ADSORPTION CHROMATOGRAPHY Muhammad Tanveer Khan
DEFINITION <ul><li>“ It is a type of chromatography in which a mobile liquid or gaseous phase is adsorbed onto the surface...
PRINCIPLE <ul><li>Principle involves competition of components of sample mixture for active site on adsorbent. These activ...
PRINCIPLE <ul><li>Separation occurs because of the fact that an equilibrium is established between molecules adsorbed on s...
TYPES Adsorption   chromatography Column  chromatography Thin layer  chromatography Gas solid chromatography
ADSORBENTS <ul><li>“ An adsorbent is a substance, usually porous in nature and with a high surface area that can adsorb su...
AN IDEAL ADSORBENT <ul><li>The Ideal adsorbent must fulfill the following requirements: </li></ul><ul><li>Insoluble in mob...
COMMON ADSORBENTS <ul><li>Hydrated silica gel </li></ul><ul><li>Silica gel G </li></ul><ul><li>Silica gel S </li></ul><ul>...
COMMON ADSORBENTS <ul><li>Modified silica gel </li></ul><ul><li>Alumina </li></ul><ul><li>Kieselghur (Diatomaceous earth) ...
THIN-LAYER CHROMATOGRAPHY <ul><li>“ The technique which involves flowing of mobile phase over a thin layer of adsorbent, a...
Instrumentation <ul><li>Chromatography jar </li></ul><ul><li>Capillary tube </li></ul><ul><li>Thin layer chromatography pl...
Instrumentation <ul><li>Chromatography jar: </li></ul><ul><li>It is made of glass and has a lid on it. Jar maintains prope...
Instrumentation <ul><li>TLC plate: </li></ul><ul><li>Borosilicate glass plates are preferred. Most commonly used </li></ul...
Instrumentation <ul><li>Mobile phase: </li></ul><ul><li>Mobile phase may be a single liquid or a mixture of liquids. </li>...
Procedure <ul><li>Clean and dried chromatography jar is taken. </li></ul><ul><li>A paper impregnated in the mobile phase i...
Procedure <ul><li>Sample is applied on TLC plate with help of capillary tube. </li></ul><ul><li>Sample spot is air dried. ...
Location of separated components <ul><li>If the sample is separated into colored components, then </li></ul><ul><li>the lo...
Documentation <ul><li>Storage of chromatogram for TLC is difficult. It is </li></ul><ul><li>usually undesirable since plat...
Applications <ul><li>It is used for separation and identification of; </li></ul><ul><li>Amino acids </li></ul><ul><li>Pept...
PARTITION  CHROMATOGRAPHY
DEFINITION <ul><li>“ This form of chromatography is based on a thin film formed on the surface of a solid support by a liq...
PRINCIPLE <ul><ul><li>Separation of components of a sample mixture occurs </li></ul></ul><ul><ul><li>because of partition....
PRINCIPLE <ul><li>The stationary phase immobilizes the liquid  surface layer, which becomes stationary phase. Mobile phase...
TYPES Partition   chromatography Liquid-liquid chromatography Gas-liquid chromatography
PAPER CHROMATOGRAPHY <ul><li>“ .” </li></ul>
Instrumentation <ul><li>Chromatography jar </li></ul><ul><li>Capillary tube </li></ul><ul><li>Stationary phase (liquid imp...
Instrumentation <ul><li>Chromatography jar: </li></ul><ul><li>It is made of glass and has a lid on it. Jar maintains prope...
Instrumentation <ul><li>Mobile phase: </li></ul><ul><li>Mobile phase may be a single liquid or a mixture of </li></ul><ul>...
Procedure <ul><li>Clean and dried chromatography jar is taken. </li></ul><ul><li>A paper impregnated in the mobile phase i...
Procedure <ul><li>Sample is applied on paper with help of capillary tube. </li></ul><ul><li>Sample spot is air dried. </li...
Location of separated components <ul><li>If the sample is separated into colored components, then </li></ul><ul><li>the lo...
Documentation <ul><li>Storage of chromatogram. </li></ul><ul><li>Calculating Rf values </li></ul>
Applications <ul><li>It is used for separation and identification of; </li></ul><ul><li>Amino acids </li></ul><ul><li>Carb...
ION EXCHANGE  CHROMATOGRAPHY
DEFINITION <ul><li>In this type of chromatography, a resin (the stationary solid phase) is used to covalently attach anion...
MECHANISM <ul><li>Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anio...
MECHANISM
SIZE EXCLUSION  CHROMATOGRAPHY
DEFINITION <ul><li>It is also known as gel permeation or gel filtration chromatography. </li></ul><ul><li>This type of chr...
<ul><li>It is generally a low-resolution chromatography technique and thus it is often reserved for the final, &quot;polis...
MECHANISM
 
AFFINITY  CHROMATOGRAPHY
DEFINITION <ul><li>This is the most selective type of chromatography. </li></ul><ul><li>It utilizes the specific interacti...
 
Upcoming SlideShare
Loading in …5
×

Partition chromatography 3

29,385 views

Published on

Published in: Education
5 Comments
44 Likes
Statistics
Notes
No Downloads
Views
Total views
29,385
On SlideShare
0
From Embeds
0
Number of Embeds
54
Actions
Shares
0
Downloads
1,389
Comments
5
Likes
44
Embeds 0
No embeds

No notes for slide

Partition chromatography 3

  1. 1. ADSORPTION CHROMATOGRAPHY Muhammad Tanveer Khan
  2. 2. DEFINITION <ul><li>“ It is a type of chromatography in which a mobile liquid or gaseous phase is adsorbed onto the surface of a stationary solid phase. The equilibration between the mobile and stationary phase accounts for the separation of different solutes.” </li></ul>
  3. 3. PRINCIPLE <ul><li>Principle involves competition of components of sample mixture for active site on adsorbent. These active sites are formed in molecule due to </li></ul><ul><li>* Cracks </li></ul><ul><li>* Edges </li></ul><ul><li>The electrostatic forces present in the molecule, which hold together the crystal lattice, are directed outward. These forces and electrostatic forces of solute molecule cause separation. </li></ul>
  4. 4. PRINCIPLE <ul><li>Separation occurs because of the fact that an equilibrium is established between molecules adsorbed on stationary phase and those which are flowing freely in mobile phase. </li></ul><ul><li>The more the affinity of the molecule of particular component, less will be its movement. </li></ul>
  5. 5. TYPES Adsorption chromatography Column chromatography Thin layer chromatography Gas solid chromatography
  6. 6. ADSORBENTS <ul><li>“ An adsorbent is a substance, usually porous in nature and with a high surface area that can adsorb substances onto its surface by intermolecular forces.” </li></ul>
  7. 7. AN IDEAL ADSORBENT <ul><li>The Ideal adsorbent must fulfill the following requirements: </li></ul><ul><li>Insoluble in mobile phase </li></ul><ul><li>Inert to solutes (adsorptive) </li></ul><ul><li>Colorless especially when work with colored mixtures </li></ul><ul><li>Suitable particle size enough to give good separation and reasonable flow rate </li></ul>
  8. 8. COMMON ADSORBENTS <ul><li>Hydrated silica gel </li></ul><ul><li>Silica gel G </li></ul><ul><li>Silica gel S </li></ul><ul><li>Silica gel GF 254 </li></ul><ul><li>Silica gel H </li></ul><ul><li>Silica gel N </li></ul><ul><li>Silica gel HF 254 </li></ul><ul><li>Silica gel PF 254 </li></ul>Instrumentation
  9. 9. COMMON ADSORBENTS <ul><li>Modified silica gel </li></ul><ul><li>Alumina </li></ul><ul><li>Kieselghur (Diatomaceous earth) </li></ul><ul><li>Cellulose MN 300 </li></ul><ul><li>Cellulose microcrystalline </li></ul>
  10. 10. THIN-LAYER CHROMATOGRAPHY <ul><li>“ The technique which involves flowing of mobile phase over a thin layer of adsorbent, applied on solid support, where separation of components occur by differential migration which occurs when solvent flows along fine powder spread on glass plates, is called thin –layer chromatography.” </li></ul>
  11. 11. Instrumentation <ul><li>Chromatography jar </li></ul><ul><li>Capillary tube </li></ul><ul><li>Thin layer chromatography plate </li></ul><ul><li>Stationary phase </li></ul><ul><li>Mobile phase </li></ul>
  12. 12. Instrumentation <ul><li>Chromatography jar: </li></ul><ul><li>It is made of glass and has a lid on it. Jar maintains proper environment that is required for separation. </li></ul><ul><li>Capillary tube: </li></ul><ul><li>It is used to apply sample mixture on TLC plate. </li></ul><ul><li>Stationary phase: </li></ul><ul><li>Adsorbents </li></ul>
  13. 13. Instrumentation <ul><li>TLC plate: </li></ul><ul><li>Borosilicate glass plates are preferred. Most commonly used </li></ul><ul><li>sizes are; </li></ul><ul><li>20 X 20cm </li></ul><ul><li>20 X 10cm </li></ul><ul><li>20 X 5cm </li></ul><ul><li>Microscopic slides are also used. </li></ul>
  14. 14. Instrumentation <ul><li>Mobile phase: </li></ul><ul><li>Mobile phase may be a single liquid or a mixture of liquids. </li></ul><ul><li>Commonly used mobile phases are; </li></ul><ul><li>Methanol </li></ul><ul><li>Ethanol </li></ul><ul><li>Ethyl acetate </li></ul><ul><li>Diethyl ether </li></ul><ul><li>Acetone </li></ul><ul><li>Chloroform </li></ul>
  15. 15. Procedure <ul><li>Clean and dried chromatography jar is taken. </li></ul><ul><li>A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the jar is saturated with solvent vapors. </li></ul><ul><li>Mobile phase is added to the jar at a length of 0.5-1cm from the bottom. </li></ul><ul><li>Jar is closed. </li></ul><ul><li>Equilibrium is allowed to be maintained. </li></ul><ul><li>Base line is marked on adsorbent. </li></ul>
  16. 16. Procedure <ul><li>Sample is applied on TLC plate with help of capillary tube. </li></ul><ul><li>Sample spot is air dried. </li></ul><ul><li>TLC plate is put in the chromatography jar and lid is closed. </li></ul><ul><li>The system is allowed to be static until the solvent move to a proper distance from baseline. </li></ul><ul><li>TLC plate is taken out and dried. </li></ul>
  17. 17. Location of separated components <ul><li>If the sample is separated into colored components, then </li></ul><ul><li>the location is dried in ordinary light. But in case of </li></ul><ul><li>colorless components following are used; </li></ul><ul><li>Uv lamp </li></ul><ul><li>Iodine crystals </li></ul><ul><li>Spraying agents </li></ul>
  18. 18. Documentation <ul><li>Storage of chromatogram for TLC is difficult. It is </li></ul><ul><li>usually undesirable since plates are employed for </li></ul><ul><li>repeated use. Various methods for separation include; </li></ul><ul><li>Rf value in TLC </li></ul><ul><li>Preservation of chromatogram by peeling off adsorbent. </li></ul><ul><li>Graphical copying i.e. tracing on transparent paper. </li></ul><ul><li>Photography </li></ul>
  19. 19. Applications <ul><li>It is used for separation and identification of; </li></ul><ul><li>Amino acids </li></ul><ul><li>Peptides and proteins </li></ul><ul><li>Alkaloids </li></ul><ul><li>Carbohydrates </li></ul><ul><li>Fats and fatty acids </li></ul><ul><li>Antibiotics </li></ul><ul><li>Narcotic analgesics </li></ul><ul><li>Glycosides </li></ul>
  20. 20. PARTITION CHROMATOGRAPHY
  21. 21. DEFINITION <ul><li>“ This form of chromatography is based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.” </li></ul>
  22. 22. PRINCIPLE <ul><ul><li>Separation of components of a sample mixture occurs </li></ul></ul><ul><ul><li>because of partition. Stationary phase is coated with </li></ul></ul><ul><ul><li>a liquid which is immiscible in mobile phase. </li></ul></ul><ul><ul><li>Partition of component of sample between sample </li></ul></ul><ul><ul><li>and liquid/ gas stationary phase retard some </li></ul></ul><ul><ul><li>components of sample more as compared to others. </li></ul></ul><ul><ul><li>This gives basis for separation. </li></ul></ul>
  23. 23. PRINCIPLE <ul><li>The stationary phase immobilizes the liquid surface layer, which becomes stationary phase. Mobile phase passes over the coated adsorbent and depending upon relative solubility in the coated liquid, separation occurs. The component of sample mixture appear separated because of differences in their partition coefficient. </li></ul>
  24. 24. TYPES Partition chromatography Liquid-liquid chromatography Gas-liquid chromatography
  25. 25. PAPER CHROMATOGRAPHY <ul><li>“ .” </li></ul>
  26. 26. Instrumentation <ul><li>Chromatography jar </li></ul><ul><li>Capillary tube </li></ul><ul><li>Stationary phase (liquid impregnated paper) </li></ul><ul><li>Mobile phase </li></ul>
  27. 27. Instrumentation <ul><li>Chromatography jar: </li></ul><ul><li>It is made of glass and has a lid on it. Jar maintains proper environment that is required for separation. </li></ul><ul><li>Capillary tube: </li></ul><ul><li>It is used to apply sample mixture. </li></ul><ul><li>Stationary phase: </li></ul><ul><li>liquid impregnated paper </li></ul>
  28. 28. Instrumentation <ul><li>Mobile phase: </li></ul><ul><li>Mobile phase may be a single liquid or a mixture of </li></ul><ul><li>liquids. </li></ul><ul><li>Commonly used mobile phases are; </li></ul><ul><li>Methanol </li></ul><ul><li>Ethanol </li></ul><ul><li>Ethyl acetate </li></ul><ul><li>Diethyl ether </li></ul><ul><li>Acetone </li></ul><ul><li>Chloroform </li></ul>
  29. 29. Procedure <ul><li>Clean and dried chromatography jar is taken. </li></ul><ul><li>A paper impregnated in the mobile phase is applied to the walls to ensure that atmosphere of the jar is saturated with solvent vapors. </li></ul><ul><li>Mobile phase is added to the jar at a length of 0.5-1cm from the bottom. </li></ul><ul><li>Jar is closed. </li></ul><ul><li>Equilibrium is allowed to be maintained. </li></ul><ul><li>Base line is marked on adsorbent. </li></ul>
  30. 30. Procedure <ul><li>Sample is applied on paper with help of capillary tube. </li></ul><ul><li>Sample spot is air dried. </li></ul><ul><li>Paper is put in the chromatography jar and lid is closed. </li></ul><ul><li>The system is allowed to be static until the solvent move to a proper distance from baseline. </li></ul><ul><li>Paper is taken out and dried. </li></ul>
  31. 31. Location of separated components <ul><li>If the sample is separated into colored components, then </li></ul><ul><li>the location is dried in ordinary light. But in case of </li></ul><ul><li>colorless components following are used; </li></ul><ul><li>Uv lamp </li></ul><ul><li>Iodine crystals </li></ul><ul><li>Spraying agents </li></ul>
  32. 32. Documentation <ul><li>Storage of chromatogram. </li></ul><ul><li>Calculating Rf values </li></ul>
  33. 33. Applications <ul><li>It is used for separation and identification of; </li></ul><ul><li>Amino acids </li></ul><ul><li>Carbohydrates </li></ul><ul><li>Tannins </li></ul><ul><li>Glycosides </li></ul><ul><li>Alkaloids etc. </li></ul>
  34. 34. ION EXCHANGE CHROMATOGRAPHY
  35. 35. DEFINITION <ul><li>In this type of chromatography, a resin (the stationary solid phase) is used to covalently attach anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are attracted to the resin by electrostatic forces. </li></ul><ul><li>Ion exchange mechanism separates analytes based on their respective charges. </li></ul><ul><li>Ion exchange chromatography is performed in columns but can also be useful in planar mode. </li></ul>
  36. 36. MECHANISM <ul><li>Ion exchange chromatography uses a charged stationary phase to separate charged compounds including anions, cations, amino acids, peptides, and proteins. In conventional methods the stationary phase is an ion exchange resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. </li></ul><ul><li>Ion exchange chromatography is commonly used to purify proteins. </li></ul>
  37. 37. MECHANISM
  38. 38. SIZE EXCLUSION CHROMATOGRAPHY
  39. 39. DEFINITION <ul><li>It is also known as gel permeation or gel filtration chromatography. </li></ul><ul><li>This type of chromatography lacks an attractive interaction between the stationary phase and solute. The liquid or gaseous phase passes through a porous gel which separates the molecules according to its size. The pores are normally small and exclude the larger solute molecules, but allows smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones. </li></ul>
  40. 40. <ul><li>It is generally a low-resolution chromatography technique and thus it is often reserved for the final, &quot;polishing&quot; step of a purification. </li></ul><ul><li>It is also useful for determining the tertiary structure and quaternary structure of purified proteins. </li></ul>
  41. 41. MECHANISM
  42. 43. AFFINITY CHROMATOGRAPHY
  43. 44. DEFINITION <ul><li>This is the most selective type of chromatography. </li></ul><ul><li>It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. </li></ul><ul><li>For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH. </li></ul>

×