HPLC Analysis of Gliadin and Glutenin

In preparation for HPLC analysis, both gliadin and glutenin extracts from the DME TILLING mutants and wild type Kronos and Express were passed through 0.45 μm Durapore^® 13 mm membrane filters. The filtered samples were then injected onto a reversed-phase C8 analytical column (Zorbax 300SB-C8, Agilent Technologies) with 5 μm particle size and 30 nm microporous silica diameter (250 mm length, 4.6 mm I.D.), using a 1200 Series Quaternary LC-System liquid chromatograph (Agilent Technologies), with a diode array UV-V detector. The column temperature was maintained at 60°C, and a linear elution gradient was implemented using two mobile solvents. The polar solvent A consisted of a mixture of 0.1% trifluoroacetic acid (TFA) (v/v) and type-I ultrapure water (18 MΩ·cm specific resistance), and the non-polar solvent B contained 0.1% TFA (v/v) and acetonitrile (ACN). Absorbance was monitored at a detection wavelength of 210 nm. The injection volumes for both the gliadin and the glutenin fractions were 30 μL, and the flow rate was adjusted to 1.0 mL min⁻¹. The elution gradient conditions were as described in Mejías et al. (38 (link)).