The way to ultrafast, high-throughput enantioseparations of bioactive compounds in liquid and supercritical fluid chromatography

Until less than 10 years ago, chiral separations were carried out with columns packed with 5 or 3 μm fully porous particles (FPPs). Times to resolve enantiomeric mixtures were easily larger than 30 min, or so. Pushed especially by stringent requirements from medicinal and pharmaceutical industries, during the last years the field of chiral separations by liquid chromatography has undergone what can be defined a “true revolution”. With the purpose of developing ever faster and efficient method of separations, indeed, very efficient particle formats, such as superficially porous particles (SPPs) or sub-2 μm FPPs, have been functionalized with chiral selectors and employed in ultrafast applications. Thanks to the use of short column (1–2 cm long), packed with these extremely efficient chiral stationary phases (CSPs), operated at very high flow rates (5–8 mL/min), resolution of racemates could be accomplished in very short time, in many cases less than 1 s in normal-, reversed-phase and HILIC conditions. These CSPs have been found to be particularly promising also to carry out high-throughput separations under supercritical fluid chromatography (SFC) conditions. The most important results that have been recently achieved in terms of ultrafast, high-throughput enantioseparations both in liquid and supercritical fluid chromatography with particular attention to the very important field of bioactive chiral compounds will be reviewed in this manuscript. Attention will be focused not only on the latest introduced CSPs and their applications, but also on instrumental modifications which are required in some cases in order to fully exploit the intrinsic potential of new generation chiral columns

The Role of Adsorption and pH of the Mobile Phase on the Chromatographic Behavior of a Therapeutic Peptide

The impact of two different stationary phases and ion-pair reagents on the retention behavior of glucagon, a therapeutic peptide consisting of 29 amino acidic residues, has been investigated under reversed-phase elution conditions. Retention of glucagon was investigated under isocratic conditions by varying the fraction of the organic modifier in the range of 28–38% (v/v). The two stationary phases have been characterized in terms of excess adsorption isotherms to understand the preferential adsorption of eluent components on them. Results suggest that the ligand characteristics and the pH of the mobile phase play a pivotal role on retention

Investigation of Mass Transfer Phenomena and Thermodynamic Properties of new Generation Porous Particles for High-Efficient Separations in Liquid Chromatography Through Experimental and Theoretical Approaches

In the chromatographic field, the continuous need of higher column efficiency and resolution and faster analysis has led to the development of stationary phases made of ever smaller porous particles and to the use of Ultra-High Performance Chromatographic systems. From a theoretical view point, on the one hand, kinetic factors (diffusion, eddy dispersion, mass transfer resistance, finite rate of adsorption/desorption process) directly affect the efficiency of a chromatographic separation and on the other, thermodynamic factors (adsorption equilibria) have an impact on retention and selectivity. It is clear that in order to achieve selective, ultra-fast and high efficient separations it is of fundamental importance to study both kinetic and thermodynamic contributions. The detailed evaluation of the different sources of band broadening of kinetic origin can be obtained through the coupling of proper models of diffusion (parallel model or effective medium theories) in porous media and experimental measurements (peak parking and van Deemter curves). Conversely, the application of the so-called inverse method and the perturbation method allows for the estimation of thermodynamic parameters (saturation capacity, binding constants) through the construction of adsorption isotherm of both analytes and mobile phase components on a stationary phase. The aim of this thesis has been the complete characterization of porous materials being both hydrophobic and chiral in terms of kinetic and thermodynamic values. In the first part of this work, mass transfer kinetics of columns designed for ultrafast high performance separations packed with both sub-2 μm and sub-3 μm fully porous particles and the second generation of core-shell particles with different functionalization have been investigated. More in detail, this work has been focused on the direct comparison between SPPs and FPPs designed for both achiral (C 18 ) and chiral separations and on a review of the most recent applications of these materials, especially in the field of high-throughput ultrafast enantioseparations in liquid (LC) and supercritical fluid chromatography (SFC).In the second part, the study of thermodynamic properties of porous materials has been carried out on different stationary phases: chiral (Teicoplanin, Whelk-O1 and Polysaccharide) designed for ultrafast chromatography, achiral (C 8 , C 18 ) designed for preparative chromatography and a resin designed for affinity chromatography. The acquired information are needed to possibly correlate chemico-physical properties of porous particles (specific loading, particle type and geometry, surface area, etc.) to kinetic (adsorption-desorption kinetics, column efficiency, etc.) and thermodynamic quantities (binding constants, saturation capacities, etc.). Moreover, thermodynamic studies may be useful for the prediction of the adsorption behavior of products of pharmaceutical interest, leading to the investigation of the feasibility of purification process through preparative and affinity chromatography. This thesis is the result of two periods of research spent at ETH Zürich (Switzerland) and at University of Pécs (Hungary) and various national and international collaborations with Prof. Gasparrini (University of Rome, “La Sapienza”, Italy) and Prof. Chankvetadze (University of Tbilisi, Georgia) for chiral chromatography, Prof. Felinger (University of Pécs, Hungary) for SFC, Prof. Morbidelli (ETH Zürich, Switzerland) and Fresenius Kabi iPSUM (Villadose, Rovigo, Italy) for affinity and preparative chromatography.In campo cromatografico la richiesta continua di elevate efficienze, risoluzione e analisi veloci ha portato allo sviluppo di fasi stazionarie costituite da particelle di diametro sempre più ridotto e all’utilizzo di strumenti cromatografici ultra performanti. Da un punto di vista teorico, da un lato, i fattori cinetici (diffusione, dispersione eddy, resistenza al trasferimento di massa, velocità finita del processo di dsorbimento/desorbimento) influenzano direttamente l’efficienza di una separazione cromatografica e dall’altro, fattori termodinamici (equilibri di adsorbimento) hanno un effetto su ritenzione e selettività. Per ottenere separazioni selettive, ultra veloci e altamente efficienti è quindi fondamentale studiare sia i contributi cinetici che termodinamici. La valutazione dettagliata delle differenti sorgenti di allargamento di banda di origine cinetica può essere effettuata attraverso l’accoppiamento di opportuni modelli di diffusione nei mezzi porosi (modello parallelo o effective medium theories) e misure sperimentali (peak parking e curve di van Deemter). Al contrario, l’applicazione del metodo inverso e del metodo delle perturbazioni permette la stima di parametri termodinamici (capacità di saturazione, costanti di binding) attraverso lo studio di isoterme di adsorbimento sia di analiti che di componenti della fase mobile adsorbiti sulla fase stazionaria. Lo scopo di questo lavoro di tesi è stato la completa caratterizzazione di materiali porosi, sia di tipo idrofobico che chiarle, in termini di valori cinetici e termodinamici. Nella prima parte del lavoro sono state indagate le cinetiche di trasferimento di massa di colonne progettate per separazioni ultra veloci e ad alta efficienza impaccate con particelle totalmente porose (FPP) con diametro sub-2 μ m e sub-3 μ m e con la seconda generazione di particelle superficialmente porose (SPP) con diversa funzionalizzazione. Più precisamente, questo lavoro è stato focalizzato nel confronto diretto tra SPP e FPP utilizzate per separazioni achirali e chirali e su una review riguardante le applicazioni più recenti per questi materiali con un maggiore dettaglio nel campo di separazioni enantiomeriche ultra veloci ad alta produttività ed efficienza in cromatografia liquida (LC) e a fluido supercritico (SFC). Nella seconda parte della tesi lo studio delle proprietà termodinamiche di materiali porosi è stato effettuato su diverse fasi stazionarie: chirali (teicoplanina, Whelk-O1 e polisaccaridiche) progettate per cromatografia ultra veloce, achirali ( C 8 e C 18 ) progettate per cromatografia preparativa e una resina progettata per cromatografia di affinità.Le informazioni ottenute sono necessarie per poter correlare le proprietà chimico-fisiche delle particelle porose (quantità di selettore presente, tipo e geometria delle particelle, area superficiale, ecc.) alle quantità cinetiche (cinetiche di adsorbimento-desorbimento, efficienza di colonna, ecc.) e termodinamiche (costanti di binding, capacità di saturazione, ecc.). Inoltre studi termodinamici potrebbero essere utili per predire il comportamento di adsorbimento di prodotti di interesse farmaceutico, rendendo così possibile lo studio dell’attuabilità del processo di purificazione attraverso la cromatografia preparativa e di affinità. Questa tesi è il risultato di due periodi di ricerca effettuati in istituzioni estere svolti presso l’ETH di Zurigo (Svizzera) e presso l’Università di Pécs (Ungheria) e di numerose collaborazioni nazionali e internazionali con il Prof. Gasparrini (Università di Roma, “La Sapienza”, Italia) e il Prof. Chankvetadze (Università di Tbilisi, Georgia) per quanto concerne la cromatografia chirale, il Prof. Felinger (Università di Pécs, Ungheria) per cromatografia a fluido supercritico, il Prof. Morbidelli (ETH di Zurigo, Svizzera) e Fresenius Kabi iPSUM (Villadose, Rovigo, Italia) per cromatografia di affinità e preparativa

Separation techniques

Chapter 3 contains the information related to the use of the most suitable separation techniques used in metabolomics research, considering also sample preparation as an important sample pre-treatment step fundamental to perform the analytical separation. General principles of the most used extraction methods and analytical separation techniques are described. In this context, the main focus will be on column chromatography, briefly discussing theoretical principles and practical issues, along with instrumental features. A section on multidimensional chromatography is also present

Modern trends in downstream processing of biotherapeutics through continuous chromatography: The potential of Multicolumn Countercurrent Solvent Gradient Purification

Single-column (batch) preparative chromatography is the technique of choice for purification of biotherapeutics but it is often characterized by an intrinsic limitation in terms of yield-purity trade-off, especially for separations containing a larger number of product-related impurities. This drawback can be alleviated by employing multicolumn continuous chromatography. Among the different methods working in continuous mode, in this paper we will focus in particular on Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) which has been specifically designed for challenging separations of target biomolecules from their product-related impurities. The improvements come from the automatic internal recycling of the impure fractions inside the chromatographic system, which results in an increased yield without compromising the purity of the pool. In this article, steps of the manufacturing process of biopharmaceuticals will be described, as well as the advantages of continuous chromatography over batch processes, by particularly focusing on MCSGP