A sample pretreatment strategy for pH-zone-refining counter-current chromatography.

A sample pretreatment method using acid-base extraction was established for pH-zone-refining counter-current chromatography (CCC) to increase the yield of the target compound. As the most important step, selection of two-phase solvent system for pH-zone-refining CCC should follow some rules. One of these rules is that when pH of two-phase solvent system is around 2 (for an acidic analyte) or 10 (for a basic analyte), partition coefficient values of K(acid) or K(base) should be much greater than 1. According to this rule, a new pretreatment method was developed that could substantially increase the yield of the target compound in pH-zone-refining CCC. The separation of the crude alkaloids extracted from the seeds of Sophora alopecuroides was selected as an example to show the advantage of this method. Before CCC separation, 20 g of crude sample was extracted 3 times with the selected two-phase solvent system composed of MtBE-CH₃CN-H₂O (2:2:3, v/v), where pH of the upper phase was adjusted with triethylamine to 10. A 10 g amount of the second basic extract thus obtained was subjected to pH-zone-refining CCC separation. As a result, 169 mg of sophoramin and 696 mg of sophoearpin were isolated from the second basic extract, where the yields have been doubled compared with those of the untreated sample (crude sample). Experimental results showed that the present method for pretreating the crude sample could produce substantially higher yields of target compounds in pH-zone-refining CCC.