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 that separates complex mixtures (in either a solid or liquid form) into different components. There are a number of different chromatography separation techniques, but they share the same basic principles, entailing a solvent called the mobile phase for its ability to move through 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), separating the compounds into their 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 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.
What are the three types of chromatography?
Chemists choose the stationary and mobile phases based on the sample to be 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 up the paper or glass slide by capillary action.
Beyond TLC, today’s labs use far more accurate automated chromatography systems that 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.
Flash chromatography pushes the mobile phase 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 technological advancements 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 greater pressure than flash chromatography. This,
along with using 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
evaporation time.
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.
What is Purge and Trap and how is it used?
Purge and Trap is a sample preparation/introduction technique for gas chromatography mass spectrometry (GCMS). Water and soil samples cannot be injected directly into the GCMS for the analysis of Volatile Organic Compounds (VOCs), so a technique such as Purge and Trap must be used.
Purge and Trap utilizes an inert gas (He/N2) to purge the VOCs from the water/soil samples. The VOCs are carried to an adsorbent trap, where they are concentrated and retained. The adsorbent trap is then heated (desorbed), allowing the VOCs to be released and transferred to the GCMS for separation and detection.
What are the key methods for Purge and Trap?
Many drinking water, wastewater, and soil methods mandate using Purge and Trap for sample preparation and introduction to the GC/GCMS. Drinking water methods consist of USEPA 524 Series and 624, while the wastewater and soil methods are outlined in USEPA 8260.
What are common Volatile Organic Compounds (VOCs)?
Disinfection byproducts from
chlorine treatment,
gasoline (made up of many volatile compounds),
ethanol (beer),
acetone (nail polish remover),
mineral spirits (paint thinner),
and hexane (glues).
