Static vs. dynamic electrostatic repulsion reversed phase liquid chromatography: solutions for pharmaceutical and biopharmaceutical basic compounds

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

Advanced techniques in separation science for unraveling the complexity of natural substances: from small chiral molecules to large proteins

Natural substances are built up or transformed into each other in living organisms. Depending on the substance class, their tasks are varied and range from simple metabolism or energy production via cell components and building materials of the organism to complex control tasks. Regarding their functions, one can distinguish between primary and secondary natural products. Primary natural products include all the compounds needed for supporting and growing organism. These include, in particular, fats and biopolymers of carbohydrates and proteins. Secondary natural products are usually smaller molecules than primary products. They are formed for reasons often unknown and are subdivided into the broad classes of terpenes, aromatics, and alkaloids. Thus, the demand to study a range of increasingly complex samples of plant and animal origin, trying to solve and identify their chemical entities with greater confidence, has prompted researchers to develop innovative analytical approaches. The work showed in this thesis has the aim to investigate new analytical proficiencies to enrich separation science, especially by using cutting-edge chromatographic and detection techniques, and their applicability in secondary natural products chemistry and proteomics. The first section of this dissertation has been focused on terpenophenolic and polyphenolic secondary metabolites deriving from Cannabis sativa L. and Malus pumila Miller (cultivar Annurca) respectively. In the former case, the attention was focused on a fascinating feature of cannabinoids (C21 terpenophenolic compounds specific of Cannabis): the chirality. Chiral natural products are usually generated in optically pure form; however, occasionally both enantiomers are biosynthesized. It becomes evident, therefore, the worth of determination of the enantiomeric purity (i.e., enantiomeric excess, e.e.) in naturally occurring samples. The problem faced in the stereoselective analysis of Cannabis plant extracts was that vegetable extracts are highly enriched complex mixtures and often the minor enantiomers or the racemates, are not available as reference samples. In order to overcome this limitation, our group has previously developed a method for the identification of enantiomeric couples and accurate quantification of the minor enantiomer in trace analysis of natural products, named the ‘‘inverted chirality columns approach’’ (ICCA). The method allows determining the e.e. of (–)-Δ9-THC in medicinal marijuana. The e.e. was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not to be underestimated, and it is worth a systematic evaluation of bioactivity. In the latter case, an improved online comprehensive two‐dimensional liquid chromatography was developed. The use of a hydrophilic interaction chromatography column in the first dimension coupled to a trapping column modulation interface, and using a high retentive fully porous monodisperse reversed-phase column in the second dimension, enabled the simultaneous separation of multiple polyphenolic classes, as well as oligomeric procyanidins up to a degree of polymerization of 10. Moreover, thanks to hyphenation with an ion trap time-of-flight mass spectrometer, the tentative identification of 121 compounds has been possible. Thus, the presented system showed it could be a powerful analytical tool for the accurate profiling of complex polyphenolic-rich matrices. The topic of the second part of this dissertation concerns the study of analytical strategies applicable in the field of proteomics. In particular, the focus has been on the development of monolithic stationary phases to be used in capillary high performance liquid chromatography. Indeed polymethacrylate-based monolithic capillary columns, prepared by γ-radiation-induced polymerization were used, and the HPLC experimental conditions, such as nature of the organic modifiers, content of acid additive, and column temperature, were optimized for the separation of nine standard proteins with different pI, hydrophobicities and a wide range of molecular weights. The high working flow and high efficiency of these columns have allowed employing a longer column (up to 500 and 1000 mm), and thus, to reach a peak capacity value up to 1000. In order to probe the capacities of the monolithic columns, the range of molecular weights of the proteins examined was further expanded (from 3000 Da of glucagon to 150 kDa of monoclonal antibodies), showing in both cases excellent results. Precisely, monoclonal antibodies are the protagonists of the last chapter of this thesis, where a sample treatment procedure and an analytical method are developed in order to characterize the oxidized/reduced state of disulfide bridges. Indeed, upon incubation of antibodies with the reducing agent tris(2‐carboxyethyl)phosphine, three reduced isoforms of light chain can be identified at different reaction times: light chain, partially reduced light chain+2H, with one of two disulfide bridges opened in the constant or variable region, and entirely reduced light chain+4H

A Compendium of the Principal Stationary Phases Used in Hydrophilic Interaction Chromatography: Where Have We Arrived?

Hydrophilic interaction liquid chromatography (HILIC) today is a well-known and largely applied technique to analyse polar compounds such as pharmaceuticals, metabolites, proteins, peptides, amino acids, oligonucleotides, and carbohydrates. Due to the large number of stationary phases employed for HILIC applications, this review aims to help the reader in choosing a proper stationary phase, which often represents the critical point for the success of a separation. A great offer is present for achiral applications in contrast to the chiral phases developed for HILIC enantioseparations. In the last case, up-to-date solutions are presented

Ultra-high performance separation of basic compounds on reversed-phase columns packed with fully/superficially porous silica and hybrid particles by using ultraviolet transparent hydrophobic cationic additives

The use of the tetrabutylammonium additive was investigated in the ultra-high performance reversed-phase liquid chromatographic elution of basic molecules of pharmaceutical interest. When added to the mobile phase at low pH, the hydrophobic tetrabutylammonium cation interacts with the octadecyl chains and with the residual silanols, thus imparting a positive charge to the stationary phase, modulating retention and improving peak shape of protonated basic solutes. Two sources of additive were tested: a mixture of tetrabutylammonium hydroxide/trifluoroacetic acid and tetrabutylammonium hydrogen sulfate. Retention and peak shape of 11 basic pharmaceutical compounds were evaluated on commercially available ultra-fast columns packed with octadecyl stationary phases (Ascentis Express C18 2.0 mu m, Acquity BEH C18 1.7 mu m, Titan C18 1.9 mu m). All columns benefit from the use of additive, especially tetrabutylammonium hydrogen sulfate, providing very symmetric peaks with reasonable retention times. Focusing on the probe compounds amitriptyline and sertraline, efficiency and asymmetry values were investigated at increasing retention factor. The trend is very different to that obtained in reversed-phase conditions and the effect lies in the complex molecular interaction mechanisms based on hydrophobic and ion exchange interactions as well as electrostatic repulsion

Exploring the potential of zwitterionic teicoplanin-based CSPs by using macroporous superficially porous silica particles 2.7 μm 160Å and 3.4 μm 400Å

During the last years, the research in the enantioselective Ultra High Performance Chromatography (eUHPC) is going to push the limits of high efficient and ultrafast analyses. In this work, novel Chiral Stationary Phases (CSPs) were prepared by covalently bonding the teicoplanin selector (TE_A2-2) on Halo 2.7μm 160Å and 3.4μm 400Å Superficially Porous silica Particles (SPP). An innovative bonding protocol allowed to obtain a zwitterionic teicoplanin based CSP, which was used to produce the already known UHPC-FPP-Titan-Tzwitt CSP based on 1.9 μm 120Å Fully Porous monodispersed silica Particles (FPP) and UHPC-SPP-Halo90-Tzwitt CSP 2.0 μm [1-3]. Columns with an internal diameter of 4.6 mm and different lengths (50 and 100 mm) were packed with all CSPs and characterized in terms of permeability, efficiency and thermodynamic under HILIC conditions. The kinetic performance was evaluated through the use of van Deemter curves. The UHPC-SPP-Halo160-Tzwitt 2.7 μm column exhibited extremely high efficiencies on both achiral (>323,000 theoretical plates/meter, N/m; hr: 1.14) and chiral compounds (>240,000 N/m; hr: 1.53) under HILIC conditions, attesting the high potential of this CSP from the kinetic viewpoint. An extreme efficiency was recorded by using the HPC-SPP-Halo400-Tzwitt 3.4 μm column which allowed to achieve an efficiency of 280’000 N/m on naphthalene (hr: 1.05). Furthermore, taking into account the thermodynamic viewpoint, the UHPC-SPP-Halo160-Tzwitt 2.7 μm exhibited the highest resolution power (Rs/tr,2) thanks to its enantioselectivity values because of the higher selector density on the silica matrix. In conclusion, in this study we demonstrate the potential of the use of SPP macroporous silica particles in the UHPLC chiral field opening an interesting scenario in the chiral chromatography area. [1] O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, J. Chromatogr. A, 1427 (2016) 55–68. [2] O.H. Ismail, M. Antonelli, A. Ciogli, C. Villani, A. Cavazzini, M. Catani, F. Gasparrini, J. Chromatogr. A, 1520 (2017) 91–102 [3] O.H. Ismail, M. Antonelli, A. Ciogli, M. De Martino, M. Catani, C. Villani, A. Cavazzini, M. Ye, D.S. Bell, F. Gasparrini, J. Chromatogr. A., 1576 (2018), 42-50

Exploring the potential of zwitterionic teicoplanin-based CSPs by using macroporous superficially porous silica particles 2.7 µm 160Å and 3.4 µm 400Å

During the last years, the research in the enantioselective Ultra High Performance Chromatography (eUHPC) is going to push the limits of high efficient and ultrafast analyses. In this work, novel Chiral Stationary Phases (CSPs) were prepared by covalently bonding the teicoplanin selector (TE_A2-2) on Halo 2.7µm 160Å and 3.4µm 400Å Superficially Porous silica Particles (SPP). An innovative bonding protocol allowed to obtain a zwitterionic teicoplanin based CSP, which was used to produce the already known UHPC-FPP-Titan-Tzwitt CSP based on 1.9 µm 120Å Fully Porous monodispersed silica Particles (FPP) and UHPC-SPP-Halo90-Tzwitt CSP 2.0 µm [1-3]. Columns with an internal diameter of 4.6 mm and different lengths (50 and 100 mm) were packed with all CSPs and characterized in terms of permeability, efficiency and thermodynamic under HILIC conditions. The kinetic performance was evaluated through the use of van Deemter curves. The UHPC-SPP-Halo160-Tzwitt 2.7 µm column exhibited extremely high efficiencies on both achiral (>323,000 theoretical plates/meter, N/m; hr: 1.14) and chiral compounds (>240,000 N/m; hr: 1.53) under HILIC conditions, attesting the high potential of this CSP from the kinetic viewpoint. An extreme efficiency was recorded by using the HPC-SPP-Halo400-Tzwitt 3.4 µm column which allowed to achieve an efficiency of 280’000 N/m on naphthalene (hr: 1.05). Furthermore, taking into account the thermodynamic viewpoint, the UHPC-SPP-Halo160-Tzwitt 2.7 µm exhibited the highest resolution power (Rs/tr,2) thanks to its enantioselectivity values because of the higher selector density on the silica matrix. In conclusion, in this study we demonstrate the potential of the use of SPP macroporous silica particles in the UHPLC chiral field opening an interesting scenario in the chiral chromatography area. [1] O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, J. Chromatogr. A, 1427 (2016) 55–68. [2] O.H. Ismail, M. Antonelli, A. Ciogli, C. Villani, A. Cavazzini, M. Catani, F. Gasparrini, J. Chromatogr. A, 1520 (2017) 91–102 [3] O.H. Ismail, M. Antonelli, A. Ciogli, M. De Martino, M. Catani, C. Villani, A. Cavazzini, M. Ye, D.S. Bell, F. Gasparrini, J. Chromatogr. A., 1576 (2018), 42-50

KINETIC AND THERMODYNAMIC INSIGHTS INTO NOVEL ZWITTERIONIC MACROCYCLIC GLYCOPEPTIDE CHIRAL STATIONARY PHASES FOR HIGH-EFFICIENCY, ULTRAFAST ENANTIOSEPARATIONS UNDER DIFFERENT ELUTION CONDITIONS

In this work, a novel type of chiral stationary phase (CSP) has been prepared by covalently bonding teicoplanin chiral selector to monodisperse 1.9 µm fully porous particles (FPPs) and - for the first time in chiral chromatography - to 2.0 µm superficially porous particles (SPPs) by means of a bonding protocol that allows to obtain the zwitterionic version of the macrocyclic glycopeptide CSP. Columns in-house packed with these CSPs have been characterized from both a kinetic and a thermodynamic viewpoint towards the enantioseparation of small molecules and amino-acid derivatives comparing chromatographic behavior in RP-LC and HILIC mode. Kinetic performance has been experimentally evaluated through the analysis of van Deemter curves while thermodynamic properties and interactions of molecules with the stationary phase have been investigated by calculating adsorption isotherms (Inverse Method was used for this purpose). Results of this study revealed that the column packed with 2.0 µm SPPs shows outstanding kinetic performance (300’000 plates/m or more) [2]. In general, however, higher efficiencies and flatter van Deemter curves can be obtained when columns are operated in HILIC mode while better resolution and higher selectivity values are typical of RP-LC. This is due to the interplay of different factors including a different retention mechanism, a different contribution of adsorption-desorption kinetics and the absence of surface diffusion in HILIC conditions. These findings suggest that HILIC mode is most suitable for ultrafast enantioseparations while RP-LC should be preferred for the resolution of complex samples containing different pairs of enantiomers and for the challenging separation of racemic mixtures usually poorly resolved. Finally, zwitterionic macrocyclic glycopeptide CSPs have been also excellently used for an application of practical relevance in pharmaceutical analysis that is the simultaneous separation of chiral active pharmaceutical ingredients (APIs) from their counterions in a single chromatographic run. This is possible thanks to the peculiar properties of the zwitterionic version of teicoplanin CSPs to retain both cations and anions, differently from the traditional version of the CSP, where the latter ones are almost unretained due to Donnan repulsion effect

Preparation of a high-density vinyl silica gel to anchor cysteine via photo-click reaction and its applications in hydrophilic interaction chromatography

: Modification of surface silanols is a topic of interest in the preparation of organo-functionalized silica particles. Herein, two novel contributions, mainly focused on separation science, were presented: i) the horizontal polymerization on silica surface by using the vinyl-triacetoxy silane and ii) a preparation of cysteine-based stationary phase via photo-click thiol-ene coupling. In the first derivatization step, the vinyl-triacetoxy silane was employed instead of conventional trichloro vinyl one. The one-step synthetic procedure needed imidazole as an activating agent in addition to the silica hydration. Modified silica particles offer a high loading of vinyl fragments and an extensive passivation of silanols such as to not require a subsequent end-capping procedure. The structural morphology of media was deeper characterized by combining infrared spectroscopy, solid-state nuclear magnetic resonance, and elemental analysis. A first application, the photo-click cysteine-based material was prepared by photo-click reaction and the stationary phase was employed in the separation of some conventional targets by hydrophilic interaction chromatography