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PubMed2000

Continuous split-flow thin cell and gravitational field-flow fractionation of wheat starch particles.

The combined employment of the SPLITT (split-flow thin) cell--a relatively new system for fast, continuous binary separation--and of gravitational field-flow fractionation (GrFFF)--a fractionation technique suitable for micron particle size distribution determination--was investigated for starch separation and characterization. Emphasis is placed on the main advantages of both techniques: operating under gentle earth gravity field, low cost and ease of maintenance. The reproducibility of GrFFF is demonstrated. Both the SPLITT separation and GrFFF fractionation results were checked by optical microscopy. Application examples of typical starch fractionation experiments are reported and discussed.

Chemical Fractionation↗

PubMed2000

Quantitative analysis in field-flow fractionation using ultraviolet-visible detectors: an experimental design for absolute measurements

In previous works, it has been shown that a standard ultraviolet-visible detection system can be used for quantitative analysis of heterogeneous systems (dispersed supermicron particles) in field-flow fractionation (FFF) by single peak area measurements. Such an analysis method was shown to require either experimental measurements (standardless analysis) or an accurate model (absolute analysis) to determine the extinction efficiency of the particulate samples. In this work, an experimental design to assess absolute analysis in FFF through prediction of particles' optical extinction is presented. Prediction derives from the semiempirical approach by van de Hulst and Walstra. Special emphasis is given to the restriction of the experimental domain of instrumental conditions within which absolute analysis is allowed. Validation by statistical analysis and a practical application to real sample recovery studies are also given.

Journal Article↗

PubMedDate not supplied

Determination of viable yeast cells by gravitational field-flow fractionation with fluorescence detection.

Vinification processing is largely related to yeast performance and depends on the initial cell viability. To optimize the quality of wine fermentation, control of the yeast quality is mandatory. The present paper describes a new method using gravitational field flow fractionation (GrFFF) with fluorescence detection for the determination of yeast cell viability before the fermentation process. A GrFFF calibration procedure was developed using commercial yeast to prepare standards of viable cells and propidium iodide (PI) as fluorescent probe for nonviable cells. The suitability of the new method was tested with several commercial yeast strains with a g/L content ranging from 1 to 3. The validation of the method was performed by comparing GrFFF viability values with those obtained using Coulter counter and flow cytometry techniques.

Cell Separation↗

PubMed1989

Application of an asymmetrical flow field-flow fractionation channel to the separation and characterization of proteins, plasmids, plasmid fragments, polysaccharides and unicellular algae.

The asymmetrical flow field-flow fractionation channel has been improved by introduction of a different sample loading method, downstream central injection, which reduces sample relaxation and focusing time considerably and allows the concentrations of material in the channel, thereby enabling the loading of very large sample volumes. The performance of the channel was demonstrated by the separation of a protein from its dimer, retention of nucleic acids, i.e., plasmids and enzymatically cleaved plasmids, hyaluronate and unicellular algae.

Chemical Fractionation↗

PubMed1980

Field-flow fractionation of alkali-liberated nuclear polyhedrosis virus from gypsy moth Lymantria dispar Linnaeus.

Rod-shaped viral particles of the gypsy moth nuclear polyhedrosis virus (NPV) were obtained through alkaline dissolution of inclusion bodies isolated and purified from infected hosts. The liberated viral fraction contains a wide assortment of enveloped aggregated forms as well as enveloped monomers. This complex mixture was separated by means of sedimentation field-flow fractionation (sedimentation FFF) to give a mass spectrum of the separated particles. From the elution pattern effective molecular weights could be assigned to the various resolved components. Through electron microscopy it was possible to characterize the components as monomers, dimers, etc. In parallel experiments the same viral mixture was separated by the more commonly used density gradient sedimentation technique. The two methods are compared as to time and convenience of manipulation, as well as to the amount of physicochemical information that can be extracted from each separation. The mixture of aggregate viral structures was dialyzed against a nonionic detergent solution. Sedimentation FFF of the dialysate revealed a uniform population of non-enveloped particles with no effective molecular weight which was lower than that of the initial monomer fraction. Electron microscopic observations confirmed that this lower molecular weight fraction did indeed contain rod-like structures which were thinner than the initial enveloped monomers indicating that most if not all of the enveloped material had been removed from the viral rod structures.

Animals↗

PubMed2005

Characterizing dissolved organic carbon using asymmetrical flow field-flow fractionation with on-line UV and DOC detection.

A method of characterizing dissolved organic carbon (DOC) by asymmetrical flow field-flow fractionation with on-line UV and DOC detection is described and applied to standards and natural water samples. Poly(styrenesulfonate) polymer standards, Suwannee River humic standards, and naturally occurring surface water and groundwater DOC were analyzed using this coupled detection technique. Molecular weight determinations in the samples and standards were 6-30% lower with DOC analysis than UV analysis. This difference was attributed to the insensitivity of the latter technique to nonaromatic carbon and suggests the molecular weight determined with the DOC detector is a more accurate representation of the actual molecular weight of the DOC. A normalized intensity comparison (NIC) method was proposed to distinguish differences in the relative amounts of aromatic and aliphatic carbon in DOC by comparing the two detector responses. The NIC method was applied to yield an average aromatic content of the bulk DOC and to detail the aromatic content over a range of molecular weights in a single DOC fraction.

Journal Article↗

PubMed1989

Harnessing electrical forces for separation. Capillary zone electrophoresis, isoelectric focusing, field-flow fractionation, split-flow thin-cell continuous-separation and other techniques.

A simple analysis, first presented twenty years ago, showed that the effectiveness of a field-driven separation like electrophoresis, as expressed by the maximum number of theoretical plates (N), is given by the dimensionless ratio of two energies N = -delta mu ext/2RT in which -delta mu ext is the electrical potential energy drop of a charged species and RT is the thermal energy (R is the gas constant and T is the absolute temperature). Quantity -delta mu ext is the product of the force F acting on the species and the path length X of separation. The exceptional power of electrophoresis, for which often N approximately 10(6), can be traced directly to the enormous magnitude of the electrical force F. This paper explores the fundamentals underlying several different means for utilizing these powerful electrical forces for separation, including capillary zone electrophoresis, gel electrophoresis, isoelectric focusing, electrical field-flow fractionation and split-flow thin continuous separation cells. Remarkably, the above equation and its relatives are found to describe the approximate performance of all these diverse electrically driven systems. Factors affecting both the resolving power and separation speed of the systems are addressed; from these considerations some broad optimization criteria emerge. The capabilities of the different methods are compared using numerical examples.

Chemical Fractionation↗

PubMed1993

Physical characterization and quantification of bacteria by sedimentation field-flow fractionation.

Studies in microbial ecology require accurate measures of cell number and biomass. Although epifluorescence microscopy is an accepted and dependable method for determining cell numbers, current methods of converting biovolume to biomass are error prone, tedious, and labor-intensive. This paper describes a technique with sedimentation field-flow fractionation to enumerate bacteria and determine their density, size, and mass. Using cultured cells of different shapes and sizes, we determined optimum values for separation run parameters and sample-handling procedures. The technique described can separate and detect 4', 6-diamidino-2-phenylindole-stained cells and generate a fractogram from which cell numbers and their size or mass distribution can be calculated. A direct method for estimating bacterial biomass (dry organic matter content) which offers distinct advantages over present methods for calculating biomass has been developed.

Journal Article↗

PubMed1996

Determination of relative amounts of ribosome and subunits in Escherichia coli using asymmetrical flow field-flow fractionation.

The 30S and 50S subunits and the 70S ribosome of Escherichia coli were separated in 6 minutes by using asymmetrical flow field-flow fractionation (FFF). The total analysis time for determination of the relative amounts of ribosomes and free subunits in a preparation from a cell suspension was 8 min. The method can detect a change in the mass fraction of ribosomes if it exceeds approx, 10%. The separation is based on differences in diffusion coefficients, i.e., hydrodynamic diameters, and these can be determined from observed retention times. The hydrodynamic diameters were in good agreement with literature values obtained from electron microscopy. The mass fraction of ribosomes changed as a function of the magnesium ion concentration which confirms previous knowledge and shows the accuracy of the method. The method appears as an alternative to ultracentrifugation analysis and avoids some of its drawbacks and artefacts. An obvious application can be the optimisation of cell design in metabolic engineering in order to maximise translation and protein production.

Chemical Fractionation↗

PubMed2001

Characterization of submicrometer aqueous iron(III) colloids formed in the presence of phosphate by sedimentation field flow fractionation with multiangle laser light scattering detection.

Iron colloids play a major role in the water chemistry of natural watersheds and of engineered drinking water distribution systems. Phosphate is frequently added to distribution systems to control corrosion problems, so iron-phosphate colloids may form through reaction of iron in water pipes. In this study, sedimentation field flow fractionation (SdFFF) is coupled on-line with multiangle laser light scattering (MALLS) detection to characterize these iron colloids formed following the oxygenation of iron(II) in the presence of phosphate. The SdFFF-MALLS data were used to calculate the hydrodynamic diameter, density, and particle size distribution of these submicrometer colloids. The system was first verified with standard polystyrene beads, and the results compared well with certified values. Iron(III) colloids were formed in the presence of phosphate at a variety of pH conditions. The colloids' hydrodynamic diameters, which ranged from 218 +/- 3 (pH 7) to 208 +/- 4 nm (pH 10), did not change significantly within the 95% confidence limit. Colloid density did increase significantly from 1.12 +/- 0.01 (pH 7) to 1.36 +/- 0.02 g/mL (pH 10). Iron(III) colloids formed at pH 10 in the presence of phosphate were compared to iron(III) colloids formed without phosphate and also to iron(III) colloids formed with silicate. The iron(III) colloids formed without phosphate or silicate were 0.46 g/mL more dense than any other colloids and were >6 times more narrowly distributed than the other colloids. The data suggest competitive incorporation of respective anions into the colloid during formation.

Chemical Fractionation↗

PubMed1998

Particle size analysis of dilute environmental colloids by flow field-flow fractionation using an opposed flow sample concentration technique.

An on-line concentration method has been developed for the analysis of dilute suspensions of macromolecules and particles by flow field-flow fractionation (FFF). This method, termed opposed flow sample concentration (OFSC), uses two opposing flowstreams to focus sample into a narrow band near the inlet of the flow FFF channel. Large sample volumes (up to 1 L) have been successfully loaded, concentrated, and separated in a 1 mL volume flow FFF channel. Concentration factors as high as 10(5) have been achieved. The effectiveness of OFSC flow FFF was demonstrated using mixtures of monodispersed standards such as proteins and polystyrene latex beads. Retention times, peak areas, and resolution are comparable for a 10 mL and a typical 10 microL injection volume. The applicability of the method to environmental studies was demonstrated by a separation of river water colloids.

Automation↗

PubMed1994

Determination of acid phosphatase in cultivation medium using asymmetrical flow field-flow fractionation.

Highly glycosylated proteins and proteins larger than 100,000 Da are difficult to analyze in complex media with standard liquid chromatography techniques. Asymmetrical flow field-flow fractionation is a simple bio-compatible method for such analysis which can also be used for on-line monitoring. A method for determination of acid phosphatase (EC 3.1.3.2), molecular weight 1,000,000, produced by a recombinant DNA yeast strain is presented.

Acid Phosphatase↗

PubMed2005

Monitoring the growth of polyoxomolybdate nanoparticles in suspension by flow field-flow fractionation.

We follow the evolution of polyoxomolybdate nanoparticles in suspensions derived from the keplerate (NH4)42[MoVI72MoV60O372(CH3CO2)30(H2O)72].ca..300H2O.ca..10CH3CO2NH4 ({Mo132}) by flow field-flow fractionation (FlFFF) to monitor the particle-size distribution in situ, atomic force and high-resolution transmission electron microscopy (AFM, SEM, and HRTEM) to confirm particle sizes, inductively coupled plasma-optical emission spectrometry (ICP-OES) to determine the Mo content of the FlFFF-separated fractions, and UV/visible spectroscopy to confirm the identity of the species in suspension. We observe the formation of 3-75-nm polyoxomolybdate particles in suspension and the dynamic growth of {Mo132} crystals.

Journal Article↗

PubMed2002

Bacteria sorting by field-flow fractionation. Application to whole-cell Escherichia coil vaccine strains.

Sorting and quantification of deactivated bacteria is an important way of quality control for whole-cell bacterial vaccines. In general, surface features of deactivated bacteria used for whole-cell bacterial vaccines affect the immunoresponse to bacteria-associated antigens. Enumeration of bacteria is also an important process development parameter for these vaccines. Field-flow fractionation (FFF) was previously applied to the separation of bacteria. For the first time, FFF is used for sorting bacteria strains of the same species on the basis of differences in bacterial membrane characteristics. Two FFF techniques, gravitational FFF (GrFFF) and asymmetrical flow FFF (AsFIFFF), are shown to be able to fractionate, distinguish, and quantify different deactivated Escherichia coli strains used for vaccines. E. coli can differ in the presence of fimbriae on the bacterial membrane. Fimbriae affect E. coli pathology and thus the use of E. coli for vaccines. GrFFF and AsFIFFF are able to fractionate fimbriated/ nonfimbriated cells in mixtures of different strains. While GrFFF is characterized by low cost and simplicity, As-FIFFF shows a higher performance in size fractionation with a high-speed separation. Coupled, on-line UV/visible turbidimetry yields the relative numbers of fractionated cells and sample recovery. Scanning electron microscopy and quasi-elastic light scattering are employed as uncorrelated techniques for size and morphology analysis of the E. coli strains.

Chemical Fractionation↗

PubMed2003

Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field-flow fractionation.

The evaluation of the translation capacity of cells that produce recombinant proteins can be made by monitoring their ribosomal composition. In a previous use of asymmetrical flow field-flow fractionation (AsFlFFF) for this purpose the overall analysis time was more than 1 h and 40 min, based on a standard protocol for cell harvest, washing, cell disruption, and the final 8-min AsFlFFF determination of ribosome and subunits. In the present work the overall analysis time was reduced to 16 min. The washing step was deleted and a time-consuming freeze-thaw cycle. Cell disruption was obtained by a time-minimized lysozyme and detergent treatment for 1.5 min, respectively. The ribosomal material was finally fractionated and quantified in only 6 min, without previous centrifugation, using AsFlFFF. The great time reduction will enable the future use of AsFlFFF at-line to a growing cell cultivation, continuously monitoring the change in ribosomal composition or in other applications requiring high sample throughput. To demonstrate the high efficiency of the method the ribosome and tRNA composition in an Escherichia coli cultivation was monitored every half an hour, giving 18 measurements across the complete growth curve, a frequency of data enough to make decisions about induction or termination of the cultivation.

Bacteria↗

PubMed2001

Hollow-Fiber flow field-flow fractionation of synthetic polymers in organic solvents.

A modified polyacrylonitrile (PAN) hollow-fiber membrane from a commercial source has been applied as the separation channel in flow field-flow fractionation (FFF). With the PAN membrane fiber, the application range of flow FFF could be extended to synthetic polymers that are soluble in a variety of organic solvents. The PAN membrane was shown to be resistant to hydrophobic solvents, such as dichloromethane (DCM), tetrahydrofuran (THF), ethyl acetate, and methyl ethyl ketone (MEK), as was illustrated by the successful fractionation of different polymer standards in these solvents. The system performance was assessed using polystyrene (PS) standards with ethyl acetate as the solvent. For a 100 kDa PS standard, the average recoverywas 57%, but for standards with a molar mass of 400 kDa and higher, 100% recovery was obtained. A linear relationship between peak area and injected mass was found. The run-to-run and fiber-to-fiber repeatability was determined using 100- and 400 kDa PS standards. The repeatability appeared to be satisfactory, with relative standard deviations < 2% for the retention times and < 5% for the recoveries of the standards. Plate numbers for the 400 kDa standard on different fibers were in the order of 110. From measurements on the fractionation of ferritin aggregates, it is concluded that the instrumental band-broadening is negligible. For an accurate determination of diffusion coefficients and molecular sizes based on retention times, calibration of the channel with standards appeared to be necessary. However, it was shown that the FFF system could be coupled to a multiangle light scattering (MALS) detector, thus providing an alternative on-line method for calibration. Expressions for the maximum obtainable plate number per unit of time have been derived for a hollow-fiber flow FFF system. It is shown that an increase in the system performance can be expected from a scaling down of the fiber diameter.

Journal Article↗

PubMedDate not supplied

Size and structure characterization of ethylhydroxyethyl cellulose by the combination of field-flow fractionation with other techniques. Investigation of ultralarge components.

Ethylhydroxyethyl cellulose (EHEC) of three different viscosity classes (EHEC I, II, and III) was analyzed by programmed cross-flow asymmetrical flow field-flow fractionation coupled to multiangle light scattering and refractive index detectors to determine their size and molar mass distribution. Two size populations were detected in the two lower viscosity classes, EHEC I and II, one high molar mass and one ultrahigh molar mass (UHM). The two covered molar masses from 10(4) up to 10(9) g X mol(-1). The highest viscosity class EHEC III was less size-dispersed covering molar masses from 5 x 10(5) to 5 x 10(7) g.mol(-1). Filtering of the EHEC II solution removed small amounts of compact UHM material. Enzyme treatments were performed on EHEC II to further characterize it. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and anion ion-exchange chromatography coupled to pulsed amperometric detection showed that the UHM component contained EHEC.

Cellulose↗

PubMed2001

Flow field-flow fractionation of poly(ethylene oxide): effect of carrier ionic strength and composition.

The effects of carrier ionic strength and electrolyte composition on the retention of poly(ethylene oxide) in aqueous flow field-flow fractionation have been investigated in this work. The study shows retention to be particularly sensitive to the presence of salts, as well as to the nature of the cation. Specifically, retention effects due to sample load are found to be very different in solutions containing potassium salts compared to those observed in solutions of the corresponding sodium salts. In a potassium-containing medium, the dependence of retention on sample mass is similar to that found previously for polyelectrolytes. This effect, which is particularly prominent for samples of low molecular mass, can be attributed to specific interactions between cation and polymer.

Chemical Fractionation↗