Test Observation Inference

I. Preliminary Test and Physical Examination

1 State i) Solid Generally high molecular weight,
usually having more than 6 carbon
atom chain.
Eg: Acids, Sugars, Amides, etc.
ii) Liquid Generally low molecular weight,.
Eg: Alcohols, Ethers, Esters,
Aliphatic amines, Aldehydes,
Ketones, Hydrocarbons, etc.
2 Colour Pale Yellow Nitro Compounds s.a. nitrobenzene,
dinitrobenzene, nitrophenol, Quinones,
Iodoform
Yellow Orange Nitro-aniline
Pink -Naphthol, -Naphthol,
resorcinol,
Red Azo compounds, methyl orange
Green Nitroso compounds
Greenish Yellow o/m-nitrobenzaldehyde
Brown-dark Phenols, Amines, (darken due
to oxidation)
Colourless Compounds not containing strong
Chromophore.
Eg: Acids, Hydrocarbons, ketones,
esters, urea, thiourea, acetamide,
acetanilide, benzamide, naphalene, etc
3 Odour Pleasant Alcohols (low mol. Wt.), Aromatic
hydrocarbons, ethers, alkyl halides s.a.
chloroform, chlorobenzene.
Deep Sweet Chloroform
Fruity Esters
Phenolic / Carbolic Phenols and cresols
Fishy Aromatic amines
Kerosene Hydrocarbons
Moth Ball Naphthalene
Bitter Almond Benzaldehyde, nitrobenzene
Pungent/Irritating Aliphatic acids, Acid chlorides,
formaldehyde, and side chain
1/ CY8281- Organic Chemistry Laboratory
 halo hydrocarbons

S.N EXPERIMENT OBSEVATION INFERENCE

o
4 Flame Test: Non-sooty Flame Aliphatic compounds
Take a small quantity of Sooty Flame Aromatic compounds
compound and put it on a Ammonical odour Urea, thiourea, amides may be
metallic spatula or in a present
porcelain dish and ignite it Chars and swells Sulphur containing compounds
directly on the flame. without melting
Melts and chars with a Carbohydrates may be present
smell of burnt sugar
Produces coughing Benzoic acid, salicylic acid etc
upon charring may be present
Chars without melting Sulphanilic acid, starch, uric
acid may be present
5 Test for Saturation/Unsaturation
a. Action of Decolourization of Unsaturated compounds
KMnO4(Baeyers test): Sub KMnO4 may be present
(solid/Liquid) + Sodium
carbonate solution + few No Decolourization of Saturated compounds may be
drops of 2% KMnO4 KMnO4 present
solution – Shake vigorously
6 Lassaigne’s Test (Elemental Analysis)
Place about a pea size of freshly cut sodium metal into a sodium fusion tube and
heat the tube gently to melt the sodium to a shining globule. Add a small quantity of the
sample (solid/liquid) into the fusion tube. Heat the tube carefully at first and then as
strongly as possible until the bottom of the tube is glowing red, holding the tube at this heat
for about 2 min. Quickly plunge the hot tube in a china dish containing about 8 ml of
distilled water and cover the china dish immediately with a wire gauge. The tube crumbles
into pieces and the mass comes out and dissolves in water. Boil the contents of the china
dish thoroughly, filter the contents and collect the filtrate (which is also called Stock
solution) in a test tube. Divide the filtrate into 3-4 portions and test each portion for the
elements separately.
Test Observation Inference
1. Test for Nitrogen:

a. Prussian Blue test Green or Blue colour Nitrogen present

Stock solution + FeSO4 No Green or Blue colour Nitrogen absent
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 solution – boil and cool. Add
conc. H2SO4

7. Group Detection: Nitogen Absent groups

Test for Group like Acids, Alcohols, Aldehydes, ketones, esters, phenols, etc

a. Test for
Aldehydes: Sub + Red ppt Aldehydes or Ketones present
2,4-dinitrophenol
ii. Tollen’s Test: Silver mirror formed
b Sub + Tollen’s reagent on the walls of the Aldehydes present (aromatic
(ammonical AgNO3 soln) – test tube/ grey- black aldehyde gives test)
warm on a water bath ppt
No Silver mirror Ketones present
c Test for Alcohols:
Alcohol present
Sub + Sodium metal in a dry Effervescence
test tube
d Test for Acids: Acids present
Effervescence
Sub + NaHCO3 solution
e Test for Esters:
Sub + NaOH solution + 1 Esters present
Pink colour disappears
drop of phenolphthalein
indicator
f Test for Phenols: Violet colour, greenish
Phenols present
Sub + Alcoholic FeCl3 solutionpurple colour, bluish
purple colour
Group Detection: Nitogen Present groups
8
Test for Group like Amine and Nitro compounds

a Test for Group : 10, 20 and 30 Amines

i. Sub + Acetyl chloride or Vigorous reaction (solid 10 and 20 Amines present
Benzoyl chloride (in dry separates)
test tube)
ii. Diazotization Test: a. Clear 10 Amines present
Sub + dil. HCl till soluble – solution/Brown oil
3/ CY8281- Organic Chemistry Laboratory
 cool in ice water. Add ice obtained – further
cold solution of NaNO2 in add ice cold -
water. naphthol in NaOH
solution - Orange
Dye
20 Amines present
b. Yellow oil separates
c. Red/Brown oil -
further add NaOH
30 Amines present
solution - green solid
separates
b. Test for Nitro Compounds

i. Sub + NaOH solution – boil No smell of NH3 Amides absent

ii. Sub + NaOH solution + No smell of carbylamines Anilide absent
CHCl3 – boil
continuously for some
time
iii. Sub + Conc. HCl + Tin Clear solution
pieces – wait till reaction - Further add ice
ceases – then boil till layer cold - naphthol
disappears – cool, filter, in NaOH in
dilute and add ice cold solution
NaNO2 solution dropwise.
Nitro compound present
- Red/Orange
dye
obtained
iv. Neutral Reduction:
Dissolve 0.1 g of sample in 2 ml
ethanol + 5 drops of CaCl2
solution + pinch of Zinc dust Grey/Black ppt
and boil the contents for 5 Nitro compound present
(NO2 group present)
mins. Filter the solution in a
test tube containing 1 ml of
Tollen’s reagent
v. Test for Aromatic nitro Dinitro compound
Pink colour
compounds: Sub + acetone +
NaOH solution Mononitro compound
No pink colour
Analysis of proteins
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Aim: Analyze the given sample protein with suitable methods.

Materials Required
a) Samples
1. Egg albumin
2. Gelatin
b)Reagents
1. NaOH solution
2. 1% CuSO4 solution
3. Conc. HNO3
4. Ninhydrin solution
5. Millon’s reagent
c) Apparatus
1. Test tube
2. Test tube holder
3. Dropper

Procedure
a) Biuret test :
Take a small quantity of the dispersion of the sample in a
test tube and add 2 ml of NaOH solution into it. Now add 4-5 drops of 1%
CuSO4 solution and warm the mixture for about 5 minutes.
S. No. Sample Observation Inference
 Egg albumin  Bluish violet colour is presence of
1.
dispersion formed. protein
 Bluish violet colour is presence of
2.  Gelatin dispersion
formed. protein

b)Xanthoproteic test:
Take about 2 ml of the sample in a test tube and add
few drops of conc. HNO3 into it and heat the test tube.

S. No Inference
 Sample  Observation
.
 Egg albumin  A yellow precipitate is
1. presence of protein.
dispersion formed.
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  A yellow precipitate is
2.  Gelatin dispersion presence of protein.
formed.

c)Ninhydrin test:
Take 2 ml of the sample in a test tube and add 3-4 drops of
Ninhydrin solution and boil the contents.
Inference
SI No.  Sample  Observation

 Egg albumin  Intense blue colour is

1. presence of protein.
dispersion formed.
 Intense blue colour is
2.  Gelatin dispersion presence of protein.
formed.

d)Millon’s test :
Take 1-2 ml of the sample in a test tube and add 2 drops of
Millon’s reagent.
SI No Inference
 Sample  Observation
.
 Egg albumin  White precipitate which
1. presence of protein
dispersion changes to brick red on boiling.
 Gelatin
2.  No characteristic change. presence of protein
dispersion

Report:
Based on the following observation the sample was conformed as
protein.
a) Bluish violet colour is formed in Biuret test.
b) A yellow precipitate is formed in Xanthoproteic test.
c) Intense blue colour is formed in Ninhydrin test.
d) White precipitate which changes to brick red in Millon’s test.

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 Qualitative Analysis Of Carbohydrates

A carbohydrate is an organic compound with the general

formula Cm(H2O)n, that is, consists only of carbon, hydrogen and oxygen,
with the last two in the 2:1 atom ratio. Carbohydrates make up the bulk of
organic substances on earth and perform numerous roles in living things.
The carbohydrates (saccharides) are divided into four chemical groups:
1. monosaccharides,
2. disaccharides,
3. oligosaccharides and
4. polysaccharides.

Materials Required
a) Samples : Carbohydrates
b)Reagents
1. Molisch’s reagent (10% α-naphthol in ethanol)
2. Conc. H2SO4
3 Fehling’s solution A
4. Fehling solution B
5. Barfoed’s reagent
c) Apparatus
1.Test tube
2. Test tube holder
3.Dropper

TESTS ON CARBOHYDRATES:
S.
N Test Observation Interference
o
1 Molisch’s Test:  A brown presence of
Take 2 ml of a known color due is carbohydrate
carbohydrate solution in a test tube, add appeared due
1 drop of Molisch’s reagent (10% α- to ch
naphthol in ethanol). Then pour 1-2 ml arring.
of conc. H2SO4 down the side of the test
tube. so that it forms a layer at the
bottom of the tube. Observe the color at
the interface between two layers and
compare your result with a control test.
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 Fehling’s Test:
Take 1 ml of Fehling’s
2 solution A (aqueous solution of CuSO4) The red
add 1 ml of Fehling solution B (solution precipitate of presence of
of potassium tartrate). Add 2 ml of the cuprous oxide carbohydrate
sugar solution, mix well and boil. is formed

Barfoed’s Test:
Take 1-2 ml of Barfoed’s reagent, add brick-red
3 an equal volume of sugar solution. cuprous oxide presence of
- Boil for 5 min. in a water bath and precipitate is carbohydrate
allow to stand. formed

Report:
Based on the following observation the sample was conformed as
carbohydrate.
a. A brown color due is appeared due to charring in Molisch’s Test.
b. The red precipitate of cuprous oxide is formed in Fehling’s Test.
c. brick-red cuprous oxide precipitate is formed in Barfoed’s Test.

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 ORGANIC SYNTHETIC PROCEDURE

1)HYDROLYSIS OF METHYL SALICYLATE

Aim
To prepare the organic compound methyl salicylate from .
THEORY
Methyl salicylate is an ester easily recognized by its odor and is known
as oil of wintergreen because of its natural source. This ester will be treated
with aqueous base.The hydrolysis reaction that occurs will form methanol,
water, and the sodium salt of salicylic acid. Salts of organic compounds
usually are soluble in water or will dissolve in water with a bit of heating.
Later in the work-up, the salt is acidified with sulfuric acid to convert the
organic salt into the protonated carboxylic acid. Therefore, the major organic
products of this reaction are methanol and salicylic acid. The salicylic acid is a
solid that can be isolated and purified by crystallization. The chemical
equations that describe this experiment are:
Because the phenolic hydroxyl group is acidic, it is also converted
to the corresponding sodium salt during the basic hydrolysis. In the
subsequent acidification, this group also becomes reprotonated.

O O
-
C-O- C-O +
Na
CH3 2 NaOH + +
+ CH3OH
+
- H2O
OH O Na

methyl
salicylate H2SO4
(dilute)

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 O

C-OH

OH

salicylic acid
PROCEDURE
APPARATUS : 1) Assemble a reflux apparatus, using a 250 mL round-
bottom flask and your watercooled condenser with the wide bore inside tube.Be sure to
use a small amount of stopcock grease to seal the glass joint between the round bottom
and condenser. Use a heating mantle to heat, setting its height so that the mantle can be
dropped down when cooling is desired.
REACTION MIXTURE Dissolve 10 g sodium hydroxide into 50 mL of
deionized water in a 250 ml Erlenmeyer flask. While this solution cools a bit, remove the
round-bottom flask from your reflux set up. Weigh 5.0 g of the methyl salicylate liquid
into a small beaker, then pour this liquid into your round-bottom. Add one or two small
boiling stones to the round bottom. Finally, add the sodium hydroxide solution to this
same round-bottom.Reassemble the apparatus for reflux.
REFLUX 3) Start water flowing through the condenser, turn on the mantle,
and bring the mixture to boil. Continue heating under vigorous reflux for 30 minutes.
Your "reflux ring" should be about . to 2/3 the way up your condenser. After the thirty
minutes of reflux, stop heating, lower the heating mantle if possible, and shut down the
water flow.
CRYSTALLIZATION AND FILTRATION
4) When the solution has cooled for about five minutes, transfer it to a 250 mL
beaker. Try to leave the boiling stones "behind" when you decant the reaction mixture
from the round-bottom to the beaker.
5) Carefully add enough 1 M sulfuric acid to make the solution acidic when
tested with litmus paper (blue litmus turns red). It may be necessary to add as much as 150
mL of the acid, so you can pour in large quantities before checking with litmus each time.
When the litmus turns pink, add an additional 15 mL of 1 M sulfuric acid to guarantee an
acidic environment. The acid addition should cause the salicylic acid to precipitate from
the solution.
6) Cool the mixture in an ice bath to a temperature of 5 °C. Set up a vacuum
filtration using a Buchner funnel and filter paper .Start the vacuum on the Buchner
system, swirl the beaker containing the precipitate to loosen it, then carry out the
filtration. Use a stirring rod or spatula to transfer as much solid as possible to the filter.
Since you will be recrystallizing the product "from water" in the
10/ CY8281- Organic Chemistry Laboratory
next step, you need not get it very dry at this time.

Results:
The yield of Methyl salicylate is ______gm.

2) Preparation of acetanilide from aniline.

Aim:
To prepare the organic compound acetanilide from aniline, glacial acetic acid/acetic
anhydride and zinc dust.

Theory:
Acetanilide is prepared from aniline when it reacts with acetic anhydride/glacial
acetic acid in the presence of zinc dust. A mixture of aniline, glacial acetic acid, acetic
anhydride and zinc dust is refluxed under anhydrous condition and then poured the
mixture into ice cold water to get acetic anhydride precipitate. The crude precipitate of
acetic anhydride is recrystallized to get pure crystals of acetanilide.
The chemical reaction is given below.

Zinc is used to prevent the oxidation of aniline during the chemical reaction.
Acetanilide is medicinally important and it is used as febrifuge. Acetanilide cal also be
prepared by acetylating aniline with acetic anhydride in the presence of concentrated
hydrochloric acid. Dissolve aniline in hydrochloric acid and add acetic anhydride stir well.
Pour the mixture to sodium acetate in water. Acetanilide is formed which can be separated
and recrystallised by ethyl alcohol.
Other names – N-phenylacetamide, N-phenylethanamide, Acetanil

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Materials Required:

1. Aniline 8. Pipette
2. Glacial acetic acid 9. Reflux condenser
3. Acetic anhydride 10.Funnel
4. Zinc dust 11.Stirrer
5. Distilled water 12.Bunsensurner
6. Round bottom flask 13.Filter paper
7. Beaker 14.Electronic balance

Procedure:

Wash all the apparatus with distilled water before starting the experiment. Take a
round bottom flask in that add 10ml of aniline and 20ml of acetic anhydride and glacial
acetic acid mixture and add zinc dust. Fix the reflux condenser with the round bottom
flask. Heat the mixture gently for about 15-20 minutes on oil bath.Pour the hot mixture in
a beaker containing ice cold water with constant stirring. Stir the mixture vigorously to
hydrolyse excess of acetic anhydride. Once all the acetanilide is precipitated collect and
filter in buchner funnel. The precipitate obtained is a crude sample of acetanilide. To get
the pure crystals crystallization should be carried out.

Crystallization:
Transfer the crude sample into a beaker containing 20ml water and heat gently. If
the solution is coloured then add a small amount of activated carbon. Filter the hot
solution with a funnel. Cool the mixture for 30 min so that white shiny crystals of
acetanilide separates out. Filter off the crystals, wash them with water and dry in the
foldes of filter paper.

Results :
The yield of Acetanilide is ______gm.

3) Preparation of m-dinitrobenzene from nitrobenzene

AIM :
To prepare m-dinitrobenzene from nitrobenzene
Apparatus/Glassware Required: 
Round-Bottom Flask, reflux condenser, beaker, volumetric flask, measuring
cylinder, suction pump and Buchner funnel
Chemicals Required:
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 Nitrobenzene, concentrated sulphuric acid, fuming nitric acid and rectified spirit
Principle:
It is prepared by nitration of nitrobenzene with concentrated nitric acid in
the presence of concentrated sulphuric acid. Here, the function of the sulphuric acid is to
convert the nitric acid into the highly reactive, electrophile, nitronium ion (NO2+), which is
the effective nitrating agent. Nitration of aromatic hydrocarbons is usually carried out
with the nitrating reagent at comparatively low temperatures. Unnecessarily high
temperatures should be avoided since poly nitration is more likely and oxidative break
down of the aromatic system may occur.

The nitration of nitrobenzene containing an electron-withdrawing -NO 2 group

does not occur readily under the above conditions, in which use forcing conditions which
requires the use of fuming nitric acid and concentrated sulphuric acid need to be
employed. The deactivating affect of the nitro group is largely the result of its mesomeric
interaction (-M effect) with the π- electron system of the benzene ring which is
supplemented by the inductive (-I) effect. The overall electron withdrawal from the ring
system results in the rate of attack of the nitronium ion being substantially retarded
compared to benzene. Moreover, the representation of the canonical forms of
nitrobenzene, the Ortho and Para-positions are subject to the greatest reduction in electron
density, so energetically unfavorable the mesomeric stabilization of this intermediate is
less than that of the corresponding intermediate resulting from attack in the m-position.
Because of this reason, the incoming nitro group oriented towards the m-position in the
nitrobenzene.
Procedure:

Place 7.0 mL of concentrated sulphuric acid and 5.0 mL of fuming nitric acid,
in a 85 mL rectified spirit. Add a few fragments of unglazed porcelain. Attach a reflux
condenser and place the apparatus in a fume cup board. Add slowly, in portions of about
1.0 mL, 4.2 mL (5.0 g) of nitrobenzene. After each addition, shake the flask to ensure
thorough mixing. Heat the mixture, with frequent shaking, on a boiling water bath for 30
minutes. Allow the mixture to cool somewhat and pour it cautiously with vigorous
stirring into about 170 mL of cold water; the m-dinitrobenzene soon solidifies. Filter with
suction, wash thoroughly with cold water and allow draining as completely as possible.
Transfer the crude m-dinitrobenzene to a 250 mL flask fitted with a reflux condenser, add
80-100 mL of rectified spirit and heat on a water bath until all the crystalline solid
dissolves. If the resulting solution is not quite clear, filter it through a fluted filter paper
on a large funnel which has previously been warmed or through a warm Buchner funnel.

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Recrystalization:
Transfer the crude sample into a beaker containing 20ml water and heat gently. If
the solution is coloured then add a small amount of activated carbon. Filter the hot
solution with a funnel. Cool the mixture for 30 min so that white shiny crystals of
acetanilide separates out. Filter off the crystals, wash them with water and dry in the
foldes of filter paper.

Result:
The yield of m-niotrobenzene is ______gm.

4) Preparation of benzoic acid and benzyl alcohol from benzaldehyde

Aim
To prepare m-dinitrobenzene from nitrobenzene
Procedure:
20 g of benzaldehyde are treated in a stoppered cylinder or a thick-walled
vessel with a cold solution of 18 g of potassium hydroxide in 12 g of water, and mixed
until a permanent emulsion is formed. The mixture is then allowed to stand over night.
The vessel is closed by a cork, and not a glass stopper, since at times a glass stopper
becomes so firmly fastened that it can be removed only with great difficulty. To the
crystalline paste (potassium benzoate) separating out, water is added until a clear solution
is obtained from which the benzyl alcohol is extracted by repeatedly shaking with ether.
After the evaporation of the ether the residue is subjected to distillation; benzyl alcohol
passes over at 206° C. Yield, about-8 g. The benzoic acid is precipitated from the alkaline
solution on acidifying with hydrochloric acid and purified from hot water yielding final
product with melting point 121° C.
Recrystalization:
Transfer the crude sample into a beaker containing 20ml water and heat
gently. If the solution is coloured then add a small amount of activated carbon. Filter the
hot solution with a funnel. Cool the mixture for 30 min so that white shiny crystals of
acetanilide separates out. Filter off the crystals, wash them with water and dry in the
foldes of filter paper.

Result:
The yield of benzoic acid is ______gm.

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