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PubMed2006

Quantifying uranium complexation by groundwater dissolved organic carbon using asymmetrical flow field-flow fractionation.

The long-term mobility of actinides in groundwaters is important for siting nuclear waste facilities and managing waste-rock piles at uranium mines. Dissolved organic carbon (DOC) may influence the mobility of uranium, but few field-based studies have been undertaken to examine this in typical groundwaters. In addition, few techniques are available to isolate DOC and directly quantify the metals complexed to it. Determination of U-organic matter association constants from analysis of field-collected samples compliments laboratory measurements, and these constants are needed for accurate transport calculations. The partitioning of U to DOC in a clay-rich aquitard was investigated in 10 groundwater samples collected between 2 and 30 m depths at one test site. A positive correlation was observed between the DOC (4-132 mg/L) and U concentrations (20-603 microg/L). The association of U and DOC was examined directly using on-line coupling of Asymmetrical Flow Field-Flow Fractionation (AsFlFFF) with UV absorbance (UVA) and inductively coupled plasma-mass spectrometer (ICP-MS) detectors. This method has the advantages of utilizing very small sample volumes (20-50 microL) as well as giving molecular weight information on U-organic matter complexes. AsFlFFF-UVA results showed that 47-98% of the DOC (4-136 mg C/L) was recovered in the AsFlFFF analysis, of which 25-64% occurred in the resolvable peak. This peak corresponded to a weight-average molecular weight of about 900-1400 Daltons (Da). In all cases, AsFlFFF-ICP-MS suggested that<or=2% of the U, likely present as U(VI), was complexed with the DOC. This result was in good agreement with the U speciation modeling performed on the sample taken from the 2.3 m depth, which predicted approximately 3% DOC-complexed U. This good agreement suggests that the AsFlFFF-ICP-MS method may be very useful for determining U-organic matter association in small volume samples. Because the pH (7.0-8.1) and carbonate concentrations of these waters are typical of many groundwaters, these data suggested that facilitated transport of U by DOC may be limited in its importance in many groundwater systems.

Benzopyrans↗

PubMed2005

Elimination of edge effects in micro-thermal field-flow fractionation channel of low aspect ratio by splitting the carrier liquid flow into the main central stream and the thin stream layers at the side channel walls.

An optimized construction of the separation channel for micro-thermal field-flow fractionation (FFF) was proposed and studied experimentally. The sample is injected in such a manner that its zone moves along the channel only in the main central stream where the flow velocity profile in the plane parallel to the main accumulation wall is practically flat. This central stream is separated from the contact with the side walls of the channel by thin flowing layers of the free carrier liquid. The retained species do not reach the thin liquid streams at the side walls where the flow rate decreases rapidly to achieve zero at the side wall according to the established 3D flow velocity profile. Such a construction of the channel allows one to reduce the aspect ratio (the ratio of the channel breadth b to its thickness w) without increasing the zone broadening. The hydrodynamic splitting of the outlet streams allows one not only to increase the concentration of the detected species but also the determination of the sign of Soret coefficient.

Fractionation, Field Flow↗

PubMed2002

Separation and selective detection of lipoprotein particles of patients with coronary artery disease by frit-inlet asymmetrical flow field-flow fractionation.

An analytical method to improve the characterization of lipoprotein fractions is presented. Human plasma samples were treated with Sudan Black B to stain the lipid component in lipoproteins, then the stained lipoproteins were separated by frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF), according to the lipoprotein particle sizes, with the selective detection of eluting lipoprotein fractions, high-density lipoproteins (HDL), low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), at 610 nm. The capability of this technique has been evaluated with plasma samples obtained from patients with coronary artery disease (CAD), and it showed that the retention profile of patients' lipoprotein samples was clearly distinct from those of healthy persons. The potential of this technique comes with the direct injection of a stained lipoprotein sample without a prior procedure such as ultracentrifugation for sample preparation, and the size calculation of lipoprotein particles from the experimental retention time by theory. Since sample relaxation was achieved hydrodynamically in an FI-AFlFFF channel, sample injection and separation processes were continuously made without stopping the separation flow. This study demonstrated the potential of the FI-AFlFFF technique to be utilized as a powerful tool for the determination of the LDL profiles of patients with CAD.

Coronary Artery Disease↗

PubMed2003

Hyperlayer hollow-fiber flow field-flow fractionation of cells.

Interest in low-cost, analytical-scale, highly efficient and sensitive separation methods for cells, among which bacteria, is increasing. Particle separation in hollow-fiber flow field-flow fractionation (HF FlFFF) has been recently improved by the optimization of the HF FIFFF channel design. The intrinsic simplicity and low cost of this HF FlFFF channel allows for its disposable usage. which is particularly appealing for analytical bio-applications. Here, for the first time, we present a feasibility study on high-performance, hyperlayer HF FIFFF of micrometer-sized bacteria (Escherichia coli) and of different types of cells (human red blood cells, wine-making yeast from Saccharomyces cerevisiae). Fractionation performance is shown to be at least comparable to that obtained with conventional, flat-channel hyperlayer FIFFF of cells, at superior size-based selectivity and reduced analysis time.

Escherichia coli↗

PubMed2006

Improved theory of cyclical electrical field flow fractionation.

Previously reported theories for cyclical electrical field flow fractionation (CyElFFF) are severely limited in that they do not account for diffusion, steric, or electric double layer effects. Experiments have shown that these theories overpredict the retention of particles in CyElFFF. In this work, we present a model for prediction of steric, diffusion, and electrical effects. The electrical double layer effects are treated using a lumped electrical circuit model that accounts for the field shielding by the electrical double layer formed at the electrode-carrier interface. The electrical effects are shown to dominate retention times and outweigh the contributions of diffusion and particle size. Detailed results from the simulations are presented in this work, and a comparison between the theoretical and experimental results obtained from the retentions of polystyrene particle standards is presented in this paper. The models are shown to correctly predict the retention of the polystyrene standards in CyElFFF with a reasonable error, while existing models are shown to have significant failings.

Electricity↗

PubMed2003

Sonication effect on cellular material in sedimentation and gravitational field flow fractionation.

Sonication procedures are generally used prior to field flow fractionation (FFF) separation in order to produce suspensions without aggregates. Yeast cells manufactured in active dry wine yeast (ADWY) were placed in an ultrasound water bath in order to disrupt possible clumps and to obtain a single-cell suspension to be used in optimal conditions during fermentation processes. In order to determine whether this sample preparation procedure meets absolute needs, different yeast samples before and after sonication were analysed by two field flow fractionation techniques. It is shown that 2 min of sonication in the sample preparation process is sufficient to obtain an optimal dispersion of the yeast cells, that is, without critical percentage of aggregates. To demonstrate this effect, photographs of the yeast cell suspensions were performed with non-sonicated and sonicated yeast sample dispersion. The resulting data are compared with the elution profiles obtained from the two different FFF techniques. It is demonstrated that fractogram profiles prove the effectiveness of sonication methodologies.

Fractionation, Field Flow↗

PubMed2004

Hollow-fiber flow field-flow fractionation for whole bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

This work proposes for the first time the use of hollow-fiber flow field-flow fractionation (HF FlFFF) for improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOFMS) of whole bacteria. HF FlFFF has proved to be able to prepurify or fractionate different species of whole bacteria. Sample preparation by HF FlFFF gives improved spectra quality because noncellular components possibly present in the sample can be separated from the cells. When a mixture of two bacteria (Bacillus subtilis and Escherichia coli) is fractionated through HF FlFFF, MALDI/TOFMS analysis of each separated bacterial species preserves the most characteristic ion signals of the species without the presence of characteristic signals of the other species. The main advantages of HF FlFFF for MALDI/TOFMS analysis of whole bacteria are miniaturization, simplicity, and low cost of the fractionator components. This low cost makes disposable usage of the fractionator possible, thus eliminating the risk of run-to-run contamination of spectra due to sample carryover. The low fractionator volume yields bacterial fractionation on the order of a few minutes, which is comparable to MALDI/TOFMS analysis time. The small fractionation volume makes sample dilution low enough so that additional sample concentration steps are not strictly required to preserve MALDI/TOFMS detection.

Animals↗

PubMed2006

Multielement characterization of metal-humic substances complexation by size exclusion chromatography, asymmetrical flow field-flow fractionation, ultrafiltration and inductively coupled plasma-mass spectrometry detection: a comparative approach.

The use of three different separation techniques, ultrafiltration (UF), high performance size exclusion chromatography (HPSEC) and asymmetrical flow field-flow fractionation (AsFlFFF), for the characterization of a compost leachate is described. The possible interaction of about 30 elements with different size fractions of humic substances (HS) has been investigated coupling these separation techniques with UV-vis absorption spectrophotometry and inductively coupled plasma-mass spectrometry (ICP-MS) as detection techniques. The organic matter is constituted by a polydisperse mixture of humic substances ranging from low molecular weights (around 1kDa) to significantly larger entities. Elements can be classified into three main groups with regard to their interaction with HS. The first group is constituted by primarily the monovalent alkaline metal ions and anionic species like B, W, Mo, As existing as oxyanions all being not significantly associated to HS. The second group consists of elements that are at least partly associated to a smaller HS size fraction (such as Ni, Cu, Cr and Co). A third group of mainly tri- and tetravalent metal ions like Al, Fe, the lanthanides, Sn and Th are rather associated to larger-sized HS fractions. The three separation techniques provide a fairly consistent size classification for most of the metal ions, even though slight disagreements were observed. The number-average molecular weight (Mn), the weight-average molecular weight (Mw) and the polydispersity (rho) parameters have been calculated both from AsFlFFF and HPSEC experiments and compared for HS and some metal-HS species. Differences in values can be partly explained by an overloading effect observed in the AsFlFFF experiments induced by electrostatic repulsion effects in the low ionic strength, high pH carrier solution. Size information obtained from ultrafiltration is not as resolved as for the other methods. Molecular weight cut-offs (MWCO) of the individual filter membranes refer to globular proteins and molecular weight information may therefore, deviate from that given by the other methods after calibration with polystyrene sulfonate (PSS) standards.

Chromatography, Gel↗

PubMed2004

Programmed field decay thermal field flow fractionation of polymers: a calibration method.

The universal calibration procedure typical of thermal field flow fractionation (ThFFF) under constant thermal field operation was extended to thermal field programming (TFP) operation. The method requires knowledge of the following: (a) the programming function, which only depends on the thermal field decay function, (b) the physicochemical properties of the solvent, and (c) the calibration plot under varying channel cold wall temperatures (T(c)). Two field flow fractionation field programming conditions, with either a constant or a variable in time carrier flow velocity, are exploited. The method is based on determination, for each retention time position, of the average lambda retention value typical of TFP ThFFF. This parameter is then used to obtain the calibration plot (i.e., the molecular weight of the species as a function of the retention time position) by using the programming function and the calibration plot under varying T(c) values. The procedure approximation errors are also derived as a function of the programming type and solute-solvent system. To properly test the procedure, the calibration plot for the system constituted by polystyrene (PS) in cis-trans Decalin was determined, under varying conditions T(c) and thermal gradients, by using a set of monodisperse PS standards of different molecular weights (M). The procedure was first validated by simulation under two typical cases of TFP ThFFF operation. The approximation errors were found acceptable (in the worse cases, the accuracy in M prediction was 3%) and are in agreement with the theory. The procedure was then experimentally validated under varying programming decay function conditions. The reproducibility and accuracy of the M determination are both better than 2%.

Journal Article↗

PubMed2006

Development of non-gel-based two-dimensional separation of intact proteins by an on-line hyphenation of capillary isoelectric focusing and hollow fiber flow field-flow fractionation.

A rapid, non-gel-based, on-line, two-dimensional separation method is introduced for proteome analysis. Protein fractionation was carried out by first exploiting the differences in their respective isoelectric points (pI) in a Teflon capillary using isoelectric focusing (IEF), followed by a molecular weight (MW)-based separation in a hollow fiber by flow field-flow fractionation (FlFFF). The method developed here (CIEF-HFFlFFF) may be a powerful alternative to two-dimensional polyacrylamide gel electrophoresis, which is currently used for the separation and purification of proteins. In CIEF-HFFlFFF, proteins can be collected as a fraction of a certain pI and MW interval without being denatured. Additionally, the ampholyte solution is simultaneously removed during separation in the hollow fiber, and the overall process time is significantly reduced. This method was applied to a human urinary proteome sample, leading to the identification of 114 proteins with the subsequent off-line use of nanoflow liquid chromatography-tandem mass spectrometry after the tryptic digestion of each collected protein fraction.

Amino Acid Sequence↗

PubMed2000

Working without accumulation membrane in flow field-flow fractionation.

Nonideal interaction of sample with the separation device is a difficulty found in chromatographic methods as well as in field-flow fractionation. However, in field-flow fractionation (FFF), greater flexibility in the choice of carrier solution composition is possible, thus reducing the need of a wide choice of surface chemistry when nonideal sample interaction is to be minimized. The use of an ultrafiltration membrane as the surface for the accumulation wall is common practice in flow field-flow fractionation. Typical membranes in use are laminates of a skin membrane onto a backing material such as woven polyester. At this point, only a limited choice of membrane chemistries is available. Many membranes have been developed for protein applications as membranes are widely used in the pharmaceutical industries. While these membranes work well for protein applications, flow field-flow fractionation is applicable to polymeric particulate as well as protein samples. Thus, sample interaction with the membrane surface is possible with nonprotein applications and these interactions can induce significant secondary effects on retention ratio and affect instrumental reliability. Also, the woven texture of membranes may detrimentally affect the FFF separation. For these reasons, the study of flow field-flow fractionation using a flat, smooth surface of controlled chemistry is of relevance. We present here the results of a new, membraneless channel that uses a bare frit as the accumulation wall and that is intended for analysis of micrometer-sized particles only. Selectivity results are comparable to those obtained with the membrane, while relative sample recovery indicates that the best quantitative performance can be obtained without the membrane. Moreover, neither sample immobilization nor losses through the frit occur when operating membraneless. On the other hand, first experimental evidence of a certain level of frit surface activity suggests that optimization of experimental conditions is required.

Journal Article↗

PubMed2004

Cytochrome c-dimyristoylphosphatidylglycerol interactions studied by asymmetrical flow field-flow fractionation.

Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c-lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.

Cytochromes c↗

PubMedDate not supplied

Asymmetrical-flow field-flow fractionation with on-line multiangle light scattering detection. 1. Application to wormlike chain analysis of weakly stiff polymer chains.

Four samples of hyaluronan in the sodium form, ranging in weight-average molecular weight, M(w), from 6.67 x 10(5) to 4.23 x 10(6) were investigated by asymmetrical-flow field-flow fractionation coupled to multiangle light scattering (FlFFF-MALS) in 0.2 M aqueous NaCl at 25 degrees C. M(w) and z-average radii of gyration, R(G)(z)(), obtained via FlFFF-MALS showed a good agreement with the results obtained by conventional static light scattering. Furthermore, the molecular weight dependence of the radius of gyration for sodium hyaluronan obtained via FlFFF-MALS was analyzed on the basis of the Kratky-Porod model for unperturbed wormlike chains combined with the Yamakawa theory for radius expansion factor, and a sufficiently good agreement was observed between the theoretical prediction and experimental data. These results show the potential usage of FlFFF-MALS regarding size separation and molecular characterization even for weakly stiff chains.

Fractionation, Field Flow↗

PubMed2004

Asymmetrical flow field-flow fractionation and multiangle light scattering for analysis of gelatin nanoparticle drug carrier systems.

The physicochemical properties of nanosized colloidal drug carrier systems are of great influence on drug efficacy. Consequently, a broad spectrum of analytical techniques is applied for comprehensive drug carrier characterization. It is the primary objective of this paper to present asymmetrical flow field-flow fractionation (AF4), coupled online with multiangle light scattering detection, for the characterization of gelatin nanoparticles. Size and size distribution of drug-loaded and unloaded nanoparticles were determined, and data were correlated with results of state-of-the-art methods, such as scanning electron microscopy and photon correlation spectroscopy. Moreover, the AF4 fractionation of gelatin nanoparticulate carriers from a protein model drug is demonstrated for the first time, proposing a feasible way to assess the amount of loaded drug in situ without sample preparation. This hypothesis was set into practice by monitoring the drug loading of nanoparticles with oligonucleotide payloads. In this realm, various fractions of gelatin bulk material were analyzed via AF4 and size-exclusion high-pressure liquid chromatography. Mass distributions and high-molecular-weight fraction ratios of the gelatin samples varied, depending on the separation method applied. In general, the AF4 method demonstrated the ability to comprehensively characterize polymeric gelatin bulk material as well as drug-loaded and unloaded nanoparticles in terms of size, size distribution, molecular weight, and loading efficiency.

Animals↗

PubMed2007

Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity.

Size-exclusion high-performance liquid chromatography (SE-HPLC, SEC) is the long-standing biopharmaceutical industry standard for quantitation of soluble protein aggregates. Recently, sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged as a possible orthogonal technique to SEC for soluble aggregate quantitation. Moreover, asymmetrical flow field flow fractionation (AF4) has shown early promise in quantifying protein aggregates, both soluble and insoluble. We report soluble aggreg ate quantities measured by SEC, AF4, and SV-AUC analyzed by SEDFIT/c(s) for acid stressed and unstressed samples of a recombinant humanized monoclonal antibody. In equivalent antibody samples, SV-AUC, and AF4 detect markedly higher total aggregate levels than SEC. Furthermore, SEC fails to detect higher molecular weight soluble aggregates apparent in SV-AUC and AF4 analyses. Pooled fractions containing soluble dimeric aggregates were purified and re-analyzed by both SV-AUC and SEC. Reinjection of purified dimer onto the SEC column induces formation of detectable quantities of monomer and trimer. All sample types show statistically significant (p-values<0.01) antibody losses through the SEC column. This incomplete mass recovery from SEC indicates probable antibody physical adsorption to gel filtration media. Analysis of the sedimentation behavior of high molecular weight components suggests increased molecular asphericity with increasing molecular weight. We present an aggregation model based on nearly linear end-to-end assembly of monomeric subunits which is shown to be consistent with SV-AUC, SEC, AF4, and dynamic light scattering (DLS) results.

Antibodies, Monoclonal↗

PubMedDate not supplied

Determination of size distribution and encapsulation efficiency of liposome-encapsulated hemoglobin blood substitutes using asymmetric flow field-flow fractionation coupled with multi-angle static light scattering.

In this study, we investigated the size distribution, encapsulation efficiency, and oxygen affinity of liposome-encapsulated tetrameric hemoglobin (LEHb) dispersions and correlated the data with the variation in extruder membrane pore size, ionic strength of the extrusion buffer, and hemoglobin (Hb) concentration. Asymmetric flow field-flow fractionation (AFFF) in series with multi-angle static light scattering (MASLS) was used to study the LEHb size distribution. We also introduced a novel method to measure the encapsulation efficiency using a differential interferometric refractive index (DIR) detector coupled to the AFFF-MASLS system. This technique was nondestructive toward the sample and easy to implement. LEHbs were prepared by extrusion using a lipid combination of dimyristoyl-phosphatidylcholine, cholesterol, and dimyristoyl-phosphatidylglycerol in a 10:9:1 molar ratio. Five initial Hb concentrations (50, 100, 150, 200, and 300 mg Hb per mL of buffer) extruded through five different membrane pore diameters (400, 200, 100, 80, and 50 nm) were studied. Phosphate buffered saline (PBS) and phosphate buffer (PB) both at pH 7.3 were used as extrusion buffers. Despite the variation, extrusion through 400-nm pore diameter membranes produced LEHbs smaller than the pore size, extrusion through 200-nm membranes produced LEHbs with diameters close to the pore diameter, and extrusion through 100-, 80-, and 50-nm membranes produced LEHbs larger than the pore sizes. We found that the choice of extrusion buffer had the greatest effect on the LEHb size distribution compared to either Hb concentration or extruder membrane pore size. Extrusion in PBS produced larger LEHbs and more monodisperse LEHb dispersions. However, LEHbs extruded in PB generally had higher Hb encapsulation efficiencies and lower methemoglobin (metHb) levels. The choice of extrusion buffer also affected how the encapsulation efficiency correlated with Hb concentration, extruder pore size, and the metHb level. The most optimum encapsulation efficiency and amount of Hb entrapped were achieved at the highest Hb concentration and the largest pore size for both extrusion buffers (62.38% and 187.14 mg Hb/mL of LEHb dispersion extruded in PBS, and 69.98% and 209.94 mg Hb/mL of LEHb dispersion extruded in PB). All LEHbs displayed good oxygen-carrying properties as indicated by their P(50) and cooperativity coefficients. LEHbs extruded in PB had an average P(50) of 23.04 mmHg and an average Hill number of 2.29, and those extruded in PBS had average values of 27.25 mmHg and 2.49. These oxygen-binding properties indicate that LEHbs possess strong potential as artificial blood substitutes. In addition, the metHb levels in PB-LEHb dispersions are significantly low even in the absence of antioxidants such as N-acetyl-L-cysteine.

Blood Substitutes↗

PubMed2003

Hybrid colloid analysis combining analytical ultracentrifugation and flow-field flow fractionation.

Ferritin, the iron storage protein, is an organic-inorganic hybrid colloid consisting of a hollow protein capsule, which is filled with ferrihydride with up to 4500 iron atoms. Owing to the varying iron content and the resulting density differences, as well as the protein oligomerization, a particle size distribution is superimposed with a density distribution, making a precise analysis of ferritin by analytical ultracentrifugation difficult. This study describes how the information of the sedimentation coefficient distribution can be combined with the diffusion coefficient distribution obtained from flow-field flow fractionation to yield the buoyant molar mass of the oligomers in the mixture, extending the information content of each individual analytical method. In addition, the sedimentation and diffusion coefficients are compatible with a simple hard-sphere aggregation model, suggesting that the ferritin oligomers up to the pentamer have a globular solution structure.

Algorithms↗

PubMed2006

Combined approach to the analysis of recombinant protein drugs using hollow-fiber flow field-flow fractionation, mass spectrometry, and chemiluminescence detection.

The impurities present in recombinant protein drugs produced by large-scale refolding processes can not only affect the product safety but also interact with the expressed protein. To relate the impurity profile to conformation and functionality of the protein drug, analytical methods able not to degrade the sample components should be preferred. In this work, an urate oxidase (uricase) drug from Aspergillus flavus expressed in Saccharomyces cerevisiae, and a reagent-grade uricase from Candida sphaerica expressed in Escherichia coli, are analyzed by combining hollow-fiber flow field-flow fractionation with matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI/TOFMS) and with chemiluminescence enzyme activity assay. Preliminary detection and identification of sample impurities is performed by means of conventional methods such as RP HPLC with electrospray ionization quadrupole-TOF MS and MALDI/TOFMS with SDS PAGE and 2D SDS PAGE. Results show that the recombinant uricase samples obtained from different microorganisms have different impurities and different enzymatic activity and that different uricase oligomers are present in solution.

Aspergillus flavus↗