analysis, which confirmed, respectively,

the identity and purity of the germanium

dioxide precipitates.
LITERATURE CITED
(1) Abel, G. J ., J r., 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., J ohnson, 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. ED. 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 fat 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
Tabl e 1. Fatty Acid Composition of a
Butter Sampl e by Gas Chromatog-
raphy of Met hyl Esters Pr epar ed by
Modi f i ed 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. 77
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., I nc. One liter of
reagent-grade methanol, in a 2-liter
flask is weighed and cooled in an ice
bath. K i th 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 fatty 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 fat 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 at 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 l ayer chromatograms of
A.
the modi f l ed BF3 methanol procedure
6.
Met hyl esters made from cottonseed oi l by
Cottonseed oi l spi ked wi th f at t y aci ds
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 an 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. Fatty 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. Lzpi d 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 (cj 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. J ordan, Pei, and J avick (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
J ordan, Pei, and J avick ( 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 at 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 DB 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