Chromatography
Doug Catron
Field Sales Engineer
Shimadzu Scientific Instruments, Inc.
www.ssi.shimadzu.com
www.ssi.shimadzu.com/LCVA
SHIMADZU
Solutions for Science
Since 1875
Content
• What is chromatography
• What is HPLC and GC
• How do they work
• What makes them quit working
SHIMADZU
Solutions for Science
Since 1875
From The Beginning
Chemistry on an empty stomach
• A little butter
• Anago – Eel Sushi
• A sip of water
• Salad Dressing
SHIMADZU
Solutions for Science
Since 1875
Why A Vinaigrette Separates
Chemical X
Chemical X
Oil Drop
Chemical X
Chemical X
Chemical X
SHIMADZU
Solutions for Science
Since 1875
Why A Vinaigrette Separates
Chemical X
Chemical X
Oil Drop
Chemical X
Chemical X
Chemical X
SHIMADZU
Solutions for Science
Since 1875
Better Than Oil
•Hexane
•Ether
•Solids?
On to the next “Phase”
SHIMADZU
Solutions for Science
Since 1875
A Little TLC
A Small Difference
•Stationary Phase
•Mobile Phase
Thin Layer Chromatography
•Strips of Paper
•Glass Slides
•Glass Tubes
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Solutions for Science
Since 1875
A Phase Of A Different Color
Defining Phases
•Liquid/Liquid
•Liquid/Solid
Gas/Liquid
Gas/Solid
Liquid
Pressure
Solid
Gas
Temperature
Mobile/Stationary
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Solutions for Science
Since 1875
Building A Better Mousetrap
•How
•How
•How
•How
to
to
to
to
control the “Mobile Phase”
inject and make it gas
keep it gas while separating
detect it
SHIMADZU
Solutions for Science
Since 1875
Playing With Blocks
Flow
Injector
Detector
Oven/Column
Basic Gas Chromatograph
SHIMADZU
Solutions for Science
Since 1875
Flow Control
The Mighty Needle Valve
Gas Regulator
Flow ~ Pressure for a given viscosity
and resistance
SHIMADZU
Solutions for Science
Since 1875
Injectors
SHIMADZU
Solutions for Science
Since 1875
Oven/Column
How size matters
Isocratic
Gradient
Tide in a tube
SHIMADZU
Solutions for Science
Since 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, ……
SHIMADZU
Solutions for Science
Since 1875
A Little Performance For LC
Pump
Injector
Column
Basic HPLC
Detector
SHIMADZU
Solutions for Science
Since 1875
Pumping
Constant flow
High pressure
Low pulsation
Positive displacement
Isocratic
Gradient
Two pumps
Mixing valve
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Solutions for Science
Since 1875
Pump cont.
Single Piston
Dual Reciprocating
Tandem Piston
SHIMADZU
Solutions for Science
Since 1875
Injector
Reproducible sample volume
Sample
Pump
Vent
Column
SHIMADZU
Solutions for Science
Since 1875
Injector
Reproducible sample volume
Sample
Pump
Vent
Column
SHIMADZU
Solutions for Science
Since 1875
Column
High pressure
Frits
Particle size
Dimensions
Temperature
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Solutions for Science
Since 1875
Detector
Ultraviolet/visible spectrum
Refractive index
Fluorescence
Electrochemical
Evaporative light scatter
Mass spectrum
Post column reagent
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Solutions for Science
Since 1875
Chromatograms
Detector energy verses time
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Solutions for Science
Since 1875
Chromatograms
Detector energy verses time
3
6
2
7
4
5
1
SHIMADZU
Solutions for Science
Since 1875
Chromatograms
Detector energy verses time
6
5
2
1
3
7
4
0
10
20
30
SHIMADZU
Solutions for Science
Since 1875
Part 2 - Troubleshooting
“This Worked Yesterday”
Be careful not to confuse with method
development
SHIMADZU
Solutions for Science
Since 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?
SHIMADZU
Solutions for Science
Since 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
SHIMADZU
Solutions for Science
Since 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
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Solutions for Science
Since 1875
Can You Swap?
Redundant system
• How essential is this system
• Commonly troublesome components
• Troubleshooting takes time
Make sure you swap back!
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Solutions for Science
Since 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
SHIMADZU
Solutions for Science
Since 1875
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?
SHIMADZU
Solutions for Science
Since 1875
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
SHIMADZU
Solutions for Science
Since 1875
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
SHIMADZU
Solutions for Science
Since 1875
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
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Solutions for Science
Since 1875
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
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Solutions for Science
Since 1875
Take It From The Top - GC
Gases
•Safety
•Purity
•The “Dregs”
•Regulators
•Filters
•Generators
– Air
– Hydrogen
– Nitrogen
Flow Control
•Flow meters
•Split flow
SHIMADZU
Solutions for Science
Since 1875
Take It From The Top - GC
Flow Control (cont.)
•Purge flow
•Plugging
•Leaks…
Injectors
•Septa
•Liners
•Cleaning
Sample needles/autosamplers
•Needle size
•Cleaning
•Pyrolysis
•Technique
SHIMADZU
Solutions for Science
Since 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
SHIMADZU
Solutions for Science
Since 1875
Take It From The Top - GC
Detectors
•Varied like HPLC
•Cleaning/Baking
•Jets
•Fuel Gases
– Purity
– Mixture
•Igniting
Data aquisition
SHIMADZU
Solutions for Science
Since 1875
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
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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
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is intended for individual organizational, non-commercial use only.
SHIMADZU
Solutions for Science
Since 1875