Size Exclusion Chromatography

Size exclusion chromatography (SEC), also known as molecular sieve chromatography, is an analytical chemistry technique in which molecules dissolved in solution are separated by their size and sometimes molecular weight.  SEC is typically applied to large molecules and macromolecule complexes such as proteins and industrial polymers, both natural and synthetic.  Size exclusion chromatography has various applications in the polymer industry due to the ability to obtain molecular weights (Mw) and Mw distributions for a multitude of compounds.

Size exclusion is one of the multiple separation modes of high performance thin layer chromatography (HPLC).  In size-exclusion HPLC, the column has a material that controls the pore size, so the sample molecules are separated by particle size.  Larger particles are flushed out of the column quickly, and smaller particles remain in the column and take longer to elute (release off the column).

SEC operates by the principle that particles of varying sizes elute (filter) through the stationary phase at different rates, ultimately separating the particles in the sample of interest by size.  Additionally, particles of similar size should elute together.  Each size exclusion column can separate a range of molecular weights.  The column’s exclusion limit sets the upper and lower boundaries for the molecular weights that can be separated.

Gel-filtration chromatography (GFC) and gel permeation chromatography (GPC) are two types of SEC.  Gel-filtration chromatography can be used to fractionate proteins and other water-soluble polymers, and gel permeation chromatography can be used to analyze the molecular weight distribution of organic-soluble polymers.  Neither of these techniques should be confused with gel electrophoresis, in which an electric field moves molecules through a gel depending on the molecules’ electric charges.

GFC uses a hydrophilic material in the column as the stationary phase and an aqueous mobile phase to separate, fractionate, and/or measure the molecular weight distribution of the water-soluble molecules, such as proteins or polysaccharides.  GPC uses a hydrophobic column material as the stationary phase and an organic solvent (or non-aqueous solution) as the mobile phase.  GPC is typically used to identify molecular weight distribution of synthetic polymers.

The gel used in GFC and GPC contains spherical beads of a particular size distribution, which facilitates the separation of molecules.  Smaller molecules diffuse into the pores of the gel and thus have a slower rate of travel through the column leading to elution.  Larger molecules do not fit into the pores of the gel, so they pass through the column at a faster rate.  Due to this size-exclusion, molecules are eluted in decreasing order of molecular weight.

 

Size exclusion chromatography confers many advantages over other types of chromatography, including good separation of large and small molecules with a minimal volume of eluent in the mobile phase; the application of various solutions without interfering with the filtration process; and the preservation of biological activity of the particles being separated.  SEC is typically combined with other techniques which can further separate the molecules by additional characteristics including acidity, basicity, charge, and affinity for certain compounds.  SEC boasts short and well-defined separation times and narrow bands, and no sample loss, because solutes do not interact with the stationary phase.  Additionally, SEC allows approximation of molecular weights of compounds.

Desalting and fractionation are common applications using size exchange chromatography. Desalting proteins or nucleic acid samples occurs when the larger salt molecules are eluted off the column, leaving the desired molecule of interest.  Fractionation occurs when the molecule(s) of interest fall in the desired fractionation range of the gel.

Size exclusion chromatography allows for identification of the various sizes of all molecules contained in the sample, not just an average.  SEC is widely used for quality control in order to identify molecular differences of molecules.  With regard to synthetic polymers, even those with identical average molecular weight will have a unique Mw distribution.  Such compounds, with the same average molecular weight may have differences in their properties. Methods such as light scattering, osmotic pressure, and viscosity only provide an average molecular weight.

Columns used for size exclusion chromatography vary widely by packing material and pore size.  Each column has a unique calibration curve that identifies the range of separation by elution volume displayed linearly for each type of column.  If the sample is unknown, mixed gel columns are available which contain a variety of pore sizes and a mixture of column packing material.  Mixed columns have been found to be accurate, particularly when analyzing globular proteins, but less accurate when analyzing DNA or other linear molecules.

SEC can be used to characterize various molecules including proteins, synthetic polymers, and natural polymers.  Biochemists currently use SEC to research protein oligomerization, aggregation, conjugation and conformation, while polymer chemists use SEC to control polymer strength, toughness and performance. Pharmaceutical chemists use size exclusion chromatography to monitor product behavior, and food scientists use it to monitor product performance and quality.