Liquid-liquid extraction is a fundamental technique used to isolate compounds based on their solubility in two distinct liquids. The process involves mixing a polar aqueous solvent with a non-polar organic solvent to allow target molecules to partition into the layer where they are most soluble. Since these two solvents are immiscible, they ultimately separate into two distinct layers, and the position of the aqueous layer is not fixed.
The Rule of Separation: Density Dictates Position
The factor that determines the layering order of any two immiscible liquids is their relative density. Density is a physical property, commonly expressed in grams per milliliter (g/mL). When two liquids are placed together, the one with the higher density will settle beneath the one with the lower density.
The aqueous layer, which is primarily water, has a density of approximately 1.0 g/mL. This value serves as the dividing line for determining the position of the organic solvent.
The organic layer’s position is therefore entirely dependent on its own density relative to this 1.0 g/mL benchmark. If the organic solvent has a density less than 1.0 g/mL, it will occupy the top layer. Conversely, if the organic solvent has a density greater than 1.0 g/mL, it will sink to the bottom, placing the aqueous layer on top.
Layer Position in Common Extraction Scenarios
The aqueous layer is on the bottom when the organic solvent used is less dense than water. Common organic solvents like diethyl ether, hexanes, and ethyl acetate all have densities below 1.0 g/mL, typically ranging from about 0.6 to 0.9 g/mL. When any of these solvents are mixed with water, the lighter organic solvent forms the upper layer, causing the aqueous layer to settle beneath it.
When the aqueous layer is on the top, it signals that the organic solvent is denser than water. This situation arises with organic solvents that contain heavy halogen atoms like chlorine. Dichloromethane (also known as methylene chloride) is a frequently used example, possessing a density of about 1.3 g/mL. Chloroform is another common, heavier solvent, with a density near 1.5 g/mL.
In an extraction using dichloromethane, the solvent is heavier than the aqueous phase and will settle at the bottom of the separatory funnel. The aqueous layer remains floating on the top of the denser organic layer. The choice of which organic solvent to use is dictated by the solubility of the compound being separated, which means the layer position must be verified for every extraction.
Simple Tests for Layer Identification
When the identity of the organic solvent is unknown, a simple method called the “drop test” can be used to quickly determine which layer is aqueous. This test involves carefully adding a single drop of pure water to the layers in the separation vessel. The behavior of the added drop will immediately reveal the nature of the two phases.
If the drop of water is added to the top layer and it dissolves or mixes into that layer, then the top layer is confirmed to be the aqueous phase. If the drop is added to the top layer and instead passes straight through it to mix with the bottom layer, the bottom layer must be the aqueous phase.
Performing this quick verification is an important step before separating the layers to ensure the correct phase is collected. This simple observation prevents the accidental discarding of the desired product.