Construction And Evaluation Of Capillary Electrochromatography Using Magnetically Immobilized Beds

With the rapid progress of life sciences and biotechnology, magnetic separation technique has become an indispensable tool. The technique offers several advantages including ease of use, rapid separation, being compatible with a variety of sample matrices and amenable to automation and minimization.Capillary electrochromatography (CEC) is a relatively new separation technique that combines the high separation efficiency of capillary electrophoresis with the superior selectivity of high-performance liquid chromatography. Despite the enormous potential of the technique that has been well recognized, several technical problems have hampered the widespread application and general acceptance of CEC. One such problem is column preparation.Magnetic particles are promising candidates for replacing traditional silica for the preparation of CEC columns because of their strong responsibility and easy manipulation in the magnetic field. In this thesis, an external magnetic field was used to immobilize the magnetic microspheres in capillary electrochromatography in attempt to construct fritless capillary columns. The major results were summarized as follows:The two methods were adapted to prepare magnetic microspheres with excellent properties. The first method involves the impregnation of porous silica with ferric-salt followed by oxidation and reduction. The second method is based on the electrostatic self assembling of magnetic nanoparticle on the silica microspheres. The magnetic particles were thoroughly characterized by a range of methods including SEM, VSM, XRD, and BET. The results showed that the magnetic microspheres were uniform, well dispersed, highly porous and magnetizable, which make them ideal for use as packing materials in chromatography.The magnetic particles were immobilized in the capillary by applying an external magnetic field transverse to the direction of electroosmotic flow. Being subjected to the interplay of fluid dragging and magnetic forces, the initial loosely packed particle assembly was compacted into a uniform packing structure. Thus, a fritless packed beds comprised of magnetically responsive octadecylsilane bonded silica particles were constructed for reversed-phase electrochromatography. The magnetically immobilized beds obtained were used for separation of neutral compounds, with retention behavior and column efficiency similar to those of slurry-packed columns. Then the preparation of magnetic immobilized beds was investigated theoretically and experimentally, such as the force balance, the control of packing length and the allowable velocity of flow. The factors that determine the separation efficicency in magnetically immobilized bed electrochromtography were studied, such as the length of packings, pH and ion intensity in buffer, the content of organic phase. Under optimized conditions, five compounds were baseline separated in 50 cm column with 7.0 cm packing and the lowest absolute plate height was 1.64. The results suggest that the magnetic attraction approach to fritless column packing may be used for construction of advanced chip-based chromatography, especially in complex architectures comprising curved and intersecting channels.In addition, several magnetic particle based chiral stationary phases and magnetic lipid were prepared, which are expected to be useful in magnetically immobilized bed chromatography and evaluation of cross-membrane properties of drug candidates.