Lesson 1System configuration and plumbing: degassing, autosampler, column switching and dwell volume impactLooks at key system parts and plumbing affecting method performance, like degassing, autosampler setup, tubing sizes, and column switching. Stresses controlling 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 choosing reversed-phase stationary phases, zeroing in on C18 types, pore and particle size, endcapping, and hybrid versus pure silica. Focuses on matching phase chemistry to 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 compatibilityTackles everyday challenges in pharma labs, covering sample throughput, sturdiness, solvent compatibility, and method lifecycle. Ties regulatory needs to smart method and instrument picks.
Balancing run time and resolutionMethod robustness and ruggedness studiesSolvent compatibility with analytes and sealsMinimizing solvent use and waste disposalRegulatory expectations for routine methodsLesson 4Principles of reversed-phase HPLC and retention mechanismsIntroduces main principles of reversed-phase HPLC, like hydrophobic interactions, partitioning, and mobile phase role. Links retention to practical method development choices.
Hydrophobic interactions and partitioningRole of organic modifier in retentionEffect of analyte polarity and logPInfluence of temperature on retentionIonizable analytes in reversed-phase HPLCLesson 5Detector selection and wavelength optimization for UV detection: spectra scanning, diode-array use, sensitivity trade-offsCovers picking UV detectors and optimising wavelengths, including fixed, variable, and diode-array types. Explains 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, when to use each. Covers gradient design, slope, run time, dwell volume effects, and tips for robust 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/basesShows how mobile phase pH versus analyte pKa controls ionisation, retention, and peak shape for weak acids and bases. Guides picking pH for better resolution, sturdiness, and column life.
Ionization 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 preparationFocuses on picking and preparing mobile phase buffers like phosphate, acetate, and ammonium types. Covers ionic strength, pH control, solubility, filtration, and detector/column compatibility.
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 methanol versus acetonitrile differences in strength, viscosity, and selectivity in reversed-phase HPLC. Discusses mixed systems, temperature effects, and practical issues 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 shapeDescribes how flow rate, column temperature, and injection volume affect efficiency, backpressure, retention, and peak shape. Gives rules for scaling flow, avoiding overload, and optimising temperature for sturdiness.
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 µmExplains how column length, internal diameter, and particle size impact efficiency, backpressure, sensitivity, and analysis time. Guides choosing 3–5 µm versus sub-2 µm columns and scaling dimensions.
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
