What is Chromatography and How Does it Work?

Biotechnology & Healthcare General Knowledge in Biotech & Healthcare
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Chromatography is a process for separating components of a mixture. To get the process started, the mixture is dissolved in a substance called the mobile phase, which carries it through a second substance called the stationary phase.

The different components of the mixture travel through the stationary phase at different speeds, causing them to separate from one another. The nature of the specific mobile and stationary phases determines which substances travel more quickly or slowly, and is how they are separated. These different travel times are termed retention time.

“Writing Color”

Chromatography gets its name from a technique first used in the late 19th century to separate pigments in a complex mixture.

If a sheet of paper or cloth contacts a container filled with water or alcohol in which a complex pigment is dissolved, capillary action will carry the mixture up the paper or cloth, but the components of the pigment will not all travel at the same rate.

The largest molecules of the mixture will travel more slowly while the smallest ones race ahead, causing the stationary phase to develop discrete bands of color corresponding to each component of the mixture. This gives the technique the name “chromatography” or “writing color.”

From Art to Science

Chromatography was initially used by artists, color theorists and artisans hoping to perfect industrial dyes for textiles. With time, it also spawned a unique branch of chemistry, and with it, the techniques used today to understand and purify mixtures.

In modern laboratories, the color aspect is no longer relevant, but the same principles apply. By dissolving a mixture of interest in a mobile phase and transporting it through a stationary phase, the components of the mixture can be separated from one another based on their different speeds of travel.

By altering the mobile phase, the stationary phase, and/or the factor determining speed of travel, a wide variety of chromatographic methods have been created, each serving a different purpose and ideal for different mixtures. Some of the most common forms of chromatography are as follows.

  • In gas chromatographythe mixture of interest is vaporized and carried through a stationary phase (usually a metal or glass separation column) with an inert gas, usually nitrogen or helium. Larger molecules in the mixture take longer to pass through the column and reach the detector at the far end.
  • In liquid chromatographythe mixture of interest is dissolved in a liquid and passed through a solid stationary phase, which is often made of a silica material. Several varieties of liquid chromatography exist, depending on the relative polarities of the mobile and stationary phases (normal-phase versus reverse-phase) and whether the mobile phase is pressurized (high-performance).
  • In thin-layer chromatography (TLC), the stationary phase is a thin layer of solid material, usually silica-based, and the mobile phase is a liquid in which the mixture of interest is dissolved. Thin-layer chromatography comes with the advantage of photographing well, making its output easy to digitize.
  • Ion exchange chromatography separates the components of a mixture based on their charge, in addition to or instead of their size. In essence, positively (cations) or negatively (anions) charged ions are separated using different stationary phases and different pH mobile phases.

Chromatography can be used as an analytical tool, feeding its output into a detector that reads the contents of the mixture. It can also be used as a purification tool, separating the components of a mixture for use in other experiments or procedures. Typically, analytical chromatography uses a much smaller quantity of material than chromatography meant to purify a mixture or extract specific components from it.

For example, solid-phase extraction is a kind of liquid chromatography in which different mobile phases are used in sequence to separate out different components of a mixture trapped in a solid phase. Chromatography as a purification technique has major roles in petrochemical and other organic chemistry laboratories, where it can be one of the more cost-effective ways to remove impurities from organic solutions, particularly if the components of the mixture are heat-sensitive.

Flexibility

The principles of chromatography also appear in other laboratory techniques. Gel electrophoresis sorts nucleic acids and proteins based on their size, drawing them through the gel via an electric field. This technique is, in effect, a kind of chromatography. Similarly, distillation sorts the components of a mixture by their boiling and condensation points, with the apparatus itself being a sort of stationary phase.

Because its core principle is so simple, chromatography leaves room for substantial refinement. This has led to a variety of more specialized chromatographic techniques, such as two-dimensional chromatography for using two different chromatography methods at once, pyrolysis gas chromatography, used as part of mass spectrometry, and chiral chromatography, which is used to separate stereoisomers that other methods cannot distinguish.

Chromatography is a simple and exceedingly flexible principle, that will continue to spawn new variations and new implementations into the foreseeable future.

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