Preparative

Chromatography
 Preparative = “More”
• More mixture
– Isolate and characterize separated compds
• More silica gel
– Larger container (column v. thin-layer plate)
• More solvent
• More time
 Agenda
• Building (packing) column
– Equipment, silica, compression + test
• Applying mixture (loading)
• Collecting (eluting) fractions
• Testing fractions
 Pack Column
• Sintered glass funnel + conical adaptors +
filter flask (+ aspirator)
• Load silica LOOSELY to brim
• Compress
1) Rubber or glass stopper 2) Apply vacuum
• Test
– Head space
– Solvent flow (filter paper protects)
– Cavities + channels
 Apply Mixture
• Make mixture
– Weigh mixture (~ 400 mg)
– Add few drops EtOAc & mix
– Dilute w/ hexane
• No vacuum
• Apply entire mixture to paper (spread)
• Apply vacuum
 Collect Fractions
• Insert clean test tube
– Funnel tip INSIDE tube
• Apply vacuum
– Squeeze (& listen)
• Pour solvent mixture (~25 mL)
– Flows into tube (don’t overfill)
• Release vacuum
• Remove tube
 Solvent Mixtures
• 0% EA: 100% H Æ 40% EA: 60% H
– Increment %EA between fractions
• Method
– Make mixture in 100 mL grad cylinder
– Pour (#1)
– Add little EA, mix & pour (#2)
– Add little EA, mix & pour (#3)
– Add little EA, mix & pour (#4)
• Copy recipes into lab notebook (LEFT)
 Test Fractions
• Collect 20 fractions
• Find HOT fractions (absorb UV)
1 2 3 4 5 6 7 8 9 10

20 19 18 17 16 15 14 13 12 11

• Elute HOT (only) vs. 5% unknown

• Must get both compds off column

7 U 8
 Lucky People Only
• Combine fractions that contain same pure
compound
• Evaporate
– Once using rotary evaporator
• Switch from large RBF to small RBF (rinse large)
• Weigh small RBF
– Again using vacuum pump
• Weigh small RBF