Size exclusion chromatography
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Size exclusion chromatography
Equipment for running size exclusion chromatography. The buffer is pumped through the column (right) by a computer controlled device
Acronym	SEC
Classification	Chromatography
Analytes	macromolecules synthetic polymers biomolecules
Other Techniques
Related	High performance liquid chromatography Aqueous Normal Phase Chromatography Ion exchange chromatography Micellar liquid chromatography
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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. It 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.

PENIS 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 — and filter 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.

The advantages of this method includes good separation of large molecule of a sample from the small molecules with a minimal volume of eluate and¹ 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 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, narrow bands which lead to good sensitivity, freedom from sample loss because solutes don't interact with stationary phase while disadvantages include only a limited number of bands can be accommodated because the time scale of the chromatogram is short and generally there has to be a 10% difference in molecular mass for there to be good resolution²

Discovery

The technique was invented by Grant Henry Lathe and Colin R Ruthven, working at Queen Charlotte’s Hospital, London.³⁴ They later received the John Scott Award for this invention.⁵ While Lathe and Ruthven used starch gels as the matrix, Porath and Flodin later introduced dextran gels;⁶ other gels with size fractionation properties include agarose and polyacrylamide. A short review of these developments has appeared.⁷

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. Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of this range and is where molecules are too large to be trapped in the stationary phase. The permeation limit defines the molecular weight at the lower end of the range of separation and is where molecules of a small enough size can penetrate into the pores of the stationary phase completely and all molecules below this molecular mass are so small that they elute as a single band⁸

A size exclusion column.

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 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

A cartoon illustrating the theory behind size exclusion chromatography

In real life situations 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.

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.

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.

Standardization of a size exclusion column.

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. It is also possible to analyse the eluent flow continuously with RI, LALLS UV and or viscosity measurements.

The elution volume (Ve) decreases roughly linearly with the logarithm of the molecular hydrodynamic volume (for globular proteins this is proportional to molecular weight). Columns are often calibrated using 4-5 standard samples (e.g., folded proteins of known molecular weight), and a sample of the very large molecule blue dextran to determine the void volume. The elution volumes of the standards are divided by the elution volume of the blue dextran (Ve/Vo) and plotted against the log of the standards' molecular weights.

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).

Polymer synthesis

SEC can be used as a measure of both the size and the polydispersity of a synthesised polymer - that is, the ability to be able to find the distribution of the 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 preparative SEC can be used for polymer fractionation on an analytical scale.

References