Achiral-phase HPLC

Self-Disproportionation of Enantiomers During the Purification by Achiral-Phase HPLC... 

Counter-ions, usually small polar or ionic compounds, are routinely used to enhance the aqueous solubility and/or stability of the API. Because of their polarity, counter-ions are rarely resolved from the chromatographic solvent front in reversed-phase HPLC and have characteristically poor chromophores which makes detection difficult. The counter-ion can be omitted from the achiral method development sample set with minimal risk when this holds true. Analysis of counter-ions is normally performed using ion chromatography.9,10 This assay is separate from the reversed-phase assay performed to measure organic impurity levels. 

In comparing normal phase to reversed phase, several general attributes can be noted. Reversed-phase HPLC provides better separation between compounds varying in alkyl carbon number and elution is in order of decreasing polarity. Normal-phase HPLC provides better resolution of compounds differing in polar substituents and for achiral isomers and elution is in order of increasing polarity. However, more substantial a priori prediction of which specific column will be most suitable for a given API and related substances has remained elusive. 

Sample preparation Isolate verapamil using achiral preparative HPLC, freeze dry an appropriate aliquot of the mobile phase, injecd an aUquot into this system. 

Direct HPLC enantioseparation techniques, which are free of many disadvantages of GC, indirect and chiral mobile phase HPLC methods, have gained unequivocal prevalence in bio-analytical studies. Several methods have been advanced so much that they allow enantiose-lective determination not only of the parent chiral drugs but also of their pharmacologically relevant metabolites [121]. As already mentioned above, a direct injection of biofluids offers several advantages in terms of analysis time and sample recovery. Precolumns packed with achiral or chiral packings, or with the recently developed so-called restricted-access packing materials, may be useful in this case. 

During the aforementioned studies, we encountered an interesting and rare phenomenon when the isolation of analytically pure samples of Michael adducts 14a and 14e was attempted by achiral normal phase HPLC (Fig. 4). 

Thus, in order to evaluate the enantiopurity of the Michael product 14a obtained during the optimization of the catalyst A (Scheme 5), the purification of the analytical quantities of the crude 14a from its diastereomer and residual p-ketoester 12a was performed by semipreparative normal phase HPLC using achiral silica-based column ( -hexanes/lPA, Zorbax Rx-SIL column). This purification provided partial separation, and the pure fractions containing major diastereomer (i.e., 14a) along with some mixed fractions containing both diastereomers were collected. Remarkably, the first collected fraction contained highly enantiopure 14a (99% ee). 

Allethrin D-allethrin has eight isomers (2 = 8), four cis (C, D, E, and F) and four trans (A, B, G, H) isomers. Mancini et al. (2004) separated cisitrans isomers from each other on an achiral silica HPLC column using n-hexane tert-butyl methyl ether (96 4) (v/v) as the mobile phase (Table Cl, Appendix C). The trans isomers were separated (G, H, A, B, respectively) from each other on a CHIRAL-CEL OJ using n-hexane-tert-butyl methyl ether (90 10) (v/v). This same colunm was used to separate the cis isomers (F, D, C, and E, respectively) using n-hexane isopropanol (99.3 0.7) (v/v). Kutter and Class (1992) were able to separate the trans allethrin isomers on a chiral p-cyclodextrin RP-HPLC column, but were unable to separate the cis isomers. 

Three amino alcohols, propranolol, clenbuterol and cycloclen-buterol, could be enantioseparated on an achiral column by reversed phase HPLC (62). These alcohols are shown in Figure 1.24. 

Chromatographic Method. Progress in the development of chromatographic techniques (55), especially, in high performance Hquid chromatography, or hplc, is remarkable (56). Today, chiral separations are mainly carried out by three hplc methods chiral hplc columns, achiral hplc columns together with chiral mobile phases, and derivatization with optical reagents and separation on achiral columns. All three methods are usehil but none provides universal appHcation. 

Guo, Z., Wang, H., and Zhang, Y., Chiral separation of ketoprofen on an achiral C8 column by HPLC using norvancomycin as chiral mobile phase additives, J. Pharm. Biomed. Anal, 41, 310, 2006. 

The approach to method development is similar to the one described for HPLC and can be characterized as a rapid stationary phase screen using column and solvent switching with gradient elution followed by development of an isocratic preparative method. SFC has been successfully applied to the analytical and preparative separation of achiral and chiral compounds. 

Three approaches can be employed to separate peptide stereoisomers and amino acid enantiomers separations on chiral columns, separations on achiral stationary phases with mobile phases containing chiral selectors, and precolumn derivatization with chiral agents [111]. Cyclodextrins are most often used for the preparation of chiral columns and as chiral selectors in mobile phases. Macrocyclic antibiotics have also been used as chiral selectors [126]. Very recently, Ilsz et al. [127] reviewed HPLC separation of small peptides and amino acids on macrocyclic antibiotic-based chiral stationary phases. 

The presence of these 12 stereoisomers was experimentally confirmed by the partial resolution of 12 peaks in HPLC chromatography using an achiral stationary phase (octadecyl polysiloxane, ODS) and different mobile phase compositions (CH3CN/THF 55 45-58 42) and temperatures (288-258 K). Figure 7 shows one of these chromatograms in which 10 partially resolved peaks are clearly distinguished. ... 

Figure 7. Representative HPLC separations of the stereoisomers of (a) dendrimer 5 (G = 1) at 298 K and (b) dendrimer 6 (G = 1) at 258 K using a HPLC achiral stationary phase of ODS and ACN/THF (55 45) as mobile phase.

Chromatography. Under certain conditions, even homochiral and het-erochiral self-assemblies can be separated by achiral methods. Thus, chromatography of partially resolved enantiomers can cause depletion or enrichment of enantiomers on achiral stationary phases with an achiral mobile phase. 14C-Labeled nicotine was first resolved into its enantiomers by high-performance liquid chromatography (HPLC) on an achiral stationary phase (Partisil-ODS or -SCX) through coinjection with optically active nicotine (59). This observation was followed by resolution of a number of chiral compounds by chromatography (<50-62) (Scheme 34). When a chiral diamide in 74% ee was separated on a Kieselgel 60... 

A. G. Adams and J. T. Stewart, A HPLC method for the determination of albuterol enantiomers on human serum usage SPE and a sumichiral-achiral stationary phase, J. Liquid Chromatogr., 16 3863 (1993). 

In recent years several sensitive and specific methods for the simultaneous determination of ephedrine alkaloids in plant material have been published. These include thin-layer chromatography (292,426), gas chromatography (251), straight-phase and reversed-phase high-performance liquid chromatography (253, 255, 302, 355, 427), isotachophoresis (303, 356), and 13C-NMR (304). Resolution of enantiomeric alkaloids by HPLC has been achieved on chiral stationary phases (417, 418) or after derivatization with a chiral agent on an achiral stationary phase (419). Chromatographic separation and analytical detection of... 

The aim of this chapter is to highlight current developments in these various fields of preparative HPLC, with particular emphasis on applications that have been developed at Chemical Analytical Development at Novartis Pharma AG. Drug substance purifications from biological and synthetic sources are presented, along with the separation of chiral and/or achiral molecules on chiral stationary phases and typical isolations of by-products. Special attention is given to the determination of adsorption isotherms and their interplay with respect to the layout of chromatographic processes as well as the choice of... 

HPLC-CSPs are based on molecules of known stereochemical composition immobilized on liquid chromatographic supports. Single enantiomorphs, diastereomers, diastereomeric mixtures, and chiral polymers (such as proteins) have been used as the chiral selector. The chiral recognition mechanisms operating on these phases are the result of the formation of temporary diastereomeric complexes between the enantiomeric solute molecules and immobilized chiral selector. The difference in energy between the resulting diastereomeric solute/CSP complexes determines the magnitude of the observed stereoselectivity, whereas the sum total of the interactions between the solute and CSP chiral and achiral, determines the observed retention and efficiency. 

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