HPLC Course (High Performance Liquid Chromatography)

Lesson 1 • Modes of Liquid Chromatography

Surveys reversed-phase, normal-phase, ion-exchange, and size-exclusion modes. Students match analyte properties to the appropriate separation mode.

Lesson 2 • Core Chromatographic Concepts

Defines retention, selectivity, resolution, and efficiency using the van Deemter equation. These terms underpin every analytical decision made in later chapters.

Lesson 3 • History and Scope of HPLC

Traces HPLC's evolution from classical liquid chromatography to modern instrumentation. Establishes why HPLC is the dominant analytical separation technique.

Lesson 1 • Sample Introduction and Autosamplers

Describes manual injection valves and automated autosampler operation, including loop volume and injection precision. Injection technique directly affects peak shape and reproducibility.

Lesson 2 • Solvent Delivery Systems

Covers reservoir design, degassing methods, and pump types including reciprocating and syringe pumps. Proper solvent delivery is prerequisite to reproducible separations.

Lesson 3 • Detectors in HPLC

Surveys UV-Vis, photodiode array, fluorescence, refractive index, and electrochemical detectors. Detector choice is matched to analyte properties and sensitivity requirements.

Lesson 4 • Data Systems and Chromatography Software

Introduces chromatography data systems for instrument control, data acquisition, and reporting. Accurate data handling is essential for regulatory compliance and result integrity.

Lesson 1 • Solvent Selection Principles

Applies polarity indices and solvent strength to choose primary organic modifiers. Solvent selection is the first lever for controlling retention and selectivity.

Lesson 2 • Isocratic Method Design

Guides students through systematic isocratic method development using solvent strength and selectivity adjustments. Isocratic methods are simpler to transfer and validate.

Lesson 3 • Buffer Selection and pH Control

Covers buffer types, concentration, and pH effects on ionizable analyte retention. Proper buffering prevents peak tailing and ensures day-to-day reproducibility.

Lesson 4 • Additives and Ion-Pair Reagents

Introduces ion-pair reagents, ion suppressors, and organic additives for challenging analytes. These tools extend reversed-phase HPLC to highly polar or ionic compounds.

Lesson 1 • Column Characterization and Selection Tools

Uses hydrophobic subtraction model and column databases to compare and select columns objectively. Systematic selection reduces trial-and-error in method development.

Lesson 2 • Specialty and Chiral Stationary Phases

Introduces HILIC, mixed-mode, and chiral phases for polar, ionic, and enantiomeric separations. These phases address analytes that reversed-phase cannot adequately retain.

Lesson 3 • Reversed-Phase Bonded Phases

Compares C18, C8, C4, phenyl, and polar-embedded phases for selectivity differences. Phase selection is the primary tool for resolving co-eluting analytes.

Lesson 4 • Silica-Based Stationary Phases

Examines bare silica and bonded silica phases, including surface chemistry and silanization. Silica remains the dominant support material in modern HPLC columns.

Lesson 1 • Systematic Scouting Strategies

Applies column and mobile phase scouting screens to rapidly identify promising starting conditions. Efficient scouting reduces development time without sacrificing thoroughness.

Lesson 2 • Sample Preparation Integration

Aligns sample preparation strategy with HPLC method requirements to protect the column and improve sensitivity. Poor sample preparation is a leading cause of method failure.

Lesson 3 • Method Documentation and Transfer

Produces complete method documentation including system suitability criteria and transfer protocols. Thorough documentation enables reproducible execution across laboratories.

Lesson 4 • Defining Method Requirements

Establishes analytical target profiles, analyte physicochemical properties, and regulatory context before instrument work begins. Clear objectives prevent costly rework later.

Lesson 1 • Statistical Evaluation of Results

Applies outlier tests, confidence intervals, and uncertainty estimation to HPLC quantitative data. Statistical rigor ensures results are defensible in regulatory and scientific contexts.

Lesson 2 • Peak Integration and Quantitation

Applies correct integration parameters to accurately measure peak area and height for quantitation. Integration errors are a primary source of quantitative inaccuracy in HPLC.

Lesson 3 • Chromatogram Troubleshooting by Data Patterns

Diagnoses common data anomalies—ghost peaks, baseline drift, and retention time shifts—from chromatogram patterns. Pattern recognition accelerates root cause identification without instrument disassembly.

Lesson 4 • Calibration Strategies

Compares external standard, internal standard, and standard addition calibration for different matrices. Calibration strategy selection directly affects accuracy and matrix bias correction.

Lesson 1 • Limits of Detection and Quantitation

Calculates LOD and LOQ using signal-to-noise and regression approaches and confirms them experimentally. These limits define the lowest reliable measurement range of the method.

Lesson 2 • Specificity, Linearity, and Range

Demonstrates specificity through forced degradation and evaluates linearity across the analytical range. These parameters confirm the method measures only the intended analyte accurately.

Lesson 3 • Validation Framework and Regulatory Context

Introduces validation parameters defined by international harmonized guidelines for analytical procedures. Understanding the framework prevents gaps that cause regulatory findings.

Lesson 4 • Robustness Testing and System Suitability

Uses Plackett-Burman designs to evaluate method robustness and sets system suitability limits from validation data. Robustness testing identifies critical method parameters before routine use.

Lesson 1 • Column Performance Degradation

Identifies causes of efficiency loss, peak tailing, and column void formation and applies corrective actions. Column health directly determines separation quality and method validity.

Lesson 2 • Systematic Troubleshooting Methodology

Introduces a structured fault-isolation approach using symptom-cause-remedy logic trees. A systematic method prevents misdiagnosis and unnecessary component replacement.

Lesson 3 • Pump and Pressure System Issues

Diagnoses high pressure, low pressure, and pressure fluctuation faults in the solvent delivery system. Pressure behavior is the most informative single indicator of system health.

Lesson 4 • Preventive Maintenance Programs

Designs scheduled maintenance intervals for pumps, seals, lamps, and autosamplers to prevent unplanned downtime. Preventive maintenance is more cost-effective than reactive repair.