Dorina Kotoni

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.

Enantioselective ultra-high and high performance liquid chromatography: a comparative study of columns based on the Whelk-O1 selector.

This paper reports on the thermodynamic and kinetic evaluation of a new ultra-high performance liquid chromatography broad-spectrum Pirkle-type chiral stationary phase (CSP) for enantioselective applications (eUHPLC). The well-known Whelk-O1 selector was covalently immobilized onto 1.7-μm high-surface-area, porous spherical silica particles to produce a totally synthetic, covalently bonded CSP that was packed into 150 mm, 100mm, 75 mm and 50mm columns, either 4.6 or 3.0mm ID. A 100 mm × 4.6 mm ID column was fully characterized from a kinetic and thermodynamic point of view, using as reference a conventional HPLC Whelk-O1 column, 250 mm×4.6mm ID, based on 5-μm porous silica particles. On the eUHPLC column, van Deemter plots generated H(min) values of 3.53 μm for 1,3-dinitrobenzene, at an interstitial mobile phase linear velocity (μ(inter)) of 5.07 mm/s, and H(min) of 4.26 and 4.17 μm for the two enantiomers of acenaphthenol, at μ(inter) of 4.85 mm/s and 4.24 mm/s, respectively. Resolution of 21 enantiomeric pairs including alcohols, epoxides, sulfoxides, phosphine oxides, benzodiazepines and 2-aryloxyproprionic esters used as herbicides, were obtained with significant advantages in terms of efficiency and analysis time. Speed gain factors were calculated for the different column geometries (150 mm, 100mm, 75 mm and 50mm, either 4.6 or 3.0mm ID), with respect to the standard HPLC column (250 mm ×4.6 mm ID), and were as high as 13, in the case of the 50-mm-long column, affording sub-minute separations of enantiomers with excellent resolution factors. In particular, trans-stilbene oxide was resolved in only 10s, while a 50 mm×3.0 mm ID column was used as a compromise between reduced mobile phase consumption (less than 1 mL per analysis) and smaller extra-column band-broadening effect. Given the relatively low viscosity in NP mode, and the excellent permeability of these eUHPLC columns, with backpressure values under 600 bar for a wide range of flow rates, the use of standard HPLC hardware is possible. In this case, however, a significant loss in resolution is observed, compared to the UHPLC instrumentation, if no modifications are introduced in the HPLC apparatus to reduce extra-column variance. The excellent efficiency and selectivity, conjugated with the very high-throughput and the ultra-fast analysis time, prove the potentials of the eUHPLC Whelk-O1 columns in the development of enantioselective UHPLC methods.

Analysis of bovine milk caseins on organic monolithic columns: An integrated capillary liquid chromatography-high resolution mass spectrometry approach for the study of time-dependent casein degradation

Casein proteins constitute approximately 80% of the proteins present in bovine milk and account for many of its nutritional and technological properties. The analysis of the casein fraction in commercially available pasteurized milk and the study of its time-dependent degradation is of considerable interest in the agro-food industry. Here we present new analytical methods for the study of caseins in fresh and expired bovine milk, based on the use of lab-made capillary organic monolithic columns. An integrated capillary high performance liquid chromatography and high-resolution mass spectrometry (Cap-LC-HRMS) approach was developed, exploiting the excellent resolution, permeability and biocompatibility of organic monoliths, which is easily adaptable to the analysis of intact proteins. The resolution obtained on the lab-made Protein-Cap-RP-Lauryl-γ-Monolithic column (270 mm × 0.250 mm length × internal diameter, L × I.D.) in the analysis of commercial standard caseins (αS-CN, β-CN and κ-CN) through Cap-HPLC-UV was compared to the one observe using two packed capillary C4 columns, the ACE C4 (3 μm, 150 mm × 0.300 mm, L × I.D.) and the Jupiter C4 column (5 μm, 150 mm × 0.300 mm, L × I.D.). Thanks to the higher resolution observed, the monolithic capillary column was chosen for the successive degradation studies of casein fractions extracted from bovine milk 1-4 weeks after expiry date. The comparison of the UV chromatographic profiles of skim, semi-skim and whole milk showed a major stability of whole milk towards time-dependent degradation of caseins, which was further sustained by high-resolution analysis on a 50-cm long monolithic column using a 120-min time gradient. Contemporarily, the exact monoisotopic and average molecular masses of intact αS-CN and β-CN protein standards were obtained through high resolution mass spectrometry and used for casein identification in Cap-LC-HRMS analysis. Finally, the proteolytic degradation of β-CN in skim milk and the contemporary formation of low-molecular-weight proteose-peptones (PP) with exact monoisotopic Mr between 9444.0989 Da and 14098.9861 Da was confirmed through the deconvolution of high resolution mass spectra and literature data.

Design and evaluation of hydrolytically stable bidentate urea-type stationary phases for hydrophilic interaction chromatography

We have developed conceptually new stationary phases containing two bidentate urea-type functions suitable for the separation of a wide variety of polar compounds by hydrophilic interaction chromatography (HILIC) through a facile one-pot two-step procedure with the aim of obtaining high hydrolytic stability in a variety of elution conditions. The preparation of the new phases involves a first reaction of 1,2-ethylendiamine with (3-isocyanatopropyl)triethoxysilane to give an intermediate bis-urea with two pendant triethoxysilane functions, followed by anchoring on the silica surface. Two stationary phases were prepared, namely an urea-type stationary phase (USP-HILIC) and an urea-type phase bearing free amino groups (USP-HILIC-NH(2)), whereas silanization with 1,2-bis(trichlorosilyl)ethane yielded USP-HILIC-sil and USP-HILIC-NH(2)-sil phases, respectively. The silanization step aimed at forming a hydrophilic stable coating through cross-linking between adjacent silanols which prevents silica dissolution at alkaline pH. A full chemical characterization of the new materials has been obtained through solid-state NMR (both (29)Si and (13)C CPMAS) spectroscopy. A major application field of the bidentate urea-type stationary phase with free amino groups USP-HILIC-NH(2)-sil was sugars analysis, usually hampered by α/β anomer peak splitting and instability of the stationary phases under conditions normally employed to suppress it. Complex mixtures of mono-, di- and oligosaccharides were successfully resolved under mild chromatographic conditions, which also allowed an easy interface with mass spectrometry. The potential of such materials was shown in the separation of other highly polar compounds, including polyols, hydroxybenzoic acids, nucleobases, and vitamins.

Experimental evidence of the kinetic performance achievable with columns packed with new 1.9μm fully porous particles of narrow particle size distribution

Fully porous particles of narrow particle size distribution (nPSD) are now commercially available. In this paper, the kinetic performance of columns packed with these particles (1.9μm, 80Å pore size) has been investigated under typical reversed phase conditions by using a mixture of benzene derivatives as probes. The columns exhibited remarkably high efficiency (in the order of 300,000 theoretical plates per meter) and the possibility to be used at relatively high flow rates without loss of performance. These results contrast with previous studies on the same columns. Indeed we have found column efficiency comparable to that reported in previous work but, on the other hand, we could not observe the same dramatic loss of performance when columns were operated at high flow rates. The results presented in this paper, based on a set of six columns with different geometries (2.1 and 3.0 internal diameter×50, 75 and 100mm length), are not consistent with the previously proposed hypothesis that the unusually low intraparticle diffusion, which would characterize these particles, is the origin of the high efficiency of the columns. In a companion paper [1], a detailed investigation of the different terms leading to band broadening will be performed to point out the major contribution to plate height on nPSD columns.

Stereodynamic Investigation of Labile Stereogenic Centres in Dihydroartemisinin

Since its identification in the early 1970s, artemisinin, as well as semi-synthetic derivatives and synthetic trioxanes, have been used in malaria therapy. Reduction of artemisinin by NaBH4 produced dihydroartemisinin (DHA), and yielded a new stereochemically labile centre at C-10, which, in turn, provided two interconverting lactol hemiacetal epimers (namely α and β), whose rate of interconversion depends on buffer, pH, and solvent polarity. Since interconversion of the two epimers occurred on a chromatographic time-scale, this prompted a thorough investigation of the phenomenon as a crucial requisite of any analytical method aimed at quantitating this family of drugs. In this critical review we discuss the current importance of the on-column epimerization of DHA in the development of analytical methods aimed at quantifying the drug, with the purpose of identifying the optimal conditions to minimize on-column epimerization while achieving the best selectivity and efficiency of the overall separation.

Chiral Supramolecular Selectors for Enantiomer Differentiation in Liquid Chromatography

Biochemical processes in living organisms rely on a plethora of molecular reactions and interactions involving chiral molecules, and these processes often show different responses to the enantiomers of exogenous or endogenous chemicals. The interaction of enantiomeric drugs with a target receptor is a paradigmatic example of chirality effects on general biological action and is directly related to the formation of a drug-receptor supramolecular complex. In particular the drug-receptor model can be used to explore the relation of chirality to at least three important issues encountered in supramolecular chemistry: complementarity, preorganization, and (enantio)selectivity. A detailed understanding of those factors governing enantioselectivity of biological receptors is facilitated by the study at molecular level of model systems with simplified structures that are amenable to physico-chemical investigations. Liquid chromatography on chiral stationary phases offers the opportunity to study enantioselective interactions between surface immobilized chiral selectors, viewed as minimalist mimics of macromolecular receptors, and a large set of chiral guests, under a variety of experimental conditions. Indeed, for a significant number of systems, close agreement has been found between retention data gathered by chromatography and association constants measured by spectroscopy in free solution. Chiral supramolecular selectors featuring highly preorganized, medium-sized macrocyclic structures are attractive in this context because they often afford high levels of enantioselectivity and yet have relatively low molecular complexity, thus facilitating the understanding of operative enantioselective recognition mechanisms from easily collected chromatographic data. In the present chapter we first illustrate the general principles of supramolecular chemistry and their integration into the design of liquid chromatographic systems, with particular focus on enantioselective variants based on chiral macrocyclic selectors of natural or synthetic origin. In the second part we discuss the elaboration of enantioselective recognition models from chromatographic data and how these models can be extended to and studied with non-chromatographic systems (free solution by nuclear magnetic resonance, gas phase by mass spectrometry).

Separation of complex sugar mixtures on a hydrolytically stable bidentate urea-type stationary phase for hydrophilic interaction near ultra high performance liquid chromatography

A stationary phase bearing both bridged bis-ureido and free amino groups (USP-HILIC-NH2 -2.5SP) for high-speed hydrophilic interaction liquid chromatography separations was prepared using a one-pot two-step procedure starting from 2.5 μm totally porous silica particles. Highly polar compounds, such as polyols, hydroxybenzoic acids, and sugars, were successfully analyzed in shorter times and with higher peak efficiency, when compared to results obtained with a bidentate urea-type column packed with 5 μm particles. Increased sugarophilicity and better peak shape were attested for the USP-HILIC-NH2 -2.5SP column (100 × 3.2 mm id) when compared with two commercially available UHPLC columns, namely an acquity BEH amide packed with totally porous 1.7 μm microparticles and a HILIC Kinetex column packed with core-shell 2.6 μm particles. Finally, the new column was employed in the separation of complex mixture of sugars (mono-, di-, and oligosaccharides) and in the analysis of beer samples. The resulting chromatograms showed good selectivity and overall resolution, while the catalyzing effect of the free amino moieties resulted in excellent peak shapes and in the absence of split peaks due to sugar anomerization phenomena.

Toward enantioselective nano ultrahigh-performance liquid chromatography with Whelk-O1 chiral stationary phase.

In this study, a Whelk‐O1 chiral stationary phase immobilized on 2.5 μm silica particles was employed in nanoLC. Two nanocolumns (180 and 250 mm long, 75 μm id) with a single polymeric organic monolithic outlet frit were packed under high‐pressure ultrasonic‐assisted packing procedure. The monolithic outlet frit was prepared by thermal polymerization of methacrylate‐based monomers affording high‐mechanical stability and high‐pressure resistance. Very efficient enantioseparations with more than 70 000 plates/m were achieved in normal phase mode by eluting (+/−) acenaphthenol. Nanocolumns were also tested in RP mode by using on‐line MS detection with nano‐spray ESI ion source. Kinetic performances of columns in RP mode were comparable to those in normal phase‐conditions.