Retention Behavior Of Biopolymers On Short Chromatographic Columns

Although the resolution of small molecule solutes separated by high performance liquid chromatography (HPLC) increases as column length increases, it is known that the resolution of biopolymers is almost independent of column length. Sometimes, a short column even exhibits a better resolution than a long one. The phenomenon that the resolution of biopolymers is almost independent of column length was explained by 'open-off mechanism' and 'all-nothing principle' which are based on 'stoichiometric displacement theory for retention' (SDT-R). Although it may be true that biopolymers either do not move at all or transfer immediately in a chromatographic column, the transfer of biopolymers has not been fully understood. Because of this, the phenomenon can only be explained qualitatively. In order to explain it quantitatively, it is essential to study the retention behavior of biopolymers in a chromatographic column further.The thesis includes the following three parts:1 Introduction. A brief review on the recent development of short column theory, SDT-R, five kinds of HPLC used in separation of biopolymers, chromatographic cake and organic polymer monolithic column was summarized.2 The retention behavior of biopolymers on short chromatographic columns. Six kinds of biopolymers, cytochrome c, myoglobin, ribonuclease A, lysozyme,α-chymotrypsin andα-amylase, were used as model proteins and their transfer behaviors in a chromatographic column of hydrophobic interaction chromatography (HIC) were studied. Their characters were found that biopolymers do not move before the concentration of displacer in mobile phase approaches to a specific value. If the biopolymers begin to move, they will move in the chromatographic column very quickly. Although column length and the flow rate of mobile phase varied, three rules were always found in the process of transfer. Besides these, two qualitative rules were discovered. One is that the longer the column, the later the biopolymers move, and the other is the larger the flow rate, the earlier the biopolymers move.3 The preparation of monolithic poly(butyl methacrylate-co-ethylene dimethacrylate) column and application. The preparations of monolithic capillary glass column using a free radical in situ polymerization within 30×1.18 mm i.d. capillary glass and their application for HPLC separations of proteins have been studied. With the capillary glass RPLC column, good separations of protein mixture consisting of ribonuclease A, cytochrome c, lysozyme, bovine serum albumin and ovalbumin were achieved in less than 3 minutes. Also, a 5-protein mixture consisting of ribonuclease A, cytochrome c, lysozyme, bovine serum albumin and ovalbumin was baseline separated.