Column Chromatography is a chromatographic technique in which the separation of the 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.
In this technique, a solid adsorbent material is a stationary phase that 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 of Column Chromatography
The principle of Column chromatography is based on 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 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) = Molar concentration of analyte in stationary
phase(Cs) / Molar concentration of analyte in mobile phase(Cm)
Retention time(Rₜ) = Time taken by the analyte to pass through the column under set
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
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 solvent
Rf = (Rt’ – Mt)/Mt
Selectivity factor(𝛼) = Factor which describes the extent of separation between 2
𝛼 = (Rt₂ – Mt)/(Rt₁ – Mt)
Rt₁ = retention time of component 1
Rt₂ = retention time of component 2
The other important principle that explains column chromatography is the Plate Theory developed by Martin and Synge. The 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 an equilibrium is produced by the sample between 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
N = number of theoretical plates
L = length of 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
The various types of Column Chromatography are:
- Adsorption Column Chromatography:- This chromatography is based on the principle of adsorption of analyte on the solid stationary phase. For example- 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. For example- 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 based on the ionic nature of the analytes.
Components of Column Chromatography
A typical column chromatography consists of:
The column in this technique is 25 cm to 3 m long with an internal diameter of 2-4 mm 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 2-4 mm internal diameter.
II. 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.
III. Mobile Phase
Depending on the type of separation technique the mobile phase used in the column chromatography can be 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, a developing agent, and an eluting agent.
IV. 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 chromatogram which shows peaks of individual components.
Procedure of Column Chromatography
The separation process in column chromatography involves:
I. Column Preparation
The glass or steel column is packed with 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 dry 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.
II. 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.
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.
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 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 that has 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.