Part I

GENERAL PROCEDURE FOR THE SYSTEMATIC QUALITATIVE

ANALYSIS OF ORGANIC COMPOUNDS

i. Preliminary investigations.

a. Physical State.

b. Color

c. Odour.

d. Ignition on china porcelain.

e. Action of 30% sodium hydroxide solution.

f. Action of hot concentrated sulphuric acid.

g. Reaction with ferric chloride.etc.

ii. Detection of elements: Give details for the detection of nitrogen, sulphur and

halogens

iii. Detection of functional groups: Give the procedure for the detection of functional

groups especially for the group(s) that is found to be present.

iv. Determination of melting (boiling) point: The Compound is found to melt (boil)

at....C.

v. Probable Compound: On the basis of the standard data of the melting (boiling)

points of substances having particular functional groups(s), the given compound is

most probably.........

vi. Special tests of the compound: Give the special test(s), if any, for the particular

compound.

vii. Derivatives: Give the methods of the preparation of at least two important

derivatives and also mention their melting points observed by you.

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Result:

Hence the given organic compound is:

1. Aromatic/Aliphatic

2. Saturated/Unsaturated

3. Special element present/Not present

4. Functional group present

5. Melting point

6. Probable compound based on melting point and derivatives prepared;

PRELIMINARY INVESTIGATIONS

1. Physical state and colour

In organic compounds the colour is generally associated with the presence of

certain functional groups i.e. particular group in the compound is responsible for a

particular shade. However, it should also be noted that several organic compounds are

colourless in the pure state but oxidized to coloured compound by atmospheric air,

e.g. amines. The different important groups along with their most usual colours are

summarized below in the solid and liquid states.

A. Solids

Colour Possible compoundsYellow Iodoform, benzoin, Benzyl anthracene,

quionones and nitro compounds.Orange Ortho-nitro aniline.Pink Naphthols.

BrownDiamines, aromatic higher amines and aminophcnols.

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B. Liquids

Colourless to red brown Amines.

Yellow to Orange Nitro compounds.

2. Odour.

Most of the organic compounds possess characteristic odowgtke- odour of

some compounds is so characteristic that it can be used even to distinguish the

particular class of compounds or sometimes even a single compounded from that of

others.

Odour Possible compounds

Carbolic or Phenolic Phenols

Naphthalene like Naphthalene, p-dichloro or p-dibromo benzene.

Mice like Acetamide.

Bitter almond like Benzaldehyde. nitrobenzene

Pleasant Alcohols, ethers, esters

Very pungent Lower aliphatic acids and their halogen derivatives.

3. Test for Aliphatic or aromatic nature:

i) Ignition

The nature of organic compound (whether aliphatic or aromatic) can be

established by the ignition test.

The compound is placed on a nickel spatula. The latter is then heated first

slowly and then strongly in a burner flame. If the compound burns with an non-smoky

flame, it is an aliphatic compound (except chloroform, chloral hydrate and ethyl

acetate which burn with a smoky flame); while if it burns with a sooty or smoky flame

the compound is aromatic in nature. The aliphatic and aromatic nature of compound

may further be ascertained by the following tests.

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i. Nitration test. Take 1 gm. (or lml.) of the compound in a test-tube. Add

2ml. Of conc. HNO3 and 2ml. of conc H2SO4. Heat the contents for about

5 minutes,cool and pour the contents in cold water- formation of a yellow,

oily liquid or solid indicates the presence of an aromatic compound ;

otherwise it is an aliphatic compound.

ii. Le Rosen test. Take 0.2 gm. (or 3-4 drops) of the organic compound,3-4

drops of formaldehyde solution and 5ml, Of con H2SO4.Shake the

contents well and then warm-formation of red, orange, violet or green

colour or precipitate indicates the presence of an aromatic compound;

otherwise it is an aliphatic compound.

iii. Ignition on china porcelain: Place a small amount of the compound on a

porcelain dish and then heat it first gently and then strongly. Note the

change in appearance, the odour produced and the residue left.

Following are the notable observations and inferences of various important

compounds.

Observation Inference

Burns with luminous flame Aromajtic or unsaturated

Smoky flame... Aliphatic compounds

Smell of ammonia Amides

Violet vapours Iodoform

Chars but does not melt Uric acid, sulphonic acid, starch

Darkens, swells, chars with

Burnt Suger Odour

Carbohydrates, lartaratcs, lactates – 4 glucose

and some citrates.

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4. Solubility

(i) Action of 20% sodium hydroxide solution:

Treat a small amount of the compound with nearly 5ml of the alkali solution

and note down the change in the cold state.

Observation Inference

Dissolves in NaOH, reprecipates on adding Aromatic acids, Phenol

Con. HCL

Mixture sets to a white solid Esters of diabasic acids

Highly coloured solution Nitrophenols, nitoracids, salicylaldehyde

Colour becomes deep on standing polyhydricphenols, some aminophenols

Yellow to brown resin Aliphatic aldehydes

Now heat the solution and note the change

Compound dissolves some esters or acid anhydrides

Ammonia is evolved Amides, imides, urea, thio-urea,

Yellow or brown solution sulphonamide sugars (except sucrose), polynitro

compounds, halogennophenols, halogenonitro

hydrocarbons.

Chloroform like odour Chloral.

ii) Action of hot concentrated sulphuric acid:

Treat a small amount of the given compound with concentrated sulphuric acid,

note the change and then warm it.

Observation Inference

Evolution of CO2 without charring urea.

Evolution of CO2 and CO.

And mixture becomes yellow

without charring.

Aliphatic hydroxy acids

Evolution of CO and /or CO2

And SO2 with immediate blackening

Carbohydrates, aliphatic hydroxy

acids.

Blackening but no effervescences. Polyhydric phenols.

Pungent vapours without charring Carboxylic acids (simple)

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iii) Action of NaHCO3:

0.l g of the substance treated with 2ml of saturated NaHCO3 soln.

Dissolves with brisk effervescence of CO2 Acid or acidchloride.

iv) Action of Dil. HCl:

0.5g of the substance treated with 2ml of dil. HCI and shake well.

Dissolves in HC1 and reappears on adding Basic substance (Amines)

10% NaOH solution.

5. Reaction with Neutral ferric chloride:

This regent is frequently used for identifying the presence of phenols and

aliphatic a-hydroxy acids., Take nearly two ml. of the compound solution either in

water or in alcohol and add to it one or two drops of neutral ferric chloride solution

and note the changes as below.

Observation Inference

Red colour but disappears Lower fatty acids, maleic acid

by adding dil.HCL. or amino acid.

Intense transitory bluish-green o-phenylene diamine

Colour.

Intense red-brown colour ortho or meta-phenylene diamine.

Or ppt.

Purple colour salicylaldehyde, salicylic acids, salicylamide

acetylsalicylic acid, phenyl salicylate.

Blue or red colour Phenols, resorcinols, cresols, arninophenols.

Greenish colour or ppt. Catechol. Quinolnes of naphthol.

Deep yellow colour aliphatic a-hydroxy acids

Pale brown ppt. dissolves in

div HCL

Dicarboxyiic acids soluble in water.

Ferric chloride may also be used for identifying certain amines. In practice, the

compound is dissolved in dilute hydrochloric acid and two drops of ferric chloride

solution are added to the solution.

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Observation Inference

Green or blue green colour diphenylarnine, aniline

N-methylaniline

Violet colour P-anisidine.

Red colour P-phenetidine.

Red ppt. O-phanylenediamine.

Blue ppt. O-naphthylamine, o-toluidine.

6. Reaction with Tollen's reagent:

0.1 g of the substance dissolved in suitable solvent and add 2ml of Tollen's

reagent. Warm on water bath.

Black ppt or bright silver mirror Presence of Aldehydes, carbohydrates,

polyhydric Phenols or other reducing agents

7. Test for saturation or Unsaturation

1. Action of potassium permanganate: Potassium permanganate is a very

important oxidizing agent in neutral, acid as well as in alkaline medium.

i. In neutral medium:

Dissolve the substance in water and then add to it 4-5 Drops of neutral

potassium permanganate solution with constant shaking -violet colour discharged in

case of olefinic compounds.

ii. In acidic medium:

Perform above test using potassium permanganate solution acidified with

dilute sulphuric acid and then warm the contents-violet colour discharged in case of

olefinic compounds, or easily oxidisable compounds (aldehydes, ketones, oxalates

and formates.)

iii. In alkaline medium:

Perform the test as in (i) but using solution made alkaline with a few drops of

dilute sodium carbonate solution -violet colour discharged in case of aldehydes,

Phenolic acids, formates and cinnamates.

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In case colour is discharged on warming –a hydroxy acids, viz, citric, lactic,

tartaric, etc. may be present.

2. 0.4 g of the substance +1 ml of water 3-4 drops of Bromine water. Shake well.

Yellow colour decolorized. Unsaturated compound

Yellow color persist Saturated compound

Yellow color decolorized & ppt

is Formed Saturated compound - amines, phenol.

8. TESTS FOR SPECIAL ELEMENTS

1. Lassaigne's test for nitrogen, sulphur and halogens:

Preparation of Lassaigne's extract of sodium fusion extract: Place a few

crystals or drops of an unknown compound in a fusion tube. Mold the test tube in an

inclined position and place a freshly cut out piece (the size of a wheat grain) of

sodium metal slightly above the substance. Mold the test tube horizontally using tongs

or wooden clamp. First heat sodium until it melts. Now set the test tube in the vertical

position so that the hot drops fall onto the test-substance. Meat the mixture to redness.

Plunge the red hot tube once in a china dish having nearly 10-15 ml. of distilled water

when the fusion tube will be torn out, if not, do so yourself with the help of tongs.

Boil the resulting product. Filter the hot solution and wash the residue with small

amount of distilled water. The filtrate so obtained is known as Lassaige's filtrate or

extract and used for testing nitrogen, sulphur and halogens. Lassaigne's extract should

be colourless. Yellow or brown colour indicates incomplete decomposition of the

original substance. If so, repeat the experiment using a new portion of the same

substance.

Na + C+N NaCN

2 Na + S Na2S

Na + C + S+N NaSCN

Na + X NaX

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i. Test for nitrogen: Take a small quantity of the extract in a test tube and make

it alkaline (if it is not so) by adding a few drops of sodium hydroxide. Add 0.1

to 0.2 gms, of solid ferrous sulphate (a black precipitate will appear if sulphuc

is present), boil the resulting solution and then acidity it with cone. H2SO4 or-

HCL. The formation of Prussian blue precipitate or green colour indicates the

presence of nitrogen.

6NaCN + FeSO4 Na4Fe (CN)6 +Na2SO4

Sodium ferrocyanide

o2

FeSO4 Fe2 (SO4)3

3Na4Fe (CN)6 + 3Fe2 (SO4)3 Fe4 (Fe(CN)6)3 + 6Na2SO4

ferric ferrocyanide

Precautions: (i) Since the yellow colour of ferric chloride fades the Prussian blue

colour, it is better to avoid the addition of ferric chloride during the above test.

Similarly, on the same ground acidification with conc. H2SO4 is preferred to

concentrated HCL.

(iii) Some compounds like urea and its derivatives although possess nitrogen, they do

not respond the above test; the reason being the deficiency of carbon in these

compounds and hence in the above test whenever there is some doubt, the whole of

the process must be repeated using mixture of the compound with some amount of

cane sugar to provide sufficient amount of carbon.

Function of conc. H2SO4 or HCL The function of the acid is to dissolve the

precipitate of ferrous or ferric hydroxide (formed by reaction with FeSO4 and NaOH)

as the ferrous or ferric sulphate or chloride.

FeSO4 + 2NaOH Na2SO4 + Fe(OH)2

Fe2(SO4)3 + 6NaOH 3Na2SO4 + 2Fe(OH)3

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Fe(OH)2 FeSO4 or FeCI2 +2H2O

2Fe(OH)3 Fe2(SO4)3orFeCl3+6H20

Test for sulphur: Following tests may be adopted for sulphur.

i. Acidify the Lassaigne's extract with dilute acetic acid and then add a few

drops of lead acetate, a black precipitate will be formed.

(CH3COO) 2 Pb + Na2S 2CH3COONa + PbS

Black

ii. Add few drops of freshly prepared dilute solution of sodium nitroprusside

(since in aqueous solution, it is unstable) to the extract, the appearance of a

purple colour confirms the presence of sulpur.

Na2 [Fe(CN)5 NO] + Na2S Na4 [Fe(CN)5NO5]

Sod.nitroprusside Sod. Sodiom Sulphorino pruside

(Violet)

iii. Test for nitrogen and sulphur when present together:

During performing the test for nitrogen if a blood red colour is developed after

adding ferrous sulphate crystals, nitrogen and sulphur seem-to be present together.

6 NaSCN + Fe (SO4)3 2Fe (SCN)3 + 3Na2SO4

ferric sulphocyanide (red colour)

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Conc.H2SO4

Or Conc. HCl

Conc. H2SO4

Or Conc HCl

9. DETECTION OF FUNCTIONAL GROUPS

The tests of various important functional group are summarized below.

1. CARBOXYLIC ACIDS

Most aliphatic carboxylic acids are soluble in cold water, while aromatic acids

are sparingly soluble in cold water but readily soluble in hot or boiling water. The

presence of carboxylic group can easily be the following tests.

(i) Litmus tests:

Like phenols, the carboxylic acids turn blue litmus solution to red. For this

test, one drop of blue litmus solution is added to the aqueous solution of the acid, the

appearance of red colour indicates the presence of a phenol or an acids.

Precaution: As sometimes (viz. in case of very weak acids) it becomes

difficult to note the change in colour thus the test must be performed on a white tile.

(ii) Sodium bicarbonate:

Add some drops of concentrated sodium bicarbonate solution to the acid in

test tube or better on a watch glass or a white tile. Evolution of effervescences of

carbon dioxide indicates the presence of a carboxylic group.

RCOOH + NaHCO3 RCOONa + H2O + CO2

Precaution: Some phenols like 2,4,6 -trinitrophenol and 2,4,6-tribromophenol

also behave like acids and hence evolve carbon dioxide on treatment with sodium

bicarbonate.

(iii) lodate-iodide test:

Grind equal amounts of the carboxylic acid, potassium iodide and potassium

iodatc, the appearance of brown colour owing to the formation of free iodine indicates

the presence of a carboxylic group.

6RCOOH+5KI+KIO3 6RCOOK+3H2O+3I2

In case the compound is liquid take a little amount of it, 5-6 drops of 2%

potassium iodide solution and 5-6 drops of 4% potassium iodate solution. Cork the

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test-tube and place in boiling water for 1-2 minutes. Cool and add about 5-10 drops of

starch solution. Blue colour indicates COOH group.

(iv) Ester test:

Warm nearly 1 ml. of the acid, 2ml. of methyl or ethyl alcohol and a small

amount of concentrated sulphuric acid for 1-2 minutes on the water bath, in a test

tube. Cool and pour the solution to a china dish-having some sodium carbonate

solution, fruity smell of ester indicates the presence of acid

RCOOH + C2H5OH R.C-OC2H5+H2O

(v) Ferric chloride test:

Neutralize 0.5 gm of the acid with excess of ammonia in a boiling test tube.

Boil the solution to remove excess of ammonia, cool and a few drops of neutral ferric

chloride solution. Note the colour of the resulting solution and take out inference

according to the following facts.

A reddish brown colour or ppt. is obtained in case of aliphatic mono-

carboxylic acids.

A buff or brownish coloured precipitate is obtained in case of aromatic

acids and some aliphatic acids like succinic and adipic acids.

An intense yellow colour is obtained in case of alpha hydroxy acids this

test may also be applied as follows. First add a few drops of ferric chloride

to a dilute aqueous solution of phenol and then add this resulting violet

coloured solution to a solution of a alpha hydroxy acid in water the violet

colour is discharged and a deep yellow tinge appears.

A violet or bluish colour is obtained in case of o-substituted aromatic

hydroxy acids.

vi. Detection of unsaturation in acid:

i. Dissolve a small amount of the acid in chloroform or carbon tetrachloride and add

to 2-3 drops of bromine solution in the same solvent (i.e. either CHCL3 or CCL4).

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O

Disappearance of bromine colour without evolution by hydrobormic acid suggests

the presence of unsaturation in the compound. On the other hand, if the bromine

colour is discharged with the evolution of hydrobromic acid, a phenol, aromatic

amino or enol is indicated. The evolution of hydrobromic acid may be easily

detected by exposing the vapours to a solution of silver nitrate in nitric acid held

on the end of a glass rod.

R.CH = CH.R + Br R.CHBr.CHBr.R

(ii) Baeyer's test:

Add one or two drops of 1-2 % KMnO4 solution to small amount of the acid

dissolved in water or acetone disappearance of purple colour indicates the presence of

unsaturation.

Precaution: Some easily oxidisable compounds like aldehydes, alcohols,

formic acid some aliphatic hydroxy acids phenolic acids also respond this test.

(vii) Test for Dicarboxylic acids: Mix 0.1 g of the substance with 0.5g of resorcinol

and 4drops of con. H2SO4 in a hard glass test tube. Heat for 3-5 minutes pour it into

dil. NaOH. An intense greenish yellow fluorescence indicates a Dicarboxylic acid.

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OH OH

BrBr

+ HBr

KmnO4 R.CH-CH.R

R.CH = CH.R OH OH(O) + H2O

KmnO4R.CHO RCOOH(O)

KMnO4

2. ACID ANHYDRIDES

i. Dissolve the compound in benzene or chloroform and add some aniline to

the resulting solution. Warm the solution for nearly 1-2 minutes, and cool.

A white precipitate of anilide is formed.

iii Anhydrides produce fruity smelling esters when warmed with n-butyl

alcohol.

3. PHENOLS

i. Litmus test: Like acids. phenols also turn blue litmus solution colour to

red

ii. Sodium bicarbonate: Except highly negatively substituted phenols

(eg.picric acid), Phenols in general do not decompose sodium bicarbonate

solution (difference from acids)

iii. Ferric chloride test: Add one or two drops of neutral ferric chloride to a

dilute aqueous or alcoholic solution of the compound in a test tube, the

appearance of a characteristic colour (violet, blue or green) indicates the

presence of phenolic group.

6C6H5OH +FeCl3 [(C6H50)6Fe]-3 +3HCL +3H+

iv. Take a small amount of the phenol in a test-tube, add to it alkali and a

small quantity of a cold solution of diazotised1 p-nitroantiine solution (p-

nitro aniline with dil HCl followed by cold-NaNO2 solution) red color.

v. Phthalein test.

Take a little (nearly 0.2gm.) of the phenol and an equal amount of phthalic

acnhydride in a dry test tube. Add nearly 2-3 drops of concentrated sulphuric acid and

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Ac2O+C6H5NH2 AcNHC6H5

CONHC6H5

COOHAnilic acid

+C6H5NH2

COOH

COOH

fuse the mixture gently for about 15 minutes. Cool and pour the contents to dilute

sodium hydroxide solution. Characteristic colours of dyes are obtained as below.

Compound Colour of the dye formed Compound Colour of the dye Formed

Phenol Red Resorcinol Green

o-Croseol Red Hydroquinone Deep violet

m-Cresol Bluish-purple α-Naphthol Green

Catechol Blue β-Napthol Very faint green

vi. Nitrous acid test: Take a little of the compound in a test iube, add nearly

1ml of Conc. H2SO4 and then add a little sodium nitrite. Shake the contents

and warm gently, appearence of green/purple or blue colour indicates the

presence of phenolic OH group. Transfer the contents in little water when

the blue colour changes to red or blue-red. The colour will again change to

blue or green on addition of a little 20% Sodium hydroxide.

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vii. Azo dye test: Dissolve 2-3 drops of aniline in one ml. of concentrated

hydrochloric acid and cool it in ice. To this solution add a few drops of

cold sodium nitrite solution and then add this solution to a cold solution of

phenol in an excess of NaOH solution. Coloured solution or precipitate

indicates the presence of a phenolic group.

Tests for polyhydric Phenols: (i) Take about 2 ml. of dilute ammonium hydroxide or

NaOH solution and add to it a crystal as large as rice -grain of the original compound.

Shake the content of the test tube -formation of yellow colour changing to red-brown

indicates polyhydric phenols.

(ii) Take about 3-4 ml. of Tollen's reagent in a test -tube and add to it 1ml of the

aqueous solution of the original compound -a grey or brown precipitate is formed

immediately in case of catechol, quinol, pyrogallol, gallic acid and tannic acid; within

30 seconds in case of orcinal and phloroglucinol; and within 2 minutes in resorcinol.

4. ALCOHOLS

Alcoholic group can be detected by the following tests.

i. Action of sodium: Take nearly 1ml. of the alcohol (in case of solid, dissolve it in

the dry benzene or ether) in a test-tube and add to it a small piece of freshly cut

sodium-evolution of brick effervescences of hydrogen suggests the presence of

alcoholic group.

2ROH+2Na 2RONa+H2

(i) Reaction with acetyl chloride.

Take about 1 ml. of acetyl chloride in a dry test tube and add to it about 0.5

ml. of the original compound -vigorous reaction occurs (i.e bubbles are freely

evolved) with in 2 minutes with the evolution of HCL fumes. Bring a glass rod dipped

in NH4OH -white fumes of ammonium chloride are produced. When the reaction

(bubbles) stops, add one ml. of water to dissolve the remaining HCL gas; separation

of an oily layer along with the ester like odour Confirms the presence of alcohol.

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ROH + CH3COCl ROCOCH3 + HCI

HCI + NH4OH NH4C +H2O

Distinction between primary, secondary and tertiary alcohols:

i. Take 1ml of pure acetone, one drop or 10 mg. of the alcohol and then one drop of

the chromic acid regent. stir the mixture immediately-greenish blue coloured ppt.

is formed within two seconds in case of primary and secondary alcohols. If no

precipitate or colour change occurs at once tertiary alcohol is indicated.

ii. Take 1 ml. of the compound in a test tube and add 5 ml. of lucas reagent with

shaking. Note the following observations formation of insoluble chloride layer at

once tertiary alcohol. Appearance of cloudiness within 5 minutes secondary

alcohol. Solution remains clear with some darkening primary alcohol.

iii. Dissolve 0.2 gm. of the compound in about 10 ml. of 0.01% solution of bromine

in carbon tetrachloride. Add about 0.2 gm. of N-bromosuccinimide and place the

test tube in a water bath at 80°C.Note the colour obtained.

Permanent orange colour Primary alcohol.

Transient orange colour Secondary alcohol.

No colour Tertiary alcohol.

5. CARBONYL COMPOUNDS (Aldehydes and ketones)

i. Aldehydes and ketones, in general, are detected by reaction with 2, 4-

dinitrophenylhyrazine it form crystalline 2.4-dinitro-phenylhyrazine.

Add 2 drops or 1.1 gm. of the substance in dilute HCL to 3ml. of the 2,4-

dinitrophenylhyrazine solution (reagent) in dilute HCL and shake the mixture-yellow,

orange or red coloured crystalline precipitate may be formed at once, or on allowing

the mixture to stand for 15 minutes, or on gentle warming on a water-bath.

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ii. Sodium bisulphite test: Most of the aldehydes and ketones react with a

concentrated aqueous solution of sodium bisulphite to form a crystalline solid.

C=O + NaHSO3

iii. Solutions of many aldehydes and ketones develop red colour when treated with

sodium nitroprusside and alkali. To a solution, of the substance add 1ml of 3%

sodium nitroprusside solution and 5 drops of 10% NaOH (Red or pink color).

iv. It is important to note that the above tests are not positive in all aldehydes and

ketones. In case of ketones only those ketones which have at least one methyl or

ethyl group attached to the carbonyl group respond these tests.

v. Add Nessier's solution to extremely dilute aqueous solution of aldehydes or

ketones voluminous precipitates. This test is given only by simpler aldehydes and

ketones.

vi. Fehling's test: Mix 2ml. of fehling solution A (copper sulphate solution) and

Fehimg solution B (alkaline tartarate solution). To the mixed solution add 3 drops

(or 0.15gm.) of the suspected compound and warm the resulting solution. The

appearance of a red precipitate of cuprous oxide indicates the presence of an

aldehydic group.

R-CHO + 2Cu(OH)2 R-COOH+Cu2O +2H2O

Note that some ketones like benzoin also reduce Fehitng solution.

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OHC

SO3Na

vii. Schiff's test: Add two drops of the compound under test to 2ml. of the reagent

(pararosaniline hydrochloride solution, the colour of which is just decolourised by

passing SO2). Shake the contents, appearance of a violet or red colour within two

minutes indicates the presence of an aldehyde.

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AR

NH

SO2H

SO2 NH NH2 +2ArCHO

ShakeC

SO3H

H2N

NHC

Ar

HSO3

H

C N C

Ar

SO3H

H

viii. Tollen's Test: Take nearly 3-4 ml. of the freshly prepared Tollen's reagent in a

clean and dry test - tube and add to this 2-3 drops (or 0.1 gm. in case of solid) of

the substance. Shake the solution and if no reaction occurs in the cold, warm (not

Boil) the tube gently in a beaker of water at 50: The formation of a black

precipitate of metallic silver or silver mirror indicates the presence of a reducing

substance like aldehydes.

Precautions: (i) The reagent must freshly prepared but it must be washed

away after use. As it forms explosive substance if stored.

CHO + Ag2O COOH + 2Ag

(iii) Some ketones like pyruvic acid reduce the Tollen's reagent.

ix. Benedict's test: Add 5ml of Benedict's solution to 0.4ml. of 2% solution of the

compound. Boil the solution for two minutes and then cool rapidly. Formation of

a red precipitate suggests the presence of a reducing sugar or an aliphatic

aldehyde.

RCHO + 2Cu++ (as citrate complex) + 4OH-

6. CARBOHYDRATES

The carbohydrates may be detected by the following tests.

i. Molisch's test: Dissolve nearly 0.005gm. of the carbohydrate in one ml. of

water, add two drops of the molisch’s reagent (10% α-napthol in alcohol)

and shake. Now add carefully 2ml. of concentrated sulphuric acid along

the side of the test-tube and allow it to stand for 2 minutes. The formation

of a reddish violet colour at the junction of two liquids indicates the

presence of carbohydrate. /'Shake' the contents of the test-tube in a stream

of cold water, a deep purple solution is obtained, which on dilution with

cold water yields a violet precipitate.

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Tollen's SilverReagent mirror

RCOOH + Cu2O + 2H2O(red)

Polysaccharides Monosaccharides Sulphuric +hydroxyfurfural

Triarylmethane compounds (Violet)

(iv). Barfoed's test: The test is used in differentiating the monosaccharides from

disaccharides. Take one ml. of the freshly prepared Barfoed's reagent (0.33 gm. of

neutral copper acetate dissolved in 5 ml. of cold 1% acetic acid) and add one ml. of

the carbohydrate solution to it. Heat the solution in a boiling water-bath, formation of

a red precipitate in the blue solution, or on the sides of the tube just above the liquid,

within two minutes indicates the presence of a monosaccharide (glucose, fructose or

galactose).

7. ESTERS

i. Esters are usually identified by their hydrolysis reaction. Take 2-3ml of ester

solution in a test tube, add some sodium hydroxide solution and a drop of

phenolphthalein, the pink colour will be developed. Heat the reaction mixture

for some time, the disappearance of pink colour indicates the presence of an

ester group.

RCOQR' +alkali + Phenolphthalein RCOOH + R'OH

8. ALIPHATIC PRIMARY AMINES

Aliphatic primary amines in general can be detected by the following tests.

i. Carbylamine reaction: Warm a small amount (0.05 - 0.1 gm. or 2 drops) of

the amine with 2 drops of chloroform and one ml. of alcoholic potash in a

test tube, a nauseating (foul) odour of isocyanide (carbylamine) indicates

the presence of a primary aliphatic or aromatic amine.

RNH2 +CHCl3+ 3KOH RNC+3KCl+ 3H2O

Note: It is always advisable to decompose the isocyanide formed by adding

concentrated hydrochloric acid before throwing it in the sink.

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heat

Pink color Pink color discharged

ii. Reaction with nitrous acid: Dissolve nearly 0.2 gm. of the amine in 5ml.

of cone hydrochloric acid, cool it in ice and then add nearly 2ml. of ice-

cold sodium nitrite solution, brisk effervescences of nitrogen will be

evolved in case the compound is primary amine. Sometimes, warming on

the water-bath is required in order to evolve the nitrogen.

RNH2+ HONO ROH+N2+ H2O

iii. Rimini's test: Take fiearly 3-drops of the amino solution in 5ml.of water,

add to it 1ml. of acetone and a drop of sodium nitropursside solution,

appearance of a violet colour within two minutes indicates the presence of

primary aliphatic amino group.

9. AROMATIC PRIMARY AMINES

i. Carbylaminc reaction: Same as in aliphatic primary amines.

ii. Reaction with nitrous acid: Dissolve nearly 0.1 gm. of the given amine in

3ml. of concentrated hydrochloric acid in a small beaker or a large hard

glass test tube. Cool it in ice to nearly 0°C and to this add 5ml. of ice cold

sodium nitrite aqueous solution with constant shaking till a slight excess of

nitrous acid predominates in the solution. When the reaction ceases, add

ice cold solution of β-naphthol in alkali; the formation of a bright red,

orange red or reddish blue precipitate or colour indicates the presence of

primary aromatic amino group. The solubility of the dye in NaOH

indicates the presence of a hydroxyl group in the amine.

10. ALIPHATIC AND AROMATIC SECONDARY AMINES

i. Reaction with nitrous acid: Dissolve nearly 1 gm. Of the compound in 3-

5 ml. of dilute hydrochloric acid and cool it is ice cold water. To the above

solution add 4-5ml. 10% sodium nitrite solution drop wise and with

constant shaking. Allow the solution to stand or 5 minutes when a yellow

or white oil precipitate is produced.

ii. Simon's test: Take a few drops of the compound and add to it dilute

solution of sodium nitroprusside followed by a few drops of dilute

acetaldehyde solution-deep blue or violet colours will be produced within

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five minutes.

11. AMINO-ACIDS

Aliphatic: The presence of one of these acids may detected by the followings

tests,

i. Add few drops of 0.2% solution of ninhydrin reagent to the aqueous solution of

the substance a blue colour is produced.

ii. Add a few drops of ferric chloride solution to aqueous or alcoholic solution of

amino acids (red colour is developed),

iii. Sorensen test: Take near about 0.5gm. of the compound in a test tube and

dissolve it in water or alcohol. Add two drops of phenolphthalein and then very

dilute sodium hydroxide solution till a pink colour is obtained, In other test tube

take the similar things in the similar order only with the difference that here the

amino acid is replaced by 40% formalin solution. Add the solution from the

second test tube to the first; the disappearance of the pink colour is indication of

the amino acid.

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O

C

C

O

C

OH

OH

H2N.CHR.COOH

O

C

C

O

C = N

O

C

C

O

CO2 + RCHO + H2O

Amino acid

Blue Color

Aromatic:

a. Aromatic amino acids are more acidic than the aliphatic amino acids cause

a vigorous evolution of carbon dioxide from sodium bicarbonate.

b. They usually give reddish colour with ferric chloride solution.

c. Like aromatic primary amines, these undergo diazotisation with nitrous

acid which couple with alkaline 2-naphthol solution to give coloured azo

dyes.

12. ALIPHA TIC NITRO COMPOUNDS

i. Dissolve a small amount of the compound in concentrated sodium hydroxide

solution and then add an excess of sodium nitrite solution. Now add some amount

of water followed by dropwise addition of dilute sulphuric acid. The appearance

of a red colour which disappears on adding excess of acid and reappears on adding

alkali indicates the presence of a primary aliphatic nitro group. Secondary nitro

group on similar treatment produces a permanent dark blue or blue green colour

soluble in chloroform. Tertiary nitro compounds do not respond this test.

ii. Reduction in neutral medium: Dissolve nearly 0.5gm. of the substance in an

equirnolar mixture of alcohol and water by warming. Add to it some amount of

zinc dust and solid NH4C1. Meat the mixture to boil and allow the reaction to

comlete. Filter the solution and add Tollen's reagent to the filtrate, a black

precipitate or mirror will be developed.

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Zn-NH4Cl

RNO2 RNHOH + H2O

RNHOH + Ag2O RNO + 2Ag + H2O

13. AROMATIC NITRO COMPOUNDS

(i) Aromatic nitro compounds give the

(ii) test above

14. AMIDES AND EVIIDES

i. Reaction with nitrous acid: Like primary amines, amides and imides evolve

nitrogen on treatment with nitrous acid.

RCONH2 + HONO RCOOH + N2+ H2O

ii. Reaction with alkali: Dissolve 0.2 gm of the amide in excess of concentrated

sodium hydroxide solution. Heat the solution to boil, smell of ammonia gas

indicates the presence of amide group.

RCONH2+NaOH RCOONa + NH3

The evolution of ammonia gas can also be detected by the following tests.

a. Bring a red litmus paper over the mouth of the test tube, it changes to blue.

b. Bring a glass red dipped with concentrated hydrochloric acid to the mouth

of the test tube-dense white fumes of ammonium chloride.

iii. Only for aromatic amides: To a little of the compound, add some hydroxylamine

hydrochloride solution in ethyl alcohol. Boil the contents for about five minutes,

cool and then add about 4-5 drops of dilute (5%) ferric chloride solution-bluish

red colour

C6H5CONH2 + NH2OHHC1 C6H5CONHOH + NH4CI

iv. Only for aromatic amides: To the aqueous solution of the aromatic amide add

about 7-8 drops of 6% hydrogen peroxide and heat the contents to boiling. Cool,

add 1-2 drops of dilute (5%) ferric chloride solution bluish red colour in cold

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within a short time or a brown colour on warming.

C6H5CONH2+H2O2 C6H6CONHOH + H2O

Tests for Urea: Urea, being the typical member of simple amides, gives certain

characteristic special tests.

i. Biuret test: Fuse a small amount of the urea in a test tube until it just melts and

ammonia is evolved. After some time when it resolidifies dissolve it in 1 ml. of

dilute sodium hydroxide solution and then add one to two drops of very dilute

copper sulphate solution, a pink colour will be developed changing to violet and

lastly deep blue. This test is also given by ox amide and malonamide.

2NA2-C-NH2 NH2CONHCONH2 + NH3

ii. Xanthydrol test: Dissolve a small amount of the compound in 50% acetic acid

and add to it 1 ml. of 5% xanthydrol solution in methanol, a precipitate will

immediately be formed. The test is given by urea as well as its derivatives.

iii. Take small amount of urea in aqueous solution and add to it sodium hypobromite

solution, brisk effervescences of nitrogen and carbon dioxide will be evolved.

iv. Urea nitrate test: 0.1g of the substance +1ml of water +1ml of con. HNO3. Shake

well. White ppt formed shows a diamide like urea. Urea oxalate test: 0.1g of the

substance shaken with 3ml of saturated solution of oxalic acid. White ppt

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Benzamide Hydroxamic acid

O

ORNHCONH2 + HO.CH RNHCONH .CH O + H2O

indicates the presence of urea.

15. SULPHONIC ACIDS

Aromatic sulphonic acids:

i. These may be detected by fusion with alkali as follows. Mix about 0.5gm.of the

substance, 3gm. of potassium hydroxide and 5 drops of water in a nickel crucible.

Heat the crucible carefully, with occasional stirring, so that the mixture melts and

remains in the molten state for about 10 minutes. Cool the melt, add about 5 ml. of

water and stir the contents to dissolve the mass (warm, if necessary). Acidify the

solution - SO2 gas is evolved which may be detected by means of a filter paper

strip impregnated with acidified potassium dichromate solution.

ii. Treat the compound with phosphorus pentachloride and then with phenol in

alkaline medium. A crystalline derivative is obtained.

Ar-SO3H ArSO2Cl ArSO2-O-C6H5

The above formed sulphonyl chloride also reacts with ammonia to form

crystalline sulphonamide.

ArSo2Cl + NH2 ArSO2NH2 +HCI

16. THIOUREAS

i. Meat a small amount of the substance with 2 ml. of aq. NaOH to boiling for about

30 seconds. Cool and add one drop of CO2 Lead acetate-dark brown or black

colour.

RNH.CS.NR.R + 4KOH 2RNH2 + K2CO3 + K2S + H2O

K2S + (CH3COO)2 pb pbs black precipitate + 2CH2COOK

ii. Take a small amount of the compound in a test tube, add to it an alcoholic solution

of yellow mercuric oxide, and warm - a black precipitate of mercuric sulphide will

or formed.

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PCl C6H5-OH

-HCl

Sulphonamide

RNH.CS.NH + HgO RNH.CO.NHR + HgS

iii. Fuse (melt) a small amount of the compound in a test tube, cool and dissolve the

product in water. Add aq. ferric chloride solution - a blood red colour will be

developed.

17. HYDROCARBONS

Although hydrocarbons have no general test, they can be detected by the

following tests.

i. Iodine test: Dissolve 0.2 gm. Or 2-3 drops of the substance in 5 ml. of benzene.

To this add 5 ml. of very dilute solution of iodine in benzene and shake; solution

remains violet.

ii. Friedel - Craft condensation test: (Only for aromatic hydrocarbons). Take

0.5gm. of anhydrous AICI3 in a dry test tube. Heat the test-tube when. AICI3

sublimes to deposit on the upper end of the tube. Now add 2-3 drops of a mixture

of equal amounts of the substance and chloroform. Allow this solution to come in

contact with aluminum chloride - appearance of red, orange, blue or green colour

due to the formation of triphenylmethane dyes indicates aromatic hydrocarbons.

Formalin Test

Sample + CCl4+HCHO+H2SO4

Observation red color with black ppt.

Esters:

1. Substance +1 drop of phenolpthalein + NaOH dorpwise while warming the pink

color disharges

2. Hydroxylamine test

Substaance + methanolic soln of hydroxylamine + HCl3 DROPS) + FEW

DROPS of Meth. Soln of KOH, boil, cool, add a drop HCl3 & acidity with CONC.

HCl

Purple / wine color ester confirmed

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