REVERSE MICELLES AND PROTEIN PURIFICATION

M.V.N. UMA MAHESH

Surfactants , Micelles and Reverse Micelles

Surface active agents , commonly designated as surfactants , are amphiphilic molecules containing nonpolar and polar parts capable of interacting with surfaces. An example is given by the anionic surfactant AOT{bis(2-ethylhexyl)sulfosuccinate .

The term micelle is used to describe the aggregates of amphiphilic molecules in aqueous solutions and refers to soluble aggregate, spontaneously and reversibly formed from amphiphiles or ions. The surfactant molecules' selfaggregation occurs only above the critical micelle concentration (CMC) which in practical sense means that the surfactant has to be added of amount exceeding its solubility

Reversed micelle solvents represent nanometer-sized aqueous droplets stabilized by surfactants inside the bulk organic solvents. The structure of a reverse micelle consists of a an aqueous micro-domain facing the polar heads of the surfactant that surrounds this core interacting with the bulk organic solvent (non-polar) through hydrophobic chains

Understanding Reverse Micelles

Formation of reverse micelles. Reverse micelle shape and size. Reverse micelle interface. Organic solvent, co-solvent, cosurfactant.

Formation of reverse micelles.

A ternary system containing two immiscible liquids (ex. water and hydrocarbon)and an amphiphile form different types of micro emulsion systems out of which stable water-in-oil micro emulsion represents the reversed miceller structure. Its structure involves a nanometer-sized aqueous droplet embraced by the hydrophilic heads of the surfactant molecules and stabilized inside a bulk organic solvent through interactions with the surfactant chains.

Reverse micelle shape and size. At constant temperature, the aqueous droplet size is a
function only of the parameter W0 (=mol[water] / mol[surfactant]), a measure of the hydration degree.

The radius of the aqueous core of the reversed micelles is given considering simple spherical geometry and assuming that all the surfactant is aggregated in a micelle form (Regalado et al., 1994):

where VAQ is the volume of a water molecule (approximately 0.03 nm3) and as is the surface area of the surfactant head group (0.5 nm2 in case of AOT).

Reverse micelle interface.

Flexibility, dynamic character of the interface is favourable to enzyme solubilization and to enzyme reactivity in the RMS.
The interfacial deformation caused by the electrostatic interaction between the protein and the bulk interface facilitates the protein transfer to the reversed micelle solvent. The key to solubilization of the hydrophobic proteins is the interfacial complex with the surfactant molecules

Organic solvent, co-solvent, co-surfactant.

Few surfactants are able to establish stable water-in-oil micro emulsions without the so called co-surfactant, another surface active additive. Surfactants, that do not need a co-surfactant, include anionic AOT. The organic solvent used influences the aggregation behavior, the reversed micelle size and shape as well as the interdroplet interactions.

The efficient reversed micelle extraction cycle consists of two successive mass transport processes
Forward Extraction: Solute solubilization into the reversed micelles characterized by high extent and selectivity . Back Extraction: Subsequent solute stripping of sufficient amount and quality.

Forward Extraction( Solubilization )

Reverse Miceller Extraction of Proteins

Factors affecting Forward Extraction

Surfactant and Protein effects -Surfactant concentration -Protein Size Electrostatic interactions -pH effect -Ionic Strength effect Hydrophobic Interactions

Back Extraction( Desolubilization )

Back Extraction( Desolubilization )

Electrostatic interactions -pH effect -Ionic Strength effect Other effects -Alcohol -Temperature -Counter Ionic Detergent

Model for solubilization in Reverse micelles

Where r is the volumetric ratio of reverse micellar phase to aqueous phase Vrm/Vaq. KPRM and KRM are equilibrium constants [S] is surfactant concentration m is aggregation number n is number of rvms needed to form rvms-protein complex

A slightly different equation based on the mass-action principle was derived by Woll and Hatton which indicated a pH dependency.

Thank You