HPLC using chiral mobile-phase additives

2 HPLC using chiral mobile-phase additives 

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 

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High Performance Liquid Chromatography. Although chiral mobile phase additives have been used in high performance Hquid chromatography (hplc), the large amounts of solvent, thus chiral mobile phase additive, required to pre-equiUbrate the stationary phase renders this approach much less attractive than for dc and is not discussed here. 

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. 

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). 

CSPs and chiral mobile phase additives have also been used in the separation of amino acid enantiomers. Another technique that should be mentioned is an analysis system employing column-switching. D-and L- amino acids are first isolated as the racemic mixture by reverse-phase HPLC. The isolated fractions are introduced to a second column (a CSP or a mobile phase containing a chiral selector) for separation of enantiomers. Long et al. (2001) applied this technique to the determination of D- and L-Asp in cell culture medium, within cells and in rat blood. 

Contrary to conventional HPLC, almost 98% of chiral resolution in CE is carried out using the chiral selector as a mobile phase additive. Again all the common chiral selectors used in NLC can also be used in NCE. But, unfortunately, few chiral molecules have been tested in NCE for enantiomeric resolution of some racemates. To the best of our knowledge only cyclodextrins and protein-based chiral mobile phase additives have been used for this purpose. Manz and coworkers discussed chiral separations by NCE in their reviews in 2004 [21] and 2006 [22], Later on, Pumera [16] reviewed the use of microfluidic devices for enantiomeric resolutions in capillary electrophoresis. Not much work has been carried out on chiral resolution in NCE but the papers that are available are discussed here. 

The computational studies described above are representative examples meant to illustrate the diversity of computational techniques used to assess chiral recognition by cyclodextrins in chiral chromatography. Other published examples include the use of molecular mechanics to describe shapes of aminoalkylphosphonic acids binding to a covalently linked acetylated cyclodextrin [72], the computation of free energies of atenolol binding to a perphenylcarbamate-p-cyclodextrin likewise covalently bound to silica gel [73], and studies of cyclodextrins used as chiral mobile phase additives in reverse-phase HPLC [74] and in capillary electrophoresis [75]. 

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. 

ABSTRACT. The copper(n) complexes of p-cyclodextrins functionalized with aliphatic or pseudoaromatic amines were used for the chiral recognition of unmodified amino acids. Molecular recognition, assisted by non-covalent interactions, was proved by means of thermodynamic and spectroscopic (c.d., e.p.r. and fluorescence) measurements. A cis-disposition of amino groups seems to assist enantiomeric selectivity. The copper(II)-p-cyclodextrin complexes can be used as mobile phase additives in HPLC to separate enantiomeric mixtures of unmodified aromatic amino acids. 

A method for the enantioseparation of atrolactic acids has been presented. HPLC is used with sulfobutyl ether-jS-cyclodextrin as a chiral mobile phase additive and a C18 reversed phase column (41). 

The enantioseparation of ten mandelic acid derivatives was done using reverse phase HPLC with hydroxypropyl-j3-cyclodextrin or sulfobutyl ether-j3-cyclodextrin as chiral mobile phase additives (43). The mandelic acid derivatives are shown in Figure 1.16. 

Also, the analytical enantioseparation of five /S-substituted 2-phenylpropionic acids using HPLC with hydrox5rpropyl-/S-cyclo-dextrin as chiral mobile phase additive has been reported (53). Hy-droxypropyl-/S-cyclodextrin is shown in Figure 1.22... 

Chiral separation of flavonoids has also been carried out by chromatographic systems by using a chemically bonded chiral stationary phase or by the addition of chiral mobile phase additives (reviewed by Yanez et al. ). These chiral polymer phases can be further subdivided into polysaccharide-derived columns, and cyclodextrin and mixed cyclodextrin columns. With regard to chiral mobile phase additives, the addition of an optically active molecule to the mobile phase can facilitate separation of enantiomers on conventional stationary phases. Cyclodextrin as a chiral additive is widely used to separate enantiomers mainly by capillary electrophoresis (CE), as discussed in Section 3.6.2.I. Table 3.7 summarizes the most habitual HPLC procedures employed for the analysis of various classes of food flavonoids. 

Sharp, V.S. and Risley, D.S., Evaluation of the macrocyclic antibiotic LY333328 as a chiral selector when used as a mobile phase additive in narrow bore HPLC, Chirality, 11,75, 1999. 

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. 

B. J. Clark, Resolution of chiral compounds by HPLC using mobile phase additives and a porous graphitic carbon stationary phase, J. Pharm. Biomed. Anal., 7 1883-1888 (1989). 

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