AbstractA two‐dimensional column‐switching system without sample loop trapping, where two columns were operated via a six‐port switching valve, was employed in the isolation and purification of five isoflavonoids from Rhizoma Belamcandae: tectoridin, iridin, tectorigenin, irigenin and irisflorentin. The introduction of the six‐port switching value, instead of a sample loop, assured 100% recovery from the first dimension to the second, and the injection volumes of the second dimension were not restricted. Two configurations were tested in this study. In the first mode, only one column was used in the second dimension and two ‘heart‐cutting’ fractions were transported to the same second‐dimensional column. In the second mode, two parallel columns were used in the second dimension and two fractions were transported to the two columns. Between the two configurations, the one with two second dimensional columns had double sample size, better resolution and one more purified compound. Both two‐dimensional configurations consumed less solvent with even greater efficiency and shorter cycle time compared with conventional gradient methods. All of the isoflavonoids were isolated at high purities of greater than 95% with yields of above 82%. Copyright © 2009 John Wiley & Sons, Ltd.