B.Sc.

HONOURS PRACTICAL MANUAL(3rd Year)

CHP: 34b
Experiment 1: Qualitative Analysis of a Single Solid Organic Compound
Sample Number:
Physical Characteristics:
i) Color:

ii) Texture:

iii) Odor:

Detection of Special Elements (N, Cl, S) by Lassaignes method:

Lassaignes Test: A pea sized dry and clean sodium was taken in a fusion tube and heated gently until
it melted with a shiny surface. A pinch of the sample was added to it and the mixture was heated
gently and then strongly to red hot on a non-luminous flame for about two minutes. Finally the fusion
tube was carefully plunged in a mortar containing distilled water (8-10 mL). The mixture was ground
thoroughly by a pestle and filtered. The following experiments were performed with this aqueous
colorless filtrate:

EXPERIMENT
Test for Nitrogen:
To a portion of the filtrate, a few crystals of FeSO4
were added. (Alternatively freshly prepared FeSO4
solution may also be added.) The mixture was
boiled gently and then acidified with dil. H2SO4.
Test for Sulfur :
To a portion of the filtrate, 1-2 drop of dilute
sodium nitroprusside solution was added.
Test for halogens (If N and/or S absent):
A portion of the filtrate was acidified with
concentrated HNO3 and AgNO3 solution was
added.
To the precipitate dilute NH4OH was added.

If N and/or S present:
A portion of the filtrate was acidified with dil.
HNO3 and gently boiled till the volume of the
solution becomes half. It was then cooled and
AgNO3 solution is added.
To the precipitate dilute NH4OH was added.

OBSERVATION

INFERENCE

A deep blue solution or precipitate

Nitrogen present

Sulfur present
Purple coloration
Curdy white precipitate.
Chlorine present

Soluble, but reappeared on

acidification with dil. HNO3.

Same inference
as above

Same observation as above

Same inference
as above

Use of Zn & Na2CO3 mixture instead of metallic Na [Middletons test]

[An intimate mixture of Zn dust and Na2CO3 (2 : 3 by weight) may be stored in a bottle.]
A pinch of organic sample and the above mixture (200 mg) was heated gently and then strongly to red
hot on a non-luminous flame for about three minutes. Then the fusion tube was plunged in a mortar
containing distilled water (6-7 mL). The mixture was ground thoroughly by a pestle and filtered. The
following experiments were performed with this aqueous filtrate:
[When an organic compound is heated with a mixture of zinc powder and Na 2CO3, the nitrogen and
chlorine are converted into NaCN and NaCl respectively, and the sulphur into ZnS (insoluble in
water). NaCN and NaCl are extracted with water and detected as in Lassaignes method, whilst ZnS
in the residue is decomposed with dilute HCl and H 2S evolved is identified with lead acetate paper
which forms black PbS.]
Solubility Classification:
Observations to be made at room temperature (scale: 100 mg of sample in 3 mL of solvent)
water

5% HCl

5% NaOH

5% NaHCO3

conclusion

NP

NP

NP

Polar compound: low MW aliphatic acids,

polyphenols, salts.
Basic : amines (As the sample is insoluble
in NaOH, it will be insoluble in Na2CO3)
Strongly acidic : acids, nitrophenols

Weakly acidic: phenols, imide

Neutral compounds:
a) carbonyl (if N absent)
b) Nitro/amide (if N present)
NP means not performed

+ means soluble;

means insoluble;

If the sample is soluble in water, then no need to check the solubility in all other aqueous solvents
mentioned above. Report NP (not performed) in the columns of other solvents.
If the sample is insoluble in 5% NaOH (strong alkali), then no need to check the solubility in 5%
NaHCO3
Detection of Functional Groups by Systematic Chemical Analysis:
[If nitrogen is present as special element then tests for all functional groups must be performed.]
If nitrogen is absent as special element, then report as given below:
Since nitrogen is absent as special element, tests for nitrogen containing functional groups
aromatic amino (-NH2), aromatic nitro (-NO2), carboxamide (-CONH2) and imide (-CONHCO-)
are not performed.
[Perform only the tests for non-nitrogenous functional groups.]

Tests for nitrogen containing functional groups:

(aromatic NH2, aromatic NO2, CONH2 and HNCONH-)
EXPERIMENT
Test for aromatic NH2 (azo-dye test):
To 1 mL dilute HCl solution / suspension of the
sample, 1 mL 2% NaNO2 solution was added under
ice cold condition. This cooled diazotized solution
was then added to a previously cooled alkaline
solution of -naphthol.
Test for aromatic NO2 (in absence of NH2)
Reduction followed by azo-dye test:
A few pieces of Zn or Sn granules were added to the
sample (~50 mg) in concentrated HCl(2 mL). The
mixture was boiled gently for 2 minutes. It was then
cooled and filtered and diluted with water. Azo-dye
test as described above was performed with it.
Test for aromatic NO2 (in presence of NH2)
Mulliken and Barker test:
To a mixture of sample (~50 mg), Zn dust (100 mg)
and solid NH4Cl (100 mg) 50% aq. Ethanol (3 mL)
was added. The mixture was boiled gently in a water
bath for 5 minutes and then filtered into freshly
prepared Tollens reagent (2 mL).
Test for amide (CONH2) and imide:
A mixture of the sample (~50 mg), a NaOH / KOH
pellet and a few drops of water was heated strongly.

OBSERVATION

INFERENCE

Red / orange azo-dye

Aromatic
NH2 present

Red / orange azo-dye

Aromatic
NO2 present

A grey / black precipitate or

silver mirror

Aromatic
NO2 present

Smell of NH3 was perceived

and the evolved vapour turned
phenolphthalein paper
temporarily pink.

Amide or
imide present

Preparation of Tollens reagent, Ag(NH3)2OH:

A few drops of dilute NaOH solution are added to 1-2 mL of AgNO3 solution. The resulting precipitate
is then dissolved by adding requisite amount of NH4OH solution.

Tests for non-nitrogenous functional groups:

(phenolicOH, >CO, COOH)

EXPERIMENT
Test for carboxylic acid (COOH):
Sample or its alcoholic solution was added to a
saturated solution of NaHCO3.
Esterification: A solution of the sample in
dehydrated alcohol (2 mL) and 2 drops of conc.
H2SO4 was warmed on a water bath for 5 minutes. It
was cooled and then poured cautiously in a beaker
containing 5% solution of NaHCO3 (10-15 mL).
Test for carbonyl (>CO or CHO)
(aldehyde & ketone):
2,4-Dinitrophenylhydrazine (DNP) reagent
(Bradys reagent) was added to a saturated
alcoholic solution of the sample (2 mL). The
mixture was shaken vigorously for few minutes and
then the inner wall of the test tube was scratched
with a glass rod.

OBSERVATION

INFERENCE

Effervescence was
observed

Acidic functionality
present

Characteristic fruity
smell of ester was
perceived.

Carboxylic acid group

(COOH) present

Orange / yellow
crystalline precipitate

Carbonyl group
( >CO) present
Distinction between
aldehyde & ketone may be
done by Tollens reagent

2. Test for phenolic OH group:

(a). To an alcoholic/aqueous solution of the

sample, few drops of neutral FeCl3 solution
are added.
(b). Back Dye Test:
To a solution of 1 drop of aniline in dilute HCl, 1
mL 2% NaNO2 solution was added under ice cold
condition. The resulting solution was then added to
cold alkaline solution of the sample.
[Any one of the above tests if positive that indicates
the presence of phenolic OH group. To show the
absence of phenolic OH, both tests must be
performed.]

Permanent or transient
purple / red / blue / green
coloration
Red precipitate or
coloration of azo dye
observed.

Phenolic OH group
present.
Phenolic OH group
present.

[According to the syllabus, however, ONLY ONE TEST FOR EACH FUNCTIONAL GROUP
is to be reported]

REACTIONS OF THE TESTS FOR DETECTION OF NITROGEN, SULPHUR AND

CHLORINE IN AN ORGANIC SAMPLE:

Organic compounds containing nitrogen, sulphur or halogens when fused with sodium metal, a
reductive decomposition of the compound takes place. The elements are converted to sodium salts of
inorganic ions like CN , SCN , S 2 and X (Cl , Br , I ).
Fusion with sodium
C, H, O, N, S, X (halogen)
NaCN, NaSCN, Na 2S, NaX
Na + C + N == NaCN
Na + C + N + S == NaSCN
2Na + S ==Na2S
Na + X == NaX (X = Cl, Br, I)
2Na + H2O == 2NaOH + H2
All these are present in the aqueous filtrate.
(i) Test For Nitrogen:
a.
Lassaignes Test:
2 ml filtrate + few drops of freshly prepared FeSO 4 solution --- a dark green precipitate of Fe
(OH)2 is obtained. If S is present in the sample, the precipitate will be black. For such cases, more
FeSO4 solution is added and the mixture is heated to boiling and then dilute H 2SO4 is added and
cooled when immediate appearance of blue or green precipitate or color of Prussian blue,
Fe4[Fe(CN)6]3 or NaFe[Fe(CN)6] indicates the presence of nitrogen.
FeSO4 + 2NaOH == Fe (OH)2 + Na2SO4
Fe (OH)2 + 2NaCN == Fe(CN)2 + 2NaOH
FeSO4 + 2NaCN == Fe (CN)2 + Na 2SO4
Fe (CN)2 + 4NaCN == Na4[Fe(CN)6]
4FeSO4 + 2H2SO4 + O2 == Fe2 (SO4)3 + 2H2O
3Na4 [Fe (CN) 6] + 2 Fe2 (SO4)3 == Fe4[Fe(CN)6]3 + 6 Na 2SO4
Prussian Blue
Or
2 Na4 [Fe (CN)6] + Fe2 (SO4)3 = 2NaFe[Fe(CN)6] + 3Na2SO4
Prussian Blue
b.
Middletons Test:
When an organic compound is heated with a mixture of zinc powder and Na 2CO3, the
nitrogen and chlorine are converted into NaCN and NaCl respectively, and the sulphur into ZnS
(insoluble in water). NaCN and NaCl are extracted with water and detected as in Lassaignes
method, whilst ZnS in the residue is decomposed with dilute HCl and H 2S evolved is identified with
lead acetate paper which forms black PbS. The test for nitrogen is thus not affected by the presence of
sulphur; this constitutes an advantage of the method. [Ref: Qualitative Organic Analysis, 2nd Edition,
A.I.Vogel,(Part-2) pages: 41-42]
(ii) Test for Sulphur: [Sodium Nitroprusside Test]
2 ml of filtrate of the sodium extract + 1 -2 drops of freshly prepared aqueous solution of sodium
nitroprusside---- appearance of violet or purple color confirms the presence of sulphur.
Na2S + Na2 [Fe (CN) 5 NO] == Na 4 [Fe(CN) 5 NOS ]
Violet or purple

(iii) Test for Halogens:

Cyanide and sulphide ions, if present must be removed, since they interfere the tests with AgNO 3
solution.
Acidify the sodium extract with conc. HNO3 and boiled to reduce the volume to half of the original to
drive off HCN and H2S.
Boiled
NaCN + HNO3 (conc)
HCN + NaNO3
Na2S + 2HNO3
H2S + 2NaNO3
The acidified solution (filtrate) was taken and AgNO 3 solution was added:
(a) Test for chlorine:
Observation: White precipitate, soluble in ammonium hydroxide solution which reappears on
addition of concentrated HNO3.
NaCl + AgNO 3 = AgCl + NaNO3
White ppt.
AgCl + 2NH4OH = [Ag (NH3) 2] Cl + 2H2O
Soluble
[Ag (NH3)2]Cl +HNO3 = AgCl +NH4NO3
White ppt.
DETECTION OF FUNCTIONAL GROUPS:
1. Carboxylic acid group: ( -COOH)
2. Phenolic hydroxyl group: ( Ar OH)
3. Carbonyl group: ( ketonic >=O or aldehydic CHO group)
4. Aromatic primary amino group: (ArNH2)
5. Nitro group: (-NO2)
6. Amido or imido group: (-CONH2 or CONHCO-)
1. CARBOXYLIC ACID GROUP [COOH]:
(a) Aqueous solution, aqueous suspension or ethanolic solution of the compound + blue litmus paper
if red, presence of COOH group can be suspected.
(b) Saturated solution of NaHCO3 + a pinch of sample/ ethanolic solution of the sample
----Effervescence of CO2, COOH may be present. Effervescence of CO2 is also possible by SO3H
group or activated phenols like nitrophenols.
RCOOH + NaHCO3 == RCOONa + CO2 + H2O.
(c) Esterification test:
Small amount of sample + 2 ml ethyl alcohol + 2 -3 drops of conc. H 2SO4 heated on a water
bath for 5 minutes poured into 20 ml saturated bicarbonate solution Fruity smell of ester COOH
group confirmed.

2. PHENOLIC HYDROXYL GROUP [ArOH]:

(a) Alcoholic solution of the sample + 2 drops of neutral FeCl 3 characteristic violet/violet green /
green color appeared.

Fe

3+

6 HO

Fe (O-C6H5)6

6H +

** -Naphthol gives green precipitate.

** Hydroquinone gives Transient green color.
** -Naphthol, o nitro phenol, o and m aminophenol, 2,4 diaminophenol, m and p
hydroxybenzoic acids do not respond to this test.
(b) Back dye test:
[ 1 drop of aniline in dil. HCl + few drops of freshly prepared aqueous NaNO 2 solution cooled
o
o
to 0 -5 C in a test tube] ---- contents of the test tube were added to cooled (in ice) alkaline solution of
the sample---------brilliant red azo dye.
O

NH2

+
N

NaNO2 / dil. HCl

N Cl

O + NaCl

0o - 5o C
Soluble red dye

3. CARBONYL GROUP (>= O) [BOTH CHO AND >CO GROUP]:

(a) Alcoholic solution of the sample + equal volume of 2,4 D.N.P. ----- heated on a water bath (if
necessary) for 5 minutes and the sides of the test tube are scratched if necessary.--------- Orange or
yellow precipitate.
R1
O
R2

H2N

NH

R1

NO2

NH

NO2

R2

NO2

NO2
Orange or yellow precipitate of
2,4 Dinitro phenyl hydrazone derivative.

4. PRIMARY AROMATIC AMINO GROUP [ArNH2]:

Sample + dil. HCl + freshly prepared aqueous NaNO 2 solution, cooled to 0 o 5 o C
(diazotized solution A); cold alkaline solution of -naphthol was prepared ( -naphthol dissolved
in dilute aqueous NaOH solution B); the diazotized solution A was poured into solution B
brilliant red azo dye.
Ar
N
O
ArNH2

NaNO2 / Dil. HCl

0o - 5oC

+
ArN2 Cl

N
OH

in aqueous NaOH
Precipitate
of Azo - dye.

5. AROMATIC NO2 GROUP:

(a) Reduction of NO2 to NH2 (should be done when ArNH2 group is absent in the sample)
[Diazo coupling reaction]:
Sample + metallic Zn + 1 ml conc. HCl-- boiled for 5 minutes, cooled, diluted with 5 ml
water, filtered and cooled to 0o to 5o C in ice + freshly prepared aqueous NaNO2 solution, cooled to 0 o
5 o C (diazotized solution A); cold alkaline solution of -naphthol was prepared ( -naphthol
dissolved in dilute aqueous NaOH solution B); the diazotized solution A was poured into
solution B brilliant red azo dye.
Ar
N
O
ArNO2

Zn / conc. HCl
[H]

ArNH2

NaNO2 / Dil. HCl

0o - 5oC

+
ArN2 Cl

OH
in aqueous NaOH
Precipitate
of Azo - dye.

(b) Partial Reduction of NO2to NHOH (can be performed in presence of NH2 group):
[Mulliken and Barker test]
Sample dissolved in ethanol + pinch of NH 4Cl + Zn dust + a few drops of water in a test tube,
heated on a water bath for 5 minutes -------- filtered hot directly into Tollens reagent (AgNO3+ 1 -2
drops of aq. NaOH solution ----dark grey / black precipitate of Ag 2O -------conc. NH4OH solution was
added till a clear solution was obtained [Ag(NH 3)2] OH). --------- black precipitate turned to silver
mirror on heating on a water bath.
6 ArNO2 (in alcohol)

Zn / NH4Cl

ArNHOH

[Ag(NH3)2]OH

ArNO + NH3 +H2O + Ag

Black ppt

6. TEST FOR AMIDE GROUP [CONH2] OR IMIDE GROUP [CONHCO]:

Sample + 2 -3 solid beads of NaOH + 2 -3 drops of water ---- heated gently ----- red litmus
paper held at the mouth of the test tube ------ turned blue or glass rod moistened with conc HCl held
at the mouth of the test tube ------- white fumes of NH4Cl.
O
C - NH2

OH /

COO

+ NH

+ H2O