Implementing 1.5 mm internal diameter columns into analytical workflows.

The use of smaller column diameters in liquid chromatography (LC) is often associated with capillary LC. Although there are many analytical benefits gained by adapting this format, routine use continues to be challenging due to column fragility and extra column dispersion. Bridging the gap between routinely used 2.

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.

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).

Fundamental to achieving fast separations with high efficiency: A review of chromatography with superficially porous particles.

Types of particles have been fundamental to LC separation technology for many years. Originally, LC columns were packed with large-diameter (>100 μm) calcium carbonate, silica gel, or alumina particles that prohibited fast mobile-phase speeds because of the slow diffusion of sample molecules inside deep pores. During the birth of HPLC in the 1960s, superficially porous particles (SPP, ≥30 μm) were developed as the first high-speed stationary-phase support structures commercialized, which permitted faster mobile-phase flowrates due to the fast movement of sample molecules in/out of the thin shells.

Importance of Particle Pore Size in Determining Retention and Selectivity in Reversed Phase Liquid Chromatography.

Column selection often centers on the identification of a stationary phase that increases resolution for a certain class of compounds. While gains in resolution are most affected by selectivity of the stationary phase or modifications of the mobile phase, enhancements can still be made with an intentional selection of the packing material's microstructure. Unrestricted mass transfer into the particle's porous structure minimizes band broadening associated with hindered access to stationary phase.

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.

Predicting the HILIC Retention Behavior of the N-Linked Glycopeptides Produced by Trypsin Digestion of Immunoglobulin Gs (IgGs).

The prediction of the retention behavior/time would facilitate the identification and characterization of glycoproteins, particularly the analytical challenges, such as the characterization of low-abundance glycoforms. This task is essential in the biotherapeutics industry, where the type and amount of glycosylation on recombinant IgG alter the efficacy, function, and immunogenicity. Models exist for the prediction of the hydrophilic interaction liquid chromatography retention of peptides and glycans.

Peptide retention prediction using hydrophilic interaction liquid chromatography coupled to mass spectrometry.

A model that predicts retention for peptides using a HALO penta-HILIC column and gradient elution was created. Coefficients for each amino acid were derived using linear regression analysis and these coefficients can be summed to predict the retention of peptides. This model has a high correlation between experimental and predicted retention times (0.

Reversed-phase chromatography with large pore superficially porous particles for high throughput immunoglobulin G disulfide isoform separation.

Reversed-phase liquid chromatography (RPLC) has been commonly used in IgG2 disulfide isoforms analysis. Recently, the columns packed with large pore superficially porous particles (SPP) have become available commercially. This work explores the application of this SPP technology in IgG2 disulfide isoforms separation.

Predicting the Retention Behavior of Specific -Linked Glycopeptides.

-Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze -glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s).

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.

The Separation and Quantitation of Peptides with and without Oxidation of Methionine and Deamidation of Asparagine Using Hydrophilic Interaction Liquid Chromatography with Mass Spectrometry (HILIC-MS).

Peptides with deamidated asparagine residues and oxidized methionine residues are often not resolved sufficiently to allow quantitation of their native and modified forms using reversed phase (RP) chromatography. The accurate quantitation of these modifications is vital in protein biotherapeutic analysis because they can affect a protein's function, activity, and stability. We demonstrate here that hydrophilic interaction liquid chromatography (HILIC) adequately and predictably separates peptides with these modifications from their native counterparts.

Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC).

The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large.

Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability.

Are sub-2 μm particles best for separating small molecules? An alternative.

Superficially porous particles (SPP) in the 2.5-2.7 μm range provide almost the same efficiency and resolution of sub-2 μm totally porous particles (TPP), but at one-half to one-third of the operating pressure.

Liquid chromatography-selected reaction monitoring (LC-SRM) approach for the separation and quantitation of sialylated N-glycans linkage isomers.

The study of N-linked glycans is among the most challenging bioanalytical tasks because of their complexity and variety. The presence of glycoform families that differ only in branching and/or linkage position makes the identification and quantitation of individual glycans exceedingly difficult. Quantitation of these individual glycans is important because changes in the abundance of these isomers are often associated with significant biomedical events.

The use of ammonium formate as a mobile-phase modifier for LC-MS/MS analysis of tryptic digests.

A major challenge facing current mass spectrometry (MS)-based proteomics research is the large concentration range displayed in biological systems, which far exceeds the dynamic range of commonly available mass spectrometers. One approach to overcome this limitation is to improve online reversed-phase liquid chromatography (RP-LC) separation methodologies. LC mobile-phase modifiers are used to improve peak shape and increase sample load tolerance.

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.

Fused-core particle technology in high-performance liquid chromatography: An overview.

The advent of superficially porous particles (SPPs) for packed HPLC columns has changed the way that many practitioners have approached the problem of developing needed separations. The very high efficiency of such columns, combined with convenient operating conditions, modest back pressures and the ability to use conventional HPLC instruments has resulted in intense basic studies of SPP technology, and widespread applications in many sciences. This report contains an overview of the SPP technology first developed in 2006 by Advanced Materials Technology, Inc.

Optimization of data-dependent acquisition parameters for coupling high-speed separations with LC-MS/MS for protein identifications.

Recent developments in chromatography, such as ultra-HPLC and superficially porous particles, offer significantly improved peptide separation. The narrow peak widths, often only several seconds, can permit a 15-min liquid chromatography run to have a similar peak capacity as a 60-min run using traditional HPLC approaches. In theory, these larger peak capacities should provide higher protein coverage and/or more protein identifications when incorporated into a proteomic workflow.

An improved approach to hydrophilic interaction chromatography of peptides: salt gradients in the presence of high isocratic acetonitrile concentrations.

Hydrophilic interaction chromatography (HILIC) for separations of peptides has been employed infrequently, particularly considering that this technique was introduced over 20 years ago. The present manuscript describes a radical departure from the traditional HILIC elution approach, where separations are achieved via increasing salt (sodium perchlorate) gradients in the presence of high isocratic concentrations (>80%) of acetonitrile, denoted HILIC/SALT. This initial study compared to reversed-phase chromatography (RPC), HILIC and HILIC/SALT for the separation of mixtures of synthetic peptide standards varying in structure (amphipathic α-helix, random coil), length (10-26 residues), number of positively charged residues (+1 to +11) and hydrophilicity/hydrophobicity.

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.

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.

High recovery HPLC separation of lipid rafts for membrane proteome analysis.

Proteomic analysis of complex samples can be facilitated by protein fractionation prior to enzymatic or chemical fragmentation combined with MS-based identification of peptides. Although aqueous soluble protein fractionation by liquid chromatography is relatively straightforward, membrane protein separations have a variety of technical challenges. Reversed-phase high performance liquid chromatography (RP-HPLC) separations of membrane proteins often exhibit poor recovery and bandwidths, and generally require extensive pretreatment to remove lipids and other membrane components.

Gene expression changes by amyloid beta peptide-stimulated human postmortem brain microglia identify activation of multiple inflammatory processes.

A central feature of the inflammatory pathology in Alzheimer's disease is activated microglia clustered around aggregated amyloid beta (Abeta) peptide-containing plaques. In vitro-cultured microglia can be activated to an inflammatory state by aggregated Abeta with the induction of a range of different neurotoxic factors and provide a model system for studying microglia Abeta interactions. Gene expression responses of human postmortem brain-derived microglia to aggregated Abeta were measured using whole genome microarrays to address the hypothesis that Abeta interactions with human microglia primarily induce proinflammatory genes and not activation of genes involved in Abeta phagocytosis and removal.