Francesco Gasparrini

High-performance liquid chromatography chiral stationary phases based on low-molecular-mass selectors

A review of HPLC chiral stationary phases (CSPs) based on low molecular mass selectors is given. The review is focused on brush- and monomeric-type CSPs obtained by covalent linkage of chiral selectors, with emphasis on those obtained by total synthesis. Emphasis is given to new, emerging aspects like enantioseparations on receptor-like chiral stationary phases and dynamic enantioselective chromatography of stereolabile compounds.

Enantiomerization barriers by dynamic HPLC. Stationary phase effects

Abstract Chiral atropisomeric naphthamides have been used as model compounds to investigate solute interconversion phenomena in free solution and during liquid chromatography on a chiral stationary phase; energy barriers measured in the mobile phase (thermal racemization of enriched samples) are 0.3–1.3 Kcal/mol lower than those measured in the stationary phase (computer simulation).

Application of a new chiral stationary phase containing the glycopeptide antibiotic A-40,926 in the direct chromatographic resolution of β-amino acids

Abstract A new enantioselective HPLC procedure for the direct resolution of β-amino acids is described, based on the use of a new chiral stationary phase (CSP) containing the macrocyclic glycopeptide antibiotic A-40,926, structurally related to teicoplanin, covalently bonded to silica gel microparticles. The new CSP shows higher enantioselectivity and broader applicability in this field compared to the parent teicoplanin phase. The potential for semi-preparative separations on the A-40,926-CSP is demonstrated for a selected cyclic β-amino acid.

A "Quasi-Flexible" Automatic Docking Processing for Studying Stereoselective Recognition Mechanisms. Part I. Protocol Validation

The main purpose of this work is the development and validation of a general scheme based on a systematic and automatic “quasi‐flexible” docking approach for studying stereoselective recognition mechanisms. To achieve our goals we explore the conformational and configurational space for small‐ or medium‐size flexible molecules in a systematic way, seeking a method that is both reasonably accurate and relatively fast from the computational point of view. In particular, we have developed a general computational protocol for the global molecular interaction evaluation (“Glob‐MolInE”) to efficiently explore the orientational and conformational space of flexible selectors and selectands used in modern chiral high‐performance liquid chromatography (HPLC); the enantioselective binding of the selector (S)‐N‐(3,5‐dinitrobenzoyl)‐leucine‐ n‐propylamide (S)‐1 towards the selectand N‐(2‐naphthyl)‐alanine methyl ester 2 has been studied; the global minimum obtained for the homochiral associate [S(1)/S(2)] (Pop. >99%) is very close (RMS≃0.20) to the crystallographically determined structure.

Chromatographic resolution of 1,2-amino alcohols on a chiral stationary phase containing N,N′-(3,5-dinitrobenzoyl)-trans-1,2-diaminocyclohexane. Theoretical and practical aspects

Abstract Optical resolution of several 1,2-diamino alcohols including some β-adrenergic blocking agents (β-blockers) was obtained by high-performance liquid chromatography on a chiral stationary phase containing 3,5-dinitrobenzoyl derivatives of trans-1,2-diaminocyclohexane (DACH-DNB) as chiral selectors. After formation of oxazolidin-2-one derivatives, racemic amino alcohols were completely resolved (α values ranging from 1.14 to 1.55 and Rs from 1.2 to 3.3) on a 250 × 4.0 mm I.D. stainless-steel column. Further, some separations on chiral and achiral, coupled columns are reported: they show diastereo- and enantioselectivity for amino alcohols with more than one chiral centre. The method allows the utilization of both spectrophotometric and spectrofluorimetric detectors; moreover the availability of the (R,R), (S,S) selectors makes it possible to evaluate enantiomeric excesses higher than 99.9%. Some separations were also carried out with microbore columns (2.0 mm I.D.), which afforded the same performance.

Synthesis of sugar-based silica gels by copper-catalysed azide-alkyne cycloaddition via a single-step azido-activated silica intermediate and the use of the gels in hydrophilic interaction chromatography

Novel sugar-based silica gels were prepared by exploiting the copper-catalysed azide-alkyne cycloaddition (CuAAC) of two different sugar alkynes, namely, ethynyl C-galactoside 1 and propargyl O-lactoside 2, with new single-step azido-activated silica gels. The fully characterised stationary phases were generally used for hydrophilic interaction chromatography (HILIC), with particular application in the stereoselective separation of monosaccharides. Dynamic HILIC (DHILIC) experiments were performed to evaluate the influence of mutarotation on the chromatographic peak shapes of two interconverting sugar anomers. The potential of such materials was shown in the separation of other highly polar compounds, including amino acids and flavonoids.

Direct chromatographic resolution of carnitine and O-acylcarnitine enantiomers on a teicoplanin-bonded chiral stationary phase

R-(-)-Carnitine (vitamin B(T)) plays an important role in human energy metabolism, by facilitating the transport of long-chained fatty acids across the mitochondrial membranes. Its (S)-enantiomer acts as a competitive inhibitor of carnitine acetyltransferase, causing depletion of the body R-(-)-carnitine stock. Consequently, the separation of carnitine enantiomers is very important both to study their biological activities and to control the enantiomeric purity of pharmaceutical formulations. In the present paper we describe an easy, fast and convenient procedure for the separation of the enantiomers of carnitine and O-acylcarnitines by enantioselective HPLC on a laboratory-made chiral column containing covalently bonded teicoplanin as selector. High enantioselectivity factors (alpha values ranging from 1.31 to 3.02) and short-time analyses characterize the analytical procedure; in addition, analytes are easily detected by evaporative light scattering with no need for preliminary derivatization. The effects of pH and ionic strength of the mobile phase and of the nature of the organic modifier on the enantioselective separations were also investigated.

Transition from enantioselective high performance to ultra-high performance liquid chromatography: A case study of a brush-type chiral stationary phase based on sub-5-micron to sub-2-micron silica particles

Three brush-type chiral stationary phases (CSPs) differing in the particle size of the starting silica particles have been prepared by covalent grafting of the pi-acidic bis-(3,5-dinitrobenzoyl)-derivative of trans-1,2-diaminocyclohexane (DACH-DNB). Starting silica particles of 4.3, 2.6 and 1.9micron were used to generate the final CSPs using an improved, highly reproducible synthetic methodology, that allowed to assemble and surface-graft the whole chiral selector in only two steps. The different CSPs have been packed in columns of various length and diameters, and fully characterized in terms of flow permeability, kinetic performances and enantioselectivity using a set of test solutes. Very high speed and high resolution applications together with stereodynamic HPLC examples are demonstrated on the columns with reduced particle diameters, on which separations of several enantiomeric pairs are routinely obtained with analysis times in the 15-40s range.

Introducing enantioselective ultrahigh-pressure liquid chromatography (eUHPLC): theoretical inspections and ultrafast separations on a new sub-2-μm Whelk-O1 stationary phase.

A new chiral stationary phase for ultrahigh-pressure liquid chromatography (UHPLC) applications was prepared by covalent attachment of the Whelk-O1 selector to spherical, high-surface-area 1.7-μm porous silica particles. Columns of varying dimensions (lengths of 50, 75, 100, and 150 mm and internal diameters of 3.0 or 4.6 mm) were packed and characterized in terms of permeability, efficiency, retention, and enantioselectivity, using both organic and water-rich mobile phases. A conventional HPLC Whelk-O1 column based on 5.0-μm porous silica particles and packed in a 250 mm × 4.6 mm column was used as a reference. Van Deemter curves, generated with low-molecular-weight solutes on a 100 mm × 4.6 mm column packed with the 1.7-μm particles, showed H(min) (μm) and μ(opt) (mm/s) values of 4.10 and 5.22 under normal-phase and 3.74 and 4.34 under reversed-phase elution conditions. The flat C term of the van Deemter curves observed with the 1.7-μm particles allowed the use of higher-than-optimal flow rates without significant efficiency loss. Kinetic plots constructed from van Deemter data confirmed the ability of the column packed with the 1.7-μm particles to afford subminute separations with good efficiency and its superior performances in the high-speed regime, compared to the column packed with 5.0-μm particles. Resolutions in the time scale of seconds were obtained using a 50-mm-long column in the normal phase or polar organic mode. The intrinsic kinetic performances of 1.7-μm silica particles are retained in the Whelk-O1 chiral stationary phase, clearly demonstrating the potentials of enantioselective UHPLC in terms of high speed, throughput, and resolution.

Evaluation of the macrocyclic glycopeptide A-40,926 as a high- performance liquid chromatographic chiral selector and comparison with teicoplanin chiral stationary phase

A new macrocyclic antibiotic of the vancomycin family, referred to by its industrial designation as A-40,926, was bonded to 5 microm silica particles and utilised as a chiral stationary phase (CSP). Since A-40,926 is structurally related to teicoplanin, the A-40,926 CSP was compared to a commercially available teicoplanin CSP. A set of 28 chiral compounds, including amino-acids and related compounds, compounds with a ring containing the stereogenic centre, compounds bearing aromatic structures near their stereogenic centres and alcohols, was tested for enantioseparation on the two CSPs. The results are compared and discussed in terms of enantioselective Gibbs energy difference. The A-40,926 CSP was able to resolve one compound that was not resolved by the teicoplanin CSP. However, it could not separate four compounds that the teicoplanin CSP did separate. It is shown that the A-40,926 CSP is complementary to the teicoplanin CSP, thereby enlarging the number of enantiomers that can be separated by the macrocyclic glycopeptide based CSPs.