Chloroform is a classic non-polar solvent in biochemistry, indispensable for lipid extractions and phase separations due to its density and selective solubility for amphipathic biomolecules.

Chemical Properties

Chloroform (CHCl₃) features a tetrahedral carbon bonded to three chlorine atoms and one hydrogen atom, forming a dense, colorless liquid with a density of approximately 1.49 g/mL at 25°C. It has a boiling point of 61.2°C and a melting point of -63.5°C. Chloroform exhibits low water solubility (about 8 g/L) but is miscible with many organic solvents such as methanol and hexane. Its dipole moment (1.04 D) enables hydrogen bond accepting interactions. The solvent is highly volatile, with a vapor pressure of about 197 mmHg at 25°C. Stabilized grades containing ethanol or amylenes are commonly used to inhibit phosgene formation under light and oxygen exposure.

Biochemical Applications

In lipid biochemistry, chloroform is a key solvent in the Folch extraction method (typically using a 2:1 chloroform:methanol ratio), enabling the partitioning of phospholipids and glycolipids into the lower organic phase for subsequent analytical techniques such as thin-layer chromatography (TLC), gas chromatography–mass spectrometry (GC-MS), or nuclear magnetic resonance (NMR) analysis of fatty acids and gangliosides. It is also used for membrane protein or viral particle extraction from aqueous lysates. In molecular biology workflows, chloroform is employed in phenol–chloroform nucleic acid purification to denature proteins while maintaining DNA and RNA structural integrity. Additionally, chloroform washing steps are applied in carbohydrate analysis to remove residual lipophilic contaminants after hydrolysis, improving sample purity for high-performance liquid chromatography (HPLC).