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:
Gas chromatography
Liquid chromatography
Paper chromatography
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:
Ascending Paper Chromatography: In this technique, the solvent moves in an upward direction.
Descending Paper Chromatography: The flow of solvent happens due to the gravitational pull and the capillary action it is directed downwards.
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.
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.
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 on Stationary Phase Chromatography
1. What is the stationary phase in chromatography and why is it essential for separation of mixtures?
The stationary phase in chromatography is the fixed material through which the mobile phase moves. It is crucial because components in a mixture interact differently with the stationary phase, leading to their separation based on varying affinities. Without the stationary phase, effective separation would not occur.
2. What are the main types of stationary phase chromatography studied in CBSE Class 12 Physics?
The main types include:
- Paper chromatography (filter paper as stationary phase)
- Thin-layer chromatography (thin solid layer as stationary phase)
- Column chromatography (solid adsorbent like silica gel or alumina)
- Gas chromatography (packed or coated column)
3. How does the interaction between the stationary phase and the components of a mixture determine their separation during chromatography?
Each component in the mixture interacts with the stationary phase to a different degree based on their chemical properties. Components that are more strongly attracted to the stationary phase move more slowly, while those with weaker affinity move faster. This differential movement causes separation.
4. Why is cellulose paper commonly used as the stationary phase in paper chromatography?
Cellulose paper is widely used because it is highly pure, has a consistent structure, and efficiently absorbs water, which acts as the stationary phase. Its composition allows for effective partitioning between the mobile and stationary phases, making it suitable for separating a wide range of substances.
5. Can you explain the principle behind ascending and descending paper chromatography techniques?
In ascending paper chromatography, the solvent moves upwards through the stationary phase due to capillary action. In descending chromatography, the solvent moves downward, aided by gravity and capillary action. Both methods rely on differential migration of mixture components over the stationary phase.
6. What are common misconceptions students have about the stationary phase in chromatography?
A common misconception is that the stationary phase causes separation simply because it stops some molecules. In reality, separation depends on how strongly each component interacts with the stationary phase, not just by being trapped or stopped. Also, students often assume only solids serve as stationary phases, but liquids (like water absorbed on paper) can also act as stationary phases in some methods.
7. How does one choose the most suitable stationary phase for a specific chromatography application in the lab?
Selection is based on the nature of the mixture, the purpose (analytical or preparative), and the type of substances to be separated. Factors considered include porosity, chemical compatibility, surface area, and stability under operating conditions.
8. What is the role of Rf value in paper chromatography using a stationary phase?
The Rf value (retention factor) is calculated as the distance traveled by the component divided by the distance traveled by the solvent front. It helps quantify the extent of separation achieved on the stationary phase and assists in identifying compounds by comparing Rf values under standardized conditions.
9. How do stationary and mobile phases work together to separate plant pigments using chromatography?
Plant pigments dissolve in the mobile phase and move over the stationary phase. Each pigment interacts differently with the stationary phase—some are retained more strongly and move slowly, while others travel further with the mobile phase. This differential movement separates the pigments into distinct bands or spots.
10. In what ways is stationary phase chromatography important for industrial and scientific research?
Stationary phase chromatography is essential for analyzing mixtures in pharmaceuticals (drug identification), food industry (testing additives), environmental studies (detecting pollutants), and forensic science (sample analysis). Its ability to provide precise, reliable separation makes it invaluable for quality control and research.
