Size exclusion chromatography (SEC) separates molecules based on their size by filtration through a gel. The gel consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix. Small molecules diffuse into the pores and their flow through the column is retarded according to their size, while large molecules do not enter the pores and are eluted in the column's void volume. Consequently, molecules separate based on their size as they pass through the column and are eluted in order of decreasing molecular weight (MW).
Operating conditions and gel selection depend on the application and the desired resolution. Two common types of separations performed by SEC are fractionation and desalting (or buffer exchange.) Here we provide an overview of SEC and general considerations.
Related Topics: Affinity Chromatography, Ion Exchange Chromatography, Hydrophobic Interaction Chromatography, Mixed-Mode Chromatography, Low Pressure Chromatography Systems and Medium Pressure Chromatography Systems.
- Desalting — A common use of SEC is for desalting protein or nucleic acid samples. The molecule of interest is eluted in the void volume, while smaller molecules are retained in the gel pores. To obtain the desired separation, the gel should have an exclusion limit significantly smaller than the molecule of interest
- Fractionation — Molecules of varying molecular weights are separated within the gel matrix. With this separation method, the molecules of interest should fall within the fractionation range of the gel
Bio-Rad offers a number of media choices for SEC:
- Bio-Gel® P polyacrylamide media for MW separations under 100 KD and for desalting.
- Bio-Beads™ S-X media for the separation of lipophilic polymers.
- Bio-Gel® A 1.5m media, process scale, for the purification for antibodies and aggregates.
Size Exclusion Chromatography Media Selection Guide
|Hydrated Bead Size, µm||Hydrated Bed
|Typical Flow Rate , cm/hr*||Molecular Weight Fractionation Range|
|Bio-Gel P–2, fine||45–90||3 ml/g||5–10||100–1,800|
|Bio-Gel P–2, extra fine||<45||<10|
|Bio-Gel P–4, medium||90–180||4 ml/g||15–20||800–4,000|
|Bio-Gel P–4, fine||45–90||10–15|
|Bio-Gel P–4, extra fine||<45||<10|
|Bio-Gel P–6, medium||90–180||6.5 ml/g||15–20||1,000–6,000|
|Bio-Gel P–6, fine||45–90||10–15|
|Bio-Gel P–6, extra fine||<45||<10|
|Bio-Gel P–6DG gel||90–180||15–20|
|Bio-Gel P–10 gel, medium||90–180||7.5 ml/g||15–20||1,500–20,000|
|Bio-Gel P–10 gel, fine||45–90||10–15|
|Bio-Gel P–30 gel, medium||90–180||9 ml/g||7–13||2,400–40,000|
|Bio-Gel P–30 gel, fine||45–90||6–11|
|Bio-Gel P–60 gel, medium||90–180||11 ml/g||4–6||3,000–60,000|
|Bio-Gel P–60 gel, fine||45–90||3–5|
|Bio-Gel P–100 gel, medium||90–180||12 ml/g||4–6||5,000–100,000|
|Bio-Gel P–100 gel, fine||45–90||3–5|
Although SEC does not discern similar MW species particularly well, it is very good at separating molecules that may not be fully resolved by other methods like IEX or HIC. As such, it is often reserved for the final "polishing" step of purification. The resolution of separation depends on particle size, pore size, flow rate, column length and diameter, and sample volume. Generally, the highest possible resolution is the ability to measure a twofold difference in MW. This is obtained with moderate flow rates , long, narrow columns, small particle size gels, small sample volumes (1–5% of the total column or bed volume), and a sample viscosity that is the same as the eluent.
- Particle and Pore Size — in general the smaller the particle size, the higher the resolution. Pore size controls the exclusion limit and the fractionation range of the media
- Column Dimensions — resolution increases with the column length, and as the column diameter increases, the capacity of the column increases due to the larger column or bed volume
- Column Packing — column packing is critical to resolution; an overpacked column can collapse the pores in the beads resulting in diminished resolution. An underpacked column increases the mixing volume outside of the pores, resulting in broader, less resolved, peaks. Dead volume at the top of the column can significantly reduce resolution as the sample is allowed to diffuse prior to entering the column bed, resulting in "band broadening" or wider peaks. Dead volume at the top of the column is possibly the most critical consideration because the loss in resolution is then multiplied as the molecules travel through the column
- Flow Rate — moderate flowrates offer the highest resolution. Flowrates are specific to the type of media being used. Moderate flow rates allow time for the molecules to fully access the surface area of the stationary phase permitting the smaller MW species the time to enter the pores, resulting in improved partitioning of the different MW species. Flowrates that are too slow will reduce resolution since the peaks or bands will diffuse too much as they travel through the column
With proper column calibration using MW standards, the molecular weights of unknown molecules can be measured. Molecular weight estimates by column chromatography can be quite accurate for globular proteins and less accurate for other, more linear, molecules like DNA.
The elution volume (Ve) decreases nearly linear with the log of the molecular hydrodynamic volume. A few proteins or standards of known MW are used for calibrating the columns. Bio-Rad offers a useful Gel Filtration Standard. This calibration standard contains thyroglobulin (670 kD), to determine the void volume (Vo) of the column, as well as four additional proteins of 158, 44, 17, and 1.35 kD respectively. Analysis using any standard is carried out by dividing the Ve of the standards by the Ve of the thyroglobulin (Ve/Vo) and plotting against the log of the MW of the standard.
For desalting applications, the difference in MW between the molecule of interest and salt or buffer molecules is so large that these peaks are less likely to overlap. Therefore sample volume can be as much as 30% of the total bed volumes, and shorter, wider columns may be used.