ISSN:0003-2654    

DOI:10.1039/AN95176FX005

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176BX007

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176FP009

出版商:RSC    

年代:1951

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95176BP015

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

FEBRUARY, I95 I Vol. 76, No. 899 MINISTRY OF FOOD (PRESERVES, ETC. IN FOOD) REGULATIONS AS a result of a joint representation of the Food Group of the Society of Chemical Industry and this Society, the Food Standards Committee, appointed by the Minister of Food in January, 1948, has set up a Sub-committee, under the chairmanship of Professor E. C. Dodds, M.V.O., M.D., D.Sc., F.R.C.P., F.R.I.C., F.R.S., Director of the Courtauld Institute of Biochemistry, with the following terms of reference- “To review the Public Health (Preserves, etc. in Food) Regulations and to make any recommendations the Sub-committee may consider desirable for the amendment of the Regulations.” The Society is represented on this Sub-committee by Dr. H. E. Cox, Ph.D., D.Sc., F.R.I.C. APPOINTMENT OF SUB-COMMITTEE TO REVIEW THE PUBLIC HEALTH 57

ISSN:0003-2654    

DOI:10.1039/AN951760057b

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

58 SMITH, COMRIE AND SIMPSON: THE EVALUATION OF LIMING [Vol. 76 The Evaluation of Liming Materials for Agricultural Purposes BY A. M. SMITH, A. COMRIE AND K. SIMPSON (Presented at the meeting of the Society on Wednesday, October 4th, 1950) Experimental evidence is submitted to show that the' value of a burnt lime for agricultural purposes may be seriously under-estimated by the method prescribed under the Fertiliser and Feeding Stuffs Act. Calcium silicates and magnesium oxide are able to neutralise soil acids, and the latter is also important as a source of magnesium in certain soils. Various methods of estimating the neutralising value have been examined and a simple technique, involving a short treatment with dilute acid and applicable to both carbonates and limes, has been found t o give results in agreement with the effects produced on soil acidity in pot and field experiments.THE question of the assessment of the agricultiural value of a burnt lime has been raised many times in the last twenty years. The official methodl makes use of the fact that a sugar solution dissolves calcium hydroxide only, and gives a value for the free calcium oxide in a sample. It is thus quite satisfactory for the analysis of a lime obtained from a properly- burned calcium carbonate limestone, where the calcium oxide is the only material of neutralising value. The method does not take iinto account any carbonate that may be left as a result of under-burning and it ignores almost entirely the presence. of any magnesiumFeb., 19511 MATERIALS FOR AGRICULTURAL PURPOSES 59 oxide, which has a greater neutralising value than calcium oxide and, incidentally, is invaluable as a source of magnesium in magnesium-deficient soils.Many b u n t limes contain other compounds that are of value in reducing soil acidity and increasing the amount of exchangeable calcium in the soil. These compounds are of the nature of calcium silicate and may be produced when limestones containing considerable amounts of siliceous material are burned. They are insoluble in sugar solution, but are so readily hydrolysed that they decompose ammonium salts and neutralise soil acids to an equivalent extent; they also dissolve easily in dilute acid and are, without doubt, of value as “liming material.” The agricultural value of many burnt limes is, therefore, seriously under-estimated by the official method.Attention was first directed to this question in 1931 in a communication to the Agricultural Education Association2 concerning the behaviour of a waste carbonate after burning. It had long been appreciated that basic slag had a “lime value” greater than that corresponding to the small proportion of free calcium oxide it contained. Indeed it has frequently been claimed that a high-grade slag has the same lime value as an equal weight of limestone. This is an exaggeration, but a slag with a high solubility in citric acid does reduce soil acidity to the same extent as about half or two-thirds of its weight of calcium carbonate, because it contains compounds which readily provide an acid soil with exchangeable calci~m.~ A large proportion of the calcium in slag occurs in combination with phosphorus and silica as a result of the reaction at a high temperature between lime and the impurities in pig iron.Similar types of siliceous compounds are probably produced in a lime kiln and so it was not surprising to find that the lime value of certain burnt limes was indeed greater than indicated by their contents of free calcium oxide. METHODS OF ANALYSIS A new chemical method of evaluating an agricultural lime must be based upon convincing data obtained from experimental work with soils. It is difficult to obtain precise results on the effects of liming materials in the field on account of sampling errors, but it is nevertheless important that the accurate observations from laboratory and pot culture experiments should be supported by figures found under natural conditions.In a preliminary approach to the question a number of burnt limes in use as liming materials were examined by five methods and the results are summarised in Table I. To measure the effect on soil acidity, a series of flasks each containing 20g of soil, 50ml of boiled distilled water. and different quantities of the burnt lime were shaken at intervals TABLE I Burnt lime A €3 C D E F G H I THE EVALUATION OF BURNT LIME BY DIFFERENT METHODS Equivalent CaO by CaO by f A I -------- Soil titration NH,Cl distillation T.N.V. Cone. acid Sugar method (a) (b) (4 % % % % 54 66 67 83 86 88 92 90 97 57 65 68 81 82 83 91 90 89 57 59 33 65 69 46 68 73 46 82 81 61 80 84 66 82 86 66 88 81 70 87 86 71 87 86 77 METHODS EMPLOYED- 0.2 g of material + 40 ml of N ammonium chloride $- 700 ml of water boiled for 90 to 120 minutes, the distillate being collected in standard acid and the NH, expressed as Cs.0.Total nezctralisiwg vdue-0.5 g of material + 50 ml of 0-5 N hydrochloric acid boiled for 3 minutes. cooled and excess of acid titrated with 0-5 N sodium hydroxide with phenolphthalein as indicator; the result is expressed as equivalent CaO. Suitable quantity of material boiled with concentrated hydrochloric acid for 10 minutes and calcium in filtrate determined by standard method. The official method of determining caustic lime. (a) (b) (c) ( d )60 SMITH, COMRIE AND SIMPSON: THE EVALUATION OF LIMING during several days until an approximate equilibrium was attained, and the final pH values of the suspensions were plotted against the amounts of lime added.A similar curve was obtained with a standard, usually precipitated calcium carbonate, and the relative value of the lime in reducing the acidity was estimated from the two curves. Each figure given in Table I is the average for three acid soils and represents the percentage efficiency or lime value of each material compared with a standard treatment. To take a particular example, 54 parts of lime (CaO) reduced the soil acidity to the same extent as 100 parts of sample A, at a point on the curve corresponding to an application of about 1 ton of lime per acre. This method is a refinement and extension of that used in the routine estimation of the “lime requirement’’ of soils, where equal portions of a particular soil are shaken overnight with equal volumes but different concentrations of lime water and the final pH values are plotted against the amount of lime added.4s6 The amount of lime required to bring the pH value of the soil to any level desired in practice can be interpolated on the curve and the dressing required in the field is calculated with or without a factor according to circum- stances.Although the method is not suitable for the routine examination of liming materials it has been employed frequently in special cases and it has given valuable evidence in testing the reliability of the measurement of total neutralising value (T.N.V.). The method cannot be as precise as a chemical determination because the time required to reach equilibrium depends upon the nature of the soil and the physical and chemical properties of the liming material concerned.But it is possible to obtain smooth titration curves (some examples are shown in Fig. 1) on which interpolations at a particular pH value may be made. [Vol. 76 Fig. 1. Soil neutralisation curves and pot results The results clearly indicated that the ability of the lime to reduce the acidity of different soils was equivalent to its power (a) to decompclse a solution of an ammonium salt and (b) to neutralise dilute hydrochloric acid. These values were in turn only slightly less than (c) the percentage of calcium oxide extracted by boiling concentrated hydrochloric acid, but were always much greater than (d) the percentage of calcium oxide obtained by the official method.For many years, the values obtained by method (d) have, without exception, been low in comparison with those obtained by methods (6) and (c). For example, the last 30 samples of “lime” examined have given the results shovin in Table 11. Many of these samples were under-burned, but in the majority the amount of carbon dioxide was’less than 3 per cent. These results fully confirm the evidence in. Table I, and show that on the average the official method has under-estimated the “liming” value or T.N.V. of the burnt lime by aboutFeb., 19511 MATERIALS FOR AGRICULTURAL PURPOSES 61 18 per cent., the range of differences being from 1 to as much as 30 per cent. However, the T.N.V. figure was always within 4 of the percentage of bases dissolved by boiling concentrated acid and expressed as CaO.TABLE I1 ANALYSES OF 30 SAMPLES OF BURNT LIME Fraction measured (1) Total CaO by concentrated acid . . .. (2) Total MgO by concentrated acid . . .. (3) CaO equivalent to (1 + 2) . . . . .. (4) Free CaO (official method) . . . . . . (5) CaO equivalent (T.N.V. method) . . .. Value (5) less value (4). . .. . . .. Value (3) less value (5). . .. . . .. Range Average 39.7 to 95.3 79.1 0 to 3.1 1.1 41.4 to 95.3 80.7 8.7 to 91.1 60.6 40.0 to 94.0 78.3 4-1 to +30 fl to +a - - Of the various methods mentioned above, that giving the so-called total neutralising value is by far the simplest. It has been employed regularly by us during the past six years as a supplement to the other methods because it was felt that it gave a reliable estimate of the agricultural value of the liming material.It can equally well be used for samples of ground limestone or waste carbonate or shell sand, the result then being calculated in terms of equivalent CaCO, instead of CaO. For example, the last 30 samples of such materials examined have given the results shown in Table 111. TABLE I11 ANALYSES OF 30 SAMPLES OF VARIOUS CARBONATES Fraction measured Range Average (1) Total CaO by concentrated acid . . . . 20.2 to 53.1 41.8 (2) Total MgO by concentrated acid . . .. 0 to 9.0 1-1 (3) CaCO, equivalent to ( 1 + 2) . . .. . . 36.0 to 94.8 77.5 (5) CaCO, equivalent (T.N.V. method) . . .. 43-4 to 96.5 77.7 (4) CaCO, equivalent to CO, . . .. .. 36.0 to 96.1 77.6 Value (3) less value (5).. .. . . .. -2.8 to +4*0 - The agreement would seem to be adequate for this type of agricultural material, since the limit of variation permitted by the Fertiliser and Feeding Stuffs Act is, for limestone, 5 per cent. of the CaCO, stated, and for quicklime, 10 per cent. of the CaO. The total neutralising value method is, therefore, applicable to both carbonates and limes and so gives a fair valuation for agricultural purposes of a badly burned or partially burned limestone ; moreover, it takes account of the neutralising value of the magnesium oxide and other basic substances. P O T EXPERIMENTS Having established that the total neutralising value was in fact a good measurement of the ability of a liming material to reduce soil acidity in the laboratory, it was used as the basis of comparison for different liming materials in pots and in the field.In one set of pot experiments, two highly unsaturated soils-a mineral soil, W, derived from glacial sand, with a pH value of 4-7 and a loss on ignition of 9 per cent., and a thin peaty soil, P, overlying acid andesite, with a pH value of 3.3 and a loss on ignition of over 80 per cent.-were each treated with calcium hydroxide, at two rates, and with fine and coarse fractions of three different limestones. The “fine” fraction consisted entirely of particles passing a 100-mesh sieve; the particles of the “coarse” fraction lay between 100-mesh and 1 mm in diameter. All the materials were used in equivalent amounts based upon the total neutralising values that are given in Table IV in terms of CaCO,; the calcium hydroxide was also used at half that rate.The liming materials were thoroughly mixed with the air-dry soils before potting, and the treatments were replicated. The pots were cropped in successive years with peas, barley and peas; the pH value of the soil in each pot was determined from time to time. For comparison, a range of treat- ments with each material was tested in soil suspensions under laboratory conditions to obtain titration curves. A selection of the results is presented in Table IV and a few typical curves are shown in Fig. 1.62 SMITH, COMRIE AND SIMPSON: THE EVALUATION OF LIMING [Vol. 76 TABLE IV EFFECT OF DIFFERENT LIMING MATERIALS ON SOILS IN POTS p1-I of soil W pH of soil P -7 r--------h-_--\ T .N.V. Weeks in pots Weeks in pots Equiv. as Lab. ,-A-, Lab. /-A-, Material CaO MgO CaCO, CaCO, expt. 35 73 116 expt. 35 73 116 A1 Ca(OH),-half-rate . . 75.6 - 136.0 131.8 6.1 5.5 5.9 5.5 4.8 5-0 4.9 4.8 A2 &(OH),-full-rate . . 75.6 - 135.0 131.i3 6.9 6.2 6.3 6.0 6.0 5.7 5.6 5.6 B1 Limestone-fine . . 40.2 6.5 87.8 90.5 6.7 6.5 6.4 6.3 6.1 5.7 5.6 5.7 B2 Limestone-coarse . . 44.6 5.2 95.8 92.0 6.2 5-9 6.3 6.4 5.3 5.7 5.7 5.8 D1 Limestone-fine . . 49-8 1.8 93.0 90.’7 6.7 6.3 6.5 6-3 6.3 5.6 5.5 5.7 D2 Limestone-coarse . . 52.6 1.8 98.0 95.!i 6-2 5-9 6.5 6.5 5.5 5.7 5.2 5.8 F1 Limestone-fine . . 31.1 20.3 106.4 105.3 6.4 6.4 6.6 6.4 6.2 5.8 5.8 6.1 F2 Limestone-coarse . . 31.7 20.1 106.8 107.0 5.7 5.7 6.1 6-3 5.3 5.5 5.4 5.9 The results show that- (a) The T.N.V.figures were substantially the same as the equivalent CaCO, calculated from the total calcium and magnesium. (b) With hydroxide and fine limestone, the soil pH values reached higher levels in the laboratory suspensions than in the pots; compare curves and points A, D1 and Fl in Fig. 1. This was possibly the result of leaching of the fine material from the pots that were kept outside throughout the experiment. With the “coarse” fractions, however, the soils attained higher pH values in the pots than in the laboratory; see points D2 and F2 in Fig. 1. This was probably because the duration of the laboratory experiment was too short for complete reaction between soil and coarse particles. In the pots, the fine and coarse fractions ultimately gave the same result but more quickly in the peaty soil than in the mineral soil.(c) All the materials, added in equivalent amounts according to their total neutralising values, produced essentially the same reduction in acidity as measured by the pH values of the soils. FIELD EXPERIMENTS It is easy to prepare an intimate mixture of soil and liming material for a pot experiment but it is very difficult to get an even distribution of a liming material to a particular depth in the field. Furthermore, sampling errors in the field are relatively large, so that a smooth curve depicting the change in pH value is not to be expected from a series of samples. The trouble may be particularly acute for an application of ground burnt lime; as a result of slaking and carbonation, small pockets of the inaterial can form quite hard lumps, which do not disintegrate easily and remain very inefficient from the point of view of neutralising soil acidity.A limestone ground to pass a 100-mesh sieve may, therefore, provide a larger surface in contact with soil particles than a ground lime, and so reduce soil acidity more rapidly. Large particles of limestone have slower rates of reaction according to their size and hardness. Eventually, however, all forms will exert an effect depending upon their relative contents of available calcium and magnesium. Waste carbonates from paper works may contain some sodium hydroxide, but the amount is generally less than I per cent. In recent field experiments, designed to compare different forms of liming materials, the dressings have been based on the total neutralising values of the materials used.The primary object of these experiments was to measure the response in crop yield, but it was sometimes possible to sample the soil of the individual plots at intervals during two or three seasons, and to compare the pH values with the results expected from the titration curve obtained in the estimation of the “lime requirement” of the original soil. A selection of average results from four experiments on differei-it soil types is given in Table V. The letters L, LS and w represent respectively ground burnt lime, ground limestone and waste carbonate, and the dressings are equivalent amounts of CaO calculated from the total neutralising values (T.N.V.). For example, 2 5 ~ means that ground burnt lime was added in amount sufficient to supply 25 cwt of CaO per acre according to its T.N.V.The reduction in acidity was responsible for large increases in the yields of barley and roots in experiment 1, but the difference between equivalent amounts of lime and limestoneFeb., 19511 MATERIALS FOR AGRICULTURAL PURPOSES 63 was not significant. Treatment did not produce a significant increase in roots in experiment 2 and only the smaller dressings produced a larger yield of beet tops and sugar in experiment 3. In experiment 4, equivalent amounts of lime and of two fractions of limestone produced the same yields. The highest pH values reached in experiments 1, 2 and 3 were quite in accord- ance with the dressings applied and with the calculated values.In experiment 4, equivalent amounts of lime and finely ground limestone had effected the same increases in pH value, but it was doubtful if equilibrium had been reached with the other materials in the period of less than 8 months. TABLE V EFFECT OF DIFFERENT LIMING MATERIALS ON SOIL AND CHOP 1. 2. 3. 4. NIDDRIE MAINS-sandy loam, drainage good, original pH value 4.7. Cwt of CaO per acre, 25.3.1947 . . . . nil 25L 50L 1947 CYOP- Barley grain (cwt/acre) . . . . 0.61 2.82 3.87 Barley straw (cwt/acre) . , . . 0.89 2.87 3.56 Turnips y t s (tons/acre) . . . . 13.3 24.0 25-6 1948 GYOP- { ops (tons/acre) . . . . 2.27 3.82 4.54 pH at 11.2.1948 . . . . . . . . 5.0 5.1 5.2 19.11.1948 . . . . . . . . 4.8 5.3 5.8 21.3.1949 . . . . . . . . 4.7 5.3 5.8 14.9.1949 .. . . .. . . 4.7 5.2 5.9 Calculated pH* . . . . . . - 5.4 6.0 Rainfall was low in 1947 and high in 1948. CAMMERLAWS-Heavy loam, drainage poor, original pH value 5.8. Cwt of CaO per acre, 18.3.1948 . . . . nil 2 0 ~ s 5 2 ~ s Swedes { pH at 16.11.1948 . . . . .. . . 5.7 6.0 6-3 22.3.1949 . . . . . . . . 5.6 6.0 6.8 Calculated pH* . . . . . . . . - 6.1 6.5 AYTON LAW-Medium loam, drainage fair, original pH value 6.1. Cwt of CaO per acre, 2.2.1948 . . . . nil 1 4 ~ s 3 0 ~ s roots (tons/acre) . . . . 22.1 23.5 21.0 tops (tons/acre) . . . . 5.37 5.11 4.95 roots (tons/acre . . .. . . 9.9 11.3 10.1 tops (tons/acre) . . . . . . 10.4 12.3 20.8 sugar (cwt/acre) . . . . . . 33.8 38.8 34.5 . . .. . . 6-2 6.5 6.9 22.3.1949 . . . . . . . . 6.1 6.5 6.8 Calculated pH* . . . . . ... - 6.5 7.0 pH at 30.11.1948 . . 1 OOL 3.59 3.21 30.4 5.22 5.3 5.8 6.4 6.6 6.6 5%- 20.7 5.48 6.6 6.7 6.5 30w 10.5 11.2 36.5 6.9 7.1 7.0 CARSLOGIE-Light gravelly loam, drainage good, original pH value 5.4. Cwt of CaO per acre, 29.3.1945 . . . . nil 3 1 . 0 ~ 3 1 . 2 ~ ~ 3 1 . 5 ~ ~ Beet { (4 (b) roots (tons/acre) . . . . . . 3.71 10.7 12.1 11.0 pH a t 18.11.1945 . . . . .. . . 5.2 6.4 6.4 6.2 tops (tons/acre) . . . . . . 4.89 17-5 20.3 19-4 Calculated pH* . . . . .. . . - 6.1 6.1 6.1 5 0 ~ s S.E. 4.16 f0.43 4.13 f0-99 27.6 f1.17 4-36 k0.17 5.2 5.6 5.9 5.9 6.0 S.E. f 1.03 - S.E. f 0.60 f 1.59 - 2 0 . 7 ~ ~ 2 0 . 4 ~ (4 9.2 15.1 17.1 5.9 6.5 5.8 5.8 (4 8-8 (a) 93 per cent. of 100 mesh. Standard error for roots -~=0.86, for tops fl.50. L = ground burnt linie, LS = ground limestone, w = waste carbonate.* Calculated pH value from the titration curve with the original soil. (b) 37 per cent. of 100 mesh, 88 per cent. of less than 2 mm. (c) Shell (d) Beet factory carbonate, 42 per cent. of water. sand, 1 per cent. 100 mesh, 62 per cent. less than 2 mm. The reliability of the final pH value calculated from the laboratory estimation of lime requirement depends, of course, upon the factor used to convert the laboratory figure to a field figure; this involves assumptions regarding the apparent density and depth of the soil concerned and the uniform incorporation of the liming material. For mineral soils with an apparent density of about 1 in the dry state, a factor of 1.7 has regularly been used to obtain an estimate of the lime required to bring the top 8 or 9 inches of soil to a particular pH value, 18 to 30 months after the application of this dressing.Special consideration must be given to soils containing more than about 10 or 12 per cent. of organic matter.[Vol. 76 The agreement between the values found and those predicted is of secondary importance, however. More important for the present discussion is the fact that different liming materials applied in equivalent amounts calculated front the simple total neutralising value have produced the same effect on the pH value of these soils. Numerous data from a much larger range of soils and liming materials would be required to decide whether this is of general application. In principle, it would appear to ble quite sound for limes from badly burned or from impure limestones, for ground limestone, for waste carbonates from paper works or sugar beet factories, and for certain types of blast furnace slags. The method might have to be modified for those less abundant waste materials that contain sulphides or other decomposable salts and are sometimes used locally for liming soils.The virtue of the method lies in its simplicity and its ability to give a measure of all those compounds of calcium and magnesium which are of value in soil amelioration. It is of interest to observe, in this respect, that in the United States the Association of Official Agricultural Chemists defines a liming material as “any material whose calcium and magnesium content is capable of neutralising soil acidity.”f’ 64 SMITH, COMRIE AND SIMPSON: THE EVALUATION OF LIMING REFERENCES 1.2. 3. 4. 6. 6. COLLEGE OF AGRICULTURE “Fertilisers and Feeding Stuffs Regulations,” 1932, p. 23 Lauder, A., and Smith, A. M., Agric. Prog., 1933, 10, 172. -,- , Ibid., 1934, 11, 93. Smith, A. M., and Coull, R., Soil Res., 1932, 3, 10. Smith, A. M., Tffans. 2nd Comrn. I.S.S.S., 1933, A, 102. “Official and Tentative Methods of Analysis,” A.O.A.C., 6th Ed., Washington, 1948, p. 896. EDINBURGH AND EAST OF SCOTLAND DISCUSSION THE PRESIDENT congratulated the authors on their paper, and mentioned that he had found high values by the Total Neutralising Value method for so-called “lime waste,’ which was essentially calcium silicate. He asked if the authors had tested such silicate materials in the course of their field work.He also asked whether, if the Total Neutralising Valuation be adopted, there should not be a statement about the magnesium content if any is present. He enquired whether the Total Neutralising Value method was efficient for very hard crystalline forms of carbonate. MR. J. G. SHERRATT asked whether a fictitiously high value would not be given by the Total Neutralising Value in the analysis of waste industrial lime containing calcium sulphide. In many places throughout the country there were huge industrial dumps consisting of lime in various forms. Calcium carbonate predominated, but sulphate, sulphide and silica also were frequently present. Farmers were encouraged to use these wastes and the material was eligible for a Government grant on the basis of its lime content.In practice, a small amount of calcium sulphide did ncrt seem to be harmful; indeed, it was claimed to be beneficial, although its value in neutralising the soil acidity was not apparent. MR. J. KING asked whether limes of high silica content were as effective as the normal carbonate limes in flocculating clay soils. MR. W. F. ETHERIDGE asked whether any experimLents had been performed on tissue tests for calcium assimilation on the various liming materials mentioned. DR. J. H. HAMENCE said that he had listened tc, the paper with considerable interest, particularly in view of the fact that the Advisory Committee of the Ministry of Agriculture and Fisheries had been revived. He felt that many lime merchants had been unfairly penalised in the past, in view of the fact that lime in the form of chalk and also combined with silica had frequently not been taken into account when selling parcels of lime.The problem was a very important one, since he had found that a very small amount of silica was capable of fixing a relatively large amount of lime, and of rendering it inert in respect of its solubility in the official sugar solution. It may be of interest to note in passing that it was his experience that roughly one molecule of silica was capable of fixing four molecules of calcium oxide. For many years he had held the view that lime loosely combined with silica in this manner was available for soil neutralisation, and i t was therefore very pleasing to find that the authors had produced substantial experimental evidence to support this view.MR. C. J. REGAN asked what was the relative value of calcium carbonate and calcium oxide from the aspect of the practical farmer. Was there any advantage in burning limestone to produce quicklime for agricultural use ? DR. SMITH replied to the President’s questions thak silicate materials had not been specially examined, the field experiments having been carried out only with liming materials in regular use. The Total Neutralising Value should be supplemented by a statement of the content of magnesium oxide or carbonate if that were more than, say, 10 per cent., because instances of magnesium deficiency in plants were becoming more frequent and the best method of correcting such acid soils was to use liming materials containingFeb., 19511 MATERIALS FOR AGRICULTURAL PURPOSES 65 magnesium.Hard crystalline forms of carbonate had not been tested, but the rate of reaction between soil and carbonate depended upon the size and hardness of the particles of the latter. In reply to Mr. Sherratt, he said that the presence of sulphide presented a difficulty that might be overcome by a double titration, but the amounts of waste materials containing sulphides were relatively small and of local interest only, and farmers who used them were advised to leave the material exposed in the open until toxic substances had decomposed. The danger incurred was probably small if the waste by-product was incorporated in the soil some weeks before sowing the crop. He informed Mr. King that no experiments had been made on the flocculating power of high-silica limes. In reply to Mr. Etheridge, he said that the assimilation of calcium from different liming materials was not known. Of the several factors involved, the state of saturation of the soil with bases would probably be the most important and all the experimental evidence pointed to the fact that there was little ultimate difference between equivalent amounts of various liming materials as far as the reduction in soil acidity and the increase in exchangeable calcium were concerned. In reply to Mr. Regan, he said that it did not matter whether quicklime or ground limestone was used provided the farmer appreciated what was meant by equivalent amounts. Contrary to the usual assumption, ground limestone might react more quickly than ground lime, which was liable to be carbonated as hard lumps on the surface of the soil and remain comparatively ineffective. There was little to be said in favour of the old practice of allowing heaps of burnt lime to slake on the field; subsequent spreading was laborious and irregular and might be very bad if rain made the heaps unworkable. Ground lime could be spread evenly, but i t was a most unpleasant job. Ground limestone could be stored indefinitely and was easy to spread but, of course, was required in larger quantities than ground lime.

ISSN:0003-2654    

DOI:10.1039/AN951760058b

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Feb., 19511 MATERIALS FOR AGRICULTURAL PURPOSES 65 The Accurate Determination of “ Phosphoric Anhydride ” by Means of Quinoline Phosphomolybdate BY H. N. WILSON (Pyesented at the meeting of the Society on Wednesday, October 4th,, 1950) Present methods of determining P,O, are critically surveyed, and the requirements of a new method considered; it should be as accurate as the “official” method or better, and much quicker. As it appears that the phosphomolybdate reaction is the most suitable, previous work on this subject is summarised, and the reaction discussed in detail. It is then shown that by precipitating quinoline phosphomolybdate instead of the ammonium salt, very accurate results can be obtained by a volumetric method. Few sub- stances present in fertilisers interfere except ammonium salts, which can be destroyed; a large excess of sulphuric acid also interferes.Results by the “official” and the new method are compared and the respective standard deviations are 0.065 and 0.024 per cent. of P,O,. Full working details are given for the accurate determination of P,O, in fertilisers. THERE are two reactions, one or both of which enter into most analytical methods for the determination of phosphate- (1) The precipitation of ammonium magnesium phosphate, which is subsequently converted to magnesium pyrophosphate and weighed. (2) The precipitation of ammonium phosphomolybdate, which may be weighed as such, titrated, or converted to ammonium magnesium phosphate. All procedures involving (l), as almost all accurate or official methods do, are slow, and none are of the highest order of accuracy.There are several reasons for this. The precipitation of magnesium ammonium phosphate is slow, and is not a simple reaction; the precipitate produced may be contaminated with Mg3(P04)2, Mg(NH4),(P0,),, or other substances.l No substances other than MgNH,P04 will be quantitatively converted to the pyrophosphate on ignition, and it is only by purifying the original precipitate by solution and re-precipitation in presence of a small excess of reagent that absence of unconverted salts can be assured. The work of Epperson2 conclusively confirms that double precipitation is essential if accurate results are to be obtained. Accurate or referee methods in which the phosphate is first isolated as ammonium phosphomolybdate invariably continue by conversion of the isolated phosphate to magnesium[Vol. 76 66 WILSON : THE ACCURATE DETERMINATION OF “PHOSPHORIC ammonium phosphate? Prior precipitation as phosphomolybdate from acid solution has the great advantage of separating the phosphate from almost all substances that would interfere with the subsequent application of the magnesia process.Many of these “combined” procedures do not include a re-precipitation of the magnesium ammonium phosphate, and it is probable that a compensation of errors is involved. Many attempts have been made to avoid the use of molybdates by precipitation of magnesium ammonium phosphate from a solution containing citric acid or ammonium citrate, which by formation of complexes prevents interference from calcium, iron or aluminium.It was stated by Epperson2 that presence of citrate caused results to be slightly low and erratic, though only three results are quoted. The “citric magnesia’’ method is accurate, however. This is substantiated by Hoffmann and Lundel14 who recommend it as a method for referee analysis. It has never been popular in the fertiliser trade, perhaps under the influence of the Fertiliser and Feeding Stuffs Act regulations, which prescribe molybdate precipitation and one subsequent precipitation as magnesium ammonium phosphate, and perhaps also because it tends to give lower results. Numerous modifications of the volumetric rnolybdate procedure have been suggested, but none are sufficiently accurate for referee analyses, and all depend on an empirical standardisation of acid and alkali solutions using some supposedly pure phosphate as a standard substance.The “factor” obtained is neither stoicheiometric, nor constant. It is usually also considered that the weighing of ammonium phosphomolybdate is only suitable for small amounts of phosphate, as the errors caused by the variable composition of the precipitate are too great to allow the application of the method to major amounts. Never- theless, it appeared to the author, following his experience with silicomolybdate,5 that a phosphomolybdic acid method could be evolved that would be at least as accurate as the “official” method, and would take only a few hours. REQUIREMENTS OF A NEW METHOD To add to the vast and unnecessary number of variants on the themes noted above, to produce yet another method of analysis for so familiar a substance as “P205”, requires some justification, and the only real justificaticln would be that the new method should have clear advantages over its predecessors.(1) At least as accurate as the “standard” methods. (2) Applicable to a large variety of samples without much modification, and free from interferences. (3) Reasonably simple in manipulation. (4) Quicker than “standard” methods. (6) Truly stoicheiometric, and not dependent on empirical factors. Requirement (4) at once cuts out any procedure in which there is a slow precipitation or re-precipitation, i.e., magnesium ammonium phosphate is inadmissible. Requirement (2) also means that precipitation of magnesium ammonium phosphate is not suitable, as all substances which are precipitated as hydroxides or phosphates in ammoniacal solution must be absent.Requirement (3) suggests tha.t double precipitation or the washing of precipitates with solvents other than water is undesirable. The only known reaction which fulfils requirement (2) is the phosphomolybdate reaction, and by use of quinoline as precipitant, requirements (1) and (5) have also been met. THE PHOSPHOMOLYBDATE METHOD As a t present carried out volumetrically, the phosphomolybdate method has not the qualities of a standard method; the composition of the precipitate varies with the temperature of precipitation, the concentration of ions other than phosphate and the amount precipitated. Usually standardisation is carried out against potassium di-hydrogen phosphate. This is not desirable; because of the influence of other ions present in the sample and not in the standard a bias is introduced (results tend to be high), the factors are empirical and vary with conditions, and the end-point is not very good.It. should be- The formula for ammonium phosphomolybdate is approximately- (NH,) [PO,. 12MoOJ .uzH20, but the precipitate tends to drag down and tenaciously hold other ions, or even acids. The older literature abounds in formulae for this salt, and it is significant that such formulae6Feb., 1951 J ANHYDRIDE” BY MEANS OF QUINOLINE PHOSPHOMOLYBDATE 67 as (NH,),P0,.12Mo0,.2HN0,.H20 are common. Sulphate ions have been said to have a very marked effect, and ammonium sulphate in particular influences the composition of the precipitate.Falk and Sugiura’ considered that a definite compound was formed, to which they gave the formula (NH4),,(P0,),S0,.53M00,, which is unlikely, but indicates the extent of contamination under their conditions. Johnsons comments on the variable quality of the precipitate produced by molybdic acid reagents from different sources, which again points to variation in composition. Allen and Gaults found high results by the volumetric method for samples that had been digested with sulphuric acid to destroy organic matter. Lunge and KeanelO summarise various modifications of procedure that lead to volumetric “factors” which, calculated in terms of 0.5 N sodium hydroxide solution, range from 1.421 to 1640mg of P20,.“As regards fertilisers . . . which contain large percentages of phosphate, the liability of the precipitate to variation under different circumstances appears to the writer to make any modification of the (volumetric method) inadequate when really accurate results are required” (Bernard Dyer, Zoc. &.lo). The rather poor end-point is due to the presence of free ammonia in the solution, according to the equation- (NH,),[P0,.12Mo03] + 26NaOH = 12Na,MoO, + N+HPO, + 3NH3 + 14H,O. Molybdic acid is a weak acid, and ammonia is a sufficiently strong base to affect the indicator used (phenolphthalein) : the extent of interference will vary with temperature and concentra- tion. That the precipitate is a non-stoicheiometric compound is far less likely than that it is impure, as the conditions under which it is formed, viz., simultaneous formation of the complex acid and precipitation of the salt from a strongly acid solution containing relatively high concentrations of other ions, are those most likely to yield an impure precipitate.Entangled nitric acid or other acid would be difficult to wash out, and the carrying down of excess molybdic acid would be even more likely. Unskilled operation can produce a precipitate visibly contaminated with molybdic acid, but who can say how often small but significant amounts of “free” molybdic oxide are carried down, and perhaps “allowed for” in the method of standardisation? A recent report by Bourdon and Cottell states that the ammonium phosphomolybdate does not contain nitric acid, but that on precipitation in presence of excess of ammonium molybdate, the compound is always contaminated by molybdic acid, presumably adsorbed.Under standard conditions the extent of adsorption is more or less constant. Finally, one must consider the solubility of the precipitate: probably even when it is allowed to stand for some time in contact with the mother liquor so that the crystals can grow, some may be lost in washing. This solubility is twofold, (a) the usual physical solubility in aqueous fluids and (b) the decomposition of the precipitate by hydroxyl ions. In gravimetric analysis this is prevented by washing the precipitate with very dilute nitric acid,3 but in volumetric analysis a neutral wash liquid must be used. Potassium nitrate solution is popular for this purpose, but is probably no better than water.So a method can only be based on the phosphomolybdate complex, with its favourable equivalent weight and freedom from interference, if a precipitate can be produced that is (a) less soluble than ammonium phosphomolybdate, (b) of constant composition, free from adsorbed or occluded impurities, and (c) free from cations that will interfere in the titration, if a volumetric process be used. The recent work5 on silica indicates how these desiderata may be attained. The complex acid should first be formed in solution, not formed and precipitated simultaneously. This means that the solution must be free or practically free from ammonium salts before the molybdate reagent is added, and that ammonium molybdate must not be used as reagent.After formation, the phosphomolybdic acid must be slowly precipitated as a salt. Quinoline seems a suitable base ; quinoline phosphomolybdate has a very low solubility and quinoline is a sufficiently weak base not to interfere in the titration. THE QUINOLINE PHOSPHOMOLYBDATE METHOD It was found that quinoline phosphomolybdate has almost all the desired properties. It contains exactly 12 moles of molybdic anhydride per mole of PO4”‘, it is very insoluble, and a volumetric method of very great accuracy has been worked out. This does not depend on empirical standardisation but simply on the reaction- (C,H,N),H3[P0,.12MoO3] + 26NaOH = Na,HPO, + 12Na2Mo0, + 3C9H,N + 14H20, which is quantitatively achieved, so that in this reaction the equivalent weight of P20, is These undesirable features are explicable.68 WILSON : THE ACCURATE DETERMINATION OF “PHOSPHORIC [Vol.76 142.08/52 = 2.732. The solubility of the quinoline phosphomolybdate is negligible and, as will be seen below, the whole procedure is of satisfactory accuracy. The quinoline phosphomolybdate produced is not absolutely pure ; attempts to work out a gravimetric method were not quite successful. Whatever the contaminant, it is neutral and has no effect on the volumetric method; it is probably water retained in the crystal lattice, or it may be a minute amount of adscrbed sodium mollybdate. For work on the usual analytical scale, the volumetric process is so good that there is little cause to regret the failure of the gravimetric process.The reactions are completely quantitative ; by the weighing of aliquots of solutions instead of measuring them a considerable potential source of error has been avoided, and by recourse to weight burettes it is likely that a further advance would be made, but so far that has not been necessary. EXPERIMENTAL The description of the experimental work is divided into three parts, (a) the establishment of the quantitative nature of the proposed method, (b) attempts to establish a gravimetric method and (c) the effect of other elements and anions, and the application to fertiliser analysis. THE QUANTITATIVE NATURE OF THE REACTION-. The best analytical methods are only quantitative within a limited range of conditions; even as simple a matter as the precipitation of sulphate ion as BaSO, is only quantitative when properly carried out and hence it is to be expected that the method underinvestigation will only be accurate if the best conditions for precipitation, etc., can be found.A preliminary experiment was performed by precipitating the phosphate from a known amount of AnalaR potassium di-hydrogen phosphate under conditions similar to those used to precipitate quinoline silicomolybdate. On dissolving the precipitate in standard sodium hydroxide solution, titrating back and calculating the PdO, present from the factor given above, 50.26 mg of P,O, were found, 50.0 mg having been present. This preliminary result was regarded as most encouraging. A series of experiments was then made to ascertain the best conditions for complex formation, precipitation, etc., and a procedure was tentatively specified.The next step was the preparation of standard solutions of phosphate, whose phosphate content should be known with the greatest possible accuracy. Two sources of P,O, seemed most suitable, either dilute orthophosphoric acid or potassium di-hydrogen phosphate. Neither is ideal: phosphoric acid is not a very strong acid, hence its reference to standard substances such as sodium carbonate is not easy; potassium di-hydrogen phosphate is not easy to analyse for all possible impurities, and there is some evidencez for the existence of a more acid compound, KH,PO,.H,PO,, that would be very difficult to detect. On the other hand it has been much used by Sarensen and others as a standard salt for preparing buffer solutions.It was therefore decided to use both these chemicals as standards and to check the accuracy of the method against both. Preparation of 0.5 M phosphoric acid solution-Dilute 56 ml of phosphoric acid (sp. gr. 1.740, and passing the AnalaR specification in every particular) to about 600ml and boil gently for half an hour to ensure that any other phosphoric acids present shall be converted to orthophosphoric acid. Standardisation-The “end-point” for the neutralisation of the first hydrogen ion of H,PO, occurs at pH 4.5, and is much sharper than the second end-point at pH 9 (see, e.g., Smith13). Experiments with indicators showed that the most definite colour change at this pH was given by a mixed indicator containing 6 parts of bromo-cresol green solution (0.4 g per litre) and 1 part of methyl red solution (0.5 g per litre).This indicator is pink at pH 4.4, green at 4.6 and blue at pH 5. Partingtons (Zoc. cit., p. 623) states that the end-point is sharper at 55” C, so titrations were made at that temperature. As comparison solution an approximately 0.5 M solution of potassium diihydrogen phosphate, containing the same amount of mixed indicator as the sample solution, was used. The standard substance was sodium carbonate, freshly prepared by heating AnalaR sodium bicarbonate to 300” C in a platinum dish until constant in weight, and cooling it in a desiccator over fresh phosphoric anhydride; between 1.26 and 1.31-g portions were weighed out, giving titrations of about 50 ml. The usual precautions were taken to remove carbon dioxide at the end-point, and a calibrated chamber burette was used for the titrations.Cool and dilute the solution to 2 litres.Feb., 19511 ANHYDRIDE" BY MEASS OF QUINOLINE PHOSPHOMOLYBDATE 69 The results were 47.40, 47.37, 47.39 and 47.34 g of per litre; average47.375 g of H3P0, per litre. So that weight aliquots could be used as conveniently as volume aliquots, the density of this solution at 20" C was carefully determined by means of a Regnault's pyknometer, of approximately 135-ml capacity, very carefully calibrated at 20" C, a similar vessel being used as counterpoise throughout. As any weighings of the solution would be made in air, the density determined is apparent (i.e., not corrected for the buoyancy of the air).The mean result of two very closely agreeing determinations was- Density at 20/4" = 1.0237. PuriJication of potassium di-hyd~~gen phosphate-According to Pascal14 this salt can be purified completely except for traces of aluminium, iron and calcium by recrystallisation from a boiling saturated solution. Accordingly, 200g of AnalaR salt were dissolved in 400ml of boiling distilled water, and the solution kept on a boiling water-bath for several hours. The clear colourless solution was rapidly cooled with constant stirring, and allowed to stand overnight. The crystals were filtered with suction on a hardened filter-paper, washed twice with ice-cold water and once with 50 per cent. alcohol, and dried in an electric oven at 105" C. The dry salt was lightly ground in a Wedgewood mortar and dried for 8 hours at 105" C with occasional stirring. It was cooled over P,O, and then regarded as pure.Even so, a 0.5 M solution after a few days deposited a few minute flocs of some white insoluble substance, possibly aluminium phosphate (compare Pascal, Zoc. cit., p. 288). This substance was readily dispersed again by shaking, and must have been present in exceedingly small amounts. For most of the work, this pure salt was weighed as such on a semi-micro balance, so avoiding any errors of volumetric measurement. The correct pH for the end-point of the titration-In the volumetric molybdate method one is titrating excess of sodium hydroxide and also hydroxyl ions from the dissociation of tri-sodium phosphate. The end-point for the phosphate part of the titration (Na,P04 to Na,HPO,) is about pH 9; from the data of Travers and Malaprade15 it can be seen that the end-point for the titration of molybdic acid is about pH 8, or rather higher.As there are 12 moles of molybdic acid present per mole of phosphoric acid, an indicator changing at about pH 8.5 is desirable. Thymol blue (pH range 8 to 9-6) is very satisfactory; it is found easier to avoid over-running the end-point by adding some phenolphthalein, which decolorises at about pH 9 and indicates the approach of neutralisation. TABLE I A faint turbidity appeared, so the solution was filtered through a pulp pad. DETERMINATION OF KNOWN AMOUNTS OF PHOSPHATE Phosphoric acid Potassium di-hydrogen phosphate r P,O, present, mg 8.15 13-76 20.67 27-64 34-72 17.13 30.68 47.44 7 I ~- P,O, found, P,O, present, mg mg 8.00 16-65 13.73 31.87 20-50 53-81 27.52 26.02 34-63 42.34 17-20 30.65 47.45 v P,O, found, mg 16.72 31-96 53.53 26-00 42-11 NoTEs-(~) In all the above experiments the amount present was unknown to the analyst.(2) The first five results are low by an average amount of 0.1 mg. This was found to be due to a blank that was too high. In subsequent tests the volumetric solutions used for the blank were one-fifth as strong as those used in the test: it can be seen that the bias in subsequent results is negligible (-0.02 mg) and that the average error is $-0-07 mg. Conditions of fvecipitation-By measuring known volumes of the 0.5 M phosphoric acid solution, precipitating under various conditions, washing and titrating the precipitate, the best acidity and temperature of precipitation were found.Acetic acid was shown to be unnecessary. The exact amount of hydrochloric acid present does not seem to be very important, as long as enough is present to prevent the precipitation of quinoline molybdate; 40 ml of diluted hydrochloric acid (1 + 1) per 100 ml of original solution is very satisfactory, and 50ml will do no harm, By addition of 50ml of concentrated acid to 100ml of the70 WILSON : THE ACCURATE DETERMINATION OF “PHOSPHORIC [Vol. 76 original solution prior to addition of the quinoline, results are somewhat low (e.g., 24.04 mg of P,O, recovered from 25 mg). The phosphomolybdate complex is readily formed at the concentration of 20 ml of concentrated hydrochloric acid per 100 ml of solution, especially when warm.Precipitation of the quinoline salt should take place slowly from boiling solution (see below under “Procedure” for details). The “blank,” which is probably due to silica from the glassware, is important. The aliquots of 0-5 M phosphoric acid solution were weighed in stoppered weighing bottles on an analytical balance, the aliquots of di-hydrogen phosphate on a semi-micro balance. It is considered that these results completely establish the validity of the volumetric method. Reszclts-Table I summarises the results on known amounts of phosphate. ATTEMPTED GRAVIMETRIC METHOD- Numerous attempts to establish a gravimetric method were made, as this would avoid all errors associated with volumetric measurements. The insoluble salt was precipitated as in the volumetric process, washed several times with 1.5 N hydrochloric acid, then washed free from acid with cold water and dried to constant weight. It was ascertained that the precipitate was slightly hygroscopic, but could ‘be dried to constant weight in 2 hours at 105” C ; further drying at 120” C did not result in any further loss of weight.The results were rather disappointing; the precipitates were not quite pure enough, and results calculated on the theoretical molecular weight were high. In Table I1 the weight of precipitate obtained from various weights of P,O, is given, also the “factor” (P,O, per gram of precipitate) and the apparent molecular weight of each precipitate. By comparing this with the theoretical molecular weight, the degree of contamination can readily be seen.TABLE I1 GRAVIMETRIC RESULTS WITH QUINOLINE PHOSPHOMOLYBDATE Weight of P,05 taken, mg 11.31 22.41 31.76 13.99 20.54 14*78* 29.46* 10.51 17-21 27.51 15*06* Average Theoretical . . . . Weight of precipitate obtained, mg 353.6 668.7 992.4 440-0 642.7 462.8 917.8 332.3 540.3 864.8 472.0 . . . . .. .. .. ‘ ‘Factor” 0.03199 0.03202 0.03200 0.03176 0.03 196 0.03194 0.03210 0.03163 0.03185 0.031 81 0.03 191 0.03 19 1 0.03210 Apparent molecular weight of precipitate 2219.8 2218.2 2219.1 2233.6 3222.1, 22 2 3.8 321 3.4 22445 2229.6 2232.5 2226.0 2225.6 2212.8 * In these experiments, the source of P,O, was potassium di-hydrogen phosphate, in the remainder it was 0.5 M phosphoric acid. The nature of the contaminant was not discovered; it cannot be quiiioline molybdate or molybdic acid, or the volumetric method would have been in error ; it is not sodium chloride or hydrochloric acid as chloride could not be detected in the precipitates.It may be water very obstinately retained, or sodium molybdate, but the difficulties of analysing accurately for excess molybdate are very great. Preliminary experiments have shown that the gravimetric method can be used in micro- chemical analysis; up to 4 mg of P,O, the precipitates are of the correct composition. It is of interest that the precipitate is very readily soluble in acetone, but not in ether. ATTEMPTS TO PREPARE CINCHONINE PHOSPHOMOLYBDATE- By using the same technique, but substituting cinchonine for quinoline, a creamy- yellow precipitate is readily obtained.It is precipitated in a very finely divided state, filters badly and is difficult to remove from the sides of the beaker. The molecular weight of 3(C,,H22N,0)H,P04. 12Mo0, is 2708-2 ; in our experiments the molecular weight foundFeb., 19511 ANHYDRIDE” BY MEANS OF QUINOLINE PHOSPHOMOLYBDATE 71 was 2352,2365 and 2353. It is suggested that because of the peculiar shape of the cinchonine molecule, the crystal lattice may be unable to accommodate the theoretical amount of the base. Pyridine was also unsatisfactory as a precipitant because pyridine phosphomolybdate is not sufficiently insoluble. APPLICATIONS OF THE VOLUMETRIC METHOD- Only those ions likely to be important in fertiliser analyses were investigated, viz., (a) lime, (b) fluorine, (c) ammonia, ( d ) iron, (e) magnesia, (f) alkali salts, (g) citric acid, (h) ammonium citrate, (i) nitric acid and ( j ) sulphuric acid.(a) Lime-The presence of 1 g of lime has no effect on the determination (50 mg of P,05 present). (b) Fluorine-In the presence of 2 ml of hydrofluoric acid, results were somewhat high (e.g., 51.5 mg instead of 50 mg). This is due to attack on the beaker, bringing silica into solution, and the effect can be avoided by adding boric acid. For example, to 50 mg of P205 were added 3.5 g of calcium carbonate, 5 g of boric acid and then 2 ml of 40 per cent. hydrofluoric acid. Hydrochloric acid was then added until the calcium carbonate dissolved and the determination was continued as usual; 49.96 mg of P205 were found in one experiment and with a larger quantity of calcium carbonate, 50.08 mg.(c) A mmonia-Ammonia, as might be expected, interferes. For example, in presence of 1 g of ammonium sulphate, only 47.83 mg of P205 were found. The ammonia can readily be destroyed by sodium nitrite, aqua regia or hypobromite. With sodium nitrite, a result of 49-96 mg was obtained; with aqua regia, 49.84mg. In the method finally adopted, hypobromite was used because of convenience and speed. One gram of pure iron was dissolved in a few milli- litres of nitric acid and added to 25 ml of a solution containing 50 mg of P20,; the determined amount of P205 was unaltered. (e) Magnesia-Two grams of MgS0,.7H20 had no effect on the determination of 50 mg (f) Alkali salts-Five grams of potassium chloride and l o g of sodium sulphate added to a 50-mg aliquot of P,05 have no effect on the determination.(g) Citric acid and ammonium citrate-These salts are important in fertiliser analysis. Half a gram of each was dissolved in a 25-ml aliquot (that is equivalent to a 2 per cent. solution) so that it would represent the solution obtained by extraction of a sample with 2 per cent. citric acid or ammonium citrate solution. The 2 5 4 aliquot (of potassium di-hydrogen phosphate solution) contained 49.64 mg of P20,, and the titrations of the solution, the citric acid and the ammonium citrate solutions were identical. (h) Nitric acid-Substitution of an equivalent amount of nitric acid for hydrochloric acid in the analysis made no difference to the results. ( j ) SuZ$huric acid-With this present instead of an equivalent amount of hydrochloric acid results are erratic and high (cj.other workers7s9). Besides the normal yellow quinoline phosphomolybdate, a white solid is also precipitated. This was not analysed, as it was impossible to separate it from the yellow precipitate. Even in the absence of quinoline and phosphate, molybdic acid is slowly precipitated when sodium molybdate solution is heated with dilute sulphuric acid; this does not occur with hydrochloric acid, at least in the concentra- tions used in this analytical method. Precipitation occurs much more readily if some ammonium sulphate is also present. The amounts of SO,” and NH,’ in the precipitate are quite small, and adsorption seems more likely than formation of compounds. If hydrochloric acid is present in amount slightly in excess of the sulphuric acid, the interference is prevented, but the total acidity should not be greatly in excess of 2 N.It is preferable to avoid large excesses of sulphuric acid, and until further investigation has been made, the volumetric molybdate method should not be applied, e.g., to determining phosphorus present in organic substances that have been oxidised by Kjeldahl’s method. COMPARISON BETWEEN THE NEW METHOD AND OTHER METHODS Before using the method it is necessary to be sure that it is free from interference. (d) Iroiz-Iron is without effect. of P,O,. Various samples have been analysed by the new and older methods. Table I11 Thirty-four samples of mixed fertiliser were analysed for water-soluble P20, by two summarises the results.[Vol.76 72 analysts working independently (but using the sarne volumetric solutions). The mean differ- ence between the duplicates was 0.027, corresponding to a standard deviation of 0-024. This may be compared with the standard deviation of the molybdate magnesia method of the Act; two analysts working independently analysed 27 samples of a similar mixture for water-soluble P,O,, and the standard deviaticln was found to be 0.065. WILSON : THE ACCURATE DETERMINATION OF “PHOSPHORIC TABLE I11 COMPARISON BETWEEN METHODS OF ANALYSIS r-__-_-_--h- - Fertiliser and Quinoline Method Sample Feeding Stuffs Act, pliosphomolybdate, Keniarks P206, % P2°5, 9’0 Morocco rock 1 . . . . 33.1 33-29 Total P& 33.29 Morocco rock 2 .. . . Superphosyhate . . . . Mixed fertiliser sample No. 1 Mixed fertiliser sample Solution containing, N, Ditto . . . . . . No. 2 P205 and K,O Calcium phosphate . . 33.0 20.15 20.12 10.72 10.72 10.67 10.69 11.61 11-64 4.88 4.89 5.60 5.27 5.57 5.28 5-56 5.29 5.59 542 44.48 44.51 44.45 44.38 44.52 44.38 32.87 20.29 20.23 10.66 10.66 11.52 11.49 4.81 4-82 5-56 5.57 44.24 44.24 44.24 Total P,O, Total PzO, 10.64 by F. & F.S. 10-66)citrate method Water-soluble P,O, Water-soluble P,O, 5.60 per cent. of P,O, present Total P,@, Finally, several samples of fertiliser were specially prepared by grinding to pass a 200-mesh sieve. Three analysts each received two portions of each sample, and made one analysis of each, with results as shown in Table IV. TABLE I:V Each was well mixed, and divided into six portions.TESTS BY VARIOUS ANALYSTS, USING QUINOLINE PHOSPHOMOLYBDATE METHOD Analyst Sample Mixed fertiliser X . . .. . . Mixed fertiliser I’ . . . . . . Mixed fertiliser 2 . . .. . . Phosphate rock . . . . . . H. N. R. P,!O,, ‘X 11:23 11-19 15.47 15.39 18.62 18.63 33-20 33.15 J . S. p2°5@ % 11.23 11.18 15.35 15.37 18.60 18-65 33-38 33.39 11,-1:3 11.14 15.38 15.34 18.59 18.60 33-23 33.17 METHOD Hydrochloric acid-Concentrated, and diluted to 1 + 9, 0.5 N and 0.1 N solutions. Sodium hydroxide-0-6 N and 0.1 N solutions, both free from carbon dioxide, made from AnalaR pellets. Sodium molybdate solution-A 15 per cent. solution of the hydrated salt in water. (See note 1 below.) Quinoline hydrochloride solution-Add 20 ml of redistilled quinoline (synthetic quinoline has been found preferable to the usual coal tar product) to 800 ml of hot water acidified with 25 ml of concentrated hydrochloric acid, and stir well. Cool to room temperature, add paper REAGENTS-Feb., 19511 ANHYDRIDE” BY MEANS OF QUINOLINE PHOSPHOMOLYBDATE 73 pulp and again stir well.Filter with suction through a pulp pad, but do not wash. Dilute t o 1 litre with water. Boric acid-Pure. Bromine water-Distilled water saturated with bromine at room temperature. Perchloric acid-AnalaR, 60 per cent. Mixed indicator solution-Mix two volumes of 0.1 per cent. phenolphthalein solution with three volumes of 0.1 per cent. thymol blue solution (both in alcohol). PROCED WRE- A. Total P20, in basic slag, phosphate rock, szcperphosphate, etc.(not containing ammonia)- (1) For samples of normal P,O, content, weigh 2.500 g into a 150-ml beaker, add 20 ml of water and 10ml of perchloric acid. (See note 2.) Warm until most of the sample is dissolved, then evaporate until fuming, and continue to heat while gently fuming with the beaker covered for a t least 15 minutes, until attack is complete. Allow to cool, rinse the cover with a little distilled water, allowing the rinsings to run into the beaker, add 20 ml of diluted hydrochloric acid (1 + 9), warm carefully and then boil until all salts are in solution. Filter through a 9-cm Whatman No. 30 filter-paper in a 2-inch funnel into a stoppered conical 125-ml flask, which has been weighed to the nearest 0.5 mg, preferably with a similar flask as a counterpoise.Transfer the contents of the beaker to the filter with the minimum of water, using a wash bottle with a fine jet and a succession of small washes, allowing each t o run through before adding the next to the filter. Wash the filter thoroughly with small washes of warm water in the same way. The total volume of washes and filtrate will be from 100 to 110ml. Discard the filter-paper. Carefully dry the outside, and weigh to the nearest 0.5 mg. Call the weight of solution A grams. Weigh a dry stoppered weighing bottle, 6 x 3 cm diameter. Transfer to the weighing bottle, by means of a pipette, an aliquot that should contain about 50 mg of P20, (but not more than 60 mg), e.g., if the material contains about 20 per cent. of P205, an aliquot of about 10 ml is desirable.Stopper the weighing bottle and weigh again, to the nearest 0.5 mg. Call the weight of the aliquot B grams; then the weight of the sample taken for analysis is 2-5 x B / A g. (3) Wash the stopper of the weighing bottle, collecting the washings in a 500-ml conical flask (this is precautionary as the stopper should not have become wet) and then quantitatively transfer the aliquot to the flask, washing the weighing bottle with about 90ml of cold water. Add 20ml of concentrated hydrochloric acid, then 30ml of sodium molybdate solution. Raise the temperature to boiling, and from a burette with a coarse jet add a few drops of quinoline solution. Swirl the solution in the flask during the addition, again heat to boiling and add quinoline solution drop by drop with constant swirling until 1 or 2 ml have been added. Again boil, and to the gently boiling solution add the reagent a few millilitres at a time, with swirling, until 60 ml in all have been added.In this way a coarsely crystalline precipitate with good filtering properties is produced. Allow the solution to stand in a bath of boiling water or on the edge of a hot plate for 15 minutes, then cool to room temperature. (4) Prepare a paper-pulp filter in a funnel fitted with a porcelain cone, and tamp well down. Decant the clear solution through the filter, and wash the precipitate twice by decantation with about 20 ml of hydrochloric acid (1 + 9). (See note 4.) Transfer the precipitate to the pad with cold water, washing the flask well, and wash the filter and precipitate with cold water, with small washes of about 25 to 30m1, letting each wash run through before applying the next, until the washings are acid-free.(Test for acidity with litmus paper; 6 washes are usually sufficient). Transfer the pad and precipitate back to the original flask (now acid-free). Insert the funnel in the flask and wash it well with water to make sure that all traces of precipitate are transferred; use about 50ml of water. Shake the flask well so that filter-paper and precipitate are completely broken up. From a calibrated burette or pipette run in exactly 50.0 ml of 0.5 N sodium hydroxide solution, swirling the flask during the addition. Shake until the precipitate is completely dissolved. Add a few drops of indicator solution and titrate with 0-5 N hydrochloric acid.The end-point is very sharp; the solution becomes pale green and at the end-point suddenly changes to pale yellow. Record the volume of hydrochloric acid used to within 0.03 ml, and subtract from the volume of 0.5 N sodium hydroxide solution (50.0 ml). (2) Cool the flask, stopper it and shake to mix the contents thoroughly. This need not be accurately measured. (See note 3.)WILSON : THE ACCURATE DETERMINATION OF “PHOSPHORIC [Vol. 76 74 Run a blank on all reagents, excluding only the aliquot of sample solution, but use 0.1 N acid and alkali solutions for the titration a i d calculate it to 0.5 N sodium hydroxide. Subtract this blank from the volume neutralised by the original precipitate (see note 5). 1 ml of 0.5 N sodium hydroxide soluticn = 1.366 mg of P205 and B.Total P,O, in mixed fertilisers containing ammonium salts- (1) Weigh 2.500 g of sample into a 150-ml beaker, add 1 g of boric acid and 20 ml of water. Warm until the boric acid is dissolved. Add 10ml of concentrated hydrochloric acid (see note 6), evaporate to dryness and bake gently for 15 minutes. Allow to cool, moisten the residue with 1 to 2 ml of concentrated hydrochloric acid and add 15 ml of hot water. Warm until the salts are dissolved and proceed as under A (1) from “Filter through a 9-cm Whatman No. 30 filter-paper . . . .” (2) Proceed as A (2) above. (3) Transfer the aliquot to a 500-ml flask a!; under A (3), but only use 70 ml of water. Add 2 g of sodium hydroxide (about 10 pellets) and swirl the solution until the pellets have dissolved.This amount of sodium hydroxide will normally be sufficient; enough should be present to liberate all the ammonia from its salts and leave an excess. Add from 20 to 40 ml of bromine water, according to the quantity of ammonia present (10 ml of bromine water destroys about 20 mg of ammonia). Mix the solution, allow it to stand for 5 minutes, acidify with concentrated hydrochloric acid added dropwise (normally about 6 ml of acid is required) and boil gently to remove excess of bromine. If necessary, adjust the volume to 100 ml, add 20 ml of concentrated hydrochloric: acid, and from this point proceed as under A (3) above. C . “ Water-soluble P205,” ammonium salts absent.- (1) Weigh 10 g of sample into a 500-ml volumetric flask (class A calibration) and add 400 ml of water a t 20” C.(See note 7.) Dilute to the mark with water, mix well and filter through a dry Whatman No. 31 filter-paper. Reject the first 20 to 30 ml and then collect the filtrate. Adjust the temperature of the filtrate to 20” C. With a calibrated pipette (25 or 50 ml, according to the amount of soluble P,O, expected), transfer a suitable sized aliquot to a 500-ml conical flask. Dilute to about 100 ml with water. (2) Add 20 ml of concentrated hydrochloric acid and from this point proceed as under A (3) above. D. LLWater-soluble P205,” ammonium salts @ese&- (1) Proceed as C (1) above. (2) Proceed to the destruction of ammonium salts as in B (3) above, and complete Shake on a machine for exactly 30 minutes. the determination as in A (3) above.NOTES ON THE PROCEDURE- 1. The sodium molybdate solution should not be kept too long, as it tends to dissolve silica from the glass bottles. 2. Some samples of phosphatic minerals have been found to be very resistant to attack by hydrochloric acid, but so far we have come across no samples that did not yield all their phosphate to an attack with perchloric acid. This procedure has also the advantage of completely removing fluorine, hence precautions to prevent later attack on beakers (addition of boric acid) are not necessary. 3. Volumetric measurements cannot be as accurate as weighing, so for work of great accuracy it seems desirable to eliminate measurement of volume as far as possible. It would be possible to weigh an aliquot of 100 mg or so direct on a semi-micro balance, but in non- homogeneous materials there is always some doubt as to how truly representative a very small sample may be.With a modern aperiodic balance taking a 200-g load, the procedure outlined here is quite speedy and reduces any error in taking the aliquot to negligible pro- portions. For referee work, it is probable that use of weight burettes for all “volumetric” work, the solutions being standardised on a kilogram basis instead of a litre basis, wouldFeb., 19511 ANHYDRIDE” BY MEANS OF QUINOLINE PHOSPHOMOLYBDATE 75 result in a further gain in accuracy at the cost of a very small loss in time and convenience. Errors due to changes in temperature would vanish. 4. The preliminary washes with acid remove most of the excess of quinoline and molybdate, and prevent errors through the precipitation of quinoline molybdate or molybdic acid in the pad or precipitate.5. The “blank” is important; it is mostly due to silica, and must be carefully determined. It should not be more than about 0-4 or 0-5ml; about 0.2ml comes from the sodium molybdate and the remainder from the sodium hydroxide and the glass apparatus. Soft soda or potash glass must not be used. Flasks that become scratched or etched must be discarded; occasionally a flask that has been satisfactory may begin to yield appreciable quantities of silica. 6. This treatment is satisfactory in rendering silica insoluble; perchloric acid is not used, as the low solubility of ammonium perchlorate in water (and its rather unstable nature) may give rise to trouble.The addition of boric acid is essential if fluorides are present, and should precede acidification with hydrochloric acid. 7. “Water-soluble P205” has only one meaning in fertiliser analysis-as defined in the Fertiliser and Feeding Stuffs Act regulations. The regulations specify 20g of sample and final dilution to 1 litre. Use of 10 g, as described, is satisfactory, and 500-ml flasks are easier to accommodate in shaking machines. The time of shaking must be adhered to, and filtration must take place immediately, because of possible slow reactions between the solid phase and the solution. We have not found that silica in appreciable quantities passes into solution during this treatment, but if a fertiliser of alkaline reaction were examined, silica might be dissolved.Acknowledgments are made to Mr. H. N. Redman, who carried out most of the experi- mental work. REFERENCES 1. Bassett, H., “Theory of Quantitative Analysis,” pp. 81-88. 2. Epperson, A. W., J . Amer. Chem. SOC., 1528 50, 321. 3. Fertiliser and Feeding Stuffs Act Regulations, 1932. 4. Hoffmann, J. I., and Lundell, G. E. F., J . Res. Nut. Bur. Stand., 1937, 19, 69. 5. Wilson, H. N., Analyst, 1949, 74, 243, 6. Partington, J. R., “Inorganic Chemistry,” 4th Ed., p. 940. 7. Falk, K. G., and Sugiura, K., J . Amey. Cliem. SOC., 1915, 37, 1507. 8. Johnson, C. M., “Chemical Analysis of Special Steels,” 4th Ed., p. 315. 9. Allen, H. R., and Gault, L., J . Ass. Off. Agric. Chem., 1947, 30, 135. 10. Lunge, G., and Keane, C. .4., “Technical Methods of Chemical Analysis,” 2nd Ed., Vol.111, 1931, 11. Bourdon, D., and Cotte, J., Bul. SOC. Chim. Fv., 1949, 16, 429. 12. Parker, E. G., J . Phys. Chem., 1914, 18, 653; Chem. Abst., 1915, 9, 168. 13. Smith, T. B., “Analytical Processes,” 2nd Ed., p. 183. 14. Pascal, P., “Traiie’ de Chimie Minkvale,” Vol. 6, 1934, p. 287. 15. Travers, A, and Malaprade, Bul. SOC. Chim. Fr., 1926, 39, 1413. pp. 536-538. IMPERIAL CHEMICAL INDUSTRIES LIMITED RESEARCH DEPARTMENT BILLINGHAM, Co. DURHAM DISCUSSION MR. L. GANELLIN asked whether the author had had any experience in the determination of phosphate in basic slag by the quinoline method, and in particular, with citric acid soluble P,O,. It was his experience that silica soluble in the citric acid extract interfered in molybdic precipitates and had t o be removed by the standard precipitation method.MR. WILSON replied that he had not had any experience with citric-soluble P,O, in basic slag by this method, but he thought that silica, if extracted, might interfere. This interference could probably be avoided. The yH range over which silicomolybdic acid was formed was narrow and, a t the acidity prescribed, conversion of silica to silicomolybdic acid would be far from complete. Moreover, it was only from fresh unpolymerised silicic acid that silicomolybdic acid was formed. If to the aliquot of citric acid solution taken for analysis, 20 ml of concentrated hydrochloric acid were added, as in the usual procedure, and the mixture was gently boiled for half an hour and then diluted to the proper volume before addition of the sodium molybdate solution, i t was improbable that silica would interfere.The author had pointed out the high standard that he had set himself, in particular in that he hoped the method would not embody any empirical factors. With this in mind, would i t not have been more desirable to offer the volumetric procedure merely as a quicker alternative to the gravimetric procedure; if SO, was MR. R. C . CHIRNSIDE complimented the author on a lucid account of an elegant piece of work.76 WILSON [Vol. 76 the constitution of the quinoline phosphomolybdate precipitate clearly known ? Had the author considered the possible application of X-ray diffraction methods to i3 study of this precipitate? It might well be that a critical examination of the patterns so obtained would throw light on the nature of the attachment of the water that the author believed the precipitate contained.It would show whether the water was in the crystal lattice or was merely firmly adsorbed, and so enable conditions for its complete removal to be found. Similarly, some light might be thrown on the nature of the cinchonine precipitates that the author mentioned earlier in the paper. MR. WILSON replied that i t was likely that X-ray diffraction methods would prove valuable in the investigation of the persistent moisture in the precipitake. He agreed that a gravimetric method would have been the most desirable but, as was clearly shown in Table 11, the gravimetric results were not quite good enough, whereas the volumetric method gave acceptable results.Whilst i t was still possible that a Favimetric procedure might be evolved, he thought t h a t the investigation of weight titration was more likely to lead to a method of the highest possible accuracy. MR. J. G. SHERRATT enquired whether the method was applicable to organic fertilisers and feeding stuffs, such as animal residues containing bone, and if so, what procedure was recommended for the destruc- tion of organic matter. Hitherto he had invariably used sulphuric acid to oxidise organic matter prior to the determination of P,O,, but this involved the su'bsequent presence of ammonium sulphate and an excess of sulphuric acid in the final solution, both of which interfered with the proposed new method. MR. WILSON replied that i t was preferable not to use sulphuric acid, but if i t were used as much as possible should be evaporated, and the remainder neu-tralised by sodium carbonate before the addition of hydrochloric acid.Although there might be hesitation about the use of perchloric acid with organic matter, this mixture had been used in the wet oxidation of organic compounds (see, e.g., J . A s s . 08. Agric. Chem., 1943, 22, 182, or Shirley Institute Memoirs, April, 1949, Vol. 23). Hamlin, in the latter of these papers, described the successful destruction of up to 5 g of cotton yarn with a mixture of 14 ml of perchloric acid, 20 ml of nitric acid and only 1 ml of sulphuric acid. MR. C . G. DAUBNEY asked, in view of the reliance placed on AnalaR chemicals, whether the author would disclose the difference between AnalaR phosphate and the recrystallised phosphate used as his standard material to prepare the standard phosphate solution.MR. WILSON said that in this investigation he had wished to neglect no possible precaution, and there- fore had purified the material. DR. J. H. HAMENCE said that workers in his laboratory had spent many years trying to evolve a really efficient volumetric method for the determination of P,O,, but without any real success, and he therefore would like to congratulate Mr. Wilson on his new method. In the absence of interfering substances, the St. Gobain method had been found to give very satisfactory results. The St. Gobain method was based on the well-known Pemberton method, with the difference that neutral formaldehyde was added for the final alkali titration; this gave a very much sharper end-point, since i t removed the ammonia radical. This procedure gave excellent recoveries with simple phosphates and with water solubles from super- phosphate, but unfortunately the process broke down in the presence of fluorine and silica. Although many modifications of the method had been tried, he had never been successful in avoiding interference from silica other than by the somewhat lengthy process of removing silica prior to solution in acid. In view of this difference, he would be pleased if the author would amplify the procedure that he employed to avoid interference by fluorine and silica, particularly in substances like basic slag and apatite phosphate. MR. WILSON said that, for compounds like apatites that were soluble only with difficulty, he thought the treatment with perchloric acid was best. He had come across no instance in which fuming a finely ground sample with perchloric acid did not bring into solution the whole of the P,O, in reasonable time. This treatment also removed hydrofluoric acid. If trea.tment with hydrochloric acid was preferred, boric acid should first be added to combine with the fluorine. Mr. Wilson added that he thought the relative freedom from interference of his method as compared with the ammonium phosphomolybdate method was explained in part by the difference in crystal structure of the precipitates; whereas ammonium phospho- molybdate was cubic, the quinoline compound was of a much lower class of symmetry] and i t might well be due to the different crystal lattice that the precipitate crystallised pure, without including or absorbing foreign ions from the solution. It was possible that a mixture of nitric and perchloric acids could be used.

ISSN:0003-2654    

DOI:10.1039/AN9517600065

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Feb., 19511 HARRISON 77 The Microbiological Assay of Growth Factors after Separation by Paper Chromatography BY J. S. HARRISON (Presented at the meeti.Pzg of the Biological Methods Group o n Thursday, M a y 26th, 1949) A general method is described for the separation, identification and estimation of growth factors by means of paper partition chromatography. The apparatus consists of two crystallisation dishes of different diameters, one placed within the other; the inner dish, containing a paper cylinder, is covered by a tall beaker or bell-jar. On completion of the chromatographic separation and after removal of the solvent by evaporation, the positions of the factors on the chromatogram are located by placing strips cut from the paper on a synthetic agar medium that is deficient in the growth factor and inoculated with a suitable organism.The identified zones are then cut out and extracted with water, made up to known volume and aliquots submitted t o microbiological assay. The technique described has been applied with success to some of the growth factors in molasses. MANY natural products contain mixtures of growth factors that have similar stimulatory effects on micro-organisms ; for instance, pyridoxine, pyridoxal and pyridoxamine all stimulate certain strains of Saccharomyces carlsbergensis to the same extent. These three compounds have been estimated in mixtures by Rabinowitz and Snel1,l who used a differential micro- biological assay with three organisms, and they have been separated qualitatively by Winsten and Eigen,2 who used paper chromatography.We are not aware that any accurate quantita- tive determinations of growth factors have been carried out after separation,* although various workers have estimated amino acid$** and other substances, including inorganic compoundsY6 by soaking the portion of the paper that contains the substance in water and carrying out chemical tests on the solution. This general method has been developed to separate and estimate growth factors by microbiological assays. In particular, the technique has been applied to molasses for the quantitative separation of pantothenic acid from p-alanine and of biotin from desthiobiotin. After chromatographic separation, the growth factors were identified qualitatively by an agar plate method that fixed the positions on the chromatogram compared with those of the chemically pure factors, which were run separately on the same sheet.Microbiological assays were then carried out on the extracts from the portions of the chromatogram containing the separated factors. EXPERIMENTAL SEPARATION- Mixed growth factors in artificial mixtures or in natural products were separated by single-dimensional paper partition chromatography. The apparatus consisted of two crystallisation dishes of different diameters, the larger holding water saturated with the solvent and the smaller, which was placed inside this, containing the solvent saturated with water. A tall inverted beaker or a bell jar was placed over the smaller dish and fitted inside the larger. A sheet of Whatnian No. 1 filter-paper, 15 inches square, treated with suitable quantities of the test sample, was rolled into a cylinder, which was held together with metal clips and stood in the smaller dish.On account of the small amounts of growth factors present in some natural materials, it was often necessary to run on the chromatogram much more raw material than could be applied as one spot. This difficulty was overcome by applying the test material in a line along the lower edge of the filter-paper by means of a capillary pipette in such a way that the * Since this paper was presented, various methods have been described in the literature, e.g., J. P. Bowden and W. H. Peterson, -1. Biol. Chevn., 1949, 178, 533; J. T. Holden and E. E. Snell, Ibid., 1949, 178, 799; and H. Yacowitz, L.C. Norris and G. F. Heuser, Proc. SOC. Ex$. B’iol. _Wed., 1949, 71, 372.78 HARRISOX : THE MICROBIOLOGICAL ASSAY OF GROWTH FACTORS [Vol. 76 total amount of solids deposited per inch was not greater than 2 mg, otherwise the concentra- tion of solids was high enough to interfere with the running of the chromatogram by holding back part of the growth factors and so causing tailing. Beyond the end of the line of test substance were placed separate marker spots of the growth factors under examination, and the chromatogram was run with the chosen solvent. Tests with mixtures of amino acids, which gave easily identifiable colour reactions, showed that the line of substance being chromatographed travelled in a band parallel to the lower edge of the paper. IDENTIFICATION- After evaporation of the solvent, vertical strips above the marker spots were cut out and a narrow vertical strip of known width was cut from the centre of the portion of filter- paper holding the sample.These strips were placed on a large plate of synthetic agar medium deficient in the growth factor and inoculated with a suitable organism. The strips were removed after 5 minutes, by which time a proportion of the growth factors had been transferred to the medium. After incubation overnight, zones of growth marked the points to which the growth factors had travelled. This step was necessary because the RF values varied somewhat, and if the zones were near together it was important to locate them exactly. The zones given by the pure sub- stances were used to identify the naturally occurring factors. If the RF value for the natural factor and that of the pure substance agreed when several solvents were used, this was taken as evidence that the two were probably identical.ESTIMATION- Horizontal strips slightly wider than the zones of growth found with the sample under test were cut from the chromatogram. These were separately extracted with water, using a Waring blendor to homogenise the paper, which was then removed from the solution by filtering or centrifuging. The solution was made up to a known volume and aliquots were used to carry out a microbiological assay. BIOTIN AND DESTHIOBIOTIN The chromatographic method was first tested with biotin and desthiobiotin, because a reliable differential assay method was available to check the results.Most strains of yeast respond to both biotin and desthiobiotin, while a smaller number of yeasts, as well as certain other organisms, such as Lactobacillus arabinosus, respond specifically to biotin. By use of typical organisms in these two groups, the amount of biotin-like and desthiobiotin-like TABLE I RF VALUES OF BIOTIN AND DESTHIOBIOTIN RF value r A 7 Growth factor Phenol Butanol Collidine - lutidine Biotin, pure . . . . . . .. . . . . 0.95 0.23 0.70 and 0.85 29 in blackstrap molasses . . . . . . 0-95 0.33 - 33 in high test molasses . . . . . . __ 0-23 - Desthiobiotin, pure . . . . . . . . . . 0.86 0.48 91 in blackstrap molasses . . . . 0.88 0.52 9 9 in high test molasses . . .. - 0-38 2 7 in refiners’ cane molasses . . . . - 0.45 0.83 - substances in natural products such as molasses can be estimated.There has naturally been some doubt as to the precise identity of these substances, particularly those that react as desthiobiotin, in the absence of any test more specific than the microbiological response. Chromatograms run in butanol, phenol and a 1 + 1 collidine - lutidine mixture showed that the biotin-like and desthiobiotin-like substances in several types of beet molasses behaved in a similar way to biotin and desthiobiotin respectively. Table I gives the R F values obtained in these tests. It was found that both natural and synthetic biotin gave two distinct zones with RF values of 0.70 and 0.85 in collidine or a mixture of collidine and lutidine. Two- dimensional chromatography with the same solvents showed that the leading fraction (RF 0-85) changed progressively during the run to the slower fraction (RF 0.70).Feb., 19511 AFTER SEPARATION BY PAPER CHROMATOGRAPHY 79 Quantitative microbiological assays using S.carkbergensis or L. arabinosus as the test organism showed that biotin and desthiobiotin could be completely recovered from filter-paper after drying. After chromatography with butanol or phenol, the recovery was also 100 per cent. A sample of blackstrap molasses was tested after separation of the biotin and desthio- biotin, with the results shown in Table 11. The values quoted are the means of three or more assays. It will be seen that the biotin and desthiobiotin contents after chromatography agree well with those obtained by differential assay.TABLE I1 BIOTIN AND DESTHIOBIOTIN CONTENT OF BLACKSTRAP MOLASSES Growth factor content f 1 Differential Chromatographic* -7 Rutanol, 0 Pg/g Pg/g P d g Pg/g Growth factor Yeast, I.. avabznosus, Phenol, Biotin . . . . . . . . 1-4 1.1 1.4 1.3 Yesthiobiotin . . . . . . 0.9 - 1.2 1.2 * Assayed microbiologically with yeast as test organism. PANTOTHENIC ACID AND P-ALANINE Substances that react microbiologically as pantothenic acid and as ,5-alanine occur in molasses, but although pantothenic acid can be estimated specifically by L. arabinos~s and other organisms, a satisfactory method of assaying /?-alanine has not been found. The reason is that a-amino acids interfere with the /3-alanine activity,6 and many natural substances contain amino acids. The chromatographic method has been used to separate and estimate the two growth factors.Qualitative tests with three solvents showed that the growth factors in blackstrap cane molasses behaved chromatographically as /3-alanine and pantothenic acid. Butanol was found to give the best separation of the two factors, the RF value of /I-alanine being 0.02 and that of pantothenic acid 0.57. TABLE I11 Table I11 gives the values for various solvents. RF VALUES OF PANTOTHENIC ACID AND ,5-ALANINE RF value \ Growth factor Ghenol Butanol Collidine - lutidine Pantothenic acid, pure . . . . . . . . 0-73 0.57 0.67 3 1 - - in molasses . . . . . . 0.74 /3-Alanine, pure . . . . . . . . . . 0.63 0.02 0.2 1 75 in molasses . . . . . . . . 0.63 0.02 - Quantitative recoveries from paper before and after chromatography with butanol were close to 100 per cent., but this solvent was found to be unsatisfactory for the estimation of TABLE IV PANTOTHENIC ACID AND /3-ALANINE IN BLACKSTRAP MOLASSES Yeast assay Chromatographic : Chromatographic Growth factor direct, phenol, butanol, P=/g P6/g P 6 / 8 /3-Alanine .. . . .. . . c 70* 71 --t Pantothenic acid . . . . .. 32 30 2.5 * Obtained by correcting for inhibition due t o sc-amino acids. t a-Amino acids invalidate this estimation. 13-alanine in natural materials, because many a-amino acids had low RF values in butano and consequently the microbiological assay was invalidated. The most satisfactory results were obtained with phenol, which gave values for blackstrap molasses of 30.4 and 71.5 pg80 HARRISON [Vol.76 per g for pantothenic acid and /3-alanine respectively. The pantothenic acid content agreed with the value of 31-7 pg per g obtained by direct assay with S. cerevisiae, and the value for /3-alanine was of the same order as that obtained by differential assays corrected for the inhibitory effect of a-amino acids. Table IV shows the amounts of pantothenic acid and /3-alanine in molasses found by various methods. It should be noted that the growth factor measured is that available to the test organism. The total /3-alanine content of this sample of blackstrap molasses, determined after alkaline hydrolysis, was 170 pg per g. The chromato- graphic behaviour of the bound form has not been investigated. Preliminary tests show that pyridoxine, pyridoxal and pyridoxamine can be studied in a similar way, using butanol as the solvent.DISCUSSION OF RESULTS The technique described has already proved useful for the examination of some of the growth factors in molasses, and it is clear that modifications of the method could be used to clarify many points of a similar nature. For instance, it would be possible to examine cell contents by separating conjugates of growth factors, and to study the properties of these. If necessary, the information so obtained could be used to devise a suitable method of separating the factors on columns, when larger quantities would be available than in paper chromatography . The author wishes to thank the Directors of the Distillers Company Limited for permission to publish this paper. REFERENCES 1. 2. 3. 4. 5 . 6. RESEARCH AND DEVELOPMENT DEPARTMENT Rabinowitz, J. C., and Snell, E. E., J . Biol. Clievn., 1948, 176, 1157. Winsten, W. A., and Eigen, E., Proc. SOC. Exp. Biol. Med., 1948, 67, 513. Woiwod, A. J., Nature, 1948, 161, 169. Naftalin, L., Ibid., 1948, 161, 763. Arden, T. V., Burstall, F. H., Davies, G. R., Lewis, J. A., and Linstead, R. P., Ibid., 1948, 162, Hartelius, V., Cowipt. R z ~ . I-ab. Carlsberg., Sdv. Physiol., 1946, 24, 185. 691. THE DISTILLERS COMPANY LIMITED EPSOM, SURREY DISCUSSION DR. G. E. FOSTER asked whether Mr. Harrison had tried any chemical methods for detecting growth factors on the chromatograms. MR. HARRISON replied that he had used chemical methods only for /3-alanine. With biotin the total amount of growth factor present was only about 2 to 3 mpg, and he doubted if so small an amount could be detected chemically.

ISSN:0003-2654    

DOI:10.1039/AN9517600077

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

Feb., 19511 HILDITCH, PATEL AND RILEY 81 The Spectrographic Determination of Linoleic and Linolenic Acids BY T. P. HILDITCH, C. B. PATEL AND J. P. RILEY The values of the extinction coefficients (EiZ) for the ultra-violet absorption bands at 234 mp and 268 mp, which are developed when linoleic and linolenic acids are subjected to isomerisation with alkali under the conditions specified by Hilditch, Morton and Riley, have been re-investigated. Pure linoleic and linolenic acids that had been isolated from natural sources by physical methods alone were employed, in addition to specimens prepared respectively by debromination of tetrabromostearic and hexabromostearic acids, For linoleic acid prepared either by physical or chemical methods, and isomerised with alkali at 180" C for 60 minutes, the mean observed value of EiL at 234 mp was 908, in close agreement with the earlier value of 906.For linolenic acid prepared by debromination, the values were also usually close to those observed in the earlier work, b u t linolenic acid isolated by physical methods of separation gave somewhat different values, namely : after isomerisation at 170" C for 15 minutes, Ei:m at 268 mp = 555, and after isomerisation at 180" C for 60 minutes, Ei$'m at 234 mp = 575. It is considered that these should be used in place of the earlier values of 532 (268mp) and 569 (234mp). IN 1945, Hilditch, Morton and Rileyf recommended that, in the spectrophotometric determina- tion of linoleic and linolenic acids after alkali isomerisation as originally proposed by Mitchell, Kraybill and Zscheile,2 isomerisation to conjugated dienes should be carried out at 180" C for 60 minutes, and isomerisation to conjugated trienes (for linolenic acid) at 170" C for 15 minutes.Employing specimens of linoleic and linolenic acids prepared by debromination of the crystalline tetrabromo- or hexabromostearic acids obtained from the natural acids, these workers observed the following values of EtL with a Hilger E3 Quartz Spectrograph with sector photometer for the pure acids- E: & h Linolenic acid, alkali-isomerised at 170" for 15 minutes . . 532 268 mp Linolenic acid, alkali-isomerised a t 180" for 60 minutes . . 569 234 mp Linoleic acid, alkali-isomerised at 180" for 60 minutes . . 906 234 mp In recent and current investigations a Beckman or a Unicam photo-electric spectro- photometer has been employed, and it is now possible to isolate natural linoleic and linolenic acids by solely physical means (crystallisation or chromatographic adsorption) without recourse to the chemical procedure of bromine addition and subsequent debromination.In view of the extensive use now made of the spectrophotometric methods for the determination of linoleic and linolenic acids in natural fats, it was considered desirable to re-investigate the above reference values for Ei yk. Accordingly, specimens of linoleic acid from sunflower, safflower and Niger seed oils, and of linolenic acid from linseed and conophor oils, have been prepared by both the older chemical and the newer physical procedures and, after the standard isomerisations with alkali, have been examined in a Beckman photo-electric spectrophoto- meter and (in one instance) in the Hilger E3 spectrograph.ISOLATION OF LINOLEIC AND LINOLENIC ACIDS (a) B Y CHEMICAL METHODS- The procedure used was that described in the earlier paperf (p. 69). Tetrabromostearic acid was prepared from concentrates of linoleic acid from sunflower or safflower seed oils and debrominated to methyl linoleate by the method of R ~ l l e t t , ~ the crude ester being then fractionated in a vacuum. Hexabromostearic acid was prepared from unsaturated concen- trates of the acids of linseed or conophor oils and purified by crystallisation from xylene followed by washing with boiling ether. The purified acid was debrominated to linolenic acid by the method of Kaufmann and Mestern.* The crude acid was methylated and thea2 HILDITCH, PATEL AND RILEY: THE SPECTROGRAPHIC [Vol.76 methyl ester fractionally distilled. Six or seven fractions of distilled esters were obtained, those of maximum iodine value being used in the further investigation. The characteristics of the specimens of esters finally obtained and subsequently used for alkali isomerisation and spectrophotometric examination are given in Tables I and 11. TABLE I LINOLEIC ACID (METHYL LINOLEATE) PREPARED BY THE CHEMICAL METHOD Methyl linoleate Tetrabromostearj c (-Ap, Linoleic acid Distilled fraction acid froin & studied fraction. Yield, ",.p.> Yield, r-A-, Iodine Source % (w/w) C yo (w/w) No. Iodine value value* Safflower seed oil . ... . . 55 115.5 33 3 171.4 180.1 Sunflower seed oil concentrates (I.V. 169) . . . . . . . . a4 116 41 4 171.0 179.2 TAEZE I1 LINOLENIC ACID PREPARED BY THE CHEMICAL METHOD Methyl Hexabromos tearic acid linolenate. Linolenic Crude Purified crude fraction studied from I o d i n e Iodine Source yo (w/w) C % (w/w) C yo (w/w) value* No. value* value* A f 1 Linolenic acid Distilled acid - -7 - Yield, y.p., Yield, F.p., Yield, Iodine fraction. Linseed acid concentrates (I.V. 231) . . 65 178 35 181 33 265.3 3 259.4 270.6 270.7 Conophor mixed acids (I.V. 210). . 50 178 29 181.4 33 264.0 4 { iii:: 269.8 Conophor mixed acids (I.V. 210). . 44 178 33 181.2 32 264.2 4 259.9 271.0 * It was noticed (cf. McCutcheone) that, at high concentrations of linolenic acid or ester, iodine values determined by the Wijs method required 3 hours contact with the Wijs reagent before a constant maximum absorption was reached.For this reason 3 hours (instead. of the usual half-hour) contact with the reagent was employed for both the linoleic and the linolenic compounds dealt with in this paper when approximate purity was being approached. These 3-hour contacts arc: recorded as "iodine value*." Theoretical iodine values : linoleic acid 181.4, linolenic acid 274.1. (b) BY PHYSICAL METHODS- Attempts were made to find conditions whereby linoleic or linolenic acids could be effectively separated from small proportions of accompanying unsaturated acids by crystallisa- tion of the lithium salts from acetone, but without success. In other experiments the use of lithium or barium hydroxides in amounts sufficient only to neutralise a portion of the fatty acids present was attempted, but in none was the salt of linoleic (or linolenic) acid obtained in a condition approaching purity, whether from.solutions in alcohol or acetone or from mixtures of the two solvents. The procedure, recommended by Nicholson and Formo,s of employing a mixture of two bases (e.g., sodium and barium hydroxide) also failed to lead to the isolation of the pure acids. Recourse was therefore had to crystallisation of the acids themselves from appropriate solvents a t low temperatures. Following the general procedure recommended by Brown and Frankel6 we were able by repeated crystallisation of acids rich in linoleic acid, first from acetone at -60" to -70" C, and finally from light petroleum at -65" to -70" C, to isolate specimens of linoleic acid of high purity; but we also confirmed the experiences of Shinowara and Brown' that linolenic acid cannot be prepared free from contamination by about 10 per cent.of linoleic acid by low-temperature crystallisation methods alone. Riemenschneider, Herb and Nichols,s however, have recently described an adsorption method for the isolation of pure linolenic acid, and we found that application of their procedure to concentrates rich in linolenic acid produced by preliminary use of low-temperature crystallisation enabled us to prepare almost pure specimens of natural linolenic acid.Feb., 19511 DETERMINATIOX OF LINOLEIC AND LINOLENIC ACIDS 83 Linoleic acid-Specimens of the pure acid were obtained from the mixed acids of sunflower, safflower and Niger seed oils by crystallisation from acetone and light petroleum (b.p.40" to 60" C) at low temperatures. As an example, the isolation of linoleic acid from the mixed acids of Niger seed oil may be briefly described. The mixed acids (100 g, iodine value 139.6) were first crystallised from 10 per cent. solution in acetone at -60" C, the deposited acids being further crystallised from acetone a t -55" C, and this process was repeated twice at -55" C. The acids left in solution a t each stage were: at -60" C, 17.9 g, iodine value 157.1; at -55" C (l), 17.8 g, iodine value 169.7; at -55" C (2), 17*0g, iodine value 173.4; at -55°C (3), lO.Og, iodine value 173.6. The first batch of acids contained most of the unsaponifiable matter from the original oil, but the three succeeding batches (44 g) were united and further crystallised as below; from this stage onwards linoleic acid was concentrated in the deposited solids and not in the acids left in solution.The acids deposited at each stage, and the conditions of crystallisation, were as follows- Cry stallisation - Deposited acids r I o d i n e v a i u e * Solvent of solute, n / Temperature, O P 6 Concentration ;'(I Acetone . . . . . . . . 10 Light petroleum . . . . . . 5 Light petroleum . . . . . . 5 Light petroleum . . . . . . 6 Light petroleum . . .. . . 6 Light petroleum . . .. . . 3 Light petroleum . . . . . . 6 L - 70 38.0 173.5 - 60 33.1 176.2 - 60 28.2 177.1 - 60 23.2 177.7 - 60 17.1 180.0 - 60 11.7 179.9 - 60 5.5 179.5 The specimens with iodine values* 180 and 179.9 (theory 181.4) were used for spectro- The specimens of purified linoleic acid thus prepared and employed for spectrophotometric photometric examination after alkali isomerisation.analysis are summarised in Table 111. TABLE I11 SPECIMENS OF LINOLEIC ACID PREPARED BY LOW-TEMPERATURE CRYSTALLISATION Source of acid Iodine value* of acid studied Sunflower seed oil . . .. .. . . . . 176.9 SafRower seed oil . . . . . . .. . . 179.3 Niger seed oil . . . . .. . . . . . . 180.0 Niger seed oil . . . . . . .. . . .. 179.9 Linolenic acid-Application of the cryst allisation procedure used to isolate linoleic acid to the mixed acids of oils (linseed or conophor) rich in linolenic acid failed to give a pure product, a mixture of more or less constant composition (about 85 per cent. of linolenic and 15 per cent.of linoleic acid) being reached, which could not be further separated by crystallisa- tion alone. Thus, the mixed acids (iodine value 215) of conophor oil (iodine value 205) left in solution in acetone at -70" C about 80 per cent. of fatty acids of iodine value 242. These were crystallised nine times from 5 per cent. solutions in light petroleum at -60" to -70" C, but the acids that separated from the last three recrystallisations remained at a practically constant iodine value* of 256. Specimens of approximately pure linolenic acid were, however, obtained by submitting the methyl esters of polyethenoid concentrates of the acids of conophor or linseed oils obtained by low-temperature crystallisation to separation by chromatographic adsorption, employing the technique of Riemenschneider, Herb and Nichols.* The process may be illustrated with reference to fatty acids from linseed oil.The mixed acids (188 g, iodine value 192) from linseed oil (iodine value 182.7) were first crystallised from 10 per cent. solutions in acetone at -60" C, when 132 g of acids (iodine value 235) were left in solution. These were converted to methyl esters (iodine value 226), which were fractionated in a vacuum through an electrically-heated and packed column : from 37.5g of esters a distilled fraction (20*3g, iodine value 231-0) was obtained for use in the separation over the silica gel adsorbent.84 HJLDITCH, PATEL AND RILEY: THE SPECTROGRAPHIC [Vol.76 The adsorbent was prepared (cf. Riemenschneider, Herb and Nichols*) by thoroughly mixing 4 parts of silica gel (previously ground to pass a 30-mesh sieve and washed with aqueous hydrochloric acid and then with water until free from acid and fine particles of gel) with 1 part of Hyflo "Supercel" in a mortar and activating the mixture by heating for 2 hours at 220" C in a gentle current of carbon dioxide. 'The prepared adsorbent was cooled and at once transferred to the adsorption apparatus (a tube 4.3 cm in diameter filled to a height of 40 to 45 cm with a column of the adsorbent). The column was electrically heated and, after filling, was kept at 72" C for 3 hours in a current of carbon dioxide. It was then cooled in the stream of carbon dioxide and the packed column was wetted with light petroleum (b.p.40" to 60" C). The apparatus was so designed that throughout the separation (which occupied several days) a slight positive pressure of carbon dioxide was maintained in the system. A solution of the unsaturated esters (1945 g, iodine value 231.0) in light petroleum (97.5 ml, b.p. 40" to 60" C) was introduced into the column and, immediately after they had been adsorbed, elution with the same quality o:F light petroleum was begun, the rate of efflux of the solvent being about 250 ml per hour. This was an extremely lengthy process and, as shown in Table IV, resulted in a long series of fractions of esters being recovered, the first being of very low iodine value, and the final fractions approaching closely in iodine value to that of methyl linolenate (261.0).When this point was reached, the eluant was changed to a mixture of 98 per cent. of the light petroleum with 2 per cent. of ether, which rapidly removed the remaining methyl linolenate. This fraction (usually about 4 to 5g) was then finally redistilled through a small vacuum-jacketed fractionation column, and the middle fractions of the distillate were converted to acid and used for the spectrophotometric analysis. TABLE I'V ELUTION OF ADSORBED METHYL LINOLENA'TE FROM THE ADSORPTION COLUMN (19.5 g of esters (iodine value 231.0) of unsaturated acid concentrates from linseed oil) Elution with digkt ;betroleurn Fraction 1 2 3 4 5 6 7 8 9 10 11 Eluate, ml 2500 2900 2500 1500 2300 2300 2300 2300 1500 2000 1500 Methyl esters & 6 I.V.0.10 66.3 0.05 83.6 0.05 - 0.06 31-2 0.05 - 0.1 1 92.0 0-17 103.6 0.33 105.0 0-36 112.4 0.62 125.0 2.40 127.7 Fraction Eluate, 12 1100 13 1000 14 1500 15 1000 16 850 17 1250 18 1000 19 1000 20 1000 21 1500 22 1000 ml Methyl esters & g I.V. 1.95 209.3 1-35 230.3 1.37 242.4 1.12 244.1 0-46 246.6 0.5 1 246.G 0-38 - 0-28 250-0 0.23 - 0.29 253.5 0.17 252.3 Elution with 98% of light petroleum f 2% of ether Fraction Eluate, /-Ap, Methyl esters ml g I.V. (3.09 0-12} 255.0 23 450 24 700 25 1000 4.98 256.7 26 1000 0.36 255.5 (Total weight recovered 18.0 g = 92.1%) Fraction No. 25 had an iodine value* (Wijs, 3 h.ours contact) of 260.1 and was fractionally distilled. The third and fourth fractions of the distilled esters (1.68 and 1.73 g, iodine values* 25943 and 261.0) were combined and hydrolysed, arid the resulting acids (iodine value* 271.9) were used for spectrophotometric analysis.From a similar sequence of operations conducted on the methyl esters (iodine value 200) of the mixed fatty acids of a specimen of conophoi: oil, there was finally obtained a distilled fraction of methyl linolenate, iodine value* 259.7, .which yielded a specimen of linolenic acid with iodine value* 271.8.Feb., 19511 DETERMINATION OF LINOLEIC AND LINOLENIC ACIDS SPECTROPHOTOMETRIC EXAhlINATION OF ALKALI-ISOMERISED LINOLEIC AND LINOLENIC ACIDS 85 The specimens of highly-purified linoleic and linolenic acids isolated as described in the preceding pages by both chemical and physical methods were submitted to isomerisation with caustic potash in ethylene glycol solution, a t 170" C for 15 minutes to develop triene TABLE V ISOMERISATION AT 180°C FOR 60 MINUTES EXTINCTION COEFFICIENTS (AT 234 mp) FOR LINOLEIC ACID AFTER ALKALI Extinction coefficient determinations Isolation f A Deviation Iodine & Origin value* 30.EiFm Mean Standard Chemical (debromination) . . Sunflower seed oil . . 179.2 6 906.3 10.1 13-1 Safflower seed oil . . 180.1 6 915-5 9.7 12.5 Physical (crystallisation) . . Sunflower seed oil . . 176.9 7 912-1 4.7 6.7 Safflower seed oil . . 179.3 2 903-1 4.3 - Jl80.0 2 906.3 1.3 - Niger seed oil . . * . 1179.9 2 910.9 4.5 - TABLE VI EXTINCTION COEFFICIENTS FOR LINOLENIC ACID Extinction coefficient determinations r-- & Iodine Isolation Origin value* Chemical (debromination) .. Linseed oil . . 270-6 Conophor oil . . 271.0 A t 268 mp, after alkali isomerisation at xo. Ey& 170" C f o r 15 nzinutes 12 539.0 6 538-3 I6 559.3 Physical (adsorption) . . Linseed oil . . 271.9 Conophor oil . . 271.8 9 358.9 6 552.0 At 234 mp, after alkali isomerisation at 180" C f o r 60 nzinutes Chemical (debromination) . . Linseed oil . . 270-6 6 560-7 Conophor oil . . 271.0 6 570-3 Conophor oil . . 270.7 8 589.7 Physical (adsorption) . . Linseed oil . . 271.9 Conophor oil . . 271.8 5 572-3 6 578-5 Mean 5.8 3.7 2.2 6.2 6.8 2.1 3.2 3.1 3.6 8.0 4.0 2.5 Standard 8.0 5.5 2.9 7.4 8.9 2.8 5.5 4.4 4-6 10.0 5.4 3.4 conjugation from linolenic acid, or at 180" C for 60 minutes to develop diene conjugation from either acid. The analytical details given in the earlier paper1 (pp.69, 70) for the preparation of the alkaline glycol reagent and for the conduct of the isomerisation were carefully followed, As therein recommended, all determinations were made on the free acids and not on their esters. In some instances we employed glycerol in place of ethylene glycol 3s the isomerisation solvent (cf. Brice and Swainlo), but in our experience this led to less concordant values and we reached the conclusion that glycol is definitely preferable to glycerol for this purpose. The spectrophotometric measurements were made throughout in a Beckman spectro- photometer, but in one series, for comparison, determinations were also made on a Hilger E3 Quartz Spectrograph (as used in the earlier work). Blank determinations with the alkaline glycol solution were carried out under exactly the same conditions as the actual determinations, the spectrographic observations of the latter being made in duplicate on each isomerised specimen.After the isomerisations at 170" or 180" C, the solutions were diluted with86 HILDITCH, PATEL AND RILEY: THE SPECTROGRAPHIC [Vol. 76 purified absolute alcohol so that the values of log I,/I recorded on the Beckman apparatus fell within the range 0.3 to 0.6. Tables V and VI give a summary of the extinction coefficients recorded, respectively, for the specimens of linoleic and linolenic acid. In these tables are given the method of isolation of the acids (chemical or physical), the oils from which they were obtained, the observed iodine value of the specimen, the number of separate alkali isomerisations made in each instance, and the mean value of the extinction coejkcients observed, together with the mean and standard deviations therefrom.DISCUSSION AND CC~NCLUSIONS LINOLEIC ACID- The mean of all the observed values (Table V) for the extinction coefficient at 234mp of alkali-isomerised linoleic acid isolated by physical means from natural sources is Et& = 908. This value is within 0.3 per cent. of that formerly de terminedl (906), and we therefore consider that the latter may continue to be employed. The final mean value for linoleic acid produced chemically by debromination of tetra- bromostearic acid was very close to this figure, although the mean values for each series and the mean and standard deviations varied to a greater extent than the corresponding figures for the linoleic acids obtained by crystallisa tion.LINOLENIC ACID- Extinction coeficients at 268 mp (triene con$%gfi:tion)-Here the values of Ei& for alkali- isomerised linolenic acid isolated by purely physical (adsorption) methods differed by about 4 per cent. from that (532) observed earlier,l the final mean value being 555 (cf. Table VI). The latter value should in our opinion now be adopted as the reference value for Ei:& at 268 mp for alkali-isomerised linolenic acid. Of three specimens of linolenic acid prepared by the debromination procedure, one gave values of the same order as the above (EiL = 555 to 559), but the other two gave values similar to those observed in the earlier work for debrominated linolenic acid, namely, EiL = 538 to 539.Matthews, Brode and Brown,ll who have contributed considerably to our knowledge of the chemical properties and beha.viour of linoleic and linolenic acids alter- natively prepared by debromination or isolated by low-temperature crystallisation, have concluded that the debrominated acids may contain 12 per cent. or more of unsaturated acids that are not the same as the natural linoleic or linolenic acids. Although some of the isomers produced will presumably be geometrical (cis-trans) isomers of the natural acids, and these may isomerise on treatment with alkali at 170" to 180" C more or less similarly to the natural (wholly cis-) acids, it seems probable that, at least when linolenic acid is con- cerned, some of the chemically regenerated acid does not react to alkali to the same extent as the natural acid.It is, nevertheless, curious that in one instance the acid obtained by debromination gave the same extinction Coefficient at 268 mp after isomerisation as that given by the linolenic acids prepared by physical means. It is also evident that, although Brown et d.ll have observed similar chemical differences between regenerated and recrystallised linoleic acid. as with the linolenic acids, linoleic acids produced by either method give similar yields of conjugated diene acids when isomerised with alkali. In one instance in Table VI it will be seen that a specimen of linolenic acid was examined, after alkali isomerisation, in both the Beckman and the Hilger instruments, with identical results, The differences in extinction coefficients that have been observed are accordingly due solely to the means employed to isolate linolenic acid, and not to any difference in the spectrographic technique.Extinction coejicients at 234 mp (diene conjugation)-The earlier figure found for the extinction coefficient at 234 mp for linolenic acid after isomerisation with alkali a t 180" C for 60 minutes was EiZ = 569. The mean value determined in the present study from eleven determinations on linolenic acid isolated from linseed or conophor oils by the adsorption procedure was 575. Although this is only about I. per cent. higher than the former figure, it is suggested that the value EiZ = 575 at 234 nip should be used for the contribution of linolenic acid to diene conjugation during alkali isomerisation.The corresponding figures for linolenic acid specimens produced by debromination are less consistent but also lead to a final mean value of EiL = 575.Feb., 19511 DETERMINATION OF LINOLEIC AND LINOLENIC ACIDS 87 We recommend that the extinction coefficients for linoleic and linolenic acids to be used in spectrophotometric determinations of these acids should be as follows- E: & h Linolenic acid, alkali-isomerised at 1TO" C for 15 minutes . . G55 (formerly 532) 268mp Linolenic acid, alkali-isonierised a t 180" C for 60 minutes . . 575 (formerly 568) 234mp L noleic acid, alkali-isomerised a t 180" C €or 60 minutes . . 906 (as formerly) 234mp On this basis, the figures based on spectrophotometric analyses recorded in our publica- tions up to the present for linolenic acid in drying oils may be about 4 per cent.too high, i e . , 2 to 3 units per cent. too high in linolenic-rich oils such as conophor or linseed, and about 1 unit per cent. high in candlenut or rubberseed oils, whilst in oils such as soya bean or hemp seed there is only a fractional difference. Similarly, the figures recorded hitherto for linoleic acid are about 1 per cent. low for oils rich in linolenic acid, but not sensibly different in other drying oils. The effect on the proportions of oleic and saturated acids is somewhat greater; in oils rich in linolenic acid (linseed, etc.) the oleic acid figures may be 4 to 5 units per cent. low, and the saturated acid figures 2 to 3 units per cent. high, as recorded on the basis of the Ei& values hitherto used. It is, however, only in the mixed acids of oils such as conophor or linseed that the differences involved exceed, at the most, about 1 unit per cent. in the component acid data. We wish to express our cordial thanks to Professor R. L4. Morton, F.R.S., for the use of the spectrographic apparatus and for his assistance and advice during the course of this work, and to Mr. R. H. Creed for assistance with many of the spectrographic measurements. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Hilditch, T. P., Morton, R. A., and Riley, J. P., Analyst, 1945, 70, 68. Mitchell, J. H., Kraybill, H. R., and Zscheile, F. P., Ind. Eng. Chem, Anal. Ed., 1913, 15, 1. Rollett, A, 2. physiol. Chem., 1909, 62, 410. Kaufmann, H. P., and Mestern, H. E., Ber., 1936, 69, ( B ) , 2684. Nicholsen, R., and Formo, M. W., J . Amer. Oil Chem. Soc., 1949, 26, 329. Brown, J. B., and Frankel, J. S., J . Amev. Chem. Soc., 1941, 63, 1483; Frankel, J. S., Stoneburner, Shinowara, G. Y . , and Brown, J. B., Ibid., 1938, 60, 2734. liiemenschneider, R. W., Herb, S. F., and Nichols, P. L., J . Awer. Oil Chem. Soc., 1949, 26, 371. McCutcheon, J. W., Ind. Eng. Chem., Anal. Ed., 1940, 12, 465. Brite, B. A., and Swain, M. L., .J. Opt. SOC. 14nzer., 1945, 35, 532. Matthews, N. L., Rrode, W. K., and Brown, J. B., J . ,4???el/. Chem. Soc., 1941, 63, 1064. W., and Brown, J. B., Ibid., 1943, 65, 259. THE UNIVERSITY LIVERPOOL June, 1950

ISSN:0003-2654    

DOI:10.1039/AN9517600081

出版商:RSC    

年代:1951

数据来源: RSC

摘要:

88 JOHNSTOX, OGSTON AND STANIER: A MODIFICATION OF THE ELSOS p o l . 76 A Modification of the Elson and Morgan Method for the Estimation olf Glucosamine BY J. P. JOHNSTON, A. G. OGSTlON AND J. E. STANIER An investigation of the stages of the Elson and Morgan method for estimating glucosamine has led to modifications of the details of its stages. It has been found that 1Opg or more of free glucosamine or combined glucosamine of hyaluronic acid can be estimated with an accuracy of 3 per cent. IN spite of the satisfactory accuracy and sensitivity of the colorimetric method for the estimation of glucosamine described by Elson and IMorgan,l a number of other workers have re-investigated and modified it: chief of these (using less than 3 mg of glucosamine) have been Palmer, Smyth and Meyer,2 Sarensen3 and B l i ~ , ~ while others have modified it for use with larger quantities.The accuracies claimed range from 2 per cent. to 10 per cent. Ogston and Stanier5 found the version of Palmer, !';myth and Meyer insufficiently accurate, in spite of minor modifications. In order to test the last method further, we made a number of parallel determinations of free glucosamine, taken from the same batch of material; we found differences of up to 6 per cent. between estimations on samples of the same solution after treatment in bulk with acetylacetone, and even larger differences between estimations on lots separately treated with acetylacetone. Investigation of the stages of the method was therefore carried out, beginning with the final stage and working backwards.From this, it appeared that considerable errors might arise from the processes of transfer and dilution, from loss of acetylacetone during heating in open tubes and from variations in the temperatures and durations of the several stages. We therefore modified the method so as to eliminate the transfer of material from one vessel to another as far as possible. At. the only remaining transfer, the whole sample is transferred and this has also the effect of reducing the amount of glucosamine that may be estimated to as little as 1Opg. Under the conditions chosen, the optimal times for some stages have been found to differ from those given by other authors. The procedure is given below, followed by comment on its stages. PROCEDURE Hydrolysis (when the glucosamine is combined in hyaluronic acid)-Seal 1 ml bf solution, containing 10 to 80 pg of glucosamine, and 1 ml of 8 N hydrochloric acid in a Pyrex tube 12 cm long and of 0.8 cm internal diameter.Mix the solution thoroughly and totally immerse the tube in a boiling water bath for 4 hours. Removal of hydrochloric acid-Cool the tube, centrifuge to remove traces of solution from the upper end and open it. Remove the hydrochloric acid and evaporate the contents of the tube to dryness by placing the tube over solid sodium hydroxide in a desiccator evacuated by an oil pump. Treatment with acetylacetone-Add 1 ml of distilled water to the tube to dissolve the contents, and then add 0.5 ml of freshly prepared acetylacetone solution (0.2 ml of acetyl- acetone in 10 ml of 0-5 N sodium carbonate).Re-seal the tube and totally immerse it in a boiling water bath for 60 minutes, then cool it in ice, centrifuge and open it. With free glucosamine, the estimation begins with the treatment with acetylacetone. Treatment with Ehrlich's reagent-Transfer the contents of the tube quantitatively by Pasteur pipette to a 5-ml graduated flask. Wash the tube three times with a total of 2.9 ml of aldehyde-free ethanol and add the washings to the flask. Stopper the flask and mix the contents; warm the flask to 37" C by immersion in a thermostat for 3 minutes. Then add 0.5 ml of Ehrlich's reagent (0.8 g of 9-dimethylaminobenzaldehyde, A.R., in 30 ml of aldehyde- free ethanol plus 30ml of concentrated hydrochloric acid) and sufficient ethanol to make the solution up to 5ml.After mixing, leave the flask in the thermostat for 60 minutes, then remove it and measure the extinction at 535 mp with a spectrophotometer. Comparison is made with standard glucosamine introduced at the acetylacetone treatment stage. COMMENTS ON PRWEDURE Hydrolysis-The yield of estimated glucosamine (Fig. 1) from ox synovial-fluid mucin rose to a maximum after 4 hours and fell slowly again with longer hydrolysis to 88 per cent.Feb., 19511 89 at 22 hours. If the hydrolysis and destruction are both unimolecular processes, the maximum yield at 4 hours would be 97 per cent. of the total glucosamine. No significant loss of pure glucosamine occurred under the same conditions up to 32 hours (Fig. 1); this allows the glucosamine standard to be introduced a t the stage of the treatment with acetylacetone.The change of reactivity of glucosamine reported by Blix4 to follow heating with 4 N hydrochloric acid was not observed. Evaporation of the hydrochloric acid in vacuo (Hadidian and Piriee) avoids the need for transfer and neutralisation. AND MORGAN METHOD FOR THE ESTIMATION OF GLUCOSAMINE L I 1 I l l 1 1 4 0 4 8 12 16 20 24 28 32 Duration of Hydrolysis, hours ' 10 20 30 40 SO 60 70 80 90 100 I10 I I I I I I 1 1 1 1 . Acetylation time. minutes Fig. 1. Effect of duration of hydrolysis Fig. 2. Effect of time of heating on extinction obtained with free glucosamine with acetylacetone on final colour develop- and with mucin. Ordinate: Elcrn a t ment. Ordinate: Elcrn a t 535 mp, as per- 535 mp as percentage of value obtained centage of value obtained after 30 minutes a t reference time.0 Extinction given by free glucosamine, as percentage of value a t start of hydrolysis. 0 Extinction given by much glucosamine as percentage of value obtained a t 4 hours Treatment with acetylacetone-This is an intrinsically unsatisfactory reaction, since both the acetylacetone and the glucosamine undergo some destruction' during its course. Blix4 has shown that a greater yield is obtainable by using larger amounts of acetylacetone and sodium carbonate and that, under his conditions, the yield is least sensitive to variations in the amounts of these reagents. We have used the amounts recommended by Palmer, Smyth and Meyer2; although these give a lower yield, the sensitivity of the reaction is sufficient, and we are satisfied that the ordinary errors of measuring the reagents will not introduce an error of more than 1 per cent.into the final estimate. We found it to be essential to carry out this reaction in sealed tubes, totally immersed at 100" C. Under these conditions, the maximum yield is at 60 minutes (Fig. 2) ; Blix4 recommends this period, but other authors have used shorter periods (Slzrrensen3 20 minutes; Palmer, Smyth and Meyer2 15 minutes; Ogston and Stanier5 30 minutes). Treatment with Ehrlich's reagent-Differences of up to 10 per cent. were found if the reagents were mixed at 0" C or room temperature and then brought to 37" C. Bringing to 37" C before mixing reduced this difference to 1.9 per cent. (standard deviation of eight determinations). A cczcracy-Fourteen parallel samples of glucosamine hydrochloride (each 30 pg of glucosamine) showed a standard deviation (single estimate) of 1.9 per cent. of the mean value, and seven parallel samples of ox synovial mucin (each containing 30 pg of glucosamine) a standard deviation of 3 per cent. One of us (J. E. S.) is indebted to the Medical Research Council for a Training S tudentship. REFERENCES 1. 2. 3. 4. Blix, G., Acta, Chem. Scand., 1948, 2, 467. 5. 6. 7. Elson, L. A., and Morgan, W. T. J., Biochenz. J . , 1933, 27, 1824. Palmer, J. W., Smyth, E. M., and Meyer, K., J . Biol. Chem., 1937, 119, 491. Slarensen, M., C.R. Lab. Carlsberg, 1938, 22, 487. Ogston, A. G., and Stanier, J. E., Biochem. J., 1950, 46, 364. Hadidian, Z., and Pirie, N. W., Ibid., 1948, 42, 260. Tracey, M. V., personal communication. DEPARTMENT OF BIOCHEMISTRY OXFORD June, 1950

ISSN:0003-2654    

DOI:10.1039/AN9517600088

出版商:RSC    

年代:1951

数据来源: RSC