that under conditions of rapid esterification other products are avoided. This was confirmed by subjecting different preparations of methyl palmitate t o GLC. Under the optimal conditions of esterification, side products were not detectable. In other cases, volatile contaminants are in, or close to, the solvent peak when conditions were set for the resolution of long chain fatty esters. The promoting or adverse effect of methanol on esterification and formation of side products depends on its concentration. This factor should be under control. Therefore, for analytical purposes, CHzNz is generated in ether and the esterification is carried out in the presence of a suitable amount of methanol in ether. Other reactions of CHzN2 besides esterification may occur with special samples of lipides, in spite of the careful control of reagent concentration and the short time of reaction involved. Catalysis of interesterification and reaction of CH2N2with carbonyl compounds, with activated double bonds, and with triple bonds have been reported. Reaction with alcoholic hydroxyl groups may be surmised from the above experiments with pure methanol. Some of the possibilities have been ruled out experimentally. Ethyl palmitate, 1hexadecanol, and tripalmitin were treated with C14H2N2 for 30 minutes. Subsequent paper chromatograms did not reveal any radioactive lipides. Other experiments were designed to test for interaction with methyleneinterrupted double bonds. Methyl linoleate was treated with C14H2N2 for 20 minutes and the solution chromatographed on paper. The absence of radioactivity in the chromatograms rules out formation of pyrazoline or Gyclopropane derivatives. The infrared spectra of soybean oil acids, after excessive treatment with CH2N2, to form the esters, remained unchanged except for variations due to the different carboxyl functions. Similarly no structural changes were observed
tested the extent of esterification of pbromobenzoic acid in ether, dioxane, and methanol after reaction periods of between 15 minutes and 60 hours. The acid reacted rapidly in ether, whereas in methanol its esterification was suppressed b y other reactions of CH2Nz. In the beginning of this work, we used ether and repeatedly failed to achieve complete esterification of fatty acids within 30 minutes. The reaction, however, should be fast since a prolonged accumulation of CHaN2 is bound to yield side products. I n particular, when the radioactive compound is being used, the side products may interfere with subsequent analytical procedures. Therefore, the effect of solvents on the rate of the esterification of fatty acids was investigated (Figure 1). Water (0.133M) and ethyl alcohol (0.267M) showed effects equal to that of methanol a t the same concentrations while ethyl acetate, chloroform, and heptane were not as effective. A relationship between the cosolvent and the amount of CHzN1 present after 1 hour could not be established. New or heavily used glassware did not influence the rate of esterification. Turbidity and finally flakes, appearing in nearly all reaction mixtures after 20 to 30 minutes, indicated the formation of polymers. h direct evaluation of side reactions of the esterification &-as possible with C“HZN2. Palmitic acid was esterified on a small scale, as described under Procedure, but R ith the solvents specified in Table I. Aliquots containing 110 pg. of the product were spotted on paper and chromatographed (6). Highly polymerized material is expected to remain in the starting point of paper chromatograms, while compounds of low molecular weight are eApected to move with the front line, or t h t y may volatilize. The paper chionintograms were assayed for C14 with a scaiinw and recorder, and then chemically assayed for acid and ester (6). Typical results (Table I), show
Table 1.
Esterification of Palmitic Acid with
C14H2Nn
By-products detected by paper chromatography yo of Total Radioactivity Found in Time in Starting Front Solvent Min. point line 10% CHsOH in ether 10 None None (V./V.) None As above 30 0.15 -4s above” None 30 0.4
Ether
CHaOH’ (I
30
30
0.5
None
Esterification tube scratched with sand.
* Yellow color did not appear in esterification tube.
1414
ANALYTICAL CHEMISTRY
1.0
None
when linolenic or arachidonic acid was esterified. For isotope analysis following esterification with C14H2N2it is important to know if methanol acts merely as a catalyst or if it participates t o some extent in the formation of methyl esters. It has been mentioned earlier that when palmitic acid was esterified with C14HzNz,the molar activity of the resulting methyl-C14 palmitate was in good agreement with that of the M-C’CNSA starting reagent. This indicates no significant participation of methanol in the reaction. Furthermore, when CH2Y2 was reacted with palmitic acid in the presence of ethyl alcohol, ethyl palmitate was not found in P C or GLC. Correspondingly, methyl palmitate was not found when C2H4N, was reacted in the presence of methanol. LITERATURE CITED
(1) deBoer, T. J., Backer, H. J., Rec. trau. chim. 73,229 (1954). (2) Eistert, B., Arndt, F., Loewe, L., Ayca, E., Chem. Ber. 84,156 (1951).
(3) hlangold, H. K., Gellerman, J. L., Schlenk, H., Federation Proc. 17, 268 (1958). (4) hlurray, A., Williams, D. L., “Organic Synthesis with Isotopes,” Pt. 1, p. 584, Interscience. New York. 1958. (5) “Organic Syntheses,”’W. S. Johnson, ed., Vol. 34, p. 96, Wiley, Sew York, 1_Q54 __-
(6) Schlenk, H., Gellerman, J. L., Tillotson, J. A., Mangold, H. K., J . Am. Oil Chemists’ Soc. 34, 377 (1957). (7) Stoffel, IT., Chu, F., Ahrens, E. H., ANAL.CHEX.31,307 (1959). (8) Stoll, A., Ruschmann, J., von Wartburg, A., Reut, J., Helv. Chim. Acta 41, 993 (1956). RECEIVEDfor review March 24, 1960. Accepted July 22, 1960. Kork supported by the National Institutes of Health (research grant 4226 C5) and by the Hormel Foundation.
Correction Spectrophotometric Determination of Chlorophylls and Pheophytins in Plant Extracts I n this article by L. P. Vernon CHEAI. 32, 1144 (1960)], On page 1147, column 3, the following should be included a t the end of the second paragraph. “In most cases the control and the converted samples must be diluted further before the absorbances at the above wave lengths are determined for other vegetables. The absorbance a t 665 mF for the nonconverted control must fall in the range of 0.4 to 0.7 in order for the results to be accurate.” [ANAL.
yo Esterification 100 100 100
80 20
