Analysis of Analgesic Tablets by Thin Layer Chromatography (TLC)

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Analysis of Analgesic Tablets by Thin Layer
Chromatography (TLC)

• Organic Chemistry Lab I
• Fall 2008
• Dr. Milkevitch
• Oct 12 14, 2009

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Separating Molecules

• Today Learn a separation technique
• Molecules can be separated by a number of
  methods
• Extraction
• Precipitation
• Chromatography
• Chromatography to write in color
• Originally developed to separate components of
  inks

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Thin Layer Chromatography

• Thin Layer Chromatography The separation of
  moderately volatile or nonvolatile substances
  based upon differential adsorption on an inert
  solid (stationary phase) immersed in a organic
  solvent or solvent mixture (mobile phase)
• Makes use of
• A stationary phase solid support
• Mobile phase a fluid
• Basic principle of separation
• Differential interactions of molecules with these
  2 phases

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How the separation works

• Molecules can move with the mobile phase
• Or, they can remain fixed on the stationary phase
• Called partitioning
• How molecules partition between the 2 phases
  depends on
• Properties of the phases
• Properties of the molecules being separated

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How this works, continued

• Molecules that partition into the mobile phase
• Will move more rapidly than molecules that
  partition into the stationary phase
• Therefore, they will separate from those that are
  partitioned into the stationary phase

Faster moving component partitioned more into
mobile phase
Solvent movement
Slower moving component partitioned more into
stationary phase
Two component mixture applied to TLC plate
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Nuts and Bolts of the Method

• You must carefully select mobile and stationary
  phases to achieve full separation of mixtures of
  molecule
• Trial and error
• Stationary phase thin layer of silica spread on
  a surface
• Mobile phase solvent system that migrates
  through the silica
• Samples are spotted on the plate
• Moved along by the solvent(s) migrating through
  the silica

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Rf Values

• Molecules that are separated will migrate as
  spots and their migration can be measured
• Migration usually reported as an Rf value
• Calculation of an Rf value
• Ratio of sample migration (how far spot moved) to
  solvent migration (how far the solvent moved)
• Can be used to identify components in a mixture
• Compared to standards
• Today look at the components of analgesic
  tablets by TLC
• Look for the actives

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Calculating an Rf Value
Solvent Front
Solvent Front 5 cm Spot 1 moved 1 cm
Rf 1/5 0. 2 cm Spot 2 moved 4 cm Rf
4/5 0. 8 cm
Origin
Spot 1
Spot 2
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Analgesic Medications

• Analgesia Greek for the deadening or absence
  of pain without loss of consciousness
• Analgesics compounds that relieve pain
• Range from aspirin to morphine, and other related
  narcotics
• Many OTC (over the counter) analgesics available
• Tylenol
• Advil
• Excedrin
• Aspirin (various preparations)

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Actives

• OTC analgesics contain many compounds
• The active is what relieves pain
• Other compounds are binders, flavorings,
  colorants, stabilizers, solubility enhancers

Paracetamol (acetaminophen)
Ibuprofen
Caffeine
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Procedure

• Obtain a silica gel plate from me
• This plate will have a UV indicator in it
• Draw a straight line (lightly, in pencil)
  approximately 2 cm from the bottom edge of your
  TLC plate. Use a ruler to do this. This is
  called the origin.
• Draw a straight line (lightly, in pencil) about 1
  cm from the top edge of your TLC plate. Again,
  use a ruler to do this. This is called the
  solvent front.
• Mark 5 spots (in pencil) along the origin.
  Distribute the spots evenly along the origin.
• Obtain the TLC standards. These are solutions of
  known components of the analgesic tablets. They
  include
• Caffeine
• Acetylsalicylic Acid (aspirin)
• Ibuprofen
• Acetaminophen

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Procedure, cont

• Spot the TLC plates with the TLC standards and 1
  unknown. Use a spotting capillary or the small
  diameter plastic dropper. Make the spots small
  (approx 1 mm in diameter).
• Before you do this, ask me or Scott/Nicole to
  demonstrate on your plate
• Preheat the TLC plate in the 50 C oven for 5 min.
  This will dry the spots. Sometimes you will
  need to dry the plates for a longer period of
  time.

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What your TLC Plate will Look Like
Solvent front (drawn lightly in pencil)
origin
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Procedure (cont)

• Make a TLC tank (called a developing tank). Use a
  250 ml beaker. Add a few ml of the mobile phase
  to the beaker
• Mobile phase ethyl acetate
• Place a piece of filter paper in the beaker
• Will allow the beaker to fill with solvent vapor
• Cover TLC tank with a watch glass/parafilm

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TLC Tank
Watch glass
Filter paper
Mobile phase
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Developing

• Place TLC plate (spotted) in the tank. Solvent
  level must be below the origin, otherwise youll
  lose your spots
• Tilt plate over a bit onto the side of the beaker
• Shouldnt touch the filter paper
• Watch the migration of the solvent
• Remove the plate from the TLC tank when the
  solvent is exactly at the solvent front line
• Let the plate dry
• Visualize the plate using the UV box
• See me to demonstrate use of the UV box
• After this, further develop the plate in the
  iodine tank
• Iodine will react with the spots
• Makes the spots yellow
• When fully developed, remove and circle the spots
  with pencil

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Results

• Measure the distance the solvent (mobile phase)
  traveled
• Bottom to the solvent front line
• Measure the distance from the center of each spot
  to the origin
• Calculate Rf values for each spot

Distance spot traveled
Rf
Distance solvent traveled
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Results II

• Attempt to identify what the spots are in your
  unknown
• Compare the Rf values of the standards versus the
  Rf values of the spots in your unknown
• If they match closely, then you have that
  component in your unknown
• Can you identify what unknown you have?
• Possibilities Advil, Tylenol, Excedrin
• Be sure to draw a figure of your TLC plate
• Identify all standards, and all spots
• Show calculations of your Rf values

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Conclusions Things to Think About

• What can we conclude?
• Did your TLC plate work?
• Were you able to spot your plate successfully,
  and develop the plate?
• Did you see the spots for the standards?
• For your unknown, did you see a spot or spots?
• Were you able to resolve the different spots?
• Did the spots separate from each other?
• This is what we are trying to do!!!!
• Could you identify your unknown?
• See me for the identity of your unknown
• If you did, then you have successfully utilized
  TLC to identify your unknown compound