Ionic Liquids And Carbon Nanotubes In The On-Line Separation And Preconcentration Of Proteins

This work focuses on the seperation and proconcentration of proteins from complex biological sample matrixes with ionic liquid and carbon nanotubes as adsorbents.Chapter 1 of this thesis is an overview of the ionic liquid and carbon nanotubes as well as their applications in the field of separation.The following chapter demonstrates the direct extraction of proteins from human body fluids with an ionic liquid/aqueous two phase system based on the hydrophilic ionic liquid 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) and K2HPO4. Bovine serium albumin (BSA) at low levels were quantitatively extracted into the [Bmim]Cl-rich upper phase with a distribution ratio of about 10 between the upper and lower phase and an enrichment factor of 5. FTIR and UV spectroscopies demonstrated that no alterations of the natural properties of BSA were observed. The partitioning of proteins in the two-phase system was assumed to have been facilitated by the electrostatic potential difference between the coexisting phases, as well as by salting out effects. The system has been applied for the quantification of proteins in human urine after on-line phase separation in a flow system.Chapter 3 explores the online selective sorption and isolation of basic proteins in biological sample matrices by solid-phase extraction by using oxidized multiwalled carbon nanotubes (MWNTs) as absorbent. The retained protein species were afterwards separated from each other by sequential elution from the microcolumn through the employment of appropriate eluents. With a sample loading volume of 2.0 mL, enrichment factors of 11 and 15 were derived for hemoglobin and cytochrome c, along with retention efficiencies of 100% for both species and recovery rates of 98 and 90%, respectively. The practical applicability of this system was demonstrated by processing human whole blood for isolation of hemoglobin, and satisfactory results were obtained by SDS-PAGE assay.Chapter 4 presents a simple procedure for assembling multiwalled carbon nanotubes (MWNTs) on the surface of silica beads using a polyelectrolyte-assisted layer-by-layer (LBL) assembly technique. The surface-assembled silica spheres with MWNTs(MWNTs/SiO2), was used as a novel solid-phase extraction sorbent for the separation/pre-concentration of basic proteins. The adsorption behavior of cytochrome c by MWNTs/SiO2 spheres agrees well with the Langmuir adsorption model. A maximum adsorption capacity of cytochrome c was derived as 112 mg/g MWNTs. A distinct feature of the MWNTs/SiO2-packed micro-column provides clear advantages of minimized flow impedance when operated in a flow system, in addition to better separation efficiency as well as the favorable enrichment capability of proteins, characterized by an enrichment factor of 30 by using 2.0 mL of aqueous solution.Chapter 5 presents a simple, non-covalent procedure for functionalization of MWNTs by wrapping of positively charged water soluble linear polymer poly(diallyldimethylammonium chloride)(PDDA). The micro-column packed with PDDA-wrapped MWNTs in sequential injection system facilitates selective sorption of acidic protein species. Proteins adsorbed onto the PDDA-MWNT composites are afterwards collected by elution with a citrate buffer as stripping reagent. With a sample loading volume of 2.0 mL and an eluent volume of 200μL, a retention efficiency of 100% and a recovery of 90% are achieved for BSA in the range 6-120μg, resulting in an enrichment factor of 14.Chapter 6 discusses the selective isolation of acidic proteins using a thin layer of MWNTs functionalized with PDDA. A suspension of PDDA-functionalized MWNTs that had been well dispersed by sonication was filtered through an MF-Millipore membrane with a pore aperture of 1.2μm, and a uniform layer of PDDA-MWNT composites with a thickness of ca.5μm formed on the membrane. A piece of the obtained membrane was introduced into a module and incorporated into a sequential injection system for performing on-line separation and preconcentration of acidic protein, i.e., bovine serum albumin(BSA). The BSA retained on the layer could be effectively eluted with a citrate buffer. In addition to a significant reduction in flow resistance, a dynamic sorption capacity of 3.8 mg/mg or 1.4 mg/cm2 for BSA was achieved using the layer-based system, i.e., a 146-fold improvement over that obtained using a packed microcolumn mode.