Lesson 1System configuration and plumbing: degassing, autosampler, column switching and dwell volume impactThis lesson reviews main system parts and piping that affect method work, including removing air bubbles, autosampler setup, pipe sizes, and column changing. It stresses controlling dwell volume and extra-column spreading.
Ways to remove air and stop bubblesAutosampler design and preventing carryoverPipe inner diameter, length, and spreading effectsColumn switching valves and arrangementsMeasuring and fixing dwell volumeLesson 2Selecting stationary phase: C18 chemistries, pore size, particle size, endcapping, hybrid vs silicaThis lesson details picking reversed-phase stationary phases, focusing on C18 types, pore and particle sizes, endcapping, and hybrid versus pure silica. It highlights matching phase chemistry to sample properties and method aims.
C18 bonding density and ligand typeEndcapped versus non-endcapped phasesPore size for small molecules versus peptidesHybrid silica versus traditional silica phasesChoosing particle size for performance needsLesson 3Practical constraints for pharmaceutical labs: sample throughput, robustness, and solvent compatibilityThis lesson tackles real-life limits in medicine labs, including sample processing speed, sturdiness, solvent matching, and method lifespan. It connects rules to practical method and instrument choices in Ghana.
Balancing run time and separation clarityMethod sturdiness and toughness studiesSolvent matching with samples and sealsReducing solvent use and waste handlingRule expectations for daily methodsLesson 4Principles of reversed-phase HPLC and retention mechanismsThis lesson introduces main ideas of reversed-phase HPLC, including water-hating interactions, sharing, and mobile phase role. It links retention ways to practical choices in method building.
Water-hating interactions and sharingRole of organic changer in retentionEffect of sample polarity and logPInfluence of temperature on retentionCharged samples in reversed-phase HPLCLesson 5Detector selection and wavelength optimization for UV detection: spectra scanning, diode-array use, sensitivity trade-offsThis lesson covers UV detector picking and wavelength fine-tuning, including fixed, variable, and diode-array types. It explains scanning spectra, checking peak purity, and balancing sensitivity with choice and noise.
Fixed versus variable versus diode-array detectorsPicking λmax from UV spectraBandpass, noise, and sensitivity balancesPeak purity check with DAD spectraLinear range and detector saturation limitsLesson 6Gradient vs isocratic choices: when to use each, gradient slope, dwell volume considerationsThis lesson compares steady and changing elution, explaining when each fits. It covers gradient shape design, slope and run time, dwell volume effects, and tips for strong gradient moving between HPLC systems.
When to pick steady versus changing elutionDesigning start and end mobile phase strengthGradient slope, run time, and separationSystem dwell volume and gradient delayMoving gradients between instrumentsLesson 7pH selection: pKa relationships, effect on retention and peak shape for weak acids/basesThis lesson explains how mobile phase pH compared to sample pKa controls charging, retention, and peak evenness for weak acids and bases, with advice on picking pH to boost separation, sturdiness, and column life.
Charging of weak acids and bases versus pHUsing Henderson–Hasselbalch for pH pickingpH effect on retention and choicepH influence on peak dragging and pushingBuffer pH limits for silica column steadinessLesson 8Mobile phase formulation: buffers (phosphate, acetate, ammonium), ionic strength, and buffer preparationThis lesson focuses on mobile phase buffer picking and making, covering phosphate, acetate, and airy ammonium buffers. It discusses ionic strength, pH control, dissolving, filtering, and matching with detectors and columns.
Picking buffer types and pH rangeBuffer capacity and ionic strength effectsPreparing, filtering, and airing buffersBuffer dissolving with high organic contentAiry buffers for MS matchingLesson 9Organic modifiers: methanol vs acetonitrile effects, solvent strength and selectivityThis lesson explains how methanol and acetonitrile differ in solvent power, thickness, and choice in reversed-phase HPLC. It discusses mixed organic setups, temperature links, and practical points like cost and safety.
Solvent power in common RP eluotropic scalesThickness, backpressure, and temperature effectsChoice differences MeOH versus ACNUsing mixed organic changers for tuningSafety, cost, and supply pointsLesson 10Flow rate, temperature, and injection volume: effects on efficiency, backpressure, and peak shapeThis lesson describes how flow rate, column temperature, and injection amount affect efficiency, backpressure, retention, and peak shape. It gives rules for scaling flow, avoiding overload, and tuning temperature for sturdiness.
Van Deemter and best flow pickingTemperature effects on retention and speedInjection amount and column overloadSolvent mismatch and peak twistingScaling flow with column ID and lengthLesson 11Column dimensions and particle size trade-offs: length, ID, 3–5 µm vs sub-2 µmThis lesson describes how column length, inner diameter, and particle size affect efficiency, backpressure, sensitivity, and analysis time. It provides guidance on choosing 3–5 µm versus sub-2 µm columns and scaling sizes between systems.
Effect of column length on separation and timeInner diameter and sensitivity points3–5 µm versus sub-2 µm efficiency and pressureScaling methods between column sizesGuard columns and frit design effects
