9%; the piobable error is 6%. Assuming that a monomolecular layer of methyl red is adsorbed, the area covered by a methyl red molecule is ca. 160 sq. A. This value is in reasonable agreement with the projected area (125 sq. A.) of the flat-lying molecule using Pauling’s values (9) for the necessary covalent and van der Waals’ radii. The above results constitute a calibration of the methyl red adsorption method. The accessible surface area of alumina-silica and silica gel can be determined with a probable error of 670 simply by measuring methyl red adsorption as described and multiplying che resulting quantity by the factor, 960 sq. meters per mmole. Fine-Pore Samples. Samples of magnesia-silica (together with one alumina and two silica gel samples) adsorbed considerably less methyl red per unit area than the 1.04 pmoles per sq. meter value cited above. Ii‘oting that the average pore diameters of the samples listed in Figure 2 ranged from 42 to 140 A. and that the samples bn question had much smaller average pore diameters, i t was concluded that adsorption was low because of the inability of methyl red to penetrate the smallest pores in such samples. The pertinent data are listed in Table I. These data suggest that a qualitative measure of the surface accessible only through small pores can be obtained from the difference between methyl red and B.E.T. surface area determinations. Effect of Sample Pretreatment. The effect of t h e pretreatment of
alumina-silica and silica gel on methyl red adsorption is shown in Table 11. These d a t a indicate t h a t there is a small drop in adsorption per unit area when samples are calcined (in air) a t 550’ C. It is, therefore, desirable to standardize t h e pretreatment when using a n adsorption method for t h e determination of surface ares. Table 111.
Methyl Red Adsorption on Alumina
Surface Brea, Sq. Meter/ Gram
Average Pore Diameter, -4.
Methyl Red Adsorption, pMoles/ Sq. Meter
189 370 2u8 331
108 36 44 18
1.91 1.37 1.02 0.42
on alumina varies considerably. Since alumina is amphoteric, i t is possible t h a t adsorbed methyl red is attached through either t h e carboxyl or t h e azo group. r o t enough d a t a are available t o determine whether i t is this property t h a t causes the wide variation in methyl red adsorption or whether the variation is caused by large differences in pore size distributions among alumina samples. ACKNOWLEDGMENT
The author expresses his appreciation to C. F. Lee for the surface area and pore volume data and to 14.0.Babcock for his help with the experimental work. LITERATURE CITED
(1) Benesi, H. A., J
Am. Chem. SOC.
78,5490 (1956).
( 2 ) Benesi, H. A., J . Phys. Chem. 61, 970
M e t h y l R e d Adsorption on Mag-
nesia. Magnesia samples t h a t had been calcined a t 550’ C. adsorbed four times as much methyl red per unit area as did alumina-silica and silica gel. This result leads t o an area of only 40 sq. -4.per molecule of methyl red, which, in turn, indicates t h a t t h e molecules are oriented perpendicular, or nearly so, to the magnesia surface, What seems likely is that dye attachment has occurred through reaction of the carboxyl group in methyl red with the strongly basic oxide ions in magnesia. M e t h y l R e d Adsorption on Alumina.
T h e d a t a (Table 111) shorr t h a t methyl red adsorption (per unit area)
(1937). (3) Benesi, H. A., Bonnar, R. U., Lee, C. F., ANAL.CHEW27, 1963 (1955) (4) Brunauer, S., Emmett, P H , Teller, E., J . A m . Chem. SOC.60,309 (1938). (5) Johnson, C. E., Division of Colloid
Chemistry, 131st lleeting, ACS, Miami, Fla., April 1957. (6) 7Mitchell, G., Poole, P., Segrove, H. D.,
A ature 176, 1025 (1955). (7) Paneth, F., Radu, A4., Be?. 57, 1221 (1924).
( 8 j Paneth, F., Vorwerk, W.,2. physik. Chem. 101, 470 (1922). (9) Pauling, L., “The Sature of the
Chemical Bond,” Cornel1 Universitv Press. Ithaca. S.’Y.. 1944. (10) Shapiro, I., Kolthoff, I. lI.,J . Am.
Chem. SOC.72, 776 (1950). (11) Walling, C., Ibid.,72, 1164 (1950). RECEIVED for review March 14, 1960. Accepted June 16, 1960.
Esterification of Fatty Acids with Diazomethane on a Small Scale HERMANN SCHLENK and JOANNE
L.
GELLERMAN
The Horrnel Institute, University o f Minnesotu, Austin, Minn.
b The use of methyl and m e t h ~ 1 - C ’ ~ esters of fatty acids in microanalysis requires a simple method for preparing them pure on a small scale. The esterification of long-chain fatty acids is instantaneous when gaseous diazomethane is introduced into a solution of the acids in ether which contains 10% methanol. When pure ether is used, the reaction is slow or even incomplete, thereby giving rise to side products.
A
for the preparation of fatty acid methyl esters by acidic interesterification has recently JIICROMETHOD
1412
ANALYTICAL CHEMISTRY
been described by Stoffel and coworkers (7). The conditions they describe are applicable, though inconvenient, for esterification of acids. The use of specially dried methanol as reactant in great excess makes the procedure impractical for the preparation of methylCY4 esters. Introducing a radioactive methyl group, however, is an obvious way to apply isotope derivative methods for the analysis of “cold” fatty acids. A routine procedure for esterifying milligram amounts of fatty acids by CH2X2 is described here. The esterification mixture, after adjustment to the desired concentration or solvent,
is ready for gas-liquid chromatography (GLC) or paper chromatography (PC). The method has been applied extensively for the radioactive methylation of analytical samples of fatty acids (S), and, therefore, the use of C14H2X2 is discussed also. Diazomethane is a particularly mild agent for methylation but its explosion and toxicity hazards have discouraged the mider use it merits. However, when proper precautions are taken, the small amount involved here minimizes these deterrents. Some of the numerous ways for preparing CH2N2have been applied to give
C14H2N2 (4). For the preparation of both nonlabeled and labeled CHYNlin amounts of 0.1 to 0.2 mmole, we chose the method of deBoer and Backer ( I ) , which has been used also by Stoll and coworkers for radioactive methylation of a phenolic glucoside (8). The intermediate A' - methyl - N - nitrosop toluenesulfonamide (M?r'SA4) reportedly is stable enough for storage. This is particularly desirable when keeping the labeled compound on hand. It is more easily obtained in pure form than other precursory nitroso compounds. The improved stability of MXSA after purification has been emphasized recently ( 5 ) .
-
PROCEDURES
I5
59
\\
.-P0 IC a r
0
.-
c L
-05
5"
5.
.
30 40 so 60 Minutes Esterification of fatty acids with diazomethane 10
- -
S-Methyl- CIC S - nitroso p toluenesulfonamide. The preparation of AI-Cl4-XSA from BaC1403 has been described (8). Commercial C 1 4 H , h " ~ (1 mmole, 1 mc.) was used here. After dilution with approximately 22 mmoles of CHBNHZ, it was subjected to the procedure described in (5) modified to accommodate the small amount of amine. The recrystallized product, m.p. 60.5-61.5' C., was obtained in a chemical yield of 64% (3.15 grams, 14.7 mmoles), while the radioactive yield was 52%. The radioactive purity of the product was established by comparing its activity with that of rneth~1-C'~palmitate derived from it. Both were measured in the form of BaC14O3 and had activitiw of 34 to 35 pc. per mmole. The radioactive compound was stored in aliquots of 150 to 250 mg. a t -15' and n-as used, over a period of one year, to prepare esters for P C and dilution analyses. Chemical decomposition was not observed during this time. Conimeicial >INS-4, Diazald, from the Aldrich Chemical Co., Inc., was also recrystallized before storage. Other Reagents. 2-(&Ethoxyethoxy)ethanol (Carbitol, practical grade) is purified by heating to 110' C. for 1 hour with 5'% K O H follon-ed by distillation a t approximately 90" C. and 12 mm. of pressure. After such treatment the solvent does not turn yellow in the presence of K O H and this facilitates visual observation of the proceeding generation of CH2Y2. Ether is freed of peroxides and dried over Ka. Apparatus. The apparatus consists of three test tubes with side arms. A stream of S 2 is saturated with ether in the first tube (16 X 150 mm.) and carries CH2N2generated in the second tube into the third tube (both 15 X 85 mm.) where the esterification takes place. The side arms (7 nim. in outer diameter) are bent downwards and reach close t o the bottom of the next tube. They are drawn out a t the ends to approximately 1 mm. in outer diameter. Rubber stoppers are used for connections. Esterification. The flow of K2 through the ether of tube 1 is adjusted to approximately 6 ml. per minute.
. .
Figure 1 .
20
Temperature, 2 1 C.; initial molarity of acids, 0.027; of CH2N2, 0.06; solvent ether, with cosolvents as specified. 1. Stearic, myristic, and linoleic acids 2. Stearic acid, 0 . 0 2 6 7 M CHaOH 3. Stearic acid, 0.133M CH30H 4. Stearic acid, 0 . 2 6 7 M CHIOH 5. Stearic acid, 2 . 6 7 M CHaOH 6. Stearic acid, 0.1 33M H 2 0 7. Linoleic acid at 1 C.
Tube 2 contains 0.7 ml. of Carbitol, 0.7 ml. of ether, and 1 ml. of a s o l u tion of 6 grams of K O H in 10 ml. of water. Between 5 and 30 mg. of f a t t y acids dissolved in 2 t o 3 ml. of ether which contains 10% methanol is placed in tube 3. About 2 mmoles of MNSA or M-C'+NSA per milliequivalent of fatty acids is dissolved in 1 ml. of ether and added to tube 2. Connection is made immediately with tube 1 while the yellow color of CHzNz is already appearing in tube 2. As soon as a yellow tinge becomes visible in tube 3 against a white background, this tube is disconnected and the slight excess of CHzK2in it is consumed by adding acetic acid diluted with ether, or removed by a stream of Kg, The whole procedure requires 10 t o 12 minutes. The yields of CH2Szand of C14HzIT2 when measured with excess benzoic acid were consistently between 68 and 72%. Excess of CI4HzNz coming from the generator may serve for esterification of another sample or may otherwise be absorbed by acid. I t is not advisable, however, to carry out the procedure with two samples in series since this unnecessarily exposes the first to the reagent needed for the second sample. The apparatus is cleaned by rinsing with solvents, and when working with C14H&2, is replaced after about 20 runs. Ethyl esters can be prepared similarly by substituting nitrosoethylurethane for IVKSA. Allowance must be made for the low volatility of CzHdr\Tz by lengthening the time for transfer or by gently warming tube 2. Solvents and Rate of Esterification. Alcohol-free solutions of CH2S2 in
ether were prepared in the usual manner from 35 grams of recrystallized commercial RINSA. They were repeatedly dried over K O H pellets and redistilled a t 30' t o 40' C. in a stream of ?.T2 and dried again over K O H . Appropriate concentrations of CH2X2in ether were mixed with fatty acids dissolved in ether and cosolvents to obtain 75 ml. of the solutione speeified under Figure 1. Samples of 5 ml. were pipetted a t timed intervals into test tubes which contained 0.1 ml. of acetic acid to consume immediately all CH2X2 still present. After removal of solvents and acetic acid, the unesterified fatty acids n-ere titrated. Figure 1 shows that the rate of esterification in ether is equal for the most common fatty acids (curve l ) , and that it is strongly influenced by temperature (curve 7 ) . HoTvever, in pure ether a t 20' C., the esterification is still incomplete after 1 hour, although a final titration with benzoic acid proves CH2X2 to be present in excess. The addition of methanol (curves 2, 3, 4, 5 ) incrrases the rate so that, a t a concentration of about 2 M , the esterification beconies too rapid t o be measured. The action of methanol appears to be essentially a catalytic one since the molar activities of MVI-C14-SSAand of methyl-CI4 palmitate, obtained under the described conditions, are in good agreement. DISCUSSION
Esterification, like other reactions with CHzK2 ( g ) , is greatly influenced by solvents. Stoll and coworkers (8) VOL. 32, NO. 1 1 , OCTOBER 1960
1413
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 by 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
1414
ANALYTICAL CHEMISTRY
tube.
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
