Vernon Shoup asked,
> We are considering buying an economical light scattering detector for
> HPLC, for characterizing protein multimers/aggregates and PEGylated
> proteins.
> Wyatt's low-end instrument is the triple-angle (45, 90, 135 degrees)
> MiniDawn. Precision Detectors' low-end instrument is the single-angle (90
> degrees) PD2010. More advanced instruments use more angles and/or QELS.
>
> I'd appreciate some feedback from light-scattering detector users. In
> particular, for my purposes, does one need multiple-angle detection, or is
> just a scattering angle of 90 degrees sufficient? How important is
> temperature control? How easy to use are the various manufacturers'
> software packages?
>
> Finally, can someone tell me what QELS (Dynamic Scattering) is?
>
Well, we work with both of these detectors routinely, primarily in
conjunction
with flow FFF separations, so let me take a stab at this one. The answer,
of course, is: "it depends." In this case, primarily on the anticipated
size of
your analytes, as well as on what you want to learn and the chromatographic
conditions you plan to use. The short answer is that if you want the size
of
modestly-sized analytes, you might want to use a multi-angle detector. If
you want only mass, or if you want to size large objects, either one can
work
(if the PD is equipped with the DLS option), and then other factors will
come
into play.
If your proteins are "small" (generally used to mean a diameter less than
1/20th the vacuum wavelength of the incident light), there will be little
angular dependence to the scattering, so the use of multiple angles will
not yield an accurate size (r.m.s. radius). In this case, the miniDawn's
three angles would merely provide a degree of redundancy in your mass
measurements, while the PD unit would give a stronger and cleaner lone
signal at 90ƒ. Both will give you the molecular mass.
Of course, you probably already know the mass of your protein monomer;
it's the oligomerization state you want to discover. As the aggregate
becomes larger, the underlying assumption that the form factor P(theta)=1
for 90ƒ-only detectors (and multi-angle detectors operated below the above-
stated size limit) becomes increasingly incorrect. Since the calculated
mass is directly proportional to this form factor, the accuracy of your mass
suffers. However, the masses are still at least ordinally correct; a
10-mer
will give you a larger mass than a 9-mer. As the oligomer size increases
beyond the lower size limit (roughly 10 nm r.m.s. radius), this size becomes
available from the miniDawn's three angles, and is then used to calculate a
more accurate value for P(theta), leading in principle to more accurate
masses. As for the size itself, the relationship between r.m.s. radius and
oligomerization state is not obvious, especially for uncharacterized
proteins,
so knowing the size of your oligomer might not be as helpful as you'd hope.
The chromatographic conditions come into play because the flow cell volumes
of the instruments differ dramatically. The Dawn family of detectors,
including
the miniDawn, has a flow cell volume of roughly 140 µL, a large value for
low-
flow applications. In such cases, a significant degree of dispersive
broadening
can occur in the cell, leading to a loss of resolution and a severe
perturbation of
the obtained results (the infamous "smiles" and "frowns"). In the PD
family, cell
volumes are closer to 10-12 µL, so that dispersive broadening is less of a
problem. The glass components of each company's detectors are also made
of different materials, so that it might be prudent to check for
compatibility with
a specific analyte before purchasing. Specifically, PEG itself is known to
coat
some types of glass, leading to refraction problems for detectors in which
the
light path is not perfectly orthogonal to the glass-liquid interface.
Both company's instruments are designed to detect elastically-scattered
light,
known as classical or Rayleigh scattering, in which the scattered light has
the
same wavelength as the incident light and is averaged over time at a
specific
angle. If the scattered light is not time-averaged, but instead the time-
dependence of the scattered light is correlated with itself, information can
be
obtained on the diffusive motion of the scattering particles. The signal
from this
quasi-elastically scattered (QELS) light can be used to calculate the
diffusion
coefficient of the scatterers, from which a molecular mass can then be
calculated (albeit with a lot of assumptions). Both families of instruments
can
be fitted with such detectors, generally at an angle of 90ƒ, but PD claims
to be
able to achieve reasonable accuracy at lower flow rates. In our hands, the
DLS
option has not proven useful for pharmaceutical-sized proteins. However, if
if the pendant PEG or other group is sufficiently large, the larger
scattering
signal and slower motion may permit accurate characterization.
As for temperature control, this feature is primarily intended for
high-temperature
GPC experiments, and is seldom used for ordinary HPLC. You might want it if
you plan on performing very high-precision work. Both companies also
produce
acquisition and processing software which is compatible with their own, and
no
other, detectors.
Hope this helps!
- greg
Greg Cauchon, Ph.D.
Analytical Research & Development
Amgen Inc., Mail Stop 25-2-A
One Amgen Center Drive
Thousand Oaks, CA 91320-1799
Office: 805-447-2631 Fax: 805-447-8673
Email: gcauchon@amgen.com
Visit us at http://www.amgen.com