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Laboratory Manual ExperimentsIN

Organic Chemistry for Biology Section

Chem 270

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General Information

LABORATORY RULES AND SAFETY PRECAUTIONS

General Policies :

1. Each student is expected to attend each laboratory session and to be in the

laboratory on time. Those students who come early, should wait inside the

laboratory but never gather in the corridors.

2. Each student must wear a clean, ironed and buttoned up laboratory coat.

3. Each student is responsible for keeping the laboratory clean and in good

order. All working areas, balances mustbe kept clean by wiping them with

a cloth or a sponge.

4. Powdered drugs, greasy or waxy materials orany insoluble waste

materials will block the sink if thrown into it. Dispose of them properly in a

waste-basket. Water-immiscible organic solvents and other liquids should be

discarded in a designated waste-solvent can but never poured into a sink.

5. Coats, books and personal belongings should be kept in your lockers. Do

not bring them along with you to the laboratory.

6. Smoking, drinking, eating or chewing gum are prohibited in the laboratory.

7. Each student is expected to work quietly and independently.

8. Familiarize yourself with the location of fire extinguishers and safety showers,

and know how and when to use these devices.

9. Do not carry reagent bottles to your desk.

10. Never return excess material to reagent bottles.

11.Students should bring two hand towels, a sponge, detergent, matches, a

marker and a calculator with them to each laboratory session.

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Safety Precautions:

1. Eye protectionmust be worn at all times in the laboratory, regardless of

what is being done. Prescription glasses (not sunglasses) are acceptable.Contact lenses provide no protection.

2. If chemicals are splashed in or near your eyes, wash immediately with clean,

cold running water (remove contact lenses). Continue washing the eyes for

10- 15 minutes. Consult a physician afterwards.

3. When inserting glass tubing or thermometers into rubber stoppers, always

lubricate both the glass tubing and the hole with glycerin and protect hands

with a towel.

4. Never taste any compound in the laboratory.

5. To determine the odor of a compound, bring the stopper of the bottle

cautiously towards the nose, do not inhale.

6. Avoid any contact of chemicals with the skin, especially the face. Wash

hands as soon as possible after making transfers or other manipulations.

7. When heating a test tube or carrying out a reaction in one, never point the

tube toward yourself or your neighbor.

8. Never, heat an organic liquid directly over a flame except under a condenser.

When refluxing a liquid, be sure that the condenser is tightly fitted. If a

temperature below 95 oC is sufficient, use a steam bath rather than a burner.

9. Before lighting a flame check to see that volatile liquids are not being poured

or evaporated in your vicinity.

10.Always turn a burner off as soon as you have finished using it.

11.As general practice, and particularly if a burner is in use, avoid loose-fitting

long sleeves and cuffs; long hair should be tied back during laboratory work.

12.Any experiment involving the use or production of objectionable (i.e.

poisonous or irritating) gases must be performed in the hoods.

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Warning Signs

Warning signs are used to properly identify and warn of possible hazards. Theyshould be black text on a yellow background and should always include a pictorialsymbol

danger slippery

surface

mind

your

head

fork lift explosive acid scull

electric

shock

trip

hazard

fire risk noise radiation non

ionizingradiation

danger of

death

COMMON LABORATORY GLASS WARE AND EQUIPMENT

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Beaker Glass rod Funnels Conical

Separation Funnel Spatula Test tubes Volumetric Flask

Test Tube Holder Test Tube Rack Round Bttomed Flask Graduated Cylinder

Bunsen Burner Water Bath Hot Plate Balance

Lab 1

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Purification Of Organic Compounds

1- Purification Of Solid Organic Compounds :

Crystallization :

Crystallization is : " The process in which a solid substance separates fromsaturated solution as crystals ".

It is a technique which used to purify solid compounds , It is based on the principlesof solubility and depends on :1) Dissolving organic compounds (solutes)in hot liquids (solvents)2) Filtration to remove impurities .3) Cooling the hot solution to precipitate the organic compounds in crystal form.

Organic solvent can be classified into:

a) polar solvent : H2O , alcohol ( CH3OH , CH3CH2OH) Acetic Acid (CH3COOH) .b) Non Polar solvent : petroleum ether , toluene , benzene and chloroform

Common solvents for recrystallization :

Solvent Structure Boiling point (T0

C)Water H2O 100

Methanol CH3OH 64.5

Ethanol CH3CH2OH 78

Petroleum Ether CH3-O-CH2CH3(-CH2-O-CH2-)

60 - 100

Acetone CH3-CO-CH3 56

Ethyl Acetate C2H5COOCH3 78

Acetic Acid CH 3COOH

Chloroform CHCl3 61

Benzene 80

Cyclo Hexane 81

Experimental

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Objective : Crystallization of Benzoic acid

Material Needed :Glass ware : 2 conical flask , funnel , beaker, glass rod , stand , wire gauzeclamp .filter paper.Chemicals : Benzoic acid , distilled water

Procedure :

1- Place 2gm of Benzoic acid in a 100ml conical flask2- heat 50ml of distilled water to boiling3- add the water to the benzoic acid until it dissolve completely using glass rod4- prepare a fluted filter paper and short stem funnel , place it in a suitable

conical flask and start to filtrate the hot solution rapidly .5- leave the filtrate to cool at room temperature

6- filter the cold solution and collect the pure crystals .

Recrystallization:

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6- Crystals usually begin forming on the bottom of the flask. It's possible to aidcrystallization by scratching the flask with a glass rod at the air-solvent junction(assuming you are willing to purposely scratch your glassware). The scratch

increases the glass surface area, providing a roughened surface on which thesolid can crystallize.

7- Crystals of purified solid are isolated by filtration. This is usually done withvacuum filtration, sometimes washing the purified solid with chilled solvent. Ifyou wash the product, be sure the solvent is cold, or else you run the risk ofdissolving some of the sample.

8- The product may now be dried. Aspiring the product via vacuum filtrationshould remove much of the solvent. Open-air drying may be used as well. In

some cases, the recrystallization may be repeated to further purify the sample.

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SublimationSublimation is :

" The transition of a substance from the solid phase to the gas phase withoutpassing through an intermediate liquid phase ". solid vapor

At normal pressures, most chemical compounds and elements possess threedifferent states at different temperatures. In these cases the transition from the solidto the gaseous state requires an intermediate liquid state.

Sublimation is a technique used by chemists to purify compounds. Typically a solidis placed in a sublimation apparatus and heated undervacuum. Under this reducedpressure the solid volatilizes and condenses as a purified compound on a cooledsurface (cold finger), leaving a non-volatile residue ofimpurities behind. Onceheating ceases and the vacuum is removed, the purified compound may be

collected from the cooling surface.

Examples:

Carbon dioxide

Solid carbon dioxide (dry ice) sublimates readily at atmospheric pressure at -78.5C(197.5 K, 104.2 F), while liquid CO2 can be obtained at pressures andtemperatures above the triple point (5.2 atm, -56.4C).

Water

Snow and ice sublimate, although more slowly, below the melting point temperature.This allows wet cloth to be hung outdoors in freezing weather and retrieved later in adry state. In freeze-drying the material to be dehydrated is frozen and its water isallowed to sublimate under reduced pressure or vacuum. The loss of snow from asnowfield during a cold spell is often caused by sunshine acting directly on the outerlayers of the snow.Ablation is a process which includes sublimation and erosivewear ofglacier ice.

Other compounds

a. Camphorbeing sublimated. Note the white purified camphor on thecold finger, and the dark-brown crude product.

b. Iodine produces fumes on gentle heating. It is possible to obtain liquid iodineat atmospheric pressure by controlling the temperature at just above themelting point of iodine.

c. Naphthalene, a common ingredient in mothballs, also sublimates easily.Arsenic can also sublimate at high temperatures.

Various substances appear to sublimate because of undergoing chemical reactionsor decomposition; for example, ammonium chloride when heated decomposes into

hydrogen chloride and ammonia.

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2- PURIFICATION OF LIQUID ORGANIC COMPOUNDS

Distillation :

Distillation is the process of heating a liquid until it boils, capturing and coolingthe resultant hot vapors, and collecting the condensed vapors.Distillation is a powerful tool, both for the identification and the purification of

organic compounds .It is used to purify a compound by separating it from a non-volatile or less-volatile material.When different compounds in a mixture have different boiling points, they separate

into individual components when the mixture is carefully distilled.

Types of Distillation :There are many types of Distillation

1- Simple distillation :

Simple distillation is designed to evaporate a volatile liquid from a solution of

non-volatile substances; the vapour is then condensed in the water condenser andcollected in the receiver.

The apparatus consists of a round-bottomed distilling flask bearing a stillhead

connected to a water condenser(Liebig condenser). This is attached via a venteddelivery bend to the receiver, also a round-bottomed flask. The stillhead has athermometer adapterwith a thermometer.

Notes:

The bulb of the thermometer is opposite the exit to the condenser. Youwant the temperature of the exit vapours since it is these that will condense. The delivery bend is vented so that when the apparatus is heated thejoints aren't pushed apart by expanding gas. Never draw a closed apparatus. Water goes in at the bottom of the condenser jacket and out at the top. Note the structure of the condenser - the water jacket is separate fromthe tube down the middle!

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Fractional Distillations:

A fractional distillation is used when Mixtures of liquids whose boiling points aresimilar (separated by less than 70C) cannot be separated by a single simple

distillation .

Fractional distillation is the separation chemical compoundsby heating them to a

temperature at which several fractions of the compound will evaporate. Generallythe component parts boil at less than 25 C from each other under a pressure of oneatmosphere (atm). If the difference in boiling pointsis greater than 25 C, a simpledistillation is used.

Fractional Distillation Set-up

The photo below is of a fractional distillation set-up. The only difference between thisset-up and that of a simple distillation set-up is the inclusion of a fractionatingcolumn (see close-up below) between the round bottom flask and the Y-adaptor. Inthe illustration, the fractionating column is a regular condenser filled with glassbeads. Note that you do not hook the tubing connectors on this fractionatingcolumn/condenser to tubing and water.

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Heat source, such as a hot plate with a bath, and ideally with amagnetic stirrer. Distilling flask, typically a round-bottom flask Receiving flask, often also a round-bottom flask Fractionating column Distillation head Thermometerand adapter if needed Condenser Vacuum adapter (not used in image to the right) Boiling chips, also known as anti-bumping granules Standard laboratory glassware with ground glass joints, e.g. quickfitapparatus.

The photo below is of two different styles of fractionating column. The upper one isan ordinary condenser filled with small glass beads. This is the style you will use inthe organic chemistry teaching labs. The lower one is a Vigreux column

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Vacuum Distillation:

Vacuum distillation is : distillation of liquids performed at a pressure lower thanatmospheric pressure to take advantage of the fact that reducing the pressurelowers the boiling point of liquids. This permits the distillation of liquids that aretemperature sensitive and avoids any degradation of such liquids

Vacuum Distillation Set-up

A vacuum distillation set-up differs from a simple distillation with the inclusion of asecurely attached round bottom flask as the receiving flask (note in the picture, it isclamped with a small pronged clamp) and a vacuum line connected to the vacuum

adaptor.

The vacuum line in this particular set-up is connected to a "water-trap", which is aside-arm flask. The water-trap itself is connected to a water aspirator (not shown).Preferably, you would connect the vacuum line from the vacuum adaptor to a

mechanical vacuum system.

Instead of a boiling chip, use a spin barinside the round bottom flask. A boiling chipcannot be used in vacuum distillation, since the reduced pressure sucks the trappedair from boiling chips and thus they do not work to prevent bumping.

A rapidly spinning spin bar does prevent bumping. Place a stir motor under the

heating source for the round bottom flask.

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Steam DistillationDistillation of 2-immiscible liquids , this technique can be used for purification of highboiling liquids which decompose near their boiling points and these liquids areimmiscible with water

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Determination of Physical PropertiesFor Organic Compounds

Melting Points

The melting point of a solid substance is :

" The temperature at which the transition from solid to liquid occurs atatmospheric pressure " ( solid liquid )

It is unaffected by changes in external pressure

A pure solid has a sharp melting point and will melt within a narrow

range of less than two degrees.

Melting Point Determination

Melting points are relatively easy and inexpensive to determine, they are handyidentification tools to the organic chemist.

If you want to use the melting point to identify a solid compound which you haveisolated in the lab, you will need to compare its melting point with that of the truecompound.

If the compounds are sold slightly impure, the melting point range will reflect this

fact.

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Boiling Points

The boiling point is :

" The temperature at which the vapor pressure of the liquid phase

of a compound equals the external pressure acting on the surface of the liquid ". Theexternal pressure is usually the atmospheric pressure.For instance, consider a liquid heated in an open flask. The vapor pressure of theliquid will increase as the temperature of the liquid increases, and when the vaporpressure equals the atmospheric pressure, the liquid will boil.

Different compounds boil at different temperatures because each has a different,characteristic vapor pressure.As a liquid is heated, the temperature is increased.

As the temperature increases, the kinetic energy increases which causesincreasing molecular motion.

Eventually the molecular motion becomes so intense that the forces of attractionbetween the molecules is disrupted to the extent the molecules break free of theliquid and become a gas. At the temperature of the boiling point, the liquid turns intoa gas. The molecules are not in contact each other in the gaseous state.

compounds with higher vapor pressures will boil at lower temperatures.

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EXTRACTION WITH SOLVENTSExtraction :

Separation and isolation of organic substances from the mixtures , it can besubdivided into :

1- Extraction of Solids:e.g. perfume essence from flowers by using solvents ( H2O , CHCl3 , acetone ,ether ,) by using Soxhelt System

2- Extraction of Solutions :Technique used for isolation dissolved substances by shaking its solution withimmiscible solvent in a separatory funnel

On standing, the two immiscible phases ( usually organic and aqueous) from twoseparate layer ( upper and lower) that can be separated by separating funnel .

Experimental

Objective :Separating a two-liquid component

Material Needed :Glass ware :separatory funnel , 2conical flask ,stand , wire gauze, clamp holder,filter paper.Chemicals :Ether ,water

Procedure1- Place 50ml of water and 50ml of ether in a separating funnel

2- Shake it well

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3- Draw off the lower aqueous layer into a flask and repeat extraction of

the lower phase

4- Draw off the other layer into another flask .

CHROMATOGRAPHY

Thin Layer Chromatography - TLC Thin layer chromatography (TLC) is a method for identifying substances and

testing the purity of compounds. TLC is a useful technique because it isrelatively quick and requires small quantities of material.

Separations in TLC involve distributing a mixture of two or more substancesbetween a stationary phase and a mobile phase. The stationary phase is athin layer of adsorbent (usually silica gel or alumina) coated on a plate. Themobile phase is a developing liquid which travels up the stationary phase,

carrying the samples with it. Components of the samples will separate on thestationary phase according to how much they adsorb on the stationary phaseversus how much they dissolve in the mobile phase.

when the Rf of a compound is compared with the Rf of a known compound(preferrably both run on the same TLC plate).

A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin layer ofa solid adsorbent (usually silica or alumina). A small amount of the mixture to beanalyzed is spotted near the bottom of this plate. The TLC plate is then placed in ashallow pool of a solvent in a developing chamber so that only the very bottom of

the plate is in the liquid. This liquid, or the eluent, is the mobile phase, and it slowlyrises up the TLC plate by capillary action.

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As the solvent moves past the spot that was applied, an equilibrium is establishedfor each component of the mixture between the molecules of that component whichare adsorbed on the solid and the molecules which are in solution. In principle, thecomponents will differ in solubility and in the strength of their adsorption to theadsorbent and some components will be carried farther up the plate than others.

When the solvent has reached the top of the plate, the plate is removed from thedeveloping chamber, dried, and the separated components of the mixture arevisualized. If the compounds are colored, visualization is straightforward. Usually thecompounds are not colored, so a UV lamp is used to visualize the plates.

Column Chromatography

In column chromatography, the stationary phase, a solid adsorbent, is placed in avertical glass (usually) column and the mobile phase, a liquid, is added to the topand flows down through the column (by either gravity or external pressure). Column

chromatography is generally used as a purification technique: it isolates desiredcompounds from a mixture.

The mixture to be analyzed by column chromatography is applied to the top of thecolumn. The liquid solvent (the eluent) is passed through the column by gravity or bythe application of air pressure.

An equilibrium is established between the solute adsorbed on the adsorbent andthe eluting solvent flowing down through the column. Because the differentcomponents in the mixture have different interactions with the stationary and mobilephases, they will be carried along with the mobile phase to varying degrees and a

separation will be achieved. The individual components, or eluants, are collected asthe solvent drips from the bottom of the column.

Column chromatography is separated into two categories, depending on how thesolvent flows down the column. If the solvent is allowed to flow down the column bygravity, or percolation, it is called gravity column chromatography. If the solvent isforced down the column by positive air pressure, it is called flash chromatography, a"state of the art" method currently used in organic chemistry research laboratories

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Gas Chromatography

In gas chromatography (GC), the stationary phase is a high-boiling liquid and themobile phase is an inert gas. GC is used as an analytical tool to find out how manycomponents are in a mixture. It can also be used to separate small amounts ofmaterial.

The GC Instrument

The process of gas chromatography is carried out in a specially designedinstrument. A very small amount of liquid mixture is injected into the instrument andis volatilized in a hot injection chamber. Then, it is swept by a stream of inert carriergas through a heated column which contains the stationary, high-boiling liquid. Asthe mixture travels through this column, its components go back and forth atdifferent rates between the gas phase and dissolution in the high-boiling liquid, and

thus separate into pure components. Just before each compound exits theinstrument, it passes through a detector. When the detector "sees" a compound, itsends an electronic message to the recorder, which responds by printing a peak ona piece of paper.