Biomedical Engineering Reference
In-Depth Information
5.5.2 E
NANTIOSELECTIVE
C
HROMATOGRAPHY
The preparative separation of the enantiomers of chiral drugs using chromatography relies on the
formation of diastereomeric complexes. This can be achieved by precolumn derivatization (using
a chiral derivatizing agent followed by separation using conventional reverse-phase chromatogra-
phy) or by using a chiral column or a chiral mobile phase additive. Direct separation of enantiom-
ers using commercially available HPLC columns containing an immobilized CSP is usually the
method of choice.
5.5.2.1 Ligand Exchange
Chiral ligand-exchange chromatography relies on the covalent binding of an optically active ligand
to a solid support. Resolution of amino acids, using a CSP of (
S
)-proline bound to the silica gel
solid support, is the main application of this technique. At i rst, copper (II) ions are passed through
the column to form a complex with the (
S
)-proline. The racemic mixture is then passed down the
column and the
R
and
S
isomers displace one of the (
S
)-proline ligands from the copper complex
resulting in the formation of transient diastereomeric complexes with different stabilities leading to
different retention times.
5.5.2.2 Crown Ethers
Resolution of nonderivatized racemic amino acids using crown-ether CSPs relies on the formation
of a diastereomeric inclusion complex formed between the ammonium ion of the primary amino
group and the oxygen atoms of the crown ether. The commercially available Crownpak column
consists of a single enantiomer of a chiral dinaphthyl crown ether (Figure 5.13) immobilized on a
stationary phase. One disadvantage of using this column is that, mobile phases containing more
than 15% methanol results in leaching of the chiral crown ether from the column and deterioration
of CSP performance. Recently, a CSP bearing structural similarity to the crown ether found in the
Crownpak column has been developed that has additional functionality allowing covalent immobi-
lization on silica gel.
O
2
N
O
NO
2
O
O
N
N
Si
O
Si
(CH
2
)
3
H
O
O
O
O
O
Crownpak CR (+)
Pirkle pi-acceptor CSP
FIGURE 5.13
Examples of crown ether and Pirkle CSP.
5.5.2.3 Pirkle Columns
Developed mainly by Pirkle and coworkers, there are two main types of these columns: a p-acceptor
phase based mainly on
N
-(3,5-dinitrobenzoyl)-phenylglycine bonded via a linker to the silica and
a p-donor phase typically based on naphthylamino acid derivatives bonded to silica. Resolution of
chiral drugs relies on preferential interactions of one enantiomer over the other with the CSP due
to formation of charge-transfer complexes, p-p bonding and steric effects. Although commercially
available and suitable for preparative separation, they can only separate aromatic compounds and
therefore precolumn derivatization of the drug of interest may be necessary.
