Subcritical solvent regeneration of contaminated HPLC columns used for separating biomolecules

The regeneration of contaminated chromatography columns using subcritical mobile phases is presented. The method employs minimal amounts of organic solvents (pressurized and heated above their boiling point) to clean strongly retained sample components from hydrophobic stationary phase particles. The process lasts approximately 1 h and is demonstrated to restore the performance of polymeric and bonded-phase columns. The technique is simple to apply and widely accessible, requiring only common laboratory equipment. Results indicate that this could be a beneficial alternative approach for cleaning contaminated columns either directly or in cases in which conventional methods have been unsuccessful.