Hydrodynamic size

The elution volume of a molecule in HPSEC is determined by its hydrodynamic size and the pore size of the column packing. In setting up an HPSEC experiment, the chromatographer must match the pore size of the column to the molecular size range of the sample. 

Proteins are separated on Zorbax GF columns based on their hydrodynamic size, which may be related to the proteins molecular weights (Fig. 3.10). Under ideal conditions, two proteins whose molecular sizes differ by a factor of 2 can be baseline separated. 

FIGURE 3.10 Under ideal conditions, proteins are separated on Zorbax GF columns based on their hydrodynamic size. 

There is usually an ionic strength above which there is no more effect on hydrodynamic size or, worse yet, there are hydrophobic interactions of the polymer with the stationary phase. Thus, the optimum 1 is usually at the low 1 end of the plateau of size vs 1. This concentration will minimize ionic strength effects while also minimizing wear on the pump seals and pistons. 

TTiis ion exclusion effect can sometimes be exploited beneficially. For example, by purposefully choosing a column with some carboxyl groups and a pH that ionizes them (greater than approximately 6.5), it may allow separation of a charged and an uncharged polymer that have the same hydrodynamic size. Alternatively, one may be able to fine-tune elution of a polymer by adjusting pH. 

Figure 22.3 shows such theoretical curves as well as experimental points of capillary columns with 0.6- to 1.4-/i.m radii. The separation ranges of these capillary columns are from 5 X 10 to 10. Most of the data points follow the modified DG model. With OTHdC, the molecular hydrodynamic size can be calculated. However, the separation range of a single capillary column is relatively narrow, only about 1.5 order of magnitudes. 

The major advantage of the capillary hydrodynamic chromatography is that the mobile phase does not need to have similar solubility parameter as the sample and packing material. (In SEC, nonsize exclusion effects may be observed if the solubility parameter of the sample, packing material, or mobile phase is considerably different.) Therefore, the hydrodynamic size of polymers can be studied in a 0 solvent and even in a solvent that is not compatible with any currently available SEC packing material (9). Figure 22.4 is an example of polystyrene separation in both THF and diethyl malonate. Diethyl malonate is the 0 solvent of polystyrene at 31-36 C. 

The larger macromolecules can be separated using larger particle size columns. However, the flow rate should be watched carefully. As the effective hydrodynamic size of the macromolecules may be reduced due to the deformation by shear (23). Figure 22.8 shows that the effective hydrodynamic size of a 12-15 X 10 MW polyacrylamide sample will not reach its maximum, or the size without shear, unless the flow rate is reduced to 0.01 ml/min. A... 

The length of monomer unit of MA is taken to be 0.25 nm [71]. The length of the polymer chain equal to the turn or the diameter of the protein globule is calculated taking into account the hydrodynamic size of the protein molecule via Stockes radius (Rsl). 

The size distribution of the PVCL microgel particles synthesised by a batch emulsion polymerisation was monomodal and reasonably narrow [177], regardless of the choice of the emulgator, SDS (El, E2) or macromonomer (E3, E4). The size distributions remain monomodal upon subsequent grafting. A typical size distribution of a PVCL microgel (El) at 20 °C is presented in Fig. 17, as well as the effect of grafting, as a second step, on the hydrodynamic size (El-g). 

The results are discussed in terms of collision rate theories where the shear rate in the system and the hydrodynamic sizes of the particles and the polymer molecules are considered. 

The technique allows fine particles to be examined in a liquid environment so that estimates can be made of their effective hydrodynamic sizes. This is not possible using other techniques. 

For many years, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) methods have been used as an essential tool to determine the hydrodynamic size, monitor product purity, detect minor product or process-related impurities, and confirm batch-to-batch consistency of protein and antibody products. ITowever, gel-based techniques have several limitations, such as lack of automation, varying reproducibility, and a limited linear range. SDS-PAGE is also labor-intensive and generates large volume of toxic waste. Most importantly, the technique does not provide quantitative results for purity and impurity determination of proteins and antibodies. 

The CE-SDS method is a size-based separation technique generally applicable to proteins from 10 to -200—300kDa. The specificity is generally tested against the formulation buffer and any other possible contaminant proteins. There is usually no interference from the formulation buffer with the assay. For samples that contain contaminant proteins with a hydrodynamic size of 10—200kDa, the method is not specific. 

In CZE, proteins are separated from each other based on the differences in their electrophoretic mobilities. The electrophoretic mobility is a function of the molecular charge and hydrodynamic size of a protein. In a given environment, the electrophoretic mobility is an intrinsic property of the protein, similar to the isoelectric point. Therefore, the mobility can be used to distinguish proteins from each other. This is the basis for using CZE as a simple identity test for final product and package labeling. As an example, the CZE profiles of six... 

In order to calculate the hydrodynamic size (Stokes size) of metal nanoparticles with the surrounding envelope, the Taylor dispersion method has been proposed (50). Since metal nanoparticles can be detected by UV-Vis absorption in this method, only the size of the particles involving metal nanoparticles can be determined (Fig. 9.1.7). 

The success of PEG-ADA led to other applications of this surface-modification strategy. In particular, the ability of PEG to increase the hydrodynamic size of IL-2... 

Note Soluble recombinant interleukin-2 (IL-2) was modified with various lengths of polyethylene glycol (PEG) polymers with average molecular weight listed. The final PEG to IL-2 ratio is listed as molar ratio, and their respective hydrodynamic size was determined based on their sendimentation coefficient. Hence the molecular weight listed in Effective Size is not the sum of the PEG and IL-2 with consideration of molar ratio of the PEG-IL-2 conjugates. 

Under a variety of conditions, plasmid DNA undergoes a dramatic compaction in the presence of condensing agents such as multivalent cations and cationic polymers. Naked DNA coils, typically with a hydrodynamic size of hundreds of nanometers, after condensation it may become only tens of nanometer in size. Contrary to proteins which show a unique tertiary structure, DNA coils do not condense into unique compact structure. Cationic polymers execute their gene carrier function by their condensation effect on gene materials and, furthermore, their protection effect on DNA from nuclease digestion. Currently, the most widely used cationic polymers in research include linear or branched PEI (poly (ethyleneimine) (161-165), polypeptides such as PLL (poly-L-lysine) (166-169), PLA (poly-L-arginine) (170). 

Figure 8 illustrates the relationship between inherent viscosity (IV) and concentration for PBI/PAr/NMP solutions. It is interesting to note that the IV of all solution blends exhibited normal polymer solution characteristics. At a fixed concentration (0.5%), it was noted that the IV of the solution blends exceeded the rule of mixtures (see Fig. 9) suggesting that PBI and PAr exhibit specific interactions in a dilute solution, such that the resulting hydrodynamic sizes of the blends were greater than that of the calculated averages based on each component. 

In theory, if the net charge, q, on a molecule is known, it should be possible to measure / and obtain information about the hydrodynamic size and shape of that molecule by investigating its mobility in an electric field. Attempts to define /by electrophoresis have not been successful, primarily because Equation 4.3 does not adequately describe the electrophoretic process. Important factors that are not accounted for in the equation are interaction of migrating molecules with the support medium and shielding of the molecules by buffer ions. This means that electrophoresis is not useful for describing specific details about the shape of a molecule. Instead, it has been applied to the analysis of purity and size of macromolecules. Each molecule in a mixture is expected to have a unique charge and size, and its mobility in an electric field will therefore be unique. This expectation forms the basis for analysis and separation by all electrophoretic methods. The technique is especially useful for the analysis of amino acids, peptides, proteins, nucleotides, nucleic acids, and other charged molecules. 

Viscosity Water binding, hydrodynamic size, shape Soups, gravies, salad dressings ... 

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