Organic Chemistry Laboratory I
Extraction of (+) and (-)-Carvone from Oil of Caraway and Oil of Spearmint

Experimental Procedure


Introduction
Students will work individually to isolate and analyze a carvone enantiomer from either oil of caraway or oil of spearmint using a combination of column chromatography (CC) and thin layer chromatography (TLC). All four students at each bench will use the same oil and isolate the same enantiomer.  (Benches A, B and C will use oil of spearmint and benches D, E and F will use oil of caraway.)  Each student will  analyze products by TLC, IR spectroscopy, and calculate his or her percent recovery of carvone.  Students will then pool their isolated carvone samples for analysis by polarimetry.  Students will be alloted two weeks to complete the experiment.  During week 1, students should complete the TLC analysis of the crude oil, column chromatography, TLC analysis of isolated fractions and evaporation of mobile phase from the fractions containing carvone and limonene.  Polarimetry and IR analysis will be done during week 2. 

Column Chromatography/Thin Layer Chromatography
TLC Analysis of Oil of Spearmint or Oil of Caraway
Prepare a silica gel TLC plate (~2cm X 5cm) by lightly drawing a line across the bottom of the plate with pencil.  Mark the plate with three short lines, perpendicular to the origin line.  Label each line as "C" for carvone, "L" for limonene and "O" for oil.  Label three test tubes as "C", "L", and "O" and dispense ~0.5ml of a 1% solution of carvone into the tube labeled "C", 0.5ml of 1% limonene into the tube labeled "L" and ~0.5ml of the oil into the tube labeled "L".  Using a microcapillary pipet, spot the plate with the appropriate solution.   Set up a small beaker containing acetone and place a KimWipe on the bench to rinse out the micropipet after each application, before proceeding to the next sample.  (Dip the micropipet into the acetone and touch it to the Kimwipe to drain it.  Repeat 2-3 times between applications of the samples to the plate to be sure that the micropipet is clean).  Develop the plates in 10% ethyl acetate : 90% hexane and mark the solvent front.  After the solvent evaporates from the plate, place the plate in an iodine chamber to stain any components on the plate.  Calculate the Rf values for carvone, limonene and the components in the oils.  Identify the components in the oil by comparison of the Rf values of components with carvone and limonene. 

Column Chromatography of Oil of Spearmint or Oil of Caraway
Column chromatography will be used to isolate carvone and limonene from caraway or spearmint oil.  Clamp an empty column (20ml syringe) to a ring stand.  Plug the outlet inside, on the bottom of the column, with a small piece of cotton or glass wool and place a 50 or 100ml beaker or Erlenmeyer flask under the column.  Set up a test tube rack with twenty 13 X 100 mm clean and dry test tubes.  Label the tubes 1-20 with a permanent marker along the top rim of the tube.  Set the rack aside.  Prepare a slurry of silica gel (7.0g) in hexane (~20ml) in a 50 or 100ml beaker.  Stir the slurry (stationary phase) and pour it into the coulmn.  Allow the silica gel to settle and the hexane to drain from the column until it just reaches the surface of the silica gel.  Gently tap the outside of the column to level the surface of the silica gel.  Measure a volume of approximately 0.5 ml into a small, pre-weighed vial.  Weigh the vial and the oil, and  determine the exact mass of the sample.  Record the mass in your notebook.  Dissolve the oil (either spearmint or caraway) in hexane (1ml).  Using a glass pipet, slowly dispense the hexane/oil solution onto the column by running the tip of the pipet around the inside diameter of the column, being careful not to disrupt the level surface of the silica gel.  Allow the solution to form a layer on the surface of the silica gel.  Drain the column until the oil/hexane solution is just adsorbed onto the silica gel.  Weigh out approximately 2g of sand and gently pour the sand onto the surface of the silica gel to create a protective layer.  Using a pipet, add hexane (mobile phase), a few ml at a time to ensure the oil is not re-dissolved in the mobile phase.  Begin collecting fractions into the test tubes.  Collect ~5ml of mobile phase (approximately a half full tube) draining from the bottom of the column into each consecutively numbered test tube (i.e., 5ml into tube #1, switch to tube #2, collect 5 ml, switch to tube #3 etc...).   Collect ten, 5 ml fractions (#1-10).  Change the mobile phase to 10% ethyl acetate:90% hexane.  Continue to collect 10 more, ~5ml  fractions into the remaining ten tubes (#11-20). 


Setting up the column



Labeled test tubes for collecting fractions
TLC Analysis of Isolated Fractions from the Column
TLC analysis is used to identify the specific components of the oil of spearmint or oil of caraway isolated from the column.  Fractions should be evaluated for the presence of both carvone and limonene by comparison with carvone and limonene standards.  Obtain four TLC plates (silica gel) approximately 2.5cm wide X 7cm long.  Using a pencil, draw an origin line across the first plate approximately 1cm from the bottom.  Mark the TLC plate
with four short lines evenly spaced along the bottom of the plate.  Label the first mark as "C" (carvone), the second as "L" and the remaining two marks as 1, and 3, corresponding to the collected fractions 1 and 3.  Mark the second plate in a similar way with four short marks along the bottom and labeled as "C" 7, 9 and 11.  Plates 3 and 4 will be marked with the carvone and fractions  C, 13, 15, and 17 (plate 3) and C, 19 and 20 (plate 4).   The carvone will serve as a reference for the components isolated from the column and must be included on all four plates.  Using a microcapillary pipet, spot each plate with the appropriate fraction.  Set up a small beaker containing acetone and place a KimWipe on the bench to rinse out the micropipet after each application, before proceeding to the next fraction.  (Dip the micropipet into the acetone and touch it to the Kimwipe to drain it.  Repeat 2-3 times between applications of the fractions to the plate to be sure that the micropipet is clean). Develop the plates in 10% ethyl acetate : 90% hexane and mark the solvent front.  After the solvent evaporates from the plate, place the plate in an iodine chamber to stain any components in the collected fractions.  Identify which fractions contain carvone by comparison of the Rf values of components in the fractions with the Rf of the standard carvone.  Combine all of the fractions that contain pure carvone in a 50ml Erlenmeyer flask. Set up a water bath on a hot plate, clamp the flask to a ring stand and immerse it in the water bath (slightly above the surface of the hot plate).  Gently warm the solution to evaporate the solvent.  When the volume in the flask reaches ~3-5ml, transfer the solution to a pre-weighed, clean, dry test tube (Record the weight in your notebook).  Clamp the tube to the ring stand and continue to warm the solution in the water bath to remove the remaining solvent (to a volume of  ~0.25-0.5ml). Wipe the exterior of the tube dry and weigh it again to determine the mass of the recovered carvone from the oil.  Calculate the percent recovery of carvone.  Repeat this process for the fractions that contain limonene. 

         
Analyzing fractions isolated from the column by TLC


Combining fractions containing carvone


Removing mobile phase solvent from combined fractions

IR Spectroscopy
After the percent recovery is determined, the carvone and limonene will be analyzed by IR spectroscopy.  Obtain a set of salt plates and set one of the plates on a Kimwipe.  Hold the plates on the sides to avoid getting fingerprints on the plates which can interfere with the analysis.  Using a disposable, glass pipet, place 1-2 drops of the liquid onto the center of the plate (Figure at right).  Set the second salt plate on top of the first, distributing the liquid evenly over the surface of the plate, creating a film.  Set the plates in the sample compartment of the IR spectrophotometer and run the sample.  Identify the characteristic peaks in the spectrum and determine if the experimental spectra obtained are  consistent with carvone and limonene.  Note the differences between these spectra. 
      


Polarimetry
Preparing the Sample for Polarimetry
At each bench, collect and combine all four isolated samples of carvone.  Determine and record the mass of the pooled sample.  Transfer the pooled carvone sample into a clean and dry 25ml volumetric flask.  Use a small amount of hexane to transfer samples (Do not exceed a total of 10ml of hexane for the transfer).  Fill the volumetric flask to the calibration line with hexane and thoroughly mix the solution.  Calculate the concentration of the solution in g/ml.  If the solution is cloudy or turbid, filter the sample through a disposable, glass pipet, fitted with a cotton plug before transferring the solution to the sample polarimeter tube.  Measure ~20ml of hexane into a graduated cylinder and transfer the hexane to the sample polarimeter tube. 
Mark the level of the solution in the tube using a permanent marker (Sharpie) (The level should be approximately 14 cm).  Using a ruler, measure the height of the solvent in the tube and record the value in decimetersReturn the tube to the polarimeter and record the rotation of the blank solvent. (See below).  Remove the tube from the polarimeter and empty the contents.  Transfer the solution from the volumetric flask to the polarimeter tube.  Mark the level of the solution in the tube using a permanent marker (Sharpie).  Using a ruler, measure the height of the solvent in the tube and record the value in decimeters. Return the sample tube to the polarimeter and measure the rotation.  FIlter the solution to remove turbidity

Transfer pooled carvone sample to volumetric flask

Removing/replacing the sample tube from
the polarimeter


Transfer carvone solution from volumetric flask to
polarimeter sample tube

Determining the Specific Rotation
Four students from each bench will pool their samples of carvone for determination of the specific rotation.  Since there can be significant variation in the observed rotation (and thus the calculated specific rotation), four readings of the same sample will be taken, with each student at the bench being responsible for taking one reading.  The students will then take an average of their four observed rotations, and use this average to calculate the final specific rotation for the sample.







Dial on the Polarimeter
        
A "rotation" must be measured for the solvent that is used in the analysis to establish a background that will then be subtracted from the observed rotation of the solvent plus the sample. With the instructor's assistance, set up the polarimeter (filter, polarizer, lamp, power source) and place the sample tube, filled with ~20ml of hexane into the polarimetercompartment. (Mark and measure the height of solvent (pathlength).  You will need this value to calculate the specific rotation.) Close the door of the compartment.  Set the dial on the top of the polarimeter to 00 and check for transmittance of light.  Rotate the dial in a clockwise direction until no light passes through (extinction) and the center of the circle viewed through the eyepiece is dark.  Move the dial back and forth a few degrees to determine the maximal point of extinction of light.  Record the value at which maximal extinct of light occurs.  Four readings for the hexane should be taken, one by each student at the bench.  Calculate an average and use this value for all calculations. Remove the sample tube from the polarimeter and empty the hexane into a clean beaker for re-use.  Reset the dial back to zero.

Transfer ~20ml of the carvone sample in hexane from the volumetric flask to the sample polarimeter tube.   (Fill the tube to the line marked previously when the rotation of hexane was determined.)  Insert the sample tube into the polarimeter.  Check the dial to ensure that it is in the zero position.  Rotate the dial clockwise until a position of maximal extinction is reached.  Record this value.  Each student at the bench should take a reading of the sample and an average value from the four readings determined.  Use the average value in all future calculations. Subtract the average value obtained for hexane from the average value obtained for hexane + sample (This may be either a positive or negative number).  Record this number as the observed rotation.  Calculate the specific rotation of the sample from the observed rotation, concentration and pathlength used.