Shimadzu Scientific Instruments, Inc. Chromatography Doug Catron Field Sales Engineer Shimadzu Scientific Instruments, Inc. www.ssi.shimadzu.com www.ssi.shimadzu.com/LCVA

Content What is chromatography What is HPLC and GC How do they work What makes them quit working

From The Beginning Chemistry on an empty stomach A little butter Anago – Eel Sushi A sip of water Salad Dressing

Why A Vinaigrette Separates Chemical X Chemical X Oil Drop Chemical X Chemical X Chemical X

Why A Vinaigrette Separates Oil Drop Chemical X Chemical X Chemical X Chemical X Chemical 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 Gas/Liquid Gas/Solid Solid Liquid Gas Temperature Pressure Mobile/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

Flow Injector Detector Oven/Column Playing With Blocks Flow Injector Detector Oven/Column Basic Gas Chromatograph

Flow Control The Mighty Needle Valve Gas Regulator Flow ~ Pressure for a given viscosity and resistance

Injectors

Oven/Column How size matters Isocratic Gradient Tide 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 Pump Injector Column Detector Basic HPLC

Pumping Constant flow High pressure Low pulsation Positive displacement Isocratic Gradient Two pumps Mixing valve

Pump cont. Single Piston Dual Reciprocating Tandem Piston

Injector Reproducible sample volume Pump Column Sample Vent

Injector Reproducible sample volume Vent Sample Column Pump

Column High pressure Frits Particle size Dimensions Temperature

Detector Ultraviolet/visible spectrum Refractive index Fluorescence Electrochemical Evaporative light scatter Mass spectrum Post column reagent

Chromatograms Detector energy verses time

Detector energy verses time Chromatograms Detector energy verses time 3 7 6 4 2 5 1

Detector energy verses time Chromatograms Detector energy verses time 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 Try it again Test column Slow erosion Snapped 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

But not always 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

Take It From The Top - HPLC Filter Frits Mechanical Degasser Low pressure solvent mixing valve Pump Check valves Isolating the right one Pump seals Plungers Bubbles Behind the seal rinse Purge valve seal Pressure sensor Frit?

Take It From The Top - HPLC Pump (cont.) Motor/gears Photo sensors Mixer Injector Vial/caps/septa Needle seal Syringe/metering pump Rotors/stators Needle Blunt Bent Rinse port Mechanicals

Take It From The Top - HPLC Column oven Column Prefilter Precolumn Void Fouled frits Degradation Particle breakdown Burst column Cleaning Water in a C18 column

Take It From The Top - HPLC Detectors Each is unique Lamps Flow cells Mirror fogging RID reference UV/Vis wavelength accuracy Photodiodes/PMTs Data collection Standards and curves EMI A/D Windows

From The Problem Point OF View Shifting peaks No peaks “Phantom” peaks Variable peaks area from the same vial Changing peak shape Not finding peaks Bad standard curves High backpressure Low backpressure Variable backpressure

Take It From The Top - GC Air Hydrogen Nitrogen Gases Safety Purity The “Dregs” Regulators Filters Generators Air Hydrogen Nitrogen Flow Control Flow meters Split flow

Take It From The Top - GC Flow Control (cont.) Purge flow Plugging Leaks… Injectors Septa Liners Cleaning Sample needles/autosamplers Needle size Pyrolysis Technique

Take It From The Top - GC Injector/detector depth Tension Column Packed vs. capillary Bake out Cut Ends Broken columns Degradation Cleaning Proper installation Injector/detector depth Tension Touching the oven Phase and dimensions

Take It From The Top - GC Purity Mixture Detectors Varied like HPLC Cleaning/Baking Jets Fuel Gases Purity Mixture Igniting Data aquisition

From The Problem Point OF View Shifting peaks No peaks “Phantom” peaks Variable peaks area from the same vial Changing peak shape Not finding peaks Bad 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.