Size- exclusion chromatography - Wikipedia, the free encyclopedia. Size- exclusion chromatography (SEC), also known as molecular sieve chromatography. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel- filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers. Applications. Either technique should not be confused with gel electrophoresis, where an electric field is used to . The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds. With size exclusion chromatography, there are short and well- defined separation times and narrow bands, which lead to good sensitivity. Gel Filtration/Size Exclusion Chromatography. We offer a wide range of gel filtration or size exclusion chromatography (SEC) media (resins).
There is also no sample loss because solutes do not interact with the stationary phase. The other advantage to this experimental method is that in certain cases it is feasible to determine the approximate molecular weight of a compound. The shape and size of the compound (eluent) determine how the compound interacts with the gel (stationary phase). In order to determine the approximate molecular weight, the elution volumes of compounds with their corresponding molecular weights are obtained and then a plot of . This plot acts as a calibration curve, which is used to approximate the desired compound. The Ve component represents the volume at which the intermediate molecules elute such as molecules that have partial access to the beads of the column. In addition, Vt is the sum of the total volume between the beads and the volume within the beads. The Vo component represents the volume at which the larger molecules elute, which elute in the beginning. Size exclusion chromatography (SEC), also called gel filtration chromatography or gel. A short review of these developments has appeared. Moore of the Dow Chemical Company published his work on the preparation of gel permeation chromatography (GPC) columns based on cross- linked polystyrene with controlled pore size. It was recognized almost immediately that with proper calibration, GPC was capable to provide molar mass and molar mass distribution information for synthetic polymers. Because the latter information was difficult to obtain by other methods, GPC came rapidly into extensive use. SEC works by trapping smaller molecules in the pores of the adsorbent materials adsorption (. The larger molecules simply pass by the pores because those molecules are too large to enter the pores. Larger molecules therefore flow through the column more quickly than smaller molecules, that is, the smaller the molecule, the longer the retention time. One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the solute volume the analytes can enter, rather than chemical or electrostatic interactions with the stationary phases. Thus, a small molecule that can penetrate every region of the stationary phase pore system can enter a total volume equal to the sum of the entire pore volume and the interparticle volume. This small molecule will elute late (after the molecule has penetrated all of the pore- and interparticle volume. At the other extreme, a very large molecule that cannot penetrate any the smaller pores can enter only the interparticle volume (~3. The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near- simultaneously, particles of the same size should elute together. However, as there are various measures of the size of a macromolecule (for instance, the radius of gyration and the hydrodynamic radius), a fundamental problem in the theory of SEC has been the choice of a proper molecular size parameter by which molecules of different kinds are separated. Experimentally, Benoit and co- workers found an excellent correlation between elution volume and a dynamically based molecular size, the hydrodynamic volume, for several different chain architecture and chemical compositions. In fact, both theory and computer simulations assume a thermodynamic separation principle: the separation process is determined by the equilibrium distribution (partitioning) of solute macromolecules between two phases - -- a dilute bulk solution phase located at the interstitial space and confined solution phases within the pores of column packing material. Based on this theory, it has been shown that the relevant size parameter to the partitioning of polymers in pores is the mean span dimension (mean maximal projection onto a line). The exclusion limit defines the molecular weight at the upper end of the column 'working' range and is where molecules are too large to be trapped in the stationary phase. The lower end of the range is defined by the permeation limit, which defines the molecular weight of a molecules that is small to penetrate all pores of the stationary phase. All molecules below this molecular mass are so small that they elute as a single band. These pores may be depressions on the surface or channels through the bead. As the solution travels down the column some particles enter into the pores. Larger particles cannot enter into as many pores. The larger the particles, the faster the elution. The filtered solution that is collected at the end is known as the eluate. The void volume includes any particles too large to enter the medium, and the solvent volume is known as the column volume. Factors affecting filtration. Also, the stationary- phase particles are not ideally defined; both particles and pores may vary in size. Elution curves, therefore, resemble Gaussian distributions. The stationary phase may also interact in undesirable ways with a particle and influence retention times, though great care is taken by column manufacturers to use stationary phases that are inert and minimize this issue. Like other forms of chromatography, increasing the column length will enhance the resolution, and increasing the column diameter increases the capacity of the column. Proper column packing is important to maximize resolution: An overpacked column can collapse the pores in the beads, resulting in a loss of resolution. An underpacked column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores. Unlike affinity chromatography techniques, a solvent head at the top of the column can drastically diminish resolution as the sample diffuses prior to loading, broadening the downstream elution. Analysis. The more similar the particles are in size the more likely they will be in the same fraction and not detected separately. More advanced columns overcome this problem by constantly monitoring the eluent. Common spectroscopy detection techniques are refractive index (RI) and ultraviolet (UV). When eluting spectroscopically similar species (such as during biological purification), other techniques may be necessary to identify the contents of each fraction. It is also possible to analyse the eluent flow continuously with RI, LALLS, Multi- Angle Laser Light Scattering MALS, UV, and/or viscosity measurements. Columns are often calibrated using 4- 5 standard samples (e. The technique can determine the quaternary structure of purified proteins that have slow exchange times, since it can be carried out under native solution conditions, preserving macromolecular interactions. SEC can also assay protein tertiary structure, as it measures the hydrodynamic volume (not molecular weight), allowing folded and unfolded versions of the same protein to be distinguished. For example, the apparent hydrodynamic radius of a typical protein domain might be 1. SEC allows the separation of these two forms, as the folded form will elute much later due to its smaller size. Polymer synthesis. If standards of a known size are run previously, then a calibration curve can be created to determine the sizes of polymer molecules of interest in the solvent chosen for analysis (often THF). In alternative fashion, techniques such as light scattering and/or viscometry can be used online with SEC to yield absolute molecular weights that do not rely on calibration with standards of known molecular weight. Due to the difference in size of two polymers with identical molecular weights, the absolute determination methods are, in general, more desirable. A typical SEC system can quickly (in about half an hour) give polymer chemists information on the size and polydispersity of the sample. The preparative SEC can be used for polymer fractionation on an analytical scale. Drawback. However, the approximate molecular weight can be calculated from SEC data because the exact relationship between molecular weight and hydrodynamic volume for polystyrene can be found. For this, polystyrene is used as a standard. But the relationship between hydrodynamic volume and molecular weight is not the same for all polymers, so only an approximate measurement can be obtained. Any interaction leads to a later elution time and thus mimics a smaller analyte size. When performing this method, the bands of the eluting molecules may be broadened. This can occur by turbulence caused by the flow of the mobile phase molecules passing through the molecules of the stationary phase. In addition, molecular thermal diffusion and friction between the molecules of the glass walls and the molecules of the eluent contribute to the broadening of the bands. Besides broadening, the bands also overlap with each other. As a result, the eluent usually gets considerably diluted.
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Format: Other Format. Categories: Psychiatric nursing- > Methods, Nurse and patient, Nurse- Patient relations. Contributors: Graham Dexter - Author. Format: Paperback. Categories: Dementia- > Patients- > Care, Psychiatric nursing, Community mental health services. Contributors: John Keady - Author. Format: Paperback. Medical Book Psychiatric Nursing. Both a classroom text and a reference for treating psychiatric disorders in any setting, it is grounded in current research and reflects current practice. Special features include: interdisciplinary treatment plans; listings of movies about psychiatric disorders; therapeutic dialogues; solid psychopharmacological content; psychoeducation checklists; and clinical vignettes. Each chapter includes key concepts, key points summary, and critical thinking questions. The first Canadian psychiatric and mental health nursing textbook. Psychiatric and Mental Health Nursing for Canadian Practice is now in its thoroughly revised. 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