摘要:

32 BLAKE : APPLICATION OF RADIO-FREQUENCIES TO CONDUCTIMETRIC [VOl. 75 Application of Radio-Frequencies to Conductimetric Analysis (Rectified Radio-Frequency Method) BY G. G. BLAKE SYNoPsIs-Small changes in the conductivity of solutions, e.g., those associated with neutralisation, as in conductimetric titration, can with advantage be measured by means of radio-frequency oscillations. A recent form of apparatus is described, in which the liquid under test is drawn up into a conductimetric tube around which are three sleeve electrodes; after traversing the liquid in the tube the oscillatory current is rectified and measured with a micro-ammeter. All complications and disturbances due to contact of electrodes with the liquid are avoided. Examples of titration curves are illustrated. THE difficulties encountered in the use of a “conductivity cell” in conductimetric analysis are mainly due to the electrodes being submerged in the solution.By operating at a radio- frequency and employing a “conductimetric tube” fitted with external electrodesl~2f in place of a cell, all such difficulties are avoided and the time and trouble involved in wetting platinised electrodes are eliminated. Colloidal solutions can be used, and precipitation is no longer a serious trouble. By this new method any amount of solution, down to a quantity as small as one millilitre, can be analysed. The method is suitable therefore both for general and for micro titration.Jan., 19501 ANALYSIS (RECTIFIED RADIO-FREQUENCY METHOD) 33 As a result of investigations carried out since the rectified radio-frequency method was published originally,l the author has acquired a considerable amount of further information, which is described in the present paper.Before giving details of the apparatus used for rectified radio-frequency analysis the new method may be outlined as follows. A radio-frequency current is applied to an electrode attached to the outside of a glass tube into which successive samples of solution are introduced. The radio-frequency current passes through the sample, emerges via a second external electrode and passes thence through a small rectifier. The rectified current from this is measured by means of a micro-ammeter or a galvanometer. Given constant frequency and a circuit having fixed values of capacitance and inductance, the micro-ammeter deflections will be proportional to the radio-frequency resistance of the circuit and as the column of solution is the only variable, the deflections will give a direct indication of the radio-frequency resistance of the solution.(1) RADIO-FREQUENCY OSCILLATOR- Any type of stable radio-frequency oscillator can be employed, the choice of frequency not being a t all critical; 2000 Kc/s. is a suitable value. When making this selection it should be borne in mind that the higher the frequency the greater will be the difficulties experienced through stray capacity, and the lower the frequency the larger will be the dimensions of the inductance, etc. Two suitable types of oscillator have already been described’. For the present application a zero-shunted micro-ammeter in the plate current feed of the oscillator is not required. (2) THE CONDUCTIMETRIC TUBE AND ITS ACCESSORIES- Fig.1 is a diagram embodying the latest developments. The conductimetric tzcbe-The dbe, G, is of thin-walled Pyrex glass 3 mm. in diameter. It has a reservoir at its head connected by rubber tubing to a pipette, P. The original conductimetric tubelJJ was fitted with two external electrodes. The new tube as seen in Fig. 1 has three external sheath electrodes. These may be of lead or tin-foil wrapped round the tube. By spreading the electric field in this way a smaller screening tube, T, can be employed. The pifwtte-In lieu of the glass syringe originally employed, now that the apparatus is smaller, a pipette, P, is used to draw successive samples of solution into the tube.As seen in Fig. 1 this is fitted with an air-inlet valve, V. In the side of the pipette there is a small hole covered by a rubber sleeve stretched over a roller. When in the position shown in the figure the air-inlet hole is closed. Air is admitted by a rotation of the roller which lifts the sleeve when in the proximity of the air-inlet hole. Use of this valve enables air to be bubbled forcibly through the solution after each addition of the reagent. The micro-burette-On the left-hand side of Fig. 1 there is a micro-burette at the head of which is a length of rubber tube, R, closed by a stopper, K. The burette is filled with the reagent by compressing and then releasing R by means of a clamp, L. The measured units of reagent are delivered as desired by gradually tightening the clamp by means of the thumb- screw, L.The Rectijer-After passing through the solution, the radio-frequency current is led to a small rectifier, Re (either a “Westector” or a “fixed crystal”). The rectified current, as modified by the radio-frequency resistance of the solution under test, passes through a radio- frequency choke, H, and its value is read by a zero-shunted micro-ammeter. Another new development is the employment of a very small pre-set condenser, W (Fig. 1); this is seen better in the enlarged drawing at the left-hand bottom corner of the figure. Its object is to by-pass a very small amount of the radio-frequency current directly through the rectifier to ensure that the latter will operate on the straight portion of its charac- teristic curve.It was found that a perfect shielding of the rectifier was liable to cause distortion of the graphs. When once this “pre-set” condenser has been properly set it should never require any further adjustment. A small hole in the side of the earthed metal screening cylinder, T, is provided through which a screw-driver may be inserted to make the needful adjustment. I t should be noted that the conductimetric tube G, choke H and the rectifier Re are all screened and the screening is earthed (see T and T’).34 BLAKE : APPLICATION OF RADIO-FREQUENCIES TO CONDUCTIMETRIC [VOl. 75 The zero-shunt circuit4-The zero-shunt may be operated from a 6-volt direct current supply or by rectified alternating current3 and is used to adjust the zero reading of the micro- ammeter either before or, if necessary, during a titration.Fig. 1. Conductimetric tube and accessories. This diagram shows several new features, including by-pass condenser, three electrodes (instead of two), micro-burette attached to the screening cylinder and a pipette with an air-inlet valve It has been shown in the previous papers to which reference has been made that adjust- ment of the zero-shunt enables any desired point on the scale of the micro-ammeter to be used as zero, whether or not the instrument has a scale with zero at its centre, also that if during a titration it becomes obvious that the subsequent deflections will out-distance the scale in either direction the needle can then be set forward or backward, as the case may require, by any desired amount, and so long as the amount of this readjustment is deducted from (or added to) all the successive readings the titration can then proceed.CoZCpZing condenser-The coupling condenser, C, which should have as good a zero as possible, is of great importance. It regulates the radio-frequency current applied to the conductimetric tube. First it is set at zero. Let us suppose that the scale of the particular micro-ammeter to It is used in the following manner.Jan., 19501 ANALYSIS (RECTIFIED RADIO-FREQUENCY METHOD) 35 1 I 1 I 4 UNITS OF HCI IN IOML. OF WATER I I 1 I I * l l I l l ~ l 2 4 6 8 NUMBER OF UNITS OF f 4 UNITSOF HCi IN 20ML. OF WATER i I I I I I t I 1 I I t I l f I I I 2 4 6 0 N E N T (NOOH) ADDED I 1 I I 1 I 1 I I I I I I I t I I I I * Normal meter zero for all graphs and coupling readjustment to 40pa.1 unit = 5 microlitres. The concentrations of both the HC1 and the NaOH were 1 M Fig. 2. The four graphs in this figure, plotted under the conditions described in the text, show how the form of the graphs acquire a more obtuse angle as the concentration of the acid solutions is reduced 40 1 AFTER FIRST NEGATIVE ZERO - REArnUSTMENT $10- I 5 - I I I - 1 - ! 0 : 1 I " ' " ~ 0 2 4 6 8 NUMBER OF UNITS C 9FTER A SECOND NEGAIWE SETBACK AND COUWNG I I l l 1 I . L 1 1 1 2 4 6 8 1 0 IM REAGENT -0 1 unit = 5 microlitres of 1 M solution Fig. 3. The two graphs in this figure are for the same low concentration as that for graph D of Fig. 2, and show how the form of the latter is improved: first after one negative zero set-back, and still more after a second negative zero set-back36 BLAKE : APPLICATION OF RADIO-FREQUENCIES TO CONDUCTIMETRIC [vol.75 be employed reads from 0 to 6Opa. The conductimetric tube having been filled with a sample of the solution under analysis, the coupling condenser, C, is adjusted until a certain pre-selected deflection, say 40 pa., is obtained. This adjustment of the coupling is maintained throughout the entire analysis, 40 pa. being taken as the starting-point of the graph. This was so for each of the graphs in Fig. 2. SOME APPLICATIONS Figs. 2 and 3 are a series of titration graphs made with the apparatus illustrated in Fig. 1. The end-point is determined by extending the two straight portions of the graph to the point of intersection.The four graphs shown in Fig. 2 illustrate the effect of reducing the concentration of the solution. It will be noted that although the correct value of the end-point is obtained in all the tests, the sensitivity of the method is reduced as the solution concentration is reduced. 1 -A V 1 I I t I I '1 I I I I I 1 I I I I I I I I t . 1 s I I * 1 . 1 I I I I 0 2 4 6 8 1 0 15 2 0 25 3 NUMBER OF UNITS OF REAGENT ADDED Fig. 4. This graph is for an aqueous solution containing smaller quantities of two different acids, hydrochloric and acetic. The mixture was titrated with 1 M ammonia solution in 5-microlitre units The graphs of Fig. 2 were all obtained with the zero-shunt adjusted to give a normal zero reading on the micro-ammeter when the conductimetric tube was empty.It was found, however, that if the meter zero was set back by adjustment of the shunt, the sensitivity could be again increased and Figs. 3, E and F, illustrate this point. They were obtained with the same concentration of solution as Fig. 2, D, but with meter ze;o set-back. First, with the zero set at its normal position, the conductimetric tube is filled with solution and the coupling condenser is adjusted to obtain a reading. If the deflection is then returned to zero by zero-shunt adjustment the actual zero will have shifted by that amount to the minus side of normal. In order to obtain larger negative zero settings the process is repeated, several times if necessary. Should the micro-ammeter have a central zero-scale, the first negative readings can, of course, be read directly from its scale, and when that limit has been reached the foregoing process should be applied. The amount of negative zero set-back can be gauged in the following manner.Jan., 19501 ANALYSIS (RECTIFIED RADIO-FREQUENCY METHOD) 37 J, in Fig.1, is the usual resistance controlling the range of the meter. When making negative zero readings the author has found it advisable to keep the meter heavily short- circuited by closing switch M, or better still by means of a key which is only released while there is solution in the conductimetric tube and actual readings are taken. Fig. 3, F, is a graph for the same solution concentration as for graphs D and E, but was plotted after a further negative zero set-back.In each case, after the negative setting had been made, the deflection for the solution was again brought forward to 40pa. by tightening the coupling. From the foregoing it will be seen that negative zero adjustment makes it possible to titrate extremely dilute solutions. It is obvious that the graphs here reproduced do not cover anything like the whole range possible; however, the graph F, Fig. 3, has already reached a sensitivity sufficient to enable the operator to dis,pense with graphs and to obtain sufficiently accurate results for many practical purposes by merely counting the number of units added before the conductivity of the solution takes an upward turn, and there is no reason why the sensitivity should not be still further increased by further negative bias. To 50 ml. of distilled water were added six 5-microlitre units of 1 M hydrochloric acid and also thirteen units of 1 A4 acetic acid. This mixture was then titrated with 1 M ammonia added in 5-micro- litre units. As can be seen, the quantities of both these acids are confirmed by the graph. Blake, G. G., “A Device for Indicating Small Changes in Electrolytic Resistance, J . Sci. Instruments, - , “Conductimetric Analysis at Radio-Frequency,” Ibid., 1947, 24, 101. - , “Micro-conductimetric Analysis and Titration Rectified Radio-Frequency Method, ” A ustra2. - , “Developments of the Zero-Shunt Circuit,” Ibid., 1944, 6, 101. Fig. 4 is a graph measuring the quantities of two acids in aqueous solution. REFERENCES 1. 2. 3. 4. 1945, 22, 174. J . Sci., 1947, 10, 80. DEPARTMENT OF PHYSICS UNIVERSITY OF SYDNEY AUSTRALIA First submitted, June, 1948 Amended, September, 1949

ISSN:0003-2654    

DOI:10.1039/AN9507500032

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

Jan., 19501 ANALYSIS (RECTIFIED RADIO-FREQUENCY METHOD) 37 Some Applications of a Modified Technique in Paper Chromatography BY L. RUTTER SYNOPSIS-A modified technique in paper chromatography whereby com- ponents of mixtures are separated into circular zones, is described, together with resultant advantages, such as speed, simplicity of apparatus and repro- ducibility. Some new methods of detecting colourless adsorbates are outlined, and examples given of the application of the technique in the analysis of dyes, biological materials and inorganic substances. The possible importance of solubility considerations as a guide to choice of developers is discussed. IN a previous communication1 a brief description was given of a new technique in paper chromatography, by means of which the substances under analysis are separated into circular zones, instead of the usual spots or bands.The present paper is intended to provide fuller details of the practice, applications and advantages of this method. According to the technique, two parallel cuts, about 2 mm. apart, are made from the same edge up to the centre of an 11-cm. circular filter paper, and the “tail” so formed is bent at the joint perpendicular to the plane of the paper and cut down to approximately 1.5 cm. in length. Care must be taken to ensure that the parallel cuts are equal in length, otherwise the “tail” will not be squarely perpendicular to the plane of the paper and development may not be truly circular. The solution to be analysed is then placed as a drop on the joint and generally air-dried.The “tail” is then immersed in developing solvent contained in a small capsule inside a Petri dish, the sides of which support the filter paper in a horizontal plane. A glass plate,38 RUTTER: SOME APPLICATIONS OF A MODIFIED [Vol. 75 which may be engraved with a suitable scale to assist in following the course of development, is then placed on the filter paper to retard evaporation. Alternatively, the filter paper is sandwiched between two glass plates, the lower one being perforated to accommodate the “tail.” The author has found it generally satisfactory to use No. 3 Whatman papers, but for special purposes No. 1 or No. 5 may be more suitable. Filter papers that are acid in their reaction have not been found satisfactory as a general rule.By capillarity, the solvent rises, and the rate of development *may be readily controlled by varying the distance between the liquid surface and the plane of the paper. Solutions containing coloured constituents, suitably developed, give sharp separations into individual zones. In the case of colourless chromatograms, after development a test sector is cut out and pressed between filter papers impregnated with reagents giving coloured products with constituents of the chromatogram, or sprayed with reagent solution. Preparation of deriva- tives on the main chromatogram, which may be undesirable, is thereby avoided. Other methods of locating colourless adsorbates are described below. After their positions have been marked on the main chromatogram and this has been dried, the various bands may be cut out as circular strips and each of these conveniently prepared for elution by placing both ends on filter paper soaked in eluent.Capillary action concentrates the adsorbate in the centre of the strip, whence it may be drawn off by paper or by capillary pipette. The apparatus is illustrated in Figs. 1 and 2, and a suitable arrangement for concentration of the adsorbates, prior to elution, is shown in Fig. 3. In the latter, the excised strip of filter paper is supported on a pile of three or four micro slides placed in a Petri dish with the ends of the strip touching a small wad of filter paper soaked in eluent. If desired, several strips may be simultaneously treated, the whole being conveniently enclosed by an upper glass plate or inverted Petri dish.In common with other paper techniques, only very small quantities of materials are necessary for analysis, the method in many cases having a sensitivity of approximately 1 pg. or less of solute. The present technique, however, frequently shows some advantages over the sheet technique of Consden, Gordon and MartinJ2 or the strip methods of Lederer,3 and Linstead? for the following reasons. (1) Speed of separations-For most separations so far investigated, development to a circle of diameter 6 cm. or less is sufficient and this seldom takes longer than 10% minutes. In contrast with other methods the rate of feed may be accurately and readily controlled by “tail” width and height of paper above the developer surface. Thus, for relatively viscous liquids, such as butyl or iso-butyl alcohol, which are slow-feeding, the capsule should be filled to bring the surface within 0.5 cm.approximately of the plane of the paper. As the plane of the paper is horizontal, the feed rate is constant and there is no danger of “water- logging” by syphoning. A 1 r r 1 , 1 1 1 J Fig. 1. Sectional view of Apparatus A = Upper engraved plate B = Lower perforated plate C = Petri dish D = Container for developer (2) Sharpness of separations-It has usually been found that zones are better defined than the spots or bands obtained by the sheet or strip technique, one reason for which is that there is no blurring of edges, since the zones are circular. Front, and frequently rear boundaries, are also usually sharp and zones are thereby more cleanly separated. (3) Testing during development-A further advantage of the method is that a number of test sectors may be removed at intervals, without upsetting the course of development; this dispenses with the necessity for duplicate or replicate runs. (4) Simplicity of appuratus-A Petri dish and filter paper are the only essentials: the upper engraved glass plate and the lower perforated plate are convenient, but the latter may be dispensed with and an inverted Petri dish serves equally well in place of the upperJan., 19501 TECHNIQUE IN PAPER CHROMATOGRAPHY 39 plate, if this is not readily available.The developer may be placed in the lower Petri dish, but a small capsule or similar container has advantages and a glass phial of the type used for tablets, cut down to a depth of 1 cm.is more convenient as a container for the developer. (5) Compactness-The apparatus occupies very little space and may thus be placed in a thermostat or refrigerator, if desired. Its small size and portability also render it suitable for enclosure in cabinets, illuminated by ultra-violet or other light. Fig. 2. Plan of apparatus, showing Chromatogram in process of development LOCATION OF ZONES AFTER DEVELOPMENT- With coloured compounds, zones are self-indicating, but the location of colourless adsorbates may be more difficult. Many useful methods have been described in the literature, among them the brush technique of Zeichmeister: spraying with specific reagents giving coloured cornpounds,6*7 examination under ultra-violet light8sg and the use of dyes or other coloured substances, whose behaviour, relative to some constituent of the mixture under analysis, is known.1° Some further methods that the author has found useful are based on the utilisation of various factors, among them the following. (1) Charring-The presence of an adsorbate affects the temperature a t which the paper chars, and by exposing a test sector to progressively higher temperatures, bands frequently become visible, particularly if examined under ultra-violet light, during heating.I t should be noted that some papers contain fluorescent material, which may be removed by a preliminary washing. (2) PH Changes-The pH values of adsorbates may differ from that of the paper, and spraying with dilute solutions of indicators, particularly Universal indicators, is frequently of assistance in showing the positions of the bands.(3) Changes in adsorptive properties of the paper-Adsorbates may alter the adsorptive properties of the paper, and by exposure of a test sector to acid or alkaline vapours, after spraying with indicator, differences in the intensity and shade of indicator colour developed a t different parts of the chromatogram, may reveal the location of zones. Exposure to dilute acid fumes, after alkaline vapour treatment, and vice versa, frequently affords a sensitive means of differentiation. Spraying with starch solution, after exposure to iodine vapour, is suggested and, if necessary, subsequent treatment with gaseous sulphur dioxide, for closer differentiation.40 RUTTER: SOME APPLICATIONS OF A MODIFIED [Vol.75 (4) Changes in light transmission and reflection-In the case of hydrophobic adsorbates, bands may sometimes be detected by spraying the paper with water: when viewed by trans- mitted light, adsorbates show as darker zones which are lighter by reflected light. Such effects may sometimes be enhanced by the use of liquids of different refractive index, such as glycerol, in place of water. The central feed technique is applicable to separations in various fields, and some typical examples (which are merely illustrative and not intended to be fully comprehensive) are given below. (a) PLANT EXTRACTS- Many constituents of plant leaves and petals can be rapidly separated by the above technique. In the case of Californian Poppy (Eschscholtzia), an extract of the petals is made with alcohol and ethyl ether and a drop, after drying on the paper, is developed with light Fig.3. Arrangement for concentration of adsorbates A = Micro slides to support excised strip B = Excised strip Wad of filter paper soaked in eluent petroleum (b.p. 40" to 60" C.). As many as seven different zones can be detected without further treatment and by exposure to iodine vapour, followed by spraying with starch solution, other bands become visible. Constituents of grasses may be extracted with a mixture of alcohol, ethyl ether and chloroform and a drop of the extract developed, after drying, with light petroleum. At least three zones become visible, namely, a central green zone consisting largely of chlorophyll, a deep orange zone close to this and a yellow zone at the solvent front.On exposure of the paper to iodine vapour followed by spraying with starch solution, other zones become visible. A rapid qualitative test for ascorbic acid in plants or artificially prepared mixtures such as vitamin tablets, may be carried out as follows. The material under test is ground with a pestle and mortar, with a little water if necessary, and a drop of the crude extract (which need not be freed from fibrous or other materials), is placed on the centre of a prepared paper and developed with water to a circle of diameter approximately 3 cm. The paper is then gently dried and sprayed with ammoniacal silver nitrate solution. If ascorbic acid is present it will react in the cold with this reagent to give a grey or black band of silver at the former position of the solvent front.Sugars do not interfere with this test, as they reduce this reagent only on heating.Jan., 19501 TECHNIQUE I N PAPER CHROMATOGRAPHY 41 (b) DYES- Many mixtures of dyes can be separated on paper chromatograms and much useful information concerning purity or identity quickly obtained. As a general rule, very dilute solutions (of about 0.1 per cent. concentration) should be used, and water or salt solutions as developers. In the case of Edicol Green (I.C.I., Ltd.), development with water failed to separate the constituents, the band moving with the solvent front, but development with 1 per cent. sodium chloride solution resolved the mixture satisfactorily into a yellow band near the centre and a blue band beyond this.Ammonium sulphate solutions of various concentrations may also prove useful in certain cases. If the dyes are very strongly adsorbed with water alone, the use of dilute acids or alkalis for development may give more satisfactory results. Sometimes solutions of more strongly adsorbed compounds may be used with advantage as developers. For example, a mixture of Chlorazol Fast Scarlet 4B150, Chlorazol Brown LF150 and Chlorazol Yellow G200 (I.C.I., Ltd.), which proved difficult to separate by other means, was satisfactorily resolved by development with a 0.025 per cent. aqueous solution of cetyltrimethyl ammonium bromide. If it is undesirable to use salt solutions, “partition” methods in many instances may be recommended ; e g ., Edicol Green developed with iso-butyl alcohol saturated with water separates well into its components. (c) INORGANIC SEPARATIONS- By taking advantage of complex formation many cations may be separated. For example, one drop of an 0.1 per cent. solution of mixed copper and iron nitrates may be developed with water to a circle of diameter approximately 2 cm. and then exposed to ammonia vapour and developed with a dilute solution of ammonia containing ammonium chloride. The iron is precipitated on exposure to the ammonia vapour, whereas the complex cuprammonium compound moves with the solvent front on further development. Spraying a test sector with potassium ferrocyanide solution reveals the position of the zones, whose presence may also be shown by “charring” a test sector, Cobalt‘and nickel may also be separated by development with dilute ammonia, test sectors being treated with the appropriate reagents.The “extraction” methods for inorganic separations described by Linstead and his colla- b o r a t o r ~ ~ ~ ~ ~ may be carried out by the central feed technique and in many instances show advantages over the use of single solvents for development. CHOICE OF DEVELOPERS- The success of chromatographic separations depends largely on the solvents used, and some guide to choice may be obtained from considerations of polarity and solubility. It is generally recommended in the literature l 2 v = that non-polar or only slightly polar liquids should be used for development, but solubility effects, which are seldom mentioned, may have an important bearing and should be considered.For example, anthracene and fluorescein may be rapidly separated by development with water containing 5 per cent. of ethyl alcohol, a mixture in which anthracene is less soluble than fluorescein, although water is highly polar. Alternatively, development with light petroleum, in which anthracene is the more soluble, also leads to separation, the fluorescein being preferentially adsorbed. Some fluorescent constituents of a sample of complex tarry material, which were eluted when development with light petroleum was attempted, were satisfactorily resolved by development with alcohol, in which they were less soluble. Certain proteins and dyes may be separated by the use of salt solutions in which they are less soluble than water, as suggested by Ti~e1ius.l~ It would appear that solvents in which the substances to be separated show the greatest difference of solubility are desirable and that for adsorption of non-polar or only slightly polar compounds a solvent in which they have a low solubility is preferable.Investigations of the physical factors affecting chromatographic separations on paper are facilitated by the reproducibility that is a feature of this method, and a study of the effects of polarity of solvent, surface tension, activation of paper and possible electrical effectslS is in progress and will be reported elsewhere. The author wishes to express his indebtedness to Mr. D. W. Wilson of the Sir John Cass Technical Institute, and Dr. J. H. Hamence for helpful discussions of this work, and to Messrs. I.C.I., Ltd., who kindly supplied various dyestuffs.42 SUTTON, MARKLAND, BARRACLOUGH AND CHAPMAN : INVESTIGATIONS [VOl. 75 REFERENCES 1. Rutter, L., Nature, 1948, 161, 435. 2. 3. Lederer, M., Nature, 1948, 162, 776. 4. 5. 6. 7. Bate-Smith, E. C., Ibid., 838. 8. 9. 10. 11. 12. 13. 14. 15. Consden, R., Gordon, A. H., and Martin, A. J . P., Biochent. J., 1944, 38, 224.. Arden, T. V., Burstall, F. H., Davies, G. R., Lewis, J. A,, and Linstead, R. P., Ibid., 1948, 162, Zechmeister, L., Cholnoky, L. V., and Ujhelji, E., BUZZ. SOC. Chinz. Biol., 1936, 18, 1885. Forsyth, W. G. C., Nature, 1948, 161, 239. Phillips, D. M. P., Ibid., 63. Good, P. M., and Johnson, A. W., Ibid., 1949, 163, 31. Brockmann, H., and Busse, A., 2. Physiol. Chem., 1937, 249, 176. Burstall, F. H., Davies, G. R., Linstead, K. P., and Wells, R. A., Nature, 1949, 163, 64. Strain, H., Chromatographic Adsorption Analysis, Interscience Publishers Inc., New York, 1945, Williams, T. I., An Intmductim to Chro?natography, Blackie and Sons Ltd., 1946. Tiselius, A., paper read before Soc. Chem. Indust., March 5th, 1948, a t Manchester. Rutter, L., Nature, 1949, 163, 487. 691. et al. THE SIR JOHN CASS TECHNICAL INSTITUTE LONDON, E.C.3 May, 1949

ISSN:0003-2654    

DOI:10.1039/AN9507500037

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

42 SUTTON, MARKLAND, BARRACLOUGH AND CHAPMAN : INVESTIGATIONS [VOl. 75 Investigations in the Examination and on Variations in the Composition of Milk Part I. The Determination of the Hortvet Freezing-Poin t BY R. W. SUTTON, J. MARKLAND, A. BARRACLOUGH AND W. B. CHAPMAN SYNoPsIs-Some difficulties in the determination of the freezing-point of milk by the Hortvet method are discussed. Possible sources of error are enumerated, some of which are thought to be inherent in the apparatus, and it is shown that these errors can be reduced to low proportions by the adoption of a carefully controlled technique, which is particularly necessary in the standardisation of the thermometer with sugar solutions. IN the examination of samples of milk the determination of the freezing-point is a test which has received considerable attention since Hortvet described an apparatus suitable for routine work, and more particularly in this country following the work of Elsdon and Stubbs.The importance of an accurate result can hardly be over-emphasised, for the test has come to be regarded as one that enables a true distinction to be made between milks naturally deficient in non-fatty solids and those containing added water. The action of a Food and Drugs Authority may therefore depend on the result. Correct figures are also important in com- piling records if these are to be of value. It is clear that if any worker is regularly recording figures for the Hortvet freezing-point depression* which are incorrect, he will either report a rate of milk adulteration which is higher than the truth, or alternatively he will report a greater proportion of milks showing a natural deficiency in non-fatty solids than would be reported with correct Hortvet results.In his original paper, Hortvetl reviews earlier work on the freezing-point of milk and describes a cryoscope suitable for routine work. He states ‘that “the thermometer should be carefully standardised and calibrated in comparison with a U.S. Bureau of Standards tested instrument,” There is no mention in this paper of the use of sugar solutions. These were apparently first used in further collaborative work whilst Hortvet was Referee on Dairy Products2 The sucrose solutions (7 and 10 per cent. w/v) originally used in collaborative work were later adopted by the A.O.A.C.as the basis for standardisation of the thermometer, and they are specified in the present official m e t h ~ d . ~ It must be assumed that the freezing- point depressions (viz., 0.422” and 0.621” C.) accepted by the A.O.A.C. for these two sugar solutions were those obtained when the most accurately calibrated thermometers available * It is usually convenient to refer to freezing-point depressions rather than actual freezing-points, and unless otherwise stated in the text, this practice will be followed in this paper.Jan., 19501 IN EXAMINATION AND VARIATIONS IN COMPOSITION OF MILK 43 were used in the Hortvet apparatus and with the Hortvet technique, ie., with no corrections for super-cooling or heat transference. Elsdon and Stubbs4 record that they ascertained that the two thermometers used would be correct to about 0.002” to 0.005” C.I t should, however, be emphasised that whilst Hortvet’s figures may not be strictly correct, they form the real basis for all “Hortvet” results. All our records rest on the assumption that 7 per cent. w/v sucrose solution when examined by the Hortvet method gives a depression of 0.422” C. and that 10 per cent. sucrose solution gives a depression of 0.621” C. If this is accepted it seems clear to us that the actual apparatus used is unimportant, provided that the thermometer is a satisfactory one and that with the apparatus chosen it is possible to obtain reproducible results. Any alterations in apparatus or technique to improve the reproducibility of the results, although possibly representing a departure from the process originally described by Hortvet, may be adopted without objection and the results may properly be classed as “Hortvet results” if in the standardisation Hortvet’s figures for 7 and 10 per cent.sucrose solutions are accepted. The object of this paper is to record our conclusions following an extensive experience in using the Hortvet method, to refer to factors which in our opinion can be responsible for some lack of agreement in the results obtained by different workers, to emphasise the need for a more comprehensive standardisation of the thermometer than is adopted by some workers and to recommend the adoption of a very rigid technique. DIFFICULTIES BND POSSIBLE SOURCES O F ERROR (a) USE OF SUGAR SOLUTIONS- In the A.O.A.C.official method, 7 and 10 per cent. sucrose solutions w/v are still specified for the standardisation, and as pointed out by Stubbs and Elsdon5 the accuracy of the correction to be applied to any recorded figure depends on the assumptions that there is equidistant spacing of the graduations between the two freezing-points recorded and that the bore of the thermometer is uniform between these two points. These assumptions may not be correct. There is further some difficulty in obtaining really concordant results for the freezing- point-of sugar solutions, and this difficulty has been referred to by previous workers. In the collaborative work undertaken when sugar solutions were first used2 there is the instruction not to include adventitious results “which are in marked disagreement with other results obtained by carefully following instructions,” and this direction is included in the official A.O.A.C.method to-day. They encountered variations up to 0.005” C. with sugar solutions, but state that by making a sufficiently large number of determinations it is possible to obtain a figure that can be accepted as correct. We agree with this but, from our conversations with other workers, we doubt if this possible source of error is generally appreciated. Further, although occasionally results are obtained which “are in marked disagreement with other results obtained by carefully following instructions” it sometimes happens that the results are spread evenly over a relatively wide range, and in these circumstances, in our opinion, an average figure cannot properly be accepted as correct.Accurate results can be obtained only if this variability is reduced to really low proportions. In our opinion this can be done by strict control in the experimental technique. At an early stage we decided that a better standardisation with sugar solutions than that specified by the A.O.A.C. was necessary, and indeed it has been our practice for many years to standardise thermometers when first used and a t about two-year intervals in con- siderable detail with sugar solutions of intermediate concentration as well as the 7 and the 10 per cent. solutions. We also check at more frequent intervals with 8.75 per cent. w/v sugar solution (A = 0.536-7” C.). “Hortvet” figures for these intermediate strength sugar solutions have been computed by Stubbs and E l ~ d o n .~ Corrections to be applied between the temperatures recorded for the seven sugar solutions are obtained by interpolation. There is obviously less error in assuming uniformity in the bore and in the graduations of the thermo- meter for the smaller intervals. In our work with these sugar solutions of concentrations ranging from 7 to 10 per cent. we have found in two separate cryoscopes that the spread of results is more marked with the stronger solutions. Indeed, there is almost a regular decrease in reproducibility with increase in the concentration of the sugar solution under examination. In Table I figures are included which show the gradual increase in the spread of the results with increasing sucrose concentration.These results were obtained after close standardisation Elsdon and Stubbs4 refer to it.44 SUTTON, MARKLAND, BARRACLOUGH AND CHAPMAN : INVESTIGATIONS [VOl. 75 of the experimental technique (referred to later), and greater variations have been recorded with insufficient attention to detail. TABLE I PER CENT. SUCROSE SOLUTIONS w/v 7.0 7.5 8.0 8.5 9.0 9-5 10.0 No. of readings . . 12 12 12 12 30 12 12 Average depression, "C. . . . . 0-4144 0.4457 0.4771 0.5095 0.5420 0.5752 0.6087 Difference between extremes, O C. . . 0.002 0.0025 0.003 0.0015 0.004 0.003 0.004 Standard deviation, O c. . . f0.0007 &0.0008 &O*OOlO j0.0005 f O * O O l l &O*OOlO &0.0013 Theory, O C.'(Stubbs and Elsd;n5) . . 0.4220 0.4543 0.4870 0.5200 0.5532 0.5869 04210 Correction, C.. . +0.0076 +0.0086 +Om0099 +0-0105 +0.0112 +0-0117 +0.0123 (b) FACTORS AFFECTING THERMOMETER READINGS- There are several uncontrolled factors which affect the thermometer readings. Their effects are to be seen in the variations in the ice-point, but clearly they are included in all readings. The factors to which we wish to draw particular attention are additional to those 3 740 750 760 770 BAROMETRIC PRESSURE, mm. H g Fig. 1 gradual changes which have been recorded in newly made thermometers and which may persist over a period of years. They may conveniently be divided into three categories- (i) Pressure e$ects-The true freezing-point of water or of an aqueous solution is slightly depressed by an increase in pressure. In practice considerable changes in the ice-point of a Hortvet thermometer do occur from one day to another, and previous workerss have recorded that this variation is correlated with atmospheric pressure.The correlation is, in fact, a positive one and is, of course, due to the thin-walled thermometer bulb being com- pressed by increase in atmospheric pressure and forcing the mercury further up the capillary. Observations of the freezing-point of distilled water in one apparatus, recorded a t differer,t atmospheric pressures, over a period of some months are illustrated in Fig. 1. The correlation coefficient is +0.68 f0.05. (ii) Temperature eflects-In the Hortvet apparatus there is a certain length of the column of mercury exposed to atmospheric temperature and any variation in this temperature willJan., 19501 IN EXAMINATION AND VARIATIONS IN COMPOSITION OF MILK 45 cause an alteration in the reading.We have calculated that for the mercury column exposed in an ice-point determination the reading will change by about 0.0003" C. per degree centigrade change in atmospheric temperature. The corresponding figure for a depression of 0.5" C. is about 0.0002" C. An ice-point determination immediately before or after the determination of the freezing-point of a milk sample will compensate for the effect of external pressure and for the bulk of the temperature effect, but it is clear that if the ice-point.and the freezing- point of a milk sample are determined at different conditions of room temperature an error will be introduced. (iii) Mechanical changes-We find that Hortvet thermometers show some variation in the ice-point throughout a period of continuous use during the day.The variation is always in the same direction and in our experience there is a gradual rise in the temperature recorded. This does not entirely accord with the experience of Elsdon and StubbsJg who found with one thermometer that there was a tendency for the ice-point to be lower a t the end of a day- even when the thermometer had been kept in ice between the readings. We have regularly observed this alteration in the ice-point during continuous use of a thermometer. The effect is more noticeable in some thermometers than in others. With one thermometer we have recorded a change in ice-point from +0.0205" C. to +0.026" C.in the course of a day. During this period 24 freezing-point determinations had been made. Most of the change takes place in the earlier stages. Later the change is more gradual and constant readings may finally be recorded. The early part of this change is probably due to a delay in contraction of the glass after being kept at room temperature. The later part may to some extent be due to an increase of room temperature. The variation may be only small but it is measurable and, in making a long series of freezing-point determinations, we now invariably include several determinations of the ice- point. These are applied to the adjoining results for the purpose of recording depressions. (c) APPARATUS- From our experience we think that variations in size of different parts of the Hortvet apparatus may indirectly be responsible for some variation in results.In this laboratory two Hortvet cryoscopes have been in use for some years. They were made by different firms and they differ considerably in the following measurements, which are thought to be important. A B 8-9 mm. Diameter of thermometer .. .. .. 9-5 mm. Internal diameter of freezing-point tube . . . , 23.2 19 27.0 99 Outside diameter of freezing-point tube . . . . 25.9 99 29.6 9 ) Inside diameter of metal tube . . .. . . 26.7 39 31.7 $9 *Clearance of bulb from bottom of tube . . .. 11 19 15 n Volume of liquid used . . .. .. . . 27.5 ml. 43.5 ml. for the period when most of the records were made. * This distance, of course, can be controlled. The figures given are correct It will be seen that the* clearance between the tubes (occupied by the alcohol layer) is 0.4mm.in apparatus A and 1.05mm. in apparatus B and, probably what is more important, the clearance between the thermometer bulb and the inside of the freezing-point tube (occupied by the solution under test) is 6.85mm. for apparatus A and 9.05 mm. for apparatus B. In use, apparatus A with the narrower tube and smaller volume of solution is much quicker than apparatus B, but it presents greater difficulty in standardising with sugar solutions. We think that the clearance between the thermometer bulb and the wall of the freezing-point tube is of importance and that a difference in this measurement may easily be the explanation of the greater difficulty in obtaining concordant results on sugar solutions in apparatus A compared with apparatus B.In apparatus A-with the wider thermometer and narrower freezing-point tube-the clearance is less and the effect of the lower temperature of the outer cooling bath is more noticeable. Further, the smaller this clearance the greater would be the effect of any slight out-of-centre position of the thermometer. That the nearness of the thermometer bulb to the outer cooling bath has an effect on the results can be readily demonstrated by altering the depth of the thermometer in the freezing-point tube. The following results were obtained with a thermometer in different positions and they are recorded in the order in which they were obtained by two workers.46 SUTTON, MARKLAND, BARRACLOUGH AND CHAPMAN : INVESTIGATIONS [vol.75 These variations i n position, of course, were chosen to exaggerate the disturbance of The greater depressions are undoubtedly due to a more pronounced effect by the results. the outer bath. Distance between thermometer bulb and bottom of tube Reading, a C. Ice-point, a C. + 0.027 r 2cm. - 0.4975 0.4965 cm* I - 0.503 +. 0-027 - 0.502 - 0,502 - 0.497 - 0.497 - 0.496 + 0.027 (a) TECHNIQUE- In the instructions given in the A.O.A.C. Methods of Analysis it is directed that three stirs shall be given as the mercury approaches its maximum. Whether or not this is intended to exclude all stirring between seeding and the three stirs is not clear, S t ~ b b s , ~ in investigating the stirring in the Hortvet apparatus, found that there was no significant difference in the result obtained whether the stirring was continuous or as laid down by the Hortvet technique.Indeed, it is suggested that the Hortvet technique might be altered so as to permit of a certain amount of stirring during the ascent of the mercury column. This led us, and it may have led others, to conclude that the actual amount of stirring and the time of stirring in the Hortvet process were not of major importance. It appears, however, that Stubbs's conclusions were reached following experiments with milk samples only. In our experience alterations in the amount of stirring have a marked effect on the results with sugar solutions and a less, though measurable, effect when examining milks. With increased stirring the effect is to give a larger depiession and the time during which the mercury remains stationary a t its maximum is reduced.The effects are more noticeable in apparatus A, with the smaller clearance between the thermometer bulb and the freezing-point tube, where stirring is likely to be more efficient. With this apparatus, if stirring is continued alternately with tapping of the thermometer it is almost impossible to record a result. The mercury rises to a maximum which is considerably lower than is obtained by the usual procedure and immediately falls rapidly. Whatever technique is followed, if one or two stirs are given after the steady temperature has been recorded, there is a quick fall of the mercury. This surely means that the outer layers of solution in the freezing-point tube are at a lower temperature and that the thermometer has recorded the temperature of an inner layer only.This cooling effect of the outer bath leading to more formation of ice and a concentration of the solution (with increased depression of the freezing-point) must in fact be going on all the time and the temperature recorded at the final reading represents a balance. It is that point where the heat produced by formation of ice is equalled (and later exceeded) by the effect of the cooling bath. With a limited amount of stirring in the Hortvet process, temperature changes due to these two opposing effects are slow and there is a point where there is no noticeable change in the reading of the thermometer. We are therefore led to the conclusion that difficulty in obtaining concordant results in the Hortvet apparatus is largely due to the cooling effect of the outer bath, and all our experience accords with the theory of Monier-Williamss that steadiness of temperature at the final reading is secured mainly by the fact that stirring is inefficient.Indeed, in our opinion it is absolutely essential to limit the amount of stirring and to adhere to a rigid technique if reasonably concordant results are to be obtained. In our early experiments we found that final temperatures were steadier (the temperature One further point may be mentioned.Jan., 19501 IN EXAMINATION AND VARIATIONS IN COMPOSITION OF MILK 47 being constant for at least a minute with sugar, solutions and for several minutes with milk) if three stirs were made immediately after seeding with ice and no further stirring done.The seeding was done at -1.6" C., the mercury probably receded to about -1.65" C. before rising, three stirs were given a t a steady rate during which time the mercury rose to about -1.3" C. Tapping of the thermometer was commenced when the movement of the mercury was quite slow. Fig. 2 Later results were somewhat more erratic and we have returned to the technique of giving three stirs at a later stage. We still seed with ice at - 1 - 6 O C. and manipulate the starter and the stirrer to secure a quick rise of the mercury (in our experience those occasional results which are readily classed as abnormal are always associated with a slow rise of the mercury), stir three times immediately after the mercury has risen past -1.0" C.and thereafter do no more stirring. With this technique we have obtained rather more concordant results with sugar solutions, but differences are still recorded. We also have the impression that the final temperatures are not quite so steady, The thermometer is tapped until the reading is constant.48 SUTTON, MARKLAND, BARRACLOUGH AND CHAPMAN INVESTIGATIONS VOl. 75 particularly with the stronger sugar solutions. In many instances the maximum rise of the mercury is not maintained for more than half a minute. We, however, prefer this later technique, because of the more concordant results and also because it tends to give a slightly higher rise of the mercury, L e . , a smaller depression. A convenient stirring device is illustrated in Fig. 2.It consists of a metal bracket attached to the back board of the apparatus. The front part of the horizontal arm is slotted to take the thermometer-this being held in place by a small metal arm. If the bracket is of the right size, the thermometer is thereby held in a safe and perfectly upright position. Part of the way along the bracket is a metal pillar mounted on a swivel fitting and carrying a pulley wheel. A small S hook is attached to the stirrer and a thread from the S hook passes over the pulley, through an eye hook in the back board, through a second eye hook on the side of the apparatus and is threaded through and secured to a metal bead somewhat larger in diameter than the eye hook. The eye hook at the side of the apparatus is fixed so that the distance above bench level is at least equal to the depth of liquid in the freezing-point tube.The thread length is arranged so that when the metal bead is at its highest level (k, next to the eye hook) the stirrer is just resting on the bottom of the freezing-point tube. Under these conditions simple pulling of the metal bead down to bench level provides for complete movement of the stirrer through the liquid being frozen. TYPICAL RESULTS In Table I are summarised results on our latest standardisation of a thermometer with sucrose solutions (apparatus A). They were obtained by three workers following the recom- mended technique and all contributed at least three results with each sugar solution. Ice- point determinations were made at intervals throughout each series of tests and applied to the appropriate individual results.It is clear that individual results for sucrose solutions ought not to be accepted for the purpose of applying corrections, but, from the standard deviations, it is also clear that if a sufficient number of determinations are made on any sugar solution the error need not be large. We prefer this comprehensive standardisation of the Hortvet thermometer since corrections ascertained at seven points should reveal any serious defect in the bore of the thermometer. After plotting the results, a table may be constructed giving the appropriate corrections to be applied to any result. It is desirable to check the standardisation occasionally and for this purpose 8.5 per cent. or 8-75 per cent.w/v sugar solution may conveniently be used. SUGGESTED FURTHER IMPROVEMENTS If we are correct in our opinion that many of the difficulties with the present Hortvet apparatus are due to the continuous contact between the freezing-point tube and the cooling bath, then it follows that the principle of removing the alcohol layer in the Monier-Williams apparatus is eminently sound. We have recently conducted further experiments with the Monier-Williams apparatus, particularly to ascertain the effect of differences in technique. We can say at once that in this apparatus the technique employed is of far less importance. The contrast between the Monier-Williams apparatus and the Hortvet cryoscope is indeed quite remarkable. In our experiments with the Monier-Williams apparatus the amount of stirring and the time when this is done are without effect.Stirring can be done immediately after seeding or as the mercury approaches its maximum or throughout the rise of temperature. Indeed, it is difficult even with the 10 per cent. sucrose solution to obtain results which are not in com- plete agreement. This difference between the two cryoscopes may, of course, be due to several factors. We feel, however, that the isolation of the freezing-point tube by removal of the alcohol layer at the time of freezing must be of chief importance. The apparatus has disadvantages for routine work of which we are conscious. Because of the larger volumes of liquid to be cooled it is much slower. In our apparatus, 60 ml. of solution are required to immerse the thermometer bulb and 50 ml.of dilute alcohol required in the space between the tubes. A determination requires 20 to 25 minutes. There is more heat loss from the instrument and therefore a greater consumption of ether. It is therefore difficult to recommend the adoption of the Monier-Williams apparatus for routine testing of milks, but we see no reason why the principle of removing the alcohol layer should not be added to the Hortvet apparatus. This, of course, has already beenJan., 19501 IN EXAMINATION AND VARIATIONS IN COMPOSITION OF MILK 49 tried by Elsdon and Stubbs4 and not recommended. We are now having a Hortvet cryoscope altered on similar lines, and feel sure that there should be some improvement in the repro- ducibility of the results with sugar solutions on which the accuracy of all other results depends so much. Provided that standardisa- tion is carried out on the assumption that in the Hortvet method 7 per cent. sucrose freezes at -0.422" C. and that 10 per cent. sucrose freezes at -O.62l0C., a modified apparatus will still give results which are quite properly compared with previous records, some of which have been collected with great care, and there will be no need to alter materially the figures which are to-day accepted as normal for genuine milk. As explained earlier, we see no objection to this in principle. REFERENCE s 1. Hortvet, J., Ind. Eng. Chem., 1921, 13, 198. 2. 3. 4. 5. 6. 7. 8. Monier-Williams, G. W., Ibid., 1933, 58, 254. - , J.A.O.A.C., 5, 470. OBcial and Tentative Methods of Analysis of the A.O.A.C., 6th Edition, 1945, p. 313. Elsdon, G. D., and Stubbs, J. R., Analyst, 1934, 59, 585. Stubbs, J. R., and Elsdon, G. D., Ibid., 1936, 61, 455. Elsdon, G. D., and Stubbs, J . R., Ibid., 1930, 55, 423. Stubbs, J. R., Ibid., 1935, 60, 147. COUNTY LABORATORY ST. MARY'S GATE DERBY July, 1949

ISSN:0003-2654    

DOI:10.1039/AN9507500042

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

Jan., 19501 IN EXAMINATION AND VARIATIONS IN COMPOSITION OF MILK The Determination of Carotene in Dried 49 Peas BY DAVID FRANKLIN, F. B. LYONS AND T. S. WHEELER SYNoPsIs-The powdered sample, containing 14 per cent. of moisture, is extracted by standing for 16 hours with a 2 + 1 mixture of light petroleum and acetone. The extract is evaporated with light petroleum to remove acetone and then passed first through dicalcium phosphate to remove unwanted pigments and afterwards through a mixture of sodium sulphate and alumina to adsorb the carotene, which is eluted with a 1 to 1.2 per cent. solution of acetone in light petroleum and estimated absorptiometrically as 8-carotene in the eluate. Statistical interpretation of the results of an experimental study of its various stages indicates that the method is efficient in tEat there is a linear relationship between the quantity of pea-flour taken and the quantity of natural carotene found.The results at the 95 per cent. level for a 4.3-g. sample are accurate to within &€I per cent. of the carotene present. There is evidence that pea-flour contains an anti-oxidant which stabilises carotene during the removal of acetone from the first extract. IN recent years much work has been carried out on the estimation of carotene in plant and food material by chemical and physical means. Less attention has been paid to the accuracy of the methods used, even when this would appear desirable, for example, with dried peas where minute quantities (about 3 to 4p.p.m.) of the pigment are involved. This paper describes a chromatographic method of estimating carotene contents of the order of 3 p.p.m.in dried peas and includes a statistical examination of the accuracy of the results. Figures for the carotene content of peas in various forms-fresh, frozen, canned and blanched-have been published by a number of a~thors,~,~~,~6,22,24,40,48, etc. and the statistical treatment of carotene contents have been disc~ssed7~s~1~~1*~22~~~~0~40~41~ etc. No attempt has been made in the present work to distinguish between the constituents of the total crude carotene obtained, which has, as is usual, been determined as /?-~arotene.~y7,18,~0 Kemmerer18,20 has suggested using the plural, carotenes, for the mixture of carotene isomers that is estimated, but we prefer the singular, carotene, which is used throughout this paper in a general sense, and includes mixtures of carotenes of unknown composition.METHOD USED FOR THE DETERMINATION OF CAROTENE A number of general discussions of the estimation of carotene have appeared in the literat~re.~,*~y~~ For the purpose of the present research there was required a rapid process50 FRANKLIN, LYONS AND WHEELER: THE DETERMINATION OF [Vol. 75 of estimation that would give with dried peas results suitable for statistical treatment. The following method, based on a combination of known and tested procedures for extraction, chromatography and photometric determination of carotene, was developed after a number of trial experiments; eight to ten results can be obtained with it in a working day. Extraction-The extraction of carotene has been made the subject of frequent discussion.ly2 93y497911a ,27,28,36,37,42,*4,49 The technique employed was similar to that described in 36 and 44.A sample (about 4 g.) of marrowfat peas, dried at 44" C. to 14 per cent. moisture content, was ground to pass a 60-mesh sieve. Except in the experiments listed under Group E below, all such samples were taken from a master sample (7 lb.) of the marrowfat peas. The moisture content (14 per cent.) was checked at intervals. The ground material was kept in the dark7,33 at room temperature for 16 hours in contact with a mixture of 40 ml. of light petroleum (b.p. 40" to 60" C.) with 20 ml. of A.R. acetone. The latter solvent was removed from the decanted extract and washings (light petroleum, b.p.40" to 60" C. ; two lots of 50 ml.) by evaporation a t 100" C. to 15 ml. ; fresh light petroleum (50 ml.) was added and concentration to 15 ml. repeated.44 This procedure causes some isomerisation of @-carotene, but the effect is sma11,3,4,35,36942945,46 as shown by the results of recovery tests (see Table I). The solution of carotene and other pigments in light petroleum (15 ml.) thus obtained was diluted with light petroleum to 40 ml. before recovery of the pigments. Chromatography-The chromatogrphic method for the separation of carotene is now more popular than the phasic meth0d.l 9 3 A two-stage processu was employed, dicalcium p h o ~ p h a t e ~ ~ s ~ ~ being used in the first, and sodium sulphate - alumina mixture7 in the second column. The additional stage was found useful for concentration of carotene prior to deter- mination, and also as a check on the activity of the first column.Dicalcium phosphate (see 26 for the use of bone meal), is satisfactory for the removal of contaminating pigments from carotene, but its activity is liable to change so that a second guard column is on the whole a d ~ i s a b l e . ~ ~ 7 ~ ~ Further, the phosphate is not specific for the removal of chlorophyll.42 In the preparation of the first column, dicalcium phosphate was placed in a glass tube, 32 cm. long by 1.0 cm. internal diameter, constricted at one end, plugged there with cotton wool and connected to. a receiver through suction.7 A slurry of the dicalcium phosphate with light petroleum was introduced and further adsorbent was added intermittently with intermediate suction.When the adsorbent reached a length of 15 cm., it was capped with a 1-cm. layer of anhydrous sodium sulphate. The level of light petroleum was maintained above the sulphate at all times. The second column, 19.0 cm. long by 1.0 cm. internal diameter, which was used as described in 7, was filled to a height of 7 cm. with a mixture of sodium sulphate and alumina; the value of alumina in avoiding the necessity for saponification is discussed in 537,17. Neither column was employed more than twice, and not if over a few hours old. Changes in activity were indicated by the behaviour of the columns; in particular the dicalcium phosphate did not adsorb all chlorophyll, and the adsorption band of carotene on the alumina appeared diffuse.The prepared extract was transferred to the first column, and two 10-ml. portions of light petroleum were used for washing; When about 1 cm. of washing liquid remained over the sodium sulphate surface, carotene and possibly some chlorophyll were washed through with light petroleum (50 ml.) ; other interfering pigments remained on the dicalcium phosphate.27 The solution of carotene in light petroleum, amounting to about 110 ml., was poured on to the second column where the pigment was adsorbed as a diffuse orange band (0.5cm.) at the top. Booth's eluting solution7 (1.2 to 2 per cent. by volume of acetone inlight petroleum) was added, and the eluate, about 7 ml., containing carotene was made up to 10 ml. with light petroleum. Usually four lots (about 4 g.each) of one sample of peas were examined at one time, using two columns of each adsorbent. Estimation of carotene-The crude carotene content20 of the resulting solution was estimated as /3-carotene by means of a Spekker photo-electric absorptiometer fitted with a filter giving maximum transmission a t 450 rnp.l29lg and calibrated with /3-carotene at various concentrations (0.2 to 2.0 p.p.m.) in light petroleum (b.p. 40" to 60" C.). Neither a visual photometer nor a colorimeter using aqueous potassium dichromate as standard was found satisfactory30p47 with the carotene concentrations involved. The standard sample of /3- carotene (Ei& = 2310 at 464 mp. in benzene)12 was kindly supplied by Messrs. Lever Brothers and Unilever Limited (Dr.G. C. Hampson and Dr. R. F. Hunter). It was stored in the dark at 0" C. in an atmosphere of carbon dioxide.Jan., 19501 CAROTENE IN DRIED PEAS 51 DETAILS OF EXPERIMENTS RECOVERY OF p-CAROTENE IN RELATION TO VARIOUS STAGES OF THE ROUTINE PROCEDURE (SEE TABLE I)- These experiments, which were preliminary to the main work (groups E and F below), A: pea-flour which had been previously stripped of pigments, B: a 2 : 1 mixture of light petroleum and acetone by volume, C : the adsorbents (dicalcium phosphate followed by sodium sulphate - alumina mixture), D: stripped pea-flour to which quinol had been added. These trials were designed to yield information in regard to losses of carotene due to- (A) incomplete extraction from the material; (B) loss during the evaporation of acetone from the pigment extract; (C) incomplete recovery from the adsorbents.In the (D) series the protective (anti-oxidant7y44) effect of quinol was examined. EXPERIMENTAL METHODS- The calibrated photo-electric absorptiometer fitted with a filter as described above was used to standardise a p-carotene solution of suitable concentration in light petroleum (b.p. 40" to 60" C.) (cf. reference 2, p. 600). This solution was employed to provide in replicate the quantities of p-carotene required for the recovery trials. A-Trials-The pea-flour used was ground to pass 60 mesh and stripped of pigments by extraction with acetone until colourless, and air-dried. Quantities of /I-carotene in light petroleum (about 10ml.) corresponding to the quantity of carotene found in the un- extracted pea-flour (about 3 p.p.m.) were mixed with the stripped air-dried flour samples (about 4 g.) contained in conical 150-ml.flasks and light petroleum and acetone were added to make up the prescribed extracting solvent. The stoppered flasks were kept overnight in the dark, and carotene was estimated by the complete routine procedure. B-Trials-Amounts of /I-carotene of the same order as present in pea-flour, and of five and ten times those amounts, were kept overnight in stoppered flasks in the prescribed quantity (60ml.) of solvent, and the routine procedure for estimation was followed from that stage onwards. C-Trials-The amounts of /I-carotene used in the A-Trials were dissolved in light petroleum (10 ml.) and added to a column of dicalcium phosphate prepared as described above, and the remainder of the routine estimation procedure was followed.D-Trials-The procedure was as described for the A-Trials with the modification that the addition of quinol in light petroleum (10 ml.) to the stripped pea-flour preceded that of /3-carotene. TABLE I involved the determination of the percentage recovery of added /I-carotene from- RECOVERY OF p-CAROTENE IN RELATION TO VARIOUS STAGES OF THE ROUTINE PROCEDURE A B1 B2 B3 C D1 D2 Number of estima- Trial series tions (Stripped pea-flour) . . 8 10 (Extracting solvent) . . 10 10 7 (Adsorbents) . . . . 10 } 7 (Stripped pea-flour and quinol) 7 J ,+Carotene added in individual estima- tions, Pg. 12.9 12.9 70.8 132.8 11.0 26.5 + (1.2 PLg. quinol) 20.3 4- quinol) (3.5 Pg.Recovery (%) of added 13-carotene I Max. Min. Mean 92.2 82.2 87.3 91-4 82.0 86.7 93.2 86.7 90.3 95.4 90.4 91.6 100.0 92.7 95.6 98.1 88.7 94.1 96.1 90.1 94.0 Average amount of ,%carotene lost 1.6 1.9 6.9 11.2 0.5 1.5 1.2 Coefficient of variation of results 4.33 3.63 2.49 1-04 2-05 3.7 1 2.3 I I t will be seen that the recoveries in trials A and B1 are substantially identical, from which we infer that the carotene loss (about 13 per cent.) is unlikely to be due to incomplete extraction from the pea-flour. From trials B1, B2 and B3 it is seen that recovery from the52 FRANKLIN, LYONS AND WHEELER : THE DETERMINATION OF solvent is to some extent a function of the amount present, and that the greater the con- centration of carotene, the better and less variable the recovery, from which it must be concluded that the accuracy of the method is necessarily limited by the low level of carotene normally present in dried peas.Trial C gave recoveries significantly higher (nearly 9 per cent.) than trials A and B1, which indicates that the excluded stage in trial C (the evaporation of the petroleum acetone) must be responsible for this difference. It also seems that chromatography accounts for more than 4 per cent. loss of carotene under these conditions. Hence the over-all loss of 13 per cent. is divided between the two main stages. But it is also seen from trials D1 and D2 that this 13 per cent. can be reduced to 6 per cent. by the addition of quinol. The protective (anti-oxidant) action of quinol and related compounds on carotene is of course These conclusions are supported by the results of application of the F- and t-test~~1,1~,~2 to the experimental data.[Vol. 75 well kno~n.6,7,31,37,38,39,43,44 ACCURACY OF ESTIMATION OF CAROTENE IN PEA-FLOUR After completion of the preliminary experiments on the various stages of the procedure, the accuracy of the method of estimation was examined in five independent sets of experiments divided into two groups, E and F. Group E, which related to one batch of peas, comprised three sets, in two of which known weights of carotene were added to weighed pea-flour samples before applying the routine analytical procedure. In the third set, carotene was not added before estimation. The second group, F, which was based on the rnaster-sample batch of peas, involved two sets only, in one of which carotene was added before estimation.Table I1 summarises the relevant data. TABLE I1 Number Range of sample of weights in set Max. r. samples 4 5-75 4-04 12 4.70 4.04 16 5.61 3.7 1 8 4.91 3.93 8 5.00 3.55 Group Set E } 2 g- g. E3 Wt. of carotene added, 8.2 5.8 0 12.8 0 Range of results for carotene originally present in p.p.m. Max, Min. Mean A r \ 4-06 3.74 3.88 4.82 3.60 3-93 4-39 3.01 3.71 3-35 2.75 3.11 3.31 2.87 3.04 The experiments included in group E differed from those in group, F in that the absorption readings for group E were made on a different absorptiometer housed in a building about 400 yards distant from the laboratory, so that it was necessary to carry the solutions of carotene in light petroleum between the buildings.The methods of regression analysis (see reference 11, p. 118) were applied to the five independent sets of experiments. It was found that the results in both E and F could be expressed by an equation of the form Y = A,, + A,X, where Y is the natural carotene content in pg., and X is the weight of the sample taken. A,, has the values 2.58 and -0.94 and A, the values 3.24 and 3.29 for group E and-group F respectively. The two values for Al do not differ significantly, but there is a significant difference in the A, values, although each of these values is not significantly different from zero. It is of course to be expected that Y and X will vanish together. The analysis also shows that, taking a 4.3g. sample as standard, the carotene content of the group E samples at the 95 per cent.level of probability is 3.8 f 0.14 p.p.m. (3-8 p.p.m. f4 per cent.) and that of the group F samples is 3.1 f 0.10 p.p.m. (3.1 p.p.m. rt3 per cent.). A simplified treatment of the results in group F in which it is assumed without regression analysis that Y = A,X may be of interest. Table I11 shows the results, in p.p.m., obtained in the group F experiments. Subtraction of the @-carotene added (column 2) from that found (column 3) gave an estimate (column 4) of that naturally present in the pea-flour. These results were found not to differ significantly (F- and t-tests) from the results (F, series) obtained with pea-flour to which carotene had not been added. The carotene that had been added was, therefore, completely recovered, and there was no loss in the process.It is readily calculated from the standard error that the accuracy of the mean (3-1 p.p.m. of carotene) is 3.1 -f 0.19 p.p.m. in 95 per cent. of cases.Jan., 19501 CAROTENE IN DRIED PEAS 63 This compares well with the result of the more elaborate analysis given above (3.1 rt 0.10 p.p.m.). The simplified method applied to the group E series yielded for the accuracy of the mean at the 95 per cent. level, 3.8 f 0.24 p.p.m. The results for group F were, as stated above, more satisfactory than the group E results. TABLE I11 SIMPLIFIED TREATMENT OF Set Fl f A -l Set F, &Carotene added to Total Carotene Carotene in pea-flour samples carotene naturally untreated -7 found present pea-flour 99 2.8 99 3.2 99 3.1 99 2.7 77 2.6 99 2.9 99 3.3 p.p.m.( 3) 6.4 6.1 6.3 5.9 5.7 5.5 6-2 6.6 p.p.m. (4) 3.2 3.3 3.1 2.8 3.0 2.9 3.3 3.3 p.p.m. (5) 3-0 3.2 3-3 2.9 2.9 3.0 2.9 3.3 RESULTS Comparison of Sets F, and F, (columns 4 and 5) Mean, Set F, = 3.11 91 7) F, = 3-04 A . Variance ratio (F) Probability Significance % - 1.69 > 10 value of t - 0-801 >10 . Standard Error of difference between means, S = 0.087 STABILISATION OF CAROTENE The fact that there is no significant loss of the carotene added in the experiments shown in Tables I1 and I11 indicates the presence in natural pea-flour of a substance that stabilises carotene during the removal of acetone by evaporation. I t is possible that tocopherol, which occurs in peas, is the protective agent during the estimation of carotene by this method.Kozin and Bessonov21 have shown that pea-flour contains an anti-oxidant which prevents rancidity in sunflower oil, though an ether extract is ineffective; Lieck and WillstaedP have determined the amount of the anti-oxidant a-tocopherol in peas; tocopherols are known to be stabilisers for c a r ~ t e n e . ~ , ~ ~ SUMMARY A rapid two-stage chromatographic method applicable to the determination of total carotene (about 3p.p.m.) in dried peas is described. The various stages in the analytical procedure were studied statistically in a series of control experiments involving the recovery of added /3-carotene. In such recovery from pea-flour previously stripped of carotene, it was found that the greater portion of the pigment was lost in the stage involving removal of acetone from a carotene solution by evaporation.This loss was reduced when traces of an anti-midant (quinol) were added to the stripped pea-flour. A statistical examination of the results of experiments in which p-carotene was added to untreated pea-flour showed that the method is efficient in that there was a linear relation- ship between the quantity of pea-flour taken and the quantity of natural Carotene found. The accuracy of the results a t the 95 per cent. level for a 4.3 g. sample is better than k 5 per cent. of the ,carotene present. The results also indicate that pea-flour contains a substance which stabilises carotene during removal of acetone by evaporation. The thanks of the authors are due to Dr. V. H. Booth for advice, to Messrs. Lever Brothers and Unilever Limited (Dr.G. C . Hampson and Dr. R. F. Hunter) for gifts of 8- carotene and to the Directors of Batchelor and Company (Ireland) Limited, for facilities afforded to one of us (F. B. L.). REFERENCES 1. 2. 3. 4. Assoc. Off. Agric. Chem., “Changes in Methods of Analysis,” J . Assoc. Off. Agric. Chem., 1947, - , Methods of Analysis, 6th Edition, Washington, 1945. Association of Vitamin Chemists, Methods of Vitamin A ssuy, Interscience Publishers, New York, Beadle, B. W., and Zschiele, F. P., J . B i d . Chem., 1942, 144, 21. 30, 84. 1947.54 FRANKLIN, LYONS AND WHEELER [Vol. 75 12. 13. 14. 16. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 5. 6. 7. 8, 9. 10. 11.Ila. Bickoff, E., and Williams, K. T., Ind. Eng. Chem., Anal. Ed., 1943, 15, 266. - - , Oil and Soap, 1946, 23, 65; Chem. Abts., 1946, 40. 2359. Booih, V. H., J . SOC. Chem. Ind., 1945, 64, 162. Brown, H. D. et al., Food Res., 1947, 12, 4. Brunius, E., and Hellstrom, V., Svensk Kem. Tid., 1945, 57, 86; Chem. Abts., 1946, 40, 2541 Charkey, L. N., and Wilgus, H. S., I n d . Eng. Chem., Anal. Ed., 1944, 16, 184. Davies, 0. L., Statistical Methods in Research and Production, Oliver and Boyd, London and Derby, J. V., and DeWitt, J. B., J . Assoc. Ofl. .4gric. Chem., 1948, 31, 704. Devine, J., Hunter, R. F., and Williams, N. E., Biochem. J . , 1945, 39, 5. Fisher, R. A., Statistical Methods for Research Workers, Oliver and Boyd, London and Edinburgh, Edinburgh, 1947.1946. Gleim, E., et al., Food Res., 1946, 11, 61. Guerrant, N. B., Vavich, M. G., and Fardig, 0. B., I n d . Eng. Chem., Anal. Ed., 1945, 17, 710. Guerrant, N. B., et al., Ind. Eng. Chem , 1947, 39, 1000; 1948, 40, 2258. Harris, L. J., et al., Eleventh International Congress of Pure and Applied Chemistry, London, July, Kemmerer, A. R., J . Assoc. Off. Agric. Chern., 1945, 28, 563. -, Ibid., 1946, 29, 18. Kemmerer, A. R., Fraps, G. S., and Meinke, W. W., Food Res., 1945, 10, 66. Kozin, N. I., and Bessonov, S. M., Voprosy Pitaniya, 1941, 10, 24; Chem. Abts., 1946, 40, 479. Lamb, F. C., Pressley, A., and Zuch, T., Food Res., 1947, 12, 273. Lieck, H., and Willstaedt, H., Svensk Kem. Tid., 1945, 57, 134; Chem. Abts., 1946, 40, 4759. Lo, T., Food Res., 1945, 10, 308.Mackinney, G., Amoff, S., and Bornstein, B. T., Ind. Eng. Chem., Anal. Ed., 1942, 14, 391. hlann, T. B., Analyst, 1944, 69, 34. Moore, L. A., I n d . Eng. Chem., Anal. Ed., 1940, 12, 726. - Ibid., 1941, 13, 600. -, Ibid., 1942, 14, 707. Nelson, W. A. G., Analyst, 1947, 72, 200. Olcovich (Olcott), H. S:, and Mattill, H. A., J . Bid. Chem., 1931, 91, 105. Paterson, D. D., Statistical Technique in Agricultural Research, McGraw-Hill, New York and Pepkowitz, L. P., J . Biol. Chem., 1943, 149, 465. Ramasarma, G. B., Hakim, D. N., and Rao, S. D., Analyst, 1947, 72, 194. Schrenk, W. G., Silker, R. E., and King, H. H., I n d . Eng. Chew., Anal. Ed.; 1944, 16, 328. Silker, R. E., Schrenk, W. G., and King, H. H., Ibid., 1944, 16, 513. --- , Ind. Eng. Chem., 1944, 36, 831. Sreehivasai, A., and Vaidya, R. M., J . Sci. Ind. Research (India), 1947, 6, 69; Chem. Abts., 1948, Stern, M. H., et al., J . Amer. Chem. SOC., 1947, 69, 869. Stimson, C. R., Tressler, D. K., and Maynard, L. A,, Food Res., 1939, 4, 475. Taylor, R. J., Analyst, 1946, 71, 566. Wall, M. E., and Kelley, E. G., Ind. Eng. Chem., Anal. Ed., 1943, 15, 18. Weier, T. E., and Stocking, C. R., Science, 1946, 104, 437; Chem. Abts., 1947, 41, 4867. Wilkes, J. B., Ind. Eng. Chem., Anal. Ed., 1946, 18, 702, Zechmeister, L., Chem. Reviews, 1944, 34, 267, Zechmeister, L., and Polgar, A., J . Amer. Chem. SOG., 1943, 65, 1522. Zschiele, F. P., and Beadle, B. W., Ind. Eng. Chem., Anal. Ed., 1942, 14, 633. Zschiele, F. P., Beadle, B. W., and Kraybill, H. R., Food Res., 1943, 8, 299. Zschiele, F. P., and Whitmore, R. A., Analyt. Chem., 1947, 19, 170. 1947. London, 1939. 42, 1022. DEPARTMENTS OF CHEMISTRY AND MATHEMATICS UNIVERSITY COLLEGE DUBLIN February, 1949

ISSN:0003-2654    

DOI:10.1039/AN9507500049

出版商:RSC    

年代:1950

数据来源: RSC

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Jan., 19501 NOTES 56 Notes AMPEROMETRIC TITRATION OF MERCAFTANS IT has recently been reported1 that the strength (purity) of mercaptans determined by ampero- metric titration under the conditions given by Kolthoff2 had given results several units less than 100 per cent., despite the fact that the total sulphur determination indicated that a pure material had been used. The authors attributed this to the fact that mercaptans easily oxidise to the disulphide (which would cause little change in the total sulphur content) and they inferred that the amperometric titration gives a correct measure of the mercaptan content. Results obtained in these laboratories by amperometric titration on /3-mercaptonaphthalene were lower than the results by iodine titration, and a short investigation was undertaken to ascertain whether or not amperometric titration produced results at the right level.Two mercaptans, 8-mercaptonaphthalene and mercaptobenzthiazok, were purified by re- crystallisation, their purities were checked by alternative methods (see below) and they were then examined amperometrically by silver nitrate titration in ammoniacal ethanol solution. The results are given in Table I. TABLE I /3-Mercaptonaphthalene Mercaptobenzthiazole O ! %I I - Strength by titration with sodium hydroxide . . - Strength by titration with iodine . . .. .. 98.9 Strength by amperometric titration . . .. 95.0 It was suspected that the low amperometric results were due to oxidation occurring during the course of the titration, and this was confirmed by stirring an ammoniacal ethanol solution of 8-mercaptonaphthalene in air for 4 hr.before titration with silver nitrate; a result of 60.7 per cent. was obtained. Further tests were carried out in order to ascertain the source of the oxidising agent ; the results, together with values obtained by iodine titration, sodium hydroxide titration and potentiometric titration3 (using aqueous silver nitrate instead of alcoholic silver nitrate) are given in Table 11. In test (a) the /3-mercaptonaphthalene solution was prepared and titrated under an atmosphere of nitrogen, and the result (compare with Table I) shows that in a normal titratios atmospheric oxidation is a negligible factor; an aliquot portion from the initial p-mercaptonaphthalene solution was also titrated with 0.01 N iodine solution, with a result 98-6 per cent., from which it is concluded that no oxidation occurs on standing in neutral alcohol solution.hr., the mercaptan was titrated and then a second aliquot portion of mercaptan solution was added and titrated. It was hoped that any oxidising agent in the ammoniacal ethanol would be used up in oxidising the first portion of mercaptan, and that the second portion of mercaptan would not be subject to oxidation from this cause. The titres on the first and second portions corresponded to purity figures of 82 and 94-4 per cent. respectively; the difference must be due to slow oxidation (during the first test) caused by an oxidising agent in the ammoniacal ethanol. TABLE I1 In test (b), the solution was stirred under nitrogen for Strength by /3-Mercaptonaphthalene Mercaptobenzthiazole ?/, % Iodine titration .... . . 1 . I . 98.9 Sodium hydroxide titration . . .. . . .. - Potentiometric titration3 . . . . . . .. 98.9 Amperometric titration : ( b ) Under nitrogen: (a) Under nitrogen (immediate titration) . . 96.2: (i) After .) hr. stirring . . . . . . 82 (ii) Second portion of mercaptan solution (c) As in (a) with silver nitrate in boiled-out added after completion of (b) (i) . . 94.4 distilled water . . .. .. .. .. 97.3 Test (c) was carried out under nitrogen, using silver nitrate that had been freshly prepared with boiled-out distilled water. The result is substantially higher than in the other amperometric tests, and is not very far short of the iodine figure and potentiometric titration figure.It is concluded that the main sources of oxidising agent are the dissolved air in silver nitrate solution56 NOTES [Vol. 75 as normally used and the ammoniacal ethanol. It will be noted that the potentiometric titration in neutral alcohol solution gives a satisfactory result, as the mercaptan is less susceptible to oxidation in neutral than in ammoniacal solution. It might be possible to develop an amperometric titration in neutral alcohol solution, but there would appear to be no advantage in doing this. In the case of mercaptobenzthiazole, agreement between the alkali titration and amperometric titration methods is rather better, presumably owing to the greater stability of the mercaptan group in mercaptobenzthiazole. REFERENCES 1. Frank, R.L., Smith, P. V., Woodward, F. E., Reynolds, W. B., and Canterino, P. J., “Mercaptan Structure and Regulator Activity in Emulsion Polymerisation,” J . of Polymer Scieice, 1948, 3, 39. Kolthoff, I. M., and Harris, W. E., “Amperometric Titration of Mercaptans with Silver Nitrate using the Rotating Platinum Electrode,” Ind. Eng. Chem., Anal. Ed., 1946, 18, 161. Tamele, M. W., and Ryland, L. B., “Potentiometric Determination of Mercaptans,” Ibid., 1936, 8, 16. 2. 3. ANALYTICAL LABORATORIES IMPERIAL CHEMICAL INDUSTRIES LTD. BLACKLEY, MANCHESTER, 9 N.STRAFFORD F. R. CROPPER A. HAMER May, 1949 THE FRUCTOSE UREA AND FRUCTOSE ACETONE REACTIONS AS SELECTIVE TESTS FOR SUGARS As is well known, urea combines with furfural in concentrated acids to form an unstable violet pigment (Schiffl).With w-hydroxymethyl furfural, and its reduction product “methyl-furil, ” the pigment is bright blue (Fentons). The furfural reaction has been used by Nakashima and Maruaka3 for estimation of urea in blood plasma, stannous chloride being added to protect the aldehyde from decomposition during the condensation. A converse form of the reaction has been introduced by Foulgel4 as a test for keto-hexoses; he uses its the reagent a 40 per cent. solution of urea in 40 per cent. sulphuric acid containing 2 per cent. of stannous chloride. The value of the reducing agent when urea is used as a sugar reagent is doubtful, since it can be shown that mild oxidation is necessary for final development of the colours in all the sugar reactions depending on formation of a furfural as intermediate.By omitting the stannous chloride and substituting hydrochloric for sulphuric acid, the fructose urea reaction can be applied as a simple and very delicate selective test for keto-hexoses. METHOD-TO about 0-6 to 1 g. of urea in a dry porcelain dish add 5 or 6 drops of concentrated hydrochloric acid and 2 or 3 drops, not more, of the sugar solution. Gently shake until the urea has dissolved. I f the sugar solution contains fructose in con- centrations of 0-5 to 1 per cent., a bright, turquoise-blue ring will appear within 5 minutes, and eventually close in to form a small pool of blue syrup. More dilute solutions require longer times, but the test will reveal fructose in 2 drops of a 0.01 per cent. solution within about 15 minutes.The test is very sensitive, and the lower limit is of the order of 5 pg. of fructose, or 15 pg. of sucrose. Of course, greater delicacy can be obtained by concentrating some of the fructose solution with the urea prior to adding the acid. Excess of urea is necessary to stabilise the pigment in the final viscid mixture. If sulphuric acid or hydrobromic acid is used instead of hydrochloric, the pigment will be destroyed. SELECTIVITY-The blue reaction is characteristic of keto-hexoses, including fructosides, sucrose and inulin, and sorbose. Non-ketose sugars only react chromatically when in relatively high concentration and after longer times. Aldo-pentoses and pentosans give yellow colours ; aldo-hexoses and hexosans give red or red-purple colours. Crystalline ascorbic acid does not react, apart from the faint blue due to contamination with sorbose.Even in presence of foreign sugars, the fructose reaction can be recognised by the early appearance of the characteristic blue ring. For exact work it is necessary to extract the pigments with ethanol, and spectrograph the mixture. As obtained in the spot test, the blue pigment is insoluble in chloroform but freely soluble in acidified alcohols and in dilute acids. When the solution in ethanol is concentrated the pigment separates out as an amorphous, indigo-like precipitate, but has not yet been obtained in crystalline form. Urea cannot be replaced by ammonium salts, acetamide, glycine, ethyl carbamate, methyl- amine, methylurea or thiourea. Guanidine salts, as Foulger has observed, give red-purple colours with aldo-hexoses as well as with fructose.Out of a variety of non-nitrogenous compounds tested, acetone was found to be the only one that reacted in a manner somewhat similar to urea in the Heat on a boiling water-bath. Water de-sensitises the reaction. It is reversibly changed to red by alkalis.Jan., 19501 REVIEWS 57 sugar tests. Owing to its volatility it cannot be used in the spot test, and, generally, it is less sensitive and less selective as a fructose reagent. As the colour reaction between acetone and the sugars does not appear to have attracted attention, it is briefly described here as a qualitative test. THE ACETONE TEST METHOD-Mix 4 or 5 drops of acetone with 5.1. of concentrated hydrochloric acid. Mix and allow the mixture to cool spontaneously, Boil vigorously for a few seconds, not long enough to expel the acetone.Add 1 or 2 drops, not more, of the sugar solution. With fructose solutions down to 1 per cent., a violet colour, changing to blue, soon develops. Concentrations down to 0.1 per cent., which is about the lower limit of the test, require 10 minutes for colour development. The test is positive with a “pin-head” (0.5 mg.) of solid fructose. Even a slight excess of water must be avoided, as it completely inhibits the test. Non-ketose sugars do not react appreciably in original concentrations below 1 per cent. ; above this, they produce colours ranging from red to purple, some of which resemble those due to fructose. This work forms part of an investigation into the composition of marine algae, carried out by the aid of a grant from the Medical Research Council of Ireland. REFERENCES 1. 2. 3. 4. Schiff, H., Ber., 1877, 10, 773. Fenton, H. J. H., Trans. Chew SOG., 1903, 83, 187. Nakashima, Y., and Maruaka, K., Deutsch. Arch. f. Klin. Med., 1924, 143, 318. Foulger, J. H., J . Biol. Chem., 1932, 99, 207. DEPARTMENT OF BIOCHEMISTRY TRINITY COLLEGE, DUBLIN WILLIAM R. FEARON J. A. DRUM April, 1949

ISSN:0003-2654    

DOI:10.1039/AN9507500055

出版商:RSC    

年代:1950

数据来源: RSC

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Jan., 19501 REVIEWS 57 Reviews BIOCHEMICAL PREPARATIONS. Volume I. Edited by H. E. CARTER. Pp. xi + 76. New York: To those who have used Organic Syntheses, this first volume of Bioclzemical Preparations will have a familiar appearance, for both series are closely similar in style and format. It is intended that this book, which will be followed by other volumes at intervals of twelve to eighteen months, will do for biochemistry what Organic Syntheses did for organic chemistry. Very wisely the editors, before launching the new series, conferred with the editors of Orgavlic Syntheses, so that they have been able to profit by the experience gained in the publication of the older series. The new volumes will certainiy meet a long-felt need; they will describe, with full details, methods of preparing compounds of natural origin not readily available commercially.This is an even more difficult task than that which confronted the editors of Organic Syntheses in view of the greater number of variables involved and the difficulty of putting into words the subtle differences of technique that sometimes make for success or failure in biochemical work. It will be interesting to see to what extent the yields and purities claimed in this volume can be reproduced by workers in different laboratories, even though each preparation submitted is invariably checked in an independent laboratory before publication. Wiley & Sons Inc. London: Chapman & Hall, Ltd. 1949. Price 15s.58 REVIEWS [Vol. 75 Although the format of Organic Syntheses has been closely followed there is one important difference ; in BiochemicaE Preparations an additional section (Properties and Purity of Product) has been included for each substance.This infomation is most helpful, as it is generally more difficult to lay down standards of purity for substances made from natural sources than for synthetic compounds, and in any event, it is frequently more important to know what impurities are present than to be assured that a substance is almost pure. The preparations listed in this volume comprise : adenosine di- and tri-phosphate, diphospho- pyridine nucleotide, the a-glucose- 1-phosphates and ~~-glyceraldehyde-3-phosphoric acid ; L-alanine, L-serine (together with three of the reagents required for making these two amino acids), 6-3 : 4- dihydroxyphenyl-L-alanine, L-glutamine, L-lysine monohydrochloride and D-tyrosine; casein, lycopene and lysozyme.The book also contains a list of the compounds of biochemical interest which have appeared in Organic Syntheses, but the reviewer was somewhat puzzled to see in this list “Casein,” the preparation of which is actually described in this first volume of Biochemical Preparations. This seems an instance of unnecessary duplication, but the two methods differ significantly in details and presumably the newer method offers some advantages over the earlier one; one would surely have expected a word of explanation, but the method described in Organic Syntheses is not even referred to. There is no doubt about the success the new series will have, and the volumes will become as indispensable in the biochemical laboratory as Orgahic Syntheses has become in the organic laboratory.The book is well printed and the binding is sufficiently substantial to withstand the constant usage that a book of this type will undoubtedly receive. Biochemists will look forward with interest to the subsequent volumes promised. F. A. ROBINSON CANNING TECHNOLOGY. By A. J. HOWARD, M.A., A.R.I.C. Pp. viii + 287. London: J . & A. Twenty years ago most sections of the canning industry in this country were in their infancy. The intervening years have been ones of rapid growth, education and discipline, with the result that the industry is now not only of considerable size, but is well instructed in the scientific principles on which it is based, and fully aware of the responsibility it must bear in matters of public health.In its early period of growth the principal textbooks available were written chiefly for the practical canner and, while dealing comprehensively with the operations required for each particular product , tended to ignore, or to give scant attention to, the fundamental principles of the preservation of food by heat. The works chemist was left to rummage through the scientific and technical journals i f he wished to equip himself with adequate knowledge of his subject. The situation improved considerably with the publication, six years ago, of a small but excellent book on the microbiology of canning, but this work was necessarily limited in its scope, and a fuller survey of the scientific aspects of canning was still required.The book now under review meets this need, and is written with the authority of one who held for a time the office of Director of Canning at the Ministry of Food. After a short historical introduction the author devotes three chapters-more than one-third of the whole book-to consideration of the tinplate container. This emphasis on the can-its origin, structure and behaviour-is welcome, as the importance of the container is apt to be over- shadowed by other factors more obviously concerned with the quality and purity of the contents. These three chapters deal with the modem methods of manufacture of tinplate, the fabrication and lacquering of cans and the types of internal and external corrosion that may develop when the cans are stored.The section concerned with the electrochemistry of corrosion is particularly clear and comprehensive. Attention is next given to problems encountered in establishing a cannery, to the methods used in examining and preparing the raw food materials, and to the operations of exhausting, closing and processing. Excellent illustrations and descriptions of modem canning equipment make it possible to follow the operations in detail, and the balance between the scientific and technological aspects of each subject is well observed. Other chapters deal with the principles of heat sterilisation, the causes and prevention of microbiological spoilage, and the metallic contamination of canned foods. It is in the chapter dealing with the principles of heat sterilisation that the reader may possibly find himself least convinced by the arguments put forward, though it must be added that these are strictly orthodox.The author stresses the view that the survival of bacterial spores is largely determined by their individual heat resistance, and that there is some point in the heat process Churchill, Ltd. 1949. Price 30s. net.Jan., 19501 REVIEWS 59 at which complete destruction of the spores can be guaranteed. The newer alternative hypothesis, that every spore has a finite chance of survival, however severe the process may be, and that processing times are related to the initial infection and to the degree of survival that can be tolerated for each type of spore, might have been mentioned with advantage.In future editions, which will no doubt be called for, rather more space might be devoted to the health aspects, including nutritional values, of canned foods. As it is there is little doubt that scientists and canning technologists will b$nefit by careful study of this book. It is pleasant to read, not only on account of the agreeable style in which it is written, but also for its attractive lay-out and printing, and for the large number of clear illustrations and diagrams with which it is provided. W. B. ADAM ABSORPTION SPECTROPHOTOMETRY. By G. F. LOTHIAN, M.A., F.1nst.P. Pp. 196. London : Hilger & Watts, Ltd. This book is a successor to Twyman and Allsopp’s The Practice of Absorption Spectro- fdzotometry (Second Edition, 1934). Considerable developments have taken place since 1934 in the techniques of photo-electric spectrophotometry in the visible and ultra regions and in equipment for infra-red spectroscopy.An increasing number of important analytical problems have been or are being solved by methods which culminate in measurements of light absorption, and the photo-electric absorptiometer is replacing visual colorimetry just as photo-electric spectro- photometry is taking the place of photographic methods. For many purposes, however, some of the older methods are far from being obsoIete and a great deal of useful work can still be done with sector photometers, quartz spectrographs and visual spectrophotometers, instruments that cost little to maintain and do not deteriorate. For other purposes, photo-electric spectrophoto- metry, more sensitive to small differences in absorption intensity, is indispensable.There are further problems which though not open to attack by the methods based on selective absorption of visible or ultra-violet rays, are readily solved by the methods of infra-red spectroscopy. In this field the older equipment is obsolete. The newer techniques are accurate and speedy and there is a growing body of fact and interpretation which progressively assists application to new problems. Many of the best instruments are American; supply has stimulated demand and demand production. In this country instrument manufacturers have been through a difficult time since 1939, and the dollar “famine” adds urgency to the hope that their great efforts to meet the needs and even outstrip their competitors will soon be successful. Mr. Lothian’s book is in three parts: (1) Principles of Spectrophotometry, (2) Applications and (3) Technique. Chapter V on methods of calculation in the application of spectrophotometry to quantitative analysis will be found particularly useful. Chapter XI1 on infra-red techniques, although excellent in its way, is inadequate, and the subject-matter perhaps needs more space in later editions. 1949. Price 26s. net (plus postage 9d.). All users of spectroscopes will find much that is of value to them in this book. R. A. MORTON

ISSN:0003-2654    

DOI:10.1039/AN950750057b

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

LOCAL SECTIONS AND SUBJECT GROUPS THE North of England Section and the Scottish Section were formed to promote the aims and interests of the Society among members in those areas. Members residing in England or Wales north of Birmingham may become members of the North of England Section, and those residing in Scotland members of the Scottish Section. The Microchemistry Group, the Physical Methods Group and the Biological Methods Group have been formed within the Society to further the study of the application of micro- chemical, physical and biological methods of analysis. All members of the Society are eligible for membership of the Groups. There is no extra subscription for membership of a Section or Group. Application for registration as a member should be made to the Secretary of the Society. The Sections and Groups hold their own meetings from time to time in differentplaces.

ISSN:0003-2654    

DOI:10.1039/AN950750060b

出版商:RSC    

年代:1950

数据来源: RSC