Column Chromatography.pptx

Column Chromatography

1. COLUMN CHROMATOGRAPHY

2. Defination Column chromatography   that in which the various solutes of a solution are allowed to travel down an absorptive column. The individual components being adsorbed by the stationary phase and then are eluted according to their affinities from the column by the mobile phase.

3. Types Column chromatography GRAVITY COLUMN FLASH COLUMN If the solvent is allowed to flow If the solvent is forced down the column by gravity down the column by positive air pressure above

4. Principle Principle : When a mixture of mobile phase and sample to be separated are introduced from top of the column, the individual components of mixture move with different rates. Those with lower affinity to stationary phase move faster and eluted out first while those with greater adsorption affinity move or travel slower and get eluted out last.

5. . Diiferent sizes of column used

6. Adsorbents Commonly used adsorbents are SILICA GEL AND ALUMINA.

7. Silica Gel "silica gel 60" or "silica gel 230-400" are a couple of examples. This number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing process Adsorbent particle size affects how the solvent flows through the column. Smaller particles (higher mesh values) are used for flash chromatography, larger particles (lower mesh values) are used for gravity chromatography. For example, 70-230 silica gel is used for gravity columns and 230-400 mesh for flash columns.

8. Alumina Alumina is quite sensitive to the amount of water which is bound to it: the higher its water content, the less sites it has to bind organic compounds, and thus the less "sticky" it is. This stickiness or activity is designated as I, II, or III, with I being the most active. Alumina is usually purchased as activity I and deactivated with water before use according to specific procedures. Alumina comes in three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most commonly employed.

9. Solvents

10. PREPARATION OF COLUMN Bottom portion of the column – packed with glass wool/cotton wool or may contain asbestos pad. Above which adsorbent is packed After packing a paper disc kept on the top , so that the adsorbent layer do not get disturbed on introduction of sample or mobile phase. PACKING CAN BE: Dry packing Wet packing

11. Dry packing Adsorbent is packed in the column in dry form Fill the solvent, till equilibrium is reached DEMERIT: Air bubbles are entrapped b/w M.P & S.P→ cracks appear in the adsorbent layer. After filling tapping can be done to remove void spaces.

12. Wet packing Ideal & common technique The material is slurried with solvent and generally added to the column in portions. Stationary Phase settles uniformly & no crack in the column of adsorbent. Solid settle down while the solvent remain upward.

13. Loading of sample Wet loading Dissolve the sample in the minimum amount of solvent (5–10 drops). Using a pipette or syringe with a thick needle, drip the sample directly onto the top of the silica. Carefully add a layer of sand (approx. 2–5 mm). This will help prevent the surface of the silica from being disturbed as more solvent is added. Use a pipette to carefully add more solvent so that the solvent level is about 10 mm above the top of the sand. Allow this solvent to drain until the solvent level is approximately 1–2 mm above the top of the sand. Repeat steps 5 and 6 once or twice more. This will ensure that your sample is absorbed onto the silica.

14. Wet loading .

15. Dry loading Dissolve the sample in an appropriate solvent. Transfer it to a round-bottomed flask Add dry silica to the dissolved sample (approx. 10–20 times the mass of the sample). Swirl or stir gently to ensure all of the silica is suspended within the solution. Gently evaporate the solvent by using a rotary evaporator until the silica is dry and free-flowing. If it is still an oil, add more silica and repeat the procedure. Carefully add solvent to your column so that the solvent level is about 2–3 cm above the top of the silica. Use a pipette to ensure the surface of the silica is not disturbed. Pour the dry silica that is saturated with your sample into the column and allow it to settle.

16. For the  dry  method, first dissolve the sample to be analyzed in the minimum amount of solvent and add about 100 mg of silica gel. Swirl the mixture until the solvent evaporates and only a dry powder remains.

17. Place the dry powder on a folded piece of weighing paper. Transfer it to the top of the prepared column

18. Elution techniques Isocratic elution technque ( Iso means – same) Same solvent composition or solvent of same polarity Used throughout the process of separation.

19. Gradient elution ( gradient – gradually) Solvents of gradually ↑ polarity or ↑ elution strength are used during the process of seperation . E.g. initially benzene, then chloroform, then ethyl acetate then chloroform

20. FACTORS AFFECTING SEPARATION FACTORS DUE TO STATIONARY PHASE - 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. In HPLC we use very fine particles to get very good separation. The flow rate problem is solved by the use high pressure pumps to push the mobile phase through the stationary phase.

21. . Uniformity of packing of the column If the stationary phase is not packed uniformly then the bands will be irregular and less uniform resulting in poor separation Concentration of the mixture The proper ratio between sample to be separated and the amount of stationary phase is very important ,too much samples resulted in bad separation

22. FACTORS DUE TO MOBILE PHASE Selection of the proper mobile phase 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. Rate of flow Slower flow rate usually resulted in a better separation and narrower bands. Consistency of flow The continuous flow of the mobile phase during the whole experiment gives better separation than interrupting the flow then continue it later.

23. FACTORS DUE TO COLUMN 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. 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.

24. Detection of components DETECTION OF COMPONENTS Colored components-Visually Colorless components- Different properties which can be used are – UV / visible detector Flourescence detector RI detector Flame ionization detector …

25. CHIRAL SEPARATIONS Chiral stationary phases can be used to separate enantiomers . By giving the stationary phase a “handedness,” one enantiomer will be specifically retained on the column. These columns are very expensive and specific to the particular type of separation, but have led to great achievements in separation science.

26. Applications Separation of different mixture of compounds Removal of impurities Isolation of active constituents Isolation of metabolite from biological fluids Estimation of drugs in formulations

27. Advantages Any type of mixture can be separated Any quantity of the mixture can be separated Wider choice of mobile phase Automation is possible It can be used in both analytical and preparative applications.

28. Disadvantages Time consuming method More amount of solvents are required which are expensive Automation technique makes complication

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