300sb c8 reverse phase analytical column

The gliadin fractions from 100 mg samples of flour and bread were extracted and analyzed by Reverse-phase high-performance liquid chromatography as described in Reference [34 (link)]. Briefly, gliadin extracts (40 μL) were applied to a 300SB-C8 reverse phase analytical column (4.6 × 250 mm, 5 μm particle size, 300 Å pore size; Agilent Technologies, Santa Clara, CA, USA) using a 1200 Series Quaternary LC System liquid chromatography (Agilent Technologies, Santa Clara, CA, USA), with a DAD UV-V detector. Absorbance was monitored with the DAD UV-V module at 210 nm.
Flour and bread samples from the low-gliadin wheat were analyzed for gluten content (parts per million) at the Centro Nacional de Biotecnología (CNB-CSIC) using a competitive ELISA, based on the monoclonal antibody R5 as described in Reference [35 ]. The assay was performed in triplicate.

Prior to RP-HPLC, a 700 μL of protein extracts was centrifuged through a 0.45 µm pore Corning^® Costar^® Spin-X nylon filter. Volumes of prolamin extracts (60 μL) were applied to a 300SB-C8 reverse phase analytical column (4.6 × 250 mm, 5 µm particle size, and 300 Å pore size; Agilent Technologies) using a 1,200 series high-resolution liquid chromatography quaternary system (Agilent Technologies) with a DAD UV-V detector, as described in Refs. (18 (link)) and (17 (link)). The elution system consisted of deionized water (A) and acetonitrile (B), both containing 0.1% (v/v) trichloroacetic acid. The proteins were eluted with a linear gradient from 24% B to 56% B in 60 min, at a flow rate of 0.5 mL/min at 50°C. Absorbance was monitored using the DAD UV-V module at 210 nm, and integration was automatically handled by the RP-HPLC system software with some manual adjustments. Absolute amounts of prolamins were determined using bovine serum albumin (BSA, 98% BSA, fraction V, Sigma-Aldrich, St Louis, MO, USA, cat No. A3294) as a protein standard.