Frit Inlet Asymmetrical Flow Field-flow Fractionation Research Articles

Characterization of dextran particle size: How frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) coupled online with dynamic light scattering (DLS) leads to enhanced size distribution

Accurate determinations of particle size and particle size distribution (PSD) are essential to achieve the clinical translation of medical nanoparticles (NPs). Herein, dextran-based NPs produced via a water-in-oil emulsification/crosslinking process and developed as nanomedicines were studied. NPs were first characterized using traditional batch-mode techniques as dynamic light scattering (DLS) and laser diffraction. In a second step, their analysis by frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) was explored. The major parameters of the AF4 procedure, namely, crossflow, detector flow, crossflow decay programming and relaxation time were set up. The sizes of the particle fractions eluted under optimized conditions were measured using DLS as an online detector. We demonstrate that FI-AF4 is a powerful method to characterize dextran-NPs in the 200 nm -1 µm range. It provided a more realistic and comprehensive picture of PSD, revealing its heterogenous character and clearly showing the ratio of different populations in the sample, while batch-mode light scattering techniques only detected the biggest particle sizes.

Evaluation of exosome separation from human serum by frit-inlet asymmetrical flow field-flow fractionation and multiangle light scattering

Exosomes are extracellular vesicles that mediate intercellular communication, immune response, and tumour metastasis. However, exosome isolation from the blood is complicated because their size and density are similar to those of blood lipoproteins. Here, we employed field programming frit-inlet asymmetrical flow field-flow fractionation (FIAF4) coupled with multiangle light scattering (MALS) for the effective separation of exosomes from free unbound proteins and lipoproteins present in serum samples using different pre-treatment methods, namely, a commercial exosome isolation kit, ultracentrifugation (UC), and a simple centrifugation followed by ultrafiltration (UF). Sizes of the eluted exosomes, as calculated by MALS signals, approximated well with the results of batch dynamic light scattering of the collected fractions and with the sizes of polystyrene particles. Exosome separation from lipoproteins was validated by western blotting with several markers of exosomes and lipoproteins, followed by proteomic analysis using nanoflow ultrahigh-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry. UC requires relatively large amounts of serum samples (at least 2 mL) but is more efficient at removing lipoproteins. The UF method with a centrifugal concentrator (300 kDa) was found to be more effective in retrieving exosomes with low serum volumes (50 μL). Altogether, this study demonstrates the application of field programming FIAF4 for the isolation/purification of exosomes from proteins and lipoproteins in the serum.

Effect of cationic monomer content on polyacrylamide copolymers by frit-inlet asymmetrical flow field-flow fractionation/multi-angle light scattering

In this study, ultrahigh-molecular-weight (MW) (>107Da) cationic polyacrylamides (C-PAMs), which are water-soluble polymers used in waste water treatment, were characterized using frit-inlet asymmetrical flow field-flow fractionation coupled with multi-angle light scattering and differential refractive detection. C-PAMs copolymerized with acryloxyethyltrimethyl ammonium chloride (DAC) were prepared by varying the feed amount of cationic monomer, polymerization method (solution vs. emulsion), and degree of branching. The MW of the copolymers prepared using emulsion polymerization (107–109Da) was generally larger than that of copolymers prepared using solution polymerization (4×107–108Da). When the amount of cationic monomer was increased from 10 to 55mol% in solution polymerization, hydrophobic contraction of the core induced formation of more compact C-PAMs. The copolymers prepared using emulsion polymerization formed highly aggregated or supercoil structures owing to increased intermolecular hydrophobic interaction when less cationic monomer was used. However, the MW decreased with increased cationic group content. In addition, C-PAMs larger than ∼108Da prepared using the emulsion method were separated by steric/hyperlayer elution mode while those in the 107–108Da range were analyzed in either normal or steric/hyperlayer mode depending on the decay patterns of field programming. Moreover, branched copolymers were found to be resolved with different elution modes under the same field decay pattern depending on the degree of branching: steric/hyperlayer for low-branching and normal for high-branching C-PAMs.

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.

Effect of Inlet Frit Lengths on the Hydrodynamic Relaxation Efficiency in Frit Inlet Asymmetrical Flow Field‐Flow Fractionation

The efficiency of a frit inlet asymmetrical flow field‐flow fractionation (FI‐AFlFFF) channel has been evaluated by varying the length of the inlet frit element. In a FI‐AFlFFF channel, a high speed frit flow is introduced through the inlet frit to provide hydrodynamic relaxation of the incoming sample stream from the channel inlet. The experimental plate heights and peak recovery values are examined for three different FI‐AFlFFF channels by varying field strengths and the channel membrane materials. It has been found that the length of the inlet frit element influences the performance of hydrodynamic relaxation, as well as peak recovery in the FI‐AFlFFF channel system. Experimental plate height data show that the hydrodynamic relaxation itself occurs more efficiently when the length of inlet frit is longer. While an FI‐AFlFFF channel having a longer inlet frit (4.2 cm long) gives a good relaxation, it shows less efficiency in considering peak recovery over a range of the field strengths examined in this study. A similar tendency is observed when channel membrane is varied. By considering the efficiency of hydrodynamic relaxation and sample recovery during elution, an FI‐AFlFFF channel of an intermediate length of inlet frit (3.2 cm) is shown, from experiments, to be optimum.

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.

Retention and Efficiency in Frit-Inlet Asymmetrical Flow Field-Flow Fractionation

Sample relaxation in the frit-inlet asymmetrical flow field-flow fractionation (FIA-FlFFF) channel is accomplished hydrodynamically. The focusing/relaxation procedure, which is essential to the efficient operation of conventional asymmetrical flow FFF, is therefore avoided. It follows that for FIA-FlFFF there is no requirement for valve switching to achieve convergent (focusing) channel flows, and sample relaxation and separation proceed without interruption. This represents a major advance in the facility of operation of asymmetrical flow FFF. In this work, we present the derivation of equations to describe the variation in mean channel flow velocity along the full length of the frit-inlet asymmetrical channel. Equations for channel void time (the time for a nonretained material to pass through the channel) are also presented. The equations are sufficiently general so as to be applicable to both rectangular and trapezoidal channel designs. On the basis of these equations, the retention time for a sample component can be calculated by applying basic flow FFF theory. Simple equations are also presented to determine conditions consistent with complete relaxation of samples within the frit-inlet region. Such conditions ensure maximization of separation efficiency during subsequent elution. The retention and resolving power of FIA-FlFFF are demonstrated using sets of standard latex spheres and proteins. Experimental data show that retention in FIA-FlFFF follows the general principles of field-flow fractionation, provided the variation in flow velocity is correctly accounted for. Finally, effects of sample load and injection volume are also discussed.

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.