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 color 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.                                                

About Adsorption Chromatography by Vedantu

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 molecules (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.                              

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.                                                        

• 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.                                                         

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 a column is 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.                   

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 column chromatography, it requires very few samples. It has two phases one is the stationary phase (adsorbent) and another is the 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.

What is Adsorption Chromatography?

Adsorption chromatography is a technique for the separation of a component in a mixture by adsorption from a mobile phase into the stationary solid surface. 

Adsorption chromatography is the type of chromatography which is one of the oldest types of chromatography technique. During this process, a mobile phase which can either be in liquid or gaseous state is adsorbed onto the surface of a stationary solid phase.

Principle of Adsorption Chromatography

In Adsorption Chromatography the analytical separation of a chemical mixture is involved based on how the adsorbate and the adsorbent interact with each other. A mixture of gas or liquid gets separated when it is passed over the adsorbent bed that has different adsorbing capacity and rate for different compounds.

Simply put, an Adsorbent is a substance which is usually porous in nature with a high surface area to adsorb substances on its surface by intermolecular forces like non-ionic attractive forces and hydrogen bonding is called adsorbent. Some commonly used adsorbents are Silica, alumina and carbon.

Adsorption Chromatography Procedure

Here's a description of the types of phases and the types of forces involved during the mixture separation process:

• Stationary phase – In adsorption chromatography, adsorbent is the stationary phase. Solutes on the surface of adsorbent are removed with various forces so that they can move with the mobile phase.

• Mobile phase – Mobile phase is either a liquid or a gas in adsorption chromatography. Forces that are involved in the process help to remove solutes from the adsorbent for its movement with the mobile phase. If a liquid is used as a mobile phase it is called LSC (Liquid-Solid Chromatography) and if a gas is used as a mobile phase it is called GSC (Gas-Solid Chromatography).

Apparatus –

Chromatography jar – The glass jar helps in maintaining a suitable environment during separation.

Thin-layer chromatography plate – A Borosilicate glass plate with size 20*20 cm, 20*5 cm, 20*10.

Capillary tube – Capillary tube is used to apply sample mixture to TLC.

Mobile phase – Either a Liquid or gas

Stationary phase – Adsorbents

Adsorption Chromatography Experiment (TLC)

• A clean and dry chromatographic jar with a lid is required for the experiment

• In order to maintain a suitable environment inside the jar, a paper soaked in the mobile phase is applied on its walls to ensure the presence of saturated vapors.

• In the next step add the mobile phase to the jar and close the lid properly.

• Wait until equilibrium is maintained.

• Carefully mark  the baseline on the adsorbent.

• Apply the sample to the TLC plate with the help of the capillary tube and allow it to dry.

• Now, Put the plates in the jar and close it.

• Wait until you see movement of the solvent from the baseline and the TLC plate is developed.

• Take out the TLC plate and let it dry.

• Now with the help of an UV lamp, the spot can be located.

• Calculate the retention factor ‘Rf’ of the sample mixture. It is calculated by recording the distance moved from the baseline by the compound and the distance moved from the baseline by the solvent front.

Adsorption Chromatography Applications

• Adsorption chromatography is used for the separation of amino acids from a mixture.

• Used in the isolation of antibiotics.

• Used in the identification of carbohydrates.

• Adsorption Chromatography is used for separating and isolating lipids, steroids, and fats.

• Used to isolate and determine various peptides and proteins.

Types of Adsorption Chromatography

These are some important types of Adsorption Chromatography.

1. Thin Layer Chromatography (TLC) – In this chromatography technique the mobile phase moves over the adsorbent. The adsorbent is a thin layer which is applied to a solid support for the separation of different components. The separation of the components takes place through differential migration which occurs when the solvent starts moving along the powder spread on the glass plates.

2. Paper Chromatography – It is a type of Chromatography technique that uses paper sheets as the adsorbent being the stationary phase through which a solution is made to pass. The solid surface of the paper is the stationary phase and the liquid phase is the mobile phase in this process.

3. Column Chromatography – In this technique, the solutes of a solution are allowed to travel down a column where the individual components are adsorbed by the stationary phase. The separation of this mixture depends upon the strong and hard affinity for the adsorbent. The component that is strongly adsorbed is visible at the top of the column.

4. Gas-Solid Chromatography – In this technique, an inert gas such as nitrogen or helium is used to separate the mixture as a mobile phase and the stationary phase is a solid such as silica or alumina. It is mainly used for solutes which have less solubility in the stationary phase. This technique has a very limited number of stationary phases available and therefore it is not commonly used.