Omar H. Ismail

Expanding the potential of chiral chromatography for high-throughput screening of large compound libraries by means of sub-2μm Whelk-O 1 stationary phase in supercritical fluid conditions

With the aim of exploring the potential of ultra-fast chiral chromatography for high-throughput analysis, the new sub-2 micron Whelk-O 1 chiral stationary phase (CSP) has been employed in supercritical fluid conditions to screen 129 racemates, mainly of pharmaceutical interest. By using a 5-cm long column (0.46cm internal diameter), a single co-solvent (MeOH) and a 7-min gradient elution, 85% of acidic and neutral analytes considered in this work have been successfully resolved, with resolution (Rs) larger than 2 in more than 65% of cases. Moreover, almost a half of basic samples that, for their own characteristics, are known to be difficult to separate on Whelk-O 1 CSP, have shown Rs greater than 0.3. The screening of the entire library could be accomplished in less than 24h (single run) with 63% of positive score. For well-resolved enantiomers (Rs roughly included between 1 and 3), we show that method transfer from gradient to isocratic conditions is straightforward. In many cases, isocratic ultra-fast separations (with analysis time smaller than 60s) have been achieved by simply employing, as isocratic mobile phase, the eluent composition at which the second enantiomer was eluted in gradient mode. By considering the extension and variety of the library in terms of chemico-physical and structural properties of compounds and numerousness, we believe that this work demonstrates the real potential of the technique for high-throughput enantioselective screening.

Ultra-fast high-efficiency enantioseparations by means of a teicoplanin-based chiral stationary phase made on sub-2 μm totally porous silica particles of narrow size distribution.

A new ultra-high performance teicoplanin-based stationary phase was prepared starting from sub-2 μm 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 278,000, 270,000, 262,000 and 232,000 plates/m in hydrophilic interaction liquid chromatography (HILIC), polar organic mode (POM), normal phase (NP) and reversed phase (RP) respectively. The lowest plate height, Hmin=3.59 μm (h(/)=1.89), was obtained under HILIC conditions at a flow rate of 1.4 mL/min. Efficiency as high as 200,000-250,000 plates/m (at the optimum flow rate) was obtained in the separation of the enantiomers of chiral probes under HILIC/POM conditions. N-protected amino acids, α-aryloxy acids, herbicides, anti-inflammatory agents were baseline separated on short (2-cm) and ultra-short (1-cm) columns, with analysis time in the order of 1 min. The enantiomers of N-BOC-d,l-methionine were successfully baseline separated in only 11s in HILIC mode. Several examples of fast and efficient resolutions in sub/supercritical fluid chromatography were also obtained for a range of chiral carboxylic acids.

Pirkle-type chiral stationary phase on core-shell and fully porous particles: Are superficially porous particles always the better choice toward ultrafast high-performance enantioseparations?

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 enantiomers of trans-stilbene oxide (TSO) in normal phase mode in less than 1s.

Rationale behind the optimum efficiency of columns packed with new 1.9μm fully porous particles of narrow particle size distribution

Columns packed with new commercially available 1.9 fully porous particles of narrow particle size distribution (nPSD) are characterized by extremely high efficiency. Under typical reversed phase conditions, these columns are able to generate very high number of theoretical plates (in the order of 300,000plates/m and more). In this paper, we investigate the origin of the high performance of these nPSD columns by performing a series of measurements that include, in addition to the traditional determination of the van Deemter curve, peak parking, pore blocking and inverse size exclusion experiments. Two nPSD columns (both 100×3.0mm) have been considered in this study: the first one, packed with particles of 80Å pore size, is commercially available. The second one is a prototype column packed with 1.9 fully porous particles of 120Å pore size. The main conclusion of our study is that these nPSD columns are characterized by extremely low eddy dispersion, while longitudinal diffusion and mass transfer kinetics are substantially equivalent to those of other fully porous particles of similar chemistry.

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.

Evaluation of two sub-2μm stationary phases, core-shell and totally porous monodisperse, in the second dimension of on-line comprehensive two dimensional liquid chromatography, a case study: Separation of milk peptides after expiration date

Milk is a rich source of bioactive peptides of great interest for their healthy properties. These peptides are usually encrypted in the sequences of proteins and are released after time dependent proteolysis as very complex hydrolysates. In order to separate and identify the bioactive sequences, we developed an on-line comprehensive two dimensional liquid chromatography approach using the high performance combined with the ultra high performance conditions. A microbore reversed phase (C18 silica, 5 μm) column was employed in first dimension, while, in second dimension, two different UHPLC columns, packed with C18 silica, were tested: a new column based on monodisperse sub-2 μm fully porous particles with high surface area (50 mm × 3.0 mm, 1.9 μm d.p., from Supelco), and a column based on sub-2 μm core-shell particles (50 mm × 3.0 mm, 1.7μm d.p, from Phenomenex(®)). Both set-ups were compared, showing high peak capacity values with respect to a high efficiency monodimensional method, maintaining the same analysis time. Satisfactory selectivity was obtained through the use of different pH between the two dimension, while a very fast continuous shifted gradient in second dimension ensured a good employment of the 2D separation space.

Future perspectives in high efficient and ultrafast chiral liquid chromatography through zwitterionic teicoplanin-based 2-μm superficially porous particles

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 2.0μm Superficially Porous Particles (SPPs). An already validated bonding protocol, which permits to achieve teicoplanin-based CSPs exhibiting zwitterionic behaviour, has been employed to prepare not only the 2.0μm version of the CSP but also two other analogous CSPs based, respectively, on 2.7μm SPPs and 1.9μm Fully Porous Particles (FPPs). The kinetic performance of these CSPs has been compared through the analysis of both van Deemter curves and kinetic plots by employing in-house packed columns of 4.6mm internal diameter and different lengths (20, 50 and 100mm). In particular on the columns packed with 2.0μm SPPs, extremely large efficiencies were observed for both achiral (>310,000 theoretical plates/meter, N/m; hr: 1.61) and chiral compounds (>290,000 N/m; hr: 1.72) in HILIC conditions. Thanks to their efficiency and enantioselectivity, these CSPs were successfully employed in ultrafast chiral separations. As an example, the enantiomers of haloxyfop were baseline resolved in about 3s, with a resolution higher than 2.0, (flow rate: 8mL/min) on a 2cm long column packed with the 2.0μm chiral SPPs.

New frontiers and cutting edge applications in ultra high performance liquid chromatography through latest generation superficially porous particles with particular emphasis to the field of chiral separations

AbstractAbout ten years after their introduction to the market (happened in 2006), the so-called second generation superficially porous particles (SPPs) have undoubtedly become the benchmark as well as, very often, the preferred choice for many applications in liquid chromatography (LC), when high efficiency and fast separations are required. This trend has interested practically all kinds of separations, with the only exception of chiral chromatography (at least so far). The technology of production of base SPPs is advanced, relatively simple and widely available. The deep investigation of mass transfer mechanisms under reversed-phase (RP) and normal-phase (NP) conditions for achiral separations has shown the advantages in the use of these particles over their fully porous counterparts. In addition, it has been demonstrated that SPPs are extremely suitable for the preparation of efficient packed beds through slurry packing techniques. However, the research in this field is in continual evolution. In this article, some of the most advanced concepts and modern applications based on the use of SPPs, embracing in particular ultrafast chiral chromatography and the design of SPPs with engineered pore structures or very reduced particle diameter, are revised. We describe modern trends in these fields and focus on those aspect where further innovation and research will be required. Graphical AbstractWord cloud of cutting edge applications of superficially porous particles in liquid chromatography

Unmatched kinetic performance in enantioselective supercritical fluid chromatography by combining latest generation Whelk-O1 chiral stationary phases with a low-dispersion in-house modified equipment

This proof-of-concept work investigates the ultimate kinetic limits reachable in chiral supercritical fluid chromatography (SFC) with modern columns and advanced technological solutions. A commercial equipment (Waters Acquity UPC2) has been in-house modified to minimize its overall extra-column variance through a series of technical adjustments including low-volume connecting tubings, reduced-volume flow cell, an in-house made external column oven, external low-dispersion injection system, and electronic temperature controller. Compared to the original (as-shipped) configuration, the extra-column variance on the low-dispersion equipment was reduced by more than 97%, from about 85 to slightly more than 2 μL2 (measured at 2.0 mL/min). This was mainly achieved thanks to the occurrence of fully developed turbulent regime with a proper selection of capillary i.d. at significantly smaller flow rates (1.5-4 mL/min; CO2/methanol 80/20, v/v; 35 °C; back pressure regulator (BPR), 105 bar) than in entry-1 configuration. Ultrahigh efficiency columns of different geometries in-house packed with latest generation sub-2 μm UHPC-FPP-Whelk-O1 Chiral Stationary Phase (CSP) have been employed under sub- and supercritical fluid conditions. By carefully modulating the length and the internal diameter of connecting tubings in the function of column geometry, state of the art efficiencies (estimated in roughly 300 000 theoretical plates/m with reduced HETP of roughly 1.85) have been obtained on 4.6 mm i.d. chiral columns. Remarkably, for 3.0 mm × 100 mm (i.d. × length) columns, the efficiency gain on the fully modified SFC system (compared to an instrumental configuration where only the standard injector was replaced by the low-dispersion one) was greater than 90% for compounds with a retention factor of 1 and as large as 25% for retention factors of 2.5.

Recent Developments in Chiral Separations by Supercritical Fluid Chromatography

Abstract The increasing interest for obtaining pure, single enantiomers of chiral organic compounds has greatly expanded the progress of new synthetic procedures, especially in the field of chiral drugs, agrochemicals, flavors, and natural products. A parallel trend has been observed in the development of stereoselective separation methods for the precise and accurate measurements of the enantiomeric purity of chiral compounds. The advent of chiral stationary phases for gas or liquid chromatographic applications in the last three decades changed the approach to determine enantiomeric purity on the analytical scale and to obtain sizeable amounts of pure enantiomers by preparative resolutions. In recent years, continuous efforts have been placed on faster analytical methods and on environment-friendly processes. Chromatographic processes using supercritical fluids, especially carbon dioxide, combine both aspects with success because of the extremely favorable properties of the eluents in terms of physicochemical parameters and of impact on the environment. An overview of the recent achievements in the field of supercritical fluid chromatography using packed column with chiral stationary phases will be given, with an emphasis on very recent studies on high-efficiency, ultrafast chiral separations.