Ep15 size exclusion / gel permeation chromatography cont. - UC San Diego - NANO 134 Darren Lipomi

This paragraph introduces the concept of size exclusion chromatography (SEC), also known as gel permeation chromatography. It explains the basic principle of SEC, which is the separation of polymer chains based on their size. Larger polymers bypass the beads in the column, while smaller ones get temporarily trapped in the pores, leading to a longer retention time. The paragraph also discusses the volume of the solvent being divided into two components: the interstitial volume and the volume within the pores. The importance of understanding the separation mechanism is emphasized, with a visual aid of a narrow band of solvent entering the column and the different behaviors of larger and smaller polymer chains.

The second paragraph delves into the retention volume equation for a specific molecular weight fraction in SEC. It introduces the concept of the distribution coefficient (KD), which is specific to the pore size and the size of the polymers. KD indicates the fraction of the internal volume accessible to a solute, and the ideal range for KD is between zero and one for effective separation. The paragraph uses a diagram to illustrate the significance of KD, considering the radius of the polymer blobs and the cylindrical pore size. It also explains the limiting cases for KD and how it relates to the solute's ability to penetrate the pores.

This paragraph discusses the concentration gradient within the pores and how it affects the accessible volume for solutes of different sizes. It explains that the concentration inside the pore is always less than outside due to the finite radius of the solute. The paragraph provides a formula for calculating KD for spherical particles and cylindrical pores, highlighting the two limiting cases where KD equals 0 (exclusion from pores) and KD equals 1 (total penetration of pores). The implications of these cases on the separation process and the detector output are also discussed.

The fourth paragraph explains the process of calibrating the SEC system using polymers with known molecular weights, often polystyrene. It describes plotting the log of the absolute molecular weight against the retention volume (V_R), which is related to the retention time by the flow rate. The paragraph outlines the different regimes observed in the plot, including the exclusion, separation, and all-pore regimes, and how they relate to the detector output. The importance of choosing appropriate standards for calibration is emphasized, as well as the assumptions made during the calibration process.

This paragraph addresses the limitations of SEC, particularly the fact that polymers do not always have predictable hydrodynamic volumes based solely on molecular weight. It discusses how different polymers with the same molecular weight can have different hydrodynamic volumes due to variations in backbone rigidity. The paragraph provides examples of different polymers, such as polyethylene, polystyrene, polythiophene, and a ladder polymer, illustrating how their rigidity affects their hydrodynamic volume and, consequently, their SEC separation.

The sixth paragraph highlights additional limitations of SEC, such as specific chemical interactions between the polymer and the matrix material, which can affect the separation. It also discusses the challenges of analyzing branched polymers with SEC, as they do not respond well to size determination by this method. The paragraph emphasizes the importance of considering these factors when selecting standards and interpreting data.

The final paragraph provides an overview of the detectors used in SEC, focusing on refractive index (RI) detectors and UV-visible absorption detectors. It explains how RI detectors measure the change in refractive index due to the concentration of the analyte, and how UV-Vis detectors can resolve different polymers based on their absorption at specific wavelengths. The paragraph also mentions the Beer-Lambert law and its application in these detectors, as well as the potential for using a monochromator to obtain a UV spectrum for each fraction of material eluting from the column.