Chiral Mobile Phase Additives CMPA

In chiral mobile phase additive mode enantiomer separation, a combination of an achiral stationary and a chiral mobile phase is employed, the latter being created by simply adding a certain amount of an appropriate SO to the eluent. On introduction of a mixture of enantiomers into this system, the individual enantiomers form diastereomeric complexes with the chiral mobile phase additive (CMPA). These transitory diastereomeric complexes may exhibit distinct association/disso-ciation rates, thermodynamic stabilities and physicochemical properties, and therefore may be separated on an appropriate achiral stationary phase. 

CMPA-based enantiomer separation techniques appear attractive from the viewpoints of conceptual simplicity and flexibility as they operate with relatively inexpensive achiral stationary phases and easy-to-prepare chiral mobile phases. In practice, the development of CMPA-based analytical assays may pose a considerable challenge for a number of reasons. In the course of the development and optimization of CMPA-based enantiomer separations a set of conditions must be identified that favor CMPA-analyte interactions and simultaneously maximize 

Despite these evident drawbacks, a broad variety of SOs have been used in CMPA-based enantiomer separations, including cyclodextrins, proteins, macro-cyclic antibiotics, chiral ion-pairing agents, amino acids in combination with transition metal salts, and crown ethers. Recent application for the separation of pharmaceutically relevant chiral compounds utilized P-cyclodextrins [46-48] charged cyclodextrins [49, 50], macrocyclic antibiotics [51, 52] and chiral ion-pairing agents [53, 54]. A more detailed discussion of CMPA-based enantiomer separation is beyond the scope of this chapter. The interested reader is referred to dedicated reviews [55, 56]. 

The most convenient and most popular analytical methodology to assess enantiomer purity is the direct separation of enantiomers on so-called chiral stationary phases (CSPs). CSPs consist of an (ideally) inert chromatographic support matrix incorporating chemically or physically immobilized SO species. CSPs may be created by a variety of SO immobilization techniques, including (i) covalent attachment onto fhe surface of suitably pre-functionalized carrier materials, (ii) physical fixation employing coating techniques, and (iii) incorporation into polymeric networks by copolymerization, or combinations of these procedures. 

It also needs to be emphasized that it was the development of robust and broadly applicable CSPs that has laid the foundations for economic chromatographic enantiomer separation on a preparative scale. Although indirect [57-62] and CMPA-based direct [63-65] chromatographic methodologies have seen some use in preparative enantiomer separation, the considerable efforts associated with chemical manipulation and/or recovery of the products render these approaches economically unattractive [66]. Preparative enantiomer separations employing CSPs are not subject to these limitations. With CSPs enantiomers can be processed directly (i.e. without prior derivatization) with readily volatile achiral mobile phases (devoid of SOs), simplifying product recovery to a trivial solvent evaporation step. 

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In recent years, for analytical purposes the direct approach has become the most popular. Therefore, only this approach will be discussed in the next sections. With the direct approach, the enantiomers are placed in a chiral environment, since only chiral molecules can distinguish between enantiomers. The separation of the enantiomers is based on the complex formation of labile diastereoisomers between the enantiomers and a chiral auxiliary, the so-called chiral selector. The separation can only be accomplished if the complexes possess different stability constants. The chiral selectors can be either chiral molecules that are bound to the chromatographic sorbent and thus form a CSP, or chiral molecules that are added to the mobile phase, called chiral mobile phase additives (CMPA). The combination of several chiral selectors in the mobile phase, and of chiral mobile and stationary phases is also feasible. 

The development of a plethora of HPLC CSPs in the 1980s and 1990s has, to a large extent, made the use of chiral mobile-phase additives (CMPAs) redundant in most modem pharmaceutical analytical laboratories [23]. Before this period, chiral selectors were used routinely as additives in HPLC, but are now only used for a small number of specific applications [23]. CMPAs are used to form... 

In contrast to the various CSPs mentioned so far, but still based on covalently or at least very strongly adsorbed chiral selectors (from macromolecules to small molecules) to, usually, a silica surface, the principle of dynamically coating an achiral premodified silica to CSPs via chiral mobile phase additives (CMPA) has successfully been adapted for enantioseparation. The so-called reverse phase LC systems have predominantly been used, however, ion-pairing methods using nonaqueous mobile phases are also possible. 

On the other hand, the direct chromatographic approach involves the use of the chiral selector either in the mobile phase, a so-called chiral mobile phase additive (CMPA), or in the stationary phase [i.e., the chiral stationary phase (CSP)]. In the latter case, the chiral selector is chemically bonded or coated or allowed to absorb onto a suitable solid support. Of course chiral selectors still can be used as CMPAs, but the approach is a very expensive one owing to the high amount of chiral selector required for the preparation of the mobile phase, and the large amount of costly chiral selector that is wasted (since there is very little chance of recovering this compound). Moreover, this approach is not successftd in the preparative separation of the enantiomers. 

Since enantiomer separation requires the Intervention of some chiral agent, one may utilize either chiral mobile phase additives (CMPA) or chiral stationary phases (CSPs). While the requirement that one add a chiral substance to the mobile phase has obvious limitations for preparative separations, it is not a serious problem for analytical separations. Indeed, for some types of compounds (e.g. amino acids) this approach may be preferred. Quite an extensive literature exists for the use of mobile phases containing chiral bidentate ligands and copper ions for the "ligand exchange" resolution of underlvatized amino acids (1, 2) and for N-dansyl derivatives of amino acids Tartaric acid derivatives have also been used as CMPAs (5). 

CyDs have been used as major chiral mobile phase additives (CMPAs) for enantio-separations in HPLC. The first application of 8-CyD as a CMPA in combination with an achiral reversed-phase material for HPLC enantioseparations was reported by Sybilska and co-workers in 1982 [27]. These authors could achieve partial resolution of the enantiomers of mandelic acid and derivatives. The CMPA method played an important role in HPLC enantioseparations before the development of effective chiral stationary phases (CSPs) but is now rarely used. The major disadvantage of this technique, together with difficulties associated with the isolation of resolved enantiomers, is the rather large consumption of chiral selector. 

As mentioned above, enantioseparations in EKC rely on a chromatographic separation principle. Despite this fact, there are significant differences between these techniques. Responsible for all differences between chromatographic and electrophoretic enantioseparations is the property of the electrophoretic mobility to be selective for the analytes residing in the same physical phase [2]. Another important point is that in chromatographic techniques, except in the case of a chiral mobile phase additive (CMPA), the analyte is virtually immobile when associated with a chiral selector. In EKC the analyte selector complex is commonly mobile. 

Chiral mobile phase additives (CMPAs) are generally used to perform direct chiral separation in thin-layer chromatography (TLC). This mode offers the advantages of flexibility and low cost as compared to the equivalent chiral stationary phase (CSP). Also, the lack of a wide range of CSPs in TLC resulted in CMPAs becoming a commonly employed approach for enantiomeric separations. 

The chromatographic methods are considered to be most useful for chiral separations. Enantiomers can be separated by two methods (a) indirect method that utilizes derivatizing agents and (b) direct method that uses chiral stationary phases (CSPs) or chiral mobile phase additives (CMPAs) [49-56]. 

Chiral resolution by HPLC can by divided into three categories (1) a direct resolution using a chiral stationary phase (CSP) (2) addition of a chiral agent to the mobile phase, which reacts with the enantiomeric analytes (chiral mobile phase additive method (CMPA)) (3) an indirect method that utilizes a precolumn diastereomer formation with a chiral derivatization reagent (Misl anova and Hutta, 2003). 

Beads = pure polymeric particles with similar chiral information to the corresponding sorbent (CSp) coated on silica gel CE = capillary electrophoresis CSP = chiral stationary phase CMPA — chiral mobile phase additive MEKC = micellar electrokinetie capillary chromatography. 

For obvious reasons CDs (and other dextrins) are potentially good chiral selectors for chromatography on the one hand they can be used as mobile phase additives (CMPA) in TLC45, HPLC46 and CE47 49 and on the other they can be covalently bonded onto solid supports50,51 and silica gei 52- 54 xhis approach can be extended to the preparative resolution of enantiomers41,55,56. 

Case II dynamically coated CSP via a chiral mobile phase additive = (S)-CMPA ... 

Although the macrocyclic glycopeptide antibiotic CSPs are very effective for the chiral resolution of many racemic compounds, their use as chiral mobile phase additives is very limited. Only a few reports are available on this mode of chiral resolution. It is interesting to note that these antibiotics absorb UV radiation therefore, the use of these antibiotics as the CMPAs is restricted. However, Armstrong et al. used vancomycin as the CMPA for the chiral resolution of amino acids by thin-layer chromatography, which will be discussed in Section 10.7. 

In addition to high-performance liquid chromatography (HPLC), the chiral resolution using CMPAs was also carried out by supercritical fluid chromatography (SFC) [91] and capillary electrochromatography (CEC) [92-98]. Salvador et al. [91] used dimethylated /1-cyclodextrin as the mobile phase additive on porous graphite carbon as the solid phase for the chiral resolution of tofizopam, warfarin, a benzoxazine derivative, lorazepam, flurbiprofen, temazepam, chlorthalidone, and methyl phehydantoin by SFC. The authors also studied the effect of the concentration of dimethylated /1-cyclodextrin, the concentration of the mobile phase, the nature of polar modifiers, outlet pressure, and the column temperature on the chiral resolution. 

The approach of CMPAs has also been used in thin-layer chromatography (TLC) for the chiral resolution of a variety of racemic compounds [100-110]. Lepri et al. [104,105] used BSA as a mobile phase additive for the chiral resolution of dansyl amino acids and other drugs by TLC. Armstrong et al. [101,102] used unde-rivatized and hydroxyethyl and hydroxypropyl /I-cyclodextrins for the chiral resolution of dansyl amino acids, alkaloids, and other compounds. Aboul-Enein... 

Analysis using a CMPA is usually resolved on a nonchiral column. A transient diastereomeric complex is formed between the enantiomer and the chiral component in the mobile phase, similar to the complexes formed with chiral stationary phases. A review by Liu and Liu (2002) cites several papers where addition of CPMAs has been used in analyzing amphetamine-related compounds. Some CPMAs include amino acid enantiomers, metal ions, proteins, and cyclodextrins. Advantages of this method of analysis include the use of less expensive columns and more flexibility in the optimization of chiral separation (Misl anova and Hutta, 2003). 

The use of CMPA is flexible and is convenient for exploring new chiral selectors. The stationary phases used for the CMPAs are less expensive than CSPs, whereas the additives are often quite expensive. Furthermore, the complex mobile phase often limits the choice of detection method (e.g., mass spectrometry [MS]) that could be used, which makes the CMPAs less commonly used than CSPs. Only a few applications have been published during the last 10 years [39,58-60]. A recent example with a chiral selector used as both the CSP and the CMPA is shown in Figure 17.2 [43]. For further reviews on the use of CMPA, see Refs. [35,40,49]. 

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