EXPERIMENT # 3: Essential Oils (EO)
實驗#3:精油實驗
WARNING NOTICE: The experiments described in these materials are potentially hazardous and require a high level of safety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. Legal Notices
警告：在這些資料中所描述的實驗是有潛在危險的，而且要求有高度的安全訓練、特別的設施和裝備，且要有適當的人員監督下才能進行的。你要獨自承擔責任， 義務，而且要為履行上述的安全手續和措施負責。麻省理工學院將沒有責任，義務，或是為滿足上述材料提及到的安全措施而負責。法律說明
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
麻省理工學院
Department of Chemistry
化學系
5.310 Laboratory Chemistry
5.310化學實驗
EXPERIMENT #3
實驗#3
ESSENTIAL OILS[1]
精油1
I. INTRODUCTION
I. 簡介
In this experiment[2] you will be working with oils prepared from caraway seeds and spearmint leaves. Each oil has a distinct and characteristic odor, yet carvone is the major component in both oils! It is amazing that the difference in odor is attributable solely to a difference in chirality of the carvone in the two oils. Due to chirality of odor receptors in the nose the R-carvone and S-carvone fit into different receptor sites, hence different odor. Can you distinguish between the odors? 8-10% of the population cannot.[3] Some physical data[4] are presented below.
在這個實驗2中，你將從葛縷子種子和綠薄荷葉中提煉出油來。兩種油各自有種與眾不同的氣味，然而香芹酮是這兩種油中的主要成分！ 令人吃驚的是氣味的不同完全是由於兩種油中香芹酮手性的不同而引起的。由於鼻子中氣味感受體的手性。R-香芹酮和S-香芹酮與不同的感受體位置結合，因而感受到不同的氣味。你能夠區別這兩種氣味嗎？ 8-10%的人不能3。以下顯示了一些物理數據4。
(S)-(+)-Carvone (R)-(-)-Carvone
(S)-(+)-香芹酮 (R)-(-)-香芹酮
FW = 150.22; bp 98-100/10 mm Fw=150.22; bp 227-230 oC
分子量=150.22；沸點98-100/10 mm 分子量=150.22；沸點227-230 oC
nD20 = 1.4970; d=0.9608 g/mL nD20 = 1.4990; d=0.9593 g/mL
[α]D20=+61.7 o (neat 96%) [α]D20=-62.5 o (neat 98%)
[α]D20=+61.7 o (純度96%) [α]D20=-62.5 o (純度98%)
major component of caraway oil major component of spearmint oil
葛縷子油的主要成份 綠薄荷油中主要成份
(Carum carvi) (Mentha spicata)
(葛樓子) (綠薄荷)
All the physical properties should be identical except for the optical rotations of the two isomers (enantiomers), which are of opposite sign. Thus, for both (+)- and (-)- carvone, the infrared, nuclear magnetic resonance spectra, the gas chromatographic retention times, the refractive indexes, and the boiling points should be identical. Hence, the only difference in properties one should observe for the two carvones are the odors and the signs of rotation in a polarimeter. However, some of the physical properties presented above are not identical because of trace impurities.
這兩個異構體(對映體)除了旋光度有相反的符號之外，其他所有的物理性質都應該是完全相同的。因此， (+)- 和 (-)-的香芹酮的紅外，核磁共振光譜，氣相色層分析的保留時間，折光率和沸點應該是完全相同的。因此，它們在性質上的唯一區別應該可通過觀測兩種香芹酮的氣味和旋光儀中旋光度的符號而得知。但是，痕量的雜質也會使上述所提到的一些物理性質不完全一樣。
The * in the formulas below denotes a chiral carbon center. Chiral or asymmetric compounds in nature exist only in living tissue or in matter that was once part of living tissue. Chirality plays a major role in the mechanisms of biological recognition. Yet it is a mystery why caraway plants, Carum carvi, produce S-(+)-carvone and spearmint plants produce its mirror image (R)-(-)-carvone. Other plants such as gingergrass produce racemic carvone. Nature goes one step further; some botanically indistinguishable plants grown in different countries can carry out complete metabolic sequences of mirror-image reactions. Presumably, the enzymes that catalyze the reactions also have a mirror-image relationship. Another example of chiral recognition[5] is found in the effect these two carvone isomers have on rates of reaction. The toxicity of (+)-carvone in rats is 400 times greater than that of (-)-carvone.
以下的分子式用*表示的是手性碳中心。在自然界中的手性或不對稱的化合物只存在於活組織或曾經是活組織的一部份的物質中。手性在生物識別的機理中扮演着一個重要的角色。這還是為什麼葛縷子植物，葛樓子，能產生S-(+)-香芹酮，而綠薄荷植物會產生它的鏡像物(R)-(-)-香芹酮的秘密所在。 其他的植物如薑草會產生外消旋的香芹酮，自然發展更進一步：一些在植物學上不可區別的植物在不同的國家都有生長，它們可以進行一系列完整的與新陳代謝有關的鏡像反應。 據推測，能催化反應的酵素應該也是有鏡像關係的。我們也發現這兩個香芹酮異構體對反應速率有不同影響的其他手性識別5的例子。 (+)-香芹酮對老鼠的毒性較(-)-香芹酮對老鼠的毒性大大超過四百倍。
Essential oils are extracts from fragrant plants. They are used extensively in the perfume and flavoring industry. Most components of essential oils are terpenes that contain multiples of a five carbon structural unit, the isoprene unit (Fig. 1).
精油是從芳香性植物提取出來的。它們廣泛用於香水和調味工業中。精油中大部份的成份是由多個五個碳結構單元，即異戊二烯單元組成的萜烯類(圖1)
(R)-(+)-Limonene (R)-(+)-α-Phelandrene (1R,5R)-(+)-α-Pinene Isoprene
(R)-(+)-檸檬烯 (R)-(+)-α-水芹烯 (1R,5R)-(+)-α-蒎烯 異戊二烯
Figure 1. Representative monoterpenes. Isoprene units are shown to indicate the common structural features.
圖1.有代表性的單萜烯類。顯示了異戊二烯單元的一般結構特點。
In addition to monoterpenes, compounds derived from two isoprene units, essential oils contain less volatile compounds derived from three and four isoprene units. These higher boiling components will be removed by vacuum distillation of the provided sample to permit facile gas chromatographic separation.
除了含有由兩個異戊二烯單元組成的單萜烯類以外，精油還含有較少揮發性的，由三個或四個異戊二烯單元組成的化合物。為了能順利地進行氣相色層分離，可以對所提供的樣品進行真空蒸餾，把較高沸點的混合物除去。
Overview of the Experiment
實驗概述
(A) You will be given a sample of either caraway oil or spearmint oil. The major component of these oils is carvone. You will separate the carvone from the higher-boiling and lower-boiling impurities (such as limonene), via a vacuum distillation.
(A) 你將會收到一個葛縷子油或綠薄荷油樣品。這些油中的主要成份是香芹酮。你將會通過真空蒸餾法，把香芹酮中較高沸點和較低沸點的雜質(如檸檬烯)分離開。
(B) You will use gas chromatography and refractometry to check the purity of your distillate and to estimate the relative concentrations of limonene and carvone in the oil.
(B) 你將用到氣相色層分析和折射儀去檢測你的蒸餾物的純度。從而估計在油中檸檬烯和香芹酮的相對濃度。
(C) You will convert the carvone to its semicarbazone for use in a polarimetric analysis.
(C) 為了作旋光度分析，你將會把香芹酮轉變為縮胺基脲。
(D) You will obtain infrared spectra of the carvone and limonene fractions and interpret the results.
(D) 你將得到香芹酮和檸檬烯餾份的紅外光譜圖。並且要解析其結果。
(E) You will also characterize the semicarbazone by melting point determination.
(E) 你也要通過測定熔點以鑑定縮胺基脲。
(R)-(+)-Limonene (S)-(-)-Limonene
(R)-(+)-檸檬烯 (S)-(-)-檸檬烯
FW = 136.24; bp 175.5-176 oC Fw=136.24; bp 175-177 oC
分子量=136.24； 沸點175.5-176 oC 分子量=136.24； 沸點175-177 oC
nD20 = 1.4730; d=0.840 g/mL nD20 = 1.4720; d=0.844 g/mL [α]D20=+123 o (neat ) [α]D19=-94 o (c=10, ethanol)
[α]D20=+123 o (純淨的 ) [α]D19=-94 o (c=10, 乙醇)
II. SAFETY
II. 安全
1. Carvone: Ketone found in caraway, dill and spearmint oils. The oils are used for flavoring liqueurs, and in perfumes and soaps. Although both enantiomers occur naturally in consumer products, both should be handled with the usual care and not ingested under any circumstances.
1. 香芹酮：在葛縷子， 蒔蘿和綠薄荷油中發現的酮類物質。這些油可用作增加利口酒的風味，也可用於香水和肥皂中。雖然它的兩個對映體都會自然地在存在於消費產品中。但還是應該小心處理，而且在任何情況下都不得攝入。
.
2. Limonene: Occurs in various oils such as Levant wormseed oil, pine needle oil and other oils. It is used as a solvent, wetting and dispersing agent. It is not considered toxic, but is an irritant. Therefore, keep it off the skin.
2. 檸檬烯： 出現在各種油如Levant土荊芥油、松針油和其他油中。它可用作溶劑， 濕潤劑及分配劑。它被視為無毒，但是它是刺激物。因此，要遠離皮膚。
3. Semicarbazide hydrochloride: Mutagen and cancer suspect agent. Do not inhale or ingest.
3. 胺基氫氯酸鹽: 誘導有機體突變的物質和懷疑致癌物質.不可吸入或攝入.
4. Ethanol: Flammable liquid. The type used in this laboratory is NOT safe to drink.
4.乙醇：可燃液體。實驗室所用的那一種是不能飲用的。
5. Sodium acetate: Irritant. Handle with usual caution.
5. 醋酸鈉：刺激物。按正常程序小心處理。
II. BACKGROUND FOR EXPERIMENTAL PROCEDURE
II. 實驗過程的背景
General References
總體參考資料
l Distillation MHSM, Chapter 11, pp 124-129
l 蒸餾 MHSM, 第11章, pp 124-129
l Vacuum Pumps TM(I), Sec. 11C.
l 真空泵 TM(I), Sec. 11C.
l Gas Chromatography MHSM, Chapter 16, pp 163-177
l 氣相色層分析 MHSM, 第16章, pp 163-177
l Polarimetry MHSM, Chapter 14, pp 140-147
l 旋光儀 MHSM, 第14章, pp 140-147
l Refractometry MHSM, Chapter 13, pp 136-140
l 折光儀 MHSM, 第13章, pp 136-140
l Infrared Spectroscopy MHSM, Chapter 18, pp 197-219
l 紅外光譜 MHSM, 第18章, pp 197-219
Distillation
蒸餾
The difference between the boiling points of carvone (230 °C @ 760 torr) and limonene (177 °C @ 760 Torr) is sufficient to permit separation of the two compounds by distillation. However, carvone thermally decomposes at higher temperatures; therefore, a vacuum distillation is necessary.
香芹酮(230 °C @ 760 陶爾)和檸檬烯(177 °C @ 760 陶爾)之間沸點的差異促以使這兩個化合物能用蒸餾法把它們分離。可是，香芹酮在較高的溫度下會發生熱分解；因此， 必需用真空蒸餾法。
Two problems are encountered in a vacuum distillation. The volume of vapor formed from a given amount of liquid is pressure dependent; i.e., the volume of vapor formed from one drop of liquid will be about 30 times as great at 25 torr as it was at 760 torr. As a result, serious bumping may occur. Boiling chips generally do not help much at the reduced pressures. Some of the bumping can be overcome with the use of a magnetic stir bar. The second problem is also related to the larger volume of vapor at lower pressure. The velocity of the vapor entering the column is greatly increased. This creates a greater pressure in the column than is registered on the manometer. Maintaining a slow, steady rate of distillation can minimize this difference in pressure.
在真空蒸餾中會遇到兩個問題：己知量的液體所形成的蒸氣體積是依賴於其氣壓的；例如一滴液體在25陶爾時所形成的蒸氣的體積是它在760陶爾時所形成的體積的30倍。因此，可能會出現嚴重的爆沸。在減壓時，沸石通常不會有多大的幫助。磁攪拌子可以克服一些爆沸的情況。第二個問題是與較低的蒸氣壓力下會有較大的蒸氣體積有關。進入柱子的蒸氣速度會大大增加，這對柱子會產生一個較壓力計所示的讀數更大的壓力，保持一個慢的，穩定的蒸餾速度可以使氣壓的增減降至最少。
Gas Chromatography and Refractometry
氣相色層分析法和折光率分析法
In Gas Liquid Chromatography a mixture of vapors is carried in a stream of helium (carrier gas) through a column. The vaporized sample components move through the column that is lined with a liquid stationary phase. The different components in the sample are retained on the stationary phase for different characteristic relative times. Each component ultimately reaches the Flame Ionization Detector, the most commonly used detector in GC (Air + Hydrogen gas, ratio 10:1). They are detected by their ability to form ions when they are burned in the H2 / air mixture. The area under a peak in a gas chromatogram is proportional to the amount of that substance in the sample.
在氣液色層分析中，蒸氣的混合物由源源不斷的氦氣(載氣)帶入柱中。蒸發了的樣品組份會慢慢經過柱子，並且會依次跟液體固定相作用。樣品中的不同組份在固定相的保留情況是不同的，因而有不同的特徵的相對時間。每個組份最後都會到達火焰離子化檢測器，這是氣相色層分析最常用的檢測器(空氣+氫氣，比率是10:1)。當它們在H2 /空氣的混合物中燃燒時，它們能形成離子，並被檢測。在氣相色層析分析譜圖中的峰面積與樣品中物質的量是成正比的。
Among the factors that influence the separation of compounds by gas chromatography are selection of liquid phase, column temperature, and flow rate of carrier gas. Two common liquid (stationary) phases are silicone oil, which separates components on the basis of boiling point, and carbowax (polyethyleneglycols), which separates components on the basis of polarity. The effect of increased column temperature is to decrease the retention time of a component. As a rough approximation, a 10-15 °C decrease in column temperature corresponds to a doubling in the retention time. For most samples, the lower the column operating temperature, the higher the partition coefficient in the stationary phase and hence the better the separation. Too low a column temperature can lead to broad, asymmetric peak shapes. The criterion for resolution of the sample is simply achieving baseline separation of the components. Varying the column temperature and selecting the appropriate liquid phase will achieve resolution of the sample into its components. Identification of retention time can be accurately obtained using a pentane peak as a standard. There will always be enough pentane in the syringe to leave a small peak on the chromatogram. The retention time of
在氣相色層分析期間，影響化合物的分離因素有液相的選擇，柱溫，載氣的流速。兩個常見的液(固定相)相是基於沸點而對組份起分離作用的矽氧烷油和基於極性對組份起分離作用的碳蠟。增加柱溫能減少組份的保留時間。粗略的估計，當柱溫減少10-15 °C時。保留時間將增加兩倍。對大部份樣品而言，柱子的操作溫度越低，固定相中的分配係數會越高，因而有較好的分離效果。太低的柱溫會引起寬闊的，不對稱的峰形。 分離度的標準是樣品中的組份要達到基線分離。改變柱溫和選擇適當的液相將會使樣品中的組份得以分離。用戊烷峰作為標準可以準確測得保留時間值。在注射器中要有足够的戊烷使其在層析圖譜中留下一個小峰。可以用戊烷峰來計算其他峰的保留時間。利用適當峰的峰面積，可以計算在每一種餾份和原油中的香芹酮和檸檬烯的含量。
EO-21
EXPERIMENT # 3: Essential Oils (EO)
the other peaks can be calculated using the pentane peak. The relative amounts of carvone and limonene in each fraction and the original oil maybe calculated by using the area under the appropriate peaks.
By measuring the refractive index of the original oil, limonene and carvone fractions, you can estimate the purity of the respective fractions and the composition of the original oil. Assuming that the actual refractive index, n, measured for the two-component mixture (limonene and carvone) is linear in the molar fraction, x, of any of the components, then one can write:
通過測量原油，檸檬烯和香芹酮餾份的折光率，你可以估計一下原油的組份和各個餾份的純度。假設實際的折光率為n，所測得的兩組份混合物(檸檬烯和香芹酮)中各組份的莫耳分數x，是線性的，那麼可以寫成：
n = (1-x carvone) * n limonene + x carvone * n carvone
Plug in your data and determine the value of xcarvone for the limonene and carvone distillation fractions and the oil itself. Compare these results with those obtained by GC.
把你的數據組合起來，然後確定在檸檬烯，香芹酮餾份和油本身的xcarvone的值，將這些數據和氣相色層分析的數據比較一下。
III. EXPERIMENTAL PROCEDURE
III. 實驗步驟
DAY 1: Distillation and Gas Chromatography: WORK in PAIRS. Split evently the limonene and carvone fractions for the derivatization step.
第一天：蒸餾和氣相色層分析：以對為單位工作，一組分一個檸檬烯一組分一個香芹酮餾份作為衍生化步驟
Part A. Distillation
A.部份 蒸餾
Before setting up the glassware as shown in Fig. 2 have your teaching assistant demonstrated how to connect to the vacuum pumping manifold. You should be able to reduce the pressure to 3-5 Torr (or less) in a closed system. At this pressure, limonene should vaporize at a temperature between 34 °C and 38 °C, while carvone should distill at about 70-80 °C. Carefully assemble the glassware as shown in Fig. 2. Lightly grease all joints as demonstrated by your TA. Be sure to include a heating mantle, a stirring plate and an ice-bath in the setup. Do not proceed until your TA has checked your setup.
在建立如圖2所示的玻璃儀器前，先由助教示範怎樣去連接真空泵的管子。你應該能夠在一個密閉的系統中將壓力減少至3-5陶爾(或更小)。在這個壓力下，檸檬烯應該會在34 °C 和38 °C間蒸發，然而香芹酮應該會在70-80 °C間蒸餾出來。小心按照圖2，所示組合玻璃儀器。按照助教的示範，在所有連接處稍微塗上油脂。在組合中應該包括一個加熱包，攪拌盤和冰浴。直至助教檢查過你的裝置後才可以進行實驗。
Pour 10 mL (save a small portion, about 5 drops, for gas chromatography and refractive index measurements) of your essential oil into the round bottom flask, and add a 1” stir bar.
將10 mL(保留一小部份，大約5滴，留作氣相色層分析和折光率的測量用的)精油倒入一個圓底燒瓶中，加入一粒1英吋長的攪拌子。
l Check if the water-in and water-out connections are set correctly (see Fig. 2).
l 檢查一下進水口和出水口的連接是否正確(參見圖2.)
l Turn the water on in the condenser.
l 把接到冷凝管的水打開
l Immerse the four pear shaped receivers flasks (connected to cow and secure with yellow Keck clamps) into a mixture of ice and water.
l 將四個梨形接收燒瓶(與多尾接液管相接，並且用黃色的有齒夾子固定)浸入冰水混合液中
l Turn on the magnetic stirrer and fully open the vacuum valve (note: pressure cannot be controlled with this valve. If an adjustment is necessary, please see the Instructor). The pressure should read less than 10 mm Hg (10 Torr) on the manometer. Determine the temperature the limonene and carvone fractions should begin to distill based on
l 打開電磁攪拌器，並且將真空閥完全打開(注意：不可以用這個閥門來控制氣壓。如果需要調整，請參看指導者)在壓力計中壓力的讀數應該不小於10 mm Hg (10 陶爾)。 利用這個實驗最後的圖表，以壓力計的壓力讀數為依據，確定檸檬烯和香芹酮餾份蒸餾時的起始溫度。 跟助教核對你的答案時不必感到拘束。
EO-21
EXPERIMENT # 3: Essential Oils (EO)
the pressure reading on the manometer, using the chart at the end of this experiment. Feel free to verify your answers with your TA.
l Use the Variac dial (starting at setting 30), increase the temperature slowly. Although some “explosive” bumping is expected, do not allow the heat to reach such a level that the column is flooded since this will dramatically decrease the efficiency of the separation.
l 用自耦合變壓器的旋鈕(起始設定為30)，慢慢地升高溫度。雖然預期會發生一些「爆沸」，別加熱到柱子完全被淹沒的程度，因為這樣會大大降低分離效能。
Note: On a warm day the room temperature may be at or above the boiling point of limonene (ca 32 ºC). If the laboratory is warm be sure to check for limonene distillation before turning on the heat source. Limonene condensation will occur efficiently due to the condenser and the ice bath in which the receiver flask is placed (ca. 0 ºC).
注意:在和暖的日子裡，室溫可能會在或超過檸檬烯的沸點(大約32 ºC)。如果實驗室是和暖的話，在打開熱源的時候要檢查一下檸檬烯的蒸餾情況。檸檬烯是否能有效地冷凝取決於冷凝管和接受瓶是否置於冰浴中(大約0 ºC)。
When carvone begins to distill, the condensation can be seen inside the thermometer adapter, thus the progress of carvone distillation can be readily monitored. The rate at which the temperature changes at the top of the column is also extremely significant. Be sure to record temperature information in your lab notebook.
當香芹酮開始蒸餾時，在溫度計轉接器內可看到有冷凝，因此能够容易地監察香芹酮的蒸餾過程。在柱子頂部溫度的改變速度也是十分明顯的。確保在你的實驗記錄本中記錄溫度資料。
To ensure an optimal separation, three fractions should be collected: the limonene fraction, an intermediate fraction that forms during the rapid increase in temperature after collection of the limonene fraction, and the carvone fraction.
應該收集三個餾份以確保理想的分離效果：檸檬烯餾份，收集檸檬烯餾份後快速升溫所產生的中間餾份以及香芹酮餾份。
To collect each fraction, rotate (under vacuum) the cow adapter such that the end of the bent outlet sits above the next empty receiver flask. Label flasks, for example, with letters A, B, and C.