Separation of basic oligopeptides by ion-pairing reversed-phase chromatography.

摘要

The present thesis consist of five chapters. Chapter I introduces background information on the ion-pairing reversed-phase chromatography and liquid chromatography in the critical condition.;Chapter II decribes our study on the isocratic separation of oligolysine (dp = 2 to 8) using a fixed content of acetonitrile (ACN) (23%) and different concentrations of HFBA in the mobile phase (0.6-30.6 mM) on a Waters XBridge Shield RP18® column. We found that the retention time of oligolysine increases as the dp increases, because of an increased number of HFBA bound to the peptides. Furthermore, when [HFBA] increased, the retention time increased at different rates. The greater the dp, the faster the rate. Based on a closed pairing model that presumes an equilibrium between an unpaired state and the paired state with a fixed number of HFBA molecules, an equation was derived for the retention factor of oligolysine.;In Chapter III, we compare retention behaviors of oligolysine (dp = 2 to 8) and oligoarginine (dp = 2 to 8) when they are separated on the Waters XBridge Shield RP18® using fixed a ACN content (23%) and difference concentrations of HFBA (0.4-30.6 mM) in the mobile phase. The retention time of oligoarginine also increased at different rates as [HFBA] increased. The greater the dp, the faster the rate. The retention time of oligolysine is shorter than that of oligarginine having the dame dp. We applied Eq.1 to analyze the plot of ln k as a function of [HFBA] for each oligopeptide component to obtain the values for n, Kip,m, and βKd,ip. For oligolysine, n increases linearly as dp increase and oligoarginine exhibits an accelerated increase in n as dp rises. The plot of ln βKd,ip against dp followed a linear relationship for both peptides.;In Chapter IV, we study the effect of mobile phase composition on the retention of oligolysine (dp = 2 to 8) on the Waters XBridge Shield RP18 ®. The ACN content was changed from 20% to 33% and the HFBA concentration from 0.7 to 38.4 mM. We investigated the effect of [HFBA] and percentage of ACN on the resolution in separating the peptides and determined the optimal mobile phase composition. We applied Eq.1 to fit the plot of ln k as a function of [HFBA] for each ACN content, which provided us values for n, Kip,m, and β Kd,ip. n. We found that β KD,ip decreases as the ACN content increases and the decrease slows down as the percentage of ACN increases, possibly caused by ACN enrichment in the stationary phase.;The study described in Chapter V used a different column, SuperAW 3000 ®, to separate an oligolysine mixture (dp = 3 to 11) in different separation modes including ion exchange, size exclusion, critical condition and reversed phase. The analysis was carried on the SuperAW 3000® column with heptafluorobutyric acid (HFBA) as an ion-pairing reagent. We changed either the percentage of ACN at a fixed concentration of HFBA or the concentration of HFBA at a fixed percentage of ACN to investigate the effects of the percentages ACN and HFBA on the retention of oligolysine in different separation modes. A low molecular weight polyethylene glycol and a low molecular weight polypropylene glycol was used as references in different conditions. We compared the reversed-phase separation on Xbridge Shield ® and SuperAW 3000®, at different concentrations of HFBA. We also found that both ion-exchange and hydrophobic interaction play a role in the separation of oligolysine on SuperAW 3000®, when [HFBA] was low. (Abstract shortened by UMI.).