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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