Column chromatography is the prototype of chromatography.
It has simple instrumentation with minimal requirements.
It works based on the principle of adsorption chromatography technique.
Column Chromatography Principle
The principle involved in this technique is the separation of components by adsorption.
The sample mixture is allowed to pass through a column of solid stationary phase under the force of a liquid mobile phase. The components in the mixture get adsorbed to the solid stationary phase during their travel. Due to the differences in the adsorption of individual components, they get separated.
Those with lower affinity and adsorption to the stationary phase move faster and come out first. While those with greater adsorption affinity move or travel slower get out of the column latter.
The solute molecules adsorb to the column in a reversible manner. So, with a constant input of the mobile phase from the top all of the sample flows out.
The rate of the movement of the components is given as follows
R= Rate of movement of a component / Rate of movement of the mobile phase. i.e. it is the ratio of the distance moved by solute to the distance moved by the solvent.
Column chromatography animation
Column chromatography instrumentation and requirements.
The instrument consists of a column and a stand.
The column chromatography contains a vertical column made of neutral glass with a knob at the bottom end.
The glass column is chosen so to avoid reactions with solvents, acids, and alkalies.
The surface of the cylindrical column is clear without graduations or readings on the surface.
Ideal dimensions of length: diameter for effective separation are 10:1 to 30:1. But sometimes even dimensions of 100:1 can be used for higher efficiency.
This is a solid material with good adsorption property and should meet the following conditions
- Particle Size and shape: Particles should be of uniform size and shape without contamination. They should be of spherical shape in the range of 60 to 200μ in diameter.
- Stability and inertness: The particles should be chemically inert and of high mechanical stability. They should not react with acids, bases, and other solvents used in the procedure.
- It should be easily available, inexpensive, and also colorless. Being colorless helps to identify chromatogram bands for easy recovery.
- Should allow free flow of mobile phase and also suitable for separation of a wide variety of compounds.
The stationary phase material is classified based on their adsorbent property as weak, moderate and strong.
|Sucrose||Calcium carbonate||Silica gel (Activated magnesium silicate)|
|Starch||Calcium phosphate||Activated Alumina|
|Inulin||Magnesium Carbonate||Activated Charcoal|
|Talc||Magnesium oxide||Activate Magnesia|
|Sodium carbonate (Na2CO3)||Calcium Hydroxide||Fullers earth (a type of clay)|
Of them, silica gel is the most commonly used adsorbent with particle size ranging between 60 to 200 microns.
This made of solvents and has varying functions in the process like
A) As the solvent to introduce sample mixture into the column
b) As developing agent to separate sample components into bands
c) As eluting agent to remove the separated components out of the column.
Many solvents are used as mobile phases based on their polarity. Examples of solvents include water, ethanol, pyridine, acetone, acetic acid, etc.
Column Chromatography Procedure
Preparation of column
The column is packed with adsorbent used as the stationary phase. First, cotton wool is placed at the bottom. Over it, the adsorbent is packed. After packing, the Whatman filter paper disc is placed on the top of adsorbent to prevent disturbance while introducing the mobile phase. The packing is done by two methods like
a) Wet packing method.
b) Dry packing method.
Wet packing method is the ideal technique as it leads to the uniform settlement of the stationary phase. The adsorbent is mixed with the mobile phase in a beaker and then poured into the column. The adsorbent settles uniformly without any air bubbles in between. There are no chances of a crack in the stationary phase leading to a uniform flow of mobile phase through it. Hence, the separation would be efficient.
In Dry packing method, the dry powdered form of adsorbent is poured into the column. Then the mobile phase solvent is allowed to pass until the equilibrium is reached. The disadvantages are, there are chances of air entrapment and also crack in the stationary phase. Hence, uniform flow and distribution cannot be achieved.
The sample mixture is mixed with the mobile phase and then placed over the stationary phase. The sample gets adsorbed on the top of the column and is ready for separation.
Development of chromatogram
The sample mixture is subjected to the separation by the constant passing of the mobile phase from the top. This is again of two types like
i) Isocratic elution and
ii) Gradient elution methods
Detection of components
The colored bands formed are collected separately and then detected by methods like
i) UV-Visible spectroscopy
ii) Fluorescence emission
iii) By refractive index detector
iv) Evaporation of solvent
The stationary phase material is suitably moistened with the mobile phase and packed sufficiently in the column with a cotton or asbestos pad at the bottom. The extracted material or sample to be separated is placed on the top of the packed stationary phase with a second cotton or asbestos pad in between.
The mobile phase is poured into the column over the sample. A collecting beaker is placed at the bottom of the column near the end to collect the elute.
The mobile phase percolates through the entire stationary phase (mixture column) reaches the bottom of the column. From there it is eluted out and gets collected in the beaker placed below.
When the mobile phase flows through, different components of the sample travel with different rates through the silica gel. This rate of travel is decided by the adsorption and affinity of molecules towards the stationary phase and mobile phase.
The fractional components of the mixture with greater affinity to the mobile phase travels fast and reach the bottom early. Those with higher affinity to stationary phase travel slow and reach bottom late.
Thus the colored bands of the sample are formed.
Each color is an indicator of one particular set of the compound in the sample mixture.
Then by differential mobile phase, different components are taken out of the column by a further flow of solvents.
This elution occurs drop by drop and the process may take a few hours to days based on the sample size, length of the column, mobile phase used and the packing material used.
1. Keep the column in a clean and dust-free place.
2. Do not disturb the column until the separation is complete.
3. Avoid gaps within the stationary phase packing.
Column chromatography Applications
It is used for purposes related to extraction and isolation to a large extent. For qualitative identification and analysis, it less preferred.
♦ For Isolation of active ingredients: Column chromatography is best suited to separate active principles from plant materials. Plants contain many constituents like alkaloids, resins, glycosides, tannins, flavonoids, and other bio-molecules. These individual constituents can be separated by using this technique. Since the plant extract is bulk this method is best to separate them.
♦ Separation of compound mixtures: After organic synthesis to obtain desired molecule column chromatography can be employed to separate the mixture.
♦ Removal of impurities: When a compound is given, the impurities from it can be removed by the appropriate section of the stationary phase and mobile phase.
♦ Drug estimation from drug formulations
♦ Isolation of metabolites from biological fluids like blood, serum, etc.
Limitations of this chromatography
- It is a time-consuming process and sometimes a single run can take a few days.
- Large volumes of solvents are required which can be expensive.
- It is a simple technique but if automated, it becomes more complex and also expensive.
Many modifications and improvements were made to column chromatography to derive advanced chromatography techniques.
These advanced forms include high-performance liquid chromatography (HPLC), Ultra Performance Liquid Chromatography (UPLC), gas chromatography (GC), ion-exchange chromatography, gel permeation chromatography, etc.
Despite so many advanced methods present, still, this method of chromatography is widely used in science, research, and industry.