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Organic Chemistry
 

Separation of Mixtures via Precipitation

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Precipitation is a technique used to separate a mixture based on the solubility of its components. The solubility of a compound depends on the ionic strength of the solution, its pH, and temperature. Manipulation of these factors can cause a compound to become an insoluble solid, and fall out of solution. This is called precipitation.

The insoluble solid, called the precipitate, initially forms a suspension, meaning that it is well dispersed in solution. The precipitate typically agglomerates, and then is separated from the liquid by sedimentation, centrifugation, or filtration. This video will introduce several methods of separating compounds using precipitation, and demonstrate a procedure in the laboratory.

A dissolved compound can be precipitated out of solution by introducing a counter ion. For example, silver can be precipitated out of solution in the reaction between silver nitrate and sodium chloride. The nitrate ion is replaced by a counter-ion, chloride, resulting in the formation of solid silver chloride.

Increasing the salt concentration of a solution can also induce precipitation. This technique, called salting-out, is common for the isolation of proteins. At high salt concentration, water molecules are more attracted to the dissolved salt, leaving fewer to stabilize the protein. As a result, the protein molecules aggregate and form a solid.

Precipitation can also be caused by a change in pH. At high and low pH, the protein is charged and attracted to the polar solution. At a certain point, the net charge of a compound becomes zero. This is the isoelectric point, or pI. The compound is unable to interact with the polar solution, causing it to aggregate and precipitate.

Temperature also affects solubility, as higher temperature increases the solubility of solids. By decreasing temperature, dissolved compounds can re-solidify. The rate of solid formation determines relative purity.

The following experiments will demonstrate the precipitation of the protein casein from milk using pH, and further separation via filtration and centrifugation methods.

To begin this procedure, add 250 mL of milk into a beaker with a stir bar. Gently warm the milk to 40 °C on a stirring hot plate. Immerse a pH meter into the warm milk, and monitor the pH. Add acetic acid drop-wise to the milk until the pH reaches the casein isoelectric point, 4.6. Insoluble milk proteins, or curds, precipitate out of solution at the isoelectric point. Remove the curds from solution by filtration. If the filter paper gets clogged, mix with a spatula to help the solution flow through. If this does not improve the filtration, change the filter paper. Transfer the wet solid from the clogged filter paper to new filter paper. This should absorb more liquid, or whey, from the solid. Continue changing the filter paper until there is minimal wetness. Pressing lightly on the solids may help the filter paper to absorb more whey.

Re-suspend the dried milk solids in 70% ethanol to wash the phospholipids out of the curds and then repeat the filtration process. As an alternative to filtration, protein solids can also be separated using centrifugation. Centrifuge 50 mL portions of the milk mixture and decant the supernatant. Re-suspend the pellet in 50 mL of 70% ethanol to help remove the phospholipids from the curds, and repeat the centrifugation process.

The milk protein solids can then be stored or re-suspended in another solution for further analysis, such as SDS-PAGE. For more information, see our video on this technique. SDS-PAGE analysis shows that precipitation enabled the removal of most impurities from the whey. All of the casein was found in the pellet, while none was found in the supernatant.

Precipitation is a commonly used technique, which can be applied to separate a variety of mixtures or solutions.

Compounds can be precipitated from a solution using a counter ion, as in this example of the precipitation of calcium carbonate.

Calcium chloride and sodium carbonate are both soluble in the aqueous phase.

When they are mixed, the calcium and carbonate form an insoluble solid, which can be separated with centrifugation. For more information on this topic, see our education video on solubility rules.

Precipitation can be utilized in the preparation of nano-scale solids that are found in a wide range of applications in nanotechnology. In this example, nano-scale seeds were used to control the growth of nano-crystals.

The precursors were heated, reacted with trioctylphosphine selenide, and then rapidly cooled. Methanol was added to the cooled solution, in order to precipitate the solids. The crystals were then recovered by centrifugation, and the crystal structure analyzed with X-ray Diffraction.

Precipitation can also be used in the preparation of polymeric ligands for drug delivery applications. In this example, a ligand is synthesized and conjugated to platinum for use as an anticancer therapy. First, the ligand was synthesized using an amide coupling reaction. It precipitated as the reaction progressed. It was then recovered using filtration.

The solid was then purified using recrystallization, and filtered again. The ligand was then complexed with the platinum compound, dried, and then purified using fractional precipitation from water with acetone. Platinum coupling was confirmed using nuclear magnetic resonance spectroscopy. The compounds could then be studied for their efficacy and side effects as anticancer agents.

You have just watched JoVE's introduction to the separation of mixtures using precipitation. You should now understand the various methods of precipitation, and how to perform these experiments in the laboratory.

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