INTRODUCTION TO HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

COURSE OUTLINE

Introduction to HPLC and Its Applications

• Overview of HPLC and its importance in analytical chemistry
• Target industries: Pharmaceuticals, food and beverage, environmental analysis, biotechnology, forensics, etc.
• Comparison with other chromatography techniques: GC, TLC, and other liquid chromatography methods
• Types of samples suitable for HPLC analysis

Sample Preparation for HPLC Analysis

• Importance of proper sample preparation
• Common HPLC sample preparation methods (e.g., filtration, dilution, derivatization, solid-phase extraction)
• Dos and Don’ts of HPLC sample preparation (e.g., avoiding contamination, proper solvent selection, solvent: sample ratio’s, sample stability considerations)

Overview of HPLC Instrumentation and Components

• Mobile phase and degassing: Role of solvents, types of degassers, and their impact on analysis
• Pumps: Types (isocratic vs. gradient), function, and performance considerations
• Injectors: Manual vs. autosamplers, sample introduction techniques
• Columns and Column Oven: Types of stationary phases, column selection, temperature control, and impact on separation
• Detectors: UV-Vis, fluorescence, refractive index, mass spectrometry, and their applications

Understanding the Chromatographic Process

Overview of the 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

Introduction to Chromatographic Performance Parameters

• Capacity Factor (k’): Measuring analyte retention time
• Selectivity (α): Differentiating structurally similar compounds
• Efficiency (N): Peak sharpness, theoretical plates, and column efficiency
• Resolution (Rs): Optimizing separation quality and peak differentiation

HPLC Mobile Phases and Additives

Mobile Phase Preparation and Optimization

Selecting solvents for optimal separation and reproducibility

Degassing techniques to prevent bubble formation and detector noise

Ensuring mobile phase purity, stability, and consistency

Effects of Organic Modifiers

Considerations for methanol, acetonitrile, and tetrahydrofuran (THF)

Impact on retention time, peak shape, and resolution

pH Control, Buffering, and Additives

Importance of pH in analyte ionization and retention

Choosing appropriate buffers and ionic strength adjustments

Role of ion-pairing agents in enhancing separation

HPLC Columns: Selection and Optimization

Understanding Column Dimensions

• Column length, internal diameter, and their impact on separation
• Pore size and particle size considerations (HPLC vs. UHPLC)

Silica-Based Supports and Stationary Phases

• Characteristics of silica-based materials in chromatography
• Porous vs. non-porous silica and their applications

Bonded Phases and Surface Chemistry

• C18, C8, phenyl, cyano, and other common stationary phases
• Hydrophobicity, end-capping, and column stability

The van Deemter Equation and Column Efficiency

• Understanding plate height (H) and optimizing flow rates
• Impact of particle size, diffusion, and mass transfer

Common Reversed-Phase Column Chemistries

Comparison of different reversed-phase chemistries

Selecting the right column for specific applications

Introduction to Alternative HPLC Separation Modes

Reversed-Phase HPLC (RP-HPLC)

Fundamentals of hydrophobic interactions and elution order

Ion Suppression and Ion-Pair Chromatography in RP-HPLC

Role of ion-pairing agents in enhancing separation of ionic compounds

Normal-Phase Chromatography (NP-HPLC)

Separation principles based on polarity differences

Common stationary and mobile phases used in NP-HPLC

Hydrophilic Interaction Liquid Chromatography (HILIC)

• Mechanism of retention for polar analytes
• Mobile phase considerations and common applications

Size-Exclusion Chromatography (SEC)

• Separation based on molecular size and hydrodynamic volume
• SEC applications in protein and polymer Analysis

Ion-Exchange Chromatography (IEX-HPLC)

• Separation principles based on charge interactions
• Cation-exchange vs. anion-exchange chromatography

Data Processing and Analysis in HPLC

Peak Integration and Quantification

• Principles of peak detection and integration
• Baseline correction and handling overlapping peaks
• Manual vs. automated integration

Standards and Calibration

• ypes of calibration curves (external, internal, and standard addition)
• Preparation of standards and validation of calibration curves
• Linearity, accuracy, and precision in quantification

System Suitability Testing (SST)

• Importance of system suitability in HPLC analysis
• Key parameters: resolution, retention time, tailing factor, theoretical plates, repeatability
• Interpretation of system suitability results

Quality Control (QC) and Data Validation

• Role of QC samples in ensuring accuracy and reproducibility
• Types of QC samples: blanks, duplicates, spiked samples, and reference standards
• Criteria for accepting or rejecting analytical runs