Handbook of Size Exclusion Chromatography and Related ...

These include the differential weight fraction distributions of both mass and size,
the corresponding cumulative distributions, the conformation plot (log Mw vs.
log rg), as well as the “calibration” curve (log Mw vs. elution volume) and related
plots. Details of these quantities are described in Shortt’s article (17).
Because the MALS software calculates both Mw and rg (for large enough
molecules) throughout the separated distributions present in the samples, for a
variety of molecular species one may calculate the eluting sample’s intrinsic
viscosity, [h], at each slice using the Flory–Fox equation (18):
pffiffiffi Mw[h] ¼ F( 6rg)
3
where F(;F0 ¼ 2:87 10 23 ) is the so-called Flory viscosity constant. In
general, the excluded volume effect is taken into account via the Ptitsyn–Eizner
equation (19) F ¼ F0(1 2:631 þ 2:861 2 ) and 1 ranges from 0 at the theta point
to 0.2 for a good solvent. The software can then calculate and plot the so-called
Mark–Houwink–Sakurada plot. An example for the NIST broad polystyrene
standard NBS706 is shown in Fig. 6.
Band broadening plays a major role in distorting the calibration curve
(log Mw vs. elution volume V ) of eluting species of extremely narrow size
distribution. This is particularly noticeable for proteins whose masses (following
separation of aggregates) should be monodisperse. Over the years, numerous
papers and books have been written on the subject of correcting for such
© 2004 by Marcel Dekker, Inc.
Figure 6 MHS plot of NBS706.
(7)