Chiral Mobile-Phase Retention Mechanisms

The PO mode is a specific elution condition in HPLC enantiomer separation, which has received remarkable popularity especially for macrocyclic antibiotics CSPs and cyclodextrin-based CSPs. It is also applicable and often preferred over RP and NP modes for the separation of chiral acids on the cinchonan carbamate-type CSPs. The beneficial characteristics of the PO mode may arise from (i) the offset of nonspecific hydrophobic interactions, (ii) the faster elution speed, (iii) sometimes enhanced enan-tioselectivities, (iv) favorable peak shapes due to improved diffusive mass transfer in the intraparticulate pores, and last but not least, (v) less stress to the column, which may extend the column lifetime. Hence, it is rational to start separation attempts with such elution conditions. Typical eluents are composed of methanol, acetonitrile (ACN), or methanol-acetonitrile mixtures and to account for the ion-exchange retention mechanism the addition of a competitor acid that acts also as counterion (e.g., 0.5-2% glacial acetic acid or 0.1% formic acid) is required. A good choice for initial tests turned out to be a mobile phase being composed of methanol-glacial acetic acid-ammonium acetate (98 2 0.5 v/v/w). 

Several scientific reports about SFC indicate that the chromatographic retention mechanisms of charged analytes in the presence of suitable ionic modifiers involve ion-pairing [14]. Ion-pairing of sulfonates with ammoninm salt additives was effectively exploited to enhance the solvating power of the mobile phase [15] and sharpen analyte peaks [16], The use of ammonium acetate produced unique results (see Figure 15.1). Ion-pairing also explained enantioselectivity when chiral analytes were analyzed with packed columns in the presence of chiral connter ions [17]. An achiral IPR under SFC conditions played a crucial role in the enantioseparation of a variety of amines [18] for reason explained in Section 13.6. 

Modern polysaccharide columns are based on cellulose or amylose derivatives coated onto silica. Chiral discrimination and applications have been extensively documented, but the mechanism of chiral discrimination is not yet fully understood. Whereas numerous phases are available within this subset, orthogonality can generally be obtained from a set of three or four columns as a first approach to method development. A typical choice of columns would be to try a set of different amylase (Chiralpak AD and AS) and cellulose (Chiralcel OD or OJ) columns and defer more extensive method development to the subset of samples not separated by these columns. The columns specified are run in the normal-phase mode and, accordingly, mobile phases are typically mixtures of hexane with small amounts of isopropanol or ethanol to control retention. However, selectivity is changed by different polar modifiers. Tailing may be minimized by the addition of 10-50 mM trifluoroacetic acid (TFA) or triethylamine (TEA). Analogue of the columns specified (AD-R, AS-R, OD-R, and OD-J) are available for reversed-phase separation. 

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]. 

The results presented in this section provide a preliminary molecular-level insight into the retention mechanism of APAs in the chiral thin layer chromatographic systems with L-arginine as chiral selector. Namely, the experimental evidence was produced, showing that the impact of L-arginine on the separation of the three pairs of the APA antimers is 2D, that is, separating these pairs in the vertical direction of the mobile phase flow, and also in the horizontal direction, perpendicular to the former one. This two-dimensionality of the antimers separations certainly enhances an overall separation effect. 

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