Papers by Claudio Villani
Molecules
trans-Resveratrol is a natural bioactive compound with well-recognized health promoting effects. ... more trans-Resveratrol is a natural bioactive compound with well-recognized health promoting effects. When exposed to UV light, this compound can undergo a photochemically induced trans/cis isomerization and a 6π electrochemical cyclization with the subsequent formation of 2,4,6-trihydroxyphenanthrene (THP). THP is a potentially harmful compound which can exert genotoxic effects. In this work we improved the chromatographic separation and determination of the two resveratrol isomers and of THP by using a non-commercial pentafluorophenyl stationary phase. We assessed the effect of natural deep eutectic solvents (NaDES) as possible photo-protective agents by evaluating cis-resveratrol isomer and THP formation under different UV-light exposure conditions with the aim of enhancing resveratrol photostability and inhibiting THP production. Our results demonstrate a marked photoprotective effect exerted by glycerol-containing NaDES, and in particular by proline/glycerol NaDES, which exerts a st...
Separations, 2021
Many efforts have been made to separate basic compounds, which are challenging to resolve in reve... more Many efforts have been made to separate basic compounds, which are challenging to resolve in reversed phase liquid chromatography. In this process, they are strongly retained and the peak shape undergoes significant distortion. The principal origin of this has been identified with the non-negligible interaction with residual deprotonated silanols. Consequently, all solutions that efficiently shield silanols are being sought. This review is an upgrade on the use of the electrostatic repulsion reversed phase (ERRP) approach: retention of bases, in protonated form, can be achieved by modulating the charge repulsion caused by the presence of positive charges in the chromatographic system. This study successfully (i) introduced fixed positive charges in the structure of stationary phases, (ii) used cationic and hydrophobic additives in the mobile phase, and (iii) used the ERRP-like approach employed at the preparative level for peptide purification.
Journal of Chromatography A, 2018
Direct analysis of chiral active pharmaceutical ingredients and their counterions by ultra high p... more Direct analysis of chiral active pharmaceutical ingredients and their counterions by ultra high performance liquid chromatography with macrocyclic glycopeptide-based chiral stationary phases.
Analytica chimica acta, Jan 17, 2018
Argentation chromatography is widely used nowadays as a powerful tool to separate complex mixture... more Argentation chromatography is widely used nowadays as a powerful tool to separate complex mixtures of analytes containing unsaturated and/or aromatic fragments. Here we present the results of chromatographic and computational studies on a silver-thiolate stationary phase, in which the silver metal is covalently bonded to mercaptopropyl silica particles. The exceptionally high selectivity displayed by this organometallic moiety prompted us to deeply investigate its molecular recognition properties. The interactions of the silver atom with a series of benzene derivatives was investigated to gain information on the mechanism by which the different ring substituents modulate retention factors and selectivity. The experimental trend was fully rationalized by means of quantum-mechanical Density Functional Theory (DFT) calculations, which allowed us to elucidate the chromatographic results in the light of unusual and unexpected substituent effects.
Journal of separation science, Jan 10, 2018
The ever-increasing need for enantiomerically pure chiral compounds has greatly expanded the numb... more The ever-increasing need for enantiomerically pure chiral compounds has greatly expanded the number of enantioselective separation methods available for the precise and accurate measurements of the enantiomeric purity. The introduction of chiral stationary phases for liquid chromatography in the last decades has revolutionized the routine methods to determine enantiomeric purity of chiral drugs, agrochemicals, fragrances, and in general of organic and organometallic compounds. In recent years, additional efforts have been placed on faster, enantioselective analytical methods capable to fulfill the high throughput requirements of modern screening procedures. Efforts in this field, capitalizing on improved chromatographic particle technology and dedicated instrumentation, have led to highly efficient separations that are routinely completed on the seconds time scale. An overview of the recent achievements in the field of ultra-high-resolution chromatography on column packed with chira...
Journal of Chromatography A, 2017
With the aim of pushing forward the limits of high efficient and ultrafast chiral liquid chromato... more With the aim of pushing forward the limits of high efficient and ultrafast chiral liquid chromatography, a new Chiral Stationary Phase (CSP) has been prepared by covalently bonding the teicoplanin selector on 2.0 µm Superficially Porous Particles (SPPs). An already validated bonding
Journal of Chromatography A, 2017
We added some comments in the text relating to the new literature references #66 and 67. 26. The ... more We added some comments in the text relating to the new literature references #66 and 67. 26. The performance of the monoliths presented in impressive for the separation of small molecules, which is generally not achieved by other research groups. But the main question is: (1) why do your monoliths perform so well (is it better homogeneity or are there no surface diffusion/gel porosity effects and why not?) and (2) if the monoliths perform so much better for small molecules does this mean that the performance of large molecule separations is also better. Discussion on these aspects is very much appreciated! We have not addressed these points in the paper, especially the relationship, if any, between the observed efficiency for small and large (bio)molecules. At this point, any comment would be speculative and we are planning to deeply investigate the interesting subject in a near future. 27. General comment: improve figure quality (4, 5, 6, 9).-labels on x and y axis, sometimes the font/size, sometimes the content!.-check the units.-check if appropriate scale are used (van Veemter curves: x-axis 5-6 mm/s does not show data points, kinetic plots y axis should be adjusted (there is no data in 10-1 and 10-2 range and 10-4 to 10-5 range, so please do not show this range). Corrected 28. Grammar last 2 highlights should be improved. Corrected Reviewer #2: Summary This work encompasses synthesis of poly(lauryl methacrylate-co-1,6-hexanediol dimethacrylate) monolithic columns in capillary diameters (75 µm, 200 µm and 250 µm i.d.) with a view to developing suitable materials for reversed-phase LC separation of small molecules, and to reinforce the suitability of the approach by demonstrating biomolecule separations. The novelty of the work is in the pre-treatment of capillary to allow a "grafting-to" approach to be used for anchoring the <gamma>-ray initiated monolithic co-polymer to the surface. While no direct comparison to columns prepared using conventional pre-treatment procedures has been made within this work, the material has been structurally characterized (IR, solid state NMR, cryogenic NMR & SEM), compared to a thermally-initiated equivalent (using AIBN), with a number of chromatographic experiments with small and large molecules also shown. Isocratic chromatographic comparison centers around evaluation of newly-synthesized materials against a column packed with 5 µm C18functionalized particles in identical dimensions to the monolithic columns. Some further examples of biomolecule separations are shown in the gradient mode with high-resolution MS (Orbitrap). The monolith prepared is primarily novel due to the pre-treatment process used. Previous publications from this group (refs [17] & [43]) contain extensive examples of peptide and protein separations including peak capacity calculations. Thus, the biomolecule work is of secondary value, except that the best material is shown to demonstrate very similar characteristics as the same stationary phase material synthesized with a conventional pre-treatment process from previous publications. However, the performance for small molecule separations is excellent and shows some behavior that is contrast to previously-published materials of similar origin. Specifically the retention-dependence of plate height seems to be almost non-existent, which is a major problem for most organic polymer monoliths used for analytical chromatography of small molecules. There are some details missing from technical aspects of the work that I believe need to be addressed before publication. This includes a full explanation of the correction of plate height data for the chromatographic comparison (including the strange behavior of the packed column in terms We changed the Highlights accordingly, emphasizing the performances for both small molecules and proteins Abstract Line 41: has "reproducibility" been assessed here? Unless a different user, location, or instrument was the focus of this part of the study, then it is "repeatability"-e.g. batch-to-batch, column-tocolumn, run-to-run, interday… etc. Please choose accordingly. Changed Line 42: again, "methacrylate-based" would be better here. Changed Line 64: "They are most commonly obtained by a single-step…" Changed Line 70: "…suitable for high efficiency separations of large biomolecules,…" Changed Line 73: "…preparation and, consequently, monolithic…" Lines 77-78: "…used to implement the sensitivity of large molecules…" does not make sense. Please revise. Changed Line 79: "The majority of recent developments have been…" Changed Line 84: "microwave-assisted polymerization process." Line 85: missing comma after "photo-induced" Changed Line 408: "…for high speed and high efficiency separations…" Changed Line 414: "inspection" Changed Lines 417-419: see later comments on retention-dependence. A useful reference for comparison is:
Journal of chromatography. A, Jan 3, 2016
Pirkle-type Whelk-O1 chiral stationary phase (CSP) was prepared on 2.6μm superficially porous par... more Pirkle-type Whelk-O1 chiral stationary phase (CSP) was prepared on 2.6μm superficially porous particles (SPPs). The chromatographic behavior of columns packed with this new CSP was compared with that of columns packed respectively with 1.8 and 2.5μm Whelk-O1 fully porous particles (FPPs). In the comparison, both thermodynamic and kinetic aspects were considered. Contrary to initial expectations, chiral columns packed with 2.6μm SPPs were quasi-comparable to those packed with 2.5μm FPPs, apparently due to larger contributions to band broadening from both eddy dispersion and, especially for the second eluted enantiomer, adsorption-desorption kinetics. These findings raise the question if SPPs, in spite of the undeniable advantages of their morphology to speed up mass transfer, are always the best choice for high-efficient ultrafast chiral separations. The last part of the work focuses on the use of short columns (10mm long) and very high flow rates to realize the separation of the ena...
Polymeric Composite Chiral Stationary Phases of Brush Type
The invention relates to novel polymeric composite chiral stationary phases of brush type suitabl... more The invention relates to novel polymeric composite chiral stationary phases of brush type suitable for the separation and/or the purification of enantiomers of organic and organometallic compounds. The invention further relates to methods for the preparation of such phases
Journal of Chromatography A, 2016
Title: Rationale behind the optimum efficiency of columns packed with new 1.9 μm fully porous par... more Title: Rationale behind the optimum efficiency of columns packed with new 1.9 μm fully porous particles of narrow particle size distribution.
Journal of chromatography. A, Jan 12, 2016
Fully porous particles of narrow particle size distribution (nPSD) are now commercially available... more 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...
Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2μm totally porous silica particles of narrow size distribution
Journal of chromatography. A, Jan 2, 2015
A new ultra-high performance teicoplanin-based stationary phase was prepared starting from sub-2μ... more A new ultra-high performance teicoplanin-based stationary phase was prepared starting from sub-2μm totally porous silica particles of narrow size distribution. Columns of different lengths were packed at high pressure and a deep and systematic evaluation of kinetic performance, in terms of van Deemter analysis, was performed under different elution conditions (HILIC, POM, RP and NP) by using both achiral and chiral probes. For the achiral probes, the efficiency of the columns at the minimum of the van Deemter curves were very high leading to some 278000, 270000, 262000 and 232000plates/m in hydrophilic interaction liquid chromatography (HILIC), polar organic mode (POM), normal phase (NP) and reversed phase (RP) respectively. The lowest plate height, Hmin=3.59μm (h(/)=1.89), was obtained under HILIC conditions at a flow rate of 1.4mL/min. Efficiency as high as 200000-250000plates/m (at the optimum flow rate) was obtained in the separation of the enantiomers of chiral probes under HIL...
International Journal of Molecular Sciences
The presence of stereogenic elements is a common feature in pharmaceutical compounds, and affordi... more The presence of stereogenic elements is a common feature in pharmaceutical compounds, and affording optically pure stereoisomers is a frequent issue in drug design. In this context, the study of the chiral molecular recognition mechanism fundamentally supports the understanding and optimization of chromatographic separations with chiral stationary phases. We investigated, with molecular docking, the interactions between the chiral HPLC selector Whelk-O1 and the stereoisomers of two bioactive compounds, the antiviral Nevirapine and the anticonvulsant Oxcarbazepine, both characterized by two stereolabile conformational enantiomers. The presence of fast-exchange enantiomers and the rate of the interconversion process were studied using low temperature enantioselective HPLC and VT-NMR with Whelk-O1 applied as chiral solvating agent. The values of the energetic barriers of interconversion indicate, for the single enantiomers of both compounds, half-lives sufficiently long enough to allow...
Cheminform, 2007
Dynamic HPLC on chiral stationary phases: A powerful tool for the investigation of stereomutation... more Dynamic HPLC on chiral stationary phases: A powerful tool for the investigation of stereomutation processes Dynamic HPLC on enantioselective stationary phases has become a well-established technique to investigate chiral molecules with internal motions that result in stereoinversion and occur on the time scale of the separation process. Kinetic parameters for the on-column interconversion phenomena can be extracted from experimental peak profiles by computer simulation or by direct calculation methods. The technique has been used in a wide range of temperatures and is complementary in scope to dynamic NMR spectroscopy.
Design and evaluation of hydrolytically stable bidentate urea-type stationary phases for hydrophilic interaction chromatography
Journal of Chromatography A, 2012
We have developed conceptually new stationary phases containing two bidentate urea-type functions... more 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.
Revealing the fine details of functionalized silica surfaces by solid-state NMR and adsorption isotherm measurements: the case of fluorinated stationary phases for liquid chromatography
Chemistry (Weinheim an der Bergstrasse, Germany), Jan 23, 2014
The structural and chromatographic characterization of two novel fluorinated mesoporous materials... more The structural and chromatographic characterization of two novel fluorinated mesoporous materials prepared by covalent reaction of 3-(pentafluorophenyl)propyldimethylchlorosilane and perfluorohexylethyltrichlorosilane with 2.5 μm fully porous silica particles is reported. The adsorbents were characterized by solid state (29)Si, (13)C, and (19)F NMR spectroscopy, low-temperature nitrogen adsorption, elemental analysis (C and F), and various chromatographic measurements, including the determination of adsorption isotherms. The structure and abundance of the different organic surface species, as well as the different silanol types, were determined. In particular, the degree of so-called horizontal polymerization, that is, Si-O-Si bridging parallel to the silica surface due to the reaction, under "quasi-dry" conditions, of trifunctional silanizing agents with the silica surface was quantified. Significant agreement was found between the information provided by solid-state NMR,...
ChemInform Abstract: Yonemitsu-Type Condensations Catalyzed by Proline and Eu(OTf) 3
ChemInform, 2015
Chirality, 1996
The copper(II) complexes of two new diastereomeric ligands, N2-(R)-and N2-(S)-2'-hydroxypropyl-(S... more The copper(II) complexes of two new diastereomeric ligands, N2-(R)-and N2-(S)-2'-hydroxypropyl-(S)-phenylalaninamide [ (R,S)-l and (S,S)-1 I, have been used as additives to the eluent in high-performance liquid chromatography (HPLC) reversed phase for the chiral separation of DNSamino acids. The aim was that of comparing the separation process obtained by the chiral eluent with that obtained by an analogous bonded stationary phase containing (S)-phenylalaninamide, previously studied [CSP-(S)-Phe-NH2]. The aftinity of the ternary complexes for the CIS column was determined by adsorption experiments in HPLC. It was shown that the two systems (chiral eluent, chiral stationary phase) work according to different mechanisms. Ternary complex formation in solution was studied by fluorescence spectroscopy. It was shown that chiral separation with the CuOI) complexes added to the eluent was determined by the relative affinities of the ternary complexes for the column-stationary phase rather than by their stabilities in solution. With CSP-(S)-Phe-NH2 the separation is accounted for by the relative stabilities of the ternary complexes, which depends mainly on the "allowed" geometry of the complex and on the steric repulsion of the amino acid side chain with the spacer. o 1996 Wiley-Liss, Inc.
Journal of Chromatography A, 2011
New monolithic HPLC columns were prepared by ␥-radiation-triggered polymerization of hexyl methac... more New monolithic HPLC columns were prepared by ␥-radiation-triggered polymerization of hexyl methacrylate and ethylene glycol dimethacrylate monomers in the presence of porogenic solvents. Polymerization was carried out directly within capillary (250-200 m I.D.) and nano (100-75 m I.D.) fused-silica tubes yielding highly efficient columns for cap(nano)-LC applications. The columns were applied in the complete separation of core (H2A, H2B, H3, and H4) and linker (H1) histones under gradient elution with UV and/or electrospray ionization (ESI) ion trap mass spectrometry (MS) detections. Large selectivity towards H1, H2A-1, H2A-2, H2B, H3-1, H3-2 and H4 histones and complete separation were obtained within 8 min time windows, using fast gradients and very high linear flow velocities, up to 11 mm/s for high throughput applications. The method developed was the basis of a simple and efficient protocol for the evaluation of post-translational modifications (PTMs) of histones from NCI-H460 human non-small-cell lung cancer (NSCLC) and HCT-116 human colorectal carcinoma cells. The study was extended to monitoring the level of histone acetylation after inhibition of Histone DeACetylase (HDAC) enzymes with suberoylanilide hydroxamic acid (SAHA), the first HDAC inhibitor approved by the FDA for cancer therapy. Attractive features of our cap(nano)-LC/MS approach are the short analysis time, the minute amount of sample required to complete the whole procedure and the stability of the polymethacrylate-based columns. A lab-made software package ClustMass was ad hoc developed and used to elaborate deconvoluted mass spectral data (aligning, averaging, clustering) and calculate the potency of HDAC inhibitors, expressed through a Relative half maximal Inhibitory Concentration parameter, namely R IC 50 and an averaged acetylation degree.
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Papers by Claudio Villani