Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography belongs to the group of methods based on extraction of material for subsequent analysis.We put together a multidetector SEC system as a more powerfult tool to characterize polymers far beyond what is possible by conventional column calibration.

Principle:, which separation of polymer particles according to their size, so that the larger particles come out first, the smaller ones being delayed between the pores of the packing materials used for the separation; one has to take care in choosing an appropriate eluent. Since the chromatographic columns separate and further dilute the polymer solution, characterization of polymers is placed into the dilute regime (1 >> 2A2cMw >> 3A3c2Mw) , and allows the use of Zimm equation

Example applications of multi-detector SEC

• Analysis of a water soluble polymer, polyacrylamide (PAM)

Raw SEC data for PAM: RI, viscometer and LS at 900 . The inset shows M both as determined directly from the LS and RI data, as well as the semi-logarithmic fit, and the concentration data, for reference.

• Effect of ionic strength on a the elution profile of a copolymeric polyelectrolyte

RI and LS90 (inset) voltages for a copolymeric polyelectrolyte (VB/Am) in solutions of NaCl at different concentrations

• Identification of sub-populations and properties of a complex polysaccharide, 'gum arabic' - a good example of the power of multi-detector SEC

Raw LS at 900 , RI and viscosity data for SEC of gum arabic. The multi-modality is best seen in the light scattering signal. (From A. Parker and W. F. Reed, unpublished results)

• Example Where Lower Mass Elutes Before Higher Mass: it illustrates how SEC columns do not necessarily separate according to M, especially in aqueous phase, and especially with complex mixtures of biological molecules of very different chemical nature. If deductions on a pure RI chromatogram were made using standard column calibration, they would be completely erroneous.

Raw RI and LS900 SEC chromatograms of proteoglycans (PG)before (0 min) and after hydrolysis by NaOH (15 min). Notice the large LS peak at 25 mL for the backbone of the hydrolyzed PG as more massive than the glycosaminoglycans (GAG) chains that elute at the 20mL peak.