Aromatic Amine Impurities in Yellow AB and Yellow OB Food Dyes WALTER
D. CONWAY and ELIZABETH J. LETHCO
National Cancer Institute, National Institutes of Health, Bethesda 7 4, Md.
Dye ( 5 grams) in ether (50 ml.)
b
The residual 2-naphthylamine determined in 10 samples of two commercial food dyes ranged from 76 to 908 p.p.m. Other amines were estimated. The method of analysis involves removal of the amines by acid extraction and spectrophotometric estimation of the 2-naphthylamine after separation by column partition chromatography. A device for continuous measurement of ultraviolet light absorption of the column eluent is described.
six 30-ml. portions 0.LY HCI combineh HC1 extracts
ether (discard)
I
two 26-1111. portions ether
! 1
two 30-ml. portions 0.1N HC1
I
I c o m b i n e d C l extracts
ether
I
T
HERE 1 as been increasing concern regarding the possible carcinogenic properties of several chemicals used as food additives (11, 13). Among the dyes for food coloring, Yellow ilB (1phenylazo-2-naphthylamine) and Yellow OB (1-0-tolylazo-2-naphthylamine) have been viewed with suspicion because they are prepared from P-naphthylamine, vhich is a potent human carcinogen. It is difficult to remove all traces of the starting material in the commercial preparation of the dye. Occupational bladder cancer has long been recognized among workers exposed to 2-naphthylamine. Bladder tumors are also produced experimentally in dogs following oral administration of the amine. Current evidence indicates that the active carcinogen involved is not 2-naphthylamine itself but a metabolite, 2-amino-1-naphthol ( I , 2, 12). The dyes, Yellow AB and Yellow OB under the names FD&C Yellow No. 3 and FD&C Yellow No. 4, respectively, were permitted until recently as food colorings in this country (4, 7 ) , and as of 1955 they were still permitted in 10 foreign countries ( 5 ) . They are still permitted in the United States under the names External D & C KO.9 and External D & C S o . 10, respectively, for use in external drugs and cosmetics ( 7 ) . The specifications for certification as External D & C dyes are identical with those formerly used for the FD&C dyes. Although the dyes are certified t o contain a total of less than 500 p.p.m. of intermediates, i t is desirable to know the actual amounts of 2-naphthylamine and possibly other amines present, to evaluate the exposure of a population to these chemicals properly. The
838
ANALYTICAL CHEMISTRY
total HCl extract Dilute to 250 ml. acid extract Figure 1.
Extraction scheme
amount of 2-naphthylamine involved here is small. However, i t should not be neglected, as i t constitutes a fraction of the total of similarly acting aromatic amines to which man is exposed in his modern environment. Two procedures for the determination of 2-naphthylamine in Yellow AB and Yellow OB have been reported. The first (6) involves extiaction of the amine from the dyes with dilute hydrcchloric acid at 60' C. Carrying out the extraction a t this temperature was considered objectionable, because the dyes are known to undergo acid hydrolysis with the release of 2-naphthylamine (9). 4 second method (8) depends upon steam distillation of 2naphthylamine from a saturated salt solution. The recovery of Znaphthylamine by this procedure was very low. RIodification as described in the experimental section resulted in better recovery of 2-naphthylaniine, but the method was still not satisfactory for samples containing less than about 800 p.p.m. of 2-naphthylamine. Seither method was designed t o indicate the presence of amine impurities other than aniline, o-toluidine, or 2-naphthylamine. I n the method of analysis described here, the free amine impurities are separated from the dye sample by acid extraction, A color test is used to measure the total aromatic amines present in this extract. After concen-
tration of the remaining extract, the 2naphthylamine is separated by partition chromatography and determined spectrophotometrically. The presence of trace amounts of other impurities is indicated by continuous measurement of the absorption of ultraviolet light by the effluent from the partition chromatogram. EXPERIMENTAL METHODS
Extraction of Sample. Five grams of dye was dissolved in 50 ml. of diethyl ether and extracted with 0.1N hydrochloric acid according t o t h e scheme indicated in Figure 1. All extractions were carried out at 0' C. t o minimize the generation of free 2-naphthylamine from the dye (9). A small volume of ether, which separated from the combined acid extracts on warming to room temperature, was removed in a stream of air before dilution to 250 ml. This solution after dilution to 250 ml. is referred to as the acid extract. This extraction procedure (Figure 1) should be sufficient to transfer over 99% of the free 2-naphthylamine to the acid extract based upon a determination of a partition coefficient of 0.1 for the amine b e h e e n ether and 0.1N HC1 at 0' C. This was confirmed experimentally by recovery of 2-naphthylamine added to 5-gram samples of purified Yellow AB. iJ7hen 1270 and 5100 y of the amine
hours: 2-butanol-2-propanol-HC1-KCl buffer p H 1.0 in the ratio 20: 10: 60, respectively, by volume. The upper layer of this system is referred to as the organic or stationary phase. The lower layer is referred to as the aqueous or mobile phase. Fifteen or 20 grams of silane-treated Celite was slurried with 100 ml. of aqueous phase and a suitable quantity (0.8 ml. per gram of Celite) of organic phase was added with stirring. After the flask containing the slurry had been evacuated to 200-mm. pressure for a few seconds to remove entrapped air, the slurry was poured into a tube (1.7 cm. in inside diameter) and alloned to settle by gravity. After settling, the bed was packed by forcing mobile phase through the column under 5p.s.i. pressure for a few minutes. To 5 ml. of the acid extract in a Finished columns measured 1.7 X 10-ml. volumetric flask, add 1 ml. of 27.6 cm. for 20 grams of Celite. I n 0.1% aqueous sodium nitrite. Mix, general, 15-gram columns gave sufficient and after 3 minutes add 1 ml. of 0.5% resolution and 20-gram columns were aqueous ammonium sulfamate. Mix, used only if the smaller column proved and after 2 minutes add 1 ml. of 1% unsatisfactory. aqueous N-(1-naphthyl)-ethylenediEluent flow rate was adjusted to amine dihydrochloride. Dilute the about 18 ml. per hour by attaching a solution to volume with 0.liV HC1 length of small-diameter polyethylene and mix. After 40 minutes, determine tubing a t the outlet of the column and the intensity a t 565 mfi, using a Beckby varying the height of the head of man DU spectrophotometer. eluent above the outlet tube. When regulated in this way, the flow rate was The color development mas maximum constant over 24-hour periods. The flow rate mas measured by inserting after 30 minutes and was stable for a t calibrated tubes a t intervals in the least 2 hours. A plot of absorbance us. collector rack. concentration was linear for standard Two milliliters of the solution to be solutions containing from 1 to 6 y of analyzed was added by pipet to the 2-naphthylamine per ml. From this top of the column and rinsed on with a plot the “total aromatic amine” content few milliliters of mobile phase. Apof the acid extract mas calculated and proximately 2-ml. fractions were colexpressed in units of 2-naphthylamine. lected for the first 8 hours of developFree 2-Naphthylamine by Partition ment. Chromatography. COXCENTRATION The light absorption of the effluent v a s continuously measured a t 254 mp, OF SAMPLE.The remaining 245 ml. using a modified Photovolt Model of acid extract was concentrated in 525 densitometer. This instrument, vacuo a t a temperature below 30” C., which is intended for measuring the using a rotating evaporator. Using density of spots on paper strips, was the “total aromatic amine” content modified by inserting a cell holder as a guide, the residue was dissolved capable of holding a silica flow cell in a volume of mobile phase, such that (Pyrocell Co., outside dimensions 1.3 X 1 ml. of the resulting solution contained 1.3 X 4.0 cm., volume 1.5 ml., light path 1.0 cm.) between the search unit approximately 125 y of 2-naphthyland the light source. A complete amine per ml. description of this adapter will be Free 2-naphthylamine was isolated published elsewhere. The output of the from a 2-ml. aliquot of this solution densitometer lvas used to drive a using the following chromatographic Varian Model G-10 recorder. system. Column effluent was collected using PREPARATION OF COLUMNS. In a Technicon fraction collector. A general, the method of Howard and single-pole single-throw microswitch was Martin was used (IO). mounted beneath the collector rack in such a position that it was momentarily closed by the rack drive each time Celite 545 (Johns-Manville) mas the rack was advanced, thereby impossoaked overnight in 1N HCl, washed ing a fraction of the voltage from a with water until washings were neutral flashlight cell upon the recorder. I n to litmus, then dried a t 120” C. The this way, a recording of light absorption dry Celite was then coated with 2,570 us. tube number was obtained. of its weight of dimethyldichlorosilane Tubes comprising the 2-naphthyl(K & K Laboratories) by passing a amine fraction were combined, rinsed, stream of dry nitrogen first through and diluted to 25 or 50 ml. with mobile the silane and then through a flask in phase. The absorbance of this solution which the Celite was stirred. The a t 275 mw was determined using a treated Celite was washed with methanol and dried a t 120” C. Beckman DU spectrophotometer. The The following solvent system was purity of the %naphthylamine fraction equilibrated by stirring for several was checked by determination of the were added, recoveries were 1270 and 5040 y, or 100 and 98.9%, respectively. The recovered amine was determined by the color test described below. When purified samples of Yellow AB and Yellow OB were extracted under these conditions, the total aromatic amines in the acid extract were 25.0 and 12.5 y , respectively. This corresponds to a 2-naphthylamine content of 5 and 2.5 p.p.m. for the Yellow AB and Yellow OB samples, respectively. Hydrolysis of the dyes can, therefore, be neglected. Total Aromatic Amines. Aromatic amines were determined in the acid extract by a modification of the method of Bratton and hiarshall ( 3 ) .
Table 1. Recovery of 2-Naphthylamine from 20-Gram Chromatographic Columns
Smine Sample
-4mine Recovered
5%
Y
251
1 2 3
229 458
255
101
92.5 94.4
212
432
Table II. Effect of Glass Wool on Recovery of Added 2-Naphthylamine (5.1 Mg.) from 1-Gram Samples of Yellow AB
Weight of Glass Wool, G.
%Naphthylamine Recovered,
8a
66.2 78.5
4 4 0
75.0 84.8
4 8 grams of wool fills approximately 3 / r of a 500-ml. round-bottomed flask.
ultraviolet absorption spectrum using a Cary spectrophotometer. If the spectrum showed any signs of contamination, the analysis FT-as repeated using a fresh column. The concentration of 2-naphthylamine was determined from a plot of absorbance us. concentration, which was linear, for standard solutions in mobile phase containing 2 to 22 y of 2-naphthylamine per ml. From this, the 2-naphthylamine content of the dye sample was calculated. Columns could be used three times. Before each run they were washed with about 300 ml. of mobile phase. Typical elution volumes for a 20gram column were 53, 57, and 80 ml. for aniline, o-toluidine, and 2-naphthylamine, respectively. Aniline and otoluidine were not determined in the chromatographic method, because we were mainly interested in the 2-naphthylamine content of the dyes and preliminary data indicated that 2-naphthylamine was the amine present in greatest amount. The ahove data indicate, however, that these materials n ill be separated f i om 2-naphthylamine. The recovery of 2-naphthylamine using the chromatographic procedure is indicated in Table I. Samples 1 and 2 represent standaid solutions of 2naphthylamine carried through the column chromatographic step only, while sample 3 represents a standard solution carried through concentration and chromatographic steps. Alternate Method. The method published by Harrow for the determination of 2-naphthylamine in Yellow AB and Yellow OB food dyes (8) involves steam distillation of the free amine from a suspension of the dye in a saturated salt solution, made alkaline with sodium hydroxide. The procedure as published calls for VOL. 32, NO. 7, JUNE 1960
839
the addition to a 500-ml. round-bottomed flask of a few boiling chips, 80 grams of sodium chloride, 5 grams of dye, enough glass wool (presoaked in water) to fill approximately three fourths of the flask, 5 ml. of 3Oy0 sodium hydroxide, and 100 nil. of water. The flask is fitted Kith a steam trap and condenser. Water is distilled into a receiver con1) (v./v.) HC1 taining 5 ml. of (1 solution until 125 ml. has been collected. One hundred milliliters of water is added to the distillation flask and the distillation continued until a total of 200 to 225 ml. has been collected. The distillate is then made alkaline and extracted with ether. The ether extract is extracted with dilute hydrochloric acid, the extract made alkaline, and the 2-naphthylamine determined spectrophotometrically in the alkaline solution.
+
It was reported that 89% of 2naphthylamine (I to 8 mg. per 5 grams of dye) added to pure samples of dye can he recovered by this procedure. I n this laboratory, using the procedure as described, only 25% of added 2-naphthylamine (7.2 mg. per 5 grams of purified Yellow AB) could be recovered On further investigation the recovery was found to depend upon the amount of glass no01 used and the weight of the dye sample. With no dye present 927, of the 2-naphthylamine was recovered, With a l-gram sample of dye, approximately 66% of the amine was iecovercd. The effect of glass wool is shown in Table 11. Although recovery is fairly good when no glass wool is used, the bumping is too violent for the distillation to be carried out safely in a routine manner. Two grams of glass wool was sufficient to prevent bumping if the wool were divided into 20 separate wads. When subdivided in this way, the smaller amount of glass wool is just as effective as packing the flask three fourths full. The recovery of 2-naphthylamine could also be improved by distillation of a greater volume of water.
Table 111.
60-
2
40,
b, 04 c '
$
20-
?
/I
The steam distillation technique for determining 2-naphthylamine can be improved by modifying Harrow's method as follows: Use a l-gram sample of dye. Use 2 grains of glass wool divided into 20 separate wads. Soak in 200 ml. of water and add the wool plus the water to the flask. This also ensures that a n equal volume of water is added for each determination. Continue the distillation until 125 ml. of distillate is obtained. Add and distill three separate 100-ml. portions of water, giving a total of 425 ml. of distillate. Carry out the extraction and spectrophotometric measurements as described by Harrow. Results obtained using this improved technique arc: 2-Naphthylamine Dye, G. Recovered, yo 91 .o 90.1
1
62.8
5
The recovery is still unsatisfactory if more than 1 gram of dye is used. When 1 gram of dye is used, the error involved in reading the spectrophotometer is about 6% for a sample containing 800 p.p.m. of 2-naphthylamine and increases appreciably as the amine content
Free Aromatic Amine Content of Food Dyes
Dye
Batch
FD & C Yellow No. 4, Yellow OB
G Hb I J
Aromatic Amine Content,' P.P.M. Total 2-Na~hthvlamine aromatic by chromatoamines graphic method
98 85
131
135 Standard deviation0 7.2 a All values are averages of 2 analyses unless otherwise noted. Average of 3 analyses. e Based on pooled variance of all samples.
840
I
ANALYTICAL CHEMISTRY
76 77
108
135 10.8
I
1
I
I
decreases. Therefore, even thib improvedmethod is not suitable for samples containing less than 800 p.p.m. of free 2-naphthylamine. RESULTS
-4typical elution curve for the acid extract of Yellow OB is shon-n in Figure 2. Ot'her dye samples showed very similar elution curves. The first peak a t 52 ml. of eluent' is not' entirely due to o-t'oluidine: as small amounts of otoluidine or aniline cannot he detected by the ultraviolet scanning system employed because of their lo^ extinction coefficient in the acidic eluent. Some aniline or o-toluidine may be present, as t'his fraction gave a positive test for aromatic amines [diazotization follon-ed l-naphthyl) ethylby coupling with S-( enediamine]. The peak a t 80 ml. of eluent is due to 2-naphthylamine. Only trace amounts of material were eluted after 2-naphthylamine. The nature of these materials was not determined. The total aromatic amine and 2naphthylamine contents of six batches of FD&C Yellow No, 3, obtained from five independent manufact'urers, and four batches of FD&C Yellov S o . 4> obtained from four independent nianufact,urers, are given in Table 111. At the time the samples were obtained Yellow AB and Yellow OB were certified as FD&C dyes, although they are now permitted in the United States only as external D&C dyes. All were certified by the U. 5. Food and Drug Administration. A comparison of the results obtained by each of the analytical methods indicates that' the major aromatic amine impurity present is 2naphthylamine. An average of all samples indicates that 2-naphthylamine represents 85% of the total aromatic amines present. I n fact, the differences between total aromatic amine content and 2-naphthylamine content are probably not as great as indicated, because of the complexity of the chromatographic method which presents several opportunit'iesfor loss of material.
I n viw- of the fact, that the 2-naphtliylaniine cont'ent det'ermined for sample F did not meet the U. S.Food and Drug .4dministrat'ion specifications for certified Yellow AB (4, 7 ) , the analysis was checked using a modification of the mrtliod of Harrow (8). Duplicate determinations on 1-gram samples of dye gave 801 and 701 p.p.m. of 2-naphthylamine. These are in reasonable agrccment with the results ohtained by 0111' nletllod, The chromatographic system descrilled may find application in the purification and estimation of other amines. ACKNOWLEDGMENT r .
1I i r authors esprrss appreciation to
W. C. Hueper for many helpful suggestions offered during the course of this work. LITERATURE CITED
(1 ) Bonser, G. M . , Bradshaw, L., Clayson,
D. B.: "Carcinogenesis, Mechanisms of ilction," Ciba Foundation Symposium, Little, Brown, Boston, 1958. (2) Bonser G. M., Clayson, D. B., Jull, J. W., Lancet 261, 286 (1951). (3) Bratton, A. C., Marshall, E. K., J . B i d . Chem 128, 537 (1939). (4) Code of Federal Regulations, Title 21, Section 9, U. S. Government Printing Office, Kashington, D. C. (5) Deutsche Forsrhungsgemeinschaft, Rommission zur Bearbcitung des Lebensmittelfarbstoffproblems, Mitteilung 6, December 1955. (6) Evenson, 0. I,., Kime, J. il., Florest,
S. S., IND.EIUG.CIIEM., ANAL. ED. 9, 74 (1937). (7) Federal Register 24, 883 (1959). (8)Harrow, L. S., J . Assoc. O$c. Ayr. Chemists 34, 131 (1951). (9) Harrow, L. S., Jones, J. H., Zbid ., 37, 1012 (1954). (10) Howard, G. .4., Martin, J. P., Biochem. J. 46, 532 (1950). (11) Hueper, W. C., Acta U n i o n Internationale contre le Cancer 13, KO.2, 219 (1957). (12) Hueper, W.C., Wiley>F. H., Wolfe, H. D., J . Ind. H y g . Tosicol. 20, 46 (1938). (13) Wilheim, R., Ivy, A . C., Gastroenterology 23, 1 (1953).
RECEIVED for review September 10, 1959. Accepted March 23, 1960. Division of Agricultural and Food Chemistry, 136th Meeting, ACP. At1:intic (:it!-, E. J., Septemb(3r 195Cl.
Determination of Oxygen-1 8 in Phosphate Ion MICHAEL ANBAR, MORDECHAI HALMANN, and BRIAN SILVER Isotope Deparfmenf, The Weizrnann Institute o f Science, Rehovoth, Israel
b A fast and accurate method for the determination of oxygen-1 8 in phosphate ion was developed io study the kinetics of the exchange reaction between inorganic phosphates and water. The method i s based on the separation of phosphate ion as trisilver phosphate, which is subsequently pyrolyzed a t 1000" C. to yield oxygen.
P
is precipitated by the addition of silver perchlorate solution. Trisilver phosphate is soluble in dilute arid and silver oxide is precipitated by the addition of alkali to solutions containing silver ion. Therefore, the phoqphate solution is adjusted to p H 5.8 to 6.2 after precipitation, and the p H of the solution is not allowed to rise above pH 8 to 9 a t any time during the precipitation procedure. The method described here is less susceptible to errors of isotopic dilution than one involving equilibration of pota5sium dihydrogen phosphate with carbon dioxide (2, 3). The procedure involving pyrolysis of potassium dihydrogen phosphate in the presence of mercuric cyanide to yield carbon dioxide (5) failed t o give reproducible results when attempted. HOSPHATE ION
PROCEDURE
A solution containing 0.2 to 0.5 mmole of orthophosphate ion in approximately 5 ml. of water was treated with slightly
less than the theoretical amount of silver perchlorate solution. A drop of bromocresol green was added t o the solution and dilute (approximately 2.47 sodium hydroxide solution was added carefully until the end point 11-as reached (pH 3.5 to 5.4). Alternate d r o p of sodium hydroxide and silver perchlorate solutions were added, thus keeping the solution in the range of pH 3 t o 5 until precipitation was complete. The solution was finally adjusted to p H 5.8 to 6.2 with the use of indicator paper. This procedure avoided the addition of alkali t o a solution containing a large concentration of unprecipitated silver ions, which might form silver oxide because of a high local concentration of alkali. The precipitate mas centrifuged off, Ivashed with n a t e r three times, and dried in a vacuum oven a t 60" t o 70" C. Then it was transferred to a platinum crucible which was suspended by a platinum mire in a borosilicate glass vessel provided with a stopcock. The vessel was evacuated t o less than 0.5 micron and the crucible then heated for 10 minutes in a Mullard H. F. induction heating generator (Type KO. F 5/2) to 1000° C. (as determined by a Leeds & Northrup optical pyrometer). DISCUSSION AND RESULTS
The yield of oxygen was approximately 1 ml. at standard temperature and pressure from 100 mg. of trisilver phosphate. The yield (approximately 10% of the total oxygen present) was directly proportional to the weight of tri-
I.
Oxygen-1 8 Determination in Orthophosphates Atom yo Atom 7 0 0 ' 8 in Phosphate
Table
0 1 8 in Water 0.462
8.5
10 _. ~.
12 95 31 79
Direct pptn. 0.456 10 77 12.68
31 06
Through Ba3P0,
10 96 12 90 31 31
silver phosphate in the range 40 to 200 mg. and n as not increased by prolonging the heating for orer 10 minutes. The vessel was attached to a vacuum line and the oxygen transferred, by the use of a Toepler pump, to an ampoule ( 4 ) having a constriction and a break-off tip. The mercury in the pump v a s allowed to rise within 1 cm. of the contriction before the ampoule was se:rled. The oxygen n as determined directly in the mass spectrometer (Consolidated Engineering Coip., Model 21-401) by scanning masses 32 to 36. The crucible n a s cleaned after each determination by leaving i t in hot nitric acid (1 to 1) for about 5 minutes, washing in distilled water, drying, and heating in the induction furnace. To test the method, samples of phusphoric arid were prepared by hydrolyzing phosphorus trichloride in HzO1* (from the separation plant of this institute) and oxidizing the resulting phosphorus acid with elementary bromine. Silver perchlorate was added VOL. 32, NO. 7, JUNE 1960
0
841
