Based on the monodisperse poly(glycidyl methacrylate-co-ethylenedimethacrylate) beads (PGMA/EDMA) with macropore as a medium, a new hydrophilic medium cation exchange (MCX) stationary phase for HPLC was synthesized by a new chemically modified method. The stationary phase was evaluated with the property of ion exchange, separability, reproducibility, hydrophilicity, effect of salt concentration, salt types, column loading and pH on the separation and retention of proteins in detail. It was found that it follows ion exchange chromatographic (IEC) retention mechanism. The measured bioactivity recovery for lysozyme was (96±5)%. The dynamic protein loading capacity of the synthesized MCX packings was 21.8 mg/g. Five proteins were almost completely separated within 6.0 min at a flow rate of 4 mL/min using the synthesized MCX resin. The MCX resin was also used for the rapid separation and purification of lysozyme from egg white with only one step. The purity and specific bioactivity of the purified lysozyme was found more than 95% and 70345 U/mg, respectively.

The monodisperse poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads with macroporous in the range of 8.0–12.0 μn were prepared by a single-step swelling and polymerization method. The seed particles prepared by dispersion polymerization exhibited good absorption of the monomer phase. The pore size distribution of the beads was evaluated by gel permeation chromatography and mercury instruction method. By using this media, a weak cation exchange (WCX) stationary phase for HPLC was synthesized by a new chemical modification method. The prepared resin has advantages of biopolymer separation, high column efficiency, low column backpressure, high protein mass recovery and good resolution for proteins. The measured bioactivity recovery for lysozyme was (96 5)%. The dynamic protein loading capacity of the synthesized WCX packing was 21.3 mg/g. Five proteins were completely separated in 8.0 min using the synthesized WCX stationary phase. The experimental results show that the obtained WCX resin has very weak hydrophobicity. The WCX resin was also used for the rapid separation and purification of lysozyme from egg white in 8 min with only one step . The purity and specific bioactivity of the purified lysozyme was found more than 92.0% and 70184 U/mg, respectively.

With the combination of the the stoichiometric displacement model for retention (SDM-R) in reversed phase liquid chromatography (RPLC) and the stoichiometric displacement model for adsorption (SDM-A) in physical chemistry, the total number of moles of the re-solvated methanol of stationary phase side, nr, and that of solute side in the mobile phase, q, corresponding to one mole of the desorbing solute, were separately determined and referred as the characterization parameters of the contributions of the adsorption mechanism and partition mechanism to the solute retention, respectively. A chromatographic system of insulin, using mobile phase consisting of the pseudo-homologue of alcohols (methanol, ethanol and 2-propanol)-water and trifluoroacetic add was employed. The maximum number of the methanol layers on the stationary phase surface was found to be 10.6, only 3 of which being valid in usual RPLC, traditionally referred as a volume process in partition mechanism. However, it still follows the SDM-R. Both of q and nr of insulin were found not to be zero, indicating that the retention mechanism of insulin is a mixed mode of partition mechanism and adsorption mechanism. When methanol is used as the organic modifier, the ratio of q/nr was 1.13, indicating the contribution to insulin retention due to partition mechanism being a bit greater than that due to adsorption mechanism. A linear relationship between q, or nr and the carbon number of the pseudo-homologue in the mobile phase was also found. As a methodology for investigating the retention mechanism retention and behaviors of biopolymers, a homologue of organic solvents as the organic modifier in mobile phase has also been explored.

With frontal analysis (FA), the dependence of adsorption isotherms of insulin on the composition of mobile phase in reversed phase liquid chromatography (RPLC) has been investigated. This is also a good example to employ the stoichiometric displacement theory (SDT) for investigating solute adsorption in physical chemistry. Six kinds of mobile phase in RPLC were employed to study the effects on the elution curves and adsorption isotherms of insulin. The key points of this paper are: (1) The stability of insulin due to delay time after preparing, the organic solvent, concentration, the kind and the concentration of ion-pairing agent in mobile phase were found to affect both elution curve and adsorption isotherm very seriously. (2) To obtain a valid and comparable result, the composition of the mobile phase employed in FA must be as same as possible to that in usual RFLC of either analytical scale or preparative purpose. (3) Langmuir Equation and the SDT were employed to imitate these obtained adsorption isotherms. The expression for solute adsorption from solution of the SDT was found to have a better elucidation to the insulin adsorption from mobile phase in RPLC.

With insulin methanol-water, and the ion-pairing agent, hydrochloric acid and trifluroacetic acid (TFA), the character of the first plateau (FP) on the elution curve of frontal analysis in reversed phase liquid chromatography (RPLC) was investigated by on-line UV-spectrometry and identified with nuclear magnetic resonance (NMR) spectrometry and mass spectrometry. The profile of the FP is the same as that of a usual elution curve of methanol in frontal analysis (FA). When the insulin concentration was limited to a certain range, the height of the FP was found to be proportional to the insulin concentration in mobile phase and its length companying to shorten. The FP profile on the intersection of two tangents reflects the components of the microstructure in the depth direction of the bonded stationary phase layer and the desorption dynamics of the displaced components. The displaced methanol was quantitatively determined by NMR and on-line UV spectrometries.

TFA with high UV absorbance can not be used as an ion-pairing agent for the investigation of the FP in RPLC, but it can be used as a good marker to investigate the complicated transfer process of components in the stationary phase in RPLC. A stoichiometric displacement process between solute and solvent was proved to be valid in both usual and FA in RPLC. From the point of view of dynamics of mass transfer, the solutes can only contact to the surface of stationary phase in usual RPLC, while solute can penetrate into it in FA of RPLC. The solvation of insulin in methanol and water solution as an example indicating the usage of the FP in the FA was also investigated in this paper.