Chromatography

Chromatography is a family of analytical chemistry techniques for the separation of mixtures. It involves passing the sample, a mixture which contains the analyte, in the "mobile phase", often in a stream of solvent, through the "stationary phase." The stationary phase retards the passage of the components of the sample. When components pass through the system at different rates they become separated in time, like runners in a mass-start foot race. Each component has a characteristic time of passage through the system, called a "retention time." Chromatographic separation is achieved when the retention time of the analyte differs from that of other components in the sample.

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 a stationary liquid or solid phase. Various techniques for the separation of complex mixtures rely on the differential affinities of substances for a gas or liquid mobile medium and for a stationary absorbing medium through which they pass; such as paper, gelatin, alumina or silica.

A chromatogram is the visual output of the chromatograph. Different peaks or patterns on the chromatograph correspond to different components of the separated mixture.

Analytical chromatography is used to determine the identity and concentration of molecules in a mixture. Preparative chromatography is used to purify larger quantities of a molecular species. Most of the following refers to analytical chromatography. This is a method used to divide/separate mixtures.

History

It was the Russian botanist Mikhail Tsvet (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 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. The phenomenon of precipitational separation was observed before Tsvet as well. His contribution was turning the phenomenon into the method of scientific analysis.

The Greek word chroma in chromatography means colour in English and refers both to Tsvet's name that is literally translated from Russian as colour and to the colour of the plant pigments he was separating at that time.

In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the Chemistry Nobel Prize for their invention of partition chromatography.

The technology of chromatography advanced rapidly throughout the 20th century. 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 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:

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 chromotography was developed by Archer John Porter Martin and Richard Laurence Millington Synge. The plate theory describes the chromotography 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 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 (tR) and retention volume (Vr) can be measured and used to calculate K.

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 placed into 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 (TLC)

In thin layer chromatography or TLC the stationary phase consists of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat carrier like a glass plate, a thick aluminum foil, or a plastic sheet.

The process is similar to paper chromatography with the advantage of faster runs, better separations, and the choice between different adsorbents. TLC is a standard laboratory method in organic chemistry. Because of its simplicity and speed TLC is often used for monitoring chemical reactions and for the qualitative analysis of reaction products.

TLC plates are made by mixing the adsorbent with a small amount of inert binder like calcium sulfate (gypsum) and water, spreading the thick slurry on the carrier, drying the plate, and activation of the adsorbent by heating in an oven. The thickness of the adsorbent layer is typically around 0.1-0.25mm for analytical purposes and around 1-2mm for preparative TLC.

Several methods exists to make colorless spots visible:

- Often a small amount of a fluorescent dye is added to the adsorbent that allows the visualization of UV absorbing spots under a blacklight ("UV254").
- Iodine vapors are a general unspecific color reagent.
- Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate.

Once visible, the Rf values of the spots can be determined. These values should be the same regardless of the extent of travel of the solvent, and in theory are independent of a single experimental run. They do depend on the solvent used, and the type of TLC plate.

Thin layer chromatography is also used in finding which pigments a plant contains. By taking extract of the plants cellulose and applying the technique, one can adequately find the pigments. It may also be used to detect pesticides or insecticides in food, or in forensics to analyze the dye composition of fibers.

Column chromatography

Column chromatography utilizes 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. Stills 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.

When applying positive pressure on top of the column, most separations could be performed in less than 20 minutes with improved separations compared to the old method. This makes flash column chromatography the method of choice for most synthetic organic chemists when purifying organic compounds.

In the modern Flash chromatography systems which can be purchased, the glass columns are replaced with pre-packed plastic cartridges. Solvent is pumped through the cartridge, which is much quicker. Systems may also be linked with detectors and fraction collectors providing automation. The introduction of gradient pumps means quicker separations and less solvent usage.

Gas-liquid chromatography

Gas-liquid chromatography is based on a partition equilibrium of analyte between a liquid stationary phase and a mobile gas. It is useful for a wide range of non-polar analytes, but poor for thermally labile molecules.

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:

- Positively charged ion exchanger (anion exchanger) interacts with anions
- Negatively charged ion exchanger (cation exchanger) interacts with cations.

Bound compounds can be eluted from the column by gradient elution or isocratic elution with a change in salt concentration or pH. Ion exchange chromatography is commonly used to purify proteins using FPLC.

Immobilized metal ion affinity chromatography

IMAC is a popular and powerful way to purify proteins. It is based on the specific coordinate covalent binding between histidine or other unique amino acids (either naturally present on the surface of the protein or grafted with recombinant DNA techniques) and various immobilized metal ions, such as copper, nickel, zinc, or iron...

Salt concentration is increased to produce later fractions.

High performance liquid chromatography (HPLC)

High performance liquid chromatography, usually referred to simply as 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. Diffusion within the column leads to broad peaks and loss of resolution. Less time on the column then translates to narrower peaks in the resulting chromatogram and thence to better resolution (it's easier to differentiate one peak from another) and sensitivity (tall, narrow peaks can be easier to discriminate from noise than shorter, broader peaks). Another way to decrease time the analyte stays on the column is to use a solvent gradient. A solvent gradient is how the composition of the mobile phase changes over a period of time and can be used to force the analyte off of the column at a faster rate.

Normal phase (NP) liquid chromatography

Normal phase HPLC (NP-HPLC) was the first kind of HPLC setup used. This method uses a polar stationary phase and a nonpolar mobile phase, and is used when the analyte of interest has a polar nature. The polar analyte associates with and is retained by the polar stationary phase. NP-HPLC has fallen out of favor recently with the development of reversed phase HPLC.

Reversed phase (RP) liquid chromatography

Reversed phase HPLC (RP-HPLC) was developed due to the increasing interest in large nonpolar biomolecules. Like the name implies the nature of the stationary phase is reversed. The RP-HPLC consists of a nonpolar stationary phase and a polar mobile phase. One common stationary phase is a normal silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17. It is the case that for a given substance the retention time is longer when the mobile phase is more polar. This is the reverse of the situation which exists when normal silica is used as the stationary phase.

Reversed phase columns are quite difficult to damage when compared with normal silica columns. But they must never be used with strong aqueous bases (alkali) as these will destroy the silica, however they can be used with aqueous acid but the column should not be exposed to the acid for too long. One reason is because the acid will corrode the metal parts of the HPLC equipment. The metal content of HPLC columns must be kept low if the best possible ability to separate substances is to be retained. A good test for the metal content of a column is to inject a sample which is a mixture of 2,2'- and 4,4'- bipyridine. Because the 2,2'-bipy can chelate the metal it is normal that when a metal ion is present on the surface of the silica the shape of the peak for the 2,2'-bipy will be distorted, tailing will be seen on this distorted peak.

Gel permeation chromatography

Gel permeation chromatography (also known as size exclusion chromatography, gel-filtration chromatography or Sephadex gel chromatography) separates molecules on basis of size. Smaller molecules enter a porous media and take longer to exit the column, whereas larger particles leave the column ealier. The elution volume decreases roughly linearly with the logarithm of the molecular hydrodynamic volume (often assumed to be proportional to molecular weight), although columns need to be calibrated using 4-5 standard samples (e.g., folded proteins of known molecular weight) to determine the void volume and the slope of the logarithmic dependence. These parameters may vary with solution conditions.

Since gel filtration is generally a low resolution chromatography (i.e., does not discern similar species well), it is often reserved for the final, "polishing" step of a purification. GPC is good for determining the quaternary structure of purified proteins, especially since it can be carried out under native solution conditions. GPC can also assay protein tertiary structure; since GPC measures the hydrodynamic volume (not molecular weight!), it can discern folded and unfolded versions of the same protein. For example, the apparent hydrodynamic radius of a typical protein domain might be 14 Г… and 36 Г… for the folded and unfolded forms, respectively; the folded form elutes much later, since it is smaller.

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 (or better: protein constructs). These constructs can be of fusion proteins with a so-called His-tag, biotinylated or possibly antigens. After purification some of these tags are usually removed and the pure protein is obtained

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