[<< wikibooks] Structural Biochemistry/Organic Chemistry/Methods of Separation and Isolation
== Liquid-Liquid Extraction ==

Extraction is a technique used for separating a compound from a mixture. An example is separating a water-insoluble organic compound from an aqueous mixture by extracting it into a water-insoluble organic solvent. These extractions are often part of the workup procedure for isolating and purifying the product of an organic reaction. Because trace amounts of water are often present at the end of an extraction process, a drying reagent is needed to ensure a dry product. The process of liquid-liquid extraction involves the distribution of a compound between two solvents that are insoluble in each other. By taking advantage of the differing solubilities of a solute in a pair of solvents, compounds can be selectively transported from one liquid phase to the other.
This occurrence is quantified by the partition coefficient (K):

             Concentration of solute in organic phase
             Concentration of solute in aqueousphase
    {\displaystyle {\text{ K}}={\frac {\text{ Concentration of solute in organic phase}}{\text{ Concentration of solute in aqueousphase}}}.}
  The larger the value of K, the solute will be found in greater amounts in the organic solvent.
In an extraction procedure, an aqueous phase, usually water, and a immiscible organic solvent known as the organic phase are generally shaken in a container. The solutes are then allowed to distribute themselves between the two layers according to their solubility, the denser layer is always on the bottom of the container. After they have distributed themselves, each layer can be removed and analyzed separately by drying the extractions. The factors that need to be considered in selecting a drying agent are its capacity for removing water, its efficiency, the speed with which it works, and its chemical inertness. Once the extraction has been dried, the solvent must be removed to recover the desired organic product. This can be achieved by heating the container of the mixture and allowing for the solvent to evaporate, leaving a solid or liquid residue or allowing the mixture in to evaporate in a fume hood, depending on the volatility of the solvent.

== Liquid-Liquid Extraction FlowChart ==
Here is an example of a simple liquid-liquid extraction problem.

You want to isolate a mixture of compounds, and you do this by taking advantage of the basicity and acidity of certain compounds. It is also important to note those structures that are rather neutral. Some key concepts are, bases will react with acid and vice versa. Also, the aqueous layer will contain a charged molecule, or ion, most of the time.
Step 1: Upon dissolving the mixture in a good solvent such as methylene chloride, a strong acid is added to the mixture. 
The strong acid is added so that it will react with any basic compounds in the mixture, which will lead it into the aqueous layer. Thus, one of the compounds has been separated. Adding base will deprotonate the proton from the nitrogen and give you the isolated product in the organic layer.
Step 2: To the remaining 3 compounds, a weak base is added, such as sodium carbonate. 
The weak base will deprotonate the most acidic of the hydrogens, which is in this case, the carboxylic acid. The carboxylic acid containing structure will be separated into the aqueous layer. Keep in mind, that the base is not limited to only deprotonating one structure, but this all just depends on the acidity of the compounds.
Step 3: To the final 2 compounds, a stronger base such as potassium hydroxide which will deprotonate the phenol. 
Generally phenols are not very acidic, that is why a strong base must be used. The cyano containing compound is left in the organic layer.
Reaction Complete. All 4 compounds have been isolated into either aqueous or organic, and as mentioned earlier, it is very easy to bring an aqueous compound back into the organic, that is by adding the complementary acid or base.

== Column Chromatography ==

Column chromatography is a preparative method for separating and isolating compounds from mixtures which can later be analyzed using thin-layer chromatography (TLC), gas chromatography, or IR. The method is used for obtaining compounds from natural sources or for purifying products from reaction mixtures. This method can be considered an upside-down version of TLC. Instead of having a thin layer of adsorbent attached to a solid support, a column is filled with a larger amount of adsorbent, often silica gel, and the mixture is loaded on top of it. While TLC depends on capillary action, column chromatography depends on a solvent or eluent moving down the column using the force of gravity. As this fluent moves down the column, it carries the soluble compounds with it and compounds having strong interactions with the adsorbent move more slowly than compounds having weaker interactions with the adsorbent. By taking advantage of the material used for the stationary and mobile phase, the compounds will separate in distinctive bands and each band will come out of the column individually. This will allow for the collection of each separate band in different vials which can then be analyzed with TLC and then dried to later be analyzed based on their melting points. 
Polarity plays a large role in the process, where the using a non-polar solvent allows for a non-polar substance to be collected first before the more polar components. As the polarity of the solvents increase, more polar compounds travel further faster. This is why it is important for the solvents used to slowly change in polarity throughout the column chromatography procedure for better separation.[Mohrig, Jerry R. "Techniques in Organic Chemistry." 2010, W.H. Freeman and Company]

== Distillation ==
One form of distillation is fractional distillation which is a simple technique for accomplishing a large number of distillation/separations, in a single continuous process. A fractional distillation column has an extensive surface area for exchange of heat between ascending vapor and descending liquid. As the condensate accepts heat from the vapor at any point in the column, the condensate is partially vaporized and the vapor is enriched in the more volatile component. At the same time, as the vapor loses heat to the condensate, part of the vapor condenses and the condensate becomes enriched in the less volatile component, returning back to the bottom of the flask. Eventually, the vapor that condenses at the top of the column is pure or enriched in the more pure, volatile component where as the condensate that has returned to the flask contains the pure and enriched less volatile component. The ability to separate these two liquids highly depends on the differences in the boiling points of the two liquids as well as other factors such as rate of distillation, insulation of the column, and the efficiency in the column. In order to achieve good separation, an even heating and low rate of distillation must be applied and maintained as much as possible, creating a high reflux ratio. The type of column also plays an important role as the process of packing the column and the material used all determine the efficiency and ability that a column has for separating the components of a liquid mixture. After the fractions have been collected, TLC can be used to determine the purity of each fraction. After spotting each fraction on a TLC plate and then developing them with a solvent, the presence of one spot in a lane is representative of a pure fraction whereas multiple spots indicate the presence of other compounds in the mixture. After isolating the pure fractions, they can then be evaporated and the pure substances analyzed.

== Solid phase Extraction ==
Solid phase extraction is a separation process by which compounds that are dissolved in a liquid mixture can be separated from other compounds by use of a stationary phase. This separation technique is different than the others in the sense that the compound is not lifted from a solution by varying polarity by means of a solvent, but instead, the adhesion to a stationary phase. 
The technique works on the basis of two phases, the mobile phase and the stationary phase. The mobile phase, or the “moving phase”, is the solvent that pushes the mixture of analysis through a stationary phase. Through means of intermolecular interactions, the various components in the mixture will rapidly bind back and forth between the mobile phase and stationary phase while the mobile phase travels up the stationary phase by capillary action. Depending on the type of compound and the interaction it has between the mobile and stationary phase, each compound will display different affinities between the respective phases and demonstrate a different adsorption pattern along the stationary phase. 
The stationary phase used most lab settings is amorphous silica gel which contains a high hydrophilic surface. Mobile phases used, examples being eythl acetate, petroleum ether and methyl tertiary butyl ether, possess different polar characteristics that allow for differential “climbing” patterns for varying compounds on a stationary phase. The combination of capillary action and surface tension allow for the mobile phase to travel up the stationary phase and provide for separation of mixtures based on whether the compound of interest would show interaction with the stationary phase or not. As the mobile phase “picks up” the mixture of compounds up the stationary phase, compounds that exhibit a stronger intermolecular interaction will spend more time binding to the stationary phase, traveling a shorter distance and vice versa. It is this differential interaction with the stationary phase that associates a Retardation factor with each compound. The retardation factor is defined as the ratio of the distance a compound travels to mobile phase front. Compounds with a lower retardation factor exhibit a stronger interaction with the stationary phase while compounds with a higher retardation factor exhibits a weaker interaction with the stationary phase.

== References ==
Mohrig, Jerry R. Techniques in Organic Chemistry. 2006, W.H. Freeman and Company
Organic Chemistry Lab, Chemistry 143A