Chapter 6
Advanced protein features
Page 6-16
MAN 0487
Copying the Debye plot
The graph can be pasted into another application (such as Microsoft Word or
Excel) by selecting the
Copy
button.
Results area
With both the table data and sample parameters entered the results will be
automatically calculated and shown alongside the graph. The results displayed are:
MW (kDa) - molecular weight
Shows the measured weight of a molecule within the sample expressed in
atomic mass units; indicated in kiloDaltons. It is calculated from the intercept
of the KC/RoP vs concentration Debye plot.
+/-
The expected error in the molecular weight, derived from the scatter in the
data about the least squares best fit line.
% Error in MW
The error in the calculated molecular weight arising from the use of only a
single angle. For isotropic scattering particles (diameter < ~50nm), this error
should be negligible.
A
2
(ml mol/g
2
) - second virial coefficient
A property describing the interaction strength between the molecule and the
solvent. This is calculated from the slope of the plot.
K (Mol cm
2
/g)
The instrument optical constant.
Molecular weight (MW) and shape estimates
The hydrodynamic size measured by Dynamic Light Scattering (DLS) is defined as
“the size of a hypothetical hard sphere that diffuses in the same fashion as that of
the particle being measured”. In practice though, macromolecules in solution are
non-spherical, dynamic (tumbling), and solvated. Because of this, the diameter
calculated from the diffusional properties of the particle will be indicative of the
apparent size of the dynamic hydrated/solvated particle. Hence the terminology,
‘Hydrodynamic’ diameter
.
If the
molecular weight
(or mass) and the partial
specific volume
(inverse
density) for the particle being measured are known, then a mass equivalent
spherical size can be calculated. The closer the particle is to being spherical, the
closer the mass equivalent spherical diameter will be to the DLS measured
hydrodynamic diameter. In fact, it is the difference in these two values, coupled
with
Perrin
theory (below), that allows
particle shape
information to be
extracted from DLS measurements.
