Retention characteristics of selected synthetic 5′-terminal phosphate absent penta-nucleotides containing adenine, guanine, and thymine were studied in relation to their sequence by hydrophilic interaction chromatography and ion-interaction reversed-phase liquid chromatography. The organic solvent content, pH, and buffer concentration in mobile phases were evaluated as influential separation conditions. Data demonstrate that both compared chromatographic modes can be used to separate synthetic penta-nucleotides according to their nucleotide composition. Moreover, reversed-phase liquid chromatography allows separation according to their sequence. We have found a simple linear additive model to describe the retention order in both separation modes in regard to their sequence. In hydrophilic interaction chromatography, the retention behavior is controlled primarily by the hydrophilicity of involved nucleotides and minimally by their sequence position. For reversed-phase liquid chromatography, the nucleotide hydrophobicity plays an important role in their retention properties and the influence of their location in sequence on the retention increases toward the center and decreases toward the termini. Our results show that the penta-nucleotide sequence, and thus its spatial arrangement induced by the surrounding environment, is highly related to the retention properties, so it may be hypothetically used to read the sequence from the retention properties acquired under particular separation conditions.