A Rapid Technique for Plant Peptides Isolation Based on their Polarity by Using BIOTAGE Isolera One Flash Chromatography

Abstract

Several years of experience performing columns chromatography have been condensed into simple guidelines useful for translating thin layer chromatography (TLC) results into either isocratic- or gradient-flash chromatography. The present study describes the use of such protocol with a silica Biotage SNAP KP-Sil 10 g cartridge (21 mm A— 55 mm, 50 I¼m particle size), to purify, in a single step, four (4) peptides from crude proteins extract of germinated Amaranthus hybridus seeds. The best solvent system used was a combination of n-hexane (non-polar solvent B from 5% to 0% final concentration) and a mixture of polar solvents (solvent A) composed of acetonitrile, n-butanol, glacial acetic acid and water in 0.9:2:1:1 ratio, respectively. The elution was done in normal-phase with a linear gradient. All the flash chromatography procedure took at most twenty-five minutes (25 min), and less than a liter (750 mL) of total solvents was used. The purified peptides named AhPA, AhPB, AhPC and AhPE showed apparent homogeneity on TLC plates, and purification yields of about 11.15%, 10.38%, 14.50% and 17.25%, respectively. This innovative technique provides an efficient alternative to researchers’ in peptides purification and participates in reducing the waste of solvents, gels, time and thus, money.