The samples were washed with water and eluted with a gradient of NaCl (0.1–0.5 M) at a flow rate of 0.1 mL/min. Cleaning (1.0 M NaCl) and re-equilibration (water) steps were performed between each elution. PEG 5000 (2 g) and (NH4)2SO4 (2 g) were weighed and added to sample A (2 mL), sample B (0.4 g) or sample C (0.4 g). The samples were dissolved in water (10 mL) and filtered. The contents were mixed thoroughly using a magnetic stirrer for 1 h for equilibration and were allowed for phase separation for 3 h. After the separation of
LY294002 mouse the two phases, the dull red bottom aqueous phase was discarded and the bright magenta top phase containing PEG and betanin was submitted to extraction with chloroform. The brown polyphenolic components present in the juice accumulate at the interface and were discarded. The PEG was regenerated after extraction with chloroform and the betanin was submitted to UV–Vis and analytical HPLC analysis (Chethana, Nayak, & Raghavarao, 2007). Reversed-phase chromatography was
performed in a Waters (Milford, MA) 600 system equipped with a UV–Vis detector (dual-wavelength, Waters 2489) and a Jupiter-15 (300 Å, 15 μm, 250 × 21.2 mm, Phenomenex, Torrance, CA) C18 column. Gradients were formed between two helium-degassed solvents: solvent A: water with 1% v/v HOAc, solvent B: 60% v/v MeCN/water with 1% v/v HOAc; linear gradient from 5% to 20% B in 60 min at 25 °C, flow rate: 10 mL/min. Samples were monitored by UV–Vis absorption at 254 nm. Absorption spectra were recorded in the UV–Vis region (200–800 nm) at 25 ± 1 °C on a Varian Cary 50
Bio spectrophotometer equipped Selleck JQ1 with a Peltier-thermostatted cell holder (Varian, Palo Alto, CA). The betanin concentration was determined by assuming a molar absorption coefficient (ε) of 6.5 × 104 L mol−1 cm−1 at 536 nm ( Schwartz & Von Elbe, 1980). Spectra were deconvoluted using the Fytik Methamphetamine analysis software ( Wojdyr, 2010). Analytical RP-HPLC separation and analysis were performed on a Waters 2695 Alliance system equipped with a UV–Vis detector (dual-wavelength, Waters 2489) and a Supelcosil LC-18 (300 Å, 5 μm, 150 × 46 mm; Supelco, Bellefonte, PA) C18 column. Solvent A was water with 0.1% v/v TFA, solvent B was 60% v/v MeCN/water with 0.1% v/v TFA; a linear gradient was performed from 5% to 95% B in 20 min at 25 °C, at a flow rate of 1 mL/min, injection volume was 10 μL, with spectrophotometric detection set at 254 and 536 nm. Due to the high polarity of most betalainic pigments (Escribano et al., 1997, Gandia-Herrero et al., 2004, Stintzing et al., 2004 and Wybraniec et al., 2010), the retention times of Bn and iBn are short under these experimental conditions. A Bruker Daltonics Esquire 3000 Plus was used for the ESI-MS analyses. Elution conditions were the same as those used in the RP-HPLC analysis. The vaporiser temperature was 325 °C and the voltage was maintained at 4.0 kV. The sheath gas was nitrogen, operated at a pressure of 26 psi (6.0 L/min).