Tetraazamacrocyclic ion exchangers tethered to Merrifield peptide resin (DTDM, TTTM) were prepared and the ion exchange capacity of these was characterized. The isotope separation of lithium was determined using breakthrough method of column chromatography. The isotope separation coefficient was strongly dependent on the ligand structure by Glueckauf's theory. We found that the isotope separation coefficients were increased as the values of distribution coefficients were increased. In this experiment the lighter isotope, 6 Li was enriched in the resin phase, while the heavier isotope, 7 Li in the solution phase. The ion radius of lighter isotope, 6 Li was shorter than the heavier isotope, Li. The hydration number of lithium ion with the same charge became small as mass number was decreased. Because 6 Li was more strongly retained in the resin than 7 Li, the isotopes of lithium were separated with subsequent enrichment in the resin phase.