5-Fluorouracil (5-FU) is pyrimidine analog which is used as anti-cancer drug. 5-Fluorouracil is very polar non-ionizable compound which is hard to retain by reversed-phase chromatography. Ion-pairing reagent cannot be employed due to non-ionizable nature of the compound. 5-fluorouracil is retained in zero organic on Primesep 100 and Primesep P columns. Both columns demonstrate perfect peak shape. 5-FU can be monitored by UV, or by ELSD, LC/MS or Corona CAD.