Thin film composite membrane having chiral selective layer was prepared by the interfacial polymerization of trans-1,4-diaminocyclohexane, piperazine and trimesoyl chloride. The composition of chiral selective layer of composite membrane was elucidated by Fourier transfer infrared spectroscopy in attenuated total reflectance (FTIR-ATR) mode. Scanning electron microscopy (SEM), atomic force microscopy (AFM) and transmission electron microscopy (TEM) demonstrate the evolutionary transition of membrane surface morphology before and after the modification of membrane. This chiral separation method has been verified using the chiral separation results of racemic amino acid. The effect of membranes pore size on enantioselectivity has also been investigated. The functional performance of membranes was determined by measuring volumetric flux, solute flux, rejection of solute and enantiomeric enrichment in cross flow closed loop mode of reverse osmosis process at 689.42 kPa pressure using aqueous solutions of racemic lysine monohydrochloride. The membranes permeated L-enantiomer preferentially resulting >78% enantiomeric excess of L-enantiomer in the permeated solution with good separation factor (alpha = 8). (C) 2012 Elsevier B.V. All rights reserved.