Optimization of the preparative separation of a chiral pharmaceutical intermediate by high performance liquid chromatography.

The prediction of optimal conditions of the preparative HPLC separation of the enantiomers of a pharmaceutical intermediate was accomplished by employing analytical chromatographic data, i.e. sample injections at low concentrations. Various temperatures and mobile phase conditions were studied. It was assumed that the sample loadability of the stationary phase is constant for a constant value of the separation factor and different mobile phase conditions and temperatures. Using this assumption, possible production rates can be compared for different method conditions. Overloading experiments were carried out to verify that the procedure employed is adequate. It was found that the optimization approach used, changing the mobile phase composition and temperature to achieve the shortest cycle time while keeping the separation factor constant, could be applied to improve the production rate of the separation.

Dependence of retention on the organic modifier concentration and multicomponent adsorption behavior in reversed-phase chromatography.

A three-parameter equation is derived to express the dependence of the logarithmic retention factor, kappa, on the volume fraction of the retention modulator, phi, in a binary eluent (such as the organic modifier in the hydro-organic eluents used in reversed-phase chromatography). It is based on the competitive binary adsorption isotherm of the eluite and the modulator generated by employing the ideal adsorbed solution (IAS) method. The equation is found to describe adequately the trends in the kappa-phi relationship experimentally observed in reversed-phase systems. Furthermore, the expression affords an estimation of the single-component adsorption isotherm of the eluite from the corresponding kappa versus phi plot and thus provides a simple means to gather data of importance in the design of separations by non-linear chromatography. For instance, the method can be used to determine whether a pair of eluite isotherms cross one another, a situation that could lead to difficulties in preparative separations. The inherent limitations of the IAS approach may restrict the usefulness of the expression in specific cases. Nevertheless, the approach presented here establishes an explicit, thermodynamically consistent link between the eluite-modulator multicomponent isotherm and corresponding plots and allows a rational description of the generally observed retention behavior in reversed-phase chromatography. The results of this work also illustrate the limitations of the competitive Langmuir isotherm, which is most frequently used to treat competitive adsorption, in the study of the kappa-phi relationship specifically and in investigating and modeling non-linear chromatography at large.

Mixed-bed ion-exchange columns for protein high-performance liquid chromatography.

Protein retention is investigated on high-performance liquid chromatography columns packed with mixtures of ion exchangers. Retention factors are measured at both low and high salt concentrations in the eluent and their dependence on the bed composition is found to be linear in some cases, but non-linear in others. The physical basis for the observed non-linear retention behavior has not been established and an empirical mixing rule is employed to express the dependence of protein retention on bed composition. Protein separations are carried out on the mixed-bed columns by using gradient elution with increasing salt concentration and the process is modelled mathematically. The retention times predicted by computer calculations correspond closely to the experimental findings. Optimal selection of the mixed-bed composition and the gradient steepness for the separation of four proteins is illustrated by using the window diagram technique. Although the experimental results presented here deal with electrostatic interaction chromatography of proteins only the applicability of mixed sorbents is expected to extend to all branches of liquid chromatography. It is anticipated that mixed-sorbent columns will find extensive use in the large-scale purification of biological compounds and in routine analysis.