What is Chromatography?

• Chromatography is a vital biophysical technique for separating, identifying, and purifying the constituents of a mixture in order to perform qualitative and quantitative analysis. It is important in many scientific disciplines and is the fundamental approach for extracting specific components from complicated mixtures.
• The capacity of chromatography to use variations in qualities such as size, binding affinities, charge, and other factors to produce separation is what makes it so effective. Using these variances, chromatography may distinguish between the many components present in a mixture and produce separate results.
• The movement of a mixture through two phases, a stationary phase and a mobile phase, is one of the fundamental principles underlying chromatography. The fixed phase is usually a solid surface, whereas the mobile phase is a liquid or gas that transports the mixture throughout the system. The mixture passes over or through the stationary phase as it is put into the mobile phase.
• Certain components of the mixture have a stronger affinity for the stationary phase and become stuck to its surface or interact significantly with it throughout this process. These components have less mobility and travel slower through the system. Other components, on the other hand, have a weaker affinity for the stationary phase and are carried along at a higher rate by the mobile phase.
• As a result, the mixture gradually separates into its constituents as it passes through the chromatographic apparatus. Components with strong interactions with the stationary phase are kept, while those with lesser interactions move on to the mobile phase. This distinct behaviour results in distinct bands or peaks that correspond to the separated components.
• It is vital to remember that different chromatographic procedures exist, each with its own set of principles and uses. Liquid chromatography (LC), for example, utilises a liquid mobile phase, whereas gas chromatography (GC) uses a gaseous mobile phase. High-performance liquid chromatography (HPLC) is a more advanced form of liquid chromatography that provides improved resolution and efficiency.
• In conclusion, chromatography is a diverse and effective separation technique that is widely employed in scientific study and analysis. To achieve separation, it takes advantage of variances in characteristics among components within a mixture. Chromatography uses a stationary phase and a mobile phase to isolate and identify individual components, providing for a better understanding of complicated mixtures in domains ranging from chemistry and biology to medicines and environmental science.

Chromatography Definition

Chromatography is a technique used to separate and analyze the components of a mixture based on their properties and interactions with a stationary and mobile phase.

What is a stationary phase?

• The stationary phase in chromatography is a critical component that plays a key role in the separation of components within a mixture. It can be either a solid or liquid particle that is selectively attached to a glass or metal surface.
• The term “stationary” indicates that this phase remains fixed or immobile while the other phase, known as the mobile phase, moves through it. The stationary phase acts as a substrate onto which the components of the mixture interact and become selectively absorbed.
• In many chromatographic techniques, the stationary phase is composed of porous materials. These pores provide a large surface area for interactions between the components of the mixture and the stationary phase, enhancing the separation process.
• The choice of stationary phase depends on various factors, including the nature of the components to be separated and the specific type of chromatography being employed. Different materials can be used as the stationary phase, such as gel beads, thin uniform paper, silica, glass, certain gases, or even liquid components.
• Each type of stationary phase has its own characteristics and interactions with the mixture components, allowing for selective adsorption and separation based on specific properties like size, polarity, charge, or affinity.
• In summary, the stationary phase in chromatography is a fixed phase, often composed of porous materials, onto which the components of a mixture selectively attach. It is a crucial element in the chromatographic process and its choice depends on the properties of the components being separated and the specific chromatographic technique being employed.

What is the mobile phase?

• The mobile phase in chromatography is an essential component responsible for transporting the mixture through the chromatographic system and facilitating the separation of its components. It can be in the form of a liquid or a gas, depending on the specific chromatographic technique employed.
• The mobile phase acts as a solvent that carries the mixture along the stationary phase, allowing the components to interact with the stationary phase and separate based on their respective affinities or interactions.
• Unlike the stationary phase, the mobile phase moves through the chromatographic system. This mobility enables the components to travel at different rates and become adsorbed to the stationary phase to varying degrees, leading to their separation.
• The selection of the mobile phase depends on factors such as the nature of the components being separated and the specific type of chromatography being conducted. Different solvents or gases are chosen to optimize the separation process and achieve desired results.
• Commonly used mobile phase solvents include alcohol, water, acetic acid, acetone, and other organic solvents. These solvents are selected based on their compatibility with the stationary phase, the properties of the components being separated, and the specific requirements of the chromatographic technique being employed.
• In summary, the mobile phase in chromatography is the phase that carries the mixture through the chromatographic system, enabling the separation of its components. It can be a liquid or a gas, selected based on the properties of the components and the type of chromatography being performed. The mobile phase plays a crucial role in achieving efficient and effective separation in chromatographic processes.

Types of Chromatography

Here is the Types of Chromatography;

1. Affinity chromatography
2. Column chromatography
3. Anion exchange chromatography
4. Cation exchange chromatography
5. Flash chromatography
6. Gas chromatography
7. Gel filtration chromatography/Size exclusion chromatography
8. High-performance liquid chromatography (HPLC)
9. Hydrophobic interaction chromatography
10. Ion exchange chromatography
11. Liquid chromatography
12. Paper chromatography
13. Reverse-phase chromatography
14. Thin-layer chromatography (TLC)

Type of Chromatography	Description
Gas Chromatography (GC)	Separates volatile compounds using a gaseous mobile phase. Commonly used for analyzing volatile organic compounds (VOCs) and gases.
Liquid Chromatography (LC)	Separates compounds using a liquid mobile phase. Includes techniques such as high-performance liquid chromatography (HPLC), where high-pressure pumps are used for improved separation efficiency.
Thin-Layer Chromatography (TLC)	Involves a thin layer of stationary phase applied to a solid support. Components of a mixture interact with the mobile phase and the stationary phase to achieve separation. Commonly used for qualitative analysis and identification of compounds.
High-Performance Liquid Chromatography (HPLC)	A type of liquid chromatography that uses high-pressure pumps for improved separation efficiency and speed. Widely used for the analysis of compounds in pharmaceuticals, environmental analysis, and food science.
Ion Exchange Chromatography	Separates charged molecules based on their interactions with an ion-exchange resin. Involves the exchange of ions between the mobile phase and the resin for separation. Used for purifying proteins, separating ions, and analyzing biomolecules.
Reverse-Phase Chromatography	Relies on hydrophobic interactions between the stationary phase and analytes in the mobile phase. Suitable for separating nonpolar and moderately polar compounds. Commonly used in pharmaceutical analysis and compound purification.
Affinity Chromatography	Separates and purifies biomolecules based on specific interactions with immobilized ligands or affinity tags on the stationary phase. Used for isolating proteins, enzymes, and antibodies.
Size Exclusion Chromatography (SEC) or Gel Filtration Chromatography	Separates molecules based on their size or molecular weight using a porous stationary phase. Larger molecules elute first while smaller molecules penetrate the pores and elute later. Often employed for the purification and analysis of proteins and polymers.
Chiral Chromatography	Separates enantiomers (mirror-image isomers) based on their interaction with a chiral stationary phase. Used for the separation of chiral compounds in pharmaceutical analysis, drug development, and research.
Supercritical Fluid Chromatography (SFC)	Utilizes supercritical fluids (e.g., carbon dioxide) as the mobile phase, offering unique solubility and selectivity properties. Often employed for separating thermally labile and nonvolatile compounds.
Paper Chromatography	Involves a specialized paper as the stationary phase, with separation occurring based on differential adsorption or partitioning of components. Widely used for qualitative analysis, such as separating ink components or detecting impurities.
Hydrophobic Interaction Chromatography (HIC)	Utilizes hydrophobic interactions between the stationary phase and hydrophobic regions of analytes. Used for separating proteins and biomolecules based on their hydrophobicity.
Expanded Bed Chromatography	Involves a stationary phase composed of rigid, porous particles that can accommodate a fluidized bed of sample and mobile phase. Enables purification of biomolecules directly from crude samples without the need for extensive sample preparation.
Flash Chromatography	A preparative chromatography technique used for fast purification of compounds in larger quantities. Typically employs a column packed with silica gel or other stationary phases, allowing rapid separation and purification.

1. Affinity chromatography

Affinity chromatography is a specialized separation technique that exploits the specific interactions between components of a mixture and a stationary phase. It is based on the principle that certain components have a particular affinity or binding affinity for specific molecules immobilized on the stationary phase.

In affinity chromatography, the stationary phase is designed to contain ligands or receptors that can selectively bind to the desired components in the mixture. These ligands can be antibodies, enzymes, nucleic acids, or other molecules with high affinity for the target component.

The process starts by introducing the mixture into the chromatographic system. As the mixture flows through the system, the target components with affinity for the immobilized ligands on the stationary phase bind specifically to these ligands. This selective binding leads to the retention of the target components, while other components that do not have affinity for the ligands pass through the system more rapidly.

After the mixture has passed through the system, the bound target components can be eluted or released from the stationary phase by altering the conditions, such as pH, temperature, or the addition of competitive molecules. Elution allows for the collection and subsequent analysis or purification of the target components.

Affinity chromatography offers several advantages in terms of specificity and purity. It allows for the isolation and purification of components based on their biological interactions, ensuring high selectivity and low contamination from other components. It is commonly used in biochemical research, protein purification, drug discovery, and diagnostics, where the affinity between target molecules and specific ligands is utilized for separation and analysis.

In summary, affinity chromatography is a powerful separation technique that utilizes the specific affinity or binding interactions between components of a mixture and the immobilized ligands on the stationary phase. By selectively retaining the target components, it enables purification and analysis based on their specific binding properties, making it a valuable tool in various fields of research and biotechnology.

Principle of Affinity chromatography

The principle of affinity chromatography is based on the selective binding affinity between components of a mixture and the stationary phase. This technique utilizes a stationary phase that is functionalized with a substrate or ligand capable of specifically interacting with the target components.

The stationary phase is prepared by immobilizing the substrate or ligand onto a solid support, such as beads or a column matrix. This immobilization ensures that the reactive sites necessary for binding are exposed and accessible to the components in the mixture.

The process begins by introducing the mixture into the chromatographic system, which contains the stationary phase. As the mixture flows through the system, the components that have binding sites complementary to the substrate on the stationary phase selectively bind to it. These components are retained on the stationary phase, while the other components that do not have affinity for the substrate continue to move with the mobile phase.

The binding between the target components and the substrate immobilized on the stationary phase is specific and strong, allowing for efficient separation. The binding affinity is often based on interactions such as antigen-antibody, receptor-ligand, enzyme-substrate, or nucleic acid-protein interactions.

After the separation has occurred, the components that are attached to the stationary phase need to be eluted or released. This is achieved by modifying the conditions of the system, such as changing the pH, ionic strength, or introducing specific elution buffers. These alterations disrupt the binding interactions, allowing the bound components to be released from the stationary phase. The eluted components can then be collected and further analyzed or utilized for downstream applications.

The principle of affinity chromatography provides a powerful tool for the purification and isolation of specific components from complex mixtures. It allows for selective binding and subsequent elution, providing high purity and specificity in the separation process.

In summary, the principle of affinity chromatography is based on the selective binding between the target components and the immobilized substrate on the stationary phase. By exploiting these specific binding interactions, this technique enables the separation of components based on their affinity for the stationary phase. Elution of the bound components is achieved by modifying the system conditions, facilitating their collection and further analysis.

Steps of Affinity chromatography

The process of affinity chromatography involves several key steps for the separation and purification of components. Here are the general steps involved in affinity chromatography:

1. Column Preparation: The chromatographic column is prepared by packing a solid support material, such as agarose or cellulose, into the column. The solid support serves as the stationary phase for the chromatographic separation. The substrate or ligand, which has affinity for the target components, is attached to the solid support via a spacer arm. This immobilization ensures that the substrate or ligand is available for interaction with the components of the mixture.
2. Loading the Mobile Phase: The mobile phase, which contains the mixture to be separated, is poured into the chromatographic column at a constant rate. The mobile phase serves as the carrier for the components and allows them to flow through the column.
3. Binding of Target Components: As the mobile phase flows through the column, the target components with specific binding sites for the substrate or ligand interact with and bind to the immobilized ligand on the stationary phase. This binding is selective, and only the components with affinity for the ligand are retained on the column, while other components pass through.
4. Washing and Removing Non-Bound Components: After the target components have bound to the stationary phase, the column is washed to remove any non-specifically bound or unbound components. This step helps to increase the purity of the bound components and reduce interference from contaminants.
5. Elution: Once the bound target components are separated from the non-bound components, the elution step is performed to release the bound components from the stationary phase. This is achieved by changing the conditions of the system, such as adjusting the pH, ionic strength, or introducing specific elution buffers. The change in conditions disrupts the binding interactions between the components and the ligand, allowing the bound components to be eluted from the column.
6. Collection and Analysis: The eluted components, now separated and purified, are collected and further analyzed or utilized for downstream applications. The collected components may undergo additional characterization, quantification, or functional assays, depending on the specific goals of the chromatographic experiment.

These steps of affinity chromatography, including column preparation, loading the mobile phase, binding of target components, washing, elution, and collection, enable the separation and purification of components based on their specific affinity for the ligand or substrate immobilized on the stationary phase. The technique is widely used in various fields of research, biotechnology, and pharmaceutical development.

Uses of Affinity chromatography

Affinity chromatography finds widespread applications in various fields due to its ability to selectively separate and purify target components. Here are some common uses of affinity chromatography:

1. Protein Purification: Affinity chromatography is extensively utilized for the purification of enzymes and other proteins. By using a ligand or substrate specific to the target protein, the technique allows for the selective binding and subsequent purification of the protein of interest from complex mixtures.
2. Antibody Production: Affinity chromatography plays a crucial role in antibody production. It is used to isolate and purify antibodies from serum or other sources by exploiting the specific antigen-antibody interactions. This technique aids in obtaining highly pure antibodies for research, diagnostics, and therapeutic applications.
3. Impurity Removal: Affinity chromatography can be employed to remove impurities from a mixture. By designing the stationary phase with ligands that selectively bind to impurities or contaminants, the technique allows for their removal, leading to higher purity of the target components.
4. Mutation and Polymorphism Detection: Affinity chromatography is utilized in the detection of mutations and nucleotide polymorphisms in nucleic acids. By incorporating complementary nucleotide sequences onto the stationary phase, the technique enables the selective binding and separation of specific DNA or RNA sequences based on their sequence complementarity.
5. Drug Discovery: Affinity chromatography plays a crucial role in drug discovery and development processes. It is used to screen and identify potential drug candidates by selectively binding and separating compounds based on their interactions with specific target receptors or enzymes.
6. Biomolecule Interaction Studies: Affinity chromatography is employed in studying biomolecular interactions. By immobilizing a ligand or receptor onto the stationary phase, the technique allows for the investigation of binding affinities, kinetics, and specificity of interactions between biomolecules, such as proteins, nucleic acids, and small molecules.
7. Diagnostic Assays: Affinity chromatography is utilized in diagnostic assays, particularly in immunoassays. It enables the selective capture and detection of specific biomarkers, antigens, or antibodies for diagnostic purposes, including disease diagnosis, monitoring, and research applications.
8. Proteomics and Genomics Research: Affinity chromatography plays a crucial role in proteomics and genomics research. It allows for the isolation and enrichment of specific proteins or nucleic acids from complex biological samples, facilitating their characterization, identification, and further analysis.

In summary, affinity chromatography is a versatile technique with numerous applications. It is widely used for protein purification, antibody production, impurity removal, mutation detection, drug discovery, biomolecule interaction studies, diagnostic assays, and proteomics/genomics research. Its ability to selectively separate and purify components based on their specific interactions makes it an invaluable tool in various scientific and medical fields.

Examples of Affinity chromatography

Affinity chromatography offers a range of examples where it has been successfully applied for the separation and purification of specific components. Here are a couple of examples:

1. Purification of coli β-galactosidase: Affinity chromatography has been employed to purify coli β-galactosidase, an enzyme commonly found in Escherichia coli bacteria. In this example, p-aminophenyl-1-thio-β-D-galactopyranosyl agarose is used as the affinity matrix. The agarose is functionalized with the substrate analog, which has a high affinity for β-galactosidase. When the mixture of proteins is passed through the column containing the affinity matrix, the β-galactosidase selectively binds to the matrix while other proteins are eluted. Subsequently, the purified β-galactosidase can be eluted and collected for further analysis or application.
2. Removal of excess albumin and α2-macroglobulin from serum albumin: Affinity chromatography has been employed for the removal of unwanted components from serum albumin preparations. In this case, a specific ligand or substrate is immobilized onto the stationary phase. The ligand is selected based on its ability to bind to the unwanted proteins, such as excess albumin or α2-macroglobulin. When the serum albumin mixture is passed through the column, the ligand selectively binds to the unwanted proteins, allowing for their removal while retaining the desired serum albumin. The purified serum albumin can then be collected for various applications, such as in research or medical diagnostics.

These examples illustrate how affinity chromatography can be tailored to separate and purify specific components from complex mixtures. By designing the stationary phase with suitable ligands or substrates, the technique allows for the selective binding and subsequent separation of target components based on their specific interactions. Affinity chromatography thus offers a powerful tool in various fields, including protein purification, removal of impurities, and isolation of specific biomolecules.

2. Column chromatography

Column chromatography is a widely used separation technique that separates components of a mixture based on their differential adsorption to the stationary phase as they pass through a column. Here is an overview of the principle, steps, uses, and examples of column chromatography:

Principle of Column Chromatography: Column chromatography operates on the principle of differential adsorption, where the components in a mixture exhibit different affinities with the stationary phase. Molecules with higher affinity remain adsorbed for a longer time, resulting in slower movement through the column. On the other hand, molecules with lower affinity move at a faster rate, leading to their separation into distinct fractions. The stationary phase, typically a solid material like silica, is packed in a column, and the mobile phase, a liquid or gas, facilitates the movement of molecules through the column.

Steps of Column Chromatography:

1. Column Preparation: A glass tube is chosen and coated with a thin, uniform layer of the stationary phase, such as cellulose or silica.
2. Sample Loading: The mixture to be separated, dissolved in the appropriate mobile phase, is introduced into the column from the top. Gravity allows the sample to pass through the stationary phase.
3. Elution: The separation of molecules occurs through an elution technique. This involves passing a solution with the same polarity (isocratic technique) or using different samples with varying polarities (gradient technique) through the column. The eluent selectively carries different components, resulting in their separation.
4. Analysis: The fractions collected during the elution process can be further analyzed for various purposes, such as identification, quantification, or downstream applications.

Uses of Column Chromatography:

1. Impurity Separation and Purification: Column chromatography is commonly used for the separation and purification of impurities from biological mixtures, allowing the isolation of desired compounds.
2. Isolation of Active Molecules: This technique is valuable for isolating active molecules and metabolites from various samples, including natural products and drug discovery research.
3. Drug Detection: Column chromatography finds applications in the detection and analysis of drugs in crude extracts, contributing to forensic science and pharmaceutical research.

Examples of Column Chromatography:

1. Extraction of Pesticides: Column chromatography can be utilized to extract pesticides from solid food samples of animal origin that contain lipids, waxes, and pigments, enabling their separation and analysis.
2. Synthesis of Pramlintide: Pramlintide, an analog of the peptide hormone Amylin used for treating type 1 and type 2 diabetes, can be synthesized and purified using column chromatography.
3. Purification of Bioactive Glycolipids: Column chromatography is employed to purify bioactive glycolipids that exhibit antiviral activity against Herpes Simplex Virus Type 1 (HSV-1).

These examples illustrate the broad utility of column chromatography in various fields, including food analysis, pharmaceutical development, and the isolation of bioactive compounds. It serves as an essential tool for separation, purification, and analysis in scientific research and industrial applications.

3. Anion exchange chromatography

Anion exchange chromatography is a separation technique used to separate negatively charged molecules based on their interaction with a positively charged stationary phase in the form of an ion-exchange resin. Here is an overview of the principle, steps, uses, and examples of anion exchange chromatography:

Principle of Anion Exchange Chromatography: Anion exchange chromatography operates on the principle of electrostatic attraction between positively charged stationary phase (ion-exchange resin) and negatively charged analytes. The stationary phase is coated with positive charges, allowing for the binding of negatively charged components in the mixture. An anion exchange resin with a higher affinity for the negatively charged analytes displaces the positively charged resin. The complex formed between the anion exchange resin and the analytes is then eluted using appropriate buffers.

Steps of Anion Exchange Chromatography:

1. Stationary Phase: A column packed with a positively charged ion-exchange resin is used as the stationary phase.
2. Sample Loading: The mixture containing negatively charged molecules is introduced into the column. The negatively charged analytes bind to the positively charged resin.
3. Anion Exchange Resin: The anion exchange resin, which has a higher affinity for the negatively charged molecules, is introduced into the column. It displaces the positively charged resin and binds to the analytes.
4. Elution: An appropriate buffer is applied to the column to separate the complex formed between the anion exchange resin and the charged molecules. The elution conditions are adjusted to achieve the desired separation.

Uses of Anion Exchange Chromatography:

1. Protein and Amino Acid Separation: Anion exchange chromatography is commonly used to separate proteins and amino acids from complex mixtures.
2. Nucleic Acid Separation: Negatively charged nucleic acids can be separated using anion exchange chromatography, facilitating further analysis of the nucleic acids.
3. Water Purification: Anion exchange chromatography can be employed for water purification, where anions are exchanged for hydroxyl ions, helping to remove contaminants.
4. Metal Separation: Anion exchange resins can be utilized to separate metals, as many metal complexes carry negative charges that bind to the anion exchangers.

Examples of Anion Exchange Chromatography:

1. Separation of Nucleic Acids: Anion exchange chromatography can be applied to separate nucleic acids from a mixture obtained after cell destruction, enabling the isolation of specific nucleic acid types.
2. Protein Separation from Serum: Anion exchange chromatography can be used to separate proteins from crude mixtures obtained from blood serum, allowing for the purification and isolation of specific proteins of interest.

These examples demonstrate the versatility and utility of anion exchange chromatography in various applications, including the separation of nucleic acids, proteins, and other charged analytes.

4. Cation exchange chromatography

Cation exchange chromatography is a separation technique used to separate positively charged molecules based on their interaction with a negatively charged stationary phase in the form of an ion-exchange resin. Here is an overview of the principle, steps, uses, and examples of cation exchange chromatography:

Principle of Cation Exchange Chromatography: Cation exchange chromatography relies on the principle of electrostatic attraction between negatively charged stationary phase (ion-exchange resin) and positively charged analytes. The stationary phase is coated with negative charges, facilitating the binding of positively charged components in the mixture. A cation exchange resin with a higher affinity for the positively charged analytes displaces the negatively charged resin. The resulting complex between the cation exchange resin and the analytes is then eluted using suitable buffers.

Steps of Cation Exchange Chromatography:

1. Stationary Phase: A column packed with a negatively charged ion-exchange resin is used as the stationary phase.
2. Sample Loading: The mixture containing positively charged molecules is introduced into the column. The positively charged analytes bind to the negatively charged resin.
3. Cation Exchange Resin: The cation exchange resin, with a higher affinity for the positively charged molecules, is introduced into the column. It displaces the negatively charged resin and binds to the analytes.
4. Elution: An appropriate buffer is applied to the column to separate the complex formed between the cation exchange resin and the charged molecules. The elution conditions are adjusted to achieve the desired separation.

Uses of Cation Exchange Chromatography:

1. Nucleic Acid Hydrolysis Analysis: Cation exchange chromatography is utilized for the analysis of products obtained after the hydrolysis of nucleic acids, enabling the identification and quantification of specific components.
2. Metal Separation: Cation exchange chromatography can be employed for the separation of metal ions, as the metal ions themselves bind to the negatively charged resins, allowing for the removal of negatively charged complexes.
3. Water Purification: Cation exchange chromatography aids in water purification by exchanging positively charged ions for hydrogen ions, resulting in the removal of impurities.
4. Inorganic Molecule Analysis: Cation exchange chromatography can be used for the analysis of rocks and other inorganic molecules, providing insights into their composition and characteristics.

Examples of Cation Exchange Chromatography:

1. Separation of Lanthanoid Ions: Cation exchange chromatography can be applied to separate positively charged lanthanoid ions obtained from the Earth’s crust, aiding in their purification and analysis.
2. Determination of Total Dissolved Salts: Cation exchange chromatography can be employed to determine the presence of calcium ions, contributing to the assessment of total dissolved salts in natural waters.

These examples illustrate the diverse applications of cation exchange chromatography, ranging from the separation of specific ions and metals to the analysis of complex mixtures in various fields such as environmental science, geology, and biochemistry.

5. Flash chromatography

Flash chromatography is a separation technique that utilizes smaller gel particles as the stationary phase and pressurized gas to drive the solvent through the column. Here is an overview of the principle, steps, uses, and examples of flash chromatography:

Principle of Flash Chromatography: Flash chromatography operates on the same principle as column chromatography, where components of a mixture are separated based on their differential adsorption to the stationary phase. The sample is passed through the column using a pressurized gas, which speeds up the process. Molecules with higher affinity for the stationary phase bind, while the solvent is eluted out by applying pressurized gas, facilitating faster separation. Flash chromatography involves a solid stationary phase, liquid mobile phase, and an additional pressurized gas.

Steps of Flash Chromatography:

1. Column Preparation: A glass tube is chosen and coated with a thin, uniform layer of the stationary phase, such as cellulose or silica. Cotton wool is packed at the bottom and top of the column to prevent the gel from escaping.
2. Sample Loading: The mixture to be separated is prepared by dissolving it in the appropriate mobile phase. The sample is introduced into the column from the top, and a pump is used to pass the sample at a constant rate.
3. Elution: The separation of molecules occurs using an elution solution. This can involve using a solution of the same polarity (isocratic technique) or different samples with varying polarities (gradient technique). The elution solvent is applied with a constant minimum pressure required to move the solute down the column.
4. Analysis: The separated fractions can be further analyzed for various purposes, such as identification, quantification, or subsequent experiments.

Uses of Flash Chromatography:

1. Rapid Separation: Flash chromatography is known for its speed and efficiency in separating components of mixtures. It is particularly useful when a quick separation is required.
2. Impurity Removal: Flash chromatography is employed for the removal of impurities from crude extracts of natural and synthetic mixtures. It helps purify target compounds and isolate them for further analysis.

Examples of Flash Chromatography:

1. Separation of Natural Products: Flash chromatography is widely used in the separation and purification of natural products, such as isolating specific compounds from plant extracts or identifying active components in traditional medicines.
2. Synthetic Chemistry: Flash chromatography finds application in synthetic chemistry, where it is utilized for the purification and isolation of intermediates and final products. It aids in ensuring the purity and quality of synthesized compounds.

Flash chromatography offers a rapid and efficient method for the separation and purification of components in various mixtures. Its ability to handle larger sample volumes and provide faster results makes it a valuable tool in research laboratories, pharmaceutical development, and chemical industries.

6. Gas chromatography

Gas chromatography is a separation technique that relies on the differential retention time of molecules on a stationary phase, based on their affinity to the stationary phase. Here is an overview of the principle, steps, uses, and examples of gas chromatography:

Principle of Gas Chromatography: Gas chromatography operates on the principle that components with a higher affinity to the stationary phase will have a longer retention time, while components with a higher affinity to the mobile phase (gas) will have a shorter retention time. The sample, which can be in the form of a liquid or gas, is vaporized at the injection point. It then mixes with the mobile phase (usually helium) and is carried through the column. Components are separated based on their affinity to the stationary phase and are detected as they come out of the column at different times.

Steps of Gas Chromatography:

1. Sample Injection: The sample is injected into the column, where it is vaporized into a gaseous state. The vaporized components mix with the mobile phase (carrier gas) and are carried through the rest of the column.
2. Separation on the Stationary Phase: The column is packed with the stationary phase, which can be a solid support coated with a liquid phase or a porous solid. The molecules in the sample are separated based on their affinity to the stationary phase. Components with higher affinity to the stationary phase have a longer retention time.
3. Detection: The components eluted from the column reach the detector at different times due to variations in their retention times. The detector measures the concentration of the separated components and generates a chromatogram, which represents the peaks corresponding to each component.
4. Analysis: The separated components can be identified and quantified by comparing their retention times with known standards or by using mass spectrometry techniques. The chromatogram provides information about the composition and concentration of the sample.

Uses of Gas Chromatography:

1. Quantitative Analysis: Gas chromatography is commonly used to determine the concentration of different chemicals in various samples, such as environmental monitoring, food analysis, and pharmaceutical analysis.
2. Environmental Applications: Gas chromatography is utilized in the analysis of air pollutants, volatile organic compounds (VOCs), oil spills, and other environmental samples to identify and quantify specific compounds.
3. Forensic Science: Gas chromatography plays a vital role in forensic science, where it is employed to identify and quantify drugs, toxic substances, and biological samples in crime scene investigations and drug testing.

Examples of Gas Chromatography:

1. Detection of Performance-Enhancing Drugs: Gas chromatography is used to identify and quantify performance-enhancing drugs in athletes’ urine samples, aiding in drug testing and anti-doping efforts.
2. Analysis of Solid Drugs in Environmental Samples: Gas chromatography is utilized to separate and quantify solid drugs in soil and water samples, providing insights into drug pollution and environmental impact.

Gas chromatography is a versatile analytical technique widely used in various fields for qualitative and quantitative analysis. Its ability to separate and detect a wide range of compounds makes it valuable in research, quality control, environmental monitoring, and forensic investigations.

7. Gel filtration chromatography/Size exclusion chromatography

Gel filtration chromatography, also known as gel permeation or size-exclusion chromatography, is a technique used to separate molecules based on their molecular sizes. Here is an overview of the principle, steps, uses, and examples of gel filtration chromatography:

Principle of Gel Filtration Chromatography: Gel filtration chromatography separates molecules based on their size by utilizing a stationary phase composed of porous polymer beads with well-defined pore sizes. Smaller molecules can enter the pores and take longer to elute, while larger molecules cannot enter the pores and elute faster. The separation occurs as the molecules are partitioned between the mobile phase and the stationary phase based on their relative sizes.

Steps of Gel Filtration Chromatography:

1. Column Preparation: The column is filled with porous polymer gel beads that have a specific range of pore sizes. The gel beads serve as the stationary phase and provide a matrix for size-based separation.
2. Sample Injection: The sample, dissolved or suspended in a suitable mobile phase, is injected into the column from the top. The mobile phase occupies the pores of the gel beads.
3. Separation: As the sample passes through the column, molecules of different sizes interact with the gel beads. Smaller molecules enter the pores and are partially or completely retained, resulting in longer elution times. Larger molecules, unable to enter the pores, are not retained and elute more quickly.
4. Elution: Elution solution is used to wash the column and facilitate the elution of the separated components. Elution can be performed using an isocratic technique with a single elution solution or a gradient technique with different elution solutions of varying polarities.

Uses of Gel Filtration Chromatography:

1. Protein and Peptide Purification: Gel filtration chromatography is commonly used for the purification of proteins and peptides from various sources. It allows for gentle separation conditions that maintain the stability and activity of the molecules of interest.
2. Nucleic Acid Separation: Gel filtration chromatography has been employed to separate nucleic acids such as DNA, RNA, and tRNA, as well as their constituent bases, providing size-based separation.
3. Recovery of Biological Activity: Gel filtration chromatography offers the advantage of high recovery of activity for fragile molecules. By avoiding molecule-matrix binding, it minimizes damage to sensitive biomolecules during separation.

Examples of Gel Filtration Chromatography:

1. Separation of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF): Gel filtration chromatography has been used to separate rhG-CSF from inclusion bodies in high yield using urea-gradient size-exclusion chromatography.
2. Separation of Hen Egg Lysozyme: Both acrylamide- and dextran-based gel columns have been employed for the gel filtration chromatography separation of hen egg lysozyme.

Gel filtration chromatography is a versatile technique widely used for size-based separation and purification of biomolecules. Its gentle separation conditions, high recovery of activity, and compatibility with various biomolecules make it a valuable tool in biochemistry, molecular biology, and pharmaceutical research.

8. High-performance liquid chromatography (HPLC)

High-performance liquid chromatography (HPLC) is a powerful separation technique used to separate the components of a mixture based on their affinity with the stationary phase. Here is an overview of the principle, steps, uses, and an example of HPLC:

Principle of HPLC: HPLC is based on the principle of differential adsorption, where different molecules in a mixture interact differently with the absorbent present on the stationary phase. Molecules with higher affinity for the stationary phase remain adsorbed for a longer time, resulting in slower movement through the column. Conversely, molecules with lower affinity move faster, leading to their separation into different fractions. HPLC differs from traditional column chromatography in that the solvent is forced under high pressures, up to 400 atmospheres, instead of relying on gravity.

Steps of HPLC:

1. Column Preparation: A glass column is prepared by coating it with a thin, uniform layer of stationary phase, such as cellulose or silica.
2. Sample Preparation: The mixture to be analyzed is prepared by dissolving or suspending it in the mobile phase. The sample is introduced into the column from the top.
3. High-Pressure Pump: A high-pressure pump is used to pass the sample through the column at a constant rate. The mobile phase carries the sample components through the stationary phase.
4. Detection: As the components elute from the column, they pass through a detector that detects molecules at a specific absorbance wavelength or other physical properties.
5. Analysis: The separated molecules can be further analyzed for various purposes, such as quantification, identification, or purity determination.

Uses of HPLC:

1. Environmental Analysis: HPLC is commonly used in the analysis of pollutants and contaminants present in environmental samples, such as water, soil, and air.
2. Industrial Quality Control: HPLC is employed for maintaining product purity and quality control in various industries, including pharmaceuticals, food and beverage, and cosmetics.
3. Separation of Biological Molecules: HPLC is utilized for separating and analyzing biological molecules like proteins, nucleic acids, carbohydrates, and lipids.
4. Research and Development: HPLC is an essential tool in research and development laboratories for analyzing complex mixtures, identifying and quantifying compounds, and studying drug metabolism.

Example of HPLC: HPLC has been used to test the efficiency of different antibodies against diseases like Ebola. By analyzing the interactions between the antibodies and viral components, researchers can determine the efficacy of potential treatments.

HPLC’s high sensitivity, versatility, and ability to handle complex samples make it a widely used technique in various scientific disciplines, including pharmaceuticals, chemistry, biology, and environmental sciences.

9. Hydrophobic interaction chromatography

Hydrophobic interaction chromatography is a separation technique that separates molecules based on their degree of hydrophobicity. Here is an overview of the principle, steps, uses, and an example of hydrophobic interaction chromatography:

Principle of Hydrophobic Interaction Chromatography: Hydrophobic interaction chromatography operates on the principle of interactions between hydrophobic groups present in molecules. The stationary phase consists of a solid support with both hydrophobic and hydrophilic groups. Molecules containing hydrophobic regions interact with the hydrophobic groups on the stationary phase, resulting in their retention. On the other hand, molecules with hydrophilic groups are less strongly retained and move through the column more quickly. Elution is achieved by applying an elution solution with a decreasing salt gradient, which weakens the hydrophobic interactions and allows the hydrophobic molecules to be separated from the stationary phase.

Steps of Hydrophobic Interaction Chromatography:

1. Column Preparation: A glass column is prepared with a solid support, such as silica gel, functionalized with hydrophobic groups like phenyl, octyl, or butyl.
2. Sample Preparation: The mixture to be separated is prepared by dissolving or suspending it in the mobile phase.
3. Injection: The sample is injected into the column from the top. Hydrophobic molecules interact with the hydrophobic groups on the stationary phase, leading to their retention, while hydrophilic molecules move through the column with the mobile phase.
4. Elution: An elution solution with a decreasing salt gradient is passed through the column. The decreasing salt concentration weakens the hydrophobic interactions, allowing the hydrophobic molecules to be released from the stationary phase.
5. Analysis: The separated molecules can be further analyzed or collected for various applications.

Uses of Hydrophobic Interaction Chromatography:

1. Protein Separation: Hydrophobic interaction chromatography is commonly used for the separation of proteins based on their hydrophobic characteristics. It is particularly effective for purifying proteins with hydrophobic regions while minimizing denaturation.
2. Separation of Organic Compounds: This technique can be applied to separate other organic compounds with hydrophobic groups, such as lipids, peptides, and small molecules.
3. Biomolecule Separation: Hydrophobic interaction chromatography allows the separation of hydrophilic and hydrophobic biomolecules from each other, facilitating their purification and analysis.

Example of Hydrophobic Interaction Chromatography: Hydrophobic interaction chromatography has been used for the separation of plant proteins from crude extracts. By utilizing the hydrophobic properties of certain proteins, they can be selectively retained on the hydrophobic stationary phase while other components are washed away.

Hydrophobic interaction chromatography is a valuable technique in various fields, including biochemistry, biotechnology, pharmaceuticals, and food analysis, enabling the separation and purification of hydrophobic molecules with high efficiency and minimal denaturation.

10. Ion exchange chromatography

Ion exchange chromatography is a separation technique used for charged molecules based on their interaction with an oppositely charged stationary phase, typically in the form of an ion-exchange resin. Here is an overview of the principle, steps, uses, and examples of ion exchange chromatography:

Principle of Ion Exchange Chromatography: Ion exchange chromatography relies on the attraction between charged analytes and a charged resin in the stationary phase. The stationary phase is coated with specific charges, and components of the mixture with opposite charges bind to it. A cation or anion exchange resin, depending on the nature of the charged components, is used to selectively bind the charged analytes, displacing the oppositely charged resin. The cation or anion exchange resin-analyte complex is then eluted using appropriate buffers.

Steps of Ion Exchange Chromatography:

1. Stationary Phase: A column containing an ion-exchange resin is chosen as the stationary phase. The resin can be either positively or negatively charged.
2. Sample Application: The mixture containing charged particles is introduced into the column and flows through the resin.
3. Ion Binding: Positively charged molecules bind to negatively charged resins (anion exchange chromatography), while negatively charged molecules bind to positively charged resins (cation exchange chromatography). The oppositely charged resin is displaced by the charged components.
4. Elution: An appropriate buffer is used to selectively elute the bound analytes from the resin. The choice of buffer depends on the specific interactions and desired separation conditions.
5. Analysis and Collection: The eluted fractions containing the separated components can be further analyzed or collected for various applications.

Uses of Ion Exchange Chromatography:

1. Water Purification: Ion exchange chromatography is commonly used in water treatment processes. Positively charged ions in water can be replaced by hydrogen ions, and negatively charged ions can be replaced by hydroxyl ions, resulting in the purification of water.
2. Nucleic Acid Hydrolysis: Ion exchange chromatography is an effective method for analyzing the products formed after the hydrolysis of nucleic acids. It allows for the separation and characterization of nucleotides and other charged fragments.
3. Separation of Metals and Inorganic Compounds: Ion exchange chromatography facilitates the separation and purification of metals and other inorganic compounds based on their charge interactions with the stationary phase.

Examples of Ion Exchange Chromatography:

1. Separation of Lanthanoid Ions: Ion exchange chromatography can be used to separate positively charged lanthanoid ions obtained from the Earth’s crust. The specific charge interactions with the resin allow for their selective binding and subsequent elution.
2. Protein Separation: Ion exchange chromatography is commonly employed for the separation of proteins from crude mixtures, such as blood serum. Proteins with different charge properties interact differently with the charged resin, enabling their separation based on charge differences.

Ion exchange chromatography is a versatile technique used in various fields, including biotechnology, pharmaceuticals, environmental analysis, and water treatment. It offers a powerful tool for separating charged molecules and purifying complex mixtures.

11. Liquid chromatography

Liquid chromatography is a versatile separation technique that utilizes a liquid mobile phase to separate components either in a column or on a plain surface. Here is an overview of the principle, steps, uses, and examples of liquid chromatography:

Principle of Liquid Chromatography: Liquid chromatography operates based on the principle of affinity between the molecules and the mobile phase. Components with a higher affinity for the mobile phase will move more quickly and elute from the column or surface faster. On the other hand, components with a lower degree of interaction with the mobile phase will move more slowly and elute later. This differential affinity allows for the separation of molecules with different polarities as they move through the stationary phase at different speeds.

Steps of Liquid Chromatography:

1. Stationary Phase Preparation: A column or paper is prepared with a solid support, and the stationary phase, such as cellulose or silica, is applied to the support.
2. Sample Application: The sample to be separated is added to the liquid mobile phase, which is then injected into the chromatographic system. The mobile phase carries the sample through the stationary phase.
3. Mobile Phase Movement: The mobile phase moves through the stationary phase, allowing for the separation of components based on their affinity with the mobile phase. Components with higher affinity move more quickly, while those with lower affinity move more slowly.
4. Elution: An elution solution is applied to the chromatographic system to separate the components from the stationary phase, allowing them to be collected or further analyzed.

Uses of Liquid Chromatography:

1. Separation of Colored Solutions: Liquid chromatography is useful for separating colored solutions as they form distinct bands after separation, making it easier to identify and analyze individual components.
2. Simplicity and Cost-Effectiveness: Liquid chromatography is often preferred over other techniques due to its simplicity and lower cost. It is a widely used method in various fields of research and analysis.
3. Insoluble Solid Molecules: Liquid chromatography can be utilized for the separation of solid molecules that are insoluble in water. The mobile phase can be selected accordingly to achieve effective separation.

Examples of Liquid Chromatography: One example of liquid chromatography is high-performance liquid chromatography (HPLC), which is a modified and advanced form of liquid chromatography. HPLC is commonly employed in research involving the separation and analysis of biological molecules, such as proteins, peptides, nucleic acids, and other biomolecules.

Liquid chromatography offers a broad range of applications in analytical chemistry, pharmaceuticals, food and beverage analysis, environmental analysis, and many other fields. Its versatility, simplicity, and efficiency make it a valuable tool for separating and analyzing complex mixtures.

12. Paper chromatography

Paper chromatography is a separation technique that involves performing the separation on specialized paper. Here is an overview of the principle, steps, uses, and examples of paper chromatography:

Principle of Paper Chromatography: Paper chromatography can be classified into two types based on different principles. The first type is paper adsorption chromatography, which relies on the varying degree of interaction between the molecules and the stationary phase. Molecules with higher affinity for the stationary phase remain adsorbed for a longer time, resulting in slower movement through the paper. Molecules with lower affinity exhibit faster movement, allowing for the separation of different fractions.

The second type is paper partition chromatography, which utilizes the moisture present in the cellulose paper as the stationary phase. The separation of molecules is based on their varying degrees of adsorption onto the stationary phase. The concept of a “retention factor” is applied during paper chromatography, calculated as the ratio of the distance traveled by a molecule to the distance traveled by the mobile phase. The retention factor can be used to differentiate between different molecules.

Steps of Paper Chromatography:

1. Selection of Stationary Phase: A high-quality cellulose paper is chosen as the stationary phase.
2. Mobile Phase Preparation: Different combinations of organic and inorganic solvents are selected as the mobile phase.
3. Sample Application: A small volume (2-200 µl) of the sample solution is injected at the baseline of the paper and allowed to air dry.
4. Development: The sample-loaded paper is carefully dipped into the mobile phase, ensuring it does not exceed a height of 1 cm. The mobile phase moves through the paper, carrying the components with it.
5. Removal from Mobile Phase: Once the mobile phase reaches near the edge of the paper, it is taken out.
6. Detection and Analysis: The separated components on the paper can be visualized and analyzed using various techniques.

Uses of Paper Chromatography:

1. Purity Testing: Paper chromatography is used to determine the purity of various pharmaceutical products.
2. Contamination Detection: It can be employed to detect contamination in samples such as food and beverages.
3. Impurity Separation: Paper chromatography is useful for separating impurities from various industrial products.
4. Chemical Lab Analysis: It is commonly used for analyzing reaction mixtures in chemical laboratories.

Examples of Paper Chromatography: One common example of paper chromatography is the separation of mixtures of inks or colored drinks. By applying the principles of adsorption or partition, the different components present in the mixture can be separated and visualized as distinct bands on the paper.

Paper chromatography is a cost-effective and widely used technique for separation and analysis in various fields, including pharmaceuticals, forensics, environmental analysis, and more. Its simplicity and versatility make it a valuable tool for qualitative and quantitative analysis of complex mixtures.

13. Reverse-phase chromatography

Reverse-phase chromatography is a liquid chromatography technique that utilizes the hydrophobic interaction between the mobile phase and the stationary phase to separate molecules. Here is an overview of the principle, steps, uses, and examples of reverse-phase chromatography:

Principle of Reverse-Phase Chromatography: The principle of reverse-phase chromatography is based on the interaction between molecules with hydrophobic groups. The stationary phase consists of a solid support with hydrophobic and hydrophilic groups. Molecules containing hydrophobic regions in the solvent interact with the hydrophobic groups on the stationary phase, causing their separation from molecules with hydrophilic groups. This interaction is reversed by applying an elution solution with a decreasing salt gradient, which separates the molecules with hydrophobic groups from the stationary phase.

Steps of Reverse-Phase Chromatography:

1. Preparation of the Column: A glass tube is chosen as the column, and a solid support such as silica gel is applied with hydrophobic groups like phenyl, octyl, or butyl.
2. Sample Preparation: The sample mixture is prepared by dissolving it in a mobile phase consisting of organic and inorganic solvents.
3. Injection of Sample: The sample is injected into the top of the column.
4. Interaction with Stationary Phase: Molecules with hydrophobic groups interact with the hydrophobic groups on the stationary phase and bind to it, while molecules without hydrophobic groups move with the mobile phase and pass through the column.
5. Elution: An elution solution with a decreasing salt gradient is passed through the column to remove the bound molecules from the stationary phase.

Uses of Reverse-Phase Chromatography:

1. Biomolecule Separation: Reverse-phase chromatography, particularly in combination with high-performance liquid chromatography (HPLC), is widely used for the separation of biomolecules such as proteins, peptides, nucleic acids, and carbohydrates.
2. Drug Analysis: It is employed in the analysis of drugs, metabolites, and active molecules in pharmaceutical research and development.
3. Environmental Analysis: Reverse-phase chromatography can be used to remove impurities and analyze environmental samples for pollutants, contaminants, and other organic compounds.

Examples of Reverse-Phase Chromatography: An example of reverse-phase chromatography is hydrophobic interaction chromatography (HIC), where the technique is used to separate proteins from complex mixtures. In HIC, the stationary phase contains hydrophobic groups, and proteins with varying hydrophobicity interact differently with the stationary phase, allowing for their separation.

Reverse-phase chromatography has extensive applications in various fields, including pharmaceuticals, biotechnology, food analysis, environmental analysis, and more. Its ability to separate hydrophobic molecules makes it a powerful tool in the purification, analysis, and characterization of diverse compounds and biomolecules.

14. Thin-layer chromatography (TLC)

Thin-layer chromatography (TLC) is a separation technique that involves applying a thin layer of stationary phase on a solid support plate and using a liquid mobile phase for separation. Here is an overview of the principle, steps, uses, and examples of thin-layer chromatography:

Principle of Thin-Layer Chromatography (TLC): Thin-layer chromatography operates on the principle that components of a mixture separate based on their affinity for the stationary phase. The stationary phase is typically a substrate or ligand bound to the solid support. Components with an affinity for the substrate bind to it, while other components are eluted with the mobile phase. As the mobile phase moves through the stationary phase, the mixture components separate and appear as spots at different locations on the plate. The separated components can be detected using various techniques.

Steps of Thin-Layer Chromatography (TLC):

1. Preparation of the Plate: The stationary phase, typically a thin layer of adsorbent material such as silica gel or alumina, is uniformly applied and dried on a solid support plate, such as glass, thin plate, or aluminum foil.
2. Sample Application: The sample is applied as spots onto the stationary phase, usually about 1 cm above the edge of the plate.
3. Mobile Phase Development: The plate is carefully dipped into a suitable mobile phase, ensuring that the mobile phase does not exceed a height of 1 cm. The mobile phase moves through the stationary phase, carrying the components with it.
4. Separation and Visualization: As the mobile phase progresses, the components separate based on their affinity for the stationary phase, resulting in spots at different locations. The separated components can be visualized using various techniques such as UV light, staining, or chemical reagents.
5. Calculation of Retention Factor: The retention factor (Rf) is calculated by dividing the distance traveled by a component spot by the distance traveled by the mobile phase. It provides information about the relative mobility of the components.

Uses of Thin-Layer Chromatography (TLC):

1. Substance Identification: TLC is commonly used in laboratories to identify different substances present in a mixture. It allows for quick and efficient separation and visualization of components.
2. Forensic Analysis: TLC plays a crucial role in the analysis of fibers in forensic investigations, helping to identify and differentiate between various fibers.
3. Pharmaceutical Analysis: TLC is used in the quality control and assay of pharmaceutical products, enabling the separation and identification of active ingredients, impurities, and degradation products.
4. Medicinal Plant Analysis: TLC is utilized in the identification and characterization of medicinal plants and their composition, aiding in the determination of the presence of specific compounds.

Examples of Thin-Layer Chromatography (TLC): Thin-layer chromatography finds applications in various fields. For example, it can be used to separate and analyze the components of a dye mixture, identify drugs and their impurities in pharmaceutical formulations, or determine the presence of specific compounds in natural extracts, such as identifying different pigments in plant extracts.

Thin-layer chromatography offers a simple, cost-effective, and rapid method for qualitative and quantitative analysis in diverse scientific disciplines.

FAQ

References

• Wilson, K., Walker, J. (2018). Principles and Techniques of Biochemistry and Molecular Biology (8 eds.). Cambridge University Press: New York.
• Ó’Fágáin, C., Cummins, P. M., & O’Connor, B. F. (2017). Gel-Filtration Chromatography. Methods in molecular biology (Clifton, N.J.), 1485, 15–25. https://doi.org/10.1007/978-1-4939-6412-3_2
• https://microbenotes.com/types-of-chromatography/