Silica with a particle size of 3–5 µm has been widely used as selector backbone material in 10–25 cm HPLC chiral columns. Yet, with the availability of 1.6 µm particles, shorter, high-efficiency columns practical for minute chiral separations are possible to fabricate. Herein, we investigate the use of two recently commercialized sub-2 µm columns with different substituents. Thus, Chiralpak ® IG-U and ID-U were used in HPLC for the fast enantioseparation of a set of drugs. Chiralpak ® IG-U [amylose tris (3-chloro-5-methylphenylcarbamate)] has two substituents on the phenyl ring, namely, a withdrawing chlorine group in the third position and a donating group in the fifth position. Chiralpak ® ID-U [amylose tris (3-chlorophenylcarbamate)] has only one substituent on the phenyl ring, namely a withdrawing chlorine group. Their applications in three liquid chromatography modes, namely, normal phase, polar organic mode, and reversed phase, were demonstrated. Both columns have similar column parameters (50 mm length, 3 mm internal diameter, and 1.6 µm particle size) with the chiral stationary phase as the only variable. Improved chromatographic enantioresolution was obtained with Chiralpak ® ID-U. Amino acids partially separated were reported for the first time under an amylose-based sub-2-micron column.