We collected volatiles from the leaves of the two morphotypes under the same growth conditions and ambient temperature, in biological triplicates. Approximately 100 g of dry leaves from the two morphotypes, were extracted with 1000 mL of reverse osmosis water using a Clevenger apparatus87 , following four hours of extraction by hydro-distillation. Samples of the essential oils extracted from the leaves were analyzed using gas chromatography with a flame ionization detector (GC-FID) (Shimadzu GC-2010 Plus) and gas chromatography coupled to mass spectrometry (GC–MS) (Shimadzu GCMS-QP2010 SE).
We conducted the analyses according to the following conditions: helium (He) as the carrier gas for both detectors, with the flow and linear speeds of 2.80 mL min−1 and 50.8 cm s−1 (GC-FID), and 1.98 mL min−1 and 50.9 cm s−1 (GC–MS), respectively; injection port temperature of 220 °C with a split ratio of 1:30; fused silica capillary column (30 m × 0.25 mm); stationary phase Rtx®-5MS (0.25 μm film thickness); oven with an initial temperature of 40 °C, maintained for 3 min, then gradually increased by 3 °C min−1 until 180 °C, where it remained for 10 min (total analysis time: 59.67 min); and FID and MS detector temperature of 240 °C and 200 °C, respectively49 (link). The used samples were taken from the vials in 1 μL of a solution containing 3% essential oil dissolved in hexane with 0.1 mol L−1 dimethylacetamide (DMA; external standard for reproducibility control).
The GC–MS analyses were performed using electron impact equipment with an impact energy of 70 eV, scanning speed of 1000, scanning interval of 0.50 fragments s−1, and fragments detected from 29 to 400 (m/z). The GC-FID analyses were carried out in a flame formed by H2 and atmospheric air at a temperature of 300 °C. Flow rates of 40 mL min−1 and 400 mL min−1 were used for H2 and air, respectively. Identification of the compounds in the essential oils was accomplished by comparing the obtained mass spectra with those available in the spectral library database (Wiley 7, NIST 05, and NIST 05 s) and retention indices (RI). To calculate the RIs, we used a mixture of saturated alkanes C7–C40 (Supelco-USA) and adjusted retention time of each compound, obtained by GC-FID. The values calculated for each compound were compared with those reported in literature88 –90 .
We calculated the relative percentage of each compound in the essential oil using the ratio between the integral area of the peaks and the total area of all sample constituents obtained via GC-FID analyses. The compounds with a relative area above 2% were identified and considered predominant if above 10%.
We conducted the analyses according to the following conditions: helium (He) as the carrier gas for both detectors, with the flow and linear speeds of 2.80 mL min−1 and 50.8 cm s−1 (GC-FID), and 1.98 mL min−1 and 50.9 cm s−1 (GC–MS), respectively; injection port temperature of 220 °C with a split ratio of 1:30; fused silica capillary column (30 m × 0.25 mm); stationary phase Rtx®-5MS (0.25 μm film thickness); oven with an initial temperature of 40 °C, maintained for 3 min, then gradually increased by 3 °C min−1 until 180 °C, where it remained for 10 min (total analysis time: 59.67 min); and FID and MS detector temperature of 240 °C and 200 °C, respectively49 (link). The used samples were taken from the vials in 1 μL of a solution containing 3% essential oil dissolved in hexane with 0.1 mol L−1 dimethylacetamide (DMA; external standard for reproducibility control).
The GC–MS analyses were performed using electron impact equipment with an impact energy of 70 eV, scanning speed of 1000, scanning interval of 0.50 fragments s−1, and fragments detected from 29 to 400 (m/z). The GC-FID analyses were carried out in a flame formed by H2 and atmospheric air at a temperature of 300 °C. Flow rates of 40 mL min−1 and 400 mL min−1 were used for H2 and air, respectively. Identification of the compounds in the essential oils was accomplished by comparing the obtained mass spectra with those available in the spectral library database (Wiley 7, NIST 05, and NIST 05 s) and retention indices (RI). To calculate the RIs, we used a mixture of saturated alkanes C7–C40 (Supelco-USA) and adjusted retention time of each compound, obtained by GC-FID. The values calculated for each compound were compared with those reported in literature88 –90 .
We calculated the relative percentage of each compound in the essential oil using the ratio between the integral area of the peaks and the total area of all sample constituents obtained via GC-FID analyses. The compounds with a relative area above 2% were identified and considered predominant if above 10%.
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