shimadzu solutions for science since 1875 since 1875chromatography doug catron field sales engineer...

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


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


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


Why A Vinaigrette Separates

Oil DropOil Drop

Chemical XChemical X






Better Than Oil

•Hexane•Ether•Solids?

On to the next “Phase”


A Little TLC

A Small Difference•Stationary Phase•Mobile Phase

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


A Phase Of A Different Color

Defining Phases•Liquid/Liquid•Liquid/Solid

SolidSolid

LiquidLiquid

GasGas

TemperaturTemperaturee

PressurePressure

Gas/LiquidGas/Solid

Mobile/StationaryMobile/Stationary


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


Playing With Blocks

FlowFlow InjectorInjector

Oven/ColumnOven/Column

DetectorDetector

Basic Gas Chromatograph


Flow Control

The Mighty Needle ValveGas Regulator

Flow ~ Pressure for a given viscosity and resistance


Injectors


Oven/Column

How size mattersIsocraticGradientTide in a tube


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, ……


A Little Performance For LC

PumpPump InjectorInjector

ColumnColumn DetectorDetector

Basic HPLC


Pumping

Constant flowHigh pressureLow pulsationPositive displacementIsocraticGradient

Two pumpsMixing valve


Pump cont.

Single PistonDual ReciprocatingTandem Piston


Injector

Reproducible sample volume

PumPumpp

ColumnColumn

SamplSamplee

VentVent


Column

High pressureFritsParticle sizeDimensionsTemperature


Detector

Ultraviolet/visible spectrumRefractive indexFluorescenceElectrochemicalEvaporative light scatterMass spectrumPost column reagent


Chromatograms

Detector energy verses time


Chromatograms


1

2

3

4

5

67


Chromatograms


0 10 20 30

1

2

3

4

5

6

7


Part 2 - Troubleshooting

“This Worked Yesterday”

Be careful not to confuse with method development


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?


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


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


Can You Swap?

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

Make sure you swap back!


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



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?



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

– Blunt– Bent

•Rinse port•Mechanicals



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

– Water in a C18 column



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


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


Take It From The Top - GC

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

– Air

– Hydrogen

– NitrogenFlow Control•Flow meters•Split flow



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



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

– Injector/detector depth

– Tension

– Touching the oven•Phase and dimensions



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

– Purity

– Mixture•IgnitingData aquisition


From The Problem Point OF View

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

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.

shimadzu solutions for science since 1875 since 1875chromatography doug catron field sales engineer...

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halls detector

sievers detector

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redundant systemhow

effect whatthe easiest