shimadzu solutions for science since 1875 since 1875 hplc method development doug catron field sales...
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SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
HPLC Method DevelopmentHPLC Method Development
Doug CatronDoug Catron
Field Sales EngineerField Sales Engineer
Shimadzu Scientific Instruments, Inc.Shimadzu Scientific Instruments, Inc.www.ssi.shimadzu.com
www.ssi.shimadzu.com/LCVAwww.ssi.shimadzu.com/LCVA
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Types Of MethodsTypes Of Methods
• PreparativePreparative• QuantitativeQuantitative• QualitativeQualitative
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
PreparativePreparative
• Sample PreparationSample Preparation• CapacityCapacity
• Big ColumnsBig Columns• High Flow RatesHigh Flow Rates
• Large Injection VolumesLarge Injection Volumes• Blinding The DetectorBlinding The Detector
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
PreparativePreparative
Heart CuttingHeart Cutting
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
QuantitativeQuantitative
• Sample PreparationSample Preparation• SeparationSeparation• DetectionDetection• CalibrationCalibration• QCQC
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
QualitativeQualitative
• Sample PreparationSample Preparation• SeparationSeparation• DetectionDetection• Data ComparisonData Comparison
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Sample PreparationSample Preparation
• SamplingSampling• HomogenationHomogenation• Sub samplingSub sampling• DissolutionDissolution• Clean UpClean Up• Chill OutChill Out
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Clean UpClean Up
• Phase SeparationPhase Separation• DigitizationDigitization• Solid Phase ExtractionSolid Phase Extraction• Solid Phase Micro extractionSolid Phase Micro extraction• CentrifugeCentrifuge• Filtering!!!Filtering!!!
Don’t Overdo Don’t Overdo It!It!
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Clean UpClean Up
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Now Lets Get To BusinessNow Lets Get To Business
Don’t Reinvent The WheelDon’t Reinvent The Wheel
•Standard MethodStandard Method
•Column ManufacturersColumn Manufacturers
Check Your SourcesCheck Your Sources
Standards vs. Real Standards vs. Real SamplesSamples
•MatrixMatrix
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Tools Of The TradeTools Of The Trade
AffinityAffinity
•Reverse PhaseReverse Phase
•Normal PhaseNormal Phase
•Strong SolventStrong Solvent
Gel PermeationGel Permeation
Ion Pairing/Ion ExchangeIon Pairing/Ion Exchange
Chiral SeparationsChiral Separations
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Move A Mountain One Peak At A Move A Mountain One Peak At A TimeTime
Lower RT = Stronger Lower RT = Stronger SolventSolvent
Higher RT = Weaker Higher RT = Weaker SolventSolvent
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Move A Mountain One Peak At A Move A Mountain One Peak At A TimeTime
Do Both With A GradientDo Both With A Gradient
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Move A Mountain One Peak At A Move A Mountain One Peak At A TimeTime
Leave Room For Uninvited Leave Room For Uninvited GuestsGuests
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
When The Mountain Won’t MoveWhen The Mountain Won’t Move
A Stationary Phase For Any A Stationary Phase For Any OccasionOccasion
•C18C18
•C8C8
•C4C4
•PhenylPhenyl
•Get NormalGet Normal
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
OptimizeOptimize
How Good Is Good EnoughHow Good Is Good Enough
•Short Is GoodShort Is Good
•ResolutionResolution
Leave A Little Wiggle RoomLeave A Little Wiggle Room
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
The Chromatographer’s TriangleThe Chromatographer’s Triangle
SpeedSpeed
ResolutionResolution CapacityCapacity
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Do My Peaks Look Fat?Do My Peaks Look Fat?
Free Mean PathsFree Mean Paths
Laminar FlowLaminar Flow
VoidsVoids
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Hot PeaksHot Peaks
1ºC ~ 1% RT1ºC ~ 1% RT
Different For Every CompoundDifferent For Every Compound
Room Temp May Not Be The BestRoom Temp May Not Be The Best
Trial and ErrorTrial and Error
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Straighten The PeaksStraighten The Peaks
Competing ChromatographyCompeting Chromatography•Size ExclusionSize Exclusion•ModifiersModifiers
OverloadingOverloading
Resonance StructuresResonance Structures
Methanol vs. AcetonitrileMethanol vs. Acetonitrile
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
DetectionDetection
SpecificitySpecificity
SensitivitySensitivity
Post And Pre Column DerivativesPost And Pre Column Derivatives
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
DetectionDetection
UV/VisUV/Vis•254nm254nm
RIDRID
FluorescenceFluorescence
ConductivityConductivity
ELSDELSD
MSMS
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
CalibrationCalibration
How Many PointsHow Many Points
Pick An EquationPick An Equation
SpacingSpacing
UncertaintyUncertainty
How Long Is The Curve ValidHow Long Is The Curve Valid
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Set’em UpSet’em Up
How Much SampleHow Much Sample•100 mg100 mg
Range Range •10 – 100 ppm10 – 100 ppm
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
ValidationValidation
Sample / Standard StabilitySample / Standard Stability
AccuracyAccuracy
PrecisionPrecision
LODLOD
LOQLOQ
ToleranceTolerance•ColumnColumn•MatrixMatrix
High ConcentrationHigh Concentration
SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875
Doing It BetterDoing It Better
Smaller ColumnsSmaller Columns
Smaller ParticlesSmaller Particles•A=A=ππr²r²
Capillary HPLCCapillary HPLC
UPLCUPLC
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