Licrochart rp 18 column

The composition and quantification of the anthocyanins released from the lees was performed by injecting 20 µL of the filtered supernatant into a Waters 2695 liquid chromatograph (Waters, Milford, PA, USA). The chromatographic conditions used in this study were those described in Busse-Valverde et al. [42 (link)]. In the chromatographic analysis, a Licrochart RP-18 column (Merck, Darmstadt, Germany), 25 × 0.4 cm, particle size of 5 μm was used. The flow (0.8 mL/min) consisted of a gradient of two different phases: acetonitrile (100%) and a solution of formic acid at 4.5% (v/v) in high purity water. A diode array detector was used. Anthocyanins were quantified at 520 nm as malvidin-3-glucoside, using malvidin-3-glucoside chloride (Extrasynthese, Genay, France) as an external standard.

Grape and wine anthocyanins were determined according to Gil-Muñoz et al. [15 (link)]. Twenty berries for each treatment and replication were randomly taken from the different clusters. Grapes were peeled with a scalpel, and the skins were stored at −20 °C until analysis. Samples (2 g) were immersed in methanol (40 mL) in hermetically closed tubes and placed on a stirring plate at 150 rpm and 25 °C. After 4 h, the methanolic extracts were filtered through 0.45 μm nylon filters and directly analyzed by HPLC. The HPLC analyses were performed on a Waters 2690 liquid chromatograph (Waters, Milford, PA, USA), equipped with a Waters 996 diode array detector and a Licrochart RP-18 column (Merck, Darmstadt, Germany), 25 × 0.4 cm, 5-µm particle size, using water plus 5% formic acid (solvent A) and HPLC grade methanol (solvent B) as solvents at a flow rate of 1 mL min⁻¹. Elution was performed with a gradient starting with 2% B to reach 32% B in 40 min, isocratic for 15 min, 50% B at 70 min, 60% B at 75 min, and then isocratic for 5 min. Chromatograms were recorded at 520 nm. Anthocyanins were quantified at 520 nm as malvidin 3-glucoside, using malvidin 3-glucoside chloride as external standard (Extrasynthèse, Genay, France).