Consequently, the milling time must be sufficient (60 min, see Tables Further understanding of the dynamic processes and interconversion of intermediates occurring at the milling stage was obtained by tracking the fate of selected short oligomers (Figure -C and Table ). The changes in content of the characteristic dimers and trimers, and yields of mixHC [8] and cycHC [8] prior to and after the aging stage were determined by HPLC-MS and HPLC-UV analyses. The collected data confirmed that coupling between the CU monomers is kinetically preferred and occurs at the initial phase of the polycondensation reaction. Thus, the quantity of CU-CU dimer drastically increased after 5 min of milling and subsequently underwent rapid decay (Figure ). Such fast dynamics and decay was absent for the biotin units reflecting a major difference in condensation between biotin and CU. In contrast to CU, the condensation of the biotin units to respective dimer B-B reached in the beginning of reaction its maximum and probably acts as a transient intermediate (Figure ). Similar behaviour was observed with the respective trimers (Table ).The yields of the macrocycles generated at the milling stage did not exceed 20%, but greatly increased during aging (Figure ).