separation techniques

Separation TechniquesDr. V. A. Chatpalliwar

Prof., Pharm. Chem.Head, Dept. Pharm. Chem.S. S. D. J. Coll. Pharm.Neminagar, ChandwadDist.: Nashik

Theory and Practice of Organic chemistry

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Acknowledgements

Sinhgad College of PharmacyVadgaon CampusPune

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Dr. V. A. ChatpalliwarDept. Pharm. Chem. Lecture Series

Management & Staff

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Separation TechniquesUsed for separating• a binary mixture of organic compounds• a racemic mixture of chiral compounds

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Chromatographic Techniques

TLCPaperHPTLCHPLCGC

Capillary Electrophoresis Super-critical Fluid Chromatography UPLCPreparative HPLC

Chiral Separation

OPEC

SSE

SLE

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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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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

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Salient points • Separation should be qualitative as well as quantitative;

if it is not achieved problems of contamination arise.

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• 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

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Scheme (systematic)Preliminary Examination of the mixture

States

Odour

Colour

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Polar components: solids

Pungent: AcidicFishy: BasicSweet/Agreeable/Aromatic: Neutral

Groups with free lone-pair of electrons/

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Element Detection Test (mixture)Lassaigne’s

Test

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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

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Separation

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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

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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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Abolish the possibility of contamination in separated components

This dissociation helps in proper interaction between component molecules and extraction solvent

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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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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

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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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Sample mixed with solvent

Clear solution

Partial solubility

Quantity of solvent be increased

Separation achievedFilter to obtain two separate components

Proceed for extraction

Washing the residue with the solvent assures proper separation

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Extraction (liquid-liquid)• dil. HCl/conc. HCl

• dil. NaOH/conc. NaOH

• dil. H2SO4/conc. H2SO4

• dil. NaHCO3

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Observation/inference of Lassaigne’s test

Nitrogen present

Amides

AminesHydrazines, Azo-

Nitro compounds

Sulphonamides

Nitrogen absent

aq. HCl (5%)

Carboxylic acid

Phenol

Carbonyl compounds

Alcohols

Hydrocarbons

Acyl halides

aq. NaOH (5%)

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Extraction (procedure)

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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

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Care to be exercisedMixing (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)


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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


Functional group identification and Derivative preparation

Result and Discussion

Neutral/acidic/basic Compound

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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


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Preparation of Reagentsaq. 2, 4-Dinitrophenylhydrazine for water-insoluble carbonyl compounds

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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

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Preparation of Samples (examples)

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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


Thiourea


Aniline

Aniline and Urea

Anthranilic acid and benzamide

Combinations in red font are not be prepared as samples

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Preparation of Samples

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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

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Casual Approach be Avoided

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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

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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


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

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Alternatives to costly methods

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Use of Silver Nitrate

Halogen presentp-Chlorobenzoic acid

Used in Lassaigne’s test for detecting presence of any halide anion/halogen in the sample

Halogen absentBenzoic acid

Loop prepared using copper wire

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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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O OH O NH2

Benzoic acid Derivative: Benzamide

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Applications (apart from synthetic chemistry)• Phytochemistry

• Formulation

• Biopharmaceutical

• Forensic analysis

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AcknowledgementsMr. S. P. Wate

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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.)

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