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Size exclusion chromatography
Size exclusion.JPG
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

Size exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated based on their size (more correctly, their hydrodynamic volume).[1] It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. 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 Mw results for polymers.

Contents

Application and usage

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..

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.

Advantages

The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate[2], and that various solutions can be applied without interfering with the filtration process, all 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 leads to good sensitivity. There is also no sample loss because solutes don't interact with the stationary phase. Disadvantages are for example that 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 to have a good resolution[3]

Discovery

The technique was invented by Grant Henry Lathe and Colin R Ruthven, working at Queen Charlotte’s Hospital, London.[4][5] They later received the John Scott Award for this invention.[6] While Lathe and Ruthven used starch gels as the matrix, Jerker Porath and Per Flodin later introduced dextran gels;[7] other gels with size fractionation properties include agarose and polyacrylamide. A short review of these developments has appeared.[8]

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 band[9]

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 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, particles in solution do not have a fixed size resulting in the probability that a particle which would otherwise be hampered by a pore passing 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. 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

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. 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.

The elution volume (Ve) decreases roughly linearly with the logarithm of the molecular hydrodynamic volume. Columns are often calibrated using 4-5 standard samples (e.g., folded proteins of known molecular weight), and a sample containing a very large molecule such as thyroglobulin to determine the void volume. (Blue dextran is not recommended for Vo determination because it is heterogeneous and may give variable results) The elution volumes of the standards are divided by the elution volume of the thyroglobulin (Ve/Vo) and plotted against the log of the standards' molecular weights.

Applications

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Biochemical applications

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.

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 in the solvent chosen for analysis (often THF). 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. .

Drawback

In SEC, mass is not measured so much as the hydrodynamic volume of the polymer molecules, that is, how much space a particular polymer molecule takes up when it's in solution. 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 arrived at.[10]

References

  1. ^ International Union of Pure and Applied Chemistry. "Size-exclusion chromatography (SEC)". Compendium of Chemical Terminology Internet edition.
  2. ^ Skoog, D. A.; Principles of Instrumental Analysis, 6th ed.; Thompson Brooks/Cole: Belmont, CA, 2006, Chapter 28.
  3. ^ Skoog, D. A.; Principles of Instrumental Analysis, 6th ed.; Thompson Brooks/Cole: Belmont, CA, 2006, Chapter 28.
  4. ^ Lathe, GH and Ruthven, CR (1955) The separation of substances on the basis of their molecular weights, using columns of starch and water. Biochem J. 60(4):xxxiv.
  5. ^ Lathe, GH and Ruthven, CR (1956) The separation of substances and estimation of their relative molecular sizes by the use of columns of starch in water. Biochem. J. 62(4): 665-674. article
  6. ^ John Scott Award
  7. ^ Porath, J and Flodin, P (1959) Gel filtration: A method for desalting and group separation. Nature 183(4676): 1657-1659.
  8. ^ Eisenstein, M (2006) A look back, adventures in the matrix. Nature Methods 3(5): 410 article
  9. ^ Skoog, D. A.; Principles of Instrumental Analysis, 6th ed.; Thompson Brooks/Cole: Belmont, CA, 2006, Chapter 28.
  10. ^ Polymer Science Learning Center (PSLC) - Size Exclusion Chromatography[1]

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