Hplc 2694 separation modules

SAE was diluted to 1 mg/mL using methyl alcohol and filtered. β-sitosterol was diluted to 0.01 mg/ml in the same manner and used for qualitative analysis. All materials were analyzed by injection into HPLC 2694 separation modules (Waters, USA) with separation flow rate of 1 mL/min through a SunFire™ C-18 column (4.6 × 250 mm, 5 μm, SunFire, Germany). In addition, the mobile phase of all materials was detected using the Dual Absorbance Detector 2487 (Waters) at 205 nm for 1 h with 5:95 water to acetonitrile.

The ethanol extract of Euryale ferox salisb was sonicated for 1 ml of distilled water per gram of the extract and centrifuged for 5 min. then, the supernatant was mixed with equal volume of Methyl alcohol, filtered, and injected into the HPLC 2694 separation modules (Waters, USA). The extract was separated through SunFire^TM C-18 column (4.6 × 250 mm, 5 μm, SunFire, Germany) with flow rate of 0.5 ml/min. The mobile phase was a binary gradient elution of (A) water and (B) acetonitrile under the following conditions: 0–40 min linear gradient from 90 to 50% A and 10 to 50% B. then, maintained 50% A and B for 5 min. the Dual λ Absorbance Detector 2487 (Waters, USA) responses at 240 nm to ESE and standard were found to be linear over range.