Adsorption Chromatography: Principle, Types, Steps, Uses microbiologystudy

Adsorption chromatography is a type of liquid-solid chromatography or liquid chromatography in which components of a mixture are separated based on their differential interaction with a solid stationary phase.

Adsorption chromatography, first discovered by Russian Botanist Mikhail Tswett in 1903 during his research on plant pigments, is one of the oldest chromatographic techniques. The analytes compete with the molecules in the mobile phase to bind to the surface of the support, leading to separation through differential elution.

Principle of Adsorption Chromatography

In adsorption chromatography, the analytical separation occurs according to the differential interaction between the components of a mixture (adsorbates) and a solid adsorbent. Upon passing over or through the adsorbent, molecules adsorb onto the surface of the solid stationary phase through weak, non-ionic attractive forces such as Van der Waals force, hydrogen bonding, and steric interactions. The type of interaction majorly depends upon 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. The stronger the binding affinity of a molecule to the adsorbent, the longer it remains attached to the stationary phase, while molecules with weaker affinity elute first. Molecules adsorb to adsorbent in a column in decreasing order of interaction with the stationary phase, i.e., molecules with higher affinity are placed higher in the column, followed by those with weaker attraction. On passing a suitable solvent, the least adsorbed molecule at the bottom of the column elutes first, followed in series by the next components, leading to the separation of components in the mixture. As solvent (eluent) constantly passes over the adsorbent (stationary phase) in the column, differences in the compound flow rate eventually lead to the separation of the organic mixture (analytes).

Phases of Adsorption Chromatography

1. Stationary phase: The adsorbent serves as the stationary phase. The adsorbent binds the solute on its outer surface using weak physical forces. 
2. Mobile phase: The mobile phase can be either a liquid (liquid-solid chromatography) or a gas (gas-solid chromatography). Criteria such as polarity, solvent strength and interaction with the stationary phase considered while selecting an appropriate mobile phase. 

Adsorbent

An Adsorbent is a porous, solid substance with high surface area that can adsorb substances on its surface through intermolecular forces of attraction. Some commonly used adsorbents include Alumina, Silica gel (type H, G, N, S), hydrated silica gel, modified silica gel, cellulose microcrystalline, alumina, modified silica gel, etc. Typical adsorbents such as alumina or silica-based resins are rigid, smaller particles. 

Generally, three types of adsorbents are used in adsorption chromatography, which are; 

1. Polar acidic adsorbents (for example: Silica is specifically used for separation of polar basic substances)
2. Polar basic adsorbents (for example: Alumina, Florisil, etc., are used for separation of polar acidic substances)
3. Non-polar adsorbents (example: Charcoal and polystyrene)

Types of Adsorption Chromatography 

There are mainly two types of adsorption chromatography on the basis of polarity of stationary phase. 

1. Normal phase Adsorption chromatography utilizes a polar stationary phase. The polar stationary phase, based on polarity, retains polar substances making it longer for them to pass through the column. During a gradient elution (a technique where the composition of the liquid phase changes during the process), the liquid becomes more polar over time to help in efficient elution of the substances.
2. 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 mobile phase over time.

Based on type of mobile phase, adsorption chromatography can be classified into two types, 

1. Solid-liquid adsorption chromatography– Uses liquid as the mobile phase  
2. Solid- gas adsorption chromatography–  Uses gas as the mobile phase 

Forms of Adsorption Chromatography

Different forms of adsorption chromatography are as follows:

1. Thin layer chromatography: Thin-layer chromatography is a chromatographic technique where the adsorbent is a thin layer applied to a solid support, and the mobile phase is passed over the adsorbent to separate the components. The separation occurs as per the differential migration of the components as the solvent passes through the thin plate via capillary action. 
2. Paper chromatography: This technique uses paper sheets or strips as the stationary phase and liquid or solution as the mobile phase. Paper chromatography is particularly useful for the separation of dissolved chemical compounds and lipids.  
3. Column Chromatography: In this chromatographic technique, the adsorbent packed in a column serves as a stationary phase, and the solutes in the solution (mobile phase) attach to the adsorbent based on their affinity as the solution flows down the column. The separation occurs based on differential interaction of solutes with the adsorbent, resulting in elution at different rates. 
4. Gas-Solid chromatography: This type of chromatography utilizes solid adsorbent (silica or alumina) as the stationary phase and inert gas (e.g., nitrogen, helium, etc.) as the mobile phase. It is useful for solutes with low solubility in the stationary phase. The limited availability of appropriate stationary phases tend to limit the use of this technique.

Procedure or Steps of Adsorption Chromatography

Process involved in adsorption chromatography (Column):

• A clean and dry glass chromatographic column is prepared and kept vertically.
• The column is packed with the adsorbent material and carefully poured down with a glass rod to prevent air bubbles from being trapped in the column.  
• An appropriate mobile phase or solvent system is selected. 
• Once the solvent is drained from the adsorbent bed, the sample is carefully applied using pipette and is allowed to run down the column.
• The mobile phase is allowed to pass through the column to remove the sample in the adsorbent bed. Sample can be applied directly using capillary tubing or roller pumps. The density of the sample can also be increased by adding sucrose in order to avoid the drainage of the column. 
• The components of the applied sample are separated by a continuous flow of the mobile phase through the column.  
• The fraction from the column is collected sequentially into a series of test tubes manually or using fraction collector. 

Process involved in adsorption chromatography (Thin-layer):

• First, a clean and dry chromatographic jar or chamber with a lid is prepared.
• The chamber atmosphere is saturated with solvent by lining the inside of the glass chamber with some filter paper soaked in the mobile phase or solvent. 
• The mobile phase is poured into the chromatographic chamber, and is closed using a lid. 
• The solvent is carefully selected based on the sample’s polarity. Commonly used solvents include ethyl ether, ethyl acetate, acetone, benzene, hexane, dichloromethane, etc
• A glass plate coated with a uniform thickness of silica is prepared.  
• The baseline is marked by drawing a thin line about 2cm from the end of the TLC plate. 
• Samples are spotted on the baseline using a capillary tube or pipette and are dried using a dryer. 
• The spotted plate is then placed into the chamber and left for some time so that, the solvent moves through the plate. The baseline should be above the line of solvent to prevent the sample from being dissolved from the TLC plate into the solvent. 
• The TLC plate is taken out and dried. 
• An Iodine chamber or UV lamp is used to visualize TLC spot after development. 
• Once the spot is spotted, the retention factor is calculated by dividing the distance travelled by the sample from the baseline by distance travelled by the solvent front from the baseline.

Factors Affecting Adsorption Chromatography

• Nature of the Adsorbent- Adsorbents having smaller particle size, and high surface areas are preferred.  Polarity should also be taken into consideration while selecting an adsorbent. 
• Nature of Solvent- The polarity, pH, and ionic strength of the solvent can be optimized for efficient separation of the components. The higher the elutropic strength of the mobile phase, the faster the elution from the column. 
• Dimension of column- Influences separation efficiency 
• Temperature- Affects adsorption strength 
• Rate of flow- The higher the flow rate, faster the separation time.    
• Forces Involved- electrostatic interactions, hydrogen bonding, van der Waals forces, and dipole-dipole interactions , etc. also affect the degree of retention. 

Applications of Adsorption Chromatography

• Used for separation of organic compounds. 
• Used for separation of non-iconic, water-insoluble compounds such as triglycerides, vitamins, and many drugs.   
• Used in biochemical research for the separation and identification of amino acids, carbohydrates, and protein purification.
• Used for detection of hormones, metabolites, and antibiotics in biological fluids such as blood, urine, etc. 
• Used for quality control and safety evaluation in food and pharmaceutical industries. 
• Used in forensic analysis and crime investigations. 
• Used to separate phytochemicals obtained from plant extracts. 
• Used to separate and identify fats, fatty acids, lipids, and steroids.
• Used in separation of enantiomer.
• Used in detection and separation of pesticides, herbicides, and other contaminants in environmental samples.

Advantages of Adsorption Chromatography

• Variety of mobile phases and their mixture can be utilized.  
• Suitable for separation of compounds usually not separated by conventional methods. 
• Effective for separating complex mixture.
• Versatile separation technique

Limitations of Adsorption Chromatography

• Time consuming. 
• Lengthy procedure. 
• Relatively more expensive and complex compared to other methods. 
• Low reproducibility 

Trouble shooting and safety considerations 

• The stationary phase should be properly packed in the column. 
• The appropriate polarity of mobile phase should be determined. 
• The solvents should be handled carefully. 
• Gloves and eyewear should be worn in order to prevent exposure to chemicals.    
• Flammable solvents should be handled carefully.
• Safe disposal of hazardous waste. 

Recent advances and innovations of Adsorption Chromatography

Recent advancements have focused on enhancing the capabilities of adsorption chromatography by following innovations, 

• Development of highly purified adsorbents for enhanced efficiency and resolution
• Improvement in column packing techniques 
• Automation and miniaturization of chromatographic equipment
• Integration of adsorption chromatography with other techniques like spectrophotometry for characterization of separated components
• Development of eco-friendly sustainable adsorbents and solvents

Conclusion 

Adsorption chromatography is one of the most versatile and efficient technique for separating compounds in a complex mixture. It has broad applicability ranging from the food industry and pharmaceuticals to analytical chemistry and other industries.

References 

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