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What exactly is size exclusion chromatography?

Since its invention in the 1950s, size-exclusion chromatography has been widely used for the analysis of proteins and other water-soluble polymers. Size-exclusion chromatography has many applications in modern science, as it is one of the main tools used by researchers to separate molecules.


What is size exclusion chromatography?

Also known as molecular sieve chromatography, size-exclusion chromatography (SEC) is a method to separate molecules for scientific and medical research. As the name implies, this chromatographic method separates molecules based on their size. Molecular weight can be used for this process as well.

The main application of size-exclusion chromatography is the analysis of large molecules, such as globular proteins and polymers. The stationary phase in size-exclusion chromatography is composed of a porous substance. 

The pores found within the stationary phase can be depressions on its surface or channels through the bead. Depending on a size-exclusion chromatography’s mobile phase, the technique can be given two different names: gel-filtration chromatography and gel permeation chromatography.

Gel-filtration chromatography

The technique receives this name when size-exclusion chromatography is performed using an aqueous material to transport the sample. The main use of gel-filtration chromatography is the fractionation of water-soluble polymers, including proteins.

Gel permeation chromatography

Gel permeation chromatography occurs when an organic solvent is used as the mobile phase. It can be used to study the molecular weight distribution of organic-soluble polymers.

This type of size-exclusion chromatography gained popularity soon after its invention because the information it provides has been difficult to obtain with other methods. 

The columns in gel permeation chromatography are based on cross-linked polystyrene and contain a controlled pore size. Gel permeation chromatography has proven capable of providing molar mass and molar mass distribution data for synthetic polymers.

Advantages of size-exclusion chromatography

A primary advantage of size-exclusion chromatography is the capacity to efficiently separate large molecules from smaller ones with a minimal volume of eluate. When performing this type of chromatography, one is capable of applying various solutions without compromising the filtration process or the biological activity of the particles being separated.

Size-exclusion chromatography exhibits short and well-defined separation times. Together with the use of narrow bands, this helps achieve good sensitivity. Another notable advantage is that, because solutes do not interact with the stationary phase, there is no sample loss.

This chromatographic method can be combined with other types of high-performance liquid chromatography to obtain more accurate results. Just as size-exclusion chromatography studies molecules through their size, other separation methods work with a molecule’s acidity, basicity, ionic charge, and more. 

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How does size exclusion chromatography work?

Molecule separations in the size-exclusion method use gel filtration to achieve chromatography. The gel contains spherical beads, which in turn contain pores that interact with molecules. Molecules that interact with this porous matrix react differently to it depending on their size.

If the molecules are larger than the pores, they will not diffuse into the beads in the gel. This will lead to faster elution. In turn, smaller molecules will enter the total pore volume and be eluted later. If a particle’s size isn’t too big or small, it will be able to penetrate the pores up to a certain degree. Due to this, pore size is specific to the intended result of the chromatography.


When should I use preparative size exclusion chromatography for protein purification?

Preparative size-exclusion chromatography can be used to isolate the components of a sample. Components divided by size-exclusion separation methods can be transferred directly to a suitable buffer. This makes size-exclusion chromatography effective for both assay and storage.

Selecting resins and columns

Column packing must be done appropriately to guarantee the success of the preparative size-exclusion chromatography. Depending on the required resolution and yield, a column bed height of up to 1 meter might be necessary. The diameter of the column is to be determined by the volume of the sample.

Optimizing preparative size-exclusion chromatography

A successful size-exclusion chromatography process depends on having good conditions for selectivity. Peak broadening effects that may happen during the separation should be counteracted.

The following steps can be taken to optimize size-exclusion-chromatography:

  1. Choosing a resin with a fractionation range that is suitable for providing a good resolution.
  2. Selecting a column with an appropriate bed height. For preparative separation, column heights between 30 and 100 centimeters are recommended.
  3. The column size must be chosen according to the volume of the sample that will be processed.

Scaling up preparative size-exclusion chromatography

If the chromatographic separation performed on a small column is successful, then larger sample volumes can be processed using larger columns in a single step. The column’s diameter and flow rate volume must be adjusted to compensate for the larger sample volume available. The scaling up of separation may be restricted by high back pressure generated by small particles.

To certify that size-exclusion chromatography scales up successfully, you should:

  1. Optimize the separation process at a small scale before upscaling operations.
  2. Maintain a congruent sample-to-volume ratio.
  3. Keep the bed height and increase the column volume. This can be achieved by increasing the cross-selectional area of the column.
  4. Maintain flow velocity across different sample size ranges.


How to read a size exclusion chromatography graph

When a polymer is eluted through a size-exclusion column, its molecular size or weight is detected by the chromatograph. A plot of time is created on the x-axis, and the number of molecules coming out of the column is measured by the y-axis.

Molecular weight distribution can be measured using elution time. The higher a molecule’s weight is, the faster it will be able to pass through the columns in size-exclusion chromatography.

Why is reversed-phase chromatography used?


How to improve size exclusion chromatography

It is important to note that the hydrodynamic volume of polymer molecules is the main focus in size-exclusion chromatography and not its mass. This means that to measure molecular weight using SEC data, one must perform calculations and approximate certain data.

Range of molecular weights

Even though size-exclusion chromatography is widely used by scientists, it is not without its limitations. Since there is no standard for molecular weight, researchers may see their analysis stunted by having nothing to compare their results to.

Estimating molecular weight

Approximations of molecular weight can be made using polystyrene as a standard. This is because the exact relationship between polystyrene’s molecular weight and hydrodynamic value is known to science. However, this relationship is not equal in all polymers. Only an approximate measurement can be achieved in certain cases.

Measuring molecular weight

Size-exclusion chromatography doesn’t provide a completely precise molecular weight. If this value is required to accomplish the goals of research, then size-exclusion chromatography may still be useful when used in tandem with other separation methods.

Improving the accuracy of size-exclusion chromatography

Size-exclusion chromatography differs from other similar methods of molecule separation because lower flow rates are favorable. Running the procedure at a lower flow rate often leads to improvements in separation and resolution (source).

The time it takes to accomplish size-exclusion chromatography may lengthen and can even take twice as long. Nonetheless, one trades speed for performance and accuracy. Due to a lower flow rate being a very simple parameter to affect, it becomes a very practical method to improve the resolution of size-exclusion chromatography.

Troubleshooting common size-exclusion chromatography issues

  • Poor resolution: Besides flow rate, other factors affecting resolution include the selectivity of the resin, particle size, and sample-to-column volume ratio. Resolution issues may also be caused by equipment.
  • Tailing peaks: These may be caused by a column packed at a very low pressure or flow rate. Tailing peaks can also be caused by an uneven sample application.
  • Fronting peaks: In contrast, an overpacked column may create fronting peaks. The resin should be evenly packed to avoid any kind of asymmetric peaks.


Valentia Analytical services

As experts in Ultra-High Performance Liquid Chromatography (UHPLC), Valentia Analytical can offer you the best pharmaceutical development services. Together with our team of experts, you can determine whether size-exclusion chromatography is the procedure you need and how to best accomplish it. From protein aggregation to plasmid DNA analysis, Valentia Analytical can help you with your research.

The specific type of chromatography needed for an analysis procedure depends on the molecules being analyzed and the goals of the research. Because of this, Valentia Analytical offers a wide range of techniques, including the most useful methods of chromatography. 

Other types of UHPLC/HPLC include: 

  • Ion exchange chromatography: This chromatographic method works by taking advantage of polarity to separate molecules based on ionic interactions. It can be implemented in the development of plasmid DNA, as it can reveal topoisometric homogeneity for the open chain, supercoiled, and linear forms.  
  • Affinity chromatography: This process separates biomolecules from a solution using macromolecular binding interactions between the analyte and other substances. It offers high selectivity and resolution. 
  • Reversed-phase chromatography: This is the most popular chromatographic method and has many implementations. Through reversed-phase chromatography, one can learn more about Active Pharmaceutical Ingredient (API) content, purity, and impurity. Likewise, many substances can be identified.

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