Exp 1 - Separation of Limonene Name ______And Carvone

CHEM 108M, Binder UCSC Experiment 1 - Separation of Carvone and Limonene Reading: Mohrig Section 18

Terpenes encompass a large family of organic compounds widespread in nature and occurring in all organisms from bacteria to mammals. They are prevalent in higher plants, where they act as volatile chemical messengers to attract insects and also to defend the plant’s territory. Mixtures of volatile and odoriferous compounds, very rich in terpenes, can be obtained by steam distillation of plant tissues (as in the isolation of citrus oils experiment). These mixtures are called essential oils. In this experiment, you will separate carvone and limonene from the essential oil of either spearmint or caraway by column chromatography, a type of adsorption chromatography. The principles of column chromatography and TLC are identical. The stationary phase is silica (SiO2) and the mobile phase is an organic solvent. In this experiment, you will pack a microcolumn with silica gel and use an eluotropic series of hexanes and acetone to separately elute carvone and limonene. As in TLC analysis, this separation is based on polarity. The only difference is in the direction of the movement of the mobile phase. In TLC, the solvent (mobile phase) moves by capillary action against gravity. In column chromatography, the mobile phase is added to the top of the column and moves down with gravity. The stationary phase (silica) is polar and will hold onto the more polar compounds longer. In other words, the less polar compounds in a mixture will elute first. The solvent collected at the bottom of the column can be collected and separated over time. You will collect limonene and carvone in separate fractions and analyze these fractions by gas chromatography (GC) and IR spectroscopy. Carvone is a naturally occurring ketone found in the essential oils of caraway, dill, and spearmint in association with other terpenoids such as limonene. Limonene is found in spearmint, caraway, lemon, and orange oils. Carvone and limonene, both monoterpenes, have only one stereogenic center and therefore they can exist in two enantiomeric forms: R and S. Enantiomers have the same physical and chemical properties, except that they interact differently with polarized light and other chiral molecules. The two enantiomers of carvone have different smells, one fresh and minty, and the other sweet and somehow unpleasant, especially at high concentrations. The olfactory receptors that line our nasal mucus are chiral ensembles with an exquisite sensitivity to the size, shape and chirality of the odorant. Their interaction with individual enantiomers is often specific, resulting in the production of distinctive smells. The odor difference between the two enantiomeric forms of carvone is obvious to most people.

(+)-(R)-Limonene (+)-(S)-Carvone d-Limonene d-Carvone [ ] = +100 [ ] = +62 ! D ! D Figure 1. Structures of limonene and carvone.

(S)-(+)-Carvone is found in association with (R)-(+)-limonene in caraway oil (Figure 1). The composition of the oil, obtained by steam distillation from dried, ripe fruits of Carum carvi L., varies according to the source of the fruits but normally contains 50-55% carvone and 40-45% 20 limonene. Thus, caraway oil has a positive specific rotation ([α]D +70 to +80). (R)-(-)-Carvone along with (S)-(-)-limonene are the constituents of spearmint oil. Spearmint oil is obtained by steam distillation from the partially dried leaves of the flowering plants Mentha spicata L.

1 CHEM 108M, Binder UCSC Spearmint oil normally contains 13-21% limonene and 46-70% carvone and minor components such as α- and β-pinene (4-6%) and dihydrocarvone (3-12%). In contrast to caraway oil, 20 spearmint has a negative sign of rotation of plane-polarized light ([α]D -48 to -59).

Notebook Preparation – see sample notebook page on eCommons Purpose: Write the purpose of the experiment in one complete sentence. Draw the process scheme and structures of limonene and carvone at the top of the page. Reagent Table: Make a table with the physical properties of silica (SiO2), carvone, limonene, hexanes (n-hexane), and acetone. Include amount to be used (mL), MM, b.p., density, and a one-word hazard description, where appropriate (flammable, corrosive, etc.). Procedure: Hand-written, step-by-step procedure with micro-column diagram (see below). Clean-up & Safety: Copy the table at the end of the procedure into your notebook.

Experimental Procedure:

Separation of Carvone and Limonene There will be a demo for the setup in the beginning of lab. A short-tipped disposable Pasteur pipet makes an appropriate column for microscale column chromatography. To prepare the column put a small piece of tygon tubing of the tip of the pipet. Clamp the tubing with a screw clamp. Clamp the microcolumn in a vertical position to a ring stand. Place a small cotton plug CHEM 108M, Binderat the bottom of the column and cover the cotton with a thin layerUCSC of sand (2mm). Add hexanes to the column and let about 1 mL of the solvent run through. This will get rid of air bubbles sand), add little bytrapped little thein the first cotton. solvent Recycle from above this solvent (use a for Pasteur the next pipet step. to doBe this).sure to Keep leave a little solvent collecting the elutentbehind in the in thevial microcolumn. labeled “fraction 1” until 2 mL has been added to the column. Do not let the column run dry in between fractions. Add the next solvent, and begin collection into “fraction 2” and soMicrocolumns on until all 5 runsolvents very fromquickly, above so ithave is important been eluted. that you Allow have the everythingcolumn to runprepared (solutions, dry when collectingpipets, “fraction and 5”.receiving containers) before starting the column. In five labeled test tubes deliver the following solvent volumes (these solvents will be used in succession to run the column): Place a boiling chip in fraction 5 and evaporate the solvent by placing the vials in a sand bath on a hot plate in the (1) fume 2 mL hood, hexanes until ; about(2) 2 0.2 mL-0.3 hexanes mL of; liquid (3) 2 remains.mL hexanes Do; not evaporate to dryness. (4) 2 mL 10% acetone in hexanes; (5) 2 mL 10% acetone in hexanes.

Cover the test tubes with a small piece of parafilm to prevent evaporation. Label 5 scintillation vials 1-5. These vials will be used to collect the column fractions. A fraction is complete when a test tube of solvent has been used up. For example, you will elute from the column into “Fraction 1” until the first 2 mL hexane portion has been delivered. You will not run the column dry so not all of that 2 mL hexane will end up in “Fraction 1.”

Packing a microcolumn:

Weigh about 1g of silica gel, or estimate the amount by comparison to the 1g sample provided. Mix the measured amount of silica gel with enough hexanes to make a pourable slurry. Use a plastic pipet with the tip cut off to suck up the slurry and pour it into your microcolumn from above. The silica gel will be free-flowing and then settle towards the bottom of the microcolumn. Bubbles can be tapped out using a microspatula. Do not apply too much pressure to the plastic Pasteur pipet, as it will cause the tygon tubing to blow off the tip of the microcolumn. Polarimetry Your TA will announce when the polarimeter is equipped with the solution of your oil. Only TAs 2 are to handle polarimeter cells. Measure the optical rotation of your oil and calculate the specific rotation using the concentration given in the lab (pathlength is 2 dm).

GC analysis Analyze the crude oil of your choice (A or B) by Gas Chromatography using a column of medium polarity at 150˚ C. Determine the percent composition of limonene and carvone in the unknown oil. Analyze fractions 1 and 5 by GC. Determine the percentage of carvone and limonene in fractions 1 and 5. Report your data in table format and attach the chromatograms to the back of the lab report.

IR Analysis Obtain the IR spectrum of fraction 5 on NaCl plates. Indicate the identifying stretch. Do not analyze every peak! Attach the IR spectrum to the back of the lab report

Clean-up Safety Liquid waste: acetone, hexanes, and fractions Acetone & hexanes are flammable Solid waste: pipets, columns, dry silica Caraway and spearmint oils are irritants Put away hotplate Silica is an irritant ALL STUDENTS IN ANY SECTION WITH GLASSWARE (INCLUDING PIPETS) IN ANY OF THE TRASHCANS WILL LOSE 5 POINTS FROM THEIR LAB REPORT

3 CHEM 108M, Binder UCSC

Repeat this process of pouring slurry into the microcolumn as many times as necessary until the adsorbent reaches the indentation level of the Pasteur pipet. A small portion of sand is added on top of the column (about 3mm high) and the column is ready to be loaded.

Loading the microcolumn: After the column has been packed, open the column outlet (screw clamp) until the solvent level comes down the top of the sand (not lower) and load the column with 150 microliters (0.15 mL) of the oil. Use a pluringe to measure this volume. Open the screw clamp and start collecting the elutent in the vial labeled “Fraction 1”. Without allowing the column to run dry, and after the sample has penetrated the column (liquid level right above the sand), add little by little the first solvent from above (use a Pasteur pipet to do this). Keep collecting the elutent in the vial labeled “fraction 1” until 2 mL has been added to the column. Do not let the column run dry in between fractions. Add the next solvent, and begin collection into “fraction 2” and so on until all 5 solvents from above have been eluted. Allow the column to run dry when collecting “fraction 5”.

Place a boiling chip in fraction 1 and fraction 5 and evaporate the solvent by placing the vials in a sand bath on a hot plate in the fume hood, until about 0.2-0.3 mL of liquid remains. Do not evaporate to dryness.

Polarimetry Your TA will announce when the polarimeter is equipped with the solution of your oil. Only TAs are to handle polarimeter cells. Measure the optical rotation of your oil and calculate the specific rotation using the concentration given in the lab (pathlength is 2 dm).

IR Analysis Obtain the IR spectrum of fraction 5 on NaCl plates. Indicate the identifying stretch. Do not analyze every peak! Attach the IR spectrum to the back of the lab report

3 CHEM 108M, Binder UCSC Introduction (Pre-lab Questions) – typed responses to be turned in at the beginning of lab. Do not re-write the question. Include with the lab report. Do not change your responses.

1. State the approximate percent composition of limonene and carvone in both spearmint and caraway oils.

2. In general, is there any correlation between R and S, and d and l enantiomers? Hint: Look at the structures and names on the first page of this handout.

3. Describe column chromatography in terms of the type of chromatography as well as the nature of stationary and mobile phases.

4. What functional groups are found in limonene and carvone (alkane is not a functional group!). Which compound, limonene or carvone, do you expect would elute first from the silica gel column? Which will have a shorter retention time on the GC? Briefly justify your answer.

5. Is it possible to separate carvone and limonene using the following series of solvents: acetone (100%), acetone:hexanes (50:50) and finally hexanes (100%)? Explain.

Results (In-lab questions) – typed and turned in with the lab report, due one week after the experiment. It is recommended that students write the in-lab questions in the notebook before lab (an abbreviated version is fine).

1. Report the observed and specific rotation of your oil. List the unknown code and the suspected identity of your oil based on polarimetry.

2. Briefly justify the elution order of limonene and carvone from the silica and GC columns.

3. In one or two sentences, comment on the success of the separation based on GC results. Report the GC results of the crude oil and fractions 1 and 5 from your separation. Calculate the percentage of carvone and limonene in the crude oil and fraction 5. Show one sample calculation each for retention time, integration, and percent composition. Report the final results in table format.

2 Sample Rt (sec) ID (Limonene/Carvone) Integration (cm ) % Composition Oil

(Continue with other samples – fraction 1 and fraction 5)

4. Report the most distinctive IR stretch found in fraction 5. List the functional group and bond responsible for this stretch.

5. Is it possible to isolate pure limonene from spearmint or caraway oil by using just hexanes as the elution solvent? Discuss.

Attach a copy of the gas chromatograms and IRs to the back of your report. Be sure that each chromatogram contains the sample name and GC conditions. If your lab partner has some or all of the GCs and IRs, be sure to state that at the top of the results section.

Abstract – see eCommons post on Technical Writing for guidelines on writing the abstract. Do not write it without consulting these guidelines!

4 CHEM 108M, Binder UCSC Exp 1 - Separation of Limonene Name ______and Carvone

Section Day _____ Time _____ TA Name ______

CHEM 108M GRADING RUBRIK - Use as cover page for report SECTION INSTRUCTOR COMMENTS POINTS ASSIGNED

IN-LAB QUIZ / 5

LAB REPORT – Staple the report together in this order before coming to lab:

ABSTRACT One paragraph, four sentences: Purpose, procedure, main result(s), and / 10 conclusion(s).

INTRODUCTION Original responses to pre-lab questions with TA initials / 30

RESULTS The main results are stated, as outlined in the in-lab questions, using complete / 30 sentences.

DISCUSSION and CONCLUSION Reflection of whether results were as NONE expected and whether it is supported by 0 / 0 the theory behind the experiment.

EXPERIMENTAL SECTION The experimental details (including final NONE amount used and obtained) are briefly 0 / 0 described in a few sentences.

NOTEBOOK PAGES Proper format: reaction scheme, chemical / 15 info table, procedure, waste and clean-up procedure.

NEATNESS AND ORGANIZATION Proper order and format (see syllabus for / 10 full descriptions of each section), spelling & grammar

LAB REPORT TOTAL / 100