Analyst, M a y , 1977, Vol. 102,13p. 367-370 367

Determination of Saccharin in Soft Drinks by a Spectrophotometric Method

Akio Tanaka, Norihide Nose, Toshimasa Suzuki, Susumu Kobayashi and Akinobu Watanabe Saitama Institute of Public Health, Kamiokubo-Higashi 639-1, Urawa, Saitama, Japan

Saccharin reacts quantitatively with phenothiazine (thiodiphenylamine) and copper(I1) acetate dissolved in 50% V / V ethanol a t 70 "C. The reaction product can be dissolved in an organic solvent and has a characteristic colour. The colour reaction is sensitive and the absorbance, a t 510 nm, of the extract in 5 ml of xylene obeys Beer's law at saccharin concentrations between 20 and 400 pg ml-1. Cyclamates, sorbic acid, benzoic acid, 4-hydroxybenzoic acid and dehydroacetic acid do not interfere. Recoveries of saccharin from soft drinks were satisfactory. A procedure suitable for routine use is proposed.

Keywords ; Saccharin determination ; soft drinks ; spectrophotometry

Saccharin has been widely used as an artificial sweetening agent in a variety of food products. However, in order to avoid excessive use, several countries have set tolerance limits. The literature contains numerous reports on the deterrnination of saccharin in food and beverages. Various techniques have been used : thin-layer chromatography,l gravimetry,2 p~larography,~ the use of an ion-selective electrode,* colorimetry,5 infrared spectrometry,6 gas - liquid chr~matography,~ ultraviolet spectrometrp and recently molecular emission cavity analysis.@ Recently, we found that saccharin reacted with a solution of phenothiazine and copper(I1) acetate dissolved in an organic solvent and produced a red colour. A colorimetric method for saccharin was reported recently by Fernandez-Flores et al.,lO but this method was not suitable for routine use because of complex formation and the vigorous reaction conditions required. In this paper, we describe the development of a simple, accurate and specific reaction for saccharin, which can be used for its routine determination in soft drinks.

Experimental Apparatus

Hitachi, Model EPS-032, spectrophotorneter.

Reagents Analytical-reagent grade reagents are t o be used throughout the determination. Phenothiazine solution.

Acetic acid solution, 0.5%. Co$$er(.II) acetate solution, 0.5%.

Ethanol, 50% V/V. Saccharin standard stock solution, 1 000 p.p.m.

Sodium hydrogen carbonate solution, 1 yo. Sadfihuric acid, 10%. Hydrochloric acid, 10%. Diethyl ether and xylene.

Dissolve 1 g of phenothiazine (thiodiphenylamine) in 100 ml of

Dissolve 0.5 g of copper(I1) acetate in 100 ml of 0.5%

Weigh accurately 100 mg of saccharin and

99.5% ethanol. Prepare the solution fresh daily.

acetic acid.

dissolve it in 100 ml of 6oy0 ethanol.

Very high purity grades.

Procedure The beverage was decarbonated by repeated shaking and pouring from one beaker to another.

A 50-g amount of sample was transferred into a 300-ml separating funnel fitted with a PTFE stopcock, 30 ml of water and 5 ml of 10% sulphuric acid were added and the mixture was extracted twice with 100 ml of diethyl ether, shaking for 3 min, and then the aqueous layer

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368 Analyst, Vol. 102 was discarded. The upper layer was extracted twice with 25 ml of 1% sodium hydrogen carbonate solution and the diethyl ether layer discarded. The aqueous layer was acidified with 10 ml of 10% hydrochloric acid, and then extracted twice with 30 ml of diethyl ether. The ether extract was washed with 10 ml of distilled water, transferred into a 100-ml flask and evaporated to a small volume at 40 "C.

The saccharin residue in the 100-ml flask was transferred into a test-tube (about 17.5 x 1.7 cm id.) by means of 5 ml of 50% ethanol, and 1 ml of copper(1I) acetate solution and 1 ml of phenothiazine solution were added, followed by 3 ml of ethanol. The test-tube was placed in a water-bath at 65-70 "C and was held at that temperature with occasional shaking for 50 min. After being cooled to room temperature, the sample solution was transferred into a 50-ml separating funnel with 2 ml of 99.5% ethanol, and 5 ml of xylene and 15 ml of water were then added. The mixture was shaken vigorously for 5-10min and the xylene layer separated and dried with 1 g of anhydrous sodium sulphate.

TANAKA et al. : DETERMINATION OF SACCHARIN IN

Preparation of Calibration Graph The maximum absorption of the colour was found to occur at 510 nm. Aliquots of the

standard saccharin solution containing 0.1 and 2 mg of saccharin were placed in test-tubes and taken through the procedure described above. The absorbances of the solutions were measured at 510 nm in 10.0-mm cells. A graph of the absorbance against concentration of saccharin was a straight line passing through the origin. Beer's law was obeyed at concentrations of saccharin in the xylene solution between 20 and 400 pg ml-1.

0.5

E & 0.4 F

v) .w

0.3 0

m f 0.2 w 0

Cx 0.1

I

0 5 10 15 20 25 30 Phenothiazine in 10 ml of reaction mixture/mg

Fig. 1. Effect of amount of phenothiazine on intensity of colour. Amount of saccharin added: 1.00 mg.

I 0.5

0.4

z In

0.3 Q1

5 0.2 -!2 w 2 0.1

0 Copper(ll) acetate in 10 ml of reaction mixture/mg

Fig. 2. Effect of amount of copper(I1) acetate on intensity of colour. Amount of saccharin added: 1.00 mg.

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May, 1977 SOFT DRINKS BY A SPECTROPHOTOMETRIC METHOD 369 Determination of Saccharin in Samples

The absorbances of both standard and sample solutions were measured in 10.0-mm cells a t a wavelength of 510nm against a blank carried through the procedure simultaneously. The amount of saccharin was calculated as follows:

B Saccharin in sample (p.p.m.) = - x 3 x 5 000 A m2

where A and B are the absorbance values of standard and sample, respectively, m, mg is the amount of saccharin per millilitre of xylene extracted from the standard solution with absorb- ance A corresponding to the corrected absorbance, and m2 g is the amount of sample present.

Results and Discussion The maximum intensity

of the colour was obtained by using 10 mg of phenothiazine, 5 mg of copper(I1) acetate and 1 ml of 0.5% acetic acid, as shown in Figs. 1 and 2 and Table I. A precipitate was obtained in a neutral or alkaline solution or if an excess of copper(I1) acetate was present. A study of the effect of reaction time showed that the optimum time for maximum sensitivity was 45-50 min at 70 "C, as shown in Table 11. Xylene, hexane, ethyl acetate, cyclohexane, 4-methylpentan- 2-one and chloroform were tried as extraction solvents. The most suitable of these solvents was xylene and the least suitable was chloroform; the absorbance of the xylene extract remained stable for a t least 1 d.

The colour reaction of saccharin with phenothiazine was studied.

TABLE I EFFECT OF ACETIC ACID CONCENTRATION ON INTENSITY OF COLOUR

Acetic acid concentration, yo . . 0.1 0.5 1.0 2.0 5.0 7.0 10.0 16.0 Absorbance at 510 nm in

10.0-mm cell . . .. . . 0.46 0.47 0.40 0.38 0.34 0.31 0.21 0.18

TABLE I1 EFFECT OF TIME AT 70 "C ON INTENSITY OF COLOUR

Timelmin . . .. .. 10 20 30 40 50 60 Absorbance at 510 nm in

10.0-mm cell . . . . 0.12 0.20 0.30 0.45 0.47 0.46

The following food additives did not interfere, as can be seen from Table I11 : cyclamates, sorbic acid, benzoic acid, 4-hydroxybenzoic acid and dehydroacetic acid.

TABLE I11 INFLUENCE OF FOOD ADDITIVES ON RECOVERY OF SACCHARIN FROM SOLUTIONS CONTAINING

1 mg OF SACCHARIN

Amount Additive addedlmg

Cyclamate . . .. 1 5

Sorbic acid . . .. 1 5

Benzoic acid . . .. 1 5

Saccharin foundlmg Recovery, yo

0.99 99 0.98 98 1 .oo 100 1.00 100 1 .oo 100 1.00 100

4-Hydroxybenzoic acid 1 1 .oo Dehydroacetic acid . . 1 1 .oo

5 1.00

5 1.00

100 100 100 100

To check the validity of the proposed procedure for determining saccharin in soft drinks, recoveries of saccharin added to various saccharin-free soft drinks were measured (Table IV).

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370 TANAKA, NOSE, SUZUKI, KOBAYASHI AND WATANABE

The recoveries ranged from 95.7 to 103.6% with an average of 99.5%. The standard deviation for the whole procedure was 0.03% for the 18 determinations given in Table IV.

TABLE IV RECOVERY OF SACCHARIN ADDED TO SACCHARIN-FREE SOFT DRINKS AND BEVERAGES

Amount of saccharin added, 1.00 mg. Saccharin found/mg

Sample I A > Water . . .. , . 1.00 1 .oo 1.00 Cola . . .. * . 0.94 0.95 0.98 Apple juice . . . . 0.97 0.97 0.98 Lemon drink . . . . 0.99 1.03 1.04 Orange drink . . . . 1.02 1.01 1.05 Carbonate drink . . 0.98 0.99 1.00

Mean recovery, yo

100.0 95.7 97.3

102.0 103.6 99.0

Mean . . .. .. Standard deviation . .

T

0.99 f0.03

99.5 f2.5

It was concluded that the reaction of saccharin with phenothiazine is quantitative and is as sensitive as previously published r n e t h ~ d s . ~ , ~ ~ * ~ Q It gives a rapid and very simple method for the determination of saccharin. The reaction is specific for saccharin and the colour is very stable.

The authors thank Mrs. Akiko Hirose for her co-operation in carrying out this work.

1. 2. 3. 4. 5. 6.

7. 8. 9.

10.

References Korbelak, T., J , Ass. Off. Analyt. Chem., 1969, 52, 487. Oakley, M. S., J . Ass. 08. Agric. Chem., 1947, 30, 492. Lasheen, A. M., Proc. Am. SOC. Hort. Sci., 1966, 77, 135. Hazemoto, N., Kamo, N., and Kobatake, Y., J . Ass. Off. Analyt. Chem., 1974, 57, 1206. Duran, J. J., J . Ind. Engng Chem., 1913, 5, 988. Coppini, D., and Albasini, A., Mitt. Geb. Lebensmittelunters. u. Hyg., 1968, 59, 239; Chem. Abstr.,

Ratchik, E. S., and Viswanathen, V., J. Pharm. Sci., 1975, 64, 133. Hussein, M. M., Jacin, H., and Rodriguez, F. B., J . Agric. Fd Chem., 1976, 24, 36. Belcher, R., Bogdanski, S. L., Sheikh, R. A., and Townshend, A., Analyst, 1976, 101, 562. Fernandez-Flores, E., Johnson, A. R., Leber, B., Larry, D., and Lerner, S., J . Ass. Off. Analyt.

Received September Znd, 1976 Accepted December 13th, 1976

1969, 70, 76503 g.

Chem., 1973, 56, 1411.

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