A Procedure for the Simultaneous Quantitative Determinaition of Glycerol and Fatty Acid Contents of Fats and Oils MICHAEL E. MASON, MARY E. EAGER, and GEORGE R. WALLER Agricultural Experiment Station, Oklahoma State University, Stillwater, Okla.
b A procedure is described which allows the simultaneous quantitative determination of glycerol as well as fatty acids of fats m d oils by gas liquid chromatography. Knowledge of the amount of both components allows a comparisorl of the weight of f a t determined b y analysis with the weight of sample IJsed for analysis. Also, comparison of the pmoles of glycerol with the pmoles of total esters found reveals additional information concerning the purify of the fat being analyzed. Consequently, glycerol and fatty acid values are expressed on an absolute rather thon relative basis.
H
et al. ( 1 ) described the use of an internal standard to correct for errors introduced into fatty acid determinations by various manipulations before and during gas liquid chromatography (GLC). Fatty acid values determined by GLC were arbitrarily corrected by a factor determined from the pel. cent recovery of the internal standard. Tinoco et al. (3) have presented ex idence indicating t h a t this procedure is valid for some lipid materials. However, internal standards cannot c o r r x t for fatty acids which are incompletely released from the lipid or incompletely converted to methyl esters. Error:, from incomplete conversions are quite probable with some procedures ( 4 ) . Obviously, the arbitrary adjustment of values for all methyl esters according to the loss incurred in the internal standard would be valid only in those cases in which the same loss occurred i n all the methyl esters present. Seemingly, this occurrence is unlikely in samples containing a diverse reprmentation of both low and high molecular weight esters. Therefore, the degree of error produced by making the arbi rary adjustment would depend on which method is used, the manipulations involved, and the type of lipid being an2 lyzed. The preparation of methyl esters from fats and oils for determination by GLC using a procedure which is extremely simple and requires little effort for the preparation of samples ORNSTEIN
was described by the authors (2). The procedure provides either relative or absolute fatty acid content of fats and oils but is especially convenient for routine analysis of large numbers of samples where only relative values are desired. The procedure described herein is a result of modifications of the former procedure but differs in the amount and kind of information obtained and the manipulations performed. Absolute quantitative determination of both fatty acid and glycerol content of fats and oils is realized. The fatty acids are determined as their methyl esters and glycerol as isopropylidene glycerol (IPG). Absolute quantities (pmoles of component per 100 mg. oil analyzed) of both moieties of a fat or oil are obtained without making adjustments calculated from losses in the internal standard. Recoveries (mg. fat by analysis per 100 mg. fat analyzed) are calculated directly from the GLC analyses. Internal standards are used only as indicators of faulty manipulations or
- 0
o
20 40 60
eo
io0 120 140
T i m e , Minutes
Figure 1. Time course-of-reaction a t room temperature using basic catalysis on a mixture of pure triglycerides
0 A
Mole per cent transesterincation of total fatty acids added as triglycerides M o l e per cent conversion of glycerol to IPG added as triglycerides
incorrect standard curves. This report discusses the modified procedure and data which establish the validity of its use. EXPERIMENTAL
Apparatus. The apparatus used in these studies were the same as those used in the similar procedure (2) with the following exceptions: chromatograms were obtained solely with ‘l4-inch by 6-foot aluminum columns of 14.5% EGS (ethylene glycol succinate) on Anakrom, 100 to 110 mesh, type A. Helium at a flow rate of 80 cc. per minute was used as the carrier gas. Reagents. The reagents used were also the same with the exception t h a t the methanolic sodium methoxide used in this procedure was approximately 2 . 0 5 . Procedures. TRAKSESTERIFICATION OF TRIGLYCERIDE MIXTURES AND FATSAh’D OILS F O R A%CCURACT A N D PRECISIOK STCDIES. Approximately 35 pmoles of each of several triglycerides were accurately weighed into a 25-ml. glass stoppered Erlenmeyer flask and reagents were added in the following order: 10.0 ml. of benzene, 4.0 ml. of DRIP, 5.0 ml. of methanol and 1.0 ml. of 2.0-V sodium methoxide. The mixture was swirled, allowed to stand a t room temperature for 5 minutes, and sufficient methanolic HC1, determined by prior titration, added to supply about 0.3 mmoles of excess HC1. The mixture was sn-irled again and allowed to stand 50 minutes. .%bout 1.5 grams of solid neutralizer was then added and the mixture swirled periodically during a 30-minute period. The mixture was allowed to settle and the supernatant decanted into a 25-ml. volumetric flask. The residue was washed tivice with two 2-ml. portions of methanol which were also decanted into the volumetric flask. The volume was adjusted to 25 ml. with methanol and 50-111. aliquots were withdrawn for GLC analysis. Fat and oil studies involved accurately weighing out approximately 200 mg. of the material and performing the analysis in the same way as that used for the triglycerides. TIMECOURSE-OF-REACTIOS STUDIES. Triglyceride mixtures and volumes of reagents used were the same for all time course-of-reaction studies as those described in the preceding section. VOL. 36, NO. 3, MARCH 1964
587
IO
0'
20
40
30
60
50
m moles H CI x 103
Figure 2. Effect of acid on mixtures of pure triglycerides that had already been subjected to basic conditions. Abscissa is mmoles HCI added to a 3ml. reaction mixture
A
Mole per cent transesterification of fatty acids added as triglycerides Mole per cent conversion to IPG of glycerol added as triglycerides
0
For the data in Figure 1, 2-ml. aliquots were periodically removed from the basic solutions, placed into 3-ml. volumetric flasks containing the exact amount of methanolic HCl needed to neutralize the aliquot, the volume was adjusted to 3.0 ml. with methanol and 50-p1. aliquots were taken by syringe for GLC analyses. Data presented in Figure 2 were obtained by adding increments of HC1 in the form of methanolic HCI to the 3-ml. reaction mixtures used to obtain data plotted in Figure 1 described in the preceding paragraph. The triglycerides in all these solutions, depending on the time under basic conditions, had undergone 65 to 80% transesterification (Figure 1). Thus, Figure 2 shows the effect of increasing acid content upon solutions which had undergone from 65 to 80% transesterification under basic conditions. The ~~
~
~~~~
~
~
Lard
4.9 fO.50 4.6 4Z0.77 12.3 f0.36 3.5 f0.42 0.6 f0.07
Palm oil Tallow Cottonseed oil Soybean oil Hardened soybean oil Safflower oil Corn oil Peanut oil 0
Precision Study:
83.7 f0.35 161.5 f3.46 85.3
f0.50 71.8 f2.89 36.8 fO.10 40.3 f1.84 25.1 f 0 . 10 41.7 f0.85 38.2 f1.20
588
~~
0
~
ANALYTICAL CHEMISTRY
5
:
V
2o
40
E 30 IO 0
0
IO
20 30 40 50 T i m e , Minuter
60
Figure 3. Time course-of-reaction study on a mixture of pure triglycerides at room temperature to determine time necessary for complete transesterification and IPG formation under acidic conditions (0.30 mmole HCI per 20 mI. reaction mixture) after 5 minutes under basic conditions
0
~~~
:
I I
A
Mole per cent transesterification of total fatfy acids added as triglycerides Mole per cent conversion to IPG of glycerol added as triglycerides
esterification and IPG formation (Figure 2) * Data in Figures 1 and 2 clearly indicated that 5 minutes under basic condit,ions followed by very mildly acidic conditions would result in complete conversion of the triglycerides to I P G and the corresponding methyl esters in a short period of time. Addition of 0.050 mmole of HC1 to the 2-ml. aliquots studied (3 ml. final dilution) was sufficient to bring transesterification and IPG formation to completion within 5 minutes. ~
Fatty Acid" Content of Various Fats and Oils
pmoles per 100 mg. and std. dev. C, CZ c= 18
C,
CIS
7.9 zk0.42 0.4 f0.04 8.3 f0.36 1.4 f0.04 0.2 fO.00
35.4 f1.68 13.9 fO.10 74.0 4Z3.68 8.0 f0.85 13.4 f0.10 26.1 f0.76
7.8 fO.00 6.1 fO.OO 8.0 51.28
141.5 f1.91 127.2 f3.36 130.9 f1.42 65.9 zk1.76 85.5 zk2.12 212.2 f6.78 47.6 f0.92 94.6 f0.99 158.8 h1.34
C, = palmitoleic; C;8 = oleic; C,; = linoleic; C z = linolenic.
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0
u
RESULTS AND DISCUSSION
~~
c 1 6
l80
Reaction Conditions. Variation of the volume of 2.ON methanolic sodium methoxide used in the procedure from 1.0 ml. to 5.0 ml. had little effect on the formation of esters from triglyceride fatty acids. I n all studies, the maximum yield of total esters (80%) was obtained within 5 minutes after adding sodium methoxide after which the total ester content decreased for a time and then increased to nearly 80% again. I P G formation was only about 40% complete and varied in the same manner as ester formation; these results are shown in Figure 1. However, incremental additions of HC1 to these same reaction mixtures resulted in progressive increases in trans-
~~~~~~
Table 1. c 1 4
acidified solutions were allowed to stand for 5 minutes and 0.25 gram of solid neutralizing agent was added. Estimations of the additional dilutions introduced by this procedure were made and 50-p1. aliquots were analyzed by GLC. Since 0.03 mmole of HCl was sufficient to cause nearly complete transesterification and I P G formation in 5 minutes when 2-ml. aliquots were acidified (Figure 2), 0.3 mmole of HC1 were used to acidify the 20-ml. reaction mixtures used to obtain data plotted in Figure 3. This HC1 was added after 5 minutes under basic conditions and periodically 2-ml. aliquots removed from the acidified reaction mixture and transferred to a 3-ml. volumetric flask containing 0.25 gram solid neutralizer. The volume was adjusted to 3.0 ml. with methanol and 50-pl. aliquots chromatographed. The reasons for choosing an intermediate acid concentration when higher concentrations would have resulted in shorter reaction times are covered in the section entitled Results and Discussion.
43.2 fO.78 36.0 f0.92 5 8 f0.36 197.9 f0.64 171.9 f2.96 44.1 f0.35 262.4 f9.06 189.9 f0.64 118.2 f0.92
C20
C22
Cza
3.05 f0.92
9.65 f0.10
2.55 f0.60
4.7 f0.50 2.8 fO.OO 3.5 4ZO.05
26.4 f1.77 2.85 10.49 1.7 f0.06 3.5 f0.05 5.6 lt1.20
Figure 3 shows the time course-ofreaction study designed to determine the time necessary for complete transesterification and IPG formation under the acidic conditions prescribed in the procedure. The curves were extrapolated from 10 minutes t o zero time (time of acidification) to intersect the ordinate a t points corresponding to those obtained after 5 minutes under basic conditions (Figure 1). Extrapolation was necessary because the course of reaction v;as not followed during the first 10 minutes after acidification. As a result of this study, a 50-minute period under the prescribed acidic conditions was chosen for the final procedure. The use of larger amounts of HC1 would obviously have resulted in shorter reaction times but this was avoided because the rate a t which IPG is destroyed increases rapidly as the hydrogen ion caoncentration increases. This is a property common to ketals and the primary reason quantitative recoveries of "PG were never obtained from the highly acidic reaction mixtures described in another procedure ( 2 ) . Thus, an intermediate level of HCl was chosen which would provide the slight acidity needed for complete IPG formation but not so strong as to result in some IPG destruction. Also, examination of Figure 3 would indicate that 30 minutes under these conditions should be sufficient time but 50 minutes Tias chosen for the final procedure to he1 3 ensure complete reaction. Precision and Accuracy Studies. F a t t y acid contents of the oils listed in T a b k I are expressed on a n absolute basis (pmoles ester pel- 100 mg. fat) and are averages of duplicate determinations. Standard deviations are also included and rarged from 0.00 to 9.06. For major coniponents, such as palmitate (Cle), oleate (C,) and linoleate (CG), the relative error ranged from about k0.3 tcl 53.0%. However, for minor components, such as myristate (CIJ, stearrite (CIS),linoleate (C,;), arachidate ( C W ) ,behenate (C,?), and lignocerate (L), the relative error ranged from 0 to ahout 30%. These larger errors Lvere ciused mainly by instrumentation problems involved with peaks that were eithei. small, diffuse, or both. The Disc Integrator introduced as much as 10% error into sharp peaks below 10 units defleclion on the chart and progressively gieater error into peaks that were equally low and progressively more diffu1,e. Since all but two (CH and CIS) of the lesser components appeared in 1 he latter part of the chromatograms where diffusion was most prominent, nearly all had a high relative error. Sevei*theless, precision of measuring the total ester content was very satisfactory as >videnced by the
Table II.
Precision and Accuracy Study with Various Fats and
Oils
pmoles total fatty acid per 100 mg. fat
pmoles glycerol per 100 mg. fat X 3
Total mg. fat determined per 100 mg. fat analyzed (yorecovery)
Lard 325.3 f 4.80 Palm oil 346.2 =t 4.52 Tallow 326.2 & 7.14 Cottonseed oil 348.5 f 6.22 Soybean oil 334.8 =t 6 . 0 1 Hardened soybean oil 325.5 & 6.24 Safflnwernil ~~..~~344.6 f 8 . 3 4 Corn oil 335.8 f i.53 Peanut oil 343.2 f 0.32
343.7 f 12.79 347.8 f 11.73 336.1 f 0.85 346.4 f 6.23 319.4 f 3.21
95.7 f 1 . 1 8 100.6 f 0.74 96.6 & 0.84 103.7 f 1.51 99.8 =t 1 . 6 5
873.1 f 32.35 862.8 f 24.26 893.1 f 3.20 865.9 f 15.52 937.6 f 9.55
326.0 & 4.95 318 8 f 4.88 32i:o f 5.09 329.0 f 8.91
98.5 f 1 . 3 6 103.1 f 2.52 i 0 3 . i f 1.44 104.2 f 0.27
920.3 f 13.86 940.7 f 14.45 934.7 & 14.98 912.1 f 24.67
~~
~~
Table 111.
Average molecular weight
Accuracy Study on Mixtures of Pure Triglycerides
% Recovery of fatty acids added as triglyceride Sample
ClC
1 2 3
95 109 100 101.3 f4.10
Mean Std. dev.
C16
106 100 101 102.3 3~3.24
average values and standard deviations listed in the first column in Table 11. The relative error ranged from about h O . 1 to =k2.3%. Thus the data in Table I and the first column of Table I1 served to establish the loaer and upper limits of precision in measuring absolute amounts of individual fatty acids as well as total fatty acids of a fat or oil. Accuracy was evaluated in m t o different ways. Three different mixtures of tripalmitin, trimyristin, triolein, and tristearin were analyzed using the prescribed procedure and the results along with mean values and standard deviations are presented in Table 111. Analytically determined values for each ester and for glycerol averaged very nearly 100% of the amounts added to the reaction mixtures as triglycerides. The standard deviations ranged from iz1.5 for oleate to 2C4.1 for myristate corresponding to relative errors of about iz1.5 to &4.1%, respectively. These data leave little doubt that the glycerides were completely converted to I P G and their corresponding methyl esters. However, the inherent error is great enough to warrant performing analyses in triplicate. Two criteria for testing the accuracy of analyses of fats and oils are inherent in this procedure. The number of pmoles total ester by analysis should equal the ,pmoles of glycerol found by analysis multiplied by three. If equality exists, the sample is probably pure triglyceride and the preciseness of measurement of glycerol and fatty acid content is further verified. Also, if equality exists and the sample is pure triglyceride, the sum of the weights of total esters and glycerol found by
Ci8
C,
IPG
95 101 101 99.0 f3.54
100 102 103 101.7 f1.58
102 97 104 101 .o f3.67
analysis minus the water of reaction should equal the weight of sample used for analysis. If so, the accuracy of the measurements is established. Application of these criteria to the data from the nine fats and oils analyzed revealed agreement between glycerol (second column of Table 11) and fatty acid contents (first column of Table 11) of the nine fats and oils analyzed within limits of precision except for lard, soybean oil, corn oil, and peanut oil. Of these, the fatty acid content of lard was a little low compared to glycerol indicating incomplete measurement of all the glyceride fatty acids present. However, fatty acid contents of the latter three oils were slightly higher indicating the presence of nonglyceride fatty acids in the oils or inaccurate measurement of glyceride fatty acids present. .kdditional information was provided by calculating for each fat and oil the weight of oil determined by analysis per 100-mg. sample analyzed. These values are listed in the third column of Table 11. Recovery of lard was about 59& low indicating incomplete measurement of fatty acids present which was in agreement with the conclusion drawn by comparing the total fatty acid and glycerol contents. However, peanut oil and corn oil recoveries were both high indicating that the relatively high total ester content was probably the result of inaccurate measurement rather than the presence of nonglyceride fatty acids. Therefore, even though the precision, as indicated by the standard deviations, is within *2%, the accuracy, as measured b y per cent recovery, is about f3%. These data established the degree of purity of VOL. 36, NO. 3, MARCH 1964
589
the fats and oils analyzed, the accuracy and precision of measuring the glycerol and fatty acid contents, and allowed the expression of these contents on absolute terms not dependent on estimations based on internal standards. The same criteria should be applicable to samples which are pure or nearly pure mono- or diglycerides. Average molecular weights calculated from the amount of glycerol determined by analysis and the weight of sample analyzed are included in the fourth column of Table I1 along with standard deviations. Because they were calculated from glycerol content, these molecular weights were no more precise or accurate than duplicate values from which they were calculated. However, the confidence placed in these values should be greater than those determined from saponification numbers because of being able t o first establish whether or not the sample is pure triglyceride. Calculations. Some minor components present in trace amounts were not included in Table I because their identities were not definitely established. Thus, tallow contained mi-
nute amounts of materials with retention times expected for tetradecenoate (CJ, pentadecanoate (&), hexadecadienoate (CJ and heptadecanoate (Cl,) while lard contained a trace amount of material corresponding to a retention time expected for heptadecanoate (C17). However, estimations were made for these trace components and included in values in the first and third columns of Table 11. Area.to-pmole relationships were obtained for each methyl ester and IPG (2) from the chromatograms of an appropriate mixture of standard methyl esters and IPG dissolved in methanol. These relationships were then used to calculate the amounts of fatty acids and glycerol in the fats, oils, and triglyceride mixtures. All samples analyzed containel the same amount of internal standard (methyl caprate). If the area of the internal standard changed by more than i 2 % , injection of the standard solution of methyl esters was repeated and new unit areas calculated: however, this was necessary only occasionally. Increments from 10 t o 50 111. were chro-
matographed to ensure linearity of response of the detector over the range of concentration being analyzed. ACKNOWLEDGMENT
The authors thank F. L. Kauffman, Swift and Co., Chicago, Ill., for supplying fats and oils and E. J. Eisenbraun, W. D. Gallup, and Ralph Matlock for helpful suggestions. LITERATURE CITED
(1) Hornstein,
Irwin, Alford, J. A., Elliott, L. E., Crowe, P. F., AKAL. CHEW32, 540 (1960). ( 2 ) Mason, Michael E., Waller, George R., Ibzd., 36, 583 (1964) (3) Tinoco, Joan, Shannon, Angela, Miljanich, Peter, Lymah, Richard L., Okey, Ruth, Anal. Biochem. 3, 514 (1962). (4) T-orbeck, Marie L., Mattick, Leonard R , Lee, Frank A Pederson, Carl S., A N A L . cHEM. 33, i k i 2 (1961). RECEIVEDfor review June 14, 1963. Accepted November 7, 1963. This work was supported in part by grants from the Southwest Peanut Research Foundation and Corn Products Institute of Nutrition. The major author is a National Institutes of Health Trainee Fellow.
The Determination of Total Carbon and Sodium Carbonate in Sodium Metal SILVE KALLMANN and ROBERT LIU Ledoux & Co., Inc., Teaneck, N. J.
b The total carbon content of sodium metal is determined b y low-temperature ignition of the sample in an argonoxygen mixture, treatment with dilute sulfuric acid, and measurement of the liberated COS in a conductometric Leco Apparatus. For the determination of the sodium carbonate, steam introducted into the combustion chamber by argon gas decomposes sodium metal with the formation of sodium hydroxide. Again, COz is liberated b y dilute sulfuric acid and measured conductometrically.
P
a n hEC Round Robin program covering the determination of carbon in sodium metal indicate wide discrepancies in results obtained by the participating laboratories (6) and emphasize the lack of reliable procedures. The literature covering the determination of various forms of carbon in sodium metal is very scanty. Stoffer and Phillips (8) published a procedure based on the ignition of a 100-mg. sample in a stream of oxygen. The carbon in the sample is converted to carbon dioxide which RELIMINARY DATA Of
590
ANALYTICAL CHEMISTRY
is then absorbed in a n AscariteDrierite micro absorption bulb. The combustion tube is made of quartz and the section containing the sample is externally heated to 950' C. It is claimed that sufficient heat is evolved to prevent combination of the carbon dioxide with the alkali oxides. An asbestos plug serves to confine the oxide fumes within the combustion area and prevents their being carried to the cooler parts of the apparatus. When this procedure was tested in this laboratory on sodium samples containing about 100 p.p.m. of carbon, recoveries of COz varied, but never exceeded 50%. The reason for the incomplete recovery of COz may be attributable to its interaction with sodium oxide, either in or outside of the combustion area. FYhile the geometry of the apparatus used in this laboratory varied somewhat from that recommended by Stoffer and Phillips, it is felt that even under ideal conditions, the reaction of C o n with ?;azo is difficult to prevent, thus causing low results. It has recently also been pointed out (4) that while dissociation of sodium carbonate begins around
700" C., the vapor pressure does no exceed 1 mm. of Hg until about 950' C. and a temperature of 1100' C. is needed to recover completely carbon from sodium carbonate in 1 hour of combustion time. The original method has been improved by using sensitive manometric ( 2 ) or gas chromatographic (4) measuring devices. A procedure for free carbon in sodium which employs !vet combustion has also been described (5, '7). The sodium sample is cut into small pieces, which are then dissolved, one by one, in a small volume of water under a stream of nitrogen. The resulting solution is neutralized with sulfuric acid until an excess of the acid is present. The mixture is evaporated to near dryness, and then Van Slyke combustion fluid (9) is added. Cpon heating, the carbon is oxidized to carbon dioxide. Hot perchloric acid has also been used for the oxidation of the carbon, and the COz, after being coldtrapped, can be measured by manometric methods, by gas chromatography, or by titration of unreacted Ba(OH)2. Recently a method was recommended based on the solution of the sample in
