Agilent 1200 hplc dad system

The concentration of the xanthophyll cycle pigments was determined by HPLC pigment analysis. The pigments were extracted from thylakoid membranes by 80% acetone and analysed using an Agilent 1,200 HPLC–DAD system (Agilent, USA) equipped with a LiChroCART RP-18 (250 × 4 mm, 5 μm) chromatographic column (Merck, Germany) applying the eluent system and gradient programme described by^{72 (link)}. Dark-adapted thylakoid membranes of appropriate pH were taken after 10 min of the dark period (control samples), and illuminated thylakoids, after 10 min of illumination (770 µmol photons m⁻² s⁻¹; after measurement of light-induced NPQ). The extent of Vx de-epoxidation was determined as de-epoxidation state of xanthophyll cycle pigments DEPS = (Zx + Ax)/(Vx + Ax + Zx) according to^{73 (link)}.

An extract of photosynthetic pigments was prepared in 100% acetone with the addition of a small amount of MgCO₃. The extract was centrifuged for 3 min at 3468 × g (EBA 20, Hettich, Germany) and diluted to a final concentration of 80% acetone. The supernatant was then filtered through a 0.22 µm PTFE filter (Whatman, UK) into vials.
The photosynthetic pigment extracts were analyzed using an Agilent 1200 HPLC–DAD system (Agilent, USA) equipped with a LiChroCART RP-18 (250 × 4 mm, 5 µm) chromatographic column (Merck, Germany). The separation was performed using two mobile phases (MPs): MP A (acetonitrile/methanol/tris, 241/30/1, v/v/v) and MP B (methanol/n-hexane, 4/1, v/v) with chromatographic column at 22 °C. For the detection of individual pigments, 440 nm light was used. To estimate the relative photosynthetic pigment quantities in A. thaliana plants, conversion factors published by Färber and Jahns (1998 (link)) were used. For more details see Materová et al. (2017 (link)).

Ascorbic acid (AA) was definite in respected of the method of Ruiz–Cruz, et al. [80 (link)]. First, samples of a homogeneous peppers suspension (2 g) of 4% metaphosphoric acid (15 mL) were added. Then, the sample was sonicated/5 min (Sonicator VWR model 150 D; VWR International., West Chester, PA, USA) and centrifuged at 1610× g/10 min at room temperature (Centrifuge Thermo Scientific Sorvall ST16, Porton Down, UK). The sample supernatant was filtered through a polyethylene cartridge of 0.45 μm of pore size (Millipore Corp., Bedford, MA) and identified and quantified using a Agilent 1200, HPLC-DAD system, coupled with an auto-sampler and a UV/Vis detector. Ascorbic acid was scanned in reversed-phase at λ = 254 nm on a Sorbaz Eclipse Plus C18 column (250 × 4.6 mm, 5 μm). Samples (20 μL) were inserted with a gradient elution isocratic and the flow rate 1 mL/min, which was kept at 25 °C. The mobile phase was programmed of 0.2 M KH₂PO₄, pH 2.3 with 85% o-phosphoric acid. Ascorbic acid was prepared, identified in pepper extracts as a standard compound, and used for quantification calibration curves of pure Ascorbic acid (Sigma-Aldrich, DEU). Each extract was analyzed three times.