Novel Preparative Electrochromatography Apparatus For The Separation Of Small Polar Compounds

The extensive progress of biotechnology and bioengineering requires the development of novel separation techniques. The combination of the existing techniques is an important way. In order to enhance the separative resolutions and efficiency, coupling liquid chromatography with electrophoresis by introducing an electric field into a column has been attempted at home and abroad recently. But up to now, only several groups are engaged in the research of preparative electrochromatography. In reported literatures, samples are confined to macromolecules, such as proteins and nucleic acids. Accordingly size-exclusion gels, ion-exchange gels and hydroxylapatite, which are commonly used for macromolecules, are chosen as the stationary phase. In the field of bio-active substances, besides macromolecules, a lot of small polar compounds with novel activities have been discovered, such as peptides, polyphenols, oligonucleotides and water-soluble antibiotics. These compounds have good biocompatibility due to their strong polarity and water-solubility, but these features induce some trouble to separation engineers. In the field of separation process, liquid-liquid extraction and RP-HPLC could be easily scaled, which have good performance for the separation of non-polar and weakly polar compounds, but they are not suitable for polar ones due to the poor retention (e. g. cephalosporin C and oligonucleotides). Since most polar compounds are chargeable, they could be well separated by capillary electrophoresis or electrochromatography. However the capillary-based methods could not be scaled, so that it is difficult to provide microgram-scale fractions. If preparative electrochromatography is explored, it may be of significance and novelty for the separation of small polar compounds. Therefore a novel kind of preparative electrochromatography apparatus was developed in this dissertation, which is especially suitble for small polar or weak polar compounds. Accordingly preparative electrochromatography techniques to enhance resolution and concentration of fractionated samples were designed and investigated. Experiments in terms of the influencing factors and the mechanisms were also investigated, which illustrated that the electric field had great effects on chromatographic performance.A novel preparative electrochromatography apparatus was constituted on the basis of column chromatography (40 cm×0.6 cm I.D., 40 cm×1.2 cm I.D., 40 cm×2.0 cm I.D.). Common problems of preparative electrochromatography, i. e. electrolytic bubbles and Joule heat, were alleviated. The construction and manipulation of this apparatus were established. Two electrode chambers containing electrode buffer and electrodes were separatedly connected with two column ends, which was similar to the reported apparatus, while the manner of their connection was newly proposed in this dissertation. In literatures, exclusive membranes or gel rods were put between the electrode chambers and the column in order to prevent bubbles from column, meanwhile electrode buffers were circulated with the reservoir to take the bubbles away. Apparently the reported apparatus are not suitable for small molecules, which may travel across the membranes or gel rods and lose in electrode buffer. Diverse from the reported apparatus, electrode chambers in this dissertation were open to free electrolytic bubbles. The outlet electrode chamber in"T"-shape with each branch of 5 mm I.D. was put at the foot of the column and connected with the column branch, so as to free bubbles without the need of membranes or gel rods. The effluent out of column could pass through the outlet chamber and then flew to online detection without loss. Therefore the novel apparatus could accommodate the separation of small compounds. In the cooling system of this apparatus, besides column water jacket and the ice-water bath for the inlet eluent, the"T"-shape outlet chamber and the connecting tubes were also cooled, where the Joule heat was prone to accumulate. Thus higher electric field of 60~120 V/cm could be applied steadily, while most electric field used in literature was only 20~50 V/cm.In the main part of the dissertation, macroporous non-polar adsorption resin usually used for the separation of small molecules, cation-exchange gel which could compose anion-exclusion chromatography with anions, and porous size-exclusion gel were chosen as the stationary phase to separate small polar compounds, including tea polyphenols, mononucleotides and water-soluble cephalosporins.Tea polyphenol monomer EGCG and ECG could be partially separated from crude tea extract containing caffeine by applying negative electric field (cathode at column inlet, anode at outlet) on the column packed with non-polar adsorptive DIAION HP20 resin. By altering the chromatographic factors, including particle size, flow rate, ethanol concentration, and the electrophoretic factor, buffer pH, solute retention could be dramatically affected. This verified the successful combination of the chromatography and electrophoresis techniques. During the separation course, the mechanisms including electrophoresis, electroosmosis and chromatography could affect separation results. Besides, electric field could affect the chromatographic performance by influencing the mass transfer from mobile phase liquid into resin particles. The electroosmotic flow could not formed in the intraparticle of the resin due to its small pore size (<30 nm), thus mobile phase flow containing sample solutes could merely travel interpartically and mass transfer into intraparticles was hindered to some extent. Especially the retention of EGCG and ECG, whose adsorptive parts are asymmetrically distributed in molecule, was more likely to be affected. The electrochromatography was amplified by column diameter while the column length kept the same. Consequently Joule heat was not apparently increased, and the coupling of electrophoresis and chromatography was observed. However the separative performances were not as good as those obtained from smaller columns, due to the loss of partial column efficiency.When applying negative electric field (cathode at column inlet, anode at outlet) on an anion-exclusive chromatographic column packed with weakly acidic gels, the carboxy groups on resin could be protonated via the electrolysis of anode, and the protonation could be enhanced with the increase of electric field applying time. Consequently, tea polyphenols (EGCG and ECG) were completely separated from caffeine by applying electric field. Studies on this novel method revealed that the electrolytic reaction in electrode chambers was the main cause of the chromatographic performance alteration. The gel at the column bottom was protonated due to electrolysis, thus the anion-exclusive interactions with polyphenols were weakened and the resolutions were improved. It is the first time to study this separative method, which is referable for the preparation of polyphenols or other weakly acidic compounds. The separation mechanisms by applying electric field on anion-exclusive chromatography and adsorptive chromatography were different, which illustrated that the electric field could affect the chromatographic performance from different aspects.Porous size-exclusion gels, which are commonly used to separate macromolecules, were first attempted to separate small polar mononucleotides and cephalosporins with electric field. Different from the above methods for tea polyphenols, gels here could slightly or not adsorb sample solutes, thus all solutes result in the same retention and could not be separated without electric field. The gels here functioned as electrophoretic media to prevent the convective effects and realize the online detection and fractionation. Solutes could be partially separated on the basis of their charges when applying electric field. Actually, this could be taken as a preparative electrophoresis system. It was discovered that the bigger the pore of the gel, the better the performance of electrophoresis. Electrophoresis with isocratic eluent could apparently diminish or enhance the retention when the direction of electrophoresis was the same or opposite to hydrodynamic flow. On this basis, isoelectric focusing electrophoresis of four zwitterionic cephalosporins was established by using pH gradient eluent. Consequently, the separative time was much shorter and sample peaks were much more concentrated than the results by ion-exchange chromatography. Resolution by isoelectric focusing electrophoresis was less affected when slightly altering the buffer pH. Moreover, the pH gradient buffers were made of dilute acetate and ammonia solution (0.01 M), which is cheap and easy to be composed. The established novel isoelectric focusing electrophoresis could be used to refine and concentrate small polar compounds.Conclusively, in this dissertation a preparative electrochromatography apparatus especially for the separation of small polar compounds, and four separation methods with applying electric field were designed and studied. In the experiments, aqueous buffers were used to separate samples in aqueous solution, including small polar and weakly polar compounds. The separative mechanisms of every established method were different, but all the separations revealed that applying electric field on chromatography could enhance resolution and fractionated sample concentration. These methods are suitable for the separation of small polar compounds, and are complemented with RP-HPLC and silica-solvent technique for non-polar compounds. These methods are promising and valuable in the investigation of novel small polar or weakly polar compounds.