Experiment 3.7 A

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Experiment 3.7 ABSolvent and Polarity Effects
in Thin-layer Chromatography
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Chromatography
"Color Writing"

• A general term for a family of lab techniques
  used for the separation of mixtures
• Comes from two Greek words, chroma (color) and
  grafein (to write)
• Invented in 1900 by a Russian botanist ( Mikhail
  Tsvet) for separating plant pigments
• It involves passing a mixture dissolved in a
  mobile phase through a stationary phase.
• The analytes in the mixture are partitioned
  differently between the stationary and mobile
  phases which causes the separation.
• Many types of chromatography - column, paper,
  thin layer, gas, high performance liquid, ion
  exchange

http//en.wikipedia.org/wiki/chromatography
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Thin Layer Chromatography
TLC plate
silica gel - silicon dioxide (SiO2)x (a common,
inexpensive stationary phase)
5 x 10 cm 250 µm silica gel layer impregnated
with a fluorescent indicator, on a foil backing
bulk (SiO2)x
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Thin Layer Chromatography

• Five Steps in a TLC Analysis
• Prepare Developing Chamber Saturate with solvent
  vapor.
• Apply Samples Capillary used to spot
  solution of each sample.
• Develop Plate This is when the separation
  actually occurs.
• Visualize Developed Plate View under UV
  light.
• Interpret Results Determine Rf values
  identify components.

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1. Prepare Developing Chamber

• 400mL beaker.
• Place a piece of filter paper against side of
  beaker. (Cut one side so its flat on the
  bottom.)
• Pour 10mL of developing solvent (mobile phase)
  into the beaker.
• Cover beaker with a watch glass and allow to
  stand undisturbed for about 15 minutes.
• Allows development chamber to become saturated
  with solvent.

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2. Apply Samples (spot the plate)
Process

• Draw starting line lightly with pencil 1
  cm from bottom
• Make light x where each spot will be
• Dissolve solid sample in CH2Cl2
• Use TLC capillary to transfer and spot dissolved
  sample (keep spots very small 1-2 mm.)

TLC plate
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3. Develop TLC Plate

• Place spotted TLC plate in developing chamber.
• The solvent is drawn up the plate by capillary
  action.
• Remove TLC plate when solvent front is 1 cm
  from top.
• Mark solvent front position with a pencil
  immediately.

NOTE During this 20 min. developing stage,
compounds in the original spots are being pulled
through the silica gel.
Developing Chamber (400 mL beaker with 10mL
solvent)
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4. Visualize Results

• Allow solvent to evaporate from surface of TLC
  plate.
• View results under UV light. Look for colored
  spots on the fluorescent green background
• Trace spots with a pencil while viewing under UV
  light.
• Mark the center of each spot.

UV
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5. Interpret Results (Rf Values)
Solvent Front
Y
T
Z
X
Starting Line
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5. Interpret Results (identify components)

• Compare Rf values of components in sample to Rf
  values of standards.

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Purpose of Experiment

• Determine factors that affect rate of elution
  (and Rf values) of organic compounds.
• Understand the relationship between polarities of
  compounds and their rates of elution.
• Understand the relationship between solvent
  polarity and rate of elution.
• Select an appropriate solvent system to separate
  and identify components of an unknown mixture.

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Solutes (Analytes) and Solvents

• Solutes

Vanillin
Vanillyl alcohol
trans-stillbene

• Solvents

Hexane, ethyl acetate, acetone
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Pre-lab Preparation

• Read Technique K (pp. 92-100)
• Prepare Notebook
• Header info
• Purpose
• Reference (use the lecture notes)
• Table of reagents
• Structures, hazards of solvents (hexane, ethyl
  acetate and acetone) and compounds to be
  separated. Appropriate physical properties.
• Data source
• Procedures (two-column format, use lecture notes
  for steps.)

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In Lab

• Part A (groups of three or two)
• Determine Rf of trans-stilbene, vanillin and
  vanillyl alcohol in hexane, ethyl acetate and
  acetone.
• Each person in group does 1 solvent.
• Determine the best solvent to use to separate a
  mixture of trans-stilbene, vanillin, and vanillyl
  alcohol
• Consider mixed solvents
• Review with lab instructor before doing part B.

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Data Collection

• Part A
• Record the solvent used for each TLC plate.
• Also, after plate development, write the solvent
  name at the top of the plate.
• Measure and record the distances that each
  compound and the solvent front traveled for each
  solvent used. (mm convenient)
• Draw a diagram of each plate in your notebook.

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Part A Summary

• Spot and develop the plates. Record the solvent
  used.
• Measure the distances and calculate the Rf
  values.
• Share the values amongst the group members.
• Draw diagrams in notebook.

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In Lab

• Part B
• Do this part individually.
• Determine components in an unknown mixture.

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Data Collection

• Part B
• Record the unknown number.
• Record the solvent (or solvent mixture) used for
  the analysis of the unknown.
• Measure and record the distances that each known
  solute, each solute in the unknown and the
  solvent front traveled.

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Part B Summary

• Spot and develop the plate. Record the solvent
  and unknown used.
• Measure the distances and calculate the Rf
  values.
• Determine the components in the mixture.
• Draw diagram in notebook.

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Thin Layer Chromatography

• TLC lab technique hints
• Do not touch the face of the TLC plate. Edge
  only.
• Write lightly on the plate. Avoid chips/gouges in
  the plate surface.
• Slide watch glass off beaker instead of lifting
  it off to maintain solvent vapor saturation in
  beaker.
• Spot the solutes in the same order each time.
• Keep spots small (2 mm maximum).
• Use a new spotter for every solute every time.
• Dont get the solutes too close to the plate
  edge.
• Separate spotting points evenly.
• Do not disturb beaker during development.
• Ideal Rfs are between 0.25 and 0.75.
• Consider using mixed solvent to achieve this

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After Lab

• Calculations
• For Part A Calculate the Rf value for each
  solute in each solvent.
• For Part B Calculate the Rf for each known
  solute and each solute in the unknown.
• Results and Discussion
• Identify the solutes in the unknown and write a
  short paragraph describing the process you used
  to figure it out.
• What conclusions did you come to regarding
  solvent and solute polarity in connection with
  TLC rate of elution? (See original purpose).