Chromatographic Separation Methods in Simple Terms

Separating mixtures of substances to analyze them qualitatively and quantitatively – these are the chromatographic methods you should know about

If you don't like the mushrooms in your salad, shove them to the side of your plate. If only everything were so easy to separate. With mixtures of chemicals, it's usually a lot more difficult.  

You might have already heard of chromatography. It is used to separate mixtures of substances into their individual components in laboratories, analytics and manufacturing. The method takes advantage of the fact that the molecules contained in a mixture of substances interact to different degrees with the selected mobile and stationary phases. If you want to know more about chromatography's physico-chemical principle, take a look here!  

Depending on which substance you want to separate from a mixture, why you are separating it, and how accurate your separation needs to be, you can select from among several chromatographic separation methods.

Some important chromatographic methods: 

  1. Paper chromatography
  2. Thin-layer chromatography
  3. Column chromatography, high-performance liquid chromatography (HPLC) and ultra-high performance LC (UHPLC)
  4. Gas chromatography


All four chromatographic methods are based on adsorption, i.e. liquid or gas molecules attaching to a solid surface.  

However, there are further methods for separating different components of a substance mixture from each other. For example, to purify water you can use ion exchange chromatography: an ion exchanger as the stationary phase captures anions or cations – again by adsorption. 

Purifying biomolecules is becoming ever more important. Affinity chromatography can be used to separate nucleic acids, proteins and blood from mixtures of substances. The stationary phase consists of a specific, high-affinity ligand of the substance to be separated. Protein mixtures can also be purified by gel chromatography (also size-exclusion chromatography).

A closer look at chromatographic separation methods – the choice is yours 

Method 1: Paper chromatography – keeping things simple 

In their chemistry lessons, most schoolchildren will have observed the process when dyes of various felt-tip inks get separated. What looks gorgeous in the classroom when the ink splits into its individual color components is often used in the lab to detect the smallest amounts of substances.  

Special chromatography papers made of cotton cellulose that differ regarding their absorbency, thickness and mass usually serve as the stationary phase, and pure organic solvents or mixtures of these as the mobile phase. To optimize the separation of specific substances, it is often necessary to change the liquid's polarity, which can be controlled via the mixing ratio. 

Depending on the flow direction of the eluent, we speak of ascending, descending or circular paper chromatography. In ascending chromatography, the substance mixture is applied to the stationary phase, i.e. the chromatography paper. However, this is unsuitable for larger quantities of substances, as this method separates only about 5 to 50 micrograms.  

Place the paper into a container with solvent so that the mixture initially does not come into contact with the solvent. The capillary action of the paper lets the mobile phase rise upwards, taking the various substances with it to different extents, depending on their polarity. This creates a chromatogram with different substance spots.

Method 2: Thin-layer chromatography – high sensitivity, little effort 

Thin-layer chromatography (TLC) generates results quickly and with little effort. In principle it works in the same way as paper chromatography. Fine-grained materials such as cellulose, silica gel, kieselguhr or aluminum oxide serve as the stationary phase and are applied to thin plates of plastic, aluminum or glass. Mixtures of non-polar and moderately polar solvents, such as petroleum ether and ethyl acetate, are used as flow agents in what is called normal-phase thin-layer chromatography (NP-TLC). When polar solvents such as acetonitrile and water are used, the chromatographic separation technique is called reversed-phase thin-layer chromatography (RP-TLC).  

Before getting started, the substance mixture is dissolved in a suitable solvent and applied to the carrier plate as dots using a thin capillary. The plate is then placed in a container containing the liquid and the lid closed to avoid evaporation as this could falsify results. 

You can't see anything? No problem: almost all organic compounds can be visualized after separation using UV light or spray reagents such as ninhydrin, silver nitrate/ammonia or acid-base indicators.

Method 3: Column chromatography (LC), high-performance liquid chromatography (HPLC) and ultra-high-performance LC (UHPLC) – separate large amounts quickly and quantitatively 

For these chromatographic separation techniques, the material for the stationary phase is packed into a column made of, for example, glass or stainless steel (for HPLC and UHPLC). The mobile phase is forced through the column material by applying pressure. The substances that flow the fastest exit the column first, so the substances can be collected separately.  

If classical column chromatography won't separate your substance mixture, then state-of-the-art HPLC or UHPLC could. However, they require more complex equipment. The stainless steel columns used in HPLC and UHPLC chromatography are packed with particularly small particles for greater separation efficiency (hence the term high performance liquid chromatography). It takes high pressures to force the mobile phase through the column. These techniques require an HPLC or UHPLC instrument, of which a wide range is available. The world's largest market overview of HPLC/UHPLC systems helps to select the right set-up. In addition to a pump and the column, today's instruments contain a detector that visualizes the sample to be separated in trajectories and chromatograms.

Method 4: Gas chromatography – separation at full steam! 

Unlike the three separation methods discussed above, gas chromatography uses an inert gas as the mobile phase, for example helium, argon or nitrogen. This method can therefore be used only to separate mixtures in which all the substances are gaseous or can be brought into the gas phase without decomposing. The stationary phase, a solid or a gas, is contained in a spiral tube. It is possible to separate the components of a substance mixture mainly because their boiling points differ.   

The solubility or adsorption properties also play a role. Most analytical laboratories use very fine capillary columns that allow you to separate even isomers that have very similar boiling points.  

In gas chromatography, a thermal conductivity detector usually detects the individual components of the substance mixture that flow through the stationary phase at different rates. It is often coupled with a mass spectrometer (GC/MS). To select the right one for an application, take a look at the world's largest market overview of mass spectrometers.