1. LU 3: Separation Technique (P2)

2. Column Chromatography
The stationary phase, a solid adsorbent, is placed in a vertical glass (usually) column.
The mobile phase, a liquid, is added to the top and flows down through the column (by either gravity or external pressure).
Column chromatography is generally used as a purification technique: it isolates desired compounds from a mixture.

3. Packed column

4. Column chromatography

5. Column Chromatography
The mixture to be analyzed by column chromatography is applied to the top of the column.
The stationary phase is held in a narrow tube and the mobile phase is forced through the tube under pressure or gravity.
An equilibrium is established between the solute adsorbed on the adsorbent and the eluting solvent flowing down through the column.

6. Column Chromatography
Because the different components in the mixture have different interactions with the stationary and mobile phases, they will be carried along with the mobile phase to varying degrees and a separation will be achieved.
The individual components, or eluents, are collected as the solvent drips from the bottom of the column.

7. The Adsorbent
Silica gel (SiO2) and alumina (Al2O3) – commonly used as adsorbents.
Sold in different mesh sizes.
E.g “Silica gel 60” or “silica gel ” - This number refers to the mesh of the sieve used to size the silica.
Adsorbent particle size affects how the solvent flows through the column. .

8. The solvent
The polarity of the solvent which is passed through the column affects the relative rates at which compounds move through the column.
Often a series of increasingly polar solvent systems are used to elute a column.
A non-polar solvent is first used to elute a less-polar compound.
Once the less-polar compound is off the column, a more-polar solvent is added to the column to elute the more-polar compound.

9. Column Chromatography
The stationary phase (column packing) in the column is very polar.
Polar compounds are going to be attracted to the polar column packing by hydrogen bonding or dipole-dipole attractions. Polar compounds are going to move slowly.
Non-polar compounds are going to come off the column first, while the polar compounds are going to come off the column last.
Usually, one starts with a less polar solvent to remove the less polar compounds, and then slowly increase the polarity of the solvent to remove the more polar compounds.

10. The expected elution order of organic classes

11. Eluting power of organic solvent

12. Column chromatography
The stationary phase is POLAR.
The more polar component interacts more strongly with the stationary phase.
The more polar component moves more slowly.
The non-polar component moves more rapidly.

13. Reverse Phase Column Chromatography
The stationary phase (column packing) is now NON-POLAR
Non-polar compounds will move more slowly because they are attracted to the column packing.
The more polar component moves more quickly down the column.
Polar solvents, such as water and methanol are used in reverse phase chromatography
Used mainly in columns, such as HPLC

14. Separation Based on a Change of State
Analyte & interferent are in the same phase. Separation is possible by inducing a change in one of their physical or chemical states.
Change in Physical State - liquid-to-gas & solid-to- gas phase transitions.
(a) Distillation
analyte & interferent are miscible liquids with significantly different boiling points.
Simple distillation – for separating liquids with large difference in boiling points.
Fractional distillation – more efficient.

15. Simple distillation

16. Separation Based on a Partitioning Between Phases
Separation techniques based on the selective partitioning of the analyte/interferent between 2 immiscible phases.
For a solute, S, in contact with a second phase, the solute partitions itself between the 2 phases: Sph1 and Sph2
At equilibrium, the distribution coefficient,
KD =
If KD is large  solute moves from phase 1 to phase 2.
If KD is small  solute remains in phase 1.
Separation is possible when KD favor for only one of the solutes.

17. Separation Based on a Partitioning Between Phases
Extractions include:
Liquid-liquid extractions
Liquid-solid extractions
Solid-liquid extractions
Gas-solid extractions

18. LIQUID SOLID EXTRACTION
The liquid sample is passed through a cartridge containing solid adsorbent.
The choice of adsorbent depends on the properties of the species being detained and the matrix in which it is found.
Eg. Sedatives can be isolated from serum using a C-18 solid adsorbent. The retained sedatives are eluted from the cartridge by a solid-liquid extraction using acetone.
Advantages: ease of use, faster extraction time, smaller volumes of solvent, and ability to concentrate the analyte.
Direction of
sample flow
Solid adsorbent

19. Solid-liquid extractions
Soxhlet Extractor
Continuous extraction by passing extracting phase through the sample until a quantitative extraction is achieved.
The extracting solvent in the lower reservoir is heated to its boiling point.
The solvent in the vapour state moves upward to the condenser where it condenses back to liquid and collects in the upper reservoir with the sample held in a porous cellulose filter thimble.

20. Soxhlet Extractor Advantages: Use only small amount of solvent;
Can remove a high percent of a solute;
Can works unattended for long periods.

21. Liquid-liquid extractions
Using separatory funnel.
The 2 liquids are shaken to increase the surface area between the phases.
The liquids are allowed to separate, with the denser phase forming the lower layer.
Eg. Pesticides in water may be extracted into hexane.

22. THE END