tWe evaluated at-column dilution (ACD) as a possible approach to reduce peak distortion caused by theinjection of strong solvents in preparative hydrophilic interaction chromatography (HILIC). Three modelcompounds, namely uridine, cytidine and guanosine were selected and injected either in conventional orACD conditions, using various proportions of water and acetonitrile in the sample diluent. Plate numberand peak capacity were systematically investigated under both isocratic and gradient elution conditions,respectively. In isocratic conditions, ACD was found to provide higher plate numbers with sample diluentcontaining more than 40% water, but lower plate numbers with injections of less than 40% water, incomparison to a conventional injection system at the preparative scale. These contrasting results wereattributed to the fact that i) efficiency was fundamentally reduced in ACD, due to the low make-up flowrate that was used to flush the injection loop, but ii) the trend was reversed for highly aqueous injectionthanks to the capacity of ACD to maintain similar efficiency regardless of the injection solvent. On theother hand, peak capacity was comparable between ACD and conventional systems in gradient elutionfor injections containing low amounts of water. However, ACD became increasingly advantageous as theproportion of water in the injection solvent and/or the injection volume increased. This was confirmedby the separation of a plant root aqueous extract. Altogether, our findings suggest that ACD is beneficialboth in isocratic and gradient modes when strongly aqueous solutions are injected, offering an attractiveapproach to increase loadability, while preserving peak shapes in preparative HILIC.