In the normal phase preparative HPLC of fermentation derived echinocandins, resolution of key impurities from the product of interest, pneumocandin B(o), is accomplished using a ternary ethyl acetate/methanol/water mobile phase with silica gel as the sorbent. In this work, previous characterization of the system is extended to define the impact and role of water content on the separation efficiency and retention of pneumocandin B(o). Experimental results indicate that column efficiency, measured using both the product of interest and small molecule tracers (compounds used for pulse tests), is good despite the use of an irregular silica and unusually high levels (greater than 6%) of water in the mobile phase. In contrast to column efficiency measurements using small molecules (MEK and toluene), measurements performed with the product itself indicate improved efficiency with increasing water content of the mobile phase. Building on these results, a scale-up/scale-down protocol was developed based on measurements of column efficiency using theoretical plate counts determined with pneumocandin B(o). Since the solubility of pneumocandin B(o) in the ternary mobile phase is relatively low, a higher strength solvent with higher levels of methanol and water is employed for dissolution of the crude product at concentrations of up to 40g/L. The mismatch between the high strength solvent used for the feed introduction and the mobile phase has the potential to affect column performance. The impact of this mismatch using plate count measurements with the product at both analytical and semi-preparative scales was found not to be significant. Finally, a van't Hoff analysis was performed to characterize the thermodynamics of adsorption of pneumocandin B(o) on silica. The analysis shows that the adsorption process for pneumocandin B(o) on silica in the ternary solvent system is endothermic (DeltaH(ads)>0), implying that the adsorption is entropically driven. Results from an overall water balance across the column indicate significant enrichment of adsorbed water on the silica surface. These results further emphasize the importance of selective partitioning of water between the bulk mobile phase and the silica as a dominant factor in controlling retention.