ADVANCED HPLC METHOD DEVELOPMENT

ADVANCED HPLC METHOD DEVELOPMENT

LIVE ONLINE INTERACTIVE COURSE · process‑driven & systematic

For the experienced chromatographer – a comprehensive, process‑driven approach to developing optimal and robust HPLC methods. Move beyond trial and error.

📌 COURSE OBJECTIVES

• Understand key principles of HPLC method development
• Apply Quality by Design (QbD) principles for robust analytical methods
• Optimize separation parameters for improved resolution, selectivity, and efficiency
• Ensure method reproducibility, sensitivity, and robustness

⚙️ QUALITY BY DESIGN (QbD) IN HPLCWhat is QbD?

• Importance of systematic method development
• Defining Critical Quality Attributes (CQAs) and Critical Method Parameters (CMPs)

Building robustness

• Identifying and controlling variables affecting method performance
• Strategies for reducing variability and enhancing reproducibility

Key factors influencing chromatographic resolution

• Retention factor (k’)
• Efficiency (N)
• Selectivity (α)

🧪 SAMPLE PREPARATION STRATEGIES

• Optimizing sample solubility: effect of solvents, pH, temperature
• Choosing the right diluent: matching polarity, avoiding solvent interactions with mobile phase

💧 MOBILE PHASE OPTIMIZATION

• Effect of sample polarity on separation
• pH control and buffer selection – impact on ionizable compounds, pH stability
• Optimizing flow rates for resolution and throughput

🧬 COLUMN SELECTION AND OPTIMIZATION

• Physical dimensions: length, internal diameter, efficiency
• Particle size and type: conventional HPLC vs. UHPLC
• Column temperature effects on retention and peak shape
• Chemically modified silica‑based phases: reversed‑phase ligands (C18, C8, phenyl, etc.)
• Hydrophobic Subtraction Model (HSM) classification for column selection

📈 GRADIENT ELUTION & METHOD OPTIMIZATION

• Isocratic vs. gradient elution: when to use each approach
• Optimizing retention factor (k’) for improved separation and peak shape
• Effect of gradient parameters: slope, dwell volume, mobile phase composition

🔧 BUILDING A PRACTICAL HPLC METHOD

• Comprehensive sample preparation techniques
• Optimizing retention and selectivity (α): screening column chemistries and mobile phase conditions
• Chromatographic optimisation strategies – resolution, peak symmetry
• Assessing linearity, range, and sensitivity
• Pre‑validation considerations: system suitability, robustness, repeatability

🗣️ Q&A AND CLOSING DISCUSSION

• Summary of key concepts
• Open discussion on challenges faced in HPLC method development
• Resources for further learning

💻 ONLINE PRACTICAL COMPONENT

• Interactive decision exercises: column screening, gradient simulations, robustness testing scenarios
• Case‑based optimisation workshops

QbD approach HSM column selection