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KARAN TRIVEDI 1Contents: Contents Principles underlying chromatographic techniques Retention mechanism Diffusion Fick’s law Types of size exclusion chromatography Gel permeation Gel filtration Applications Reliability of results KARAN TRIVEDI 2Objective of Separation: Objective of Separation Proteins are extracted from animals and humans as a mixture in a serum of body fluids. When immunologists want to study a specific protein, like an antibody, hormone, or enzyme, they need to separate it from the mix. KARAN TRIVEDI 3Some examples of separative techniques: Some examples of separative techniques Solvent extraction Chromatography Precipitation Recrystallisation Electrophoresis KARAN TRIVEDI 4Some examples of chromatographic techniques: Some examples of chromatographic techniques Ion-exchange chromatography Size-exclusion chromatography Paper chromatography Thin layer chromatography Affinity chromatography KARAN TRIVEDI 5Paper Chromatography of Inks: Paper Chromatography of Inks Inks from pens are chromatographed on paper using water as the mobile phase . KARAN TRIVEDI 6Chromatography of spinach extract: Chromatography of spinach extract Spinach extract is separated by thin layer chromatography into chlorophyll and B-carotene KARAN TRIVEDI 7Slide 8: Type Stationary phase Mobile phase Ion-exchange Based on charge Polymeric matrix – bonded with functional groups e.g. carboxylic acids, quarternary amines Liquid ionic solutes are retained by forming electrostatic chemical bonds Size-exclusion Based on size Polymeric substance with numerous pores Liquid or gaseous small solutes diffuse into pores, big solutes remain in mobile phase Affinity Based on biorecognition ( ligand specificity) A specific ligand e.g. antibody is bound to stationary phase A mixture of solute containing an antigen, will bind strongly to the ligand KARAN TRIVEDI 8Stationary Phase: Stationary Phase semi-permeable, porous beads with well-defined range of pore sizes . beads are crosslinked polymers Degree of crosslinking is controlled carefully to yield different pore sizes How & why? Property includes a fractionation range (due to the different pore sizes) molecules within that molecular weight range can be separated. KARAN TRIVEDI 9Nature of Porous Material (stationary phase): Nature of Porous Material (stationary phase) Porous material must swell up and imbibe/absorb the liquid phase The created solvent-filled ‘sponge’ allows diffusion of molecules Therefore, stationary phase may be hydrophilic to imbibe aqueous media, or lipophilic to imbibe non-polar organic solvents. KARAN TRIVEDI 10Types of Stationary Phase: Types of Stationary Phase Soft gels e.g. Polyacrylamide gels, dextran (natural glucose polymer) Separation of proteins Semirigid or rigid gels e.g. 1) Polystyrene gels Separation of non-polar polymers in non-polar solvents e.g. 2) Porous glass gels Separation of polar systems ..give an example... KARAN TRIVEDI 11Soft gels: Soft gels Before column is packed, gel is imbibed by enough liquid to completely swell. These gels are used with aqueous mobile phase . Once column is packed, the composition of the mobile phase cannot be altered to prevent shrinkage or bursting of the packed column. Because of low structural strength, they cannot be used under high pressure. classified as gel filtration . KARAN TRIVEDI 12Semirigid Gels: Semirigid Gels Made from crosslinked polystyrene, glass beads or alkylated dextran. Used for separation of organic-soluble polymers. Non-aqueous mobile phases e.g. chloroform, acetone, pyridine or tetrahydrofuran. Classified as gel permeation. KARAN TRIVEDI 13Mobile Phase: Mobile Phase The mobile phase contains a mixture of solutes. Small solutes will diffuse in and out of the pores (obeying Fick’s law) Their path through the column is longer The elution time will be longer KARAN TRIVEDI 14Chromatogram: Chromatogram extent of retention depends on size of the included molecules relative to the pores. Smallest molecules will enter all pores . Intermediate molecules, due to velocity of mobile phase , will not be able to diffuse into the pores that they may fit, thus will be retained less effectively. Initial peak contains totally EXCLUDED solutes . Final peak contains totally included solutes . KARAN TRIVEDI 15Slide 16: KARAN TRIVEDI 16 Porous beads pores Totally excluded –eluted first Partially included Totally included –eluted last columnIllustrative description of separation in SEC. (From Introduction to Modern Liquid Chromatography, 2nd edition by L. Snyder and J. J. Kirkland, © 1979 by John Wiley & Sons, Inc. ): Illustrative description of separation in SEC. (From Introduction to Modern Liquid Chromatography, 2nd edition by L. Snyder and J. J. Kirkland, © 1979 by John Wiley & Sons, Inc. ) KARAN TRIVEDI 17Common terms in size exclusion chromatography: Common terms in size exclusion chromatography KARAN TRIVEDI 18Advantages of Gel Filtration: Advantages of Gel Filtration Can handle biomolecules that are sensitive to changes in pH, concentration of metal ions or harsh environmental conditions. Separations can be performed in the presence of essential ions , detergents, urea,, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment. KARAN TRIVEDI 19 Lecture ends hereSeparation of proteins by gel filtration: Separation of proteins by gel filtration SMALL proteins can fit inside all the pores in the beads : included . have access to mobile phase inside the beads & mobile phase between beads and elute last LARGE proteins that cannot fit into any of the pores: excluded have access only to the mobile phase between the beads: elute first Proteins of intermediate size are partially included - can fit inside some of the pores in the beads. These proteins will then elute between the large ("excluded") and small ("totally included") proteins. KARAN TRIVEDI 20Separation based on size: Separation based on size Proteins are separated according to their molecular weight because this is the major contribution to molecular size. However, the shape will affect its apparent size in solution. Hence, gel filtration is NOT recommended for separating proteins with only a small difference in molecular weight . KARAN TRIVEDI 21Effect of Shape on Size: Effect of Shape on Size Myosin and cytochrome C are proteins which have approximately the same molecular weight ca. 530 kD . However, myosin is a long rod-shaped molecule and cytochrome C is globular with Stokes radii 194.6 and 69.2 respectively. KARAN TRIVEDI 22Exercise: Exercise Consider the separation of a mixture of glutamate dehydrogenase (MW 290,000), lactate dehydrogenase (MW 140,000), serum albumin (MW 67,000), ovalbumin (MW 43,000), cytochrome c (MW 12,400) on a gel filtration column: fractionation range 15,000 - 150,000). KARAN TRIVEDI 23Slide 24: The separations can be described by this equation V r = V o + KV i V r is the retention volume of the protein , V 0, void volume, is the volume of mobile phase between the beads of the stationary phase inside the column V i , included volume, is the volume of mobile phase inside the porous beads K is the partition coefficient (the extent to which the protein can penetrate the pores in the stationary phase, with values ranging between 0 and 1). KARAN TRIVEDI 24Slide 25: In the mixture of proteins given, the partition coefficient K=0 for glutamate dehydrogenase (totally excluded), K = 1 for cytochrome c (totally included) 0 > K > 1 for the other proteins, which are within the fractionation range for the column. KARAN TRIVEDI 25Slide 26: KARAN TRIVEDI 26Applications : Applications Desalting Concentration Molecular weight determination Fractionation KARAN TRIVEDI 27Desalting: Desalting Necessary for purification of biochemicals. Due to techniques involving buffers and precipitating reagents. Gel with low exclusion limit MW 1000-2000 is used. Short column and high flow rate can be used because of the vast difference in size of solutes and contaminants. Macromolecules will be eluted with little dilution and salts retained on the column. KARAN TRIVEDI 28Concentration of Dilute Solutions: Concentration of Dilute Solutions Exclusion limit of gels less than MW of solutes. Solution is mixed with a small quantity of dry gel that will absorb 10 to 20 times its weight in water. Some salts and small molecules are taken up also. Final macromolecules in a solution of almost unchanged pH and ionic strength but significantly decreased volume. KARAN TRIVEDI 29Molecular Weight Determination: Molecular Weight Determination Size is approximately proportional to molecular weight, M. Elution time , V E , can be expressed by: V E = a + b log M a and b are constants dependent on the mobile and stationary phases. KARAN TRIVEDI 30To separate a protein sample by gel filtration chromatography: To separate a protein sample by gel filtration chromatography KARAN TRIVEDI 31Equipment for running size exclusion chromatography: Equipment for running size exclusion chromatography The buffer is pumped through the column (right) by a computer controlled device. KARAN TRIVEDI 32Columns and Detectors : Columns and Detectors Detection of the solute zones as they emerge from the column can be achieved by spectrophotometric monitoring of the eluate by measurement of refractive index of eluate Collection of aliquots for later analysis Mobile phase is allowed to flow by gravity Very high flow rate not suitable for soft gels KARAN TRIVEDI 33Types of Column: Types of Column exclusion range for some common gel filtration chromatography media. Sephadex G-50 1-30 kD Sephadex G-100 4-150 kD Sephadex G-200 5-600 kD Sephadex is a trademark of Pharmacia. KARAN TRIVEDI 34Choice of columns: Choice of columns The first column contains Sephadex G-75, which separates blue dextran and hemoglobin. The second column contain Sephadex G-10, which separates hemoglobin and riboflavin. Because there is a difference in the two packing materials, the hemoglobin molecule runs very differently in the two columns. KARAN TRIVEDI 35 Column 2Column - example: Column - example Trisacryl GF 05: Particle size 40-80 µm exclusion limit 3,000 Da fractionation range 200-2,500 Da Physical form: Aqueous suspension in 1 M NaCl and 20% ethanol Application: Highly hydrophilic beaded poly(N-tris[hydroxymethyl]methyl acrylamide) suitable for medium pressure separations of small molecules and peptides. Highly resistant to acid environments, sensitive to strong alkaline agents. KARAN TRIVEDI 36How to check reliability?: How to check reliability? Calibrate Use standards Choice of standards depends on application Available in low and high molecular weight ranges supplied lyophilized in individual vials. Kits include Blue Dextran 2000 to determine the column void volume and to check column packing. KARAN TRIVEDI 37Calibration for MW Determination: Calibration for MW Determination Calibrate using large molecule such as blue dextran to establish void volume of the system. Use deuterium oxide or sucrose to determine retention time for a totally included solute . Use a series of standard proteins or polymers to calibrate regions between these two limits. KARAN TRIVEDI 38Calibration Kits : KARAN TRIVEDI 39 Calibration Kits Type Molecular Weight Gel Filtration LMW Calibration Kit Ribonuclease A 13 700 Chymotrypsinogen A 25 000 Ovalbumin 43 000 Bovine Serum Albumin 67 000 Blue Dextran 2000 » 2 000 000 Gel Filtration HMW Calibration Kit Aldolase 158 000 Catalase 232 000 Ferritin 440 000 Thyroglobulin 669 000 Blue Dextran 2000 » 2 000 000Calibration for large fractionation range: Calibration for large fractionation range For media that have very large fractionation range, blue dextran cannot be used to determine a void volume. Alternative methods include the use of raw dextrans (MW 50 MDa) polystyrene microspheres KARAN TRIVEDI 40Summary: Summary KARAN TRIVEDI 41 Source: from the internet – TQ to contributor CHIRAL CHROMATOGRAPHY: CHIRAL CHROMATOGRAPHY KARAN TRIVEDI 42Slide 43: Introduction:- Chiral Chromatography is a branch of chromatography that is oriented towards the exclusive separation of chiral substances. Certain stereoisomers that differ only in the spatial arrangement of their atoms and in their capacity for rotating the plane of polarized light are termed optically active or chiral and the individual isomers are called enantiomers. Enantiomeric separations are achieved in chiral chromatography by the judicious use of chiral phases. The mobile phase can be a gas or liquid giving rise to chiral gas chromatography and chiral liquid chromatography. 43 KARAN TRIVEDISlide 44: Principal of Enantiomer separation:- On transfer of a pair of enantiomers into asymmetric environment . To diastereomeric species are formed with distinct physicochemical property profile On the basis of which physical separation into individual enantiomers may be achieve. 44 KARAN TRIVEDISlide 45: Chiral selector (SO):- Capable of undergoing covalent or non-covalent interaction with the individual enantiomer ( selectands, SAs ). Depending on the nature of the interaction stabilizing the respective diastereomer SO-SA species 45 KARAN TRIVEDISlide 46: Enantiomer separation strategies:- Indirect enantiomer separation Direct enantiomer separation Indirect enantiomer separation:- Chiral Derivatization agent (CDAs):- Transformation of the SAs of interest into covalent diastereomer by conversion with suitably reactive SOs. Followed by separation of diastereomeric product with achiral chromatographic techniques 46 KARAN TRIVEDISlide 47: Applicable only to enantiomer presenting a single or more but selectively addressable functional group suitable for derivatization. Direct enantiomer separation:- 1) Chiral mobile phase additive 2) chiral stationary phase mode 1) Chiral mobile phase additive (CMPA): A combination of an chiral stationary and a chiral mobile phase is employed. On introduction of a mixture of enantiomers into this system. 47 KARAN TRIVEDISlide 48: The individual of enantiomers form diastereomeric complex with the chiral mobile phase additive. This diastereomeric complex may exhibit distinct association / dissociation rate, thermodynamics stability, and physiochemical property therefore may be separated on an appropriate a chiral stationary phase. 2) chiral stationary phase mode: Consists of an inert chromatographic support matrix incorporating chemically or physically immobilized SO species. 48 KARAN TRIVEDISlide 49: CSPs may be created by a variety of SO a immobilization technique. Covalent attachment on to the surface of suitably prefunctionalized carrier materials. Physical fixation employing coating technique. Incorporation into polymeric network by copolymerization or combination of this procedures. 49 KARAN TRIVEDISlide 50: CSPs provide several operational advantages over CMAs based enantiomers separation. stability of CSPs more and flexibility with respect to method optimization parameter. used wide range of mobile phase solvent and modifiers. also used for gradient elution and variable temp. protocol. 50 KARAN TRIVEDISlide 51: Classification of Chiral Stationary Phase: Type-1 Chiral Organic Polymer -Pure -Polymer coating on inorganic support -Grafted polymer. Type-2 Carrier material modified with Chiral moieties -inorganic material mainly silica gel modified on the surface. -organic polymer network grafted with chiral molecules. 51 KARAN TRIVEDISlide 52: Type-3 Imprinted material -imprinted polymer -inorganic material imprinted on the 52 KARAN TRIVEDISlide 53: Polymeric phase Polysaccharide based CSPs : -Applied as pure polymers in a form adequate for chromatographic purposes or as coating on an inert achiral support to confer mechanical stability. Polyacrylamide CSPs: -cross linked optically active polyacrylamides and polymethacrylamides constitute. -Improvement in the mechanical performance of these CSPs was achived by polymerization of the acrylic monomer on the surface of silica gel,yielding a grafted polymer 53 KARAN TRIVEDISlide 54: Polymeric chiral phase derived from Tartaric acid : -Prepared by polymerization of N,N’-diallyl derivatives of tartardiamide and grafted onto silica gel. -As this phase are cross linked and bonded to silica gel they are insoluble in organic solvent and there is no limitation regarding the choice of mobile phase. -Normal phase, reversed phase and supercritical fluid condition have been applied. -They show good mechanical stability but relatively high content of achiral silica gel in these CSPs reduces their loading capacity. 54 KARAN TRIVEDISlide 55: Brush type CSPs Π -acidic and Π -basic phases : - Π -acceptor phases are derived from the amino acids phenylglycine or leucine covalently or ironically bonded to 3-aminopropyl silica gel. -These chiral phase are commercially available for analytical or preparative separation of enatiomers. -Amine chiral selector such as valine, phenylalanine, tyrosine and 1,2-trans –diaminocyclohexane and 1,2-trans-diphenylethylene diamine. 55 KARAN TRIVEDISlide 56: Cyclodextrin based CSPs -It is cyclic olygosaccharides that can form inclusion complex in their highly hydrophobic chiral cavity with a large variety of molecules. -The size of cavity differs for α -, β -, γ - cyclodextrins and the substitute on cyclodextrin play a determining role in the ability to complex a defined molecule. -Cyclodextrin CSPs were prepared by immobilizing CD in polymeric structures or on silica gel which give good performance on an analytical scale. 56 KARAN TRIVEDISlide 57: Chirobiotic CSPs -These phase have been prepared by covalent immobilization of the glycopeptides (vancomycin, teicoplanin, and restocetin) to silica gel according to standard procedures. -The chiral selectors are characterized by the presence of several chiral cavities providing different environments for enantioselective interaction. 57 KARAN TRIVEDISlide 58: Chiral ion-exchange stationary phases -Developed a series of anion- exchange CSPs based on quinine and quinidine as chiral selectors. -This phase are perticularly appropriate for the separation of the enantiomers of chiral acidic compounds. Aptamer type CSPs -DNA and RNA aptamer use as powerful target specific affinity probe for many analytical application. 58 KARAN TRIVEDISlide 59: Antibody type CSPs -Generation of sterioselective recombinant antibody fragment and which is immobilization to polystyrene-based macro porous perfusion resin. -immobilized monoclonal anti-D and anti-L-phenylalanine antibodies as target-specific CSPs -To avoide antibody denaturation enantiomer separation were carried out with phosphate buffer saline at pH 7.4 59 KARAN TRIVEDISlide 60: Application:- -Quinine and Quinidine -Atropine and Hyoscyamine -Cetrizine and Levo-cetrizine -Omeprazole and Esomeprazole(more effective in GERD) -Dopamine and levodopamine -D-Amphetamine and Amphetamine -Dextromethorphan and Levo-methorphan 60 KARAN TRIVEDIReferences: References Peter Atkins & Julio de Paula, Atkin’s Physical Chemistry 7 th Ed., Oxford, New York (2002) Chapter 22 AR Gennaro, Remington: The Science and Practice of Pharmacy 20 th Ed., Lippincott Williams & Wilkins (2000) Part 4 DG Peters, JM Hayes, GM Hieftje, Chemical Separations and Measurements, Saunders, Philadelphia(1974) Chapter 17 http://www.chromatography.amershambiosciences.com/ And others….. 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