Handbook of Size Exclusion Chromatography and Related ...

therefore, is to remove any lingering doubts about the power of the method. A
summary of some of the key historical events of the light scattering method are
mentioned in the next section. This is followed by a brief description of the theory
and its implementation via modern instrumentation. Next follows an explicit
explanation of the significance of absolute measurements and the importance of
the multi-angle, traditional measurement itself.
Discussions follow of the many problem areas relating to chromatographic
separations in general and their effects on light scattering. Included here are
discussions of band broadening and mobile phase preparation.
2 SOME BRIEF HISTORICAL NOTES
Although light scattering techniques were well known and understood in many
respects in the 19th and 20th centuries, it was not until the seminal works of
Einstein (2), Raman (3), Debye (4), and Zimm (5,6) were all brought together by
the mid 1940s that the true power of the technique became recognized. By the
1930s, the possibility that proteins were distinct macromolecules was resolved by
the early 908 light scattering experiments of Putzeys and Brosteaux (7). Their
measurements appeared to confirm this hypothesis since the scattered light
intensity, from light scattering theory, was known to be directly proportional to the
weight-average molar mass times the molecular concentration.
The first commercial light scattering photometer incorporating a laser was
introduced by Wyatt and Phillips (8) in 1970. The early applications of these
instruments were directed almost entirely to measurements of colloids and
microorganisms. In about 1972, Beckman Instruments introduced instrumentation,
with the primary focus of measuring macromolecules, incorporating a laser
to make measurements at very small scattering angles (9,10). The instrumentation
(referred to as low-angle laser light scattering, or LALLS) was developed further
and fully commercialized by the Chromatix. As discussed further in the next
section, such low-angle measurements permitted the deduction of the molar mass
of light-scattering molecules directly.
Although size exclusion chromatography (SEC) was developed by Moore
(11) in 1964, it was not until the early 1970s that Ouano and Kaye (12) showed
how the combination of SEC separation and LALLS could produce a quantitative
distribution of molar mass. Whereas until that time, the classical light scattering
methods of Zimm could yield at best a weight-averaged molar mass, here at last
was a remarkable result that showed finer details of the samples so examined.
3 SOME ELEMENTS OF THE THEORY
In Zimm’s earlier papers, he showed the relationship between the light scattering
quantities measured and the physical elements of the measurement itself. In
© 2004 by Marcel Dekker, Inc.