摘要:

JULY, 1968 THE ANALYST Vol. 93, No. I108 Amperometric Titration of Copper and Cadmium Presence of Zinc, Cobalt and Nickel with Sodium Die th yldit hiocar bamate in the BY A. BROOKES AND A. TOWNSHEND (Chemistry Departmewt, Birmingham University, P.O. Box 363, Birmingham 15) The reaction of some metal ions with diethyldithiocarbamate in 0.1 M sodium hydroxide solution is investigated polarographically. Methods are presented for the amperometric titration of cadmium in the presence of zinc and cobalt, and of cadmium and copper in admixture. MANY metals, including copper, cadmium and zinc, have been titrated amperometrically with sodium diethyldithiocarbamate.1 However, because numerous metal ions react with this ligand, the use of masking agents or pH control is necessary to achieve selectivity.For instance, copper(I1) can be titrated in the presence of zinc, nickel and iron when EDTA is present2 or in the presence of manganese(I1) and chromium(II1) when tartrate is added.3 Copper can be titrated in the presence of zinc in ammoniacal solution at pH 11, and the zinc can subsequently be titrated after acidification to pH 6 with acetic acid.* Other examples are also kn0wn.l It has been shown5 that zinc, manganese(II), nickel and cobalt do not complex with disodium ethylene-l,2-bisdithiocarbamate in 0.1 N sodium hydroxide solution, whereas copper(II), cadmium and lead do form complexes. Thus, under these conditions, it would be possible to titrate copper or cadmium amperometrically in the presence of the non- complexing metals by using sodium ethylene-l,2-bisdithiocarbamate without adding a masking agent.However, aqueous solutions of this reagent are not stable for more than a few hours,6 so sodium diethyldithiocarbamate was investigated as an alternative titrant. Solutions of this compound in 0-01 M sodium hydroxide solution are stable for at least 3 days6 M) did not complex with diethyldithiocarbamate in 0.05 M sodium hydroxide solution. Addition of titrant to the metal-ion solution immediately produced an anodic wave of the ligand. Copper(I1) gave the expected 2 : 1 ligand-to-metal complex. No anodic wave appeared until this ratio had exceeded 2: 1. M) was titrated, the first increment of titrant produced a small anodic wave, but the height of this wave hardly increased until the ligand-to-metal ratio was 2: 1.A smaller concentration of nickel (lo-* M), however, showed no evidence of complexing with diethyldithiocarbamate in 0.1 M sodium hydroxide solution. When ligand was added to a cadmium solution, the anodic wave of the ligand appeared when the ligand-to-metal ratio was greater than 1.0, whereas in the reverse titration, the ligand wave had disappeared when the ratio was 2.0. Addition of ligand to cadmium until it was in appreciable excess over cadmium gave the titration graph shown in Fig. 1. It shows that the 1: 1 species is converted into the 2: 1 complex when a large amount of ligand is present. Moreover, the anodic wave of a 1-fold excess of uncomplexed ligand in the presence of the 1 : 1 complex decreased to half- height after 30 minutes but showed little change thereafter.Polarographic measurements showed that zinc, manganese(I1) and cobalt(I1) (6 x When nickel (5 x Cadmium showed rather more complicated behaviour. 0 SAC and the authors. 426;426 BROOKES 0.15 a 0.IC J 0 L 3 -0 3 0.05 W 6 U 0 4ND TOWNSHEND : AMPEROMETRIC TITRATION [Ana&St, VOl. 93 0 0.2 0.4 0.6 0.8 1.0 1.2 10-3~ Diethyldith iocarbamate solution added, mi Fig. 1. Amperometric titration of 4 x lo-' moles of cadmium(I1) with 1 0 - 3 ~ sodium diethyldithiocarbamate solution The results can be interpreted on the basis of competition for the metal ions between M(0€1)2 + 2L- + ML, + 2 OH- (M = Co(II), Mn(II), Cd or Cu) (1) For cobalt(II), manganese(I1) and zinc, the equilibria lie almost completely towards the left- hand side of the equations, whereas for copper(II), it is completely to the right-hand side; nickel is an intermediate example, and small amounts of ligand are insufficient to send the equilibrium towards the right-hand side.These results enable limits for the values of the solubility products of these metal dithiocarbamates to be calculated, on the basis of the diethyldithiocarbamate (L-) and hydroxide- . . or 2110,~- + 2L- + 2H20 + ZnL, + 4 OH- . . .. .. . . (2). TABLE I SOLUBILITY PRODUCTS OF METAL DITHIOCARBAMATES Metal Cobalt (11) Manganese (11) Zinc Nickel Copper (11) log KgO (M(OH),)' . . .. - 15 - 13 - 17 - 15 - 20 log KBO (ML,) by Hulanickis . . - - log K B O (ML,) * * .. .. >-16 >-14 >-19 -19.7 f0.5 <-28 - 17 - 23 - 30 solubility products of the metal hydroxides.These results do not take into account the effect of supersaturation phenomena and possible slow reactions between diethyldithiocarbamate and the metal hydroxides. These values are given in Table I. The reaction of cadmium can best be described as follows- rapid slow Cd(OH), + L- 7 Cd(0H)L + OH- . . * - (3) Cd(0H)L + L- 7 CdL, + OH-. . .. . . (4). Equilibrium in equation (3) is displaced markedly and rapidly to the right-hand side, but equilibrium in equation (4) needs an excess of ligand to drive it to this side. The latter reaction is also slow, and is responsible for the slow decrease in ligand wave-height in the presence of the 1 : 1 complex. This slow decrease could not have arisen from decomposition of either free or complexed ligand to give sulphide ions because addition of further ligand still gave the 2: 1 end-point on extrapolation of the steep part of the titration graph.Complexes of the type Cd(0H)L have also been postulated' when L represents C1-, POg3-, P,OIo5- or nit rilo t riacet ate. The investigation indicated that it should be possible to titrate amperometrically cadmium or copper in the presence of zinc, cobalt or manganese, in a hydroxide solution.July, 19681 OF COPPER AND CADMIUM WITH SODIUM DIETHYLDITHIOCARBAMATE 427 Table I1 shows that the titration of 2 x 10-4 M cadmium was satisfactory in the presence of a 50-fold excess of cobalt(I1) and a 5-fold excess of zinc. A 50-fold excess of zinc or man- ganese( 11) , however, gave low results, presumably because of co-precipitation of cadmium with manganese hydroxide or as cadmium zincate.It was also possible to titrate copper in the presence of similar amounts of nickel (Table 11). Care should be taken, however, not to spatter nickel hydroxide at the top of the polarographic cell during de-aeration, because the precipitate is difficult to dislodge from the glass, and it is likely to entrain copper. TABLE I1 TITRATION OF CADMIUM AND COPPER Other ions added, moles x lo7 * moles x lo7 Other ions found, Cadmium added, moles x lo7 Cadmium found, moles x lo7 2.0 2.0 2.0 2.0 2.0 2.0 1.0 3.0 - - - - - Zn 100 Zn 10 Mn 100 Mn 10 co 100 cu 1.0 cu 1.0 cu 4.5 Ni 0.5 c u 5.0 Ni 5.0 cu 5.0 Ni 10.0 * Poor graph. Non-linear graph, - 1.9 1.5 2.0 0*1* - --t 2.1 1.0 c u 3.0 c u - cu - - - - - cu - c u therefore no intercept.The formation of the two cadmium complexes enabled a method to be devised for the determination of cadmium and copper in admixture. The sum of the metals is first deter- mined by adding increments of titrant in the range appreciably in excess of that required to form CdL, and CuL,. The titration is then repeated, with lesser amounts of titrant so that the inflection for the CdL complex can be detected, as in Fig. 1. This inflection occurs when CuL, and CdL have been formed. EXPERIMENTAL- The polarographic curves were recorded in a Kalousek vessel with a standard calomel reference electrode and a Cambridge pen-recording polarograph. The titrant was a loh3 M solution of analytical-reagent grade sodium diethyldithiocarbamate in water, freshly made up every 3 days.The total anodic current of the diethyldithiocarbamate was measured at -0.3 volt versus S.C.E., except when the concentration of free ligand was sufficiently large M) for the adsorption wave to be separate from the main wave,6 when measure- ments should then be made at -0.2 volt, so that the total wave-height is measured. PROCEDURE DETERMINATION OF CADMIUM, OR COPPER, IN THE PRESENCE OF ZINC AND COBALT- M cadmium solution to 8 ml of 0.1 M sodium hydroxide solution in the polarographic cell. De-oxygenate with nitrogen, and titrate with diethyldithiocarbamate in 0.2-ml increments. Correct the wave-height for dilution effects, and plot the corrected wave-height vewus volume of titrant ; extrapolate the straight line to zero wave-height to obtain the end-point.DETERMINATION OF COPPER AND CADMIUM IN ADMIXTURE- Dilute a volume of the unknown solution, containing about 10-7 moles of cadmium and copper, to 10 ml with 0.1 M sodium hydroxide solution, and proceed as above. The end-point obtained gives the end-point for the formation of CuL, and CdL,. Repeat the titration with five times the volume of unknown solution, by adding the titrant in O-l-ml increments. The plot of corrected wave-height versus volume of titrant (as in Fig. 1) gives Add 2 ml of about428 BROOKES AND TOWNSHEND the end-point for the formation of CuL, and CdL at the point of first appearance of the anodic current. The concentration of copper and cadmium can readily be calculated. If the approximate concentration of copper plus cadmium is known, the first titration can be omitted, and the two end-points obtained from the second graph. In this instance, the titration should be carried out rapidly, to avoid the slow changes in wave-height with time noted above. The authors thank Professor R . Belcher and Dr. P. Zuman for their interest, Cambridge Instruments Ltd. for the loan of a polarograph and the S.R.C. for provision of a maintenance grant for A.B. REFERENCES 1. 2. 3. 4. 6. 6. 7. 8. Stock, J . T., “Amperometric Titrations,” Interscience Publishers, New York, 1965, p. 441. Usatenko, Yu. I., and Tulyupa, F. M., Izv. Vysh. Ucheb. Zavedenii Khim. i. Khim. Tekhn.oZ., 1958, -,- , Zav. Lab., 1959, 25, 280. Stricks, W., and Chakravarti, S. K., Analyt. Chewz., 1962, 34, 508. Halls, D. J., Townshend, A., and Zuman, P., Analytica Chim. Acta, 1968, 41, 63. -,-,- , Ibid., 1968, 41, 61. Sill&, L. G., and Martell, A. E., Editors, “Stability Constants of Metal-ion Complexes,” The Hulanicki, A., Acta Chim. Hung., 1961, 27, 41. Received January 21st, 1968 No. 3, 56; Chem. Abstr., 1959, 53, 1991d. Chemical Society, London, 1964.

ISSN:0003-2654    

DOI:10.1039/AN9689300425

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Analjst, July, 1068, Vol. 93, $9. 429432 429 The Rapid Dissolution of Plutonium Dioxide by a Sodium Peroxide = Sodium Hydroxide Fusion, Followed by Determination of the Plutonium Content by Controlled-potential Coulometry BY G. W. C. MILNER AND D. CROSSLEY (Analytical Sciences Division, U. K.A .E.A . Research Group, Atomic Energy Research Establishment, Harwell) A method is described for the dissolution of plutonium dioxide, followed by determination of the plutonium content by controlled-potential coulo- metry. The plutonium dioxide is brought into solution by fusion with a mixture of sodium peroxide and sodium hydroxide at 600" C for 15 minutes in an alumina crucible. The cold melt is leached with water, which is then acidified with sulphuric acid. The solution is heated for 15 minutes to de- compose hydrogen peroxide and, after cooling, diluted to a suitable volume.The plutonium content of an aliquot containing about 4 mg of plutonium is determined by controlled-potential coulometry. A potential of + 0.30 volt versus a S.C.E. is used for reduction to plutonium(III), whereas +Om70 volt veuus a S.C.E. is used for the quantitative oxidation to the plutonium(1V) state. Mean recoveries on 100-mg amounts of plutonium dioxide that had been ignited at 850" C were 99.95 per cent., with a coefficient of variation of 0.11 per cent. For the complete dissolution of samples previously ignited at higher temperatures (about 1600" C), an increase in the ratio of the weight of fusion mixture to sample is necessary. Mean recoveries on 50-mg amounts of plutonium dioxide that had been ignited at 1600" C were 99-85 per cent., with a coefficient of variation of 0.54 per cent.IN earlier work from this laboratory a sodium peroxide sinter technique1 was described for the rapid dissolution of plutonium dioxide, including high-fired material. The plutonium content of the resulting solution was then determined by differential spectrophotometry. Subsequent experience with this method has shown that occasionally inconvenience can result from the existence of small amounts of undissolved sample in the final solution. This problem was traced to the difficulty of obtaining satisfactory mixing of the sodium peroxide and the sample under glove-box conditions. It was considered that this difficulty might disappear on modifying the sinter so that fluid conditions occurred; these conditions would be produced by increasing the temperature to above 500" C.In addition, reduction of the size of the sample required for analysis would help to improve and speed up the dissolution procedure. This objective could be achieved if the differential spectrophotometric method for plutonium was replaced by an electrochemical method. For example, it is possible to determine as little as 1 mg of plutonium to within k0.2 per cent. by controlled-potential coulometry, as compared with the 30mg of plutonium needed for a single determination by differential spectrophotometry. Another advantage is that by handling smaller sample weights, the final determination can be carried out in a fume cupboard, instead of a glove-box, with a consequent improvement in the speed of analysis.It was expected that a peroxide fusion would give a rapid method for the dissolution of plutonium dioxide, either alone or in mixtures with certain other materials. It should, for 0 SAC; Crown Copyright Reserved.430 MILNER AND CKOSSLEY: THE RAPID DISSOLUTION OF PLUTONIUM [Ana&St, VOl. 93 example, be much quicker than the ammonium hydrogen sulphate fusion,2 which takes 4 hours to effect the dissolution of refractory plutonium dioxide. Moreover, it should also be satisfactory for the dissolution of other materials mixed with plutonium dioxide, particularly those which are not dissolved by fusion with ammonium hydrogen sulphate. These materials include ruthenium metal, ruthenium dioxide, silica and chromium oxides.In view of these possible advantages, the peroxide fusion technique has been examined in some detail. EXPERIMENTAL SELECTION OF A SUITABLE CRUCIBLE- Although a platinum crucible is satisfactory for sodium peroxide sinters carried out at about 450" C, it is not suitable for fusions at temperatures in excess of 500" C. In considering suitable crucible materials, it was thought that a metal crucible would be preferable to one made from a ceramic because of such factors as ease of handling and the absence of porosity, difficulties usually associated with ceramics. Specimens of various metals were, therefore, tested by fusing small pieces with 0.5 g of sodium peroxide plus 0-5 g of sodium hydroxide for 15 minutes at 600" C in alumina crucibles.By this means it was hoped to identify those metals undergoing negligible attack in the fusion process, and a summary of the results is shown in Table I. These results indicated that, of the metals examined, zirconium was the most suitable for further study. Several zirconium crucibles were made, therefore, by a cold-drawing process. Initially, they had rough internal surfaces, which led to losses caused by melts creeping up the crucible walls, but this effect was reduced by polishing the internal surfaces of each crucible. Evidence of the removal of some zirconium from the crucible walls by the fusion process was detected. Moreover, this attack caused the formation of a grey deposit on the bottom of each crucible, which proved difficult to remove.At this stage, it was concluded that a satisfactory metal crucible would be difficult to obtain. In spite of known difficulties with ceramics, crucibles made only from this type of material were left for consideration. Experimental fusions were carried out, therefore, in thoria, magnesia and alumina crucibles. Thoria crucibIes were completely unattacked by the peroxide melt but, unfortunately, they had a poor resistance to thermal shock, which resulted in severe cracking. Magnesia crucibles were badly attacked during the fusion, and the melt crept up the wall surfaces. Fortunately, alumina crucibles withstood the fusion much better, and were only slightly attacked, 20 mg being a typical average loss in weight for a 15-minute fusion. Moreover, each crucible appeared to be usable for about four fusions, provided that careful drying of the crucible walls was carried out after each fusion. Also, problems connected with the creeping of the melt did not occur with the crucibles tested.On this evidence, an alumina crucible appeared to be the only satisfactory ceramic crucible readily available. The small amounts of aluminium passing into solution would not cause any interference in the coulometric determination of plutonium. Alumina crucibles of 15-ml capacity, as supplied by Thermal Syndicate Ltd., were, therefore, used exclusively in this investigation. DISSOLUTION OF PLUTONIUM DIOXIDE- Experimental fusions were carried out initially on a plutonium dioxide sample that had been ignited at 850" C. The mesh size of the material was less than 100 B.S.S.The sample (100 mg) was mixed with 0-5 g of sodium peroxide in an alumina crucible, 0-5 g of sodium hydroxide then added and the crucible heated at 600" to 620" C for 15 minutes. After cooling, the melt was leached by the method already described,l and the resulting solution acidified by adding it, dropwise, to 10 ml of water @us 4 ml of sulphuric acid (spgr. 1.84) contained in a 50-ml beaker. After warming for 15 minutes, the solution was cooled and diluted to 50 ml with water. The plutonium content was determined on a suitable aliquot by controlled- potential coulometry. The coulometric determination appeared to be fairly normal, with little trouble arising from any residual peroxide in solution. The only noticeable effect was that a slightly longer than normal reduction time (about 30 minutes) was required to reach a constant background current of less than 10 PA.A total of eight fusion experiments was carried out, and the behaviour in each instance was identical. Clear brown solutions were obtained without any trace of undissolved material. The mean recovery for plutonium from these determinations was 99.95 per cent., with a coefficient of variation of 0.11 per cent.July, 19681 431 Experimental fusions were next attempted on a plutonium dioxide sample that had been fired at 1600" C. A series of eight dissolutions with 100-mg portions of material was carried out, followed by coulometric determination of the plutonium. These experiments gave recoveries for plutonium that were up to 2 per cent.low. Further work showed that this bias was not caused by interference in the coulometry, but to slightly incomplete dissolution of the sample. Further dissolutions were then carried out with 50-mg portions of the plutonium dioxide sample to determine whether the higher ratio of fusion mixture to sample would improve the situation. Three fusions were carried out initially, and these gave good recoveries on subsequent coulometric titration. Ten more determinations were completed, and the mean recovery for the thirteen determinations was 99.85 per cent., with a coefficient of variation of 0.54 per cent. DIOXIDE BY A SODIUM PEROXIDE - SODIUM HYDROXIDE FUSION TABLE I OBSERVATIONS ON FUSING VARIOUS METALS WITH SODIUM PEROXIDE &US SODIUM HYDROXIDE Material Nickel .. .. .. Manganese - nickel alloy Stainless steel . . .. Zirconium (clean surface) Zirconium (oxidised surface) Silver . . .. .. Gold . . .. .. Observations .. . . Badly attacked .. . . Badly attacked .. . . -10% weight loss .. .. 2% weight loss .. .. 4% weight loss .. . . -30% weight loss .. . . Badly attacked METHOD APPARATUS- Thermal Syndicate Ltd. furnace. 1010-2, Solartron Laboratory Instruments Ltd., Chessington, Surrey). REAGENTS- Alumina crucibles-Recrystallised alumina crucibles, 15-ml capacity, as supplied by Mufle furnace-A "Hotspot," obtainable from A. Gallenkamp & Co. Ltd., or similar Controlled-fiotential coulomete@ p4-This was fitted with a digital voltmeter (Type LM Electrolysis cell for coulometry-As described previously.6 All reagents were of AnalaR grade.Sodium peroxide. Sodium hydroxide. Sul#huric acid, 18, 1 and 0-5 M. Distilled water. RADIOCHEMICAL SAFETY- Operation on dry samples containing plutonium dioxide, up to the point of complete dissolution, should be conducted in a glove-box. Aliquot portions for completion of the analysis can be handled in a fume cupboard with an efficient extraction and filtration system. PROCEDURE- Weigh 50 to 100 mg of sample ground to less than 100 B.S.S. mesh size (50 mg for samples of plutonium dioxide that have been ignited at temperatures above 1000" C) and transfer it into an alumina crucible containing 0.5 g of sodium peroxide. Mix by rotating the crucible by hand at an angle of 45", then add 0.5 g of sodium hydroxide in pellet form. Heat the crucible in a muffle furnace at 600" to 620" C for 15 minutes, keeping the crucible covered with an alumina (or silica) lid.Then remove the crucible from the furnace and allow it to cool. Add 1 ml of water to the crucible and allow the dissolution reaction to proceed for 5 to 10 minutes. Then add a further 0 6 m l of water, and gently swirl the contents of the crucible. If any of the melt is left undissolved, warm the crucible cautiously on a hot-plate, but avoid prolonged heating. (All of the melt should dissolve to give a brown to black- coloured suspension.) Transfer the extract, dropwise, with a Pasteur pipette into a 50-ml432 MILNER AND CROSSLEY beaker containing 10 ml of water fiulus 4 ml of sulphuric acid. Mix the solution during the addition by gently swirling the contents of the beaker.Wash the crucible by transferring about 2 ml of the acidic solution back into the crucible, and rinse the crucible walls with this solution. Wash the crucible with three further 2-ml portions of 0.5 M sulphuric acid and then with 2 mi of water. Combine the washings with the solution in the beaker, then warm on a hot-plate €or 15 minutes, or until all de-gassing has ceased. Cool the solution and dilute to 50 ml with M sulphuric acid. Use suitable aliquots, containing about 4 mg of plutonium, for determination by cont rolled-po t en t ial coulomet ry. Couulometric determination-Transfer the aliquot of sample solution to the coulometer cell, and add sufficient M sulphuric acid to cover the working electrode. Remove oxygen from the solution by passing a stream of nitrogen through it.Then reduce the plutonium to the tervalent state by electrolysing at a potential of +0.30 volt veysus a S.C.E. until the current attains a low constant value (10 pA or less). After adjusting the coulometer to zero, carry out the quantitative oxidation of plutonium to the quadrivalent state by electrolysing at +0.70 volt veysus a S.C.E. until the cell current reaches its previous low value (10pA or less). Correct the digital-voltmeter reading, Q, for a blank determination, carried out in exactly the same way with an aliquot of solution from a blank sodium peroxide-sodium hydroxide fusion. Calculate the weight of plutonium from the expression- Q (corrected) x F x 239.1 x I' 96,487 x A plutonium, mg = where F is the calibration factor in millicoulombs per millivolt for the coulometer range used, A is the volume of the aliquot taken for analysis and I' is the total volume of the sample solution.RESULTS The procedure was tested on two samples of plutonium dioxide, one having been ignited at 850" C and the other at 1600" C. The results, which are shown below, are expressed as percentage recoveries, assuming PuO,.,, stoicheiometry. Any error in this assumption is very small and is negligible relative to the precision obtained. Temperature of sample ignition 850" C; sample weight 100 mg; recovery, per cent. 99.83, 100916, 100.04, 100.05, 100*01, 99.89, 99434 and 99.93 (mean 99.95); coefficient of variation 0.11 per cent. Temperature of sample ignition 1600" C; sample weight 50 mg; recovery, per cent.99-95, 100-23, 99.70, 99.15, 100.95, 100.04, 99.50, 100.29, 99.05, 99.60, 100.25, 99-27 and 100*08 (mean 99435); coefficient of variation 0-54 per cent. CONCLUSIONS The sodium peroxide - sodium hydroxide fusion technique is a rapid and simple method for the complete dissolution of refractory plutonium dioxide, and it is by far the most rapid method for dissolving high-fired material. The sulphuric acid solution obtained on dissolving the melt is suitable for the direct determination of the plutonium content by controlled- potential coulometry. Although a glove-box is necessary for the fusion and dissolution of the melt, the resultant solution can be transferred into a fume cupboard and the determination completed there because of the small amount of plutonium needed for coulometry. This method represents an improvement and a simplification over the method involving a sodium peroxide sinter followed by differential spectrophotometry. The analysis by this latter method must be carried out entirely in a suite of glove-boxes because of the high concentration of plutonium involved. We thank Mr. D. Wicks for carrying out some of the coulometric determinations. REFERENCES 1. 2. 3. 4. 5. Milner, G. W. C., Crossley, D., Jones, I. G., and Phillips, G., Analyst, 1965, 90, 732. Milner, G. W. C., Wood, A. J., Weldrick, G.. and Phillips, G., Ibid., 1967, 92, 239. Rockett, J . J., U.K. Atomic Energy Authority Report AERE-R 3784, H.M. Stationery Office, Milner, G. W. C., and Edwards, J. W., U.K. Atomic Energy Authority Report AERE-R3772, Phillips, G., and Milner, G. W. C., in Shallis, P. W., Editor. "Proceedings of the SAC Conference, Received February 7th, 1968 London, 1961. H.M. Stationery Office, London, 1961. Nottingham, 1965," W. Heffer and Sons Ltd., Cambridge, 1965, p. 240.

ISSN:0003-2654    

DOI:10.1039/AN9689300429

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Analyst, July, 1968, Vol. 93, jbjb. 433435 433 A Stable, Solid-state, High Voltage Source for Electrode Polar isa tion BY E. BISHOP (Chemistry Department, University of Exeter, Stocker Road, Exeter, Devon) A simple, cheap, versatile, solid-state, high voltage source is proposed as a replacement for high tension battery supplies. It is capable of providing up to 70 pA a t 1000 volts, with drift and noise less than 0.1 per cent., and can be used for electrode polarisation, radiation counting tube supplies and other small-current applications. THE obsolescent high tension radio battery of 120 volts has been commonly used in high voltage sources for the supply of small currents for polarisation of electrodes in techniques such as differential electrolytic p0tentiometry.l The high cost and bulk of, e g ., a 1000-volt source built up from such batteries, and the short shelf life and increasing scarcity of the tapped batteries, have initiated a search for a simple, cheap alternative. A Cockroft - Walton voltage multiplier driven by a square-wave generator2 offered a promising start. SOURCE VOLTAGE AND CURRENT STABILITY- In differential electrolytic pot entiometry and other cons tant-curren t techniques, the electrode potential is a function of current density, and if the potential difference between the electrodes is EA volt, the source voltage is Vs volt, the stabilising resistance in series with source and cell (the ballast resistance) is RB, and the cell resistance plus the internal resistance of the source is Rint, then the current is given by- .... .. .. . . (1). vS - E A I = RB + Rint In a differential electrolytic potentiometric titration, for instance, EA will vary from zero to a maximum value dependent on I and, if the electrode processes are fast, on the Q of the t i t r a t i ~ n . ~ A maximum EA of 100 mV would require a Vs of 100 volts to restrict variation of I to 0.1 per cent., while a maximum EA of 1 volt would require a 1000-volt source for the same stability. RB must be correspondingly large, not only to minimise the effect of any change in Rint, but also to give the proper value of I . Partial differentials emphasise the dependences so, plainly, the larger RB the better. Ballast load (the product Vs x RB) is a significant parameter in differential electrolytic potentiometry, and partial differentiation with respect to this product- shows that the larger Vs and the ballast load the better within the limits of Johnson noise.As EA changes rapidly in the equivalence point region and also changes greatly with current density, inadequate stabilisation leads to erratic and drifting potentials. In view of these requirements it was decided to turn attention to a variable voltage source capable of supplying up to 50pA at 1000 volts. 0 SAC and the author.434 BISHOP: A STABLE, SOLID-STATE, HIGH VOLTAGE [Arta&St, VOl. 93 CIRCUIT PRINCIPLES- The circuit, shown in Fig. 1, consists of an inverter and a voltage multiplier. The inverter is a push - pull amplifier with two driver transformers. The output is fed back via C, to the primary of T, at such a level as to overdrive the transformer, giving a clipped waveform that approaches a fast rise square wave.The output of T, carries a nominal 100-volt peak ax. signal and is isolated, so that consistent earthing of positive or negative battery terminals can be used. The multiplier is a conventional series of voltage doublers, and the number of stages can be reduced or increased if a lower or higher maximum output voltage is required. Any silicon diodes capable of handling 200 peak inverse volts can be used, and the capacitors should be generously rated. Regulation is effected by connecting miniature neons across any suitable taps. As shown in Fig. 1, three 70-volt neons in series are connected across adjacent 200-volt taps, and give a nominal 1000-volt output in steps of 100 volts.Connected over two taps (e.g., -300 to -700) the output would be 500 volts in steps of 50 volts, while a single neon in place of three as in Fig. 1 would give a 350-volt output in steps of 35 volts. The unit consumes less than 500mW and is conveniently powered by small dry batteries, of from 6 to 15 volts; the output voltage will vary with supply voltage. Fig. 1. Circuit diagram of inverter multiplier. T, and T, are Rex LT44, 20KO/lKOCT Details of the obtained from Alpha Radio Ltd. circuit are given in the text Leads: R red, B black, W white, G green. CONSTRUCTION- The circuit can be laid out as in the circuit diagram and occupies about 4 x 2 inches on a piece of drilled paxolin. The board can be mounted in a small box, together with the battery and a ceramic wafer switch connected to the various taps so that ten steps of output voltage can be selected.A small square electrical conduit joint box makes a convenient container, and reduces the effect of temperature variations. The neons serve as indicator lamps, and glow steadily when operating satisfactorily; near overload they flicker, while if too large a current is drawn the lamps are extinguished. PERFORMANCE- For a 12-volt supply a typical maximum output is 70pA at 900 volts. Stability was examined by connecting the output to a ballast resistor and a standard (+O.Ol per cent.)July, 19681 SOURCE FOR ELECTRODE POLARISATION 435 resistor in series, monitoring the voltage drop across the standard resistor by means of an E.I.L. 39A pH meter, and recording the difference from a standard voltage on a Honeywell 513 X17 strip chart recorder.The stability of the output is a function of the stability of the supply to the inverter. With a raw 12 volt d.c. laboratory supply, produced by a TABLE I OUTPUT STABILITY OF 1000-VOLT SOURCE Current, RB, RS, Duration, Drift $lus noise, P A n a minutes per cent. 1 109 IW 100 <0.1 2 5 x 108 1 06 60 0-08 5 2 x 108 106 35 0-04 10 1 08 106 100 0-06 20 5 x 107 1 0 4 120 0.09 0.5* 2 x 109 105 70 2.0 2* 5 x 108 l@ 75 1-5 * Raw 12-volt d.c. supply f 10 per cent., 200 to 400-mV noise. Remainder with battery or regulated d.c. supply. 3-phase transformer rectifier, regulated to 10 per cent. and containing 200 to 400mV of noise, an output stability of 1 to 2 per cent. was obtained. With batteries or a regulated d.c. supply (Solartron AS 1411) regulation of the output was 0.1 per cent. or better, and high frequency noise was of the same order. Typical results are given in Table I. The author thanks Mr. M. Riley who carried out the performance tests. REFERENCES 1. 2. 3. Bishop, E., Analyst, 1968, 83, 212. Bollen, D., Wireless Wodd, 1965, 71, 381. Bishop, E., in Shallis, P. W., Editor, “Proceedings of the SAC Conference, Nottingham, 1965,” Received November 7th. 1967 W. Heffer & Sons Ltd., Cambridge, 1965, p. 291.

ISSN:0003-2654    

DOI:10.1039/AN9689300433

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

436 Analyst, July, 1968, Vol. 93, pp. 436440 A Semi-automatic Timed End-point Karl Fischer Titrator BY B. COPE (“Shell” Research Limited, Carrington Plastics Laboratory, Urmston, Manchester) The construction, operation and evaluation are described of a low-cost instrument for providing semi-automatic analysis of the water content of liquids and gases by means of a Karl Fischer titration. THE Karl Fischer titration is usually carried out manually by adding small volumes of titrant at regular intervals to the titrand until a given current is flowing between the indicating electrodes. If the end-point current is held for a given time, then the titration is complete. However, if the current falls below the given value during this period, then more titrant is added until a permanent end-point is reached.The instrument described does this automatically. DESCRIPTION- The instrument operates by means of a standard Karl Fischer dead-stop indicator circuit,l with a moving-coil relay replacing the micro ammeter. The moving-coil relay operates as a switch, in conjunction with a second relay, to de-activate (at the end-point), and to activate (when water is present) an automatic titrant dispenser, which dispenses a given volume of titrant into the titrand each time it is activated. End-point detector circuit D.C. power unit Burette unit End-point timer and alarm unit t +- MS 2 I + neutral 4 240 V neutral Reaction cell Fig. 1. Semi-automatic Karl Fischer water-content titrimeter circuit diagram In the end-point condition, the given interval of time allowed for the end-point is automatically measured, and an audible alarm energised when this condition is fulfilled.An impulse counter fitted to the dispenser mechanism counts the number of additions of titrant dispensed, from which the amount of water present in the titrand can be calculated. A circuit diagram is given in Fig. 1, the location of the components is shown in Figs. 2 and 3 and the components are listed in the Appendix. 0 SAC and the author.Fig. 2. Location of components, rear view [To face poge 436Fig. 3. Location of components, front view To face page 4371COPE: A SEMI-AUTOMATIC TIMED END-POINT KARL FISCHER TITRATOR 437 END-POINT DETECTOR CIRCUIT- A standard Karl Fischer “dead-stop” circuit is used, except that the micro ammeter normally used to detect the end-point current is replaced by a moving-coil relay, fitted with a “make” contact at 90pA.This relay, RLYl (Fig. l), provides a means of switching the reagent dispenser on and off via the second relay, RLY2, by breaking the electrical supply to the motor-driven syringe. THE D.C. POWER UNIT- 12-volt d.c. for the operation of the relay, RLY2, and the “Bleeptone” alarm, A. The d.c. power unit consists of a transformer, TR1, and rectifier, REC1, to provide THE BURETTE UNIT- The burette unit is made from a Fison’s automatic dispenser with several modifications. ELECTRICAL MODIFICATIONS- The control unit provided with the dispenser has been built into the burette unit as shown in Fig. 1. The microswitch, MS2, has been modified to provide an impulse source for the burette counter (Fig.1). DISPENSER-VALVE MODIFICATIONS- The dispenser valve, as supplied by the manufacturer, is made from polytetrafluoro- ethylene (PTFE) and stainless steel. However, as Karl Fischer reagent attacks the metal portion of the valve, this has been replaced by a PTFE section. PTFE inlet and outlet lines have been fitted to the valve, and full details are shown in Fig. 4. The unit is fitted with l-ml tuberculin syringe, with Luer fitting, and set to deliver about 0.05ml per stroke. n J - d ,PTFE block .Adjusting screw , ,e PTFE nozzle - t PTFE tubing I mm bore 2mm 0.d. Fig. 4. Burette valve construction438 COPE: A SEMI-AUTOMATIC TIMED END-POINT KARL FISCHER TITRATOR [A%&?ySt, VOI. 93 END-POINT TIMER AND ALARM UNIT- The end-point timer and alarm are connected as shown in Fig.1. The timer is auto- matically re-set to the given end-point time each time the burette is activated. If the burette is not activated and the timer allowed to traverse its cycle, then the “Bleeptone” alarm is activated signalling the completion of the titration. OPERATION- the mode of operation of the instrument. The following procedure for the determination of water in methanol is given to illustrate APPARATUS- .Karl Fischer semi-automatic titrimeter. Titration cell (Fig. 5 ) . Syringe, 10 pl. REAGENTS- Karl Fischer reagent (1 ml 2: 5 mg of water). Water. Reagent inlet B I9 joint 6” B 19 joint B 24 joint Nitrogen c-s & outlet Stirrer Plat i nu m electrodes Fig. 6. Karl Fischer titration cell PROCEDURE- Introduce 100 ml of dry methanol into the titration cell, switch on the stirrer and set the variable resistance, R, of the titrimeter at 125 ohms and the end-point timer at 2 minutes.Switch on cell current, S, and burette power supply, S,. Allow the titrator to operate until the “Bleeptone” alarm sounds.July, 19681 CALIBRATION- Set the burette counter at zero and add 5 pl of water to the titrand, by means of the syringe. Allow the titrator to operate until the “Bleeptone” alarm sounds, and note the burette counter reading. SAMPLE ANALYSIS- the titration cell, the burette counter reading (C, counts). COPE : A SEMI-AUTOMATIC TIMED END-POINT KARL FISCHER TITRATOR 439 5 pl of water = 5 mg of water = C counts. Set the burette counter at zero and transfer by pipette a volume of sample (V ml) into Allow the titrator to operate until the “Bleeptone” alarm sounds and note c, x 5 x 100 c x V x 1000‘ Percentage w/v of water in sample = EVALUATION- The instrument was evaluated with the above technique.Volumes of water in the range 1 to lop1 were introduced into the cell, under steady The conditions, and the number of injections of Karl Fischer reagent dispensed recorded. results are shown in Table I. TABLE I EVALUATION OF SEMI-AUTOMATIC KARL FISCHER TITRIMETER Volume of water added, 1 2 3 4 5 6 7 8 10 Pl Number of injections dispensed 6, 5, 6, 6, 6, 5 10, 10, 10 16, 15, 16, 15 21, 21 27, 27, 27, 27, 27, 26, 27, 28, 27, 27 32, 33 37, 37 44, 43, 43 54, 54 The standard deviation of the number of injections is 0.23 injection, i.e., k0.46 injection at 95 per cent. probability.As less than 1 injection is not practicable, the error of a single determination must be 0 or 1 injection. This corresponds to better-than 99.9 per cent. confidence limits. For the Karl Fischer reagent used in this evaluation (1 ml II 5 mg of water), 5-4 injections are equivalent to 1 pl of water and the coefficient of variation (99.9 per cent. + probability), based on an error of + 1 count, is given by- 100 5.4 x w where Wmg is the amount of water present in the titrand. titrand under the conditions quoted. a dilute Karl Fischer reagent should be used. For a 5 per cent. coefficient of variation, at least 4 mg of water should be present in the To determine smaller amounts of water at this accuracy, PERFORMANCE- A determination of the type illustrated takes 5 minutes, during which time the operator is available to carry out other work, such as preparation of samples.The instrument can also be used for the determination of moisture in gases. For example, in the method of Reid and Turner,2 for the determination of water in plastics, the water present in the polymer is vaporised into a nitrogen stream and swept into a Karl Fischer cell. Whereas previously the titration of the water was carried out manually, it may now be carried out automatically. The instrument has been in use in the laboratories of this Company for more than a year, and has given satisfactory service during this period. The cost of components for this instrument is about fl100 and the over-all cost fl200.440 COPE: A SEMI-AUTOMATIC TIMED END-POINT KARL FISCHER TITRATOR [Analyst, Vol.93 Appendix COMPONENTS LIST (Instrument assembly by I.C.A.M. Ltd., Northop, Mold, Flintshire) Item s1 .. .. .. s 2 .. .. .. TI and T2.. .. N1 .. .. .. TRl .. .. RECl .. * . RLY2 .. .. D1 .. .. .. PI .. .. RLYl .. .. PL1 and SOCl . . MS1 andMS2 . . v1 .. .. .. v 2 .. .. .. A .. .. .. Burette unit . . PTFE valve block Counter . . .. Timer . . .. Case .. .. Description Manufacturer Switch SPDT . . .. .. .. SwitchSPDT .. .. .. Insulated terminals . . .. Panel, neon clear, 240-volt . . Transformer, Hygrade, 240-volt 60 cycles 2 X 6*3-~0lt . . .. .. Rectifier Rec 20 . . .. . . Relay, type 1, 12-volt d.c., 120 ohms . . Diode 10 DE type, REC50A . . Potentiometer, Model A, 10 turns, 500 ohms and Duo-Dial Model RB .. Beckman, Glenrothes, Scotland S170 d.c. relay make at 90 PA, resistance 3300 ohms, Specification S170/1/457 . . 6-Pin plug and socket, Part No. P194 . . Microswitch, type HA1 . . . . . . Crouzet Ltd., Brentford, Middlesex .. .. :I . . Radiospares Limited, London, W. 1 Sangamo Weston, Enfield, Middlesex A. F. Bulgin, Barking, Essex - 1.Ei-Volt d.c. battery . . .. .. 240-Volt a.c. supply . . . . .. - Audible alarm, “Bleeptone”, 12-volt d.c. Fisons automatic dispenser includes It1 4700-ohm resistor and C1 0.4-pF con- denser . . .. .. . . . . Fisons Ltd., Loughborough, Leicester- I.C.A.M. Ltd., Northop, Mold, Flintshire Veeder-Root, Croydon, Surrey A. P. Besson Ltd., Hove, Sussex shire - Re-set vending counter, Part No. KK1441 Chronoset CF, 0-36 minutes direct-clutch model . . .. .. .. . . Technical Representations Ltd., Stock- port, Cheshire Type DA 40168 . . .. . . . . Bedco Ltd., Harpenden, Herts. REFERENCES 1. 2. Vogel, A. I., “A Text-book of Quantitative Inorganic Analysis, including Elementary Instrumental Reid, V. W., and Turner, L., Analyst, 1961, 86, 36. Received December 22nd, 1967 Analysis,’’ Third Edition, Longmans, Green & Co. Ltd., London, 1961, p. 945.

ISSN:0003-2654    

DOI:10.1039/AN9689300436

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Analyst, July, 1968, Vol. 93, $9. 441-442 441 The Rapid Determination of Tungsten in Ores by X-ray Fluorescence Analysis BY K. G. CARR-BRION AND K. W. PAYNE ( Warren Spring Laboratory, Stevenage, Herts.) An X-ray fluorescence powder method involving the use of tungsten Ka radiation enables tungsten to be rapidly determined in ores of different mineralogical composition. Good agreement with chemical assays is obtained. WHEN tungsten is determined in powdered ores by X-ray fluorescence analysis, marked heterogeneity effects1 can occur with the particle sizes produced by normal grinding methods. These effects are caused by tungsten being found as two minerals with markedly different X-ray absorption coefficients in the tungsten L wavelength region. The minerals are scheelite (CaWO,) and wolframite (Fe,Mn)WO,.The difference in tungsten La intensity per unit concentration from scheelite to wolframite containing samples was found to be about 20 per cent. in the middle of the concentration range examined. If the ratio of the two minerals is known and remains effectively constant, analyses can still be carried out with tungsten L radiation by using mineral powder standards. However, if the ratio of the two minerals varies, considerable errors can result. These effects can be overcome by fusion or minimised by ultra-fine grinding. Fusion is time consuming and may prove difficult with some ores, for instance, those containing large amounts of arsenic. The fineness of grinding required to eliminate these effects is beyond the range of rapid conventional grinding equip- ment : the samples examined had already been ground to less than 300 mesh in a “swing mill.” A third method of overcoming the effect is to use tungsten Ka radiation for the determination.The much lower X-ray absorption coefficients encountered enable the determination to be made directly on the powdered ores without measurable heterogeneity effects. The use of hafnium as an internal standard guards against any matrix effects with both the tungsten L and K radiations. EXPERIMENTAL INSTRUMENTAL CONDITIONS- A Phillips P.W. 1212 X-ray spectrometer, equipped with a gold tube operating at 100 kV, 20 mA and a lithium fluoride 220 analysing crystal was used: 28 angles used were tungsten Kq,, 8-39’, hafnium‘ Kcc,,, 8.92’ and background 8.09’: counting times for each position, 30 seconds (in two increments of 15 seconds).SAMPLE PREPARATION- Samples were ground to less than 300 mesh in a “swing mill.” Those expected to contain more than 5 per cent. of tungsten were diluted 1 + 7 w/w with a potassium sulphate buffer powder containing 5 per cent. of hafnium dioxide; those expected to contain less than 5 per cent. of tungsten were diluted 1 + 1 with the buffer. Two-gram buffered samples were examined directly by hand tamping in the sample holders. RESULTS Some typical results obtained are shown in Table I. TABLE I EXAMINATION OF ANALYSED SAMPLES Tungsten found by X-ray method, Sample per cent. 6516 53.1 5543 43.5 5544 16.3 6371 2-07 8344 0.84 4618 0.52 0 SAC; Controller, H.M. Stationery Office. Accepted tungsten concentration, per cent.52.5 42.8 15.8 1.92 0.73 0.5 1442 CARR-BRION AND PAYNE REPRODUCIBILITY AND ACCURACY- The reproducibility obtained on successive sampling was better than 1 per cent. relative. The error relative to the composition determined by standard chemical methods was 1.8 per cent. for ten samples in the concentration range 16 to 60 per cent. of tungsten, and 6 per cent. for six samples in the concentration range 0.5 to 2 per cent. The exact mineralogical com- position of these samples was unknown, but wolframite and scheelite standards gave equal intensities per unit concentration. Few samples were examined in the intermediate range. CONCLUSIONS The proposed method enables tungsten in ores of widely varying mineralogical com- position to be determined rapidly. The concentration range covered is between 0.5 and 60 per cent. of tungsten. The sensitivity is limited by the relatively poor signal-to-background ratio found in the tungsten K region of the spectrum. The use of tungsten L radiation would give a much lower limit of determination, but would require the use of a fusion technique to eliminate heterogeneity effects. REFERENCE 1. Claisse, F., and Samson, C., Adv. X-ray Analysis, 1962, 5, 335. Received December 20tk, 1967

ISSN:0003-2654    

DOI:10.1039/AN9689300441

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Autalyst, July, 1968, Vol. 93, pp. 443444 443 A Rapid Method for the Determination of Malathion in Wheat Grains BY E. WEISENBERG, S. GERTNER AND J. SCHOENBERG (Institute of Control and Standardisation of Drugs, Ministry of Health, Jerusalem, Israel) A simple method for the determination of malathion in wheat grains is described. The insecticide was extracted with chloroform and the extract treated with a Celite - Nuchar - sodium sulphate mixture to absorb impur- ities selectively. The hydrolysis was carried out with ethanolic sodium hydroxide solution and the copper complex extracted into cyclohexane. The method is applied to amounts of 50 to 250 pg. THE use of malathion, which has been widely used for the control of pests in stored grains, has been based on its relatively strong pesticidal qualities and its low toxicity to mammals.The method described by Norris, Easter, Fuller and Kuchar,l based on the alkaline decom- position of malathion, has been recommended for the determination of residues in cereals and oil seeds.2 Bates, Rowlands and Harris3 used chromatography on Fuller’s earth for the purification of the plant extracts before analysis. In a later publication, Bates and Rowland9 found that acceptable recoveries were being obtained without the preliminary use of an absorbent column. Uphams reported a modification of this method for the determination of malathion in formulations, in which carbon tetrachloride was replaced by cyclohexane, and the colour stability was found to have improved. In an attempt to improve the accuracy and precision of the method, a critical study was made by Orloski.6 We adapted and modified this method in order to determine amounts as low as 50 pg.Chloroform was selected as the extracting solvent; according to Upham,s the descending order of solvent power for malathion was found to be chloroform, carbon tetrachloride and carbon disulphide. As a preliminary purification of the chloroform extract was found necessary, we effected selective absorption of the impurities by Celite - Nuchar - sodium sulphate as recommended by Koivistoinen, Karinpaa, Kononen and Roine.7 This treatment eliminated the need for repeated washings of the plant extract, which were found to be criticaL2 REAGENTS- METHOD Chloroform, B.P. quality. Acetonitrile, analytical-reagent grade.Cyclohexane, analytical-reagent gmde. Ethanolic sodium hydroxide solution, about 0.5 N-Dissolve 1 g of analytical-reagent grade Iron(II1) chloride - hydrochloric acid solutiow---Dissolve 0.2 g of iron(II1) chloride in Copper sulphate solution, 1 per cent. w/v. Absorbent for clean-up-This consisted of 1 part of Celite 545 (Johns-Manville) , plus 2 parts of Nuchar C-190-N (West Virginia Pulp and Paper Co., Covington, Virginia) plus 1 part of anhydrous analytical-reagent grade sodium sulphate. Ethanol, absolute, analytical-reagent grade. PROCEDURE- Shake mechanically 150 mg of coarsely ground wheat grains with 300 ml of chloroform for 24 hours. Filter the mixture through a Buchner funnel and wash the residue in the funnel with three 50-ml portions of chloroform.Evaporate the extract and washings to 200 rnl in a Rinco vacuum evaporator. Transfer a 100-ml aliquot into a 250-ml Erlenmeyer flask, add 4 g of the Celite - Nuchar - sodium sulphate mixture and shake it for 5 minutes. sodium hydroxide in 50ml of absolute ethanol by heating under reflux. 8 ml of hydrochloric acid (sp. gr. 1-18) and make up to 1 litre with water. 0 SAC and the authors.444 WEISENBERG, GERTNER AND SCHOENBERG Filter through paper, wash the first flask and the residue on the filter six times with 10-ml portions of chloroform and add these washings to the filtrate, then evaporate the filtrate in a vacuum evaporator to about 10 ml. Transfer it to a small beaker, evaporate to dryness with a current of hot air and dissolve the residue in 8 ml of ethanol; transfer to a 125-ml separating funnel and wash the beaker with 5 ml of cyclohexane and add to the ethanol.To the contents of the separating funnel add 0.2 ml of acetonitrile and 1 ml of ethanolic sodium hydroxide solution; swirl the funnel gently (do not shake) for 5 to 10 seconds and let it stand for 2 minutes. Add 25 ml of iron(II1) chloride solution (cooled to 10” C), mix well by swirling it for 10 seconds and let it stand for 5 minutes to allow the phases to separate; discard the cyclohexane. Add exactly 10 ml of cyclohexane and 1 ml of copper sulphate solu- tion and shake the separating funnel immediately for 1 minute. Allow the phases to separate and, as soon as separation occurs, discard the aqueous phase and filter the cyclohexane solution through a small filter containing 0.1 g of anhydrous sodium sulphate.Measure the optical density of the yellow solution, within 15 minutes from the beginning of the hydrolysis, at 420 mp in a cell of l-cm path length. Prepare a standard graph from pure malathion to cover the range of 50 to 250pg of malathion. The line has a slope of 1.48 optical density units for 1 mg of malathion. RESULTS AND DISCUSSION In the samples of wheat grains analysed the malathion content was found to vary The reliability of the method was studied by recovery tests performed on an untreated from 0 to 4 p.p.m. sample, and the results are presented in Table I. TABLE I RECOVERY OF MALATHION FROM WHEAT GRAINS Amount added, p.p.m. Amount found, p.p.m. Recovery, per cent. 1.04 0.96 92 2-05 2.03 97 4.16 3.96 95 6.24 5-73 92 8.67 8.33 96 From these results we found that satisfactory recoveries were obtained.We found that the hydrolysis with ethanolic sodium hydroxide in the presence of cyclo- hexane was advantageous. The last traces of coloured impurities remained in the cyclohexane layer after the addition of the iron(II1) chloride solution, and the aqueous solution remained colourless and clear. The stability of the copper complex in cyclohexane has been found satisfactory. It is possible that the stability of the colour is connected with the fact that in our method a clear solution free from impurities was obtained directly after the hydrolysis. The calibration graph was found to be reproducible and, therefore, it was unnecessary to run daily standards. It is proposed to study the applicability of the method to a range of foodstuffs in which malathion residues may be present. We gratefully acknowledge the contribution made by Mrs. S. Gershson, in adapting the Orloski method for the determination of micro amounts of malathion. REFERENCES 1. 2. 3. 4. 5. 6. Orloski, E. J., Ibid., 1964, 47, 248. 7. Norris, M. V., Easter, E. W., Fuller, L. T., and Kuchar, E. J., J . Agric. Fd Chem., 1958, 6, 111. Analytical Methods Committee, “The Determination of Malathion Residues in Cereals and Oil- Bates, A. N., Rowlands, D. G., and Harris, A. H., Ibid., 1962, 87, 643. Bates, A. N., and Rowlands, D. G., Ibid., 1964, 89, 286. Upham, S. D., J . Ass. Off. Agric. Chem., 1960, 43, 360. Koivistoinen, P., Karinpaa, A., Kononen, M., and Robe, P., J . Agric. Fd Chem., 1964, 12, 561. Received December 6th, 1967 seeds,” Analyst, 1960, 85, 915.

ISSN:0003-2654    

DOI:10.1039/AN9689300443

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Analjst, July, 1968, Vol. 93, $9. 445452 446 A One-step Extraction and Clean-up Procedure before Gas = Liquid Chromatographic Determination of Some Organochlorine Pesticide Residues in Blood BY G. CZEGLI?DI-JANKO* AND V. CIELESZKY (Institute of Nutrition, Budapest I X , Gydli ut 3/a, Hungary) An apparatus and a method are described for the extraction and clean-up of organochlorine pesticides, e.g., DDT, DDE, a- and y-BHC (lindane) and dieldrin, from heparin-treated and lyophilised blood samples for gas-chromato- graphic determinations. Extraction and clean-up are camed out in one step on a suitable column with amounts of solvent as small as 25 to 30 ml. Two types of column are used according to the type of organochlorine pesticide to be determined; for acid-stable substances a sulphuric acid - diatomaceous earth column is used, and for alkali-stable pesticides an alkaline column, on which saponifi- cation of fat takes place simultaneously with extraction.The method has been applied successfully in worker as well as in general population surveys. THE accumulation and storage of organochlorine pesticides, particularly DDT, in the human organism is well established.l~2s3s4 Investigation of this phenomenon, however, has been carried out mainly on human fatty tissue and milk samples. Methods for the identification and determination of organochlorine residues are exten- sively reviewed by Beynon and Elgar.6 In general, extraction and clean-up of the active agent are carried out separately. A one-step procedure for the two operations on a conditioned Florisil column was recently described by Langlois, Stemp and Liska.6 Experimental results recording the occurrence and level of organochlorine residues in human blood are scarce.Moreover, the few investigations of this kind that have been carried out were concerned primarily with alkali-stable active agents in relation to occupationally exposed p e ~ p l e . ~ . ~ However, DDT and DDE could not be determined separately by the methods used in these investigations. For the assessment of organochlorine pesticides in the blood of experimental animals, a method was published by Jain, Fontan and Kirk,g and hexane-extractable organochlorine insecticides in human-blood samples were determined by Dale, Curley and Cueto.lo In these tests, as well as in the experiments of Radomski and Fiserova-Bergerova,ll no clean-up was used before analysis. For the determination of organochlorine pesticide residues in blood by gas - liquid chromatography we tried to develop a procedure with which substantially lower pesticide levels in blood originating from nutritional intake could be assessed, not only in occupationally exposed people but also in the general population. For this purpose, efficient clean-up of the blood extracts seemed necessary, particularly as the sensitivity of the tritium-foil electron- capture detector, which was also used in our investigations, becomes seriously impaired by lipid co-extractives present in blood extracts.12 The present paper describes an apparatus and a method by which the extraction of small amounts of organochlorine (DDT, DDE, E- and y-BHC and dieldrin) residues in heparin- treated and lyophilised human-blood samples, and the clean-up of the extract, can be achieved in a single step by using only 25 to 30 ml of solvent.The resulting extract is suitable for gas - liquid chromatographic analysis. The addition of the anti-coagulant to the blood samples became necessary because in this method, even when only incipient clotting occurs, the extractability of the organo- chlorine pesticides considerably decreases and, generally, these compounds cannot be extracted at all from clotted blood. Lyophilisation in pesticide residue analysis has been recently mentioned by Branden- berger and Miiller.13 Also, in the course of other investigations in this Institute, good results * Present address : State Institute of Hygiene, Department of Disinfection, Budapest IX, GyAli ut 2-4, Hungary.0 SAC and the authors.446 [Analyst, Vol. 93 have been obtained by using lyophilised samples in various types of analysis. The procedure had already been successfully applied to the investigation of organochlorine residues in foods, as well as to the determination of other agents, e.g., phosphate esters. EXPERIMENTAL REAGENTS- CZEGL~DI- J A N K ~ AND CIELESZKY : GAS-CHROMATOGRAPHIC All materials should be of analytical-reagent grade, unless otherwise stated. Florisil, 60 to 100 mesh-This was washed with light petroleum, air-dried and condi- tioned by the method of Langlois, Stemp and Liska.6 It was kept for 10 to 12 hours at 140" C, then mixed with 5 per cent. of water; it may be stored for 1 week in a glass-stoppered bottle.Sodium sulphate, anhydrous. Diatomaceous earth , commercial grade. Sulphuric acid mixture-Prepare by mixing equal volumes of concentrated sulphuric Potassium hydroxide. Alumina (Merck)-This was kept at 450" C for 3 hours and then mixed with 10 per cent. Sodium chloride. Heparin solution. Saline (sodium chloride) solution, 0.9 per cent. Hexane, re-distilled, boiling-range 62" to 64" C. Light petroleum, re-distilled, boiling-range 32" to 37" C. Methylene chloride, re-distilled, boiling-point 42" C. Benzene, re-distilled, boiling-range 80" to 81" C. Olive oil. acid and fuming sulphuric acid containing 20 per cent. of sulphur trioxide. of water. LYOPHILISATION OF SAMPLE- Freshly drawn blood (2 to 10 ml), depending on the expected amount of pesticide, is placed into a glass tube containing 1 drop of heparin solution.By centrifuging the blood sample, plasma and red blood cells can be examined separately. For this purpose the cellular components must be washed thoroughly with saline and the washings added to the plasma. After mixing the sample with 50 per cent. of air-dried Florisil, lyophilisation occurs. In our experiments, an apparatus (supplied by Labor Co., Budapest, Hungary) operated by an oil vacuum pump was used, the schematic representation of which can be seen in Fig. 1. Hg vacuum A = Flask B = Receivers C = Freezing mixture at -70" C Fig. 1. Schematic representation of the apparatus for the lyophilisation of blood samples The Florisil- blood mixture is spread on the inner wall of flask A so that a uniform layer is formed.The flask is then immersed in a freezing mixture consisting of solid carbon dioxide and light petroleum until the layer becomes frozen. During this procedure a thin layer of ice is formed on the outer wall of the flask. For subsequent lyophilisation, flask AJuly, 19681 DETERMINATION OF ORGANOCHLORINE PESTICIDE RESIDUES IN BLOOD 447 is placed into the apparatus and the vacuum pump started. Vapours of sublimating ice are condensed in receiver B, which is cooled to -70" C by the freezing mixture, and thus do not reach the vacuum pump. Lyophilisation is completed when the layer of ice on the outer wall of flask A melts away. The water-free layer adhering to the inner wall of the flask is now scraped off and pulverised.mm of mercury. Under these con- ditions, no organochlorine residues, except lindane (see Table I), could be extracted in recovery experiments by methylene chloride (which is used generally in this laboratory in water analysis14), in determinable amounts from the ice accumulated in the receiver after thawing, indicating that DDT, DDE and dieldrin do not escape from blood during lyophilisa- tion. In some instances, however, when a higher vacuum is needed, it may become essential to re-check the lyophilisation procedure for eventual loss of pesticide residues. EXTRACTION AND CLEAN-UP OF SAMPLE- adsorbent (Fig. 3). The vacuum used in this experiment was 5 x For extraction and clean-up, column A, in the apparatus shown in Fig.2, is filled with D A = Column containing adsorbent B = Flak Fig. 2. C = Condenser D =ilnsulated tube Apparatus for extraction of organochlorine pesticides and clean-up of the extract With acid-stable active agents, such as DDT and its metabolite DDE, or BHC, a modified Davidow column15 (Fig. 3-Acid) can be used, and with alkali-resistant dieldrin an alkaline column (Fig. 3-Alkaline), developed by the authors, on which saponification of fat takes place simultaneously with extraction. An alkaline column of different composition has been described by Albert .16 In the apparatus shown in Fig. 2 a small amount of light petroleum, containing 20 per cent. of methylene chloride for dieldrin, circulates through the lyophilised sample placed on top of the adsorbent in column A.The amount of solvent used (25 to 30 ml) should be such that, after percolating through the column, about 6 to 8ml would accumulate in flask B. Water is then circulated in condenser C. The water jacket around column A, and condenser C (Fig. 2), prevents the vapour pressure of the low-boiling solvent from disrupting the column, which would counteract the continuous circulation of the solvent. The solvent is made to circulate by gently warming flask B on a suitably regulated water-bath. The solvent vapours enter through insulated tube D into condenser C, where condensation occurs, and the con- densed solvent again percolates through the sample and column. The warming of flask B is regulated so that above the sample there should always be a solvent layer of 0.5 to 1.0 cm.With correct column filling the solvent flow-rate is about 3 ml per minute.448 AND CIELESZKY : GAS-CHROMATOGRAPHIC [A ?Za&St, VOl. 93 Acid Alkaline Fig. 3. Acid and alkaline columns: A, lyophilised blood sample; B, sulphuric acid - diatomaceous earth mixture (3.5 ml of fuming sulphuric acid and 4 g of diatomaceous earth) ; C, 3 g of pulverised potassium hydroxide, wetted with methylene chloride saturated with water; D, 0.6 g of diatomaceous earth; E, 0.5 g of alumina; F, 3 g of Florisil, conditioned; G, 0.5 g of sodium sulphate; H, glass-wool After 3 hours' circulation the contents of flask B are transferred into a conical centrifuge tube aad the solvent evaporated. The residue is dissolved in 0.25 to 1.0 ml of hexane and is ready for gas - liquid chromatographic analysis.With alkaline column filling the extract in flask B is transferred into a separating funnel, washed twice with water and dried over anhydrous sodium sulphate. GAS - LIQUID CHROMATOGRAPHIC ANALYSIS OF PURIFIED EXTRACTS- A Perkin-Elmer gas chromatograph, Model 452, was used, and a Pyrex-glass column, 2 feet long and $-inch diameter, filled.with Chromosorb W, 60 to 100 mesh, with 2-5 per cent. of Apiezon L and 0.75 per cent. of Epikote resin 1001. The conditions used were: carrier gas, nitrogen; inlet pressure, 1.4 kg per cm2; flow-rate, 200 ml per minute; column temperature, 170" C; injection temperature, about 230" C; detector, electron capture and applied potential, 45 volts; amplifier gain, &th to Ath; and injection syringe, Hamilton microlitre syringe, 701-N.In experiments reported here, 10-pl aliquots of the purified and dissolved extract were injected into the gas chromatograph and chromatograms obtained by selecting the appropriate amplifier gain according to active-agent content. Insecticides were identified by comparing their retention times with those of compounds used for reference.* Thin-layer chromatography, as described by K o v ~ c s , ~ ~ was used as a complementary technique. The quantitative determination of the pesticide was carried out by multiplying the peak height in the chromatogram by the peak width at half the peak height. Calibration graphs were prepared by injecting 1 to 10 p1 of the standard solutions into the gas chromatograph containing 0.5 to 1.0 pg of pure pesticide per ml of solvent.Amounts of blood extracts and reference substances must be chosen so that comparative measurements can be carried out with the same amplifier gain. For DDT, with both test and reference substances, the ratio of the peak heights of DDT, and of DDD originating from a minor decomposition of DDT under the working conditions already described, must be considered. * All organochlorine compounds used for reference were recrystallised several times and controlled by their melting-points.July, 19681 DETERMINATION OF ORGANOCHLORINE PESTICIDE RESIDUES IN BLOOD 449 RECOVERY FROM COLUMNS- The recovery of known amounts of organochlorine pesticides in pure solution from the columns described has been checked. It was found that on both types of column, acidic as well as alkaline, 94 to 96 per cent.of the active agents added in amounts of 0.05 pg (equiva- lent to 0-01 p.p.m. of agent in 5ml of blood) was recovered after 10 minutes’ circulation. After 20 minutes the recovery was 98 to 100 per cent. EXTRACTION TIME- To determine the extraction time necessary to obtain the maximum of extractable residual material, extraction times of 1 to 4 hours were carried out. It was found that whereas 2 hours were insufficient for optimum results, the highest residue yields could be achieved with certainty in 3 hours. With the solvent flow-rate already described, extraction for 3 hours with only 25 to 30 ml of solvent corresponds to a conventional extraction carried out with 550 to 600 ml of solvent.RECOVERY OF ADDED PESTICIDES- Because of lyophilisation, it was necessary to devise special techniques of adding various known amounts of pesticides to blood. The reference substances could be added to blood only in those solvents that do not interfere with the freezing of the Florisil- blood mixture before lyophilisation. In addition, the complete incorporation of the added material into the sample was necessary to avoid. substantial evaporation under vacuum. All of these requirements could be met by dissolving the reference substances in benzene and by mixing known portions of the benzene solution with olive oil containing (from previous experiments) no appreciable amounts of organochlorine residues. One microgram of each pesticide, viz., DDT, DDE, lindane and dieldrin, dissolved in 1 ml of benzene containing 5 drops of olive oil, was added to 10ml of blood (equivalent to 0.1 p.p.m.of pesticide in the blood). The mixture was vigorously shaken and then allowed to stand for a few hours, during which period the shaking was frequently repeated. Subse- quently, an amount of Florisil equal to 50 per cent. w/w of the blood sample was added and the mixture lyophilised. Recoveries are shown in Table I. TABLE I RECOVERY OF ORGANOCHLORINE PESTICIDES ADDED TO BLOOD Amount of pesticide in blood after adding 0.1 p.p.m. of reference Amount of pesticide Pesticide p.p.m. p.p.m. 0.105 DDT .. .. .. 0.0 104 0.107 0.105 0.119 DDE .. .. .. 0.0172 0.1 16 0-113 0.025 Lindane .. .. .. 0-0061 0.025 0.017 0.094 Dieldrin . . .. ..<0-0001 0.097 0.098 in blood, substance, Recovery, per cent. 96.2 97.1 94.7 102.0 98.8 96-3 20.0 20.5 11.9 94.5 97-2 98-3 It is noted that although the recovery of added DDT, DDE and dieldrin was almost complete, that of lindane was only partial. This might arise from the considerable solubility in water and volatility of lindane, in consequence of which most of this substance, when added to blood samples, escapes during lyophilisation and can be recovered in receiver B. Of the lindane originally present, however, no trace could be detected in the receiver, and we found no explanation for this, but the assumption of Dale, Curley and Hayes,ls according to which organochlorine pesticide residues in blood are probably bound to proteins, deserves attention. Therefore, the results must be evaluated with respect to recovery experiments.450 CZEGL~DI- J A N K ~ AND CIELESZKY: GAS-CHROMATOGRAPHIC [Analyst, Vol.93 RESULTS AND DISCUSSION With the apparatus described, extraction of organochlorine pesticides (DDT, DDE, dieldrin, lindane and a-BHC) from blood, and purification of the extract before gas - liquid chromatographic examination can be performed in one single step with a minimum of solvent. The clean-up of extracts containing acid-stable or alkali-stable pesticides is carried out by using columns with different fillings. With an alkaline column, a partial decomposition of methylene chloride present in the solvent mixture can occur. Our experiments, however, proved that neither did interfering peaks appear on the chromatogram, nor did interference with quantitative extraction or recovery of added reference substance occur as a consequence of this decomposition. Under these experimental conditions, clear-cut peaks appeared in the course of gas - liquid chromatographic determinations separately carried out with DDE, dieldrin, lindane and a - B H C , indicating that no decomposition occurred.DDT, however, became slightly decomposed to DDD in the gas - liquid chromatographic procedure, thus giving two peaks. Identification of the small DDD peak was achieved by using DDD as a reference sub- stance. When establishing the DDT calibration graph, the peak area of DDT and the small one of the DDT breakdown product were considered. In measurements of the peak areas of the sample, DDT and DDD were compared with corresponding peak areas in the chromato- gram of pure DDT solutions used for the preparation of the calibration graph. The decomposition of DDT depends also on the condition of the gas-chromatographic column.With a freshly filled column increased decomposition was observed, and the rate of decomposition is also affected by the absolute amount of DDT present, for instance, a larger proportion of 1 ng than of 10 to 15 ng is decomposed. Therefore, comparison of sample and reference DDT must be made as far as possible with similar amounts, with the same amplifier gain and also by preparing sample and reference chromatograms in rapid succession. In Fig. 4 the gas chromatogram of the acid-treated blood extract of a healthy person, with only the usual environmental exposure to insecticides, is shown, together with the thin-layer chromatogram of the same extract and of reference compounds. Although the presence of #I-BHC is revealed by the thin-layer chromatographic plate, the peak of this compound cannot be identified in the gas chromatogram, because, under the conditions used for these measurements, the peak of P-BHC and other unidentified peaks partially overlap.It is noteworthy that the small, unidentified peak after the DDE peak can be seen only in the chromatogram of acid-treated blood extracts and was not noticed, for instance, in fat extracts. In the chromatogram of blood samples other peaks also appear, the identities of which are not yet established. Fig. 5 shows the gas and thin-layer chromatograms of the blood extract of a worker who was employed on a plant that produced aldrin-treated fertiliser.The blood extract was purified by an alkaline column before gas - liquid chromatography, and the thin-layer chromatogram clearly shows two spots corresponding to dieldrin and DDE, the latter including also DDT converted into DDE by the alkaline treatment. Dieldrin is a metabolite of aldrin formed in the organism. The method has also been used in general population surveys for the determination of the DDT, DDE, a- and y-BHC (lindane) and dieldrin contents of blood, and in monitoring the dieldrin concentration of the blood of workers engaged in the production of aldrin-treated fertilisers. TABLE I1 ORGANOCHLORINE PESTICIDE RESIDUE LEVELS IN THE BLOOD OF THE GENERAL Results are expressed in p.p.m.number DDT DDE a-BHC (lindane) Dieldrin POPULATION IN BUDAPEST, HUNGARY, 1967 Serial y-BHC 1 0.0 104 0.0172 0.0004 0-0051 <0~0001 2 0.0085 0.0061 0*0007 0-0063 <0-0001 3 0.0081 0.0052 0.0003 0.0041 < 0.000 1 4 0.0124 0.0462 0~0002 0-0032 <0-0001 5 0.0255 0.0253 0~0001 0.0035 t0~0001July, 19681 DETERMINATION OF ORGANOCHLORINE PESTICIDE RESIDUES IN BLOOD 451 Table I1 shows results from material continuously collected by us from among the general population; the presence of a considerable amount of lindane is remarkable. It is pointed out by Hayesls that the presence of lindane in blood is generally a consequence of some unusual exposure to this substance. Our findings, however, show that with the method 0 oDDE ODieldrii. 0 0 DDE (a) 0 0 DDT 0 c a - B H C 0 0 O y - B H C B - B H C DDT DDE DDD a 7 Blood Start -extract X DE Fig.4. (a) Thin-layer chromatogram and (b) gas chromatogram of an acid-treated blood extract (general population blood sample). X = unidentified peak. On gas chromatogram, section A was recorded a t 30 inches per hour, with an amplifier gain of &th and section B a t 15 inches per hour with an amplifier gain of &th. The diagram is reduced to one third of the original size Dieldrin 1 X 1 I Fig. 5. (a) Thin-layer chromatogram and (b) gas chromatogram of an alkali-treated blood extract (aldrin-exposed worker's blood), X = unidentified peak. The gas chromatogram was recorded a t 15 inches per hour, with an amplifier gain of h t h . The diagram is reduced to one third of the original size452 CZEGL~DI- J A N K ~ AND CIELESZKY described above, 0.01 p.p.m.of lindane can be detected regularly in the blood of the general population at present in Hungary. In Table I11 some representative results are shown, as selected from material collected among workers on a plant producing aldrin-treated fertiliser, corresponding to two different levels of exposure. The first three values relate to transport personnel working in the store- house of the plant, the other three to workers operating the mixing machine inside the plant. It can be seen that the difference between heavy and moderate exposures also reveals itself in the dieldrin levels in blood. TABLE I11 DIELDRIN LEVELS IN THE BLOOD OF WORKERS EMPLOYED IN A PLANT PRODUCING ALDRIN-TREATED FERTILISER Serial number Dieldrin in blood, p.p.m.0.017 1 Transport workers 0.024 0.020 0.195 5 Workers in the mixing room 0.103 6 0-123 In Table IV some results are presented relating to the distribution of DDT and DDE in plasma and erythrocytes in the blood of the general population. TABLE IV DDT AND DDE CONTENTS IN PLASMA AND RED BLOOD CELLS OF PEOPLE NOT OCCUPATIONALLY EXPOSED TO PESTICIDES, BUDAPEST, 1966 I In plasma p.p.m.* Serial & number DDT DDE 1 0.005 0-016 2 0.008 0.026 3 0.008 0.039 4 0.007 0.030 5 0.003 0.018 2 3 In whole blood, as calculated from 1 and 2, In red blood cells, p.p.m.* p.p.m. & - DDT DDE DDT DDE 0.002 0.007 0-007 0.023 0.004 0.009 0.012 0.034 0.003 0.011 0.011 0.050 0.003 0,016 0.010 0,046 0.003 0-007 0.006 0.025 * Calculated to whole blood.4 In whole blood, as determined, p.p.m. DDT DDE 0.006 0.021 0.010 0.032 0.010 0.046 0.009 0-051 0-006 0.028 The results reported in this paper show the suitability of the method for the purposes outlined above. Results and evaluation of our monitoring work will be published elsewhere. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. REFERENCES Quinby, G. E., Hayes, W. J., Armstrong, J. F., and Durham, W. F., J . Amer. Med. Ass., 1965, Maier-Bode, H., Medna Exp., 1960, 1, 146. DCnes, A., Die Nahrung, 1962, 6, 48. Robinson, J., Richardson, A., Hunter, C. G., Crabtree, A. N., and Rees, H. J., Brit. J . Ind. Med., Beynon, K. I., and Elgar, K. E., Analyst, 1966, 91, 143. Langlois, B. E., Stemp, A. R., and Liska, B. J.. J . Agric. Fd Chem., 1964, 12, 243. Brown, V. K. H., Hunter, C. G., and Richardson, A., Brit. J . Ind. Med., 1964, 21, 283. Richardson, A.. Robinson, J.. Bush, B., and Davies, J. M., Archs Enuir. Hlth, 1967, 14, 703. Jain, N. C., Fontan, C. R., and Kirk, P. L., J . Pharm. Pharmac., 1965, 17, 362. Dale, W. E., Curley, A., and Cueto, C . , jun., Life Sciences, 1966, 5, 47. Radomski, J. L., and Fiserova-Bergerova, V., Ind. Med. Surg., 1965, 34, 934. McCully, K. A., and McKinley, W. P., J . Ass. Off. Agric. Chem., 1964, 47, 652. Brandenberger, H.. and Muller, S., Mit. Geb. Lebensmittelunters. u. Hyg., 1965, 56, 281. Cieleszky, V., Egdszsbgtudomdny, 1967, 11 , 93. Davidow, B. J., J . Ass. Ofl. Agric. Chem., 1950, 33, 130. Albert, R. A., Ibid., 1964, 47, 659. Kovacs, M. F., Ibid., 1963, 46, 884. Dale, W. E., Curley, A., and Hayes, W. J., jun., Ind. Med. Surg., 1967, 36, 275. Hayes, W. J., jun., “Scientific Aspects of Pest Control,” Publication No. 1402. National Academy First received November 17th, 1966 Amended January 26th, 1968 191, 175. 1965, 22, 220. of Sciences, National Research Council, Washington, D.C., 1966, p. 314.

ISSN:0003-2654    

DOI:10.1039/AN9689300445

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

Analyst, July, 1968, Vol. 93, Pfi. 453455 453 Determination of the Number of Oxygen Substituents of Steroids by Chromatography BY D. J. H. TRAFFORD AND R. W. H. EDWARDS (Institute of Child Health, University of London, 30 Guilford Street, London, W.C. 1 ) ARm values obtained by difference of Rm values in the presence and absence of formaldehyde are shown to group in a manner determined by the number of polar functional groups and, to a lesser degree, by the nature of the steroid skeleton. It is proposed that determination of the AR, value provides a means of characterising steroids from natural sources. PARTITION chromatography gives ARm values that are constant for each substituting func- tional group in a particular family of solvent mixtures (reviewed by Bush1 and Edwards2). The present observations stem from the use of formaldehyde in the stationary phase in an attempt to cause strong association with hydroxylic and ketonic substituents, and thus to alter the AR, values.Paper chromatography of steroids with mixtures of aqueous formaldehyde, light petro- leum and benzene gave excessive tailing of the chromatographed spots. Incorporation of methanol eliminated tailing, and the mixture effectively became a modification of the Bush3 system. EXPERIMENTAL The general apparatus, methods, reagents and details of steroid chromatography are described elsewhere2 and only special points will be elaborated. REFERENCE STEROIDS- These were generally purchased from Steraloids Ltd., Croydon, Surrey, and were checked for correctness of melting-point, chromatographic properties and chemical reactions.Other steroids were kindly provided by Professor W. Klyne from the Medical Research Council reference steroid collection. CONDITIONS OF CHROMATOGRAPHY- Whatman No. 2 paper was used at 37" C, with 3 hours' equilibration. The solvent front was allowed to proceed 40 cm by descent, and steroids were located by chemical and physical procedures. SOLVENT MIXTURES- The stationary phase consisted of a mixture of methanol and saturated aqueous formalde- hyde (1 + 1). Tailing occurred at lower concentrations of methanol and higher concen- trations lead to monophasic conditions. The mobile phase consisted of light petroleum and benzene mixtures. As the ARm values for replacement of light petroleum by benzene were proportional to the benzene concentration, as reported for the Zaffaroni systems4 and Bush systemsI3 it is not necessary to define precise solvent mixtures.Rmo for the family is defined as the Rm value determined for the mobile phase of light petroleum alone. For precision, observations were made within the limits +O-6 to -0.6 Rm units. In the Bush systems3 the benzene concentration factor was 0-0195 Rm units. It was found that R,, = observed Rm + 0.026 (benzene concentration, per cent. v/v, when preparing), 0 SAC and the authors.454 TRAFFORD AND EDWARDS: DETERMINATION OF THE NUMBER OF [ArtahySt, VOl. 93 RESULTS The Rmo values obtained by chromatography of ninety-eight steroids are summarised in Tables I and 11. TABLE I KEY STEROID Rmo VALUES Steroid* P4-3 : 20-one 5 /3P-3a-ol-20-one P5-3 /3-01-20-0ne 5aA-3a-ol-17-one 5 /3A-3a-ol-l 7-one As-3 8-01-1 7-one 5PP-3a : 17a : 2Oa-01 P4-3 : 11 : 20-one-17a: 21-01 P4-3 : 20-one-l1/3 : 17a: 21-01 Trivial name Progesterone Pregnanolone Pregnenolone Androsterone Aetiocholanolone Dehydro-epi-androsterone Pregnanetriol Cortisone Cortisol Rmo t - 0.45 - 0.37 -0.18 + 0-02 + 0.27 + 0.37 + 1-34 + 2.25 -j- 2-54 * Bush1 abbreviated nomenclature.t R,, = Rm of substance in methanol - aqueous formaldehyde - light petroleum mixture (1 + 1 + 2), obtained by calculation if necessary; see text. TABLE I1 MEAN AR, VALUES IN FORMALDEHYDE SYSTEMS Substituent ARm 1/3-01 . . .. . . 1.20 la-ol .. .. . . 1-40 2a-01 .. .. . . 0.81 6a-01 .. .. . . 1.81 6/3-01 . . .. . . 1.44 7-one .. .. . . 1.19 701-01 .... . . 1.84 ll-one . . .. . . 0.91 lla-ol . . .. . . 1-52 1201-01 . . .. . . 1-49 16/3-01 . . .. . . 2.03 17-one . . .. . . 1.65 17a-01 (P) . . .. . . 1.03 20-one . . . . . . 0.89 21-01 .. . I . . 1.04 11p-01 . . .. . . 1.09 CH, . . .. .. . . 0.22 5a-+5/3 . . .. . . 0.12 3a-ol+ 3/3-01 . . . . 0.29 %one + 3/3-01 . . . . 0.58 4-ene-3-one + 3a-ol-5p . . 0.24 1 l-one +- 11 /?-01 . . . . 0.26 20-one + 2OP-01 . . . . 0.33 20-one +- 20a-01 . . . . 0.57 Number of examples 3 1 1 1 2 1 1 8 3 12 1 3 1 9 1 6 4 9 2 2 10 7 5 5 &Standard deviation 0.15 - - 0.17 0.16 0.15 - - 0.07 0.07 0.14 0.15 DISCUSSION On plotting the steroid Rmo values obtained with and without formaldehyde, the points were found to group about three parallel lines. Each group shared a common number of functional groups, being di-, tri- and tetra-, or more, oxygenated.An alternative method of plotting is summarised in Fig. 1 , where the plot of ARm value caused by the formaldehyde is plotted against the number of polar functional groups. In this figure the observations are plotted as the mean value of each group and the standard error. It is clear that the observed values cluster in a manner useful in characterising steroids from natural sources into one of the following groups: tetra-, or more, oxygenated; tri-oxy- genated, with indications at the upper and lower ends of androstane or pregnane skeletons; di-oxygenated androstane or pregnane derivatives, with no ambiguity.July, 1968] OXYGEN SUBSTITUENTS OF STEROIDS BY CHROMATOGRAPHY 455 Q Number of polar functional groups Fig.1. Correlation of ARm when formaldehyde is incor- porated with several polar functional groups. The mean, number of steroids in each group, standard error and the prob- abilities of significance of differences are indicated. Groups marked A are androstane derivatives and P, pregnane The above characterisation of steroids by number of functional groups was carried out initially on simple hydroxylic and ketonic steroids without vicinal effects. On extension to vicinally substituted steroids no marked divergence was found for 2,3-, 16,17-, 17,220- and 20,21-di-substitution, provided that the substances were not penta-substituted. Double bonds in isolation, or in conjugation with carbonyl groups, had no effect, and the acetoxy group behaved as &-functional.A further examination of the results was carried out separately in the androstane and pregnane groups by multiple regression to distinguish between the effects of hydroxyl groups and ketones and assess each standard error. The pairs of values were not significantly different, providing justification for the empirical procedure used above, in which the A& effects of hydroxyl and ketone groups were assumed to be equal. The present procedure has been applied in the characterisation of substances from the urine of a new-born child5 and indicated that certain oxidation products were tri-oxygenated androstane derivatives. This was in agreement with other observations on these substances. Although the present study is limited to steroids it may be presumed that similar results would be obtained with other groups of substances that do not react chemically with formaldehyde. REFERENCES 1. 2. 3. 4. 5 . Bush, I. E., “The Chromatography of Steroids,” Pergamon Press Ltd., Oxford, London, New York Edwards, R. W. H., in Smith, I., Editor, “Chromatographic and Electrophoretic Techniques,” Bush, I. E., Biochem. J., 1952, 50, 370. Kasabakalian, P., and Basch, A., Analyt. Chem., 1960, 32, 459. Edwards, R. W. H., and Trafford, D. J. H., Biochem. J., 1968, 108, 185. and Paris, 1961. Third Edition, Volume 1, William Heinemann Ltd., Publishers, London, 1968. Received October 31sl, 1967

ISSN:0003-2654    

DOI:10.1039/AN9689300453

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

456 Analyst, July, 1968, Vol. 93, pp. 456457 The Use of Depleted Cells as Inocula in Vitamin Assays BY L. GARE (Beecham Research Laboratories, Vitamins Research Station, Walton Oaks, Dorking Road, Tadworth, Surrey) Preparation of assay inocula in such a way that the cells contain a reduced level of the vitamin to be assayed gives low background growth in plates, and the greater contrast enhances zone definition. Steps required to be taken to find the optimum conditions for preparation of inocula are described. The assays of nicotinamide, pantothenic acid, folic acid and vitamin B, can be improved in this way. A FAMILIAR problem in microbiological assays is high “blank” growth. In our early attempts to assay folic acid by a plate (agar-diffusion) method, in which Lactobacillus helveticus was used, there was insufficient contrast between background growth and response zone.The inoculum was prepared by growing cells in “enriched culture medium”l for 18 hours and then washing twice with 0-85 per cent. saline. (The levels of the relevant B vitamins in the “complete” medium used were established by microbiological assay and are shown in Table I.) Further washing of the cells with 0.85 per cent. saline reduced the background growth, but only slowly and incompletely, as if the cells could store folic acid. In our first attempt at depletion, the cells were washed, re-suspended and incubated in a volume of folic acid free medium equal to that of folic acid rich medium (“enriched culture medium”) in which they were grown; this treatment gave no reduction in background growth.When cells grown in folic acid rich medium were diluted and incubated in a volume of folic acid free medium such that considerable increase in cell numbers could take place, background growth in plates was reduced, and the contrast was sufficiently great for zones to be well defined. The depleted cells are prepared by growing in “enriched culture medium” at 37” C for 18 hours, and a small volume is diluted fifty times in folic acid free medium, incubated for 7 hours and used without washing. It appears that cells growing in vitamin-rich media can accumulate vitamins to such an extent that, on being diluted and incubated in a medium deficient of one vitamin, they can divide until they are depleted of the vitamin absent in the medium.Because this treatment of L. helveticus cells in folic acid assay improved zone quality, attempts were made to improve other assays in the same way. The plate assays of vitamin B, with Saccharomyces carlsbergensis (with an inoculum grown initially on malt extract - agar), and pantothenic acid and nicotinamide with LactobaciZZus arabinosus (with an inoculum grown initially on “enriched culture medium”), have been improved in this way (see Table I). The suspensions can be stored in the refrigerator (about +5” C) for at least 1 week and are suitable for use in tube assays. The details of techniques for producing depleted inocula would probably differ between organisms, strains, media and laboratories. The steps required to be taken to find the optimum conditions for depletion are as follows.PROCEDURE- (1) Dilute a small volume of culture grown in a vitamin-rich medium with a medium free from the appropriate vitamin, incubate and determine, by taking viable counts, at intervals, when multiplication has ceased. (2) Dilute graded volumes of culture grown in a vitamin-rich medium with a medium free from the appropriate vitamin, incubate for the length of time indicated in (1) and measure the extent of multiplication by taking viable counts. This will indicate the minimum volume of culture which, on dilution and incubation, gives the maximum number of cells, These cells will be fully depleted. (3) Determine the optimum concentration of depleted cells for use in an assay plate. 0 SAC and the author. This is the depletion time.GARE TABLE I VITAMIN LEVELS IN MEDIA FOR INOCULA Amounts per ml of medium 457 r Pantothenic acid Nicotinamide Folic acid B‘, 2.3 ng - - 0.14 pg “Enriched culture medium” 0.27 p g 1.5 rg Malt extract - agar* ..- - * Difco malt extract 4-6 per cent.; agar 1-5 per cent. That micro-organisms can accumulate vitamins and then multiply in the absence of one of them has been known for some time,2s3s4 and the incubation of cells in media low in, or free from, vitamin has been advocated for assay inocula. Gibbons and E ~ ~ g l e , ~ for example, to demonstrate menadione qualitatively, prepared menadione-deficient Bacteroides melanino- genicus cells by incubating the organism in broth free from menadione “where limited growth occurred.” Simpson6 recommends growing L.helveticus for 16 to 18 hours in 100ml of single-strength assay medium containing riboflavin, washing and re-suspending the cells in the same volume of single-strength assay medium, without riboflavin, and incubating for 16 to 18 hours. In our experience, the cells must be able to multiply in order to deplete, and to effect this the cells should be diluted before they are incubated in the vitamin-free medium. Strohecker and Henning’ put forward the idea of depletion, but in no instance do they incubate in the absence of the vitamin to be assayed, and they describe no plate assays. The mechanism of the accumulation and depletion of vitamins is not yet known in detail or fully understood. Work has begun towards a better understanding of this phenomenon, and it is hoped to publish the results in due course. I thank Mr. S. A. Price for helpful criticism of the text. REFERENCES 1 . 2. 3. Meissel, M. N., Nature, 1947, 160, 269. 4. 5. 6. 7 . The Association of Vitamin Chemists Inc., Editors, “Methods of Vitamin Assay,” Third Edition, Knight, B. C. J. G., Vitam. Horm., 1945, 3, 139. Toennies, G., Das, D. N., and Feng, F., J . Bact., 1966, 92, 707. Gibbons, R. J., and Engle, Lois P., Science, N.Y., 1964, 146, 1307. Simpson, J. S., in Kavanagh, F., Editor, “Analytical Microbiology,” Academic Press Inc., New Strohecker, R., and Henning, H. M., “Vitamin Assay; Tested Methods,” Verlag Chemie, GmbH, Received September 19th, 1967 Interscience Publishers, New York, London and Sydney, 1966, p. 52. York and London, 1963, p. 115. Weinheim, 1965, p. 22.

ISSN:0003-2654    

DOI:10.1039/AN9689300456

出版商:RSC    

年代:1968

数据来源: RSC

摘要:

458 Analyst, July, 1968, Vol. 93, $9. 458460 Species Identification of Cooked Fish by Disc Electrophoresis BY I. M. MACKIE (Ministry of Technology, Torry Research Station, P.O. Box 31, 135 Abbey Road, Aberdeen) The present objective methods of identifying fish species are based on the species-specific protein-separation patterns obtained on electrophoresis of the water-soluble sarcoplasmic proteins of fish muscle. As the proteins must be in their native undenatured state, electrophoretic identification of fish species has, so far, been restricted to raw fish. An extension of the electrophoretic method to the identification of cooked fish is described. The protein fragments extractable in 6 M urea from the denatured proteins of cooked muscle can also be separated by electrophoresis into species' characteristic patterns that could be used for species identifica- tion.The separation patterns obtained on polyacrylamide gel for the urea extracts of cooked herring, halibut, plaice, salmon, cod and haddock are presented. In its present form the method does not apply to canned fish. THE identity of a fish species is usually readily determined from the physical appearance of the whole fish, and it is only when the distinguishing features normally used for identification have been removed, as in a fillet, that the identity of the species can be in doubt. After further processing, such as cooking, and subsequent incorporation into fish cakes and fish pastes, identification by sensory means is often impossible. There is, therefore, a need for a reliable non-sensory method of identifying the species in fish products whenever there can be doubt as to its authenticity.This is of importance commercially because of the possibility of substituting cheaper for more expensive varieties of fish. An objective method of species identification is based on the species characteristic protein separation patterns obtained after electrophoresis of the water-soluble sarcoplasmic proteins of the muscle.1 Zone electrophoresis on starch gel2 has been used by Thompson3 as a routine analytical method of species identification, and, more recently, disc electrophoresis on poly- acrylamide4 has been used by PayneY5 Mancuso,6 Torry Research Station' and Thompson.s These methods of identifying species can only be applied to raw undenatured muscle as the separation patterns are obtained from the proteins in their native, undenatured state.If the fish is cooked or dried the proteins become denatured by heating and coagulate to form precipitates, which can no longer be examined by this method. Partially cooked products such as fish fingers can, however, be examined provided there is still sufficient undenatured flesh to give a satisfactory protein separation. This paper describes a possible extension of species identification to cooked fish products. METHODS PREPARATION OF COOKED FISH FILLETS- Heat fish fillets on a steam-bath for 30 minutes in covered casseroles. 0 SAC; Crown Copyright Reserved.(4 (b) (4 (d) (4 (f (€9 Fig. 1. Electrophoretic patterns of urea extracts of cooked fish (a to f ) of (a) herring, (b) halibut, (c) plaice, (d) salmon, (e) haddock and (f) cod; (g) is the pattern for the sarcoplasmic proteins of cod in 6 M urea.Acrylamide gel concentration is 7.5 per cent. w/v Fig. 2. Electrophoretic patterns of water extracts (the sarcoplasmic proteins) of raw fish of (a) herring, (b) halibut, (c) plaice, (d) salmon, (e) haddock and (f) cod. Acrylamide gel concentration is 6.0 per cent. w/v [To face page 458MACKIE: SPECIES IDENTIFICATION OF COOKED FISH 459 UREA EXTRACT OF FISH- urea solution. fish residue by centrifuging for 20 minutes at 6000 x g. for electrophoresis. DISC ELECTROPHORESIS- Tris - glycine bzq$er solzdiow-Weigh 28.8 g of glycine and 6.0 g of tris(hydroxymethy1) aminomethane and dissolve it in water. Make the solution up to 1 litre and adjust the pH to 8.6.Dilute 1 to 10 for use as the solvent for the gel reagents and as the electrolyte solution for electrophoresis. AcryZamide gel rods-To prepare a 7-5 per cent. w/v acrylamide gel, dissolve 3.0 g of “Cyanogum 41”” in 20 ml of tris - glycine buffer solution. Add 10 ml of 1.60 per cent. w/v /3-dimethylaminopropionitrileg and 10 ml of 0.20 per cent. w/v ammonium persulphate. Transfer the solution quickly to the gelling tubes (7.5 x 0.5 cm), fill to a depth of 6.5 cm, overlayer with water according to the procedure of Ornstein and Davis,4 and set aside to polymerise at room temperature for about 20 minutes. Prepare a 6 per cent. gel by reducing the amount of “Cyanogum 41” to 2.40g. Method of electrophoresis-After polymerisation is complete transfer the tubes to a disc electrophoresis apparatus? in a chilled room at 1” C.Carry out a pre-run for 20 minutes at 200 volts to remove any discontinuities in the gel. Apply 30 to 60 pl of the urea extracts of the fish directly to the tops of the gels by dipping a syringe through the upper electrolyte compartment. Carry out the electrophoresis for 55 minutes at 280 volts. Staining and developing the gels-When the run has been completed remove the gels from the tubes4 and stain them for half an hour in a 0.1 per cent. solution of Amido black in 7 per cent. acetic acid solution. Wash out the excess of dye with the acetic acid solution and allow to stand overnight in a methanol - acetic acid - water solvent (21 + 3 + 96). Examine the developed gels the following morning.Break up a 25-g portion of fish fillet or fish product and suspend it in 50 ml of 10 M After allowing it to stand overnight at room temperature, remove the insoluble Use the supernatant solution directly RESULTS AND DISCUSSION In Fig. 1 are given the electrophoretic separation patterns of the protein residues extracted into 6 M urea from cooked herring, halibut, plaice, salmon, cod and haddock. For comparison the patterns of the corresponding water extracts from raw fish (the sarco- plasmic proteins) are given in Fig. 2. In general, the urea-extracted protein residues have fewer slow moving components and there is a greater over-all similarity of the pattern that makes differentiation more difficult. For example, the patterns for the closely related species cod and haddock, are similar and are more likely to be confused than are the corresponding sarcoplasmic protein patterns.Nonetheless, the patterns obtained for all six species are sufficiently different to allow an unequivocal identification of the species to be made. Preliminary examination of some cooked fish products was carried out. When the urea extracts from canned herring were examined by this method there were no well identifiable protein zones as are obtained from herring steam-cooked at atmospheric pressure. A fish “chip” preparation made from a homogenised mixture of cod flesh, potato powder and starch (6 + 7 + l ) , which had been cooked in vegetable oil at 260” C, gave a pattern identifiable as that of cod. There was no contribution to the pattern from the small amount of vegetable protein present.A commercial white fish cake of unspecified fish content gave a pattern easily recognisable as that of cod. Urea is well known as an agent for splitting hydrogen and hydrophobic bonds in native ProteinslO and, as such bonds are believed to form when proteins are denatured by heating, it is not surprising that fragments of these denatured proteins are extracted with strong urea solutions. In fact, when raw fish were examined by this method the separation patterns of the urea extracts were the same as the corresponding ones from urea extracts of cooked fish, suggesting that non-covalent bonds of this type are formed on cooking and that they are subsequently broken by urea. As shown in Fig.1 (f and g), the sarcoplasmic proteins contribute to only part of the pattern of the urea extract of the fish flesh as a whole. The remainder of the zones must derive from the myofibrillar and connective tissue proteins. monomer and 5 per cent. of bisacrylamide. * “Cyanogum 41” is obtainable from British Drug Houses Ltd. t Supplied by the Shandon Scientific Company Ltd., London. It contains 95 per cent. of acrylamid460 MACKIE SPECIES IDENTIFICATION OF COOKED FISH CONCLUSION Electrophoretic examination of the protein fragments in urea extracts of fish appears to be a promising method for the identification of species in cooked products. A more extensive survey would, however, be necessary before it could be used as an objective method. In its present form it is not applicable to the identification of canned products. It is restricted to fish cooked under atmospheric pressure. Mr. B. W. Thomson assisted in the experiments described in this paper. The work described was carried out as part of the programme of the Ministry of Technology. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Connell, J. J., Biochem. J., 1953, 55, 378. Smithies, O., Ibid., 1959, 71, 585. Thompson, R. R., J . Ass. Off. Agric. Chem., 1960, 43, 763. Ornstein, L., and Davis, B. J ., “Disc Electrophoresis,’’ pre-printed by Distillation Products Payne, W. R., J . Ass. Ofl. Agric. Chem., 1963, 46, 1003. Mancuso, V. M., Ibid., 1964, 47, 841. Torry Research Station Annual Report, H.M. Stationery Office, Edinburgh, 1966, p. 53. Thompson, R. R., J . Ass. Off. Analyt. Chem., 1967, 50, 282. Barka, T., J . Histochem. Cytochem., 1961, 9, 542. MacKenzie, H. A., Smith, M. B., and Wake, R. G., Biochim. Biophys. Acta, 1963, 69, 222. Industries, Rochester, New York, U.S.A. Received November 22nd, 1967

ISSN:0003-2654    

DOI:10.1039/AN9689300458

出版商:RSC    

年代:1968

数据来源: RSC