Db wax

Extracts were analysed using a Hewlett Packard (HP-6890) GC with a non-polar (HP-1) and a polar (DB-WAX) column (J & W Scientific, Folsom, CA, USA). HP-1 (50 m × 0.32 mm; film thickness, 0.52 μm) and DB-WAX (30 m × 0.32 mm; film thickness, 0.5 µm) columns were fitted with a cool-on-column (COC) injector, hydrogen carrier gas and a flame ionisation detector (FID). The oven temperature was maintained at 30 °C for 0.5 min and then programmed at 5 °C/min to 150 °C, held for 0.1 min, then 10 °C/min to 230 °C and then held for 35 min.
Compounds within the samples were quantified approximately by comparison with an injection (1 μL) of 100 ng/μL solution of n-alkanes (C₇-C₂₅) in hexane, and then concentrated to 50 µL so that the largest peak was at approximately 100 ng/μL, using a gentle flow of purified nitrogen before being re-analysed. As each extract was concentrated 10-fold, from 500 uL to 50 uL, the extracts now contained 33.6 BBEXP per uL. Retention indices (RI) were calculated following Bartle⁶¹ . An aliquot (30 μL) of each of the eight volatile extracts were combined for further analysis.

The pooled MR3 headspace extract was analysed on a combined gas chromatograph and mass spectrometer (GC-MS; 6890 GC and 5975 MS; Agilent Technologies), operated in the electron impact ionization mode at 70 eV. The GC was equipped with fused silica capillary columns (60 m × 0.25 mm, 0.25 μm film thickness), coated with DB-wax (J&W Scientific, Folsom, CA, USA) or HP-5MS (Agilent Technologies). Helium was used as the mobile phase at an average linear flow rate of 35 cm s⁻¹. Two micro-litres of the sample were injected. The temperature programmes were the same as for the GC-EAD analysis. Compounds were identified according to retention times (Kovat’s indices) and mass spectra, in comparison with custom made and NIST05 libraries (Agilent), and confirmed by co-injection of authentic standards: (±)-α-pinene (CAS no. 7785-70-8; Aldrich, 98%), (−)-β-pinene (CAS no. 18172-67-3; Sigma, 99%), 3-carene (CAS no. 13466-78-9; Aldrich, 90%), (±)-limonene (CAS no. 5989-27-5; Sigma, 97%), nonanal (CAS no. 124-19-6; Aldrich, 95%), decanal (CAS no. 112-31-2; Aldrich, 92%), β-caryophyllene (CAS no. 87-44-5; Sigma, 98.5%) and sulcatone (6-methyl-5-hepten-2-one; CAS no. 110-93-0; Fluka, 96%). For quantification, 100 ng of heptyl acetate (99.8% chemical purity; Aldrich) was added as an internal standard to a 20 μl aliquot out of the total 400 μl headspace extract.

This was carried out following the method reported by Nuzzi et al. with minor modifications [39 (link)]. The qualitative and quantitative analyses of volatile compounds were performed using an Agilent 7890A gas chromatograph coupled with an Agilent model 7000B series mass spectrometer (GC–MS) and desorbed for 7 min in a split/splitless GC injection port, which was equipped with an inlet linear specific for SPME use (Agilent Technologies, USA). The volatiles were separated on DB-5 and DB-Wax (30 m × 0.25 mm i.d., 0.25 µm, J & W Scientific) silica capillary columns.
The oven temperature was initially set at 40°C, held for 3 min, then increased to 200°C at a rate of 5°C/min, further increased to 230°C at a rate of 10°C/min and held for 3 min and finally held at 250°C for 3 min. The injection port and ionizing source were kept at 250°C and 230°C, respectively. Helium was used as the carrier gas at a flow rate of 1.2 ml min⁻¹. The injector mode was splitless. Electron-impact mass spectra were generated at 70 eV, with an m/z scan range from 35 to 350 amu. Compounds were identified using NIST 14.0 mass spectra libraries installed in the GC–MS equipment.

The BH-660 pollen headspace extract was injected (2 µl) and analysed on a combined gas chromatograph and mass spectrometer (GC–MS; 6890 GC and 5975 MS; Agilent Technologies), operated in the electron impact ionization mode at 70 eV. The GC was equipped with fused silica capillary columns (30 m × 0.25 mm, 0.25 µm film thickness), DB-wax (J&W Scientific, Folsom, CA, USA) or HP-5MS (Agilent Technologies). Helium was used as the mobile phase at an average linear flow rate of 35 cm s⁻¹. The temperature programmes were the same as for the GC-EAD analysis. Compounds were identified according to retention times (Kovat’s indices) and mass spectra, in comparison with custom made and NIST05 libraries (Agilent), and confirmed by co-injection of authentic standards: (±)-α-pinene (Cas no. 7785-70-8; Aldrich, 98%), (±)-limonene (Cas no. 5989-27-5; Sigma, 97%), nonanal (Cas no. 124-19-6; Aldrich, 95%), benzaldehyde (Cas no. 100-52-7; Aldrich, 99%), p-cymene (Cas no. 99-87-6; Aldrich, 97%). For quantification, 100 ng of heptyl acetate (99.8% chemical purity; Aldrich) was added as an internal standard.

Major aroma compounds such as isobutanol, isoamyl alcohol, methionol, β-phenylethanol, and acetic acid were determined using a variation of the method published by Ortega et al. (2001 (link)). The strategy followed a liquid-liquid microextraction with dichloromethane and uses several internal standards to correct for matrix effects (recoveries above 95% in all cases). 2-Butanol was used as internal standard for isobutanol, 4-methyl-2-pentanol for isoamyl alcohol, and benzyl alcohol and 4-hydroxy-4-methyl-2-pentanone for methionol, and β-phenylethanol, all of them spiked at 1.5 mg L⁻¹ to the wine. Analyses were carried out using a GC-3800 from Varian (Walnut Creek, CA) equipped with a flame ionization detector (FID). The column used was a DB-WAX from J&W (Folsom, CA) 30 m × 0.32 mm × 0.5 mm film thickness, preceded by a silica precolumn from Agilent Technologies (Santa Clara, CA), 3 m × 0.32 mm i.d. The carrier gas was He at 2.2 mL min⁻¹. Two microliters were injected in split mode (1:20). Injector and detector were both kept at 250°C. The temperature program: 40°C for 5 min, then raised at 4°C min⁻¹ up to 102°C, 2°C min⁻¹ up to 112°C, 3°C min⁻¹ up to 125°C, this temperature was kept for 5 min, 3°C min⁻¹ up to 160°C, 6°C min⁻¹ up to 200°C, and this temperature was kept for 30 min.

Oenological parameters as alcohol degree, pH, volatile acidity, total acidity, reducing sugars, glycerol, malic acid, and lactic acid were measured by Fourier transform infrared spectroscopy in the laboratories of Liec Agroalimentaria S.L. (Manzanares, Spain). An accredited laboratory for physico-chemical analysis in wines to conform to UNE-EN ISO/IEC 17025:2005 rules. YAN was determined in must by the formol titration method (Gump et al., 2002 ).
Quantification of major volatile compounds was carried out in a GC Agilent 6850 with a FID detector equipped with a column DB-Wax (60 m × 0.32 mm × 0.5 μm film thickness) from J&W Scientific (Folsom, CA, United States). Analyses were done according to Gil et al. (2006) (link) and Balboa-Lagunero et al. (2013) (link).