- Email: [email protected]
Identification and determination of parts-per-million formaldehyde and lower carbonyl homologs by gas chromatography
MICROCHEMICAL
JOURNAL
35, 296-304 (1987)
Identification and Determination of Parts-per-Million Formaldehyde and Lower Carbonyl Homologs by Gas Chromatography DAVID G. OLLETT AND E. DAVID MORGAN~ Department of Chemistry, University of Keele, Keele, Staffordshire ST5 5BG, United Kingdom Received November 5, 1986; accepted November 19, 1986 Formaldehyde, acetaldehyde, acetone, and propionaldehyde may be identified and determined in concentrations of parts per million by reduction to the corresponding alcohol followed by gas chromatography of the alcohols on porous polyaromatic resin beads. The method may be used for determining the position of double bonds in, e.g., vinyl and isopropylidene groups by prior ozonolysis. The method is limited in sensitivity chiefly by the levels of these lower carbonyl compounds in the atmosphere. o 1987 Academic PXSS, IIIC.
INTRODUCTION
While gas chromatography remains the most sensitive method for determining most volatile organic compounds, it singularly fails for the analysis of formaldehyde because of the low flame response of this compound (1, 2). To use the thermal conductivity detector means a loss of sensitivity of about loo-fold. The demand for increasingly sensitive methods for determining atmospheric pollutants and the need for sensitive methods for detecting and quantifying small carbonyl compounds produced in the micro-ozonolysis of pheromones has caused us to seek new methods for determining them (3-7). The chief difficulty is to determine formaldehyde alone or to find the relative amounts of formaldehyde and the other carbonyl compounds produced by ozonolysis and to find a system where these substances are not hidden by a large solvent peak. We have recently described a method (8) for separating and determining nanogram amounts of formaldehyde, acetaldehyde, propionaldehyde, acetone, etc., by conversion to their O-benzyloximes in the small scale device, the Keele micro-reactor (9), and separating and determining the derivatives on a packed or capillary GC column. We now describe an alternative method, by which the carbony1 compounds can be reduced efficiently to the corresponding alcohols and determined with a flame ionization detector on a porous polyaromatic resin bead column. Methanol gives a much better flame response than formaldehyde and good peak shape with little possibility of interference by the other compounds and, with this method, no interference from solvent. EXPERIMENTAL Apparatus and chromatographic conditions. The analytical work was performed with a Pye PU4500 gas chromatograph with a flame ionization detector (FID) using a 1.5 m x 4 mm column tilled with Porapak Q (120- 150 mesh, Waters r To whom correspondence
should be addressed. 296
0026-265X187 $1.50 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
DETERMINATION
OF FORMALDEHYDE
297
Associates, Inc.). The oven temperature was 15O”C, with nitrogen used as the carrier gas at 50 ml/min. The preparative work was performed with a Pye 104 gas chromatograph with a flame ionization detector using a 2.75 m x 4 mm column of 10% PEG 20M on Chromosorb W (loo-120 mesh). Nitrogen was used as the carrier gas at a flow rate of 40 ml/min. Trapping technique. The gas chromatograph effluent was passed through a splitter of the design of Baker et al. (10) to give a 95:5 (outlet:FID) split ratio. Trapping of the alkenes was then carried out according to the method of Attygalle and Morgan (II) using a glass capillary (45 x 0.5-mm o.d., 0.45-mm i.d.) threaded through two holes in a polyethylene sample tube cap (15-mm diameter) filled with liquid nitrogen. Ozonolysis. A glass tube was drawn down to a narrow capillary so that it would Ozonized oxygen I
-
-
Trapping
capillary
Alkene
-I P 0.45 mm
FIG. 1. Apparatus for ozonolysis of nanogram quantities of alkene collected from a gas chromatograph. Dimensions are approximate.
298
0LLEl-T
I 10
AND
MORGAN
I 5
1 0
min.
FIG. 2. Gas chromatogram showing the separation of (a) water, (b) methanol, (c) acetaldehyde, (d) ethanol, (e) acetone, and (f) isopropanol on a Porapak Q column.
fit into the capillary containing the alkene (Fig. 1). A fine stream of ozonized oxygen from an ozone microgenerator (2) was then passed through the capillary to form the ozonides. These were then reduced to alcohols using aqueous sodium borohydride solution. Sodium borohydride reduction. A solution of sodium borohydride (1 ~1 of 0.1 g in 10 ml water) was added to the ozonide in the glass capillary using a 5~1 syringe (SGE, London) fitted with a 75 x 0.23-mm-o.d. steel needle (II). The capillary was then broken carefully into pieces and these pieces were sealed in another glass capillary for solid sampling. After 5 min in the injection port of the gas chromatograph, the reaction was complete and the glass capillary was crushed. The alcohols were then analyzed using the Porapak Q column. Reaction blank. The nitrogen carrier gas was passed through an empty capillary cooled in liquid nitrogen as it emerged from the gas chromatograph. Ozonized oxygen from a micro-ozone generator was then passed down the capillary followed by 1 cl.1 of the aqueous sodium borohydride solution. This was then sealed in another capillary and reinjected onto the gas chromatograph at the same
DETERMINATION
299
OF FORMALDEHYDE
I
I
1
10
5
0
min
FIG. 3. Gas chromatogram showing the methanol obtained by ozonolysis and reduction of safrole. The large peak (a) consists of a mixture of water and borane and (b) methanol.
attenuation as that used for the reduction of the alkenes. This served as the control blank determination. Isolation OffarneSeneSfrom ants. The Dufour glands from three worker ants of Myrmica aloba were dissected out, sealed in a glass capillary, and injected without solvent by the method of Morgan and Wadhams (12) onto a 25 m x 0.32 mm OV-I fused silica column using helium as carrier gas at 1 ml min- l. The individual compounds were trapped through a metal splitter into glass capillaries held in a polyethylene cap tilled with liquid nitrogen. The trapped farnesenes were each ozonized and reduced as described above. The mean amount of these compounds in the Dufour glands was determined by gas chromatography of 10 individual samples. RESULTS AND DISCUSSION
Pheromones often contain vinyl or isopropylidene end groups which upon ozonolysis yield formaldehyde and acetone, respectively. The molecules, being very small, are not easy to determine directly using a packed column or capillary GC. However, using a porous polyaromatic resin bead column such as one of the
300
OLLETT
AND MORGAN
0
5 min.
min.
FIG. 4. (A) Gas chromatogram showing the products obtained on ozonolyzis and reduction of &lododecadienyl acetate: (a) water and borane, (b) methanol (produced from the C-9 carbon atom), (c) acetaldehyde, and (d) ethanol. (B) Gas chromatogram of reaction blank showing amounts of (a) water and borane, (b) methanol, (c) acetaldehyde, and (d) ethanol produced under the conditions of the experiments.
Porapak or Chromosorb series, these molecules can easily be determined at moderate temperatures. Formaldehyde, however, still causes a problem because of its low flame response (due to its high state of oxidation), but reduction with borohydride to methanol produces a greater flame response. This, together with the good peak shape of methanol and short retention time, makes it amenable to GC with an FID detector. Sensitivity of detection of formaldehyde is of primary importance. This method is carried out in situ and uses only 1 t.~l of water as solvent in total. The sample is directly trapped from the GC, ozonized, and then reacted with 1 ~1 of an aqueous solution of sodium borohydride. The reaction is then completed by being left in the injection port of the GC for 5 min. This method therefore avoids the loss of sample by poor extraction or evaporation of excess solvent. The method was found to be almost quantitative and reproducible for as little as lOpa g of formaldehyde. Greater sensitivity was limited by the size of peaks in the blank determinations.
DETERMINATION
Sodium Borohydride
301
OF FORMALDEHYDE
Reduction
Ozonolysis is a widely used method for the determination of double bond position. In this method solventless ozonolysis is carried out to form the ozonide, which is then reduced to give the alcohol with an aqueous solution of sodium borohydride. Previous work using sodium borohydride to reduce aldehydes and ketones to their respective alcohols has been carried out (13). Also, samples of 1 kg have been reduced in the syringe barrel and products larger than hexanol have been identified (14). The advantage of using an aqueous solution of sodium borohydride is that water gives virtually no response with an FID detector. A peak, apparently due to borane, is found at very short retention time but methanol is eluted after it, and no important peaks are obscured. Though the method was developed for formaldehyde it can be used equally well for acetaldehyde, acetone, and propanal and with less sensitivity for butanal and butanone. In each case the corresponding alcohol is observed in the gas chromatogram (Figs. 2-4). As an example of the application of the method to the determination of end groups in natural products, the homologous group of farneseries found in Myrmica ants (1.5) has been examined. The structures VI to VIII were originally assigned to these compounds from mass spectral evidence, but with the isolation of insect juvenile hormones of similar structure but not the
OH
CHp=C :I Q
\c
0
O-f
\
(IV)
(III)
CHO (“I)
(VI
: R*=
R3=
(“II)
R’=
R’
R3,
Me.
(VIII)
R’=
632:
EL.
Me R2= RJ:
Et Me
302
OLLET-T
AND MORGAN
same carbon skeleton for VII, and the later isolation of the allofarnesene IX (26), confirmation of these structures was required. Attygalle and Morgan showed the formation of acetone and 4-oxopentanal from VI after ozonolysis, of acetone and 4-oxohexanal from VII, and of butanone and 4-oxopentanal from VIII, confirming the left-hand portions of these molecular structures. We have now been able to isolate formaldehyde (determined as methanol) from all three compounds obtained from M. aloba workers, confirming the terminal double bond and eliminating structures such as IX. Isopropanol (from the isopropylidene end group) was also found for compounds VI and VII. The 2-butanol that would be formed from VIII has a much longer retention time and hence poor peak shape under the conditions suitable for methanol determination, 2-Butanol can be determined by this method at somewhat greater concentrations. The mean amounts of the farnesene homologs found in the ants (together with their standard deviations) and an approximate value of the amount ozonized, assuming a trapping efficiency similar to that obtained for nerolidol, are shown in Table 1. The farnesenes are very unstable in air and are rapidly polymerized. This may partly account for the low yields obtained. Other compounds tested are shown in Table 2. The method is equally useful for determining formaldehyde and the other lower carbonyl compounds as atmospheric pollutants, if the compounds are trapped in the manner described here or in any other convenient way and reduced with sodium borohydride as described. Nanogram quantities can be determined easily and quickly. The sensitivity of the method, like that of the earlier method we described for simple carbonyl compounds (8), is limited chiefly by the high levels of acetone and formaldehyde found in the laboratory atmosphere. Capillaries made from clean glassware showed blank determinations containing approximately 5 ng of acetone when oxygen-free nitrogen was passed through a column of 10% PEG 20M at 150°C and then through the liquid nitrogen-cooled capillaries. The method of methoxymercuration-demercuration followed by mass spectrometry is very useful in the location of double bond positions in monoalkenes (17), but as four ions are produced for each double bond, the method becomes too complicated with polyenes. The iodine-catalyzed addition of dimethyl disultide to alkenes (18, 19) gives only two mass spectral ions per double bond and is a more convenient method to use but even this becomes unmanageable in the analysis of TABLE 1 of 10 Individual Samples of Dufour Glands of 10 Myrmica aloba Workers
Results of Gas Chromatography
Compound
Mean amount per insect (ng)
Standard deviation (ng)
Amount0 collected (ng)
Theoretical yieldb of methanol (ng)
Famesene Homofamesene Bishomofamesene
641 768 277
2 546 k-600 2 250
796 944 340
117 130 44
(1Assuming 41% efficiency of trapping (as for nerolidol). b Compare with actual yield in column 5 of Table 2.
DETERMINATION
OF FORMALDEHYDE
303
TABLE 2 Results of Ozonolysis and Borohydride Reduction of Some Selected Alkenes (800 ng of Each Injected)
Compound Nerolidol (I) Eugenol (II) Limonene (III) Safrole (IV) Citronella1 (V) 8, IO-Dodecadienyl acetate Farnesene” (VI) Homofarnesene” Bishomofamesene”
Trapping efficiency (%)
Ozonolyzis products determined
Amount found (ng)
Yield on analysis and reduction (%)
41 20 52 38 35 40
Methanol and isopropanol Methanol Methanol Methanol Isopropanol Ethanol
30 & 52 23 78 40 59 49
64 73 80 67 54 77
28” 65’ 36’ 67’ 14”
24 28 28 26 32
b
(VII)
b
b
Methanol Isopropanol Methanol Isopropanol Methanold
Note. The products were examined on a Porapak Q column at 150°C. a Trapped from three Dufour glands of M. aloha. b Efficiency assumed to be similar to that of nerolidol. c Poor yield could be due to inefficient trapping, cyclization of the trapped material, or small quantity in the ant glands. d Methanol only was detected; 2-butanol, the other product, was not detected.
polyenes such as the farnesene, homofarnesene, and bishomofarnesene series. Here ozonolysis and identification of key fragments unambiguously locates the double bonds and the nature of the side chains (15). The present method has been used with these compounds trapped from the glands of workers of the myrmicine ant, M. aloba. ACKNOWLEDGMENTS We thank the Royal Society for a grant for gas chromatography for providing and identifying the M. aloha ants.
equipment and C. A. Collingwood
REFERENCES Moore, B. P., and Brown, W. V., J. Chromatogr. 60, 157-166 (1971). Beroza, M. A., and Bierl, B. A., Anal. Chem. 39, 1131-1135 (1967). Fung, K., and Grosjean, D., Anal. Chem. 53, 168-171 (1981). Lipari, F., and Swarin, S. J., J. Chromarogr. 247, 297-306 (1982). Magin, D. F., J. Chromatogr. 178, 219-227 (1979). Magin, D. F., J. Chromarogr. 202, 255-261 (1980). Kobayashi, K., Tanaka, M., and Kawai, S., J. Chromatogr. 187, 413-417 (1980). Attygalle, A. B., Morgan, E. D., and Ollett, D. G., J. Chromatogr. 367, 207-212 (1986). Attygalle, A. B., and Morgan, E. D., Anal. Chem. 58, 3054-3058 (1986). Baker, R., Bradshaw, J. W. S., Evans, D. A., Higgs, M. D., and Wadhams, L. J., J. Chromatogr. Sci. 14, 425-427 (1976). 11. Attygalle, A. B., and Morgan, E. D., J. Chromatogr. 290, 321-330 (1984). 12. Morgan, E. D., and Wadhams, L. J., J. Chromatogr. Sci. 10, 528-529 (1972). 13. Hoff, J. E., and Feit, E. D., Anal. Chem. 36, 1002-1008 (1964). 1.
2. 3. 4. 5. 6. 7. 8. 9. 10.
OLLETT
304
AND MORGAN
14. Fredricks, K. M., and Taylor, R., Anal. Chem. 15. Attygalle, A. B., and Morgan, E. D., J. Chem. 16. Williams, H. J., Strand, M. R., and Vinson, S. 17. Abley, P., McQuillan, F. J., Minniken, D. E., Chem. 18. 19.
Sot.
Chem.
Commun.,
348-349
38, Sot.
1961-1962 (1966).
Perkin Trans. I 949-951 (1982). B., Experientia 37, 1159-1169 (1971). Kusamran, K., Masken, K., and Polgar, N., J.
(1970).
Francis, G. E., and Veland, K., J. Chromarogr. 219, 379-384 (1981). Billen, J. P. J., Evershed, R. P., Attygalle, A. B., Morgan, E. D., and Ollett, D. G., J. Chem. Ecol. 12, 669-685 (1986).
JOURNAL
35, 296-304 (1987)
Identification and Determination of Parts-per-Million Formaldehyde and Lower Carbonyl Homologs by Gas Chromatography DAVID G. OLLETT AND E. DAVID MORGAN~ Department of Chemistry, University of Keele, Keele, Staffordshire ST5 5BG, United Kingdom Received November 5, 1986; accepted November 19, 1986 Formaldehyde, acetaldehyde, acetone, and propionaldehyde may be identified and determined in concentrations of parts per million by reduction to the corresponding alcohol followed by gas chromatography of the alcohols on porous polyaromatic resin beads. The method may be used for determining the position of double bonds in, e.g., vinyl and isopropylidene groups by prior ozonolysis. The method is limited in sensitivity chiefly by the levels of these lower carbonyl compounds in the atmosphere. o 1987 Academic PXSS, IIIC.
INTRODUCTION
While gas chromatography remains the most sensitive method for determining most volatile organic compounds, it singularly fails for the analysis of formaldehyde because of the low flame response of this compound (1, 2). To use the thermal conductivity detector means a loss of sensitivity of about loo-fold. The demand for increasingly sensitive methods for determining atmospheric pollutants and the need for sensitive methods for detecting and quantifying small carbonyl compounds produced in the micro-ozonolysis of pheromones has caused us to seek new methods for determining them (3-7). The chief difficulty is to determine formaldehyde alone or to find the relative amounts of formaldehyde and the other carbonyl compounds produced by ozonolysis and to find a system where these substances are not hidden by a large solvent peak. We have recently described a method (8) for separating and determining nanogram amounts of formaldehyde, acetaldehyde, propionaldehyde, acetone, etc., by conversion to their O-benzyloximes in the small scale device, the Keele micro-reactor (9), and separating and determining the derivatives on a packed or capillary GC column. We now describe an alternative method, by which the carbony1 compounds can be reduced efficiently to the corresponding alcohols and determined with a flame ionization detector on a porous polyaromatic resin bead column. Methanol gives a much better flame response than formaldehyde and good peak shape with little possibility of interference by the other compounds and, with this method, no interference from solvent. EXPERIMENTAL Apparatus and chromatographic conditions. The analytical work was performed with a Pye PU4500 gas chromatograph with a flame ionization detector (FID) using a 1.5 m x 4 mm column tilled with Porapak Q (120- 150 mesh, Waters r To whom correspondence
should be addressed. 296
0026-265X187 $1.50 Copyright All rights
0 1987 by Academic Press, Inc. of reproduction in any form reserved.
DETERMINATION
OF FORMALDEHYDE
297
Associates, Inc.). The oven temperature was 15O”C, with nitrogen used as the carrier gas at 50 ml/min. The preparative work was performed with a Pye 104 gas chromatograph with a flame ionization detector using a 2.75 m x 4 mm column of 10% PEG 20M on Chromosorb W (loo-120 mesh). Nitrogen was used as the carrier gas at a flow rate of 40 ml/min. Trapping technique. The gas chromatograph effluent was passed through a splitter of the design of Baker et al. (10) to give a 95:5 (outlet:FID) split ratio. Trapping of the alkenes was then carried out according to the method of Attygalle and Morgan (II) using a glass capillary (45 x 0.5-mm o.d., 0.45-mm i.d.) threaded through two holes in a polyethylene sample tube cap (15-mm diameter) filled with liquid nitrogen. Ozonolysis. A glass tube was drawn down to a narrow capillary so that it would Ozonized oxygen I
-
-
Trapping
capillary
Alkene
-I P 0.45 mm
FIG. 1. Apparatus for ozonolysis of nanogram quantities of alkene collected from a gas chromatograph. Dimensions are approximate.
298
0LLEl-T
I 10
AND
MORGAN
I 5
1 0
min.
FIG. 2. Gas chromatogram showing the separation of (a) water, (b) methanol, (c) acetaldehyde, (d) ethanol, (e) acetone, and (f) isopropanol on a Porapak Q column.
fit into the capillary containing the alkene (Fig. 1). A fine stream of ozonized oxygen from an ozone microgenerator (2) was then passed through the capillary to form the ozonides. These were then reduced to alcohols using aqueous sodium borohydride solution. Sodium borohydride reduction. A solution of sodium borohydride (1 ~1 of 0.1 g in 10 ml water) was added to the ozonide in the glass capillary using a 5~1 syringe (SGE, London) fitted with a 75 x 0.23-mm-o.d. steel needle (II). The capillary was then broken carefully into pieces and these pieces were sealed in another glass capillary for solid sampling. After 5 min in the injection port of the gas chromatograph, the reaction was complete and the glass capillary was crushed. The alcohols were then analyzed using the Porapak Q column. Reaction blank. The nitrogen carrier gas was passed through an empty capillary cooled in liquid nitrogen as it emerged from the gas chromatograph. Ozonized oxygen from a micro-ozone generator was then passed down the capillary followed by 1 cl.1 of the aqueous sodium borohydride solution. This was then sealed in another capillary and reinjected onto the gas chromatograph at the same
DETERMINATION
299
OF FORMALDEHYDE
I
I
1
10
5
0
min
FIG. 3. Gas chromatogram showing the methanol obtained by ozonolysis and reduction of safrole. The large peak (a) consists of a mixture of water and borane and (b) methanol.
attenuation as that used for the reduction of the alkenes. This served as the control blank determination. Isolation OffarneSeneSfrom ants. The Dufour glands from three worker ants of Myrmica aloba were dissected out, sealed in a glass capillary, and injected without solvent by the method of Morgan and Wadhams (12) onto a 25 m x 0.32 mm OV-I fused silica column using helium as carrier gas at 1 ml min- l. The individual compounds were trapped through a metal splitter into glass capillaries held in a polyethylene cap tilled with liquid nitrogen. The trapped farnesenes were each ozonized and reduced as described above. The mean amount of these compounds in the Dufour glands was determined by gas chromatography of 10 individual samples. RESULTS AND DISCUSSION
Pheromones often contain vinyl or isopropylidene end groups which upon ozonolysis yield formaldehyde and acetone, respectively. The molecules, being very small, are not easy to determine directly using a packed column or capillary GC. However, using a porous polyaromatic resin bead column such as one of the
300
OLLETT
AND MORGAN
0
5 min.
min.
FIG. 4. (A) Gas chromatogram showing the products obtained on ozonolyzis and reduction of &lododecadienyl acetate: (a) water and borane, (b) methanol (produced from the C-9 carbon atom), (c) acetaldehyde, and (d) ethanol. (B) Gas chromatogram of reaction blank showing amounts of (a) water and borane, (b) methanol, (c) acetaldehyde, and (d) ethanol produced under the conditions of the experiments.
Porapak or Chromosorb series, these molecules can easily be determined at moderate temperatures. Formaldehyde, however, still causes a problem because of its low flame response (due to its high state of oxidation), but reduction with borohydride to methanol produces a greater flame response. This, together with the good peak shape of methanol and short retention time, makes it amenable to GC with an FID detector. Sensitivity of detection of formaldehyde is of primary importance. This method is carried out in situ and uses only 1 t.~l of water as solvent in total. The sample is directly trapped from the GC, ozonized, and then reacted with 1 ~1 of an aqueous solution of sodium borohydride. The reaction is then completed by being left in the injection port of the GC for 5 min. This method therefore avoids the loss of sample by poor extraction or evaporation of excess solvent. The method was found to be almost quantitative and reproducible for as little as lOpa g of formaldehyde. Greater sensitivity was limited by the size of peaks in the blank determinations.
DETERMINATION
Sodium Borohydride
301
OF FORMALDEHYDE
Reduction
Ozonolysis is a widely used method for the determination of double bond position. In this method solventless ozonolysis is carried out to form the ozonide, which is then reduced to give the alcohol with an aqueous solution of sodium borohydride. Previous work using sodium borohydride to reduce aldehydes and ketones to their respective alcohols has been carried out (13). Also, samples of 1 kg have been reduced in the syringe barrel and products larger than hexanol have been identified (14). The advantage of using an aqueous solution of sodium borohydride is that water gives virtually no response with an FID detector. A peak, apparently due to borane, is found at very short retention time but methanol is eluted after it, and no important peaks are obscured. Though the method was developed for formaldehyde it can be used equally well for acetaldehyde, acetone, and propanal and with less sensitivity for butanal and butanone. In each case the corresponding alcohol is observed in the gas chromatogram (Figs. 2-4). As an example of the application of the method to the determination of end groups in natural products, the homologous group of farneseries found in Myrmica ants (1.5) has been examined. The structures VI to VIII were originally assigned to these compounds from mass spectral evidence, but with the isolation of insect juvenile hormones of similar structure but not the
OH
CHp=C :I Q
\c
0
O-f
\
(IV)
(III)
CHO (“I)
(VI
: R*=
R3=
(“II)
R’=
R’
R3,
Me.
(VIII)
R’=
632:
EL.
Me R2= RJ:
Et Me
302
OLLET-T
AND MORGAN
same carbon skeleton for VII, and the later isolation of the allofarnesene IX (26), confirmation of these structures was required. Attygalle and Morgan showed the formation of acetone and 4-oxopentanal from VI after ozonolysis, of acetone and 4-oxohexanal from VII, and of butanone and 4-oxopentanal from VIII, confirming the left-hand portions of these molecular structures. We have now been able to isolate formaldehyde (determined as methanol) from all three compounds obtained from M. aloba workers, confirming the terminal double bond and eliminating structures such as IX. Isopropanol (from the isopropylidene end group) was also found for compounds VI and VII. The 2-butanol that would be formed from VIII has a much longer retention time and hence poor peak shape under the conditions suitable for methanol determination, 2-Butanol can be determined by this method at somewhat greater concentrations. The mean amounts of the farnesene homologs found in the ants (together with their standard deviations) and an approximate value of the amount ozonized, assuming a trapping efficiency similar to that obtained for nerolidol, are shown in Table 1. The farnesenes are very unstable in air and are rapidly polymerized. This may partly account for the low yields obtained. Other compounds tested are shown in Table 2. The method is equally useful for determining formaldehyde and the other lower carbonyl compounds as atmospheric pollutants, if the compounds are trapped in the manner described here or in any other convenient way and reduced with sodium borohydride as described. Nanogram quantities can be determined easily and quickly. The sensitivity of the method, like that of the earlier method we described for simple carbonyl compounds (8), is limited chiefly by the high levels of acetone and formaldehyde found in the laboratory atmosphere. Capillaries made from clean glassware showed blank determinations containing approximately 5 ng of acetone when oxygen-free nitrogen was passed through a column of 10% PEG 20M at 150°C and then through the liquid nitrogen-cooled capillaries. The method of methoxymercuration-demercuration followed by mass spectrometry is very useful in the location of double bond positions in monoalkenes (17), but as four ions are produced for each double bond, the method becomes too complicated with polyenes. The iodine-catalyzed addition of dimethyl disultide to alkenes (18, 19) gives only two mass spectral ions per double bond and is a more convenient method to use but even this becomes unmanageable in the analysis of TABLE 1 of 10 Individual Samples of Dufour Glands of 10 Myrmica aloba Workers
Results of Gas Chromatography
Compound
Mean amount per insect (ng)
Standard deviation (ng)
Amount0 collected (ng)
Theoretical yieldb of methanol (ng)
Famesene Homofamesene Bishomofamesene
641 768 277
2 546 k-600 2 250
796 944 340
117 130 44
(1Assuming 41% efficiency of trapping (as for nerolidol). b Compare with actual yield in column 5 of Table 2.
DETERMINATION
OF FORMALDEHYDE
303
TABLE 2 Results of Ozonolysis and Borohydride Reduction of Some Selected Alkenes (800 ng of Each Injected)
Compound Nerolidol (I) Eugenol (II) Limonene (III) Safrole (IV) Citronella1 (V) 8, IO-Dodecadienyl acetate Farnesene” (VI) Homofarnesene” Bishomofamesene”
Trapping efficiency (%)
Ozonolyzis products determined
Amount found (ng)
Yield on analysis and reduction (%)
41 20 52 38 35 40
Methanol and isopropanol Methanol Methanol Methanol Isopropanol Ethanol
30 & 52 23 78 40 59 49
64 73 80 67 54 77
28” 65’ 36’ 67’ 14”
24 28 28 26 32
b
(VII)
b
b
Methanol Isopropanol Methanol Isopropanol Methanold
Note. The products were examined on a Porapak Q column at 150°C. a Trapped from three Dufour glands of M. aloha. b Efficiency assumed to be similar to that of nerolidol. c Poor yield could be due to inefficient trapping, cyclization of the trapped material, or small quantity in the ant glands. d Methanol only was detected; 2-butanol, the other product, was not detected.
polyenes such as the farnesene, homofarnesene, and bishomofarnesene series. Here ozonolysis and identification of key fragments unambiguously locates the double bonds and the nature of the side chains (15). The present method has been used with these compounds trapped from the glands of workers of the myrmicine ant, M. aloba. ACKNOWLEDGMENTS We thank the Royal Society for a grant for gas chromatography for providing and identifying the M. aloha ants.
equipment and C. A. Collingwood
REFERENCES Moore, B. P., and Brown, W. V., J. Chromatogr. 60, 157-166 (1971). Beroza, M. A., and Bierl, B. A., Anal. Chem. 39, 1131-1135 (1967). Fung, K., and Grosjean, D., Anal. Chem. 53, 168-171 (1981). Lipari, F., and Swarin, S. J., J. Chromarogr. 247, 297-306 (1982). Magin, D. F., J. Chromatogr. 178, 219-227 (1979). Magin, D. F., J. Chromarogr. 202, 255-261 (1980). Kobayashi, K., Tanaka, M., and Kawai, S., J. Chromatogr. 187, 413-417 (1980). Attygalle, A. B., Morgan, E. D., and Ollett, D. G., J. Chromatogr. 367, 207-212 (1986). Attygalle, A. B., and Morgan, E. D., Anal. Chem. 58, 3054-3058 (1986). Baker, R., Bradshaw, J. W. S., Evans, D. A., Higgs, M. D., and Wadhams, L. J., J. Chromatogr. Sci. 14, 425-427 (1976). 11. Attygalle, A. B., and Morgan, E. D., J. Chromatogr. 290, 321-330 (1984). 12. Morgan, E. D., and Wadhams, L. J., J. Chromatogr. Sci. 10, 528-529 (1972). 13. Hoff, J. E., and Feit, E. D., Anal. Chem. 36, 1002-1008 (1964). 1.
2. 3. 4. 5. 6. 7. 8. 9. 10.
OLLETT
304
AND MORGAN
14. Fredricks, K. M., and Taylor, R., Anal. Chem. 15. Attygalle, A. B., and Morgan, E. D., J. Chem. 16. Williams, H. J., Strand, M. R., and Vinson, S. 17. Abley, P., McQuillan, F. J., Minniken, D. E., Chem. 18. 19.
Sot.
Chem.
Commun.,
348-349
38, Sot.
1961-1962 (1966).
Perkin Trans. I 949-951 (1982). B., Experientia 37, 1159-1169 (1971). Kusamran, K., Masken, K., and Polgar, N., J.
(1970).
Francis, G. E., and Veland, K., J. Chromarogr. 219, 379-384 (1981). Billen, J. P. J., Evershed, R. P., Attygalle, A. B., Morgan, E. D., and Ollett, D. G., J. Chem. Ecol. 12, 669-685 (1986).