JEE Organic Chemistry: Qualitative &

Quantitative Analysis Complete Guide

TABLE OF CONTENTS
1. Purification Methods of Organic Compounds
1.1 Sublimation
1.2 Crystallisation
1.3 Distillation
1.4 Differential Extraction
1.5 Chromatography
2. Qualitative Analysis of Organic Compounds
2.1 Detection of Carbon and Hydrogen
2.2 Detection of Nitrogen (Lassaigne's Test)
2.3 Detection of Sulphur
2.4 Detection of Halogens
2.5 Detection of Phosphorus
3. Quantitative Analysis of Elements
3.1 Carbon and Hydrogen Estimation
3.2 Nitrogen Estimation (Dumas & Kjeldahl Methods)
3.3 Halogen Estimation (Carius Method)
3.4 Sulphur Estimation
3.5 Phosphorus Estimation
3.6 Oxygen Estimation
4. Qualitative Tests for Functional Groups
4.1 Alcohols
4.2 Phenols
4.3 Aldehydes vs Ketones (Including Sodium Bisulfite Test)
4.4 Carboxylic Acids
4.5 Esters, Acid Halides, Anhydrides
4.6 Amines (Primary, Secondary, Tertiary)
4.7 Nitriles
4.8 Halides
4.9 Sulfur-Containing Compounds
5. Qualitative Tests for Biomolecules
5.1 Carbohydrates Tests
5.2 Protein Tests
5.3 Lipid Tests
6. Quick Reference Tables
1. PURIFICATION METHODS OF ORGANIC
COMPOUNDS
1.1 Sublimation
Aspect Details
Some solid substances change from solid to vapour
Principle
without passing through liquid state
Separation of sublimable compounds from non-
Use
sublimable impurities
Suitable
Compound Naphthalene, benzoic acid, iodine, camphor, dry ice
s
Heat the impure compound under reduced pressure;
Procedure
pure solid sublimes and is collected on a cold surface
Advantage Quick purification with minimal decomposition
Only applicable to compounds with high vapour
Limitation
pressure at solid state

1.2 Crystallisation
 Aspect Details
Based on difference in solubilities of compound
Principle
and impurities in suitable solvent
Purifying solid organic compounds; most common
Best For
method
Choose solvent: sparingly soluble at room
Solvent
temperature, appreciably soluble at high
Selection
temperature
1. Dissolve impure compound in hot solvent
2. Concentrate solution
General
3. Cool gradually
Procedure
4. Pure crystals separate
5. Filter and dry
Mixed Solvent Use mixture of two solvents when compound is
Crystallisation highly soluble in one and poorly soluble in another
Removing Add activated charcoal to hot solution for
Coloured adsorption of colouring agents; filter through hot
Impurities filter paper
Repeated Necessary for compounds with comparable
Crystallisation solubility impurities
Purity Check Sharp melting point indicates pure compound

1.3 Distillation
1.3.1 Simple Distillation
 Aspect Details
Principl Liquids with different boiling points vaporise at different
e temperatures
Separate volatile liquids from non-volatile impurities;
Use
compounds with sufficient BP difference
Apparat Round bottom flask, thermometer, condenser, receiver,
us heating apparatus
1. Place liquid mixture in flask
Procedu 2. Heat gently
re 3. Collect vapours in condenser
4. Collect distillate separately
Exampl
Separation of chloroform (BP 334 K) and aniline (BP 457 K)
e
Observa
Lower boiling component distils first
tion

1.3.2 Fractional Distillation

Aspect Details
Vapours pass through fractionating column before
Principle
condensation
Separate liquids with small differences in boiling
Use
points
Additional fractionating column with packing (glass
Apparatus
beads, Raschig rings, bubble plates)
Principle of Provides multiple surfaces for heat exchange; vapours
Column become richer in more volatile component as they rise
Crude oil fractionation into different petroleum
Application
fractions
Fractionatin Measured in theoretical plates; commercial columns
g Efficiency have hundreds of plates
Separation Better separation than simple distillation; more
Quality volatile component distils first
1.3.3 Distillation Under Reduced Pressure

Aspect Details
Princi At lower pressure, liquids boil at lower temperatures:
ple Liquid boils when vapour pressure = external pressure
Purify liquids with very high boiling points; compounds that
Use
decompose at normal BP
Formu
p (liquid) + p (if water) = P (external)
la
Appar Special distillation setup with water pump or vacuum pump;
atus manometer for pressure measurement
Exam
Glycerol separation from spent-lye in soap industry
ple
Advan Prevents thermal decomposition of heat-sensitive
tage compounds

1.3.4 Steam Distillation

Aspect Details
Principl Steam volatile compounds immiscible with water are
e separated using steam
Separate volatile compounds from non-volatile impurities
Use
when both immiscible with water
Boiling Mixture boils at below normal BP because: p(organic) +
Point p(water) = P(atmospheric)
1. Pass steam through heated flask
Procedu 2. Collect steam + organic vapour mixture
re 3. Condense the vapours
4. Separate organic layer using separatory funnel
Example Aniline separation from aniline-water mixture
Advanta Lower temperature prevents decomposition; useful for
ge thermolabile compounds
1.4 Differential Extraction
Aspect Details
Organic compound transferred from aqueous to
Principle
organic solvent based on different solubilities
When organic compound in aqueous medium needs
Use
separation
Requirement Two immiscible solvents; organic solvent chosen for
s higher solubility of compound
Apparatus Separatory funnel with stopcock
1. Add organic solvent to aqueous solution
2. Shake well (solvents remain immiscible)
Procedure 3. Allow layers to separate
4. Drain lower layer through stopcock
5. Collect organic layer separately
Continuous Used when compound sparingly soluble in organic
Extraction solvent; same solvent used repeatedly
Distil or evaporate organic solvent to recover
Final Step
compound

1.5 Chromatography
1.5.1 Principles and Classification
 Aspect Details
Mixture applied to stationary phase; mobile phase
Principle
moves over it causing separation
Stationary
Solid or liquid that doesn't move
Phase
Mobile Pure solvent, solvent mixture, or gas that moves
Phase slowly over stationary phase
Basis of Components move different distances based on
Separation differential adsorption/partition
Chromatogr Paper/plate after development showing separated
am components
Classificatio 1. Adsorption chromatography
n 2. Partition chromatography

1.5.2 Adsorption Chromatography

Column Chromatography
 Aspect Details
Differential adsorption of mixture components on
Principle
adsorbent
Stationary Silica gel or alumina packed in glass tube with
Phase stopcock
1. Pack adsorbent in column
2. Apply mixture at top
3. Allow eluant (appropriate solvent) to flow down
Procedure
slowly
4. Components separate based on adsorption
strength
Most adsorbed substances stay near top; others move
Observation
down various distances
Collection Different fractions collected at bottom
Adsorbent Silica gel or alumina; choose based on compound
Selection polarity
Eluant
Varies based on separation requirements
Selection

Thin Layer Chromatography (TLC)

 Aspect Details
Thin layer (0.2 mm) of silica gel or alumina on
Setup
glass plate
Mixture spotted 2 cm above lower edge of
Application
plate
Components adsorb differently; move different
Principle
distances as eluant rises
Eluant rises via capillary action through
Mobile Phase
stationary phase
Detection -
Coloured Visible due to original colour
Compounds
(a) UV light (for fluorescent compounds)
Detection -
(b) Iodine vapour (brown spots)
Colourless
(c) Spray reagents (e.g., ninhydrin for amino
Compounds
acids)
Retardation factor = Distance moved by
Rf Value
substance / Distance moved by solvent front
Single spot indicates purity; multiple spots
Purity Check
indicate mixture
Advantage Quick, requires small samples

1.5.3 Partition Chromatography

Paper Chromatography
 Aspect Details
Stationary
Water trapped in chromatography paper
Phase
Mobile
Appropriate solvent or solvent mixture
Phase
Strip of chromatography paper spotted at base with
Setup
mixture; suspended in solvent jar
Mechanis Solvent rises by capillary action; components separate
m by differential partition
Chromatog Developed paper strip showing separated spots at
ram different heights
Detection Same as TLC (UV light, iodine, spray reagents)
Rf Value Same calculation as TLC; helps identification
Applicatio Qualitative analysis; separation of amino acids, sugars,
n dyes

2. QUALITATIVE ANALYSIS OF ORGANIC

COMPOUNDS
2.1 Detection of Carbon and Hydrogen
 Parameter Details
Principle Oxidation of C and H in organic compound using CuO
2C + 2CuO → 2Cu + CO₂↑
Reactions
2H + CuO → Cu + H₂O
Detection
CO₂ + Ca(OH)₂ → CaCO₃↓ (white turbidity)
of Carbon
Detection
of H₂O + CuSO₄ (anhydrous) → CuSO₄·5H₂O (turns blue)
Hydrogen
Heat organic compound with CuO in combustion tube;
Procedure pass vapours through lime-water for CO₂; collect water
in anhydrous CuSO₄
Positive Turbidity with lime-water; blue colour with anhydrous
Test CuSO₄
Carbon and hydrogen present in all organic compounds
Note
(confirmed for both)

2.2 Detection of Nitrogen (Lassaigne's Test)

 Step Details
1. Sodium Heat organic compound with sodium metal in
Fusion combustion tube
C + Na → NaCN (if N present)
N + Na → NaCN (if N present)
Reactions
S + Na → Na₂S (if S present)
X + Na → NaX (if halogens present)
Cool fused mass; boil with distilled water; filter if
2. Extraction
necessary
Product Sodium Fusion Extract containing NaCN, Na₂S, NaX
3. Test for Add sodium fusion extract to FeSO₄ solution; acidify
Nitrogen with conc. H₂SO₄
6CN⁻ + Fe²⁺ → [Fe(CN)₆]⁴⁻ (sodium hexacyanoferrate
Reaction for N II)
[Fe(CN)₆]⁴⁻ + 4Fe³⁺ → Fe₄[Fe(CN)₆]₃ (Prussian blue)
Positive Test Prussian blue colour (deep blue precipitate)
Some Fe²⁺ oxidised to Fe³⁺ by conc. H₂SO₄; Fe³⁺ reacts
Mechanism
with [Fe(CN)₆]⁴⁻
Iron(III) hexacyanoferrate(II) = ferriferrocyanide
Formation
·xH₂O (Prussian blue)

2.3 Detection of Sulphur

 Test Procedure Observation
Add sodium fusion extract to Black precipitate
Test 1: Lead
lead acetate solution after of PbS confirms
Acetate Test
acidifying with acetic acid sulphur
Reaction S²⁻ + Pb²⁺ → PbS↓ (black) -
Test 2:
Sodium Add sodium fusion extract to Violet colour
Nitroprussid sodium nitroprusside solution indicates sulphur
e Test
S²⁻ + [Fe(CN)₅NO]²⁻ →
Reaction -
[Fe(CN)₅NOS]⁴⁻ (violet)
Special Case: Sodium thiocyanate formed: Blood red colour;
N & S both Na⁺ + C≡N⁻ + S²⁻ → NaSCN + no Prussian blue
present [Fe(CN)₆]⁴⁻ (no free CN⁻)
If excess Na used in fusion: Reformed N and S
Decompositio
2NaSCN + 2Na → NaCN + Na₂S + ions give separate
n
C tests

2.4 Detection of Halogens

 Step Procedure Observation
If N or S also present, boil sodium
Prepar fusion extract with conc. HNO₃ to
-
ation destroy NaCN/Na₂S (interfere with
test)
Acidifi
Acidify extract with dilute HNO₃ -
cation
Addin
Add AgNO₃ solution to acidified
g Precipitate forms
extract
AgNO₃
Chlori White precipitate
ne AgNO₃ + Cl⁻ → AgCl↓ (white) soluble in NH₃
Test (forms [Ag(NH₃)₂]⁺)
Yellowish
Bromi
precipitate
ne AgNO₃ + Br⁻ → AgBr↓ (yellowish)
sparingly soluble
Test
in NH₃
Iodine Yellow precipitate
AgNO₃ + I⁻ → AgI↓ (yellow)
Test insoluble in NH₃
Solubi
Confirms halogen
lity AgCl > AgBr > AgI in ammonia
identity
Order

2.5 Detection of Phosphorus

 Parameter Details
Heat organic compound with Na₂O₂ (strong oxidising
Oxidation
agent)
Product
Phosphorus oxidised to phosphate (PO₄³⁻)
Formed
1. Heat compound with Na₂O₂
2. Cool and dissolve in water
Procedure
3. Boil solution with dilute HNO₃
4. Add ammonium molybdate solution
Positive Yellow colouration or yellow precipitate indicates
Test phosphorus
PO₄³⁻ + Mo-containing reagent → Yellow product
Reaction
(ammonium phosphomolybdate complex)
Alternative: Add magnesia mixture to get MgNH₄PO₄; on
Note
ignition yields Mg₂P₂O₇ (white)

3. QUANTITATIVE ANALYSIS OF ELEMENTS

3.1 Carbon and Hydrogen Estimation (Combustion
Analysis)
Principle and Setup
 Param
Details
eter
Princi Complete oxidation of organic compound; measuring CO₂
ple and H₂O produced
Reacti 2C + 2CuO → 2Cu + CO₂↑
ons 2H + CuO → Cu + H₂O
Combustion tube, CuO pellets, sample in platinum boat, pure
Appar
dry O₂ supply, condenser, anhydrous CaCl₂, KOH solution in
atus
U-tubes
1. Place weighed sample in platinum boat
2. Heat in combustion tube with CuO in O₂ atmosphere
Proced 3. Pass produced gases through anhydrous CaCl₂ (absorbs
ure H₂O)
4. Pass remaining gases through KOH solution (absorbs CO₂)
5. Weigh U-tubes to find mass increase
Tempe Heat until all C and H oxidised; copper gauze may reduce
rature any NO₂ to N₂

Calculations

Parameter Formula
Molar mass of CO₂ 44 g/mol
Molar mass of H₂O 18 g/mol
Carbon in CO₂ 12 g per 44 g CO₂
Hydrogen in H₂O 2 g per 18 g H₂O
% Carbon (12 × m₂ × 100) / (44 × m) = (27.27 × m₂) / m
% Hydrogen (2 × m₃ × 100) / (18 × m) = (11.11 × m₃) / m
m = mass of organic compound (mg)
Variables m₂ = mass of CO₂ produced (mg)
m₃ = mass of H₂O produced (mg)
Example Problem
Given: 0.246 g compound → 0.198 g CO₂ + 0.1014 g H₂O

Element Calculation
% Carbon (0.198 × 12 / 44) × 100 / 0.246 = 22.09%
% Hydrogen (0.1014 × 2 / 18) × 100 / 0.246 = 4.56%

3.2 Nitrogen Estimation

Method 1: Dumas Method

Paramete
Details
r
N-containing compound heated with CuO in CO₂
Principle
atmosphere; N₂ gas produced collected
Reactions Organic compound + CuO → N₂ + CO₂ + H₂O + Cu
General
CₓHᵧNᵤCuO → N₂ + CO₂ + H₂O + Cu
Equation
By- Traces of NO₂ reduced to N₂ by passing over heated
products copper gauze
Gas
Over aqueous KOH solution (absorbs CO₂); N₂ collected in
Collectio
upper part of graduated tube
n
Apparatu Combustion tube, CuO, organic compound, heated copper
s gauze, nitrometer tube, KOH solution, mercury seal
Measure Volume of N₂ collected (V mL) at temperature T and
ment pressure P

Dumas Method Calculations

 Parameter Formula / Details
Molar mass of N₂ 28 g/mol
At STP 28 mL of N₂ ≡ 28 g N₂ at STP = 22,400 mL
(28V / 22,400m) × 100 where V = volume of N₂ at
% Nitrogen
STP (mL), m = mass of compound (g)
Correction for V(at STP) = [V(observed) × (P - P_aqueous) / 760]
Aqueous Tension × (273 / T)
V = volume of N₂ collected (mL)
P = atmospheric pressure (mm Hg)
Variables P_aqueous = aqueous tension at temperature T
T = absolute temperature (K)
m = mass of compound (g)

Example Problem
Given: 0.3 g compound, 50 mL N₂ at 300 K, 715 mm Hg, aqueous tension 15 mm

Calculation
Actual pressure = 715 - 15 = 700 mm
V(at STP) = [50 × 700 / 760] × (273 / 300) = 41.9 mL
% N = (28 × 41.9 × 100) / (22,400 × 0.3) = 17.46%

Method 2: Kjeldahl Method

 Parameter Details
N converted to NH₄⁺ by heating with conc. H₂SO₄;
Principle
ammonia evolved with NaOH collected in excess H₂SO₄
Step 1: Heat N-compound with conc. H₂SO₄
Digestion Organic N → (NH₄)₂SO₄
Reaction 1 R-N + conc. H₂SO₄ → (NH₄)₂SO₄
Step 2:
Add excess NaOH; heat; ammonia evolved and distilled
Distillation
Reaction 2 (NH₄)₂SO₄ + 2NaOH → Na₂SO₄ + 2NH₃↑ + 2H₂O
Step 3: Ammonia absorbed in known volume of standard
Absorption H₂SO₄
Reaction 3 2NH₃ + H₂SO₄ → (NH₄)₂SO₄
Step 4:
Excess H₂SO₄ titrated with standard NaOH; volume of
Back-
H₂SO₄ used = used volume - back-titration volume
titration
Kjeldahl flask, digestion setup, distillation apparatus,
Apparatus
standard acid and alkali
Not applicable for:
- Nitro compounds (-NO₂)
- Azo compounds (-N=N-)
Limitations
- Nitrogen in rings (e.g., pyridine)
These N forms don't convert to (NH₄)₂SO₄ under
conditions

Kjeldahl Method Calculations

 Parameter Formula
Molar mass of N 14 g/mol; 1 M NH₃ solution ≡ 17 g/L NH₃ ≡ 14 g N/L
% Nitrogen (14 × M × (2V₁ - V₂) × 100) / (1000 × m)
Simplified % N = 1.4M(2V₁ - V₂) / m
M₁ = molarity of H₂SO₄ taken
V₁ = volume of H₂SO₄ taken (mL)
M₂ = molarity of NaOH used in back-titration
Variables
V₂ = volume of NaOH used in back-titration (mL)
m = mass of compound (mg)
(2V₁ - V₂) = mL of H₂SO₄ actually used by ammonia

Example Problem
Given: 0.5 g compound, 10 mL of 1 M H₂SO₄ neutralised

Calculation
1 M H₂SO₄ 10 mL ≡ 20 mL 1 M NH₃
1 M NH₃ 20 mL contains: (14 × 1 × 20) / 1000 = 0.28 g N
% N = (0.28 × 100) / 0.5 = 56%

3.3 Halogen Estimation (Carius Method)

 Parameter Details
Halogen in compound converted to halide ion;
Principle
precipitated as silver halide
C-X + AgNO₃ → AgX↓ + ... (where X = halogen)
Reactions
AgCl, AgBr, AgI are insoluble
1. Mix organic compound with AgNO₃ in a sealed tube
2. Heat tube in bomb furnace at 573 K
3. At high temperature and pressure, C-X bond
Procedure
breaks; Ag⁺ precipitates halide
4. Cool; AgX precipitate forms
5. Filter, wash, dry, and weigh AgX
573 K (sealed tube prevents escape of volatile
Temperature
products)
Halide
AgCl (white), AgBr (cream), AgI (yellow)
Compounds
Atomic Cl = 35.5; Br = 80; I = 127; Ag = 108; AgCl = 143.5; AgBr
masses = 188; AgI = 235

Carius Method Calculations

Halogen Formula
For % Cl = (35.5 × m₁ × 100) / (143.5 × m) where m₁ = mass
Chlorine AgCl, m = mass compound
For
% Br = (80 × m₁ × 100) / (188 × m) where m₁ = mass AgBr
Bromine
For
% I = (127 × m₁ × 100) / (235 × m) where m₁ = mass AgI
Iodine
% X = (Atomic mass of X / Molar mass of AgX) × (m₁ / m) ×
General
100
3.4 Sulphur Estimation
Parameter Details
Sulphur oxidised to SO₄²⁻; precipitated as barium
Principle
sulphate
C-S + HNO₃ (oxidation) → H₂SO₄
Reactions
H₂SO₄ + BaCl₂ → BaSO₄↓ (white)
1. Heat organic compound with fuming HNO₃ in a
beaker
2. Sulphur oxidised to H₂SO₄
Procedure 3. Cool; dilute with water
4. Add excess BaCl₂ solution
5. Filter, wash, dry precipitate at 373 K
6. Weigh white BaSO₄
Atomic
S = 32; Ba = 137; SO₄ = 96; BaSO₄ = 233
masses

Sulphur Estimation Calculations

Parameter Formula
% Sulphur % S = (32 × m₁ × 100) / (233 × m)
m₁ = mass of BaSO₄ precipitate (g)
Variables
m = mass of organic compound (g)

Example Problem
Given: 0.481 g compound → 0.664 g BaSO₄

Calculation
% S = (32 × 0.664 × 100) / (233 × 0.481) = 18.86%

3.5 Phosphorus Estimation

Method 1: As Ammonium Phosphomolybdate

Parameter Details
Phosphorus oxidised to phosphate; precipitated with
Principle
molybdate reagent
Organic P + HNO₃ (fuming) → H₃PO₄
Reactions PO₄³⁻ + MoO₄²⁻ + NH₄⁺ → (NH₄)₃[PO₄(MoO₃)₁₂]
(yellow)
1. Heat compound with fuming HNO₃
2. Phosphorus oxidised to H₃PO₄
3. Cool; add excess NH₄⁺ and molybdate solution
Procedure 4. Yellow precipitate forms: ammonium
phosphomolybdate
5. Filter, wash, dry at 373 K
6. Weigh yellow precipitate
Molecular
(NH₄)₃PO₄·12MoO₃
formula
Molar mass 1877 g/mol

Calculations for Phosphomolybdate Method

Parameter Formula
% Phosphorus % P = (31 × m₁ × 100) / (1877 × m)
m₁ = mass of (NH₄)₃PO₄·12MoO₃ (mg)
m = mass of organic compound (mg)
Variables
31 = atomic mass of P
1877 = molar mass of ammonium phosphomolybdate

Method 2: As Magnesium Pyrophosphate

 Parameter Details
Phosphate precipitated as MgNH₄PO₄; ignited to
Principle
Mg₂P₂O₇
PO₄³⁻ + Mg²⁺ + NH₄⁺ → MgNH₄PO₄↓
Reactions
2MgNH₄PO₄ → Mg₂P₂O₇ (on ignition) + 2NH₃ + H₂O
1. Get PO₄³⁻ from compound oxidation (as above)
2. Add magnesia mixture (contains Mg²⁺ and NH₄⁺)
3. White precipitate MgNH₄PO₄ forms
Procedure
4. Filter, wash, dry, weigh
5. Heat in crucible (ignite)
6. Weigh white Mg₂P₂O₇
Magnesia
Contains MgCl₂, NH₄Cl, NH₃·H₂O
Mixture

Calculations for Magnesium Pyrophosphate Method

Parameter Formula
% Phosphorus % P = (62 × m₁ × 100) / (222 × m)
m₁ = mass of Mg₂P₂O₇ (mg)
m = mass of organic compound (mg)
Variables
62 = mass of 2 P atoms
222 = molar mass of Mg₂P₂O₇

3.6 Oxygen Estimation

 Parameter Details
Principle Determined by difference from other elements
Calculation % O = 100 - (% C + % H + % N + % S + % X + % P)
Note Oxygen is always determined indirectly as difference
Only when: (a) Compound contains only C, H, O
When used (b) Percentage of other elements known
(c) Direct estimation not needed
1. Determine C and H by combustion
2. Determine N by Dumas/Kjeldahl
Procedure
3. Determine S, halogens, P if present
4. Oxygen = 100 - (sum of all other elements)

4. QUALITATIVE TESTS FOR FUNCTIONAL

GROUPS
4.1 Alcohols
 Comp
Procedur Observatio Mechanis
Test Reagent ound
e n m
Type
Sodi Vigorous
Add small All
um reaction; H₂ 2R-OH +
Sodium piece of alcoho
Met gas evolved; 2Na → 2R-
metal Na to ls (1°,
al heat ONa + H₂↑
alcohol 2°, 3°)
Test produced
1° Alcohol:
No turbidity
(or cloudy
Mix
after hours) Forms
alcohol Differ
2° Alcohol: alkene: R-
with entiat
Luc Conc. Turbidity in OH → R⁺
Lucas es 1°,
as HCl + 5-15 min → R-Cl
reagent; 2°, 3°
Test ZnCl₂ (warm if (via
observe alcoho
needed) carbocatio
at room ls
3° Alcohol: n)
temp
Immediate
turbidity at
room temp
1° Alcohol:
Orange →
green
(alcohol
oxidised to
Add aldehyde, Differ
Chr
K₂Cr₂O₇/ alcoholic then acid) entiat Redox
omi
conc. solution 2° Alcohol: es reaction;
c
H₂SO₄ to Orange → 1°/2° Cr(VI) →
Acid
(orange) chromic green from Cr(III)
Test
acid (oxidised to 3°
ketone)
3° Alcohol:
No colour
change (no
oxidation)
Este Carboxy Warm Pleasant All R-OH + R'-
rific lic acid alcohol fruity smell alcoho COOH ⇌
 Comp
Procedur Observatio Mechanis
Test Reagent ound
e n m
Type
atio (e.g., with of ester ls R'-COO-R +
n acetic carboxyli (e.g., ethyl H₂O
acid) + c acid and acetate) (Fischer
conc. conc. esterificati
H₂SO₄ H₂SO₄ on)

4.2 Phenols
 Reage
Test Procedure Observation Mechanism
nt
Complexation:
Ferric Violet/blue/gree
C₆H₅-OH + FeCl₃
FeCl chlori Add FeCl₃ to n colour
→ Coloured
₃ de phenol depending on
complex; lone
Test soluti solution phenol
pair on O
on structure
donates to Fe³⁺
Bromi Instant Electrophilic
Bro
ne decolourisation; aromatic
min Add bromine
water white substitution: OH
e water to
(oran precipitate group activates
Wat phenol
ge- (2,4,6- ring; Br₂ adds to
er solution
yello tribromophenol ortho and para
Test
w) ) or yellow oil positions
Explosion risk
Warm
(high temp); Electrophilic
phenol
Conc. yellow oily aromatic
Nitr carefully
HNO₃ liquid (mixture substitution;
atio with dilute
+ of phenol highly
n HNO₃ or (for
conc. nitrophenols); activated; gives
Test vigorous
H₂SO₄ explosive ortho, meta,
test) use
compound para products
conc. HNO₃
forms
First: Blue
Formation of
colour
Heat phenol quinonoid
Lieb (phenoxide ion
with CHCl₃ structure;
erm CHCl₃ complex) when
and KOH formation of p-
ann + KOH heated; On
solution; benzoquinone
Test acidification:
acidify type
Red/brown
intermediate
colour
vs NaHC Add both to Phenol: No Phenol is
Car O₃ or phenol and reaction; weaker acid
box Na₂CO carboxylic solution than H₂CO₃;
ylic ₃ acid remains RCOOH +
Acid separately colourless; NaHCO₃ →
doesn't dissolve
 Reage
Test Procedure Observation Mechanism
nt
in NaHCO₃ RCOONa + H₂O
Carboxylic acid: + CO₂
Reacts; CO₂ gas
evolved;
solution turns to
salt

4.3 Aldehydes vs Ketones

 Reage Mechani Procedur
Test Aldehydes Ketones
nt sm e
RCHO +
2[Ag(NH₃
Silver
)₂]⁺ + H₂O Add
mirror on
→ compound
Ammo test tube
No RCOONH to freshly
niacal wall or
reaction; ₄ + 2Ag↓ + prepared
Tolle AgNO₃ black
solution 3NH₃ Tollen's
n's (Tollen precipitate
remains Aldehyde reagent;
Test 's of Ag;
colourles oxidised warm in
reagen clear
s to water
t) solution
carboxyla bath (5-10
becomes
te; Ag⁺ min)
cloudy
reduced
to Ag
Fehlin RCHO +
g's 2Cu²⁺
Brick-red No
reagen (basic) →
precipitat precipita
Fehli t (Cu²⁺ RCOO⁻ +
e of Cu₂O; te;
ng's in Cu₂O↓
blue solution
Test alkalin Aldehyde
solution → remains
e oxidised;
colourless blue
tartrat Cu²⁺ →
e) Cu⁺
Brick-red No
Benedi
precipitat reaction;
ct's Same as
e of Cu₂O; remains
Bene reagen Fehling;
colour blue (or
dict' t slightly
changes: green/yel
s (simila different
blue → low
Test r to Cu²⁺
green → dependin
Fehlin complex
yellow → g on
g)
brick-red conc.)
Schif Schiff' Purple/ma No RCHO +
f's s genta colour H-
Test reagen colour; change; C(=NH)-
t colourless remains C₆H₃(SO₃
(fuchsi solution H)₂
 Reage Mechani Procedur
Test Aldehydes Ketones
nt sm e
n dye becomes colourles (fuchsin)
in SO₂) coloured s → Purple
adduct
Aldehyde
reacts
with free
amine of
dye
R-CHO +
NaHSO₃
→ R-
Satura
No CH(OH)S
ted Crystallin
reaction O₃Na
sodiu e white
Sodi or very Carbonyl
m precipitat
um slow reacts
bisulfit e forms;
Bisu (bulky with
e water-
lfite ketones nucleophi
(NaHS soluble
Test don't lic
O₃) bisulfite
react bisulfite;
solutio adduct
easily) forms
n
stable
crystallin
e adduct
2,4- 2,4- Yellow/or Yellow/o R₂C=O +
DNP dinitro ange range H₂N-NH-
Test phenyl precipitat precipita C₆H₃(NO₂
hydraz e of 2,4- te of )₂ →
ine dinitrophe DNPH R₂C=N-
(2,4- nylhydraz NH-
DNP) one C₆H₃(NO₂
in (DNPH) )₂
metha (Hydrazo
nol ne
formation
)
Nucleoph
ilic
 Reage Mechani Procedur
Test Aldehydes Ketones
nt sm e
addition;
both
aldehyde
s and
ketones
form
DNPH
CH₃-CO-R
+ I₂ +
2NaOH →
Yellow CHI₃↓
Meth No
precipita (yellow) +
yl reaction
te of RCOONa
Keto Iodine (acetaldeh
iodofor + 2H₂O
ne + yde is
m (CHI₃); Methyl
Test NaOH exception:
character ketones
(Iod / KOH HCHO + I₂
istic have CH₃-
ofor + NaOH →
pungent CO group;
m) CHI₃)
smell iodine
oxidises
and CHI₃
forms
Resistant
to
Oxid
Most oxidatio
atio
easily n (no
n
oxidised simple
Stre
- (aldehyde oxidation -
ngth
→ to acid
Com
carboxylic under
pari
acid) mild
son
condition
s)
Sodium Bisulfite Test (Detailed)
Principle:
Aldehydes and methyl ketones (and some other ketones) react with saturated aqueous
sodium bisulfite (NaHSO₃) to form crystalline, water-soluble bisulfite addition compounds
(aldol condensation products). This test is useful for:
Confirming the presence of carbonyl group
Purification of carbonyl compounds (can separate from non-carbonyl impurities)
Reaction Mechanism:

Procedure:

1. Take a saturated solution of sodium bisulfite in a test tube

2. Add 1 mL of organic compound to be tested
3. Shake well and allow to stand for 15-20 minutes at room temperature
4. Observe for precipitate formation
Observation:
Positive Test: White/crystalline precipitate forms; compound becomes turbid
Aldehyde: Reacts readily; white precipitate forms quickly
Methyl Ketone (CH₃-CO-R): Reacts; white precipitate forms
Bulky Ketones: Slow or no reaction (steric hindrance prevents nucleophilic attack)
No Carbonyl: Remains clear; no precipitate

Key Features:
Crystalline product is water-soluble (unlike most organic compounds)
Can be used to purify carbonyl compounds by precipitating them away from
impurities
Reversible reaction: adding acid releases original carbonyl compound
Does not distinguish between aldehydes and ketones (both can react)
Use Tollen's, Fehling's, or Schiff's tests to differentiate
Important Note:
On acidification, the adduct decomposes to regenerate the carbonyl:

This property makes it useful for purification without loss of compound.

4.4 Carboxylic Acids

 Reagen
Test t/Proce Observation Mechanism Notes
dure
Carboxylic
Brisk
Sodium acids are
Add effervescence RCOOH +
Bicarb strong
NaHCO₃ ; CO₂ gas NaHCO₃ →
onate enough to
solution evolved; RCOONa +
(NaHC displace CO₂
to acid solution H₂O + CO₂↑
O₃) Test from
becomes clear
bicarbonate
Vigorous Confirms
Sodium
Add effervescence 2RCOOH + presence of
Carbon
Na₂CO₃ ; CO₂ evolved; Na₂CO₃ → acid;
ate
solution solution 2RCOONa + stronger
(Na₂CO
to acid becomes clear H₂O + CO₂↑ reaction than
₃) Test
as salt forms NaHCO₃
Phenol: No Phenol (pKa
reaction; ~10) too
remains weak;
Add
colourless; doesn't react
NaHCO₃
doesn't with Key
vs or
dissolve NaHCO₃ differentiati
Phenol Na₂CO₃
Carboxylic (pKa ~6.4) on test
to
acid: Reacts; RCOOH (pKa
phenol
CO₂ evolved; ~4-5) is
dissolves as strong
salt enough
React
Fischer
with
Pleasant fruity RCOOH + R'- esterification
Esterifi alcohol
odour; ester OH ⇌ RCOO- ; confirms
cation + conc.
formed R' + H₂O carboxylic
H₂SO₄;
acid
heat
Ferric Add Reddish- Acid -
Hydrox FeCl₃ brown neutralised
ide Test then precipitate of by formed
NH₃ to Fe(OH)₃; Fe(OH)₃ or
confirms acid
 Reagen
Test t/Proce Observation Mechanism Notes
dure
carboxy forms
lic acid complex

4.5 Esters, Acid Halides, and Anhydrides

Comp
ound Test Reagent Observation Mechanism
Type
RCOO-R' +
On NaOH →
Heat with acidification: RCOONa +
Sapon
NaOH/KOH; Oily layer R'OH
Esters ificati
acidify with separates On HCl:
on
HCl (carboxylic acid RCOONa + HCl
formed) → RCOOH +
NaCl
Convert to
Ferric hydroxamic
[RCONHOH +
Hydr acid:
Burgundy/wine Fe³⁺ →
oxam RCOOR' +
-red colour Complex with
ate NH₂OH →
colour
Test RCONHOH
Add FeCl₃
Vigorous
Acid RCOCl + H₂O →
reaction; heat;
Halide Add water RCOOH + HCl↑
Hydr acidic odour
s (Acyl or aqueous RCOCl + 2NH₃
olysis (HX gas
Halide ammonia → RCONH₂ +
evolved); fumes
s) NH₄Cl
with ammonia
Electrophilic
Fried Aromatic
aromatic
el- React with ketone forms
substitution;
Crafts benzene + (aromatic
forms C₆H₅-CO-
Acylat AlCl₃ nucleus
R (aromatic
ion substituted)
ketone)
Forms
Acid Add water; carboxylic acid;
Hydr (RCO)₂O + H₂O
Anhyd warm if acidic solution;
olysis → 2RCOOH
rides needed characteristic
odour
Amm Add aqueous Ammonia (RCO)₂O +
onoly ammonia absorbs CO₂; 2NH₃ →
 Comp
ound Test Reagent Observation Mechanism
Type
sis white fumes at RCONH₂ +
mouth of tube RCOONh₄

4.6 Amines (Primary, Secondary, Tertiary)

Hinsberg Test (Best Test for Differentiating 1°, 2°, 3°)
 Secondary Tertiary
Test Procedure Primary (1°)
(2°) (3°)
No
React with Forms N- Forms N- reaction
benzenesulph benzenesulph benzenesul ;
Hins
onyl chloride onamide phonamide remains
berg
(C₆H₅SO₂Cl) in (white ppt): (white ppt): colourles
Test
presence of Insoluble in Insoluble in s; no
excess NaOH dilute HCl dilute HCl precipita
te
C₆H₅SO₂Cl +
C₆H₅SO₂Cl + R-
R₂NH +
React NH₂ + NaOH → No
- NaOH →
ions C₆H₅SO₂-NH-R reaction
C₆H₅SO₂-NR₂
+ NaCl + H₂O
+ NaCl + H₂O
No No
With
Add dilute dissolution dissolution No
HCl
HCl to (product (product product
(dilut
product insoluble in insoluble in formed
e)
dilute HCl) dilute HCl)
Dissolves to
Remains
With form soluble
Add excess undissolved No
KOH salt: C₆H₅SO₂-
KOH solution (product not product
(exce NH-R + KOH →
to product soluble in formed
ss) K⁺[C₆H₅SO₂-N⁻-
KOH)
R] (soluble)
Differ
Soluble in
entia No
KOH but Insoluble in
tion - precipit
insoluble in both
Sum ate
HCl
mary

Other Amine Tests

 Applies
Test Procedure Observation
to
1° Amine: Yellow oily liquid
(diazonium salt) or diazo
Nitrous Add NaNO₂ compound forms; on warming
Acid to amine in → phenol/alcohol + N₂ gas All
(HNO₂) HCl or 2° Amine: Yellow oil (N- amines
Test acetic acid nitroso compound) stable
3° Amine: No reaction;
remains clear
1° Amine: Characteristic
Carbyla Heat amine pungent, unpleasant odour Confirm
mine with CHCl₃ (R-N≡C, isocyanide) s
(Isocyani + excess 2° Amine: No odour (or much primary
de) Test NaOH weaker) amines
3° Amine: No odour
Detectio
Add FeCl₃ Coloration or complex
FeCl₃ n of
solution to formation (varies with amine
Test aromatic
amine structure)
amines

4.7 Nitriles
 Test Procedure Observation Mechanism
Ammonia evolved
R-C≡N + 2NaOH
Hydr Heat nitrile (pungent smell); solution
+ H₂O → R-
olysis with NaOH becomes alkaline;
COONa + NH₃↑
Test and water eventually forms
(on heating)
carboxylic acid salt
Acidi
Heat nitrile R-C≡N + 2H₂O
c Carboxylic acid formed;
with H₂SO₄ (+ H₂SO₄) → R-
Hydr ammonia may be detected
and water COOH + NH₃↑
olysis
Add FeSO₄ Nitrile
Pruss After oxidation and
then converted to
ian complex formation:
acidify CN⁻; reacts as
Blue Prussian blue colour forms
with HCl; in Lassaigne's
Test if N detected
warm test

4.8 Halides
Hal
ide Rea Mechani
Test Procedure Observation
Typ gent sm
e
Heat
compound
Cl⁻: White ppt
Sodi with Na;
soluble in NH₃ X⁻ + Ag⁺
um extract
All Lassai Br⁻: Yellowish → AgX
fusi with
Hal gne's ppt sparingly (colour
on; water;
ides Test soluble in NH₃ identifies
AgN acidify
I⁻: Yellow ppt halogen)
O₃ with
insoluble in NH₃
HNO₃; add
AgNO₃
SN2 AgN
Pri Reacti O₃ Add AgNO₃ 1° Halide: Slow SN2: R-X
ma on in to halide in precipitation + Ag⁺ →
ry (Nucle etha ethanol; (after few min to R⁺ + AgX↓
Hal ophilic nol warm if hours); forms (slow for
ides Substi or needed AgX precipitate 1°)
tution) KI
Sec AgN 2° Halide: Mix of
SN1/S
ond O₃ Add AgNO₃ Moderate speed SN1 and
N2
ary in to halide in (forms SN2;
Comp
Hal etha ethanol precipitate in intermed
etition
ides nol few minutes) iate rate
3° Halide:
SN1 Immediate SN1: R-X
Reacti AgN precipitation at → R⁺ + X⁻
Tert
on O₃ Add AgNO₃ room (fast); R⁺
iary
(Nucle in to halide in temperature; + Ag⁺ →
Hal
ophilic etha ethanol white/cream ppt R-Ag⁺ →
ides
Substi nol forms instantly; AgX↓ + R⁺
tution) solution may transfers
become warm
4.9 Sulfur-Containing Compounds (Thiols, Thioethers,
Disulfides)
Com
poun Procedur
Test Observation Mechanism
d e
Type
Vigorous reaction;
Sodium Thiol Add small H₂ gas evolved (but 2R-SH + 2Na
Metal s (R- Na piece slower than → 2R-S-Na⁺ +
Test SH) to thiol alcohols); heat H₂↑
produced
Black precipitate
Add lead
Lead of PbS; R-SH + Pb²⁺
Thiol acetate
Acetate characteristic → PbS↓
s solution to
Test rotten egg smell (black) + H⁺
thiol
intensifies
Slower darkening;
Add lead
less intense; may Disulfide less
Disul acetate to
not give reactive than
fides disulfide
precipitate thiol
(R-S-S-R')
immediately
Add Deep
Sodium SH⁻ +
sodium purple/violet
Nitropr Thiol [Fe(CN)₅NO]²⁻
nitropruss colour;
usside s → Violet
ide characteristic of
Test complex
solution thiol
Warm Disulfide
Perma disulfide Colour fades oxidised to
Disul
nganat with (from purple to sulphonic
fides
e Test dilute colourless/brown) acid; MnO₄⁻
KMnO₄ reduced
Add cold Thiol rapidly
Immediate
Thiol dilute oxidised;
decolourisation
s KMnO₄ to very easily
(very rapid)
thiol oxidised
5. QUALITATIVE TESTS FOR
BIOMOLECULES
5.1 Carbohydrates Tests
Reducing Sugars (Monosaccharides & Disaccharides like Lactose, Maltose)

Te Reage Procedur Observatio Compoun Mechani

st nt e n ds Tested sm
Fehlin Add Monosacc
Brick-red RCHO
Fe g's Fehling's harides
precipitate (aldehyd
hli reagen A & B to (glucose,
of Cu₂O; e) +
ng' t (Cu²⁺ reducing fructose);
blue solution 2Cu²⁺ →
s + sugar; Disacchari
changes to RCOO⁻ +
Te tartrat warm in des
colourless/w Cu₂O↓
st e+ water (maltose,
hite ppt (red)
NaOH) bath lactose)
Mix
Tollen' Tollen's RCHO +
Silver
s with Aldoses 2[Ag(NH₃
Tol mirror on
reagen reducing (glucose, )₂]⁺ + OH⁻
le test tube
t sugar; galactose); →
n's walls; or
(ammo warm ketoses [RCOO(N
Te black/grey
niacal gently can also H₄)]⁺ +
st Ag
AgNO₃ (avoid reduce 2Ag↓ +
precipitate
) overheati 3NH₃
ng)
Brick-
Add red/orange
Benedi
Be Benedict's precipitate
ct's
ne to of Cu₂O; Same as
reagen
dic reducing colour Fehling's; Same as
t (Cu²⁺
t's sugar; changes: more Fehling's
+
Te boil in blue → sensitive
citrate
st water green →
)
bath yellow →
red
Non-Reducing Sugars (Sucrose)

Test Procedure Observation Mechanism

No precipitate Sucrose lacks free
Add or colour aldehyde/ketone
Direc
Fehling/Benedict/ change; (both anomeric
t Test
Tollen's to sucrose remains blue; carbons involved in
no reduction glycosidic bond)
Hydrolysis: C₁₂H₂₂O₁₁
(1) Heat sucrose Brick-red
After + H₂O → C₆H₁₂O₆
with dilute HCl; precipitate
Acid (glucose) + C₆H₁₂O₆
(2) Neutralise forms (if
Hydr (fructose)
with NaOH; (3) hydrolysis
olysis Both are reducing
Add Fehling's successful)
sugars

Molisch Test (All Carbohydrates)

Tes
Procedure Observation Mechanism
t
Carbohydrate
dehydrated to
(1) Add α-naphthol furfural/hydro
Purple/violet ring
Mol solution to xymethylfurfu
at interface
isc carbohydrate; (2) ral by H₂SO₄
between solutions
h Carefully pour conc. Furfural
(characteristic);
Tes H₂SO₄ down the side condenses
solution becomes
t (don't mix with α-
warm
immediately) naphthol →
purple
complex
Pos All carbohydrates
itiv (monosaccharides,
- -
e disaccharides,
for polysaccharides)
Seliwanoff Test (Ketoses)

Test Procedure Observation Mechanism

Ketose
(fructose)
Red/brown dehydrated →
Seli Heat carbohydrate
precipitate (deep hydroxymethylf
wan with dilute HCl and
red solution); urfural (HMF)
off resorcinol in water
forms quickly for HMF +
Test bath (boil 1-2 min)
ketoses resorcinol → red
condensation
product
Time Ketoses: 0.5-1 min
Differentiates Ketoses
to (quick)
ketoses from dehydrate faster
colo Aldoses: 1-2 min
aldoses than aldoses
ur (slower)
Fructose, sorbose
Posit
(ketoses); aldoses
ive - -
also give red but
for
more slowly

Barfoed Test (Monosaccharides vs Disaccharides)

 Test Procedure Observation Mechanism
Monosacchari
Monosaccharides
des reduce
(glucose, fructose):
faster due to
Red precipitate
Add Barfoed's free
Bar Cu₂O forms quickly
reagent (Cu²⁺ + acetic aldehyde/keto
foe (within 1-2 min)
acid) to reducing ne
d Disaccharides
sugar; heat gently in Disaccharides
Test (maltose, lactose):
water bath (2-3 min) reduce slowly
No precipitate or
(partial
slower formation
opening of
(after 5+ min)
ring needed)
Key Quick red ppt =
Diff Speed of monosaccharide;
-
ere precipitation Slow/no ppt =
nce disaccharide

Iodine Test (Starch & Polysaccharides)

Test Procedure Observation Mechanism

I₂ enters helical
structure of
Deep blue/black
Add iodine starch (amylose)
colour
Iodi solution (I₂ + KI in → I₂@starch
(characteristic);
ne water) to starch complex (blue)
colour fades on
Test solution; warm if Heat: I₂ escapes;
heating; returns on
needed colour fades
cooling
Cool: I₂ re-enters;
colour returns
Pos
itiv Starch (most
- -
e characteristic)
for
Neg Reducing sugars Monosaccharides
Remains yellow-
ativ (glucose, & some
brown; no blue
e fructose, maltose, disaccharides lack
colour
for sucrose) helical structure
5.2 Protein Tests
Biuret Test (Peptides & Proteins)

Test Procedure Observation Mechanism

Cu²⁺ complexes
with peptide
(1) Add NaOH Violet/purple bonds (-CO-
Biur
solution to protein; colour NH-)
et
(2) Add CuSO₄ (characteristic of Cu²⁺ + 2(-CO-
Test
solution dropwise peptide bonds) NH-) →
Complex
(violet)
Proteins & peptides
Posit
(>3 amino acids);
ive - -
requires 2+ peptide
for
bonds
No peptide
Neg
Single amino acids bonds to
ativ Remains blue
(no peptide bonds) complex with
e for
Cu²⁺

Ninhydrin Test (Amino Acids & Proteins)

 Test Procedure Observation Mechanism
Ninhydrin
oxidatively
Heat
Purple/violet decarboxylates
Nin protein/amino
colour develops amino acids
hyd acid with
(intensity indicates Product
rin ninhydrin solution
protein/amino acid condenses with
Test (in ethanol) in
content) excess ninhydrin
water bath
→ Ruhemann's
purple (violet)
All proteins &
Posi amino acids
tive (primary amine - -
for group -NH₂
required)
Quantitative
Use analysis of amino
- -
d in acids; detection in
chromatography

Xanthoproteic Test (Aromatic Amino Acids: Tryptophan, Tyrosine,

Phenylalanine)
 Test Procedure Observation Mechanism
Nitration colour: Aromatic rings of
Yellow tryptophan/tyrosi
precipitate or ne/phenylalanine
yellow solution undergo nitration
Xant (1) Add conc. HNO₃
(after HNO₃) Aryl-NO₂ groups
hopr to protein; (2) Heat
After NaOH formed (yellow
oteic gently; (3) Cool; (4)
addition: under acidic
Test Add excess NaOH
Solution conditions)
becomes NaOH converts to
orange/golden orange (salt
yellow formation)
Proteins
Posit
containing Trp,
ive - -
Tyr, or Phe
for
residues
Free aromatic
Note amino acids also - -
give positive test

Millon's Reaction (Tyrosine - Phenolic Amino Acid)

 Test Procedure Observation Mechanism
Tyrosine contains
phenolic -OH;
Add Millon's Red/brick-red Millon's reagent
Mill reagent precipitate or (mercuric species)
on's (mercuric colour reacts with aromatic
Reac nitrate in HNO₃) (characteristic of -OH
tion to protein; phenolic Forms complex →
warm gently compounds) red colour
On heating: intensity
increases
Posit Proteins
ive containing - -
for tyrosine (Tyr)
Nega
Proteins lacking
tive No colour change -
tyrosine
for

Hopkins-Cole Test (Tryptophan)

Test Procedure Observation Mechanism

Tryptophan's
indole ring
reacts with
Violet/purple
Hopki (1) Add glyoxalic acid glyoxalic acid
ring at
ns- to protein; (2) Add H₂SO₄ acts as
interface
Cole conc. H₂SO₄ carefully dehydrating
(characteristic
Test (layer it); cool agent
)
Indole-glyoxal
condensation →
purple complex
Positi Proteins containing
- -
ve for tryptophan (Trp)
No colour
Negat
Proteins lacking change;
ive -
tryptophan remains
for
yellow-brown
Sakaguchi Test (Arginine)

Observat
Test Procedure Mechanism
ion
Arginine
contains
guanidinium
(1) Add α-naphthol solution Red/oran group (-NH-
Sak
to protein; (2) Add NaOH ge-red C(=NH)-NH₂)
agu
solution slowly; (3) Add colour Reacts with α-
chi
sodium hypobromite (Br₂ in (characte naphthol +
Test
NaOH) dropwise ristic) hypobromite→
red
condensation
product
Posi
Proteins containing arginine
tive - -
(Arg)
for

5.3 Lipid Tests

Sudan Stain Test (Fats & Lipids)

Test Procedure Observation Mechanism

Sudan III is
lipophilic
Suda Add Sudan III Orange-yellow
(dissolves in lipids)
n III (lipophilic dye) colour in Sudan III;
Colours lipids
Stai to lipid; warm if substance becomes
orange-yellow;
n needed coloured
used for staining
lipid droplets
Suda Sudan IV similar to
Add Sudan IV Red/orange-red
n IV Sudan III but
(lipophilic dye) colour; lipids
Stai produces more
to lipid; warm coloured
n intense colour
Posit Fats, oils,
ive cholesterol, - -
for other lipids
Ethanol Emulsion Test (Oils)

Test Procedure Observation Mechanism

Oil dissolves in ethanol
(1) Add absolute White (organic solvent)
Etha
ethanol to oil; turbidity/em When mixed with
nol
shake well; (2) ulsion forms water: oil precipitates as
Emul
Add to distilled (milky tiny droplets →
sion
water appearance) suspension (emulsion) =
milky appearance
Fats & oils
Posit
(distinction from
ive - -
water-soluble
for
compounds)

Acrolein Test (Fats - Glycerol)

 Test Procedure Observation Mechanism
Fat
(glyceride)
undergoes
thermal
decompositio
(1) Heat fat with Pungent n
Acr potassium bisulfate characteristic Glycerol:
olei (KHSO₄) or K₂SO₄ + conc. odour of acrolein C₃H₈O₃ - H₂O
n H₂SO₄ strongly; (2) (irritating to eyes → C₃H₄O
Test Collect vapours over & nose); white (acrolein =
glass rod fumes CH₂=CH-
CHO)
Acrolein has
characteristi
c pungent
smell
Pos
itiv Fats & oils (confirms
- -
e presence of glycerol)
for
Neg
ativ Liquid paraffin, mineral No glycerol
No acrolein odour
e oil (not glycerides) to release
for

Salkowski Test (Cholesterol)

 Test Procedure Observation Mechanism
Cholesterol
undergoes
dehydration/ox
Lower layer: Red idation by
colour H₂SO₄
(1) Dissolve
Salk (characteristic); Conjugated
cholesterol in CHCl₃;
ows Upper layer: diene system
(2) Add conc. H₂SO₄
ki Green-yellow formed →
carefully (layer it);
Test colour initially, coloured
cool
may change to complex
brown Red in lower
(acidic) layer;
Yellow-green at
interface
Cholesterol
Posit
specifically
ive - -
(characteristic test
for
for sterols)

Liebermann-Burchard Test (Cholesterol)

 Test Procedure Observation Mechanism
Acetic anhydride
acetylates
cholesterol
hydroxy group
Colour
H₂SO₄ causes
(1) Dissolve change: Blue
dehydration &
Lieber cholesterol in CHCl₃; → Green →
oxidation→
mann- (2) Add acetic Red
conjugated
Burcha anhydride; (3) Add (characteristi
system
rd Test conc. H₂SO₄ c sequence)
Blue complex
carefully; cool Final colour:
forms initially;
Red or brown
oxidation →
green, then red
quinone-like
structure
Positiv Cholesterol (very
- -
e for specific)
More
Specifi Distinguishes
c than cholesterol from - -
Salkow other sterols
ski

6. QUICK REFERENCE TABLES

Summary of Purification Methods by Compound Type
 Compound
Best Method Reason
Properties
Volatile liquid +
Difference in boiling
non-volatile Simple Distillation
points
impurities
Separation via
Liquids with close
Fractional Distillation fractionating
boiling points
column
Solid with high Solid → vapour
Sublimation
vapour pressure directly
Solid general Most common; uses
Crystallisation
purification solubility difference
Organic in Solubility difference
Differential Extraction
aqueous solution between solvents
Mixture of
Differential
complex Chromatography
adsorption/partition
components
Distillation under Lower temperature
Heat-sensitive
reduced pressure or prevents
compound
steam distillation decomposition

Element Detection Summary (Lassaigne's Test)

 Eleme Positive
Test Procedure Product
nt Observation
Heat with CuO; test
White turbidity
C vapours with lime CaCO₃
in lime-water
water
Heat with CuO; test
H vapours with Blue colour CuSO₄·5H₂O
anhydrous CuSO₄
Na fusion + FeSO₄ +
N Prussian blue Fe₄[Fe(CN)₆]₃
conc. H₂SO₄
Na fusion + Pb(OAc)₂
S Black ppt PbS
(in acetic acid)
Na fusion +
Violet colour Complex
Na₂[Fe(CN)₅NO]
Cl: White ppt
(sol in NH₃)
X Br: Yellow ppt
Na fusion + AgNO₃ AgCl, AgBr,
(Halo (sparingly sol in
(after HNO₃) AgI
gens) NH₃)
I: Yellow ppt
(insol in NH₃)
Ammonium
Heat with Na₂O₂; add Yellow ppt or
P phosphomoly
(NH₄)₂MoO₄ colouration
bdate

Quantitative Analysis Methods

 Elem Product
Method Formula
ent Weighed
C& Combustio % C = (27.27 × m_CO₂) / m;
CO₂ & H₂O
H n Analysis % H = (11.11 × m_H₂O) / m
N Dumas N₂ gas volume % N = (28V / 22,400m) × 100
H₂SO₄ used by
Kjeldahl % N = 1.4M(2V₁ - V₂) / m
NH₃
Cl, % Cl = (35.5 × m_AgCl) /
Carius AgX precipitate
Br, I (143.5 × m); similar for Br, I
Combustio % S = (32 × m_BaSO₄) / (233
S BaSO₄
n × m)
(NH₄)₃PO₄·12Mo
P Oxidation % P = (31 × m) / (1877 × m)
O₃ or Mg₂P₂O₇
By % O = 100 - (% C + % H + %
O -
difference N + % S + % X + % P)

Aldehyde vs Ketone Quick Test

Test Aldehydes Ketones Comment
✓ Silver
Tollen's ✗ No reaction Best for aldehydes
mirror
Fehling/Be ✓ Red ppt Reducing sugars
✗ No ppt
nedict (Cu₂O) aldoses
Sensitive for
Schiff ✓ Purple ✗ No colour
aldehydes
✓ Yellow Both give
2,4-DNP ✓ Yellow ppt
ppt hydrazone
✓ If CH₃-CO Methyl ketones +
Iodoform HCHO only
group acetaldehyde
✓ Easy → ✗ Resist. to mild
Oxidation Chemical difference
RCOOH oxidation
Alcohol Differentiation
Test 1° Alcohol 2° Alcohol 3° Alcohol
Lucas No turbidity / Turbidity in 5-15 Immediate
Test slow (hours) min turbidity
Orange → Green No change
Chromi Orange → Green
(oxidised to (remains
c Acid (oxidised to acid)
ketone) orange)
H₂ evolved
Sodium H₂ evolved H₂ evolved
(slower)

Amine Classification (Hinsberg)

Test 1° Amine 2° Amine 3° Amine
White ppt; Soluble White ppt; No ppt
Hinsberg
in KOH Insoluble in KOH formed
Carbyla
Pungent odour No odour No odour
mine
Nitrous Diazonium (α- No
N-Nitroso (yellow)
Acid coupling) reaction

Carbohydrate Tests Summary

 Sugar Fehli Tolle Molis Seliwan Barf
Iodine
Type ng n ch off oed
✓ ✓
Glucos ✓ ✓
Mirro Purpl ✓ Slow ✗ No
e Red Quick
r e
✓ ✓
Fructos ✓ ✓✓ Very ✓
Mirro Purpl ✗ No
e Red fast Quick
r e
✓ ✓
Maltos ✓ ✓
Mirro Purpl ✓ Slow ✗ No
e Red Slow
r e
✓ ✓
✓ ✓
Lactose Mirro Purpl ✓ Slow ✗ No
Red Slow
r e
✓
Sucros
✗ No ✗ No Purpl ✗ No ✗ No ✗ No
e
e
✓
✓✓ Blue-
Starch ✗ No ✗ No Purpl ✗ No ✗ No
black
e

Protein Tests Summary

 Hopki
Amino Acid Biur Ninh Xantho Mill Saka
ns-
Component et ydrin proteic on guchi
Cole
✓
Peptide
Viol - - - - -
bonds
et
✓
Any amino
- Purpl - - - -
acid
e
✓ ✓
Tryptophan - - - -
Yellow Violet
✓ ✓
Tyrosine - - - -
Yellow Red
Phenylalani ✓
- - - - -
ne Yellow
Arginine - - - - - ✓ Red

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