Standard High speed CCC Technique for Chiral Separation

The separations are performed using a commercial high speed CCC centrifuge equipped with a set of multilayer coil separation columns. The two-phase solvent system is selected in such a way that the target analyte has a partition coefficient ranging from 0.5 to 1 in the CS-free solvent system. When the organic phase is used as the stationary phase, the chiral selector may be made hydrophobic by attaching a long hydrocarbon chain to enhance both solubility and retention in the stationary phase.

In each separation, the column is first filled about half way with the CS-free stationary phase. This is followed by introduction of the CS-containing stationary phase (about 60% of the total column capacity) by discharging the excess amount of CS-free stationary phase from the other end of the column. In this way some amount of CS-free stationary phase (about 10% of the stationary phase retained in the column) remains at the end of the column during separation to absorb carried-over CS from the mobile phase which would contaminate the eluted fractions. After the sample solution is injected through the sample port, the mobile phase is pumped into the column while the column is rotated at the required speed, usually 800-1000 rpm. If desired separation can be carried out by successive injection of samples without replenishing the CS-containing stationary phase.

Figure 1 shows the separation of four pairs of di-nitrobenzoyl (DNB)-amino acid enantiomers by the standard CCC technique using a two-phase solvent system composed of hexane/ethyl acetate/ methanol/10 mm HCl with N-dodecanoyl-l-3,5-dimethylanilide as a CS in the organic stationary phase. Because of its high hydrophobicity (K > 100), this CS has high solubility in the organic stationary phase and is almost entirely partitioned into the stationary phase. All analytes were well resolved in 1-3 h. This chiral selector is similar to the 'Pirkle'

Figure 1 Separation of four racemic pairs of DNB-amino acids by the standard high speed CCC technique. Experimental conditions: apparatus: multilayer coil high speed CCC centrifuge with a semipreparative column of 1.6 mm i.d. and 330 mL capacity; solvent system: hexane/ethyl acetate/methanol 10 mM HCl (8:2:5:5, v/v/v/v), A/-dodecanoyl-L-proline-3,5-dimethylanilide (CS) (2 g) was added to the organic stationary phase (200 mL) as a chiral selector; samples: from left to right, ( + )-DNB-phenylglycine, ( + )-DNB-phenylalanine, (+)-DNB-valine, and (+)-DNB-leucine, 5-10 mg of each dissolved in 5 mL of solvent consisting of equal volumes of each phase; flow rate: 3.3 mL min-1; revolution: 800 rpm; analysis of fractions: optical rotation and circular dichroism; stationary phase retention: 65% of the total column capacity.

Figure 1 Separation of four racemic pairs of DNB-amino acids by the standard high speed CCC technique. Experimental conditions: apparatus: multilayer coil high speed CCC centrifuge with a semipreparative column of 1.6 mm i.d. and 330 mL capacity; solvent system: hexane/ethyl acetate/methanol 10 mM HCl (8:2:5:5, v/v/v/v), A/-dodecanoyl-L-proline-3,5-dimethylanilide (CS) (2 g) was added to the organic stationary phase (200 mL) as a chiral selector; samples: from left to right, ( + )-DNB-phenylglycine, ( + )-DNB-phenylalanine, (+)-DNB-valine, and (+)-DNB-leucine, 5-10 mg of each dissolved in 5 mL of solvent consisting of equal volumes of each phase; flow rate: 3.3 mL min-1; revolution: 800 rpm; analysis of fractions: optical rotation and circular dichroism; stationary phase retention: 65% of the total column capacity.

Figure 2 Effects of the amount or concentration of CS on the separation of DNB-amino acid racemates. In all resolved chromatograms, the first peak represents (-)-enantiomer and the second peak (#)-enantiomer. Experimental conditions: apparatus and column: see the Figure 1 caption; sample: racemic DNB-amino acid mixture consisting of DNB-valine and DNB-leucine, each 5-10 mg dissolved in 2 mL of solvent (1 mL of each phase): solvent system: hexane/ethyl acetate/methanol/10 mM HCl (8:2:5:5, v/v/v/v); stationary phase: upper organic phase with CS ranging from 0 to 4 g in 200 mL as indicated; mobile phase, lower aqueous phase; flow rate: 3 mL min-1; revolution: 800 rpm.

Figure 2 Effects of the amount or concentration of CS on the separation of DNB-amino acid racemates. In all resolved chromatograms, the first peak represents (-)-enantiomer and the second peak (#)-enantiomer. Experimental conditions: apparatus and column: see the Figure 1 caption; sample: racemic DNB-amino acid mixture consisting of DNB-valine and DNB-leucine, each 5-10 mg dissolved in 2 mL of solvent (1 mL of each phase): solvent system: hexane/ethyl acetate/methanol/10 mM HCl (8:2:5:5, v/v/v/v); stationary phase: upper organic phase with CS ranging from 0 to 4 g in 200 mL as indicated; mobile phase, lower aqueous phase; flow rate: 3 mL min-1; revolution: 800 rpm.

chiral stationary phase which has been introduced for the HPLC separation of racemic DNB-amino acid t-butylamides. For CCC separation an N-dodecanoyl group was covalently attached to the CS molecule to increase its hydrophobicity so that it is almost entirely partitioned into the organic stationary phase.

The effect of the CS concentration in the stationary phase on the peak resolution was investigated by a series of experiments as shown in Figure 2. As the CS concentration was increased, the separation factor and peak resolution were increased as indicated by eqn [6]. The results clearly imply an important technical strategy for the present method: the best peak resolution is attained by using a saturated solution of the CS in the stationary phase in a given column, and the resolution is further improved by using a longer and/or wider-bore coiled column which can hold a greater amount of CS in the stationary phase.

The preparative capability of the system was investigated on the separation of DNB-leucine enan-tiomers by varying the CS concentration in the stationary phase. The results shown in Figure 3 indicate that the sample loading capacity is mainly determined by the CS concentration in the stationary phase, i.e. the higher the CS concentration, the greater the peak resolution and sample loading capacity. As mentioned earlier, the standard HSCCC column (typically 300 mL in capacity) can be used for both analytical and preparative separation simply by adjusting the CS concentration in the stationary phase.

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