SHIMADZU Solutions for Science Since 1875 Since 1875 HPLC Method Development Doug Catron Field Sales Engineer Shimadzu Scientific Instruments, Inc

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


Types Of MethodsTypes Of Methods

• PreparativePreparative• QuantitativeQuantitative• QualitativeQualitative


PreparativePreparative

• Sample PreparationSample Preparation• CapacityCapacity

• Big ColumnsBig Columns• High Flow RatesHigh Flow Rates

• Large Injection VolumesLarge Injection Volumes• Blinding The DetectorBlinding The Detector


PreparativePreparative

Heart CuttingHeart Cutting


QuantitativeQuantitative

• Sample PreparationSample Preparation• SeparationSeparation• DetectionDetection• CalibrationCalibration• QCQC


QualitativeQualitative

• Sample PreparationSample Preparation• SeparationSeparation• DetectionDetection• Data ComparisonData Comparison


Sample PreparationSample Preparation

• SamplingSampling• HomogenationHomogenation• Sub samplingSub sampling• DissolutionDissolution• Clean UpClean Up• Chill OutChill Out


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!


Clean UpClean Up


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


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


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



Do Both With A GradientDo Both With A Gradient



Leave Room For Uninvited Leave Room For Uninvited GuestsGuests


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


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


The Chromatographer’s TriangleThe Chromatographer’s Triangle

SpeedSpeed

ResolutionResolution CapacityCapacity


Do My Peaks Look Fat?Do My Peaks Look Fat?

Free Mean PathsFree Mean Paths

Laminar FlowLaminar Flow

VoidsVoids


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


Straighten The PeaksStraighten The Peaks

Competing ChromatographyCompeting Chromatography•Size ExclusionSize Exclusion•ModifiersModifiers

OverloadingOverloading

Resonance StructuresResonance Structures

Methanol vs. AcetonitrileMethanol vs. Acetonitrile


DetectionDetection

SpecificitySpecificity

SensitivitySensitivity

Post And Pre Column DerivativesPost And Pre Column Derivatives


DetectionDetection

UV/VisUV/Vis•254nm254nm

RIDRID

FluorescenceFluorescence

ConductivityConductivity

ELSDELSD

MSMS


CalibrationCalibration

How Many PointsHow Many Points

Pick An EquationPick An Equation

SpacingSpacing

UncertaintyUncertainty

How Long Is The Curve ValidHow Long Is The Curve Valid


Set’em UpSet’em Up

How Much SampleHow Much Sample•100 mg100 mg

Range Range •10 – 100 ppm10 – 100 ppm


ValidationValidation

Sample / Standard StabilitySample / Standard Stability

AccuracyAccuracy

PrecisionPrecision

LODLOD

LOQLOQ

ToleranceTolerance•ColumnColumn•MatrixMatrix

High ConcentrationHigh Concentration


Doing It BetterDoing It Better

Smaller ColumnsSmaller Columns

Smaller ParticlesSmaller Particles•A=A=ππr²r²

Capillary HPLCCapillary HPLC

UPLCUPLC

This product was funded by a grant awarded under the President’s High Growth Job Training Initiative as implemented by the U.S. Department of Labor’s Employment & Training Administration. The information contained in this product was created by a grantee organization and does not necessarily reflect the official position of the U.S. Department of Labor. All references to non-governmental companies or organizations, their services, products, or resources are offered for informational purposes and should not be construed as an endorsement by the Department of Labor. This product is copyrighted by the institution that created it and is intended for individual organizational, non-commercial use only.

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SHIMADZU Solutions for Science Since 1875 Since 1875 HPLC Method Development Doug Catron Field Sales Engineer Shimadzu Scientific Instruments, Inc