Depending on the type of the enantiomeric interaction, a large number of chiral
stationary phases for high-performance liquid chromatographic methods are available and can be separated according to their “mode-of-action” (Mangelings and
Vander Heyden 2008; Schurig and Ciogli 2013; Scriba 2013):
1. Diastereomeric metal complex (chiral ligand exchange chromatography).
2. Charge-transfer interactions.
3. Multiple attractive interactions (Pirkle’s brush type stationary phases).
4. Inclusion into chiral cavities (cyclodextrin stationary phases, crown ethers).
5. Protein as a chiral stationary phase; complexes are based upon combinations of
hydrophobic and polar interactions (e.g. α-acid glycoproteins).
For today’s enantioselective environmental analysis, the largest number of applications is documented for modified cyclodextrin as chiral stationary phase (CSP) for
both HPLC and GC methods. α-, β- and γ-Cyclodextrins are cyclic polysaccharides
containing 6, 7 or 8 α-glycopyranose units, respectively (see Fig. 3.2). The primary
hydroxyl groups are located at the narrow opening of the ring system, whereas
secondary OH-groups are to be found at the wider opening and along the torus axis
of the cyclodextrin (Fig. 3.2). The diameter and the length of the cavity vary
depending on the type and position of the substituent introduced into the cyclodextrin molecule. Cyclodextrin stationary phases gained their importance as chiral
selector from the modified cavity of their toroidal molecular structure. In the same
way as for crown ethers, the analyte is introduced into the cavity by a reversible
enantiomer-selective inclusion mechanism.
For a more detailed description of properties, limitations and advantages of the
various stationary phases available today, we refer to the textbooks mentioned in the
reference list (Aboul-Enein and Ali 2003; Ates et al. 2013; Schurig and Ciogli 2013;
Scriba 2013; Hegade et al. 2017; Ferretti et al. 2018).
3.3 Indirect Methods
As already introduced above, indirect methods are based on the reaction of a racemic
mixture with a chiral reagent to form a pair of diastereomers. For example, a mixture
of organic acid enantiomers reacts with an optically active organic base to give two
theoretically separable diastereomeric salts (Fig. 3.1). Furthermore the acid chloride
of (S)-(–)-N-trifluoroacetylproline is a widely used derivatising agent and has been
applied to the resolution of racemic amines and alcohols by HPLC and cGC (Adams
et al. 1982; MacFarlane et al. 1991; Martins et al. 2006). A distinctive advantage of
this type of derivatisation is, once the diastereomers have been resolved and
characterised, the enantiomers can be recovered again by reversing the derivatisation
procedure, for example, by hydrolysis of the diastereomeric amides or esters, for
further characterisation.
Although such indirect methods have been used extensively for analyses in the
past and are still being used at the process scale, they have a number of severe
34
3 Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography
stationary phases for high-performance liquid chromatographic methods are available and can be separated according to their “mode-of-action” (Mangelings and
Vander Heyden 2008; Schurig and Ciogli 2013; Scriba 2013):
1. Diastereomeric metal complex (chiral ligand exchange chromatography).
2. Charge-transfer interactions.
3. Multiple attractive interactions (Pirkle’s brush type stationary phases).
4. Inclusion into chiral cavities (cyclodextrin stationary phases, crown ethers).
5. Protein as a chiral stationary phase; complexes are based upon combinations of
hydrophobic and polar interactions (e.g. α-acid glycoproteins).
For today’s enantioselective environmental analysis, the largest number of applications is documented for modified cyclodextrin as chiral stationary phase (CSP) for
both HPLC and GC methods. α-, β- and γ-Cyclodextrins are cyclic polysaccharides
containing 6, 7 or 8 α-glycopyranose units, respectively (see Fig. 3.2). The primary
hydroxyl groups are located at the narrow opening of the ring system, whereas
secondary OH-groups are to be found at the wider opening and along the torus axis
of the cyclodextrin (Fig. 3.2). The diameter and the length of the cavity vary
depending on the type and position of the substituent introduced into the cyclodextrin molecule. Cyclodextrin stationary phases gained their importance as chiral
selector from the modified cavity of their toroidal molecular structure. In the same
way as for crown ethers, the analyte is introduced into the cavity by a reversible
enantiomer-selective inclusion mechanism.
For a more detailed description of properties, limitations and advantages of the
various stationary phases available today, we refer to the textbooks mentioned in the
reference list (Aboul-Enein and Ali 2003; Ates et al. 2013; Schurig and Ciogli 2013;
Scriba 2013; Hegade et al. 2017; Ferretti et al. 2018).
3.3 Indirect Methods
As already introduced above, indirect methods are based on the reaction of a racemic
mixture with a chiral reagent to form a pair of diastereomers. For example, a mixture
of organic acid enantiomers reacts with an optically active organic base to give two
theoretically separable diastereomeric salts (Fig. 3.1). Furthermore the acid chloride
of (S)-(–)-N-trifluoroacetylproline is a widely used derivatising agent and has been
applied to the resolution of racemic amines and alcohols by HPLC and cGC (Adams
et al. 1982; MacFarlane et al. 1991; Martins et al. 2006). A distinctive advantage of
this type of derivatisation is, once the diastereomers have been resolved and
characterised, the enantiomers can be recovered again by reversing the derivatisation
procedure, for example, by hydrolysis of the diastereomeric amides or esters, for
further characterisation.
Although such indirect methods have been used extensively for analyses in the
past and are still being used at the process scale, they have a number of severe
34
3 Enantiomer-Selective High- and Ultra- High-Performance Liquid Chromatography