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476
Analyst, July, 1975, Vol. 100, pp. 476-481
The Determination of Unsulphonated Primary Aromatic Amines in Water-soluble Food Dyes and Other Food Additives E. J. Dixon and D. M. Groffman Department of Industry, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SE19NQ
EEC directives prescribe limits for the amount of unsulphonated primary aromatic amines present in food colouring matters in general and prohibit the presence of particular carcinogenic amines in food dyes and other food additives. Integrated methods based on solvent extraction and thin-layer chromatography have been devised for identification of these amines, followed by the spectrophotometric determination of their condensation products with 4-dimethylaminocinnamaldehyde. The method is also extended to the determination of primary aromatic amines in biphenyl and to the determination of aminoazobenzenes in the dye Fast Yellow. An additional thin-layer chromatographic method is given for the differentiation between the isomers of naphthylamine as their tosyl derivatives.
Directives in force within the European Economic Community (EEC)l require synthetic organic colouring matters for use in foods not to contain more than 0.01 per cent. of free aromatic amines nor more than 0-5 per cent. of intermediate synthetic products other than free aromatic amines. Further, they should not contain 2-naphthylamine, benzidine or 4-aminobiphenyl (xenylamine) or their derivatives. In Britain, the use of these three compounds is prohibited,2s3except for special research purposes, and as a result authentic reference samples are difficult to obtain. In general, for purposes of identification the analyst has to rely on the physical data for these substances of unspecified purity quoted in the literature.194.5 Examples of such data are given in Tables I-IV. Few up-to-date methods for the analysis of food dyes have been published in recent years and those previously available6-s are considered to lack sensitivity and specificity. Several gas-chromatographic methodsg910are available but are unsuitable because of the difficulty of expressing mixtures of amines as “aniline,” as required in the EEC directives, and because gas - liquid chromatography by itself would not give an unambiguous identification. Methods that depend on the production of a diazonium compound, followed by addition of a coupling agent in order to produce a colour, require specific conditions for each amine under test and for each individual coupling agent. Several thin-layer chromatographic method~~9~~912 are suitable for the separation of the amines under consideration; some of these methods have been adapted to the determination of these amines in dyes, as also have some spectrophotometric4J3 met hods. The spectrophotometric methods adopted depend on the formation of Schiff’s bases and afford quantitative determinations of individual amines that had previously been identified by thin-layer chromatography. Mixtures of amines are more difficult to analyse spectrophotometrically but a semi-quantitative estimate can be made following separation with thin-layer chromatography by comparing the intensities of individual spots with standards. However, the method is not sufficiently sensitive for a fully quantitative determination to be made by scraping off the spots and eluting the amines with solvent in order to give a solution of a known volume. Section 1 of this paper describes a basic procedure for the identification and determination of unsulphonated primary aromatic amines. However, it is not possible by this procedure to distinguish between 1- and 2-naphthylamines; a suitable method for this purpose is given in section 4. Section 2 describes the modifications necessary to make section 1 applicable t o the examination of biphenyl, and section 3 extends the general procedure to the determination of aminoazobenzenes in Fast Yellow. Crown Copyright.
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DIXON AND GROFFMAN
477
1. Determination of Unsulphonated Primary Aromatic Amines in Published on 01 January 1975. Downloaded by University of Prince Edward Island on 26/10/2014 10:29:59.
Water-soluble Food Dyes Apparatus Separating funnels. 100-ml capacity, with a PTFE stopcock and glass stopper. Capillary tubes or micropipettes. 5-p1 capacity, for spotting samples on to thin-layer chromatographic plates, and 100-pl capacity, for dispensing 3 M hydrochloric acid. PiPettes. 1- and 5-ml capacity. Pasteur ca9illary pipettes. Filter-paper. Strips of a Whatman low-ash grade filter-paper, approximately 20 x 50 mm. Cellulose thin-layer chromatographic Plates. Prepare the plates as follows: shake 20 g of microcrystalline cellulose powder with 60 ml of methanol for 2 min, then immediately spread the mixture on 200 x 200-mm glass plates to a thickness of 0.25 mm. After air-drying them for 30 min, dry the plates for 10 min at 105 "C. Up to five plates can be prepared in one application by using the above amount of mixture. Equally suitable are 200 x 200-mm ready-made plates of 0-1-mm thickness, which are obtainable from E. Merck. Test-tubes. 10-ml capacity, pointed and graduated, with ground-glass necks and stoppers. Recording spectrophotometer. Suitable for scanning in the range 350-600 nm. Spectrophotometer cells. 10-mm path length.
Reagents Sodium hydroxide solution, 1 M. Toluene. Analytical-reagent grade. Hydrochloric acid, 3 M. Methanol. Analytical-reagent grade. Propan-2-ol. Analytical-reagent grade. Mobile solvent for thin-layer chromatography. Dissolve 1.0 g of sodium acetate in about 10ml of distilled water and add 0.10ml of glacial acetic acid. Transfer the mixture, with aqueous washings, into a 100-ml calibrated flask and make the volume up to the mark with water. 4-Dimethylaminocinnamaldehyde ( D A C ) solution A . Dissolve 0.1 g of DAC in about 50 ml of methanol, carefully add 5 ml of concentrated sulphuric acid, transfer the mixture plus washings with methanol into a 100-ml calibrated flask and make the volume up to the mark with methanol. DAC solution B. Dissolve a mixture of 0.1 g of DAC and 0.1 g of P-toluenesulphonic acid in about 10ml of propan-2-01, transfer the mixture plus washings with propan-2-01 into a 100-ml calibrated flask and make the volume up to the mark with propan-2-01. 4-Dimethylaminobenzaldehyde solution ( D A B ) . Dissolve 1 g of DAB in about 10 ml of methanol, add 4 ml of concentrated hydrochloric acid, transfer the mixture plus washings with methanol into a 100-ml calibrated flask and make the volume up to the mark with methanol.
Procedure Weigh accurately about 3 g of dye into a separating funnel containing 50ml of water. Shake the funnel in order to dissolve the sample. Add 10 ml of 1 M sodium hydroxide solution and mix, then add 5 ml of toluene and shake the funnel for 1 min. Allow the layers to separate and discard the lower aqueous layer. Add another 5-ml portion of 1 M sodium hydroxide solution and shake the funnel for 1 min. If on separation the lower layer is coloured (caused by partition of the dye between the organic and inorganic phases) discard the lower layer and repeat the above extractions with alkali. Finally, wash the toluene extract with two 10-ml portions of water and discard the washings. Dry the inside of the stem of the separating funnel, then insert a 20 x 50-mm rolled-up strip of Whatman low-ash filter-paper. Transfer the toluene extract into a graduated, pointed ground-glass test-tube containing 100 & 2 pl of 3 M hydrochloric acid, stopper the tube and shake it vigorously for about 30 s. Allow the acid to sink to the bottom of the tube (it may be helpful to tap the side of the tube) and carefully remove as much as possible of the toluene with a Pasteur capillary pipette, the toluene being discarded. Spot two 5-pl portions of the remaining acidic solution about 100 mm apart, and each 20 mm from the bottom of a cellulose thin-layer chromatographic plate, and spot 5 p 1 of appropriate standards alongside each spot under test. Run the
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478
DIXON AND GROFFMAN
: DETERMINATION
OF UNSULPHONATED PRIMARY
Analyst, VoE. 100
chromatogram in a tank containing the prescribed mobile solvent until the solvent has travelled about 150 mm above the base-line. Allow the plate to dry, cover one set of standards and sample with a glass plate and spray the remaining set with DAC solution A. Cover the other half of the plate, then spray with DAB solution. Compare the positions and colours of spots in the sample with the standards. The chromatographic behaviour of some amines that may be present is shown in Table I.
TABLE I CHROMATOGRAPHIC PROPERTIES OF DAB AND DAC
DERIVATIVES OF SOME AMINES
Colour produced with
RF value on Aniline . . .. p-Toluidine .. l-Naphthylamine 2-Naphthylamine 4-Aminobiphenyl Benzidine ..
cellulose TLC plate .. 0.88 .. 0.86 .. 0.47 .. 0.46
.. ..
..
.. ..
.. ..
..
0.34
0.32
1
~ A spray B Yellow Yellow Yellow Yellow Yellow Orange - pink
DAC spray Pink Orange - pink Purple - pink Bluish pink Bluish pink Blue
By using this technique it is not possible to differentiate between 1- and 2-naphthylamines, for which purpose a separate method is described in section 4. The absolute detectionlimitsfor the DAC spray are 10 ng of 4-aminobiphenyl and 2-naphthylamine and 2 ng of benzidine, which are equivalent to 0.07 and 0.02 pg g-l,respectively, when 5 pl are spotted from 3-g samples.
Spectrophotometric Determination of Amines The identity of the amines may have been indicated by the thin-layer chromatographic method described in the second paragraph of Procedure and the operator can then refer to the appropriate standards, the spectrophotometric properties of which are shown in Table 11. Confirmation of identity can be made by spectrophotometric analysis as described below. To the remainder of the acidic solution (or to a 5-p1 aliquot of this solution if the presence of large amounts of aniline or toluidine has been indicated) and to another tube containing 100 p1 of 3 M hydrochloric acid (to be used as a blank), add 1 ml of DAC reagent B and make the volumes up to a total of 6 ml with methanol. Mix and allow the solution to stand for 20 min. Scan the absorption spectrum of the colour produced (if any) in the range 350-600 nm by means of a recording spectrophotometer, using 10-mm cells with the reagent blank as reference. Note the absorbance at the wavelength of maximum absorption and determine the amount of amine present by comparison with appropriate standards, as shown in Table 11. TABLE I1 SPECTROPHOTOMETRIC PROPERTIES OF THE AMINE STANDARDS Aniline . . .. p-Toluidine . l-Naphthylamine 2-Naphthylamine 4-Aminobiphenyl Benzidine ..
.
.. .. .. .. ..
..
.. ..
.. .. .. ..
Amax. for DAC derivativelnm 618 621 612
Absorbance of 10 pg in 6 ml of final solution (10-mm cell)
634 534
0.79
676
0-46
0.76 0.33 0.62 0-64
The results should be expressed as aniline unless another amine is known to be present. With a mixture of amines the results should be expressed in terms of the predominant amine.
Discussion of Experimental Details I n the final stage of the extraction procedure it was established that the amines should be extracted as their hydrochlorides so as to prevent losses caused by the relatively high volatilities of free aniline and toluidine. Extraction into a large volume of hydrochloric acid followed by evaporation down to a small volume proved laborious and gave rise to several unknown interferences in both the thin-layer chromatographic and spectrophotometric determinations. As far as possible it is necessary to exclude water from the amine - DAB and amine - DAC reactions, which are of the condensation type. The spectrophotometric method
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JZCZY, I975
AROMATIC AMINES IN WATER-SOLUBLE FOOD DYES AND ADDITIVES
479
with DAC can tolerate the presence of up to 2 per cent. of water, above which content the sensitivity4 is markedly reduced. Several acids were tried in the spectrophotometric procedure before finally deciding on the use of 9-toluenesulphonic acid. Mineral acids such as hydrochloric or sulphuric acid caused great changes in sensitivity with different acid concentration^,^ as well as producing unstable complexes. They were, however, satisfactory for use in the DAB and DAC spray reagents. Trichloroacetic acid produced more stable colours but only a t very high acid concentrations. 9-Toluenesulphonic acid, although producing less sensitive colours, formed very stable solutions at low acid concentrations with all the amine complexes tested, reaching a maximum colour intensity within 20 min.
2. Determination of Unsulphonated Primary Aromatic Amines in Biphenyl Biphenyl, which is often used as a preservative for oranges and other citrus fruits, is liable t o contain trace amounts of amines. An EEC directive14 requires that biphenyl shall contain not more than 2 mg k g l of aromatic amines, expressed as aniline. The proposed method is essentially the same as that described in section 1 for dyes, apart from a variation in the extraction procedure. Procedure Dissolve 3 g of biphenyl in 5 ml of toluene and transfer the solution into a separating funnel with the aid of a further 5 ml of toluene. Add 2 ml of 3 M hydrochloric acid and 50 ml of water and shake the mixture for 1 min. Allow the layers to separate and run the lower aqueous layer into a second separating funnel containing 10 ml of 1 M sodium hydroxide solution and 5 ml of toluene. Proceed as in the first and second paragraphs of the procedure in section 1. 3. Determination of 2- and 4-Aminoazobenzenes in Fast Yellow Fast Yellow dye (C.I. Number 13015), also known as Acid Yellow 9 and Food Yellow 2, is manufactured by the disulphonation of 4-aminoazobenzene which, with the 2-isomer, occurs as an impurity in the dye. Apart from the general requirement, the EEC directive1 specifically requires that Fast Yellow (E105) shall contain not more than 10 mg k g l of unsulphonated aromatic amines. In the procedure for the determination of amines in dyes (section l ) ,these substances exhibit some properties that may mistakenly be taken as evidence for the presence of benzidine. The following method permits individual spectrophotometric determinations to be made of the extracted isomers after separation by column chromatographySeveral improvements in the method described in the EEC directive1 have been made, namely an approximate ten-fold reduction in the amounts of sample and solvent, and the use of toluene instead of chlorobenzene. Toluene has two advantages over chlorobenzene for use in this method. Firstly, it does not form emulsions when shaken with aqueous solutions as does the latter. Secondly, it is more convenient to use a solvent that has a lower density than water when several washes with water are required.
Apparatus Chromatographic column. Glass, with PTFE stopcock and Quickfit joint at the neck, length at least 200 mm and internal diameter approximately 15 mm. Separating funnel. Cylindrical, 100 ml in capacity, with ground-glass spout to fit the neck of the chromatographic column. Reagents AZumina. Brockmann activity 11, equivalent to a 3 per cent. water content. For preparation of the alumina column for chromatography, weigh accurately 20 g of alumina into a beaker, add a few millilitres of toluene, mix and pour the slurry into the chromatographic column fitted with a small cotton-wool plug at the bottom. Run off the toluene until the meniscus coincides with the top of the column of alumina. Procedure Dissolve 2.0 g of Fast Yellow in 20 ml of water and transfer the solution into a separating funnel with the aid of a further 30 ml of water. Proceed thereafter as in the first paragraph
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480 DIXON AND GROFFMAN : DETERMINATION OF UNSULPHONATED PRIMARY Analyst, V d . 100
of the procedure in section 1. Transfer the toluene extract into a test-tube and reduce the volume of the solution to about 0.5 ml by evaporation. Transfer the solution and a small amount of toluene used for washings on to the prepared alumina column and elute it with toluene. Collect separately the first 30 m l of eluate, which contains the 2-aminoazobenzene, and the next 40 ml containing the 4-isomer. Make the volume of each fraction up to 50 ml with toluene and scan each solution on a spectrophotometer over the range 300-500nm. Note the wavelength of maximum absorbance of each solution and the absorbance at that wavelength and compare the values with the standards shown in Table 111.
TABLEI11 SPECTROPHOTOMETRIC PROPERTIES OF 2-Isomer 4-Isomer
.. ..
..
..
THE AMINOAZOBENZENES
Amax./nm Absorptivity (1 mg ml-l, 10-mm path length) 414 376
40 110
4. Detection and Identification of 1- and 2-Naphthylamines in Water-soluble Food Dyes and Other Food Additives With neither the cellulose thin-layer chromatographic method nor the spectrophotometric method is it possible to distinguish satisfactorily between 1- and 2-naphthylamines in the presence of each other or in the presence of large amounts of other amines. However, their tosyl derivatives can be resolved by the following method, which affords further confirmation of the identity of amines determined by the thin-layer chromatographic and spectrophotometric methods. Of other amine derivatives that were investigated for suitability for thinlayer chromatography, only the dansyl derivative gave reasonable separation of 1- and 2-naphthylamines. The spots produced by the dansyl derivatives were more compact and more strongly fluorescent than those of the tosyl derivatives but the presence of hydrolysis and other products gave a chromatogram that was too complex for positive identification.
Apparatus Alumina thin-layer chromatographicplate. Dimensions 200 x 200 mm and 0.1 mm thick, with a pH of approximately 9 (obtainable from Eastman Organic Chemicals). Reagents Mobile solvent. n-Hexane - chloroform (2 1 V / V ) . p-Toluenesul~honylpuoride. Procedure Prepare the amine mixture in toluene as described in the first paragraph of the procedure in section 1. Transfer this solution into a graduated, pointed test-tube containing approximately 10 mg of 9-toluenesulphonyl fluoride. Hold the tube over a steam-bath and slowly pass air into the tube until the volume of the solution is reduced to about 0.1 ml. Spot 6 p1 of this solution about 20 mm from the bottom of an alumina thin-layer chromatographic plate and spot 5 p1 of available appropriate standards alongside. Run the chromatogram in the hexane - chloroform mobile solvent until the solvent front has travelled about 150 mm from the base-line. Allow the plate to dry and then observe the spots under ultraviolet radiation. Note the positions and colours of the spots and compare them with standards (Table IV). TABLE IV
+
PHYSICAL PROPERTIES OF AMINE TOSYLATES
RF value on alumina Aniline . . .. p-Toluidine .. l-Naphthylamine 2-Naphthylamine 4-Aminobiphenyl Benzidine ..
.. .. .. ..
.. ..
.. .. .. ..
.. ..
0.62 0.57 0.59
0.11
Fluorescence under ultraviolet radiation Not observed Not observed Sky blue Dark blue Dark blue Dark blue
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JdY, 1975
AROMATIC AMINES IN WATER-SOLUBLE FOOD DYES AND ADDITIVES
481
Discussion Although the tosyl derivatives of 1- and 2-naphthylamine are not completely resolved by the alumina thin-layer chromatographic method, either isomer can be observed in the presence of at least a ten-fold excess of the other as they give different fluorescent spectra. Large excesses of aniline or j+toluidine, which are liable to interfere in 1- and 2-naphthylamine determinations by the cellulose thin-layer chromatographic method, do not interfere under the reaction conditions described in section 4; 4-aminobiphenyl or benzidine would not normally be expected to occur with the naphthylamines and in any event would have already been identified by the latter method. We thank the Government Chemist, Department of Industry, for permission to publish this paper. References 1. EEC Directive 2645/62, 23 October, 1962, Official Journal No. 115, 11.11.62. 2. “Factories Act, The Carcinogenic Substances Regulations 1967,” S.I. 1967 No. 879, H. M. Stationery Office, London. 3. “Factories Act, The Carcinogenic Substances (Prohibition of Importation) Order 1967,” S.I.1967 No. 1675, H.M. Stationery Office, London. 4. Sakai, S., Mori, H., Suzuki, K., and Fujino, M., Ja#an Analyst, 1960, 9, 862. 5. Parihar, D. B., Sharma, S. P., and Tewari, K. C., J . Chromat., 1966, 24, 443. 6. Butt, L. T., and Strafford, N., J. A$$l. Chem., 1956, 6, 525. 7. Caemmerer, A. B., J . Ass. 08. Agric. Chem., 1948, 31, 592. 8. Caemmerer, A. B., J . Ass. Off. Agric. Chem., 1949, 32, 613. 9. Pisano, J . J ., “Theory of Application of Gas Chromatography to Industrial Medicine,” Hahnemann Symposium, First Edition, Grune and Stratton, New York, 1966 (published 1968), p. 147. 10. Clarke, D. D., Wilk, S., and Gitlow, S. E., J . Gus Chromat., 1966, 4, 310. 11. Seiler, N., J . Chromat., 1971, 63, 97. 12. Yasuda, K., J. Chromat., 1971, 60, 144. 13. Feigl, I?., “Spot Tests in Inorganic Chemistry,” Seventh Edition, Elsevier Publishing Company, Amsterdam, London and New York, 1967, p. 243. 14. EEC Directive 67/428/EEC, 27 June, 1967, Official Journal No. l48/10, 11.7.67. Received January 13th, 1975 Accepted February 5t12, 1975
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476
Analyst, July, 1975, Vol. 100, pp. 476-481
The Determination of Unsulphonated Primary Aromatic Amines in Water-soluble Food Dyes and Other Food Additives E. J. Dixon and D. M. Groffman Department of Industry, Laboratory of the Government Chemist, Cornwall House, Stamford Street, London, SE19NQ
EEC directives prescribe limits for the amount of unsulphonated primary aromatic amines present in food colouring matters in general and prohibit the presence of particular carcinogenic amines in food dyes and other food additives. Integrated methods based on solvent extraction and thin-layer chromatography have been devised for identification of these amines, followed by the spectrophotometric determination of their condensation products with 4-dimethylaminocinnamaldehyde. The method is also extended to the determination of primary aromatic amines in biphenyl and to the determination of aminoazobenzenes in the dye Fast Yellow. An additional thin-layer chromatographic method is given for the differentiation between the isomers of naphthylamine as their tosyl derivatives.
Directives in force within the European Economic Community (EEC)l require synthetic organic colouring matters for use in foods not to contain more than 0.01 per cent. of free aromatic amines nor more than 0-5 per cent. of intermediate synthetic products other than free aromatic amines. Further, they should not contain 2-naphthylamine, benzidine or 4-aminobiphenyl (xenylamine) or their derivatives. In Britain, the use of these three compounds is prohibited,2s3except for special research purposes, and as a result authentic reference samples are difficult to obtain. In general, for purposes of identification the analyst has to rely on the physical data for these substances of unspecified purity quoted in the literature.194.5 Examples of such data are given in Tables I-IV. Few up-to-date methods for the analysis of food dyes have been published in recent years and those previously available6-s are considered to lack sensitivity and specificity. Several gas-chromatographic methodsg910are available but are unsuitable because of the difficulty of expressing mixtures of amines as “aniline,” as required in the EEC directives, and because gas - liquid chromatography by itself would not give an unambiguous identification. Methods that depend on the production of a diazonium compound, followed by addition of a coupling agent in order to produce a colour, require specific conditions for each amine under test and for each individual coupling agent. Several thin-layer chromatographic method~~9~~912 are suitable for the separation of the amines under consideration; some of these methods have been adapted to the determination of these amines in dyes, as also have some spectrophotometric4J3 met hods. The spectrophotometric methods adopted depend on the formation of Schiff’s bases and afford quantitative determinations of individual amines that had previously been identified by thin-layer chromatography. Mixtures of amines are more difficult to analyse spectrophotometrically but a semi-quantitative estimate can be made following separation with thin-layer chromatography by comparing the intensities of individual spots with standards. However, the method is not sufficiently sensitive for a fully quantitative determination to be made by scraping off the spots and eluting the amines with solvent in order to give a solution of a known volume. Section 1 of this paper describes a basic procedure for the identification and determination of unsulphonated primary aromatic amines. However, it is not possible by this procedure to distinguish between 1- and 2-naphthylamines; a suitable method for this purpose is given in section 4. Section 2 describes the modifications necessary to make section 1 applicable t o the examination of biphenyl, and section 3 extends the general procedure to the determination of aminoazobenzenes in Fast Yellow. Crown Copyright.
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DIXON AND GROFFMAN
477
1. Determination of Unsulphonated Primary Aromatic Amines in Published on 01 January 1975. Downloaded by University of Prince Edward Island on 26/10/2014 10:29:59.
Water-soluble Food Dyes Apparatus Separating funnels. 100-ml capacity, with a PTFE stopcock and glass stopper. Capillary tubes or micropipettes. 5-p1 capacity, for spotting samples on to thin-layer chromatographic plates, and 100-pl capacity, for dispensing 3 M hydrochloric acid. PiPettes. 1- and 5-ml capacity. Pasteur ca9illary pipettes. Filter-paper. Strips of a Whatman low-ash grade filter-paper, approximately 20 x 50 mm. Cellulose thin-layer chromatographic Plates. Prepare the plates as follows: shake 20 g of microcrystalline cellulose powder with 60 ml of methanol for 2 min, then immediately spread the mixture on 200 x 200-mm glass plates to a thickness of 0.25 mm. After air-drying them for 30 min, dry the plates for 10 min at 105 "C. Up to five plates can be prepared in one application by using the above amount of mixture. Equally suitable are 200 x 200-mm ready-made plates of 0-1-mm thickness, which are obtainable from E. Merck. Test-tubes. 10-ml capacity, pointed and graduated, with ground-glass necks and stoppers. Recording spectrophotometer. Suitable for scanning in the range 350-600 nm. Spectrophotometer cells. 10-mm path length.
Reagents Sodium hydroxide solution, 1 M. Toluene. Analytical-reagent grade. Hydrochloric acid, 3 M. Methanol. Analytical-reagent grade. Propan-2-ol. Analytical-reagent grade. Mobile solvent for thin-layer chromatography. Dissolve 1.0 g of sodium acetate in about 10ml of distilled water and add 0.10ml of glacial acetic acid. Transfer the mixture, with aqueous washings, into a 100-ml calibrated flask and make the volume up to the mark with water. 4-Dimethylaminocinnamaldehyde ( D A C ) solution A . Dissolve 0.1 g of DAC in about 50 ml of methanol, carefully add 5 ml of concentrated sulphuric acid, transfer the mixture plus washings with methanol into a 100-ml calibrated flask and make the volume up to the mark with methanol. DAC solution B. Dissolve a mixture of 0.1 g of DAC and 0.1 g of P-toluenesulphonic acid in about 10ml of propan-2-01, transfer the mixture plus washings with propan-2-01 into a 100-ml calibrated flask and make the volume up to the mark with propan-2-01. 4-Dimethylaminobenzaldehyde solution ( D A B ) . Dissolve 1 g of DAB in about 10 ml of methanol, add 4 ml of concentrated hydrochloric acid, transfer the mixture plus washings with methanol into a 100-ml calibrated flask and make the volume up to the mark with methanol.
Procedure Weigh accurately about 3 g of dye into a separating funnel containing 50ml of water. Shake the funnel in order to dissolve the sample. Add 10 ml of 1 M sodium hydroxide solution and mix, then add 5 ml of toluene and shake the funnel for 1 min. Allow the layers to separate and discard the lower aqueous layer. Add another 5-ml portion of 1 M sodium hydroxide solution and shake the funnel for 1 min. If on separation the lower layer is coloured (caused by partition of the dye between the organic and inorganic phases) discard the lower layer and repeat the above extractions with alkali. Finally, wash the toluene extract with two 10-ml portions of water and discard the washings. Dry the inside of the stem of the separating funnel, then insert a 20 x 50-mm rolled-up strip of Whatman low-ash filter-paper. Transfer the toluene extract into a graduated, pointed ground-glass test-tube containing 100 & 2 pl of 3 M hydrochloric acid, stopper the tube and shake it vigorously for about 30 s. Allow the acid to sink to the bottom of the tube (it may be helpful to tap the side of the tube) and carefully remove as much as possible of the toluene with a Pasteur capillary pipette, the toluene being discarded. Spot two 5-pl portions of the remaining acidic solution about 100 mm apart, and each 20 mm from the bottom of a cellulose thin-layer chromatographic plate, and spot 5 p 1 of appropriate standards alongside each spot under test. Run the
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478
DIXON AND GROFFMAN
: DETERMINATION
OF UNSULPHONATED PRIMARY
Analyst, VoE. 100
chromatogram in a tank containing the prescribed mobile solvent until the solvent has travelled about 150 mm above the base-line. Allow the plate to dry, cover one set of standards and sample with a glass plate and spray the remaining set with DAC solution A. Cover the other half of the plate, then spray with DAB solution. Compare the positions and colours of spots in the sample with the standards. The chromatographic behaviour of some amines that may be present is shown in Table I.
TABLE I CHROMATOGRAPHIC PROPERTIES OF DAB AND DAC
DERIVATIVES OF SOME AMINES
Colour produced with
RF value on Aniline . . .. p-Toluidine .. l-Naphthylamine 2-Naphthylamine 4-Aminobiphenyl Benzidine ..
cellulose TLC plate .. 0.88 .. 0.86 .. 0.47 .. 0.46
.. ..
..
.. ..
.. ..
..
0.34
0.32
1
~ A spray B Yellow Yellow Yellow Yellow Yellow Orange - pink
DAC spray Pink Orange - pink Purple - pink Bluish pink Bluish pink Blue
By using this technique it is not possible to differentiate between 1- and 2-naphthylamines, for which purpose a separate method is described in section 4. The absolute detectionlimitsfor the DAC spray are 10 ng of 4-aminobiphenyl and 2-naphthylamine and 2 ng of benzidine, which are equivalent to 0.07 and 0.02 pg g-l,respectively, when 5 pl are spotted from 3-g samples.
Spectrophotometric Determination of Amines The identity of the amines may have been indicated by the thin-layer chromatographic method described in the second paragraph of Procedure and the operator can then refer to the appropriate standards, the spectrophotometric properties of which are shown in Table 11. Confirmation of identity can be made by spectrophotometric analysis as described below. To the remainder of the acidic solution (or to a 5-p1 aliquot of this solution if the presence of large amounts of aniline or toluidine has been indicated) and to another tube containing 100 p1 of 3 M hydrochloric acid (to be used as a blank), add 1 ml of DAC reagent B and make the volumes up to a total of 6 ml with methanol. Mix and allow the solution to stand for 20 min. Scan the absorption spectrum of the colour produced (if any) in the range 350-600 nm by means of a recording spectrophotometer, using 10-mm cells with the reagent blank as reference. Note the absorbance at the wavelength of maximum absorption and determine the amount of amine present by comparison with appropriate standards, as shown in Table 11. TABLE I1 SPECTROPHOTOMETRIC PROPERTIES OF THE AMINE STANDARDS Aniline . . .. p-Toluidine . l-Naphthylamine 2-Naphthylamine 4-Aminobiphenyl Benzidine ..
.
.. .. .. .. ..
..
.. ..
.. .. .. ..
Amax. for DAC derivativelnm 618 621 612
Absorbance of 10 pg in 6 ml of final solution (10-mm cell)
634 534
0.79
676
0-46
0.76 0.33 0.62 0-64
The results should be expressed as aniline unless another amine is known to be present. With a mixture of amines the results should be expressed in terms of the predominant amine.
Discussion of Experimental Details I n the final stage of the extraction procedure it was established that the amines should be extracted as their hydrochlorides so as to prevent losses caused by the relatively high volatilities of free aniline and toluidine. Extraction into a large volume of hydrochloric acid followed by evaporation down to a small volume proved laborious and gave rise to several unknown interferences in both the thin-layer chromatographic and spectrophotometric determinations. As far as possible it is necessary to exclude water from the amine - DAB and amine - DAC reactions, which are of the condensation type. The spectrophotometric method
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JZCZY, I975
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with DAC can tolerate the presence of up to 2 per cent. of water, above which content the sensitivity4 is markedly reduced. Several acids were tried in the spectrophotometric procedure before finally deciding on the use of 9-toluenesulphonic acid. Mineral acids such as hydrochloric or sulphuric acid caused great changes in sensitivity with different acid concentration^,^ as well as producing unstable complexes. They were, however, satisfactory for use in the DAB and DAC spray reagents. Trichloroacetic acid produced more stable colours but only a t very high acid concentrations. 9-Toluenesulphonic acid, although producing less sensitive colours, formed very stable solutions at low acid concentrations with all the amine complexes tested, reaching a maximum colour intensity within 20 min.
2. Determination of Unsulphonated Primary Aromatic Amines in Biphenyl Biphenyl, which is often used as a preservative for oranges and other citrus fruits, is liable t o contain trace amounts of amines. An EEC directive14 requires that biphenyl shall contain not more than 2 mg k g l of aromatic amines, expressed as aniline. The proposed method is essentially the same as that described in section 1 for dyes, apart from a variation in the extraction procedure. Procedure Dissolve 3 g of biphenyl in 5 ml of toluene and transfer the solution into a separating funnel with the aid of a further 5 ml of toluene. Add 2 ml of 3 M hydrochloric acid and 50 ml of water and shake the mixture for 1 min. Allow the layers to separate and run the lower aqueous layer into a second separating funnel containing 10 ml of 1 M sodium hydroxide solution and 5 ml of toluene. Proceed as in the first and second paragraphs of the procedure in section 1. 3. Determination of 2- and 4-Aminoazobenzenes in Fast Yellow Fast Yellow dye (C.I. Number 13015), also known as Acid Yellow 9 and Food Yellow 2, is manufactured by the disulphonation of 4-aminoazobenzene which, with the 2-isomer, occurs as an impurity in the dye. Apart from the general requirement, the EEC directive1 specifically requires that Fast Yellow (E105) shall contain not more than 10 mg k g l of unsulphonated aromatic amines. In the procedure for the determination of amines in dyes (section l ) ,these substances exhibit some properties that may mistakenly be taken as evidence for the presence of benzidine. The following method permits individual spectrophotometric determinations to be made of the extracted isomers after separation by column chromatographySeveral improvements in the method described in the EEC directive1 have been made, namely an approximate ten-fold reduction in the amounts of sample and solvent, and the use of toluene instead of chlorobenzene. Toluene has two advantages over chlorobenzene for use in this method. Firstly, it does not form emulsions when shaken with aqueous solutions as does the latter. Secondly, it is more convenient to use a solvent that has a lower density than water when several washes with water are required.
Apparatus Chromatographic column. Glass, with PTFE stopcock and Quickfit joint at the neck, length at least 200 mm and internal diameter approximately 15 mm. Separating funnel. Cylindrical, 100 ml in capacity, with ground-glass spout to fit the neck of the chromatographic column. Reagents AZumina. Brockmann activity 11, equivalent to a 3 per cent. water content. For preparation of the alumina column for chromatography, weigh accurately 20 g of alumina into a beaker, add a few millilitres of toluene, mix and pour the slurry into the chromatographic column fitted with a small cotton-wool plug at the bottom. Run off the toluene until the meniscus coincides with the top of the column of alumina. Procedure Dissolve 2.0 g of Fast Yellow in 20 ml of water and transfer the solution into a separating funnel with the aid of a further 30 ml of water. Proceed thereafter as in the first paragraph
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480 DIXON AND GROFFMAN : DETERMINATION OF UNSULPHONATED PRIMARY Analyst, V d . 100
of the procedure in section 1. Transfer the toluene extract into a test-tube and reduce the volume of the solution to about 0.5 ml by evaporation. Transfer the solution and a small amount of toluene used for washings on to the prepared alumina column and elute it with toluene. Collect separately the first 30 m l of eluate, which contains the 2-aminoazobenzene, and the next 40 ml containing the 4-isomer. Make the volume of each fraction up to 50 ml with toluene and scan each solution on a spectrophotometer over the range 300-500nm. Note the wavelength of maximum absorbance of each solution and the absorbance at that wavelength and compare the values with the standards shown in Table 111.
TABLEI11 SPECTROPHOTOMETRIC PROPERTIES OF 2-Isomer 4-Isomer
.. ..
..
..
THE AMINOAZOBENZENES
Amax./nm Absorptivity (1 mg ml-l, 10-mm path length) 414 376
40 110
4. Detection and Identification of 1- and 2-Naphthylamines in Water-soluble Food Dyes and Other Food Additives With neither the cellulose thin-layer chromatographic method nor the spectrophotometric method is it possible to distinguish satisfactorily between 1- and 2-naphthylamines in the presence of each other or in the presence of large amounts of other amines. However, their tosyl derivatives can be resolved by the following method, which affords further confirmation of the identity of amines determined by the thin-layer chromatographic and spectrophotometric methods. Of other amine derivatives that were investigated for suitability for thinlayer chromatography, only the dansyl derivative gave reasonable separation of 1- and 2-naphthylamines. The spots produced by the dansyl derivatives were more compact and more strongly fluorescent than those of the tosyl derivatives but the presence of hydrolysis and other products gave a chromatogram that was too complex for positive identification.
Apparatus Alumina thin-layer chromatographicplate. Dimensions 200 x 200 mm and 0.1 mm thick, with a pH of approximately 9 (obtainable from Eastman Organic Chemicals). Reagents Mobile solvent. n-Hexane - chloroform (2 1 V / V ) . p-Toluenesul~honylpuoride. Procedure Prepare the amine mixture in toluene as described in the first paragraph of the procedure in section 1. Transfer this solution into a graduated, pointed test-tube containing approximately 10 mg of 9-toluenesulphonyl fluoride. Hold the tube over a steam-bath and slowly pass air into the tube until the volume of the solution is reduced to about 0.1 ml. Spot 6 p1 of this solution about 20 mm from the bottom of an alumina thin-layer chromatographic plate and spot 5 p1 of available appropriate standards alongside. Run the chromatogram in the hexane - chloroform mobile solvent until the solvent front has travelled about 150 mm from the base-line. Allow the plate to dry and then observe the spots under ultraviolet radiation. Note the positions and colours of the spots and compare them with standards (Table IV). TABLE IV
+
PHYSICAL PROPERTIES OF AMINE TOSYLATES
RF value on alumina Aniline . . .. p-Toluidine .. l-Naphthylamine 2-Naphthylamine 4-Aminobiphenyl Benzidine ..
.. .. .. ..
.. ..
.. .. .. ..
.. ..
0.62 0.57 0.59
0.11
Fluorescence under ultraviolet radiation Not observed Not observed Sky blue Dark blue Dark blue Dark blue
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Discussion Although the tosyl derivatives of 1- and 2-naphthylamine are not completely resolved by the alumina thin-layer chromatographic method, either isomer can be observed in the presence of at least a ten-fold excess of the other as they give different fluorescent spectra. Large excesses of aniline or j+toluidine, which are liable to interfere in 1- and 2-naphthylamine determinations by the cellulose thin-layer chromatographic method, do not interfere under the reaction conditions described in section 4; 4-aminobiphenyl or benzidine would not normally be expected to occur with the naphthylamines and in any event would have already been identified by the latter method. We thank the Government Chemist, Department of Industry, for permission to publish this paper. References 1. EEC Directive 2645/62, 23 October, 1962, Official Journal No. 115, 11.11.62. 2. “Factories Act, The Carcinogenic Substances Regulations 1967,” S.I. 1967 No. 879, H. M. Stationery Office, London. 3. “Factories Act, The Carcinogenic Substances (Prohibition of Importation) Order 1967,” S.I.1967 No. 1675, H.M. Stationery Office, London. 4. Sakai, S., Mori, H., Suzuki, K., and Fujino, M., Ja#an Analyst, 1960, 9, 862. 5. Parihar, D. B., Sharma, S. P., and Tewari, K. C., J . Chromat., 1966, 24, 443. 6. Butt, L. T., and Strafford, N., J. A$$l. Chem., 1956, 6, 525. 7. Caemmerer, A. B., J . Ass. 08. Agric. Chem., 1948, 31, 592. 8. Caemmerer, A. B., J . Ass. Off. Agric. Chem., 1949, 32, 613. 9. Pisano, J . J ., “Theory of Application of Gas Chromatography to Industrial Medicine,” Hahnemann Symposium, First Edition, Grune and Stratton, New York, 1966 (published 1968), p. 147. 10. Clarke, D. D., Wilk, S., and Gitlow, S. E., J . Gus Chromat., 1966, 4, 310. 11. Seiler, N., J . Chromat., 1971, 63, 97. 12. Yasuda, K., J. Chromat., 1971, 60, 144. 13. Feigl, I?., “Spot Tests in Inorganic Chemistry,” Seventh Edition, Elsevier Publishing Company, Amsterdam, London and New York, 1967, p. 243. 14. EEC Directive 67/428/EEC, 27 June, 1967, Official Journal No. l48/10, 11.7.67. Received January 13th, 1975 Accepted February 5t12, 1975
