Chiral mobile phase additives enantiomers

Resolution of enantiomers of amino acids through MR has been found to be simple and rapid on RP TLC plates without using impregnated plate or a chiral mobile phase additive. Enantiomers of glutamate and aspartate... 

An alternative model has been proposed in which the chiral mobile-phase additive is thought to modify the conventional, achiral stationary phase in situ thus, dynamically generating a chiral stationary phase. In this case, the enantioseparation is governed by the differences in the association between the enantiomers and the chiral selector in the stationary phase. 

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. 

In recent years, for analytical purposes the direct approach has become the most popular. Therefore, only this approach will be discussed in the next sections. With the direct approach, the enantiomers are placed in a chiral environment, since only chiral molecules can distinguish between enantiomers. The separation of the enantiomers is based on the complex formation of labile diastereoisomers between the enantiomers and a chiral auxiliary, the so-called chiral selector. The separation can only be accomplished if the complexes possess different stability constants. The chiral selectors can be either chiral molecules that are bound to the chromatographic sorbent and thus form a CSP, or chiral molecules that are added to the mobile phase, called chiral mobile phase additives (CMPA). The combination of several chiral selectors in the mobile phase, and of chiral mobile and stationary phases is also feasible. 

Prbpy, 5-(2-MePr)bpy and 5-(2,2-Mc2Pr)bpy have been prepared and characterized. The mer-and /uc-isomers of each complex have been isolated by use of cation-exchange column chromatography as the steric requirements of the R group increase, the percentage of the /uc-isomer decreases. Enantiomers of [Ru(5-Prbpy)3] + were separated on SP Sephadex C-25. Electro-kinetic chromatography has been used to separate the enantiomers of [Ru(104)3] " anionic carboxymethyl-/ -cyclodextrin was employed as the chiral mobile phase additive. ... 

On the other hand, the direct chromatographic approach involves the use of the chiral selector either in the mobile phase, a so-called chiral mobile phase additive (CMPA), or in the stationary phase [i.e., the chiral stationary phase (CSP)]. In the latter case, the chiral selector is chemically bonded or coated or allowed to absorb onto a suitable solid support. Of course chiral selectors still can be used as CMPAs, but the approach is a very expensive one owing to the high amount of chiral selector required for the preparation of the mobile phase, and the large amount of costly chiral selector that is wasted (since there is very little chance of recovering this compound). Moreover, this approach is not successftd in the preparative separation of the enantiomers. 

One of the classical approaches of liquid chromatography, paper chromatography, was used for chiral resolution about 50 years ago but is not part of modem practice. In paper chromatography, the stationary phase is water bonded to cellulose (paper material), which is of course chiral and hence provides a chiral surface for the enantiomers. However, some workers used chiral mobile phase additives also in paper chromatography [73,74]. In 1951 some research groups independently [73,75-77] resolved the enantiomers of amino acids. Simultaneously, numerous interesting publications on chiral resolution by paper chromatography appeared [70]. 

In view of the importance of chiral resolution and the efficiency of liquid chromatographic methods, attempts are made to explain the art of chiral resolution by means of liquid chromatography. This book consists of an introduction followed by Chapters 2 to 8, which discuss resolution chiral stationary phases based on polysaccharides, cyclodextrins, macrocyclic glyco-peptide antibiotics, Pirkle types, proteins, ligand exchangers, and crown ethers. The applications of other miscellaneous types of CSP are covered in Chapter 9. However, the use of chiral mobile phase additives in the separation of enantiomers is discussed in Chapter 10. 

The use of chiral mobile phases has both advantages and disadvantages. For example, the multiple equilibria occurring in the mobile phase and in the stationary phase complicates elucidation of the separation mechanism. The presence of the chiral mobile phase additive can also complicate detection. For instance, additives with relatively high UV absorbance decrease the detection limit of the separated enantiomers when using UV detection. Furthermore, resolved enantiomers enter in the detector cell in the form of complexes with the chiral resolving ligand. These complexes are diastereomers and therefore may differ in molecular absorptivity, as well as other properties. As a consequence, it is necessary to have a separate calibration curve for each enantiomer. 

The numerator in equation (22-26) represents the processes occurring in the mobile phase, while the denominator represents the processes occurring in the stationary phase. Such a situation can be realized by combining a chiral stationary phase in a push-pull mode with a chiral mobile phase of opposite con-hguration, where two enantiomers of the chiral selector are involved, one for the chiral stationary phase and the other for the chiral mobile phase. The most selective chiral chromatographic system should be encountered when one enantiomer binds to the immobilized chiral selector in the stationary phase, whereas the other enantiomer predominantly associates with the chiral mobile-phase additive [158]. The above treatment is applicable to all applications regarding the use of chiral mobile phases. 

It is well known that a chiral environment is essential for the enantiomeric resolution of racemates. In CE, this situation is provided by the chiral compounds used in the BGE and is known as the chiral selector or chiral BGE additive. Basically, the chiral recognition mechanisms in CE are similar to those in chromatography using a chiral mobile-phase additive mode, except that the resolution occurred through different migration velocities of the diastereoisomeric complexes in CE. The chiral resolution occurred through diastereomeric complex formation between the enantiomers of the pollutants and the chiral selector. The formation of diastereomeric complexes depends on the type and nature of the chiral selectors used and the nature of the pollutants. 

A chiral resolving agent is a chiral mobile-phase additive or a chiral stationary phase that preferentially complexes one of the enantiomers. 

Such mirror images are called enantiomers. Either chiral mobile-phase additives or chiral stationary phases are required for these separations. Preferential com-plexation between the chiral resolving agent (additive or stationary phase) and one of the isomers results in a separation of the enantiomers. The chiral resolving agent must have chiral character itself to recognize the chiral nature of the solute. 

Direct enantiomer separation methodologies circumvent the rather laborious formation of covalent diastereomers, but instead exploit subtle energetic differences of reversibly formed, noncovalent diastereomeric complexes for chiral recognition. Direct chromatographic enantiomer separation may be achieved in two different modes, the chiral mobile phase additive and the chiral stationary phase mode. 

In chiral mobile phase additive mode enantiomer separation, a combination of an achiral stationary and a chiral mobile phase is employed, the latter being created by simply adding a certain amount of an appropriate SO to the eluent. On introduction of a mixture of enantiomers into this system, the individual enantiomers form diastereomeric complexes with the chiral mobile phase additive (CMPA). These transitory diastereomeric complexes may exhibit distinct association/disso-ciation rates, thermodynamic stabilities and physicochemical properties, and therefore may be separated on an appropriate achiral stationary phase. 

In practice, separation of enantiomers by the use of chiral stationary phases is not free from problems. Chiral stationary phases are difficult to prepare reproducibly, are sometimes of lower chromatographic efficiency than expected, and optimization of separation conditions is restricted by the fixed nature of the chiral centres. Chiral mobile phases are free from many of these problems, optimization of the separation is more convenient, and conventional reversed-phase columns may be used. Thus N-(2, 4-dinitrophenyl)-L-alanine-n-dodecyl ester has been used as a non-ionic chiral mobile phase additive for the resolution of 1-azahexahelicenes by reversed-phase chromatography. The resolution obtained was found to be a function of the mobile phase polarity and the concentration of chiral additive used. 

Zeng, S. Zhang, L. Liu, Z.Q. [Quantification of the enantiomers of ofloxacin in human urine by RP-HPLC with chiral mobile phase additive]. Yao Hsueh Hsueh Poo, 1994, 29, 223-227 Israel, D. Gillum, G. TXirik, M. Harvey, K. Ford, J. Dalton, H. Towle, M. Echols, R. Heller, A.H. Polk, R. Pharmacokinetics and serum bactericidal titers of ciprofloxacin and ofloxacin following mul-... 

Chiral mobile phase additives provide a more versatile and cost-effective approach for enantiomer separations in thin-layer chromatography. Typically, chemically bonded layers with cyclodextrin and its derivatives, bovine serum albumin, or macrocyclic glycopeptides are used as chiral additives in the reversed-phase mode [59,60,172-178]. For [5- and y-cyclodextrins and their derivatives, a 0.1 to 0.5 M aqueous methanol or acetonitrile solution of the chiral selector is used as the mobile phase. Bovine serum albumin is generally used at concentrations of 1-8 % (w/v) in an aqueous acetate buffer of pH 5 to 7 or in a 0.5 M acetic acid solution, in either case with from 3-40 % (v/v) propan-2-ol (or another aliphatic alcohol), added to control retention. Enantioselectivity usually increases with an increase in concentration of the chiral selector, and may be non existent at low concentrations of the chiral selector. 

Since enantiomer separation requires the Intervention of some chiral agent, one may utilize either chiral mobile phase additives (CMPA) or chiral stationary phases (CSPs). While the requirement that one add a chiral substance to the mobile phase has obvious limitations for preparative separations, it is not a serious problem for analytical separations. Indeed, for some types of compounds (e.g. amino acids) this approach may be preferred. Quite an extensive literature exists for the use of mobile phases containing chiral bidentate ligands and copper ions for the "ligand exchange" resolution of underlvatized amino acids (1, 2) and for N-dansyl derivatives of amino acids Tartaric acid derivatives have also been used as CMPAs (5). 

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. 

---