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.