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LABORATORY EXPERIMENTS

ORGANIC CHEMISTRY

DEGREE IN BIOTECHNOLOGY COURSE 2016-2017

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Chemistry laboratory rules and regulations………………………………………………………………………3

Safety in the laboratory……………………………………………………………………………………………………4 Laboratory material…………………………………………………………………………………………………… …….7 Laboratory notebook ………………………………………………………………………………………… …………….9 Sample taking……………………………………………………………………………………….…………………………….10 Mounting glassware………………………………………………………………………………………………… ………16 Isolation and purification of products…………………………………………………………………………20 Calculating the reaction yield…………………………………………………………………………………………25 EXPERIMENT 1: Structure of Organic Compounds. Use of molecular models …28 EXPERIMENT 2: Recrystallization……………………………………………………………………………….33 EXPERIMENT 3: Distillation of wine..............................................................................38 EXPERIMENT 4: Liquid liquid extraction.......................................................................41 EXPERIMENT 5: Thin Layer Chromatography…………………………………………………………..47 EXPERIMENT 6: Synthesis of aspirin............................................................................53 EXPERIMENT 7: Synthesis of turt-butyl chloride.....................................................57 EXPERIMENT 8: Synthesis of borneol by reduction of camphor............................60

INDEX

Biotechnology 2: Organic Chemistry Laboratory Regulations

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The experiments corresponding to the course of Organic Chemistry consist of 7 sessions each lasting two hours. Attendance is mandatory for all of them.

Each student must bring with them from the very first day of the laboratory:

LABORATORY COAT (in good condition with long sleeves) SAFETY GOGGLES (even if you wear eye glasses) GLOVES ALUMINUM SPATULA (or a micro spoon) SCISSORS LABORATORY NOTEBOOK

Wear your lab coat, buttoned and with the sleeves properly rolled down Always wear your safety goggles Wear gloves The experiments are to be carried out in pairs Eating and drinking within the laboratory are prohibited Leaving the laboratory without prior notification of the instructor is prohibited You may not abandon your work station Your must keep your work station clean, as well as the laboratory’s common areas, scales,

cabinets, sinks, etc. You must be punctual

Grading the experiments For grading the experiments, the following will be kept in mind: When grading the laboratory work itself, the instructor will keep the students’ ability

in the laboratory in mind, as well as their interest and attitude. The laboratory report: within which assessed are the order, clarity, correctness, rigor,

and quantity of information collected regarding the experiments conducted. The laboratory examination will take place simultaneously along with the chemistry

lecture exam. As the experiments are required: One unjustified absence for any experiment will suppose a failing grade for the

chemistry lab. Two absences, justified or otherwise, will suppose a failing grade for the chemistry

lab. The experiments may be made up, always and when the absence has been justified if

space is available in the laboratory or when the instructor has been warned of the change beforehand.

CHEMISTRY LABORATORY RULES AND REGULATIONS

Biotechnology 2: Organic Chemistry Safety in the Laboratory

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A chemistry laboratory is always a dangerous place (flammable, explosive and toxic substances abound). A vigilant and attentive attitude is essential at all times, even when you are not doing anything. Thinking and acting in a sure manner is an integral portion of chemistry education. No experiment may be begun before you are sure you fully understand what will occur, and only after answering these two questions: What is the worst thing that could happen? How could I remedy it? Asking the instructors, in cases of doubt, is a good habit. The following regulations are obligatory for all who enter or remain within the laboratory:

1) SMOKING IS NOT ALLOWED, nor is lighting any flame or lighter inside the laboratory.

2) DO NOT CONNECT electronic devices without being sure that there is no danger from nearby solvent vapors. Never place flammable products near heat sources. Many flammable organic substances produce heavier than air vapors capable of moving considerable distances across laboratory tabletops.

3) SAFETY GOGGLES ARE ALWAYS REQUIRED even if you are not conducting an experiment (someone else nearby could cause an accident affecting you). Do not wear contact lenses inside the laboratory (organic vapors could damage them; moreover, caustic reagents cannot be eliminated from the eyes if lenses are being worn). Learn where the laboratory eyewash stations are located.

4) AVOID PHYSICAL CONTACT WITH ORGANIC SOLVENTS. Do not breathe solvent vapors. (Consult the section on Risks associated with reagents and solvents.)

5) WEAR PROTECTIVE GLOVES AT ALL TIMES. In the event of eye or skin contact with corrosive or irritating materials, wash the affected area immediately with abundant quantities of water (several minutes in the case of the eyes), and urgently inform the person responsible in the laboratory. Avoid any initiative that you have not previously consulted with the person responsible.

6) EATING AND DRINKING IN THE LABORATORY IS PROHIBITED, as well as introducing food and drink.

7) All CHEMISTRY ACTIVITIES involving REAGENTS, AND TOXIC, LACHRYMATORY AND FOUL-SMELLING SOLVENTS, must take place in a FUME HOOD with its extractor running.

8) USED SOLVENTS are not to be POURED DOWN THE DRAIN; rather, they are to be stored in the appropriate recipients designed for them. Concentrated acids and bases are to be neutralized, and the resulting saline solution disposed of with the faucets running. Sodium residues are to be destroyed with methanol before disposing of it. In the event of doubt, ask the person responsible about how to proceed.

9) NEVER LEAVE THE LABORATORY WITHOUT AUTHORIZATION by the person responsible for it, and never without leaving somebody else in charge of the experiments taking place. When the laboratory session finishes, carefully review and disconnect devices and reactions, as well as closing water faucets. No reactions or processes may be left in motion overnight without express authorization and supervision by the person responsible for the laboratory.

SAFETY IN THE LABORATORY


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10) LEARN ABOUT THE FIRST AID KIT CONTENTS AND ABOUT THE LOCATIONS OF THE FIRE EXTINGUISHERS AND EMERGENCY EYEWASHES AND SHOWERS, AND LEARN HOW THEY FUNCTION.

11) Cleanliness and order are essential in the laboratory at all times. This helps prevent accidents.

Specific regulations in case of accidents 1) In case any accident occurs, INFORM, or have somebody else do it, THE NEAREST

LABORATORY INSTRUCTOR. 2) If a chemical product comes into contact with your eyes, go the nearest emergency

eyewash and wash them with abundant quantities of water. Be careful not to use too much water pressure to avoid injuring the eyes that way. Immediately inform, or have somebody else do it, the nearest laboratory instructor.

3) If a fire results, the best advice is to move away from it and allow the laboratory instructor to take charge of the situation. Do not panic! Use your best judgment! If the fire is small, it can be extinguished by placing a fire blanket over it. If the fire is concentrated within a flask or beaker, it can be extinguished by simply covering the opening of the container with a watch glass or a larger glass. If these solutions are not successful, or if the fire is large enough, the CO2 extinguishers need to be employed. Never use water. Always inform, or make somebody else do it, the nearest laboratory instructor immediately.

4) If your clothing catches fire, do not run, because this will simply fan the flames. Advance determinedly towards the fire blanket or nearest emergency shower. Covering the area in flames with the blanket should be enough to extinguish the flames. Inform, or have somebody else do it, the nearest laboratory instructor immediately.

5) Should you burn yourself by touching a hot object, cool the affected area with abundant cold water. Approach and inform the laboratory instructor, for there are creams and lotions in the first aid kit for these cases.

6) In case of burns from chemical products, they must be neutralized. If that responsible is an acid, a diluted solution of sodium bicarbonate can be employed; in the case of bases, 2% acetic acid can be used. Inform, or have somebody else do it, the nearest laboratory instructor immediately.

7) In case of cuts, wash the wound with abundant water. Inform, or have somebody else do it, the nearest laboratory instructor immediately.

Risks associated with reagents and solvents

Remember that the correct handling of reagents and solvents is essential if accidents, as well as contamination from these products, are to be avoided. Contamination from a reagent or solvent will cause errors in the results of everything they contact. The labels on bottles, and whatever other information supplied about them, must be read carefully. The labels on solvents and reagents contain symbols that refer to the hazards associated with them that are in accordance with current European Union legislation. Keep the labels in mind when these substances are handled.

T+

VERY TOXIC. Substances and preparations that from inhalation, ingestion or skin penetration, even in very small quantities, can cause acute or chronic effects, even death.


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T

TOXIC. Substances and preparations that from inhalation, ingestion or skin penetration, even in very small quantities, can cause acute or chronic effects, even death.

Xn

HARMFUL. Substances and preparations that from inhalation, ingestion or skin penetration, even in very small quantities, can cause acute or chronic effects, even death.

Xi

IRRITANT. Non-corrosive substances and preparations that from brief, prolonged or repeated contact with the skin or mucous membranes, can cause an inflammatory reaction.

C

CORROSIVE. Substances and preparations that, from contact with living tissue, can cause a destructive action within them.

F+

EXTREMELY FLAMMABLE. Liquid substances and preparations that have an extremely low flash point and a low boiling point; gaseous substances and preparations that at a normal temperature and pressure are flammable in air.

F

HIGHLY FLAMMABLE. Substances and preparations that can heat up and burst into flame in the air at room temperature without any additional contribution of energy; solids that can burst into flame easily after brief contact with a source of flame and that will continue burning and consuming themselves after that source has been removed; liquids whose flash point is very low, or that, in contact with water or humid air, release extremely flammable gases in dangerous quantities.

O

OXIDIZING. Substances and preparations that when in contact with other substances, especially with flammable substances, produce a strongly exothermic reaction.

E

EXPLOSIVE. Solid, liquid, pasty and/or gelatinous substances that, even when lacking oxygen, can react exothermically, rapidly forming gases and that, under certain test conditions, detonate, deflagrate rapidly and/or, under the effects from heat and in cases of partial confinement, explode.

N

DANGEROUS FOR THE ENVIRONMENT. Substances and preparations that possess or may possess an immediate or future threat for one or more components in the environment.

Other symbols exist that are not included here and they too are important. There is a

series of lettered phrases pronouncing the specific risks of the product (R-phrases), and advice for their safe handling, for example, for their storage (S-phrases).

Biotechnology 2: Organic Chemistry Laboratory material

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In order to work in a laboratory, it is essential to learn and become familiar with the names and forms of the pieces comprising commonly used material. This material is shown in the following diagram:

BEAKER STILL HEAD

“PIG” ADAPTOR ELBOW EXTENDER

ADAPTER

PLUG

LABORATORY MATERIAL

ROUND BOTTOM FLASK ERLENMEYER KITASATO FLASK

BÜCHNER FUNNEL

CONICAL FUNNEL

REFLUX CONDENSER THERMOMETER GRADUATED

CYLINDER

CONDENSER GRADUATED PIPETTE BURETTE

Biotechnology 2: Organic Chemistry Laboratory material

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DECTANTATION FUNNEL

TEST TUBE

WATER ASPIRATOR PIPETTE SUPPORT

CRYSTALLIZING DISH TUBE RACK WASH

BOTTLEALCOHOLOMETER BRUSH

HOT PLATE/STIRRER

MICRO SPOON

NIPPLE

CORK RING

CLIP

CONE

MAGNET

CALCIUM CHLORIDE

TUBE

NUT

BURETTE CLAMP

SPATULA

METALLIC RING

Biotechnology 2: Organic Chemistry Laboratory Notebook

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Researchers consider their laboratory notebook as one of their most valuable tools. The lab notebook is where we reflect on what we have been doing in the laboratory, along with notes and incidents about what is going on. Although it is somewhat personal, the lab notebook should always reflect sufficient information so that the experimental process described in it can be repeated in a reliable manner.

The lab notebook serves to take immediate note of all experimental observations, in a

brief but clear and concise manner. For this, it must be placed on the very laboratory table and notes taken at the same time the experiments are taking place. Taking notes about the experiment on loose paper for subsequent entry into the notebook is never recommended, for they can get lost in the meantime. Entries must be made directly into the notebook, and never in a rough or incomplete fashion for later cleaning up or organizing of them. Quantitative and qualitative data must never be omitted.

Examples of how a laboratory notebook can serve you might be the following:

Name of the experiment and date it takes place. Chemical reaction occurring (if there is one), adjusted and with the molecular masses

of all compounds involved. Also appearing in an outline form are the solvents used, the conditions of temperature, catalyst, and type of atmosphere if necessary.

A summary of the quantities added from each of the products, along with their molar

equivalence, order in which they were added, as well as a brief description of the glass material used: size and number of flasks, etc.

Changes produced during the addition or reaction, like changes in color, appearance,

or disappearance of precipitates, changes in turbidity, spontaneous heating of the reaction following the addition of any of the reagents, etc.

Manner in which the reaction is stopped, for example cooling to room temperature or in an ice bath, adding some product…and what happens when we stop: if a solid appears upon cooling, various phases appear…

Manner in which the product is isolated: if filtering occurs to eliminate impurities or in

order to obtain a solid product, if a specific solvent is extracted, how many times the process is repeated, if the solvent is eliminated in the rotary evaporator…

Manner of purification, and with what solvent(s) this is done. Types of purity tests conducted. Weight of the isolated and dried product, calculation

of the theoretical and actual reaction yields. Any other incidents, like power outages or water stoppages… Making an assembly diagram of the glassware employed during the reaction is a good

idea.

LABORATORY NOTEBOOK

Biotechnology 2: Organic Chemistry Sample taking

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Generally, all chemical procedures begin by acquiring the necessary quantities of reagents and solvents, and so knowing how to do this correctly is very important. Two situations must be distinguished for this: either the reagent is ready to use, in which case the corresponding quantity must be simply measured, or a solution must be prepared for it. 1) Ready-to-use reagents 1.1) Solid reagents

These types of reagent are weighed in a balance. To do this, place the recipient that the product will be added to on the balance (never weigh directly on the balance pan), and the weight of the empty recipient is recorded. If the balance has a tare button, push it to set the weight showing to zero. For a recipient, you must use either suitable glass material (having a wide mouth that allows scraping) or a piece of foil paper (do not use filter paper because as it is rough some material could get lost) that does not hang over the balance pan. Next, add the product with a spatula (never with your hand) a little at a time, trying at all times not to add too much. This will avoid having to return the product back to its original recipient. 1.2) Liquid reagents and solvents

If a mass of a liquid must be added, it is generally preferable to convert this mass into a volume by way of its density. If this is not possible because this data is lacking, the liquid must be weighed, like in the case of solid reagents, on a balance. The procedure is to attempt to adjust the weighed quantity as closely as possible to the theoretical quantity, weighing directly inside the reaction flask. Once this is done, redo all the calculations of the remaining reagents and solvents in function of the actual quantity of liquid. For example, we must add 3 g of a liquid whose density is unknown to 2 g of a solid. The procedure would be the following: weigh the liquid, attempting to adjust to 3 g, but the actual weight is 2.9 because if one additional drop is added the weight will become 3.2. Now, recalculate the quantity of solid necessary to react with 2.9 of liquid, because this is the actual quantity of liquid reagent that we have added. This solid quantity results as 1.93 g. The theoretical reaction yield must be calculated in function of the real quantities.

If the density is known, once the mass has been converted to volume the measuring

system must be chosen; between pipette, graduated cylinder, burette or flask, depending upon the available capacities and of the liquid quantity being measured. Pipettes and burettes are used for transferring liquid volumes whose measurement requires some accuracy. Flasks are employed to prepare specific volumes of solutions in known concentrations with some accuracy.

For volume measurements, the liquid’s level is compared with the tick marks on the wall of the measuring instrument. This level is read at the bottom of the meniscus that the liquid forms. Exact measurements are obtained by placing your eyes at the level of the meniscus.

SAMPLE TAKING


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Pipette: This is usually used to measure liquid volumes smaller than 10 mL whose measurement requires some accuracy. Always use a propipette, never your mouth, for pipetting. Never place it in a horizontal position because the liquid could move up into the bulb or the propipette, and this could damage it. With the goal of obtaining the correct quantity, and not having to return liquid back to the bottle, the pipette will be marked with the following calculation:

For example, if a 10 mL pipette is used to add 6.4 mL of a liquid, the pipette mark must be moved from 10 – 6.4, which is 3.6 mL. Graduated cylinder: Volumes transferred with graduated cylinders are less exact than those transferred with a pipette. The cylinder’s precision diminishes as their capacity increases. Never prepare solutions in these. Burette: This is employed exclusively to measure volumes in assessments as the stopcock allows controlling the flow of liquid. Flask: Measures volumes with great precision, but only measures the volume given by its capacity. Solutions must be prepared in a beaker and then transferred to the flask.

2) Prepared reagents If the reagent is not ready-to-use, or if it is not given to us already prepared, it must be prepared. In order to do this, we must know the most common units of concentration and how to convert from one to another. 2.1) Most common units of concentration In the following definitions, the abbreviations have these meanings: mp = Product mass mS = Solvent mass ms = Solution mass

Mp = Molar mass of the product VS = Solvent volume Vs = Solution volume

Mark = Pipette capacity – Quantity to add

Correct mark Incorrect mark


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S = Solvent density s = Solution density np = Number of moles of the product n = Valence % m = Percentage in mass

% m/v = Percentage in mass/volume M = Molarity N = Normality m = Molality

% Mass (%): Mass of the product divided by the mass of the solution and multiplied by 100. For example, a solution with 30% of its mass in one product means that for each 100 g of the solution, 30 are product and 70 solvent.

Example: We want to prepare 120 g of a solution with 28% of its mass in product A (Mp(molar mass)) = 34 g/mol) in a solvent with density S = 1.3 g/cm3. The solution’s density obtained is 1.4 g/cm3. Compound A is a diprotic acid.

mp + mS = 120 g

By resolving the equation, it turns out that mp = 33.6 g, and mS = (120-33.6) = 86.4 g. % Mass/Volume (% m/V): Mass of the product divided by the volume of the solvent and then multiplied by 100. For example, a solution of 20% mass/volume means that for each 100 mL of solvent, there are 20 g of product.

Example: We need to prepare 40 mL of a solution with a concentration of 20% in mass/volume of product A (Mp = 34 g/mol) in a solvent of density D = 1.3 g/cm3. The density of the solution obtained is 1.4 g/cm3. Compound A is a diprotic acid.

In these cases, the 40 mL of solution is used as the starting point, equaling it to those of

the solvent in such a way that by directly solving the formula, the mp results as 8 g. The result of this strategy is that a quantity of solution is prepared that is slightly larger than what is strictly necessary. If the exact quantity is to be prepared, the following equation must be solved:

mp + VS x S = Vs x s (mp/VS) x 100 = % m/V

In such a system, the only unknown quantities are mp and VS, precisely the data

necessary. By solving it, it turns out that mp = 7.46 and VS = 37.3 mL. Molarity (M): Moles of a product divided the volume of solution in liters. For example, a 0.5 M solution of a product means that for 1 liter of solution there will be 0.5 moles of product.

p

s

m% mass (%) x 100

m

p

S

m% mass/volume (% m/V) x 100

V

p

s

n (moles)Molarity (M)

V (L)

p

p S

mx 100 28%

m m


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Example: We wish to prepare 50 mL of a 2 M solution of product A (Mp = 34 g/mol) in a solvent of whose density is S = 1.3 g/cm3. The density of the solution obtained is 1.4 g/cm3. Compound A is a diprotic acid.

By directly applying the formula to calculate molar concentrations: Solving for mp, we obtain mp = 3.4 g.

I.e., to prepare such a solution, weigh 3.4 g of product A and add solvent until reaching 50 mL. Normality (N): Equivalents of the product divided by the volume of a solution in liters. The equivalent is defined as moles of a compound multiplied by the valence. This unit of concentration is used above all to give concentrations of acids, in whose case, the valence is simply the number of protons of such acid. It would be 1 In HCl, while in H3PO4, the valence would be 3. For acids with only one proton, like HCl, the normality coincides with the molarity. For example, a 0.1 N solution of HCl would be one containing 0.1 equivalents of pure HCl in l liter of solution, and as the valence of such acid is 1, it would contain 0.1 moles of HCl.

Example: We want to prepare 50 mL of a solution whose concentration is 2 N of product A (Mp = 34 g/mol) in a solvent of density S = 1.3 g/cm3. The solution’s density is 1.4 g/cm3. Compound A is a diprotic acid.

By directly applying the formula to calculate normal concentrations:

Solving for mp, mp = 1.7 g.

In order to prepare such a solution, 1.7 g of product A is weighed and solvent is added to it until reaching 50 mL. Molality (m): Moles of a product divided by the mass of a solvent in Kg. For example, a 3 m solution of a product means that there are 3 moles of product per Kg of solvent.

Example: We need to prepare 200 mL of a solution whose concentration is 3 m of product A (Mp = 34 g/mol) in a solvent of density S = 1.3 g/cm3. The solution’s density is 1.4 g/cm3. Compound A is a diprotic acid.

The easiest way is to prepare a quantity of solution that is slightly superior to that

needed, and so we start with 200 mL of solvent. If it is done this way, the solvent’s volume must be converted to mass, multiplying by its density, obtaining 260 g.

p

s

equivalentNormality (N)

V (L)

p

S

n (moles)Molality (m)

m (Kg)

p

p

s

mM

M x 1000V (in mL)

p

p

s

mM

N x 1000 x nV (in mL)


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By directly applying the formula to calculate molal concentrations: Solving for mp, mp=26.52 g.

In order to prepare such solution, 26.52 g of product A are weighed and 200 mL of solvent are added to it.

One problem that is proposed when working with solutions is to have one whose concentration is known in one unit and have to convert it to a distinct unit. Look at the following table to see how this is done. All the masses appear in grams and all the densities are in g/cm3:

p

p

S

mM

m x 1000m (in g)

Biotechnology 2: Organic Chemistry Toma de muestras

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KNOWN

CONCENTRATION IN

CONVERTS TO

% m % m/V M N m

%m 1

S

% mx100

100 % mρ

p

s

%mM

0.1ρ

p

s

%mM

x n0.1ρ

p

% mM

100-% m1000

% m/V S

% m/Vx100

% m/V 100 x ρ

1 p

S

s

% m/Vx 1000

M

100 x ρ % m/V

ρ

p

S

s

% m/V x 1000

Mx n

100 x ρ % m/V

ρ

p S

% m/V 10 x

M ρ

M p

s

M x M

10 x ρ

p

s p

S

M x Mx100

1000 x ρ M x M

ρ

1 M x n s p

M x 1000

1000 x ρ M x M

N

(always convert to molarity)

p

s

M x M

10 x ρ

p

s p

S

M x Mx100

1000 x ρ M x M

ρ

n

N 1 s p

M x 1000

1000 x ρ M x M

m 100 x M x m1000

M x m

p

p

S

p

ρm x M x

10 p

s

m x 1000

1000 m x M

ρ

p

s

m x 1000x n

1000 m x M

ρ

1

Biotechnology 2: Organic Chemistry Mounting glassware

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Almost all chemical processes in a laboratory employ different glassware mountings, and so knowing how to assemble them is essential. Although many variations exist, the most common are those of distillation and reflux, and these will be used in these experiments.

1) General considerations

The pieces comprising mountings are practically always assembled using male and female connections of ground glass. If precautions are not taken, it is common for these pieces to become stuck together, which makes taking them apart difficult, and this difficulty can result in broken glass causing cuts to the skin.

Therefore, with a system with ground glass joints, Teflon tape or vacuum grease must be applied to the male connections in order to eliminate this problem. Teflon tape must cover the male ground glass entirely and if possible using only a single layer in order to obtain a good seal between the pieces.

If grease is applied, it must be extended with the finger in the ground area farthest away from the edge of the piece, in such a way that the contact between the grease and the possible solvents being used in the reaction is minimized. Once both pieces have been joined, rotate them in both directions to uniformly distribute the grease throughout the joint. In cases where grease has been applied, it must be eliminated from the mouth of the flask once the reaction has finished before extracting the liquid. This prevents the solvent from contacting and mixing with the grease, and possibly contaminating the product.

Before continuing with the mounting, the magnet must be introduced into the flask. This prevents it falling from the mouth, which could possibly break it.

2) Holding the pieces

All pieces must be perfectly held in order to avoid falls and breaks. As a rule, a clamp must always hold the reaction or distillation flask. The hold must be firm, but not too tight, and the clamp’s arms must be placed in such a way that contact between them and the glass is complete.

MOUNTING GLASSWARE

CORRECT HOLD INCORRECT HOLD INCORRECT HOLD


17

Another clamp must be placed to hold either the reflux, or distillation condenser. The connections between the pieces must be secured with red plastic clips for large mouths, and yellow if the mouth is small. These clips are essential if the piece being joined is not mounted vertically.

3) Mounting order

As a rule, the system being mounted must be in such a way so that a single person can accomplish this, and with the assurance that the pieces comprising it are correctly held. The following diagram may be useful. 3.1) Reflux system

First, position the hot plate/stirrer along with its support and metallic base, on top if the support is flat, and between the legs if it is a tripod.

Next, place the water or oil heating bath on top of the hot plate according to the

reaction temperature necessities that are going to take place.

Secure the reaction flask (with the magnet already inserted) with the clamp and adjust its height so the flask is as deep inside the bath it can be without touching the bottom. Once the height has been adjusted, tighten the clamp that secures the flask to the base at the ideal height so there is no danger from it falling.

Now, simply introduce the reflux condenser into the flask mouth, supporting it. Then

secure the clamp holding the reflux condenser, making sure to place it as high up as possible in such a way that when the system is opened once the reaction finishes, or to expel the

1

2

3

4

5

E ntra dade agu a

Sal id ade agua

1) Placa calefactora2) Baño de calefacción3) Matraz de reacción4) Refrigerante de

5

1. Hot plate/stirrer 2. Heating bath 3. Reaction flask 4. Reflux condenser 5. Holding clamps

Water entrance

Water exit


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reagents or solvents, we simply have to loosen the condenser clamp and not move it all along the support.

Once you are sure that the assembly is secure, connect the refrigeration hoses. The one from the water faucet is connected to the condenser’s lower fitting (water entrance), and that connected to its upper fitting (water exit) continues on to the drain. Before beginning heating, slowly open the tap until you see the condenser begin to fill with water. Never open the tap too much, because this increases the danger from flooding were the hoses to detach or break.

The addition of the products can be done from on top of the reflux condenser if they

are liquids, or by lifting it up and directly adding the products to the flask mouth if they are solids.

Once the reaction has finished, disconnect the heat source, loosen the condenser’s

clamp, loosen the clamp from the base supporting the flask while holding it, and raise the entire assembly in such a way that the reaction begins to cool. Now, tighten the flask’s clamp to the base again, raise the condenser, and tighten it again. Only shut the tap water off now, and continue working with the reactions according to how the manual indicates. 3.2) Distillation system

In distillation system assemblies, in which not all pieces are found in the same vertical

plain, two supports must be used along with the necessary clamps to secure the entire system.

Begin by placing the hot plate/stirrer upon the support, or between the legs of it if is a tripod, and the heating bath above it.

Next, by holding the distillation flask with a clamp, introduce it as deep as possible

into the bath without touching the bottom, and afterwards tighten the clamp to the support.

Place the still head is upon the flask’s mouth and the distillation thermometer above it. On some occasions, the thermometer will not have a ground glass joint to introduce into the still head, but rather a threaded plug with an orifice into which the thermometer is introduced. If the system you use is this type, the thermometer must be in place before inserting the still head upon the flask. The plug is unscrewed and the thermometer placed through the rubber

2

3 4

5

6

7 8

9

10

11

En t rad a d e ag u a

Sali d a d e ag ua 1) Placa calefactora2) Baño de calefacció n 3) Matraz de destilació n 4) Pinza de sujeción5) Cabeza de destilaci ó n 6) Termómetro de des t i lació n 7) Refrigerante de des ti l aci ó n8 ) Pinza de sujeción9) Codo10) Matraz de recogid a11) Clips para uniones es m erilad as

1. Hot plate/stirrer 2. Heating bath 3. Distillation flask 4. Holding clamp 5. Still head 6. Distillation thermometer 7. Distillation condenser 8. Holding clamp 9. Elbow 10. Receiving flask 11. Clips for ground joints

Water entrance

Water exit

1


19

gasket at the edge of the still head, adjusting its height in such a way that the thermometer bulb is the same height as the stem through which the vapor passes. The plug is placed again, but without turning it, and the still head is united with the distillation flask. Finish by adjusting the thermometer height, and then firmly tighten the plug holding the thermometer.

To unite the condenser, begin by placing the second support alongside the first,

placing a completely open clamp at an approximate height and inclination. Afterwards, connect the condenser to the still head, and without letting go of it, position the clamp in such a way that the condenser rests upon it. Once this is done, the support clamp is tightened until it secures the condenser. Once this is done, it will be safely fastened without a risk of it falling.

The elbow and receiving flask are connected with a clip, and afterwards the elbow is

united to the condenser with another, in such a way that the assembly is finished.

Lastly, before beginning heating, very slowly open the water tap until a minimum quantity of water passes through the condenser.

When distillation finishes, the heating is stopped and then wait until no condensation

of vapor is observed in the distillation condenser entrance. When this is occurs, first loosen the clip joining the receiving flask with the elbow in such a way that the distillate, which is of great interest to us, is kept safe from possible accidents. Then proceed how the manual indicates.

To disconnect the system, begin by emptying the water from the condenser; for this,

remove the hose from the tap. Generally, this operation is sufficient to ensure emptying, but if not, separate the other hose from the condenser exit and allow a little air to enter. Once the condenser is empty, loosen the two rubber hoses and remove the clip joining the elbow. Then, loosen the clamp joining the condenser and disconnect it. Lastly, disconnect the still head from the distillation flask, and once separated, loosen the plug and take out the thermometer. If this system is equipped with a ground male, first remove the thermometer and the still head afterwards. All material must be washed and rinsed perfectly, and allowed to dry overnight.

Biotechnology 2: Organic Chemistry Isolation and purification

20

Büchner funnel

Kitasato

Vacuum hose

Water aspirator

Tap

All organic reactions finish with the isolation of the reaction product, and if necessary, with its purification. Therefore, it is very important to know how to proceed in each case. We will begin by seeing common isolation techniques, and then comment about those for purification.

1) Isolation of products

The first thing to keep in mind when choosing the method to employ is the physical form of the product we wish to isolate, whether it is in the solid or liquid phase. 1.1) Solid products When the product being isolated is found as a solid along with a solution, the method to follow is filtration with a Büchner.

Filter paper is cut so that all the Büchner holes are covered, but without excess paper climbing up its sides:

The vacuum hose is connected to the Kitasato hose barb, the water tap completely opened, and some solvent that our product is mixed with is added on top of the paper so it moistens and sticks to the Büchner. Once this first liquid has filtered, our mixture of solid and liquid is added to it after being stirred. When all the liquid has been filtered, wash our product with the solvent the lab manual indicates. When the filtration has finished, it is very important to remember always to first disconnect the rubber hose from the Kitasato, and only afterwards close the water tap. If this is done in reverse order, water may enter the Kitasato and, depending upon the filtrate composition, could even be dangerous.

ISOLATION AND PURIFICATION OF PRODUCTS

Paper

Correct Incorrect

Paper


21

1.2) Isolation of liquids or solutions When we want to isolate a liquid within which some type of solid particle exists, we must proceed in two distinct ways. If it is necessary to recover both the solid and liquid, a conical funnel with filter paper

adequately folded into a cone shape is used. Once inserted into the filter interior, the paper is moistened with the washing liquid so the paper totally adheres to its walls.

If the solid does not need to be recovered, pleated filter paper and a Büchner filter are

used. The pleated filter paper must not be previously moistened. Once filtered, a rotavap is usually used to eliminate the solvent. This consists of a bath that allows heating the flask the product is contained within and a serpentine condenser for the water to pass through, cooling the vapors and making them condense. The flask is kept within the rotavap until no condensation from the solvent is observed above the serpentine.


22

2) Purification of products

Once our product is obtained via the isolation method of choice, it will generally have to be purified. Three methods are used most for this: distillation, recrystallization and extraction. Although we must use the method indicated by the manual for each product, a general criterion may be the following: if the product is a liquid, purification can be tried with distillation. If it is a solid, recrystallization is usually tried first. Extraction is an interesting method if our compound and the impurities accompanying it might have different solubilities in aqueous and organic media. For example, if we have an acid, we can make an extraction with an organic solvent and an aqueous solution with a basic pH, in such a way that the acid forms a carboxylate, transforming itself into an ionic compound whose solubility in water will be much greater than the initial acid. Once extraction is carried out, the acidification of the aqueous medium will protonate the acid again, precipitating, and we can isolate it by filtration like how we saw earlier.

With these general criteria, we are going to see what each method consists of. 2.1) Recrystallization

If this purification method is chosen, the first thing that must be done is to search for a good recrystallization solvent, one that dissolves the compound at a hot temperature but not at a cold one. Therefore, collect a spatula tip-size quantity of the product, add it to a test tube and add about one finger’s worth of solvent, then observe if there is dissolution. If it dissolves, the solvent is no good and another must be tried. If it does not dissolve, it must be heated and shaken until the solvent boils, then observe what occurs. If the product does not dissolve at this high temperature it is not any good, and another must be found. If on the other hand, the opposite occurs, and it does dissolve, it must be cooled, even by sticking the tube into ice, until the compound crystallizes.

To try solvents, it is common to begin by trying with water, as it is cheap and non-toxic. If this is unsuccessful, continue with solvents, beginning with those that are inexpensive and not very toxic.

Once a suitable solvent has been found, our product is dissolved using the minimum quantity possible of such solvent and when this solvent is either boiling or total dissolution is observed, it is filtered with a pleated filter and the filtrate cooled. Once the product has crystallized, it is filtered in a Büchner funnel to obtain the solid.

2.2) Extraction

If extraction is the chosen method, our product is dissolved in a solvent, either aqueous or organic, and this solution is added to a decantation funnel. Next, another solvent is added to the funnel, it is covered with a plug, the funnel is placed with the stopcock pointing upwards, and opened to liberate gases. It is then closed, lightly shaken, and opened again to liberate gases. This operation is repeated a few times, after which it can be shaken strongly. After each shaking, opening the stopcock carefully to eliminate gases is convenient; make sure it is not pointed at anybody in case there is a projection of liquid. Once the process has finished, the funnel is placed above the ring, the plug removed, and the phases are allowed to separate. The procedure to follow depends upon which phase the product of interest has remained within, and whether we are trying to eliminate impurities from the phase the product


23

is found in or whether we are trying to remove our product from the phase it was initially found in.

P Agitación

DecantaciónP

I

VaciadoI0 I1

P I1

Adición de

disolventeI1P

Decantación

AgitaciónI2

I

P

I2P

Vaciado

I0 > I1 > I2 > ..... > In

Esquema para eliminar impurezas mediante extracción

Esquema para separar producto mediante extracción

P0 > P1 > P2 > ..... > Pn

Vaciado

I

IAgitación

DecantaciónP

IAdición de

disolventeI

II Vaciado

PDecantación

AgitaciónP0 P1 P2

P1

P1

P2

A common problem with this technique is knowing at all times which phase is the organic and which the aqueous. The easiest way to find this out is by adding a drop of water when the phases are clearly separated, and seeing which phase it joins. Generally, the great majority of organic solvents used in extractions, like ether, ethyl acetate or hexane, have densities less than water, and so they will normally constitute the superior phases. Exceptions to this are halogenated solvents, like chloroform or dichloromethane, that have densities greater than water, and so in extractions with these solvents, they will constitute the inferior phase.

Once the phases are separated, the procedure to follow depends upon which phase is of interest to keep working with, and always keep in mind that it is not advisable to throw anything away until our product has been recovered. If the phase of interest is superior, keep removing the lower one after each shaking and decantation stage, and then add new solvent, until we consider that the purification process is sufficient. If on the other hand, the phase of interest to us is inferior, then there is no other choice besides removing this one first, then removing the filter’s superior phase, and inserting the phase of interest into the filter again, adding new solvent again and repeating the process. Once the extraction has finished, all the phases we have generated are joined into those our product is in, and work is done with them. If the product is in the aqueous phase, we must make it precipitate by adding a suitable reagent (a mineral acid if the product is an acid type or a soda solution if the product is a basic type) in order to be able to isolate the product by filtration afterwards. If the product is in the organic phase, it must be dried with a drying agent, like sodium sulfate or anhydrous magnesium, before eliminating the solvent in the rotavap. For this, such desiccant is added in the organic phase, until observing that there is nonsetting desiccant. Time is allowed to pass, for example ten minutes, and it is filtered next with a pleated filter and conical funnel, into a round tared flask. The filtrate is inserted into the rotavap, the solvent eliminated, and the product weighed directly within the flask.


24

2.3) Distillation The purification process by distillation coincides completely with the distillation

method previously seen, and so it is not repeated here.

Biotechnology 2: Organic Chemistry Reaction yield

25

All chemical reactions have the objective of obtaining the greatest possible quantity of the product of interest to us. This intuitive idea of the greatest quantity is mathematically translated into the calculation of the reaction yield.

This is always defined as the quotient between the actual quantity obtained and the

quantity that should have been had everything gone perfectly. The first value is obtained experimentally once the product is isolated without doing any more than weighing or measuring its volume. Therefore, the maximum theoretical value is that what must be calculated. Two situations must be distinguished in order to do this.

Purification yield

In this case, the product will not undergo any chemical transformation, so the calculation of the maximum quantity that can be obtained is reduced to measure the quantity we purify. Therefore, when calculating the yield, simply divide the quantity of product obtained following purification by the quantity of product put to purification. In such a simple case, it is possible to calculate the yield in any unit, and without the necessity of keeping the compound’s molar mass in mind.

Beginning quantity

Maximum possible quantity

Quantity obtained following

purification

Purification yield

3 mL 3 mL 2.7 mL (2.7/3) x 100 = 90% 4 g 4 g 3 g (3/4) x 100 = 75% 1 mol 1 mol 0.6 mol (0.6/1) x 100 = 60%

As a rule, yields are given without decimals, so it is essential to apply rounding

regulations. For example, if after making the quotient, a value of 50.6% results, the yield is rounded up to 51%, while if the quotient returns a value of 50.4%, it must be rounded down to 50%.

Yield of a chemical reaction

In order to explain the process, we will use an example to clarify. 3.8 g of benzaldehyde are made to react with 1 mL of acetone in a basic medium, obtained by dissolving 2 g of sodium hydroxide in 10 mL of water. Following the reaction, the product is filtered and dried, and 2.7 g of dibenzylideneacetone (1,5-diphenyl-1,4-pentadien-3-one) result. Calculate the reaction yield. The density of acetone is 800 Kg/m3. The steps to follow are the following:

CALCULATING THE REACTION YIELD


26

2.1) Write the reaction, adjust it, and calculate the molar mass of

reagents and products 2.2) Calculate the number of moles per reagent

benzaldehyde

3.80.035 moles

106

mn

M

33

acetone 6 3

800 1 mL 1000 g 1 m0.013 moles

58 1 Kg 10 cm

Kgm V mnM M

2.3) Verify the stoichiometry of the reaction

2 benzaldehyde: 1 acetone → 1 dibenzylideneacetone 2.4) Check which is the limiting reagent

To do this, the number of moles that was previously calculated is divided by the respective stoichiometric coefficient, and the quotient whose value is smaller corresponds to the limiting reagent.

Benzaldehyde: 0.035/2 = 0.0175 Acetone: 0.013/1 = 0.013 → Limiting reagent

The physical significance of the limiting reagent is the following: it is the first reagent to be consumed when a reaction takes place, and so no matter the quantity of the remaining reagents, the reaction cannot continue.

2.5) Calculate the number of theoretical moles of the product obtained

This is done by dividing the stoichiometric coefficient of the product by the stoichiometric coefficient of the limiting reagent and then multiplying by the number of moles of the limiting reagent.

theoretical of the product limiting reagent

theoretical of the product

Stoichiometric coefficient of the productn = n

Stoichiometric coefficient of the limiting reagent

1n = 0.013 moles = 0.013 moles of dib

1

enzylideneacetone

C O

H H 3C C

O

CH3

O

NaOH2 1 1+

Molar mass = 106 Molar mass = 58 Molar mass = 234


27

2.6) Calculate the number of theoretical grams of the product

mn = m = n M = 0.013 234 = 3.042 theoretical grams of dibenzylideneacetone

M

2.7) Calculate the reaction yield

experimental grams 2.7Yield = 100 100 88.8%

theoretical grams 3.04289%

Biotechnology 2: Organic Chemistry Experiment 1: Molecular models

28

OBJECTIVES Build molecular models in order to observe them in three dimensions and try to see the

spatial dimensions of the atoms in a molecule. Try to understand, using models of geometric isomerism, the shapes and absolute

configuration.

THEORETICAL BASIS If we are asked what organic chemistry is, the most common definition we find is that organic chemistry is the chemistry of carbon compounds. There are several characteristics about these carbon compounds that we must know about: -Carbon forms strong bonds with itself as well as with other elements; most of the elements commonly found in organic compounds, aside from carbon, are hydrogen, oxygen, and nitrogen. -Carbon atoms are generally tetravalent. This means that carbon atoms in most organic compounds are connected to adjacent atoms by four covalent bonds. -Organic molecules are three-dimensional and they occupy space. The covalent bonds that carbon atoms make to adjacent atoms are at discrete angles to each other. Depending upon the type of organic compound, these angles may be 180º, 120º, or 109.5º. Such angles correspond to compounds having triple, double, or single bonds, respectively. -Organic compounds may have a limitless variety in their composition, shape, and structure. While the molecular formula indicates the number and type of atoms present in a compound, it says nothing about its structure. Molecular models help obtain a three-dimensional representation of a molecule that shows the sequence wherein atoms are connected and the type of bond. For example, the molecular formula C4H10 can be represented by two different structures: n-butane and 2-methylprone (isobutane). Observe the following figures:

Also, consider the molecular formula C2H6O. Two structures correspond to this formula: dimethyl ether and ethanol (ethyl alcohol).

EEXXPPEERRIIMMEENNTT 11 SSttrruuccttuurree ooff oorrggaanniicc ccoommppoouunnddss.. UUssee ooff

mmoolleeccuullaarr mmooddeellss..


29

In the preceding examples, each structure represents a different compound and each possesses distinct physical and chemical properties. Compounds having the same molecular formula but different structural formulas are called isomers.

Stereochemistry is the part of organic chemistry that deals with three-dimensional structures of molecules. Analyzing the stereochemistry of a molecule enables studying the spatial relationships between one carbon atom and the carbon atoms adjacent to it. Open-chain molecules are able to rotate around single bonds. The different three-dimensional arrangements that the molecule can adopt due to the rotation about C-C bonds produce conformational isomers. A specific conformation is called a conformer. While individual isomers can be isolated, conformers cannot because their interconversion by rotation is very rapid. Conformers may be represented in projections. Such projections try to show the three-dimensional molecules on a flat surface. A sawhorse projection uses thick solid lines to indicate bonds that project outward and forward out of the plane (above the plane of the drawing) and normal dotted lines for backward-projecting bonds (below the plane). All that is on the plane of the paper is drawn with normal continuous lines. Shown next is a sawhorse projection for propane.

One simple alternative to perspective representations is a Newman projection. In this type, one of the molecule’s C-C bonds is observed head on, with the leading carbon atom covering the one directly behind it, but the bonds leading out from each of them are clearly visible. The leading carbon atom is represented by the point of intersection of the three bonds attached to it, one of which is usually drawn vertically and pointing upwards. The trailing carbon atom is


30

a circle and its bonds are drawn leaving the outer edge of that circle. In Newman projections, conformers are easily shown. Two conformers for propane are shown in the following figure.

Other stereoisomers include geometric isomers, which are those that are generated due to the rigidity of the double bond, and are of two classes: cis (or Z), with the two higher-priority substituents on the same side, and trans (or E) with the two higher-priority substituents on opposing sides. In order for geometric isomers to exist, the groups linked to the same double-bond carbon atom must be different. Cis and trans isomers are also present in cyclic compounds.

One final type of stereoisomerism remains, that which is related to chirality. Chemical compounds possessing non-superimposable mirror images of each other (such as our hands) are said to be chiral. The pair of mirror images that do not superimpose are called enantiomers. Chirality generally arises from the presence of an asymmetric carbon atom (chiral carbon atom). An asymmetric carbon atom is that presenting four different atoms or groups attached to it. The arrangement of these groups around chiral carbon is called absolute configuration and can be described as either (R) or (S). Molecules that are mirror images contain identical physical properties. The lone difference that enantiomers possess is that they rotate the plane of linearly polarized light in opposite directions. Enantiomers possess optical activity. What does optical activity consist of? Natural light possesses waves that vibrate in all directions. Plane polarized or linearly polarized light is formed by waves that only vibrate in one plane. When polarized light passes through a solution that contains an optically active compound, it makes the plane of light rotate a certain angle either towards the right or the left. If a molecule presents this characteristic, it is said to possess optical activity. Another type of representation, the Fischer projection, is used often, above all for substances that contain many chiral centers. In each center of chirality in a Fischer projection, horizontal lines are considered to be leaving the page, while those vertical are behind it.


31

d

C

c

a

b

d

b

a c

d

b

ca

In order to establish the absolute configuration of chiral carbon, one must use the Cahn-Ingold-Prelog priority rules.

MATERIAL Molecular models

EXPERIMENTAL PROCEDURE

Questions about conformational isomers 1. With the molecular models, depict ethane in staggered and eclipsed conformations. Draw the wedge formulas and Newman projections. Represent the energy profile for ethane conformers.

2. With the molecular models, depict 1,2-dichloroethane. Obtain the singular conformations and represent them. Draw the Newman projections and arrange them in terms of relative stability.

3. With the molecular models, depict butane. Obtain the singular conformations, represent them, and make the Newman projections. Represent the energy profile for butane conformers.

Questions about stereoisomers 4. Which of the following compounds exhibit geometric isomerism? Draw the possible isomers.

a) CH3-CH=CH2

b) CH3-CH=CH-Cl

c) CH3-CH=CH-CH3

5. With the molecular models, depict 2-chloropropane and confirm that its mirror image is superimposable.

6. With the molecular models, depict 2-chlorobutane. Draw the enantiomers and assign the configuration to the stereocenters. Confirm that in order to transform a compound into its enantiomer, bonds must be broken.

7. With the molecular models, depict the enantiomers of lactic acid (2-hydroxypropanoic). Draw the sawhorse and Fischer projections and assign the configurations.


32

8. With the molecular models, depict the isomers of the following compounds. Draw them and assign the E/Z configuration of the double bonds. Confirm that the isomers are distinct compounds, and that in order to transform from one to another, bonds must be broken.

a) 1-propene

b) 2-pentene

c) 2-methyl-2-butene

d) 1-chloro-2-methyl-1-butene

9. How many stereocenters does 2-bromo-3-chlorobutane have? Depict them with the molecular models and indicate the relationship they maintain. Confirm that they are not superimposable. Draw them and assign the configurations to the stereocenters. Make the Newman and Fischer representations.

10. How many stereocenters does 2,3-dychlorobutane have? Depict them with the molecular models and indicate the relationship they maintain. Are they all optically active? Reason your answer. Confirm that they are not superimposable. Draw them and assign the configurations to the stereocenters. Make the Newman and Fischer representations.

11. With the molecular models, depict the stereoisomers of 2-chlorocyclobutanol. Draw them and assign the R or S configuration. Indicate which are enantiomers and which are diastereoisomers, and differentiate between cis and trans isomers.

12. With the molecular models, depict cis- and trans-1,2-dichlorocyclobutane. Indicate which stereoisomer has a plane of symmetry and which an axis of symmetry. Produce the mirror images and confirm whether they are superimposable. Are they all optically active? Draw them and assign the configurations of the stereocenters.

13. With the molecular models, depict cis- and trans-1,3-dychlorocyclobutane. Do they have any element of symmetry? Produce the mirror images and confirm whether they are superimposable. Are they optically active? Draw them.

14. Write the formulas of five chiral molecules that did not appear in the preceding exercises. Use the molecular models to try to depict them.

Biotechnology 2: Organic Chemistry Experiment 2: Recrystallization

33

OBJECTIVES Purification of a solid compound by recrystallization.

THEORETICAL BASIS

Recrystallization is one of the most suitable and simple procedures for purifying solid substances still today.

In general, purification by recrystallization is based on the fact that most solids are more soluble in a hot solvent than in a cold one. The procedure consists of:

i) Dissolving the solid to be purified in the smallest possible quantity of hot solvent (generally to boiling);

ii) Filter the hot solution to eliminate insoluble particles and impurities (in case they exist); iii) Allow the hot solution to cool, allowing the dissolved substance to crystallize this way; iv) Separate the crystals from the water stock (dissolution). Ideally, the majority of the

desired substance must be separated in crystalline form while all the soluble impurities must remain dissolved in the water stock.

The most important factor for this purification technique is obviously choosing a suitable solvent. Two basic characteristics must be met:

1) A large difference in solubility must exist between low (room temperature or 0 oC) and

high temperatures (boiling) in the compound being purified. Logically, this property will most easily be met by solvents having relatively high boiling points, allowing for a large temperature interval when the solutions are cooled.

2) It must be chemically inert in relation to the substance being purified.

If two or more solvents are suitable for recrystallizing a compound, the final choice

will depend upon factors like toxicity, handling ease, flammability, and/or cost.

The most common solvents used in recrystallization are shown in the table in decreasing order of polarity.

EEXXPPEERRIIMMEENNTT 22 RReeccrryyssttaalllliizzaattiioonn


34

COOH

OH

SOLVENT BOILING POINT (ºC) CHARACTERISTICS

Water 100 Used in any situation

Methanol 64.5 Flammable. Toxic

Ethanol 78 Flammable

Acetone 56 Flammable

Ethyl acetate 78 Flammable

Glacial acetic acid 118 Not very flammable. Irritating vapors

Dichloromethane 41 Not flammable. Toxic

Chloroform 61 Not flammable. Toxic vapors

Diethyl ether 35 Flammable

Benzene 80 Flammable. Toxic

Dioxane 101 Flammable. Toxic

Carbon tetrachloride 77 Not flammable. Toxic

Cyclohexane 81 Flammable

Table 1. Most common solvents used in recrystallization.

It is frequent to find that a substance is too soluble in one solvent and not soluble enough in another to perform recrystallization. In this case, a mixture of both solvents (as long as they are always miscible) is usually used with good results.

The compound being recrystallized in today’s experiment is salicylic acid. Salicylic acid is a key additive in many skin care products designed for treating acne, psoriasis and calluses (hardening of the skin due to persistent pressure), goose bumps and warts. It treats acne by causing skin cells to fall off more easily, which prevents pores from clogging. This effect on skin cells also makes salicylic acid be an active ingredient in several shampoos designed for treating dandruff. Salicylic acid’s medicinal properties (primarily to reduce fever) have been known since 1763.

The recrystallized salicylic acid will be subsequently used in the aspirin synthesis experiment.

MATERIAL Aluminum bucket Rubber cone Funnel Büchner funnel 250 mL Erlenmeyer flask Test tube rack Kitasato Nut

Filter paper Stirring piece (magnet) Clamps Plastic Pasteur pipettes (4) Hot plate/stirrer Support Test tubes (4)


35

REAGENTS AND SOLVENTS ●Acetone ●Ethanol ●Salicylic acid ●Hexane ●Distilled water

PRECAUTIONS Acetone: Danger associated with inhalation, ingestion and by absorption through

the skin. Irritant. Contact with eyes may cause severe injury. Salicylic acid: Danger associated with inhalation, ingestion and by absorption through

the skin. Irritant. Ethanol: Irritating to the eyes and skin. Its ingestion may cause nausea, vomiting

and intoxication. Prolonged use may have severe consequences for life. Hexane: Danger associated with inhalation, ingestion and by absorption through

the skin. Irritant. Prolonged exposure may result in infertility.

EXPERIMENTAL PROCEDURE 1) Place the bucket full of water being heated upon the hot plate. 2) Choose the most suitable solvent for conducting recrystallization.

2.1) Place a spatula tip-size quantity of salicylic acid that the 250 mL Erlenmeyer holds into each of the 4 test tubes.

2.2) To each test tube, add 1 mL (approximately) of the solvents provided to test their solubility: hexane, acetone, ethanol and water (a different one for each test tube).

2.3) Shake each one vigorously for a few seconds and observe the degree of dissolution at room temperature.

a) If the problem solid completely dissolves, or does so almost completely, it is

not suitable for recrystallization. b) If, on the other hand, the problem solid is insoluble or not very soluble,

proceed by heating the bucket (that was previously filled with water and begun heating) until the solvent boils. Hold the test tube with a clamp and do not aim it at your partner while heating. If the solid dissolves, the solvent is suitable for recrystallization.

The suitable solvent will be that which totally dissolves the solid when heated, as well as when an abundant formation of crystals is observed after the test tube has been cooled to room temperature or is in an ice bath. NOTE: In the event that the compound does not crystallize when cooled, it is sometimes useful to scrape the test tube walls with a spatula. This may induce crystallization (very small fragments of glass can act as crystallization germs).


36

3) Make your solvent choice known to a person responsible in the laboratory for recrystallizing the compound.

4) Recrystallize the compound. 4.1) Weigh the product that remains in the 250 mL Erlenmeyer. 4.2) Add 100 mL of the chosen solvent. 4.3) Heat the Erlenmeyer, which is held by a clamp bound to the support, directly on the

hot plate/stirrer (without the water bucket) until the solvent boils. 4.4) If the solid has not dissolved once the solvent reaches its boiling point add more

solvent in 5-mL portions a maximum of 4 times. 4.5) When the solid has dissolved or has practically done so, proceed by filtering the hot

solution as rapidly as possible with a pleated filter and funnel, like Figure 1 indicates. If the solid is completely dissolved this step is not necessary.

4.6) Next, allow the solution to slowly cool in an ice bath. The crystals’ sizes generally depend upon the rate of cooling. The higher this is the smaller the crystals are.

4.7) Lastly, all that remains is to separate the crystals from the water stock. This operation

is performed by vacuum filtration in a Büchner, just as the figure indicates. To recover the last crystals remaining in the Erlenmeyer, a small quantity of the solvent employed is usually added when cold and poured into the Büchner, which moreover allows washing the crystalline mass.

NOTE: Using a water aspirator

1) Completely open the tap.

2) Join the Kitasato to the water aspirator with the rubber vacuum hose.

3) Pour the crystalline mass into the Büchner.

4) Wait for all liquid to filter.

5) Disconnect the rubber hose from the Kitasato.

6) Close the tap.

Büchner funnel

Kitasato

Vacuum hose

Water aspirator

Tap


37

Now remove the crystals from the Büchner and place them on dry filter paper. Allow them to dry overnight before weighing them and calculating the recrystallization yield.

QUESTIONS

1) Complete the following table in the notebook.

2) Weigh the crystals obtained following recrystallization. 3) Calculate the recrystallization yield.

SOLUBILITY SOLVENT

HEXANE ETHANOL ACETONE WATER

COLD SOLUBILITY

HOT SOLUBILITY

Biotechnology 2: Organic Chemistry Experiment 3: Distillation

38

OBJECTIVES Use of a simple distillation device. Estimation of the purity of the alcohol obtained.

THEORETICAL BASE

Simple distillation is a separation method that consists in heating a mixture in order to evaporate its volatile substances and subsequently condense them by cooling into a collecting recipient. Simple distillation is used when the mixture contains only one volatile component, or when there are two whose boiling points are very different.

In order to obtain complete separation of two substances, successive distillations must

take place with both the distillate and the distillation residue. In practice, this process employs a fractionating column where vapors are condensed and distilled repeatedly, achieving that the most volatile component exits the column at the upper end while the least volatile is condensed on the distillation trays. This process is known as fractional distillation.

Ethyl alcohol, or ethanol, which is the compound being distilled in today’s

experiment, is not only the oldest synthetic organic chemical employed, but it is also one of the most important in the chemical industry. It is used as a solvent for lacquers, varnishes, perfumes and condiments, as well as a medium for chemical reactions and recrystallization.

Ethyl alcohol is prepared as much for hydration of ethylene (a sub product from

“cracking” petroleum) as it is for fermentation of sugar molasses (primarily from sugar cane). Alcoholic beverages contain ethyl alcohol. It is classified medicinally as a hypnotic (it produces drowsiness) and is less toxic than other alcohols. For example, methanol is very poisonous: its ingestion, inhalation for prolonged periods, as well as contact with the skin can lead to blindness and even death.

Most industrial alcohol is denatured to avoid paying taxes, because then it becomes

inadequate for producing spirits and is not subject to such high fees. As for denaturing agents, those most commonly used are methanol and high-octane gasoline. Pure ethyl alcohol (absolute alcohol), used for chemical purposes, is strictly controlled by governments.

In any of the acquisition methods, ethanol results mixed with water and is then

concentrated by simple or fractional distillation. However, the lowest boiling point corresponds to a binary azeotrope (a solution that boils at a constant temperature, producing vapor at the same composition as the liquid), which contains 95% alcohol and 5% water, and is unable to become concentrated above this composition no matter the fractional column’s

EEXXPPEERRIIMMEENNTT 33 DDiissttiillllaattiioonn ooff wwiinnee


39

efficiency. The acquisition of absolute ethanol, although more expensive, can be achieved by treating the preceding azeotrope with metallic magnesium. Water reacts with this and forms insoluble magnesium hydroxide, which is separated from the alcohol.

MATERIAL ● Elbow ● Clamps (2) ● Still head ● Hot plate/stirrer ● Crystallizing dish ● 25mL graduated cylinder ● Silicone gaskets (2) ● Distillation condenser ● 100 mL flask, B29 ● Supports (2) ● 250 mL flask, B29 ● Plastic plug, B29 ● Nuts (2) ● Thermometer ● Stirring piece (magnet)

REAGENTS AND SOLVENTS Table wine

PRECAUTIONS Ethanol: Irritating to the eyes and skin. Its ingestion may cause nausea, vomiting and

intoxication. Prolonged use may have severe consequences for life.

EXPERIMENTAL PROCEDURE Add 100 mL of table wine to a 250 mL flask. Assemble the distillation apparatus like the figure shows. ASK A PERSON RESPONSIBLE IN THE LABORATORY TO REVIEW THE ASSEMBLY. The wine is heated and stirred with the magnet.

As it is heated, ethanol vapors will condense on the thermometer and begin to produce

a constant trickle. The condensed vapor will displace toward the condenser (cooled with

2

3 4

5

6

7 8

9

10

11

En t rad a d e ag u a

Salid a d e ag ua 1) Placa calefactora2) Baño de calefacci ó n 3) Matraz de destilació n 4) Pinza de sujeción 5) Cabeza de destilaci ó n 6) Termómetro de des ti laci ó n 7) Refrigerante de d es til ació n8 ) Pinza de sujeción9) Codo10) Matraz de recog i da11) Clips para uniones es m eril ad as

1. Hot plate/stirrer 2. Heating bath 3. Distillation flask 4. Holding clamp 5. Still head 6. Distillation thermometer 7. Distillation condenser 8. Holding clamp 9. Elbow 10. Receiving flask 11. Clips for ground joints

Water entrance

Water exit

1


40

countercurrent water) until reaching the receiving flask. When a constant trickle exists in the receiving flask, record the thermometer’s temperature (it will be between 78-85 ºC). This value corresponds to the boiling point of ethanol at normal pressure.

The distillation is finished when the person responsible in the laboratory says so. The

contents in the receiving flask are poured into a clean and dry graduated cylinder to measure the volume of distilled alcohol.

To measure the actual quantity of alcohol, wait for the person responsible for the

experiment to pass by with an alcoholometer. This alcoholometer is introduced into a 250 mL graduated cylinder within which the distilled alcohol has previously been added to and leveled off with water to 200 mL. These devices are prepared to measure in 100 mL volumes, and as we do so with a volume of 200 mL, the degree of alcohol in the wine is directly read in the alcoholometer’s rod and multiplied by two.

QUESTIONS 1) Fill in the following results table in your laboratory notebook:

Distillation temperature (ºC) Ethanol-distilled water mixture volume (mL) Degree of alcohol in the distilled mixture (% by volume)

Biotechnology 2: Organic Chemistry Experiment 4: Liquid-liquid extraction

41

OBJECTIVES

Separating organic compounds based on their acidic or basic properties using the extraction technique.

THEORETICAL BASIS

Liquid-liquid extraction is the distribution of a compound or compounds between two inmiscible solvents. In order to be used effectively as a separation technique, the compounds must show large solubility differences between the two solvents. This can be obtained by exploiting the acid-base properties of the organic compounds.

Organic acid compounds: carboxylic acids and phenols. Organic basic compounds: amines. Organic neutral compounds: aldehydes, ketones, esters, amides, alcohols, ethers,

nitriles.

Extraction is a technique utilized for separating an organic compound from a reaction mixture. Generally, when there is an organic reaction, an aqueous treatment is subsequently necessarily, isolating the organic compounds from these media by extraction. The procedure consists in shaking the aqueous solution with an inmiscible organic solvent in water (for example, CH2Cl2, CHCl3, CH3CO2Et, EtOEt, etc.). The organic compounds will dissolve better in the organic solvent than in the aqueous solution, while the inorganic salts will do so in the aqueous phase.

This technique is based on the different distribution coefficient that the solute presents between the two solvents (water and organic solvent). Therefore, with the aim of assuring the product’s total extraction, it is better to extract several times with not very large quantities of solvents instead of one single time with a large quantity of solvent.

The requirements the extraction solvent must meet are:

a) To be inmiscible in water. b) To dissolve the solute that is wanted extracted.

EEXXPPEERRIIMMEENNTT 44 LLiiqquuiidd--lliiqquuiidd eexxttrraaccttiioonn


42

PRACTICAL ASPECTS a) Manipulating the decantation funnel The decantation funnel is suitable for carrying out this operation. Its stopcock must be

greased and the plug perfectly adjusted to prevent losses. The decantation funnel is secured to a support by a metallic ring. An Erlenmeyer is placed below it and a conical funnel above (see Figure 1a). Add the organic solution containing the products to extract and then the aqueous phase

in which the different compounds are going to be extracted. Take away the conical funnel and position the plug. Separate the funnel from the ring, and then with both hands, one holds the plug and

the other manipulates the stopcock (see Figure 1b). With the funnel tipped on its side, shake lightly to mix the phases. While holding the plug with one hand, open the stopcock to allow gases to escape. Once you have done this a few times, shake it vigorously and then allow it to rest on

the support. Remove the plug and wait for the phases to separate. The lower phase is allowed to exit from the lower portion, while the superior is

emptied by the upper one. The organic phase will be either up or down, depending upon the chosen solvent’s density. For example, solvents denser than water, like CH2Cl2, CHCl3 and Cl4C will be in the lower phase. Solvents less dense than water, like CH3CO2Et, benzene, toluene or ethyl ether, will appear in the upper phase. In any case, if you are unsure which one is the organic phase, you may add a little water and observe which phase increases.

Figure 1a Figure 1b


43

In this experiment, a mixture composed of salicylic acid and ethyl p-aminobenzoate, whose compounds are acidic and basic, respectively, will be separated.

MATERIAL Metallic ring Büchner funnel Aluminum bucket Rubber cone Conical funnel 250 mL decantation funnel 250 mL Erlenmeyer flask 100 mL Erlenmeyer flask (2)

50 mL Erlenmeyer flask (2) Kitasato Nuts (2) Filter paper pH paper 4 plastic Pasteur pipettes 50 mL graduated cylinder Support

REAGENTS AND SOLVENTS Ethyl acetate Salicylic acid Water Ice Ethyl p-aminobenzoate

8% HCl solution 16% HCl solution 10% NaOH solution 20% NaOH solution

PRECAUTIONS Ethyl acetate: Danger due to ingestion. Irritant. Very flammable. Chlorohydric acid: Extremely corrosive. Danger due to inhalation and absorption.

Irritating to the eyes and skin. Salicylic acid: Danger due to inhalation, ingestion and absorption via the skin.

Irritant. Ethyl p-aminobenzoate: Irritating to the skin, eyes and respiratory system. Sodium hydroxide: Very corrosive. Causes severe burns, above all to the eyes. Very

dangerous due to inhalation, ingestion, and contact with the skin.

SALICYCLIC ACID

COOH

OH NH2

O

C2H5

ETHYL P-AMINOBENZOATE


44

EXPERIMENTAL PROCEDURE 1. Pour 30 mL of the problem solution into a decantation funnel. 2. Separation and isolation of the basic compound (see diagram on page 45)

2.1. Add 15 mL of 8% HCl to the decantation funnel containing the problem organic

phase and perform a first extraction (see practical aspects) Make sure you do not point this at any of your classmates when opening the stopcock to release pressure. Do not point it at any teachers, either. Empty aqueous phase I into an Erlenmeyer, and proceed to extract organic phase I again with another 15 mL of 8% HCL. Pour aqueous phase II again into the same Erlenmeyer that contains aqueous phase I. DO NOT THROW OUT ORGANIC PHASE II.

2.2. In an ice bath, cool the acidic aqueous phase (aqueous phase I + aqueous phase II), and slowly add 20% NaOH until the pH becomes basic (pH paper blue in color) and precipitate the ethyl p-aminobenzoate. Leave the Erlenmeyer in the ice bath for 10-15 minutes to allow for precipitation of the amine. An oil sometimes appears first and solidifies over time. Filter the solid at a reduced pressure with a Büchner funnel, and wash the two portions with 4 mL of water. Place the crystals on a piece of filter paper and allow them to dry overnight to be able to calculate the yield.

3. Separation and isolation of the acidic compound (see diagram on page 46)

3.1. Pour organic phase II that resulted from the previous extraction into the decantation funnel, and perform two extractions using 15 mL of 10% NaOH each time. Collect aqueous phases III and IV from the basic extractions into an Erlenmeyer. DO NOT THROW OUT ORGANIC PHASE IV.

3.2. Cool the Erlenmeyer in an ice bath and slowly add sufficient 16% HCl to reach an

acidic pH (pH paper red in color) and precipitate the salicylic acid. Leave the Erlenmeyer in the ice bath for 10-15 minutes to allow for precipitation of the acid. Filter the solid at a reduced pressure with a Büchner funnel, and wash the two portions with 4 mL of water. Place the crystals on a piece of filter paper and allow them to dry overnight to be able to calculate the yield.

QUESTIONS 1. Weigh the quantities of each of the products obtained. This will be done the following

day. 2. Calculate the yield for each of the products obtained knowing that the concentration of

each of the reagents in the problem solution was 33 g/L for salicylic acid and 49,5 g/L for ethyl p-aminobenzoate.


45

30 mL solution problem

1 5 m L H C l 8 %

Organic phase

Aqueous phase

Shake and leave still

H 3N C OEt

O

Aqueous phase I 1 5 m L H C l 8 %

Organic phase I

Shake and leave still

Aqueous phase II Aqueous phase II Aqueous phase I

Solution NaOH 20%

H3N C O Et

O +NaOH H2N C OEt

O

+ H 2O + N aC lprecipitate

Büchner filtration and allow to dry

C l

Organic phase I

H3N C OEt

OCl

C l

Basic pH

Organic phase II

ISOLATION OF THE BASIC COMPOUND


58

15mLNaOH 10%

Organicphase II

Aqueousphase

Shake and

leave still

Aqueous phase III

15mLNaOH 10%

Shake andleave still

Aqueous phaseIVAqueous IVAqueous III

HCl 16%Solution

+ HCl + H2O + NaCl

precipitate

Büchnerfiltrantion and

allow to dry

Organicphase II

COO

OH

COO

OH

COOH

OH

ISOLATION OF THE ACID COMPOUND

Na

Organicphase III

COO

OH

Na

Organicphase III

Organicphase IV

Na

Acid pH

Biotechnology 2: Organic Chemistry Experiment 5: Thin layer chromatography

58

OBJECTIVE Determine the purity of a group of compounds and calculate their Rf.

THEORETICAL BASIS Chromatography is a technique that permits separating the components from a mixture due to the influence of two opposing effects: Retention: the effect exerted on the mixture components by a stationary phase. Displacement: the effect exerted on the mixture compounds by a mobile phase.

The mixture to separate is deposited in the stationary phase, and during the mobile phase, it moves through the system, with the components of the mixture displacing at distinct velocities that depend upon the magnitudes of their relative interactions with both phases. This migration phenomenon during the stationary phase is called elution. Solid-liquid adsorption chromatography is characterized by employing a solid stationary phase (adsorbent) polar in nature and a liquid mobile phase (eluent). The stationary phase is constituted by a finely granular polar solid. Its surface contains polar active centers capable of adsorbing polar molecules basically thanks to dipole-dipole interactions or hydrogen bonds. The adsorbent used most is silica gel (SiO2 . xH2O), whose interactions are established between surface SiOH and Si-O-Si groups and the polar functional groups of the organic compounds.

The mobile phase is constituted by a solvent in which the components of the mixture must be at least partially soluble. A compound’s elution rate increases by increasing the solvent polarity.

O

SiO

O

O

H

SiSi

OSi

OH

O

O

OR'R

RO

H

SiO

R X

Silica gel particle

EXPERIMENT 5 Thin Layer

Chromatography


58

Order of eluent strength Hexane dichloromethane diethyl ether ethyl acetate acetonitrile methanol water

Retention can be explained on the basis of the competition established between the solute to separate and the molecules from the mobile phase by adsorbing the active centers from the stationary phase. The more polar the compound is, more strongly adsorbed will it remain, and need a more polar eluent to be displaced. As such, the polar compounds will advance more slowly through the stationary phase, separating from those less polar, which will advance more quickly due to their weak adsorption.

General elution order of organic compounds on polar adsorbents:

Alkanes alkenes ethers halogenated hydrocarbons aromatic compounds aldehydes and ketones esters alcohols carboxylic acids

THIN LAYER CHROMATOGRAPHY (TLC)

This is a simple and very useful laboratory technique. It is used to learn the number of components in a mixture, control the progress of a reaction, check the effectiveness of purification, and even for identifying substances by comparison with a standard. The adsorbent is deposited on a rigid plate, forming a thin uniform layer. The mixture being analyzed is deposited with a capillary a short distance from the bottom edge of the plate, and it is introduced into a beaker that contains the mobile phase, which ascends by capillary action, moving the compounds at different rates. When the solvent front is near the top edge of the plate, it is removed from the beaker and allowed to dry. If the compounds absorb visible light, they will be directly visible. Chromatographic plates usually have a fluorescence indicator that permits visualizing compounds that absorb ultraviolet light (UV). The indicator absorbs UV light and emits visible light. The presence of a compound that absorbs UV prevents the indicator from absorbing light, and appears as a dark stain on the plate. If the compounds absorb neither visible nor UV light, visualization agents that react with the adsorbed compounds, producing colored compounds, must be resorted to. The most common visualizing agents used are iodine (for unsaturated and aromatic compounds), phosphomolybdic acid (for easily oxidizable compounds), ninhydrin (for amines, amino acids, and sugars), 2,4-dinitrophenylhydrazine (for aldehydes and ketones), and potassium permanganate (for unsaturated compounds and alcohols). The relationship between the distance traveled by a given compound and that by the solvent, from the origin of the chromatogram, is known as the retention factor and abbreviated as Rf (see Figure 1). Its value is constant for each compound under specific chromatographic conditions (adsorbent, eluent, temperature, size of the beaker, etc.). The more polar a compound is, the greater it will be retained, and the smaller its Rf will be. For the same compound, the more polar the eluent is, more rapidly will it travel, and the greater its Rf.


58

Choosing an eluent in which the mixture components have an average Rf around 0.3-0.5 is recommended, and the search for an ideal eluent requires testing with various solvents having different polarities or mixtures. When a compound elutes with an Rf either less than 0.2 or greater than 0.7, it could be that what appears to be a unique compound is actually a mixture of various ones. In such cases, it must be changed for another solvent more or less polar, respectively. For scarcely polar compounds, which move from the origin very easily, an apolar solvent, such as hexane, must be used. In cases of medium-polarity compounds, using hexane and ethyl acetate mixtures in different proportions is advisable.

Figure 1

MATERIAL 3 fine glass capillaries Beaker Silica gel plate 4 test tubes REAGENTS AND SOLVENTS Phthalocyanine Perylene tetraester Phenanthroquinone Hexane Ethyl acetate PRECAUTIONS Ethyl acetate: Danger due to ingestion. Irritant. Very flammable. Hexane: Irritant for the respiratory system and skin. Central nervous

system depressant. EXPERIMENTAL PROCEDURE TLC will be performed with three products. Such products are three pigments synthesized

in the research laboratory.

Origin

X AX B

Y

AB

Rf (A) =

XA

Y

XB

YRf

(B) =

Rf (B) Rf

(A)

B is more polar than A

Solvent front


58

The first molecule is a phthalocyanine of zinc, the second a perylene tetraester, and the third a diketone of phenanthrene, phenanthrenequinone.

With a pencil and without pressing very hard, make the following marks on the TLC plate.

Put 1 mL of the green solution into a test tube. Put 1 mL of the yellow fluorescent solution into a test tube. Put 1 mL of the yellow solution into a test tube. Introduce a fine glass capillary into test tube 1, wet it with the solution inside it, and place

a few drops on stain number 1 that you marked on the chromatography plate (see Figure 1a). Do the same with the remaining two test tubes with their respective contents with the other two capillaries.

After allowing the solvents to evaporate on the plate, place it inside the glass beaker, within which a small quantity of eluent has been added (the depth of the eluent should be approximately 0.5 cm). In any case, the surface of the eluent must always be below the line upon which the products were placed on the chromatographic plate (see Figure 1b). Capillary action causes the eluent to rise rapidly, and if the product is colored, a stain from

N

N N N

N N

N

N

Zn

O

O

OO

OO

O

O

144 176 8 8Chemical formula:

Molecular Weight.: 2212.42

C H N O Zn

48 60 8

Mol. W t.: 764.9852

C H O14 8 2

M ol. W t.: 208.2121

C H O

1. Phthalocyanine 2. Perylene tetraester 3. Phenanthrenequinone


58

the ascending product will also be visible; in cases with a mixture of two colored products and a suitable solvent, two stains will be visible.

Before the leading edge of the solvent reaches the other edge of the plate, remove it from the beaker and use a pencil to mark the levels reached by the eluent. Allow the solvent to completely evaporate from the plate, place it under an ultraviolet lamp, and use a pencil again to mark the stains of the observed products. Then, the retention factor (Rf) of the compound is calculated, which is the relationship between the distance the compound advances (A or B) and that reached by the solvent (Figure 2).

for substance AA

CfR

Figure 2

Rf is a physical property of the compound under given conditions of the stationary phase conditions and of the composition of the mobile phase.

Figure 1a

Figure 1b

Solvent edge

Substance B

Substance A B C

A

Initial solvent level

1 cm

Problem substance stain (less than 1 mm in diameter)


58

Example calculation of the Rf for substance A: we use silica gel as stationary phase and ethyl acetate as mobile phase.

A 11R 0.27

C 41f

For the three samples, the following will be used as eluents:

Hexane

Hexane/Ethyl acetate: 10/1



Ethyl acetate

C = 41 cm A = 11cm

RfA = 0.27 (SiO2, AcOEt)

Biotechnology 2: Organic Chemistry Experiment 6: Synthesis of aspirin

58

OBJECTIVE Acetylation and purification of an organic compound.

THEORETICAL BASIS

The history of aspirin began on June 2, 1763 when Edward Stone, a clergyman, presented a paper to the Royal Society of London concerning the treatment of agues (used to reference fevers associated with malaria) using extract from willow bark (this does not actually cure these ailments, it just reduces fever symptoms). Almost a century later, a Scottish doctor discovered that these same extracts relieved symptoms from acute rheumatism. It was finally discovered that the extract from willow bark (the same as a specific plant that grows on prairies, the Spirea) possesses analgesic properties to relieve pain, is antipyretic (reduces fever), and is anti-inflammatory.

Shortly afterwards, organic chemists were able to isolate and identify the active

principal in these extracts, which they named salicylic acid (salix is Latin for willow). From then on, it was possible to produce large quantities of these products chemically for medical uses. However, it was soon confirmed that its use was seen as being limited due to its acidic character (it produced irritation in the membranes of the mouth, esophagus and stomach). The first solution to this problem was to use the sodium salt of acid, but this had to be discarded because this product has an unappealing flavor.

In 1893, Felix Hoffman, a chemist at the Germany company Bayer, designed a route

for obtaining large quantities of acetylsalicylic acid. It was soon discovered that this compound possessed all the medicinal properties of the precursor acid without its disadvantages (irritating to mucous membranes or questionable flavor). The company decided to call this compound aspirin, with a for acetil and spir for the Latin word for spirea (spiraea). Aspirin tablets contain approximately 0.32 g of acetylsalicylic acid, pressed with a small quantity of starch to give them some cohesion.

Salicylic acid (o-hydroxybenzoic or 2-hydroxybenzoic) is a difunctional compound

and can experience reactions typical of the carboxylic and phenolic groups. In this experiment, we will deal with the reactivity of the phenolic group. During the acetylation of the salicylic acid, a small quantity of polymer forms due to the difunctional character within the very molecule. Acetylsalicylic acid reacts with sodium bicarbonate to form the corresponding sodium salt, soluble in water. The purification of aspirin can be justified due to the difference in behavior between the salt and the polymer. However, the most common impurity in the proposed synthesis is the very acid started with, due to incomplete acetylation or partial hydrolysis during its isolation.

EEXXPPEERRIIMMEENNTT 66 SSyynntthheessiiss ooff aassppiirriinn ((AAcceettyyllssaalliiccyylliicc aacciidd))


58

In this experiment, an ester will be obtained (acetylsalicylic acid) by reaction of a phenol with an anhydride in the presence of an acid catalyst.

(Salicylic acid) (Acetic anhydride) (Acetylsalicylic acid)

MATERIAL Aluminum bucket Rubber cone Büchner funnel 2 rubber gaskets Test tube rack Kitasato 100 mL round bottom flask, B-29 2 nuts Filter paper

Stirring piece (magnet) 2 clamps 3 plastic Pasteur pipettes Hot plate/stirrer 50 mL graduated cylinder Reflux condenser Support Plug, B-29 4 test tubes

REAGENTS AND SOLVENTS Salicylic acid Concentrated sulfuric acid Distilled water Acetic anhydride Commercial aspirin 1% FeCl3 solution Phenol Ice

C

O

OH

OH

CH3 CO

OC

OCH3

C

O

OH

O C

O

CH3

+H2SO4

H2O

ácido salicílico anhídridoacético

ácido acetilsalicílico

+ CH3-COOH


57

PRECAUTIONS Salicylic acid: Danger associated with inhalation, ingestion and by absorption

through the skin. Irritant. Sulfuric acid: Extremely corrosive and toxic. Very dangerous if inhaled, ingested or

makes contact with the skin. Causes severe burns. Prolonged exposure may cause cancer.

Acetic anhydride: Toxic. Corrosive. Dangerous if inhaled. Irritating if it makes contact with the eyes.

Sodium bicarbonate: Irritating for the eyes. Phenol: Highly toxic if inhaled. It can be absorbed through the skin. Iron trichloride: Corrosive. Dangerous if inhaled, ingested or makes contact with the

skin.

EXPERIMENTAL PROCEDURE 1) Place 4.0 g of salicylic acid and 10 mL of acetic anhydride (97%; d=1,082g/mL) into a 100

mL round-bottom flask, and constantly shake the mixture to prevent a paste from forming. 2) Add 5 drops of concentrated sulfuric acid and connect the flask to a reflux condenser. 3) When all the salicylic acid has dissolved, heat the mixture in a water bath for 15 minutes

(set the plate to 100 ºC). 4) After allowing it to dry at room temperature, acetylsalicylic acid crystals will appear. If

this is not the case, cool it in an ice bath. 5) Add 50 mL of water (DO NOT ADD THE 50 ML OF WATER UNTIL THE

ACETYLSALICYLIC ACID CRYSTALS HAVE FORMED), and strongly shake the flask (conveniently covered).

6) Recover the precipitate that has formed by vacuum filtration with a Büchner funnel (the filtrate may be used to drag the crystals that may have remained in the Erlenmeyer).

7) Wash the crystals with small portions of cold water (the same as from the ice bath), and dry by suction.

8) Dry the crystals from the Büchner and let dry at room temperature until the following day.

Verifying the aspirin purity Place some phenol crystals and 1 mL of distilled water in a test tube. Place some salicylic acid crystals and 1 mL of distilled water in a test tube. Place some synthesized aspirin crystals and 1 mL of distilled water in a test tube Place some commercial aspirin crystals and 1 mL of distilled water in a test tube.

Wait for a person responsible in the laboratory to pass by before adding two or three drops of 1% iron trichloride (recently prepared) to each of the tubes. The formation of a


58

phenol-Fe (III) complex produces a color that varies from red to violet, depending upon the phenol we are dealing with.

QUESTIONS ABOUT THE EXPERIMENT 1) What is the role of the sulfuric acid in the reaction? 2) Weigh the quantity of the aspirin obtained. 3) Calculate the reaction yield.

Biotechnology 2: Organic Chemistry Experiment 7: Synthesis of borneol

57

OBJECTIVE Obtaining borneol by the reduction of commercial camphor using sodium borohydride.

THEORETICAL BASIS Camphor is a semisolid and waxy crystalline substance with a strong and penetrating

acrid odor. It is a carotenoid with the chemical formula C10H16O. Camphor can be obtained from the camphor tree (Cinnamomum camphora), an enormous perennial originating in Asia. Industrially, camphor is used in the manufacture of cellulose plastics, as well as explosives, pyrotechnics, lacquers and varnish, balsamic fluids, drugs and cosmetics. Camphor is quickly absorbed by the skin, causing a cooling sensation similar to that of menthol, acting like a light local anesthetic and an antimicrobial. It may be administered in small quantities (50 mg) for fatigue and minor cardiac symptoms. It is used as a sweet flavor in India and Europe. It is thought that camphor was used to flavor food similar to ice cream in China during the Tang Dynasty (618-907 AD).

Borneol has a multitude of uses: it aids the digestive system, stimulating the

production of gastric juices, tones the heart and improves circulation, treats bronchitis, constipation and colds, relieves pain from rheumatic diseases and sprains, reduces swelling, relieves stress, can be used as a relaxing tonic and to reduce fatigue. In some parts of the world, it is even used as an insect repellent.

(+)-borneol is present in several medicinal plants, such as valeriana, lavender and

chamomile. It is also used in traditional Chinese and Japanese medicine for sedation, analgesia and anesthesia. It is thought that (+)-borneol can contribute to the sedating and relaxing effects from valeriana and these other medicinal plants.

Sodium borohydride is a metal hydride that by nucleophilic addition adds a hydride

ion from a boron atom to a carbon that is deficient in electrons from a ketone. This process repeats itself with three other ketone molecules, until all the hydrogen atoms from the boron are transferred. Subsequently, the boron complex can decompose with water to form alcohol. Borohydride is a weak reducing agent that can only react with aldehydes, ketones and acyl chlorides. It reacts very slowly with water and alcohol and can be used with ease in these solvents without the occurrence of a significant loss of this reagent. However, it decomposes in acid solutions, releasing gaseous hydrogen.

EEXXPPEERRIIMMEENNTT 77 Synthesis of borneol by reduction of camphor


58

Two alcohols, borneol and isoborneol, are formed by the reduction of camphor due to the borohydride ion being capable of attacking from below or above the carbonyl group. The attack from above the carbonyl is usually referred to as an exo attack, while from below it is called endo. In this example, the exo attack is not very favored, due to steric repulsion produced by the methyl groups in the bridge of the bicyclic camphor. Therefore, 14% borneol and 86% isoborneol are formed.

EXO and ENDO attacks

O

CH3H3C

H3C

CH3H3C

H3C

CH3H3C

H3C

AtaqueEXO

AtaqueENDO

BORNEOL

ISOBORNEOL

EtOH/H2O

OH

H

H

OH

EtOH/H2O

MATERIAL Aluminum bucket 2-100 mL Erlenmeyers 50 mL Erlenmeyer Silicone gaskets 100 mL round bottom flask, B-29 2 Nuts Hot plate/stirrer

10 mL graduated cylinder Reflux condenser Kitasato Stirring piece (magnet) 2 clamps Büchner funnel Cone

REAGENTS AND SOLVENTS Camphor Ethanol Sodium borohydride

Distilled water Ice


59

PRECAUTIONS

Ethanol: Irritating to the eyes and skin. Its ingestion may cause nausea, vomiting and intoxication. Prolonged use may have severe consequences for life.

Sodium borohydride: Flammable. Toxic if ingested. Causes burns.


In a 100 mL round bottom flask, dissolve 3.0 g of camphor in 10 mL of ethanol. To this solution, slowly add 1.5 g of sodium borohydride in small portions (BE CAREFUL, EFFERVESCENCE MAY RESULT). After adding the sodium borohydride, add it to the reflux condenser and slowly heat to boiling for 20 minutes. Stop heating and empty the hot mixture into approximately 50 g of ice and water. Wash the flask with small quantities of distilled water to try to remove the remaining product residue. When the ice has melted, perform vacuum filtration to the solution to collect the precipitate formed.

Introduce the filtered product into a clean, 100-mL round bottom flask again, and add

the minimum quantity of hot ethanol necessary to recrystallize it. Add it to the reflux condenser again and heat, in reflux, until the solid dissolves. If impurities remain without dissolving, filter with a pleated filter above a 100 mL flask. After filtering, heat again and add hot water slowly until the solution becomes turbid, then add a little hot ethanol to redissolve the solid formed. Once the solid is dissolved proceed to slowly cool the solution with stirring (first air and then water bath and finally ice water bath). Filter the resulting crystals in a Büchner.

QUESTIONS ABOUT THE EXPERIMENT

1) Weigh the borneol crystals obtained. 2) Calculate the reaction yield.

Biotechnology 2: Organic Chemistry Experiment 8: Synthesis of tert-butyl chloride

60

OBJECTIVES Obtaining alkyl chloride from a nucleophilic substitution reaction THEORETICAL BASIS

Nucleophilic substitution is a very common and useful reaction in organic synthesis. It consists in an attack by a molecule or ion (nucleophilic reagent comes from the Latin nucleus, and the Greek phillos, for lover) upon another molecule, with expulsion of a fragment from the latter (the leaving group).

Depending whether the expulsion is produced beforehand or at the same time as the

attack by the nucleophile, these types of reactions can be divided into two large groups: unimolecular nucleophilic substitution (SN1) and bimolecular nucleophilic substitution (SN2).

In SN2 reactions, the attack by the nucleophile and the expulsion of the leaving group

are simultaneous.

With SN1, the reaction proceeds in two stages. In the first, the leaving group is expelled, forming a carbocation. This, in a second stage, suffers the attack by the nucleophile.

Depending upon the conditions under which the reaction occurs (presence of polar solvents able to solvate the charged intermediate species) and the reagents used (nucleophilicity of the reagents, stability of the generated carbocation), it will pass through one or another mechanism.

C Br

CH3CO2-

BrCH3CO2 CCH3CO2

+ Br-

H3CH3C

CH3

Br

CH3

CH3H3C

Br-

Br+

HO-CH2-CH3

H3CH3C

CH3

OCH2CH3

H

H3CH3C

CH3

OCH2CH3

H-

H+

EEXXPPEERRIIMMEENNTT 88 Synthesis of tert-butyl

chloride


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In this practice, tert-butyl chloride will be synthesized, starting from the

corresponding alcohol, using the chloride ion as nucleophile. As the OH- group is a poor leaving group, the reaction will be conducted in an acid medium in such a way that the hydroxyl group is protonated and transformed into a good leaving group (H2O).

MATERIAL Extension or bent connecting adapter ● 50 mL graduated cylinder Still head ● 10 mL graduated cylinder 2 silicone gaskets ● Conical funnel 250 mL flask, B29 ● Distillation condenser 50 mL flask, B29 ● 2 supports 2 nuts ● Thermometer Stirring piece (magnet) ● Hot plate/stirrer 2 clamps

REAGENTS AND SOLVENTS Concentrated hydrochloric acid Tert-butanol Anhydrous calcium chloride Distilled water

PRECAUTIONS Hydrochloric acid: Extremely corrosive. Danger due to inhalation and absorption.

Irritating to the skin and eyes. Tert-butanol: Dangerous if inhaled. Irritating to the skin and eyes.


Add 35 mL of the concentrated hydrochloric acid (37%; d= 1,190 g/mL), 10 mL of tert-butanol (99%; d = 0.78 g/mL) and a magnet to a 250 mL round bottom flask. Shake vigorously for some 10 minutes for the reaction to take place. Assemble the distillation system. The receiving flask (50 mL) must be dry and tared. REQUEST SOMEONE

H3CH3C

CH3

OH

CH3

CH3H3CH3C

H3C

CH3

OH

HH3C

H3C

CH3

ClH-

H+ - H2O+ Cl


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RESPONSIBLE IN THE LABORATORY FOR HIM OR HER TO REVISE THE ASSEMBLY. Heat this in a water bath to approximately 60-65 ºC. When a constant drip begins, record the distillation temperature. When the drip into the receiving flask stops, the distillation is considered finished.

QUESTIONS ABOUT THE EXPERIMENT

1) Distillation temperature 2) Weigh the quantity of tert-butyl chloride obtained 3) Calculate the reaction yield


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