Direct HPLC Enantioseparations

direct HPLC enantioseparation has been established as an analytical method of choice for determining the enantiomeric composition. This technique is characterized with high precision, sensitivity, speed, easy automation, tolerance to impurities, and byproducts, etc. 

The most important advantages of direct chromatographic enantioseparations for preparative purposes include the presence of resolved enantiomers in original and high optically pure form in different volume fractions of the mobile phase, no loss of chiral selectors, and amost no risk of contamination of desired enantiomers with the chiral selector. Parallel to enantioseparations other impurities may be removed from chiral compounds during this process. 

In principle, any chiral compound possessing an ability to interact non-covalently and enantioselectively with chiral molecules may be used more or less successfully as a chiral selector in liquid chromatography. There is a set of characteristics which a chiral selector has to meet, depending on the goal of the separation, the mode, and technique used. 

Several books are devoted to the chiral separations in HPLC [69,93,104,108,110]. Among several hundreds of CSPs described, ligand-exchange [105], Pirkle-type [113], protein and peptide [114], polysaccharide [115], macrocyclic [93,116], and synthetic polymeric [117-119] CSPs are most widely used. 

The diameter of HPLC columns may vary from several tens of micrometers to several tens of centimeters. Depending on the size of the separation column - nano-, capillary-, narrow-bore - analytical and preparative separations may be distinguished. A current trend in HPLC instrument development provides the hope that in time it may become feasible to use a column of any size with a single instrument. 

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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. 

Capillary electrophoresis and its most popular hybrid technique - capillary electrochromatography - are complementary to HPLC, offering rapid analysis, low consumption of sample and solvents, and usually a higher efficiency of separation (due to a larger number of theoretical plates). Similar to HPLC, enantioseparation with the use of electrophoretic methods can be conducted by direct (chiral phase... 

In addition to the miniaturization of HPLC enantioseparations, another current trend occurs in the opposite direction, namely the scaling-up of separations. The techniques of preparative-scale enantioseparations using liquid chromatography (LC) are described below. 

The reversal of enantiomeric elution order for the polysaccharide CSP was first reported by Okamoto et al. in 1991. They found that the reversal of the elution order of the enantiomers on a modified cellulose column was associated with changes in the mobile phase modifiers during the investigation of the direct chromatographic enantioseparation of pyriproxyfen, an insect growth regulator. If one can find such phenomena, although very rare in HPLC, it will be important to understand the reasons for this behavior and to anticipate when such inversions of elution order are likely to occur. 

Cass et al. [71] described a direct injection HPLC method, with column-switching, for the determination of omeprazole enantiomers in human plasma. A restricted access media of bovine serum albumin octyl column has been used in the first dimension for separation of the analyte from the biological matrix. The omeprazole enantiomers were eluted from the restricted access media column onto an amylose tris (3,5-dimethylphenylcarbamate) chiral column by the use of a columnswitching valve and the enantioseparation was performed using acetonitrile-water (60 40) as eluent. The analytes were detected by their UV absorbance at 302 nm. The validated method was applied to the analysis of the plasma samples obtained from 10 Brazilian volunteers who received a 40-mg oral dose of racemic omeprazole and was able to quantify the enantiomers of omeprazole in the clinical samples analyzed. 

Chromatographic separation of two antimers, most often referred to as enantiosep-aration, can be carried out following either a direct or an indirect strategy. It is perhaps noteworthy that this duality of alternative options is characteristic not only of enantioseparations by means of thin layer chromatography (TLC), which is the primary subject of this book, but also of high-performance liquid chromatography (HPLC) and gas chromatography (GC). 

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