Column chromatography

6,652 views
6,396 views

Published on

Chromatographic Method

Published in: Education
2 Comments
7 Likes
Statistics
Notes
No Downloads
Views
Total views
6,652
On SlideShare
0
From Embeds
0
Number of Embeds
9
Actions
Shares
0
Downloads
437
Comments
2
Likes
7
Embeds 0
No embeds

No notes for slide

Column chromatography

  1. 1. <ul><li>COLUMN CHROMATOGRAPHY </li></ul><ul><li>by Mr. Shaise Jacob Faculty, Nirmala College of Pharmacy Muvattupuzha Kerala, India </li></ul>
  2. 2. COLUMN CHROMATOGRAPHY Column of stationary phase is used » Solid – S.P » Liquid – M.P PRINCIPLE ◊ Adsorption Mixture of components dissolved in the M.P is introduced in to the column. Components moves depending upon their relative affinities.
  3. 3. PRACTICAL REQUIREMENTS <ul><li>1.STATIONARY PHASE </li></ul><ul><li>Adsorbent in C.C should meet following criteria </li></ul><ul><li>◘ Spherical in shape </li></ul><ul><li>◘ Mechanical stability must be high </li></ul><ul><li>◘ They shouldn’t react chemically </li></ul><ul><li>◘ It should be useful for separating for </li></ul><ul><li>wide variety of compounds </li></ul><ul><li>◘ It should be freely available & inexpensive </li></ul>
  4. 5. SELECTION OF S.P <ul><li>Success of chromatography -> proper selection of S.P, it depends on the following. </li></ul><ul><li>Removal of impurities </li></ul><ul><li>No. of components to be separated </li></ul><ul><li>Length of the column used </li></ul><ul><li>Affinity differences b/w components </li></ul><ul><li>Quality of adsorbent used </li></ul>
  5. 6. ◊ MOBILE PHASE <ul><li>They act as </li></ul><ul><li>▫ Solvent </li></ul><ul><li>▫ Developer </li></ul><ul><li>▫ Eluent </li></ul><ul><li>◊ COLUMN CHARACTERISTICS </li></ul><ul><li>Column - Neutral glass </li></ul><ul><li>Column dimensions - length & diameter ratio (10:1,30:1 or 100:1) </li></ul>
  6. 7. PREPARATION OF THE COLUMN <ul><li>» Bottom portion of the column – packed with glass wool/cotton wool or may contain asbestos pad, </li></ul><ul><li>» Above which adsorbent is packed </li></ul><ul><li>» After packing a paper disc kept on the top </li></ul><ul><li>Two types of packing techniques are there. </li></ul><ul><li>1.Dry packing </li></ul><ul><li>2.Wet packing </li></ul>
  7. 8. DRY PACKING
  8. 10. Dry Packing Technique <ul><li>Adsorbent is packed in the column in dry form </li></ul><ul><li>Fill the solvent, till equilibrium is reached </li></ul><ul><li>DEMERIT: Air bubbles are entrapped b/w M.P & S.P-> cracks appear in the adsorbent layer. </li></ul><ul><li>Wet Packing Technique </li></ul><ul><li>» ideal & common technique </li></ul><ul><li>Adsorbent + M.P in a beaker & poured in to column </li></ul><ul><li>◊ S.P settles uniformly & no crack in the column of adsorbent </li></ul>
  9. 13. U
  10. 15. INTRODUCTION OF THE SAMPLE <ul><li>Samples dissolved in M.P & introduced into the column at once -> eluted </li></ul><ul><li>ELUTION PROCEDURES </li></ul><ul><li>Two techniques </li></ul><ul><li>1)Isocratic elution techniques </li></ul><ul><li>2)Gradient elution techniques </li></ul><ul><li>Isocratic elution techniques </li></ul><ul><li>(Iso means – same) </li></ul><ul><li>Same solvent composition or solvent of same polarity used throughout the process of separation </li></ul>
  11. 16. Gradient elution techniques <ul><li>( gradient – gradually) </li></ul><ul><li>Solvents of gradually ↑ polarity or ↑ elution strength are used during the process of seperation. </li></ul><ul><li>E.g. initially benzene, then chloroform, then ethyl acetate then chloroform </li></ul><ul><li>DETECTION OF COMPONENTS </li></ul><ul><li>Colored components- Visually </li></ul><ul><li>Colorless components- Different properties which can be used are – uv / visible detector, flourescence detector, RI detector, Flame ionization detector… </li></ul>
  12. 17. RECOVERY OF COMPONENTS <ul><li>◘ ELUTION </li></ul><ul><li>FACTORS AFFECTING COLUMN EFFICIENCY </li></ul><ul><li>1) Nature of solvents </li></ul><ul><li>2) Dimension of columns </li></ul><ul><li>3) Particle size of column packing </li></ul><ul><li>4)Temperature of the column </li></ul><ul><li>APPLICATIONS </li></ul><ul><li>► Seperation of mixture of components </li></ul><ul><li>► Purification process </li></ul><ul><li>► Isolation of metabolites </li></ul><ul><li>► Estimation of drugs in formulations </li></ul>
  13. 18. Advantages of C.C <ul><li>» Any type of mix. can be separated </li></ul><ul><li>» Any quantity of mix. can be separated </li></ul><ul><li>» Wider choice of M.P </li></ul><ul><li>» Automation is possible </li></ul><ul><li>Disadvantages of C.C </li></ul><ul><li>» Time consuming </li></ul><ul><li>» ↑ amounts of M.P required </li></ul><ul><li>» Automation makes the techniques more complicated & expensive </li></ul>
  14. 19. <ul><li>THANK YOU </li></ul>

×