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WELCOME TO:

WELCOME TO:. Course 724. Chromatography of Biomolecules. Textbooks. Chromatography by Synder & Kirkland Books on HPLC are also available in the Library. EXAMS and Quizzes. 2 COURSE EXAMS (20 pts each). Assignments (10 pts). Comprehensive Examination (50). TOTAL = 100 pts.

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  1. WELCOME TO: Course 724 Chromatography of Biomolecules

  2. Textbooks Chromatography by Synder & Kirkland Books on HPLC are also available in the Library

  3. EXAMS and Quizzes 2 COURSE EXAMS (20 pts each) Assignments (10 pts) Comprehensive Examination (50) TOTAL = 100 pts

  4. Lectures Test Jan. 08, 09 Jan.15, 16 Jan. 22, 23 Jan. 29 Jan. 29, 30 Feb. 05, 06 Feb 12 Feb. 12, 13 Feb. 19, 20 Feb. 26, 27 March 12

  5. Grade/Exams Students must take all exams at the scheduled time

  6. 100 A+ 90% A 80 B 71% 50 40 D 50% 20

  7. Course content

  8. Biomolecules Molecules produced by any living organism. These molecules may occur as monomers, oligomers or polymers.

  9. Primary metabolites are directly involved in normal growth, development, and reproduction. Secondary metabolites are not directly involved in normal growth, development, and reproduction but usually have an important ecological function

  10. Types of Biomolecules • A diverse range exists • Amino acids, Peptides, Proteins • Carbohydrates • Nucleic acids, DNA, RNA • Lipids, Phospholipids, Glycolipids, Glycerolipids, Sterols • Vitamins • Hormones, Neurotransmitters • Metabolites arising from degradation of biomolecules

  11. Chromatography History of chromatography, basic concepts, classical methods, liquid chromatography on plane surfaces, Chromatographic packing materials- synthesis and design Separation technique based on the different interactions of compounds with two phases, a mobile phase and a stationary phase, as the compounds travel through a supporting medium.

  12. Why separate biomolecules?

  13. Even molecules with same molecular formula can differ in structure (shape) and show different chemical properties different biological functions 6carbons 6carbons 6carbons

  14. Form affects function Structural differences imparts important functional significance amino acid alanine L-alanine used in proteins but not D-alanine medicines L-version active but not D-version sometimes with tragic results… stereoisomers

  15. C h r o m a t o g r a p h y The word comes from Greek language Chroma  Color and Graphien  to write

  16. Chromatography It is a very powerful and versatile technique It can separate a mixture into its individual components in a single step process and simultaneously provide an quantitative estimate of each constituent. Samples may be gaseous, liquid or solid in nature and can range in complexity from a simple blend of two entantiomers to a multi component mixture containing widely differing chemical species.

  17. Development of chromatography over one century

  18. Chromatography • Basic concepts • Classical methods • High performance liquid chromatography, • Techniques commonly used in separation of macromolecules - Size exclusion, ion exchange, affinity, reversed phase

  19. What is Chromatography? Physical separation method based on the differential migration of analytes in a mobile phase while moving along a stationary phase. Why one needs to make a chromatographic experiment? Interested in looking at characteristics of a particular compound

  20. Classification of chromatography • Principle of Separation • Modes of separation • Elution technique • Scale of separation • Type of analysis

  21. Types of Chromatographic experiment Column Chromatography – the stationary phase is held in a narrow tube through which the mobile phase is forced under pressure or by gravity. Planar Chromatography – the stationary phase is supported on a flat plate or the interstices of a paper and the mobile phase moves through the stationary phase by capillary action or by gravity.

  22. What do we need for performing a column chromatography experiment? Column Stationary Phase Mobile Phase Detection system Recording

  23. Column Glass Plastic Stainless Steel

  24. Stationary Phase Composition Particle size Pore size Morphology

  25. Stationary Phase Most common a) For small molecules - Silica, Alumina, Polyamide etc. b)For large molecules – Sephadex, Sepharose, Cellulose, etc.

  26. Types of Chromatography: - chromatography can be classified based on the type of mobile phase, stationary phase and support material

  27. Mechanisms of Separation: • Partitioning • Adsorption • Exclusion • Ion Exchange • Affinity

  28. Components Supporting medium: a solid surface on which the stationary phase is bound or coated Stationary phase: a layer or coating on the supporting medium that interacts with the analyte Mobile phase: a solvent that flows through the supporting medium

  29. Paper Chromatography Ascending Descending Thin Layer Chromatography – Continuous development Column Chromatography (low pressure) Column Adsorption Column Partition Gas Chomatography High Performance Liquid Chromatography

  30. Stationary Phases for chromatography

  31. Characteristic features of analyte and stationary phase interactions • Relatively weak intermolecular attraction • High discriminating power in recognizing analytes according to their chemical and physical structure ( molecular sensor) • Rapid mass transfer and interaction kinetics

  32. Gas phase separations • Applying molecular sieve type of adsorbents and using pressure swing and temperature variation (Sorbex technology) Liquid Phase separations • Applying selective adsorbents and using elution gradient and displacement techniques

  33. Desired chromatographic properties of stationary phases, packings and columns • High resolution • Fast analysis • High mass and bio-recovery • High reproducibility • Long life time • Adaptation to operational conditions (adequate pressure – flow rate dependency, fast regeneration etc.)

  34. Choices • Cheap bulk adsorbents • Large particle size analytical packing with identical surface chemistry • Tailor made adsorbents specially designed for isolation purposes

  35. Stationary phase The general approach in stationary phase design involves a carefully balanced combination of • Theory • Practice • Originality and • Common sense. Much of the experience utilized in stationary phase design stems from knowledge accumulated in material and surface science. The key issue is to combine this experience in the most intelligent way.

  36. An optimization strategy needs to include both the physical and chemical structure parameters of the support and stationary phase Physical structure parameters • Particle shape and size • Porosity, pore shape and size • Surface area Chemical Structure parameters • Phase composition, distribution and transition • Type, strength, density and distribution of functional groups (ligands)

  37. Primary goals in view of the application • Defined surface functionality • Surface homogeneity • Uniformity of pore size and particle size • Variable and reproducible surface chemistry, particle size and pore size

  38. Historical review of column packing development

  39. Developments in packing manufacture • Rigid polymeric organic packing • Inorganic packing e.g. Oxides with improved pH stability (pure silicas, alumina, zirconia) • Composites: inorganic core, organic layer or coating

  40. Novel concepts in particle texture & pore structure • Non-porous particles • Entirely macroporous or mesoporous particles • Particles with a distinct bimodal pore size distribution (macro + mesoporous)

  41. Modes of manufacture of polymer beads • Emulsification polymerization • Suspension polymerization • Swollen emulsion polymerization (Ugelstad process)

  42. Dispersion of colloidal silica Silica hydrogel Silica Xerogel drying Gelling in two phase system Dehydrated Silica Silica + polymer burning Agglutination Silica Xerogel Spray drying Silica Xerogel Colloidal Silica particles Aggregates of colloidal silica particles Silicate solution Manufacture of spherical silicas Basic processes

  43. Highly cross-linked Permanent porosity Cross-linked swelling porosity Pore size >>2 nm Pore size in the swollen state -0.5-2.0nm Manufacture of rigid macro-porous organic supports Microporous gels Macroporous gels microsphere

  44. Particle morphology and texture • Shape of particles • Texture How the particles built up? (agglomerates, aggregates, structures obtained by leaching and dissolution, conversion of liquid droplets into solid spheres) • Method of examination: Light and electron microscopy

  45. Particle size and size distribution • Magnitude of particle size dp (dp= 10,20,40,80 mm) • Particle size distribution How is the size distribution measured, calculated and presented? • Changes of the particle size as a result of pressure and flow

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