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Types of Chromatography – Classification of Chromatography

What is Chromatography?

  • Chromatography stands as a fundamental method in the realm of scientific analysis, instrumental in the separation, identification, and purification of mixture components. This technique is pivotal across various scientific fields, facilitating both qualitative and quantitative assessments of complex mixtures.
  • The essence of chromatography lies in its diverse methodologies, each tailored to exploit distinct characteristics such as molecular size, binding affinities, and electrical charge among others. This versatility renders chromatography an indispensable tool across numerous scientific disciplines, often serving as the sole viable option for dissecting complex mixtures into their constituent elements.
  • Central to the chromatographic process are two key elements: the mobile phase and the stationary phase. The mobile phase, typically a liquid or gas, carries the mixture through the stationary phase, usually a solid or viscous liquid immobilized on a solid support. The interaction between the mixture components and the phases leads to differential migration rates, thereby effectuating the separation. As the mobile phase progresses, components with lesser affinity for the stationary phase move more swiftly, whereas those with greater affinity linger, thus achieving the segregation of the mixture’s constituents based on their intrinsic properties.
  • In summary, chromatography’s broad applicability and ability to discriminate based on a spectrum of molecular characteristics make it a cornerstone technique in scientific inquiry, underpinning advancements in research and development across a myriad of scientific domains.

Definition of Chromatography

Chromatography is a laboratory technique for the separation of a mixture by passing it in a solution or suspension or as a vapor (as in gas chromatography) through a medium in which the components move at different rates.

How does Chromatography work?

  • Chromatography works based on the principle of separation of components of a mixture through differential migration as they move through two phases: a stationary phase and a mobile phase. The mobile phase consists of a fluid that carries the mixture to be separated, while the stationary phase is a solid or liquid supported on a solid that stays fixed in place.
  • When the mixture is introduced into the system, it travels with the mobile phase across the surface of the stationary phase. The separation occurs because each component of the mixture interacts differently with the stationary phase due to differences in properties like polarity, size, or affinity. Some components may adhere strongly to the stationary phase and move slowly, while others interact less and move more quickly with the mobile phase.
  • As the components proceed at different rates, they separate from each other, allowing for their individual identification and analysis. The specific conditions and materials used for the stationary and mobile phases can be varied to optimize the separation process for different kinds of mixtures, making chromatography a versatile tool for chemical analysis and purification.

Applications Of Chromatography

Chromatography is a versatile analytical technique with numerous applications across various fields due to its ability to separate, identify, and quantify components within a mixture. Some of the key applications include:

  1. Pharmaceutical Industry: Chromatography is extensively used in the pharmaceutical sector for the purification of drugs, quality control, and the analysis of pharmaceutical compounds. It helps in ensuring the purity of active pharmaceutical ingredients and in the analysis of formulations for their chemical composition.
  2. Environmental Testing: It is employed to detect and quantify pollutants in environmental samples, such as water, air, and soil. Chromatography can identify trace levels of contaminants like pesticides, heavy metals, and toxins, contributing significantly to environmental monitoring and cleanup efforts.
  3. Food and Beverage Industry: In food analysis, chromatography helps in identifying additives, preservatives, and contaminants such as aflatoxins or pesticides. It is also used to ensure the quality and authenticity of food products by analyzing flavor compounds, vitamins, and nutritional components.
  4. Research and Development: Chromatography plays a crucial role in scientific research, including the fields of chemistry, biology, and materials science. It is used for analyzing complex mixtures, studying reaction processes, and in the development of new materials or chemicals.
  5. Forensic Science: Forensic analysts use chromatography to analyze substances found at crime scenes, including drugs, poisons, and explosive residues. It is vital in the investigation of criminal cases, helping to link evidence to suspects.
  6. Clinical and Medical Research: Chromatography is used in the analysis of biological samples, such as blood and urine, for the presence of drugs, hormones, and other biomolecules. It supports diagnostics, therapeutic drug monitoring, and biomedical research.
  7. Petrochemical and Energy Industry: In the petrochemical sector, chromatography is utilized for analyzing crude oil and its fractions to determine their composition and to monitor processes. It is also important in the development of biofuels and in the quality control of gas and energy products.

What are Mobile phase and stationary phase?

Stationary phase
  • In chromatography, the separation of mixture components is achieved through the interaction between two distinct phases: the stationary phase and the mobile phase. Understanding these two elements is crucial for comprehending the fundamentals of chromatographic techniques.
  • The stationary phase is a key component in the chromatographic process, characterized by its fixed position within the system. It can be composed of either solid particles or a liquid layer that is coated onto a solid support, such as glass or metal. This phase acts as a medium for the selective adsorption or interaction of the mixture’s components. The choice of stationary phase material is influenced by the specific characteristics of the mixture being analyzed and the chromatographic method employed. Common materials for the stationary phase include silica gel, alumina, or even specialized gels and papers designed for this purpose. The porous nature of these materials plays a vital role in facilitating the separation process by providing a surface for the mixture’s components to interact with.
  • On the other hand, the mobile phase is the dynamic component of chromatography that transports the mixture through the stationary phase. It can be a liquid or a gas, depending on the chromatographic technique in use. This phase serves as a carrier, moving the mixture along the stationary phase and enabling the differential migration of its components. The selection of the mobile phase is determined by the nature of the mixture and the separation requirements, with common choices including solvents like water, alcohol, and acetone for liquid chromatography, or gases such as helium or nitrogen in gas chromatography.
  • The interplay between the mobile and stationary phases is fundamental to the chromatographic separation process. As the mobile phase flows through or past the stationary phase, it carries the mixture’s components at varying speeds. These differences in migration rates result from the varying degrees of interaction between each component and the stationary phase, leading to the effective separation and analysis of the mixture’s constituents.

Types of Chromatography

Chromatography encompasses a range of techniques designed for the separation of substances within a mixture. These methods are differentiated mainly by the interaction between the mobile and stationary phases used in the process.

  1. Affinity Chromatography: This technique leverages the specific interactions between a molecule and a partner that it binds with, such as an enzyme and its substrate or an antibody and its antigen. The stationary phase contains a ligand to which specific molecules from the mixture will bind. Unbound substances are washed away, and the bound molecules are later released under specific conditions.
  2. Ion Exchange Chromatography: This method is particularly useful for separating ions and polar molecules based on their charge. It utilizes a charged stationary phase to attract and bind compounds of opposite charge. By altering the pH or ionic strength of the mobile phase, the bound components can be eluted.
  3. Size Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography: SEC separates molecules based on their size and shape. This method uses a porous stationary phase; smaller molecules enter the pores and take longer to travel through the column, whereas larger molecules bypass the pores and elute more rapidly.
  4. Partition Chromatography: Here, separation is based on differential partitioning between the mobile and stationary phases. The stationary phase is a liquid that coats a solid support, and compounds in the mixture distribute themselves between the two phases according to their solubilities.
  5. Adsorption Chromatography (or Normal Phase Chromatography): In this form, the stationary phase is typically a solid material on which the sample components adsorb. The degree of adsorption depends on the physical or chemical properties of the analyte, such as polarity. Nonpolar solvents are used to elute the analytes from the column.
  6. Reverse Phase Chromatography (RPC): This is essentially the opposite of normal phase chromatography. The stationary phase is nonpolar, so polar compounds elute first, and nonpolar compounds have a stronger interaction and elute later.
  7. Gas Chromatography (GC): GC is used for volatile compounds. The mobile phase is a carrier gas (usually helium, nitrogen, or hydrogen), and the stationary phase is a high-boiling liquid coated inside a column. Separation is achieved based on the compounds’ boiling points and affinity for the stationary phase.
  8. High-Performance Liquid Chromatography (HPLC): This is an advanced form of column chromatography used extensively in biochemistry and analytical chemistry to separate, identify, and quantify compounds. HPLC utilizes high pressure to push the solvent through the column, allowing the use of finer particle size for the stationary phase, which provides a greater surface area for interactions between the stationary phase and molecules in the sample.
  9. Thin-Layer Chromatography (TLC): TLC involves a stationary phase immobilized on a glass or plastic plate as a thin layer. The sample is spotted near the base of the plate and is developed by moving a solvent front up the plate via capillary action, causing the compounds to move at different rates.
  10. Paper Chromatography: This method uses a strip of paper as the stationary phase, with the mobile phase moving through the paper by capillary action. Compounds within the mixture move at different rates, leading to their separation.
  11. Hydrophobic Interaction Chromatography (HIC): This technique separates molecules based on their hydrophobicity. The stationary phase is typically coated with hydrophobic groups, and the mobile phase often includes high salt concentrations to promote hydrophobic interactions. Molecules with greater hydrophobic surfaces adhere more strongly and elute later when the salt concentration is gradually reduced.
  12. Hydrophilic Interaction Liquid Chromatography (HILIC): HILIC is a variant of normal phase chromatography that’s particularly useful for separating polar compounds. In HILIC, the stationary phase is polar, and the mobile phase is a mixture of water and a less polar solvent, encouraging polar compounds to interact with the stationary phase and be retained longer.
  13. Chiral Chromatography: This form of chromatography is used to separate enantiomers, which are mirror images of each other and cannot be separated by ordinary methods due to their identical physical and chemical properties in a symmetrical environment. Chiral chromatography uses a chiral stationary phase that interacts differently with the enantiomers, leading to their separation.
  14. Supercritical Fluid Chromatography (SFC): SFC uses a supercritical fluid (a substance at a temperature and pressure above its critical point, where it displays properties of both gases and liquids) as the mobile phase. This method combines the high diffusion rates of gas chromatography with the high solvating power of liquid chromatography, making it effective for a wide range of compounds, especially those that are thermally labile or have a high molecular weight.

1. Gas chromatography

Gas chromatography
Gas chromatography | Source:
  • Gas chromatography is an analytical technique widely employed for separating and analyzing compounds that can be vaporized without decomposition. This method is particularly effective for the analysis of volatile substances and relies on the time it takes for each component to travel through the chromatography column—known as retention time—as a key differentiating factor.
  • Underlying Principle: The core principle of gas chromatography is the separation of compounds based on their relative affinities to the stationary phase within the column. Substances with greater affinity to the stationary phase will spend more time interacting with it, thus having longer retention times. Conversely, substances that prefer the mobile phase, which is a carrier gas like helium or nitrogen, will pass through the column more quickly and exhibit shorter retention times. Each substance’s passage through the column is monitored and quantified using a detector.
  • Procedure: The gas chromatography process unfolds in these primary stages:
    • Sample Introduction: The sample, either in liquid or gaseous form, is vaporized at the injection point.
    • Carrier Gas Transport: The mobile phase, which is the inert carrier gas, moves the vaporized sample through the column.
    • Separation: Inside the column, the sample’s components separate due to differing interactions with the stationary phase.
    • Detection: As the components exit the column, they pass through a detector that records their retention times.
    • Data Analysis: The detector signals are analyzed, leading to the identification and quantification of the components within the sample.
  • Applications: Gas chromatography is applied in various fields due to its high precision and capability to analyze complex mixtures:
    • Chemical Analysis: It is used to measure the purity of substances or to separate and analyze mixtures of chemical compounds.
    • Environmental Monitoring: This technique plays a crucial role in detecting pollutants in air, water, and soil.
    • Forensic Investigations: In forensics, gas chromatography can be used to identify substances present at a crime scene, including drugs or flammable liquids.
  • Practical Examples: Instances where gas chromatography is employed include:
    • Doping Control: Detecting performance-enhancing drugs in athletes’ urine samples.
    • Environmental Testing: The analysis of soil or water samples for the presence and concentration of pollutants or drugs.

2. Affinity chromatography

Affinity chromatography
Affinity chromatography
  • Affinity chromatography is a sophisticated method used to purify and separate substances within a mixture, leveraging the specific interactions between molecules. This technique is invaluable in biochemical and analytical fields, offering high specificity and efficiency in isolating desired compounds.
  • Principle of Affinity Chromatography
    • The fundamental concept behind affinity chromatography revolves around the selective interaction between a molecule of interest and a stationary phase that possesses a complementary binding partner, often referred to as a ligand. The stationary phase is designed so that only target molecules with a high affinity for the ligand are retained, while others are washed away with the mobile phase. The selective binding is reversible, allowing for the elution of the bound substances by altering the environmental conditions, such as pH or ionic strength, to weaken the interaction between the ligand and the target molecule.
  • Procedure – The process of affinity chromatography involves several key steps:
    • Column Preparation: A solid support, like agarose or cellulose, is packed into a column and modified to present the specific ligand that will selectively bind the target molecule.
    • Sample Application: The mixture containing the target molecules is introduced into the column, where it flows over the stationary phase.
    • Washing: The column is then washed with a buffer solution to remove unbound or weakly bound components.
    • Elution: Finally, the target molecule is recovered by changing the buffer conditions to disrupt the interaction between the ligand and the target molecule, allowing it to be eluted from the column.
  • Applications: Affinity chromatography has a wide array of applications, particularly in the purification of biological molecules:
    • Protein Purification: It is extensively used to purify enzymes and other proteins with high specificity.
    • Antibody Isolation: The technique is crucial for isolating antibodies from serum or other sources, exploiting the specific antigen-antibody interactions.
    • Biomedical Research: It aids in studying molecular interactions and is employed in diagnostics for identifying mutations and polymorphisms in nucleic acids.
    • Pharmaceutical Industry: Affinity chromatography is instrumental in the purification of biologically active compounds for therapeutic use.
  • Examples
    • The purification of the enzyme β-galactosidase from a complex protein mixture using an affinity matrix specifically designed to bind to this enzyme.
    • The selective removal of certain proteins like albumin and α2-macroglobulin from serum samples, enhancing the purity of therapeutic proteins.

3. Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography

Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography
Gel filtration chromatography/ Gel permeation chromatography/ Size exclusion chromatography/ Molecular sieve chromatography | Source:
  • Gel filtration chromatography, known by several other terms such as gel permeation, size exclusion, or molecular sieve chromatography, is a chromatographic method that separates molecules based on size. This technique is frequently employed in biochemistry and polymer science to sort molecules according to their molecular weight.
  • Principle: The underlying principle of gel filtration chromatography is the separation of molecules as they pass through a column packed with porous beads. The beads create a matrix with pores of defined sizes that discriminate molecules based on their ability to enter these pores. Larger molecules, being excluded from the pores, travel more rapidly through the matrix and elute first. Smaller molecules, which penetrate the pores, have a longer path and therefore elute later.
  • Process: The process of gel filtration chromatography unfolds in distinct steps:
    • Column Packing: A chromatographic column is packed with a gel matrix of porous polymer beads. The pore size of the beads is selected based on the range of molecular sizes expected in the sample.
    • Sample Introduction: The sample mixture is introduced at the top of the column along with the mobile phase.
    • Elution: As the sample travels through the column, molecules are separated based on their size. The mobile phase may be a simple buffer or an organic solvent, depending on the nature of the sample.
    • Collection: Fractions are collected as they exit the column, with the assumption that each fraction contains molecules of similar size.
  • Applications: Gel filtration chromatography is advantageous for several reasons:
    • Maintaining Molecular Integrity: It allows for the separation of molecules under gentle conditions that preserve their structural integrity and biological activity.
    • High Recovery Rates: Since there is minimal interaction with the stationary phase, delicate molecules are less likely to be damaged, leading to high recovery rates.
    • Versatility: The technique is widely used for purifying proteins, peptides, nucleic acids, and even for analyzing the molecular weight of polymers.
  • Practical Examples: Real-world applications of gel filtration chromatography are diverse:
    • Biotechnology: It has been employed to purify recombinant proteins, such as human granulocyte colony-stimulating factor, from complex mixtures like inclusion bodies.
    • Biochemical Analysis: Researchers have used this method to separate and study proteins like hen egg lysozyme, taking advantage of different gel matrices to achieve optimal resolution.

4. Ion exchange chromatography

ion exchange chromatography
ion exchange chromatography
  • Ion exchange chromatography is a method widely used in biochemistry and analytical chemistry for separating charged particles, such as proteins, amino acids, or nucleic acids, based on their charge.
  • Principle of Ion Exchange Chromatography: The core principle of ion exchange chromatography is the reversible adsorption of charged molecules to a stationary phase that carries an opposite charge. Molecules in a mixture are differentiated by how strongly they are attracted to the ion exchange resin in the stationary phase. Those with a greater affinity will bind more strongly and will be eluted later when the conditions, such as ionic strength or pH of the mobile phase, are altered.
  • Steps of Ion Exchange Chromatography:
    • Column Preparation: A column is packed with an ion exchange resin, which may carry either a positive (anion exchange) or negative (cation exchange) charge.
    • Sample Introduction: The sample mixture is introduced into the column. Charged molecules will interact with the oppositely charged resin.
    • Binding: Molecules with charges opposite to the resin’s charges will bind to the resin.
    • Elution: Bound molecules are then eluted with buffers of varying ionic strength or pH. This step may involve a gradient to progressively displace bound molecules.
    • Collection: The eluted fractions are collected and can be analyzed further.
    • Applications of Ion Exchange Chromatography
    • Water Purification: It is used for demineralizing water, exchanging calcium and magnesium ions for hydrogen and hydroxyl ions to soften water.
    • Biochemical Analysis: Ion exchange chromatography is essential for analyzing the by-products of nucleic acid hydrolysis and for separating proteins and other biomolecules.
    • Industrial Processing: In metallurgy, it assists in the purification and separation of metal ions.
  • Examples of Ion Exchange Chromatography
    • Environmental Science: The technique is employed for isolating lanthanoid ions from geological samples.
    • Biotechnology: Proteins can be separated from complex mixtures, such as those derived from blood serum, with high specificity.

5. Anion exchange chromatography

Anion exchange chromatography
Anion exchange chromatography
  • Anion exchange chromatography stands as a pivotal technique in the realm of biochemical and molecular analysis, particularly for isolating molecules that bear a negative charge. This method employs a stationary phase imbued with positive charges, typically in the form of an ion-exchange resin, to selectively attract and bind negatively charged species from a mixture.
  • Fundamental Principle: The underlying principle of anion exchange chromatography hinges on the electrostatic attraction between negatively charged analytes and a positively charged stationary phase. This attraction facilitates the selective binding of negatively charged molecules to the stationary phase, allowing for their separation from the rest of the mixture. The process involves an ion exchange mechanism, where positively charged ions on the resin are displaced by the negatively charged molecules from the sample, leading to their temporary immobilization on the resin.
  • Operational Steps: The operational sequence of anion exchange chromatography can be delineated into several key steps:
    • Preparation of the Stationary Phase: A chromatography column is packed with a positively charged resin, which serves as the stationary phase.
    • Sample Introduction: The mixture containing negatively charged molecules is introduced and passed through the column. These molecules interact with and bind to the positively charged stationary phase.
    • Washing and Elution: Subsequently, the column is washed with a buffer solution. This step may involve the use of buffers with varying pH or ionic strength to selectively elute bound molecules based on their differential affinities to the resin.
    • Collection of Eluates: The molecules that elute from the column are collected in fractions and can be further analyzed or purified as needed.
  • Applications: Anion exchange chromatography finds utility in a broad spectrum of applications, owing to its efficacy in separating negatively charged biological molecules:
    • Protein and Amino Acid Purification: This method is adept at isolating proteins and amino acids, which often carry negative charges under physiological pH conditions.
    • Nucleic Acid Isolation: The technique is invaluable for the separation and purification of negatively charged nucleic acids, facilitating downstream genetic analysis.
    • Water Purification: In environmental applications, anion exchange chromatography is employed for the removal of anions from water, contributing to water purification processes.
    • Metal Separation: The method also extends to the separation of metals, particularly those that form negatively charged complexes, further underscoring its versatility.
  • Examples: The practical application of anion exchange chromatography is best illustrated through specific examples:
    • Nucleic Acid Purification: Following cellular lysis, the method can be used to separate nucleic acids from the resultant mixture, aiding in genetic analysis.
    • Protein Isolation: Proteins can be isolated from complex mixtures, such as blood serum, through this chromatography technique, enabling further biochemical characterization.

6. Cation exchange chromatography

  • Cation exchange chromatography is a widely utilized technique in analytical chemistry, providing an efficient approach to separating positively charged molecules—cations—based on their interaction with a negatively charged stationary phase.
  • Fundamental Principle: Cation exchange chromatography operates on the principle that cations in a mixture are attracted to a negatively charged stationary phase, typically a resin. The resin’s negative charge is poised to bind with positively charged particles in a solution. Once bound, the cations can be selectively released or eluted by altering the conditions of the mobile phase, typically by varying the pH or ionic strength.
  • Operational Procedure: The procedure for cation exchange chromatography can be broken down into a series of systematic steps:
    • Column Preparation: A column is prepared by packing it with a resin that carries a negative charge, creating the stationary phase.
    • Sample Introduction: The sample mixture, containing the cations, is introduced into the column. The positively charged cations are attracted to and bind with the negatively charged resin.
    • Washing: To remove unbound or loosely attached molecules, the column is washed with a buffer solution.
    • Elution: Bound cations are then eluted by altering the buffer conditions to disrupt their interaction with the resin, allowing for their collection.
  • Practical Applications: Cation exchange chromatography is a versatile technique with applications in various sectors:
    • Biochemical Analysis: It is instrumental in the purification of cations such as amino acids, peptides, and even entire proteins.
    • Water Purification: The method is used for softening hard water by removing calcium and magnesium ions, which are responsible for water hardness.
    • Metal Separation: It is essential in geochemical analysis for isolating specific metal ions from complex geological samples.
  • Real-world Examples: This chromatography technique is exemplified in scenarios such as:
    • Nucleic Acid Processing: Post-hydrolysis, cation exchange chromatography can be employed to analyze the resultant nucleic acid products.
    • Geochemical Exploration: The separation of metal ions, especially lanthanides, from earth samples is facilitated by this method.
    • Environmental Monitoring: Determining the total dissolved salts in water bodies involves analyzing cations such as calcium, which cation exchange chromatography can efficiently achieve.

7. Paper Chromatography

Paper Chromatography
Paper Chromatography| Source:
  • Paper chromatography is a simple yet effective technique for separating and identifying compounds within a mixture, utilizing specialized paper as the stationary phase.
  • Principle of Paper Chromatography: Paper chromatography operates on two main principles, depending on the type of analysis:
    • Adsorption Chromatography: This type relies on the different degrees of adsorption of various molecules to the cellulose paper. Molecules with greater affinity to the paper will move more slowly, while those with lesser affinity will travel faster along the paper strip.
    • Partition Chromatography: Here, the moisture inherent in the cellulose paper serves as the stationary phase, with the separation of molecules occurring based on their differential solubility in the mobile phase and the stationary water phase.
    • A key concept in paper chromatography is the retention factor (Rf), which quantifies the migration of a substance relative to the solvent front. The Rf value is a ratio of the distance traveled by the substance to the distance traveled by the solvent, offering a way to identify different substances.
  • Steps of Paper Chromatography: The process involves several systematic steps:
    • Preparation: A strip of high-quality cellulose paper is chosen as the stationary phase.
    • Application of the Sample: A small volume of the sample is applied near the base of the paper and allowed to dry.
    • Development: The paper is then placed in a container with a suitable solvent, ensuring the sample spot is above the solvent level. As the solvent ascends the paper by capillary action, it carries the sample compounds with it.
    • Drying and Analysis: After the solvent front approaches the top of the paper, the strip is removed and dried. The separated components can then be visualized and analyzed, often using the Rf value for identification.
  • Applications of Paper Chromatography: This method is versatile, with applications spanning various fields:
    • Pharmaceuticals: Used for assessing the purity of drugs and identifying their components.
    • Food and Beverages: Employed to detect contaminants or analyze the composition of food products.
    • Industrial Products: Helpful in identifying impurities in chemicals and other manufactured goods.
    • Chemical Analysis: Utilized in laboratories for analyzing reaction mixtures and tracking the progress of chemical reactions.
  • Examples in Practice: A common application of paper chromatography is in the separation and identification of colored substances, such as inks or dyes in drinks, allowing for a visual representation of the components within a mixture.

8. Flash chromatography

Flash chromatography
Flash chromatography | Source:
  • Flash chromatography is a streamlined and rapid technique in the chromatographic family, designed for the swift separation of components within a mixture. It uses smaller particles in the stationary phase and applies pressurized gas to expedite the movement of the solvent through the column.
  • Fundamental Principle: Flash chromatography is grounded in the principle of differential adsorption, akin to traditional column chromatography. It is the differing affinity of each component in the mixture for the stationary phase that allows for separation. With the aid of pressurized gas, the process is significantly accelerated, resulting in a quicker separation.
  • Operational Steps: The execution of flash chromatography involves these critical steps:
    • Column Setup: A glass column is packed with a finely divided stationary phase such as silica gel, bracketed by cotton wool at both ends to secure the media.
    • Sample Preparation and Introduction: The sample is dissolved in a mobile phase and introduced at the top of the column.
    • Elution: A solvent system, selected based on the properties of the sample components, is used to elute the sample through the column. Pressurized gas is applied to maintain a steady flow rate.
    • Collection: Fractions containing separated components are collected as they elute from the column’s bottom.
  • Applications: The use of flash chromatography spans several practical applications:
    • Purification: It is extensively used for the rapid purification of chemical substances, including both natural and synthetic compounds.
    • Impurity Removal: In both research and industrial settings, flash chromatography helps in the efficient removal of impurities from crude extracts.
  • Advantages: The primary benefits of flash chromatography include:
    • Speed: As the name implies, it is a much faster method compared to traditional gravity-fed chromatography.
    • Efficiency: The use of pressurized gas enables a more efficient separation process, often resulting in cleaner separations.

9. Liquid chromatography

Liquid chromatography
Liquid chromatography | Source:
  • Liquid chromatography is a fundamental analytical technique used for separating components within a mixture through a liquid mobile phase.
  • Principle of Liquid Chromatography: The foundation of liquid chromatography rests on the differential affinity of molecules for a mobile phase and a stationary phase. Molecules with a higher affinity for the mobile phase elute more quickly, while those with greater affinity for the stationary phase move more slowly. The interaction between the phases leads to the separation of molecules based on differences in polarity, charge, or other chemical properties.
  • Process of Liquid Chromatography: The following steps outline the typical liquid chromatography procedure:
    • Preparation: A solid support, such as cellulose or silica, is prepared as the stationary phase either in a column or on a plane surface.
    • Sample Introduction: The sample is dissolved in the mobile phase and introduced into the chromatography system.
    • Separation: As the mobile phase passes through the stationary phase, different components of the sample move at varying rates depending on their interaction with the mobile phase.
    • Elution: A solvent or mixture of solvents is used to facilitate the separation of the components from the stationary phase.
    • Detection and Collection: The separated components are detected, often by UV-vis spectrophotometry, and collected for further analysis.
  • Applications of Liquid Chromatography: Liquid chromatography is versatile and can be adapted for a wide range of applications:
    • Complex Mixtures: It’s used to separate and analyze complex solutions, particularly when the components have different colors or absorbance properties.
    • Cost-Effective Analysis: It provides a relatively simple and economical approach to separation science.
    • Non-Aqueous Systems: It can be employed to separate substances that are not water-soluble.
  • Examples in Practice:

10. Column chromatography

Column chromatography
Column chromatography | Source:
  • Column chromatography is a foundational technique in chemical analysis and purification, which distinguishes compounds based on their differential adsorption properties. Utilizing a solid stationary phase and a fluid mobile phase, this method is an indispensable tool in various scientific fields.
  • Core Principle: The method is grounded in the principle of differential adsorption. Molecules within a mixture interact differently with the stationary phase, a solid material typically made of silica or alumina. Some molecules cling more tenaciously to the stationary phase, traversing the column at a slower rate, while others, with weaker interactions, move more rapidly. This variance allows the separation of the mixture into discrete bands or fractions.
  • Process Steps: Implementing column chromatography involves a series of carefully conducted steps:
    • Column Preparation: A glass column is packed with a solid stationary phase, ensuring even distribution.
    • Introduction of the Sample: The mixture, dissolved in a suitable solvent, is applied atop the prepared column.
    • Elution: The mobile phase is passed through the column, either by gravity or external pressure, facilitating the movement of the sample through the stationary phase.
    • Separation and Collection: Different components of the mixture are eluted at varied rates and collected as distinct fractions.
  • Applications: Column chromatography is utilized across a spectrum of applications:
    • Purification: It’s a go-to method for purifying chemical substances, from small organic molecules to large biomolecules.
    • Isolation: Active ingredients in pharmaceuticals and metabolites in biological samples are often isolated using this technique.
    • Analysis: Detecting and quantifying drugs or pesticides in complex mixtures is another area where column chromatography is extensively employed.
  • Practical Examples: Real-world applications of column chromatography include:
    • Food Safety: It’s used to extract and purify pesticides from food samples, ensuring the safety of the food supply.
    • Medical Therapeutics: The synthesis of medical peptides like Pramlintide, used in diabetes treatment, involves purification steps that rely on this method.
    • Antiviral Research: The isolation of bioactive compounds, such as glycolipids with antiviral properties, exemplifies its role in biomedical research.

11. High-performance liquid chromatography (HPLC)

High performance liquid chromatography
High performance liquid chromatography | Source:
  • High-performance liquid chromatography (HPLC) is an advanced analytical technique derived from traditional column chromatography. It is designed to improve upon the efficiency and resolution of separation processes by utilizing high-pressure conditions.
  • Core Principle: The principle of HPLC lies in the selective adsorption of components in a mixture to a stationary phase while being propelled by a liquid mobile phase. Molecules interact differently with the stationary phase based on their chemical properties. Those with a higher affinity to the stationary phase will adhere longer and move slowly through the column, while those with less affinity will pass through more quickly. This differential retention leads to the separation of the mixture into distinct fractions.
  • Process of HPLC: The steps involved in HPLC are as follows:
    • Column Preparation: A high-quality column is packed with fine particles of the stationary phase, typically silica or polymer-based, designed to offer a large surface area for interaction.
    • Sample Introduction: The sample is dissolved in the mobile phase and introduced into the column using an injector.
    • High-Pressure Elution: A high-pressure pump propels the mobile phase and the dissolved sample through the column.
    • Detection: As each component elutes from the column, it is detected, often by absorbance at specific wavelengths.
    • Analysis: The resulting chromatogram is analyzed to determine the retention times and concentrations of the components.
  • Applications: HPLC has a wide array of applications across various fields:
    • Environmental Analysis: It is utilized to detect and quantify pollutants in water, soil, and air samples.
    • Industrial Quality Control: HPLC plays a critical role in ensuring the purity of pharmaceuticals, food products, and other manufactured goods.
    • Biological Research: The technique is pivotal in the separation and characterization of complex biological samples, including proteins and nucleic acids.
  • Speed and Effectiveness: One of the significant advantages of HPLC is its speed, made possible by the high-pressure system, which accelerates the separation process, and its effectiveness in separating and quantifying mixtures with high precision and resolution.
  • Example of Use: An instance of HPLC application is in the pharmaceutical industry, where it has been employed to assess the potency and purity of antibodies developed for treating diseases, exemplified by the evaluation of therapeutic candidates for conditions like Ebola.

12. Hydrophobic interaction chromatography

Hydrophobic interaction chromatography
Hydrophobic interaction chromatography| Source:
  • Hydrophobic interaction chromatography (HIC) is a chromatographic method that distinguishes molecules based on their hydrophobicity—their aversion to water.
  • Principle of Hydrophobic Interaction Chromatography: HIC operates on the premise that hydrophobic molecules will have a tendency to associate with similarly hydrophobic entities. In this context, the stationary phase within the chromatography column is coated with hydrophobic groups, which interact with the hydrophobic regions of the molecules in a sample. The strength of these interactions depends on the degree of hydrophobicity of the molecules. Elution is achieved by using a solvent with a decreasing salt gradient, which weakens the hydrophobic interactions and releases the molecules from the stationary phase.
  • Chromatographic Process: The process of HIC involves several structured steps:
  • Applications: HIC is particularly significant for its gentle handling of biological molecules:
    • Protein Separation: It is highly effective for separating proteins, especially those with hydrophobic surfaces, without causing denaturation.
    • Organic Compound Isolation: HIC is also suitable for isolating organic molecules that exhibit hydrophobic characteristics.
  • Advantages: HIC is noted for its ability to separate molecules under conditions that are close to their native state, thus minimizing the risk of denaturation or loss of biological activity. It’s an especially beneficial technique when working with sensitive biomolecules.
  • Example of Use: An application of HIC is seen in the separation of plant proteins from crude extracts, where it enables the isolation of proteins based on hydrophobicity while preserving their functional integrity.
  • Column Preparation: A column is packed with a stationary phase that includes a solid support like silica gel, modified to present hydrophobic groups such as phenyl or alkyl chains.
  • Sample Introduction: A sample solution is prepared and introduced into the column. The hydrophobic components in the sample interact with the hydrophobic stationary phase.
  • Elution: Molecules are eluted by introducing a solvent with a decreasing salt gradient, which diminishes the hydrophobic interactions, allowing the molecules to be washed out of the column.
  • Collection: Fractions are collected as they elute from the column, with each fraction containing molecules of similar hydrophobic properties.

13. Reverse-phase chromatography

Reverse-phase chromatography
Reverse-phase chromatography | Source:
  • Reverse-phase chromatography is a variant of liquid chromatography that employs a nonpolar stationary phase and a polar mobile phase, making it well-suited for the separation of hydrophobic molecules.
  • Principle of Reverse-Phase Chromatography: This technique is predicated on the hydrophobic interactions between the analyte molecules and the stationary phase. In reverse-phase chromatography, the stationary phase is coated with hydrophobic groups (such as phenyl, octyl, or butyl groups), which interact with the hydrophobic regions of the molecules in the sample. Molecules with greater hydrophobicity tend to have stronger interactions with the stationary phase and therefore elute more slowly than their hydrophilic counterparts. The separation process can be modulated by changing the polarity of the mobile phase, often through a gradient of organic solvents.
  • Steps of Reverse-Phase Chromatography
    • Column Preparation: A column is packed with a silica-based stationary phase modified with hydrophobic groups.
    • Sample Introduction: The sample dissolved in the mobile phase is injected into the column.
    • Elution: As the mobile phase passes through the column, molecules interact with the stationary phase to varying degrees based on their hydrophobicity. A gradient of solvents is often used to facilitate the elution of tightly bound molecules.
    • Detection: The eluted molecules are detected, typically using UV absorption, fluorescence, or mass spectrometry.
    • Analysis: The retention times and peak areas are analyzed to identify and quantify the components of the sample.
  • Uses of Reverse-Phase Chromatography: Reverse-phase chromatography is particularly useful in the analysis of:
    • Biomolecules: It is widely used for the separation and purification of peptides, proteins, and other biomolecules.
    • Pharmaceuticals: The technique is essential for the analysis and quality control of drugs and their metabolites.
    • Environmental Samples: It can be employed to detect and quantify pollutants in environmental matrices.
  • Examples: A practical application of reverse-phase chromatography is in the field of biochemistry, where it is used for the purification of proteins. By exploiting the hydrophobic interactions between the proteins and the stationary phase, reverse-phase chromatography can effectively separate proteins from complex mixtures, aiding in the study of their function and structure.

14. TLC, or thin-layer chromatography (TLC)

Procedure of Thin Layer Chromatography (TLC)
Procedure of Thin Layer Chromatography (TLC)
  • Thin-layer chromatography (TLC) is a widely used analytical technique in chemistry for separating the components of a mixture. It involves a stationary phase affixed to a solid support and a liquid mobile phase to facilitate the movement of the components
  • Principle of Thin-layer Chromatography (TLC): TLC operates on the principle of selective adsorption. The components in a mixture are separated based on their affinity towards the stationary phase. Those with a higher affinity bind more strongly to the stationary phase and move more slowly, whereas components with a lesser affinity to the stationary phase are carried along more swiftly by the mobile phase. This differential migration results in the separation of components as distinct spots along the TLC plate.
  • Steps of Thin-layer Chromatography (TLC): The TLC process involves several key steps:
    • Preparation: A thin layer of stationary phase, typically silica gel or alumina, is spread over a solid support like a glass plate, plastic, or aluminum foil.
    • Application: The mixture to be analyzed is applied as small spots near the bottom of the plate.
    • Development: The bottom of the plate is placed in a shallow pool of solvent, the mobile phase, which then ascends the plate by capillary action, carrying the sample mixture with it.
    • Detection: Once the solvent has reached a predetermined height, the plate is removed, dried, and the separated components are visualized, often using UV light or chemical stains.
    • Analysis: The retention factor (Rf) of each spot is calculated by measuring the distance traveled by the substance relative to the solvent front. This factor helps in identifying the components.
  • Uses of Thin-layer Chromatography (TLC): TLC is a versatile technique with a broad range of applications:
    • Substance Identification: It is used to identify components within a mixture by comparing their Rf values to those of known substances.
    • Forensic Analysis: In forensic science, TLC can analyze fibers and other materials found at crime scenes.
    • Pharmaceutical Analysis: It is employed to assay the composition of pharmaceutical products, ensuring their purity and detecting any impurities.
    • Herbal and Medicinal Plant Analysis: TLC is useful in identifying the compounds present in medicinal plants, contributing to the understanding of their therapeutic properties.


  1. Wilson, K., Walker, J. (2018). Principles and Techniques of Biochemistry and Molecular Biology (8 eds.). Cambridge University Press: New York.

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