Abstract
Two chromatographic narrow‐bore columns, a novel 2.6 μm particle‐packed Kinetex™ C~18~ core–shell (50×2.1 mm id) and monolithic Chromolith^®^ FastGradient RP‐18e (50×2 mm id), were evaluated for the analysis of diastereoisomers of the flavonolignans silybin and 23‐O‐acetylsilybin under isocratic conditions. The main advantages of the core–shell column are markedly higher efficiency (h~min~=2.8 versus 5.6 for silybin A) and better peak symmetry. The Kinetex column exhibits only a slight change in the height equivalent of the theoretical plate with a higher linear velocity of the mobile phase. The monolithic column shows notably higher selectivity in terms of selectivity factor (1.21 versus 1.12) in the analysis of critical‐pair of diastereoisomers (silybin A and silybin B) and enables shorter run duration (approx. twofold) together with lower backpressure. The resolution power was found to be comparable, but the Kinetex column required a higher pressure of the mobile phase that, together with the higher chance of clogging, can be a disadvantage in the separation of biological samples. Successful baseline separation of silybin diastereoisomers in real pharmaceutical sample on monolithic column was accomplished.