Gel Filtration, also known as Gel Chromatography or Size Exclusion Chromatography (SEC), is a highly reliable separation technique used to separate molecules based on their size and molecular weight. Unlike other forms of chromatography that depend on charge, polarity, or adsorption, gel filtration separates molecules purely based on their ability to penetrate the pores of a stationary phase. Larger molecules elute first because they cannot enter the pores, while smaller molecules penetrate the gel matrix and travel slowly.
This technique is widely used in protein purification, desalting, buffer exchange, separation of polymers, and analysis of complex mixtures. Because it is a non-destructive method, gel filtration is ideal for biomolecules that require gentle handling.
Theory
The principle of gel filtration chromatography is simple: molecules are separated according to their hydrodynamic volume. The stationary phase consists of porous gel beads with carefully controlled pore sizes. When a sample mixture is passed through a column:
- Large molecules are excluded from entering the pores and move around the beads, eluting early.
- Medium-sized molecules partially enter the pores and elute at an intermediate rate.
- Small molecules enter most of the pores and elute last.
Elution volume is directly related to molecular size, making the technique useful for estimating molecular weight and purifying biomolecules without altering their structure or activity.
Instrumentation
A typical gel filtration apparatus includes:
- Column (glass or plastic)
- Gel matrix (stationary phase)
- Pumps or gravity flow systems
- Fraction collector
- Detector (usually UV–Visible)
The entire system must allow gentle separation to maintain biological activity of purified molecules.
1) Column
The column is a vertical tube made of glass or durable plastic. Columns vary in length and width, depending on:
- Resolution required
- Sample volume
- Nature of the gel matrix
Long columns offer better resolution, while wide columns allow larger sample volumes. The column must be clean, uniform, and free of air bubbles to ensure smooth flow and reproducible results.
2) Gel (Stationary Phase)
The gel serves as the heart of gel filtration chromatography. Commonly used gels include:
- Sephadex (Dextran-based)
- Sepharose (Agarose-based)
- Bio-Gel P series (Polyacrylamide-based)
Each gel type is available in different grades and pore sizes. The selection depends on the molecular weight range of interest. Gels must be chemically inert, stable, and capable of swelling uniformly in the solvent system.
3) Packing of the Column
Proper packing ensures consistent separation. The steps typically include:
- Allowing the gel to swell fully in the solvent
- Degassing to remove trapped air
- Slurry preparation and gentle pouring into the column
- Avoiding turbulence or air bubble formation
- Allowing the gel bed to settle uniformly
Any voids or uneven packing can cause peak distortion and poor resolution.
4) Preparation of Sample
Samples must be prepared carefully to ensure optimal separation. Requirements include:
- Dissolving sample in appropriate buffer
- Filtration or centrifugation to remove particulates
- Sample volume not exceeding 5–10% of column volume
- Avoiding viscous or highly concentrated solutions
Well-prepared samples improve resolution and prevent blockages in the column.
5) Application of the Sample
The sample is applied gently on top of the gel bed without disturbing the surface. Methods include:
- Using a pipette for small volumes
- Layering sample under buffer using a narrow tube
A narrow, uniform application zone ensures sharp bands and accurate separation.
6) Solvents (Mobile Phase)
The mobile phase is typically a buffer solution or an aqueous solvent. The choice of solvent depends on the stability and solubility of the sample molecules. Ideal solvent characteristics include:
- Maintaining pH stability
- Preventing aggregation of biomolecules
- Ensuring compatibility with the gel matrix
Common solvents include phosphate buffer, Tris buffer, or simple saline solutions.
7) Elution and Flow Rate
Elution may occur under gravity flow or controlled pumping. Important considerations include:
- Consistent, slow flow rate improves resolution
- Too high flow rate causes band broadening
- Elution volume is used to estimate molecular size
Isocratic elution is typically used, meaning a constant solvent composition throughout the run.
8) Detector
UV–Visible detectors are commonly used to monitor separated components emerging from the column. Detection wavelength is selected based on absorbance characteristics of the analyte, such as 280 nm for proteins and 260 nm for nucleic acids.
Data from the detector can be plotted as chromatograms showing elution volume vs. absorbance.
Applications of Gel Chromatography
- Purification of proteins, enzymes, and nucleic acids
- Separation of polysaccharides and synthetic polymers
- Desalting and buffer exchange
- Determination of molecular weight
- Removal of small impurities from biomolecules
- Fractionation of complex mixtures
- Analysis of serum proteins or antibodies
Advantages of Gel Chromatography
- Non-destructive technique—ideal for sensitive biomolecules
- Good reproducibility and reliability
- No adsorption or chemical interaction with the stationary phase
- Ability to work with large sample volumes
- Simple, isocratic operation
Limitations of Gel Chromatography
- Limited to molecules that differ significantly in size
- Long analysis time compared to other chromatographic techniques
- Flow rate must be slow for high resolution
- Columns may clog with viscous samples
- Not suitable for separating molecules with similar molecular size
Detailed Notes:
For PDF style full-color notes, open the complete study material below: