Chromatography Fall 09


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Chromatography Fall 09

  1. 1. Thin-Layer Chromatography and Column Chromatography
  2. 2. Background <ul><li>Chromatography: refers to several related techniques for analyzing, identifying, or separating mixtures of compounds. </li></ul><ul><li>Two-part operation: </li></ul><ul><ul><li>The sample mixture is placed into a moving liquid or gas: mobile phase </li></ul></ul><ul><ul><li>The mobile phase carries the sample through a solid support: stationary phase </li></ul></ul><ul><ul><li>Compounds move through the stationary phase at different rates due to different attractions from the mobile and stationary phases </li></ul></ul>
  3. 3. <ul><li>Alumina and silica gel are the most commonly used stationary phase (adsorbents): </li></ul><ul><ul><li>They are extremely polar, so they have strong attractions to compounds </li></ul></ul><ul><li>Organic solvents with different polarities make the mobile phase (eluent): </li></ul><ul><ul><li>The more polar an eluent, the greater is its eluting power, that is, its ability to move compounds through the adsorbent. </li></ul></ul>cyclohexane petroleum ether hexane toluene dichloromethane ethyl acetate ethanol acetone methanol least polar most polar TLC vs. Column Chromatography
  4. 4. TLC vs. Column Chromatography <ul><li>TLC </li></ul><ul><ul><li>TLC plate: a plastic plate coated with a thin layer of silica gel. </li></ul></ul><ul><ul><li>The silica gel ( adsorbent ) adsorbs the mixture compounds strongly. </li></ul></ul><ul><ul><li>As the solvent ( eluent ) travels up through the adsorbent, the compounds in the mixture move with the solvent. </li></ul></ul><ul><li>Column </li></ul><ul><ul><li>Tube instead of a plate. Eluent travels down. </li></ul></ul><ul><li>Adsorbent vs Eluent: </li></ul><ul><ul><li>Adsorbents are typically much more polar than eluents The more polar a compound from the mixture is, the more strongly it adheres to the adsorbent and the slower it moves. </li></ul></ul><ul><ul><li>The more polar the eluent is, the less strongly a compound adheres to the adsorbent and the faster the compound moves . </li></ul></ul>
  5. 5. Thin-Layer Chromatography
  6. 6. Spotting, Developing, and Visualizing <ul><li>Spotting: </li></ul><ul><ul><li>Mark the origin by with a pencil (1 cm from the edge). </li></ul></ul><ul><ul><li>Mark the spot(s) where sample(s) will be spotted. </li></ul></ul><ul><ul><li>Dissolve sample in a volatile solvent such as acetone or dichloromethane. </li></ul></ul><ul><ul><li>Use a glass capillary tube to apply a small amount of sample onto the plate (too much sample leads to tailing). </li></ul></ul>
  7. 7. Spotting, Developing, and Visualizing <ul><li>Developing: </li></ul><ul><ul><li>Place the plate in a developing chamber (beaker). Solvent in the chamber should be lower than the origin on the plate . </li></ul></ul><ul><ul><li>When the eluent front reaches about 1cm from the top of the plate, remove the plate and mark the eluent front immediately. Then let it dry. </li></ul></ul>
  8. 8. Spotting, Developing, and Visualizing <ul><li>Visualizing: </li></ul><ul><ul><li>Not necessary for colored compounds </li></ul></ul><ul><ul><li>UV for fluorescent compounds </li></ul></ul><ul><ul><li>Iodine vapor to form a colored complex with many compounds. </li></ul></ul><ul><ul><li>Note: Mark the spots with a pencil. </li></ul></ul>UV Light Iodine
  9. 9. Retention Factor (R f ) <ul><li>Definition: </li></ul><ul><li> distance traveled by compound, mm </li></ul><ul><li> R f = ———————————————————— </li></ul><ul><li> distance traveled by eluent front, mm </li></ul>Eluent Front S A 1 Origin A 1 Rf 1 = ———— S <ul><li>As long as the stationary and mobile phases and temperature are kept constant, R f of individual compound is reproducible. </li></ul><ul><li>An increase in eluent polarity increases the R f . </li></ul><ul><li>Two different compounds can sometimes have the same R f in a given eluent, but they are unlikely to have the same R f in another eluent of different polarity . </li></ul>1 2 A 2 A 2 Rf 2 = ———— S
  10. 10. What You Need for TLC Experiment <ul><li>6 TLC Plates </li></ul><ul><li>5 micropipets (one for each solution, do not mix them !) </li></ul><ul><li>4 clean test tubes </li></ul><ul><ul><li>0.5 mL of “stock solution” </li></ul></ul><ul><ul><li>0.5 mL of benzophenone </li></ul></ul><ul><ul><li>0.5 mL of biphenyl </li></ul></ul><ul><ul><li>0.5 mL of benzhydrol </li></ul></ul><ul><li>Vial with unknown </li></ul>
  11. 11. TLC Experiment <ul><li>Omit Part 1. </li></ul><ul><li>Part 2: Al foil is used instead of plastic wrap in Fig. 4. </li></ul><ul><li>Part 4: Only ethyl acetate, hexane and toluene are used (so only 3 plates are needed). </li></ul><ul><li>Part 5: Use only 2 plates (ask TA how). </li></ul><ul><li>Part 7: All solvents go into a waste bottle. </li></ul><ul><li>Turn in ALL plates with your report (in an envelope or zip lock bag). </li></ul>
  12. 12. Column Chromatography
  13. 13. 1- Preparing a Dry Pack Column <ul><li>In a short stem Pasteur pipet place: </li></ul><ul><ul><li>A small cotton plug at the tip (don’t make it too tight otherwise eluent won’t flow). </li></ul></ul><ul><ul><li>Attach the column to a support stand using a piece of paper towel ( make sure it is vertical ). </li></ul></ul><ul><ul><li>Pour a little bit of sand, then the alumina powder (tap the side of the pipet to pack firmly). </li></ul></ul><ul><ul><li>Add 90 mg of the already prepared mixture sample: ferrocene/acetylferrocene/alumina. </li></ul></ul><ul><ul><li>Add a little bit more of alumina to protect the surface of the sample from disruption when adding the solvent. </li></ul></ul>
  14. 14. 2- Eluting Ferrocene <ul><li>Label a beaker hexane, fill with hexane </li></ul><ul><li>Place an empty beaker or erlenmeyer flask (labeled hexane) under the column </li></ul><ul><li>Add hexane to the top of the column (allow the liquid to flow down the side of the column, taking care not to disturb the alumina bed) </li></ul><ul><li>Collect the hexane as it elutes from the column </li></ul><ul><li>Switch to a flask (labeled ferrocene) to collect ferrocene (yellow) </li></ul>
  15. 15. 3- Eluting Acetylferrocene <ul><li>Label a beaker TBME ( t -butyl methyl ether), fill with TBME </li></ul><ul><li>Place an empty erlenmeyer flask (labeled TBME) under the column </li></ul><ul><li>Add TBME to the top of the column </li></ul><ul><li>Collect the TBME as it elutes from the column </li></ul><ul><li>Switch to a flask (labled acetylferrocene) to collect acetylferrocene when it comes out (orange). </li></ul><ul><li>After all the acetylferrocene has eluted, stop adding solvent </li></ul><ul><li>Note: If crystals form at the tip of the column, use TMBE to rinse into the flask </li></ul>
  16. 16. Observations <ul><li>Observe and record the color of the solutions containing the ferrocene and the acetylferrocene </li></ul><ul><li>Check with TA and get his approval </li></ul><ul><li>Pour both solutions into the waste bottle </li></ul>Ferrocene in hexane Acetylferrocene in TBME
  17. 17. Experiment <ul><li>Part 1: It’s unnecessary to weigh the flasks. Larger Erlenmeyer flasks may be substituted for the 50 ml flasks. </li></ul><ul><li>Part 3: “Preparing the Sample” has already been done. Weigh out 90 mg of the prepared ferrocene/acetylferrocene/alumina sample. </li></ul><ul><li>Part 7: Omit. Only observe and record the color and show your TA. </li></ul>
  18. 18. Waste Disposal <ul><li>Waste solvents are collected in the hood. </li></ul><ul><li>Column glass pipets will be collected in the hood for proper disposal. </li></ul><ul><li>Good to know if you want to have a good grade: </li></ul><ul><ul><li>Your grade will suffer if you do not submit your TLC plates . 50% of your grade is based upon the results of your lab work and 50% upon your answers to lab questions and the write-up of your report. </li></ul></ul><ul><li>Have fun! </li></ul>