Precipitation (chemistry)

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The Process

Introduction

The process by which a dissolved chemical species comes out of solution as a solid is called precipitation. It is often employed in bioseparation applications for protein purification and concentration, although it may be used to isolate other compounds, such as nucleic acids and other cell components. Precipitation is achieved by altering the solubility of a target protein in a solution to cause it to become insoluble and thus precipitate, allowing for this concentrated solid to be more easily separated from the liquid phase (supernatant).

Solubility of Macromolecules

Solubility is the property of a substance (solute) – in either gas, liquid, or solid phases – to dissolve in a liquid (called the solvent) and form a homogenous (single-phase) solution. The extent of which a solute dissolves in a solvent is its solubility, and is based on factors such as temperature, compatibility between the solute’s net charge and the solvent’s polarity, the formation of hydration layers, the presence and concentration of other solutes dissolved in the solvent, and solvent’s ionic strength and dielectric constant.

Macromolecules (such as proteins and nucleic acids) are typically present in aqueous solvents. They are able to dissolve as a result of assuming a stable conformation in the solvent and then being surrounded by hydration layer(s). When placed in an aqueous solution, the macromolecules adopt a structure in which most hydrophobic (nonpolar) portions of the molecules gather inwards, and most hydrophilic (charged/polar) portions surround the exterior, guided to a final conformation that has the lowest Gibbs free energy. The attraction between the hydrophilic surface of the macromolecule and the polar water molecules creates an interface in which similarly-oriented water molecules associate with, and surround, the solute’s surface to form a highly ordered layer, called the hydration layer (also called the interfacial double layer). The formation of hydration layers enhances the solubility of macromolecules by greatly reducing inter-macromolecular dipole-dipole attraction and thus preventing their association with other solutes.

Effects of Solute Net Charge

In an aqueous solvent solutes with a higher net charge are more soluble, due to their enhanced ability to associate with water molecules and create a hydration layer, and their lessened inter-solute interactions as a result of increased repulsion from like-charges.

Effects of Solvent Ionic Strength

Increasing a solvent’s ionic strength (the concentration of ions in solution) decreases the availability of unassociated water molecules, which are required for the formation of hydration layers. This increases intermolecular attraction between solutes and promotes coalescence, thus decreasing the solute’s solubility.

Common Methods of Precipitation

Salting Out

A common method of protein precipitation is called salting out, and is achieved by increasing an aqueous solvent’s ionic strength by dissolving a salt. Increases in salt concentrations steadily decrease the solubility of proteins in the solution, until the proteins become insoluble and precipitate out of solution. Since proteins generally differ in their solubility, slowly adding salt allows for selective precipitation of dissolved proteins in order of lowest to highest solubility. This process is described empirically by the Cohn equation:
$ln(S)=\beta -K_{s}^{'}*I$
where $S$ is the solubility of the protein in mol/L, $\beta$ is the solubility at zero ionic strength in mol/L, $K_{s}^{'}$ is the protein’s characteristic salting-out constant (no units), and $I$ is the concentration of ions in solution in mol/L.
The most commonly used salt for this procedure is ammonium sulfate, (NH₄)₂SO₄ due to its high solubility, lack of buffering capacity, minimal cost, and the low density of the resulting solution relative to other salts, which aids in centrifugation separation.

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