Protein Migration Characteristics On The Liquid-solid Interface

The purity and separation of recombinant proteins are the technical bottleneck in down-stream technology of biotechnology. High performance liquid chromatography (HPLC) is the best way for purifying and separating the proteins, especially in preparative scale, it is also an important analytical tool in pharmaceutical industry. Due to the distinction of proteins and small molecular mass, the retention character of proteins during separation process by HPLC is quite different from that of small solutes. The changes in the molecular conformation of proteins during this process may change and bring in more difficulties. So it is important to optimize the separation parameter and to understand molecular mechanism of protein retention. Besides thermodynamic factors, the migration rule of solute involving in dynamic factors is the other important thing for this optimization. The thesis is to investigate the latter instance. The thesis contains five parts as follow:From the recent development of protein retention model was reviewed. When the retention mode of small solutes is employed for the prediction of protein retention, a great deviation occurs. This fact indicates that some problems scientists have net been found yet. 148 references were cited, the recent 3 years references account for 16%.Take hydrophobic interaction chromatography (HIC) column as an example, the migration rule of proteins on the interface between the stationary phase of HIC and mobile phase was investigated. It is firstly found that during gradients elution and the molecular conformation of proteins remains invariance, proteins retention is independent of the injection time until to a specific value for each protein. Thus, the retention of protein can be separated two parts: protein almost does not move as mobile phase strength is weaker than a certain value and protein starts to migrate until it is eluted out. In the latter instance, a quantitative relationship between the protein migration and the mobile phase composition was found. Based on this fact, a new concept of proteins discontinuous migration is firstly proposed.For reversed phase liquid chromatography (RPLC), using acetonitrile-water as mobile phase, when proteins were separated under the condition of molecular conformation of proteins changes, even denature, the foregoing discontinuous migration was also found. Small solutes were found to move faster than proteins when velocity of mobile phase increase, and also found a log-log linear relationship between relative retention time and flow-rate.Four commercial weak-anion exchange chromatography (WAX) columns were tested whether HIC retention exists, or not. The experimental result indicates that the existence of HIC mechanism in all of WAX column is a universal phenomenon, i.e., each of WAX column has a mixed-retention mode of WAX and HIC, although the extent is different. The phenomenon was explained by molecule mechanism and the stoichiometric displacement theory (SDT) was used to test this phenomenon.The HIC mechanism existed in WAX was used to on-line two-dimensional liquid chromatography using a single column (2DLC-1C) to separate industryα-amylase firstly, and buffer exchange was carried out also with on-line manner. Compared to commercial HIC, the purity efficiency of 2DLC-1C is superior to either a single HIC column, or a WAX IEC column.