Practical Applications of Asymmetrical Flow Field-Flow Fractionation (AF4): A Review

Characterization of macromolecules and colloids is an area of considerable interest. Asymmetrical flow field-flow fractionation (AF4) has become a well-established method, but many potential users possess limited knowledge of its capabilities, or how it can provide additional information or serve as validation to the traditional analytical techniques. This review article highlights several practical applications where AF4 should be given special consideration, and discusses benefits and drawbacks of the different methods

Size separations of starch of different botanical origin studied by asymmetrical-flow field-flow fractionation and multiangle light scattering.

Asymmetrical-flow field-flow fractionation combined with multiangle light scattering and refractive index detection has been revealed to be a powerful tool for starch characterization. It is based on size separation according to the hydrodynamic diameter of the starch components. Starch from a wide range of different botanical sources were studied, including normal starch and high-amylose and high-amylopectin starch. The starch was dissolved by heat treatment at elevated pressure in a laboratory autoclave. This gave clear solutions with no granular residues. Amylose retrogradation was prevented by using freshly dissolved samples. Programmed cross flow starting at 1.0 mL min(-1) and decreasing exponentially with a half-life of 4 min was utilised. The starches showed two size populations representing mainly amylose and mainly amylopectin with an overlapping region where amylose and amylopectin were possibly co-eluted. Most of the first population had molar masses below 10(6) g mol(-1), and most of the second size population had molar masses above 10(7) g mol(-1). Large differences were found in the relative amounts of the two populations, the molar mass, and hydrodynamic diameters, depending on the plant source and its varieties

Analysis of Proteins, Biologics, and Nanoparticles in Biological Fluids Using Asymmetrical Flow Field-Flow Fractionation

With the increasing interest in biopharmaceuticals such as proteins, antibodies, and nucleic acids, there is a corresponding increase in the need for characterizing such components. Much effort is spent on characterization in the early drug development phases as well as during formulation development and quality control. One parameter that is commonly investigated is the size distribution of the macromolecular components to deduce if there is aggregation or degradation occurring, if conformational changes occur, or if there are interactions with excipients. While the properties of the protein drug in the buffer system or in the pharmaceutical formulation are important, possibly even more interesting are the properties of the drug once it enters the body. Size characterization of macromolecules in biological fluids has traditionally been an area hampered by the complexity of the matrix. The large amount of indigenous components can interfere with commonly applied analytical techniques for size characterization. However, the separation technique asymmetrical flow field-flow fractionation (AF4) has recently shown increasing applicability for the characterization of components in blood plasma and serum. This article reviews some aspects of applying AF4 to plasma, serum, milk, and cerebrospinal fluid in the field of analysis and characterization of proteins, biologics, and nanoparticles in biological fluids

Programmed cross flow asymmetrical flow field-flow fractionation for the size separation of pullulans and hydroxypropyl cellulose

Different functions for the programming of the cross flow in asymmetrical flow field-flow fractionation were studied with the aim to find the flow conditions most suitable for the molar mass distribution analysis of high molecular weight polysaccharides. A mixture of four differently sized pullulans covering the molar mass range 5.8 x 10(3)-1.6 x 106 g mol(-1) were used as a model sample. Two types of programs were studied, linear and exponential decays, both with and without initial periods of a constant cross flow. For comparison, nonprogrammed runs, i.e. using constant cross flow, were studied. It was found that exponentially decaying cross flow gave the most uniform molar mass selectivity across the fractogram. The programmed cross flow was applied to the molar mass distribution analysis of a technical quality of hydroxypropyl cellulose. (c) 2006 Elsevier B.V. All rights reserved

Competitive adsorption of a polydisperse polymer during emulsification: Experiments and modeling

In this paper we study the selective adsorption of a high molar mass polymer, OSA-starch, at the cyclohexane/water interface during emulsification. This was made possible through the use of AsFlFFF-MALS-RI which enables us to characterize the size and molar mass of polydisperse ultrahigh molar mass polymers. The results show that the high molar mass components in the molar mass distribution of the polymer were selectively adsorbed. The selective adsorption is most likely due to convective mass transport in the turbulent flow fields, during formation of the emulsion, which favors transport to the interface of the high molar mass polymers in the sample. The rapid adsorption that occurs during emulsification is likely to give rise to nonequilibrium effects and jamming at the interface. Furthermore, we describe the adsorption process and illustrate its selective nature through a turbulent flow collision model

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 rrms 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)

Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow field-flow fractionation (AF4)

The analysis of aggregates of therapeutic proteins is crucial in order to ensure efficacy and patient safety. Typically, the analysis is performed in the finished formulation to ensure that aggregates are not present. An important question is, however, what happens to therapeutic proteins, with regard to oligomerization and aggregation, after they have been administrated (i.e., in the blood). In this paper, the separation of whole blood, plasma, and serum is shown using asymmetric flow field-flow fractionation (AF4) with a minimum of sample pre-treatment. Furthermore, the analysis and size characterization of a fluorescent antibody in blood plasma using AF4 are demonstrated. The results show the suitability and strength of AF4 for blood analysis and open new important routes for the analysis and characterization of therapeutic proteins in the blood

Revisiting the dynamics of proteins during milk powder hydration using asymmetric flow field-flow fractionation (AF4)

The dynamics of β-casein and casein micelles in the reconstitution of skim milk were revisited in this study. β-casein migrates into casein micelles upon an increase in temperatures due to an increase in the hydrophobic effect and lower calcium-phosphate cluster solubility. This process can be reversed upon cooling. These phenomena are well known in fresh milk and are not yet clearly established for reconstituted milk powder. As milk powder is commonly used as a functional ingredient in food products, it is of interest to investigate the migration of casein micelle β-casein to and from the serum phase in reconstituted milk. This study aimed to use asymmetrical flow field flow fractionation (AF4) in combination with various detectors to revisit the dynamics of β-casein when reconstituting skim milk at different temperatures. Fluorescence-labelled β-casein was added to fresh and reconstituted skim milk and rapid transport of β-casein into the outer shell of the casein micelles could be observed already after 5 ​min of reconstitution at 50 ​°C. This process stabilized after approximately 5 ​h, which indicates that an equilibrium of β-casein between the serum and the micellar phase was reached. Similar results were found for fresh milk. The apparent density of the casein micelles in the skim milk samples was also found to increase during reconstitution at 50 ​°C. During cold reconstitution of milk powders, the migration of β-casein to the serum was not observed. The results suggest that β-casein was already present in the serum phase upon reconstitution at 6 ​°C. When a sample was reconstituted for 180 ​min at 50 ​°C, the migration of β-casein back into the serum was observed upon cooling the same sample to 6 ​°C. The size of casein micelles in reconstituted milk at 6 ​°C was larger compared to reconstitution at 50 ​°C. With AF4 and the multi-detector approach, the change in concentration and size of casein micelles can be readily investigated and the migration of β-casein can be tracked simultaneously. Therefore, the method is a valuable tool for studies of the properties and changes in various milk samples

Investigating the effect of powder manufacturing and reconstitution on casein micelles using asymmetric flow field-flow fractionation (AF4) and transmission electron microscopy

Milk powders are commonly used for a variety of food products in which among others the milk proteins add to the properties of the products. Processing of milk can, depending on the processing parameters, change the size and structure of the proteins. These changes can be difficult to measure due to the polydispersity of milk components, which makes it a challenge to obtain direct information about the individual proteins. In this paper, the results from an investigation of casein micelle size,size distribution, and structure in reconstituted skim milk and the comparison with raw and pasteurized skim milk are reported. The investigation used asymmetrical flow field-flow fractionation (AF4) in combination with online UV, multi-angle light scattering (MALS), and refractive index (RI) detection and the results were confirmed by transmission electron microscopy (TEM). The results show that there is a difference in casein micelle size distribution between the differently processed milk samples. The casein micelles of the reconstituted milk were found to have a z-average radius of gyration of 72 nm and the casein micelles in the raw and pasteurized skim milk were 58 and 62 nm respectively. The AF4 and TEM data suggest that the cause of the larger casein micelle size is a layer of aggregated whey proteins associated with the casein micelles surface. Moreover, the TEM investigation showed that a larger proportion of the casein micelles are aggregated in reconstituted milk compared to raw and fresh skim milk. Investigation of the effect of reconstitution time shows that the amount of aggregated casein micelles decreases during the first 20 min of reconstitution. The results show that the AF4-method can provide detailed insights into the reconstitution process and properties of different milk samples. Hence, it can be used as a reference or validation for more indirect methods to track the reconstitution of milk powders

Uniform Pores in Cross-Linked Polymers by Dispersed Fumed Silica Templating

Hydrophobic fumed silica dispersions in organic monomers were explored as a pore-forming system in polymer synthesis. The method developed provides a simple and effective way of controlling the pore size in highly cross-linked polymers. Fumed silica suspensions in divinylbenzene were polymerized with subsequent etching of the silica particles, therefore creating the porosity in the polymer. The resulting polymers are mesoporous materials, exhibiting an extremely narrow pore size distribution with an average pore size of about 100 angstrom, replicating the size of the nanofiller. BET surface areas were found appreciably high (similar to 350 m(2)/g). Furthermore, the rheological behavior of the prepolymerization mixtures was studied to elucidate the formation of the porous network and showed that a tridimensional network of particles is formed at a minimum silica fraction (Phi(v)) of 0.08