Rapid Preparation of Methyl Esters from Lipids, Alkyd Paint Resins, Polyester Resins, and Ester Plasticizers John C. West Division of Analytical Laboratories, P.O. Box 162, Lidcombe, 2141, N.S. W., Australia
The preparation of methyl esters from lipids has been achieved by alkali or acid transesterification, or by saponification followed by methylation of the fatty acids. Methanolysis with potassium or sodium methylate in anhydrous methanol is a simple and convenient method for the quantitative preparation of methyl esters of glyceride fats ( I , 2). Methanolic HC1 ( 3 ) or HzS04 ( 4 ) can also be used to convert lipids directly to fatty acid methyl esters. Mason and Waller ( 5 ) used dimethoxypropane to improve the methanolic HC1 method. Alkaline transesterification is mild and rapid but is limited to samples with low acid values, whereas acid catalyzed methanolysis is often a lengthy process. Therefore, saponification followed by methylation is most popular. Metcalfe et al. (6) published a quick method in which lipids are saponified with methanolic sodium hydroxide followed by refluxing with BF3 in methanol. Catalysts such as BC13 and HC1 in methanol can be used in place of BF3. Subsequently, Hartman and Lago ( 7 ) reported a readily prepared catalyst system which employs a solution of ammonium chloride and sulfuric acid in methanol, wherein HC1 is formed as primary esterification reagent. In another method ( 8 ) ,tetramethylammonium hydroxide was used to saponify fats and the methyl esters were formed by pyrolysis in the injector of the gas chromatograph. Similar methods have been used to determine the acid components in alkyd paint resins, plasticizers, and polyester resins. Although the most common method is methanolysis with potassium or sodium methylate in methanol (9, IO), saponification followed by esterification is also frequently used (11-13). Transesterification of these polyester materials with methylate is also limited to samples with relatively low acid numbers. Recently, Greeley (14) reported an extremely mild method for preparing alkyl esters of acidic substances by reaction with alkyl iodides in the presence of tetramethylamdimethylacetamide/methanol monium hydroxide and N,Nsolvent. This paper describes a rapid and quantitative method for preparing methyl esters from glycerides and alkyd resins and the method can be used with ester plasticizers and polyester resins. The method employs a methanolic solution of tetramethylammonium hydroxide for saponification and methyl iodide as esterification reagent. N,N-Dimethylformamide is used to provide a polar solvent for esterification.
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Figure 1. Gas chromatograms of methyl esters prepared from a vegetable oil and chromatographed at 190' on a stainless steel column (6 ft) containing 10% SP216PS on Supelcoport ( A ) Esters prepared by t h e
method of Hartman and Lago (7). (5)Esters prepared by the method described in this paper. (C) Esters prepared by the method described in this paper and extracted with hexane (20 ml). The numbered peaks correspond to the methyl esters of the following acids: (1) palmitic, (2)stearic, (3)oleic, and (4) linoleic acids
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Chemicals. Tetramethylammonium hydroxide 24% in methanol was from Analabs, methyl iodide from Ajax Chemicals, and N,Ndimethylformamide was from May and Baker. Procedure. Approximately 100 to 200 mg of fatty material, alkyd resin, plasticizer, or polyester resin is added to a test tube (18 X 150 mm). If desired an internal standard such as pentadecanoic acid (approximately 30 mg per 100 mg of ester material) can be added a t this stage. Tetramethylammonium hydroxide 24% in methanol (0.5 ml) and methanol (0.5 ml) are added to the mixture which is heated in a steam bath for 10 minutes. Fat globules normally go into solution in about 5 minutes but with some materials, especially solids such as polyethylene terephthalate fiber, hydroly1708
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sis may take longer (15 minutes). N,N-Dimethylformamide ( 5 ml) is added to the test tube, the mixture shaken, and methyl iodide (0.5 ml) added. The mixture is shaken and tetramethylammonium iodide precipitates. The precipitate is allowed to settle and the supernatant solution is injected into the gas chromatograph. Alternatively, if the methyl esters are to be separated from the reaction mixture, then the saponification and methylation steps are conveniently carried out in a 50-ml volumetric flask. After esterification, hexane or petroleum ether (5-20 ml) is added, and the hexane layer is floated up into the neck of the volumetric flask by addition of water or saturated NaCl solution if low molecular weight acids are present. The hexane solution is dried over anhydrous sodium sulfate and can be injected into the gas chromatograph or the hexane can be evaporated off leaving the methyl esters. If evaporation is necessary, care should be taken to avoid oxidation of unsaturated fatty esters. Evaporation in an inert atmosphere or under a jet of nitrogen is generally satisfactory. Alternatively, evaporation may be avoided if the reaction mixture is scaled down. For small samples (1-20 mg) a tenfold reduction in reagents and reaction in a 10- X 90-mm test tube is possible. A more polar extraction solvent such as butyl acetate is required when phthalic acid is present.
RESULTS AND DISCUSSION The reliability of this method was tested by comparing it with the method of Hartman and Lago ( 7 ) .Gas chromatograms obtained by both methods with a sample of vegetable oil are shown in Figure l. There is no significant difference between the chromatograms obtained by the two methods. The quantitative nature of the reaction was determined by examining known mixtures of trimyristin, tripalmitin, and tristearin (Table I). Results for a mixture of tripalmitin and polyethylene terephthalate are also included in Table I. The method is mild as the methylation is carried out a t room temperature leaving an essentially neutral solution which can be injected directly into the gas chromatograph. There is no residual strong acid or base catalyst to be neu-
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Table I. Results of Analyses for Acids in Synthetic Mixtures of Triglycerides and Polyestersa Amount found Sample
Composition
Amount used, mg
in DMF solution
in hexane extract
32.8 33.7 Trimyristin 32.0 Tripalmitin 25.9 . ... 37.6 36.5 37.6 Tristearin 63.0 64.5 61.4 2 Trimyristin Tripalmitin 33.2 ... 52.8 52.9 54.1 Tristearin 3 Trimyristin 56.6 55.8 57.8 Tripalmitin 32.9 ... Tristearin 83.4 83.2 84.1 4" Tripalmitin 50.8 ... Polyethylene terephthalate 39.5 39.7 a Hydrolyzed for 10 minutes according to the method described in the Experimental section. Calculated with tripalmitin as internal standard. Both the DMF reaction mixture and a hexane extract (5 ml of hexane used) were analyzed. Hydrolysis was for 1
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15 minutes.
tralized before gas chromatography. Also, since the material is saponified before methylation, samples with high acid numbers are easily analyzed. Greeley (14) used N,N-dimethylacetamide as polar solvent to promote esterification and now N,N-dimethylformamide has been found to be an effective solvent. The maximum allowable methanol concentration in the solvent appears to be about 20%. At higher methanol concentrations, tetramethylammonium iodide does not precipitate and esterification is unsatisfactory. N,N- Dimethylformamide is used in place of N,N-dimethylacetamide because it is readily available and has a lower boiling point. Therefore, interference during gas chromatography is reduced when low boiling acids such as succinic and maleic acids are present. Interference from N,N-dimethylformamide is eliminated if the esters are extracted. Fatty acid esters are readily extracted with hexane or petroleum ether (Figure 1) but a more polar solvent such as butyl acetate is required when phthalic acid is present. The effectiveness of butyl acetate for extraction is shown in chromatograms of methyl esters obtained from a phthalate alkyd paint (Figure 2). Alkyd paints can be examined directly as the solvents and pigments do not interfere with the esterification or gas chromatography. Fatty acid methyl esters can be extracted into petroleum paint solvents whereas dimethyl phthalate cannot and, therefore, extraction with butyl acetate is recommended whenever a solvent layer separates out of the reaction mixture. In methods based on BF3 catalysis, esterification of the isomeric phthalic acids occurs under reflux and much more slowly than esterification of aliphatic carboxylic acids ( 1 1 ) . However, by contrast, rapid esterification of both aliphatic and aromatic carboxylic acids is obtained at room temperature by this method. An additional feature of this method is the formation of soluble tetramethylammonium salts of the phthalic acids during saponification. Saponification with sodium or potassium hydroxide in methanol forms insoluble salts which can cause bumping and sometimes loss of sample. In the method of Greeley (14), any alkyl iodide can be used to form the corresponding ester, and it was hoped that
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Figure 2. Gas chromatograms of methyl esters prepared from an alkyd paint resin. Chromatography was at 200' on the same column as used in Figure 1. ( A ) Esters prepared by the method described in this paper. (8) Esters prepared by the method described in this paper and extracted with butyl acetate (20 ml). Peaks 1 to 4 are the same as in Figure 1. Peaks 5 and 6 correspond to the methyl esters of linolenic and phthalic acids respectively
derivatives other than methyl esters could be prepared after saponification with tetramethylammonium hydroxide. Unfortunately, some methylation occurs during saponification and only qualitative analysis of higher esters such as the butyl esters would be possible. Perhaps the most important reason for preparing methyl esters of total acids in unknown paint resins, polyester resins, and plasticizers is to identify the acids present and, subsequently, to determine the amount of each. This method is particularly suited to this type of work because the methyl esters are prepared rapidly and the relative proportions of the various acids are easily determined by gas chromatography of the reaction mixture. In conclusion, a rapid method for preparing methyl esters of acids in lipids, alkyd resins, polyester resins, and ester plasticizers is described. The method is based on rapid saponification with tetramethylammonium hydroxide in methanol followed by esterification with methyl iodide in the presence of N,N- dimethylformamide/methanol solvent.
LITERATURE CITED (1) F. E. Luddy, R. A . Barford, S. F. Herb, and P. Magidam, J. Am. OilChem. SOC.,45, 549 (1968). ( 2 ) S. W . Christopherson and R. L. Glass, J. Dairy Sci., 52, 1289 (1969). (3) M. L. Blank, B. Verdino, and 0. S. Privett, J. Am. Oil Chem. SOC.,42, 87 (1965). (4) K. V. Peisker, J. Am. 0ilChem. SOC.,41, 87 (1964). (5) M. E. Mason and G. R. Waller, Anal. Chem., 36, 583 (1964). (6) L. D. Metcalfe, A. A. Schmitz. and J. R. Pelka, Anal. Chem., 38, 514 (1966). (7) L. Hartman and R. C. A. Lago, Lab. Prac., 22, 475 (1973). (8) J. B. F. Lloyd and B. R. G. Roberts, J. Chromatogr., 77, 228 (1973). (9) J. Rawlinson and E. L. Deeley. J. Oil Cobur Chem. Assoc., 5 0 , 373 (1967). (10) G. G. Esposito and M.H. Swann. Anal. Chem., 34, 1048 (1962). (1 1) A. Krishen, Anal. Chem., 43, 1130 (1971). (12) D. F. Percival, Anal. Chem., 35, 236 (1963). (13) J. K. Haken, Aust. PaintJ., 11 (1967). (14) R. H. Greeiey, J. Chromatogr., 88, 229 (1974).
RECEIVEDfor review November 8, 1974. Accepted April 25, 1975. The author is grateful to the Director and Government Analyst and to the Health Commission of N.S.W. for permission to publish this paper.
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