Development and Optimization of a Reversed-Phase HPLC Method to Separate Pulse Proteins

Abstract

Due to the demand for new sources of non-GMO and allergen-free plant proteins in food formulations that have not previously existed, there is a growing need for analytical methods to characterize these proteins. A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and optimized to separate the proteins from yellow pea, lentil, chickpea, great northern beans, and a commercial pea protein isolate. A response surface methodology (RSM) model was used for final method optimization. The optimized separation conditions were 0.089% trifluoroacetic acid (TFA) in water (mobile phase A) and acetonitrile (mobile phase B) with a gradient of 20% B to 30% B for 10 min, 30% B to 39% B for 20 min, 39% B to 60% B for 10 min, and keeping 60% B for the last 5 min of the run. The optimized sample concentration was 12.8 mg/mL, using a C18 column at 55.5 °C. These conditions for yellow pea sample resulted in a good resolution (116 peaks with model predictability of 0.96) and high recovery (106% recovery with model predictability of 0.92). It was shown that the optimized method has great potential to be used for quantitation of total protein content in pulse samples using pea protein isolate as a standard (R² of 0.9985 for the standard curve), and values obtained using this method were comparable with those obtained through nitrogen combustion.