Preparation Of Novel Capillary Columns And Its Related Applications

Column technology, which is the heart of the capillary separation process, is also the bottle neck technology. In the field of column preparation, the primary task of capillary separation is to prepare novel and applicable columns.Sol-gel technology offers a versatile and simple means of creating stationary phase with desired chromatographic and surface characteristics to column fabrication for microseparation techniques.The combination of sol-gel technology and preparation of hybrid LC packing material is a novel intersection point. Polymer-coated silica, which is prepared by covering a thin layer of organic polymer onto the surface of inorganic support has the advantages of both high efficiency and rigidity of bonded phases, as well as chemical stability of polymer phases. However, the cross-linking reactions used in the conventional polymer immobilization process are difficult to control to ensure the same degree of cross-linking in different columns with the same stationary phase, and may lead to significant changes in the polymer structure, and chromatographic properties of the resulting immobilized polymer. Therefore, it is a new and significant research work for the use of sol-gel technique as a method of depositing and immobilizing a polymer layer onto the pore surfaces of silica support for the preparation of GC stationary phase-coated or encapsulated stationary phases with different selectivity.The combination of sol-gel technique and capillary packed-column to prepare narrow-bore particle-entrapped monolithic columns has been applied to capillary electrochromatography separations. Based on the pioneer work, the feasibility of making large-bore particle-entrapped monolithic column is promising. Therefore, the preparation of large-bore particle-entrapped monolithic columns and application of it as a preconcentration column for on-line peptides enrichment are innovative and practical.Proteomics is playing more and more important roles in the life sciences after the sequencing of the human genome was completed. Because a biological system is very complex and diverse, the study and application of technological platform for the separation and analysis with high resolution, high sensitivity, high throughput and broad dynamic range have become one of the important and hot spots. Consequently, it is important to develop a novel platform to circumvent the throughput problem incurrent on-line 2D-LC/MS/MS system for complex proteome analyses. In addition, the application of new capillary columns to proteomics platform is also gaining increasing attention.Therefore, the core of this dissertation is exploring the related investigation and application based on the preparation of the novel and practical capillary columns using sol-gel technology. This dissertation is divided into five parts.In the first chapter, advances in stationary phases of capillary separations, sol-gel-based capillary columns and multidimensional liquid separation techniques were summarized with details.In chapter 2, a sol-gel chemistry-based polymer coating approach was developed for the preparation of a novel polysiloxane-coated silica stationary phase for capillary liquid chromatography. SE-30, a commercial polysiloxane stationary phase used in gas chromatography, was incorporated into the properly designed sol solution. Then the sol-gel mixture was introduced into a silica gel-packed capillary column by pressure. A thin film of sol-gel SE-30-coating is chemically bonded to the surface of silica gel particles by hydrolytic poly condensation under mild conditions without any free radical cross-linking procedures, therefore the sol-gel approach offers a simple and effective pathway to create a hybrid polymer-coated silica stationary phase. Various factors affecting column making were optimized and discussed in this report. The resulting stationary phase showed good permeability, mechanical robustness, high durability to alkaline mobile phase and satisfactory chromatographic performance in separations of polar and non-polar aromatic compounds. The results also indicate that the stationary phase has a reversed-phased character with SE-30 providing chromatographic functionality.In chapter 3, The retention behaviors of polymer-coated silica stationary phase prepared by using different polysiloxane (SE-30, SE-54, OV-1701) were studied by means of linear salvation energy relationships (LSERs) and compared with ODS stationary phase. On the four stationary phases, 21 kinds of compounds were tested in mobile phase of methanol and water. Good relationships between log k' and LSERs parameters are obtained for these phases. According to percent variance, retention on the polysiloxane-coated silica is dominated by four major factors: the solute size, dipolarity/ polarizability, hydrogen bond donor acidity and the solute hydrogen bond acceptor basicity. This result indicates that the resulting stationary phase has different selectivity and retention mechanism compared with ODS, and the selectivity ofstationary phase can be varied by appropriate choice of polymer for coating to realize the required separation.Chapter 4 describes the preparation and characterization of large-bore particle-entrapped monolithic precolumns, which are suitable for incorporation into a 2D-LC system for proteome analysis. The fritless precolumns with different inner diameter (320 μm and 530 μm) were rapidly and successfully prepared by entrapping octadecylsilica (ODS) particles prepacked into fused silica capillaries with a sol-gel network which was formed by hydrolysis and polycondensation of methyltriethoxysilane (MTES). Enough mechanical strength and permeability of the resulting large-bore monolithic precolumns were achieved by optimizing the percentage of MTES in the sol solution, and No cracking or shrinkage of the column bed was observed even in 530-μm-i.d. capillary. The performance of the particle-entrapped monolithic precolumns used for preconcentration and desalting of proteolytic digests was evaluated by on-line coupling the large-bore precolumns with a capillary reversed-phase liquid chromatographic column followed by UV detection. The laboratory-made monolithic precolumns with 320 and 530 μm i.d. were characterized by using BSA digested peptides as the analytes with respect to sample loading capacity, linearity, recovery and reproducibility, etc. The results indicated that the large-bore and short precolumns (5 mm×320 μm i.d or 5 mm×530 μm i.d.) allow sample fast loading and have a mass loading capacity for BSA peptides of about 70μg. Good linear calibration curves (R~2>0.99) were obtained and the limits of detection were improved by 60-fold even with a UV absorbance detector. The total recovery was found to be approximately 90%. With good reproducibility for column-to-column and batch-to-batch, no significant degradation or decrease in precolumn performance was showed even after -150 preconcentration /desorption cycles. The precolumns also proved to be resistant to salt buffer with high concentration and low-pH mobile phase. The large-bore particle-entrapped monolithic precolumns were further used in a high-throughput 2D-LC array system coupled with tandem matrix assisted laser desorption/ionization-time of flight-time of flight-mass spectrometry (MALDI-TOF-TOF-MS) detection for proteome analysis.In chapter 5, an automated on-line capillary array-based 2D-LC system coupled with MALDI-TOF-TOF-MS proteomics analyzer for comprehensive proteomic analyses has been developed, in which one capillary strong cation-exchange (SCX) chromatographic column was used as the first separation dimension and 18 parallelcapillary RPLC columns were used as the second separation dimension. 18 particle-entrapped monolithic precolumns, which were made using sol-gel technique in our laboratory, were placed individually prior to each parallel capillary RP column to interface the two dimensions for desalting and enrichment of peptide fractions eluted from capillary SCX column. Peptides bound to the SCX phase were "stepped" off using multiple salt pulses followed by sequentially loading of each subset of peptides onto the corresponding precolumn. After salt fractionation, with a home-built 18-channel liquid splitter, peptide fractions were concurrently back-flushed from the precolumns and separated simultaneously by 18 capillary RP columns. By using a home-made LC-MALDI interface, LC effluents was directly deposited on the MALDI target through an array of capillary tips at a 15 s time-interval for further MS analysis. Therefore, with simplified equipment, this new system allows an 18-fold increase in throughput for complex proteome separation compared with serial-based 2D-LC system. The high efficiency of the overall system was demonstrated by analysis of tryptic digest of proteins extracted from normal human liver tissue.