Open tubular nano-liquid chromatography with a new polylysine grafted on graphene oxide stationary phase for the separation and determination of casein protein variants in milk.

In this study, a novel narrow capillary (i.e., 20 µm i.d.) coating procedure was reported for the preparation and application of open-tubular (OT) column for the separation of casein (CN) protein variants in milk. Based on this approach, a new poly-L-lysine (PLL) grafted on graphene oxide stationary phase was developed for OT nano-liquid chromatography (OT-nano LC). In this preparation, methacryloyl graphene oxide nanoparticles (MGONPs) were synthesized and used as reactive monomer. Fused silica capillaries with 20 µm i.d. were used for the preparation of OT column. After silanization of the capillary column, the stationary phase was prepared by MGONPs graft polymerization and PLL modification. It was shown that the use of MGONPs amount was important for the capillary coating. A column without PLL modification was also prepared for comparing the effect of PLL. SEM images of the column showed that MGONPs were bound to the surface of capillary in a dense monolayer. Alkylbenzenes (ABs) were used for the chromatographic characterization of the OT column. The results showed that the modification was successfully performed and allowed satisfactory separation of ABs. The developed OT column in the ProFlow nano-LC was applied to the separation of casein (CN) protein variants, including αs1-CN, αs2-CN, β-CN and Ƙ-CN. Results indicated that the column provided a promising separation of casein protein variants due to both hydrophobic interactions and repulsive electrostatic interactions. Run-to-run and column-to-column reproducibilities were satisfactory (i.e., ≤3.12%). Real sample application of the OT column was demonstrated using a milk sample. Several isoforms of αs1-CN could be detected. All results demonstrated that the developed OT column with a novel coating procedure was promising for advanced nano-liquid chromatographic separations.