Specifically, we need R(U) at zero angle, U = 0°, and infinite dilution, c = 0. The fundamental parameter of interest in obtaining the molecular weight and the size of macromolecules is the intensity of the scattering R(U) that depends on the angle U and on the concentration c. However, other than A, dn/dc also depends a little on the solvent (salt, buffer) used and in general it is better to measure it. The dn/dc value for hyaluronan is well known: 0.15 mL/g, in 0.15 M NaCl solvent, at 25 ☌ and A = 632.8 nm.
#Dynamic light scattering size exclusion Offline
Often the dn/dc of the polymer may be found in the literature, otherwise the value must be measured, generally by an offline refractometer at the same wavelength, solvent and temperature of the LS experiment. All the parameters of the K optical constant are known, only the dn/dc of the polymer is unknown. The constant K puts together all the physical and optical parameters. Modern LS photometer uses coherent light, that is a laser (often a He-Ne laser with wavelength A = 632.8 nm), and vertical polarization. Where AR(ff) is the scattering excess (Rayleigh factor) at angle U of the solution with regard to the pure solvent, U the angle between the incident light and the detector, c the concentration, A2 the second virial coefficient, P(U) the form factor, K = (4p2n2(dn/dc)2)/(NaA4) an optical constant, n0 the refractive index of the solvent, dn/dc the refractive index increment of the polymer, A0 the wavelength of light in a vacuum, NA the Avagadro's number. On the contrary, in a quasi-elastic LS experiment (also known as dynamic or photon correlation spectroscopy) we measure the fluctuations of the intensity of the scattering due to the Brownian movement of the macromolecules.įollowing Zimm (67) the intensity of the scattering of a solution of macromolecules is related to the molecular weight M of the sample by the following equation Kc 1 In this case, we assume that the scattered light has the same wavelength and polarization of the incident light. In an elastic LS experiment (also known as static or total intensity or Rayleigh scattering) we measure the intensity of the scattering. For the characterization of macromolecules (molecular weight and size) only elastic and quasi-elastic LS are of interest. Depending on the type of scattering analyzed (elastic, quasi-elastic, Raman, etc.) different information may be obtained. The interaction of light with matter is a very complex topic. LS concerns the interaction of light with matter in the specific case with macromolecules in solution. As a consequence, LS is a fundamental method for the characterization of hyaluronan. Figure kindly provided by P DeAngelis and W Jing. S: a mixture of 5 different monodisperse SelectHA preparations with indicated Mw determined by SEC-MALS C and C0: commercial hyaluronan samples D: DNA standards, Bioline Hyperladder 1, containing DNA of 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5 kb D0: DNA standards, BioRad 1 Kilobase Ruler, containing DNA of 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 kb. Gel was 0.7% agarose in Tris-acetate-EDTA (minigel format), stained with Stains-All by the method of Lee and Cowman (61). Figure kindly provided by P DeAngelis and W Jing.įigure 7 Agarose gel electrophoresis of nearly monodisperse hyaluronan standards and commercial hyaluronan. Gel was 0.7% agarose in Tris-acetate- EDTA (minigel format), stained with Stains-All by the method of Lee and Cowman (61). However, only the LS technique can be used online to a SEC system in obtaining the wholeįigure 7 Agarose gel electrophoresis of nearly monodisperse hyaluronan standards and commercial hyaluronan. LS and a few other methods such as osmometry, sedimentation, and mass spectrometry are absolute techniques. A primary method in estimating the molecular weight and the size of macromolecules is LS. Fundamental properties of hyaluronan, such as viscoelasticity and flow behavior primarily depend on the MWD, size, and conformation of the macromolecules.
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