Waters autosampler

Standard diosgenin (SD) was procured from Sigma Aldrich (USA). An aqueous extract of Helicteres isora, extracted diosgenin, and standard diosgenin were undertaken for thin layer chromatography on pre-coated silica gel plates with solvent combinations of N-hexane: ethyl acetate (7.5: 2.5). The plates were heated in an oven at 90 °C for three to 5 min to observe the spots after being sprayed with a solution of ethanol, sulfuric acid, glacial acetic acid, and anisaldehyde reagent (135:5:1:3.7) [28 (link)]. Waters High Performance Liquid Chromatography system equipped with Waters 1525 Binary HPLC Pump, Waters 2998 - Photodiode Array Detector (PDA) and Waters Autosampler was used for chromatographic determination of diosgenin on Waters C-18 column. Waters Breeze 2 Software system was used for analysis and data acquisition.
ED and SD were dissolved in acetonitrile and filtered via a 0.10 μm syringe filter before use. Samples were injected using a rheodyne injector fitted with a 20 μl fixed loop. The mobile phase reported by Deshpande and Bhalsing, (2014) was referred to wherein acetonitrile: water (90:10, v/v) was used as a solvent system with a flow rate of 0.7mL/min maintaining the ambient temperature [29 (link)]. The PDA detector was set at 273 nm to acquire the chromatogram.

SQAd/VitE NPs with different VitE content were formulated in 5% dextrose solution and subsequently washed with water twice using an ultrafiltration device (molecular weight cutoff, 100,000). NPs were dissolved in ethanol and measured by HPLC to determine VitE content. HPLC Waters Alliance 1695 (Waters, Milford, MA), equipped with a Waters DAD 2996 photodiode array detector, Hewlett-Packard computer with a Waters Empower 3 software, and a Waters autosampler with a 50-μl loop, was used, with simultaneous spectra detection wavelengths from 200 to 600 nm recorded for all peaks. A Waters XSelect LC-18 column (2.1 mm by 150 mm, 3.5 μm) was used with a nongradient mobile phase of acetonitrile and methanol (50:50, v/v) at a constant flow rate of 1 ml/min. The VitE peak was measured at a wavelength of 216 nm and quantitatively determined by comparing with a standard curve.