NOTE Agr. Biol. Chem., 39 (3), 723•`724, 1975

collected in the separator. The lower layer was dis

carded and the n-hexane extract was drained into a Colorimetric Determination of volumetric flask through a 10cm•~10mm I. D. column of granular anhydrous sodium sulfate. The

Biphenyl in Fruits separator was washed with n-hexane and washings were

passed through the column and collected in the flask. Akio TANAKA and Yoshinori FUJIMOTO Citrus oils were removed by alumina column chro matography. A 20cm•~15mm I. D. column was pre Institute of Public Health in Saitama, pared with 1.0g of activated alumina topped by 0.3g of Kamiokubo, Urawa, Japan anhydrous sodium sulfate suspended in n-hexane.

Received July 10, 1974 After damping the content of the column with n- hexane, 5ml of the extract was applied to the column

and eluted with n-hexane at a rate of 1.5 to 2.0ml/min. The colorimetric analysis of biphenyl is based upon The first 4 to 4.5ml fraction of the effluent was dis the red-purple color development by the addition of carded and the successive effluent was collected. More acetone and sodium hydroxide to the nitro-compound n-hexane was supplied until the effluent volume reached of biphenyl in isobutyl alcohol.1) This color reaction 20ml. can be practically applied to the determination of bi The effluent from the alumina column was transferred phenyl in citrus fruits if interferences are removed. In to a 50-ml separatory funnel and 3ml of 97% (w/w) order to remove citrus oils such clean-up procedures as sulfuric acid was added. The mixture was well shaken alumina column chromatography2) and sulfuric acid for S min and the acid layer was transferred to a 50-ml treatment3•`10) have been effectively applied, and thus centrifuge tube with a stopper. To the upper layer biphenyl in grape-fruit, and was quanti remained in the funnel, 3ml of sulfuric acid was again tatively measured by the calorimetric method. added and the operation was repeated. Finally all The reagents and used in the present study were the same as described in a previous paper.1) Al portions including the n-hexane layer were transferred to the same centrifuge tube. The combined solution though chloroform,3,9) cyclohexane,4•`6,8,13) n-hep was centrifuged at 3000rpm for 5min. Then the acid tane2,11,12,14) and isooctane10,14) have been used for ex layer was discarded, and the purified extract was placed traction of biphenyl by other investigators, n-hexane in a 16cm•~3cm I. D. test tube for the determination was used in the present work. Since biphenyl in n of biphenyl. hexane has a well-marked absorption band at 248nm, The procedures of biphenyl determination were de the removal of interfering substances was checked by scribed in detail in the previous paper.1) UV after each step of clean-up using a Hitachi EPS-3T

quartz spectrophotometer. Effect of clean-up

Determination procedures When the n-hexane extract was passed through the Five fruits of the average size were chopped on a alumina column, an yellowish band was found, which mainly consisted of citrus oils and was particularly plate and ground by a blender to a slurry. Fruits were marked in the case of lemon. Biphenyl in 5ml of the not peeled but whole fruits were used. Fifty g of slurry of grape-fruit, orange or lemon was placed in a 500-ml n-hexane extract was not separated from an yellowish band with 0.5g of alumina. However, through the round bottomed flask, into which 100 to 150ml of distilled water and 1ml of sulfuric acid were added. In column with 1g of alumina, only an essential oil was found in the first 4 to 4.5ml fraction of the effluent and order to avoid foaming or bumping, a few drops of silicon antifoam and several boiling chips were added. biphenyl was collected from the successive effluent. The ultraviolet spectrum of biphenyl in the distillate Then the flask was connected to an extraction unit, which is recommended by the Pharmaceutical Society from citrus fruits was not detectable as shown by curve A in Fig. 1, whereas the spectrum of the extract treated of Japan15) and also is similar to that recommended by A. O. A. C.12) The side arm of the extraction unit was on the alumina column partially represented a charac teristic curve of biphenyl as shown by curve B. filled with distilled water to overflowing and ca. 3 ml of The clean-up procedure with concentrated sulfuric n-hexane was placed on the top of the side arm and acid is effective to remove interfering substances,3,3,8) cooled with a very rapid flow of cold water. The flask was gradually heated with a mantle to boiling, and then but it causes a loss of biphenyl owing to sulfonation.5,7,10) Rajzman,9) however, pointed out that no loss of bi vigorous boiling was maintained. was continued for 1hr from the time of boiling. The dis phenyl was observed after rigorous purification of three tillate was transferred to a 100-ml separatory funnel. volumes of a chloroform extract by one volume of concentrated sulfuric acid. When 0.5 or 1.0mg of The cold finger and the extraction unit were washed with biphenyl dissolved in 20ml of n-hexane was treated a small amount of n-hexane, Successive washings were 724 A. TANAKA and Y. FUJIMOTO

TABLE ‡T. RECOVERY OF BIPHENYL ADDED TO GRAPE-FRUIT, ORANGE AND LEMON

AT THE 10 OR 20ppm LEVEL

a) Average of 2 data.b) Average of 3 data. c) Average of 4 data.

93.0%,respectively. The detection limits were ca. 4ƒÊg FIG. 1. Ultraviolet Spectra of Extracts in n-Hexane for biphenyl in grape-fruit and orange, and ca. 5ƒÊg on Each Step of Clean-up Procedures. in lemon. The results indicate that the method pro A, distillate from grape-fruit; B, extract treated on posed here is simple and sensitive, and that the precision alumina column chromatography; C, extract treated was satisfactory. with 97% (w/w) sulfuric acid; D, authentic sample of biphenyl, 5ppm in n-hexane. REFERENCES with 3ml of 97%. (w/w) sulfuric acid, the loss of 1) A. Tanaka and Y. Fujimoto, Agr. Biol. Chem., 38, biphenyl was not found. In a separatory funnel the 1575 (1974). n-hexane part was not distinctly separated from the acid 2) W. O. Winkler, J. Ass. Offic. Anal. Chem., 42, layer, and therefore, the separation was performed by 554 (1959). centrifugation. The absorption spectrum of the ex 3) R. G. Tomkins and I. A. Ischerwood, Analyst, tract after the acid treatment is shown by curve C in 70, 330 (1945). Fig. 1, suggesting that most of the interfering sub 4) H. E. Cox, ibid., 70, 373 (1945). stances was removed. 5) A. P. Steyn and F. Rosselet, ibid., 74, 89 (1949). 6) H. Bohme and L. Bertling, Z. Lebensmitt. Unter Recoveries such., 105, 311 (1957). Grape-fruits which contain biphenyl were treated by 7) B. Koether, ibid., 108, 158 (1958). the method proposed in the preceding parts. After 8) F. A. Gunther, R. C. Blinn and J. H. Barkley, clean-up the extracts were nitrated and the products Analyst, 88, 36 (1963). were identified to be 2, 2•L,4, 4•L-tetranitrobiphenyl by the 9) A. Rajzman, ibid., 88, 117 (1963). examinations described in the previous paper.1) And 10) S. Norman, G. L. Rygg and A. W. Wells, J. Ass. it was confirmed that biphenyl was stable throughout Offic. Anal. Chem., 49, 590 (1966). the procedures. 11) P. E. Corneliussen, ibid., 50, 934 (1967). Since fruits free from biphenyl were not obtained, the 12) Official Methods of Analysis of the A. O. A. C., 11th recovery tests were carried out using citrus fruits treated ed., p. 490 (1970). with biphenyl, and the amount of biphenyl quanti 13) T. Hayashi, S. Kato, H. Watanabe, Y. Hara and tatively added to fruits at the 10 or 20ppm level were A. Tanimura, J. Food Hyg. Soc. Japan, 13, 78 analyzed. The recoveries of biphenyl added to grape (1972). fruit, orange and lemon are presented in Table ‡T. 14) J. G. Kirchner, J. M. Miller and R. G. Rice, J. At the 10ppm level the average values of 4 data for Agr. Food Chem., 2, 1031 (1954). grape-fruit, orange and lemon were 94.0, 91.5 and 15) Pharmacopoeia Japonica, 8th ed., vol. I, B-78 96.0%, and those at the 20ppm level 95.0, 95.5 and (1971).