Chiral mobile phase additives chromatographic separation

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. 

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. 

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. 

Conversely, the use of chiral mobile phases has advantages which make it very appealing. Many chiral additives are readily available or can be easily synthesized. Achiral stationary phases, which are significantly cheaper than chiral phases, can be used. The approach also offers more flexibility than direct separation with chiral stationary phases because the chiral mobile phase additives can often be easily washed out of the chromatographic system and replaced with another additive for subsequent separations. 

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 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. 

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. 

As mentioned above, enantioseparations in EKC rely on a chromatographic separation principle. Despite this fact, there are significant differences between these techniques. Responsible for all differences between chromatographic and electrophoretic enantioseparations is the property of the electrophoretic mobility to be selective for the analytes residing in the same physical phase [2]. Another important point is that in chromatographic techniques, except in the case of a chiral mobile phase additive (CMPA), the analyte is virtually immobile when associated with a chiral selector. In EKC the analyte selector complex is commonly mobile. 

The chromatographic methods are considered to be most useful for chiral separations. Enantiomers can be separated by two methods (a) indirect method that utilizes derivatizing agents and (b) direct method that uses chiral stationary phases (CSPs) or chiral mobile phase additives (CMPAs) [49-56]. 

The enantiomeric separation with chiral mobile phases consists of the addition of an active compound in the mobile phase which is constantly pumped though the chromatographic system. The active ingredient contributes to a specific secondary chemical equilibrium, interacting with the enantiomers in the mobile phase as well as in the stationary phase, leading to the formation of diastereomeric complexes potentially in both phases. This affects the overall distribution of the analyte between the stationary phase and the mobile phase, affecting its retention and the overall enantiomeric separation. The rates of formation of the diastereomeric complexes should be similar to the diffusion rates to minimize excessive chemical contribution to the band-broadening. 

If some fields may be empty in the sublevels, all the fields in the main level are required for each entry. A new chiral separation record can be added in CHIRBASE solely if the authors correctly identify both sample and CSP. Since the beginning of the project, our policy has been to contact the authors of all publications containing incomplete, ambiguous or inconsistent data and to ask for additional information. Providing the separations with unique case numbers helps us considerably in this essential task, and also facilitates avoiding redundancies in the database. When chiral separations are reported for the second time in a new publication with exactly the same chromatographic conditions, this is stated in a footnote added in the field comments . In this field, miscellaneous information that cannot appear elsewhere are listed (detection limit, description of a reported chromatogram, racemization study, mobile phase limitations, etc.). 

Mixing the additive in the eluent used as a mobile phase can also modify the chromatographic system (dynamic modification), but the use of modified adsorbents has led to an improvement of resolution. Example works include that by Armstrong and Zhou [11], who used a macrocyclic antibiotic as the chiral selector for enantiomeric separations of acids, racemic drugs, and dansyl amino acid on biphenyl-bonded silica. 

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