffiness” became noticeable on entering from outside, although acclimatisation was very rapid. After a little experience it was quite possible to tell on going into a cinema whether the concentration was above or below this point. The main ventilation systems found in public halls are generally either “plenum” in the more modern buildings, or exhaust, in the older ones. They can be roughly distinguished as “push” and “pull” systems.In the plenum cinema arrangement, air is drawn through a fine wire mesh, washed by a water spray, and injected through grilles alongside the screen. It is exhausted through grilles situated under the balcony, in the roof at the back of the balcony and often high up on the back and side walls. The air distribution is good and few dead areas are found, these being generally in front of draught screens and at the back of the balcony. With such systems there is little difficulty in keeping the carbon dioxide content below the suggested limit, even with a packed house. Inefficient ventilation in such buildings means that the fans have been slowed down or stopped, which may have been done to allow the house to warm up. With the exhaust system, air is admitted or finds its way through doors, windows or grilles, and is withdrawn by exhaust fans from the walls or roof.Dead areas are plentiful, particularly in the centre of blocks of seats anti under balconies, and there is considerable difficulty in maintaining a good atmosphere with a full house. A cinema with an exhaust system was inspected at 9 p.m. one evening when the house was full, and concentrations of carbon dioxide ranging from 21 to 25 parts per 10,000 were found (outside air 3.5). Next morning, with a wind blowing and all the doors and windows open, the air on the ground floor was at the same concentration as the street air, but at the back of the balcony there was still 11 parts per 10,000. The same evening, again with a full house and the fans at full speed, the results ranged from 13 to 18 parts per 10,000-obviously an unsatisfactory system.A plenum system cinema, with a full house, gave figures from 12.1 to 17.6 (outside air 3.3). Next morning, the doors having been kept closed, the atmosphere was the same inside as outside the cinema. Re-inspected by arrangement with the management, the carbon dioxide ranged from 9.7 to 10-7, showing that the system was quite efficient. Samples taken from the inlet and exhaust ducts showed that the washing did not affect the carbon dioxide content of the incoming air, although it removed a surprising amount of dirt. In the main extraction duct the concentration was 11.3 parts per 10,000 and in one from under the balcony, 8-9. The hand pump and bladders proved very convenient for taking air samples from ducts and otherwise inaccessible places. A final point concerns a question which is often raised-the effect of smoking on the carbon dioxide content of the atmosphere.The amount of carbon dioxide produced from one cigarette is of the order of 0.02 cu. ft., and it is doubtful if the consumption of cigarettes exceeds on the average one per person per hour. Thus the additional carbon dioxide produced Some results from typical cinemas with these systems may be of interest.October, 19501 CONDITIONS I N THEATRES AND CINEMAS 529 by smoking is of the order of 3 per cent., which is far less than the individual variation in expired air. Much more significant than the increase in carbon dioxide is the haze produced, which is composed of minute droplets of tarry matter.In the darkness of a cinema this is kept down to a satisfactory level by the suggested ventilation rates, but it is worth remarking that countries which allow smoking in cinemas are, in my experience, in the minority. In well-lit theatres and concert halls where smoking is permitted, ventilation can almost be left to look after itself, since any system which will keep the atmosphere satisfactorily clear of tobacco smoke will ensure that the concentration of carbon dioxide is well below the suggested maximum limit. REFERENCE 1. Frederick, Robert C., ‘‘Ventilation Conditions-Xormal and Abnorimal, and Their Investigation, ” Royal Institute of Chemistry Monograph, 1929. CITY ANALYST’S DEPARTMENT, MOUNT PLEASANT, LIVERPOOL, 3. DISCUSSION MR. C.H. MANLEY said that, with regard to the question of smoking in cinemas, he understood (and he noted that Mr. Clark agreed) that in Australia smoking was forbidden in the auditorium, there being a half-time interval during which patrons could smoke and partake of refreshments. DR. H. E. Cox noticed that the author expressed the opinion that the proper limit for carbon dioxide was about 15 parts per 10,000, and he wondered whether i t could really be valid to arrive a t such a figure on a chemical basis alone. He thought the limits both of carbon dioxide and moisture needed to be related to their physiological effects. Much data on this had been accumulated in relation to submarines. The present problem should be studied in the light of experience in mines and submarines. MR.J. G. SHERRATT referred to the type of carbon dioxide absorption tube used by Mr. Clark and asked by what method the author had proved that it was fully effective. In the questioner’s experience there was risk of incomplete absorption because of the relatively large volume of air enclosed within each bubble, and he had discarded this type of tube in favour of one utilising glass beads moistened with barium hydroxide solution. MR. F. L. OKELL, in reply to Mr. Sherratt, said that the efficiency of a bubbler of the type used by Mr. Clark did not depend on the size of the gas bubbles, but on the narrow space, about half a millimetre, between the wet walls of the bulb and the outer vessel through which the gas had to pass in a thin layer. Between each “bubble,” the surfaces of the gas passage were re-moistened by the absorbent and the result was very efficient absorption.The original designer was G. Nevi11 Huntly, who was for many years a well- known member of the Society. MR. CLARK, in reply to Dr. Cox’s point, said that the carbon dioxide had no physiological significance in the concentrations ordinarily found in theatres and cinemas. It merely served as an indicator of the ventilation by showing the extent of dilution of exhaled air. The suggested limit of 15 parts per 10,000 had been decided on as a reasonable and attainable compromise between over-ventilation causing draughts and under-ventilation causing stuffiness. Mr. Frederick’s paper, to which he had referred, went much more thoroughly into the physiological effects of extremes of ventilation, and the data there had mostly been accumulated in connection with Admiralty work, with particular reference to atmospheres in confined spaces. If the bulbs of the bubbler fitted closely to the sides of the tube, a second bubbler in series did not absorb a determinable amount of carbon dioxide, and a known amount of carbon dioxide was completely recovered within the limitations of accuracy of the method. The bubbler had been successfully used for other carbon dioxide estimations such as the testing of self-raising flour and baking powder. It was, however, inadvisable to exceed about two-fifths of the absorptive power of the baryta. What would the physiologist say? His experience confirmed Mr. Okell’s statements regarding the efficiency of the bubbler.

ISSN:0003-2654    

DOI:10.1039/AN9507500525

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

630 GADDUM: HORMONE ASSAY: INTRODUCTION [Vol. 75 Hormone Assay The following four papers were read at a joint meeting of the Biological Methods Group and the Society for Endocrinology on Thursday, October ZOth, 1949. Introduction BY J. H. GADDUM CANDIDATES in pharmacological examinations are sometimes asked why certain substances are assayed by biological methods. The correct answer is, of course, that no one would do bio-assays if he could help it, but that some drugs, such as insulin, cannot be controlled in any other way, and some drugs, such as acetylcholine, are commonly present in concentrations that are too low to be detected in any other way. The motives that drive us to bio-assays can, however, also be classified pragmatically. Some of us wish to estimate the potency of conimercial preparations, so that they may be safe to use and so that we may assess their comniercial value.For these purposes, methods of bio-assay have been developed that can give almost any required degree of accuracy. The members of the Biological Methods Group of the Society are expert in such matters. The members of the Society for Endocrinology, 011 the other hand, are interested in bio-assays mainly as a means of discovering new facts. 'Their problems are complicated and their arguments often involve assumptions and approximations that are open to criticism. The object of both should be that the endocrinologists should learn accuracy and that the analysts should learn something of the difficulties of the endocrinologists. Methods of hormone assay can be classified according to the kind of effect observed, though some hormones can be assayed by means of several different kinds of effect.Table I summarises most of the established methods of assay for hormones and gives some indication TABLE I[ Adrenaline, etc. Thyroid . . Androgens . . Oestrogens . . Progesterone. . Corticoids . . Insulin . . Parathyroid . . Secretin . . Oxytocin . . Vasopressin . . Antidiuretic . . Growth . . Prolactin . . Thyrotrophin Gonadotrophins Corticotrophin .. .. .. . . . . 1 . .. .. . . . . .. .. .. . . . . . . Smoked Animal drum weight 1 1 Organ Fluid Bio- weight volume chemical 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 quantitative. 2 quantal. Mor- Death, phology etc. 2 1 2 1 2 1 2 2 2 2 1 2 2 1 2 of the effects. A few of these are quantal in the sense that the response of each animal is not measured, but merely recorded as positive or negative.These latter are denoted in the table by the figure 2 ; other things being equal, they require for an'y given degree of accuracy about twice as many animals as the first type of test. Under the heading of morphology are included observations of the presence of legs in tadpoles, of the appearance of vaginal smears and of histological preparations of sections of tissues. Such results are difficult to assess quantitatively and the test is generally quantal. Most of the tests are based on a quantitative measure of the effect.October, 19501 GADDUM: HORMONE ASSAY: INTRODUCTION 531 The heading, “death, etc.,” includes the symptoms produced by insulin in mice.It may be asked,how death can be quantitative. The answer is that some tests depend on the duration of survival. For example, when gonadotrophins are administered to males they act on the testes, which release androgens, and these androgens then act on the prostate. It might be expected that assays based on the weight of the testes would be more accurate than assays based on the weight of the prostate, but this is not necessarily so. We probably agree about the principles upon which assays should ideally be founded- (1) Each assay should involve a comparison between an unknown preparation and a standard preparation. (2) Each of these preparations should contain the same single active principle and no other substances that modify its action in the test. (3) Each assay should be complete in itself; it should involve no arbitrary assumptions about such things as the slope, or even the shape, of the dose - effect curve.(4) The limits of the error should be calculated from the internal evidence of each assay. If the first two conditions are fulfilled, the test must give an estimate of the concentration of a single substance, and the results of parallel quantitative tests of the same solution by a series of different methods will all agree, within the errors of the tests. If the substance used as a standard is not the same as the substance present in the unknown solution, it is generally found that the results of parallel quantitative tests do not agree. This circumstance forms the basis of a valuable method for identifying substances and distinguishing them from other closely related substances.By means of such tests it has been shown in recent years that adrenaline is generally accompanied, or replaced, in the body by the closely related substance noradrenaline. It follows from these facts that the biological response on which the result of an assay depends need have no relation to the effect that the preparation is expected to produce. The requirements of an ideal assay are seldom, if ever, completely fulfilled. Even in careful assays of insulin the assumption is generally made that the dose - effect curve can be transformed into a straight line by mathematical devices that have been successful in the past. In practice, it is necessary to compromise in order to make any progress at all.We may discuss how far this compromise should be allowed to go. A standard preparation is not always obtainable. There is no international standard preparation for many of the hormones, and in many instances it is not yet clear how to make a standard preparation. A later paper will tell, for example, something about human pituitary gonadotrophins, which are only available at present as variable mixtures of unknown substances that increase one another’s effects to unknown extents and cannot be separated from one another. Oestrogens should present a simpler problem since their chemical formulae are known, but even so it is impossible a t present to make satisfactory assays of mixtures of oestrogens. The reasons for this have been discussed fully by Emmens,l but let us consider some of the facts. Biological fluids may contain variable proportions of three oestrogens, oestradiol, oestrone and oestriol.The first of these substances is the most active and the last the least active, but the ratios of the activities depend on the details of the experimental technique and may vary enormously. For example, Emmens quotes figures varying from 1 to 250 for the ratio of the activity of oestrone to that of oestriol. In his own experiments, the ratio was 2-4 when the drugs were given in four doses, and 70 when they were given in two doses. These figures alone are enough to show that it is impossible to attach any real meaning to the results of assays of unknown mixtures of oestrogens. Estimates which purport to give “oestrogenic activity’’ of such mixtures are liable to vary over a 30-fold range or more, according to the way the test is done.A control sample of urine contains 1 mg. of oestrone and 10 mg. of oestriol. The patient is then given a dose of medicine, and another sample of urine is collected containing 2 mg. of oestrone and 4 mg. of oestriol. Two observers both estimate the oestrogenic activity of these samples of urine in terms of oestrone. Observer A gives the oestrogens in two doses and observer B gives them in four doses. In the first test, according to Emmens, the ratio of activity of oestrone to activity of oestriol is 70, and it may be calculated that the conclusion will be that the medicine causes The last three hormones in the list act primarily on other glands.Consider an imaginary example.532 GADDUM: HORMONE ASSAY : INTRODUCTION [Vol. 75 a rise in the oestrogenic activity of the urine from 1-14mg. of oestrone equivalent to 2.06. In the second test, the ratio is 2.4, and the conclusion will be that the medicine causes a fall in the oestrogenic activity from 5.17 to 3-67. The two methods give opposite results and they are both wrong; their results have no meaning. The variation of ratios can, of course, be reduced by standardising the technique, but if this is done the interpretation of the results becomes very artificial. In the example just discussed, any conclusions about the effect of the medicine would depend on which system of doses had been arbitrarily chosen as the standard one. The position is further complicated, as Emmens pointed out, by the presence in urine of variable quantities of substances, not oestrogenic themselves, but having the property of increasing the effect of substances that are.The discovery of the synthetic oestrogens has made it unnecessary t o use biologically standardised mixtures of oestrogens in therapeutics. If it had not been for this discovery, as much time and labour might have been wasted in the study of methods of standardisation of mixtures of oestrogens as has been wasted on the same problem in connection with extracts of digitalis. These are known to contain a variable mixture of active substances, and enormous amounts of scientific energy have been devoted to the search for a reliable method of predicting their effect on man.Such experiments serve a practical purpose, but rest on an insecure logical basis and scarcely deserve the name of assays at all. It is hoped that they will soon become unnecessary because doctors will use pure active principles. The only possible way of getting results with any intelligible meaning in bio-assays of mixtures of oestrogens seems to depend on removing the interfering substances and separating the oestrogens from one another before the biological tests are made. Chemical methods give results that are easier to interpret, since the three main oestrogens have equal effects in the Kober test generally used, so that it is possible to estimate the total amount of oestrogen present. This test is, however, not sensitive enough to estimate the oestrogens in non-pregnant blood.If a biological test could be discovered in which all the oestrogens were equally effective, progress could be made, but sufficiently sensitive chemical methods would be even more valuable. Accuiate tests for single oestrogens have, of course, been available for years, but they have played only a small part so far in advancing knowledge. The researches that have led to our present detailed information about the chemistry of these substances were controlled by biological tests in which all the laws of bio-assay were neglected. This shows that it sometimes pays to take risks, but does not justify all the risks that have been taken by endocrinologists. In some instances it may still be necessary to speak of mouse units and to assume that all the mice in the world are equal, but every effort should be made to get away from such dangerous assumptions as soon as possible.There can be no justification for speaking, as some still do, of mouse units of the chorionic gonadotrophin present in the urine of pregnant women. An international standard preparation of this substance is available and should be used. Results should be given in international units. Unfortunately, this standard cannot be safely used for assays of other gonadotrophins, such as those present in the urine after the menopause. Apart from the provision of reliable preparations of hormones, the contribution that bio-assays can make to clinical endocrinology depends on the estimation of hormones in blood and urine. The concentration in the blood presumably gives an indication of the concentration with which the tissues are in equilibrium. The concentration in the urine may provide an indirect estimate of the daily output of hormone, provided that all the urine excreted during 24 hours is collected.If, for example, it is found that 5 per cent. of a hormone appears in the urine when it is injected, it may be justifiable to make the provisional assump- tion that the daily output by the gland is equal t o the amount excreted in the urine multiplied by 20. In such instances it is important to remember that changes observed in the urinary output may represent either a change in the amount of hormone released into the blood stream or a change in the proportion of the hormone reaching the urine. So long as this fact is borne in mind, estimations on the urine may be expected to give valuable information not given by estimations on blood.The time will probably come when provision for assays of this type will be an essential part of the equipment of all large hospitals, but that time is not yet. Very few methods of assay available at present are sensitive enough for this purpose; the discovery of more sensitive methods is a most important objective. Chorionic gonadotrophin is present in such high concentrations during pregnancy that its concentration in both blood and urineOctober, 19501 SCHILD: GENERAL APPROACH TO BIOLOGICAL ASSAYS 533 can be accurately estimated by bio-assay, but this is not true of any other hormone. Until more sensitive tests are discovered the amount of hormone available is too little for a perfectly designed assay.REFERENCE 1. Emmens, C . W., ( ‘ Variables affecting the Estimation of Androgenic and Oestrogenic hctivity,” Spec. Rep. Ser., No. 234, Medical Research Council, London, 1939. PHARMACOLOGICAL LABORATORY UNIVERSITY NEW BUILDINGS EDINBURGH, 8 General Approach to Biological Assays BY H. 0. SCHILD SYNopsrs-The term ‘‘ bio-assay ” is used for two different procedures. In the first, an unknown sample is assayed in terms of a standard of the same composition, the aim being to determine the amount of some chemical substance present; this may be termed “ analytical assay.” In the second, the test sample is assayed in terms of a substance or mixture of different composition, the aim being to determine the biological activity of the test sample.The paper briefly reviews some of the principles underlying the analytical type of assay. BIOLOGICAL assays may be used for two purposes, which must not be confused. Either they may be used analytically, as substitutes for a quantitative chemical analysis, or they may be used to determine the biological activity of a substance or a mixture of substances. In the first case the aim of the assay is to detect the concentration of a certain chemical substance in a sample. The only essential condition is that the standard and unknown should have the same composition. If they are chemically identical, then it does not matter what species or what reaction is used in the test; the final result is always the same and the best method is that by which an accurate result is produced with the greatest economy of time and labour.An entirely different situation arises where the two samples differ in chemical composition. The assay then ceases to be an analytical method and becomes a comparison of biological activity in which species, end-point and experimental conditions become all-important. When two substances that are chemically different, say cocaine and procaine, are com- pared, it is impossible to assign to them an activity ratio in general terms, since their relative activity depends on experimental conditions. Further, even under constant experimental conditions, the results of successive experiments are not necessarily identical, as the relative sensitivity of a preparation to different substances is not constant.Not only major chemical differences, but even minor differences in composition and purity may completely distort an assay and change it from an analytical method into a comparison of activity valid only for the particular species and method of administration used. For example, Gold, Cattel, Kwit, Kramer, Modell and Zahml found that by oral administration in man, 1.25 mg. of digitoxin produced the same effect as 1.25 g. of digitalis, when tested by a fall in heart rate in patients with auricular fibrillation. By the intravenous cat method, however, the former dose corresponds to 3 to 4 lethal “ cat units” whilst the latter corresponds to 12.5 cat units. Although digitoxin is the main active constituent of digitalis, yet in this instance differences in purity, in the method of administration and in the species used led to entirely different results by the two methods.COMPARATIVE ASSAYS NECESSITY FOR A STABLE STANDARD- It is generally believed that biological assays, at least those of the analytical kind, should be comparative and should be made by reference to a stable standard. This belief is based on two assumptions: first, that it is not possible to keep a biological reaction under534 SCHILD : GENERAL APPROACH TO BIOLOGICAL ASSAYS [Vol. 75 “statistical control’’ in different laboratories and that statistically significant variations in the activity of so-called animal units occur between different laboratories and also within the same laboratory at different times; secondly, that international standard preparations are stable and do not deteriorate. A good example of the variability of animal units is provided by the collaborative digitalis assay done for the U.S.P.under the guidance of B l k 2 In this assay the intravenous lethal cat dose of digitalis was determined, using the same standard preparation in various laboratories and at various periods. The assay technique was completely standardised and in each experiment the standard was compared with an unknown consisting of a dilution of the standard preparation. When the results were analysed it was found that the lethal dose of standard-the cat unit-would stay constant, within the limits of error of the assay, only for periods of about 8 or 10 days in any one laboratory. During that period there was therefore no need, in assaying the unknown, to refer back more than once to the standard preparation.When, however, the assay was repeated in the same la‘boratories a few months later, the cat unit had altered significantly in about half of the laboratories. Table I, which gives some data TABLE X Degrees of Variance ratio, Variation freedom Mean square F Between collaborators . . . . . . 9 0,03162 4.99 Between preparations . . .. .. 1 0.1 1491 18.15 Collaborators x preparations . . . . 9 0-00463 0.73 Error . . . . . . .. . . . . 160 0.00633 1.00 taken from Bliss’s paper, shows that significant variation in the lethal dose occurred between different laboratories (collaborators), but it is of interest that the mean square for “ interaction” of laboratories on preparations was no greater than that for error.As in this instance inter- action measures the relative potency of standard and unknown as determined in different laboratories, the result signifies that although the absolute activity as determined in different laboratories varied, the relative activity of standard and unknown remained the same within statistical limits in the hands of different investigators. In other words, whilst the absolute values could not be brought under statistical (control the relative activities had been so controlled. This is a good demonstration of the superiority of comparative assays over assays relying on animal units. DETERIORATION OF STANDARD- In using comparisons with a stable standard, rather than animal units, for biological standardisations, one assumes of course that the variation in activity of the stable standard is negligible compared with the variation of the animal unit.Unfortunately, there is no way of determining whether this is true or whether a so-called stable standard is deteriorating. This problem arose, for example, in 1942, when the standard for posterior pituitary at Hampstead was replaced by a fresh standard. The new standard was compared in a number of laboratories against the old standard by the three methods, oxytocic, vaso-pressor and antidiuretic, and was generally found to be about 15 per cent. more active than the old one. The Biological Standards Commission, however, decided to adopt as a unit the same weight of the new standard as had been used for the old standard, taking no account of the higher activity of the new one.Both standards had been prepared by the methods of Voegtlin, who had himself assumed that an extract prepared from fresh glands strictly according to his instructions would always have the same activity. This may well have been so, and, in that event, one has to assume that the old standard had slightly deteriorated. I do not wish to suggest, however, that everyone should now prepare his own standards by Voegtlin’s methods for fear that the international standard may have deteriorated. It may be expected that workers less skilled than Professor Voegtlin would produce extracts varying in activity by much more than 15 per cent. STANDARDISATI~ON ON MAN In many biological standardisations one: is dealing with impure extracts whose identity with the standard cannot be guaranteed.Since relative activity may then depend on the species and end-point used for the assay, should these standardisations be done on man?October, 19501 SCHILD : GENERAL APPROACH TO BIOLOGICAL ASSAYS 535 The answer can only be found experimentally by comparing standardisations done on animals and on man. The most thorough attempt at standardisation on man has probably been that of Gold, Cattel, Otto, Kwit and KrameI.3 in New York who tried to standardise digitalis on man. These workers used two reactions: the slowing of the heart rate in auricular fibrillation and an inversion of the T-wave in the electrocardiogram. The first method is preferable because it measures a therapeutic action, but the second gives more accurate results.In one series, using the slowing of the heart rate, these workers concluded that the result of comparisons for activity of the standard powder with an unknown in man agreed fairly well with those of the cat assay, although the agreement between the human assay and the frog assay was not as close. In another series, however, using the T-wave test, they found marked discrepancies between the human assay and the cat assay, amounting sometimes to over 50 per cent. Kamm separated the oxytocic and the vaso-pressor principles, using the guinea-pig uterus as test object. Pitressin had only a very small action on the guinea-pig uterus, and this was usually attributed to contamination with pitocin. Later on it was found that pitressin did affect the uterus in some species; for instance, in the cat it may produce a strong inhibition of the uterus (Robson and Schild*) and more recently it has been shown by Chassar MoiI-5 that in man pitressin produces under certain conditions a greater contraction of the uterus than pitocin.Obviously a pituitary extract standardised by the guinea-pig method gives no adequate indication of activity in the human uterus. The routine assay of pituitary extract on the parturient human uterus is probably impractical, but it would be interesting to know whether at that stage an extract of pituitary standardised by the usual methods for oxytocic activity would be more active than purified oxytocin standardised in the same way. It would therefore seem that in dealing with mixed extracts, animal standardisations are a useful safeguard rather than a reliable method of assessing activity.It is important that animal standardisations of these extracts should occasionally be checked by human standardisation, but even this is no complete safeguard, as pointed out by Gaddum (previous paper), since all human beings do not react alike. Apparently these experiments have not been repeated. Posterior pituitary hormone is another interesting example. STATISTICAL METHODS The simplest type of graded assay is one for which no statistics are required, inwhich the standard and unknown are matched by a trial-and-error process until they produce equal effects. This method has one great advantage: it does not depend on the assumption of a mathematical relation between dose and effect.However, it also has several disadvan- tages. First, it is inefficient because the preliminary effects are not utilised in the final assessment. Secondly, it is subjective, depending on the judgment of the experimenter as to what constitutes a satisfactory match. Thirdly, its experimental error cannot be determined from the assay itself but only by separate experiments. And fourthly, since the effects are matched at only one dose level, it gives no indication of whether the con- centration - action curves of standard and unknown are parallel. It thus fails to show up qualitative differences between standard and unknown. Assays that involve statistical methods are usually based on the assumption of some mathematical relation between dose and effect.Experience has shown that frequently the relation between dose and effect is expressed by an S-shaped curve when the dose is plotted on a logarithmic scale. Thus, if the reaction is of the all-or-nothing type (quantal), an S-shaped curve results if the percentage of animals reacting is plotted against log dose, whilst if the reaction is of the graded type (quantitative) an S-shaped curve results when the size of the effect is plotted against log dose. Usually they are interpreted as integrated normal curves when the reaction is quantal and as Langmuir adsorption curves when it is quantitative. In order to obtain approximately straight lines only the middle part of the graded response curve may be utilised. With all-or-nothing reactions the ordinates may be transformed to normal equivalent deviations or probits.The transformed curves are usually approximately linear, especially in the range above 20 per cent. mortality. Other transformations have been used, such as the logit transformation derived from the logistic curve. Finney6 has shown that practically identical results may be obtained in an assay using any one of four different transformations. These S-shaped curves may be variously interpreted.536 SCHILD: GENERAL APPROACH TO BIOLOGICAL ASSAYS [Vol. 75 Gaddum has outlined three characteristics for a satisfactory assay, viz., (1) a linear relation between x and y over the relevant range, (2) a standard deviation (this applies to graded responses only) independent of effect and (3) a small value of s/b.The latter is a good measure of variability, since it measures the variability of response in terms of the dose. Bliss has suggested another important characteristic of a good assay, namely, that it should furnish an estimate of its variability as an integral part of the assay. The statistics of quantal and quantitative assays have evolved on rather different lines. This is due to a mathematical peculiarity of quantal assays, the need for weighting the response. In quantitative assays, on the other hand, it can usually be assumed that all points on the curve have the same variance and therefore the same weight. Owing to this fact, Fisher’s analysis of variance could be readily applied to quantitative assays with all its implications of planning experiments in such a way that sources of variation not attributable to experimental error may be eliminated from the final comparison. Statistical methods are becoming increasingly complex, and we must leave it largely to the statisticians to work out their mathematics.Biologists should, however, keep in mind the basic assumptions on which these mathematics are founded. For example, the limits of error of an assay involving two straight parallel lines may be calculated by one of two formulae, one giving what are usually called the approxirnate limits of error, and the other giving exact fiducial limits. The reason why one is called “approximate” and the other “exact” is that in the derivation of one formula a mathematical approximation is used, namely, the variance of the ratio of two variables, whilst the derivation of the other is mathematically exact.It would, however, be wrong to assume t‘hat, because the formula is mathematically exact, the actual fiducial limits as calculated in an individual assay are necessarily correct. This would only be so if the assumptions on which the calculations are based were exact. These, however, we know to be only approximations; for example, we know that the assump- tion of linearity in quantitative assay is an approximation. One may, of course, test the data for deviations from linearity and parallelism, but lack of statistically significant deviation from linearity does not prove a linear relation in a particular assay. Thus the fiducial limits as calculated from a given experiment must always be taken with a grain of salt.This applies particularly to research work where new situations constaqtly arise and where the basic assumptions have not been tested by extensive previous experience. REFERENCES 1. 2. 3. 4. 6. 6. UNIVERSITY COLLEGE, LONDON Gold, H., Cattell, McK., Kwit, N. T., Kramer, IM. L., Modell, W., and Zahm, W., J . Pharmacol., Bliss, C. I., J . Amer. Pharm. Assoc. Gcielztific Edition), 1944, 33, 225. Gold, H., Cattell, McK., Otto, H. L., Kwit, N. T., and Kramer, M. L., J . Pharmacol., 1942.75, 196. Robson, J. M., and Schild, H. O., J . Physiol., 1938, 92, 1. Chassar Moir, J . Obstet. and Gyn., 1944, 51, 247. Finney, D, J., J . Roy. Statist. Soc., 1947, 9, Supplement, p. 46. 1944, 82, 187. DEPARTMENT OF PHARMACOLOGYOctober, 19501 SOMERS : THE MEASUREMENT OF THYROIDAL ACTIVITY 637 The Measurement of Thyroidal Activity BY G.F. SOMERS SYNoPsIs-After an account of methods used or proposed for evaluating thyroid activity in natural and synthetic products, this paper includes a brief description and comparative assessment of the method based upon comparing the resistance of mice to anoxia, which is reduced by thyroid treatment. Assay methods based on the antagonistic action of anti-thyroid drugs and thyroid preparations are also discussed. r. I H E evaluation of thyroid preparations is a problem not yet satisfactorily solved. The official method of standardisation (British Pharmacopoeia, 1948)l is based on Harington and Randall’s estimation2 of acid-insoluble organically combined iodine. The United States Pharmacopoeia, 1947,3 makes use of the total iodine present in organic combination.Neither method gives values in close accord with physiological results4 ; on chemical considerations the B.P. method is to be preferred, on the assumption that thyroxine is the only source of acid- insoluble iodine. The thyroid contains two distinct organic iodine compounds, thyroxine and di-iodo- tyrosine; only the former is physiologically active. The relative amounts of the two com- pounds differ considerably in different samples of thyroid. The method of Harington and Randall2 appears satisfactory for those thyroid preparations in which all the acid-insoluble iodine is in thyroxine, but it is unsatisfactory for iodinated proteins.These contain a large amount of acid-insoluble iodine, of which only a small and variable proportion has thyroid activity.5 Chemical analysis of these preparations being therefore of little value, biological assays are necessary. A modification of Harington and Randall’s method was developed by Leland and Foster6; in this, after the preliminary alkaline hydrolysis, the thyroxine fraction is extracted with butyl alcohol. Reineke, Turner, Kohler, Hoover and Beezley7 have claimed excellent agreement between the results of biological assays and a chemical extraction method modified from that of Blau.8~9 Other chemical methods suggested involve absorptiometric measurementslO of the red colour formed with sodium nitrite in ammoniacal solution,ll of the red colour formed with diazobenzene sulphonic acid in sodium carbonate solution,l2 or of the purple colour formed with diazotised N1-diethylsulphanilamide in alkaline solution.13 Polarographic methods have also been emp10yed.l~ J5$16 They not only determine the total iodinated diphenyl ethers present, but can also distinguish between thyroxine and 3 : 5-di-iodotyrosine. It should be remembered that the optical form of thyroxine is all-important, the physiological activity of DL-thyroxine residing largely, if not entirely, in the laevorotatory component.Thyroxine in thyroid extracts and in iodinated proteins is solely the L-form. BIOLOGICAL ASSAYS Although chemical methods are reasonably satisfactory for standardising gland extracts, there is occasionally a need to evaluate the thyroid activity of new materials, such as iodinated protein, thyroxine analogues and other organic compounds.In testing for thyroid activity, the accepted principles of biological assay must be applied. Simultaneous examinations of a standard preparation and the test material should be made; accurate assays are only possible when the active principle of both is the same. In comparing new compounds with old, quantitative assessments should be made with caution and results obtained with one species should not be assumed to apply to another. METHODS Numerous biological methods have been suggested for the assay of thyroidal activity ; some of them bear no obvious relationship to the physiological action of thyroxine in mammals, e.g., the acetonitrile test in mice.17 No one of them has been generally accepted, possibly because of their practical difficulties and inherent inaccuracy.Most procedures have not been developed on a reasonably quantitative basis; few workers have accompanied their reports on the comparison of two thyroid preparations with information about the precision538 SOMERS : THE MEASUREMENT OF THYROIDAL ACTIVITY [Vol. 75 of the estimates attainable. it has often been found that in fact the precision claimed has not been achieved. The commoner methods employed will now be briefly considered. INCREASE OF METABOLISM- Thyroid preparations cause an increase in metabolic rate ; the associated changes occurring in oxygen consumption and carbon dioxide production can be used for the estimation of thyroidal activity.Such methods are most closely related to the physiological actions of thyroid in man, but they tend to be too laborious and troublesoqe for routine assays. For the measurement of oxygen consumption the animal is usually enclosed in a chamber immersed in a constant-temperature water-bath.ls 9 1 9 Expired carbon dioxide is absorbed in soda-lime and the oxygen consumed is measured by a valve or from a Benedict spirometer. By this method, Wokes2* compared thyroid extracts on guinea-pigs, and obtained limits of error, P = 0-95, of & 30 per cent., using two dose levels of the standard and test preparations with six guinea-pigs on each dose level. The guinea-pig is very suitable for this type of assay because of its sensitivity and rapid response. The apparatus is possibly over-elaborate, and a simpler method for measuring the oxygen consumption of mice, which should be readily adaptable to the assessment of thyroid preparations, has recently been described by Maclagan and Sheahan.21 is usually employed.Mice are placed in a closed container through which air is drawn after previous removal of carbon dioxide with soda-lime. The carbon dioxide evolved by the animals is absorbed in a train containing standard sodium hydroxide, the amount being subsequently determined by titration with standard hydrochloric acid. According to March, the method is one of the most accurate for determining thyroidal activity, but a simpler and less time- consuming method is desirable for routine assays. Gaddum and Hethe~ington~~ compared the activities of a number of thyroid preparations by this method, but the results were only roughly proportional to the thyroxine contents determined by the method of Harington and Where data susceptible to statistical analysis have been given , For the estimation of carbon dioxide production the method of INCREASED SUSCEPTIBILITY TO ANOXIA- The thyroid gland has an influence on the susceptibility of mammals to anoxia, their resistance being decreased by previous administration of thyr0xine.2~ y25 p26 $27 Smith, Emmens and Parkes28 have developed a method of assay for thyroid preparations based on this decreased survival time of mice enclosed in jars.We have utilised the method extensively for com- paring the activity of thyroxine and a number of its derivatives.29 For calculation of the results we used the logarithms of the survival times, because the distribution within each group of the survival times themselves was skew.The relative activities were calculated by the method of Bliss and Marks30 and errors in the slopes provided for according to 1rwi1-1.~~ The slope, b, for thyroxine sodium by the subc:utaneous route was -20.8 with a standard error (P = 0.95) of s 3 . 1 , b being calculated as the increase in log survival time per 10-fold increase in the dose. The value of s/b = 0.35; to attain a precision of 5 20 per cent. (P = 0.95) it would therefore be necessary to ernploy 240 animals on both the standard and test preparations and to use three dose levels for each. The slopes were even less steep by the oral than by the subcutaneous route (b = 14-8, s/b = 0.48).The precision of the method therefore leaves something to be desired, but the assays are relatively easy to carry out. LOSS OF BODY-WEIGHT- The administration of thyroid to mice, rats and guinea-pigs causes a loss in body- weight.2°~22~32~33~34 Gaddumlg found that adult rats lost weight pari passu with the rise in their metabolic rate. WokesJ20 however, found wide variations in the susceptibility of mice to thyroxine and considered that large numbers would be required for an assay based on weight changes. Hutcheon,% using rats, showed that the slope of the line relating the loss in weight to the log dose of thyroxine was 9.1 per unit log dose with a standard deviation of & 4.4 g. The precision of the estimates is thus not good; further, the method cannot be specific, the presence of toxic factors influencing the result.There is also a wide variation in the response of guinea-pigs to a given dose, so that large numbers must be employed under carefully controlled conditions, or the results obtained will be far from precise.3qeas The value of s/b is therefore 0.49.October, 19501 SOMERS : THE MEASUREMENT OF THYROIDAL ACTIVITY 539 DECREASE IN SUSCEPTIBILITY TO TOXIC SUBSTANCES- Hunt37 observed that previous administration of thyroid by mouth increased the resistance of mice to acetonitrile poisoning. A method for assaying thyroid preparations, based on this observation, was developed by Hunt and Seidell,38 and this was improved by Hunt39 and by Haffner and K ~ n y a m a .~ ~ Unfortunately, the susceptibility of mice to acetonitrile is extremely variable and is affected by numerous known and unknown factors. Gaddum and Hetheringt~n~~ controlled these factors in some experiments, but the variation in the response of the mouse to acetonitrile alone was so great that they rejected the procedure as a possible test method. VVokes4O found that at ordinary room temperatures the dose - mortality curve for acetonitrile became very flattened. A t higher temperatures the dose - mortality curve was steeper, but thyroid no longer afforded any protection. Laland and St0a41 obtained more favourable results with the method and considered it serviceable for routine tests, but they have not stated its precision. INCREASE IN THE RATE OF AMPHIBIAN METAMORPHOSIS- G~dernatsch~~ showed that a thyroid preparation accelerated the rate of metamorphosis of amphibian larvae, and caused a rapid shrinking in size, if suspended in the water in which they were living.Several of the effects on tadpoles have been utilised for testing the activity of thyroid preparations, These include: time to first appearance of the leg buds, time to produce death, length of gut, body-weight, body volume and total length. A number of Anura have been employed, Rana pipiens and Rana temporaria being mostly used. The chief difficulty with the tadpole test has been a regular supply of tadpoles; this can be over- come by the use of Xenopus Zaevis if they are bred in the laboratory.43 As a method of assay, the use of amphibian larvae can be criticised on the grounds of unspecificity, particularly because of the wide difference in species from mammals and in the mode of administration Di-iodotyrosine and D-thyroxine are active in amphibia43 but relatively inactive in mammals.Quantitative methods have been described by numerous workers. woke^,^ using tail length in tadpoles from Rana temporaria, obtained results giving an s/b value of 0.38, and sub- stantially similar values are shown by the results of Deanesly and Parkesm using eruption of the front legs in XenopzGs laevis. RESTORATION AND MAINTENANCE OF GROWTH IN YOUNG THYROIDECTOMISED RATS- The retardation of growth in young thyroidectomised rats is prevented by the daily injection of a thyroid powder suspension or thyroxine. The possibility of using the response for measuring the biological activity of iodinated caseins and other allied substances was investigated by Rowlands.@ The method, however, as an assay procedure, presents difficulties in practice.The curve relating dose to response is so flat that a very large number of animals would have to be used, 6100 for an error of & 10 per cent. at P = 0.95. The time needed for the test, 90 days, is also too long. ANTAGONISM OF THYROXINE TO ANTI-THYROID DRUGS- Certain anti-thyroid compounds, such as ally1 thiourea, phenyl thiourea, sulphaguanidine, thiourea and thiouracil, prevent the formation of the active thyroid p r i n ~ i p l e . ~ g ~ ~ 947 The subsequent decrease in the amount of circulating hormone in the blood causes an increased production of thyrotrophic hormone by the anterior lobe of the pituitary; this, through its stimulating action, causes enlargement and histological changes in the thyroid gland.The hyperplasia and histological changes induced by the feeding of anti-thyroid drugs is inhibited by simultaneous administration of thyroxine, but not by and the ability of thyroidal substances to reduce the enlargement of the thyroids of chicks49 and rats48t60 treated with thiouracil may be used for the assay of thyroidal activity. The method gives results in accord with those obtained by the standard metabolic method.5* According to Hutcheon,% the method with rats is reasonably precise, the s/b value being 0.16. The chick has advantages over the rat in its cheapness, greater sensitivity, availability and ease of handling.The anti-thyroid drugs also cause a decrease in the iodine content of the thyroid glands, which can be prevented by the simultaneous administration of thyroxine.51 The iodine concentration in the glands can be measured chemically or by radio-active tracer techniques using 1311, and can therefore be employed as an index of thyroidal activity.540 SOMERS: THE MEASUREMENT GF THYROIDAL ACTIVITY [Vol. 75 CONCLUSIONS In spite of considerable effort, no wholly satisfactory method for measuring thyroidal activity has yet been evolved and discrepancies arise in comparing biological with chemical results. While chemical methods based on the estimation of acid-insoluble iodine (British Pharmacopoeia, 1948) may be preferred to those based on total iodine, neither appears to give a reliable indication of the physiological activity of various thyroid preparations.* 923 Discrepancies may be large, and indeed evidence is accruing that the published estimates for the thyroidal activity of iodo-casein preparations, which has been stated to be equivalent to about 3 per cent.of L-thyroxine,52 were in ‘The chief difficulty with biological assays is the extent of variation in the response of the individual animals, which necessitates the employment of relatively large numbers before an adequate degree of precision can be attained. While thyroxine is undoubtedly the active principle present in thyroid extractsa and in iodinated proteins,= the form in which it is administered plays an important part in its activity, especially by the oral Differences in the availability of thyroxine in various preparations may account for the discrepancies.Similarly, the same calorigenic substances may be utilised differently by different organisms and results obtained with one species may not apply to another. We can say that neither chemical analysis nor physiological assay will give a true index of the activity of a preparation on man; this can only be settled by clinical trial. In any event the development: of a new method for the synthesis of L- thyroxine60 may reduce considerably the need for bio-assays. ,67 y5* 959 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.REFERENCES “British Pharmacopoeia,’’ General Medical CoLtncil, London, 1948. Harington, C. R., and Randall, S. S., J . Yharw. Pharmacol., 1929, 2, 501. United States Pharmacopoeia, U.S. Pharmacopoeia1 Convention, Washington, 1947. Wokes, F., J. Pharm. Pharmacol., 1938, 11, 521. Deanesley, R., and Parkes, A. S., J . Endocrinol., 1945, 4, 324. Leland, J. P., and Foster, G. L., J . Biol. Chern., 1932, 95, 165. Reineke, E. P., Turner, C. W., Kohler, G. O., Hoover, R. D., and Beezley, M. B., Ibkd., 1945, Blau, N. F., Ibid., 1933, 102, 269. -, Ibid., 1935, 110, 351. Reineke, E. P., and Turner, C. W., Agv. Exp. Sta. Mo., Res. Bull., 1942, 355. Koche, J., and Michel, R., Biochem. Biuphys. Acta, 1947, 1, 335. Moser, H., Experientia, 1947, 3, 119. Winikoff, D., and Trikojus, V.M., Biochem. J., 1948, 42, 475. Simpson, G. K., and Traill, D., Ibid., 1946, 40, 116. Simpson, G. K., Johnston, A. G., and Traill, I)., Ibid., 1947, 41, 181. Borrows, E. T., Hems, B. A., and Page, J. E., J. Chem. Soc., 1949, S 204. Hunt, R., Arch. Intern. Med., 1925, 35, 671. Richards, A. N., and Collison, L. W., J . Physiol., 1928, 66, 299. Gaddum, J . H., Ibid., 1930, 68, 383. Wokes, F., “A Comparison of Different Methods of Estimating the Biological Activity of the Thyroid Hormone,” Ph.D. Thesis, University of London, 1938. Maclagan, N. F., and Sheahan, M., Proc. 1st Internat. Congr. Biochem., 1949, 413. Mclrch, J. R., J . Physiol., 1929, 67, 221. Gaddum, J. H., and Hetherington, M., J . Pharm. Pharmacol., 1931, 4, 183. Duran, M., Biochem. Z., 1920, 106, 254.Rydin, H., C.R. SOC. Biol., Paris, 1928, 99, l(585. Campbell, J. A., J . Physiol., 1938, 92, 2 9 ~ . Leblond, C. P., Proc. SOG. Exp. Biol., N . Y., 1944, 55, 114. Smith, A. U., Emmens, C. W., and Parkes, A. S., J . Endocrinol., 1947, 5, 186. Basil, B., Somers, G. F., and Woollett, E. A., Brit. J . Phavmacol., 1950, 5, 315. Bliss, I. C., and Marks, H. P., J . Pharm. Pharmacol., 1939, 12, 82 and 182. Irwin, J. O., J . Roy. Statist. SOG., 1937, 4, Supplement, 1. Cameron, A. T., and Carmichael, J., J . Biol. Chem., 1920, 45, 69. Haffner, F., and Konyama, T., Arch. Exp. Path. Pharmak., 1925, 107, 69. Kreitmair, H., 2. ges. exp. Med., 1928, 61, 202. Hutcheon, D. E., J . Pharmacol., 1948, 94, 308. Rotter, G., and Mecz, M., Arch. Exp. Path. Pharmak., 1932, 166, 649.Hunt, R., J. Bid. Chem., 1905, 1, 33. Hunt, R., and Seidell, A., Hyg. Lab. Bull., 1909, 47. Hunt, R., Amer. J . Physiol., 1923, 63, 257. Wokes, F., J . Pharm. Pharmacol., 1935, 8, 54. Laland, P., and Staa, K. F., Acta Pharmacol. Toxicol., 1949, 5, 1. Gudernatsch, J. F., Amer. J . Anat., 1912, 15, 431. Deanesly, R., and Parkes, A. S., J . Endocrinoi., 1945, 4, 324 and 356. 161, 599.541 October, 19503 SOMERS: THE MEASUREMENT OF THYROIDAL ACTIVITY 44. Rowlands, I. W., Ibid., 1945, 4, 305. 45. Mackenzie, C. G., and Mackenzie, J. B., Endocrinology, 1943, 32, 185. 46. Astwood, E. B., Sullivan, J., Bissell, A., and Tyslowitz, R., Ibid., 1943, 32, 210. 47. Astwood, E. B., J . Pharmacol., 1943, 78, 79. 48. Dempsey, E. W., and Astwood, E. B., Endocrinology, 1943, 32, 509.49. Mixner, J. P., Reineke, E. P., and Turner, C. W., Ibid., 1944, 34, 168. 50. Reineke, E. P., Mixner, J. P., and Turner, C. W., Ibid., 1945, 36, 64. 51. Astwood, E. B., and Bissell, A., Ibid., 1944, 34, 282. 52. Reineke, E. P., “Vitamins and Hormones,” Academic Press, New York, 1946, 4, 207. 53. Bailey, G. L., Bartlett, S., and Folley, S. J., Nature, 1949, 163, 800. 54. Kendall, E. C., Trans. Assoc. Amer. Phys., 1915, 30, 420. 55. Reineke, E. P., and Turner, C. W., J . Biol. Chern., 1943, 149, 563. 56. Schittenhelm, A., and Eisler, B., 2. ges. ex?. Med., 1932, 80, 569. 57. Thompson, W. O., Thompson, P. K., Dickie, L. F. N., and Alper, J. M., Arch. Intern. Med., 1933, 58. Elmer, A. W., “Iodine Metabolism and Thyroid Function,” Oxford University Press, London, 59.Monroe, R. A., and Turner, C. W., Amer. J . Physiol., 1949, 156, 381. 60. Chalmers, J. R., Dickson, G. T., Elks, J., and Hems, B. A., J . Chem. Soc., 1949, 3424. 52, 809. 1935. RESEARCH DIVISION GLAXO LABORATORIES LIMITED GREENFORD , MIDDLESEX DISCUSSION DR. H. 0. J. COLLIER said that for the test of thyroidal activity, using protection against toxic agents as a criterion, it might be expected that certain toxic agents would be more suitable than others. For example, some of the substances that were known to undergo a marked chemical detoxication in the body might be suitable for this test, since the test material could be expected to stimulate the general level of activity of the cells. Had Dr. Somers conducted any comparative experiments on different types of toxic agents for use in this test of thyroidal activity, and, if so, did they lend any substance to the foregoing suggestion ? DR.SOMERS replied that other chemical substances could be and had been tried, but he had only used acetonitrile. He thought Dr. Collier’s suggestion a reasonable one that probably explained the mode of action of thyroxine in lowering the toxicity of acetonitrile. MR. W. A. BROOM said that in his laboratory they had had experience with three of the methods described by Dr. Somers, namely, the method based on the change in metabolism in guinea-pigs, the closed vessel mouse method and the tadpole method. In their hands, the metabolism method was most unreliable and time consuming, and the mouse method, though workable, was less precise than the tadpole method.They preferred the tadpole method, which gave results that agreed very well with the experience of veterinary workers studying the effect of iodinated casein on the milk yield of cows. DR. R. PITT-RIVERS pointed out that experimental evidence was available that D-thyroxine was active when tested on myxoedematous patients; it had one-eighth to one-tenth of the activity of L-thyr0xine.l She did not consider it justifiable to compare a D- with a DL-amino acid and to deduce the relative activities of D- and L-forms because, in the presence of the natural isomer, the very slight activity of the unnatural isomer might be masked or lost owing to experimental error. REFERENCE TO DISCUSSION 1. Pitt-Rivers, R., and Lerman, J., J . Endocrinol., 1948, 5, 223.542 STEWART : THE ASSAY OF POSTERIOR PITUITA4RY LOBE EXTRACTS [Vol.75 The Assay of Posterior Pituitary Lobe Extracts BY G. A. STEWART SYNOPSIS-A review is given of the methods of assaying the oxytocic, vasopressor and antidiuretic hormones of posterior pituitary lobe extracts. The difficulties of assaying the oxytocic hormone by the guinea-pig uterus method are described in detail, and descriptions are given of the rat uterus and chicken blood-pressure assays, which enable the hormone t o be determined with a much greater degree of accuracy. Assays of the vaso- pressor hormone on the blood pressure of the spinal cat and anaesthetised dog are described, and mention is made of the more recently developed assays of this hormone on the blood pressure of the rat.Assays of the antidiuretic hormone on the rat and dog are described. The relative merits of the assays referred to are compared and discussed. The variability of commercial extracts in respect to their contents of these three hormones is considered. THREE of the active principles of posterior pituitary lobe extracts are used clinically, namely, the oxytocic, vasopressor and antidiuretic hormones, and of these, as a rule, only the oxytocic and vasopressor hormones are assayed commercially. Generally an extract containing all three hormones is assayed for its oxytocic activity only. THE ASSAY OF THE OXYTOCIC HORMONE- The “in vitro” method of Dale and Laidlawl using the guinea-pig uterus for the assay of the oxytocic principle is beset by many practical difficulties, notably that of obtaining a suitable uterine preparation.The uterus usually employed is that from a virgin guinea-pig in dioestrus. Some uteri contract very slowly when stimulated by posterior pituitary lobe extract, some fail to give good differentiation between graded doses of the same extract, whilst others may react to the same dose of the pituitary extract with alternate large and small contractions. As a result of these many difficulties, different workers have sought to find conditions under which the guinea-pig uterine assay may work to advantage in their own laboratories, whilst others have investigated the “in vitro” assay using the uteri of different animals. Morell, Allmark and Bachinski2 described a method using eight small longitudinal strips of uterus, obtained from both uterine horns of a guinea-pig, suspended in Van Dyke and Hastings’ s~lution.~ The number of strips responding to a dose of pituitary extract was recorded on a smoked kymograph paper. Three doses of the standard solution and three doses of the unknown solution, whose potency was assumed to be equivalent to that of the standard, were administered in various volumes.The larger doses produced responses from between 50 and 87.5 per cent. of the strips. The medium doses produced responses from about 50 per cent., and the smaller doses, responses from about 25 per cent. of the strips. Each dose was given three times in a restricted randomised design. The injections of the standard doses alternated with those of the unknown solution. They observed that it was preferable to give the higher concentrations first.From these quanta1 responses the potency of the unknown solution could be calculated and a standard error determined. There was no significant difference in the results obtained with uterine strips from the uteri of guinea-pigs in oestrus and in dioestrus. Later, these workers4 transformed the same technique into a quantitative assay by measuring all responses over 1 mm. produced by the eight uterine strips. Of 23 assays on solutions whose potencies were known, 16 assays produced a range of percentage standard errors that did not include the known potency and thus showed the fallibility of the method. Hamburger5 studied the assay of the oxytocic hormone on the isolated uterus of avirgin guinea-pig and used four concentrations of both the standard and the unknown solutions.The ratio between successive concentrations was 2 to 1. Two experimental designs for the injection of doses into the bath were considered, one in which the four concentrations of standard were given in diminishing order, followed likewise by the four doses of the unknown solution. The second design was to give The response was related to the logarithm of the dose. This dose sequence was given three times in all.October, 19501 STEWART : THE ASSAY OF POSTERIOR PITUITARY LOBE EXTRACTS 543 doses of the standard alternately with doses of the unknown twice at each of the four dose levels, the latter being randomised. Responses were measured and related to the logarithm of the dose. From these data the potency of the unknown solution could be determined.No complete analysis of the results was given, but Hamburger claimed that a posterior pituitary lobe extract could be standardised with " sufficient accuracy" by the employment of three or four suitable guinea-pig uteri. Some workerss s 7 have attempted to improve the guinea-pig uterine assay by modifying the magnesium and calcium ion concentrations of the Ringer's solution. They claim thereby to have improved the assay. Fig. 1 shows the tracing of a test carried out by the assay procedure adopted in our laboratories. The uterus of a virgin guinea-pig was suspended in the physiological salt solution described in the British Pharmacopoeia,s maintained at a temperature of 37" C.and aerated with compressed air. Recordings were made on a smoked kymograph paper with a frontal writing lever giving a magnification of x 2. Two different submaximal responses to an extract of the standard were obtained. Next a dose of the unknown solution giving a response intermediate between the two standard responses was found, and the three doses were repeated in the reverse order. The maximum ratio we allow for the two doses of standard is 1.33. The interval between doses was 10 minutes. GUINEA-PIG 210 G. S = 0.0167 UNIT/rnl. DOSES IN mi. T = UNKNOWN 11600 0.8 0.6 0.7 0.7 0.6 0.8 S U S T T S S MIN Fig l.* Typical example of the isolated guinea-pig uterine assay of a posterior pituitary extract. S = ml. of Extract of the International Posterior Pituitary Standard 2 I.U.per ml.; dilution 1 : 120. T = ml. of Unknown; dilution 1 : 600. * Fig. 1 and all the tables in this paper are reproduced from the Journal of Pharmacy and Pharmacology, 1949, 1, 436-453, by permission of the Editor. Gaddums studied the precision of the guinea-pig uterine assay when four doses were employed, e.g., standard, unknown, unknown, standard, until the unknown is found to have an activity greater than one particular potency and less than another potency. He discarded results in which the ratio of the two limiting activities was greater than 1.5, and found that the standard error on 43 assays was 7.73 per cent. HoltonlO described a method employing the isolated uterus of a non-pregnant rat of weight 120 to 200g., suspended in Locke's solution having one-quarter of the calcium and one-half of the glucose concentrations normally employed.Recordings of the uterine con- tractions were made with an isotonic and linear lever of Schild's design.ll The bath was aerated with a mixture of 95 per cent. of oxygen and 5 per cent. of carbon dioxide and kept at a constant temperature of about 32" C. A four-point assay was conducted by the method described by Schild.12 Doses were administered regularly at intervals of three or four minutes and the mean experimental time of an assay was 3.75 hours, with a mean percentage error of544 2.16 on eight satisfactory assays: We have confirmed that this is the most precise of the isolated-ute:rus methods for the assay of the oxytocic hormone. STEWART THE ASSAY OF POSTERIOR PITUITARY LOBE EXTRACTS Wol.75 Fig. 2 is a tracing of such a test. RAT 175 G. A = 0.06 UNI B = 0.045 UN F* 40 r 8. 4. 49 DOSE INTERVAL 4 MINS. C = 0.3 ml. UNKNOWN /300 D -- 0.3 ml. UNKNOWN /400 A BC D BA DCC DA BDC B A Fig. 12. Bachinski and Allmark13 have conducted “in. vitro” assays on the uterus of the guinea-pig, rat and rabbit and on strips of human uterine muscle excised during Caesarean sections at or near full term. They found the sensitivity of the rabbit uterine muscle to “Pitocin” and “ Pitressin” (Parke Davis preparations of the relatively pure extracts of the oxytocic and vaso- pressor principles) more closely resembled that of human uterine muscle than did those of the uterine muscles from other animals. A sample of Pitocin assayed against an extract of the Canadian standard posterior pituitary powder gave results on the normal guinea-pig uterus that differed significantly from the assay conducted on the normal rabbit uterus or human uterine muscle.They employed Van Dyke and Hastings’ solution3 throughout their assays, because its magnesium ion concentration approximates to the serum magnesium ion level of man as well as of pregnant woman. that Pitressin exerts a variable oxytocic activity depending upon the concentration of magnesium and calcium ions in the physiological salt solution : the observations on variations in the concentration of magnes- ium confirmed the results of Fraser.15 It was also shown14 that the sensitivity of guinea-pig uteri to stimulation by Pitocin and Pitressin depends upon the hormonal state of the guinea- pig, Table 1-shows that the sensitivity of virgin guinea-pig uteri to stimulation by Pitocin TABLE I It has recently been THE SENSITIVITY OF UTERI TO PITOCIN AND PITRESSIN FROM VIRGIN GUINEA-PIGS TREATED WITH PROGESTERONE AND STILBOESTROL Uterine sensitivity r Sensitivity ratio, Pitressin, J- Pitocin/Pitressin rnilIiunits/ml.milliunits/ml. Progesterone-treated guinea-pigs . . . . 0.04-0.33 0.08-0.26 1.08 Stilboestrol-treated guinea-pigs . . . . 0*01-0*04 0*002-0.018 2-60 Sensitivity ratio of progesterone-treated guinea-pigs to stilboestrol-treated guinea - .. .. .. .. 0.138 0.058 - pigs . . .. is much greater when they have undergone a treatment with stilboestrol than with progesterone. The uteri of The same was found for the oxytocic effect of Pitressin.October, 19501 STEWART: THE ASSAY OF POSTERIOR PITUITARY LOBE EXTRACTS 545 stilboestrol-treated guinea-pigs are more sensitive to Pitocin than to Pitressin, whilst the uteri of progesterone-treated guinea-pigs are almost equally sensitive to both.Because of the complex nature of a pituitary extract and because the ratio of the amount of the oxytocic hormone to the amount of the vasopressor hormone in the extract may not be the same as in an extract of the international standard, exact details of the conditions of assay should be stated when a sample is being compared in different laboratories. Con- fusion has often arisen over the potency of a particular commercial sample assayed on the guinea-pig uterus in different laboratories. Table I1 shows the results of the assay of commercial extracts prepared by six different firms and examined for oxytocic activity under the same conditions on the guinea-pig uterus TABLE I1 RESULTS OBTAINED FOR THE OXYTOCIC ACTIVITY OF COMMERCIAL POSTERIOR PITUITARY PER ML., WHEN ASSAYED BY THE GUINEA-PIG UTERUS METHOD USING RINGER’S SOLUTION EXTRACTS, LABELLED TO CONFORM WITH THE B.P.REGULATIONS OF 10.0 OXYTOCIC UNITS B.P. 1948 Sample Potency, Limits, u./ml. u./ml.* A 10.0 8.G11.4 B 8.8 7.5-10.0 C 7.8 6.7- 8.9 D 11.0 10.0-12.2 E 8.7 7.5-10.0 F 7.7 6.7- 8.9 * Range of upper and lower doscs used in the bracketting arrangement described for the uterine assay. in the physiological salt solution described in the British Pharmacopoeia.8 All the ampoules were labelled 10 units per ml.The samples did not possess equal oxytocic activity. Table I11 shows the results obtained for the assays of six different mixtures of Pitocin and Pitressin in which the ratio of Pitocin to Pitressin ranged from 4.0 to 0.125. The assays TABLE Irr TYPICAL DIFFERENCES IN GUINEA-PIG UTERINE ASSAY RESULTS USING TWO USUAL FORMULAE FOR THE PHYSIOLOGICAL SALT SOLUTION Ratio, Pitocin Pitressin 4.00 2.00 1.00 0.50 0.25 0,125 Actual total oxytocic potency, * u./ml. 10.1 10.2 10.4 10-8 11.6 13.2 Ringer’s solution, B.P., 1948, potency found, u./ml. 8.0 9.2 10.0 9.3 11.2 13.0 Ringer’s solution, with added magnesium (Hsu, 1948)t 10.6 10.6 10.6 13-6 13.6 19.2 * Allowance has been made for 4 per cent. of oxytocic hormone impurity in Pitressin and for 4 per cent.OlE vasopressor hormone impurity in Pitocin in computing the actual total ox*ocic potencies of the solutions. t Hsu (1948) Ringer contains 0-045 per cent. of magnesium chloride. were conducted in two physiological salt solutions, one of which contained the magnesium ion concentration stated in the British Pharmacopoeia8 and the other a much higher con- centration.’ The assays conducted in the latter solution produced much higher results than those expected when the ratio of Pitocin to Pitressin was 0.5 or less. Gaddumle studied under various conditions the stability of the oxytocic hormone of the British standard of 1926, which later became the 1935 International Standard Powder. We have confirmed his results for powders of the potency required by the United States Pharmacopoeia XIII, i.e., not less than 1000 oxytocic units per gram.In recent years, however, commercial samples have very rarely attained this minimum unitage, and in a comparison of the stability of commercial powders of oxytocic potency less than 1000 units per g. with powders of potency similar to that of the International Standard Powder we obtained different results. Also the ratio of the vasopressor hormone to the oxytocic hormone was not always unity in the less potent powders. McClosky, Miller and Le Messurier17 have found that in whale hypophysis the ratio of the vasopressor hormone to the oxytocic hormone546 STEWART : THE ASSAY OF POSTERIOR PITUITARY LOBE EXTRACTS is greater than unity. For various reasons, therefore, assays are sometimes attempted in which the ratio of the oxytocic hormone to the vasopressor hormone in the standard differs from that in the sample under test.Because of these many difficulties with “in vjtro” assays we decided to study an “in vivo” assay. GaddumlS showed that the depressor effect of posterior pituitary extract on the blood pressure of the fowl was due to the oxytocic hormone. Coonlg conducted assays on the blood pressure of a cockerel anaesthetised with sodium phenobarbitone. The procedure was to match a response of the standard with an equal response of the unknown solution. Tachyphylaxis gradually develops and thus any method based on matching responses will entail certain inaccuracies. Blackwell Smith, jun., and Vos, jun.,20 employing the cockerel, performed, according to the design of Bliss and Marks,21$22 a four-point assay with a dose injected every 10 minutes in a random arrangement.The latent sources of variation due to the influence of the height of the blood pressure immediately preceding a given response and the size of the preceding dose were ignored. An analysis of the data from several assays by partial regression co- efficients showed that small fluctuations in blood pressure and the effect of the preceding dose could be ignored without markedly influencing the accuracy of the assays. Errors up to 18.2 per cent. with an average error of 6.9 per cent. were obtained in their assays of posterior pituitary solutions of known strength. Thompson,23 however, pointed out that the main difficulty was to eradicate from experi- mental comparisons the effect of changing sensitivity and this was only achieved in part by the four-point random dose method, as any change in sensitivity occurring during the time required for the injection of, and response to, the four doses causes variations in the responses, which would contribute to the error of the assay. This can be great in the chicken blood-pressure assay.Thompson therefore used a statistical design similar to that used by Vosa for the assay of ergometrine. When White Leghorns were in short supply we tried Rhode Island Red cockerels, but the sensitivity of these birds to posterior pituitary extract was extremely small. On two occasions no response was elicited. Light Sussex cockerels appeared to be suitable subjects and we finally adopted them for our assays.The weight range of these birds was from 1.8 to 2-3 kg., and the birds were injected intravenously with 180 mg. of sodium phenobarbitone per kg. We tried other barbiturates but none was as good as sodium phenobarbitone in maintaining a high blood pressure in the bird. The cockerels were prepared for assay according to the description given by Coon. The blood pressure was recorded from the ischiadic artery by means of a capillary mercury manometer, and 8.5 per cent. of sodium citrate was used as anticoagulant. An extract of the International Standard, 2 oxytocic units per ml., was generally diluted to 1 in 10 and occasionally to 1 in 20 with 0.9 per cent. saline. Doses not exceeding 0.4 ml. were injected rapidly into the crural vein regularly every 3, 4 or 5 minutes.The unknown was diluted to a strength assumed to‘be that of the standard dilution, and assays were carried out on the same bird according to the Schild12 experimental design and also that described by V O S . ~ ~ If the standard and test materials are similar to each other, the experi- mental design qf VosM gives the more accurate results. Should the standard and unknown materials be dissimilar then by the analysis of variance possible with Schild’s experimental design12 one can say whether the assay is valid or not. Fig. 3 illustrates the V o P experimental design. The dose of standard is kept constant whilst the three different doses of the unknown are given, so as to produce one response greater than, one equal to, and one less than that produced by the standard dose.We have found that falls in blood pressure of 30 to 60 mm. of mercury give responses linearly related to the logarithms of the doses. When the blood pressure falls to a low level, ephedrine hydrochloride, 4 to 8 mg. per kg., may be administered to raise it. After this has risen it will remain elevated for a period of 1 to 3 hours and falls only very gradually. If the bird becomes insensitive to posterior pituitary extract then it may be rested for an hour or two, after which the sensitivity has usually returned. Table IV shows a comparison of the results of six different solutions when assayed by the guinea-pig uterine method, using the physiological salt solution described in the British Pharmacopoeia,8 and by the chicken blood-pressure method.The column on the extreme right shows the good agreement between the two methods. Fol. 75 > Both Coon19 and Thompson23 used White Leghorn cockerels.October, 19501 STEWART: THE ASSAY OF POSTERIOR PITUITARY LOBE EXTRACTS TABLE IV COMPARISON OF THE GUINEA-PIG UTERUS AND CHICKEN BLOOD-PRESSURE METHODS FOR THE MEASUREMENT OF OXYTOCIC ACTIVITY 547 Chicken depressor u./g. & S.E. (C) Sample method, A 677 -J= 52 B 872 f 70 C 762 f 49 D 792 f 68 E 921 & 49 F 785 f 65 G 467 f 44 Guinea-pig uterus, 700 875 637 700 900 824 600 u-/g. (GI Ratio, C/G 0.967 0.997 1.196 1.131 1.023 0.950 0.945 Coon,l9 using White Leghorn cockerels, showed that the vasopressor hormone produced a potentiating effect on the depressor response of the bird to oxytocic hormone when the ratio 5.7. 49 COCK 2.3 Kg. DOSE INTERVAL 4 MINS. S = 0.06 UNIT B = 0.3 ml. UNKNOWN 1/50 A = 0.2 ml. UNKNOWN 1/50 C = 0.4 ml. UNKNOWN 1/50 S A S B S C S B S C S A S C S [ZERO 6.r Fig. 3. of the oxytocic to the vasopressor hormone was less than 0.4. Table V shows the results obtained on the assays of mixtures of Pitocin and Pitressin in Light Sussex cockerels with the TABLE V THE INFLUENCE OF DIFFERENT RATIOS OF PITOCIN TO PITRESSIN ON THE ASSAY OF THE OXYTOCIC PRINCIPLE OF THE POSTERIOR PITUITARY LOBE GLAND BY THE CHICKEN DEPRESSOR METHOD Ratio, Pitocin Pitressin 4-00 2-00 1.00 0.50 0.25 0.125 0.100 Actual total oxytocic potency,* u./ml. 10.1 10.2 10.4 10.8 11.6 13.2 14.4 Potency found, u./mI. 10.3 11.2 9.7 10.8 11.1 11.1 18.6 Limits of error ( p = 0.95) 9.3-1 1.3 8.9-13-5 8 .6 1 1-0 10.0-1 1.6 10- 1-1 2.1 8.3-13.9 16.0-2 1.6 * Allowance has been made for 4 per cent. of oxytocic hormone impurity in Pitressin and for 4 per cent. of vasopressor hormone impurity in Pitocin in computing the actual total oxytocic potencies of the solutions ratio of Pitrocin to Pitressin from 4.00 to 0.100. Only when the ratio of Pitocin to Pitressin is 0.100 or less does the vasopressor hormone produce any appreciable potentiating effect. The chicken assay is more reliable and more informative in that fiducial limits can be calculated; it requires less skill and experience on the part of the laboratory assistant and less time, 1 to 1.75 hours for an assay involving 10 doses of the unknown solution, compared with 1 to 2 days for the ordinary guinea-pig uterine assay, and it is less expensive than the guinea-pig uterine assay.548 STEWART : THE A S S U OF POSTERIOR PITUITARY LOBE EXTRACTS Pol.75 THE ASSAY OF THE VASOPRESSOR HORMONE- Two methods have been widely used for the assay of the vasopressor hormone. Hogben, Schlapp and MacDonald25 used the spinal cat method. Burn26 described the preparation of the cat by Elliot’s method. The blood pressure was recorded from the left common carotid artery and injections of the drug were made into the right external jugular vein. The blood pressure falls about an hour after the operation and can be maintained at that level for long periods of time. We have found that it is best to leave the preparation at least 2 hours after the operation before beginning the assay.The standard extract of 2 units per ml. is diluted to 1 in 10 with 0.9 per cent. saline and the unknown solution is diluted to a potency assumed to be equal to that of the standard dilution. Injections do not exceed 0.5ml. Although Burn stated that in some cats the interval between doses can be shortened to half an hour or even 20 minutes, we have found that hourly intervals are better, since this reduced the likelihood of tachyphylaxis. After 10 hours the preparation becomes more sensitive to the vasopressor hormone. Doses used are equal to half what is required to produce maximum response, and responses are matched by the null method. Doses differing by 10 per cent. or more can be distinguished. Since the time required to assay a sample by the cat method is long, and since there is an acute shortage of experimental cats, we have used the anaesthetised dog in the method described by Kamm, Aldrich, Grote, Rowe and Bugbee.27 Medium-sized healthy dogs are deeply anaesthetised with an intraperitoneal injection of 0.4 g.of chlorbutol per kg. Cannulas are inserted in a femoral vein and a carotid artery. Injections are made every 15 minutes and doses are kept as small as possible. The method proved to be as accurate as the spinal cat method. Fig. 4 shows the tracings obtained in assays of a solution by the cat and dog methods. Simon2* used the effect of the vasopressor hormone on the blood pressure of the decapi- tated rat and found this to be an extremely sensitive preparation, responding to 2.5 to 5.0 milliunits. Shipley and Tilden29 have used the pithed rat with satisfactory results. Stephenson30 described a preparation of the hind limbs 6f the rat perfused a t room temperature with Ringer - Locke solution through the abdominal aorta. He designed a simple and sensitive outflow recorder for measuring the changes in pressure produced by the pressor hormone.After 4 to 5 hours the preparation is said to respond to 2 milliunits or even 1 milliunit of posterior pituitary extract. Some preparations are known to last 24 hours. In our experience it was found after a few experiments that the preparation failed to recover after the second or third injection of the drug into the perfusate. Histamine in small doses was without. action. THE ASSAY OF THE ANTIDIURETIC HORMONE- Although most types of animals have been used at one time or another for assay of the antidiuretic hormone, the method described by Burn31 has been widely employed. Sixteen male rats, weighing 140 to 240g., are divided into two groups.Each rat is given 5ml. of water per 1OOg. of body-weight by stomach tube. The posterior pituitary extracts, standard and unknown, are injected subcutaneously into the two groups of rats respectively, and the urine excreted is collected. The assay is based upon the time interval between the administration of water and the maximum excretion of urine during periods of 15 minutes. The time elapsing is greater the larger the dose of the extract. The potency is calculated from a previously determined curve relating dose to time elapsing before the maximal rate of excretion occurs.The estimated results, according to Burn, differed from the true results as much as 23 per cent. and the average variation was 12 per cent. Recently, Theobald, Graham, Campbell, Gange and Driscoll32 have made reference to assays of the antidiuretic hormone by the method described by Verney and his Theobald has shown that the intravenous injection of from 0.5 to 10 milliunits of “ Infundin” (Burroughs Wellcome preparation) inhibited water diuresis in the dog and man and in woman during the last few weeks of pregnancy. The amount of antidiuretic activity necessary to inhibit water diuresis was remarkably constant for each dog, and the water-diuresis curves obtained over a period of several months could be almost superimposed.He adds that the antidiuretic activity affords the most delicate and possibly the most precise method of assaying posterior pituitary extracts, and he assumes that the vasopressor hormone and the antidiuretic hormone are one and the same principle.October, 19501 STEWART : THE ASSAY OF POSTERIOR PITUITARY LOBE EXTRACTS 649 Heller,= ?36 however, has prepared from the vasopressor fraction an extract that contains a high proportion of antidiuretic activity and very little vasopressor activity. Wokes37 found in a series of assays of commercial posterior pituitary lobe preparations, assayed for oxytocic potency on the guinea-pig uterus and for antidiuretic potency on the rat, that four out of 20 assays gave results showing a disproportion of the two active principles that he claimed was significant.We have found disproportionate amounts of the oxytocic and vasopressor hormones in commercial pituitary extracts. DOG 8 5.0 Kg. B.P. 70 m.m. Hg. DOES IN mls. 3. 5. 49 DOSE INTERVAL 15 MINS. S = 0.2 UNITS/rnl. T = UNKNOWN 1/50. Q*4 0.5 0.3 0.25 0.4 0.3 0.3 0.4 0.4 0.5 S S T T S S 7 T s S 0.25 T. E 0.4 S. CAT $2 2.8 Kg. B.P. 70 m.m. Hg. DOSES IN mls. 5. 5. 49 DOSE INTERVAL 60 MINS. S = 0.2 UNITS/ml. T = UNKNOWN 1/50. 0.5 0,4 0.3 03 0.45 0.3 0.2 S 5 T T S T 5 0.3 T. E 0.45 S. Fig. 4. When the potency of one of the posterior pituitary hormones in a pituitary extract is required, a method should be employed that measures only that active principle. It should not be assumed, for example, that if the extract contains 10 vasopressor units per ml.when assayed against the International Standard, it will necessarily contain an equivalent unitage of the oxytocic and antidiuretic principles. I wish to thank the Directors of the Wellcome Foundation Ltd. for permission to publish this paper. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. Dale, H. H., and Laidlaw, P. P., J . Pharmacol., 1912, 4, 75. Morell, C. A., Allmark, M. G., and Bachinski, W. M., Ibid., 1940, 70, 440. Van Dyke, H. B., and Hastings, A. B., Awier. J . Physiol., 1928, 83, 563. Bachinski, W. M., Allmark, M. G., and Morrell, C. A,, Canad. J . Res., 1945, E23, 126. Hamburger, C., Acta Pharmacol. Toxicol., 19-15, 1, 112. Garcia de Jalon, P., Farwzacoter. Act., 1947, 4, 177. Hsu, Yu-C., Quart.J . Pharm. Pharmacol., 1948, 21, 146. “British Pharmacopoeia,” 1948, p. 812. Gaddum, J. H., Quart. J . Pharm. Pharmacol., 1938, 11, 697. Holton, P., Brit. J . Phannacol., 1948, 3, 328. Schild, H. O., Ibzd., 1947, 2, 189. Bachinski, W. M., and Allmark, M. G., J . Amer. Pharm. Ass., Sci. Ed., 1947, 36, 73. Stewart, G. A., J . Pharm. Phavmacol., 1949, 1, 436. Fraser, A. M., J . Pharmacol., 1939, 66, 85. Gaddum, J. H., Biochenz. J., 1930, 24, 941. McClosky, W. T,, Miller, L. C., and LeMessurier, D. N., J . Pharmacol., 1936, 57, 132. -, J . Physiol., 1942, 101, 115.550 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. STEWART : THE ASSAY OF POSTERIOR PITUITARY LOBE EXTRACTS [Vol. 75 Gaddum, J . H., J . Physiol., 1928, 65, 434. Coon, J. M., Arch. Int. Pharmacodyn., 1939, 62:, 79. Blackwell Smith, R., jun., and Vos, B. J., jun., J . Pharmacol., 1943, 78, 72. Bliss, C. I., and Marks, H. P., Quart. J . Pharna. Pharmacol., 1939, 12, 82. Thompson, R. E., J . Pharmacol., 1944, SO, 373. Vos, B. J., jun., J . Amer. Pharm. Ass., Sci. Ed., 1943, 32, 138. Hogben, L. T., Schlapp, W., and MacDonald, A. D., Quart. J . Exp. Physiol., 1924, 14, 301. Burn, J. H., “Biological Standardisation,” Oxford University Press, London, 1937, p. 67. Kamm, O., Aldrich, J. B., Grote, I. W., Rowe, L. W., and Bugbee, E. P., J . Amer. Chem. SOC., Simon, A, Arch. Exper. Path. Pharmak., 1937, 187, 678. Shipley, R. E., and Tilden, J. H., Proc. Soc. Ex$. Biol. Med., 1947, 64, 453. Stephenson, R. P., J . Physiol., 1948, 107, 162. Burn, J. H., Quart. J . Pharm. Pharmacol., 1931, 4, 517. Theobald, G. W., Graham, A., Campbell, J., Gange, P. D., and Driscoll, W. J., Brit. Med. J., Klisiecki, A., Pickford, M., Rothschild, P., and Verney, E. B., Proc. Roy. SOC., B., 1933, 112, 496. Verney, E. B., Ibid., B., 1947, 135, 25. Heller, H., J. Physiol., 1939, 96, 337. Wokes, F., Quart. J . Pharm. Pharmacol., 1932, 5, 390. , , Ibid., 1939, 12, 182. -- 1928, 50, 573. 1948, 2, 123. -, Ibid., 1940, 98, 405. WELLCOME BIOLOGICAL CONTROL LABORATORIES DARTFORD, KENT DISCUSSION PROFESSOR GADDUM enquired whether it was possible to obtain preparations of human uterus that were more sensitive to the vasopressor than to the oxytocic hormone. MISS MACAULAY enlarged on Professor Gaddum’s mtmtion of the anaesthetised rat method1 for assaying the vasopressor activity of the posterior lobe of the pituitary. In their experience the chief advantages of the preparation were (a) its sensitivity-it responded t o 0.001 i.u. of vasopressor activity, while 0.005 i.u. gave a rise in blood pressure of 30 to 40 mm.; (b) it did not respond to the oxytocic fraction: (c) 10 per cent. discrimination was easily obtained; ( d ) no tolerance cleveloped to injections made every 10 minutes- usually 30 to 40 injections were made into the same animal; (e) it was completely insensitive to the histamine concentrations encountered in the worst commercial powders. DR. F. J. DYER asked the author if he would favour making routine assays on low potency commercial pituitary powders by two methods, oxytocic and vasopressor (or antidiuretic) and expressing the answer as a ratio of the one to the other. The International Standard was assumed (by B.P. definition) to possess an oxytocic to vasopressor ratio of 1 to 1. MR. STEWART said that Russell2 had claimed that Pitressin had a much greater oxytocic effect than Pitocin on human uterine strips excised a t term. More recently, Bachinski and Allmark conducted assays on a statistical basis and found that Pitocin and Pitressin were equally active in oxytocic potency on an isolated uterine strip taken a t the peak of pregnancy. He himself had not conducted assays with this type of preparation. Replying to Dr. Dyer, Mr. Stewart said that the use of the rabbit uterine assay and the expression of the answer in terms of one hormone only was possibly the most suitable method. At least Bachinski and Allmark had shown that assays conducted on the normal rabbit and parturient human uteri were comparable. However, the best method could only be stated after further clinical researches had been made into the mode of action of these hormones. REFERENCES TO DISCUSSION 1. 2. Landgrebe, F. W., Macaulay, M. H. I., and Waring, H., Proc. Roy. SOC. Edin., B., 1946,62, 202. Russell, C. S., J . Obstet. Gynaecol. Brit. Empire, 1943, 50, 287.

ISSN:0003-2654    

DOI:10.1039/AN9507500530

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

October, 19501 NOKWITZ: DIRECT ELECTROLYSIS OF COPPER Direct Electrolysis of Copper from Silver Chloride Filtrates BY GEORGE NORWITZ SYNoPsIs-Copper can be deposited directly from silver chloride filtrates if an excess of hydrogen peroxide is present. The procedure saves 3 hours in the analysis of alloys containing silver and copper, as it avoids the necessity for evaporating the sulphuric acid until it fumes to remove the chloride ion. 551 IN the analysis of alloys containing silver and copper, it is the usual practice to dissolve the sample in nitric acid, determine silver as silver chloride, and determine copper electrolytically in the silver chloride filtrate. Before depositing the copper, however, it is necessary to evaporate the sulphuric acid until it fumes1 to remove the hydrochloric acid, since hydro- chloric acid interferes with the electrolytic determination of copper.3 This evaporation takes about 3 hours, and obviously increases the chances of error.The author has found that by adding an excess of hydrogen peroxide to the silver chloride filtrate, the copper can be deposited directly in the presence of the hydrochloric acid. Hydrazine sulphate has been used for the electrolytic determination of copper in the presence of hydrochloric acid.2 Attempts by the author, however, to use this reagent for the electrolytic determination of copper in silver solders failed. This failure can be traced to the fact that hydrazine sulphate cannot be used in the presence of more than very small amounts of nitric acid.2 EXPERIMENTAL A silver nitrate solution was prepared by dissolving 15.7 g.of silver nitrate in water and diluting to 1 litre. Ten millilitres of this solution contained approximately 0.1 g. of silver. A standard copper nitrate solution was prepared as follows: 5.000 g. of pure electrolytic copper were dissolved in 40 ml. of diluted nitric acid (1 + 1) by warming on a steam bath. The oxides of nitrogen were boiled off and the solution was cooled and diluted to 500 ml. in a volumetric flask. Varying amounts of silver nitrate solution and standard copper nitrate solution were measured into 300-ml. beakers. Measuring cylinders were used to measure the silver nitrate solution, and pipettes for the copper nitrate solution. The solutions were diluted to 150 to 175 ml. with water, and were treated with 3 ml.of 70 per cent. nitric acid. The silver was precipitated as the chloride according to the A.S.T.M. method,l and the solutions filtered through Gooch filters. Four millilitres of 70 per cent. nitric acid and 30 ml. of 3 per cent. hydrogen peroxide were added to the filtrates, and the copper was electrolysed at 2 amperes for 1 hour with platinum gauze cathodes, 60mm. in height and 50mm. in diameter, and platinum spiral anodes. The results obtained for copper, as can be seen from Table I, were excellent. The deposits were in- variably bright and adherent. The above experiment was repeated on two samples without the addition of the hydrogen peroxide. The copper plated out was so dark that the electrolysis was discontinued after 10 minutes for fear of injuring the platinum.During the electrolysis the solutions were stirred. TABLE I RESULTS FOR COPPER IN SYNTHETIC MIXTURES OF COPPER AND SILVER Silver present, g. 0-25 0.25 0.50 0.50 0-75 0.75 0.10 0.10 Copper present, g. 0.7500 0.7500 0.5000 0-5000 0.2500 0.2500 0*1000 0~1000 Copper found, €5 0-7499 0.7496 0.4996 0.5001 0.2498 0.2498 0.0997 0.1001552 NOKWITZ : DIRECT ELECTXOLYSIS OF COPPER [Vol. 75 A typical silver solder was analysed by the R.S.T.M. procedure,l and found to contain 15.47 per cent. of copper, 49.90 per cent. of silver, 17.97 per cent. of cadmium and 16.60 per cent. of zinc. This sample was analysed for copper four times by the following modification of the method described here. A l-g. sample was dissolved in 20 ml. of diluted nitric acid (I + 4), and the silver precipitated as the chloride according t o the A.S.T.M.procedure.1 The silver chloride filtrate was treated with 4 ml. of ‘70 per cent. nitric acid and 30 ml. of 3 per cent. hydrogen peroxide, and electrolysed for copper. The results obtained for copper are shown in Table 11. The method shows high accuracy and precision. TABLE I1 RESULTS FOR COPPER IN A TYPICAL SILVER SOLDER Copper present, Copper found, % 73 15.47 16-52 15.47 15.50 15.48 Average 15.49 + 0.02 To find out how much hydrochloric acid could be present during the electrolysis the following experiment was performed. Six 25-ml. portions of standard copper solution were pipetted into 300-ml. beakers, and 100 ml. of water and 7 ml. of 70 per cent. nitric acid were added to each portion.Amounts of 36 per cent. hydrochloric acid ranging from 0 to 5 drops were added, followed by 30 ml. of 3 per cent. hydrogen peroxide. The solutions were diluted to 200 ml. and electrolysed for copper. The results obtained for copper, as shown in Table 111, were satisfactory, but the deposits obtained in the presence of 4 to 5 drops of hydrochloric acid were slightly discoloured. No attempt was made to use more than 5 drops of hydrochloric acid. TABLE I11 RESULTS FOR COPPER USING VARIOUS AMOUNTS OF HYDROCHLORIC ACID Hydrochloric acid present, drops . . 0 1 2 3 i 5 Copper found, g . . . .. .. . . 0.2498 0.2501 0.2496 0.2495 0.2499 0.2496 Two platinum anodes that had been used throughout the experiments with solutions containing hydrogen peroxide were weighed before and after the series of electrolyses. One was found to have lost 0.3 mg. after eight runs and the other 0.2 mg. after ten runs. The average loss was 0.028 mg. per run. This is approximately the usual loss obtained in electro- lysing copper from nitric acid solutions which do not contain hydrochloric acid. REFERENCES 1. “A.S.T.M. Methods of Chemical Analysis of Metals,” American Society for the Testing of Materials, 2. Diehl, H., “Electrochemical Analysis with G.raded Cathode Potential Control,” G. Frederick 3. Kolthoff, I. M., and Sandell, E. B., “Textbook of Quantitative Inorganic Analysis,” Macmillan & Philadelphia, Pa., 1946, p. 253. Smith Chemical Co., Columbus, Ohio, 1948, pp. 36, 39. Co., New York, 1943, p. 422. 3353 RIDGE AVENUE PHILADELPHIA 32, PA. Afiril, 1950

ISSN:0003-2654    

DOI:10.1039/AN9507500551

出版商:RSC    

年代:1950

数据来源: RSC