# Chromatography

A chemist is shown using column chromatographic apparatus in the mid-1950s to separate constituents in a coal tar color analysis
Pictured is a sophisticated gas chromatography system. This records concentrations of acrylonitrile in the air at various points throughout the chemical laboratory.

Chromatography (from Greek χρώμα: chroma, colour) is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture which contains the analyte through a Stationary Phase, which separates it from other molecules in the mixture and allows it to be isolated.

## History

It was the Russian botanist Mikhail Semyonovich Tsvet who invented the first chromatography technique in 1901 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 XI 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 "Tsvet" 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 Nobelprize.org: The Nobel Prize in Chemistry 1952. 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 increasingly similar molecules to be resolved

## Chromatography terms

• The analyte is the substance which is to be purified or isolated during chromatography
• Analytical chromatography is used to determine the identity and concentration of molecules in a mixture
• A chromatogram is the visual output of the chromatograph. Different peaks or patterns on the chromatogram correspond to different components of the separated mixture
• A chromatograph takes a chemical mixture carried by liquid or gas and separates it into its component parts as a result of differential distributions of the solutes as they flow around or over the stationary phase
• The mobile phase is the analyte and solvent mixture which travels through the stationary phase
• Preparative Chromatography is used to purify larger quantities of a substance
• The retention time is the characteristic time it takes for a particular molecule to pass through the system
• The stationary phase is the substance which is fixed in place for the chromatography procedure and is the phase to which solvents and the analyte travels through or binds to. Examples include the silica plate in thin layer chromatography.

## Chromatography theory

Chromatography is a separation method that exploits the differences in partitioning behavior between a mobile phase and a stationary phase to separate the components in a mixture. Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption. 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:

$R_f = \frac{distance\ moved\ by\ compound} {distance\ moved\ by\ solvent}$

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

### 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:

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

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 $(t_R)$ and retention volume $(V_r)$ can be measured and used to calculate K.

## Capillary-action chromatography

### Paper chromatography

This is an older technique which involves placing a small spot of sample solution onto a strip of chromatography paper. The paper is dipped into a suitable solvent and placed in a sealed container. 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 identify an unknown substance.

### 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, aluminum oxide, or cellulose on a flat, inert carrier. 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 (the stationary phase) and how soluble they are in the mobile phase. This allows the calculation of an Rf value and can be compared to standard compounds to identify an unknown substance.

## Column chromatography

A diagram of a standard column chromatography and a flash column chromatography setup

Column chromatography encompasses a number of techniques based around utilizing a vertical glass column filled with some form of solid support, with the sample to be separated placed on top of this support. The rest of the column is filled with a solvent which, under the influence of gravity, moves the sample through the column. Similarly to other forms of chromatography, differences in rates of movement through the solid medium are translated to different exit times from the bottom of the column for the various elements of the original sample.

In 1978, W. C. Still introduced a modified version of column chromatography called flash column chromatography (flash). 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.

### 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.

### High performance liquid chromatography

High performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. The analyte is forced through a column (stationary phase) by a liquid (mobile phase) at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to diffuse within the column. 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.

### Ion exchange chromatography

Ion exchange chromatography is a column chromatography that uses a charged stationary phase. It is used to separate charged compounds including amino acids, peptides, and proteins. The stationary phase is usually 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

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

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 labeled 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.

## Gas-liquid chromatography

Gas chromatography (GC) is based on a partition equilibrium of analyte between a liquid stationary phase and a mobile gas. 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 biochemistry, it is well suited for use in the petrochemical, environmental monitoring, and industrial chemical fields. It is also used extensively in chemistry research.

## Countercurrent Chromatography

Countercurrent Chromatography (CCC) is a type of liquid-liquid chromatography, where both the stationary and mobile phases are liquids. It is perhaps one of the most robust preparative chromatographic techniques available today, however its use is only just now beginning to become wide-spread. In CCC components are separated as a result of repeated partitioning between two phases of a bi-phasic solvent system. (See Countercurrent Chromatography)

• Library 4 Science online books about chromatography.
• GC Help website with basic gas chromatography information and troubleshooting