Experimental organic chemistry laboratory manual

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International Program UAM-Boston University Laboratory Manual Organic Chemistry I 2013-2014 Departamento de Química Orgánica Ernesto Brunet Romero Ana María Martín Castro Ramón Gómez Arrayás Laboratory Manual Table of Contents ............................................................................... 1 Introduction ............................................................................... 2 Prelab preparation ............................................................................... 2 Notebook ............................................................................. 3 Safety .............................................................................. 3 Laboratory Practices and Safety Rules ............................................................. 4 Accidents and injuries ........................................................................... 5 Fires ............................................................................. 5 Chemical Wastes ............................................................................. 6 Cleaning Responsibilities ............................................................................. 6 Lab cleanliness ............................................................................. 6 Laboratory Equipment ............................................................................. 7 Proper use of glassware ............................................................................. 8 Some techniques in lab experiments Heating, cooling and stirring ............................................................................. 9 Measurements ............................................................................. 10 Extraction ............................................................................. 10 TLC chromatography ............................................................................. 13 Recrystallization ............................................................................. 18 Common organic solvents ............................................................................. 23 Experiment 1: Using Extraction and NMR to isolate and identify the analgesic active principles of a sample ............................................................................. 24 Experiment 2: Preparation of an azoic dye: Para-red ........................................... 35 Annex to Experiment 2: Acetanilide nitration: a 1H-NMR exercise ……. 43 Experiment 3: Synthesis of a flavoring principle: Isopentyl Acetate ................... 47 1 Introduction In this laboratory course you should be prepared to apply and understand what you have learned in General Chemistry and the Organic lecture course to real situations. You should never expect to walk into lab and see something illustrated from class. That is not the way science works. Laboratory is for discovery You will expand your theoretical understanding (microscopic) to the macroscopic scale. Whether or not organic chemistry is your idea of fun, there is no reason for you not to enjoy your organic laboratory experience. Lecture and lab are, of course, related. Chemistry is an experimental science. Everything you learn in lecture was originally discovered in a laboratory. When asked what ethanol is, many chemists, both students and teachers, are satisfied with the answer “CH CH OH”. This answer is wrong, or at best, 3 2 incomplete. To answer the question “What is ethanol?” you have to know that it is a clear liquid, flammable and a powerful intoxicant. Ethanol is an actual substance and “CH CH OH” is simply one convenient model 3 2 we use for describing it. Never forget the substance behind the formula Is it a solid, liquid, or gas? Is it pure or a mixture? Is it life-sustaining or lethal? There are two overriding principles that guide this organic laboratory curriculum: (1) Laboratory should present the opportunity for you to learn how to extract knowledge from an experimental result, because that is the heart of science. (2) Laboratory should engage you into learning about chemistry. Organic reactions rarely lead to a single clean product in stoichiometric yield, so the ability to find out what the product of a reaction is depends on your ability to purify the material. Much of the new learning is centered on methods of purification and analysis. All of this requires learning new laboratory techniques. Upon completion of this laboratory course, you should have an understanding of a selection of tools and techniques used by organic chemists. Aside from good laboratory technique you should strive to learn how to take data carefully, record relevant observations, and use your time effectively. You will be asked to assess the efficiency of your experimental method and to plan the preparation, isolation, and purification of organic substances. The development of your scientific writing and record keeping skills is an important aspect of this course. It is anticipated you will improve your skills in communicating analytical results in a clear and concise manner. Finally, you should understand the need for safe laboratory practices involving chemicals and their conditions for use. Prelab preparation Advance preparation for lab is one key to success in the organic lab. Unprepared students waste time their own and the teaching assistant’s (TA) and can be a hazard in the laboratory. Before the start of lab, - You should read appropriate sections in the lab manual and online sources. - You should be certain of the purpose of the experiment. - Information regarding references to any literature used should be prepared in the laboratory notebook. 2 - A plan of action should be outlined taking into consideration reactions, hazards and important physical data. Notebook Use a bound notebook with pages pre-numbered by the manufacturer. Leave a few blank pages at the beginning of the book to build a table of contents when you finish the lab. What You Should Write in Lab You should record the following while an experiment is progress: procedure, data, observations, and those conclusions on which an immediate procedure decision is based. All notebook information should be as complete as possible. If necessary, you or another student should be able to refer to your notebook and repeat the experiment exactly, comparing observations to those on record. The body of the notebook must be written as simultaneously as possible with your performance of the lab work. What You Should Write After Lab Occasionally you will analyze data, find information in reference books, share data with another student, interpret spectra, and calculate stoichiometry, etc. after lab is over. This information, along with the answers to post-lab questions, should be kept in your notebook. The Mechanics of Keeping a Lab Record For the part of your notebook to be kept in lab, all entries should be made directly into it and not copied from another source. NEVER record elsewhere information that belongs in the notebook  neither on handy scraps of paper, margins of the lab manual, filter paper, or paper towels. NEVER temporarily memorize information for later transcription into your notebook. Do not be overly concerned with the cosmetic appearance of your notebook. While certain amount of neatness and organization is necessary, legibility and comprehensibility are the essential qualities. Be careful not to render conclusions in your notebook (e.g., “the boiling point is…”) without the supporting data or observations, i.e., “Bubbling slowed as the temperature dropped to 120-115 °C. Bubbling stopped and liquid entered the tube at 114.5 °C. Tube full of liquid at 113 °C.” Safety Safety in the laboratory is an extremely important element in the chemistry program at this University. Failure to follow safe practices can cause laboratory accidents that may result in personal injury or, at the least, loss of time, damage to clothing and other property. By following suitable precautions, you can anticipate and prevent situations that could lead to accidents. You must become thoroughly familiar with the information in the following sections, as well as the specific information provided for each experiment. You must also sign the Laboratory Safety Rules and Practices Contract that you will receive in the first scheduled laboratory meeting. Eye protection (safety goggles) is mandatory for all occupants of the laboratory when anyone is performing lab work. 3 Dispose of all chemicals properly. Liquid and solid waste should be disposed of in the designated WASTE bins and/or tanks. The lab has a special waste bin or bottle for each special type of waste. Please be aware of any waste instructions in the pre-lab lectures. If you are unsure, please do ask your Teaching Assistant. Should a chemical spill occur, please clean it up at once with the appropriate technique. We all get mad when we hear about some chemical company that creates a spill and is slow at solving it. This is true at the undergraduate laboratory as well. Spills occur frequently at the balance area. You ought to make sure that you do clean up your chemical spill. If you do not know how to clean up a particular spill, notify your lab TA. Fire is of maximum concern in the organic laboratory. While heat guns are available in the lab, use them only in the hood after ensuring the area is clear of flammable liquids. Be aware of your surroundings at all times. Look around your lab room and make sure that you are aware of the location of the fire extinguishers, showers, and eye washers. Laboratory Practices and Safety Rules A. Personal Protection 1. You are only allowed to work in the laboratory if, and only if, the teaching assistant is present. 2. You must work only on authorized experiments. 3. You must wear proper eye protection in the laboratory whenever any laboratory work is in progress. 4. You must wear shoes that do not have open spaces; sandals, flip-flops or any peep toe shoes are not acceptable. 5. You may not eat, drink or smoke in the laboratory. You must not even bring food or drink into the laboratory. 6. You must confine long hair and neckties. Loose jewelry may also be a hazard. 7. You must not engage in acts of carelessness while in the laboratory. 8. You must work carefully with a full awareness of what you are doing in order to avoid ruining equipment or spilling chemicals. B. Proper Laboratory Practices 1. Carefully read TWICE the label on a bottle before using its contents. 2. Take only the quantity of reagent needed. NEVER return an unused reagent to its container. 3. Mix reagents only when specifically directed to do so. 4. NEVER place chemicals directly on the balance pan. Weigh reagents using a beaker, flask or weighing paper. 5. If instructed to observe the odor of a chemical, do so by fanning air with your hand over the container toward your nose. DO NOT directly smell any substance. 4 6. The fume hood is for your personal protection. You must leave the hood at the indicated working level for your protection and the protection of others. Do not lock the hood in the full-open position. The air-flow velocity is insufficient when the hood sash is in the fully-raised position. 7. NEVER taste reagents. 8. Avoid handling chemicals directly with your hands. Protect your hands with gloves. If contact occurs, immediately flush the area with plenty of water. 9. Use a bulb or a pipetting device to draw liquids into a pipette. NEVER do pipetting by sucking with your mouth. 10. When diluting strong acids or strong bases, the acid or base should be added to the water, not the other way around. 11. Try to avoid using heat guns but before turning it on, make ALWAYS sure no flammable liquids or vapors are close in the area. 12. Heat test tubes at the surface of the liquid. Agitate the tube. Be sure to slant its open end away from yourself and other people. 13. Stay clear of an open vessel in which a process is occurring that could produce spattering. 14. Keep reagents and equipment away from the edge of the lab bench. 15. Do not use cracked glassware, as it may break when even slightly stressed. Accidents and Injuries You must report all accidents and injuries to the TA as soon as possible. Band-aids and first aid kits with some simple medical supplies and latex gloves are located in the laboratory. Wear gloves when helping with an open wound. In the event of an injury, some basic first aid procedures should be immediately carried out as follows: - Skin Burns or ocular lesions: There are more than 25,000 chemicals likely to cause skin or ocular lesions and burns (after a single or repeated contact), such as acids, bases, oxidizers, reducing agents, and solvents. The affected tissues must be rinsed as quickly as possible with ® DIPHOTERINE solution (BE AWARE of its location in the lab) as a first-aid treatment in emergency situations at the workplace (or at the scene of the accident). The sooner the first-aid is applied, the lower the probability of any serious after-effects. It stops the development of chemical burns and allows a rapid return to a physiological state. - Hair or Clothing Fires: Use quickly the safety shower to extinguish flames. Fires - If the fire is contained in a beaker, try to smother it with a fire blanket placed over the beaker. For a larger fire, discharge the fire extinguisher at the base of the flame. - In the event of a large or uncontrollable fire, TA’s must direct students to immediately evacuate the room, according to the following evacuation procedure: a) Direct students to leave the building (BE AWARE where the emergency exits are 5 located) b) Shut down all equipment in the laboratory, if possible, and close all doors c) Activate the fire alarm in the hallway a) Report the fire to the authorities, or call emergency (112) Chemical Wastes Special instructions for waste disposal are given at the end of each laboratory procedure. Properly dispose of all wastes: the trash can, sink, glass disposal box, solid waste disposal box, or hazardous liquid waste bottle will be properly designated. Please, BE AWARE of their location. Never pour organic solvents or toxic wastes, such as solutions containing chromium, mercury or lead, into the sink. Cleaning Responsibilities 1. You are responsible for cleaning any equipment used in the experiment, cleaning your immediate work area, and returning equipment to the proper places. 2. Additional responsibilities for cleaning designated areas of the laboratory will be assigned by the TA. 3. Clean all glassware before storing it. Soap solution and squeeze bottles of acetone for cleaning are provided at the large sinks. 4. Neutralize acid, basic or neutral (organic) spills with the apprpriate solid absorption agent before cleaning the area. For large chemical spills on the bench or floor, immediately alert your neighbors and the TA. Clean the spill as directed. 5. Dustpans, brooms and brushes are available in the lab for sweeping broken glass from the benches and floor. Place broken glass in the special cardboard containers provided (glass disposal boxes). 6. Remove any paper, broken glass or any other debris from the sinks. If you behave in an unsafe manner in the laboratory you will be elegible for immediate expulsion from the laboratory. Unsafe behavior includes, but is not limited to, failure to wear proper goggles and proper lab attire including proper shoes. If you are expelled in this manner you will not receive credit for the experiment and will not be allowed to make up the experiment. Lab Cleanliness Clean-up begins 15 minutes before the scheduled end of the period. You are responsible for cleaning up your personal work area. This includes returning all equipment and supplies (hot plates, ring stands, clamps, etc.) to the proper place, correctly disposing of any waste, and cleaning the bench-top. Failure to do this will result in loss of technique points. 6 Laboratory equipment Short path distillation Vigreux Reflux Distillation Graduated head distillation condenser receiver cylinder Three-necked round- Erlenmeyer Prolonged Solid addition Separatory bottom flask Flask clamp funnel funnel Liebig Volumetric Büchner funnel Kitasato flask Addition funnel condenser flask 7 Fritted glass Distillation Cold finger Dessicator Beaker funnel termometer Reducing joint Chromatography TLC developing Round bottom Clamp holder adapter column tank flask Rings/clamps Ring clamp Hot plate stirrer Keck clips Stir bars Stand Proper use of glassware Most of the experiments in this manual are described on microscale or miniscale. This generally means working with between 50 mg and 2 g of material. For comparison purposes, a regular aspirin tablet contains 325 mg of acetylsalicylic acid. Working with small amounts of materials highlights the importance of working with clean glassware. If you prepare 50 mg of a product, which then picks up 10 mg of foreign material from dirty glassware, the product is now significantly contaminated. Transferring small amounts of material from one container to another requires care. You can avoid unnecessary transfers with careful planning. When transferring solids between containers, losses are unavoidable. Do the best job you can to scrape material out of the original container. Quantities of liquids 8 less than 5 or 10 mL should be transferred using a Pasteur pipette. Always hold a pipette right side-up. Never ever invert a pipette. This contaminates the rubber bulb and the sample you are holding. Do not attempt to pour small amounts of liquids. If the liquid in question is a solution of your product in an organic solvent (for example, 100 mg of benzophenone dissolved in 2 mL of hexane), you can make a very efficient transfer by pipetting the solution to a new container, then rinsing the original container with a bit of the original solvent (hexane). Combine this wash hexane with the material first transferred. Most scientific glassware can be viewed simply as a container with a specialized purpose. When choosing glassware, keep in mind what will happen later. Does the material need to be heated, cooled, refluxed? Will you be adding more material? Choose an appropriate-sized container. It makes no sense to store 5 mL of liquid in a 250 mL beaker. Cleaning glassware Always wash glassware before the end of lab. That way, when you return to lab later, it will be clean, dry, and ready to use. Usually, soap, water and a little elbow grease are all that is necessary. For highly water-insoluble materials, it may be necessary to rinse the item with a bit of wash acetone, located in squeeze bottles by the sink. While working in lab, if you find it necessary to wash and re-use a piece of glassware, determine if the piece must be dry before use. Many students have used a considerable amount of lab time carefully drying a piece of glassware only to then use it to hold water. Heating, Cooling and Stirring Efficient cooling is performed in an ice bath, which really means an ice-water bath. If you are cooling a small container that can tip over, clamp the container in place. If it can tip over, it will tip over. Please heed this warning. Heating is performed on a hot plate. A heat gun is never used to heat an organic solvent. Flat- bottomed containers (beakers, and Erlenmeyer flasks) can be heated directly on the surface of the hot plate. Round-bottomed items such as test tubes and flasks are best heated in the hole of an aluminium block placed on the hot plate. The hot plates surfaces respond slowly to changes made in dial setting. This surface will warm and cool very slowly. The most effective way to stop heating a container is to raise it above the surface of the plate or remove the container entirely. Be careful: the high setting on a hot plate is often extremely hot. Do not pick up an aluminum block after it has been resting on the top of a hot plate. You will receive a nasty burn. At the end of lab, leave the aluminum block in the hood with the heater, not in your drawer. The hot plates in lab are also equipped with a magnetic stirrer. Below the center surface of the hot plate is a strong magnet mounted on an electric motor. Solutions on the heater-stirrer can be mixed by adding a Teflon-coated magnetic stir bar or spin vane. Small amounts of materials in a large test tube can be effectively mixed by rapid agitation in small up and down strokes. 9 Bumping Organic liquids have a tendency to super-heat when being heated in a glass container. The super- heated liquid will then boil violently in a sudden fashion: a condition known as “bumping.” Bumping will usually spray the hot liquid around the laboratory and on surrounding people. Bumping is easy to avoid. To avoid bumping one of the following solutions can be applied: 1. Add one or two boiling stones to any liquid you are boiling. Boiling stones, also called boiling chips or Boilezers® are small chips of an inert porous material such as porcelain or carbon. They act as a nucleation source for boiling to occur. 2. Magnetically stir the liquid. 3. In some cases you can constantly attend the boiling of a small amount of liquid in a large container, where constantly swirling of the solution is possible. Measurements It is important to understand whether a measurement needs to be accurate or whether an approximation will suffice. Reagents affecting the stoichiometry of a reaction are generally made accurately. Solvent, solution, and reagent quantities used in gross excess can often be approximated (e.g., organic solvents or aqueous solutions used for extractions). Precise measurements are best made on a balance. Small amounts of liquids are either weighed or measured by volume with a syringe. A graduated cylinder does not deliver sufficient accuracy for small volumes of an organic liquid (less than a few milliliters). Hamilton's precision syringe, which you may have in the lab, are designed to deliver highly accurate and precise volumes of liquids, but are quite expensive and indicated for special cases. Electronic balances are a great convenience, but are also expensive and fragile. To maintain calibration, never move the balance. Keep the balance area clean, removing any spills immediately. Before use, depress the “zero” or “tare” button. Place an item to be weighed on the pan, and record the mass in your laboratory notebook. Because you cannot place chemicals directly on the balance pan, you will weigh reagents on a weighing paper or directly into the container in which you plan to use the chemical. In this case, the mass of the empty container must be subtracted from the gross weight of container and contents. This process is known as a tare. The balance can provide this feature electronically. Place the empty container on the balance, and again, press the “tare” or “zero” button. The weight of the container is automatically subtracted. Do not use this feature, however, if you need to re- weigh the container and contents later in the experiment. The graduations on the sides of beakers and test tubes provide a nice method for the estimation of volumes. Extraction When something is extracted, it is pulled away from something else. For example, a dentist extracts a tooth by pulling it out of your mouth. In chemistry, extraction is the physical process by which a compound (or a mixture of compounds) is transferred from one phase into another. When you make tea or coffee, an 10 extraction takes place: the water-soluble components in the tea leaves or the coffee grounds are transferred from a solid phase into a liquid phase (the boiling water). This is an example of a solid-liquid extraction. It is also possible to partition the components of a mixture between two immiscible liquids (i.e., liquids that will not dissolve in each other and form two distinct phases when combined). This process is called a liquid-liquid extraction. There are two general types of liquid-liquid extractions: - An organic solvent extraction in which an organic solvent with a high affinity for the desired compound is used to extract the compound from another solution. - An acid-base extraction, in which an organic acid or base is extracted from an organic solvent by using an aqueous solution of an inorganic base or acid, respectively. A neutralization occurs which converts the compound into an ionic, water-soluble salt, causing it to transfer from the organic phase to the aqueous phase. The choice of apparatus for an extraction is determined by the volumes of the solution being extracted and the extracting solutions. Typical extractions in the laboratory are done in a separatory funnel, while microscale extractions are done in a conical vial. Extraction with organic solvents Liquid-liquid extractions usually involve water and an organic solvent. Most common organic solvents (diethyl ether, ethyl acetate, toluene, dichloromethane) are immiscible in water. If you place 50 mL of ethyl acetate and 50 mL of water in a flask and stir the solution to mix it, you will not obtain a homogeneous solution. Rather, if the solution is allowed to stand after stirring, two distinct liquid phases will form in the flask: the more dense solvent as the lower layer and the less dense solvent as the upper layer. Most organic solvents are much less polar than water. A general rule of thumb for solubility states that “like dissolves like.” Polar compounds are more soluble in polar solvents than in nonpolar solvents, and vice versa. The selective solubility of different compounds in polar versus nonpolar solvents allows the separation of the compounds in a mixture by liquid-liquid extraction. Suppose that we add compound X to a flask containing ethyl acetate and water, and stir the contents of the flask to mix them. After mixing, the ethyl acetate and water will separate into two distinct phases, and compound X will be found dissolved in both the ethyl acetate layer and in the water layer. How compound X distributes between the two solvents is based on the solubility of X in each of the two solvents: more of compound X will be found in the solvent in which it is more soluble. The ratio of the concentrations of X in each of the immiscible solvents is called the distribution coefficient or the partition coefficient K , d where: 11 The value of the distribution coefficient depends on the solubility of the compound in the two solvents in the system. In the above system, if compound X has a higher solubility in ethyl acetate than in water, at equilibrium the concentration of X in ethyl acetate will be greater than the concentration of compound X in water, and the value of the distribution coefficient K , will be greater than 1. If instead d compound X has a higher solubility in water than in ethyl acetate, at equilibrium the concentration of X in water will be greater than the concentration of compound X in ethyl acetate, and the value of the distribution coefficient K will be less than 1. d The efficiency of a liquid-liquid extraction depends on the distribution coefficient of the desired compound between the two solvents. If we want to extract an organic compound from an aqueous solution into an organic solvent, it is desirable to use a solvent that has a much higher affinity for the compound than does water. For example, at 25°C, the solubility of benzoic acid in water is 3.4 g per liter while the solubility of benzoic acid in chloroform (CHCl ) is 222 g per liter. Water and chloroform are immiscible solvents. If a 3 solution of 1 g of benzoic acid in 400 mL of water is extracted with 400 mL of chloroform, we would expect most of the benzoic acid to be transferred to the chloroform layer in which it is more soluble. The benzoic acid will distribute itself between the two solvents in the ratio (approximately) of the solubilities in each solvent: No matter how much benzoic acid is present in the system, it will always be distributed between the chloroform and water so that the ratio of the concentration in each solvent is 65.3. From this estimate of the distribution coefficient, we can calculate how much benzoic acid is present in the chloroform and water layers after the extraction. Let x = grams of benzoic acid in the water layer and y = grams of benzoic acid in the chloroform layer. Since we started with 1 g of benzoic acid, x + y = 1. Using this equation along with the value for the distribution coefficient calculated above, we can determine the concentration of benzoic acid in each layer. Or, since the volumes of both solvents are the same: The total amount of benzoic acid present is (x + y = 1). Rearranging this equation and substituting in for the previous equation gives: 12 Solving this equation for y gives 0.015 g (15 mg) of benzoic acid in the water layer, and, since the total amount of benzoic acid is 1 g, there is 0.985 g (985 mg) of benzoic acid in the chloroform layer. Multiple extractions In the previous example, one extraction with 400 mL of chloroform removed 98.5% of the benzoic acid from the aqueous solution. If we divide the 400 mL of chloroform used in half and do two successive extractions of the aqueous phase, the amount of benzoic acid extracted will increase. The equation for the distribution coefficient for two 200 mL chloroform extractions of the 400 mL aqueous solution of benzoic acid is: In the first extraction, 1 g of benzoic acid is distributed between the phases, so (x + y) as before. Solving the two equations in two unknowns gives x = 0.97 g in CHCl3 and y = 0.03 g in H2O. When the aqueous phase is extracted a second time with a fresh 200 mL of chloroform, only 0.03 g of benzoic acid is left in the aqueous phase to distribute between the two solvents. In this extraction the equation for the distribution coefficient is the same but (x + y) = 0.03, and solving for x and y, the amount of benzoic acid in each layer after the second extraction gives x = 0.0291 g in CHCl3 and y = 0.0009 g in H2O. Combining the amounts of benzoic acid found in the two chloroform extracts gives 99.91% (0.9991 g of the original 1 g) of the benzoic acid extracted into the chloroform layer by using two 200 mL extractions instead of 98.5% removed with one 400 mL extraction. In general, it is always more efficient to carry out several extractions using a small volume of solvent each time than to carry out a single extraction using a large volume of solvent. Acid-base extraction Organic compounds are classified as being neutral, acidic, or basic depending on the types of functional groups they contain. Many organic compounds, although just slightly polar overall, contain functional groups that can act as a Bronsted-Lowry acid or base (i.e., they can donate or accept a proton, respectively). Carboxylic acids, phenols, and thiols are examples of acidic functional groups; substituted amines (including anilines) are examples of basic functional groups. Although the water-solubility of these compounds is often limited because of their overall nonpolar character, their aqueous solubilities can be dramatically increased through an acid-base neutralization reaction. This changes the compound into an ionic salt that is very water soluble and will distribute almost completely into the aqueous layer. Experiment 1 has been designed to illustrate how an acid-base extraction works. Thin layer chromatography (TLC) Chromatography: A General Introduction Chromatography is the most versatile technique for separating mixtures. The technique derives its name from early experiments in which plant pigments were separated into individual components such as 13 chlorophylls and xanthophylls by passing the mixture through a column packed with calcium carbonate. Bands of different colors appeared on the column. Chromatography is not limited to colorful materials, however. Many different types or classes of chromatography exist and are used not only to separate, but also to isolate and identify both in a qualitative and quantitative manner. What all forms of chromatography have in common is that each employs a stationary phase and a mobile phase. Components of the mixture are carried past the stationary phase by the flow of the mobile phase. The components distribute, or partition, between the two phases, and separation occurs based on the average time a component spends in each of the phases. For example, materials that are strongly attracted to the stationary phase will flow slowly; whereas components that are highly soluble in the mobile phase will travel quickly. Different classifications of chromatography are made based on the identity of each of the phases (for example, whether the mobile phase is a gas or a liquid) and the operating conditions of the system. Compounds partition between the two phases based on polarity. Adsorbents used for solid-liquid chromatography are generally polar, so polar solutes adsorb strongly onto the stationary phase. They will elute (move with the mobile phase) slowly. Nonpolar compounds are not as strongly adsorbed, and therefore spend more time, on average, in the mobile phase and thus elute rapidly. For routine organic laboratory work, silica gel (SiO •nH O) is the most common adsorbent. 2 2 Thin Layer Chromatography (TLC) In TLC the adsorbent is spread in a thin layer over a solid support such as a sheet of aluminium (or glass) to make a TLC plate. Samples are spotted at the bottom of the plate which is developed by placing it in a chamber containing small amount of the mobile phase. The mobile phase (a solvent) wicks up the plate by capillary action. TLC is used as an analytical technique, and provides qualitative information about a sample. TLC is the most common and widely used method of analysis in a synthetic organic laboratory. It is not, however, the most powerful technique for analysis. It is used because of low cost, rapid analysis time, convenience, and simplicity. As already mentioned, the most common stationary phase used is silica gel mixed with plaster of Paris (calcium sulfate) to harden and support the adsorbent. You must handle TLC plates carefully and only by the edges of the plates. The adsorbent coating is fragile and should remain clean prior to use. 14 The sample to be analyzed must be prepared as a dilute solution. The solvent chosen for this should dissolve the compound well, and must evaporate rapidly. A small sample of this solution is spotted near the bottom of a TLC plate with a fine glass capillary tube. The plate is typically prepared in advance by drawing a light pencil line 1 cm from the bottom of the plate, then making light cross marks to indicate where the sample will be spotted. TLC is most often used to compare samples, so each plate will have several spots. The plate is placed in a development chamber. Solvent moves up the plate by capillary action. The plate is removed from the chamber when the solvent has risen to within one centimeter of the top of the plate. A pencil line is immediately drawn across the top to indicate the height the solvent front travelled. The technical name for a substance being analyzed is “analyte”, but we rarely use that term. It will be referred to as “the solute” (because it is in solution) or, more roughly, “the stuff you are analyzing,” or “the stuff that you spotted,” or “the sample.” We will often relate molecular structure of the analyte to different types of intermolecular forces. This will allow us to relate the structure of a molecule to a property we can observe–the distance it travels along the plate in a TLC analysis. The mobile phase is also referred to as the developing solvent, or “eluent” from the verb, “to elute”. Changing the developing solvent will change the result of an analysis. Again, the types of intermolecular forces present are important and will be related to how the mobile phase behaves. Finally, YOU MUST DILUTE a sample before you can spot it on a TLC plate. A solution between 2% and 5% is usually sufficient. Place a few milligrams of the sample in a small disposable vial and add approximately 0.5 mL of solvent. The solvent you use for this has no special name, and if selected correctly, has no effect on the outcome of your TLC analysis. This solvent is simply used to dilute the sample and must evaporate before the plate is developed. Select a solvent that dissolves a wide range of samples and has a low boiling point. Diethyl ether or dichloromethane are often good choices. Shown below are typical results from TLC analyses. These results illustrate the effect from developing solvent polarity, and the effect of analyte polarity. 15 Effect of eluent polarity on TLC analysis Effect of sample polarity on TLC analysis TLC can be used to establish if two materials are definitely different (they travel different distances) or possibly the same. Two different materials can coincidentally behave the same in TLC. Illustrated below are some typical problems encountered in TLC. Eluent polarity problems Sample concentration problem 16 Experimental design problem Visualization Unless the materials being analyzed are colored (absorb visible light), a method for visualizing the resulting spots is required. Several methods are available. Materials that absorb ultraviolet light are easily visualized because the adsorbent has a fluorescent indicator added to it. When UV light at 254 nm is shined on the plate, the adsorbent surface will fluoresce brightly and the compounds that absorb UV light will be seen as dark spots. These spots may then be traced on the surface of the plate with a pencil. Caution UV light is harmful. Minimize exposure to skin and do not shine in eyes. This method is limited to materials that absorb UV light. Aromatic rings and conjugated ketones are two important functional groups you will see in laboratory that absorb UV light. The second method of visualization takes advantage of the fact that iodine will form a complex with a wide variety of organic compounds. Place the plate to be visualized in a wide-mouth jar with iodine and warm the bottom of the jar gently to fasten the process of sublimation. Yellow or brown spots will appear. This method is temporary, so a permanent record should be made by tracing the outline of the spots on the plate. Other visualization reagents are available and are generally prepared as a solution that is sprayed on the plate or in which the plate is dipped. Methods for preparing these reagents can be found in handbooks of organic chemistry or chromatography. Retention factor (R ) f Under a fixed set of conditions, a given compound always travels the same distance relative to the distance the solvent front travels. This ratio of distances is called the retention factor and is abbreviated R . f A sample calculation is shown below. It is customary to measure from the center of the spot. 17 Calculating a retention factor The polarity of the mobile phase (eluent, developing solvent) also determines the speed at which substances being analyzed will elute. As a general rule of thumb, any solute will elute more rapidly in a more polar eluent. This is a rough guideline. The polarity guidelines are based on intramolecular forces between adsorbent and solute, or adsorbent and solvent, including van der Waals force, dipole-dipole, hydrogen bonding, coordination, and salt formation. Recrystallization The most common method of purifying solid organic compounds is by recrystallization. In this technique, an impure solid compound is dissolved in a solvent and then allowed to slowly crystallize out as the solution cools. As the compound crystallizes from the solution, the molecules of the other compounds dissolved in solution are excluded from the growing crystal lattice, giving a pure solid. Crystallization of a solid is not the same as precipitation of a solid. In crystallization, there is a slow, selective formation of the crystal framework resulting in a pure compound. In precipitation, there is a rapid formation of a solid from a solution that usually produces an amorphous solid containing many trapped impurities within the solid's crystal framework. For this reason, experimental procedures that produce a solid product by precipitation always include a final recrystallization step to give the pure compound. The process of recrystallization relies on the property that for most compounds, as the temperature of a solvent increases, the solubility of the compound in that solvent also increases. For example, much more table sugar can be dissolved in very hot water (just below the boiling point) than in water at room temperature. What will happen if a concentrated solution of hot water and sugar is allowed to cool to room temperature? As the temperature of the solution decreases, the solubility of the sugar in the water also decreases, and the sugar molecules will begin to crystallize out of the solution. (This is how rock candy is made.) This is the basic process that goes on in the recrystallization of a solid. The steps in the recrystallization of a compound are: 1. find a suitable solvent for the recrystallization; 2. dissolve the impure solid in a minimum volume of hot solvent; 3. use decolorizing carbon (if necessary); 4. remove any insoluble impurities by filtration; 5. slowly cool the hot solution to crystallize the desired compound from the solution; 18 6. filter the solution to isolate the purified solid compound. 1. Choosing a Solvent The first consideration in purifying a solid by recrystallization is to find a suitable solvent. There are four important properties that you should look for in a good solvent for recrystallization. First is that the compound should be very soluble at the boiling point of the solvent and only sparingly soluble in the solvent at room temperature. This difference in solubility at hot versus cold temperatures is essential for the recrystallization process. If the compound is insoluble in the chosen solvent at high temperatures, then it will not dissolve. It the compound is very soluble in the solvent at room temperature, then getting the compound to crystallize in pure form from solution is difficult. For example, water is an excellent solvent for the recrystallization of benzoic acid. At 10°C only 2.1 g of benzoic acid dissolves in 1 liter of water, while at 95 °C the solubility is 68 g/L. The second desirable property of a good recrystallization solvent is that the unwanted impurities should be either very soluble in the solvent at room temperature or insoluble in the hot solvent. This way, after the impure solid is dissolved in the hot solvent, any undissolved impurities can be removed by filtration. After the solution cools and the desired compound crystallizes out, any remaining soluble impurities will remain dissolved in the solvent. A third important property of the recrystallization solvent is that it must not react with the compound being purified. The desired compound may be lost during recrystallization if the solvent reacts with the compound. Finally, the recrystallization solvent should be volatile enough to be easily removed from the compound after it has crystallized. This allows for easy and rapid drying of the solid compound after it has been isolated from the solution. Finding a solvent with the desired solubility properties is a search done by trial and error. First, test the solubility of tiny samples of the compound in test tubes with a variety of different solvents (water, ethanol, methanol, ethyl acetate, diethyl ether, hexane, toluene, etc.) at room temperature. If the compound dissolves in the solvent at room temperature, then that solvent is unsuitable for recrystallization. If the compound is insoluble in the solvent at room temperature, then the mixture is heated to the solvent's boiling point to determine if the solid will dissolve at high temperature, and then cooled to see if it crystallizes from the solution at room temperature. 2. Dissolving the Solid Once a suitable solvent is selected, place the impure solid in an Erlenmeyer flask and add a small volume of hot solvent to the flask. Erlenmeyer flasks are preferred over beakers for recrystallization because the conical shape of an Erlenmeyer flask decreases the amount of solvent lost to evaporation during heating, prevents the formation of a crust around the sides of the glass, and makes it easier to swirl the hot solution while dissolving the solid without splashing it out of the flask. Keep the solution in the Erlenmeyer flask warm on a hot plate or in a water bath, add small volumes of hot solvent to the flask until the entire solid just dissolves. Swirl the solution between additions of solvent 19