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Three major active components of the traditional Chinese medicinal herb  Salvia miltiorrhiza  Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B, and protocatechualdehyde, are separated and purified from a crude water extract in one step by isocratic hydrogen bond adsorption chromatography on cross-linked 12% agarose (Superose 12 HR 10/30). Separation is achieved by stepwise elution with mobile phases composed of mixtures of ethanol and acetic acid: 0–50 mL, 5% ethanol, 5% acetic acid; 50–100 mL, 20% ethanol, 20% acetic acid; and 100–200 mL, 30% ethanol, 30% acetic acid. The 3,4- dihydroxyphenyllactic acid is obtained with a purity of 97.3% and with a recovery of 88.1%. The corresponding figures for protocatechualdehyde are a purity of 99.4% with a recovery of 90.7%, and for salvianolic acid B a purity of 90.4% with a recovery of 50.3%, respectively. At a sample load of 40 mg crude extract dissolved in 0.5 mL mobile phase (corresponding to a load of 1.6 mg/mL gel), a 3,4-dihydroxyphenyllactic acid purity of approximately 94% with a recovery of 80.2% is obtained.

journal of chromatographic science

One-Step Purification of 3,4-Dihydroxyphenyllactic Acid, Salvianolic Acid B, and Protocatechualdehyde from  Salvia miltiorrhiza  Bunge by Isocratic Stepwise Hydrogen Bond Adsorption Chromatography on Cross-Linked 12% Agarose