Gel
Chromatography
By: Sakshi Jagadi , B Pharm 7th sem
Guide: Mr. Uttam Kumar, Dept of Pharmaceutical Analysis
Chromatography :
▸ Chromatography is an advanced analytical and
separation technique used in instrumental
analysis to separate, identify, and quantify the
individual components present in a complex
mixture.
▸ The principle of chromatography is based on the
differential migration of components between
two phases — a stationary phase (solid or liquid
supported on a solid) and a mobile phase (liquid
or gas) that moves relative to the stationary phase.
▸ Chromatography serves as a crucial tool in
pharmaceutical analysis for the purity testing,
qualitative and quantitative analysis, and
determination of drug stability. Various
chromatographic techniques include Thin Layer
Chromatography (TLC), High-Performance
Liquid Chromatography (HPLC), Gas
Chromatography (GC), and Paper
2 Chromatography.
Introduction
▸ Gel Chromatography, also known as Size Exclusion Chromatography (SEC) or Gel Filtration Chromatography, is
an important instrumental analytical technique used to separate molecules based on their molecular size and shape.
▸ It is a physical separation method that does not rely on adsorption, ion exchange, or affinity interactions, but on
the differential penetration of molecules into the pores of a stationary phase. In this technique, the stationary phase
consists of porous gel particles made of materials like dextran (Sephadex), agarose, or polyacrylamide, and the
mobile phase is an inert solvent or buffer that flows through the column.
▸ When a solution containing a mixture of molecules is passed through the column, larger molecules are excluded
from entering the pores and move rapidly through the column, while smaller molecules enter the pores and
therefore take a longer time to elute.
▸ Thus, separation occurs purely on the basis of molecular size and hydrodynamic volume. The elution order is from
largest to smallest molecules.
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Gel Chromatography Principle
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Principle of Gel Chromatography
(Size Exclusion Chromatography)
▸ Basis of Separation:
▹ The separation depends entirely on the molecular size and hydrodynamic volume of the solute
molecules.
▸ Stationary Phase:
▹ It consists of porous gel beads made up of materials like dextran (Sephadex), agarose, or
polyacrylamide.
▹ These gels act as a molecular sieve.
▸ Mobile Phase:
▹ A buffer or solvent that flows continuously through the column, carrying the sample molecules.
▸ Mechanism of Separation:
▹ When the sample mixture is introduced into the column, molecules migrate through the gel.
▹ Large molecules are excluded from the pores and move quickly through the column.
▹ Smaller molecules enter the pores and take a longer path, hence move slowly.
▸ Order of Elution:
▹ Molecules elute in the decreasing order of molecular size:
Large molecules → Intermediate molecules → Small molecules
▸ No Chemical Interaction:
5 ▹ There is no adsorption, ion exchange, or affinity binding; separation occurs purely on physical
Theory of separation:
▸ A column is made up of swollen gel particles and solvent used to swell the gel in a suitable
tubular container Analytes that are too large will not be retained; on the other hand, Analytes
that are too small will be entirely retained.
▸ An equation is given below:
▸ Vt = Vg + Vi + Vo
Where,
▸ V t = the total volume of the column
▸ Vg = the volume of gel matrix
▸ Vo = the volume of liquid outside the gel matrix
▸ Vi = the volume of liquid inside the matrix
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Instrumentation
Mobile Phase Reservoir
↓
Pump
↓
Sample Injector
↓
Column (with porous gel)
↓
Detector
↓
Recorder / Data System
↓
Fraction Collector
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1. Mobile Phase
Reservoir
•The mobile phase is a solvent or buffer that
moves through the column, carrying the
sample with it.
•It maintains a stable pH, ionic strength, and
temperature, preventing protein denaturation
or aggregation.
•Commonly used mobile phases:
a. Phosphate buffer (pH 7.0)
b. Tris buffer
c. Saline solutions (e.g., 0.9% NaCl)
•Example: In protein purification using
Sephadex G-100, phosphate buffer is used as
the mobile phase.
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2. Pump or Flow
Control System
•Provides a steady and controlled flow rate
of the mobile phase through the column.
•In manual systems, gravity or a simple
peristaltic pump is used.
•In advanced systems (HPLC–SEC), high-
pressure pumps ensure precise flow and
reproducibility.
•Typical flow rate: 0.5 – 1.0 mL/min.
•Example: HPLC-SEC systems use dual
piston pumps for consistent mobile phase
delivery.
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3. Sample Injector
•Used to introduce the sample solution into
the top of the column without disturbing
the gel bed.
•The sample volume should be small
(generally 1–5% of the column volume) to
obtain sharp peaks.
•Sample may be applied using:
a. A syringe (manual system)
b. An autosampler (automated system)
•Example: In protein separation, 1 mL of
enzyme solution may be injected into a 100
mL gel column.
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4. Chromatographic
Column
▸ The main part of the system where separation occurs.
▸ It is a vertical glass or stainless-steel tube packed with
porous gel beads (stationary phase).
▸ Common gels used:
a. Sephadex (Dextran-based gel)
b. Sepharose (Agarose-based gel)
c. Biogel P (Polyacrylamide-based gel)
▸ The gel pores act like molecular sieves — large
molecules pass around the beads, small molecules
enter the pores.
▸ The column length and diameter determine resolution
and capacity.
▸ Example: A Sephadex G-75 column can separate
proteins in the molecular weight range 3,000–80,000
Da.
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5. Detector
•The detector monitors the concentration of
components as they elute from the column.
•Converts the physical/chemical property of
solute (like absorbance or refractive index) into
an electrical signal.
•Common detectors used:
a. UV–Visible detector → for proteins and
nucleic acids (absorbing at 280 nm or 260
nm).
b. Refractive Index Detector (RID) → for
sugars, polymers, and compounds that
don’t absorb UV.
c. Fluorescence Detector → for highly
sensitive biomolecule detection.
•Example: In protein analysis, a UV detector
at 280 nm is typically used.
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6. Recorder
▸ Connected to the detector to record the
output as a chromatogram.
▸ The chromatogram shows peaks
corresponding to different molecules
in the sample.
▸ Each peak’s position (elution volume)
relates to molecular size, and peak
area gives quantitative data.
▸ In modern systems, this is displayed
and analyzed using computerized data
acquisition software.
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7. Fraction Collector
▸ Collects the eluted fractions (liquid
portions) in separate tubes or vials.
▸ Allows for further testing or
purification of specific fractions.
▸ Automated fraction collectors can
collect samples based on time
intervals or detector response.
▸ Example: Fractions containing high-
molecular-weight proteins are
collected first, followed by smaller
peptides.
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Applications of Gel
Chromatography
•Used to separate and purify biomolecules by size.
•Determines molecular weight of proteins and polymers.
•Performs desalting and buffer exchange.
•Used for enzyme and protein purification.
•Analyzes polymer size distribution.
•Studies protein aggregation and interactions.
•Ensures purity in biopharmaceutical products.
•Separates polysaccharides and glycoproteins.
•Removes impurities and aggregates from samples.
•Examines molecular shape and conformation.
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Than
k
16 you!
