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LABORATORY MANUAL

FOR
CHEMICAL EXPERIMENTATION I

(COURSE NO. CHEM F242)

DEPARTMENT OF CHEMISTRY

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE, PILANI K. K. BIRLA


GOA CAMPUS, GOA
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Team of Chemical Experimentation I, 2014-2015


Department of chemistry,
BITS, Pilani- K K Birla Goa Campus
All rights reserved.
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Table of Contents
INDEX
PART A

Page no.

Introduction

General Laboratory Safety

Preparation of notebook

Common laboratory techniques

12

Experiment 1:

Elemental analysis of organic compounds.

38

Experiment 2:

Functional group analysis of organic compounds

49

Experiment 3:

Qualitative analysis of unknown compounds including derivatization.

60

Experiment 4:

Chemical separation and identification of mixture of two compounds

80

Experiment 5:

Single step synthesis:


a) reduction of benzophenone by NaBH4

83

b) reduction of camphor by NaBH4

85

c) paracetamol to another analgesic drug phenacitin

88

d) synthesis of 3-Methylpyrazol-5-one

90

e) synthesis of Methyl Orange

91

f) synthesis of benzylideneacetophenone

93

g) Preparation of Dinitrobenzene

94

h) Diels-Alder reaction between furan and maleic acid

98

i) Synthesis of dibenzalpropanone (Dibenzylideneacetone)

100

j) Preparation of 1, 1-bis-2-naphthol

102

k) Synthesis of dihydropyrimidinone

104

l) Synthesis of biodiesel: Glycerol

106

PART B

Experiment 6:

Multistep syntheses:
a) IBX from anthranilic acid

108

b) Benzaldehyde to Dilantin (i.e. Phenytoin)

110

c) Benzaldehyde to benzilic acid

115

Experiment 7:

Extraction of natural products: extraction of caffeine from tea leave

Experiment 8:

Purification techniques: Separation of known mixture by 120


chromatography, purification of one of compound from single
step/multistep synthesis, resolution of enantiomers

Experiment 9:

Quantitative analysis: Estimation of Glucose

130

Operation of software related to organic chemistry: chemdraw, chem3D


etc.

135

Experiment 10:

Appendix

117

136

PART A
INTRODUCTION

Welcome to the Organic Chemistry lab. You are about to embark on a journey that will be both
challenging and interesting. The principle objective of this course is to give students hands-on
experience on various basic and advance laboratory methods of organic chemistry. This is an
attempt to integrate students theoretical knowledge and concept on organic chemistry to
practical experience. Here, you will be introduced to techniques chemists use to carry out in
day-to-day operations all over the world from the academy to industry. In this course you will
be introduced to chemical methods as well as instrumentation used by the organic chemist. The
sequence of experiments in this Laboratory Manual is designed to follow the lecture
curriculum. Prior to each lab period, you will need to spend some time reading the Laboratory
Manual. This reading will provide background information and an outline of the procedures to
be performed. Precision is key as many of the outcomes of the reaction will depend on how
well you measure, stir, heat, observe, etc. As such, in this course a large part of your grade will
depend on your technique (how well you carry out the reaction). If you are careful and come to
lab prepared, you will do just fine.
We hope you find this laboratory manual helpful in your study of chemistry.

ACKNOWLEDGMENTS

This manual is the culmination of the efforts of many individuals. Many faculty members of the
Department of Chemistry have provided inputs in preparation and successful implementation
of this laboratory course. Particularly, the efforts of the core team members of Chemical
Experimentation I (Drs. Mainak Banerjee (IC), Amrita Chatterjee, Tincy Lis Thomas and
Subhadeep Banerjee) are sincerely acknowledged.
The experiments described in this laboratory manual are mainly variations of similar
experiments that may be found described in the laboratory manuals of other universities or in
commercially produced lab texts. Most of the experiments have been modified and rewritten,
keeping the particular needs of BITS, Pilani K. K. Birla Goa Campus students in mind.
Ph.D. students of the departments and laboratory staffs have had a role in helping to develop
these experiments and, in particular, helping to ensure that the experiments are tailored to our
laboratories here at BITS, Pilani K. K. Birla Goa Campus. The team of CHEM F242 thanks all of
them.
This is the 2nd edition of the earlier version of 2012-2013. In this version, many new Green
experiments have been added to enrich the content and to give a hands-on exposure of one of
the prime needs of our society, what will call the Sustainable Chemistry.
The following online resources are gracefully acknowledged.
http://www.umsl.edu/~orglab/
http://www.bluffton.edu/~bergerd/classes/CEM221/Handouts/LabManual.pdf
http://ocw.mit.edu/courses/chemistry/5-301-chemistry-laboratory-techniques-january-iap2012/labs/MIT5_301IAP12_comp_manual.pdf
http://www.aircleansystems.org/ductlessfumehoods.htm
http://www.dst.gov.in/green-chem.pdf

Mainak Banerjee
Instructor-In-Charge
Semester II 2014-2015
Department of Chemistry

LABORATORY SAFETY
Safety First, Last, and Always
The organic chemistry laboratory is potentially one of the most dangerous of undergraduate
laboratories. That is why you must have a set of safety guidelines. Do consult/inform your
instructor when you have any doubts regarding safety.
Disobeying safety rules will be penalized by deduction of performance marks.
Attire:
1. Eye injuries are extremely serious and can be mitigated or eliminated if you keep your
safety goggles on at all times. The wearing of contact lenses in the laboratory is strongly
discouraged.
2. Lab coats and shoes must be worn in the Lab. Avoid very loose fitting clothes. Long hair
must be tied back.

Safety Equipment
A set of safety rules is written below. Careful observation of these rules will help to prevent
accidents in the laboratory. However, from time to time accidents can occur. Therefore, safety
equipment is installed for his eventuality in the laboratory. Safety equipment should include:
An eye wash
A safety shower
Fire extinguishers
First-aid kit.

a) Eye Wash
The eyewash is designed to flush irritating chemicals from the eyes. It should be capable of
providing a stream of water for at least 15 minutes. In the event of an eye accident, you
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should proceed to the eyewash at once and wash the eye for at least 15 minutes. During
this process, the eye should be kept open. The eyes are the most vulnerable part of the
body. In the event of any eye injury notify the instructor at once. All eye injuries should be
immediately examined by a health professional.
Never use the eyewash for anything other than its intended purpose.

b) Safety Shower
The safety shower is designed for two purposes, namely, to extinguish clothing fires and to
provide a whole body wash if a large chemical spill occurs.
i. Clothing Fires: If your clothing catches fire, perhaps the best rule is to fall and roll. Never
run to a shower with your clothes on fire, it will only fan the flames. Use the shower
afterwards to squelch any residual embers.
ii. Large Chemical Spills: Large chemical spills on clothing or exposed parts of the body
should be removed at once using the deluge shower. Contaminate clothing should be
removed, and the affected body areas should be thoroughly washed to remove any
chemical traces. Do not reuse contaminated clothing until it has been completely washed!
Serious and avoidable injuries have resulted from wearing contaminated clothing.

c) Fire Extinguishers
In the laboratory, you will sometimes work with flammable materials. Several fire
extinguishers should be placed in the laboratory. Learn their location. In case of any
accidental fire you should use it or immediately inform your instructor.

d) First Aid Kits


First aid kits are used for minor injuries. Report all cuts and bums to the instructor, and at
his/her discretion, visit the school physician for further treatment.

Handling of Chemicals and Equipment:


1. Consider all chemicals to be hazardous. Know what chemicals you are using and their
properties.
2. Avoid contact of chemicals with your skin or eyes. In case such contact does occur, flush
immediately with profuse amounts of water, and inform the instructor immediately.
3. Do not bring flammable regents near open flames.
4. Never point a test tube are heating towards yourself or a neighbor, or vertically
upwards.
5. Always pour acids into water, and not the other way around.
6. Never taste or directly smell chemicals. To detect odor, by means of your cupped hand,
waft a small sample of vapour towards your nose.
7. Dispose off chemical waste properly as directed by the instructor.
8. Follow directions carefully while using instruments.
9. Do not leave burners unattended. Turn them off when you leave. If the burner goes off,
turn off the supply valve immediately. Open again only while relighting the burner.
Never use paper torches for lighting burner.
10. Beware, hot glass looks just the same as cold glass.

Conduct:
1. No food or beverages are allowed in the laboratory.
2. No unauthorized experiments are to be performed.
3. Keep your work area clean. Put paper trash and broken glass, if any, in the dustbins.
Clean your work area before you leave.
4. Avoid spills. If you do spill something, clean up the area immediately taking adequate
precautions. Inform the instructor.
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5. All instruments are very sensitive. Handle them with care. Keep the area around
instruments clean and free of trash paper.
6. If, during the course of a laboratory, you are injured, no matter how minor, you should
bring this to the attention of the instructor or teaching assistant.
7. Always wash your hands thoroughly before you leave the laboratory.

ACCIDENTS WILL NOT HAPPEN


That's an attitude you should hold while working in the laboratory.

ADVANCED PREPARATION AND THE NOTEBOOK


Before coming to the laboratory you will find that some advanced preparation can save
significant time and effort in the laboratory. The advanced preparation will vary depending on
the nature of the experiment, but some general rules will be applicable to all experiments. Read
the experiment first and identify all of the reagents and substances that will be used and
prepared during the course of the experiment. Next, collect the physical properties of these
materials before coming to the laboratory. Physical properties that will be useful include
melting point and boiling points (if available) of all the reagents and expected products, their
densities (if liquid) and some general solubility properties. Molecular weights should be
calculated for all the reagents. This information should be recorded in your notebook which will
be described below.
http://doc.chemnetbase.com/dictionarysearch.do;jsessionid=44C8A37A0B563BB3698013E89F08E997?method=view&id=7152670&si=

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"Aldrich Catalog Handbook of Fine Chemicals", Aldrich Chemical Co.


"Merck Index", Windholz, M. editor, Rahway N.J.
The laboratory notebook is the permanent record documenting what you did in the
laboratory during a specific period of time. It should contain sufficient details and
documentation that an individual with similar training could repeat your work months or years
later. The specific requirements for entries will vary from experiment to experiment and from
instructor to instructor. The following is intended to serve as a guideline of what to include in
the notebook. A substantial part of the grade earned in this course will be derived from the
contents of your notebook.
i)

Use a bound notebook. Notebooks with a spiral binding are not acceptable nor
are any that allow for insertion or removal of pages.

ii) Write in ink. It is expected that you will make mistakes. Just draw a line through the
material you wish to delete. Neatness is always appreciated.
iii) Leave the first few pages of the notebook blank for a table of contents so that each
experiment can be readily located. Pages should be numbered consecutively.
iv) Some notebook preparation should be completed before you come to the
laboratory.
What is required will vary from instructor to instructor. However, the following will apply to all.
1. Title: Each reaction must contain an appropriate title. The title for each experiment can be
found in this manual.

2. Equation: This section only applies to those experiments where you are carrying out a
reaction. Not every experiment will have an associated reaction.
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3. Table of Reagent: This table will contain all of the amounts of reagents used in the reaction
as well as their physical properties (molecular weight, melting point (solids), boiling point
(liquids), density (liquids), etc.).

4. Experimental Procedure: This is where you will write out the procedure so you can follow it.
That means you need to include appropriate details without including everything contained in
the manual. Do not simply copy the procedure from the manual. Again, you should follow
closely the style of the example.

5. Data/Observations: You should record all pertinent observations that you note for each step
of a reaction. If you add a reagent and the color changes, you should note the change of colors.
In addition to any observable changes during the reaction, you will need to record any
calculated values (mass, percent yield/recovery, retention factor (Rf), melting point, boiling
point, etc.) Any calculations that you carry out need to be written in the notebook.

6. Signature/Date: Each experiment must have your signature and the date it was completed.
This is standard procedure and authenticates your work.

Notebook Calculations
The number of calculations you will need to perform in the organic laboratory is quite small but
those that you will be required to perform are very important. The calculations are similar to
the calculations you performed in introductory chemistry and be sure to review them if you
have forgotten the details and theory behind them. What follows is a brief summary of some
important definitions.

12

The calculations associated with conversion of the starting materials to product are
based on the assumption that the reaction will follow simple ideal stoichiometry. For example,
in the preparation of aspirin from salicylic acid and acetic anhydride, in calculating the
theoretical and actual yields, it is assumed that all of the starting material is converted to
product, even though some of the starting material actually forms a polymer as a consequence
of the reaction conditions and catalyst that is used.
The first step in calculating yields is to determine the limiting reagent. The limiting
reagent in a reaction that involves two or more reactants is simply the reagent which is present
in lowest molar amount based on the stoichiometry of the reaction. This reagent will be
consumed first and will limit any additional conversion to product. In the reaction of salicylic
acid with acetic anhydride, the latter reagent is used in excess. There are several reasons for
doing so. An important consideration whenever any reagent is used in excess is to determine
how this reagent will be separated from the product at the end of the reaction. In this case, the
acetic anhydride which is not water soluble can be allowed to stand in water a while during
which time it will slowly hydrolyze to acetic acid. The acetic acid which is water soluble can be
separated from the aspirin by filtration.
The salicylic acid will be the limiting reagent. As far as the calculation of the theoretical
yield is concerned, we assume that every mole of salicylic acid will be converted to aspirin or
acetylsalicylic acid. According to the stoichiometry of the reaction, one mole of salicylic acid will
be converted to one mole of aspirin. Therefore, from the number of moles of salicylic acid we
used, we evaluate the maximum amount of aspirin that can be obtained. Multiplying the
number of moles of aspirin by its molecular weight results in the theoretical yield which is
usually reported in grams. In all cases, the calculation is performed similarly, regardless of how
many products are formed. However, we can now evaluate the actual yield by determining
how much aspirin we have actually isolated, experimentally. The % yield is simply the ratio of
the actual yield divided by the theoretical yield times 100.
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COMMON LABORATORY TECHNIQUES

Crystallization is used to purify a solid. The process requires a suitable solvent. A suitable solvent is one
which readily dissolves the solid (solute) when the solvent is hot but not when it is cold. The best
solvents exhibit a large difference in solubility over a reasonable range of temperatures. (eg, Water can
be a crystallization solvent between 0-100oC).

Characteristics of a solvent:
a. chosen for solubilizing power-- solubility usually increases with increasing temperature.
b. polarity is important--like dissolves like; polar compounds are more soluble in polar solvents;
nonpolar compounds in nonpolar solvents
c. almost all solvents are COMBUSTABLE--avoid flames
d. mixed solvents (eg; 1:1 water/methanol) provide a huge range of possible solvents but they must be
soluble in one another

Use solvent to get solids into solution but to get them out of solution:
a. lower the temperature--solute will be less soluble
b. concentrate the solution by removing solvent with a hot plate, heating mantle (flasks), steam bath
(use in hood) or with the Roto-Evaporator.

To remove solvent:
1. You must have ebullation to concentrate at atmospheric pressure--use a boiling stone.
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2. If you used reduced pressure to concentrate solution, use the water aspirator with a TRAP in the line.
DO NOT turn off the water until the pressure is released. In general, CLAMP any flask that could
conceivably trip over.

Crystallization (review)
Used to obtain pure crystalline solid
Use the proper solvent or solvents--test if necessary; a proper solvent will exhibit a big solubility
difference over a small temperature range.

Re-crystallization or crystallization
a. use an Erlenmeyer flask, it is specifically designed for this purpose
b. dissolve solid in minimum amount of boiling solvent - add solvent in small amounts. For example, if
you add 5 mL and approx. half of the solid dissolves, it should take only another 5 mL to dissolve the
remaining half. If some of the solid does not dissolve then....
c. are remaining particles your compound or insoluble material (eg, sand, old boiling stones)?
d. to determine this, add ca. 10% more hot solvent. If insoluble material, you can decant (carefully
transfer solution into another flask leaving the insoluble material behind) or filter.
e. if filtering is necessary, do so to remove suspended solids, the faster the better, keep solution warm
so crystallization does not occur (this may require filtering on a hot plate or other heating device).
f. to decolorize, use a small amount of charcoal and filter with filter aid (see below). For both (e) and (f),
rinse filter paper with a small amount of hot solvent.
g. let the filtered liquid (filtrate) cool to room temperature slowly in the Erlenmeyer flask
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h. cool the filtrate in an ice-water bath


i. if crystals have not formed
1. seed with a small crystal of product, or
2. scratch the flask with a glass rod which has not been fire polished at the end (ask for a
demonstration), or
3. add a second solvent dropwise until the cloud point is reached; the cloudiness suggests that the
solute has reached a saturation point in this new mixed solvent and will start to come out of solution.
j. if material oils out, you must redissolve by heating the solution and then proceed again from part (g)
k. if material precipitates out, it is time to filter.

Decolorization:
Most organic compounds are colorless. Highly conjugated compounds (e.g., polar polymers) will absorb
light in the visible region of the spectrum and thus be colored. If these highly polar, large molecules are
impurities, they can be removed by use of finely granulated activated charcoal (Norit). Polar compounds
(eg, polar impurities) adsorb to the charcoal which is insoluble in the solvent and can be filtered away
from solution. Unfortunately, some of your compound will also adsorb if there is enough charcoal so
the trick is to use just the right amount. Usually, a very small amount of charcoal will suffice (there is a
lot of surface to these particles). The Norit is added in small amounts to the hot (but not boiling)
solution until sufficient decolorization has occurred. CAUTION--trapped air in the Norit can cause rapid
frothing when it hits the hot solution. The Norit can be filtered from the hot solution using a fluted filter
paper.

Filtering
Used to remove insoluble solids suspended in solution.
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Use a GRAVITY FILTER FUNNEL when you DONOT want the solid
Use a BUCHNER funnel with vacuum when you do want the solid but....
Never use a Buchner funnel with a hot solution unless suggested by your instructor.
For the Buchner funnels, use a piece of round filter paper which fits the funnel. A proper fit is a piece of
paper that just covers the holes but does not touch the sides of the funnel (ask for a demo). You will
need to use reduced pressure, usually via a water aspirator (be certain that the TRAP is clean). For
aqueous solutions, you can use the house vacuum line (be certain that the TRAP is clean).
Just prior to pouring the precipitate onto the filter paper, wet the filter paper with a few mL of solvent
and apply the vacuum. This will cause the filter paper to stick to the filter.
For the gravity funnel, you can prepare a suitable filter from round filter paper by folding into quarters
or by folding it into more than quarters (fluted like a fan). Ask for a demo. Another trick is to use a
piece of cotton loosely wedged into the cone of the funnel. It must be tight enough to not move and to
trap the solid particles but loose enough to allow the solution to flow through. This procedure is often
used to remove drying agents from the solution.
Receiver Flasks: For gravity filtration, you can collect the filtrate in any receiver, including the
concentration flask you plan to use immediately thereafter. Be certain to secure the receiver since it
will contain your important compound.
For a Buchner funnel, you will need a filter flask with a sidearm for a rubber tubing connection to the
TRAP and the reduced pressure source. Use an appropriately sized filter flask, one that will not fill more
than halfway and secure this flask so that it does not tip over with that expensive funnel containing your
valuable crystals.
TRAP: For suction filtration, you want a clean glass trap in between your filter flask and the suction
source. One reason is obvious--in the event that your filtrate is sucked out of the filter flask, it can be
trapped and recovered before it goes down the drain or into the house vacuum line. Another reason is
that a changing flow of water affects the pressure in the water aspirator so that water can back up and
flow towards your filter flask. This way, the trap will fill first and prevent dilution of your filtrate.
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Rinsing: You should rinse the gravity funnel with a small amount of solvent to wash down the remaining
solution that adheres to the filter paper and funnel. Remember, you want the solution; you do not want
the solid. Use a small amount of solvent to prevent excess dilution of the filtrate.
You should rinse the crystals in the Buchner and Hirsch funnel with a minimum amount of cold solvent.
Remember, you want the crystals and do not want to dissolve the crystals with excess solvent which will
wash them into the filtrate.
The proper way to wash the crystals is to SHUT OFF the vacuum, add a minimum amount of cold solvent
so that the crystals are barely sitting in solvent for about 5 seconds (the solvent will not drip through
quickly) and then apply the vacuum. The solvent will be sucked into the filtrate. Do these one or two
times for each solid. You can air dry the solid by sucking air through the solids. To more rapidly dry a large
amount of solid, press another piece of filter paper on top of the solid.

Filtrate from Buchner filtration: The filtrate will probably still contain some of your desired compound
and is called the mother liquor. Often, by concentrating this solution further and cooling the solution, one
obtains more crystals. This may happen while you are drying the original crystals under reduced pressure.
To collect this second batch of crystals, filter as before but do not combine with the first batch of crystals.
The purity of this second crop may be different from the first batch. Use melting point or other methods
(eg, thin layer chromatography) to determine whether the purity of the second crop is equal to that of the
first. If so, they can be combined. If not, keep them separate.

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Solvent Polarity in decreasing order:


water
amides (N,N-dimethylformamide)
alcohols (methanol, ethanol)
ketones (acetone, methyl ethyl ketone)
esters (ethyl acetate)
chlorocarbons (methylene chloride, chloroform)
ethers (diethyl ether)
aromatics (toluene)
alkanes (hexanes, petroleum ether)

Heating:
There are different methods used for heating material in the laboratory. Flames are used only when it is
instructed. Electric hot plates and heating mantles are most commonly used. Be careful not to turn this
equipment to its highest setting which can burn it out. It does take several minutes for these instruments
to reach the desired temperature. The heating mantles are plugged into a variable rheostat which
provides a temperature control. Heating mantles are used for round-bottom flasks (rbf); choose an
appropriate size to fit the flask you plan to use.
Steam is often used for heating volatile, non-aqueous, flammable solvents in the fume hoods. This is
preferable to using hot plates which can lead to flash fires. The 100oC maximum temperature from steam
is sufficient for most commonly used organic solvents including many mixed aqueous solvents.
Ask your instructor which technique is to be used for a particular point of time

19

Melting Point Determination:


The standard physical property of a solid is its melting point. The melting point is actually a melting point
range. It is used to help determine the purity of a solid and to help verify the identity of the compound. A
pure compound should melt over a narrow temperature range. Impurities usually cause the melting point
range to widen and lower in value. To obtain the melting point range, you record the temperature at
which the first crystals begin to melt (solid to liquid phase) and the temperature at which the last crystal
melts, eg; m.p. 124-126oC.
Preparing the sample in a capillary tube: The sample must be dry or else you cannot get it into the
capillary tube. The capillary tube should be open at one end only. Tamp the open end onto your crystals
until a few collect inside the mouth of the capillary. Now tap the crystals to the closed bottom end by
bouncing the capillary (bottom end down) through the open space of a large piece of glass tubing. Ask for
a demo. Follow the instructions given above for the rate of heating.

Extraction
Extraction is a method for moving a compound from one medium to another. For example, if you make
coffee from coffee beans, you are extracting some flavorful components of the bean and some caffeine
into the water. The remainder of the beans (grounds) are left behind and discarded. This is called a solidliquid extraction. If you are trying to move a compound from one liquid phase (solvent 1) into another
liquid phase (solvent 2), this is liquid-liquid extraction but the two solvents must be immiscible or
insoluble to the extent that they form two distinct layers. The compound is now distributed mostly into
one layers which can be separated.
Liquid-liquid extractions are common in organic chemistry. Usually, one of the solvents is water and the
objective is to remove a component from an aqueous solution into a solvent such as ether, methylene
chloride, or ethyl acetate (all of which have low water solubility). Often, water-insoluble organic solvents,
such as ether, methylene chloride and hexane, may contain some undesirable water soluble components
(like HCl). In that case, we would extract those components out of the organic solvent by using water as
the second solvent. That is often called a water wash. You will have an opportunity to do several
extractions and water washes. For these purposes, you will use a separatory funnel.
20

The rule of thumb for liquid-liquid extractions is that several small extractions are more efficient than one
big extraction.
Separatory Funnel: This glass equipment is very cleverly designed to carry out the task of separating two
immiscible liquids (which form two distinct layers). Work with this equipment in a proper fashion and it
will perform remarkably well. However, read the instructions first and follow the steps carefully.
Use of the Separatory funnel: First, check that the stopper fits and that the stopcock works properly.
Glass stopcocks must be greased; Teflon stopcocks do not need grease.
2. Close the stopcock and support the funnel using a ring clamp.
3. Place a beaker underneath the funnel to catch any spills or leaks.
4. Fill the funnel with the two solvents. Do not fill the funnel more than 75% of capacity (so plan ahead in
choosing the proper size funnel)
5. Stopper. Remove the funnel from the stand, hold properly and invert (IMPORTANT, ask for a demo),
release any pressure buildup by opening the stopcock repeatedly BEFORE shaking (and then after
shaking).
6. After returning the funnel to the stand where it is secured, loosen the stopper immediately--shaking
builds up pressure
7. When the two phases separate, draw off the lower layer.
8. Pour out the upper layer if necessary. NOTE: The funnel is designed to retain the last few drops.
9. Save both upper and lower layers until you are certain that you have the compound you want. If you do
not throw it away, it is not lost. I repeat, never throw away a layer unless you are certain that you will
never need it.

Percent Yield: Although you may have obtained the product you desired, the amount of material you
obtained (in grams), compared to the amount you could have obtained (in grams), is a valuable piece of
information. It can suggest that you did or did not run the reaction efficiently or that other products may
21

have been formed via other reactions. To calculate % Yield you must first calculate the theoretical yield.
You then take actual yield/theoretical yield x 100 = % yield. The % yield can never exceed 100%; if it does,
it may mean that your sample is wet or contains extraneous material, such as a boiling stone.

Labels
Properly label your sample vials when you submit your products for grading.
e.g.
BENZOIC ACID (appropriate, unambiguous name of the compound)
mp 120-121oC (melting point observed for this sample)
3.2 g (amount in vial)
Students name and ID
Date of submission

Distillation
Distillation is an important commercial process that is used in the purification of a large variety of
materials. All substances regardless of whether they are liquids or solids are characterized by a
vapor pressure. The vapor pressure of a pure substance is the pressure exerted by the substance
against the external pressure which is usually atmospheric pressure. Vapor pressure is a measure
of the tendency of a condensed substance to escape the condensed phase. The larger the vapor
pressure, the greater the tendency to escape. When the vapor pressure of a liquid substance
reaches the external pressure, the substance is observed to boil. If the external pressure is
atmospheric pressure, the temperature at which a pure substance boils is called the normal
boiling point.
22

Simple Distillation
Although all of us have brought water to a boil many times, some of us may have not
realized that the temperature of pure boiling water does not change as it distills. This is why
vigorous boiling does not cook food any faster than a slow gentle boil. The observation that
the boiling point of a pure material does not change during the course of distillation is an
important property of a pure material. The boiling point and boiling point range have been
used as criteria in confirming both the identity and purity of a substance. For example, if we
synthesized a known liquid that boiled at 120-122 C, this value could be used to confirm
that we prepared what we were interested in and that our substance was reasonably pure.
Of course, additional criteria must also be satisfied before the identity and purity of the
liquid are known with certainty. In general, a boiling point range of 1-2 C is usually taken as
an indication of a pure material. You will use both of these properties later in the semester
to identity an unknown liquid.
Occasionally, mixtures of liquids called azeotropes can be encountered that mimic
the boiling behavior of pure liquids. These mixtures when present at specific concentrations
usually distill at a constant boiling temperature and can not be separated by distillation.
Examples of such mixtures are 95% ethanol-5% water (bp 78.1 C), 20% acetone-80%
chloroform (bp 64.7 C), 74.1% benzene, 7.4% water, 18.5 % ethanol (bp 64.9).

The

azeotropic composition sometimes boils lower the than boiling point of its components and
sometimes higher. Mixtures of these substances at compositions other than those given
above behave as mixtures.
Returning to our discussion of boiling water, if we were making a syrup by the
addition of sugar to boiling water, we would find that the boiling point of the syrup would
increase as the syrup begins to thicken and the sugar concentration becomes significant.
Unlike pure materials, the boiling point of an impure liquid will change and this change is a
reflection of the change in the composition of the liquid. In fact it is this dependence of
boiling point on composition that forms the basis of using distillation for purifying liquids.
23

We will begin our discussion of distillation by introducing Raoult's Law, which treats liquids in
a simple and ideal, but extremely useful manner.

Figure 1.

The apparatus used in a simple distillation. Note the position of the

thermometer bulb in the distillation head and the arrangement of the flow of the cooling
water.
PAobs = PAo ,

where PAobs is the observed vapor pressure of A


= nA/(nA + nB + nC +...)
PAo = vapor pressure of pure A

nA, nB, nC ... : number of moles of A, B, C, ...


24

This relationship as defined is capable of describing the boiling point behavior of


compound A in a mixture of compounds under a variety of different circumstances. Although
this equation treats mixtures of compounds in an oversimplified fashion and is not applicable
to azeotropic compositions, it does give a good representation of the behavior of many
mixtures.
Let's first consider a binary system (2 components) in which only one of the two
components is appreciably volatile. Our previous discussion of sugar + water is a good
example. Raoult's law states that the observed vapor pressure of water is simply equal to the
product of the mole fraction of the water present and the vapor pressure of pure water at
the temperature of the mixture. Once the sugar-water mixture starts to boil, and continues
to boil, we know that the observed vapor pressure of the water must equal one atmosphere.
Water is the only component that is distilling. Since the mole fraction of water in a mixture
of sugar-water must be less than 1, in order for the observed vapor pressure of water ( PAobs)
to equal one atmosphere, PAo must be greater than one atmosphere. As the distillation of
water continues, the mole fraction of the water continues to decrease thereby causing the
temperature of the mixture to increase. Remember, heat is constantly being added. If at
some point the composition of the solution becomes saturated with regards to sugar and the
sugar begins to crystallize out of solution, the composition of the solution will become
constant; removal of any additional water will simply result in the deposit of more sugar
crystals. Under these set of circumstances, the composition of the solution will remain
constant and so will the temperature of the solution although it will exceed 100 C.
During the course of the distillation, the water vapor which distilled was initially at
the temperature of the solution. Suspending a thermometer above this solution will record
the temperature of the escaping vapor. As it departs from the solution, the temperature of
the vapor will cool by collisions with the surface of vessel until it reaches 100 C. Cooling
below this temperature will cause most of the vapor to condense to a liquid. If cooling to 20
C occurs in the condenser of a distillation apparatus, then by using the appropriate
geometry as shown in Figure 1, it would be possible to collect nearly all of the liquid. The
25

only vapor that would be lost to the environment would be that small amount associated
with the vapor pressure of water at 20 C. Since the vapor pressure of water at 20 C is
roughly 2.3 kPa, then 2.3/101.325 or 0.023 would be the fraction of water that would not
condense and would pass out of the condenser. This is why the distillate is frequently chilled
in an ice bath during the distillation.
The distillation of a volatile material from non-volatile is one practical use of
distillation which works very well. However, often there may be other components present
that although they may differ in relative volatility, are nevertheless volatile themselves. Let's
now consider the two component system you will be using in the distillations you will
perform in the laboratory, cyclohexane and methylcyclohexane. The vapor pressures of
these two materials in pure form are given in Table 1. As you can see from this table,
although cyclohexane is more volatile than methylcyclohexane, the difference in volatility
between the two at a given temperature is not very great. This means that both materials
will contribute substantially to the total vapor pressure exhibited by the solution if the
distillation is carried out at 1 atmosphere. The total pressure, P T, exerted by the solution
against the atmosphere according to Dalton's Law of partial pressures, equation 2, is simply
the sum of the observed vapor pressures of cyclohexane, Pcobs, and methylcyclohexane, Pmobs:
PT = Pcobs + Pmobs.

As before, boiling will occur when the total pressure, P T , equals an atmosphere. However
since we have two components contributing to the total pressure, we need to determine the
relative contributions of each. Again we can use Raoult's Law but we need more information
about the system before we can do so. In particular we need to know the composition of
the solution of cyclohexane and methylcyclohexane. For ease of calculation, let's assume
that our original solution has equal molar amounts of the two components. What we would
like to determine is whether it would be possible to separate cyclohexane from
methylcyclohexane by distillation. By separation, we would like to determine if it would be
possible to end up with two receiver flasks at the end of the experiment that would contain
26

mainly cyclohexane in one and mainly methylcyclohexane in the other. It is clear that at
some point we will need to intervene in this

Table 1.

Vapor pressures of cyclohexane and methyl cyclohexane as a function of

temperature.
cyclohexane

T/K

methylcyclohexane

P/kPa

T/K

P/kPa

300

14.1

300

6.7

305

17.6

305

8.5

310

21.7

310

10.6

315

26.5

315

13.2

320

32.2

320

16.2

325

38.8

325

19.8

330

46.5

330

24.0

335

55.3

335

28.9

340

65.4

340

34.6

345

77.0

345

41.2

350

90.0

350

48.7

354

101.3

354

55.4

360

121.3

360

66.9

362

128.5

362

71.1

365

139.9

365

77.9

370

160.5

370

90.2

27

373

174.0

373

101.3

380

208.8

380

119.3

385

236.7

385

136.4

390

267.3

390

155.3

395

300.9

395

176.2

400

337.5

400

199.1

experiment. Otherwise, if we were to collect the entire contents of the original distilling
flask, called the pot, into one receiver flask, we would end up with the same composition as
we started. Initially the mole fractions of both cyclohexane and methylcyclohexane are 0.5.
From Raoult's Law (equation 1), Dalton's Law (equation 2) and the information in Table 1, we
can estimate that boiling will occur at approximately 362 K when the total pressure of the
two components equals one atmosphere or 101.3 kPa.:
PT = Pcobs + Pmobs = 0.5(128.5 kPa) + 0.5(71.1 kPa) 101.3 kPa
The first thing that we should note is that the initial boiling point is higher than the lowest
boiling component and lower than the highest boiling component. Next, we should inquire
about the composition of the vapor. Is the composition of the vapor the same as the initial
composition of the pot or is it enriched in the more volatile component? If the composition
of the vapor is the same as that of the original mixture, then distillation will not be successful
in separating the two components. However, we should ask, "What is the composition of the
vapor?" Before the vapor is cooled and condenses on the condenser, we can treat the vapor
as an ideal gas. Recalling that: PV = nRT, where P is the pressure of the gas or vapor, V is the
-

volume it occupies, n is the number of moles of gas, R is the Gas Constant (0.0821 L . atm.K
1

.mol-1) and T is the temperature, we can determine the composition of the vapor by taking

advantage of the following factors. First we note that:

28

PTV = nTRT so that

PcobsV = ncRT and

PmobsV = nmRT

where nT refers to the total

number of moles, since ( Pcobs + Pmobs ) = (nc + nm)RT.


If the total vapor can be treated as an ideal gas, then according to Dalton's Law, so can each
of the components. Since the two components are in thermal contact and are distilling
together, we can expect them to be at the same temperature. We don't necessarily know
the volume of the container, but since it is assumed that the volumes of the molecules are
very small in comparison to the total volume the gas occupies, whatever the value of V, it is
the same for both components. This means we can establish the following ratio:
Pcobs/ Pmobs =

nc/nm

which in turn allows us to determine the composition of the vapor from the observed partial
pressures of the two components. If we use the experimental values found in Table 1, we
conclude that the composition of the vapor is 1.8/1, and is indeed enriched in the more
volatile component.
This simple treatment allows us to understand the principles behind distillation.
However it is important to point out that distillation is far more complex than our simple
calculation indicates. For example, we just calculated the composition of the vapor as soon
as the solution begins to boil and we have correctly determined that the vapor will be
enriched in the more volatile component. This means that as the distillation proceeds, the
pot will be enriched in the less volatile component. Since the composition of the pot will
change from the initial 1:1 mole ratio and become enriched in the less volatile component;
the new composition in the pot will introduce changes in the composition of the vapor. The
composition of the vapor will also change from the initial ratio we just calculated to a new
ratio to reflect the new composition of the pot. The consequences of these changes are that
the temperature of both the pot and the distillate will slowly increase from the initial value
to a value approaching the boiling point and composition of the less volatile component. If
we are interested in separating our mixture into components, we are left with the task of
29

deciding how much material to collect in each receiver and how many receivers to use.
Obviously this will depend on the quality of separation we are interested in achieving.
Generally, the more receivers we use the less material we will have in each. It is possible to
combine fractions that differ very little in composition but this requires us to analyze each
mixture. While it is possible to do this, in general, we really want to end with three receivers,
one each enriched in the two components of our mixture and a third that contains a
composition close to the initial composition.
It is difficult to describe how much material to collect in each receiver since the
volume collected will depend on the differences in the boiling points of the components. As
a general rule, the receiver should be changed for every 10 C change in boiling point. Each
fraction collected can be analyzed and those with compositions similar to the initial
composition can be combined. The main fractions collected can then be fractionated a
second time if necessary.
The experiment we have just discussed is called a simple distillation. It is an
experiment that involves a single equilibration between the liquid and vapor. This distillation
is referred to as involving one theoretical plate. As you will see, it is possible to design more
efficient distillation columns that provide separations on the basis of many theoretical
plates. Before discussing these columns and the advantages offered by such fractionating
columns, it is important to understand the basis of the advantages offered by columns with
many theoretical plates. The following is a simplified discussion of the process just described
involving a column with more than one theoretical plate.
Vacuum Distillation
Elevation of the boiling point with an increase in external pressure, while important
in cooking and sterilizing food or utensils, is less important in distillation. However, it
illustrates an important principle that is used in the distillation of many materials. If the
boiling point of water is increased when the external pressure is increased, then decreasing
the external pressure should decrease the boiling point. While this is not particularly
30

important for the purification of water, this principle is used in the process of freeze drying,
an important commercial process. In addition, many compounds cannot be distilled at
atmospheric pressure because their boiling points are so high. At their normal boiling points,
the compounds decompose. Some of these materials can be distilled under reduced
pressure however, because the required temperature to boil the substance can be lowered
significantly. Rewording the "rule of thumb" described above so that it is applicable here
suggests that the boiling point will be lowered by 10 C each time the external pressure is
halved. For example, if the external pressure above a substance is reduced to 1/16 of an
atmosphere by mean of a mechanical pump, the boiling point will have been reduced four
times by 10 C for a total reduction of 40 C (1 atm x (1/2)(1/2)(1/2)(1/2) =1/16 atm).
A nomograph is a useful device that can be used to estimate the boiling point of a
liquid under reduced pressure under any conditions provide either the normal boiling point
or the boiling point at a some given pressure is available. To use the nomograph given the
normal boiling point, simply place a straight edge at on the temperature in the central
column of the nomograph (b). Rotating the straight edge about this temperature will afford
the expected boiling point for any number of external pressures. Simply read the
temperature and the corresponding pressure from where the straight edge intersects the
first and third columns. As an example lets choose a normal boiling point of 400 C. Using
the nomograph in Figure 2 and this temperature for reference, rotating the straight edge
about this temperature will afford a continuous range of expected boiling points and the
required external pressures necessary to achieve the desired boiling point. At a pressure of 6
mm, the expected boiling point would be 200 C. Likewise, our compound boiling at 400 C
at 1 atm would be expected to boil at 145 C at 0.1 mm external pressure.

31

Pressure-Temperature Nomograph for Vacuum Distillations

Boiling Point

Pressure (mm)
Boiling Point

at pressure P

at 1 atm

(mm)

1 atm=760
mm;

(760 mm)

(a)

(b)

(c)

Figure 2. A nomograph used to estimate boiling points at reduced pressures. To use, place a
straight edge on two of the three known properties and read out the third. Column c is in
mm of mercury. An atmosphere is also equivalent to 101.3 kPa and will support a column of
mercury, 76 cm (760 mm).

Fractional Distillation
We have just seen that starting with a composition of 1:1, cyclohexane:
methylcyclohexane, the composition of the vapor was enriched in the more volatile
32

component. Suppose we were to collect and condense the vapor and then allow the
resulting liquid to reach equilibrium with its vapor. Lets call this liquid, liquid 2. The
properties of liquid 2 will differ from the original composition in two ways. First, since the
composition of liquid 2 is higher in cyclohexane than the initial one; the temperature at
which liquid 2 will boil will be lower than before (what is the approximate boiling point of a
1.8/1 mixture of cyclohexane/methylcyclohexane? see Table 1). In addition, the composition
of the vapor, vapor 2, in equilibrium with liquid 2 will again be enriched in the more volatile
component. This is exactly what happened in the first equilibration (first theoretical plate)
and this process will be repeated with each new equilibration. If this process is repeated
many times, the vapor will approach the composition of the most volatile component, in this
case pure cyclohexane, and the liquid in the pot will begin to approach the composition of
the less volatile component, methylcyclohexane. In order for this distillation to be successful,
it is important to allow the condensed liquid which is enriched in the less volatile component
relative to its vapor, to return to the pot. In a fractional distillation, the best separation is
achieved when the system is kept as close to equilibrium as possible. This means that the
cyclohexane should be removed from the distillation apparatus very slowly. Most fractional
distillation apparati are designed in such a way as to permit control of the amount of
distillate that is removed from the system. Initially the apparatus is set up for total reflux,
(i.e. all the distillate is returned back to the system). Once the distillation system reaches
equilibrium, a reflux to takeoff ratio of about 100:1 is often used (about 1 out of every 100
drops reaching the condenser is collected in the receiver).
A column which allows for multiple equilibrations is called a fractionating column and
the process is called fractional distillation. An example of a fractionating column is shown in
Figure 4. Each theoretical plate is easy to visualize in this column. The column contains a
total of 4 theoretical plates and including the first equilibration between the pot and
chamber 1 accounts for a total of 5 from pot to receiver. As you might expect, a problem
with this column is the amount of liquid that is retained by the column. Many other column
designs have been developed that offer the advantages of multiple theoretical plates with
33

low solvent retention. Typical spinning band columns often used in research laboratories
offer fractionating capabilities in the thousand of theoretical plates with solute retention of
less than one mL. Commercial distillation columns have

Figure 4. A fractionating column which contains four chambers, each with a center opening
into the chamber directly above. The vapor entering the first chamber cools slightly and
condenses, filling the lower chamber with liquid. At equilibrium, all chambers are filled with
34

distillate. A portion of the liquid condensing in the first chamber is allowed to return to the
pot. The remaining liquid will volatilize and travel up the column condensing in the second
chamber and so on. As discussed in the text, the composition of the vapor at each
equilibration is enriched in the more volatile component. The heat necessary to volatilize the
liquid in each chamber is obtained from the heat released from the condensing vapors
replacing the liquid that has been removed. The vacuum jacket that surrounds the column
ensures a minimum of heat loss.
Columns have been designed for gasoline refineries that are multiple stories high and are
capable of separating compounds with boiling points that differ by only a few degrees.
In addition to performing a fractional distillation at one atmosphere pressure, it is
also possible to conduct fractional distillations at other pressures. This is often avoided
when possible because of the increased difficulty and expense in maintaining the vacuum
system leak free.

Steam Distillation
The concentration and isolation of an essential oil from a natural product has had a
dramatic impact on the development of medicine and food chemistry. The ability to
characterize the structure of the active ingredient from a natural product has permitted
synthesis of this material from other chemicals, resulting in reliable and often cheaper
sources of the essential oil.

The process often used in this isolation is called steam

distillation. Steam distillation is an important technique that has significant commercial


applications. Many compounds, both solids and liquids, are separated from otherwise
complex mixtures by taking advantage of their volatility in steam. A compound must satisfy
three conditions to be successfully separated by steam distillation. It must be stable and
relatively insoluble in boiling water, and it must have a vapor pressure in boiling water that is
of the order of 1 kPa (0.01) atmosphere. If two or more compounds satisfy these three
35

conditions, they will generally not separate from each other but will be separated from
everything else. The following example, expressed as a problem, illustrates the application of
steam distillation:
Suppose we have 1 g of an organic compound present in 100 g of plant material composed
mainly of macromolecular material such cellulose and related substances. Let's assume that
the volatile organic material has a molecular weight of 150 Daltons, a vapor pressure of 1
kPa and is not soluble in water to an appreciable extent. Examples of such materials
characterized by these properties include eugenol from cloves, cinnamaldehyde from
cinnamon bark or cuminaldehyde from cumin seeds. How much water must we collect to be
assured we have isolated all of the natural oil from the bulk of the remaining material?
We can simplify this problem by pointing out that the organic material is not
appreciably soluble in water. We know from previous discussions that boiling will occur
when the total pressure of our system equals atmospheric pressure. We can also simplify the
problem by assuming that the essential oil in not appreciably soluble in the macromolecular
material. While in reality this does not have to be correct, this assumption simplifies our
calculation. Boiling of our
obs
obs
PT = Pwater
+ Porg
;

o
o
PT = water Pwater
+ org Porg

mixture will occur close to 100C. Remember that very little oil is soluble in water which
makes the mole fraction of water near unity. Similarly for the volatile oil, its mole fraction is
also close to one according to our assumption. The total pressure, PT, is the sum of the vapor
o
pressure of water, 100 kPa, and the essential oil, Porg
, 1 kPa. Boiling will occur very close to

the boiling point of pure water. Treating the water vapor and the organic vapor which are
miscible as ideal, the PV ratio for both vapors is given by the following:
PwaterV/ PorgV = nwaterRT/norgRT;
Pwater/ Porg = nwater/norg and
36

nwater = wtwater/18; norg = 1/150; rearranging:


wtwater = (100/1)(18/150) = 120 g water or 120 mL
Our calculation suggests that we can be assured that most of the 1 g of the organic mater
has been transferred by the steam if we condense and collect 120 mL of water. The basis of
the separation by steam distillation is that while the water and organic condensed phases
are immiscible, the vapors of both are miscible. Once condensed, the two separate again
allowing for an easy separation. As noted above, both liquids and solids can be distilled by
steam.

37

PART B
Here, you will be made familiar with three major parts of practical organic chemistry namely
a) qualitative analysis of known and unknown organic samples, b) quantitative estimation of
a few organic substances and c) single and multi step syntheses of several organic
compounds. Each of these parts consists of number of experiments and will take several lab
hours. The following section will describe the experimental procedure.

38

EXPERIMENT - 1

ELEMENTAL ANALYSIS OF ORGANIC COMPOUNDS

Objective:
To carry out the detection of inorganic elements in an organic compound i.e. nitrogen, sulfur
and halogens (chlorine, bromine and iodine).

Principles:
In an organic compound carbon, hydrogen and oxygen are assumed to be present. Elements
other than these elements i.e. nitrogen, sulfur and halogens (chlorine, bromine and iodine)
may also be present in an organic compound. As you have known earlier that chemical
analysis is of two types: Qualitative analysis and Quantitative analysis. The detection of extra
elements in a given organic compound is a type of qualitative analysis since here we are
dealing with the composition of the compound. This experiment is to be done very carefully
as further analysis of the organic compound is based on the extra element present in it.

LASSAIGNES TEST
These extra elements are usually detected by Lassaignes test. In this test, the organic
compound is fused with metallic sodium to convert these elements into water soluble
sodium salts. And then this extract is used to perform the tests.

Experimental Procedure

Preparation of Lassaignes extract


1. Take a small piece of sodium metal with a metallic spatula and dry it by pressing between
the folds of filter paper and put this piece of sodium metal into an ignition tube.
2. The ignition tube is heated slowly till the sodium metal turns into shining globule.
39

3. Remove it from flame and add the compound to be tested (a pinch of solid compound or
2-3 drops of liquid compound). First heat it gently after compound addition and then
strongly until it becomes red hot.
4. Plunge it into 10 ml of distilled water contained in a china dish and cover the china dish
immediately with wire gauze to stop the flames. The same procedure is repeated by 2
more ignition tubes. The ignition tubes if not broken completely, are crushed with the
help of a glass rod.
5. Boil the contents of the china dish for about 5 minutes and filter. The filtrate is called
Lassaignes extract or sodium extract. The filtrate should be colorless. Filtrate will be
colored when fusion is incomplete. So, if it is colored, whole procedure should be
repeated again.

The reactions involved in the preparation of Lassaignes extract are:

Nitrogen, Na + C + N NaCN (sodium cyanide). So, nitrogen will be present in the form of
sodium cyanide in the Lassaignes extract.

Sulfur, 2Na + S Na2S (sodium sulfide). Thus, sulfur will be present in the form of sodium
sulfide in the Lassaignes extract.

Nitrogen and sulfur both, Na + C + N + S NaSCN (sodium thiocyanate). So, when nitrogen
and sulfur are present together in the organic compound, they will be present in the form of
sodium thiocyanate in the Lassaignes extract.

Halogens, Na + X NaX (X=Cl, Br and I) (sodium halide). So, halogens will be present in the
form of sodium halide (if X=Cl, then sodium chloride, if X= Br, then sodium bromide and if X=
I, then sodium iodide) in the Lassaignes extract.

40

Reactions of Nitrogen test:


1. Ferrous sulfate reacts with the NaOH present in the extract to give a dirty green color
precipitate. FeSO4 + 2NaOH Fe(OH)2 + Na2SO4
(Ferrous hydroxide, dirty green ppt)
NaOH is formed in the Lassaignes extract by the reaction between unreacted sodium
metal and water. Thus, if no green precipitate is obtained that means NaOH is not
present in the extract so few drops of it is added in the test tube to get the precipitate.
2. Ferrous ions react with sodium cyanide (ionic form of nitrogen in the extract) to give
sodium ferricyanide as per the equation Fe(OH)2 + 6NaCN Na4Fe(CN)6 + 2NaOH
(sodium ferrocyanide)
On boiling the alkaline solution of ferrous salt, some ferric ions are inevitably produced by
the aerial oxidation. And ferrous and ferric hydroxides are dissolved on the addition of dilute
sulfuric acid. Sodium ferrocyanide reacts with ferric salt to give ferriferrocyanide complex
which is Prussian blue color complex.
3Na4Fe(CN)6 + 2Fe2(SO4)3 Fe4[Fe(CN)6]3 + 6Na2SO4
(Ferri-ferrocyanide, Prussian blue)

Rection of Sulfur test:


Sodium sulfide (ionic form of sulfur in the extract) reacts with sodium nitroprusside to give
sodium sulphonitroprusside which is violet in color.
Na2S + Na2 [Fe(CN)5NO] Na4[Fe(CN)5NOS]
(sodium nitroprusside) (sodium sulphonitroprusside) (violet color)

Reaction of Nitrogen and Sulfur test together:


Sodium thiocyanate (ionic form of nitrogen and sulfur together in the extract) reacts
with ferric chloride to give ferric thiocyanate, a blood red color complex.
3NaSCN + FeCl3 Fe(SCN)3 + 3NaCl
(ferric thiocyanate) (blood red color)
41

During the preparation of Lassaignes extract, if the organic compound containing both
nitrogen and sulfur is fused with an excess of sodium metal, instead of sodium
thiocyanate, sodium cyanide and sodium sulfide are formed.

NaSCN + 2Na NaCN + Na2S

In such cases, the tests for NaCN and Na2S can be performed. Thus in such cases
individual test for nitrogen and sulfur will be positive.

Reaction of Halogens:
Sodium halide (ionic form of halogen in extract) reacts with silver nitrate to give the
precipitate of silver halide. NaX + AgNO3 AgX + NaNO3
If X=Cl, then AgCl (silver chloride) will be formed which is white color precipitate soluble in
Ammonium hydroxide.
If X=Br, then AgBr (silver bromide) will be formed which is pale yellow color precipitate
soluble in excess of Ammonium hydroxide.
If X=I, then AgI (silver iodide) will be formed which is yellow color precipitate insoluble in
Ammonium hydroxide.

Reaction of Layer test:


Chlorine water is used as an oxidizing agent and Br-(ionic form of bromine in extract)
gets converted into Br2 as per the following equation
2NaBr + Cl2 2NaCl + Br2 and Br2 gives orange color to organic layer.
Chlorine water oxidises I- (ionic form of iodine in extract) into I2
2NaI + Cl2 2NaCl + I2 and I2 gives violet color to organic layer.

SUMMARY
We have learnt about the detection of following extra elements in the given organic
compound:
42

nitrogen, sulfur, chlorine, bromine and Iodine.

HOW TO RECORD

AIM: To detect the presence of extra element(s) in the given organic compound.

APPARATUS REQUIRED: China dish, funnel, test tubes, ignition tubes, capillary tubes, glass
rod, wire gauze, pair of tongs, test tube holder, dropper, and spatula.

CHEMICALS REQUIRED: Organic compound whose extra element is to be determined,


sodium metal, ferrous sulfate, sodium nitroprusside, ferric chloride solution, silver nitrate,
aq. NaOH, dil. Sulfuric acid, dil. Nitric acid, dil. Hydrochloric acid, conc. Nitric acid

OBSERVATION TABLE:

Sl.No. Experiment

Observation

Inference

Dirty green

Nitrogen is

Test for Nitrogen

To the Lassaignes extract (1-2 ml), precipitate

present

add ferrous sulfate (50-75mg).


[If no precipitate is formed, add a
few drops of dilute sodium
hydroxide solution to get the
precipitate.]

Prussian blue color

Now heat this mixture gently with


shaking for 1 minute and add
dilute sulfuric acid into it.

Test for Sulfur

Violet color
43

Sulfur is

To the sodium extract (1-2 ml) add formation

present

1ml of freshly prepared sodium


nitroprusside solution.
3

Test for Nitrogen and Sulfur Blood red color

Nitrogen and

present together

Sulfur are

Take 1-2 ml of sodium extract and

present

acidify it with dilute hydrochloric

together

acid. Add 2-3 drops of ferric


chloride solution.

Test for Halogens:

White precipitate

Silver Nitrate Test When nitrogen soluble in

present

and / or sulfur are absent

Ammonium

Acidify 1-2ml of sodium extract

hydroxide solution

with dilute nitric acid. Add silver


nitrate solution (0.5ml)

Chlorine is

or
Pale yellow

Bromine is

precipitate soluble

present

in excess of
Ammonium
hydroxide solution
or
Yellow precipitate

Iodine is

insoluble in

present

Ammonium
hydroxide

Silver Nitrate Test When nitrogen White precipitate

Chlorine is

and / or sulfur are present

present

soluble in
44

Acidify 1 ml of sodium extract

Ammonium

with dilute nitric acid (2-3ml).

hydroxide solution

Boil the mixture to reduce the

or

original volume and now perform

Pale yellow

Bromine is

the silver nitrate test.

precipitate soluble

present

in excess of
Ammonium
hydroxide solution
or
Yellow precipitate

Iodine is

insoluble in

present

Ammonium
hydroxide

When the silver nitrate test is Carbon tetrachloride

Bromine is

positive (precipitate is obtained) or chloroform layer

present

then the layer test is to be (bottom layer) turns


performed for identification of orange in color
the correct halogen
Take 1-2 ml of sodium extract in a Carbon tetrachloride

Iodine is

test tube and add 1 ml of carbon or chloroform layer

present

tetrachloride or chloroform. Now, (bottom layer) turns


add 1 ml of chlorine water and violet in color
shake the test tube well.
No coloration of the

Chlorine is

bottom layer is seen

present

RESULT: The given organic compound no. --- contains --------- as an extra element.
45

PRECAUTIONS:

1) Do not touch the sodium metal with your fingers; always use forceps to handle it.
2) Never throw the sodium metal into sink.
3) Unreacted sodium metal should be carefully decomposed by adding small amount of
ethyl alcohol into it.
4) Always use freshly prepared ferrous sulfate and sodium nitroprusside solution.
5) Fuse 3-4 ignition tubes so that the extract should be concentrated.
6) Remove sodium cyanide and sodium sulfide before performing the test for halogens by
using nitric acid before performing the silver nitrate test.
7) In layer test, shake the test tube by putting thumb on the mouth of the test tube.
8) When the liquid compound gets evaporated very rapidly before its fusion with sodium

metal, a paste of the compound with sodium carbonate may be used or after the
addition of liquid compound in the ignition tube, solid sodium carbonate may be added.

Alternative Green Procedures of LASSAIGNES TEST


In the present practice, the use of metallic sodium for fusion with organic compound is
terribly hazardous and is a cause of great worry and concern in a student laboratory. The
idea of fusion with sodium metal is to convert the water insoluble organically bound extra
elements to water-soluble sodium salts which can be easily detected by various tests.

46

A non hazardous and safe procedure:


Use of zinc and sodium carbonate instead of metallic sodium
Organic sample (about 10 mg) is thoroughly mixed with an intimate mixture of Zn dust (40
mg) and Na CO (60 mg) powder in a fusion tube [no need to weigh any of the chemicals,
2

take very small amount of sample and approximately 5 times of Zn and 5 times of Na2CO3],
heated first gently and then strongly in the flame till it becomes red hot and kept at red-hot
condition for two minutes. The bottom part of the fusion tube is plunged into 5 ml of
distilled water taken in a mortar, ground well with the pestle and filtered. With the filtrate
tests for S, N and Cl / Br / I are carried out as usual as in the case of Lassaignes Test.
Notes: 1. The fusion tube must be heated VERY STRONGLY, KEEPING AT RED HOT CONDITION
THROUGHOUT FOR AT LEAST TWO MINUTES. If not properly heated, fusion is not properly
done (as in case of sodium also), and thus expected observation (colour change) may not be
made. In that case, it is advised to repeat the fusion.
2. The amount of water taken in the mortar must be within 5 ml.; otherwise, the

solution will be too dilute to respond to tests, described below.


3. While carrying out the test for nitrogen, ferrous sulphate crystals are to be added; not the
solution. This is to avoid excessive dilution.
4. Acidification must be carried out with dilute sulfuric acid, not with HCl.
5. No ferric chloride should be added.
6. This method works well with covalently bound nitrogen of aromatic compounds (except pnitrophenol and p-aminophenol).

S. No.
1.

Experiment
0.5 ml filtrate + FeSO

Observation
Prussian blue color

Inference
N present

Violet color

S present

Black ppt

S present

crystal heat + dil. H SO


2

2.

3.

a) 0.5 ml filtrate + Sodium


nitroprusside
b) filtrate + dil acetic acid +
Lead acetate
filtrate + FeCl

Blood red
47

N+S both present

4.

filtrate + 2 drops
Conc HNO boil
3

cool + AgNO

Curdy white ppt


(Soluble in
NH OH)
4

Chlorine present
Bromine present
Iodine present

Pale yellow ppt


(Partly soluble in
NH OH)
4

Yellow ppt
(Insoluble in
NH OH)
4

Green context:
This experiment totally eliminates the risk of explosion and fire hazard which are
often met while carrying out the same experiments using metallic sodium.
The aforesaid zinc-alkali mixture (prepared by intimately mixing 2 parts by weight of
zinc dust and 3 parts by weight of sodium carbonate can be stored in a stoppered
bottle for more than a month.

48

EXPERIMENT - 2

DETECTION OF FUNCTIONAL GROUPS IN KNOWN ORGANIC COMPOUNDS

I Objective:
To carry out the detection of reactive functional groups in a known organic compound
containing functional groups such as alcohol, aldehyde, ketone, nitro, amine, etc.

II Principles:
In an organic compound one or more reactive functional groups could be present giving the
compound its characteristic properties. Generally these are present due to the heteroatoms
(nitrogen and oxygen) found in the molecules giving rise to functional groups like alcohols,
aldehydes, ketones, amines, etc. These functional groups impart a particular reactivity to the
molecule towards oxidizing/reducing agents or can for addition products using which, we will
detect them.

III Procedures

The following procedures describe the methods of detection of various functional groups
in a molecule based on their reactivities.

Detection of unsaturation in a molecule using Bromine test: Alkenes and alkynes will add
bromine across the multiple bonds unless there are electron withdrawing groups on the
multiple bonds. One observes the rapid disappearance of the red-brown bromine color.
Aromatic compounds can react with bromine more slowly to give bromine substitution and
the formation of HBr, which can sometimes be observed by placing a piece of wet litmus
paper over the mouth of the test tube.

Warning: the reagent deteriorates with the

formation of HBr so compare the results with a blank.


49

Experiment

Observation

Inference

Into a dry, clean test tube, dissolve 0.1 mL of a The bromine color
liquid (or 50 mg of a solid) in 1 mL of

should disappear. To

methylene chloride. Add a 2% solution of

continue addition

bromine in a dichloromethane dropwise with

until the bromine

agitation.

color disappears to

A positive test requires five or more drops of

ensure all

bromine solution to reach a persistent red-

unsaturation has

brown color.

reacted.

Detection of unsaturation using Permanganate test:

Aqueous permanganate rapidly

oxidizes double and triple bonds while being reduced to MnO2, a brown precipitate.
Therefore, disappearance of the purple color and formation of a brown precipitate in
minutes is a positive test. However, other compounds react slowly with the reagent
including alcohols, aldehydes, phenols, and aromatic amines so interpret your results
carefully and look for corroboration from the other tests.

Experiment

Observation

Into a clean test tube, dissolve 0.1 mL of a liquid (or The

Inference

purple

50 mg of a solid) in 1 mL of 95% ethanol or 1,2- color

of

dimethyoxyethane. Add a 1% solution of aqueous permanganate


potassium permanganate dropwise with agitation.

disappears

Detection of Aldehydes and Ketones using 2,4-DNP:

Hydrazines such as 2,4-

dinitrophenylhydrazine react with the carbonyl group of aldehydes and ketones to give
colored precipitates. Normally the reaction is fast but heating may be necessary. The test
solution is prepared using sulfuric acid and 95% ethanol. Later, if you wish to make a
derivative of your compound, you can use a different 2,4-DNP solution prepared with HCl
50

and methanol. This usually gives a slower forming precipitate which often provides a
derivative of higher purity (and higher mp). However, the slow formation of the precipitate
is not desirable when looking for a qualitative test signal
Experiment

Observation

Dissolve only 1 drop of your liquid compound (or Yellow

Inference
to

10 mg of your solid) in a minimum number of orange or red


drops of 95% ethanol in a test tube. Add 1 mL of precipitate
the 2,4-DNP test solution and agitate.

If a formation

precipitate does not form in 10 minutes, heat on a


water or steam bath for a few minutes.

Tollens Test for Aldehydes and other easily oxidized functional groups. In this test, a
stabilized silver ion is reduced to elemental silver by an easily oxidized compound, such as an
aldehyde. The aldehyde is oxidized to a carboxylic acid. Prepare the Tollens reagent
immediately before you plan to use it
Experiment

Observation

Preparation of Tollens reagent:


Mix 3 mL of Tollens solution A (aqueous silver
nitrate) with 3 mL of Tollens solution B (10%
aq. NaOH) resulting in the formation of solid
silver oxide. Add 10% ammonium hydroxide
solution dropwise, with agitation, until the
silver oxide just dissolves. This produces a
silver ammonium complex and is the Tollens
solution you will use for the test.

Into each of 3 clean, dry test tubes, add 2 mL


51

Inference

of the Tollens reagent which is freshly A positive test is the


prepared as above. Dissolve 10 mg of a solid formation of a silver
(or 1 drop of a liquid) unknown in the mirror as the elemental
minimum amount of bis(2-ethoxyethyl)ether silver adheres to the
required to give a clear solution (less than 1 wall of the glass tube.
mL).

Add the test compound solution

dropwise, with agitation, to the first test tube. This test is to be


Mix vigorously and allow the solution to performed only after
stand. For the blank, simply add 0.5 mL of getting a positive result
bis(2-ethoxyethyl)ether.

for the 2,4-DNP test.

Warning: Wash any minor amounts of residual Tollens reagent into a sink and flush with
water. The reagent forms silver fulminate which is very explosive. The test solutions can be
disposed of in a jar labeled for that purpose. The silver mirror can usually be washed clean
with soapy water and a scrub brush. If not, see your instructor.

Detection of alcohols using Chromic Acid Test: In this test, a chromium oxidant is used to
oxidize alcohols and aldehydes and is visually detectable by a color change of the chromium
oxidant. Aliphatic aldehydes are oxidized in less than a minute, aromatic aldehydes take a bit
longer. Since the condition of the acetone is critical, it is wise to carefully run the blank with
just acetone to be certain that the acetone itself is not giving a false positive. Warning:
Cr(VI) compounds are considered suspect carcinogens and should be handled carefully.
Experiment

Observation

Dissolve 10 mg of a solid (or 1 drop of a liquid) The orange color of the


compound in reagent grade acetone in a clean, Cr(VI) ion is replace by
dry test tube. Add a few drops of chromic acid the green color of Cr(III)
52

Inference

solution one drop at a time with shaking. as

the

chromium

is

Aldehydes and primary and secondary alcohols reduced indicating that


are oxidized very quickly. Tertiary alcohols are oxidation
not oxidized.

functional

of

the

group

has

occurred.
The chromic acid solution is prepared by
dissolving 1.0 g of CrO3 in 1.0 mL of
concentrated sulfuric acid and then carefully
diluting with 3 mL of water.

Detection of esters using Ferric Hydroxamate Test: If you have a carbonyl compound which
is not an aldehyde or ketone or carboxylic acid, it could be an ester. One test for esters is the
ferric hydroxamate test whereby the ester is converted to a hydroxamic acid (HOHN-C=O)
which will give a positive ferric chloride test. Since enols can give a positive ferric chloride
test, first test your compound with ferric chloride solution.
Experiment

Observation

Dissolve 10 mg of solid (or 1 drop of liquid) A deep burgundy color


compound in 1 mL of 95% ethanol, add 1 mL of 1 is positive.
N HCl, and then a 1-2 drops of 5% ferric chloride
solution. If you obtain a color other than yellow,
the test cannot be used. Otherwise, the test is
conducted as follows: dissolve 50 mg of solid or 2
drops of liquid in 1 mL of 0.5N hydroxylamine
hydrochloride in 95% ethanol and 0.2 mL 6N
NaOH. Heat to boiling for 2-3 minutes, then cool
and add 2 mL 1N HCl. If the solution becomes
cloudy, add 1-2 mL of 95% ethanol to clarify. Add
1 drop of 5% ferric chloride solution. If a color
53

Inference

forms and then fades, add additional drops of 5%


ferric chloride until the color persists. The color is
due to a complex between the hydroxamic acid
and the ferric ion.

Detection of Phenols using Ferric Chloride test: Just as enols can form colored complexes
with ferric ion, phenolate ions can as well. Therefore, this test is designed to convert the
weakly acidic phenols to their conjugate base which can then complex with ferric ion. Not all
phenols will give a positive test.
Experiment

Observation

Inference

If the phenol is water soluble, add a few drops of A deep red, green,
2.5% aqueous ferric chloride solution to a 3% or blue color is
aqueous solution of the phenol.

positive.

If the phenol is not water soluble, dissolve 20 mg of An intense color is


the solid (or 1 drop of the liquid) in 1 mL of a

positive

test.

methylene chloride and add 1 drop of pyridine. Add Use phenol as a


3 drops of 1% ferric chloride in methylene chloride.

known.

Detection of methyl ketones using Iodoform test: In this test you will convert the methyl
ketone to a triidomethyl ketone which is then cleaved to form iodoform, HCI 3, a yellow solid.
Acetone gives a nice positive test so be certain that no traces of acetone are in your
glassware. The color of the iodine will disappear more slowly in the later additions.
Experiment

Observation

In a large clean test tube or a vial, place 100 mg

After standing for 15

of a solid or 5 drops of a liquid compound. Add 2

minutes, a pale yellow

mL bis(2-ethoxyethyl)ether, 5 mL of water, and 1

precipitate

54

Inference

of

mL of 10% NaOH, and mix well. Add a total of 3

iodoform (mp 119-

mL of iodine-potassium iodide solution in six

121oC) is a positive

equal portions, stopper and shake well after each

test

addition. Caution: seal the tube carefully and

ketone.

for

methyl

avoid skin contact with the iodine solution.


The solution should be slightly yellow. Heat if
necessary and shake again to force the iodine to
react. When the color is slightly yellow, add
water to nearly fill the test tube or container,
stopper, and shake vigorously.
The iodine-potassium iodide solution is prepared
from 10 g of iodine and 20 g of potassium iodide
in 100 mL of water.

Detection of Amines using Hinsburg Test: If you have a basic compound which you believe
to be an amine, you can corroborate your suspicion and determine if you have a primary,
secondary, or tertiary amine using the Hinsberg test. You will react the amine with a sulfonyl
chloride forming an insoluble sulfonamide of a primary or secondary amine or the soluble
salt of a tertiary amine. The insoluble sulfonamide of a primary amine will be made soluble
in base (via removal of the slightly acidic proton on N) but that of a secondary amine will not
(no proton on N to remove).
Experiment

Observation

Add 100 mg of a solid or 0.1 mL of a liquid Formation of a


compound,

200

mg

of

p-toleuenesulfonyl precipitate under acidic

chloride, and 5 mL of 10% KOH solution to a clean conditions suggests


test tube.

that the previously


soluble sulfonamide
55

Inference

Stopper the tube and shake it for several was of a primary


minutes.

Remove the stopper and heat the amine.

mixture on a steam bath for 1 minute. Cool the


solution and if it is not basic to pH paper, add
additional KOH solution.

If a precipitate has formed, add 5 mL of water If the precipitate does


and shake vigorously. If there is no precipitate, not re-dissolve in the
add 5% HCl until the solution is just acidic when basic solution, it is
tested by pH paper.

indicative of a
sulfonamide of a
secondary amine.
If no precipitate has
formed at any stage,
the initial amine could
have been a tertiary
amine.

ALTERNATE SET OF FUNCTIONAL GROUP TESTS


Experiment

Observation

Alcohols

If the alcohol is

Ritters test

tertiary, the purple

This test, based on the ability of primary and permanganate color


secondary alcohols to be oxidized by an acetic will persist as a rose
acid solution of potassium permanganate, color after 1 or 2 drops
distinguishes these alcohols from tertiary have been added.
alcohols. The permanganate ion is purple, but
56

Inference

when reduced the color changes to brown; a If the alcohol is primary


positive test is the disappearance of the purple or secondary, the
color.

solution will decolorize

To 3 mL of glacial acetic acid (caution- the permanganate and


corrosive!) contained in a small test tube, add remain clear until
2 drops of your liquid (or about 20 mg of a sufficient
solid) and mix thoroughly. Add dropwise, with permanganate has
swirling to mix the contents after each been added to oxidize
addition, a saturated aqueous solution of all of the alcohol.
potassium permanganate and note any change
in the color of the solution.
Lucas Test

Tertiary alcohols give

The Lucas test distinguishes between primary, an immediate


secondary, and tertiary alcohols and is based separation (emulsion)
on the rate of formation of the insoluble alkyl of the chloride,
chloride. To be reliable the alcohol should be secondary alcohols
soluble in water.

require about 5 min,

To 0.5 mL of the alcohol add quickly 3 mL of but most primary


the hydrochloric acid-zinc chloride reagent at, alcohols do not react
room temperature. Close the tube with a cork significantly in less than
and shake it; then allow the mixture to stand.

an hour.

Aldehydes and Ketones

Development of purple

Schiff's Fuchsin Test

color.

The intensely colored triphenylmethane dye


fuchsin reacts with bisulfite (a source of SO2) to Ketones do not
produce the colorless "leuco" form of the dye. respond to this test
Aldehydes, but not ketones, react with this when perfectly pure,
"leuco"

dye

to

produce

new but the color reaction


57

triphenylmethane dye possessing a similar is very sensitive and


fuchsia color.

responds to mere

To a few drops of the unknown to be tested, in traces of an aldehyde


4-5 mL of water, add about 1 mL of the fuchsin
test reagent
Aromatic Hydrocarbons

Benzene and its

Confirmation can be obtained from the Friedel- derivatives give an


Crafts alkylation test described here.

orange-red color;

Place about 100 mg of anhydrous aluminum naphthalene and


chloride in a small, dry Pyrex test tube and phenanthrene as well
heat it strongly with the tube held almost as their derivatives give
horizontally so as to sublime the chloride onto blue-purple colors; an
the cooler wall of the tube. While the tube is anthracene ring
cooling, prepare in the hood in another small produces a green color.
test tube a solution of about 20 mg of
unknown in 10 drops of chloroform (cautionchloroform is toxic). Add this solution to the
test tube containing the freshly sublimed
aluminum chloride by dropping in directly onto
the salt and note the color, if any, where they
meet.

Carbohydrates
Osazone test
Place 0.2 g of the sample in a test tube and add
0.4 g of phenyl hydrazine hydrochloride, 0.6 g Needle-shaped yellow
of crystallized sodium acetate, and 4 mL of osazone crystals will be
distilled water. Place the test tube in a beaker observed for glucose
58

of boiling water. Note the time that the test and fructose whereas
tube was immersed and the time of the lactosazone shows
precipitation. After 20 min, remove the test mushroom shaped and
tube from the hot water bath and set it aside maltose produces
to cool. A small amount of the liquid and solid flower-shaped crystals.
is poured on a watch glass. Tip the watch glass
from side to side to spread out the crystals,
and absorb some of the mother liquid with a
piece of filter paper, taking care not to crush or
break up the clumps of crystals.
The formation of tarry products due to
oxidation of the phenylhydrazine may be
prevented by the addition of 0.5 mL of
saturated sodium bisulfite solution. This should Observe the violet
be done before heating if it is desired to isolate colour at the junction
the osazone and determine its melting point.

of the two liquids.

Molisch Test
In

test

tube,

add

ml

of A blue black colour is

the test carbohydrate solution and 2 drops of observed which is


-naphthol solution. Carefully incline the tube indicative of presence
and pour dropwise conc. H2SO4, using a of polysaccharides.
dropper,along the sides of the tube.
Iodine Test
Add 2 drops of iodine solution to about 2 mL of
the carbohydrate containing test solution.

Results: Please write out your results in a tabular format as described in this handout.

59

EXPERIMENT - 3

COMPLETE ANALYSIS OF UNKNOWN COMPOUNDS

I Objective:
To carry out the detection of elements and reactive functional groups in a unknown organic
compound leading up to its identification through derivatization and melting point
determination.

II Principles:
In an organic compound one or more reactive functional groups could be present giving the
compound its characteristic properties. Generally these are present due to the heteroatoms
(nitrogen and oxygen) found in the molecules giving rise to functional groups like alcohols,
aldehydes, ketones, amines, etc. These functional groups impart a particular reactivity to the
molecule towards oxidizing/reducing agents or can for addition products using which, we will
detect them. In addition to functional group detection, analysis of heteroatoms present will
be examined through the Lassaignes test to fully identify the unknown compound.

III Procedures
Solubility tests:

The first thing that is important is to evaluate the solubility of the compound in range of nonpolar to polar solvents. This will give you an idea as to the polarity of the molecule and what
solvents you can use for the various tests.

For these tests, you should use approx. 30 mg of your compound in 1 mL of solution to give
you a 3% solution. We will use 3% as a determination of solubility. If your compound is not
completely soluble as a 3% solution, you can call it insoluble or partially soluble. Moreover,
some compounds which are not soluble at room temperature may be soluble at higher
60

temperature. Therefore, after obtaining data at room temperature, heat your solution in
water bath at 70C or higher and observe any further changes. The results you record may
have more meaning later when you know more about your unknown compound. You will
test the solubility/reactivity of your compound in 4 different solutions.

V. SCHEME OF ORGANIC ANALYSIS

The Scheme of Analysis may be divided into five parts


1.Preliminary tests
2.Detection of elements
3.Detection of Characteristic groups
4.Confirmatory tests
5. Determination of melting point (in case of solid)
6. Confirmation by preparing a solid derivative for identifying the organic compound.
Procedure: Into 4 labelled test tubes, add 1 mL of: water; 5% aq HCl; 5% aq NaHCO3; and 5%
aq. NaOH. To each tube, add approx. 30 mg of the unknown. Shake or stir for a few seconds.
Record your observations. It may take several minutes for the unknown to appear to dissolve
or react. After suitable time, heat the four test tubes in a water bath and observe. Look for
colour changes, evolution of gas, any evidence of reaction such as precipitates, and
enhanced solubility.
Record your observations.

61

1) Tests to find whether aliphatic or aromatic

Experiment

Observation

(i)Ignition test.

Inference

(a) Burnt with a non-

A small quantity of the

smoky flame

substance is ignited on nickel

(b) Burnt with a smoky

spatula

luminous flame

Presence of aliphatic
substance
Presence of aromatic
substance

(ii)Nitration test:
A little of the substance is
added to a mixture containing

Presence of aliphatic
Colorless solution

substance

Yellow solution or precipitate

Presence of aromatic

2mL con. Sulfuric acid and 1mL


conc. Nitric acid taken in test
tube. It is then heated on a

substance.

boiling water bath for about


half an hour and then poured
into cold water taken in the
beaker
(2 Test to find out whether unsaturated or saturated
i)Action of dilute potassium

(a) Immediate de-

Presence of

permanganate: A little of the

colorization

unsaturated compound

substance is shaken with water


and one or two drops of dil.

Presence of easily
b) Slow de-colorization

potassium permanganate solution

oxidizable substance
like phenol, nitro
phenol, amines,
benzaldehyde, etc.

ii)Action of bromine water: A little

Presence of

of the substance is dissolved in

Decolorization without the

suitable solvent( alcohol/ water)

formation of a precipitate.

then a little bromine water is


added

unsaturated substance

Presence of saturated
No Decolorization

62

compound.

(iii) Action of bromine in


Carbon tetrachloride: A

Presence of

little of the substance is dissolved

(a) Decolorization without

in carbon tetrachloride and

the evolution of hydrogen

bromine in carbon tetra chloride

bromide

unsaturated substance

Presence of saturated

is added and shaken

substances
(b)Decolorization with the
evolution of hydrogen

Presence of easily

bromide

brominated
compounds like

(c) Decolorization with

phenols, aromatic

formation of a precipitate.

amines etc.

3 Action of conc. Sulphuric acid: A dissolves gradually on

Presence of aromatic

little of the substance is warmed

hydrocarbon

heating

with conc. H2SO4


Presence of basic

4.Action of sodium hydroxide

White precipitate which

substance like aromatic

dissolved in excess of acid

amines

(a) Ammonia is evolved

Presence of amide

(b) substance dissolved

Presence of acidic

solution:
1) A little of the substance is
boiled with dilute sodium

substances like acids

hydroxide solution

phenols and their


derivatives

(c)Separation of oil or

Presence of anilides

formation of an emulsion

(d)Solution turns deep yellow

Presence of

in colour

nitrophenols

(2)The given compound is boiled


63

with20% sodium hydroxide for


half an hour then cooled and

Presence of aromatic

acidified with dil HCl

esters and amides

5. Action of Sodalime:
A little of the substance is mixed

a) Ammonia gas is evolved

with thrice its mass of dry


sodalime in a dry test tube and

Presence of amides and


amines

(b) Kerosene like smell

Presence of acids

6 Action of sodium bicarbonate:

Brisk effervescence of carbon

Presence of acids

to one mL saturated solution of

dioxide

heated. The smell of the issuing


gas is noted.

sodium bicarbonate solution little


of the substance is added
7 Action of Metallic Sodium:

Brisk effervescence

To a little of the substance ( if

Presence of alcohols,
acids and phenols.

solid dissolve in dry benzene) in a


dry test tube a small piece of
metallic sodium is added
8 Action of ferric chloride

(a) Violet color

Presence of phenol

substance in Water or alcohol a

(b) A flocculent white

Presence of -naphthol

few drops of neutral ferric

precipitate

solution: To a little of the

chloride is added
(c) green color changing to a

Presence of -naphthol

white precipitate

(d) Buff colored precipitate

Presence of benzoic
acid, cinnamic acid or
phthalic acid

64

9 Action of Borsches reagent


(2,4-DNP test)
: To one mL of Borsches reagent a A yellowish orange

Presence of aldehydes

little of the substance is added

precipitate is obtained

or ketones

: A little of the substance is added

Violet color developed within

Presence of aldehydes

to 1mL Schiffs reagent taken in

two minutes

and heated over a water bath for


five minutes. Cooled and little
water is added
10 Action of Schiffs reagent

test tube and shaken well

11 Action of Tollens reagent


: A little of the substance is added

(a) Black or brown

Presence of polyhydric

to about 2ml Tollens reagent in a

precipitate

phenol

(b) Bright silver mirror is

Presence of aldehydes,

formed

reducing sugars such as

clean test tube and heated in a


boiling water bath

glucose, fructose,
maltose etc.
12 Action of Fehlings solution
: Fehlings solution A and Fehlings
solution B are mixed in equal

Reddish brown precipitate is

Presence of aldehydes,

volumes. To 1 mL of this reagent a

formed

polyhydric phenols, and

little of the organic compound is

reducing sugars.

added and heated on a boiling


water bath

13 Action of Molishs reagent


To a solution of substance in
water added a few drops of

Violet ring is formed at the


65

Presence of

alcoholic solution of -naphthol.

junction

Carbohydrates.

Then added about 1mL of


con.H2SO4
along the sides of the test tube
without disturbing

IV Confirmatory Tests
A If nitrogen is present is present as detected from Lassaignes test, the following tests are
conducted. Besides the following tests for those groups for which indications are got are also done.

1 Action of sodium hydroxide solution. A

(a) Ammonia is evolved Presence of amides

little of the substance heated with sodium


hydroxide

(b)Separation of oil

Presence of anilides

and formation of an
emulsion

2 Action of Soda-lime: A little of the organic

(a) Ammonia is evolved Presence of amides and

substance is heated with excess of dry sodalime

amines
(b) Amine is produced
Presence of amino
acids, toluidines and
anilides

3 Biuret test
A little of the substance is gently heated in a
dry test-tube until it melts and then

A violet colour is

Presence of diamide like

solidifies. The residue is dissolved in a little

produced.

urea.

4 Action of nitrous acid: A little of the

(a) Liberation of

Presence of aliphatic

substance is dissolved in dilute hydrochloric

nitrogen with the

primary amines.

water and a dilute solution of copper


sulphate is added followed by sodium
hydroxide solution drop by drop.

66

acid, cooled in ice water and a 10%solution

formation of alcohol.

of sodium nitrite is added with shaking till it


is slightly in excess

Presence of secondary
(b) Separation of

amines

yellow oil.
Presence of tertiary
(c) Reddish brown

amines

solution is obtained.
5 With the solution obtained above the
following tests are done.
(i)To one portion of the solution an alkaline
solution of -naphthol is added.

A scarlet red

Presence of aromatic

precipitate isformed.

primary amines.

water. The ether is evaporated off and

Blue or green solution

Presence of secondary

Liebermanns nitroso reaction is conducted

is obtained.

amines.

(ii)A portion of the solution is extracted


with ether. The ether extract is washed with
sodium hydroxide solution and then with

with the residual oil.

(iii)To another portion, dilute sodium


hydroxide solution is added and then
shaken with little ether.

Ether layer becomes


deep green.

Presence of
tertiaryamines.

7. Mulliken and Barkers reaction. A little of

Bright silver mirror or

the substance is dissolved in alcohol. A few

black precipitate is

drops of calcium chloride solution is added

obtained.

and pinch of zinc dust. Boiled for five


67

Presence of nito group.

minutes, cooled and filtered into a tube


containing Tollens reagent.
8 Reduction of nitro group to amino group.
A little of the substance is treated with few
ml of dilute hydrochloric acid and a pinch of
zinc dust. Heated for some time and
filtered. With the filtrate the following tests
are done.
(b) To another portion of the filtrate dil.

An offensive smell is

hydrochloric acid is added, cooled in ice and

produced

sodium nitrite solution is added in excess.

Presence of nitro group

Then alkaline -naphthol solution is added


A scarlet precipitate is
obtained

Presence of aromatic
nitro group.

B If halogen is present, the following tests are conducted. Besides the following tests, tests for those
groups for which indications are got also done
1 Action with litmus. A little of the

(a) soluble and acidic to

Aliphatic halogen

substance is shaken with hot water and

litmus

substituted acids

tested with litmus


Presence of aromatic
(b) Insoluble and acidic

halogen substituted
acids

(c) Insoluble and neutral

Presence of halogen
substituted
hydrocarbons, ketone
etc.

2 Action with silver nitrate solution. A

(a) Precipitate of silver

Halogen is in the side

little of the substance is boiled with

halide is formed

chain

68

sodium hydroxide solution for


15minutes. Cooled, acidified with dil.

(b) No precipitate of

nitric acid and then added silver nitrate

silver halide

Halogen is in the ring

3 Action of alcoholic silver nitrate. To a

(a) Precipitate of silver

Presence of halogen in

little of the substance 2 ml of alcoholic

halide is obtained

the side chain

(b) No precipitate of

Presence of halogen in

silver halide

the ring.

solution.

silver nitrate solution is added and


warmed gently

C If sulphur is present, the following tests are conducted. Besides the following tests, the tests for
those groups which indications are got also done.

1 Action of alcoholic sodium

Ammonia is evolved

hydroxideTo a little of the

Presence of thiourea or
sulphonamide

substance 2 ml of alcoholic
sodium hydroxide solution
isadded and warmed gently.
2 Action of con. hydrochloric

Pungent smell

Presence of

acid. To alittle of the substance

substitutedthiourea.

2 ml con. HCl is added and


warmed gently.
3 Fusion with alkali. A little of

(a) Hydrogen sulphide is

the substance is fused with

evolved

sodium hydroxide dissolved in

Presence of thio urea

Presence of sulphonic acid

water and hydrochloric acid is

(b) Sulphur dioxide is evolved

added

with the formation of phenol


Presence of aminosulphonic
69

(c) No phenol is formed but

acid

precipitate of barium sulphate


when barium chloride is added
(d) Ammonia is evolved during

Presence of sulphonamide

fusion. No phenol is formed.


Sulfur dioxide is evolved on
adding acid

D. If nitrogen, halogens and sulphur are absent, tests for the following groups for which indications
are got, are done.
Aldehydes

1.Schiffs reagent test is conducted

Violet colour is obtained

Presence of aldehydes.

2 Borsches reagent test is conducted.

A yellow precipitate is

Presence of aldehydes.

obtained.
3 Tollens reagent test is conducted

Bright mirror or black

Presence of aldehydes

precipitate is obtained.

4 Fehllings solution test is conducted.

Red precipitate is

Presence of aldehydes.

obtained.
5 Sodium bisulphite test is conducted:

White crystalline

A little of the substance is added to a

precipitate is obtained.

Presence of aldehydes.

saturated solution of sodium bisulphite


and shaken well.

6 Semicarbazide test

White crystalline

: Dissolved 0.5g of semicarbazide

precipitate is obtained.

hydrochloride in5ml of water and added


0.5g of anhydrous sodium acetate. It is
warmed to get a solution. Then added a
70

Presence of aldehydes.

small quantity of the substance and


warmed on a water bath.

II. Ketones.

1 Borsches reagent test is conducted.

An yellow precipitate is

Presence of ketones.

obtained
2 Semicarbazide test is conducted.

White crystalline

Presence of ketones.

precipitate is obtained
3 Sodium bisulphate test is conducted.

White crystalline

Presence of ketones

precipitate
4 Iodoform test is conducted

Yellow precipitate with

Presence of ketones

characteristic odour is

containing the CH3-CO-

formed

group

Effervescence

Presence of acids

Kerosene smell obtained

Presence of acids.

III.Acids

1 Tested with sodium bicarbonate


solution.

2 Sodalime test is conducted


3 Ester formation test is conducted
About 0.5g of the substance is heated
gently with about 1 ml of ethanol and

Pleasant fruity smell.

few a drops of conc. sulphuric acid for

Presence of acids

about 1minute. Cooled and poured into


a few ml of water in test-tube.
4 s-Benzylthiouronium salt test

White crystalline

:About 0.25 g of the acid is dissolved in

precipitate

2 ml of warm water. The acid is


71

Presence of acids

neutralised by adding a few drops of


NaOH solution(phenolphthalein can be
used asan indicator) .Then 2 drops of
NH4Cl are added followed by 0.5 gof sBenzylthiouronium chloride in 2ml
water. It is cooled in ice.
5 Fluorescein reaction. Fused together
in a dry test-tube a small quantity of the
substance with an equal amount of

A reddish solution having

Presence of

resorcinol after moistening the mixture

an intense green

dicarboxylic acids.

with two drops of conc. Sulphuric acid.

fluorescence is produced.

Cooled, dissolved in water and then


added excess of sodium hydroxide
solution.

IV.Phenols
1

(a) Violet blue or green

Neutral ferric chloride solution test is

colour.

Presence of phenol

conducted. A little of the substance is


treated with neutral ferric chloride

(b) A flocculent white

Presence of -

solution

precipitate.

naphthol.

2.Liebermanns nitroso reaction

Red solution which turned

Presence of phenol.

: To two drops of melted phenol, added

to green or blue on adding

little solid NaNO2. Heated gently for 1

sodium hydroxide

minute. Cooled and added 4 drops of

solution.

conc.H2SO4. Diluted cautiously with


water.
3Phthalein fusion reaction:

Red, bluish-purple, blue


72

Presence of phenol.

About 2 drops of melted phenol is mixed

green fluorescene, green

with a small quantity of phthalic

or very faint green

anhydride in a dry test-tube. 2 drops of

colouration.

conc. H2SO4
are added. The mixture is heated at
about 150 oC for2 min. Cooled and
excess of 10 %NaOH solution is added.
4 Benzoylation (Schotten

Crystalline white

Baumannreaction) is conducted

precipitate.

Presence of phenol.

: Dissolve about 0.25 g of phenol in


about 5mlof 10 % NaOH solution
contained in a boiling tube. About 1 ml
of benzoyl chloride is added. The boiling
tube is corked and shaken vigorously for
about 15 min.

5 Azo-dye formation reaction

Orange, scarlet, dark red,

: Dissolve 2 drops of aniline in 1 mldil.

brownish red solution or

HCl and well cooled in ice. A few drops

precipitate is obtained.

Presence of phenol.

of saturated NaNO2
solution is added. The diazonium
solution thus obtained is added to a well
cooled solution of phenol in aqueous
NaOH solution.
VI. Esters.
1 Hydrolysis. A little of the substance is

White precipitate is

refluxed with concentrated solution of

formed.

Presence of ester.

sodium hydroxide and then acidified


with conc. Hydrochloric acid.
2 Hydroxamic acid formation.

A violet or a deep red-

To a few drops of the substance, added

brown color developed


73

Presence of ester

0.2g of hydroxylamine hydrochloride

immediately

and about 5ml of 10% sodium hydroxide


solution and the mixture gently boiled
for 2 minutes, cooled and acidified with
dilute hydrochloric acid and then added
a few drops of ferric chloride solution.
VII Carbohydrates.
1 Concentrated sulphuric acid test is

Charring with smell of

Presence of

conducted. Warmed a little of the

burnt sugar

carbohydrate.

2 Sodium hydroxide test is conducted. A little

Solution turned yellow or

Presence of

of the substance is boiled with sodium

brown. Caramel smell is

carbohydrate.

hydroxide solution

emitted

3 Molischs test is conducted.

A deep violet ring is

Presence of

formed.

carbohydrate.

4 Treated with

Bright silver mirror or

Presence of reducing

Tollens reagent

black precipitate.

sugar.

5 Fehlings solution test

Red precipitate is formed.

Presence of reducing

substance with conc. Sulphuric acid.

Is conducted: Warmed with Fehlings

sugar.

solution.
6 Osazone test is conducted

Yellow crystals are formed. Presence of


carbohydrate.

VIII. Hydrocarbons.
1 Odor is noted.

Kerosene like smell

Presence of

observed.

hydrocarbons

.2Sulphonation is conducted

Substance has gone into

Presence of

: To 1 ml of fuming H2SO4

solution

hydrocarbon.

contained in a test tube, 2drops of the


substance are added and shaken well
74

for 3 min.

3Nitration is conducted

4Picrate test is conducted :

Red or yellow precipitate.

Presence of

Saturated solutions of naphthalene and

polynuclear

picric acid, both in benzene are

hydrocarbons.

prepared separately. These two


solutions are mixed in a watch-glass and
allowed to evaporate.

Confirmation by preparing a solid derivative.


The final step in the analysis of a sample organic compound is the preparation of a suitable
solid derivative.

Preparation of Derivatives
1. Derivatives for Aldehydes and Ketones.
a) Semicarbazone derivative:
Procedure: Dissolve a small amount of the compound (approx. 200 mg) in ~2 mL 95% Ethanol.
Add aqueous solutions of 0.5 g semicarbazide hydrochloride (in 0.5 ml) followed by aqueous
sodium acetate (0.7 g in 0.5 ml). Mix the liquids thoroughly. If and only if two liquid layers form,
add 95% ethanol dropwise while shaking the tube to just obtain a clear single phase. Heat the
tube in a 60-70 water bath for about 10 minutes, and then cool the mixture to room
temperature, and then in an ice bath. If no crystals have formed on cooling in ice, add a few
drops of water to the tube and scratch the inside wall of the tube under the liquid surface with
a glass rod. Collect the solid by suction filtration on filter paper in a Hirsch funnel, wash the
crystals with several mL cold water, and then dry the solid on filter paper. Record MP.
75

b) Oxime Derivative Preparation


Disolve 0.2 gm. hydroxylamine hydrochloride in 2 mL. of water and 1 mL. of 3M NaOH. Add 0.2
gm. of your unknown to the above solution. Warm the mixture in a boiling water bath for 10
minutes. Cool in an ice bath and collect the crystals using vacuum filtration. Dry overnight and
determine the compounds melting range.
2. Derivatives for Aromatic amines, Phenols, Alcohols.
Procedure-- Add 2 mL of aqueous sodium hydroxide solution into a large test tube. Add the
amine, about 0.20 g if its a solid. Shake the solution vigorously and add about 5 drops of
benzoyl chloride. Stir vigorously for 10 minutes, and then few drops of aqueous HCl (this helps
the amide to crystallize). Cool on ice, filter the lumpy product through the Buchner funnel and
wash with 3x5 mL of cold water, then 2 x 3 mL of aq. HCl (to wash off unreacted amine), and
then 2 x 3 mL of NaOH/water (to wash off unreacted benzoyl chloride). Recrystallize using a
minimum of ethanol, perhaps adding water as necessary. Dry under suction and record MP.
(the filtrate may contain excess benzoyl chloride, decompose it by adding excess NaOH solution
and discard it after 15 min.)
3. Derivatives for Alcohols, Phenols.
3,5-Dinitrobenzoate Derivative Preparation
Dissolve 0.25 gm. Of sample in 0.25 g of 3,5-dinitrobenzoyl chloride and heat gently for few
minutes, cooled and poured into 10 ml of cold water taken in a beaker. The ppt. is filtered and
washed with cold water, dried, and subjected to MP determination.
76

4. Bromo derivatives for Phenols, Acids, Amines, Amino acids.


In a large test tube, add 0.2 mL of Bromine in 1 ml of glacial acetic acid. This solution is added to
a mixture of 0.2 gm of unknown dissolved in 1 mL glacial acetic acid and shaken vigorously.
Continue adding brominating solution dropwise, with mixing, until the orange bromine color
persists. Warm the solution on a water bath for few minutes. Add 2 mL of H 2O and shake
vigorously. Collect the precipitate by filtration. Wash the precipitate with cold water.
Recrystallize from an appropriate Methanol: H2O mixture.
5. Derivatives for Acids.
a) Ethyl Ester derivative:
200 mg of compound is dissolved in 2 ml of ethanol, 2 drops of conc. H 2SO4 is added and the
solution is heated to reflux for 5 min. The solution is allowed to stand for 10 min, 5 ml of water
is added and the solid is filtered.
6. Preparation of derivatives by Hydrolysis (for amides, anilides, esters etc.)
0.5 g of O.S. and 10 ml of 20% NaOH solution are boiled in a beaker for 15 min. and then cooled.
The mixture is then acidified with dil HCl. The solid is filtered, washed with cold water,
recrystallized from water of alcohol. Determine MP.

77

7. Derivatives for Aromatic Hydrocarbons. The main reactions carried out for the preparation
of derivatives for aromatic hydrocarbons are (a) nitration or (b) side chain oxidation.
(a) Nitration.
Nitro derivatives can be prepared for benzene, toluene etc. About 1 ml of fuming nitric acid and
1 ml of conc. sulphuric acid are mixed and cooled in ice-water. About 0.25 ml of benzene or
toluene (or other hydrocarbon) is added to the nitrating mixture. Then the mixture is heated on
a boiling water bath for half an hour, till a drop of mixture poured into water crystallizes
immediately. The mixture is then poured into cold water taken in beaker and stirred well. The
crystals are filtered at the pump, recrystallised from dilute alcohol, dried and then melting point
is noted.

(b) Side chain oxidation.


For aromatic hydrocarbons containing side chain like toluene or sidechain like xylenes, side chain
oxidation can be effected for the preparation of their derivatives. About 0.25 ml (0.25g) of the
substance is mixed with 0.25 g of sodium carbonate in 10 ml of water was taken in a 100 ml
beaker and heated to boiling (keep it on a wire gauze over a burner) and portionwise add 1 g of
potassiumpermanganate under gentle boiling until slight permanent pink colour is obtained.
The excess pink colour of permanganate is decomposed by addition of excess sodium
bisulphate. The mixture is allowed to cool and is carefully acidified with dilute sulphuric acid.
The mixture is then again refluxed for 5 min and then cooled. The excess of MnO 2 is removed

78

by addition of little sodium hydrogen sulphite, if required. The white solid is filtered,
recrystallised from warm water or alcohol, dried and MP was determined.

Note: you can also prepare some derivative which is specific to a particular compound like
anthraquinone for anthracence and phthalic anhydride for phthalic acid etc. Please follow
known standard methods for preparation of those derivatives.
Results: Report the unknown number, your name, the results of the solubility tests mp or bp and
how you distinguished between these possibilities and arrived at your conclusion.

79

EXPERIMENT - 4

CHEMICAL SEPARATION OF A MIXTURE OF TWO COMPOUNDS

Objective:
To carry out the separation of two compounds with different functional groups.

I Principles:
Often in organic chemistry, we are required to separate and purify two or more different
chemical compounds present in a mixture. This can be achieved by utilizing the differing
reactivities of the functional groups present in them.
For example, a mixture of a carboxylic acid and a phenol can be separated using their varying
acidities. Both these molecules have acidic hydrogen atoms, but a carboxylic acid has greater
acidity than phenol. Hence, a weak inorganic base will only react with the organic carboxylic
acid making its corresponding carboxylate salt, while not reacting with the phenol of lower
acidity. In this way, the acid can be separated from the phenol.

II Experimental Procedure

Case 1: Mixture of a carboxylic acid and a phenol

Experiment

Observation

Take 100 mg of the mixture in a test tube


and dissolve in 10 mL dichloromethane
solvent
Transfer the contents to a 50 mL
separating funnel and allow for the layers
to separate
80

Inference

To this add a 10% aqueous sodium Brisk

effervescence

bicarbonate (a weak inorganic base) very should be observed


carefully, with gentle shaking

with separation of
layers

Collect the top and bottom layers


separately in test tubes and label the test
tubes as organic and aqueous; DO
NOT MIX THEM.
The bottom layer is of dichloromethane
containing the unreacted phenol and the
top layer is the aqueous layer containing
the sodium salt of the carboxylic acid
Repeat the bicarbonate treatment of the
dichloromethane layer 2 more times in
the separating funnel and combine all
organic and aqueous extracts
To the aqueous layer containing the Precipitate formation
carboxylate salt, add 6 M HCl CAREFULLY occurs
dropwise and swirl the test tube. Check
with blue litmus paper to ensure acidity
The test tube containing the precipitate is White Crystals

This is the

boiled on a water bath with a boiling appear, which are

carboxylic acid

stone until precipitate has gone into isolated by filtration

form the original

solution.

mixture

Allow the test tube to cool in an ice-bath.


Organic

layer

of

dichloromethane A layer separation is

containing the phenol is treated with an observed, whereby


aqueous solution of a strong base (5% once again the
81

NaOH solution) and checked for basicity bottom layer is the


using red litmus paper

organic layer

Separate the layers into test tubes and to If a precipitate


the aqueous layer, add 6 M HCl appears, heat the
CAREFULLY dropwise, check with blue solution with a
litmus paper for acidity.

boiling stone until all


solids dissolve

Cool the solution in an ice-bath

Crystal formation is

This is the phenol

observed

of the original
mixture

RESULT: The given organic mixture no. --- contains --------- as constituents.

82

EXPERIMENT - 5

SINGLE STEP SYNTHESIS

Objective:
To provide hand on experience on the synthesis of organic compounds, purification etc.

a) Reduction of Benzophenone by NaBH4

Principle: Ketones are compounds reducible to secondary alcohols by the action of a


reducing agent like sodium borohydride (NaBH4). The reaction proceeds by activation of the
carbon oxygen double bond followed by nucleophilic addition of the hydride attached to
boron to form the secondary alkoxide. Subsequently, addition of water and acid to the
alkoxide generates the desired alcohol product. In this example, benzophenone an aromatic
ketone is to be reduced to biphenyl methanol.

Procedure:
1. In a 25-mL Erlenmeyer flask, dissolve 1.35 g of benzophenone in 9 mL of methanol. If
available, place a magnetic stir bar into the flask, and place the flask on a stir plate.
2. In a second 25-mL Erlenmeyer flask, dissolve 0.3 g of sodium borohydride in 4.5 mL of cold
distilled water.
3. Use a Pasteur pipette to add the aqueous solution of sodium borohydride one drop at
a time to the solution of benzophenone. Swirl the reaction mixture between the additions of
each drop in order to disperse any cloudiness. Do not add more sodium borohydride until
the cloudiness caused by the previous drop has disappeared.
83

4. When all the sodium borohydride has been added, use a magnetic stirrer to stir the
reaction mixture until a heavy slurry of diphenylmethanol crystals has formed.
5. Decompose the excess sodium borohydride by slowly adding the slurry of crystals and
solvent to a mixture of 30 g of crushed ice and 3 mL of concentrated hydrochloric acid in a
250 mL beaker. (CAUTION: Prepare the latter by adding the concentrated hydrochloric acid
to the crushed ice, not vice versa. Do this step in a fume hood. Wear gloves and protect
your eyes.)
6. Collect the diphenylmethanol by suction filtration. Wash the crystals with two 15- mL
portions of water. Leave the aspirator (or vacuum pump) running for about 30 minutes in
order to dry the crystals as best you can.
7. Re-crystallize the diphenylmethanol using hexane as the solvent.
(Hint: About 25-30 mL of solvent will be required, use 50 oC water bath to warm solvent.)
After the crystals have been dried and weighed, place about 0.1 g of the diphenylmethanol
in each of two clean, dry test tubes (13 75 mm) and stopper the tubes with corks. These
small samples will be used in Parts B and C.
9. Determine the yield, melting point, mixed melting point with authentic standard (if
available), and %yield of the pure diphenylmethanol. Store your crystals in a suitably labeled
glass vial and hand it to your instructor for grading.

Wt. of Product X Mol. Wt. of benzophenone


% yield = ------------------------------------------------------------ X 100
Wt. of benzophenone X Mol. Wt. of Product

84

b) Reduction of camphor

Principle: Ketones are compounds reducible to secondary alcohols by the action of a


reducing agent like sodium borohydride (NaBH4). The reaction proceeds by activation of the
carbon oxygen double bond followed by nucleophilic addition of the hydride attached to
boron to form the secondary alkoxide. Subsequently, addition of water and acid to the
alkoxide generates the desired alcohol product. In this example, camphor, a naturally
occurring aliphatic ketone to be reduced to its corresponding secondary alcohol.

Procedure:
1. Place 0.0132 mole of your assigned camphor in a 50 mL round-bottom flask (RBF). Add 8
mL of methanol and cool the flask in ice in a 250 mL beaker ice bath. While the flask is
cooling, weigh 0.016 mole of NaBH4 in a vial and keep it covered. Crush any large lumps with
a spatula.
2. Add the NaBH4 to the camphor solution in 3 portions with cooling, as described in steps ac below.
a. Remove the flask from the ice bath, and add about 2 scoopula-tips full of NaBH4. Swirl the
mixture for 5-6 minutes. Crush any solid lumps that form with a thin, bent spatula.
Whenever the bubbling becomes vigorous and/or the flask no longer feels cool, return it to
the ice bath. Continue to swirl and crush lumps.
b. After about 6 minutes, cool the flask in the ice bath again; repeat step a.
c. Repeat step b, adding the remaining NaBH4.
3. After about 5 minutes, let the solution warm to room temperature. Swirl it occasionally
for another 10 minutes. Meanwhile, clamp the flask to a ring stand with the flask in contact
with a steam bath (STEAM OFF) and equip it with a reflux condenser. (Cooling water tubing

85

will not be needed for this short reflux time.) When bubbling becomes slow, heat the
solution to boiling, and reflux it for about 5 minutes.
4. Put about 30 mL of ice into a 125 mL Erlenmeyer flask, cool the reaction mixture for a
minute or two, and pour it with stirring onto the ice. Rinse the reaction flask twice with 2 mL
portions of methanol, and add the washes to the reaction mixture.
5. Stir the mixture until the ice melts. Collect the solid by suction filtration. Wash the filtered
solid with cold distilled water, and press it dry with a glass stopper.
6. Transfer the crude reaction product to the same 125 mL Erlenmeyer flask. Save a few
small crystals of crude product to use as seed crystals, in case the product oils out as the
re-crystallization solution cools.
7. Dissolve the crude product in a minimum volume of hot 1:1 methanol-water mixture. (The
flask containing the solvent and the flask containing the crude product should be on the
steam bath.) Then add cold water dropwise until a permanent turbidity is seen. Add hot
methanol again until the solution just becomes clear. Set aside to cool slowly. Then cool in
ice, and filter the re-crystallized product using suction filtration. Use two pieces of filter
paper.
8. Cover the Buchner funnel with parafilm and store it in a beaker.

Second Lab Period


9. Weigh your dry product and determine the yield.
10. Functional Group Tests
Two classical tests will be performed in order to determine whether or not the product is an
alcohol and whether or not it is contaminated by unreacted camphor. For a and b below,
record your observations and results in tabular form, and give general equations for
positive test results.
a. Chromic Acid Test for Alcohols
In the chromic acid test, 1 and 2 alcohols are oxidized by solutions of chromic acid (H2CrO4)
to form carboxylic acids and ketones, respectively; tertiary alcohols do not react with H2CrO4.
A positive test is indicated by the formation of a green-blue precipitate.
86

Add a match head size of solid or 1 drop of liquid to 1 mL acetone in a test tube. Then add 1
drop of chromic acid reagent and shake the mixture. The appearance of an opaque bluegreen color within 2 seconds is a positive test. Perform the test on your product.
b. 2,4-Dinitrophenylhydrazine (2,4-DNP) Test for Ketones
In the 2,4-DNP test, ketones (and aldehydes) react rapidly with 2,4-dinitrophenylhydrazine to
give brightly colored precipitates. Alcohols do not react with this reagent.
Different concentrations of reactants will be used to increase the sensitivity of the test, so
that small amounts of unreacted starting material (camphor) can be detected in the product
from your reduction reaction.
Knowns
Perform this test on the following substances: isoborneol and camphor. For each test, put 1
mL of 95% ethanol in a small test tube. Add (and mix) a spatula-tip full or 5 drops of the test
compound, followed by 1 mL of 2,4-DNP reagent. Mix well. If a precipitate does not form,
boil the solution in the test tube in a steam bath for 1 minute. Cool it in ice. A colored
precipitate is a positive test for a ketone.

Reaction product
Repeat the 2,4-DNP test as above, but use about 0.1 g of your product. If a positive test is
observed, explain why this occurred.

87

c) Synthesis of Phenacetin

Principle: Analgesics are a class of compounds that are used every day for the alleviation of
pain (e.g. head-ache, muscle sprains, fever, etc). The most widely known analgesic
compound, aspirin, has recently come under scrutiny for some of its side-effects. A more
suitable analgesic, acetaminophen (Tylenol), has gained in popularity, owing to few side
effects.
Analogs of acetaminophen also have properties of analgesics, although they are not widely
used because of liver toxicity and carcinogenic properties. Phenacetin is one such analog.
Phenacetin was discovered in the late 19th century and was widely used to reduce fever. The
US FDA, however, banned the use of phenacetin in any commercial products due to its toxic
properties.
The synthesis of phenacetin has not changed much from the initial synthesis of the
compound. Starting from acetaminophen, the alcohol of the phenol is converted into an
aromatic ether. Owing to the increased acidity of the phenolic proton, the deprotonation can
be carried out using a fairly mild base. This type of reaction is called the Williamson Ether
synthesis and, oftentimes, leads to the product in moderate yields.

Experimental Procedures
Obtain a clean dry 25 mL round bottom flask equipped with a stir bar. Place 500 mg of
acetaminophen into the flask. Add 7 mL of 0.5 M ethanolic sodium hydroxide. Stir the
reaction mixture and to reflux. Reflux the mixture for 20 minutes and add 0.40 mL of
iodoethane. Allow the mixture to reflux for an additional 20 minutes. While the mixture is
still hot, filter through a Buchner funnel into a flask containing 20 mL of ice water (ice chunks
should be visible). Filter off the phenacetin and dry. Obtain a crude mass of the product. Re88

crystallize the product from hot water and obtain the mass of the dried product. Calculate
the percent yield for the reaction and obtain a melting point of the pure material.

89

d) Synthesis of 3-Methylpyrazol-5-one
Principle: This reaction is an example of synthesis of a heterocyclic molecule. In this case the
ethyl acetoacetate starting material forms an imine intermediate with its keto functional
group and one of the amine groups of hydrazine (H2N-NH2). In the final step, an amide bond
formation occurs between the second amine group of the attached hydrazine and the ethyl
ester group of ethyl acetoacetate. This leads up to formation of the heterocycle.
Me
O

H2N-NH2
N

OEt

N
H

Procedure:
Place 6.5 g of ethyl acetoacetate in a conical flask and stir magnetically during the slow
dropwise addition of a solution of 2.5 g (0.5 mol) of hydrazine hydrate in 4 mL of absolute
ethanol. The temperature rises during this addition which should be regulated so that a
temperature of about 60oC is maintained. A crystalline deposit separates. After further
stirring for 1 hr at r.t., cool the reaction mixture in an ice bath to complete the crystallization.
Filter it, and washed it with ice-cold ethanol. Take the melting point of the product and
report to the instructor. The pure product melts at 222oC.

90

e) Synthesis of Methyl Orange


Principle: Azo dyes comprise the largest and most important class of dyes. These synthetic
compounds are used as clothing and food dyes, inks for printers and, paint pigments. Azo
dyes can exhibit a wide range of colours (yellow, red, orange, violet, blue) oftentimes
dependent upon the pH of the solution. The generation of colour arises from the high
amount of conjugation through the system. Azo dyes are easily synthesized from the
reaction of an aromatic amine with sodium nitrite and hydrochloric acid to form the
intermediate diazonium salt which, upon treatment with an activated aromatic compound,
yields the product azo dye in good yield. This process allows for the ready formation of a
series of dye compounds through substitution of either the aniline or the activated aromatic
compound. For this experiment, you will be carrying out the synthesis of the azo dye Methyl
Orange which is used as indicator of acids and bases. You will use your synthesized dye to
determine the molar absorptivity () and max .
.

Experimental Procedures
Add 1.0 g of sulfanilic acid monohydrate to a 50 mL Erlenmeyer flask equipped with a stir
bar. Add 11 mL of 0.25 M sodium carbonate and heat to dissolve. Once the entire solid has
dissolved cool the solution to room temperature and add 400 mg of sodium nitrite. Stir at
room temperature until all the solid dissolves, then pour the solution into a100 mL beaker
containing 10 g of ice and 1.0 mL of conc. HCl. Gently swirl the solution until a white
precipitate forms (this is the diazonium salt). Set this beaker aside until needed. In a test
tube, add 0.8 mL of N,N-dimethylaniline and 0.5 mL of acetic acid. Thoroughly mix the
solution together with a pipette and add to the stirred suspension of diazonium salt. Stir the
91

mixture until a thick, coloured paste forms (5 10 minutes) and add 8 mL of 3.0 M NaOH.
Heat the mixture until the solid dissolves and then place into an ice bath. Allow the solid to
crystallize undisturbed (10 15 minutes). Collect the solids via vacuum filtration and wash
with saturated NaCl followed by 5-6 mL of ethanol. Thoroughly dry the solid, obtain the mass
and calculate the percent yield. Dissolve 5 mg of the solid in 50 mL of 1.0 M HCl. Dilute this
solution in a 10 mL volumetric flask to a final concentration 1.0 X 10-5 M. Obtain the max
and calculate the molar absorptivity ().

92

f) Synthesis of Benzylideneacetophenone

Principle: This is an example of aldol condensation.


O

O
OH

CHO
+

Procedure: Place a solution of 1.1 g of NaOH in 10 mL of H2O and 6.5 mL of rectified spirit in
a 100 mL beaker. Immerse the beaker in crushed ice mixture bath, pour 2.6 g of
acetophenone, start the stirring and add 2.3 g of Ph-CHO. Keep the temperature of the
mixing at about 25 oC. Stir vigorously until the solution is so thick that stirring is no longer
effective. Cool the reaction mixture in refrigerator weeklong. Filter the product under
suction, wash with cold water, until the washings are neutral to litmus. The crude chalcone is
air dried, and its mp is recorded.
NB: The substance should be handled with great care as it acts as a skin irritant.

93

g) Preparation of Dinitrobenzene
Principle: In this experiment you will prepare meta-dinitrobenzene from nitrobenzene by
means of a nitration reaction. The overall reaction is shown below.

Figure 1. Nitration of Benzene meta-dinitrobenzene or 1,3-dinitrobenzene

Procedure:
Add 1 mL of nitrobenzene to a 100 mL Erlenmeyer flask and then add 3 mL concentrated
sulfuric acid. Swirl the contents to ensure mixing [CAUTION: Do not touch nitrobenzene
with your hands. It can be irritating to the skin. If you get any on your hands, wash them
immediately and thoroughly under cold running water]. Prepare an ice-water bath for
cooling. After the nitrobenzene has dissolved, add 2 mL of concentrated nitric acid
DROPWISE USING THE DROPPER. It is important that the reaction not get too hot. After
every adding watch the temperature closely. It should not go ABOVE about 70 C but it is
also important that the temperature does go to at least 60 C. Ensure mixing by swirling the
flask gently. Keep the ice bath handy in case the temperature gets too hot.
DO NOT ALLOW THE TEMPERATURE TO RISE ABOVE 70 C. A POTENTIALLY DANGEROUS
REACTION COULD OCCUR.

94

After the temperature in the Erlenmeyer flask starts dropping, place the flask on the water
bath(placed inside the fume hood). Heat at boiling for 15 minutes. Stir the contents with
your glass stirring rod.
Again, if you see any brown gas being evolved, please stand back and notify your
instructor. In some cases there can be a delayed exothermic reaction and there might be
splattering of nitric acid out of the Erlenmeyer.
After 15 minutes of heating, remove the Erlenmeyer flask containing your product and pour
its contents into a beaker containing 100 mL of ice cold water. Stir the solution as you pour it
into the water. The product should form a solid yellow precipitate. You will collect the solid
its contents into a beaker containing 100 mL of ice cold water. Stir the solution as you pour it
into the water. The product should form a solid yellow precipitate. You will collect the solid
product by suction filtration, use your Buchner funnel with a piece of the appropriately sized
filter paper. Wet the filter paper first with a few mL of water and then connect the sidearm
of the filter flask to one of the water aspirators in the lab. Filter the solid.
The yellow crystals must now be washed three times with 15 mL portions of water to
remove all traces of the nitric and sulfuric acids contaminating the product. Remember that
you must disconnect the rubber tubing of the vacuum pump from the filter flask before
adding the wash water. If the product forms into big lumps, transfer it to the watch glass,
break it up into tiny pieces with the spatula and return it to the Buchner funnel for the
washings.
You will purify your product using the technique of recrystallization, using ethanol as the
solvent.
95

Recrystallization
Procedure for Recrystallization of Dinitrobenzene
Place all of your solid dinitrobenzene inside a medium test tube (or small Erlenmeyer flask).
Use your spatula to scrape as much of it as possible from the damp filter paper. Cover the
solid material with ethanol and then heat the test-tube in your boiling water bath so that the
ethanol just begins to boil. Stir the mixture vigorously to aid in the dissolving process. Add
more ethanol, a few drops at a time using your dropper and each time you add more solvent
be sure to bring the solution back to the boiling point by re-heating in the water bath.
Dinitrobenzene is quite soluble in ethanol and you will need only about 2 mL ethanol per
gram of product.
Work quickly and do NOT leave the test tube in the bath so long that your solvent begins to
boil away. Otherwise you will start to DECREASE the volume of ethanol and your compound
will not dissolve.
When all the solid material has dissolved (the solution should now be clear though it will not
be colorless) remove it from the water bath and place it in your test tube rack. Allow it to
cool slowly to room temperature. Do not stir the solution. The most pure crystals form when
the solution is allowed to cool slowly, undisturbed.
When the solution has cooled to room temperature, place the test tube in an ice bath for a
few minutes. Filter your product using the Buchner funnel. You can use a few mL of ice-cold
ethanol to aid in the transfer of the solid from the test tube. Do not use more than 3-5 ml of
the ethanol or you will lose some of your product.

96

If you do not see a precipitate forming, then this means that you have used too much of the
ethanol. You will have to reduce the total volume by placing the test-tube back in the boiling
water bath. After the crystals dry, weigh your compound and calculate the percent yield.
Turn in your sample and Yield Report to your instructor. Be sure to get a melting point range
and include this on your report sheet.

97

h) Diels-Alder reaction between furan and maleic acid


Principle: This is a green approach to conventional [4+2] cycloaddition reaction. In
conventional method, reaction is carried out by refluxing in benzene, which is a very toxic
and carcinogenic solvent. In the given method reaction carried out in aqueous medium
avoiding benzene. This reaction is efficient at room temperature itself and 100% atom
efficient.
O
COOH
O

COOH

H2O

COOH

RT
COOH

Chemicals required:
Furan : 200 mg (3 mmol)
Maleic acid : 420 mg (3.6 mmol)

Procedure: A mixture of furan (200 mg, 3 mmol) and maleic acid (420 mg, 3.6 mmol) in
water (4 ml) was shaken or stirred for 2 hrs at room temperature. The adduct formed, was
filtered, washed with water, dried and recrystallized from aqueous ethanol, m.p. 138-140 oC
Yield =
Green Context:
Reaction carried out in aqueous medium avoiding benzene
Efficient at room temperature itself
100% atom efficient

98

i) Synthesis of dibenzalpropanone (Dibenzylideneacetone): A greenchemical


approach
This is an example of aldol condensation.

Chemicals Required:
Acetone: 0.81 mL (11 mmol)
Benzaldehyde: 2.3 mL (20 mmol)
LiOH.H2O: 42 mg (1 mmol, 10 mol%))
Procedure: In a 25 mL round bottom flask containing a small magnetic bar, the aldehyde and
ketone were taken with ethyl alcohol (5 ml) and lithium hydroxide (42 mg) monohydrate was
added into it. The reaction mixture was magnetically stirred vigorously for 8-10 minutes. The
pale yellow solid precipitated out, 5 g of crushed ice was added and the solid was allowed to
99

settle down. The precipitated pale yellow solid was filtered, washed with water, air dried and
recrystallized with ethanol. The solid was dried and its melting point was measured
(Literature M.p. 120 - 121 C).
Yield =
Precaution: The aldehyde should be free from acid.

Green Context:
Hazardous organic solvents are avoided.
Lithium hydroxide is easy to handle as it is comparatively less hygroscopic than other alkali
metal hydroxide.
Use of catalytic amount of the base.

S. Bhagat, R. Sharma, and A.K. Chakraborti, J. Mol. Cat. A: Chemical 2006, 260, 235-240.
14.

100

j) Preparation of 1, 1-bis-2-naphthol
Principle: This is an example of RADICAL COUPLING REACTION. This demonstrates the
concepts of oxidative coupling, free radical and C-C bond formations.

Chemicals Required:
2-Naphthol - 2.88 g
Iron(III) chloride - 0.7 g
Water - 2 drops
Toluene (for recrystallization)
101

A mixture of of 2-naphthol (1.44 g) and iron(III) chloride (0.35 g) with 1 drop of water in an a
porcelain mortar pestle was grinded for about 20 minutes. The mixture was allowed to stand
for about 2 hrs with a little grinding now and then. The mixture was transferred with water
(40 ml) into a 100 ml beaker and boiled for 10-15 minutes. The mixture was cooled and the
solid was filtered, washed with boiling water (10 ml), dried and recrystallized from toluene.
o

m.p. of the product = 214-217 C.


Yield =
Green Context:
Efficient method
Easily available catalyst
Reaction is performed with simple grinding at room temperature without any solvent
Work up of the reaction involves aqueous medium.

A. I. Vogel, Textbook of Practical Organic Chemistry, Fifth Edition, 1989.

102

m) Synthesis of dihydropyrimidinone: A green THREE component coupling


Principle: This is an example of a multi-component reaction in organic chemistry involving an
active methylene group, aldehyde, and urea derivative.

Mechanism:

103

Chemicals Required:
Ethyl acetoacetate 1.3 g
Benzaldehyde - 1.1 g
Urea 0.7 g
Procedure: A mixture of benzaldehyde (1.1 g), ethyl acetoacetate (1.3 g) and urea (0.7 g),
taken in a round bottom flask was shaken by hand for 2 minutes. The reaction mixture was
o

then heated in a water bath at 90 C for one hour. With progress of the reaction a solid
started to deposit and after one hour the flask is full of solid. The solid was taken out
carefully with a spatula or spoon in a conical flask. The yellow solid was washed with cold
water (1 ml) and then recrystallized from rectified spirit to give a colourless solid, mp 201o

202 C.
Yield =
Green context:
Use of no hazardous organic solvents
No requirement of catalyst
Faster reaction
A. C. Ranu, A. Hajra and S. S. Dey, Org. Proc. Res. Dev. 2002, 6, 817

104

n) TRANSESTERIFICATION REACTION
Synthesis of biodiesel

Mechanism:

Chemicals Required:
Vegetable oil - 100 ml
Methanol - 20 ml
Sodium hydroxide - 3 pellets
Green Procedure:
The finely ground anhydrous NaOH was added into pure (99% or higher purity) methanol (20
ml) in a 250 ml Erlenmeyer flask and stirred vigorously until all the NaOH was dissolved. The
o

pure vegetable oil (100 ml) was warmed to about 40 C in a 250 ml beaker. The warmed up
oil was poured into the methoxide solution with continuous stirring. At first the mixture
would become cloudy, but should soon two layers would separate. This was stirred for 15-20
105

minutes. The contents of the flask were transferred into a 250 ml separatory funnel. The
mixture will separate into two different layers. The glycerol will fall to the bottom, and the
methyl ester (biodiesel) will float to the top. Allow the experiment to sit for an hour. The
stopcock of the separatory funnel was opened and the glycerol was allowed to drain into a
small beaker.

Green Context:
This lab experiment demonstrates three key green principles: the use of renewable
feedstock, catalysis and design for degradation. Vegetable oil is a renewable starting
material as it is derived from growing plants, rather than irreplaceable material like the
earths petroleum and natural gas supplies. The reaction is catalyzed by NaOH making this
process economically viable for the industrial scale production of biodiesel. Biodiesel is an
excellent product as it is environmentally friendly.
Safety:

Methanol: Flammable and poisonous. Dispose excess by allowing it to evaporate


in the fume hood.

NaOH: Very corrosive. Causes severe burns. May cause permanent eye damage.
Very harmful by ingestion.

106

EXPERIMENT - 6

MULTI STEP SYNTHESIS

1. Preparation of 2-iodoxybenzoic acid (IBX) from anthanilic acid


Objective:
To give you idea about how a target molecule is planned and synthesized using a series of
organic reactions.

Principle: In this example we will be preparing an oxidizing agent (IBX) using a two-step
procedure starting from anthranilic acid. The first step begins with the diazotization reaction
of anthranilic acid followed by its conversion to the iodo derivative. Subsequently this iodo
derivative is oxidized using potassium bromate (KBrO3, an inorganic oxidant) to generate IBX.
The noteworthy point here is that iodine is capable to higher oxidation states and hence it
can take up two moles of oxygen in addition to being attached to a carbon atom of the
phenyl ring. The final step is the ring closure between the carboxylate group and the iodine
atom resulting in the IBX structure as shown below.

107

STEP-1
Preparation of 2-iodobenzoic acid from anthranilic acid
NH2
CO2H

NaNO2, HCl
KI

I
CO2H

Procedure:
1. Anthranilic acid (7 g) is dissolved in 50 mL of water containing C. H2SO4 (7 mL) in a
250 mL Erlenmeyer flask. The mixture is swirled to dissolve the anthranilic acid. The
undissloved material is filtered and the filtrate is cooled to 4 oC in an ice-water bath.
2. NaNO2 (3.5 g) is dissolved in water (15 mL) and the solution is cooled in an ice bath.
The cold solution of NaNO2 is gradually added to the cold solution of anthranilic acid.
3. KI (13 g) is dissolved in 1M H2SO4 (25 mL). The solution is added to the diazotized
solution with stirring keeping the mixture in the ice-water bath.
4. The mixture is heated in water bath for 15 min and then cooled in ice bath for 10
min. The solid is collected by filtration under suction. The crude solid is transferred
into another Erlenmeyer flask and Na2S2O3 solution (60 mL) is added and the mixture
is swirled. The solid is filtered and washed with cold water (50 mL). The product is recrystallized from hot water.
5. Purity of the product can be checked by melting point measurement.

108

STEP 2
Synthesis of IBX
O

CO2H

CO2H

I
O

I
O

OH

KBrO3 (7.6 g) is added over a half-an-hour period to a vigorously stirred mixture of 2iodobenzoic acid (8.5 g) and 73 mL of 0.73M H2SO4. During the addition the reaction mixture
is kept below 55 oC. The mixture is warmed to 65 oC and stirred for 2.5 hrs. Cooling to 0 oC,
filtering and washing with 100 mL of water and two 10 mL portions of ethanol give the
product.
Note: IBX is a mild and effective oxidizing agent.

109

2. Conversion of Benzaldehyde to Dilantin (i.e. Phenytoin)


Principle: Dilantin (in the form of its sodium salt) is used as an anticonvulsant for the control
of epilepsy. The step is benzion condensation, which is typically catalyzed by cyanide ion, but
we will use a biological catalyst, thiamine hydrochloride (vitamin B1). This is followed by
oxidation of benzoin to benzil followed by condensation with urea to give the desired
product.

110

STEP 1
Thiamine catalyzed Benzoin condensation
O
CHO

Thiamine-HCl
OH
NH2
N

Me

Me

Cl+
N

OH

. HCl

Procedure:
1. Thiamine.HCl (0.84 g) was taken in a 50mL Erlenmeyer flask. Water (2.5mL) was
added and the mixture was swirled until all the material was dissolved.
2. EtOH (95%; 6mL), NaOH solution (2M, 2.5mL) and benzaldehyde (2.5 mL) were added
to the solution of thiamine hydrochloride. The mixture was swirled thoroughly after
the addition of each component to ensure through mixing of chemicals.
3. The mixture was allowed to stand for seven days. The crystals of benzoin were
collected by suction filtration. Crystals were washed with cold ethanol/water mixture
(5:1, 6mL), dried and kept for subsequent step.

Alternative Procedure:
Thiamine hydrochloride (1.1 g) was dissolved in water (1.5 mL) and diluted with 95% ethanol
(10 mL) and the solution was cooled in an ice-water bath. Cold 3 M sodium hydroxide
solution (2.2 ml) was slowly added over a 5-min period while swirling to ensure thorough
mixing. Benzaldehyde (6.0 mL) was added and the reaction mixture was heated in a waterbath to reflux for 90 min. Upon cooling to room temperature, the product was precipitated
by cooling in an ice water bath to about 10 oC. The crude product was collected by vacuum
filtration and washed with 25 mL cold water. The crude benzoin was re-crystallized from 95%
111

ethanol. After drying on a filter paper the crystals were weighed giving a yield of ____%, and
a sample melted at ____oC.

Note: Thiamine must be fresh and stored cold. Benzaldehyde should be recently purchased
or
distilled before use. The experiment will not work properly if these precautions are not
taken.

Safety Precautions (read before starting the experiment):


Avoid contact between skin, eyes and clothing with all chemicals used in this experiment. Do
not breathe any vapors or dust from these chemicals. Wash hands and any other body parts
that come into contact with any chemicals thoroughly. Benzaldehyde is and irritant and is
toxic (wear gloves). Sodium hydroxide solutions are corrosive and cause burns.

STEP 2

Synthesis of Benzil
This reaction is an oxidation using nitric acid; under acidic conditions, nitric acid produces
nitronium ion. You have seen nitronium in the lecture course, as an intermediate in the
nitration of benzene. Nitronium ion can also oxidize alcohols. In this reaction, it is reduced to
nitrous acid (HONO) and the alcohol in benzoin is oxidized to a carbonyl.

Procedure:
Nitric acid (5 mL) was added slowly to benzoin (2.0 g) in glacial acetic acid (10 mL). Provision
was made to allow the NO2 evolved during reaction to be trapped in a water-ice mixture
(think about how to set up an apparatus to accomplish this). The reaction mixture was
heated at 85-95 oC for 1h. The reaction mixture was allowed to cool to room temperature
before adding water (40 mL) and some ice chips. The pale yellow crystals of benzil were
collected by vacuum filtration and washed with 3 portions (5 mL) of cold water. The product
112

was re-crystallized from methanol by allowing crystallization to begin at room temperature


before cooling in an ice-water bath. The crystals were pressed by a filter paper to remove as
much solvent as possible. The product was obtained in ___% yield, and the crystals melted at
___ oC.

Safety Precautions (read before starting the experiment):


Avoid contact between skin, eyes and clothing with all chemicals used in this experiment. Do
not breathe any vapors or dust from these chemicals. Wash hands and any other body parts
that come into contact with any chemicals thoroughly. Glacial acetic acid and nitric acid are
corrosive (wear gloves) Chloroform and dichloromethane are volatile and toxic. Methanol is
volatile, toxic and flammable.

STEP 3
Synthesis of 5,5-diphenylhydantoin (Dilantin)
Diphenylhydantoin can be synthesized by treating the benzil that you made last week with
urea, in the presence of a strong base. The first step is a nucleophilic attack by a nitrogen
atom of urea. Following deprotonation of the intermediate, migration of a phenyl group
occurs. This process is similar to the [1,2] shifts of carbocations that you have seen in lecture.
The carbonyl carbon atom is not a carbocation, but it does have a partial positive charge due
to the polarized C=O bond.

Procedure:
Benzil (0.5 g) and urea (0.24 g) in 95% ethanol (10 mL) were treated with 9.4 M KOH (1.4 mL)
and heated under reflux for 1.5 h. Any precipitate that formed was removed by vacuum
filtration and diluted with water (35 mL). 10% Hydrochloric acid solution was added
dropwise to this solution until the pH was 4-5 (use pH paper, should take 8-10 mL of acid).
The reaction mixture was cooled in an ice-water bath for about 10 min and collected by
vacuum filtration. 5,5- Diphenylhydantoin was re-crystallized from ethanol and the
precipitate was dried on filter paper.
113

The product (___% yield) melted at ____ oC.

Safety Precautions (read before starting the experiment):


Avoid contact between skin, eyes and clothing with all chemicals used in this experiment. Do
not breathe any vapors or dust from these chemicals. Wash hands and any other body parts
that come into contact with any chemicals thoroughly.
Potassium hydroxide solution causes severe burns and eye damage. 5,5-Diphenylhydantoin
is a potent anti-convulsive drug. Wear gloves during the entire work-up procedure.

114

2A. Synthesis of benzilic acid from benzaldehyde


This is again a three-step procedure. First two steps are common to dilintin.
CHO

KOH

HNO3

Thiamine-HCl

OH

Grind

OH
COOH

Step 3A:
Benzil Benzilic acid rearrangement
(Preparation of Benzilic Acid in Solid State under Solvent-free Condition)
O
OH
O

HO

Mechanism:

Chemicals Required:
Benzil : g
Sodium hydroxide or potassium hydroxide: g
Conc. Hydrochloric acid
Procedure : Benzil ( g) was thoroughly grounded with solid NaOH or KOH ( g) in a dry
mortar with the help of a pestle to make an easy flowing powder. This material was
115

subsequently taken in a dry conical flask fitted with a piece of cotton at its mouth and heated
on a boiling water-bath for 20 minutes. Then it was cooled to room temperature, dissolved
in minimum amount of water (unreacted benzil, if any, was removed simply by filtration) and
the aqueous solution was acidified with conc. HCl with thorough cooling in ice. The
precipitated benzilic acid was filtered, washed with cold water and crystallized from hot
water, if needed.
M.p. 149-151 oC
Yield : 0.86 g (80%.)

Green Context:
Solvent-free procedure
Atom efficient

116

EXPERIMENT 7: EXTRACTION OF NATURAL PRODUCTS: EXTRACTION OF CAFFEINE FROM


TEA LEAVE
Objective: Caffeine is a minor constituent of tea, coffee, and other natural plant materials.
The major constituent of tea is cellulose which is not water soluble. Caffeine is water soluble
but so are some tannins and gallic acid which is formed in the process of boiling tea leaves.
The latter two components can be converted to their calcium salts which are insoluble in
water. The caffeine can then be extracted from the water by methylene chloride in almost
pure form. Some chlorophyll is often extracted at the same time.
Procedure:

Place 6 g of tea leaves, 2 g of calcium carbonate powder and 75 mL of water

into a 500 mL beaker. Boil the solution on a hot plate for 20 minutes with occasional stirring.
Cool the solution but, while it is still warm, vacuum filter through a Buchner funnel using a
fast filter paper, if available. Normally, hot solutions are not vacuum filtered. Rinse the
leaves with 20 mL of water. Carefully press out as much filtrate as possible since the caffeine
is in the aqueous layer. Rinse again with 20 mL of water.
Cool the solution to room temperature and pour it into a 500 mL separatory funnel.
Extract with 20 mL of methylene chloride. In a departure from normal procedure, it will be
necessary to vigorously shake the separatory funnel in order to extract the caffeine. First,
relieve the pressure buildup as soon as you mix the two liquids. Then shake vigorously for 10
seconds and relieve pressure, repeat the shaking two more times. An emulsion will probably
form.
To break the emulsion formed in the methylene chloride layer, slowly drain the
methylene chloride layer through a small amount of anhydrous magnesium sulfate in a
powder funnel with a loose cotton plug (a tight plug will prevent drainage).
Extract the aqueous solution once again with a 20 mL of methylene chloride,
repeating the steps above to collect the lower layer. Combine the methylene chloride
extracts and, if necessary, dry further with additional anhydrous sodium sulfate.
117

The methylene chloride solution will be concentrated on a water bath. Tare weigh a
100-mL r.b. flask and transfer the dried methylene chloride solution to it. Be certain that
there is no sodium sulfate in the solution. Stripping this solution to dryness will take less
than 5 minutes. You will be left with a small amount of residue with a greenish tinge. Obtain
the weight of crude caffeine by difference.
Add 5-8 mL of hot acetone to dissolve the crude caffeine and transfer the solution to
a 50 mL Erlenmeyer flask for re-crystallization. Add a few drops of petroleum ether until you
reach the cloud point (caffeine is less soluble in this mixed solvent and is just beginning to
precipitate) and then cool the solution. If you do not get a precipitate, you may have used
too much acetone, carefully boil off the excess on a steam bath using a boiling stick for
ebullation.
Suction filter the caffeine using a small Bruckner funnel and petroleum ether as a
transfer/rinse solvent. A second crop of caffeine may form in the filtrate as the solvent
evaporates. This second crop can also be collected by vacuum filtration but keep it separate
from the first crop. After air drying, weigh each crop and record your % caffeine recovered
from tea.
The sublimation will be performed as described by your instructor. You will use 50
mg of your caffeine to make a salicylate derivative and sublime the remainder (which should
be at least 50 mg).
You will not take a mp of the purified caffeine which would require a sealed capillary
to prevent sublimation near the melting point.
Caffeine is a base which can react with acids to form salts. A well characterized salt
of caffeine is caffeine salicylate formed by using salicylic acid. This derivative of caffeine has
an accurate melting point. Later this semester, you will be required to make solid derivatives
of other compounds.

118

Preparation of Caffeine Salicylate. Using an analytical balance weigh 25 mg of caffeine and


20 mg of salicylic acid (both can be plus or minus 1-2 mg) and dissolve them in 2 mL of
toluene in a small 25 mL Erlenmeyer flask by warming on a steam bath. Add 0.5 mL
(dropwise) of petroleum ether and allow the mixture to cool and crystallize. If necessary,
cool in an ice-water bath. Collect the crystals by vacuum filtration, air dry, weigh, record the
o

yield, and take a mp (lit mp 137 C).

119

EXPERIMENT 8: PURIFIVATION TECHNIQUES

Objective: To isolate the desired product from the reaction mixture by using various
techniques.

1. COLUMN CHROMATOGRAPHY
THEORETICAL BACKGROUND

The most common approach for separations of compounds from a mixture of compounds is
column chromatography, where the adsorbant (usually silica gel or alumina) is packed into a
long glass tube, called column, and a continuous flow of solvent (eluent) is passed through
the adsorbant. As with TLC, the different compounds in the sample are carried along, going
back and forth between solvent and adsorbant. The relative rates of these movements are
dependent upon the relative strengths of the attraction of these compounds to the
adsorbant and upon their solubilities in the eluent. With a polar adsorbant, the more polar
compounds in the sample will be held more tightly to the stationary phase and move more
slowly down the column. Therefore, when the same adsorbant and solvent system are used
in both, TLC and column chromatography will yield the same order of elution for a given
series of compounds. So, TLC is a valuable tool for rapidly devising appropriate solvent
systems for column chromatography separations.
Unlike TLC, column chromatography is not limited to only one solvent system for a given
separation. Since the solvent is continuously supplied at the top of the column, it is possible
to change the eluting solvent at any point in the separation. This advantage allows us to
separate complex mixtures containing compounds of widely varying polarity. For separations
of such samples, the column is prepared in a relatively non-polar solvent. As the less polar
components are eluted from the column, the polarity of the solvent can be progressively
increased, thus allowing selective removal of compounds in order of increasing polarity.

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In addition to this versatility for separating complex mixtures, column chromatography has
the advantage that it can be used for preparative scale separations and purification, while
TLC is usually limited to analytical procedures. The amount of material to be separated can
be varied from milligrams to kilograms by increasing the size of the column and the amount
of adsorbant. For convenience and economy, the separation of a mixture of ferrocene and
acetylferrocene in our experiment will be done on a semi-micro scale.

EXPERIMENTAL PROCEDURE

1. Sample Preparation
Weigh out about 0.20 g the given mixture in a 25 mL conical. Dissolve the sample in 1 mL of
CH2Cl2. Add about 0.5 g silica gel (add little more if required) to the sample solution and stir
continuously with a glass rod unless it becomes a dry slurry.

2. Column Preparation
a. Obtain about 30 mL of hexane in a clean and dry 100 mL flask. Add some silica and make a
wet slurry.
b. Fill about 2/3 of the column with hexane.
c. Pour the silica slurry in hexane into the column using a funnel. Tap the column gently as
you pour the silica.
d. When all of the alumina has been added, open the stopcock and allow the solvent to drain
until its level is about 1 cm above the top of the silica. Use the excess solvent in the flask to
wash down any silica adhering to the inside column walls.
e. Carefully transfer the sample prepared in step 1 to the column. Rinse the beaker with a
few drops of hexane, and add this to the column. Allow the sample to run onto the column.
Insert a small piece of cotton by a stick and firmly place it above the sample slurry.

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3. Chromatography Procedure
a. Label three 100 mL beakers as 1, 2, 3 and so on and take their initial weights. These
beakers will be used to collect the components (fractions) of the mixture as they elute from
the column. After the sample has been applied to the column, begin eluting as described
below.
b. Fill the top of the column with hexane. Open the stopcock and allow the column to run
continuously. DO NOT CLOSE THE STOPCOCK and DO NOT ALLOW THE LEVEL OF ELUTING
SOLVENT TO FALL BELOW THE COTTON AT THE TOP OF THE COLUMN. (Add solvent as
needed.) Collect the initial eluent in a waste beaker. When the first colored band is about 1
cm from the bottom of the column, start collecting the fraction in the beaker labeled 1, and
continue eluting with the same solvent mixture until the entire first colored band has been
eluted from the column.
c. Change the polarity of the eluting solvent and collect the intermediate fraction in the
beaker labeled 2, until the second colored band is about 1 cm from the bottom of the
column,
d. Continue this operation until all the compounds come out of the solution.

4. Isolation and Identification of Products


a. Put a small boiling chip and place the conical in a water bath and concentrate the
solutions.
b. Take the final weight of the conical containing the solids. Find the mass of solid recovered
from each fraction.
c. Take little solid out and spot each of these solutions on the TLC plate.
d. Spot standard solutions of standard compounds on the same TLC plate.
e. Run the TLC plate in the eluent (as suggested by your instructor) and check the purity of
the compounds you eluted out as well as identity which one is which by matching the spots
with the spot of the reference product.

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Clean-Up: At the end of the period, clean the chromatography column as demonstrated by
an instructor.

CALCULATIONS

Calculate the total % recovery.


= (weight of eluted product / weight of starting mixture) x 100%

2. Resolution of Racemic
1Phenylethylamine via Diastereomer Formation with (2R, 3R)Tartaric Acid (or vice versa)
PURPOSE
To separate the enantiomers of phenylethylamine and to learn the technique of measuring
optical rotation with a polarimeter.
INTRODUCTION
The separation of the enantiomers, resolution, of phenylethylamine can be achieved by
exploiting the very different solubilities in methanol of their diastereomeric complexes with
a single diastereomer of tartaric acid. Recall that enantiomers have identical physical
properties, while those of diastereomers differ. When a hot solution of racemic (R,S)1
phenylethylamine and an equivalent amount of (R,R)tartaric acid in methanol is allowed to
cool, the less soluble (S)amine (R,R)tartrate complex crystallizes out of solution (see
Scheme 1, below).

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Scheme 1. Solubility of (R,R)-tartrate salt complexes in methanol.

Isolation of this crystalline material and treatment with excess aqueous sodium hydroxide
allows liberation of the (S)amine from the tartrate. Just as with the extraction experiment
you have already done, we now have a situation where we can separate a relatively non
polar organic material (the amine) from a polar one (the tartrate salt). After extraction the
pure (S)amine is obtained by evaporating the methanol solvent on a rotary evaporator. In
theory, the more soluble diastereomeric complex that remains dissolved in the filtrate can
also be isolated by treating the filtrate with sodium hydroxide solution, extracting the
liberated amine into an organic solvent to separate it from tartrate, and evaporating the
methanol solvent. Scheme 2 details the steps involved in this resolution experiment.

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Scheme 2. Resolution and isolation of phenylethylamine enantiomers.

It is very rare for a single crystallization of this kind to prove sufficient to fully separate two
enantiomers. Invariably, the complex containing the (S) enantiomer crystallizes along with
some of the complex containing the (R) enantiomer, and not all of the (S) enantiomer
complex crystallizes. Thus we are left with a mixture of some of the (S) containing complex
and most of the (R) containing complex in solution. If it was required to achieve absolute and
complete separation of a pair of enantiomers the crystallization experiment would be

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performed several times, in sequence, gradually enriching the percentage of pure single
enantiomer.

PROCEDURE
Work in pairs throughout this experiment. Though the first part of this twoweek
experiment is rather short, the best results are obtained by being careful at
this stage and performing the simple operations cautiously.
Safety Considerations: The reacemic amine mixture has a strong and irritating odor.
Dispense this material and perform all manipulations in the hood. The sodium hydroxide
solution you will use during week 2, will cause serious burns if it comes into contact with your
skin. Wear gloves and eye protection at all times when handling this material.
Lab 1: Preparation of the tartrate salt complexes.
Weigh 6.25 g (41.6 mmol) of (R,R)tartaric acid into a 250 mL Erlenmeyer flask, add 100 mL
of methanol, and heat the mixture gently on a hotplate for a few minutes. To the warm
solution slowly add 5 g (5.3 mL, 41.2 mmol) of (R,S)1phenylethylamine. Care! Too rapid
addition will cause the mixture to boil over. Once the addition is complete, let the solution
boil very gently on the hotplate for at least 15 minutes (use a boiling stick). Let the solution
cool to room temperature, seal the flask with parafilm, label it with your names and
laboratory section, and place it in the location indicated by your TA or instructor. The
solution must now be left to slowly crystallize until next week.

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Lab 2: Resolution of enantiomers.


Part 1: Regenerating the S-amine
1. Collect the crystals by vacuum filtration and wash the crystals with methanol.
Dry the crystals on a piece of large filter paper and record the yield. Do not
discard the filtrate!
2. Dissolve the crystals in 50 mL of distilled water in a 100 mL beaker and add 4.5 mL of 50 %
NaOH solution. Caution: concentrated NaOH is very caustic. Ensure that the solution is basic
by testing with pH paper.
3. Transfer the aqueous solution to a separatory funnel, add 30 mL of diethyl
ether (the density of diethyl ether: 0.71 g/mL). Perform the extraction
technique.
Diethyl ether is extremely volatile and flammable. When using it for
extractions release the pressure after just a few shakes of the funnel
and do extractions in the hood.
4. Return the aqueous layer to the separatory funnel and repeat the extraction
with a second 30 mL portion of ether.
5. Collect the second portion of ether, combine with the ether extract from the
first extraction, and then add more anhydrous Na2SO4. Dry the ether layer for
10 min. NOTE: Diethyl ether will form H-bonds with water, so there is more
water in the ether layer. We have to add more drying agent and wait for a
longer time to remove water.
6. Weigh a dry, clean, and empty 250 mL round-bottomed flask and record the
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weight. Decant the dried extracts into this flask and give it to your TA who will demonstrate
the operation of the rotary evaporator to remove the solvent.
7. Weigh the round bottomed flask again after removing all the solvent and record the
weight. The difference of the weights is the amount of your Samine.
8. Prepare the following solution of S-amine in a clean vial: 0.1 g S-amine and 9.9 g methanol.
9. Measure the optical rotation (the specific rotation).
Part 2: Regeneration of the R-amine:
1. Weigh a dry and clean 250 mL round bottomed flask and record the weight.
Transfer the filtrate to this flask and evaporate the solvent on the rotary evaporator.
2. Weigh the round bottomed flask again after removing all the solvent and record the
weight. The difference of the weights is the amount of your Ramine-(R, R) tartrate.
3. Add 50 mL of distilled water and 4.5 mL of 50 % NaOH solution. Caution:
concentrated NaOH is very caustic. Ensure that the solution is basic by testing with pH
paper.
4. Repeat steps 3-7 of the Part 1 procedure for regenerating the S-amine.
5. Prepare the following solution of R-amine in a clean vial: 0.1 g R-amine and 9.9 g
methanol.
6. Measure the optical rotation (the specific rotation).
With the assistance of your instructor record the optical rotation of your two samples using
the polarimeter in the central instrument room. In the same manner, also record the optical
rotation of the original racemic mixture, (R,S)1phenylethylamine.

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CLEAN UP
Your purified amines, methanol filtrate, and any ether left over from the extraction should
be disposed of in the nonchlorinated waste solvent container. The basic layers from the
extraction should be placed in the aqueous waste container.
REPORT
In the observations/data section describe the crystals you obtained. Record the dried weight
of the crystals, and the weights of the two samples of amine you isolated. The preparation of
each solution for the optical measurement should be described and the optical rotation for
each sample clearly stated. In the discussions/conclusions section calculate the percentage
yield for the crystalline material you isolate and the yield of the free amine. With reference
to the optical rotations you recorded on the polarimeter, discuss the effectiveness of your
attempt to separate the two amine enantiomers (optical rotations for the pure single
enantiomers, in other words the theoretical values, are: (S) 38.2 and (R) +38.2).

QUESTIONS
1. The (S) enantiomer is isolated via a crystalline salt and, as crystals grow in a pure form,
this enantiomer can potentially be isolated in a very pure state. Isolating the (R) enantiomer
from the residue left in solution inevitably involves isolation of that portion of the (S) amine
that did not crystallize. The presence of this (S) amine has an effect on the optical rotatory
power of the sample isolated in this part of the experiment. What effect?
2. It is possible to separate the two enantiomers comprising a racemic mixture using the
various forms of chromatography you have met (TLC, column, GC, HPLC). These techniques
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share a common operational basis, but what modification is required if separation of


enantiomers is to be achieved?

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EXPERIMENT NO. 9: QUANTITATIVE ANALYSIS: ESTIMATION OF GLUCOSE

Objective: Estimation of glucose by DNSA method.

Principle: This method tests for the presence of free carbonyl group (C=O), the so called
reducing sugar. This involves the oxidation of the aldehyde functional group present; for
example, in glucose and the ketone functional group in fructose. Simultaneously 3,5
dinitrosalicylic acid (DNS) is reduced to 3-amino-5-nitrosalicylic acid under alkaline
conditions.

The above reaction scheme shows that one mole of sugar will react with one mole of 3,5dinitrosalicylic acid. Different reducing sugars generally yield different colour intensities; thus
it is necessary to calibrate for each sugar. In addition to the oxidation of the carbonyl groups
in the sugar, other side reactions such as the decomposition of sugar also competes for the
availability of 3,5 dinitrosalicylic acid.
Although this is a convenient and relatively inexpensive method, due to the relatively Low
specificity, one must run blanks diligently if the colorimetric results are to be interpreted
correctly and accurately.
When the effects of extraneous compounds are not known one can effectively include a socalled internal standard by first fully developing the colour for the unknown sample, then a
known amount of sugar is added to this sample. The increase in the absorbance is equivalent
to the incremental amount of sugar added.

REQUIRMENTS:
1 Test tubes, Pipettes, Spectrophotometer
2 NaOH
3 DNSA (i.e. 3,5-Dinitrosalicylic acid)
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4 Standerd giucose solution

PROCEDURE:
1. Add 3mL of DNSA reagent to 3 mL of glucose sample in a lightly capped test tube (To avoid
the loss of liquid due to evaporation, cover it with parafilm).
2. Heat the mixture at 90 C for 5-15min to develop the red brown colour.
3. Add 1ml of 40% potassium tartarate (Rochelle salt) solution to stabilize the colour.
4. After cooling at room temperature in a cold water bath, record the absorbance with a
spectrophotometer at 575 nm.

RESULT:
Concentration of unknown was found to be ___________mg/ml.

METHOD B:

Standardisation of Fehlings Solution:

Prepare a solution (known standard solution) of glucose AR by weighing accurately 1.25gm


and dissolving it in 250 mL standard flask in water. Make up the volume to the mark.
Pipette out 20 mL each of Fehlings A & B in a dry conical flask and shake thoroughly. Pipette
out 20 mL of this freshly mixed Fehlings solution in a clean conical flask and dilute it with 20
mL water. Heat the solution up to 70 C on a water bath. Take the standard solution of
glucose prepared in a burette and run this solution slowly into the boiling Fehlings solution
until the blue colour has completely disappeared. Take care to maintain this temperature for
every addition of glucose solution. Repeat the above titration by running the glucose
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solution steadily into the boiling Fehlings solution until the end point is approached and
then cautiously add glucose solution drop-by-drop till the end point is reached.
Alternatively to detect the end point more accurately, 5-6 drops of methylene- blue
indicator may be added to the Fehlings solution and then glucose solution added drop by
drop. However, if methylene-blue is used as indicator the Fehlings solution should not boil
for more than 2-3 minutes at a stretch. The end-point here also is marked by the
disappearance of the blue colour.
Repeat the same titration procedure with unknown sugar solution.
Simulator Procedure:
1. Choose the titrant.
2. Choose the titrate.
3. Select the normality of the titrate.
4. Select the volume of the titrate.
5. Start titration.
6. When the blue colour just fades select the indicator.
7. Continue the titration.
8. End point is noted at the colour change of the solution.
9. From the final reading the normality of titrant can be calculated by the equation:

10. After finding the normality, the amount of substance in the whole of the given
solution can be calculated by the equation:

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Note: 10 times dilute the stoke solution and that is used as titrant.

Atomic weight of Glucose=180.1559 g/mol.

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EXPERIMENT NO. 10: OPERATION OF SOFTWARE RELATED TO ORGANIC CHEMISTRY

Objective: To demonstrate students how to draw structures of organic molecules, how to


calculate their energy minimized structures etc.
Procedure: The operation of software will be demonstrated to you by the instructors on spot
followed by hands-on experience for you on those software.

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Appendix A: Table of Solid Unknowns

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