Final choice of which solvent is to be used is based on their elution position on a 100% water ( buffered to pH 6.8 with 10mM ammonium formate ) to 100% acetonitrile; reverse phase hplc gradient. The L. trifolia extract corresponded to region B, which suggests mid-polarity compounds, and a preferred choice of a hexane: ethyl acetate: methanol: water biphasic solvent system. Next research was undertaken to find the best solvent ratios in order to develop a gradient system."
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