Department of Chemistry and Chemical Laboratory
Chongqing University of Medical Sciences

余瑜
尚京川
孙立力
周丽平
王驰
于明安
胡雪原
杨晓兰
母昭德
陈志琼
李伟
陆巍
黄维
汤渝
景淑华
潘黎军
张冀平

With the goal of bilingual teaching we compile this book in January, 2006.
This book contains thirteen experiments whose purpose is to help you learn the basic operations by applying them to a preparation or to the solution of a specific problem.
This book is compiled by Yu yu( 余瑜 ), Shang Jingchuan( 尚京川 ), Sun Lili( 孙立力 ), Zhou
Liping( 周丽平 ) , Yu Ming’an( 于明安 ), Hu Xueyuan( 胡雪原 ) ， Wang Chi( 王驰 ), Yang
Xiaolan( 杨晓兰 ), Mu Zhaode( 母昭德 ), Chen Zhiqiong( 陈志琼 ), Li Wei( 李伟 ), Lu Wei( 陆
巍 ) ， Huang Wei( 黄维 ), Tang Yu( 汤渝 ) ， Jing Shuhua( 景淑华 ), Pan Lijun( 潘黎军 ) and Zhang
Jiping( 张冀平 ).
The draft is examined and revised by Yu yu( 余瑜 ), Sun Lili( 孙立力 ) and Zhou Liping( 周丽
平 ).
We will be pleased to receive any constructive comments, suggestions for improvements, or criticisms from those who have used this book. We will be very glad to thank you.
January, 2006

Laboratory Safety ............................................................................................................. (1)
Glassware and Equipment in the Laboratory.................................................................... (7)
1 Determination of Melting point ................................................................................ (10)
2 Determination of Boiling point................................................................................. (13)
3 Molecular Model ...................................................................................................... (20)
4 Preparation of Cyclohexene...................................................................................... (22)
5 Preparation of Acetylsalicylic Acid .......................................................................... (24)
6 Isolation of Caffeine from Tea .................................................................................. (26)
7 Extraction of Lecithin and identification of its compositions ................................... (30)
8 Thin Layer Chromatography (TLC) ......................................................................... (33)
9 Paper Chromatography of Amino Acids ................................................................... (37)
10 Cellulose Acetate Membrane Electrophoresis of Serum Proteins .......................... (41)
11 The Chemical Property of Carbohydrates .............................................................. (43)
12 The Chemical Property of Proteins ........................................................................ (46)
13 Identification of Organic Compounds .................................................................... (49)
14 Determination of Optical Rotation of Glucose ....................................................... (52)
References ...................................................................................................................... (53)

LABORATORY SAFETY 1
Preventing Laboratory Accidents
You must observe a few basic safety rules:
1. Wear appropriate glasses or safety goggles in the laboratory at all times. Prescription glasses should have safety lenses, if possible. Contact lenses should not be worn in the laboratory since corrosive fumes or chemicals may get underneath them. The location and operation of the eyewash fountain should be learned at the first laboratory session.
2. Never smoke in the laboratory or use open flames in operations involving low-boiling flammable solvents. Ethyl ether and petroleum ether are particularly hazardous, but other common solvents such as acetone and ethanol can be dangerous as well. The location and operation of the fire extinguishers, fire blanket, and safety shower must be learned at the first laboratory session. Smoking is prohibited in chemistry laboratories because of the fire hazard.
3. Consider all organic chemicals hazardous to your health and minimize your exposure to them. Do not inhale the vapors of volatile chemicals and your skin, eyes, and clothing; some chemicals cause burns or severe allergic reactions, and others can be absorbed through the skin and cause poisoning. Do not taste any chemicals, and wash your hands thoroughly after handling them. To prevent accidental ingestion of any toxic chemicals, do not bring food or drink into the laboratory or use mouth suction for pipetting. Read the Safety Precautions section for each experiment, and use protective gloves or a fume hood when indicated.
4. Handle strong acids or bases, bromine, and other corrosive chemicals with great care and never allow them in contact with skin or eyes. Spills of such chemicals must be cleaned up immediately, using an appropriate neutralizing agent and plenty of water.
5. Use safe techniques for inserting and removing glass tubing. Great care must be exercised when inserting or removing thermometers and glass tubing from stoppers and thermometer adapters, since severe lacerations can result from accidental breakage.
6. Wear appropriate clothing. Serviceable shoes (not sandals) should always be worn in the laboratory to provide adequate protection against spilled chemicals and broken glass. Hairnets for long hair may be advisable if burners are in use (human hair is very flammable!) clothing should be substantial enough to offer protection against accidental spills of corrosive

2 ORGANIC CHEMISTRY EXPERIMENTS chemicals, but the laboratory is obviously not the place to wear your best clothes.
7. Dispose of chemicals in the proper containers and in the manner specified by your instructor. Most organic chemicals, for reasons of safety and environmental protection, should not be washed down the drain. Whenever possible, they should be disposed of in waste crocks
(for solids) or in labeled waste-solvent cans. Some dilute aqueous solutions can be safely washed down the drain, but the instructor should be consulted if there is any question as to the best method for disposing of particular chemicals or solutions.
8. Never work alone in the laboratory or perform unauthorized experiments. If you find it necessary to work in the laboratory when no formal lab period is scheduled, you must obtain permission form the instructor and be certain that others will be present while you are working.
Reacting to Accidents: First Aid
Any serious accident involving poisoning or injury should be treated by a competent physician. To minimize the damage from such an accident, all students and instructors should be familiar with some basic first-aid procedures. If you have an accident that requires quick action to prevent permanent injury (flushing chemical burns with water, etc.), take the appropriate action as describe below and then, as soon as possible, see that the instructor is informed of the accident. If you witness an accident you should summon the instructor immediately and leave the first aid to him or her unless (1) no instructor or assistant is in the laboratory area; (2) the victim requires immediate attention because of stopped breathing, heavy bleeding, etc.; (3) you have had formal training in emergency first-aid procedures and the instructor consents.
If an accident victim has difficulty breathing or goes into shock as a result of any kind of accident, standard procedures for artificial respiration and treating shock should be applied.
Eye Injuries .
If any chemical enters your eyes, they should immediately be flooded with water from an eyewash fountain, with the eyelids kept open. If contact lenses are being worn, they must be removed before irrigation. Irrigation should continue for at least 15 minutes, and the eyes should then be examined by a physician. The use of boric acid or other neutralizing solutions is not recommended for eye injuries, since this sometimes causes more damage than no

LABORATORY SAFETY 3 irrigation at all.
If foreign bodies such as glass particles are propelled into an eye, the injured person should get immediate medical attention. Removal of such particles is a job for a specialist.
Chemical Burns.
The affected area should immediately be flushed with water, using a safety shower if the area of injury is extensive or if it is inaccessible to washing from a tap. Speed in washing is the most important factor in reducing the extent of injury. Water should be applied continuously for 10~15 minutes and the burned area should then be covered with sterile gauze or other clean cloth. Neutralizing solutions, ointments, or greases should not be used on chemical burns unless specifically called for in a first-aid procedure. Unless the skin is merely reddened over a small area, a chemical burn should be examined by a nurse or physician.
If the burn is extensive or severe, the victim should be made to lie down with the head and chest a little lower than the rest of the body. If the victim is conscious and able to swallow, he or she should be provided with plenty of nonalcoholic liquid to drink until a physician arrives.
Thermal Burns.
For small thermal burns such as those inflicted by handling hot glass, a sterile gauze pad should be soaked in a baking soda solution (2 tablespoons of sodium bicarbonate to a quart of lukewarm water, about 2.5%NaHCO
3
), placed over the burn, then bandaged loosely in place.
If the skin is not broken, the burned part can be immersed in clean, cold water, or ice can be applied to reduce the pain. Blisters should not be opened. Unless the skin is merely reddened over a small area, the burn should be examined by a nurse or physician.
In case of an extensive thermal burn, the burned area should be covered with the cleanest available cloth material and the victim should be made to lie down, with the head and chest lower than the rest of the body, until a physician arrives. If the injured person is conscious and able to swallow, he or she should be provided with plenty of nonalcoholic liquid to drink
(water, tea, coffee, etc.).
Bleeding, Cuts, and Abrasions.
In case of a minor cut or abrasion, the wound and surrounding skin should be cleansed with soap and lukewarm water that is applied by wiping away from the wound. A sterile pad should

4 ORGANIC CHEMISTRY EXPERIMENTS be held over the wound until bleeding stops and then be replaced by a clean pad, which should be secured loosely with a triangular or rolled bandage. The pad and bandage should be replaced as necessary with clean dry ones. Contact between the wound and the mouth, fingers, handkerchiefs, or other unsterile objects should be avoided. Antiseptics should not be used on an open wound.
In case of a major wound that involves heavy bleeding, you should not waste time cleansing the wound; apply pressure immediately directly over the wound. Use a pad (clean handkerchief or other cloth), and press firmly with one or both hands to reduce the bleeding as much as possible. The victim should be made to lie down with the bleeding part higher than the rest of the body, and the pad should be held in place with a strong bandage (a necktie may be adequate, or cloth strips torn form a shirt). A physician should be called as soon as possible, and the victim should be kept warm with a blanket or coat in the meantime. If the victim is conscious and able to swallow, he or she should be provided with plenty of nonalcoholic liquid to drink.
Poisoning.
Chemical poisoning may have occurred whenever, after chemicals have been handled or accidentally ingested, there is a pain or burning sensation in the throat, discoloration of lips or mouth, stomach cramps, nausea and vomiting, confusion, or loss of consciousness.
If poison is swallowed, a physician or ambulance should be called immediately, and the victim should be given 2~4 glasses of water or milk to drink. If it is safe to do so, vomiting should be induced by placing a finger at the back of the victim’s throat or by having the victim drink a glass of warm water in which 2 teaspoons of table salt have been dissolved (10 g in 200 mL), or one ounce (30 mL) of ipecac syrup. Vomiting should not be induced if the patient is unconscious, in convulsions, or has severe pain and burning sensations in the mouth or throat, or if the poison is a petroleum product or a strong acid or alkali. When vomiting begins, the victim should be placed face down with head lower than hips, and vomiting should continue to be induced until the vomitus is clear. When there is time, the poison should be identified (if possible) and an appropriate antidote given. If the poison cannot be identified and a physician is not present, a heaping tablespoon (15g) of the following universal antidote can be given in a half glass of warm water: 2 parts activated charcoal, 1 part magnesium oxide
(milk of magnesia), 1 part tannic acid. A sample of the poison should be saved for the

LABORATORY SAFETY 5 physician if possible; if the poison is unknown, the vomitus should be saved for examination.
If a poison has been inhaled, the victim must be taken to fresh air and a physician called immediately. If the victim shows evidence of difficulty in breathing, mouth-to-mouth resuscitation should be administered. The victim should be kept warm and as quiet as possible until professional help is available. If the poison is a highly toxic gas such as hydrogen cyanide, hydrogen sulfide, or phosgene, the persons attempting to rescue the victim should wear gas masks while they are in contact with the vapors.
In case of skin contamination by toxic substances, the same general procedure as for chemical burns should be followed.
In case of a serious fire, one’s first reaction should be to get away from it as quickly as possible. If the fire is small and confined to a container such as a flask or beaker, it can often be extinguished by quickly placing an asbestos pad or watch glass over the mouth of the container. Otherwise, you should get out of the area and let the instructor or an assistant extinguish it. If no instructor or assistant is in the vicinity, you may obtain a fire extinguisher of the appropriate type (dry chemical extinguishers are best for most flammable liquid fires) and attempt to put out the fire by aiming the extinguisher at the base of the fire (maintain a safe distance). Be prepared to call the fire department and arrange to evacuate the area if there is a chance that the fire cannot be controlled.
If your hair or clothing should catch on fire, go directly to the nearest fire blanket or safety shower and attempt to extinguish the fire by wrapping hair or clothing has caught on fire, try to prevent panic and wrap the person in a coat or fire blanket or lead him or her to a safety shower.
Chemical Hazards.
For your own health and safety, it is essential that you exercise caution while handling chemicals and minimize your exposure to them. Recent information about the health hazards posed by certain chemicals and more restrictive governmental regulations for their disposal have caused most academic chemistry departments to enact stringent policies for dealing with hazardous chemicals. Your instructor will inform you of any departmental or institutional rules relating to the safe handling and disposal of such chemicals. Most of the experimental procedures in this book describe specific chemical hazards and handling precautions under the heading Safety Precautions, and some may also include reminders regarding the disposal of

6 ORGANIC CHEMISTRY EXPERIMENTS certain chemicals. The hazard descriptions are meant to inform you of the potential danger posed by certain chemicals when they are not handled properly. If you observe the third safety rule listed above and follow the recommendations in the Safety Precautions sections, there is little likelihood that you will suffer any ill effects from working with any chemical.

GLASS EQUIPMENT 7
A typical set of standard-taper glassware is shown in Figure 1-1-1, and consists of: round-bottomed flasks for reactions, distillations three-neck flasks for more complicated reaction set-ups (two-neck flasks are also available) addition funnel for adding liquids to reaction mixtures (may be cylindrical or pear-shaped) separatory funnel for extractions and reaction work-up condenser for refluxing reaction mixtures, distillations air condenser for high boiling liquids (can also be packed and used as a fractionating column) drying tube for filling with a drying agent and attaching to the apparatus to reduce the ingress of water stoppers recuction/expansion adapters for connecting equipment with different-sized joints still head for distillation
Claisen adapter for distillation or converting a simple round-bottomed flask to a two-neck flask
Distillation adapter for distillation vauum distillation adapter for distillation under reduced pressure
Take off adapter for attaching to tubing
Thermometer/tubing adapter for inserting thermometer or glass tube into apparatus
A typical set of nongraduated glassware without ground glass joints are shown in Figure
1-1-2: beakers for temporary storage or transfer of materials, reactions
Erlenmeyer flasks for recrystallization, collecting solutions after extraction funnel for transfer of liquids, filtration powder funnel for transfer of solids stemless funnel for hot filtration filter flask ( Buchner flask ) for suction filtration
Hirsch funnel for suction filtration of small quantities
Pasteur pipet for transfer of smaller quantities of liquid graduated cyclinders for measuring liquids by volume

8 ORGANIC CHEMISTRY EXPERIMENTS graduated pipets for accurate measurement of liquids.
Figure 1-1-1 Glass equipment with standard-taper ground glass joints

GLASS EQUIPMENT 9
Figure 1-1-2 Other glass equipment

10 ORGANIC CHEMISTRY EXPERIMENTS
：
Purpose
1. To be familiar with the principles of determination of melting point.
2. To master the method of determination of melting point with capillary tube.
3. To know the significance of the determination of physical constant on identification of organic compounds.
Principles
The true melting point of a crystalline substance is defined as the temperature at which the solid phase exists in equilibrium with the liquid phase. At the melting point the vapor pressure of the solid phase equals the vapor pressure of the liquid phase (melt).
The temperatures recorded as the observed melting point are the temperatures at which the substance commences to liquefy and that at which it becomes completely liquefied. The melting point range (the interval between the beginning of liquefaction and complete liquefaction) and the actual temperature of the melting point are valuable indications of the purity of the substance.
A pure crystalline organic substance usually possesses a sharp melting point; that is, melts completely over a very short temperature range not more than 0.5~1.0℃. The melting point range is influenced not merely by the purity of the material but also by factors such as the size of the crystal, the amount of material, rate of heating, etc.
The presence of even small amounts of miscible, or at least partially miscible, impurities will usually produce a marked increase in the melting point range and a depression of the actual temperature at which fusion begins to occur. These facts are made use of in establishing the identity of organic compounds and in determining the purity of compounds by taking the melting points of mixtures, a method commonly known as “mixed melting points”.
Apparatus and Reagents
Capillary Tube; Thermometer (200℃); Alcohol Burner; Watch Glass; Glass Tube; Rubber
Band.
Paraffin Oil; Urea (A.R.); Benzoic Acid (A. R.); Mixtures of Equal Mole of Urea and

EXPERIMENT 1 DETERMINATION OF MELTIING POINT 11
Benzoic Acid.
Procedures
① Sealing capillary tubes
Put one end of a prepared capillary tube to the edge of a small hot flame and rotate the capillary tube slowly so that the end is sealed. To ensure the capillary tube sealed compactly and smoothly, you should operate it carefully and not make it bended or into a small ball.
② Filling capillary tubes
Place a small sample of the solid pulverized finely on a clean watch glass, then collect the solid into a small mound and push the open end of the capillary tube down into the sample.
The solid may be forced down by dropping the tube (sealed end downward) through a long length of ordinary glass tubing onto the desktop. Further increments of the sample are introduced in the same way until the material forms a compact column 3~5 mm high at the bottom of the tube after repeated dropping. It is essential that the material be packed firmly and densely into the end of the tube.
Follow the method above; fill two tubes with benzoic acid, urea, mixtures and unknown sample, respectively.
③ Arranging assembly
Introduce some bath liquid into a 50mL beaker and keep the bath level at the position of the two thirds of the beaker. The capillary tube containing the sample is attached to a thermometer by means of a small rubber band. The rubber band must be kept well above the level of the hot oil, or the oil could melt the rubber and break the band. For accurate reading, the sample compound in the capillary tube is kept close to and at the level of the thermometer bulb, which is fully submerged and centered in the oil bath, as shown in Figure 2-1-1.
④ Taking melting point
When the apparatus has been arranged, properly heat the bottom portion of the side arm with burner. Convection currents carry the heated oil up through the side arm and down the main shaft of the apparatus for uniform heating. Apply heat at a moderately rapid rate until the bath liquid is within 15~20℃ of the melting point. Continue the heating with a very small flame adjusted so that the temperature rises slowly and at a uniform rate (about 1℃ per minute). If necessary, hold the burner by its base and move it back and forth under the bath.
Observe carefully the samples in the melting point tube and the thermometer reading. Record

12 ORGANIC CHEMISTRY EXPERIMENTS as the observed melting point the range between the thermometer reading when sample starts to liquefy and that when the melt is clear.
Figure 2-1-1 Capillary tube method for determination of melting point a. Apparatus of capillary tube method b. Position of the capillary tube adhering to thermometer
After the samples have melted, extinguish the flame and allow the bath to cool. Melting point tube is discarded into waste crock after a single use.
When taking the melting points of a mixture or an unknown, you’d better make a rough preliminary determination.

EXPERIMENT 2 DETERMINATION OF BOILING POINT 13
：
Purpose
1. To be familiar with the principles of determination of boiling point.
2. To master the method of determination of boiling point.
3. To know the fundamental processes and the proper manipulation of distillation.
Principles
In a liquid the molecules are in constant motion and have a tendency to escape from the surface and become gaseous molecules, even at temperatures far below the boiling point.
When a liquid is placed in an enclosed space, the pressure exerted by the gaseous molecules increases until it reaches the equilibrium value for that particular temperature. The equilibrium pressure is known as the vapor pressure and is a constant characteristic of the material at a specific temperature. Although vapor pressures vary widely with different materials, vapor pressure always increases as the temperature increases. The vapor pressure is commonly expressed as the height, in millimeters, of a mercury column that produces an equivalent pressure (mmHg). The addition of soluble substances to a pure compound alters the measured vapor pressure.
In ordinary glass flasks, there are microscopic pockets of air trapped in the pores and crevices of the walls. With a liquid in the container the pockets are filled with vapor of the liquid at its equilibrium vapor pressure. When the temperature of the liquid is raised, the vapor remains compressed until the vapor pressure exceeds the applied pressure (the pressure at the liquid surface plus the hydrostatic pressure), whereupon the trapped vapor rapidly expands to form bubbles that rise to the surface and expel their vapor. The resulting agitation
(boiling) churns more air bubbles into the liquid where they continue the process after receiving new charges of vapor. Liquids heated in containers that have been degassed do not boil, although they vaporize explosively if heated to a sufficiently high temperature. To avoid the hazards associated with sudden, irregular boiling (bumping) a dependable source of bubbles should always be introduced into a flask before its contents are heated to boiling.
When a liquid is boiled at atmospheric pressure, the bubbles should always be introduced into a flask before its contents are heated to boiling. When a liquid is boiled at atmospheric pressure, the bubble source is customarily a boiling chip; with vacuum distillations boiling

14 ORGANIC CHEMISTRY EXPERIMENTS chips do not work as reliably and other sources are frequently used.
Boiling Point and Boiling Temperature.
The boiling point of a liquid is defined as the temperature at which its vapor pressure equals the atmospheric pressure. By convention, boiling points are reported in the scientific literature at a pressure of 1atm except when otherwise specified. The boiling temperature is the actual observed temperature when boiling occurs and is generally a few hundredths to a few degrees above the boiling point because of experimental difficulties involved in the measurement.
Distillation of a Pure Compound.
Distillation consists of boiling a liquid and condensing the vapor in such a manner that the condensate (distillate) is collected in a separate container. A simple apparatus assembly for this operation is shown in Figure 2-2-1.
Figure 2-2-1 Apparatus assembly for simple distillation
When a pure substance is distilled at constant pressure, the temperature of the distilling vapor will remain constant throughout the distillation, provided that sufficient heat is supplied to insure a uniform rate of distillation and superheating is avoided. In actual practice these ideal conditions are not realized; drafts in the laboratory can cause momentary condensation of vapors before they reach the thermometer. A certain amount of superheating of vapors occurs almost invariably under ordinary conditions. Because of these contrary effects a distillation range of 1℃ actually represents an essentially constant boiling point. With somewhat more refined apparatus and technique a distillation range of 0.1℃ can be observed for a pure compound.

EXPERIMENT 2 DETERMINATION OF BOILING POINT 15
The temperature reading of a thermometer in the distilling vapor represents the boiling point of that particular portion of the distillate. This temperature will be the same as the boiling point of the liquid in the distilling flask only if the distilling vapor and the boiling liquid are identical in composition. Since a pure liquid fulfills this condition, a constant thermometer reading is sometimes used as a criterion of purity of a liquid. It should be noted, however, that certain mixtures (such as azeotropes) give constant thermometer readings.
Occasionally two liquids have such similar boiling points that no appreciable change in thermometer reading will be observed when a mixture of them is distilled.
Apparatus for Simple Distillation.
A simple distillation apparatus suitable for distillation of samples greater than 5 mL in volume is shown in Figure 2-2-1. This consists of a round-bottom flask connected by means of a distillation adapter to a water-cooled condenser. A thermometer is held in place in the vertical arm of the distillation adapter by a special rubber connector at a height adjusted so that the top of the mercury bulb is even with the bottom of the opening of the side arm. A vacuum adapter is connected to the lower end of the condenser.
The distilled liquid is collected in a clean, dry receiver, commonly a round-bottom flask with its ground-glass joint mated to the lower joint of the distillation adapter. It is permissible to use an Erlenmeyer flask or a graduated cylinder as a receiver if vapor losses and fire hazards are minimized by inserting the lower end of the adapter well into the mouth of the receiver. A distilling assembly must have an opening to the atmosphere to avoid development of a dangerously high pressure within the system when heat is applied. When a mated round-bottom flask is used as the receiver, the side arm on the distillation adapter becomes the opening, and this arm must not be sealed.
If a burner is used as a heat source, the distilling flask should rest on a piece of wire gauze
(preferably one with a heat-dispersing center), which is supported on an iron ring. The main purpose of the wire gauze is to prevent superheating and decomposition of the liquid or vapor that might result from heating the sides and upper portion of the flask. An alternative, used almost exclusively in advanced work, is to heat the flask in an oil bath or with a heating mantle. Hot oil baths have the advantage of being more even heat sources, but they can cause severe burns if the hot oil spills on the skin. Heating mantles are safer if they are grounded properly and used with the proper electric-current-controlling device.

16 ORGANIC CHEMISTRY EXPERIMENTS
The size of the distilling flask chosen should be such that the material to be distilled occupies one-thirds of the bulb. If the bulb is more than two-thirds filled, there is danger that some of the liquid may splash into the distillate. If the bulb is less than one-third filled, there will be an unnecessarily large loss resulting from the relatively large volume of vapor required to fill the flask. This loss is particularly serious with compounds of high molecular weight.
The only exception to the one-third to two-thirds rule is with liquids, such as cyclohexanol, that foam badly on distillation. These liquids require a much larger distillation flask to contain the foam.
Whether a pure compound or a mixture is distilled, a small portion of liquid will always be left in the flask upon cooling. The flask containing the material to be distilled should never be heated to dryness with a flame since there is a possibility that the flask will crack.
Distillation
The proper method of carrying out a distillation is to supply just enough heat at the distilling flask so that the liquid distills regularly at a uniform rate. Insufficient supply of heat will arrest the distillation temporarily and permit the bulb of the thermometer to cool below the distilling temperature, resulting in erratic temperature readings. Overheating and unsteady application of heat increase the opportunity for superheating the liquid and result in bumping.
Even under the proper conditions of heating it is necessary to introduce one or two tiny boiling chips of porous substance or some other antibump agent into the liquid before heat is applied.
Superheating occurs because the transformation of a liquid into the vapor phase will not take place immediately, even at the boiling point, unless the liquid is in contact with a gaseous phase. Consequently, in a distilling flask, the liquid can vaporize only at the surface unless gas bubbles are introduced into the body of the liquid. Boiling chips consists of porous material containing a large amount of air, which expands on heating and furnishes bubbles that initiate vaporization throughout the liquid. Boiling chips lose their effectiveness after a single use and must be discarded; indeed, fresh boiling chips should be added before resuming a distillation that has been interrupted. It is dangerous to introduce boiling chips into a liquid that is at or near its boiling point, as this will induce sudden and violent bumping.
When the distillation assembly has been completed it is checked for tightness of all connections and for physical stability. The liquid to be distilled is introduced through the neck

EXPERIMENT 2 DETERMINATION OF BOILING POINT 17 of the distilling flask with the aid of a funnel to prevent it from contaminating the ground-glass joint. When a condenser is being used, the flow of water through the jacket is started before heat is applied. The water should enter the lower end of the jacket and flow in a direction opposite to that of the organic vapor (countercurrent cooling). The rate of flow through the condenser should not be excessive but adequate to keep the jacket cool; this may be tested from time to time by carefully touching the underside of the adapter through which the distillate is running to see if the distillate is too warm.
The rate of heating must be adjusted so that the liquid boils gently and distills slowly at a uniform rate, generally between 30 and 60 drops (1~2mL)/min for simple distillation. Heating should be stopped just before the last traces of liquid have been vaporized to avoid decomposition and charring in the flask. The thermometer reading is recorded when the first drops of distillate appear at the end of the side arm or on the walls of the condenser; this is called the “initial boiling point”. Thereafter the temperature and the volume of the distillate are recorded at frequent intervals. If the purpose of the distillation is to determine the composition of the liquid, many temperature-volume readings are required, and it convenient to collect the distillate directly in a graduated cylinder. The results should be recorded in a tabular form.
It is useful to plot a temperature-volume curve, from which the presence and amount of low-boiling impurities, the approximate distilling range of constant-boiling components of a mixture, and so on, can be determined. When a substance containing small amounts of impurities is distilled, the first portion of distillate (called the forerun or low-boiling fraction) will contain the more volatile impurity and a certain amount of the main liquid that is carried with it. As the temperature continues to rise, the bulk of the principal liquid will distill over a short temperature range, usually 2~3℃ (called the principal fraction or main fraction). After this fraction has distilled, the boiling point will rise, owing to the presence of the less volatile impurity. The next fraction (called the afterrun or high-boiling fraction) will consist of a mixture of the principal liquid and the less volatile impurity. The residual liquid in the distilling flask will contain the less volatile impurity. The residual liquid in the distilling flask will contain the less volatile impurity along with some of the principal liquid, which it holds back from distilling. However, even a pure substance will always leave a small amount of residual liquid.
When the distillation is being carried out to purify a liquid, it is better to use tared flasks to

18 ORGANIC CHEMISTRY EXPERIMENTS collect the different fractions. If the distillation behavior is known or can be estimated (as when a liquid of known boiling point is being purified), it is a simple matter to use three receivers and to collect the forerun, the main fraction, and the afterrun over the proper temperature ranges. When a liquid with unknown properties is being purified and a sufficient sample is available, it is a good strategy to determine first the temperature-volume distillation curve. If the losses of two distillations cannot be tolerated, it is necessary to deduce the boiling behavior of the sample as the distillation proceeds. This requires close attention to the thermometer readings; it is desirable that several extra tared flasks be available in case the collection of the main fraction is begun or ended prematurely.
Apparatus and Reagents
Round Bottom Flask (50mL); Condenser; Adapter; Distillation Head; Thermometer (100℃);
Receiver; distilled water
Assembly and Manipulation
A simple distillation assembly mainly consists of a distilling flask, a condenser and a receiver.
Arrange all the apparatus in order, usually from bottom to top and left to right.
Introduce 25mL of distilled water into a dry 50mL distilling flask. To help prevent bumping, add two or three tiny boiling chips to the distilling flask. (It is dangerous to introduce boiling chips into a liquid that is at or near its boiling point, as this may induce sudden violent ebullition, and after a single use these boiling chips must be discarded; indeed, fresh boiling chips should be added before resuming a distillation that has been interrupted.) the mouth of the flask is provided with a distillation head connected to the water-cooled condenser set at a downward angle and supplied with a rubber bung holding a thermometer, which is placed so that the top of the mercury bulb is just parallel to the lower wall of the side arm of the distillation head and this ensures that the bulb will be in the vapor stream and will give a correct reading.
When the distillation assembly has been completed, the flow of water through the condenser jacket is started (the water should enter from the bottom and exit from the top. The rate of flow through the condenser should not be excessive but adequate to keep the jacket cool.). Then heat the flask gently on wire gauze, using a small flame, until the liquid begins to boil, and record the temperature reading when the first drop of distillate fall in the receiver.

EXPERIMENT 2 DETERMINATION OF BOILING POINT 19
Adjust the rate of heating so that the liquid boils gently and distills slowly at a uniform rate, generally 1~2 drops per second for simple distillation. When 2~3mL of the liquid is remained, record the thermometer reading and discontinue the distillation (when the distillation has been finished, the burner flame should be first extinguished, then the flow of water is cut off.).

20 ORGANIC CHEMISTRY EXPERIMENTS
Purposes
1. To be familiar with the chair conformation.
2. To master to express the method of cis-trans isomer and Fischer projection.
3. To know the conformation and configuration of molecule.
Contents
1. Alkanes
(1) Build up the models of methane and dichloromethane.
(2) Build up the models of ethane, ethane and ethyne. Compare the sp 3 , sp 2 and sp hybrid orbitals.
(3) To know the conformation of ethane.
① What is conformation?
② Draw the stable conformation of ethane (in Newman projection).
③ Draw the instable conformation of ethane (in Newman projection).
④ Which sport form induce the transformation of conformations.
⑤ Draw the energy change curve of the conformation transformation of ethane.
(4) The isomer models of butane.
Draw four primary conformations of n-butane (in Newman projection).
2. Aromatic hydrocarbon
Watch the model of benzene.
3. Conformation of cyclohexane and its ramification.
(1) Build up the models of chair conformation of cyclohexane
， and along one C-C bond watch the model of Chair conformation of cyclohexane.
①
The bonds between two carbon atoms are across or overlap?
②
Find out the six equatorial bonds (e bonds) and six axial bonds (a bonds).
③
Draw the conformation formula of chair conformation of cyclohexane, line out a bonds and e bonds.
(2) Build up the models of boat conformation of cyclohexane. Along C
2
-C
3
bond and C
5
-C
6 bond watch the model of Chair conformation of cyclohexane.
① T he bonds between the two C-C atoms are across or overlap?

EXPERIMENT 3 MOLECULAR MODEL 21
②
The arrangement of C-H bond on the C
1
－ C
4
atoms.
③ Why the chair conformation of cyclohexane is more stable than boat conformation of cyclohexane?
④ Rotate the C-C single bond, change the conformation.
(3) The conformation of 1,2-dichlorocyclohexane
4. cis-trans isomers
(1) Build up the molecular model of 1,2-dichloroethene. How many models can you build up?
Draw their configuration formula and name them. What is the condition of isomerization?
(2) Build up the molecular models of cis-butenedioic acid and trans-butenedioic acid. The trans-butenedioic acid does not dehydrate intermolecule easily?

22 ORGANIC CHEMISTRY EXPERIMENTS
Purposes
1. To master the operation of fractionation and distillation.
2. To know the principle and method of synthesizing cyclohexene.
Principles
In laboratory you will produce cyclohexene through the acid catalyzed elimination of water from cyclohexanol (dehydration). Because the boiling point of cyclohexene is lower than that of corresponding alcohol, you can separate the cyclohexene from the mixture.
OH concentrated H
2
SO
4 b.p.161 ¡æ b.p.83 ¡æ
Apparatus and reagents
Fractionation apparatus, distillation apparatus, tap funnel, conical flask, glas funnel.
Cyclohexanol, concentrated sulfuric acid, anhydrous calcium chloride, sodium chloride, 5% sodium carbonate.
Procedures
Figure 2-4-1 Apparatus of fractionation

EXPERIMENT 4 PERPARATION OF CYCLOHEXENE 23
Add 20g of cyclohexanol (21mL, about 0.2mol), 1mL of concentrated sulfuric acid and several zeolites into 100mL dry round flask. Shake the mixture well.
Heat up the flask gently till the mixture boil. Control the temperature at the top of the fractionating column not more than 90 ℃ (cyclohexanol can form azeotrope with water, boiling point is 97.5
℃ , contain 80% water). You’d better control the rapid of distillation at
2~3 seconds per drop. When there is several milliliter of solution and produce white fog, stop heating immediately. The outputs contain cyclohexene and water. It needs about one hour for finishing the distillation. After reaction, dismantle the fractionation apparatus. Wash the flask with cold water and pour the remains into the waste liquid tank. Clean the flask with chloroform.
The distilled substance should be saturated with 2g NaCl and the residual acid should be neutralized with 3~4mL of 5% sodium carbonate. The output is separated in the tap funnel.
Shake the tap funnel and place it for delamination. The upper is rough cyclohexene.

24 ORGANIC CHEMISTRY EXPERIMENTS
Purpose
1. To be familiar with the principle of aspirin’s synthesizing via acetylating reaction.
2. To master method of aspirin’s synthesizing via acetylating reaction.
3. To know the recrystallizing technique.
Principles
Acetylsalicylic acid (aspirin) is a kind of antifebrile and antalgic medicine. Its structure is showed below:
3
Acetylsalicylic acid (aspirin) can be usually synthesized by acylation of salicylic acid. In this experiment, acetic anhydride is used as an acylating agent and concentrated sulfuric acid is used as a catalyst. The action of H
2
SO
4
is to break the intramolecular hydrogen bonding between carboxyl (-COOH) and hydroxyl (-OH) in salicylic acid so as to accelerate the reaction.
COOH
OH
+
CH
3
CH
3
O
C
O
C
O concentrated sulferic acid or phosphorus acid
COOH
O C
O
CH
3
+ CH
3
COOH
The crude products may contain a small amount unreacted salicylic acid which can be removed by recrystallizing technique.
Apparatus and reagents
Water bath, alcohol burner, Buchner funnel, filter flask, filter paper, platform balance, glass spoon, ice, 0.1
％ FeCl
3
solution, conical flask, beaker (50mL), graduated flask (10mL, 25mL), thermometer (150 ℃ ).
Salicylic Acid, acetic anhydride, 85% phosphorus acid, 95% alcohol.
Procedures

EXPERIMENT 5 PREPARATION OF ACETYLSALLICYLIC ACID 25
1. Preparation of acetylsalicylic acid
Place 2.1g (0.015mol) of salicylic acid crystals in a 100mL drought conical flask. Add 4mL
(0.03mol) of acetic anhydride followed by 5 drops of 85% H
2
SO
4
from a dropper, and stir carefully with a glass rod. Heat the flask on the hot water bath (70~80℃) for 15 minutes, and shake the flask sometimes during the reaction.
Cool the mixture for a while after the reaction has completed, then pour the mixture into a
100mL beaker containing 60mL cold water while stirring. Cool the mixture in an ice-cold water bath until the crystallization has completed. Collect the product by suction on a Buchner funnel and wash the crystals with cold water twice, then the crude product (about 2.5g) are obtained.
Dissolve a few crystals of salicylic acid or the crude product in 2mL of 95% ethanol in a beaker. Gently heat up the mixture on water bath (50~60℃) till the product dissolve. Add a little ice-cold water and cool the mixture (don’t shake). After the crystal separated out completely, vacuum filter, and wash the crystal with a little ice-cold water. Dry the purified material. Weight and calculate the percentage. Identify the purity of product with 0.1% ferric chloride (FeCl
3
) solution.
Calculate the yield of the acetylsalicylic acid:
Yield ＝（ real output/ theoretical output ） ×100 ％
Discussion
1. When we prepare acetylsalicylic acid, the apparatuses used must be anhydrous, why?
2. Why do we use the coned H
2
SO
4
in the acetylation reaction?
3. What side reaction will occur in the experiment?

26 ORGANIC CHEMISTRY EXPERIMENTS
Purposes
1. To be familiar with the principle and method of isolating alkaloid form plant.
2. To master the operation technique of isolating organic compounds by solvent method.
3. To know the properties of alkaloid.
Principles
Caffeine is a member of the class of compounds organic chemists call alkaloids . Alkaloids are nitrogencontaining basic compounds that are found in plants. They usually taste bitter and often are physiologically active in humans. The names of some of these compounds are familiar to you even if the structures aren’t: nicotine, morphine, strychnine, and cocaine. The role or roles these compounds play in the life of the plants in which they are found is not well understood. In some cases they may act as pesticides; nicotine is found in tobacco and has been sprayed onto other plants, in which it is not found, to function as an insecticide. The structure of caffeine is shown to the right. It can be considered to be constructed from the purine ring system, which is important biologically, being found in nucleic acids and elsewhere.
Caffeine is found in a number of things ingested by people. The table to the right lists some of them. Caffeine acts as a stimulant. It stimulates the heart, respiration, the central nervous system, and is a diuretic. Its use can cause nervousness, insomnia and headaches. It is physically addictive. A person who drinks as few as 4 cups of coffee a day and who attempts to stop “cold turkey” may experience headache, insomnia, and possibly nausea as the result of withdrawal.
Tea has been consumed as a beverage for almost 2,000 years, starting in China. It is a beverage produced by steeping in freshly boiled water the young leaves and leaf buds of the tea plant, Camellia sinensis . Today, two principal varieties are used, the small-leaved China plant ( C. sinensis sinensis ) and the large-leaved Assam plant ( C. sinensis assamica ). Hybrids of these two varieties are also grown. The leaves may be fermented or left unfermented.
Fermented teas are referred to as black tea, unfermented teas as green tea, and partially fermented teas as oolong. As trade routes opened to Asia in the 17th century, tea was imported to Europe. Today, you are going to make a small but strong cup of tea and extract

EXPERIMENT 6 ISOLATION OF CAFFEINE FROM TEA 27 the caffeine from it.
H
3
C
N
O
N
CH
3
O N
CH
3
N
Tea leaves consist mostly of cellulose, a water-insoluble polymer of glucose, which is a simple sugar (a monosaccharide). Cellulose performs a function in plants similar to that of fibrous proteins in animals: it is structure building material. Along with the cellulose are found a number of other things including caffeine, tannins (phenolic compounds, compounds that have an -OH directly bonded to an aromatic ring) and a small amount of chlorophyll. The idea in this experiment is to extract the water soluble materials in the tea leaves into hot water.
[The solubility of caffeine in water is 22 mg∙mL -1 at 25 ℃ , 180 mg∙mL -1 at 80 ℃ , and 670 mg∙mL -1 at 100 ℃ .] The hot solution is allowed to cool and the caffeine is then extracted from the water with dichloromethane (methylene chloride), which is an organic solvent that is insoluble in water. Since caffeine is more soluble in dichloromethane (140 mg∙mL -1 ) than it is in water (22 mg∙mL -1 ), it readily dissolves in the dichloromethane. However, the tannins are slightly soluble in the dichloromethane. But we want to separate the caffeine from the tannins by having the caffeine dissolve in the dichloromethane and the tannins remain in the water.
We can do this by taking advantage of the fact that phenols are acidic enough to be converted to their salts (deprotonation of the -OH group) by reaction with sodium carbonate. So, we will add sodium carbonate to the water and the tannins will be converted to phenolic anions, which are not soluble in the dichloromethane but are soluble in highly polar water. There is one practical disadvantage in converting the tannins to their salts – they become anionic surfactants. Detergents and soap are surfactants. It is the purpose of surfactants to cause materials that do not dissolve in water (like oil, grease and dichloromethane) to form an emulsion with water. We want to be able to separate the aqueous phase from the dichloromethane phase, so the last thing we want is an emulsion of the two. Consequently, as you extract the caffeine from the water into the dichloromethane do not shake the separatory funnel vigorously.
The flow diagram below summarizes the extraction portion of the experiment.

28 ORGANIC CHEMISTRY EXPERIMENTS tea leaves hot water
¹ý ÂË insoluble material: cellulose, etc.
tannin salts, water-soluble water-soluble material: mainly tannins,
Pb(AC)
2 filter caffeine aqueous phase filtrate concentrate chloroform extraction chloroform
phase caffeine boil off chloroform
Caffeine can be identified by murexide reaction and bismuth potassium iodide reagent.
Apparatus and reagents
Beaker, evaporating dish, tap funnel, filter flask, magnetism spoon, glass funnel, cotton, glass spoon, wood clamp.
Tea leaves, 10 ％ Pb(Ac)
2
, chloroform, NaCl, concentrated hydrochloric acid, potassium chlorate crystal, concentrated ammonia, 5 ％
H
2
SO
4
, bismuth potassium iodide reagent.
Procedures
1. Extraction of caffeine.
(1) Place 5g of tea leaves and 60mL of hot water into the bottom of a clean, but not necessarily dry, 250 mL beaker. Boil the contents of the beaker for 15 minutes (retain volume of water). Filter the mixture by the glass funnel with cotton. Add dropwise about 10mL of Pb(Ac)
2
solution into the hot filtrate until there is no settle to produce.
(2) Heat up the mixture for 5 minutes, then vacuum filter. Transfer the filtrate to the

EXPERIMENT 6 ISOLATION OF CAFFEINE FROM TEA 29 evaporating dish, and heat up it directly on the fire to boil off one half water, cool. If settle is separated out, it can be discard by vacuum filtering.
(3) Transfer the concentrated solution to separatory funnel that is supported by a ring on a ring stand. Add 15mL chloroform and 10mL of saturated salt solution. Stopper the funnel and grab the neck of the funnel with one hand, holding the stopper into the neck of the funnel.
With your other hand grasp the stopcock in such a way that you can turn the plug in the barrel to open and close the stopcock. While holding the stopper tightly into the neck of the funnel, invert the funnel so the liquid no longer is in contact with the stopcock. Pointing the stem of the funnel away from everybody, open the stopcock to release any pressure that may have built up inside the funnel. Close the stopcock and agitate the material in the funnel without shaking it vigorously – you want the contents to mix, but you do not want to generate an emulsion. Point the stem of the funnel upward, and making sure no liquid is in contact with the stopcock open it to again relieve any pressure build-up. Close the stopcock. Agitate again as before and reclamp the separatory funnel to the ring stand. Allow the contents of the separatory funnel to settle. There should be two distinct mostly clear layers. If there is an emulsion (cloudy) layer between two clear layers it is sometimes possible to break the emulsion by swirling the contents of the funnel or stirring the contents using a glass rod. If the emulsion persists seek your instructors help. Carefully drain the lower (dichloromethane) layer into a 50mL beaker. Try to not include any of the aqueous (upper) layer. If there is a lot of emulsion, include it in the beaker. Boil off the chloroform in the water bath, you will get caffeine.
2. Identification of caffeine.
(1) Murexide reaction
Place several grains of caffeine crystal into a magnetism spoon, and add potassium chlorate crystal as big as a mung bean and 2~3 drops of concentrated hydrochloric acid. Boil off the liquid on the alcohol burner, cool and add a drop of ammonia. If purple appear, it shows caffeine exist.
(2) Bismuth potassium iodide reagent reaction
Place the residual raw product crystal of caffeine into 1mL of 5% H
2
SO
4
solution, and stir it to dissolve. Add 4 drops of bismuth potassium iodide solution into it. If there is saffron settle, it contains caffeine.

30 ORGANIC CHEMISTRY EXPERIMENTS
Purpose
1. To be familiar with the principle and method of extraction of virtual components.
2. To master the extraction and identification of Lecithin.
3. To know the composition of Lecithin.
Principle
Lecithin, or phosphatidyl choline, is a kind of phosphatide exists in all kinds of histiocytes of animals. It is called lecithin because it is extracted from vitelline initially.
Vitelline is composed of protein (20%), lipid (fat 20%, lecithin 8%, a few of cephalin) and water (50%). The solubilities of the compositions in different solvents are listed in table
2-7-1.
Table 2-7-1 The solubility of lecithin in different solvent protein fat lecithin cephalin alcohol chloroform acetone insoluble insoluble insoluble soluble soluble soluble soluble soluble insoluble insoluble soluble insoluble
According to the solubility of each component, alcohol can be used as solvent to extraction, so fat and lecithin can be separated from other components. Lecithin can be separated from fat because of their different solubility in acetone. The procedures are showed as follows: residuum (protein¡¢ cephalin) vitelline distill by ethanol filtrate protein fat lecithin cephalin ethanol solution fat lecithin boil off ethanol oily chloroform chloroform matter solution lecithin acetone fat fat lecithin
Lecithin is a kind of lipoid and has the structure of esters. Lecithin can be hydrolyzed as

EXPERIMENT 7 EXTRACTION AND IDENTIFICATION OF LECITHIN 31 glycerol, fatty acid, phosphonic acid and choline with the existence of base.
In basic solution, fatty acid change can react to form soap, which separate out free acid after acidification and react with Pb 2
＋
to form lead soap of various fatty acids, a kind of white sediment.
Glycerol reacts with copper hydroxide to form navy blue copper glycerol solution.
Choline reacts with Kraut’s reagent which consists of basic bismuth nitrate and potassium iodide to form red sediment.
Phosphonic acid can react to form yellow sediment.
Apparatus and reagents
Mortar, evaporating dish, Buchner funnel, filter flask, glass funnel, glass stick, test tube holder, water bath.
95 ％ ethanol, chloroform, acetone, 10 ％ sodium hydroxide, 5 ％ bluestone, ammonium molybdate reagent, Kraut’s reagent 、 concentrated nitric acid, concentrated sulfuric acid, 10 ％ lead acetate solution, litmus blue test paper, boiled vitelline, water.
Procedures
1. Distill of lecithin
(1) Grind a cooked vitelline in a mortar, add 15mL of 95% ethanol, grind and beat the mixture until smooth. Vacuum filter, and gather filtrate. Transfer the residuum from the filler to the mortar, then add 15mL of 95% ethanol, grind and filter (the filtrate should be transparent). Both the filtrates are placed in an evaporating dish.
(2) Place the evaporating dish on the water bath, boil off ethanol and get yellow oily matter.
(3) After cooling, add 2~3mL of chloroform, stir till the oily matter dissolved.
(4) Add 10~15mL of acetone slowly with stirring, lecithin separate out. Stir the lecithin so that they can adhere to the glass stick as conglobation. The solution should be dumped to the recycling bottle.
2. Hydrolysis of lecithin and identification of its compositions.
(1) Hydrolysis
Add the distilled lecithin and 5mL of sodium hydroxide solution into a clean tube, heat up for10 minutes in boiling water bath with stirring and the lecithin hydrolyze. After cooling, the mixture is filtered by a glass funnel with a little cotton. The filtrate is used to the

32 ORGANIC CHEMISTRY EXPERIMENTS identification.
(2) Identification of fatty acid.
Take a little sediment on the cotton and add 5mL of water, stir and watch if there is bubbles.
Filter off the clear liquid; add concentrated nitric acid and a few drops of 10% lead acetate solution. Watch the phenomenon.
(3) Identification of glycerin.
Add a drop of 5% bluestone solution and 2 drops of 10% sodium hydroxide solution in a little tube, shaking the tube, and you will get sediment of copper hydroxide. Add digest into the tube, the sediment will dissolve and you will get navy blue copper glycerin solution.
(4) Identification of choline.
Add 1mL of filtrate in a tube, add dropwise concentrated sulfur acid to neutralize (tested by litmus blue test paper). Add a drop of Kraut’s reagent, and watch if there is brick red sediment.
(5) Identification of phosphoric acid.
Add 10 drops of filtrate and 5 drops of 95% ethanol, then add 2 drops of nitric acid to acidification. Add 1mL of ammonium molybdate reagent, and watch the phenomenon. Heat up the tube on the fire directly for 5~10 minutes, and you will get yellow ammonium phosphomolybdate.

EXPERIMENT 8 THIN LAYER CHROMATOGRAPHY (TLC) 33
Purposes
1. To be familiar with the principle of TLC
2. To master the basic technique of TLC.
Principles
Chromatography is a term applied to several separation techniques bases on differential migration. It makes use heterogeneous equilibrium established during the flow of a solvent called the mobile phase through a fixed (stationary) phase to separate two or more components from material carried by the solvent. Chromatography can be classified as adsorption chromatography, distribution chromatography, and ion-exchange chromatography based on different relationship of substance between the two phases of the system in use.
Basing on the different support of operation it can be named as: thin layer chromatography
(TLC), paper chromatography (PC), and column chromatography (CC).
Thin layer chromatography is a sensitive, fast, simple, and inexpensive analytical technique that will be used repeatedly in carrying out organic experiments. It is a true micro technique; as little as 10 -9 g of material can be detected, although the usual sample size is from 1 to
100×10 -6 g.
TLC involves spotting the sample to be analyzed near one end of a sheet of glass, plastic, or aluminum coated with a thin layer of an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered jar containing a shallow layer of solvent. As the solvent rises b capillary action up though the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent and the stationary adsorbent
Figure 2-8-1 The diagram of TLC Figure 2-8-2 The diagram of spot sample

34 ORGANIC CHEMISTRY EXPERIMENTS phase. The more strongly a given component of the mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the TLC plate.
The two most common coatings for thin-layer chromatography plates are alumina, Al
2
O
3
, and silica, SiO
2
. Of the two, alumina, when anhydrous, is the more active; that is, it will adsorb substances more strongly. It is thus the adsorbent of choice when the separation involves relatively nonpolar substrates. To separate the more polar substrates, silica gel, is used. A polar solvent will carry along with it polar substrates, and nonpolar solvents will do the same with nonpolar compounds, another example of the generalization “like dissolves like”.
The R f
value of the ration of the distance the spot travels from the point of origin to the distance the solvent travels. The best separations are achieved when the R f
value falls between 0.3 and 0.7 without units.
f
It indicates that sample cannot mobile if R f
value is zero. The more different of R f
value between species of sample is, the better separation of mixture is.
Apparatus and reagents
Glass plate (7.5×2.5cm); medicine dropper; TLC tank; mortar and pestle; oven; capillary tube; sprayer; graduated cylinder; pencil; ruler; platform balance.
Silica Gel G; 0.1g·.L
-1 cholesterol in chloroform; 0.1 g·.L
-1 cortisone acetate in chloroform;

EXPERIMENT 8 THIN LAYER CHROMATOGRAPHY (TLC) 35
0.5 g·.L
-1 molybdic acid; developing solvent (benzene: ethyl acetate=7:3 v/v).
Procedures
1. Coating plates
The aqueous slurry [1] of silica gel G [2] is prepared by mixing about 3 g powder with 8 mL distilled water in mortar with pestle. Coat the slurry evenly with medicine dropper in 1~3 mm thick on four to six 3×10 cm plates. Allow the plates to air dry at room temperature in the horizontal position, when the surface of the slides has become dull, then active them in an oven at 110℃ for 30 min before using.
2. Sample application
Following the procedure outlined above, draw a light pencil line about 1 cm from the end of some chromatographic plates and on this line spot cholesterol, cortisone acetate, and their mixture [3] . Make each spot as small as possible, preferably less than 0.5 mm in diameter. If it is necessary to apply a larger amount [4] , let the first spot dry completely and then touch the capillary again at the same place. You should take care to make sure that each of the spots is the same distance from the bottom of the plate.
3. Elution [5]
Pour 10 mL developing solvent of mixed benzene-ethyl acetate into the tank. Place the plate upright in the developing tank so that the sample end is immersed in the developing solvent to a depth of 0.5 cm. Don’t put the spot immersed in the surface of solvent; the solvent should be introduced into the tank at least 15 minutes before commencing the separation. Allow the solvent to creep up the TLC plate until it is about 1.0 cm from the top.
The plate is then removed from the developing tank, and mark [6] the solvent front immediately with a small scratch. The solvent allowed evaporating.
4. Visualization
The position of the separated solutes can be located by spraying the plate with a visualization Reagent (phosphomolybdic acid), which produces colored areas in the regions which samples occupy. In this case, phosphomolybdic acid can be sprayed the plates after solvent evaporating completely. Put it in an oven until the spots show.
5. Determination of R f
Value
Remove the chromatogram from the oven, and take it back to your desk. Circle each spot, and measure the distance from the origin to the center of each spot as well as the distance

36 ORGANIC CHEMISTRY EXPERIMENTS from the origin to the solvent front with ruler. Calculate all R f
values for any spots that are visible, and determine the identity of the components of your unknown. Record the chromatogram diagram and submit it to your instructor with your report.
Annotation
[1] The object in plate coating is to spread a smooth, uniform layer that dries without bubbles, pits, or cracks.
[2] Silica gel is a pore molecular polymer with larger surface areas, on which exchange of molecules between the solid and a solvent flowing over the surface occurs by successive adsorption and desorption.
The separation process on silica is somehow a function of the surface hydroxyl content by means of the strength of the hydrogen bond formed with other compounds. G is abbreviated from gypsum which is binder. Adsorbents with a binder are usually designated by the name of a hard layers, without a binder are called soft layers,
[3] You should make capillary tube just contact with a layer, rather than heavy spot, so as to avoid destroying the layer when the samples are applied.
[4] The amount of each sample should be suitable. Overloaded sample applications produce comet-like vertical streaks or overlap on the chromatogram. While insufficient sample give poor separation which spots of some components can not appear.
[5] Selecting solvent mainly depends on its polarity and that of the sample, solubility of the sample, and adsorptity of adsorbent, etc. You can refer to other books about system of selecting solvent in TLC.
[6] You should make the solvent front immediately after taking the layer out of TLC tank. If you forget to do so, you cannot determinate the location of solvent after it evaporate.
Questions
1. Why can we use the R f
values of components to distinguish substances under certain condition?
2. What’s the effect on thin layer chromatography if the spot is immersed in the surface of the solvent?

EXPERIMENT 9 PAPER CHROMATOGRAPHY OF AMINO ACIDS 37
Purposes
1. To be familiar with the principle of paper chromatography;
2. To master the method of separating amino acids.
Principles
When a small spot containing a mixture of amino acids is placed near the bottom of a piece of filter paper, and the filter paper is placed in a covered jar containing a small amount of suitable solvent, the solvent moves up the filter paper, carrying each different amino acid in the mixture up the paper to a different extent. This results in a series of spots on the paper, each spot corresponding to a different compound. This is the steps of paper chromatography, and is showed in Figure 2-9-1.
Solvent front
Chromatography tank
Chromatographic paper
Sample spot
Solvent
Figure 2-9-1 Apparatus of paper chromatography
Color spot
Initial line
Figure 2-9-2 Diagram of paper chromatography
A
： alanine B
： leucine C
： mixture of A and B
The water absorbed on the filter paper [1] is called the stationary phase, and the solvent is called the mobile phase. Paper chromatography is a partition process between the two phases, the paper being the solid support for the aqueous stationary phase. Paper chromatography can be used for the separation of mixture. The separation can be due to the different distribution coefficient of the components in stationary phase and mobile phase.
Chromatography was primarily applied to separate colored substances, and now it may also be applied to separate colorless compounds, such as amino acids. The compound ninhydrin can react with all amino acids to produce purple products. So after developing the

38 ORGANIC CHEMISTRY EXPERIMENTS chromatogram, ninhydrin solution should be sprayed so that the spots, corresponding to the different amino acids, could be showed. You will then determine a R f value for each compound. The R f
value is the ration of the distance traveled by the compound to the distance traveled by the solvent in a specified time period.
O
O
2
O
OH
+ R CHCOO
OH
NH
3
O
H
OH
+ RCHO+NH
3
+CO
2
+3H
2
O
O
O
OH O
H
O +NH
3
+
HO
N
O
O O O purple
Apparatus and reagents
Capillary, chromatography jar, developer, filter paper for chromatography (Xinhua No.1), pencil, ruler.
Alanine, leucine, mixture of alanine and leucine (1:1), solvent (n-butanol: glacial acetic acid: water= 4: 1.5: 1), 0.5% ninhydrin anhydrous alcohol solution
Procedures
1. Choose a precut (16cm×6cm) sheet (making sure to touch it only along the top edge) and place it on a clean sheet of notebook paper. With the short (6cm) way aligned to the left and right, using a straightedge, draw a light pencil line (not ink!) from left to right. Parallel to and up from the bottom edge by 2 cm, place 3 small pencil marks at 1 cm intervals along this line.
Label the three marks with A, A+B and B.
2. Using a spare sheet of ordinary filter paper, you will practive using a capillary tube to make a spot between 0.2 and 0.3 cm in diameter. Then dip the capillary tubes into the alanine solution and make the spot on the sheet, applying it at the position marked with A, leucine at spot B and the mixture of the two at the spot A+B (Figure 4-9-2). Allow the paper to dry for a few minutes. If the spot is not big enough then make second application at the same positions as the first. Allow the paper to dry.

EXPERIMENT 9 PAPER CHROMATOGRAPHY OF AMINO ACIDS 39
3. Pour the developer into the chromatography jar with the depth about 1.5 cm. Allow it to stand for few minutes before the separation, in order to form the saturated vapor in jar. Hang the strip in the jar with the marked end in the lower, and dip into the developer (insuring the spot do not dip into the developer and the edges of the paper not be allowed to touch the inside wall of the chromatography jar). Cover the jar tightly.
4. Allow the chromatogram to develop for at least an hour. When development is finished (the solvent front is about 8~10 cm away from the initial line), you should remove the chromatogram and immediately mark the location of the solvent front before it has a chance to dry.
5. Dry it and spray it with ninhydrin solution. Then have your paper dried by hair dryer, and leave it there for 10 minutes.
6. Remove the chromatogram, and take it back to your desk. Circle each spot and measure the distance from the origin to the center of each spot as well as the distance from the origin to the solvent front. Calculate the R f
values, and report them to your instructor.
Distance from start to center of substance spot
R f
=
Distance form start to solvent front
Reference values of R f
: alanine: 0.35~0.45 leucine: 0.7~0.8
Notes
[1] Filter paper is nearly pure cellulose, a carbohydrate. The surface of the paper is normally covered with water molecules, which is attracted there by the many –OH (hydroxyl) groups of the cellulose molecules. Both water and the hydroxyl groups are polarity and can form hydrogen bonds.
Caution
1. When the chromatogram is being developed, the jar shouldn’t be moved anyway.
2. Avoid getting ninhydrin on your hands or clothing, it will cause stains that are difficult or impossible to remove.
Discussion
1. The baseline is the line upon which the spots of amine acids are applied. Why is it important that the depth of solvent in the bottom of the large jar not greater than the distance

40 ORGANIC CHEMISTRY EXPERIMENTS from the bottom of the filter paper to the baseline?
2. Why does one use pencil but not ink in marking the paper?
3. You are cautioned to avoid the edges of the paper touching the jar wall. Why?

EXPERIMENT 10 CELLULOSE ACETATE MEMBRANE ELETROPHORESIS OF SERUM PROTEINS 41
Purposes:
1. To be familiar with the principle of cellulose acetate membrane eletrophoresis.
2. To master the separation and identification of amino acid by cellulose acetate membrane eletrophoresis.
Principles:
An ion, which has position or negative charges, will move towards the electrode which has opposite charges. The quality is called electrophoresis. As we know, the exact structure of an amino acid depends on the pH. The pH at which an amino acid exists as zwitterions is known as the isoelectric point (pI). At the pI, amino acids have a zero net charge and are electrically neutral and without removal in the electric field. When the pH of solution is great than the pI of the compound, the amino acid will carry a net charge (anionic form), and will remove to the anode. Conversely, when pH of the solution is below the pI value, the amino acid exists predominantly in the cationic form (not positive charge), and to the cathode.
According to the difference of removal direction and rate of different amino acids ions, we can separate and identify the amino acid.
Apparatus and reagents electrophoresis apparatus, culture dish (diameter 15cm), Xinhua filter paper, nipper, Cellulose acetate membrane (Chop the Cellulose acetate membrane into strips of 8 × 2cm, and immerge the strips in barbital buffer solution), rubber stopper for spot sample.
Barbital buffer solution (pH6.86, ionic strength 0.06); barbital 0.83g, sodium barbital 6.38g, dissolved in water and diluted to 500mL.
Dyeing solution: amino black 10B 0.5, methanol 50mL, glacial acetic acid 10mL, distilled water 40mL.
Poaching solution: alcohol 45mL, glacial acetic acid 5mL, distilled water 50mL.
Procedures
1. Assembly apparatus
Join the output stage of electrophoresis apparatus with the platinum wire of electrophoresis

42 ORGANIC CHEMISTRY EXPERIMENTS trough. Immit barbital buffer solution to the 2/3 of the volume of trough and adjust the oil level are equivalent on each cistern. Adhibit four pieces of filter paper on the clapboards between the anode and cathode as salt bridge.
2. Spot sample
Take out the cellulose acetate membrane with nipper and place it on the filter paper. Absorb the odd buffer solution. Dip a little serum with rubber stopper, and absorb odd serum by touching on the filter paper. Spot sample on the cellulose acetate membrane about 1.5cm from one end of frosting. After the serum filter the membrane, cling the cellulose acetate membrane on the filter paper bridge with the frosting adown (the spotting end close with cathode), then hood the glass cover.
3. Electrify
Turn on the power, and adjust voltage between 90volt and 120volt, 0.4~0.6mA/cm 2 . About
60 minutes later, turn off the power.
4. Dyeing
When electrophoresis is finished, remove the strips out of the electrophoresis tank with a pair of tongs, and dip it directly in the dyeing solution for 5~10 min. Wash the strips three times, decolor it till the background is achromatous. You will see five stained protein strips. The farthest strip is albumin, then
α
1
, α
2
, β and γ globulin.
You can determine the relative content of the five components according to the depth of color of their strips.

EXPERIMENT 11 THE CHEMICAL PROPERTY OF CARBOHYDRATES 43
Purposes
1. To master the method of identifying carbohydrates chemically.
2. To know the chemical properties of carbohydrates.
Principles
Monosaccharides and Disaccharides with hemiacetal hydroxyl can reduce weaker basic oxidant, such as Tollons, Fehing’s and Benedict’s reagents. Those sugars are classified as reducing sufars. Disaccharides, oligosaccharides and polysaccharides which contain acetal or ketal units can not react with Tollons’s, Fehing’s and Benedict’s reagents. Nonreducing sugars are stable and do not mutarotate.
Reducing sugars are treated with an excess of phenylhydrazine, they are converted into highly crystalline compounds called osazones. Sugars that have identical configurations at the other carbons give the same osazone, except for the first two carbon atoms. Sugars that have different configurations at the other carbons give the different osazone, except for the first two carbon atoms. Osazone formation was one of the reactions used for identication purposes.
The important identification methods of sugars are reaction made by Mollish and
Sheliwanoff. Saccharides, glycosides and other sugary substances react with Alpha-naphthol and concentrated sulfuric acid to produce pruple-red ring. The reaction is used to identify sugars and other substances. Red color appeared quickly by reaction of the ketohexose with the m-dihydroxybenzene, concentrated hydrochloric acid when the mixture is heated. The reaction speed of aldoses is slower than that of ketoses in the identical condition. The reaction is used to identify aldoses and ketoses.
Nonreducing sugars yield reducing monosaccharide on hydrolysis when it is heated with aqueous acid solution. The starch undergoes hydrolysis to yield violadextrin step by step, and then yield erythrodextrin, achroodextrin, maltose and glucose at last. The solution of iodine is added to the solution as a test for the process of hydrolysis. After hydrolysis completely,
Benedict’s reagent can identify it.
Apparatus and reagent
Test tubes, water bath, alcohol burner, beakers, burettes, Erlenmeyer flask, glass tubes, hob,

44 ORGANIC CHEMISTRY EXPERIMENTS iron clamp.
2% glucose solution, 2% saccharose solution, 2% fructose solution, 2% maltose solution,
20% sulfuric acid, 10% sodium hydroxide, 1% starch solution, iodine solution, Benedict reagent, Molisch reagent (α-naphthol ethanol solution), concentrated sulfuric acid, litmus red test paper, Tollen reagent, Fehling reagent.
Procedures
1. The reduction of sugars
Samples: 2% glucose solution, 2% saccharose solution, 2% fructose solution, 2% maltose solution, 1% starch solution
(1) React with Fehling reagent
Mix 2.5mL of Fehling reagent solution A and 2.5mL of Fehling reagent solution B, average it into five test tubes. Add 0.5mL sample solutions separately into five test tubes and hot up them in water bath. Watch and compare the result.
(2) React with Benedict reagent
Add 2mL of Benedict reagent into each test tube and then add 0.5mL of sample solutions separately. Boil them for 2~3 minutes in boiling water bath, cool them and watch if there is red or yellow settle.
(3) React with Tollen reagent
Add 1mL of Tollen reagent into each test tube and then add 1mL of sample solutions separately into the five tubes. Immerse them into 60 ℃ ~80 ℃ hot water bath for several minutes. Watch and compare the result.
2. The hydrolysis of sugars
(1) The hydrolysis of saccharose
Add 1mL of 2% saccharose solutions into two test tubes separately, and add 2 drops of 20% sulfuric acid into one test tube. Boil the two test tubes into boiling water bath for about five minutes. Cool them and add 10% sodium hydroxide solution till the solution to be alkali (test with the litmus red test paper). Add Bendict reagent into the two test tubes and then boil them in the water bath. Watch the results.
(2) Iodine test and hydrolysis of starch
① Iodine test of starch
Add 1mL of starch solution into a test tube, add one drop of Iodine solution. Watch the

EXPERIMENT 11 THE CHEMICAL PROPERTY OF CARBOHYDRATES 45 result. Heat up the test tube directly on the fire, watch the result. After cooling, if the blue recur?
② Acid hydrolyses of starch
Add 10mL of starch solution into a 50mL beaker and then add 1mL of 20% sulfuric acid.
Boil it for 15 minutes with gentle fire. Add 2mL of cooled digest into two test tubes separately, add 10% sodium hydroxide solution till the digest be alkali. Add iodine reagent into one test tube and Benedict reagent into another test tube. Heat up them in water bath. Watch the results.
(3) The color reaction of sugars (Molisch test)
Samples: 2% glucose solution, 2% saccharose solution, 2% fructose solution, 2% maltose solution, 1% starch solution
Add 1mL of sample solutions into five test tubes separately and add 4 drops of newly prepared Molisch reagent. Lean the test tubes for 45˚ angle and add slowly 1mL of concentrated sulfuric acid along the test tube wall (don’t shake the test tubes). Watch purple-red ring when hold these test tubes upright and stabilized. If the purple-red ring failed to appear, heating the reaction mixtures for 1~2 min, and looking at carefully.

46 ORGANIC CHEMISTRY EXPERIMENTS
Purposes
1. To master the method of identifying proteins chemically.
2. To know the chemical properties of proteins.
Principles
Proteins are complex, high-molecule-weight compounds which contain nitrogen. Natural proteins are made up of 20 Alpha-amino acids which belong to L configuration. In the action of acid, base or enzyme, protein hydrolysis to yield Alpha-amino acid at last. Some special groups of amino acids in protein react with chemical reagents to produce all kinds of color reaction. The color reaction can be used to identify and determine the amount of proteins or amino acids. Though a kind of protein react with a reagent to produce color, not all proteins react with the identical reagent to produce color, because of different chemical structure and different amino acids properties. Pay attention to other substances contain identical groups react with identical reagent to produce color.
Protein contain both acidic and basic groups in the same molecule, they exist primarily as the dipolar ion. They react with acids as well as with bases. Proteins undergo drastic changes in its physical and biological properties because of their unstable structure. The phenomenon is brought about by a variety of conditions and reagents such as organic solvent, heavy metals, and so on. The solubility of proteins also varies considerably. The solubility of proteins in water undergoes change which is brought about by a variety of physical and chemical agent.
The change leads to formation of precipitate. We can take advantage of the theory to separate a mixture of amino acids into pure constituents.
Apparatus and reagents
Test tubes, beaker, glass rod.
Protein solution, saturated ammonium sulfate solution, solid ammonium sulfate, 1% lead acetate, 2% copper sulfate, 1% silver nitrate, saturated picric acid solution, 5% acetic acid, saturated digallic acid solution, 3% sulfosalicylic acid, 10% trichloroacetic acid, 1% lysine, ninhydrine, concentrated nitric acid, 10% sodium hydroxide, Millon reagent, 0.5% phenol solution, urea crystal, litmus red test paper, 1% copper sulfate, 20% sodium hydroxide, water.

EXPERIMENT 12 THE CHEMICAL PROPERTY OF PROTEINS 47
Procedures
1. Salting out of protein
Add 1.5mL of protein solution and 1.5mL of saturated ammonium sulfate solution
(concentration is 50%) into a test tube. Shake gently the test tube, stand for several minutes.
The globin will separate out as flocks (if no settle separate out, add a little saturated ammonium sulfate solution). Filter the mixture and add solid ammonium sulfate into the upper solution till the solid can not dissolve any more. The albumin separate out. Add water and watch if the settle disscolve.
2. Deposition of protein
(1) Deposition of protein by heavy metallic salt
Add 1mL of protein solution into three test tubes and add 6% lead acetate, 2% copper sulfate and 1% silver nitrate separately. Watch the protein separating out.
(2) Deposition of protein by organic acid.
Prepare two test tubes and add 1mL of protein solution and 2 drops of 5% acetum into each test tube. Add dropwise saturated picric acid and saturated digallic acid till the settle separate out.
Do the experiment with 10% trichloroacetic acid and 3% sulfosalicylic acid separately.
(3) The color reaction of protein
① The protein-yellow reaction
Add 1mL of protein solution and 10 drops of concentrated nitric acid, and then boil it on the fire directly. Watch the color of solution and settle. Cool it and add 20 drops of sodium hydroxide solution. Watch the color change.
② Ninhydrine reaction
Add 1mL of 1% lysine into a test tube and 1mL of protein solution into another test tube.
Add 2 drops of ninhydrine solution into the test tubes separately and heat the mixture till it reaches boiling point.
Watch the phenomenon.
③ React with mercury nitrate (Millon reaction)
Millon reagent is the mixture of nitric acid, nitrous acid, mercury nitrate and mercury nitrite and it can reaction with phenol and some phenols.
Add 1mL of 0.5% phenol into a test tube and add 1mL of protein solution into another test tube, and then add 0.5mL of Millon reagent into each test tube. The phenol solution appears rose color and the heated protein solution separate out settle. The settle is brick red or red.

48 ORGANIC CHEMISTRY EXPERIMENTS
(4) Biuret reaction
Add a little urea crystal (0.2g~0.3g) into a dry test tube and heat up it gently. The ureas melt and produce biuret. The ejective ammonia can be determined by litmus red test paper.
When there is white solid in the test tube, stop heating.
Cool the outcome and add 1mL of 10% sodium hydroxide solution. Shake the test tube and add 2 drops of 2% copper sulfate solution. Shake up and watch if purple appear.
Add 1mL of albumin solution and 1mL of copper hydroxide solution into a test tube, and then add 1~2 drops of 2% copper sulfate solution. Heat up the mixture. The protein reacts with copper sulfate and yield purple complex.
Compare the phenomenon with 1% lysine solution and watch the color change.

EXPERIMENT 13 IDENTIFICATION OF ORGANIC COMPOUNDS 49
Purposes
1. To master identification methods of organic compounds.
2. To know some major reactions of organic compounds.
Principles
It is difficult to determine the molecular structure of a newly founded chemical. Usually the systemic organic qualitative analysis method is used to determine the molecular structure of an unknown sample. However, if the unknown samples are restricted to a certain range, they can be classified by using some reagents firstly and then distinguished by special reactions.
Some physical properties can provide exhaustive information for determination of unknown compounds, such as boiling point, melting point, infrared spectra, etc.
We will distinguish eight kinds of organic compounds, including alkanes, alkenes, haloalkane, alcohols, aldehydes, ketones, carboxylic acids and amines.
Soluble carboxylic acids can make litmus blue test paper turn red. Soluble amines can make litmus red test paper turn blue. Both ketones and aldehydes contain carbonyl groups, which react with 2,4-dinitrophenylhydrazine (DPNH) solution and produce yellow sediment. Fatty aldehydes can react with benedict reagent but ketones can’t. Most of alcohols react with chromic acid solution to form cyan suspension in 2~3 seconds. Alkenes can make the potassium permanganate solution discolor and produce brown sediment. Green flame will appear when haloalkanes combust in the flame of brass wires (Beilstein test). All the tests for alkanes are negative.
The procedure is shown as Figure 2-13-1.
Apparatus and reagents
Brass traveler, spot plate, water bath pot.
Acetaldehyde, acetone, n-butanol, acetic acid, chloroform, paraffin, triethylamine, cyclohexene, 2,4-dinitrophenylhydrazine, litmus blue test paper, litmus red test paper, 95% ethanol, chromic anhydride-sulfuric acid reagent, 1% potassium permanganate, Benedict reagent, water.
Procedures

50 ORGANIC CHEMISTRY EXPERIMENTS
1. Acidityalkalescence test
Sample (5drops or 0.1 g solid) is placed in a test tube and 3mL of water added to the test tube. Pendulate, and used the litmus blue test paper and the litmus red test paper to test the sample solution respectively. Watch the color change. eight kinds of compounds litmus paper litmus blue test paper turns red (+) litmus red test paper turns blue (+) alcohol, aldehyde, alkane, alkene, haloalkane, ketone carboxylic acid
2,4-dinitrophenylhydrazine
(£- ) alcohol, alkene, haloalkane
(+) aldehyde, ketone
(£- ) chromic acid alkane, alkene, haloalkane
(+) alcohol potassium pomanganate amine
(£- )
Benedict reagent
(+) ketone aldehyde
(£- ) alkane, haloalkane
Beilstein's test
(+) alkene
(£- ) (+) alkane haloalkane
Figure 2-13-1 The flow chart of the identification tests for eight compounds
2. 2,4-dinitrophenylhydrazine (DPNH)
Sample (5 drops or 40mg solid in 1mL of 95% alcohol) is placed in a test tube, DPNH reagent (1~2 drops) added to the sample, pendulate, lay it for about 15 minutes, watch if there is yellow selling, or use the glass stick to scrub the tube wall gently and then watch the phenomenon.
3. Chromic acid test
Sample (5 drops or 30mg solid) is placed in a test tube, acetone (1mL, solvent) added to the sample. Then chromic anhydride- sulfuric acid reagent (10 drops) is added, pendulate, watch the color change in two minutes.

EXPERIMENT 13 IDENTIFICATION OF ORGANIC COMPOUNDS 51
Primary alcohol and secondary alcohol will react and produce opaque bluish- green emulsion in 2 seconds. Aromatic aldehyde makes the reagent change color in 20 ~120 seconds or longer. If the solution is still saffron or a little black, the result is negative.
4. Potassium permanganate test
Sample (5 drops) is dissolved in 1 mL of water, and then (1%) is dropped, watch the color change. If the purple fade, potassium permanganate solution (1%) is dropped. When the solution does not change color, count the drops of added solution. If the reaction does not happen immediately, watch it after 5 minutes.
If the sample can make more than one drop of potassium permanganate solution fade, and produce brown MnO
2
settle, the result is positive. All unsaturated hydrocarbons contain double bond or triple bond produce positive result. Moreover, compounds which are easy oxidation such as aldehyde, aromatic aldehyde, phenol, formic acid and formate produce positive result. Liquid alcohols contain impurities which easy to oxidation react with a few solutions. So the potassium permanganate solution (1%) should be added dropwise. If the sample can only make the first drop of solution change color, the result is negative. Most of pure alcohols can’t react with potassium permanganate solution in 5 minutes. Conjugated hydrocarbon and aromatic hydrocarbon which contain side chain can not react with neutral potassium permanganate solution without heating.
5. Beilstein test
Heat the brass traveler by the alcohol burner till the brass traveler turn red. When the green or cyan flame disappears, cooling, a few samples adhered on the brass traveler, and burn it on the edge of the flame. Watch the color of the flame.
If green or blue flame appears, the result is positive. Alkyl halide decomposed to CuX
2
but fluoride can’t react.
6. Benedict test
Place 10 drops of sample in 2mL of water, and then add 2mL of Benedict reagent. Heat up in boiling water bath. If settle appear, watch the color of settle.
Fatty aldehydes produce saffron or brick red settle. Other compounds such as reductive sugars and

-hydroxyketones can also react. Ketones and aromatic ahdehydes can’t react.

52 ORGANIC CHEMISTRY EXPERIMENTS
Purposes
1. To master the determination method of optical rotation.
2. To know the determination principle of optical rotation.
Principles
Polarimeter is a kind of optical instrument which can be used to determine the angle that active substance circumrotate the polarized light. It consists of the following four parts:
1. Natrium lamp: it is a lamp-house which can produce a light of 589nm.
2. Polarizer: a piece of fixed Nicol’s prism which can change the light to be plane polarized light.
3. Sample cell: place the sample.
4. Analyzer: a piece of rotatable Nicol’s prism which can be used to detect the polarized light. polarized light
¦Á solution
Figure 2-14-1 Diagram of polarized light circumrotating lamp-house
¦Á polarizer sample cell analyzer
Figure 2-14-2 Diagram of determination
¦Á eye

EXPERIMENT 14 DETERMINATION OF OPTICAL ROTATION OF GLUCOSE 53
Apparatus and reagents
Polarimeter (WZZ-1S self indicating polarimeter), measuring flask, 2dm rotatary tube.
Glucose (pharmaceutical), glucose (A. R.), 5% glucose injection, ammonium solution.
Procedures
1. Dispense of standard glucose solution
Dispense a series of standard glucose solutions. The concentrations of the solutions are
0.015g.mL
-1 , 0.030g.mL
-1 , 0.045g.mL
-1 , 0.060g.mL
-1 and 0.075g.mL
-1 . Add corresponding quantity of ammonium solution. Shake up and place for 10 minutes.
2. Protract of standard line
Determine the optical rotation of the standard solutions. Protract optical rotation- concentration standard line.
3. Determination of content of sample solution
Determine the optical rotation of the glucose injection. According to the standard line, determine the content of glucose in the injection.

54 ORGANIC CHEMISTRY EXPERIMENTS
1. Vogel’s: Text book of practial Organic Chemistry, Fourty Edition, 1978
2. Stuart J Baumeta; Laboratory Exercisesin Organicand Biological Chemistry Second Edition, 1981
3. John W Lehman; Operational Organic Chemistry, A Laboratory Course (second edition),
1988
4. Charles F. Wilcox, Jr. Experimental Organic Chemistry, Theory and Practice. 1984
5. Charles F. Wilcox, Jr. Experimental Organic Chemistry, A Small-Scale Approach. 1988
6. Laurence M. Harwood, Christopher J. Moody. Experimental Organic Chemistry, Principles and Practice. 1989
7. Xi’an Jiaotong University. Organic Chemistry Experiments. 2002
8. The Department of Chemistry of Chongqing University of Medical Sciences. Medical
Chemistry Experiments. 2005.