MEAN-SQUARE RADIUS OF MOLECULES AND SECONDARY INSTRUMENTAL BROADENING

In a chromatographic separation such as size-exclusion chromatography, the concentrations of the injected molecules are generally so low by the time they reach the light-scattering (LS) detector that terms involving the second virial coefficient may be neglected in the equations which relate the measured Rayleigh excess ratio to the derived molecular weights and sizes. For sufficiently large molecules (root mean square radius greater than about 10 nm for 633 nm incident light wavelength), the root mean square radius may be calculated independently of the molecular concentration from the Rayleigh ratios measured as a function of scattering angle. Precise measurements of the root mean square radius are presented for some nearly monodisperse polystyrene standards. These measurements confirm that the eluting molecules have a nearly constant size over a relatively broad range of elution volumes, yet the corresponding mass values are not constant. This inconsistency is shown to be due to a secondary instrumental broadening (IB) of the sample which occurs primarily in the refractive index detector which follows the LS detector. This secondary IB, which may be calculated from the distorted mass versus elution volume curves, is shown to vary with molecular mass.