Lesson 1System configuration and plumbing: degassing, autosampler, column switching and dwell volume impactExamines vital system parts and connections that influence method results, covering degassing, autosampler features, tube sizes, and column changes. Highlights managing dwell volume and extra dispersion outside the column.
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 silicaOutlines selecting reversed-phase stationary phases, emphasizing C18 types, pore and particle dimensions, endcapping, and hybrid versus pure silica. Stresses 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 compatibilityTackles everyday challenges in pharmaceutical settings, such as sample processing speed, method durability, solvent matching, and lifecycle handling. Connects rules to real method and equipment decisions.
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 mechanismsPresents fundamental ideas of reversed-phase HPLC, including hydrophobic bonds, partitioning, and mobile phase role. Relates retention processes to hands-on method creation 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-offsDiscusses UV detector choices and wavelength tuning, including fixed, variable, and diode-array types. Covers spectrum scanning, peak purity evaluation, and trading sensitivity for selectivity and low 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 considerationsContrasts isocratic and gradient elution, noting suitable uses. Addresses gradient shape design, slope and duration, dwell volume impacts, and tips for reliable gradient shifts across HPLC setups.
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 compared to analyte pKa manages ionization, retention, and peak form for weak acids and bases, with advice on pH picks to boost resolution, durability, 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 preparationCenters on choosing and making mobile phase buffers like phosphate, acetate, and ammonium types. Covers ionic strength, pH management, solubility, filtering, and fit 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 selectivityClarifies differences in methanol and acetonitrile regarding strength, viscosity, and selectivity in reversed-phase HPLC. Examines mixed organics, temperature links, and 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 shapeExplains influences of flow rate, column heat, and injection amount on efficiency, pressure, retention, and peak form. Offers guidelines for scaling flow, preventing overload, and tuning heat 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 µmOutlines how column length, inner diameter, and particle size impact efficiency, pressure, sensitivity, and time. Guides on picking 3–5 µm versus sub-2 µm columns and adjusting sizes 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
