analysis, which confirmed, respectively, the identity and purity of the germanium dioxide precipitates.

LITERATURE CITED

(1) Abel, G. J., Jr., ANAL. CHEM. 32, 1886 1960 ).

\ - - - - I ___.

(2) Cheng, K. L., Goydish, B. L., Zbid.,

(3) Duval, C., “Inorganic Thermogravi- 35, 1273-5 (1963).

metric Analysis,” p. 293, Elsevier, Amsterdam, 1953.

(4) Hillebrand, W. F., Lundell, G. E. F., Bright, H. A., Hoffman, J. I., “Applied Inorganic Analysis,” 2nd ed., pp. 299-300, Wiley, New York, 1953.

(5) Kodama, K., “hlethods of Quantita- tive Inorganic Analysis,” pp. 209-11, Interscience, New York, 1963.

(6) Krause, H. H., Johnson, 0. H., ANAL. CHEM. 25, 134 (1953).

( 7 ) Rlueller, J. H., J. Am. Chem. SOC. 44, 2493 (1922).

(8) Mueller, J. H., Eisner, A., IND. ENG. CHEM., AN.4L. E D . 4, 134 (1932).

(9) Willard, H. H., Zuehlke, C. W., Zbid., 16, 322 (1944).

(10) Zuber, J. R., Radio Corporation of America. Somerville. N. J.. Drivate

I .

communications, 1965. W. W. WHITE

Commercial Receiving Tube and Semiconductor Division

Radio Cor oration of America Harrison, 8. J. 07029

Rapid Preparation of Fatty Acid Esters from Lipids

for Gas Chromatographic Analysis

SIR: This paper describes a very rapid technique for preparing methyl esters from triglycerides and other lipids. The method should find wide interest, particularly in commercial fa t and oil laboratories. We had long been interested in extending the BF3- methanol esterification procedure (3) to the direct formation of methyl esters from triglycerides, phospholipids, poly- esters, and other lipids. Interesterifica- tion techniques were first explored. However, even after refluxing a triglyc- eride one hour with BF3-methanol, the transesterification was still incomplete. A transesterification technique was recently reported that uses benzene as a solvent combined with BF3-methanol (4).

A new approach using a rapid saponification technique that does not cause isomerization was used (1). This

Table 1. Fatty Acid Composition of a Butter Sample by Gas Chromatog- raphy of Methyl Esters Prepared by

Modified BF3-Methanol Procedure Hil-

GLC, ditch CSDA Acid 7c (2) ( 5 )

Saturated C4

c13 c14

Cl6 CIS

c11 ClS c20

Unsaturated ClO 1 = c12 1 = c14 1 = Cl4 2 = C16 1 =

ClS 1 = cis 2 = C18 3 = (220-22

c16 2 =

3 . 9 1.1 1 . 0 2.4 0 . 3 2 . 7 0 . 1 9 . 7 0 . 9

26.0 0 . 5

15.4 0 . 5

0 . 1 0 . 1 1 . 4 0 . 3 1 . 4 0 . 3

28.8 1 . 3 1 . 8 . . .

3.72 1.20 0 . 7 7 1.09

2.76

8.11

. . .

. . .

22:41

ii:6i 0.87

0.12 0.20 0.84

2.95

36:72 5.68

0.72

. . .

. . .

3 . 8 1 . 8 0 .8 1 . 4

2 . 5

8 . 3

24.4-28.8

. . .

. I .

9.8-13.9 . . .

. . .

2.1

27.6-39.2 1.1-5.0 0.4-1.2

0 . 3

is followed by boiling the soaps with BF3-methanol in the same vessel for 2 minutes. This procedure resulted in quantitative conversion of the fatty acids to methyl esters in a 10-minute operation. The methyl esters may then be floated out of the mixture using a saturated salt solution. This salting out technique almost completely overcomes the objectionable loss of lower fatty acids in the water layer of the original procedure. Quantitative recoveries of fatty acid esters even from butter have been made.

EXPERIMENTAL

Reagents. B F 3 - l I ~ ~ ~ ~ ~ ~ ~ REA- GENT. BF3 gas was obtained from the Matheson Co., Inc. One liter of reagent-grade methanol, in a 2-liter flask is weighed and cooled in an ice bath. K i t h the flask still in the bath, BF3 is bubbled through a glass tube into the methanol until 125 grams is taken up. This operation should be performed in a good fume hood, and the gas should not flow so fast that white fumes emerge from the flask. (The BF3 must be flowing through the glass tube before it is placed in and until it is removed from the methanol, or the liquid may be drawn into the gas cylinder valve system.) This reagent has an excellent shelf life and has been used up to two years after preparation.

Preparation of Methyl Esters from Triglycerides and Other Lipids. Bp- proximately 150 mg. of fa t ty ma- terial is added to a 50-ml. volumetric flask. Four milliliters of 0.5W meth- anolic sodium hydroxide is added to the mixture which is heated on a steam bath until the fa t globules go into solution. This step will take about five minutes. With some lipid materials it may take somewhat longer. Five milliliters of BF3-methanol is added to the flask and the mixture is boiled for 2 minutes. Enough of a satu- rated sodium chloride solution is added to the flask to float the methyl esters up into the narrow neck of the flask where they may be readily withdrawn with a syringe. If the esters are solid or it is desired to recover the anhydrous acids, then the entire mixture is transferred

to a separatory funnel. About 20 ml. of petroleum ether (b.p. 30-60” C. reagent grade redistilled) is added to the separatory funnel. The funnel is shaken vigorously for 1 minute and the layers are then allowed to separate. The lower aqueous layer is drained off and discarded. The petroleum ether layer is drained through filter paper into a 50-ml. beaker. The solvent is then evaporated on a 60” C. water bath or removed by a stream of air a t room temperature. The esters are now ready for GLC analysis.

The above procedure is readily applicable to free fatty acids by adding 3 ml. of BF3-methanol to a 150-mg. sample of acids.

DISCUSSION

When fatty acids are esterified, a simple titration for free fatty acid is a good indication of the completeness of esterification. However, when a tri- glyceride or other lipid is converted to methyl esters, the amount of conversion is difficult to determine. Thin layer chromatography was found to be an

Figure 1 . lipids

Thin layer chromatograms of

A. the modifled BF3 methanol procedure 6.

Methyl esters made from cottonseed oil by

Cottonseed oil spiked with fatty acids

514 ANALYTICAL CHEMISTRY

excellent technique for determining the relative amounts of methyl ester, fatty acid, and triglyceride or other lipid in the reaction product. This thin layer chromatograph technique was dem- onstrated by converting a commercial cottonseed oil to the methyl esters by the described saponification-esterifica- tion procedure. A thin layer chromato- graph was run on the methyl esters. The original triglyceride, methyl oleate and a fatty acid were also run by thin layer chromatography to show the comparative R, values. Figure 1 shows the results of these thin layer chromatograms using silica gel G plates. The solvent system was petroleum ether, diethyl ether, and acetic acid 9O:lO:l on a volume basis. The conversion of the triglyceride fatty acids to the methyl esters appear l o be quantitative by TLC. The amount of conversion was far more complete than with any short period interesterification procedures we have tried. The gas chromatography of these esters gave excellent results.

One of the objections raised against esterification procedures that required the addition of water was the possible loss of lower molecular weight fatty acid esters. This loss occurs because of the much greater water solubility of these

lower esters and their low volatility. With acids below Cs this loss precludes the quantitative recovery of the esters. However, i t was found that if a saturated salt solution is added to the esterification mix, a quantitative re- covery of acids down to the C4 range can be effected.

Butter is a n example of a fat with a very wide range of acids. Table I shows the quantitative results obtained with methyl esters of fatty acids ob- tained from butter. These results are compared to literature values generally accepted for butter (2 , 5 ) . The results compare very favorably in the lower acid range. The higher acids vary, but are still in the expected range. This variation is dependent on the diet of the animal. This was not an attempt to make an exhaustive analysis of butter, The BF3-methanol esterification pro- cedure for obtaining methyl esters has been extended to include fatty acids, soaps, triglycerides, polyesters, lipo- proteins, phospholipids, and other esters of fatty acids. Fat ty acid amides have also been converted to methyl esters with the procedure, though not quantitatively. The procedure as written is not applicable to phenols, rosin acids, and dimer acids.

I n conclusion, a rapid procedure for preparing methyl esters from lipids based on a rapid saponification followed by esterification with BF3-methanol in the same vessel was developed. The technique has been applied to a number of lipid systems. We have on occasion scaled it down to handle micro amounts of fats. The greatest use for the procedure will probably be in industry for the control analysis of fats and oils.

LITERATURE CTED

(1) Ast, H. J., ANAL. CHEM. 35, 1539 (1963).

(2) Hilditch, T. P., “The Chemical Con- stitution of Xatural Fats,’’ Wiley, p. 591, New York, 1956.

(3) Metcalfe, L. D., Schmitz, A. A., ANAL. CHEM. 33,363 (1961).

(4) Morrison, W. R., Smith, L. M,, J. Lzpid Res. 5 , 600 (1964).

( 5 ) U. S. Dept. of Agriculture, “Fatty Acids in Animal and Plant Products,” 1959.

L. D. METC~LFE A. A. SCHMITZ

J. R. PELHA

Research Laboratory Armour Industrial Chemical Co. McCook, Ill. 60529

Thermometric Titration of Cetylpyridinium Chloride

SIR: d number of analytical methods have been developed for the deter- mination of quaternary ammonium surfactants. These include: (a) spectro- photometric determination of a colored complex; ( b ) formation and separation of a derivative by the addition of a reagent and subsequent determination of the unreacted reagent; and (c j titra- tion with an anionic surfactant, usually in the presence of an indicator dye, in a two phase system.

.ill of these methods are subject to some criticism. Method (a) requires standardization by the particular sur- factant involved; method (b) requires time-consuming extractive procedures and standardization of both the reagent and titrant solutions; and method (c) is a relatively slow and tedious tech- nique requiring dropwise addition of the titrant as the end point is approached. After the addition of each drop of titrant, the phases must be mixed and examined for dye transfer or color change.

Quaternary ammonium surfactants are known to interact with a variety of high molecular weight species. Because alnioqt all reactions exhibit detectable enthalpy changes, it was expected that thermometric titrations could be utilized for the determination of these sur-

factants. Jordan, Pei, and Javick (1) demonstrated the applicability of this technique to the determination of alkylbenzenesulfonates. These workers reported that a titration employing a quaternary ammonium surfactant solu- tion as titrant could be completed in approximately 5 minutes with a re- sultant average relative error of about 1% for titrate solutions in the con- centration range of 10-3X. However, the typical enthalpogram reported ex- hibited considerable curvature, which makes accurate extrapolation to the end point difficult.

The purpose of this study was to adapt thermometric titrations to the determination of quaternary ammonium surfactants. The use of alkylbenzenesul- fonates as the titrant gave enthalpo- grams similar to those reported by Jordan, Pei, and Javick ( I ) , and the extrapolations necessary to obtain end points were subject to error. Further- more, the problem of obtaining pure alkylbenzenesulfonates limited the use- fulness of this procedure.

I n an attempt to improve the linearity of the enthalpogram and to eliminate the difficulty of obtaining pure reagents, other titrants were considered. Orange 11, an anionic dye which has been shown to react with quaternary am-

monium surfactants in the ratio of 1 : 1 was selected. The reaction products of Orange I1 and quaternary ammonium surfactants were isolated and sho\m to be insoluble salts having K,, values in the order of to a t 25’ C. (3). Orange I1 is available in relatively pure form; aqueous solutions are stable for long periods of time; and standardized solutions are easily prepared.

EXPERIMENTAL

The purification of the sodium salt of p - (2 - hydroxy - 1 - naphthy1azo)- benzenesulfonic acid (Orange 11) and cetylpyridinium chloride (CPC) are reported elsewhere ( 3 ) . The silver nitrate used was reagent grade.

Solutions of CPC, Orange 11, and AgT\TO3 in deionized water were pre- pared. The concentration of the Orange I1 solution was determined spectrophotometrically with a Beck- man Model D B spectrophotometer. A value for the molar absorptivity at 488 mp of 2.097 X lo4 (3) was used.

The thermometric titration appara- tus used is described by Raffa and Stern ( 2 ) . Titrant was delivered at a rate of 0.83 ml./minute. The end points were determined by extrapolation of the straight line portions of the curves as illustrated in Figure 1.

VOL. 38, NO. 3, MARCH 1966 51 5