Capacity factor cyclodextrin mobile phases

The use of cyclodextrins as the mobile phase components which impart stereoselectivity to reversed phase high performance liquid chromatography (RP-HPLC) systems are surveyed. The exemplary separations of structural and geometrical isomers are presented as well as the resolution of some enantiomeric compounds. A simplified scheme of the separation process occurring in RP-HPLC system modified by cyclodextrin is discussed and equations which relate the capacity factors of solutes to cyclodextrin concentration are given. The results are considered in the light of two phenomena influencing separation processes adsorption of inclusion complexes on stationary phase and complexation of solutes in the bulk mobile phase solution. 

One form of the psuedophase retention equation is shown below (14). It relates LC retention (as the capacity factor, k ) to the binding of a solute to cyclodextrin, K., and to the concentration of cyclodextrin in the mobile phase, [CD]. 

Figure 3 shows the effect of temperature on the capacity factor of p-nltroanillne, from 0°C to 77°C. A mobile phase consisting of 10 methanol/water was employed. The retention at 0°C was 23.62 min. while at 77°C was 2.28, a ten fold decrease. This decrease in retention may be attributed to many factors such as increased solubility of the p-nitroaniline with increase in temperature, which results in less solute-stationary phase, and an increase in solute-mobile phase Interactions increase in mass transfer, and decrease in the pressure. Also the binding constant of any solute with cyclodextrin goes to zero at 80°C (11). 

Figure 1. Effect of temperature on the capacity factor of naphthalene (—o—) and biphenyl (-0-) using a 6 cyclodextrin column, ii.6 x 100 mm, and a mobile phase of methanol/water at a flow rate of 1 ml/min.

In conventional reversed phase HPLC, differences in the physicochemical interactions of the eluate with the mobile phase and the stationary phase determine their partition coefficients and, hence, their capacity factor, k. In reversed-phase systems containing cyclodextrins in the mobile phase, eluates may form complexes based not only on hydrophobicity but on size as well, making these systems more complex. If 1 1 stoichiometry is involved, the primary association equilibrium, generally recognized to be of considerable importance in micellar chromatography, can be applied (11-13). The formation constant, Kf, of the inclusion complex is defined as the ratio of the entrance and exit rate constants between the solute and the cyclodextrin. Addition of organic modifiers, such as methanol, into the cyclodextrin aqueous mobile phase should alter the kinetic and thermodynamic characteristics of the system. This would alter the Kf values by modifying the entrance and exit rate constants which determine the quality of the separation. 

Typically plots of In k versus methanol content of the mobile phase is shown in Figure 8. The plots are linear, and exhibit a high degree of correlation based on the respective regression analysis given in Table V. These data indicate that the capacity factor of these test solutes on nonpolar C-18 and cyclodextrin-bonded stationary phases decrease exponentially with the methanol content of the mobile phase. Therefore, the elution behavior obtained with these three types of columns can be explained in a similar manner, and the CD columns behave in the same fashion as conventional reversed phase columns. 

The tetrahydrophthalic ester of prednisolone, an anti-inflammatory drug, consists of two diastereoisomers. Olszewska et al. [66] describes the effect of addition of an optically active compound (amino acids, cyclodextrins, camphosulfonic acid) on the capacity ratios, k, and separation factors of these diastereoisomers by TLC. They used both mobile phases containing a chiral additive and stationary phases impregnated with an optically active compound, for example, silica gel plates impregnated with amino acids. The best resolution was obtained by impregnation of the stationary phase with o-camphosulfonic acid and copper acetate, and use of dichloromethane-isopropanol (90+10, v/v) as mobile phase. The addition of chiral compound to the mobile phase had less effect on the resolution of diastereoisomers. 

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