Chromatography
- This article should material from Paper chromatography of amino acids
Chromatography is an analytical chemical process for the separation of mixtures involving passing a sample (the analyte) in the "mobile phase", often in a stream of solvent, through the "stationary phase", some form of material that will provide resistance by virtue of chemical interactions (not reactions) between the components of the sample and the material. Usually, each component has a characteristic separation rate that can be used to identify it and thus the composition of the original 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 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 adsorbing medium through which they pass; such as paper, gelatin, or magnesium silicate gel.
Analytical chromatography is used to determine which chemicals are in a mixture and their concentrations. Preparative chromatography is used to purify larger quantities of a chemical. Most of the following refers to analytical chromatography.
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Paper chromatography
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A small spot of solution containing the sample is applied to a strip of chromatography paper about one centimetre from the base. This sample is adsorbed onto the paper. This means that the sample will contact the paper and may form interactions with it. Any substance that will react with (and thus bond to) the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent (such as ethanol or water) 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. Paper chromatography takes some time and the experiment is usually left to complete for some hours.
The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes. Compounds can be identified by calculating Rf values which can be compared with values in a data book. These can be calculated by:
Rf = (distance moved by spot) / (distance moved by solvent)
Two-way paper chromatography involves using two solvents and rotating the paper 90o inbetween. This is useful for separating complex mixtures of similar compounds.
Thin layer chromatography
The adsorbent (the solid which the sample is adsorbed to) is applied in a thin layer on the surface of a glass plate (aluminium foil or a plastic sheet can also be used as base materials). The adsorbent (e.g. silica gel or calcium sulfate) is pasted on to the glass and baked. The process is the same for paper chromatography. The advantage is that wider separations can be achieved in less distance, and different adsorbents can be used.
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.
Column chromatography
Immobilized Metal Ion Affinity Chromatography (ion exchange)
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 or high pressure liquid chromatography
Frequently referred to simply as HPLC, this form of column chromatography is used frequently in biochemistry. The analyte is forced through a column by liquid at high pressure, which decreases the time the separated components remain on the stationary phase and thus the time they have to spread out within the column, leading to broader peaks. Less time on the column then translates to narrower peaks in the resulting chromatogram and thence to better selectivity (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). Solvents used include any miscible combination of water or various organic liquids (alcohols, acetonitrile, dichloromethane). Often, a gradient over time in the solvent composition passing through the column is used to separate analyte mixtures, as a function of how well the changing solvent composition differentially mobilizes the analyte. For instance, using a water/methanol gradient, the more hydrophobic components will elute under conditions of relatively high methanol, whereas the more hydrophilic will elute under conditions of relatively low methanol. Whether one starts with high methanol or low methanol depends on the nature of the stationary phase.
Reversed phase liquid chromatography
Traditionally HPLC stationary phases are polar, whereas so-called "reverse" phase (RP-HPLC) stationary phases are hydrophobic. On an RP-HPLC column, then, hydrophobic analytes would tend to be retained on the column, eluting more readily as the proportion of the hydrophobic component of the stationary phase is increased. RC-HPLC has lower resolution than GC.
Gel filtration chromatography (also called size exclusion chromatography)
Separates molecules on basis of size. Smaller molecules enter a porous media and take longer to exit the column. SEC is good for determining polymer MW but is low resolution.
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.
Affinity Chromatography 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.
History
It was the Russian botanist Mikhail Tsvet (Mikhail Semyonovich Tsvet) who invented adsorption chromatography in 1901 during his research of chlorophyll. He used liquid-adsorption columns 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 chlorophylle in the German botanical journal, Berichte der Deutschen botanischen Geselschaft. In 1907 he demonstrated his chromatogaph for the German Botanical Society.
The phenomenon recipitational separation was observed before Tsvet as well. His contribution was turning the phenomenon into the method of scientific analysis.
In 1952 Archer John Porter Martin and Richard Laurence Millington Synge were awarded the Chemistry Nobel Prize "for their invention of partition chromatography". [1] (http://www.nobel.se/chemistry/laureates/1952/)
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