Prostar 335 photodiode array detector

High-performance liquid chromatography was performed for the quantification of phytochemicals accumulated in micro-shoot cultures of A. integrifolia. Galaxie version 1.9.3.2 software with Varian HPLC system (Varian Prostar 230 pump, Varian Prostar 410 autosampler and Varian Prostar 335 Photodiode Array Detector) was used to separate the components. Reading was measured at 320 nm. Two HPLC grade solvents i.e., HCOOH/ H₂O, pH = 2.1 (A) and CH₃OH (B) were used in the mobile phase with a flow rate 1 ml/min. Composition of mobile phase varied according to nonlinear gradient i.e., 8% B (0 min), 12% B (11 min), 30% B (17 min), 33% B (28 min), 100% B (30– 35 min), 8% B (36 min). Ten min of equilibration time after each run was applied. Phytochemicals were quantified based on retention times and UV spectra compared with authentic standards.

After extraction, each extract was centrifuged at 3000 rpm for 15 min and the resultant supernatant was filtered with a syringe filter (0.45 µm, Millipore, Molsheim, France) prior to HPLC analysis. Separation was done by HPLC (High-Performance Liquid Chromatography) by the use of a complete Varian HPLC system consisting of: Prostar 230 pump, Metachem Degasit, Prostar 410 autosampler, Prostar 335 Photodiode Array Detector (PAD) and driven by Galaxie version 1.9.3.2 software (Varian, Les Ulis, France). An RP18 column (Purospher RP-18; 250 × 4.0 mm²; internal diameter: 5 µm; Merck Chemicals, Molsheim, France) was used for separation at a temperature of 35 °C. The mobile phase consisted of a mixture of two solvents A (HPLC grade water with 0.2% (v/v) acetic acid) and B (HPLC grade methanol). The non-linear gradient applied for separation was: 8% B (0 min), 12% B (11 min), 30% B (17 min), 33% B (28 min), 100% B (30–35 min), 8% B (36 min) at a flow rate of 1 mL/min. A re-equilibrating time of 10 minutes was applied between each injection. Compound detection was set at 295 and 325 nm (corresponding to the λmax of the main compounds) [6 (link)].