Quantitative Analysis of Some Cl0 Dibasic Acids and Associated Monobasic Acids by High Temperature Gas Chromatography R. C. BARTSCH, F. D. MILLER, and F. M. TRENT U. S. Industrial Chemicals Co., Division o f National Distillers and Chemicals Corp., Cincinnati, Ohio
b A rapid method was developed for the quantitative analysis of mixtures of three aliphatic Cl0 dibasic acids and two CS monobasic acids by gas chromatography. Samples were quantitatively converted to methyl esters via diazomethane. The esters were resolved at a temperature near 2 3 0 " C. on a 12-foot column of specially prepared high vacuum silicone grease or on a &foot co~umnof a polyester. Data are given for the accuracy and precision of the method. Retention times are given for a number of aliphatic acids.
articles suggested that a similar method might be applicable to the analysis of the isomeric C Smono- and isomeric C ~ O dibasic acids. Furthermore, the availability of a new reagent for the preparation of diazomethane offered attractive possibilities for the rapid, quantitative preparation of the methyl esters (1, 9). The physical properties of these acids and their methyl esters have been d e termined in this laboratory (11). The characteristic cracking patterns and a method of analysis for these acids and their methyl esters by mass spectrometry have also been described (10). EXPERIMENTAL
I
ACID (U.S.Industrial Chemicals Co.) is a mixture of three Crodibasic acids: 2,5-diethyladipic (12 to l8Q/, Zethylsuberic (72 to 80%), and sebacic (6 to 10%) acids. The method of preparation has been described (3). Small amounts of pelargonic and Z-ethylheptanoic acids are produced as by-products. The usual methods for the analysis of mixtures of acids, such as neutral equivalent, adsorption chromatography, and liquid-liquid partition chromatography, were too time-consuming or not applicable. Esterification of the acids followed by analytical vacuum fractionation on a spinning band column was satisfactory, but too lengthy for practical use. The gas-liquid chromatography of aliphatic acids and their methyl esters has been studied rather extensively. I n general, these investigations have considered only members of homologous series and the esterifications were standard, requiring 4 to 6 hours. James and Martin (6) successfully separated high boiling monobasic isomers when the isomerization was a single methyl group. Nowakoska (7) resolved a homologous seriesof either mono- or dibasic acids, but did not attempt to separate a mixture. Unsaturated acids, which are expected to have greater differences in polarity, particularly when conjugated, have been resolved by James (6). The analytical scheme presented in these SOSEBACIC
Apparatus. The chromatographic column was either a 12-foot (silicone) or a 4-fOOt (polyester) copper spiral, l/4 inch in outer diameter. A Beckman Model GC-2 gas chromatograph was used, but any instrument capable of operating a t temperatures of 220' t o 240" C. would be satisfactory. Column Packing. One column packing was a mixture of 30- to 60mesh untreated Celite (Fischer Column-Pak) and Dow Corning HiVacuum silicone grease which had been treated in a manner suggested by. Cropper and Heywood (2). The finished column material contained %yoby weight of extracted grease. The following procedure was adopted for the preparation of the packing: One part by weight of grease was added, in increments, to ll/z parts of ethyl acetate with constant agitation. Agitation was maintained for 5 minutes after the mixture became homogeneous. The mixture was washed five or six times with ethyl alcohol to coagulate the grease and remove the ethyl acetate. The amount of ethyl alcohol per wash was one half the amount of grease used. The extracted grease was dispersed next in sufficient toluene to form a free-flowing mixture when stirred. The toluene-grease mixture was then poured evenly over the Celite, and the solvents were evaporated on a hot plate. Solvent removal was facilitated by spreading the wet material over as large an area as possible. The polyester column was prepared by dissolving the polyester in acetone, pouring the mixture over untreated
30- to 60-mesh Celite, and evaporating the solvent. The finished column contained %yoby weight of polyester. Diazomethane Preparation and Esterification. A rapid, quantitative procedure for the esterification of the acids involved the preparation and utilization of diazomethane. Diazald ( N - methyl - N - nitroso - p - toluenesulfonamide) and directions for preparing diazomethane from this compound can be obtained from the Aldrich Chemical Co., 3747 Booth St., Milwaukee 12, Wis. A general esterification procedure is also given by Smith ( 9 ) . The esterification must be carried out a t or below 0" C. in clean, smooth surface glassware to prevent the formation of polymethylene or loss of diazomethane. In addition, care must be exercised during the process to prevent the introduction of water, m-hich produces tailing of the peaks. Instrument Calibration. Impure acid samples (90 to 98% purity) were prepared in quantity by acid-catalyzed esterification with methanol, and the methyl esters were isolated and hydrogenated to remove the last traces of unsaturation. After repeated vacuum fractional distillation, their purities, as estimated by this method, exceeded 99.8y0. Primary standards containing all five esters were prepared for use in instrument calibration. Analysis and Calibration were based on peak height measurements. Day to day drift in calibration was not serious, but for accurate results, daily calibration was desirable. All chromatograms were obtained a t 236" f 0.2" C. with a helium flow rate of 45 ml. per minute (soap bubble determination). Procedure. T o avoid the necessity for highly reproducible sample introduction, ratios of the dibasic acids were determined. By using a standard containing equal percentages of each of the Clo acids, the ratio of sensitivities could be obtained from the ratio of peak heights (sensitivity of dimethyl sebacate = 1.000). The peak heights in the sample were then corrected by dividing the sample peak height by the proper sensitivity obtained from the standard. The resulting corrected peak heights were then normalized. This method was usually sufficiently accurate VOL 32, NO. 9, A U G U S T 1960
1101
Table I. Analysis of Samples of Known Composition
Found,
Present,
70
I3-f
II.
Retention Times and Sensitivity Coefficients for Some Mono- and Dibasic Acid Esters
Deviation,
/O
%
2,ti-Diethyladipic Acid 19.9 20.0 -0.1 37.0 37.7 -0.7 16.5 16.0 $0.5 33.0 33.4 -0.4 15.7 16.0 -0.3 14.7 14.9 -0.2 7.7 8.2 -0.5 14.8 14.9 -0.1 29.6 31.2 -0.6 33.4 0.0 33.4 32.7 32.8 -0.1 2-Ethylsuberic Acid 78.3 78.0 +0.3 30.8 30.6 +0.2 77.4 78.0 -0.6 33.4 33.5 -0.1 64.1 63.6 $0.5 59.8 59.3 +0.5 75.6 75.3 +0.3 59.0 59.3 -0.3 29.8 29.3 +0.5 33.8 33.5 $0.3 33.7 34.0 -0.3 Sebacic Acid 2.0 1.8 -0.2 -0.4 31.7 32.1 +0.1 6.1 6.0 -0.5 33.6 33.1 -0.2 20.2 20.4 +0.2 19.2 19.0 +O.l 7.9 8.0 19.0 +0.5 19.5 +0.7 29.9 30.6 $0.9 33.1 31.0 +0.8 32.8 33.6 Abs. av. dev 10.4
for isosebacic acid, which has a minimum Clo content of 98.5y0. hlonobasic acids were determined on an absolute basis through volume measurement with some sacrifice in accuracy n-hich was not serious, as they were present only in trace amounts. As Little as 25 p.p.m. of a monobasic acid or 100 p.p.m. of dibasic acids could be detected by using samples as large as 0.05 ml., although the normal sample was 0.01 to 0.02 ml. For samples containing less than 98.5% Clo acids, an absolute method employing an internal standard was developed. A known amount of adipic acid (a rare impurity) was added to each sample as the internal standard. The calibration curves used in the analyses were plots of peak height ratios (component to internal standard) us. concentration ratios (component to internal standard). Graphing in this manner eliminated the requirement that all systems contain the same amount of internal standard, so that tedious weighings or unnecessary calculations were avoided. Within experimental error, these calibration curves were linear over the entire concentration range. The peak height ratiosobtained from the sample chromat-
1102
Table
ANALYTICAL CHEMISTRY
2-Ethyl heptanoate Adipate Pelargomte 2,5-Diethyl adipate 2-Ethyl suberate Tridecanoate Sebacate Myristate Palmitate Tetradecanedioate Stearate Theoretical platese Resolving powerd
2.8 3.4 3.6 7.4 10.1 12.8 13.2 18.4 34.8 50.0 65.2 550
1.12
a
Time measured from air peak.
d
Calculated on methyl sebacate peak (4). Calculated on Cppeaks (4).
1.7 2.8
4.5 3.8 3.5 1.8 1.5 1.1 1.2 0.80 0.40 0.28 0.21
...
7.2 11.8
... ...
18.1
...
... ...
642 1.54
9.1 6.8
...
3.1 1.9
...
1.2
...
...
... ...
* Chart divisions per milliliter of sample (1mv. = 100 chart divisions).
ograms were converted to concentration ratios from the calibration curve. With a knowledge of the concentration ratio and the concentration of internal standard in the sample, the absolute percentage of each component could be calculated. I n addition, unknown impurities could be quantitatively estimated from a curve of sensitivity coefficient us. retention time (Figure 1). This curve was plotted from data obtained on the silicone column, but analogous results were obtained on a polyester column. Because these columns operate nonselectively for esters, retention time is a function of boiling point. Consequently, for the retention time of an unidentified peak, one obtains from this curve a sensitivity coefficient in scale divisions per milliliter of component. From the peak height of the unidentified material and the total volume introduced] the percentage of a n unknown component can be calculated. Using this method recoveries of 98.5% or better were achieved for complex mixtures. DISCUSSION
Mixtures of all five acids (two CS monobasic and three Cl0 dibasic acids) have been successfully resolved. The method has been in use in these laboratories for two years and has been successfully applied as a control procedure for product purity. A large variety of samples resulting from pilot plant and plant operations as well as products from laboratory studies on separation methods have been successfully analyzed. Some data on the accuracy of the method are given in Table I. The average deviation of all determinations is =k0.470 absolute for the major components.
The method has been extended to the separation of some other mono- and dibasic acids. Retention times and approximate sensitivities are given for these as well as for the Cg mono- and Ciodibasic acids in Table IT.
11I
2
3
4 RETENTION 5 6 7 O I TIME 20MIMJTE 3$ 40
6070 Id0
Figure 1. Sensitivity coefficients for some mono- and dibasic acid esters
Also included in Table I1 are the number of theoretical plates in each column, calculated from the methyl sebacate peak, and the resolving power of the column, calculated from the two Cp peaks. A complete analysis for five components can be obtained inabout 1.5 hours’ elapsed time, including the time necessary for the esterification and removal of the solvent. Five to seven samples may be esterified in approximately 1 hour and the first sample is introduced into the chromatograph while the processing of the remainder is completed. About hour of instrument time is required for each sample.
Orr and Callen (8) described the separation of the methyl esters of fatty acids on a Reoplex 400 plasticizer substrate (Geigy Chemical Co.). Investigation n i t h this material indicated that resolution of the CS monoand Clo dibasic acids could be achieved, but that the column was unstable a t the temperature necessary for resolution. I t is possible that the Reoplex 400 varies in temperature stability from lot to lot. This use of a polyester substrate led to a polyester with a molecular weight of ca. SO00 (from 1,2-propylene sebacic acid), which mas availglycol able in this laboratory. A 4foot column containing 35y0 by weight of this polyester on Celite gave excellent resolution and stability. I n contrast t o the silicone grease the polyester substrate is polar, and the polyester column is not as versatile as the silicone grease. For example. the silicone column will yield acceptable resolution of octadienes, amines, hydrocarbons, and alcohols. The polyester column, on the other hand, gives the best results only n-hen the sample components have some
+
polarity. However, the decrease in substrate preparation time and improved resolution on shorter columns were sufficient to recommend it for this analysis. Two facts concerning the sensitivity coefficient curve (Figure 1) should be mentioned. First, the graph as shown is a log-log plot. If the data are plotted on Cartesian coordinates, two lines are produced, one each for mono- and dibasic acids. However, such a two-curve plot is not as useful as a single curve, as retention time alone cannot reveal the nature of the acid. Secondly, the graph presented is not linear even on the loglog plot. The sensitivity apparently reaches a maximum value at low retention times. Because the width of the base of the first CS peak is more than twice the recorder speed, one possible explanation for this curvature is slow detector response. The method described here is sufficiently accurate, precise, and rapid to serve as a routine analytical method. Moreover, it should be adaptable to a variety of mono- and dibasic acids with minor modifications.
LITERATURE CITED
(1) de Boer, T. J., Backer, H. J., Rec. trav. chim. 73. 229 11954). (2) Cropper, F.’R., Heywood, A., Suture
174, 1083 (19%). (3) Frank, C. E., Abstracts of Papers, Division of Industrial and Engineering Chemistry, 133rd Meeting, ACS, San Francisco, Calif., April 1958. (4)International Union of Pure and Applied Chemistry, Special Committee, Analytical Section, D. Ambrose, Chairman, JUIY1959. (5) James, A. T., Biochem. J . 6 6 , 515 (1957). 16) James. A. T.. Martin. A. J. P., l b i d . , 50, 679 (1952).‘ ( 7 ) Nowakoska, J., Melvin, E. H., Wiebe, R., J . Am. Oal Chemists’ SOC. 34,411 (1957). (8) Orr, C. H., Callen, J. E., J . Am. Chem. Soc. 80, 249 (1958). (9) Smith, L. I., Chem. Revs. 23, 193 (1938). (10) Trent, F. M., M. S. thesis, University of Cincinnati, Cincinnati, Ohio, 1958. (11) Trent, F. M., Miller, F. D., Brown, G. H., J . Chem. Eng. Datu 5 , 110 (1960). RECEIVED for review December 10, 1959. Accepted M a y 20, 1960. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 2 to 6, 1959. \
,
Gas-Liquid Partition Chromatography of Methylated Sugars HENRY W. KIRCHER Deparfment o f Agricultural Biochemistry, College of Agriculture, University o f Arizona, Tucson, Ariz.
,The methyl glycosides of tri-0methylpentoses and tetra-0-methylhexoses are separated on a number of commercially available gas-liquid partition columns. Better results are obtained with columns prepared from certain methylated polysaccharides. A mixture of the fully methylated methyl 01- and @-pyranosides of Darabinose, D-xylose, D-glucose, D-
mannose, and D-galactose separates into eight distinct peaks when put through a methylated hydroxyethylcellulose column. The methyl glycosides of di- and tri-0-methylhexoses also pass through the columns. Starch, cellulose, guar gum, and dextran are used as examples of the applicability of gas-liquid partition chromatography to polysaccharide structural studies.
of methylated sugars are volatile entities and should, thcrefore, be subject to analysis by gas-liquid partition chromatography. JlcInnes et al. (10) described the separation of a number of fully methylated pentose and hexose pyranosides on an hpiezon 11 column, and showed that these sugar derivatives pass through the column unchanged. Subsequent 11-ork by this group consisted of the separation of partially methylated glucoses and sugar alcohols whose free hydroxyl groups were acetylated (2).
This work was initiated to see if 2,3,4,6-tetra-O-methyl-~-$lucosecould be separated from the corresponding mannose derivative. Prior work on a glucomannan (6) had failed to discover a solvent system for the separation of these two methylated sugars by payer partition chromatography. The tetra-0methylglucose did not pass through the gas chromatograph, but the methyl glycoside of this sugar separated into two peaks, corresponding to the 01- and p-anomers. 4 number of methyl glycosides were
ETHYL GLYCOSIDES
obtained (Table I). They were exaniined by paper chromatography on several solvents and each yielded only one spot. Of all the anonieric pairs, only methyl a- and p-n-mannopyranoside separated from each other (Table I). The methyl glycosides were fully methylated by the Kuhn procedure (9). The columns that were examined are listed in Table 11. Their efficiency was gaged by their ability to separate the fully methylated derivative of p-Darabinopyranoside from p-D-glucopyranoside and that of e-D-mannopyranoside from a-D-glucopyranoside. Columns n-ere also prepared from methylated and acetylated cellulose, but would not pass the carrier gas. The films and hard clumps that formed by evaporation of solvent from the cellulose derivative-Chromosorb mixtures required extensive trituration t o pulverize them. The resulting particles were then too small and packed too closely in the column. VOL. 32. NO. 9, AUGUST 1960
1103
