This document summarizes Dr. V. A. Chatpalliwar's lecture on separation techniques. It discusses various chromatographic and physical separation methods that can be used to separate binary mixtures and chiral compounds. These include TLC, HPLC, GC, and techniques based on differences in solubility such as extraction with acidic or basic solutions. The document provides guidance on properly selecting and applying separation methods, including considering the properties of the compounds involved and avoiding approaches that could damage or contaminate the samples.

1. Separation Techniques
Dr. V. A. Chatpalliwar
Prof., Pharm. Chem.
Head, Dept. Pharm. Chem.
S. S. D. J. Coll. Pharm.
Neminagar, Chandwad
Dist.: Nashik
Theory and Practice of Organic chemistry

2. Acknowledgements
Sinhgad College of Pharmacy
Vadgaon Campus
Pune
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Management & Staff

3. Separation Techniques
Used for separating
• a binary mixture of organic compounds
• a racemic mixture of chiral compounds
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Chromatographic Techniques
 TLC
Paper
HPTLC
HPLC
GC
 Capillary Electrophoresis
 Super-critical Fluid
Chromatography
 UPLC
Preparative HPLC
 Chiral Separation
OPEC
 SSE
 SLE

4. Binary Mixture (containing two components)
 Physical means of separation
 Preliminary test with solvents
Try to separate on the basis of
solubility in acidic/basic solution,
viz. dil./conc. HCl, dil./conc. H2SO4,
dil./conc. NaOH/dil. NaHCO3
 Re-precipitate by neutralization
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Usual Practice Consequence
 Only one component of the mixture is
collected in pure form/in manageable
quantity
 Exhaustion of sample before separating
it satisfactorily due to loss is performing
unwanted tests/steps/techniques for
achieving separation
Just Three Hours No Gain after lot of pain

5. Salient points
• Separation should be qualitative as well as quantitative;
if it is not achieved problems of contamination arise.
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
• Process used for separating the components should not
induce a drastic chemical change in either component of
sample
• Rational/separable mixtures should be prepared while
designing the practical

6. Scheme (systematic)
Preliminary Examination of the mixture
 States
 Odour
 Colour
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Polar components: solids
Pungent: Acidic
Fishy: Basic
Sweet/Agreeable/Aromatic: Neutral
Groups with free lone-pair of electrons/

7. Element Detection Test (mixture)
Lassaigne’s Test
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
3 tests instead of 6
Absence of nitrogen in mixture rules out possibility of amines, amides,
nitro, hydrazines, and sulphonamides
• Conservation of time
• Guides in skipping few steps of separation

8. Separation
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
On the basis of
 physical state does not allow proper separation
 chemical separation based on molecular properties allows
proper separation; presence of acidic/basic functional groups
should be exploited to separate the components of mixture
Toluene and Acetic acid

9. Dissolution (prior to extraction)
Allows dissociation of molecules from each other which otherwise
are strongly held to each other by intermolecular forces
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Abolish the possibility of contamination in separated components
This dissociation helps in proper interaction between component
molecules and extraction solvent

10. Choice of Solvent
• Should be able to dissolve wide
range of organic compounds
• Should not trigger unwanted
reactions with components
• Sample/components should be
recoverable after separation
• Should be immiscible with other
solvent; extractable
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Diethyl ether
√
√ ethers are less reactive; next to alkanes
√ boiling point in 35 ° C
√ immiscible with water
√ Far less dense (0.78 g/mL) so as to
form separable bi-layers
inflammable, flash point - 45 ° C, creeping nature

11. Method
• All possible source of
flame/s and/or hot
surfaces must be
removed from the
vicinity to diethyl
ether.
• Fast working is
required in order to
avoid loss of solvent
due to evaporation.
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Sample mixed with solvent
Clear
solution
Partial
solubility
Quantity of solvent be
increased
Separation achieved
Filter to obtain two separate
components
Proceed for
extraction
Washing the residue with the solvent assures
proper separation

12. Extraction (liquid-liquid)
• dil. HCl/conc. HCl
• dil. NaOH/conc. NaOH
• dil. H2SO4/conc. H2SO4
• dil. NaHCO3
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Observation/inference of
Lassaigne’s test
Nitrogen present
Amides
Amines
Hydrazines, Azo-
Nitro compounds
Sulphonamides
Nitrogen absent
aq. HCl (5%)
Carboxylic acid
Phenol
Carbonyl compounds
Alcohols
Hydrocarbons
Acyl halides
aq. NaOH (5%)

13. Extraction (procedure)
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Time for
interaction
aq. HCl (5%)/aq. NaOH
Two solutions
Neutralizing
solvent
Neutral/acidic/basic
Compound
Filter/Separate
Wash and Dry
Acidic/Basic Compound
Salt/polar
compound
Evaporation

14. Care to be exercised
Mixing (for extraction with immiscible solvent) is advisable to be performed in beaker
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Accident-prone
Selecting proper-sized separating funnel
Extraction should be performed repeatedly for complete separation ,
till further neutralization (with double-strength solution) does not
result in precipitation/phase separation
Small volume at interface be discarded/collected else-where
Washings should be carried till acid/base is completely removed
Drying (vacuum/desiccator/desiccant)
Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series

15. Identification
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Acidic/Basic Compound A B
Observation
/inferences
from
separation
process
Lassaigne’s test Lassaigne’s test
Aromaticity Aromaticity
Water insoluble
&
Acid/base soluble
Water insoluble
&
Ether soluble
Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Functional group identification and Derivative
preparation
Result and Discussion
Neutral/acidic/basic
Compound

16. Preparation of Lassaigne’s Solution
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Fusion tube Fusion assembly Decomposing the
protecting material
Exposed Sodium metal
Extraction with water
Filtration through
soaked filter paper
Clear (not dark)
Lassaigne’s solution
Addition of sample
directly over exposed
metal
Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series

17. Preparation of Reagents
aq. 2, 4-Dinitrophenylhydrazine for water-insoluble carbonyl compounds
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
aq. HCl (5%) aq. NaOH (5%)
Stored Schiff’s reagent On adding sodium
metabisulphite
N1V1 = N2V2
Schiff’s reagent with
sample, Benzaldehyde
Schiff’s reagent with
sample Benzaldehyde on
proper mixing
Schiff’s reagent with
p-Hydroxybenzaldehyde
Schiff’s reagent

18. Preparation of Samples (examples)
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Benzoic acid and p-Aminophenol
Benzoic acid and Anthracene
Benzoic acid and m-Dinitrobenzene
Phthalic acid and Anthracene
Salicylic acid and Acetanilide
Benzoic acid and Acetanilide
α-Naphthol and Anthracene
Phthalic acid and Benzamide
Salicylic acid and Anthraquinone
α-Naphthol and Anthraquinone
p-Nitrobenzoic acid and α-Naphthol
Benzoic acid and β-Naphthol
Benzoic acid and α-Naphthol
Cinnamic acid and β-Naphthol
Salicylic acid and β-Naphthol
Salicylic acid and Resorcinol
β-Naphthol and p-Nitrophenol
Diphenylamine and Acetanilide
Diphenylamine and α-Naphthol
Urea and Toluene
Thiourea and Toluene
Benzamide and Nitrobenzene
Aniline and Nitrobenzene
p-Chlorobenzoic acid and
Diphenylamine
Salicylic acid and Urea
p-Chlorobenzoic acid and
Thiourea
p-Chlorobenzoic acid and
Aniline
Aniline and Urea
Anthranilic acid and benzamide
Combinations in red font are not be prepared as samples

19. Preparation of Samples
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Should be separable by undergraduate students
Should not be prepared with quantities out-of-proportion
Should be mixed well so as to avoid easy jump to the conclusion
Should contain mono-functional compounds
Should not contain water soluble compounds

20. Casual Approach be Avoided
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
 Samples should be prepared by technical person
 Curiosity/interest of students should be respected and
preserved; their memory should not be useful in the context
 Enough/sufficient/rational time should be allowed for proper exercise
Compound as supplied Compound precipitated
during experiment
 Physical appearance of sample/compounds often mislead
Compound collected after
Precipitation during experiment

21. Casual Approach
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Ammoniacal Silver nitrate
 Not stored due to short shelf-life
 prepared just when it is needed
 It is never heated; gentle warming is required for
reaction with some carbonyl compounds
Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
 Risk formation of silver fulminate which
explodes dangerously on drying
 All apparatus in which reagent has been used
should be carefully & immediately after use should
be washed with dil. HNO3

22. Alternatives to costly methods
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Use of Silver Nitrate
Halogen present
p-Chlorobenzoic acid
 Used in Lassaigne’s test for detecting presence
of any halide anion/halogen in the sample
Halogen absent
Benzoic acid
Loop prepared using
copper wire

23. Discussion (example of Benzoic acid)
The solid, white, odourless compound, melted at 119-20 °C (thermometer no. 07, calibration + 1 °
C), did not contain nitrogen, sulphur or any halogen (Lassaigne’s test) dissolved in aq. NaOH (5 %).
It decomposed sodium bicarbonate in a saturated aqueous solution to evolve carbon dioxide with
effervescence, confirming the presence of a carboxyl group. The compound was converted to an
amide derivative which melted at 127-28 °C (thermometer no. 07, calibration + 1 ° C).
The foregoing description proves the given organic compound is benzoic acid (lit. m. p. 121 ° C); its
amide derivative, benzamide, was prepared (lit. m. p. 129 ° C), both having following structures.
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
O OH O NH2
Benzoic acid Derivative: Benzamide

24. Applications (apart from synthetic chemistry)
• Phytochemistry
• Formulation
• Biopharmaceutical
• Forensic analysis
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series

25. Acknowledgements
Mr. S. P. Wate
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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series
Dr. P. B. Khedekar
Dr. V. V. Parashar
Mrs. A. J. Asnani
Late (Miss) V. R. Rabra
Dr. A. A. Joharapurkar
Late Dr. R. A. Fursule
Prof. S. R. Tambe
Dr. C. D. Upasani
Mr. Pawan Porwal
Mr. Santosh S. Chhajed
All Students of M. Pharm.
(Pharm. Chem.)
DELEGATES

26. Feel Free to Contact any Hour
9922546062
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mail: vchatpalliwar@yahoo.co.in