Dynamic vs static light scattering

Philip J. Wyatt phil at wyatt.com
Thu Nov 30 19:50:41 EST 1995

In an earlier reply by to Dr. James Fethiere, discussing DLS and SEC  
disparities, I discussed a classical light scattering approach per the  

>  The amount of light scattered by a solution of solvated molecules in  
excess of the pure solvent scattering (baseline) is directly proportional  
to the weight average molecular weight times the concentration. (This is  
true also for DLS)  Although aggregates of very high molecular weight yet  
very small concentration often produce very high light scattering signals,  
they rarely show up on a UV or RI detector. The events you described are  
very typical.  In general, one must know the concentration to get the  
molecular weight, yet for these very low concentration aggregates, without  
a signal (by which to divide the light scattering signal) you cannot get a  
molecular weight.  However, even without a concentration signal,  
multiangle LS will calculate a rms radius from which one can make some  
pretty good guesses as to the number of contributing molecules. DLS yields  
a different size result (hydrodynamic radius) which is then used,  
following an assumption of spherical shape, to questimate a molecular  
weight based on a rather crude calculation based on interpolation of the  
measured diffusion coefficient against a set of experimentally "measured"  
proteins to yield a "guestimate" of the MW. Your example shows you how  
poorly DLS determines mass in the presence of any aggregation.  Even  
without aggregation, DLS is often in error (by means of this interpolation  
technique) by 20 to 30% or more.  A far more accurate weight average  
molecular weight (if you wanted to look just at the unseparated sample)  
would be obtained by the classical multiangle LS technique since such  
weights are absolute and do not depend on standards. It's very obvious  
that you knew exactly what you were doing when you tried SEC.  What you  
might not have known is that the contributing aggregates often produce no  
concentration signal. 
There is a very important related discussion in the recent paper by George  
and Wilson at Mississippi State University entitled: PREDICTING PROTEIN  
D50, pp 361-5. The dilute solution property they refer to is obtained from  
static light scattering measurements as a predictor for protein  
crystallation experiments.  They provide another interesting discussion of  
the limitations of DLS.

Phil Wyatt

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