Gas-Liquid Chromatographic Separation of Chlorophenols and Determination of Pentachlorophenol as Methyl Ethers* BY Jun KANAZAWA

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1 [Agr. Biol. Chem., Vol.27, No.3, p.153,-158, 1963] Gas-Liquid Chromatographic Separation of Chlorophenols and Determination of Pentachlorophenol as Methyl Ethers* BY Jun KANAZAWA Agricultural Chemicals Inspection Station, Ministry of Agriculture and Forestry, Tokyo Received August 20, 1962 Chlorophenols are readily converted to methyl ethers by the reaction with diazomethane. Mixtures of methyl ethers of mono-, di-, tri-, tetra- and pentachlorophcnols can be separated completely by gas-liquid chromatography on silicone high vacuum grease and sodium alkylbenzene sulfonate columns at 150 `190 Ž. The peaks of chlorophenol methyl ethers are sharper than that of free chlorophenols. Pentachlorophenol and tetrachlorophenol in technical products and commercial herbicide formulations can be determined by internal standard method with dibutyl phthalate. Pentachlorophenol (PCP) is commonly used as a preplanting herbicide on the field of rice plant in recent years. The technical products of PCP contain several other chlorophenols as impurities. Therefore, the separation of these chlorophenol by-products and the determination of PCP in the technical products and herbicide formulations are needed. The separation and analysis of chlorophenols have been performed by three following techniques: ultraviolet spectrophotometry1); infrared spectrophotometry2); and ion exchange chromatography3) up to this date. While ultraviolet spectrophotometry and infrared spectrophotometry are likely to be difficult to apply without at least partial separations beforehand; ion exchange chromatography is complicated and take time. Recently, Barry et al.4) proposed that gasliquid chromatography (GLC) is the most convenient method for analytical separation * Gas Chromatography of Agricultural Chemicals. Part VI. 1) J.B. Laclair, Anal. Chem., 23, 1760 (1951). 2) R.T. Scheddel, ibid., 29, 1553 (1957). 3) N.E. Skelly, ibid., 33, 271 (1961). 4) J.A. Barry, R.C. Vasishth and F. J. Shelton, ibid., 34, 67 (1962). of chlorophenols. However, the peaks of chlorophenols obtained by their procedure were broad, and little work has been carried out on the quantitative determination of PCP in herbicide formulations. EXPERIMENTAL Apparatus. The apparatus used in this work was a Shimadzu Model GC-2A gas chromatograph, equipped with a hot wire detector and a 2-mv. recorder. The column used was a copper spiral tube of 3 m long and 4mm internal diameter. Column Preparation. Two stationary phases used in this work were silicone high vacuum greases (HVG) as nonpolar liquid and sodium alkylbenzene sulfonate (ABS) as polar liquid. HVG column packing was prepared by dissolving 10g of silicone high vacuum grease (Dow Corning) in ethyl acetate, adding 50g of 32- to 48-mesh Celite 545, and evaporating the ethyl acetate with gentle heating and stirring. ABS column packing was prepared by dissolving 10g of sodium alkylbenzene sulfonate (Toho Chem. Co.) in methyl alcohol, adding 50g of 35- to 42-mesh Fire brick C-22, and evaporating the solvent. The column was filled with these coated packings by applying an electrical vibrator along the column. Reagents. 2-Chlorophenol (2MP), 4-chlorophenol

2 JIM KANAZAWA (4MP), 2,4-dichlorophenol (24DP) and 2,6-dichlorophenol (26DP) of commercial extra pure grade were used without further purification. 2,4,5-Trichlorophenol (245TrP), 2,4,6-trichlorophenol (246TrP), 2,3,4,6-tetrachlorophenol (2346TeP) and PCP were purified by recrystallizing with benzene. Hexachlorophenol (HCP) was synthesized by further chlorination of PCP according to the method of Ohta and Imamura5). The diazomethane ethereal solution was prepared according to Garratt's method6) from N- methyl-n-nitroso-p-toluene sulphonamide and potassium hydroxide. Dibutyl phthalate (DBP) was purified by vacuum distillation. Purity of DBP was ascertained by the proposed GLC with normalized peak area. Ten per cent DBP solution in toluene was prepared for the internal standard solution. Preparation of Methyl Ethers of Phenols. It is known that the phenols can be readily separated and determined as their methyl ethers or trimethylsilyl ethers by GLC7,8). Although various methods have been carried out for methylation, diazomethane method was adopted because of ease to handle. The methylations of chlorophenols were carried out by the following procedure: To one gram of chlorophenol dissolved in a minimum amount of anhydrous ethyl ether was added ethereal solution of diazomethanc until yellow colour persists. The solvent was carefully evaporated on a steam bath. The chlorophenol methyl ethers were dissolved into 2 to 3ml of toluene and injected into the gas chromatograph. Gas Chromatographic Procedures. The column temperature was adjusted to 150 Ž for the separation of anisole, mono- and dichlorophenol methyl ethers, to 190 Ž for the separation of tri-, tetra- and pentachlorophenol methyl ethers, and to 225 Ž for the determination of 2346TeP and PCP for the rapid analysis. The carrier gas (helium) was sent at a flow rate of 70ml. per minute, which was measured by a soap-film flow meter. The detector cell current was adjusted to 180mA. The chart speed was 1cm per minute. The retention time was calculated from the distance between the summits of the sample and air peaks. Retention volume is defined as the product 5) N. Ohta and J. Imamura, Tokyo Kogyo Shikenjo Hokoku, 52, 142 (1957). 6) D.C. Garratt, Analyst, 86, 367 (1961). 7) W. Carruthers, R.A.W. Johnstone and J.R. Plimmer, Chem. and Ind., 1958, ) S.H. Langer, P. Pantages and I. Wender, ibid., 1958, of the retention time and the flow rate of the carrier gas. The optimum sample size was in the range of 20 to 30.J. Determination of PCP and 2346 TeP in Technical Products and Herbicide Formulations. (A) Technical Product. A sufficient sample containing about 300mg of PCP was weighed into a 50ml Erlenmeyer flask and dissolved into a minimum amount of ethyl ether. This ethereal solution of PCP was methylated with diazomethane according to the above mentioned procedure. A known amount (about 300mg) of DBP was added to the sample solution as an internal standard, then ethyl ether was evaporated in a water bath at about 60 Ž. Twenty to 30 Al of the resulting toluene solution was injected into gas chromatograph. (B) Water Soluble Formulation. A sufficient sample containing about 300mg of PCP was weighed, transfered to a 100ml separatory funnel, and dissolved into a minimum amount of water. Ten millilitres of 2 N hydrochloric acid was added to the separatory funnel and the whole was shaked vigorously; PCP separated was extracted with 10ml of ethyl ether. Allowing the layers to separate, the aqueous layer was transfered into a second separatory funnel containing 10ml of ethyl ether and extraction of PCP was repeated. These ethyl ether solutions were combined and transfered to a 50ml Erlenmeyer flask. The following procedures were corresponded to the case of technical product. (C) Granular Formulation. After the sample was crushed with a mortar, a sufficient sample containing about 600mg of PCP (about 3g for 25% PCP granules) was weighed into a 200ml Erlenmeyer flask. The sample was soaked with exactly 100ml of 50% methyl alcohol, allowed to extract PCP by shaking from time to time for 1hr, at room temperature. The extract was filtered through dry filter paper, then exactly 50ml of filtrate was transfered to a 100ml Erlenmeyer flask, and methyl alcohol was evaporated in a water bath at about 80 Ž. The residue was dissolved by a minimum amount of water, transfered into a 100ml separatory funnel; the following procedures were corresponded to the case of water soluble formulation. Calibration Curve. For the accurate analytical results, it is necessary to establish calibration curve by use of internal standard. DBP was found to be most suitable for internal standard. The calibration curve

3 Gas-Liquid Chromatographic Separation of Chlorophenols was obtained by plotting the ratio of the peak area a function of the ratio of the amount of PCP to a of PCP methyl ether to the peak area of the DBP as standard amount of DBP. The calibration curve of FIG. 1. Chromatograms of Chlorophenol Methyl Ethers.

4 Jun KANAZAWA 2346TeP was also obtained by the same method. Calculation of Results. The areas of the PCP methyl ether and DBP peaks was calculated by multiplying the peak height by the width at half height. If A is area of PCP methyl ether, As area of DBP, C a slope of calibration curve, W weight of sample taken, and Ws weight of DBP taken for determination. The weight % of PCP is calculated from the following formula. The weight % of 2346TeP can be also calculated by the above formula. RESULTS AND DISCUSSION The typical chromatograms of chlorophenol methyl ethers obtained by the proposed method are shown in Fig.1, and the relative retention volumes of chlorophenol methyl ethers are given in Table I. Mixtures of lower chlorophenol methyl ethers including phenol, mono- and dichlorophenols can be separated by using HVG and ABS column at 150 Ž. On the HVG column, the elution of chlorophenol methyl ethers are methyl ethers. Whereas ABS column can separated those, but though the separation of anisol and 24DP methyl ether are not perfect. Mixtures of more highly chlorinated phenol methyl ethers including tri-, tetra- and pentachiorophenol can be conveniently separated using two columns at 190 Ž. The peak forms obtained by HVG column were sharper than that obtained by ABS column. The PCP methyl ether (m.p. 106 `7 Ž, lit. 108 Ž9)) obtained by treating PCP with diazomethane was ascertained by infrared absorption spectra. The yield of PCP methyl ether obtained from the reaction with diazomethane was essentially quantitative (the mean of per cent of recovery was 100.6). The variance of peak area ratio (PCP methyl ether/dbp), obtained by analyzing repeatedly a single sample is shown in Table II. The coefficient of variance of peak area ratio was 1.18%. TABLE II. VARIANCE OF AREA RATIO ordinarily faster than on the ABS column; they are eluted approximately in the order of their boiling points. It is interesting that the position of anisole peak is reversed by the polarity of stationary liquid. HVG column does not completely separate 4MP and 26DP TABLE I. RELATIVE RETENTION VOLUMES OF CHLOROPHENOL METHYL ETHERS As shown in Fig.2, the calibration curves of PCP and 2346TeP in the range of 0.5 to 2.5 and 0.3 to 1.5 weight ratio respectively are practically straight lines and passed through the zero point. The slopes are and respectively. These slopes of calibration curves were adopted as a calibration factor which converts the area ratio to the weight ratio. 9) I. Heilbron et al., Dictionary of Organic Compounds ", Oxford Univ. Press., 1953, P. 58.

5 Cas-Liquid Chromatographic Separation of Chiorophenols FIG. 4. Chromatogram of Hexchlorophenol Methyl Ethers. Column and conditions arc the same as in Fig.3. TABLE III. ANALYTICAL RESULTS OF TECHNICAL PRODUCTS FIG.2. Calibration Curves of PCP and 2346 TeP. - PCP - - œ Tep The typical chromatogram of PCP technical products obtained by the proposed method is shown in Fig.3. From this chromatogram, 2346TeP and highly chlorinated phenols are main impurities detectable in commercial PCP technical products. It seems that highly chlorinated phenols are chiefly HCP (Fig.4). The results of determination of PCP and 2346TeP in PCP technical products by the TABLE IV. ANALYTICAL RESULTS OF COM- MERCIAL WATER SOLUBLE FORMULATIONS TABLE V. ANALYTICAL RESULTS OF COMMERCIAL GRANULES FIG.3. Chromatogram of Technical PCP Methyl Ethers. Column, HVG, Column temperature, 190 Ž, Heflow, 70ml/min. S Solvent Tep methyl ether 2 PCP V 3 DBP (Jnt. Std.) proposed method are shown in Table III. The analytical values of PCP agreed well and

6 J un KANAZAWA the standard deviation were 0.73 to 1.00%. Tables IV and V show the analytical results of commercial water soluble formulations and granules. According to Tables III and IV, results obtained by the proposed method is perfectly in agreement with that by infrared spectrophotometry. These results indicate that the proposed GLC is an excellent method for the separation and detection of chlorophenols, and also a specific method of determining PCP and 2346TeP in technical products and herbicide formulations. Acknowledgement. The author wishes to thank Dr. R. Sato, director of this department, for his interest and support of this work. He is also indebted to Mr. S. Goto of this department, for the infrared absorption spectrometry and to Mr. T. Kitukawa of Kanesp5 Chem. Co. for kind offer of hexachlorophenol.