Column chromatography is a method used to separate mixtures based on differences in how components partition between a stationary and mobile phase. Components travel down an absorptive column at different rates based on their affinity for the stationary phase, with those having less affinity eluting first. Key factors that affect separation include the stationary phase properties like particle size, the mobile phase used and flow rate, and column dimensions and temperature. Common stationary phases are silica gel and alumina, while mobile phases vary in polarity. Separated components can be detected visually or using instruments like UV/Vis detectors. Column chromatography has applications in purification, isolation, and analysis across various fields.

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

29. THANK YOU