The Column - June 2008 - (Page 18) Tips & Tricks: GPC/SEC The Column www.thecolumn.eu.com June 2008 LWA/Getty Images Tips & Tricks: GPC/SEC How Do I Calibrate a GPC/SEC System? Daniela Held, PSS Polymer Standards Service, Mainz, Germany. GPC/SEC is the method of choice for characterizing macromolecules by distribution (MMD). It is an easy to use method and the measurements can be done using standard LC equipment, proper high resolution GPC/SEC columns or column combinations, and dedicated GPC/SEC software for data capture and analysis. Why does GPC/SEC require a calibration? The primary information obtained from GPC/SEC standard detectors (e.g., UV, ELSD or RI) is not the molar mass but the apparent concentration at a certain elution volume. It is only with the GPC/SEC calibration curve and the concentration profile from the concentration detector that all molar mass averages and the molar mass distribution can be obtained.1,2 GPC/SEC is, therefore, a relative method. The GPC/SEC calibration is based on assigning the elution volume to a molar mass. This is in contrast to HPLC, where the detector response (signal intensity, peak area) is calibrated and assigned to a concentration. molecules irrespective of their size elute at the same volume, in the exclusion limit of the column/column combination. Region II is the optimum separation range of this column/column combination. Molecules are separated according to their size in solution. Large particles with high molar masses elute first, molecules with lower molar masses elute at higher elution volumes. Region III is the region where separation is influenced by interaction with the column material and by the size of the Figure 1: Typical GPC/SEC calibration curve with three distinct regions. The recommended sample concentration for calibrating depends on the molar mass and is shown on the y-axis. What are the general characteristics of a GPC/SEC calibration curve? For a GPC/SEC calibration curve the logarithm of the molar mass is plotted against the elution volume. Most calibration curves, even the ones for linear or mixed-bed columns, have a sigmoidal shape that is in agreement with the fundamental separation characteristics. This is in contrast to other calibrations in chromatography where linear calibration curves for the peak area plotted versus the concentration are obtained. A GPC/SEC calibration curve can be divided into three distinct regions, as shown in Figure 1. Region I is the region where no separation takes place. The pores are too small to separate the molecules according to their hydrodynamic volume. All Author: Daniela Held E-mail: dheld@polymer.de 18 http://www.thecolumn.eu.com
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