Short DNA and RNA sequences like therapeutic anti-sense oligonucleotides (ASO), microDNA (miDNA) and microRNA (miRNA) are predominantly analyzed using liquid chromatographic techniques or capillary gel electrophoresis. The current demand for improved analysis of these nucleotides gives incentives to reconsider old truths, such as the need for gel-based capillary electrophoresis for DNA and RNA analysis. The present study shows the applicability of capillary zone electrophoresis using acidic background electrolytes for the separation of ASO-, miDNA- and miRNA-sized nucleotides. Using the different pKa of the nucleobases as a foundation, different nucleotides with same size, but varying sequence, has been successfully separated with high efficiency and short analysis time. Furthermore, the ratio of adenosine and cytidine to guanosine and thymidine nucleobase content, AC/GT, is shown as a valid parameter to assess single-stranded nucleotide migration and separation.