Exercise K.1 Thin Layer Chromatography Thin Layer Chromatography of Analgesics and Natural Products

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Exercise K.1Thin Layer ChromatographyThin
Layer Chromatography of Analgesics and Natural
Products
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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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Effects of a Polar Stationary Phase

• Polar solutes are held more tightly than
  non-polar solutes
• So, will polar or non-polar solutes travel faster
  up the TLC plate?
• _______________________
• Always? Always.

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Effects of Solvent Polarity

• Polar solvents compete better with the polar TLC
  plate so all solutes (even non-polar solutes)
  elute faster with polar solvents
• We say that polar solvents have greater eluting
  power than non-polar solvents.
• Always? Always.

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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)
TLC plate
Process

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

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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

• To observe the effects of solvent polarity on
  order and rate of elution
• To identify components in an unknown using TLC
  data

caffeine
acetaminophen
vanillin
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Pre-lab Preparation

• Read Technique K (pp. 92-97)
• Omit Resolution and Column Chromatography
• Prepare Notebook
• Header info
• Purpose statement
• Procedural reference
• Table of reagents
• Formula, structure, MW and hazards of ethyl
  acetate, hexane, and the compounds to be
  separated
• Remember your data source(s)

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Pre-lab Preparation

• Notebooks Procedures
• Use the steps outlined earlier to write your
  procedures (2-column format)
• In addition, incorporate the following
• Check the newly spotted TLC plate under the UV
  before you develop it.
• Be sure you can see the spots before you start!!
• Two solvent systems will be used (run the known
  compounds in both of these)
• 100 ethyl acetate
• 50/50 mixture of ethyl acetate and hexanes

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Procedures continued

• Part One using Known Compounds
• Work in pairs. Each student will run one of the
  two solvents.
• Spot all three known compounds on each plate and
  develop.
• Compare and share Rf data for each solvent. Write
  the data and a sketch of the TLC plate in your
  notebook.
• Part Two using Unknown Mixtures
• Work individually. Each student gets an unknown
  containing at least one of the knowns.
• Spot the unknown and all three known compounds.
  You choose your development solvent system based
  on your observations from part one.

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In Lab

• Follow your procedures to collect the Rf data for
  Parts 1 and 2.
• Recap
• Part One pairs, share data.
• Part Two individually, identify unknown.

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TLC Plates
Part One
Part Two
?
?
1 cm.
1 cm.
CAF ACE VAN
UNK CAF ACE VAN
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In/After Lab

• Calculations
• Calculate the Rf value for each known compound in
  both solvents systems. (6 calcs)
• Calculate the Rf value for each compound in the
  unknown mixture. (4-6 calcs)
• Results
• Discuss your observations concerning the known
  compounds in the two solvent systems.
• Identify the compounds in your unknown and the
  rationale for your choice.

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Thin Layer Chromatography

• TLC lab technique hints
• 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).
• Never double dip spotting pipette.
• 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