Why Take CHM 312

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Why Take CHM 312?

• How to make use of analytical instruments for the
  organic chemist
• Applicable for natural product chemistry,
  environmental organic chemistry and synthetic
  chemistry to name a few
• Whole premise is to take an unknown and figure
  out what it is

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CHM 312 Spring 2009 CHROMATOGRAPHY
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Chromatography Who Cares?

• Uses
• Purification
• Isolation
• Quantitative
• Qualitative
• Types
• Planar (e.g. TLC)
• Column (e.g. GC, HPLC)
• Common Traits
• Mobile phase forced through immiscible
  stationary phase
• Sample is distributed between phases to varying
  degree
• Moves only with mobile phase
• Thus separation occurs

TLC of Black Ink
Column Chromatography
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THIN LAYER CHROMATOGRAPHY (TLC)
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THIN LAYER CHROMATOGRAPHY

• Thin layer chromatography (TLC) is an important
  technique for identification and separation of
  mixtures of organic compounds. It is useful in
• Identification of components of a mixture (using
  appropriate standards)
• following the course of a reaction,
• analyzing fractions collected during
  purification,
• analyzing the purity of a compound.
• In TLC, components of the mixture are partitioned
  between an adsorbent (the stationary phase,
  usually silica gel, SiO2) and a solvent ( the
  mobile phase) which flows through the adsorbent.

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THIN LAYER CHROMATOGRAPHY
In TLC, a plastic, glass or aluminum sheet is
coated with a thin layer of silica gel. A very
small amount of a solution of the substance to be
analyzed is applied in a small spot with a
capillary tube, 1cm from the bottom of the TLC
plate
The TLC is developed in a chamber which contains
the developing solvent (the mobile phase). A
truncated filter paper placed in the chamber
serves to saturate the chamber with mobile phase.
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THIN LAYER CHROMATOGRAPHY
As the mobile phase rises up the TLC plate by
capillary action, the components dissolve in the
solvent and move up the TLC plate. Individual
components move up at different rates, depending
on intermolecular forces between the component
and the silica gel stationary phase and the
component and the mobile phase.
http//www.instructables.com/id/EW1YDCYF4REC0IU/
The stationary phase is SiO2 and is very polar.
It is capable of strong dipole-dipole and
H-bond donating and accepting interactions with
the analytes (the components being analyzed).
More polar analytes interact more strongly with
the stationary phase and move very slowly up the
TLC plate. By comparison, the mobile phase is
relatively nonpolar and is capable of
interacting with analytes by stronger London
forces, as well as by dipole-dipole and
H-bonding. More nonpolar analytes interact
less strongly with the polar silica gel and more
strongly with the less polar mobile phase and
move higher up the TLC plate.
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THIN LAYER CHROMATOGRAPHY

• Once the solvent is within 1-2 cm of the top of
  the TLC sheet, the TLC is removed from the
  developing chamber and the farthest extent of the
  solvent (the solvent front) is marked with a
  pencil.
• The solvent is allowed to evaporate from the TLC
  sheet in the hood.
• The spots are visualized using a UV lamp.
• A fluorescent compound, usually
  Manganese-activated Zinc Silicate, is added to
  the adsorbent that allows the visualization of
  spots under a blacklight (UV254). The adsorbent
  layer will fluoresce light green by itself, but
  spots of analyte quench this fluorescence and
  appear as a dark spot.

http//orgchem.colorado.edu/hndbksupport/TLC/TLCpr
ocedure.html
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THIN LAYER CHROMATOGRAPHY - Visualization

•   
• As the chemicals being separated may be
  colorless, several methods exist to visualize the
  spots
• Visualization of spots under a UV254 lamp. The
  adsorbent layer will thus fluoresce light green
  by itself, but spots of analyte quench this
  fluorescence.
• Iodine vapors are a general unspecific color.
• Specific color reagents exist into which the TLC
  plate is dipped or which are sprayed onto the
  plate.
• Once visible, the Rf value of each spot can be
  determined

Chromatogram of 10 essential oils, Stained with
vanillin reagent.
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THIN LAYER CHROMATOGRAPHY Calculation of Rfs

• The Rf is defined as the distance the center of
  the spot moved divided by the distance the
  solvent front moved (both measured from the
  origin)

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THIN LAYER CHROMATOGRAPHY Calculation of Rfs

• The Rf is defined as the distance the center of
  the spot moved divided by the distance the
  solvent front moved (both measured from the
  origin)

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THIN LAYER CHROMATOGRAPHY Rfs

• Rf values can be used to aid in the
  identification of a substance by comparison to
  standards.
• The Rf value is not a physical constant, and
  comparison should be made only between spots on
  the same sheet, run at the same time.
• Two substances that have the same Rf value may
  be identical those with different Rf values are
  not identical.

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THIN LAYER CHROMATOGRAPHY Rfs
Absorption of Solutes The adsorption strength of
compounds increases with increasing polarity of
functional groups, as shown below -CHCH2,
-X, -OR, -CHO, -CO2R, -NR2, -NH2, -OH,
-CONR2, -CO2H. (weakly adsorbed)
(strongly adsorbed) (nonpolar)
(more polar)
Elution Strength of Mobile Phase (e) Elution
strength is generally considered to be equivalent
to polarity. A solvents elution strength depends
on Intermolecular Forces between the solvent and
the analytes and between the solvent and the
stationary phase. A more polar (or more strongly
eluting solvent) will move all of the analytes to
a greater extent, than a less polar, weakly
elution solvent. For example, the elution
strength of hexane is very low e 0.01.
the elution strength of ethyl acetate is
higher e 0.45 the elution strength
of ethanol is even higher e 0.68
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Solvent Properties and Elution Strengths
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Elution Strength of Mixed Solvents
The elution strength of the mixture is assumed to
be the weighted average of the elution strengths
of the components eonet eoA (mole A)
eoB (mole B) where mole A (moles A)
/ (moles A moles B) Thus, to determine the
eonet of a solvent mixture, the molar ratio of
the solvents must first be calculated. For
example, the eonet of a solvent mixture prepared
from 1.0 mL of ethyl acetate plus 9.0 mL of
hexanes is calculated as shown below eonet
eoEtOAc (moles EtOAc)/(moles EtOAcmoles
hexane) eohexane (moles
hexane)/(moles EtOAcmoles hexane) where
moles EtOAc (volume EtOAc) (density EtOAc) /
molecular weight of EtOAc thus eonet
0.45(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)0.01
(9.0mLhexane)(0.659g/mL)/86.18g/mole) (1.0
mLEtOAc)(0.902g/mL)/88.11g/mole) (9.0
mLhexane)(0.659g/mL)/86.18g/mole) and eonet
0.067
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Resolution The separation between two analytes
on a chromatogram can be expressed as the
resolution, Rs and can be determined using the
following equation Rs (distance between
center of spots) (average
diameter of spots) In TLC, if the Rs value is
greater than 1.0, the analytes are considered to
be resolved.
x
x
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• Improving Resolution
• For two closely migrating components, optimum
  resolutions are usually obtained when the Rfs of
  both compounds are between 0.2 and 0.5
• To Improve Rs, change the elution strength of
  the solvent to optimize Rfs
• change eonet ( in capacity factor), all
  compounds will be effected similarly.
• Alter the composition of the solvent system so
  that the components affinity for the mobile phase
  vs. the solid phase are differentially changed (
  change in selectivity).
• Changing the chemical nature of the solvent
  system, such as changing a hydrogen bonding
  solvent to a solvent which cannot hydrogen bond
  to the analyte, is often the most effective.
• Improve Rs by decreasing the diameter of the
  analyte spots. This can be achieved by applying
  smaller and less concentrated spots.

http//orgchem.colorado.edu/hndbksupport/ TLC/TLCp
rocedure.html
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Optimize Rfs
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TLC Stationary Phases
www.vwr.com
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www.vwr.com
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PREPARATIVE TLC (PTLC)
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TLC - Optimizing for column chromatography
Optimum 0.2 lt Rf lt 0.5