Asymmetrical Flow Field-flow Fractionation Channel Research Articles

Chip-Type Asymmetrical Flow Field-Flow Fractionation Channel Coupled with Mass Spectrometry for Top-Down Protein Identification

A chip-type design asymmetrical flow field-flow fractionation (AF4) channel has been developed for high-speed separation of proteins and top-down proteomic analysis using online coupled electrospray ionization mass spectrometry (ESI-MS). The new miniaturized AF4 channel was assembled by stacking multilayer thin stainless steel (SS, 1.5 mm each) plates embedded with an SS frit in such a way that the total thickness of the channel assembly was about 6 mm. The efficiency of the miniaturized AF4 channel at different channel lengths was examined with the separation of protein standards by adjusting flow rates in which an identical effective channel flow rate or an identical void time can be maintained at different channels. Detection limit, overloading effect, reproducibility, and influence of channel membrane materials on separation efficiency were investigated. Desalting and purification of proteins achieved during the AF4 operation by the action of an exiting crossflow and the use of aqueous mass-spectrometry-compatible (MS-compatible) buffer were advantageous for online coupling of the chip-type AF4 with ESI-MS. The direct coupling of AF4 and ESI-MS capabilities was demonstrated for the high-speed separation and identification of carbonic anhydrase (29 kDa) and transferrin (78 kDa) by full scan MS and for the first top-down identification of proteins with AF4-ESI-MS-MS using collision-induced fragmentation (CID). The presence of intact dimers (156 kDa) of transferrin was confirmed by AF4-ESI-MS via size separation of the dimers from monomers, followed by multiply charged ion spectral analysis of the dimers and molecular mass determinations. It was also found from these experiments that AF4-ESI-MS analysis of transferrin exhibited an increased signal-to-noise ratio compared to that of direct ESI-MS analysis due to online purification of the protein sample and size separation of dimers with AF4.

Effect of asymmetrical flow field-flow fractionation channel geometry on separation efficiency

The separation efficiencies of three different asymmetrical flow field-flow fractionation (AF4) channel designs were evaluated using polystyrene latex standards. Channel breadth was held constant for one channel (rectangular profile), and was reduced either linearly (trapezoidal profile) or exponentially (exponential profile) along the length for the other two. The effective void volumes of the three channel types were designed to be equivalent. Theoretically, under certain flow conditions, the mean channel flow velocity of the exponential channel could be arranged to remain constant along the channel length, thereby improving separation in AF4. Particle separation obtained with the exponential channel was compared with particle separation obtained with the trapezoidal and rectangular channels. We demonstrated that at a certain flow rate condition (outflow/inflow rate = 0.2), the exponential channel design indeed provided better performance with respect to the separation of polystyrene nanoparticles in terms of reducing band broadening. While the trapezoidal channel exhibited a little poorer performance than the exponential, the strongly decreasing mean flow velocity in the rectangular channel resulted in serious band broadening, a delay in retention time, and even failure of larger particles to elute.

Molecular weight and structure characterization of sodium hyaluronate and its gamma radiation degradation products by flow field-flow fractionation and on-line multiangle light scattering

A combined flow field-flow fractionation (FlFFF)/multiangle light scattering (MALS)/differential refractive index (DRI) detection method has been utilized for the size fractionation and characterization of ultrahigh molecular weight (MW) sodium hyaluronate (NaHA) samples. Separation of broad MW NaHA polymers was carried out by a frit inlet asymmetrical FlFFF channel employed with a linear field programming method followed by the on-line monitoring of light scattering at multiple angles for the calculation of MW and for the study of the conformation of NaHA samples. NaHA samples examined were: (1) two different viscosity fractions of NaHA obtained by a refinement process and (2) NaHA products of gamma radiation degradation. While the NaHA samples of two different viscosity fractions exhibited clearly different MW distributions and similar conformation, the radiation degraded NaHA samples showed a clear difference in both MW distribution and polymer structure.

Characterization of functionalized styrene–butadiene rubber by flow field-flow fractionation/light scattering in organic solvent

Flow field-flow fractionation (FlFFF) using an organic solvent as mobile phase has been effectively utilized for the separation and characterization of functionalized styrene–butadiene rubbers (SBR) that are polymerized and followed by coupling reaction in solution. Separation of broad molecular weight SBR was accomplished by an asymmetrical FlFFF channel in THF under field programming and the molecular weight distribution (MWD) of the SBR sample was determined by on-line measurement of light scattering. In this study, FlFFF has been utilized to characterize high-MW functionalized SBR from the low-MW non-functionalized molecules which were used for coupling reaction to produce high-MW functionalized SBRs, and to determine the coupling number of the functionalized SBRs depending on the type of the coupling reagents. The resulting MWD of the SBR samples prepared by the different coupling reagents (SnCl 4 and a polydimethylsiloxane compound) were compared.

Elution Behavior of Protein and Pullulan in Asymmetrical Flow Field-flow Fractionation (AsFlFFF)

An AsFlFFF channel was designed and built, and then tested for analysis of pullulans and proteins. Pullulans and proteins having various nominal molecular weights were injected at various conditions of the cross-flow rate (<TEX>$F_c$</TEX>) and the channel-out flow rate (<TEX>$F_{out}$</TEX>). The retention (measured by the retention ratio R) and the zone broadening (measured by the plate height H) were measured, and then compared with theory. When the incoming flow rate, <TEX>$f_{in}$</TEX> (and thus <TEX>$F_{out}$</TEX>) was varied with <TEX>$F_c$</TEX> fixed at 2.5 mL/min, the plate height measured for the pullulan with nominal molecular weight (M) of about 100,000 showed the trend expected by the longitudinal diffusion theory (H decreases with increasing flow rate). In contrast, when <TEX>$F_{out}$</TEX> was varied with the flow rate ratio, <TEX>$F_c/F_{out}$</TEX>, fixed constant at 5, the plate height measured for the same sample showed the trend expected from the non-equilibrium theory (H increases with increasing flow rate). Calibration plots (log D vs. log M) obtained with pullulans and proteins were not coincide, probably due to the difference in molecular conformation, suggesting the analysis of pullulans and proteins using AsFlFFF requires independent calibration. It was found that the linearity of the protein-calibration plot was improved by using a buffer solution as the carrier.

Mechanical Degradation and Changes in Conformation of Hydrophobically Modified Starch

In this paper, we study the mechanical degradation and changes in conformation of a branched ultrahigh molar mass biomacromolecule, hydrophobically modified starch, as caused by high-pressure homogenization. The characterization was performed with asymmetrical flow field-flow fractionation (AsFlFFF) with multiangle light scattering (MALS) and refractive index detection. The starch which had been chemically modified with octenyl succinate anhydride (OSA) proved to be very large and polydisperse. Upon high-pressure homogenization, the molar mass and rms radius (r(rms)) decreased, and the extent of these changes was related to the turbulent flow conditions during homogenization. The treatment also induced an increase and scaling with size in the apparent density of the macromolecules. To further study the changes in conformation, it was necessary to calculate the hydrodynamic radii (r(h)). This can be determined numerically from the elution times in the analysis and the flow conditions in the AsFlFFF channel. The results showed that the treatment can cause a dramatic decrease in the quotient between r(rms) and r(h), suggesting major conformational changes. These results together could be interpreted as degradation and "crumpling" of the macromolecule, which would give a decrease in r(rms) and an increase in apparent density, together with a "fraying" of more outer parts of the macromolecule, which could give rise to the increase in r(h).

Laser-induced breakdown detection combined with asymmetrical flow field-flow fractionation: application to iron oxi/hydroxide colloid characterization

The combination of asymmetrical flow field-flow fractionation (AsFlFFF) with the laser-induced breakdown detection (LIBD) is presented as a powerful tool for the determination of colloid size distribution at trace particle concentrations. Detection limits (Dl) of 1, 4, and 20 μg/L have been determined for a mixture of polystyrene reference particles with 20, 50, and 100 nm in size, respectively. This corresponds to injected masses of 1, 4, and 20 pg, which is lower than found in a previous study with the symmetrical FlFFF (SyFlFFF). The improvement is mainly due to the lower colloid background discharged from the AsFlFFF channel. The combined method of AsFlFFF–LIBD is then applied to the analysis of iron oxi/hydroxide colloids being considered as potential carriers for the radionuclide migration from a nuclear waste repository. Our LIBD arrangement is less sensitive for iron colloid detection as compared to reference polystyrene particles which results in a detection limit of ∼240 μg/L FeOOH for the AsFlFFF–LIBD analysis. This is superior to the detection via UV-Vis absorbance and comparable to ICP-MS detection. Size information (mean size 11–18 nm) for different iron oxi/hydroxide colloids supplied by the present method is comparable to that obtained by sequential ultrafiltration and dynamic light scattering. A combined on-line ICP-MS detection is used to gain insight into the colloid-borne main and trace elements.

Modeling of sample dynamics in rectangular asymmetrical flow field-flow fractionation channels

We model the evolution of the concentration field of macromolecules in a rectangular asymmetrical flow field-flow fractionation channel using center manifold techniques. The deviation of the primary flow from a parabolic profile influences the concentration field and this is investigated to complement previously known results. The long-term evolution of the components of the sample is shown to be well described by a one-dimensional advection-diffusion equation. The coefficients of this equation are determined by the rigorous analysis of the complete set of equations governing the two-dimensional fluid flow. This model gives quantitative predictions of the elution time of the samples, the width of the concentration pulse, and the resolution of the apparatus. The influence of initial sample width and effects of the secondary relaxation from focusing to elution conditions are discussed. Reported theoretical predictions are in agreement with experimental results published previously.

Design of an asymmetrical flow field-flow fractionation channel for uniform channel flow velocity

A new design for an asymmetrical flow field-flow fractionation (FFF) channel, having a breadth that decreases exponentially along its length, is presented. It is shown that such a channel may be operated under certain flow conditions to obtain a constant mean channel flow velocity throughout its effective length. This is not possible with trapezoidal channels, which were themselves introduced with the objective of reducing the variation of mean flow velocities along the channel length. It is also shown that when operated under other flow conditions, the resulting variation in mean flow velocity is reduced in comparison to that predicted for trapezoidal channels. © 1997 John Wiley & Sons, Inc. J Micro Sep9: 459–467, 1997

Separation speed, retention, and dispersion in asymmetrical flow field-flow fractionation as functions of channel dimensions and flow rates

The theory for the asymmetrical flow field-flow fractionation channel was evaluated experimentally. The assumption of a constant crossflow along the channel was satisfactory. The zone-broadening theory was extended to take into a account the longitudinal diffusion and the width of the starting sample zone. In the present apparatus the longitudinal diffusion was negligible but may have to be considered for lower outlet flow rates. The axial flow velocity gradient has the advantage that the contribution by the width of the starting sample zone to the final zone width decreases

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 concentration 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.

Improved separation speed and efficiency for proteins, nucleic acids and viruses in asymmetrical flow field flow fractionation

The performance of the asymmetrical flow field-flow fractionation channel has been improved by the use of a much thinner (0.12-mm) channel than before and by flow programming (stepwise gradient elution). The thinner channel contributes to a decreased zone broadening which enables complete resolution in a shorter time. Three protein peaks, representing molecular weights from 12 000 to 136 000, were completely resolved within 3 min. Flow programming speeds up the elution of the high-molecular-weight materials which occur late in a fractogram. This enabled separation of a small plasmid fragment (700 base pairs) from the large amounts of a big fragment (4600 base pairs) in 30 min and improved detection of a presumed trimer of albumin. Two viruses (1.8 · 10 6 and 50 · 10 6 daltons) were eluted as narrow peaks within 5 min.

Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTProperties of an asymmetrical flow field-flow fractionation channel having one permeable wallKarl Gustav. Wahlund and J. Calvin. GiddingsCite this: Anal. Chem. 1987, 59, 9, 1332–1339Publication Date (Print):May 1, 1987Publication History Published online1 May 2002Published inissue 1 May 1987https://doi.org/10.1021/ac00136a016RIGHTS & PERMISSIONSArticle Views1669Altmetric-Citations379LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts