摘要:

Nov., 19501 RIBOFLAVINE IN YEAST AND YEAST PRODUCTS 613 A 24-Hour Plate Assay Technique for the Vitamin B6 Complex of Yeast BY A. JONES AND S. MORRIS (Read at the meeting of the Biological Methods Grot@ on Tuesday, December 13th, 1949) SYNOPSIS-A plate assay technique for the vitamin B, complex has been developed using Saccharomyces carlsbergensis. For the complete u tilisation of pyridoxamine in the plate assay, tryptophan appears to be an essential metabolite for the organism. Indications are also given that a fourth, hitherto unknown, factor of the B, complex may be present in certain yeasts. IN a recent publication1 it was shown that, by certain modifications of the original method of Stokes, Larson, Woodward and Foster2 in which Neurospora sitophila was used, a satis- factory degree of accuracy could be obtained for the assay of the vitamin B6 complex in yeast and certain other foodstuffs.Unfortunately, the incubation period for the mould is 5 days, which entails considerable delay in obtaining the assay results. The turbidimetric method of Atkin, Schultz, Williams and F r e ~ , ~ using Snccharomyces carlsbergensis, has the advantage of a short incubation period of 24 hours, but requires highly specialised apparatus. For these reasons, the possibility of obtaining a plate-assay technique, using S. carlsbergensis, was investigated. The plate assay has been used for other vitamins of the B compIex4J~6~7 but, so far as is known, no attempt has been made to assay the vitamin B, complex by this method. Throughout this study parallel assays were carried out, the mould technique being used for control puxposes. In one case, with the assistance of Dr.E. R. Dawson of Distillers614 Company, Ltd., Great Burgh, Epsom, a comparative assay was carried out using S. cads- bergensis and the technique in which the yeast is hydrolysed in 0.055 N sulphuric acid. Throughout the present paper, the term vitamin B, is used to include the three known constituents pyridoxine, pyridoxal and pyridoxamine, and the assay values are stated as pg. of vitamin B, per g. or as pg. of pyridoxine, pyridoxal or pyridoxamine, not as their hydrochlorides. JONES AND MORRIS: A 24-HOLJR PLATE ASSAY TECHNIQUE [vol. 75 METHOD OF ASSAY BASAL MEDIUM- The basal medium (Table I) is essentially that used by Atkin et aZ.3 with the addition of nicotinic acids and tryptophan.Although the inclusion of 2.25pg. each of nicotinic acid and calcium pantothenate per ml. has been recommended for the assay of vitamin B, using S. carlsbergensis, for the plate-assay technique 1000 pg. per 900 ml. of medium was found to be adequate. The medium, with the addition of 2 per cent. of agar was filled into tubes in amounts of 20 ml., steamed for 20 minutes and stored a t laboratory temperature until required. STOCK CULTURE AND PREPARATION OF THE INOCULUM- The growth medium was 4 per cent. malt - agar at a pH of 5 to 5.5, and incubation took place for 18 to 24 hours at 28" C. The organism, S. carZsbergepzsis 4228, was grown in 1-ounce screw-capped bottles. Sub-culturing was carried out every 14 days.TABLE I BASAL MEDIUM Glucose .. . . .. .. .. .. Potassium di-hydrogen phosphate . . .. * . Potassium citrate . . .. .. .. .. Citric acid . . .. .. .. .. .. Casein hydrolysate . . .. .. .. .. Nicotinic acid .. .. .. .. .. hneurine hydrochloride . . * . . . .. Calcium pantothenate . , .. . " .. Biotin . . .. .. .. .. .. .. Inositol .. .. .. .. .. .. Salt solution*. . . . * . .. .. .. DL-Tryptophan . . .. . . .. .. Volume made up to . . .. pH adjusted to . . .. .. Agar (0x0) . . .. .. .. .. .. 50 g. 0.55 g. 5.0 g. 1.0 g. 1000 pg. 1000 pg. 4.0 g. 250 pg. 8 0-025 g. 25 ml. 0.1 g. 900 ml. 18 g. 4.6 * The salt solution contains 1.70 g. of KCI, 0.5 g. of CaCl,.GH,O, 0.5 g. of MgSO4.7H,O, 0.01 g. of To prepare the inoculum, the growth of yeast from the surface of a malt - agar slope was washed off with 10 ml.of sterile saline. The suspension was centrifuged and the yeast washed once with sterile saline and finally suspended in 10 ml. of saline for use as inoculum. With increasing age of the sub-culture, the zone of growth increased in size and diminished in clarity. FeCl,.GH,O and 0.01 g. of MnS0,.4H20 per 100 ml. Sub-cultures older than 14 days were never used for preparation of the inoculum. PREPARATION OF THE PETRI PLATES- The Petri plates were prepared in a manner similar to that used for the assay of ane~rine.~ One millilitre of inoculum was used for each 20 ml. of medium. Preliminary drying of the plates and incubation with the lids raised, essential for the assay of aneurine, were found to be unnecessary for the assay of vitamin B,.The contents were melted by immersing the tubes in a boiling water-bath. The tubes were cooled to a temperature of 48" to 50" C. and maintained at that temperature, and 1 ml. of inoculum was added to each tube. Immediately after addition of the inoculum, the contents of each tube were thoroughly mixed by rotation to distribute the bacterial suspension evenly throughout the medium. The contents of the tubes were poured severally into sterile Petri Five tubes of medium were used for the standard and for each test sample.Nov., 19501 FOR THE VITAMIN B, COMPLEX OF YEAST 615 dishes, 9 cm. in diameter, on a flat, even surface and allowed to set. Five holes were cut in each plate, each hole 10mm. in diameter, the agar discs removed and the holes sealed with the addition of a drop of melted medium. PREPARATION OF THE YEAST SAMPLES- Two grams of dried yeast and 0.3 g.of takadiastase were added to 20 ml. of 1 per cent. sodium acetate buffer solution a t pH 4.5. When necessary, the pH of the mixture was adjusted to 4.5, then 2 drops of benzene were added and the whole was incubated at 37" C. for 18 hours. After incubation, the mixture was steamed for 10 minutes, cooled, diluted to 25 ml. and filtered. The filtrate was diluted 1 to 2, 1 to 4, 1 to 8 and 1 to 16 with glass-distilled water and 0.1 ml. of each dilution was put into the appropriate hole of the Petri plates. The Petri plates were finally incubated at 28" C. for 18 hours. For the preparation of the standard curve, the assay range was 0.125, 0.25, 0.50, 1.0 and 2.Opg.of pyridoxine, pyridoxal or pyridoxamine per ml. The mean values of the diameters in millimetres of the zones of growth were plotted against the logarithms of the concentrations of the standard pyridoxine, pyridoxal or pyridoxamine. Under the test conditions, the effect of doubling the concentration of pyridoxine, pyridoxal or pyridoxamine was to increase the zone diameters by 2.5 mm. Since the activities of the three members of the vitamin B, complex were equal for S. carlsbergensis on this medium, pyridoxine was mostly used as a standard. A series of different brewers' yeasts has been assayed by this method and, for comparative purposes, by the Neurospora technique. The results are shown in Table 11, and a statistical evaluation of the results is given in Table 111.TABLE I1 COMPARISON O F VITAMIN B, CONTENT O F YEASTS ASSAYED BY THE PLATE AND Neurospora METHODS Vitamin B, Yeast A B C D E Y 449 YEAST 449. Neurospora method A r I Mean P?5 Per g. P.g* 18. 21.7, 22.5, 21.7, 23-0, 21.5, 21-4, 21-31 22.6, 20.8, 20.3, 20.2, 18.7 20-1, 20.5, 21.2, 20.9, 18.2 20.18 18.8, 16.1, 17.0, 18.5, 17.6, 18-6, 20.76 23.5, 23.1, 23.0, 23.6, 24.1, 22.7, 23.3 17.4, 18.4, 16.9, 16.9, 17.3, 14.9, 18.35 18.6, 17.1, 19.4, 18.0, 20.8, 21-0, 20.0, 20-5 17.0, 17.7, 20.1, 18.0, 17-9, 18-3, 18-24 18.3, 17.7, 18.3, 19.1 19.8, 20.4, 20.8, 20.3, 21.3, 17-7, 19-15 19.1, 20.3, 19-5, 20.3, 18.1, 18.2, 19.1, 18.2, 17.1, 16.2 17.7, 18.7, 16.9, 16-5, 17.2. 17.0, 18.33 19.8, 18.3, 19.1, 19.6, 18.1, 18.7, 19.7, 19.2, 18.5 Assay carried out with S.carlsbergensis 4228- Plate method A r P!z* Per g. 29.2, 31.2, 29*4,26*0, 29.4 18-8, 20*6,22.1, 18.0, 21.7, 21.4 24.4, 24.6, 24.5, 25.7, 24.0, 24-6, 25.4 23*6,21*7, 21.2, 21.2 23-6,22.2,20.2, 204, 22.3 22.8, 23.8, 24.0, 24.4, 20.5, 20.5, 21.7,20.5 28.8, 29.9, 29.8, 30.1, 29.8, 29.8, 29.8 1 7 Mean 29.04 20.43 24.74 Pg-lg. 21.92 21.74 22.27 29-71 100 mg. of dried yeast were treated with 25 ml. of 0.055 N sulphuric acid for 5 hours at 20 lb. pressure. The amounts of vitamin B, found were 33, 29 and 34 (mean 32) pg. per g. THE EFFECT O F THE TRYPTOPHAN CONTENT OF THE BASAL MEDIUM ON THE ASSAY OF VITAMIN Be- The effect of the tryptophan content of the basal medium was examined by preparing two media, one with and the other without it. Otherwise the test was carried out as above.Dose - response curves were obtained for pyridoxine, pyridoxal and pyridoxamine. Whereas the curves for the two former were identical] irrespective of the medium, those for616 JONES AND MORRIS: A 24-HOUR PLATE ASSAY TECHNIQUE [Vol. 75 TABLE I11 STATISTICAL EVALUATION OF THE RESULTS SHOWN IN TABLE I1 No. of Mean value Coefficient Yeast Method Samples 4 standard error of Variation A Neurospora 11 21.31 f 0.377 6.9 Plate 5 29.04 f 0.842 6.5 B Neurospora 5 20.18 f 0.528 5.9 Plate 6 20.43 & 0.681 8.2 *C Neurospora 13 (20-76 f 0-828)* (14-4)* Plate 7 24.74 f 0.225 2.4 D Neurospora 14 18.36 f 0.469 9.6 Plate 4 21.92 & 0.571 6-2 E Neurospora 10 18-24 & 0-269 4.7 Y Neurospora 16 19-15 f 0-361 7.5 Plate 6 21-74 0.638 6.6 Plate 8 22-27 f 0.596 7.6 449 Neurospma 15 18-33 -& 0.277 6.9 Plate 7 29-71 & 0.158 1-4 * The results of the Neurospora assay on yeast C clearly fall into two groups. The second group of seven have a mean value of 23-33 with a standard error of 50.173.The first six samples have a range of 16.1 to 18.8, the second seven from 22.7 to 23.6. It is not permissible to treat all thirteen as a homogeneous group. (C.V. = 2.0.) Like the values for all other samples of yeast except 13, this is significantly lower than that given by the plate assay. For sample 449 the plate assay gave a mean value more than 60 per cent. higher than the Neurospora method. pyridoxamine varied markedly according to the medium. In the absence of tryptophan, the responses for pyridoxamine were always 50 to 60 per cent.lower than those for pyridoxine and pyridoxal. On the other hand, the activity of the pyridoxamine was constant through- out the assay range in terms of pyridoxine and pyridoxal. Further, the zones of growth obtained with pyridoxamine and with yeast samples on a tryptophan-deficient medium were not as sharp and clearly defined as those from pyridoxine and pyridoxal. With the medium containing 0.1 g. of DLAryptophan per 900 ml., the dose - response curve for pyridoxamine was identical with those for pyridoxine and pyridoxal (Fig. 1). The clarity and ease of measurement of the growth zones with pyridoxamine and yeast was improved by using the tryptophan medium. Several attempts were made to raise the lower response with pyridoxamine on the tryptophan-deficient medium, various additions or substitutions being made to the basal medium.TABLES IV COMPARISON OF RESULTS OBTAINED BY SUBSTITUTING OTHER AMINO ACIDS FOR TRYPTOPHAN IN THIS BASAL MEDIUM Tryptophan . Asparagine . . Histidine . . Arginine . . Lysine Phen ylalanine Valine Serine Aspartic acid Glutamic acid Tyrosine . . Cystine , . * . .. .. . . .. .. .. .. .. .. .. .. 7 7 0.125 24-3 23.4 24.7 23.2 24.0 26.2 26.7 25.5 27.0 25.6 26.5 26.2 Zone diameters in mm. A Pyridoxine, pg. / i d . Pyridoxamine, pg. /ml. A v r A 0.25 0.50 1.0 2.0 0.125 0.25 0-50 1.0 26.9 29.4 31.9 34.4 24.8 27.3 29.8 32.5 26.4 29-1 31.9 34.4 20.8 24.5 26.1 28.6 27.4 29.7 32-3 34.7 21.4 24.1 26.8 29.4 25.7 28.2 30.7 35.2 20.9 23.2 26.0 28-4 26.5 29.0 31.5 34.0 20.6 23.7 26.4 28.9 29.0 31.3 33.8 36.2 24.1 26.5 20.3 31.7 29.4 31.6 34.4 37.0 24.2 27.1 29.7 32.2 28.5 31.2 33.7 35.9 22.5 25-2 28.1 30.6 30.1 32.2 34.4 36.9 23.7 26.5 29-1 31.8 28.2 30.9 33.5 36.0 22.8 25.6 28-2 30.6 29.0 32.1 34.5 36.6 23.3 25.9 28.4 30.9 29.0 31.9 34.1 36.8 23.3 26.4 28.8 31.3 1 7 2.0 34-9 31-1 31.9 30-9 31.4 34.7 34-8 33-3 34.1 33.6 334 33.8Nov., 19501 FOR THE VITAMIN B, COMPLEX OF YEAST 617 In Table IV are shown the results obtained by substituting other amino acids for trypto- phan in the basal medium.In some experiments, the zone diameters obtained both with pyridoxine and with pyridoxamine were greater than those with the tryptophan medium. But in none was the zone diameter the same for pyridoxine and pyridoxamine; that for pyridoxine was always the greater.34 3; 3c 21 € E it 2, w k w I a 24 W Z 2; R 2c PYRIDOXINE OR PYRIDOXAMINE,yg per mi. Fig. 1. Dose response curve for pyridoxine and pyridoxamine on basal media with and without tryptophan 0 = Pyridoxine on basal medium with or without tryptophan X = Pyridoxamine on basal medium without tryptophan A = Pyridoxamine on basal medium with tryptophan With certain amino acids, rather irregular increases in zone diameters with increasing levels of pyridoxine and pyridoxamine were found. An enzymatically-prepared casein digest wits substituted for the acid-hydrolysed casein, the nicotinic acid and calcium pantothenate contents of the medium were increased to 3.5 pg. per ml., five different samples of agar were used and finally the medium was used at double strength. On no occasion was the pyridoxamine response curve identical with those for the other vitamin B, members, and assays of yeast gave low values.In this connection, it is interesting to note that Rabinowitz and Snell,lo using the original yeast assay technique, found that the activity of pyridoxamine, in terms of pyridoxal or pyridoxine, was not constant over the assay range used. In these experiments the assay range was low, being 0.002 to 0-2 pg. METHODS OF EXTRACTING VITAMIN B, FROM YEAST- In a series of experiments investigating methods for the extraction of vitamin B, from yeast for assay purposes, Rabinowitz and Snellll found that the most effective method was to autoclave the yeast at 20 lb. pressure for 5 hours with 0-055 N hydrochloric acid. When 2 N acid was substituted for 0.055N, lower results were obtained and could be increased by a second digestion with 0-055 N acid.Digestion with clarase also gave maximum extraction of vitamin B,.618 For the plate-assay technique, the minimum necessary amount of vitamin B, is much greater than for the method of Atkin, Schultz,, Williams and Frey. For this reason the extraction of yeast samples containing 2 pg. of vitamin B, in 180 ml. of 0.055 N acid, as advocated by Rabinowitz and Snell, was not feasible. It was necessary to investigate other extraction methods. The results are given in Table V. JONES AND MORRIS: A 24-HOu~: PLATE ASSAY TECHNIQUE [Vol. 75 METHODS I I Yeast449 . . .. I I I I TABLE V OF EXTRACTION OF VITAMIN B, FROM YEAST Extraction method 2 N HCI autocIaved for 5 hours a t 20 Ib.3 N HC1 autoclaved for 5 hours a t 20 lb. 4 N HCI autoclaved for 5 hours a t 20 lb. 0.3 g. of takadiastase 2 N HC1 autoclaved for 5 hours at 20 lb. Takadiastase- 0.05 g. 0.10 g. 0.20 g. 0.15 g. 0.30 g. 0.50 6. Extraction rate for A c 7 Medium without Medium containing tryptophan, tryptophan, CLg. Per g* 20.3, 20.1, 23.3 23.0, 24.7, 23.9, 23.9, 22.9, 26.8, 28-2, 26.6, 28.0 CLg. Per 6- 18.8 19.2 - 28-8, 29.9, 29-8, 30-1, 29.8, 29.8, 29.8 18.9 19-7, 20.1, 25.2, 22.0, 22.8 6.8 17-9, 20.1 10.2 20.9 12.0 24.0 24.4 12.6, 14.9, 15.7, 24.4, 23.8, 20.5, 15-1, 16.8 20.5, 21.7, 20.5, 24.4, 24.0 24-4 By using takadiastase, higher values were obtained than by autoclaving the yeast for 5 hours with 2 N acid. Although acid treatment yielded values as high as those obtained with takadiastase in some assays, this did not occur regularly.Different samples of takadiastase appeared to vary in their ability to release vitamin B, from yeast. For all samples tested, however, 0.3 g. of the enzyme was found to be adequate. The vitamin content of the takadiastase was always less than 0.2 pg. per g., it being impossible to assay this amount by either the plate or the mould technique. It appeared possible that the lower values recorded after hydrolysis with 2 N acid were due to the excessive amount of sodium chloride present after neutralisation of the acid. The possible inhibitory effect of salt on S. carlslwrgensis was studied, using a standard solution of pyridoxine and a takadiastase digest of yeast. Sodium chloride was added to these solutions to give final concentrations of 2.5, 5 and 10 per cent.On assay there were noted no differences in zone diameters from those obtained with the normal standard or yeast to which salt had not been added. It would appear that concentrations of sodium chloride up to 10 per cent. have no inhibitory action on the growth and activity of S. cadsbergensis on the solid medium. THE EFFECT OF THE AGE OF THE CULTURE OF S. cadsbergensis- In the assay of vitamin B, using Neurosfiora sito?hila,l the mould was found to retain its activity up to at least 5 months. On the other hand, Streptococcus faecalis cultures, after sub-culturing on certain media, partly failed to respond to folic acid.g For this reason it was decided to study the effect of the age of the S.carlsbergensis culture on the assay of vitamin B,. Cultures on malt - agar were tested over a period of 5 months.Nov., 19501 FOR THE VITAMIN B, COMPLEX OF YEAST 619 From the results given in Table VI it will be seen that, with cultures older than 2 weeks, the growth zones became progressively larger and less distinct. With cultures older than 2 months, no growth zones were obtained. TABLE VI EFFECT OF AGE OF CULTURE USED IN ASSAY Zone diameters at concentrations of pyridoxine, pg./ml., of I - mm. mm. mm. mm. mm. Age of culture 0.125, 0.26, 0.50, 1.0, 2.0, 1 week . . . . . . 24.5 27.0 29.5 32.0 2 weeks . . . . . . 24.5 27.0 29.6 32.0 :::!} Sharp 1 month . . . . 27.2 29.8 32.1 34.6 37-1 Zones 2 months . . . . 25.0 29.0 36.0 39.0 44.0 Zones 3 months Pmonths .. .. growth around the holes . . . . } No zones of growth-only slight diffuse Remarks zones not so sharp diffuse and irregular THE USE OF TAKADIASTASE FOR THE EXTRACTION OF VITAMIN B, IN THE ASSAY OF VITAMIN B, BY USE OF Neurospora sitophila- It will be seen from Table I11 that, with the majority of yeasts, the mould method gave lower results than those found with S. carlsbergensis. Since takadiastase appeared to release maximum amounts of vitamin B, with the yeast-assay technique, it was decided to investigate the effect of this treatment on the results with Neurospora sitophila. In the latter method it was essential to destroy all the aneurine before assaying vitamin B,. Three yeasts were treated for 18 hours with takadiastase and then sodium hydroxide was added to the mixture to give a final concentration of 4 per cent.The alkaline solution was autoclaved a t 151b. pressure. The results are given in Table VII. TABLE VII COMPARISON OF VITAMIN B, ASSAY VALUES BY USE OF Neurospora OR s. carlsbergensis Assay and treatment , Yeast A . . .. . . Yeast C . . . . .. Yeast 449 . . . . .. Newospora ; Neurospoya ; takadiastase NaOH only. followed by NaOH. Vitamin B,, Vitamin B,, r*g* Per g - Pg- per g. 21.3 21.8 20.8 22.5 18.3 23.9 1 S carlsbergeizsis (plate assay) ; takadiastase. Vitamin B,, Pg- Per g- 29.0 24.7 29.7 Although treatment with takadiastase appears to increase the amount of vitamin B, found, the increase is very small and the results obtained were still below those found with S. carlsbergensis. It was possible that the treatment with sodium hydroxide had destroyed part of the vitamin B,, although it is known1?l2 that pyridoxine, pyridoxal and pyridoxamine and their phosphates are unaffected by this treatment.Further evidence to support the view that alkaline hydrolysis may destroy a fraction of the vitamin comes from the results obtained after hydrolysis of yeast with sodium hydroxide followed by acid hydrolysis, or vice versa. There was no increase in the amount of vitamin B, found by these treatments. To confirm this view, the effect on two yeasts of alkaline hydrolysis following treatment with takadiastase was observed, with S. carlsbergensis as the test organism. From the results, given in Table VIII, it would seem either that the Neurospora assay measures an entity that does not affect S.carlsbergensis in the plate assay, and is not destroyed by sodium hydroxide, TABLE VIII COMPARISON O F VITAMIN B, CONTENT OF YEASTS BY THE PLATE METHOD BEFORE AND AFTER TREATMENT WITH SODIUM HYDROXIDE Takadiastase Takadiastase treatment alone followed by N NaOH Yeast 449 .. . . .. 29.7 16.6, 10.0 Yeast C . . .. .. 24.7 16-0620 or, more probably, that an inhibiting agent for 5. carlsbergensis is formed during treatment with sodium hydroxide. In the NeurosPora assay, the aneurine must be removed without destroying part of the vitamin B, group, and in the plate assay, the higher level of testing limits the use of efficient extraction methods. In a preliminary series of experiments, using a casein solution treated with sodium hydroxide as diluent for pyridoxine, pyridoxal and pyridoxamine at a high level (0.4 g.of treated casein per flask) in the Neurospora assay, growth appeared to be restricted-especially so with pyridoxamine, less so with pyridoxal, and least of all with pyridoxine. However, when the casein solution was added in smaller amounts corresponding to the protein level of yeast present in an assay, no effect upon the growth of Neurospora was found. But the same high levels added in the S. carlsbergensis plate assay appeared to have no effect. JONES AND MORRIS: A %&HOUR PLATE ASSAY TECHNIQUE [Vol. 75 So far, it has been difficult to show which is correct. CONCLUSIONS FROM RESULTS From the results of the plate-assay technique with S. carlsbergensis, it appears that a satisfactory method has been devised, having a precision closely approaching that of the original yeast method of Atkin and his associates.The advantages of the plate method are that no specialised apparatus is necessary and that measurements of growth zones are more easily made and are as accurate as by turbidity measurements. It has the advantage over the Neurospora method of being much more rapid. That the results obtained with the plate technique are generally higher than with Neurospora sitophila is a finding discussed below. In the plate-assay method, the effect of tryptophan on the assay of pyridoxamine is most marked. Tryptophan was found to be an essential growth factor in the assay of the vitamin B, complex using Saccharomyces cereviseae.13 No distinction was made between the various members of the complex and it is probable that, had this been done, results similar to those recorded in the present paper would have been obtained.It is peculiar, however, that no such finding was noted by Snell and the various w0rkers~@~~4 who have used the original yeast method. In this connection the possibility exists that, owing to the very low assay level of the original method, sufficient tryptophan was present in the basal medium for assay purposes. On the other hand, Snell found that the pyridoxamine and pyridoxal growth - response curves were dissimilar, and this dissimilarity may be the expression, at the low level of assay, of a tryptophan deficiency. In the present work, the effect of tryptophan appears to be highly specific in that absence of it affects only the utilisation of pyridoxamine by S.carlsbergensis. Further work is clearly essential to determine the exact role of tryptophan in the metabolism of pyridoxamine and whether the effect is specific for tryptophan. Attention must be drawn to the marked. discrepancy, which was found with some of the yeasts, between the results obtained by the mould and the yeast assay techniques. With certain yeast samples assayed, the mould, Neurospora sitophila, yielded results down to 30 per cent. lower than the yeast S. carlsbergensis. It has been shown1 that pyridoxine, pyridoxal and pyridoxamine are equally effective for the growth of Neurospora sitophila. Further, the phosphates of pyridoxal and pyridoxamine are released from yeast by treatment with N sodium hydroxide, as prior treatment of the yeast with acid, known to release the phosphates, followed by treatment with alkali, does not yield higher results.It appears possible that the difference in the results obtained with the mould and yeast is due to the presence of a fourth member in the vitamin B6 complex. It seems that certain yeasts do not contain this factor in measurable quantities. Whether it is related to the known members of the vitamin B, complex cannot yet be shown. In this connection it is interesting to note that Melnick, Hochberg, Himes and Oser15 mentioned the possible existence of a fourth factor in the B6 complex, but it was shown that the idea arose from a faulty extraction of the complex. Further work is being carried out with special reference,to the extraction of this factor and to its effect on the growth and development of vertebrates.Preliminary chromato- graphic analyses confirmed Snell’s thesis that most of the vitamin B, of yeasts was present as pyridoxamine. No trace of a possible fourth factor was noted when butanol - acetic acid was used as extractant. The authors thank the Directors of Beecharn Research Laboratories, Ltd., for permission The authors also thank Dr. E. R.. Dawson of Distillers Co., Ltd., Epsom, for his to publish.Nov., 19501 FOR THE VITAMIN B, COMPLEX OF YEAST 621 co-operation, Merck & Co., U.S.A., and Roche Products, Welwyn, for the samples of pyridoxal and pyridoxamine, and Miss A. Braddick and Miss M. Copus for assistance in the Newosfiora assays.REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. Morris, S., Herwig, G., and Jones, A., Analyst, 1919, 74, 37. Stokes, J . L., Larson, A., Woodward, C. R., and Foster, J. W., J . Biol. Chem., 1943, 150, 17. Atkin, L., Schultz, A. S., Williams, W. L., and Frey, C. N., Ind. Eng. Chem., Anal. Ed., 1943, Price, S. A., Nature, 1948, 161, 20. Jones, A., and Morris, S., Analyst, 1949, 74, 333. Genghof, D. S., Partridge, C. W. H., and Carpenter, F. H., Arch. Biochem., 1948, 17, 413. Bacharach, A. L., and Cuthbertson, W. F. J., Analyst, 1948, 73, 334. Hopkins, R. H., and Pennington, R. J., Biochem. J . , 1947, 41, 110. Jones, A., and Morris, S., Analyst, 1949, 74, 29. Rabinowitz, J. C., and Snell, E. E., J . Biol. Chem., 1948, 176, 1157. -- , J . Biol. Chew., 1947, 169, 643. Siegil, L., Melnick, D., and Oser, B. L., Ibid., 1943, 149, 361. Rubin, S. H., Scheiner, J.. and Hirschberg, E., Ibid., 1947, 167, 599. Melnick, D., Hochberg, M., Himes, H. W., and Oser, B. L., Ibid., 1945, 160, 1. 15, 141. 8 , I n d . Eng. Chem., Anal. Ed., 1947, 19, 277. -- BEECHAM RESEARCH LABORATORIES LIMITED BROCKHAM PARK, BETCHWORTH, SURREY DISCUSSION MR. J. S. HARRISON and MR. S . A. PRICE both expressed surprise a t the statement that there was no Mr. Harrison had observed losses in the Saccharomyces curls- MR. A. L. BACHARACH said that surely there must be a more elegant method for removal of aneurine. MR. S. A. PRICE wondered whether a thiaminase, e.g., from carp viscera, could be used for removal of DR. MORRIS said that vitamin B, in yeast was mainly in the form of pyridoxamine, which would be loss on treatment with sodium hydroxide. bergensis method of assay. What was the effect of nitrous acid? aneurine from extracts prepared by acid hydrolysis. destroyed by nitrous acid.

ISSN:0003-2654    

DOI:10.1039/AN9507500613

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

Nov., 19501 FOR THE VITAMIN B, COMPLEX OF YEAST 621 Picrolonates and Calcium Semi-Micro Volumetric Determination with Cetyl Pyridinium Bromide BY C. C. WASHBROOK SYNoPsIs-Picrolonates are determined by titration with cetyl pyridinium bromide solution by two partition methods, methylene blue and bromophenol blue being used as indicators. In the first method, the methylene blue - picrolonic acid complex is extracted by shaking with a mixture of nitrobenzene and trichloroethylene (1 : 4). On subsequent titration with cetyl pyridinium bromide solution the blue dye returns t o the aqueous phase when the layers are shaken together. Complete transfer indicates the end-point. In the second method the aqueous picrolonate solution is titrated with cetyl pyridinium bromide in the presence of bromophenol blue and chloroform.The formation of a blue dye complex, soluble in chloroform, indicates the end- point. The titrations are stoicheiometric and the methylene blue procedure is recommended for the determination of calcium. This determination is made by the back titration of lithium picrolonate solution after precipitation and removal of the sparingly soluble calcium picrolonate. MANY of the methods available for the micro and semi-micro volumetric estimation of calcium depend to a greater or less degree on specialised manipulative techniques. These are not readily adapt able to routine work by operators unskilled in microchemical techniques nor suitable for use in laboratories without specialised equipment. A simple method that is free from these disadvantages has been described by Bo1liger.l It is based on the back622 WASHBROOK : PICROLONATES AND CA.I,CIUM.SEMI-MICRO VOLUMETRIC [VOl. 75 titration of lithium picrolonate with methylene blue solution after precipitation of the sparingly soluble calcium picrolonate by addition of the corresponding lithium salt. The methylene blue - picrolonic acid complex is removed by extraction with chloroform, the end-point being indicated by the chloroform extracts becoming colourless. Experimental work has shown that this procedure is tedious and lengthy. A detailed investigation of the titration procedure has been made by Cohn and K ~ l t h o f f , ~ , ~ who devised suitable conditions for amperometric titration. A rapid and simple titration procedure has now been devised which is an improve- ment on the visual technique; it is based on the following considerations.The ability of certain anionic surface-active substances to form chloroform-soluble complexes with methylene blue has been demonstrated by Jones: and Auerbach5 has described complexes, soluble in chloroform, formed by the union of bromophenol blue with paraffin chain cation-active substances. Simple methods for the direct titration of dilute solutions of anion-active substances with dilute solutions of cation-active substances have been described by Epton,6 and by Barr, Oliver and Stubbings.’ These methods use partition end-points in a mixture of chloroform and aqueous solution, methylene blue or bromophenol blue being used as indicator. Thus, chloroform solutions of the methylene blue anion-active complexes can be titrated with aqueous cetyl pyridinium bromide solution with consequent liberation of the methylene blue.Similarly, aqueous solutions of anion-active substances can be titrated with aqueous cation-active solutions in the presence of bromophenol blue, the end-point being indicated by the formation of the blue cation-active substance with the bromophenol blue. Picrolonic acid and its salts form methylene blue complexes which are similarly soluble in organic solvents8 and it has been found that a chloroform solution of the complex is decom- posed by titration with an aqueous solution of cetyl pyridinium bromide. A superior solvent, however, is a mixture of nitrobenzene and trichloroethylene, which removes the dye complex in a single extraction; as this solvent separates readily from aqueous solutions, its use enables titrations to be carried out rapidly and the former tedious extraction procedure is avoided.In the first, the aqueous picrolonic acid solution is treated with an aqueous solution of methylene blue hydrochloride, and the dyestuff complex is separated by partition with the solvent. The mixture is titrated with aqueous cetyl pyridinium bromide solution anti when sufficient has been added, the dyestuff is liberated and is transfered to the aqueous layer when the two phases are shaken together. The end-point, which is observed in the solvent phase, is indicated by the complete transfer of methylene blue to the aqueous phase. Detection of the transfer of the last traces of methylene blue may be aided by the addition of a small quantity of an oil-soluble red dyestuff to the solvent phase.In the second procedure an aqueous solution of picrolonic acid is titrated with cetyl pyridinium bromide solution to form a complex which is extracted with chloroform as a yellow solution. The end-point of the titration is then determined in the manner used by Barr et al.,’ with bromophenol blue as the indicator. Thus, excess of cetyl pyridinium bromide forms a complex with the bromophenol blue and passes into the chloroform layer which then becomes green. Two alternative procedures have been devised. DETERMINATION OF PICROLONIC ACID REAGENTS- Picrolonic acid-0.002 M aqueous solution. Prepared from the “Spot Test” grade by recrystallisation from benzene containing a trace of methyl alcohol.Cetyl pyridinium bromide-0.001 M or 0.005 M aqueous solution. Prepared from Fixanol C (Imperial Chemical Industries, Ltd.) twice recrystallised from methyl-ethyl ketone. Methylena blue indicator-Methylene blue, zinc-free salt, 0.5 per cent. alcoholic solution, 6.0 ml. ; 2 N sulphuric acid, 120 ml. ; sodium sulphate, anhydrous A.R., 50 g. ; distilled water to 1 litre. Bromophenol blue indicator-Bromophenol blue, 0.1 per cent. alcoholic solution, 20 ml. ; sodium sulphate, anhydrous A.R., 50 g. ; s0diu.m carbonate, anhydrous A.R., 5-0 g. ; distilled water to 1 litre. Solvent (a)-A mixture of trichlorethylene, 75 ml., freshly distilled nitrobenzene, 20 ml., and oil-soluble red dyestuff, colour index No.258, 0.01 per cent. solution in trichlorethylene, 5.0 ml. Solvent (b)-Chloroform B.P.Nov., 19501 DETERMINATION WITH CETYL PYRIDINIUM BROMIDE METHOD A WITH METHYLENE BLUE AS THE INDICATOR- 623 Transfer a suitable volume of the picrolonic acid solution to a chemically-clean stoppered reagent bottle (all traces of any detergent used for washing must be removed); add 20 ml. of methylene blue solution, and 20 ml. of solvent. Shake the bottle vigorously with a rocking motion of the hand and then stand to allow the two phases to separate. At this stage the solvent should be a deep blue colour and the aqueous phase should be almost colourless. Titrate the mixture with cetyl pyridinium bromide solution ; shake the mixture vigorously after each addition and stand to allow the phases to separate.As the end-point is approached the solvent layer becomes pale green and the methylene blue is concentrated in the aqueous layer; the end-point is indicated by a colour change from pale grey-green to amber in the solvent phase. Carry out a preliminary titration adding the cation-active solution in 1-ml. stages, followed by a second titration in which additions of 0.1 ml. or less are made as the end-point is approached. METHOD B WITH BROMOPHENOL BLUE AS INDICATOR- Transfer the picrolonate solution to the stoppered reagent bottle, add 20 ml. of indicator solution and 20 ml. of chloroform. Titrate the mixture with cetyl pyridinium bromide solution in the manner described for use with methylene blue. The end-point is indicated by a change of colour of the chloroform phase from yellow to pale green; further additions of cation-active solution intensify the green colour. Stoicheiometry of the titrations-Experimental work has indicated that the relationship between picrolonic acid and cetyl pyridinium bromide is virtually stoicheiometric. In this investigation a purified sample of picrolonic acid was used for checking the relationship between cetyl pyridinium bromide and picrolonic acid.A solution of the picrolonic acid of exactly 0.002 M concentration was prepared and its strength was assessed by two methods, viz., (a) titration with carbonate-free sodium hydroxide using phenolphthalein indicator, (6) titration with cetyl pyridinium bromide by means of the two procedures described above. The titrations were made in replicate with 0.005 M cetyl pyridinium bromide and 0.005 M sodium hydroxide in aqueous solutions.TABLE I CONCENTRATION OF 0.002 M PICROLONIC ACID SOLUTION Titration with cetyl pyridinium bromide A r Titration with (a) Methylene blue ( b ) Bromophenol blue Sodium hydroxide Picrolonic acid titrated, ml. . . 10.0 10-0 10.0 10.0 10.0 10.0 80.0 100.0 Titre, ml. .. . . . . 4-43 4.43 4.43 4-28 4.30 4.28 27.5 34.3 Concentration titrating solution Picrolonic acid concentration x 1 0 - 3 ~ . . .. . . 4.63 4.63 5.807 x 1 0 - 3 ~ . . .. . . 2.05 2.05 2.05 1-98 1.98 1.98 1.98 1.99 Table I shows that the bromophenol blue method gives results that are strictly stoicheio- metric and the methylene blue method gives results approximately 3 per cent. higher. These findings are comparable with those of Barr et aZ.,' who found that in determinations of anionic surface-active agents the methylene blue technique gave results varying from 102 to 106 per cent.of those obtained with the bromophenol blue method. They attributed this divergence to the necessity of establishing a minimum concentration of cation-active material by partition in the solvent phase before release of methylene blue occurs. In the procedure described by Eptoq6 methylene blue is used as the indicator, and the end-point is taken when the dye concentration in each phase is equal. This technique is stated to give strictly stoicheiometric results, but it was not readily applicable to this investigation because of the differing hues of the two phases. Reference standards-In the work described by Barr et aZ.,' highly purified anion-active substances, e.g., potassium $-benzyl-o-sulphonate, were used to standardise the cetyl pyridinium bromide solution.For the present investigation the procedure described by Eptone has been used, whereby the cetyl pyridinium bromide solution is standardised against624 WASHBROOK PICROLONATES AND CALCIUM. SEMI-MICRO VOLUMETRIC [VOl. 75 potassium dichromate using sodium thiosulphate as an intermediate. The cetyl pyridinium bromide solution is treated with excess of potassium dichromate solution of a suitable con- centration and the mixture is heated to coagulate the insoluble quaternary dichromate. This is removed by filtration and the concentration of dichromate in the filtrate determined iodimetrically .The present work has shown a strictly stoicheiometric relationship between picrolonic acid and cetyl pyridinium bromide when the bromophenol blue titration procedure is used. Picrolonic acid is therefore an acceptable primary substance for the standardisation of cetyl pyridinium bromide solutions. Hence, the broinophenol blue technique forms a direct and analogous procedure for standardisation when t'he halide solution is to be used for partition titrations. DETERMINATION OF CALCIUM The titration procedures described above have been applied to the estimation of calcium with picrolonic acid. The general technique using lithium picrolonate described by Bolligerl has been used, and' the concentration of the picrolonate has been determined by titration with cetyl pyridinium bromide.Experience has shown that for this application the methylene blue procedure is to be preferred, as the approach of the end-point is clearly indicated, and if necessary the determination may be made wit'h a single titration. The higher values given by this procedure do not cause erroneous results for calcium, because the determination is based on the difference between two titrations. The interference of the alkali metals with the picrolonate procedure has been thoroughly investigated by Cohn and Kolthoff3; and the calcium solution should therefore be treated either to eliminate or reduce the concentration of alkali metal ions before the determination is made. Barium, strontium, lead, copper, thorium, iron, manganese, zinc and some organic bases form sparingly soluble picrolonates and should be absent from the test solution. METHOD REAGENT- PROCEDURE- 40 mg.of calcium per litre. Lithiam ~icrolonate--0~05 M aqueous solution. Adjust the concentration of the neutral calcium solution so that it does not exceed Add 2.00 ml. of lithium picrolonate solution to 25 ml. of calcium TABLE: I1 DETERMINATION OF KNOWN AMOUNTS OF CALCIUM BY TITRATION WITH 0.00464 kf CETYL PYRIDINIUM BROMIDE Concentration of calcium Volume of solution Calcium Calcium solution Titre x 1 0 - 3 ~ taken, recovered, titrated, difference, Error, mg. mg. ml. ml. % 1-04 1.04 1.01 5.0 2.02 - 2.9 1.04 1-02 5.0 2-04 - 2.9 0.842 0.83 0.83 5.0 1-67 0 0.83 0.82 5.0 1.64 - 1.2 0.8 1 0.81 5.0 1.63 0 0-886 0.65 0.66 5.0 1.33 + 1.5 0.65 0.65 5.0 1.30 0 0.510 0.49 0.49 10.0 1.93 0 0.49 0.48 10.0 1.88 -2.1 0.49 0.48 10.0 1-86 -2.1 0.328 0.3 1 0.31 15.0 1.86 0 0.166 0.16 0.16 15.0 0.93 0 solution, stopper the flask and allow to stand overnight in a refrigerator at 35" F.Immediately after removal from the refrigerator, filter the solution through a No. 3 sintered-glass filter- stick. Allow the filtrate to return to room ternperature and remove an aliquot portion for titration by the methylene blue technique previously described. Simultaneously carry out a blank determination in which 25 ml. of distilled water are used instead of the calciumNov., 19501 DETERMINATION WITH CETYL PYRIDINIUM BROMIDE 626 solution. The difference between the blank and test titres is equivalent to the amount of calcium present.The determination of known quantities of calcium was made by taking aliquot portions of a standard solution of calcium prepared by dissolving A.R. calcium carbonate in dilute acetic acid. The new technique was compared with the original Bolligerl titration procedure. TABLE I11 COMPARISON OF THE CETYL PYRIDINIUM BROMIDE TITRATION WITH BOLLIGER’S METHOD Calcium taken, mg. 1.00 1.00 0.63 0.63 0.80 0.80 0.49 0-16 C.P.B. method Titration with C.P.B. Calcium recovered, *g. 0.99 1-00 0.63 0.63 0.81 0.80 0.49 0.16 Bolliger’s method Titration with methylene blue Calcium recovered, mg- 1.01 0.99 0.64 0.63 0.80 0.79 0.49 0.16 The method has been applied to the determination of calcium in used lubricating oil. The oil is ashed and the ash is suitably treated to remove interfering metals using the A.S.T.M.proced~re.~ An alternative method involves the precipitation of lead and barium as sulphates followed by extraction of zinc, copper and iron as thiocyanates in amyl alcohol. This alterna- tive method is to be described separately at a later date. The conditions that usually apply necessitate the determination of amounts of calcium varying between 0.2 and 1-mg., and the method has been devised for this range. Typical values obtained and the effect of addition of specific quantities of calcium are shown in Table IV. TABLE IV DETERMINATION OF CALCIUM IN USED LUBRICATING OIL Sample KO. Oil taken, Calcium added, 1 5.00 nil g. mg- - 0.20 - 0.40 - 0.30 - 0.50 - 0.10 - 0.050 - - 2 5-00 nil - - 3 5-00 nil - - Calcium found, Calcium found, mg.Yo 0.32 0.0064 0.51 - 0.73 - 0.54 0.01 1 0.84 - 1.02 - 0.93 0.019 1.02 - 0.99 - The errors involved are of the same order as those observed by Cohn and Kolthoff2 and by Bolliger,l who recorded a maximum error of 3 per cent. CONCLUSIONS Picrolonic acid forms a complex with a paraffin chain cation-active substance, cetyl pyridinium bromide, that is soluble in both chloroform and a mixture of nitrobenzene and trichlore thylene. Solutions of picrolonic acid and picrolonates can be directly titrated with cetyl pyridinium bromide solutions, with methylene blue or bromophenol blue as indicators in partition end- points. The bromophenol blue titration procedure gives strictly stoicheiometric results, but the methylene blue method gives results approximately 3 per cent. too high. The methylene blue method is conveniently applied to the volumetric semi-micro determination of calcium with lithium picrolonate. It is preferable to the bromophenol blue technique for this purpose, the approach of the end-point being more easily observed. This procedure is more convenient than the older extraction method.626 NOTES [Vol. 75 Picrolonic acid is a convenient primary substance for standardisation of cetyl pyridinium bromide solution when the bromophenol blue titration procedure is used. Acknowledgment is made to the Chief Scientist of the Ministry of Supply for permission to publish this communication. 1. 2. 3. 4. 5. 6. 7. 8. 9. REFERENCES Bolliger, A., Austral. J . Exp. Biol., 1935, 13, 75. Cohn, G., and Kolthoff, I. M., J . Biol. Chem., 1943, 148, 711. -- , Ibid., 1943, 147, 105. Jane's, H. H., J . Ass. On. Agric. Chem., 1945, 28, 398. Auerbach, M. E., I n d . Eng. Chem., Anal. Ed., 1943, 15, 492. Epton, S. R., Trans. Faraday SOC., 1948, 44, 226. Barr, T., Oliver, J., and Stubbings, W. V., J . SOC. Chewz. Ind., 1948, 67, 45. Bolliger, A., Analyst, 1939, 64, 416. American Society for Testing Materials, Standards on Petroleum Products and Lubricants, Method D, 811-441.. MXNISTRY OF SUPPLY CHEMICAL INSPECTORATE Febmayy, 1960

ISSN:0003-2654    

DOI:10.1039/AN9507500621

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

626 NOTES [Vol. 75 Notes PREPARATION OF 9-NITROBENZYL DEIRIVATIVES OF BARBITURIC ACIDS ON A SEMI-MICRO SCALE BARBITURIC acids extracted from viscera during toxicological examinations are often exceedingly difficult to identify, because (a) they will not crystallise readily, e.g., 5-allyl-5-secamyl barbituric acid (seconal), and (b) they are not sufficiently pure to give good melting-points. Very often neither repeated vacuum sublimation, nor charcoal. purification will give adequate purity. Then one course is the preparation and subsequent identification of some derivative from the barbituric acid. The p-nitrobenzyl derivatives seem best for this purpose as their melting-points, in general, are well spaced in the range 100" to 250' C. - -Dro p-i n -B19 Joint Condenser Bulb sf 30 ml.capacity However, the literature1s2 describes their preparation on a large scale. About 0.5 g. is taken and the derivative isolated by washing with water, to free from inorganic material, followed by alcohol, to free from surplus P-nitrobenzyl chloride or bromide. Owing to the solubility of the derivatives themselves in alcohol, the starting quantity cannot in general be reduced below 0.25 g. Unfortunately, quantities as large as this are rarely found during toxicological examinations. Improved yield of derivative has been found to result by removal of the surplus p-nitrobenzyl chloride or bromide by vacuum sublimation followed by leaching of the derivative from the residual inorganic material. The apparatus shown is useful for performing nearly all the steps in one vessel, thus avoiding losses due to transference.Procedure-Heat the sodium salt of the acid with an alcoholic solution of p-nitrobenzyl chloride or bromide under reflux for 1 hour, with the condenser in and the side tube open. Then, with the condenser out and the side tube still open, dry the resulting mixture. Replace the condenser, connect the side tube to the vaciium line and carry out complete vacuum sublimation at 100" C. of the p-nitrobenzyl chloride or bromide. Leach out the residue with hot dry chloroform, filter the chloroform solution, and evaporate the filtrate to dryness. Recrystallise the derivative from a suitable solvent such as alcohol or an alcohol - benzene or alcohol - light petroleum mixture.Nov., 19501 NOTES 627 This method has been found to yield adequate amounts for identification with starting quantities of about 10mg.The writer is indebted to Mr. E. B. Parkes, Director of this laboratory, for permission to publish. REFERENCES Allport, N. L., “Colorimetric Analysis,” Chapman & Hall Ltd., London, 1947, p. 189. “Organic Reagents for Organic Analysis,” Hopkin & Williams Ltd., London, 1944, p. 68. SOUTH-WESTERN FORENSIC SCIENCE LABORATORY B. LILLIMAN 1. 2. BRISTOL April, 1950 ESTIMATION OF BROMIDES BY AN ACTIVATION - EXCHANGE METHOD IN the method of activation analysis,l the sample is bombarded by suitable particles and one or more of the original elements are identified or determined by the radiation characteristics of the unstable nuclides to which they give rise. The analysis of mixtures may be very difficult because of the complex nature of the decay schemes involved; their analysis requires many observations over a long period of time. This difficulty may be partly met by the addition of a carrier followed by separation and specific activity measurements of the radio-nuclide being determined. This separation may be tedious and unreliable on account of such dangers as co-precipitation of con- taminating radio-activities out of all proportion to the chemical contamination.If, however, the radio-nuclide A* to be determined, e g . , radio-active bromide produced by the neutron activation of natural bromide in the Pile, will exchange isotopically with an added compound RA, e.g., methyl bromide, which is a gas a t room temperature and is therefore easily separated, then these difficulties are obviated.The exchange may be represented- A* + RA $ RA* + A (Unknown weight (known weight to be determined) added) The asterisk denotes radio-active material. The experimental procedure for bromide in a mixture of salts is then as follows. Procedure-Irradiate a known weight of the mixture (e.g., 100mg.) in the Pile at a known flux for a given time. On removal from the Pile, transfer the sample to a flask fitted with a head and vacuum tap, together with an ampoule containing a known weight of pure methyl bromide (e.g., 500 mg.) and 1 ml. of aqueous acetone, which serves as a common solvent. Partly evacuate the flask, close the tap, break the ampoule and heat the mixture at 100” C. to facilitate exchange.Then connect the flask to a similar flask that has been evacuated. Open both taps so that some of the methyl bromide, which will now be radio-active according to the bromide content of the original sample, is drawn into the second flask. Decompose the methyl bromide sample by drawing a suitable reagent (e-g., monoethanolamine) into the second flask, precipitate the bromide formed as silver bromide after acidification and measure its radio-activity under a thin end-window Geiger counter. When the ratio RA/A is large, it can be shown that if w be the weight in grams of element giving rise t o A* under bombardment, then, where a is the atomic weight of the element being assayed, d the observed number of disintegrations per second due to A* in a separated fraction 0 of the total weight of compound RA with which the sample was allowed to exchange after bombardment, h is the disintegration constant in reciprocal seconds, t is the time in seconds between determining d and removing the sample from the target area, t’ is the irradiation time, t” is the time allowed for exchange, F is the effective particle flux in particles per sq.cm. per second, (I is the particle capture cross-section in barns for the formation of A*, and K is the exchange velocity constant in reciprocal seconds. F and 0 may not be accurately known in practice, in which case a second sample containing a known added amount of the required element is bombarded under identical conditions and used as a reference. K need not be known accurately provided the exchange is allowed to proceed to virtual equilibrium. Starting with known concentrations of bromide (17 p.p.m.) in tobacco leaf the method has628 NOTES [Vol.75 been applied alternatively to the determination of K for the exchange between the bromide42 formed by direct neutron irradiation of an untreated sample of the leaf and the bromine of pure methyl bromide subsequently applied; 8.2 and 2 8 4 per cent. respectively of the bromine432 found had exchanged at 20" C. and at 35" C. respectively in 24 hours. Other experiments indicated that the exchange between radio-active bromides (and iodides) and the corresponding inactive methyl halides was virtually complete within an hour or so a t 100" C. in acetone. The method appears therefore to be reasonably rapid, specific arid sensitive ; e.g., assuming an available neutron flux of 5 x 10l1 neutrons per sq.cm. per second it would be possible to determine 10-8 g . of bromine in the presence of any amount of the other halogens to within a few per cent. with typical counting equipment. The method should be similarly applicable to the ultra-micro determination of iodine and chlorine by making use of the C13? (n, 7 ) CP8, and the 1127 (n, 7) 1128 reactions, but then the assays would have to be completed within a few hours of removal from the Pile because of the short half-lives of these isotopes. This note is published by permission of the Department of Scientific and Industrial Research. REPERE,NCE 1. Tobias, C. A., and Dunn, R. W., Science, 1949, 109, 109. DEPARTMENT OF SCIENTIFIC AND INDUSTRIAL -SEARCH PEST INFESTATION LABORATORY SLOUGH, BUCKS.F. P . W. WINTERINGHAM February, 1960 THE DECIPHERING OF CHARRED DOCUMENTS A PAPER by Grant1 summarised the methods available for deciphering charred documents. Some further observations may be of interest to those who have to decipher documents damaged by fire. The charred remains are carefully "painted" with a 25 per cent. solution of gum arabic and allowed to dry, after which it is lightly coated, by the aid of a soft camel hair brush, with a white paint made by ball-milling pure titanium oxide in 25 per cent. gum arabic solution. When dry, the document should appear a uniform light grey or white. It is then placed in a desiccator containing chlorine or bromine vapour, in heavy concentration, for about 30 minutes, after which it is removed and set aside in moist air for an hour.A "gassing" in a desiccator containing ammonium sulphide will cause the development of any iron left as residue of iron gall ink, and if the writing so produced is only faint, the bromine and moist air treatments should be repeated until a satisfactory development is obtained when the document may be photographed. If a permanent reproduction on the document is desired, the final ammonium sulphide treatment may be omitted and the document lightly "painted" with a 5 per cent. solution of potassium ferrocyanide containing 2 per cent. nitric acid, when the remains of iron gall ink writing develop on the pale background. The effect of iron in the paper due to loading materials, etc., is reduced to a minimum and the contrast between the writing and the background increased to a maximum by this process.A modification of Murray's method2 has proved very successful with printing, typing, carbon copies, iron gall writing inks and lithographic prints, In the modified method, the 5 per cent. silver nitrate solution is treated with ammonia until the precipitate first formed is dissolved, and the charred or incinerated document is immersed in this solution, which is held at a temperature between 80" and 95" C. The actual temperature between these limits does not appear to be critical for obtaining good results. Within a few ininutes a silvery outline of the printed, typed or written matter appears on the darker background of the rest of the document, or the writing, etc., may appear black on a silvery background.Whether the writing4will appear as silver on black, or black on silver, appears unpredictable and fortuitous; in some instances, both the phenomena appear in different parts of the same document. The reproduced matter is best read or photographed while the document is immersed under water. Address, stamp and cancellation marks, together with any printed matter, are readily reproduced on burnt envelopes and this modification appears to be more universal in its application than any so far examined, providing that the documents have been burnt with rkasonably free access of air and not kept in- the charred condition for more than two or three days. Documents charred whilst packed into a closed box, or those which have been lying in the charred state for more than three days have not been examined. The effect on pencil writing is very erratic; sometimes excellent results have been achieved, but on other occasions only fair or poor results have been attained.Nov., 19501 BRITISH STANDARDS INSTITUTION REFERENCES 1. 2. 87, SCHOOL ROAD TILEHURST Grant, J., Analyst, 1942, 67, 42. Murray, M. D., Nature, 1941, 148, 199; Analyst, 1941, 66, 440. READING, BERKS. 629 J. A, RADLEY April, 1950

ISSN:0003-2654    

DOI:10.1039/AN9507500626

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

Nov., 19501 BRITISH STANDARDS INSTITUTION 629 British Standards Institution DRAFT SPECIFICATIONS A FEW copies of the following draft specifications, issued for comment only, are available to interested members of the Society, and may be obtained on application to the Secretary, Miss D. V. Wilson, 7-8, Idol Lane, London, E.C.3. Draft Specifications prepared by Technical Committee DS/l3--Upholstery and Bedding Fillings. CM(DS) 4759-Draft B.S. for Woollen Felt for Bedding Upholstery and similar purposes. CM(DS) 468ARevision of B.S. 1425, Cleanliness of Fillings and Stuffings for Bedding, etc CM(LBC) 5402-Draft B.S. for Filters made of Sintered Materials. Draft Specification prepared by Technical Committee LBC/l3-Filtration Apparatus.

ISSN:0003-2654    

DOI:10.1039/AN950750629c

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

630 REVIEWS [Vol. 75 Reviews PHENOMENA, ATOMS, MOLECULES. By IRVING LANGMUIR. Pp. xi + 436. New York: The The first is to set out the author’s opinions on science and topics of related interest, such as scientific legislation, the provision of incentives to further progress in research, the limitation of the scientific method when applied to human affairs and the world control of atomic energy. The second is to present the important results of Dr. Langmuir’s forty years of research, mainly by reprinting, with little alteration, his most significant papers. The result is an uneven book, in which there i s a certain amount of repetition, but also a most stimulating book, which its purchasers will value, and which many non-purchasers, who may be in this category on sheer grounds of price, will regret not having on their shelves.To the specialist in the field of surface chemistry the book will be welcomed as a distillation of Dr. Langmuir’s contribution to the subject, which is by no means solely of historic interest, though most of the work was published over twenty-five years ago. To the non-specialist, the volume will provide as good an introduction to the subject as is to be found. If some of the sections may be rather detailed for him, this will be more than offset by the clarity and simplicity of expression and thought that characterise Dr. Langmuir. The general sections of the book, occupying one-tenth, are thought-provoking, as may best be shown by quoting some of the author’s conclusions. “The net result of the modem principles of physics has been to wipe out almost completely the dogma of causation.” “Human affairs are characterised by a complexity of a far higher order than encountered ordinarily in the field of science.I believe the field of application of science to such problems to be extremely limited.” “In Russia they are frankly incorporating into their communist government the best features of our capitalist system, while we are tending to put into our democracy some of the worst features of communism.” “I believe Russia is planning to embark on a programme of research and develop- ment greater than that contemplated by any other government.” The sections covering Dr. Langmuir’s research, occupying some 350 pages, embrace a wide variety of topics in surface chemistry. His classical work on the interactions of gases with heated metal filaments, and on the adsorption of electropositive layers, such as caesium or thorium on tungsten, is described in detail.In addition, there are sections on molecular orientation in the surfaces of pure liquids, on intermolecular forces, on interactions in liquid - liquid mixtures, on monolayers adsorbed on water surfaces, on the condensation and evaporation of gas molecules, and of course on the familiar adsorption isotherm. In particular one admires the fruitful study of caesium layers on tungsten, which gave the roughness factor of the filament, the condensation coefficient of the caesium, the variation of the surface dipole with adsorbed amount and quantitative information on the surface migration of the caesium, and also showed that, except for a few atoms, the surface was uniform in adsorptive power.Altogether the reader is left with the feeling that Dr. Langmuir’s work is of even greater weight than he had previously thought. Philosophical Library, Inc. 1950. Price $10.00. Dr. Langmuir’s book seeks to do two things. B. M. W. TRAPNELL ELSEVIER’S ENCYCLOPAEDIA OF ORGANIC CHEMISTRY. Edited by F. RADT. Volume 12B, Part I. Series 111, Carboisocyclic Condensed Compounds. Pp. xxx + 344 + index. New York and Amsterdam : Elsevier Publishing Co. London Distributors : Cleaver-Hume Press. The editorial aims of this encyclopaedia, the way in which they are being achieved and certain shortcomings were commented upon in connection with volume 13, the first to appear after the war (see Analyst, 1948, 73, 53).The remarks then made still apply, save that the proposed interval of four years between literature references and publication has now been attained; as before, the text has been opened occasionally for the inclusion of additional information, especially where structure is concerned. The present part deals with the hydrocarbons containing one naphthalene nucleus (including their hydrogenated derivatives) and the halogen derivatives (including iodoso- and iodoxy- compounds) of such hydrocarbons. A careful check: on the many methylnaphthalenes has disclosed no omission of any reference of importance. The collection of references by years reveals that considerable interest in this group of compounds has been shown over the past thirty years; indeed, this interest is being maintained and more recent work will probably necessitate amendment of the data on physical properties attributed to some of the less simple naphthalene hydrocarbons.1948. Price k6 5s. for subscribers to the whole work.Nov., 19501 REVIEWS 631 In the accumulation of matter for a work of such expanse, occasional discrepancies in the literature are inevitably encountered. Part of the Elsevier service is to investigate these dis- crepancies and several reports on such have already appeared in Rec. trav. chim. ; reprints of these are available for circulation with the appropriate volume if the results have not already been incorporated therein. Finally, it may be added that the second part of Volume 12B, dealing with the nitrogen derivatives of the compounds appropriate to Part I, has now been published.B. A. ELLIS METHOUES MODERNES D’ANALYSE QUANTITATIVE MIN~RALE. By G. CHARLOT and D. BI~ZIER. This is the second edition of a book first published in 1944 by Professor Charlot and his colleague of 1’Ecole de Physique et de Chimie industrielles. In the introduction, Professor Charlot states that the book has been entirely re-set in order to take note of various criticisms and suggestions that were made concerning the first edition. Its general character, however, remains the same and its declared objective is to serve as a guide rather than as an analytical treatise, something which he considers inevitable having regard to the breadth of the subject. The book is divided into two main sections, the first headed “G6nkralites,” comprises some 25 chapters; the second, “Dosage de Principaux Elkments,” over 350 pages, gives in alphabetical order a survey of the methods available for the determination of the elements from aluminium to zirconium.Section I covers a great deal of ground and it may be useful to itemise some of the main chapter headings. Beginning with a fairly elementary treatment of volumetric processes and techniques, the book goes on to deal with oxidimetry and oxidation - reduction processes. Chapter VI is on “Titrimktrie par formation de Complexes” ; Chapter VII, “Titrimktrie par precipitation” ; Chapter VIII, “Acidimktrie dans les solvants autres que l’eau.” Chapter IX deals with reactions in the presence of two solvents and with extraction processes.In Chapter X there is a brief review of the apparatus and techniques of gravimetric analysis and a very short section on Sampling. Chapter XI is a page and a half only of general discussion of the growing importance of Physical Methods, while XI1 and XI11 deal with Absorption Spectroscopy and Emission Spectro- scopy respectively. In the introduction to this edition, the authors say that they have given “une plus grande place” to instrumental methods, in particular to absorption spectrophotometry ; it is therefore disappointing to find this section, and that on emission spectroscopy, so brief and superficial. Absorption in the ultra-violet and in the infra-red are each covered in six lines. Some of the information about photo-electric cells is out of date, no examples of the applications of the methods are given and there is hardly a reference to any British instrument. At the end of the chapter on Emission Spectroscopy, X-rays (presumably used for diffraction techniques) are dismissed in three lines.Then follow chapters on Potentiometric Titration, Electrolysis, Polarography, and one on miscellaneous methods. This last comprises Conductimetry, Dielectric Constant (4 references only), Measurement of High Frequency Current, Absorption of Neutrons (1 reference), Chromato- graphy (1 page), Capillary Analysis, Methods involving Radio-activity, Mass Spectrometry, Magnetic Permeability, Raman Spectra, and three or four more techniques, none of which receives more than a few lines. This section is almost useless and falls far short of what the authors promised. The usefulness of the next chapter, on Organic Reagents, is increased by the references and the bibliography that are given; indeed, one of the good features of this book is the inclusion of a comprehensive bibliography and a number of references in each chapter.There is a chapter on the Properties of Precipitates and one on Separations, a short one on the Determination of Traces and a still shorter one on Semi-micro and Micro Determinations. Methods of Solution, Destruction of Organic Matter prior to Determination of Inorganic Constituents and Gas Analysis complete the first part. In the second part there is a chapter for each element, and the general pattern of the work will be illustrated by quoting the section headings for Silver: Rappel de quelques propriktks, S&parations, Volumktrie, GravimCtrie, Potentiometrie, glectrolyse, Colorimktrie, Cas Particuliers, Dosage des Mktaux prkcieux par voie seche.This is the more useful section of the book and its value, as before, is enhanced by the number of up-to-date references. Some elements, unfortunately, receive very scant treatment ; for rubidium and caesium, for example, there are only a few references. While other elements Second Edition. Pp. viii + 685. Paris: Masson et Cie. 1949. Price 2000 fr.; 44s.632 PUBLICATIONS RECEIVED [Vol. 75 are dealt with more comprehensively, there is sometimes a lack of critical selection and a number of methods given are described as “tr&s rapide mais assez peu prkcise.” None of these criticisms should obscure the fact that the book, despite its title, is declared to be a guide rather than a treatise, and that in it is collected together a great deal of very useful information, not least the copious and up-to-date references and the general bibliography at the end. It is noted that Abstracts C does not appea:r in the list of “PCriodiques donnant la biblio- graphie compl&te.” To sum up, this book disappoints in many respects, and yet contains so much that is valuable as perhaps to deserve a place on the bookshelf of the inorganic analyst. It attempts too much, so that inequality of treatment was bound to ensue. The paper covers will not stand up to the amount of handling required even for review purposes, and the reader must also be prepared to cut his way into the pages, no mean task with a book of this length. One must be critical of the binding, as of most French bindings. R. C. CHIRNSIDE

ISSN:0003-2654    

DOI:10.1039/AN9507500630

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

632 PUBLICATIONS RECEIVED [Vol. 75 Publications Received REPORTS ON BIOLOGICAL STANDARDS : VI. THE DESIGN OF TOXICITY TESTS. (Medical Research Council, Special Report Series No. 270.) 13y W. L. M. PERRY. Pp. vi + 51. London: H.M. Stationery Office. 1950. Price 1s. (id. THORPE'S DICTIONARY OF APPLIED CHEMISTRY. Fourth Edition. Vol. X : Plagioclase-Sodium. Pp. xi + 913. London: Longmans, Green & Co. 1950. Price L5. COLONIAL PLANT AND ANIMAL PRODUCTS. Vol. I, No. 1, January-March, 1950. Editor: G. T. BRAY, F.R.I.C. Pp. vi + 94. London: H.M. Stationery Office. Price 5s. This quarterly journal i s the successor to the Plant and Animal Products Department section of the Bulletin of the Imperial Institute. Vol. I, No. 1, 1950. Editor: E. H. BEARD, B.Sc. Pp. xvi + 120. London: H.M.Stationery Office. Price 5s. The quarterly bulletin of the Colonial Geological Surveys. COLONIAL GEOLOGY AND MINERAL RESOURCES. PRACTICAL SPECTROSCOPY. By G. R. HARRISON, Ph.D., Sc.D., R. C. LORD, Ph.D., and J. R. LOOFBOUROW, Sc.D. Pp. xiv + 605. Lortdon: Blackie & Son, Ltd. 1950. Price 35s. STRUCTURAL CHEMISTRY OF INORGANIC COMPOUNDS. Vol. I. By W. HUCKEL, D.Phi1. Pp. xii + 437. London: Cleaver-Hume Press, Ltd. New York and Amsterdam: Elsevier Publishing Co., Inc. 1950. Price 70s.; $9.00. ANALYTICAL ABSORPTION SPECTROSCOPY. Edited by M. G. MELLON. Pp. vii + 618. London: Chapman & Hall. New York: John Wiley & Sons, Inc. 1950. Price 72s.; $9.00. AN INTRODUCTION TO LABORATORY TECHNIQUE. Second Edition. By A. J. ANSLEY. Pp. xv + 288. London: Macmillan & Co., Ltcl. 1950. Price 16s. INTERIM REPORT OF THE MINISTRY OF HEALTH DEPARTMENTAL COMMITTEE ON DETERIORATION OF CAST IRON AND SPUN IRON PIPES. London: H.M. Stationery Office. 1950. Price 3s. 6d. This Report covers the scientific and technical aspects of corrosion and the extent of the problem and its implications. It suggests measures for protecting pipes in areas where the soil formation i s known to be corrosive. THE 1951 PITTSBURGH CONFERENCE ON ANALYTICAL CHEMISTRY AND APPLIED SPECTROSCOPY THIS conference will be held at the William Penn Hotel, Pittsburgh, Pennsylvania from March 5th to 7th, 1951. It is jointly sponsored by the Analytical Chemistry Group of the Pittsburgh Section of the American Chemical Society and the Spectroscopy Society of Pittsburgh. An Exposition of Modern Labor'atory Equipment will be a feature of the conference.

ISSN:0003-2654    

DOI:10.1039/AN9507500632

出版商:RSC    

年代:1950

数据来源: RSC