CHROMATOGRAPHY ppt use and its application

Chromatography

1. 2.4.CHROMATOGRAPHY 1

2. C HROMATOGRAPHY : It is a technique used to separate and identify the components of a mixture. Works by allowing the molecules present in the mixture to distribute themselves between a stationary and a mobile medium. Molecules that spend most of their time in the mobile phase are carried along faster. 2

3. I NTRODUCTORY P RINCIPLES Chromatography is a combination of two words; Chromo – Meaning color Gr a p h y – writ i n g / r e pre s e ntation of so m ethi n g on paper/ Prin c i p le : th e samp les ar e subjected to fl o w by mobi le liquid onto or through the stable stationary phase . 3

4. Chromatography 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 r etaine d and s e parated b y i t . 28

5. M i x ture Separate Analyze Identify Purify Q u a n t i fy 5 Components C H R O MAT O GRAP H Y

6. Mobile Phase – gas or liquid that carries the mixture of components through the stationary phase. Stationary Phase – the part of the apparatus that holds the com p o nent s a s they m o v e th r oug h it , s e parating the m . ` T h e sta t i o na r y phase m a y b e a soli d , o r a li q uid suppo r ted o n a soli d o r g e l . 6

7. U s e s f o r C h r omato g rap h y 7 Chromatography is used by scientists to : Ana l y z e – e xam i n e a mi x tu r e , it s components, and relations to one another Identify – determine the identity of a mixture or components based on known components their P u rify – s e parate components i n o r d e r to isolat e on e of interest for further study Quanti f y – dete r mine the amo u n t o f the a mi x tu r e and/or the components present in the sample

8. Pharmacy Company Hospital Law Enforcement Environmental Agency Food Manufacturing Plant 8

9. Chromatogram: It is the visual output of the chromatograph. Chromatograph: It is equipment that enables a sophisticated Separation. S t a tiona r y p h ase ( b ounde d p h ase ) : It is a phase that is covalently bonded to the support particles or to the insi d e w all of the colu mn tubin g .

10. M o bil e p h ase: It is the phase which moves in a definite direction. A n a l y te ( S am ple): It is the substance to be separated during chromatography. Eluate: It i s the mo b il e pha s e le a ving the colu m n . 10

11. R e te n tion t i m e : It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. Eluent: It is the solvent that will carry the analyte. 11

12. R e ta r dation f a c t o r ( R ): Fraction of an analyte in the mobile phase of a chromatographic system. 12

13. 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) 39

14. GEL FILTRATION Gel filtration separates molecules according to the differences in size as they pass through the filtration medium packed in the column. It is well suited for biomolecules that are sensitive to pH ,concentration and harsh environment. Parameters that affects gel filtration are, particle size, flow rate, packaging density, porosity of the particle and viscosity of the mobile phase. 40

15. C ONT ’ D ….. Gel permeation chromatography 41

16. Cross linked dextrans (sephadex) A g a r os e ( s e pha r ose) Polymer /Polyacrylamide/ P o r ou s g lass g el. APPLICATIONS Fractionation (purification of the desired protein using suitable gel) Molecular weight determination 16 M A TERIA L S REQUIRED ARE :

17. ION EXCHANGE Ion e x c ha n g e c h r omat o g rap h y i s u se d to r e m o v e i on s o f o n e ty p e f ro m a mixt u r e and r e place them b y ion s of another type. The basic principle is reversible competitive binding 17

18. ION EXCHANG E RS Cation exchangers (negative ions – stationary) Anion exchangers (positive ions - stationary) Four types of polymers are commonly used. They are, Synthetic hydrophobic polymer resins cross linked with d i vinel y benzene. Naturally occurring as well as synthetic polymers(cellulose) Synthetic hydrophilic polymers Silic a g el 44

19. AFFINITY CHROMATOGRAPHY: Affinity chromatography includes bioaffinity, dye- ligand affinity and immobilized metal ion afffinity techniques. It is based on the formation of the specific and reversible complexes between a pair of biomolecules. 19

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