CHROMATOGRAPHY WITH ITS TYPES, APPLICATION AND MOST

ADVANCED TECHNIQUES

ASSIGNMENT NO # 2

(BTH-201) BIOCHEMISTRY-2

SUBMITTED BY:

UMAR ZEESHAN

ROLL NO:

23014160-029

SUBMITTED TO:

MISS ROBINA

DEPARTMENT OF BIOTECHNOLOGY (EVENING)

UNIVERSITY OF GUJRAT

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Category Type of Mobile Stationary Subtypes Applications
Chromatography Phase Phase
Based on Liquid Liquid Solid or - High
Phase Chromatography liquid film Performance Separation of organic
(Mobile (LC) Liquid compounds, proteins,
and Chromatography and ions. Purification
Stationary) (HPLC) and analysis of
- Thin Layer complex mixtures.
Chromatography
(TLC)
Gas Gas Solid or - Capillary GC Analysis of volatile
Chromatography (Helium, liquid film - Packed compounds,
(GC) N₂) Column GC environmental
monitoring,
pharmaceutical
analysis, forensic
analysis.
Supercritical Supercritical Solid or - None Separation of non-
Fluid fluid (CO₂) liquid (generalized volatile compounds,
Chromatography stationary SFC technique) lipids, complex
(SFC) phase organic molecules.

Based on Adsorption Liquid or Solid (e.g., - Thin-layer Separation based on

Separation Chromatography gas silica gel) Chromatography adsorption
Mechanism (TLC) differences. Used in
- Column TLC and column
Chromatography chromatography for
organic mixtures.
Partition Liquid Liquid - Liquid-Liquid Separation based on
Chromatography film Partition solubility differences
Chromatography between two phases.
Applied in biological
and chemical analysis.
Ion Exchange Liquid Ion- - None (standard Purification of
Chromatography exchange ion exchange proteins, nucleic
resin technique) acids, ions. Used in
biochemical and
pharmaceutical
applications.
Size Exclusion Liquid Porous - None (standard Separation based on
Chromatography material SEC technique) molecular size. Used
(SEC) (e.g., gel for polymers,
beads) proteins, nucleic
acids, and other
biomolecules.
Affinity Liquid Ligand- - None (standard Protein purification,
Chromatography bound affinity enzyme isolation,
material technique) receptor-ligand
binding studies.

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Advanced High- Liquid Solid or - Reverse-phase High-speed, high-
Techniques Performance liquid film HPLC efficiency separation.
Liquid - Normal-phase Widely used in
Chromatography HPLC pharmaceutical and
(HPLC) chemical industries
for analysis.
Ultra-High Liquid Solid or - None Faster and more
Performance liquid film (advanced efficient than HPLC.
Liquid HPLC) Used in advanced
Chromatography pharmaceutical,
(UHPLC) chemical, and life
science research.
Gas Gas Solid or - None Separation +
Chromatography- liquid film (combines GC identification of
Mass with MS) compounds. Used in
Spectrometry forensic science,
(GC-MS) environmental
analysis, and drug
testing.
Capillary Electric Thin - None High-resolution
Electrophoresis field (fluid capillary (electrophoretic separation of small
(CE) movement) tube (gel, method) molecules, proteins,
liquid) nucleic acids. Applied
in genomics,
proteomics, etc.
Supercritical Supercritical Solid or - None (standard Used for separation of
Fluid CO₂ liquid SFC) lipids, non-volatile
Chromatography stationary organic compounds.
(SFC) phase

1. Chromatography separates mixtures based on interactions with a stationary phase

while moving through a mobile phase.
2. It has diverse applications in chemical analysis, biological sciences,
pharmaceuticals, environmental testing, and forensics.
3. Advanced techniques like HPLC, GC-MS, and SFC provide high efficiency and
precision for specific analytical needs.

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 Chromatography Overview
Chromatography is a laboratory technique used to separate mixtures into their individual
components. It works by exploiting differences in the rate at which components of a mixture
move through a stationary phase when dissolved in a mobile phase. The technique is widely
used in chemical analysis and purification.

Basic Principles
1. Mobile Phase: This is the fluid (liquid or gas) that moves through or over the stationary
phase. It carries the mixture to be separated.
2. Stationary Phase: This is a solid or a liquid film that is fixed in place and through which the
mobile phase moves. It interacts differently with various components of the mixture.

Types of Chromatography
1. Based on the Phase (Mobile and Stationary)

1. Liquid Chromatography (LC):

 Stationary Phase: Solid or liquid coating on solid support.
 Mobile Phase: Liquid.
 Applications: Used for separating a variety of compounds, including organic
compounds, proteins, and ions.
 Subtypes:
 High-Performance Liquid Chromatography (HPLC): Uses high
pressure to push the mobile phase through a packed column for
faster separation.
 Thin-Layer Chromatography (TLC): Uses a thin layer of stationary
phase (usually silica gel or alumina) on a flat plate for separation.
2. Gas Chromatography (GC):
 Stationary Phase: Solid or liquid film coated on an inert solid support.
 Mobile Phase: Gas (usually helium or nitrogen).
 Applications: Used for volatile and semi-volatile substances, especially in
environmental, pharmaceutical, and forensic analysis.
 Subtypes:
 Capillary GC: Uses capillary columns for higher efficiency.
 Packed Column GC: Uses columns filled with stationary phase
material.
3. Supercritical Fluid Chromatography (SFC):
 Stationary Phase: Solid or liquid stationary phase.
 Mobile Phase: Supercritical fluid (a state of matter between liquid and gas, often
CO₂).
 Applications: Used for separating non-volatile compounds, particularly lipids and
complex organic molecules.

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2. Based on Separation Mechanism

1. Adsorption Chromatography:
 Separation occurs due to differences in the adsorption of components onto a solid
stationary phase (e.g., silica gel).
 Application: TLC and column chromatography often use this mechanism.
2. Partition Chromatography:
 Separation is based on the differential solubility of the components between the
stationary phase and the mobile phase (liquid-liquid partitioning).
 Application: Used in liquid-liquid chromatography.
3. Ion Exchange Chromatography:
 Based on the exchange of ions between the stationary phase (usually resin beads)
and the sample ions in the mobile phase.
 Applications: Used in the purification of proteins, peptides, and nucleic acids.
4. Size Exclusion Chromatography (SEC):
 Separation is based on the size of the molecules. Larger molecules pass through the
column faster, while smaller molecules interact with the stationary phase and take
longer to elute.
 Application: Used for separating polymers, proteins, and nucleic acids.
5. Affinity Chromatography:
 Based on the specific interaction between a target molecule and a ligand attached to
the stationary phase.
 Application: Commonly used for protein purification and enzyme isolation.

Applications of Chromatography
1. Chemical Analysis:
 To separate and analyze chemical mixtures.
 Identification of unknown compounds.
 Purification of compounds.
2. Pharmaceutical Industry:
 Drug analysis and quality control.
 Purification of active pharmaceutical ingredients.
 Screening for contaminants.
3. Biological and Biochemical Studies:
 Protein purification, enzyme analysis, and identification.
 Separation of biomolecules (e.g., nucleic acids, lipids, carbohydrates).
4. Environmental Analysis:
 Detection of pollutants (e.g., pesticides, heavy metals, hydrocarbons).
 Monitoring air and water quality.
5. Forensic Science:
 Analysis of blood, urine, and hair samples for drugs or toxins.
 Detection of substances in criminal investigations.

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 Most Advanced Techniques in Chromatography
1. High-Performance Liquid Chromatography (HPLC):
 Uses a high-pressure pump to pass the mobile phase through a column at high flow
rates, achieving rapid and efficient separations.
 Subtypes:
 Reverse-phase HPLC: The stationary phase is hydrophobic, and the mobile
phase is polar.
 Normal-phase HPLC: The stationary phase is polar, and the mobile phase is
nonpolar.
2. Ultra-High Performance Liquid Chromatography (UHPLC):
 An advanced version of HPLC with even smaller particle sizes and higher pressure,
enabling even faster separations.
3. Gas Chromatography-Mass Spectrometry (GC-MS):
 Combines the separating power of gas chromatography with the identifying power
of mass spectrometry.
 Applications: Used in drug testing, environmental analysis, and identifying unknown
compounds.
4. Capillary Electrophoresis (CE):
 Separation occurs based on the differential migration of ions in an electric field. It's a
high-efficiency technique often used for small sample volumes.
 Applications: Used for protein, DNA, and RNA analysis.
5. Supercritical Fluid Chromatography (SFC):
 Uses supercritical CO₂ as the mobile phase, offering the advantages of both liquid
and gas chromatography. It's especially useful for separating lipids and organic
compounds that are poorly soluble in liquids.

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