ADVANCED HPLC METHOD DEVELOPMENT

COURSE OUTLINE

Course Objectives

• Understanding key principles of HPLC method development
• Applying Quality by Design (QbD) principles for robust analytical methods
• Optimizing separation parameters for improved resolution, selectivity, and efficiency
• Ensuring method reproducibility, sensitivity, and robustness

Quality by Design (QbD) in HPLC

What is QbD?

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

Building Robustness in Method Development

• 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, and temperature on solubility

Choosing the Right Diluent

• Matching diluent polarity with analyte properties
• Avoiding solvent interactions with mobile phase

Mobile Phase Optimization

• Effect of Sample Polarity on Separation
• pH Control and Buffer Selection
• Impact of pH on ionizable compounds
• Choosing the right buffer for pH stability
• Optimizing Flow Rates for Resolution and Throughput

Column Selection and Optimization

Physical dimensions

• Column length, internal diameter, and 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 and Method Optimization

Isocratic vs. Gradient Elution

• When to use each approach

Optimizing Retention Factor (k’)

• Controlling k’ for improved separation and peak shape

Effect of Gradient Parameters on Separation

• Impact of gradient slope, dwell volume, and mobile phase composition

Building a Practical HPLC Method

Comprehensive Sample Preparation Techniques

Optimizing Retention and Selectivity (α)

• Screening different column chemistries and mobile phase conditions

Chromatographic Optimization Strategies

• Adjusting parameters for better resolution and peak symmetry

Assessing Linearity, Range, and Sensitivity

Pre-Validation Considerations

Evaluating system suitability, robustness, and repeatability

Q&A and Closing Discussion

• Summary of Key Concepts
• Open Discussion on Challenges Faced in HPLC Method Development
• Resources for Further Learning