SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

ChromatographyChromatography

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

ContentContent

• What is chromatographyWhat is chromatography• What is HPLC and GCWhat is HPLC and GC• How do they workHow do they work• What makes them quit workingWhat makes them quit working

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

From The BeginningFrom The Beginning

Chemistry on an empty stomachChemistry on an empty stomach• A little butterA little butter• Anago – Eel SushiAnago – Eel Sushi• A sip of waterA sip of water• Salad DressingSalad Dressing

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Why A Vinaigrette Separates

Oil DropOil Drop

Chemical XChemical X

Chemical XChemical X

Chemical XChemical X

Chemical XChemical X

Chemical XChemical X

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Why A Vinaigrette Separates

Oil DropOil Drop

Chemical XChemical X

Chemical XChemical X

Chemical XChemical X

Chemical XChemical X

Chemical XChemical X

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Better Than Oil

•Hexane•Ether•Solids?

On to the next “Phase”

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

A Little TLC

A Small Difference•Stationary Phase•Mobile Phase

Thin Layer Chromatography•Strips of Paper•Glass Slides•Glass Tubes

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

A Phase Of A Different Color

Defining Phases•Liquid/Liquid•Liquid/Solid

SolidSolid

LiquidLiquid

GasGas

TemperaturTemperaturee

PressurePressure

Gas/LiquidGas/Solid

Mobile/StationaryMobile/Stationary

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Building A Better Mousetrap

•How to control the “Mobile Phase”•How to inject and make it gas•How to keep it gas while separating•How to detect it

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Playing With Blocks

FlowFlow InjectorInjector

Oven/ColumnOven/Column

DetectorDetector

Basic Gas Chromatograph

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Flow Control

The Mighty Needle ValveGas Regulator

Flow ~ Pressure for a given viscosity and resistance

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Injectors

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Oven/Column

How size mattersIsocraticGradientTide in a tube

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Detectors

Detectors for every need•Thermal Conductivity Detector – TCD•Flame Ionization Detector – FID•Electron Capture Detector – ECD•Mass Spectrum - MS•NPD, PDID, FPD, Halls Detector, Sievers Detector, ……

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

A Little Performance For LC

PumpPump InjectorInjector

ColumnColumn DetectorDetector

Basic HPLC

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Pumping

Constant flowHigh pressureLow pulsationPositive displacementIsocraticGradient

Two pumpsMixing valve

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Pump cont.

Single PistonDual ReciprocatingTandem Piston

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Injector

Reproducible sample volume

PumPumpp

ColumnColumn

SamplSamplee

VentVent

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Injector

Reproducible sample volume

PumPumpp

ColumnColumn

SamplSamplee

VentVent

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Column

High pressureFritsParticle sizeDimensionsTemperature

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Detector

Ultraviolet/visible spectrumRefractive indexFluorescenceElectrochemicalEvaporative light scatterMass spectrumPost column reagent

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Chromatograms

Detector energy verses time

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Chromatograms

Detector energy verses time

1

2

3

4

5

67

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Chromatograms

Detector energy verses time

0 10 20 30

1

2

3

4

5

6

7

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Part 2 - Troubleshooting

“This Worked Yesterday”

Be careful not to confuse with method development

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Troubleshooting

LET GO OF THAT WRENCH!!!!•Confirm you have a problem

– Try it again– Test column

•What Changed– Slow erosion– Snapped– Identify the problem

•Who touched it last?

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Before You Have A Problem

Know your system•Backpressure•Test chromatograms•How is it configured•Instrument log•Manuals•What is that thing!?•Do preventative maintenance

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Time For The Wrench?

Not Quite

Isolate the problem•Know what effect what•The easiest answer is usually it

– But not always•Rarely do two things break at once*•Eliminate

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Can You Swap?

Redundant system• How essential is this system• Commonly troublesome components• Troubleshooting takes time

Make sure you swap back!

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - HPLC

Solvent•Properly mixed•Correct chemicals

– Purity grades

– Reagent Alcohol ≠ Ethanol•Buffers

– Proper pH

– Proper concentration•Modifiers•Contamination•Particles•Degassing•Microbes

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - HPLC

Filter FritsMechanical DegasserLow pressure solvent mixing valvePump•Check valves

– Isolating the right one•Pump seals•Plungers•Bubbles•Behind the seal rinse•Purge valve seal•Pressure sensor•Frit?

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - HPLC

Pump (cont.)•Motor/gears•Photo sensorsMixerInjector•Vial/caps/septa•Needle seal•Syringe/metering pump•Rotors/stators•Needle

– Blunt– Bent

•Rinse port•Mechanicals

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - HPLC

Column ovenColumn•Prefilter•Precolumn•Void•Fouled frits•Degradation•Particle breakdown•Burst column•Cleaning

– Water in a C18 column

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - HPLC

Detectors•Each is unique•Lamps•Flow cells•Mirror fogging•RID reference•UV/Vis wavelength accuracy•Photodiodes/PMTsData collection•Standards and curves•EMI•A/D•Windows

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

From The Problem Point OF View

Shifting peaksNo peaks“Phantom” peaksVariable peaks area from the same vialChanging peak shapeNot finding peaksBad standard curvesHigh backpressureLow backpressureVariable backpressure

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - GC

Gases•Safety•Purity•The “Dregs”•Regulators•Filters•Generators

– Air

– Hydrogen

– NitrogenFlow Control•Flow meters•Split flow

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - GC

Flow Control (cont.)•Purge flow•Plugging•Leaks…Injectors•Septa•Liners•CleaningSample needles/autosamplers•Needle size•Cleaning•Pyrolysis•Technique

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - GC

Column•Packed vs. capillary•Bake out•Cut Ends•Broken columns•Degradation•Cleaning•Proper installation

– Injector/detector depth

– Tension

– Touching the oven•Phase and dimensions

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

Take It From The Top - GC

Detectors•Varied like HPLC•Cleaning/Baking•Jets•Fuel Gases

– Purity

– Mixture•IgnitingData aquisition

SHIMADZUSHIMADZUSolutions for ScienceSolutions for Science Since 1875Since 1875

From The Problem Point OF View

Shifting peaksNo peaks“Phantom” peaksVariable peaks area from the same vialChanging peak shapeNot finding peaksBad standard curves

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