Distillation is laboratory technique used for separating and purifying liquids. It is powerful tool, both for the identification and the purification of organic compounds. Simple Distillation and fractional distillation are often used for isolation of mixture of liquids; however with simple distillation volatile organic compounds that are immiscible or insoluble in water such as oil can be separated. Therefore, in this experiment, steam distillation is used to separate carvone from caraway seeds and spearmint leaves. For steam distillation, distill liquids boiling point has to be less than 100° C, compound mixed with water has to be stable at 100° C and its vapor pressure should be greater than 5 Torr at 100° C. In this process, volatile liquids and oils co distill with water. The principles of steam distillation focus on Raoult’s law and Dalton’s law. According to Raoult’s law partial pressure of compound equals mole fraction of that compound multiply by vapor pressure. If Raoult’s law is applied for immiscible component (X) in a heterogeneous mixture with water, then partial pressure of X equals vapor pressure of X. Since X is not soluble in water, it does not depend on its mole fraction in the mixture. Therefore, Raoult’s law does not apply to steam distillation because the compounds are immiscible, so they do not depend on each other’s mole fraction. According to Dalton’s law, total vapor pressure equals sum of individual vapor pressures. So, total vapor pressure is higher than most volatile component and boiling point mixture is lower than the lowest boiling component due to the higher total vapor pressure. Normally, distillation process of these liquids would require high temperature; however, with presence of water other components of mixture boil below its normal boiling point.
Steam distillation apparatus requires same glassware similar to other distillation methods such as still pot flask, a condenser tube, a thermometer adapter, and a separatory funnel as well. Separatory funnel is important because in steam distillation, water must be continuously added to the stillpot. This is because the water is being vaporized along with the sample, so water is added at the rate that is distills out so the volume stays nearly constant. The process begins by placing the sample in still pot with hot water. As the sample is boiling, it vaporizes and passes through the condenser tube. The sample gets condensed along with water vapor and then collected as distillate. Then, as the distillate is received the separatory funnel is opened to allow water to drip in the still pot. After obtaining the distillate, it is extracted with methylene chloride. Once it is extracted, it will then be separated from the water by using a chemical that is miscible; in this experiment, it is Na2SO4.
Figure 2: Steam Distillation Apparatus
After the process of steam distillation, Thin-layer Chromatography can be applied as an analytical tool for rapid analysis of small quantities of samples. Thin layer chromatography (TLC) is like column chromatography except that the stationary phase is bound to a layer of silica. Tiny spots of the different molecules are placed on a baseline. The solvent is then added and it travels up the piece of plate. The folded piece of filter paper can be used for solvent equilibrium. The molecules attach to the solvent, which serves as mobile phase. The farther the compounds move, the more non-polar they are because they have a higher retention factor. When the solvent stops moving up the solid, so do the molecules. They leave a visible mark where they stopped moving. The retention factor (Rf) can be measured with the following formula: Rf = (distance traveled by the substance) / (distance traveled by the solvent). The higher the retention factor is then the lower the retention time. Therefore, if the solvent is in the mobile phase longer, then it will have a higher retention factor. Besides determining which substances are more polar, TLC can also be used to determine is two compounds are different.
Figure 3: Thin-Layer Chromatography Chamber
Lastly, Infrared spectrometry is used to verify the separation. Infrared spectrometry can determine the different types of functional groups in a molecule. When increasing energy is sent through the molecule, radiation is absorbed by different functional groups and this corresponds to different transitions among vibrational-rotational levels. The bonds between the atoms vibrate at a frequency that is read by the IR spectrometer. As this frequency increases, so does the wave number of the groups and radiation energy. Hooke’s law can be used to calculate the stretching frequency of the bonds: ΰ= √(k)/(2πc√(m*)) where c= speed of light, k= force constant of bond, and m*= reduced mass of atoms joined by bond (mAmB)/( mA + mB). Different functional groups exhibit different frequencies and intensities. Alkenes have a frequency around 1600-1660cm-1 with variable intensities. Carbonyl groups have a variety of different frequencies and intensities depending on the other groups in the molecule or where the carbonyl group is. Ketone has a frequency of around 1705-1725cm-1 with strong intensity. However, because carvone has a ketone attached to six membered ring with a double bond, the frequency is lowered. Therefore, the frequency for carbonyl group in carvone is about 1675cm-1. Since (S)-(+)-Carvone and (R)-(-)-Carvone are enantiomers, IR spectra for both enantiomers of carvone are identical. The Rf value of a compound is a physical constant for a given set of chromatographic conditions.
By performing these various methods, (S)-(+)-Carvone from caraway seeds and (R)-(-)-Carvone from spearmint leaves were isolated. It was determined that the enantiomers have the same boiling point, IR spectrum, and retention factor. However, each enantiomer has unique smell and they differ in their optical rotation direction.
Experimental Procedures
Part I: Steam Distillation
In a group of two experimenters, the distillation of each compound was assigned between experimenters. Prior to Setting up apparatus, 5 grams of caraway seeds and 5 grams of spearmint leaves were obtained in a watch glass and transformed into 500 mL round bottom flask. Then, the two steam distillation apparatus were assembled: one for caraway seeds and one for spearmint leaves using the guidelines provided accordingly. The 500 mL round bottom flask was equipped with claisen adapter, stillhead west condenser (thin column), bent vacuum adapter, separatory funnel, thermometer, and thermometer adapter. The equipments were tightly clipped with Keck Clips to prevent vapor from escaping apparatus. The separatory funnel was placed on top of claisen adapter to add water. A thermometer was placed into a stillhead. It was instructed to keep the thermometer slightly below the entrance of condenser so the mercury bulb of thermometer is immersed thoroughly in vapors. Then, two water tubing were obtained and were hooked with west condenser. One of the water tubing allowed the water into the condenser and the other was there to drain it. Then, 150 mL of boiled water was added to the round bottom flask with 5 grams of caraway seed, and then the flask was heated. The faucet was turned on to initiate water flow. The distillation was produced at the maximum rate without foaming into condenser. When the rate became steady, the stopcock of the separatory funnel was opened to replace the water that distilled in the graduated cylinder in order to keep the volume of water in the round flask constant. About 60 mL of distillate was collected in graduated cylinder. During process of steam distillation, the temperature and distillate volume were recorded at 10 mL interval. Then the distillate was examined carefully and the observations were recorded. Afterwards, using the separatory funnel, the distillate was extracted three times with 7 mL methylene chloride. The extraction was carried out by inverting the stopper of separatory funnel ten times, without shaking vigorously. The extracted liquid was transferred into 50 mL Erlenmeyer flask and 1 gram of anhydrous sodium sulfate was added and the flask was swirled to remove the water. The drying agent was removed by filtration into second 50 mL beaker. Then, most of the solvent was removed by steam bath or using heating mantle and approximately 0.5 mL of solvent was left. The left over solvent was clear sample of the essential oil dissolved in a small amount of methylene chloride.
Part II. IR Spectroscopy
Using the 0.5 mL of samples left from the distillation process, IR spectrum was obtained for each sample using the FTIR equipment. First, the crystal surface of the equipment was cleaned using couple drops of isopropanol and dry Q-tips. Then it was allowed to dry for couple of seconds. Afterwards, using the computer connected to the equipment, a background scan was done. Then, liquid retainer was placed onto the crystal plate with the Teflon gasket facing toward the crystal. The pieces were arranged in a way that the crystal was visible through the opening in the liquid retainer then 203 drops of caraway sample were applied onto the crystal through the opening. The volatiles cover was placed over the opening. Then, the pressure clamp was pressed upon the arrangement after pressing the switch in located in the back of the clamp. The sample was scanned afterwards, baseline and peaks were adjusted, title was added and the spectrum was printed then. The same procedure was followed for spearmint sample as well as the pure sample.
Part III. Thin-Layer Chromatography
In order to do TLC analysis, first a TLC plate was prepared using a capillary tube. A baseline was drawn on the TLC plate about 2 cm from bottom and a mark was drawn about 8 cm from the baseline. Then three marks were drawn: pure sample of (R)-(-)-carvone and (S)-(+)-carvone, co-spot mixture, and oil extraction obtained. A capillary tube was placed in pure sample and spotted onto the plate far right side on the first mark. Then, the mixture of pure sample and carvone extraction of spearmint leaves was spotted onto the second mark – in the middle. Then, on the third spot (left side), the carvone extracted from spearmint leaves was spotted. Another TLC was also made using same three spots procedure, except instead of putting spot from the extraction of spearmint leaves one will put spot from the carvone extraction from caraway seeds. Afterwards, a prepared solvent of ethyl acetate and hexanes (ratio 1:4) was obtained in the two beakers, which was served as developing chamber as shown in figure 3. The prepared TLC plates were then placed in the two separate beakers containing prepared solvent ratio of ethyl acetate and hexanes – 1:4. The solvent was allowed to travel up the mark that was about 8 cm away from the baseline for both plates. Then, the TLC plates were removed from the beaker.
Part IV: Baeyer’s Test
A UV light detector was used to determine movement of the compound on TLC plate. Then, the TLC plate was stained using Potassium Permanganate KMnO4 stain, and was dried using a heating drier. Lastly the retention factor was calculated for all spots.
Data Acquisition/ Presentation
Relevant Equations:
Steam Distillation
Distillate Observations:
Caraway Seeds (5 grams) Spearmint leaves (5 grams)
Color: Brown Color: Green
Smell: Strongly earthy Smell: Minty
Sample: Somewhat clear Sample: Somewhat cloudy
More than one phase: Yes More than one phase: Yes
During steam distillation of caraway seeds and spearmint leaves, recorded temperature and volume for 10 mL interval is listed in table below.
As one can see in the table, the temperature of distilled solvent is around the boiling point of water of 100° C. Why is the temperature of distillation around 100° C? According to Raoult's law partial pressure of compound equals mole fraction of that compound multiply by vapor pressure. Applying Raoult's law for immiscible component (X) in a heterogeneous mixture with water, partial pressure of X equals vapor pressure of X. Since X is not soluble in water, it does not depend on its mole fraction in the mixture. According to Dalton's law, total vapor pressure equals sum of individual vapor pressures. Therefore, the total vapor pressure is always higher than most volatile component-water in steam distillation. Thus, boiling point of mixture is always lower than the lowest boiling component-always water in steam distillation. Because of the presence of water another component of mixture boils below its normal boiling point. This has important advantage in separating molecules mixing with water. According to our results, the temperature at distillation is little higher than 100° C. The reason for that might be boiling the mixture at higher temperature, cause temperature to go up a little. Also if position of thermometer is too low, it records higher temperature than its normal temperature.
Infrared Spectroscopy
The scanned spectrums for caraway seeds, and spearmint leaves are attached at the back. Following are the values observed:
Spearmint leaves:
1667.06 cm-1 → Alkenes C=C
Caraway seeds:
1713.29 cm-1 → Ketones C=O
1669.01 cm-1 → Alkenes C=C
Thin-Layer Chromatography and Baeyer’s Test
After the spots were observed on the TLC plate, their retention factor was calculated. Retention factor was observed after the collection of spots was measured from the baseline. Also, the solvent was prepared by 1:4 ratios of ethyl acetate and hexane, respectively.
Rf = Distance traveled by substance
Distance traveled by solvent
Spearmint leaves:
Caraway Seeds
Conclusion
The objective of this lab experiment was to isolate (S)-(+)-Carvone from caraway seeds and (R)-(-)-Carvone from spearmint leaves through process of steam distillation. By performing TLC analysis, IR Spectroscopy and Baeyer’s test, the characteristics of the two enantiomers were observed. The steam distillation process was done successfully because the compound mixed with water has to be stable at 100° C, and temperature noted at every 10 mL was constant around 101° C. Also, the temperature of distilled solvent is around the boiling point of water 100° C which can be explained by Dalton’s Law. According to Dalton’s law, total vapor pressure equals sum of individual vapor pressures. So, total vapor pressure is higher than most volatile component and boiling point of mixture is lower than the lowest boiling component due to the higher total vapor pressure. Due to presence of water, another component of mixture boils below its normal boiling point and close to water’s boiling point – which is an important advantage in separating molecules mixing with water.
When the distillates were observed, the smell and color were very distinct. Knowing that a stereochemical difference is detected in the odors of the two enantiomers of carvone, it gave better understanding of their physical properties and optical activity. IR Spectroscopy and TLC analysis were done to examine their characteristics furthermore. From IR spectrums obtained from both of the carvones, two main functional groups were quite obvious: alkene C=C and carbonyl C=O. For caraway seeds, C=C were observed around 1669.01cm-1, and C=O around 1713.29 cm-1. However, for spearmint leaves, only C=C was observed at around 1667.06 cm-1. This is may be due to errors caused by impurity of extracted oils which can affect absorption frequencies. Moreover, it looks like that the vibrational frequency values, especially that of alkene group C=C, are very close to each other. This shows that the stem distillation results were quite accurate because the IR spectra for both enantiomers of carvone and for any two enantiomeric compounds are identical.
Additionally, a TLC analysis and Baeyer’s test was done on distilled samples and Rf was calculated. The resulting spots from all three samples traveled about same distance. There were three big spots muddy brown in color. Out of the three main muddy spots, there were also some yellowish spots observed for the spearmint leaves extraction sample and co-spot mixture sample. Knowing that both of the enantiomers contain Limonene, these yellowish spots probably indicate presence of Limonene. The location of the spots indicates the affinity of the compound to specific phase. The muddy spots observed were close to each other at similar distance from the baseline. This indicates that the compounds were less polar and had greater affinity to the mobile phase and quickly moved up the plate with larger retention factors. Also, more polar compounds in the sample have greater affinity to the stationary phase and therefore move slowly with smaller retention factors. This findings suggest that Carvones are less polar compared to limonene, which is somewhat more polar because it was found in the middle of baseline and muddy spots. This reasoning is supported by the calculated retentions factor. The Rf for the Spearmint leaves for the three samples were calculated to be: pure (R)-Carvone sample = 0.71, Spearmint extraction = 0.73, and Co-Spot Mixture = 0.73. The limonene observed in spearmint sample had Rf = 0.60 for spot 1 and Rf =0.47 for spot 2, and the co-spot mixture had Rf = 0.62 for spot 1 and Rf = 0.49 for spot 2. For the caraway seeds, the Rf for the pure carvone sample is 0.75, co-spot mixture = 0.76, and seed extraction = 0.75. When looking at the main muddy carvone spots specifically, the retention factors are almost identical polarities among the enantiomers.
Thus far, by performing various methods and comparing results, the physical properties of enantiomers were identified, which are identical in boiling point, melting point, dipole moments, IR frequencies, and retention factors as supported by the results. Enantiomers are non-superimposable mirror images with identical physical and chemical properties in an achiral environment. However, an important exception was observed through district smell of each carvone. Due to opposite optical rotation of enantiomers and chirality of olfactory receptors (S)-(+)-Carvone enantiomer has a caraway smell, while the (R)-(-)-carvone enantiomer has a spearmint smell.
Reference
Gilbert, John C. and Martin, Stephan F. Experimental Organic Chemistry: A Miniscale and
Microscale Approach, fourth edition. United States: Thomson Brooks/Cole, 2006
Landrie, Chad L. Organic Chemistry Lab I (233): Lab Manual and Course Materials. 2010.
by
nighatali (student)
