Superficially Porous Particles Research Articles

Understanding the Transition from High-Selective to High-Efficient Chiral Separations by Changing the Organic Modifier with Zwitterionic-Teicoplanin Chiral Stationary Phase.

The retention behavior of small molecules and N-protected amino acids on a zwitterionic teicoplanin chiral stationary phase (CSP), prepared on superficially porous particles (SPPs) of 2.0 μm particle diameter, has shown that efficiency and enantioselectivity, and so enantioresolution, dramatically change depending on the employed organic modifier. In particular, it was found that while methanol permits the boost of enantioselectivity and resolution of the amino acids, at the cost of efficiency, acetonitrile allows for the ability to reach extraordinary efficiency even at high flow rates (with reduced plate height <2 and up to 300,000 plates/m at the optimum flow rate). To understand these features, an approach based on the investigation of mass transfer through the CSP, the estimation of the binding constants of amino acids on the CSP, and the assessment of compositional properties of the interfacial region between bulk mobile phase and solid surface has been adopted.

Evaluation of Kinetic Performance of Reversed-Phase Columns for Protein Separations by Gradient Kinetic Plots

The increasing importance of protein biopharmaceuticals has triggered the development of new, highly efficient stationary phases for reversed-phase liquid chromatography (LC) of proteins. They typically have C4 ligands or phenyl surfaces for weak hydrophobic interactions and are based on various morphologies, such as silica monolith, sub-2-µm fully porous particles (FPPs), or superficially porous particles (SPPs). Selection of the best column based on physical parameters provided by vendors may sometimes be unequivocal. Simple performance evaluation tools, such as the gradient kinetic plot methodology reported by Desmet and co-workers, can help to shed light on this issue and allow for a more differentiated view on column performance. This article compares the performance of wide-pore silica monolithic, sub-2-µm FPP, and SPP columns. It also addresses the question of whether 1000 Å or 400 Å SPP columns are more suitable for reversed-phase LC-type protein separations and presents a kinetic performance comparison of a number of wide-pore core–shell particle columns.

Boosting the enantioresolution of zwitterionic-teicoplanin chiral stationary phases by moving to wide-pore core-shell particles

A novel zwitterionic-teicoplanin chiral stationary phase (CSP), based on superficially porous particles (SPPs) of 2.7 µm particle diameter and 160 Å pore size, has been prepared and evaluated towards the enantioseparation of important classes of compounds, including chiral drugs, pesticides, and N-derivatized amino acids. The comparison with two analogous CSPs prepared on SPPs with 2.7 and 2.0 µm particle diameter and 90 Å pore size has revealed that the use of large-pore particles allows to dramatically improve both the enantioselectivity and the resolution-per-analysis-time, at the point that the column prepared with the new CSP outperformed the one packed with the finest particles. On the novel wide-pore CSP, the separation of fifteen racemates of pratical importance was significantly improved in terms of both enantioselectivity and resolution-per-analysis time-compared to the CSPs based on SPPs with smaller pores (90 Å). Such a CSP would be suitable for very fast enantioseparations allowing the saving of solvent for greener high-efficiency/high-throughput applications.

Developing a Fast Purification Method for a Natural Product with a Preparative LC Column Packed with Superficially Porous Particles

A fast and simple purification method for withaferin A (WFA) in ashwagandha extract was successfully developed using a preparative column packed with superficially porous particles (SPPs). The SPP column provided better resolution and a 45% decrease in run time over a traditional preparative column packed with totally porous particles. The performance benefits of SPP preparative columns, especially at high flow rates, align well with the needs of pharmaceutical discovery laboratories and other high-throughput environments.

Mass transfer kinetics on modern Whelk-O1 chiral stationary phases made on fully- and superficially-porous particles

In this work, a detailed study of mass transfer properties of trans-stilbene oxide (TSO) enantiomers on two Whelk-O1 chiral stationary phases (CSPs) has been performed. The CSPs were prepared by using both fully-porous silica particles of 2.5 μm particle diameter and superficially-porous ones of 2.6 μm particle diameter as base materials. By combining stop-flow and dynamic measurements in normal-phase conditions, the different contributions to mass transfer have been estimated. The study of intraparticle diffusion has revealed that the adsorption of both enantiomers is localized (i.e., characterized by absence of surface diffusion). The determination of thermodynamic binding constants (measured through adsorption isotherms) supports this finding.

Enantioseparation performance of superficially porous particle vancomycin-based chiral stationary phases in supercritical fluid chromatography and high performance liquid chromatography; applicability for psychoactive substances

Novel psychoactive substances (NPS) are synthetic compounds that have been designed to produce the physiological and psychological effects of known recreational drugs, while circumventing current drug control laws and scheduling guidelines. Such “designer drugs” pose problems in detection and prevention of use, and they are no less dangerous than known controlled substances. Among the various classes of NPS, many are chiral. As they are synthetic products, most are racemates. Not unexpectedly, there is limited information about different the pharmacological and toxicological properties of their pure enantiomers. Hence, fast and reliable enantioselective methods are of great interest.In this work, superficially porous particle (SPP) vancomycin-based chiral stationary phases were used for development of fast enantioselective separation methods for various classes of NPS in supercritical fluid chromatography and liquid chromatography. The NPS tested included pyrovalerones, benzofurans, phenidines and phenidates. The effect of mobile phase composition on the retention and resolution of NPS in supercritical fluid chromatography was examined. The amount as well as the ratios of additives used is crucial for enantioseparation efficiency. Results showed the high enantioselective potential of vancomycin-based columns in both chromatographic techniques; 88% of NPS tested were enantioseparated in supercritical fluid chromatography and 69% of NPS tested were enantioseparated in liquid chromatography. Moreover, under optimized conditions, simultaneous enantioseparations of some NPS were achieved, which indicates great suitability of vancomycin-based columns for this purpose. The proposed methods can serve as guides for method development and for enantioseparation of further upcoming NPS.

Development of a rapid reversed-phase liquid chromatographic method for total free thiol quantitation in protein therapeutics

Free thiols, or unpaired cysteines, are important product quality attributes in the therapeutic proteins due to their potential impact on the protein structure, bioactivity and stability. While many free thiol quantitation methods were developed for specific therapeutic formats, an unmet need still exists for a multiproduct, high-throughput method for free thiol quantitation. In this study, a workflow was established that combines N-cyclohexylmaleimide (NcHM) derivatization and high-throughput reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) separation with superficially porous particle (SPP) column for quantitating total free thiols in monoclonal antibodies (mAbs), fragment antigen-binding (Fab), and bispecific antibodies (BsAbs). The NcHM derivatization increases the hydrophobicity of the free thiol variants and allows the further separation and quantitation with RP-UHPLC. A thorough evaluation of sample preparation, column selection, chromatographic condition and LC–MS peak identification was performed to optimize and characterize the method outputs. Method optimization resulted in a 42-minute total analysis time. Method qualification demonstrated suitable accuracy, precision, linearity, specificity and robustness. This high-throughput method is not only used for quantitation of total free thiols for both in-process testing and drug substance/drug product batch testing, but also for provide the positional distribution of the free thiols in the protein.

High–throughput enantioseparation of Nα–fluorenylmethoxycarbonyl proteinogenic amino acids through fast chiral chromatography on zwitterionic-teicoplanin stationary phases

In this study, 31 racemates of Nα-FMOC (fluorenylmethoxycarbonyl) amino acids (AAs) with different chemico-physical characteristics (neutral nonpolar, neutral polar, acidic and basic) have been successfully resolved in fast enantioselective chromatography on recently-developed zwitterionic-teicoplanin chiral stationary phases (CSPs). The CSPs were prepared by covalently bonding the teicoplanin selector on fully-porous particles of narrow dispersion particle-size distribution (particle diameter 1.9 µm) and superficially-porous particles (2.0 µm). Both the zwitterionic-teicoplanin CSPs have proved to be ideal media for the separation of this important class of compounds. In particular, the zwitterionic CSP prepared on superficially-porous particles exhibited superior enantioselectivity and resolution, compared to that made of fully porous particles, in virtue of more favorable thermodynamics. The zwitterionic nature of these CSPs allowed avoiding the annoying effect of Donnan's exclusion of enantiomers from the stationary phase. This effect, on the opposite, was frequently observed on a commercial teicoplanin CSP (Teicoshell) employed for comparative purposes. Noticeably, on the zwitterionic-teicoplanin CSPs, by using either acetonitrile- or methanol-rich mobile phases (MPs), it was possible to favor speed over enantioresolution and vice versa. This work gives further replies to the request for rapid determination of enantiomeric excess of Nα-FMOC proteinogenic (and non–proteinogenic) AAs, typically used as preferred chiral synthons in the solid-phase synthesis of therapeutic peptides.

Enantioselective potential of teicoplanin- and vancomycin-based superficially porous particles-packed columns for supercritical fluid chromatography

Application of the superficially porous particles (SPPs) grafted with chiral selectors can substantially improve resolution in chromatographic techniques. In this work, we carried out a deeper study on supercritical fluid chromatography systems with 2.7 µm SPPs bonded with teicoplanin and vancomycin. Fast separations of the majority of enantiomers of phytoalexins, substituted tryptophans, and ketamine derivatives, as representatives of important biologically active and structurally diverse chiral compounds have been achieved. The chromatographic behavior of the structurally different analytes served to characterize these separation systems. The influence of separation conditions, namely mobile phase composition, i.e. type of co-solvent and additive on retention, enantioselective resolution and enantioselectivity was examined. The success rate of baseline and partial separations in individual groups of compounds differed with the chiral stationary phase and also with mobile phase composition. The best, baseline separations for the phytoalexins were achieved on the TeicoShell column using methanol as a co-solvent and trifluoroacetic acid as an additive if used. Mostly partial separations were achieved on the vancomycin-based column for all groups of analytes. Complementary separation behavior of these CSPs was confirmed for the majority of the chiral compounds examined in this work.

Synthesis and optimization of SiO2@SiO2 core-shell microspheres by an improved polymerization-induced colloid aggregation method for fast separation of small solutes and proteins

Recently, superficially porous particles (SPPs) have been intensively studied and employed for highly efficient and fast separations. In this paper, the SiO2@SiO2 SPPs were synthesized by an improved polymerization-induced colloid aggregation (PICA) method using urea-formaldehyde polymer as the template. The agglomeration of silica core during modification with ureidopropyltrimethoxysilane (UPS) can not only be avoided by reflux in neutral ethanol solution, but also the secondary nucleation of the colloidal silica sol can be inhibited via optimizing the reaction conditions including pH, temperature, colloidal silica sol concentration and the reaction time. The shell thickness and pore size of SPPs can be controlled successfully by adjusting the weight ratio of silica core/colloidal silica sol and the particle size of colloidal silica sol, respectively. The SPP-C18 columns packed using octadecyltrichlorosilane (ODS) modified SPPs with different pore sizes were employed to separate small solutes and proteins. The baseline separations of 6 kinds of alkyl benzenes and 5 kinds of aromatic alcohol homologues were achieved within 4 min by the SPP-C18 column with 8 nm pore size. Compared with the commercial BEH-C18 column, more than 50,000/m of the plate number of propylbenzene was obtained, and the former provided higher column efficiency to separate small solutes than the latter. Meanwhile, 6 kinds of proteins were also separated completely within 2 min using the SPP-C18 column with 40 nm pore size. In addition, the SPP-C18 capillary column was applied to separate and identify the BSA/HeLa/mouse liver digests with capillary LC-MSMS, respectively. The results indicate that more proteins and peptides can be identified using SPP-C18 capillary column compared with commercial silica-C18 capillary column. The result demonstrates that the prepared SPP-C18 column provides higher column efficiency and the SPPs synthesized with the improved PICA method shows a great potential application for the fast separation of small solutes and proteins.

Fast super/subcritical fluid chromatographic enantioseparations on superficially porous particles bonded with broad selectivity chiral selectors relative to fully porous particles

Superficially porous particles (SPPs) have shown advantages in enantiomeric separations in HPLC by conserving selectivity while providing higher efficiency separations with significantly reduced analysis times. The question arises as to whether the same advantages can be found to the same extent in super/subcritical fluid chromatography. In this work, the low viscosity advantage of carbon dioxide/MeOH mixtures is coupled with high-efficiency 2.7 μm superficially porous particles for enantiomeric separations. Given the fact that the viscosity of the mobile phase is typically ten times lower than liquid mobile phases it is possible to use flow rates as high as 14 mL/min on 5 cm packed columns. Superficially porous particles (SPPs) were grafted with teicoplanin (TeicoShell), a chemically modified macrocyclic glycopeptide (NicoShell), vancomycin (VancoShell), and isopropyl derivatized cyclofructan-6 (LarihcShell-P). One hundred chiral analytes were separated in a very short time frame, as little as 0.2 min (13 s). Even shorter separations can be obtained with advances in SFC instrumentation. The LarihcShell-P is the only chiral crown ether-based selector which showed high selectivity for primary amines. The Teicoshell column offered unique separations for acidic and neutral analytes. The NicoShell and the VancoShell were useful in separating amine (secondary and tertiary) containing pharmaceutical drugs and controlled substances. By chemically modifying a macrocyclic glycopeptide (NicoShell) we report the first enantiomeric separation of nicotine under SFC conditions within 3 min with a resolution of >3. Additionally, van Deemter plots are constructed comparing the fully porous particles and superficially porous particles bonded with the same chiral selectors. In toto the SPP advantages also were found for SFC. However instrumental shortcomings involving extra column effects and pressure limitations need to be addressed by instrument manufacturers to realize the full advantages of SPPs and other smaller particle supports.

Evaluation of superficially porous particle based zwitterionic chiral ion exchangers against fully porous particle benchmarks for enantioselective ultra-high performance liquid chromatography

Zwitterionic chiral ion-exchange selectors (ZWIX) obtained by conjugation of quinine and 2-aminocyclohexanesulfonic acid via a carbamate bond were immobilized on three different silica particle types, viz. 120 Å 3 μm fully porous particles (FPP), 200 Å 3 μm FPP and 160 Å 2.7 μm superficially porous particles (SPP). Selector densities were determined by elemental analysis and the porosities of packed columns measured by inverse size exclusion chromatography with polystyrene standards. Liquid chromatographic tests with a set of chiral zwitterionic, acidic and basic analytes showed that the surface chemistry was successfully transferred to the distinct particle morphologies. The chromatographic performance of the three columns was evaluated by acquiring van Deemter curves. The results showed that the column packed with the SPP particles gives the best performance and kinetic plots further demonstrated that they represent the most favorable compromise in terms of speed, efficiency and pressure drop. Sub-minute separations could be accomplished at much lower pressure drop on the core-shell column, e.g. 2-amino-2-phenylbutyric acid was baseline separated in less than 15 s on a 5 cm long column. The Maxwell effective medium theory with second order approximation was applied to calculate effective diffusion in the mesoporous zones of SPP and FPP, which allowed eventually to deconvolute the individual peak dispersion contributions (ha, hb, hc,m, hc,s, hc,ads). The efficiency gain of the 160 Å SPP column compared to the 120 Å FPP (benchmark) column was mainly due to lower eddies (ha), smaller c-term accounting for slow adsorption-desorption kinetics in enantioselective chromatography (hc,ads), and also due to lower stationary mass transfer resistance (hc,s). Enhanced effective diffusion (Deff) in the SPP column contributed to a lower longitudinal diffusion (hb). In contrast, the mobile phase mass transfer coefficient was similar in the two columns leading to comparable hc,m contributions. This study discloses some options for improvement of the efficiency of ZWIX-type chiral columns such as replacing narrow pore (120 Å) by wide pore (200 Å) particles, substituting FPP by SPP and reducing the selector density on the surface.

Measuring porosities of chromatographic columns utilizing a mass-based total pore-blocking method: Superficially porous particles and pore-blocking critical pressure mechanism

Experimentally determined total, interstitial and intraparticle porosity values are necessary to equate theory, simulation and experimental column performance. This paper reports a study of a mass-based technique for determining total, interstitial and intraparticle porosity measurements based on the total pore-blocking (TPB) method. Commercially available superficially porous particle (SPP) columns, in a variety of small-pore and wide-pore materials, with both hydrophobic and hydrophilic surfaces, are utilized as samples. The results are compared with previously determined literature values for a number of columns and contrasted with HPLC-based elution methods. This method uses only a high-precision balance and an HPLC pump.A simple theoretical analysis of the TPB method using the Young-Laplace equation shows the pressure bounds and flow rate constraints of the method which ensure pore blocking stability. The results suggest that particles with small-pore diameters can be analyzed over a range of solvent clearing pressures and flow rates. However, wide-pore materials, typically with pore diameters in excess of 400 Å, have very low critical pressures and are difficult to determine without losing the pore blocking component. Small mass differences between clearing solvents are shown to present a challenge for measuring the interstitial volume.

Cyclofructans as Chiral Selectors: An Overview.

Cyclofructans are cyclic oligosaccharides made of β-2,1-linked fructofuranose units. They have been utilized as chiral selectors, usually after derivatization, with high-performance liquid chromatography (HPLC), gas chromatography (GC), capillary electrophoresis (CE), and supercritical fluid chromatography (SFC). The focus herein will be directed to their development and applications as chiral selectors in various chiral separation techniques. Discussion of their use in hydrophilic liquid interaction chromatography (HILIC) will be limited. Their use in liquid chromatography, especially their improvements with the use of superficially porous particles (SPPs) will be emphasized. Method parameters and future directions are also discussed.

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.

On the effect of chiral selector loading and mobile phase composition on adsorption properties of latest generation fully- and superficially-porous Whelk-O1 particles for high-efficient ultrafast enantioseparations

The adsorption isotherms of trans-stilbene oxide (TSO) enantiomers have been measured under a variety of normal phase (NP) mobile phases (MPs) on three Whelk-O1 chiral stationary phases (CSPs), prepared respectively on 1.8 μm and 2.5 μm fully porous particles (FPPs) and 2.6 μm superficially porous particles (SPPs). Specific loading of chiral selector (moles per square meter) of the two FPPs was about 20% smaller than that of SPPs (even if they were prepared under exactly the same experimental conditions).Regardless of particle size or format, adsorption was described by means of a Bilangmuir model with ethanol/hexane MPs. On the other hand, in pure hexane, the Tóth isotherm was employed. Interestingly, it was found that selective and nonselective Henry's constants vary in opposite directions by increasing the percentage of strong MP modifier (between 3 and 10%, v/v). Saturation capacity of SPPs (referred only to the porous zone of the particle) was remarkably smaller than those of FPPs. On the other hand, binding constants on both selective and nonselective sites were significantly larger on SPPs. Finally, a correlation between the specific loading of chiral selector and the binding constants of enantiomers was suggested by data, which can be important also to understand the kinetic behavior of these particles in chiral ultrafast applications.

Mass spectrometry detection of basic drugs in fast chiral analyses with vancomycin stationary phases

Current trends in chiral analysis of pharmaceutical drugs are focused on faster separations and higher separation efficiencies. Core-shell or superficially porous particles (SPP) based chiral stationary phases (CSPs) provide reduced analysis times while maintaining high column efficiencies and sensitivity. In this study, mobile phase conditions suitable for chiral analyses with electrospray ionization LC-MS were systematically investigated using vancomycin as a representative CSP. The performance of a 2.7 µm SPP based vancomycin CSP (SPP-V) 10 cm × 0.21 cm column was compared to that of a corresponding 5 µm fully porous particles based analogue column. The results demonstrated that the SPP-V column provides higher efficiencies, 2–5 time greater sensitivity and shorter analysis time for a set of 22 basic pharmaceutical drugs. The SPP-V was successfully applied for the analysis of the degradation products of racemic citalopram whose enantiomers could be selectively identified by MS.

Comparison of small size fully porous particles and superficially porous particles of chiral anion-exchange type stationary phases in ultra-high performance liquid chromatography: effect of particle and pore size on chromatographic efficiency and kinetic performance

Recent advancements in particle design are common in reversed-phase liquid chromatography (RPLC), but in chiral separations their use is still sporadic in commercially available chiral stationary phases (CSPs). Due to reported lower mass transfer resistance, they might be a promising opportunity to increase efficiency and reduce time of analysis since the relatively higher mass transfer resistance term of CSPs caused by slow adsorption-desorption kinetics is the most performance-limiting factor in enantioselective chromatography. This study was dedicated to the evaluation of new support materials for tert-butylcarbamoylquinine (tBuCQN) based CSP to provide highly efficient and fast enantioseparations. As the main focus of this study, the chiral selector tBuCQN was immobilized on sub-2 μm fully porous particles (FPPs) and 2.7 μm superficially porous particles (SPPs) and their column performance in enantioseparation was evaluated in comparison to 5 μm FPPs by van Deemter and Knox analyses as well as kinetic plots using racemic Fmoc-Phe. Both new particle types outperformed the 5 μm FPP benchmark in terms of speed and efficiency, with wider pore materials (160 or 200 Å) being advantageous (over 90 or 120 Å). Basically decisive for the performance gain was the 10-times smaller mass transfer resistance. Furthermore, 2.7 μm 160 Å SPPs outperformed their fully porous sub-2 μm 120 Å counterpart (HminR = 4.64 μm vs. HminR = 8.94 μm) due to various parameters affording reduced plate height h of 1.7. Caused by the inaccessible core, separations were about 2-times faster. Packing of 2.7 μm core-shell particles provided a very homogeneous column bed, and, owing to its higher permeability, the column backpressure was much lower. It enables packing of longer columns providing theoretically separation efficiencies of up to 106 plates per m (as indicated by kinetic plots) and versatile use without the necessity of UHPLC systems. Investigating the effect of particle size reduction (FPPs: 5 μm, 3 μm, 1.7 μm; SPPs: 2.7 μm, 2 μm) and wider pores (FPPs: 120 Å, 200 Å; SPPs: 90 Å, 160 Å), a significantly reduced mass transfer resistance was the driving force for performance gain. Individual contributions of peak dispersion were deconvoluted for 5 μm FPP CSP and confirmed that slow adsorption-desorption kinetics is the most significant contribution to peak broadening in this chromatographic system.

Transport properties and size exclusion effects in wide-pore superficially porous particles

The effects of hydrodynamic radius on the transport of solute molecules in packed beds of wide-pore superficially porous particles (SPP) are studied using pore-scale simulation. The free molecular diffusion rate varies with radius through the Stokes-Einstein relation. Lattice Boltzmann and Langevin methods are used to model fluid motion and the transport of an ensemble of solute molecules in the fluid, providing statistics on solute concentration, flux, molecule age and residence time, as a function of depth in the SPP. Intraparticle effective diffusion and bed dispersion coefficients are calculated and correlated with the hydrodynamic radius and accessible porosity.The relative importance of convection and diffusion are found to depend on the molecule (tracer) size through the diffusion rate, and convection effects are more significant for larger, slower-diffusing molecules. When larger molecules are utilized, the intraparticle concentration is reduced in proportion to the local particle porosity, leading to a natural definition of the accessible porosity used in size exclusion chromatography (SEC). Although the pore shape is complex, the SEC constant K can be calculated directly from simulation. Simulation demonstrates that the effective diffusion coefficient is elevated near the particle hull, which is largely open to interstitial flow, and decreases with depth into the particle. All molecules studied here have transport access to the entire particle depth, although the accessible volume at a given depth depends on their size. The first passage time into the particle is well predicted by the diffusion rate, but residence time is influenced by convection, shortening the average visit duration. These results are of interest in “perfusion” chromatography where convection is thought to increase separation efficiency for large biomolecules.

Performance of Optimized Wide Pore Superficially Porous Particles for Separation of Proteins and Immunoglobulin G Antibodies.

In this study, we studied the chromatographic performance of this newly developed wide pore superficially porous particles (SPPs) with 3.5 μm particle size and 450 Å pore size, for the separation of proteins and Immunoglobulin G antibodies. We studied the selectivity of different phases (C4, SB-C18 and Diphenyl), the effect of temperature, column carryover and column chemical lifetime. We also compared our SPPs with other wide pore SPPs in similar particle sizes and sub 2 µ wide pore totally porous particles by van Deemter studies and gradient separations of proteins and immunoglobulin G antibodies. The results showed that the SPPs containing larger pore size gave better chromatographic performance.