Gel Permeation Chromatography (GPC) – Definition, Principle, Instrumentation, Steps, Applications, Advantages & Limitations

Gel Permeation Chromatography (GPC) – Definition, Principle, Instrumentation, Steps, Applications, Advantages & Limitations

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Introduction to Gel Permeation Chromatography

  • Gel Permeation Chromatography (GPC), also called Size Exclusion Chromatography (SEC) or Gel Filtration Chromatography, is a powerful separation technique widely used in biochemistry, biotechnology, and polymer science.
  • The technique separates molecules based on size (hydrodynamic volume), rather than charge or affinity.
  • The stationary phase is a porous matrix (like dextran, agarose, polyacrylamide, or polystyrene), while the mobile phase is a suitable liquid solvent.
  • Molecules move through the column filled with porous beads:
    • Large molecules are excluded from the pores and elute first.
    • Smaller molecules enter the pores, travel longer paths, and elute later.
  • This creates a molecular sieve effect, allowing efficient separation.

In simple words: GPC separates molecules by size – big molecules come out first, small molecules later.

Figure: Gel Permeation Chromatography (GPC). Image Source: www.priyamstudycentre.com

Historical Background

  • The technique was first developed in 1955 by Lathe and Ruthven, who used dextran gels to separate proteins.
  • Later improvements introduced synthetic gels like polyacrylamide and cross-linked polystyrene, making GPC highly versatile.
  • Today, GPC is widely used in protein purification, polymer analysis, and molecular weight determination.

Principle of Gel Permeation Chromatography

  • The separation is based on size exclusion.
  • The stationary phase consists of microporous beads with defined pore sizes.
  • The mobile phase flows continuously through the column, carrying molecules along.

Mechanism:

  1. Large molecules are too big to enter pores → move around beads → elute faster.
  2. Medium molecules partially enter pores → take longer → elute later.
  3. Small molecules fully penetrate pores → travel longest path → elute last.

The efficiency depends on:

  • Pore size and distribution.
  • Flow rate of mobile phase.
  • Shape and hydration of molecules.

Components of Gel Permeation Chromatography (GPC)

Components of Gel Permeation Chromatography
Figure: Components of Gel Permeation Chromatography. Image Source: www.priyamstudycentre.com

GPC instruments consist of several key components:

1. Stationary Phase (Gel Beads)

  • Semi-permeable, porous polymer gel beads.
  • Properties: chemically inert, mechanically stable, uniform pore size.
  • Examples:
    • Dextran gels (Sephadex) – natural polysaccharide.
    • Agarose gels – for large biomolecules.
    • Polyacrylamide gels – synthetic, adjustable pore sizes.
    • Polystyrene gels – widely used for synthetic polymers.

2. Mobile Phase

  • Solvent that carries molecules through the column.
  • Must dissolve analytes well and not interact chemically with them.
  • Common solvents: buffers for proteins, organic solvents (like THF) for polymers.

3. Columns

  • Packed with gel beads.
  • Types:
    • Analytical columns (7.5–8 mm diameter).
    • Preparative columns (22–25 mm diameter).
    • Narrow-bore columns (2–3 mm, for fast analysis).
  • Length: 25–60 cm.

4. Pumps

  • Maintain constant flow of mobile phase.
  • Types: syringe pumps, reciprocating pumps.

5. Detectors

  • Record elution of molecules.
  • Common detectors:
    • Refractive index detector (RI) – universal, widely used.
    • UV/Vis detectors – for absorbing compounds.
    • Light scattering detectors – for molecular weight distribution.
    • Viscometer detectors – measure intrinsic viscosity.

Steps in Gel Permeation Chromatography

Step 1: Preparation of Column

  • Gel is swollen in solvent.
  • Column packed with uniform bead suspension.
  • Washed with buffer to remove air bubbles.

Step 2: Sample Loading

  • Sample dissolved in mobile phase.
  • Introduced into column via syringe or injector.

Step 3: Elution

  • Mobile phase flows through column.
  • Molecules elute based on size.
  • Larger molecules → first, smaller → last.

Step 4: Detection & Analysis

  • Detectors record elution peaks.
  • Chromatogram is generated (plot of signal vs retention time).
  • Peak position indicates molecular size; peak area indicates concentration.

Applications of Gel Permeation Chromatography (GPC)

1. Biological Applications

  • Protein purification and separation.
  • Isolation of enzymes, antibodies, polysaccharides.
  • Determination of protein quaternary structure.

2. Medical Applications

  • Analysis of blood serum proteins.
  • Detection of abnormal proteins in disease diagnosis.

3. Polymer Science

  • Determination of molecular weight (Mn, Mw) of synthetic polymers.
  • Study of molecular weight distribution (polydispersity index, PDI).
  • Used in quality control of plastics, rubbers, and fibers.

4. Pharmaceutical Applications

  • Purification of peptides and drugs.
  • Formulation analysis for protein-based drugs.

5. Environmental and Food Science

  • Separation of natural polysaccharides.
  • Detection of biopolymers in food.
  • Analysis of pollutants and toxins.

Advantages of Gel Permeation Chromatography

  • Short analysis time.
  • Well-defined, reproducible separation.
  • Narrow bands, high resolution.
  • No sample loss (non-destructive method).
  • Requires small volume of solvent.
  • Can handle fragile biomolecules (no harsh conditions).
  • Flow rate can be controlled easily.

Limitations of Gel Permeation Chromatography

  • Limited peak resolution if molecular sizes are too close.
  • Requires filtration of samples to avoid clogging.
  • Columns are expensive and delicate.
  • Works best only within a specific molecular weight range.
  • Not suitable for molecules with similar hydrodynamic volumes but different shapes.

Conclusion

  • Gel Permeation Chromatography (GPC) is a highly valuable separation technique in biology, medicine, polymer science, and biotechnology.
  • It separates molecules purely on the basis of size, making it simple and reproducible.
  • Despite limitations like column cost and limited resolution, its advantages in protein purification, polymer analysis, and drug development make it indispensable.

In short: GPC is a molecular sieve chromatography method that helps scientists study proteins, polymers, and biomolecules with precision.

Frequently Asked Questions (FAQs) on Gel Permeation Chromatography (GPC)

Q1. What is Gel Permeation Chromatography (GPC)?
Ans: GPC is a size-based separation technique where molecules are separated according to their molecular size and weight using a porous gel-filled column.

Q2. Is Gel Permeation Chromatography the same as Size Exclusion Chromatography?
Ans: Yes. GPC is a type of Size Exclusion Chromatography (SEC). The term “GPC” is often used when analyzing polymers in organic solvents, while “SEC” is common in biological applications.

Q3. Who discovered Gel Permeation Chromatography?
Ans: GPC was developed in 1955 by Lathe and Ruthven, using dextran gels for protein separation.

Q4. What is the principle of GPC?
Ans: The principle is size exclusion:

  • Large molecules cannot enter pores → elute first.
  • Small molecules enter pores → travel longer → elute later.

Q5. What is the stationary phase in GPC?
Ans: Porous polymer beads such as:

  • Sephadex (dextran gels)
  • Agarose gels
  • Polyacrylamide gels
  • Cross-linked polystyrene

Q6. What is the mobile phase in GPC?
Ans: The mobile phase is a solvent that carries molecules through the column. Example:

  • Aqueous buffers for proteins/enzymes.
  • Organic solvents (like THF) for synthetic polymers.

Q7. What detectors are used in GPC?
Ans: Common detectors include:

  • Refractive Index Detector (RI) – universal detector.
  • UV/Vis Absorbance Detector – for chromophores.
  • Light Scattering Detector – for molecular weight determination.
  • Viscometer Detector – for intrinsic viscosity.

Q8. What is the difference between GPC and Ion Exchange Chromatography?
Ans:

  • GPC: separates by size.
  • Ion Exchange Chromatography: separates by charge.

Q9. What is the difference between GPC and Affinity Chromatography?
Ans:

  • GPC: based on size exclusion.
  • Affinity Chromatography: based on specific binding interactions (e.g., antigen-antibody).

Q10. What is the retention volume in GPC?
Ans: Retention volume is the volume of solvent required to elute a molecule from the column. It depends on molecular size.

Q11. What is polydispersity index (PDI) in GPC?
Ans: PDI measures the molecular weight distribution of a polymer.

  • PDI = Mw / Mn (Weight average molecular weight / Number average molecular weight).

Q12. Can GPC be used for protein purification?
Ans: Yes. It is widely used for separating proteins, enzymes, and antibodies based on size.

Q13. What are the applications of GPC?
Ans:

  • Protein purification
  • Enzyme isolation
  • Determination of polymer molecular weight
  • Quality control in plastics and fibers
  • Detection of abnormal proteins in medical diagnostics

Q14. What are the advantages of GPC?
Ans:

  • Gentle and non-destructive
  • High reproducibility
  • Works for both biological molecules and synthetic polymers
  • Short analysis time

Q15. What are the limitations of GPC?
Ans:

  • Expensive columns
  • Limited resolution if molecular sizes are very close
  • Requires filtered samples to avoid clogging
  • Not suitable for molecules of the same size but different shape

Q16. Is GPC qualitative or quantitative?
Ans: GPC provides both:

  • Qualitative data – molecular size separation.
  • Quantitative data – molecular weight distribution.

Q17. What is calibration in GPC?
Ans: Calibration is performed using standards of known molecular weight to create a calibration curve for accurate measurement.

Q18. Can GPC be used for nanoparticles?
Ans: Yes. It is used for studying nanoparticles, micelles, and colloids by determining their size distribution.

Q19. How is GPC different from SDS-PAGE?
Ans:

  • GPC: column-based, liquid chromatography, separates molecules by size in solution.
  • SDS-PAGE: gel electrophoresis, separates proteins by size using an electric field.

Q20. Can GPC analyze both natural and synthetic polymers?
Ans: Yes. It is used for biopolymers (proteins, polysaccharides) as well as synthetic polymers (plastics, resins, fibers).

Q21. Why do large molecules elute first in GPC?
Ans: Large molecules are too big to enter the pores of gel beads, so they move quickly around the beads and elute earlier.

Q22. What is the role of flow rate in GPC?
Ans: The flow rate of mobile phase affects resolution:

  • Too high → poor separation.
  • Too low → long analysis time.

References

  1. https://www.slideshare.net/asabuwangwa/gel-permeation-chromatography-gpc
  2. http://www.materials-talks.com/blog/2016/08/30/an-introduction-to-gel-permeation-chromatography-in-30-minutes/
  3. https://microbenotes.com/gel-permeation-chromatography/
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/

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