Gel Filtration Chromatography

In this gel filtration chromatography principle and procedure post we have briefly explained gel filtration chromatography’s principle, components, gel filtration chromatography method procedure, applications, advantages and limitations.

Gel filtration chromatography method is a type of partition chromatography in which molecules are partitioned between a mobile and stationary phase based on their fractionation range. The fractionation range or exclusion limit is represented by resins or gel beads with a predefined size range.

Molecules that are larger than the fractionation range elute faster and enter the stationary phase more quickly. The molecules in the fractionation range, on the other hand, move slowly. As a result, the gel filtration chromatography method is important in the size analysis and determination of various biomolecules.

Gel filtration Definition

Gel filtration chromatography method is a chromatography method that isolates components based on their molecular sizes using porous gel beads of varying porosity. Gel filtration chromatography method primarily retains or excludes particles based on size, hydrophobicity, and molecular charges. It primarily aids in the isolation of biomolecules such as proteins, peptides, and oligonucleotides.

A porous polymer gel matrix embedded in a chromatographic column is used in size exclusion chromatographyOnce the aqueous buffer has passed through the column, the liquid that has entered the pores of the gel matrix serves as the stationary phase, and the liquid that has not entered the pores serve as the mobile phase.

Gel filtration Principle

The principle of size exclusion chromatography is based on the isolation of biomolecules based on differences in molecular weight or size. As the packing material in the chromatography column, spherical gel beads with definite porosity are used in the gel filtration technique.

A liquid mixture is passed through a column of porous gel beads, where some molecules elute earlier or later depending on the elution limit. The elution limit is a factor that determines whether molecules are retained or excluded through the packing material.

Molecules that possess high molecular weight compared to the elution limit will elute early. In contrast, the molecules with low molecular weight or size than the elution limit will elute later. In this way, the particles are separated in the gel filtration chromatography.

Components in Gel filtration

a) Stationary Phase

The stationary phase is a column packed with microporous gel beads. Dextran (Sephadex), polyacrylamide (Bio-Gel P), dextran-polyacrylamide (Sephacryl), and agarose (Sepharose and BioGel A) are the most commonly used gel filtration chromatography media. They are available in a wide range of pore sizes for the separation of macromolecules of various sizes.

b) The Mobile Phase

The mobile phase is the solvent that runs through the column. To begin, dilute the test sample in appropriate organic solvents. The test sample is then filtered and passed through the column. The column is where a multi-component mixture is separated.

Gel filtration chromatography

c) The Columns

The dimensions of the column are critical for effective purification. The resolution of gel filtration chromatography increases with column length. While the column diameter increases the column’s capacity as the bed volume increases. In general, a gel filtration column should be longer but smaller in diameter than other types of chromatography.

Analytical columns with diameters of 7.5–8mm are the most commonly available column dimensions. Preparative columns have a diameter of 22–25mm, standard column lengths are 25, 30, 50, and 60 cm, and narrow-bore columns have a diameter of 2–3mm.

d) The Pump

Using a chromatography system, a peristaltic pump, or a syringe, apply samples directly to the column. The size of the column, the type of gel filtration medium, and the sample volume all influence the equipment selection. A chromatography system, for example, will be required for a Superdex column, whereas a syringe can be used with small prepacked columns like HiTrap Desalting.

e) Detectors

The differential UV photometer is the most sensitive detector, and the differential refractometer is the most common detector (DRI). When characterising copolymer, two detectors in series are required.

Steps in Gel filtration

a) Column Preparation

Gel filtration chromatography

b) Swelling

According to the manufacturer’s recommendations, add an appropriate amount of filtered ddH2O to the gel matrix based on the swelling capacity of the beads. Decant the ddH2O on top of the gel once it has swollen and settled in the bottom of the beaker.

Then, add the appropriate buffer to the gel, gently mix it, and wait for it to settle. Decant and resuspend the gel in an equal volume of buffer, then degas the gel to remove any trapped air. Heating the slurry at 90oC for 5 hours results in rapid gel swelling. Swelling at room temperature is much slower and takes about 72 hours.

c) Packing

Adjust the bottom adapter and place the glass column vertically on a metal stand. Add some buffer to the empty column and allow it to pass through the outlet tube to remove any trapped air bubbles in the path.

Now, using a glass rod, block the outlet tubing and pour the gel slurry to fill the column to the required height. The glass rod should be positioned so that it touches the inner wall of the column.

This prevents the introduction of any air bubbles. To ensure smooth and continuous pouring of the gel, a gel reservoir should be used. Allow it to settle before decanting the excess buffer and gel and inserting the top adapter.

d) Equilibration

Connect the peristaltic pump and buffer reservoir to the top tubing. Keeps the bottom tubing connected to the waste. The operating pressure should be set in accordance with the gel matrix’s manufacturer’s recommendations. It is always preferable to keep the flow rate slower. Choose a buffer that meets your purification requirements and equilibrate the column with 2-3 bed volumes of it.

e) Sample Loading

The sample should be clear, free of any debris; therefore it should be filtered prior to loading. To load the sample manually, remove the upper adapter and remove the excess of the buffer on the top of the gel without disturbing the gel surface. Then, gently pipette in the sufficient amount of sample and allow the sample to pass through the surface.

At the time, take care that the surface of the gel does not dry. Finally, add little buffer to the column and set the upper adapter. The above processes can also be done by automated sample injector or pump.

f) Elution

A fraction collector is attached to the system to collect the fractions of the elution. The sample is eluted isocratically using a single buffer system. The length of tubing connected to fraction collector should be small and small fractions of size 1mL or less should be collected to avoid mixing of peaks.

The detector connected to the fraction collector analyses the separation. The total elution should be equal to or more than one bed volume. The volume of the buffer that elutes from the column before a particular peak in the elution profile appears is called elution volume (Ve). The distribution coefficient (Kd) of an analyte in gel filtration chromatography can be mathematically defined as

Kd =Ve –V0/ Vt – V

After completion of each run, the column should be washed thoroughly to remove any analytes left in the gel. Ideally, it should be done by 1-2 bed volumes of buffer. For long term storage, antimicrobial agents should be added to the buffer.

Gel filtration Applications

  1. Gel filtration chromatography method is a reliable method for purifying biomolecules such as enzymes, polysaccharides, nucleic acids, proteins, and so on.
  2. Renaturation of denatured proteins is possible with a gel filtration unit. Gel filtration chromatography method is beneficial in protein fractionation experiments.
  3. Using gel filtration chromatography method, one could determine the molecular weight of the separated particles. Gel filtration chromatography method is a technique for determining the quaternary structure of purified proteins.

Gel filtration Advantages

  1. Gel filtration chromatography method can separate biomolecules that are sensitive to changes in pH, temperature, metal ion concentration, and so on.
  2. Unlike in ion-exchange chromatography, the particles do not adhere to the chromatography medium.
  3. Gel filtration chromatography method takes very little time to explain the outcome. It creates a clear separation. A small amount of test sample is sufficient to draw conclusions. The flow rate could be adjusted.

Gel filtration Limitations

  1. To prevent particles from clogging the instrument, the test sample must be filtered before being passed through the gel filtration chromatography method column.
  2. Any dust or particulate matter within the gel filtration chromatography method instrument may interfere with the interpretation of the results or produce erroneous results.