WHAT IT IS
Gel Permeation Chromatography (GPC) and Size-Exclusion Chromatography (SEC) are LC techniques that separate molecules by size. GPC usually refers to polymer separations in organic solvents, while SEC applies to biomolecules such as proteins and nucleic acids in aqueous buffers. Both rely on porous stationary-phase particles: large molecules elute first because they cannot enter the pores, while smaller molecules penetrate the pores and elute later.
HOW IT WORKS
GPC/SEC follows the same flow path as conventional LC but with a size-based stationary phase:
Mobile Phase Delivery - Runs are usually isocratic. GPC uses organic solvents (e.g., tetrahydrofuran) for polymers, while SEC employs aqueous buffers for biomolecules.
Sample Injection - Small volumes are introduced to avoid peak broadening.
Column Separation - Porous beads with defined pore sizes control retention. Molecules elute in order of decreasing size.
Detection - UV absorbance is standard for proteins; refractive index, light scattering, or viscosity detectors are used for polymers. Multi-detector setups can provide molecular weight and distribution.
ADVANTAGES
Size-Based Separation: Direct measurement of molecular size distribution.
Non-Destructive: Preserves protein conformation and polymer integrity.
Broad Applicability: Suitable for polymers, proteins, nucleic acids, and nanoparticles.
Multi-Detector Compatibility: Light scattering, viscosity, or MS add molecular weight and structural data.
Simple Operation: Typically isocratic, without the need for solvent gradients.
APPLICATION AREAS
Polymer Science (GPC): Molecular weight averages (Mn, Mw), polydispersity, and chain length distributions.
Biochemistry (SEC): Protein purification, desalting, and aggregation analysis.
Biopharmaceuticals: Characterization of monoclonal antibodies and therapeutic proteins.
Nanomaterials: Analysis of nanoparticles and macromolecular assemblies.
Nucleic Acids: Size separation of DNA, RNA, and oligonucleotides.
CHALLENGES AND LIMITATIONS
Resolution: Limited ability to distinguish molecules of similar size.
Calibration: Accurate molecular weight data require standards, especially in polymer analysis.
Sample Dilution: Broad elution peaks may require concentration before downstream use.
Column Sensitivity: Prone to clogging by particulates or strongly adsorbing compounds.
Solvent Dependence: Requires solvents that preserve analyte stability and conformation.