AppliCations No. 6 Size-Exclusion Chromatography for purification of biomolecules

Size-exclusion chromatography (SEC) is a popular method to separate biomolecules based on their size. Primarily, it is applied to the separation of biopolymers such as proteins and nucleic acids, i.e. water-soluble polymers. This system is also called Gel Filtration or Gel Permeation Chromatography (GPC), typically with beads of dextran or agarose serving as gel matrix. Smaller molecules pass significantly slower through the column than larger molecules. Not to be mixed up with gel electrophoresis, there are big differences in terms of the separation principle. SEC does not require electric current and the sieving effect will not separate small molecules first. It is indeed correct that smaller molecules pass more slowly through the matrix than larger molecules. This is due to the longer path the smaller molecules must travel. The longer path arises from the pores of the beads. The smaller molecules can enter the pores and go inside the beads. Entering the pores creates a longer path for the smaller molecules. There is a relationship between the delay this causes and the molecular size. The larger molecules can not enter the pores and therefore have a shorter path. The larger molecules therefore travel faster through the column than smaller molecules.
The main applications are desalting, buffer exchange and purification of proteins and nucleic acids (e.g. dye terminator removal).

An advantages of SEC / Gel Filtration is that the separation of the large molecules from the small ones is performed under mild conditions which means that biomolecules retain their biological activity. The volume of the eluate may be kept small and therefore the technique is suited for concentrated samples.

AppliChem's size-exclusion chromatography columns are packed with AppliXchange-G25 M, a beaded composite material composed partially of polymerized dextran. It exhibits high selectivity, high resolution and chemical stability. Molecules purified with AppliXchange-G25 M are separated according to size. The chemical interaction of gel matrix and molecules to be separated is negligible. Little or no absorption or binding takes place. Buffer and pH effects on resolution are kept minimal. Therefore, buffer conditions, pH value and temperature during the filtration process may be adapted according to the needs of the molecules but not the column material. Essential co-factors or ions necessary for the function or stability of proteins may be included in the eluent as well, since they largely don't interfere with the separation. The size exclusion cut-off for AppliXchange-G25 M is set at 10 kD for proteins and 10 bp for nucleic acids. Purified biomolecules are not significantly diluted when processed using AppliXchange-G25 M.

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ready-to-use Size-Exclusion Chromatography Columns from AppliChem:

DescriptionProduct No.QuantitySample Volume
DextraSEC NA10A88702 or 50 columns0.5 - 1 mL
DextraSEC NA2A85902 or 50 columns0.15 - 0.25 mL
DextraSEC 96WA85952 or 25 Platesmax.15 µL/well
DextraSEC PRO10A88222 or 50 columns0.5 - 1 mL
DextraSEC PRO2A87102 or 50 columns0.15 - 0.25 mL