High Performance Hydrophobic Charge Induction Chromatography For Protein Analysis And Separation

Currently, High Performance Liquid Chromatography (HPLC) technology with the highresolution becomes one of the most commonly used techniques for the protein separation andanalysis, providing effective support for protein conformation and structure study. However,the single patter of HPLC technology still can’t meet the requirements for the separation andanalysis of the mixed commonents to reach the crystal purity. In recent years, a newdule-mode chromatography: Hydrophobic Charge Induction Chromatography (HCIC) wasintroduced to separate themacromolecules successfully under atmospheric press. Here HCICis combined into HPLC to form a new chromatography mode of High PerformanceHydrophobic Charge Induction Chromatography (HPHCIC), which is recognized as highresolution, dual interation mode andhigh biocompatibility. In the present work, a new type ofHPHCIC media was prepared and the retention and separation behavior of model proteinwere investigated. Moreover, the structure of the model proteins were studied in theChromatographic condition to show the theoretical basis for the possible mechanism of theprotein retention behavior. The main works was carried out as follows:(1) Preparation and characteristic of HPHCIC media. The ligand4-Mercapto pyridinewas bonded to the spherical silica gel with the diameter of10microns and the pore size of300angstroms using KH-560as the intermediate coupling agent. The results show that thehighest utilization ratio of KH-560can be reached at the reaction temperature and time of75℃and10h, respectively. Meanwhile the activation density on the media can be obtained in awide range at the pH, reaction temperature and reaction time of7.5,40℃and6h,respectively, resulting in a wide range of ligand density on the media through the control of4-mercaptopyridine. The HPHCIC media is proving by the infrared spectrometer (FTIR) andThermofravimetry (TG).(2) The retention behaviors of model protein on the HPHCIC media. Bovine serumalbumin (BSA) and lysozyme with different isoelectric points were selected as the modelproteins to investigate the retention behaviors on the HPHCIC media with the ligand densityof75μmol·g-1. The effect of pH, acetonitrile and salt concentration (C(NaCl)andC(Na2SO4)) ofmobile phase on the retention factor was examed and the minimal and maximal retentionfactors for BSA and lysozyme are found at the certain condition of pH, acetonitrile and saltconcentration. Moreover, the possible mechanism is discussed for the interaction of the modelprotein and the HPHCIC media under the condition of chromatography.(3) Structure study of model proteins under different mobile phase condition. Thesecondary structure of BSA and lysozyme is studied under different mobile phase by themethod of circular dichroism (CD) spectrometer. The results showed that the secondarystructure of model protein changes significantly as the function of pH, acetonitrile, NaCl andNa2SO4, respectively. The molecular conformation of BSA and lysozyme is investigated byFluorescence spectra. The results showed that the endogenous fluorescence of BSA could besignificantly quenched when the pH value is lower than7, the acetonitrile content is higherthan30%, C(NaCl)is higher than0.1mol·L-1,C1(Na2SO4)is higher than0.05mol·L-, respectively. and the endogenous fluorescence of lysozyme could be significantly quenchedwhen C(NaCl)is higher than0.5mol·L-1,C(Na2SO4)is higher than0.05mol·L-1, respectively. Theresults of CD and fluorescence spectra correspond to retention behavior of model protein indifferent mobile phase conditions, providing a theoretical mechanism for HPHCIC.(4) Separation of model protein by HPHCIC. The effect of single mobile phase pH,mobile phase pH gradient (ΔpH) on the resolution of BSA and lysozyme was investaged. Andwhen ΔpH is1.8, without organic solvents, the resolution of BSA and lysozyme reaches2.315.Moreover, salts and acetonitrile were introduced to improve the resolution. The resultsshowed the better resolution of BSA and lysozyme could be obtained when NaClconcentration is0.1mol·L-1, the acetonitrile content is30%and Na2SO4concentration is0.005mol·L-1, the acetonitrile content is60%.