Column chromatography


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Column chromatography

  1. 1.  The separation of a mixture by distribution of its components between a mobile and stationary phase over time  It is a solid - liquid technique in which the stationary phase is a solid & mobile phase is a liquid.  The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for column chromatography is Silica Gel, followed by Alumina Oxide.  The mobile phase or eluent is a liquid. It is either a pure solvent or a mixture of different solvents.  It can be used for molecules whose molecular weight is < 2000 g/mol
  2. 2. Detector Signal Chromatogram - Detector signal vs. retention time or volume 1 2 time or volume
  3. 3.  when a mixture of components dissolved in the mobile phase is introduced into the column , the individual components move with differentiates 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.
  4. 4.   During the separation process, the solute takes part in mass transfer between the two phases {Stationary phase and mobile phase} Components separate depends on the differences in their mass transfer capacities Factors influencing mass transfer differences Hydrogen bonding  Ion exchange  Solubility  Various types of polar fores and interactions 
  5. 5. Gravity Columns: The mobile phase move through the stationary phase by gravity force. Flash Columns (Air or nitrogen pressure): The mobile phase is pushed by stream of air or nitrogen using special using (Adaptors special values
  6. 6. Low and Medium Pressure Columns (pumped): The movement of mobile phase is accelerated by using pumps that generate low or medium pressure. The increase in the flow rate shorten the time of separation. Vacuum Columns [Vacuum liquid chromatography (VLC)]: The adsorbent is applied dry into a sintered glass funnel. The sample is applied by dry method or as solution. Then the mobile phase is added portion by portion and vacuum is applied after each portion to collect each fraction.
  7. 7. IDEAL PROPERTIES  Particles should have uniform size and spherical     shape [60-200] microns. Should have high mechanical stability, inert, insoluble in mobile phase used. Colorless Should allow free flow of mobile phase. Freely available and in expensive
  8. 8. Adsorbents require activation before use.  This can be achieved by heating, where adsorbent losses water and other adsorbed materials  Eg.,  Alumina is activated in an oven at 200 0C or at 4000C  Silica Gel is activated by heating at 160 0C
  9. 9. Dry Packing Method :  Add dry silica / Alumina to the column and apply to the bottom of the column. This will compress the silica gel and keep it compressed for the next steps. Packing can be improved by tapping the column.  While applying vacuum; pour solvent in it.  Allow the solvent to move though the column until reaches to the bottom. At this stage vacuum is not require.  Allow 5–6 columns value of solvent to flow through the column to make sure it is complete packed.  Drain the solvent till the solvent level is just even with the surface of the stationary phase
  10. 10.  DEMERITS OF DRY PACKING:  Air bubbles are entrapped between mobile phase and stationary phase and the column may not be uniformly packed.  Cracks appear in the adsorbent present in the column. Hence flow characteristics and clear band of the separated component may not be obtained.
  11. 11. Wet Packing Method:          Fill the column about one third with solvent In a beaker, measure out the required amount of silica / alumina. In another beaker, take solvent approximately one and a half times the amount of silica / alumina. Add silica/alumina to the solvent while swirling in small quantity at a time. Use a glass rod to mix the slurry. Pour some of the slurry into column & allow solvent to drain to avoid overflowing. Tap the column carefully to encourage bubbles to rise and the silica to settle Continue to move the slurry to the column until all the silica or alumina is added. Wash the inside of the column by pouring solvent down the inside edge. Drain the solvent till the solvent level is just even with the surface of the stationary phase
  12. 12. Silica Gel Alumina Oxide Chemical For mula SiO2 (Silicon Dioxide). Chemical Formula Al2O3 (Aluminium Trioxide ). it is acidic in nature which we are making it to neutral It can be Acidic, Basic & Neutral Silica Gel has Higher Surface area as compare to Alumina i.e. 350-550 m2/gm Alumina’s Surface Area is less than Silica Gel i.e. 140-160 m2/gm Sizes available: 35-70, 60-120, 70230, 100-200, 230-400 mesh Size Available: 100-300 mesh & 200400 mesh Bulk Density: 040-0.65 gm/ml Bulk Density: 0.90-1.2 gm/ml Pore Dia: 20, 60, 100, 300, 1000A Pore Dia: 50-60A
  13. 13. Normal Phase Chromatography Reverse Phase Chromatography It uses a polar stationary phase and a It uses a non polar stationary phase and non-polar (low Polarity Solvents) a polar mobile phase. mobile phase. Non-polar compounds elute faster than polar compounds. Polar compounds elute faster than non polar compounds. When we increase polarity of mobile phase elution time will increase. When we increase polarity of mobile phase elution time will decrease. It can not be reused / reproducible It Can reused / reproducible Mobile phase are non polar i.e. IPA, hexane, dichloromethane, chloroform, ethyl ether, and isopropyl alcohol (IPA). Mobile phase are polar compounds such as water, acetonitrile, methanol
  14. 14.  To introduce the mixture into column as solvent.  To develop the zones for separation as developing agent.  To remove pure component out of the column as eluent.
  15. 15. Three Considerations must be made in choosing Mobile phase  Solvent should satisfy the practical factors such as suitable viscosity, stability, Solubility with respect to the sample, purity.  Solvent should provide maximum resolution for the separation of sample.  Polarity
  16. 16.  Transfer of a mixture to adsorbent column – Non polar solvents e.g.., ether, petroleum etc.  Development of chromatogram – Intermediate polar solvents e.g.., acetone, benzene.  Elution of the solute – strongly polar solvents eg., alcohols
  17. 17. Chromatography Mobile Phases Increasing Polarities                      Helium Nitrogen Petroleum ether (pentanes) Ligroin (hexanes) Cyclohexane Carbon tetrachloride* Toluene Chloroform* Dichloromethane (methylene chloride) t-Butyl methyl ether Diethyl ether Ethyl acetate Acetone 2-Propanol Pyridine Ethanol Methanol Water Acetic acid *Suspected carcinogens.
  18. 18.  Column material is made of good quality neutral glass since it should not be affected by solvents, acids or alkalies.  Column dimensions are important for effective separations.  Length: diameter – 10:1 to 30:1  For more efficient separation Length :diameter -100:1
  19. 19.  The sample which is usually a mixture of components is dissolved in minimum quantity of the mobile phase.  The entire sample is introduced into the column at once and get adsorbed on the top portion of the column.  From this zone, individual sample can be separated by a process of elution.
  20. 20. After the introduction of the sample, by the elution techniques the individual components are separated out from the column. Two types of elution techniques  Isocratic elution technique  Gradient/Stepwise elution technique
  21. 21. Isocratic elution technique   Iso means same/ similar. In this elution technique, the same solvent or solvent system of same polarity is used throughout the process of separation. Ex- chloroform only, petroleum: ether=1:1
  22. 22. Gradient/Stepwise elution technique   The solvents of gradually increasing polarity or increasing elution strength are used during the process of separation. Initially low polar solvent is used followed by gradually increasing the polarity. Ex-Initially Benzene then Chloroform, Ethyl acetate, Methanol etc.
  23. 23.      It was assumed that the chromatographic system composed of number of “distribution systems” or “equilibrations” called “Theoretical Plates”. Each theoretical plate is composed of stationary phase and mobile phase. The height of each plate is called “Height equivalent to Theoretical Plate” (HETP). The number of theoretical plates “N” is important for separation. Increasing “N” resulted in narrower bands and better separation.
  24. 24. Factors due to Stationary Phase: 1. Particle size of the stationary phase. Reducing the particle size increases the surface area and improve separation. However, reduction of the particle size will decrease the flow rate of the mobile phase. 2. Adsorbent activity: The choice of the suitable adsorbent is very important. 3. Uniformity if packing of the column: 4. Concentration of the mixture: If the stationary phase is not packed uniformly then the bands will be irregular and less uniform resulting in poor separation. the proper ratio between sample to be separated and the amount of stationary phase is very important too much samples resulted in bad separation.
  25. 25.  Factors due to Mobile Phase: 1. Selection of the proper mobile phase: 2. Rate of flow: 3. Consistency of flow: Very polar mobile phase will wash out all components without any separation. On the other hand very non polar mobile phase will result in broad band and poor separation. Slower flow rate usually resulted in a better separation and narrower bands. The continuous flow of the mobile phase during the whole experiment gives better separation than interrupting the flow then continue it later
  26. 26.  Factors due to Columns: 1. Column dimensions: Increasing the length of the column improve separation. However, that usually leads to slower flow rate. Also increasing the column length some times is impractical. 2. Column temperature: Increasing the temperature usually reduces the adsorption power of the stationary phase and increase elution speed. This may leads to decrease in the efficiency separation.
  27. 27. ►Separation of mixture of compounds ►Purification process ►Isolation of active constituents ►Estimation of drugs in formulation ►Isolation of active constituents ►Determination of primary and secondary glycosides in digitalis leaf. ► separation of diastereomers