Combined differential light scattering with various liquid chromatography separation techniques

Abstract

Measurements of scattered light intensity as a function of angle (often called differential [l] or total intensity light scattering) from an eluting sample separated into its particulate/molecular constituents by various chromatographic means permit the improvement of subsequent analyses {in the Rayleigh-Gans-Debye (RGD) approximation [2]} in three distinct ways. First, the ability to make repeated simultaneous measurements at several angles improves the precision of the derived weight averaged mass values. Secondly, for particles/molecules which are sufficiently large, the z-average square radius ('radius of gyration' squared) may be derived [3], as well. Finally, if a distribution of particles/molecules is present in the sample and they have been sufficiently separated so that both mass and radius may be derived at each elution fraction, then by plotting the root z-average square radius against the weight averaged mass on a log-log scale, the molecular/particulate configuration (rod, sphere, coil, etc.) may be deduced [4]. For particles that do not satisfy the RGD criteria, a series of other deductions from the recorded data are possible, including size, refractive index, and structural non-homogeneity.For macromolecules, and other particles of mean radius u satisfying the RGD approximation criteria:Im-ll*l and the inversion of the scattering data to yield mass and structural information is straightforward [ 51.Here, the incident wavelength in vacuum is A,, and the medium refractive index no. The relative refractive index of the particles, n/no = m. The inversion is far more difficult when the RGD criteria are not satisfied. The broad range of particles separable by various field flow fractionation and gradient phase techniques further confirms the power of differential light-scattering measurements. For molecules such as heparin and pectin, the light-scattering measurements indicate the presence of features that cannot be noticed by conventional gel permeation chromatography techniques.

A Structural Model for the Damage-sensing Complex in Bacterial Nucleotide Excision Repair

“…Light scattering and refractive index signals were measured using a Wyatt Optilab and Dawn EOS system. Scattering curves were processed using the provided Astra software package (15,16).…”

Molecular flexibility of citrus pectins by combined sedimentation and viscosity analysis

“…Analytical fractionation was carried out using a series of SEC columns TSK G6000PW and TSK G4000PW protected by a similarly packed guard column (Tosoh Bioscience, Tokyo, Japan) with on-line MALLS (Harding, Vårum, Stokke & Smidsrød, 1991;Wyatt, 1992) detectors. The eluent (0.1 M NaCl) was pumped at 0.8 ml/min (PU-1580, Jasco…”

Global conformation analysis of irradiated xyloglucans

“…Molecular weights (weight averages, number averages and distributions) were estimated using SEC-MALLs (Wyatt, 1992). The SEC system used consisted of a Jasco HPLC pump (Model PU-1580, Jasco Corporation, Tokyo, Japan), a Phenomenex guard column (Phenomenex, Macclesfield, UK), and TSK (Tosoh Biosciences, Tokyo, Japan) Gel G4000 PW and Gel G3000 PW columns connected in series.…”