Chromatography and its types
- 1. PRESENTER: TUBA NAFEES CHROMATOGRAPHY & IT’S TYPES 1
- 2. CHROMATOGRAPHY Chromatography is a technique that separates components of a mixture • The Russian botanist Mikhail Tsvet coined the term chromatography in 1906. • Archer Martin and Richard Synge during the 1940s and 1950s, established the principles and basic techniques of partition chromatography, and their work encouraged the rapid development of several chromatographic methods 2
- 3. CHROMATOGRAPHIC TERMS Adsorbent: A material which will allow components of the mixture to adhere to itself The common adsorbents used are alumina (Al2O3)and silica gel (SiO2). Both are polar. Adsorption is the process of molecules 'adhering' to one another, without the making of chemical bonds. 3
- 4. PRINCIPLE OF CHROMATOGRAPHY The mixture is dissolved in the mobile phase, which carries it through a structure holding another material called the stationary phase. Components of sample most strongly adsorbed Components of sample least strongly adsorbed Eluent STATIONARY PHASE Original sample placed here 4
- 5. Affinity ------ Adsorption & Solubility High adsorption-------- slow movement through a column High solubility----------- Faster movement through a column Polarity of the compounds dictate their affinities towards Stationary & mobile phase. 5
- 6. TYPES OF CHROMATOGRAPHY CHROMATOGRAPHIC BED SHAPE Planar e.g. TLC & PAPER Column Mobile Phase Liquid HPLC, FPLC. Gas Supercritical Fluid Separation Mechanism Ion exchange Size exclusion / Gel permeation/ GEL Filtration Adsorption Chromatography Partition Chromatography Affinity Chromatography Stationary Phase Normal Phase Reverse Phase 6
- 7. Least hydrophobic components elute first Most hydrophobic components elute last Product elutes 7
- 8. ADSORBTION CHROMATOGRAPHY Separation of components depends upon the degree of adsorption by the adsorbent and solubility in the solvent 8
- 9. PARTITION CHROMATOGRAPHY Based on the partitioning of compounds between a liquid stationary phase and a liquid mobile phase. The liquid stationary phase can be held on any solid support. Based on solute partition between two liquids 9
- 10. Based on the exchange of charged components of the mixture with charges on stationary phase • Allows separation of charged/ ions/ polar molecules • Cation exchange= Column retains cation • Anion exchange= Column retains anion A change in pH affects the charge on the particular molecules and therefore alter binding ION EXCHANGE CHROMATOGRAPHY 10
- 11. CATION EXCHANGE CHROMATOGRAPHY 11 Positively charged molecules replace cations attached to negatively charged functional groups
- 12. List of ion exchangers used in IE Chromatography S. No ION EXCHANGERS Functional group 1 DEAE Cellulose (Anion exchanger) DEAE (Diethylaminoethyl) 2 QAE Sephadex (Anion exchanger) QAE (Quaternary aminoethyl) 3 Q Sepharose (Anion exchanger) Q (Quaternary ammonium) 4 CM- Cellulose (Cation exchanger) CM (Carboxymethyl) 5 SP Sepharose (Cation exchanger) SP (Sulfopropyl) 6 SOURCE S (Cation exchanger) S (Methyl sulfate) 12
- 13. Affinity chromatography is a method of separating biochemical mixture based on a highly specific interaction between target molecule and ligand attached to stationary phase. AFFINITY CHROMATOGRAPHY 13
- 14. GEL PERMEATION CHROMATOGRAPHY The smaller analytes can enter the pores more easily and therefore are eluted last Conversely, larger analytes lesser time in the pores and are eluted quickly. The size of the pores for the packing should be chosen according to the range of molecular weight of analytes to be separated. Separation of molecules on the basis of their size and shape 14
- 15. HPLC 15
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- 18. PICTURES CREDITS • https://microbenotes.com/chromatography-principle-types-and- applications/ • http://www.dynamicscience.com.au/tester/solutions1/chemistry/analyt ical%20chem/columnchromatography.htm • https://chembam.com/techniques/chromatography/ • https://link.springer.com/chapter/10.1007/978-981-15-1547-7_11 • https://en.wikipedia.org/wiki/Gel_permeation_chromatography • https://makeagif.com/gif/affinity-chromatography-protein-seperation- OkCLDs • https://gfycat.com/joyousvictoriouslabradorretriever • https://www.researchgate.net/figure/Representation-of-a-HPLC- system-with-mobile-phase-solvent-The-system-is-connected-to- a_fig5_306375471 • http://faculty.sdmiramar.edu/fgarces/LabMatters/Instruments/HPLC/HP LC.htm 18
- 19. REFERENCES https://en.wikipedia.org/wiki/Ion_chromatography https://chromatography.conferenceseries.com/events-list/applications-of- chromatography https://www.brainkart.com/article/Column-chromatography--Types,-Principle_27838/ http://www.biologydiscussion.com/biochemistry/chromatography-techniques/top-12- types-of-chromatographic-techniques-biochemistry/12730 http://library.umac.mo/ebooks/b28050630.pdf https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206469/ Kumar. P. (2018). Fundamentals and Techniques of Biophysics and Molecular Biology. New Delhi: Pathfinder Publication. p. 7. ISBN 978-93-80473-15-4. https://microbenotes.com/chromatography-principle-types-and-applications/ https://byjus.com/chemistry/column-chromatography/ https://www.google.com/amp/s/www.azolifesciences.com/amp/article/Life-Science- Applications-of-Chromatography.aspx 19