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

Experiment Description & Background


Introduction
Students will purify and isolate limonene and either (+)-carvone from oil of caraway or (-)-carvone from oil of spearmint using column chromatography.  The isolated carvone and limonene will be identified by comparison with standards using thin layer chromatography (TLC) , infrared (IR) spectroscopy, and the optical rotation of the isolated carvone will be determined by polarimetry.



Oil of Spearmint and Oil of Caraway

Spearmint
Essential oils, derived from natural products,  have been used for centuries as flavorings, fragrances and for medicinal purposes.  These oils typically contain multiple organic components, generally with one or more compounds dominating the mixture to provide characteristic odor and properties.  Essential oils are typically isolated from plant sources through distillation or extraction.  Oil of spearmint is derived from the leaves of the spearmint (Mentha spicata) plant.  A major constituent of oil of spearmint is the R enantiomer of carvone.  Minor amounts of limonene, a metabolic precursor of carvone is also present in oil of spearmint.  Caraway oil is extracted from caraway seeds (Carum carvi) and contains manily the S enantiomer of carvone along with higher levels of limonene. The limonene in oil of caraway is also a precuror to S-carvone.  Specific carvone enantiomers can be isolated in pure form from oil of spearmint or oil of caraway using column chromatography.  Limonene, a less polar constituent of these essential oils can be separated from the carvones during chromatography.  Purity of the isolated samples can be evaluated by thin layer chromatography and spectroscopic analysis, and the optical purity of the enatiomers can be evaluated using polarimetry. 

Caraway

Column Chromatography and Thin Layer Chromatography














Analytical TLC Analysis

Chromatography is an experimental method that is used in the laboratory to separate and characterize organic compounds.  Chromatography may be used preparatively or analytically.  Preparative chromatography is used to physcially separate components of a mixture for further use and characterization.  Thus, preparative chromatography is a form of purification.  Analytical chromatography measures the relative proportions of components in a mixture and may be used to chracterize specific components by comparison with standards or to characterize the mixture.  There are many types of preparative and analytical chromatography, including thin layer chromatography (TLC), column chromatography (CC), gas chromatography (GC) and high pressure liquid chromatography (HPLC).

All chromatographic techniques, whether preparative or analytical, involve a two component system: 1) a stationary phase and 2) a mobile phase.  In TLC, the stationary phase is a plastic, glass or aluminum plate (usually 2.5cm X 10cm) coated with a material that serves as the stationary phase.  The term stationary phase is used to describe the material on the plate becasue it does not move during the analysis, or it remains "stationary".  Silica gel is most commonly used as a stationary phase for simple TLC analysis, but numerous other stationary phases can be employed for more sophisticated experiments. The mobile phase is a solvent that moves during the analysis and carries the analyte(s) (compound or compounds of the mixture) along the stationary phase.  The mobile phase of a TLC analysis is also called the developing solvent.  The developing solvent may be a single organic solvent or a mixture of two or more organic solvents.  When binary (two solvents) or tertiary (three solvents) mixtures are used, they must be completely miscible in each other.  Usually the solvents are of different polarities.  Aqueous solvents are rarely used for simple TLC analyses.  For CC, the stationary phase is the column packing material (often silica gel or alumina) and the mobile phase is the solvent that runs through the column carrying the analytes.  For both TLC and CC, different components of the mixture will adhere to the stationary phase to different degrees depending on the relative polarity between the stationary phase and the specific component of the mixture. Polar components adhere strongly to a polar stationary phase; non-polar components adhere weakly to a polar stationary phase.  For example, silica gel, the stationary phase used in this experiment is very polar.  Very polar components of the mixture will adhere strongly to the silica gel, while less polar constituents have a weaker attraction.  When the plate in a TLC analysis is developed, the polar components will tend to stay at the bottom of the plate (bound to the silica gel) and the non-polar components will tend to move with the relatively less polar mobile phase (developing solvent).  The polar components will have a smaller Rf value than less polar components.  For CC, the more polar component will remain at the origin (or top of the column) and the less polar components will move down the column at a faster rate.  Thus polar components have longer retention times (rt) and non-polar components have shorter retention times.  Non-polar stationary phases are hydrocarbon-based and are usually desginated by the number of carbons associated with the packing material.  Non-polar columns (CC) and plates (TLC) are often referred to as C18 or C22 to indicate ther number of carbons in the hydrocarbon making up the non-polar stationary phase.









Preparative Column Chromatography

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How to Choose the Mobile Phase (Developing Solvent)
The purpose of the mobile phase is to move components of the mixture up the TLC plate or down the column and away from each other.  The degree to which a component of the mixture moves with the mobile phase as opposed to staying adhered to the stationary phase (closer to the origin) depends on the component's polarity relative to each of these two phases.  If  the component has a polarity more like the mobile phase, then it will dissolve in the mobile phase and move (up the plate or down the column).  If the component has a polarity more like the stationary phase, it will remain adhered to the stationary phase (at the bottom of the plate or at the top of the column).  The polarity of the stationary phase is fixed.  For example, silica gel is polar while C18 stationary phases are non-polar.  However, the polarity of the mobile phase can be adjusted if more than one solvent is used.  Binary (two solvents) or tertiary (three solvents) mixtures are usually used as a developing solvent for simple TLC and CC analyses.  Typically the polarities of the solvents used in binary or tertiary mixtures are different.  The overall polarity of the mobile phase can then be adjusted by changing the ratio of the polar solvent relative to the non-polar solvent of the mobile phase.  Some typical solvent mixtures used as mobile phases in TLC and CC analyses are given in the table below.  The more polar solvent of each mixture is given first. 

Ethyl Acetate-Hexane
Ether-Pentane
Acetone-Petroleum Ether
Ethanol-Chloroform
Ethanol-Chloroform-Hexane
Acetic acid-Methanol-Benzene

Solvent Combinations for Use as Mobile Phase in TLC Analysis

Determining an appropriate mobile phase to achieve maximal separation of components in a mixture is a trial and error process.  Ideally, all components of the mixture should be cleanly resolved (separated) from each other with no overlapping.  All the components should also be located in the bottom/middle two thirds of a TLC  plate after it has been developed.  The only way to find a mobile phase that will result in meeting these criteria is to try a solvent mixture of a specific ratio and see what happens.  If the desired results are not achieved, then adjust the solvent ratios.  Consider some simple scenarios for guidance in how to adjust the ratios of solvent of binary or tertiary mobile phases to get the results you want.

For example, let's say you have a mixture of three compounds, A, B, and C.  You decide to use silica gel as the stationary phase and a binary mobile phase of ethyl acetate-hexane in a 50:50 ratio.  The result of the TLC analysis looks like the illustration in Figure 2.5.  None of the components of the mixture moved, suggesting A, B and C are all very polar and adhere strongly to the polar silica gel.  A more polar solvent system is needed to move at least some of the components up the plate.  You decide to increase the ratio of ethyl acetate to hexane to 75:25 resulting in a plate that looks like the one depicted in Figure 2.6.  Two of the three components of the mixture have been resolved, but not the third. You then decide to increase the polarity of the mobile phase even more (90:10 ethyl acetate-hexane) to move the components further away from each other.  The desired result is achieved as shown in Figure 2.7.

Figure 2.5:  Developed in 50:50
Ethyl acetate-hexane

Figure 2.6:  Developed in 75:25
Ethyl acetate-hexane



Figure 2.7:  Developed in 90:10
Ethyl acetate-hexane

In an alternative scenario, using 50:50 ethyl acetate-hexane with silica gel, TLC analysis of a mixture of compounds X, Y and Z gave a developed TLC plate shown in Figure 2.8.   All of the compounds moved very high on the plate suggesting they are all non-polar.  It is necessary to make the components less soluble in the mobile phase.  Increasing the polarity of the mobile phase will make the components less soluble and force them to remain lower on the plate.   TLC analysis of the mixture with 75:25 ethyl acetate-hexane, then 90:10 ethyl acetate-hexane, gave the results shown in Figures 2.9 and 2.10.

Figure 2.8:  Developed in 50:50
Ethyl acetate-hexane


Figure 2.9:  Developed in 75:25
Ethyl acetate-hexane


Figure 2.10:  Developed in 90:10
Ethyl acetate-hexane

Column chromatography (CC)  and thin layer chromatography (TLC) are often used together in an experiment.  CC is used to preparatively separate components of a mixture into fractions, where each fraction ideally contains only one component of the mixture.  Multiple fractions are collected as the mobile phase moves down the column, carrying different components down the column at a different rate.  TLC is used to analyze the fractions and identify the component(s) of the fraction and to evaluate the effectiveness of the separation/purification by CC.  Test tubes are commonly used to collect fractions from the column and the solution in each test tube is spotted onto a TLC plate next to a known standard of the desired component.  Spots on the TLC plate with Rf values similar to the standard are identified. 

Calculating the Retention Factor (Rf) in TLC Analysis
The retention factor is  (Rf) a unitless value that is used to indicate how far a compound moves on a TLC plate.  Rf values are calculated for each spot that
appears on a  plate using the formula given below .and illustrated in Figure 2.11.  Distances are typically measured in cm, from the starting line at the bottom of the plate to the center of the spot.

Rf     =  distance traveled by the spot
                      distance traveled by the solvent front

Figure 2.11:  Calculating retention factors
    (adapted from Feiser & Williamson, p. 126)


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Infrared Spectroscopy
In IR spectroscopy, a sample molecule is exposed to radiation with a frequency in the infrared region of the electromagnetic spectrum.  Only a portion of the infrared region of the electromagnetioc spectrum(2.5 X 10-6 - 2.5 X 10-5m) is used in conventional IR spectroscopy.  Specific frequencies of radiation within the infrared region of the spectrum are absorbed by individual bonds in the molecule, causing the bonds to vibrate.  The frequency of the radiation that is absorbed by the molecule is recorded as a peak or absorbance in the spectrum.  These peaks are characteristic of certain types of bonds (or functional groups).  The table below lists various functional group classes, specific bonds associated with those functional groups, and the characteristic position of the peak in an IR spectrum corresponding to that bond.  Click on the link to view a sample spectrum containing peaks corresponding to the bond type/functional group indicated.

 
Absorbance Range
FunctionalGroup/Bond Type
Intensity of Absorption
2850-2960 cm-1
Csp3-H of alkanes and alkyl groups
medium-strong
3020-3100 cm-1
Csp2-H of alkenes and alkenyl groups
medium-strong
1640-1680 cm-1
Csp2=Csp2 of alkene
medium
3300 cm-1
Csp-H of alkynes
strong
2100-2260 cm-1
CºC of alkynes
CºC of nitriles
weak-medium
500-800 cm-1
Csp3-X, X = halogen of alkyl halide
strong
3200-3650 cm-1
O-H of alcohol, phenol, carboxylic acid
strong, broad
1660-2000 cm-1
1450-1600 cm-1
Csp2=Csp2 of aromatic
weak
medium
1680-1850 cm-1
C=O of carbonyls 
ketones, aldehydes, esters, carboxylic acids, amides
See more specific ranges
strong
3300-3500 cm-1
1030-1230 cm-1
N-H of amines
C-N of amines
medium
medium
1540 cm-1
N=O of nitro group (-NO2)
strong

Characterisic IR Absorbances Ranges for Various Bond Types

Interpretation of the IR Spectrum
Interpretation of IR spectra involves correlating peaks in an experimentally generated spectrum with known ranges for specific bond or functional group types present in the structure of the compound that is being analyzed..  There are some general guidelines that can be used to interpret an IR spectrum. 

Focus on Peaks between 1500-3600cm-1.  Peaks below 1500cm-1 generally cannot be specifcally interpreted.
Focus on the major peaks in the spectrum with absorbances of 50% of scale or greater.  Weaker absorbances generally cannot be specifically interpreted. 
The most characteristic regions of the IR spectrum are 3000-3500 cm-1where OH (very broad and intense) and NH (sharper than OH) peaks appear and 1650-1850cm-1 where carbonyl peaks appear. 
The absence of peaks can be just as characteristic as the presence of peaks.  For example, a peak at 1750cm-1 indicates a carbonyl containing functional group is present in the molecule.  If no peak appears between 1650-1850cm-1, then no carbonyl containing functionl groups are presen

Sample Preparation and the Infrared Spectrophotometer
There are a variety of methods for preparing samples for IR analysis.  The most common methods of sample prepartion for IR analysis are listed in the table below. Liquid samples will typically be run in this course using sodium chloride plates (i.e. salt plates).  The sample is prepared by placing a few drops of the liquid compound on one salt plate and placing a second salt plate on top of the first.  Solid samples will be prepared for IR analysis using potassium bromide pellets.  Pellets are prepared by mixing the solid sample with KBr, placing the mixture in a mini-press and compressing the mixture for form a small disc or pellets.  ATR analysis is a faster and simpler way to run solid samples.  Other sample preparation methods (mull, solution) are available but will not be used in this course.  A mull is prepared by grinding a solid sample with mineral oil to form a paste which is then placed between salt plates for analysis.  The solutions of samples, either liquids or solids can analyzed using special solution cells.

Sample Prep Method
Compound State
Neat (undiluted, salt plates) 
Liquids
Mull
Solid
KBr Pellet or ATR
Solid
Solution 
Liquid or Solid

Sample Preparation Methods for IR Spectroscopy


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Stereochemical Designation by Optical Rotation
                                     
Enantiomers
Chiral or assymmetric molecules arise due to the presence of an sp3 carbon that is directly bonded to four unique substitutents.  The four substituents may be four different atoms or they may be the same atom, each with different hybridization or secondary substituents.   Molecules that contain chiral carbon atoms give rise to stereoisomers.  If the molecule contains only one chiral carbon atom, two stereoisomers, called enantiomers, are possibleEach enantiomer is a unique molecule, however the enantiomeric pair differs only by the specific spacial arrangement of the four substituents around the chiral carbon atom. R- and S- carvone are examples of an enantiomeric pair.  Enantiomers are often defined as non-superimposable, mirror image isomers.  Enantiomers have exactly the same physical properties (boiling point, melting point, density, Rf values, retention times etc) except, thay have optical rotations that are equal in magnitude but opposite (+ or -) in the direction that each isomer rotates a plane of polarized light. A racemic mixture is defined as a 50:50 mixture of an enatiomeric pair. Many drugs that contain chiral carbon atoms are sold as racemic mixtures since separation of the two compounds is very expensive.  However, enantiomers often do not have the same biological and toxicological activity.  For this reason, the Food and Drug Administration (FDA) requires pharmaceutical companies to separate and test each enantiomer for its specific biological activity and toxicity before recieving approval for sale in the United States. 
Optical Rotation
Enantiomers are often referred to as optical isomers since one isomer of the enantiomeric pair rotates a plane of polarized light in a right-handed direction (+) and the other enantiomer of the pair rotates a plane of polarized light in a left-handed (-) direction.  For the enantiomeric pair, the rotational directions are opposite, but the magnitude of the rotation is approximately the same.  The enantiomer of the pair that rotates light to the right is referred to as the (+) or dextrorotatory isomer (abbreviated as d-) and the isomer that rotates light to the left is called the (-) or levorotatory isomer (l-).  The d- and (+) designation are interchangeable as are the l- and (-) designation.   Often both the d-/l- and the (+)/(-) designations are included in the name of an appropriately designated enantiomer.  If both d- and l- (or (+)/(-)) are part of the name, or if no d- or l- (or (+)/(-)) designation is included in the name, it implies that the sample is a racemic mixture of the two enantiomers. 

The optical rotation of an enantiomer is measured using an instrument called a polarimeter.   A light source in the polarimeter (sodium lamp) emits light which travels through a polarizer (Nicol prism).  The polarizer collects a portion of the light emitted from the source and aligns the light in a single direction to create a plane of polarized light.  The polarized light then passes through a tube containing a solution of the sample.  If the sample is optically active, the plane of polarized light will rotate from its original orientation.  The direction of the rotation is described as + if the rotation is clockwise and it is described as - if the rotation is counterclockwise.  The magnitude of the rotation is described by an angle of displacement from the original plane of polarized light.  The schematic diagram below illustrates the principles of the polarimeter. 


Schematic Diagram of Polarimeter
Samples for polarimetry are typically dissolved in a solvent (eg. CHCl3, hexane).  Appropriate solvents are not optically active and completely dissolve the compound to be analyzed.  To ensure consistency in measurement, optical rotation of a sample is reported as specific rotation to correct for variability in sample tube length, temperature, solvent wavelength of the light source and concentration of the sample.  The specific rotation of a sample is given by the equation on the right.  Liquid samples may also be run "neat (i.e., no solvent).  In these cases, the concentration term, c, is replaced with the density of the liquid.  Pure enantiomers will give rise to a specific rotation that is consistent with a literature value for that enantiomer.  The specific rotation of a racemic mixture will be zero, since the magnitude and direction of a specific enantiomer will be "cancelled out" by the other enantiomer of the pair.


Optical Purity and Enantiomeric Excess (ee)
Optical purity refers to how accurately the measured specific rotation of an enantiomer is to the reported literature value of that enantiomer.  The optical purity is calculated using the equation given below.  For example, the specific rotation of (+)-2-bromobutane reported in the literature is +23.10.  If the experimental specific rotation is determined to be +9.20, then the optical purity would be calculated as 40%. 



A compound that is x% optically pure contains x% of a pure enantiomer and (100-x)% of a racemic mixture. 


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