6Column chromatography Compounds are separated throughdifferential intermolecular forces between the components of the mixture with the mobile phase and between the components with the stationary phase.
7COLUMN CHROMATOGRAPHY 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.
8Column chromatography Applications:Column chromatography (CC) is an extremely valuable technique for purification ofSynthetic orNatural products.
9Size Exclusion Chromatography Size Exclusion chromatography is a separation based on size. It is also called molecular exclusion, Gel Filteration or gel permeation chromatography.
10Size 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 stepsEquilibrationSample loadingElution
12Size Exclusion Chromatography Applications:Fractionation and molecular weight determination of proteins,nucleic acid separation,plasmid purification,polysaccharide fractionation
13Affintiy 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).
14Affinity 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.
16Affinity Chromatography Applications:It is used to separate and purify theProteinsPeptidesDNA fragments, etc
17Ion 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.
18Ion Exchange chromatography Applications:It is used to separate the4 Heamoglobin variantsProteins, etc
19High 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.
21High Performance Liquid Chromatography Applications:HPLC has many uses includingMedical.Detecting vitamin DConcentrations in blood serumLegalDetecting performance enhancement drugs in urine
22High Performance Liquid Chromatography Applications:ResearchPurifying 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
23Fast 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 matrixA type of liquid chromatography where the solvent velocity is controlled by pumps. The pumps control the constant flow rate of the solvents
24Fast Performance Liquid Chromatography Applications:It is used forPurification of proteinsRapid purification of RNAAnalyzing lipoprotein profilesIn pharmaceutical (eg, Drugs)The analysis of chemical components of Chinese herbal medicines
25Gas ChromatographyThe 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.
26Gas Chromatography Applications: Analyze the contents of chemical productsVolatile mixtures are separatedQuality assessment of alcoholic beveragesAnalysis of foodAnalysis of biomolecules(eg, melanins)
27Other Types of Chromatography Hydroxyapatite chromatographyReverse-Flow ChromatographyHydrophobic interaction chromatographyImmobilized metal ion affinity chromatography
28Hydroxyapatite chromatography Ca5(PO4)3(OH)2 is a form of calcium phosphate that can be used for the separation and purification ofproteinsenzymesnucleic acidsviruses, and other macromolecules.
29Reverse-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.
30Hydrophobic 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.
31Immobilized metal ion affinity chromatography Most widely used – nickel: also zinc and cobaltNickel binds molecules rich in electrons – such as histidine (from the His-tag fame!)IMAC uses the affinity of histidine’s imidazole side chains for metal ions.
33Electrophoresiss 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.
34Types of Electrophoresis Gel ElectrophoresisSDS-PAGE ElectrophoresisImmuno ElectrophoresisCapillary Electrophoresis
35Gel ElectrophoresisGel 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.
36How does gel electrophoresis work? The gel is made from agarDNA is a negative moleculesMolecules sort based onChargeSizeshape
37The movement of molecules is impeded in the gel so that molecules will collect or form a band according to their speed of migration.% agarose: 2% % %500 bp200 bp50 bp500 bp200 bp50 bp500 bp200 bp50 bpThe concentration of gel/buffer will affect the resolution of fragments of different size ranges.
38Gel Electrophoresis Types: Slab Gel Electrophoresis (SGE) Polyacrylamide Gel Electrophoresis (PAGE)Agarose Gel Electrophoresis (AGE)Pulsed Field Gel Electrophoresis (PFGE)
39Slab 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
40Slab Gel Electrophoresis Slab gel electrophoresis can have either a horizontal or vertical format.Sample is introduced into wells at the top of the gel.
41Slab Electrophoresis Limitations: Slow Labor intensive Difficult to automateDoes not yield very preciseQuantitative informationJoule heating
42Polyacrylamide Gel Electrophoresis Acrylamide, in combination with a cross linker, methylene bis-acrylamideSynthetic, consistent polymerPolymerization catalysts: ammonium persulfate (APS) plus N,N,N',N'-tetramethylethylenediamine (TEMED), or light activation
43PAGE 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.
44Native PAGE (non-denaturing PAGE) Types of PAGENative PAGE (non-denaturing PAGE)The proteins are resolved in the absence of SDSSDS-PAGE (Denaturing PAGE)The proteins are resolved in the presence of SDS
45Feature of PAGE High resolving power. Acceptance relatively large sample size.Minimal interaction b/w migrating molecules & matrix.Physical stability of matrix.Separation basisMolecular sievingElectrophoresis mobility
46Agarose 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.
48SDS-PAGE What is SDS-PAGE? Based on the migration of charged molecules in an electric fieldSeparation techniqueUses the Polyacrylamide gel as a “support matrix”. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run.
49Role of SDSDenatures 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 acidsIn doing so, SDS creates a large negative charge to the polypeptide in proportion to its length
50ConclusionSDS 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 resultsCannot validate the experimental technique
51Pulsed Field Gel Electrophoresis Very Large DNA Molecules are Separated by Pulsed Field Gel Electrophoresis (PFGE).
58SummaryElectrophoresis 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.
60MicrofiltrationSeparates soluble contaminants remaining within the supernatantSupernatant may include:Other proteinsBio-moleculesUn-used growth mediaMicrofiltration image from:
61MicrofiltrationMF 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.
62How does Microfiltration work? Pressure driven processSeparates:Components in a solution or suspension based on molecular sizeParticles size range:10mm (starches) to aprx mm (DNA, Viruses, and globular proteins)Microfiltration image from:
64Microfiltration Proteins act as the permeate Applications:Proteins act as the permeateSeparates larger particlesFor example-ColloidsFat globulesCells
65Microfiltration Applications: Pharmaceutical, Biotechnology, Medical app’s (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.)