Chromatography types

Chromatography and its types

1. CHROMATOGRAPHY

2. Presenters Tahreem Khawar Afifa Khizar Arfa Khizar Farah Masood Amna Shehzadi

3. Introduction of Chromatography

4. DEFINITION It is a physical separation method of separation in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it . It is used for extraction and purification of molecules in downstreaming processing

5. Mixture Separate Analyze Identify Purify Quantify Components CHROMATOGRAPHY

6. Glossary… Chromatograph: Instrument employed for a chromatography. Eluent : Fluid entering a column. Eluate : Fluid exiting the column. Elution: The process of passing the mobile phase through the column. Flow rate: How much mobile phase passed / minute (ml/min).

7. Uses of Chromatography

8. Uses for Chromatography Real-life examples of uses for chromatography: Pharmaceutical Company Hospital Environmental Agency Manufacturing Plant

9. Types of Chromatography Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) Gas Chromatography – separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Paper Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography – separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

10. Separation will based on Size Charge Hydrophobicity Charge

11. Size Exclusion chromatography

12. Introduction: Invented by grant henry and colin . does not require an “interaction” to achieve separation. It is a chromatographic method in which molecules in solution are separated by their size . applied to large molecules or macromolecular complexes such as proteins and industrial polymers.  Also known as “gel filtration chromatography” or “gel permeation chromatography”.

13. Principle: The sponge-like gel beads with pores serve as molecular sieves for separation of smaller and bigger molecules. A solution mixture containing molecules of different sizes (e.g. different proteins) is applied to the column and eluted. The smaller molecules enter the gel beads through their pores and get trapped. On the other hand, the larger molecules cannot pass through the pores and therefore come out first with the mobile liquid

14. Principle:

15. Instrumentation:

16. advantages Short analysis time. Well defined separation. Narrow bands and good sensitivity. There is no sample loss. Small amount of mobile phase required. The flow rate can be set. disadvantages Limited number of peaks that can be resolved within the short time scale of the GPC run. Filtrations must be performed before using the instrument to prevent dust and other particulates from ruining the columns and interfering with the detectors. The molecular masses of most of the chains will be too close for the GPC separation to show anything more than broad peaks

17. Thin layer chromatography(TLC)

18. TLC is one of the simplest, fastest, easiest and least expensive of several chromatographic techniques used in qualitative and quantitative analysis to separate organic compounds and to test the purity of compounds.

19. TLC is a form of liquid chromatography consisting of: A mobile phase (developing solvent) and A stationary phase (a plate or strip coated with a form of silica gel) Analysis is performed on a flat surface under atmospheric pressure and room temperature

20. Principle of TLC It is based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employed The components with more affinity towards stationary phase travels slower. Components with less affinity towards stationary phase travels faster

21. TLC consists of three steps - spotting, development, and visualization. Spotting consists of using a micro pipet to transfer a small amount of this dilute solution to one end of a TLC plate. Development consists of placing the bottom of the TLC plate into a shallow pool of a development solvent

22. Visualization The silica gel on the TLC plate is impregnated with a fluorescent material that glows under ultraviolet (UV) light.

23.   Separation of mixtures in microgram quantities by movement of a solvent across a flat surface; components migrate at different rates due to differences in solubility, adsorption, size or charge.

24. Where TLC used Pharmaceutical and drugs Clinical chemistry, forensic chemistry, and biochemistry Cosmetology Food analysis Environmental analysis Analysis of inorganic substances

25. Advantages of TLC Short analysis time Spot can easily visualized Adoptable the most pharmaceuticals Low cost Reliable and quick

26. HYDROPHOBIC INTERACTION CHROMATOGRAPHY

27. Initally it is called salting out bead polymers were created that are suitable for HIC chromatographic purposes . The surface of the beads was modified by hydrophobic alkyl or aryl groups. Such media include the polysaccharide-based Butyl- Sepharose , Octyl-Sepharose and Phenyl- Sepharose materials that are derived from polymers that had proven to be suitable for chromatographic separation of proteins.

28. Why HIC Different basis of separation Weaker interactions Less structural damage Maintain high activity

29. Principal Separation of substances is based on their varying strength of interaction with hydrophobic groups attached to an uncharged gel matrix Hydrophobic groups on proteins are sufficiently exposed to bind to the hydrophobic groups on the matrix . High salt concentration promote binding of Proteins with Ligand Low salt concentration promotes unbinding of protein with ligand

30. HYDROPHOBIC INTERACTION CHROMATOGRAPHY HAS 4 STAGES Equilibrium Sample application and wash Elution Regeneration

31. HIC Resin Protein

32. Factors Affecting Hydrophobic interaction Chromatography The type and density of the hydrophobic ligand on the matrix surface The quality of the salt used and its concentration pH Temperature Additives

33. Application of Hydrophobic interaction chromatography Purification of a monoclonal antibody for clinical studies of passive immunotherapy of HIV- 1. Purification of recombinant human Epidermal Growth Factor (h-EGF) from yeast. Assessment of purity and quantification of plasmid DNA in process solutions using high-performance hydrophobic interaction chromatograph Separation of biomolecules Economical