What is an important use of chromatography

Chromatography or. Chromatography (Greek, in German Color writing) In chemistry, a process is called that allows the separation of a mixture of substances through the different distribution of its individual components between a stationary and a mobile phase. This principle was first described in 1901 by the Russian botanist Michail S. Tswett, in 1903 it was described in public for the first time, and in 1906 he first used the term "chromatography". He examined colored plant extracts, for example from leaf material, and was able to isolate various dyes from them by means of chromatography. This method is used in practice on the one hand in production for the isolation or purification of substances (= preparative chromatography), on the other hand in chemical analysis in order to separate mixtures of substances into ingredients that are as uniform as possible for the purpose of identification or quantitative determination. Chromatography has become an integral part of today's organic chemistry, biochemistry, biotechnology, microbiology, food chemistry, environmental chemistry and also inorganic chemistry.

Description of the chromatography process

The easiest way to explain chromatography is to make a comparison:

A raging river can carry quite a bit of floating debris. The speed at which the floating debris is moved depends on

  • the type of floating debris (grains of sand are transported faster than pebbles),
  • the nature of the river bed (rough surfaces increase the friction of the floating debris and thus reduce the speed of removal) and
  • on the flow velocity.

In chromatography, mixtures of substances (= floating debris) are used in the so-called Mobile phase (= Water) on one Stationary phase (= River bed) transported on. Due to the interactions (see the classification under separation principles) between the sample, the stationary phase and the mobile phase, the individual components are transported on at different speeds and can thus be separated from one another: A mixture of sand, very small and slightly larger pebbles is created in one place brought in by the river; after a hundred meters, for example, all the sand arrives first (distributed over a few meters) and after a certain waiting time all the smaller pebbles and much later the larger ones, each pulled apart a certain distance.

This graphic comparison is useful for a first introduction. In fact, the process (in chromatography) is more reminiscent of a "digital" process (stop and go). The sample molecules are either carried along with the mobile phase (at the speed of the mobile phase - analogy would be a raft that is passively carried in a stream) or they adhere to the stationary phase (speed equal to zero). They switch back and forth between these two possibilities very quickly (due to the movement of heat they constantly receive shocks). The comparison with the river bed could also lead to another misunderstanding: the delays that the various sample molecules suffer on their way through the chromatographic system nothing with friction phenomena to do. The basis for understanding are differences in the distribution (of the different types of molecules A, B, C etc.). They correspond to differences in the proportion of time (which the individual molecules of type A, B, C etc. spend on average in the mobile phase). Chromatography manages to convert these differences into speed differences and thus make them useful for a separation. This could also be called the “trick” or the principle of chromatography. Otherwise these often very small differences could hardly be used, neither for separation and cleaning processes nor for analyzes.

It is easier to understand using an example: If 45% of the A molecules are in the mobile phase (on average), due to the dynamic equilibrium, the individual A molecules are also in the mobile phase 45% of the time Spend phase (on average). Therefore, their speed will be 45% of the mobile phase speed (on average). For good results in chromatography, it is crucial that the exchange of substances between the two phases takes place very quickly, i.e. the individual sample molecules should switch back and forth between the two phases very often (diffusion processes, heat movement). A prerequisite for this is that the paths that the molecules have to cover from the stationary phase to the mobile phase are very short. If the stationary phase contains a powder, the grain size of this powder should be very small (for example only a few micrometers). For certain reasons, the powder grains should also be shaped as uniformly as possible and have as uniform a size as possible (narrow grain size distribution).



For the chromatography, the establishment of the flow of the mobile phase, the injection of the sample to be separated, the actual separation and the detection are necessary. The Flow of the mobile phase is achieved either by means of pressure (hydraulic pump, gas pressure), capillary force or by applying an electrical voltage.

The injection (= Introduction of the substance mixture into the chromatographic system) takes place either before the flow of the mobile phase is established (e.g. thin-layer chromatography) or while the mobile phase is already flowing. With a large number of samples, so-called autosamplers (together with their own data acquisition systems) are used with automatable types of chromatography, which inject the samples fully automatically.

Then the actual Separation of the substance mixture on the isolating distance. Without Detection Chromatography is not conceivable (= making it clear when a substance passes a certain part of the chromatography system or where a substance comes to a standstill after the process has ended). Different detection systems are used for each type of chromatography, either by using physical properties (absorption of light, fluorescence, light scattering, thermal conductivity ...) of the substances or by obtaining a signal through chemical reactions. By means of chemical reactions, e.g. a coloration is achieved in planar chromatography (e.g. amino acids using ninhydrin) or reactions are carried out before separation (pre-column derivatization) or after separation (post-column derivatization) in column chromatography.

In preparative chromatography, a Faction collectors required to collect the separated substance.

Due to the design, chromatographic purification processes are always batch processes. This means that only a certain amount of substance can be applied and separated before proceeding with the next amount. This is particularly problematic when working up large amounts, so that some processes have been developed in order to be able to operate chromatography continuously: Annular chromatography, TMB (True Moving Bed) chromatography and SMB (Simulated Moving Bed) chromatography.

Definition of some terms

Stationary phase

Phase that interacts with the individual substances of the substance mixture and does not move. The stay of the analytes during their retention alternates between the mobile and stationary phase (random walk) and causes the substance-characteristic retention time.

Mobile phase

Phase in which the substance mixture is introduced at the beginning of the separation system and which is moved (phase on a solid or liquid substance). Mobile phases differ in their elution ability ("Strength" see below "Elutrope range"), this requires different retention times and often also different selectivities.


Delay of individual substances of the substance mixture through interaction with the stationary phase.

Retention time

Time that the molecules of a pure substance need to travel through the column (from injection to detection).

Flow time (dead time)

The flow-through time (formerly also "dead time") indicates the time that the mobile phase or a substance that is not retained needs to migrate through the column. A substance not retained (Inert substance) is only in a negligibly low concentration in the stationary phase and therefore passes through the column in the same time as the mobile phase.


Elution (from lat. eluate "Wash out") is the leaching or displacement of adsorbed substances from solid or liquid-soaked adsorbents and ion exchangers by continuously adding a solvent (Eluent = mobile phase). The solution flowing out of the separation column becomes Eluate called.

This process is of particular importance in solid phase extraction.


Mobile phase that has passed the isolating distance.

Eluotropic series

Arrangement of the solvents commonly used as mobile phases according to their elution power in the case of a reference substance (usually silica gel or aluminum oxide). The order can be selected in ascending or descending order.


Column: In chromatography, a column is a hollow tube with a diameter of a few micrometers to several meters. This tube is either completely filled with the stationary phase (packed column) or thinly coated on the inside (capillary column).

Reversed phase mechanism

There are two ways of separating a substance mixture in adsorption chromatography:

  1. Normal phase: polar stationary phase (such as silica gel, aluminum oxide), non-polar to medium-polar mobile phase (such as HC substances, dioxane, ethyl acetate ...) or
  2. Reversed phase (Reverse phase): non-polar stationary phase (like modified silica gel) and polar mobile phase (like buffered water).

In the first case, lipophilic substances are easily eluted, polar substances are difficult, in the reverse case polar substances are easily eluted ("similia similibus solvuntur").

In HPLC, gradient elution is often used (reversed phase), in which the composition of the solvent is slowly changed (e.g. from 80% to 20% water content). Alkanes emerge from the column very late and amino acids emerge very early, and these fractions can be cut out.

The chromatographic separation methods can be classified according to different aspects:

Classification according to the separation principle

The basic principle of all chromatographic processes is the often repeated establishment of an equilibrium between a stationary phase and a moving phase. The equilibrium can develop due to various physico-chemical effects.

  • Adsorption Chromatography - Here there is a separation of the various components due to the different strengths of the adsorptive bonds to the resting phase. The moving phase can be a more or less polar solvent or, in the case of gaseous substances, a carrier gas. In the case of liquid chromatography, one imagines a competition between the various sample molecules and the molecules of the mobile phase (solvent, solvent) for the adhesion points on the (large) surface of the stationary phase.
  • Partition chromatography - Similar to the extraction process, the different solubility of the components to be separated is used here. In chromatography, however, the solvent remains as a static phase on a carrier material. The moving phase can again be a solution or a carrier gas.
  • Ion exchange chromatography (see also anion exchange chromatography and cation exchange chromatography) - The moving phase is usually a solution of the ions to be separated. The resting phase is a solid ion exchanger. Ion exchangers form bonds of different stability between the various ions in the moving phase.
  • Sieving effect - In the resting phase, substances are used that separate the components based on their size. Essentially, a distinction is made between three methods.
With a sieve, particles have “advantages” that are fine enough to penetrate the pores of the sieve. It is exactly the opposite with the corresponding chromatography methods. Sufficiently fine particles are able to "get lost" in the cavities of the stationary phase and are therefore slower to travel than molecules that are excluded from these cavities (more or less or entirely) due to their size. If they are of sufficient size, they migrate without delay, since they are only located in the moving flow of liquid and never in the stationary part of the liquid (in the cavities - e.g. the gel).
  • Affinity chromatography - A chemical compound specific for each analyte is used as the stationary phase, which causes a separation due to non-covalent forces. It is a highly selective method.
    • IMAC (Immobilized Metal Ion Affinity Chromatography) - Metal ions such as Fe, Co, Ga, etc. bound to a matrix. The separation is achieved via the different interactions between the analyte and the metal ions. This method has established itself particularly in the purification of proteins by phosphorylation. Mainly Fe and Ga are used as metal ions. For some time now, enrichment using titanium dioxide columns has proven to be a competitive process. There are also various IMAC methods for the purification of poly-histidine-labeled proteins. Above all, Ni and Co ions are used for the enrichment.
  • Chiral Chromatography - For the separation of chiral molecules. The stationary phase contains an enantiomer which enters into a diastereomeric interaction of different strengths with the two enantiomers of the racemate. The two enantiomers are retarded to different degrees.

Classification according to the phases used

Due to the mobile phases, chromatography can be divided into three areas, which can be subdivided into different groups according to the carriers of the stationary phases or the physical state of the stationary phases.

  • Liquid chromatography (English liquid chromatography, LC)
    • Planar chromatography
      • Paper chromatography - The solid phase used is paper that is either lying or (mostly) standing vertically in a glass container. As with thin layer chromatography, the mobile phase is moved due to capillary forces.
      • Thin-layer chromatography - The solid phase are e.g. B. silica particles applied in a fine layer on a flexible carrier film made of aluminum or plastic or a glass plate. A variant is the circular TLC, with a rotating, coated circular disk (especially suitable for preparative purposes).
    • Column chromatography
      • Low pressure chromatography - The columns used here have a diameter of one to several centimeters. This form of liquid chromatography is mainly used for preparative separations.
      • High performance chromatography (The expression High pressure chromatography; engl. HPLC High Performance (Pressure) Liquid Chromatography) - It is the most widespread separation method used in analytics today, the actually incorrect (outdated) designation High Pressure Liquid Chromatography refers to the pressures that this method differs from the low pressure or other Different types of chromatography. After all, up to 400 bar are generated here with a flow rate of the mobile phase of up to 5 ml / min, which, however, has nothing to do with the separation performance, but only serves to move the eluent mixture in the column.
      • Electrochromatography - In this case, the mobile phase is moved by applying voltage. This method is still in the development stage and is not used in routine operation. Not to be confused with electrophoresis.
    • Membrane chromatography
      • Instead of a column filled with a chromatographic matrix, a single or multi-layer membrane is used as the solid phase in a suitable housing. The mobile phase is pumped through the membrane at low pressures of up to 6 bar and at around 20 times higher flow rates than is usual in column chromatography.
  • Gas chromatography
    • Packed pillars - The inside of a column (long tube) is filled with a fine-grained material. As a rule, the stationary phase consists of a thin film of a largely inert and high-boiling liquid that coats the powder grains.
    • Capillary columns - Only the column wall is covered with a thin layer of stationary phase.
      • Liquid stationary phase
      • Solid stationary phase
  • Supercritical fluid chromatography (Eng. SFC supercritical fluid chromatography) - A substance in its supercritical phase (state between gas and liquid) is used as the mobile phase. This is mostly carbon dioxide. In this method, only columns are used to support stationary phases.

Chromatography parameters

  • u is called the linear Flow rate the mobile phase through the column, it is defined as:
  • the Retention factork is defined by
  • The Separation factor α indicates the quality of the separation of two substances. It is based on the retention times tR. of the components in the column. The retention time is the time that the component under consideration needs to traverse the column and is shown at the peak maximum:
  • R., the chromatographic resolution (resolution) of two peaks is calculated from:

  • N, the Number of separation stages or Number of trays describes the number of equilibrium settings of the substance to be separated between stationary and mobile phase in the column. The larger N, the more equilibrium adjustments can be made in a certain length, which results in a better separation performance of the column. N is calculated using the formula:

bB. : Baseline width

F.W.HM. : "Full Width at Half Maximum" Fwhm

n: Peak capacity; Indicates how many peaks within an interval between t0 and the k-value of a particular peak can theoretically be separated from one another with a resolution of R = 1.

  • H denotes the Separation step height (or theoretical floor height) of a theoretical tray (HETP) and is the relationship between the length of the columns and the number of trays:
Practical values ​​are in the range from 0.1 to 0.5 mm.

Partition height H

The separation step height of a chromatographic column is a measure of the separation efficiency of the column. The separation stage can be imagined as the imaginary section of the separation column on which the chromatographic equilibrium is once established. The more such equilibrium settings "have space on the column", the lower the height of the separation stage and the higher the separation efficiency of the column. To achieve a low separation step height are under analyticalconditions the following requirements are necessary:

  1. Rapid equilibrium of adsorption or distribution is expected. Therefore, the particle diameter should be as small as possible.
  2. Constant temperature throughout the column. A column thermostat can be used for this.
  3. Constant flow rate: A piston pump with up to 400 bar is used for this.
  4. Linear adsorption range: The stationary phase should not be overloaded during the course of the chromatography.
  5. Negligible diffusion would be desirable, but unfortunately cannot be achieved experimentally. Therefore, as regular as possible packings with particles of particularly small diameter are used.

The so-called Van Deemter equation for HPLC can be used to determine the height of the separation stage as a function of the flow rate of the eluent:

in which:

  • H the separation step height,
  • ux is the linear flow rate.
  • A.-Term takes into account the eddy diffusion caused by different flow paths through the packing. The following applies: in which
    • p the packing factor,
    • d denotes the particle diameter.
  • The B.-Term takes into account the longitudinal diffusion. The longitudinal diffusion is the diffusion of the analyte molecules in both directions of the separation stage. The following applies: in which:
    • D. the diffusion constant in the mobile phase and
    • L. is the maze factor. The labyrinth factor takes into account the pore structure of the stationary phase.
  • the C.-Term takes into account the peak broadening due to the slow establishment of equilibrium between the mobile and stationary phase. Here is also the diffusion constant D.s to be observed along the pores of the stationary phase. It applies

Peak symmetry

Theoretically, every substance should leave a chromatography column as a sharp eluting line. However, for various reasons, chromatographic peaks always have a certain width. Ideally, they have the shape of a Gaussian bell curve. In practice, however, it often happens that the peaks deviate from this ideal shape and appear more or less asymmetrical. An asymmetry in which the front rise of the peak is steeper than the peak fall is referred to as “tailing”, while the effect that the rise is less steep than the fall is referred to as “fronting”. The tailing factor, which is a measure of the peak symmetry, is determined by dropping the perpendicular from the peak maximum to the baseline, and at a certain height, usually 10% of the peak height, the distances to the peak front (a) and to the peak end (b ) determined. Then the quotient of the two values ​​is formed, whereby different calculation formulas (e.g. according to IUPAC or according to USP) are in use:

An ideal "Gaussian peak" reaches the value 1, values ​​above 1 mean "tailing", while values ​​below 1 mean "fronting".

Practical experiment

Chromatography can be performed at home using commercially available means. You need:

  • a filter bag, if possible white or black
  • some colored pencils of the water-soluble kind and definitely marker pens that are not water-soluble.

Paint is applied to the lower edge of the coffee filter and the paper is placed in a bowl with water so that the paper soaks up with water.

Since the color of the crayons is water-soluble, the water now transports the color upwards.

A red pencil, for example, is basically not red, but consists of a mixture of different colors that together appear red. In the experiment, the different color pigments interact with the paper to different degrees and are therefore transported more or less quickly by the water.

Therefore, you can soon see several different colored spots, the crayon colors are separated chromatographically.

In order to test the dependence of the separation on the solvent, one can carry out this experiment again with the same pen with vodka, cleaning gasoline or denatured alcohol and observe the different results. (However, this should only be done in well-ventilated rooms, as the fumes from cleaning gasoline are poisonous.)

See also

Categories: Chromatography | Separation process