Identification of an Unknown ALdose

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Identification of an Unknown ALdose

• CHEM 203

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In this experiment, you will determine the
identity of an unknown aldose by

• Bial test to differentiate aldopentose and
  aldohexose.
• Measurement of the specific rotation of the
  aldose.
• Reducing the aldose to its alditol and
  determining its optical activity.
• Converting the aldose to its phenylosazone and
  determining its melting point.

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Possible Aldoses
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Part 1 Bial test

• This test is used to differentiate aldopentoses
  and aldohexoses.
• Aldopentoses
• react very rapidly
• give a blue-green color
• Aldohexoses
• react slower
• result in reddish-brown solutions

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Part 1 Bial Test

• Bial Reagent is FeCl3, HCl and orcinol
• Experimental Dissolve a small amount (about 5
  mg) of your unknown aldose in 1 mL of water in a
  test tube. Add 1 mL of Bials reagent and a
  boiling stone. Heat the test tube on a steam
  bath until it just boils. Remove from the heat
  and record the color.

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Part 2 Optical Activity

• Remember that the Fisher Projection represents
  chiral centers
• Each aldopentose and aldohexose will have its own
  unique combination of /- optically active
  centers resulting in a net rotation

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Part 2 Optical Activity

• Using a polarimeter you will determine the aobs
  for your aldose
• Remember from our discussion of optical activity
  that the magnitude of rotation is cell length and
  concentration dependent

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Part 2 Optical Activity

• Once you have your aobs from our polarimeter you
  will need to convert that value to a specific
  rotation or aD
• aD aobs / (l x c) where l path length (1
  dm) c concentration in g/mL
• You are using a 1 decimeter cell that holds 10
  mL

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Part 2 Optical Activity
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Part 3 Reduction to Alditol

• By reducing the aldehyde with NaBH4 you produce a
  new compound with unique properties optical
  activity / melting point

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Part 3 Reduction to Alditol

• For some aldoses, the introduction of a plane of
  symmetry will cause loss of optical activity!

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Part 3Reduction to Alditol

• Experimental Dissolve 1.0 grams of the unknown
  aldose in 5 mL of water in a small flask by
  stirring. Dissolve 0.1 grams sodium borohydride
  in 2.0 mL of 1M NaOH in a test tube. Cool both
  solutions in an ice/water bath for 5 minutes or
  more.
• Remove both solutions from the ice bath. Using a
  Pasteur pipette, add the sodium borohydride
  solution to the aldose solution drop-by-drop with
  constant stirring over a 10-minute period. Do
  not let the temperature rise over 25C, if it
  does cool the flask in an ice/water bath for a
  minute or so, then continue your addition. When
  the addition is complete, let the solution stir
  at room temperature for 20 minutes. Add 6M HCl
  to the beaker dropwise until the foaming stops
  and the solution turns blue litmus paper red.
• Cool the reaction mixture in an ice/water bath to
  crystallize the product it may be necessary to
  scratch the side of the beaker to induce
  crystallization. For best results, put Para film
  over the top of the beaker and place it in the
  refrigerator until next lab period
• Once crystallization is complete, collect the
  crystals by vacuum filtration wash the crystals
  with two portions of cold 95 ethanol. Dry the
  crystals as well as you can by pressing them
  between two pieces of filter paper. Prepare an
  aqueous solution of the alditol (0.5 grams in 10
  mL of water) and determine its optical activity.

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Part 4 Preparation of phenylosazone

• Derivatives of compounds offer another unique set
  of physical properties to further ascertain the
  identity of a compound
• Aldehydes and ketones react with hydrazines to
  form hydrazones

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Part 4 Preparation of phenylosazone

• Careful both D-glucose and D-mannose give the
  SAME phenylosazone. When the phenylosazones are
  formed both C-1 and C-2 form new C-N double
  bonds. Therefore, the difference in
  configuration at C-2 in glucose and mannose is
  lost. The remaining structural unit, C-3 through
  C-6, (shown in the boxes above) is the same for
  both glucose and mannose

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Part 4 Preparation of phenylosazone

• Experimental Dissolve 0.5 grams of the unknown
  aldose in 10 mL of water in a 50 mL Erlenmeyer
  flask, stir in 1.0 grams of phenylhydrazine
  hydrochloride, 1.5 grams sodium acetate
  trihydrate and 1 mL of saturated aqueous sodium
  bisulfite. Heat the flask on a boiling water
  bath for 30 minutes with occasional swirling.
  Add 15 mL of water to the flask and cool in and
  ice/water bath. Collect the crystals by vacuum
  filtration, wash the crystals with a small amount
  if ice-cold methanol. Allow the crystals to dry
  in your drawer until next lab period. Once they
  are dry, obtain a weight and melting point for
  your crystals

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Part 4 Preparation of phenylosazone

• Of the 12 D-aldohexoses, there are only 6
  possible phenylosazones whose melting points are
  in the handout

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Part 5 Putting it together

• With experimental error or possibly a failed
  experiment, not all of your data may match
  perfectly
• Keep good observations here is where the
  notebook is key so that if you are forced into a
  3 of 4 or worse yet 2 of 4 situation, you know
  which of your experiments you can rely on most!

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Part 5 Putting it together

• Your report should have the following elements
• Introduction
• Data from each test
• Discussion discuss your logic in applying the
  data into solving the identity of the unknown
• Conclusion