1. SHIMADZU Solutions for Science Since 1875 Since 1875 HPLC Method Development Doug Catron Field Sales Engineer Shimadzu Scientific Instruments, Inc. www.ssi.shimadzu.com www.ssi.shimadzu.com/LCVA

2. SHIMADZU Solutions for Science Since 1875 Since 1875 Types Of Methods PreparativePreparative QuantitativeQuantitative QualitativeQualitative

3. SHIMADZU Solutions for Science Since 1875 Since 1875Preparative Sample PreparationSample Preparation CapacityCapacity Big ColumnsBig Columns High Flow RatesHigh Flow Rates Large Injection VolumesLarge Injection Volumes Blinding The DetectorBlinding The Detector

4. SHIMADZU Solutions for Science Since 1875 Since 1875Preparative Heart Cutting

5. SHIMADZU Solutions for Science Since 1875 Since 1875Quantitative Sample PreparationSample Preparation SeparationSeparation DetectionDetection CalibrationCalibration QCQC

6. SHIMADZU Solutions for Science Since 1875 Since 1875Qualitative Sample PreparationSample Preparation SeparationSeparation DetectionDetection Data ComparisonData Comparison

7. SHIMADZU Solutions for Science Since 1875 Since 1875 Sample Preparation SamplingSampling HomogenationHomogenation Sub samplingSub sampling DissolutionDissolution Clean UpClean Up Chill OutChill Out

8. SHIMADZU Solutions for Science Since 1875 Since 1875 Clean Up Phase SeparationPhase Separation DigitizationDigitization Solid Phase ExtractionSolid Phase Extraction Solid Phase Micro extractionSolid Phase Micro extraction CentrifugeCentrifuge Filtering!!!Filtering!!! Don’t Overdo It!

9. SHIMADZU Solutions for Science Since 1875 Since 1875 Clean Up

10. SHIMADZU Solutions for Science Since 1875 Since 1875 Now Lets Get To Business Don’t Reinvent The Wheel Standard MethodStandard Method Column ManufacturersColumn Manufacturers Check Your Sources Standards vs. Real Samples MatrixMatrix

11. SHIMADZU Solutions for Science Since 1875 Since 1875 Tools Of The Trade Affinity Reverse PhaseReverse Phase Normal PhaseNormal Phase Strong SolventStrong Solvent Gel Permeation Ion Pairing/Ion Exchange Chiral Separations

12. SHIMADZU Solutions for Science Since 1875 Since 1875 Move A Mountain One Peak At A Time Lower RT = Stronger Solvent Higher RT = Weaker Solvent

13. SHIMADZU Solutions for Science Since 1875 Since 1875 Move A Mountain One Peak At A Time Do Both With A Gradient

14. SHIMADZU Solutions for Science Since 1875 Since 1875 Move A Mountain One Peak At A Time Leave Room For Uninvited Guests

15. SHIMADZU Solutions for Science Since 1875 Since 1875 When The Mountain Won’t Move A Stationary Phase For Any Occasion C18C18 C8C8 C4C4 PhenylPhenyl Get NormalGet Normal

16. SHIMADZU Solutions for Science Since 1875 Since 1875Optimize How Good Is Good Enough Short Is GoodShort Is Good ResolutionResolution Leave A Little Wiggle Room

17. SHIMADZU Solutions for Science Since 1875 Since 1875 The Chromatographer’s Triangle Speed Resolution Capacity

18. SHIMADZU Solutions for Science Since 1875 Since 1875 Do My Peaks Look Fat? Free Mean Paths Laminar Flow Voids

19. SHIMADZU Solutions for Science Since 1875 Since 1875 Hot Peaks 1ºC ~ 1% RT Different For Every Compound Room Temp May Not Be The Best Trial and Error

20. SHIMADZU Solutions for Science Since 1875 Since 1875 Straighten The Peaks Competing Chromatography Size ExclusionSize Exclusion ModifiersModifiersOverloading Resonance Structures Methanol vs. Acetonitrile

21. SHIMADZU Solutions for Science Since 1875 Since 1875Detection SpecificitySensitivity Post And Pre Column Derivatives

22. SHIMADZU Solutions for Science Since 1875 Since 1875Detection UV/Vis 254nm254nmRIDFluorescenceConductivityELSDMS

23. SHIMADZU Solutions for Science Since 1875 Since 1875Calibration How Many Points Pick An Equation SpacingUncertainty How Long Is The Curve Valid

24. SHIMADZU Solutions for Science Since 1875 Since 1875 Set’em Up How Much Sample 100 mg100 mgRange 10 – 100 ppm10 – 100 ppm

25. SHIMADZU Solutions for Science Since 1875 Since 1875Validation Sample / Standard Stability AccuracyPrecisionLODLOQTolerance ColumnColumn MatrixMatrix High Concentration

26. SHIMADZU Solutions for Science Since 1875 Since 1875 Doing It Better Smaller Columns Smaller Particles A=πr²A=πr² Capillary HPLC UPLC 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.