Size-Exclusion Chromatography of Macromolecules: A Brief Tutorial Overview on Fundamentals with Computational Tools...

Size-exclusion chromatography (SEC) is presently a widely used and very informative technique for the characterization of macromolecules in solution. Beyond the first implementations of SEC-which required cumbersome column calibrations and were mainly intended for the determination of molecular weights-the modern SEC approach involving multiple detectors (md-SEC) is based on solution properties such as intrinsic viscosity and light scattering. Thus, md-SEC enables the direct and more efficient determination of molecular weights, as well as the determination of relationships between property and molecular weight, which can be quite useful in structural studies. Here, we first present a review of the fundamental aspects of the dilute-solution properties of macromolecules-particularly the differential refractive index, intrinsic viscosity, and scattering-related properties-on which the various detectors involved in md-SEC are based. Then, we developed SECtools, a suite of public-domain, open-source computer programs, which allow for the full analysis of md-SEC chromatograms. These analyses range from just the recorded raw signals (mV) of the detectors to a full determination of molecular weight averages and distributions. The use of these programs is illustrated through experimental studies using various samples.