Adsorption Chromatography

What is adsorption chromatography?

A Russian botanist, Mikhail Tsvet in the year 1901, invented the process called adsorption chromatography while doing his research on plant pigments. He separated chlorophyll and carotenoid pigments of a different colour by passing the sample of leaves through a column of calcium carbonate, sucrose, and alumina, allowing them to rinse with petroleum, ether/ethanol mixtures. The term "chromatography" was coined from the Greek words chroma meaning "color" and graphos meaning "to write" in a 1906 publication.
                                                                                     Mikhail Tsvet

In the process of adsorption chromatography, the adsorbent which adsorbs the sample components (solute) via van der Waal forces and steric interactions is called the stationary phase which is a solid phase. The mobile phase may be either a gas (gas-solid chromatography) or a liquid (liquid-solid chromatography). 

TLC is the abbreviation of Thin-layer chromatography which is an extensively employed technique of adsorption chromatography where the mobile phase is a liquid, and the stationary phase is a planar, in the form of a solid-supported on an inert glass plate. 

Adsorption chromatography can be defined as a technique for the separation of the component in a mixture by adsorption from a mobile phase into the stationary solid surface. 
For a good separation of the sample mixture, adsorption chromatography depends on the following condition:

  •  The selection of the adsorbent

  •  The choice of the solvent for the sample mixture

  •  The rate of flow of the solvent

  •  The temperature

  •  The selection of the column size

  • Principle of adsorption chromatography

    Adsorption Chromatography is based on the principle that some solid substances, which are known as adsorbent, have the power to hold molecules at their surface. This holding force is due to weak, non-ionic attractive forces of the van der Waals' and hydrogen bonding, which only occur at specific adsorption beds. These adsorbent beds can differentiate between the organic molecule (adsorbate) based on their strength of binding. As solvent (eluent) is constantly passed over the adsorbent (stationary phase) in the column, differences in the compound flow rate eventually lead to the separation of the organic mixture (analytes). The most commonly used adsorbents are silica, alumina, and carbon.

                                                                             Figure 1: Adsorption chromatography

    Procedure of adsorption chromatography

    The process involved in TLC is as follows:

  •  Glass plate coated with a uniform thickness of silica is selected. Here silica serves as the stationary phase.

  •  The solvent is carefully selected based on the polarity of the sample. Here solvent serves as the mobile phase. Commonly used solvents are ethyl ether, ethyl acetate, acetone, benzene, hexane, dichloromethane, etc.

  •  Developing chamber: In order to develop the TLC, a glass chamber with a lid is taken. Then to keep the chamber atmosphere saturated with solvent, line the inside of the glass chamber with some filter paper as shown in the figure.

                                                                 Figure 2: Developing chamber for TLC

  •  Samples are spotted on the baseline using a capillary tube.

  •  Iodine chamber or UV lamp: It is used to visualize the TLC spot after developing. The iodine chamber is prepared by adding a few crystals of iodine in a dry lid jar.

  • Adsorption Chromatography Experiment (TLC)

  •  The test sample is first dissolved in a suitable solvent.

  •  The baseline is marked, usually by drawing a very thin line about 1 cm from the end of the TLC plate.

  •  With the help of a capillary tube, mark the sample on the baseline in a very small area as possible and then evaporate it using a dryer.

  •  Put the spotted TLC into the development chamber. It is to be noted here the baseline should always be kept higher than the level of the solvent to prevent the sample from dissolving from the TLC plate into the solvent.

  •  Wait for some time until the solvent phase moves from the baseline, and the TLC plate is developed.

  •  Take out the TLC place and dry it.

  •  Now using the help of the Iodine chamber or UV lamp, the spot can be located.

  •  Once spotted, calculate the Rf "retention factor" of the sample mixture. It is calculated by recording the distance moved from the baseline by the compound and distance moved from the baseline by the solvent front, as shown in the figure.

                                                                                    Figure 3: Thin-layer chromatography

    Types of Adsorption Chromatography

    There are three main types of adsorption chromatography - 

  •  Column chromatography

  •  Thin Layer chromatography

  •  Gas-solid chromatography

  •  Column chromatography is an analytical technique in which column packed with a solid, which serves as a stationary phase, and the liquid (the mobile phase or eluent) runs through this column. Separation of the mixture depends upon the strong affinity for the adsorbent. The compounds that absorb stronger will be at the top of the column, and the movement will be very slow. Compounds that absorb weakly will be moving fast and collect in a beaker at the bottom.

                                                                          Figure 4: Column Chromatography

  •  Thin Layer Chromatography or TLC is a method for analyzing mixtures by differentiating the adsorption of the component in the given mixture. It has three steps i.e. spotting, development, and visualization. In comparison with the column chromatography, it requires very less amount of sample. It has two phases one is stationary phase (adsorbent) and another mobile phase (solvent). The process by which the component mixture moves upward in TLC by passing the solvent is called elution. The component, which rises upward from the baseline of the mixture sample, is called eluate.

  •  Gas-Solid chromatography or GSC is an analytical technique to separate the mixture using an inert gas (nitrogen or helium) as the mobile phase, and the stationary phase is a solid (silica or alumina). This technique is not very popular as it has a limited number of stationary phases.

  •    Adsorption Chromatography Applications

  •  This technique is used for separating unsaturated hydrocarbons from the mixture of organic compounds.

  •  This technique is used for separating and isolating lipids, steroids, and fats.

  •  This technique is helpful in the identification of carbohydrates.

  •  This technique is used for separating diastereomers like threo and erythro isomers.