APPLICATION OF DYNAMIC CHROMATOGRAPHY METHODS WITHIN EXTREME OPERATING CONDITIONSEXTREME OPERATING CONDITIONS

CHIRAL SEPARATIONS. CHIRAL DYNAMIC CHROMATOGRAPHY

5. APPLICATION OF DYNAMIC CHROMATOGRAPHY METHODS WITHIN EXTREME OPERATING CONDITIONSEXTREME OPERATING CONDITIONS

The great advantage in using DC techniques is promptly highlighted when they are compared with classical kinetic determinations based on batchwise procedures. Typically, in the latter case, isomerization rate constants are obtained by monitoring the amount variations of one of the reacting species as a function of time. Frequently, a chromatographic technique is employed as the monitoring tool, so that the progressive residual amount of a reacting species or the increasing amount of the formed product is evaluated by the areas underlying the registered chromatographic peaks. When the studied isomerization involves chiral molecules, as necessarily occurs in enantiomerizations, the variation of enan- tiomeric excess is monitored as a function of time by off-line enantioselective chromatography. In a typical experiment, pure or enriched samples are allowed to equilibrate into an isolated and thermo- statted system in the presence of reaction solvent, and the progression of the isomerization is monitored by chromatography under conditions of suppressed interconversion. Although rigorous and of general applicability, batchwise approaches are usually laborious, time-consuming, and expensive in terms of the amount of product to process. Moreover, a preliminary collection of pure or highly enriched reactants at preparative or semipreparative scale must be accomplished, starting from the equilibrated mixture of the couple of interconverting species to be analyzed (a racemic mixture, for the case of enantiomerizations). All these drawbacks are completely overcome by using the DC approach.

However, two main limitations should also be taken into account in this case: (1) the limited range of solvents that can be used and (2) the perturbing effect of the SP (seeSection 7).

A quite great number of enantiomerizations/diastereomerizations of chiral species have been studied by DC approaches in the past two decades. In several cases, the chromatographic results were supported and/or compared to alternative kinetic methods, such as DNMR spectroscopy (Gasparrini et al., 1995, 2002a; Dell’Erba et al., 2002; Dalla Cort et al., 2005) and stopped-flow gas

O P

O N

N MeO

MeO

Me Me

NO2

CH3

NH2 N

N H N

O O

O H

O O O

O H

OH S

Me Me

CH2Ph

CH2Ph I

Conformational isomerizations

Compound ∆S^≠ Compound

(e.u.)

∆G^≠ (kcal mol^–1)

∆S^≠ (e.u.)

∆G^≠ (kcal mol^–1) Configurational isomerizations

(diastereomerization) DHPLC

0.5 14.8 (–68 °C) –19

–9.9 22.7 (50 °C)

2.0 15.0 (–70 °C)

–3.0 19.8 (–10 °C) –40 22.0 (50 °C)

–40 18.5 (–5 °C)

–31 ÷ –20 22.1 (25 °C) –44 24.0 (45 °C)

–4.0 19.5 (–5 °C)

5.0 19.0 (–5 °C)

29.8 (160 °C)

–35 29.8 (160 °C)

–37 29.3 (160 °C) (enantiomerization)

DHRGC

(enantiomerization) DHPLC

(diastereomerization) DHPLC

(enantiomerization) DHRGC

(enantiomerization) DHPLC Second-order process

Second-order process

(epimerization) DHPLC First-order process

(different pHs) (enantiomerization)

DHPLC First-order process

FIGURE 3.6

Activation entropies (DS^s, e.u.) and free energies (DG^s, kcal/mol) of conformational and configurational isomerizations calculated by DHPLC and DHRGC.

N N

(enantiomerization) DHPLC

(enantiomerization) DHPLC –9.2

–16.9 21.9 (30 °C) –55

–

– –59

33.6 (190 °C)

17.7 (– 20 °C)

18.7 (– 20 °C)

18.1 (– 20 °C)

15.7 (– 55 °C) 38.0 (220 °C) 21.8 (30 °C)

–2.9 21.9 (30 °C)

–2.0

–

21.7 (30 °C)

14.3 (–70 °C)

– 14.9 (–60 °C)

O O H N

N H C O

CI N

N O

N F O N

H C H C O

CI N

N O

CI N N H O

* O

HO OH

OCH O

O N

N O

O N HO OH

(diastereomerization) DHPLC

Conformational isomerizations Conformational isomerizations

diazepam

prazepam

tetrazepam

flunitrazepam (enantiomerization)

DHPLC

(enantiomerization) DHPLC (enantiomerization)

DHRGC

(enantiomerization) DHRGC

(enantiomerization) DHPLC

(enantiomerization) DHPLC (enantiomerization)

DHPLC

(enantiomerization) DHPLC

FIGURE 3.6 Cont’d

O O

iPr

iPr iPr

MeN Me

O P

Me N

Me Me N Me

MeN Et

O P

Et N

Me Me N Et

MeN iPr

O P

iPr N

MeO

P OMe

OMe Me N iPr

EtO

P OEt

OEt

EtO

a b

R R

R R = NO R = COOH R = OCH R =H

R = NH R = CH OCOR R = H R =NH EtO

H OH O O OHC O

O N NO

O O Ru

CI N N

OEt P OEt OEt

OEt O Ru

CI N N

(diastereomerization) DHPLC

–

–15

–12 23.7 (30 °C) 20.3 (–10 °C)

19.9 (15 °C)

–21.5 17.2 (–25 °C) –18.5 16.3 (–50 °C)

–12.0 16.8 (–50 °C)

– 23.7 (50 °C) – 20.5 (15 °C)

–12 24.8 (35 °C)

– 18.7 (–10 °C)

– 17.9 (–10 °C)

–

– –

17.9 (–10 °C)

a 21.9 (24 °C) b 19.7 (–5 °C) (enantiomerization)

DHPLC

(enantiomerization) DHPLC

(enantiomerization) DHPLC (enantiomerization)

DHPLC

(enantiomerization) DHPLC

(epimerization) DHPLC

(enantiomerization) DHPLC

(enantiomerization) DHPLC (enantiomerization)

DHPLC

FIGURE 3.6 Cont’d

chromatography (Wolf, 2005; Trapp and Schurig, 2000). It was therefore found out that the DC methods, in the whole of their different forms, can ensure the coverage of a very wide range of activation energies, from the very small value of about 15 kcal/mol (Gasparrini et al., 2000) to the much greater value of 38 kcal/mol (Trapp et al., 2002). A schematic representation of the optimal range of applicability of DC [including gas chromatography, high-performance liquid chromatography (HPLC), and UHPLC] and DNMR techniques in the determination of activation barriers for isomerization processes is given inFig. 3.7.

InFig. 3.6some representative examples of isomerizations are collected whose activation barriers determined by DC methods range between the extreme values of 14.7 and 38.0 kcal/mol. Both temperature and residence time of the interconverting species inside the column are parameters that can be tuned to approach the upper or lower limits of the above range. The residence time may be effectively modulated by changing the flow rate, but also by drastically improving the chromatographic efficiency. Very high efficiencies, in fact, allow better resolutions in shorter times, and this strongly reduces the residence time of stereolabile species having extremely low activation barriers to interconversion. A second strategy is to reduce the column temperature, and several examples of cryo- chromatography on chiral stationary phases (CSPs) at temperatures ranging from 50C down to80C are listed inFig. 3.6. The chemical and stereochemical diversity of the solutes investigated, together with the values of energy barriers spanning from 14.8 to 24.8 kcal/mol, indicate that the DC approach is well suited to study a broad range of intriguing chiral molecules with labile stereogenic elements. The implementation of novel and more efficient chromatographic materials, columns, and improved hardware, such as in the UHPLC approach, may introduce additional resolving power and speed of analysis, thus extending the application range of the dynamic technique.

Dalam dokumen Handbook of Advanced Chromatography/Mass Spectrometry Techniques (Halaman 114-118)