Chromatography

Chromatography is a laboratory technique used for the separation, identification, and

purification of components in a mixture. It relies on the differential partitioning of substances
between a stationary phase (a solid or liquid) and a mobile phase (a liquid or gas) as the
mixture moves through the system.
Mobile Phase
The mobile phase is the fluid (liquid or gas) that carries the sample mixture through the
chromatographic system.
Properties:
1. State: Can be a liquid (in liquid chromatography) or a gas (in gas chromatography).
2. Role: Moves the analytes along the stationary phase.
3. Interaction: Determines the speed at which components travel based on their solubility
or volatility.
Examples:
• In Liquid Chromatography (LC): Water, organic solvents (e.g., methanol, acetonitrile).
• In Gas Chromatography (GC): Inert gases like helium, nitrogen, or hydrogen.
Stationary Phase
The stationary phase is the fixed material (solid or liquid-coated solid) through which the
mobile phase flows. It interacts with the components of the mixture, causing separation based
on differences in affinities.
Properties:
1. State: Solid (e.g., silica gel, alumina) or liquid-coated solid (e.g., polymers, liquid films).
2. Role: Retains analytes based on physical or chemical interactions (e.g., adsorption,
partitioning).
3. Interaction: Components with a stronger affinity to the stationary phase move slower,
leading to separation.
Examples:
• In Liquid Chromatography (LC):
• Silica gel (polar stationary phase) for normal-phase LC.
• C18-bonded silica (non-polar stationary phase) for reverse-phase LC.
 • In Gas Chromatography (GC):
• Porous solids (e.g., activated carbon) in gas-solid chromatography.
• Liquid films coated on inert supports (e.g., polyethylene glycol) in gas-liquid
chromatography.
Types of Chromatography
Chromatography is broadly classified into different types based on the physical state of the
mobile phase. Below are the major types with a focus on liquid chromatography and gas
chromatography.
1. Liquid Chromatography (LC)
Liquid chromatography uses a liquid mobile phase to separate components. It is further
categorized based on the interaction between the analyte, stationary phase, and mobile phase.
Types of Liquid Chromatography
1. Partition Chromatography:
• Based on the solubility of components between two liquid phases (stationary
and mobile).
• Example: Separation of polar compounds like sugars or amino acids.
2. Adsorption Chromatography:
• Separation occurs through adsorption of solutes onto a solid stationary phase.
• Example: Separation of dyes and pigments.
3. Ion-Exchange Chromatography:
• Separates charged molecules like ions or proteins based on their affinity to the
oppositely charged stationary phase.
• Common in protein purification and water treatment.
2. Gas Chromatography (GC)
Gas chromatography uses a gaseous mobile phase (inert gases like helium or nitrogen) and a
liquid or solid stationary phase to separate volatile compounds.
Types of Gas Chromatography
1. Gas-Liquid Chromatography (GLC):
• The stationary phase is a liquid film coated on a solid support.
 • Separates compounds based on differences in volatility and interaction with the
stationary phase.
2. Gas-Solid Chromatography (GSC):
• The stationary phase is a solid adsorbent (e.g., activated charcoal or molecular
sieves).
• Used for separating gases like oxygen or nitrogen.
Key Differences Between Liquid and Gas Chromatography
Aspect Liquid Chromatography (LC) Gas Chromatography (GC)
Mobile Phase Liquid Gas
Stationary Phase Solid or liquid Solid or liquid-coated solid
Analyte Type Non-volatile, polar, or biomolecules Volatile or semi-volatile
Temperature Operates at ambient or controlled temperatures Requires high temperatures
Applications Pharmaceuticals, biomolecules, food analysis Petrochemicals, VOCs, and
environmental testing
1. Paper Chromatography (PC)
• Description: A simple and cost-effective method where filter paper acts as the stationary
phase. The mobile phase (solvent) moves through the paper by capillary action,
separating components based on their solubility and interaction with the paper.
• Applications: Used for separating polar compounds like amino acids, sugars, and
pigments.

Planar Chromatography
Planar chromatography involves separation techniques where the stationary phase is
laid on a flat surface, and the mobile phase moves across it by capillary action or gravity.
2. Column Chromatography (CC)
• Description: A technique where the stationary phase (solid or liquid-coated solid) is
packed into a vertical column, and the mobile phase (liquid) flows through it under
gravity or pressure. Components are separated based on their interaction with the
stationary phase.
• Applications: Purification of compounds, isolation of natural products, and
pharmaceutical applications.
3. Thin-Layer Chromatography (TLC)
• Description: A type of planar chromatography where a thin layer of adsorbent (e.g.,
silica gel or alumina) is coated on a flat surface like glass or plastic. The mobile phase
ascends by capillary action, separating components based on their interaction with the
stationary phase.
• Applications: Rapid analysis of small samples, monitoring reaction progress, and testing
purity.
4. Packed Column Chromatography
• Description: A column is tightly packed with a stationary phase (e.g., silica gel, alumina,
or polymer beads). It is used in techniques like liquid chromatography and gas
chromatography. Separation occurs as the mobile phase flows through the packed bed.
• Applications: Widely used in industrial and analytical applications, including HPLC and
GC for separating complex mixtures.
5. Open Tubular Chromatography
• Description: The stationary phase is coated on the inner walls of a hollow, narrow tube,
leaving the center open for the mobile phase. This is the primary type of column used in
gas chromatography.
• Types: Includes wall-coated (liquid film on walls) and support-coated (solid particles on
walls).
• Applications: High-efficiency separation of volatile compounds, such as gases and low-
boiling-point liquids.

Applications of Chromatography
a.Column chromatography is a versatile technique
1. Medical Applications
Qualitative Applications
• Identification of biomolecules: Separation and identification of proteins, peptides, and
nucleic acids from biological samples.
• Diagnosis: Isolating specific markers or compounds for diagnosing diseases (e.g.,
biomarkers for cancer or metabolic disorders).
 • Pharmacokinetics: Separation of drug metabolites to study the metabolism of
pharmaceuticals.
Quantitative Applications
• Drug Purity Analysis: Quantitative analysis of pharmaceutical ingredients and detection
of impurities in drugs.
• Therapeutic Drug Monitoring: Measuring the concentration of drugs in biological fluids
for patient management.
• Hormonal Studies: Quantification of hormones (e.g., insulin or cortisol) in plasma.

2. Analytical Applications
Qualitative Applications
• Compound Identification: Separation and characterization of unknown compounds in
mixtures.
• Forensic Analysis: Identification of drugs, toxins, or poisons in forensic samples.
• Environmental Testing: Identifying trace contaminants in water or soil samples.
Quantitative Applications
• Pollution Monitoring: Quantification of pollutants, such as pesticides or heavy metals, in
environmental samples.
• Food Quality Control: Measuring specific compounds like vitamins, preservatives, or
flavoring agents in food products.
• Chemical Standardization: Ensuring accurate concentrations in chemical formulations.

3. Industrial Applications
Qualitative Applications
• Process Development: Identifying intermediates during chemical or pharmaceutical
production.
• Quality Assurance: Verifying the consistency and composition of raw materials and
finished products.
 • Natural Product Isolation: Separation of plant-derived compounds like alkaloids or
flavonoids.
Quantitative Applications
• Pharmaceutical Manufacturing: Measuring the yield and purity of drugs during
production.
• Petrochemical Industry: Quantification of hydrocarbons or additives in fuel products.
• Biotechnology: Estimating the concentration of proteins or enzymes in bioprocessing.

b.planar chromatography other technique

1. Paper Chromatography (PC)
Medical Applications
Qualitative Applications
• Diagnosis of Diseases: Identification of amino acids or sugars in biological fluids to
diagnose metabolic disorders.
• Separation of Biomolecules: Analysis of plant and animal pigments or metabolites in
biological research.
• Forensic Medicine: Identifying toxins, drugs, or blood composition in forensic
investigations.
Quantitative Applications
• Drug Testing: Quantifying small amounts of pharmaceuticals in urine or blood samples.
• Hormonal Analysis: Measuring hormone levels in research and clinical diagnostics.
• Blood Plasma Studies: Quantification of amino acids or small peptides in medical
samples.

Analytical Applications
Qualitative Applications
• Compound Identification: Separation of dyes, pigments, or unknown chemicals in
mixtures.
• Quality Control: Checking for contaminants in pharmaceutical or food products.
 • Forensic Analysis: Detecting inks, dyes, or drugs in forensic samples.
Quantitative Applications
• Food Safety Testing: Measuring food preservatives or pesticide residues.
• Pollution Monitoring: Quantifying trace contaminants in water or soil samples.
• Color Analysis: Estimation of dye concentration in textile and paint industries.

Industrial Applications
Qualitative Applications
• Natural Product Analysis: Identifying plant-derived alkaloids, essential oils, or other
compounds.
• Raw Material Testing: Checking the purity of components in the production of chemicals.
• Ink Composition: Determining ink formulations in the printing industry.
Quantitative Applications
• Product Development: Measuring the composition of products like perfumes or food
colorants.
• Quality Assurance: Ensuring the consistency and purity of industrial chemicals.
• Pesticide Quantification: Estimating pesticide levels in agricultural samples.

2. Thin-Layer Chromatography (TLC)

Medical Applications
Qualitative Applications
• Drug Detection: Screening for illicit drugs in urine or blood samples.
• Metabolite Identification: Separation of metabolites for metabolic or pharmacological
studies.
• Pathogen Detection: Identifying microbial lipids or toxins in diagnostic microbiology.
Quantitative Applications
• Pharmaceutical Analysis: Determining the active ingredients in drug formulations.
• Vitamin Quantification: Measuring vitamin content in biological or food samples.
 • Hormonal Studies: Quantifying hormones in endocrine research.

Analytical Applications
Qualitative Applications
• Compound Purity: Checking the purity of chemical substances.
• Food Adulteration Detection: Identifying contaminants or adulterants in food products.
• Environmental Testing: Identifying pollutants or toxins in air, water, or soil.
Quantitative Applications
• Residue Analysis: Measuring pesticide or herbicide residues in agricultural samples.
• Pollution Quantification: Estimating toxic compounds in industrial effluents.
• Nutritional Analysis: Quantifying vitamins or antioxidants in food samples.

Industrial Applications
Qualitative Applications
• Process Monitoring: Identifying intermediates in chemical manufacturing.
• Natural Product Isolation: Analysis of plant extracts for bioactive compounds.
• Pigment Analysis: Studying the composition of dyes and pigments.
Quantitative Applications
• Pharmaceutical Manufacturing: Measuring active pharmaceutical ingredients during
production.
• Food Industry: Quantifying artificial colorants, preservatives, or flavors.
• Biotechnology: Estimating enzyme or protein concentrations in industrial processes.