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PubMed1991

Selection of motile nonchemotactic mutants of Escherichia coli by field-flow fractionation.

We have developed a chromatographic method for isolating bacterial cells that are motile but nonchemotactic. Separation of strains of different phenotype occurs along a thin horizontal channel between two stirred chambers, the lower one containing a chemical attractant. The channel is bounded above and below by rigid filters, permeable to the attractant but not to the bacteria. The lower part of the channel is occupied by a porous plate comprising a vertical array of capillary tubes. An aliquot of cells is injected at one end of the channel and eluted by continuous flow of cell-free medium. Fluid leaving the other end of the channel is collected in a fraction collector. Cells that respond to the gradient swim to the bottom of the channel where they are retarded by the capillary array. Nonmotile cells sink to the bottom and are trapped in a similar manner. Motile cells that fail to respond to the gradient diffuse across the full height of the channel and, thus, travel through the apparatus at the average velocity of the eluent. When mixed with wild-type cells at a ratio of 1:1000 and subjected to an aspartate gradient, aspartate-blind cells were recovered quantitatively. The enrichment was approximately 200 to 1. The wild-type cells that survived the selection had a poorly motile phenotype.

Cell Separation↗
PubMed2005

Natural sample fractionation by FlFFF-MALLS-TEM: sample stabilization, preparation, pre-concentration and fractionation.

Two flow field flow fractionation (FlFFF) systems: symmetrical (SFlFFF) and asymmetrical (ASFlFFF) were evaluated to fractionate river colloids. Samples stability during storage and colloids concentration are the main challenges limiting their fractionation and characterization by FlFFF. A pre-fractionation (<0.45 microm) and addition of a bactericide such as NaN3 into river colloidal samples allowed obtaining stable samples without inducing any modification to their size. Stirred cell ultra-filtration allowed colloidal concentration enrichment of 25-folds. Scanning electron microscope (SEM) micrographs confirmed the gentle pre-concentration of river samples using the ultra-filtration stirred cell. Additionally, larger sample injection volume in the case of SFlFFF and on channel concentration in the case of ASFlFFF were applied to minimize the required pre-concentration. Multi angle laser light scattering (MALLS), and transmission electron microscope (TEM) techniques are used to evaluate FlFFF fractionation behavior and the possible artifacts during fractionation process. This study demonstrates that, FlFFF-MALLS-TEM coupling is a valuable method to fractionate and characterize colloids. Results prove an ideal fractionation behavior in case of Brugeilles sample and steric effect influencing the elution mode in case of Cézerat and Chatillon. Furthermore, comparison of SFlFFF and ASFlFFF fractograms for the same sample shows small differences in particle size distributions.

Colloids↗
PubMed2003

Development of asymmetrical flow field-flow fractionation-multi angle laser light scattering analysis for molecular mass characterization of cationic potato amylopectin.

The goal of this study is to investigate the applicability of asymmetrical flow field-flow fractionation (AsFlFFF)-multi angle laser light scattering (MALLS), and to develop a method for analysis of cationic potato amylopectin (CPAP) having ultrahigh molecular mass (UHMr). Use of the aqueous carrier having low salt content (3 mM NaN3) resulted in a distortion in AsFlFFF fractograms of CPAP with a general pattern of a sharp rise at the beginning of the elution followed by a long tailing, probably due to combination of attractive and repulsive charge interactions (attractive interaction between CPAP molecules and the channel membrane, and repulsion among cationic CPAP molecules). As the cross flow-rate (Fc) increases, the tailing tends to increase, and the repeatability of the AsFlFFF retention data tends to decrease, which is an indication of the presence of the charge interactions. The tailing gradually decreased, and the repeatability of the AsFIFFF retention data increased, as the salt content of the carrier increased. The distortion of the fractogram finally disappeared at Fc of about 0.2 ml/min and the channel flow-rate (F(out)) of about 1 ml/min with the aqueous carrier having the salt content of 40 mM (3 mM NaN3 +37 mM NaNO3). The weight-average molecular mass (Mw) and the z-average radius of gyration ((rg),) determined by MALLS were 5.2 x 10(7) and 34 x 10(1) nm, respectively. With the flow-rate ratio, Fc/F(out) kept constant, the degree of the charge interactions (and thus the distortion of fractogram) seems to increase with the cross flow-rate (Fc) and with the sample injection mass. AsFIFFF-MALLS was applied for determination of molecular mass distributions (MrDs) and the sizes of CPAPs prepared by various cooking procedures.

Amylopectin↗
PubMed2005

On-line hollow-fiber flow field-flow fractionation-electrospray ionization/time-of-flight mass spectrometry of intact proteins.

Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods. Flow field-flow fractionation (FlFFF) is characterized by the particularly "soft" separation mechanism, which is ideally suited to maintain the native structure of intact proteins. This work describes the original on-line coupling between hollow-fiber FlFFF (HF FlFFF), the microcolumn variant of FlFFF, and electrospray ionization/time-of-flight mass spectrometry (ESI/TOFMS) for the analysis and characterization of intact proteins. The results show that the native (or pseudonative) structure of horse heart myoglobin and horseradish peroxidase is maintained. Sample desalting is also observed for horse heart myoglobin. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI/TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.

Chromatography, High Pressure Liquid↗
PubMed2006

Effect of dissolution temperature on the structures of sodium hyaluronate by flow field-flow fractionation/multiangle light scattering.

Molecular weight distribution (MWD) and structural deformation of ultrahigh molecular weight (MW) sodium hylaluronate (10(5)-10(8) g/mol) were studied under different sample dissolution temperature conditions, using on-line flow field-flow fractionation (FlFFF) and multiangle light scattering (MALS). Sodium hyaluronate (NaHA) materials from sarcoma fluid have been studied by dissolving them in water at three different temperature conditions (5 degrees C, 50 degrees C, and 90 degrees C). Frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF), with field programming, was utilized for the separation of NaHA by MW, and on-line observation of light scattering of fractionated NaHA by MALS was performed in order to determine the MWD and molecular conformation. In these experiments, NaHA molecules exhibited an extended structure from a formerly rather compact geometry when the dissolving temperature was raised to 90 degrees C. This study also showed a clear difference in the MWD of NaHA when a preliminary filtration process was applied.

Fractionation, Field Flow↗
PubMed2006

Size characterization of green fluorescent protein inclusion bodies in E. coli using asymmetrical flow field-flow fractionation-multi-angle light scattering.

The goal of this study was to investigate the applicability of asymmetrical flow field-flow fractionation-multi-angle light scattering (AsFlFFF-MALS) for size analysis of green fluorescent protein inclusion bodies (GFPIBs). The size distributions of GFPIBs prepared by various culture conditions were determined. For GFPIBs prepared at 37 degrees C the peak maximum hydrodynamic diameter (d(H)) first increased and then decreased with the increase of the induction times in the presence of 0.1 and 2 mM isopropyl-beta-D-thiogalactoside (IPTG). For GFPIBs prepared at 30 degrees C the peak maximum d(H) was constant at about 700 nm irrespectively of the induction times and IPTG concentrations.

Dose-Response Relationship, Drug↗
PubMed2005

Application of flow field flow fractionation-ICPMS for the study of uranium binding in bacterial cell suspensions.

Field flow fractionation (FFF) is a size-based separation technique applicable to biomolecules, colloids, and bacteria in solution. When interfaced with ICPMS on-line, elemental data can be collected concurrent with size distribution. We employed hyperlayer flow FFF (Fl FFF) methodology to separate cells of Shewanella oneidensis strain MR-1 from exopolymers present in washed cell suspensions. With a channel flow of 4 mL min-1 and a cross-flow of 0.4 mL min-1 cells eluted with a retention time of 4.7 min corresponding to an approximate equivalent spherical cell diameter of 0.8 microm. Cell suspensions were amended with increasing concentrations of U to establish an adsorption isotherm and with fixed U concentrations at varying pH to establish the pH dependence of sorption. A linear sorption isotherm was determined for U solution concentrations of 0.2-16 microM, maximum U sorption occurred at pH 5. A high molecular weight compound, presumably a cell exudate, was identified by Fl FFF-ICPMS. This cell exudate complexed U, and at elevated pH, the exudate appeared to have a greater affinity for U than cell surfaces. Thus, Fl FFF interfaced with ICPMS detection is a powerful analytical technique for metal sorption studies with bacteria; analysis can be carried out on small sample volumes (25 microL) and additional speciation information can be gained because of the versatile Fl FFF separation range and multielement detection capabilities of ICPMS.

Adsorption↗