Column Chromatography: Principle, Types, Working & Applications
Column Chromatography is a chromatographic technique in which the separation of constituents of a substance is carried out in a column packed with an adsorbing material. It is a type of adsorption chromatography in which the constituents separate at differential rates in fractions.
It is a technique that uses a solid adsorbent material as its stationary phase which is packed in the column and the mobile phase (gas or liquid) runs through it. The most commonly used stationary phase materials are silica gel or alumina whereas organic solvents are the widely used mobile phase in column chromatography.
Principle or Column Chromatography
Column chromatography is based on the principles of the rate of adsorption of the analyte on the stationary phase according to its affinity with the adsorbent.
It states that when a sample is loaded at the top of the column along with the mobile phase, the components of the sample separate at differential rates in fractions depending upon their affinity with the stationary phase.
If a component has a higher affinity with the stationary phase it will take more time to separate whereas if the component has less affinity with the stationary phase, then the separation of that component will be quick. The components that move fast are removed first whereas the components that move slowly are eluted out last.
The separation of a component is calculated in the form of partition coefficients, retention time, retardation factor, and selectivity factor. They are given by:
Partition Coefficient(K) = Cs/Cm
Where, Cs is known as the Molar concentration of analyte in stationary phase
Cm is known as Molar concentration of analyte in mobile phase
Retention time(Rₜ) = the time taken by the analyte to pass through the column under set conditions.
However, the accurate result is given by adjusted retention time which indicates the retention time of only analyte and not the mobile phase. It is indicated by:
Rt’ = Rt – Mt
Where, Rt’ = Adjusted retention time
Rt = retention time of the mixture
Mt = Dead time, which is the time of the mobile phase to reach the detector
Retardation factor(Rf) = Distance traveled by solute / Distance traveled by the solvent
Rf = (Rt’ – Mt)/Mt
Selectivity factor(𝛼) = the factor which describes the extent of separation between two components.
𝛼 = (Rt₂ – Mt)/(Rt₁ – Mt)
Rt₁ = retention time of component 1
Rt₂ = retention time of component 2
The other important principle which explains column chromatography is the Plate Theory developed by Martin and Synge.
The Plate theory states that, “A chromatographic column consists of a series of discrete yet continuous hypothetical horizontal layers which are termed as the theoretical plates. The separation of components of the sample takes place at these plates when equilibrium is produced by the sample between the stationary and mobile phases. The equilibrium directs a stepwise separation of sample components by transferring them to each plate one by one.“
The theoretical plate number is an index that indicates column efficiency based on a calculation in which the larger the theoretical plate number the sharper the peaks will be obtained. It is represented as:
N = L/H
Where, N = number of theoretical plates
L = length of the column
H = height equivalent of theoretical plates (HETP)
HETP is the height of a layer of the column such that the solution leaving the layer is in equilibrium with the average concentration of the solute in the stationary phase throughout the layer.
Types of Column Chromatography
There are various types of column chromatography that are used:
- Adsorption Column Chromatography – This chromatography is based on the principle of adsorption of analyte on the solid stationary phase. Its examples are solid-gas chromatography and solid-liquid chromatography.
- Partition Column Chromatography – This technique is based on the partition of the analyte between the liquid stationary and mobile phases. Its examples are liquid-liquid chromatography and liquid gas chromatography.
- Gel Permeation Chromatography – This technique is also called size-exclusion chromatography which uses a porous gel material as the stationary phase.
- Ion-Exchange Chromatography– In this technique, the sample is separated on the basis of the ionic nature of the analytes.
Components of Column Chromatography
Typical column chromatography consists of:
1. Column
The column is 25 cm to 3 m long with an internal diameter of 2-4 mm and is made up of stainless steel or glass. The column of liquid chromatography is 25-50 cm long with an internal diameter of 2 mm and made up of stainless steel. For gas chromatography, the column is made up of glass which is 1-3 m long with a 2-4 mm internal diameter.
2. Stationary Phase
The stationary phase used in column chromatography is usually a solid adsorbent material that is inert and does not create any hindrance in the flow of the mobile phase. Usually, the adsorbent used is silica or alumina. It is packed in the column.
3. Mobile phase
Depending on the type of separation technique the mobile phase used in column chromatography can be either liquid or gas. The liquid used is most of the time an organic solvent and the gases used are the usual inert gases. The mobile phase acts as a solvent, developing agent as well as eluting agent.
4. Detector and Recorder
The separated components of the sample are detected by different detectors depending on the type of column chromatography used. The final result is depicted in the form of a chart called a chromatogram which shows peaks of individual components.
Working of Column Chromatography
The separation process in column chromatography involves the following:
1. Column Preparation
The glass or steel column is packed with a solid adsorbent material. A cotton wool or glass wool pad is placed at the bottom of the column to avoid leakage. To avoid any disturbance during the introduction of samples in the column, the top of the column is covered with a paper disc. Before use, the column should be cleaned and dried thoroughly to avoid any disturbance.
The packing techniques are classified as:
- Dry Packing– The dried finely powdered adsorbent material is packed in the column and then the mobile phase is allowed to run through it.
- Wet Packing– A slurry of the adsorbent material with mobile phase is prepared and filled in the column.
2. Sample Introduction
The sample is mixed with the mobile phase and introduced in the column at the top portion of the column. This is the moment when the separation process begins.
3. Elution
Elution is the process of passing the mobile phase through the column. This leads to the separation of the sample components in the column. The elution technique can be of two types:
- Isocratic Elution– In this technique, the solvent of the same polarity or same composition is used throughout the process.
- Gradient Elution– In this technique, solvents of gradually increased polarity or increased elution strength are utilized throughout the procedure.
4. Detection
The eluted components collected after elution are examined visually if they are colorful. But if the elute is colorless, then thin-layer chromatography is used to analyze each component.
Column Efficiency
The column efficiency elucidates how efficiently a column can separate the columns. The efficiency of the column is affected by:
- Width of peaks
- Retention time
- Temperature
- Dimensions of the column
- Pressure
- The particle size of adsorbent
- Nature of solvent
Advantages of Column Chromatography
- Easy to handle
- A wide range of solvents can be used
- All types of mixtures can be separated
- Automation can be done
- The sample can be reused
Disadvantages of Column Chromatography
- It is a time-consuming procedure
- Contamination of the column is possible
- A large amount of solvent is required for the process
- Since automation is possible it makes the procedure quite complicated
- Noise can be produced by the instrument
Applications of Column Chromatography
- It is useful to separate and purify the chemical compounds
- In forensic toxicology, column chromatography can be used to analyze various poisons, alkaloids, pesticides, and drugs.
- The various metabolites of blood and other biological samples can be separated by column chromatography.
- Diastereoisomers can be isolated by column chromatography.
Conclusion
Column Chromatography is a type of separation technique used to isolate and purify a compound. It uses a column as the main site for the separation hence the name is such. It is a useful technique having wide application in the chemical, agricultural and pharmaceutical industries.
In forensic science, the technique is used to examine various pieces of evidence of chemical and biological origin.
