Column chromatography.pptx

Prepare by : Pawan Dhamala
Topic: Column Chromtography
M.Pharmacy (Pharmaceutics)
RR College of Pharmacy,
Bangalore

1. 08-06-2022 © R R INSTITUTIONS , BANGALORE 1 RR COLLEGE OF PHARMACY MODERN PHARMACEUTICAL ANALYTICAL TECHNIQUES COLUMN CHROMATOGRAPHY SUBMITTED BY : SUBMITTED TO: PAWAN DHAMALA ASSOCIATE PROF. SYED NIZAMMUDIN 1 ST SEM , M.PHARMACY DEPARTMENT OF PHARMACEUTICAL ANALYSIS

2. 08-06-2022 © R R INSTITUTIONS , BANGALORE 2 CONTENT Introduction – Definition and Principle Common terminologies used in column chromatography Types of column chromatography Practical steps involved in column chromatography Applications of column chromatography Advantages and disadvantages of column chromatography

3. INTRODUCTION OF CHROMATOGRAPHY Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact with a selectively absorbent stationary phase . OR It is a technique that enables the separation, identification and purification of the components of a mixture for qualitative and quantitative analysis . It was developed by the American petroleum chemist D. T. Day in 1900 . In 1906 , M. S. Tswett , Polish Botanist used the adsorption columns to detect the plant pigments. Martin and Synge introduced partition column chromatography in 1941. 08-06-2022 © R R INSTITUTIONS , BANGALORE 3

4. INTRODUCTION-COLUMN CHROMATOGRAPHY Column chromatography may be defined as a separation process involving the uniform percolation of a liquid solute through a column packed with finely divided material . If solid is used as stationary phase then it is Adsorption CC ( solid – liquid chromatography ) If liquid is used as stationary phase then it is Partition CC ( liquid – liquid chromatography ) 08-06-2022 © R R INSTITUTIONS , BANGALORE 4

5. COMMON TERMINOLOGIES Stationary phase : It is also called as adsorbent . It is a solid or liquid. Mobile phase : It is also known as Eluent . Eluent : Mobile phase used to elute the solutes from the bed of column. Sample : It is also known as adsorbate which get adsorbs. Elution : process of removing the components from the bed of column using mobile phase. Eluate : Fraction coming out of column containing separated component. 08-06-2022 © R R INSTITUTIONS , BANGALORE 5

6. Types of column chromatography Adsorption column chromatography The stationary phase used normally are in Solid state and the mobile phase/s are in liquid or gaseous state. Example: CCC, Ion Exchange chromatography, Gel Chromatography, Affinity Chromatography, HPLC, GSC Partition column chromatography Both the stationary phase and the mobile phase are in Liquid state . Example: CCC, HPLC, GLC 08-06-2022 © R R INSTITUTIONS , BANGALORE 6

7. 1.Adsorption column chromatography 08-06-2022 © R R INSTITUTIONS , BANGALORE 7

8. Principle When a mixture of components dissolved in the mobile phase is introduced into the column, the individual components move with different rates depending upon their relative affinities . The compound with lesser affinity towards stationary phase moves faster and it is eluted out of the column first . The one with greater affinity towards stationary phase moves slower down the column and hence it is eluted latter . Thus the compounds are separated. 08-06-2022 © R R INSTITUTIONS , BANGALORE 8

9. The type of interaction between SP (Adsorbent) and solute is reversible in nature . Example: Ion-dipole, Dipole-dipole (Hydrogen bond, Van-deer waal’s ), London forces, Debye forces, H-bond donor, H-bond acceptor, - interactions. 08-06-2022 © R R INSTITUTIONS , BANGALORE 9

10. PRACTICAL STEPS INVOLVED 08-06-2022 © R R INSTITUTIONS , BANGALORE 10

11. 1. COLUMN CHARACTERISTICS AND SELECTION 08-06-2022 © R R INSTITUTIONS , BANGALORE 11

12. 2. STATIONARY PHASE/ADSORBENTS Requirements of an ideal stationary phase: 1. Particles should have uniform size and spherical shape , Particle size :60-200 µ  2. Should have high mechanical stability . 3. They should be insoluble in mobile phases. 4. Should be chemically inert . 5.Should be colourless to facilitate observation of zones. 6. Should have reproducible properties from batch to batch. 7. Should allow free flow of mobile phase 8. Should be useful for separating wide variety of solutes . 9. Should be inexpensive and freely available . 08-06-2022 © R R INSTITUTIONS , BANGALORE 12

13. The most commonly used chromatographic adsorbent is silica or Silicic acid or silica gel (80-100 mesh or 100-200 mesh size, which has a particle size of 60-200 µm) . They adsorbs polar and unsaturated substances by the formation of hydrogen bonds with the hydroxyl groups on the silicon atom. 08-06-2022 © R R INSTITUTIONS , BANGALORE 13 Type of mesh Size in microns 60/120 mesh 120-250 micron 100/200 mesh 75-150 micron 70/230 mesh 63-200 micron 70/ 325 mesh 45-200 micron

14. Classification of Stationary Phase 08-06-2022 © R R INSTITUTIONS , BANGALORE 14 Weak activity Medium activity Strong activity Sucrose Calcium carbonate Activate magnesium silicate Starch Calcium phosphate Activated alumina Inulin Magnesium carbonate Activated charcoal Talc Magnesium oxide Activated magnesia Sodium carbonate Calcium oxide Activated silica

15. 3. MOBILE PHASE Mobile phase act as an eluent to remove the pure component out to the column. Strain (in 1942) has arranged the solvents in order of eluting power. A grouping of solvents in order of chromatographic strength/Elution strength/polarity index is known as Eluotrophic series . 08-06-2022 © R R INSTITUTIONS , BANGALORE 15

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17. 4. PREPARATION (PACKING) OF COLUMN 08-06-2022 © R R INSTITUTIONS , BANGALORE 17

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19. 4. SAMPLE APPLICATION Wet application: Dissolve the sample in the initial mobile phase and apply by pipette to the top of the column. This is very good method but in most of cases the samples are not soluble in the initial mobile phase . 08-06-2022 © R R INSTITUTIONS , BANGALORE 19

20. 2. Dry loading: Dissolve sample in any volatile solvent. The sample solution is then adsorbed an small amount of adsorbent and the solvent is allowed to evaporate. The dry adsorbent loaded with the sample is then applied to the column. 08-06-2022 © R R INSTITUTIONS , BANGALORE 20

21. 6. DEVELOPMENT TECHNIQUES 1. FRONTAL ANALYSIS :  This technique was developed by Tiselius in 1940 .  In this method, the solution of sample mixture is added continuously on the column . No mobile phase (solvent) is used for development of column.  A mixture containing A,B,C is added on the column.  If component A is least adsorbed, component B is adsorbed to intermediate extent and component C most strongly to adsorbent material.  08-06-2022 © R R INSTITUTIONS , BANGALORE 21

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23. 2. DISPLACEMENT ANALYSIS :  In this method, a small volume of mixture (A, B & C) is added to the column along with the solute-D (most strongly adsorbed than any component of the mixture) .  The adsorbed constituents of mixture are displaced by the solute (D).  Each solute in the mixture in turn displaces another substance which is less firmly adsorbed.  The least adsorbed constituent is pushed out of the column first.  The solute-D used is called as displacer.  08-06-2022 © R R INSTITUTIONS , BANGALORE 23

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25. 3. ELUTION ANALYSIS :  It is a common method used in column chromatography .  In this method a small volume of mixture to be separated is added on the top of column & mobile phase is allowed to flow through the column.  The mixture introduced on the column gets separated into individual as the components of mixture are adsorbed to the column material to different extent.  Each component of mixture is eluted out as separated components (called eluate ). 08-06-2022 © R R INSTITUTIONS , BANGALORE 25

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27. 7. DETECTION OF COMPOUNDS  The detection of coloured components can be done visually. Absorption of UV-Visible light using UV-Vis detector like Photo Multiplier Tube (PMT ).  Emitted light or Fluorescence is measured using Fluorescence detectors. Flame ionization Detector and Refractive index detector. Evaporation of the solvent of an eluate and weighing the residue. Monitoring the fractions/ Eluates by TLC. 08-06-2022 © R R INSTITUTIONS , BANGALORE 27

28. 8. RECOVERY OF COMPOUNDS 08-06-2022 © R R INSTITUTIONS , BANGALORE 28

29. 08-06-2022 © R R INSTITUTIONS , BANGALORE 29 2. Partition column chromatography

30. It is similar to adsorption column chromatography except that, the stationary phase is liquid . A solid support like silica gel or cellulose is used to hold the liquid stationary phase like water, buffer solutions, oils, polysiloxanes are used as thin film on the surface. Mobile phase is similar to that of column chromatography. The gradient elution technique is not used since the equilibrium will be disturbed. This technique is not widely used. 08-06-2022 © R R INSTITUTIONS , BANGALORE 30

31. APPLICATIONS Separation and identification of dyes [Methyl orange and methylene blue] in mixture. Separation of mixture of compounds: Separation of alkaloids, glycosides, amino acids, plant extracts. Separation of Active Pharmaceutical Ingredients (API) in formulation. It is used to isolate the active constitutes (like flavonoids, saponins ) of plant extract. It is used to remove impurities and helpful in purification process. 08-06-2022 © R R INSTITUTIONS , BANGALORE 31

32. Estimation of drugs in formulations and crude extracts. Determination of Betamethasone in tablets and injections Determination of primary and secondary glycosides in Digitalis leaf Separation of geometrical isomers and diastereomers Separation of Tautomers and racemates Isolation of metabolites from biological fluids: 17-ketosteroids from urine and Cortisol from blood 08-06-2022 © R R INSTITUTIONS , BANGALORE 32

33. ADVANTAGES  Any type of mixture can be separated by column chromatography.  Any quantity of the mixture can also be separated.  Wider choice of mobile phase.  In preparative type, the sample can be separated and reused.  Automation is possible.  08-06-2022 © R R INSTITUTIONS , BANGALORE 33

34. DISADVANTAGES  Time consuming method.  More amount of solvents are required, which are expensive.  Automation makes the technique more complicated and expensive. 08-06-2022 © R R INSTITUTIONS , BANGALORE 34

35. B.K.SHARMA , Instrumental methods of chemical analysis, Goel publishing house, Twenty fourth edition , Pg no: C-96 to C-108. S.RAVI SHANKAR, Text book of pharmaceutical analysis, Rx publications, 2001.Pg no : 13-4 to 13-13. INSTRUMENTAL METHODS OF CHEMICAL ANALYSIS by GURUDEEP R. CHATWAL 08-06-2022 © R R INSTITUTIONS , BANGALORE 35 REFERENCES

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