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1. **Introduction to Chromatography: Principles and Techniques** 2. **Chromatography is a physical separation method inv

Chromatography Principles, Separation Techniques, Stationary Phase, Mobile Phase, Column Chromatography

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1. **Introduction to Chromatography: Principles and Techniques** 2. **Chromatography is a physical separation method inv

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  1. Chromatography • A physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (the stationary phase) while the other (the mobile phase) moves in a definite direction (IUPAC) • Term ‘chromatography’ was coined by Mikhael Tswett in 1906 (Chroma – colour, Graphos – writing) • Invented by M. Tswett in 1906 • Partition and Paper chromatography was introduced by Martin and Synge in 1941 • Gas Chromatography was introduced by Martin and his colleagues in 1952

  2. Chromatography: Principles • Partition/ Distribution Coefficient (Kd) (Kd= Concentration in solvent A/Concentration of solvent B) • Effective distribution coefficient Total amount of substance present in one phase to total amount in other phase • Stationary Phase: Immobilized (Solid, gel, liquid or solid/liquid mixture) • Mobile Phase: flows through stationary phase (liquid or gas)

  3. Chromatography: Theories Two Theories: 1. Plate Theory 2. Rate Theory

  4. Plate Theory • Developed by Martin and Synge A/Plate theory, • Chromatographic column consists of a series of theoretical plates • Equilibrium of solute between stationary and mobile phases at each of these plates • Migration of solute by series of stepwise transfers between plates • Efficiency of separation increases with increase in number of theoretical plates N = L/H (N: No. of theoretical plates; L: Length of Column; H: Height Equivalent of Theoretical Plates)

  5. Rate Theory • Explains the effect of variables such as mobile phase velocity and adsorbabilities which determine the width of elution band • Proposed by Van Deemter et al. (1956) to explain the main parameters which influence band broadening H = A + B/u + Cu (Van Deemter equation ) H: Height equivalent of theoretical plates A: Eddy Diffusion B: Longitudinal diffusion constant C: Mass transfer coefficient u: linear velocity • A molecule alternates rapidly between adsorbed and desorbed states

  6. Eddy Diffusion Eddy Diffusion • Result of multitude of pathways by which a molecule can find its way through a column • Length of pathways differ, thus, residence time for molecules of same species in column are variable • Can be related to particle size, geometry and tightness of packing of stationary phase • Independent of flow rate

  7. Longitudinal Diffusion • Results from tendency of molecules to migrate from concentrated center part of band toward more dilute regions • Can occur both in mobile and stationary phase • Most important where mobile phase is gas because of higher diffusion rates in gases • Amount of diffusion increases with time • Longitudinal diffusion is inversely proportional to flow rate

  8. Non – Equilibrium Mass Transfer • Flow rate of mobile phase is ordinarily so rapid that true equilibrium between phases can’t be realized causing broadening of chromatographic band. • Directly proportional to flow rate

  9. Separation on Column 1. Rates of solute migration u = L/tm (u: average linear rate of migration of mobile phase, L: length of column, tm: retention time of mobile phase ) v = L/tR (v: average linear rate of migration of solute, L; length of column, tR: retention time of solute )

  10. …Continued • Relationship between retention time and partition coefficient v = u x (no. of moles of solute in mobile phase/total number of moles of solute) v = u{CMVM/ (CMVM+ CSVS)} v = u[1/{1 + (CSVS/CMVM)}] v = u[1/{1 + K(VS/ VM)}] k’ = K(VS/VM) [k’ = capacity factor]

  11. …Continued Resolution • Resolution is a measure of the separation of two peaks in terms of their average peak width at baseline Rs= 2(tr2− tr1) /(wb1+ wb2) (Rs: Resolution, tr: Retention time, wb1and wb2are the peak widths at baseline of the first and second closely eluting peaks)

  12. Development of Chromatogram • Frontal Analysis • Elution Analysis • Displacement Analysis

  13. Chromatography: General Steps • Adsorption or retention of a substance or substances on stationary phase • Separation of adsorbed or retained substances by mobile phase • Recovery of separated substances by continuous flow of mobile phase (Elution) • Qualitative and Quantitative Analysis

  14. Types of Chromatographic Techniques S. No. Technique Stationary Phase Mobile Phase 1. Column/Adsorption Chromatography Solid Liquid 2. Partition Chromatography Liquid Liquid 3. Paper Chromatography Liquid Liquid 4. Thin Layer Chromatography Liquid or Solid Liquid 5. Gas Liquid Chromatography Liquid Gas 6. Gas Solid Chromatography Solid Gas 7. Ion Exchange Chromatography Solid Liquid

  15. Classification of Chromatographic Methods Stationary Phase Mobile Phase Chromatographic Methods Solid Liquid Plane Chromatography Paper Chromatography Thin Layer Chromatography Adsorption Column Chromatography Solid (Ion exchange resin) Liquid Ion Exchange Chromatography Solid Gas Gas Solid Chromatography Solid Matrix Liquid Gel Permeation Chromatography Liquid Gas Gas Liquid Chromatography Liquid Liquid Liquid Liquid Chromatography Solid Liquid High Chromatography Performance Liquid

  16. References • Chatwal, G.R., Anand, S.K. (2019). Instrumental methods of chemical analysis. Himalaya Publishing House. Lundanes, E., Reubsaet, L., Greibrokk, T. (2012). Chromatography: Basic principles, sample preparationsand related methods. Wiley-VCH. Clatworthy, A.J., DeBree, P.K., Edwards, R.W.H., Ersser, R.S., Goodwin, H.J., Jackson, J.V., Lake, B.D. (1976). Chromatographic and electrophoretic techniques. A William Heinemann Medical Books Publication. Chicago. Zweig, G., Whitaker, J.R. (1971). Paper chromatographic and electrophoresis. Academic Press. New York. Wolstenholme, R., Jickells, S., Forbes, S. (2021) Analytical methods in Forensic Sciences. Wiley. DFS Manual NOTE: These slides are only for academic purposes • • • • •

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