Paper Chromatography- Definition, Types, Principle, Steps, Uses
Paper chromatography is a type of planar chromatography that uses a paper as the stationary phase and a liquid solvent as the mobile phase. It is a simple, inexpensive and widely used technique for separating and identifying the components of a mixture based on their different affinities for the two phases.
Paper chromatography can be used for qualitative or quantitative analysis of organic and inorganic compounds, such as amino acids, sugars, dyes, drugs, pesticides, etc. It can also be used for testing the purity of substances, detecting adulterants or contaminants, and studying biochemical reactions.
The principle of paper chromatography is mainly partition rather than adsorption. The paper contains cellulose fibers that hold water molecules as the stationary phase. The solvent moves up the paper by capillary action, carrying the sample with it. The sample components will separate according to how strongly they bind to the water molecules versus how readily they dissolve in the solvent. The more polar a component is, the more it will stay close to the water phase and move slowly up the paper. The less polar a component is, the more it will prefer the solvent phase and move faster up the paper.
The separation of sample components in paper chromatography can be visualized by different methods, such as staining with reagents, exposing to ultraviolet light, or measuring radioactivity or fluorescence. The degree of separation can be quantified by calculating the retention factor (Rf) for each component, which is the ratio of the distance traveled by the component to the distance traveled by the solvent front. The Rf value is a characteristic property of a component under specific experimental conditions, such as the type of paper, solvent, temperature, etc.
Paper chromatography has several advantages over other chromatographic methods, such as simplicity, low cost, high resolution, and minimal sample and solvent requirements. However, it also has some limitations, such as low accuracy, sensitivity, and reproducibility compared to more advanced techniques like high-performance liquid chromatography (HPLC) or gas chromatography (GC).
Paper chromatography can be classified into different types based on the direction and mode of solvent movement:
- Ascending chromatography: The solvent moves upward against gravity. The paper is suspended vertically and the spots are applied near the bottom edge. The solvent reservoir is at the bottom of the chamber. This is the most common type of paper chromatography.
- Descending chromatography: The solvent moves downward due to gravity and capillary action. The paper is suspended vertically and the spots are applied near the top edge. The solvent reservoir is at the top of the chamber. This type of paper chromatography is faster than ascending chromatography.
- Ascending-descending chromatography: The solvent moves upward first and then downward after reaching the top of the paper. The paper is folded into a cylinder or a loop and the spots are applied near the fold. The solvent reservoir is at the bottom of the chamber. This type of paper chromatography allows longer separation distance and better resolution.
- Radial or circular chromatography: The solvent moves radially from the center to the periphery of a circular paper. The paper is laid horizontally and the spots are applied at the center. The solvent reservoir is at the center of the paper. This type of paper chromatography produces circular chromatograms that can be cut and developed further.
- Two-dimensional chromatography: The solvent moves in two perpendicular directions on a rectangular paper. The paper is laid horizontally and the spots are applied near one corner. The first solvent reservoir is at the opposite corner and the second solvent reservoir is at a right angle to the first one. This type of paper chromatography can separate complex mixtures of compounds with similar polarity.
The principle of paper chromatography is partition. In paper chromatography, the paper acts a stationary phase and organic solvent or mixture of solvents is mobile phase. The paper is made of cellulose fibers that contain water molecules in their pores, which act as a polar stationary phase. The solvent or solvents are chosen to have different polarity and solubility for the compounds to be separated.
The sample mixture is applied as a small spot on a baseline near one end of the paper. The paper is then placed in a chamber with a shallow layer of the mobile phase at the bottom, making sure that the spot is above the solvent level. The mobile phase rises up the paper by capillary action and carries the sample components with it. The different components of the sample have different affinities for the stationary and mobile phases, depending on their polarity and solubility. Therefore, they move at different rates along the paper and get separated into distinct spots.
The separation of the components depends on the balance between their solubility in the mobile phase and their adsorption on the stationary phase. The more soluble a component is in the mobile phase, the faster it moves up the paper. The more strongly a component adsorbs on the stationary phase, the slower it moves up the paper. The degree of separation can be quantified by calculating the retention factor (Rf) for each component, which is defined as the ratio of the distance travelled by the component from the baseline to the distance travelled by the solvent front from the baseline. The Rf value is a constant for a given component under fixed experimental conditions, such as type of paper, composition of solvent, temperature, etc.
Paper chromatography is based on the principle of partition between two phases, one stationary and one mobile, where different components of a mixture are separated according to their relative affinities for both phases.
Paper chromatography is a simple and inexpensive technique that does not require sophisticated equipment. The necessary equipment includes a development chamber or tank, fine quality cellulose paper, and a suitable liquid solvent or mixture of solvents.
The development chamber should be a closeable container, such as a screw-capped glass bottle or a jar, with a wire or clip to support the paper strip. The chamber should be large enough to accommodate the paper without touching the walls or the bottom. It should also have a tight-fitting lid to prevent evaporation and maintain a saturated atmosphere of solvent vapor.
The paper used as the stationary phase should be of fine quality, with defined porosity, high resolution, negligible diffusion of the sample, and favoring good rate of movement of solvent. The paper can be modified by washing with acid or base, impregnating with silica or alumina, or coating with hydrophilic or hydrophobic substances to alter its properties and suitability for different types of samples.
The mobile phase is a suitable liquid solvent or mixture of solvents that can dissolve the components of the sample and carry them along the paper. The choice of solvent depends on the nature and polarity of the sample and the desired separation. For example, hydrophilic solvents such as water, methanol, or isopropanol are used for polar compounds, while hydrophobic solvents such as kerosene, cyclohexane, or dimethyl ether are used for nonpolar compounds. The solvent should be pure and free from impurities that may interfere with the separation.
The sample should be prepared by dissolving it in a minimum amount of solvent and applying it as a small spot on the baseline of the paper using a micropipette or a capillary tube. The spot should be allowed to dry before placing the paper in the chamber. It should be positioned above the level of solvent in the chamber to avoid washing off the sample.
The detection of the separated components can be done by various methods depending on their nature and properties. Some components may be visible as colored spots on the paper, while others may require staining with reagents such as iodine vapor, ninhydrin, or fluorescein. Some components may be detected by measuring their radioactivity or fluorescence if they are labeled with radioactive or fluorescent tags. The Rf values of the components can be calculated by dividing the distance traveled by the component from the baseline by the distance traveled by the solvent front from the baseline.
In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is immersed in a solvent, and the solvent moves up the paper by capillary action. The basic steps include:
- Selection of Solid Support: Fine quality cellulose paper with defined porosity, high resolution, negligible diffusion of the sample, and favoring good rate of movement of solvent is chosen as the solid support. The paper can be modified or impregnated with different substances to enhance its properties.
- Application of Sample: The sample to be analyzed is usually dissolved in a suitable solvent and spotted onto the paper strip or sheet using a capillary tube or a micropipette. The spot should be small and concentrated to avoid spreading and overlapping. Many such spots (of different materials) may be placed side by side on the same piece of paper.
- Development of Chromatogram: The spotted paper is carefully dipped into a small amount of a suitable solvent (mobile phase) in a chromatographic chamber, in such a way that only the bottom edge of the paper is submerged in the liquid, not the spots. The chamber should be saturated with solvent vapor to prevent evaporation. The solvent then slowly rises up the paper by capillary action, reaches the spots, and begins to dissolve them and carry the substances up the paper with it. Different types of development techniques can be used, such as ascending, descending, ascending-descending, or circular.
- Visualization of Separated Components: After the development, the solvent front is marked and the paper is left to dry. The separated components are then detected by different methods depending on their nature. For example, colored components can be seen directly, while colorless components can be stained with reagents or exposed to ultraviolet light.
After the sample is applied on the paper, the paper is placed in a chamber containing a suitable solvent or a mixture of solvents as the mobile phase. The solvent moves up the paper by capillary action and carries the sample components along with it. The separation of the components depends on their relative affinity for the stationary phase (water) and the mobile phase (solvent).
There are different types of development techniques that can be used in paper chromatography, depending on the direction of the solvent flow and the shape of the paper. Some of the common techniques are:
- Ascending development: This is the conventional type of paper chromatography, where the solvent flows against gravity. The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the bottom. This technique is suitable for samples that are soluble in the solvent and have low Rf values.
- Descending development: This is carried out in a special chamber where the solvent holder is at the top. The spot is kept at the top and the solvent flows down the paper. This technique is suitable for samples that are less soluble in the solvent and have high Rf values.
- Ascending-descending development: This is a hybrid of ascending and descending techniques, where the paper is folded into a cylindrical shape and both ends are dipped in the solvent. The solvent first ascends and then descends through the paper. This technique increases the length of separation and improves the resolution.
- Radial or circular development: This technique uses a circular paper with a small hole at the center. The spot is applied at the center and a wick is inserted through the hole. The solvent flows radially from the center to the periphery of the paper. This technique allows uniform distribution of the solvent and simultaneous separation of multiple samples.
- Two-dimensional development: This technique uses a rectangular paper that is developed in two different directions using two different solvents. The paper is first developed horizontally with one solvent, dried, and then developed vertically with another solvent. This technique is useful for separating complex mixtures of compounds having similar polarity, such as amino acids.
After the development, the paper is dried and marked with the solvent front. The separated components can be detected by various methods, such as staining, fluorescence, radioactivity, or spectrophotometry. The Rf values of each component can be calculated by dividing the distance traveled by the component from the origin by the distance traveled by the solvent from the origin. The Rf values are characteristic for each component under specific conditions of solvent, temperature, and pH.
After the development of the chromatogram, the separated components need to be detected on the paper. Some components may be visible as colored spots, but others may be colorless and require special reagents or techniques to reveal them. For example, iodine vapor can be used to stain some organic compounds, ninhydrin can be used to stain amino acids, and UV light can be used to detect fluorescent compounds.
The position and distance of each component on the paper can be measured by calculating the retention factor (Rf) value. The Rf value is a ratio of the distance traveled by the component from the application point to the distance traveled by the solvent from the same point. The Rf value is a constant for a given component under the same experimental conditions, such as the type of paper, solvent, temperature, etc. The Rf value can be used to identify an unknown component by comparing it with the Rf values of known standards.
The Rf value can be calculated using the following formula:
Rf = ds / df
where ds is the distance traveled by the solute (component) and df is the distance traveled by the solvent (mobile phase).
The Rf value is usually expressed as a decimal number between 0 and 1. A higher Rf value means that the component is more soluble in the solvent and less adsorbed on the paper. A lower Rf value means that the component is less soluble in the solvent and more adsorbed on the paper.
The Rf values can be used to compare the components with known standards or reference tables. For example, if component A has an Rf value of 0.2 and a standard compound X has an Rf value of 0.2 under the same conditions, then it is likely that component A is compound X. However, Rf values alone are not conclusive evidence for identification, and other methods such as spectroscopy or mass spectrometry may be needed to confirm the identity of a component.
Paper chromatography has various applications in different fields:
- Separating colored pigments from a mixture, such as plant pigments, ink samples, food dyes, etc. This can help identify the composition and origin of the pigments.
- Monitoring chemical reactions by spotting the reactants and products on the paper at different time intervals. This can help determine the progress and completion of the reaction.
- Qualitative analysis of organic and inorganic compounds by comparing their Rf values with known standards. This can help identify unknown substances, such as drugs, pollutants, antibiotics, etc.
- Isolation and purification of components from a mixture by cutting and dissolving the separated spots on the paper. This can help obtain pure samples for further analysis or characterization.
- Pathology and forensic science by detecting biomolecules, such as amino acids, proteins, nucleic acids, etc., in biological samples, such as blood, urine, saliva, etc. This can help diagnose diseases, determine genetic profiles, or identify suspects.
Paper chromatography has many advantages over other chromatographic methods, such as simplicity, low cost, rapidity, and minimal sample requirement. However, it also has some limitations, such as low accuracy, low resolution, low sensitivity, and difficulty in quantification. Therefore, paper chromatography is mainly used as a preliminary or screening technique before applying more sophisticated methods.
Paper chromatography has both advantages and limitations that should be considered:
Advantages of Paper Chromatography
- It is a simple and inexpensive technique that does not require sophisticated equipment or specialized skills.
- It requires a minimal amount of sample and solvent, which reduces the cost and environmental impact of the analysis.
- It can identify both organic and inorganic compounds, as well as mixtures of different types of substances, such as food colorings, inks, dyes, or plant pigments.
- It can be used for qualitative and quantitative analysis, by comparing the spots with reference standards or measuring their Rf values.
- It has a high resolving power, which means it can separate closely related compounds that have similar physical and chemical properties.
- It can be performed in various modes, such as ascending, descending, circular, or two-dimensional, depending on the desired separation and resolution.
Limitations of Paper Chromatography
- It cannot handle large quantities of sample or complex mixtures that contain many components, as it may result in overlapping or indistinct spots.
- It has a long separation time compared to other chromatographic techniques, such as high-performance liquid chromatography (HPLC) or gas chromatography (GC).
- It has low accuracy and precision for quantitative analysis, as it may be affected by factors such as temperature, humidity, solvent quality, paper quality, spot size, and solvent front position.
- It cannot separate volatile compounds, as they may evaporate during the development or detection process.
- It is difficult to maintain uniformity and reproducibility in paper chromatography, as different batches of paper may have different properties or impurities that affect the separation.
- It is not suitable for scaling up or automation, as it requires large equipment and manual intervention for each step.
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