The Separation Principle Behind Chromatography in Simple Terms

An overview of the physical and chemical basics and more

There's more to the color green than meets the eye: back in the early 20th century, the Russian botanist Mikhail Semyonovich Tsvet discovered that the green pigment chlorophyll in leaves consists of several colored components. When he dissolved a chlorophyll extract in ligroin (light petroleum) and ran it over a sugar mixture that he had poured into a glass tube, the green extract separated into two components: blue-green chlorophyll a and yellow-green chlorophyll b. Tsvet had actually discovered a separation principle in chemistry that enables mixtures of substances to be separated into their individual components.  

To this day, it is one of the most important methods of separating substances. Tsvet called the process chromatography. This term is derived from the Greek words "chroma = color" and "graphein = to write". Incidentally, the Russian word for color is also pronounced "tsvet" (Цвет), so the researcher's name turned out to be telling.  

Somewhat earlier than Mikhail Semyonovich Tsvet, the German chemist Ferdinand Friedlieb Runge, who lived from 1794 to 1867, observed that concentric rings formed when he dripped certain solutions onto absorbent paper – in doing so, he was actually preforming paper chromatography. Runge, who is best known for his discovery of aniline, phenol and caffeine, however, had more of an artistic than analytical interest in the phenomenon – he used his technique to "paint" color pictures, as it were, as a pastime. 

If you want to know exactly how chromatography works, you'll have to learn a little about the various chromatographic separation techniques and delve a little into the physico-chemical background. 


  1. The basics: Chromatography's separation principle
  2. The process: How do chromatographic separations work?
  3. Classification: Based on physico-chemical effects and phases
  4. Applications: Where chromatography is used

The basics: Chromatography's separation principle

Chromatography is a method for separating the substances contained in mixtures. The separation into individual components is based on their different interactions with two phases: a mobile and a stationary phase. These two phases must not be mixable with each other.  

What sounds complicated is actually quite simple. Figuratively speaking, you can imagine chromatography as a river carrying along objects like sand, small pebbles and larger stones. Depending on the flow rate of the river's water (the mobile phase) and the nature of the objects (the analyte), the objects flow at different speeds over different rough surfaces of the riverbed (the stationary phase), so they're all in a flux! 

But we'll have to look into the separation principle of chromatography in a bit more detail. This is because the individual components of the substance mixture, the analytes, switch between the mobile and the stationary phase, depending on their properties – this is called a "random walk". The analytes interact either with the mobile phase or with the stationary phase. Switching back and forth between these two possibilities very quickly is crucial for good chromatography results. So it's chemical mix & match! 

This constant switching is made possible by diffusion processes and the thermal motion that keeps the molecules moving. A dynamic equilibrium is established between the mobile and stationary phases. Depending on whether an analyte interacts strongly with the stationary phase or not, it is transported by the mobile phase through the stationary phase at different rates. This leads to substance-specific retention times. The result is a temporal or spatial separation of the individual components of the substance mixture. 

The process: How do chromatographic separations work?

The chromatographic separation principle is one aspect, but how does the process work in practice? After preparing the mixture of substances – how depends on the chromatographic separation method to be used – the mobile phase is set in flow. This is done either by pressure, capillary force or applying an electrical voltage, again depending on the chromatographic method. Subsequently the sample mixture is applied to or injected into the stationary phase, in some cases before the flow of the mobile phase is stopped. If chromatographic separation is used only on a few samples, this step is performed manually. If a large number of samples need to be separated or analyzed, so-called autosamplers help by automating injection.  

Detection systems are added to the setup to make the individual components visible or to detect them after going through chromatographic separation. The signals they detect depend on certain physical properties of the components to be separated, for example absorption of light, fluorescence, light scattering, thermal conductivity or mass. Almost all organic compounds can also be made visible by means of chemical reactions. This can be done by spraying certain reagents at them.   

To separate the individual components of a substance mixture preparatively, a fraction collector is needed. Numerous instruments and instrument combinations are available for modern chromatographic separation methods. For the popular HPLC/UHPLC techniques in particular, a detailed market overview can help.

Classification: Based on physico-chemical effects and phases

If you have read this far, you will know by now that not all chromatography is the same. There are many different chromatographic separation techniques. How do you keep track of them all?  

Irrespective of the general chromatographic separation principle, all methods can be classified according to their type of mobile phase. Different stationary phases can divide the methods even further into subgroups.  

Group 1: Liquid chromatography (LC) 

The mobile phase is a liquid (solvent, solvent mixture) – subgroups: paper, thin layer, column and membrane chromatography. 

Group 2: Gas chromatography (GC) 

The mobile phase is an inert (unreactive) gas – subgrouped depending on separation column: a) columns fully packed with solid, inert support material or b) capillary columns with support material finely coated on the inside.   

Group 3: Supercritical fluid chromatography (SFC)  

Here the mobile phase, usually carbon dioxide, is in a supercritical state between gaseous and liquid.  

Another way of classifying chromatographic techniques is to consider the physico-chemical effects and properties on which the separation is based: adsorption, distribution, ion exchange, sieving, affinity, or even chirality.

Applications: Where chromatography is used

From the early color experimentations on absorbent paper, to the first "real" chromatographic separation of chlorophyll using something like column chromatography, to present-day sophisticated technologies, much has happened in the field of chromatography. Today's organic chemistry, biochemistry, biotechnology, microbiology, food chemistry, environmental chemistry and inorganic chemistry wouldn't be the same without this separation method.  

Preparative chromatography helps to isolate or purify substances industrially on a production scale. Water purification also uses ion exchange chromatography on a vast scale. A variety of applications in chemical analysis and laboratory medicine use chromatography to identify or quantify substances.  

Chromatography is here, there, everywhere – despite its complex separation principle, it is universally used in chemistry and science.