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– of Proteins, Antibodies, Viruses, Polymers and Nano-/Microparticles

Many applications are based on the specific properties of these materials. For this reason, an exact knowledge of the molar mass or particle sizes distribution is essential for later application as well as for optimization of the production processes or quality control. In addition the knowledge about the chain structure of macromolecules or biopolymers as well as about the shape of particles is often necessary. For this reason those samples, which often can be very complex systems, have to be separated according to parameters such as e. After the separation process the resulting narrow dispersed fractions are detected with different techniques.

Thus, an optimal separation is necessary to ensure that the obtained values are correct, otherwise the best detector will deliver wrong or at least partially erroneous results [].

SC2000 Modular SEC

So far the separation of very large or highly adsorbing material was partially realized by chromatographic techniques such as e. The chromatographic methods are mostly based on the separation of the analyte inside a porous stationary phase, this leads to particularly high shear forces and there is a permanent risk of adsorption present. The most chromatographic techniques show high performance for low or medium size material but for higher molar masses or increased particle diameters they are strongly limited and deliver insufficient or even no separation.

The characterization of polymers and particles by field flow fractionation offers the possibility of a universal separation with high resolution [4]. Also very large structures can be analyzed [5] under shear free conditions and without filtration by porous material or unwanted adsorption on the surface of a stationary phase.

The analyte species, which are dissolved or dispersed in the solvent, will be separated in an empty channel. This separation can be according to size, chemical composition or density. An external field force, which acts perpendicular to the carrier-flow, is the source of sample separation. The cross-flow in AF4 leads to an accumulation of the analyte species at the channel bottom.

The size-dependent diffusion abilities of particles or molecules lead to an arrangement in different layers of the parabolic flow profile inside the channel. As a result the analyte components with small hydrodynamic diameter will elute first and the larger molecules or particles will elute later. In Thermal FFF a temperature gradient is used for the separation of the sample. Since the thermal diffusion coefficient also depends on the chemical nature of the analyte this method can be used for separation according to both, size and chemical composition. As a result components with the same hydrodynamic radius but different composition can be separated by TF3.

This method allows an improved separation according to size and density. Consequently the separation of particles with the same diameter and different density can be performed. The high selectivity for dense material causes an improved retention also of very small particles despite their high diffusion coefficients. These advantages make the CF3 to an ideal method for the analysis of compact particles. All FFF separation fields can be adjusted in their strength. A gradient function of any shape can be applied.

Thus the separation efficiency can individually be adapted with regard to each separation problem. A tailor-made calibration curve can be applied. In FFF the separation is not limited like it is the case in SEC where the separation can only be in the range of the default calibration curve which is defined by the given column-combination. In addition FFF can be used together with various detector combinations.

The most important setup is multi angle light scattering MALS together with concentration sensitive devices such as RI, UV or IR, which enables to detect the molar mass as well as the radius of gyration simultaneously [6]. The AF4 technique can be used for characterization of both, polymers and particles.


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The separation depends on the hydrodynamic radius of the species which is a very universal character of the most samples. The fractogram in Fig. A separation with high resolution can easily be performed over a huge size range within a relatively short analysis time. The performance of the AF4 compared with conventional methods can easily be demonstrated by the example of the separation of e. In Fig. For both samples the high molar mass is combined with an increased degree of branching which leads to important application properties such as high stability and low viscosity of the melt or solution.

The radii and molar masses were determined by MALS detection. The comparison of SEC and AF4 separation impressively shows the magnitude of the FFF principle and the variety of additional information such as the correct molar mass or the complete branching information from AF4.


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On the contrary, a correct evaluation of the SEC-MALS-data is not possible due to the high amount of shear degradation and the high partial retention of large macromolecules. Thus, the method can be applied for the analysis of the most polymeric materials. Important commodity polymers, like e. Also rubbers can be fully analyzed by AF4, while the SEC provides more or less insufficient information.

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In the analysis of the rubber sample which is shown in Fig. Since the samples are not filtered by a column during AF4 analysis, a high molar mass shoulder gets visible in the Light scattering signal of the rubber in Fig. This shoulder represents the gel content of the sample. The low slope of the Rg-M-relationship indicates highly branched or cross linked material with very compact coil structures in solution. Thus AF4 often offers a new perspective on many interesting samples, which were claimed to be already well understood in the past. In a similar way like for synthetic polymers also natural material such as proteins, starches and alginates can be separated.

In addition AF4 is also well suited for characterization of higher biological structures such as micelles or viruses as well as for dense particulate samples like e. In addition to the advantages of the shear-free und universal separation which is realized by the adjustable cross-flow gradient, AF4 offers also other possibilities for the optimization of the sample analysis. A focusing of the sample at the beginning of the separation process allows e. The injected sample is focused in a narrow zone at the channel entrance by a second inlet stream focus flow which counteracts the carrier flow.

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Analysis of complex polymers by multidetector field-flow fractionation. Ashwell Makan. Advanced detector combi- native to column-based polymer fractionation methods such nations are discussed, most prominently the very recently as size-exclusion chromatography SEC or interaction chro- developed coupling to 1H NMR. Finally, analysis of polymer matography IC. The most common polymer fractionation nanocomposites by asymmetric flow field-flow fractionation method, SEC, has its limitations when polymers with very AF4 —FTIR is presented.

Another limitation of all column-based methods is that the Keywords Field-flow fractionation. Polymer samples must be filtered before analysis and shear degradation nanocomposites. Finally, the separation of very polar polymers may be a challenge because such polymers Introduction interact very strongly with the stationary phase, causing irre- versible adsorption or other negative effects.

This article re- Complex polymers have two or more distributions in molec- views the latest developments in field-flow fractionation of ular properties, for example molar mass distribution MMD complex polymers. It is demonstrated that some of the limita- and chemical composition distribution CCD , functionality tions of column-based chromatography can be overcome by type distribution FTD , or distribution in molecular topology FFF.

When appropriate, results from column-based fraction- MTD. The comprehensive characterization of a complex ations are compared with those from FFF fractionations to polymer requires quantitative analysis not only of each of highlight the specific merits and challenges of each method.

In these distributions but also the correlation between them. In addition to the fractionations themselves, various detector all cases, quantitative analysis of a specific distribution must setups are discussed to show that different polymer distribu- be based on fractionation according to the specific molecular tions require different experimental procedures.

Examples are property.

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The most widely used technique for analysis of the given of the analysis of molar mass distribution, chemical molar mass distribution of complex polymers is size-exclusion chromatography SEC. SEC is the most common and popular Published in the topical collection Field-Flow Fractionation with guest method for molecular size separation but, as with all other editors S.

Kim R.

Publication details

Williams and Karin D. These relate to converting molecu- H. Makan : H. Chirowodza : N.

Ngaza lar size into molar mass for complex polymers by use of Department of Chemistry and Polymer Science, University of suitable elution volume vs. Stellenbosch, Private Bag X1, Matieland, South Africa Other problems are encountered with polymers having very e-mail: hpasch sun. Makan position or topology distributions.