Ecological Archives E095-139-A5

Amy M. Trowbridge, Ryan W. Daly, Detlev Helmig, Paul C. Stoy, Russell K. Monson. 2014. Herbivory and climate interact serially to control monoterpene emissions from pinyon pine forests. Ecology 95:1591–1603. http://dx.doi.org/10.1890/13-0989.1

Appendix E. Description of monoterpene chemical analysis of pinyon foliar samples.

Sub-samples of current and one year old pinyon pine needles were removed from the sample branches after all volatile and physiological measurements were taken. The needles were immediately flash frozen and stored in liquid nitrogen, transported to lab, and placed in a -80 °C freezer. For monoterpene extraction, needles were removed from the -80 °C freezer and immediately ground in liquid nitrogen using a chilled mortar and pestle to minimize monoterpene loss. Frozen needle powder was then weighed into 2 dram glass vials, exact weights (between 0.4 and 0.6 g) were recorded, and 2 mL of GC-grade n-hexane (Fisher Scientific) containing 0.1 µL mL-1 (+)-fenchone (Sigma-Aldrich) as an internal standard was added to the vial. Vials were immediately closed with teflon lined caps, mixed with a vortexer, and allowed to soak for 7d at ambient temperature. After the 7d soaking period, 100 µL the solution from each monoterpene sample was injected into micro-inserts in small mouth clear vials capped with PTFE liners (Alltech Associates, Deerfield, IL) for chemical analysis. Remaining samples were then refrigerated for future use.

One µL of each sample was then injected on an Agilent Technologies 6890 gas chromatograph-5975 mass spectrometer (GC-MS) fitted with a Cyclodex-B chrial column (30 m  250 µm   0.25 µm; J&W Scientific). Helium was used as the carrier gas at a flow rate of 1.0 mL/min with a split flow ratio of 25:1. Injector temperature was set at 230 °C. The oven profile consisted of an initial temperature of 60 °C followed by a ramp of 5 °C min-1 to 200 °C, then a second ramp of 25 °C/min to 230 °C. Monoterpene enantiomers were identified by comparing retention times of known standards and mass spectra using the NIST library and MSD Chemstation software. Concentrations for each compound were calculated using 4-point calibration curves with injections of known amounts of pure standards and the internal standard, fenchone. All standards were purchased from Sigma Aldrich (Saint Louis, MO) with the exception of β-phellandrene (Glidco Organics, Jacksonville, FL).


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