Laboratory Manual Experiments
IN
Organic Chemistry for Biology Section
Chem 270
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 must be kept clean by wiping them with
a cloth or a sponge.
4. Powdered drugs, greasy or waxy materials or any 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.
Safety Precautions:
1. Eye protection must 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.
Warning Signs
Warning signs are used to properly identify and warn of possible hazards. They
should be black text on a yellow background and should always include a pictorial
symbol
danger slippery
surface
electric
shock
trip
hazard
mind
your
head
fire risk
fork lift explosive
noise
radiation
acid
scull
non danger of
ionizing death
radiation
COMMON LABORATORY GLASS WARE AND EQUIPMENT
Beaker
Glass rod
Funnels
Separation Funnel
Spatula
Test tubes
Test Tube Holder
Test Tube Rack
Bunsen Burner
Water Bath
Conical
Volumetric Flask
Round Bttomed Flask Graduated Cylinder
Hot Plate
Balance
Lab 1
Purification Of Organic Compounds
1- Purification Of Solid Organic Compounds :
Crystallization :
Crystallization is : " The process in which a solid substance separates from
saturated solution as crystals ".
It is a technique which used to purify solid compounds , It is based on the principles
of 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
Water
Methanol
Ethanol
Petroleum Ether
Acetone
Ethyl Acetate
Acetic Acid
Chloroform
Benzene
Cyclo Hexane
Structure
H2O
CH3OH
CH3CH2OH
CH3-O-CH2CH3(-CH2-OCH2-)
CH3-CO-CH3
C2H5COOCH3
CH3COOH
CHCl3
Boiling point (T0C)
100
64.5
78
60 - 100
56
78
61
80
81
Experimental
Objective : Crystallization of Benzoic acid
Material Needed :
Glass ware : 2 conical flask , funnel , beaker, glass rod , stand , wire gauze
clamp .filter paper.
Chemicals : Benzoic acid , distilled water
Procedure :
1- Place 2gm of Benzoic acid in a 100ml conical flask
2- heat 50ml of distilled water to boiling
3- add the water to the benzoic acid until it dissolve completely using glass rod
4- 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:
Recrystallization is used to purify solids. Usually this method works best when there
is only a small amount of impurity in the solid.
Recrystallization depends on different solubilities of the target compound and other
compounds present in the impure mixture.
The goal of this method is to have one compound dissolved in a solvent while the
other compound is not dissolved. If one compound is an un dissolved solid, it can be
filtered out of the solution in order to separate it from all the other things that are in
solution.
Solubility in a solvent is a physical property of a material, just like its boiling point or
melting point. Sodium chloride (table salt) has a particular solubility in cold water
(35.7 g will dissolve in 100 mL) while sodium oleate (found in some soaps) has a
different solubility in cold water (10 g per 100 mL). That difference can be exploited
to separate these two compounds.
Summary of Recrystallization Steps
1.
2.
3.
4.
Add a small quantity of appropriate solvent to an impure solid.
Apply heat to dissolve the solid.
Cool the solution to crystallize the product.
Use vacuum filtration to isolate and dry the purified solid.
Recrystallization process:
1- Choose a solvent such that the impure compound has poor solubility
at low temperatures , yet is completely soluble at higher temperatures.
the point is to fully dissolve the impure substance when it is heated, yet
have it crash out of solution upon cooling.
2- Add as small a quantity as possible to fully dissolve the sample.
More solvent can be added during the heating process, if necessary.
3- Heat the suspension to fully dissolve the sample. Usually a hot water bath or
steam bath is used, since these are gentle, controlled heat sources. A hot plate or
gas burner is used in some situations.
4- One the sample is dissolved, the solution is cooled to force
crystallization of the desired compound...
5- Slower cooling may lead to a higher purity product, so it's
common practice to allow the solution to cool to room temperature before
setting the flask in an ice bath or refrigerator.
6- Crystals usually begin forming on the bottom of the flask. It's possible to aid
crystallization 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 the
solid can crystallize.
7- Crystals of purified solid are isolated by filtration. This is usually done with
vacuum filtration, sometimes washing the purified solid with chilled solvent. If
you wash the product, be sure the solvent is cold, or else you run the risk of
dissolving some of the sample.
8- The product may now be dried. Aspiring the product via vacuum filtration
should 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.
Sublimation
Sublimation is :
" The transition of a substance from the solid phase to the gas phase without
passing through an intermediate liquid phase ". solid↔ vapor
At normal pressures, most chemical compounds and elements possess three
different states at different temperatures. In these cases the transition from the solid
to the gaseous state requires an intermediate liquid state.
Sublimation is a technique used by chemists to purify compounds. Typically a solid
is placed in a sublimation apparatus and heated under vacuum. Under this reduced
pressure the solid volatilizes and condenses as a purified compound on a cooled
surface (cold finger), leaving a non-volatile residue of impurities behind. Once
heating ceases and the vacuum is removed, the purified compound may be
collected from the cooling surface.
Examples:
1- Carbon dioxide
Solid carbon dioxide (dry ice) sublimates readily at atmospheric pressure at -78.5°C
(197.5 K, −104.2 °F), while liquid CO2 can be obtained at pressures and
temperatures above the triple point (5.2 atm, -56.4°C).
2- 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 a
dry state. In freeze-drying the material to be dehydrated is frozen and its water is
allowed to sublimate under reduced pressure or vacuum. The loss of snow from a
snowfield during a cold spell is often caused by sunshine acting directly on the outer
layers of the snow. Ablation is a process which includes sublimation and erosive
wear of glacier ice.
3- Other compounds
a. Camphor being sublimated. Note the white purified camphor on the cold
finger, and the dark-brown crude product.
b. Iodine produces fumes on gentle heating. It is possible to obtain liquid iodine
at atmospheric pressure by controlling the temperature at just above the
melting 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 reactions
or decomposition; for example, ammonium chloride when heated decomposes into
hydrogen chloride and ammonia.
2- PURIFICATION OF LIQUID ORGANIC COMPOUNDS
Distillation :
Distillation is the process of heating a liquid until it boils, capturing and cooling
the 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 nonvolatile 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 and
collected 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 vented
delivery bend to the receiver, also a round-bottomed flask. The stillhead has a
thermometer adapter with a thermometer.
Notes:
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The bulb of the thermometer is opposite the exit to the condenser. You want
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 the joints
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 from the
tube down the middle!
Fractional Distillations:
A fractional distillation is used when Mixtures of liquids whose boiling points are
similar (separated by less than 70°C) cannot be separated by a single simple
distillation .
Fractional distillation is the separation chemical compounds by heating them to a
temperature at which several fractions of the compound will evaporate. Generally
the component parts boil at less than 25 °C from each other under a pressure of one
atmosphere (atm). If the difference in boiling points is greater than 25 °C, a simple
distillation is used.
Fractional Distillation Set-up
The photo below is of a fractional distillation set-up. The only difference between this
set-up and that of a simple distillation set-up is the inclusion of a fractionating
column (see close-up below) between the round bottom flask and the Y-adaptor. In
the illustration, the fractionating column is a regular condenser filled with glass
beads. Note that you do not hook the tubing connectors on this fractionating
column/condenser to tubing and water.
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Heat source, such as a hot plate with a bath, and ideally with a magnetic
stirrer.
Distilling flask, typically a round-bottom flask
Receiving flask, often also a round-bottom flask
Fractionating column
Distillation head
Thermometer and 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. quickfit
apparatus.
The photo below is of two different styles of fractionating column. The upper one is
an ordinary condenser filled with small glass beads. This is the style you will use in
the organic chemistry teaching labs. The lower one is a Vigreux column
Vacuum Distillation:
Vacuum distillation is : distillation of liquids performed at a pressure lower than
atmospheric pressure to take advantage of the fact that reducing the pressure
lowers the boiling point of liquids. This permits the distillation of liquids that are
temperature 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 a
securely attached round bottom flask as the receiving flask (note in the picture, it is
clamped 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 a
side-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 bar inside the round bottom flask. A boiling chip
cannot be used in vacuum distillation, since the reduced pressure sucks the trapped
air 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.
Steam Distillation
Distillation of 2-immiscible liquids , this technique can be used for purification of high
boiling liquids which decompose near their boiling points and these liquids are
immiscible with water
Determination of Physical Properties
For Organic Compounds
1- Melting Points
The melting point of a solid substance is :
 " The temperature at which the transition from solid to liquid occurs at
atmospheric 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 handy
identification tools to the organic chemist.
If you want to use the melting point to identify a solid compound which you have
isolated in the lab, you will need to compare its melting point with that of the true
compound.
If the compounds are sold slightly impure, the melting point range will reflect this
fact.
2- 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 ". The
external pressure is usually the atmospheric pressure.
For instance, consider a liquid heated in an open flask. The vapor pressure of the
liquid will increase as the temperature of the liquid increases, and when the vapor
pressure 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 causes
increasing molecular motion.
Eventually the molecular motion becomes so intense that the forces of attraction
between the molecules is disrupted to the extent the molecules break free of the
liquid and become a gas. At the temperature of the boiling point, the liquid turns into
a gas. The molecules are not in contact each other in the gaseous state.
compounds with higher vapor pressures will boil at lower temperatures.
EXTRACTION WITH SOLVENTS
Extraction :
Separation and isolation of organic substances from the mixtures , it can be
subdivided 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 with
immiscible solvent in a separatory funnel
On standing, the two immiscible phases ( usually organic and aqueous) from two
separate 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
Procedure
1- Place 50ml of water and 50ml of ether in a separating funnel
2- Shake it well
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 is relatively
quick and requires small quantities of material.
Separations in TLC involve distributing a mixture of two or more substances
between a stationary phase and a mobile phase. The stationary phase is a thin layer
of adsorbent (usually silica gel or alumina) coated on a plate. The mobile phase is a
developing liquid which travels up the stationary phase, carrying the samples with it.
Components of the samples will separate on the stationary phase according to how
much they adsorb on the stationary phase versus 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 of
a solid adsorbent (usually silica or alumina). A small amount of the mixture to be
analyzed is spotted near the bottom of this plate. The TLC plate is then placed in a
shallow 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 slowly
rises up the TLC plate by capillary action.
As the solvent moves past the spot that was applied, an equilibrium is established
for each component of the mixture between the molecules of that component which
are adsorbed on the solid and the molecules which are in solution. In principle, the
components will differ in solubility and in the strength of their adsorption to the
adsorbent 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 the
developing chamber, dried, and the separated components of the mixture are
visualized. If the compounds are colored, visualization is straightforward. Usually the
compounds 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 a
vertical glass (usually) column and the mobile phase, a liquid, is added to the top
and flows down through the column (by either gravity or external pressure). Column
chromatography is generally used as a purification technique: it isolates desired
compounds from a mixture.
The mixture to be analyzed by column chromatography is applied to the top of the
column. The liquid solvent (the eluent) is passed through the column by gravity or by
the application of air pressure.
An equilibrium is established between the solute adsorbed on the adsorbent and
the eluting solvent flowing down through the column. Because the different
components in the mixture have different interactions with the stationary and mobile
phases, 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 as
the solvent drips from the bottom of the column.
Column chromatography is separated into two categories, depending on how the
solvent flows down the column. If the solvent is allowed to flow down the column by
gravity, or percolation, it is called gravity column chromatography. If the solvent is
forced 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
Gas Chromatography
In gas chromatography (GC), the stationary phase is a high-boiling liquid and the
mobile phase is an inert gas. GC is used as an analytical tool to find out how many
components are in a mixture. It can also be used to separate small amounts of
material.
The GC Instrument
The process of gas chromatography is carried out in a specially designed
instrument. A very small amount of liquid mixture is injected into the instrument and
is volatilized in a hot injection chamber. Then, it is swept by a stream of inert carrier
gas through a heated column which contains the stationary, high-boiling liquid. As
the mixture travels through this column, its components go back and forth at
different rates between the gas phase and dissolution in the high-boiling liquid, and
thus separate into pure components. Just before each compound exits the
instrument, it passes through a detector. When the detector "sees" a compound, it
sends an electronic message to the recorder, which responds by printing a peak on
a piece of paper.