Chiral separation of β-blockers is performed by utilizing the supercritical fluid chromatographic method. The chiral columns utilized were Chiralpak IG and Chiralpak IBN-5. The finest mobile phase was CO2 −0.2% TEA in methanol (60:40). The values atenolol enantiomers retention factors were 6.39 and 8.98. These values for propranolol enantiomers were 3.39 and 4.06. These values for betaxolol enantiomers were 4.08 and 4.68. The separation and resolution factor values for atenolol, propranolol, and betaxolol were 1.41 and 3.33, 1.19 and 2.23, and 1.15 and 1.87, separately and respectively. By comparison, it was observed that Chiralpak IG column is better than Chiralpak IBN-5 column. Supercritical fluid chromatography has been found as the best analytical technique due to its high speed, being eco-friendly, and being economic. The various most probable interactions responsible for the chiral resolution are hydrogen bonding, dipole–dipole interactions, steric effect, and π–π interactions. The reported methods are effective, efficient, and reproducible and may be used to separate and identify atenolol, propranolol, and betaxolol in any unknown samples.
