Phenol extraction is a processing technology used to prepare
phenols
In organic chemistry, phenols, sometimes called phenolics, are a class of chemical compounds consisting of one or more hydroxyl groups (— O H) bonded directly to an aromatic hydrocarbon group. The simplest is phenol, . Phenolic compounds are ...
as raw materials, compounds, or additives for industrial
wood processing
Wood processing is an engineering discipline in the wood industry comprising the production of forest products, such as pulp and paper, construction materials, and tall oil. Paper engineering is a subfield of wood processing.
The major wood pro ...
and for chemical industries.
Phenol
Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it ...
extraction is also a laboratory process to purify
DNA and RNA contained biological samples.
Process
A
mixture of TE (or
Tris
Tris, or tris(hydroxymethyl)aminomethane, or known during medical use as tromethamine or THAM, is an organic compound with the formula (HOCH2)3CNH2, one of the twenty Good's buffers. It is extensively used in biochemistry and molecular biology as ...
-
Ethylenediaminetetraacetic acid
Ethylenediaminetetraacetic acid (EDTA) is an aminopolycarboxylic acid with the formula H2N(CH2CO2H)2sub>2. This white, water-soluble solid is widely used to bind to iron (Fe2+/Fe3+) and calcium ions (Ca2+), forming water-soluble complexes ev ...
) and
phenol
Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it ...
is combined with an equal volume of an aqueous
DNA and RNA sample. After
agitation and
centrifugal separation, the aqueous layer is extracted and further processed with
ether
In organic chemistry, ethers are a class of compounds that contain an ether group—an oxygen atom connected to two alkyl or aryl groups. They have the general formula , where R and R′ represent the alkyl or aryl groups. Ethers can again be ...
and the DNA is concentrated by
ethanol precipitation Ethanol precipitation is a method used to purify and/or concentrate RNA, DNA, and polysaccharides such as pectin and xyloglucan from aqueous solutions by adding ethanol as an antisolvent.
DNA precipitation Theory
DNA is polar due to its ...
.
The phenol extraction technique is often used to purify samples of
nucleic acids
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main cl ...
taken from cells. To obtain nucleic acid samples, the cell must be lysed and the nucleic acids separated from all other cell materials. Phenol is a useful compound for breaking down superfluous cell materials that would otherwise contaminate the nucleic acid sample.
Phenol extraction of nucleic acids works as a result of its
non-polar
In chemistry, polarity is a separation of electric charge leading to a molecule or its chemical groups having an electric dipole moment, with a negatively charged end and a positively charged end.
Polar molecules must contain one or more polar ...
nature and its higher density than water (1.07 g/cm
3 compared to water’s 1.00 g/cm
3). This means in a water-phenol solution,
denatured proteins and other cell components will be dissolved in the phenol, leaving nucleic acids dissolved in the water. The solution can then be centrifuged to separate the phenol from the water into corresponding organic and aqueous phases. The nucleic acid containing aqueous phase can then be extracted.
Phenol is often used in
combination with chloroform.
The purpose of adding chloroform along with phenol is to ensure a clear separation between the aqueous and organic phases. Chloroform and phenol are miscible, unlike phenol and water. The density of chloroform is 1.47 g/cm
3, higher than that of water and phenol. Mixing chloroform and phenol creates a denser solution than phenol alone, therefore the separation of the organic from the aqueous phase is even clearer than if only phenol was added to a cell sample. There is less cross-contamination from the organic phase in the aqueous phase. This is useful when the aqueous phase is removed from the solution to obtain a pure nucleic acid sample.
For phenol to be effective, the pH of the solution must vary according to what is being extracted. In the case of DNA purification, a pH of 7.0–8.0 is used. If an experiment aims to obtain samples of purified
RNA, a pH of around 4.5 is used. Due to the negative charge on the backbone of DNA from phosphates, decreasing the pH of a solution will lead to neutralization. A pH of 4.5 has a higher concentration of H+ ions that would neutralize the negative phosphate charges and cause DNA to dissolve in the organic phase, while RNA has an additional
hydroxyl group in pentose sugar which allows the RNA to remain in the water phase.
See also
*
Phenol-chloroform extraction
References
{{Reflist
External links
Physiology.med.cornell.edu
Molecular biology