Gel permeation chromatography

From Academic Kids

Gel permeation chromatography (GPC) also known as size exclusion chromatography (SEC) is a chromatographic method in which molecules are separated based on their size. This method is most widely used in the analysis of polymer molecular weights (or molar mass). The term GPC was used in the beginning of polymer analysis when people used glass columns filled with gels to perform GPC. Nowadays more and more automated and high pressure liquid chromatographic columns are used. Therefore GPC is an old terminology and size-exclusion chromatography (SEC) is the correct expression for the determination of molecular weights.

In SEC, a column (steel cylinder typically 10 mm in diameter and 500 to 1000 mm in length) is packed with a porous material (typically silica or crosslinked polystyrene) and solvent is forced through the column (at rates typically 1 ml/min and pressures of 50 to 200 bar). A sample is dissolved in the same solvent that is running through the column and is then introduced into the solvent stream going through the column. A detector monitors the concentration of sample exiting the end of the column. Inside the column, molecules are separated based on their hydrodynamic volume (the volume the molecule occupies in a dilute solution). For polymers this can vary greatly with the particular solvent and the temperature. By studying the properties of polymers in particular solvents and by calibrating each column setup with samples of known molecular weight, it is possible to get a relative distribution of molecular weights for a given polymer sample. Using this data, it is possible to calculate number average molecular weight, weight average molecular weight, polydispersity, as well as higher order molecular weights to within a useful level of accuracy.

Inside the column, molecules are separated by whether or not they can fit within the pore size of the packing material. When columns are created they are packed with porous beads with a specific pore size so that they are most accurate at separating molecules with sizes similar to the pore size. As a molecule flows through the column it passes by a number of these porous beads. If the molecule can fit inside the pore then it is drawn in by the force of diffusion. There it stays a short while and then moves on. If a molecule can not fit into a pore then it continues following the solvent flow. For this reason, in a GPC column, molecules with larger size will reach the end of the column before molecules with smaller size. The effective range of the column is determined by the pore size of the packing. Any molecules larger than all the pores in a column will be eluted together regardless of their size. Likewise, any molecules that can fit into all the pores in the packing material will elute at the same time.

It is important to remember that the only absolute measure in SEC is volume of the molecule (hydrodynamic volume), and even that measurement has certain error built into it. Interactions between the solvent, packing, and or the sample will affect the measurement as will concentration due to sample-sample interactions. Calculating the molecular weight from this molecular size introduces even more error into the system. SEC is a useful tool for determining molecular weight in polymers, but it is essential that the column and instrumentation be carefully equilibrated and properly calibrated for the results to be trusted.cs:Gelová chromatografie fr:Chromatographie d'exclusion stérique sv:Gelfiltrering


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