Dr SAJAL KUMAR DAS, DEPT.

OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

Solubility-based separation of binary mixture of organic compounds and

identification of special element(s) and functional group(s) present in the
separated compounds

The supplied sample will be a mixture of two solid compounds. First you must separate the two
compounds based on their solubility in water, 5% NaHCO3, 5% NaOH or 5% HCl (see below).
Then, you have to identify the special element(s) and functional group(s) present in each of the
two separated compounds.

1. PHYSICAL CHARACTERISTICS

First you note the physical state, color and odor of the given sample.

2. SOLUBILITY TESTS

SOLUBILITY TABLE

REAGENT AND TEST CLASS GROUP OF COMPOUNDS

Low Mol. Wt. compounds having polar
functional groups
Neutral, acidic or slightly
Neutral or slightly basic, e.g. urea,
Soluble in cold or hot basic. (check it with litmus
thiourea, acetamide, acetanilide etc.
water. or universal indicator
Acidic, e.g. carboxylic acids, nitro
paper)
phenols, di- and trihydroxybenzenes etc.
Basic, e.g. amines
Aromatic amines (aliphatic amines will
Soluble in dil. HCl Basic
not be given as unknown samples)
Soluble in dil. NaOH Acidic Most acids, most phenols.
Soluble in NaHCO3 Strongly acidic Most carboxylic acids.
Naphthalene, anthracene,
Insoluble in water, acid
Neutral phenanthrene,
and alkali
Aromatic aldehydes and ketones

A. Performing the water solubility test:

A. Checking water solubility: Add small amount (~25 mg) of a solid sample to 0.5 mL of
distilled water in a test tube. Tap the tube with your finger to mix or stir gently with a glass
stirring rod. Record the sample as soluble or insoluble. If the unknown sample is water-soluble,
test the solution with pH paper. Also test the pH of water as a control. A solution at pH 4 of lower

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

suggests the presence of a carboxylic acid, nitrophenol or any other sufficiently acidic
compound. A solution at pH 8 or higher suggests an amine.

If your compound is insoluble in water at rt, check the solubility in hot water and the test the pH.

If your sample is water-insoluble, proceed further as described below.

B. Performing the 5% NaHCO3 solubility test (for water-insoluble compounds): Put about
25 mg of solid sample in a dry test tube. Add 0.5 mL of 5% sodium hydrogen carbonate
(NaHCO3). Do not stir. Watch for bubbles at the interface of the phases. Then tap the tube with
your finger to mix or stir gently with a glass stirring rod. Record the sample as soluble or
insoluble. The generation of bubbles or solubility indicates a carboxylic acid, nitrophenol or any
other sufficiently acidic compound.

C. Performing the 5% NaOH solubility test (for water- and 5% NaHCO3-insoluble

compounds): Add about 25 mg of a solid sample to 0.5 mL of 5% NaOH in a test tube. Tap the
tube with your finger to mix or stir gently with a glass stirring rod. Record the sample as soluble
or insoluble. To verify that a compound has dissolved, add 5% HCl to the NaOH mixture until the
solution is acidic to pH paper. Look for a precipitate, indicating that the water-soluble salt has
converted back into the water-insoluble compound. If the compound is not soluble in NaHCO3
but is soluble in NaOH, it is likely a phenol. Confirm the presence of phenol with a phenol
classification test.

D. Performing the 5% hydrochloric acid solubility test (for compounds insoluble in water,
5% NaOH or NaHCO3):

Add about 25 mg of a solid sample to 0.5 mL of 5% HCl in a test tube. Tap the tube with your
finger to mix or stir gently with a glass stirring rod. Record the sample as soluble or insoluble. If
the compound is soluble in 5% HCl, it is most likely an aromatic amine.

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

SUMMARY OF THE SOLUBILITY TEST:

The solubility of the unknown sample in the following reagents provides very useful
information. Once the solubility is confirmed, you can use the major part of the remaining
amount of the sample for solubilizing one part and then separating the insoluble part by
filtration. Remember the followings:

Based upon the positive and negative results from the above experiments you should now be
able to narrow the possibilities for the functional group(s) present in your unknown
sample. You should now carefully decide which experiments are needed for the
identification of the functional group(s) present.

3. QUALITATIVE ANALYSIS FOR SPECIAL ELEMENTS (N, S, Cl, Br and I):

In organic compounds the elements commonly occurring along with carbon and hydrogen,
are oxygen, nitrogen, sulfur, chlorine, bromine and iodine. The detection of these elements
depends upon converting them to water-soluble ionic compounds and the application of specific
tests.

A. Lassaigne's sodium fusion test

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

PROCEDURE

CAUTION: You must use hand gloves and mask.

Cut a small piece (about the size of a pea) of sodium and then dry it by pressing between the
folds of filter paper cuttings. Put the sodium piece into a dry fusion tube. Add a little (50 mg) of
the given organic sample (must be anhydrous) into the tube. Hold the tube by a tong and heat it
gently and carefully (exposing the mouth of the tube away from you) on the Bunsen flame when
solid melts. The tube has to be shaken carefully in the flame to avoid excessive local heating and
to ensure mixing of the sample with the melted sodium (it may be temporarily drawn out of the
flame in the case of violent action). Then, heat the bottom of the tube to dull redness for about 3-
5 minutes and finally plunge the tube, while still hot, into a clean beaker containing cold distilled
water (5-7 mL). Crush the tube completely with the help of a glass rod, boil the contents of the
beaker for few minutes and then filter. The 'fusion' filtrate which should be clear and
COLORLESS, is used for the SPECIFIC TESTS DESCRIBED BELOW:

Test for NITROGEN

To a portion (2 mL) of the 'fusion' filtrate, add 2 mL of freshly prepared aq. FeSO4 solution. Boil
the mixture for a half a minute and then cool the test tube. Add 2–3 drops of FeCl3 and acidify by
adding conc. HCl dropwise. Formation of a bluish-green precipitate/coloration (Prussian blue)
or a blue solution indicates that the original substance contains nitrogen. Note that the presence
of sulphur may tend to obscure the test due to reduction of Fe+3 to Fe+2 ions (formation of black
precipitate of FeS). In that case, this test can be performed successfully by adding excess FeSO4
solution to precipitate black FeS. The mixture is then boiled, filtered and the filtrate is treated
with FeCl3 and conc. HCl.

OR

To a portion (2 mL) of the 'fusion' filtrate, add 2 mL of freshly prepared aq. FeSO4 solution. Boil
the mixture for a half a minute, cool and acidify by adding dilute sulfuric acid dropwise.
Formation of a bluish-green precipitate (Prussian blue) or a blue solution indicates that the
original substance contains nitrogen.

When the alkaline ferrous salt solution is boiled, some ferric ions are produced by the action of
air on ferrous salts and on acidification with dilute acid the hydroxides of ferrous and ferric
dissolves and the sodium ferrocyanide reacts with ferric cations to produce ferric ferrocyanide
having a Prussian blue color. But because a very small amount of this complex is formed the
solution is either green or blue in color.

Na + C + N → NaCN

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

6NaCN + FeSO4 → Na4[Fe(CN)6] + Na2SO4

Sodium ferrocyanide

Na4[Fe(CN)6] + Fe3+ → Fe4[Fe(CN)6]3

Ferric ferrocyanide

In Lassaigne's test, a fresh solution of FeSO4 must be used. Otherwise, it is oxidized to ferric sulfate
due to aerial oxidation. HCl is added to convert ferrous hydroxide, a green precipitate to ferrous
chloride, which is soluble in water. Otherwise the green precipitate may interfere with Prussian
blue color.

Fe(OH)2 + 2HCl --------------> FeCl2 + 2H2O

Tests for SULPHUR

(a) Sodium nitroprusside test:

To the cold 'fusion' filtrate (1 mL) add a few drops of cold, freshly prepared, dilute solution of
sodium nitroprusside. The latter may be prepared by adding a small crystal of the solid to 2 mL
of water. Production of a rich purple color indicates that the original substance contains sulphur.
This test is very sensitive. Only strong positive results are significant.

Na + S → Na2S
Na2S + Na2[Fe(CN)5NO] → Na4[Fe(CN)5NOS]
sodium nitroprusside violet color

(b) Lead acetate test:

Acidify 2 mL of sodium extract with acetic acid and add 1 ml of lead acetate solution when a
black precipitate of lead sulfide is produced confirming the presence of sulfur.

Why is it necessary to use acetic acid and not sulfuric acid for acidification of sodium extract for
testing sulfur by lead acetate test?

Tests for NITROGEN and SULPHUR

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

This test should be performed only when the presence of N and S have already been confirmed
separately from the above tests.

When both N and S are present in the sample, fusion filtrate may not respond to the Prussian
blue test. In such case proceed as follows. Acidify 1 mL of sodium fusion extract with dil. HCl and
add 1-2 drops of FeCl3 solution, a blood red coloration will indicate the presence of sulfur and
nitrogen.

When both nitrogen and sulphur are present in an organic compound, sodium fusion will
convert it into sodium thiocyanate which then react with Fe3+ to form blood colour complex
[Fe(SCN)]2+

Na + C + N + S → NaSCN
Fe3+ + 2Na → [Fe(SCN)]2+ (red color)

Tests for CHLORINE, BROMINE AND IODINE

Case 1: When nitrogen and sulfur absent:

Add 1 mL of conc. HNO3 to 2 mL of the fusion filtrate and boil the solution for few minutes. After
cooling the test tube to rt, add AgNO3 solution. A precipitate of silver halide (curdy white for Cl,
pale yellow for Br and yellow for I) indicates the presence of chlorine, bromine and iodine in the
sample. Filter the precipitate with minimum exposure to light on a funnel, wash with water, and
then with 1 mL of concentrated ammonia solution. If the precipitate is white and readily soluble
in ammonium hydroxide solution it is AgCl; if it is pale yellow and not readily soluble it is AgBr;
if it is yellow and insoluble it is AgI.

Case 2: When nitrogen and sulfur present:

The tests of halogens have to be modified as follows if nitrogen and sulphur are present.

A portion of the filtrate is rendered acidic with dilute HNO3 and the solution is evaporated to half
its original volume to expel HCN or H2S in a fume hood. The residual solution is then diluted with
an equal volume of distilled water and then used for performing the tests of halogens as
described above.

4. TESTS FOR SELCTED FUNCTIONAL GROUPS

There are many functional groups in organic molecules, but compounds containing selected
functional groups will be given to you.
(a) Unsaturated compounds (compounds containing C=C)
(b) C, H and O containing groups
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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

(i) –COOH (ii) Ar-OH (phenolic -OH) (iii) –CHO (aldehyde) and (iv) –CO (Keto group)
(c) N containing functional groups:
(i) Ar-NH2 (ii) Ar-NO2 (iii) –CONH2 or –CSNH2 (iv) –CONHAr (anilido group)
(d) Aromatic hydrocarbons like anthracene and phenanthrene (no functional group)

C. Tests for nitrogen containing functional groups

If the given sample does not contain nitrogen, tests for nitrogen containing functional groups are
not required to be performed.

Tests for Ar-NH2

(i) Azo-dye test

Dissolve small amount of the sample in dil. HCl (2.0 mL) and water (3 mL) and cool the solution
to 0–5 oC in an ice-bath for 5 minutes. Add a cold solution (ice-bath) of sodium nitrite (0.5 g) in
water (2.0 mL) from a dropper, with swirling of the test tube, still keeping the mixture in the ice-
bath. Add the cold diazonium solution (with swirling) to a cold solution of 2-naphthol (0.2 g) in
5% NaOH solution (2 mL). Formation of red or orange-red azo dye confirms the presence of Ar–
NH2 group (formation of brown/reddish purple/violet dye indicates the presence of
diaminobenzene and generation of soluble dye indicates the presence of aminophenol)

(ii) Carbylamine test: DO NOT perform this test (isocyanides are highly poisonous)

Tests for Ar-NO2

(i) Reduction test

Add 2.5 mL of conc. HCl to 0.25 g of the sample in a test tube. To this add 2 mL ethanol and few
pieces of granulated tin or zinc. Warm the test tube on a water bath for 10 min with occasional
shaking. After cooling the test tube to rt, decant the mixture. Cool the filtrate in ice water. Then
add an ice cooled solution of sodium nitrite until the reaction ceases. To this add now an ice
cooled solution of 2-naphthol in 2N NaOH. Red, orange or brown dye will be formed. The test is
not applicable if the sample contains Ar-NH2 or RCONHAr group.

(ii) Mulliken-Barker test: Take small amount of the sample in a test tube, add 4-5 ml of
ethanol, add a solution of NH4Cl in water (0.2 g in 2 mL) and zinc dust (100 mg) (if your sample
is a nitrobenzoic acid, PERFORM the test without NH4Cl). Boil the solution for 2-3 minutes and
then filter the solution and with the filtrate, perform the following tests.

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

Add Tollen’s reagent to a portion of the filtrate and gently warm the test tube in a water
bath. A white precipitate turning grew (mostly you will see the grey precipitates because of
excess of the filtrate) on standing indicates the presence of Ar-NO2 group. The test is not
applicable if the sample contains –CHO group.
Mix the remaining portion of the filtrate with a small amount of Fehling’s solution. The
formation of a red precipitate indicates the presence of Ar-NO2 group.

Test for anildo (-CONHAr) group

Hydrolysis test

Hydrolyze the sample by heating a test tube containing small amount of the sample in ~3 mL of
conc. HCl and ~2 mL of water or ~5 mL of 50% conc. H2SO4. Cool the test tube to rt, dilute the
content with adding small amount of water and then perform the azo dye test. Formation of a
red or orange-red azo dye confirms the presence of -CONHAr group. The test is not applicable if
the sample contains Ar-NH2 group.

Tests for –CONH2 group

(i) Nitrous acid test

Add sodium nitrite (0.5 g) in water (2.0 mL) to a solution of small amount of the sample and cool
the test tube in ice water. Next, add ice-cooled dil. HCl (2.0 mL) to the test tube. Effervescence
due to the evaluation of nitrogen gas indicates the presence of –CONH2 group.

(ii) Hydrolysis test

Heat a test tube containing a small amount of the sample and ~2 mL of 50% NaOH solution. A
characteristic smell of NH3 which turns mercurous nitrate paper black indicates the presence of
–CONH2 group.

C. Tests for non-nitrogenous functional groups

Tests for –CO2H

(a) Litmus test:

Test a solution or suspension of small amount of the sample in distilled water with a blue litmus
paper. If the sample has –CO2H group or any other sufficiently acidic group (like –OH in ortho-

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

and para-nitrophenols, ortho- and para-hydroxybenzaldehydes etc.), the blue color of the litmus
paper will turn into red.

(b) NaHCO3 test

Add 1 mL of 5% NaHCO3 solution to a small amount of sample taken in a test tube. If the sample
has –CO2H group or any other sufficiently acidic group (like –OH in ortho- and para-
nitrophenols, ortho- and para-hydroxybenzaldehydes etc.), there will be effervescence due to the
evaluation of CO2 gas.

(c) Esterification test:

To 1 ml of ethanol in a test tube, add small amount of the sample followed by a few drops of
concentrated sulfuric acid (catalyst). Heat the test tube in a water bath for five minutes. Pour the
product into a beaker containing 20 ml of sodium carbonate solution (to neutralize any excess
acid). If the product smells of ‘glue’ or ‘pear drops’ an ester has been formed and the sample is
confirmed as a carboxylic acid.

Tests for Phenols

(i) Ferric chloride test

Most phenols react with iron (III) chloride to form colored complexes. The colors vary - red,
purple, blue or green - depending on various factors, e.g. the phenolic compound used, the
solvent, concentration. Since some phenols do not give colors, a negative test must not be
taken as significant without supporting information.

Dissolve 0.05 g of the compound in 2 mL water (or a mixture of water and ethanol if the
compound is not water-soluble) and add an aqueous solution of ferric chloride dropwise.
Observe any color changes which may occur. Many of these complexes are short-lived; the color
may fade soon after it forms. Some phenols may not react at all, so a negative iron(III) chloride
test is inconclusive. Aldehydes or ketones with significant enolic character can also give colored
complexes with FeCl3. Also, aliphatic acids give a yellow solution; aromatic acids give a tan
precipitate. A negative test must not be taken as confirming the absence of a phenol without
additional supporting evidence.

(ii) Back dye test

Dissolve 3-4 drops of aniline in dil. HCl (2.0 mL) and water (3 mL) and cool the solution to 0-5 oC
in an ice-bath for 5 minutes. Add a cold solution (ice-bath) of sodium nitrite (0.5 g) in water (2.0
mL) from a dropper, with swirling of the test tube, still keeping the mixture in the ice-bath. Add
this cold diazonium solution and with swirling to a cold solution of your sample (10 mg) in 2N
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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

NaOH solution (2 mL). An orange-red azo dye is formed. When the sample is ortho- or para-
nitrophenol, this test must be performed with 4-nitroaniline instead of aniline.

(iii) Phthalein test: This test is useful when para position of a phenol (without an electron
withdrawing group) is not substituted.

In a dry test tube, put about 0.1 gm of the sample and equal amount of phthalic anhydride, mix
well and add 1-2 drops of conc. H2SO4. Heat gently on a direct flame for 2-3 minutes until the
crystals of the mixture get melted and fused. Then cool the test tube and pour the content of the
test tube in beaker containing 20 mL of 10% NaOH solution. A characteristic color (green, pale
green, red, pink, violet) with or without fluorescence will be observed.

Tests for aldehydes and ketones

(i) 2,4-DNP (2,4-dinitrophenylhydrazine or Brady's reagent) Test

Both aldehydes and ketones react with 2,4-dinitrophenylhydrazine reagent to form yellow,
orange, or reddish-orange precipitates. Formation of a precipitate therefore indicates the
presence of an aldehyde or ketone. Procedure: Dissolve 20 mg of a solid unknown (1 or 2 drops
of a liquid) in 0.5 mL of ethanol in a small test tube. Add 1 mL of the 2,4-dinitrophenylhydrazine
test reagent. Shake the test tube vigorously. Formation of a large amount of yellow to red,
insoluble 2,4-dinitrophenylhydrazone indicates a positive test. If no precipitate forms, heat the
mixture to boiling for 30 s and shake the tube again. If there is still no precipitate, allow the tube
to stand for 15 min. As a control, try the test on a known aldehyde and ketone. Most aromatic
aldehydes and ketones produce red dinitrophenylhydrazones, whereas many nonaromatic
aldehydes and ketones produce yellow products. If an orange precipitate forms, no definite
conclusions can be drawn from the color.

Preparation of Brady’s reagent:

1. Add 3 g of 2,4-dinitrophenylhydrazine into a conical flask containing 20 mL of water

and 70 mL of ethanol. Place the flask in an ice bath in a beaker.
2. Add 15 mL of concentrated sulfuric acid very carefully and slowly. Make sure that
temperature remains within 10 °C. If the temperature goes above 20 °C, stop addition
until the temperature goes back to 10 °C.
3. Leave the mixture overnight to allow the compound to dissolve completely.
4. Filter through a fritted funnel, if necessary. This gives the yellow-brown stock
solution.

(ii) Test with Tollen's reagent

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 Dr SAJAL KUMAR DAS, DEPT. OF CHEMICAL SCIENCES, TEZPUR UNIVERSITY

Fresh reagent should be prepared each time the test is conducted. The reagent is prepared by
placing 2 mL of a 5% aqueous solution of silver nitrate in a small test tube. A drop of 10% NaOH
is added, producing brown silver oxide precipitate. This precipitate is dissolved by adding just
enough dilute (2%) ammonia to dissolve the silver oxide precipitate. The test is carried out by
adding a few drops of this reagent to a small amount of the unknown sample. Frequently, a silver
mirror or colloidal silver appears instantly. If not, gently warm the tube for a few minutes in a
75°C water bath or a steam bath.
Tollen’s reagent gives a fast positive test with the simplest aldehydes. Some -hydroxyketones,
such as fructose, also give a positive test. The reactions are sometimes characterized by a brief
induction period, probably due to the limited water solubility of the organic unknown and to the
necessity of silver metal formation as an autocatalytic reagent.

(iii) Test with Fehling's solution

Mix 1 mL of each of the solutions “Fehling I” and “Fehling II” to obtain the Fehling reagent. Add
3–5 drops of your sample (if necessary, dissolve the sample in EtOH) to this reagent, and warm
the mixture in the water bath for 2–3 min. A yellow to orange precipitate of Cu2O indicates an
aldehyde. The test is positive for aliphatic aldehydes but is often indecisive for aromatic
aldehydes.

Test for unsaturation

Potassium permanganate test

Dissolve 0.2 g (or 0.2 mL) of the substance in 2 mL of water or acetone. Add the potassium
permanganate solution dropwise and observe the result.

For a blank determination, count the number of drops added to 2 mL of acetone before the color
persists. A significant difference in the number of drops required in the two cases is a positive
test for unsaturation.

Because KMnO4 is a strong oxidizing agent, aldehydes, some primary and secondary alcohols,
phenols, and aromatic amines can also react. So, a negative or positive test must not be taken
as confirming the absence or presence of an unsaturation without additional supporting
evidence.

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