ISSN:0003-2654    

DOI:10.1039/AN95075FX045

出版商:RSC    

年代:1950

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95075BX047

出版商:RSC    

年代:1950

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95075FP091

出版商:RSC    

年代:1950

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN95075BP097

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

DECEMBER, I950 Vol. 75, No. 897 THE ANALYST PROCEEDINGS OF THE SOCIETY OF PUBLIC ANALYSTS AND OTHER ANALYTICAL CHEMISTS AN Ordinary Meeting of the Society was held at 7 p.m. on Wednesday, October 4th, 1950, in the Meeting Room of the Chemical Society, Burlington House, London, W.l. The chair was taken by the President, Mr. George Taylor, O.B.E., F.R.J.C. The following papers were presented and discussed : “The Evaluation of Liming Materials for Agricultural Purposes,” by A. M. Smith, Ph.D., D.Sc., F.R.I.C., A. Comrie, B.Sc., A.R.I.C., and K. Simpson, B.Sc., A.R.I.C. ; “The Accurate Determination of ‘Phosphoric Anhydride’ by Means of Quinoline Phosphomolybdate,” by H. N. Wilson, F.R.I.C. ; “The Determination of Potassium in Fertilisers by Flame Photometry,” by L. Brealey, B.Sc. NEW MEMBERS Alec Harold Adams, BSc., MSc. (Lond.), A.R.I.C. ; James Bernard Attrill, B.A. (Cantab.) ; Douglas Bryan, B.Sc. (Notts.) ; William Edwin John Field, B.Sc. (Lond.), F.R.I.C. ; Albert William Harrington; Gordon Hopkins, B.Sc., M.Sc. (Lond.), A.R.I.C. ; Roy James MacWalter, B.Sc., Ph.D. (Lond.), F.R.I.C., A.M.1.Chem.E.; Peter Morries, B.Sc. (Birm.); Donald Pickles, BSc. (Lond.), A.R.I.C.; Kenneth Albert Proctor, B.Sc. (Notts.); K. R. Srinivasan, M.A., F.R.I.C.; Robert Guy Stuart, B.Sc. (Lond.), F.R.I.C.; Robert Arthur Sutton, B.Sc. (Liv.), A.R.I.C. ; William Leon David Diaper, B.Sc.Tech. (Manc.) ; James Robert Embleton, A.R.I.C. ; John Brian Dudley Robinson, B.Sc. (Reading). DEATHS WE regret to record the deaths of George Mason Hills Charles Arthur Hallas. 633

ISSN:0003-2654    

DOI:10.1039/AN9507500633

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

634 RIDYARD : THEORETICAL AND PRACTICAL CONSIDERATIONS {Vol. 75 Theoretical and Practical Considerations in the Determination of Aneurine (Vitamin B1) with Special Reference to the Recovery Factor BY H. N. RIDYARD SYNoPsI+-An analysis is made of the factors affecting the “recovery” of aneurine added to extracts in the determination of this substance. The effects of variation of sample and errprs in volume measurements are shown to be considerable. The combination of aneurine during digestion, the possibility of effects a t the oxidation stage, and interference with the emission of fluorescent light in the fluorimeter are considered. Results obtained in practice are given to illustrate the arguments and an investigation of the effects in the fluorimeter cell is included in some detail.The losses that take place in base-exchange methods are mentioned. The remarkable stability of the thiochrome method is showr, both by the expressions developed theoretically and by the practical results. Some of the considerations raised may have application to other estima- tions that involve similar raw materials and methods. THE addition of pure aneurine to extracts prepared for the fluorimetric determination of this substance has frequently been used as a criterion of the efficiency of the analytical pr0cedure.l s 2 y3 s4 s6 For this purpose, two similar extracts of the same sample, to one of which a known amount of aneurine has been added, are submitted to the same analytical procedure. The amount of aneurine found in the extract to which no addition has been made is then sub- tracted from the amount found in the other portion and the difference, “aneurine recovered,” is divided by the amount of aneurine added, to give the “recovery factor.” The added aneurine has commonly been assumed to be subject to the same losses as that already present in the material under examination, and it is this assumption and the accuracy of the method in relation to the physical and chemical factors involved that form the subject of the present investigation.Recovery experiments of this kind are commonly used for checking various analytical procedures and are often of great value, but examination of their theoretical bases appears to have been neglected, although their complete adequacy has been questioned.6 When recovery experiments are applied to a complex system such as we have in the fluorimetric estimation of aneurine they have definite limitations and conceal sources of error, which, if overlooked, can lead to fallacious conclusions.Some of the considerations brought forward in this paper are applicable to a wide variety of recovery experiments, others are specific to the estimation of aneurine. Furthermore, although additions are commonly used as a check on analytical procedure, in the estimation of aneurine the “recovery factor” has sometimes been used to correct the amount found-a procedure much more open to question. It appeared therefore that an analysis of the various factors involved would be of value to workers in this and similar fields, especially as the remarkable stability of the thiochrome method becomes more understandable in the course of the investigation. The fluorimetric determination of aneuririe may be considered in four successive stages- I.Extraction of the aneurine from the raw material either by steeping in acid water or by heating with dilute acid t o boiling-point, adjusting to pH 4.5 and digesting with takadiastase or other source of phosphatase according as aneurine pyrophosphate (co- carboxylase) is absent or present. The extract is sometimes passed over base-exchange materials. 11. Oxidation of the aneurine to thiochrome with alkaline ferricyanide, a process said t o be only about 70 per cent. efficient.’ 111. Extraction of the thiochrome by means of isobutyl alcohol. IV. Fluorimetric examination of this extract.Dec., 19501 I N THE DETERMINATION OF A4NEURINE (VITAMIN B1) I.FACTORS INVOLVED AT THE EXTRACTION STAGE I a . THE MODE OF ADDITION AND THE ERRORS CONCERNED IN THIS- Aneurine can be added to extracts in two ways- 635 I a 1. A definite weight of material can be extracted with acid water or digested in a buffer solution as is necessary, and a similar weight extracted in the same manner with a precisely similar fluid containing a definite amount of aneurine. I u 2. An extract can be prepared, centrifuged, filtered or otherwise clarified, and divided into two or more portions, to one of which is added a solution of aneurine. It will be seen that any factor, chance or otherwise, which affects the two extracts or portions of extracts in a different manner may appear as a considerably increased error in the recovery factor owing to the method of calculation. I a 1.In the first method, the homogeneity of the sample is obviously a factor in the type of result obtained, and many materials remain heterogeneous in spite of careful mixing, or may indeed segregate in the process of mixing. In extracts of samples of imported flour, enriched by synthetic aneurine, a small proportion showed deviations from the mean B, content which were as high as 0.05 pg. per ml. or even more, and this condition was not improved by further mixing of the flour. If only one extract without aneurine and one with were taken, such deviations would frequently act in opposition. If the mean quantity of aneurine in the flour extract should be 0.6 pg.per ml. (a normal level) the level without addition could be 0-55 or 0.65 and that with (say) 0-5 pg. per ml. of added aneurine could be 1-15 or 1.05 pg. per ml. The first pair of results would give a recovery of 0-6/0.5 = 120 per cent. and the second pair 0.4/06 = 80 per cent. For samples less homogeneous, the difficulties of obtaining similar fractions and exact weights of material increase, and the chance of error is increased accordingly.8 The question of volumes is of less importance than it is in method I a 2, for the added aneurine is measured as a standard solution and no error in concentration is involved; even a cylinder should measure 50ml. to within &0,25ml. Treated as above this could give recoveries of 101 or 99 per cent. In good work the deviation from the mean value is less than 0.02 pg.per ml. For the great majority of samples it rarely reaches 0.03 pg. per ml. and is of the same order in solutions of any con- centration between 0 and 1.0 pg. per ml. If with a solution containing 0.6 pg. per ml., the greatest error given above operated in opposite ways at the levels with and withod an addition of 0.5 pg. per ml., the results could be 0-57 or 0-63 without addition, and 1.13 or 1-07 with addition, which lead to recoveries of 0*56/0-50 = 112 per cent. and 044/060 = 88 per cent. Both percentage errors will increase rapidly as the quantity of added aneurine decreases. In rare and extreme cases these factors could all operate the same way to give recoveries of 120 x 101 x 112 = 136 per cent.or 80 x 99 x 88 = 70 per cent. These considerations show that to rely upon two extracts only (one with and one without addition) is useless. Nevertheless, because of the accuracy of the volume factor the method has commonly been used in this laboratory when recovery data were desired. The precaution has always been taken of using at least five extracts in all: two without additions, one with a moderate addition and two with larger additions, the results being plotted on squared paper.* If any point diverged seriously from a straight line drawn through the points, the matter was further investigated. I a 2. In the second procedure an extract is prepared, divided into two or more portions and aneurine added to one or more of these. This method is at once subject to the criticism that the extraction stage remains unchecked, although valuable informatiofi can obviously be obtained.The aneurine must be dissolved in the same buffer solution or dilute acid as is used in the extraction (see Table VI below). The manner of volume measurement is important. One mode of addition known to have been used was to add very small volumes of a strong aneurine solution from a graduated pipette and to neglect the increase in volume of the extract. Two different 1-ml. graduated pipettes were found to deliver 0.1 ml. with a standard deviation of 0.002 ml. for six successive deliveries from each, and of 0.008 ml. for six separate deliveries each; the maximum error being 0-011 ml. and the first deliveries always high. A 10 per cent. error in the recovery factor can thus easily be made, and the errors due to determination will still be present to The remaining error to be considered at this point is that of analysis.The mode of addition will be the critical factor.636 RIDYARD : THEORETICAL AND PRACTICAL CONSIDERATIONS [Vol. 75 affect the result. As the volume error decreases with better technique or larger volumes, it is usually preferable to add several ml. at least of aneurine solution, with care to add the same volume of acid water or buffer to the unfortified levels. The question of blank determination (fluorescence of a solution treated with caustic soda but not ferricyanide) must also be considered. This is advisedly determined in extracts with and without additions, and is, subject to the usual error of determination, normally the same in both.But if their independent blanks are subtracted before calculating the recovery factor, the blank errors could be added to those given above; hence in the determina- tion of the recovery factor the aneurine values obtained should be subtracted from one another before subtraction of the blank for determination of the aneunne level in the solution with no addition. The blank may of course be partly oxidised when ferricyanide is added.g*10 I b. COMBINATION OF ADDED ANEURINE DURING DIGESTION- One other factor must be considered at this stage when digestion processes are carried out during extraction of the raw material. Enzymic digestion sets up a state of equilibrium between free and combined forms of aneurine. Aneurine is considered to be phosphorylated by adenosine triphosphatell ; the aneurine pyrophosphate so formed combining with a protein to form carboxylase.In the following treatment no particular mechanism is assumed, but the equilibrium states of one reaction involving aneurine and of three successive reactions, are considered in respect of the effect of adding aneurine to the system. I b 1. ONE EQUILIBRIUM- A + B + C where A and C are free and combined forms of aneurine respectively. If now a is the total concentration of A free and combined, b the concentration of B free and combined and y the amount of C formed, we have at equilibrium .. .. . . . . (I b 1.1) Hence, neglecting the term y2 as very small indeed (since a is of the order g. mol. per litre) . . . .. . ?(I b 1.2) . . . . ab ’’ K’ + a + b * ’ From (I b 1.2) we see that for y to be just detectable (Le., = 10-8g. mol. per litre), if a = 10-6 g. mol. per litre then -# + lo-,. The free aneurine measured by the thiochrome b K method will be a - y = a ). If now we add aneurine of a concentration a’ b to the solution, the free aneurine measured will be (a + a’) recovery factor determined will be the difference between these two observed values divided by a’, i.e., (I b 1.3) (a + a’)b ab Recovery factor = 1 - _____ a’(K’ + a + a’ + b) a’(K’ + a + b) Since a and a’ are of the order the denominators of the two fractions in (I b 1.3) are virtually the same unless both K‘ and b are less than lo4, and the expression becomes b 1 - - and is independent of aneurine concentration.K’ + b 1 b 2. THREE EQUILIBRI-4- D + E + B A + B + C + F C + H - + M Let d , e, a, f, h, be the initial total concentrations of D, E, A, F, H, and at equilibrium let x of D and E be transferred, y of A, and x of H; K,, K,, K,, being the (reciprocal) equilibrium constants of the three reactions. Solving for x and z in the equilibrium equations of theDec., 19501 IN THE DETERMINATION OF ANEURINE (VITAMIN B1) 637 first and third reactions (rejecting terms involving second powers of x , y or x) and substituting in the second equation, we find that- . . . . + 12) = K,KJ(K, + d + e) + (K, 3. h)(ad + ae + de) - ride (I b 2.1) The first and third reactions have a buffering effect, and from considerations similar to those advanced above in the case of one equilibrium it seems unlikely that the recovery factor will be noticeably affected by aneurine concentrations, although the possibility must be borne in mind.Although these theoretical aspects have been considered for several years, it is only recently that practical results have been obtained which indicate that added aneurine may be combined to a marked extent at the digestion stage in the case of two or three materials. As these observations were made during investigations with a new adaptation of the sand absorption m e t h ~ d , ~ which will be described in a forthcoming paper, and very little work has yet been done on the phenomenon, further comment is not possible here. I c. BASE-EXCHANGE PURIFICATION AND RECOVERY- The use of zeolitic materials to purify extracts needs special mention.These usually lessen the optical interferences (see Table VII, Eluates), but may lead to losses by irreversible adsorption of aneurine, displacement by other basic material (organic or inorganic) in the extracts used, or inadequate adsorptive capacity. Irreversible adsorption, always found with De~also,~ might be proportional t o the weight of zeolite used, the concentration of aneurine, the time of contact, or to all three. Hence it cannot be assumed that recovery is thesame at the levels of addition and no addition. One example has been met with in which a 96 to 100 per cent. recovery was obtained with a lower analysis value than that obtained by another method giving a similar recovery. This might have been due to a chance combination of factors mentioned above, or to a genuine separation of aneurine from another substance fluorescing after oxidation. Safeguards lie in recovery experiments at more than one level of extract concentration and total aneurine - zeolite ratio and by comparison with other methods (see also I11 and IV, wheats, below).11. ERRORS AT THE OXIDATION STAGE Little special information has been obtained concerning any factor affecting recovery at the oxidation stage, and, with the possible exception of a few materials which have given serious and complicated difficulties in other ways, there is no experimental reason to suspect differences in the proportion of aneurine oxidised to thiochrome in solutions with and without addition, or indeed that the recovery at this stage is less than with pure aneurine solutions. The oxidation stage with pure aneurine solutions probably includes not only the main reaction, aneurine -+ thiochrome, but also a side reaction.If complex extracts introduce a substance that can combine with the aneurine during the oxidation process, the three reactions might be represented thus- Kl A + B K2 A + C K3 A + D + E aneurine -+ thiochrome side reaction Let the initial concentrations of A and D be a and d respectively, and let the amounts of B, C and E formed at time t be p , q and Y respectively, and let 9 + q = y. Then 3 + dq - = - dY = (K, + K2)(a - y - r) . . .. . . (I1 1) dt dt dt = K,(a - y - r)(d - Y ) . . . . ar dt -____ K3 (d - r) andr = d (1 - e ''1 7 Kz ") (compare R. Wegescheider12). Hence - = _____ dr dY Kl + K2638 RIDYARD THEORETICAL AND PRACTICAL CONSIDERATIONS [Vol.75 This value of r is expanded in series, terms involving y to powers greater than 1 being Hence, on integration, discarded (since y is of the order 10-6), and substituted in (I1 1). transferring to the exponential form and expanding, we find that- K3 y*- . . .] 1 . . (I1 3) The stability of this oxidation reaction, in spite of all kinds of foreign substances in the various extracts used, is well expressed by this equation, since p and q will not vary appreciably from their values with pure solutions unless dK,/(K, + K,) is comparable with 1, i.e., either d must be of the order a x lo4, or, if d is of the same order as a, K, must be of the order (K, + K2)1O4. 111. THE Z’SOBUTANOI, EXTRACTION STAGE In isobutanol extraction, interferences are less likely, with the important exception of the effect of high concentrations of dissolved materials in the aqueous solution changing the proportion of aneurine passing to the isobutanol layer.This is most likely to occur when using base-exchange methods with eluates containing high concentrations of a salt, for changes in the concentration of the salt may lead to serious error^.^^^^ IV. FACTORS INVOLVED IN THE FLUORIMETRIC MEASUREMENTS Although it has been upon this stage that attention has been concentrated in published references to recovery, the somewhat complicated process has not been adequately treated. Fluorescence is considered to be due to a molecule of the fluorescing substance absorbing a quantum of light of short wavelength and correspondingly high energy content, and sub- sequently emitting a quantum of longer wavelength and lower energy content ; the difference in energy due to the change in wavelength being dissipated as heat.Three factors can interfere with this simple relationship between absorbed and emitted light- IVa. Short wavelength light may be absorbed by some substance other than that fluorescing. IV b. The molecule of fluorescent substance may lose its acquired energy by collision with other molecules, either of the same substance, or other substances. The latter process is tenned “quenching,” but in the literature concerned with aneurine estimation the term appears to be used loosely to cover all forms of loss of output of fluorescent light.IVc. The fluorescent light may be absorbed before it leaves the solution. IV a. ABSORPTION OF SHORT WAVELENGTH LIGHT IN THE CELL BY SUBSTANCES OTHER THAN In view of the complex nature of the extracts used for aneurine estimation, it is obvious that this effect is likely to be present in the fl~orirneter.~ Now the addition of aneurine to an extract will ultimately involve a change in the total light absorbed in the cell, of which the light absorbed by the thiochrome is only a part. As the light absorbed varies logarith- mically as compared with the concentrations of the absorbing substances and since, further, in dilute solution the fluorescence is proportional to the light absorbed by the thiochrome and is measured at some distance up the cell, it can be seen that the results of changing the concentration of the aneurine are not simple. A detailed examination of the light absorption in the cell, and a determination of the extinction coefficients concerned, was made in order to investigate the matter.In the Spekker fluorimeter a narrow band of light passes vertically through a rectangular cuvette, the fluorescent light emitted being collected by a circular selenium cell from a circular patch of indefinite extent; an area in the centre of the cuvette having the greatest effect (Fig. 1). Let a beam of light incident on the base of the cuvette have a width w and an intensity I,; and the circle from which light falls on the photo-cell a radius Y and centre situated a t a height h above the base of the cuvette. THIOCHROME-Dec., 19501 IN THE DETERMINATIOX OF ANEURINE (VITAMIN B1) 639 From the Beer - Lambert law, the light absorbed in this circular region by the thiochrome in a solution containing other absorbing substances will be- (IV a 1) where K,C, are the absorption coefficient and concentration of thiochrome in the cuvette, and K,C, the absorption coefficient and concentration of another absorbing substance.Other factors, K,C,, etc., could be added if necessary without changing the argument. Front view of cuvecte Side VICW of cuvettc Fig. 1. Optical effects in fluorimeter cuvette This expression, rearranged, integrated in series, and simplified (terms beyond the second . . ( I V a 2) The light absorbed by a corresponding thiochrome solution free from other absorbing agents will be given by (IV a 2) with the omission of KbCb wherever it occurs .. . . ( I V a 3 ) The relative amount of light absorbed by the thiochrome in the two will be given by (IV a 2) divided by (IV a 3), i.e., will be e } .. . . . . .. . . ( I V a 4 ) In a particular solution, the actual aneurine concentration, multiplied by this factor, gives the apparent aneurine concentration that would be obtained in the analysis if light absorption by substances other than thiochrome were the only disturbing condition. The factor (IV a 4) would then be the “recovery factor”; it is denoted by “R” in the remainder of this discussion. being discarded as extremely small), reduces to K,C, m%Ioe - h(KaCa 4- K*cb){ 1 + Q[r(K,C, + K,Cb)]e) . . V Fig. 2. Arrangement of apparatus for measurement of light absorption640 RIDYARD : THEORETICAL AND PRACTICAL CONSIDERATIONS [Vol.75 Experimental deteymination of absorption coeficients-The determination of the absorption coefficients was undertaken with the simple apparatus shown in Fig. 2. The right-hand side block of the Spekker fluorimeter was removed, and the light from the mercury discharge lamp A, after passing a filter B (Chance’s 0x1, UV), the lens C normally mounted on the fluorimeter, and an 8mm. cardboard diaphragm D, was further concentrated by another lens E, on to a series of cardboard diaphragms F, G, H, with holes 16mm. in diameter, extended over a distance of about 1 metre. By this means a reasonably parallel beam was obtained, which passed through either of the cells used without touching the side.The cells K (9.2 and 29.2 cm. long, and 26.0 mm. diameter) were glass tubes with flanged ends and side tubes. The end plates were squares of photographic plate sealed on with Seccotine. The light, after passing through the cell fell on a selenium cell M, immediately in front of which was placed another Chance filter, 0 x 1 , N. A further filter P (Chance OV1, purple 8), was interposed in the beam before the cell. The two glasses together give a narrow trans- mission band in the 365 mp. region, as shown in Fig. 3 (manufacturers’ curves). SI’ECT‘tCIY Fig. 3. Manufacturers’ curves for transmission through Chance Glass Filters OX1 (ultra-violet) and OV1 i:purpIe) The solutions were prepared by precisely the same oxidation method as is used in normal procedure, but larger tubes were used, and all quantities multiplied by ten.The isobutanol extract (250ml.) was syphoned off from the aqueous layer by means of a syphon bent upwards at the tip to prevent contamination. The first attempts to determine the trans- mission of these solutions failed, as cloudiness caused a larger loss of light than the true absorption. The normal, or increased, amounts of ethyl alcohol added delayed the formation of cloud, but did not prevent it. The formation of minute crystals of sodium carbonate was found to be the main cause. Finally, a technique was adopted of syphoning the isobutanol extract into 350-ml. stoppered bottles and storing these in the refrigerator overnight. This threw out of solution a certain amount of water with dissolved sodium hydroxide. The next day the bottles were transferred to the instrument room (in darkness) and allowed to warm up without undue disturbance.Immediately before the determination of its transmission the isobutanol extract was transferred to a beaker containing 10 ml. of absolute alcohol and drawn up into the absorption cell. By thia means clear solutions that gave remarkably reproducible results were obtained. The extracts were later found to have ff uorescences indistinguishable from those of corresponding extracts prepared in the normal manner immediately before making the comparison. Two or more blanks, made by oxidising acid water only, and one or two standards made by oxidising solutions of pure aneurine only, were used in each run, and all determinations, including the extraction of any raw material, were repeated on several different days.The absorption coejicient of solutions derived from pure aneurine-From the results obtained with the isobutanol extracts prepared from standard solutions of pure aneurine,Dec., 19501 IN THE DETERMINATION OF ANEURINE (VITAMIN B ~ ) 641 used in each day's work, the absorption coefficient was calculated using the formula I loge I'" K,= - c, x L where the intensities I, and I were given by the galvanometer deflections obtained when the cell was filled with isobutanol extracts obtained by the oxidation of acid water and an aneurine solution respectively, C, was the concentration of the aneurine solution used in pg. per ml., and l was the length of the cell.The results obtained after the adoption of the technique described are shown in Table I. It was the agreement of the values of this coefficient which was used as the final check on the clarity of the solutions and the correctness of other dis- posit ions. It should be mentioned here that the aneurine used throughout this work, and in terms of which all aneurine concentrations are expressed, was a good commercial crystalline sample kept in a screw-topped bottle in the open laboratory. At a later date and for another purpose this was repeatedly and very carefully compared with the international standard aneurine by the thiochrome method and found to be equal to the international standard x 0.91. K, for the international standard would thus be 0.0087 per pg.per ml. per cm. TABLE I LIGHT ABSORPTION COEFFICIENTS OF SOLUTIONS DERIVED FROM PURE ANEURINE 7 Date 22.11.45 13.12.45 28.12.45 22. 1.46 25. 1.46 Y9 1 ) 29.2 cm. cell Concentration, pg./ml. 0.8 2.0 1.0 2.0 1.0 2.0 0.8 A Mean 0.00814 0.00807 0.00745 0.00837 0.00755 0.00780 0.00794 0.00790 7 Date 30.11.45 2. 2.46 8. 2.46 15. 2.46 15. 3.46 11 Y Y 9.2 cm. cell Concentration, 4.0 2.0 1.2 2.0 2.0 1.6 2.0 A w / m l . Mean 0.00780 0.00793 0.00807 0.00797 0.00797 040790 0-00826 0.00796 Mean of all results 0.00793. Standard deviation 0.00026. Absorption in solutions derived from extracts of bran-Turning to solutions in which light was likely to be absorbed by substances other than thiochrome, bran was taken as a materia1 with a low recovery which varies with the concentration of bran in the extract.The first bran chosen, B.1045, was made up in concentrations of 2, 3, 4, 5, 6, 7, 8 g. in 50 ml., with,five Fig. 4. Bran B. 1045. Observed aneurine concentration642 RIDYARD : THEORETICAL AND PRACTICAL CONSIDERATIONS [Vol. 75 separate extracts a t each concentration. Two of these contained no added aneurine, the others 0.2, 0.4, 0.6 pg. per ml. respectively. On analysis these yielded the values shown in Fig. 4. The highest value at 8 g. bran concentrations with 0.6 pg. per ml. addition was off the normal scale of readings and hence was somewhat uncertain. Blank values were neglected as largely destroyed on oxidati~n.~ Absorption measurements were carried out on isobutanol extracts prepared as described above. These extracts were prepared twice for work on different days and with a different arrangement of the apparatus, two different cells employed and by different disposition of the diaphragms, etc., the light intensities were changed.A provisional value for the aneurine concentration was obtained from the 3 g. bran concentration as the most reliable, the mean value of 0.365 pg. per ml. at no addition divided by recovery interpreted from the graph as 87.5 per cent. giving a level of 0.4,17 pg. per ml. as the true value, or 0-139 pg. per ml. per g. per ml. I t was assumed for this purpose that the “recovery” at this bran concentration was the same for the “no addition” level as for that with additions; the justification for this assumption lying only in the reasonableness of the subsequent results as a whole.It was provisionally assumed also that the concentration of the absorbing substance would be proportional to the bran concentration, which was used for the term Cb in the expression I = 1,e - z(KaCu -t KbCb), where I, is the incident intensity and I the intensity of light leaving the cell, which has the length 1. K, is the absorption coefficient considered previously, C, the concentration of aneurine, and K,Cb the corresponding values relating to the component derived from bran. From this it follows that I loge f - K,C, x I C b x 1 K, = and values of this are given in Table 11. TABLE I1 LIGHT ABSORPTION COEFFICIENTS, BRAN B. 1045 Concentrations Cell length, cm. ----7 7- Bran in Added aneurine in ,---*------, extract aneurine extract Galvanometer AbsorDtion Galvanometer Absomtion A \ Total 29*2* 9.2t -.Tg./50 ml.) (pg./rnl.) (pg. /rnl.), deflection coeffikent deflection coeffiiient Ca Kb Kb 0 2 3 3 4 5 5 6 7 7 8 0 0.0 0.0 0-6 0.0 0.0 0.6 0.0 0.0 0.6 0.0 0 0.278 0.417 1.017 0.556 0.695 1.295 0.834 0.973 1.573 0.813 35.1 38.0 24.7 E;*O 0.0212 20.7 4:*6 0.0205 19-9 16.2 1:5 0.0204 13.6 l.5 0.0200 11.1 0.6 0.0197 9.0 0.4 0.0201 10.3 7.5 0.0222 0.0208 0-0206 0.02 19 0.021 1 0.0210 0.021 1 0.0160 0.0208 Means . . 0.0203 0.0206 * Date of determination, 22.11.45. t Date of determination, 30.11.45. The remarkably good agreement of these figures was encouraging, especially in view of the very small deflections obtained with the long cell. Hence notice was taken of the general decline in the absorption coefficient with increase in concentration of bran, and therefore of aneurine, and particularly the very low level recorded with 7 g.+ 0.6, which actually gave a larger deflection than 7 g. + 0. To test if this were real, two pairs of solutions were prepared, 6 g. per 50 ml. + 0.0 and + 0.6, and 8 g. per 50 ml. + 0.0 and + 0.6. No more of the original bran was available, so some bran from a fresh milling of the same wheat was used, and in view of this the results agreed well with those obtained earlier. As theapparent fall in absorption might be caused by blue fluorescent light passing the OX1 filter in frontDec., 19501 IN THE DETERMINATION OF ANEURINE (VITAMIN B ~ ) 643 of the selenium cell, a second filter of the same type was interposed as an extra precaution, In spite of this the results were more definite than before (Table 111).TABLE I11 EFFECT OF ANEURINE CONCENTRATION r- -----7 LIGHT ABSORPTION COEFFICIENTS, BRAN SIMILAR TO B. 1045 Concentrations 13.12.45 Cell length, 9.2 cm. Galvanometer Absorption Bran, Added aneurine, deflection coefficient 6 0.0 13.7 0.0208 6 0-6 16.1 0.0190 8 0.0 10.3 0.0197 8 0.8 13.4 0.0163 g./50 nil. pg./ml. The probable value for the absorption coefficient Kb at each concentration was obtained by plotting the values obtained at no addition and at 0.6 pg. per ml. addition on a graph, drawing lines passing evenly between the points, and reading from these the values required at each bran concentration. The values for 0.2 and 0.4 pg. per ml. addition were obtained by symmetrical interpolation between the values for 0.0 and 0.6 pg.per ml. obtained from the curves. From these, the values of R given in column 4, Table IV, are derived (formula (IV a 4) above, Y taken as 1-5 cm., see Table XI). A calculation was then made of the apparent aneurine concentration C,R that would be given by each of the bran extracts if this light absorption were the only interference; and these values are given in column 5, TABLE IV EFFECT OF LIGHT ABSORPTION ON APPARENT ANEURINE CONCENTRATION IN BRAN EXTRACTS , AND OF LIGHT ABSORPTION + QUENCHING Concentrations r 3 Bran Aneurine Total extract, added, aneurine, g./50 ml. pg./ml. pg./ml. cb Ca 2.0 0.0 0.278 0.2 0.478 0.4 0.678 0.6 0.878 3.0 0.0 0.417 0.2 0.617 0-4 0.817 0.6 1-017 4.0 0.0 0.556 0.2 0.756 0.4 0-956 0.6 1.156 5.0 0.0 0.695 0-2 0.895 0.4 1.095 0.6 1.295 6.0 0.0 0.834 0.2 1434 0.4 1 *234 0.6 1.434 7-0 0.0 0-973 0.2 1-173 0.4 1.373 0.6 1.573 8.0 0.0 1.112 0.2 1-312 0.4 1.512 0.6 1.712 * h Factor, R 0.9175 0.9186 0.9193 0.9201 0.8804 0.8820 0.8837 0.8857 0.8457 0.8485 0.8 Fi 1 9 0.8545 0.8140 0.8181 0.8221 0.8262 0.7838 0.7894 0.7951 0.8008 0-7564 0- 7 6 2 6 0.7697 0.7768 0.7305 0.7388 0.7466 0.7557 Effect of light absorption only, CaK K./ml* 0.25.5 0.440 0.623 0.8 10 0.364 0*t546 0.721 0.900 0.471 0.642 0.824 0.987 0.566 0.7 3 3 0.900 1.070 0-653 0.816 0.982 1.150 0.736 0.896 1.057 1-222 0.814 0.970 1-130 1.297 .heurine content observed, pg. /ml. 0.25 0.17 0.43 0-60 0.80 0.38 0.35 0.53 0-69 0.90 0.48 0.45 0.63 0.77 0.95 0.56 0.54 0-72 0.85 1.02 0.65 0.64 0.78 0.91 1-06 0.69 0-69 0.79 0.93 1.06 0.75 0.73 0-91 1.01 1-18? Effect of light absorption + quenching Y (IV b 4) (IV b 6) Kc = 0.11 Ka = 0.0098 0.250 0.431 0.611 0.794 0.363 0-530 0.700 0.871 0-453 0-617 0.783 0.949 0.254 0.438 0.619 0.804 0.361 0.538 0.710 0.884 0.463 0.627 0.790 0.952 0.539 0.548 0-700 0-703 0.857 0.854 1.019 1.005 0.616 0.770 0.926 1.085 0.688 0-837 0.988 1.142 0.754 0.898 1.046 1.200 0-618 0.762 0-904 1.043 0.676 0.807 0.934 1.057 0.738 0.866 0.990 1.114644 RIDYARD : THEORETICAL AND PRACTICAL CONSIDERATIONS [Vol.75 Table IV. For comparison the values actually observed are given in colunin 6. Each observed aneurine concentration is the mean of three or four determinations on one extract. The duplicate values given are such means from two separate extracts.It will be seen from these figures that the observed values are in almost every result lower than those calculated. Hence, as would be expected, there is another form of inter- ference, possibly quenching. IV b. THE EFFECT OF QUENCHING IN ADDITION TO LIGHT ABSORPTION- It was assumed in the first instance that one molecule of thiochrome, activated by absorption of one quantum of ultra-violet light, is; de-activated by collision with one molecule of some substance derived from the bran whose concentration would be proportional to that of the bran itself. A stationary state will be reached when the rate of formation of activated thiochrome molecules will be equal to their rate of destruction. Now let [T] be the actual thiochrome Fig. 5 . Bran B.1045.Effect ot light absorption and quenching proportional t o bran C R concentration. Curves of the expression ---.-?--. Observed values in circles. KaC, -f 1 concentration, [TIB the apparent thiochrome concentration in presence of substances derived from bran, [T*l0 , [T*IB the stationary state concentration of activated thiochrome molecules in absence and presence of bran respectively; [labs]o and [I,ba]B the corresponding rates of absorption of light by the thiochrome. In addition, Ka KP Then T + hv --+ T*, T* -+ T + Izv'. a molecule of substance derived from bran. .. .. .. .. . . (IV b 1) .. .. .. .. . . (IV b 2 ) Since it has been assumed that [B] is proportional to C b , this may be transformed to give .. .. .. . . CaR K,Cb + 1 C, observed = . * (IV 64) and . .(IV b 5 ) C,R - C, observed K a = % = .. .. . . .. Kp C, observed x Cb . 'Dec., 19501 IN THE DETERMINATION OF ANEURINE (VITAMIN B ~ ) 646 The value of K8 will best be obtained from a high bran and aneurine concentration, and 5 g. per 50 ml. + 0-6 pg. per ml. was chosen as the most reliable high value. Substitution of the appropriate values for C,R and C, observed gave Ks = 0.0098. This value wasused for the calculation of the apparent aneurine concentrations given in column 7, Table IV, and plotted as the curves in Fig. 5 , on which the observed values are indicated by the circles. 1 4 - E z 4 z z V . ?I2 0 10 W 8 08 W d z 3 06 4 I- $ 0 4 4 0 U ' 02 (z ADDED ANEURINE Fig. 6. Bran B.1045. Curves of the expression CaR - 0.11 @ x Ca.>': 3- 1. Observed values in circles The differences between the observed and calculated results show means of +0.014 (16) and -0.019 (19).Although this could be regarded as satisfactory, the fact that all the results at 7 g. concentration are low is disturbing. This peculiar change in slope of the curves of observed aneurine concentration when passing from 6 to 7 g. of bran per 50ml. was too great to be due to normal experimental error, and could be a real effect--e.g., the concentration of some component could reach a maximum at 7 g. of bran, or all the molecules of thiochrome in the fluorimeter cell could be brought into some kind of association with molecules of a disturbing substance. Only a deviation proportional to the square of the aneurine concentra- tion would account for the changes in slope.Various provisional hypotheses were considered, and as a result of examining these it was found that the expression CUR- Kg $ x CUR (" >' .. . . (IV b 6) gave values in excellent agreement with the experimental results. the concentration 7 g. per 50 ml. + 0.6 pg. per ml., and found to be 0.11. Kt; was determined from Values for the TABLE V LIGHT ABSORPTION COEFFICIENTS, BRAN B. 1388 Concentrations Bran, Added aneurine, /ml. 0.0 0.6 0-0 0.0 0.6 0.0 0.0 0.6 Total aneurine; tLg. /ml. (C,) 0.54 1-14 1.08 1.62 2.22 1.89 2.16 2-76 8.2.46 9.2 cm. cell Absorption coefficient 0.0242 0.0252 0.0233 0-0246 0.0238 0.0242 0.0247 0.0245 Mean 0.0243646 RIDYARD : THEORETICAL AND PRACTICAL CONSIDERATIONS [Vol. 75 above expression are given in column 8, Table IV, and are plotted as curves in Fig.6, the observed values being indicated by circles. A second bran, B.1388, gave absorption coefficients very similar to those obtained from €3.1045 (Table V). In this material the aneurine content was greater than in B. 1045, which indicates the presence of more germ, whereas the drop in absorption coefficient with increase in aneurine concentration was not noticeable, possibly owing to the high initial aneurine level. The losses due to light absorption were again found to be insufficient to account for the low recoveries, and an additional factor of the same form as before was found to do this with great precision. C, apparent = C,R - 0.055 (2 x CaR)e 2 > 1 as is shown in the accompanying graph (Fig. 7). It will be noticed that the bran appears Fig.7. Bran 1388. Curves of expression C,R - 0.056 (5 x CaR)’ c_b 3- 1. 4 ’ 4 to exert its maximum effect on the solution at ,a concentration of 4 g. per 50 nil., while the constant KZ is one half of the value found with bran B.1045. An expression of this form is not easily accounted for in view of the very low aneurine concentrations involved, and further data are obviously required. The formation of “dimers” has been suggested as a cause of quenching at higher concentrations.14 Efect of acid concentration on absorption coefficient-The effect of the concentration of the acid used in extraction of the bran was also examined, with the following results- TABLE VI EFFECT OF ACID CONCENTRATION ON ABSORPTION COEFFICIENT Absorption coefficient uncorrected for aneurine concentration BRAN CONCENTRATION, 7 G .PER 50 ML. Hydrochloric acid Absorption coefficient 0.12 N 0.0 184 0.24 N 0.0220 0.36 N 0.0255Dec., 19501 IN THE DETERMINATION OF ANEURINE (VITAMIN BJ 647 Wheats-The absorption coefficients, I log, f - K,C, x I K, = I of a number of wheats were determined, and found to be surprisingly similar to one another (Table VII). Together with these, the eluates derived from sand adsorptiong were examined, and the results illustrated the remarkable degree of purification effected by this method. TABLE VII LIGHT ABSORPTION OF EXTRACTS DERIVED FROM WHEATS AND ELUATES OF WHEAT (Where the variety is mentioned more than once it was grow%at a different location) ' Corrected for aneurine concentration c, x I Concentrations L r- -7 Lab. Xo.€3.1054 1060 B. 1073 1076 1079 1303 1308 1400 1421 1441 H. 1390 7 7 ,f 39 B. 1320 Added Variety Wheat, aneurine, CW PQ. /ml. Little Joss 5 g./50 ml. Juliana, 9, 9, 7, ,Y 1, 79 7, Despres 80 S Steadfast 79 Conditioned Manitoba 9 9 Manitoba Y, Y, 5, Y Y 77 I f Y, Y, 10 g. /50 ml. 7, 5 g./50 ml. 3, 7, Mixed Grist >9 (70% Manitoba) 10 g. /50 ml. Y, Y, ?? 7, Ehates Manitoba 5 g./50 ml. 0.0 Mixed Grist 5 g./50 ml. Y9 I 9 10 g. /50 ml. Y Y 7, 10 g./50 ml. YY * In the eluate. Total aneurine, ca Pg. /ml. 0.35 0.35 0.35 0.35 0.39 0.3 8 0.44 0.40 0.43 0.42 0-46 0.86 0.88 0.47 1.07 0.375 0-75 0.75 0.375 0*305* 0*565* 0*278* 0*555* Cell length, 1 29.2 5 , Y Y 3, 9, 9, ,, 1) 9) 97 3, 1, Y Y 9-2 29.2 9, Y I 3, Y, 9, Y Y f Y n Absorption coefficient, % 0.00'742 0.00600 0.00548 0-00455 0.00573 0.00615 0.00564 0.00540 0.00534 0.00483 0.00555 15.2.46 0.00556 0.00548 ::::::} 27.2.46 0.00559 0.00558 0*00610 0.00617 } When it is considered that more than half of the total absorption of these eluates was due to the thiochrome, and that it had to be calculated from the separately determined aneurine absorption coefficient and concentrations and deducted before calcul&ion of the absorption coefficient of the eluate, the agreement is quite remarkable and leads to greatly increased confidence in the methods employed.The difference between the eluates from the two wheats is interesting in view of the similarity of the coefficients for the plain extracts. The difference is much too great to be error in galvanometer reading (I$ = 447/35.6 for log.per 50ml. of Manitoba; 43-0/36.6 for log. per 50ml. of mixed grist), is too great to be attributed to an error in the estimation of thiochrome concentration, and is unlikely to be caused by cloudiness in the solutions in view of the excellent agreement of the pairs of results, and of all other results after the first few days' work early in the investigation. The values for the thiochrome absorption coefficient were particularly good (0.00794 and 0.00790) on the two days on which these eluates were examined. The factor R for the Manitoba wheat was calculated and as with bran found to be inadequate to account for the low recovery found. The difference between C,R and observed aneurine concentration was accounted for well by a linear correction plus a square law correc- tion to allow for 15 per cent.of bran. Results are given in Fig. 8. Blank values have again been neglected.648 RIDYARD : THEORETICAL AND PRACTICAL CONSIDERATIONS [Vol. 76 IVC. ABSORPTION OF BLUE LIGHT- The absorption coefficients for blue light of isobutanol extracts derived from Manitoba wheats and from pure aneurine were determined, and are given in Table VIII. Since the fluorescent light passes only 0-7 cm. of the solution when leaving the cell, this factor has been neglected. TABLE VIII ABSORPTION COEFFICIENTS FOR BLUE LIGHT isoButano1 extract Absorption coefficient Aneurine (1.0 pg./ml.) . . .. .. . . 0.00037/pg./ml. Manitoba wheat, 10 g./50 ml. + 0.0 . . . . 0*000278/g./50 ml. Manitoba wheat, 10 g./60 ml.+ 0.4 . . . . O.O00219/g./50 ml. B Fig. 8. Manitoba wheat. Curves of the expression C,R - 0.056 C,K x b ”)*- 0.01 C, x C,R. Observed values in circles ( 4 x 100 Yeast-The ultra-violet absorption of isobutanol extracts prepared from digests of bakers’ yeast was also examined, with the results shown in Table IX. TABLE IX ABSORPTXON COEFFICIENTS OF iSOBUTANOL EXTRACTS DERIVED FROM YEAST, (FOR ULTRA-VIOLET LIGHT) Concentrations WL 7 Added Total Yeast, aneurine, aneurine, Buffer 1 0.0 0.22 Acetate 1 0.5 0.72 1, 2 0.0 0-44 1 0.0 0.22 Phosphate 1 0.5 0.72 I 9 2 0.0 0.44 18 g./50 ml. CLQ. /ml. Pg*/ml. (PH 4.5) ,, Absorption coefficient, per g./50 ml. 0.0 153 0.0145 0.0117 0.0141 0.0133 0.0138Dec., 1950j IX THE DETERMINATION OF ANEUKINE (VITAMIN BJ 649 The J1uorescence of solutions derived from pure arzeurine-The fluorescence of solutions derived from pure aneurine was compared with the light absorbed, which was calculated from the absorption coefficient relating to pure aneurine (0.00793 per pg.per ml.). Solutions of pure aneurine of various concentrations were oxidised and extracted with isobutanol in the usual manner, and the extract placed in the fluorimeter cuvette. The selenium cell receiving the fluorescent light was connected directly to the galvanometer with the same series-shunt arrangement as was used with the absorption measurements to diminish sensitivity, and the deflections given by the various solutions noted. The relative amount of light absorbed was calculated from the formula given above (IV a 3), r being the radius of a circle which includes about 100 per cent.of the light collected (1.5 cm., see Table XI). The factor relating deflection to light absorbed was calculated from the deflection at one concentration. The results are given in Table X, and it will be seen that there is good agreement between the observed and calculated figures a t concentrations much above those used elsewhere in this work. As was assumed before this study was undertaken, quenching due to interaction of activated thiochrome molecules can be neglected where C, is of the order TABLE X LIGHT ABSORBED RELATIVE TO FLUOKESCENCE For solutions derived from pure aneurine Galvanorne ter deflection (cm. ) Coilcentration o f aneurine, Relative light pf5 Iml. a.bsorbed 0.2 0.00158 0.4 0-003 15 0.6 0.00472 0.8 0.00626 1.0 0.00771 2.0 0.01538 4.0 0.0297% 6.0 0.04328 8.0 0.05594 10.0 0*0677!$ 20.0 0.1 1630 14.2.49 m- 7 Calculated Observed 0.379 0.44 0-755 0.88 1-130 1-17 1.502 1.45 1-85 1-83 3.54 3.62 7-13 7.08 10.39 10.18 13.40 13.32 16.30 16-33 (correlation factor a t 1.0 pg.) 22.2.49 1 Calculated Observed 0.346 0.36 0.690 0.69 1.032 1.03 1,372 1-35 1-69 1-73 3.37 3-38 6-52 6.49 9.48 9.38 12-25 12-15 14.84 14.84 25.44 24-95 (correlation factor at 10.0 pg.) Radius of circle from which is derived the light falling on the selenium cell in the SPekker juorimeter-To gain some idea of the radius of the circle from which the fluorescent light measured in the Spekker fluorimeter is derived, tinfoil diaphragms were placed in turn behind the fluorirneter cuvette, and the deflection of the galvanometer noted, the left-hand (com- parison) cell of the instrument being cut off frox'n the light source by a screen.The following results were obtained (Table XI). TABLE XI LIGHT PASSING DIAPHRAGMS PLACED IMMEDIATELY BEHIND THE FLUORIMETER CUVETTE Diaphragm radius, cm. 0.500 0.625 0.750 0.900 0.975 1.500 30 diaphragm Deflection, Percentage passed cm. 0-5 22 0.8 35 1.0 45 1.4 61 1.8 78 ".3 100 2-3 DISCUSSION OF RESULTS I t will be seen from the foregoing considerations that the determination of aneurine involves an exceedingly complex series of chemical reactions and physical processes. The addition of pure aneurine to extracts under examination is a valuable method of investigation if carried out with meticulous care, but neither recovery of added aneurine nor consistency of results is an adequate measure of the agreement of a result with the amount of aneurine650 RIDHARD [Vol.76 actually present in the sample. Nor is agreement with biological estimation a fully satis- factory criterion, since utilisation may very well be affected by other substances present (cf. vitamins A and E153), and important biochemical effects may easily be obscured. The fullest possible theoretical understanding of the determination is a great safeguard. It will be seen from the present study that factors affecting recovery of added aneurine are not, strictly speaking, linearly related to the total concentration of aneurine present in an extract, but that the deviations from linearity may be very small owing to the very low concentrations under consideration. Nevertheless, in bran and probably other materials, part of each deviation appears to be proportional to the square of the aneurine concentration, but this needs confirmation, and if confirmed, satisfactory theoretical explanation. Some support for this is given by the common experience in this laboratory that the quotient- amarent aneurine concentration recovery factor increases with the factor and therefore with concentration of + starting material. Base- exchange methods of purifying extracts do not completely remove the need for this kind of study, for, although as a rule they greatly diminish optical interferences, they also add some new complications. The writer’s thanks are due to Mr. G. G. Grindley for assistance in the absorption measurements and aneurine determinations. 1. 8. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. REFERENCES Wang, Y. L., and Hams, L. G., Biochem. J., 1939, 33, 1366. Booth, R. G.. J . Soc. Chem. Ind., 1940, 59, 181. Dawson, E. R., and Martin, G. W., Ibid., 1942, 61, 14. Vitamin B, Sub-committee of the Medical Research Council and the Lister Institute, Biochem. J., Wokes, F., and Organ, J. G., Ibid., iii. Wood, E. C., Analyst, 1946, 71, 1. Williams, R. R., and Spies, T. D., “Vitamin B1,” Macmillan Co., New York, 1939, p. 218. Ridyard, H. N., Analyst, 1949, 74, 18. -, J . SOC. Chem. Ind., 1946, 65, 93. Bouman, J., 2. V’itaminforsch., 1948, 19, 391. Weil-Malherbe, H., Biochem. J., 1939, 33, 1997: Wegescheider, R., 2. physikal. Chem., 1902, 41, 56. Herd, C. W., Mundy, L. M., and Ridyard, H. N., Analyst, 1943, 68, 174. Weil-Malherbe, H., and Weiss, J., Nature, 1942, 167, 471. Moore, T., Biochem. J . , 1940, 34, 1321. Davies, A. W., and Moore, T., Nature, 1941, 147, 794. 1943, 37, 438. THE RESEARCH ASSOCIATION OF BRITISH FLOUR MILLERS CEREALS RESEARCH STATION, ST. ALBANS July. 1949

ISSN:0003-2654    

DOI:10.1039/AN9507500634

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

Dec., 19501 BAKER, DOBSON AND MARTIN 661 Analysis of Penicillin Mixtures by Paper Chromatography of the Hydroxamic Acid Derivatives BY P. B. BAKER, F. DOBSON AND A. J. P. MARTIN* (Read at the meeting of the Society on Wednesday, AfiriL 6th, 1949) SYNOPSIS-A rapid method that is independent of biological assays and suitable as a routine procedure is described for estimating one or more species of penicillin in a mixture. This method requires less than 8 hours for completion; it is based on the fact that the relatively stable hydroxamic acid derivatives of the various penicillins show different partition coefficients between isopropyl ether - isopropyl alcohol and phthalate buffer, at a given pH, and can therefore be separated by paper chromatography. A novel apparatus for use with volatile solvents and heavily buffered papers, is described.A direct, qualitative result is obtained by developing the chromatograms with dilute ferric chloride solution. A quantitative result is attained by extracting the iron complexes of the various hydroxamic acids with butyl alcohol, measuring the degree of extinction in a colorirneter and reading the penicillin concentration from standard curves. GOODALL and Lev9 have described a method of analysis of penicillin based on partition chromatography on buffered paper strips and using bacteria-sown plates to detect and estimate the various penicillins. When the present work was carried out that was the only published method capable of routine application to give a reasonably complete analysis of a mixture of several different penicillins.The accuracy of the method is essentially that of the biological assay, which is sufficient for most purposes when the content of one species of penicillin is high, but barely adequate when more than one penicillin is present in considerable amount. A further disadvantage of the method is the length of time, three days, that it takes. Although we have had but little experience of the Goodall and Levi method, we believe that considerable experience is required before results comparable with those obtained by its originators can be obtained. For these reasons it seemed worth while to reinvestigate the possibilities of other methods that use paper chromatography. Little progress was made, however, until the colorimetric method of Ford2 was adapted to our requirements.Ford made hydroxamic acids by mixing penicillin and hydroxylamine- S CH, S CH, / \ / -i- NH,OH -+ R.CO.NH.CH/:H C / \/ R.CO.NH. CH-CH I I PCHs CO-Y __- CH.COONa ~ H O H and showed that the Fe"' complexes with the hydroxamic acids could be used for colori- metric estimations. These hydroxamic acids are relatively stable substances that can be separated on paper chromatograms at room temperature without serious loss. After separation the paper is sprayed with ferric chloride to give reddish-brown spots on a buff background at the positions of the individual hydroxamic acids. These spots can be extracted from the paper and the various penicillins estimated colorimetrically in the extracts. It is suitable for crude or pure salts of penicillin, but not for culture fluids without a preliminary extraction.A The colorimetric method is far less sensitive than the biological. * Present address. National Institute for Medical Research, Mill Hill, London.652 BAKER, DOBSOh' AND MARTIN: ANALYSIS OF PENICILLIN MIXTURES BY [VOl. 75 strongly coloured spot is given by 100 pg. of penicillin. A convenient amount for quantitative estimation is 1 mg. With buffer-loaded paper chromatograms it is difficult to keep control of the humidity of the paper during development, and this difficulty is accentuated when a volatile solvent is used. Goodall and Levi, by rigorous attention to the routine of handling the paper, succeeded in reproducing their conditions, even though their paper was loaded with strong phosphate solution and their chamber had walls covered with cloth that was wet with water.It is evident that no eaquilibrium can be attained in such a system, and it is to be expected that the chromatographic behaviour will vary with every change in the previous history of the paper, with the shape and size of the vessels and with the temperature and time of running. An attempt was made to arrange conditions so that the chromatograms could run under equilibrium conditions. The air within the chamber was kept saturated with respect to both phases on the paper. To do this was found to be difficult, and not until both phases were pumped continuously over the walls of the chamber were reproducible results obtained. No method of simply stirring the air or the liquid, or both, was adequate.With the apparatus described below, however, it was found that the paper came to substantial equilibrium in half an hour and that the temperature was comparatively un- important, a variation of as much as 10" C. during the run being without any deleterious effect. It is believed that this type of apparatus will be of general utility when volatile solvents (e.g., ethyl ether) and heavily buffered papers must be used, If the stationary phase is an aqueous solution of a non-volatile substance, it is of course not necessary that it should be circulated round the vessel, but it can be replaced by anj. other solution with the same vapour pressure, and this may be advantageous when expensive or corrosive solutions are concerned. The role of the buffer with which the payer is loaded is worthy of some discussion.Satisfactory chromatograms are obtained with either citrate or phthalate buffers, but not with oxalate or phosphate of the same pH value. With oxalate or phosphate the spots of the hydroxamic acid complex are elongated and the RF values are small. With citrate or phthalate the spots are compact and the RI: values much greater. This may be explained by the assumption that the hydroxamic acids exist as dimers in the mobile phase (a mixture of isopropyl ether and isopropyl alcohol) when phosphate or oxalate buffers, which are insoluble in the mobile phase, are used. When citrate or phthalate buffers are used, citric or phthalic acid dissolves in the mobile phase to an extent that is large compared with the amount of hydroxamic acid present and chelation between the hydroxamic acid and the buffer acid occurs.Since the buffer is in excess, the amount of chelated product is pro- portional to the concentration of hydroxamic acid and not, as with the dimer, to the square of the concentration. As a result the partition coefficient is more in favour of the mobile phase and is comparatively insensitive to the concentration of the hydroxamic acid. Hence the spots on the chromatograms are faster running and more compact with phthdate or citrate than with oxalate or phosphate buffers. METHOD Paper-Whatman No. 4 paper is used. It is dipped in 0.10M potassium hydrogen yhthalate solution and air-dried. The paper comes finally into equilibrium with 0.50 M buffer solution and should be dry enough to gain rather than lose water when it is placed in the chromatogram box.To retain heptyl penicillin hydroxamiq acid on a reasonable length of paper while the other penicillins are adequately developed, the lower third of the paper should be dipped in phthalate buffer of pH 6.2. Whatman No. 1 paper will give satisfactory chromatograms but a much longer time of development is then required. Preparation of hydroxamic acids-In 1 ml. of a mixture of equal volumes of 4 N hydroxyl- amine hydrochloride and 3 N sodium hydroxide are dissolved 10 to 40 mg. of penicillin salt. Ten p1. of this solution are applied as a spot to the chromatogram and air-dried. Ten sqch spots can be accommodated on a sheet 15 cm. wide. A single spot suffices for a qualitative analysis, ten for a quantitative analysis.Mobile phase-The mobile phase is isopropyl ether containing 15 per cent. v/v of isopropyl alcohol. To each 100ml. of the mixture are added 2.4ml. of O.lOIW potassium hydrogen phthalate to give approximate saturation with respect to the stationary phase. The mixtureDec., 19501 PAPER CHROMATOGRAPHY OF HYDROXAMIC ACID DERIVATIVES c G 1 I L A B C D Scale Fig. 1. Diagram of chromatogram 110s for usc ith .i-olatile solvents ,I. Front clevation (trough absent) B. Side elevation (trough present) C . Plan 2). Isometric view of lid. The box is constructed of 3/16-inch Perspex. (a) Flanged, gabled lid, with stoppered opening for filling trough; ( b ) rods for supporting cloth lining; (c) perforated tube delivering pumped liqnid to cloth lining; (d) outlet (with gauze filter) for return of liquid to pump: (e) support for trough; ( 1 ) stainless stcel trough (end view alone shown); (6) wooden base 653654 BAKER, DOBSON AND MARTIN: ANALYSIS OF PENICILLIN MIXTURES BY [VOl.75 should be free from aldehydes or peroxides: distillation of the solvents from saturated bisulphite or 5 N sodium hydroxide ensures this. Apparatus-The apparatus used, Fig. 1, consists of a Perspex box 15 x 25 x 60 cm. with a gabled lid, so that liquid condensing on the top does not drip on to the chromatograms. The walls of the box are lined with cotton cloth, over which both phases are pumped con- tinuously by a diaphragm pump delivering 1500ml. per minute. The liquids are drawn from the bottom of the box, and, in setting up the apparatus, special attention is paid to ensuring that both phases are circulated.The papers, already spotted with hydroxarnic acids and hanging from an cmpty trongh, are placed in the chromatogram box and the pump is started. After thirty minutes the trough is filled with 50 ml. of the mobile phase. Six hours later the paper is removed, air-dried and sprayed with 2 per cent. ferric chloride solution in 0.01 N hydrochloric acid. Inspection of the sheet now permits a qualitative analysis. K group "MiXTURE" ''INTS OF ORIGIN Benzyl Pent ctlli n ~- Benzyl Penicillin Fig. 2. Chromatograni showing various penicillin types, 100 pg. per "spot" Extraction techniqwe-The chromatograms are cut across into strips, each containing the 10 spots from one species of penicillin, which will have an average area of 75 sq.cm. Each strip is macerated with 1 ml. of 20 per cent. ferric chloride in 0.1 N hydrochloric acid, 10 ml. of n-butanol, 2 g. of anhydrous sodium sulphate and 0.6 g. of sodium chloride. The tubes are held in a water-bath at 20" C. until measured. The paper and salt can be packed at the bottom of the tube with a glass rod so that 7 to 8 ml. of the butanol can be poured off for the colorimetric measurement. The measurements were made in a photo-electric colori- meter (Evans Electroselenium Ltd.) with a "tricolour" green filter, No. 404. A more elaborate instrument would no doubt give greater accuracy.Dec., 19503 PAPER CHROMATOGRAPHY OF HYDROXAMIC ACID DERIVATIVES 655 The extinction of the butanol solution prepared in this way increases with temperature and the extraction and measurement should be made at a constant temperature.The colour appears to be stable for many hours and the solution obeys Beer’s law within the error of measurement. Satisfactory extraction of the hydroxamic acid from the paper required a somewhat polar solvent, which unfortunately also dissolved enough ferric phthalate to make a large and variable blank. To displace the hydroxamic acid from an aqueous solution into, say, butanol, without multiple extractions The reagent mixture was arrived at in the following way. 0.5 I .o I .5 2.0 1.5 AMMONIUM PENlClLLtN, mg. PER 10 ml. OF BUTANOL Fig. 3. Standard curves from pure penicillins Curve G, benzyl penicillin; curve F, pent-2-enyl penicillin; curve D, B-amyl penicillin; curve K, n-heptyl penicillin, etc.it was necessary to “salt out” with sodium sulphate or some other salt. The depth of colour of a given butanol solution of hydroxamic acid is dependent upon the amount of ferric chloride in it. Since the amount of ferric chloride sprayed on the paper is liable to variation, a large excess of ferric chloride was added to the extraction mixture. In the presence of saturated sodium sulphate, the ferric chloride is almost completely converted to ferric sulphate, which is not extracted by the butanol; hence no colour is obtained. By saturating the solution with sodium chloride also, the ferric chloride activity is raised and good colour development of the hydroxamic acid is obtained with a small and constant ferric chloride blank.The high concentration of chloride and sulphate in the aqueous phase, by competing with the relatively small amount of phthalate, so reduces the amount of ferric phthalate dissolved in the butanol layer that the blank remains independent of the area of paper taken. By this procedure no dilution or making up to known volume is required and practically the whole of the hydroxamic acid is dissolved in the 10 ml. of butanol, of which a large proportion is available for use in the colorirneter. RESULTS A typical chromatogram is shown in Fig. 2. The RF values3 of the hydroxamic acids Standard curves from pure penicillins are given in Fig. 3, showing the relation between If the gradient for penicillin G Pure penicillin from different penicillins are shown in Table I.log current and amounts of penicillins of different kinds. is taken as 100, the gradients of the other curves are F 95.5, D 86.3, K 75.8.656 BAKER, DOBSOS ,4ND MARTIN [Vol. 75 X has not been available and it is uncertain whether the K penicillin is wholly 92-heptyl. Other K varieties have not been available. TABLE I RF VALUES OF HYDROXAMIC ACIDS FROM T*rlHIOUS PENICILLINS Parent penicillin 13, value $2-Heptyl (K) . . . . . . . . . . 0.57 Pent-2-enyl (1;) . . . . . . . . 0.20 Benzyl (G) . . . . . . . . . . 0.13 w-Amy1 (D) . . . . . . . . 0.27 p-Hydroxy benzpl (X) . . . . . . 0.0i Tables I1 and I11 show analyses of control mixtures of pure penicillins, Table IV analyses of an unknown mixture. T-SBLIS 11 ,!XALYSIS OF A CONTROL MIXTURE CONTAISING 75 PER CENT. OF BENZYL PEKICILLIN AND 25 PER CENT. OF n-mYJ; PENICILLIN 73.2 7 2 4 76.3 76.6 75.2 26.8 37.6 23.i 23.4 24.8 TABLE 111 ANALYSIS OF CONTROL MIXTURE CONTAINING 50 PEK CENT. OF BENZYL, 8 PER CENT. OF PENT-2-ENYL, 22 PER CENT. OF n--kMYL AND 20 PER CENT. OF PE-HEPTYL PENICILLINS Benzy 1, Pent-2-eny1, +z-Xniyl, ?a-Heptyl, 48.8 9.3 14.7 27.2 49.1 7-7 19.6 23.6 46.8 7.7 22.1 23-2 47.9 7.2 23.0 91-7 % % Y O % TABLE IV SAMPLE 316 AMMONIUM SALT Benzyl, % 43.7 44.8 46.0 47.0 41.0 44-0 43.8 44.0 47.6 44.1 Pent-2-eny1, 0 1 / O 14.6 14.0 17.0 16.0 15.5 17.8 16.3 16.4 13.5 l5.8 I<-group, % 17.3 16.3 9.7 12.0 18.8 13.1 14.9 14.3 13.5 13.8 The authors wish to thank Sir Jack Drummond, F.R.S., Director of Research, Boots Pure Drug Co., for his encouragement and for permission to pubIish this work. REFERENCES 1. 3. Ford, J. H., Anal, Chew., 1947, 19, 1004. 3. Goodall, 13. R., and Levi, A. A,, ..Imdyst, 1947, 72, 277. Consden, R., Gordon, A. H., and '1\Iartin, -4. J. P., 13ioclieiJf. J . , 1944, 38, 224. RESEARCH DEPARTMENT BIOCHEMISTRY DIVISION HOOTS PURE DRUG COMPANY LIMITED First submitted, May, 1949 NOTTINGHAM Amended, J u ~ , 1050

ISSN:0003-2654    

DOI:10.1039/AN9507500651

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

Dec., 19503 ALBANS AND BAKER 667 Paper Chromatography in Penicillin Production Control BY J. W. ALBANS AND P. B. BAKER (Read at the meeting of the Society on Wednesday, April 6th, 1949) SYNoPsIs-The application of a method for estimating penicillin species as described by Baker, Dobson and Martin in the previous paper, to culture filtrates and similar process samples, is discussed. The changes in com- position that take place during fermentation and the use of the technique for examining such fermentation variables as the composition of the medium, the rate of aeration, the effect of precursors and of the fermentation period are also discussed. Some confirmation has been obtained of the existence of substances previously indicated, but not yet characterised, by the micro- biological chromatographic technique.The construction of a large vessel for multiple analyses is described. Esamples of the degree of reproducibility of the results are shown. As knowledge of penicillin therapy has increased and as methods of fermentation and processing penicillin have developed, there has been a demand for a rapid and accurate method of estimating the different penicillin species in fermentation liquors and in the fractions obtained during extraction and purification. Of the several methods proposed, the differential assays of Schmidt, Ward and Coghilll and of Higuchi and Peterson2 are of limited application unless a wide range of suitable organisms is available for the tests. The inherent error in biological assays of this kind is then multiplied and the method becomes cumbersome.The Craig3 counter-current distribution technique has been found suitable for a small number of samples, but it is slow and laborious. A paper chromatographic method developed by Goodall and Levi4p5 gives a direct qualitative estimate of the various penicillins in mixtures, but the quantitative treatment proposed is open to criticism. Moreover, the delay in obtaining the results, 72 hours, renders the method of limited use for production control when the results are required with a minimum of delay. The foregoing paper by Baker, Dobson and Martin,6 which describes a method for the separation and estimation of the penicillin hydroxamic acids, refers mainly to work on the crystalline penicillins that were obtained from fermentation liquors by various methods of extraction and purification.The method is claimed to be suitable for the differential estima- tion of penicillin species in mixtures; and with those penicillins that have been isolated and for which a standard curve can be constructed it is claimed that the results for specific penicillins are reliable. The present paper deals with applications of this basic technique to the estimation of the penicillins in culture fluids and similar process samples obtained during the extraction of penicillin. Any method of analysis that is to be useful as a control test for a production plant must fulfil certain conditions. When the processing of a large quantity of material is dependent on the results of a control test, the test should be speedy.The method must be one that can easily be carried out by unqualified assistants and should require the minimum of special apparatus. The technique to be described has been employed successfully by two assistants for more than a year. Throughout this period, supervision has been reduced to a minimum. The results obtained have been satisfactory and periodical checks on known mixtures of penicillins have shown that accuracy has been maintained. The only special apparatus used is the tank made of Yerspex and this will last indefinitely. The diaphragm pump, mentioned in the previous paper,6 has been replaced by a cheap centrifugal pump readily available from stock. The other apparatus consists of items normally used in a biochemical laboratory. On only one point does the method fail to fulfil the conditions mentioned above, viz., that the minimum period for dealing with a sample of liquor is about 12 hours and for a solid, 8 hours, but this rather long delay has not caused any trouble.Moreover, it often happens that the constitution of a sample is known before the normal bio-assay is available. Further, the method of calculating the results is very simple.658 ALBANS AND BAKER : PAFER CHROMATOGRAPHY IN [Vol. 75 THE ESTIMATION OF PENICILLIN SPECIES IN FERMENTATION LIQUORS AND PROCESS SAMPLES- Owing to the low concentration of penicillin compared with other solids, the normal method of separating and estimating the hydroxamic acids of the penicillins6 cannot be applied directly to culture filtrates and similar process samples.Thus, for a culture fluid of 600 units per ml., in order to achieve the desired loading of 1 mg. of each penicillin species, at least 300mg. of inactive material would be applied at each spot. To overcome this difficulty the penicillins are extracted into ether at about pH 2 and then converted into the hydroxamic acids. Removal of ether and subsequent concentration of the aqueous residue are carried out under such conditions that inactivation is reduced to a minimum. METHOD Extract 200 ml. of culture filtrate (or the equivalent of any other sample) , previously clarified with the aid of kieselguhr, with two 100-ml. portions of ether at pH 1-5 to 2.5, adjusting with 20 per cent. phosphoric acid. Transfer the ether extract to a 6-in. crystallising dish containing 0.5 ml.of 4 M hydroxylamine hydro- chloride and 0-7 ml. of 3 M sodium hydroxide. Mix well and see that the aqueous layer does not become too acid; adjust it to pH 6-2 with N sodium hydroxide, if necessary. Remove the ether by means of a gentle stream of air over the surface. Concentrate the aqueous residue (4 to 6 ml.) to about 0.5 to 1.0 ml. by means of a blast of warm air from a hair-drier fixed about 12 in. above the dish. Remove any insoluble matter by means of a filter-stick and apply the clear filtrate to buffered paper as previously described.6 (A check on the concentration achieved can be obtained by spotting samples of the concentrate and a control of known strength on to a filter-paper soaked in 2 per cent. ferric chloride.) In the earlier stages of the work the results were rather erratic, owing in the main to insufficient control of the extraction and conversion stages.Attempts to avoid emulsifica- tion led to incomplete extraction, e.g., only 25 per cent. of added penicillin could be extracted from solution in a corn-steep-liquor medium, whereas with an aqueous solution, where no emulsification occurred, the recovery was more than 90 per cent. A centrifuge had been used quite successfully to break the emulsions, but it was thought that an alternative method that involved no fire risk was desirable. The use of surface-active compounds showed some promise, but to work out the exact conditions would probably have required a consider- able amount of study and this idea has not yet been pursued further.If the fermentation liquor, free from mycelium, is heated to 60' C. and rapidly cooled before clarification, emulsification is less troublesome. The precipitation of proteins by tannic acid is unsatis- factory as the blue stain produced when the papers are sprayed with ferric chloride masks the bands due to penicillin. The acid extraction should, of course, be carried out rapidly, and it is of more importance to separate the ether extract and add it to the hydroxylamine reagent quickly than to attempt to make fine adjustments in the degree of acidity. In experiments with solutions of penicillin salts, it has been shown that, under the conditions recommended, there is no differential extraction of any of the penicillin species. It is vital that traces of emulsion or of aqueous layer be not added to the dish or the mixture will be too acid for conversion to hydroxamic acids.For the concentration of the hydroxamic acid solutions, lyophilic drying has been used with success, but the stability of these compounds has made it possible to use the quicker method described earlier as a routine. Break the emulsion by centrifuging. LARGE APPARATUS FOR MULTIPLE ANALYSES- As the development of this method of analysis progressed it became necessary to construct a larger vessel. In view of the critical humidity conditions necessary for good separation, a prototype (10 x 16 x 24 in. high) , consisting essentially of three of the smaller vessels, was built. However, it was eventually found that spa-rgers and curtains round the outer walls of the vessel were sufficient to achieve the desired degree of humidification in this size of vessel.With the arrangement indicated in Fig. 1, movement of the zones on paper 3b was rather slower than on the other papers and, in fact, fairly wide variations in the distances travelled were shown on the various papers (see Table I). The spargers and curtains are shown in Fig. 1.Dec., 19503 PEXICILLIN PRODUCTION CONTROL TABLE I 659 DISTANCES OF TRAVEL, MM., OF ZONES IN 64 HOURS Papers 7 A -I Iu 1b 2a 2b 3a 3b G 66 55 69 64 74 49 F 7s 79 97 93 100 76 D 102 107 128 I24 132 99 K 840 240 300 289 285 264 This difference in the distance of travel is attributed to striation in the spargers. In order to obtain similar separation on the different papers in the tank, it is advisable to have b Box 2 I , 3 t Fig.1. Large apparatus for multiple analyses the spray evenly distributed round the walls of the tank. This refinement is not absolutely essential, however, for, although the absolute distances of travel vary, the relative movements are constant, provided that the conditions specified6 are satisfied. This constancy of relative movement is shown by Tables I1 and 111. TABLE 11 RATIOS OF DISTANCE OF TRAVEL G F D K Papers l a lb 2n 9h 3 n 3b 1.00 1.00 1.00 1-00 1-00 1.00 1-39 1-44 1.41 1 4.4 1.35 . 1.53 1.83 1.94 1-85 1 *92 1.78 2-05 4.28 1.36 4-34 4.46 3.85 5-17 A 7 7 TABLE 111 MEAN VALUES OF RATIOS UNDER VARYING HUMIDITY CONDITIONS G F D K Spargers All in B out B, C out B, C, D out 7 A \ 1.00 1-00 1.00 1 -00 1-43 1.40 1.46 1-42 1.89 1-91 1.96 1.91 4.41 4-90 5-30 4.56 Mean 1-00 1.43 1.89 4.41660 ALBANS AND BAKER : PAPER CHROMATOGRAPHY IN Wol.75 This constancy of relative movement for penicillins G, F and D is of very great importance in determining the identity of the bands, as will be seen later. The variation shown by penicillin K is due to the fact that this band is in the pH 6.2 region of the paper, and the relative movements of bands in the two regions are not comparable. The degree of reproducibility attained with this vessel is indicated by the two examples given in Table IV. TABLE IV Mean REPLICATE ANALYSES OF A MIXTURE Operator A G F D K f- 47.5 44.1 46.0 44.0 47.3 45.4 45.7 per cent. w/w 13-5 25.5 15.8 26.6 14.2 28.0 14.5 27-8 11.’7 27.8 14.13 27.4 14.1 2’7.16; --A G Operator B F I) per cent.w/w Mean 48.7 49.4 45.0 43-2 47.5 45.4 46.5 14.6 15.7 15.2 13.1 15.8; 15.1 14.9 23.6 22.7 25.3 23.9 24.8 26.2 24-4 7 13.5 13.5 11.8 14.0 13.2 12.4 13.07 K 7 13.1 12.2 14.5 19.8 11.9 13.4 14-1 For the vessel the following details are regarded a s essential- (i) The return line to the circulating pump should not be of greater diameter than Q in.; otherwise, with a centrifugal pump, there is a tendency for the lighter ether layer to be circulated in preference to the salt solution, and this will lead to streaky and indistinct zones. (ii) To prevent warping of the longer sides of the vessel it is advisable to fix metal strips along the top. In addition, to prevent warping and leakage of vapour, flanges should be screwed to the lid. (iii) The spraying system should form a continuous ring round the vessel, with the pump feeding at two diametrically opposite points.In this way the sprays deliver at ail even pressure and the two phases remain emulsified. (iv) If the pump or any part of the apparatus is made of brass or other material attacked by the phthalate solution, the latter can be replaced by a 12 per cent. w/v solution of anhydrous sodium sulphate, which has approximately the same vapour pressure as 0.6 M phthalate. (v) With the increased surface area of the lid condensation occasionally occurs. If the droplets fall on the paper strips the strips become waterlogged and separation does not take place. A piece of filter-paper fastened to the lid to absorb the moisture, or a gabled lid to induce the drops to run to the side walls, overcomes this difficulty.APPLICATIONS TO FERMENTATION STUDIES- It is possible to investigate how the production of the different penicillins is affected by such factors as precursors, composition of the medium, aeration and culture variation. For example, a high rate of aeration favours the production of penicillin K, as is shown in Table V, where the proportion of penicillin K increases as the volume of medium in “shaken flask” cultures decreases. The simplicity of this technique has led t o its use in problems of fermentation.Dec., 19501 PENICILLIN PRODUCTION CONTROL TABLE V 661 Volume . . .. 100ml. 80 ml. 60 ml. 40 ml. K, per cent. . . . . 14.4 49.3 63.3 90.2 G, per cent. . . .. 85.6 50.7 36.7 9.8 The changing picture during fermentation has been followed on a small scale by the use of cultures that produce a mixture of penicillins predominantly G, F, D and K.Under the conditions used, the G content rose rapidly to a maximum and then decreased. This latter fall in G content was associated with an increasing production of penicillin K and an increasing biological titre. In fermentations of this kind, in which a mixture of penicillins is produced, the biological assay is of little value and can, in fact, be misleading. Detection of “newJJ penicillins-During some work recently carried out on fermentation liquors, samples taken during the early stages of fermentation have shown a number of bands riot associated with any of the major penicillins, X, G, F, D, K, but corresponding in position on the chromatograin to the minor constituents detected by the Goodall and Levi technique.6 The entities responsible for these bands have not been isolated nor has their biological activity been determined; but, since they are decomposed by penicillinase one is led to suppose that they are similar in structure to the known penicillins. TABLE VI POSITIONS OF UNIDENTIFIED ZONES x ? G F D (4 0.08 1 -00 1-50 2.13 - ( b ) - 0.55 1 -00 1.49 2.05 One species has been detected between the bands due to X and G, but, as this was in a sample from a single shaken flask culture, it has not been possible to follow its production or ultimate fate.A second “unknown” species, lying between D and K on the chromatogram, has been produced in the early stages of some stirred fermentations.The proportion rises to a maximum and eventually the band disappears as the-fermentation is prolonged. Tables VI and VII indicate the positions of these two fractions, and the figures show the relative distances travelled by the various penicillins. In Table VI, the values in column (a) are the means of 64 determinations. In column (b) the existence of a compound producing a band between those due to penicillins X and G is indicated. In Table VII the presence of a penicillin that produces bands between those due to penicillins D and K is shown. As the fermentation proceeds this band disappears. TABLE VII 60 hours 72 hours 84 hours G 1 4 0 1.00 1.00 F 1.47 1.49 1.46 L) 1.92 1.99 1.97 ? 2.25 2.24 - K 5.50 5.61 5.94 Thirdly, in many instances the K zone has been separated into its three components. In this connection a recent paper7 on the estimation of the penicillins as their “R-group acids,” indicates the heterogeneity of penicillin K, but the side chains of the individual compounds have not been established.A fourth mauve band appearing at the lower end of the pH 4 paper has been identified as due to phenylacetic acid; it also disappears as fermentation is prolonged. These points indicate the complexity of the problems confronting those investigating fermentation. At this stage we offer no theories about the constitution of compounds that appear as minor constituents nor on their role or fate during fermentation, but it seems likely that further investigation of them would give useful information about the mode of production of the various penicillins.NICHOLAS AND POLLAK: THE ISOLATION OF THE LINES [Vol. 76 662 Our thanks are due to numerous colleagues for providing samples for this investigation and to Boots Pure Drug Company Limited for permission to publish this paper. RBFErm %CES 1 . 2 . 3. 4. 5 . 6. 7. Schmidt, \V. H., \Yard, C . J,:., and Coghill, 1i P., ./. JJucf., 1945, 49, 1 1 1 Higuchi, I<., and Petcrson, I!‘. H., A m l . Chei,i., 1947, 19, 68. Craig, L. C., J . Biol. Chem., 1944, 155, 519. Goodall, R. K., and Lei4, A. A., Xalzur, 1946, 158, 678. -,- , Analyst, 1947, 72, 277. Baker, P. B., Dobson, I?., and Martin, A. J. P., Ibid., 1950, 75, 6.>1. Higuchi, K., and Peterson, My. €I., ,3nal. Chuui., 1949, 21, 659. BOOTS PURE DRUG Com.my LI~IITEI) TECHNICAL DEVELOPMEXT DEPARTMEST .IND BIOCHEMISTRY DIVISIOX 017 THE KESEARCII I h i ) i~ri-vi<\ I ISLAM, STREET 1;ii y t bubmitted, May, 1949 h-OTTIKGHASI -hiended, June, 1960

ISSN:0003-2654    

DOI:10.1039/AN9507500657

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

662 NICHOLAS AND POLLAK: THE ISOLATION OF THE LINES [Vol. 76 The Isolation of the Lines of the Mercury Arc by Filters With Especial Reference to Photo-electric Absorptiometrj- BY J. W. NICHOLAS . ~ N D F. I;. POLLAK SYNoPsIs-The light filters normally used or recommended for use in England with the mercury vapour lamp photo-electric absorptiometer do not isolate monochromatic light, with the exception of the 577/9 nip. lines. An improved series is described, which give monochromatic light in the more frequently used regions of thc mercury spectrum, and an approxima tion to it in some regions which ha1.e not hitherto been used, i.e., infra-red, red, orange and blue-green. THE use of filter photometers in place of spectrophotometers for absorptiometric determina- tions has certain advantages such as lower cost, greater speed and greater suitability for routine use by relatively unskilled operators, but normally there are several disadvantages.Filter photometers are not suitable for measurements of more than one unknown in the same coloured solution and no specific extinctions can. be determined and published for use by other workers on other instruments. Sensitivity and accuracy are also reduced by the use of mixed light, and apparent deviations from Beer’s law occur when dealing with substances having sharp absorption bands (cf. Miillerl) . States and Anderson2 find that with a substance obeying Beer’s law a curved calibration graph is to be expected when there is stray light, with the exception of the special case where the solution has equal densities for the stray light and the desired wavelength.Lothian3 has shown that when a finite waveband is used instead of monochromatic light the effective wavelength varies with the absorption curve of the solution being examined, and that the measured density bears no easily predictable relationship to the true density. Some of these difficulties can be obviated by the use of a mercury vapour lamp as light source, and this was available in the Zeiss Pulfrich photometer (Heilmeyefl) with filters said to give isolation of the 436, 546 and 577 mp. 1ines.l Vaughan5 used the mercurv arc in conjunction with the Hilger Spekker photo-electric absorptiometer. Mullerl points out that with a mercury arc and a highly selective filter, a filter photometer need not give results any less accurate than a spectrophotometer.In applying a photo-electric absorptiorneter to spectrophotometric procedures we found certain discrepancies which led us to believe that, with the exception of Ilford No. 606 for the 577/579 mp. lines, truly monochromatic light was not obtained with the filters normally advised or supplied for the purpose. It has been suggested (e.g., Stross6) that interference filters might have an application in absorptiometry because of their greater transmission. It should be noted, however, that although their transmission maxima are sharp, the positions of these maxima are subjectDec., 19501 OF THE MERCURY ARC BY FILTERS 663 to a tolerance of A10 mp., both between different filters and between different places on the same filter (Miiller7).This tolerance makes it impossible for them to be used for spectro- photometric procedures with a continuous source, and may well lead to considerable difficulties with a line source. At their present stage of development it would appear that they present no advantages for the 365, 436, 546 and 577 mp. lines. S t a a t ~ ~ , ~ has described the design of filters for the isolation of all the main lines of the mercury arc. She employed an exclusively mathematical treatment based on published data for light source, filters and receivers. Her figures take no account of background spectrum immediately adjacent to the required line, and the region 532 to 5614mp. is considered as identical with the 546mp. line, and 561-5 to 595 mp.with the 577/9 mp. lines. She also fails to mention the 3906.4 A. line, which would be passed by both her 365 and 405mp. filters. Unfortunately, Corning and Jena glasses only are used, and neither of these is available to the English analyst. In the course of his development of absorptiometric methods for metallurgical analysis, Vaughan5 has also examined a small number of filters spectrographically with the mercury arc. He gives a plate in which only Ilford 606 and Wratten 74 appear to transmit an adequate quantity of monochromatic light, but in the text gives Wratten 62 and Ilford 604 (omitted from the plate) as suitable alternatives to Wratten 74, and also suggests the use of Ilford 601 in the violet region, although his own plate shows this to be far from mono- chromatic.He mentions a suggested use of Wratten 36 + Wratten 2A for the isolation of the 436 mp. line, but does not record any experiments with this combination. Unfor- tunately his plate does not include the red end of the spectrum, although he states that red and orange filters can also be used. He gives no details of spectrographic or photographic technique, but it appears from the absence of background and the suppression of the line at 4960-3 A. that his spectra are under exposed and that his filters have therefore been tested too leniently. PRESENT PRACTICE FOR THE ISOLATION OF LINES For convenience, the groups of closely adjacent lines that would not normally be resolved by a monochromator are considered as single lines and given a single nominal wavelength.Thus we shall refer to the 3650, 3654, 3662 and 3663 A. group as the 366 rnp. line. 365 mp.- Messrs. Hilger and Watts Ltd.l0J1J2 and Haywood and Wood13 recommend Wood's glass (supplied by Hilger as H556) without any supplementary filter. This glass is apparently identical with Chance 0 x 1 , as is Ilford 828; the material appears to vary considerably from melt to melt. Our spectrogram, Fig. l b , shows that it is not monochromatic in the ultra- violet when used alone, and in addition it transmits red and infra-red. 405 mp.- Hilger and also Haywood and Wood advise Chance 8 (OV1) +- Wratten 2 for isolating the 406 mp. line. Haywood and Wood describe OV1 as a heat absorbing filter, which it certainly is not, having a transmission a t 750 mp. of 46 per cent.14 Gentry and Sherrington,l51ls by using this combination, obtained curved calibration graphs for tungsten and therefore substituted Ilford 601 for Wratten 2.Although this combination is a great improvement, we found appreciable transmission of the 365, 391 and 436mp. lines. 436 mp.- For 436 mp., Hilger and also Haywood and Wood recommend Chance 6 (OB2) and IYratten 50. Hadleyl' used Calorex (ON3) and Wratten 50, but found fluctuations due to heat effects on the gelatin filter. For this reason he preferred Calorex and Chance OBI." As an alternative he suggests modifying the Spekker so that the right-hand filter is on the right-hand side of the drum. This difficulty has been overcome in the new model H760 of the Spekker (cf. Isbellla). Rogers,lg Harrison,20 de Lippa21 and Lennardn used Ilford 601, as did Davis,Z3 who obtained a curved calibration graph with the silicomolybdate colour (cf.Fig. 11). 546 mp.- Calorex and Ilford 605, as advised by Hilger and by Haywood and Wood, were used by Kogers,lg British Iron and Steel Research AssociationM and Parker.% Calorex and Ilford 604, recommended by Vaughan, have been used by Edwards and Gailer,26 Edwards and Robinson2? * Manufacture now discontinued-Chance Bros. (personal communication)664 NICHOLAS AND POLLAK: THE ISOLATION OF THE LINES KEY TO SPECTOGRAMS Fig. 1. 366mp. (a) Unfiltered light source (control) , . ,. .. . . .. .. (b) Wood's glass, 2 mm. (Hilger, H556) . . .. .. .. .. .. combination) . . . . . . . . .. . . .. .. . . (c) Chance OB2, 1 mm. + Wratten 17 4- Chance 0x1, 3 mm.(recommended Fig. 2. 405mp. (a) Control . . ,. .. .. .. . . . . . . .. . . (b) Chance OV1, 2mm. + Wratten 2 . . . . .. .. .. . . (c) Chance OV1, 2 mm. + Ilford 601 . . .. .. .. .. . . (d) Chance OV1, 4 mm. + Wratten 86C + Chance OB2, 1 mm. (the recom- mended combination (see Table 11) gives an identical spectrogram, but is preferred because of lower red and infra-red transmission) . . Pig. 3. 436mp. (a) Control . . . . .. .. . . . . . . .. . . (b) Chance OB2, 2mm. + Wratten 50 . . . . . . . . . . (c) Chance ON13, 2mm. + Ilford 601 . . .. . . .. .. (recommended combination) . . .. . . . . .. . . (d) Chance OB2, 2 mm. + Wratten 36 -t Wratten 2A (two thicknesses) (e) Chance OB2, 2mm. + Wratten 2A (three thicknesses) + Wratten Fig. 4. 492mp. (a) Control .. . . . . . . . . . . . . . . . . (6) Ilford 603 + 804 . . .. .. .. .. . . . . . . (c) Ilford 603 + Wratten 5 + Ilford 302 + Ilford 804 (recommended com- bination) . . .. . . . . . . . . . . . . .. . . .. .. .. .. .. 36 .. .. .. Fig. 5. 546mp. (a) Control . . a . . . .. . . .. . . . . . . . . (b) Chance ON13, 2mm. + Ilford 605 . . .. . . .. . . .. (c) Chance ON13 + Ilford 604 . . .. .. .. .. .. . . (d) Wratten 16 + 74 + Ilford 504 (recommended combination) . . . . [Vol. 75 Relative" exposure, sec. 1 3 24 1 11 48 252 1 19 8 21 28 16 216 684 8 256 1420 260 * Exposures are calculated for a slit width of 0.04mm., although for the longer exposures a slit of 0.08mm. and half the exposure time was actually used. It should be noted that with intense lines necessitating a control exposure of 1 sec.there was some suppression of the background and the 4960-3 A. line. Spectrograms have also been taken with ten times the exposure and they do not show any foreign line.Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Figs. 1-5. Spectrograms showing the isolating properties of various filter combinations (see Table 11, p. 660, and Key on p. 664)Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10 Figs. 6-10. Spectrograms showing the isolating properties of various filter combinations (see Table 11, p. 669, and Key on p. 665)Dec., 19503 OF THE MERCURY ARC BY FILTERS 666 KEY TO SPECTOGRAMS-CO~~~~~C~~. Fig. 8. 577 mp. (a) Control . . . . .. . . . . . . . . . . (b) Chance ON13, 2mm. + Ilford 606 . . . . . . . . (c) Ilford 812 -+ 804 (recommended combination) .. . . Fig. 7. 607mp. (a) Control . . . . .. .. . . ,. . . . . (b) Chance 0x13, 2 mm. + Ilford 607 . . .. . . . . (c) Ilford 202 + Wratten 25 + 804 . . . . . . . . (a) Wratten 26 +- Ilford 804 (recommended combination) . . (e) Wratten 25 $. Ilford 801 (“orange-red” combination) . . Fig. 8. 691 mp. (u) Control . . .. .. .. .. .. .. .. . . . . . . . . . . . . . . . . .. . . .. .. .. .. .. . . .. .. ( b ) Chance ON13, 2mm. f Ilford 206 + Chance ON13, 2.6mm. (recom- mended combination) . . . . .. .. .. .. ,. .. Fig. 9. 735 + mp. .. .. .. .. . . .. . . .. (a) Control . . .. (b) Wratten 88A (recommended filter) . . .. . . . . . . .. Fig. 10. Uniformity of filters. Spectrograms of a number of finished filters intended to isolate the 365 mp. Control exposure, 1 sec.Relative exposure for filters, 25 to 28sec. line. Relative* exposure, sec. 8 182 270 30 288 1494 1614 66 30 514 30 48 * Exposures are calculated for a slit width of 0.04 mm., although for the longer exposures a slit of 0.08mm. and half the exposure time was actually used. I t should be noted that with intense lines necessitating a control exposure of 1 sec. there was some suppression of the background and the 4960.3 -4. line. Spectrograms have also been taken with ten times the exposure and they do not show any foreign line.666 and Lennard.22 Harrison28 abandoned 604 in favour of 605 because the latter was not sensitive to a yellow interfering colour. This suggests that some light of shorter wavelength than desired was passed by 604 (cj. Fig. 5). The possibility of using Wratten 74 has been mentioned by Vaughan and by Haywood and Wood.Fig. 5b shows the considerable yellow transmission of Ilford 605, and Fig. 5c shows the high transmission of blue-green background by Ilford 604. NICHOLAS AND POLLAK: THE ISOLATION OF THE LINES [Vol. 75 SILICON, ?:, Fig. 11. Calibration curves for the estimation of silicon in aluminium as silicomolybdate. Curve A, with traditional combination of Chance 6 and Wratten 50; curve R, with proposed 436 mp. combination. Final concentrations were : aluminium (containing 0 to 14-4 per cent. of silicon added as sodium silicate), 0.32 g. per litre; nitric acid, 0.128 X; ammonium molybdate, 10 g, per litre 557 mp.- For this h e , workers have been almost unanimous in using Ilford 606, usually with Calorex as recommended by Hilger, by Vaughan and by Haywood and Wood, e.g., Harrison,20 British Aluminium C O . , ~ ~ Bairstow, Francis and Wyatt." Most workers report straight line graphs. It is difficult to see the advantage of using such a combination (see Fig. 74. Payneal has used Ilford 607 with Calorex. GENERAL CONSIDER4TIONS It has been suggested that glass filters are more satisfactory than gelatin. The usual cause of failure of filters appears to be heat by conduction and convection, and with careful use the effect of radiation is small. Filters are not exposed to heat in the H760 Spekker absorptiometer, and we assume that any user of the H560 instrument will have modified it as recommended by Hadley.1' Some gelatin filters are markedly fluorescent, and if placed immediately in front of the photo-cell large errors occur.When placed in front of the solution under test the error will be very small, but we have nevertheless tried to eliminate the effects of fluorescence (8.g. , by absorbing the exciting or fluorescent radiation, or both, by non-fluorescent filters) and the filters recommended will give consistent results irrespective of their distance from the photo-cell. Fluorescent elements are marked ( f ) in Table 11. It has been usual to place a heat-absorbing filter between the lamp house and the gelatin filter to protect the latter from radiant heat. Since a filter can onl57 be heated by radiation being absorbed, the dyed gelatin films will scarcely be heated at all by the infra-red, although they will be heated by the strong visible lines.We have therefore allowed other considerations to determine the position of the heat absorber, and our filter combinations are intended for use in one direction only. The components in Table I1 are listed in the order in which light passes through them.Dec., 19501 OF THE MERCURY ARC BY FILTERS 667 EXPERIMENTAL METHODS Probable filter combinations were selected from the publications of Messrs. Kodak,32 IlfordS and Chancel4 to ensure that filters commercially available in England were used, The following filters were examined in various combinations- Wratten: 2, 2A, 4, 5, 8, 9, 16, 17, 21, 32, 25, 26, 29, 32A, 35, 36, 44, 44A, 45, 50, 62, 70. Ilford: 201, 202, 206, 302, 601, 603, 602, 606, 606, 607, 608, 801, 802, 803, 804, 805, 806, Chance: OB2, OB10, ON12, ON13, OX1 and OV1.Densities of individual filters were measured with a double-cell photo-electric absorptio- meter that will be described elsewhere.34 The filter combinations used on this instrument were improved as we went along, and for the final measurements were those we recommend later. The exposure required for any filter combination was determined from the sum of the densities of its elements at the required line, the aim bGing to make the photographic density the same as in a spectrum of the unfiltered light source in which the region of the required line showed considerable background. Spectrograms were taken of the mercury arc (Siemens lamp MB/D, 125 watt, as used in the Spekker) in the Hilger medium quartz spectrograph E498.The light source at a distance of 38 cm. from the slit was focussed on the collimating lens by the Hilger quartz condensing lens F1026. Slit widths of 0.04 and 0-08 mm. were used. A two-step sputtered metal filter (Hilger F1219), having densities at 4Fio mp. of 0.0 and 0.98, was placed over the slit. Spectrograms were taken on Kodak 111-L plates, which have a fairly uniform sensitisation extending as far as 900 mp. Development was in Kodak D 19b developer, undiluted. Messrs. Kodak’s statement32 that “all dyed gelatine film filters” transmit freely in the infra-red” indicated that this region required full investigation. In view of the difference between the spectral sensitivity of the plate and photo-cell, and in view also of the relatively wide range over which an integrated value was required, measurements were made on the absorptiometer already mentioned rather than on the plate.The absorptiometer was used as a single-cell instrument to measure the fraction of the light transmitted by the filter that would pass through Ilford 608, this value being corrected for the density of Ilford 608 itself. This method takes into accdunt the spectral response curve of the photo-cell and the result is referred to as the efective red and infra-red transmission. The effect of this transmission can best be shown by considering a hypothetical extreme case of a substance with a density of 0.801 (15.812 per cent. transmission) at the required n-avelength and a density of zero in the red and infra-red. Let us assume that a density reading of 04300 is actually obtained, corresponding to a transmission of 15.849 per cent.Let the effective percentage of red and infra-red light be R and let the intensity of the required line on the photo-cell at the zero setting be I ; then at the zero setting the red and infra-red intensity will be IR/100, and the total intensity will be I ( 1 1 R/100). During the reading the 73, 74, 75, 86C, 87, 88, 88A and 89. 807, 808, 809 and 812. intensity of the line on the photo-cell will be m9 I , and the intensity of red light will be ( 15*81 ”> ;’ RI :,. m). Assthe totals at the zero setting and during the reading will be equal, and therefore R = 0.044 per cent. We have therefore tried to reduce R below this level by the incorporation of one or other of the following heat-absorbing elements: Chance 0x13, OB2, Ilford 801, 802, 803 or 804.In the H76O model of the Spekker absorptiometer there is a built-in heat absorbing filter. This will normally be unnecessary, and in the case of the 691 mp. and infra-red filters will interfere. As an example of the effect of this stray radiation we show in Table I the density readings of a 1 in 100 dilution of blood as oxyhaemoglobin with various filter combinations intended * Ilford Ltd. manufacture a series of gelatin filters absorbing in the near infra-red (Sos. 801 to 804). These contain a copper salt, not an organic dye.668 NICHOLAS -4ND POLLAK: THE ISOLATION OF THE LINES [Vol. 75 to isolate the 577 mp. line. low red and infra-red absorption. The solution has a sharp absorption band near 577 mp.and very TABLE I EFFECT OF VARIOUS FILTER COMBINATIONS INTENDED TO ISOLATE THE 577 ~ p . LINE Optical density Alone . . .. . . + Chance ON13, 2mm. -+ Chance OB2, 2mm. + Chance OB2, 4mm. + Ilford 801 . . . . + Ilford 802 . . . . + Ilford 803 . . . . + Ilford 804 . . . . . . . . , * . . . . . . . . . . . . . . . . . . Ilford 812 0.950 0.960 0.980 0.980 0.959 0.971 0.972 0.980 Ilford 868 0.727 0.763 0-917 0.964 0.785 0.865 0.884 0.937 A water-cell only absorbs radiation of wavelength greater than 1 . 4 ~ . and its effect on the efective infra-red transmission was found to be negligible. It was also found that a thermometer placed in a position corresponding to the left-hand photo-cell of the Spekker absorptiometer showed no significant rise in temperature.Moreover, Messrs. Evans Electro- selenium Ltd. (the makers) state that infra-red is not harmful to their photo-cells. The left- hand water-cell is placed very near the lamphouse and its rise in temperature owing to conduction and convection causes the formation of air-bubbles with consequent drift in the zero setting. We have therefore abandoned the use of this cell, and where difference methods are employed the right-hand water-cell has also been dispensed with. In the estimation of the sensitivity of the Spekker absorptiometer with our recommended filters, the left-hand aperture was completely closed by a shutter and the drum was opened to give a full-scale deflection (about 0.5 p-amp.) on the Cambridge spot galvanometer at maximum sensitivity.Following Vaughan’s nomenclature this gives the air-to-air setting that should give full sensitivity. RESULTS- In general, we used one basic filter to absorb as many foreign lines as possible, and one or more supplementary filters to obtain complete isolation. In addition, heat and red- absorbing filters were incorporated. The Cambridge spot galvanometer normally used with the Spekker absorptiometer has a sensitivity of 170mm. per p-amp., and will not give a satisfactory response with some combinations. For these the substitution of a more sensitive galvanometer, e.g., Tinsley type VS6/45, is recommended. In view of the importance of the red region, which has not previously been used, an alternative filter combination of higher transmission but wider waveband is given.In all the red combinations the lines are much less intense in relation to the background and the filters do not give such strictly mono- chromatic light as the others. Following the practice of many workers with the tungsten lamp we shall refer to each region by its dominant wavelength, which in both cases is a line (607 and 691 mp). There is no commercially available filter that will cut off the infra-red on the long wave side, so it is necessary to rely on the falling off in sensitivity of the photo-cell with increasing wavelength. Unfortunately different photo-cells vary in their response to infra-red, but no doubt the makers would be able to select cells having the desired characteristics. It should be noted that the response in the infra-red is more sluggish than in the visible and ultra-violet regions, but not enough to interfere unduly with the convenience of taking readings.PREPARATION 01; FILTERS- For convenience and to reduce the density by the avoidance of surface reflections we made compound filters containing all the gelatin films between glass plates, which were clear glass or glass filters according to requirements. Filters are usually cemented with Canada Balsam.32 The yellowing of this with age may interfere with the transmission of wavelengths below 492 mp., and it is possible that other media such as damrnar or plastics may be preferable here (see Nicholas and Pollak=). I t is not essential for colourless glass t o be optically worked, as the For some wavelengths as many as five constituent filters were used.Dec., 19501 OF THE MERCURY ARC BY FILTERS 669 light already has to traverse the optically imperfect silica and glass walls of the lamp, and we have found the glass of photographic plates perfectly satisfactory.Glass filters must be plane-parallel to avoid the introduction of density gradients. TABLE I1 RECOMMENDED FILTER COMBINATIONS I\’ave- Recommended Individual length, combination densities* mb. 365 405 43 6 492 546 577 “607” “891” 735 + ‘ ‘Orange red” OB2, lmm. Wratten 17 (f) 0x1, 3mm. OV1, 4mm. Wratten 86Ct OB10, 1-43 mm. OB2, 2mm. Wratten 2A (treble thick- ness) (f) Wratten 36 Ilford 603 Wratten 5 Ilford 302 Ilford 804 Wratten 16 Wratten 74 Ilford 804 Ilford 812 Ilford 804 Wratten 26 (f) Ilford 804 ON13, 2 m n ~ Ilford 206 ON13, 2.5mm.Wratten 88A Wratten 25 (f) Ilford 801 0.627 0.607 0.221) 1.180 0.902 0.242 0.369 0.354 0-852 1.030 0.343 0.110 0.122 0-170 1 -085 0.239 0.967 0.627 0.290 1.119 0.333 0.365 0.499 Effective red and Over-all infra-red density transmis- (cemented) sion, O/ / O 1.411 < 0.03 2-272 0.2 1.410 <0*03 1.496 0.1 1-411 .:0*03 1.527 <0.03 1.374 __ 1.239 0.197 - 0.357 .- Air-to-air setting on Spekker for full deflection 0.985 1.28 1.52 1.30 1.46 Remarks Requires sensitive galvano- Combination amended meter November, 1950; spectro- gram of this new combina- tion is identical with Fig. 2d. See Fig. 11 Requires sensitive galvano- meter Satisfactory results also ob- tained with Ilford 606, 812 or Wratten 73 with any heat absorber Includes 607 to 623 mp. lines.Requires sensitive galvano- meter Transmits 672 to 709 mp. Requires sensitive galvano- meter Requires sensitive galvano- meter Density measured at 607 mp. Not monochromatic ; in- tended for use when only Cambridge spot galvano- meter available XoTEs-Filters marked (f) are fluorescent. Filters not marked Wratten or Ilford are made by Chance Bros. * The density of many glass filters differs from melt to melt and the thickness is not therefore a complete We have therefore given the density of elements in proved satisfactory combinations in specification. addition. t Messrs. Kodak have recently changed the composition of Wratten 86C without changing its designa- It is therefore probable that recent batches will not give Suitable batches should correspond with the published absorption curve,32 This combination is recorded in the hope that the original tion or giving any other indication to the user. results identical with ours.and have a density at 405 nip. of about 0.9. filter will again be made available.6iO 1. 3 . 4. * L . r ) . ti. 7. 8. 9. 10. 11. 12’. 13. 14. 15. 16. 17. 1s. l!). 20. 21. 23. 2s. 23. 2’6. 27. 28. 29. 30. 31. 32. 33. 34. 35. .) 8 -“. SILVER NICHOLAS AND POLLAK KEFEKENCES [Vol. 75 hliiller, It. H., f l i t ? . Iztig. Chein., .-lizal. Ed., 1941, 13, 667. States, 31. S., and Anderson, J . C., J . Opt. SOC. Amer., 1942, 32, 659. Lothian, G. F., “Absorption Spcctropliotometry,” Hilger and \Vatts Ltd., London, 1949. Heilmcyer, Ludwig, “Medizijzischc .S~ektl.ophotonietrie; aiasgewtihlte Methoden und neure r l ~ t t t ’ ~ , - szrc~~u~tgse~,ueb?~isse am Korperfavbstofjee?r ztnd Iiorpevfliissi~:keiiE,I,“ C.Fischer, Jena, 1933 ; EngliGh translation by Jordan and Tippell, Hilger, I-ondon, 1943. Vaughan, I3. J., “Further Advances in the Use of the Spekker Photo-electric Absorptiometer in Rletallurgical halysis,” Institute of Chemistry, London, 1942. Stross, \Y., Aiiulyst, 1949, 74, 622. Miiller, 1s. H., I n d . Eng. Chenz., Anal. Ed., 1946, 18, So. 11, p. 21 of advertising section. Staats, E. M., J . Opt. SOC. Auier., 1938, 28, 112. -, Ibid., 193‘3, 29, 221. Hilgcr and IVatts Ltd., “The Hilger Photo-electric Absorptiometer,” London, 1947. .~ , “Instructions for Use of Absorptiometer,” London, 1944. .- , “Hilger Spekker Xbsorptiometer Type H.760,” London, 1949. Haywood, F. IV., and iVood, A. A. It., “Metallurgical Analysis by Means of the Spekker Photo- Chancc Rros. Ltd., “Chance Coloured Glass for Scientific l’urposes,” Smethwick, not dateci. Gentry, C. H. It., and Sherrington, I,. G., .41ztclyst, l94ri, 71, 432. -- __ , Ibid., 1948, 73, 57. Hadiey, \V. H., Ibid., 1945, 70, 43. Isbell, R. A. L., Ibid., 1949, 74, 618. Rogers, B., ,’l.letallui~gia, 1945, 33, 13. Harrison, T. S., .4nal*yst, 1945, 70, 362. de Lippa, 31. Z., Ibid., 1946, 71, 34. Lcnnard, G. J., Ibid., 1949, 74, 253. Davis, 13. C., “The Photometric Analysis of Steel and Aluminium Alloys,” Royal Aircraft Estab- British Iron and Steel Research Association, Il.letnlLzcrgin, 1948, 39, 105. Parker, C. ;I., -4wdyst, 1949, 74, 112. Edwards, F. H., and Gailer, J . W., Ibid., 194.5, 70, 365. Edwards, F. H., and Robinson, A. hl., Ibid., 1946, 71, 379. Harrison, T. S., J . SOC. Cheiti. Iizd., 1944, 63, 347. British Aluniinium Co. Ltd., “Chemical Analysis of :Iluminium and its Alloys,” London, 1947. Hairstow, S., Francis, J . , and Wyatt, G. H., Analyst, 1947, 72, 340 Payne, S. T., “Colorimetric and Polarographic ’Inalysis of Some Non-Ferrous Metals and Alloys, ‘’ Eastman ICodak Co., “\Vratten Light Filters,” Rochester, N.Y., 1945. Ilford Ltd., “Ilford Colour Filters,” London, not dated. Nicholas, J. W., Metallurgin, in the Press. Nicholas, J . W., and Pollak, F. F., J . Sci. Ins&., 1951, 28, 23. electric Xbsorptiometer,” Hilger, London, 1944. lishment, Farnborough, Report No. M.7880, not dated. British Non-Ferrous Metals Research Association, Report No. 729, London, 1947. I h D 1;irst submitted, Febvuarv, 1950 1f-1 r H AM , ESSE s Amended, Jz&; 1950

ISSN:0003-2654    

DOI:10.1039/AN9507500662

出版商:RSC    

年代:1950

数据来源: RSC

摘要:

Dec., 19501 OSBORN 67 1 The Use Of Polarographic Methods for the Analysis of Fine Chemicals BY G. H. OSBORN (Read at the meeting of the Physical Methods Group 092 Friday, October 'ith, 1949) SYNoPsIs-'rhe function of polarographic methods in a laboratory dealing with the analysis of fine chemicals is discussed at length and followed by outlines of the methods at present in me. Detailed procedures are given for new methods. THE analyst in charge of a laboratory controlling the production of thousands of different fine chemicals is faced with what appears to be an almost insuperable task. The vast variety of the samples to be analysed, together with all the various possible impurities in them and the ever-growing demand by customers for very pure chemicals of known analysis, sets the analyst a very difficult problem.It is only by the use of every possible modern tool that he is able to attempt to cope with the problem, and the polarograph is one of the most essential of these tools. It must first be said that the application of the polarograph to any problem in such a laboratory is only justified if there exists no simpler or more rapid method by other techniques. Each particular application of the polarograph calls for very careful investigation before the method can be put into routine use, and it will be obvious, therefore, that the amount of research required would render its wholesale application to the analysis of thousands of different chemicals out of the question. The polarograph is, therefore, only used in our laboratory where it is justified on the grounds of accuracy, speed or simplicity, or where no other method is known to exist.There are many hundreds of polarographic methods recorded in literature, but this paper deals mainly with those found to be useful in one particular fine chemical laboratory. We find the polarograph to be of most use for the estimation of trace amounts of elements or groupings, either organic or inorganic; it is seldom used for the determination of the major constituents in a chemical. In the determination of trace elements or groupings in fine chemicals, it may be stated as a general rule that, if the half-wave potential of the trace to be determined precedes the half-wave potential of the compound under test, then the determination of the trace is usually a simple matter.Solution of the compound in water, in which the compound itself acts as a supporting electrolyte, followed by taking a polarogram should theoretically give the desired result. If, however, the half-wave potential of the compound under test precedes that of the trace being determined, then interfering elements or groupings must either be complexed or removed by some method such as electrolysis. We find the most expedient method of measurement to be that using internal standards.l This greatly decreases the time required for a determination and avoids the use of thermostats. We find it advisable to make two separate additions of known amounts of the substance being determined and to take a polarogram after each addition to ensure that the increases in wave heights are linear.INORGANIC TRACES ZINC- The polarograph is especially valuable for the estimation of zinc owing to the difficulty of estimating traces of this element by other methods. Zincgives very well-defined waves both in acid and in alkaline solutions and can readily be estimated, e.g., in ferrous sulphate,l cadmium salts: aluminium salts3 and copper salts. In the determination of zinc in ferrous sulphate it is not necessary to remove the iron, and this transforms what was a very difficult problem into a very simple one. For the determination of zinc in cadmium, it is necessary to remove the cadmium by electrolysis and we have found that in doing so there is no loss of zinc. This electrolysis must be from a solution made just acid with either sulphuric or672 OSBORN: THE USE OF POLAROGRAPHIC METHODS [Vol.76 perchloric acid, and the current should be made 3 to 4 amps., copper-plated platinum electrodes4 being used. Similarly, in copper salts, the copper is removed by electrolysis prior to the polarographic estimation. The determination of zinc in organic compounds falls into two classes, (a) those compounds, such as insulin and thiouracil, which must first be subjected to wet oxidation because they yield interfering waves, and (b) those substances that do not give interfering waves. Zinc may be determined directly in phenol after treatment with excess of caustic soda. If an organic sample is wet oxidised with sulphuric and nitric acids before the polarographic estimation of zinc, care must be taken to remove all traces of residual nitrate by treatment with ammonium oxalate because the nitrate group has a half-wave potential in alkaline solutions close to that of zinc and can cause serious interference.Kolthoffb states that the nitrate group gives no interference in alkaline solution, but this is not our experience. Another interesting procedure recently developed is for zinc in thorium salts, when the thorium is complexed with sulphosalicylate at pH 8-5 in the presence of a small amount of gelatin. The method is excellent for the range of 20 p.p.m. to 1.0 per cent. of zinc in thorium.6 It is better to use sulphuric and perchloric acids whenever possible. NICKEL- There are a number of elegant methods for the determination of nickel in the presence of many elements, but most of these are unsuitable for the determination of small quantities of nickel in the presence of large amounts of cobalt.Traces of nickel can be determined in cobalt salts by the simple expedient of neutralising and then complexing the cobalt with ammonium thiocyanate.' When dealing with complex amino-cobalt compounds or organic salts of cobalt it is preferable to convert them to cobalt sulphate by wet oxidation with sulphuric acid before neutralisation. The colorimetric determination of nickel in the presence of large amounts of cobalt was extremely tedious, even by those methods that were accurate, whereas by aid of the polaro- graph it is simple and rapid. This effectively separates the cobalt and nickel waves. COBALT- The problem of determinating small traces of cobalt in nickel has been for a long time unsolved, although polarographic methods were known by which it was possible to separate large traces of cobalt from nickel, e.g., those involving the use of ammonium oxalate as a supporting electrolyte.8 None of the methods proposed, however, was found to be satis- factory for the determination of small traces of cobalt in nickel.A method has been proposed lately involving the use of Trilon B (ethylene diamine tetra-acetic acid) as a base solution.9 We have tried the method suggested, but quite apart from the fact that the sensitivity claimed by the authors is poor, not indicating less than 0.4 per cent. of cobalt in nickel, we have not succeeded in obtaining good waves for low concentrations of cobalt.We feel that the application of Trilon B to this particular problem is far more likely to be solved on a photometric basis. We are, however, proceeding with further polarographic experiments with this reagent. LEAD- Lead is not normally determined polarographically in our laboratories as other methods exist, but in certain cases where it is tedious to estimate by these other methods, such as in ferrous sulphate,l it is rapidly determined polarographically, using the salt as the ground solution. Lead can be determined directly in nickel salts using the nickel salt as supporting electrolyte.l* Another useful application is the determina- tion of traces of lead in zinc salts simply by dissolving the salt in dilute hydrochloric acid and taking a polarogram.ll Lead is also determined in cadmium salts by using a cyanide supporting electrolyte when the lead wave appears about -0.4 volt before the cadmium wave.We have also found that we can determine lead directly in phenol by adding excess of sodium hydroxide and then taking the polarogram. Lead may be determined in the presence of large amounts of tin, aluminium and iron since at pH 6-5 to 7-0 these elements are not reduced, although lead gives a wave at about -0.5 volt. Nitrates and free hydro- chloric acid must be eliminated as they give drawn-out waves.12 The supporting electrolyte Reliable results are obtained.Dec., 19501 FOR THE AN-4LYSIS OF FINE CHEMICALS 673 is a 30 per cent. solution of calcium chloride. Lead can be determined in aluminium salts in a sodium carbonate ground solution.Interference from tin is prevented by oxidation, from iron by reduction in alkaline solution and from copper by precipitation with potassium thiocyanate. l3 COPPER- Trace amounts of copper are not normally determined on the polarograph since the colorimetric method using sodium diethyldithiocarbamate is so rapid and simple. The polarographic method is, therefore, only used where it is difficult to apply other methods. For example, with ferrous salts we have found it advantageous to use the polarographic method which does not involve the removal of the ir0n.l As in the case of lead, copper can be determined directly in nickel salts by using the nickel salt as supporting electrolyte.lo Copper may also be determined polarographically in the presence of cadmium, nickel, zinc and manganese in lead and its salts.14 Davies and Key15 have described a method in which copper is determined in the presence of iron with N potassium fluoride or sodium potassium tartrate as supporting electrolyte.CADMIUM- The polarographic estimation of this element is most usefully applied in our laboratories to the determination of traces in zinc salts. Since the cadmium wave precedes the zinc wave by about 0.4 volt it is only necessary to dissolve the zinc salt in water and take a polaro- gram ; the salt itself serves as supporting electrolyte.ll IRON- Iron is not normally determined polarographically as so many other methods exist, but we find it useful to determine this element in cobalt salts by dissolving the salt in alkaline tartrate solution when, if all the iron is present in the ferric state, its wave appears well before the cobalt wave.We have not found this method much use below 0.1 per cent. of iron, although at and above this figure good waves are obtained. This procedure has also been recommended for the determination of iron in manganese salts.lB A method has recently been proposed whereby iron may be determined in an acidic oxalate supporting electrolyte at pH 5 provided the iron is first reduced to the ferrous state. With the exception of copper, which must be removed, no other ion soluble in dilute acid solution containing sulphur dioxide gives an interfering wave. It is claimed that for small amounts of iron this procedure is more accurate than either L-olumetric or gravimetric procedures.lC VANADIUM- oxidation,la although it is not usually determined polarographically in fine chemicals. Vanadium is sometimes determined polarographically in organic materials after wet TELLURIUM- We find it convenient to determine small amounts of tellurite in the presence of a large amount of selenitels by addition of a slight excess of ammonia, de-oxygenation with sodium sulphite and polarography over the range -0.5 to -1.0 volt. It is advisable to add a little gelatin to improve the wave shape. ?. I IN- A useful method for the determination of tin in phenol has been published,20 but normally we do not determine this element polarographically. ANTIMOPU’Y AND BISMUTH- Page and Robinson21 have described methods for the micro-estimation of antimony and bismuth in inorganic and organic compounds.Tervalent antimony may be directly determined; quinquevalent antimony must first be reduced. In N sulphuric acid the half- wave potential of antimony and bismuth are respectively -0-34 and -0.02 volt and in N nitric acid they are -0-17 and -0.03 volt. Thus it is possible to estimate traces of both in sulphuric674 OSBORN : THE USE OF PO1,AROGRAPHIC METHODS [Vol. 75 acid; the wave, however, coalesces in hydrochloric or nitric acid. Bismuth can be determined in copper salts by polarography in tartrate and citrate media.% In acid tartrate of pH 4.5, bismuth can be determined in the presence of large amounts of lead and cadmium. ,iLKALINE EARTHS- The alkaline earths are reduced at large negative potentials.Thus their trace estimation in the presence of most other elements is impossible without some preliminary chemical separation, so that a polarographic method has no advantage over the standard technique, Zlotowski and Kolthoff,s however, claim that barium, strontium and calcium can be deter- mined in the presence of each other in alcohol - water media. When present in approximately the same amounts, the barium wave appears first followed by the strontium wave and finally the calcium wave. Magnesium interferes with the calcium determination. In our laboratories, however, we have found the flame photometer to be the ideal instrument for determination of trace amounts of alkalies and alkaline earths, and we do not, therefore, see any reason for application of the polarograph.NITRATE- The determination of nitrate in sodium nitrite can be readily accomplished by means of the polarograph. Haslam and Cross% have described a method in which the nitrite is first decomposed with sodium azide in hydrochloric acid solution and, after concentration, the solution is polarographed in a lanthanum base solution over the voltage range -1.2 to -2.1 volts. This method is satisfactory over the range 0.04 to 1 per cent. of sodium nitrate. NITRITE IN NITRATE- In the presence of uranyl ions and dilute hydrochloric acid, nitrite and nitrate ions are both reduced at approximately -0.9 volt (versus the saturated calomel electrode). The diffusion current is proportional to the nitrite concentration when the ratio of uranyl ion to nitrite is greater than unity.Under these conditions, the reduction of nitrite involves three electrons, indicating a reduction to nitrogen. Analysis of the wave shows that the reduction is irreversible. A solution can be analysed for both nitrate and nitrite ions in two polarographic experiments. The diffusion current due to the two constituents in the original solution is first measured. The nitrite in a second sample is oxidised to nitrate by hydrogen peroxide in acid solution, the excess of peroxide is destroyed catalytically by manganese dioxide in alkaline solution, and the diffusion current is measured.% IODATES IN IODIDE- We have found it very convenient to determine iodates in iodides when both are water- soluble, e.g., the potassium salts. For potassium iodide, the solution is made slightly alkaline with sodium hydroxide, de-oxygenated with hydrogen and a polarogram is taken, when the iodate wave appears at about -1.2 volts.BROMATE IN BROMIDE- Bromate in bromide may easily be determined in the same way as iodate in iodide. It is, unfortunately, not possible to determine chlorate in chloride in a similar manner owing to the fact that the reduction potential of the chlorate is higher than that of the supporting electrolyte . ORGANIC APPLICATIONS In the field of fine organic chemical analysis, the use of the polarograph has been less well developed than in the inorganic field. Quantitative organic polarography in general has from the beginning lagged behind inorganic developments. However, among other applications of polarographic methods to organic analysis the following are valuable methods in the analysis of fine chemicals.ALDEHYDES I N ALCOHOL- The rapid and accurate determination of acetaldehyde in ethyl alcohol has become possible by means of the following procedure, which is an application of the method by Adkins and Cox26 for the polarographic measurement of aldehyde.Dec., 19501 FOR THE ANALYSIS OF FINE CHEMICALS 675 The alcohol is mixed in equal proportions with a I M solution of ammonium chloride and, after de-oxygenation of the solution, a polarogram is taken, when the acetaldehyde wave occurs in the range - 1.0 to - 3.0 volts. Internal standards are then added and measure- ment made as usual. We have found this method to have great advantages over the usual method with Schiff's reagent.FURFURALDEHYDE IN FORMALDEHYDE- Reduction of furfuraldehyde occurs in acid, neutral and alkaline media giving a single wave in acid and alkaline solutions and two waves in solutions of pH 4.0 to 7.0. Within this range, with increasing acidity the first wave gets smaller and the second larger, but the total wave-height remains unchanged. With pH 7, the reduction potential is 0.25 to 0.30 volt more positive than that of hydrogen, and under these conditions formaldehyde does not interfere.27 NITROBENZENE IN ANILINE- Small quantities of nitrobenzene in aniline may very conveniently be determined by the method of Haslam and Cross.28 To a known amount of the sample is added a little con- centrated hydrochloric acid containing nigrosine and a polarogram is taken.From 0.01 to 0.05 per cent. of nitrobenzene in aniline can be determined with an accuracy closer than 4 per cent. GAMMA ISOMER IN GAMMEXANE (BENZENE HEXACHLORIDE)- A useful method of assay as distinct from the trace determinations so far discussed is the determination of the gamma isomer of benzene hexachloride as described by Dragt.29 The gamma isomer is the only one of the five isomers reduced at the dropping mercury electrode under the conditions described. The method consists of taking a solution of the isomers in acetone, alcohol and water, buffered with a potassium chloride - sodium acetate buffer and, after de-oxygenating, taking a polarogram through the range -0.5 to -2.0 volts. Other methods exist for the determination of this isomer, but this is by far the most rapid with the possible exception of the infra-red method.MALEIC - FUMARIC ACID MIXTURES- It is claimed that there exists no satisfactory method for the estimation of these acids in the presence of each other, except by a recent polarographic method.aO This method enables the analyst to solve the problem in a simple manner by polarography in an ammonium hydroxide - ammonium chloride buffer solution of pH 8-2 as supporting electrolyte. The maleate wave precedes the fumarate wave. If interfering substances are present, maleic and fumaric acids must be precipitated as their barium salts in alcoholic solution. The precipitate is soluble in the base solution. We find that the method works quite well when maleic and fumaric acids are present in roughly equal quantities.It fails entirely when there is an overwhelming proportion of one or other constituent. Thus we were quite unable to detect 6 per cent. of fumaric acid added to maleic acid. The authors of this method refer throughout their work to roughly equal quantities of both and make no reference to unequal proportions. We find that 25 per cent. of fumaric acid had to be present before we could detect the slightest sign of a second wave. Our experience with this method has not been particularly fortunate. PEROXIDES AND ALDEHYDES IN ETHER- The method normally employed in our laboratories for the determination of peroxides in ether is based on the oxidation of ferrous thiocyanate and subsequent measurement of colour. Recently, however, we have been trying out the polarographic method of G ~ s m a n .~ ~ Peroxide and aldehyde are extracted from the ether by shaking with an equal volume of 0.01 N lithium hydroxide and determined by polarography of the aqueous layer. The peroxide wave occurs at -1.3 volts and the acetaldehyde wave at -1.8 volts. It is claimed that this method shows waves where chemical methods have failed to detect any peroxide. The method has the disadvantage that not all the peroxide or aldehyde is extracted in one shaking with the lithium hydroxide. From the partition coefficients, which are 0.45 for676 OSBORN THE USE OF POLAROGRAPHIC METHODS [Vol. 75 peroxide and 0.63 for aldehyde, it is possible to calculate the amount originally present in the ether. The waves are measured by comparison with the increases in wave height obtained by the addition of dilute solutions of hydrogen peroxide and acetaldehyde.In spite of its apparent disadvantage, it appears that the method is a promising one. PEROXIDE IN DIOXAN (DIETHYLENE DIOXIDE)-- Peroxide may be determined in dioxan very easily by the following technique. The diosan is mixed with an equal volume of M lithium hydroxide solution and, after de-oxygena- + - // ' \CHLOROFORM ELECTRIC HOT PLATE Fig. 1 tion with hydrogen, a polarogram is taken over the range -0.5 to 2.0 volts. To estimate the peroxide content a known volume of a standard diluted hydrogen peroxide solution is added and polarography repeated.32 HYDROGEN PEROXIDE- The polarographic determination of hydrogen peroxide can be carried out by making use of the reduction step at -1.0 volt versus the saturated calomel electrode. The method is limited to peroxide concentrations of less than 0.15 per cent., owing to the oxidation of mercury by hydrogen peroxide a t higher concentrations. If a stationary platinum micro- electrode is used in place of the dropping mercury electrode, current - voltage curves in the range from 0 to -0.6 volt versus the Saturated calomel electrode show a reduction step whose height is proportional to the hydrogen peroxide concentration over a much widerDec., 19501 FOR THE -4NALYSIS OF FINE CHEMICALS 677 range, and determinations can be carried out simply by measuring the limiting current at -0.6 volt.The upper limit of peroxide concentrations that can be measured depends on the concentration of the supporting electrolyte, and if saturated potassium chloride solution is used, concentrations up to 0.9 per cent.can be determined.% THXOMERSALATE- Page and WallersP have recently described an interesting polarographic method for the estimation of thiomersalate. A well-defined wave at abott -0.5 volt is obtained in N hydrochloric acid. This method is very useful for the determination of this antiseptic in vaccines and pharmaceutical preparations. THE DETERMINATION OF BENZANTHRONE AND ANTHR.4QUINONE IN THE PRESENCE OF EACH OTHER- An interesting application has been suggested35 for these two chemicals since both give a good wave in 70 per cent. methyl alcohol containing 0.1 N sulphuric acid. Benzanthrone c. 5 a a 3 0 +C i 0 -0.5 -1.0 -I *s -2.0 VOLTS Fig. 2 has a half-wave potential at 0.96 volt while anthraquinone has a half-wave potential at -0-36 volt.The wave height is proportional to the concentration and this simplifies the detennina- tion of small quantities of one in the presence of large amounts of the other. DETERMIN.4TION OF PHENYL MERCURIC ACETATE IN A GELATIN BASE- Some recent interesting experimental work in the B.D.H. laboratories has led to the development of a method by L. J. J. Hillman for the determination of small quantities of phenyl mercuric acetate in a gelatine base. Page34 had reported two waves for this compound in dilute hydrochloric acid base solution and this has been confirmed by us. Our problem was complicated by the presence of the large amount of gelatin, which made the solution very viscous at room temperature, and consequently it gave an erratic polarogram owing to the mercury drop not falling at a steady rate.This difficulty was overcome by conducting the experiment at an elevated temperature in a specially constructed polarographic cell. Details of this cell are shown in Fig. 1. It consists of a graduated cell made from a burette barrel by sealing one end. The cell is heated by immersion in the refluxing vapour of an organic liquid. The solvent used to produce a constant temperature was chloroform (AnalaR) and the working temperature was found to be 58" & 0.2" C. It is essential that the temperature be kept constant. Preearation of calibration cuyve-A standard solution of phenyl mercuric acetate was made up as follows.Take 6 g . of glycerol and dissolve in it 0.15 g. of phenyl mercuric acetate, add 2.25g. of triethanolamine and make up to 100ml. with water. Take various known678 OSBORN THE USE OF POLAROGRAPHIC METHODS [Vol. 75 amounts of this solution and to each add 0-5 ml. of 0.01 per cent. methyl red solution, 0.4 g. of gelatin and 2 crystals of sodium sulphite (AnalaR). Make the volume of each portion to 10 ml. with distilled water. Place each solution in turn in the polarographic cell and heat in the vapour of the refluxing chloroform for about a quarter of an hour so as to allow a uniform temperature to be achieved. Then set the potentiometer at -1.05 volts, adjust PHENYL MERCURIC ACETATE, mg. Fig. 3 the zero current and condenser current to give a good deflection, and take the reading on the galvanometer scale.Disconnect the lead to the polarographic cell so that it is no longer in circuit and take another reading; the difference between these two readings gives the diffusion current from the cell at -1.05 volts, which is proportional to the phenyl mercuric acetate concentration. I t is necessary to adopt this procedure since an anodic wave due to sulphite just before the first phenyl mercuric acetate wave makes it impossible to, estimate the residual current (see Fig. 2). Plot the diffusion current obtained from the above solutions against the respective concentrations of phenyl mercuric acetate when a straight line graph (Fig. 3) is obtained. Sodium sulphite was used as it was not possible to obtain a satisfactory degree of de-oxygena- tion in a reasonable time by the use of hydrogen.The graph does not pass through the origin since the current measured by this method includes the residual plus the diffusion current at -1-05 volts. It will be found that the current in microamps represented by XY in Fig. 3 corresponds to that represented by CD in Fig. 2, and their values correspond to the residual current at -1.05 volts. Accuracy-By means of the calibration graph so constructed, a series of twenty samples containing known amounts of phenyl mercuric acetate were estimated by an operator who did not know the true contents. All the results obtained were found to be within k5.0 per cent. of the true value. Thanks are due to Mr. A. Jewsbury, BSc., A.R.I.C., and to Mr.L. J. J. Hillman of these laboratories for assistance and constructive advice in the preparation of this review, and to the Directors of the British Drug Houses, Ltd., for permission to publish. 1. 3. 4. 5. 9 d. REFERENCES Jewsbury, A., and Osborn, G. H., Analyst, 1948, 73, 506. B.D. H. Laboratories-unpublished work. Stross, W., and Osborn, G. H., Light Metals, July, 1944, 7, 323-7, and Stross, W., Metallurgin, 1947, Osborn, G. H., Metallurgia, 1949, 39, 111. Kolthoff, I. Rf., and Lingane, J. J., “Polarography,” Interscience Publishers, Tnc., New York, 36, 163-6 and 223-5. p. 314.Dec., 19501 FOR THE ANALYSIS OF FINE CHEMICALS 679 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 65. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. Patterson, J .H., and Ranks, C . V., Anal. Chern., 1948, 20, 897. Lingane, J. J., and Kerlinger, H., Ind. Eng. Chem., Anal. Ed., 1941, 13, 77. Prajzler, J., Coll. Czech. Chew. Comnz., 1931, 3, 406. Souchay, P., and Faucherre, J., .-3naZ. Chim. A d a , 1949, 3, 252. Korchunov, I. A., and Sazanova, L. N., Zavod. Lab., 1948, 14, 621. Terai, Y., Bull. Inst. Phys. Chem. Research, Tokyo, 1938. Mhlopen, 3. Ya., J . Anal. Chenz. Russ., 1947, 2, 55. Stross, W., Metallwgia, 1947, 37, 49-51. Urotschmann, C., Metallwirtshaft, 1944, 23, 343. Davies, W. C., and Key, C., Ind. Chemist, 1943, 19, 555. Verdier, E. T., Coll. Czech. Chern. Comm., 1939, 11, 340. Meites, L., Anal. Chem., 1948, 20, 895. Page, J. E., and Robinson, F. A., Analyst, 1943, 68, 269. Schwaer, L., and Sucky, K., Coll. Czech. Chem. Comm., 1935, 7, 75. Wilson, H. N., and Hutchinson, W., Analyst, 1947, 72, 149. Page, J. E., and Robinson, I;. A., J . SOC. Chem. Ind., 1942, 61, 93. Sucky, K., Coll. Czech. Chem. Coinm., 1931, 3, 354. Zlotowski, I., and Kolthoff, 1. M., J. Phys. C h e w , 1945, 49, 386. Haslam, J., and Cross, L. H., .I, SOC. Chem. Ind., 1945, 64, 259. lieilin, B., and Otvos, J . W., J . Amer. Chem. SOL, 1946, 68, 2665. Adkins, H., and Cox, F. W., Ibid., 1938, 60, 1151. Malyugina, N. I., and Korshunov, I. A., J . Anal. Chem. Rzkss., 1947, 2, 341. Haslam, J., and Cross, L. H., J . Soc. Chem. Ind., 1944, 63, 94. Dragt, G., Anal. Chenz., 1948, 20, 737. W'arskowsky, B., Eloing, P. J., and Mandel, J., Anal. Chenz., 1947, 19, 161. Gosman, I3. A., Coll. Czech. Chem. Comin., 1935, 7, 467. Unpublished work-B.D.H. Laboratories. GiguCre, P. A., and Jaillet, J . B., Canad. J . Res., 1948, B, 26, 767. Page, J. E., and W a l k , J. G., Analyst, 1949, 74, 292. Yu, I. Vainshtein, Zavod. Lab., 1949, 15, 411. ANALYTICAL DEPARTMENT THE BRITISH DRUG HOUSES, LTD. POOLE, DORSET (B.D.H. LABORATORY CHEMICALS GROUP) First submitted, Novembev, 1949 Amended, April, 1950

ISSN:0003-2654    

DOI:10.1039/AN9507500671

出版商:RSC    

年代:1950

数据来源: RSC