Handbook of Size Exclusion Chromatography and Related ...

calibration constant for the detector k 0 that converts the signal into a change in
refractive index, such that for each chromatogram slice,
ci ¼ k0
dn=dc hi
(23)
This avoids the problems with the peak mass not corresponding to the injected
mass and thus increases the measurement precision, but it means that dn=dc for the
sample must be known. Because dn=dc must be known for the light-scattering
calculation, this clearly does not require any additional work for SEC-light
scattering. Furthermore, once the concentration detector is calibrated, dn=dc for
unknown polymers can be determined using Eq. (23) if the injected mass is
known. With this approach it is best to use a monochromatic light source for the
refractometer having the same wavelength as the light source used for the lightscattering
experiment.
3.4 Interdetector Delay Volume
When a molecular weight sensitive detector is added as a second detector to an
SEC system, it is essential that the dead volume in the connecting tubing between
the measurement points of the two detector cells be known precisely. If this is not
done, the calculated values contain significant errors. In particular, the measured
polydispersity and Mark–Houwink coefficients are extremely sensitive to errors
that may be incurred in the interdetector dead volume.
A number of approaches can be used to determine the interdetector volume.
The obvious procedure is to calculate the geometric offset volume from the
connection volume between detectors. As discussed by Bruessau (31) and
Lecacheux and Lesec (32), however, these calculated values are not correct
because they do not take into account peak shape changes that can occur. The most
commonly used approach for determining interdetector volume for either
viscometers or light-scattering detectors is to measure peak maxima differences of
a narrow molecular weight distribution polymer standard or a monodisperse
solute, such as a protein. In a viscometer, a solute, such as methanol, can be
employed for aqueous SEC. Measurement of peak onset difference, as well as the
peak maxima difference of an excluded polymer peak, has been reported (33).
A different procedure was used by Lecacheux and Lesec (32) for
determining interdetector volume for both a viscometer and a light-scattering
detector. In this approach, an excluded monodisperse polymer standard is injected.
When the correct interdetector volume is selected, the calculated intrinsic
viscosity, or molecular weight, is equal to the expected value and remains constant
as a function of elution volume.
To determine the interdetector delay volume for a viscometer, a broad
molecular weight distribution standard can be injected and a Mark–Houwink plot,
© 2004 by Marcel Dekker, Inc.