The flower scents were collected through dynamic headspace collection method at 12:00–14:00 p.m. Branches with full bloom flowers were clipped in distilled water, and then instantly transported to the laboratory at 25 °C for volatile collection. In each of three experimental replicates, 0.2–0.3 g whole blooming flowers was collected and placed into 100 mL injection vials, which were held for 10 min before using extraction fiber to adsorb the volatiles for 30 min at 30 °C. SPME fiber coated with divinylbenzene/carboxen/polydimenthylsiloxane (50/30 μm DVB/CAR/PDMS) was selected to collect volatiles, as reported in previous studies [31 ]. The emitted volatiles were analyzed using GC-MS, carried out by Shimadzu QP2010 (Shimadzu, Kyoto, Japan) equipped with a DB-5MS capillary column (30 × 0.25 mm, 0.25 μm thickness, Shimadzu, Kyoto, Japan). The injection temperature was held at 250 °C. Helium was the carrier gas in the split mode with the split ratio at 20 and column flow at 27.0 mL min−1. The GC oven temperature started at 40 °C, maintained 2 min, and then increased to 250 °C by 5 °C min−1, holding for 6 min. The mass spectrometer interface temperature was 250 °C and the electron potential was set to 0.9 KV with mass scan range of 30 to 300 m/z units. The solvent cut time was 3.7 min.
The peak area of every scent compound was integrated to obtain the total ion current, with removing the peaks presented in control sample. Individual compound was tentatively identified by comparing the mass spectra with NIST11 library (the National Institute of Standards and Technology 2011, Shimadzu, Japan). Main compounds (relative amount more than 1%) were confirmed by comparing with authentic standard samples. 20 mg of standard compounds (benzaldehyde, benzyl alcohol, benzyl acetate, estragole, eugenol, methyl benzoate and propionic acid benzyl ester) were diluted using 1 mL of hexane. Then, 20 mg of standard compounds of cinnamyl alcohol, methyleugenol, cinnamyl acetate and benzyl benzoate were diluted using 1 mL of methanol. Further, 50 μL of each diluted solution was then co-added to a total volume of 1ml of hexane to make a mixed solution. Then, 5 μL of this mixed solution was placed into 100 mL capped vial for measurement. In addition, propionic acid benzyl ester as internal standard was added into capped vial in every sample. To improve the repeatability of the internal standard in every sample, 5 μL of propionic acid benzyl ester diluted solution was placed into the same position of the injection vial wall.
The peak area of every scent compound was integrated to obtain the total ion current, with removing the peaks presented in control sample. Individual compound was tentatively identified by comparing the mass spectra with NIST11 library (the National Institute of Standards and Technology 2011, Shimadzu, Japan). Main compounds (relative amount more than 1%) were confirmed by comparing with authentic standard samples. 20 mg of standard compounds (benzaldehyde, benzyl alcohol, benzyl acetate, estragole, eugenol, methyl benzoate and propionic acid benzyl ester) were diluted using 1 mL of hexane. Then, 20 mg of standard compounds of cinnamyl alcohol, methyleugenol, cinnamyl acetate and benzyl benzoate were diluted using 1 mL of methanol. Further, 50 μL of each diluted solution was then co-added to a total volume of 1ml of hexane to make a mixed solution. Then, 5 μL of this mixed solution was placed into 100 mL capped vial for measurement. In addition, propionic acid benzyl ester as internal standard was added into capped vial in every sample. To improve the repeatability of the internal standard in every sample, 5 μL of propionic acid benzyl ester diluted solution was placed into the same position of the injection vial wall.
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