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  1. 1. Presented by, Malika nischal 1
  2. 2.  Chromatography is an analytical method usd for the seperation ,identification and determination of chemical components in complex mixture. Or  Chromatography is the ability to seperate molecules using the partition characterisitics of molecules to remain in a stationary phase verse mobile phase. Why chromatography called chromatography?  It was first put forward into application in 1903 by M.S Tswett fot the separation of plant pigments since components had different color ―green chromatos—color― Can chromatography identify components?  Chromatography is the process of seperation but with detector it can identify manual cases, eyes and nose can be detector. 2
  3. 3.  STATIONARY PHASE:- Phase that stay inside the column and it can be a solid, gel or highly viscous liquid.  MOBILE PHASE:- It is the solvent moving through the column.  ELUENT:- Fuild entering the column( equilibration and elution buffers).  ELUATE:- Fluid existing the column (fractions separated).  ELUTION:- The process of passing the mobile phase through the column.  CHROMATOGRAM:- Graph showing detector response as a function of a time.  FLOW RATE:- Amount of mobile phase that passes through column per minute (ml/min).  LINEAR VELOCITY:- Distance passed by mobile phase per min in the column (cm/min). 3
  4. 4.  The resolution expresses the extent of separation between the components from the mixture. Retention time It is the time between injection and detection of the analyte. Retention volume It is the volume of mobile phase required to elute a solute to a maximum from a column. • Retention time tell as about compound identity qualitatively • Peak Area or height tell about compound quantitatively. 4
  5. 5. General classification Liquid chromatography This separation is based on the interactions of the sample with mobile and stationary phase Gas chromatography This separation is based on the interactions of the sample with mobile and stationary phase High pressure liquid chromatography This separation is based on the interactions of the sample with mobile and stationary phase 5
  6. 6.  Adsorption chromatography: It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.  Partition chromatography: This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid. 6
  7. 7.  Ion Exchange chromatography : In this type of chromatography the use of a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. 7
  8. 8.  Molecular Exclusion Chromatography (gel permeation/gel filtration): This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. 8
  9. 9.  Affinity chromatography : this technique separates proteins on the basis of a reversible interaction between a protein or group of proteins and a specific ligand coupled to a chromatography matrix. The technique can be used to separate active bio molecules from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants. 9
  10. 10. ENZYME PURIFICATION Centrifugation Cross flow filtration This is the step followed after harvesting the crude medium in a rotor centrifuge at 6000 rpm for 30 min at 40C. This step is followed after centrifugation when supernatent is collected and filtered through a muslin cloth and then centrifuged again to concentrate the filtrate. Anion exchange chromatography Hydrophobic interaction chromatography DEAE(Diethylaminoethyl) Sepharose fast flow column (XK 50, GE Healthcare) . Source 30Q sepharose flow column XK 16, GE healthcare Phenyl‐Source fast flow column (XK 16, GE Healthcare 10
  11. 11.  This is a weak anion exchange chromatography which follows the principle of ion exchange i.e. at low ionic strength competition between the buffer ions and proteins for charged groups on the ion exchange is minimum so protein being more strong negatively charged bind strongly. Then by gradually increasing the ionic strength competition between protein and counter ions increase that tend the protein to elute. 11
  12. 12. Properties Description Ion exchange type Weak anion Total ionic capacity 0.11–0.16 mmol/ml media Exclusion limit 4 x 106 (globular proteins) Matrix Cross-linked agarose 6% Bead form Spherical, 45–165 μm Flow rate 300–600 cm/h* Chemical stability All commonly used aqueous buffers 1 M NaOH 8 M urea 6 M guanidine hydrochloride 70% ethanol The following should be avoided Oxidizing agents Long exposures (1 week, 20 C) to pH <4 12
  13. 13. Suggested buffers for use with DEAE Sepharose Fast Flow column 13
  14. 14.  In column chromatography the target molecule is bound while contaminants pass through the column, or vice versa. In gel filtration , which separates sample components based on size, the components travel through the column at different speeds, giving different elution positions. Therefore type of elution differs as:  GRADIENT ELUTION: The eluent composition is changed continuously toward conditions favoring dissociation from the chromatography medium. Elution position differs between substances depending on their binding strength. 14
  15. 15.  STEPWISE ELUTION: The eluent composition is changed stepwise at several intervals and each substance may be eluted in each step.  COMBINATION OF GRADIENT AND STEPWISE ELUTIONS: Gradient and stepwise elution can be combined by doing part of the elution in gradient mode and part of it in stepwise mode.  ISOCRATIC ELUTION:The composition of the eluent is unchanged during the entire purification (binding and elution). The target molecule passes through the column slower or fast than impurities. This type of elution is used in GF. 15
  16. 16.  This column provide Separation using Hydrophobic interaction between a protein and the hydrophobic ligand bound to the chromatography matrix. The technique is an ideal next step when samples have been subjected to ammonium sulfate precipitation or after separation by ion exchange chromatography.  The interaction between hydrophobic proteins and a HIC medium is influenced significantly by the presence of certain salts in the running buffer. A high salt concentration enhances the interaction while lowering the salt concentration weakens the interaction. In this example, all three proteins interact with the hydrophobic surface of the HIC medium, but, as the ionic strength of the buffer is reduced, the interaction is reversed and the protein with the lowest degree of hydrophobicity is eluted first. The most hydrophobic protein elutes last, requiring a greater reduction in salt concentration to reverse the interaction. 16
  17. 17. Equilibration HIC medium equilibrated with high-salt start buffer Wash Final ―salt-free‖ wash removes any hydrophobically bound proteins before re-equilibration. Sample application Start buffer causes hydrophobic proteins bind to hydrophobic ligands on the medium, becoming concentrated on the column. Proteins with insufficient hydrophobicity elute during or just after sample application. Elution 3 Elution 1 Decreasing salt content (using a linear gradient) causes hydrophobic proteins to elute. The least hydrophobic proteins elute first. Elution 2 Further decreases in salt displace the more hydrophobic proteins (more tightly bound). 17
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  19. 19. Q Source Sepharose Fast Flow ion exchangers include media that are called strong ion exchangers. strong exchanger are used in those cases where maximum resolution occurs at an extreme pH and the proteins of interest are stable at that pH . And the big advantage of using this column is its high binding capacity for selected proteins moreover this is a process of polishing that removes trace impurities or closely-related substances giving sample condition almost pure. Q SOURCE: R-O-CH2-CH-OH-CH2-O-CH2-CH-OH-CH2-N+(CH3)3 19
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  21. 21.            D.C.Harris, Exploring Chemical Analysis, 3rd ed, W.H. Freeman & Co. 2004 Scott, R. P. W. Introduction to Analytical Gas Chromatography; 2nd ed.;Marcel Dekker, 1998. Jennings, W. G.; Mittlefehldt, E.; Stremple, P. Analytical Gas Chromatography; 2nd ed.; Academic Press, 1997. McNair, H. M.; Miller, J. M. Basic Gas Chromatography; Wiley, 1997. Grant, D. W. Capillary Gas Chromatography; Wiley, 1996. Fowlis, I. Gas Chromatography; 2nd ed.; Wiley, 1995. Scott, R. P. W. Techniques and Practices of Chromatography; 2nd ed.; Marcel Dekker, 1995. Grob, R. L. Modern Practice of Gas Chromatography; 3rd ed.; Wiley, 1995. Baugh, P. E. Gas Chromatography: A Practical Approach; Oxford, 1994 Hinshaw, J. V.; Ettre, L. S. Introduction to Open Tubular Column Gas Chromatography; Advanstar, 1994. 21
  22. 22.  Grob, K. Split and Splitless Injection in Capillary Gas Chromatography; 3rd  ed.; Hüthig, 1993.  Hill, H. H.; McMinn, D. G. Detectors for Capillary Chromatography; Wiley,  1992.  Grob, K. On-Column Injection in Capillary Gas Chromatography; 2nd ed.;  Hüthig,1991.  Poole, C. F.; Poole, S. K. Chromatography Today; Elsevier, 1991.  Baars, B.; Schaller, H. Fehlersuche in der Gaschromatographie; VCH, 1994.  Kolb, B. Gaschromatographie in Bildern; Wiley-VCH, New York, 1999.  Kenndler, E.; Huber, J. F. K. In Analytiker Taschenbuch; Springer, 1989. 22
  23. 23. Thank you for your kind attention 23