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 invo
lving the second virial coefficient may be neglected in the equations
which relate the measured Rayleigh excess ratio to the derived molecul
ar weights and sizes. For sufficiently large molecules (root mean squa
re radius greater than about 10 nm for 633 nm incident light wavelengt
h), the root mean square radius may be calculated independently of the
molecular concentration from the Rayleigh ratios measured as a functi
on of scattering angle. Precise measurements of the root mean square r
adius 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 th
e corresponding mass values are not constant. This inconsistency is sh
own to be due to a secondary instrumental broadening (IB) of the sampl
e which occurs primarily in the refractive index detector which follow
s the LS detector. This secondary IB, which may be calculated from the
distorted mass versus elution volume curves, is shown to vary with mo
lecular mass.