Waters alliance 2690 separations module

To conduct our experimental work, samples of the extracts were reconstituted in an acetonitrile/water mixture to a concentration of 18.2 mg/mL. After that, samples were filtered with a 0.22 μm filter and submitted to chromatographic analysis at 28 °C using a Waters Symmetry (3.9 × 150 mm, 5 μm) protected with a Waters Symmetry pre-column (3.9 × 20 mm, 5 μm). The gradient elution was performed with a 0.8 mL/min flow rate using solvent A (0.05% v/v trifluoroacetic acid (TFA) in water (H₂O)), solvent B (acetonitrile (MeCN)), and solvent C (MeOH) and the gradient program presented in Table 6. Peaks were detected at the maximum intensity and maxplot chromatograms were generated using Waters Millenium 32 software. HPLC-UV/DAD analysis was performed using a Waters Alliance 2690 Separations Module (Waters Corporation, Milford, MA, USA) coupled with a Waters 996 photodiode array detector (UV/DAD) (Waters Corporation, MA).

The concentration of fructose, glucose and sucrose in carrot juices was determined by using High Performance Liquid Chromatography system (HPLC) equipped with Waters Alliance 2690 Separations Module (Waters Inc., Rydalmere, NSW, Australia) and Waters Refractive Index Detector 410 (Waters Inc., Rydalmere, NSW, Australia). Prior to sugar analysis carrot juice samples were mixed with 80% ethanol in the ratio 1 to 4, and centrifuged at 20,000× g, 20 °C for 10 min using Eppendorf temperature-controlled centrifuge (Eppendorf, Macquarie park, NSW, Australia). The isocratic separation of sugars in the supernatants was performed on Shodex Asahipak NH2P-50 4E column (4.6 × 250 mm) (Phenomenex Australia, Lane Cove, NSW, Australia) fitted with guard column Shodex Asahipak NH2P-50G (4.6 × 50 mm) (Phenomenex Australia, Lane Cove, NSW, Australia). The column temperature was maintained at 30 °C during the operation. The mobile phase (68% acetonitrile) was isocratically delivered at flow rate of 1 mL/min. The total run time was 15 min. The quantification of the sugars was based on the calibration curves obtained for each sugar, after injecting known concentrations of a standard.

The extracts were analyzed by HPLC using a Waters Alliance 2690 Separations Module (Waters Corporation, Milford, MA, USA) coupled with a Waters 996 photodiode array detector (UV/DAD) (Waters Corporation, Milford, MA, USA). The Purospher RP-18 end-capped HLPC column (particle size 5 µm, 250 × 4 mm) was connected to a pre-column with the same stationary phase. A mixture of water + 0.1% formic acid (solvent A) and acetonitrile (solvent B) was used as the mobile phase. The injection volume was 25 µL with a flow rate of 1 mL/min. A solvent gradient of 95:5 to 0:100 in 60 min was used.
Before the analysis, samples were solubilized in water (10 mg/mL) and filtered through a polytetrafluoroethylene syringe filter (0.2 µm). Data were collected and analyzed using Waters Millennium^® 32 Chromatography Manager (Waters Corporation, Milford, MA, USA). The chromatogram was monitored and registered on Maxplot wavelength (240–650 nm).