Chromatography
Chromatography is a technique used to separate components of a mixture, and also purify compounds. The name of the technique comes from the Greek word Chroma, meaning color.
In 1903, Tswett discovered this technique for separating the colored components found in plants. The principle of separation of substances in this technique is similar to solvent extraction (i.e., distribution of the solutes in two phases).
In chromatography, we use two phases for separation: (a) Stationary phase and (b) Mobile phase. This technique is based on the difference in rates at which components in the mixture move through the stationary phase under the influence of the mobile phase. First, the mixture of components is loaded at one end of the stationary phase, and then the mobile phase (a pure solvent or a mixture of solvents) is allowed to move over the stationary phase. Depending on the relative affinity of the components toward the stationary phase and mobile phase, they remain on the surface of the stationary phase or move along with the mobile phase, gradually getting separated.
The stationary phase can be a solid or a liquid. Depending on the stationary phase, chromatography is classified into Adsorption Chromatography and Partition Chromatography.
Adsorption Chromatography
This type of chromatography is based on the principle of differential adsorption, where different solutes adsorb to varying degrees on a solid stationary phase. Adsorption chromatography is broadly divided into two primary types:
1. Column Chromatography
Column chromatography involves separating components over a stationary phase packed inside a cylindrical column. Common stationary phase materials include alumina and silica gel. A slurry of the adsorbent is filled into a long glass tube equipped with a stopcock and a glass wool plug at the bottom to hold the packing in place.
Column Chromatography: Different stages of separation
The mixture to be separated is dissolved in a minimal amount of a suitable solvent and loaded onto the top of the adsorbent column. A mobile phase—a pure solvent or a mixture of solvents—is then continuously passed through the column. As the mixture travels down, solutes adsorb onto the stationary phase according to their affinity. The most strongly adsorbed components remain near the top, while weakly adsorbed components move further down, separating into distinct bands.
The component with the lowest affinity for the stationary phase desorbs and elutes (leaves the column) first, while more strongly bound components elute later. The separated fractions are collected individually, and the pure solutes are recovered by evaporating the solvent.
2. Thin Layer Chromatography (TLC)
In thin layer chromatography, a thin layer (approximately 0.2 mm thick) of adsorbent—such as silica gel or alumina—is spread uniformly over a glass, plastic, or aluminum plate, forming the stationary phase. This prepared plate is known as a TLC plate or chromplate.
Stages in Thin Layer Chromatography
The sample mixture is applied as a small spot roughly 2 cm above the bottom edge of the plate. Once dried, the plate is placed vertically inside a sealed developing chamber containing the mobile phase, ensuring the solvent level remains below the sample spot. As the mobile phase ascends the plate via capillary action, the components travel at different rates depending on their degree of adsorption.
Developed chromatogram
Colored components appear as distinct visual spots. Uncolored components can be visualized using several techniques:
- UV Light: For compounds that fluoresce or absorb ultraviolet light.
- Iodine Chamber: Organic compounds adsorb iodine vapors to form temporary brown spots.
- Visualization Reagents (Spraying Agents): Specific chemicals are sprayed onto the plate to induce color reactions (e.g., spraying ninhydrin solution to detect amino acids as purple spots).
Partition Chromatography
In partition chromatography, both the stationary phase and the mobile phase are liquids. Separation occurs through the continuous differential partitioning of components between the two immiscible liquid phases based on their relative solubilities.
Paper Chromatography
Paper chromatography is a common application of partition chromatography:
- Stationary Phase: Water molecules bound within the cellulose fibers of a specialized filter paper (such as Whatman No. 1 paper) act as the liquid stationary phase.
- Procedure: The sample solution is spotted near the baseline (about 2 cm from one edge) using a fine capillary tube. Once dried, the paper strip is suspended vertically inside a sealed developing chamber containing the mobile phase, ensuring the sample spot remains above the solvent level.
- Separation Mechanism: The mobile phase ascends the paper via capillary action. As it moves over the sample spot, components distribute themselves continuously between the stationary water phase and the mobile organic solvent. Solutes with a higher affinity for the mobile phase travel faster and further up the paper.
Different Stages in Paper chromatography
- Visualization: The developed paper strip is called a chromatogram. Colored components appear directly as visible spots. Colorless components can be visualized under UV light, by exposure to iodine vapors, or by spraying specific chemical reagents (e.g., ninhydrin).
Retention Factor (Rf Value)
The relative rate of movement of a solute compared to the mobile phase solvent front is expressed as its Retention Factor (Rf). Under identical experimental conditions (temperature, solvent, stationary phase), the Rf value is a constant characteristic of a given compound.
$$\text{R}_f = \frac{\text{Distance traveled by the solute from the baseline}}{\text{Distance traveled by the solvent front from the baseline}}$$
Note: Since the solute never travels further than the solvent front, the Rf value is always less than 1.
Retention factor