High-Performance Thin-Layer Chromatography (HPTLC)
- 1. H P T L C "Hig h Perform anc e Thin L ayer Chromatography" Prof. Mohammad Abuzar
- 2. What is HPTL C High-Performance Thin-Layer Chromatography (HPTLC) is a sophisticated analytical technique used for separating and analyzing compounds in a mixture. It is an advanced version of Thin- Layer Chromatography (TLC) that offers higher resolution, sensitivity, and reproducibility.
- 3. Princ ip le of HPTL C 1. SEPARATION MECHANISM HPTLC operates on the principle of partitioning or adsorption between a stationary phase (usually a thin layer of adsorbent on a glass, plastic, or aluminum plate) and a mobile phase (a solvent or solvent mixture). 2. SEPARATION P RO C ES S Sample components migrate at different rates due to differences in their affinity for the stationary phase and the mobile phase. This results in the separation of compounds as distinct spots or bands on the plate.
- 4. Ins trum entation High-Performance Thin-Layer Chromatography (HPTLC) involves several key components that work together to achieve precise separation and analysis of compounds. Here are the main components: 1. STATIONARY PH ASE Thin Layer Plate The stationary phase is typically a thin layer of adsorbent material coated onto a flat, inert support, such as glass, aluminum, or plastic plates. Common adsorbents include silica gel, aluminum oxide, and cellulose. Coating Thickness The thickness of the adsorbent layer is usually between 0.1 mm and 0.5 mm, depending on the application.
- 5. 2. MOBILE PHA SE Solvent System The mobile phase consists of one or more solvents or solvent mixtures that move up the plate by capillary action. The choice of mobile phase depends on the nature of the compounds being analyzed and the stationary phase used. Solvent Chamber A developing chamber or tank where the solvent system is contained and the thin- layer plate is placed for development. 3. SAMPLE APPLICATION SYSTEM Applicator A device used to apply small volumes of the sample solution onto the stationary phase. This can be in the form of a manual applicator or an automatic sample applicator.
- 6. Application Forms Samples are applied as spots or bands, depending on the method and the nature of the analysis. 4. DEVELOPMENT CHAMBER Chamber Setup The development chamber is where the thin-layer plate is placed during the chromatographic process. It must be kept at a controlled temperature and humidity to ensure consistent results. Mobile Phase The solvent or solvent mixture in the chamber allows for the separation of compounds as it moves up the plate. 5. DETECTION & VISUALIZATIONSYSTEM Detection Methods After development, separated compounds are detected using various methods such as UV-visible light, fluorescence, or specific chemical reagents.
- 7. Visualization Compounds can be visualized as spots or bands using UV light, fluorescence imaging, or by chemical derivatization techniques. Densitometer An instrument used to measure the intensity of the spots or bands on the developed plate for quantitative analysis. 6. DATA ANALYSIS TOOLS Software HPTLC systems often come with software for data acquisition and analysis, which helps in interpreting the results and quantifying the compounds. Each of these components plays a crucial role in the effectiveness and accuracy of the HPTLC technique. The careful selection and maintenance of these components ensure reliable and reproducible results in analytical applications.
- 8. Proc ed ure 1. SAMPLE APPLICATION Small volumes of the sample solution are applied as spots or bands onto the coated plate. 2. DEVELOPMENT The plate is placed in a developing chamber containing the mobile phase. The solvent moves up the plate by capillary action, carrying the sample components with it. 3. DETECTION After development, the plate is dried, and the separated components are visualized using various detection methods like UV light, specific chemical reagents, or by visual inspection.
- 9. D etec tion & Analysis 1. VISUALIZATION Compounds are visualized using UV light or specific reagents. Some compounds can be detected directly, while others require derivatization. 2. QUANTIFICATION Densitometry is used to measure the intensity of the spots or bands. The data can be analyzed to determine the concentration of the components in the sample.
- 10. A p p lic ations of H P T L C A. FOOD & B E V E R AG E INDUSTRY For quality control and identification of food additives and contaminants. B. ENVIRONMENTAL ANALYSIS Used to detect pollutants and contaminants in air, water, and soil. C. PHARMACEUTICAL INDUSTRY For the analysis of drugs and their formulations, including the determination of purity and identification of compounds. D. FORENSIC SCIENCE To analyze substances in forensic samples.
- 11. A d vantag es o f H P T L C 1. Resolution HPTLC offers high resolution and separation efficiency compared to traditional TLC. 2. Sensitivity It can detect trace amounts of compounds due to improved sensitivity. 3. Quantitative Analysis Provides quantitative analysis with high precision and reproducibility. 4. Versatility Suitable for a wide range of applications, including pharmaceutical analysis, environmental testing, and food quality control.
- 12. L im itations of H P T L C 1.Complexity in Method Development Requires optimization of various parameters such as the stationary phase, mobile phase, and detection methods. 2. Limited to Non Volatile Compounds Best suited for compounds that are stable and non-volatile.
- 13. Conclusion HPTL C is a powerful technique for detailed and accurate chemical analysis, offering enhanced performance over traditional TLC methods.