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Size-Exclusion Chromatography
Bij size-exclusion chromatography (SEC), ook bekend als moleculaire-exclusion of gel-permeation chromatography, worden moleculen gescheiden op basis v…
Size-exclusion chromatography, or SEC, involves separating molecules based on size.
Here, the stationary phase can be silica particles or cross-linked polymer resin beads that contain a network of uniform pores.
Large molecules are excluded from entering the pores and eluted first, whereas very small molecules are retained the longest and eluted last.
Intermediate-sized molecules are separated in the retention window and undergo fractionation.
The retention time of the molecules is directly influenced by their size.
The experimental calibration curve obtained using standard substances is then used to estimate the unknown molecular mass.
Size exclusion chromatography using hydrophilic packing is known as gel filtration and is applied to water-soluble samples. Hydrophobic packing materials are used in gel permeation chromatography for substances soluble in less-polar organic solvents.
Both these methods are useful for the separation of polymers or macromolecules.
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Q1: How does size-exclusion chromatography separate molecules?
Size-exclusion chromatography separates molecules based on their size using a stationary phase containing uniform pores. Large molecules cannot enter the pores and elute first, while small molecules penetrate deeply into the pores and elute last. Intermediate-sized molecules are separated in the retention window, with retention time directly influenced by molecular size.
Q2: What are the differences between gel filtration and gel permeation chromatography?
Gel filtration uses hydrophilic packing materials and is applied to water-soluble samples, while gel permeation chromatography uses hydrophobic packing materials for substances soluble in less-polar organic solvents. Both methods separate polymers and macromolecules effectively. The choice of packing depends on the solvent compatibility and sample polarity requirements.
Q3: What types of stationary phases are used in size-exclusion chromatography?
Size-exclusion chromatography uses two main stationary phase types: silica particles and cross-linked polymer resin beads. Silica particles offer rigidity and stability with various solvent compatibility. Polymer beads come in different pore sizes and can be hydrophilic for aqueous separations or hydrophobic for non-polar solvents like polystyrene-divinylbenzene.
Q4: How is molecular mass determined using size-exclusion chromatography?
An experimental calibration curve is prepared using standard substances with known molecular weights. This curve relates retention time or volume to molecular size. The calibration curve is then used to estimate the unknown molecular mass of sample molecules by comparing their retention behavior to the standards.
Q5: What is the relationship between solute size and retention volume in SEC?
Smaller solutes spend more time within the pores of the stationary phase, resulting in longer elution times and greater retention volumes. The retention volume for a solute depends on its distribution ratio, which ranges from 0 at the exclusion limit to 1 at the inclusion limit. This reflects how well the solute penetrates the pores.
Q6: Can size-exclusion chromatography be performed using conventional HPLC equipment?
Yes, size-exclusion chromatography can be carried out using conventional high performance liquid chromatography instrumentation by replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is typically used to obtain chromatograms. This approach allows researchers to adapt existing equipment for SEC applications.
Q7: Why are silica particles deactivated in size-exclusion chromatography?
Silica particles used for size exclusion are deactivated to prevent unwanted interactions between the sample molecules and the stationary phase surface. Deactivation ensures that separation is based solely on molecular size and pore penetration rather than chemical interactions. In contrast, polymer resins are synthesized without exchange sites to achieve the same goal.