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

Reversed Micelles Extraction 

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

Hydrophilic group

(Water loving)

Hydrophobic group

Air liquid interface

(Water hating)

Amphiphilic molecule

Aqueous phase

Surfactants arranged at interface

O O H3C

S

Na O

Molecular Formula = C12 H25 Na O3 S

Sodium dodecyl sulfate (SDS) H3C

Na

O

O

CH3

O O S O O

O

CH3

H3C

Molecular Formula = C32 H62 Na2 O10 S2

Sodium bis (2-ethylhexyl) sulphosuccinate (AOT)

+

N

CH3

Molecular Formula = C17 H30 N

Cetyl pyridinium bromide O H3C

O

H Molecular Formula = C12 H26 O2

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

Critical micelle concentration (CMC)

Conc(surfactant)

• Reduction in surface tension takes place

• No change in surface tension after CMC • Micelles formed only above the CMC

P < 0.33 Spherical/ellipsoidal

P = 0.33-0.5 Cylindrical/rodlike

P  alc •  = Surfactant molecular volume

• a = Area per polar head group of surfactant

• lc = length of the hydrocarbon chain

P = 0.5-1.0 bilayers/vesicles

P > 1 Reverse/Inverted micelles

• nanometer scale (3-5nm)droplet of aqueous solution stabilized in an apolar environment by the presence of surfactant layer at the interface. Aqueous core Oil phase

• 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.

Empty micelle

Filled micelle

• Surfactants reduce surface tension when adsorbed at the interface at low concentration

Organic Phase

• Surfactant molecules selfassociate to form aggregates

Protein

Aqueous phase

Mechanism of protein solubilization in reverse micelles

• The concentration above which the aggregates are formed is called critical micelle concentration (CMC)

• Reverse micelles should be capable of selective solubilization of proteins, called forward extraction

Water

Oil

• 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. Forward Extraction Backward Extraction

• Aqueous phase parameters • pH • Ionic strength

• Type of salt

• Organic phase parameters

• Protein characteristics

• Type of surfactant/solvent

• pI

• Presence of co-surfactant

• Size and Shape

• Others • Temperature

• Hydrophobicity/charge distribution

• Those which can form a reverse micelle (AOT, CTAB, TOMAC etc)

Anionic/Cationic ? pH < pI Protein positively charged

pH > pI Protein negatively charged

Electrostatic interaction mainly responsible for protein extraction

Surfactant Concentration

Forward Extraction (FE)

• Increasing favours FE

Backward Extraction (BE)

conc.

• Increasing conc. does not favor BE

• At higher conc. precipitation of proteins may also occur.

• Optimum concentration need to be found out.

Effect of Salt (Ionic strength)

Forward Extraction (FE)

• Lower salt concentration favorable (less debye screening effect)

Backward Extraction (BE)

• 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. 2. 3. 4.

What was the objective of this experiment ? What were the different parameters studied? Outline the extraction procedure What were the optimum results obtained and critically evaluate the results.