# Chromatography

Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]

Pictured is a sophisticated gas chromatography system. This instrument records concentrations of acrylonitrile in the air at various points throughout the chemical laboratory.

Chromatography (from Greek χρώμα:chroma, colour and γραφειν:"grafein" to write) is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture dissolved in a "mobile phase" through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated.

Chromatography may be preparative or analytical. Preparative chromatography seeks to separate the components of a mixture for further use (and is thus a form of purification). Analytical chromatography normally operates with smaller amounts of material and seeks to measure the relative proportions of analytes in a mixture. The two are not mutually exclusive.

## Explanation

Look up chromatography in
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An analogy which is sometimes useful is to suppose a mixture of bees and wasps passing over a flower bed. The bees would be more attracted to the flowers than the wasps, and would become separated from them. If one were to observe at a point past the flower bed, the wasps would pass first, followed by the bees. In this analogy, the bees and wasps represent the analytes to be separated, the flowers represent the stationary phase, and the mobile phase could be thought of as the air. The key to the separation is the differing affinities among analyte, stationary phase, and mobile phase. The observer could represent the detector used in some forms of analytical chromatography. A key point is that the detector need not be capable of discriminating between the analytes, since they have become separated before passing the detector.

## History

It was the Russian botanist Mikhail Semyonovich Tsvet who invented the first chromatography technique in 1900 during his research on chlorophyll. He used a liquid-adsorption column containing calcium carbonate to separate plant pigments. The method was described on December 30 1901 at the 11th Congress of Naturalists and Doctors (XI съезд естествоиспытателей и врачей) in St. Petersburg. The first printed description was in 1903, in the Proceedings of the Warsaw Society of Naturalists, section of biology. He first used the term chromatography in print in 1906 in his two papers about chlorophyll in the German botanical journal, Berichte der Deutschen Botanischen Gesellschaft. In 1907 he demonstrated his chromatograph for the German Botanical Society. Interestingly, Mikhail's surname "Цвет" means "color" in Russian, so there is the possibility that his naming the procedure chromatography (literally "color writing") was a way that he could make sure that he, a commoner in Tsarist Russia, could be immortalized.

In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the Chemistry Nobel Prize for their invention of partition chromatography.[1] Since then, the technology has advanced rapidly. Researchers found that the principles underlying Tsvet's chromatography could be applied in many different ways, giving rise to the different varieties of chromatography described below. Simultaneously, advances continually improved the technical performance of chromatography, allowing the separation of increasingly similar molecules.

## Chromatography terms

• The analyte is the substance which is to be separated during chromatography.
• Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.
• A bonded phase is a stationary phase that is covalently bonded to the support particles or to the inside wall of the column tubing.
• A chromatogram is the visual output of the chromatograph. in the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.
Plotted on the x-axis is the retention time and plotted on the y-axis a signal (for example obtained by a spectrophotometer, mass spectrometer or a variety of other detectors) corresponding to the response created by the analytes exiting the system. In the case of an optimal system the signal is proportional to the concentration of the specific analyte separated.
• A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.
• Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
• The effluent is the mobile phase leaving the column.
• An immobilized phase is a stationary phase which is immobilized on the support particles, or on the inner wall of the column tubing.
• The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). A better definition: The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column. In one case of HPLC the solvent consist of a carbonate/bicarbonate solution and the sample is the anions being separated. The mobile phase moves through the chromatography column (the stationary phase) where the sample interacts with the stationary phase and is separated.
• Preparative chromatography is used to nondestructively purify sufficient quantities of a substance for further use, rather than analysis.
• The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
• The sample is the matter analysed in chromatography. It may consist of a single component or it may be a mixture of components. When the sample is treated in the course of an analysis, the phase or the phases containing the analytes of interest is/are referred to as the sample whereas everything out of interest separated from the sample before or in the course of the analysis is referred to as waste.
• The solute refers to the sample components in partition chromatography.
• The solvent refers to any substance capable of solubilizing other substance, especially the liquid mobile phase in LC.
• The stationary phase is the substance which is fixed in place for the chromatography procedure. Examples include the silica layer in thin layer chromatography.

## Chromatography theory

Chromatography is method of separating mixtures and identifying their components i.e. it's a separation method that exploits the differences in partitioning behavior of analytes between a mobile phase and a stationary phase to separate components in a mixture. The interaction of the components of a mixture with the two phases is influenced by several different intermolecular forces, including ionic, dipolar, nonpolar, and specific affinity and solubility effects. There are two theories of chromatography, the plate and rate theories.

### Retention

The retention is a measure of the speed at which a substance moves in a chromatographic system. In continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the retention is usually measured as the retention time Rt or tR, the time between injection and detection. In interrupted development systems like TLC the retention is measured as the retention factor Rf, the run length of the compound divided by the run length of the eluent front:

${\displaystyle R_{f}={\frac {distance\ moved\ by\ compound}{distance\ moved\ by\ solvent}}}$

The retention of a compound often differs considerably between experiments due to variations of the eluent, the stationary phase, temperature, sample matrix and the setup. It is therefore important to compare the retention of the test compound to that of several standard compounds under absolutely identical conditions.

During the chromatographic process the analyte experiences zone broadening as a result of diffusion. Two analytes with different retention times yet with large broadening do not resolve and this is why in any chromatographic system broadening needs to be minimized. This is done by selecting the proper stationary and mobile phase, the eluent velocity, the track length and temperature. The Van Deemter's equation gives an ideal eluent velocity taking into account several physical parameters.

### Plate theory

The plate theory of chromatography was developed by Archer John Porter Martin and Richard Laurence Millington Synge. The plate theory describes the chromatography system, the mobile and stationary phases, as being in equilibrium. The partition coefficient K is based on this equilibrium, and is defined by the following equation:

${\displaystyle K={\frac {Concentration\ of\ solute\ in\ stationary\ phase}{Concentration\ of\ solute\ in\ mobile\ phase}}}$

K is assumed to be independent of concentration, and can change if experimental conditions are changed, for example temperature is increased or decreased. As K increases, it takes longer for solutes to separate. For a column of fixed length and flow, the retention time ${\displaystyle (t_{R})}$ and retention volume ${\displaystyle (V_{r})}$ can be measured and used to calculate K.

## Techniques by chromatographic bed shape

### Column chromatography

For more details on this topic, see Column chromatography.
A diagram of a standard column chromatography and a flash column chromatography setup

Column chromatography is a separation technique in which the stationary bed is within a tube. The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube (packed column) or be concentrated on or along the inside tube wall leaving an open, unrestricted path for the mobile phase in the middle part of the tube (open tubular column). Differences in rates of movement through the medium are calculated to different retention times of the sample.[2]

In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash).[3] The technique is very similar to the traditional column chromatography, except for that the solvent is driven through the column by applying positive pressure. This allowed most separations to be performed in less than 20 minutes, with improved separations compared to the old method. Modern flash chromatography systems are sold as pre-packed plastic cartridges, and the solvent is pumped through the cartridge. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps resulted in quicker separations and less solvent usage.

In expanded bed adsorption, a fluidized bed is used, rather than a solid phase made by a packed bed. This allows omission of initial clearing steps such as centrifugation and filtration, for culture broths or slurries of broken cells.

### Planar Chromatography

Thin layer chromatography is used to separate components of chlorophyll

Planar chromatography is a separation technique in which the stationary phase is present as or on a plane. The plane can be a paper, serving as such or impregnated by a substance as the stationary bed (paper chromatography) or a layer of solid particles spread on a support such as a glass plate (thin layer chromatography).

#### Paper Chromatography

For more details on this topic, see Paper chromatography.

Paper chromatography is a technique that involves placing a small dot of sample solution onto a strip of chromatography paper. The paper is placed in a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.

#### Thin layer chromatography

For more details on this topic, see Thin layer chromatography.

Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.

## Techniques by physical state of mobile phase

### Gas chromatography

For more details on this topic, see Gas chromatography.

Gas chromatography (GC), also sometimes known as Gas-Liquid chromatography, (GLC), is a separation technique in which the mobile phase is a gas. Gas chromatography is always carried out in a column, which is typically "packed" or "capillary" (see below) .

Gas chromatography (GC) is based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in analytical chemistry; though the high temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them), frequently encountered in biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.

### Liquid chromatography

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. Liquid chromatography can be carried out either in a column or a plane. Present day liquid chromatography that generally utilizes very small packing particles and a relatively high pressure is referred to as high performance liquid chromatography (HPLC).

In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure. HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases. Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and the opposite (e.g. water-methanol mixture as the mobile phase and C18 = octadecylsilyl as the stationary phase) is called reversed phase liquid chromatography (RPLC). Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more.

Specific techniques which come under this broad heading are listed below. It should also be noted that the following techniques can also be considered fast protein liquid chromatography if no pressure is used to drive the mobile phase through the stationary phase. See also Aqueous Normal Phase Chromatography.

### Supercritical fluid chromatography

For more details on this topic, see Supercritical fluid chromatography.

Supercritical fluid chromatography is a separation technique in which the mobile phase is a fluid above and relatively close to its critical temperature and pressure.

## Techniques by separation mechanism

### Ion exchange chromatography

For more details on this topic, see Ion exchange chromatography.

Ion exchange chromatography utilizes ion exchange mechanism to separate analytes. It is usually performed in columns but the mechanism can be benefited also in planar mode. Ion exchange chromatography uses a charged stationary phase to separate charged compounds including amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. Ion exchange chromatography is commonly used to purify proteins using FPLC.

### Size exclusion chromatography

For more details on this topic, see Size exclusion chromatography.

Size exclusion chromatography (SEC) is also known as gel permeation chromatography or gel filtration chromatography and separates particles on the basis of size. Smaller molecules enter a porous media and take longer to exit the column, whereas larger particles leave the column earlier. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of a purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins, especially since it can be carried out under native solution conditions.

### Affinity chromatography

For more details on this topic, see Affinity chromatography.

Affinity chromatography is based on selective non-covalent interaction between an analyte and specific molecules. It is very specific, but not very robust. It is often used in biochemistry in the purification of proteins bound to tags. These fusion proteins are labelled with compounds such as His-tags, biotin or antigens, which bind to the stationary phase specifically. After purification, some of these tags are usually removed and the pure protein is obtained.

## Special techniques

### Reversed-phase chromatography

For more details on this topic, see Reversed-phase chromatography.

Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase.

### Two-dimensional chromatography

By using an additional physico-chemical (Chemical classification) criterion for separation of the mixture of analytes (sample), the resolution and quality of chromatographic separation can be increased. As a result, higher specifity concerning the separational capability of the chromatographic technique is obtained, allowing separation and preparation or analysis of compounds indistinguishable by One-dimensional chromatography.

In Gas-Phase Chromatography, two dimensional separation is achieved by coupling a second, short column to the first long column. Coupling is achieved by different techniques, for example shock-freezing the elutes in order of elution from the first column at fixed time-intervals, and then reheating them in order of elution, releasing them into the second column. The time of traversal through the second column needs to be shorter than the time remaining until the next sample is reheated to prevent compound buildup and to fully exploit the separational capability.

In addition, a Time-of-Flight Mass Spectrometer (TOFMS) can be used as the second dimension of a 2-D gas chromatoghraph. TOF-Mass Spectrometers used in gas chromatography can be very short, as there is a somewhat limited range of molecules that can be separated. In this technique, one column is used to separate analytes, followed by TOFMS detection. This allows for a greater number of analytes to be separated in one experimental run.

### Fast protein liquid chromatography

For more details on this topic, see Fast protein liquid chromatography.

Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques which are used to purify proteins. Many of these techniques are identical to those carried out under high performance liquid chromatography.

### Countercurrent chromatography

For more details on this topic, see Countercurrent chromatography.

Countercurrent chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids. It involves mixing a solution of liquids, allowing them to settle into layers and then separating the layers.