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Note 41: Hydrocarbon Production in Pine by Direct Thermal Extraction

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By Santford Overton and John J. Manura
1999

INTRODUCTION

Metablolic links have been made between isoprene emission, a precursor of terpenoid compounds, and the photosynthetic and photorespiratory pathways and the influence of environmental conditions on terpene production among woody, tree species. There exixts a significant amount of evidence to indicate that water and nutrient deficiencies or excess ozone and acid rain levels are presently reducing pine productivity, and may reduce growth to a greater extent in future climate change scenarios. It is, therefore, critical that the pathways through which growth is influenced be identified and understood. More sensitive analytical techniques are needed to identify and quantitate the volatile organics to help elucidate these pathways. The purpose of this project is to examined the effect of the environment on hydrocarbon production in white pine and several other species and how this relates to plant productivity. Tree bark and core samples were taken 3 to 4 feet above the base of the tree and analyzed by "Direct Thermal Extraction". This new technique utilizes a thermal desorption apparatus attached to the injection port of a GC/MS system and permits the direct thermal extraction of volatile and semi-volatile organics directly from small sample sizes (mg) without the need for solvent extraction or other sample preparation. The samples are ballistically heated and together with the carrier gas flow through the samples the volatiles are outgassed into the injection port and onto the front of the GC column for subsequent analysis via the GC and/or GC/MS. This proposed study will add considerably to the present level of knowledge regarding the impact of environmental stresses on the growth of individual trees, and provide a linkage to stand and ecosystem level studies.

Instrumentation

A new technique called "Direct Thermal Extraction" which utilizes a thermal desorption apparatus attached to the injection port of a GC/MS system permits the direct thermal extraction of volatile and semi-volatile organics directly from small sample sizes (mg) without the need for solvent extraction or other sample preparation. The sample is placed inside a preconditioned glass-lined stainless steel desorption tube between two glass wool plugs which simply hold the sample in place. The desorption tube containing the sample is attached to the Short Path Thermal Desorption System and a syringe needle attached. The desorption tube with sample is injected into the GC injection port, ballistically heated and together with the carrier gas flow through the sample the volatiles are outgassed into the injection port and onto the front of the GC column for subsequent analysis.

All experiments were conducted using a Scientific Instrument Services model TD3 Short Path Thermal Desorption System accessory connected to the injection port of an HP 5890 Series II GC interfaced to an HP 5971 Mass Selective Detector. The mass spectrometer was operated in the electron impact mode (EI) at 70 Ev and scanned from 35 to 550 daltons during the GC run for the total ion chromatogram.

A short 0.5 meter by 0.53 mm diameter fused silica precolumn was attached to the injection port end of a 30 meter x 0.25 mm i.d. DB-5MS capillary column containing a 0.25m film thickness. The GC injection port was set to 260 deg C and a 10:1 split was used. The head of the column was maintained at -70C using a GC Cryotrap model 951 (Scientific Instrument Services, Ringoes, NJ) during the desorption and extraction process and then ballistically heated to 200 deg C after which the GC oven was temperature programmed from 35 deg C (hold for 5 minutes) to 80 deg C at 10 deg C/min, then to 200 deg C at 4C/min and finally to 260 deg C at a rate of 10 deg C/min.

Experimental

Several individual white pine species as well as 3 other tree species (Blue Spruce, Birch & Maple) were analyzed to identify and compare the volatile organics present to determine the variation among a single tree species as well as between different tree species. Tree bark and core samples were taken 3 to 4 feet above the base of the tree and analyzed by "Direct Thermal Extraction". Core samples from the individual trees measuring 5-10 mg were placed into an inert thermal desorption tube on top of a glass wool plug. The desorption tube with sample was then attached to the Short Path Thermal System and a syringe needle attached. The desorption tube was injected into the GC injection port at a desorption block temperature of 170 deg C. The extracted organics are subsequently cryo-trapped at the front of the GC injection port using the GC cryotrap at a temperature of -70 deg C. After the 5 minute desorption period, the Cryotrap was heated to 200 deg C to elute the volatiles and begin the GC analysis and identification via the mass spectrometer.

Results and Discussion

Core samples from 4 individual white pine species and 3 other tree species including blue spruce, birch and maple were analyzed by "Direct Thermal Extraction" to identify and compare the volatile organics present. Over 100 volatile organics were identified in the trees studied. Most of the trees studied produced 50 or more volatile organics which were identified in addition to many more that were either too weak to identify or in which a good NBS library match was not achievable. The trees possessed numerous mono- and sesquiterpenoid compounds as well as straight and branched chain hydrocarbons, aldehydes, alcohols and esters.

Figure 1

Figure 1 - Direct Thermal Extraction Of White Pine Trees

The predominant terpenes identified in the white pines (Fig. 1) included the monoterpenes -thujene, -pinene, camphene, sabinene, 1--pinene, 2--pinene, cymene, limonene, -terpinene, -terpinolene and 1--terpineol as well as the sesquiterpenes trans-caryophyllene, -humulene and -cadinene in varying concentrations. The white pine species were also found to contain the compounds endobornyl acetate and manoyl oxide in addition to many other higher molecular weight compounds which were not identifiable by the NBS library. Although these individual white pines possessed many common compounds, each tree had its own distinct fingerprint chromatograph.

Figure 2

Figure 2 -Direct Thermal Analysis Of Blue Spruce

In addition to the above terpenoid compounds identified in white pine, high concentrations of the monoterpene -3-carene were also detected in the blue spruce species (Fig. 2). Other volatile organic compounds identified in blue spruce included citronellyl acetate and the higher molecular weight compounds cembrene, isopimaradiene and 1-naphthalenepropanol, .alpha. -eth. Birch species were found to contain high concentrations of -bergamotene, tricyclo [2.2.1.02,6] heptane, 1,7-d, farnesene, trans--farnesene and -sesquiphellandrene and in lesser concentrations -himachalene, -santalene, (-)-AR-curcumene and -bisabolene (Fig. 3). Numerous straight and branched chain hydrocarbons as well as the monoterpenes -pinene, camphene and 1--pinene were the predominant volatile organics detected in maple (Fig. 4). As can be concluded from the above study, there exists not only variation between separate tree species, but also within the species such as was observed in the white pine. In addition to species variation, these differences may also be attributed to the effect of enviromental stresses on the growth of individual trees, and in the future may provide a linkage to stand and ecosystem level studies.

Figure 3

Figure 3 - Direct Thermal Extraction of Birch

Figure 4

Figure 4 - Direct Thermal Extraction Of Maple

Conclusion

Two primary stresses of interest, ozone and moisture, are highly influenced by climate, while the third, nutrient deficiency, is not a climatic factor but can be exacerbated by certain climatic conditions. There exists a significant amount of evidence to indicate that water and nutrient deficiencies as well as excess ozone and acid rain levels are presently reducing pine productivity, and may reduce growth. It is critical that the pathways through which growth is influenced be identified and understood. One such technique that can be used to identify and compare the volatile organics to help elucidate these pathways is "Direct Thermal Extraction". This new technique uses the Short Path Thermal Desorption System attached to the injection port of a GC/MS system and permits the direct thermal extraction of volatile and semi-volatile organics directly from small sample sizes without the need for solvent extraction or other sample preparation. The thermal extraction of volatiles and semi-volatiles using the "Direct Thermal Extraction" technique is thorough and complete with no trace of foreign contaminants from the extraction technique, solvents or other memory effects contributing to the chromatogram. This technique can be easily incorporated into a troubleshooting technique to detect problems in plant productivity which may arise from environmental stresses and/or disease.