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Adsorption and partition column chromatography.pptx
- 1. Unit III Adsorption andpartition column chromatography Mrs Vandana Sharma Assistant professor
- 2. Previous years questions Explain partition chromatography with reference to the principle, procedure, different types and applications in pharmacy field. 16 1. Answer the following questions in very short (Maximum 20 Words) a) Partition coefficient . 2 b) Significance of Rf value 2
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- 4. Contents Methodology Advantages Disadvantages applications
- 5. Definition Chromatography- Colorwriting Chromatography---- group of methods-----for separating ------ molecular mixtures-----that depends upon the differential affinities of the solute between the 2 immiscible phase. One phase--- Stationary phase Other phase--- fluid or gas---- Mobile phase Stationary Phase--- Porous or finely divided solid Or Liquid that has been coated as thin layer on an inert support Mobile Phase--- May be pure liquid or mixture of solutions or may be gas or mixture of gases
- 6. Classification Stationary Phase-Solid or Liquid Mobile phase- Liquid or gas Basis of separation of components ---- result of combination of two or more factors example Migration rate These factors rely on stationary and Capillary action the mobile phase employed extent of adsorption etc S. No. Classes Examples 1. Adsorption Chromatography Columnar methods Gas solid chromatography 2. Partition Chromatography Paper chromatography Thin layer chromatography (TLC) Reverse phase partition extraction chromatography
- 7. Classification S. No. classes Examples 3. Ionexchange chromatography Cation exchange chromatography Anion exchange chromatography 4. Exclusion Chromatography Gel Chromatography 5. Electrochromatography Electrohoresis S. No. Classes Details 1. Preparative Chromatography Is a type of purification Separation mixture components for further used 2. Analytical Chromatography Determines the relative proportions of mixture analytes Require small amounts of
- 8. General Principles ofchromatography Principle of chromatography involves ----- separation of components into varied bands (or color graphs) and their identifications. Preferential separation occurs based on the differential affinities of compounds for stationary and mobile phase. Upon separation compounds are identified by different methods of detections . The variation in affinities among components occurs due to relative adsorption or partition coefficient in both the phases • Adsorption • Partition
- 9. Adsorption Based onthe polarity of the component with respect to the stationary phase If stationary phase---polar polar component- will migrate slowly--- Sample component--- polar separate from sample at last In the same manner stationary phase-nonpolar nonpolar component- will migrate slowly--- sample component –nonpolar separate from sample at last Result- hence- the stationary and mobile phase--- are opposite---ie., Stationary phase ----polar Mobile phase ---- nonpolar or Vice versa Eluent--- Solvent used in the column as mobile phase Eluate-The liquid reaching the column end with the separated components
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- 11. Partition Partition depends on thecomponents solubility in different liquids--- hence--- mobile phase and stationary phase ---- should be liquid. The liquid stationary phase is a thin film on a solid support in the column. Thus , if the components of a sample mixture have differences in their solubility in different liquids And are of same nature (i.e., either polar or nonpolar) they split into two liquid phases (i.e., Mobile and Stationary phases) This is based on their partition coefficient between two liquids. Definition of Partition or distribution coefficient ( Kd ) --- Distribution of compound among immiscible phases. Constant at given temperature It is basis of separation by chromatographic methods
- 12. Continue…… Eg. A compoundthat distributes itself between two immiscible solvents (A and B) at given temprature Kd = Conc. In solvent A Conc. In Solvent B If Kd = value is 0.5 (for a substance distribution b/w silicic acid and benzene)= means (conc. of substance in benzene is twice than that in silicic acid) Theories of Chromatography • Plate theory– depends on Migration rate of solutes • Rate or kinetic theory- depends on the development of peaks in the chromatograms
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- 14. Development of Chromatograms Thefollowing methods are used for development of chromatograms Frontal Analysis Elution Analysis Displacement Analysis
- 15. A+B In carrier solvent Saturated adsorbent A+ B A VR determine the conc. of A in mobile phase Frontal Analysis 1. Mixture of sample (A+B) --- is continuously introduced in the adsorbent packed column 2. This results in complete adsorption of the sample and saturation of the adsorbent. 3. On further adding the sample solution, A (the least adsorbed component) elutes out first in pure form, accompanied by the mixture of A+B (the strongly adsorbed component ) that elutes out slowly. Component A have low affinity for adsorbent -- separated from component B having high affinity for adsorbent
- 16. A. Frontal Analysis- Preparative technique- Is a type of purification Separation mixture components for further used 1. The sample solution is passed through the adsorbent column. 2. The solute having high affinity for adsorbent ---- occupy the active sites of adsorbent column or the solute having low affinity for adsorbent---- weakly adsorbed or accumulated in the migrating solvent front Result---the minimum adsorbed solute first elute out of the column followed by the other solutes based on their degree of adsorption. Uses--- This method is used for eliminating traces of strongly adsorbed unwanted substances, while retaining the least adsorbed desirable components.
- 17. B. Elution analysis Widely used for developing chromatograms In this method 1. Small amount of sample solution is added on the column top. 2. The pure solvent (Mobile phase or eluent ) is made to run down the column. 3. This leads to differences in the migration of solutes in mobile phase. 4. Different solute elute out the column at different rates depending on their partition coefficient. 5. Differences in partition coefficient of the solutes in a sample lead to separation mixtures that appear as bands or zone. Note- Gradient elution 1. Is a technique of separating the components (having variable affinity for the stationary phase) by changing the eluent composition. 2. The eluting power of mobile phase --- can be increased by changing the ratio of two or more eluting components.
- 18. C A+C 4. The Components eluteout on the basis of their degree of affinity but their migration depends on the mobile phase. B+C Mobile Phase (Eluent C) C C A+ B 2. A mobile phase = having lower affinity than the components for the stationary phase) allow to run through the column. 3. Migration rate of components is slower than that of mobile phase. 1. A small amount of Mixture (A+B components) is added at the column top. 5. The components completely separate from each other by eluting out with another mobile phase ( Eluent C). 6. Note - This elution method can be improved by changing the eluent after specific time period 7. For example – in step wise elution, a combination of eluents (with increased eluting powers ) is used to elute out the components having greater affinity for the stationary phase and also to aid their movement throughout the system. C Elution Analysis
- 19. c. Displacement Analysis- Inthis method 1. A small amount of sample solution is introduced at the column top. 2. The mixture components are separated by the downward flow of displacing agent--- which is solution of substance having stronger affinity than the other components for the stationary phase 3. Column adsorbs the displacing agent and form zone or band at the top. 4. This band of displacing agent moves downwards and displaces out the sample components from the adsorption sites.
- 20. 5. Thus, thecomponent bands are formed farther from the band of displacing agent. 6. The components continuously displace each other from the column. 7. The component having minimum affinity will first elute out of the column, followed by the elution of other components depending on their adsorption degree. Advantage- 1. Column can be heavily loaded ( The amount of sample solution added in the column termed loading.) Disadvantages- 1. After one flow the displacing agent saturates the column.
- 21. Eluent C or Displacing agentC 1. A sample mixture (A and B components)is introduced at the column top, where it gets adsorbed on the stationary phase. C 2. Then displacing agent ( C ) is made to run through the column. Because ( C ) that adsorbs on the stationary phase more strongly than the sample components (A+B) B 3. This agent (C )while moving down the column displaces the components (A and B) from the stationary phase and elutes them out. A 4. This leads to separation of components depending on the differences in their partition coefficient and adsorption. Disadvantage-Displacement analysis does not give complete separation of components as the zones of pure components as the zones of pure components have regions of mixtures between them that can be collected for further treatment. Displacement Analysis
- 22. Retention factor (RF) ------------- ----------- --------------- 4 c m 5.5 cm Solvent front Compoud spot Baselin (Origin) Detection of RF value Distance travelled by the compound Distance travelled by the solvent front Retention factor (RF ) = (RF ) = 4.0 cm = 0.73 5.5 cm If all the component move such a distance from baseline that RF values lays between 0.15 and 0.85, the solvent used is considered adequate
- 23. Importance----- This indicatesthat the distance travelled by the compound and the RF value of the same compound are directly proportional to each other. This also indicates that when RF values of different compounds are compared under the same chromatographic conditions, the compound with larger RF value is less polar due to its weak interaction with polar adsorbent on the plate.
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