ISSN:0003-2654    

DOI:10.1039/AN97499FX009

出版商:RSC    

年代:1974

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97499BX011

出版商:RSC    

年代:1974

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97499FP025

出版商:RSC    

年代:1974

数据来源: RSC

ISSN:0003-2654    

DOI:10.1039/AN97499BP031

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

MARCH, 1974 THE ANALYST Vol. 99, No. 11 76 Coulometric Microtitration of Arsenic(II1) and Isoniazid Using a Vitreous Carbon Generating Electrode BY V. J. JENNINGS, A. DODSON AND A. HARRISON (Department of Chemistry and Metallurgy, Lanchester Polytechnic, Priory Street, Coventry, C V l 5FB) A small-scale coulometric titration cell and apparatus is described in which a vitreous carbon rod anode is used to generate bromine. The amount of the latter required to titrate from 3 to 15 pg of arsenic(II1) or isoniazid with a precision of 2 per cent. is found by using an integrating digital milli- ammeter. The titration end-points were obtained by use of a form of differential electrolytic potentiometry. PREVIOUS work has shown that bromine can be generated at a vitreous (or glassy) carbon electrode with a current efficiency of at least 99.9 per cent.1 In the present work results obtained by using a vitreous carbon electrode to generate bromine in the coulometric micro- titration of arsenic( 111) and isoniazid (isonicotinylhydrazide) are reported.There is general interest in analytical methods for determining microgram amounts of both substances, arsenic because of its toxicity and isoniazid because of its use as a chemo- therapeutic agent for the treatment of tuberculosis.2 Conventional oxidimetric titration methods have been reported for the determination of arsenic(I1I)s and isoniazid4 in which the oxidising agent is potassium bromate, occasionally in the presence of added bromide ions. For the latter condition the chemical reactions are- for arsenic(II1) and for isoniazid.Coulometric titrations with electrolytically generated bromine at a platinum anode have been reported for determining a r s e n i ~ ( 1 I I ) ~ ~ ~ and isoniazid.' A major difficulty in micro-titrations in which the initial concentration of the titrand is approximately 10-5 M is in the location of the end-point, as the detection system must be sensitive to concentration levels of about lo-' M in the vicinity of the equivalence point if an end-point precision of 1 per cent. is to be achieved. In the present work, a form of differential electrolytic potentio- metry has been used to locate the end-point because it is known to provide an end-point detection method of high sensitivity in, for example, the coulometric determination of hydrazine with bromate.* In differential electrolytic potentiometry, the potential difference between two platinum electrodes that have been polarised by the passage of microampere currents is measured.For arsenic(II1) and isoniazid titrations it is expected that the complete titration graph of potential versm amount of bromine generated would show a relatively high potential plateau region and a sharp fall in this potential at the end-point. However, in order to decrease the time required to complete a titration in the present work, it was decided that instead of attempting to plot a complete graph, the amount of titrant required to produce an arbitrarily chosen decrease in potential of 1oOmV from the initial start potential would be taken as the end-point value.In a conventional coulometric titration, the current is maintained at a constant value and the elapsed time from the start to the end-point is used to determine (from Faraday's laws of electrolysis) the amount of titrant generated. After using some commercially available coulometric titration apparatus, we have found that it is not always a simple matter to main- tain a constant current and there are difficulties in synchronising the current flow with the 145; As3+ + Br, = As5+ + 2 B r .. .. * * (1) (2) C5H,N.CONHNH, + 2Br, + H,O = C,H,N.COOH + 4 B r + 4H+ + N, @ SAC and the authors.146 [Analyst, Vol. 99 timing system. There have been attempts to use a low-inertia integrating meter as a coulo- nieter in series with the titration cell9 in order to measure the charge directly. Integrating electronic digital milliammeters have recently become commercially available and such an instrument was used successfully in this work.JENNINGS et al. : COULOMETRIC MICROTITRATION OF ARSENIC(III) EXPERIMENTAL REAGENTS- Reagents of analytical-reagent grade quality were used when possible. The isoniazid (isonicotinylhydrazide pure, Koch-Light Laboratories Ltd.) was recrystallised from solution in ethanol and dried at 110 "C for 2 hours. Its melting-point after recrystallisation was 170 to 171 "C. E 0- F' P -N -M H A B C D E F G H J K L & M N 0 P Q R Griffin constant-current, 1 - to 10-mA supply unit (S576-130) Time Electronics Digital Integrator Type TS1 OOA (1 00-mV range) Beckman Research pH meter (mV mode) (also used to measure the potential across L and M) ALMA Type SIT 50sZ f 0.05 per cent, resistor Le Carbone (Great Britain) Ltd.3-mm diameter vitreous carbon rod anode (V25) Johnson Matthey Ltd. thermopure platinum wire cathode, 0.5-mm diameter Glass cathode compartment Porous plug (coil of Whatman No. 541 filter- Magnetic stirrer bar Bottom half of glass cell, capacity 3 ml D.E.P. 0.5-mm diameter p!atinum wire elec- trodes Sovirel bored cap for sliding joint, size 22 mm PTFE wrapped silicone rubber sealing ring (22 x 16) Top half of glass cell White-spot nitrogen inlet Nitrogen outlet (also used for adding sample with hypodermic syringe) paper) Fig. 1. The cell and coulometer circuitAIarCh, 197-11 water and making the volume up to 500 ml in a Grade A calibrated flask.previously described.1 AKD ISOXIAZID USING A VITREOUS CARBON GENERATING ELECTRODE 147 Isoniazid solution, 0.01 &I-This was prepared by dissolving 0.2 g of the solid in distilled Arscnic(II1) oxide solzttioiz, 0.01 nf-This was prepared in a similar manner to that APPARATCS- The cell and couloiiieter circuit are sliowii in Fig. 1. The integrator display, which was a six-digit totalising magnetic counter with maiiual zero re-set, was calibrated by passing a known (2 mA) constant current for a given length of time (measured on a stopwatch to h O . 1 s). The mean value of 1 integrator unit was equivalent to a charge of 6-652 x C (theoretical value, 6.666 x lo-* C) and the relative standard deviation from the mean value was 0.1 per cent. for timed periods oi 100 s.The surface area of the vitreous carbon rod exposed to the electrolyte solution was 0-16 cm2 with a current density of 12 mA cm-2. A for the differential electrolytic potentiometric platinum electrodes was supplied by a 90-V Exide Dymax dry battery (DM 256) in series with a W7elwyn Electric Ltd. 100 MQ (&5 per cent.) carbon film resistor. The current density a t the differential electrolytic potentiometric electrodes was about 12 x A cm-2. The potential across the electrodes was measured on a Beckman Research pH meter used in its millivolt measuring mode. The polarising current of about PROCEDURE- For carrying out titrations tlie method of successive aliquot additions, as described previously,1° was used. To the cell was added 2 ml of electrolyte solution that was 0.2 M in potassium bromide and 1-0 nr in sulpliuric acid.White-spot nitrogen was bubbled through the cell for 5 minutes so as to remove any titratable volatile impurity, then an aliquot of titrand was added by use of a 10-pl precision syringe (S.G.E. Pty. Ltd.). The potential across the two differential electrolytic poteiitionietric indicator electrodes was measured and the amount of charge, in integration units, required to decrease this potential by 100 mV was found. ,4 further aliquot of titrand was tlieii added and the above procedure repeated. Naturally, the result for the first aliquot included any blank value, that is, the charge required to cause the 100-mV potential change in the absence of sample. This result was therefore high and was rejected.With the isoniazid titrations it was found that the result for the second aliquot could also be high so that this result was again rejected. It is believed that some pre-conditioning of the diff ereniial electrolytic potentiometric electrodes is necessary for isoniazid titration, as has been found by other workers in the coulonietric titration of sulphur dioxide with 1)romine.ll The results are given in Tables I to IV. It was observed that there was a slow downward drift of the initial differential electrolytic potential after the addition of each aliquot. TABLE I COULOXETRIC TITRATIOKS OF 10 p1 o b 0.01 M ARSENIC(III) OXIDE SOLUTION 10 p1 of solution zz 15.13 pg of arsenic(II1) Experiment series . . .. * . .. A r, C D E Nuiiiber of individual results in series ..8 8 8 1Ican amount 01 arscnic(II1) fouIid/pg . . 15.16 15.31 15.19 15.39 15.18 Relative staiidard deviation, per cent. . . 2.1 1-8 1.0 1-8 1-9 Error, per cent. . . .. . . .. . . t o e 1 f l - 2 +0*4 +1.4 + 0-4 c 10 DISCUSSION AND CONCLUSION A RS E x IC ( I I I) - The results in Tables I and 11 show that the accuracy and precision are of the order of 1 and 2 per cent., respectively, except for the srnallest amount of sample (3 pg). At that level there is an accumulation of random errors, for example, the fact that there is an experimental limit to the minimum incremental amount of bromine that can be generated. A general limit on tlie precision level is imposed by the use of a microsyringe, with which the volume delivered is subject to a probable random error of 1 per cent.148 JENNINGS, DODSON AND HARRISON TABLE I1 COULOMETRIC TITRATIONS OF 2 TO 8 pl OF A 0.01 M ARSENIC(II1) OXIDE SOLUTION Volume of hs,O, solution taken/pI .. . . 2 4 G 8 Number of individual results in series .. 8 8 8 8 Amount of arsenic(II1) presentlpg . . . . 3-03 6.05 9-08 12-11 Mean amount of arsenic(II1) found/pg . . 3.15 6.14 9.04 12.22 Relative standard deviation, per cent. . . 2.5 1.5 0.8 1.9 Error, per cent. .. .. .. .. . . f 4 . 2 +1*4 -0.4 + 1.0 A least-squares fit of the results given in Tables I and I1 shows that if x pg of arsenic(II1) are taken, then the y pg of arsenic(II1) found is given by the equation- I SON I AZI D- The results in Tables I11 and IV are similar to those for arsenic(II1) although the precision is lower and there is an appreciable increase in the error.A least-squares fit of the results given in Tables I11 and IV shows that if xpg of isoniazid are taken, then the ypg of isoniazid found is given by the equation The error may be partly due to the possibility that the solid, although recrystallised, was not absolutely pure. However, it is also likely that at these concentration levels the rate of reaction of isoniazid with bromine and the instrumental response of the end-point detection system limit the precision and accuracy that can be attained. TABLE I11 COULOMETRIC TITRATIONS OF 5 pl OF 0-01 M ISONIAZID SOLUTION 5 pl of solution = 6.88 pg of isoniazid Experiment series . . .. .. .. A B C D E Relative standard deviation, per cent. . . 2.0 1.6 4.0 3.1 2.2 Error, per cent... .. . . .. . . +1*4 4 3 . 5 $7.3 +5*6 +5.5 It is concluded that the apparatus and technique described in this paper are suitable for the coulometric titration of down to 3-pg amounts of arsenic(II1) and isoniazid and that the method could be applied to the determination of large numbers of samples as a result of the speed with which these titrations can be carried out. y = 1.002~ + 0.07 y = 0.981~ + 0.390 Number of individual results in series .. 8 8 8 9 8 Mean amount of isoniazid found/pg . . . . 6-97 7.1 1 7-35 7.24 7.23 TABLE IV COULOMETRIC TITRATIONS OF 2 TO 10 pl OF 0.01 M ISONIAZID SOLUTION Volume of isoniazid solution talrenlpl . . 10 8 6 4 Number of results in series . . .. .. 8 7 8 8 Amount of isoniazid takenlpg .. . , 13.75 11-00 8.25 5.50 Mean amount of isoniazid found/pg . . . . 13.85 11-22 8.58 5.56 Relative standard deviation, per cent. . . 1.6 1.3 1.9 2.8 Error, per cent. .. .. . . . . . . +0*7 +2-0 +4.1 +1-1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. REFERENCES 2 8 2.75 3.16 2.7 + 14.8 Jennings, V. J., Dodson, A., and Atkinson, A. M., Analyst, 1972, 97, 923. Ellard, G. A., Gammon, P. T., and Wallace, S. M., Biochem. J., 1972, 126, 449. Bishop, E., Analyst, 1958, 83, 212. Vulterin, J., and Z$ka, J., Talanta, 1963, 10, 891. Myers, R. J., and Swift, E. H., J . Amer. Chem. Soc., 1948, 70, 1047. Lee, J. K., and Adams, R. N., Analyt. Chem., 1958, 30, 240. Kawamura, F.. Momoki, K., and Suzuki, S., Bull. Fac. Engng Yokohama Natn. Univ., 1955,4, 123. Bishop, E., Mikrochim. Acta, 1960, 803. Smythe, L. E., AnaZyst, 1957, 82, 228. Jennings, V. J., Dodson, A., and Tedds, G., Talanta, 1973, 20, 681. Bailey, P. L., and Bishop, E., Analyst, 1972, 97, 311. Received Octobev 5th. 1973 Accepted November 12th, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900145

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

Analyst, March, 1974, Vol. 99, pp. 149-152 149 Gel Supports for Electrophoresis with Pyridine - Acetic Acid Media BY D. LEIGHTON, G. J. MOODY AND J. D. R. THOMAS (Chemistry Department, University of Wales Institute of Science and Technology, Cardif, CFl 3N U, Wales) Agar gels that are suitable for use with an 80 per cent. solution of pyridine in water and cross-linked polystyrene gels in pyridine - acetic acid are effective support media for electrophoresis. They provide the means for widening the application of electrophoresis t o materials themselves insoluble in aqueous media but soluble in pyridine - acetic acid. Illustrative examples of the electrophoresis of methylene blue, rhodamine €3, alizarin red, alizarin blue and eosin are given. ZONE electrophoresis, although usually performed in aqueous buffer - electrolyte systems, has also been effected in non-aqueous systems, and the work recorded to date indicates a wider perspective for electrophoresis.For example, Paul and Durruml have separated oil- soluble dyes by electrophoresis in absolute ethanol, nitromethane - glacial acetic acid and pyridine - glacial acetic acid systems on filter-paper supports. Also, the electrophoretic behaviour of arsenazo I11 and its analogues on filter-paper in glacial acetic acid and mixed formic acid - chloroacetic acid media has been reported,2 and several inorganic ions that were difficult to separate in aqueous solutions were readily resolved in a methanol - acetone - hydrochloric acid m e d i ~ m . ~ Apart from filter-paper, thin-layer supports (as prepared for chromatography) are suitable for electrophoresis in non-aqueous media (Leighton, D., Moody, G.J., and Thomas, J. D. R., unpublished work), but the enormous capacity for resolution achieved with the various gel supports in aqueous systems4 cannot always be extended to non-aqueous media because of gel solubility and stability problems, especially with polyacrylamide. The characteristics of electrophoretic systems based on agar gels for aqueous pyridine media, and cross-linked polystyrene gels for pyridine - acetic acid buffer electrolytes, are described in this paper. EXPERIMENTAL PREPARATION OF AGAR GELS- The appropriate amount of special Noble Agar (Difco Laboratories, Detroit), but ideally 2.25 g, was dissolved in 30 cm3 of water by heating the mixture on a boiling water bath and 120 cm3 of pyridine were added.After cooling it on ice until it became viscous, the mixture was poured into a O.6-cm deep horizontal trough formed by an aluminium frame sealed to a 20 x 16-cm glass plate with high-vacuum silicone rubber grease. The agar surface was levelled flush with the top of the aluminium frame with a glass rod and the whole kept on ice until the gel was firm. The frame was removed and slits were cut midway along the gel layer for sample application, PREPARATION OF CROSS-LINKED POLYSTYRENE GELS- A mixture of styrene and divinylbenzene (50 to 60 per cent. m/m in ethylvinylbenzene) monomers in appropriate proportions, and containing 0-60 g of benzoyl peroxide initiator, was added to 120 cm3 of a pyridine - acetic acid - perchloric acid mixture (Table I) and the whole poured into a trough similar to that described above for agar gels, except that the top side of the aluminium frame was now covered with a glass plate.In order to facilitate filling of the trough, one 16-cm edge of the frame was cut away. For gelling, the assembly, which was held together by strong rubber bands, was placed in a boiling water bath with the open end just above the water level. Between 1 and 3 hours were usually required for gelling, depending on the concentration of the divinylbenzene. After gelling, the top glass plate and the aluminium frame were removed, thus leaving the gel resting on the other plate. Samples for electrophoresis were placed into slits cut midway along the gel layer.@ SAC and the authors.150 LEIGIITON d U l . : GEL SUPPORTS FOR ELECTROPHORESIS [A?UdySt, VOl. 99 ELECTROPHORESIS- Electrophoresis was carried out in tlie same way as for aqueous systems by using a Shandon power pack (Catalogue No. SAE 2525) and a Bairct and Tatlock horizontal electro- phoresis tank (Catalogue No. C40/4000) constructed of silica. Contact between the gel and buffer - electrolyte was effected with wicks made of filter-paper. The test solutions used were 1 per cent. solutions of the dye in the appropriate buffer. TABLE I PARAMETERS OF POLYSTYRICSE GEL FORMATION Gel constituents r 1 Buffer composition : Divinyl- pyridine to acetic Styrene benzene Renzoyl acid ratio, V / V monomer/ monomer/ peroxicle/ (120 cn13 usctl in I3uffcr GelNo. cm3 1 1 2 3 4 5 6 7 8 9 36 10 48 11 60 12 72 13 84 18 24 19 ;: J 22 cm3 19.2 - 14.4 12.0 9.6 7.3 6.0 4.8 3.6 ] 7.2 4.8 7.2 1 i 4*8 J .. g each instance ) pH partof 71 t o 73 per cent. 6.45 perchloric acid i Om6 I, 100 : 0 J 100 : 1.0 100: 7.16 100 : 19.2 100 : 33.8 100 : 40.6 100 : 48.7 100 : 66.7 9.2 8-46 6.7 5-98 5.46 5.33 5.09 4.73 Gelling time/ hours Gel character 1.0 Cloudy and 1.25 } rubbery 1.5 Yellow and 1.5-2.0 rather brittle Clear yellow but no gel 1.6 -) 1.0 1.0 0.75 0.75 1-0 2-0 1.0 1.0 1.6 + Clear yellow 1.5 J 1.5 Translucent 1.0 White opaque 0.5 } precipitate * Following 4-t-butylcatechol inhibitor extraction (by shaking with four times the monomer volume o 0.06 M sodium hydroxide solution) from styrene and divinylbenzene monomers, the gelling times were 1.25 (a) 2 (b) and 6 (c) hours.RESULTS AND DISCUSSION AGAR GELS- The development of agar gels was directed towards a purely non-aqueous pyridine system but the agar would not dissolve in water containing more than 80 per cent. of pyridine; the study of agar gel parameters was therefore based on varying the amount of agar in a mixture of 30 cm3 of water and 120 cnl3 of pyridine. The qualities of gels obtained after 1 hour, with the various amounts of agar used parenthesised, were as follows: no gel formed even after 8 hours (0.9 g), very soft gel (1.2 g), firm gel (1.5, 2.25, 3.0 and 3.75 g), very firm gel (4.5 g). Firm and very firm gels were suitable for testing by electrophoresis and typical results for dye systems are summarised in Table 11. Migration of methylene blue and rhodamine I3 to the cathode, and of alizarin blue and eosin to the anode, is as expected from the cationic and anionic residues of these respective pairs of dyes.Although the zero migration of alizarin red in the buffers with high pyridine contents is rather surprising, agar gels based on aqueous pyridine mixed solvent media clearly provide suitable supports for zone electrophoresis in buffer media containing very substantial amounts of pyridine, especially for the gel based on 2-25 g of agar in 30 cm3 of water plus 120 cm3 of pyridine (Table 11).TABLE I1 ,Y % Migration distanceslcm *% ELECTROPHORETIC MOBILITIES OF DYES ON GEL SUPPORTS I N PYRIDINE - ACETIC ACID MEDIA I A Buffer Gel constituents Buffer PH Agar gels- (1 + 4 v/v) (4 + 1 v / v I Water (30 cm3) + agar (1.5 g) -+ pyridine (120 cm3) Water (30 cm3) + agar (2.25 g) + pyridine (120 cm3) Water (30 cm3) + agar (4.5 g) $- pyridine (1 20 cm3) Water (120 cm3) + agar (2.25 g) + pyridine (30 cm3) Pyridine (120 cm3) + acetic acid (1.2 cm3) -t 71-73 per cent.perchloric acid (0.6 cm3) + Water - pyridine 8.8 Water - pyridine 8.5 Cross-linked colystyrene gels- benzene (6.0 cm3) f- benzoyl Pyridine (120 cm3) 4- acetic 5-45 styrene (24 cm3) -k divinyl- peroxide (0.6 g) (gel No. 6) acid (2.25 d -t 71-73 ver cent. 9.2 i 5*45 Pyridine - acetic 1 5.45 Pyridine - acetic 5-45 Pyridine - water 8.6 (1 4- 4 v/v) acid (3 + 1 V / V ) * Tailing. i perchloric :&id (0.6 cm;) + styrene (24 cm3) + divinyl- benzene (4.8 cm3) + benzoyl peroxide (0.6 g) (gel No. 7) Pyridine (120 cm3) + styrene (24 cm3) + divinylbenzene (4.8 cm3) -+ benzoyl peroxide (0.6 g) (gel.No. 14) Pyridine (120 cm3) + styrene (24 cm3) + divinylbenzene (7.2 cm3) + benzoyl peroxide (0.6.g). (gel No. 15) Pyridine (90 cm3) + acetic acid (30 cm3) + styrene (24 cm3) f divinylbenzene acid (3 + 1V/V) (4-8 cm3) + benzoyl peroxide (0.6 g) (gel No. 19) Pyridine Silica gel- { Silica gel G (Merck) Constant voltage/ V 1000 1000 1000 300 1000 600 600 600 600 600 1000 300 Starting current/ mA 35 35 35 5 8 7 4 3 5 5 1 4 Time/ minutes 40 45 45 180 180 180 45 45 90 240 240 180 Cathode Origin Anode - 1 Methylene blue 0.5 5.5 0.5 2.0 3.5 3.5 9.0 1.8 6.5 9-6t 0.1 0.4 Rhoda- mine B 0.3 1.5 0.3 1.2 2.0 2.0 2.2 0.4 1.6 4.8 0 0.8 Aliza- rin red 0 0 0 0.8 (to anode) 0 0 0 0 0 0 0 0 Aliza- rin blue 0.3 1.6 0-3 0.8 0.3 1.0 5.7* 0.7 2.0 6.5 0.6 0.3 Eosin 0.8 3.0 0.6 1.2 0.4 0.8 4*5* 0-9 1.5 4.5 0.8 1.9 t M o d to end of gel (9.6 cm) in 166 minutes.152 LEIGHTON, MOODY AND THOMAS CROSS-LINKED POLYSTYRENE GELS- The several parameters examined for polystyrene gel formation are summarised in Table I.An alternative photopolymerisation process that involved gelling under an ultraviolet fluores- cent lamp gave gels of similar quality to those given by the thermal method described above but the required gelling times of at least 3 days are rather too long for convenience. The general features for successful gel formation are bound by the requirements that the ratio of divinylbenzene to polystyrene should not be less than 1 : 5 (gel No. 7) or as high as 2 : 5 (gel No.3), although the former stipulation can be relaxed when the amount of poly- styrene monomer is increased (gels Nos. 9 to 13). With respect to pH (a relative, empirical quantity only for the mixed solvent media used in this study), stable gels were obtained for the pH range 9.2 to 5-45 (gels Nos. 14 to 19), but under more acidic conditions a precipitate formed during the thermal treatment stage (gels Nos. 21 and 22). Despite the presence of 4-t-butylcatechol inhibitor in the styrene and divinylbenzene monomer constituents, gelling times were of the order of 1 to 3 hours, but removal of the inhibitor reduced the times by about one third [see (a) and (b) in Table I for gels Nos. 5 to 71. Such a reduction was considered to be an insufficient advantage for the removal of the inhibitor to be introduced into the procedure for the preparation of standard gels.A further variant assessed with the view of simplifying gel preparation was the pre- forming of cross-linked polystyrene slabs followed by soaking in buffer electrolyte. Even though translucent slabs were obtained, these slabs, when swelled by floating on the buffer electrolyte, became brittle and snapped readily and were therefore unsuitable as supports for electrophoresis. Gels Nos. 4 to 7 and 9 to 19 (Table I) were of suitable quality for electrophoresis and the most economical in terms of monomer materials are gels Nos. 6 and 7, and 14 to 19. Repre- sentative samples of these gels were tested for the electrophoresis of dyes and typical results are summarised in Table 11.The migration behaviour is the same as for agar gels, with methylene blue and rhodamine B migrating to the cathode, alizarin blue and eosin migrating to the anode and alizarin red remaining immobile. Migration distances are rather less for the more cross-linked gel No. 6 than for gel No. 7 and also less for gel No. 15 than for gel No. 14. The conditions corresponding to gel No. 14 provide for the efficient separation of all five dyes. Reducing the pH from 9.2 (gel No. 14) to 5.45 (gel No. 19) lowers the migration rates, especially for the anionic dyes, but the separation is still effective. Migration rates were even less for a buffer of similar acidity on a silica gel G support, This feature and the reduced diffusion for the polystyrene gel confirm the suitability of the polymer gel as a support medium. CONCLUSION Although not as convenient or as elegant to use as purely aqueous systems, agar gels make suitable stable supports for electrophoresis in media of high pyridine content. Similarly, cross-linked polystyrene gels that are stable in pyridine - acetic acid provide another electro- phoresis gel support medium for the separation of materials that are difficultly soluble in water and for which pyridine and acetic acid can emphasise the differences in the polar character of such materials. The authors thank the Esso Petroleum Company Ltd. for financial support, including a research award (to D.L.). REFERENCES 1. 2. 3. 4. Paul, M. H., and Durrum, E. L., J , Amer. Chew. Soc., 1952, 74,4721. Savvin, S. B., Akimova, G., Krysin, E. P., and Davydova, M. M., Zh. Analit. Khim., 1970, 25, 430. Maki, M., Japan Analyst, 1955, 4, 156. Moody, G. J., and Thomas, J. D. R., “Practical Electrophoresis,” Merrow Press, Watford, to be Received July 6th, 1973 Accepted October 17th, 1973 published.

ISSN:0003-2654    

DOI:10.1039/AN9749900149

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

Analyst, March, 1974, Vol. 99, fq5. 153-155 153 Detection of Curcuma zedoaria and Curcuma aromatica in Curcuma longa (Turmeric) by Thin-layer Chromatography BY A. R. SEN, P. SEN GUPTA AND N. GHOSE DASTIDAR (Central Food Laboratory, 3 Kyd Street, Calcutta-16, India) It is difficult to determine the genuineness or otherwise of powdered turmeric (Curcuvna longa) when it is admixed with C. zedoaria or C. aromatica. A simple and rapid thin-layer chromatographic technique is described that involves a three-step colour sequence for the detection of camphor and camphene, the active principles of these adulterants, which are absent in turmeric. TURMERIC is the dried rhizome or bulbous root of Curcuma Zonga Linn., a perennial herb of the ginger family (Zingiberaceae), which is extensively cultivated in India, as well as in China and the East Indies.It is not a true spice, but rather a condiment that is used to a very large extent in the preparation of curries, pickles and many spicy Indian foods. It is also one of the chief ingredients of curry powder. The adulteration of ordinary spices and condiments is exceedingly prevalent in India and probably the most subject to admixture is turmeric. The fact that this condiment is fre- quently offered for sale in a ground condition furnishes an opportunity for the incorporation of various cheaper vegetable substances. The use of a microscope is important for the detection of these additions, but when the adulterants belong to the same genus (Curcuma), even experts in microscopy find it difficult to decide whether or not a sample of turmeric is genuine.In general, turmeric can readily be identified by the brilliant greenish yellow colour of the “paste balls,” oleoresin cells that contain the valuable yellow colouring principle, curcumin. Curcumin is, however, also found in several other species of Curcuma,l e.g., C. aromatica Salisb. and C. domestica Valeton. “The Wealth of India”2 and the “Glossary of Indian Medicinal plant^",^ however, appear to be confused and class C. domestica with C. Zonga, while Watt* and Bailey5 did not mention it. Guenther6 categorically differentiated C. domestica as a different species. It may be mentioned here that similar confusion had also arisen when Guibourt’ classed C. caesia Roxb. as C. Zoaga. Watt4 suspected that so-called forms of C.Zonga may prove to be the tubers of different species. “The Wealth of India”8 has mentioned its substitution with C. aromatica, as well as its close resemblance to C. zedoaria Roscoe, and from long experience we have found that both of these species, particularly the latter (local name Kachura, Soti), are being used extensively as adulterants. Although the latter species do not contain curcumin, this fact is of little significance when they are mixed with C. Zonga in the powdered form. Parry9 suggested that because the starch grains are usually more or less gelatinised as a result of the method of preparation, the presence of some well defined starch grains may indicate the presence of an adulterant. However, although most of the starch is swollen, grains may occasionally be found that still exhibit the characteristic scitaminaceous shape; moreover, while scalding is a very commonly used process, it is not obligatory and hence the presence of long, lens-shaped unaltered starch grains cannot be taken as indicative of adult eration.The statutory chemical standards prescribedlO are of minor importance, as all of these species belong to the same genus. Consequently, the detection of adulteration of turmeric will remain very difficult until their characteristic differences are established. The flavouring substances present in the essential oils of C. aromatica and C. zedoaria contain sufficient amounts of camphor and camphene (both of which are absent in C. Zonga) to permit their identification, thus providing a useful indication of the presence of C.aromatica @ SAC and the authors.154 SEN et al. : DETECTION OF Curcuma xedoaria [Analyst, Vol. 99 or C. xedoaria. The relatively simple and rapid thin-layer chromatographic technique des- cribed below is suitable for differentiating these compounds and is of value for quality control purposes. A number of solvent systems, as well as different spraying reagents, have been tried. Although the solvent with which optimum separation was achieved is not new,ll the detection by means of a three-step colour sequence has not previously been attempted. EXPERIMENTAL AND RESULTS SOLVENT SYSTEM- Ethyl acetate - n-hexane (3 + 17). (1) Concentrated sulphuric acid (50 ml) - nitvic acid (as oxidising agent) (0.5 ml). (2) Anisaldehyde - sulphuric acid reagent-A freshly prepared mixture containing 0.5 ml of anisaldehyde, 9 ml of ethanol (95 per cent.), 0.5 ml of concentrated sulphuric acid and 0.1 ml of glacial acetic acid.THIN-LAYER CHROMATOGRAPHY- Any convenient type of applicator can be used. Clean 10 x 20-cm thin-layer chromato- graphic plates with detergent and rinse them thoroughly. Coat them to a thickness of 500 pm with a 1 + 2 slurry of silica gel G in water. (An adsorbent layer of this thickness results in higher sensitivity and less background, and does not tend to peel when sprayed.) Air-dry the coated plates in a vertical position for half an hour and then dry them for 1 hour at 110 "C so as to activate the layers. Cool, wipe the backs and edges of the plates free from excess of silica gel G and store them in a desiccating storage cabinet until needed.The adsorbent layer must be uniform from plate to plate in order to obtain reproducible results. Steam distillation (as recommended by the British Phannacopoeia12) for 4 hours is the most convenient method of separating the essential oils from other plant constituents. The separated oils (from 20 to 25 g of sample) are mixed with a volatile solvent (benzene) so as to make an approximately 1 per cent. solution and a 10-pl spot is applied to the plate 2 cm from the bottom edge. Spots are identified by com- parison with volatile oils from authentic materials run alongside. The developing tank should be prepared at least 3 hours before use, so as to allow the tank to become saturated and to shorten the developing time.The solvent level should not be higher than the spotting line, a depth of 1 cm being generally sufficient. As the solvent travels rapidly (10 to 12 minutes), the plate must be removed from the tank as soon as the solvent front reaches the previously marked line, 10 cm from the origin. IDENTIFICATION- The identification of the substances was achieved by means of a three-step sequence: colour produced with sulphuric acid - nitric acid (chromogenic reagent No. l), fluorescence and the subsequent colour produced with anisaldehyde - sulphuric acid (chromogenic reagent No. 2), as described below. After allowing the solvent to evaporate, the chromatogram is sprayed with chromogenic reagent No. 1. The entire chromatographed portion must be uniformly sprayed in order to achieve optimum results (10 ml of reagent are sufficient).The plate is allowed to stand for 1 minute, so as to enable the layer to become saturated with the reagent. Although the patterns obtained are somewhat similar near the origin, the spots obtained with C. longa with R, values of 0.55 and above are different from those obtained with the other two varieties (see Table I). Inspection under ultraviolet light (365 nm) showed that with C. zedoaria and C. aromatica the spots fluoresce at R, 0.55 while with C. longa there is no such fluorescence in that zone, hence both species can be clearly differentiated from turmeric in this way. If the chromatogram is then further sprayed with chromogenic reagent No. 2, a contrasting pattern of colour differentiation is revealed.Orange (R, 0.72) and deep pink (R, 0.55) spots of camphene and camphor are characteristic of C. xedoaria and C. aromatica. Neither of these spots is given by oils of C. longa, which show only one bluish violet spot (R, 0.75) and another dirty brown spot (R, 0.60) closer to the region of the camphene and camphor spots (see Table I). CHROMOGENIC REAGENTS- Different samples can be spotted 2 cm apart.March, 19741 AND Curcuma aromatica IN Curcuma Zonga 155 The spots can also be made visible by spraying the plate with chromogenic reagent No. 2 alone, a similar pattern being observed after heating the plate for 2 minutes at 110 “C. The colour reactions described generally depend on the substance, amount of reagent used, temperature and duration of heating.The R, values obtained should not be regarded as absolute as they may be influenced by the presence of other extractives from the sample. For this reason, the identity of an isolate should be confirmed by re-chromatographing the suspected material against known standards. For identification purposes, a 5 per cent. ad- dition of C. zedoaria or C. aromatica to C. Zonga can be clearly distinguished from the C. Zonga by the R, values, the colour of the spots and their fluorescence. TABLE I COLOUR REACTIONS IN THE DIFFERENT DETECTION METHODS Figures in parentheses are R, values Chromogenic reagent No. 1 Daylight Ultraviolet C. aromatica C. longa C. avomatica C. longa A A I \ t \ C. zedoaria and Violet (0.77) Bluish violet Pink (0.72) Light pink (0.64) Orange (0.60) Brown (0-60) C.zedoaria and (0.75) Dirty green (0.55) Greenish violet (0.46) Violettish pink (0.40) Bluish violet (0.33) Pink (0.23) Bluish fluorescence (0.55) Light blue Violet (0.40) Bluish green Light pink (0.23) (0:46) (033) Chromogenic reagents No. 1 and No. 2 (daylight) r A 1 C. zedoaria and C. avomatica C. longa Violet (0.77) Bluish violet Orange (0.72) Light Pink (0-64) Blue (0.60) Dirty brown Deep pink (0.55) Violet Greenish violet (q.46) (0.46) Light pink Pinkish brown (0.40) (0.40) Bluish violet Bluish green (q.33) (0.33) Violet (0.23) Brown (0.23) (0.75) (0.60) It may be further added that the above test can be applied to check for the presence of C. caesia and C. domestica,6 as both of these species contain camphor, and the former also contains camphene.The authors are grateful to Shri T. V. Mathew, Director, Central Food Laboratory, Calcutta, for his keen interest in this study and kind permission to publish this work. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. REFERENCES Rao, B. S., and Shintre, V. P., J . SOC. Chem. Ind., Lond., 1928, 47, 54T. “The Wealth of India,” Volume 11, “Raw Material,” Council of Scientific and Industrial Research, Chopra, R. N., Nayar, S. L., and Chopra, I. C., “Glossary of Indian Medicinal Plants,” Council of Watt, G., “A Dictionary of the Economical Products of India,” Volume 11, Department of Revenue Bailey, L. H., “Manual of Cultivated Plants,” The Macmillan Co., New York, 1949. Guenther, E., “The Essential Oils,” Volume V, Van Nostrand Co. Inc., New York, 1952, p. 123. Watt, G., op. cit., p. 658. “The Wealth of India,” Volume 11, “Raw Material,” Council of Scientific and Industrial Research, Parry, E. J., “Food and Drugs,’’ Volume 1, Scott, Greenwood and Sons, London, 1911, p. 245. “The Prevention of Food Adulteration Rules, 1955,” Government of India, Ministry of Health Miller, J. M., and Kirchner, J. G., Analyt. Chem., 1953, 25, 1107. “The British Pharmacopoeia 1968,” The Pharmaceutical Press, London, 1968, p. 1275. New Delhi, 1950, p. 402, Scientific and Industrial Research, New Delhi, 1956, p. 85. and Agriculture, Government of India, 1889, p. 665. New Delhi, 1950, pp. 401 and 406. Publication, Appendix B, Item 05.20.01 (Substituted by GSR 1633, dated July Sth, 1968). Received June 21st, 1973 Accepted September 25th, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900153

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

156 Anallysf, March, 1974, Vol. 99, $9. 156-162 A Rapid Method for the Semi-quantitative Determination of Volatile N-Nitrosamines in Alcoholic Beverages BY M. CASTEGNARO, BRIGITTE PIGNATELLI AND E. A. WALKER (Unit of Environmental Carcinogens, International Agency for Research on Cancer, 150 Cours Albert Thomas, 69008 Lyon, France) A method is described for the rapid extraction of N-nitrosamines from strong alcoholic drinks with methylene chloride after saturating the water - ethanol phase with magnesium perchlorate. The nitrosamines are then determined by oxidation to nitramines and clean-up of the oxidation products by adsorption chromatography on a column containing two layers of different grades of alumina , followed by gas chromatography with an electron-capture detector. The method is equally applicable to beverages with low alcohol content such as beer.THE N-nitrosamines have been widely investigated in studies on carcinogenesis, but while certain of them are carcinogenic to all animal species investigated, there is still no definite evidence of their carcinogenicity in man. In areas of Northern France, epidemiology studies are currently being carried out by us on the relationship between high alcohol consumption and cancer of the oesophagus.1 In order to further these studies, an investigation of analytical methods for the determination of nitrosamines in home-distilled spirits is being undertaken, particular attention being paid to reduction in the time of analysis. In a similar study of home-produced beers conducted in East Africa, Collis, Cook, Foreman and Palframan2$3 found no evidence of the presence of nitrosamines in concen- trations above 100 pg k g l , which was their minimum level of detection.However, such reliable evidence as is available on the presence of nitrosamines in foodstuffs usually indicates levels in the order of 1 to 10 pg kg-l. It would seem reasonable to require a useful screening method for determining nitrosamines in spirits to be capable of detecting similar levels. Isolation of nitrosamines from solutions containing appreciable amounts of ethanol presents problems in extraction and separation, as the lower homolopes of the dialkyl- nitrosamines are freely soluble in water, ethanol and solvent phases. Distillation also presents problems, which arise from the formation of azeotropes.Crosby, Foreman, Palframan and Sawyer4 attempted to concentrate the nitrosamines by using a spinning band column, but the concentration factor obtained was only modest and the technique is slow. An alternative method has been proposed by Sen and Dalpe5 in which the alcohol is removed as an azeotrope with either benzene or toluene. The method, however, is somewhat lengthy for screening large numbers of samples. The authors report a method of analysis for nitrosamines in alcoholic beverages, which involves the use of a simple liquid-liquid extraction of nitrosamines and is suitable for screen- ing large number of samples. The final determination of the nitrosamines is made by gas chromatography of the nitramine derivatives according to the method of Sen,6 after clean-up on a column of alumina.EXPERIMENTAL As methylene chloride has been found by most workers to be the most suitable solvent for the extraction of nitrosamines, attempts were first made to extract, with methylene chloride, a 1 + 1 water - ethanol mixture containing known amounts of seven different nitrosamines. After separating it, the solvent phase was dried over sodium sulphate and distilled in a Kuderna-Danish apparatus. The collected fractions of the distillate and the residue were then analysed for nitrosamines by gas chromatography. As expected, the co-extracted ethanol carried into the solvent phase an appreciable amount of water, with @ SAC and the authors.CASTEGNARQ, PIGNATELLI AND WALKER 157 the result that the nitrosamines passed into the distillate and were lost.The problem, therefore, was to prevent extraction of water and ethanol into the methylene chloride phase. Calcium chloride is known to bond loosely with ethanol; therefore, extraction was attempted with simulated spirit (1 + 1 water - ethanol), after first saturating the solution with calcium chloride. However, on shaking the mixture in a separator, emulsions formed that were difficult to break down. As an alternative, magnesium perchlorate, which is soluble in ethanol and has a high affinity for water, was tried, with success. On separation, the volumes of the two layers were only slightly changed and the solvent could be completely distilled off without significant increase in the temperature of distillation.Ethanol was mainly retained in the water. A minor problem was encountered in differentiating the layers when the concentration of ethanol was varied about the ratio 1 : 1. As the difference in density between the perchlorate-saturated water - ethanol solution and methylene chloride is small, for relatively small variations in the percentage of ethanol, the methylene chloride may be found as either the upper or the lower layer. On addition of sufficient ethanol to bring its content to about 55 per cent., the two layers separate cleanly, with methylene chloride as the bottom layer. All subsequent extractions were made on solutions containing 55 per cent. of ethanol. Alternatively, if concentrations of ethanol are adjusted with water to be less than 45 per cent., the phases readily separate with methylene chloride as the upper layer.The method can thus be varied to suit the type of product to be analysed. The partition between the two phases was checked on 10 p.p.m. solutions of nitrosamines in 1 + 1 water - ethanol and by measuring those in the methylene chloride extract in an ultraviolet spectrophotometer. Three extractions with methylene chloride were sufficient to remove all the nitrosamines from the perchlorate-saturated water - ethanol phase. This extraction technique was applied to solutions of ethanol in water containing 10 pg k g l of each of seven nitrosamines and the methylene chloride extract analysed for the nitrosamines. The recoveries were generally found to be of a similar order to those found by Sen and Dalpe.5 The method was then applied to samples of nitrosamine-free calvados, both with and without addition of the nitrosamines. Accurate analysis was difficult owing to the presence of a large number of interfering peaks.Attempts to reduce their number by distillation of the calvados from alkaline and acidic solution were ineffective and, furthermore, led to lower recoveries, particularly of the higher-boiling nitrosamines. The problem was overcome by using an alumina column similar to that used by Telling' for the determination of nitrosamines in foodstuffs. The method required some modification as the neutral-grade alumina used by this author did not remove all the interfering peaks in the chromatograms originating from the calvados. Those peaks which remained were removed by using an alkaline grade, and an effective composite column of the two types of alumina was devised .Fig. 1 illustrates a chromatograni obtained from a calvados sample that contained 10 pg k g l of each of seven nitrosamines before clean-up. Figs. 2 and 3 illustrate the chromatograms obtained from the same calvados sample with and without addition of the nitrosamines after clean- up on the alumina column. METHOD OF ANALYSIS FOR NITROSAMINES IN CALVADOS MATERIALS- N-Nitroso cosqbounds-Nitrosodimethylamine, nitrosodiethylamine , nitrosodipropylamine and nitrosodibutylamine were obtained from Eastman Kodak Ltd. and nitrosomethylpentyl- amine from Schuchardt, Munich, and all were distilled before use. Nitrosopyrrolidine was kindly supplied by Dr.Eisenbrand of the Krebsforschungszentrum, Heidelberg, and nitroso- ethylmethylamine by Mr. J. F. Palframan of the Laboratory of the Government Chemist, London. All samples contained gas-chromatographic impurities in concentrations of less than 1 per cent. SOLVEXTS- Methylew chloride-Merck, analytical-reagent grade, was redistilled from sodium car- bonate in 2-litre batches, the first 100 ml being rejected. All other solvents were of analytical- reagent grade. REAGENTS- Hydrogen peroxide solution, 50 per cent. m/V.158 CASTEGNARO et al. : SEMI-QUANTITATIVE DETERMINATION OF [Analyst, Vol. 99 Trijuoroacetic acid-Merck, analytical-reagent grade, was redistilled before use. Magnesium perchlorate-Prolabo, laboratory grade. Sodium sulphate, adzydrous-Merck, analytical-reagent grade.50 40 30 20 10 Time/minutes I Fig. 1. Chromatogram illustrating the masking of nitramine peaks by other oxidation products : arrows indicate the position of the peaks for dimethylnitramine (a) ; ethylmethyl- nitramine (b) ; diethylnitramine (c) ; dipropylnitramine (d) ; methylpentylnitramine (e) ; dibutylnitramine (f) ; and pyrrolidylnitramine (g) EXTRACTION- The approximate percentage of ethanol in the calvados is first measured by means of a hydrometer. To a 50-ml aliquot of sample sufficient ethanol is added to bring its concen- tration to about 55 per cent. To this solution is then added sufficient anhydrous magnesium perchlorate to saturate the solution, the magnesium salt being added in successive amounts of about 10 g and the solution cooled under a tap after each addition (interaction between water and anhydrous magnesium perchlorate is exothermic).The saturated solution is then extracted with three successive 50-ml portions of methylene chloride, which are bulked and shaken in a second separator with 35 to 40 g of anhydrous sodium sulphate so as to remove any of the water - ethanol phase that may be carried through in suspension, and the methylene chloride layer is run off into a suitable apparatus for removal of the solvent. The sodium sulphate is finally washed with 1 O m l of methylene chloride, which is added to the bulked extracts. Two methods of evaporation have been carried out, (a) by using the Kuderna-Danish apparatus, and (b) by evaporation from a 250-ml round-bottomed flask immersed in a water- bath at 60 "C and flushed with a gentle current of nitrogen.In each instance the volume of solvent is reduced to between 0.5 and 1 ml. The results shown in Table I indicate that better recoveries for the more volatile nitrosamines are obtained by using the Kuderna-Danish apparatus. The second method, however, is much more rapid and shows little disadvantage for the higher nitrosamines.March, 19741 VOLATILE N-NITROSAMINES IN ALCOHOLIC BEVERAGES 159 PREPARATION OF NITRAMINES- The nitrosamines in the residue are oxidised to nitramines with a mixture containing 5 ml of trifluoroacetic acid and 4 ml of 50 per cent. hydrogen peroxide solution.6 After gently shaking it overnight at room temperature, the solution is cooled in an ice-bath, the pH adjusted to between 10 and 11 by dropwise addition of a 20 per cent.solution of potassium carbonate and then extracted with three successive 50-ml volumes of methylene chloride. The extracts are bulked in a separator and dried by shaking with 15 to 20 g of anhydrous sodium sulphate. The methylene chloride is then transferred into a flask as before. The sodium sulphate is washed with 20 ml of methylene chloride, which is added to the bulked extracts, and the volume of solvent reduced in the water-bath at 60 "C to slightly less than 10ml. The concentrate is then transferred, with washing, into a graduated test-tube and its bulk is further reduced in a current of nitrogen to slightly more than 6 ml (it is important not to reduce the volume below this level as nitramines may be lost); 2 ml of hexane are then added and the bulk further reduced to between 0-7 and 06ml (again, care must be taken not reduce the volume below 0.5ml).The volume is finally adjusted to 5ml with pentane and the solution is ready for the final clean-up by column chromatography. I I I I I U 50 40 30 20 10 0 Time/rninutes Fig. 2. Chromatogram illustrating the seven nitramines a to g (Fig. l), gas chromatographed a t 130 "C, as indicated by the arrows, after clean-up on the alumina column COLUMN CHROMATOGRAPHY- The column consists of three layers: a bottom layer of 3 g of basic aluminium oxide, activity 11; a middle layer of 3 g of neutral aluminium oxide, activity 111; and, at the top, a layer of 1 g of anhydrous sodium sulphate. A glass column, 1 em in diameter and 25 cm in length, is used.Each type of oxide is prepared by first activating the appropriateMerck grade (ie., basic or neutral) of activity I by heating it in an oven for 3 hours at 240 "C. After cooling in a desiccator, 100 g of the activated material are shaken in a stoppered flask for 3 hours with the appropriate amount of water (3g for basic aluminium oxide, activity 11, and 6 g for neutral aluminium oxide, activity 111). The stoppered flask is then left to stand overnight so as to enable the mixture to equilibrate. Three grams of the basic aluminium oxide (activity 111) are then agitated gently for half an hour with a sufficient volume of a 1 per cent. solution of &ethyl ether in pentane160 CASTEGNARO et al. : SEMI-QUANTITATIVE DETERMINATION OF [Analyst, Vol.99 I I I I Time/m i nu tes Fig. 3. alumina column. (Fig. 1) would be found Chromatogram obtained for a blank calvados after clean-up on the Arrows indicate the positions a t which the nitramines a to g to cover the solid and desorb gases from the alumina. The washed alumina is then added to the glass column and solvent run off from the bottom of the column until the top of the solvent layer is level with the upper surface of the alumina. This procedure is repeated with the neutral aluminium oxide (activity 11) and then finally 1 g of anhydrous sodium sulphate is added to the top of the column. The pentane solution containing the nitramines is then added to the column and liquid run out until the upper level coincides with the upper surface of the column.The column is eluted successively, first with 25 ml of a 1 per cent. solution of diethyl ether in pentane, then with 40ml of a 25 per cent. solution of diethyl ether in pentane and finally with diethyl ether alone. The first 25 ml of eluate are rejected, after which it is collected in 5-ml fractions. From each fraction a 5-pl portion is injected into the gas chromatograph. Finally, the solutions are bulked and the volume is reduced to 5ml by evaporation on a water-bath. A suitable aliquot is then injected into the gas chromatograph. The minimum level of detection is about 1 pg k g l of nitrosamine in the original sample. The volume of ether can be further reduced to 0.5 ml without loss of nitramine, giving a factor of 10 on the peak size. This step is preferable for accurate measure- ments of the concentrations below 10 pg k g l .So far it has not been possible to adjust conditions so as to obtain complete separation of the individual nitramines on the alumina column. However, as the retention volume of each nitramine is reasonably reproducible, analysis of each fraction separately increases confidence in identification. The nitramines are eluted from the column in order of polarity, the least polar being eluted first, i.e., in the order: dibutyl, dipropyl, diethyl, methylpentyl, ethylmethyl, dimethyl and, finally, the pyrrolidyl compound. The total volume of eluate is normally about 55 ml but varies slightly from batch to batch of the prepared alumina, and it is therefore necessary to run nitramine standards for each batch.The variation has been found not to be more than 5 ml for the nitramines that have the longest retention times when using freshly prepared alumina. Recovery of the nitramines after having passed through the column, the fractions having been bulked and the volume reduced, is 100 per cent.March, 19741 VOLATILE N-NITROSAMINES IN ALCOHOLIC BEVERAGES 161 GAS CHROMATOGRAPHY- Pye 104 gas chromatographs fitted with nickel-63 electron-capture detectors were used with 6 foot x 4 inch 0.d. glass columns packed with 10 per cent. Carbowax 20M on DMCS- treated Chromosorb W. The columns were conditioned at 220 "C for 2 days before use. The determination of nitramines in the series dimethyl to the methylpentyl derivative was performed isothermally a t 130 "C with a nitrogen flow of 50 ml min-l through the column and a scavenge flow of 20mlmin-l through the detector.The injection temperature was 170 "C and the chart speed 5 mm min-1. The determination of the dibutyl- and pyrrolidyl- nitramines was performed on a second chromatograph at 160 "C. With a second instrument available this procedure was found to be more convenient than temperature programming. RESULTS The recoveries obtained in the replicate analyses of the aqueous ethanolic solution containing 10 pg k g l each of seven nitrosamines are illustrated in Table I, where the results shown were obtained by using both methods for evaporation of the solvent. Table I1 shows the recoveries for the same concentration of nitrosamines in four different samples of calvados; in this instance only the rapid method of evaporation in the water-bath was used.Recoveries were good, with the exception of nitrosodimethylamine and nitrosopyrrolidine, losses of which, even when using a Kuderna-Danish apparatus, still tended to occur. TABLE I RECOVERY OF THE SEVEN NITROSAMINES FROM THE WATER - ETHANOL MIXTURE CONTAINING 10 pg kg-1 OF EACH NITROSAMINE Recovery of each nitrosamine, per cent. A f 1 (4 Nitrosamine - Nitrosodimethylamine . . .. .. 33 49 46 Nitrosoethylmethylamine . . .. 52 64 89 Nitrosodiethylamine . . .. .. 86 78 76 Nitrosodipropylamine . . .. .. 83 98 82 Nitrosodibutylamine . . .. .. 96 110 100 Nitrosomethylpentylamine . . .. 97 92 79 Nitrosopyrrolidine . . . . .. 70 70 43 (4 7 - 42 26 48 38 22 79 33 60 47 43 71 95 64 64 57 79 83 100 100 72 75 86 100 100 95 104 100 89 100 100 40 30 60 70 60 (a) Methylene chloride removed by using a Kuderna-Danish apparatus.(b) Methylene chloride removed by evaporation on a water-bath. As acidic and alkaline distillation stages are frequently used in the clean-up of nitrosamines extracted from food, a number of analyses of both water - ethanol and calvados containing nitrosamines were carried out with the inclusion of a distillation stage. Distillation was carried out nearly to dryness and the distillate analysed by using the method described above. The results shown in Table I11 indicate considerable losses of higher nitrosamines, which, we feel, indicates the value of a liquid - liquid extraction technique in analysing alcoholic samples for nitrosamines. TABLE I1 RECOVERY OF SEVEN NITROSAMINES, ADDED AT THE 10 pg k g l LEVEL, FROM FOUR DIFFERENT SAMPLES OF CALVADOS* Nitrosamine Nitrosodimethylamine .. .. .. .. Nitrosoethylmethylamine . . .. .. Nitrosodiethylamine . . .. .. .. Nitrosodipropylamine . . .. .. .. Nitrosomethylpentylamine . . .. .. Nitrosodibutylamine . . .. .. .. Nitrosopyrrolidine . . .. .. .. Recovery of each nitrosamine, per cent. it3 82 2s 2; 76 83 76 73 45 71 44 49 88 99 93 83 99 102 80 98 102 103 63 68 40 40 26 50 * Methylene chloride was removed by simple evaporation on a water-bath.162 CASTEGNARO, PIGNATELLI AND WALKER TABLE I11 RECOVERY OF NITROSAMINES FROM THE DISTILLATE OF A WATER - ETHANOL Each sample was initially spiked with nitrosamines at the level of 10 pg k g l MIXTURE AND FOUR DIFFERENT SAMPLES OF CALVADOS Nitrosamine Nitrosodimethylamine .. Nitrosoethylmethylamine Nitrosodiethylamine . . Nitrosodipropylamine . . Nitrosomethylpentylamine Nitrosodibutylamine . . Nitrosopyrrolidine . . Recovery of each nitrosamine, per cent. A f- > (4 (b) - f 1 .. .. 20 31 34 14 85 16 8 .. .. 21 83 58 26 84 77 106 .. . . 29 Trace 59 Trace 71 19 27 I . . . 33 Trace 66 Trace 91 28 32 .. .. 45 65 75 Trace 90 18 22 .. . . 22 Trace Trace Trace 100 31 29 .. . Trace Trace Trace Trace Trace 25 37 (a> Water - ethanol mixture. (b) Four different samples of calvados. As an example of samples with low alcohol content, the liquid - liquid extraction technique was used for the analysis of a number of beers spiked with the seven nitrosamines as before. Recoveries again were in the same range as those for calvados. However, as interference from other oxidation products in the gas-chromatographic analysis was considerably less than for calvados, it was possible to eliminate the column clean-up procedure, thus considerably diminishing the time of analysis. REFERENCES 1. Tuyns, A. J., I d . J . Cancer, 1970, 5, 152. 2. Collis, C. H., Cook, P. J., Foreman, J. K., and Palframan, J. F., Lancet, 1972, ii, 441. 3. ---- , Gut, 1971, 12, 1015. 4. Crosby, N. T., Foreman, J. K., Palframan, J. F., and Sawyer, R., in Bogovski, P., Preussmann, R., and Walker, E. A., Editors, “N-Nitroso Compounds, Analysis and Formation. Proceedings of a Working Conference held a t the Deutsches Krebsforschungszentrum, Heidelberg, 13-15 October, 1971,” IARC Scientific Publication No. 3, International Agency for Research on Cancer, Lyon, France, 1972, p. 38. 5. Sen, N. P., and Dalpe, C., Analyst, 1972, 97, 216. 6. Sen, N. P., J . Chromat., 1970, 51, 301. 7. Telling, G. M., Ibbid., 1972, 73, 79. Received July 17tk, 1973 Accepted August 29tk, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900156

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

Analyst, March, 1974, Vol. 99, @. 163-165 163 A Combustion Method for the Determination of Total Sulphur in Limestones BY L. M. RUNDLE (Geochemical Division, Institute of Geological Sciences, 64-78 Gray's Inn Road, London, WClX SNG) A method is described for the determination of total sulphur in limestones. The sample is mixed with tungsten trioxide and quartz flour and placed in a boat of refractory material. The boat and contents are inserted into an aluminous porcelain combustion tube maintained at a temperature of about 1200 "C in a resistance-type tube furnace. The combustion tube carries a coil of copper gauze at the outlet end to reduce sulphur trioxide. Nitrogen is used as carrier gas and the sulphur dioxide evolved is absorbed in dilute hydrochloric acid containing potassium iodide and starch as indicator, titra- tion with standard potassium iodate solution being carried out as the analysis proceeds.SINCE this Institute undertook a feasibility study for a survey of resources of limestones, the need for a rapid method for the determination of sulphur in such material became apparent. The classical method of precipitating sulphur as barium sulphate is time consuming and susceptible to errors. Many combustion method~l-~ have been used, all of which involved somewhat different conditions. That proposed by Coller and Leiningerl requires a high tem- perature and equipment that is not readily available in this laboratory. The method of Sen Gupta2 in which vanadium pentoxide was used as a flux was found to give low recoveries when applied to the analysis of limestones, and tests made on the residues from the com- bustion boats in this method showed that some sulphur remained in them. Results improved when the heating time was increased from 30 to 50 minutes or longer, but in view of the number of samples to be analysed, this heating time was not practicable.In an attempt to overcome these problems, a flux composed of tungsten trioxide mixed with quartz flour, and a furnace temperature of 1200 "C were used. Results were slightly improved but still tended to be low. Examination of the residue showed sulphur to be absent, so it was assumed that part of the sulphur had been lost as sulphur trioxide. Intro- duction of a piece of copper gauze, about 40 x 120 mm, in the form of a 40-mm long coil into the outlet end of the furnace tube where the temperature lay in the range 750 to 850 "C overcame the losses.Results obtained with the modified method compared favourably with certificate values and gravimetric determinations. Samples other than limestones were also analysed and the results indicated that the method could be used for a wide variety of geo- logical materials regardless of the form in which sulphur was present. The presence of the copper gauze had no detrimental effect if sulphur were present as sulphide. EXPERIMENTAL APPARATUS- The apparatus used is shown in Fig. 1 and consists of the following: a furnace capable of maintaining a temperature of 1200 "C; an aluminous porcelain combustion tube, 0.6 mm long and of 20 mm i.d.; combustion boats of high-alumina fireclay with over-all dimensions of length 72 mrn, width 16 mm and depth 10 mm; oxygen-free (white-spot) nitrogen from a cylinder (further purification is unnecessary) ; a 100-ml tall-form measuring cylinder; and a multi-hole bubbler that reaches the bottom of the measuring cylinder.REAGENTS- Tungsten trioxide. Dilute hydrochloric acid (1.5 + 98.5). Potassium iodide solution, 3.0 per cent. m/V. Quartz flour, 240 mesh B.S.S. Starch solution, 2.0 per cent. m/V. @ SAC and the author.164, RUNDLE A COMBUSTION METHOD FOR THE DETERMINATION [Analyst, VOl. 99 Standard potassium iodate solution, 0.006 25 N-Disso1ve 0.223 g of potassium iodate (previously dried for 1 hour at 110 "C) in distilled water, dilute the solution to 1 litre in a calibrated flask and mix thoroughly. 1 ml of solution = 0-10 mg of sulphur.Copper gauze, 40 mesh. PROCEDURE- Mix, in a small bottle, 1 g of tungsten trioxide, 0.5 g of quartz flour and a known suitable amount of sample. To a measuring cylinder containing 80 ml of dilute hydrochloric acid (1.5 + 98.5) add 1 ml of potassium iodide solution and 1 ml of starch indicator. Place the measuring cylinder in position at the end of the combustion train. Pass nitrogen through the system at a steady rate (approximately 150 ml min-l) and titrate the absorbing solution with the standard potassium iodate solution to a faint blue colour. (The intensity of the blue colour can be adjusted to suit individual needs.) Resistance furnace I . :; r'..' . . .. . . ,,:, the red zone) Soda- Magnesium asbestos perch lorate Fig.1. Apparatus for the determination of total sulphur Transfer the pre-mixed sample into a combustion boat (which has been ignited previously and then cooled) made of refractory material and insert the boat quickly into the hot zone of the furnace (1200 "C). Continue to pass nitrogen through the system and titrate the absorbing solution so as to maintain the blue colour. Continue passing the nitrogen for about 15 minutes or until the colour of the absorbent remains unchanged for 2 to 3 minutes. Wash the inside of the bubbler by closing the screw-tap and again opening it. Any condensa- tion appearing on the glass tube should be vaporised either by increasing the nitrogen flow-rate at the end of the run or by heating the tube gently with a burner.If the colour of the absorbent fades, add another drop of titrant and ensure that the colour remains unchanged after a further 2 minutes. Carry out a blank test by using tungsten trioxide and quartz flour and make any necessary changes to the volume of titrant used. Sulphur, per cent. = - 100 x w Note the volume of potassium iodate solution required. 7) where v ml is the volume of potassium iodate solution used after correcting for the blank and w g the amount of sample taken. RESULTS Results obtained on standard materials by using the above method are shown in Table I. Selected limestone samples covering the whole range of sulphur contents encountered in the feasibility study were analysed by both the combustion method and the barium sulphate gravimetric method, which was applied after decomposition of the sample by fusion with sodium carbonate.The results of these determinations are shown in Table 11.March, 19741 OF TOTAL SULPHUR I N LIMESTONES TABLE I RESULTS OBTAINED ON STANDARD MATERIALS Total sulphur, per cent. 165 r 1 Value by Sample Certificate value combustion meth3d Argillaceous limestone N.B.S. No. la 0.27 0.28 Portland cement N.B.S. No. 177 . . 0.637 0.623 B.C.S. iron ore “A” . . .. .. 0.063 0.055 U.S. Geological Survey: Standard rock BCR-1 . . .. 0-04 0.040 Standard rock GSP-1 . . .. 0.03 0.033 The coefficients of variation obtained for the argillaceous limestone and Derbyshire limestones Nos. 4 and 11 were 6-07, 6-02 and 3.15 per cent., respectively, based on twelve determinations in each instance.The procedure described is satisfactory for all types of samples tested, although the heating period can be varied slightly, depending upon the form in which the sulphur is present. For example, if the sulphur is present entirely as sulphide the heating period can be reduced to less than 15 minutes. The copper gauze turns black after three or four runs and should be replaced; it can, however, be re-used after the oxide layer has been removed. In order not to melt the copper, it is important to place the gauze just outside the red zone, otherwise its purpose is not served. TABLE I1 RESULTS OF ANALYSIS OF LIMESTONE SAMPLES Total sulphur, per cent, by gravimetric combustion A I ’I Sample method method Derbyshire limestone No. 4 .. 0.05 0.04 No. 11 .. 0.50 0.45 No. 12 . . 1.95 1-92 Calcareous marl . . .. .. 0.22 0.22 CONCLUSIONS A rapid combustion - titrimetric method has been developed for the determination of sulphur in limestones. By using a flux composed of a mixture of tungsten trioxide and quartz flour, good sulphur recoveries are obtained over a wide range of sample compositions. Inclusion of a coil of copper gauze in the cooler part of the furnace ensures that any sulphur trioxide evolved is reduced to dioxide. The method is comparable in accuracy with the barium sulphate gravimetric method but has the advantage of greater speed. The author thanks Mr. P. J. Moore for helpful discussion with this paper, and the Director of the Institute of Geological Sciences for permission to publish this work. REFERENCES 1. 2. 3. Coller, M, E., and Leininger, R. K., Anulyt. Chem., 1955, 27, 949. Sen Gupta, J. G., Ibid., 1963, 35, 1971. - , Analytica Chim. Acta, 1970, 49, 519. Received July 9th, 1973 Accepted October 16t12, 1973

ISSN:0003-2654    

DOI:10.1039/AN9749900163

出版商:RSC    

年代:1974

数据来源: RSC

摘要:

166 Analyst, March, 1974, Vol. 99, $+. 166-167 A Comparison of Two Procedures for the Determination of Organobromine by the Schoniger Oxygen-flask Method BY RONALD C. DENNEY AND PHILIP A. SMITH (School of Chemistry, Thames Polytechnic, London, SE18 6PF) A statistical comparison of the argentimetric and mercurimetric methods of finish in the oxygen-flask determination of organobromine has been carried out. While no statistical difference was found to exist, the mercurimetric method is recommended for use in laboratories that lack the facilities required for the potentiometric determination. THE oxygen-flask method for the determination of organically bound bromine still follows the basic combustion method developed for micro-scale work by Schoniger.lS2 In all analyses, assuming that total combustion takes place, accuracy is dependent upon conversion of the organobromine into a suitable form for determination by a well established method of finish.Various methods of finish have been recommended for the determination of bromine in organic compounds by the oxygen-flask m e t h ~ d . ~ , ~ Iodimetric, argentimetric and mercuri- metric titrations have all been extensively used but controversy remains over their relative merits. There is also disagreement over the need to add hydrogen peroxide to the absorbing solution.3~5 Childs, Meyers Cheng, Laframboise and Balodis,6 for example, demonstrated that for bromine determinations absorption of combustion products is rapid if a solution of either hydrazinium sulphate or hydrogen peroxide in sodium hydroxide solution is used.We have, therefore, carried out a statistical comparison of the argentimetric and mercurimetric titra- tions, both with and without the use of hydrogen peroxide. EXPERIMENTAL APPARATUS- The oxygen-flask combustion unit manufactured by Thomas and Co., Philadelphia, USA., was used because of the safety features it incorporates. We have found the method involving ignition with an infrared lamp by remote control to be of particular value in intro- ducing inexperienced workers to oxygen-flask procedures. ABSORPTION SOLUTION- For each determination in the statistical study, the absorption solution consisted of 10 ml of approximately 0.1 M sodium hydroxide solution containing 0.3 ml of 100-volume hydrogen peroxide. After ignition, the flask was left to cool for 10 minutes before being shaken in order to assist absorption of the combustion products in the absorption solution.METHODS OF FINISH- Argentimetric jnish-Titration of the neutralised solution with 0.01 M silver nitrate solution was carried out potentiometrically by using essentially the procedure recommended for chlorine determinations by the Analytical Methods Committee.' For our determinations, the absorption solution was transferred from the combustion flask into a 100-ml beaker with 40 ml of distilled water, The pH of the solution was adjusted to between 6.0 and 6.2 measured with a glass - calomel pH electrode. The titration was carried out by using a glass - silver electrode system with the potential measured on a millivoltmeter.Mercwimetric jnish-The traditional method of titration of bromide ions with standard mercury( 11) nitrate, using diphenylcarbazone with bromophenol blue as a screen, gives poor results in aqueous solution. The modified procedure developed by ChengS and applied extensively by White: which involves the use of an 80 per cent. ethanolic solution, was used for our determinations. @ SAC and the authors.DENNEY AND SMITH 167 Following the combustion procedure the flask was washed with 10 ml of distilled water and acidified with 0.1 M nitric acid to the yellow colour of bromophenol blue; 100 ml of absolute ethanol and 0.5 ml of 0-1 M nitric acid were added and the pH was adjusted to 3-6. After addition of diphenylcarbazone solution (0.5ml of a 0.1 per cent. ethanolic solution), the solutions were titrated with 0.01 M mercury(I1) nitrate solution to the first appearance of a purple colour.RESULTS AND DISCUSSION All determinations were carried out on micro-analytical reagent grade 3-bromobenzoic acid (Br content 39.75 per cent.) with 10 to 20-mg samples. Corrections for blanks were applied to all results. An initial set of results obtained in the absence of hydrogen peroxide in the absorption solution were discarded as they were found to be irregular and valueless from an analytical point of view. This finding was in sharp contrast with Steyermark’s views,5 but confirmed those expressed by Schoniger,lV2 Macdonald3 and Childs et aZ.6 Argentimetric finish 39.68, 39-35, 39.78, 39.85, 39-57, 39.57, 39-86, 39.36, 39.13, 39.56 Mean .. . . 39.57 Standard deviation . . . . 0.237 Mercurimetric finish 40.00, 39.71, 39.56, 39-84, 39.75, 39-51 Mean .. . . 39.71 Standard deviation . . . . 0.202 The results obtained for bromine, per cent., were as follows: (ten determinations) (six determinations) STATISTICAL COMPARISON OF RESULTS- Student’s &testlo was used and for the above calculated standard deviations t was found to be 1.21. As the value of t obtained lies between + 1.76 and -1.76, there is no statistically significant difference between the two means. At this level, P = 0.2 to 0.3. From a comparison of precision by using the F distribution with the above values, F is 1.38. At the 5 per cent. level, Fo.05 is given as 4.77 and as the value found for F is less than 4.77, there is no significant difference in the precision of the means obtained by the two methods.CONCLUSIONS This investigation has shown that for argentimetric and mercurimetric finishes it is essential to include hydrogen peroxide in the absorbent solution. Our initial results were wholly unrelated to each other in the absence of hydrogen peroxide. The statistical analysis of the results supports Steyemark’s contention5 that there is no significant difference between the results obtained by these two methods. It is, therefore, apparent that even a poorly equipped laboratory that lacks the facilities for potentiometric titrations can obtain reproducible results for the determination of bromine by using the less sophisticated mercurimetric finish. Of the two methods it has, in any event, the great ad- vantage of being more rapidly carried out. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. REFERENCES Schoniger, W., Mikrochim. Acta, 1955, 123, -- , Ibid., 1956, 869. Macdonald, A. M. G., in Reilley, C. N., Editor, “Advances in Analytical Chemistry and Instrumenta- tion,” Volume 4, Interscience Publishers, New York, London, Sydney and Toronto, 1965, p. 75. Belcher, R., Gawargious, Y. A,, Gouverneur, P., and Macdonald, A. M. G., J . Chem. Soc., 1964,3560. Steyermark, A., J . Ass. 08. Agric. Chem., 1965, 48, 709. Childs, C. E., Meyers, E. E., Cheng, J., Laframboise, E., and Balodis, R. B., Microchem. J., 1963, Analytical Methods Committee, Analyst, 1963, 88, 415. Cheng, F. W., Microchem. J., 1959, 3, 537. White, D. C., Mikrochim. Acta, 1961, 449. Spiegel, M. R., “Theory and Problems of Statistics,” Schaum’s Outline Series, McGraw-Hill Book Received July 29th. 1971 Amended October l s f , 1973 Accepted October 4th, 1973 7, 266. Co., New York, Toronto, London, Sydney and San Francisco, 1961.

ISSN:0003-2654    

DOI:10.1039/AN9749900166

出版商:RSC    

年代:1974

数据来源: RSC