What is Adsorption Chromatography?
It is one of the oldest chromatography types and is most widely used in organic laboratories in the form of column chromatography and thin-layer chromatography. TLC is used to analyze the compounds and to check reactions progress and column chromatography performs separation and purification of compounds. In adsorption chromatography, the stationary phase is a fine solid partitioned to maximize surface area. The analytes are interacting differently with the adsorbent since each has a different affinity. Hence, they separate different retention times.
Adsorption Chromatography Principle:
Adsorption chromatography works on the principle that certain solid materials, called adsorbents can hold compounds onto their surface. The adsorbent beds will distinguish between the adsorbate based on their binding strength as eluent is continuously run over the stationary phase in the column; variations in the flow rate of the compound ultimately lead to separation of the analytes. The most widely used adsorbents in adsorption chromatography include silica, alumina, and charcoal. Based on their relative polarity the elution order of components from the absorptivity are different.
Types of Adsorption Chromatography:
There are three main types of adsorption chromatography, such as column chromatography, thin-layer chromatography (TLC), and gas-solid chromatography.
Column chromatography is a commonly used technique of separating the compounds from the sample mixture. It is used on small or large scans to separate and purify compounds. The mixture of samples moves through the stationary phase with the mobile phase and based on various degrees of adhesion it separates the compounds.
Thin-layer chromatography is an analytical method of separation used for qualitative analysis and monitoring of the reaction as well as identifying unknown compounds.
Gas-solid chromatography (GC) is an analytical method for the separation of volatile compounds in which the mobile phase is gas and the stationary phase is suitable for solid support. The gas-solid chromatography, work on the principle of separation is adsorption and generally used for samples that had less solubility in the stationary phase.
Procedure of Adsorption Chromatography:
Before beginning an adsorption chromatography experiment,
we must recognize the various components essential to perform the process.
Stationary phase: The stationary phase of adsorption
chromatography is adsorbent.
Mobile phase: In adsorption chromatography, either a liquid
or a gas is used as a mobile phase.
Apparatus for TLC method:
TLC
jar: It helps to keep the atmosphere proper
during isolation.
TLC
plate: It is used as the stationary phase coated
with a thin layer of silica, alumina, on glass plastic or aluminum.
Iodine
chamber or UV chamber: It helps visualize the spots on the thin
layer chromatography plate.
Capillary
tube: It is used to apply the sample mixture.
Thin Layer Chromatography Diagram:
Experimental Procedure of Adsorption
Chromatography:
The
following steps are involved in the TLC process.
- Prepare the developing container: Pour the solvent to a depth just below 0.5 cm inside the chamber to further saturate with the
- Prepare the TLC plate: TLC plates are usually commercially available; just you need to cut them to the required size.
- Spot the TLC plate: Dip the micro-capillary into the sample solution, and subsequently touch its end gently on the plate at a suitable place.
- Develop the plate: In the chamber, place the prepared plate, cover it with the lid and leave it untouched. Via capillary action, the solvent will raise the TLC plate.
- Visualize the spots: Remove the plate from the chamber, and if the plate has some colored spots, mark them. Most of the analytes are not colored and should be visualized by placing them in an iodine chamber or UV cabinet. Finally, calculate the Rf values for all the separated compounds.
Adsorption Chromatography Applications:
- It is used for the separation of amino acids.
- In this technique, the analytes are purified from sample mixtures according to certain physicochemical properties.
- It is used to determine the concentration of a molecule.
- It is used for the separation and identification of isomers.
- The adsorption technique is useful for the identification of carbohydrates.
- It is used for the separation and identification of various mixtures of volatile compounds.
The advantages of adsorption chromatography are as follows.
- Adsorption chromatography is an important tool for separating many molecules that cannot be distinguished from other methods.
- Adsorption chromatography uses a broad range of mobile phases.
- Very few types of equipment are used as opposed to other separation methods.
- The molecules in the complex mixture can be easily separated.
The disadvantages of adsorption chromatography are as follows.
- Automation is making it more complicated and expensive.
- The major disadvantage of adsorption chromatography is that some solutes have longer retention times.
- It can cause catalytic variations in the sample.
- Results obtained by some methods of adsorption chromatography are difficult to reproduce.
Commonly
asked questions on chromatography are as follows.
What is the basic principle of ion-exchange chromatography?
The exchange of ions is the basic principle of ion-exchange chromatography. Cationic and anionic exchangers are two forms of ion-exchange chromatography are used in this process.
What is column chromatography?
Column chromatography is a commonly used method of separating the compounds from complex mixtures. The chromatography, when performed in a column, is called column chromatography.
What is the major difference between affinity and ion-exchange chromatography?
The major difference between affinity and ion-exchange chromatography is that affinity chromatography is used to separate charged or uncharged molecules, while ion-exchange chromatography is used to separate charged molecules.
What type of paper is used in paper chromatography?
Filter paper, chemically modified papers, acetylated papers, and pure cellulose papers of different grades like No.1, 2, 3, 4, 7, 17, 20, 40, 42, etc are used as stationary phases in paper chromatography.
What is the major advantage of HPLC?
One of the major advantages of high-performance liquid chromatography is that it has the ability to test a wide variety of samples.
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