Lesson 1System configuration and plumbing: degassing, autosampler, column switching and dwell volume impactThis lesson reviews essential system parts and connections that influence method performance, covering degassing, autosampler design, tubing sizes, and column switching. It stresses controlling dwell volume and extra-column spreading.
Ways to degas and prevent bubblesAutosampler design and preventing carryoverTubing inner diameter, length, and spreading effectsColumn switching valves and arrangementsMeasuring and adjusting dwell volumeLesson 2Selecting stationary phase: C18 chemistries, pore size, particle size, endcapping, hybrid vs silicaThis lesson details selecting reversed-phase stationary phases, emphasising C18 types, pore and particle sizes, endcapping, and hybrid versus pure silica. It highlights matching phase chemistry to analyte traits and method aims.
C18 bonding density and ligand typesEndcapped versus non-endcapped phasesPore size for small molecules versus peptidesHybrid silica versus traditional silica phasesChoosing particle size for performance requirementsLesson 3Practical constraints for pharmaceutical labs: sample throughput, robustness, and solvent compatibilityThis lesson tackles real-life challenges in pharmaceutical labs, such as sample processing speed, method durability, solvent compatibility, and lifecycle handling. It connects regulatory standards to practical method and instrument decisions.
Balancing run time and separation qualityMethod durability and toughness studiesSolvent compatibility with analytes and sealsReducing solvent use and waste managementRegulatory standards for everyday methodsLesson 4Principles of reversed-phase HPLC and retention mechanismsThis lesson introduces key principles of reversed-phase HPLC, including hydrophobic interactions, partitioning, and mobile phase composition's role. It links retention mechanisms to real-world choices in method development.
Hydrophobic interactions and partitioningOrganic modifier's role in retentionAnalyte polarity and logP effectsTemperature's influence on retentionIonizable analytes in reversed-phase HPLCLesson 5Detector selection and wavelength optimization for UV detection: spectra scanning, diode-array use, sensitivity trade-offsThis lesson covers choosing UV detectors and optimising wavelengths, including fixed, variable, and diode-array types. It explains scanning spectra, checking peak purity, and balancing sensitivity with selectivity and noise.
Fixed versus variable versus diode-array detectorsSelecting λmax from UV spectraBandpass, noise, and sensitivity trade-offsPeak purity assessment with DAD spectraLinear range and detector saturation limitsLesson 6Gradient vs isocratic choices: when to use each, gradient slope, dwell volume considerationsThis lesson compares isocratic and gradient elution, explaining suitable uses. It covers gradient profile design, slope and run time, dwell volume impacts, and strategies for reliable gradient transfer between HPLC systems.
Choosing isocratic versus gradient elutionDesigning initial and final mobile phase strengthGradient slope, run time, and resolutionSystem dwell volume and gradient delayTransferring gradients between instrumentsLesson 7pH selection: pKa relationships, effect on retention and peak shape for weak acids/basesThis lesson explains how mobile phase pH compared to analyte pKa manages ionisation, retention, and peak symmetry for weak acids and bases, with tips on selecting pH to enhance resolution, durability, and column life.
Ionisation of weak acids and bases versus pHUsing Henderson–Hasselbalch for pH selectionpH impact on retention and selectivitypH influence on peak tailing and frontingBuffer pH limits for silica column stabilityLesson 8Mobile phase formulation: buffers (phosphate, acetate, ammonium), ionic strength, and buffer preparationThis lesson focuses on selecting and preparing mobile phase buffers, including phosphate, acetate, and volatile ammonium types. It discusses ionic strength, pH control, solubility, filtration, and compatibility with detectors and columns.
Choosing buffer types and pH rangeBuffer capacity and ionic strength effectsPreparing, filtering, and degassing buffersBuffer solubility with high organic contentVolatile buffers for MS compatibilityLesson 9Organic modifiers: methanol vs acetonitrile effects, solvent strength and selectivityThis lesson explains differences between methanol and acetonitrile in solvent strength, viscosity, and selectivity in reversed-phase HPLC. It covers mixed organic systems, temperature interactions, and practical factors like cost and safety.
Solvent strength in common RP eluotropic scalesViscosity, backpressure, and temperature effectsSelectivity differences MeOH vs ACNUsing mixed organic modifiers for tuningSafety, cost, and supply considerationsLesson 10Flow rate, temperature, and injection volume: effects on efficiency, backpressure, and peak shapeThis lesson describes how flow rate, column temperature, and injection volume affect efficiency, backpressure, retention, and peak shape. It offers guidelines for scaling flow, avoiding overload, and optimising temperature for durability.
Van Deemter and optimal flow selectionTemperature effects on retention and kineticsInjection volume and column overloadSolvent mismatch and peak distortionScaling flow with column ID and lengthLesson 11Column dimensions and particle size trade-offs: length, ID, 3–5 µm vs sub-2 µmThis lesson describes how column length, internal diameter, and particle size impact efficiency, backpressure, sensitivity, and analysis time. It provides advice on selecting 3–5 µm versus sub-2 µm columns and scaling dimensions between systems.
Effect of column length on resolution and timeInternal diameter and sensitivity considerations3–5 µm vs sub-2 µm efficiency and pressureScaling methods between column dimensionsGuard columns and frit design impacts
