Polysaccharide-type phase

From the list in Table 3.9, cellulose and amylose-based phases are by far the most often used in preparative and, especially, SMB applications. These adsorbents offer good productivities because of their high loadabilities (Fig. 3.22). In addition, the four most commonly used CSP of this type separate a broad range of different race-mates. The major problem of these adsorbents is their limited solvent stability, especially towards medium-polar solvents such as acetone, ethyl acetate or dioxane. In the past their use in conjunction with aqueous mobile phases was not recommended by the manufacturer as well. However, this limitation was successfully overcome by recent studies, in which amylose- and cellulose-based CSPs are transferred to the reversed phase (RP) mode with aqueous mobile phases. The first results for the use of polysaccharide-type phases with aqueous solvents were reported by Ishikawa and Shibata (1993) and McCarthy (1994). The stability of the adsorbent after switching to RP conditions has been reported by Kummer et al. (1996) to be at least 11 months and by Ning (1998) to be 3 years. No peak deviation is observed after switching to RP mode. Novel developments have led to polysaccharide-based adsorbents dedicated to use with nearly all organic solvents (Cox and Amoss, 2004). 

Girod, M., Chankvetadze, B., and Blaschke, G. (2000) Enantioseparations in non-aqueous capillary electrochromatography using polysaccharide type chiral stationary phases, J. Chromatogr. A 887, 439-455. 

K. Tachibana and A. Ohnishi, Reversed-phase hquid chromatographic separation of enantiomers on polysaccharide type chiral stationary phase, J. Chromatogr. A 906 (2001), 127-154. 

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. 

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. 

Fig. 7.5 Su mma7 of the chemical structures and tradenames of the most important cellulose and amylose derivatives incorporated in polysaccharide-type chiral stationary phases.

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]).

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. 

Chankvetadze, B.,Yamamoto, C., Okamoto,Y. Enantioseparation of selected chiral sulfoxides using polysaccharide-type chiral stationary phases and polar organic, polar aqueous-organic and normal-phase eluents,/. Chromatogr. A,... 

Chankvetadze, B., Kartozia, 1., Yamamoto, C., Okamoto, Y. Comparative enantioseparation of selected chiral drugs on four different polysaccharide-type chiral stationary phases using polar organic mobile phases,/. Pharmaceut. Biomed., 2001, 27, 467-478. 

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. 

Zhou, L., Welsh, C., Lee, C., Gong, X., Antonucci, V., Ge, Z. (2009) Development of LC chiral methods for neutral pharmaceutical related compounds using reversed phase and normal phase liquid chromatography with different types of polysaccharide stationary phases./. Pharm. Biomed. Anal., 49, 964—969. 

Fig. 8. From left to right schematic representation of pure and coated polysaccharide phases, brush-type phase, protein phase.

Zhe X, Wenwei X, Hua H, Lirui P, Xu X (2008) Direct chiral resolution and its application to the determination of the pesticide tetramethiin in soil by high-performance liquid chromatography using polysaccharide-type chiral statitmary phase. J Chromatogr Sci 46 783-786... 

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