sepharose and acetonitrile

This paper describes the establishment of an immunoaffinity chromatography (IAC) for selective extraction of fenvalerate from vegetable samples. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against fenvalerate to CNBr-activated Sepharose 4B and packed into a cartridge. The extraction conditions were carefully optimized, including loading, washing and eluting solutions. Under the optimal conditions, the IAC column was able to capture fenvalerate with the maximum capacity of 4000 ng. An average recovery of 94.5% and a RSD of 8.8% were obtained with six IAC columns prepared on six different days. Three vegetable samples spiked with fenvalerate at four different concentrations were extracted with IAC column and determined by gas chromatography with electron capture detection (GC-ECD). Chromatograms of final extracts were clean and fenvalerate could be easily detected without the interferences. The extraction recoveries and RSD were 74.7-96.5% and 2.5-5.2%, respectively, and the calculated limit of detection of the whole method was 0.008-0.012 ng g(-1).

Topics: Acetonitriles; Antibodies, Monoclonal; Chromatography, Affinity; Chromatography, Gas; Immunosorbent Techniques; Mass Spectrometry; Methanol; Nitriles; Pesticide Residues; Pyrethrins; Reproducibility of Results; Sensitivity and Specificity; Sepharose; Vegetables

The highly cross-linked 12% agarose gel Superose 12 10/300 GL causes retardation of glycine peptides when mobile phases containing varying concentrations of acetonitrile in water are used. An investigation has been made into the retention mechanism behind this retardation using the glycine dipeptide (GG) and tripeptide (GGG) as models. The dependence of retention times of analytical-size peaks under different experimental conditions was interpreted such that the adsorption most probably was caused by the formation of hydrogen bonds but that electrostatic interactions cannot be ruled out. Thereafter, a nonlinear adsorption study was undertaken at different acetonitrile content in the eluent, using the elution by characteristic points (ECPs) method on strongly overloaded GG and GGG peaks. With a new evaluation tool, the adsorption energy distribution (AED) could be calculated prior to the model selection. These calculations revealed that when the acetonitrile content in the eluent was varied from 0% to 20% the interactions turned from (i) being homogenous (GG) or mildly heterogeneous (GGG), (ii) via a more or less stronger degree of heterogeneity around one site to (iii) finally a typical bimodal energy interaction comprising of two sites (GG at 20% and GGG at 10% and 20%). The Langmuir, Tóth and bi-Langmuir models described these interesting adsorption trends excellently. Thus, the retardation observed for these glycine peptides is interpreted as being of mixed-mode character composed of electrostatic bonds and hydrogen bonds.

Topics: Acetonitriles; Adsorption; Buffers; Chromatography; Cross-Linking Reagents; Entropy; Gels; Glycine; Glycylglycine; Hydrogen-Ion Concentration; Models, Chemical; Peptides; Sepharose; Sodium Chloride; Temperature; Water

Following its market introduction in 1982, the cross-linked 12% agarose gel media Superose 12 has become widely known as a tool for size exclusion chromatography of proteins and other biological macromolecules. In this review it is shown that, when appropriate mobile phases are used, Superose possesses adsorption properties similar to that of traditional media for hydrophilic interaction liquid chromatography (HILIC). This is illustrated by the separation and purification of low molecular weight compounds such as polyphenols including active components of traditional Chinese medicinal herbs and green tea. Structural features of the cross-linked agarose that likely cause the observed adsorption effects are discussed as well. These are identified as being primarily ether bonds acting as strong hydrogen bond acceptors as well as hydrophobic residues originating from the cross-linking reagents.

Topics: Acetonitriles; Adsorption; Catechin; Chromatography, Liquid; Fallopia japonica; Flavonoids; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Microscopy, Electron, Scanning; Phenols; Plant Extracts; Plant Roots; Polyphenols; Salvia miltiorrhiza; Sepharose; Tea

(-)-Epigallocatechin gallate (EGCG) was purified in one step from a green tea polyphenol (GTP) crude extract by adsorption chromatography on a Superose 12 HR 10/30 column. The mobile phase used was a mixture of acetonitrile and water with an optimum mobile phase compositions regarding purity, recovery and separation time of 78/22 (v/v). Maximum practical sample loading was 100 mg GTP per run (corresponding to 4.2 mg/ml Superose). An EGCG purity of 99% with recoveries in the range 60-65% was achieved in one step directly from the crude GTP extract. Full column regeneration was obtained using solvents in the following order: 0.5 M NaOH, distilled water and 30% acetic acid.

Topics: Acetonitriles; Catechin; Chemical Fractionation; Chromatography, High Pressure Liquid; Molecular Structure; Plant Extracts; Sepharose; Solvents; Tea; Water

The adsorption behaviour of (-)-epigallocatechin gallate (EGCG), the major polyphenolic substance in green tea extracts, on the cross-linked agarose gel Superose 12 HR 10/30, has been studied using a variety of solvent systems and shown to be based on a mixture of hydrogen bonding and hydrophobic interaction. The hydrogen bonding was studied in acetonitrile in the presence of different co-solvents possessing varying hydrogen bond donor (HBD) and/or hydrogen bond acceptor (HBA) characteristics. The HBA-value of the co-solvent had the highest effect whereas the HBD-value played a subordinate role. Retention due to hydrophobic interaction could be demonstrated when mobile phases containing high water content were applied. The retention of EGCG, and its analogues (-)-epigallocatechin (EGC) and (-)-catechin (C) were thus shown to be dependent on the polarity of the organic modifiers added. However, the elution order of EGC and C, was inversed to that observed in reversed phase chromatography, indicating that some hydrogen bonding was still in effect. The retardation of EGCG in the presence of a wide concentration range of acetonitrile in water confirmed the interpretation that the retention mechanism is of mixed-mode character based on both hydrogen bonding and hydrophobic interaction.

Topics: Acetonitriles; Adsorption; Alcohols; Catechin; Chromatography, Agarose; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Sepharose; Solvents; Tea