sensitivity and resolution offered by the separate flow into the ionization chamber. Since the change in sensitivity of the ionization chamber where several inexpensive gases are used is slight, reducing the time constant of the chamber to quite low values is practical as well as feasible merely by increasing the purge gas flow. Reducing the electrical time constant of the measuring circuit is somewhat more difficult a t these low current ranges. However, faster responses than those reported here have been obtained through use of increased feedback with commercially available equipment (3). The number of disintegrations that occurs within the chamber as a result of the passage of a radioactive compound through it is a function of the time spent by the compound in the chamber; this time is determined solely by the ratio of chamber volume to flow rate. The sensitivity of the chamber and the resolution are therefore both controlled by the flow rate of "purging gas." Using the column conditions listed
above, a 275-cc. ionization chamber and a flow rate of 1000 cc. per minute, the carbon-14 radioactivity in methyl myristate emerging from a column 8 minutes after injection was measurable when the injected activity was only 445 cl.p.m. With this gas flow and chamber volume, the peak height of components emerging before this was not higher, while the height of peaks emerging later was diminished as a function of the ratio of their retention times to that of the myristate. These data were successfully used to predict the response for a given fatty acid methyl ester emerging from the column after a given length of time. LITERATURE CITED
(1) Borkowski, C. J., U. S. At. Energv Comm.Rept. MDDC-1009 (1947). (2) Cacace, F., Inam-ul-Haq, Science 131, 732 (1960). (3) Cary-Loenco Chromatography-Radioactivity Analysis System. Preliminary Data Sheet 203. Baird-Atomic, Inc., Bethesda, Md., 1961. (4) Come, J., Teranishi, R., J. Lipid Res. 1, 191-192 (1960).
(.5 .) Dutton. H. J.. Pittsburgh Conference
on Analytical Chemistry- and Applied Spectroscopy, Feb. 28, 1961. (6) Farquhar, J. W., Insull, W., Jr., Rosen, P., Stoffel, W., Ahrens, Jr.. E. A.; Nutrition Reviews 17, S o . 8 (Supplement) 1-30 (1959). (7) Fredrickson, D. S., Ono, K., J. Lab.
Clan. Med. 51, 147-151 (1958). (8) Guinn, V. P., Wagner, C. D., Sj-m-
posium on Ionization Chamber Measurements of Radioactivity and Radiation, San Francisco, Nov. 13, 1959. (9) Haahti, E., Academic Dissertation, Dept. of Med. Chem., Univ. of Turku, Finland, 1961. (10) James, A. T., Piper, E. .4.,J. Chio-
matog. 5,265-270 (1961). (11) Karmen, -4,, Giuffrida, L., Bowman, R. L., Natuie 191,906-907 (1961). (12) Karmen, A., Tritch, 'H. R., Ibid., 186, 150-151 (1960). (13) Lovelock, J. E., J . Chromalog. 1, 35-46 (1958). (14) Mason, L. H., Dulton, H. J., Bair, L. R., Ibid., 2,322-323 (1959). (15) Popjak, G., Lowe, A. E., Moore, D., Brown, L., Smith, F. A., J. Lipid Res. 1, 1,29-40 (1959). (16) Stoffel, W., Chu, F., Ahrene, E., Jr., ANAL.CHEM. 31, 307-308 (1959).
RECEIVED for review Februa,ry 15, 1962. Accepted June 13, 1962.
Determination of Thymol Isomers by Gas-Liquid Chromatography Using Lanolin PETER J. PORCARO and V. D. JOHNSTON Analytical laboratory, The Givaudan Corp., Delawanna, N.
-
b A gas liquid chromatographic method is presented for evaluating mixtures of thymol isomers. Thymol and vic-thymol are difficult to separate, but all four isomers are clearly separated using lanolin as substrate. Application is made to synthetically prepared thymol mixtures and to naturally' occurring thyme oil.
G
chromatography is presented as a means of evaluating thymol and its isomers. The classical methods of analysis cannot distinguish the isomers in mixtures without length\separations by distillation and crystallization (1). The thymols, excluding thymol itself which is well known and the subject of considerable study, AS-LIQUID
A. thymol
Iic
-thymol
J.
I
$OH sym -thymol
p -thymol
include vic-, sym-, and p-thymols as they are commonly known. Chemical Abstracts lists the above isomers as 3-p-cymenol, 3-o-cymenol, 5-m-cymenol, and 5-0-cymenol, considering them as cymene derivatives. Carpenter and Easter (1) have included melting point data for purified isomers as follows: thymol, 51.5' C.; vic-thymol, 70-71' C.; sym-thymol, 50-50.5' C.; p-thymol, 112-113° C. They mention the order of volatility to be vic > thymol > sym > para, but no boiling point data are available on all the isomers. The isomers elute in the above order when they are completely separated (Figure 1). Thymol has a wide variety of commercial uses. Its anthelmintic, anti-
bacterial, and antifungal properties are employed in many drug and cosmetic preparations, in preservatives for anatomical specimens and urine, in mildew, antimold, and herbarium parasite preparations (3). The percentage composition of thymol isomers in a mixt,ure is of practical importance as well as academic. EXPERIMENTAL
Apparatus. A Perkin-Elmer Model 154D Vapor Fractometer equipped
with 8000-ohm thermistors was used in this work. Column Preparation. Lanolin, deodorized U.S.P., 10% by weight, mas used on 60- to 80-mesh acid-washed Chromosorb - IF- (Johns - Manville). After slurrying with acetone, evaporating, drying, and sieving, 16 grams of the support was packed into 3 meters of l/Anch copper tubing. The isomers used were thymol (m.p. 51' C.), vic-thymol (m.p. 69.5" t o 70.5' C.), sym-thymol (m.p. 48" t o 50' C.), and p-thymol (m.p. 111' to VOL. 34, NO. 9, AUGUST 1962
1071
80 to 100 mesh, because of a more pronounced support interaction. In an effort to decrease tailing, the particle sizes were increased to 60 to 80 and two supports, Chromosorb-W and GasChrom P (Applied Science Labs.), were investigated. We found Gasvic-thymol thymol ''6
19.2
Figure 1 .
A
Four thymol isomers
112" C.) which were prepared in our own laboratories. The thymols were injected in acetone solution. Instrument Parameters. Column: lanolin lo%, 3 meters. Temp.: 162' C. Pressure, p.s.i.: 2 0 , 4 5 ml. per minute inlet flow. Sample size: 1 t o 2 pl. Recorder range: 0 to 2 niv. Attenuation: as marked. Bridge voltage: 8 volts. Chart speed: l/z inch per minute -4ir peak elution: 0.8 minute. Gas : helium. RESULTS AND DISCUSSION
Separating thymol from vic-thymol was difficult. The two eluted together
on a variety of polar and nonpolar materials tried (Table I). Our efforta were thus concentrated on the use of a suitable substrate, varying column preparation, and instrument parameters which would thus yield the best victhymol-thymol separation. During this investigation, lanolin was tried as a substrate. Lanolin and Apiezon AI gave indications of yielding the best separation. With Apiezon 11, its concentration was first varied in stages from 30y0to 5%. Although the resolution became more pronounced a t lower concentrations, the tendency to tail became greater with particle sizes of
Figure 2. Vic-thymol and thymol
Chroni P had a more pronounced tailing effect than Chromosorb-W in this application. Columns 4 meters long caused excessive pressure drop, hence long elutions. Three meters gave the best compromise yielding fair separations in less than a half hour but with noticeable tailing for the Apiezon M columns. The separation obtained on the four thymols using 25% lanolin as
thymol 19.2
A
I I
p-thyml
diisopropyl cresol
\ Figure 3.
1072
ANALYTICAL CHEMISTRY
sym-thymol
23.6
Mixture resulting from alkylation of m-cresol
m-cresol
Figure 4.
Commercial stabilizer mixture
Figure 5.
Thyme oil, N.F. grade
Table 1. Substrates Investigated Carbowax 20M (20,000) Union Carbide Chemicals Co. Tricresyl hosphate Monsanto Chemical Co. m-Tolyl pkosphate, pure Eatman Organic Chemicals Diethylene glycol succinate (Polyester A) F & M Scientific Co. Sucrose octaacetate Union Carbide Chemicals Go. Hyprose SP-80 Dow Chemical Co. Silicone rubber SE-30 General Electric (Silicone Products Dept.) DC Antifoam A Dow Cornin Corp. Asphaltum, quick drying Matheson, zoleman, & Bell Apiezon L, M, & W James G. Biddle Co. Lanolin Robinson- Wagner
substrate on 30- to 60-mesh acid-washed Chromosorb-W in a 2-meter length showed no tailing, but the vic-thymolthymol pair was only poorly resolved. On subsequently reducing the con-
centration to lo%, but on 60- to 80-mesh acid-washed Chromosorb-W in 3 meters column length, the separation occurs m shown in Figure 1. Satisfactory chromatograms free of
tailing were thus obtained with 10% lanolin on 60- to 80-mesh acid-washed Chromosorb-W in 3 meters column length. Figure 2 shows a separation on the wic-thymol-thymol pair at 162’ C. The other isomers presented no problem as their boiling points must have sufficient spread. On a known mixture containing approximately equal amounts by weight of each of the isomers (Figure l), the quantitative results obtained using the method of triangulation are within experimental error varying to less than 2% from known. By applying these findings to various mixtures of thymols encountered in industry, we may correlate our knowledge of the given product with the various isomeric ratios observed. The resulting mixture in an alkylation of mcresol is in Figure 3. Some thymol mixtures have been used as stabilizers in the fixed oil industry t o retard oxidation. A typical mixture is shown in Figure 4. Thymol occurs naturally in white thyme oil (2, 4). Figure 5 shows a white thyme oil (N.F. grade) with carvacrol, 2-methyl 5-isopropyl phenol, appearing as a peak (5). Lanolin columns are stable at 210’ C. in 25% concentrations. There is very little bleed-off at this temperature, but it is noticeable at 240’ C. on prolonged usage. Column stability is good. No preheating or conditioning was necessary. Columns heated daily to around 200” C. have lasted as long as four months. Lanolin behaves predominantly nonpolar with the wide variety of materials found in our perfume and flavor industry. Separations, in general, are made in the order of increasing boiling points. Perhaps it may also find application t o other problems in other industries. We may suggest the chlorinated phenols as a n example, as we have used lanolin with success in a limited investigation of isomeric mixtures. LITERATURE CITED
(1) Carpenter, M. S., Easter, W. M., J . Org. Chem. 20, 401 (1955). (2) Givaudan Corp., Delawanna, N. J.,
“Givaudan Index,” 2nd ed. 1961. (3) Merck, and Co., Inc., Rahway, N. J. “Merck Index,” 6th ed., 1952. (4) National Formulary, 11th Ed., 1960, ublished by American Pharm. Assn., baahington. (5) Naves, Y. R., France Parfums 8 , 23 (1955). RECEIVEDfor review March 12, 1962. Accepted May 28, 1962.
VOL. 34, NO. 9, AUGUST 1962
1073
