Lesson 1System setup and pipes: removing air, auto sampler, column changing and dwell volume effectsLooks at key machine parts and pipes that change how well the method works, like removing air, auto sampler build, pipe sizes, and column changing. Stresses dwell volume and extra pipe spread control.
Ways to remove air and stop bubblesAuto sampler build and carryover stopPipe inside size, length, and spread effectsColumn changing valves and setupsMeasuring and fixing dwell volumeLesson 2Picking fixed phase: C18 types, hole size, particle size, end covering, mixed vs silicaExplains how to choose backward-phase fixed phases, looking at C18 kinds, hole and particle size, end covering, and mixed vs pure silica. Stresses matching phase chemistry to what is being tested and method aims.
C18 bond strength and link typeEnd covered vs not covered phasesHole size for small bits vs chain bitsMixed silica vs old silica phasesPicking particle size for work needsLesson 3Real limits for medicine labs: sample speed, strength, and liquid matchDeals with real limits in medicine labs, like sample speed, strength, liquid match, and life management. Connects rule needs to useful method and machine picks.
Balancing run time and clear splitMethod strength and tough testsLiquid match with tests and sealsLess liquid use and waste throwRule needs for daily methodsLesson 4Rules of backward-phase HPLC and stay mechanismsStarts core rules of backward-phase HPLC, like water-hating links, sharing, and moving liquid mix role. Links stay mechanisms to useful picks in method building.
Water-hating links and sharingRole of mix changer in stayEffect of test wetness and logPWarmth effect on stayChargeable tests in backward-phase HPLCLesson 5Picker for detector and wave tune for UV see: wave scan, diode-array use, feel trade-offsCovers UV detector pick and wave tune, like fixed-wave, change-wave, and diode-array detectors. Explains wave scan, peak clean checks, and balancing feel with pick and noise.
Fixed vs change vs diode-array detectorsPicking top wave from UV wavesBand pass, noise, and feel trade-offsPeak clean check with DAD wavesStraight range and detector full limitsLesson 6Slope vs straight choices: when to use each, slope rise, dwell volume thinksCompares straight and slope push, explaining when each fits. Covers slope shape build, rise and run time, dwell volume effects, and useful ways for strong slope move between HPLC machines.
When to pick straight vs slope pushBuilding start and end moving liquid strengthSlope rise, run time, and clear splitMachine dwell volume and slope waitMoving slopes between machinesLesson 7pH pick: pKa links, effect on stay and peak shape for weak acids/basesExplains how moving liquid pH next to test pKa controls charge, stay, and peak even for weak acids and bases, with help on picking pH to better split, strength, and column life.
Charge of weak acids and bases vs pHUsing Henderson–Hasselbalch for pH pickpH hit on stay and pickpH pull on peak drag and frontBuffer pH limits for silica column steadyLesson 8Moving liquid mix: buffers (phosphate, acetate, ammonium), charge strength, and buffer readyLooks at moving liquid buffer pick and ready, covering phosphate, acetate, and air ammonium buffers. Talks charge strength, pH hold, solve, filter, and match with detectors and columns.
Picking buffer kind and pH rangeBuffer power and charge strength effectsReady, filter, and air remove buffersBuffer solve with high mix contentAir buffers for MS matchLesson 9Mix changers: methanol vs acetonitrile effects, liquid strength and pickExplains how methanol and acetonitrile differ in liquid strength, thick flow, and pick in backward-phase HPLC. Talks mixed mix systems, warmth links, and useful thinks like cost and safe.
Liquid strength in common back push scalesThick flow, back push, and warmth effectsPick differ MeOH vs ACNUsing mixed mix changers for tuneSafe, cost, and supply thinksLesson 10Flow speed, warmth, and put volume: effects on work, back push, and peak shapeTells how flow speed, column warmth, and put volume change work, back push, stay, and peak shape. Gives rules for scale flow, stop overload, and best warmth for strength.
Van Deemter and best flow pickWarmth effects on stay and speedPut volume and column overloadLiquid not match and peak twistScale flow with column inside and lengthLesson 11Column sizes and particle size trade-offs: length, inside, 3–5 µm vs under 2 µmTells how column length, inside round, and particle size change work, back push, feel, and check time. Gives help on picking 3–5 µm vs under 2 µm columns and scale sizes between systems.
Column length effect on split and timeInside round and feel thinks3–5 µm vs under 2 µm work and pushScale methods between column sizesGuard columns and net design hits
