Size exclusion chromatography (SEC) is the separation column chromatography. Gel permeation chromatography (GPC), Gel filtration chromatography (GFC), and Size exclusion chromatography (SEC) are interchangeably used to describe the same liquid column chromatographic technique.

Size exclusion chromatography is a fast, reliable, and inexpensive tool for the identification of a variety of samples. Some advantages which make it preferable to others are given below:

  1. Short analysis time
  2. It can effectively separate sugars, polymers, elastomers, and rubber
  3. Sample loss
  4. A small amount of mobile phase is required

GPCSEC High Resolution Gradient analysis of polymer

Principle

The principle of size exclusion chromatography is the separation of molecules on the basis of size or molecular weight.  The small size particle reaches the interior of the pores in the stationary phase, whereas the large size particles are excluded, being unhindered completely.

Theoratical Chromatogram of bimicro molecules using size exclusion chromatography

Working

It is a sophisticated technique that separates the components based on the difference in weight. The stationary phase is porous material while the mobile phase is the solvent. The sample is pumped through columns containing microporous gel.

The large-sized molecules thus pass through unhindered whereas, the smaller molecules get stuck or penetrate into the gel.

In size exclusion chromatography, the sample volume should be approximately 0.3% of the mobile phase volume to achieve optimal results. Moreover, the sample volume should not be greater than 2% to achieve high resolution.

Category Description
Sample ID PM101
Sample type polymer product
Sample volume 5μL
Column superdex®
Flow rate 0.50mL/min
Detector PMT

Components of size exclusion chromatography

  1. Stationary phase
  2. The columns
  3. The mobile Phase
  4. The pump
  5. Detectors

Stationary phase

It is made up of porous material, a gel-like semi-permeable substance. The materials are available in a variety of pores sizes.

Properties of good stationary phase

Some important features of the stationary phase are given below:

  • Chemically and mechanically stable substance
  • Homogeneous porous structure
  • Inert to used solvent and sample chemicals

Examples of stationary phase

  1. Glucose natural gel
  2. Galactose natural gel
  3. Acrylamide gel

Size exclusion columns

Size exclusion chromatography columns are used to separate molecules w.r.t to their size or molecular weight. They work only when the correct size of pore size resin is used. These columns are centrifugal tubes in classical times which are relatively inexpensive. They have limited applications as they can only be used when the analyte molecules are known.

Now the columns become advanced and more sophisticated. They come with machines in which they are fitted in such a way that a supply of mobile phase is provided through pumps. The sample is injected with a syringe in the flowing mobile phase. The eluted molecules fraction is detected using a spectrophotometer.

Types of size exclusion chromatography columns 

The columns are classified into the following types regarding their functions:

  • Organic and aqueous columns
  • Analytical columns
  • conventional and efficient solvent columns
  • High-speed GPC columns

Gel and its types, used in SEC

Dextran gel: It is α 1,6 polymer of glucose. The fermentation of sucrose forms it.

Galactose natural gel: It is a natural gel. It is composed of repeating units of β- D galactose and anhydrous α-L-galactose

Acrylamide gel: It is a synthetic gel. It is made up of polymerized acrylamide.

Types of gels based on the swelling process

  1. Soft gels: They are used for the separation of protein and polymers in an aqueous medium. For example, polyacrylamide gel, dextran, and agarose.
  2. Rigid gels: They are further divided into two types based on the separating analyte.

Examples

  • Polystyrene gel: It is used for the separation of nonpolar polymers with the aid of nonpolar solvents.
  • Porous gel: Separation of the polymer system.

Mobile phase

The mobile phase is the solvent polar or nonpolar depending upon conditions. The eluent or the mobile phase solvent should have a minimal response in the detectors. It has the ability to buffer to some extent and can wet the stationary bed.

The most common solvents (eluents) used in SEC can dissolve the analyte at room temperature. For example, tetrahydrofuran, chloroform, and dimethyl formamide.

Selection of solvent (mobile phase) for SEC

The solvents used for a mobile phase of SEC must fulfill the criteria given below:

  1.  The solvent must completely dissolve the sample analyte at room temperature.
  2.  The solvent has different properties with solute to be separately identified at the detector.
  3. The solvent should be HPLC/GPC grade.
  4. It should not denature the solute.
  5. The solvent is not corrosive to any metallic component of the instrument.

Pumps

A pump is used to constantly maintain the flow in size exclusion chromatography. The flow has great importance in SEC analysis as an error of 0.1% in the flow rate can produce a 10% error in the molecular weight of the analyte.

Types of SEC pumps

  • Syringe pumps
  • Reciprocating pumps
  • Pneumatic pumps

Detectors

Eluted material detection has a pivotal role in SEC. Detectors act as the eyes of the instrument. The detector is opted as per the type of analyte. Some important types of detectors and their capabilities are given below:

Concentration sensitive detectors

 1. Bulk Property Detectors: Refractive Index (RI) Detector

2Solute Property Detectors: Ultraviolet (UV) Absorption Detector

3. Evaporative Detectors (ELSD): Evaporative Light Scattering Detector

4. Molar mass-sensitive detectors

  • Light Scattering Detectors: Low Angle Light Scattering (LALS) Detectors and Multiangle Light Scattering (MALS) detectors
    Viscosity Detectors: Differential Viscometers
  • Mass spectrometer

Related Resources

Size Exclusion Chromatography Rules

The rules are made to be followed during the lab analysis to get the best possible results. Some of them are given below:

  1. The polymer should be dissolved in minimum quantity. (0.1% w/w)
  2. The polymer solution under consideration should pass through cross-linked organic gels.
  3. Larger molecules do not fit into the pores and are eluted first.
  4. By principle, there should be no absorption of analyte or polymer on the packed materials(gels)
  5. Every polymer should be eluted between the Vo and Vt

How to improve peak resolutions in chromatography?

Peak resolution is important in mixture analysis. To improve the resolution, the following steps can help:

  • By using longer column
  • Decreasing particle size such that filter to remove bigger particles
  • By reducing peak tailing
  • Increasing temperature
  • Using minimum sample

Applications

Size exclusion has a wide range of applications for the purification and analysis of natural, man-made, and biological polymers. For example, proteins, polysaccharides, and nucleic acids can be quantified in the sample.

1. Analysis of Proteins: Purification, identification, and fractionation.

  •  Desalting
  • Copolymerisation studies

2. Molecular weight determination of protein polymers, fats, rubber, resins, etc.
3. Separation: The substances such as sugar, proteins, rubbers, and others can be conveniently separated based on the size of their molecules.

key Takeaways

  • Size exclusion chromatography (SEC) is a separation technique that uses size to separate molecules. It’s also known as gel filtration or molecular sieve chromatography.
  • SEC involves passing a sample through a column packed with porous material, such as beads of cross-linked polymers or agarose.
  • Smaller molecules can enter the pores of the beads while larger molecules are excluded.
  • Depending on their size, the separated molecules emerge from the column at different times and can be collected and analyzed by using UV-Vis spectroscopy.
  • SEC is commonly used in biochemistry and molecular biology for protein purification and characterization.

Concepts Berg

Why did the liposomes fluoresce during size-exclusion chromatography?

Size exclusion usually uses UV visible detectors. Liposomes do not absorb UV light so they are impossible to detect with that detector. Therefore, the size of beads is set in a way that the liposomes can retain in the gel for a long time and then be analyzed afterwords with fluorescence spectroscopy.

How does size exclusion chromatography work?

Size exclusion chromatography separates the molecules based on their sizes. The analyte passes through the column which consists of beads of various sizes. Large-size molecules pass through without much hindrance while small particles get stuck in the gel and thus elute later.

What elutes first in size exclusion chromatography?

Molecules larger in size are eluted first in size exclusion chromatography. As the small-size proteins are more susceptible to being stuck in pores, therefore, they spend relatively more time in the column.

Where is size exclusion chromatography (SEC) used?

SEC is used for the separation and identification of proteins. Furthermore, it can be applied to separate the mixtures of sugars, rubbers, and other macromolecules on the basis of molecular weight.

How many samples to load size exclusion chromatography?

In analytical size exclusion chromatography, the samples can be loaded manually or with an autosampler. In the autosampler 100 sample can be loaded at once.

Can size exclusion chromatography be used to purify proteins?
Size exclusion chromatography is the simplest method of protein separation. It is considered the final step during protein purification.