(Table V). The marked effect of acetylation of a C-3 equatorial hydroxyl on the Alog r value for a change in configuration a t (3-5, shows the potentialities of esterification for separating isomers. These preliminary results illustrate that data obtained from gas chromatography of steroids can be interpreted in the same systematic way as RM values in paper chromatography. The ability, for instance, to predict the approximate relative retention times of compounds should greatly increase the effectiveness of gas chromatography in its application to the identification of steroid metabolites. ACKNOWLEDGMENT
We thank Sir Solly Zuckerman for hie interest and encouragement. Steroids were kindly provided by I. E. Bush (Birmingham University), C. Djerksi (Stanford University), D. D. Evans (Parke Davis & Co.), E. Forchielli (Worcester Foundation), J. Fried (Squibb Institute for Medical Research), and W. Klyne (Westfield College). We
also thank the London Rubber Co. for funds to support a University Research Fellowship for one of us (B.A.K.) and the Caroline Harrold Research Fund for a grant t o cover the purchase of a gas chromatograph. LITERATURE CITED
(1) Bate-Smith, E. C., Westall, R. G., Biochim. Biophys. Acta 4, 427 (1950). (2) Beerthuis, R. K., Recourt, J. H., Nature 186,372 (1960). (3) Brooks, S. G., Hunt, J. S., Long, A. G., Mooney, B., J. Chem. SOC.1957, 1175. (4) Bush, I. E., Biochem. SOC.Symp. 18, 1 (1960). (5) Bush, I. E., “Chromatography of
Steroids,” Pergamon Press, London,
1961. (6) Clayton, R . B., Nature 190, 1071 (1961). (7) Zbid:, 192, 524 (1961). (8) Eglinton, G., Hamilton, R. J., Hodges, R., Raphael, R. A., Chem. & Znd. (London) 1959, 955. (9) Haahti, E. 0. A., VandenHeuvel, W. J. A,, Homing, E. C., Anal. Biochm. 2,182 (1961). (10) Zbid., 2, 344 (1961). (11) Haahti, E. 0. A., VandenHeuvel,
W. J. A,, Horning, E. C., J . Org. Chem.
26, 626 (1961). (12) Kabasakalian, P., Basch, A., ANAL. CHEM.32, 458 (1960). (13) Lipsky, 8. R., Landowne, R. A., Ibid., 33,818 (1961). (14) O’Neill, H. J., Gershbein, L. L., Zbid., p. 182. (15) Sweeley, C. C., Homing, E. C., Nature 187. 144 (1960). (16) VandenHeuvej, W.’ J. A., Haahti,
E. 0. A., Homing, E. C., J. Am, Chem.
SOC.83, 1513 (1961). (17) VandenHeuvel, W. J. A., Homing, E. C., Biochem. Biophys. Res. Commun. 3, 356 (1960). (18) VandenHeuvel, W. J. A., Homing, E. C., J. Org. Chem. 26, 634 (1961). (19) VandenHeuvel, W. J. A,, Homing, E. C., Sato, Y., Ikekawa, N., Zbid., p. 628. (20) VandenHeuvel, W. J. A,, Sjovsll, J., Homing, E. C., Biochim. Biophya. Acta 48, 596 (1961). (21) VandenHeuvel, W. J. A,, Sweeley, C. C., Homing, E. C., J. Am. Chem. Soc. 82, 3481 (1960). (22) Wotiz, H. H., Martin, H. F., J. Biol. Chem. 236, 1312 (1961). (23) Ziffer, H., VandenHeuvel, W. J. A.,
Haahti, E. 0. A., Homing, E. C., J. Am. Chem. SOC.82, 6411 (1960).
RECEIVED for review November 13, 1961. Accepted April 30, 1962.
identification of Carboxylic Acids in Alkyd and Polyester Coating Resins by Programmed Temperature Gas Chromatography G. G. ESPOSITO and M. H. SWANN Coating and Chemical laboratory, Aberdeen Proving Ground, Md.
,A gas chromatographic procedure is proposed for identifying dicarboxylic and monocarboxylic acids present in alkyd and polyester coating resins. The method was effectively used to identify 19 of the most frequently encountered acids used in the production of synthetic resins. The technique involves transesterification of the resin with lithium methoxide to form methyl esters and subsequent separation by programmed temperature gas-liquid chromatography (PTGLC) on polar and nonpolar columns and identification by their relative retention.
A
of mono- and dicarboxylic acids are used in the manufacture of alkyd and polyester resins where they finally occur as esters, either partially or completely reacted with a variety of polyhydric alcohols. The importance of these acids in synthetic resin production has resulted in the development of a number of analytical methods. ChemWIDE VARIETY
1048
m w r n c A i CHEMISTRY
ical and instrumental methods for nine dicarboxylic acids are discussed in Parker excellent reviews by Jones (4, (6),and Shreve (7). In general, the chemical methods required, a prior separation of the potassium salts of the dicarboxylic acids by nonaqueous saponification which is somewhat timeconsuming. The ultraviolet and infrared spectroscopic methods were limited primarily to aromatic type acids and none of the methods possessed the broad scope necessary for the range of acids encountered. A microscopic method (2) is available but is not suitable for dicarboxylic acid mixtures. Since the advent of gas chromatography, a number of analytical methods applying this technique to coating analysis have appeared. Zielinski, Moseley, and Bricker (8) presented a detailed method to characterize oils used in coating resins. A gas chromatographic method (6) was devised for the identification of fatty acids in vegetable oils using Apiezon L and polyester columns; only those dicar-
boxylic acids that are formed during fatty acid degradation studies were included. More recently (1) the principle of programmed temperature gasliquid chromatography (PTGLC) was used to identify the polyols present in alkyd and polyester resins and is an excellent companion method to this one for examining coating materials. Because the separation of the methyl esters of fatty acids has been studied extensively, emphasis was placed in this work on the separation of acids not found in drying oils. For gas-liquid chromatography (GLC) analysis, the material being examined must have a sufficiently high vapor pressure. In the case of polymeric materials, analyses have been conducted on products of pyrolysis and volatile derivatives. Methyl esters of carboxylic acids are congruous for GLC studies, and a rapid, general transesterification technique applied directly to resin samples was used to prepare the materials for chromatographic separation here. The method
C
4
K
Figure 1 . Separation of methyl esters obtained from lithium methoxide transesterification on a polyesterCarbowax column A. 6. C. D. E. F. G. H. I. 0
IO
I5
20
25
1. Triacetin
Pelargonic Succinic Benzoic Maleic and fumaric Lauric Adipic Itaconic Diglycolic Myristic
K.
Azelaic Palmitic Sebacic N. o- and lsophthalic 0. Stearic P. Oleic Q. Linoleic R. Linolenic
1. M.
30
MINUTES
is suitable for a wide range of dirarboxylic acids. A study of monocarboxylic acids that are frequently encountered in alkyd and polyester resins is also included. The acids investigated were o-phthalic, isophthalic, fumaric, maleic, itaconic, succinic, adipic, azelaic, sebacic, diglycolic, pelargonic, and benzoic. Acids derived from drying oils such as lauric, myristic, palmitic, stearic, oleic, linoleic, and linolenic can be identified along with the acids mentioned above. The relative amounts of fatty acids separated from the samples were used to identify tentatively the oil or source of fatty acids used to prepare the alkyds. Confirmation was by an alternate method (8). The samples were analyzed by transesterifying the esters with lithium methoxide in methanol; the resulting methyl esters were isolated. Boron trifluoride was substituted for lithium methoxide only when it was necessary to distinguish fumaric acid from maleic acid. The methyl esters formed were separated on a two-part polyester-Carbowax column and a silicone grease column and the relative retention time data were compared to tables obtained from knowns. APPARATUS AND MATERIALS
Chromatographic Unit. The instru-
ment used to obtain the chromatograms was a Model 500 Linear Programmed Temperature Gas Chromatograph (F & M Scientific Co.) equipped with a Brown Electronik Recorder (Minneapolis-Honeywell Regulator Co.). Operating Conditions
3etector cell temperature, C. 300 ?etector cell current, ma. 150 .njection port temperature, C. 330 Telium flow at exit, cc. per minute 85 ?rogrammed temperatureodetails Column heating rate, C. per minute 4.0 Startin column temperature,
8.
C
,
,
I
5
0
10
20
15
25
30
35
4 0
45
MINUTES
Figure 2. Separation of methyl esters obtained from lithium methoxide transesterification on a silicone grease column A. B. C. D.
E.
F. G.
Succinic Benzoic Diglycalic, maleic, and fumaric Pelargonic, itaconic, and adipic Triacetin o-Phthalic lraphthalic
Silicone grease 75 Polyester-Carbowax 125 FinisFmg column temperature, C.
Silicone grease Polyester-Carbowax
250 225
Column Preparation. Both column packings were made with 20% by weight of liquid phase on 60- t o 80mesh Chromosorb W. A 6-foot length of l/rinch copper tubing was packed with silicone grease on acid-washed Chromosorb W. To prepare the polar column, a 6-foot length of copper tubing was bent into a U-shape and one side was filled with Carbowax 20M on Chromosorb W and the other side with diethylene glycol succinate on Chromosorb W. Constant vibration was maintained and small amounts of packing material were added alternately to each side. The column was mounted
H. 1. 1. K. I.
M.
Lauric and azelaic Sebacic Myristic Palmitic Oleic, linoleic, and linolenic Stearic
so that the sample pctssed first through the polyester section of the column. Reagents. Lithium methoxide in methanol (0.5N)was prepared by adding small pieces of metallic lithium, about the size of a small pea, to a flask containing absolute methanol which was kept chilled in an ice bath. During the addition of lithium, a few milliliters of reagent were titrated periodically until the normality reached 0.5 or more. If the normality exceeded 0.5, the correct amount of absolute methanol was added to adjust the solution to 0.5N. The reagent was filtered before each use. When kept tightly stoppered it lasted for a t least one month. The boron trifluoride reagent was prepared by bubbling the gas into a flask containing absolute methanol in an ice bath. The addition is discontinued when one milliliter of the reagent, diluted with 25 ml. methanol, uses 11 to VOL 34, NO. 9, AUGUST 1962
1049
I
0
5
10
I5
,
I
I
25
20
dissolved. It was then ready for chromatographic analysis. A 6foot polyester-Carbowax column wm mounted in position and heated to the starting temperature. About 5 pl, of sample were introduced onto the column along with about 0.2 pl. of triacetin, and the mechanism for increasing column temperature was immediately engaged. After the maximum column temperature was reached, isothermal heating was maintained until all of the volatile components emerged. The analysis was repeated using a 6foot silicone g r e w column in accordance with the operating conditions previously described for that column. If the presence of maleic or fumaric acids is indicated, repeat the transesterification with boron trifluoride catalyst on another sample of approximately 0.3 gram as before, using 5 ml. of the reagent and boiling gently for 5 minutes. Transfer t o a separatory funnel with 50 ml. of water and 35 ml. of methylene chloride and shake vigorously. Filter the methylene chloride layer (no washing is necessary) and evaporate. Chromatographing of this sample will show the presence or absence of fumaric acid in accordance with Table I.
I
30
35
MINUTES
Figure 3. Methyl esters from an alkyd resin separated on a polyester-Carbowax column A. B. C.
D.
E.
Benzoic Triacetin Palmitic Iro- or o-phthalic
122ml. of 0.5N KOH in methanol, titrating to the yellow end point of thymol blue indicator. EXPERIMENTAL
A resin solution containing approximately 0.3 gram of nonvolatile material was poured into a 125-ml. flask, and 15 nil. of the 0.5N lithium methoxide were added along with a boiling stone. A short air condenser (about 10 inches) was attached to the flask which was placed on a steam bath. The sample was swirled while being heated until solution was effected and then boiled
F. G.
H.
Stearic Oleic Linoleic Linolenic
for 2 minutes. The flask was removed from the steam bath promptly a t the end of the 2-minute period, the condenser removed, and 5 ml. of 6N sulfuric acid were added a t one. The contents of the flask were then transferred to a separatory funnel and diluted to 50 ml. with water. Thirty-five milliliters of methylene chloride were added followed by vigorous shaking. The methylene chloride layer was separated and washed with 15-ml. portions of water until all the sulfuric acid was removed. The beaker was placed in a warm water bath and removed as soon as all of the solvent had been expelled. If insoluble methyl esters were present, tetrahydrofuran was added dropwise with warming until all or most of the sample had
RESULTS
The methyl ester peaks were identified by calculating their retention, relative to triacetin, and comparing this to the calibration chart in Table I. Values in Table I were obtained by examining methyl esters formed by lithium methoxide catalyzed transesterification of known esters. Figures 1 and 2 show the separation of methyl esters on a polyester-Carbowax column and on a silicone grease column, respectively. A more complete separation was possible with the polar column but i t was not possible to distinguish isophthalic from
Table 1. Relative Retention lime Data for Methyl Esters of Carboxylic Acids
(Formed by Lithium Methoxide Transeaterification) Methyl 6-Ft. 6Ft. Sicone Este; Pol esterGrease0 of Acid Carcoowaxa 0.83 Pelargonic 0.29 0.49 Succinic 0.44 0.62 Benzoic 0.47 Fumaric 0.63(0.38)’ 0.69 (0.49)b Maleic 0.64(0.52)a 0.68 (0.47)b 1.29 Lauric 0.66 0.83 Adipic 0.71 0.83 Itaconic 0.74 0.70 Diglycolic 0.79 1.55 Mqtic 0.90 1.31 Azelax 1.08 1.79 Palmitic 1.14 1.43 Sebacic 1.19 1.17 Orthophthalic 1.26 1.26 Isophthalic 1.26 2.02 Stearic 1.36 1.98 Oleic 1.41 1.98 Linoleic 1.49 1.98 Linolenic 1 .60 0 Relative retention time (triacetin 1). * When transeaterified with boron tnfluoride in methanol.
-
1050
ANALYTICAL CHEMISTRY
D
cl
B
I 0
I
I
I
I
I
1
I
I
I
5
10
15
20
25
30
35
40
45
MINUTES
Figure 4. Methyl esters from an alkyd resin separated on a silicone grease column A. 8. C.
Benzoic Triacetin Orthophthalic
D.
E.
F.
lrophthalic Pahnitii Stearic, oleic, linoleic, and linolenic
I
0
,
5
I
!
IO
15
2 0
30
25
1
I
0
5
,
15
10
MINUTES
C.
D.
A.
Triacetin lrophthallc or 0-Phthalic
o-phthalic and, in addition, maleic, fumaric, lauric, and adipic acids are grouped close together. The silicone grease column will separate isophthalic from o-phthalic and change the relative retention times of most of the other methyl esters. The identification of a complex mixture of acids in an alkyd resin is illustrated in Figures 3 and 4. In Figure 3 methyl benzoate was easily recognized in the early part of the chromatogram, but o-phthalic and isophthalic acids emerged together. In Figure 4 the same type of analysis is shown on a silicone column with the methyl benzoate peak located adjacent to the solvent peaks, but the methyl isophthalate and orthophthalate peaks were completely resolved. Esters of fumaric and maleic acids reacted similarly when treated with lithium methoxide and were not separated from each other. Fumaric was identified in the presence of maleic by using an alternate transesterification technique. Both esters were readily converted to thei! methyl esters with concentrated boron trifluoride in methanol. Figures 5 and 6 were obtained from the analysis of a polyester known to contain fumaric, pelargonic, and isophthalic acids. After lithium methoxide transesterification, pelargonic and isophthalic acids were easily identified, but the fumaric peak mas not distinguished from maleic. When the same polyester was treated with boron trifluoride in methanol and re-chromatographed, the position of the pelargonic and isophthalic peaks remained unaltered; fumaric formed a methyl ester having a retention time different from that produced by the alkali catalyzed transesterification and was easily recognized. The presence of modifying resins
25
30
Figure 6. Separation of methyl esters, from a polyester resin, on a silicone grease column
Figure 5. Separation of methyl esters, from a polyester resin, on a polyester-Carbowax column A. Pelargonic B. Maleic or fumaric
r7
1
20
M I N U T E S
8.
D.
Triacetin lrophthalic
tion. Only the high-boiling hydrocarbon solvents occasionally found in alkyd resins interfered with the low boiling methyl ester peaks, and this occurred only with the silicone grease column. These solvbnts were easily detected in the chromatogram and were removed by drying the sample before transesterification under a vacuum a t 85" C. for 1.5hours. When maleate, fumarate, and itaconate esters were treated with lithium methoxide, the corresponding methyl esters did not form and the peaks that resulted gave retention times considerably different from those of the pure methyl esters. This suggests a rearrangement involving the double bond and dicarboxylic acid groups peculiar to all three acids. Sodium or potassium could be substituted for lithium but they were more
such as phenol, rosin, nitrocellulose, melamine- and urea-formaldehyde did not interfere with the proposed method. The identification of orthophthalic acid in a nitrocellulose-alkyd lacquer is illustrated in Figure 7 using a silicone grease column. A preliminary separation on a polyester-Cerbowax column was performed but is not shown. Tetrachlorophthalic and chlorendic acids could not be identified, but their presence did not interfere with the identification of the other acids investigated. DISCUSSION
The &foot 2-part polyester-Carbowax column separated most of the methyl esters studied but the &foot silicone grease column was also used to achieve a more definite identifica-
I
C.
Maleic or fumaric Pelargonic
II
D
I
I
I
I
I
I
5
10
I5
20
25
0
I
30
I 35
'
-7-
40
45
MINUTES
Figure 7. Separation of methyl esters, from an alkyd resin modified with nitrocellulose, on a silicone grease column A. 8.
Triacetin O-PhIhalic
C.
D.
Palmitic Stearic, oleic, linoleic, and linolenic
VOL. 34, NO. 9, AUGUST 1962
1051
difficult to handle and the yields were lower. Although the transesterification conditions described appear to be optimum for the qualitative identification of a wide range of acids, they are not ideal for quantitative purposes. Changes in time of reaction and reagent concentration affect esterification yields and these conditions are currently being studied to obtain quantitative determination of individual acids, particularly phthalic, in alkyds by GLC. Boron trifluoride is selective as a transesterification catalyst and cannot be used in a general transesterification technique for resin analysis. Rosin and esters of rosin do not form methyl esters when treated with lithium methoxide or boron trifluoride in methanol. The analysis for rosin would have t o be achieved by a n indirect approach involving high temperature saponifica-
tion, isolation of rosin acids by extraction, and methyl ester formation using diaeomethane. Hudy (3) formed methyl esters of resin acids with diaeomethane in his GLC separation of the various resin acids. This analytical method can be expanded to include other acids. Tetrahydrophthalic acid was detected in a new polyester shortly after this work was completed and exhibits a relative retention time that does not coincide with those illustrated. Other noncoating uses of this technique are easily recognizable-e.g., detection of the presence of vegetable oil plasticizers in plastics, and identification of polyesters in urethane foams. ACKNOWLEDGMENT
The advisory assistance of C. F. Pickett, director of the laboratory, is acknowledged and appreciated.
LITERATURE CITED
(1) Esposito, G. G., Swann, M. H., ANAL.CHEM.33, 1854 (1961). (2) Esposito, G. G., Swann, M. H., OJic. Dig. Federation Paint CYC Varnish Prod. Clubs 30, 1059 (1958). (3) Hudy, J. A,, ANAL. CHEM.,31, 1754 (1959). ( 4 ) Jones, J. R., Jr., in “Analytical Chemistry of Polymers,” G. M. Kline, ed., Vol. XI1 of “High Polymers,” p. 17, Interscience, New York-London, 1959. (5) Miwa, T. K., Mikolajczak, K. L., Earle, F. R., Wolff, I. A., ANAL.CHEM. 32, 1739 (1960). (6) Parker, E. E., in “Analytical Chemistry of Polymers,” 0. M. Kline, ed., Vol. XI1 of “High Polymers,” p. 295, Interscience, New York-London, 1959. (7) Shreve, 0. D., in “Chemical Analysis of Resin-Based Coating Materials,” C. P. A. Kappelmeier, ed., p. 125, Interscience, New York-London, 1959. (8) Zielinski, W. L., Moseley, W. V., Bricker, R. C., Oqc. f i g . Federation SOC.Paint Technol. 33, 622 (1961). RECEIVEDfor review April 33, 1962. Accepted June 7, 1962.
Theory and Practice of Low-Loaded Columns in Gas Chromatography DONALD T. SAWYER and JAMES K. BARR Department of Chemistry, University of California, Riverside, Calif.
b The advantages and limitations of low-loaded columns for gas chromatography have been investigated from theoretical as well as experimental considerations. The effect upon column efficiency and separation efficiency of lowering the loading on Chromosorb W has been established. A number of the practical problems of low-loaded columns are discussed, particularly adsorption by the support material and its prevention. Several examples of separations using lowloaded columns are presented which confirm the advantages of such columns; namely, analyses of mixtures at temperatures up to 200’ below the boiling point of the mixture are possible without loss of column efficiency and with a general increase in the ease of separation. Thus, thermally unstable mixtures and mixtures outside the hightemperature limits of conventional columns can be analyzed with lowloaded columns.
T
of lightly loaded columns for gas chromatographic separations was first suggested by Cookr in 1960 (4). Examples were presented of a number of separations using glass microbeads coated wit,h O.lO’% to 0.15% liquid phase. More recently a series HE USE
1052
ANALYTICAL CHEMISTRY
of separations using low-loaded glassbead columns has been discussed by Hishta, Messerly, and Reschke (8). Both of these studies demonstrated that low-loaded columns permit studies at much lower temperatures for a given sample mixture than are normally used with conventional packed columns. I n fact, separations are routinely possible with such columns operating 100” below the boiling point of the mixture. Thus, low-loaded columns extend the range of samples that may be analyzed for a given liquid phase or for a given instrument. Another advantage of operating at lower temperatures has been suggested by Cooke (6); namely, the separation of a given mixture should require fewer theoretical plates. In June 1961 Frederick and Cooke (7) discussed the use of low-loaded columns with Johns-Manville Chromosorb P as the support material and showed that the separation efficiency of the column was not affected by lowering the loading down to 3.U% liquid. Below this level serious tailing was caused from adsorption by the support. The authors have been engaged in a detailed study of low-loaded columns since the latter part of 1960. The present discussion is concerned with the theoretical justification for the apparent advantages of such columns, some prac-
tical considerations and problems of using such columns, and some examples of separations which support the theory and the conclusions based on it. One of the major problems with using small percentages of liquid phase on supports has been the attendant adsorption by the support (4, 7). Therefore, a study of various support materials and the effect of treating them with deactivating reagents has been a necessary part of our investigation; the results of this study are the basis of a separate study (14). The adsorption data indicate that Johns-Manville Chromosorb M’, when treated with hexamethyl disilaeane (2), gives the most satisfactory support material when coated with a liquid phase. The experimental portion of the present discussion is based on the use of this material as a support for the liquid phases. THEORETICAL CONSIDERATIONS
Bohemen and Purnell (3) have recently established that the extended rate equation, originally proposed by van Deemter, Zuiderweg, and Klimkenberg (6) can be written
H
=
A
+ B- + UO
C
g
~
+ Clzi o
(1)
