Reversed-phase and polar-organic modes

This relatively new class of CSPs incorporates glycopeptides attached covalently to silica gel. These CSPs can be used in the normal phase, reversed phase, and polar organic modes in LC [62]. Various functional groups on the macrocyclic antibiotic molecule provide opportunities for tt-tt complexation, hydrogen bonding, and steric interactions between the analyte and the chiral selector. Association of the analyte... 

Because plasma and urine are both aqueous matrixes, reverse-phase or polar organic mode enantiomeric separations are usually preferred as these approaches usually requires less elaborate sample preparation. Protein-, cyclodextrin-, and macrocyclic glycopeptide-based chiral stationary phases are the most commonly employed CSPs in the reverse phase mode. Also reverse phase and polar organic mode are more compatible mobile phases for mass spectrometers using electrospray ionization. Normal phase enantiomeric separations require more sample preparation (usually with at least one evaporation-to-dryness step). Therefore, normal phase CSPs are only used when a satisfactory enantiomeric separation cannot be obtained in reverse phase or polar organic mode. 

Separations in the Reversed-Phase and Polar-Organic Modes.523... 

Chiral Separations in Biological Matrices Using the Reversed-Phase and "Polar-Organic" Mode ... 

Only a limited number of biomedical applications have been published in the normal-phase mode, as can be seen in Table 17.4. However, the sensitivity of the methods seems to be comparable to the reversed-phase and polar-organic mode applications, although a detailed comparison is not feasible since the LOQ data are missing for the few substances that have been analyzed in both modes. The majority of the methods are based on MS detection, and APCl seems to be the predominant ionization mode for the applications in normal-phase mode. 

As discussed in Sect. 3.3, the chiral recognition mechanisms in different HPLC modes on CD-based CSPs vary remarkably. Consequently, derivatized n-acidic and K-basic CD CSPs that are applicable to all three LC modes are able to resolve different classes of chiral compounds in reversed-phase and normal-phase modes. Underivatized CD CSPs are mainly used in reversed-phase and polar organic modes, but less likely in normal-phase mode. It is common for aromatic compounds with multiple H-bonding sites to be separated on CD CSPs in both RP and POM [73,79, 82]. In these cases, the U-shaped retention behavior is typically observed, i.e., the analytes are more strongly retained under high aqueous content and high organic content mobile phases. An example is presented in Fig. 19 [78]. 

Comparisons of LC and SFC have also been performed on naphthylethylcar-bamoylated-(3-cyclodextrin CSPs. These multimodal CSPs can be used in conjunction with normal phase, reversed phase, and polar organic eluents. Discrete sets of chiral compounds tend to be resolved in each of the three mobile phase modes in LC. As demonstrated by Williams et al., separations obtained in each of the different mobile phase modes in LC could be replicated with a simple CO,-methanol eluent in SFC [54]. Separation of tropicamide enantiomers on a Cyclobond I SN CSP with a modified CO, eluent is illustrated in Fig. 12-4. An aqueous-organic mobile phase was required for enantioresolution of the same compound on the Cyclobond I SN CSP in LC. In this case, SFC offered a means of simplifying method development for the derivatized cyclodextrin CSPs. Higher resolution was also achieved in SFC. 

Cass et al. [66] used a polysaccharide-based column on multimodal elution for the separation of the enantiomers of omeprazole in human plasma. Amylose tris (3,5-dimethylphenylcarbamate) coated onto APS-Hypersil (5 /im particle size and 120 A pore size) was used under normal, reversed-phase, and polar-organic conditions for the enantioseparation of six racemates of different classes. The chiral stationary phase was not altered when going from one mobile phase to another. All compounds were enantioresolved within the elution modes with excellent selectivity factor. The separation of the enantiomers of omeprazole in human plasma in the polar-organic mode of elution is described. 

Chiral separations can be considered as a special subset of HPLC. The FDA suggests that for drugs developed as a single enantiomer, the stereoisomeric composition should be evaluated in terms of identity and purity [6]. The undesired enantiomer should be treated as a structurally related impurity, and its level should be assessed by an enantioselective means. The interpretation is that methods should be in place that resolve the drug substance from its enantiomer and should have the ability to quantitate the enantiomer at the 0.1% level. Chiral separations can be performed in reversed phase, normal phase, and polar organic phase modes. Chiral stationary phases (CSP) range from small bonded synthetic selectors to large biopolymers. The classes of CSP that are most commonly utilized in the pharmaceutical industry include Pirkle type, crown ether, protein, polysaccharide, and antibiotic phases [7]. 

Lipka, E., Glacon, V., Mackenzie, G., Ewing, D., Len, C., Postel, D., Vaccher, M. P., Bonte, J. P. and Vaccher, C. HPLC Separation and Determination of Enantiomeric Purity of Novel Nucleoside Analogs, on Cyclodextrin Chiral Stationary Phases, Using Reversed and Polar Organic Modes. Anal. Lett 37 385, 2004. 

Derivatized polysaccharide CSPs are operational, quite well, in all three HPLC separation modes as well as under super- or sub-critical fluid conditions [153, 160, 166, 170, 176-178]. A previous study based on a collection of more than 100 pharmaceutically important compounds with diverse structures clearly showed that polysaccharide CSPs generally had much higher success rate in resolving enantiomers under normal-phase and SFC conditions, followed by RP and polar organic modes (Fig. 17) [167]. This study also revealed that amylose tris(3,5-dimethylphenylcarbamate) AD phase was more effective than the other studied polysaccharide CSPs in polar organic mode and SFC, whereas cellulose tris(3,5-dimethylphenylcarbamate) phase is more applicable in reversed-phase mode. This observation is consistent with two other studies [159, 160]. 

The macrocyclic glycopeptides CSPs arc capable of operating in three different mobile phase systems reversed phase, normal phase, and the new polar organic mode. The new polar organic mode refers to the approach when methanol is used as the mobile phase with small amounts of acid and/or base as the modifier to control... 

In the new polar organic mode, the ratio of acid/base in the mobile phase affects the selectivity and the concentration of acid and base controls the retention. It is suggested to start the method development with a medium concentration (0.1 %) for both acid and base. If retention is too long or too short, the concentration can be increased to 1 % or reduced to 0.01 %. If no selectivity is observed in this mode, reversed phase is recommended as the next step in the protocols. 

When analytes lack the selectivity in the new polar organic mode or reversed-phase mode, typical normal phase (hexane with ethanol or isopropanol) can also be tested. Normally, 20 % ethanol will give a reasonable retention time for most analytes on vancomycin and teicoplanin, while 40 % ethanol is more appropriate for ristocetin A CSP. The hexane/alcohol composition is favored on many occasions (preparative scale, for example) and offers better selectivity for some less polar compounds. Those compounds with a carbonyl group in the a or (3 position to the chiral center have an excellent chance to be resolved in this mode. The simplified method development protocols are illustrated in Fig. 2-6. The optimization will be discussed in detail later in this chapter. 

Similar to the new polar organic mode, the retention of analytes in normal phase is not difficult to predict. For all the compounds, the average of the retention on individual columns is fairly close to the retention on the coupled columns. The selectivity of most compounds on coupled columns is an average of the selectivities of individual columns (Fig. 2-9). However, it was found that the elution order for some compounds was reversed on ristocetin A and teieoplanin or vancomycin. As a result. 

In achiral-chiral LC-LC, the mobile phases used with the achiral and chiral columns must be miscible with one another. Since the enantiomeric separation is usually the most difficult to optimize, it is usually the separation that dictates the mode of operation of the total analysis. Thus, it makes sense that a chiral column that operates in the normal phase mode would require an achiral column that also works in the normal phase mode. Polar organic mode chiral separations are universal in that they can be paired with an achiral column that operates in either the reverse phase or normal phase mode. The choice of the achiral column is always determined after selecting the chiral column and the mode of operation. As with traditional liquid chromatography, different achiral columns will give different selectivity. 

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