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Premium member Presentation Transcript Fast Protein Liquid Chromatography (FPLC) : Fast Protein Liquid Chromatography (FPLC) Andhra University College of Pharmaceutical sciences Presented By M.Siva Prasad II/II M.Pharmacy Pharmaceutical Analysis and Quality Assurance Under The Guidence of Prof. Dr.D.GOWRI SANKAR M.Pharm., Ph.D., F.I.C.CONTENTS: CONTENTS INTRODUCTION PRINCIPLE INSTRUMENTATION ADVANTAGES DRAWBACKS APPLICATIONS CONCLUSIONIntroduction : Introduction FPLC is basically a “protein friendly” HPLC system. FPLC is an intermediate between classical column chromatography and HPLC. It is a complete system for chromatographic separations of proteins and other biomolecules such as nucleic acids. It is commonly used in biochemistry and enzymology .Principle: Principle Size(size exclusion chromatography) Charge distribution(ion exchange) Affinity chromatographySize exclusion chromatography: Size exclusion chromatography chemistry.adelaide.edu.au Ion exchange chromatography: Ion exchange chromatography Waters.comAffinity chromatography : Affinity chromatography Matrix Spacer arm Bound Protein Ligand Unbound Materials en.wikibooks.orgInstrumentation : Instrumentation 1. Stationary Phase : It is typically a resin composed of cross-linked agarose beads with varying surface ligands. 2. Mobile Phase : Mostly buffers, organic solvents. 3. Pump : Constant controlled flow by peristaltic pumps. The flow rate is varied based on scale of preparation ie; analytical or preparative chromatography. 4 . Mixer: Powered and controlled by the pump. Especially important when forming gradients between buffer sources. The flow rate is for bench-top systems- ml/min, for industrial scale –litres/min.PowerPoint Presentation: The mixer ensures that the buffers used are in the correct proportion relative to each other during the course of the FPLC run. MixerPowerPoint Presentation: 5.Injection valve: Pumps are connected to valves which send the buffers in the desired direction. The Load position is for filling a loop with sample or for running buffer through a column. The Inject position is for injecting contents of the sample loop onto a column. The Purge position is for priming the biologic workstation gradient pump and purging lines to waste without removing the column from the system.PowerPoint Presentation: 6. Column: large [internal diameter,mm] tubes. contains small [ 13–15 µ] particles or gel beads. Typical column materials are PEEK,inert plastic or inert fluid surfaces like Teflon, titanium, and glass. It is designed to operate at not more than 580 psi. Pre-packed columns are also available. Columns should be stored in 24% ethanol/H2O when not in use.PowerPoint Presentation: Column tubings: These are rigid/semi-rigid tubing. For medium and high-pressure applications use two different outside diameters (OD). OD with 1/8’’(0.125’’) usually PTFE(Teflon)-used for upstream of pump. OD with 1/16’’(0.0625’’) usually PEEK-used for downstream of pump. For most purposes OD-1/16’’(0.0625’’) ID-1/50’’(0.020’’).FPLC columns: FPLC columns Column MW range Type Bed volume (ml) pH range Mono Q up to 10 7 Anion exchange 1 3-11 Superdex 200 1 x10 4 to 6 x10 5 gel filtration 120 3-12 Superose 6 5 x10 3 to 5 x10 6 gel filtration 24 3-12 Amersham Biosciences, www.amershambiosciences.comPowerPoint Presentation: 7. Fraction collector: Allows fixed volume fractionation. 8. Flow Restrictor: Generates a steady back-pressure to prevent air bubbles being formed after the columns in the flow cells. 9. On-line Filter: Rejects sample particulates that may clog the fluidic system by generating a maximum back-pressure of 0.5 MPa. 10. Detection system : UV or UV/Vis spectrophotometer, Conductivity detector and Refractive Index (RI) Detector based on characteristics of the analyte of interest.PowerPoint Presentation: www.mnstate.edu/biotech/chrom_fplc.pdfPowerPoint Presentation: 1 2 3 5 4 No air bubbles (Priming) Use degassed buffers Injector Module Column Inlet Detector Fraction Collector www.pharmacia.comHow does it differ from HPLC ??: How does it differ from HPLC ?? FPLC differs from HPLC in that the columns used for FPLC can only be used up to maximum pressure of 3-4 MPa (435-580 psi). Stainless steel components replaced with glass and plastic. Inert surfaces are necessary since many of the resolving buffers contain high concentrations of halide salts that attack and corrode stainless steel surfaces. FPLC pump delivers a solvent flow rate in the range of 1-499ml/hr HPLC pump gives a rate 0.010-10ml/min. FPLC system can use FPLC columns only but in HPLC system we can use both.Advantages : Advantages Reproducible with excellent resolution. Very simple system programming. Inert construction against the very high salt concentrations and corrosive liquids hence columns have longer lifetime. Since lower pressures are used in FPLC than in HPLC, a wider range of column supports are possible. The wide flow range makes it suitable both for analytical and preparative chromatography.Drawbacks: Drawbacks Needs glass columns. Can not handle high pressure. Instrument does not support HPLC columns. Purifying thermolabile (heat sensitive) proteins is a tough task.Applications: Applications Protein analysis. Lipoprotein separation by FPLC. Purification of animal venoms. Separation of Plasma Proteins in Urine and Cerebrospinal Fluid. Isolation of porphobilinogen deaminase from human erythrocytes.PowerPoint Presentation: Rapid purification of RNA. The Analysis of nitrogenous constituents of beer. Coupled to a double focusing inductively coupled plasma mass spectrometer for trace metal speciation in human serum and detectable levels of Cr, Al, Pb and Sn were present in uraemic sera. FPLC method could be used as a cost-effective method for routine β-thalassaemia diagnosis.Conclusion : Conclusion Thus FPLC SYSTEM is a very efficient system mainly for the separation of proteins and other bio- molecules. Recent advances in this system makes it to conquer the field of protein analysis.References: References High Performance Liquid Chromatography- HPLC. URL: http://www.chemguide.co.uk/analysis/chromatography/hplc.html#top . Accessed: June 15, 2007. High Performance Liquid Chromatography (HPLC) Primer.URL: http://www.waters.com/WatersDivision/Conten td.asp?Watersit =JDRS-6UXGZ4. Accessed: June 22, 2007. Kazakevich, Y; McNair, H.M. Basic Liquid Chromatography . URL: http://hplc.chem.shu.edu/HPLC/index.html . Accessed: June 22, 2007. Boyer, Rodney. Modern Experimental Biochemistry.3rd Ed. Addison Wesley Longman. San Frisco, CA. 2000. 87-100. Robertson, James W.; Frame, Eileen M.; Frame, George M. Undergraduate Instrumental Analysis . Marcel Dekker: New York, NY. 2005.797-836.PowerPoint Presentation: F.W.M. de Rooij, Department of Internal Medicine II, Erasmus University, Rotterdam The Netherlands. Rao VB, Natrajan PG, Institution Research Society, B. J. Wadia Hospital for Children, Parel, Bombay, India. Choice E , Ayyobi AF , Pritchard PH , Madden TD , Department of Pharmacology & Therapeutics, University of British Columbia, 2176 Health Sciences Mall, Vancouver, British Columbia, V6T 1Z3, Canada. A.T. Andrews , Food Research Institute (Reading), Shinfield, Reading RG2 9AT U.K. Tibor Kremmer, National Oncological Institute, Department of Biochemistry, H-1525 Budapest Hungary. Debont T , Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, University of Leuven, Belgium You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.