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CHROMATOGRAPHY Chromatography is a technique for separating and identifying the components of a mixture. Many different forms of chromatography are used but they all work on the same principle. The components of the mixture have different affinities for two phases: a stationary phase and a mobile phase and so are separated as the mobile phase moves through the stationary phase. A component which has a strong attraction for the mobile phase will move quickly, whereas a component with a strong attraction for the stationary phase will be held back. If the stationary and mobile phases are carefully chosen, the different components will move at different speeds and so be separated effectively. Polar compounds, for example, are more likely to move quickly when the mobile phase is a polar solvent. Two main types of chromatography There are two main types of chromatography: partition and adsorption chromatography. Partition chromatography Chromatography using a non-volatile liquid stationary phase held on an inert solid surface is known as partition chromatography. The components distribute themselves between the two phases according to their relative solubility. Paper chromatography and gas liquid chromatography are examples. The more soluble or volatile the component, the faster it will move. Adsorption chromatography Chromatography which uses a solid stationary phase and a mobile liquid or gas phase is known as adsorption chromatography. Some components of the mixture are attracted to the solid surface and the other components which are less strongly bonded travel faster with the mobile phase. Thin-layer chromatography is an example. As the stationary phase is generally a polar solid, the more polar solutes are more readily adsorbed than the less polar solutes. Thin-layer chromatography (TLC) Thin-layer chromatography is an example of adsorption chromatography. It follows the same procedure as paper chromatography, with small spots of the test solutions placed on the base line using a capillary tube. The stationary phase is a thin layer of absorbent particles of alumina or silica supported on a glass or thin plastic plate and the mobile phase is a liquid solvent. The different components separate and can be identified. The technique is used in qualitative analysis to determine whether a substance is pure. TLC is a type of planar chromatography.
Types of Chromatography Adsorption Chromatography
Adsorption chromatography is probably one of the oldest types of chromatography around. It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibriation between the mobile and stationary phase accounts for the separation of different solutes.
(2010) Introduction to Chromatography
An introduction to chromatographic analysis, describing the different types of chromatography including TLC, GLC, HPLC and ion chromatography, their application areas and basic principles of operation. It is illustrated with simple diagrams, photographs of equipment and chromatograms illustrating practical aspects of the technique.
Journal of Chromatography A, 1988
This investigation deals with protein retention behaviour in high-performance anion-exchange chromatography in terms of the average distance of approach between the protein solute and the positively charged anion-exchange stationaryphase surface. The theoretical treatment is based on a modified Debye-Hiickel theory for spherical impenetrable ions, where the electrostatic potential energy has been related to the chromatographic capacity factor, k'. Results are presented for three globular proteins, eluted isocratically from a Mono-Q strong anion-exchange resin with sodium choride as the displacer salt by a mobile phase with pH in the range 5.5&9.60. Analysis of experimental retention data indicates that topographically predelined, charged regions on the protein surface, called ionotopes, control the orientation and approach distance of the protein solute.
Chromatography can be defined as a method of separating mixture of components into individual components through equilibrium distribution between two phases.
Journal of Chromatography A, 2005
This article describes a new complementary peptide separation and purification concept that makes use of a novel mixed-mode reversedphase/weak anion-exchange (RP/WAX) type stationary phase. The RP/WAX is based on N-(10-undecenoyl)-3-aminoquinuclidine selector, which is covalently immobilized on thiol-modified silica particles (5 m, 100Å pore diameter) by radical addition reaction. Remaining thiol groups are capped by radical addition with 1-hexene. This newly developed separation material contains two distinct binding domains in a single chromatographic interactive ligand: a lipophilic alkyl chain for hydrophobic interactions with lipophilic moieties of the solute, such as in the reversed-phase chromatography, and a cationic site for anion-exchange chromatography with oppositely charged solutes, which also enables repulsive ionic interactions with positively charged functional groups, leading to ion-exclusion phenomena. The beneficial effect that may result from the combination of the two chromatographic modes is exemplified by the application of this new separation material for the chromatographic separation of the N-and C-terminally protected tetrapeptide N-acetyl-Ile-Glu-Gly-Arg-p-nitroanilide from its side products. Mobile phase variables have been thoroughly investigated to optimize the separation and to get a deeper insight into the retention and separation mechanism, which turned out to be more complex than any of the individual chromatography modes alone. A significant anion-exchange retention contribution at optimal pH of 4.5 was found only for acetate but not for formate as counter-ion. In loadability studies using acetate, peptide masses up to 200 mg could be injected onto an analytical 250 mm × 4 mm i.d. RP/WAX column (5 m) still without touching bands of major impurity and target peptide peaks. The corresponding loadability tests with formate allowed the injection of only 25% of this amount. The analysis of the purified peptide by capillary high-performance liquid chromatography (HPLC)-UV and HPLC-ESI-MS employing RP-18 columns revealed that the known major impurities have all been removed by a single chromatographic step employing the RP/WAX stationary phase. The better selectivity and enhanced sample loading capacity in comparison to RP-HPLC resulted in an improved productivity of the new purification protocol. For example, the yield of pure peptide per chromatographic run on RP/WAX phase was by a factor of about 15 higher compared to the standard gradient elution RP-purification protocol.