Evaluation of two sub-2μm stationary phases, core–shell and totally porous monodisperse, in the second dimension of on-line comprehensive two dimensional liquid chromatography, a case study: Separation of milk peptides after expiration date

•Online comprehensive LC×UHPLC.•Separation of peptide fraction of milk after expiration date.•Evaluation of totally porous monodisperse and core–shell sub-2μm columns in 2D. Milk is a rich source of bioactive peptides of great interest for their healthy properties. These peptides are usually encrypted in the sequences of proteins and are released after time dependent proteolysis as very complex hydrolysates. In order to separate and identify the bioactive sequences, we developed an on-line comprehensive two dimensional liquid chromatography approach using the high performance combined with the ultra high performance conditions. A microbore reversed phase (C18 silica, 5μm) column was employed in first dimension, while, in second dimension, two different UHPLC columns, packed with C18 silica, were tested: a new column based on monodisperse sub-2μm fully porous particles with high surface area (50mm×3.0mm, 1.9μm d.p., from Supelco), and a column based on sub-2μm core–shell particles (50mm×3.0mm, 1.7μm d.p, from Phenomenex®). Both set-ups were compared, showing high peak capacity values with respect to a high efficiency monodimensional method, maintaining the same analysis time. Satisfactory selectivity was obtained through the use of different pH between the two dimension, while a very fast continuous shifted gradient in second dimension ensured a good employment of the 2D separation space.