Wider Pore Superficially Porous Particles for Peptide Separations by HPLC (original) (raw)
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Superficially porous silica particles with wide pores for biomacromolecular separations
Journal of Chromatography A, 2012
Since 2006, columns of superficially porous particles (SPP), often called Fused-core®, porousshell or core-shell particles, have had serious impact on HPLC separations. These particles have pore diameters of about 100 Å designed for separating small molecules. More recently, SPP with 160-200 Å pore diameter have been made available for separating peptides and small proteins. This report describes the effects of fused-core particle size, pore size, shell thickness and ligand type for the rapid, efficient separation of larger molecules such as intact proteins and other biomacromolecules up to at least 400 kDa. Optimization of these parameters resulted in particles that show no restricted diffusion that would compromise separating efficiency for large biomolecules. The thin porous shell provides excellent mass transfer (kinetics) for these large molecules, resulting in superior separations compared to conventional totally porous particles. Sample loading capacity can be adjusted to allow good detection sensitivity for minor components in a complex mixture. Strong particle strength ensures the loading of stable, high-efficiency columns. Stationary phases with different alkyl ligands were tested to provide data on retention, column efficiency and peak shapes for proteins. The development of these new wide-pore fusedcore particles now allows the HPLC separation of a wide range of molecules of different sizes with advantages of the SPP configuration.
Journal of Chromatography A, 2014
The challenges in HPLC are fast and efficient separation for a wide range of samples. Fast separation often results in very high operating pressure, which places a huge burden on HPLC instrumentation. In recent years, core-shell silica microspheres (with a solid core and a porous shell, also known as fused-core or superficially porous microspheres) have been widely investigated and used for highly efficient and fast separation with reasonably low pressure for separation of small molecules, large molecules and complex samples. In this review, we firstly show the types of core-shell particles and how they are generally prepared, focusing on the methods used to produce core-shell silica particles for chromatographic applications. The fundamentals are discussed on why core-shell particles can perform better with low back pressure, in terms of van Deemter equation and kinetic plots. The core-shell particles are compared with totally porous silica particles and also monolithic columns. The use of columns packed with core-shell particles in different types of liquid chromatography is then discussed, followed by illustrating example applications of such columns for separation of various types of samples. The review is completed with conclusion and a brief perspective on future development of core-shell particles in chromatography.
Journal of Separation Science, 2007
Fused-core particle technology as an alternative to sub-2-lm particles to achieve high separation efficiency with low backpressure Fused-Core particles have recently been introduced as an alternative to using sub-2lm particles in chromatographic separations. Fused-Core particles are composed of a 1.7 lm solid core surrounded by a 0.5 lm porous silica layer (d p = 2.7 lm) to reduce mass transfer and increase peak efficiency. The performance of two commercially available Fused-Core particles (Advanced Materials Technology Halo C18 and Supelco Ascentis Express C18) was compared with sub-2-lm particles from Waters, Agilent, and Thermo Scientific. Although the peak efficiencies were only L80% of those obtained by the Waters Acquity particles, the 50% lower backpressure allowed columns to be coupled in series to increase peak efficiency to 92 750 plates. The low backpressure and high efficiencies of the Fused-Core particles offer a viable alternative to using sub-2-lm particles and very-high-pressure LC instrumentation.
Prototype sphere-on-sphere silica particles for the separation of large biomolecules
Journal of chromatography. A, 2015
The goal of this study was to evaluate the possibilities offered by a prototype HPLC column packed with ∼2.5μm narrow size distribution sphere-on-sphere (SOS) silica particles bonded with C4 alkyl chains, for the analytical characterization of large biomolecules. The kinetic performance of this material was evaluated in both isocratic and gradient modes using various model analytes. The data were compared to those obtained on other widepore state-of-the-art fully core-shell and fully porous materials commonly employed to separate proteins moreover to a reference 5μm wide pore material that is still often used in QC labs. In isocratic mode, minimum reduced plate height values of hmin=2.6, 3.3 and 3.3 were observed on butylparaben, decapeptide and glucagon, respectively. In gradient elution mode, the SOS column performs very high efficiency when working with fast gradients. This prototype column was also comparable (and sometimes superior) to other widepore stationary phases, whatever...
Journal of Chromatography A, 2012
The separation range of superficially porous particles (Fused-core®) has been extended by design of particles with 160 Å pores. These particles show superior kinetics (lower resistance to mass transfer), allowing fast separations of peptides and small proteins (molecular weights of <15,000). The high efficiency and relatively low back pressure of these 2.7 μm fused-core particles has been maintained so that separations can be performed with conventional HPLC instruments. Longer columns can be used for higher resolution of complex mixtures of peptides, such as proteolytic digests. Highly reproducible separations of peptides at elevated temperatures with low pH mobile phases are maintained as a result of a stable bonded stationary phase. The utility of such highly stable materials is exemplified by separations of problematic amyloid peptides at low pH (TFA mobile phase) at an operational temperature of 100 °C. To address the issue of poor peptide peak shape in formic acid-containing mobile phases we show that the addition of 10-20 mM ammonium formate improves peak shape, retention and load tolerance of peptides. Use of the Fused-core particle materials for separations of synthetic peptides and tryptic digests yields peak capacities that are comparable to those obtained using columns packed with sub-2-μm particles, but with less than one-half of the operating back pressure. A peak capacity of 530 was obtained in 150 minutes on coupled columns of HALO Peptide ES-C18 with a combined length of 250 mm.
Superficially porous particles columns for super fast HPLC separations
Biomedical Chromatography, 2012
Superficially porous silica particles columns (SPSPCs) are manufactured by different companies. The most common have the brand names Halo, Ascentis Express and Kinetex. These columns provide super fast, sharp peaks and moderate sample loading and back pressure. These are available in different chemistries such as C 8 , C 18 , RP Amide and Hilic. Normally, the silica gel particles have 2.7 and 1.7 mm total and inner solid core diameters with 0.5 mm thick outer porous layer, 90 Å pore size and 150 m 2 /g surface area. They have been used for the separation and identification of low and high molecular weight compounds. The present article describes the state of the art of superficially porous silica particles based columns with special emphasis on their structures, mechanisms of separation, applications and comparison.
Core-shell particles lead the way to renewing high-performance liquid chromatography
TrAC Trends in Analytical Chemistry, 2015
In this review, we present the latest highlights and trends in the use of core-shell particles as stationary phases in columns for liquid chromatography (LC). These highly homogeneous particles have a diameter of 1.3-5 μm with a solid silica core and a porous surface that make their performance excellent compared to fully-porous particles. Use of these columns has been growing exponentially since their first largescale manufacture in 2006, and they are an emerging trend in the analysis of biological, toxicological and pharmaceutically interesting compounds. We review the main theoretical aspects responsible for their surprisingly good chromatographic behavior and analytical features. We also summarize state-ofthe-art analytical applications taking advantage of this column technology that is leading the current revolution in LC.
Journal of chromatography. A, 2017
To facilitate mass transport and column efficiency, solutes must have free access to particle pores to facilitate interactions with the stationary phase. To ensure this feature, particles should be used for HPLC separations which have pores sufficiently large to accommodate the solute without restricted diffusion. This paper describes the design and properties of superficially porous (also called Fused-Core(®), core shell or porous shell) particles with very large (1000Å) pores specifically developed for separating very large biomolecules and polymers. Separations of DNA fragments, monoclonal antibodies, large proteins and large polystyrene standards are used to illustrate the utility of these particles for efficient, high-resolution applications.
Cheap C18-modified Silica Monolith Particles as HPLC Stationary Phase
Columns packed with silica-based bonded phases have been used in reversed-phase liquid chromatography because of their high porosity, large surface area, compatibility with different solvents, mechanical stability, ease of surface modification, and economic feasibility. Bulk monolithic columns have attracted a considerable amount of interest in the last decade, and both monolithic and packed chromatographic columns have their own advantages and disadvantages. 1-5 The columns packed with particles have a high efficiency but they are accompanied with a high column back pressure due to lower permeability, while the monolithic columns have a high permeability but they result in inferior separation efficiency for the analysis of small molecules in HPLC. In our laboratory, we have been using the pseudo-monolithic silica particles with C-18 ligand 1,14,15 or polystyrene film 1,2,16-18 as packing materials. In the current study, porous partially sub-2 μm silica monolith particles have been prepared by a sol-gel process with a six times increased production scale compared with our previous work. These particles have been chemically modified with a C18 reagent and end-capped with a mixture of trimethylchlorosilane (TMCS) and 1,1,1,3,3,3 hexamethyldisilazane (HMDS) and packed in HPLC columns (1.0 mm × 300 mm). Five analytes (phenol, acetophenone, 4-methyl-2-nitroaniline, benzene, and toluene) are separated with good separation efficiency and resolution by using 60/40 (v/v) ACN/ H 2 O containing 0.1% TFA as the mobile phase with a flow rate of 25 μL/min.