Size Exclusion Chromatography
Size exclusion chromatography, also known as gel filtration chromatography, separates molecules by size. The gel matrix consists of spherical beads with pores of a specific size distribution. Separation occurs when molecules of different sizes are excluded from the pores. As small molecules diffuse into the pores, their flow through the column is retarded, while large molecules bypass the pores and are eluted in the column’s void volume. Consequently, molecules are separated according to size and elute in order of decreasing molecular weight.
Operating conditions and selection of media depend on the application and the desired resolution. A common method used in size exclusion chromatography is group separation, which includes desalting and fractionation.
Desalting — The molecule of interest is eluted in the void volume, while smaller molecules are retained in the gel pores; to achieve the desired separation, the matrix should have an exclusion limit smaller than the molecule of interest.
Fractionation — Molecules with a range of molecular weights are separated within the gel matrix; with this method, the molecules of interest should fall within the fractionation range of the gel.
Common applications include protein fractionation and molecular weight determination, nucleic acid separation, plasmid purification, and polysaccharide fractionation.
Resolution depends on particle size, pore size, flow rate, column dimensions, and sample volume. Generally, the highest resolution is obtained with low flow rates (2–10 cm/hr), long, narrow columns, small-particle-size gels, small sample volumes (1–5% of the total bed volume), 2-fold difference in molecular weights, and a sample viscosity that is the same as the eluent. For desalting, the sample volume can be as much as 30–40% of the total bed volume, and shorter, wider columns may be used.
For a list of antibody purification applications, request bulletin 1801. Protein applications are given in bulletin 1802.
Bio-Gel P Polyacrylamide Gels
Bio-Gel P polyacrylamide gels, for high-resolution gel filtration, are prepared by copolymerization of acrylamide and N,N'-methylene-bis-acrylamide. Bio-Gel P gels:
- Are available in several particle size ranges with molecular weight exclusion limits ranging from 100 to 100,000
- Are extremely hydrophilic and essentially nonionic
- Provide efficient, gentle gel filtration of sensitive compounds
- Do not support microbial growth or leach carbohydrates (due to their synthetic composition), as dextrose and agarose gels can
Bio-Gel P gels are autoclavable at pH 5.5–6.5 and operate over a pH range of 2–10 at room temperature. Flow rate and resolution increase with increasing temperature in the range of 4–80°C.
The gels are compatible with dilute organic acids, 8 M urea, chaotropic agents, detergents, and miscible organic solvents. Alcohol, up to 20% v/v, can be used to enhance the solubility of nucleotides, peptides, and tannins without altering the exclusion properties of the gels. Formamide may also be used at full strength since Bio-Gel P gels are completely swollen in this solvent.
Bio-Gel P-6DG gel, with a molecular weight exclusion limit of 6,000, is recommended specifically for desalting and buffer exchange applications. It provides rapid results with high sample recovery and minimal sample dilution. Its hydrophilicity ensures little or no interaction between the gel and sample molecules.
For more information, request bulletin 2068.
|
| | Hydrated Bead
Size (µm) | Hydrated Bed
Volume Mass Dry Gel | Typical Flow
Rates (cm/hr)* | Molecular Weight
Fractionation Range |
|
Bio-Gel P-2 gel, fine
Bio-Gel P-2 gel, extra fine | 45–90
<45 | 3 ml/g | 5–10
<10 | 100–1,800 |
Bio-Gel P-4 gel, medium
Bio-Gel P-4 gel, fine
Bio-Gel P-4 gel, extra fine | 90–180
45–90
<45 | 4 ml/g | 15–20
10–15
<10 | 800–4,000 |
Bio-Gel P-6 gel, medium
Bio-Gel P-6 gel, fine
Bio-Gel P-6 gel, extra fine
Bio-Gel P-6DG gel | 90–180
45–90
<45
90–180 | 6.5 ml/g | 15–20
10–15
<10
15–20 | 1,000–6,000 |
Bio-Gel P-10 gel, medium
Bio-Gel P-10 gel, fine | 90–180
45–90 | 7.5 ml/g | 15–20
10–15 | 1,500–20,000 |
Bio-Gel P-30 gel, medium
Bio-Gel P-30 gel, fine | 90–180
45–90 | 9 ml/g | 7–13
6–11 | 2,500–40,000 |
Bio-Gel P-60 gel, medium
Bio-Gel P-60 gel, fine | 90–180
45–90 | 11 ml/g | 4–6
3–5 | 3,000–60,000 |
Bio-Gel P-100 gel, medium
Bio-Gel P-100 gel, fine | 90–180
45–90 | 12 ml/g | 4–6
3–5 | 50,000–100,000 |
|
* Flow rates determined in a 1.5 x 70 cm column using a hydrostatic pressure head-to-bed ratio of 1:1. 
