Chromatography is a laboratory technique for mixed separation. The mixture is dissolved in a liquid called the mobile phase, which carries it through a structure holding another material called stationary phase. Various mixed constituents move at different speeds. , causing them to separate. Separation is based on the differential partition between mobile and stationary phases. The subtle differences in the partition coefficient of the compound result in differential retention in the stationary phase and thus affect the separation.
Chromatography can be either preparative or analytical. The purpose of preparative chromatography is to separate the mixed components for later use, and thus constitute a form of purification. Analytical chromatography is performed normally with a smaller amount of material and to establish the presence or measurement of the relative analytical proportions in a mixture. The two are not mutually exclusive.
Video Chromatography
Etymology and pronunciation
Chromatography, pronounced , comes from the Greek ????? chroma , meaning "color", and ??????? graphein , which means "write".
Maps Chromatography
History
Chromatography was first used in Russia by the Italian-born scientist Mikhail Tsvet in 1900. He continued to work with chromatography in the first decades of the 20th century, mainly for the separation of plant pigments such as chlorophyll, carotene, and xanthophylls. Because these components have different colors (green, orange, and yellow, respectively) they give the technique its name. New chromatographic types developed during the 1930s and 1940s make this technique useful for many separation processes.
Chromatographic techniques developed substantially as a result of the work of Archer John Porter Martin and Richard Laurence Millington Synge during the 1940s and 1950s, where they won the 1952 Nobel Prize in Chemistry. They set the basic principles and techniques of partition chromatography, and their work encourages the rapid development of several chromatographic methods: paper chromatography, gas chromatography, and what are known as high performance liquid chromatography. Since then, technology has grown rapidly. The researchers found that the main principles of Tsvet chromatography can be applied in various ways, resulting in the different types of chromatography described below. Progress continues to improve technical performance of chromatography, enabling an increasingly similar molecular separation.
Chromatography requirements
- analte is the substance separated during chromatography. It's also usually what is needed from the mix.
- Analytical chromatography is used to determine the presence and possibly also the concentration of the analyte in the sample.
- The bound phase is a stationary phase which is covalently bonded to the support particles or to the inner wall of the column tubing.
- The chromatogram is the visual output of chromatography. In the case of optimal separation, different peaks or patterns on the chromatogram correspond to different components of a separate mixture.
- Mapped on the x-axis is the retention time and plotted on the y-axis of the signal (eg obtained by spectrophotometer, mass spectrometer or other detector) according to the response made by the analyte coming out of the system. In the case of an optimal system, the signal is proportional to the specific concentration of the analyte being separated.
- A chromatography is a device that enables advanced separation, such as gas chromatography or liquid chromatography separation.
- Chromatography is a physical separation method that distributes components to separate between two phases, one stationary (the stationary phase), the other (mobile phase) moves in a definite direction.
- The eluat is the mobile phase that leaves the column. This is also called effluent.
- The eluent is the solvent that carries the analyte.
- eluite is an analyte, a dissolved solute.
- A series of eluotropics is a list of solvents rated according to their eluting power.
- The immobilized phase is a stationary phase immobilized on the support particles, or on the inner wall of the column tubing.
- The mobile phase is the phase that moves in the exact direction. It may be liquid (LC and Capillary Electrochromatography (CEC)), gas (GC), or supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of a separated/analyzed sample and a solvent that drives the sample through a column. In the case of HPLC the mobile phase consists of a non-polar solvent such as hexane in a normal phase or a polar solvent such as methanol in reversed phase chromatography and the sample is separated. The mobile phase moves through the chromatography column (stationary phase) in which the sample interacts with the stationary phase and is separated.
- preparative chromatography is used to purify the amount of substances sufficient for further use, rather than analysis.
- The retention time is the characteristic time required for a given analytes to pass through the system (from inlet column to detector) under specified conditions. See also: Kovats' retention index
- Sample is a problem analyzed in chromatography. These may consist of a single component or perhaps a mixture of components. When samples are treated in the course of an analysis, the phases or phases containing the interest analytes are referred to as samples whereas everything that comes out of interest separated from the previous sample or in the analysis process is referred to as waste.
- The dissolved refers to the sample component in the partition chromatography.
- The solvent refers to a substance capable of dissolving other substances, and especially the aqueous phase in liquid chromatography.
- The stationary phase is the substance that remains in place for the chromatographic procedure. Examples include silica coatings in thin layer chromatography
- detector refers to the instruments used to detect analytes qualitatively and quantitatively after separation.
Chromatography is based on the concept of partition coefficient. Each partition of solute between two solvents that can not mix. When we make a moving solvent (with adsorption on a solid support matrix) and other phones, this results in the most common chromatographic applications. If matrix support, or stationary phase, is polar (eg paper, silica, etc.) phase chromatography forward, and if non-polar (C-18) it is the reverse phase.
Techniques with chromatographic bed shape
column chromatography
Chromatographic column is a separation technique in which a stationary bed is in a tube. The solid phase solid phase particles or support coated with a stationary stationary phase can fill the entire volume inside the tube (packed column) or concentrated on or along the inner tube wall leaving an unrestricted open lane for the mobile phase in the center of the tube (open tubular column). The difference in movement rate through the medium is calculated to different sample retention times.
In 1978, W. Clark still introduced a modified version of column chromatography called flash column chromatography (flash). This technique is very similar to traditional column chromatography, except that the solvent is pushed through the column by applying positive pressure. This allows most of the separation done in less than 20 minutes, with better separation compared to the old method. Modern flash chromatography systems are sold as pre-packaged plastic cartridges, and solvents are pumped through the cartridges. The system can also be connected to detectors and collector fractions that provide automation. The introduction of gradient pumps results in faster separation and fewer solvent usage.
In the expanded bed adsorption, the fluidized bed is used, rather than the solid phase made by the packaged bed. This allows the removal of the initial cleaning steps such as centrifugation and filtration, for the culture broth or the damaged cell porridge.
Phosphocellulosic chromatography utilizes the affinity of binding of many DNA binding proteins to phosphocellulose. The stronger the protein interaction with DNA, the higher the salt concentration needed to elute the protein.
Planar Chromatography
Planar chromatography is a separation technique in which the stationary phase is present as or on the plane. The aircraft may be paper, serving as such or impregnated by a substance as a stationary bed (paper chromatography) or a solid particle layer dispersed on a support such as a glass plate (thin layer chromatography). Different compounds in the sample mixture traveled a different distance according to how strongly they interacted with the stationary phase compared to the mobile phase. Specific Retention Factors (R f ) of each chemical can be used to help identify unknown substances.
Paper chromatography
Paper chromatography is a technique involving the placement of small dots or sample solution lines on chromatographic paper strips . The paper is placed in a container with a superficial and sealed solvent layer. As the solvent rises through the paper, it fills the mixture of samples, which begin to crawl the paper with solvents. The paper is made of cellulose, polar substances, and compounds in the mixture moving further if they are non-polar. More polar substances are bound with cellulose paper faster, and therefore have not traveled so far. Thin layer chromatography
Thin layer chromatography (TLC) is a widely used laboratory technique used to separate different biochemicals based on their size and is similar to paper chromatography. However, instead of using stationary stationary paper, it involves the stationary phase of a thin layer of adsorbent such as silica gel, alumina, or cellulose on a flat inert substrate. TLC is very versatile; multiple samples can be separated simultaneously on the same layer, making them particularly useful for screening applications such as drug level testing and water purity. The likelihood of cross contamination is low as each separation is done on a new layer. Compared to paper, it has the advantage of faster processing, better separation, better quantitative analysis, and choice between various adsorbents. For better resolution and faster separation using fewer solvents, high performance TLC can be used. The older popular use is to distinguish chromosomes by observing the distance in the gel (separation is a separate step).
Moving chromatography
The basic principles of displacement chromatography are: A molecule with a high affinity for a chromatographic matrix (displacer) competes effectively to bind the site, and thus removes all molecules with a lower affinity. There is a clear distinction between elution displacement and chromatography. In elution mode, a substance usually appears from a column at a narrow Gaussian peak. A broad peak separation, preferably to the baseline, is desirable for maximum purification. The speed at which each mixed component moves down the column in the elution mode depends on many factors. But for two substances to travel at different speeds, and thus can be solved, there must be substantial differences in some interactions between biomolecules and chromatographic matrices. The operation parameters are adjusted to maximize the effect of this difference. In many cases, the basic separation of the peaks can be achieved only by gradient elution and low column load. Thus, two drawbacks to elution mode chromatography, particularly on the preparative scale, are operational complexity, due to gradient solvent pumping, and low throughput, due to low column loads. The displacement chromatography has an advantage over elution chromatography in components that are settled into consecutive zones of pure substance rather than "peak". Because the process takes advantage of the isotherm's nonlinearity, larger column feeds can be separated on specific columns with purified components recovering at significantly higher concentrations.
Technique with the physical state of the mobile phase
Gas chromatography
Gas chromatography (GC), also sometimes known as gas-liquid chromatography, (GLC), is a separation technique in which the gas phase is a gas. Gas chromatographic separation is always done in columns, which are usually "packed" or "capillary". The packed columns are regular gas chroatographic work horse, cheaper and easier to use and often provide adequate performance. Capillary columns generally provide a much more superior resolution and although more expensive are becoming widely used, especially for complex mixtures. Both types of columns are made of non-adsorbent and inert chemicals. Stainless steel and glass are common materials for packed columns and quartz or fused silica for capillary columns.
Gas chromatography is based on equilibrium partition analyzer between solid or viscous liquid phase (often liquid silicone based material) and mobile gas (most often helium). The stationary phase is attached to the inside of a small diameter (usually 0.53 - 0.18 mm in diameter) of glass or silica-fused tube (capillary column) or solid matrix in larger metal tubes (packed columns). It is widely used in analytical chemistry; although the high temperatures used in GC make it unsuitable for biopolymers or high molecular weight proteins (heat to silence them), often found in biochemistry, particularly suitable for use in petrochemical, environmental monitoring and recovery, and industrial chemistry. It is also used extensively in chemical research.
Liquid chromatography
Liquid chromatography (LC) is a separation technique in which the mobile phase is fluid. This can be done either in a column or plane. Current liquid chromatography which generally uses very small packing particles and relatively high pressures is referred to as high performance liquid chromatography (HPLC).
In HPLC, the sample is forced by a liquid at high pressure (mobile phase) through a column packed with a stationary phase comprising irregularly shaped particles or spheres, porous monolithic layers, or porous membranes. HPLC has historically been divided into two distinct sub-classes based on mobile and stationary phase polarities. The method in which the stationary phase is more polar than the mobile phase (eg, toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC) and vice versa (eg, methanol-water mixture as cell phone). phase and C18 (octadecylsilyl) as a stationary phase) is called reversed phase liquid chromatography (RPLC).
Specific techniques under this broad heading are listed below.
Affinity chromatography
Affinity chromatography is based on selective non-covalent interaction between the analyte and the specific molecule. It's very specific, but not too strong. It is often used in biochemistry in the purification of proteins that are bound to the tags. These fusion proteins are labeled with compounds such as his tag, biotin or antigen, which bind the stationary phase in particular. After purification, some of these tags are usually removed and pure protein is obtained.
Affinity chromatography often uses the affinity of biomolecules for metals (Zn, Cu, Fe, etc.). Columns are often set up manually. Traditional affinity columns are used as a preparatory step to clean up unwanted biomolecules.
However, HPLC techniques exist that utilize the nature of affinity chromatography. Immobilized Metal Affinity Chromatography (IMAC) is useful for separating these molecules based on relative affinity for metals (ie Dionex IMAC). Often these columns can be loaded with various metals to create columns with targeted affinity.
Supercritical fluid chromatography
Supercritical fluid chromatography is a separation technique in which the mobile phase is fluid above and relatively close to its critical temperature and pressure.
Technique with separation mechanism
ion exchange chromatography
Ion exchange chromatography (commonly referred to as ion chromatography) uses an ion exchange mechanism to separate the analyte based on each cost. Usually done in columns but also can be useful in planar mode. Ion exchange chromatography uses a stationary phase of charge to separate charged compounds including anions, cations, amino acids, peptides, and proteins. In the conventional method the stationary phase is an ion exchange resin carrying a charged charge group that interacts with the group of compounds with the opposite charge to be maintained. There are two types of ion exchange chromatography: Cation Exchange and Anion-Exchange. In Stationary phase Cation Chromatography the stationary phase has a negative charge and the exchange ion is cation, whereas, in Anion-Exchange Chromatography the stationary phase has a positive charge and the exchange ion is an anion. Ion exchange chromatography is commonly used to purify proteins using FPLC.
Exclusion size chromatography
Sole exclusion chromatography (SEC) is also known as gel permeation chromatography (GPC) or gel filtration chromatography and separates molecules according to their size (or more precisely according to their hydrodynamics). diameter or hydrodynamic volume). Smaller molecules can enter the pores of the media and, therefore, the molecules are trapped and removed from the flow of the mobile phase. The average residence time in the pores depends on the effective size of the analyte molecule. However, molecules larger than the average pore size of the packaging are excluded and thus essentially have no retention; Such species are the first to elute. This is generally a low-resolution chromatographic technique and is therefore often used for the final purification step. It is also useful for determining the tertiary structure and quaternary structure of the purified proteins, especially since it can be carried out under the conditions of the original solution.
Separate layer adsorption chromatography separation
The expanded column of columnation adsorption column (EBA) for the biochemical separation process comprises a fluid distribute distribution distributor having its own cleaning function under a porous blocking screen plate at the bottom of the expanded bed, assembling the top nozzle having a backflush cleaning function at the top of the site expanded sleep, better distribution of liquor raw materials added to the expanded bed ensuring that the liquid passes through the expanded bed layer showing the piston flow state. The expanded bed layer shows the piston flow state. The extended bed chromatography separation column has the advantage of increasing the separation efficiency of the expanded bed.
Bed-bed chromatography is a convenient and effective technique for capturing proteins directly from unclassified raw samples. In EBA chromatography, the bed is initially expanded with upward flow of the equilibrium buffer. Raw feed, a mixture of dissolved proteins, contaminants, cells, and cell debris, then passed up through an expanded bed. Target proteins are captured on the adsorbent, while particulates and contaminants pass through it. The change in the buffer elution while maintaining upward flow results in the desorption of the target protein in the extended-bed mode. Alternatively, if the flow is reversed, the adsorbed particles will quickly settle and the protein can be absorbed by the elution buffer. The mode used for elution (sleep-expanded versus fixed-bed) depends on the characteristics of the feed. After elution, the adsorbent is cleaned with a predetermined clean-in-place (CIP) solution, with cleaning followed by column regeneration (for further use) or storage.
Custom techniques
Reversed phase chromatography
Reversed-phase chromatography (RPC) is a liquid chromatographic procedure where the motion phase is significantly more polar than the stationary phase. Named so because in normal phase liquid chromatography, the motion phase is significantly less polar than the stationary phase. Hydrophobic molecules in the motion phase tend to absorb into relatively hydrophobic stationary phases. Hydrophilic molecules in the motion phase will tend to elicit first. Separating columns usually consist of C8 or C18 carbon chains attached to the silica particles substrate.
Hydrophobic interaction chromatography
Hydrophobic interactions between proteins and chromatographic matrices can be utilized to purify proteins. In hydrophobic interaction chromatography matrix material is lightly replaced by hydrophobic groups. These groups may range from the methyl, ethyl, propyl, octyl, or phenyl groups. [] At high salt concentrations, non-polar sidechains on the surface of the protein "interact" with the hydrophobic group; ie, both types of groups are excluded by polar solvents (hydrophobic effects coupled with increased ionic strength). Thus, the sample is applied to a column in a very polar buffer. The eluant is usually an aqueous buffer with decreased salt concentration, increasing the concentration of detergent (which interferes with hydrophobic interactions), or changes in pH.
In general, Hydrophobic Interaction Chromatography (HIC) is advantageous if the sample is sensitive to pH or rough solvent changes commonly used in other types of chromatography but not high salt concentrations. Generally, it is the amount of salt in the buffer that varies. In 2012, MÃÆ'¼ller and Franzreb describe the effect of temperature on HIC using Bovine Serum Albumin (BSA) with four types of hydrophobic resins. This study changed the temperature to affect the affinity of binding of the BSA to the matrix. It was concluded that cycling temperatures from 50 degrees to 10 degrees would not be sufficient to effectively wash all BSAs from the matrix but could be very effective if the columns would only be used multiple times. Using temperature changes to effect allows the lab to cut costs to buy salt and save money.
If high salt concentrations along with temperature fluctuations want to be avoided you can use more hydrophobic to keep up with your samples to deceive. [source] This independent HIC method of salt shows direct isolation of Human Immunoglobulin G (IgG) from serum with satisfactory results and uses Beta-cyclodextrin as a competitor to replace IgG from the matrix. This mostly opens the possibility of using HICs with salt-sensitive samples because we know that high salt concentrations precipitate proteins.
Two-dimensional chromatography
In some cases, the chemistry in the given column is not sufficient to separate some analytes. It is possible to direct a series of unresolved peaks to the second column with different physico-chemical properties (chemical classification). Since the retention mechanism of these new solids differs from the separation of the first dimension, it is possible to separate the compounds by two-dimensional chromatography indistinguishable by one-dimensional chromatography.
The sample is seen in one corner of a square plate, developed, dried with air, then rotated 90 Â ° and usually rebuilt in a second solvent system.
Motion-moving chromatography simulation
The sleep moving simulation technique (SMB) is a variant of high performance liquid chromatography; this is used to separate the particles and/or chemical compounds that would be difficult or impossible to solve otherwise. This increased separation is caused by the valve-and-column arrangement used to extend the stationary phase indefinitely. In sleep chromatography techniques the preparative feed entries and recovery analytes are simultaneous and continuous, but due to practical difficulties with the bed moving continuously, the sleep moving simulation technique is proposed. In the simulation technique to move the sleep instead of moving the bed, the sample entry hole and the exit position of the analyte are moved continuously, giving the impression of a moving bed. True moving sleep chromatography (TMBC) is only a theoretical concept. This simulation, SMBC is achieved by the use of various columns in complex valve settings and settings, providing solvent examples and feed, as well as proper wastewater analyzes and disposal of any column, where it allows redirection at regular intervals. sample entries in one direction, solvent entries in opposite directions, while changing the analyte and removing the exact takeoff position as well.
Pyrolysis gas chromatography
Mass spectrometry of pyrolysis gas chromatography is a method of chemical analysis in which the sample is heated to decomposition to produce smaller molecules separated by gas chromatography and detected using mass spectrometry.
Pyrolysis is the decomposition of thermal materials in an inert or vacuum atmosphere. The sample is put into direct contact with a platinum wire, or placed in a quartz sample tube, and rapidly heated to 600-1000 Â ° C. Depending on the application, higher temperatures are used. Three different heating techniques are used in actual pyrolyzers: isothermal furnaces, inductive heating (Curie Point filaments), and resistive heating using platinum filaments. Large molecules divide at their weakest points and produce smaller and more volatile fragments. These fragments can be separated by gas chromatography. Pyrolysis GC chromatograms are usually complex due to different decomposition products formed. Data can be used as fingerprints to prove the identity of materials or GC/MS data used to identify individual fragments for structural information. To increase the volatility of polar fragments, various methylating reagents can be added to the sample before pyrolysis.
In addition to the use of special pyrolysis, the GC pyrolysis of solid and liquid samples can be carried out directly within the Programmable Temperature Vaporizer (PTV) injector which provides rapid heating (up to 30 ° C/s) and a maximum maximum temperature of 600-650 ° C. This is sufficient for some pyrolysis applications. The main advantage is that no special instrument should be purchased and pyrolysis can be performed as part of routine GC analysis. In this case a quartz inlet GC liner should be used. Quantitative data can be obtained, and good results from derivatization in PTV injectors are also published.
Fast protein liquid chromatography
Fast protein liquid chromatography (FPLC), is a liquid chromatographic form often used to analyze or purify a mixture of proteins. As in other chromatographic forms, separation is possible because different components of the mixture have different affinities for two materials, mobile fluid ("mobile phase") and porous solids (stationary phase). In FPLC, the mobile phase is an aqueous solution, or a "buffer". The buffer flow rate is controlled by the positive-displacement pump and is usually kept constant, while the buffer composition can be varied by drawing fluid in different proportions of two or more external reservoirs. The stationary phase is a resin consisting of beads, usually of a cross-linked agarose, packed in a cylindrical glass or plastic column. FPLC resins are available in various bead sizes and surface ligands depending on the application.
Counter chromatography
Current counter chromatography (CCC) is a liquid-liquid chromatography type, in which the stationary and moving phases are fluid. The principle of operation of CCC equipment requires a column consisting of a circular open tube around the coil. The spindle is rotated in a double axis gyratory movement (cardioid), which causes the variable gravity field (G) to act in the column during each rotation. This movement causes the columns to see one partition step per revolution and the components of the samples separated in the columns due to their partition coefficients between the two unused liquid phases. There are many types of CCCs available today. These include HSCCC (High Speed ​​â € <â €
Unlike Time Counter Chromatography (see above), periodic current counter chromatography (PCC) uses a solid phase of silence and only a liquid phase of motion. Thus it is much more similar to conventional affinity chromatography than opposing chromatography. PCC uses several columns, which during the loading phase are connected in queue. This mode allows to burden the first column in this series without losing the product, which has broken through the column before the resin is fully saturated. This breakthrough product is captured on the next column (s). In the next step, the columns are disconnected from each other. The first column is washed and eluted, while other columns are still loaded. After the first column (initially) re-equilibrates, it is re-introduced to the loading stream, but as the last column. The process then proceeds cyclically.
Chiral chromatography
Chiral chromatography involves the separation of stereoisomers. In the case of an enantiomer, it has no chemical or physical difference apart from a three-dimensional mirror image. Conventional chromatography or other separation processes are unable to separate them. To allow chiral separation to take place, the mobile phase or stationary phase must be chiralally created, providing a distinct linkage between the analyte. Chromatographic Chromatography The HPLC columns (with chiral stationary phase) in normal phase and turning phase are commercially available.
Normal aqueous phase chromatography
Normal phase chromatography (ANP) is characterized by elution behavior from the classic normal phase mode (ie, where the mobile phase is significantly less polar than the stationary phase) where water is one component of the solvent system of the mobile phase. It is distinguished from liquid hydrophilic interaction chromatography (HILIC) where the retention mechanism is largely due to adsorption rather than partitioning.
See also
References
External links
- IUPAC Nomenclature for Chromatography
- Overlapping Change Program - Learn by Simulation
- Video Chromatography - MIT OCW - Digital Lab Techniques Manual
- Chromatography Equation Calculator - MicroSolv Technology Corporation
Source of the article : Wikipedia
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