Factor four vf 1

The headspace analysis by SPME of samples were carried out using a Clarus 500 model Perkin Elmer (Waltham, MA, USA) gas chromatograph coupled with a mass spectrometer and equipped with a FID (flame ionization detector) [19 (link),29 (link)]. The capillary column used for the separation of compounds, was a Varian Factor Four VF-1. The operative chromatographic and spectrometric conditions were as follows: the oven GC temperature program was: isothermal at 60 °C for 5 min, then ramped to 220 °C at a rate of 6 °C min⁻¹, and finally isothermal at 220 °C for 20 min. The carrier gas was He at flow rate of 1.0 mL min−1 in constant mode. The mass spectra were obtained in the electron impact mode (EI), at 70 eV, in scan mode in the range 35–450 m/z. For the identification of compounds, the matching between their mass spectra with those stored in the Nist 02 mass spectra library database, was performed. Further, the linear retention indices (LRIs), were calculated using a series of alkane standards (C₈–C₂₅n-alkanes) and compared with those available in the literature. Relative amounts of compounds, expressed as percentage, were calculated in relation to the total area of the chromatogram by normalizing the peak area without the use of an internal standard and any factor correction. All analyses were carried out in triplicate.

The analyses were performed by using a gas chromatograph coupled with a mass spectrometer Clarus 500 model Perkin Elmer (Waltham, MA, USA), equipped with an FID (flame detector ionization). The used capillary column was a Varian Factor Four VF-1 [19 (link),20 (link)]. The oven temperature was programmed as follows: initially 55 °C, then increased to 220 °C at 6°/min and finally held for 15 min. Helium was used as carrier gas at a constant rate of 1 mL/min. The mass spectrometer was operated at 70 eV (EI) in full scan mode in the range 40–450 m/z. The ion source and the connection parts’ temperature was 200 °C.
The identification of volatile compounds was performed by matching their mass spectra with those stored in the Wiley 2.2 and Nist 02 mass spectra library databases and by calculating the linear retention indices (LRIs) using a series of alkane standards analyzed under the same conditions as those of the samples. LRIs were then compared with available retention data reported in the literature. The peak areas of the FID signal were used to calculate the relative concentrations of the components expressed as a percentage without the use of an internal standard or any factor correction. All analyses were carried out in triplicate.

To describe the chemical composition of spirulina, a derivatization reaction was performed. For this purpose, ~2 mg of spirulina was added to 300 µL of pyridine and 100 µL of bis-(trimethylsilyl) trifluoroacetamide (BSTFA) (Sigma-Aldrich, Steinheim, Germany), with heating at 80 °C for 30 min. Then, 1 μL of the silylated sample was manually injected at 270 °C into the GC injector in splitless mode. The analysis was performed using the same apparatus GC-FID/GC-MS and the same capillary column (Varian Factor Four VF-1). The oven temperature program was as follows: 60 °C, then a gradient of 7 °C/minute to 170 °C for 1.0 min and a gradient of 8 °C/min to 250 °C for 25 min. Mass spectra were acquired in electron impact mode. The identification of compounds was based on the percentage of similarity plus comparison of mass spectra (MS) using the software NIST data library, with the percentage of total ion chromatograms (TIC%). Relative percentages for the quantification of the components were calculated through electronic integration of the GC-FID peak areas, and no response factors were calculated.

To investigate the volatile composition of encapsulated LEO over time, a Perkin-Elmer (Waltham, MA, USA) Headspace Turbomatrix 40 autosampler connected to a Clarus 500 GC-MS was used. For the separation, a Varian Factor Four VF-1 (60 m × 0.32 mm, 1.0 μm of film thickness) capillary column was used. The GC temperature program was as follows: 40 °C to 220 °C at a rate of 5 °C/min, held for 10 min. Helium was used as carrier gas at a flow rate of 1 mL/min. The mass spectra were recorded at 70 eV (EI) and were scanned in the range of 40–450 m/z. The ion source and the connection parts temperature was 220 °C. The headspace procedure was performed following Garzoli et al. with slight modifications [33 (link)]. The peak areas of the FID signal were used to calculate the relative percentages of the components without the use of an internal standard or any factor correction. The identification of the components was performed by comparing the mass spectra for each compound with those reported on the MS library search (Wiley and Nist 11). Furthermore, linear retention indices (LRIs) of each compound were calculated using a mixture of n-alkanes (C₈–C₃₀, Ultrasci) at the same operating conditions reported above. All analyses were repeated twice.

To analyze the volatile composition of UM, an SPME device from Supelco (Bellefonte, PA, USA) with 1 cm fiber coated with 50/30 μm DVB/CAR/PDMS (divinylbenzene/carboxen/polydimethylsiloxane) was used. The operative conditions for the sampling were the following: equilibration time of 30 min, and sampling time of 60 min at 35 °C. Lastly, the SPME fiber was inserted into the injector of the GC-MS system, maintained at 250 °C and operating as below. A gas chromatograph equipped with a FID and coupled with a mass spectrometer (Clarus 500 model Perkin Elmer-Waltham, MA, USA) was used. The capillary column was a Varian Factor Four VF-1, and the optimized temperature program was the following: from 70 °C to 120 °C at 6 °C/min; from 120 °C to 220 °C at 7 °C/min and held for 10 min. The components were identified by comparison between their calculated linear retention indices (LRIs) and those relating to a mix of n-alkanes. Furthermore, the matching of their mass spectra against commercial libraries (NIST) was performed. All analyses were conducted in triplicate and the results were expressed as average percentages calculated by peak area normalization from GC-FID chromatograms, without the use of an internal standard or correction factors.

To describe the non-volatile content of the solution, a derivatization reaction was performed. For this purpose, 20 mL of the hydroalcoholic solution were dried under reduced pressure at 37 °C to obtain 43.0 mg of solid residue. The solid material was washed 3 times with 2.0 mL of acetone, and the extract combined and dried under reduced pressure at 30 °C to obtain 15.0 mg of residue.
Subsequently, 1 mg of extract was added to 300 µL of pyridine and 100 µL of bis-(trimethylsilyl) trifluoroacetamide (BSTFA) with heating at 60 °C for 30 min. One μL of the silylated sample was manually injected at 270 °C into the GC injector in the splitless mode. The analysis was performed using the same apparatus GC-FID/GC-MS and the same capillary column (Varian Factor Four VF-1). The oven temperature program was as follows: 60 °C then a gradient of 7 °C/min to 170 °C for 1.0 min and a gradient of 8 °C/min to 250 °C for 25 min. Mass spectra were acquired in an electron impact mode. The identification of compounds was based on the percentage of similarity plus comparison of mass spectra (MS) using software NIST data library, with the percentage of total ion chromatograms (TIC%). Relative percentages for quantification of the components were calculated by electronic integration of the GC-FID peak areas, and no response factors were calculated.

To characterize the chemical composition of EO and Hy from exocarp, a Clarus 500 model Perkin Elmer (Waltham, MA, USA) gas chromatograph coupled with a mass spectrometer and equipped with an FID (flame detector ionization) was used. Chromatographic separation was performed using a Varian Factor Four VF-1 capillary column and the gas carrier was He at flow rate of 1.0 mL min⁻¹ in constant flow mode. The analytical conditions were applied following [22 (link)], with some modifications. For MS detection, an electron impact ionization (EI) system was used at 70 eV in scan mode in the range 35–400 m/z. The volatile separated compounds were identified by matching their mass spectra with those stored in the Wiley 2.2 and Nist 02 mass spectra libraries database and by comparison of their linear retention indices (LRIs), relative to C₈–C₃₀ n-alkanes analyzed under the same conditions, with those available in the literature. Relative concentrations of individual compounds were expressed as a percentage of the relative peak area to that of the total peak area without the use of an internal standard and any factor correction. All analyses were carried out in triplicate.