Asymmetric flow field-flow fractionation with multiangle light scattering detection for characterization of cellulose nanocrystals

Cellulose nanocrystals (CNCs) were analyzed by asymmetric flow field-flow fractionation (AF4) coupled with multiangle light scattering (MALS) detection. Small fractions were collected from the output of the AF4 apparatus for investigation by transmission electron microscopy (TEM). The influence of CNC injection amount, the number of passes through a high-pressure homogenizer, and different CNC sources on the elution behavior and particle size distribution was investigated. The AF4-MALS results on crystal length were compared with those from TEM. Peak distortion and variation in elution profiles with the increase in sample load were observed. Good resolution was obtained when the injection mass varied from 20 to 40 μg, corresponding to injections of 4-8 μL at a starting concentration of ～5 μg/μL; concentrations during the separation process and at the detector were significantly lower. As the number of homogenization treatments increased, the peak shape became narrower and more symmetrical. This indicates a narrowed crystal length distribution, but regardless of source or homogenization treatment, no CNC preparation was as uniform as tobacco mosaic virus, a well-known rigid rod model structure, whose length was found by AF4-MALS to be in agreement with literature values. CNCs derived from cotton contained longer crystals than those derived from microcrystalline cellulose, as shown by both AF4-MALS and TEM techniques. An advantage of AF4-MALS compared to TEM is the ability to sample large numbers of rodlike particles, which is challenging and time-consuming for TEM image analysis, especially without the presorting afforded by AF4. The good TMV results suggest a high degree of accuracy will pertain to the CNC size distribution measurements.     

Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field-flow fractionation and multiangle light scattering techniques

A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF-MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF-MALS analysis are discussed.     

Branching features of amylopectins and glycogen determined by asymmetrical flow field flow fractionation coupled with multiangle laser light scattering

The aim of this work was to characterize starch polysaccharides using asymmetrical flow field flow fractionation coupled with multiangle laser light scattering. Amylopectins from eight different botanical sources and rabbit liver glycogen were studied. Amylopectins and glycogen were completely solubilized and analyzed, and high mass recoveries were achieved (81.7-100.0%). Amylopectin Mw, RG, and the hydrodynamic coefficient nuG (the slope of the log-log plot of RGi vs Mi) were within the ranges 1.05-3.18 x 10(8) g mol(-1), 163-229 nm, 0.37-0.49, respectively. The data were also considered in terms of structural parameters. The results were analyzed by comparison with the theory of hyperbranched polymers (Flory, P. J. Principles of Polymer Chemistry; Cornell University Press: Ithaca, NY, 1953; Burchard, W. Macromolecules, 1977, 10, 919-927). This theory, based upon the ABC model, has been shown to underestimate the branching degrees of amylopectins. However, quantitative agreement with the data in the literature was found for amylopectins when using the ABC model modified by the introduction of a multiplying factor, determined from previously described amylopectin structures in terms of the number of branching point calculations.     

Conformational change and aggregation of κ-carrageenan studied by flow field-flow fractionation and multiangle light scattering

The relatively novel combination of flow field-flow fractionation (FFF) and multiangle light scattering (MALS) was employed to study a nondegraded κ-carrageenan in different 0.1M salt solutions. The applicability of the technique was tested, and the effects of salt type and salt composition on the molar mass and radius of gyration were studied. A conformational ordering was induced at room temperature by switching the solvent from 0.1M NaCl (coil form) to 0.1M NaI (helix form). An approximate doubling of the average molar mass and an increase in radius of gyration was then observed, in agreement with results obtained previously using size exclusion chromatography–MALS. This increase in size was attributed to conformational ordering and to the formation of double helices. Severe aggregation was observed above 40% CsI in the 0.1M mixed salt solution of CsI and NaI. This was ascribed to the association of helices into large aggregates. For these large associates, having molar masses of several millions, a reversal of the elution order in flow FFF was detected. © 1998 John Wiley & Sons, Inc. Biopoly 45: 85–96 1998     

Flow field-flow fractionation/multiangle light scattering of sodium hyaluronate from various degradation processes

Sodium hyaluronate (NaHA) is an ultrahigh molecular weight polysaccharide that is found in body tissues, synovial fluid, the vitreous humor, and the umbilical cord, and the size characterization of NaHA is important in pharmaceutical applications. On-line field-flow fractionation/multiangle light scattering/differential refractive index (FlFFF/MALS/DRI) has been applied for the study of degradation efficiency of sodium hyaluronate (NaHA). A NaHA raw sample was degraded by different chemical or physical methods and the degraded NaHA samples were separated using field-programming FlFFF, in which separation is achieved by differences in diffusion coefficients or hydrodynamic diameters. Separation was followed by serial detection using MALS and DRI. Molecular weight distribution (MWD) and information relating to the radius of gyration of the NaHA samples were examined for the raw and degraded NaHA samples. Samples studied include: two different products of ultrasonic degradation, two products of alkaline degradation, and four different products of enzymatic degradation. While alkaline degradation showed a moderate degradation compared to ultrasonic and enzymatic methods in reducing average MW, the latter two degradation methods showed significant changes in average molecular weight and in conformation of NaHA.     

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.     

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.     

Vesicle sizing: Number distributions by dynamic light scattering

A procedure is described which optimizes nonnegative least squares and exponential sampling fitting methods for analysis of dynamic light scattering (DLS) data from aqueous suspensions of vesicle/liposome systems. This approach utilizes a Rayleigh-Gans-Debye form factor for a coated sphere and yields number distributions which can be compared directly to distributions obtained by freeze-fracture electron microscopy (EM). Excellent agreement between the DLS and EM results are obtained for vesicle size distributions in the 100-200-nm range.     

Cell discrimination by multiangle light scattering.

Measurement of the light scattered by biological cells as a function of scattering angle provides information that can be correlated with cell type. Two flow systems that provide multiangle scattering data from cells have been constructed and tested. The first utilizes two narrow-aperture detectors positioned at different angles; the second utilizes the motion of the cell to generate complete scatter patterns of individual cells over a 67 degrees range of scattering angle.     

Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid

Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150 mmol L^?1 sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.     

Characterization of seed nuclei in glucagon aggregation using light scattering methods and field-flow fractionation

Background  

Glucagon is a peptide hormone with many uses as a therapeutic agent, including the emergency treatment of hypoglycemia. Physical instability of glucagon in solution leads to problems with the manufacture, formulation, and delivery of this pharmaceutical product. Glucagon has been shown to aggregate and form fibrils and gels in vitro. Small oligomeric precursors serve to initiate and nucleate the aggregation process. In this study, these initial aggregates, or seed nuclei, are characterized in bulk solution using light scattering methods and field-flow fractionation.     

Molecular characterization of multivalent bioconjugates by size-exclusion chromatography with multiangle laser light scattering

The degree of substitution and valency of bioconjugate reaction products are often poorly judged or require multiple time- and product-consuming chemical characterization methods. These aspects become critical when analyzing and optimizing the potency of costly polyvalent bioactive conjugates. In this study, size-exclusion chromatography with multiangle laser light scattering was paired with refractive index detection and ultraviolet spectroscopy (SEC-MALS-RI-UV) to characterize the reaction efficiency, degree of substitution, and valency of the products of conjugation of either peptides or proteins to a biopolymer scaffold, i.e., hyaluronic acid (HyA). Molecular characterization was more complete compared to estimates from a protein quantification assay, and exploitation of this method led to more accurate deduction of the molecular structures of polymer bioconjugates. Information obtained using this technique can improve macromolecular engineering design principles and help to better understand multivalent macromolecular interactions in biological systems.     

Quantitative analysis of virus-like particle size and distribution by field-flow fractionation

Asymmetric flow field-flow fractionation (AFFFF) coupled with multiple-angle light scattering (MALS) is a powerful technique showing potential for the analysis of pharmaceutically-relevant virus-like particles (VLPs). A lack of published methods, and concerns that membrane adsorption during sample fractionation may cause sample aggregation, have limited widespread acceptance. Here we report a reliable optimized method for VLP analysis using AFFFF-MALS, and benchmark it against dynamic light scattering (DLS) and transmission electron microscopy (TEM). By comparing chemically identical VLPs having very different quaternary structure, sourced from both bacteria and insect cells, we show that optimized AFFFF analysis does not cause significant aggregation, and that accurate size and distribution information can be obtained for heterogeneous samples in a way not possible with TEM and DLS. Optimized AFFFF thus provides a quantitative way to monitor batch consistency for new vaccine products, and rapidly provides unique information on the whole population of particles within a sample.     

Cortical cell elution by sedimentation field-flow fractionation.

As a cell sorter, Sedimentation field-flow fractionation (SdFFF) can be defined as an effective tool for cell separation and purification, respecting integrity and viability as well as providing enhanced recovery and purified sterile fraction collection. The complex cell suspension containing both neurons and glial cells of all types, obtained from cerebral cortices of 17-day-old rat fetuses, is routinely used as a model of primary neuronal culture. Using SdFFF, this complex cell mixture was eluted in sterile fractions which were collected and cultured. SdFFF cell elution was conducted under strictly defined conditions: rapid cell elution, high recovery (negligible cell trapping), short- and long-term cell viability, sterile collection. After immunological cellular type characterization (neurons and glial cells) of cultured cells, our results demonstrated the effectiveness of SdFFF to provide, in less than 6 min, viable and enriched neurons which can be cultured for further investigations.     

Rapid protein separation and diffusion coefficient measurement by frit inlet flow field-flow fractionation.

In this study three flow field-flow fractionation (flow FFF) channels are utilized for the separation of proteins and for the simultaneous measurement of their translational diffusion coefficients, D. One channel has a traditional sample inlet, whereas the other two incorporate a frit inlet design that permits more convenient and rapid sample introduction. The dependence of retention time on D, which leads to differential elution and the opportunity to measure D for protein peaks purified by the flow FFF process, is described theoretically and examined experimentally. Factors affecting band broadening, resolution, and optimization are also examined. The separation of proteins is achieved in the time range 4-20 min. Partial resolution is achieved in multiple runs requiring 2 min each. Values of D calculated from retention times are reported for 15 proteins. These include two protein dimers (bovine serum albumin and gamma-globulin) not ordinarily accessible to measurement. The D values from the three channels are compared with one another and with literature data. Reasonable consistency (within 3-4%) is found. High-speed repetitive runs can be used to acquire multiple values of D in time intervals as short as 1 min.     

Monitoring DNA/poly-L-lysine polyplex formation with time-resolved multiangle laser light scattering

Nonviral DNA complexes show promise as alternative and attractive gene delivery vectors for treating genetic diseases. Nonviral DNA complexes are typically formed by combining DNA with various condensing/complexing agents such as lipids, polyelectrolytes, polymers, polypeptides, and surfactants in solution. DNA/poly-L-lysine polyplex formation kinetics are probed by time-resolved multiangle laser light scattering (TR-MALLS), which yields the time evolution of the supramolecular complex mass and geometric size. Primary polyplexes whose geometric size is smaller than individual DNA molecules in solution are formed very rapidly upon mixing DNA and poly-L-lysine. Over time, these primary polyplexes aggregate into larger structures whose ultimate size is determined primarily by the relative concentrations of DNA and poly-L-lysine. This final polyplex size varies with the DNA/poly-L-lysine mass ratio in a non-monotonic fashion, with the maximum polyplex size occurring at a DNA/poly-L-lysine mass ratio of approximately two to three (charge ratio near unity). The utility of TR-MALLS for monitoring the temporal evolution of DNA loading and supramolecular complex size growth (mean square radius and molar mass) throughout the DNA/poly-L-lysine polyplex formation process is demonstrated. The polyplex DNA loading and size, both geometric and molar mass, are key to understanding the transfection process and for developing optimal gene therapy vectors.     

Effects of normalization errors on size distributions obtained from dynamic light scattering data.

This paper presents a study of the influence of normalization errors on size distributions obtained from the analysis of intensity fluctuations by photon correlation spectroscopy. The effects of these errors are demonstrated by means of computer-generated autocorrelation functions simulating light scattered from a monomodal Schulz distribution of small, spherical, unilamellar lipid vesicles. The calculations show that even small errors in the baseline, modifying the data upon normalization systematically, will cause serious errors in the estimated size distribution. As it turns out this is due to the peculiar characteristics of normalization errors in data of the first order autocorrelation function. The errors introduced there are described in parts by functions of the delay time having positive exponents. Such components are not considered in the integral equations commonly used to analyze the measured data. The error's property to be a function of delay time in turn enables us to obtain the relative baseline error from the inversion of the data. The new method for its determination is described in some detail. Here, it has been realized with a modified version of the size distribution algorithm CONTIN.     

Aggregation of amphiphilic pullulan derivatives evidenced by on-line flow field flow fractionation/multi-angle laser light scattering

Size-exclusion chromatography (SEC) is a useful steric separation technique for the analysis of water-soluble polysaccharides in aqueous solution. However, in the case of amphiphilic derivatives, the usefulness is limited because of interactions between hydrophobic segments and the stationary phase. Alkyl-bearing pullulans differing from the extent and the length of alkyl groups were characterized using flow-field flow fractionation with on-line coupling multi-angle laser light scattering (F⁴/MALLS). Comparison of SEC and F⁴ is presented and the interest of F⁴ in the field of amphiphilic derivatives is demonstrated.     

Determination of size and charge distributions by combination of quasi-elastic light scattering and band transport

A convenient method for the determination of size and charge distributions of polydisperse samples is described. It includes three steps: (a) band transport to disperse the components of the sample in a scattering cell; (b) total intensity scattering to determine the concentration and mobility at each location; and (c) quasi-elastic light scattering (QLS) to characterize the hydrodynamic sizes. Combining the results of (b) and (c) yields size and charge distributions. Since complete resolution of the various components of the sample cannot occur in step (a), a method is described to estimate the average form factor, from the local mean size and the local degree of polydispersity obtained by QLS. The potential of this method is demonstrated by the results of its application to two radiocolloids which have broad size distribution (50- to 600-nm diameters).     

Characterization of yeast cultivations by steric sedimentation field-flow fractionation.

The characterization and quantification of biomass is often time consuming and dependent on the cultivation media and gives no detailed information between cell size and shape and their productivity. By monitoring the bioprocess with steric sedimentation field-flow fractionation (Sd/StFFF) in combination with laser light scattering, not only cell growth, but also the variation of cell size and shape during the cultivation, can be observed. In this work, the feasibility of separating and characterizing cell populations by steric sedimentation field-flow fractionation is demonstrated by its application to three different yeast cultivation broths. For this purpose samples which were collected at different cultivation times were injected into an FFF system. Fractograms were obtained in less than 4 min. Due to the relatively high resolution of the method, a cell sample could be fractionated in several subpopulations differing in their size as well as in their number of buds.