Journal of Chromatography B (v.845, #2)

by Cécile Cren-Olivé (185).

Chiral resolution of histidine using an anti-d-histidine l-RNA aptamer microbore column by Josephine Ruta; Catherine Grosset; Corinne Ravelet; Jennifer Fize; Annick Villet; Anne Ravel; Eric Peyrin (186-190).
In this paper, we report a new anti-amino acid aptamer chiral stationary phase (CSP). The enantiomers of histidine were separated using an immobilized histidine-specific l-RNA aptamer (40-mer) and an aqueous buffer as mobile phase. The effects of the variation of different operating parameters, including the mobile phase pH and the MgCl2 concentration as well as the column temperature, on the solute retention were assessed. The results suggested that (i) the protonated form of histidine was involved in the stereospecific RNA binding and (ii) Mg2+ was essential for the target enantiomer binding to the specific aptamer sites. From a practical point of view, it appeared that the baseline resolution in a minimum analysis time can be achieved at a column temperature of 35 °C for an eluent containing 10 mM of MgCl2, pH 5.5.
Keywords: Aptamer; Histidine; Chiral separation;

Evaluation of radial chromatography versus axial chromatography, practical approach by Charlotte Cabanne; Marcel Raedts; Emil Zavadzky; Xavier Santarelli (191-199).
Developments in packing and packing port design of radial columns in recent years have resulted in a claimed significant increase in performance of this process chromatography technology. In this first study, the main chromatographic parameters as efficiency, capacity factor, asymmetry and resolution were evaluated in a unique one-to-one comparison between a 120 ml bed-volume and 6 cm bed length radial chromatography mini-process column against a 50 mm diameter, 6 cm bed height and 120 ml bed-volume axial chromatography column. Radial chromatography showed an increase in efficiency by 31% in the number of plates per meter while the equilibration could be reduced by 0.4–0.5 column volumes. The asymmetry factor for bovine serum albumin in radial chromatography showed a reduction of 20% while the reduction of the asymmetry factor of the smaller protein ovotransferrin decreased even by 46% in comparison to the performance of the comparative axial chromatography column. Therefore in radial chromatography resolution improved up to 20%. The retention volume was similar in both cases. For radial chromatography, the decrease in “width at half height” at Height Equivalent of Theoretical Plates (HETP) measurements was 40% while the decrease of the over-all width of the peak was 27%. For adsorbed/desorbed proteins, the elution peak showed similar results: “width at half height” decreased to 45% while the over-all width of the peak decreased by 28%. The concentration of the non-retained protein in the flow-through (lysozyme), increased by 35% while the concentration of the eluted fraction (serum albumin bovine), increased with 40% in the radial chromatography columns. The better results obtained with the radial column were probably the consequence of the geometrical design of this device (larger inlet surface area and small outlet surface area which concentrate the eluted fraction).
Keywords: Radial chromatography; Axial chromatography;

A supramolecular system involving host–guest interactions between immobilized β-cyclodextrin (β-CD) cavities and adamantyl groups was evaluated for the preparation of immunosorbents which can be regenerated after use. First a dextran layer bearing both adamantyl groups and carboxylic functions is immobilized onto β-CD-modified porous silica particles (400 nm) by formation of inclusion complexes. Then, antibody molecules are grafted to the polymer layer. The stationary phases can be prepared in batch or directly in the column. They are stable in aqueous media and are able to trap specifically the corresponding antigen. In case of alteration of the antibody layer, it is possible to remove it by passing a SDS solution through the column. The feasibility of the procedure was evaluated, using the anti-HSA/HSA system.
Keywords: β-Cyclodextrin; Adamantane; Immunoaffinity chromatography; Human serum albumin;

Centrifugal partition chromatography as a tool for preparative purification of pea albumin with enhanced yields by Serge Bérot; Elisabeth Le Goff; Alain Foucault; Laurence Quillien (205-209).
A new procedure including the use of centrifugal partition chromatography (CPC) is proposed to purify PA1b and its isoforms. These pea (Pisum sativum L.) seed proteins are toxic against weevils and can be used as an environment-friendly insecticide. CPC was applied to a whole albumin fraction prepared from pea flour. The butanol:aqueous TFA system used in CPC allowed the separation of PA1b from other albumins and a degree of purification above 95%. Compared to analytical procedures based on methanol extraction, anion exchange and then reversed-phase chromatography (RPC), CPC recovered PA1b in much better yield, which is indispensable for large-scale purification of a biodegradable insecticide.
Keywords: Pea albumin; Insecticide; Centrifugal partition chromatography;

Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we had isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp β-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we have developed a novel affinity column procedure using an mAb prepared against α-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl and 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then prewashed with a 0.04% sodium dodecyl sulfate (SDS)–PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS–PBS, pH 11, and collected in tubes containing 1 M Tris–HCl, pH 6.8. As a result, the isolated Hp was devoid of apoA-I and was able to retain the biological function by forming an Hp–hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1, or 2-2 was greater than 95% with an yield greater than 50%. The procedure described here is significantly improved in time consumption, recovery, and purity. The rationale, design, and optimization for each step are described in detail.
Keywords: Human haptoglobin; Affinity column; Hemoglobin; Apolipoprotien A-I; Monoclonal antibody; Sodium dodecyl sulfate;

Application of microfluidic chip with integrated optics for electrophoretic separations of proteins by Julien Vieillard; Radoslaw Mazurczyk; Christophe Morin; Benjamin Hannes; Yann Chevolot; Paul-Louis Desbène; Stanislas Krawczyk (218-225).
This paper describes the fabrication, the characterization and the applications of a capillary electrophoresis microchip. This hybrid device (glass/PDMS) features channels and optical waveguides integrated in one common substrate. It can be used for electrophoretic separation and fluorimetric detection of molecules. The microfluidic performance of the device is demonstrated by capillary zone and gel electrophoresis of proteins.
Keywords: Lab-on-a-chip; Integrated optics; Microfluidic; Capillary electrophoresis;

Thiophilic adsorption revisited by Julie Hardouin; Magalie Duchateau; Ludovic Canelle; Céline Vlieghe; Raymonde Joubert-Caron; Michel Caron (226-231).
Specific and efficient selection of serum immunoglobulins, but not other proteins, on T-gel remains difficult. T-gel capacity was determined for different activation conditions and serum loadings. Mass spectrometry analysis was used to identify the proteins found in the flow-through and in the eluted fractions. Alpha-2-macroglobulin and albumin were the major contaminants of the eluates. The influence of the competition between immunoglobulins and the other serum proteins on the adsorption was also studied. Using a serum depleted in immunoglobulins (flow-through of a first chromatography on T-gel), many serum proteins were retained on the T-gel, including albumin. We conclude that T-gel selectivity is less than absolute and may reflect for a large part the experimental conditions of the adsorption.
Keywords: Immunoglobulin; Mass spectrometry; Serum; Thiophilic adsorption;

Purification and characterization of a low molecular weight multifunctional cytotoxic phospholipase A2 from Russell's viper venom by Gargi Maity; Somnath Mandal; Amitabha Chatterjee; Debasish Bhattacharyya (232-243).
A basic toxin from Russell's viper venom of 7.2 kDa (RVV-7) has been purified to homogeneity after partial unfolding by 4 M urea followed by filtration through Centricon-30 membrane. Its N-terminal sequence showed strong homology with snake venom cytotoxins. Cytotoxic activity of RVV-7 has been demonstrated with B16F10 melanoma cells. PLA2 activity was observed in cytotoxin (CX3) from Naja kauthia bearing sequence homology with RVV-7. Phospholipase A2 and trypsin inhibitory activities were also observed with RVV-7. Chemical modification and inhibition studies suggested independent functional sites for these activities. A qualitative assessment of tumor growth inhibition by RVV-7 has been made.
Keywords: Phospholipase A2; Low molecular weight enzyme; Russell's viper venom; Cytotoxicity; Trypsin inhibitor; Tumor growth suppression;

Matrix effects on mass spectrometry (MS) response were investigated with three atmospheric pressure ionization (API) sources after on-line solid-phase extraction (SPE) of human plasma. On-line SPE was evaluated with one restricted access material (RAM), two large particle supports (LPS) and one monolith. A sample protein precipitation (PP) with acetonitrile (2:1) and a direct injection were tested. Principal component analysis (PCA) was performed to simplify data presentation and interpretation. Protein precipitation was found to be mandatory for reducing signal modification. Regarding sensitivity towards matrix effects after PP, atmospheric pressure photoionization (APPI) was globally the least sensitive ionization mode while electrospray ionization ESI was the most sensitive.
Keywords: Matrix effects; APPI; Human plasma; On-line SPE; Post-column infusion;

HPLC columns partition by chemometric methods based on peptides retention by Bogusław Buszewski; Sylwia Kowalska; Tomasz Kowalkowski; Katarzyna Rozpędowska; Monika Michel; Tobias Jonsson (253-260).
In recent years, multivariate techniques have been utilized to evaluate reversed-phase high-performance liquid chromatographic data. In the present study, 11 high-performance liquid chromatography (HPLC) columns were divided into several groups according to the retention factors of 12 peptides. Principal component analysis (PCA) and cluster analysis (CA) were used in column and peptides’ comparison and grouping. CA results indicated that all stationary phases may be generally grouped into several clusters, due to stationary phase structure and properties. On the other hand, interesting results were obtained with the use of PC. There is almost linear relationship between classified HPLC columns in the space of new PCs, which is connected with meaning of the PC's reflected in their loading values. The first component describes non-polar properties of peptides, whereas the second component is loaded with polar peptides having much lower log  P values. PCA and CA were also used in peptides comparison however, complete explanation of peptides grouping still remains unclear.
Keywords: Peptides properties; HPLC; Stationary phase type; Column grouping; Cluster analysis; Principal component analysis;