Adsorption chromatography is a type of liquid-solid chromatography or liquid chromatography.
It separates components of a mixture based on their differential interaction with a solid stationary phase.
First discovered by Russian Botanist Mikhail Tswett in 1903 during his research on plant pigments.
One of the oldest chromatographic techniques.
Analytes compete with molecules in the mobile phase to bind to the surface of the support.
Separation occurs through differential elution.
Principle of Adsorption Chromatography
In adsorption chromatography, analytical separation occurs based on the differential interaction between mixture components (adsorbates) and a solid adsorbent.
Molecules adsorb onto the surface of the solid stationary phase through weak, non-ionic attractive forces, such as Van der Waals forces, hydrogen bonding, and steric interactions.
The type of interaction mainly depends on the nature of the interacting molecules.
As the mobile phase flows through the system, compounds interact differently with the stationary phase based on their polarity and binding affinity.
Molecules with stronger binding affinity to the adsorbent remain attached to the stationary phase longer, while weaker-affinity molecules elute first.
Adsorption onto the stationary phase occurs in decreasing order of interaction, with higher-affinity molecules placed higher in the column.
Upon passing a suitable solvent, the least adsorbed molecule at the bottom of the column elutes first, followed sequentially by other components.
Continuous solvent (eluent) flow over the stationary phase results in differences in compound flow rates, ultimately leading to the separation of the organic mixture (analytes).
Phases of Adsorption Chromatography
Stationary phase: The adsorbent serves as the stationary phase, binding the solute on its outer surface using weak physical forces.
Mobile phase: Can be either a liquid (liquid-solid chromatography) or a gas (gas-solid chromatography).
Selection criteria for mobile phase: Factors such as polarity, solvent strength, and interaction with the stationary phase are considered.
Adsorbent: A porous, solid substance with a high surface area that adsorbs substances through intermolecular forces of attraction.
Common adsorbents: Alumina, Silica gel (types H, G, N, S), hydrated silica gel, modified silica gel, cellulose microcrystalline, alumina, and silica-based resins.
Adsorbent characteristics: Typically rigid, with smaller particles, ensuring efficient adsorption.
Types of adsorbents in adsorption chromatography:
Polar acidic adsorbents: Example – Silica, used for separating polar basic substances.
Polar basic adsorbents: Examples – Alumina, Florisil, used for separating polar acidic substances.
Types of adsorption chromatography are based on the polarity of the stationary phase:
Normal phase adsorption chromatography:
Utilizes a polar stationary phase.
Retains polar substances, causing them to take longer to pass through the column.
Uses gradient elution, where the liquid phase becomes more polar over time for efficient elution.
Reversed phase adsorption chromatography:
Utilizes a less or non-polar stationary phase.
Non-polar substances interact strongly with the stationary phase, taking longer to pass through.
Gradient elution involves decreasing the polarity of the mobile phase over time.
Types of adsorption chromatography based on the type of mobile phase:
Solid-liquid adsorption chromatography – Uses a liquid as the mobile phase.
Solid-gas adsorption chromatography – Uses a gas as the mobile phase.
Forms of Adsorption Chromatography
Thin-layer chromatography (TLC):
Adsorbent is applied as a thin layer on a solid support.
The mobile phase passes over the adsorbent to separate components.
Separation occurs due to differential migration of components as the solvent moves through the thin plate via capillary action.
Paper chromatography:
Uses paper sheets or strips as the stationary phase.
A liquid or solution serves as the mobile phase.
Particularly useful for separating dissolved chemical compounds and lipids.
Column chromatography:
The adsorbent is packed in a column and serves as the stationary phase.
Solutes in the mobile phase interact with the adsorbent based on their affinity as the solution flows down.
Separation occurs due to the differential interaction of solutes with the adsorbent, leading to elution at different rates.
Gas-solid chromatography (GSC):
Uses a solid adsorbent (e.g., silica or alumina) as the stationary phase.
An inert gas (e.g., nitrogen, helium) acts as the mobile phase.
Useful for solutes with low solubility in the stationary phase.
Limited by the availability of suitable stationary phases.
Procedure or Steps of Adsorption Chromatography
Procedure for Adsorption Chromatography (Column Chromatography):
A clean and dry glass chromatographic column is prepared and placed vertically.
The adsorbent material is packed carefully into the column using a glass rod to prevent air bubbles.
An appropriate mobile phase or solvent system is selected.
Once the solvent is drained from the adsorbent bed, the sample is applied carefully using a pipette and allowed to run down the column.
The mobile phase is passed through the column to remove the sample from the adsorbent bed.
The sample can be applied using capillary tubing or roller pumps. Sucrose can be added to increase density and prevent drainage.
Components of the sample separate due to continuous mobile phase flow through the column.
The fractions from the column are collected sequentially into test tubes, either manually or using a fraction collector.
Procedure for Adsorption Chromatography (Thin-Layer Chromatography - TLC):
A clean and dry chromatographic jar or chamber with a lid is prepared.
The chamber atmosphere is saturated with solvent by lining the inside with filter paper soaked in the mobile phase.
The mobile phase is poured into the chromatographic chamber, and the chamber is closed with a lid.
A suitable solvent is selected based on the sample's polarity (e.g., ethyl ether, ethyl acetate, acetone, benzene, hexane, dichloromethane).
A glass plate coated with silica is prepared.
A baseline is marked about 2 cm from the end of the TLC plate.
The sample is spotted on the baseline using a capillary tube or pipette and dried with a dryer.
The spotted plate is placed into the chamber, ensuring the baseline remains above the solvent level to prevent dissolution.
The TLC plate is removed and dried after the solvent moves through it.
The spots are visualized using an iodine chamber or UV lamp after development.
The retention factor (Rf value) is calculated as:
Rf = Distance travelled by the sample/Distance travelled by the solvent front
Factors Affecting Adsorption Chromatography
1. Nature of the Adsorbent:
Adsorbents with smaller particle sizes and higher surface areas enhance separation efficiency.
The polarity of the adsorbent plays a crucial role in interaction with the analytes.
2. Nature of the Solvent:
Polarity, pH, and ionic strength of the solvent influence the separation process.
A solvent with higher elutropic strength leads to faster elution of components from the column.
3. Dimension of the Column:
The length and diameter of the column directly affect separation efficiency and resolution.
4. Temperature:
Higher temperatures can reduce adsorption strength, leading to faster elution and lower retention times.
Lower temperatures may enhance adsorption, improving separation but slowing the process.
5. Rate of Flow:
Higher flow rates result in faster separation but may decrease resolution.
Lower flow rates enhance interaction with the stationary phase, improving separation quality.
6. Forces Involved in Adsorption:
Electrostatic interactions
Hydrogen bonding
Van der Waals forces
Dipole-dipole interactions
These forces influence retention time and separation efficiency of analytes in the chromatographic process.
Applications of Adsorption Chromatography
Used to isolate and purify organic molecules based on their interaction with the stationary phase.
Efficient for the separation of non-ionic, water-insoluble compounds such as triglycerides, vitamins, and pharmaceuticals.
Used in biochemical research for the separation and identification of amino acids, carbohydrates, and proteins.
Helps detect hormones, metabolites, and antibiotics in biological fluids such as blood and urine.
Ensures product purity, safety, and standardization in food and pharmaceutical industries.
Used in forensic analysis and crime investigations for detecting chemical evidence, toxins, and drugs.
Isolates plant-derived bioactive compounds for medicinal and scientific applications.
Used for the separation and identification of fats, fatty acids, lipids, and steroids.
Helps analyze lipid profiles in food and medical industries.
Used in pharmaceutical and chemical industries to separate optical isomers of drugs.
Detects and separates pesticides, herbicides, and other contaminants from environmental samples like water and soil.
Advantages of Adsorption Chromatography
Allows the use of a wide range of mobile phases and their mixtures for optimized separation.
Suitable for separating compounds that are difficult to isolate using conventional methods.
Efficient for separating complex mixtures with diverse chemical properties.
Highly versatile and adaptable for various applications in research, industry, and analytical sciences.
Limitations of Adsorption Chromatography
Time-consuming process due to multiple steps involved.
Lengthy procedure requiring careful preparation and execution.
Relatively more expensive and complex compared to other chromatographic methods.
Low reproducibility due to variations in adsorption conditions and sample interactions.
Trouble shooting and safety considerations
Ensure the stationary phase is properly packed in the column to prevent uneven flow and poor separation.
Determine and use the appropriate polarity of the mobile phase for effective separation.
Handle solvents with care to avoid spills, contamination, and exposure.
Wear gloves and protective eyewear to prevent direct contact with chemicals.
Exercise caution when handling flammable solvents to prevent fire hazards.
Dispose of hazardous waste safely according to proper chemical disposal guidelines.
Recent advances and innovations of Adsorption Chromatography
Development of highly purified adsorbents for improved efficiency and resolution.
Advancements in column packing techniques for better separation performance.
Automation and miniaturization of chromatographic equipment for faster and more precise analysis.
Integration of adsorption chromatography with techniques like spectrophotometry for enhanced characterization.
Development of eco-friendly and sustainable adsorbents and solvents to reduce environmental impact.
Conclusion
Adsorption chromatography is a versatile and efficient technique for separating compounds in complex mixtures.
It has broad applications in industries such as pharmaceuticals, food, and analytical chemistry.
Continuous advancements, including automation, improved adsorbents, and eco-friendly innovations, enhance its efficiency and sustainability.
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