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9/8/2014
1
Chromatography
Chromatography
• Major types of chromatography
– Liquid Chromatography
– Thin Layer Chromatography
– Gas Chromatography
• Major purposes
– Purification
– Characterization
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Chromatography Basics
• Separation!
• Based on different affinities (IMF) for stationary and mobile phases
• Stationary phase: silica gel particles, polymer particles
• Mobile phase: flowing gas, flowing organic liquid
Characterization of Purified Substance
Big questions in organic chemistry lab:
Was it purified? What was purified?
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TLC
• Thin layer chromatography
• Stationary phase
• Mobile phase
Separation AND Characterization
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Polarity and IMF
• Silica gel: polar, water‐covered surface
– Compound(s)
• Polar: _______ affinity for plate, travels _______
• Nonpolar: _______ affinity for plate, travels ______
– Developing solvent
• Polar: higher affinity for plate, travels slower, displaces compound more (compound travels __________)
• Nonpolar: lower affinity for plate, travels faster, displaces compound less (compound travels ________)
Test your Understanding
• Which spot represents a more polar compound?
• What would happen to each spot if a less polar solvent were used?
• Why should you ALWAYS report your developing solvent with any TLC data?
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Quantitative Characterization
• Retention factor
• Distance traveled/ solvent front distance
• Unitless
• For silica gel TLC, based on polarity of the compound(s) Must report solvent!
Solvent effect on Rf• Polar solvents outcompete compounds, drive them up plate
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Choosing a Developing Solvent
• Adjust solvent to give Rf values around 0.4
• Common mixtures– Ether/Hex
– EtOAc/Hex
– CH2Cl2/methanol
• Determined experimentally
Visualization
• Most compounds are invisible on TLC
• UV lamp
• Stains
• Iodine chamber
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Application of TLC
• Purity
• Identity
• Reaction Progress
What can we determine about the identity of the unknown?
Column 1 is your target compound; column 2 is an expected impurity. What can you determine about your reaction (column 3)?
Common Problems
• Overspotting
• Underspotting
• Wrong solvent
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Column Chromatography
• Similar to TLC in separation
• Preparative process
– 0.1g to 5 g scale
• Purify small quantities of liquids/solids (advantage over recrystallization?)
• Not for characterization!
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• Mobile phase: eluent similar to TLC
• Stationary phase: silica gel similar to TLC
• Column is upside down from TLC, so a larger Rf for a compound means it comes out ____
Practical Considerations
• Preparing the column
• Loading the sample
• Choosing the solvent
• Separation capacity
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Practical Considerations
• Preparing the column
• Loading the sample
• Choosing the solvent
• Separation capacity
Practical Considerations
• Preparing the column
• Loading the sample
• Choosing the solvent
• Separation capacity
Most common:Hexane/ethyl acetateCH2Cl2/methanol
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Practical Considerations
• Preparing the column
• Loading the sample
• Choosing the solvent
• Separation capacity
– Effect of diameter
– Effect of length
– Effect of Silica gel grade
Flash Chromatography
• Faster separation
• Tighter separations
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HPLC
Gas Chromatography
• Chromatography
– TLC (analytical)
– Column (preparative)
– GC (analytical)
• Why do we need GC?
– Strengths of TLC
– Weaknesses of TLC
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GC Basics
Gas‐Phase Separation
• Partitioning between mobile gas and stationary column
• Highly effective separations
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GC
• Injection
• Column
• Detector
• Chromatogram
GC
• Injection
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GC
• Column
Column Material
• Nonpolar – For nonpolar material
– vDW
– Follows bp
• Polar– For polar material
– Follows polarity
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GC
• Detector
– FID
– TCD
GC
• Chromatogram
– Retention time
– Area under peak
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GC Parameters and Retention Time
• Length of column
• Type of column
• Temperature profile
• Gas flow rate
Important GC Questions
• What is each peak? (identity)
– Characterization
– Determination of Unknown
• How much of each compound? (Quantity)
– Absolute quantity
– Relative quantity (product distribution)
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Identity
• Retention time corresponds to Rf
• Spike with authentic sample
Gas Chromatography/Mass Spec
• Identifying components in mixtures
• Separation then identification
• GC‐MS
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Quantitation
• Area under peak corresponds to amount of compound causing signal
• Use standard plot to determine absolute concentration
• Use percent of total area to determine product distribution– Assumes same response factor
Review of Techniques
• Purification– Extraction– Recrystallization– Distillation– Column chromatography
• Identification– MS– IR– NMR– mp, bp– GC– TLC
• Purity analysis– Elemental analysis– Mp, bp– GC– TLC– Proton NMR
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