ABSTRACTThis paper reports planar chromatographic behavior and multivariate characterization of three types of macrocyclic compounds: cyclodextrins, calixarenes and macrocyclic antibiotics. Additionally two non-macrocyclic chemicals (estriol and pyrene) were analyzed as the reference retention markers. Target compounds were chromatographed using normal and reversed-phase systems and involving mono as well as binary water-organic liquids mobile phases based on methanol, ethanol and acetonitrile. Non-forced flow TLC/HPTLC elution was performed under isothermal (elevated temperature = 303 K) and mobile phase vapor saturated conditions using 10 cm long glass plates positioned vertically inside temperature controlled Dewar type chambers. It is hoped that presented retention data, which were collected for the whole range of binary mobile phases compositions (0–100%) may be useful for designing of hybrid active layers for extraction, separation and detection systems involving host-guest interactions. Retention profiles were inspected using multivariate statistics (principal component analysis, PCA). Based on reported data various extraction and separation systems can be created where weak or strong interactions of macrocycles with chromatographic stationary phases or given micro device active supports should be expected. Particularly, studied macrocycles can be applied as selective additives for solid phase adsorption (SPE, SPME), microfluidic paper devices (μPAD) as well as micro-total analytical systems (μTAS), where bars formed active layers are fabricated.