Lesson 1System configuration and plumbing: degassing, autosampler, column switching and dwell volume impactExamines main system parts and plumbing that influence method performance, such as degassing, autosampler design, tubing sizes, and column switching. Stresses control of dwell volume and extra-column dispersion.
Degassing methods and bubble preventionAutosampler design and carryover controlTubing ID, length, and dispersion effectsColumn switching valves and setupsMeasuring and adjusting dwell volumeLesson 2Selecting stationary phase: C18 chemistries, pore size, particle size, endcapping, hybrid vs silicaExplains selection of reversed-phase stationary phases, emphasising C18 types, pore and particle sizes, endcapping, and hybrid versus pure silica. Focuses on aligning phase chemistry with analyte traits and method aims.
C18 bonding density and ligand typeEndcapped vs non-endcapped phasesPore size for small molecules vs peptidesHybrid silica vs traditional silica phasesChoosing particle size for performance needsLesson 3Practical constraints for pharmaceutical labs: sample throughput, robustness, and solvent compatibilityDiscusses real-world limits in pharmaceutical labs, covering sample throughput, reliability, solvent compatibility, and method lifecycle. Connects regulatory needs to practical method and instrument decisions.
Balancing run time and resolutionMethod robustness and ruggedness studiesSolvent compatibility with analytes and sealsMinimising solvent use and waste disposalRegulatory expectations for routine methodsLesson 4Principles of reversed-phase HPLC and retention mechanismsIntroduces fundamental principles of reversed-phase HPLC, including hydrophobic interactions, partitioning, and mobile phase role. Relates retention mechanisms to practical method development choices.
Hydrophobic interactions and partitioningRole of organic modifier in retentionEffect of analyte polarity and logPInfluence of temperature on retentionIonisable analytes in reversed-phase HPLCLesson 5Detector selection and wavelength optimisation for UV detection: spectra scanning, diode-array use, sensitivity trade-offsDiscusses UV detector choices and wavelength optimisation, covering fixed-wavelength, variable-wavelength, and diode-array detectors. Covers spectra scanning, peak purity checks, and balancing sensitivity with selectivity and noise.
Fixed vs variable vs 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 considerationsCompares isocratic and gradient elution, explaining suitable scenarios. Addresses gradient profile design, slope and run time, dwell volume impacts, and strategies for reliable gradient transfer across HPLC systems.
When to choose isocratic vs 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/basesDescribes how mobile phase pH relative to analyte pKa affects ionisation, retention, and peak symmetry for weak acids and bases, with tips on selecting pH to enhance resolution, reliability, and column life.
Ionisation of weak acids and bases vs 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 preparationConcentrates on mobile phase buffer choices and preparation, including phosphate, acetate, and volatile ammonium buffers. Covers ionic strength, pH control, solubility, filtration, and compatibility with detectors and columns.
Choosing buffer species 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 selectivityExplains differences between methanol and acetonitrile in solvent strength, viscosity, and selectivity in reversed-phase HPLC. Discusses mixed organic systems, temperature effects, 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 shapeOutlines how flow rate, column temperature, and injection volume impact efficiency, backpressure, retention, and peak shape. Offers guidelines for scaling flow, preventing overload, and optimising temperature for reliability.
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 µmDescribes impacts of column length, internal diameter, and particle size on efficiency, backpressure, sensitivity, and analysis time. Guides selection of 3–5 µm versus sub-2 µm columns and scaling dimensions across 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
