HPLC Workshop Report PDF

Workshop on HPLC, Mass and ICPMS 1. HPLC High-Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture. It involves passing a liquid sample through a column packed with a stationary phase, with separa...

Workshop on HPLC, Mass and ICPMS 1. HPLC High-Performance Liquid Chromatography (HPLC) is an analytical technique used to separate, identify, and quantify components in a mixture. It involves passing a liquid sample through a column packed with a stationary phase, with separation achieved based on interactions between the sample components and the stationary phase. Basic Working Principle of HPLC Invention of chromatography by M Tsvet Chromatography is the separation technique which separates the mixture of compounds based on partition coefficient. Main purpose is to separate and quantify the components. Substance having more affinity to the stationary phase elutes slowly and those who have less affinity to stationary phase/more affinity to the mobile phase elutes faster. Chromatogram Area under the curve and height will give concentration and quantity of the compound. Retention time (Rt) identifies the compound. Rt depends on stationary phase, mobile phase and its composition, column parameters (diameter, length, particle size), flow rate, column temperature etc. Types of chromatography 1. Based on modes: Normal Phase Reverse Phase 2. Based on elution technique: Isocratic separation Gradient elution 3. Based on type of analysis: Qualitative analysis Quantitative analysis  4. Based on the scale of the operation: Analytical HPLC Preparative HPLC Separation modes of HPLC Normal Phase chromatography Reverse Phase Chromatography Ion exchange chromatography Size filtration (Gel permeation and Gel filtration) Uses of HPLC – identification, quantification, and purification of components. Components of HPLC Mobile Phase – Degassing unit – Solvent delivery pump – Injector and Sample vials – Column – Detector – Drain/Fraction collector Representative HPLC Detectors UV-visible absorbance detector – sensitivity is more compared to photodiode array Photodiode array detector - Can scan entire wavelength from 200 – 800 nm Fluorescence detector - highly sensitive, but the compound should be fluorescent Refractive index detector - Universal detector; least sensitive ELSD - Evaporative Light Scattering detector Mass spectrometer Applications Pharmaceutical applications – assay of API, content uniformity, related substance analysis Biopharmaceutical applications – peptide mapping, charge variant analysis, glycan analysis, size exclusion chromatography Food industry – analysis of pesticides, vitamins, amino acid etc. Chemical industry – quantitative and qualitative analysis of synthetic compounds Environmental applications – water and soil analysis Columns Two types of columns are available: Polar columns - Made of silica and are used in normal phase HPLC. Non-polar columns - Made of C18 and are used in reverse phase HPLC Steps involved in HPLC a. Mobile Phase Preparation i. Selecting the Mobile Phase Components Purpose: Choose solvents and additives that will effectively separate the analytes. Considerations: o Polarity: Match the polarity of the mobile phase to the analytes and stationary phase. o Solvent Strength: Adjust to achieve the desired retention times. o Compatibility: Ensure solvents are compatible with the HPLC system and detectors. o Additives: Include buffers, pH adjusters, or ion-pairing agents if necessary. ii. Measuring and Mixing Solvents Purpose: Accurately measure and mix solvents to create the mobile phase. Steps: o Use high-purity HPLC-grade solvents to avoid impurities. o Measure solvents using graduated cylinders, volumetric flasks, or automated mixing systems. o Mix solvents thoroughly to ensure a homogeneous mobile phase. iii. Adding Buffers and Additives Purpose: Add any necessary buffers or additives to control pH and improve separation. Steps: o Buffers: Prepare buffer solutions at the desired concentration and pH.  o Additives: Measure and add ion-pairing agents, pH adjusters, or other additives. o Dissolution: Ensure all components are fully dissolved. iv. Adjusting the pH Purpose: Adjust the pH of the mobile phase to optimize analyte separation. Steps: o Measure pH using a calibrated pH meter. o Adjust pH using acid (e.g., phosphoric acid) or base (e.g., sodium hydroxide) as needed. o Re-check pH to confirm it is within the desired range. v. Filtration Purpose: Remove particulate matter to prevent clogging and ensure smooth operation. Steps: o Use a vacuum filtration setup with a membrane filter (typically 0.2 µm or 0.45 µm pore size). o Filter the mobile phase to remove any particulates. o Alternatively, use pre-filtered solvents or inline filtration systems. vi. Degassing Purpose: Remove dissolved gases to prevent bubble formation and ensure detector stability. Methods: o Vacuum Degassing: Apply vacuum to the mobile phase container. o Sparging: Bubble an inert gas (e.g., helium) through the solvent. o Ultrasonic Degassing: Use ultrasonic waves to degas the solvent. vii. Storing and Handling Purpose: Properly store and handle the mobile phase to maintain its quality. Steps:  o Store in clean, appropriately labelled glass or plastic containers. o Avoid prolonged exposure to light and air. o Use amber bottles for light-sensitive solvents. viii. Connecting to the HPLC System Purpose: Ensure a seamless connection to the HPLC system. Steps: o Prime the solvent lines with the prepared mobile phase. o Ensure there are no air bubbles in the lines. o Monitor the pressure and flow rate to detect any issues. b. Degassing Purpose: Remove dissolved gases from the mobile phase to prevent bubble formation, which can cause noise and instability in the detector signal. Methods: o Vacuum Degassing: Using a vacuum to remove gases. o Sparging: Bubbling an inert gas like helium through the solvent. o Ultrasonic Degassing: Using ultrasonic waves to remove gases. c. Washing (System Flushing) Purpose: Clean the system to remove contaminants and residues from previous runs. Steps: o Flush the system with a strong solvent (e.g., methanol or acetonitrile) to remove impurities. o Follow with a wash using the mobile phase to equilibrate the system. d. Priming Purpose: Prepare the pump and ensure the mobile phase is evenly distributed throughout the system. Steps: o Pump the mobile phase through the system to fill all tubing, pump, and detector with the solvent.  o Ensure the mobile phase reaches a stable flow rate and pressure. e. Sample Preparation Purpose: Prepare samples for injection to ensure accurate and reproducible results. Steps: o Dissolution: Dissolve the sample in a suitable solvent. o Filtration: Filter the sample to remove particulates (using syringe filters or centrifugation). o Dilution: Dilute the sample to the desired concentration. f. Placing the Stationary Phase (Column Preparation) 1. Purpose: Install and prepare the HPLC column which contains the stationary phase. 2. Steps: o Column Installation: Connect the column to the HPLC system. o Column Equilibration: Flush the column with the mobile phase until the baseline is stable. o Ensure the column is compatible with the mobile phase and sample. g. Detectors Purpose: Detect and quantify the analytes as they elute from the column. Types: o UV/Vis Detector: Measures absorbance of analytes at specific wavelengths. o Refractive Index Detector: Measures changes in refractive index of the eluent. o Fluorescence Detector: Detects analytes that can fluoresce. o Mass Spectrometer: Provides molecular weight and structural information. h. Column Wash (Post-Run Cleaning) Purpose: Remove residual analytes and mobile phase to maintain column performance and longevity. Steps: o Flush the column with a strong solvent to remove bound analytes.  o Use a gradient wash (starting with a high percentage of organic solvent) to ensure thorough cleaning. o Store the column in a suitable solvent, often a mixture of water and organic solvent (e.g., 50:50 water). 2. MASS SPECTROMETRY Mass spectrometry is an analytical technique used to measure the mass-to-charge ratio of ions. It identifies and quantifies molecules by ionizing chemical compounds, separating the ions based on their mass and charge, and detecting them. This method provides detailed information on molecular structure and composition. Key Components of Mass Spectrometry: 1. Ionization Source: The sample is first ionized to produce charged particles (ions). Common ionization methods include Electron Ionization (EI), Electrospray Ionization (ESI), and Matrix-Assisted Laser Desorption/Ionization (MALDI). Different ionization techniques are used depending on the type of sample and the desired analysis: a. Electron Ionization (EI): Common in gas chromatography-mass spectrometry (GC-MS), where electrons are used to ionize gas-phase molecules. b. Electrospray Ionization (ESI): Frequently used in liquid chromatography-mass spectrometry (LC-MS) for large biomolecules like proteins and peptides. c. Matrix-Assisted Laser Desorption/Ionization (MALDI): Useful for large biomolecules like proteins and polymers, where a laser ionizes the sample embedded in a matrix. 2. Mass Analyzer: The ions are then separated based on their mass-to-charge ratio. Different types of mass analyzers include Quadrupole, Time-of-Flight (TOF), and Orbitrap. Each analyzer has its own resolution, accuracy, and application suitability. a. Quadrupole: Uses oscillating electric fields to filter ions by m/z. It is compact and widely used for routine analysis. b. Time-of-Flight (TOF): Measures the time it takes for ions to reach the detector; ions with different m/z ratios travel at different speeds. c. Orbitrap: Provides high resolution by trapping ions in an electric field and measuring their oscillation frequency, offering high accuracy and precision.  3. Detector: After separation, the ions are detected, and their abundances are recorded. The detector generates a spectrum representing the mass-to-charge ratio of the ions present in the sample. 4. Data System: The data collected is processed to produce a mass spectrum, which is a plot of ion signal as a function of the mass-to-charge ratio. The peaks in the mass spectrum provide information about the molecular weight and structure of the sample components. Operation Modes 1. Product Ion Scanning: In product ion scanning, a specific precursor ion (parent ion) is selected by the first mass analyzer (MS1) and fragmented in a collision cell (using collision-induced dissociation, CID). The second mass analyzer (MS2) then scans the resulting fragment ions (product ions). 2. Precursor Ion Scanning: The second mass analyzer (MS2) is set to detect a specific fragment ion, while the first mass analyzer (MS1) scans for precursor ions that produce this fragment upon fragmentation. 3. Neutral Loss Scanning: Both mass analyzers (MS1 and MS2) scan ions, but they are set with a fixed mass difference corresponding to the mass of a neutral molecule that is lost during fragmentation (neutral loss). 4. Multiple Reaction Monitoring (MRM): Also known as Selected Reaction Monitoring (SRM), MRM involves selecting a specific precursor ion in MS1, fragmenting it, and then monitoring a specific product ion in MS2. This mode is typically used in triple quadrupole mass spectrometers. Applications of Mass Spectrometry Mass spectrometry has various applications across various fields due to its versatility, sensitivity, and ability to analyze a wide range of substances. Here are some key applications: 1. Proteomics and Genomics: a. Protein Identification and Characterization: MS is extensively used in proteomics to identify and characterize proteins and post-translational modifications, such as phosphorylation.  b. DNA and RNA Analysis: MS can be used to sequence DNA and RNA, as well as to study nucleic acid modifications. 2. Pharmaceutical and Drug Development: a. Drug Discovery and Metabolism: MS is employed to identify potential drug candidates, study their metabolic pathways, and understand their interactions with biological systems. b. Quality Control: MS ensures the purity and potency of pharmaceutical products by analyzing active ingredients and contaminants. 3. Clinical Diagnostics: a. Biomarker Discovery: MS helps in identifying biomarkers for diseases, which can be used for early diagnosis and personalized medicine. b. Toxicology: MS detects and quantifies toxic substances, drugs, and metabolites in biological samples. 4. Environmental Analysis: a. Pollutant Detection: MS is used to identify and quantify environmental pollutants like pesticides, heavy metals, and organic compounds in air, water, and soil samples. b. Climate Studies: Analyzing isotope ratios in atmospheric gases helps in understanding climate change and environmental processes. 5. Food and Beverage Testing: a. Contaminant Analysis: MS detects contaminants such as pesticides, mycotoxins, and heavy metals in food and beverages, ensuring safety and compliance with regulations. b. Authentication and Adulteration: It is used to authenticate food products and detect adulteration by identifying unique chemical fingerprints. 3. INDUCTIVELY COUPLED PLASMA MASS SPECTROMETRY (ICP-MS) Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a powerful analytical technique used to detect and quantify trace elements and isotopes in various types of samples. It combines an inductively coupled plasma source with a mass spectrometer to measure the mass-to-charge ratio (m/z) of ions. ICP-MS is widely used in environmental monitoring, food safety, clinical analysis, and geochemistry due to its high sensitivity, low detection limits, and ability to analyze multiple elements simultaneously. Principle of ICP-MS The principle of ICP-MS involves three main steps: atomization, ionization, and detection. a. Atomization and Ionization: A liquid sample is introduced into a nebulizer, where it is converted into an aerosol. This aerosol is then transported into the inductively coupled plasma (ICP) torch, where the sample is subjected to temperatures of 6,000-10,000 K. At these high temperatures, the sample is atomized and ionized, producing a stream of positively charged ions of the elements present in the sample. b. Mass Spectrometry: The ions generated in the plasma are extracted into the mass spectrometer, where they are separated based on their mass-to-charge ratio (m/z) using a mass analyzer, typically a quadrupole or a time-of-flight (TOF) analyzer. Detection: The separated ions are detected by a detector (usually an electron multiplier), which converts the ion signal into an electrical signal that is proportional to the concentration of the elements in the sample. Key Components of ICP-MS 1.Sample Introduction System: Nebulizer and Spray Chamber: Converts the liquid sample into an aerosol and removes large droplets to produce a fine mist that can be efficiently ionized in the plasma. 2.ICP Torch: Consists of three concentric quartz tubes through which argon gas flows. A high-frequency radiofrequency (RF) generator creates an electromagnetic field that ionizes the argon gas, generating a plasma that can reach temperatures up to 10,000 K. 3.Interface Region: The interface region contains a series of cones (sampler and skimmer cones) that allow the transfer of ions from the high-temperature plasma to the low-pressure mass spectrometer. 4.Mass Analyzer: a. Quadrupole Analyzer: The most common type, which separates ions based on their m/z ratio using oscillating electric fields. b. Time-of-Flight (TOF) Analyzer: Provides high resolution and the ability to detect a wide range of ions simultaneously. 5.Detector: Typically an electron multiplier detector that converts ions into an electrical signal. The detector amplifies the signal to measurable levels, allowing for the quantification of trace elements. Metals and Elements Analyzed by ICP-MS ICP-MS can analyze a wide range of elements across the periodic table, from lithium (Li) to uranium (U), with very low detection limits (down to parts per trillion, ppt). Some common metals and elements analyzed include: Heavy Metals: Lead (Pb), Cadmium (Cd), Mercury (Hg), Arsenic (As), Chromium (Cr), Nickel (Ni) Nutrient Metals: Iron (Fe), Zinc (Zn), Copper (Cu), Manganese (Mn), Selenium (Se) Rare Earth Elements: Lanthanides such as Cerium (Ce), Neodymium (Nd), and Europium (Eu) Alkali and Alkaline Earth Metals: Sodium (Na), Potassium (K), Calcium (Ca), Magnesium (Mg) Applications of ICP-MS ICP-MS is widely used in: a. Environmental Analysis: Monitoring of water, soil, and air samples for trace metals and pollutants. b. Clinical and Pharmaceutical Analysis: Determination of trace elements in biological fluids and tissues, and monitoring metal contamination in pharmaceuticals. c. Food and Beverage Testing: Detecting toxic elements in food products and ensuring compliance with safety standards. d. Geochemistry and Metallurgy: Analysis of rocks, minerals, and ores to study geological processes and mineral composition.