Gas Chromatographic Separation of Volatile Organic Acids in Presence of Water IRVING R. HUNTER, VICTOR H. ORTEGREN, and JAMES W. PENCE
U. S.
Western Regionol Research loborofory, Agriculfurol Research Service,
Deportment of Agriculture, Albony, Calif.
b Volatile organic acids including acetic, propionic, isobutyric, butyric, isovaleric, valeric, isocoproic, caproic, and caprylic acids have been separated by gas-liquid chromatogrophy using an adipote polyester of diethylene glycol os the liquid phase and helium os the carrier gos. Fractions emerging from the column were oxidized to carbon dioxide which was then measured in thermal conductivity cells. Neorly complete resolution of most of the acids was readily obtained, even in the presence of water in amounts up to 50% of the mixture.
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of volatile fatty acids by gas chromatography was first reported by James and Martin in 1952 (2). These investigators worked with anhydrous mixtures of volatile organic acids and a column in which the liquid phase was DC 550 silicone oil containing 10% of stearic acid. The eluted fractions were measured by a recording buret coupled to a photoelectric control circuit,. Until very recently this procedure, or its modifications ( I ) , appears to have been the only one developed for the separation of organic acids by gas chromatography, despite the widespread use and rapid growth of this technique. Two factors which undoubtedly contributed to the lack of progress were the need for strict maintenance of anhydrous conditions and the corrosiveness of acid vapors on metallic elements of the usual types of sensitive measuring equipment. At the 1956 Institute of Petroleum (London) symposium on vapor phase chromatography successful separations of 20% aqueous solutions of mixed organic acids were mentioned, but with no description of the equipment or techniques used (4). More recently Smith (6) described the separation of aqueous mixtures of formic, acetic, propionic, isobutyric, and n-butyric acids using Tween 80 (5%) on Celite 545. He also described the separation of formic, acetic, isobutyric plus propionic as a single peak, and wbutyric by the use of Carbowax 400 on Celite. This paper describes a technique for the analytical separation of mixtures of organic acids up through CS,whether anhydrous or in aqueous solution. EPARATION
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ANALYTICAL CHEMISTRY
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detectors), column, flowmeter, vapor jacket combustion furnace, drying agent, and the necessary circuitry for detecting, amplifying, and recording the detector signal. Column. The chromatographic column, constructed of 8 feet of tubing 4 mm. in inside diameter, was mound in the form of a helix about 1.5 inches in diameter. The first and last 2 inches of the column were made of heavy-walled tubing (3 mm. i d . ) sealed into the top of a vapor jacket provided with a 50/50 standard taper joint. This was cut to a 15mm. length to provide a large enough opening for the glass-blowing work. Attached to the inlet of the column was a short side arm shaped to receive a rubber serum-bottle cap through which samples could be injected with a syringe. The outlet of the column ended in a 12/5 outer spherical joint which was heated electrically during a run by several windings of glass-covered Nichrome wire (24-gage B & S, with a resistance of 1.67 ohms per foot) to prevent condensation of acids as they emerged from the column. The column packing was 35- to 40mesh Johns-Manville C-22 firebrick coated with 15 grams of the adipate polyester of diethylene glycol per 100 grams of brick. This column packing was prepared by dissolving the ester in chloroform and then mixing in the firebrick. The solvent was then evaporated under vacuum in a laboratory evaporator. Column Temperature Control. The
rapor jacket of the column was 2 inches in diameter and about 16 inches long. It was wrapped with asbestos paper, wound with 24-gage B & S Sichrome wire, and then wrapped again with asbestos paper and aluminum foil. Electrical heating of the wire was controlled by a variable transformer A 200-ml., roundbottomed flask, which held the reflux liquid, was heated by a Glas-Col heating mantle, The jacket temperature and consequently the column temperature (125' C.) used in the experiments reported here was obtained by refluxing the monomethyl ether of ethylene glycol (methyl Cellosolve) in the boiling flask and jacket. Combustion Train. The combustion tube consisted of a '/r-inch glass U-tube 6 inches long closely wound with Sichrome wire and enclosed in a metal container filled with magnesium oxide. The tube was filled with 20mesh copper oxide and was connected to a 12-inch length of '/,-inch diameter glass tube filled with magnesium perchlorate to remove water of combustion. The inlet of the combustion tube was connected to the column by a 12/5 ball joint held in place by spring clamps. The heating wires surrounding the combustion tube and the column outlet were in series and were connected by porcelain wire nuts. REAGENTS
All chemicals were reagent grade unless otherwise specified. The acetic, propionic, butyric, $0butyric, valeric, isovaleric, caproic, isocaproic, and caprylic acids were commercial samples. All yielded single peaks when tested individually in the chromatographic apparatus. The adipate polyester of diethylene glycol, which is a commercial plasticizer known as LAC-1-R 296 (Cambridge Industries Co., Inc., 101 Potter St., Cambridge 42, Mass.), was used. Ethylene glycol monomethyl ether (methyl Cellosolve). Helium, welding grade, 99.5% pure.
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PROCEDURE
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Separations were made in a column containing the adipate polyester of diethylene glycol (LAC-1-R 296) a t a column temperature of 125' C., as established by the refluxing ethylene glycol monomethyl ether. (The plasticizers LAC-2-R 446, an adipate polyester of diethylene glycol that had been partially cross-linked with pentaerythritol, and LAC-3-R1 composition unknown, were also found to be acceptable liquid phases.) The helium flow rate was 106 ml. per minute. The detector block R'as kept in air a t ambient temperatures, and the filaments were operated a t 200 ma. The following separations were made. A sample containing about 0.2 rng. each of acetic, propionic, butyric, isobutyric, valeric, isovaleric, caproic, isocaproic, and caprylic acids was injected VOL. 32, NO. 6, MAY 1960
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onto the column to obtain the chromatogram tracing illdstrated in Figure 2. The similar tracing shown in Figure 3 was obtained by injection of an aliquot of the eame mixture of acids diluted with an equal volume of water. Because the longer chain acids are soluble in water to only a limited degree, it was necessary to shake the aqueous mixture vigorously during withdrawal of the sample. Acid mixtures of unknown compoaition are in many cases obtained as water solutions of alkali salts extracted from biological materials. After acidification, these solutions could be sampled and treated as described above. To simulate such a situation, an aliquot of a solution of sodium acetate and propionate containing about 20% by weight of the salts waa chromatographed after it was acidified to p H 2 with 85% phosphoric acid (Figure 4). RESULTS AND DISCUSSION
Figures 2 and 3, illustrating the separation of anhydrous and hydrated organic acids, respectively, are closely similar in that the peaks occur a t very nearly corresponding points in both chromatograms. Admixture of water with the organic acids used does not
significantly affect the resolving power of the column under the conditions described nor does the resolving power of the column noticeably change with continued use. At the 125' C. temperature of separation selected for these runs, the more volatile components emerge as sharp, closely spaced peaks, whereas the high-boiling components are low, broad, and widely separated. This situation could be improved by operating the column a t slightly higher temperatures; however, higher temperatures will cause a decrease in the resolution of the lower-boiling components. For quantitative work two determinations a t different temperatures could be made. One could be run a t a temperature below 125' C. to improve the separations of the Co through CS acids, and another separate determination made at a higher temperature to improve the separation of the acid homologs above CS. Figure 4 illustrates that the separation procedure described is applicable to the identification of simple volatile organic acids derived from plant tissues or other biological materials. Since the liquid phases used in this
work have been applied successfully in the separation of the esters of fatty acids (S), higher acids than caprylic could be analyzed on the same column a t the same operating temperature by converting them to esters. ACKNOWLEDGMENT
The authors thank E. B. Kester of this laboratory for technical advice. LITERATURE CITED
(1) Hardy, C. J., Pollard, F. H., Chromatog. 2, 22 :1959).
J.
( 2 ) James, A. T.,, Martin, A . J. P., Biochem. J. 50, 679 (1952j. (3) Lipsky, S. R., Landowne, R. A., Bzochzm. et Bzowhus. Acta 27, 666 I\A1 aKQ) Q"",.
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4) McInnes, A., in "Va our Phase Chro-
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IS'.Desty, ed., tterworths London, 1957. Iartin, A. h,,Smart, J., Nature 175, (1955).
mith Bength, Acta Chem. Scand. A
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RECEIVED for review September 14, 1959. Accepted December 21, 1959. Mention of specific products does not imply recommendation by the Department of Agriculture over others of a similar nature not mentioned.
Separation of PoIycycIic Aromatic Hydrocarbons in Complex Mixtures Chromatog ra phic Determination of Trace Amounts in Petroleum Waxes WILLIAM LlJlNSKY Division o f Oncology, The Chicago Medical School, Chicago, 111.
b Four polycyclic aromatic hydrocarbons-benzo[a]pyrene, dibenz[a,h]anthracene, benz [alanthracene, and chrysene-added to petroleum waxes both individually and combined, have been recovered at as low a concentration as 0.01 p.p.m. Recovery ranged from 35% for benzo [alpyrene to 94% for dibenz[a,h]anthracene. The analytical method was a combination of adsorption chromatography on magnesiaCelite and subsequent paper chromatography using different solvent systems.
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ANY natural and commercial prod-
ucts are of interest in studies of environmental cancer because they may contain very small quantities of polycyclic aromatic hydrocarbons, some 684
ANALYTICAL CHEMISTRY
of which are carcinogenic. As some petroleum products may contain traces of polycyclic aromatic hydrocarbons (not necessarily carcinogenic), methods for the isolation and estimation of those hydrocarbons added to such largely aliphatic material were studied. As representative of the more difficult type of substance which might be encountered, two petroleum waxes were chosen; it has been difficult to determine trace components in waxy materials, because of bheir low solubility. Five polycyclic aromatic hydrocarbons-dibenz [qhlanthracene, benzo [a]pyrene, benz [a]anthracene, chrysene, and anthracene-were added to wax a t three concentrations-1, 0.1, and 0,Ol p.p.m.-both separately and as a mixture, and their separation and quantitative recovery were attempted. Adsorption chromatography seemed
the best means of separating the polynuclear hydrocarbons from the nonaromatic material. The most suitable adsorbent for such a separation was magnesium oxide (calcined magnesite), mixed with Celite to permit easier filtration ( 9 ) . Magnesium oxide is an extremely powerful adsorbent; benzene is unable to elute tetracyclic or pentacyclic aromatic hydrocarbons from it. This is important because large volumes of benzene had to be washed through the chromatographic columns to remove the wax. The very high adsorption affinity of magnesia for the polynuclear hydrocarbons prevented their quantitative removal from the adsorbent by simple elution with polar solvents (acetone and ethyl alcohol), Thus, from 20 grams of magnesia on which were adsorbed 100 y of dibenz[a,h]anthracene, only 10 to
