Chromatography Techniques PDF

CHROMATOGRAPHY I. Chromatography General Concept: Chromatography is a laboratory technique used to separate, identify, and analyze the components of a mixture. The separation is based on the differential interaction of components between a stationary phase (a solid or liquid fixed in place) and a mo...

CHROMATOGRAPHY I. Chromatography General Concept: Chromatography is a laboratory technique used to separate, identify, and analyze the components of a mixture. The separation is based on the differential interaction of components between a stationary phase (a solid or liquid fixed in place) and a mobile phase (a liquid or gas that moves through the stationary phase). Principle of Chromatography: Separation Mechanism: Different components in a mixture travel at varying speeds due to differences in their: 1. Affinity to the stationary phase (adsorption or partitioning). 2. Solubility in the mobile phase. Components with stronger interactions with the stationary phase move slower, while those more soluble in the mobile phase move faster. Types of Chromatography Chromatography is classified based on the nature of the stationary and mobile phases, and the mechanism of separation: 1. Liquid Chromatography (LC): Stationary Phase: Solid or liquid. Mobile Phase: Liquid. Applications: Analysis of pharmaceuticals, biomolecules, and environmental samples. Subtypes: o High-Performance Liquid Chromatography (HPLC): ▪ High-pressure system for rapid, precise separation. o Size-Exclusion Chromatography: ▪ Separates molecules based on size. o Ion-Exchange Chromatography: ▪ Separates ions and polar molecules based on charge. 2. Gas Chromatography (GC): Stationary Phase: Liquid or solid coating inside a column. Mobile Phase: Inert gas (e.g., helium, nitrogen). Applications: Volatile and thermally stable compounds, such as hydrocarbons or fragrances. 1 Dr. Haitham ALhakimi 3. Thin-Layer Chromatography (TLC): Stationary Phase: Thin layer of silica gel or alumina on a flat surface. Mobile Phase: Solvent or solvent mixture. Applications: Quick, qualitative analysis of mixtures. 4. Paper Chromatography: Stationary Phase: Filter paper (cellulose). Mobile Phase: Solvent (aqueous or organic). Applications: Separating pigments or small polar molecules. 5. Affinity Chromatography: Stationary Phase: Specific ligands that bind to target molecules. Mobile Phase: Buffer or solution. Applications: Purification of proteins, enzymes, and antibodies. 6. Column Chromatography: Stationary Phase: Packed column (solid adsorbent). Mobile Phase: Solvent or mixture. Applications: General purification of compounds. 2 Dr. Haitham ALhakimi >> The modern types of chromatographic methods can be classified in the following ways given below the table, Chromatography technique Technique Method Abbreviation Columnar method LC Adsorption chromatography Gas solid chromatography GSC Liquid-liquid partition LLPC Paper chromatography PC Partition chromatography Thin-layer chromatography TLC Reverse phase partition RPPC Cation exchange CEC Anion exchange AEC Ion exchange chromatography Inorganic exchanger IE Liquid exchanger LIE Zone electrophoresis ZE Boundary layer method BLE Electrophoresis Curtain electrophoresis CC Capillary electrophoresis CZE II. Extraction General Concept: Extraction is a process used to separate a desired substance from a mixture using a solvent. It exploits differences in solubility or chemical affinity between the components. Types of Extraction: 1. Liquid-Liquid Extraction (LLE): Principle: Partitioning of a solute between two immiscible liquids (e.g., oil and water). Example: Extraction of caffeine from tea using an organic solvent. 2. Solid-Liquid Extraction (SLE): Principle: Dissolution of a solute from a solid matrix into a solvent. Example: Extraction of essential oils from plants. 3. Supercritical Fluid Extraction (SFE): Principle: Using supercritical CO₂ as a solvent to extract compounds. Example: Extraction of flavors and fragrances. 3 Dr. Haitham ALhakimi 4. Soxhlet Extraction: Principle: Repeated solvent reflux for thorough extraction. Example: Extraction of lipids from biological materials. 5. Ultrasonic-Assisted Extraction (UAE): Principle: Ultrasonic waves enhance solvent penetration into the sample. Example: Rapid extraction of bioactive compounds from herbs. Applications of Chromatography and Extraction: Pharmaceuticals: o Chromatography: Purification of drugs and active ingredients. o Extraction: Isolating plant-derived bioactive compounds. Food Industry: o Chromatography: Identifying food additives, preservatives, and contaminants. o Extraction: Obtaining flavors, colors, and nutrients. Environmental Science: o Chromatography: Detecting pollutants in air, water, and soil. o Extraction: Isolating toxic substances for analysis. Biotechnology: o Chromatography: Protein purification. o Extraction: Recovery of biomolecules from fermentation processes. Applications of Chromatography in Pharmaceuticals: 1. Drug Purity Testing: o HPLC is used to check the purity of active pharmaceutical ingredients (APIs). 2. Identification of Active Compounds: o TLC is used for initial screening in drug discovery. 3. Quantitative Analysis: o GC quantifies volatile impurities in formulations. 4. Biomolecule Isolation: o Affinity chromatography isolates therapeutic proteins, antibodies, and enzymes. 5. Quality Control: o Chromatography ensures consistency across batches of pharmaceutical products. 4 Dr. Haitham ALhakimi Applications of Extraction in Pharmaceuticals 1. Natural Product Isolation: o Extraction of bioactive compounds, such as alkaloids or glycosides, from plants for drug development. 2. API Purification: o Removing unwanted byproducts or impurities from drug formulations. 3. Preparation of Herbal Medicines: o Extracting essential oils, polyphenols, and terpenoids from medicinal plants. 4. Recovery of Antibiotics: o Solvent extraction of antibiotics from microbial fermentation. Integrated Use of Chromatography and Extraction in Pharmaceuticals 1. Extraction as a Precursor to Chromatography: o Extract raw plant or microbial material to obtain a crude mixture. o Purify and analyze the mixture using chromatographic methods. o Example: Isolating penicillin (extraction) and verifying purity (HPLC). 2. Quality Assurance: o Extraction isolates analytes; chromatography confirms their identity and concentration. 3. Formulation Development: o Extract bioactive compounds; separate, purify, and incorporate them into therapeutic drugs. Conclusion Chromatography and extraction are indispensable tools in pharmaceutical sciences. They complement each other to ensure the purity, quality, and efficacy of drugs, playing a critical role from drug discovery to manufacturing. 5 Dr. Haitham ALhakimi  Detailed Methods and Pharmaceutical Examples in Chromatography and Extraction I. Advanced Chromatographic Methods and Applications 1. High-Performance Liquid Chromatography (HPLC): Principle: Separates compounds based on their polarity and interaction with the stationary phase under high pressure. Key Features: o Uses high-pressure pumps for faster separation. o Highly sensitive and precise. o Ideal for heat-sensitive compounds. Pharmaceutical Example: o Drug Purity Testing: HPLC is used to determine the purity of paracetamol and to detect degradation products in formulations. o Dissolution Testing: Measures the release rate of active ingredients from tablets during quality control. 2. Gas Chromatography (GC): Principle: Separates volatile compounds based on their boiling points and interaction with the column material. Key Features: o Requires samples to be volatile or derivatized. o Equipped with detectors like Flame Ionization Detector (FID) or Mass Spectrometry (MS). Pharmaceutical Example: o Residual Solvent Analysis: Determines the presence of organic solvents (e.g., ethanol, methanol) in pharmaceutical products. o Essential Oil Analysis: Identifies and quantifies volatile oils in herbal medicines. 3. Thin-Layer Chromatography (TLC): Principle: Components travel across a silica or alumina-coated plate at different rates based on polarity. Key Features: o Simple, cost-effective, and rapid. o Provides a visual representation of separation. Pharmaceutical Example: o Active Ingredient Detection: TLC is used to identify caffeine or aspirin in pharmaceutical mixtures. o Herbal Medicine Testing: Detects secondary metabolites like flavonoids and alkaloids. 4. Affinity Chromatography: Principle: Utilizes specific interactions between ligands on the stationary phase and target biomolecules. Key Features: o Highly selective for proteins, antibodies, or enzymes. Pharmaceutical Example: o Monoclonal Antibody Purification: Isolates antibodies for cancer immunotherapy drugs. o Insulin Purification: Used to extract recombinant insulin from fermentation cultures. 6 Dr. Haitham ALhakimi 5. Ion-Exchange Chromatography: Principle: Separates ions and charged molecules based on their affinity to charged groups in the stationary phase. Key Features: o Effective for ionic compounds like amino acids or nucleotides. Pharmaceutical Example: o Vaccine Development: Purifies viral particles or DNA fragments. o Protein Purification: Used for therapeutic proteins like growth hormones. II. Advanced Extraction Methods and Applications 1. Liquid-Liquid Extraction (LLE): Principle: Partitions solutes between two immiscible liquids based on their solubility. Key Features: o Ideal for separating polar and non-polar compounds. Pharmaceutical Example: o Alkaloid Extraction: Extracting morphine from opium. o Antibiotic Recovery: Isolates penicillin from fermentation broth using organic solvents. 2. Supercritical Fluid Extraction (SFE): Principle: Utilizes supercritical fluids (e.g., CO₂) as solvents to extract non-polar compounds. Key Features: o Environmentally friendly and efficient for thermolabile compounds. Pharmaceutical Example: o Essential Oil Extraction: Extracts volatile oils from plants without thermal degradation. o Sterol Extraction: Isolates phytosterols for cholesterol-lowering drugs. 3. Soxhlet Extraction: Principle: Repeatedly extracts solutes using a heated solvent. Key Features: o Provides thorough extraction of non-volatile compounds. Pharmaceutical Example: o Lipid Extraction: Isolates fatty acids for formulation studies. o Plant Compound Extraction: Extracts curcumin from turmeric. 4. Ultrasonic-Assisted Extraction (UAE): Principle: Uses ultrasonic waves to enhance solvent penetration and solute dissolution. Key Features: o Fast and effective for fragile compounds. Pharmaceutical Example: o Polyphenol Extraction: Isolates antioxidants from green tea leaves. o Terpenoid Extraction: Extracts terpenes from medicinal plants for anti-inflammatory drugs. 7 Dr. Haitham ALhakimi III. Integrated Pharmaceutical Applications Drug Discovery: 1. Herbal Drugs: o Example: Extracting bioactive compounds from Cinchona bark (quinine) for anti-malarial drugs. o Process: Ultrasonic-Assisted Extraction → HPLC for purity testing. 2. Synthetic Drugs: o Example: Identifying and purifying synthetic intermediates in antiviral drug synthesis. o Process: LLE → GC-MS for component identification. Formulation Development: Application: Ensuring the stability and uniformity of drug formulations. o Example: Monitoring preservatives in syrups using GC. Quality Assurance: 1. API Consistency: o Example: Checking the concentration of ibuprofen in tablets using HPLC. 2. Impurity Analysis: o Example: Detecting trace levels of toxic solvents in injectables using GC. Case Study Example Production of Antibiotics (e.g., Penicillin): 1. Fermentation: o Antibiotic is produced in large-scale fermenters. 2. Extraction: o Solvent extraction isolates penicillin from the broth. 3. Purification: o Chromatography (Ion-Exchange) separates penicillin from impurities. 4. Quality Control: o HPLC ensures the final product meets purity standards. Conclusion Chromatography and extraction techniques are indispensable in the pharmaceutical industry for drug development, quality control, and formulation studies. Advanced methods like HPLC, GC, and SFE, combined with robust extraction processes, ensure precise, efficient, and safe production of medicines. 8 Dr. Haitham ALhakimi