Lesson 1System configuration and plumbing: degassing, autosampler, column switching and dwell volume impactWe review key system parts and plumbing dat affect method performance, including degassing, autosampler design, tubing sizes, and column switching. We stress dwell volume and extra-column dispersion control.
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 silicaWe detail how to choose reversed-phase stationary phases, focusing on C18 types, pore and particle size, endcapping, and hybrid vs pure silica. We stress matching phase chemistry to analyte properties and method goals.
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 compatibilityWe address real-world constraints in pharmaceutical labs, including sample throughput, robustness, solvent compatibility, and lifecycle management. We link regulatory expectations to practical method and instrument choices.
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 mechanismsWe introduce core principles of reversed-phase HPLC, including hydrophobic interactions, partitioning, and de role of mobile phase composition. We connect retention mechanisms to practical choices in method development.
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-offsWe cover UV detector selection and wavelength optimization, including fixed-wavelength, variable-wavelength, and diode-array detectors. We explain 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 considerationsWe compare isocratic and gradient elution, explaining when each is appropriate. We cover gradient profile design, slope and run time, dwell volume effects, and practical strategies for robust gradient transfer between 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/basesWe explain how mobile phase pH relative to analyte pKa controls ionization, retention, and peak symmetry for weak acids and bases, with guidance on choosing pH to improve resolution, robustness, and column lifetime.
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 preparationWe focus on mobile phase buffer selection and preparation, covering phosphate, acetate, and volatile ammonium buffers. We discuss 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 selectivityWe explain how methanol and acetonitrile differ in solvent strength, viscosity, and selectivity in reversed-phase HPLC. We discuss mixed organic systems, temperature interactions, and practical considerations such as 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 shapeWe describe how flow rate, column temperature, and injection volume influence efficiency, backpressure, retention, and peak shape. We provide rules for scaling flow, avoiding overload, and optimizing temperature for robustness.
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 µmWe describe how column length, internal diameter, and particle size affect efficiency, backpressure, sensitivity, and analysis time. We provide guidance on choosing 3–5 µm vs 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
