摘要:

,522 HIGH AND PLACITO : THE SPECTROPHOTOMETRIC DETERMINATION [Vol. 83 The Spectrophotometric Determination of Iron in High-purity Bismuth BY J. H. HIGH AND P. J. PLACITO (Hawker Szddeley h’uclear Power Co. Ltd., Sutton Lane, Langley, nr. Slough, Bucks.) A routine method for the determination of traces of iron in 99.95 per cent. pure bismuth is described, based on measurement of the absorption of ferric chloride solutions in the near ultra.-violet region. By a suitable choice of wavelength for optical-density measurement, the interfering effect of up to a millionfold excess of bismuth is avoided. PUBLISHED methods for determining small amounts of iron in bismuth are based on the use of 0-phenanthroline,~3~9~ but are complicated by the fact that, at the pH required forSept., 19581 OF IRON IN HIGH-PURITY BISMUTH 523 colour development, insoluble oxy salts are formed.Holmesl complexes bismuth with ethylenediaminetetra-acetic acid and citric acid before colour development, but, as these reagents seem to form stable complexes with iron, colour development is retarded. Taylor, Ward and Sheldon2 use only citric acid, but the optical density must be measured before colour development is complete. Booth and Evett3 first reduce iron with stannous chloride and then add 4 : 7-diphenyl-l : 10-phenanthroline before the complexing and pH-adjusting reagents; colour development is rapid and complete. These methods are not well suited to routine work and an alternative method was sought. Investigation of other methods for determining small amounts of iron4t5 showed that bismuth seriously interferes with procedures based on the use of thiocyanate, thioglycollic acid and 2 : 2'-dipyridyl.CHLORIDE METHOD Snell and %ell4 note that ferric chloride solutions in hydrochloric acid have a pronounced absorption in the ultra-violet region. Desesa and Rogersa recommend that this absorption be measured at 342.5 mp in 6 M hydrochloric acid. They state that, under these conditions, the method has one-tenth of the sensitivity of methods in which o-phenanthroline is used. For practical reasons, described later, it is necessary to measure optical density at 390 mp, but, even at this wavelength, absorption is cf the order of 0.009 per cm for a solution containing 5 pg of iron per 25 ml. Some variation in optical density with acidity has been found, so colour is developed in constant-boiling hydrochloric acid, prepared by distillation from an all-glass apparatus, as this both reduces the blank value for iron and ensures the production of an acid of reasonably constant composition.Desesa and Rogersa found that the coefficient of variation of optical density with temperature is 0-13 per cent. per "C. The spectrophotometer in use in this laboratory is fitted with a thermostatically controlled absorption-cell compartment, and all readings are made at 25" i 0.5" C. ISTERFERENCE FROM BISMUTH- Solutions of bismuth chloride in hydrochloric acid have an absorption peak at 330 mp, and, in consequence, will interfere in the measurement of iron absorption at 342.5 mp.6 A study of the absorption spectra of bismuth and ferric chlorides indicates that the absorption of bismuth decreases as the wavelength increases much more rapidly than does that of iron.At about 390 mp the bismuth absorption curve flattens out, but there is some residual absorption. That this absorption is not due to bismuth is indicated by the fact that its magnitude varies from sample to sample. The most probable explanation is that it is due to small amounts of iron present in the bismuth as an impurity. If this is so, it is to be expected that the apparent iron content, as determined by this procedure, will decrease as the wavelength increases until a point is reached at which it remains steady. That this does, in fact, occur was shown by a series of determinations made at 2-mp intervals from 380 to 400mp; the results were as follows- IVavelength, mp .. . . 380 382 384 386 388 390 392 394 396 398 400 Apparent amount of iron in first sample, p.p.m. . . 21 14 11 10 9 5 5 5 5 4 4 Apparent amount of iron in second sample, p,p.m. . . 66 88 55 55 53 50 50 50 49 50 49 In view of these results, the assumption was made that, at 390 mp, absorption due to bismuth at concentrations of 1 to 2 g per 25 ml ceases to be significant, and this wavelength was accordingly chosen for optical-density measurements. With a less selective spectrophoto- meter it may be necessary to choose a somewhat longer wavelength. It should, however, be noted that the absorption due t o iron also decreases as the wavelength increases, and instruments that operate with a half-energy band-width greater than about 5 to 10 mp are not likely to give satisfactory results.That 390 mp is a suitable wavelength under the conditions used is shown in Table I, which also demonstrates the good recovery of added iron.524 HIGH AND PLACITO : THE SPECTROPHOTOMETRIC DETERMINATION TABLE I RECOVERY OF ADDED IRON IN THE PRESENCE OF BISMUTH was carried out in the presence of 1 g of bismuth per 25 ml of final solution [Vol. 83 Optical densities were measured in 1-cm cells at wavelength 390 mp. Each recovery Optical density in Amount of iron added, Optical density in Amount of iron found, absence of bismuth p g per 25 ml presence of bismuth p g per 25 ml 0.018 10 0.020 11 0.045 25 0.043 24 0.090 50 0.092 52 0.134 75 0.135 74 0.179 100 0.176 98 0.269 150 0,271 151 0.314 175 0-314 175 0.358 200 0,356 I99 0.403 225 0.406 226 0.537 300 0.539 301 0.627 350 0.630 352 0.716 400 0.721 403 0.806 450 0.816 456 0.894 500 0.890 497 INTERFERENCE FROM NITRIC ACID- Nitric acid and its reduction products have been stated to interfere? As nitric acid is used to dissolve bismuth, tests were made on samples that had been evaporated once, twice and three times with hydrochloric acid, and also on samples to which 1 drop of con- centrated nitric acid had been added to the final solution.The results showed that special precautions to remove the last traces of nitric acid are not necessary. INTERFERENCE FROM OTHER ELEMENTS- Silver and other cations that form chlorides insoluble in hydrochloric acid will interfere by producing precipitates or suspensions.SDver is not detectable in bismuth obtained from Mining and Chemical Products Ltd., and it was found that up to 5 pg of silver per 25 ml has no measurable effect. The presence of 500 pg of tin, 2000 pg of lead, 250 pg of copper, 50 pg of nickel and 250 pg of zinc per 25 ml of final solution produced no measurable interference. According to data from Mining and Chemical Products Ltd., these are at least twenty times the amounts present in high-purity bismuth as supplied. Although ruthenium chloride absorbs strongly at 390 mp, it has a minor absorption peak at 475 mp, at which wavelength iron has no measurable absorption at a concentration of 50 pg per 25 ml. It has, however, been found that, if bismuth containing ruthenium is dissolved in nitric acid, the ruthenium forms a very stable colourless complex with the acid or its reduction products, and thus causes no interference in the method.METHOD APPARATUS AND REAGENTS- All glassware must be carefully cleaned and rinsed with hydrochloric acid before use. The nitric acid must be of at least analytical-reagent grade, and the constant-boiling hydro- chloric acid must be prepared by distillation of diluted analytical-reagent grade acid from an all-glass apparatus. Beakers in which other reagents have been used should be boiled for a few minutes with hydrochloric acid before use. The instrument used in this laboratory is a Beckman DU/G4700 spectrophotometer - photomultiplier combination. Measurements are made in optically matched quartz 1-cm cells at a slit width of 0.015 mm.This corresponds to a half-energy band-width of about 0.15 mp at 390 mp. The wavelength-calibration scale of the instrument has been checked against the mercury lines. The deviations from the lines at 334.1 and 404.7 mp were 0.0 and 0.2 mp, respectively. PROCEDURE- Dissolve a weighed sample (about 10 g if possible) of bismuth in concentrated nitric acid. Cse 3.5 ml of acid for each gram of sample. When dissolution is complete, boil for a few minutes, cool and make up to a suitable volume with distilled water. This volume must not be so large that bismuth oxy salts are precipitated when the solution is made up to the mark. For a 10-g sample, 100 ml is a convenient volume.Sept., 19581 O F IRON I N HIGH-PURITY BISMUTH 525 Place an aliquot of this solution, equivalent to 0.7 to 1-5 g of the original sample, in a suitable beaker and evaporate just to dryness on a hot-plate. Allow to cool to room temperature and add 10 ml of constant-boiling hydrochloric acid.Again evaporate to dryness, cool, add 5 to 10ml of constant-boiling hydrochloric acid and bring the solution to the boil. Remove the beaker from the hot-plate, cool and then use constant-boiling hydrochloric acid to transfer the solution to a 25-ml calibrated flask and to dilute to the mark. Measure the optical density of the solution at 390 mp against a blank solution prepared in the same way. Include a suitable standard iron solution, containing, say, 50 pg of iron per 25m1, with each batch of samples. RESULTS The absorption obeys Beer’s law up to a concentration of at least 5OOpg of iron per 25ml of final solution, the equation of the calibration graph being as follows- micrograms of iron per 25 ml = 559 x optical density The maximum observed deviation from linearity was k2.5 per cent.a t concentrations above 10 pg per 25 ml. Below this concentration, the error was of the order of i: 10 per cent., and increased to 1 2 5 per cent. or more below a concentration of 3 to 4 pg per 25 ml. Duplicate optical-density readings for individual samples are reproducible to better than i 1 per cent. Reference has been made in Table I to the excellent recovery of added iron by the method. A number of analyses on a sample of hydrogen-blown pharmaceutical-grade bismuth were carried out by seven methods in different laboratories.The proposed method, Booth and Evett’s m e t h ~ d , ~ a spectrographic method and Holmes’s methodl all gave a result of 1 p.p.m. of iron. A method based on o-phenanthroline, a modified thiocyanate method and a second spectrographic method gave results of 2, 2-5 and less than 4 p.p.m. of iron, respectively. Solution-rate studies and measurements of the solubility of iron in bismuth at different temperatures have given results in close accord with those of other workers a t HarwelP and at Brookhaven. CONCLUSIONS No separations or complexing of bismuth are required and the use of a multiplicity of reagents is avoided. In consequence, reagent blank solutions have an extremely small absorption, and there is less chance of contamination by extraneous iron, provided that the normal precautions are taken.The method is well suited to routine work and has been in use here for over 1 year, during which time more than fifteen hundred analyses have been made. Desesa and Rogers6 noted that the hydrochloric acid method has about one-tenth of the sensitivity of the o-phenanthroline procedure, and it is possible that for the proposed method this ratio is even smaller. The proposed method, however, can be used to determine extremely small amounts of iron if a suitable spectrophotometer - photomultiplier combina- tion is used for optical-density measurement. With other materials, for which iron absorption can be measured a t a lower wavelength, an increase in the sensitivity of the method could be expected. An increase in sensitivity might also be achieved by using 4-cm cells for optical-density measurements. We thank Mr. A. Hall and Miss L. Bell for technical assistance, Mr. E. Booth, A.E.A. (Woolwich), and Mr. G. 1%’. C. Milner, A.E.R.E. (Harwell), for advice and many helpful suggestions, Mr. A. G. Ward, A.E.R.E. (Harwell), for arranging the comparative analyses of the samples of pure bismuth, Mr. G. J. Metcalfe, Chief Metallurgist, for his interest and advice and the Chief Executive and Directors of the Hawker Siddeley Nuclear Power Company Ltd., for permission to publish this paper. REFERENCES The proposed method is reasonably rapid and simple in technique. 1. 2. 3. 4. 5. 6. Holmes, D. G., Analyst, 1957, 82, 528. Taylor, S. W., Ward, A. G., and Sheldon, R., Atomic Energy Research Establishment Report, Booth, E., and Evett, T. W., Analyst, 1958, 83, 80. Snell, F. D., and Snell, C. T., “Colorimetric Methods of Analysis,” Third Edition, D. \-an Nostrand Sandell, E. B., “The Colorimetric Determination of Traces of Metals,” Second Edition, Interscience Desesa, M. A,, and Rogers, L. B., Anal. Chinz. Ada, 1952, 6, 534. M/R 2295, Harwell, 1957. Co. Inc., Princeton, N. J., 1949, Volume 11, Chapter 17. Publishers Inc., New York, 1950, Chapter 23. Received December loth, 1957

ISSN:0003-2654    

DOI:10.1039/AN9588300522

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

526 IRVISG ASD COX: THE PREPARATIOS OF METAL-FREE ACIDS, [Vol. 53 The Preparation of Metal.-free Acids, Alkalis and Buffer Solutions of High Purity BY H. IRVIKG AND J. J. COX (The Inorganzc Chemistry Laboratory, South Parks Road, Oxford) Many buffer solutions can be freed from metallic impurities by exhaustive extraction with dithizone, but concentrated metal-free solutions of ammonia and hydrochloric acid are best prepared by isopiestic distillation. A range of metal-free buffer solutions is provided by suitable admixtures of these components. IX the absorptiometric determination of traces of metals with sensitive reagents such as dithizone, it is essential to work with metal-free reagents if high blank values are to be avoided. Buffer components, such as sodium or ammonium acetate, potassium sodium tartrate, diammonium hydrogen citrate and trisodium or triammonium citrate, can be freed from metallic impurities by repeated extraction with successive portions of a concentrated solution of dithizone in chloroform, until a green colour persists in the (diluted) organic phase; the comparatively small amount of dissolved dithizone can then be removed by extraction with pure organic solvents.lI2 The same procedure is sometimes applied to the purification of acids, but it will clearly be most effective for removing those metals that, like mercury and silver, form dithizonates stable in acid solution: it will be a comparatively inefficient and sometimes a very ineffective way of removing metals, such as lead or thallium, that do not form dithizonates except in solutions of high pH.Basic solutions cannot readily be "cleaned up" by this procedure, for the partition coefficient of dithizone now favours the aqueous, phase, and the high concentration of dithi- zonate ion inevitably remaining after the extraction of metallic impurities can only be removed by laboriously repeated extractions with successive portions of organic solvent. It is virtually impossible to obtain metal-free solutions of sodium hydroxide in this way, and, whenever possible, it is customary to substitute concentrated solutions of ammonium hydroxide prepared by heating ammonium hydroxide, spgr. 0.880, with solid sodium hydroxide, or ammonium chloride with lime, and absorbing the gas obtained in this way (or gas from a cylinder of liquid ammonia) in ice-cold water.IC3:etal-free hydrochloric acid has been prepared in a similar manner from the gas obtained by dropping concentrated sulphuric acid on to solid ammonium ~hloride.~ Fractional distillation has also been used to prepare metal-free hydrochloric acid4 and acetic acid, but in all such instances it is necessary to use apparatus made of quartz or borosilicate glass, which has been carefully freed from adhering trace-metals. A very simple method of preparing certain metal-free solutions by isopiestic distillation has been in use in this laboratory for some time. Although this has certainly been described before for ammonium h y d r o ~ i d e ~ ~ ~ ~ ~ ~ ~ and hydrochloric a ~ i d , ~ ~ ' the original papers are not generally accessible,B p 8 or they give such scant details5 ,6 that it seemed worth while to make the procedures more widely known.PREPARATION OF METAL-FREE HYDROCHLORIC ACID- The method of preparing a metal-free solution of hydrochloric acid is as follows. A desiccator of capacity 4 to 6 litres is cleaned and dried, and then 500 ml of hydrochloric acid, sp.gr. 1.18, are added. (The exposed surface of the acid varied from 200 to 300 sq. cm in different vessels.) The usual platform of perforated zinc is replaced by a grill of glass rods, which supports an open polythene vessel containing 250 ml of conductivity water prepared either by redistillation of laboratory distilled water in a quartz or all-Pyrex-glass apparatus equipped with a special spray-trap, or by passing distilled water through a mixed-bed ion- exchange resin.Screw-top polythene vessels of capacity about 300 ml, in which the surface area of exposed liquid is about 30 sq. cm, have proved very serviceable. The lid of the desiccator is replaced (no grease is necessary or Idesirable) and the apparatus is left at room temperature (18" to 20" C) for several days. The results of a typical experiment are shown in Fig. 1. Within 2 days, 250 ml of 2 N hydrochloric acid are readily obtainable, and, in about 4 days, the concentration rises to 3 N . If a smaller volume of water, e g . , 50 ml, is placed in the polythene bottle, the uptake of acid is faster, and, as shown in Table I, a moreSept., 19581 ALKALIS AKD BUFFER SOLUTIONS OF HIGH PURITY 527 concentrated product results, viz., 10 N after 3 days.The solution has now nearly reached its equilibrium concentration, which, for this ratio of acid to water, would be 10.3 N . Since the density of hydrochloric acid increases monotonically with its concentration, the concentrated acid formed by the absorption of hydrogen chloride in the surface layer tends to sink, and the resulting convection currents promote good mixing and ensure a distillate of uniform Time of equilibration, hours Fig. 1 . Preparation of metal-free solutions of ammon- ium hydroxide and hydrochloric acid by isopiestic distil- lation; curve A, 50 ml of water and 500 ml of ammonium hydroxide, sp.gr. 0.880; curve B, 250 ml of water and 500 ml of hydrochloric acid, sp.gr. 1.185 TABLE I TYPICAL RESULTS SHOWING THE RATE OF INCREASE OF COXCENTRATION OF AMAIOXIUM HYDROXIDE AND HYDROCHLORIC ACID DURING ISOPIESTIC DISTILLATION Time, hours .. . . . . 20 40 60 80 100 135 150 Concentration of distillate, X* . 2.1 4.2 6.3 7.7 8.5 8.7 8.7 - - ’{ [i] 5.0 8.2 9.6 10 10 (a) The desiccator contained 500 ml of ammonium hydroxide, sp.gr. 0.880, and the polythene vessel 250 mi of water. ( b ) The desiccator contained 500 ml of hydrochloric acid, sp.gr. 1.18, and the polythene vessel 50 ml of water. * The figures given are rounded values from a large number of experiments, which were closely reproducible. PREPARATIOX OF METAL-FREE AMMONIGM HYDROXIDE- Fig. 1 shows the result of an isopiestic distillation from 500 ml of ammonium hydroxide, sp.gr. 0.880, into 50 ml of water. A concentration of over 9.5 N is reached after 2 days.If 250 ml of metal-free water are used to absorb the gas, saturation requires 4 days only and provides alkali of concentration 8.7 N (see Table I). Since the density of aqueous solutions of ammonia decreases with increasing concentration, and since the absorption of ammonia only occurs in the surface layer, rapid mixing can only occur by thermal currents (which are negligible if the desiccator is kept a t a uniform temperature), or by mechanical agitation (which is inconvenient for this type of preparation). That stratification does occur was shown by taking samples from various positions in the solution contained in the polythene bottle (see Table 11). Such stratification really presents no serious drawbacks to the isopiestic method, although mixing is greatly facilitated by gently rocking the desiccator and its contents two or three times a day, or leaving it where it will be exposed to changes of temperature.Isopiestic distillation is familiar on a smaller scale in the so called microdiffusion pro- cedures elaborated by Conwayg ; the large-scale preparation of acids and alkaline solutions by this procedure ought similarly to proceed faster as the ambient temperature is increased.528 RYAN : A S IMPROVED MICRODIFFCSION PROCEDURE [Vol. 83 This was not tested experimentally. The use of a water pump in an attempt to lower the partial pressure of the confined air and thus t o increase the partial pressure and rate of transfer of ammonia vapour was found to be ‘of little practical value. Not only was the rate of equilibration effectively unaltered, but the loss of ammonia during the preliminary pumping down led to lower values for the final concentration of the distillate.TABLE I1 STRATIFICATIOS DURING THE ISOPIESTIC DISTILLATION OF AMMONILX HYDROXIDE Concentration of ammonium hydroxide in distillate A 7 -7 Time, Bulk concentration after hours admixture, N 35.8 6.75 0.96 3.63 60.5 8.28 3.GO 5.34 In view of their lower vapour pressures, the preparation of pure acetic or nitric acid by the isopiestic method is unlikely to prove of value. On trial, equilibration of 250 ml of water with 500 ml of nitric acid, sp.gr. 1.425, or with 500 ml of glacial acetic acid gave, after 1 week, solutions of concentration 0.05 and 1.09 N , respectively. Hydrobromic and hydriodic acids, however, are well suited to the isopiestic method, and by obvious adaptations it can be used to prepare metal-free solutions of sulphurous acid, of ammonium salts, or of chlorides.No metals are introduced in the course of isopiestic distillations and the concentration of heavy metals in the resulting solutions was never found to exceed that in the samples of redistilled or de-ionised water used in their preparation; this was below the limit of detection by dithizone on a 100-ml sample. By a suitable combination of metal-free ammonium hydroxide or hydrochloric acid with salts of weak acids (which can readily be freed from metallic impurities as described above), it is a simple matter to prepare buffer mixtures that cover a very wide range of pH values. REFEREXES 1. 2. 3. 4. 5. 6. 7. 8. 9. Analytical Methods Committee, “The Determination of Zinc in Foodstuffs,” Analyst, 1948, 73, 304. Analytical Methods Committee, “The Determination of Lead in Foodstuffs,” Ibid., 1954, 79, 397. Hogl, O., and Sulser, H., Mitt. Lebensmitt. Hyg., Bern., 1951/1952, 42/43, 286. Biefeldt, K., and Ganssle, A., Angew. Clzem., :l954, 66, 563. Abrahamczik, E., Mikrochemie, 1938, 25, 228. Koroleff, F., Merentutkimuslait. Julk., 1950, 145, 7. Xbrahamczik, E., Nikrochemie, 1951, 36/37, 104. Geiger, R. W., Dissertation, University of Minnesota, 1951; Dissevt. Abstv., 1952, 12, 249. Conway, E. J., “Microdiffusion Analysis and Vclumetric Error,” Second Edition, Crosby Lockwood Received March 3rd, 1958 & Son Ltd., London, 1947.

ISSN:0003-2654    

DOI:10.1039/AN9588300526

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

528 RYAN : A S IMPROVED MICRODIFFCSION PROCEDCRE [Vol. 83 An Improved Microdiffusion Procedure for the Determination olf Lactic Acid BY H. RYAN (Department of Biochemistry artd Phavmacology, University College, Dublin) A modified microdiffusion procedure for the determination of lactic acid is described. The acetaldehyde liberated from lactic acid by ceric sulphate oxidation in the outer chamber of a Conway unit is absorbed in a buffered solution of semicarbazide, and the optical 'density of the acetaldehyde - semicar- bazide complex is measured with a Beckman DU spectrophotometer a t a wavelength of 224mp. The mean recovery from 48 determinations with amounts of lactic acid from 20 to 100 pg was 98.4 per cent. A coefficient of variation of 10.72 per cent. was found for replicate determinations with 60 pg of lactic acid.The method has been used successfully for determining lactic acid in the leg muscles of rats. After injection with Myanesin, the mean lactic acid content was 18.0 mg per 100 g of muscle. A MICRODIFFUSION procedure for determining lactic acid was first proposed by 'Iliinnick,l who based his oxidation on the principles of the method devised by Gordon and QuasteL2Sept., 19581 FOR THE DETERMIXATION OF LACTIC ACID 529 The lactic acid was oxidised to acetaldehyde with ceric sulphate in the outer chamber of a Conway unit. The liberated acetaldehyde was absorbed in sodium hydrogen sulphite solution in the centre chamber and was determined iodimetrically. Conway and RiIcCarvilP modified the method by carrying out the preliminary oxidation in stoppered tubes.After 30 minutes a t 37" C, 1-ml samples of the incubated mixture were transferred to the outer chambers of the units. Acetaldehyde was absorbed in sodium hydrogen sulphite solution in the centre chambers, and was determined iodimetrically. Although the latter procedure at first appeared to be reliable, subsequent investigation with various concentrations of lactic acid showed that recovery was not consistent. Elsden and Gibson4 criticised microdiffusion procedures on the grounds that the acetalde- hyde formed was partly oxidised to acetic acid, owing to prolonged contact with the ceric sulphate solution. Elsden and Gibson devised a method in which the acetaldehyde was removed as rapidly as it was formed by means of steam distillation. The acetaldehyde was trapped in sodium hydrogen sulphite solution and was determined in the usual manner.Although Elsden and Gibson's method has been used successfully in this laboratory, it is rather tedious if large numbers of deter- minations are required. It was decided to investigate the microdiffusion procedure further, in order to ascertain the exact cause of the inconsistent recoveries. When pure acetaldehyde standards were used in the outer chambers of the units, recoveries were between 80 and 90 per cent. The recovery was the same whether ceric sulphate was present or not, and it was concluded, therefore, that the loss could not be attributed to a conversion of acetaldehyde to acetic acid. The lack of appreciable oxidation of acetaldehyde can be attributed to the relatively low temperature a t which the oxidation of lactic acid is carried out and to its rapid diffusion from the oxidising mixture as fast as it is formed.The low recovery was attributed to some acetaldehyde being trapped in the outer chambers of the units by back-diffusion of sulphur dioxide. The proposed method was suggested by the procedure of Burbridge, Hine and Schicka for determining free acetaldehyde in blood. As in Winnick's procedure, lactic acid is oxidised with ceric sulphate solution in the outer chamber of a unit. The liberated acetaldehyde is absorbed, however, in a buffered solution of semicarbazide. At the end of the diffusion period, the optical density of the acetaldehyde - semicarbazide complex is measured with a Beckman DU spectrophotometer at 224 mp.When this method was used, consistent recoveries of about 98 per cent. were obtained, the only drawback being the necessity for an ultra-violet spectrophotometer. METHOD REAGEKTS- Semicarbazide reagent solution-Prepare a 0,0067 M solution of semicarbazide hydro- chloride in a phosphate buffer solution of pH 7 (0.06 M in sodium dihydrogen orthophosphate and 0-14 M in disodium hydrogen orthophosphate). Sulphuric acid, approximately 6 N-Dilute 162 ml of sulphuric acid, sp.gr. 1.84, to 1 litre. Ceric sulphate solution, saturated-Grind ceric sulphate to a fine powder in a clean dry Add an excess of the powder to 6 N sulphuric acid in a test-tube, and shake well. This solution must be freshly Standard lactic acid solution-Dissolve 1.065 g of pure lithium lactate in water containing In support of this, they quoted the experiments of Long5 An alternative absorbent for acetaldehyde was therefore sought.mortar. Remove the undissolved ceric sulphate by centrifugation. prepared. 1 ml of sulphuric acid, sp.gr. 1.84, and dilute the solution to 1 litre. 1 ml E 1 mg of lactic acid. Perchloric acid solution, 15 per cent. Potassitlm hydroxide solution, 40 per cent. w / v . Trichloroacetic acid solutions, 5 and 22 per cent. w / v . Fixative for wzits-IIelt 3 parts of petroleum jelly with 1 part of paraffin wax (m.p. 55" C), and allow to cool. PREPARATION OF CALIBRATIOS GRAPH FOR LACTIC ACID- (Conway KO. 1). Introduce 2 ml of semicarbazide reagent solution into the centre chamber of each unit To the outer chamber of each unit add 0.4 ml of saturated ceric sulphate530 RYAN : AN IMPROVED MICRODIFFUSION PROCEDURE [Vol.83 solution and then 2 ml of lactic acid solution. Seal the units by means of a paraffin wax - petroleum jelly fixative, and, after mixing the contents of the outer chambers, place them in an incubator at 37" C. '4fter 2 hours, open the units and remove 1-ml portions from the centre chamber of each. Dilute each portion to 10 ml with distilled water, and measure the optical densities of the diluted solutions with a Beckman DU spectrophotometer at 224mp. Use a blank solution, prepared by substituting 2 ml of distilled water for the lactic acid solution in one of the units, to set the instrument a t zero. The graph is linear when standard lactic acid solutions in the concentration range 10 to 70 pg per ml are used.The amount of lactic acid in a solution of unknown concentration can be read directly from such a graph, or, alternatively, can be calculated from a single standard determined simultaneously. The latter procedure is to be preferred unless strictly standard conditions are adhered to from day to day. PROCEDURE FOR DETERMIKING LACTIC ACID IN BLOOD- Add 2 ml of freshly collected blood to a 15-ml centrifuge tube containing 2 ml of 15 per cent. perchloric acid solution and 7 ml of distilled water. Mix the suspension thoroughly and spin in a centrifuge. Treat a measured amount of the supernatant liquid (8 to 8.5 ml) with sufficient 40 per cent. potassium hydroxide solution (0.2 to 0.4 ml) to precipitate the excess of perchloric acid.(The precipitate settles rapidly and leaves a clear solution.) Place 2-ml samples of this solution in the outer chambers of the units and determine lactic acid as described previously. Carry out a blank determination on a further 2 ml of this solution by substituting 0.4 ml of 6 AT sulphuric acid for the ceric sulphate solution. RESULTS The percentage recovery of lactic acid was determined from a graph similar to that described by Burbridge, Hine and Schick,* prepared in the following manner. Three milli- litres of the buffered semicarbazide reagent solution were placed, by pipette, in each of six 10-ml calibrated flasks, and to each was added different amount of acetaldehyde between 10 and 30pg. Each solution was diluted to the mark with distilled water, and a blank solution containing no acetaldehyde was prepared.The optical densities of these solutions were measured with a Beckman DU spectrophotometer a t 224 m p and the values were plotted. Acetaldehyde values read from such a graph can be converted to equivalent lactic acid values by multiplying by 2.0455. Forty-eight determinations of amounts of lactic acid between 20 and 100 pg had an average recovery of 98.4 per cent., with a coefficient of variation of F1.8 per cent. for a duplicate determination. Average recoveries for different amounts of lactic acid were as follows- Number of determinations . . . . . . 4 5 7 10 8 Amount of lactic acid present, pg . . . . 25.0 40.0 50.0 60.0 100.0 Average amount of lactic acid found, pg .. 25.1 39.0 48.8 58.2 98.6 These figures are based on results assembled over a period of several months, and, in some instances, the experimental conditions were slightly different from those described. The variations shown are greater than may be expected when standard conditions apply. The coefficient of variation for the group of ten determinations with 60 pg of lactic acid was only $_Om72 per cent. The precipitation of blood proteins can be carried out with trichloroacetic acid in place of perchloric acid. Five samples of human blood were analysed; when perchloric acid was used as precipitant, the results were 19.1, 10.4, 6.6, 6.1 and 3.8 (mean 9.2) mg of lactic acid per 100 ml. When trichloroacetic acid was used as precipitant, the results were 18.7, 10.2, 6.8, 5.9 and 3.8 (mean 9.1) mg per 100 ml, respectively.The differences can be considered to lie within the limits of experimental error. Whefi trichloroacetic acid was used, 2 ml of the blood sample were added to a mixture of 2 ml of 22 per cent. trichloroacetic acid solution and 7 ml of distilled water, which gave a final trichloroacetic acid concentration of 4 per cent. After centrifugation, the supernatant liquid was used directly for analysis. Ten freshly collected samples of human blood were analysed by the proposed method and also by the method of Elsden and G i b ~ o n , ~ perchloric acid being used in both methods to precipitate the proteins. An internal standard was used for the analyses; this precautionSept., 19581 FOR THE DETERMINATIOS OF LACTIC ACID 531 is unnecessary unless extremely accurate results are required.There was close agreement between the two methods; the results were as follows- Lactic acid by proposed method, mg per 100 ml . . . . . . 29.1 16.9 15.6 10.3 9.6 8.9 8.7 7.8 7.9 7.1 (mean 12.2) Lacticacid by Elsden and Gibson's method, mg per 100 ml . . , . 26.4 15.4 14.3 9.5 10.9 9.3 9.0 6.8 7.7 6.9 (mean 11.6) APPLICATION OF THE METHOD DETERMINATION OF LACTIC ACID IN RAT LEG MUSCLES- In order to maintain the lactic acid content of the muscle at as low a level as possible, the following procedure was adopted. Rats were injected intraperitoneally with 2 ml of a 2 per cent. solution of Myanesin, in a manner similar to that described by Bate-Smith and Bendall.' After 30 minutes, the rats were killed by a sharp blow on the back of the head Parts of the leg muscles were removed as quickly as possible and frozen in liquid oxygen.The frozen muscles were ground to a fine powder and approximately 1-g samples were trans- ferred to weighed beakers containing 20 ml of 5 per cent. trichloroacetic acid solution. The beakers were re-weighed to find the exact amount of muscle added. The suspensions were mixed thoroughly and set aside for 5 to 10 minutes before centrifugation. Lactic acid in the supernatant liquid was determined by the proposed method. Blank values were determined by using 2 ml of the trichloroacetic acid extract and 0.4 ml of 6 N sulphuric acid in the outer chambers of the units. The results of analyses on muscles of five rats were 27.6, 19.3, 16.6, 15.2 and 12.2 (mean 18.0) mg of lactic acid per 100 g of muscle.The high lactic acid content of the first sample was probably caused by slight twitching of the muscles, which occurred after death. CONCLUSIONS The proposed procedure has several advantages over other methods for lactic acid. A large number of determinations can be carried out simultaneously with comparatively little labour, and, since the only standard solution required is one of lactic acid, sources of error are reduced to a minimum. In addition, under the experimental conditions described, glucose does not interfere with the determination, so that preliminary treatment of biological samples with copper - lime reagent is unnecessary. The fact that a trichloroacetic acid medium is suitable for the determination is a further advantage, as trichloroacetic acid is widely used in clinical methods as a precipitant for proteins.The method has been used satisfactorily for determining lactic acid in blood. The principal source of error with blood is the time lapse between removal of the sample and precipitation of the blood proteins, which should be carried out as rapidly as possible. The results quoted were obtained from analyses on human blood samples removed at random from a number of different normal individuals. It is hardly necessary to add that variation in the lactic acid levels are largely due to the amount of exercise the subject had been taking before the sample was taken. The lactic acid concentrations in rat leg muscle may be taken as somewhat lower than the average normal value, since Myanesin inhibits all movement for 30 minutes. I thank Professor E. J, Conway, F.R.S., for his kind interest and helpful criticism with respect to this work. 1. 2. 3. 4. 5. 6. 7. REFERENCES Winnick, T., J . Bid. Chew., 1942, 142, 451. Gordon, J. J.. and Quastel, J. H., Biochem. J ~ , 1939, 33, 1332. Conway, E. J., and hlccarvill, M., 1st International Congress of Biochemistry, Cambridge, 1949, Elsden, S. R., and Gibson, Q. H., Biochenz. J., 1954, 58, 154. Long, C., Ibid., 1946, 40, 27. Burbridge, T. N., Hine, C. H., and Schick, A. F., J . Lab. Cliiz. Med., 1950, 35, 983. Bate-Smith, E. C., and Bendall, J. R., J . Physiol., 1949, 110, 47. Abstract No. 11712. Received March 7th. 1958

ISSN:0003-2654    

DOI:10.1039/AN9588300528

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

532 BUSH AND GALE: BLIJE TETRAZOLIUM AS [Vol. 83 Blue Tetrazolium as a Reagent for Reducing Steroids BY I. E. BUSH AND MURIEL 3f. GALE (Department of the Regius Professor of Medicine, The Radcliffe InJirmary, Oxford) Blue tetrazolium is a valuable reagent for the determination of many reducing substances, it being reduced in alkaline solution to a formazan of intense blue colour. Most commercial samples give rise to different propor- tions of pigments with reddish or purple hues, which have previously been supposed to be products of the partial reduction of the tetrazolium base. This phenomenon was investigated because i t prevented the accurate deter- mination of reducing steroids with this reagent on paper chromatograms. It is shown that the reddish reduction products can be separated, are not altered in colour by adding further reducing agent and can be oxidised to colourless substances that form the same coloured products when the reduction is repeated.Extremely pure samples of blue tetrazolium give rise only to the characteristic indigo-blue formazan on reduction. It is concluded that the reddish products are not partial reduction products, but typical formazans derived from tetrazolium salts other thim blue tetrazolium. Ways in which these and other impurities in blue tetrazolium can be avoided are suggested. THE tetrazolium salts are readily reduced by various steroids in alkaline solution and blue tetrazolium [3 : 3’-dianisolebis-4 : 4’-(3 : 5-diphen yl) tetrazolium chloride] was introduced by Chen, Wheeler and Tewelll as a more sensitive reagent for cr-ketolic steroids than is red tetrazolium (2 : 3 : 5-triphenyltetrazolium chloride).A standard quantitative method of determining this class of steroids with blue teti:azolium was also introduced by Mader and Buck2 and found wide favour. Chen, Wheeler and Tewelll quoted Rutenberg, Gofstein and Seligman3 to the effect that purple or red colours often occurred instead of the full deep blue given by the reagent under most conditions. They attributed this to the formation of partially reduced (formazan) derivatives, but gave no evidence for this view. Other complications of the use of this reagent have been observed by Jimbor,4-6 such as colour changes induced by light, colourless or abnorm.ally coloured reduction products under some conditions, and the effects of colloids.When b:lue tetrazolium was used for the quantitative determination of reducing steroids on paper chromatograms,e it was found a t first that the slopes of the calibration curves varied considerably, but predictably, with the solvent in which the chromatograms had been run. (The reagent was, of course, applied after the chromatogram had been dried.) This phenomenon was attributed at first to colloidal effects similar to those observed by Jimbor,6 but it was subsequently shown that extremely pure samples of blue tetrazolium failed to behave in this way, and, in the course of investigating the problem, several features of blue tetrazolium were discovered. These are described, as they may be helpful to those who use the reagent.EXPERIM:ENTAL BEHAVIOUR OF DIFFERNT SAMPLES OF BLUE TETRAZOLIUM- Four samples of blue tetrazolium were testsed with reducing steroids on chromatograms and were observed to give different shades of purple and blue with only slight dependence on the amount of steroid present. The same colour differences were observed when the four samples were reduced by an alkaline solution of ascorbic acid in the cold in test-tubes. It seemed unlikely that these differences in colour could be due to colloidal effects or to partially reduced products, and other evidence suggested that the samples were in fact of widely different degrees of purity. The samples that produced the reddest colour, for instance, formed some green scum when the ethanolic solution of blue tetrazolium was added to the aqueous alkali, whereas the sample that produced a deep indigo formazan formed no scum. The solution of formazans produced by reducing the worst sample of blue tetrazolium with alkaline ascorbate so1utio.n contained a mixture of coloured products, the presence of which was easily demonstrated by extracting either the aqueous solution or the precipitated formazans with immiscible solvents.The indigo-coloured product was readily extracted with ethylene dichloride, but was only slightly soluble in acetone or n-propyl alcohol; reddish products were readily soluble in both acetone and n-propyl alcohol.Sept., 19581 A REAGEKT FOR REDUCIXG STEROIDS 533 Further, it was found that no change in colour occurred when a purplish solution of formazans from the reduction of one of the impure samples of blue tetrazolium was treated with further alkaline ascorbate solution.These findings suggested that the reddish products of reduction were not intermediates in the reduction of blue tetrazolium to a formazan, but formazans produced by the reduction of tetrazolium salts other than blue tetrazolium, which were present as impurities. It was assumed that blue tetrazolium gave rise to the indigo-coloured product, since no other product worthy of the term “blue” was found among the formazans observed by solvent extractions and because the sample that gave rise to an apparently pure indigo-coloured product was the purest by a variety of criteria. In order to be more certain of this supposition, the worst and best samples were reduced under identical conditions with alkaline ascorbate solution and the formazans were extracted from the aqueous mixture with ethylene dichloride.The extract was evaporated to dryness under reduced pressure, and the residue was dissolved in a mixture of n-propyl alcohol and ethylene dichloride (9 + 1, v/v) and chromatographed on an alumina column. The products from the worst sample gave rise to two easily separated bands of deep indigo and pinkish purple, together with other fainter bands of colour. The product from the best sample, however, gave rise to one band only, which was a deep indigo colour. The bands were eluted separately, evaporated under reduced pressure and dissolved in ethanol. When alkaline ascorbate solution was added, no change in colour, other than that owing to slight dilution, was observed with any of the samples.This was taken to be good evidence that the worst sample was in fact an extremely impure mixture of various tetrazolium salts, which gave rise to a mixture of fully reduced formazans. Partial separation of these tetrazolium salts was achieved by solvent extractions and fractional crystallisation, but a completely pure specimen of blue tetrazolium chloride was difficult to obtain by these means from any of the commercial samples. SYNTHESIS OF BLUE TETRAZOLIUM CHLORIDE- When the method of Rutenberg, Gofstein and Seligman3 was followed exactly, a satis- factory sample of blue tetrazolium chloride was obtained at the first attempt, with no previous experience in the synthesis of this class of substance.It became clear, during the preparation of subsequent batches from our stock of crude product from the first synthesis, that the most important step in the preparation of a pure product is the exhaustive washing of the formazan before its oxidation to the tetrazolium salt, i.e., exactly as de~cribed.~ The final product is also greatly improved by several crystallisations from a mixture of ethyl acetate and methanol (1 + 1, v/v).l The green scum formed when the commercial products are dissolved in alkali is probably the starting material, tetra-azotised o-dianisidine, which is not removed from the formazan unless the exhaustive washing described by the originators is carried out in full (compare Ried and Gick’). DIFFERENT SAMPLES OF BLUE TETRAZOLIUM- One commercial sample (No.l), as marketed, and three other samples (Nos. 2, 3 and 4) were investigated. The research samples Nos. 2 and 3 were kindly donated for trial by firms who were investigating the synthesis of blue tetrazolium, and sample No. 4 was donated by Dr. J. Walker (National Institute for Medical Research, London). A 1 per cent. w/v solution of each sample was prepared in absolute ethanol (Burroughs, R.R. ethanol). Paper chromatograms of cortisol (32.0 pg) were run in the system benzene - methanol - water (2 + 1 + 1, v/v) for 34 hours at room temperature (approximately 19” C), dried and dipped in a fresh mixture of 2.5 ml of ethanolic blue tetrazolium and 7.5 ml of 2 N aqueous sodium hydroxide at room temperature. Sample No. 1 formed an immediate pinkish purple colour with cortisol, whereas sample No.4 formed a deep indigo colour. Samples No. 2 and 3 formed intermediate colours. Sample No. 1 produced a large amount of green scum, which separated when the ethanolic solution was mixed with the aqueous sodium hydroxide. Samples Nos. 2 and 3 produced less scum and sample No. 4 none. Five millilitres of an approximately 5 per cent. w/v solution of ascorbic acid in 2 N sodium hydroxide were then added to 1-0-ml portions of the alkaline aqueous ethanolic solutions of samples No. 1, 2 , 3 and 4 (proportions as above) at room temperature. Reduction was instantaneous, and the same colour differences were observed between the formazans from the four samples as had been seen with cortisol on paper chromatograms.534 BUSH AND GALE: BLUE TETRAZOLIUM AS [Vol.83 The formazans produced from the worst sample of blue tetrazolium (No. 1) were extracted with ethyl acetate (1 volume) and ethylene dichloride (2 volumes), the extracts were washed separately with 3 N hydrochloric acid and water, combined and then evaporated to dryness under reduced pressure at 45" C. The residue was dissolved in ethanol and made up to the same final proportions of 2 N sodium hydroxide and ethanol. The solution was then treated with an equal volume of alkaline ascorbate solution, but no further change in colour was observed during the following 10 minutes. PARTIAL SEPARATION OF TETRAZOLIUM DERIVATIVES- Five millilitres of a 1 per cent. ethanolic solution of blue tetrazolium (sample No. 1) were dissolved in about 15 ml of water and extracted with three 10-ml portions of n-butyl alcohol.The extracts were diluted separately with 20ml of benzene and extracted with 20 ml of 3 N hydrochloric acid. The acid extracts were made alkaline with sodium hydroxide solution in the presence of a little ice and extiacted with an equal volume of ethyl acetate. The three extracts were acidified with a slight excess of acetic acid and evaporated to dryness under reduced pressure at 45" C. The residues were dissolved in 15 ml of water and, together with the original solution of blue tetrazolium, diluted to 20 ml with 2 N sodium hydroxide. Two millilitres of 5 per cent. alkaline ascorbate solution were added to each solution and the colours produced were compared with that of a similar solution of blue tetrazolium that had not been extracted with a-butyl alcohol.The extracts all formed pink colours when alkaline ascorbate solution was added ; the extracted solution of blue tetrazolium formed a much more intense blue colour than the control solution of sample No. 1, but was still not as blue as the colour formed by sample No. 4 with alkaline ascorbate solution. A few milligrams of sample No. 1 were dissolved in 5.0 ml of a 1 per cent. v/v solution of methanol in benzene and put on to an alumina column (unstandardised), 13.5 cm x 1.2 cm. The chromatogram was developed with 10 ml of benzene and then 20 ml of benzene - acetone mixture (9 + 1, v/v), 20 ml of benzene - acetone mixture (8 + 2, v/v), 20 ml of benzene - acetone mixture (6 + 4, v/v), 40 ml of acetone, 35 ml of acetone - methanol mixture (85 + 15, v/v) and 20 ml of acetone - methanol mixture (7 + 3, V/V) were passed through the column.Five-millilitre fractions, including the 10 ml of developing solvent, were taken and 1 ml of each fraction was evaporated to dryness with a jet of air; the residue was dissolved in 1 ml of ethanol and mixed with 4 ml of 2 N sodium hydroxide and 1 ml of a 5 per cent. solution of ascorbic acid in 2 N sodium hydroxide to detect the eluted tetrazolium derivatives. Apart from minor components that gave colours in this test (not necessarily formazans), three main products were found in the eluates. Fractions 26 to 28 formed a slowly developing pink colour with alkaline ascorbate solution; fractions 30 to 32 formed a rapidly developing in- digo colour that was soon precipitated; fractions 31 to 34 formed a rapidly developing pink colour (different from that of fractions 26 to 2811, which was detectable in fractions 31 and 32 as soon as the main indigo-coloured product had precipitated.Fraction 30 behaved exactly as the best sample of blue tetrazolium (No. 4) in both colour and speed of precipitation of the indigoid pigment. Fifty microlitres of the ethanolic 1 per cent solution of sample No. 1 were run on a paper chromatogram with water-saturated n-butyl alcohol, as in the method described by Ried and Gick.7 A slowly developing pink colour appeared at the solvent front, a rapidly developing pink colour at the origin and a deep indigo spot at R, 0.7. Further 1-ml portions of the eluates from the alumina column were treated by making two successive additions of alkaline ascorbate s801ution.Xo change of colour occurred with the second addition in any fraction. The presence of excess of available ascorbate was confirmed by adding 1 ml of a 1 per cent. ethanolic solution of sample No. 4 ; a deep indigo colour developed immediately. SEPARATION OF REDUCTION PRODUCTS- Five millilitres of a 1 per cent. ethanolic solution of sample KO. 1 were treated with 10 ml of alkaline ascorbate solution and 10 ml of 2 N sodium hydroxide, and the precipitate was collected on a Whatman No. 2 filter-paper. After being washed with water, the precipitate was extracted with acetone. A deep purple solution was quickly obtained, and successive extractions left an insoluble indigo residue, which dissolved moderately easily in ethylene dichloride.No change of colour occurred when either extract was dissolved in ethyl acetate The strip was then dipped in alkaline ascorbate solution. Sample No. 4 produced only the indigo spot.Sept., 19681 A REAGEST FOR REDUCING STEROIDS 535 and diluted to a faint colour. A small portion of each extract was evaporated under reduced pressure a t 45' C and dissolved in ethanol. No change in colour occurred when excess of alkaline ascorbate solution was added. The presence of available excess of ascorbate was proved by adding 1 ml of a 1 per cent. ethanolic solution of sample No 4; a deep indigo precipi- tate formed rapidly. One volume of ethylene dichloride was added to 9 volumes of a solution of the acetone- soluble product in n-propyl alcohol, and the solution was put on to an alumina column (un- standardised), 15 cm x 1.2 cm.The purple product was not retained on the column and was rapidly eluted with n-propyl alcohol. When 1 volume of an ethylene dichloride solution of the indigo product was diluted with 9 volumes of n-propyl alcohol and put on a similar column, the product was retained as a narrow band a t the top of the column and was not eluted with large volumes of n-propyl alcohol. RE-OXIDATION OF SEPARATED PRODUCTS- A few milligrams of the indigo-coloured reduction product that had been separated on alumina were dissolved in 5 ml of ethylene dichloride and shaken with 10 ml of a 10 per cent. w/v solution of ceric sulphate in N sulphuric acid in a separating funnel.As soon as the colour had disappeared, the ethylene dichloride layer was run off and 100 ml of absolute ethanol were added to the aqueous layer. The suspension was filtered on a No. 3 sintered- glass filter, and the filtrate was mixed with an equal volume of 4 N sodium hydroxide. Ten millilitres of the slightly turbid alkaline solution were then treated with 5 ml of 6 per cent. alkaline ascorbate solution and extracted with 3 ml of ethylene dichloride. A deep indigo- coloured solution was obtained, the colour of which was not changed on dilution with ethylene dichloride. Although it is not recommended as a reliable route to the tetrazolium salt, the important finding was that in no instance did the re-oxidised indigo-coloured formazan give rise to a mixture of different coloured products when the reduction was repeated with alkaline ascorbate solution. SYKTHESIS OF BLUE TETRAZOLIUM- The procedure of Rutenberg, Gofstein and Seligman3 was followed exactly, starting with 406 g of tetra-azotised o-dianisidine (Brentamine fast blue B, Imperial Chemical Industries Ltd.), and 110 g of crude formazan were obtained.The final tetrazolium salt (286 g) produced a colour with alkaline ascorbate solution that was slightly inferior to that produced by sample No. 4. This was improved by twice recrystallising the salt from a mixture of ethyl acetate and methanol (1 + 1, v/v), after which the colour produced by alkaline ascorbate solution was indistinguishable from that produced by sample No.4. Only one coloured band was detected when a small portion of reduction product was chromatographed on alumina. CONCLUSIONS Many workers have investigated the use of blue tetrazolium for determining reducing steroids, e g . , Meyer and Lindbergs and Izzo, Keutmann and B ~ r t o n , ~ but these studies have dealt mainly with problems of specificity and of establishing conditions for repro- ducible results. The reduction of other tetrazolium salts has received more attention because of their use as redox indicators with enzyme systems (reviewed by Smithlo). Chen, Wheeler and Tewelll commented in 1953 on the poor quality of some commercial samples, but we were nevertheless surprised by the poor quality of most of the samples we tested. It is obvious from the studies of JAmbor4,5 and others that the reactions of tetrazolium salts are complex, but we are not satisfied that all the effects observed by JAmbor5 would be obtained with really pure samples of blue tetrazolium.Our own investigations started from the assumption that Chen, Wheeler and Tewell'sl supposition was correct, namely, that red or purple colours produced by alkaline reduction of blue tetrazolium were due to partially reduced intermediate products. The peculiar effects observed by Bush and Willoughby during the course of unpublished work on chromatograms were interpreted as being due to optical effects caused by structural alterations in the paper, or to an effect of paper on the proportion of partially reduced products obtained. It was only when sample No. 4 was obtained that we began to suspect that impurity of the reagent might explain these findings.Although we have not carried these investigations further, and it is obvious that the reduction of tetrazolium salts requires much further study, we thought it worth while to report No trace of red products could be detected on an alumina chromatogram. This experiment was repeated several times with rather variable yield.536 NOTES [Vol. 83 our findings, so that others may be able to avoid the irregular behaviour of impure samples of blue tetrazolium in their work. It migh be held that the products we separated on alumina columns were, in fact, the partially reduced products described by JLimbor5 and Chen Wheeler and Tewe1l.l Against this, it should be emphasised that such products from impure samples of blue tetrazolium were incapable oi further reduction to other coloured products; the purest samples never gave rise to them, and if, after separation from the mixture of reduction products, they were re-oxidised with ceric sulphate, subsequent reduction always gave the same coloured product and not the mixture obtained by reducing the original sample under similar conditions.Again, reversible light-induced colour changes similar to those reported by JBmbor5 were seen with the impure samples, but not with the pure samples. In our experience, it is easier to purify the formazan from which the tetrazolium salt is obtained than to obtain really pure samples from the seriously impure tetrazolium salts available commercially. Although it is desirable that such a useful reagent should be available commercially in good quality, it seeins better a t present, as in 1953,l to synthesise one’s own reagent. The worst sample in the series tested contained about one-third of its supposed content of the correct tetrazolium salt. As novices in this field, it took one of us 10 days’ part-time work, and materials worth seventy shillings, to prepare an excellent sample worth forty-six pounds at the current retail price of seriously impure samples. We are grateful for the support and interest of Professor Sir George Pickering and St. Mary’s Hospital Medical School during this work. We are also very grateful to Dr. J. Walker (National Institute for Medical Research, London) for a gift of sample S o . 4, and to two firms who supplied test samples, but who wish to remain anonymous. REFEF.ENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Chen, C., Wheeler, J., and Tewell, H. E., J . Lab. Clin. Mad., 1953, 42, 749. Mader, W. J., and Buck, R. R., Anal. Chem, 1952,24, 666. Rutenberg, A. M., Gofstein, R., and Sehgman, 4. M., Cancer Res., 1950, 10, 113. JAmbor, B., Nature, 1954, 173, 774. - , ?bid., 1955, 176, 603. Bush, I. E., and Willoughby, M., Biochem. J , 1957, 67, 689. Ried, W., and Gick, H., Annalen, 1953, 581, 16. Meyer, A. S., and Lindberg, M. C., Anal. Cham, 1953, 27, 813. Izzo, A. J., Keutmann, E. H., and Burton, R. B., J . Clin. Endocrin. ;IIetabolzsm, 1957, 17, 889. Smith, F. E., Sczence, 1951, 113, 751. Received Xovenzber 15th, 1957

ISSN:0003-2654    

DOI:10.1039/AN9588300532

出版商:RSC    

年代:1958

数据来源: RSC

摘要:

536 NOTES [Vol. 83 Notes THE MICROBIOLOGICAL ASSAY OF MERCURIALS IN PHARMACEUTICAL PRODUCTS THE method devised by Kleinl for the assay of mercury in small amounts is tedious and sometimes not dependable, although certain modifications are claimed to make i t more reliable.2 In order, therefore, to determine the concentration of organic mercurials, and particularly of phenylmercuric nitrate and thiomersal, when used as preservatives in pharmaceutical and cosmetic preparations, microbiological methods of assay were investigate’d. The methods described are mainly applicable to the control of preparations of known composition; they cannot be used reliably with other preparations until the possibility of interference from other constituents has been ascertained. To our knowledge, there is no published account of any such methods.Two basic techniques were open to investigation, the serial-dilution and the plate-diffusion methods. At first, our main interest was in the: normal tube-dilution method, the end-point of the test being determined after a prolonged incubation period of 48 to 72 hours. Subsequently, it was centred on the plate-diffusion method and a.n adaptation of the normal tube-dilution assay, referred to in the text as the “rapid-dilution’’ method, and these proved to be more satisfactory, largely because of their greater precision. On ‘balance, plate-diffusion is probably the method of choice, especially for those practised in this type: of assay, but the others are of value, particularly when dealing with formulations containing other possibly antibacterial substances, because they are more sensitive and respond at much lower levels of concentration of the mercurials, thus allowing interfering substances to be more effectively diluted out.It was realised from the outset that, at the very low concentrations of mercury used in any of these assays, quite small amounts of other added substances, particularly if they are of anSept., 195Sj SOTES 537 organic nature, might significantly alter the end-point of the assay, and so a control solution made to the same basic formulation as the test material was included in each assay, the test material and the control being treated as nearly identically as possible a t all stages of the assay. NORMAL TUBE-DILUTION AND RAPID TUBE-DILUTION METHODS These two methods have many factors in common, so that it is convenient to consider them together and to note the differences where necessary, The principle of both methods is to pre- pare serial dilutions of the test material and of the control solution in a liquid nutrient medium, inoculate with a chosen test organism, incubate a t 37°C and note the concentration at which growth is just inhibited.I n the normal method, incubation is for 48 to 7 2 hours, and growths are assessed by direct observation; in the rapid method, incubation is only for 4 hours in a water bath (to bring the tubes quickly and uniformly to 37" C), the growths again being assessed by direct observation. By comparison of the end-points with the test material and the control solution, the concentration of the mercurial present can be calculated.The medium-Because all mercurials are readily inactivated by organic matter, a culture medium was chosen containing the minimum amounts of nutrients needed to support the growth of the chosen test organism. An ordinary nutrient medium diluted tenfold with water was found to be satisfactory, and a simple peptone medium was preferred to a digest broth because of the greater apparent uniformity in its nutrient properties and the smaller consequent fluctuations in the end-point of the test. The composition of the medium is as follows- Oxoid peptone . . . . . . .. . . I g Glucose . . . . . . . . . . . . 2 g Sodium phosphate, B.P.. . . . . . . . 5.5 g Solution of bromocresol purple, B.P. . . . . 5ml ITater .. . . . . . . . . . . t o 1000 ml It is adjusted to pH 7.2 with citric acid, distributed in 5-ml portions in $inch x &inch test-tubes and sterilised in steam a t 121" C for 10 minutes. The system is weakly buffered to balance any slight acidity or alkalinity in the test material; the indicator is added to note the acid produced in those tubes in which growth of the organism has occurred, and thus to assist in determining the end-point with preparations such as creams, which, by their nature, give turbid dilutions before incubation. The culture-The culture recommended is a strain of Escherichia coli (N.C.T.C. 5934) grown for 24 hours a t 37' C in nutrient broth. For the normal tube-dilution method it is diluted 1 + 99 with sterile saline immediately before use.PROCEDVRE- A preparation is made of similar formulation to the test material, but containing no mercury. Sufficient of the mercurial under assay is added, in as small a volume of solvent as possible, to give the theoretical amount of mercury in the formulation; this constitutes the control solution. If the basic test material possesses antibacterial properties, which would disturb the otherwise linear relationship between the growth response in the test and the amount of mercury present, then additional control solutions containing progressively smaller amounts of mercury may be required. (If the mercurial to be assayed is phenylmercuric nitrate, it can be added as a 0.1 per cent. solution in glycerol, without any apparent interference in the assay from the glycerol.) Treating the test and the control preparations identically, each sample is diluted suitably with sterile distilled water, and, from this, a series of dilutions is prepared in 10 per cent.stages in sufficient numbers to span the expected end-point of the assay (about 8 such dilutions are usually adequate). Five-millilitre portions of each dilution are transferred to each of 4 tubes containing 5 ml of the assay medium. For the normal method, 0.2ml of the diluted culture is added to each tube; they are then incubated a t 37" C for 48 to 7 2 hours, and the growths are recorded. The end-point is the greatest dilution a t which no growth occurs. For the rapid method, 1 standard drop (approximately 0.03 ml) of the undiluted culture is added to each tube; the tubes are transferred to a water bath a t 37" C, and, after 4 hours, they are examined for growth.The end-point is the greatest dilution a t which there is marked inhibition of growth, as shown by a significant decrease in turbidity (this is usually quite sharp). With preparations that give initially turbid dilutions, a slightly longer incubation is given so that the end-point can be read by the colour change of the indicator. With either method, the mercury content of the test material is calculated by com- parison of its mean end-point in the assay with that of the control solution.538 NOTES [Vol. 83 In order to obtain the approximate end-point in the assay, it may be necessary to make a preliminary ranging test. This is done in the manner described above, but with use of a much more widely spaced series of dilutions.For phenylmercuric nitrate and thiomersal, the end-point under the conditions given is a t about 0.2 p.p.m. by the normal tube-dilution method and 0.05 p.p.m. by the rapid method. THE PLATE-DIFFUSION METHOD This assay follows in principle the Heatley cup assay as devised originally for penicillin. The medium-The composition of the recommended medium is as follows- Oxoid peptone . . * . . . . . l o g Agar powder (Davis) . . . . . . l 5 g Lab-Lemco . . . . . . .. 1 5 g Water . . . . .. .. . . t o 1000 ml It is adjusted to pH 7.2 and sterilised in steam at 121" C for 15 minutes. The rather higher concentration of agar (normally, 1.1 per cent. is adequate) gives more clearly defined zones of inhibition.The culture-A culture of Staphylococcus auwus is used, a 24-hour growth in nutrient broth being employed for seeding the agar. The strain chosen must be sufficiently sensitive to the mercurial under assay, and must give a well defined zone of inhibition; in this respect there is a considerable variation between strains. PROCEDURE- As with the tube assays described, a control solution, or solutions, of similar formulation to the test material is required, which is used as the standard in each assay. The same procedure is followed as in the British Pharmacopoeia1 method for the microbiological assay of penicillin, preferably cut-out cups intead of cylinders being used; satisfactory zone responses are obtained with concentrations of 10 and 2 p.p.m. of the phenylmercuric compounds and thiomersal.The assay is sensitive only down to about 1 p.p.m. TABLE I COMPARATIVE ASSAYS OF PHENYLMERCURIC NITRATE AND THIOMERSAL BY THREE METHODS Amount found by- Mercurial Amount added, p.p.m. Phenylmercuric nitrate . . 175 130 Thiomersal . 75 35 15 7 2.5 1.0 0.6 225 115 100 25 10 3.5 1.7 0.5 normal tube-dilution method, p.p.m. 140 50 13 - - 7.4 2.9 - - 235 117 - - 10.5 3.3 1.5 - rapid tube-dilution method, p.p.m. 140 - - 33.3 - 3 plate-diff usion method, p.p.m. 179 128 69 34.5 12.7 6.8 2.35 1.02 n.a. 206 115 106 28.2 10 3.4 ma. n.a. ma. = not assayable. RESULTS Most of the experimental work has been carried out with solutions and preparations containing phenylmercuric nitrate or thiomersal, although some tests have also been made with mersalyl and mercurochrome.Various aqueous solutions, the concentrations of which were unknown to the assayist, were examined by one or more of the methods described and the results are shownSept., 19581 NOTES 539 in Table I. over the normal tube-dilution method. determined, and the results are shown in Table 11. They show the greater precision of the rapid tube-dilution and plate-diffusion methods The organic mercurial contents of several different pharmaceutical preparations were also TABLE I1 ASSAYS OF MERCURIALS IN PHARMACEUTICAL PREPARATIONS Amount of -4mount of Preparation mercurial present, mercurial found, Method of assay p.p.m. (2) Said to contain 10 Water-miscible ointment base with (1) 6 phenylmercuric nitrate Tragacanth paste (experimental) (1) 50 with phenylmercuric nitrate (2) Said to contain 100 Experimental solution in polythene (1) Said to contain 20 bottle with phenylmercuric nitrate (2) Same solution after (1) Said to contain 10 2 months Experimental solution in polythene bottle containing boric acid and (2) Same solution after thiomersal 1 month REFERENCES .1. 2. STANDARDS DEPARTMENT Klein, ,4. K., J . Ass. Off. Agric. Chem., 1952, 35, 537. .4bbott, D. C., and Johnson, E. J., Aitalyst, 1957, 82, 206. MICROBIOLOGICAL DIVISION BOOTS PURE DRUG Co. LTD. XOTTINGHBX p.p.rn. 5.2 15 16.8 47 145 165 156 25.6 8.5 10.5 -1 Plate-diff usion Plate-diff usion Rapid tube-dilution Plate-diff usion Plate-diffusion Plate-diff usion Rapid tube-dilution Plate-diff usion Plate-diff usion Rapid tube-dilution Rapid tube-dilution D.V. CARTER G. SYKES Received March 25th, 1958 BRUSHITE NODULES AND HYDROGEN SWELLS IN DEFECTIVE CANNED CHEESE PASTE As a result of complaints, the Dunedin Branch of the Dominion Laboratory received three 4-02 cans of cheese paste. The New Zealand Food and Drug Regulations, 1946,' require cheese paste to contain not less than 75 per cent. of cheese, together with other wholesome foodstuffs and condiments. The samples were found to contain 27.0 per cent. of fat, 46.7 per cent. of water and 4-70 per cent. of ash, which is consistent with a paste containing 75 per cent, of cheese. Two of the cans contained several dozen hard rounded nodules, up to 1.5 mm in diameter, but, in the third can, nodules of diameters up to 5 mm were found. The nodules were insoluble in both hot and cold water, but were soluble in dilute hydrochloric acid.They were found to contain 14.5 per cent. of phosphorus (present as phosphate) and 19-1 per cent. of calcium, i.e., equimolar proportions of calcium and phosphorus. Microscopical examination of the crushed nodules showed that they consisted of radial aggregates of microcrystals. Less than 1 per cent. of the nodules consisted of small spherulitic aggregates of crystals with a high refractive index (about 1.6 and above) and high birefringence. It is possible that these were calcite. The remaining crystals showed little variation, with minimum refractive index about 1.539 and maximum refractive index about 1.555. This is consistent with brushite, hydrated dicalcium orthophosphate, CaHP0,.2H,O (a = 1.539, fl = 14545, y = 1.551),2 but excludes monetite, anhydrous dicalcium orthophosphate, CaHPO, ( a = 1.600, fl = 1.614, y = 1.631),s calcium a-pyrophosphate, a-Ca,P,O, ( a = 1.585, 6 = 1.60, y = 1*605), and calcium 8-pyrophosphate, 8-Ca,P,O, (a = 1.624, y = 1.628).4 As the crystals were in the form of fine complex aggregates, it was impossible to obtain any values for optic axial or extinction angles. From X-ray powder diffraction examination with a Phillips X-ray diffractometer, lattice spacings calculated for the most prominent lines were as shown in Table I.Corresponding lattice spacings for brushite, reported by Hanawalt, Rinn and Frevel,6 and Bale and co-workers,4 and calculated from the data of Hill and Hendricks,B are also shown for comparison.Weaker lines in the pattern also show excellent agreement with those previously reported. Hence, i t is clear that the crystalline component of the nodules is brushite. From this identification and the analysis it was deduced that the nodules consisted of 82 per cent. of hydrated dicalcium ortho- phosphate, together with cheese paste and water.540 NOTES [Vol. 83 TABLE I COMPARISON OF LATTICE SPACINGS OF SAMPLE AND BRUSHITE Spacing of sample, 7.83 4.23 3.04 2.615 2.425 2.170 A 7- reported by Hanawalt, Rinn and Frevel,s 7.6 4.24 3.04 2.62 2 4 2 2.16 A Spacing of brushite- reported by Bale and co-w~rkers,~ 7.7 4.20 3.02 2.60 2.42 2.15 - A A - calculated from data of Hill and Hendricks,6 A - 4.24 3.07 2.63 2.44 2.19 Rank and Siebenlist’ have reported s a n d i n s in processed cheese packaged in cans and tubes, owing to formation of grains of calcium phosphate.Their analyses were: phosphorus, 18.8 per cent., present as phosphate; calcium, 16.9 per cent.; loss on ignition, 24.5 per cent., which appeared to be mostly water of crystallisation. I’rom the composition of the emulsifier used and the analysis, they deduced that the grains were probably a mixture of calcium metaphosphate and pyrophosphate, together with organic impurities. Rank and Siebenlist attribute formation of the grains to high temperatures during processing and sterilising and to prolonged storage, especially when there is a high calcium content in the raw cheese or the emulsifier. In the example reported here, it is probable that the soluble phosphate added as an emulsifier or “cheese melting salt” during manufacture OF the paste has reacted with calcium present in the cheese to form nodules.Calcium tartrate crystals, formed in a similar way from tartrate emulsifiers in processed cheese, have been reported by Blanchard* and Leather.9 White specks in mature Cheddar cheese have been identified a!; calcium lactate by McDowall and McDowell’O and as tyrosine with calcium phosphate as an iripurity by Dorn and Dahlberg.ll Palmer and Sly12 have reported two occurrences of sodium phosphate crystals in processed cheese owing to crystallisation of the emulsifier during storage a t a low temperature. In addition to the nodules, the three cans of cheese paste contained 18, 33 and 40 ml of gas, respectively. The gas was found to be a mixture of 93.5 per cent.of hydrogen and 6.5 per cent. of carbon dioxide. According to Jones,I3 “hydrogen swells,” which are caused by corrosion of the tin-plate of the can, are “almost entirely confined to acid fruits.” The interiors of these cans were extensively etched and pitted. The surface of the cheese paste in contact with the metal was stained grey and, in one sample, contained 1880 p.p.m. of tin and 800 p.p.m. of iron. It is probable that both of the defects reported here have been aggravated by prolonged storage. I thank Mr. J . D. Raeside, Soil Bureau, Duriedin, for assistance with the microscopy, Pro- fessor D. S. Coombs, Geology Department, University of Otago, for assistance with X-ray crystallography, Mr. 0. H. Keys, Government Analyst, Dunedin, for advice and criticism and the Director, Dominion Laboratory, New Zealand, for permission to publish this Note. The pH of the cheese paste was 5.6. 1. 2. 3. 4. 5. 6. 7 . 8. 9. 10. 11. 12. 13. REFERENCES “The Food and Drug Regulations 1946,” Govi:rnment Printer, Wellington, N.Z., 1946, p. 36. Larsen, E. S., and Berman, H., “The Microscopic Determination of the Nonopaque Minerals,’’ _ _ ~ . 0.6. cit.. D. 111. Second Edition, U.S. Geol. Survey Bull. No. 848, 1934, p. 104. Bale: W. F., Bonner: J. F., Hodge, H. C., Adler, H., Wreath, A. R., and Bell, R., Ind. Eng. C h e w , Hanawalt, J. D., Rinn, H. W., and Frevel, L. K., Ibid., 1938, 10, 457. Hill, W. L., and Hendricks, S. B., Ind. Eng. Chew., 1936, 28, 440. Rank, B., and Siebenlist, E.. Dtsch. MoZk.-Ztf., 1941, 62, 1036. Anal. Ed., 1945, 17, 491. Blanchard, J. F., Food Ind., 1949, 21, 51. Leather, A. N., Analyst, 1949, 74, 51. McDowall, F. H., and McDowell, A. K. R., J . Dairy Res., 1939, 10, 118. Dorn, F. L., and Dahlberg, A. C., J . Dairy Sci., 1942, 25, 31. Palmer, H. J., and Sly, W. H., J . Soc. Chew. Ind., 1944, 63, 363. Jones, O., “Canning Practice and Control,” Third Edition, Chapman & Hall Ltd., London, 1949, p. 52. THE DOMINION LABORATORY P.O. Box No. 562 DUNEDIN, KEW ZEALAND D. F. NELSON First received March 13th, 1957 Amended, April 22nd, 1958

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DOI:10.1039/AN9588300536

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年代:1958

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Sept., 19581 BOOK REVIEWS 54 1 Book Reviews A TEXTBOOK OF PHARMACOGNOSY. By G. E. TREASE, B.Pharm., D. de l’U., F.P.S., F.R.I.C., F.L.S. Seventh Edition. Pp. viii + 808. London: BailliBre, Tindall & Cox Ltd. 1957. Price 42s. Every author faced with the problem of preparing a new edition of a standard textbook has to choose between increasing the size of his book and deleting some of the older material in order to find room for the new. Dr. Trease, in the seventh edition of “A Textbook of Pharmacognosy,” is to be congratulated on having succeeded, not merely in maintaining the size of the volume, but actually reducing it by a few pages, although admittedly his subject has not expanded very much since the sixth edition was published in 1952. The same pattern is followed as in the previous editions, and the bulk of the book still comprises a systematic treatment of the vegetable drugs with a description of their constituents, modified when necessary in the light of recent advances in the field.Such revision is particularly evident in the monographs on “Veratrum” and “Rauwolfia,” the constituents of which have of course assumed considerable importance in recent years. In the new edition the section dealing with the history of drugs, their cultivation and collection remains unchanged, except that the chapter on enzymes has been expanded to include so called “pharmaceutical enzymes,” such as hyaluronidase, papain, streptokinase and streptodornase. Unfortunately, the two new formulae of alkaloids on p. 596 both contain errors. Rather surprisingly, the chapter on chromatographic analysis remains unchanged, but there is a new chapter entitled “Tracer Technology and Pharmacognosy,” which should be of great use to students.It comprises a brief description of the elementary principles of the subject followed by references to several applications in which labelled carbon dioxide has been used to produce drugs such as digitoxin and morphine labelled with I3C. The remaining chapters have not been altered except the one on “Fibres and Surgical Dress- ings,” which now includes a useful description of man-made fibres. The maps that were a feature of previous editions have been deleted, but there is a new appendix listing the plants or plant products referred to in the different National pharmacopoeias.This is included in order to make the book more useful to non-British students. The style of printing and binding remains unchanged and the new “Trease” will undoubtedly continue to serve the needs of students of pharmacognosy, as its predecessors have done for over 20 years. “A Textbook of Pharmacognosy” remains basically unchanged. Part 4, on chemistry, has been expanded slightly, but it is still rather sketchy. F. A. ROBINSON THE DEVELOPMENT OF TITRIMETRIC ANALYSIS TILL 1806. By E. RANCKE MADSEN. Pp. 239. At a time when so many great developments in analytical chemistry are taking place and new and unexplored frontiers are opening before us, it is stimulating to find one contemporary who can pause to look back and re-assess our great debt to the past. Dr.Rancke Madsen rightly points out that no modern text makes more than passing reference to the historical development of titrimetric analysis (or, for that matter, to any other branch of classical analytical chemistry). The reason clearly enough is that no complete history is in existence from which a concise summary can be made, and “the history of analytical chemistry is still waiting for its author.” In the meantime, Dr. Rancke Madsen has done a signal service in treating in great detail one major section of the complete story. I t is well known that the colour change of acid - base indicators was first observed by Robert Boyle in 1663, but perhaps less generally known is that one of Boyle’s indicators belonged to the class now called fluorescent indicators and which did not come into use until 1926.The first attempt to measure solutions by volume in analysis seems to have been made by the Scottish physician Francis Home (1756), who used a teaspoon for measuring the amount of nitric acid (1 : 6) required to neutralise a known amount of plant ash. “I took a drachm of blue pearl ashes and poured on it . , , the acid mixture, An effervescence arose and before i t was finished, 12 teaspoonfuls of the mixture were required.” One of the most interesting facts unearthed by Dr. Rancke Madsen is the contribution of the Englishman, William Lewis (1767), whose work has received little notice. Lewis used what is undoubtedly the first M-eight burette. He titrated ashes, first noting the decrease in effervescence to mark the approach of the end-point, and then indicator paper to obtain the end-point exactly.Copenhagen: G. E. C. Gads Forlag. 1958. Price Dan. kr. 20.00.542 BOOK KE:VIE\YS iVol. 83 Lewis used paper impregnated with lacmoid or blue archil or the “blue paper used for wrapping sugar in.” He standardised the acid against pure potassium carbonate “perfectly dried by fusion in an iron ladle.” To avoid over-titrating, he reserved a little of the test solution in another vessel, which was finally washed into the titration vesse:l. Hence to Lewis must go the credit of being the first to use colour indicators (incorrectly ascribed to Lampadius, 1801), a weight burette and a standardised solution, and for absolute determination by titrimetry. Dr. Rancke Madsen has taken his researches much further than delving in the ancient litera- ture; he has also attempted to reproduce the experiments used by these early investigators.Thus he finds an error of Among other titrimetric methods that have been examined in this way are de Morveau’s method for determining carbon dioxide in water and chloride with lead nitrate solution, Home’s method for water-hardness and Gadolin’s method for iron. This attempt to reconstruct and check the past enhances the value of the book and adds considerably to its interest. The only suggestion that the reviewer has to offer is that, in future editions, a list of the relevant important dates in chronological order be given, so that the various developments can be seen a t a glance. This is a fascinating book that should interest all engaged in the practice of chemistry; it is to be hoped that the author will be encouraged sufficiently by its reception to turn his attention to the history of other branches of analytical cbemistry.5 per cent. when Home’s method is used for detecting the end-point. R. BELCHER CHAKACTERISATIOK OF ORGAKIC COMPOUNDS. :By F. WILD, M.A., Ph.D., F.R.I.C. Second In the past i t has been considered desirable when identifying an organic substance to proceed Edition. Pp. viii + 306. Cambridge University Press. 1958. Price 37s. 6d. along the following lines- (i) To establish presumptive evidence that. the substance is pure. (ii) To determine the elements present and then to find the class to which the substance (iii) To assign the position of the substance in this class.(iv) To complete the identification by preparing a crystalline derivative of the compound The second edition of Wild’s book deals almost exclusively with item (iv), i.e., with the methods of preparation of crystalline derivatives of organic substances, and their melting-points. There are eleven chapters, two of which deal with the selection of reagents and the general classification of organic substances ; the remaining chapters deal with the preparation of suitable derivatives of hydrocarbons, halides, hydroxyl compounds, mercaptans, etc., carbonyl compounds, acids, acid halides and anhydrides, etc., amines and amino compounds and nitro, cyano and nitroso compounds, etc. For example, for halides, the chemical reactions involved in the preparation of suitable derivatives are given in detail, after which full working details of the procedures for derivative preparation are described. Finally, there is a set of Tables that show a t a glance the melting-points of all those derivatives of the halides that are considered t o be of value in identification work.The main point about this new edition, as compared with the previous one, is that many new derivatives and their physical constants are described and certain of the melting-points have been revised in the light of recent determinations. The book is intended for use by final-year students in advanced practical classes and also by research students, but should appeal to a much wider section of readers. Many organic chemists today have to carry out identifications on much smaller amounts of substances than are envisaged in this book, and hence a great deal of importance is attached to ultra-violet and infra-red spectra, etc.Nevertheless, the ability to prepare a suitable derivative of an organic compound as a final step in its identification is still part of the equipment of the modern analytical chemist, and this book, so well written, should be of great help to him in this aspect of his work. belongs. of authentic melting-point. The layout of each chapter is very similar. J. HASLAX BRITISH PHARMACOPOEIA 1958. Published under the direction of the General Medical Council. The new edition of the British Pharmacopoeia contains 110 more pages than its predecessor The number of drugs that were not included in that edition or in the 1955 Addendum Among the casualties are many well known remedies, including Pp.xxvi + 1012. London: The Pharmaceutical Press. 1958. Price 63s. of 1953. is 66, but 62 have been deleted.Sept., 19581 BOOK REVIEWS 543 arsenic trioxide, barbitone, the carbonate, salicylate and subgallate of bismuth, chenopodium oil, carbon tetrachloride, cinchophen, mercurous chloride, neoarsphenaniine, pepsin, sulphanilamide and sulphatbiazole. Amylobarbitone, probably the most widely used hypnotic with the exception of phenobarbitone appears for the first time, but perhaps the most significant additions are two radioactive compounds, sodium radio-iodide (1311) and sodium radio-phosphate (“P). Elaborate tests invoh-ing the use of a Geiger - Muller counter are given for the determination of half-life, radiochemical purity and potency.The Pharmacopoeia has indeed gone far since the time when it was considered that all tests should be capable of being performed by the pharmacist a t the back of his shop. Counting only drugs with a definite pharmacological action and ignoring salts and derivatives, the number of synthetic drugs is 134 and of drugs of natural origin 97, a ratio of 1.38. In the 1953 edition, the numbers were 107 and 85, respectively, a ratio of 1.26. The trend towards the use of synthetic drugs is not therefore as great as might be expected. Many assays now depend on a non- aqueous titration, which is used for barbiturates and a number of other drugs. Potentiometric titration with sodium nitrite and the dead-stop end-point is used for sulphonamides and other compounds.More assays depend on the spectrophotometric determination of the extinction a t a specified wavelength. One complexometric titration is described for the determination of calcium in calcium gluconate injection. The name sodium edetate has been coined for the disodium salt of ethylenediaminetetra-acetic acid. Although this appears to be an excellent choice, it is un- fortunate that an additional name should be introduced at a time when the use of EDTA is becoming firmly established among analysts. The use of standard preparations of compounds for use in chemical tests as well as for biological methods has been extended, following the example of the U.S.P. The limits tests for arsenic and lead are practically unchanged. The chance of these elements obtaining access to chemicals in therapeutic quantities nowadays must be so small that one wonders whether the thousands of tests that are performed annually in analytical laboratories are really necessary; for some of the compounds for which limits are given, a test for strontium-90 would appear to be as logical.Among the new tests noted are the determination of the jelly strength of gelatin with the Bloom Gelometer, and a limit test for noradrenaline in adrenaline; a chromatographic method is described for the detection of hydrocarbons in cetostearyl alcohol. I t is, of course, essential that the analytical methods of the B.P. should be reliable and well established. Bearing this in mind, i t appears that the analyst member of the British Pharma- copoeia Commission, Dr.J. R. Nicholls, and the many analysts serving on its numerous sub- The additions include five new antibiotics. Many changes have been made in analytical methods. committees have made a wise selection. hTORhlAN EVERS STANDARD METHODS OF CLINICAL CHEMISTRY. Volume 11. By the American Association of Clinical Chemists. Edited by DAVID SELIGSON. Pp. xii + 217. New York and London: Academic Press Inc. 1958. Price $5.50; 44s. Volume I of this series was favourably reviewed in this Journal four years ago; it was then hoped that these volumes would become as important for the clinical chemist as “Organic Syn- theses,” “Biochemical Preparations” and “Official and Tentative Methods of Analysis” are for chemists in other fields.As in the previous volume, each is prefaced by a description of its scientific basis and is followed by an account of special difficulties that may arise, as well as of practical applications. Each technique is docu- mented by a valuable series of references. Although methods for the estimation of calcium, cholesterol, phosphatase and lipase were included previously, new methods are now presented either on account of greater accuracy or convenience. This implements the intention of the publishers to produce a series of small volumes, which would permit revision of new methods from time to time without the delay necessitated by a new edition. Thus serum calcium was originally estimated by the usual permanganate titration, but, in \*ohme 11, a compleximetric titration has been described; chloride in Volume I was estimated by a mercurimetric titration-in the new volume there is a very complete description of the titration with silver nitrate, dichlorofluorescein being used as an adsorption indicator.The methods for phosphatase and lipase have been improved, the latter in respect of a better substrate of greater stability, the former in respect of the final colour It would now appear that this hope is being fulfilled. Twenty-one techniques are described in detail in the present volume.544 BOOK REVIEWS reaction in order that Beer’s law may more rigorously be obeyed. In the previous volume, estima- tion of sodium and potassium by flame photometry was discussed in particular relation to the expensive American Berkeley instrument, not readily available here.The chapter on the same subject in the present volume is presented in a much more general way and provides a really useful account of the subject, without reference to any particular make of instrument. There is a useful account of the measurement of the blood pH. Wew methods described are Van der Kamer’s estimation of total fatty acids in the stools, Friedman’s fractionation estimation of gamma globulin in serum, the o-phenanthroline estimation of iron in serum. The method for estimating free a d conjugated 17-hydroxycorticosteroids in plasma is a modification of the original one for free steroids first described by Nelson and Samuels. The oxyhaemoglobin, cyanmethaemoglobin and total iron methods for estimating haemoglobin are given with full practical details.It is surprising that the compilers have included the cephalin - cholesterol flocculation test as a liver function test, since, owing to the difficulties encountered in preparing a suitable preparation of reproducible sensitivity, the necessary reagent needs to be purchased from a commercial source. One would have thought a test such as Maclagan’s thymol turbidity test would have been preferred. The reviewer regrets that aqueous alkali is recom- mended in the method of estimating 17-oxosteroids in urine by the Zimmerman reaction. The experience of most people in this country is that the specificity is decreased by the use of aqueous rather than the ethanolic reagent. Other methods described include nitrogen by the Kjeldahl method, non-protein nitrogen by Nesslerisation and phosphatides in plasma. A fluorimetric method is given for porphyrins in urine, but the more general availability of good spectrophotometers rather than fluorimeters would have suggested that, in a work such as this, a spectrophotometric method would have been of more use. Protein-bound iodine in serum is measured by a modification of the Chaney method, and urobilinogen in urine and faeces by a modification of that originally described by Watson, With the exception of those described in the chapters on pH measurement and flame photometry, all these methods have been submitted to checking by an independent chemist, and their efficacy is thus assured. Volume I1 of Standard Methods of Clinical Chemistry, like its predecessor, will undoubtedly be a well thumbed reference book for use on the laboratory bench. Even those workers to whom many of these methods are well known will find some aspect of technique with which they may not be familiar. C. H. GRAY

ISSN:0003-2654    

DOI:10.1039/AN9588300541

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年代:1958

数据来源: RSC

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544 BOOK REVIEWS Publications Received GENERAL BIOCHEMISTRY. By JOSEPH S. FRUTON and SOFIA SIMMONDS. Second Edition. Pp. xii + 1077. New York: John Wiley & Sons Inc.; London: Chapman & Hall Ltd. 1958. Price $18.00; 144s. By FRED BASOLO and RALPH G. PEARSON. New York: John Wiley & Sons Inc.; London: Chapman & Hall Ltd. 1958. Price $11.75; 94s. By F. FEIGL, Eng., DSc. Translated by R. E. OESPER, Ph.D. Fifth Edition. Pp. xiv + 600. Amsterdam: Elsevier Publishing Co.; London: Cleaver-Hume Press Ltd.; New York and Toronto: D. Van Nostrand Co. Inc. 1958. Price 65s. ANNUAL REPORT 1957-8. Pp. 272. London: British Standards Institution. 1958. Price 7s. 6d. INDUSTRIAL HYGIENE AND TOXICOLOGY. Volume I . GENERAL PRINCIPLES. Edited by FRANK A. PATTY. Second Edition. Pp. xxviii + 830. New York and London: Interscience Publishers Inc. 1958. Price $17.50; 132s. MECHANISM OF INORGANIC REACTIONS: A STUDY OF METAL COMPLEXES IN SOLUTION. Pp. xiv + 426. SPOT TESTS IN INORGANIC ANALYSIS.

ISSN:0003-2654    

DOI:10.1039/AN9588300544

出版商:RSC    

年代:1958

数据来源: RSC