Reverse Micelles Introduction
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Transcript of Reverse Micelles Introduction
PROTEIN EXTRACTION USING
Reverse Micelles (RMs)
Liquid-liquid extraction is a useful method to separate
components (compounds) of a mixture.
The success of this method depends upon the
difference in solubility of a compound in various solvents
Surfactant form a kind of aggregate in
non-polar organic solvent.
◦ When the concentration of surfactant exceed the
critical micellar concentration, surfactant can form a
kind of aggregate in water solution.
◦ micelles and reversed micelles
Reversed Micelles Extraction
Aqueous
phase
Air liquid
interface
Surfactants arranged at interface
Hydrophobic group
Hydrophilic group
(Water hating)
(Water loving)
Amphiphilic molecule
CH3 S
O
O
O
Na
Molecular Formula = C12
H25
Na O3 S
S
O
OO
Na
O O
O
CH3
CH3
O CH3
CH3
Molecular Formula = C32
H62
Na2 O
10 S
2
Sodium dodecyl sulfate (SDS)
Sodium bis (2-ethylhexyl) sulphosuccinate (AOT)
N+
CH3
Molecular Formula = C17
H30
N
CH3 OO
H
Molecular Formula = C12
H26
O2
Cetyl pyridinium bromide
Polyoxyethylenes
Brij-30
Classification of surfactants
Classified by the hydrophilic group:
Anionic
• Carboxylic
• Sulfuric esters
•Alkane sulfonic acids
•Alkyl aromatic sulfonic acids
• Others: Phosphates, phosphoric acids, …
Cationic
• Amine salts (primary, secondary, and tertiary)
• Quaternary salts
Nonionic
• Ethers
• Esters
• Amides
• Amphoteric
• Class of aggregates formed from amphiphilic
molecules (contains both polar and apolar group)
Surface
tension
Conc(surfactant)
Critical micelle concentration
(CMC)
• Micelles formed only above
the CMC
• Reduction in surface
tension takes place
• No change in surface
tension after CMC
P < 0.33 Spherical/ellipsoidal P = 0.33-0.5 Cylindrical/rodlike
P = 0.5-1.0 bilayers/vesiclesP > 1 Reverse/Inverted micelles
claP
• = Surfactant molecular volume • a = Area per polar head group of surfactant
• lc = length of the hydrocarbon chain
Oil phase
Aqueous
core
• nanometer scale (3-5nm)droplet of
aqueous solution stabilized in an apolar
environment by the presence of
surfactant layer at the interface.
• Aggregates form in such a way that the surfactant polar heads
orient inwards encapsulating an aqueous micro-domain
• Reverse micelles are also called w/o micro-emulsions.
Aqueous phase
Organic Phase
Empty micelle Filled micelle
Protein
Mechanism of protein solubilization in reverse micelles
• Surfactants reduce surface
tension when adsorbed at the
interface at low concentration
• Surfactant molecules self-
associate to form aggregates
• The concentration above
which the aggregates are formed
is called critical micelle
concentration (CMC)
Water Oil
Forward Extraction
Backward Extraction
• Reverse micelles should be capable of
selective solubilization of proteins, called
forward extraction
• Also it should be able to release the
purified protein back into the organic
phase, called backward extraction.
• Forward and backward extraction of
proteins is governed by several
parameters.
• Aqueous phase
parameters
• Organic phase
parameters• Protein
characteristics
• Others
• pH
• Ionic strength
• Type of salt
• Type of surfactant/solvent
• Presence of co-surfactant
• pI
• Size and Shape
• Hydrophobicity/charge
distribution
• Temperature
• Those which can form a reverse micelle (AOT, CTAB, TOMAC etc)
Anionic/Cationic ?
pH < pI pH > pI
Protein positively
charged
Protein negatively
charged
Electrostatic interaction mainly responsible for protein extraction
• Increasing conc.
favours FE
• At higher conc.
precipitation of
proteins may also
occur.
• Increasing conc.
does not favor BE
• Optimum concentration need to be
found out.
Surfactant
Concentration
Forward
Extraction
(FE)
Backward
Extraction
(BE)
Effect of Salt
(Ionic strength)
Forward
Extraction
(FE)
Backward
Extraction
(BE)
• Lower salt
concentration
favorable (less
debye screening
effect)
• Higher salt
concentration
favorable.
• Optimum concentration need to be
found out.
Reverse micellar extraction of Hen Egg Lysozyme
Refer to this paper
Work in groups 2 people per group
Discuss the following:
1. What was the objective of this experiment ?
2. What were the different parameters studied?
3. Outline the extraction procedure
4. What were the optimum results obtained and
critically evaluate the results.