The mechanisms of structure selective and enantioselective retentions of amines and acids on two chiral stationary phases based on wild type cellobiohydrolase I (CBH I) and its mutant D214N have been investigated. All the amino alcohols tested had an enantioselective site that overlaps with the catalytically active site of CBH I, whereas the enantioselectivity of prilocaine was not affected by the mutation. The hydroxyl group of the amino alcohols did not seem to be an important contributor to the total binding strength whereas a bromo substituent in the aromatic ring promotes a high enantioselectivity (alpha=7.05). Interestingly, the chiral recognition site of the acid warfarin overlaps with the binding site of the amino alcohols. Di-p-toluoyltartaric acid and dibenzoyltartaric acid were strongly retained probably due to electrostatic attraction, but no enantioselectivity was observed. The difference in retention characteristics for the amino alcohols on the two stationary phases was strongly pH-dependent. A change in elution order of different amino alcohols occurred when changing the pH from 5.0 to 7.0. The difference between the two phases was lower at low pH. The retention times could also be affected by ionic strength and by use of cellobiose as a mobile phase additive but no indication of ion-pair retention of the amines was observed, when adding hexanesulphonate as counter ion to the mobile phase. The temperature dependence of the retention of the enantiomers of propranolol at pH 7.0 on the mutant D214N was similar to what was earlier observed on the wild type CBH I at lower pH.