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High Performance Liquid Chromatography

THE THEORY OF HPLC• IntroductiontoHPLC• ChromatographicParameters• BandBroadening• Columnchemistry• Reversephase(partition)chromatography• Ion-PairChromatography• Normalphase(absorption)chromatography• GradientHPLC• QuantitativeandQualitativeHPLC• FastHPLC• HILIC• SupercriticalFluidChromatography• IntroductiontoIonChromatography

INSTRUMENTATIONOFHPLC• MobilePhaseConsiderations• HPLCSolventPumpingSystems• Autosamplers• Detectors


Stator

Rotor Seal

IsolationSeal

Stator Face

It is necessary to divert the flow of mobile phase away from the sampling system (i.e. the syringe) when aspirating the sample and / or filling the sam-ple loop prior to injection. This is achieved using the injection valve containing a Rotor Seal.

The interface between the HPLC capillaries and the stator is known as the Stator Face...

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Gas Chromatography

THEORYANDINSTRUMENTATIONOFGC

• IntroductiontoGC• ChromatographicParameters• BandBroadening• GasSupplyandPressureControl• SamplingTechniques• SampleIntroduction• GCColumns• GCTemperatureProgramming• GCDetectors• SupercriticalFluidChromatography

1. Aims&Objectives2. Introduction3. Supercritical Fluids 4. Modes of Chromatography5. SFCApplications6. SFCInstrumentation7. Packed&CapillarySFC8. The Mobile Phase9. Exhaust Gases10.OrganicModifiers11.PumpingIssues12. SFC Columns13.SFCStationaryPhases&ColumnSelection14.Detection15. Pressure Regulators16.AdvantagesandDisadvantagesofSFC


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FUNDAMENTALGC-MS• Introduction• GCConsiderations• GC-MSInterfaces

MSINTERPRETATION• GeneralInterpretationStrategies• FundamentalsofMSProteomicsResearch


Corona Electrode Pin

e+

e+e+

e+ e+e+

e+

e+e+e+

e+e+

M+ M++++++

M+++++

MM+ M++++++

M

Non-Volatile Analyte is Charged in Solution

+++++

M+++++

M+++++

M

Nebulising Gas

Sample Preparation

SOLIDPHASEEXTRACTION• MolecularProperties

1. LearningAims&Objectives2. Functional groups3. Molecular Properties4. Functional group interactions5. HydrophobicorNon-PolarGroups6. HydrophobicInteractions-Solubility7. HydrophobicInteractions-Sorbents8. Polar Groups9. PolarInteractions-Solubility10.PolarInteractions-Sorbents11.Ionicgroups12.Ionicgroups-pH|Ka|pKa|Ionicstrength13.IonicInteractions-Solubility|Sorbents14.IonicInteractions-ChelatingGroups15. Chelating Groups 16.ChelatingInteractions17. Solubility18. Sorbents19.ProteinBinding20.Assessment

• SPEOverview• SPEMechanisms• MethodDevelopment• PrimarySamplePreparationTechniques• Liquid/LiquidExtractionTechniques• ApproachestoAutomationforSPE

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6more

a

a

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2

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Select up to 3 Chromatographic Symptoms

Positive baseline drift Negative baseline drift Irregular baseline drift Cycling Baseline (Short Term) Cycling Baseline (Long Term) Erratic baseline Regular baseline noise Irregular baseline noise Baseline Spikes Not the Chromatogram I was expecting! Loss of resolution (some peaks)Loss of resolution (all peaks)No Peaks Additional peaks (ghost peaks) Negative peaks (One or more peaks) Peak broadening (Loss of Efficiency)Peak fronting (one or more peaks)Peak shouldering (one or more peaks)Peak splitting (one or more peaks)Peak tailing (one or more peaks)Rounded peaksChange in selectivityDecreasing retention timesIncreasing retention timesToo much retention

Selectupto2Instrument Symptoms

Low pressureHigh pressureContinuously decreasing pressure (no sample injection)Continuously decreasing pressure (after sample injection)Continuously increasing pressure (no sample injection)Continuously increasing pressure (after sample injection)Pressure fluctuationLeaks in the solvent reserviour connective tubingLeaks at the degasser / fittingsLeaks around the pump headLeaks around the check valves or fittingsLeaks in the autosampler or connective tubingLeaks around the sampling valveLeaks at the column fittingsLeaks from the detector fiitingsLeaks within the detectorPoor quantititive reproducibilityPoor linearity of response

Possible Causes

Solution

Resources

Mobile phase pH affects retention of ionisable analytes and the selectivity of separations in reversed phase HPLCSome methods require a very precise pH in order to remain robustLow pH tends to increase retention of acidic an-alytes and decrease retention of basic analytes

1. Incorrect/non-optimalmobilephasepH2. Incorrect column geometry / dimensions3. Buffer precipitation4. Incorrect buffer / non-optimal buffer concentration5. Analyte-stationary phase secondary interaction (e.g. silanol interactions)6. Incorrect mobile phase7. Incorrect mobile phase temperature

BuffersandpHMeterThis video explains how to prepare a buffer and use a pH meter.BufferpreparationThis useful tool will help you to prepare the cor-rect buffer for your HPLC analysisHow to calibrate pH meterThis video describes how to calibrate your pH

Normal

Problem

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