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Thin layer chromatography of leaf pigments

Chromatography , chromatography ( Greek , χρῶμα chroma "color" and γράφειν graphein "write", in German color writing ) is a process called in chemistry that allows the separation of a mixture of substances through different distribution of its individual components between a stationary and a mobile phase. This principle was first used in 1901 by the Russian botanist Michail Semjonowitsch Zwetdescribed, in 1903 it was described in public for the first time, 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 on the one hand in production for the purification of substances (= preparative chromatography), on the other hand in chemical analysis to separate mixtures of substances into ingredients that are as uniform as possible for the purpose of identification or quantitative determination. Chromatography is used in organic chemistry , pharmacy , biochemistry , biotechnology , microbiology, food chemistry , environmental chemistry and also in inorganic chemistry .

Principle of chromatography

Relationships in chromatography and comparisons

The easiest way to explain chromatography is by comparing it:

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)
  • on the flow velocity.

In chromatography, different substances (= floating debris) are transported in the so-called mobile phase (= water) on a stationary phase (= river bed). Due to the interactions (see the division under separation principles) between the sample, the stationary phase and the mobile phase , individual substances are transported at different speeds and thus separated from one another: A mixture of sand, very small and somewhat larger pebbles is introduced at one point in the river ; after a hundred meters, for example, all the sand arrives first (spread 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 comparison is suitable 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 a further misunderstanding: the delays that the various sample molecules suffer on their way through the chromatographic system have nothing to do with friction phenomena . 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), the dynamic equilibrium means that 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 very often switch back and forth between the two phases (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).


Schematic representation

For 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 are used with automatable types of chromatography (together with their own data acquisition systems), which inject the samples fully automatically.

The actual separation of the substance mixture then takes place on the separating section. Chromatography is inconceivable without detection (= making it visible 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 z. B. a coloration achieved in planar chromatography (e.g. amino acids using ninhydrin ) or reactions carried out before separation (pre-column derivatization) or after separation (post-column derivatization) in column chromatography.

In the case of preparative chromatography, a fraction collector is then 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 methods 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.

Terminology and principle

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. In gas chromatography, the stationary phase consists of a liquid (separating liquid) or a gel with which the inside of the capillary is coated. In liquid chromatography, the stationary phase is normally solid, but it can also be a liquid which is immiscible with the mobile phase and which wets the powdery carrier . Finally, the stationary phase can also consist of molecules chemically bound to the carrier.

Mobile phase

Phase in which the substance mixture is introduced at the beginning of the separation system and which is moved. In liquid chromatography, the mobile phase is liquid. In gas chromatography, carrier gases such as hydrogen , helium or nitrogen are used, in thin layer chromatography one speaks of a flow agent . Mobile phases differ in their elution ability ("Strength" see below " Elutrope range "), this requires different retention times and often different selectivities.


Retention is understood to mean the delayed flow of individual substances in the substance mixture of the mobile phase through interaction with the stationary phase.

The retention of a substance by the stationary phase is essentially determined by three aspects:

  • Strength of the interaction of the substance with the stationary phase ("tendency to remain in the stationary phase")
  • Boiling point of the substance ("tendency to remain in the mobile phase")
  • Diffusion properties of the substance ("mobility in the stationary and mobile phase")

In many cases, a special interaction between the substance to be analyzed and the stationary phase is used to separate substances. The strength of the interactions between the sample components and the stationary phase is determined both by their structure and by their functional groups. In the case of non-polar substances, only dispersion interactions ( van der Waals bond ) occur, while polar separation phases can also enter into polar interactions, such as hydrogen bonds or donor-acceptor bonds. The latter separate according to the principle: opposites attract. This means that separation phases that are able to take up hydrogen for hydrogen bonding, for example, separate substances that can provide hydrogen for bonding (such as alcohols). Enantiomers , for example , which do not differ in their boiling points and would therefore have the same retention times, can also be separated due to their interactions of different strengths with special derivatives of cyclodextrins.

Retention time

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

In contrast to the carrier gases, most chemical substances interact with the stationary phase, i.e. that is, they stay in the stationary phase for a certain time. The duration of your stay in the stationary phase is added to the duration of your stay in the mobile phase (dead time), so you need longer overall to pass the entire GC column. The term retention is originally derived from the fact that the stationary phase holds back the analyte for a certain period of time. Nowadays, however, the term retention time is used in a simplified manner for the time that the analyte needs to pass through the column and this includes the dead time. Therefore the terms are defined as follows:

  • Retention time ( t R ): is the total time it takes for an analyte to pass through the column. This corresponds to the time between injection and detection of the analyte.
  • Dead time ( t 0 ): is the time in which an analyte stays in the mobile phase without interacting with the stationary phase; thus corresponds to the time it takes for the mobile phase to run through the column.
  • Net retention time or reduced retention time ( t N ): Is the difference between retention time and dead time. It therefore corresponds to the time in which an analyte is in the stationary phase. t N = t R - t 0

Flow time (dead time)

The flow time (also called "dead time") indicates the time that the mobile phase or a substance that is not retained needs to travel through the chromatography apparatus from the injection via the column to the detector (see also flow volume ). The flow time can be determined by injecting a substance that has not been retained (“inert substance”). This substance only interacts with the stationary phase to a very small extent. It therefore passes through the apparatus in the same time as the mobile phase. The flow time is then identical to the time at which the peak appears in the detector.

Flow volume

The flow volume can be derived directly from the flow time. It results from the simple formula flow volume = flow of the mobile phase · flow time. The flow volume is very important for numerous calculations in high performance liquid chromatography (HPLC), e.g. B. for method transfer between columns with different volumes.


(Elution lat . Eluère "wash out") is the dissolving out or displace the adsorbed substances from solid or liquid impregnated adsorbents and ion exchangers by continuous addition of a solvent ( eluent = mobile phase ). The solution flowing out of the separation column is called the eluate .

This process is of particular importance in solid phase extraction .

Eluotropic series

Arrangement of the solvents commonly used as mobile phases according to their elution power for a reference substance (usually silica gel or aluminum oxide).


When bleeding an effect on chromatography columns is referred to, in which the column loses small amounts of their matrix. One also speaks of column bleeding. In gas chromatography, the cause of increased column bleeding can be an excessive thermal load on the column and in high-performance liquid chromatography (HPLC) the use of, for example, unsuitable pH values ​​of the eluent or the eluents (too strongly acidic or alkaline). Column bleeding also occurs to a small extent during operation and is normally not a problem there. The column bleeding results, among other things, in the aging of separation columns. Heavy column bleeding, however, causes a lot of signal noise and a high background value during detection or downstream analysis methods such as a mass spectrometer .


In chromatography, a column , or separation column , is a hollow tube with a diameter of a few micrometers to several meters. The length also varies from a few centimeters to 150 meters. In this tube, either only the inner wall is coated (capillary column) or the column is filled with the stationary phase (packed column). It differs from the column in construction in that it does not have to be straight or vertical, but can also be rolled up like a hose.

Reversed-phase mechanism

In adsorption chromatography, there are two ways of separating a substance mixture:

  1. Normal phase : polar stationary phase (such as silica gel , aluminum oxide), non-polar to medium-polar mobile phase (such as hydrocarbons, dioxane, ethyl acetate ...) or
  2. Reversed 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 high-performance liquid chromatography , gradient elution is often used, 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.

Classification according to the separation principle

The fundamental 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 physical-chemical effects.

  • Adsorption chromatography - the various components are separated 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 (flow agent, 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 the case of 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 - the moving phase is mostly 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.
    • Chelating agent chromatography is a special form of cation exchange chromatography. The resting phase selectively binds polyvalent cations via complex-forming, functional groups. The selectivity for heavy metal ions compared to alkali and alkaline earth ions is high.
  • Sieve effect - In the resting phase, substances are used that separate the components based on their size. There are essentially three different procedures.
With a sieve, particles have “advantages” that are fine enough to penetrate the pores of the sieve. It is exactly the opposite for the corresponding chromatography processes. Sufficiently fine particles are able to “get lost” in the cavities of the stationary phase and therefore travel more slowly 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 only stay 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) - Here, metal ions such as Fe, Co, Ga and the like are produced via complex bonds. a. 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, enrichment using titanium dioxide columns has proven to be a competitive process. Various IMAC methods are also available for the purification of poly-histidine-labeled proteins. Above all, Ni and Co ions are used for the enrichment.
  • Thiol - disulfide exchange chromatography uses firmly bound thiol groups on the resting phase in order to reversibly bind the thiol groups on the molecules in the moving phase as covalent disulfides. Those thiol groups that are on the outside of the protein molecules mainly participate in these bonds.
  • 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 divided according to the carriers of the stationary phases or the density

  • Liquid chromatography (Engl. 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 used is 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 diameters from one to several centimeters. This form of liquid chromatography is mainly used for preparative separations.
      • High-performance liquid chromatography (the term high-pressure chromatography is incorrect, but also very common; HPLC High Performance (Pressure) Liquid Chromatography) - It represents the separation method most widely used in analytics today, the actually incorrect (outdated) term High Pressure Liquid Chromatography refers to the pressures that distinguish this method from low pressure or other types of chromatography. After all, up to over 1000 bar are generated here at 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 corresponding housing. The mobile phase is pumped through the membrane at low pressures of up to 6 bar and at about 20 times higher flow rates than is usual in column chromatography.
  • Gas chromatography
    • Packed Columns - 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 (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

  • Column length
  • is called the linear flow rate of the mobile phase through the column, it is defined as:
  • the retention factor is defined by
  • The selectivity coefficient α indicates the quality of the separation of two substances. It is based on the retention times of the components in the column. The retention time is the time that the component under consideration needs to traverse the column and is plotted at the peak maximum:
  • That chromatographic resolution ( Resolution ) of two peaks calculated as follows:
The factor 1.18 results from the ratio of the half-width to the base width of a Gaussian bell curve ((2 · ln 2) 0.5 ).
  • , the number of plates or plates describes the number of equilibrium settings of the substance to be separated between the 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:
 : Baseline width
 : "Full Width at Half Maximum" Fwhm
  • : Peak capacity; Indicates how many peaks within an interval between and the k-value of a certain peak can theoretically be separated from one another with a resolution of R = 1.5 (baseline separation).
  • refers to the plate height (or theoretical plate height ) of a theoretical plate (HETP -, height equivalent of a theoretical plate ", English , height equivalent to a theoretical plate" ) and the relationship between column length and number of plates :
Practical values ​​are in the range from 0.1 to 0.5 mm.

Partition height H

The separation stage height of a chromatographic column is a measure of the separation efficiency of the column. The imaginary section of the separation column on which the chromatographic equilibrium is established can be imagined as the separation stage. 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 plate height, the following requirements are necessary under analytical conditions :

  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 high-performance liquid chromatography can be used to determine the height of the separation stage as a function of the flow rate of the eluent :

in which:

  • the separation step height,
  • is the linear flow rate.
  • -Term takes into account the eddy diffusion , which is caused by different flow paths through the packing. The following applies: where
    • the packing factor,
    • denotes the particle diameter.
  • The 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: where:
    • the diffusion constant in the mobile phase and
    • is the maze factor. The labyrinth factor takes into account the pore structure of the stationary phase.
  • the term takes into account the peak broadening due to the slow establishment of equilibrium between the mobile and stationary phases. The diffusion constant along the pores of the stationary phase must also be taken into account. It applies

Peak symmetry

Theoretically, every substance should leave a chromatography column as a sharp eluting line. For various reasons, however, chromatographic peaks always have a certain width. In the ideal case, 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" or " leading ". 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, with different calculation formulas (e.g. according to IUPAC or USP ) being used:

An ideal “Gauss peak” reaches the value 1, values ​​above 1 mean “tailing”, values ​​below 1 mean “fronting”.



Web links

Wiktionary: Chromatography  - explanations of meanings, word origins, synonyms, translations