Anal. 'Chem. 1983,
55, 1379-1384
1379
compounds was significantly weak compared to model compounds. The heavier the fraction, the weaker the retention. Thus, in the separation of heavy petroleum fractions, even polar compounds are not retained as strongly as expected from the behavior of model compounds.
LITERATURE CITED
1
I
I
10
I
0
20
30
Time(min.)
Flgure 3. HPLC separation of a heavy petroleum distillate: column,
Nucleosll NO,: eluent, as In Table IV; sample, Kuwait 350-500 O C distillate; (a) original sample, (b) fractions obtained by preparative alumina chromatography of (a). centrated Iby chromatography on ion exchangers (16,17), and collected fractions were injected into the HPLC system. Although cross-contamination was observed, the attribution of two peaks proved to be proper. The retention of these
(1) Snyder, L. R.;BuelU, B. E. Anal. Chem. 1988, 40, 1295-1302. (2) Rubedo, R. 0.;Jevvell, D. M.; Jensen, R. K.; Cronaues, D. C. Prepr. Pap.-Am. Chem. Soc., Div. Fuel Chem. 1974, 19 (2), 258-2!30. (3) Farcasiu, M. Fuel 1977, 58, 9-14. (4) Schiller, J. E.; Mathiason, D. R. Anal. Chem. 1977, 49, 1225-1228. (5) Suatoni, J. C.; Garber, H. R.; Davis B. E. J . Chromatogr. Sci. 1975, 13, 367-371. (6) Suatonl, J. C.; Garber, H. R. J . Chromatogr. Sci. 1978, 74, 546-5418, (7) Suatoni, J. C.; Swab, R. E J . Chromafogr. Scl. 1975, 13, 361-366. (8) Suatoni, J. C.; Swab, R. E. J . Chromatogr. Sci. 1978, 14, 535-537. (9) Selucky, M. L.; Rue, T. C. S; Strausz, 0. P. Fuel 1978, 57, 585-591. (IO) Wise, S. A.; Chesler, S. N.; Hertz, H. S.; Hllpert, L. R.; May, W. E. Anal. Chem. 1977, 49, 2306-2310. (11) Mourey, T. H.; Slggia. S.; Uden, P. C.; Crowley, R. J. Anal. Chem. 1980, 52, 885-8911, (12) Dark, W. A.; McFadden, W. H.; Bradford, D. L. J . Chromatogr. 86. 19'17, 15,454-460. (13) Matsunaga, A.; Yagi, M. Anal. Chem. 1978, 50, 753-756. (14) Schabron, J. F.; Hurtublse, R. J.; Silver, F. Anal. Chem. 1977, 49, 2253-2260. (15) Majors, R. E. Anal. Chem. 1972, 4 4 , 1722-1726. (18) McKay, J. F.; Weber, J. H.; Latham, D. R. Anal. Chem. 1978, 48, 891-888. (17) McKay, J. F.; Cogswell, T. E.; Weber, J. H.; Latham, D. R. Fuel 1975, 54, 50-61.
RECEIVED for review September 28, 1982. Accepted March 17, 1983. Presented in part a t the National Meeting of the American Chemical Society, Atlanta, GA, April 1981.
Reaction Gas Chromatography without Solvent for Identification of Nanogram Quantities of Natural Products Athula B. Attygalle and E. Davld Morgan" Department of Chemistty, University of Keele, Keele, Sta ffordshlre, ST5 SBG, United Kingdom
A method Is described for trapplng nanogram quantities of compounds or mixtures from a gas chromatography column with approximately 85 % efficiency and relnjecting the sample on the same or a different column, with or without carrylng out a reaction, and all without contamlnatlon wlth solvent. The method includes a solvent-free ozonolysis technlque for determinlng the positlon of double bonds In alkenes by using 200 ng of sample, even when low molecular weight carbonyl compounds are produced. Characterlratlon of carbonyl compounds by sodlum borohydride reduction, hydrogenatlon of alkenes, and cleavage of epoxldes by periodic acid at this scale, wlthout the use of solvents, are also descrlbed.
For some years we have been developing our methods for examining the small quantities of chemicals (pheromones anal hormones) found in insect tissues. Despite the great advances in spectroscopic methods, the only spectral technique generally applicable to nanogram quantities of compound is mass spectrometry, and a mass spectrum alone cannot always provide complete identification of a compound. The conventional method for investigating those chemicals present
in plant or animal materiL-, below the microgram lev6 is to extract large quantities and to concentrate by chromatography for subsequent spectroscopic identification (1,2). It may ble very difficult to accumulate the required quantity of insect material. We have therefore concentrated our attention on methods for separating, quantifying, and identifying substances directly at the nanogram level by chromatography and mass spectrometry. In 1972 we described a method for direct solid injection of a piece of biological tissue, such as an insect gland without introducing any solvent (3). The method included simple reactions, such as treatment with sulfuric acid or bromination. Later we have extended the applicability by showing that low molecular weight compounds down to C1could be examined in the same way and that other reactions could be applied for the recognition of alcohols, aldehydes, and ketones and the method could be extended for use with capillary columns (4, 5). Though there is nothing fundamentally original about these methods [solid sampling was first suggested in 1958 (6) in the infancy of gas chromatography], we have used them regularly in our research on insects. An example of their use is in the identification of the trail pheromone of eight species of Myrmica ants, using less than 100 ants, and the determi-
0003-2700/83/0355-1379$01.50/00 1983 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 8. JULY 1983
nation of the amount of the substance in individual worker ants (7,8). When it is required to trap a single suhstance for bioassay or for rechromatography in a different system, normally a solvent is required, into which the substance is trapped, or with which to wash out the tiny amount of substance from the trap. The resulting solvent peak and its impurities can completely obscure the desired compound when only nanograms of it are available, and the whole of the sample cannot he efficiently rechromatographed. Reaction gas chromatography can he a useful technique to supplement or confirm the evidence from mass spectrometry (9,101. For this too, trapping in a solvent is required, which prevents reinjection of the whole sample, hence loss of sensitivity, and the solvent peak may hide reaction product peaks. We have therefore sought to improve our techniques by finding a method for trapping such small samples and reinjecting them efficiently and totally onto the column, with or without reactions on-column or in the injection port. We descrihe here methods for trapping into a glass capillary and subsequent ozonolysis, hydrogenation or reduction, cleavage, and rechromatography, that we have found useful and efficient. EXPERIMENTAL S E C T I O N Apparatus. A Pye 104 series gas chromatograph with a flame ionization detector (FID) was used for GLC, using one of the following columns: (A) 1.5 m X 4 mm (i.d.1, glass column packed with 5% SE-30 on Chromosorb W, 100-120 mesh; (B) 2.75 m X 4 mm (i.d.), glass column packed with 10% PEG 20M on Chromosorh W, 100-120 mesh; (C) 2.75 m X 4 mm (i.d.1, glass column packed with 10% PEGA on Chromosorh W, 100-120 mesh; and (D) 1.5 m x 4 mm (id.), glass column packed with Porapak Q (Waters h i a t e s , Milford, MA), 1-150 mesh. The attenuation of the amplifier of the GC system was not changed during any given experiment. Unless otherwise stated nitrogen was used as the carrier gas at a flow rate of 45 mL/min. Insect tissue or glandular contents extracted with a glass capillary, weighing up to 1pg, were sealed in a 35 mm X 1.8 mm soft glass tube, heated to approximately 150 "C for 3 rnin in the injection area of the GC, and then crushed in the carrier gas stream, as described by Morgan and Wadhams (3),and thus avoiding the use of solvent. Trapping and Rechromatography. An effluent splitter (11) was made from thick-walled glass capillmy tubing (6 mm 0.d. and 0.4 mm i.d.1, with a 6 mm metal screw sealed to the outlet (collector) end and a metal capillary fitted with a restrictor sealed to the end for connection to the detector. The restrictor was a piece of fine wire inside the metal capillary, of such a diameter and length to give a 955 (outletFID) split ratio. The outlet heater was maintained at 200 'C and the outlet kept closed with a 6 mm Pye hexagonal coupling nut with a silicone rubber septum. Approximately 5 s before the desired GLC peak under study appeared, the outlet was opened, and a new nut with a 45 mm x 0.5 mm (0.45 mm i.d.1 glass capillary going through the silicone rubber septum and a 15 mm polyethylene specimen tube cap was screwed in, and the specimen tube cap was filled with liquid nitrogen (Figure 1). Once the peak had eluted, the hexagonal nut was unscrewed and the capillary was immediately removed, broken into three equal pieces, and dropped into a 35 mm X 1.8 mm 0.d. (1.75 mm id.) soft glass tube closed at one end, and the open end was then sealed in a microflame. This was reintroduced into the gas chromatograph by the solid injection technique (3). When rapid change-over of traps is required, for collecting compounds eluted close together, the splitter illustrated may he replaced with one ending in a male Luer fitting and the glass capillaries attached with a female Luer joint holding a soft silicone rubber septum. The glass capillaries and tubes were baked at 230 "C for 30 min before use. It was important to avoid contamination of the tube by handling with greasy hands. The efficiency of the procedure was determined by injecting 0.5 pL of a standard solution of tetradecane and pentadecane in hexane (500 ng each/pL), onto column A at 145 "C. The pentadecane peak was trapped, the sample was rechromatographed, and peak areas were compared.
A
G Flgure 1. Schematic representation of peak kapping system: (A) liqua nilrcgen; (B)silicone rubber septum: (C) glasslmetal seal; (D) ailglass splier; (E) ferrule with restrictor to flame ionization detector; (F) Swageiok union; (G)glass column: (H) outlet heater; (I) hexagonal nut; (J) glass capillary coliection tube.
To demonstrate the use of the technique to determine the homogeneity of a GLC peak, a Dufour gland of the ant Myrmica rubrn was diasected out, sealed up in a 35 mm X 1.8mm soft glass tube, and chromatographed on column C, with the oven temperature programmed from 125 to 162 "C, at 2 OC min, and the effluent between 13.5 and 15 rnin was collected and rechromatographed on column A with the temperature programmed from 140 to 192 "C a t 4 'C/min. Ozonolysis. A standard solution of n-heotadecane and (ZWheptadecene in hexane (700 ng each/pL) was used for efficiency determination. A sample (0.5 pL) was injected onto column B, with the oven temperature held a t 130 O C for 8 min, and then programmed at 40 OC/min to 170 "C, both hydrocarbon peaks were trapped together, and the collection tube was broken into three pieces and dropped into the larger soft glass tube with one end sealed as described above. A fine stream of ozone (10 mL/min) from a micro ozone generator (12) was passed for 20 s through a 0.5 mm hard glass capillary, extending to the bottom of the larger soft glass tube cooled in ice. The open end was sealed immediately and kept in the injection area a t 200 OC for 5 min before crushing, for the pyrolytic cleavage of the ozonides. This was applied to a variety of alkenes (Table I) by trapping 200-300 ng of sample from columns B and D, and the products were identified on either column B or D. Ozonolysis of Polyenes. Farnesene (200 ng), homofarnesene (200 ng),and hishomofamesene (200 ng) were collected separately from the ant Myrmica scabrinodis by injecting a single Dufour gland on column C at 140 "C. 0-and (Z)-nerolidoI(ZWng each) were obtained by trapping from a commercial sample of mixed isomers. Each of these compounds was ozonized separately and the products were analyzed on column D at 160 "C. NaBH, Reduction. A whole head of the garden ant Myrmica rughodis was chromatographed on column B at 125 OC, to show the volatiles of the mandibular gland. The 3-octanone (500 ng) was trapped, and the three pieces of the trapping capillary were dropped into the soft glass tube, open at one end which contained finely powdered solid NaBH, (1mg or less). The other end was sealed immediately and the tube was kept at room temperature for 15 min with occasional shaking. This was reintroduced into the gas chromatograph and kept for 5 rnin at 200 "C in the injection area hefore crushing. Hydrogenation. A standard solution (0.5 pL) containing heptadecane and (Z)-bheptadecene (700 ng each/pL) was chromatographed, and the two hydrocarbons were trapped as a single fraction and then rechromatographed on column B, with 1cm of 1%palladium catalyst (131,on 100-120mesh Chromhsorh W, as a precolumn packing between two silanized glass wool plugs. Hydrogen was used as the carrier gas with the flow rate adjusted to produce the same retention time for the petadecane peak as when using nitrogen. The oven temperature was held at 130 "C for 8 rnin and then raised at 40 "C/min to 170 OC. Cleavage of Epoxides. The epoxides not commercially available were synthesized from the corresponding alkenes by
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
1381 I
Table I. RLesults of Ozonolysis of Alkenes no.
source
compound a
1 2-methyl-2-butene
products and comments lower mol wt productsd
higher mol wt produictsC
ethanal t acetone (minor product, acetic acid) acetone ethanal t acetone (minor product, acetic acid) ethanal t pentanal (minor product, acetic acid)
C
2 3
2,3 .dimethyl-2-butene (2).2-penI,ene
C C
4
(2)-2-heptene
C
( 2 ) 8 -heptadecene
octanai t nonanal nonanal + decanal nonanal + tetradecanal nonanal t aldehyde ester benzaldehyde t f acetophenone t f benzaldehyde t g C benzaldehyde C benzaldehyde t C 2-phenylpropanal N (Myrmica scabrinodis)e 4-oxopentanal + f , g N (Myrmica scabrinodis)e 4-oxohexanal t f , g f,g N (Myrmica scabrinodis)e 4-oxohexanal i4-oxopentanal + f , g C
5 6 7 8 9 10 11 12 13
(2).9-nonadecene (&).9-tricosene methyl (2)-9-octadecenoate phenylethene 1-methyl-1-phenylethene (E)-3-phenyl-2-propenal (2)-1,2diphenylethene 1,3-diphenyl-l-butene
14 15 16 17
(2,E)-a-farnesene (I) homofarnesene (11) bishomofarnesene (111) (2)or (E)-nerolidol (IV)
N (Myrmica rubra)e N (Myrmica rubra)e N (Solenopsis geminata)e C C C
acetone acetone butanone acetone
C = commercial, N = natural (source). Products larger than hexanal were identified on a Sample size 200-350 ng. Products smaller than pentanal were identified on Porapak Q column. e Samples collected from 10%PEG 20M column. Sample not the Dufour glands of the ants by preparative GLC. f Methanal not observed due to its ]poor flame response. analyzed for other products.
reacting with rn-chloroperbenzoic acid (14). The juvenile hormones were obtained from Sigma Chemical Co. (St. Louis, MO). Periodic acid purchased from Fluka, Switzerland, supplied as HJO,was dried to a constant weight in an evacuated drying pistol at 100 OC and ground to a fine powder in a mortar. The epoxides were trapped separately (200-300 ng) from column B and the pieces of the capillary were dropped into the soft glass tube, open at one! end which contained solid periodic acid (0.5 mg). The other end was sealed immediately and the tube was kept at room temperature for 3 min. This was reintroduced into the gas chromatograph and the products were identified either on column B or D.
RESULTS AND DISCUSSION Since carrying out our experiment on trapping into a fine glass capillary for reinjection, we have discovered that this part of our method was largely anticipated by Stanley, who in 1972 described a similar method of trapping, adding a reactive gasi, and rechromatography (15). His method has apparently been forgotten, or its usefulness unrecognized, perhaps because Stanley did not indicate how efficient his method of trapping was, nor did he attempt the submicrogram samples we have used. We therefore record the efficiency with which we find this simple process can be effected in the nanogram range. Efficiency of Trapping and Rechromatography. Figure 2 shows typical chromatograms of trapping and rechromatography. The split ratio was 95:5 as determined by the peak areas with and without the splitter. The trapped material can be rechromatographed with 85 f 5% efficiency as found by five replicate determinations of n-pentadecane. But obviously the trapping efficiency will depend on the boiling point of the sample. The size of the glass capillary used for trapping is critical. A narrower capillary alters the flow rate and split ratio, and mLore effluent is vented to the FID. A larger capillary introduces problems of efficient trapping, and it is not possible to rechromatograph all the material in a large, long capillary. The optimum size of the capillary is 45 mm X 0.5 mm i.d. Slower flow rates of the carrier gas give better trapping efficiences. Flow rates above 50 mL/min decrease the trapping efficiency and no trapping was observed a t 80 mL/min.
I
-
/
4 1 31
0
I
,
4 4
I1
0
4 4
0
min
Flgure 2. Chromatograms on SE-30 column at 145 'C: (a) 250 ng each of tetradecane and pentadecane In 0.5 I.LL of hexane; (b) sama amount Injected and effluent between the two arrows was trapped: (c) total trapped material was reinjected.
Nanogram to microgram amounts of material were made available by this technique, for rechromatography on a different stationary phase, to check the homogeneity of a peak, and subsequent reaction gas chromatography. Figure 3 shows the application of the technique to demonstrate the heterogeneity of a particular GLC peak in a chromatogram obtained from a single Diifour gland of the ant Myrmica rubra. The peak, corresponding to 200 ng at retention time 14.0 min on a PEGA column, gave two peaks when rechromatographed on a SE-30column. Ozonolysis. Alkene double bonds are present in abundance in insect hormones (16,17), pheromones (10, 18-20), and a variety of biologiically important natural products (21, 22). Considerable progress has been made recently to develop microtechniques to determine the location of the unsaturation. Although a mass spectrum of such a compound could be obtained with a few nanograms of material the double bond position is difficult to determine even with chemical ionization technique (23). Derivatives can often be prepared and the location of the double bond can be subsequently determined by mass spectrometry if 1 Mg or more of the substance is available. These met hods include methoxymercuration-
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
Table 11. Results of Periodic Acid Cleavage of Epoxides no. 1 2 3 4 5 6
I 8 9 10 11 12 13
14 15 16 11
products and comments higher mol wt products lower mol wt productsC
compound a
1,2-epoxypropane l,2epoxybutane (2)-2,3epoxypentane (Z)-2,3epoxyhexane 2,3-epoxy-2-methylbutane 2,3-epoxy-2,3-dimethylbutane (2)-8,9epoxyheptadecane octanal t nonanal (E)-8,9epoxyheptadecane octanal + nonanal (2)-9,1O-epoxynonadecane nonanal + decanal (E)-9,lo-epoxynonadecane nonanal -t decanal (2)-9,1O-epoxytricosane nonanal + tetradecanal methyl (2)-9,lO-epoxyoctadecanoatenonanal + aldehyde ester 1,2epoxyethylbenzene benzaldehyde + phenylacetaldehyde (minor product) + d (E)-2,3epoxy-3-phenylpropanal benzaldehyde + e juvenile hormone I ( V ) e juvenile hormone I1 (VI) e juvenile hormone I11 (VII) e
ethanal t acetic acid (trace) + d propanal + d ethanal + propanal + acetic acid (trace) ethanal + butanal + acetic acid (trace) ethanal + acetone + acetic acid (trace) acetone
butanone butanone acetone
a Sample size 200-300 ng. Products larger than hexanal were identified on 10%PEG 20M column. Products smaller than pentanal were identified on Porapak Q column. Methanal not observed due to its poor flame response. e Sample not analyzed for other products. ,-
-
c Y
Y L Y
-
e
I Y
I
Y
Flgure 4. Chromatograms on 1 0 % PEG 20M column, temperature held at 130 OC for 8 min and programmed 40 'Clmln to 170 OC; (a) 350 ng each of heptadecane and 8-heptadecene in 0.5 pL of hexane. The effluent between the arrows was trapped and (b) reinjected, (c) reinjected and hydrogenated, (d) ozonized and relnlected.
I
1
I
0.0
14.0
a
mln
Flgure 3. (a) Chromatogram of one Dufour gland of Myrmica rubra , with the oven temperature programmed from 125 to 162 OC at 2 OC/min, on 10% PEGA column. (b) Effluent between the two arrows of (a) was trapped and rechromatographed on 10% PEGA column. (c) Same as (b) but rechromatographed on 5 % SE-30 column with temperature prrogrammed from 140 to 192 OC/min.
demercuration (24, 25), deuteration (26,27) and formation of 0-isopropylidene derivatives (28),silyl ethers (29),methyl ethers (30),Nfl-dimethylhydroxyamines (31),ketones (32), and epoxides (33, 34). The majority of these methods are lengthy, require 1pg or more of the alkene, and sometimes involve solvent extraction of the final product before GLC. Epoxidation has been performed on 0.5-pg samples (34),but because the reaction is done in a solvent, the epoxides had to be collected by preparative GLC before obtaining the mass spectrum. Ozonolysis of alkenes has been a particularly useful technique, but the main drawback of ozonolysis in a solvent, as pointed out by others who have sought alternative methods (34), is that it is difficult to identify the small molecules
formed. A variety of solvent systems has been studied to find the best solvent for ozonolysis and subsequent analysis of the products by GLC (12, 35). We have collected the alkene substance in a capillary after chromatographing a mixture, such as is obtained from a pheromone mixture, treated it with ozone, and immediately rechromatographed the ozonides. Decomposition of the ozonides in the gas chromatograph gave quantitative yield of the corresponding carbonyl compounds. Figure 4 shows ozonolysis of trapped heptadecane and (Z)-%heptadecene. The (2)-8-heptadecene peak has completely disappeared to yield the corresponding octanal and nonanal, while the heptadecane peak remained unreacted. Though 350-ng samples were conveniently used, analysis of samples as small as 50 ng was readily achieved. This technique was used to determine the double bond positions in a number of known and natural alkenes isolated from ants. The results were summarized in Table I. Beroza et al. (12,35) identified the products of ozonolysis of 1pg of methyl oleate and many other compounds in solution by injecting 20 pL of solution into the gas chromatograph. White et al. (36) determined the structures of vulpinic acid by ozonolysis of 25 pg in 100 pL of ethyl acetate and injecting
ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
h
1383
I
1
a
m
Y
z 0
z c < s m
m I
I 4.5
8.5
I
mr
0
min Figure 5. Chromatograms on Porapak Q column at 160 O C . Ozonolysis products of 200 ng each of (a) homofarnesene and (b) bishomofarneseiie.
10 KL into the gas chromatograph. The huge solvent peak thus obtained extends over 12 min. Such large solvent peaks, even if the solvent is scrupulously pure, would render the method not particularly useful to identify the small molecules produced. The solventless ozonolysis technique overcomes these difficulties and allows total identification of the products. Alkylideiie Analysis. Determination of alkylidene groups (the part of molecule between its end and the first double bond) is difficult by ozonolysis in solvent, when small molecules are produced. Solventless ozonolysis readily demonstrated the presence of the isopropylidene groups in 200 ng each of farnesene (I), homofarnesene (11)and (2)-and (E)nerolidol (IV) by the production of acetone. The presence of an isobutylidene group in bishomofarnesene (111)was shown by the production of butanone (Figure 5 and Table I). As no solvent is used the sensitivity of this technique can be increased to the maximum detection limit of the chromatograph, and it is quite valuable for alkylidene group analysis in large
terpenoid molecules. The loss of material is minimum as no evaporation is involved and the product is introduced to the chromatograph as ozonides rather than the more volatile carbonyl compounds. NaBH4Reduction,, The identification of aldehydes and ketones has been aided by their reduction to alcohols with sodium borohydride and subsequent GLC analysis of the products (37). A saturated solution of NaBH4 in ethanol has been used to reduce 1 pg samples in the syringe barrel, and products larger than hiexanol have been identified (38). As a solvent is involved this method cannot be used to monitor small molecules. Solid NaBH, has been used to reduce the total mixture of components from three heads or six poison reservoirs of the ant Myrmica rubra (4, 5 ) and for micaosynthesis of alcohols in a preparative scale (39). Surprisingly, no hydrolysis step or additional moisture for hydrolysis is required. The 3-odanone peak in Figure 6 represents approximately 400 ng (40); this was quantitatively reduced to 3-octanol in 15 min. The minor component 3-heptanol which appeared as a shoulder on the 3-sctanone peak was trapped with it, but this minor peak is clearly visible once the major 3-octanone
. Y =
. I
e c m
a
1
74
-
I
4.B
C
E
-+
-
1
4.9
1
1.1
I I
rnin
Figure 6. Chromatograms on 10% PEG 20M column at 125 OC: (a) the total volatiies of the mandibular gland of Myrmlca ruginodis from one whole head; (b) the effluent between the two arrows was trapped and reinjected; (c) the effluent was trapped and reinjected with NaBH,.
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ANALYTICAL CHEMISTRY, VOL.
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peak was reduced to 3-octanol. Hydrogenation. The characterization of alkenes can be facilitated by hydrogenation to saturated analogues which provide simpler mass spectra. Hydrogenation of unsaturated compounds has been readily achieved by injecting the compounds in a solvent onto a column with a precolumn catalyst and using hydrogen as carrier gas (9,13). As described before, a solvent limits its applicability to larger molecules. Hydrogenation of (Z)-a-heptadecene is shown in Figure 4, the yield is quantitative and can be applied to a few nanograms of material conveniently. By combination with the trapping technique, hydrogenation can be used to determine the degree of unsaturation and the number of rings present in the molecule. Cleavage of Epoxides. Epoxide groups are found in many biologically important natural products [e.g., insect juvenile hormones (16)) sex attractants (41)],and techniques to locate their positions in nanogram amounts are of great importance to the natural product chemist. Position of epoxide groups or olefinic double bonds (by converting them to epoxides) can be determined by reacting the compound with periodic acid. Bierl et al. performed this by cleaving 1-100 p g samples in chlorinated solvent, and subsequent examination of the solution for carbonyl compounds by GLC (42). Schwartz et al. (43)used a column of periodic acid on calcium sulfate to cleave micromole amounts of epoxides to aldehydes. We have determined the epoxide position in 17 known compounds, by trapping 200-300 ng samples and rechromatographing with solid periodic acid. Table I1 summarizes the results obtained. The technique clearly demonstrated in 200 ng samples the presence of an ethyl and a methyl group attached to the epoxide ring of juvenile hormones I and I1 (structures V and VI) by the production of butanone, whereas juvenile hormone I11 (structure VII) produced acetone to indicate the presence of two methyl groups. The cleavage was quantitative and little or no side reaction products were observed. When acetaldehyde was a product, trace amounts of acetic acid were observed due to oxidation. Epoxyethylbenzene gave phenylacetaldehyde as a minor product, along with the expected major product benzaldehyde. The amount of side reaction products increased with prolonged reaction time or excess periodic acid. It is prudent to keep a separate column for cleavage of epoxides as the column becomes contaminated with periodic acid in time or else the fiit few inches of packing should be removed and replaced with fresh packing.
ACKNOWLEDGMENT The authors thank R. P. Evershed for the synthesis of 8-heptadecene and 9-nonadecene.
Registry No. I, 28973-98-0; 11, 73690-00-3; 111, 70234-77-4; (Z)-IV, 3790-78-1; (E)-IV, 40716-66-3; V, 13804-51-8;VI, 3421861-6; VII, 22963-93-5; 2-methyl-2-butene, 513-35-9; 2,3-dimethyl-2-butene, 563-79-1; (Z)-2-pentene,627-20-3;(Z)-2-heptene, 6443-92-1; (2)-8-heptadecene, 16369-12-3; (Z)-g-nonadecene, 51865-02-2; (Z)-g-tricosene,27519-02-4; methyl (Z)-g-octadecenoate, 112-62-9;phenylethene, 100-42-5;1-methyl-1-phenylethene, 14371-10-9; (Z)-l,2-diphenyl98-83-9; (E)-3-phenyl-2-propenal, ethene, 645-49-8; 1,3-diphenyl-l-butene77614-93-9; 1,a-epoxypropane, 7556-9; 1,2-epoxybutane, 106-88-7;(Z)-2,3-epoxypentane, 3203-99-4; (2)-2,3-epoxyhexane,1192-32-1;2,3-epoxy-2-methyl5076-20-0; butane, 5076-19-7; 2,3-epoxy-2,3-dimethylbutane,
(Z)-8,9-epoxyheptadecme9 85267-93-2;(E)-8,9-epoxyheptadecane, 85267-94-3; (Z)-9,1O-epoxynonadecane,85267-95-4; (E)-g,lO-epoxynonadecane, 85267-96-5; (2)-9,10-epoxytricosane,66640-79-7; methyl (2)-9,1O-epoxyoctadecanoate,2566-91-8; 1,2-epoxyethylbenzene, 96-09-3; (E)-2,3-epoxy-3-phenylpropanal, 71403-94-6.
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RECEIVED for review November 4,1982. Accepted March 11, 1983. The British Council provided partial financial assistance to A.B.A.
