the components is indeed a pesticide. When artifacts do obscure a pesticide peak, it would be useful to be able to quantitate unknowns using the peak area of one particular degradation product. Although this cannot be done with the present technique, work in this area is in progress. Since photolytic derivatization, either as a solid film or in solution, is feasible with any material that is light sensitive or can be made to be by proper choice of wavelength, radiation
intensity, or photosensitizer, it is not restricted to pesticide residues. Other applications should readily be found. RECEIVEDfor review December 28, 1970. Resubmitted February 15, 1972. Accepted February 15, 1972. Mention of commercial products does not constitute an endorsement by the United States Department of Agriculture over others of a similar nature not mentioned.
Application of a Sequential Reduction Regimen to Fractionation of Essential Oils P. A. Hedin, A. C. Thompson, and R. C. Gueldner Entomology Research Division, Agriculture Research Service, USDA, State College, Miss. 39762 The application of reductions in sequence with sodium borohydride, lithium aluminum hydride, and platinum oxide to the fractionation of cotton bud essential oil is discussed. The use of saponification and alcohol dehydration with neutral alumina was also investigated. GLC and TLC were employed to monitor the reactions and fractionations. The methods were also evaluated with a synthetic mixture of terpenes.
IN THE COURSE of our investigations of the essential oil of the cotton plant (Gossypium hirsutum L. var. Deltapine Smooth Leaf) for constituents attractive to the boll weevil, Anthonomus grandis Boheman, we found mostly hydrocarbons and alcohols, but also several aldehydes and ketones ( I ) , at least one epoxide (2), and several esters and oxides. IR, NMR, and MS spectra normally provide sufficient information for the identification of the CB-C,Ocompounds. However, this is generally not possible with the sesquiterpenes because there is often a question about the functional group and its site in the compound. In this case, standard reductive procedures are among the most favored methods of gathering additional structural information because they are usually simple and do not usually initiate rearrangements. Thus, this approach to structure elucidation is widely practiced, and reaction gas chromatography (3) is a typical example of the application of catalytic reduction for this purpose. The present report describes several reductive procedures, used in a particular sequence, to fractionate cotton essential oil. Also, a simple synthetic mixture of monoterpenoids was investigated. EXPERIMENTAL
Reagents. The following reagents were employed : sodium borohydride (98 %), No. 8486, h!atheson, Coleman and Bell, Norwood, Ohio; lithium aluminum hydride (95+%), Metal Hydrides, Inc., Beverly, Mass. ; platinum oxide(85 %), Engelhard Industries, Newark, N.J. ; Woelm's neutral alumina, Alupharm Chemicals, New Orleans, La. The terpenes that comprised the standard mixture were procured as follows: d-a-pinene, K and K Laboratories, Plainview, N.Y. ; d-limonene epoxide from d-limonene, Matheson, (1) J. P. Minyard, J. H. Turnlinson, A. C. Thompson, and P. A. Hedin, J. Agr. Food Chem., 15, 517 (1967). (2) J. P. Minyard, D. D.Hardee,R. C. Gueldner, A. C. Thompson, G. Wiygul, and P. A. Hedin, ibid., 17, 1093 (1969). (3) M. Beroza and R. A. Coad, J. Gas Chromatogr.,4, 199(1966).
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
Coleman and Bell, Norwood, Ohio, reacted in ethyl ether at Alroom temperature with m-chloroperbenzoic acid (85 drich Chemical Co., Milwaukee, Wis. ; cineole and citronellal, Eastman Organic Chemicals, Rochester, N.Y. ; a-terpineol, Matheson, Coleman and Bell, Norwood, Ohio; and linalyl acetate (90%), Norda, New York, N.Y. Chromatographic Procedures. The terpenes were purified by preparative GLC on a thermal conductivity instrument with an 0.0064- X 6.145-m column packed with 30% Carbowax 4000 on 60/80 mesh Chromosorb P treated with HMDS (carrier gas flow He at 125-150 ml/min, column temperature 160 "C, injector 170 "C, detector 175 "C), or with an 0.0064- X 6.145-m column packed with 10% SE-30 on 60/80 mesh Chromosorb W treated with HMDS (GLC operating conditions were the same). The purified terpenoids were formulated into equal weight mixtures. Also, several terpenoids that had been hydrogenated with platinum oxide were collected from these columns for PMR analysis. Analytical GLC separations were performed on a FID instrument with (A) an 0.0032- X 6.145-m stainless steel column packed with 3 0 z Carbowax 4000 on 60/80 mesh Chromosorb P treated with HMDS (carrier gas flow NP at 48 ml/min, column temperature 175 "C, injector 180 "C, detector 190 "C), and (B) an 0.0032- x 6.145-m stainless steel column packed with 10% SE-30 on 60/80 mesh Chromosorb W treated with HMDS (carrier gas flow Nz at 50 ml/min, column temperature 160 and 175 "C, injector 185 "C, detector 190 "C). Thin layer chromatographic (TLC) separations were performed on a support consisting of 9 parts of silica gel (plain) (Warner-Chilcott Laboratories, Richmond, Calif.) and 1 part of calcium sulfate (anhydrous powder). The irrigant was 10% EtzO in pentane. For monitoring, color development was achieved by heating at 100 "C for 3-5 minutes after spraying with 3 vanillin in 0.5 % concentrated HzSO~ absolute methanol. Alcohols that were formed by the reductive processes were removed by banding the reaction mixture on the plates. Location of the alcohols after irrigation was aided by UV light. Cotton Bud Essential Oil "Oxide" Fraction. Cotton bud essential oil was fractionated on a 2.0- x 20-cm water jacketed column. The support was silica gel coated with, Carbowax 20M (0.75%) wjw ( I ) . The hydrocarbons were removed by elution with pentane. The least polar oxygenated fraction containing chiefly carbonyls, esters, and oxides (2.5 of the oil) was eluted with 2 % EtzO in pentane. Progress of the elution was monitored by TLC to exclude tertiary alcohols. Reductive and Other Processes. Sodium borohydride reductions were performed in absolute methanol at room
z),
-
Table I. Gas Chromatographic Data for the Model Terpenes and Their Reduction and Dehydration Products, lk Values0
0.510LIAlHd.
bLCGUOLS
0
7
6 MINUTIS
I
O
1
1
6
8
*MUTES
Figure 1. GLC profiles of a synthetic mixture ( A ) , after reaction with sodium borohydride ( B ) , the nonpolar fractions after reaction with lithium aluminum hydride (C), and the polars after reaction with lithium aluminum hydride ( D ) Analysis with a 6.145-m SE-30 column at 160 “C. 1 = a-pinene, 2 = cineole, 3 = citronellal, 3A = citronellol, 4 = d-limonene epoxide, 4A = p-menth-8-en-1-01, 4B = p-menth-8-en-2-01, 5 = a-terpineol,6 = linalyl acetate, 6A = linalool temperature for 1 hr with stirring. The alcoholic reaction mixture was evaporated to a minimum (ca. 3-5 ml) and extracted from added water (ca. 15 ml) with ethyl ether. The lithium aluminum hydride reduction was performed by refluxing for ca. 4 hr in anhydrous ethyl ether. Ten per cent aqueous sodium hydroxide was added dropwise to decompose the remaining reagent, and the contents were extracted with ethyl ether. The platinum oxide reduction was performed in absolute methanol at 50 psi H2. Saponification was done with 7.5% potassium hydroxide in methanol for ca. 2 hr. Dehydrations were performed by refluxing with Woelm’s neutral alumina in o-xylene for ca. 2 hr. Spectral Data on Monoterpenoid Compounds. Alpha-pinene. PMR analysis in c c l 4 showed 6 (ppm) 0.84 (s, 3, methyl), 1.27 (s, 3, methyl), 1.65 (m, 3 vinyl methyl), 1.80-2.50 (m, 6 methylenes and methinyls), 5.17 (s, br, 1 vinyl). Cineole. PMR analysis in CC14 showed 6 (ppm) 1.05 (s, 3 CH,-CR’R”0-R”’), 1.25 (s, 6, gem dimethyls), 1.30-2.20 (m, 9, methylenes and methinyl). (+)-I ,2-Epoxy-p-menth-S-ene(d-limonene epoxide). Mass spectrum mje 41 (loo), 43 (99, 39 (70), 91 (20), 59 (18), 57 (16), 55 (14), 119 (9), 95 ( 5 ) , 152 (4). PMR analysis in CC14 1.54 (s, 3, showed 6 (ppm) 1.15 (s, 3, CHI CR””-OR”’), vinyl methyl), 1.20-2.20 (m, 7 methylenes and methinyl), 2.72 (d, 1, epoxy proton), 4.51 (s, 2, terminal methylene). p-Menth-8-en-1-01. PMR analysis in CCl4 showed 6, (pprn) 0.95 (s, 1,-OH), 1.12 (s, 3, CHK-OHR’R”), 1 6 7 (s, 3, vinyl methyl), 0.85-1.80 (m, 9, methylenes and methinyl), 4.59 (s, 2, terminal methylene). p-Menthan-1-01. PMR analysis in CC14 showed 6 (ppm) 0.87 (d, 6, isopropyl methyls), 1.12 (s, 3, CH3 C-OHR’R”), 1.OO-2.00 (m, 11, methylenes, methinyls, -OH). p-Menth-8-en-2-01 (dihydrocarveol). PMR analysis in CCl, showed 6 (ppm) 1.05 (d, 3, CHCHR’R”, 1.65 (s, 3, vinyl methyl), 1.20-2.50 (m, 9, methylenes, methinyl, -OH), 3.75 (s, br, 1, R’R”CHOH), 4.55 (s, 2, terminal methylene).
Compound SE-30 C4000 a-Pinene 970 1072 ( +)cis-Pinane 1002 1103 Myrcene 984 1182 2,6-Dimethyloctane 932 1032 Cineole 1127 1280 Citronellal 1146 1533 Citronellol 1225 1765 3,7-Dimethyl-l-octanol 1190 1685 3,7-Dimethyl-l-octene 963 1265 Linalyl acetate 1250 1548 Linalool 1082 1558 3,7-Dimethyl-3-octanol 1091 1412 3,7-Dimethyl-2-octene 922 1238 plus 3,7-Dimethyl-3-octene a-Terpineol 1205 1698 p-Menthan-8-01 1162 1569 p-Meth-4(8)-ene 998 1220 d-Limonene 1051 1242 d-Limonene oxide 1148 1562 p-Meth-&en-1-01 1156 1731 p-Methan-1-01 1156 1650 p-Meth-8-en-2-01 1208 1812 p-Methan-2-01 1205 1732 p-Meth-1-ene 985 1213 plus p-Meth-2-ene Z k (Kovats indices) values for conditions described in Experimental, e.g., pentadecane = 1500, hexadecane = 1600 ( 4 ) .
p-Menthan-2-01. PMR analysis in Ccl4 showed 6 (ppm) 0.85 (d, 9, C H E H R ’ R “ and isopropyl methyls), 1.00-2.20 (m, 10, methylenes, methinyls, -OH), 3.75 (s, 1, R’R”CHOH). Citronellol. PMR analysis in CC14 showed 6 (ppm) 0.87 (d, 3, CH3CHR’R”), 1.62 (d, 6, vinyl methyls), 1.10-2.30 (m, 7, methylenes and methinyl), 3.54 (t, 2, RCHzOH), 3.85 (s, 1, -OH), 5.09 (t, 1, CH3CH3 C 4 H R ) . 3,7-Dimethyl-l-octanol (dihydrocitronellol). PMR analysis in CC14 showed 6 (ppm) 0.86 (d, 9, CH3CHR’R“ and isopropyl methyls), 1.05-2.10 (m, 10, methylenes and methinyls), 3.54 (t, 2, RCH,OH), 3.85 (s, 1, -OH). Linalool. PMR analysis in CCl4 showed 6 (ppm) 1.15 (s, 3, CH,CR’R”OH), 1.55 (d, 6, vinyl methyls), 2.48 (s, 1, -OH), 1.20-2.30 (m, 4 methylenes), 4.95 (m, 1, CH3 CH,C==CHR), 5.13 (9, Jtrans = 14.5, Jcis = 11.5 HZ 2, R C H S H ? ) , 5.82 (q, J,,,,, = 17.0, Jcis= 10.0 Hz, 1, RCH=CHz). 3,7-Dimethyl-3-octanol (tetrahydrolinalool). PMR analysis in CCl4 showed 6 (ppm) 0.87 (t, 9, methyls), 1.25 (s, br, 3, R’R”CH3C-OH), 1.20-2.50) m, 9, methylenes and methinyl), 2.70 (s, 1, -OH). a-Terpineol. PMR analysis in CC14 showed 6 (ppm) 1.12 (s, 6, RCH3CHd2-OH), 1.58 (s, 3, vinyl methyl), 1.40-2.20 (m, 7, methylenes and methinyl), 2.68 (s, br, 1, -OH), 5.30 (s, br, 1, R’CH3C=CHR“). p-Menthan-8-01 (dihydro-a-terpineol). PPdR analysis in cc14 showed 6 (ppm) 0.85 (t, 3, R’R”CH3CH), 1.05 (s, 6, RCH3CH3C-OH), 1.15-2.10 (m, 10, methylenes and methinyl), 1.17(~,1,-OH). RESULTS AND DISCUSSION
The Model Terpenoids. The GLC profiles are shown in Figure 1, A-D, and additional GLC retention data are presented as Ik (Kovats indices) values (4)in Table I. The pro(4) E. s ~KovBts, . Fresenius’ 2. Anal. Chem., 181, 351 (1961). ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
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A. OXlMS
I
I
0
2
4
8
6
10
12
14
8. OXlMS
0 OXIDES
0
2
4
6
8
K)
12
16
18
22
?o
24
26
20
+ NaBHq
+ EASE
14
16
10
70
27
74
26
28
Figure 2. GLC profiles of the cotton bud essential oil “oxide” fraction ( A ) , after reaction with sodium borohydride ( B ) , after reaction with lithium aluminum hydride (0,and after saponification(D) Analysis with a 0.0032- X 6.145-m Clo0ocolumn at 175 “C file for the original mixture of a-pinene, cineole, citronellal, d-limonene epoxide, a-terpineol, and linalyl acetate is shown in Figure 1A. Treatment of the mixture with sodium borohydride reduced citronellal (3) to citronellol ( 3 4 (Figure 1B). Reduction of the sodium borohydride treated mixture with lithium aluminum hydride produced 2 alcohols from d-limonene epoxide and linalool from linalyl acetate. Preparative TLC of the reaction mixture yielded a-pinene (1) and cineole ( 2 ) in the nonpolar fraction (Figure 1C) and the alcohols (linalool = 6A, p-menth-8-en-1-01 = 4A, p-menth-8-en-2-01 = 4B, a-terpineol = 5, citronellol = 3A) in the more polar fraction (Figure 1D). When the sodium borohydride reaction mixture was saponified rather than reduced with lith1256
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
ium aluminum hydride, the only change was the conversion of linalyl acetate to linalool. While no effort was made to determine the stereochemistry of the pinane produced, Cocker et al. (5) reported that with either platinum oxide or 5 % platinum on carbon, 93-96 % of the (+)cis isomer is formed from (+)-a-pinene. No alcohols were formed from cineole by hydrogenolysis with platinum oxide. An examination of the literature indicates that saturated furan and pyran systems undergo metal catalyst induced ( 5 ) W. Cocker, P. V. R. Shannon, and P. A. Staniland, SOC.C,1966,41.
J. Chem.
the purpose of this report to identify all the GLC maxima, hydrogenolysis with extreme difficulty if at all. Sodium but several are known, and the identification of the others is in bisulfite (6) and lead tetraacetate (7) have been shown to progress. All the maxima except peak 1 (a mixture) are effect hydrogenolysis of the pyran ring of cineole; however, probably sesquiterpenoids. Peak 4 is p-caryophyllene, the no metal catalyst induced ring opening was reported. most abundant CI6hydrocarbon in the cotton bud essential We had previously isolated a constituent of cotton essential oil, 4,8-dimethyl-2-(2-methylpropenyl)-l-oxaspiro[4.5]dec-7- oil ( I I ) , which apparently was not completely eluted with the rest of the hydrocarbon fraction. Peaks 5 and 6 may be CYene, which yielded tetrahydro-P-bisabolol when reduced with humulene and cis-7-bisabolene. Peak 7 is a mixture of 2 chloroplatinic acid in isopropanol (8). We also were able to oxides with m/e 218, CI5H2*0.The structure 4,8-dimethyl-2effect a ring opening of linalool oxide (a mixture of cis and (2-methyl-propenyl)-l-oxa-spir0[4,5]dec-7-ene has been protrans-2-vinyl-2-methyl4 (1 ’-hydroxy-1’-methylethyl-) tetraposed for one of the two known components in peak 12 (8). hydrofuran) with R02 in methanol; however, the reaction Peak 15 is P-caryophyllene oxide (2), and peak 19 is P-bisaproducts were not characterized. bolo1 (12). Also, Isoe et al. (9) converted matatabiether [(lS,4S,5S)Treatment with sodium borohydride (Figure 2B) removed (+)-1,4-dimethyl-8-methylene-2-oxabicyclo[3.2.l]octane] to peaks 5 and 9. Previously, the presence of 15 C3-Cl0 car~,2,3-trimethylcyclopentaneethanolby hydrogenation with bonyls ( I ) and of @-ionone (13) has been reported. Reduc10% Pd/charcoal in methanol. Brooks and Campbell (IO) reduced caparrapi oxide (1,3,7,7-tetramethyl-3-vinyl-2-oxa- tion with lithium aluminum hydride (Figure 2C) removed or diminished peaks 1 , 2 , 13, 15, and 17; peak 15 was previously bicyclo[4.4.0]decane)and obtained the related tertiary alcohol, reported as caryophyllene oxide (2). Saponification (Figure 1,3,3-trimethyl-2-(3-methyl-3-pentenyl)cyclohexanol. All of 2 0 ) diminished peaks 1, 2, 7, 10, 11, and 12, while 2 new these 4 examples represent systems which contain a double peaks, 16a and 18a, appeared. The enhancement of peak 6 bond allylic to the oxygen. The hydrogenolysis is quite is probably explained by alkaline isomerization of the terminal facile, and is directed to the carbon-oxygen bond adjacent methylene groups of the oxides of peak 7, which are known to to the unsaturation. be present from IR evidence. Peaks 16a and 18a may be The individual alcohols (Figure 1 0 ) and the mixture were tertiary alcohols of such low polarity that they remained in quantitatively reduced with PtOz in methanol for subsequent the oxide fraction during the preparative TLC procedure structural confirmation by PMR analysis. All except one which was performed after the saponification. From inspecpair, p-methan-8-01 and p-menthan-1-01, were resolved on the SE-30 column; however, this pair was resolved on the C ~ O O O tion of the GLC profiles of the lithium aluminum hydride reduction and the saponification products, peaks 13, 15, and column (Table I). Finally, the saturated alcohols were dehydrated by refluxing 17 are inferred to be epoxides; the components in peaks 1, with Woelm’s neutral alumina in o-xylene. GLC analysis 2, 10, 11, and 12 may be esters. Catalytic reduction and showed that the conversions were complete in 2-4 hr. 3,7dehydration probably would be most advantageously applied Dimethyl-3-octanol (tetrahydrolinalool) apparently gave only to isolated components though, if the total oxide fraction were the 2 “endo” unsaturated hydrocarbons since the terminal treated, the skeletal types would be delineated. methylene isomer was not observed as indicated by the longer The desirability of coupling this regimen to mass specGLC retention time. Similarly, no terminal methylene isotrometry is obvious. Also, analysis of the alcohols formed during the reductions would give information about the nummer was observed after dehydration of p-methan-1-01, pmethan-2-01,and p-methan-8-01. ber of unsaturations and rings and the location of the hydroxyl The cotton bud essential oil “oxide” fraction. Figures 2, groups. A-D give the GLC profiles of the original fraction ( A ) , after RECEIVED for review September 24,1971. Accepted March 1, sodium borohydride reduction (B), after lithium aluminum 1972. Paper presented in part at the 160th National Meeting, hydride reduction (C), and after saponification ( D ) . It is not ACS, Division of Agricultural and Food Chemistry, Chicago, Ill., Sept. 1970. The mention of a pesticide or proprietary (6) I. Ogura, Yakagaku Zasshi, 78 932 (1958); Chem. Abstr., 52 product in this paper does not constitute a recommendation 20227a. or an endorsement of this product by the U.S. Department (7) T. Aratani, J. Sci. Hiroshima Unio., 24A, 131 (1960); Chem. of Agriculture. Abstr., 55 7461d. (8) P. A. Hedin, A. C. Thompson, R. C. Gueldner, and J. M. Ruth, 160th National Meeting, ACS, Div. of Agr. and Food Chemistry, Chicago, Ill., No. 84 (1970), Phytochemistry, in (11) J. P. Minyard, J. H. Tumlinson, A. C. Thompson, and P. A. Hedin, J. Agr. Food Chem., 14,332 (1966). press, 1972. (9) S. Isoe, T. Ono, S. B. Hyeon, and T. Sakan, Tetrahedron Lett.. (12) J. P. Minyard, A. C. Thompson, and P. A. Hedin, J. Org. No. 51, 5319 (1968). Chem.. 33, 909 (1968). (10) C. J. W. Brooks and M. M. Campbell, Phytochemistry, 8, (13) P. A. Hedin, A. C . Thompson, R. C. Gueldner, and J. P. 215 (1969). Minyard, Phytochemistry, 10, 3316 (1971).
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