Analytical Chromatography - fSEC
FSEC resources and user guide ⬇︎
Size-Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography separates macromolecules in solution by size and shape. Size-exclusion columns are packed with fine, porous beads composed of dextran polymers, agarose, polyacrylamide, or silica. The bead pore sizes determine the size of macromolecules separated by the column. Proteins are separated by hydrodynamic radius, with large proteins eluting before small proteins. Particles that are larger than the pore size will pass through the column quickly and elute first, in the “Void peak”. The smallest molecules, including sample solvent, will elute last, because they can fully diffuse into the smallest of the pores, therefore spending the most time in the column before eluting, in the “Solvent peak.” For globular proteins, hydrodynamic radius, and molecular weight (MW) are proportional, so SEC is often used to estimate the mass and oligomeric state of protein particles by comparison to a set of globular MW standards.
Fluorescent Size exclusion chromatography (fSEC) is used for analytical size-exclusion chromatography with fluorescence and UV detection. This can be done with small sample quantities and short columns for a very rapid analysis of protein aggregation and oligomeric state.
The CMI offers training, access and select data collection services on analytical fluorescent size-exclusion chromatography (fSEC).
The CMI has an Agilent 1260 Infinity Isocratic Liquid Chromatography System with autosampler (5-100 μl) and variable wavelength UV and fluorescence detectors. The same Agilent Infinity 1260 system also used for Size exclusion chromatography with multi-angle static light scattering (SEC-MALS).
FSEC Resources
FSEC Supplies
- Sample filters, 0.02-0.2 μm filters (recommended)
- Autosampler vials (provided by the CMI)
- Analytical SEC Column:
- A range of analytical SEC columns can be used including silica and agarose columns
- Analytical columns
- e.g. SEPAX SRT SEC-300, 7.8 mm x 50 mm, guard column size
- maximum pH of 7.5
- Make sure that the column is compatible with your sample and buffer. Some detergents and membrane proteins will require special columns designed for hydrophobic samples and buffers.
FSEC Sample Preparation
Assay Buffers/Solvents
- Running buffer should always be chosen to be compatible with both the SEC column and the protein sample.
- Make sure you know the buffer compatibility of the SEC column you are using.
- Prepare sufficient buffer (500ml - 1L is recommended)
- Recommended Buffer: PBS or HBS (25 mM HEPES pH 7-7.5, 150 mM NaCl), filtered.
- Most silica columns will not tolerate pH above 7.5.
- CMI chromatography systems are tolerant of Aqueous Solvents Only.
- CMI systems are NOT compatible with organic solvents, including acetone, acetonitrile, DMAc, DMF, DMSO, THF, TCE or with concentrated acids.
Samples
- Know the UV extinction coefficient (in ml/mg•cm) and concentration (in mg/ml) of your protein.
- Concentration should be accurately measured to assess column recovery.
- Filter or centrifuge samples before use.
- Protein aggregates can damage the column.
- Recommended protein concentration varies depending on fluorescence intensity.