Nanoparticle-based Capillary Electrochromatography - Separation of Small Molecules and Proteins

Abstract: The use of nanoparticle-based pseudostationary phases (PSPs) in capillary electrochromatography (CEC) for separation of small molecules and proteins is demonstrated. A continuous full filling approach is used where the nanoparticles are suspended in the electrolyte. A new interaction phase is introduced for every analysis, which eliminate problems with stationary phase-based carry-over effects. Highly efficient separations of small molecules (700 000 plates /m) were achieved using dextran-coated polymer nanoparticles as PSP in CEC with mass spectrometry (MS) detection (Paper I). The analytes were detected by using an orthogonal electrospray interface to prevent the nanoparticles from entering the mass spectrometer. The methodology (PSP-CEC) was used for protein separation utilizing soft lipid-based liquid crystalline nanoparticles as PSP (Paper II-III and V-VI). These nanoparticles are known for their high biocompatibility and porosity. A one-step procedure based on lipid self assembly was used for their preparation and the surface chemistry is thereby easily alternated by changing the composition of the lipid preformulation. To detect proteins in the presence of high concentrations of lipid-based nanoparticles, laser induced fluorescence (LIF) detection was used. Green fluorescent protein (GFP) and its variants were used to evaluate the separation system. Single amino acid substituted GFP variants differing in charge, could be separated by conventional CE. To separate GFP variants having the same charge use of anionic lipid-based nanoparticles enabled separation. At high buffer salt concentrations the lipid-based nanoparticles were adsorbed to the capillary wall, which suppressed the electroosmotic flow (EOF). In addition, it is suggested that the high concentration of buffer salt induced interactions between the lipid-based nanoparticles and GFP variants, which provided selectivity and thereby enabled separation. Three different PSP-CEC methods were developed based on either cationic or anionic nanoparticles or a combination of both types of nanoparticles. To enable UV detection in a nanoparticle-based PSP-CEC system pegylated silanized gadolinium nanoparticles were used due to their UV transparency (Paper IV). In addition, the nanoparticles could be evaluated concerning their interaction with proteins. The developed nanoparticles-based system were transferred to a polymeric capillary (Paper V) and microchip (Paper VI) where the nanoparticles appear to prevent protein adsorption to the capillary wall. Highly efficient separation of GFP variants were demonstrated in these systems demonstrating the potential use of these methods and particles in future mass fabricated chip formats. This work demonstrates the potential of various novel nanoparticle-based separation in various capillary analytical formats for highly efficient separation of small and large molecules (such as proteins) with potential applications within the pharmaceutical or proteomic field.