Chromatography
Chromatography
• Major types of chromatography
– Liquid Chromatography
– Thin Layer Chromatography
– Gas Chromatography
• Major purposes
– Purification
– Characterization
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?
TLC
• Thin layer
chromatography
• Stationary phase
• Mobile phase
Separation AND Characterization
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?
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
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
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
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!
• 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
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 acetate
CH2Cl2/methanol
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
HPLC
Gas Chromatography
• Chromatography
– TLC (analytical)
– Column (preparative)
– GC (analytical)
• Why do we need GC?
– Strengths of TLC
– Weaknesses of TLC
GC Basics
Gas-Phase Separation
• Partitioning
between
mobile gas
and
stationary
column
• Highly
effective
separations
GC
•
•
•
•
Injection
Column
Detector
Chromatogram
GC
• Injection
GC
• Column
Column Material
• Nonpolar
– For nonpolar
material
– vDW
– Follows bp
• Polar
– For polar
material
– Follows polarity
GC
• Detector
– FID
– TCD
GC
• Chromatogram
– Retention time
– Area under
peak
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)
Identity
• Retention time
corresponds to Rf
• Spike with authentic
sample
Gas Chromatography/Mass Spec
• Identifying
components
in mixtures
• Separation
then
identification
• GC-MS
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