EXPERIMENT 5.

SEPARATION OF THE ORGANIC COMPONENTS OF A

MIXTURE. IDENTIFICATION OF UNKNOWN COMPOUNDS.

INTRODUCTION AND THEORETICAL BACKGROUND

The isolation, purification and identification of organic compounds is an important task in the
field of organic chemistry. At present the components of a mixture are separated using
chromatographic techniques (HPLC, GC) and organic compounds are identified using
spectroscopic techniques (FTIR, UV-VIS, NMR), spectrometric techniques (MS) and
elemental analysis. However, traditional procedures are still very useful for separating
products at a preparatory laboratory level. Likewise, the identification tests of functional
groups, solubility behaviour and the determination of physical constants continue to be
widely used because they are simple and enable us to decide on, the most appropriate
technique for a complete characterization later on.

SEPARATION OF THE COMPONENTS OF A MIXTURE

The components of a mixture are separated by using their different solubilities, which
depend on their functional groups and structure. The procedure is shown in the following
scheme and involves a series of extractions in a specific order.
In an extraction, a component is separated from a mixture by shaking an immiscible organic
solvent with water (or aqueous solution) in a separatory funnel, which separates the two
layers. Depending on their relative solubility, the solutes are distributed between the
aqueous and organic phases.
The following protocol provides a series of six possible residues, each of which belongs to a
family of organic compounds with characteristic functional groups. The detection of a
residue, therefore, will allow us to classify the unknown product, which will guide the tests to
carry required to confirm its structure.

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Residue 1: salts, sulphonic acids, polyhydroxylated compounds, amino acids.
Residue 2: aliphatic and aromatic amines
Residue 3: amphoteric compounds
Residue 4: carboxylic acids and nitrophenols
Residue 5: phenols and ketoenolic compounds
Residue 6: ethers, alcohols, ketones, esters, nitriles, halogenated compounds, nitro
derivatives, aldehydes and hydrocarbons

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The choice of extraction solvent is crucial for separation and must
 be immiscible in water,
 dissolve the substance to be extracted better than water,
 have a low boiling point,
 not react, and
 (if possible) be economic but not flammable or toxic.
Separation of acid or basic compounds
If these kinds of compound are dissolved in an organic solvent, they can be taken to the
aqueous phase by being transformed into the corresponding salt when they are reacted with
an acidic or basic solution. The salt formed is soluble in water and insoluble in the organic
solvent. Once the compound is in the aqueous phase, it can be recovered with the acid-base
reverse reaction that will transform the salt into the original organic compound, which is
water-insoluble.

IDENTIFICATION OF A PURE SAMPLE

To identify an unknown substance by chemical methods, a certain sequence of tests is
usually followed which give information about its structure and the functional groups present.
a) Preliminary classification by physical state, colour and odour

b) Melting-point or boiling-point determination. Other physical data.

c) Elemental analysis. When an organic compound containing nitrogen, sulphur or halide
atoms is fused (300-400 °C) with sodium metal, there is a reductive decomposition of the
compound, which converts these atoms to the sodium salts of the inorganic ions CN-, S2-
and X-. When the fusion mixture is dissolved in distilled water, the cyanide, sulphide and
halide ions can be detected by standard qualitative inorganic tests (This experiment will not
be performed).
d) Solubility tests. The solubility of a small amount of the compound with different solutions
is tested in a test tube. The tube needs to be shaken well for a while or even warmed. If the
substance to be identified is one of the separation residues of a mixture, the solubility tests
are not necessary, since the solubility of the compounds is already known.
e) Flame tests
1. Place a small amount of compound on a spatula and put it in the flame of a Bunsen
burner. Observe the calcination. Compounds giving a sooty yellow flame have a high degree
of unsaturation and may be aromatic. A blue flame with little smoke indicates an oxygenated
aliphatic compound. Amino acids do not burn well and smell of burned hair. Carbohydrates
do not burn well, either, and smell of caramel. Compounds that burn with a green flame

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contain boron.
2. Beilstein test: place a small amount of substance on the end of a clean copper wire
and burn directly in the flame. Green flame colouration indicates the presence of a
halogenated compound.

f) Recognition of functional groups

Place a small amount of the unknown substance in a test tube and add the corresponding
reagent drop by drop, while shaking the mixture well. Changes in colouration and solubility or
the formation of precipitates indicate that a particular functional group is present. Do not use
a large excess of the reagent, so that you can accurately appreciate the colouration
changes. The most common tests and reagents are those indicated in the experimental part.
The information provided by these tests reveals the characteristic functional group of the
problem sample. Then, compare the experimental melting point or boiling point with those
published in the books for the identification of organic compounds. In the published list, there
may be several products with similar melting or boiling points. Thus, you must prepare a
derivative of the product in order to unequivocally discern its identity.

g) Preparation of a derivative
In order to prepare the derivatives, a small-scale synthesis is usually performed following the
recommended standard procedures for each functional group, which are described in the
books for the identification of organic compounds.

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TIMING OF THE EXPERIMENT: 4 sessions

REAGENTS TO BE USED
Diethyl ether TOXIC AND HIGHLY FLAMMABLE
Bromine solution TOXIC, IRRITANT
KMnO4 solution ---
Copper wire ---
Fehling solutions (A and B) ---
Brady reagent ---
Tollens reagent ---
Cr (VI) oxidant solution CORROSIVE, OXIDANT
Lucas reagent CORROSIVE
Methanol solution of FeCl3 ---
Picric acid, saturated solution EXPLOSIVE
Sodium nitrite ---
-Naphthol TOXIC
Phenylhydrazine hydrochloride TOXIC
Anhydrous sodium acetate ---
0.1M NaOH CORROSIVE
Phenolphthalein solution ---
Potassium hydrogen phthalate ---

LIST OF TECHNIQUES AND BASIC PROCEDURES

Selective solubilization of a mixture
Separation by reactive extraction
Solvent extraction
Drying of an organic phase with anhydrous MgSO4
Rotary evaporation
Preparation of derivatives
Recrystallization
Melting point
Base-acid evaluation

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EXPERIMENTAL PROCEDURE
You will have an unknown mixture consisting of two pure products. The constitution of the
mixture will be different for each group. At the end of the experiment, you will have to explain
to the teacher the procedure you used to separate the two compounds and how you have
identified them. You will have to explain how you have assigned each structure on the basis
of the tests carried out and the evidence obtained.

A) SEPARATION OF A MIXTURE OF COMPOUNDS

To separate the unknown mixture, follow the scheme above with approximately half the
sample (it might be necessary to repeat the analysis). The mixture may be made up of any of
the 6 residues. For this reason, follow the procedure until both compounds have been
separated. You need not carry out the whole process, only the part that allows both
components to be separated.
The first washing with ether should be performed in an Erlenmeyer flask. If there is an
insoluble product, decant the ether with care into the separatory funnel (the solid should not
pass into the separatory funnel) and repeat the washing and decantation once or twice more
to make sure that there is no remaining soluble product in residue 1. Separate the rest of the
residue by extraction operations. To ensure complete separation, perform extractions three
times, shake the separatory funnel gently and leave enough time for the acid-base reactions
to be completed. This is especially important in the case of washing with NaHCO3 (CAUTION
WITH OVERPRESSURE). The separation between the two phases also needs to be good to
prevent the separated residue from being contaminated.
Dry the final ethereal solutions with anhydrous magnesium sulphate and concentrate them in
the rotary evaporator to obtain the corresponding product. If necessary, dry them on the
stove but don’t forget that its melting point is still unknown so carry out a previous test to
prevent melting.
Once you have separated the two organic compounds, dry them well and then identify them
individually. Before you make the identification, give the results to the teacher, tell him/her
the residues that you have obtained and wait for their agreement.

B) IDENTIFICATION OF ORGANIC COMPOUNDS. HOW TO PROCEED:

- Physical observation: state, colour, odour

- Determination of melting point (if solid) or boiling point (if liquid).

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- Determination of solubility behaviour. The solubility has already been partially observed
during the separation of the compounds. So not all the solubility tests need to be repeated,
only those that you consider necessary. The general behaviour of the solubility of organic
compounds is as follows:
i) Water. Positive for polyhydroxylic compounds, sulphonic acids and high polar
compounds. Look at the pH of the solution. Compounds that contain five or fewer carbons
and also contain O, N or S atoms are often soluble in water.
ii) 10% NaHCO3 solution. Positive for strong acids: carboxylic acids, sulphonic acids and
nitrophenols.
iii) 5% NaOH solution. Positive for weak acids; phenols and ketoenolic compounds.
iv) 5% HCl solution. Positive for aliphatic and aromatic amines.
v) Cold concentrated H2SO4. Hydrocarbons, aryl or alkyl halides are not soluble. The
sulphuric acid test reveals whether a neutral compound has a functional group that contains
an oxygen, a nitrogen or a sulphur atom or a different functional group that can be
protonated (alcohols, aldehydes, ketones, esters, alkenes, etc).
- Flame tests: combustion and the Beilstein test.
- Elemental analysis: To simplify the experiment, the elemental analysis will not be carried
out. Instead, the data from this analysis will be provided by the teacher when giving the
results of the separation.
- Identification of functional groups. The tests of suitable functional groups will be carried out
depending on the family of compounds to which the residue belongs. These tests are:

TESTS FOR UNSATURATION

1) Bromine decolouration when a solution of the sample is added.
2) KMnO4 decolouration (or change from violet to brown) when a solution of the sample is
added.
Colour changes indicate the presence of unsaturations (C=C and C≡C). Products that are
easily brominated (phenols) can give a false positive result, as can those compounds that
oxidize or easily enolise (aldehydes, ketones, thioethers).

RECOGNITION OF ALDEHYDES AND KETONES

Both aldehydes and ketones give a positive result with Brady reagent (2,4-
dinitrophenylhydrazine). Only aldehydes react positively with Tollens reagent (reduction of
Ag+) and Fehling reagent (reduction of Cu2+). Some aromatic aldehydes, however, give a
negative result in the Fehling test.

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1) Brady test
Dissolve a small portion of the sample in ethanol (1-2 mL) in a test tube and add 3 mL of
Brady reagent. If a precipitate does not appear immediately, scratch the inner walls of the
tube with a glass rod and heat the bath for about 5 minutes. If a precipitate appears when
cooling or adding diluted sulphuric acid the test has been positive.

2) Fehling test
In a test tube with 1.5 mL of the Fehling A reagent solution, add the Fehling B solution
dropwise until the blue precipitate of copper hydroxide that forms initially has dissolved.
Afterwards, add a few drops of an ethanol solution of our compound and gently boil the
mixture for two minutes. A red precipitate of Cu2O indicates a positive test.

3) Tollens test
To a test tube with 2.5 mL of Tollens reagent (ammonium silver nitrate), add a few drops of
ethanol solution of the sample. Heat the mixture gently without boiling. If a mirror of silver is
deposited on the inner walls of the test tube, the test is positive.

RECOGNITION OF ALCOHOLS

1) Oxidation
In a test tube with 2 mL of oxidizing solution (aqueous solution of potassium dichromate and
sulphuric acid), add a small amount of the compound to be tested. Shake the tube and
observe possible warming and a change from initial orange to green.

2) Lucas test
Place 1 mL of ethanol solution in a test tube and add 2 mL of Lucas reagent (concentrated
zinc chloride solution in hydrochloric acid). Shake the tube while heating the solution.
Tertiary, benzylic and allylic alcohols react quickly to form an insoluble oil. Secondary
alcohols react after 5-10 minutes. Primary alcohols do not react.

RECOGNITION OF PHENOLS AND ENOLS

Add a 1% methanol solution of FeCl3 drop by drop to 1 mL of diluted ethanol solution of the
sample. A coloured complex usually forms immediately if the test is positive.

RECOGNITION OF CARBOXYLIC ACIDS

1) Sodium bicarbonate test. Add 2 mL of aqueous sodium bicarbonate solution to 2 mL of
a sample ethanol solution in a test tube. Observe the solution carefully. If the compound is
an acid, you will see bubbles of carbon dioxide form.

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2) Neutralization equivalent. Accurately weigh (three significant figures) 100 mg of the
sample and place in an Erlenmeyer flask. Dissolve the acid in water and titrate the acid,
using a solution 0.1M NaOH and a phenolphtalein indicator. The NaOH solution must be
standardized with potassium hydrogen phthalate. Calculate the neutralization equivalent
(NE). The NE is identical to the equivalent weight of the acid. If the acid has only one
carboxyl group, the NE and the molecular weight of the acid are identical. If not, the NE
equals the molecular weight of the acid divided by the number of carboxyl groups. After the
NE has been determined, the possible molecular weights of the compound can be deduced.

RECOGNITION OF CARBOHYDRATES

Osazone formation. Since carbohydrates do not have a defined melting point (they
decompose), they are characterised by the formation rate of the osazone. Each osazone has
a characteristic formation time. The crystal structure of the osazone is also often distinctive.
To carry out the assay, follow the procedures described in the organic analysis books in the
section on derivative preparation methods.

RECOGNITION OF AROMATIC HYDROCARBONS

Picrate formation. Prepare a saturated solution of picric acid by stirring 3 mL of ethanol and
a sufficient amount of acid. Decant the supernatant solution into a test tube containing 2 mL
of ethanol solution of the sample. Boil the mixture and allow to cool slowly. Filter the picrate
crystals and wash with ethanol. Determine the melting point.

RECOGNITION OF PRIMARY AROMATIC AMINES

Colorant formation. In a test tube, add 1mL of NaNO2 solution and some drops of HCl to
2mL of the ethanol solution sample, which must be cooled in a gel bath. To this solution, add
an aqueous solution of -naphthol in NaOH. The formation of orange or red colouration
indicates a primary aromatic amine.

PREPARATION OF A DERIVATIVE
Once the organic product has been identified, prepare a derivative to confirm its structure.
Each type of functional group has its characteristic derivatives. Choose the derivative to
synthesize and find the corresponding procedure in the organic analysis books (section on
derivative preparation).

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QUESTIONS AND TASKS
1. Indicate the components of your mixture and identify the structure of the products.
2. Complete the following table with the help of the bibliography:

Compound A B

Acid-base behaviour
Physical state
Colour
Melting (or boiling) point (ºC)
Recrystallization solvent
Other features

3. Propose and justify the separation procedure and discuss it with the teacher.
4. What is the reaction of the alkenes with the bromine solution?
5. What is the reaction of aldehydes and ketones with Brady reagent?
6. What is the reaction of tertiary alcohols with Lucas reagent?
7. What is the ratio between the experimental equivalent weight of a carboxylic acid and its
molecular weight?
8. What is the structure of osazones? What is the mechanism of their formation?
9. What interactions are involved in picrate formation?
10. Show the separation scheme, isolation and purification of the following mixtures:

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11. Given the following mixtures of compounds, explain how you would perform the
separation in each case, and how you would recover each product of the mixture in its pure
state.
a)
OH OCH3 OMe OH

NO2 Cl N N
H3C CH3

b)
O OH OH OH
OH
HO

NO2 N OH
H3C CH3

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