Stationary Phase Chromatography

Introduction to Chromatography

Chromatography was first introduced to separate the pigments of plants that include chlorophyll, xanthophyll, and carotenes. It was first devised by an Italian scientist in 1900 in Russia, he by developing the technique, coined the term chromatography. These pigments were separated by different colours in different bands, indirectly this got the name for the technique. Furthermore, types of chromatography were introduced for the separation processes. The technique used in the laboratory for the separation of the mixture is called chromatography. There are two phases of chromatography that include stationary phases and mobile phases. 

There are four main types of chromatography:

  1. Gas chromatography

  2. Liquid chromatography

  3. Paper chromatography

  4. Thin-layer chromatography


Mobile Phase and Stationary Phase

The mixture used in the technique is dissolved in a fluid, this is known as the mobile phase. This mobile phase is carried through a system that consists of a fixed material on it, which is known as a stationary phase. For the stationary phase, the different constituents present in the mixture have different affinities. Depending on the stationary phase the different molecules stay shorter or longer depending on the interaction between the molecules and the surface sites. The difference in the partitioning between the mobile and stationary phase is the base for the separation of a mixture. The chromatography can happen with the preparation or analytically. A form of purification and the separation of the mixture that happens for later use is preparative chromatography. For the smaller amount of mixture, analytical chromatography is done.


Mobile and Stationary Phase in Paper Chromatography

The separation of a mixture is done by passing the solvent through a chromatographic paper which is called paper chromatography. In the stationary phase chromatography, the water is absorbed that is present in the cellulose whereas the mobile phase consists of organic solvent that is immiscible with the stationary phase. This type of chromatography mainly works on the principle of partition and absorption

Paper is used as a stationary phase, the filter paper is selected depending on the four factors, which are the thickness of the paper, purity of the solvent, flow rate, and strength of the paper. There are different types of filter papers that are available commercially, the chemical composition of this paper includes 99% of alpha-cellulose, 0.3-1% of beta cellulose, 0.5-0.8% of pentose, 0.0-0.07% of ash. The widely used type of filter paper is Whatman filter paper. The mobile phase in paper chromatography is selected depending on the Rf value, if the Rf value ranges from 0.2-0.8, then it is selected for the process. 


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There are different types of paper chromatography:

  1. Ascending Paper Chromatography: In this technique, the solvent moves in an upward direction.

  2. Descending Paper Chromatography: The flow of solvent happens due to the gravitational pull and the capillary action it is directed downwards.

  3. Ascending-Descending Paper Chromatography: After a particular point the movement of solvent occurs in a two-direction way. Initially, it travels in an upward direction on the paper that is folded around the rod. When the solvent crosses the paper it travels in a downward direction. 

  4. Circular or Radial Paper Chromatography: The sample is present on the filter paper that is circular, it is allowed to dry and once the sample is dried it is tied horizontally on the solvent containing petri dish.

  5. Two-Dimensional Paper Chromatography: Substances that have the same Rf value can be separated by using this technique.


Gas Chromatography Stationary Phase

Gas chromatography is the analytic technique used in industrial laboratories and many research for the control of the quality and the identification and quantitation of the mixture. It is frequently used in forensic and environmental laboratories as it allows for the detection of small quantities. The mobile phase consists of inert gas and the stationary phase consists of the packed column. Here the whole solid or packing itself acts as a stationary phase. In most of the analytical gas chromatography, capillary columns are used, the wall of the small-diameter tube is coated with the stationary phase. The mixture is separated depending on the different strengths of interaction between the compounds on the stationary phase. 


Stationary Phase in Column Chromatography

Column chromatography is used to isolate the chemical compound from that of a mixture. The main advantage of this type of chromatography is the stationary phase used here is of low cost and is disposable. The stationary phase in this is solid. The most commonly used stationary phase is silica gel and alumina. There is a wide range of stationary phases that are used to perform different types of chromatography. Usually, the stationary phase used is finely grounded or gel these are microporous that increases the surface area. The important ratio between the stationary phase weight and dry weight of the analyte mixture has to be applied to the column. For the silica column chromatography, the ratio should be 20:1 or 100:1. 


Conclusion:

Chromatography is a technique used to separate the different mixtures. It contains two phases they are stationary and mobile phase. Here the stationary phases are kept fixed and the mobile phase is passed through this stationary phase. Chromatography also helps to catch the criminals, in proteomic analysis, drug testing, etc. Even though it is widely used for many applications it has some disadvantages as it takes a lot of time for the separation of the mixture, the equipment is to be handled with care, high knowledge is required or most experienced persons are required to perform the process.

FAQs (Frequently Asked Questions)

1. Mention the Applications of Chromatography.

Ans: Chromatography is widely used in different industries,

  • Pharmaceutical Industry: To analyze or identify the chemicals present in the sample. It is used to develop new drugs.

  • Food Industry: Used to determine the shelf life of food substances, the presence of chemical additives in food is also determined by this process, and also nutritional value in the food is also determined by this technique. 

  • Chemical Industry: Testing of purity of water and air, toxic contaminants present in the oil, and pesticides can be detected. 

  • Field of Molecular Biology: It is used for the study of metabolomics and proteomics. Used in nucleic acid research. 

2. What are Chromatography and Mention its Types?

Ans: The process of separation of a mixture by using the different techniques is called chromatography. There are different types of chromatography they are:

  • Gas chromatography

  • Liquid chromatography

  • Paper chromatography

  • Thin-layer chromatography