Ultra-fast separation of wheat glutenin subunits by reversed-phase HPLC using a superficially porous silica-based column

A relatively new, unique column packing material for reversed-phase high-performance liquid chromatography (RP-HPLC) was evaluated for rapid separation of wheat glutenin protein subunits. The product named “Poroshell” by the manufacturer consists of a solid core and a porous coat instead of solid silica spheres used in conventional RP-HPLC column packing. This architecture favours rapid mass transfer, facilitating faster reversed-phase separations of biomolecules compared to conventional silica columns. The main objective of this study was to evaluate the quality of separations of glutenin subunits (GS), as well as to optimize conditions to produce the fastest possible run times without sacrificing resolution using a Poroshell 300SB-C₈ 2.1×75 mm column. The stability of GS separations over time was also assessed. Two different bread wheat genotypes were used for optimization of separation conditions and six more common and durum wheat genotypes possessing different subunit combinations were used for further evaluation. Glutenin protein was extracted with 0.08 M Tris–HCl buffer (pH 7.5) containing 50% 1-propanol under reducing conditions after pre-extraction of soluble proteins with 50% 1-propanol. Optimization of GS resolution and sample throughput by RP-HPLC was assessed in response to variation in eluent flow rate, acetonitrile (ACN) gradient, and column temperature. The best resolution of both HMW- and LMW-GS was obtained in 13 min using a 23–44% ACN gradient with a flow rate of 0.7 mL/min at 65 °C. Subunit elution times and integrated areas were highly repeatable even after several hundred injections. Highly satisfactory separation of HMW-GS and quantification of ratio of HMW- to LMW-GS were achieved in less than 4 min per sample using a modified HPLC gradient. Ratio of HMW- to LMW-GS was unaffected by the speed of the separations. As well, the elution order of HMW- and LMW-GS was unaffected by the rapid analysis, compared to conventional RP-HPLC separations, so no new learning was required for interpreting chromatograms and classification of subunits. The rapid RP-HPLC method using the Poroshell column appears to be very well suited for routine quantification of HMW-GS and LMW-GS especially for purposes of wheat quality screening and wheat cultivar development activities where large numbers of samples are typically encountered.