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Encyclopedia > HPLC

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 marathon. Ideally, each component has a characteristic time of passage through the system, called a "retention time." Analytical chemistry is the analysis of material samples to gain an understanding of their chemical composition and structure. ... In chemistry and chemical engineering, a separation process is a process that transforms a mixture of substances into two or more compositionally-distinct products. ... An Analyte is the substance or chemical constituent that is undergoing analysis. ... A solvent is a liquid that dissolves a solid, liquid, or gaseous solute, resulting in a solution. ...


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. A liquid will assume the shape of its container. ... Gas (actually, as), the GNU assembler, is the default GCC back-end. ... A substance is soluble in a fluid if it dissolves in the fluid. ... In chemistry, adsorption of a substance is its concentration on a particular surface. ... Piece of paper Paper is a thin, flat material produced by the compression of fibres. ... Gelatin (also gelatine) is a translucent brittle solid substance, colorless or slightly yellow, nearly tasteless and odorless, which is created by prolonged boiling of animal connective tissue. ...


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.

Contents


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 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 chlorophyll in the German botanical journal, Berichte der Deutschen botanischen Geselschaft. In 1907 he demonstrated his chromatogaph 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. Botany is the scientific study of plant life. ... Mikhail Semyonovich Tsvet (Михаил Семенович Цвет, 1872-1919) was the Russian botanist who invented adsorption chromatography. ... 1901 was a common year starting on Tuesday (see link for calendar). ... Chlorophyll is a green photosynthetic pigment found in plants, algae, and cyanobacteria. ... In biology, pigment is any material resulting in color in plant or animal cells which is the result of selective absorption. ... 1903 has the latest occurring solstices and equinoxes for 400 years, because the Gregorian calendar hasnt had a leap year for seven years or a century leap year since 1600. ... Warsaw (Polish: Warszawa, see also other names, in full The Capital City of Warsaw, Polish: Miasto Stołeczne Warszawa) is the capital of Poland and its largest city. ... 1906 was a common year starting on Monday (see link for calendar). ... 1907 was a common year starting on Tuesday (see link for calendar). ...


The Greek word chroma in chromatography means color in English and refers both to Tsvet's name that is literally translated from Russian as color and to the color 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". [1] 1952 was a leap year starting on Tuesday (link will take you to calendar). ... Archer John Porter Martin was a British chemist and Nobel Prize winner. ... Richard Laurence Millington Synge (born Liverpool, October 28, 1914, died Norwich, August 18, 1994) was a British biochemist, and winner of the 1952 Nobel Prize in Chemistry for the invention of partition chromatography. ...


The technology of chromatography advanced rapidly throughout the 20th century. Researchers found that the priciples underlying Tsvet's chromatography could be applied in many different ways, giving rise to the different varieties of chromatography described below. Simultaneously, advances continally 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: Archer John Porter Martin was a British chemist and Nobel Prize winner. ... Richard Laurence Millington Synge (born Liverpool, October 28, 1914, died Norwich, August 18, 1994) was a British biochemist, and winner of the 1952 Nobel Prize in Chemistry for the invention of partition chromatography. ...

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

See the article paper chromatography Paper chromatography is an analytical technique for separating and identifying pigments and other molecule from extracts that contain a complex mixture of molecules. ...

In paper chromatography, chemical interactions with the paper make compounds travel at different rates.
In paper chromatography, chemical interactions with the paper make compounds travel at different rates.

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. Diagram of TLC tank drawn by Theresa Knott. ... Diagram of TLC tank drawn by Theresa Knott. ... A solvent is a liquid that dissolves a solid, liquid, or gaseous solute, resulting in a solution. ... Ethyl alcohol, also known as ethanol or grain alcohol, is a flammable, colorless chemical compound, one of the alcohols that is most often found in alcoholic beverages. ... Water (from the Old English word wæter) is a colorless, tasteless, and odorless substance that is essential to all known forms of life and is known also as the most universal solvent. ... A chemical compound is a chemical substance formed from two or more elements, with a fixed ratio determining the composition. ...


The final chromatogram can be compared with other known mixture chromatograms to identify sample mixes. 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 (TLC)

Separation of black ink on a TLC plate.
Separation of black ink on a TLC plate.

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. Separation of black ink on TLC plate. ... Separation of black ink on TLC plate. ... Some examples of silica gel sachets Silica gel is a granular, porous form of silica made synthetically from sodium silicate. ... Aluminium oxide or aluminum oxide is a chemical compound of aluminium and oxygen with chemical formula Al2O3. ... Cellulose (C6H10O5)n is a long-chain polymer polysaccharide carbohydrate, of beta-glucose. ... The materials definition of a glass is a uniform amorphous solid material, usually produced when a suitably viscous molten material cools very rapidly, thereby not giving enough time for a regular crystal lattice to form. ...


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. Organic chemistry is the scientific study of the structure, properties, composition, reactions, and synthesis of organic compounds. ... A chemical reaction is a process involving one, two or more substances (called reactants), characterized by a chemical change and yielding one or more product(s) which are different from the reactants. ...


TLC plates are made by mixing the adsorbent with a small amount of inert binder like calcium sulfate (gypsum) and water, spreading the a 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 0.1–0.25 mm for analytical purposes and around 1–2 mm for preparative TLC. Inert is the state of doing little or nothing. ... Gypsum is a very soft mineral composed of calcium sulfate dihydrate, with the chemical formula CaSO4·2H2O. Chemical structure Gypsum from New South Wales, Australia Heating gypsum above approximately 150°C (302°F) partially dehydrates the mineral, by driving off exactly 75% of the water contained in its chemical structure. ...


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 spots can be quantified by way of calculating their Rf values. These values should be the same regardless of the extent of travel of the solvent, and in theory are independant of a single experimental run. They do depend on the solvent used, and the type of TLC plate. Fluorescence induced by exposure to ultraviolet light in vials containing various sized cadmium selenide (CdSe) quantum dots. ... Note: Ultraviolet is also the name of a 1998 UK television miniseries about vampires. ... Note: Ultraviolet is also the name of a 1998 UK television miniseries about vampires. ... General Name, Symbol, Number iodine, I, 53 Series halogens Group, Period, Block 17 (VIIA), 5, p Density, Hardness 4940 kg/m3, no data Appearance violet-dark grey, lustrous Atomic properties Atomic weight 126. ...


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. Gas-liquid chromatography (GLC), or simply gas chromatography (GC) is a type of chromatography in which the mobile phase is a carrier gas, usually an inert gas such as helium or nitrogen, and the stationary phase is a microscopic layer of liquid on an inert solid support. ...


Ion exchange chromatography

Ion exchange chromatography is a method to separate molecules such as proteins by their charge in a process of ion exchange. Ion exchange chromatography uses ion exchange resins as stationary phase. Ion-Exchange Chromatography The ion-exchange chromatography process allows the separation of ions and polar molecules based on the electrical properties of the molecules. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... Charge is a word with many different meanings. ... Ion exchange is a process in which ions are exchanged between a solution and an ion exchanger, an insoluble solid or gel. ... An ion-exchange resin is an insoluble matrix (or support structure) normally in the form of small (1-2mm diameter) beads, fabricated from an organic polymer substrate on the surface of which are sites with easily trapped and released ions. ...


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. A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... A coordinate covalent bond (also known as dative covalent bond) is a special type of covalent bond in which the shared electrons come from one of the atoms only. ... Histidine is one of the 20 most common natural amino acids, coded for in DNA. Nutritionally, in humans, histidine is considered an essential amino acid, but mostly only in children. ... In chemistry, an amino acid is any molecule that contains both amino and carboxylic acid functional groups. ... Recombinant DNA technology adds/replaces DNA in an organism resulting in the recipient organism containing exogenous DNA. Recombinant proteins are proteins that are produced by different genetically modified organisms following insertion of the relevant DNA into their genome. ...


Salt concentration is increased to produce later fractions.


High performance liquid chromatography (HPLC)

Modern HPLC systems are highly automated
Modern HPLC systems are highly automated

High performance liquid chromatography, frequently referred to simply as HPLC, is a form of column chromatography 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 (the most common are methanol or acetonitrile). 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. HPLC Modern HPLC systems are highly automated User talk:Emilio8#Image:HPLC.jpg File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... HPLC Modern HPLC systems are highly automated User talk:Emilio8#Image:HPLC.jpg File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... Biochemistry is the chemistry of life. ... Methanol, also known as methyl alcohol or wood alcohol, is a chemical compound with chemical formula CH3OH. It is the simplest alcohol, and is a light, volatile, colourless, flammable, poisonous liquid that is used as an antifreeze, solvent, fuel, and as a denaturant for ethyl alcohol. ... Acetonitrile is an organic molecule, often used as a solvent, with the chemical formula of CH3CN. Also known as methyl cyanide, it is the simplest of the organic nitriles. ...


Reversed phase (RP) 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 mobile phase is increased. RP-HPLC has lower resolution than GC.


Gel permeation chromatography

Gel permeation chromatography, also known as size exclusion chromatography or Sephadex gel chromatography, separates molecules on basis of size. Smaller molecules enter a porous media and take longer to exit the column, hence larger particles leave the column first. GPC is good for determining polymer molecular weight distribution, but is low resolution. Gel permeation chromatography (GPC) also known as size exclusion chromatography (SEC) is a chromatographic method in which molecules are separated based on their size. ... The word resolution has several meanings, depending on context. ...


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. Affinity chromatography is a biochemical separation method that combines size fractionation capability of gel permeation chromatography with the ability to design a stationary phase that reversibly binds to a known subset of molecules. ... Referring to the health, strength and durability of something. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... A fusion protein is a protein created through genetic engineering from two or more proteins/peptides. ... Contents // Categories: Biochemistry stubs | Molecular biology ... Biotin, also known as vitamin H or B7 and C10H16N2O3S (Biotin; Coenzyme R, Biopeiderm), is a water-soluble B-complex vitamin which is important in the catalysis of essential metabolic reactions to synthesize fatty acids, in gluconeogenesis, and to metabolize leucine. ... An antigen is a molecule that stimulates the production of antibodies. ... TaG stands for Touch and Go, which is arguably the fastest growing class in karting. ...


Countercurrent chromatography

Countercurrent chromatography This article is in need of attention from an expert on the subject. ...


See also

This is a description of how to use paper chromatography of amino acids (to identify amino acids). ...

External Links

  • Library 4 Science online books about chromatography.
  • HPLC Find - A directory of HPLC sites on the web, an HPLC search, instrument auctions, conference calendar, liquid chromatography resources, discussion groups, and magazines.

  Results from FactBites:
 
A Guide to HPLC (138 words)
HPLC is a popular method of analysis because it is easy to learn and use and is not limited by the volatility or stability of the sample compound.
history section illustrates the HPLC's evolution from the 1970's to the 1990's.
Modern HPLC has many applications including separation, identification, purification, and quantification of various compounds.
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