please explain chromatography in detail...........

Asked by  | 8th Jun, 2008, 11:58: AM

Expert Answer:

it is a broad range of physical methods used to separate and or to analyze complex mixtures. The components to be separated are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed. A mixture of various components enters a chromatography process, and the different components are flushed through the system at different rates. These differential rates of migration as the mixture moves over adsorptive materials provide separation. Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates. The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column. In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production line. Today, there exists a wide market of methods in which industries can accomplish these goals. Chromatography is a very special separation process for a multitude of reasons! First of all, it can separate complex mixtures with great precision. Even very similar components, such as proteins that may only vary by a single amino acid, can be separated with chromatography. In fact, chromatography can purify basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed. Second, chromatography can be used to separate delicate products since the conditions under which it is performed are not typically severe. For these reasons, chromatography is quite well suited to a variety of uses in the field of biotechnology, such as separating mixtures of proteins.

Gas chromatography makes use of a pressurized gas cylinder and a carrier gas, such as helium, to carry the solute through the column. The most common detectors used in this type of chromatography are thermal conductivity and flame ionization detectors. There are three types of gas chromatography that will be discussed here: gas adsorption, gas-liquid and capillary gas chromatography.

Gas adsorption chromatography involves a packed bed comprised of an adsorbent used as the stationary phase. Common adsorbents are zeolite, silica gel and activated alumina. This method is commonly used to separate mixtures of gases.

Gas-liquid chromatography is a more common type of analytical gas chromatography. In this type of column, an inert porous solid is coated with a viscous liquid which acts as the stationary phase. Diatomaceous earth is the most common solid used. Solutes in the feed stream dissolve into the liquid phase and eventually vaporize. The separation is thus based on relative volatilities.

Capillary gas chromatography is the most common analytical method. Glass or fused silica comprise the capillary walls which are coated with an absorbent or other solvent. Because of the small amount of stationary phase, the column can contain only a limited capacity. However, this method also yields rapid separation of mixtures

There are a variety of types of liquid chromatography. There is liquid adsorption chromatography in which an adsorbent is used. This method is used in large-scale applications since adsorbents are relatively inexpensive. There is also liquid- liquid chromatography which is analogous to gas-liquid chromatography. The three types that will be considered here fall under the category of modern liquid chromatography. They are reverse phase, high performance and size exclusion liquid chromatography, along with supercritical fluid chromatography.

Reverse phase chromatography is a powerful analytical tool and involves a hydrophobic, low polarity stationary phase which is chemically bonded to an inert solid such as silica. The separation is essentially an extraction operation and is useful for separating non-volatile components.

High performance liquid chromatography (HPLC) is similar to reverse phase, only in this method, the process is conducted at a high velocity and pressure drop. The column is shorter and has a small diameter, but it is equivalent to possessing a large number of equilibrium stages.

Size exclusion chromatography, also known as gel permeation or filtration chromatography does not involve any adsorption and is extremely fast. The packing is a porous gel, and is capable of separating large molecules from smaller ones. The larger molecules elute first since they cannot penetrate the pores. This method is common in protein separation and purification.

Supercritical fluid chromatography is a relatively new analytical tool. In this method, the carrier is a supercritical fluid, such as carbon dioxide mixed with a modifier. Compared to liquids, supercritical fluids have solubilities and densities have as large, and they have diffusivities and viscosities quite a bit larger. This type of chromatography has not yet been implemented on a large scale.

Ion exchange chromatography is commonly used in the purification of biological materials. There are two types of exchange: cation exchange in which the stationary phase carries a negative charge, and anion exchange in which the stationary phase carries a positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution of varying pH or ionic strength is passed through it. Separation by this method is highly selective. Since the resins are fairly inexpensive and high capacities can be used, this method of separation is applied early in the overall process

Affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds. The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or "affinity tails", that are inserted into the matrix can be genetically engineered to possess a specific affinity. In a process similar to ion exchange chromatography, the desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Fouling of the matrix can occur when a large number of impurities are present, therefore, this type of chromatography is usually implemented late in the process

Paper chromatography is an analytical technique for separating and identifying mixtures that are or can be colored, especially pigments. This can also be used in secondary or primary schools in ink experiments. This method has been largely replaced by thin layer chromatography, however it is still a powerful teaching tool. Two-way paper chromatography, also called two-dimensional chromatography, involves using two solvents and rotating the paper 90° in between. This is useful for separating complex mixtures of similar compounds, for example, amino acidsA small concentrated spot of solution that contains the sample is applied to a strip of chromatography paper about 2 cm away from the base of the plate, usually using a capillary tube for maximum precision. This sample is absorbed onto the paper and may form interactions with it. Any substance that reacts or bonds with the paper cannot be measured using this technique. The paper is then dipped in to a suitable solvent, such as ethanol or water, taking care that the spot is above the surface of the solvent, and placed in a sealed container. The solvent moves up the paper by capillary action, which occurs as a result of the attraction of the solvent molecules to the paper,also this can be explained as differential absorption of the solute components into the solvent. As the solvent rises through the paper it meets and dissolves the sample mixture, which will then travel up the paper with the solvent solute sample. Different compounds in the sample mixture travel at different rates due to differences in solubility in the solvent, and due to differences in their attraction to the fibers in the paper. Paper chromatography takes anywhere from several minutes to several hours

Answered by  | 9th Jun, 2008, 08:30: AM

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