HPLC High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC) is an advanced analytical technique used to separate, identify, and quantify components in a mixture. It is widely utilized in various fields, including pharmaceuticals, biochemistry, environmental science, and food industry, due to its high resolution, sensitivity, and precision.

1. HPLC "High Performance Liquid Chromatography" Prof. Mohammad Abuzar

2. What is HPLC ? High-Performance Liquid Chromatography (HPLC) is an advanced analytical technique used to separate, identify, and quantify components in a mixture. It is widely utilized in various fields, including pharmaceuticals, biochemistry, environmental science, and food industry, due to its high resolution, sensitivity, and precision.

3. Principle of HPLC When each component dissolved in the mobile phase passes through the stationary phase, the size and strength of each component's interaction with the stationary phase (adsorption, distribution, exclusion, affinity) are different. Therefore, the residence time of these components in the stationary phase is different, flowing out from the stationary phase at different times one after another.

4. SOLVENT RESERVOIR Hold the mobile phase, which is the solvent or solvent mixture used to carry the sample through the column. PUMP Delivers the mobile phase at a constant and precise f l o w r a t e a n d p r e ss u r e , t y p i c a ll y r a n g i n g f r o m 1 t o 1 mL/min. INJECTOR Introduces the sample into the mobile phase stream. This can be done manually or automatically using an autosampler. Key Components of HPLC

5. COLUMN The heart of the HPLC system, packed with stationary phase material. The column's properties determine the separation process. DETECTOR Monitors the eluent coming out of the column and provides data on the components based on their interaction with the detector. Common types include UV-Vis, fluorescence, and mass spectrometry (MS) detectors. DATA SYSTEM Collects and processes the data from the detector, often displayed as a chromatogram, which shows the separation of compounds over time.

6. Types of HPLC NORMAL PHASE HPLC Uses a polar stationary phase (e.g., silica) and a non- polar mobile phase (e.g., hexane). Suitable for separating non-polar or weakly polar compounds. REVERSE PHASE HPLC Uses a non-polar stationary phase (e.g., C18 or C8 columns) and a polar mobile phase (e.g., water- methanol or water-acetonitrile mixtures). The most common type of HPLC, ideal for separating polar compounds.

7. ION EXCHANGE HPLC Uses a charged stationary phase to separate ionic compounds based on their charge. Commonly used for proteins, peptides, and nucleotides. SIZE EXCLUSION HPLC Separates molecules based on their size using a porous stationary phase. Useful for large biomolecules like proteins and polymers. AFFINITY HPLC Uses a stationary phase with specific binding sites to separate compounds based on their biological affinity. Often used for purifying proteins and antibodies.

8. Steps in HPLC Analysis PREPRATION OF MOBILE PHASE The solvent or solvent mixture is prepared, often requiring degassing to remove air bubbles. SAMPLE PREPARATION The sample is filtered and dissolved in a suitable solvent to prevent clogging the column. SYSTEM SETUP The HPLC system is set up, and the column is equilibrated with the mobile phase.

9. SAMPLE INJECTION The sample is injected into the mobile phase stream. SEPARATION As the sample passes through the column, its components separate based on their interactions with the stationary and mobile phases. DETECTION The separated components are detected as they elute from the column. DATA ANALYSIS The detector signals are processed to produce a chromatogram, which is analyzed to identify and quantify the compounds.

10. Applications of HPLC FOOD & BEVERAGE INDUSTRY Analysis of additives, preservatives, and nutritional content. Detection of contaminants and adulterants. BIOTECHNOLOGY & BIOCHEMISTRY Protein and peptide analysis. Analysis of nucleic acids and other biomolecules. ENVIRONMENTAL STUDIES Detection of pollutants and contaminants in water, soil, and air samples.

11. PHARMACEUTICAL INDUSTRY Quality control and purity testing of drugs. Separation and analysis of complex mixtures of pharmaceutical compounds. CLINICAL CHEMISTRY Analysis of blood, urine, and other biological samples for diagnostics and research.

12. Advantages of HPLC HIGH RESOLUTION Can separate complex mixtures with high precision. SPEED Faster analysis compared to traditional methods. SENSITIVITY Capable of detecting low concentrations of compounds. VERSATILITY Applicable to a wide range of substances, including both organic and inorganic compounds. 5. QUANTITATIVE & QUALITATIVE ANALYSIS Provides both identification and quantification of components.

13. Limitations of HPLC COST Equipment and maintenance can be expensive. COMPLEXITY Requires technical expertise to operate and interpret results. SAMPLE PREPARATION Often requires extensive preparation to avoid column damage. SOLVENT USE Can be costly and requires proper disposal due to environmental concerns.

14. Conclusion HPLC is a powerful and versatile analytical technique essential for modern scientific research and industrial applications. Its ability to separate, identify, and quantify compounds with high accuracy makes it invaluable in various fields.

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