Polysaccharide-type CSPs

The observation of enantioselective binding properties of polysaccharides dates back to the early SOs. At this time Kotake [95] and Dalgliesh [96] achieved thin layer chromatographic separation of amino acid enantiomers on cellulose carriers. However, the poor chiral recognition capacity of native polysaccharides hampered further developments. 

Expansion of these investigations to other types of polysaccharide derivatives led to the development of silica-coated versions of cellulose and amylose carbamates and benzoates [100-102]. These CSPs showed particularly versatile chiral recognition profiles, resolving an extremely broad assortment of chiral analytes. Several of these CSPs have been commercialized by Daicel Chemical Industries, Ltd., the most important ofwhich are depicted in Fig. 7.5. 

Polysaccharides-type CSPs are excellently compatible with SFC conditions 

Recently, organic sulfonic acids have been suggested as normal-phase and SFC additives to improve peak shapes for basic analytes [125]. These strongly acidic additives proved particularly beneficial for the separation of a broad variety of amines on a CHIRALPAK AD CSP under normal-phase conditions. The addition of ethanesulfonic and methanesulfonic acid allowed successful separation of a selection of amines which had failed to resolve wifh less acidic additives.The enantiomer separation of a basic drug compound employing ethanesulfonic acids and trifluoroacetic acid is shown in Fig. 7.8. 

The origin of the favorable effects of these strongly acidic additives remains to be established. The authors suggested that fhe strongly acidic additives may play an essential role in the formation of stable ion pairs, or promote a local pH-effect enhancing the enantioselective interaction wifh fhe binding sites at the polysaccharide-type CSP. 

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Polysaccharide type CSPs as well as most synthetic polymeric type CSPs have no ionic interaction sites and thus are primarily operated in the normal-phase mode. Proteins, in contrast, have several (positively and negatively) charged adsorption sites for strong ionic interactions, which have to be balanced by buffered mobile phases. The system must take into account that denaturation of the proteins must not occur, which limits the amounts of organic mridifiers that can be used as part of the aqueous mobile phase. 

ENANTIOSEPARATION OF PHARMACELTICALLY RELEVANT CHIRAL COMPOLNUS USING POLYSACCHARIDE TYPE CSPs... 

For all the polysaccharide type CSPs, their primary mode of operation, panicularly for preparative separations, is the normal-phase mcxle. Usually, w-heptane or n-hexane-isopropanol, resp. ethanol, mixtures are employed as mobile phases. For the separation of acids, small quantities of acids, e.g. trifluoroacetatic acid, are added to the mobile phase [147], The tailing of basic SAs on the other hand can be reduced with addi-... 

One considerable disadvantage of coated polysaccharide type CSPs, however, is the high solubility of the SO in many organic solvents, e.g. chloroform, ethylacetate, and tetrahydrofuran, restricting the choice of mobile phases that can be used. Accordingly, inflexibility in the optimization of separations and enantioselectivity is a considerable drawback this counts in particular for preparative separations, where often the solubility of the SAs in the mobile phase is limited and thus loadability and finally the productivity rate is reduced. 

Enantioseparations in SEC have been reported for several CSPs. including native and derivatized cyclodextrin-based CSPs [427-432. Pirkle-concept CSPs [77,336-338,347,348,363,365,433,434], polysaccharide type CSPs [137.435-438], macrocyclic antibiotic type CSPs [436], and others. 

Fig. 7.7 Effect of various types of additives on the reversed-phase enantiomer separation of neutral, acidic and basic analytes on a polysaccharide-type CSP. Column CH I RALCEL AD-RH (150 x 4.6 mm i.d.), mobile phase aqueous mobile phase containing modifier indicated in the figure/acetonitrile (60/40 v/v), flow rate 0.5 mb min" , temp. 25 °C, detection UV 254 nm. (Reprinted with permission from [116]).

One of the major hmitations of coated polysaccharide-type CSPs is their incompatibility with so-called non-standard solvents. Specifically, the exposure of coated CSPs to dichloromethane, chlororform, ethyl acetate, tetrahydrofuran, dioxane, toluene and acetone, induces swelling and/or dissolution of the physically adsorbed polymer films and thus destruction of fhe columns. To address this serious drawback, a considerable amount of research has focused on the development of immobihzed versions with global solvent compatibility. In the last two decades numerous immobilization strategies have been reported [126], and the quest for solvent-stable versions capable of reproducing with fidelity the excellent separation characteristics of their coated congeners is still an active field of study [100, 127-130]. Reported immobilization approaches capitalize on (i) surface at-... 

CSPs tolerate ethers, ketones, esters, chlorinated solvents and aromatic solvents, enhancing the scope of application of the coated congeners immensely [143, 144]. Figure 7.9 presents a comparison of the enantiomer separations achieved for bupi-vacaine on the coated CHIRALPAK AD and the corresponding immobilized CHIRALPAK IA employing standard and non-standard mobile phase mixtures [144]. Excellent durability and inertness of these stabilized CSPs have been demonstrated (see Fig. 7.10), making them an invaluable complement to the established repertoire of coated polysaccharide-type CSPs [144]. 

However, the (undisclosed) proprietary immobilization process appears to modify the enantiomer separation characteristics as compared to the coated versions [145, 146]. Ghanem et al. compared the chiral recognition profile of a coated CHIRALPAK AD CSPs with that of the immobilized version, employing hexane/ 2-propanol containing TEA (0.1%) as mobile phase [145]. They reported superior enantiomer separation for the coated CSP, with some analytes failing to resolve on the immobilized version. These differences in the enantiomer separation capacity of coated and immobilized polysaccharide-type CSPs may complicate attempts at direct method transfer. 

The separation of the enantiomers of the chiral p-blocker propranolol in capillary HPLC using a polysaccharide type CSP is shown in Fig. 4 [120]. The capillary format requires aproximately 106 times less stationary and mobile phases, while offering separation characteristics at least adequate or better than that achieved with common-size columns. 

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