Use of an Ionization Chamber for Measuring Radioactivity in Gas Chromatography Eff Iuents JAMES WINKELMAN and ARTHUR KARMEN National Heart Institute, National institutes of Health, Bethesda 7 4, hid.
b Highly sensitive measurement of radioactivity in the effluent of a gas chromatography column has been performed using an ionization chamber. The organic materials leaving the chromatography column were first subjected to combustion over heated copper oxide. The carbon dioxide produced was then conducted to an ionization chamber for carbon-1 4 assay. The water produced was either trapped or was reacted with heated iron to release hydrogen gas for tritium assay. The ionization chamber used for the measurement was 275 cc. in volume, permitting highly efficient assay. An additional gas flow was supplied to the chamber to reduce its response time. The sensitivity of the method was such that 202 micromicrocuries of carbon-14 or 227 micromicrocuries of tritium in a compound emerging 8 minutes after injection could be distinguished from baseline fluctuations. The sensitivity of the ion chamber to unlabeled compounds, which limited its applicability to measurements at this order of Sensitivity, was eliminated by including the water trap in the system.
Two
KINDS of method8 have been used for measuring radioactivity in effluents of gas chromatography columns. I n the first of these, components are condensed out of the gas stream for subsequent radioassay; in the second, the radioassay is performed during the course of the analysis. In general, the first method is adaptable to measurement of smaller quantities of radioactivity while the second generally offers higher resolution. The techniques for condensing the radioactive components out of the gas stream for subsequent radioassay include two that have been made automatic. By using an automatic fraction collector, high boiling compounds have been condensed by passing the effluent gas through a succession of cartridges containing anthracene crystals coated vith silicone oil. The radioactivity condensed in each cartridge is then measured by scintillation counting (12). Alternatively, the components have been condensed by bubbling the column effluent continuously through liq-
uid scintillator solution. Aliquots of the solution are taken automatically for scintillation counting (6). Scintillation counting has also been employed for monitoring the radioactivity in the column effluent as it leaves the column. This has been accomplished by the device described by Popjak and coworkers (16),in which the entire hot column effluent is injected into and bubbled continuously through a solution of liquid scintillator. The scintillation rate in this solution is continuously monitored. The scintillation rate of a single cartridge filled with coated anthracene into which the effluent flows, and in which the high boiling components condensed, has also been monitored. The result of both approaches is a cumulative record of eluted radioactivity, in which the emergence of a radioactive compound from the column is seen as an increment in the counting rate of the scintillator. Techniques for measurement of radioactivity in the gas phase have also been applied to gas chromatographic effluents. Proportional counters have been constructed suitable for operation a t high temperatures (7), and also have been operated a t room temperature on column effluents previously subjected to combustion to carbon dioxide (10). High temperature ionization chambers have been constructed for use with gas chromatographs (14), and ionization chambers have been used a t room temperature following combustion of the sample. A record of eluted radioactivity that resembles the output of the conventional gas chromatography “mass” detector may be obtained from these detectors. Each of the detectors employing measurement of radioactivity in the gas phase has high efficiency and low noise. Because high sensitivity is required in many biochemical problems, the use of an ionization chamber was studied to determine which factors limited the sensitivity, and how these limitations might be overcome. I n preliminary work with several different kinds of ionization chambers, it became apparent that when these chambers were used in flowing gas systems, a response of the ionization chamber could be obtained by injecting unlabeled material into the gas stream.
The response obtained varied with the kind of material injectdd as well as its quantity. The response could be obtained without polarizing the chamber, and indeed, often indicated current flow in a direction against the applied potential gradient. It was hypothesized that these effects resulted either from changes in the dielectric constant of the gas in the chamber or from poorly understood reactions of the vapors a t the surfaces of the chambers. Whatever the explanation, it was evident that the occurrence of this effect limited the sensitivity. To avoid this limitation, a method suggested by James and Piper (IO) and Cacace and Inam-ul-Haq (2) was investigated. The column effluent was subjected to combustion so that the gas entering the ionization chamber was composed only of carbon dioxide (or hydrogen) and carrier gas. Secondly, a large chamber was used and a brisk dilution flow of carrier gas was added directly to the chamber to keep the composition of the gas in the chamber effectively constant. In the experiments reported here, the sensitivity of an ionization chamber used in this way and the factors influencing the sensitivity were defined by the response of the system to mixtures of radioactivc compounds in which the radioactivity was determined by liquid scintillation counting. EXPERIMENTAL
Apparatus and Reagents. The gas chromatographic system utilized a Barber Coleman I D S Model 20 column oven, modified to accept a 6 foot long coiled glass column similar to that described by Haahti (9). These columns were .packed with ethylene glycol adipate polyester 2075, coated on Chromosorb W 80% (Johns-Manville and Co., New York). The polyester was treated by the method of Corse and Teranishi (4), to remove esterification catalysts. The combustion unit used was a Von Czoernig-Alber Electric Furnace No. 5679-A (Fisher Scientific Corp., Pittsburgh, Pa.). The combustion tube was an 8 inch long quartz tube, inch i.d. filled with cupric oxide (wire form) and stoppered on both ends, with flow through type silicone rubber stoppers. For detection of tritium VOL. 34, NO. 9, AUGUST 1962
1067
,.,Coiled
Column
The anthracene and the DPO-T : hyamine were assayed in the Packard Tri-Carb (R) Liquid Scintillation Spectrometer. The use of a mixture of 1 cc. hydroxide of hyanline 10 - X [p-(diisobutylcresoxyethoxyethyl) dimethyl benzylammoniuni hydroxide] Rohm & Haas 5%, ethanol, 95%, and 10 cc. of DPO in toluene (500 mg. per 100 ml.) has been reported by Frederickson and Ono to be effective in trapping carbon-14 labeled carbon dioxide from gases for scintillation counting (7). The counting efficiency of this system a t the voltage setting used was SOYo, which was the same as that of the anthracene cartridge a t this setting. A 1 inch long cartridge filled with anthracene coated with Dow Corning 550 silicone oil retains the vapors of methyl laurate in the face of a stream of flowing, heated gas for a t least 1 hour (12). The division of radioactivity between the anthracene cartridge and the DPOtoluene-hyamine following injection of methyl esters of lauric, myristic, and stearic acids is shown in Table I. Sirice material passing through the combustion chamber was not retained in the anthracene cartridge, but was trapped in DPO-toluene with hyamine added, fragmentation of the material to more volatile products, if not cornbution to carbon dioxide, ww assumed to be complete. Characteristics of Ionization Chamber. Background current was 3.7 X 10-18 amp., with argon flowing. This was recorded as 0.37 mv. across 1Ol2 ohms input resistance. This background current may be compared with amp. reported by the values 3 x Guinn and Wagner (8) using similar equipment. The electrical time constant was estimated as 8.3 seconds vhen 10'2 ohms input resistance was used. Peak-to-peak variation in the bsckground current over a 5-minute period, measured with this time constant was 1.2 X amp. Voltage. A polarizing potential of 90 volts was on the ionization chamber
Argon
7.1 Streom
Flow Adjust
-
Combustion
.-
t
t
Figure 1. Arrangement of gas chromatography system components-gas flow indicated with solid lines and electrical connection indicated with dashed lines
labeled materials, the second half of the combustion tube was filled with iron filings, which effected the conversion of water to hydrogen gas. The "mass" detector used was an ionization detector cell constructed as described previously (11) to which a tritium source (100 mc.) had been added (13). The potential across this cell was obtained from a Keithley model 2004A voltage supply. The current signal was amplified using a Leeds & Northrup microvolt d.c. indicating amplifier, modified to measure current by placing a 33 kilohm resistor across its input. Zero suppression was obtained using dry cells in a bucking circuit as described by Farquhar et al. (6). The output of this amplifier was recorded using a Leeds & Northrup Model G Speedomax recorder, or alternatively, one channel of a dual channel Texas Instrument Co. Servo potentiometric recorder. Radioactivity was measured using a Cary Ionization Chamber Model 31-31v, which has a volume of 275 cc., and a Cary Vibrating Reed Electrometer Model 31. Polarizing potential was obtained using a 90volt battery. Scintillation counting of samples was performed using a Packard-Tri Carb Model 314 Automatic Liquid Scintillator Spectrometer. Labeled fatty acids were obtained from New England Nuclear Corp., Washington, D. C. Methyl esters were prepared by the method of Stoffel, Chu, and Ahrens (16). Procedure. Components were arranged as shown in Figure 1. The gas flow through the column was between 40 to 60 cc. per minute. A supplementary argon flow of 200 cc. per minute was introduced into the gas stream a t the end of the column. This combined flow was divided in a 7 to 1 ratio using a T connection made of unequal lengths of stainless steel capillary tubing. The smaller portion was led to the argon ionization detector for mass detection. The larger portion wm led to the end of the quartz combustion tube through a '/I6 inch 1068
ANALYTICAL CHEMISTRY
diameter pipe, electrically heated so that the temperature of the end of the pipe and the beginning of the tube was not less than 200" C. Additional gas was introduced into the stream through a glass T-tube at the inlet of the ionization chamber. RESULTS
Combustion. To determine the adequacy of the combustion train, the samples of methyl esters of long chain fatty acids were introduced directly into a heated empty tube in line with the combustion tube. This was to test the ability of the tube t o combust the sample. If combustion were complete using this system, it was assumed to be complete for a sample of the same size delivered over the longer time interval that occurred in a chromatographic analysis. The effluent from the combustion tube was passed first through a 2 inch long, 8 mm. i.d. glass tube filled with anthracene crystals coated with silicone oil. The effluent of this cartridge was then bubbled through 25 cc. of diphenyloxazole (DPO), toluene (T) : hyamine (1O:l).
Table 1.
Combustion of
C14
Fatty Acid Methyl Esters"
Methyl Laurate
,Methyl Myristate
Methyl Stearate
Radioactivity in'ected, d.p.m. 34,600 39,700 47,950 Counts in coated anthracene, d.p.m. 470 960 230 Counts DPO-T: Hyamine, d.p.m. 34,300 38,950 47,880 Conditions of experiment. Temperature of injection cartridge, 245' C. Tem erature pyrolyzer, 725-800' C. Gaa 8ow rate, 30 cc. argon per minute Time of collection, 30 minutes. Under these conditions, methyl laurate C14 placed directly on the coated anthracene waa not eluted, Activity of samples waa obtained in DPO-to1uene:Hyamine (10: 11, with 63% counting efficiency. 4
plateau of this chamber. Increase in voltage to 180 volts increased neither the background current nor the response to labeled compounds. Effect of Gas Composition. Changing the purge gas had an effect on the response of the chamber. The total charge produced by injected samples was measured by the rate of charge method. The resistance on the ionization chamber was open (a). Full scale on the vibrating reed electrometer was 10 volts. The recorder indicated the integrated charge from the entire sample. The proportionality of total voltage to sample size was independent of variations in the rate of purge gas flow. l h e ion current n as greztest v ith argon anti smallest with helium aniong the gases tried (Table I t ) . l'he results ncre reproducj ble and thc chaige car] ird R as proportional to radioactivitv nith each of the gases tcsterl. Performance. Since more current flows through the chamber when a compound bearing radioactivity stays in the chamber longer, the lower the flow rate of the "purging" gas, the greater the peak heights and peak areas. The resolution decreases directly with decreases in flow rate. The purge gas flow rate of 1 liter per minute, yielding a chamber time constant of 23 seconds, represented a compromise between sensitivity and resolution. The sensitivity of the chamber, in terms of current yield per radioactive disintegration in the chamber. was coulomb for estimated as 1.25 x carbon1' disintegrations and 2.2 X 10-17 coulomb for tritium disintegrations. This compares with values of 1.64 x coulomb per carbon14 disintegration found by Guinn and Wagner (8) using a 500-ml. chamber and Cor, and 1.0 X 10-16 coulomb per CI4 disintegration calculated from data obtained by Borkowski using a 300-cc. chamber and argon ( I ) . The results of injection of different amounts of C14 labeled methyl myristate are in Table 111. The peak produced by 445 d.p.m. gave a response 5.1 x 10-16 amp. high, which may be compared with a peak-to-peak baseline fluctuation of 1.6 X 10-'6 amp., with approximately the same periodicity. The areas of the peaks were more linearly related to the quantity of radioactivity injected than the peak height listed in Table 11. This proportionality is shown in Figure 2. The peak areas produced by six coilsecutive injections of 120,000 d.p.m. of CI4 methyl myristate had a standard deviation of 4.1%. The expected standard error for samples of that size due to fluctuations in decay rate alone is less than 0.3%. Samples of only 5 0 d.p.m. counted for 1 minute nil1 have a minimum standard error of nearly 5%.
Table II. 9 t
Effect of Purge Gas on Ion Current
Total Charge, Relative to Argon
Gas Areon C6:
1 .o(;
0.68
K2 Air
0.53 0.4i 0.15
He
't
o;*'o
,I,
5bo
DPM
IAO
CI4
IAO
,bo
INJECTED
,JC
Conditions. C" Methyl niyristnte, 103,500 d.p.m. was int,rodured into the chromatographic system varying only the ionization chamber purge gas.
Figure 2. Relation of d.p.m. C14 to peak area
Table 111.
The deviations from absolute proportionality with small samples were within the limits established by random statistical deviation, plus an additional 5% (see Figure 2). The sensitivity of the system for the detection of tritium labeled fatty acid methyl esters was also determined. The area of the peak produced by tritium labeled palmitate was linearly proportional to the radioactivity. As was the case with CI4, the peak area was proportional to the quantity of tritium injected (Table IV). The record in Figure 3 shows the chromatography of ai1 artificial mixture consisting of methyl esters of soap and a mixture of C14 labeled methyl esters of decanoic, dodecanoic, tetradecanoic, hexadecanoic, and octadecanoic acids. Simultaneous recordings of the mass and radioactivity on a dual channel recorder are shown in this figure. Approximately 17,500 d.p.m. total, or 3500 d.p.m. per fatty acid methyl ester coniponent gave these results. Adjacent peaks are resolved with a moderate amount of tailing a t the flow rates employed. The area of the peaks would have been predicted on the basis of the data from which Figure 2 was constructed. A lag of 16 seconds is apparent between the mass and the radioactivity peaks. The same lag is present for all
I
Current Yield of Ionization Chamber for CL4
Methvl Myristace Injected, D.P.M..
Height of Peak Produced, Amperes
34 ,800 25,000 6,250 890 445
8 X lo-" 4 x 10-14 1 . 2 x 10-1' 1 . 2 x 10-15 5 . 1 x 10-15
C14
a Activity of samples in c.p.m. was determined in DPO-toluene; counted with SO% efficiency.
Table IV. Detection of Tritium by Ionization Chamber
Ha Methyl
Palmitate" Injected, D.P.M.
Peak Area, 3q. Cm.
Per Disintegration
27,000 54,000 108,000
4.38 8.70 17.50
2.23 X 10-17 2.18 X 10-17 2.20 x 10-17
a Activity of samples in c.p.11~.waa determined in DPO :toluene counted with 20.3% efficiency.
the peaks an represents the transit time of the gas\through the volume of the combustion unit and its connections. At lower flow rates through t,his portion
7
-~
1
-, _ _- -_
I
-
-
-- :i , +--' +
~~
-'+ 'i"4 -T , -
-
L-
- ,
'7
-1
I-
-
1
-
-x +-ir -:---
-
-L-- 4- TI-+
-
I
-8 -
- - - - -
-
--
____ i-
-
n
n-
- +
j=T:z z - - . - - - r'---- - - . r - --- :--
--
Coulomb
C?
Q.8
-
- -
- - -
L4
d 4 1
-..-
4-1 I
._ n
- *- n
- - Q
10
Figure 3. Simultaneous mass and radioactivity record obtained from mixture of CI4 labeled methyl esters and methyl esters of soap A small "solvent peak" and "alpha peaks" of characteristic shape can b e seen in record of radioactivity VOL. 34,
NO. 9,
AUGUST 1962
1069
effluent in DPO-T :hyamine a t different points in the system. The results are shown in Table VI. The radioactivity eluted from the entire system, 250 d.p.ni., accounted for only 18% of the peak area produced by the solvent injection in which i t was found. The major contribution of the solvent peak is the effect of mass on the ionization chamber. Water, iso-octane, or other solvents delivered directly to the ionization chamber produce a rapid decrease followed by a longer increase in ion current. The response of the ionization chamber to solvent which has passed through the combustion unit appeared only as an increase in ion current. This effect can be completely eliminated by removing the water produced by the combustion of solvent by interposing magnesium perchlorate cartridge before the ionization chamber. “Alpha peaks” are also apparent in Figure 3. These have been attributed to contaminants in the chamber material and nonaged gases (4). Individual alpha particles give rise to bursts of current which can be distinguished by their characteristic shape from the response to the C’402 delivered from the combustion tube.
Table V. Response of Ionization Chamber of Unlabeled Solvent
Solvent, ~ 1 . Peak Area, Equivalent in Ieo-octane Sq. Cm. D.P.M. CY4 10 5
2 1 0.5 10 10
13.0 7.1 4.1 3.1 2.3 8.8 9.6
1940 1062 G20 463 350 ...
1355 1440
of the system, the lag becomes longer. At higher flow rates only slight shortening of the lag was achieved, but the decreased sensitivity a t these flows worsened the record. Effect of Unlabeled Compound. The ionization chamber responded t o the solvent peak or other concentrated sample peaks if no magnesium perchlorate trap was placed between the combustion tube and the ionization chamber during C14 analyses. This effect wm negligible during tritium analyses when iron filings were in the second half of the combustion tube. The response of the ionization chamber ~ a not a directly proportional to the volume of unlabeled solvent, and it was found that the response to a given amount of solvent depended upon the immediate prior experience of the system. Table V shows the equivalent of disintegration per minute of C14 of the peaks produced by serial injection of unlabeled solvent into the chromatographic column. A small portion of this solvent peak was due to radioactivity eluted from the system. A source of this radioactivity in one instance was localized to the connecting tubing between the column and the combustion unit by trapping of
Table VI.
DISCUSSION
The kinds of organic substances analyzed by gas chromatography encompass both high boiling and low boiling materials, polar materials that are adsorbed strongly on metal surfaces and materials that are relatively inert, materials present in high specific radioactivity and materials present in low specific radioactivity. The problems of designing an ionization chamber that will perinit radioassay of each of these kinds of materials are largely avoided by
Contribution of Radioactivity to Solvent Peak 5 Minute Collection Collection of Effluent after of Effluent I Injection before H amine Hyamine of 10 pl. Solvent *IO pl. dthing Added, Iso-octane, Injection, Iso-octane, D.P.M. D.P.M. D.P.M. D.P.M.
5 Minute
Conditions 1. Total system. Has had CI4sam-
ples injected previously. Effluent collected beyond ion chamber 2. Column only. Effluent collected from end of column 3. Column and connectors only. Effluent collected before combustion chamber 4. Combustion chamber and ion chamber only. Solvent delivered directly to combustion chamber through heated T-tube. Effluent collected beyond ion chamber
1070
ANALYTICAL CHEMISTRY
121
126
128
380
135
131
127
309
108
112
dombwting the effluent gas to carbon dioxide, and/or hydrogen prior to delivering it to the ionization chamber. All problems involved with constructing insulators capable of maintaining stable, high resistance values a t temperatures in excess of 200’ C. are avoided, and the same chamber may be used, for example, in analyses of cholesterol as well as acetic acid. We were particularly interested in developing a procedure for fatty acid methyl esters, and so the majority of the tests were performed using these materials. Combustion of many materials has been studied in connection with the performance of elemental analyses. It is probable that compounds requiring specific procedures for quantitative combustion for elemental analyses will require them here BS well. The use of a relatively larger ionization chamber purged by a brisk dilution flow possesses the further advantage that the composition of the gas in the chamber is changed relatively little by the entrance of a peak of unlabeled carbon dioxide into the chamber. Since a larger concentration of carbon dioxide decreases the ionization current only slightly from the value obtained in argon, the effect obtained after dilution with argon is not detectable. Combusting the sample to carbon dioxide also eliminated another source of difficulty in the operation of an ionization chamber. In other work, we noted that injection of a variety of unlabeled organic materials into the gas stream flowing directly into an ionization chamber caused current to flow through the chamber. Because the signals produced by injecting a single eompound were sometimes diphasic, apparently indicating flow of current against the potential gradient of the polarizing potential, as well as with it, these effects may have been attributable a t least in part to changes in the dielectric constant of the gas. They were, however, not always diphasic, so that this explanation may be inadequate. Combusting the effluent of the chromatograph, however, almost entirely eliminated this effect. It was noted only when the solvent peak, usually 5 mg. of material, passed into the chamber. It w&srecorded only on the most sensitive ranges and was eliminated entirely by including a magnesium perchlorate trap for water in the line. Argon was added directly to the column effluent to reduce the time lag introduced by the volumes of the connecting tubing and the combustion tube while allowing the column to be operated at near optimal conditions. While it is possible to increase the flow of gas and thus do away with the necessity for adding gas directly to the ionization chamber, i t was decided not to do so in order to preserve the control over
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
