Zb 5ht inferno capillary column

Around 200 mg of each sampled oil was placed in a 10 mL propylene tube. Then, 200 μL of an internal standard solution (5α-cholestan-3β-ol, Sigma-Aldrich) in hexane was added. The analysis of the unsaponifiable fraction was performed by GC, without a preliminary thin-layer chromatography fractionation. This approach was used for the analysis of sterols and squalene in olive fruit and olive oil by many authors [30 (link),31 (link)]. Alkaline hydrolysis was performed by adding 2 mL of KOH 2%, then tubes were soaked in a water bath at 80 °C for 15 min, and the unsaponifiable fraction was extracted by vortexing with 1 mL hexane and 1.5 mL NaCl 1%. The upper hexane layer was transferred to 2 mL glass vials. Samples were conserved at −20 °C until analysis, within 24 h from preparation.
Analyses were performed on a GC-FID, using a ZB-5HT Inferno capillary column (15 m × 0.32 mm × 0.10 μm film thickness, Phenomenex, Torrance, CA, USA). The carrier gas was helium (column flow 1.5 mL/min), and the split ratio was 1:100. The oven temperature was programmed as follows: 0.5 min at 150 °C, from 150 °C to 240 °C at 8 °C/min and from 240 °C to 370 °C at 25 °C/min held for 5 min, at 370 °C, followed by 320 °C for the injector and 350 °C for the detector (FID). The quantification was performed by external standards of squalene and sterols purchased from Sigma-Aldrich (St. Louis, MO, USA).

The extraction of sterols and squalene from 400 mg of pulp was performed starting from [63 (link)] protocol, by alkaline hydrolysis with 2 mL of KOH 2%, in a water bath at 80 °C for 15 min and using 5α-cholestan-3β-ol (Merck, Darmstadt, Germany) as internal standard. The unsaponifiable fraction was extracted with 1 mL hexane and 1.5 mL NaCl 1 %. The upper hexane layer was analyzed by GC-FID (Agilent Technologies 7820A, Santa Clara, CA, USA), using a ZB-5HT Inferno™ capillary column 30 m × 0.25 mm × 0.10 μm film thickness (Phenomenex). The quantification of each identified component has been carried out using the internal standard method.

Free amino acids were purified via SCX columns according to the manufacturer's protocol (Strata SCX, 55 μm, 70A, 500 mg/3 mL, Phenomenex, USA). Samples were derivatized according to the method by Husek and Sweeley (20 (link)). In brief, the amino acids residue was diluted with a mixture of water/ethanol/pyridine (60:32:8) and then, 5 μL of ethyl chloroformate was added to the mixture. The tube was shaken for 5 s and 100 μL of chloroform (containing 1% ethyl chloroformate) was added to each tube. An aliquot of chloroform layer was taken for the analysis. Separation of amino acids was conducted on a 30-m Zebron ZB-5HT Inferno capillary column of 0.25 mm i.d. and 0.25 μm film thickness (Phenomenex, USA) using nitrogen 5.0 as the carrier gas (Siad, Czech Republic). The injection volume was 1 μL and the flow rate was set as 3 mL/min. The injector temperature was 250°C with a split ratio of 20:1 and the FID temperature was 220°C. The oven temperature was programmed as follows: the column was held initially at 140°C for 1 min, then raised 40°C/min to 300°C. The temperature was held for 1 min. Chromatographic data were recorded and integrated using Clarity software (Data Apex, Czech Republic).