Experiment 1

Purification of an Impure Acetanilide Sample by

Recrystallization and Determination of Purity by
Melting Point.
Objectives
To illustrate the concept of recrystallization and its application
To demonstrate the proper techniques in recrystallizing an impure organic compound
To illustrate the principles and proper techniques in the determination of melting point, one of the
physical properties of organic compounds

Reagents

Acetanilide (N-Phenylacetamide, C8H9NO), Impure Acetanilide (Acetanilide-sodium chloride-congo

red mixture), activated carbon, acetone (2-propanone, C3H6O), Ethyl alcohol (ethanol, C2H6O), and
toluene (Methylbenzene, C7H8).

Materials
For Recrystallization
test tubes, beaker, graduated cylinders, medicine dropper, hot plate, Bunsen burner wire
gauze, iron ring, iron stand, test tube holder, stirring rod, short stem funnel, steam bath,
Büchner funnel with stopper, vacuum flask, crucible tongs and an ice water bath,

For Melting Point Determination

Mel-Temp apparatus, Glass plates, Spatula

Procedure

A. Determination of an Appropriate Solvent for Recrystallizing Acetanilide

Perform the succeeding procedure in testing the solubility of acetanilide in water [H 2O],
ethanol [CH3CH2OH], acetone {(CH3)2CO] and toluene [C6H5CH3]. Determine which solvent is most
suitable for recrystallizing acetanilide [C6H5NHCOCH3].

Place approximately 25 mg of acetanilide into a test tube containing 1 mL of solvent being

tested. Shake the tube and observe. If the substance fails to dissolve, heat the test tube gently
under a hot water bath. Do not heat longer than necessary to avoid undue evaporation of the
solvent. If undissolved solid still remains, add 1 mL more solvent and warm. Shake the test tube
and observe for the dissolution of acetanilide. Test for the solubility of acetanilide in water,
CH3CH2OH, (CH3)2CO and C6H5CH3.

Cool the contents of four test tubes (i.e. H2O, CH3CH2OH, (CH3)2CO and C6H5CH3) and
observe for any reappearance of the crystals. If crystals do not appear in any of the test tubes, add
a pin head-size acetanilide crystal into the solution or scratch the inside wall of the test tube
beneath the solvent surface. Which solvent do you observe will be most suitable for recrystallizing
acetanilide? Why? Report your findings to the instructor.
B. Recrystallization of Impure Acetanilide

Accurately weigh 1 g of impure acetanilide. Place it in an Erlenmeyer flask and add 50 mL

of the solvent that was observed to be suitable for recrystallizing the compound. Heat the mixture,
stirring it uniformly until it almost boils. If the solution is colored, add a pinch of activated charcoal
and continue heating to almost boiling. Check if the suspension is still colored by spotting a drop of
the mixture on filter paper. Add more activated charcoal if necessary. Meanwhile, prepare to filter
the hot suspension.

Prepare a filtration set-up (Figure 1) by folding a fluted filter paper and placing it on a
funnel. Use a 125-mL Erlenmeyer flask as the receiver. Pre-heat the set-up just prior to use by
pouring hot solvent into the filter paper. Why? Empty the flask and filter the hot suspension. It is
essential that this operation be completed as quickly as possible in order that cooling is minimized.
Why? It is also advisable to place only a few mL of the hot solution into the funnel, keeping the
remainder of the solution in the flask hot by heating over the Bunsen flame until it is transferred to
the funnel.

Figure 1: Filtration Set-up using a Fluted filter paper.

If the filtrate remains to be colored, add a pinch of activated charcoal and heat. Refilter by
gravity filtration.

Allow it to cool slowly to room temperature undisturbed. Observe the appearance of

crystals. To induce crystallization of more acetanilide, chill the flask in an ice-water bath and
scratch the walls of the flask in contact with the solution.

2
 While waiting for most of the acetanilide to recrystallize, assemble the suction filtration
apparatus (Figure 2) as follows: cut a circular piece of filter paper of such diameter that it exactly
covers the flat surface of the perforated portion of the Büchner funnel filter plate. Determine the
weight of this paper and place it in the Büchner funnel. Fit the Büchner funnel to a suction flask.

Figure 2: Suction Filtration Apparatus

When the suction filtration apparatus has been set-up, moisten the paper in the funnel
with a few drops of the solvent, (why?) and draw the solvent down through the suction. Now, filter
the crystals out of the solution by pouring it in the Büchner funnel and the solvent will eventually be
drawn inside the vacuum flask through suction. When the crystals are almost dry, release the
vacuum by disconnecting the tube from the suction flask (Do not turn the pump off!) and wash the
crystals with 5-10 mL cold solvent with stirring (use a stirring rod) for half a minute. Reconnect the
tube to dry the crystals.

To dry the crystals, transfer the crystals together with the filter paper into a watch glass,
cover it with another filter paper and keep it in the locker until the next laboratory period and
experiment (melting point determination). Weigh the dried crystals and filter paper to the nearest
0.01 gram. Determine the percentage of the pure crystals recovered from the impure sample.

C. Melting point determination

Determine the melting point of the recrystallized and impure acetanilide from the previous
experiment. Using the melting point apparatus, adjust the temperature setting from 10-20°C. Do
not set the temperature greater than this range. (Why?) Place very few amounts of the acetanilide
sample on the glass plate and place this on the heating plate of the apparatus. Observe the
crystals and record the temperature range at which the crystals start to melt until all of it has
melted. Remove the glass plate from the apparatus and clean it for the next person to use.

NOTE: Do not discard the glass plate. Properly dispose of its contents and return it to the
technician.

3
Questions:

1. Among the following solvents-water, acetone, ethanol, and toluene, which one was
observed to b e most suitable for recrystallization of acetanilide? Why?

2. Why was a water bath used instead of direct heating in the choice of solvent?

3. Why was activated charcoal added into the mixture of acetanilide, sodium chloride and
congo red? What is adsorption?

4. Would it be wrong to dissolve the original impure material in a large excess of solvent?
Why?

5. In gravity filtration, what is the advantage of using a short-stemmed funnel over a long-stem
funnel?

6. Why were the funnel and the flask preheated before filtering the impure acetanilide?

7. Why was the filter paper in the Büchner funnel moistened before pouring in the mixture
containing the acetanilide to be recrystallized?

8. What is the purpose of washing the acetanilide crystals with a few mL of cold solvent? What
do you think would happen if the washing solvent was not cold?

9. Why must the rubber tubing of the suction flask be disconnected before turning off the water
aspirator?

10. Why is it necessary to use thick-walled tubing in connecting the suction flask to the
aspirator?

11. At what steps in the recrystallization procedure did you lose some of the product? What can
you do in the future to avoid this losses?

12. What is the effect of impurities on the observed melting point of acetanilide?

13. Given the solubilities of the following compounds:

Water Ethanol Benzene
cold hot cold hot cold hot
Acetamide s s s s ss s
Aspirin i s s s ss ss
Sample Y i s s s i ss
where s means soluble; i means insoluble; and ss means slightly soluble

(A) Which of the solvents would be suitable for recrystallization of acetamide? Of aspirin?
(B) Could acetamide be separated from acetanilide by recrystallization from water? Why or
why not?
(C) Could aspirin be separated from acetanilide by recrystallization from water? Why or why
not?
(D) Devise a recrystallization scheme for the purification of aspirin contaminated with small
amount of acetamide and sample Y.

4
Experiment 2
Separation of a Binary Mixture by Simple and
Fractional Distillation
Objectives
To present the concept of distillation and its applications
To illustrate proper techniques in distillation

Reagents
n-hexane (C6H14), toluene (C7H8)

Materials
50-mL pear-shaped flask, simple and fractional distillation set-ups, thermometer, graduated
cylinder, small test tubes (10x75mm), 2-3 utility clamps, hot plate or burner, oil bath, wire gauze,
beaker, capillary tubes, rubber band, iron ring and iron stand.

Procedure

Separation of Hexane and Toluene by Simple Distillation

Acquaint yourself on the parts of an apparatus for simple distillation (Figure 1). Start with
the burner, followed by an iron ring and wire gauze placed 4-5 cm above the burner. Place a 100-
mL beaker on top of the wire gauze. Clamp a 100-mL pear-shaped flask (or a round-bottom flask)
to an iron stand protecting the neck of the flask with a few layers of tissue paper. A semi-
microscale set-up for distillation may have a one-piece attachment that already includes the air
column, the distilling head, the thermometer holder, the condenser, and the receiver. In this case,
place a thin film of grease crosswise at the male ground-glass joint (upper portion) of the column
and insert it to the female ground-glass joint of the pear-shaped flask. Note: Do not use excess
grease, as it will contaminate the sample. Prior to the attachment of the distilling section, make
sure that the delivery tubes of the condenser are attached properly (water inlet is near the receiving
end and the outlet near the Claisen head, Why?) and securely. Support the condenser with another
utility clamp making sure that the condenser is padded with tissue paper. Place a few drops of oil
at the thermometer holder before placing the thermometer in. Note: these instructions are based on
the available set-up. If you are provided with individual parts, consult any literatures on
experimental organic chemistry for the set-up of a simple distillation apparatus. Make sure that all
connections are secured. Have a slow stream of water circulate through the condenser and use a
clean and dry 10-mL graduated cylinder as a receiving flask to collect the first few mL to be
discarded (forerun). Have the instructor check your apparatus at this point.

5
 Figure 1: Actual Apparatus for Simple Distillation

Place 30 mL of n-hexane and 30 mL of toluene into the 100 mL round bottom flask. Make
sure to do this while the round bottom flask is not placed in the set-up, avoiding any spill onto an oil
bath/water bath.

Bring the mixture to a boil. When liquid begins to drop into the receiver, adjust the heat so
that the drops come steadily at a rate of about one second per drop. Discard the first 1mL and
record the temperature every 1 mL fractions as the distillation proceeds until 20 mL of the distillate
are collected. Transfer all distillates having the same boiling point into a test tube.

If distillation is thru, turn off the burner and lower the oil bath. Never heat the distilling flask
to dryness and do not stop the circulating water until the mixture is no longer hot.

6
Separation of Hexane and Toluene by Fractional Distillation

Acquaint yourself on the parts of an apparatus for fractional distillation (Figure 2): Actual
Apparatus for Simple Distillation. The only difference in set-up is the type of column used. Place a
100-mL beaker on top of the wire gauze. Clamp a 100-mL pear-shaped flask (or a round-bottom
flask) to an iron stand protecting the neck of the flask with a few layers of tissue paper. A semi-
microscale set-up for distillation may have a one-piece attachment that already includes the
fractional column, the Claisen head, the thermometer holder, the condenser, and the receiver. In
this case, place a thin film of grease crosswise at the male ground-glass joint (upper portion) of the
column and insert it to the female ground-glass joint of the pear-shaped flask. Note: Do not use
excess grease as it will contaminate the sample. Prior to the attachment of the distilling section,
make sure that the delivery tubes of the condenser are attached properly (water inlet is near the
receiving end and the outlet near the Claisen head, Why?) and securely. Support the condenser
with another utility clamp making sure that the condenser is padded with tissue paper. Place a few
drops of oil at the thermometer holder before placing the thermometer in. Note: these instructions
are based on the available set-up. If you are provided with individual parts, consult any literatures
on experimental organic chemistry for the set-up of a simple distillation apparatus. Make sure that
all connections are secured. Have a slow stream of water circulate through the condenser and use
a clean and dry 10-mL graduated cylinder as a receiving flask to collect the first few mL to be
discarded (forerun). Have the instructor check your apparatus at this point.

Figure 2: Actual Apparatus for Fractional Distillation

Place 30 mL of n-hexane and 30 mL of toluene into the 100 mL round bottom flask. Make
sure to do this while the round bottom flask is not placed in the set-up, avoiding any spill onto an oil
bath/water bath.

Bring the mixture to a boil. When liquid begins to drop into the receiver, adjust the heat so
that the drops come steadily at a rate of about one second per drop. Discard the first 1 mL and

7
 record the temperature every 1 mL fractions as the distillation proceeds until 20 mL of the distillate
are collected. Transfer all distillates having the same boiling point into a test tube.

If distillation is thru, turn off the burner and lower the oil bath. Never heat the distilling flask
to dryness and do not stop the circulating water until the mixture is no longer hot. Compare data
gathered from simple and fractional distillation.

Questions

1. Plot the boiling point vs volume distilled for both the distillation of hexane-ethyl acetate mixture
with and without column on the same graph. Label the curve On the basis of these curves,
which procedure was more efficient at separating the mixture into its components?

2. Would a longer fractionating column be more efficient in separating mixtures of liquids than a
shorter column? Why? Give advantages and disadvantages of using a longer column over a
shorter one.

3. Why is better separation of two liquids achieved by slow rather than fast distillation?

4. In a fractional distillation, is the composition of the vapor just above the surface of the liquid
the same as the vapor near the thermometer bulb? Explain.

5. Why is it important to have the cooling water enter the condenser jacket at the lower end and
exit at the upper end rather than have it flow in the opposite direction?

6. What is the purpose of adding boiling chips to the distilling mixture?

7. Why should it be dangerous to heat an organic compound in a distilling apparatus that was
closed tightly at every joint and had no vent or opening to the atmosphere or to vacuum
pump?

8. Why should a distilling flask at the beginning of distillation be filled to not more than two-thirds
of its capacity?

8
Experiment 3
Extraction: Determination of its Efficiency &
Calculation of the Distribution Coefficient
Objectives
To illustrate the concept of extraction and its application

Reagents
Acetic Acid (CH3COOH), Toluene, sodium hydroxide, and phenolphthalein

Materials
Burette, Pipette, Aspirator, 125-mL Erlenmeyer flasks, Separatory Funnel, beaker, Iron ring and
Iron stand, volumetric flask.

Procedure

Measure 2mL Acetic Acid and put it in a 50-mL beaker, add 20-mL distilled water and stir.
Place the contents of the beaker in a 250mL volumetric flask then dilute up to the mark with
distilled water. Stopper the flask then mix the solution thoroughly. Into three (3) separate 125mL
Erlenmeyer flasks, pipet 20mL of the Acetic Acid solution into each flask. Label the flasks as 1, 2
and 3.

Determination of the Number of Grams of Acetic Acid in a 20-mL Aliquot

Fill a burette with 0.1 M standard NaOH solution. Withdraw enough solution to remove the
air from the jet tip and bring the liquid into a graduated region of the burette. Record the initial
volume of the NaOH solution in the burette. Add 2 drops of phenolphthalein into flask 1 and then
titrate with the 0.1 M standard NaOH solution. Swirl the flask while titrating. Add the base solution
drop by drop near the end of the operation, until the last drop of base turns the solution in the flask
to pink. Record the final burette reading. Calculate the number of grams of Acetic Acid that are
dissolved in the 20-mL aqueous solution.

Determination of the Number of Grams of Acetic Acid extracted by one 20-mL portion of Toluene

Place the contents of flask 2 in a separatory funnel (Figure 1), and then add 20-mL
Toluene. Shake the funnel with intermitted release of pressure for several minutes. Place the
funnel upright, with the stopcock closed and the stopper removed. Allow the two layers to separate
completely.

9
 s

Figure 1: Separatory Funnel used for Extraction”

Drain the lower aqueous layer into a clean 125-mL Erlenmeyer flask, add 2 drops of
phenolphthalein and again titrate the solution with the 0.1 M standard NaOH solution. Dispose the
other layer from the top of the funnel (Why?) and into the organic waste disposal bottle.

Calculate the number of grams of Acetic Acid that remained in the aqueous layer. By
subtracting the calculated grams of Acetic Acid in the aqueous layer from the results obtained in
the first part, determine the weight of the acid that was extracted into the toluene layer then
determine the distribution coefficient.

Determination of the Number of Grams of Acetic Acid extracted by two 10-mL portions of Toluene

Repeat the procedures from the second part, but this time extract the remaining 20-mL
aliquot of Acetic Acid solution in flask 3, first with only 10-mL Toluene. Collect the lower aqueous
layer in a clean beaker and dispose the other layer from the top of the funnel and into the organic
waste disposal bottle. Put the aqueous layer that is in the beaker back into the separatory funnel
and extract with another 10-mL portion of fresh toluene. Now drain the lower aqueous layer into a
clean 125-mL Erlenmeyer flask then add 2 drops of phenolphthalein. Titrate the contents of the
flask till the endpoint (pink). Perform the same calculations that were done in the second part.

10
Experiment 4
Isolation of Nicotine From Tobacco
Objectives
To measure the amounts of nicotine present in a cigarette sample by Soxhlet extraction

Reagents
dichloromethane (Methylene chloride, CH2Cl2), anhydrous alcohol (ethyl alcohol; ethanol,
CH3CH2OH), methanol (Methyl alcohol, MeOH, CH3OH), Picric acid (2,4,6-Trinitrophenol,
C6H3N3O7).

Materials
Cigarette sample (to be provided by the student), Soxhlet extractor, utility clamps, Bunsen
burner, wire gauze, iron ring, iron stand, distilling flask, Buchner funnel, vacuum flask,
condenser, rubber tubings, thread and filter paper.

Procedure
Weigh 6 g sample and place it on a filter paper. Roll the filter paper with the
sample then fold the upper and lower flaps. Tie a thread around the filter paper roll to
prevent the sample from escaping. The roll should fit the mouth of the Soxhlet extractor
and its length should not exceed the length of the arm of the extractor. This should be
observed to ensure that all leaves will be immersed in the solvent during extraction.

Assemble the Soxhlet extraction apparatus (Figure 1). Start with the burner
followed by the iron ring and a wire gauze positioned 3-4 cm away from the mouth of the
burner. Place a beaker large enough for a 200 mL distilling flask on top of the wire gauze.
Fill the beaker with tap water until up to a quarter and add boiling chips. This would serve
as a water bath for the setup.

11
 Figure 1: Actual Apparatus for “Soxhlet Extraction”

Fill a 200-mL distilling flask with 100 mL CH2Cl2 and add 2-3 pieces of boiling
chips. Cover the neck of the flask with tissue paper and then clamp it to the iron stand.
Slowly lower the flask into the beaker until the underside of the flask is about a centimeter
away from the bottom of the beaker or until the utility clamp almost touches the mouth of
the beaker. Clamp the mouth of the Soxhlet extractor to the iron stand and carefully fit the
extractor to the mouth of the distilling flask. Be extra careful in handling the Soxhlet
extractor. It is very expensive glassware. Push the filter paper roll filled with the sample
into the mouth of the extractor until it reaches the bottom. Fit a condenser on to the
extractor and have running water pass through the lower arm of the condenser. Have
another rubber tubing attached to the upper arm of the condenser and have it emptied into
the sink. Place a cotton bulb at the opening of the condenser to prevent evaporating gases
to escape.

Turn on the burner and heat the water bath gently. CH 2Cl2 has a low boiling point
and it would only require a small amount of heat. It is advisable to cover the arms of the
Soxhlet extractor with aluminum foil during extraction. Continue extracting the sample until
the color of the solvent in the Soxhlet extractor becomes very pale. After extraction,
remove the distilling flask from the apparatus and evaporate the solvent under a hood
using a hot water bath. Transfer the oil to a pre-weighed test tube and weigh.

12
Experiment 5
Identification of Common Analgesic Drugs by Thin
Layer Chromatography
Objective
To perform thin-layer chromatography and calculate Rf values
To utilize thin-layer chromatography to identify the analgesic compound(s) present in an unknown
sample of an over-the-counter painkiller preparation
To learn concepts of chromatography, polarity of molecules and intermolecular forces of attraction.

Reagents
Solutions of analgesics in methanol (aspirin, paracetamol, ibuprofen, caffeine), unknown OTC
analgesic tablet, methanol, solvent mixture (25 parts ethyl acetate: 1 part ethanol: 1 part acetic
acid)

Materials
Small beaker (50-mL), aluminum foil or plastic wrap to cover beaker, test tubes, stirring rod, glass
capillary tubes for spotting, plastic TLC sheet, about 5 cm  10 cm, ultraviolet lamp, ruler and
pencil

Procedure

Get the TLC plate from the stock room. This plate consists of silica gel on a flexible plastic
or aluminum sheet. Handle it with care (do not touch the silica face, handle it on the sides) and lay
it over a notebook. Mark lightly with a pencil a straight line 0.5 cm from one end. Make a light pencil
mark or a dot on the straight line you drew.

Dip the spotter to the corresponding sample solution so that it partly fills with liquid. Spot
the solution on the dot previously marked on the TLC plate. The spot should not exceed 1mm in
diameter. The amount of liquid coming out of the capillary can be controlled by holding the
uppermost end of the capillary. (General rule: the smaller the spot the better). You can make the
spot concentrated by repeatedly touching the plate. Allow the spot to dry thoroughly. The capillary
is cleaned by spotting the excess liquid on a sheet of tissue paper and rinsed with technical grade
acetone.

Add enough of the solvent mixture (25 Ethyl acetate: 1 Ethanol: 1 Acetic acid) to give a
thin layer of solvent in the bottom of the chromatographic chamber (50-mL beaker). To provide
an atmosphere saturated with solvent inside the container, place a piece of filter paper around the
inside surface of the container, extending into the solvent. Then cover the container with the
plastic wrap, foil or screw cap and set it aside while preparing the chromatographic sheet.

Place the TLC plate in the chromatographic chamber (Figure 1) in such a way that the
spot(s) is/are not immersed in the solvent and the sheet is in an upright position (The pencil mark is
located at the bottom). Return the cover on the mouth of the beaker (Why?). When the solvent
front in the plate is about 0.5 cm from the top, remove the plate from the chamber and mark the
solvent front with a pencil. View the spots on the TLC plate by using the appropriate visualizing
agent. (UV lamp/Iodine chamber). Calculate the Rf value of the spot(s) in your TLC profile.
13
 Rf = distance traveled by the substance = distance to center of spot
distance traveled by the solvent distance to solvent front

Figure 1: Chromatographic chamber for Thin Layer Chromatography

Caution: UV radiation is harmful to your eyes. Do not stare directly at the UV lamp.

QUESTIONS:

1. Suggest possible advantages and disadvantages of using a longer (taller) TLC sheet?

2. Why do you think it was important to use a very small amount of sample when spotting the plate?

3. The relative movement of components is controlled partially by the polarity of the molecules. The
TLC sheet is coated with a highly polar substance, whereas the solvent mixture has a much lower
polarity. From your chromatographic results, predict the relative polarities of aspirin, paracetamol,
ibuprofen and caffeine by arranging them in order of increasing polarity. Explain your reasoning.

14
Experiment 6
Column Chromatography of Food Dye.

Objectives
To illustrate the concepts of chromatography and its applications
To illustrate the proper techniques involved in thin layer chromatography (TLC).

Reagents
Water saturated 2-butanol with acetic acid, ammonia in butanol, 1 part 1-butanol 1 part acetic acid,
2 parts methanol 1 part water, dye mixture (Indigo dye)

Materials
Thin Layer Chromatography plates (Silica plates), capillary tubes, small test tubes (10x75mm),
watch glass, beaker, 10-mL graduated cylinder, TLC plates, medicine droppers, filter paper.

Procedure
To choose an appropriate solvent for Column Chromatography, choose a solvent system
that gives a TLC profile where majority of the spots (or the target spot to be separated) is within a
Rf value of 0.3-0.5 (range). The choice of solvent must then have a lower polarity than this solvent
that gave an Rf value of 0.3-0.5 for its TLC profile. Why?

Separation of Pigments by Column Chromatography with TLC monitoring

Clamp the column upright (Figure 2), and insert a very small wad of cotton and tap it
gently into a constriction. You may add a small amount of sand to level bottom. Place 2 g of
alumina/silica on a piece of filter paper and pour it into the column in a slow stream. (Make sure
that no alumina/silica adheres to the side of the column where it is dry. You may tap the column
gently with an aspirator to make it compact. Make sure that the column bed is flat. Add enough
sand to cover the surface area of the column bed (~3mm).

In separate test tube, place about 10-15 mL of the chosen solvent. In another test tube,
place 10-15 mL of the chose solvent but with higher polarity. To be able to do this, mix 75% of the
chosen solvent with 25% of another substance with higher polarity. Prepare the next set of solvents
by adjusting the percentages of each substance in the mixture. (50:50, 75:25 and 100:0)

Place approximately one-half milliliter (~0.5 mL) of the dye solution into the column when
ready. Drop the sample mixture with a medicine dropper against the side of the column in a circular
motion. Do not directly drop the sample onto the column bed (Why?). Add the chosen solvent in
such a way that as the liquid extract touches the column bed, it is replenished immediately with the
solvent, thereby avoiding drying up of the column. The solvent used for elution should be changed
only when there is little or no separation of bands on the column. Moreover, the solvent must be
used in order of increasing polarity, taking note of the total volume used for each.

15
 Figure 2: Actual Apparatus for Column Chromatography

As bands begin to separate and move down the column, place the empty test tube under
the column to collect the separated pigment solution as they are eluted. Change test tube as
pigment start to come out or when a pigment has completely been collected. After collection, take
the TLC profile of the pigments collected in a single TLC plate. Make sure that they are sufficiently
apart (when you spot) so that they don't mix when developing your TLC.

16
Experiment 7
Analysis of Hydrocarbons
Objective

To illustrate the different tests involve in differentiating saturated and unsaturated aliphatic
hydrocarbons, and aromatic hydrocarbons.

Reagents
soda lime, anhydrous sodium acetate, concentrated sulfuric acid, absolute ethyl alcohol, 10%
sodium bicarbonate, calcium carbide, toluene, cyclohexane, bromine water, alcoholic iodine, 1%
alkaline potassium permanganate, ammoniacal cuprous chloride, bromine in chloroform,
concentrated nitric acid.

Materials
test tubes (10x75mm), cork fitted with a delivery tube, distilling flask, thermometer, gas generator,
test tube holder, evaporating dish, Bunsen burner, wire gauze, iron ring, iron stand, suction flask
and separatory funnel.

Procedure

Methane, ethylene and acetylene are used to illustrate the reactions of the
saturated and unsaturated hydrocarbons. The alkene is expected to exhibit the same reactivity as
the alkyne towards reagents that test for unsaturation.

Preparation of Acetylene: Set-up a gas generator with a 250-mL suction flask clamped to an iron
stand. Attach a separatory funnel fitted with a cork or rubber stopper to the mouth of the flask.
Connect a delivery tube to the side arm of the suction flask and immerse the other end of the
rubber tubing in a trough filled with water. Have five test tubes with fitting stoppers already in
trough.

Place solid calcium carbide in the generator and fill the separatory funnel with water.
Slowly, drop water into the calcium carbide. Let a few bubbles of a gas escape. Then collect five
test tubes of gas by water displacement. Label the test tubes.

Analyses
One sample tube from each gas will be used for the following tests: Flammability test,
reaction with bromine water, reaction with alcoholic iodine, Baeyer’s test, and formation of Copper
acetylide.

Flammability of Aliphatic and Aromatic Hydrocarbons: The way in which an organic

compound combust can often provide clues as to the kind of organic compound it is. This is
determined by how quickly they ignite, the color of the flame produced and whether the smoke
produces soot.

17
 Hold a tube of gas using a test tube holder. Bring its mouth downward near a non-
luminous Bunsen flame. Remove the stopper as the mouth of the tube nears the flame. Observe
the color and other characteristics of the flame. Compare the 3 gases.

Place about 3 drops of toluene on a clean evaporating dish and carefully ignite it. What
kind of flame was produced? Repeat the test using cyclohexane instead of toluene. Compare both
results with the 3 gases. How would you differentiate aromatic hydrocarbons from aliphatic
hydrocarbons?

Reaction with Bromine Water: Add to the second set of test tubes 1 mL of bromine
water. Stopper immediately then shake. Describe and interpret results.

Reaction with Alcoholic Iodine: Add about 2 drops of alcoholic iodine to the third set of
tubes of different gases. Shake and note any noticeable change(s). Repeat the same procedure
with naphthalene solution and cyclohexane instead of the three gases. Compare the results.

Baeyer's Test for Unsaturation: The test for unsaturation when performed with
potassium permanganate in the presence of sodium carbonate or bicarbonate to ensure slight
alkalinity is known as Baeyer's test.

Add 1 mL 1% alkaline potassium permanganate solution to a fourth set of tubes of

different gases. Observe the color and nature of the solution.

Place in two different test tubes 1 mL each with naphthalene solution and cyclohexane.
Add about 2-3 drops of 1% alkaline potassium permanganate to each test tube and shake. Note
any noticeable change(s) and compare the results with the three gases. Can potassium
permanganate oxidize saturated hydrocarbons and an aromatic ring?

Formation of Copper Acetylide: To the fifth set of test tubes, add 5 mL ammoniacal
cuprous chloride. . Stopper immediately then shake. Observe.

Reaction of Bromine with Aromatic Hydrocarbons: Place 1 mL of toluene in two

separate test tubes and add dropwise to each tube a solution of bromine in chloroform until a faint
color is obtained. Cover both test tubes with a stopper then place one test tube in sunlight, and the
other in the dark. Compare the appearance of both test tubes after 20 minutes.

Action of Nitric Acid with Aromatic Hydrocarbons: Prepare in a test tube a mixture of
2 mL concentrated sulfuric acid and 1 mL concentrated nitric acid. Add 6 drops of toluene, and
place the tube in a water bath heated to a constant temperature at about 55-60ºC. Keep the
mixture in a bath for about 10 minutes, shaking it at short intervals. Cool the tube and pour the
contents into 10 mL of ice water. Observe. Repeat the test with cyclohexane and compare it with
toluene.

18
Experiment 8
Alcohols and Phenols
Objectives
To illustrate some general chemical properties of alcohols and phenols.
To present the different tests involved in distinguishing alcohols and phenols from other
organic compounds.

Reagents
95% ethyl alcohol, isopropyl alcohol, tert-butyl alcohol, acetyl chloride, Lucas' reagent,
anhydrous magnesium sulfate, 1% potassium permanganate, litmus paper, sodium metal,
bromine water, 1% potassium dichromate, 1% ferric chloride solution, ceric nitrate, phenol,
resorcinol, hydroquinone, salicylic acid, glacial acetic acid, 6M sodium hydroxide solution,
6M sulfuric acid, 10% sodium bicarbonate solution, chromic acid, acetone

Materials
medicine droppers, test tubes, test tube rack, test tube holder, beakers, graduated
cylinder, crucible tongs, cork stopper, and sand bath.

Procedure
Alcohols and Phenols

The activity of alcohols is mainly due to the relative reactive hydroxyl group rather
than those of the comparatively inert alkyl substituents.

Sodium Detection for Acid Hydrogen: Prepare 2 mL of dry alcohol by adding a

pinch of anhydrous magnesium sulfate into 95% ethanol. Shake the mixture well and
decant the liquid into a clean and dry test tube. Verify the presence of acid hydrogen in the
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alcohol by dropping a small piece of sodium metal wiped clean from kerosene. Allow the
reaction to proceed to completion then evaporate the solution to dryness over a sand bath.
Allow the test tube to cool then add 1 mL of water to the residue. Test the solution with
litmus paper and interpret the results.

Note: Place 1-2 pieces of boiling chips into the test tube and, remember, no
flames! Never put metallic sodium into water! Add alcohol to destroy excess sodium.

Comparison of Oxidizing Conditions: Determine in which condition oxidation

occurs most rapidly by arranging in a test tube rack three labeled test tubes containing 5
drops of ethyl alcohol and 2 mL of water. Introduce into test tubes two and three a drop of
6M sulfuric acid and a drop of 6M sodium hydroxide solutions, respectively.
Simultaneously add into each test tube 2 drops of 1% potassium permanganate solution
then observe.

Comparison of Oxidation Rates of Alcohols: Place 1 mL of ethyl alcohol,

isopropyl alcohol and tert-butyl alcohol in three separate test tubes. Compare the ease by
which each of the alcohols are oxidized by simultaneously introducing 5 drops of 6M
sulfuric acid and 5 drops of 1% potassium dichromate solutions into each test tube. Repeat
the test with phenol and determine if it undergoes oxidation as well.

Ceric Nitrate Test for Alcohols: The ceric nitrate test uses cerium ammonium
nitrate in nitric acid to test for alcohols. Alcohols cause the reagent to change from yellow
to red. However, it can only be used for alcohols with ten or fewer carbons.

To each of three small test tubes, add approximately 1 mL of ceric nitrate solution.
Add 10 drops of ethyl alcohol into the first tube and into test tubes two and three, add
isopropyl and tert-butyl alcohols, respectively. Mix thoroughly and note if the yellow color
changes to red. Compare the results with a fourth test tube containing 0.5 mL of water with
1 mL ceric nitrate.

Chromic Acid Test for Distinguishing 1 and 2 Alcohols from Tertiary

Alcohols: The Chromic acid test is a rapid method for distinguishing primary and
secondary alcohols from tertiary alcohols. The reagent is very corrosive and is often used
as a cleaning solvent. It is prepared by combining solutions of sodium dichromate and
sulfuric acid. Also giving positive results with aldehydes and some enols, the reagent gives
dark-colored solutions with phenols.

Transfer 5 drops or 50 mg of test sample into 1 mL of reagent grade acetone.

Cautiously add 4 drops of chromic acid reagent to the resulting solution and observe for
the discoloration of the reagent. The appearance of a green to blue coloration within 5
seconds verifies a positive result indicating the alcohol has been oxidized. Try using
phenol as a test sample instead of the three alcohols. Give chemical equations for the
chromic acid oxidation of primary and secondary alcohols.

Lucas Test for Distinguishing 1 and 2 and 3 Alcohols: The Lucas test
makes use of ZnCl2 in concentrated hydrochloric acid to differentiate primary, secondary

20
and tertiary alcohols. It only works for samples that are soluble in the reagent. This
generally means that it may not be applied for alcohols exceeding 6 carbons.

To each of three small test tubes add approximately 1 mL of Lucas reagent. Add
into the first tube, 10 drops of ethyl alcohol. Into test tubes two and three, add isopropyl
and tert-butyl alcohols, respectively. Stopper the test tubes and then shake them
vigorously. Note the length of time it takes for the mixture to become cloudy or separate
into two layers. A tertiary alcohol reacts rapidly and thus gives observable results quickly.
Secondary alcohols usually show signs of a reaction within five minutes: while primary
alcohols remain clear for several hours. Present chemical equations for the reactions and
explain the rate by which each alcohol formed an immiscible layer.

Phenols, unlike alcohols, have hydroxyl groups bound to a carbon atom that forms
part of the aromatic ring. This feature greatly alters the properties of the phenolic hydroxyl
and serves to distinguish it from hydroxyl groups of alcohols.

Acidity of Phenols: Dissolve 50 mg or 5 drops of phenol into 0.5 mL of distilled

water and test the solution with litmus. Pour the solution into 1 mL of 10% sodium
bicarbonate solution and observe for the evolution of a gas. Repeat the test using 10 drops
of glacial acetic acid and ethyl alcohol instead of phenol and determine which of the
sample is most acidic.

Reaction of Phenols with Bromine water: Slowly add 10 drops of bromine water
to 5 drops of phenol in a test tube and observe the appearance of a precipitate. Give the
structure of the precipitate that readily formed.

Ferric Chloride Test for Water Soluble Phenols: Dissolve a drop of phenol in 1
mL of ethyl alcohol. Transfer 4 drops of freshly prepared 1% ferric chloride into this
solution and observe. Repeat the test using resorcinol, salicylic acid, hydroquinone, acetic
acid and ethyl alcohol instead of phenol.

21
Experiment 9
Aldehydes and Ketones
Objectives
To illustrate the chemical properties of aldehydes and ketones.
To distinguish aldehydes and ketones from other organic compounds by qualitative analysis.

Reagents
95% ethyl alcohol, isopropyl alcohol, tert-butyl alcohol, acetyl chloride, Lucas' reagent, anhydrous
magnesium sulfate, 1% potassium permanganate, litmus paper, sodium metal, 1% ferric chloride
solution, phenol, resorcinol, hydroquinone, acetic acid, concentrated hydrochloric acid, 6M sodium
hydroxide solution, 3M sulfuric acid, 10% sodium bicarbonate solution, 1% sodium dichromate,
chromic acid, acetone, methanol, 2,4-Dinitrophenylhydrazine, Schiff's reagent, Tollen's reagent,
Benedict's solution, Fehling's solution A and B, Iodoform reagent, sodium bisulfite solution, 5%
sodium nitroprusside solution, 3% sodium hydroxide solution, 0.1M potassium permanganate,
ferric chloride, 5% ammonium hydroxide solution, benzaldehyde, cyclohexanone and
formaldehyde.

Materials
medicine droppers, test tubes, test tube rack, test tube holder, filter paper, beakers, graduated
cylinder, crucible tongs, cork stopper, hot plate, sand bath, and Bunsen burner.

Procedure

Aldehydes and Ketones

The activity of aldehydes and ketones depend mostly on the reactive carbonyl group and
α-hydrogen, but the oxidative properties of aldehydes is what differentiates it from ketones. While
many ketones are easily oxidized, various tests are available to distinguish the two carbonyl
compounds from one another. In the succeeding activities, the general properties of aldehydes and
ketones will be illustrated. The test samples will include: acetone, formaldehyde, cyclohexanone
and benzaldehyde.

Oxidation of Aldehydes with Potassium Permanganate: Place 5 drops of 1%

potassium permanganate into a small test tube containing 1 mL of distilled water. To this solution,
add 4 drops of test sample. Check for the discoloration of the permanganate solution after
intermittent shaking for 5 minutes. If the solution remains unreacted, introduce 3 drops of 6M
sodium hydroxide. Explain your observations and write chemical equations for the reactions.

2,4-Dinitrophenylhydrazine Test for Aldehydes and Ketones: Aldehydes and ketones

readily react with 2,4-dinitrophenylhydrazine to give the yellow phenylhydrazone derivative. The
melting points of some of these derivatives are available in handbooks or manuals. These may be
compared with the derivatives of an unknown carbonyl compound to serve as confirmatory test.
22
 Measure 1 mL of 2,4-dinitrophenylhydrazine and transfer it to a small test tube. Add 2
drops of test sample to the test tube and shake. Observe for the appearance of a yellow
precipitate. Repeat the procedure using acetic acid and ethanol as test samples. Write a general
equation for the reaction.

Schiff's Test for Aldehydes: The Schiff's reagent is an aqueous solution of an organic
dye, p-rosaniline hydrochloride, which is then acidified with HCl to form a colorless bis-N-sulfinic
acid. It reacts with the aldehyde to give an unstable complex that liberates a pink or purple dye.

Measure 1 mL of Schiff's reagent into 4 separate small test tubes. Add 4 drops of test
sample to the reagent and swirl. Observe for the appearance of a pink to purple coloration. If the
compound is insoluble or immiscible in the reagent, stopper the tube with cork and shake it
vigorously. Record and interpret the results. Write a general chemical equation for the reaction.

Tollen's Test for Aldehydes: Tollen's reagent is a solution of silver nitrate in

concentrated ammonium hydroxide. Aliphatic and aromatic aldehydes reduce the reagent to
metallic silver. A Silver mirror or a black precipitate of silver constitutes a positive result. Because it
decomposes on standing and deposits as a highly explosive residue, the reagent should be
prepared just before use and it should not be stored.

Examine the reaction of Tollen's reagent with 4 drops of the test sample. If no reaction
occurs at room temperature for 2 minutes, heat the test tube at 60C. Observe the appearance of a
black precipitate after 3 minutes. Write a chemical equation for the reaction.

Fehling's Test for Distinguishing Aromatic Aldehydes from Aliphatic Aldehydes:

Fehling's reagent is a solution of a complex copper tartrate salt in aqueous base. It reacts with
aliphatic aldehydes to form the reddish brown precipitate cuprous oxide or a copper mirror on the
walls of the tube.

Obtain 1 mL of Fehling's solution A and another 1 mL of Fehling's solution B. Mix them in

a small test tube and add 4 drops or 50 mg of test sample. Swirl the mixture gently and observe.
The mixture may require heating in a hot water bath to facilitate the formation of a precipitate.
Repeat the test with glucose. What is the precipitate that formed?

Iodoform Test for Methyl Ketones. The reagent for the test contains iodine in potassium
iodide. It gives the yellow iodoform derivative with methyl ketones. However, due to its reactivity
towards acetaldehyde and compounds that are oxidizable to methyl carbinols the test is only valid
if the compound in question is undoubtedly a ketone.

Prepare the iodoform derivative of acetone by introducing 4 drops of the sample into a
test tube containing 2 mL of 6M sodium hydroxide. Add enough iodine-potassium iodide solution to
cause a persistent pink coloration. Heat the tube at 60C for a minute and check if the solution is
decolorized. Add more of the reagent to keep the solution colored. Continue heating and carefully
observe for any signs of a precipitate. Repeat the test using isopropyl alcohol as the test sample.
Write chemical equations for the reactions.

Reaction with Sodium Bisulfite for Aldehydes and Aliphatic Methyl Ketones: Into a
small test tube containing 4 drops of test sample, transfer 1 mL of sodium bisulfite solution. Shake
the mixture for 1-2 minutes and let it stand. Carbonyl compounds exhibit the precipitation of a
bisulfite addition complex but not all ketones form such a derivative. Explain.

23
 Chromic Acid Test for Aldehydes: The Chromic acid reagent is very corrosive and is
used in testing for alcohols. Prepared by combining solutions of sodium dichromate and sulfuric
acid, it also oxidizes aldehydes and some enols.

In this test, acetone is used as a solvent and it occasionally gives a positive test with the
reagent. It is therefore necessary to check if the solvent is of sufficient purity. Test 1 mL of acetone
with 5 drops of chromic acid reagent and allow the mixture to stand for three minutes. If a reaction
occurs after this period, acetone may not be used as a solvent and it must be changed with a
solvent of higher purity.

If not, proceed as follows: Into 1 mL of acetone, introduce 2 drops of test samples and 5
drops of chromic acid. Shake and observe for the discoloration of the reagent. Write a chemical
equation for the reaction.

24