Measurement of Dynamic Light Scattering Intensity in Gels
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Abstract:
In the scientific literature little attention has been given to the use of dynamic light scattering (DLS) as a tool for extracting the thermodynamic information contained in the absolute intensity of light scattered by gels. In this article we show that DLS yields reliable measurements of the intensity of light scattered by the thermodynamic fluctuations, not only in aqueous polymer solutions but also in hydrogels. In hydrogels, light scattered by osmotic fluctuations is heterodyned by that from static or slowly varying inhomogeneities. The two components are separable owing to their different time scales, giving good experimental agreement with macroscopic measurements of the osmotic pressure. DLS measurements in gels are, however, tributary to depolarized light scattering from the network as well as to multiple light scattering. The paper examines these effects as well as the instrumental corrections required to determine the osmotic modulus. For guest polymers trapped in a hydrogel the measured intensity, extrapolated to zero concentration, is identical to that found by static light scattering from the same polymers in solution. The gel environment modifies the second and third virial coefficients, providing a means of evaluating the interaction between the polymers and the gel.Keywords:
Static light scattering
Intensity
Osmotic pressure
Light intensity
Sedimentation equilibrium
Static light scattering
Protein crystallization
Sedimentation
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The behavior of monoclonal antibodies at high concentrations is important in downstream processing, drug formulation, and drug delivery. The objective of this study was to evaluate the osmotic pressure of a highly purified monoclonal antibody at concentrations up to 250 g/L over a range of pH and ionic strength, and in the presence of specific excipients, using membrane osmometry. Independent measurements of the second virial coefficient were obtained using self-interaction chromatography, and the net protein charge was evaluated using electrophoretic light scattering. The osmotic pressure at pH 5 and low ionic strength was >50 kPa for antibody concentrations above 200 g/L. The second virial coefficients determined from the oncotic pressure (after subtracting the Donnan contribution) were in good qualitative agreement with those determined by self-interaction chromatography. The second virial coefficient decreased with increasing ionic strength and increasing pH due to the reduction in intermolecular electrostatic repulsion. The third virial coefficient was negative under all conditions, suggesting that multi-body interactions in this system are attractive. The virial coefficients were essentially unaffected by addition of sucrose or proline. These results have important implications for the analysis of protein-protein interactions in downstream processing at high protein concentrations.
Osmotic pressure
Osmometer
Vapor pressure osmometry
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Static light scattering
Bovine serum albumin
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Static light scattering
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Abstract The effects of attenuation and secondary scattering on the determination of molecular weights and second virial coefficients by light scattering are investigated. The correction to the molecular weight due to these effects is small unless the reference standard has a high turbidity. The correction to the second virial coefficient is negligible in good solvents but becomes relatively more important as the ⊖‐condition is approached. Determination of the ⊖‐temperature for a polymer‐solvent system by light scattering can be in error by as much as several degrees under certain conditions, viz., large cells, large values of the constant H = (32π 3 n 2 0 /3 N a λ 4 )( dn dc ) 2 , and small values of the Flory‐Krigbaum entropy parameter ψ1.
Static light scattering
Turbidity
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Turbidity
Polystyrene
Static light scattering
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Abstract The method of Flory and a modification of the method proposed by Elias and Cornet and van Ballegooijen were used to determine theta conditions for samples of dextran and poly(vinyl pyrolidone). Light scattering and viscosity measurements made at the theta condition for dextrans of widely ranging molecular weights showed the second virial coefficient to be zero and were in agreement with the theory of Zimm and Kilb for branched polymers. Viscosity and osmotic pressure measurements were made on various mixtures of dextran and poly(vinyl pyrolidone) in water and the theta solvents determined by the above methods. Osmotic pressure measurements for various mixtures of high‐molecular‐weight dextrans and poly(vinyl pyrolidone) made in water showed a minimum in the number‐average molecular weight and osmotic second virial coefficient at all three temperatures. This minimum did not occur in blends of a lower molecular weight dextran and poly(vinyl pyrolidone).
Osmotic pressure
Osmometer
Molar mass distribution
Vinyl polymer
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Static light scattering
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This investigation examines the source of the disparity between experimental values of the light scattering second virial coefficient [Formula: see text] (mL.mol/g2) for proteins and those predicted on the statistical mechanical basis of excluded volume. A much better theoretical description of published results for lysozyme is obtained by considering the experimental parameters to monitor the difference between the thermodynamic excluded volume term and its hydrodynamic counterpart. This involves a combination of parameters quantifying concentration dependence of the translational diffusion coefficient obtained from dynamic light scattering measurements. That finding is shown to account for observations of a strong correlation between [Formula: see text] (mL/g), where M2 is the molar mass (molecular weight) of the macromolecule and the diffusion concentration parameter [Formula: see text] (mL/g). On the grounds that [Formula: see text] is regarded as a hydrodynamic parameter, the same status should be accorded the light scattering second virial coefficient rather than its current incorrect thermodynamic designation as [Formula: see text] (mL.mol/g2), or just B, the osmotic second virial coefficient for protein self-interaction.
Static light scattering
Molar mass
Virial mass
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Hydrodynamic radius
Static light scattering
Aggregation number
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