Agr. Biol. Chem., 38 (9), 1575•`1579, 1974 Colorimetric Determination of Biphenyl Based on the Janovsky Reaction Akio TANAKA and Yoshinori FUJIMOTO Institute of Public Health in Saitama, Kami-okubo, Urawa, Japan Received January 28, 1974 The quantitatively nitrated product of biphenyl with potassium nitrate and sulfuic acid shows a characteristic red-purple color on reaction with isobutyl alcohol, acetone and alkali; this is the Janovsky reaction. The color reaction was sensitive, and the absorbance at 550nm obeyed Beer's law at biphenyl concentrations between 2 and 40ƒÊg in 3.5ml of the reaction mixture. A procedure suitable for routine use is proposed. The nitro-compound derived from biphenyl was identified as 2,2•L,4,4•L-tetranitrobiphenyl by Rf on TLC, as well as by mixed melting point, IR and mass spectroscopy. Biphenyl has been widely used as a fungistat Melting points were determined with an electro to prevent rotting in citrus fruit during storage thermal capillary melting point apparatus. Infrared spectra were recorded on a Jasco infrared spectro and transport. However, in order to avoid photometer, Model IR-G. Mass spectra were ob excess biphenyl residues in treated fruit, several tained with a Hitachi RMU-6M mass spectrometer. countries have set tolerance limits. Besides the ultraviolet absorption method, Reagents and solvents. Acetone, benzene, iso butyl alcohol, chloroform, ethyl acetate, n-hexane and such as that proposed by A.O.A.C.,1) the colori D-limonene were all of analytical grade, obtained from metric method was also proposed by many Wako Pure Chemical Industries. Sulfuric acid, cal investigators.2•`7) Janovsky8•`9) found that aro cium carbonate and sodium hydroxide were of high matic nitrocompounds treated with alkali, ace purity, obtained from Kanto Chemical Co. The con tone and alcohol show a characteristic color. centration of sodium hydroxide solution was adjusted to 0.025N (F=0.9 to 1.1) for general use. Silica gel Recently Nakamura10) has applied this reaction HF254 was obtained from Mallinckrodt Chemical to a colorimetric determination for benzoic acid Works. in food. Biphenyl and 2,2•L,4,4•L-tetranitrobiphenyl were of In this paper a colorimetric determination of high purity, obtained from Wako Pure Chemical biphenyl based on the Janovsky reaction was Industries. Standard solutions of biphenyl were pre pared by dissolving 100mg biphenyl in 100ml n-hexane. proposed and a nitro-compound derived from The standards thus prepared were stable at ambient biphenyl was discussed. temperature over a period of several weeks and were diluted to the desired concentration with n-hexane for MATERIALS use. Analytical equipment. Absorbance measurements RESULTS AND DISCUSSION were made with a Hitachi spectrophotometer, Model EPS-032, with a rapid sampler attachment (10mm 1. Proposed determination procedure for bi path length). phenyl For the identification of nitro-compounds of bi Evaporation of solvent. A suitably diluted phenyl, thin-layer chromatography (TLC) was carried out on precoated silica gel plates. Silica gel HF254 biphenyl standard solution or purified extract was heated to 110•Ž for 2hr prior to use. The solvent was placed in a test tube (3cm i.d•~16cm system used was ethyl acetate-benzene (1:5), and the length) with a tapered capillary tube (2mm i.d. solvent front was 10cm above the origin in all cases. •~ ca. 17cm length) sealed to the upper end Detection was done by viewing under short-wave UV and fitted to a filtration jar with a rubber light. 1576 A. TANAKA and Y. FUJIMOTO stopper. The tip of the tapered capillary was set ca. 0.5mm from the bottom of the test tube. Dried air passed through sulfuric acid placed in a filtration jar was permitted to flow through the test tube to evaporate the n- hexane. The n-hexane was carefully evaporat ed in dry air at room temperature to avoid vaporization of biphenyl. After the removal of the n-hexane, the stopper with the tapered capillary was removed. As the biphenyl re sidue in the test tube tended to sublime, nitra tion was carried out within 2min after the evaporation of the solvent. FIG. 1. Absorption Spectra of the Compounds Produced by Color Reaction. Nitration of biphenyl. The biphenyl residue A, nitro-compound of biphenyl; B, 2,2•L,4,4•L-tetra in the test tube was dissolved in 0.5ml of sul nitrobiphenyl. furic acid and 0.1 to 0.2g of potassium nitrate. With occasional shaking, the test tube was diluting the stock solution with n-hexane. placed in a water bath and kept in boiling water Aliquots were taken in test tubes to give a- for 45min. After being cooled to room tem mounts of 0.1, 0.3, 0.5, 0.7, 1.0 and 1.5mg of perature, the sample was transfused with 20 to biphenyl. According to the procedure des 30ml of distilled water into a 100-m1separating cribed above, 20ml of isobutyl alcohol extract funnel to which 20ml of isobutyl alcohol was was obtained in each case. An aliquot of 0.5 then added. The mixture was well shaken for ml was taken for color formation. When 1 ml 3 to 5min and the layers were separated. The of the standard stock solution was taken, there isobutyl alcohol extracts were washed with 20 fore, 3.5ml of reaction mixture, which consist ml of 1% sulfuric acid and transfered to a 50-ml ed of 0.5ml of the extract, 1ml of alkali and centrifuge tube with a stopper. 2ml of acetone, contained 25ƒÊg of biphenyl. The extracts were neutralised with 0.1g of The graph obtained gave a straight line, as calcium carbonate and then centrifuged until shown in Fig. 2. Beer's law was obeyed at separation was complete. The neutralized concentrations between 2 and 40ƒÊg of biphenyl upper isobutyl alcohol layer was used for the colorimetric determination. Colorimetric determination. Upon addition of 2ml of acetone and 1ml of 0.025N sodium hydroxide to 0.5ml of the isobutyl alcohol ex tract, the mixture quickly developed a red purple color. The absorption spectrum is shown in Fig. 1. Since the maximum absorp tion was found at 550nm, quantitative meas urements were carried out at 550nm, 10, 15 and 20min after the color development. The maximum reading was adopted to determine the biphenyl content. Calibration graph. A series of working FIG. 2. Standard Curve for Biphenyl Determination. standard biphenyl solutions were prepared by Wavelength, 550nm; cell thickness, 10mm. Colorimetric Determination of Biphenyl Based on the Janovsky Reaction 1577 in 3.5ml of the reaction mixture. removal of the n-hexane. 2. Comments on the proposed procedure Conditions for nitration. For the nitration of biphenyl, 0.05, 0.1, 0.2, 0.3 or 0.4g of potas Before the proposal of the above procedure for determining biphenyl, the following points sium nitrate was added to 1mg of biphenyl with 0.5ml of sulfuric acid. The nitration was were examined. almost complete in 30min on boiling water Influence of evaporation of the solvent on the and no difference was found with any of the biphenyl recovery. Prior to the nitration of above amounts of potassium nitrate. After biphenyl it was necessary to evaporate off the the nitration, the resulting nitro-compound was n-hexane. As biphenyl is volatile, possible loss stable for at least 3 days at room temperature during or after evaporation of the solvent as isobutyl alcohol solution. should not be overlooked. 0.5 or 15ml of n-hexane which contained 0.5 or 1.0mg of Color development of the nitro-compound. biphenyl was evaporated by dry air flow at Color development depended significantly upon room temperature. The loss of biphenyl dur the mixing rate of reagents, alkali concentra ing the evaporation was scarcely observable. tion and temperature. Figures 3 and 4 show One of experiments, where 15ml of n-hexane the time curves of color formation under vari was evaporated for ca. 40min, is shown in ous conditions. The volume of isobutyl alco Table I. However, after the n-hexane had been hol was unchanged and the relative amounts of acetone and alkali were varied from 2 to 4 TABLE I. INFLUENCE OF THE EVAPORATION times. The absorbances at various combina OF THE SOLVENT ON BIPHENYL RECOVERY tions are illustrated in Fig. 3. Where the pro The amount of n-hexane was 15ml. portions of isobutyl alcohol, acetone and alkali were 1:2:4, the absorbance was most intense and the time required to reach the maximum was short. As illustrated in Fig. 4, when 0.05 N sodium hydroxide was used with this combi nation, color development was rapid, but fading was significant. With 0.075N sodium hydro xide, similar phenomena were observed. How ever, with 0.025N sodium hydroxide, color development was gradual and the color was stable for 30 min. The effect of temperature on color develop removed and biphenyl remained as the residue, ment was marked. Although the develop the decrease of biphenyl was significant, as ment and fading of color were accelerated at shown in Table II. Therefore, the sample higher temperature, the value of the maximum should be nitrated within 2min after the absorbance was constant, at least in the range TABLE II. DECREASE OF BIPHENYL RESIDUE AFTER EVAPORATION OF THE SOLVENT Biphenyl content before evaporation was 1.00mg. The volume of n-hexane was 1ml for I and 15ml for II, and the tests were carried out at room temperature. 1578 A . TANAKA and Y. FUJIMOTO of room temperature. In order to avoid com plexity in keeping the temperature constant, for routine work, the absorbance was observed 5, 10 and 15min after color development.