Nova pak c18 reverse phase column

Compounds 1 and 2 (each about 0.5 mg) were heated separately in 1 mL of 6 N HCl at 100 °C for 16 h, followed by removal of the excess HCl under vacuum. To each dry hydrolysate, 200 µL of 1% solution of 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide (FDAA, Marfey’s reagent) [24 (link)] in acetone and 40 µL of 1.0 M NaHCO₃ were added. The reaction mixture was heated at 45 °C for 1.5 h, cooled, and acidified with 20 µL of 2.0 M HCl. Similarly, standard amino acids (d and l) of phenylalanine and leucine were derivatized separately. The derivatized standard amino acids and hydrolysates of 1 and 2 were subjected to HPLC on Nova-Pak C18 reverse-phase column (150 × 3.9 mm i.d., 4 mm particle size; Waters, Milford, MA, USA) using the following gradient program. Solvent A was a 50 mM triethylamine–phosphate buffer (pH 3.5) containing 25% (v/v) MeOH, and solvent B was the same buffer containing 70% MeOH. The mobile phase was a linear gradient from 0 to 100% B (100 to 0% A) in 40 min, at a flow rate of 0.65 mL/min at 25 °C. The eluted peaks were monitored at 340 nm. The retention times for FDAA derivatives of standards and compounds 1 and 2 were as follows: (l)-leucine (t_R 27.2 min), (d)-leucine (t_R 36.0 min), compound 1 (t_R 27.2 min) and compound 2 (t_R 27.2 min) (Figure S39).

Extracted pigments were loaded on to an Agilent‐1200 series HPLC system in conjunction with a Nova‐Pak C18 reverse phase column (4 μm particle size, 3.9 mm × 150 mm; Waters, USA).