Profiling of endogenous peptides by multidimensional liquid chromatography: On-line automated sample cleanup for biomarker discovery in human urine.

A simple and flexible system, employing a column switching technique, has been designed to allow the analysis of peptides and proteins smaller than 15 kDa by molecular weight in filtered urine samples by performing a direct on-column injection utilising simultaneous sample clean-up and trace enrichment. The positively charged peptides and small proteins in the sample are attracted to the inner, negatively charged pore structure of the RAM-SCX column while the larger proteins and uncharged or negatively charged compounds are excluded. After preconditioning with the biological sample, large amounts of sample can be injected. Several important and adjustable parameters for the proper use of a …

Die Charakteristika der Optimierung in einzelnen HPLC‐Modi

ChemInform Abstract: Liquid Chromatography - Its Development and Key Role in Life Science Applications

Liquid chromatographic methods cover the broadest range of appli- cations imaginable today. Nowhere is this more evident and relevant than in the life sciences, where identification of target substances relevant in disease mechanisms is performed down to the femtomole level. On the other hand, purification of therapeutic drugs on a multi- ton scale is performed by process LC. The complexity and abundance range of biological systems in combination with the extreme purity requirements for drug manufacturing are the challenges that can be mastered today by chromatography, after more than a century of research and development. However, significant improvement is still required for a better unde…

Automated multi-dimensional liquid chromatography

A comprehensive on-line sample clean-up with an integrated two-dimensional HPLC system was developed for the analysis of natural peptides. Samples comprised of endogenous peptides with molecular weights up to 20 kDa were generated from human hemofiltrate (HF) obtained from patients with chronic renal failure. The (poly-)peptides were separated using novel silica-based restricted access materials with strong cation-exchange functionalities (SCX-RAM). The size-selective sample fractionation step is followed by cation-exchange chromatography as the first dimension. The subsequent second dimension of separation is based on hydrophobic interaction using four parallel short reversed-phase (RP) co…

Monolithic silica columns of various format in automated sample clean-up/multidimensional liquid chromatography/mass spectrometry for peptidomics.

The following particulate and monolithic silica columns were implemented in a fully automated and flexible multidimensional LC/MS system with integrated sample clean-up, to perform the analysis of endogeneous peptides from filtered urine and plasma samples: restricted access sulphonic acid strong cation-exchanger (RAM-SCX) for sample clean-up, RP 18 Chromolith guard columns as trap columns and 100 microm I.D. monolithic RP 18 fused silica capillary columns as last LC dimension. The results show sufficient overall system reproducibility and repeatability. Implementation of monolithic silica columns added an additional flexibility with respect to flow rate variation and adjustment due to the …

Screening of oxazepine indole enantiomers by means of high performance liquid chromatography with imprinted polymer stationary phase.

Chromatographic enantiomer separations of different oxazepine indole derivatives were performed using a molecularly imprinted polymer. A 5aR,12R,13S-trans-6,6-dimethyl-12,13-dihydro-6H-5a, 1 3-methanoindolo[2,1-b][1,3]naphthoxazepine-12-carboxamide enantiomerderivative was used as a template and the resultant polymer has shown enantiomer recognition for series of template related compounds. The mechanistic description of the chiral discrimination process is scrutinised, comparing the discrimination between the different conformations and substituents of the oxazepine indoles.

Chromatography

Column technology in liquid chromatography

This chapter deals with the most important part of the liquid chromatography (LC) system: the column enabling the efficient and fast resolution of complex mixtures. It is divided into seven sections under the overall aspects of manufacture, operation, and evaluation of analytical columns for the user. The first three sections highlight the column design and hardware, followed by a thorough treatment of the properties of microparticulate silica adsorbents as packing material, stationary phases performed by surface functionalization, and the column filling process. Then, the implementation of the column into the LC system is discussed, leading into chromatographic column testing as a C18-bond…

Liquid chromatography--its development and key role in life science applications.

Liquid chromatographic methods cover the broadest range of appli- cations imaginable today. Nowhere is this more evident and relevant than in the life sciences, where identification of target substances relevant in disease mechanisms is performed down to the femtomole level. On the other hand, purification of therapeutic drugs on a multi- ton scale is performed by process LC. The complexity and abundance range of biological systems in combination with the extreme purity requirements for drug manufacturing are the challenges that can be mastered today by chromatography, after more than a century of research and development. However, significant improvement is still required for a better unde…

Multidimensional Column Liquid Chromatography (LC) in Proteomics – Where Are We Now?

Flüssigkeitschromatographie - ihre Entwicklung und Bedeutung für die Lebenswissenschaften

Flussigkeitschromatographische Methoden umfassen den breitest heute vorstellbaren Anwendungsbereich. Dies wird nirgendwo so deutlich wie in den Lebenswissenschaften, wenn es darum geht, Krankheitsmechanismen aufzuklaren und komplexe Gemische bis in den Femtomol-Bereich zu analysieren. Auf der anderen Seite besteht Bedarf an hochreinen therapeutischen Wirkstoffen, die als Pharmaka auf den Markt kommen und hohen Reinheitsanforderungen genugen mussen. Diese Aufreinigung wird mithilfe der Prozesschromatographie im Tonnenmasstab bewerkstelligt. Die hohe Komplexitat und breiten Konzentrationsbereiche biologischer Systeme in Kombination mit den extremen Reinheitsanforderungen der Arzneimittelprodu…

Enrichment of proteinaceous materials on a strong cation-exchange diol silica restricted access material: protein–protein displacement and interaction effects

A study of size exclusion and enrichment of proteins employing strong cation-exchange diol silica restricted access material (SCX-RAM) under saturation conditions is presented. Experiments were carried out with bacitracin, protamine, ribonuclease, lysozyme and bovine serum albumin as individual proteinaceous analytes as well as comprehensive binary mixtures and with human urine samples. Protein size dependent capacity features of the SCX-RAM column was observed. Bacitracin demonstrated the highest capacity followed by protamine while adsorption capacities of both ribonuclease and lysozyme were found smaller by a factor of 10. Applying binary protein samples occurring displacement effects we…

Characteristics of Optimization in Individual HPLC Modes: Sections 2.2– 2.5

Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogeneous peptides in multidimensional liquid chromatography

Abstract In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm ( n -octadecyl derivative). Additional characterisations were performed on restricted access materials with nitr…

Selective protein removal and desalting using microchip CE.

Abstract This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip™). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP–CZE could not be used for the rem…

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Bo…