Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems.

This article describes the use of a new prototype column hardware made with 1.5 mm internal diameter (i.d.) and demonstrates some benefits over the 1.0 mm i.d. micro-bore column. The performance of 2.1, 1.5 and 1.0 mm i.d. columns were systematically compared. With the 1.5 mm i.d. column, the loss of apparent column efficiency can be significantly reduced compared to 1.0 mm i.d. columns in both isocratic and gradient elution modes. In the end, the 1.5 mm i.d. column is almost comparable to 2.1 mm i.d. column from a peak broadening point of view. The advantages of the 1.5 mm i.d. hardware vs 2.1 mm i.d. narrow-bore columns are the lower sample and solvent consumption, and reduced frictional heating effects due to decreased operating flow rates.

New wide-pore superficially porous stationary phases with low hydrophobicity applied for the analysis of monoclonal antibodies.

The article describes the development of new stationary phases for the analysis of proteins in reversed phase liquid chromatography (RPLC). The goal was to have columns offering high recovery at low temperature, low hydrophobicity and novel selectivity. For this purpose, three different ligands bound onto the surface of superficially porous silica-based particles were compared, including trimethyl-silane (C1), ethyl-dimethyl-silane (C2) and N-(trifluoroacetomidyl)-propyl-diisopropylsilane (ES-LH). These three phases were compared with two commercial RPLC phases. In terms of protein recovery, the new ES-LH stationary phase clearly outperforms the other phases for any type of biopharmaceutical sample, and can already be successfully used at a temperature of only 60°C. In terms of retention, the new ES-LH and C1 materials were the less retentive ones, requiring lower organic solvent in the mobile phase. However, it is important to mention that the stability of C1 phase was critical under acidic, high temperature conditions. Finally, some differences were observed in terms of selectivity, particularly for the ES-LH column. Besides the chemical nature of the stationary phase, it was found that the nature of organic modifier also plays a key role in selectivity.

Rapid analysis of polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons are a continuing environmental and health concern. The analytical methods developed to analyze this class of compounds have relied on reversed phase liquid chromatography and are often on the order of tens of minutes. Reduction in analysis times through the application of sub-2 µm fully porous and superficially porous support materials can increase the throughput of these LC separations. Herein, we demonstrate similar selectivity between a fully porous 1.8 µm and a 2.7 µm superficially porous material. Separations were individually developed with in silico modeling for a given flow rate determined by the fully porous column's backpressure requirements. Since the 2.7 µm superficially porous materials inherently require less backpressure to achieve similar levels of efficiency as the 1.8 µm fully porous materials, a marked increase in throughput is possible with elevated flow rates. Good resolution for a standard 16-component sample mixture is demonstrated in a sub-minute separation.

Improving selectivity and performing online on-column fractioning in liquid chromatography for the separation of therapeutic biopharmaceutical products.

A novel column-coupling approach is suggested to improve both the selectivity and efficiency of protein separations in liquid chromatography. Protein separations often suffer from limited selectivity or not appropriate resolving power. For a new biopharmaceutical product, the identification of the main and minor variant species is required. For that purpose, often offline collection fractioning is applied which is time consuming and regularly dilute the samples to an unacceptable extent. By serially coupling columns in the order of their increasing retentivity and applying "multi-isocratic" elution mode, indeed any (arbitrary) selectivity can be attained. Moreover, if a protein peak is trapped at the inlet of a later column segment - of a coupled system -, its band will be refocused and elute in unprecedented sharp peak. Furthermore, it becomes possible to perform online on-column fractioning of protein species within a very short analysis time (∼ 1 min) and without sample dilution. Two-, three- or multiple column systems can be developed and applied for complex sample separations (such as antibody mixtures). This new methodology can be particularly useful to improve the analysis (and therefore, safety and quality) of therapeutic mAbs and related products and offers benefits compared to offline fractionating. It is also demonstrated in this proof of concept study, that methyl (C1) modified RP phase has a great potential for protein separations despite it is not commercially available in state-of-the-art wide pore superficially porous particle format..

Performance characteristics of new superficially porous particles.

Superficially porous particles (also called Fused-Core, core shell or porous shell particles) show distinct advantages over comparable totally porous particles for separating small molecules. Columns of Fused-Core particles exhibit very high efficiency because of superior eddy dispersion properties (smaller van Deemter A term). The efficiency for columns of 2.7 µm Fused-Core particles actually rivals that for sub-2 µm totally porous particles with only about one-half the back pressure. These Fused-Core particles show special advantages with larger molecules for fast separations at high mobile phase velocities because of superior mass transfer (kinetic) properties (smaller van Deemter C term). This report describes the effect of different particle size and porous shell thicknesses on chromatographic performance for Fused-Core particles. Particle characteristics can significantly affect factors of separation importance. For example, the reduced plate height of packed columns is affected by particle diameter. Interestingly, larger Fused-Core particles show smaller reduced plate heights than smaller Fused-Core particles. Also, porous shell thickness has a strong effect on solute retention as well as separation efficiency, and particle surface area has a direct influence on sample loading characteristics. Fused-Core particles with a wide range of physical characteristics have been developed that allows the preparation of stable, efficient packed columns.