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Fast protein liquid chromatography (FPLC) - Term Paper Example

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Fast Protein Liquid Chromatography Background Chromatography is a method of separation utilized in laboratories for separation of chemicals dissolved within the same solvent. This is otherwise known as a mixture. It was developed by a Russian-Italian scientist (Mikhail Tsvet) in 1900s…
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Fast protein liquid chromatography (FPLC)
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Download file to see previous pages Furthermore, it supposes that the chromatographic column contains an infinite number of separate layers (theoretical plates). Separate equilibrations of the sample between the stationary and mobile phase occur in these layers. The analyte moves down the column by transfer of equilibrated mobile phase from one ‘plate’ to the next. There is a more convincing theory, ‘the rate theory.’ This theory depends on the speed of elution and thus speeds of diffusion of the dissolved particles. The analysis and application of this theory leads to the Van Deemter equation. This equation relates the variance per unit length of a separation column to the linear mobile phase velocity by considering several factors. They are physical, kinetic, and thermodynamic properties of a separation. The physical factors are such as; A) Eddy diffusion. B) Longitudinal diffusion C) Resistance to mass transfer It (chromatography) is thus seen to exploit the differences in partitioning behavior between a mobile phase and a stationary phase to separate the components in a mixture. These components contained within a mixture may interact with the stationary phase based on charge, differing solubility or adsorption capability. Several terminologies are associated with the process of chromatography; a) The analyte- this is the substance to be separated during chromatography. b) Bonded phase- this is a stationary phase that is covalently bonded to the support particles or to the inside wall of the tube being utilized.. c) A chromatogram is the visual output of the chromatograph. d) The eluate is the mobile phase that is leaving the separation column. e) The eluent is the solvent that carries/dissolves the analyte. f) The immobilized phase is a stationary phase that is immobilized on the support particles, or on the inner wall of the column tubing. It is similar to the bonded phase g) The mobile phase is the phase that moves in a definite direction. h) The solute refers to the sample components in a solvent. i) The solvent refers to any substance capable of solubilizing another substance. This is important especially in the liquid mobile phase in liquid chromatography. Several methods of chromatography exist as well (singh). They include; 1) Chiral chromatography 2) Countercurrent chromatography 3) Pyrolysis gas chromatography 4) Simulated moving bed chromatography 5) Reversed phase chromatography 6) Two dimensional chromatography 7) Expanded bed adsorption chromatography 8) Size exclusion chromatography 9) Ion exchange chromatography 10) Supercritical fluid chromatography 11) FPLC The FPLC is the method of interest in this case. The FPLC method was developed and marketed in Sweden by the Pharmacia Company in 1982. It was originally called fast performance liquid chromatography. Principle of functioning The purpose of purifying proteins with FPLC is to deliver quantities of the target protein at sufficient purity. This is done in a way that ensures the protein is in a biologically active state to suit its further use. Furthermore this can mean pure enough that the biological activity of the target is retained. This high level of purity requires preliminary preparation of the sample. This is mostly by IEC. In most FPLC systems, there are two solvents/ buffers (A, B). There is also a resin that is chosen so that the protein of interest will bind to it by a charge interaction. When the sample and mix of buffer (100% A) and protein is introduced, the protein will bind to ...Download file to see next pagesRead More
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