Course outline
📘 1. Introduction to HPLC and its applications
- Overview of HPLC and its importance in analytical chemistry
- Target industries: Pharmaceuticals, food & beverage, environmental, biotechnology, forensics
- Comparison with other chromatography: GC, TLC, other LC methods
- Types of samples suitable for HPLC analysis
🧪 2. Sample preparation for HPLC analysis
- Importance of proper sample preparation
- Common HPLC sample preparation methods (filtration, dilution, derivatization, SPE)
- Dos and Don’ts: avoiding contamination, solvent selection, solvent:sample ratio, stability
⚙️ 3. HPLC instrumentation & components
- Mobile phase & degassing: solvents, degasser types, impact on analysis
- Pumps: isocratic vs. gradient, function, performance
- Injectors: manual vs. autosamplers, sample introduction
- Columns and column oven: stationary phases, column selection, temperature control
- Detectors: UV-Vis, fluorescence, RI, MS – principles and applications
📊 4. Understanding the chromatographic process
🔹 Overview of reversed-phase HPLC process
- Fundamental principles and practical applications
- Interaction between stationary and mobile phases
🔹 Key concepts in chromatography
- Role of polarity, solubility, and diluents in separation
- Mechanisms of retention in reversed-phase HPLC
🔹 Chromatographic performance parameters
- Capacity factor (k’): measuring analyte retention
- Selectivity (α): differentiating structurally similar compounds
- Efficiency (N): peak sharpness, theoretical plates
- Resolution (Rs): optimizing separation quality
💧 5. HPLC mobile phases and additives
Mobile phase preparation & optimization
- Selecting solvents for separation and reproducibility
- Degassing techniques – prevent bubbles and detector noise
- Purity, stability, consistency
Effects of organic modifiers
- Methanol, acetonitrile, THF – impact on retention, peak shape, resolution
pH control, buffering, and additives
- Importance of pH in analyte ionization and retention
- Choosing buffers & ionic strength
- Ion-pairing agents for enhanced separation
🧬 6. HPLC columns – selection & optimization
- Column dimensions: length, ID, pore size, particle size (HPLC vs UHPLC)
- Silica-based supports: porous vs non-porous, characteristics
- Bonded phases: C18, C8, phenyl, cyano – hydrophobicity, end‑capping, stability
- van Deemter equation: plate height (H), flow rate optimization, mass transfer
- Common reversed-phase chemistries: comparison & selection
🔄 7. Alternative HPLC separation modes
- Reversed-phase (RP-HPLC): hydrophobic interactions, elution order
- Ion suppression & ion-pair chromatography in RP-HPLC
- Normal-phase (NP-HPLC): polarity‑based separation, stationary/mobile phases
- Hydrophilic Interaction LC (HILIC): retention mechanism for polar analytes, mobile phase
- Size-Exclusion (SEC): molecular size, applications for proteins & polymers
- Ion-Exchange (IEX-HPLC): charge interactions, cation vs anion exchange
📈 8. Data processing, integration & validation
Peak integration and quantification
- Peak detection, baseline correction, overlapping peaks
- Manual vs automated integration
Standards and calibration
- External, internal, standard addition – calibration curves
- Linearity, accuracy, precision
System suitability testing (SST)
- Resolution, retention time, tailing factor, theoretical plates, repeatability
- Interpretation of SST results
Quality control and data validation
- QC samples: blanks, duplicates, spiked samples, reference standards
- Criteria for accepting or rejecting analytical runs
