Spd 6av

Gel permeation chromatography (GPC) was carried out on a GPC system with UV-Vis detector SPD-6AV (Shimadzu, Duisburg, Germany) in hexafluoroisopropanol (HFIP; containing 4% ammonium trifluoroacetate) at room temperature using a PFG column (100 Å, 1000 Å; PSS, Mainz, Germany). The flow rate was set to 1 mL/min. The endpoint of the measurements was determined with an internal standard (toluene). Alternatively, GPC measurements were performed on a Polymer Standard Service (PSS, Mainz, Germany) System (MDS, RI detector) running under Win GPC software and using a 50 mm PFG precolumn and three 300 mm PFG columns (Mixed Bed PSS PFG linear M, 7 μm PSS, Mainz, Germany) for measurements in HFIP (containing 5 mmol/L ammonium trifluoroacetate). Columns were kept at 40 °C and the flow rate was set to 1 mL/min. Prior each measurement, samples were filtered through 0.2 μm PTFE syringe filters (Roth, Karlsruhe, Germany). Calibration of both GPC-Systems was performed using poly(methyl methacrylate) standards (PSS, Mainz, Germany) with molar masses from 0.8 kg/mol to 1600 kg/mol.

AsA was assayed as described previously [56 (link)]. AsA was oxidized into dehydro-AsA, derivatized into its osazone, then assayed using a high-performance liquid chromatography (HPLC) system: a Shimadzu HPLC apparatus, comprising LP-6A pumps, an SPD-6AV UV-visible detector, a CTO-6V column oven, and a CDS ver. 5 chromato-data processing system (LAsoft, Ltd., Chiba, Japan). Each sample (20 μL) was applied on a normal-phase HPLC column (Senshu pak, φ 6.0 × 150 mm) and eluted with acetic acid/hexane/ethyl acetate (1:4:5, v/v/v) at 40 °C. The osazone derivatives were monitored by measuring their absorbance at 495 nm. The flow rate was 1.5 mL min⁻¹.

High-performance liquid chromatography (HPLC) technology can be used to directly measure the levels of steviol glycosides (rebaudioside A and stevioside) in Stevia rebaudiana Bertoni^{36 (link)}. The levels of stevia sweetener compounds were estimated at the Central Laboratory, Faculty of Science, Alexandria University. Leaf extracts were separated and identified by HPLC according to³⁷ as follows: the stevioside solution was filtered through a Millipore membrane (13 mm diameter, 0.5 μm pore size) and analyzed using HPLC with a stevioside standard as an internal standard (10 mg/ml). Different extracts of stevia leaves were injected into an HPLC instrument (Shimadzu, Tokyo, Japan; model SPD-6AV) equipped with an LC-GA UV-vis detector and an Alex C-R 4 A recorder. The separation was carried out on a Zorbax NH2 column (25 cm × 0.4 mm I.D.; Dupont, Wilmington, DE, USA) with acetonitrile (HPLC grade, Fisons Co., England) as the mobile phase (acetonitrile: water (80: 20 v/v), adjusted to pH 5 with H₃PO₄). The flow rate was 2 ml/min; the UV detection wavelength was 210 nm; the recorder chart speed was 20 nm/min; and the analysis was performed at ambient temperature (25 °C). Two samples per variety were analyzed, and the quantities of stevioside and rebaudioside A were calculated from the area under each peak.