Peptide nucleic acids (PNAs) are synthetic biopolymers consisting of nucleobase side chains attached to an amino ethyl glycine backbone. At present this family of compounds enjoys a well deserved popularity in biomedical research, due to a number of favorable biological and chemical properties of PNAs compared to conventional oligonucleotides. PNAs are basically peptides, and are synthesized, purified and analyzed by traditional peptide chemistry, chromatography and mass spectrometry techniques. In the current report, we analyzed factors that influence the elution behavior of 29 PNAs on reversed-phase high-performance liquid chromatography using a water-acetonitrile-trifluoroacetic acid gradient elution system on C18 columns. We found that increasing the temperature from 25 degrees C to 55 degrees C resulted in improved peak shape and resolution. The retention times of the PNA analogs were dependent upon the length of the polymers with longer PNAs eluting later. Mixtures of PNAs with length, originating from inefficient monomer coupling during the polymer assembly, could be separated by single chromatographic runs. The retention time also depended upon the cytosine, thymine, adenine and guanine contact of the polymers. These differences in the contribution to the retention times could be explained by simple hydrophobicity features of the monomer side chains at pH 1.8. Based on all data, a linear equation was generated which predicted the retention time of any synthetic PNA based on composition and length. Comparison of the predicted and observed retention times showed a remarkable reliability of the algorithm.