Online combination of hydrophilic interaction chromatography (HILIC) and RP chromatography for separation of tryptic peptides is a challenging approach due to the incompatibility of direct loading HILIC fractions on the RP trapping column. High amounts of organicmodifiers in loading solvents decrease the binding efficiency of tryptic peptides on C18 phases and lower the number of identifications. A 500 mu L loop upfront of the trapping column filled with aqueousmobile phase was employed as amixing chamber and enabled direct injections and improved saliva protein identification rates of HILIC fractions.