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 × 55 mm, 50 µm 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.

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