Shimpack gws c18 column

Analytical RP-HPLC was performed on a Shimadzu Prominence HPLC (Shimadzu) using a Shimadzu Shimpack GWS C₁₈ column (5-micron; 4.6 mm i.d. × 150 mm). Analytes were eluted using a binary gradient of mobile phase A (100% water and 0.1% trifluoroacetic acid) and mobile phase B (30% water, 70% acetonitrile, and 0.1% trifluoroacetic) using the following chromatographic method: 10% B to 100% B in 14 min; flow rate—1 mL/min. Preparative RP-HPLC was performed on a Tri Rotar-VI HPLC system (JASCO) using a Phenomenex Jupiter C18 column (10 micron; 21.2 mm i.d. × 250 mm) using the following chromatographic method: 0% B to 90% B in 45 min; flow rate—14 mL/min. Pure RP-HPLC fractions (>95%) were combined and lyophilized. Electro-spray ionization mass spectrometry (ESI-MS) was performed using a Bruker Esquire 3000+ instrument equipped with an electro-spray ionization source and a quadrupole ion trap detector (QITD).

HIV‐1 protease activity was evaluated as described previously (Boso et al., 2015). For external HIV‐1 protease‐processing reactions, 0.4 μL recombinant HIV‐1 protease (approximately 16 pmol, ab84117, Abcam, Cambridge, MA) and 4 μL of 1 mm HIV‐1 protease substrate (Lys‐Ala‐Arg‐Val‐Nle‐p‐nitro‐Phe‐Glu‐Ala‐Nle amide, Sigma‐Aldrich, St. Louis, MO) were incubated with/without betanin or amaranthin in 30‐μL reaction volumes using phosphate buffer (25 mm NaCl, 25 mm Na₂HPO₄, 1 mm dithiothreitol, pH 4.7) at 25 °C for 2 h. Betalains used in the reaction were added in 10‐, 50‐ and 100‐ fold molar excess over HIV‐1 protease. Saquinavir mesylate (Sigma‐Aldrich) was used as a positive control. After incubation, the HIV protease substrate was quantified using HPLC.
A Shimadzu LC‐20AD system was used for the analytical HPLC separations. Samples were separated on a Shim‐pack GWS C18 column (5 μm; 200 × 4.6 mm i.d.; Shimadzu GLC), and linear gradients were established from 0% B to 50% B over 50 min using 0.05% TFA in water (solvent A) and 0.05% TFA in acetonitrile (solvent B) at a flow rate of 0.5 mL/min and temperature of 25 °C, with elution of the HIV‐1 protease substrate and its degradation products being monitored by absorbance at 214 nm.

Pigments were extracted from N. benthamiana leaves and BY‐2 cells and analysed as described previously (Hatlestad et al., 2012) and extracts were concentrated using a centrifugal concentrator (CC‐105, Tomy Seiko Inc., Tokyo, Japan).
A Shimadzu LC‐20AD system (Kyoto, Japan) was used for analytical HPLC separations. Samples were separated on a Shim‐pack GWS C18 column (5 μm; 200 × 4.6 mm i.d.; Shimadzu GLC, Tokyo, Japan), and linear gradients were run from 0% B to 45% B over 45 min using 0.05% trifluoroacetic acid (TFA) in water (solvent A) and 0.05% TFA in acetonitrile (solvent B) at a flow rate of 0.5 mL/min at 25 °C, with elution being monitored by absorbance at 536 nm. For evaluation of the biological activities of betalains, amaranthin‐ or betanin‐containing HPLC fractions were collected, evaporated to dryness, and the residues were dissolved in water and stored at −20 °C until needed.