Lc 30ad liquid chromatograph

The extracts obtained from plant sample were prepared in HPLC-grade methanol for quantitative analysis. Standard scopolamine (0.6 mg/5 mL) and atropine (1.0 mg/5 mL) were prepared in HPLC-grade methanol. Analysis was performed with a Shimadzu (Nexera) HPLC system equipped with LC-30AD Liquid Chromatograph, SIL-30AC Autosampler, SPD-M20A Detector, CBM-20A Communication Bus Module, CTO-20AC Column Oven and Lab solutions software. All samples were filtered through 0.45 μM (Millipore, Bedford, MA) filter. Extracts were separated on a RP-18 (4 × 250 mm, 5 μm; Merck, Bangalore, India) column. The mobile phase consisted of 0.05 M KH₂PO₄ in water (A) and acetonitrile (B) delivered at a flow rate of 1.0 mL/min. The gradient system was as follows: at 0 minutes 10% B, at 20 minutes 60% B, at 23 minutes 60% B, and at 25 minutes 10% B. The samples were analyzed at 30°C to provide efficiency to the peaks and the UV chromatograms were recorded at 210 nm.

A Shimadzu LC-30AD liquid chromatograph equipped with a RID-10A refractive index detector and Shimadzu CTO-20AC column oven were used for the size-exclusion chromatography analysis. First, 100 μL of the filtered samples were passed through an OHpak SB-G guard column and two Shodex OHpak SB-806MHQ columns in series. The elution was conducted using 0.1 M NaNO₃ solution as the mobile phase at a flow rate of 0.8 mL/min. The column oven temperature was maintained at 60 °C. A calibration curve obtained from 12 pullulan standards (Mp values of 2400, 1560, 710, 380, 200, 106, 45.9, 22.0, 11.2, 5.6, 1.08, and 0.342 kDa) was used for the estimation of the peak average, weight average, and number average molecular weights by the LabSolutions software.

The Spectrophotometer Ultraviolet-Visible V-530 was purchased from Jasco (Tokio, Japan). The HPLC Nexera2 is composed of the following units: an LC-30AD liquid chromatograph, an SIL-30AC autosampler, a CTO-20AC prominence column oven, an SPD-M30A diode array detector, and a DGU-20A5R degassing unit, purchased from Shimadzu (Kyoto, Japan). The gas chromatograph (GC) CP3800 was purchased from Varian (Palo Alto, CA, USA).