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Laboratory Technologies Chromotography – What is it?

Author / Editor: Myles Davis / Ahlam Rais

Chromatography is a physical separation process. Here, the separation of a mixture of substances is allowed by different distribution of the individual components between a stationary and mobile phase. The stationary phase does not move. The sample and a running medium result in the mobile phase. Chromatography is nowadays an integral part of biochemistry, food chemistry, environmental chemistry, etc. Chromatographic analysis methods are used for qualitative and quantitative analysis.

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Paper chromatography
Paper chromatography

Mobile phase: The mobile phase is one of the two phases that exist in chromatography. It passes through the stationary phase as a solvent and transport medium together with the substances to be separated. Thus, it is instrumental in chromatographic separation. It is usually a liquid or, as in gas chromatography, a propellant gas.

Stationary phase: The stationary phase, on the other hand, is usually solid and does not move. However, it interacts with the substances in the sample mixture from the mobile phase. The mixture of substances is separated into their components by a process known as absorption. Absorption describes the process in which different substances have different degrees of attraction to the stationary phase. This means that components with different properties, such as molecular size or polarity, move at different speeds. Chemically similar substances to the stationary phase have a stronger attraction to them. This causes these substances to move more slowly through the separation column.

The size of the substances also plays a role. The larger the substances are, the more they adhere to the stationary phase. However, this is not the case with size rejection chromatography. In this, the column is made with a porous material that contains pores of various sizes. The smaller the molecules, the more likely they are to migrate through the small pores and have a longer path to the detector than larger molecules. In gas chromatography, for example, the stationary phase is the coating on the column.

What is a chromatogram?

The result of a chromatographic separation is called a chromatogram. A distinction is made here between an inner and an outer chromatogram.

Inner chromatogram

In the inner chromatogram, the result can be seen directly in the stationary phase. This is the case, for example, in thin-layer chromatography or paper chromatography. There, for example, the position of radioactively labeled compounds can be determined with the aid of a photographic plate.

Outer chromatogram

This type of chromatogram is used in analytical chemistry. Here, the individual substances are recorded outside the separating section through a detector. The substances eluted from the chromatographic column generate electrical signals in the detector. The detector thus measures the mixture and precisely reproduces it in a diagram. The Y-axis describes the concentration of the component. The X-axis describes the time at which this substance arrived. The peak visualizes the concentration of the component as a function of time. The mobile phase creates the baseline between the peaks. The outer chromatogram is used mainly in gas chromatography and HPLC (High-Pressure Liquid Chromatography). Due to the wide range of data, it is very well suited for quantitative analysis.

Chromatographic methods

Paper chromatography

Paper chromatography is already a very old method. Nowadays, however, it is hardly ever used. In schools, this method is often used as an introductory experiment. With this process, it can be determined if substances are pure or if they are a mixture of substances.

The classic introductory experiment proceeds as follows.

Materials needed:

  • 1x sharpie (black is best)
  • 2x filter paper
  • water
  • 1x glass

Cut a hole of about 1 centimeter in one of the two filter papers. Around this hole, you draw a circle with the black sharpie. Fold the second filter paper into a wick and put it through the hole. So that the circle points upwards. Hang the other end of the wick in a glass filled with water.

Resolution: This is now an internal chromatogram. The filter paper is the stationary phase and the water in combination with the dye mixture of the black sharpie defines the mobile phase. The water as solvent causes the substances to be transported to the outside at different speeds. As described for the chromatogram, the velocity depends on the running speed of the dyes in the filter paper.

High-performance liquid chromatography

HPLC is a method in which a liquid sample is transported at high pressure over a stationary phase. It is used for the identification, quantification, purification, and separation of substances. Molecules are reversibly bound to the stationary phase by interactions. Since different sample substances react differently with the stationary phase, the substances contained in the sample are separated from each other.

Thin-layer chromatography

Thin-layer chromatography is a fast and inexpensive method for separating substances from one another. Specially coated plates are used for this purpose. The running medium transports the sample up the stationary phase (coating) by capillary forces. The stationary phase here usually consists of diatomaceous earth. The components interact to varying degrees with the running medium and stationary phase. This is how separation of the individual components occurs.

Gas chromatography

Another popular chromatography method is gas chromatography. This is used, for example, in the food and pharmaceutical industries to determine the purity of various substances. The purity of drugs can also be determined with this test.

To be able to test the substances here, they must be gaseous. The mixture to be tested is dissolved with the solvent and placed in the apparatus. A carrier gas is also added. Usually, nitrogen or helium, since these do not react with the mixture. This is heated. The substances thus migrate through a temperature-controlled column. A few mm thick, but up to 200 m long. At the end of the column is a detector, which produces an external chromatogram.

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