Aqueous two phase system
Encyclopedia
Aqueous biphasic systems (ABS) or aqueous two phase systems (ATPS) are clean alternatives for traditional organic
-water
solvent
extraction
systems.
ABS are formed when 2 polymer
s, one polymer and one kosmotropic
salt
, or two salts (one chaotropic
salt
and the other a kosmotropic salt) are mixed together at appropriate concentrations or at a particular temperature. The two phases are mostly composed of water and non volatile components, thus eliminating volatile organic compounds. They have been used for many years in biotechnological
applications as non-denaturing
and benign separation media. Recently, they have been used for metal ion separations, environmental remediation, metallurgical applications and as a reaction media.
, a water-soluble polymer, with soluble starch
or gelatine. Upon mixing, they separated into two immiscible phases
.
Subsequent investigation has led to the determination of many other aqueous biphasic systems, of which the polyethylene glycol
(PEG) - dextran
system is the most extensively studied. Other systems that form aqueous biphases are: PEG - sodium carbonate
or PEG and phosphates, citrates or sulfates. Aqueous biphasic systems are used during downstream processing
mainly in biotechnological and chemical industries.
and water
are poured into the same container, they separate into two phases or layers, because they are immiscible. In general, aqueous (or water-based) solutions, being polar, are immiscible with non-polar organic solvents (chloroform
, toluene
, hexane
etc) and form a two-phase system. However, in an ABS, both immiscible components are water-based.
The formation of the distinct phases is affected by the pH
, temperature
and ionic strength
of the two components, and separation occurs when the amount of a polymer present exceeds a certain limiting concentration (which is determined by the above factors).
than the "lower phase," consisting of the more hydrophilic and denser dextran solution.
Although PEG is inherently denser than water, it occupies the upper layer. This is believed to be due to its solvent 'ordering' properties, which excludes excess water, creating a low density water environment.http://www.lsbu.ac.uk/water/biphasic.html The degree of polymerisation of PEG also affects the phase separation and the partitioning of molecules during extraction.
Specificity may be further increased by tagging ligands specific to the desired enzyme, onto the polymer. This results in a preferential binding of the enzyme to the polymer, increasing the effectiveness of the extraction.
One major disadvantage, however, is the cost of materials involved, namely high-purity dextrans employed for the purpose. However, other low-cost alternatives such as less refined dextran
s, hydroxypropyl starch derivatives
and high-salt solutions are also available.
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
-water
Water
Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...
solvent
Solvent
A solvent is a liquid, solid, or gas that dissolves another solid, liquid, or gaseous solute, resulting in a solution that is soluble in a certain volume of solvent at a specified temperature...
extraction
Liquid-liquid extraction
Liquid–liquid extraction, also known as solvent extraction and partitioning, is a method to separate compounds based on their relative solubilities in two different immiscible liquids, usually water and an organic solvent. It is an extraction of a substance from one liquid phase into another liquid...
systems.
ABS are formed when 2 polymer
Polymer
A polymer is a large molecule composed of repeating structural units. These subunits are typically connected by covalent chemical bonds...
s, one polymer and one kosmotropic
Kosmotropic
Solutes are defined as kosmotropic if they contribute to the stability and structure of water-water interactions. Kosmotropes cause water molecules to favorably interact, which also stabilizes intermolecular interactions in macromolecules such as proteins....
salt
Salt
In chemistry, salts are ionic compounds that result from the neutralization reaction of an acid and a base. They are composed of cations and anions so that the product is electrically neutral...
, or two salts (one chaotropic
Chaotropic agent
A denaturating agent is a substance which disrupts the three dimensional structure in macromolecules such as proteins, DNA, or RNA and denatures them. A denaturating agent is a chaotropic agent, but chaotropic agents aren't necessarily denaturating agents...
salt
Salt
In chemistry, salts are ionic compounds that result from the neutralization reaction of an acid and a base. They are composed of cations and anions so that the product is electrically neutral...
and the other a kosmotropic salt) are mixed together at appropriate concentrations or at a particular temperature. The two phases are mostly composed of water and non volatile components, thus eliminating volatile organic compounds. They have been used for many years in biotechnological
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
applications as non-denaturing
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
and benign separation media. Recently, they have been used for metal ion separations, environmental remediation, metallurgical applications and as a reaction media.
Introduction
In 1896, Beijerinck first noted an 'incompatibility' in solutions of agarAgar
Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...
, a water-soluble polymer, with soluble starch
Starch
Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store...
or gelatine. Upon mixing, they separated into two immiscible phases
Phase (matter)
In the physical sciences, a phase is a region of space , throughout which all physical properties of a material are essentially uniform. Examples of physical properties include density, index of refraction, and chemical composition...
.
Subsequent investigation has led to the determination of many other aqueous biphasic systems, of which the polyethylene glycol
Polyethylene glycol
Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. It has also been known as polyethylene oxide or polyoxyethylene , depending on its molecular weight, and under the tradename Carbowax.-Available forms:PEG, PEO, or POE refers to an...
(PEG) - dextran
Dextran
Dextran is a complex, branched glucan composed of chains of varying lengths...
system is the most extensively studied. Other systems that form aqueous biphases are: PEG - sodium carbonate
Sodium carbonate
Sodium carbonate , Na2CO3 is a sodium salt of carbonic acid. It most commonly occurs as a crystalline heptahydrate, which readily effloresces to form a white powder, the monohydrate. Sodium carbonate is domestically well-known for its everyday use as a water softener. It can be extracted from the...
or PEG and phosphates, citrates or sulfates. Aqueous biphasic systems are used during downstream processing
Downstream processing
Downstream processing refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste. It is an...
mainly in biotechnological and chemical industries.
The two phases
It is a common observation that when oilOil
An oil is any substance that is liquid at ambient temperatures and does not mix with water but may mix with other oils and organic solvents. This general definition includes vegetable oils, volatile essential oils, petrochemical oils, and synthetic oils....
and water
Water
Water is a chemical substance with the chemical formula H2O. A water molecule contains one oxygen and two hydrogen atoms connected by covalent bonds. Water is a liquid at ambient conditions, but it often co-exists on Earth with its solid state, ice, and gaseous state . Water also exists in a...
are poured into the same container, they separate into two phases or layers, because they are immiscible. In general, aqueous (or water-based) solutions, being polar, are immiscible with non-polar organic solvents (chloroform
Chloroform
Chloroform is an organic compound with formula CHCl3. It is one of the four chloromethanes. The colorless, sweet-smelling, dense liquid is a trihalomethane, and is considered somewhat hazardous...
, toluene
Toluene
Toluene, formerly known as toluol, is a clear, water-insoluble liquid with the typical smell of paint thinners. It is a mono-substituted benzene derivative, i.e., one in which a single hydrogen atom from the benzene molecule has been replaced by a univalent group, in this case CH3.It is an aromatic...
, hexane
Hexane
Hexane is a hydrocarbon with the chemical formula C6H14; that is, an alkane with six carbon atoms.The term may refer to any of four other structural isomers with that formula, or to a mixture of them. In the IUPAC nomenclature, however, hexane is the unbranched isomer ; the other four structures...
etc) and form a two-phase system. However, in an ABS, both immiscible components are water-based.
The formation of the distinct phases is affected by the pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
, temperature
Temperature
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...
and ionic strength
Ionic strength
The ionic strength of a solution is a measure of the concentration of ions in that solution. Ionic compounds, when dissolved in water, dissociate into ions. The total electrolyte concentration in solution will affect important properties such as the dissociation or the solubility of different salts...
of the two components, and separation occurs when the amount of a polymer present exceeds a certain limiting concentration (which is determined by the above factors).
PEG–dextran system
The "upper phase" is formed by the more hydrophobic polyethylene glycol (PEG), which is of lower densityDensity
The mass density or density of a material is defined as its mass per unit volume. The symbol most often used for density is ρ . In some cases , density is also defined as its weight per unit volume; although, this quantity is more properly called specific weight...
than the "lower phase," consisting of the more hydrophilic and denser dextran solution.
Although PEG is inherently denser than water, it occupies the upper layer. This is believed to be due to its solvent 'ordering' properties, which excludes excess water, creating a low density water environment.http://www.lsbu.ac.uk/water/biphasic.html The degree of polymerisation of PEG also affects the phase separation and the partitioning of molecules during extraction.
Advantages
ABS is an excellent method to employ for the extraction of proteins/enzymes and other labile biomolecules from crude cell extracts or other mixtures. Most often, this technique is employed in enzyme technology during industrial or laboratory production of enzymes.- They provide mild conditions that do not harm or denatureDenaturation (biochemistry)Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
unstable/labile biomolecules - The interfacial stressStress (physics)In continuum mechanics, stress is a measure of the internal forces acting within a deformable body. Quantitatively, it is a measure of the average force per unit area of a surface within the body on which internal forces act. These internal forces are a reaction to external forces applied on the body...
(at the interfaceInterface (chemistry)An interface is a surface forming a common boundary among two different phases, such as an insoluble solid and a liquid, two immiscible liquids or a liquid and an insoluble gas. The importance of the interface depends on which type of system is being treated: the bigger the quotient area/volume,...
between the two layers) is far lower (400-fold less) than water-organic solvent systems used for solvent extraction, causing less damage to the molecule to be extracted - The polymer layer stabilizes the extracted protein molecules, favouring a higher concentration of the desired protein in one of the layers, resulting in an effective extraction
- Specialised systems may be developed (by varying factors such as temperature, degree of polymerisation, presence of certain ions etc ) to favour the enrichment of a specific compound, or class of compounds, into one of the two phases. They are sometimes used simultaneously with ion-exchange resins for better extraction
- Separation of the phases and the partitioningLiquid-liquid extractionLiquid–liquid extraction, also known as solvent extraction and partitioning, is a method to separate compounds based on their relative solubilities in two different immiscible liquids, usually water and an organic solvent. It is an extraction of a substance from one liquid phase into another liquid...
of the compounds occurs rapidly. This allows the extraction of the desired molecule before endogenousEndogenousEndogenous substances are those that originate from within an organism, tissue, or cell. Endogenous retroviruses are caused by ancient infections of germ cells in humans, mammals and other vertebrates...
proteases can degrade them. - These systems are amenable to scale-ups, from laboratory-sized setups to those that can handle the requirements of industrial production. They may be employed in continuous protein-extraction processes.
Specificity may be further increased by tagging ligands specific to the desired enzyme, onto the polymer. This results in a preferential binding of the enzyme to the polymer, increasing the effectiveness of the extraction.
One major disadvantage, however, is the cost of materials involved, namely high-purity dextrans employed for the purpose. However, other low-cost alternatives such as less refined dextran
Dextran
Dextran is a complex, branched glucan composed of chains of varying lengths...
s, hydroxypropyl starch derivatives
Modified starch
Modified starch, also called starch derivatives, are prepared by physically, enzymatically, or chemically treating native starch, thereby changing the properties of the starch. Modified starches are used in practically all starch applications, such as in food products as a thickening agent,...
and high-salt solutions are also available.