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Bryanna Tanase David and Mark Liquid CO2 Extraction of D-Limonene from Orange Rind Christopher Cain CHM 2210L-022

Introduction Essential oils are organic compounds that come from everyday things found in nature such as mint leaves, lemons, or even flowers 1. They are used in many commercial products such as soaps, herbal medicines, or creating natural flavors for food. Essential oils are part of a family of compounds called terpenes and terpenoids (terpenes containing oxygen)1. Terpenes and terpenoids are made up of structural units called isoprenes. An isoprene unit is simply the carbon skeleton of a molecule called isoprene (pictured below) without the double bonds2. Isoprene units have the general molecular formula (C5H10) and are joined head to tail2. Terpenes are named depending on the number of isoprene units they have2. For example, a monoterpene (C10H16) has two isoprene units, a sesquiterpene (C15H24) has three isoprene units, and so on2. Limonene, or specifically D-Limonene, is a monoterpene and the major component of orange oil, that is found in the rinds of orange peels1. Essential oils can be extracted from their source in many different ways including steam distillation, organic solvent extraction, or liquid CO 2 extraction3. In the past few years, technology has advanced to the point where supercritical or liquid carbon dioxide is used in place of organic solvents3 . CO2 is considered a useful green solvent because of the many environmental and safety advantages it possesses. For example, it is nonflammable, nontoxic, and environmentally benign3. Although CO2 is considered a greenhouse gas, it is captured from the atmosphere when used as a solvent rather than generated, resulting in no net environmental harm3. Because of its flexible solubility, low toxicity, and easy removal, CO 2 has led to widely used techniques for the purpose of extracting many natural products 3. One of the major benefits of CO2 as a solvent is its ability to quickly change phase3. Relatively low temperatures and pressures can be used to form liquid or supercritical CO2. In the phase diagram below, CO2

sublimes at a pressure of 1.01 bar, the triple point where the solid, liquid, and gas phases coexist occurs at 5.2 bar, -56.6º C.3 Close to this point dry ice melts and forms liquid carbon dioxide, and if the temperature is increased to the critical point (73.8 bar and 31º C) the CO 2 exists as a supercritical fluid with properties from both liquid and solid phases 3. Dry ice sublimes at atmospheric pressure and temperatures above -78ºC, and if it is kept contained during sublimation, the internal pressure increases3. Once the temperature and pressure increase enough, liquid carbon dioxide forms, and because this phase change is simple to create, liquid CO 2 is used in many different extractions3. D- Limonene is also classified as an optically active enantiomer. An enantiomer is a

substance that is a mirror image of another, similar to the way one’s hands are exact copies4. Figure 1: Phase Diagram for Carbon Dioxide3

Despite being mirror images, enantiomers are not superimposable, meaning that if one is placed on top of the other, they will not match4. Because enantiomers are not superimposable, they possess “handedness” and exist in left and right versions4 This handedness can be compared to the idea that if one is trying to put on gloves, the left hand glove will not fit on the right hand and vice versa4 . Compounds that have this handedness are said to be chiral4. A chiral atom is any atom which has a tetrahedral geometry and four different groups called substituents attached to it4. The center of the atom with four different groups attached is called a stereocenter4.

The idea that D-Limonene is an optically active compound means that it can rotate plane polarized light, and in order to do this it must have molecules with a chiral center4. This is because chirality is a property that arises from the way molecules interact with light4. Compounds that rotate the polarized light to the right are called dextrorotatory, and those that rotate it to the left are levorotatory4. Polarimetry studies the extent to which optically active molecules can rotate plane polarized light and is measured with a device called a polarimeter1. The polarimeter contains a light source and two prisms, one that produces light and one that detects how much the light is rotated1. A tube containing the experimental sample is placed between the two prisms and the operator looks through the viewing space on the device to determine how far the plane polarized light has been rotated1. The results from the polarimeter are expressed as enantiomeric excess, which is the observed rotation divided by the rotation given in the literature and multiplied by 1001. The amount of plane polarized light molecules that can rotate depends on the wavelength of light, length of the cell through which light passes, concentration of the optically active compound, and the specific rotation of the compound4. The specific rotation reflects the relative ability of a compound to rotate plane polarized light, and so every compound has its own specific rotation4. The refractive index is a constant that can be used to characterize liquids, and it is the ratio of the velocity of light in air to the velocity of light in liquid1. Because it is a constant, every 20

compound has its own refractive index. For example, the refractive index of limonene is n D

1.4727. The superscript 20 means that this is the refractive index measured at 20 degrees and the D references the D line from a sodium vapor lamp1. The measurement for refractive index is made using a device called a refractometer using some drops of a liquid sample, and often a

correction must be made due to the fact that the instrument uses white light instead of yellow light1. An additional correction must also be made to the observed reading by adding 0.00045 for each degree above 20º C1. These corrections are represented in the equation

n20 D =

t n D +0.00045(t−20 ° C) .1.

The objectives for this experiment were to extract limonene from orange peels using CO2 extraction and to use polarimetry and refractometry to obtain the observed rotation, enantiomeric excess, and refractive index of the collected sample. Experimental Before beginning the extraction, water was heated to 60-65ºC in a 100-mL beaker on a hot plate to create a hot water bath. Then a 50-mL centrifuge tube was weighed and prepared to hold the orange rinds as shown below3. Once the trap for the orange peel was constructed, 8 grams of grated orange peel were placed inside the centrifuge tube, followed by approximately 30 grams of dry ice (solid CO 2), and then capped as tightly as possible to keep the dry ice gasses from leaking out. A 100-mL plastic cylinder was filled with the heated water, and the centrifuge tube was placed inside to observe the extraction of D-Limonene from the orange rinds. As the extraction was taking place, it was noted that the cap on the tube did not hold the gases from the dry ice effectively, and one student had to hold their finger on the cap to allow the reaction to proceed. At this point, the CO 2 began to turn to liquid and evaporate down the sides of the tube as the pressure increased. Once the extraction had finished and no more gas was escaping, the tube was left in the water for 10 minutes to relieve the pressure that built up. When all the pressure was released, the centrifuge tube was removed from the water and carefully unscrewed, the solid trap and rinds were removed, and the mass, smell, and color of the extracted limonene were recorded.

The next step of the procedure was to use a polarimeter to obtain the observed rotation of limonene in order to determine enantiomeric excess and specific rotation. Following the individual group extractions of limonene, the total amount of limonene for the whole class was weighed, recorded, and then combined (save a few drops) with 3 mL of ethanol in a 50 mL Erlenmeyer flask. The solution was shaken vigorously to make it clear, and pipetted into a 10 mL volumetric flask. The volumetric flask was then filled with ethanol to the calibration mark and after was inverted to mix the contents. Before placing the limonene sample into the polarimeter, it was ensured that the device was set to 0, standardized with a known solution, and that there were no air bubbles in the solution. The sample was then placed into the polarimeter and the optical rotation was observed and recorded. The specific rotation and the enantiomeric excess were then calculated and compared with the literature value. The final piece of the procedure involved finding the refractive index of limonene using a refractometer. To do this, two to three drops of the leftover limonene were dropped onto a measuring prism using a pipette, and the prism was placed inside the refractometer. Then the lamp on the refractometer was positioned for maximum brightness and the index knob was turned so that the line separating light and dark was at the crosshairs 1. When the demarcation line was sharp and colorless, the refractive index was read, recorded, corrected to 20ºC and compared with the literature value.

Figure 2: Setup of Centrifuge Tube for Extraction3

Table of Chemicals

Chemical Molar Mass (g/mol) Refractive Index Optical Rotation (º) Color and smell Toxicity/ Hazards

D-Limonene 136. 24 1.4727 n20 D 115.5 Clear color, light citrus scent Do not inhale, ingest or put in contact with skin or eyes. Eye irritant. Prolonged exposure may lead to respiratory inflammation. Wear PPE.

Carbon Dioxide 44

Ethanol 46.07

Colorless, odorless Do not inhale, ingest, or put in contact with skin/eyes. Contact with liquid may cause burns or frostbite.

Clear, alcohol-like Do not inhale, ingest, or put in contact with skin or eyes. Skin and eye irritant. Wear PPE

Table 1: Physical and Chemical Properties

Mass of Limonene (g) Odor Results Percent Recovery Observed Rotation (º) Specific Rotation (º) Refractive Index Enantiomeric Excess (ee)

Table 2: Experimental Results

Individual: 0.05 Whole class: 0.67*/ 10 ml citrusy 0.625% 7 104.5 1.4432 90%

Calculations1,5 Percent Recovery:

mass of limoneneextracted (g) x 100= mass of orange peel grated (g)

Specific Rotation:

[ α] D =

T

α obs = ¿ c l

0.05 x 100= 0.625% 8

7 = 104.5 0.067 x l

α obs = observed rotation c= concentration of solution in grams per milliliter l= length of light path (l decimeter) T= temperature of measurement in Celsius. total mass of limonene ¿ How to calculate c: ¿ class(g) volume( mL ) 0.67 g c= = 0.067 g/ml 10 mL

Refractive Index: n20 D =

ntD +0.00045(t−20 ° C)

n20 D = refractive index of known compound t

n D = unknown refractive index of collected compound t = temperature above 20º t 1.4727=nD +0 .00045(28-20)

n28 D =1.4432 Enantiomeric excess (ee)=

[ α ] obs [ a] pure

x 100=

104.5 115.5

x 100= 90%

Discussion There were several benefits involved with using this CO2 extraction method over the steam distillation. For one, cleanup was much less tedious because not much waste was generated. Students also avoided becoming nauseated from chemicals because the strong citrus scent from the limonene overpowered the smell from the ethanol, and ethanol was sparingly used in the first place. Yet another advantage of using this method was that it extracted the limonene from the rinds in minutes as opposed to hours with steam distillation6. It should also be noted that by using CO2, the essential oils retain their natural properties6. Using this green extraction method is

also more cost effective because there is no need to purchase an apparatus to perform the extraction6. In comparing the safety of using liquid CO2 and steam distillation for extraction, one reason why using CO2 is safer is because it is nonflammable and heating to boiling temperatures is not required. Thus, using CO2 reduced the risk of heat related injuries such as burns. It should also be mentioned that because no glass was used during the extraction, the potential for injury from shattered glass was minimized. The optical (measured by polarimeter) and specific rotation of the extracted limonene were 7º and 104.5º respectively (Table 2). In comparing these values with the optical rotation of standard pure limonene, which is 115.5º (Table 1) it is noticeable that the values are close, but not as close as preferred when trying to assess the purity of an extracted compound. Thinking back to the factors that affect rotation, it could be surmised that these values are not as close as they should be because there was not a high concentration of limonene that was extracted by the class. Knowing that concentration of a particular substance and amount of product are directly proportional, if the class had been able to extract more limonene during the experiment, the concentration of limonene would have been greater, and therefore yielded a greater specific rotation. The refractive index of the collected limonene was 1.4432 (Table 2) and the value as stated in the literature was 1.4727 (Table 1). Reasoning as to why these values are slightly off from each other could be that the limonene samples that were extracted contained impurities. It should also be noted that the amount of limonene obtained by the entire class was inflated, and that perhaps these values would be more precise if that had not been the case. In addition, the fact that the refractive index of the experimental limonene needed to be corrected for temperature

also affected the data received for the refractive index because it is dependent on temperature7. Most literature values are taken at 20ºC and the lab was at 28ºC. Having to subtract the temperature difference and then multiply by a decimal undoubtedly made the experimental refractive index value smaller than that in the literature. Conclusion The objectives for this experiment were to extract limonene from an orange rind using CO2 extraction and then to determine the specific rotation, enantiomeric excess, and refractive index of the collected sample. The experiment achieved what it set out to do because the supercritical point of carbon dioxide was observed during the extraction and this illustrates that the extraction performed was successful. In addition, the values for specific rotation, enantiomeric excess, and refractive index were not too far off from the literature values, indicating that there were only small impurities in the collected sample. If this experiment were to be performed again, it would be beneficial to see what the results would be like if the total mass of limonene had not been inflated. The techniques in this experiment could be applied to extract peppermint oil from mint leaves for the creation of peppermint candies because peppermint oil is the essential oil found in mint leaves.

References [1] Weldegirma, S. Experimental Organic Chemistry Laboratory Manual, 7th ed.; Procopy Inc: Tampa, Florida [2] 26.7 Terpenes: The Isoprene Rule. [3] Green Isolation of Limonene. [4] Chirality and Optical Activity http://chemed.chem.purdue.edu/genchem/topicreview/bp/1organic/chirality.html (accessed Sep 30, 2017). [5] Polarimetry http://www.chem.ucla.edu/~bacher/General/30BL/tips/Polarimetry.html (accessed Oct 1, 2017). [6] Anitescu, G.; Deneau, C.; Radulescu, V. Isolation of Coriander Oil: Comparison Between Steam Distillation and Supercritical CO2 Extraction, 1996. [7] Refractive Index...