1260 infinity system

Ascorbate was extracted using the method described by Carvajal et al. (2015) [7 (link)]. It was then quantified by HPLC in an Agilent 1260 Infinity system equipped with an Agilent ZORBAX Eclipse plus C18 column (150 mm × 4.6 mm id, 3.5 μm) (flow rate: 0.4 mL min⁻¹; isocratic conditions: 85% Milli-Q water pH 3 and 15% methanol, run time of 7.5 min; detection: 254 nm). Results were calculated according to a calibration curve and expressed as mg ascorbate per kg of fresh weight.

The phenolic compounds were extracted from semolina, olive paste flour (OPF), spaghetti CTRL and spaghetti enriched with 10% OPF in two separated fractions: soluble free and bound, following the method reported by Mattilla [20 (link)] without acid hydrolysis. The difference between total and free polyphenols represented the bound phenolic compounds. HPLC-DAD analysis was performed using the Agilent 1260 infinity system, equipped with a 1260 binary pump, 1260 HiP Degasser, 1260 TCC Thermostat, 1260 Diode Array Detector and Agilent Open Lab Chem Station Rev C.01.05 (35) software. The UV–visible absorption chromatogram was detected at 280 nm, 325 nm and 360 nm. The separation was performed by gradient elution on a 4.6 × 250 mm reversed phase Luna C-18 (5 μm) column (Phenomenex Torrance, California, USA). The elution was performed using methanol (eluent A) and water/acetic acid 95:5 (eluent B). The gradient profile was: 85–60% B (0–25 min), 60% B (25–30 min), 60–37% B (30–45 min), 37% B (45–47 min), 37–0% B (47–52 min). The flow rate was 1 mL/min and the injection volume was 25 μL. Phenolics were identified and quantified by the retention time, spectra and response factors of the pure standards.

¹H, ¹³C and ¹⁹F NMR spectra were recorded at 400 MHz (100 MHz) or 500 MHz (125 MHz) using either Bruker AVANCE-400 or JEOL JNM-ECZR-500 NMR spectrometers, respectively. Chemical shifts are reported in ppm and referenced to residual NMR solvent peaks (¹H NMR: δ 3.62 ppm for THF, δ 7.26 ppm for CDCl₃; ¹³C NMR: δ 77.2 ppm for CDCl₃). High-resolution mass spectra were determined with a Bruker Daltonics APEXIV 4.7 Tesla FT–ICR–MS using ESI or DART ionization. The MALDI-MS spectra were acquired in linear and reflection modes in the Koch Institute at MIT using a Bruker Microflex MALDI-MS spectrometer. UV–Vis absorption spectra were measured using an Agilent Cary 4000 Series UV–Vis spectrophotometer. Gel permeation chromatography (GPC) measurements were performed in tetrahydrofuran using an Agilent 1260 Infinity system and calibrated with a polystyrene standard. ATR–FTIR spectra were acquired using a Thermo Scientific Nicolet 6700 FT–IR with either a Ge or ZnSE crystal for ATR and subjected to the ‘atmospheric suppression’ correction in OMNIC™ Spectra software. Raman spectra were collected with excitation at 633 nm laser using a Horiba LabRAM HR800 Raman spectrometer.

Size exclusion chromatography (SEC) experiments were conducted on a PSS^® Agilent Technologies 1260 Infinity system utilizing a SUPREMA^® column system consisting of a precolumn (8 mm × 50 mm, particle size: 5 µm) and three analytical columns (column 1: 8 mm × 300 mm, particle size of 5 µm, mesh size of 1000 Å; column 2: 8 mm × 300 mm, particle size of 5 µm, mesh size of 1000 Å; and column 3: 8 mm × 300 mm, particle size of 5 µm, mesh size of 30 Å). All columns were always kept at a constant temperature of 50 °C. A 0.1 M NaNO₃ aqueous (Milli-Q^® quality) solution with an added 0.05 wt % NaN₃ served as eluent, while concentrations of PMAAm varied between 1 and 2 mg mL⁻¹. The measurements were conducted at a flow rate of 1 mL min⁻¹ by applying an isocratic PSS^® SECcurity pump and ethylene glycol (20 µL per 100 mL of solvent) as internal standard. Poly(ethylene glycol) was used as reference, and the PSS^® SECcurity differential refractometer detector was operated at 50 °C. Subsequent data analysis was performed with the software WinGPC UniChrom^® V8.10.

The dynamic viscosity of liquid PF resins was determined by a Fungilab Viscolead Adv meter (Fungilab S.A., Barcelona, Spain) equipped with a suitable spindle. The non-volatiles content (solid content) was determined according to DIN EN ISO 3251:2019 [29 ]. The pH value was determined using a digital pH meter (GPH 114 Greisinger, Regenstauf, Germany) by inserting the pH meter electrode into the PF resins. The pH meter was calibrated with buffer solutions at pH 4.0 and 10.0 prior pH measurements. Free formaldehyde content was determined by the hydroxylamine hydrochloride method according to DIN EN ISO 9397:1997 [30 ].
For gel permeation chromatography (GPC) analysis, a 1260 Infinity system (degasser, isocratic pump, automatic liquid sampler, heatable column compartment, RID, MWD @ 280 nm, Agilent (Santa Clara, CA, USA) was used, where: column: 3 × PLgel 5 µ (50 Å, 100 Å, 1000 Å), 7.5 × 300 mm; solvent: tetrahydrofuran (THF); flow rate: 0.6 mL/min; flow rate marker: acetone; calibration: polystyrene standard.
Approximately 40 mg of resin was dissolved in 5 mL of THF. If the resin did not completely dissolve, it was sonicated with slow addition of H₂SO₄ (5% in methanol) until neutral. If the resin was dissolved, but precipitate from additives (such as salts) remained, the mixture was filtered with a syringe filter.

Samples were dissolved in 0.1 M NaOH containing 1 M NaCl (pH 12.5), and centrifuged at 20,400×g and 4 °C for 5 min to remove insoluble materials before separation by anion exchange HPLC under the following conditions: column, TSKgel SAX column (6.0 mm × 15.0 cm) (Tosoh Bioscience, Tokyo, Japan); linear gradient of 100 to a 60:40 ratio of aqueous 0.1 M NaOH containing 1 M NaCl (pH 12.5) to 50% acetonitrile in 0.1 M NaOH; column temperature, 30 °C; flow rate, 1.0 mL min^− 1. Folic acid, PA, DFA and DPA were detected in eluates as absorption at 254 nm using a 1260 Infinity system equipped with a photodiode array detector (Agilent Technologies, Santa Clara, CA, USA).