Column chromatography separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allows them to get separated in fractions. This technique can be used on a small scale as well as large scale to purify materials that can be used in future experiments. This method is a type of adsorption chromatography technique. The technique is widely applicable, as many different adsorbents (normal phase, reversed phase, or otherwise) can be used with a wide range of solvents. The technique can be used on scales from micrograms up to kilograms. The main advantage of column chromatography is the relatively low cost and disposability of the stationary phase used in the process.
Column Chromatography Principle
- When the mobile phase along with the mixture that needs to be separated is introduced from the top of the column, the movement of the individual components of the mixture is at different rates.
- The components with lower adsorption and affinity to the stationary phase travel faster when compared to the greater adsorption and affinity with the stationary phase.
- The components that move fast are removed first whereas the components that move slowly are eluted out last.
- The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as:
- Rf = the distance travelled by solute/ the distance travelled by the solvent
- Rf is the retardation factor.
Column preparation
- A column is prepared by packing a solid adsorbent into a cylindrical glass or plastic tube.
- The size will depend on the amount of compound being isolated.
- The base of the tube contains a filter, either a cotton or glass wool plug, or glass frit to hold the solid phase in place.
- A solvent reservoir may be attached at the top of the column.
- Two methods are generally used to prepare a column: the dry method and the wet method.
- For the dry method, the column is first filled with dry stationary phase powder, followed by the addition of mobile phase, which is flushed through the column until it is completely wet, and from this point is never allowed to run dry.
- For the wet method, a slurry is prepared of the eluent with the stationary phase powder and then carefully poured into the column.
- The top of the silica should be flat, and the top of the silica can be protected by a layer of sand.
- Eluent is slowly passed through the column to advance the organic material.
Column Chromatography Procedure
Before starting with the Column Chromatography Experiment let us understand the different phases involved.
Mobile phase – This phase is made up of solvents and it performs the following functions:
- It acts as a solvent-sample mixture that can be introduced in the column.
- It acts as a developing agent – helps in the separation of components in the sample to form bands.
- It acts as an eluting agent – the components that are separated during the experiment are removed from the column
- Some examples of solvents used as mobile phases based on their polarity are – ethanol, acetone, water, acetic acid, pyridine, etc.
Stationary phase – It is a solid material which should have good adsorption properties and meet the conditions given below:
- Shape and size of particle: Particles should have a uniform shape and size in the range of 60 – 200μ in diameter.
- Stability and inertness of particles: high mechanical stability and chemically inert. Also, no reaction with acids or bases or any other solvents was used during the experiment.
- It should be colourless, inexpensive and readily available.
- Should allow free flow of mobile phase
- It should be suitable for the separation of mixtures of various compounds.
Column Chromatography Experiment
The mixture to be separated is applied on the top of the column. The solvent is poured on the top of the column. The components of the mixture move down along with the solvent.
The effluent coming out from the column is collected into a series of test tubes. Each test tube contains the separated components of the mixture.
Application of Column Chromatography
- Column chromatography is used in the purification of compounds.
- Separation of molecules for a mixture and using it in the formation of a new substance.
- Used by a chemist to know the drug estimate in a drug solution.
- It is used to separate diastereomers, isolate racemate and separate geometrical isomers
- Used for the isolation of metabolic fluid from a biological fluid.
Advantages of Column Chromatography
- The process involves the separation of any mixture
- Separation of impurities from any mixture.
- Any variety and quantity separation are possible.
- The process involves low cost and simple understanding.
- All varieties of solvents can be used in the process which will give you the required result.
- The process can be fully automated, which is done by big laboratories.
Disadvantages of Column Chromatography
- It is a lengthy and time-consuming process.
- Small quantities become inadequate in the separation process; the only substantial amount can be used for separation.
- The process is expensive in comparison to the thin paper column chromatography process.
- It is a long process, so attention and focus are required throughout the process.
- If the process is automated, then the process becomes costly.
Thin Layer Chromatography (TLC)
