Lesson 1System configuration and plumbing: degassing, autosampler, column switching and dwell volume impactReviews key system components an plumbing dat affect method performance, including degassing, autosampler design, tubing dimensions, an column switching. Emphasizes dwell volume an extra-column dispersion control.
Degassing methods an bubble preventionAutosampler design an carryover controlTubing ID, length, an dispersion effectsColumn switching valves an setupsMeasuring an adjusting dwell volumeLesson 2Selecting stationary phase: C18 chemistries, pore size, particle size, endcapping, hybrid vs silicaDetails how fi choose reversed-phase stationary phases, focusing pon C18 variants, pore an particle size, endcapping, an hybrid vs pure silica. Emphasizes matching phase chemistry to analyte properties an method goals.
C18 bonding density an ligand typeEndcapped vs non-endcapped phasesPore size fi small molecules vs peptidesHybrid silica vs traditional silica phasesChoosing particle size fi performance needsLesson 3Practical constraints for pharmaceutical labs: sample throughput, robustness, and solvent compatibilityAddresses real-world constraints in pharmaceutical labs, including sample throughput, robustness, solvent compatibility, an lifecycle management. Links regulatory expectations to practical method an instrument choices.
Balancing run time an resolutionMethod robustness an ruggedness studiesSolvent compatibility wid analytes an sealsMinimizing solvent use an waste disposalRegulatory expectations fi routine methodsLesson 4Principles of reversed-phase HPLC and retention mechanismsIntroduces core principles of reversed-phase HPLC, including hydrophobic interactions, partitioning, an di role of mobile phase composition. Connects retention mechanisms to practical choices in method development.
Hydrophobic interactions an partitioningRole of organic modifier in retentionEffect of analyte polarity an logPInfluence of temperature pon retentionIonizable analytes in reversed-phase HPLCLesson 5Detector selection and wavelength optimization for UV detection: spectra scanning, diode-array use, sensitivity trade-offsCovers UV detector selection an wavelength optimization, including fixed-wavelength, variable-wavelength, an diode-array detectors. Explains spectra scanning, peak purity checks, an balancing sensitivity wid selectivity an noise.
Fixed vs variable vs diode-array detectorsSelecting λmax from UV spectraBandpass, noise, an sensitivity trade-offsPeak purity assessment wid DAD spectraLinear range an detector saturation limitsLesson 6Gradient vs isocratic choices: when to use each, gradient slope, dwell volume considerationsCompares isocratic an gradient elution, explaining when each is appropriate. Covers gradient profile design, slope an run time, dwell volume effects, an practical strategies fi robust gradient transfer between HPLC systems.
When fi choose isocratic vs gradient elutionDesigning initial an final mobile phase strengthGradient slope, run time, an resolutionSystem dwell volume an gradient delayTransferring gradients between instrumentsLesson 7pH selection: pKa relationships, effect on retention and peak shape for weak acids/basesExplains how mobile phase pH relative to analyte pKa controls ionization, retention, an peak symmetry fi weak acids an bases, wid guidance pon choosing pH fi improve resolution, robustness, an column lifetime.
Ionization of weak acids an bases vs pHUsing Henderson–Hasselbalch fi pH selectionpH impact pon retention an selectivitypH influence pon peak tailing an frontingBuffer pH limits fi silica column stabilityLesson 8Mobile phase formulation: buffers (phosphate, acetate, ammonium), ionic strength, and buffer preparationFocuses pon mobile phase buffer selection an preparation, covering phosphate, acetate, an volatile ammonium buffers. Discusses ionic strength, pH control, solubility, filtration, an compatibility wid detectors an columns.
Choosing buffer species an pH rangeBuffer capacity an ionic strength effectsPreparing, filtering, an degassing buffersBuffer solubility wid high organic contentVolatile buffers fi MS compatibilityLesson 9Organic modifiers: methanol vs acetonitrile effects, solvent strength and selectivityExplains how methanol an acetonitrile differ in solvent strength, viscosity, an selectivity in reversed-phase HPLC. Discusses mixed organic systems, temperature interactions, an practical considerations such as cost an safety.
Solvent strength in common RP eluotropic scalesViscosity, backpressure, an temperature effectsSelectivity differences MeOH vs ACNUsing mixed organic modifiers fi tuningSafety, cost, an supply considerationsLesson 10Flow rate, temperature, and injection volume: effects on efficiency, backpressure, and peak shapeDescribes how flow rate, column temperature, an injection volume influence efficiency, backpressure, retention, an peak shape. Provides rules fi scaling flow, avoiding overload, an optimizing temperature fi robustness.
Van Deemter an optimal flow selectionTemperature effects pon retention an kineticsInjection volume an column overloadSolvent mismatch an peak distortionScaling flow wid column ID an lengthLesson 11Column dimensions and particle size trade-offs: length, ID, 3–5 µm vs sub-2 µmDescribes how column length, internal diameter, an particle size affect efficiency, backpressure, sensitivity, an analysis time. Provides guidance pon choosing 3–5 µm vs sub‑2 µm columns an scaling dimensions between systems.
Effect of column length pon resolution an timeInternal diameter an sensitivity considerations3–5 µm vs sub‑2 µm efficiency an pressureScaling methods between column dimensionsGuard columns an frit design impacts
