Design of Experiments (DoE) for the development of a chromatographic method to separate the constitutional isomers of keto-boswellic acids (KBAs) and acetyl-keto-boswellic acids (AKBAs). (a) Three-factorial central composite design (CCD) with the starting concentration of eluent B as variable A, the slope of the gradient as variable B, and the eluent´s flow as variable C. Whereby, the central composite design is composed of a full two-level factorial (green dots), axial points (blue dots), and a center point (red dot). (b) Effect of variables A and B on the chromatographic resolution R(KBA) between 11-keto-α-boswellic acid (α-KBA) and 11-keto-β-boswellic acid (β-KBA). (c) Effect of variables A and B on the chromatographic resolution R(AKBA) between acetyl-11-keto-α-boswellic acid (α-AKBA) and acety-11-keto-β-boswellic acid (β-AKBA). Black dots represent the level configurations of the individual experiments. Only the level configurations of experiment 5 (*) enabled a separation of all four isomers. The level configurations of experiment 11 (**) allowed the separation of KBA´s isomers, but the isomers of AKBA could not be eluted under these conditions.