Reverse Mi Celle

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    REVERSE MICELLES AND

    PROTEIN PURIFICATION

    M.V.N. UMAMAHESH

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    Surfactants , Micelles and

    Reverse Micelles

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    Surface active agents , commonlydesignated as surfactants , areamphiphilic molecules containing non-polar and polar parts capable of

    interacting with surfaces. An exampleis given by the anionic surfactantAOT{bis(2-ethylhexyl)sulfosuccinate .

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    The term micelle is used todescribe the aggregates ofamphiphilic molecules in aqueoussolutions and refers to solubleaggregate, spontaneously andreversibly formed from

    amphiphiles or ions.

    The surfactant molecules' self-aggregation occurs only abovethe critical micelle concentration(CMC) which in practical sense

    means that the surfactant has tobe added of amount exceeding itssolubility

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    Reversed micelle solventsrepresent nanometer-sizedaqueous droplets stabilized bysurfactants inside the bulkorganic solvents.

    The structure of a reversemicelle consists of a anaqueous micro-domain facingthe polar heads of thesurfactant that surrounds thiscore interacting with the bulkorganic solvent (non-polar)through hydrophobic chains

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    Understanding Reverse Micelles

    Formation of reverse micelles.

    Reverse micelle shape and size.

    Reverse micelle interface.

    Organic solvent, co-solvent, co-

    surfactant.

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    A ternary system containingtwo immiscible liquids (ex. waterand hydrocarbon)and anamphiphile form different typesof micro emulsion systems out ofwhich stable water-in-oil micro

    emulsion represents thereversed miceller structure.

    Its structure involves ananometer-sized aqueousdroplet embraced by the

    hydrophilic heads of thesurfactant molecules andstabilized inside a bulk organicsolvent through interactions withthe surfactant chains.

    Formation of reverse micelles.

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    At constant temperature, the aqueous droplet size is afunction 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 assumingthat 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 asis the surface area

    of the surfactant head group (0.5 nm2 in case ofAOT).

    Reverse micelle shape and

    size.

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    Flexibility, dynamic character of the interface isfavourable to enzyme solubilization and to enzymereactivity in the RMS.

    The interfacial deformation caused by the electrostaticinteraction between the protein and the bulk interfacefacilitates the protein transfer to the reversed micellesolvent.

    The key to solubilization of the hydrophobic proteins is theinterfacial complex with the surfactant molecules

    Reverse micelle interface.

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    Few surfactants are able to establish stable water-in-oilmicro emulsions without the so called co-surfactant,

    another surface active additive. Surfactants, that do notneed a co-surfactant, include anionic AOT.

    The organic solvent used influences the aggregationbehavior, the reversed micelle size and shape as wellas the interdroplet interactions.

    Organic solvent, co-solvent, co-surfactant.

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    The efficient reversed micelle

    extraction cycle consists of twosuccessive mass transport processes

    Forward Extraction:Solute solubilization into the reversedmicelles characterized by high extentand selectivity .

    Back Extraction:Subsequent solute stripping of sufficientamount and quality.

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    ForwardExtraction( Solubilization )

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    Reverse MicellerExtraction ofProteins

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    Factors affecting Forward

    ExtractionSurfactant and Protein effects-Surfactant concentration-Protein Size

    Electrostatic interactions-pH effect-Ionic Strength effect

    Hydrophobic Interactions

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    Back Extraction( Desolubilization )

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    Back Extraction( Desolubilization )

    Electrostatic interactions-pH effect-Ionic Strength effect

    Other effects-Alcohol-Temperature-Counter Ionic

    Detergent

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    Model for solubilization

    in Reverse micelles

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    Wherer is the volumetric ratio of reverse micellarphaseto aqueous phase Vrm/Vaq.KPRM and KRM are equilibrium constants

    [S] is surfactant concentrationm is aggregation numbern is number of rvms needed to form rvms-proteincomplex

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    A slightly different equation based on themass-action principle was derived by Wolland Hatton which indicated a pHdependency.

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    Thank You