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.

由于对食品配方中非转基因和不含过敏原的植物蛋白的新来源的需求，对表征这些蛋白的分析方法的需求日益增长。开发并优化了反相高效液相色谱（RP-HPLC）方法，以从黄豌豆，小扁豆，鹰嘴豆，大北豆和市售豌豆蛋白分离物中分离蛋白质。响应面方法（RSM）模型用于最终方法优化。最佳的分离条件是水（流动相A）和乙腈（流动相B）中的0.089％三氟乙酸（TFA）梯度为20％B至30％B 10分钟，30％B至39％B 20分钟分钟，从39％B到60％B持续10分钟，并在运行的最后5分钟保持60％B。最佳样品浓度为12.8 mg / mL，在55.5°C下使用C18色谱柱 黄豌豆样品的这些条件导致了良好的分离度（116个峰，模型可预测性为0.96）和高回收率（106％回收率，模型可预测性为0.92）。结果表明，以豌豆蛋白分离物为标准品，优化后的方法可用于定量测定豆类样品中总蛋白含量。对于标准曲线，R ²为0.9985），并且使用该方法获得的值与通过氮气燃烧获得的值相当。