Quantitative Hydrolysis of Sodium Cyclamate and CaIcium Cycla mate to CyclohexyIa mine, Followed by Colorimetric Analysis Darryl E. Johnson, Helmut B. Nunn, and Stanley Bruckenstein' Food and Drug Administration, Minneapolis, Minn. 55401 The hydrolysis of sodium cyclamate to cyclohexylamine was studied in aqueous 1.3N HCI at 125" C (15 psi). The rate of hydrolysis is first order in cyclamate, with a half time of 1.17 hours. Using sodium hydroxide to adjust the p H of the hydrolysis solution to 12, cyclohexylamine was extracted into chloroform and reacted with ethanolic p-quinone (60" C for 2 hours) to form 2-(cyclohexylamine)-1,4-benzoquinone which was determined spectrophotometrically at 493 mp. Sodium cyclamate and calcium cyclamate yielded average recoveries of 99.9 i 2.1% and 99.7 f 0.8%, respectively, compared to a cyclohexylamine standard.
THEVARIOUS titrimetric, gravimetric, spectrophotometric, and gas chromatographic procedures being used to determine sodium cyclamate have been reviewed by Richardson ( I ) . Ichikawa et al. ( 2 ) describe a hydrolysis technique for sodium cyclamate in acid medium in the presence of hydrogen peroxide, followed by a colorimetric detection of the reaction product of p-quinone or quinhydrone and the cyclohexylamine produced by acid hydrolysis. Their procedure was intended only for qualitative purposes. Derse and Daun (3) also hydrolyzed the cyclamate in acid in the presence of hydrogen peroxide but used G L C for the quantitative determination of cyclohexylamine. The increasing use of cyclamate salts as sweeteners in food products has made it necessary to have a specific method for their determination. While searching for a method, we felt that the approach of Ichikawa et a/. ( 2 ) seemed promising. However, we found that acid hydrolysis in the presence of hydrogen peroxide does not produce an equivalent amount of cyclohexylamine. Therefore, we studied the hydrolysis of the cyclamates at several temperatures without adding any hydrogen peroxide, varying acid concentration and hydrolysis time. We found that quantitative conversion of cyclamate to cyclohexylamine is possible by heating a solution containing 1.3N HCl at 125" C in an autoclave for at least 7 hours. The rate of hydrolysis is first order in cyclamate, and the half time, = 1.17 hours. The qualitative colorimetric procedure of Ichikawa et at. ( 2 ) was investigated, and experimental conditions were found which yielded satisfactory quantitative results. We did not pursue the gas chromatographic approach of Derse and Daun (3). It appeared from their results and O'Donnell and Mann's gas chromatographic studies of amines ( 4 ) that the colorimetric procedure would be about an order of Chemistry Department, University of hfinnesota, bIinneapolis, AIinn. 55455. (1) 11.L. Richardson, Tuluntu, 14, 385 (1967). (2) 11. Ichikawa, S. Kojima, and H. Ichibangase, Yakugaku Zusshi, 84, 563 (1964). f3) ~, P. H. Derse and R. J. Daun. J . Assoc. Ofic. Anal. Chernisrs, 49, 1090 (1966). (4) J. F. O'Donnell and C . K. Llann, ANAL.CHEM.,36, 2097 (1964).
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magnitude more sensitive. Future developments in the gas chromatography of amines could make this technique the preferred determinative step for cyclohexylamine. EXPERIMENTAL
Apparatus. Absorbance measurements were made with a Cary Model 15 recording spectrophotometer using 1-cm cells. Hydrolysis was carried out in an American sterilizer (Model 57CR) operating at about 15 psi (125°C). Reagents. Sodium cyclamate, code 69281, lot 754-7483 and calcium cyclamate, code 69278, lot 757-7558, were obtained from Abbott Laboratories, North Chicago, Ill. CYCLOHEXYLAMINE (Eastman Kodak, White Label No. 2469). Titration with HCI in water yielded a purity of 99.4%. A 60 p g p l chloroformic solution was prepared. (One microgram of cyclohexylamine is equivalent to 2.029 pg of sodium cyclamate and 1.999 pg of calcium cyclamate.) The rest of the reagents were of reagent grade quality. Procedure. The following procedure was developed on the basis of the work reported in this paper. Cyclamate is hydrolyzed to cyclohexylamine, the cyclohexylamine is extracted into chloroform, and (I) is produced by reacting the chloroform solution of cyclohexylamine with ethanolic p-quinone (2).
I Stable absorbance readings due t o (I) are obtained provided (I) is not exposed to direct light. Weigh about 30 mg sodium or calcium cyclamate into a 100-ml beaker and dilute to 60 ml with 1.3N HC1. Put the 100-ml beaker inside a 400-ml beaker, cover the large beaker with a watch glass and place it in the pressurized sterilizer a t 125" C for at least 7 hours. Dilute the cooled solution t o 250 ml. Pipet a 25-ml aliquot into a separatory funnel, adjust p H t o 12 using 10N NaOH and add 4 drops excess base. Add 25 ml of chloroform and shake vigorously 1 minute. Pass the chloroform layer through a tight glass wool p!ug in the stem of the separatory funnel and pipet a 20-ml aliquot into a 50-ml volumetric flask. Into a second 50-ml volumetric flask, pipet 20 ml of a 60 pg/ml of cyclohexylamine in chloroform standard solution. Into a third 50-ml volumetric flask, pipet 20 ml of chloroform which serves as a reference blank. Pipet into each flask 10 ml of a freshly prepared 0.30 solution ofp-quinone in absolute ethanol. Place all the volumetric flasks into the same 60" C water bath for 2.0 hours away from direct light. Dilute the cooled solutions to volume with chloroform and determine the absorbance of the sample solution and the standard solution at 493 mp (wavelength of maximum absorption) against the reference blank. This blank is identical to that obtained by carrying a blank through the entire procedure.
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