Development and Optimization of a Reversed-Phase HPLC Method to Separate Pulse Proteins
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Development and Optimization of a Reversed-Phase HPLC Method to Separate Pulse Proteins Mostafa Taghvaei 1 & Brennan Smith 1 Received: 27 February 2020 / Accepted: 10 May 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
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. Keywords RP-HPLC . Pulse crops . Proteins . Method development . RSM
Introduction Pulses, such as chickpeas, lentils, yellow peas, great northern beans, and green peas, are gaining more popularity in North America due to the consumer’s demand for new sources of plant proteins that are non-GMO and allergen-free (DePalma et al. 2019). Many pea protein concentrates and isolates are commercially available showing a great potential for applications as food ingredient (Arntfield and Maskus 2011). However, such protein isolates are far from having desired characteristics, most importantly water solubility (Pietrysiak et al. 2018). Separation and characterization of pulse proteins by a fast and user-friendly chromatographic method, like HPLC, could help with studying the effect of different processes, such as extraction and drying, on pulse proteins. The RP-HPLC technique has been previously applied for separation of different kinds of proteins (Katzenstein et al. 1986; Aguilar 2004; Hearn et al. 2002; Turula and de * Brennan Smith [email protected] 1
School of Food Science, University of Idaho, Moscow, ID, USA
Haseth 1996; Bobaly et al. 2015). But since the introduction of HPLC technique, less work was done on separation of pulse proteins, probably because proteins from soy (Glycine max) have predominated. However, the RP-HPLC technique was shown to be a very efficient way of separating lentil (Lee et al. 2007) and c
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