Gel permeation chromatography
gel permeation chromatography
(analytical chemistry) Analysis by chromatography in which the stationary phase consists of beads of porous polymeric material such as a cross-linked dextran carbohydrate derivative sold under the trade name Sephadex; the moving phase is a liquid.
Gel permeation chromatography
A separation technique involving the transport of a liquid mobile phase through a column containing the separation medium, a porous material. Gel permeation chromatography (GPC), also called size exclusion chromatography and gel filtration, affords a rapid method for the separation of oligomeric and polymeric species. The separation is based on differences in molecular size in solution. It is of particular importance for research in biological systems and is the method of choice for determining molecular weight distribution of synthetic polymers.
The separation medium is a porous solid, such as glass or silica, or a cross-linked gel which contains pores of appropriate dimensions to effect the separation desired. The liquid mobile phase is usually water or a buffer for biological separations, and an organic solvent that is appropriate for the sample and is compatible with the column packing for synthetic polymer characterization. Solvent flow may be driven by gravity, or by a high-pressure pump to achieve the desired flow rate through the column. The sample to be separated is introduced at the head of the column (see illustration). As it progresses through the column, small molecules can enter all pores larger than the molecule, while larger molecules can fit into a smaller number of pores, again only those larger than the molecule. Thus, the larger the molecule, the smaller is the amount of pore volume available into which it can enter. The sample emerges from the column in the inverse order of molecular size; that is, the largest molecules emerge first followed by progressively smaller molecules. In order to determine the amount of sample emerging, a concentration detector is located at the end of the column. Additionally, detectors may be used to continuously determine the molecular weight of species eluting from the column. The volume of solvent flow is also monitored to provide a means of characterizing the molecular size of the eluting species.
Gel permeation chromatograph.
As the sample emerges from the column, a concentration detector signal increases above the baseline, passes through a maximum, and returns to the baseline. This signal provides a relative concentration of emerging species and is recorded as a function of elution volume. Molecular-weight detectors based on light scattering or viscometry produce a signal, independent of molecular weight, that increases with increasing molecular weight for a given sample concentration. The molecular-weight chromatograms therefore are skewed with respect to the concentration detector signal. Typically, constant-volume pumps are used, and the time axis is transformed into a volume axis. Devices such as siphons that empty after a known volume of solvent has collected, or electronic circuitry that measures the transit time of a thermal pulse between two points in the flowing solvent, have been used to provide a measurement of the volumetric flow rate.
Gel permeation chromatography has had widespread applications. For determining the molecular weight of synthetic polymers, at least 50 types of polymers have been characterized. These include alkyd resins, natural and synthetic rubbers, cellulose esters, polyolefins, polyamides, polyesters, polystyrenes, polyacrylates, uncured epoxy, urethane and phenolic resins, and a wide variety of oligomeric materials. Additionally, the ability to determine the molecular-weight distribution and changes in distribution has led to many applications in areas such as blending distributions, chain-length studies in semicrystalline polymers, interactions in solution, radiation studies, mechanical degradation studies, mechanisms of polymerization research, polymerization reactor control, and evaluation of the processing of polymers. In the field of natural and biological polymers, numerous systems have been separated and analyzed. Among these are acid phosphatases, adrenalin, albumin, amino acids and their derivatives, enzymes, blood group antibodies, collagen and related compounds, peptides, and proteins.
Additionally, gel permeation chromatography is capable of making separations of low-molecular-weight compounds. This is particularly important when both low- and high-molecular-weight species are present in the same sample. See also Chromatography; Polymer.