Equipment for running size exclusion chromatography. The buffer is pumped through the column (right) by a computer controlled device.

Size exclusion chromatography (SEC) is a chromatographic method in which particles are separated based on their size, or in more technical terms, their hydrodynamic volume. Its is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. When an aqueous solution is used to transport the sample through the column the technique is known as gel filtration chromatography. The name gel permeation chromatography is used when an organic solvent is used as a mobile phase. The main application of gel filtration chromatography is the fractionation of proteins and other water-soluble polymers, while gel permeation chromatography is used to analyze the molecular weight distribution of organic-soluble polymers. Either technique should not be confused with gel electrophoresis, where an electric field is used to "pull" or "push" molecules through the gel depending on their electrical charges. Image File history File linksMetadata Download high resolution version (1704x2272, 839 KB) Summary Photo taken by User:Kjaergaard. ... Image File history File linksMetadata Download high resolution version (1704x2272, 839 KB) Summary Photo taken by User:Kjaergaard. ... Pictured is a sophisticated gas chromatography system. ... The radius of gyration describes the distribution of particles (or infinitesimal elements) in a D-dimensional space by relating it to an equivalent distribution in a D-dimensional sphere, usually a circular (D=2) or spherical (D=3) distribution. ... In science, a molecule is a group of atoms in a definite arrangement held together by chemical bonds. ... DNA electrophoresis apparatus. ...

SEC is a widely used technique for the purification and analysis of synthetic and biological polymers, such as proteins, polysaccharides and nucleic acids. Biologists and biochemists typically use a gel medium, usually polyacrylamide, dextran or agarose, filtering under low pressure. Polymer chemists typically use either a silica or crosslinked polystyrene medium under a higher pressure. These media are known as the stationary phase. A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... Polysaccharides (sometimes called glycans) are relatively complex carbohydrates. ... Highly simplified diagram of a double-stranded nucleic acid. ... Polyacrylamide is an acrylate polymer formed from acrylamide subunits that is readily cross-linked. ... Dextran is a complex branched polysaccharide made of many glucose molecules joined into chains of varying lengths. ... An agarose is a polysaccharide polymer material, generally extracted from seaweed. ... The chemical compound silicon dioxide, also known as silica, is the oxide of silicon, chemical formula SiO2. ... Polystyrene is a polymer made from the monomer styrene, a liquid hydrocarbon that is commercially manufactured from petroleum by the chemical industry. ...

The advantage of this method is that the various solutions can be applied without interfering with the filtration process, while preserving the biological activity of the particles to be separated. The technique is generally combined with others which further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds.

Theory and method

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.

This is usually achieved with an apparatus called a column, which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes. 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 less overall volume to traverse over the length of the column, and the faster the elution.

The filtered solution that is collected at the end is known as the eluent. The void volume consists of any particles too large to enter the medium, and the solvent volume is known as the column volume.

Factors affecting filtration

A cartoon illustrating the theory behind size exclusion chromatography

In real life situations particles in solution do not have a constant, fixed size, resulting in the probability that a particle which would otherwise be hampered by a pore may pass right by it. 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 which are inert and minimize this issue. Image File history File links Download high resolution version (1200x800, 76 KB) Summary Made by Isaac Yonemoto using Inkscape. ... Image File history File links Download high resolution version (1200x800, 76 KB) Summary Made by Isaac Yonemoto using Inkscape. ... The normal distribution, also called Gaussian distribution by scientists (named after Carl Friedrich Gauss due to his rigorous application of the distribution to astronomical data (Havil, 2003)) is a probability distribution of great importance in many fields. ...

Like other forms of chromatography, increasing the column length will tighten 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.

Analysis

In simple manual columns the eluent is collected in constant volumes, known as fractions. 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.

The collected fractions are often examined by spectroscopic techniques to determine the concentration of the particles eluted. Three common spectroscopy detection techniques are refractive index (RI), evaporative light scattering (ELS), 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. The elution volume decreases roughly linearly with the logarithm of the molecular hydrodynamic volume (often assumed to be proportional to molecular weight). Columns are often calibrated using 4-5 standard samples (e.g., folded proteins of known molecular weight) to determine the void volume and the slope of the logarithmic dependence. This calibration may need to be repeated under different solution conditions. Extremely high resolution spectrum of the Sun showing thousands of elemental absorption lines (fraunhofer lines) Spectroscopy is the study of matter and its properties by investigating light, sound, or particles that are emitted, absorbed or scattered by the matter under investigation. ... Logarithms to various bases: is to base e, is to base 10, and is to base 1. ... The radius of gyration describes the distribution of particles (or infinitesimal elements) in a D-dimensional space by relating it to an equivalent distribution in a D-dimensional sphere, usually a circular (D=2) or spherical (D=3) distribution. ... The molecular mass of a substance (less accurately called molecular weight and abbreviated as MW) is the mass of one molecule of that substance, relative to the unified atomic mass unit u (equal to 1/12 the mass of one atom of carbon-12). ...

Applications

Proteomics

SEC is generally considered a low resolution chromatography as it does not discern similar species very well, and is therefore often reserved for the final "polishing" step of a purification. The technique can determine the quaternary structure of purified proteins which 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 14 Å and 36 Å for the folded and unfolded forms respectively. SEC allows the separation of these two forms as the folded form will elute much later due to its smaller size. Alternatively, folded and unfolded versions of the same metalloproteins can be separated according to their different isoelectric points by using quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE). In biochemistry, many proteins are actually assemblies of more than one protein (polypeptide) molecule, which in the context of the larger assemblage are known as protein subunits. ... Dissolving table salt (NaCl) in water This article is about a chemical solution; for other uses of the term solution, see solution (disambiguation). ... In biochemistry, the tertiary structure of a protein is its overall shape. ... The hydrodynamic radius of a collection of particles (typically belonging to a polymer) is defined as where is the distance between particles and , and where the angular brackets represent an ensemble average. ... In biochemistry, a metalloprotein is a generic term for a protein that also contains a metal cofactor. ... The isoelectric point sucks (pI) is the pH at which a molecule carries no net electrical charge. ... QPNC-PAGE (Abbr. ...

Polymer synthesis

SEC can be used as a measure of both the size and the polydispersity of a synthesised polymer - that is, the distribution of sizes of polymer molecules. 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. Alternatively, 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 generally 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 polydispersity index, or PDI, is the ratio of the weight average molecular weight to the number average molecular weight. ... A calibration curve is a graphical display of the functional relationship between the expected value of the observed signal to the analyte amount. ...

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