"Dry-column" chromatography of plant pigments

Separation of plant pigments which can be accomplished on thin-layer silica plates with mixture of petroleum ether, halocarbon, acetone, and polar solvent can be readily translated into dry-column technique that yields reproducible chromatograms after elution in fashion of liquid chromatography with fluorimeter as detector. Best solvent system was found to be mixture of petroleum ether, dichloromethane, acetone, and ethyl acetate

Complete gradient-LC-ESI system on a chip for protein analysis

This paper presents the first fully integrated gradient-elution liquid chromatography-electrospray ionization (LC-ESI) system on a chip. This chip integrates a pair of high-pressure gradient pumps, a sample injection pump, a passive mixer, a packed separation column, and an ESI nozzle. We also present the successful on-chip separation of protein digests by reverse phase (RP)-LC coupled with on-line mass spectrometer (MS) analysis

Calibration of ultrafiltration membranes against size exclusion chromatography columns

Using the extension of the concept of universal calibration parameter, yielding a relation between the hydrodynamic volume of molecules and the elution volume in sizeexclusionchromatography (SEC), to retention coefficients in ultrafiltration (UF), we propose a direct calibration of UF membranes against chromatography columns. Plotting the retention coefficient by one given UF membrane of a series of probe molecules versus their elution volume in SEC chromatography provides a calibration curve for this membrane. For a wide range of retentions, such calibration can be directly used to predict the retention of any molecule: one only needs to measure its exclusion volume by the SEC column, and read the retention by the calibrated membrane on the calibration curves. The method has been tested with dextran and PEG for the calibration, and milk proteins as test molecules, for three different membranes. The predicted values of the retention are in rather good agreement with those experimentally measured in a UF cell

Absolute Configuration of 2,2\u27,3,3\u27,6-Pentachlorinatedbiphenyl (PCB 84) Atropisomers

Nineteen polychlorinated biphenyl (PCB) congeners, such as 2,2′,3,3′,6-pentachlorobiphenyl (PCB 84), display axial chirality because they form stable rotational isomers, or atropisomers, that are non-superimposable mirror images of each other. Although chiral PCBs undergo atropselective biotransformation and atropselectively alter biological processes, the absolute structure of only a few PCB atropisomers has been determined experimentally. To help close this knowledge gap, pure PCB 84 atropisomers were obtained by semi-preparative liquid chromatography with two serially connected Nucleodex β-PM columns. The absolute configuration of both atropisomers was determined by X-ray single-crystal diffraction. The PCB 84 atropisomer eluting first and second on the Nucleodex β-PM column correspond to (aR)-(−)-PCB 84 and (aS)-(+)-PCB 84, respectively. Enantioselective gas chromatographic analysis with the β-cyclodextrin-based CP-Chirasil-Dex CB gas chromatography column showed the same elution order as the Nucleodex β-PM column. Based on earlier reports, the atropisomers eluting first and second on the BGB-172 gas chromatography column are (aR)-(−)-PCB 84 and (aS)-(+)-PCB 84, respectively. An inversion of the elution order is observed on the Cyclosil-B gas chromatography and Cellulose-3 liquid chromatography columns. These results advance the interpretation of environmental and human biomonitoring as well as toxicological studies

Protein separation with mathematical modeling for chromatographic operation

We have performed experiments and derived mathematical models for packed bed columns used for liquid phase chromatographic separations of proteins with impulse input of feed solutions. These models can now be used to describe the relationships between the elution characteristics (peak height, peak position, and shapes) and the operating conditions (flow rate and buffer conditions) of ion exchange and gel permeation column chromatography for protein separations. The surface adsorption model was discussed relative to the nature of the mobile and stationary phases in ion exchange column chromatography for two distinct cases: with and without pore diffusion. For large solute molecules, such as proteins and enzymes, the surface adsorption model without pore diffusion is adequate for prediction of elution profiles from ion exchange columns. This model is shown to be sufficient, since the solute molecules cannot readily diffuse into the solid matrix of column packings. For smaller solute molecules, such as amino acids and peptides, one must consider both the pore diffusion in the solid matrix and the axial dispersion in the mobile phase. A separate gel permeation model for chromatography was developed to focus on the diffusion of solute molecules involving no adsorption on solid phase. The retention times of the large solute molecules are less than that of smaller molecules because of the lower probability for diffusion into the solid matrix of column packings.Thus, the application of a specific model depends on the origin of packing materials in the chromatography column, the size of the solute molecules, and the interactions between the solid and mobile phases. Effects of model parameters (column length, cross sectional area, flow rate, effective contact area, void fraction, particle size, axial dispersion, mass transfer coefficient, equilibrium constant, and pore diffusivity) on the calculated elution profiles are discussed based on the series mass transfer mechanism . These effects are incorporated to describe the transport behaviors of solute molecules between the solid and liquid phases. The model protein system of hemoglobin and an albumin mixture was experimentally separated by cycling the change of pH in ion exchange column chromatography experiments, in order to study the transport relationship between the protein elution profile and transient pH wave. A pH phase lag within the column is needed to define for the pH cyclic zone operation in order to verify the elution characteristics between the experimental and predicted elution profiles. The success of our cycling techniques and models is further shown on the real protein system where we purified alkaline phosphatase from human placenta on an ion exchange packed bed with cycling of the buffer concentration. The optimal protein separation technique resulted in a high recovery and high purity product for this real protein enzyme system. The concentration phase lag and iso-ionic points are defined and combined with the relationships between the buffer concentrations and model parameters in order to predict the elution characteristics. The calculated and the experimental profiles are shown to be in good agreement when using the surface adsorption model without pore diffusion. The derived models can also be applied to determine the Number of Theoretical Plates (N) and Height Equivalent to Theoretical Plates (HETP) from the calculated profiles (peak height, peak width, retention time, and retention volume). The model parameters can be obtained from the limited experimental data for the desired operating conditions (mobile phase composition, flow rate, and column dimensions) in order to evaluate the column efficiency and optimization of column operation

Advances in DNA Affinity Chromatography

Different aspects of DNA affinity chromatography such as DNA complexity heparin elution, the Bi-column method and the oligonucluotide trapping method were studied. The complexity (length) of a DNA sequence attached to an affinity chromatography column affects column retention, and the purity of transcription factors obtained. T18: A18 tailed DNA affinity columns were better suited for purification of most of the transcription factors than either the discrete or concatemeric DNA affinity columns. A novel method using heparin for eluting transcription factors from DNA Sepharose columns was characterized. The amount of the lac repressor chimera which eluted from the column was shown to increase with increases in the mobile phase heparin concentration. The elution of the protein was also shown to be dependent on the amount of DNA coupled to the column and more protein eluted from columns containing lesser amounts of DNA. These data suggest that heparin and DNA compete for binding to the protein; this competition causes elution. Comparison of heparin- and salt-eluted protein demonstrated the heparin-eluted fraction of lac repressor was significantly purer than that eluted with salt and comparable to that obtained by elution with the specific ligand IPTG, a lactose analog. A novel Bi-column method was developed in which lac repressor is eluted from the Op1-Sepharose with a low heparin concentration and trapped on a Op1T18-Sepharose column because of its higher affinity for the lac repressor protein. Elution of the latter column with buffer containing a high salt concentration gives significantly purer transcription factor than the conventionally used single column methods and removes residual heparin. Highly pure CAAT enhancer binding protein(C/EBP) and the B3 transcription factor are also obtained by using variants of this Bi-column method. A new oligonucleotide trapping method in which a short oligonucleotide coupled to Sepharose is used to trap a complex of the transcription factor and its corresponding specific DNA sequence was developed. Highly purified transcription factor B3 was obtained using the oligonucleotide trapping method

Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk.Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column.Results: There were 12 hybridoma strains selected into the polyol responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA.Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and  gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.Keywords: tPA, Immunoaffinity chromatography, PR-mAb, ELISA-elution, Antibody, Thrombolytic activit

SP-Sephadex equilibrium chromatography of bradykinin and related peptides: Application to trypsin-treated human plasma

An analytical method is deseribed for the separation of bradykinin, Lys-bradykinin, and Met-Lys-bradykinin by equilibrium chromatography on SP-Sephadex C-25 eluted in 0.02 Tris-HCl buffer, pH 8.10, 0.12 NaCl. A second elution buffer, 0.02 Tris-HCl buffer, pH 7.70, 0.06 NaCl, serves as a second parameter for the identification of bradykinin and also separates the hormone from plasma bradykinin-potentiating peptides. Ten to one-hundred nanomoles of each peptide can be recovered in high yields, identified by elution position, and measured by bioassay with the isolated guinea pig ileum. The identification of bradykinin as the peptide released by trypsin acting on acid-denatured plasma is documented as an illustration of the method

Leveraging large data sets in continuous chromatography applications: Monitoring critical process parameters using MVDA

Continuous multicolumn chromatography is gaining momentum in the industry as an enabling technology to establish continuous biomanufacturing platforms for monoclonal antibodies and recombinant proteins. Some companies are exploring continuous multicolumn chromatography processes as a stand-alone unit operation to improve specific productivity and/or reduce buffer consumption, while other companies are considering an integrated continuous downstream process based on continuous multicolumn chromatography. One of the key features of continuous multicolumn chromatography is the amount of data generated during a single manufacturing batch or campaign. Traditional chromatography processes typically yield one (or a few) elution peaks per batch. Continuous chromatography processes, on the other hand, generates 30 to 100 elution peaks per batch. In addition to this, multicolumn chromatography systems typically have four to five times more sensors and hence collect much more information on the process. The enormous amount of information generated during a single batch and/or throughout a campaign provides an opportunity to monitor the process consistency in many ways. In this presentation, we will discuss the use of multivariate data analysis for monitoring the column performance throughout a prolonged series of BioSMB experiments with Protein A chromatography. In the presentation we will demonstrate how multivariate data analysis allows immediate identification of deviations in the process. Malfunctioning of the equipment and/or columns will immediately show up in the principal component analysis of various sensor signals. This strategy will also allow trending of the column performance and establish a means to determine column lifetime in a continuous process. In addition to this, the principal component analysis will be correlated with critical quality attributes such as the impurity profile of the eluate. The presentation will discuss the potential application of the outcome of multivariate data analysis to establish control strategies for multicolumn chromatography processes