Handbook of Size Exclusion Chromatography and Related ...

sensitivity for many samples compared with viscometry, this approach is
rarely used.
3.2.6 Right-Angle Laser Light Scattering
Haney et al. (11) used a right-angle light-scattering (LS) detector combined with
SEC-viscometry to measure directly both the intrinsic viscosity and molecular
weight of each elution slice. For molecules with molecular weights less than about
100,000 g/mol, there is no measurable scattering asymmetry and the right-angle
intensity provides a good measurement of the molecular weight. For higher
molecular weights, the Flory–Fox equation [Eq. (16)] is used in an iterative
procedure to correct for any asymmetry in the scattering and thus determine a
good approximation to the correct molecular weight. Thus, with this approach,
both molecular weight and the radius of gyration [Eq. (16)] can be determined.
The method gave accurate molecular weights for polystyrene in THF up to
3 10 6 g/mol.
3.3 Concentration Measurement
One of the advantages of conventional SEC is that the absolute concentration of
the sample at each elution slice is not required to calculate the MWD. With both
SEC-LS and SEC-viscometry it becomes necessary to determine an absolute
concentration measurement if the MWD is to be determined.
There are two approaches to determining the concentration: one is to use the
injected sample mass, and the other is to calibrate the concentration detector. In
the following discussion it is assumed that a refractometer is being used to
determine concentration, but the same applies to ultraviolet (UV) detectors, except
that the UV absorbance of a sample replaces the dn=dc value.
In the first method, the area under the concentration detector chromatogram
is taken to be proportional to the total sample mass injected m:
m
k ¼
DV P hi
(22)
and thus the concentration at each elution slice ci may be calculated from the
detector output at each slice hi by ci ¼ khi.
The advantages of this method are that it is straightforward and is not
affected by different dn=dc values for different samples. The disadvantage is that
the injected amount of sample must be known accurately. This implies that the
injection volume is known accurately.
In the second method, the concentration detector is calibrated with a series of
solutions of different concentrations and known refractive indices. This provides a
© 2004 by Marcel Dekker, Inc.