What is chromatography and how is it used? What are the 3 types of chromatography?
Chromatography was invented by botanist Mikhail Tsvet in 1900 to separate plant pigments. As these are colored compounds, Tsvet coined the word “chromatography” from “chroma,” meaning “color,” and “graphy,” meaning “to write.” Just as today, his method involved a solid phase (stationary phase) and a liquid phase (mobile phase). While his method worked using capillary action, today there are a number of chromatographic methods, including reversed phase high-performance liquid chromatography and others.
Chromatography is a chemical analysis or purification technique used to separate complex mixtures (in either a solid or liquid sample matrix) into their different components There are a number of different chromatography separation techniques, but they all share the same basic principles entailing a solvent called the mobile phase for its ability to move through a system on a fixed stationary phase. The mobile phase may be either a liquid, supercritical fluid (usually carbon dioxide) or inert gas, while the stationary phase is a solid phase, or a liquid phase coated on a solid phase. The mobile phase moves over a stationary phase (commonly silica gel) resulting in a separation of the compounds into its different components, based on the affinity of each component’s molecules for the mobile and stationary phases. The varied mass of the molecules and their varying propensity to adhere to the chromatography matrix (stationary phase) causes the compounds to migrate up the paper in paper chromatography, or a column in automated forms of chromatography, at different speeds.
Chemists choose the stationary phase and mobile phase for separation chromatography technique based on the sample being analyzed. Various solvents (polar solvent or non-polar solvent) are used for the mobile phase. The stationary phase in thin layer chromatography (TLC) may be chromatography filter paper or, most often, silica gel applied to a glass plate. The mobile phase is pulled by capillary action up the paper or glass slide.
Beyond TLC, today’s labs use automated systems for chromatography that are far more accurate and allow for dividing the individual components of a sample into separate vials using a UV detector, mass spectrometer or other method, and a fraction collector.
In column chromatography, the chemist applies a sample on top of a glass column or stainless steel column packed with silica gel or other material (the stationary phase). Teledyne ISCO’s disposable RediSep® columns for flash chromatography are precision-packed for high resolution and reproducibility. They come in a variety of sizes.
In flash chromatography, the mobile phase is pushed through the stationary phase using a solvent pump. Flash is a popular technique for purifying pharmaceutical intermediates, as well as final organic products. It is also widely used in natural products research.
Further advancements in technique include gas chromatography, supercritical fluid chromatography (SFC), and high-performance liquid chromatography (HPLC). They differ from flash chromatography in the solvents used, the particle size of the stationary phase, and the pressure used. HPLC and SFC send the mobile phase through the stationary phase under far greater pressure than in flash chromatography. This, and the use of smaller particles for the stationary phase in the column, gives HPLC and SFC superior resolution to distinguish between compounds and elute purer results.
In supercritical fluid chromatography (SFC), the common mobile phase is supercritical carbon dioxide. Carbon dioxide is normally a gas (people and animals exhale it) and when it is under high pressure at a certain temperature, the CO2 becomes supercritical, where a difference between the gaseous and liquid states can no longer be observed. This offers the ability to work at higher flow rates, decreasing purification time and resulting in a fraction where a majority of the mobile phase disappears, as the CO2 evaporates when it returns to atmospheric pressure, greatly reducing the time of evaporation.
HPLC is divided into analytical chromatography and preparative chromatography. Flash and prep HPLC are used to purify large sample quantities; in analytical HPLC, the goal is to achieve the highest resolution possible to observe each unique compound in the sample.
When should you use chromatography?
Chromatography is used for chemical analysis or purification by separating different components of a compound, or for use in the production of pharmaceuticals, other products and compounds. TLC, Flash, SFC and HPLC are used for different purposes, and each has its benefits and ideal use cases.