We present improvements on a previously reported method (Vernille JP, Schneider JW. 2004. Biotechnol Prog 20(6):1776-1782) to purify DNA oligomers by attachment of peptide nucleic acid amphiphiles (PNAA) to particular sequences on the oligomers, followed by their separation from unbound oligomers using hydrophobic interaction chromatography (HIC). Use of alky-modified HIC/media (butyl and octyl sepharose)over phenyl-modified media (phenyl sepharose) reduced the elution time of unbound DNA while not affecting the elution time of the PHAA/DNA complex. Modifying the alane tail lenght for PNAA from C-12 to C-18 increased slightly the retention of PNAA/DNA duplexes. By combning these two refinements, we show that sequence-specific purifications of DNA oligomers 60 bases length or more can be achieved with high resolution even when the PNAA alkane is attached to the centre of the target strand. The insensitivity of the PNAA/DNA duplex binding to choice of HIC media appers to be due to a surface-induced aggregation DNA. We also report on the use of batch HIC as an adequate predictor of elution profiles in linear gradient HIC, and its potential to considerably reduce purification times by applying step gradients.