CHEM 223.....Liquid-Liquid Extraction; Infrared Spectroscopy..............

Next to distillation for the separation of liquid organic products, liquid-liquid extraction is one of the most common preparative laboratory separation method for both liquids and solids. The separation of very complex mixtures such as toluene, benzoic acid, benzyl amine, and phenol can be effected by simple liquid extraction methods where the differential solubility of the molecules is used to rapidly resolve the mixture. The procedure involves a simple partitioning of a compound between two, immiscible solvents. The polarities of the solvents are usually significantly different to allow the almost total transfer of the compound to be separated into one layer. Quantitative transfers can be effected by extracting the mixture several times with small aliquots of the solvent and then pooling the solvent extracts to recover the compound quantitatively.

If water is one of the solvents, and it usually is, the small amount of water remaining in the organic phase after partitioning can be removed by drying the organic layer over anhydrous sodium sulfate for several minutes. The water remaining in the organic layer after extraction is taken into the waters of hydration of the crystalline solid which is virtually insoluble in the organic solvent. The solid is removed by pipetting the liquid away from the solid salt or the mixture can be filtered (by gravity or by vacuum filtration) to obtain the dry solvent containing the extracted compound.

A liquid-liquid extraction can be done on any scale necessary, with the limiting factor being the volume and cost of the solvents used. The normal method involves the use of a separatory funnel (of several possible designs) where the two layers can be mixed intimately by shaking or swirling. The quantitative separation of the desired compound from a mixture depends on its solubility in the extracting solvent (as measured by the partition coefficient) and by how completely the two layers are mixed. The nature of the mixing method used (shaking or swirling) is governed by the tendency of the materials in the two solvent to form an emulsion at the interface between the two layers and thus never separate completely. If an emulsion forms when the mixture is violently shaken in a separatory funnel, simple swirling can be more effective.

If an intractable emulsion is obtained, it can be broken by several techniques such as (a) salting out (if one solvent is water), (b) by simple mechanical mixing of the emulsion with a glass stirring rod, or (c) by centrifugation of the mixture. Each of these methods will resolve simple emulsions, but it is best to avoid the formation of an emulsion in the first place. Emulsions are normally a problem when bio-organic material containing some naturally occurring surfactant material is extracted with organic solvents. These surfactants are partially soluble in both layers and therefore help to stabilize the emulsion. Please note that mayonnaise is a almost permanent emulsion of water and oil which is stabilized by the surfactant phosphatidyl choline from egg yolks which are added while the oil and water are mixed in a blender. This particular emulsion is very difficult to resolve, unless microwave energy or centrifugation is used.

EXTRACTION

For this lab we will use liquid-liquid extraction to remove a flavoring agent, peppermint extract, from a commercially available alcohol-water mixture. The peppermint compounds will be partitioned between the aqueous-alcohol layer and a petroleum ether extracting solvent (mostly pentane and hexane isomers boiling between 30 and 60 degrees Celsius). The artificial food dyes present in the commercial preparation are polar and thus will remain in the alcohol-water phase and not affect the separation. The petroleum ether layer will be removed, dried over anhydrous sodium sulfate, and partially evaporated to obtain a reasonably clean preparation of menthol, menthyl acetate, and menthone.

INFRARED SPECTROPHOTOMETRY

To identify the compounds present in the peppermint extract you will obtain an infrared spectrum by simply evaporating a small aliquot of the petroleum ether extract on a sodium chloride window and placing the window in a sample cell. The b -cell is assembled and the spectrum obtained on the Beckman AccuLab 9 spectrometer. The instructions for operating the Beckman instrument are found next to the instrument and should be followed for the first time with the help of the instructor or a laboratory assistant. You should become familiar enough with the instrument to take a routine spectrum using the sodium chloride windows and b -cell with any liquid product you might make in the lab .

Infrared spectra can be obtained for any sample; all you would need is an appropriate sample cell for gases, liquids, or solids. There are many more sophisticated techniques available, especially if you have access to the modern Fourier transform infrared instruments, however for routine analysis on the older dispersive instruments, all you would use would be a small gas cell for gas samples, a small cell sodium chloride windows and Teflon spacers for neat (without solvent) liquids and solids dissolved in an infrared transparent liquid, sodium chloride windows for Nujol mulls (mixtures of fine solid particles suspended in mineral oil or Nujol) and evaporative films, and a small KBr pellet press for the preparation of KBr mulls of solid samples (see list below).

SAMPLING METHODS AVAILABLE IN INFRARED SPECTROSCOPY

SOLIDS:

 

LIQUIDS

 

GASES

 

Infrared spectroscopy is valuable in identifying functional groups that may be present in an organic sample. The compounds present in peppermint extract have different functional groups contained on the same basic cyclohexane ring structure. The major absorption peaks (stretching vibrations) for alcohols, ketones, and esters are all unique and should be present in the extract.

Functional Groups Expected in Peppermint Extract

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GROUP ..............C=O stretch............... C-O stretch.................. O-H stretch

peaks at cm-1 .......1700 cm-1 ................1100 cm-1..................... 3500 cm-1

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ALCOHOL absent present present

ESTER present present absent

KETONE present absent absent

Reference spectra can be found in the compendia in the lab (Aldrich Library of Infrared Spectra) and compared to your spectrum. IR spectra are additive and thus if the commercial preparation has a mixture of the menthol compounds, you should see peaks from all the compounds present in the spectrum of your extract. You may also notice three peaks due to the residual amount of the hydrocarbon extracting solvent (pentane,hexane) used to extract the menthol derivatives.

EXPERIMENTAL LABORATORY PROCEDURE

1. Obtain 2.0 mL of the peppermint extract and place it in a screw-capped test tube with a Teflon-lined cap.

2. Add 2.0 mL of petroleum ether to the test-tube and tighten the cap.

3. Shake the test tube for one minute and then allow the layers to separate. Note the colors of the two layers.

4. Carefully pipette the organic layer (petroleum ether layer) into a clean reaction tube containing about 100 mg of anhydrous sodium sulfate. Allow the petroleum ether extract to sit for 2-4 minutes while the residual water is removed from the organic phase. Note any change in the appearance of the sodium sulfate crystals.

5. Carefully remove the petroleum ether from the reaction tube and place it in a screw-capped vial. Allow the ether to evaporate by placing the vial next to the holes in the small fume hood on your lab bench.

6. When about half of the ether has evaporated, cap the vial and use the concentrated extract solution to obtain an infrared spectrum of your sample. Note the color, odor, and viscosity of the concentrated extract.

7. Obtain an IR beta-cell and two NaCl windows (25 mm x 4 mm). Carefully remove the windows from their protective sleeves and handle them only by the edges. Place one window on top of the IR source cooling vents in the Beckman IR-9 instrument where the material will be warmed slightly. Carefully drip the liquid sample on the window and allow any remaining petroleum ether to evaporate leaving behind an oily film with the characteristic odor of peppermint. When the window is covered with a thin layer of the sample, place the other window on top of the first and place the "sandwich" in a beta-cell. Assemble the cell and put it into the sample beam (one closest to you in the sample compartment) and follow the instructions for obtaining a spectrum at the medium scanning speed. [Be sure to record your use of the instrument in the log book next to the instrument]

8. Prepare a spectral parameters table in your lab book (and on the edge of the spectrum) where you list the following:

 

[It is a good idea to put the kind of information listed above in your lab book whenever you use an instrument in the course of the lab. This information should also appear in your lab report if you are writing up the lab.]

FURTHER LABORATORY EXPERIMENTATION

An alcohol can be quantitatively converted to its acetate ester by simple reaction with acetic anhydride. The menthol in the artificial extract can be converted to mentyl acetate by evaporating the petroleum ether and adding 10 drops of acetic anhydride to the extract and allowing it to react for a few minutes. If 2.0 mL of water are added carefully to the mixture and the acetic acid layer extracted with 2.0 mL petroleum ether, the menthyl acetate can be recovered from the petroleum ether after drying over anhydrous sodium sulfate, and evaporating the petroleum ether extract. An infrared spectrum obtained for the initial extract and for the extract reacted with acetic anhydride can be compared to see if all of the alcohol O-H peak disappears following reaction. The C=O peak should also get larger if the ester is formed in the reaction.

A solution of pure menthol dissolved in an alcohol/water (approx. 50% ethanol) solvent along with some green food coloring can serve as the "artificial flavoring" for this laboratory experiment. The use of this solution coupled with the acetic anhydride conversion to the actetate ester can be used to further illustrate the use of infrared spectroscopy to confirm the structure of reactants and products.

Other commercial flavoring agents (orange, rum) could also be used, but the composition of the ester, ketone, and aldehydes used is not as clear as for peppermint extract. Laboratory solutions of specific esters, aldehydes, and ketones could be made and used in the extraction process.

The extraction of fresh peppermint leaves and the separation of the menthol derivatives by TLC or HPLC could also be incorporated into the laboratory. The main problems with natural product isolation is the complexity of the mixture of compounds that are extracted into chloroform, ethyl acetate, acetone, or alcohol solvents.