Chromatography is a powerful laboratory technique used to separate complex chemical mixtures into their individual components. The fundamental principle involves distributing the mixture between two distinct media: a stationary phase that remains fixed and a mobile phase that moves through the system. Components travel at different speeds based on their varying degrees of interaction with these two phases, allowing for the physical separation of molecules.
The science of chromatography dates back to the early 20th century with the work of Russian botanist Mikhail Tswett. He used a glass column packed with calcium carbonate to successfully separate the different color pigments found in plants. Tswett named the technique “chromatography,” derived from the Greek words for “color” and “writing,” after observing the colored bands separating on the column.
Classification by Physical Setup (Gas vs. Liquid Systems)
Chromatographic methods are primarily categorized by the physical state of the mobile phase used to carry the sample through the system. This distinction dictates the necessary hardware and the types of samples that can be analyzed. The two main classifications are Gas Chromatography (GC) and Liquid Chromatography (LC).
Gas Chromatography employs an inert gas, such as helium or nitrogen, as the mobile phase (carrier gas). This technique is limited to analyzing samples that can be easily converted into a gaseous state without decomposing. GC is best suited for volatile compounds and substances that are thermally stable at the high operating temperatures required (150°C to 300°C).
Liquid Chromatography uses a liquid solvent or a mixture of solvents as its mobile phase, which is pumped through a column containing the stationary phase. LC is far more versatile than GC because it can analyze a wide range of compounds, including large biomolecules, polymers, and heat-sensitive substances. Since separation occurs at ambient or slightly elevated temperatures, the risk of thermal degradation is reduced.
Separation by Differential Partitioning
Chromatography is also classified by the chemical mechanism that drives the separation of components. Separation by differential partitioning relies on how strongly a component prefers one phase over the other, and this mechanism is broadly divided into adsorption and partition chromatography.
Adsorption Chromatography uses a solid material, typically silica gel or alumina, as the stationary phase. Separation occurs because components of the mixture adsorb to the surface of this solid stationary phase with varying strengths. Compounds with a stronger affinity for the solid surface, usually due to higher polarity, are retained longer and move more slowly.
Partition Chromatography relies on the differential solubility of the sample components. The stationary phase is a liquid coated onto or chemically bonded to an inert solid support. Separation is governed by the components’ distribution coefficient (the ratio of solubility in the stationary liquid phase versus the mobile phase). Components more soluble in the stationary liquid phase will spend more time immobilized and are eluted later. This distribution process is analogous to a liquid-liquid extraction. Paper chromatography, where water trapped in cellulose fibers acts as the stationary liquid, is a classic example.
Separation by Specialized Interactions
Some chromatographic techniques rely on highly specific molecular interactions for separation, moving beyond simple solubility or surface attraction. These specialized methods are important for analyzing complex biological molecules like proteins and nucleic acids.
Ion Exchange Chromatography separates molecules based on their net electrical charge. The stationary phase consists of an insoluble resin with fixed, covalently bound charged groups. For instance, a positively charged stationary phase attracts and temporarily binds negatively charged analyte molecules (anions). Neutral molecules or molecules with the same charge as the resin pass through quickly. The bound components are released (eluted) by changing the mobile phase composition, typically by gradually increasing the salt concentration. The salt ions compete with the sample molecules for the charged sites, weakening the electrostatic attraction and releasing the bound molecules.
Size Exclusion Chromatography (Gel Filtration) separates molecules based purely on their physical size and shape. The stationary phase is composed of porous beads or a gel matrix with controlled pore sizes. Large molecules are physically excluded from entering the internal pores and travel around them, following the shortest path. Smaller molecules penetrate the pores and take a longer, more tortuous path. This causes the largest molecules to elute first, while the smallest molecules are retained the longest.
Affinity Chromatography represents the most selective separation mechanism, utilizing a specific biological interaction (often described as “lock-and-key” binding). The stationary phase has a specific binding molecule, or ligand, chemically attached. When the sample passes through, only the target molecule (such as an enzyme, antibody, or receptor) that recognizes and binds to the ligand is retained. The target molecule is then recovered by applying a solution that breaks the ligand-target bond.