Document O1B19VBMJ7xqkv9xqbOGNzznK

'. .- 330 1 TOMKINS AND ISHERWOOD: THE ABSORPTION OF DIPHENYL AND (2). The nature of the coupling reactions between the phenols and diazotised />-nitraniline is discussed, and a modified test is described of considerably greater sensitivity and reliability; the test involves the removal of soaps from the saponified mixture by salting out, distillation of the acidified filtrate, and coupling the distillate with stable /(-nitrobenzene diazotate in alkaline buffer soln. , ' (3) The test is a general one for the lower phenols and their esters; the azo-colour formed can be separated and identified by pptn. as lakes on magnesium hydroxide. I am obliged to the Director, Medical and Health Department, Mauritius, for permission to publish this paper. ' _ Reference 1. Analyst, 1938, 63, 813. Government Chemist's Laboratory Reduit, Mauritius - - January, 1945 .- .^ r __ . __ The Absorption of Diphenyl and o-Phenyl-phenol by Oranges from Treated Wraps By R.-G. TOMKINS and F. A. ISHERWOOD Rotting of citrus fruits due to green mould can be reduced by wrapping the fruit in paper treated with the volatile fungicides diphenyl1'2-3 and o-phenyl-phenol.4*5'8 Methods of estimating the small amounts of these fungicides absorbed by oranges from treated wraps have now been devised and some of the factors affecting the extent of absorption are discussed below. Oranges were wrapped in papers impregnated with diphenyl, or o-phenyl-phenol and hexamine, and then stored in partly-open tins at constant temperature. The amount of diphenyl (or o-phenyl-phenol) taken up was estimated at intervals. In most of the expts. each treated wrap contained about 100 mg of diphenyl or o-phenyl-phenol, which is more than is required to reduce wastage. Consequently the quantities of the fungicides absorbed by the fruit were probably greater in our experiments than would be expected under com mercial conditions. '. ' Estimation of Dipiienyl in Oranges--A sample (4 or 5) of the oranges which had been N' ' stored in diphenyl-treated wraps was weighed and the peel was separated from the pulp. . ^ The tissue (peel or pulp) finely minced, was then steam-distilled in the usual manner until S" about 2 litres of distillate had been collected (2 hr.). The distillate was extracted three times i with chloroform (90 ml), which removed both the diphenyl and any orange oil present. Of the chloroform soln. 45 ml, containing about 2 ml of oil, were shaken in a separating funnel with . sulphuric acid (sp.gr. 1-84, 45 ml). Moderately vigorous shaking of the mixture was necessary for the efficient destruction and removal of the orange oil, but the formation of a too persistent emulsion should be avoided. After standing for 20 min. the mixture separated into two ' layers, the upper light yellow and the lower dark red. The latter (sulphuric acid) was re moved, and the upper layer (chloroform) again shaken with an equal vol. of sulphuric, acid (sp.gr. 1-84). This procedure was repeated four times, chloroform being added to the upper layer when necessary to maintain its vol. at 45 ml. After the last treatment the chloroform soln. was shaken with water (120 ml in two portions), dried over anhydrous calcium chloride and filtered. The colour of the resulting soln. was a very pale yellow, indicating the almost complete destruction of the orange oil. The diphenyl in this solution was then estimated according to Mulliken I904,7 "Drop a hard lump of sublimed aluminium chloride weighing - 0-2-0-3 g into a clean 6-8 in. test-tube that has just been taken from a hot drying-oven. Stopper the tube loosely. Hold it in a nearly horizontal jwsition, and by means of a small flame placed under one end slowly sublime the chloride until it forms a thin iight yellow coating covering a considerable portion of the glass surface. Allow to cool. Drop in 0-5 ml of a soln. containing 0-05 g of the hydrocarbon dissolved in 2*5 ml of chloroform. Stopper the tube tightly. Lay it on to its side upon a sheet of white paper that rests upon and partly covers . the colour standard. Then roll it back and forth so that the solution shall flow over and wet all parts of the sublimate. Observe the colour after a few seconds, and again after 15-20 minutes." The colour given by diphenyl was blue. (Orange oil, if present, completely masked the colour given by the diphenyl.) The chloroform soln. of the diphenyl obtained from the oranges was diluted until 0-5 ml gave a.similar colour intensity under standard conditions to that produced by 0-5 ml of chloroform containing 0-05 mg of diphenyl. Dis tilled dry chloroform was used, and every precaution taken to exclude traces of moisture. DSW 330563 STLCOPCB4077207 !f O-PHENYL-PHENOL BY ORANGES FROM TREATED WRAPS . 331 It was found convenient to take a large fragment of aluminium chloride and break it into smaller pieces under chloroform to ensure fresh substance for the test. The method must be considered only roughly quantitative, the results being subject to an error of about 20%, as shown in Table I. Table I . Amount of Amount of Procedure diphenyl added mg diphenyl recovered mg 1. Added to CHC1,, treated with H,S04> etc. .. .. .. .. "' 6 10 4-6 10 2. Added to 2 ml* of oil, the oil dissolved in CHClg and then treated with H,S04, etc. . 20 5 10 .16 4-5 8-4 20 14 3. Added to H,0, steam-distilled, and distillate extracted with CHC1,, etc. 10 8-9 4. Added to orange oil* (4 ml) steam-distilled and distillate extracted with CHC1,, etc. .. .. .. .. .. .. .. ., 10 9-5 5. Added to minced peel from 6 oranges, steam-distilled, and distillate extracted with CHCl,, etc.- ' 6 10 20 4-8 8-9 18-2 6. Orange* oil (2 ml) steam-distilled, etc. .. .. .. .. nil nil * The orange oil had been previously prepared from orange peel by steam-distillation. Wraps--The treated wraps (2 or 3, each approx. 10 in. x 10 in.) were extracted with chloroform (3 x 30 ml) and the amount of diphenyl present was determined as described above. As no orange oil was present, the treatment with cone, sulphuric acid was omitted. Estimation of o-Phenyl-phenol in Oranges--A sample (4 or 5) of the oranges which had been stored in o-phenyl-phenol treated wraps was weighed and the peel was separated from the pulp. The finely-minced tissue (pulp or peel) was then steam-distilled in the usual manner until the volume of the distillate collected was about two litres (2 hr.). The distillate was - acidified (5 ml of 2 N hydrochloric acid) and then shaken vigorously with light petroleum (10 ml, B.P. 40-60 C.) in a separating funnel. After standing a short time the mixture separated into two layers; the light petroleum layer was removed and the aqueous layer was again Shaken with light petroleum (10 ml). It was found that at least ten extractions with light petroleum were necessary to remove all the o-phenyl-phenol from the aqueous phase. The combined light petroleum extracts were then shaken vigorously with sodium hydroxide soln. (Ar/10, 33 ml) in a separating funnel. After the mixture had stood for a short time for it to separate, the sodium hydroxide was removed, and the light petroleum soln. was again , shaken with fresh sodium hydroxide (iY/10, 33 ml). This procedure was repeated once more. The o-phenyl-phenol now' in solution in the sodium hydroxide soln., was estimated colorimetrically by coupling with diazotised sulphanilic acid as described by Hanke and Kressler8 for phenols and related compounds The colour was compared with that produced by a standard solution of o-phenyl-phenol (0-01 mg/ml) under similar conditions. - Table II shows the degree of accuracy which may be expected by this method: Table II Procedure 1. Added to light petroleum (100 ml.) and extracted with sodium hydroxide soln. * 2. Added to water (2 litres), and extracted with light petroleum. The light petroleum extracted with sodium hydroxide soln. 3. Added to water, steam distilled, and the distillate extracted with light petroleum, etc. 4. Added to orange oil (4 ml) dissolved in light petroleum and ex tracted with sodium hydroxide soln. 6. Added to the peel (minced) from 5 oranges (allowed to stand over night) and then steam-distilled, etc., as described in the full procedure. 6. Untreated orange peel (5 oranges). Amount of Amount of o-phenyl- o-phenyl- phenol added phenol found mg mg -5 10 '5 10 20 . 20 10 8-6 6 . 40 10 8-3 20 16-8 65 10 10 20 20 6 3-76 10 9-1 20 16-9 nil nil 0S\N *>056* STLCOPCB4077208 332 TOMKINS AND ISHERWOOD: THE ABSORPTION OF DIPHENYL AND Wraps--The treated wraps (2 or 3, approx. 10 in. X 10 in.) were extracted with sodium hydroxide soln. (N/10, 100 ml in two portions). The aqueous soln., which contained the sodium salt of o-phenyl-phenol, was acidified and extracted with light petroleum as described above. Amounts of Diphenyl or o-Phenyl-phenol Recovered from Oranges Wrapped in Paper Impregnated with Diphenyl or o-Phenyl-phenyl and Hexamine--The effects of temperature of storage and of the amount of fungicide in the wrap are shown in Tables III, IV, VI and VII. Practically no diphenyl or o-phenyl-phenol (less than 0-1 mg) was recovered from the pulp, the amounts recorded below having been recovered from the peel. . These and other expts. showed that both fungicides are absorbed by/oranges from treated wraps in quantities which were related to the concentration in the wraps, and duration of storage. In most expts. absorption increased with temperature. The amounts absorbed also varied with oranges from different countries and was about 4 to 20 mg of diphenyl or 20 to 30 mg of o-phenyl-phenol per 100 g. Aeration of the fruit after removal from treated wraps resulted in a very slow loss of the absorbed fungicides (Tables V and VIII). Table III Uptake (mg per orange) of Diphenyl by Jaffa Oranges (Each wrap containing 100 mg of diphenyl) Temperature of storage cc. 18 10 5 Days i---------------------------------------- ------------------------ 1------------------14 27 34 48 - 62 71 3-2 10-5 4-8 6-4 7-2 8-0 40 6-4 8-0 8-9 9-4 2-4 3-9 4-5 6-5 7-3 . . Table IV Uptake of Diphenyl (mg per orange) at 10 C. from Wraps containing Different Amounts of Diphenyl Amount of diphenyl in wraps 14 Days 36 48 62 71 100 mg per wrap .. 4-0 6-4 ' 8-0 8-9 9-4 50 ,, ,, .. 2-4 4-0 5-2 5-6 6-7 ' 20................... 1-9 2-8 2-8 2-9 2-8 Table V Loss of Diphenyl from Fruit Previously Wrapped in Diphenyl Treated Wraps (The fruit was stored in a desiccator through which a slow stream of air was passing) Days in air 0 11 17 23 Diphenyl, mg per orange 7-6 7-2 4-6 4-6 The odour of diphenyl soon disappeared from these oranges. 36 4-8 __ Table VI ' Uptake (mg per orange) of o-Phenyl-phenol by Oranges at Different Temperatures " Each wrapeontained 95 mg o-phenyl-phenol plus 25 mg hexamine Temperature of storage C. 18 10 5 17 4-8 2-4 3-0 Days \- ` 42 57 68 77 9 1 11-4 10-0 10-0 4-6 5-0 8-9 8-0 4-0 4-0 4-4 5-0 Table VII Uptake of o-Phenyl-phenol by Oranges, from Wraps containing Different Amounts of o-Phenyl-phenol and Hexamine. Temperature 10 C. Amount of o-phenyl-phenol and hexamine per wrap 96 mg of o-phenyl-phenol -f 25 mg of hexamine 48 ,, 24 ,, ,, + 125 ,, +6-2 ,, ,, 86 it * *f 30 ft t------17 2-4 3-0 1-6 4-3 ;. 41 4-6 6-7 2-9 8-1 Days ________ A.________ 57 .5-0 33 2-5 8-3 68 8-9 4-2 3-3 '--- -----76 8-0 4-4 3-4 8-5 DSW 330565 STLCOPCB4077209 If "-iFsT-^i ,- =-.<fcy?v-3v? 0-PHENYL-PHENOL BY ORANGES FROM TREATED WRAPS 333 Table VIII Loss of o-Phenyl-phenol from Fruit Previously Stored in o-Phenyl-phenol Treated Wraps (the fruit was stored in a desiccator through which a slow stream of air was passing) .- Days in air .Amount of o-phenyl-phenol, mg per orange . 0 6-7 12 7-7 16 22 6-7 10-0 37 6-7 . . ' Table IX Examples of Amounts of Diphenyl or o-phenyl-phenol found in the Peel and Pulp (mg per orange) Diphenyl D-Phenyl-phenol Peel 6-1 4-8 4-7 4-6 4-2 3-4 1-8 Pulp nil 0-1 nil 0-11 0-1 017 nil < Peel 17-4 12-8 10-0 10-0 6-65 5-7 Pulp - nil 0-04 nil 0-03 nil Commercial Storage with Treated Wraps--Three cases of Jaffa oranges (each orange wrapped in a paper containing diphenyl) were sent from Palestine to England. The amount of diphenyl originally included in the papers is not known. No diphenyl was found in the papers on arrival in this country, but the amounts recovered from the fruit taken from each of the three cases was 3-9, 4-2 and 3-8 mg per orange respectively. The average weight of the oranges was 230 g and the average weight of the peel 109 g. The pulp was found to contain no diphenyl. Thus the concentration for the whole orange was 17 p.p.m., while the peel contained about 37 p.p.m. - Marmalade from Oranges Previously Stored in Treated Wraps--Sour oranges were stored for three weeks in treated wraps, and then made into marmalade (8 sour oranges, 2 lemons, 7 lb. of sugar and 8 pints of water made 12 lb. of marmalade). The amount of diphenyl-originally present in the 8 sour oranges (1265 g) was estimated to be 48 mg; of this, less than 0-6 mg remained in the marmalade (1 part per 10,000,000). . In a similar expt. with o-phenyl-phenol, 25 mg remained of 84 mg originally present in the 8 sour oranges, which weighed 1253 g (4-5 parts per 1,000,000). Summary--Methods have been devised for the estimation of diphenyl and o-phenyl- phenol absorbed by oranges from treated wraps. The amounts of diphenyl and o-phenyl- phenol found to be absorbed from wraps containing 100 mg of these substances were 4-20 mg of diphenyl or 20 to 30 mg of o-phenyl-phenol per 100 g of peel (= approx. 1 orange). The work described above was carried out as part of the programme of the Food Investigation Board. It is published by permission of the Department of Scientific and Industrial Research. We are indebted to Dr. J. R. Nicholls and Mr. A. G. Grimwade, of the Government Laboratory, for advice and help in devising the methods for the determination of diphenyl and o-phenyl-phenol, Mr. I. Sharman helped with the experimental work. . ' ' % . References 1. Tomkins, R. G., Report of Food Investigation Board for the Year 1635, p. 129. 2. Farkas, A;, Hadar. 1938, 11, [9],' 241; Id., 1939, 12, [8-10], 227. 3. Ramsey, G. B,, Smith, M. A., and Herberg, B. G., Bot. Gaz., 1944, 106, 74. 4. Tomkins, R. G., Report of Food Investigation Board for the Year 1936, p. 146. 6. ------- Report of Food Investigation Board for the Year 1938, p. 186. ` 6. Van der Hank, J. E,, Rattray. J. M,, and Van Wyk, G. FPom. and Hort. Set., 1940,18, 135. 7. Mulliken, S. P., "The Identification of Pure Organic Compounds," Vol. I, 1941, John Wiley & Sons, New York. - - 8. Hanke, M. T., and Kressler, K. K,, /. Biol, them., 1927, 50, 235-269. ,, Low Temperature Station for Research in Biochemistry and Biophysics, University of Cambridge, and Department of Scientific and Industrial Research June, 1945 DSW 330566 STLCOPCB4077210