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Humaira Rafique 2011-ag-1158 Slide 2 Contents: Chromatography Electrophoresis Microfilteration Slide 3 Chromatography Slide 4 Technique to separate components of a mixture by passing them through a matrix. Separation occurs because each compound in a mixture interacts differently with the matrix. Slide 5 Types of Chromatography: Column Chromatography Size Exclusion Chromatography Affinity chromatography Ion Exchange chromatography High Performance Liquid Chromatography Fast Performance Liquid Chromatography Gas Chromatography Slide 6 Column chromatography Compounds are separated through differential intermolecular forces between the components of the mixture with the mobile phase and between the components with the stationary phase. Slide 7 Polar components (b) adsorb more strongly to the polar silica and elute after the less polar components (a), which move more quickly with the non-polar (relative to silica) solvent. COLUMN CHROMATOGRAPHY Slide 8 Column chromatography Applications: Column chromatography (CC) is an extremely valuable technique for purification of Synthetic or Natural products. Slide 9 Size Exclusion Chromatography Size Exclusion chromatography is a separation based on size. It is also called molecular exclusion, Gel Filteration or gel permeation chromatography. Slide 10 Size Exclusion Chromatography Separation mechanism is not based on adsorption. Resolution depends on particle size, pore size, flow rate, column length and diameter, and sample volume. Three steps Equilibration Sample loading Elution Slide 11 Slide 12 Size Exclusion Chromatography Applications: Fractionation and molecular weight determination of proteins, nucleic acid separation, plasmid purification, polysaccharide fractionation Slide 13 Affintiy Chromatography Affinity chromatography (AC) is a technique enabling purification of a biomolecules with respect to biological function or individual chemical structure. The substance to be purified is specifically and reversibly adsorbed to a ligand (binding substance), immobilized by a covalent bond to a chromatographic bed material (matrix). Slide 14 Affinity Chromatography Two important things to consider; Finding a ligand specific enough to allow step elution. Finding conditions for safe binding and release within the stability window of the target molecule and the ligand. Slide 15 Slide 16 Affinity Chromatography Applications: It is used to separate and purify the Proteins Peptides DNA fragments, etc Slide 17 Ion Exchange chromatography Separates compounds based on their net charges. Negatively or positively charged functional groups are covalently bound to a solid support, yielding either a cation or anion exchanger, respectively. Slide 18 Ion Exchange chromatography Applications: It is used to separate the 4 Heamoglobin variants Proteins, etc Slide 19 High Performance Liquid Chromatography (HPLC) HPLC is also called high-pressure liquid chromatography. It is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture. Slide 20 High Performance Liquid Chromatography Slide 21 Applications: HPLC has many uses including Medical.Detecting vitamin D Concentrations in blood serum Legal Detecting performance enhancement drugs in urine Slide 22 High Performance Liquid Chromatography Applications: Research Purifying substances from a complex biological sample Separating similar synthetic chemicals from each other Manufacturing (e.g. during the production process of pharmaceutical and biologic products Slide 23 Fast Performance Liquid Chromatography (FPLC) Liquid chromatography is a term which refers to all chromatographic methods in which the mobile phase is liquid. The stationary phase may be a liquid or a solid, in the form of a matrix A type of liquid chromatography where the solvent velocity is controlled by pumps. The pumps control the constant flow rate of the solvents Slide 24 Fast Performance Liquid Chromatography Applications: It is used for Purification of proteins Rapid purification of RNA Analyzing lipoprotein profiles In pharmaceutical (eg, Drugs) The analysis of chemical components of Chinese herbal medicines Slide 25 Gas Chromatography The mobile phase of a chromatographic system is gaseous and stationary phase is a liquid coated on inert solid particles, the technique is called Gas Liquid Chromatography or simple Gas chromatography. Slide 26 Gas Chromatography Applications: Analyze the contents of chemical products Volatile mixtures are separated Quality assessment of alcoholic beverages Analysis of food Analysis of biomolecules(eg, melanins) Slide 27 Other Types of Chromatography Hydroxyapatite chromatography Reverse-Flow Chromatography Hydrophobic interaction chromatography Immobilized metal ion affinity chromatography Slide 28 Hydroxyapatite chromatography Ca 5 (PO 4 ) 3 (OH) 2 is a form of calcium phosphate that can be used for the separation and purification of proteins enzymes nucleic acids viruses, and other macromolecules. Slide 29 Reverse-Flow Chromatography Flow to the column is then reversed and the bound proteins elute from the top of the column in very concentrated form, which helps prevent denaturation. Especially useful when purifying antibodies using Protein A or other affinity columns. Slide 30 Hydrophobic interaction chromatography Separates molecules based on their hydrophobicity. Sample molecules containing hydrophobic and hydrophilic regions are applied to an HIC column in a high-salt buffer. Usually a decreasing salt gradient is used to elute samples from the column in order of increasing hydrophobicity. Slide 31 Immobilized metal ion affinity chromatography Most widely used nickel: also zinc and cobalt Nickel binds molecules rich in electrons such as histidine (from the His-tag fame!) IMAC uses the affinity of histidines imidazole side chains for metal ions. Slide 32 Electrophoresis Slide 33 s Electrophoresis is a separation method based on the differential rate of migration of charged species in a buffer solution across which has been applied a dc electric field. The rate of migration of a given species depends upon charge and size of ions. dc electric is a separation method based on differential rate of migration of charged species in a buffer solution across which has been applied a dc electric field. Slide 34 Types of Electrophoresis Gel Electrophoresis SDS-PAGE Electrophoresis Immuno Electrophoresis Capillary Electrophoresis Slide 35 Gel Electrophoresis Gel electrophoresis separates molecules on the basis of their charge and size.The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to retard the passage of molecules according to their size and shape. Slide 36 How does gel electrophoresis work? The gel is made from agar DNA is a negative molecules Molecules sort based on Charge Size shape Slide 37 The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration. 500 bp 200 bp 50 bp % agarose: 2% 4% 5% 500 bp 200 bp 50 bp 500 bp 200 bp 50 bp The concentration of gel/buffer will affect the resolution of fragments of different size ranges. Slide 38 Gel Electrophoresis Types: Slab Gel Electrophoresis (SGE) Polyacrylamide Gel Electrophoresis (PAGE) Agarose Gel Electrophoresis (AGE) Pulsed Field Gel Electrophoresis (PFGE) Slide 39 Slab Gel Electrophoresis Slab separation are carried out on a thin flat layer or slab of a porous semisolid gel containing an aqueous buffer solution within its pores Slide 40 Slab Gel Electrophoresis Slab gel electrophoresis can have either a horizontal or vertical format. Sample is introduced into wells at the top of the gel. Slide 41 Slab Electrophoresis Limitations: Slow Labor intensive Difficult to automate Does not yield very precise Quantitative information Joule heating Slide 42 Polyacrylamide Gel Electrophoresis Acrylamide, in combination with a cross linker, methylene bis-acrylamide Synthetic, consistent polymer Polymerization catalysts: ammonium persulfate (APS) plus N,N,N',N'- tetramethylethylenediamine (TEMED), or light activation Slide 43 PAGE Resolves 1 bp difference in a 1 kb molecule (0.1% difference), Tracking and loading dyes are used. Different buffers are used for protein separation. Tris Borate EDTA (TBE), Tris Acetate EDTA (TAE), Tris Phosphate EDTA (TPE) used most often for DNA. Slide 44 Types of PAGE Native PAGE (non-denaturing PAGE) The proteins are resolved in the absence of SDS SDS-PAGE (Denaturing PAGE) The proteins are resolved in the presence of SDS Slide 45 Feature of PAGE High resolving power. Acceptance relatively large sample size. Minimal interaction b/w migrating molecules & matrix. Physical stability of matrix. Separation basis Molecular sieving Electrophoresis mobility Slide 46 Agarose Gel Electrophoresis Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. It is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. We will be using agarose gel electrophoresis to determine the presence and size of PCR products. Slide 47 Agarose Gel Electrophoresis Slide 48 SDS-PAGE What is SDS-PAGE? Based on the migration of charged molecules in an electric field Separation technique Uses the Polyacrylamide gel as a support matrix. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run. Slide 49 Role of SDS Denatures proteins by wrapping around the polypeptide backbone. SDS binds to most proteins in amount roughly proportional to molecular weight of the protein-about one molecule of SDS for every two amino acids In doing so, SDS creates a large negative charge to the polypeptide in proportion to its length Slide 50 Conclusion SDS PAGE is a useful method for separating and characterising proteins, where a researcher can quickly check the purity of a particular protein or work out the different number of proteins in a mixture. Since we did not obtain results for the experiment, we have to rely on sample results Cannot validate the experimental technique Slide 51 Pulsed Field Gel Electrophoresis Very Large DNA Molecules are Separated by Pulsed Field Gel Electrophoresis (PFGE). Slide 52 Immuno Electrophoresis/ID Ag-Ab reaction Affinity based Agarose 1 % Slide 53 Capillary Electrop horesis Capillary electrophoresis yields high-speed, high-resolution separation on exceptionally small sample volumes Slide 54 Modes of Capillary Electrophoresis Capillary Zone Electrophoresis Capillary Gel Electrophoresis Capillary Isotachophoresis Capillary Isoelectric Focusing Slide 55 Capillary Electrop horesis Slide 56 Electrophoresis Applications: It has been applied to a variety of difficult analytical separation problems Inorganic anions and cations Amino acids Vitamins Drugs Slide 57 Electrophoresis Applications: Carbohydrates Peptides Nucleotides Proteins Polynucleotides Slide 58 Summary Electrophoresis is used to separate molecules by size and/or charge. Nucleic acid fragments can be resolved on agarose of polyacrylamide gels. PFGE is used to resolve very large DNA fragments. CGE is more rapid and automated than slab gel electrophoresis. The choice of electrophoresis method depends on the type and size of sample. Slide 59 Microfilteration Slide 60 Microfiltration Separates soluble contaminants remaining within the supernatant Supernatant may include: Other proteins Bio-molecules Un-used growth media Microfiltration image from: http://www.aaflow.de/filtertech/index.html http://www.aaflow.de/filtertech/index.html Slide 61 Microfiltration MF membranes are easily tested by direct examination, as their pores can be observed by electron microscopy. Large areas of microfiltration membrane can be tested and verified by a bubble test: Pores of the membrane are filled with liquid, then a gas is forced against the face of the membrane. Slide 62 How does Microfiltration work? Pressure driven process Separates: Components in a solution or suspension based on molecular size Particles size range: 10mm (starches) to aprx. 0.04mm (DNA, Viruses, and globular proteins) Microfiltration image from: http://www.faireymicrofiltrex.com/Vokes%20 Microfiltration/media/images/e-fluor.gif http://www.faireymicrofiltrex.com/Vokes%20 Microfiltration/media/images/e-fluor.gif Slide 63 Microfiltration Slide 64 Applications: Proteins act as the permeate Separates larger particles For example- Colloids Fat globules Cells Slide 65 Microfiltration Applications: Pharmaceutical, Biotechnology, Medical apps (used on the vents of sterile water tanks to prevent microbial contamination; protect autoclaves and freeze-dryers during the admission of gas after the duty cycle; etc.) Slide 66 THANKS