Inlet Asymmetrical Flow Field-flow Fractionation Research Articles

Investigation of steric transition with field programming in frit inlet asymmetrical flow field-flow fractionation

Steric transition in flow field-flow fractionation (FlFFF) was investigated under field programming by varying the channel thickness of a frit inlet asymmetrical FlFFF (FI-AF4). Steric transition is a typical inversion in sample elution mode from the increasing order of diameter (normal mode) to the opposite order (steric mode). Owing to the co-elution of two different-sized particles in the steric transition region where particles elute by the combination of the two elution modes, a loss of information in determining the accurate size of sample components in field-flow fractionation occurs. In this study, the effect of field programming on the steric transition in FI-AF4 was examined with the increase in channel thickness in order to increase the diffusional contribution of particle retention with the simultaneous reduction of steric contribution. This study demonstrated that the steric inversion diameter can be increased to >1 μm by programming the crossflow rate and by increasing the channel thickness to 350 and 490 μm. The present study also investigated the effects of outflow rate and initial field strength on the particle separation in field-programmed FI-AF4.

Characterization of ultrahigh-molecular weight cationic polyacrylamide using frit-inlet asymmetrical flow field-flow fractionation and multi-angle light scattering

In this study, frit inlet asymmetrical flow field-flow fractionation (FlFFF) with multi-angle light scattering (MALS) and differential refractive index (DRI) detection is utilized for size separation, determination of molecular weight (MW), and conformation of ultrahigh-MW (107–109 g/mol) cationic polyacrylamides (C-PAMs), a class of water-soluble copolymers based on acrylamide and vinyl-type comonomers with quaternary ammonium cations that are widely used in wastewater treatment and in paper industries. Linear and branched C-PAM copolymers prepared in two different polymerization methods (solution and emulsion) from varying amounts of crosslinking agent and initiator were size fractionated by FlFFF with field-programming. It was found experimentally that the linear copolymers from both polymerization methods were less than 108 g/mol in MW with compact, nearly spherical structures, while the branched C-PAM copolymers from the emulsion polymerization showed a significant increase in average MW up to ∼109g/mol, which was about 20-fold greater than those from the solution method, and the branched copolymers had more compact or shrunken conformations. While both linear and branched copolymers less than 108 g/mol MW were well resolved in an increasing order of MW (normal mode), it was noted that branched copolymers prepared through emulsion polymerization exhibited significantly larger MWs of 108–109 g/mol and eluted in the steric/hyperlayer mode, in which the elution order is reversed in an extreme run condition (strong initial field strength followed by a fast field decay during programming).

A Soft Preparative Method for Membrane Proteome Analysis Using Frit Inlet Asymmetrical Flow Field-Flow Fractionation: Application in a Prostatic Cancer Cell Line

Membrane proteins participate in a number of important biological functions such as signal transduction, molecular transport, and cell-cell interactions. However, due to the nature of membrane proteins, the development of a preparative method that produces a sufficient yield of purified membrane proteins from the cell remains a challenge. In the present study, frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) was employed to fractionate membrane fragments containing membrane proteins from free cytoplasmic proteins of prostatic cancer cell (DU145 cell) lysates. The isolated membrane proteins were then digested and analyzed by nanoflow liquid chromatography/tandem mass spectrometry (nLC-ESI-MS-MS). Since fractionation of the cell lysate mixtures containing membrane fragments and cytoplasmic proteins could be achieved based on the differences of their sizes in FI-AFlFFF, membrane fragments were partially isolated from the cytoplasmic proteins and collected. The performance of FI-AFlFFF for prefractionation of the membrane proteome was examined by comparing the number of membrane proteins that were identified with the number identified using an ultracentrifugation method. The application of FI-AFlFFF to membrane proteomics produced an increased yield of purified membrane proteins with fewer cytoplasmic proteins compared to a conventional ultracentrifugation method.

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.

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 °C, 50 °C, and 90 °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 °C. This study also showed a clear difference in the MWD of NaHA when a preliminary filtration process was applied.

Size Characterization of Sodium Hyaluronate by Field Programming Frit Inlet Asymmetrical Flow Field-Flow Fractionation/Multiangle Light Scattering

Sodium hyaluronate (NaHA), water soluble polymer having ultra-high molecular weight, is characterized by using on-line frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) and multiangle light scattering (MALS). This study demonstrates the capability of power programming FI-AFlFFF for the separation of NaHA and the applicability of FI-AFlFFF with MALS for the characterization of molecular weight distribution and their structural information. Since sample injection and relaxation in FI-AFlFFF are achieved by using hydrodynamic relaxation, separation of high molecular weight polymers can be achieved smoothly without halting the separation flow. Experiments are carried out with the two different NaHA products (a raw NaHA sample and a thermally degraded NaHA product) and molecular weight distribution and conformations in solution are determined. Influence of sample filtration on the change of molecular weight distribution is also discussed.碏�⨀؀က碏�⨀邏�⨀਀邏�⨀႐�⨀က႐�⨀ꂏ�⨀Ԁꂏ�⨀࢐�⨀؀က࢐�⨀ₐ�⨀਀ₐ�⨀‘�⨀က‘�⨀ゐ�⨀਀ゐ�⨀�⨀؀က�⨀႑�⨀਀႑�⨀邑�⨀က邑�⨀ₑ�⨀Ԁₑ�⨀袑�⨀؀က袑�⨀ꂑ�⨀਀ꂑ�⨀䂓�⨀Ԁက䂓�⨀낑�⨀낑�⨀碒�⨀☀碒�⨀ᄐ돀㡧�⨀堘�⨀悕�⨀돐肕�⨀��⨀袕�⨀섏덐颕�⨀쀏ꀏ
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Field programming in frit inlet asymmetrical flow field-flow fractionation/multiangle light scattering: Application to sodium hyaluronate

The capability of field-programmed separation in frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been examined for separating a high molecular weight sodium hyaluronate (NaHA) by varying the field programming parameters. Experiments were performed with on-line coupling of the field programming FI-AFlFFF and multiangle light scattering (MALS) detection. Sample relaxation, a pre-requisite step to establish equilibrium states of sample materials prior to the beginning of separation in most forms of FFF techniques, is obtained by hydrodynamically in FI-AFlFFF without stopping the migration flow. Thus, the procedures of sample injection – hydrodynamic relaxation – separation in FI-AFlFFF are continuously achieved without halting the sample migration. In this study, field programming in FI-AFlFFF was investigated for the separation of NaHA, water-soluble polysaccharides, by examining the influence of field decay pattern, initial field strength condition, and ionic strength of carrier solution on the successful separation of a degraded NaHA sample. Results were compared with molecular weight calculations of eluting materials among different field programming conditions from multiangle light scattering (MALS) signals. It was found that when the field programming was utilized in FI-AFlFFF, a proper selection of initial cross-flow rate, the field decay pattern, and an appropriate control of final field strength needed to be carefully selected in achieving a successful separation of a broad molecular weight water-soluble polymer sample.

Separation of carbon nanotubes by frit inlet asymmetrical flow field-flow fractionation.

Flow field-flow fractionation (flow FFF), a separation technique for particles and macromolecules, has been used to separate carbon nanotubes (CNT). The carbon nanotube ropes that were purified from a raw carbon nanotube mixture by acidic reflux followed by cross-flow filtration using a hollow fiber module were cut into shorter lengths by sonication under a concentrated acid mixture. The cut carbon nanotubes were separated by using a modified flow FFF channel system, frit inlet asymmetrical flow FFF (FI AFIFFF) channel, which was useful in the continuous flow operation during injection and separation. Carbon nanotubes, before and after the cutting process, were clearly distinguished by their retention profiles. The narrow volume fractions of CNT collected during flow FFF runs were confirmed by field emission scanning electron microscopy and Raman spectroscopy. Experimentally, it was found that retention of carbon nanotubes in flow FFF was dependent on the use of surfactant for CNT dispersion and for the carrier solution in flow FFF. In this work, the use of flow FFF for the size differentiation of carbon nanotubes in the process of preparation or purification was demonstrated.

Miniaturization of frit inlet asymmetrical flow field-flow fractionation.

A miniaturized frit inlet asymmetrical flow field-flow fractionation (mFI-AFlFFF) channel has been constructed and tested for the separation of proteins. By scaling down the geometrical channel dimension of a conventional FI-AFlFFF system, flow rate ranges that can be manipulated were decreased to 20-30 microL/min, which reduces the injection amount of sample materials. The end effect contribution to plate height was evaluated by varying the inner diameter of the connection tubing between the injector and the channel inlet at various injection flow rates, and the results showed that the use of silica capillary tubing of the shortest possible distance is essential in reducing the initial band broadening prior to the sample injection to the microscale channel. The capability of the microFI-AFlFFF system was demonstrated with the separation of protein standards, polystyrenesulfonates, and ssDNA strains and for the characterization of replication protein A-ssDNA binding complex regulated by redox status.

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.

Field and flow programming in frit-inlet asymmetrical flow field-flow fractionation

The separation of wide molecular mass ( M r) ranges of macromolecules using frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been improved by implementing a combination of field and flow programming. In this first implementation, field strength (governed by the cross flow-rate through the membrane-covered accumulation wall) is decreased with time to obtain faster elution and improved detection of the more strongly retained (high M r) materials. The channel outlet flow-rate is optionally held constant, increased, or decreased with time. With circulation of the flow exiting the accumulation wall to the inlet frit, the dual programming of cross flow and channel outlet flow could be implemented using just two pumps. With this flow configuration, the channel outlet flow-rate is always equal to the channel inlet flow-rate, and these may be programmed independently of the cross flow-rate through the membrane. FI-AFlFFF retains its operational advantage over conventional asymmetrical flow FFF (AFlFFF). Unlike conventional AFlFFF, FI-AFlFFF does not require time consuming, and experimentally inconvenient, sample focusing and relaxation steps involving valve switching and interruption of sample migration. The advantages of employing dual programming with FI-AFlFFF are demonstrated for sets of polystyrene sulfonate standards in the molecular mass range of 4 to 1000 kDa. It is shown that programmed FI-AFlFFF successfully expands the dynamic separation range of molecular mass.

HYDRODYNAMIC VS. FOCUSING RELAXATION IN ASYMMETRICAL FLOW FIELD-FLOW FRACTIONATION

Two different sample relaxation methods (focusing relaxation and hydrodynamic relaxation) in asymmetrical flow field-flow fractionation (AFlFFF) are examined by using a conventional asymmetrical channel and a frit inlet asymmetrical channel. In this report, sample recovery using a protein standard is studied, comparing focusing relaxation and hydrodynamic relaxation process at various field strengths and outflow rate conditions. It is shown that a conventional AFlFFF channel provides a better resolution than a frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) channel, with sample recoveries up to 80% under moderate field strength conditions. Focusing relaxation appears to provide flexibility in selecting high speed run conditions without losing resolution, since sample relaxation is expected to be achieved more completely than hydrodynamic relaxation. The FI-AFlFFF channel utilizing the hydrodynamic relaxation provides a relatively poor resolution and separation speed; however, it performs well under higher field strength conditions with higher sample recoveries than the conventional channel and it is remarkably convenient in system operation by using stopless flow sample injection method. The low separation speed can be overcome by a simple application of a field programming technique to FI-AFlFFF.

Stopless Separation of Proteins by Frit-Inlet Asymmetrical Flow Field-Flow Fractionation

The frit inlet asymmetrical flow field-flow fractionation is applied to the separation of proteins by using a stopless flow injection procedure. By utilizing a small permeable frit near the injection point in an asymmetrical flow FFF channel, sample materials injected to the flow streams can be hydrodynamically relaxed by the compressing action of high speed frit flow and the focusing/relaxation procedure can be bypassed. The separation efficiency of the frit inlet asymmetrical flow FFF channel is demonstrated with few protein standards by examining the influence of the ratio of injection flow rate to frit flow rate on the band broadening during relaxation.