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Fast Protein Liquid Chromatography - Term Paper Example

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This term paper "Fast Protein Liquid Chromatography" focuses on a method of separation utilized in laboratories for separation of chemicals dissolved within the same solvent. The purpose of purifying proteins with FPLC is to deliver quantities of the target protein at sufficient purity…
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Fast Protein Liquid Chromatography
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Extract of sample "Fast Protein Liquid Chromatography"

Fast Protein Liquid Chromatography Background Chromatography is a method of separation utilized in laboratories for separation of chemicals dissolvedwithin the same solvent. This is otherwise known as a mixture. It was developed by a Russian-Italian scientist (Mikhail Tsvet) in 1900s. It was further developed and improved by Archer John Porter Martin and Richard Laurence Millington Synge in 1940s and 1950s period. They described the plate theory of chromatography. It states that, in the chromatography system, the mobile and stationary phases exist in equilibrium. 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 the resin first leaving the aqueous state in solvent A. It will then dissociate and re- dissolve in solvent B as it passes through the column. This is achieved by alternating the flowing solvent within the column. That is, protein-solvent ‘A’ mixture passes first and the protein separates out. The concentration of solvent ‘B’ then increases gradually to 100% as it dissolves the protein. This gradual increase is termed as ‘gradient’. The resulting solution (The effluent) then passes through two main detectors which measure salt concentration by conductivity and the protein concentration by its absorption of ultraviolet light (Sheehan and osulivan). The effluent then proceeds to be collected into the collecting tubes. The diagram below is a basic schematic of the entire process. It has been borrowed from “Protein Purification Protocols” (Sheehan and osulivan). The above procedure is standard for most proteins, but may be varied for different proteins by manipulation of the resin type, or the solvent type. (Sheehan and osulivan) Components of the system A typical FPLC machine consists of one or two high-precision pumps, a control unit, a chromatographic column and a detection system. It also has a fraction collector. 1) Pump. The two pumps in the apparatus draw solvent from two different bottles, and combine them into a single flow that goes to the mixing chamber. The pumps are highly precise in their functioning to reduce errors in the process. 2) Injection loop. This is a segment of tubing of known volume that gets filled with the sample to be analyzed. Volumes are small for lab machines. (25 or 100 microliters) 3) Injection valve: This is a valve which links the mixer and sample loop to the column. The valve has the ability to load the sample, wash out the pumps and injecting the sample to the column. 4) The column: The column is a glass or plastic cylinder packed with beads of resin and filled with the buffer solution. It is mounted vertically with the buffer flowing downward. It normally has a mesh below to keep the beads in the column. Most columns contain silica matrix and sianol groups tightly packed. The beads in the column vary from 5-10micrometres in diameter. 5) Fraction collector: The fraction collector is typically a rotating rack that can be filled with test tubes. It allows for the samples to be collected. 6) Monitor/Recorder. This may be a chart recorder or a computer. 7) Flow Cells: The effluent from the column passes through flow cells to measure the concentration of protein in the effluent (Sharma). This is a small quartz chamber where light is allowed to pass through. It is measured by UV light absorption at 280nm or 540nm (Rao). Ion Exchange Chromatography This is a method of separation based on a molecule’s intrinsic charge. It has two main types; cation exchange chromatography and anion exchange chromatography. In the cation exchange, the positively charged particles are retained. In Anion exchange, the negative particles are maintained. This method of separation is often employed first in the purification processes required for obtaining super pure samples for analysis. An example is for that of X-ray crystallography that requires >99% purity. It is then followed by procedures such as the FPLC or HPLC. It has the advantage of being able to separate virtually any component (molecule or ion) that bears a charge. These include proteins, amino acids and a wide array of ions. Applications Apart from being very useful in purification of DNA and amino acids for biochemical analyses, the principle can be applied in other fields. They include; metallurgy. Here, the process is used for the separation of actinides, PUREX, water purification and softening, semiconductor preparation, and beverage production. Principle of Function It follows the same general principle of function of the FPLC where a sample is introduced to the column where separation occurs. The column contains cellulose or agarose or cellulose beads that are coated by charged molecules. This causes the separation of the required charged molecules or ions. The “wash off” from the beads is instead done by aqueous salt solution. The basis of this is ion displacement due to varying affinities. This is in comparison to that in FPLC which was based on varying solubility. The diagram shows the general principle of the procedure. To optimize binding of all the introduced charged molecules, the mobile phase is generally a low to medium conductivity solution. This is normally a salt solution with controlled concentration. The adsorption of the molecules to the solid phase (resin) is due to the ionic interaction between the oppositely charged ionic groups. The strength of the interaction is determined by the valence and molecule ionization. It is also a factor of location of the charges on the molecule in complex molecules (folded proteins). By increasing the salt concentration introduced, the molecules with the weakest ionic interactions start to elute from the column first. The molecules that have a stronger ionic interaction with the resin bound ions require higher salt concentrations and therefore elute later in the gradient. The binding capacities of ion exchange resins are generally quite high. When considering the eluent characteristics, it is important to note the three main properties; concentration of competing ion, the nature of competing ion and the pH. The pH of the mobile phase buffer should be between the isoelectric point and acid dissociation constant of the charged molecule. It should also be close to the pKa of the charged group on the resin. This is to prevent pH related disassociation, denaturing or ionization state changes in the sample. (jackson). When evaluating the retention mechanisms when dealing with protein separation, it is important to note that each protein has differing amphoteric properties. The net charge of a protein is normally affected by the pH of the solution it is dissolved in, and thus important to maintain the correct parameters as aforementioned. At a point far from the isoelectric point of a protein, the binding to resin is much stronger than if it was close to it (E.Heftman). This may affect the speed of elution of the protein, or the requirement of very high salt concentrations to achieve it. References Rajbir Singh. (2002) Chromatography. Mittal Publications. 197. Print Sheehan…O’Sulivan. (2004). Protein Purification Protocols(second edition). Springer . Page 254. Print. Sharma, BK. Chromatography. Krishna Prakashan Media. Print TS Amar Anand Rao. (2012). HPLC and FPLC: Troubleshooting and Standardizing Chromatogram Purification Profiles. GRIN Verlag. Print Peter E Jackson. (1990). Ion Chromatography. Elsevier. Page 19. Print E.Heftman. Chromatography: Fundamentals and applications of chromatography and related differential migration methods(6th ed). Elsevier. Pg 712-714. Print Read More
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