What Is Column Chromatography?
In chemistry, Column chromatography is a technique which is used to separate a single chemical compound from a mixture dissolved in a fluid. It separates substances based on differential adsorption of compounds to the adsorbent as the compounds move through the column at different rates which allow 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.
Column chromatography is a technique in which the substances to be separated are introduced onto the top of a column packed with an adsorbent, passed through the column at different rates that depend on the affinity of each substance for the adsorbent and for the solvent or solvent mixture, and are usually collected in solution as they pass from the column at different times.
It is a solid – liquid technique in which the stationary phase is a solid & mobile phase is a liquid or gas.
It was developed by the American chemist D.T Day in 1900 while M.S. Tswett, the Polish botanist, in 1906 used adsorption columns in his investigations of plant pigments.
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 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.
In column chromatography the stationary phase is packed into a glass or metal column.
The mixture of analytes is then applied and the mobile phase, commonly referred to as the eluent, is passed through the column either by use of a pumping system or applied gas pressure.
The stationary phase is either coated onto discrete small particles (the matrix) and packed into the column or applied as a thin film to the inside wall of the column.
As the eluent flows through the column the analytes separate on the basis of their distribution coefficients and emerge individually in the eluate as it leaves the column.
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 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:
1.It acts as a solvent – sample mixture can be introduced in the column.
2.It acts as a developing agent – helps in the separation of components in the sample to form bands.
3.It acts as an eluting agent – the components that are separated during the experiment are removed from the column
4.Some examples of solvents used as mobile phase based on their polarity are – ethanol, acetone, water, acetic acid, pyridine, etc.
Stationary phase – It is a solid material which should have good adsorption property and meet the conditions given below:
1.Shape and size of particle: Particles should have uniform shape and size in the range of 60 – 200μ in diameter.
2.Stability and inertness of particles: high mechanical stability and chemically inert. Also, no reaction with acids or bases or any other solvents used during the experiment.
3.It should be colourless, inexpensive and readily available.
4.Should allow free flow of mobile phase
5.It should be suitable for the separation of mixtures of various compounds.
Column Chromatography Experiment:
The stationary phase is made wet with the help of solvent as the upper level of the mobile phase and the stationary phase should match. The mobile phase or eluent is either solvent or mixture of solvents. In the first step the compound mixture that needs to be separated, is added from the top of the column without disturbing the top level. The tap is turned on and the adsorption process on the surface of silica begins.
Without disturbing the stationary phase solvent mixture is added slowly by touching the sides of the glass column. The solvent is added throughout the experiment as per the requirement.
The tap is turned on to initiate the movement of compounds in the mixture. The movement is based on the polarity of molecules in the sample. The non-polar components move at a greater speed when compared to the polar components.
For example, a compound mixture consists of three different compounds viz red, blue, green then their order based on polarity will be as follows blue>red>green
As the polarity of the green compound is less, it will move first. When it arrives at the end of the column it is collected in a clean test tube. After this, the red compound is collected and at last blue compound is collected. All these are collected in separate test tubes.
Factors Affecting Column Chromatography:
Dimensions of the column
Particle size of the adsorbent
Nature of the solvent
Temperature of the column
Pressure
Types of Column Chromatography:
1. Adsorption column chromatography – Adsorption chromatography is a technique of separation, in which the components of the mixture are adsorbed on the surface of the adsorbent.
2. Partition column chromatography – The stationary phase, as well as mobile phase, are liquid in partition chromatography.
3. Gel column chromatography – In this method of chromatography, the separation takes place through a column packed with gel. The stationary phase is a solvent held in the gap of a solvent.
4. Ion exchange column chromatography – A chromatography technique in which the stationary phase is always ion exchange resin.
Column Chromatography Applications:
Column chromatography is one of the most useful methods for the separation and purification of both solids and liquids. Its major application includes:
Separation of mixture of compounds.
Removal of impurities or purification process.
Isolation of active constituents.
Isolation of metabolites from biological fluids.
Estimation of drugs in formulation or crude extracts.
Column Chromatography is used to isolate active ingredients.
It is very helpful in Separating compound mixtures.
It is used to determine drug estimation from drug formulations
It is used to remove impurities.
Used to isolation metabolites from biological fluids.
Advantages:
Any type of mixture can be separated by column chromatography.
Any quantity of the mixture can also be separated.
Wider choice of mobile phase.
In preparative type, the sample can be separated and reused.
Automation is possible.
Limitations:
Time consuming method.
More amounts of solvents are required which may be expensive.
Automation makes the technique more complicated and costly.
Frequently Asked Questions on Column Chromatography:
What is the principle involved in column chromatography?
The basic principle involved in column chromatography is to adsorb solutes of the solution with the help of a stationary phase and further separating the mixture into discrete components.
What is column chromatography?
It is a precursory technique used in the purification of compounds based on their hydrophobicity or polarity. In this chromatography process, the molecule mixture is separated depending on its differentials partitioning between a stationary phase and a mobile phase.
What is the main advantage of column chromatography?
The main advantage of this chromatography technique is that the stationary phase is less expensive and can be easily disposed of as it undergoes recycling.
How are the compounds separated in this technique?
The separation is similar to that of TLC where the compound mixture is carried by a mobile phase via a stationary phase.
Which compounds elute out first in column chromatography technique?
Non-polar compounds. The polar compounds will strongly commune with the silica when compared to the non-polar compounds.