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Salting out (also known as salt-induced precipitation, salt fractionation, anti-solvent crystallization, precipitation crystallization, or drowning out) is a purification technique that utilizes the reduced solubility of certain molecules in a solution of very high
ionic strength The ionic strength of a solution Solution may refer to: * Solution (chemistry) Image:SaltInWaterSolutionLiquid.jpg, upMaking a saline water solution by dissolving Salt, table salt (sodium chloride, NaCl) in water. The salt is the solute and the ...
. Salting out is typically used to precipitate large biomolecules, such as proteins or DNA. Because the
salt Salt is a mineral In geology and mineralogy, a mineral or mineral species is, broadly speaking, a solid chemical compound with a fairly well-defined chemical composition and a specific crystal structure that occurs naturally in pure fo ...

salt
concentration needed for a given protein to
precipitate Precipitation is the process of conversion of a chemical substance A chemical substance is a form of matter In classical physics and general chemistry, matter is any substance that has mass and takes up space by having volume. All ever ...
out of the
solution Solution may refer to: * Solution (chemistry) Image:SaltInWaterSolutionLiquid.jpg, upMaking a saline water solution by dissolving Salt, table salt (sodium chloride, NaCl) in water. The salt is the solute and the water the solvent. In chemistry ...
differs from protein to protein, a specific salt concentration can be used to precipitate a target protein. This process is also used to concentrate dilute solutions of proteins.
Dialysis In medicine, dialysis (from Greek Greek may refer to: Greece Anything of, from, or related to Greece Greece ( el, Ελλάδα, , ), officially the Hellenic Republic, is a country located in Southeast Europe. Its population is approximate ...
can be used to remove the salt if needed.


Principle

Salt compounds dissociate in aqueous solutions. This property is exploited in the process of salting out. When the salt concentration is increased,
some of the water molecules are attracted by the salt ions
some of the water molecules are attracted by the salt ions
, which decreases the number of water molecules available to interact with the charged part of the protein. There are
hydrophobic In chemistry Chemistry is the scientific Science () is a systematic enterprise that builds and organizes knowledge Knowledge is a familiarity or awareness, of someone or something, such as facts A fact is an occurrence ...
amino acid Amino acids are organic compound In , organic compounds are generally any s that contain - . Due to carbon's ability to (form chains with other carbon s), millions of organic compounds are known. The study of the properties, reactions, a ...

amino acid
s and
hydrophilic A hydrophile is a molecule File:Pentacene on Ni(111) STM.jpg, A scanning tunneling microscopy image of pentacene molecules, which consist of linear chains of five carbon rings. A molecule is an electrically neutral group of two or more atoms ...
amino acid Amino acids are organic compound In , organic compounds are generally any s that contain - . Due to carbon's ability to (form chains with other carbon s), millions of organic compounds are known. The study of the properties, reactions, a ...

amino acid
s in
protein Proteins are large biomolecule , showing alpha helices, represented by ribbons. This poten was the first to have its suckture solved by X-ray crystallography by Max Perutz and Sir John Cowdery Kendrew in 1958, for which they received a No ...

protein
molecules. After protein folding in aqueous solution, hydrophobic amino acids usually form protected hydrophobic areas while hydrophilic amino acids interact with the molecules of
solvation Solvation (or dissolution) describes the interaction of solvent A solvent (from the Latin language, Latin ''wikt:solvo#Latin, solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a solution. A solvent is usual ...

solvation
and allow proteins to form hydrogen bonds with the surrounding water molecules. If enough of the protein surface is hydrophilic, the protein can be dissolved in water. When salt is added to the solution, there is more frequent interaction between solvent molecules and salt ions. As a result, the protein and salt ions compete to interact with the solvent molecules with the result that there are fewer solvent molecules available for interaction with the protein molecules than before. The protein–protein interactions thus become stronger than the solvent–solute interactions and the protein molecules associate by forming
hydrophobic interactions thumbnail, 250px, A droplet of water forms a spherical shape, minimizing contact with the hydrophobic leaf. The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in an aqueous solution and exclude water#Chemical and p ...
with each other. After dissociation in a given solvent, the negatively charged atoms from a chosen salt begin to compete for interactions with positively charged molecules present in the solution. Similarly, the positively charged cations compete for interactions with the negatively charged molecules of the solvent. This process is known as salting out. Soaps are easily precipitated by concentrated salt solution, the metal ion in the salt reacts with the fatty acids forming back the soap and
glycerol Glycerol (; also called glycerine in British English and glycerin in American English) is a simple polyol compound. It is a colorless, odorless, viscous liquid that is sweet-tasting and non-toxic. The glycerol backbone is found in lipids known ...
.


Application

As different proteins have different compositions of amino acids, different protein molecules precipitate at different concentrations of salt solution. Unwanted proteins can be removed from a protein solution mixture by salting out as long as the solubility of the protein in various concentrations of salt solution is known. After removing the precipitate by
filtration Filtration is a physical separation process A separation process is a method that converts a mixture or solution of chemical substances into two or more distinct product mixtures. At least one of results of the separation is enriched in one or m ...
or
centrifugation Centrifugation is a mechanical process which involves the use of the centrifugal force In Newtonian mechanics, the centrifugal force is an inertial force (also called a "fictitious" or "pseudo" force) that appears to act on all objects when v ...
, the desired protein can be precipitated by altering the salt concentration to the level at which the desired protein becomes insoluble. One demerit of salting out in purification of proteins is that, in addition to precipitating a specific protein of interest, contaminants are also precipitated as well. Thus to obtain a purer protein of interest, additional purification methods such as ion exchange chromatography may be required. Excess ethanol in vitro post consumption is frequently combined with high salt, high fat foods culminating, in the failed effort to end drunkenness colloquially called salting out. Which can typically only be reduced with time, the human liver can process 1 standard (14g pure ethanol) drink per hour.


See also

*
Ionic strength The ionic strength of a solution Solution may refer to: * Solution (chemistry) Image:SaltInWaterSolutionLiquid.jpg, upMaking a saline water solution by dissolving Salt, table salt (sodium chloride, NaCl) in water. The salt is the solute and the ...
* Protein precipitation *
Salting in Salting in refers to the effect where increasing the ionic strength The ionic strength of a solution is a measure of the concentration of ions in that solution. Ionic Chemical compound, compounds, when solubility, dissolved in water, Dissociation (c ...
*
Ammonium sulfate precipitationAmmonium sulfate precipitation is one of the most commonly used methods for large and laboratory scale protein purification and fractionation that can be used to separate proteins by altering their solubility in the presence of a high salt concentrat ...
*
Hofmeister series The Hofmeister series or lyotropic series is a classification of ions in order of their ability to salt out or salt in proteins. The effects of these changes were first worked out by Franz Hofmeister, who studied the effects of cations and anion ...


References

*Physical biochemistry, David Sheehan
Wiley Blackwell(2009)
p 285 * *C. Beldie Activity coefficients in ternary systems : non-electrolyte – water – electrolyte, Revue Roum. Chim. 15, 221–228, 1970


External links


Make The Most of Antisolvent Crystallization

Salting out
on UC Davis ChemWiki {{DEFAULTSORT:Salting Out Laboratory techniques Separation processes