Apparatus for Ozonolysis of Microgram to Milligram Amounts of Compound Morton Beroza and Barbara A. Bierl, Entomology Research Division, Agricultural Research Service, U. S. Department of Agriculture, Beltsville, Md. 20705
OZONE generators, which may be assembled quickly and easily from readily available inexpensive materials and a high-frequency Tesla coil type vacuum tester (e.g., Fisher Scientific Co. KO, 1-179), have been used to determine by ozonolysis the positions of ethylenic bonds in microgram or milligram amounts of compound (e.g., those obtained by gas or thin-layer chromatography) * TWO
EXPERIMENTAL
Syringe Ozonizer. No originality is claimed for the apparatus of Figure 1, which will be referred to as the
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Figure 1. Cross-sectional view syringe ozonizer
of
A, needle stock; 6, injection-port septum; C, lo-ml. hypodermic syringe; D, aluminum foil; E, glass-cloth tape; F, ground; G, copper wire (extends down length of borrel wiihin aluminum foil); H, chain or cord
1976
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ANALYTICAL CHEMISTRY
syringe ozonizer. I t is very similar to that described by Hoff and Feit @), but our method of using the apparatus is a refinement of their technique, and it has some significant advantages. The syringe ozonizer mas made as follows: A 20-gauge copper wire ( G ) was laid back and forth once between layers of a sheet of aluminum foil (D,31/4 X 6 X 0,001 inch) that was wrapped several times snugly around the barrel of a 10-ml. hypodermic syringe (C); the foil, which serves as the outer electrode, was taped on the barrel with two layers of glass-cloth electrical tape ( E ) . A 5-inch length of 21-gauge stainless-steel needle stock (A) (Vita Needle Co., Needham, Mass.), sharpened to a point a t its lower end, served as the inner electrode. It was held on the Luer end of the syringe (C) with a half-pierced injection-port septum ( B ) (0.239 inch 0.d. X 0.305 inch) so that the needle-stock tubing could be slid in and out of the syringe easily. (The syringe could be stoppered by pressing the sharpened tip into a silicone rubber septum.) Wire (G) was grounded ( F ) during ozone generation by attaching its distal end t o a mater tap with an alligator clip. A chain or cord ( H ) prevented the barrel from being withdrawn beyond the 10-nil. mark. The syringe ozonizer was evacuated and filled with pure oxygen 3 times and then stoppered. (We found it convenient to add and withdraw oxygen through an injection-port septum held in one leg of a tee tube, which had a source of vacuum and oxygen (5 p.s.i.g.) connected to the other two legs.) The inner end of the needle stock tubing was set on or near the 5-ml. mark of the syringe and the electrode of the vacuum tester with current flowing was held on the needle stock next to the septum for 30 seconds (periods up to 4 minutes produced little or no additional ozone). At this point the syringe contained about 0.01 meq. of ozone, and its contents were ready for injection into the solution being analyzed. Flow-Through Ozonizer. The apparatus shown in Figure 2 can generate ozone continuously and will be referred to as the flow-through ozonizer. It was constructed as follows: A 20-gauge copper wire ( F ) was laid back and forth once between layers of a sheet of aluminum foil (D,31/4 X 6 X 0.001 inch) that was wrapped several times snugly around a 4-inch length of 8 mm.-o.d., 6 mm.-id., borosilicate glass tubing (C) within inch of the ends; the foil was held on the tubing with a 3I/z-inch length of rubber tubing ( E , 6/8 inch o.d., 3//s inch i.d.). A 51/2-in~hlength of 21-gauge stainless-steel needle stock tubing was pushed through an injec-
tion-port septum (0.239 inch 0.d. X 0.305 inch) that was fitted into one end of the glass tube. Oxygen or nitrogen was supplied by means of a 3-way Teflon stopcock ( I ) to the other end of the glass tube, as necessary, through inch 0.d. gum rubber tubing ( H , and inch i.d.). The wire ( F ) was grounded (G) during ozone generation. Oxygen was led into the flow-through ozonizer with the inner end of the needle stock tube (A) just above septum ( B ) . When the system had been purged of air, the inner end of the needle stock tube was set about halfway into the glass tube. The needle stock tube was then lowered into the solution to be analyzed (kept cold) with the oxygen flowing, and ozone was gen-
Figure 2. Cross-sectional view flow-through ozonizer
of
A, needle stock tubing; 8, injection-port septum; C, glass tubing; D, aluminum foil; E, rubber tubing; F, copper wire (extends down length of barrel); G, ground; H, rubber tubing; I, three-way Teflon stopcock
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? NINUTES Figure 4. Gas chromatogram of ozonolysis products from 1 pg. of methyl oleate in 20 p l . of methylene chloride Figure 3. Milliequivalent of ozone generated in flow-through ozonizer vs. oxygen flow rate with 1-minute contact time of Tesla coil
erated by touching the needle stock tubing with the energized vacuum tester for an appropriate time interval. At a flow rate of 10 ml./min. ozone was generated a t the rate of 0.01 meq. per minute. The amount of ozone generated was determined as described by Guenther et al. (1). A plot of oxygen flow rate us. ozone generated is shown in Figure 3. The flow rate was adjusted with a soap-bubble flow meter or a rotameter. Methodology. The method of analyzing for ethylenic-bond position followed generally that of Stein and Nicolaides (3) exceDt that we used about one thousandth the quantities of sample they used and we employed flame ionization detection. The compound, in a solvent such as methylene chloride, was held a t -60" C. during ozone introduction. Because excess ozone causes sidereaction products to appear, the solution was placed in a test tube stoppered with a septum pierced by the ozonizer needle (gas inlet) and by a Teflon tube (gas outlet) through which the gas emerging from the solution was led into 4 ml. of 5% potassium iodide and starch in 5% aqueous sulfuric acid contained in a 10-ml. Erlenmeyer flask. Ozonization was terminated as soon as the starch-potassium iodide-sulfuric acid solution indicated that ozone was coming through (blue color formed). Nitrogen was then bubbled through the solution of the ozonized compound to remove oxygen and ozone (with the flow-through ozonizer, the Teflon stopcock need only be turned to switch to nitrogen), a small crystal of triphenyl phosphine was added to reduce the ozonides to carbonyl compounds, and
A: CH3(CH&CHO; 6: CHsOOC(CH2);CHO. Chromatographic conditions: 12-ft., '/r-inch 0.d. copper column containing 5% Carbowax 20M on 60-80 mesh Gas Chrom P, flow rate 30 ml./minute, column temperature held at 50' C. for 6 minutes then programmed upward at 4.6' C./minute to 2 0 0 ' C.
an aliquot of the solution was injected into the gas chromatograph for analysis. A blank run was made with solvent alone in order to recognize peaks due to solvent interference. RESULTS AND DISCUSSION
With the apparatus and methodology described, ozonolysis of milligram or microgram amounts of methyl oleate, linoleate, and linolenate was readily accomplished, and the expected products were obtained. See Figure 4. Side-reaction products are greatly diminished by ozone addition at low temperature, and their formation is further diminished by avoiding the addition of excess ozone. The avoidance of side-reaction products made it possible to analyze for double-bond position with as little as a few niicrograms of the C18 unsaturated methyl esters. I n the niethodology of Hoff and Feit, ozonolysis of a compound is carried out within the syringe at room temperature and side-reaction products prevent the analysis of compounds a t such low levels. Their analysis also requires that compounds being analyzed have a certain degree of volatility (heptenes were the heaviest compound on which they reported ozonolysis), whereas the present procedure is applicable to any compound giving fragments that can be gas chroniatographed or otherwise determined. Hoff and Feit used sodium arsenite to remove ozone and reduce the ozonides to carbonyl compounds.
The flow-through ozonizer appears to be much superior to the syringe ozonizer. It is useful for the analyeis of niilligram or microgram amounts of compound simply by employing longer or shorter periods of ozone generation, while the syringe ozonizer has a limited capacity. The flow-through ozonizer is more convenient to use in that purging of the solution with nitrogen may be accomplished without removal of the apparatus from the solution of ozonide. In designing the ozonizers the following parameters were varied: the oxygen flow rate and the diameter and length of the glass tube in the flow-through ozonizer; the length of the aluminum foil and the diameter and position of the needle stock tubing, the time of contacting the needle stock tubing of the syringe ozonizer with the vacuum tester, and the oxygen inlet pressure with both ozonizers. Only the designs and conditions giving maximum or near maximum generation of ozone with ease of manipulation are described. LITERATURE CITED
(1) Guenther, K. F., Sosnovsky, G., Brunier, R., ANAL. CHEW 36, 2508
(1964).
(2) Hoff, J. E., Feit, E. D Ibid., p. 1002. (3) Stein, R. A., Nicoddes, Nicholas, J . Lipid Res. 3, 476 (1962).
PRESENTED at the 152nd National ACS Meeting, New York, N. Y . Sept. 14, 1966. Mention of proprietary products is for identification only and does not constitute endorsement by the U. S. Department of Agriculture.
VOL. 38, NO. 13, DECEMBER 1966
1977
