Kinetic Liquid Chromatography

With the rapid development in life science, a larger number of hot researching topics for modern separation science have been reported. Chromatographic separation technology is still the key process of down-stream biotechnology. How to obtain the best LC resolution has been an important investigated hot point. In recent years, a novel chromatographic separation method basing on hydrodynamic factors, such as mobile flow rate, the shape and size of packing being different from the traditional equilibrium thermodynamics was explored. A particular feature of this chromatography is firstly named as dynamic chromatography. Many correlative publications have been concentrated in theoretical research, the practical applications are often limited to the separation of some standard small molecules and DNA molecular fragments, so more jobs should be need to promote its further development and expand its application. In this paper, the rule of solute retention by dynamic chromatography in reverse phase liquid chromatography (RPLC) has been studied. An equation that is obeyed universally by solutes is found and two parameters used to describe the dynamic chromatography behavior are firstly proposed. In the end, the method is used for the separation of the actual samples.Papers include the following six parts:1. Literature Review: A brief discussion about the development, basic theory and the main contents of chromatographic dynamics is introduced. It includes the plate theory, rate theory, non-equilibrium theory and mass balance theory. The main difference between Chromatographic dynamics and dynamic chromatography is explained. Two dynamic chromatographies, hydrodynamic chromatography (HDC) and slalom chromatography (SC) were introduced and reviewed in this paper, mainly for the recent development of separation principle, theoretical model, applications and . 75 references are cited.2. By taking the peptide mapping of cytochrome-c as an example, the effect of dynamic factors in RPLC is investigated. It is mainly for the effect on the resolution, peak capacity and solute elution order. To study the relationship between the flow rate of mobile phase and retentions, we found the logarithm of the relative retention time (RRT) of peptides to be proportional to the logarithm of the flow rate of mobile phase (υ), i.e.log (RRT) = a + b log (ν) (1)The two linear parameters were also found to obey the equation:a = c + d b (2)Where, a, b are empirical constants.The similarities and differences of log-log linear relationship with the two linear equations in stoichiometric displacement theory (SDT) are also compared.3. The dynamic chromatographic behavior of small solutes, peptides and the proteins in RPLC is also studied. The Eq. (1) is also tested and found it to be universal and discussed in detail. Both a, b are the two dynamic chromatographis parameters that have not any thermodynamics meaning. A specific relationship between the molecular mass of peptides and retention values is not found and the solute elution order does not obey the mechanism of SC or HDC. Both a and b have a little linear relationship, but the correlation coefficient is not very good.4. In this chapter, the optimization flow rate of mobile phase for the separation of four components of Lys peptide mapping is investigated and found that it is difficult to separate using common method. However, the separation of solutes at different flow rates of mobile phase is predicted from the two dynamic chromatographic parameters, a and b. The optimization flow rate of mobile phase was also predicted and it verify the meaning about a, b in separation.5. The experiment confirmed that the b value of is general smaller than that of protein macromolecules. Therefore, the small molecules' RRT change is greater than that of macromolecular proteins as the flow rate of mobile phase changed. The move velocity of small molecules is greater than that of macromolecular proteins in column. According to this principle, two dynamic chromatographic strategies for the separation of complex samples are presented. The one is that the complex samples will be quickly divided into two major components, the small molecules group and the macromolecules group, when the flow rate of mobile phase is increase up to a certain value during gradient elution and the two groups will simplify the separated. The small molecules has a width elution concentration range but that of macromolecules is very narrow that can be simply regarded as "a point", so the other dynamic chromatographic strategy can be adapted: A suitable concentration for isocratic elution to elute small molecules firstly, and subsequently a gradient elution to separate macromolecules. Components of hoptoad ovum cell were rapidly separated and purified by this way. High abundance of peptide components were removed with the low-enriched proteins. It would be expected that the two dynamically chromatographic strategies will be a new practical method used for proteomics.6. The two dynamically chromatographic parameters a, b has more stabile character in different thermodynamic conditions. A dynamically chromatographic finger Chromatogram was conceived simply.