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.

Superficially porous particles with 1000Å pores for large biomolecule high performance liquid chromatography and polymer size exclusion chromatography.

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.

Optimized superficially porous particles for protein separations.

Continuing interest in larger therapeutic molecules by pharmaceutical and biotech companies provides the need for improved tools for examining these molecules both during the discovery phase and later during quality control. To meet this need, larger pore superficially porous particles with appropriate surface properties (Fused-Core(®) particles) have been developed with a pore size of 400 Å, allowing large molecules (<500 kDa) unrestricted access to the bonded phase. In addition, a particle size (3.4 µm) is employed that allows high-efficiency, low-pressure separations suitable for potentially pressure-sensitive proteins. A study of the shell thickness of the new fused-core particles suggests a compromise between a short diffusion path and high efficiency versus adequate retention and mass load tolerance. In addition, superior performance for the reversed-phase separation of proteins requires that specific design properties for the bonded-phase should be incorporated. As a result, columns of the new particles with unique bonded phases show excellent stability and high compatibility with mass spectrometry-suitable mobile phases. This report includes fast separations of intact protein mixtures, as well as examples of very high-resolution separations of larger monoclonal antibody materials and associated variants. Investigations of protein recovery, sample loading and dynamic range for analysis are shown. The advantages of these new 400 Å fused-core particles, specifically designed for protein analysis, over traditional particles for protein separations are demonstrated.

Superficially porous silica particles with wide pores for biomacromolecular separations.

Since 2006, columns of superficially porous particles (SPPs), often called Fused-core(®), porous-shell or core-shell particles, have had serious impact on HPLC separations. These particles have pore diameters of about 100Å designed for separating small molecules. More recently, SPPs with 160-200Å 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 fused-core particles now allows the HPLC separation of a wide range of molecules of different sizes with advantages of the SPP configuration.

Fast high performance liquid chromatography separations for proteomic applications using Fused-Core® silica particles.

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 min on coupled columns of HALO Peptide ES-C18 with a combined length of 250 mm.