Nano-LC with new neutral monolith for biomolecule separation

• The development of a new monolithic nano-column is described. • The separation of biomolecules is presented. • 2- napthylmethacrylate was used for the first time, for the preparation of monolithic column with 100µm i.d.. • The developed hydrophobic monolith consumes less solvent and provide faster separation. A new capillary monolith based on the co-polymerization of 2-napthylmethacrylate (NMM) as the functional monomer and trimethylolpropane trimethacrylate as the crosslinker was introduced for nano-LC. Porogenic solvent was composed of cyclohexanol and 1-dodecanol. The influence of both monomer and porogenic solvent on the monolithic structure was investigated. The characterization of NMM monolithic column was performed using scanning electron microscopy and chromatographic analyses. It was shown that the developed NMM monolith produced over than 43.280 plates/m. Homologous alkylbenzenes and polyaromatic hydrocarbons were chromatographed on the final NMM monolith, allowing promising hydrophobic and π-π interactions. Column-to column and run-to-run and reproducibility values were found as 2.4-3.2% and <2.47%, respectively. The developed monolith was further applied for biomolecule separation. Three dipeptides (e.g. Ala-Tyr, Gly-Phe and L-carnosine) and five standard proteins, namely ribonuclease A, α-chymotrypsinogen, lysozyme, cytochrome C, Myoglobine were separated using the monolith in order to evaluate its potential. Both peptides and proteins were well separated using the NMM monolith in nano-LC, indicating promising alternative for sensitive chromatographic analyses.