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Chromatography is a laboratory technique for the separation of a mixture. The mixture is dissolved in a fluid called the mobile phase, which carries it through a system on which is fixed a material called the stationary phase. The different constituents of the mixture have different affinities for the stationary phase. The different molecules stay longer or shorter on the stationary phase, depending on their interactions with its surface sites. So, they travel at different apparent velocities in the mobile fluid, causing them to separate. The separation is based on the differential partitioning between the mobile and the stationary phases. Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus affect the separation.
Ultraviolet–visible spectroscopy or ultraviolet–visible spectrophotometry refers to absorption spectroscopy or reflectance spectroscopy in part of the ultraviolet and the full, adjacent visible regions of the electromagnetic spectrum. This means it uses light in the visible and adjacent ranges. The absorption or reflectance in the visible range directly affects the perceived color of the chemicals involved. In this region of the spectrum, atoms and molecules undergo electronic transitions. Absorption spectroscopy is complementary to fluorescence spectroscopy, in that fluorescence deals with transitions of electrons from the excited state to the ground state, while absorption measures transitions from the ground state to the excited state.
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. The chromatography column is packed with fine, porous beads which are composed of dextran polymers (Sephadex), agarose (Sepharose), or polyacrylamide. The pore sizes of these beads are used to estimate the dimensions of macromolecules. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers.
High-performance liquid chromatography (HPLC), formerly referred to as high-pressure liquid chromatography, is a technique in analytical chemistry used to separate, identify, and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out of the column.
Gel permeation chromatography (GPC) is a type of size exclusion chromatography (SEC), that separates analytes on the basis of size, typically in organic solvents. The technique is often used for the analysis of polymers. As a technique, SEC was first developed in 1955 by Lathe and Ruthven. The term gel permeation chromatography can be traced back to J.C. Moore of the Dow Chemical Company who investigated the technique in 1964 and the proprietary column technology was licensed to Waters Corporation, who subsequently commercialized this technology in 1964. GPC systems and consumables are now also available from a number of manufacturers. It is often necessary to separate polymers, both to analyze them as well as to purify the desired product.
Gas chromatography (GC) is a common type of chromatography used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. Typical uses of GC include testing the purity of a particular substance, or separating the different components of a mixture. In preparative chromatography, GC can be used to prepare pure compounds from a mixture.
Column chromatography in chemistry is a chromatography method used to isolate a single chemical compound from a mixture. Chromatography is able to separate substances based on differential adsorption of compounds to the adsorbent; compounds move through the column at different rates, allowing them to be separated into fractions. The technique is widely applicable, as many different adsorbents can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process. The latter prevents cross-contamination and stationary phase degradation due to recycling. Column chromatography can be done using gravity to move the solvent, or using compressed gas to push the solvent through the column.
In linear polymers the individual polymer chains rarely have exactly the same degree of polymerization and molar mass, and there is always a distribution around an average value. The molar mass distribution describes the relationship between the number of moles of each polymer species (Ni) and the molar mass (Mi) of that species. The molar mass distribution of a polymer may be modified by polymer fractionation.
Thin-layer chromatography (TLC) is a chromatography technique used to separate non-volatile mixtures. Thin-layer chromatography is performed on a sheet of an inert substrate such as glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase.
Fast protein liquid chromatography (FPLC), is a form of liquid chromatography that is often used to analyze or purify mixtures of proteins. As in other forms of chromatography, separation is possible because the different components of a mixture have different affinities for two materials, a moving fluid and a porous solid. In FPLC the mobile phase is an aqueous solution, or "buffer". The buffer flow rate is controlled by a positive-displacement pump and is normally kept constant, while the composition of the buffer can be varied by drawing fluids in different proportions from two or more external reservoirs. The stationary phase is a resin composed of beads, usually of cross-linked agarose, packed into a cylindrical glass or plastic column. FPLC resins are available in a wide range of bead sizes and surface ligands depending on the application.
A refractometer is a laboratory or field device for the measurement of an index of refraction (refractometry). The index of refraction is calculated from Snell's law while for mixtures, the index of refraction can be calculated from the composition of the material using several mixing rules such as the Gladstone–Dale relation and Lorentz–Lorenz equation.
An analyser or analyzer is a tool used to analyze data. For example, a gas analyzer tool is used to analyze gases. It examines the given data and tries to find patterns and relationships. An analyser can be a piece of hardware or software.
Static light scattering is a technique in physical chemistry that measures the intensity of the scattered light to obtain the average molecular weight Mw of a macromolecule like a polymer or a protein in solution. Measurement of the scattering intensity at many angles allows calculation of the root mean square radius, also called the radius of gyration Rg. By measuring the scattering intensity for many samples of various concentrations, the second virial coefficient A2, can be calculated.
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.
Absolute molar mass is a process used to determine the characteristics of molecules.
Polymer characterization is the analytical branch of polymer science.
Multiangle light scattering (MALS) describes a technique for measuring the light scattered by a sample into a plurality of angles. It is used for determining both the absolute molar mass and the average size of molecules in solution, by detecting how they scatter light. Collimated light from a laser source is most often used, in which case the technique can be referred to as multiangle laser light scattering (MALLS). The insertion of the word “laser” was intended to reassure those used to making light scattering measurements with conventional light sources such as Hg-arc lamps that low angle measurements could now be made. Until the advent of lasers and their associated fine beams of narrow width, the width of conventional light beams used to make such measurements prevented data collection at smaller scattering angles. In recent years, since all commercial light scattering instrumentation use laser sources, this need to mention the light source has been dropped and the term MALS is used throughout.
A chromatography detector is a device used in gas chromatography (GC) or liquid chromatography (LC) to detect components of the mixture being eluted off the chromatography column. There are two general types of detectors: destructive and non-destructive. The destructive detectors perform continuous transformation of the column effluent with subsequent measurement of some physical property of the resulting material. The non-destructive detectors are directly measuring some property of the column eluent and thus affords greater analyte recovery.
Capillary electrochromatography (CEC) is a chromatographic technique in which the mobile phase is driven through the chromatographic bed by electroosmosis. Capillary electrochromatography is a combination of two analytical techniques, high-performance liquid chromatography and capillary electrophoresis. Capillary electrophoresis aims to separate analytes on the basis of their mass-to-charge ratio by passing a high voltage across ends of a capillary tube, which is filled with the analyte. High-performance liquid chromatography separates analytes by passing them, under high pressure, through a column filled with stationary phase. The interactions between the analytes and the stationary phase and mobile phase lead to the separation of the analytes. In capillary electrochromatography capillaries, packed with HPLC stationary phase, are subjected to a high voltage. Separation is achieved by electrophoretic migration of solutes and differential partitioning.
The Charged Aerosol Detector (CAD) is a detector used in conjunction with high-performance liquid chromatography (HPLC) and ultra high-performance liquid chromatography (UHPLC) to measure the amount of chemicals in a sample by creating charged aerosol particles which are detected using an electrometer. It is commonly used for the analysis of compounds that cannot be detected using traditional UV/Vis approaches due to their lack of a chromophore. The CAD can measure all non-volatile and many semi-volatile analytes including, but not limited to, antibiotics, excipients, ions, lipids, natural products, biofuels, sugars and surfactants. The CAD, like other aerosol detectors, falls under the category of destructive general-purpose detectors.
References
- ↑ Undergraduate Instrumental Methods of Analysis. James W. Robinson, Eileen M. Skelly Frame, George M. Frame II. Marcel Dekker, 2005, p. 810.
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