Gel Permeation Chromatography

Gel Permeation Chromatography Analysis

Gel permeation chromatography (GPC) or gel filtration chromatography is an example of size exclusion chromatography (SEC) that analysis substances largely according to their molecular size. However, gel filtration is a special type of liquid-solid elution chromatography. GPC instrument is used widely in chemistry and biology labs to analyze polymers like proteins, nucleic acids (DNA and RNA), sugars, etc.

Gel permeation chromatography is a popular technique in the molecular-weight distribution of a polymer due to its low price, simplicity in use, and ability to provide accurate, reliable information.

Gel Filtration Chromatography Principle

The principle of gel filtration chromatography or gel permeation photography (GPC) is based upon the different permission rates of each solute molecule into the interior of the gel particles. It is different from other chromatographic technique which depends on the physical and chemical interactions of analytes.

Separation by gel filtration depends on the porous beads packed on the column.

1. By the flow of liquid, smaller analytes enter the pores more easily. Therefore, they spend more time in pores beds.
2. Similarly larger molecules spend less time in the porous beads packed on the column. Hence larger molecules appear first in the elute.

Separation is possible due to the size barrier that exists on the gel particle. Like ion exchange chromatography and column chromatography flow rate usually increases with an increase in particle size of an analyte. The smaller the particle size the lower will be the flow rate.

The flow rate is further affected by the varying nature of the porosity of the gel. Therefore, the choice of the gel depends upon the molecular size and the chemical properties of the substances to be separated.

For example, Bio-Gel 0-10 operates for substances whose molecular weight is between 5000 to 17000 units.

Gel Permeation Chromatography (GPC) Instrument

The instrumentation of gel permeation chromatography in the GPC instrument is given below the picture,

Good gel permeation chromatographic instrument contains the following important components,

1. Gels
2. Gel chromatography column
3. Eluent or mobile phase
4. Solvent delivery pump
5. Chromatographic detector

Gels

The choice of gel largely depends on the molecular size and chemical properties of analytes which can be separated by the GPC technique. Xerogels are commonly used as a stationary phase in gel permeation chromatography instruments.

All the gels used in the GPC technique are organic in nature. Different types of gels which are available in the market have the trade names,

1. BioGel p-2 (polyacrylamide)
2. Sephadex G-10-200 (Dextran)
3. Styrogel (modified polystyrene gels)
4. Agarose

The gel agarose is sold under the trade names Sepharose and BioGel A. Such gels contain hydrogen bonding and are stable under the temperature range of 0 to 30 °C and pH range between 5 to 8. The gels used in the gel filtration technique have the following common properties,

• They are chemically inert.
• They are mechanically stable.
• They contain a uniform particle and pore size.

Gel chromatography column

The column used in the GPC instrument is filled with semi-permeable, porous polymer material and eluent allowed to flow under gravity. The gels used in the chromatographic column have a well-defined range of pore sizes. Washing the column before testing with buffer solution to remove any air bubbles.

Eluent

The eluent or mobile phase used in gel permeation chromatography should be a good solvent for polymers. Generally, organic solvents used as an eluent in the GPC instrument may include,

1. tetrahydrofuran (THF)
2. o-dichlorobenzene
3. trichlorobenzene
4. polyalkenes
5. m-cresol
6. o-chlorophenol

Pump

Piston or peristaltic pumps are used in the GPC technique for uniform delivery or constant flow rate of the mobile phase.

Chromatographic detector

As in other chromatographic methods, here also a suitable physical property like refractive index, absorbance, fluorescence intensity and other electrical properties of effluent can be measured by the detector.

There are many types of detectors used in the GPC analysis. They are divided mainly into two categories,

• The first type is concentration-sensitive detectors like UV absorption detectors, refractive index (RI) detectors, infrared (IR) absorption, and density detectors.
• The second category contains molecular weight-sensitive detectors like low-angle light scattering (LALLS) detectors and multi-angle light scattering (MALLS) detectors.

Applications of gel permeation chromatography

• Gel permeation chromatography (GPC) is used principally for the analysis of a mixture of polymers with different molecular weights.
• It is also applicable to separate molecules of the same molecular weight by proper selection of gel and column height.
• Desalting is another application of gel permeation chromatography that involves the removal of salts from macromolecules. Gels of small size are suitable for such applications.
• Gel filtration chromatography is applicable to copolymerization studies.
• Proteins, dextrans, and oligomers of ethylene glycol have been handled by GPC instrument.
• Low lactose milk can be purified by the gel filtration technique.
• Nucleic acids like DNA or RNA can be separated from nucleotides by gel filtration technique.
• Polysaccharides have been separated from sugar and proteins from low molecular weight substances can be separated by gel permeation or gel filtration instrument.