Analysis of Terpene Hydrocarbons by Thin Layer Chromatography SIR:Thin layer chromatography (TLC) is a very sensitive technique for identifying volatile organic compounds separated from natural products by gas chromatography ( I , 2). Using specific color producing sprays we have been able to detect quantities as small as 1 nanogram and to make some conclusions regarding structure (1-3). TLC is also a powerful separatory tool, and compounds have been assigned to particular chemical classes on the basis of their R, values using solvent systems of varying polarity (I, 2 ) . Consequently, it has frequently been found to be more useful than gas chromatography on a second column phase for the secondary separation and identification of particular fractions. The analysis of terpene hydrocarbons using TLC required the use of a nonpolar solvent. The choice of a volatile hydrocarbon solvent was obvious. However, perfluorinated hydrocarbons, compounds in which all hydrogens have been replaced by fluorine, also have nonpolar characteristics (4-7'). The evaluation of these materials for this application will be discussed in this report and compared with results obtained using a hydrocarbon solvent. EXPERIMENTAL
The hydrocarbon solvent used was Skellysolve B (hexanes), a product of the Skelly Oil Co., Kansas City, Mo. It was allowed to ascend 10 cm. during development. Two fluorocarbon solvents, low boiling perfluorokerosine (PFK), a product of the Columbia Organic Chemicals Co., Columbia, S. C., and perfluoroalkane boiling 7080' C. (PFA), furnished by Peninsular ChemResearch of Gainesville, Fla., were used. The fluorocarbon solvents were allowed to ascend 5 and 7.5 cm., respectively. Chromatoplates used with the hydrocarbon solvent were 8 X 8 inches and were prepared in the standard manner from Silica Gel G and Aluminum Oxide G. Chromatoplates for use with the fluorocarbon solvents were 2 X 8 inches and had 20- to 40-micron layers of adsorbent. They were developed in a cylindrical chamber designed for this purpose by the Research Specialties Co., Richmond, Calif. Detection sprays were either vanillin/ HzS04 or KMn04/H2S04as described in previous publications (1-3). RESULTS AND DISCUSSION
R, values, Tables I and 11, and color reactions with detection sprays, Table 111, were useful for the identification of terpenes. The fluorocarbon solvents were extremely specific for the analysis
Table 1.
R/ Values of
Sesquiterpenes, Azulenes, and Polyterpenes Using Fluorocarbon Solvents
Silica Compound Alumina, PFAa Alumina, P F K b gel, PFA 7-Bisabolene 0.50 0.33 0.10 0.71 0.35 0.20 SCadinene @-Caryophyllene 0.65 0.44 0.30 0.44 a-Cedrene 0.73 0.56 0.49, 0.58 0.18, 0.28 Guaiene 0.34, 0.44 0.12 a-Humulene 0.30 0.35 0.20 @-Santalene 0.64 0.48 @-Selinene 0.41 0.33 0.17 Valencene 0.51 0.32 0.21 0.87 0.64 0.40 Y langene 0.57 0.30 @-Zingaberene 0.18 0.19 Azulene 0 0.05 0.10 Guaiazulene 0.05 0 0.22 Pinene dimer 0.38 0.54 0 Squalene 0 0 0 @-Carotene 0 0 PFA = perfluoroalkane, boiling range 70-80" C. PFK = low boiling perfluorokerosene.
of sesquiterpene hydrocarbons. Except for myrcene, ocimene, and yterpinene, which occasionally seemed to diffuse slightly above the origin on development with the fluorocarbon solvents, all 19 monoterpene hydrocarbons tested had R, values of zero, while all 11 sesquiterpenes had significant Rs values. These values showed sufficient variation to be combined with color reaction data and used to identify individual compounds. A diterpene (pinene dimer), a triterpene (squalene,) a tetraterpene (@-carotene), and two azulenes (azulene, guaiazulene) were also studied, but only the pinene dimer left the origin. I n contrast to their behavior with the fluorocarbon solvents, the .monoterpenes and sesquiterpenes showed considerable overlap in their R/ values when hydrocarbon solvents were used, and no distinction could be made between the two classes. However, some individual mono- and sesquiterpenes gave significant differences in Rj value and the hydrocarbon solvent can be helpful in the identification of these. Linear terpenes and aromatic hydrocarbons gave the lowest R, values while the pinenes gave the highest R/ values. The pinene dimer had exactly the same Rs value as the pinene monomers. Azulene, guaiazulene, squalene, and @-carotene showed a dommvard progression in Rs value which served to differentiate them from other classes. The two adsorbents, silica gel and alumina, gave significantly different results. When using the hydrocarbon solvent, the adsorbent activity of alumina was too low to effect good separations of individual terpenes and, consequently, the use of silica gel was
Silica gel, PFK 0.08
0.12 0.16 0.36 0.14, 0.21 0.08
0.15 0.13 0.17 0.32 0.13 0.03 0
0.14
0 0
Table 11. Rt Values of Terpenes with Skellysolve B (Hexanes)
Silica gel
Compound
Alumina
MONOTERPENES
Camphene 6-3-Carene p-Cymene p-Isopropenyltoluene &Limonene 2,4(8)-Methadiene R-Mvrcene a-O;imene t-@-Ocimene @-Phellandrene a-Pinene &Pinene a-Pyronene @-Pyronene Sabinene Terpinolene a-Terpinene 7-Terpinene a-Thujene Verbenene
0.83 0.85
0.67 0.62 0.76 0.76 0.74 0.71 0.71 0 79 0.90 0.88 0.80 0.80
0.75 0.75 0.76 0.76
0.80
0.81
0.95 0.95 0.89 0.84 0.93 0.93 0.92 0.91 0.91 0.93 0.96 0.95 0.94 0.94 0.93 0.92
0.93
0.93 0.95 0.95
SESQUITERPENES
r-Bisabolene SCadinene @-Caryophyllene a-Cedrene Guaiene a-Humulene @-Santalene @-Selinene Valencene Y langene @-Zingaberene
0.71 0.72 0.74 0.89 0.76, 0.82 0.65 0.78 0.78 0.76 0.75
0.93 0.93 0.94 0.96 0.94 0.90 0.94 0.94 0.93 0.96 0.94
0.48 0.46
0.80
0.88
AZULENES
Azulene Guaiazulene
0.77
DITERPENE
Pinene dimer
0.87
0.95
TRITERPENE
Squalene
0.48
0.85
TETRATERPENE
@-Carotene
0.16
VOL. 37, NO. 10, SEPTEMBER 1965
0.43
1289
Table 111.
Compound
Color Reactions with Vanillin/H&Od Spray . . Reagent Color after Color after Color after 1 minute 30 minutes 4 hours MONOTERPENES
Camphene 8-3-Carene p-Cymene &Limonene 2,4(8)-Menthadiene p-M yrcene a-Ocimene 2-P-Ocimene p-Phellandrene a-Pinene @-Pinene a-Pyronene p-Pyronene Sabinene Terpinolene a-Terpinene 7-Terpinene a-Thujene Verbenene p-Isopropenyltoluene
Light tan Russet None Greenish brown Greenish brown Purple Purple Purple Purple Greenish brown Greenish brown Dark pink Dark pink Greenish brown Greenish brown Greenish brown Greenish brown Dark blue Lavender Lavender
Light orange Green None Bluish green Bluish green Blue Blue Blue Blue Blue Blue Blue Blue Blue Bluish green Bluish green Bluish green Blue Blue Pink
Weak Blue None Blue Blue Yellowish green Yellowish green Yellowish green Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Blue Lavender
SESQUITERPEKES
7-Bisabolene 8-Cadinene p-Caryophyllene a-Cedrene Guaiene Humulene p-Santalene p-Selinene Valencene Ylangene p-Zingaberene
Purple Sky blue Red Red Purple-maroon Purple-maroon Bluish purple Dark green Pur le D a r t blue Bluish green
Azulene Guaiazulene
Red Pink
Pinene dimer
Purple
Squalene
Bark pink
,%Carotene
Dark green
When Carbowax 20M columlls are used for the over-all analysis of complex natural mixtures with wide boiling ranges, certain terpene pairs frequently are poorly resolved by one pass through the column. This is particularly true when one member of the pair is present in a much greater quantity than the other. Notable among these pairs are a-pinene (Ythujene, P-pinene sabinene, and myrcene 6-3-carene. When these mixtures were condensed from the gas chromatographic effluent and subjected to TLC they could be completely resolved, and this operation required less time than repassing each through the GC column.
+
+
+
ACKNOWLEDGMENT
Appreciation is expressed to the Glidden Co., Jacksonville, Fla., the Kava1 Stores Laboratory, Olustee, Fla., and the Fruit and Vegetable Products Laboratory, Winter Haven, Fla., for the donation of some samples of monoterpenes and sesquiterpenes. Appreciation is also expressed to Dr. George K. Butler, Department of Chemistry, University of Florida, for furnishing a sample of the pinene dimer, and to Peninsular ChemResearch, Inc. of Gainesville, Fla., for donating the perfluoroalkane solvent.
Brown Dark blue Purple Purple Dark blue Lavender Dark blue Dark green Maroon Lavender Dark green
Brownish green Purple Purple Purple Dark purple Brown Dark green Dark blue Dark grey Dark pink Green
Dark red Dark pink
Dark red Red
Purple
Blue
LITERATURE CITED
Tan
(1) Attaway, J. A,, Hendrick, D. V., Wolford, R. W., Proc. Florida State Ho_rt: _S_oc., Miami Beach, Fla., Nov.
Dark blue
(2) Attaway, J. A., Wolford, R. W.,
AZULENES
DITERPENE
TRITERPENE
Tan TETRATERPENE
a
3-0, lY04.
Proc. Fifth Intern. Symp. Gas Chromatography, Brighton, England, Sept.
mandatory. However, when using the fluorocarbon solvents, the high adsorbent activity of the silica gel prevented the sesquiterpenes from ascending a sufficient distance for separation, and alumina was preferred. The most satisfactory results for sesquiterpenes mere obtained using alumina plates and PFA. As it was more volatile than P K F , it ascended the plates faster and gave higher R, values. PFA traveled 7 . 5 cm. in 45 minutes while P F K required 2 hours to ascend this same distance. The colors produced by different terpenes with the detection sprays are shown in Table 111. The color produced immediately upon application of the vanillin/H2S04 spray had usually
1290
Dark green
ANALYTICAL CHEMISTRY
changed after 30 minutes a t room temperature, and frequently showed further change after a four-hour period had elapsed. These colors and. color changes were characteristic and reproducible. An earlier publication (3)showed that esters could be detected with the vanillin/H2S04 spray in quantities as low as 0.5 nanoliter per spot. The sesquiterpenes were found to be equally sensitive to the spray, but the limit of detection for some of the monoterpenes, particularly CY- and P-pinene, was considerably higher. Use with Gas Liquid Chromatography. This technique was useful as a n adjunct t o gas liquid chromatography because of the ease of separation of some key terpene pairs.
8-10. 1964. (3) Atlawax J. A,, Wolford, R. W., Edwards, G. J., ANAL.C n m . 37, 74 (1965). (4) Grosse, A. V., Cady, G. H., Znd. Eng. Chem. 39, 367 (1947). (5) Haszeldine, R. N., Smith, F., J . Chem. SOC.1951, 603. (6) Simons, J. H., Block, L. P., J . Am. Chem. SOC. 61, 2962 (1939). (7) Simons, J. H., Dunlap, R. D., J. Chem. Phys. 18, 335 (1950).
Jon3 A. ATTAWAY LEONARD J. BARABAS RICHARD W. WOLFORD Florida Citrus Commission Lake Alfred, Fla. COOPERATIVE research by the Florida Citrus Commission and the Florida Citrus Experiment Station. Presented to the Meeting-in-Miniature, Florida Section, ACS, Gainesville, Fla., May 13-14, 1965.
