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Minimization Of Diffusion Of Solutes In HPLC

Posted on:2007-04-22Degree:MasterType:Thesis
Country:ChinaCandidate:P LiuFull Text:PDF
GTID:2121360182495014Subject:Analytical Chemistry
Abstract/Summary:PDF Full Text Request
In high performance liquid chromatography (HPLC), when small solute was separated, the resolution is increase with column length, but for bio-macromolecules, according to short column theory in HPLC, resolution is almost independent of column length. Sometimes a short column even has a better resolution than a long one. Based on dynamics of mass transfer, compared to a longer column, the separation of short one has less diffusion inside column. It is important to investigate the diffusion rule of small solutes, which can provide an important information to the separation of bio-macromolecules with short column. This paper includes three parts.1. Separation of proteins by short column of HPHICUsing the same kind of hydrophobic interaction packings, PEG-600s with large particles packed in longer column, i.e., L-L combination and small particles packed in a short column, i.e., S-S combination, five kind standard proteins were separated, respectively. Compare the two kinds of columns, it was found that both have good resolution. Because the diffusion of mass transfer of the S-S combination is less than that of the L-L combination, a better resolution for former is than that for the latter.A series of column lengths from 1mm to 5mm with S-S combination were taken to investigate and compare the obtained resolution. It was found that when column length was reduce to just lmm, five kinds standard proteins can be separated completely. Finally, a short column of 2.0 mm was employed to separate rhIFN- γ(recombination human interferon-γ) successfully. The rhIFN-γ was eluted in 10min and its peak purity is 92%.2. Diffusion of small solutes in RPLCColumn length, 3.0, 5.0, 10.0, 15.0, 20.0, and 25.0cm of reversed phased high performance liquid chromatography (RPLC) was selected as the range of column length changes, methanol-water as the typical mobile phase, benzene, toluene,naphthalene, diphenyl as the model solutes, the relationship among the half-peak width, retention time, flow-rate of mobile phase, and column length was investigated under the conditions of isocratic elution, different flow-rates of mobile phase, and various methanol concentrations. The obtained results show: (1) Good linear relationship between half-peak width and retention time;(2) Half-peak width and the square root of flow-rate reciprocal value;(3) Half-peak width and the square root of column length were found to exist.Based on the changeable rule obtained above, the half-peak width was theoretically predicted. Further, the quantitative relation between the slope of linear equation and solutes molecular structural parameter was also studied. The obtained result shows: (1) The slope of linear relation between half-peak width and retention time at different flow rates of mobile phase exists;(2) Two convergences between both of van der waals volume and van der waals area of the four kinds solutes and the methanol, concentration were found to exist and their convergence coordinates were also calculated.3. TheoryIn this part, based on random walk model, these relationships found in part 2 were derived theoretically and the existence of the convergence was explained also.
Keywords/Search Tags:HPLC, hydrophobic interaction chromatography, reversed phase chromatography, protein, half-peak width, retention time, flow-rate, column length
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