Group IA

As a general rule, most applications concern the brush-type CSPs having a rc-electron acceptor charac ter. This is because many compounds of pharmaceutical interest contain a rc-donor group.

CSPs derived from N-(3,5-dinitrobenzoyl)amino acids are among the most widely used for enan-tiomeric separations of numerous compounds. The early commercialization of the well-known (R)-N-(3,5-dinitrobenzoyl)phenylglycine-derived CSP ((R)-DNBPG), designed by Pirkle and co-workers in 1980, and the easy and inexpensive preparation of this type of CSP, has prompted many researchers to design new rc-acid CSPs. Although the scope of applications of these CSPs does not vary very much, all workers agree that small structural changes in the phases can have significant effects on the chromatographic behaviour. Our laboratories have been involved in the development of CSPs derived from tyrosine. Among them, a 'broad-spectrum' CSP has been marketed under the registered name ChyRoSine-A and an improved version of this has been described. Their enantiomer-recognition abilities have been evaluated both by LC and SFC and the scope of applications including numerous racemates such as benzodiazepines, sulfox-ides, phosphine oxides, lactams and ft-blockers demonstrated.

An anthrylamine derivative adsorbed onto porous graphitic carbon has been used to separate two commercial anti-inflammatory agents (ibuprofen and flurbiprofen) and a series of racemic tropic acid derivatives. The enantioselective properties of this material were compared with the corresponding silica-based CSP and it was concluded that the former was more efficient.

re-Basic CSPs, deriving from tyrosine and bearing two stereogenic centres, were designed and successfully applied to the enantioseparation of pharmaceutical compounds using SFC. Warfarin and ICI 176334 (a potential nonsteroidal antiandrogen used in the treatment of prostate cancer) were baseline-resolved on these phases without any prior derivatization step into 3,5-dinitrobenzoyl derivatives. Several re-donor CSPs, with (_R)-N-pivaloylnaphthylethylamide as the chiral selector group, have been applied to SFC.

Valine-diamide phases have been used in SFC for the enantioseparation of racemic N-4-nitroben-zoylamino acid isopropyl esters. The enantioselectiv-ity in SFC was comparable to that in LC using the mixture 2-propanol/«-hexane as mobile phase but the time required for analysis was less than 5 min by SFC.

As a general rule, the use of SFC does not improve enantioselectivity for type I CSPs. The selectivities obtained in LC and SFC are identical, showing that the chiral recognition mechanisms are the same for hexane and carbon dioxide. In this case, the advantage of SFC over LC is of a kinetic nature, giving higher efficiency per unit time and therefore faster analysis. Figure 2 illustrates the kinetic advantage of SFC over LC by showing the separation of the enan-tiomers of Oxazepam on ChyRoSine-A in both LC and SFC. At constant resolution, the analysis time by SFC is 6 min, and 24 min by LC. However, it must be emphasized that in some cases different selectivities between LC and SFC are encountered.

The first of these cases concerns the noncon-ventional separation of re-acceptor solutes on re-acceptor CSPs. In such a case, re-re charge transfer interactions cannot take place during the chiral no2

CONH(CH2)3CH3 NO2

CONH(CH2)3CH3 NO2

Figure 2 LC (A) and SFC (B) separations of the enantiomers of oxazepam using a ChyRoSine-A CSP: a comparison of analysis time at constant resolution (Rs = 3.5). Operating conditions: 150 x4.6 mm i.d. column packed with 5 ^m ChyRoSine-A CSP. LC: mobile phase, hexane/ethanol (90:10); flow rate, 2mLmin~1. SubSFC: mobile phase, carbon dioxide/ethanol (92:8); flow rate, 4.5 mL min-1 at 0°C; outlet pressure 200 bar. Temperature, 25°C; UV detection at 229 nm. (Reproduced from Bargmann-Leyder N, Tambute A and Caude M (1992) Chiralite et chromatographie en phase supercritique. A review. Analysis 20: 189-200.)

Figure 2 LC (A) and SFC (B) separations of the enantiomers of oxazepam using a ChyRoSine-A CSP: a comparison of analysis time at constant resolution (Rs = 3.5). Operating conditions: 150 x4.6 mm i.d. column packed with 5 ^m ChyRoSine-A CSP. LC: mobile phase, hexane/ethanol (90:10); flow rate, 2mLmin~1. SubSFC: mobile phase, carbon dioxide/ethanol (92:8); flow rate, 4.5 mL min-1 at 0°C; outlet pressure 200 bar. Temperature, 25°C; UV detection at 229 nm. (Reproduced from Bargmann-Leyder N, Tambute A and Caude M (1992) Chiralite et chromatographie en phase supercritique. A review. Analysis 20: 189-200.)

Figure 3 Influence of the nature of the mobile phase on the resolution of A/-(3,5-dinitrobenzoyl)phenylglycinol on (R)-DNBPG. LC conditions: mobile phase, hexane/ethanol (85:15, v/v) (k'(r) = 3.9, k'(s)=5.6) or hexane/chloroform (10:90, v/v) (k'(r) = 4.3, k'(s) = 16.3); flow rate 2mLmin~1; temperature, 25°C; UV detection at 254 nm. SubSFC conditions: mobile phase, carbon diox-ide/ethanol (93:7, w/w); flow rate 4.5mLmin~1 at 0°C; average column pressure, 200 bar; temperature, 25°C; UV detection at 254 nm. (Reproduced from Macaudiere P, Lienne M, Caude M, Rosset R and Tambute A (1989) Resolution of rc-acid racemates on rc-acid chiral stationary phases in normal-phase liquid and subcritical fluid chromatographic modes. A unique reversal of elution order on changing the nature of the achiral modifier. JournalofChromatography 467:357-372, with permission from Elsevier Science.)

Figure 3 Influence of the nature of the mobile phase on the resolution of A/-(3,5-dinitrobenzoyl)phenylglycinol on (R)-DNBPG. LC conditions: mobile phase, hexane/ethanol (85:15, v/v) (k'(r) = 3.9, k'(s)=5.6) or hexane/chloroform (10:90, v/v) (k'(r) = 4.3, k'(s) = 16.3); flow rate 2mLmin~1; temperature, 25°C; UV detection at 254 nm. SubSFC conditions: mobile phase, carbon diox-ide/ethanol (93:7, w/w); flow rate 4.5mLmin~1 at 0°C; average column pressure, 200 bar; temperature, 25°C; UV detection at 254 nm. (Reproduced from Macaudiere P, Lienne M, Caude M, Rosset R and Tambute A (1989) Resolution of rc-acid racemates on rc-acid chiral stationary phases in normal-phase liquid and subcritical fluid chromatographic modes. A unique reversal of elution order on changing the nature of the achiral modifier. JournalofChromatography 467:357-372, with permission from Elsevier Science.)

recognition mechanism. This is why the main mechanism may vary depending on the mobile phase composition, sometimes resulting in a reversal of the elution order. As shown in Figure 3, important discrepancies in the selectivity values are noted between hexane/ethanol in LC and the supercritical mobile phase carbon dioxide/ethanol. The chromatographic behaviour observed in SFC is somewhat similar to that observed in LC with hexane/methylene chloride/chloroform mobile phases.

The second major exception concerns the separation of ^-blockers using ChyRoSine-A as CSP. Surprisingly, the direct separation of a series of blockers was achieved on commercially available ChyRoSine-A CSP and on its improved version, whereas these solutes appear to be unresolved or poorly resolved by normal-phase liquid chromatogra-phy (Figure 4; Table 2). The chromatographic behaviour (both in SFC and LC) of various propranolol analogues has been thoroughly studied and further spectroscopic investigations carried out. Starting from these data, detailed chiral recognition mechanisms have been proposed, based on molecular modelling. The solute conformations are selected by taking into account the information provided by the 1H NMR spectra and it appears that the solvating

Solution Propranolol

Figure 4 Comparative chromatograms of the resolution of propranolol on ChyRoSine-A CSP by LC (A) and SFC (B). Operating conditions: 150 x4.6 mm i.d. column packed with 5 ^m ChyRoSine-A CSP. LC. mobile phase/hexane/ethanol containing 1 % v/v of n-propylamine (95 : 5, v/v); flow rate 1 mL min~1. SFC: mobile phase, carbon dioxide/ethanol containing 1 % v/v of n-propylamine (90 : 10); flow rate, 4mL min~1 at 0°C, outlet pressure 200 bar. Room temperature; UV detection at 224 nm. (Reproduced with permission from Siret L, Bargmann N, Tambute A and Caude M (1992) Direct enantiomeric separation of p-blockers on ChyRoSine-A by supercritical fluid chromatography: supercritical carbon dioxide as transient insituderivatizing agent. Chirality 4: 252-262.)

Figure 4 Comparative chromatograms of the resolution of propranolol on ChyRoSine-A CSP by LC (A) and SFC (B). Operating conditions: 150 x4.6 mm i.d. column packed with 5 ^m ChyRoSine-A CSP. LC. mobile phase/hexane/ethanol containing 1 % v/v of n-propylamine (95 : 5, v/v); flow rate 1 mL min~1. SFC: mobile phase, carbon dioxide/ethanol containing 1 % v/v of n-propylamine (90 : 10); flow rate, 4mL min~1 at 0°C, outlet pressure 200 bar. Room temperature; UV detection at 224 nm. (Reproduced with permission from Siret L, Bargmann N, Tambute A and Caude M (1992) Direct enantiomeric separation of p-blockers on ChyRoSine-A by supercritical fluid chromatography: supercritical carbon dioxide as transient insituderivatizing agent. Chirality 4: 252-262.)

effect of carbon dioxide induces a change in conformation of propranolol (Figure 5). This change occurs in the presence of carbon dioxide but only if the solute bears both an amino proton and an ether function separated by three carbon atoms. Without carbon dioxide, (R)- and (S)-propranolol conformers have geometrical structures such that the chiral recognition process is poor: the chiral centre of the solute cannot develop stereoselective interactions with the CSP and the interactions involved are the same for both enantiomers (Figure 6). On the other hand, the conformation of propranolol in the presence of carbon dioxide is geometrically favourable to the chiral discrimination. The conformations of the chiral stationary phase, (R)-solute, (S)-solute and their respective associations are shown in Figure 7. In this case, the (R)-propranolol conformer involves higher energy interactions with (S)-CSP than the (S) conformer.

High speed chiral separations (analysis duration <1.5 min) of p-blockers have been achieved using a short packed column and a high mobile phase flow rate. The use of high speed chiral separations allows a decrease in solvent consumption (CO2 and polar modifier), and by minimizing band broadening in the column gives better detectability. As an example, Figure 8 shows the enantioseparation of propranolol and pindolol. These results again demonstrate the kinetic superiority of the SFC over LC. Moreover, in the case of p-blockers, the better kinetics of SFC is combined with enhanced thermodynamics owing to the favoured chiral recognition provided by the conformation of the molecules in the presence of carbon dioxide.

Finally, it should be noted that type I CSPs have been successfully applied to preparative SFC.

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