Gel Filtration Chromatography

Table of Contents

Gel Filtration chromatography is a type of size-exclusion chromatography (SEC) that separates analytes on the basis of molecular size, generally using an aqueous solvent as a carrier.

An alternative name for gel filtration chromatography is molecular sieve chromatography.

This technique is employed to separate large molecules, for instance, proteins and/or polymers from a complex mixture, as it passes through a column packed with the gel filtration medium.

In this article, we will discuss everything you need to know about gel filtration chromatography, so continue reading!

What is gel Filtration Chromatography?

Gel Filtration technique was developed by Grant Henry Lathe and Colin Ruthren in 1955. It is a type of partition chromatography, a subtype of liquid chromatography in which the components are partitioned between two phases, i.e. a stationary and a mobile phase.

In gel filtration chromatography, the exclusion of molecules occurs on the basis of differences in sizes via filtration through a gel containing spherical beads. Therefore, this chromatographic type is named gel filtration.

Gel filtration chromatography is applied for the analysis and fractionation of hydrophilic polymers, such as proteins using a hydrophobic stationary phase and an aqueous solvent as a mobile phase.

What is the stationary and mobile phase in gel filtration chromatography?

Stationary phase: A gel polymer matrix with pores in the shape of spherical particles is used as a stationary phase in gel filtration chromatography.

A polymer matrix is selected due to its inertness and physical and chemical stability. Agarose gel, dextran and cross-linked polyacrylamide, among several other variants of this medium, are commercially available in a variety of pore sizes in the beads.

Mobile phase: Water-based solvents or buffers are used as mobile phases in gel filtration chromatography.

Basic working principle of gel filtration chromatography

 Once the aqueous buffer flows through the chromatographic column packed with the gel, the liquid enters into the pores of the matrix. It acts as a stationary phase. On the other hand, the buffer flowing between beads, outside the pores serves as a mobile phase.

The gel beads have a size exclusion limit which indicates that the pores in beads are of a precise size and the sample molecules above a specific molecular weight cannot fit into these pores.

Therefore, molecules with sizes above the exclusion limit are unable to enter the pores. These are thus eluted out of the column comparatively early. Contrarily, the smaller molecules, which can stay in pores, take a longer path to elute out. This is how different sample molecules are separated according to their sizes in gel filtration chromatography.  

 Gel filtration is unique and differs from affinity and ion exchange chromatography, where molecules chemically interact with the medium. Therefore, buffer composition does not impact resolution, i.e. the extent of the degree of separation between chromatographic peaks.

How to perform gel filtration chromatography?

The following step-by-step guide will lead you through the process of performing gel filtration chromatography.

Step I: Column Packing

The column is packed by a gel filtration medium constituting spherical beads. A slurry of gel and buffer solution is poured, filling one-third of the column.

Step II: Washing of Matrix

Once the column is packed, several volumes of buffer are passed to eliminate any air bubbles and/or to test the homogeneity of the column.

 Step III: Sample loading

Sample in solution form is loaded into the column via a syringe.

The sample, when applied takes the form of two measurable liquid volumes. It includes the volume of liquid between the beads, which is referred to as external volume, also known as void volume (Vo). In contrast, the liquid within the beads is known as the internal volume, while the sum of both volumes is termed total volume (Vt).

Step IV: Analyte Separation

Molecules larger in size than the size of the porous stationary phase are not retained by internal volume. Rather, these quickly flow through the column and emerge at Vo.

Molecules of small and intermediate sizes equilibrate between the internal and external liquid volumes, resulting in a comparatively slower elution. Theses thus emerge at elution volume Ve , which is greater than Vo.

Step V: Elution

The components of sample elute isocratically out of the column, which implies separation can be done with a single buffer system.

Step VI: Detection

In GFC, a wide range of detectors are used to detect the final product, such as refractive index, UV absorption, IR absorption detector, etc. The detector is selected keeping in mind the sample’s nature.

Step VII: Calibration curve

A calibration curve is then plotted with the data obtained by performing gel filtration. The molecular weight of an unknown analyte present in the sample can be determined by comparing it to the molecular weight of a reference or known analyte.

Step VIII: Buffer wash for new run

At the end of the separation, a wash step is performed by running the buffer to remove any excess molecules that may get retained into the column, unwanted. This rejuvenates the column for a fresh analysis.

What is gel filtration chromatography used for ?-Applications

Some of the most useful applications of gel filtration chromatography are:

  • Fractionation

Gel Filtration is employed to operate in fractional mode to separate components that differ in size, such as different types of proteins present in a complex sample.

  • Molecular weight determination

Gel filtration is a simple method and requires less amount of sample; therefore, it facilitates determining the preliminary molecular weight of proteins.

The calculation formula for this particular analysis is only valid for spherical proteins and not for a few elongated proteins such as fibrin.

  • Purification of proteins

GFC helps in the purification of proteins, nucleic acids, enzymes, polysaccharides and other biological macromolecules.

This method can also be used for isolation purposes, such as for separating whey protein from raw milk.

  • Removal of pyrogenic substances

Pyrogenic substances are large molecular weight compounds that can heat the human body. These can be separated from smaller molecules through the exclusion property of gel filtration.

  • Desalting

Gel filtration can be used to separate small molecules, such as the removal of excess salt (desalting), and for buffer exchange.

  • Biomedical Field

An upgraded version, high-performance gel filtration chromatography (HPGFC), can on-line determine the distribution of lipoprotein cholesterol within a short period of 40 min.

This also facilitates the clinical determination of LDL-cholesterol.

  • Environmental Analysis

Gel filtration chromatography is applied for the isolation of metal-containing soluble organic fractions from water bodies near a radioactive waste disposal site.

This technique can also explain the effect of organic fractions with different molecular weights in trace metal mobilization.

Why is gel filtration chromatography important?

  • Gel filtration chromatography serves as a versatile technique used to separate biological molecules, majorly proteins, by discriminating according to their molecular sizes.
  • The conditions can be adjusted as per the sample’s nature without impacting separation.
  • Biomolecules that are sensitive to pH changes and extreme environmental conditions can utilize the gel filtration technique.

What is the difference between gel filtration and gel permeation chromatography?

Gel filtration and gel permeation are both sub-types of size exclusion chromatography. Both employ a gel matrix as a stationary phase. A primary difference between the two is that:

  • Gel filtration chromatography uses an aqueous-based mobile phase.
  • Gel permeation chromatography uses organic solvents as mobile phases.

Here is a visual representation of gel filtration chromatography for you to revise your concepts.

You may also like our article: What are those 10 different chromatography techniques.


1. Ó’Fágáin, C., P.M. Cummins, and B.F. O’Connor, Gel-filtration chromatography. Protein chromatography: Methods and protocols, 2011: p. 25-33.

2. Hagel, L., Gel-filtration chromatography. Curr Protoc Mol Biol, 2001. Chapter 10: p. Unit 10.9.

3.Duong-Ly, K.C. and S.B. Gabelli, Gel filtration chromatography (size exclusion chromatography) of proteins, in Methods in enzymology. 2014, Elsevier. p. 105-114.

4. Striegel, A.M., et al., Modern Size‐Exclusion Liquid Chromatography. 2009: John Wiley & Sons, Inc.

Organic Spectroscopy

Organic spectroscopy can be used to identify and investigate organic molecules. It deals with the interaction between electromagnetic radiation (EMR) and matter. These waves travel

Read More »