Corticosteroids

Separations of structurally similar compounds can readily be fine tuned using electrochemically modulated liquid chromatography. The challenge presented by the separation of four corticosteroids, [I]-[IV], at a porous graphitic carbon stationary phase is a particularly intriguing example.

Figure 4 Plots of log kvs. applied potential (Eapp) for the separations of the mixture of aromatic sulfonates (see Table 1) between # 0.50 V and -1.00 V. Note that the values of HBS extend only from # 0.50 V to -0.10 V because of its elec-troreduction which occurs at an Eapp between -0.10 and - 0.20 V. (Reproduced with permission from Ting EY and Porter MD (1998) Analytical Chemistry70: 94-99. Copyright ACS Publications.)

Hydrocortisone [IV]

The complication with this mixture is the discrimination between structures containing differing numbers of double bonds (e.g. prednisone vs. cortisone) and/or small differences in the identity and number of substituents (e.g. cortisone vs. hydrocortisone). On bonded reversed phases, compounds with subtle differences in functionality generally exhibit greater differences in retention than those that differ by the number of double bonds. The converse is often more important for separations on porous graphitic carbon, which possesses ^-electron sensitivity superimposed upon reversed phase characteristics. Thus,

Figure 4 Plots of log kvs. applied potential (Eapp) for the separations of the mixture of aromatic sulfonates (see Table 1) between # 0.50 V and -1.00 V. Note that the values of HBS extend only from # 0.50 V to -0.10 V because of its elec-troreduction which occurs at an Eapp between -0.10 and - 0.20 V. (Reproduced with permission from Ting EY and Porter MD (1998) Analytical Chemistry70: 94-99. Copyright ACS Publications.)

strategies that would enhance the sensitivity of carbonaceous materials toward substituent differences, would improve their applicability as packings especially as they are more stable than many of the bonded phases when exposed to strongly acidic and strongly alkaline solutions.

The chromatograms in Figure 6 present the separations of the four compounds as a function of applied potential using a porous graphitic carbon stationary phase. At the open circuit potential, the separation is only marginally effective in resolving the four compounds. Changes in the applied potential, however, have a marked influence on the separation. Interestingly, the retention of prednisone and prednisolone increase as the applied potential becomes more negative, with that for prednisone becoming greater than that for prednisolone. This difference transposes the elution order for the two compounds. The same general dependencies for the retention of cortisone and hydrocortisone are observed. In contrast to the aromatic sulfonates, the supporting electrolyte can compete in some instances with the analyte for binding sites on the stationary phase, leading to retention dependencies that are nonlinear with respect to changes in applied potential. As a result of the combined effect of these dependencies, all four components are fully resolved at — 700 mV, with a total elution time of & 10 min. Thus, separations based on manipulating the applied potential provide an effective and

Figure 5 Separation of the six-component mixture of aromatic sulfonates (see Table 1) on a porous graphitic carbon stationary phase obtained by applying a linear voltage ramp from # 0.30 V to - 1.00 V. The voltage ramp was initiated immediately upon sample injection at 10mVs-1. The mobile phase consisted of aqueous 0.1 M LiCIO4 with 1.5% acetonitrile. The flow rate was 0.75 mL min~1 and the analyte concentrations were & 200 ppm. The shift in the baseline reflects the gradual decrease in the interfacial concentration of the CIO- anion of the supporting electrolyte in the mobile phase as the applied potential becomes more negative. The background baseline gradually returns to its initial value in & 10 min. (Reproduced with permission from Dein-hamer RS, Ting E and Porter MD (1993) Journal of Electroanalyti-cal Chemistry 362 : 295-299. Copyright Elsevier Science SA.)

of a variety of stationary-mobile phase combinations. This separation can be realized by the reversible elec-trosorption of a chiral compound added to the mobile phase, a process that converts an achiral column into a chiral column. The concept is illustrated in Figure 7, using ^-cyclodextrin as the chiral mobile phase additive. The choice of this additive reflects the notable enhancement of the interactions between hydroxyl functionalities and a carbonaceous packing as the applied potential becomes increasingly positive.

Results are presented in Figure 8 for the separations with and without the chiral selector present as a mobile phase additive at several different applied

Figure 5 Separation of the six-component mixture of aromatic sulfonates (see Table 1) on a porous graphitic carbon stationary phase obtained by applying a linear voltage ramp from # 0.30 V to - 1.00 V. The voltage ramp was initiated immediately upon sample injection at 10mVs-1. The mobile phase consisted of aqueous 0.1 M LiCIO4 with 1.5% acetonitrile. The flow rate was 0.75 mL min~1 and the analyte concentrations were & 200 ppm. The shift in the baseline reflects the gradual decrease in the interfacial concentration of the CIO- anion of the supporting electrolyte in the mobile phase as the applied potential becomes more negative. The background baseline gradually returns to its initial value in & 10 min. (Reproduced with permission from Dein-hamer RS, Ting E and Porter MD (1993) Journal of Electroanalyti-cal Chemistry 362 : 295-299. Copyright Elsevier Science SA.)

facile means to tackle the challenges posed by the strong structural similarity of these compounds.

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