Size exclusion chromatography is based on separating different-sized molecules according to their molecular weights or sizes. Unlike other chromatography methods, the former technique uses mild conditions and is usually employed for improved purity at the end of the downstream process. As all our protein purification techniques, Size Exclusion Chromatography (SEC) can be included in our protein expression services.

When should you use Size Exclusion
Chromatography for Protein Purification?

At the earliest stages of your project, ProteoGenix establishes your full protein expression strategy, from gene design to protein purification. In this context, SEC purification can be recommended when:

  • You ask for contaminant-free protein: Your project requires a highly pure product, free from small-size contaminants and aggregates. SEC chromatography can be used as a further step in the downstream process to ensure improved purity.
  • Your protein of interest is sensitive: SEC uses mild conditions making it suitable for proteins that cannot withstand harsh conditions.
    • Buffer: No buffer changes are required which is advantageous for pH change-sensitive proteins.
    • Temperature: SEC can be performed at a large range of temperatures: From 4°C to 30°C.
    • Denaturing agents: Flexible denaturation concentrations can be applied.
  • You required high resolution: Do you need a sharp separation between other molecules? SEC purification offers high resolution suited for your goals. SEC resolution is dependent on the efficiency of the resin (matrix, beads molecules…).

What is Protein purification by
Gel-Filtration Chromatography?

What is the principle of size exclusion chromatography?

Size exclusion chromatography, also known as Gel-Filtration chromatography is a type of liquid chromatography. It allows the separation of molecules based on their size or molecular weight.

Gel-filtration chromatography is equipped with a resin composed of porous particles/beads. Different in size, these pores grant the size separation property of this technique. These beads are chemically and physically stable. Besides, they do not interact with the surrounding solution. However, thanks to their pores they enable the entry or not of molecules based on their size.

Gel-filtration is composed of a mobile phase as well as a stationary phase:

  • Mobile phase: Consists of all the liquid outside the particles.
  • Stationary phase: Consists of all the liquid inside the porous beads.

Steps to gel-filtration chromatography:

  • Sample injection: A mixture of different molecule sizes is introduced into the chromatography column.
  • Size separation: 2.The mixture flows down the column. Depending on the size of molecules, their moving rate differs from one another. The small and medium size molecules are trapped in their respective size pores. Larger elements like aggregates and complexes will stay in the mobile phase and continue moving down the column.
  • Elution: 3.Large-size molecules are eluted in the first place since they are out of the pores. Then, medium molecules, and finally, smaller molecules.
Size exclusion chromatography process

Applications of gel-filtration

Size exclusion chromatography is a versatile method. The flexibility of the conditions allows to use it for several applications, for instance:

  • To Increase Purity: Protein Purification SEC chromatography can be proposed as a further purification step to increase the final purity of the product. In this case, preparative size exclusion chromatography is applied.
  • To Analyze Purity: SEC-HPLC method is effective for protein analysis. It allows size-efficient profiling of protein samples and examining the purity of the product.
  • In Research Studies: Gel-filtration is ideal for purification and separating molecules according to their molecular weights. Moreover, SEC can maintain the stability and activity of the protein under specific conditions. Eventually, allowing protein characterization and other protein-related research studies.
  • In Endotoxin Removal: Removal of endotoxin is crucial in certain pre-clinical and clinical studies since they induce pyrogenic reactions. In this context, gel-filtration chromatography can be used to eliminate them.
  • In Buffer Exchange: This technique is sometimes used to place the purified molecule under a more appropriate buffer. It can be performed before, between, or/and after purification.
  • In Desalting: Separate small molecules from large molecules and small contaminants like salt, and other small contaminants originating from crude cell lysate.
  • To Monitor Aggregates: Size exclusion chromatography permits monitoring aggregates and quaternary structures and separates them from small-size molecules.

How to improve size exclusion chromatography performance?

  • Sample size and molecular weight: These factors largely influence the resol-ution. To achieve the highest resolution, it is recommended to use SEC at the final stages of purification. At this step, the protein mixture contains mainly small-size and low molecular weights molecules along with your protein of interest.
  • Choice of eluent: In gel-filtration, a high ionic strength elution buffer is utilized to minimize the electrostatic interactions created between the molecules together and the matrix
  • Effect of flow rate: The flow rate is dependent on the type of matrix chosen. In addition, a moderate flow rate usually offers high resolution.

In conclusion, size exclusion chromatography can result in yield loss due to sample dilution, but it remains an efficient method for the separation of molecules based on their size. For this reason, this method is recommended to be used at the end of the purification process. SEC purification is also well-known to increase the product’s purity in mild conditions and operate molecule separation under flexible conditions.