Chromatography- Definition, Principle, Types, Applications

Chromatography is a powerful and versatile technique that allows the separation, identification, and purification of different components of a mixture. It is based on the principle of differential distribution of the components between a moving fluid phase (the mobile phase) and a stationary phase that is either solid or liquid. The components that have a higher affinity for the stationary phase will move slower than those that have a lower affinity, resulting in their separation along the chromatographic system.

The term chromatography was coined by the Russian botanist Mikhail Tswett in 1906, who used it to separate plant pigments using a column of calcium carbonate. Since then, chromatography has evolved into a diverse family of methods that can separate molecules based on various physical and chemical properties, such as size, shape, charge, polarity, hydrophobicity, and affinity. Chromatography can be performed in different modes, such as liquid or gas chromatography, and with different configurations, such as column, thin layer, or paper chromatography.

Chromatography has numerous applications in biological and chemical fields. It can be used for qualitative and quantitative analysis of complex mixtures, such as environmental samples, pharmaceutical products, food additives, biochemical reactions, and metabolic pathways. It can also be used for isolation and purification of target molecules, such as proteins, nucleic acids, hormones, and drugs. Chromatography is often the only means of separating components from complex mixtures that cannot be separated by other methods.

In this article, we will discuss the history of chromatography, the principle of chromatography, the components of chromatographic techniques, the types of chromatography, the commonly employed chromatographic techniques, and the applications of chromatography in various industries.

Chromatography is a technique that was first developed by the Russian botanist Mikhail Tswett in 1906. He used it to separate the pigments of plant extracts by passing them through a column of calcium carbonate. He coined the term chromatography from the Greek words chroma (color) and graphein (to write), because he observed the colored bands on the column. Tswett`s work was largely ignored until the 1930s, when Richard Willstätter and his students applied chromatography to study chlorophyll and carotenoids. They also improved the technique by using different adsorbents and solvents.

In the 1940s and 1950s, chromatography was further developed by several scientists who introduced new methods and principles. For example, Archer Martin and Richard Synge invented partition chromatography, which uses a liquid stationary phase and a liquid mobile phase. They won the Nobel Prize in Chemistry in 1952 for this discovery. Martin also collaborated with Anthony James to develop gas chromatography, which uses a gas as the mobile phase and a solid or liquid as the stationary phase. Gas chromatography became widely used for the analysis of volatile organic compounds.

Another important milestone in chromatography was the introduction of high-performance liquid chromatography (HPLC) by Csaba Horváth and his colleagues in the 1960s and 1970s. HPLC uses high-pressure pumps to force the mobile phase through a column packed with small particles of the stationary phase. This allows for faster and more efficient separation of complex mixtures. HPLC is now one of the most widely used chromatographic techniques in various fields.

Chromatography has also been combined with other analytical techniques, such as mass spectrometry (MS), nuclear magnetic resonance (NMR), and infrared (IR) spectroscopy, to provide more information about the separated components. These hyphenated techniques have enabled the identification and quantification of trace substances in biological, environmental, pharmaceutical, and forensic samples.

Chromatography is still evolving as new methods and applications are being developed. Some of the recent advances include multidimensional chromatography, which uses two or more columns with different separation mechanisms; nano-chromatography, which uses nanoscale materials and devices for separation; and biochromatography, which uses biological molecules or cells as the stationary phase.

Chromatography is based on the principle of separation of compounds into different bands or peaks and the identification of those bands or peaks. The separation is done due to differential affinities of compounds towards a stationary phase and a mobile phase. The stationary phase is a solid or a liquid that is fixed in place, while the mobile phase is a liquid or a gas that flows through or over the stationary phase.

The sample to be separated is introduced into the system in a narrow zone, where it interacts with both phases. Depending on their polarity, solubility, charge, size, and shape, different compounds will have different degrees of attraction to the stationary phase and the mobile phase. The more a compound is attracted to the stationary phase, the longer it will stay in contact with it and the slower it will move along the system. The more a compound is attracted to the mobile phase, the faster it will be carried away by it and the faster it will move along the system.

As a result, compounds with different affinities will travel at different rates and will be separated from each other over time. The separation can be detected by various methods, such as color, fluorescence, UV absorption, or mass spectrometry. The separation can also be quantified by measuring the area or height of each peak in a chromatogram, which represents the amount of each compound in the sample.

By choosing appropriate stationary and mobile phases, chromatography can separate and identify a wide range of compounds based on their molecular characteristics and interaction types. Chromatography can also be classified into different types according to the shape and size of the system, such as column, thin layer, paper, gas, or liquid chromatography.

Chromatography is a technique that involves the separation of a mixture into its components based on their relative affinities for a stationary phase and a mobile phase. The three components of the chromatography technique are:

• Stationary phase: This is the phase that does not move and remains fixed in the system. It can be either a solid or a liquid that is coated on a solid support. The stationary phase acts as a selective barrier that retains some components of the mixture more than others, depending on their interactions with its surface sites. For example, in paper chromatography, the paper acts as the stationary phase, while in column chromatography, the solid particles packed in a tube act as the stationary phase.

• Mobile phase: This is the phase that moves through the system and carries the mixture along with it. It can be either a liquid or a gas that flows over or through the stationary phase. The mobile phase acts as a driving force that transports the components of the mixture at different rates, depending on their partition coefficients. For example, in thin-layer chromatography, the solvent acts as the mobile phase, while in gas chromatography, an inert gas acts as the mobile phase.

• Separated molecules: These are the components of the mixture that are separated by the chromatography technique based on their differential migration in the mobile and stationary phases. The separated molecules can be detected, identified, quantified, or collected for further analysis or use. For example, in high-performance liquid chromatography, the separated molecules can be detected by a UV detector, while in affinity chromatography, the separated molecules can be collected by eluting them from the stationary phase.

The type of interaction between the stationary phase, mobile phase, and separated molecules is the key factor that determines the separation efficiency and selectivity of the chromatography technique. Different types of chromatography techniques use different mechanisms of interaction, such as adsorption, partition, ion exchange, size exclusion, and affinity.

Chromatography can be classified into different types based on various criteria, such as the nature of the stationary and mobile phases, the method of sample introduction, and the mode of separation. Some of the common types of chromatography are:

• Column chromatography: This type of chromatography uses a solid stationary phase packed in a glass or metal column and a liquid or gas mobile phase. The sample is applied at the top of the column and then eluted with the mobile phase. The components of the sample separate based on their different affinities for the stationary phase. Column chromatography can be further divided into subtypes such as ion-exchange, gel-filtration, affinity, and reverse-phase chromatography .
• Gas chromatography (GC): This type of chromatography uses a gas mobile phase and a solid or liquid stationary phase coated on a capillary tube or packed in a column. The sample is vaporized and injected into the column, where it is carried by the gas stream. The components of the sample separate based on their different boiling points and interactions with the stationary phase .
• High-performance liquid chromatography (HPLC): This type of chromatography uses a liquid mobile phase and a solid stationary phase packed in a high-pressure column. The sample is injected into the column and eluted with the mobile phase at a high flow rate. The components of the sample separate based on their different polarities and interactions with the stationary phase .
• Thin-layer chromatography (TLC): This type of chromatography uses a solid stationary phase coated on a glass or plastic plate and a liquid mobile phase. The sample is spotted at the bottom of the plate and then placed in a chamber containing the mobile phase. The mobile phase rises up the plate by capillary action and carries the sample along with it. The components of the sample separate based on their different solubilities and adsorptions on the stationary phase .
• Paper chromatography: This type of chromatography uses a paper sheet as the stationary phase and a liquid mobile phase. The sample is spotted at one end of the paper and then placed in a chamber containing the mobile phase. The mobile phase moves along the paper by capillary action and carries the sample along with it. The components of the sample separate based on their different solubilities and affinities for the paper .

These are some of the widely used types of chromatography, but there are many other variations and combinations that can be employed for specific purposes. Chromatography is a versatile technique that can be adapted to suit different analytical needs.

There are many types of chromatography techniques that are used for different purposes and applications. Some of the most commonly employed chromatography techniques are:

• Column chromatography: This technique uses a column filled with a solid stationary phase, such as silica gel or alumina, and a liquid mobile phase, such as an organic solvent or water. The mixture to be separated is applied to the top of the column and the mobile phase flows through it. The components of the mixture have different affinities for the stationary phase and thus travel at different rates along the column. The components are collected at the bottom of the column as they elute with the mobile phase. Column chromatography is widely used for the purification and separation of organic compounds, natural products, and biomolecules .

• Ion-exchange chromatography: This technique uses a stationary phase that has charged groups attached to it, such as sulfonic acid or quaternary ammonium. The mobile phase is a buffered solution that contains ions of opposite charge to the stationary phase. The mixture to be separated contains molecules that have different charges or different degrees of ionization. The molecules bind to the stationary phase based on their charge and their affinity for the counter-ions in the mobile phase. By changing the pH or the ionic strength of the mobile phase, the molecules can be eluted from the column in order of their charge or affinity. Ion-exchange chromatography is commonly used for the separation and analysis of proteins, nucleic acids, amino acids, and metal ions .

• Gel-permeation (molecular sieve) chromatography: This technique uses a stationary phase that consists of porous beads made of cross-linked polymers, such as agarose or dextran. The mobile phase is a solvent that can permeate through the pores of the beads. The mixture to be separated contains molecules that have different sizes or shapes. The molecules enter the pores of the beads based on their size and shape, and thus experience different degrees of obstruction by the stationary phase. The smaller molecules enter more pores and travel slower than the larger molecules, which enter fewer pores and travel faster. The molecules are separated by their size or shape and elute from the column in order of their molecular weight. Gel-permeation chromatography is often used for the determination of molecular weights and molecular weight distributions of polymers, proteins, and other macromolecules .

• Affinity chromatography: This technique uses a stationary phase that has a specific binding agent attached to it, such as an antibody, an enzyme, a receptor, or a ligand. The mobile phase is a buffered solution that contains the mixture to be separated. The mixture contains molecules that have different affinities for the binding agent on the stationary phase. The molecules that have high affinity for the binding agent bind to it and are retained on the column, while the molecules that have low affinity for the binding agent pass through it and are eluted with the mobile phase. By changing the pH, the ionic strength, or adding a competitive ligand to the mobile phase, the bound molecules can be released from the column in order of their affinity. Affinity chromatography is widely used for the purification and isolation of specific biomolecules, such as enzymes, antibodies, hormones, and drugs .

• Paper chromatography: This technique uses a paper strip as the stationary phase and a liquid solvent as the mobile phase. The mixture to be separated is spotted on one end of the paper strip and then placed in a container with a small amount of solvent. The solvent rises up through capillary action along the paper strip and carries along with it the components of the mixture. The components have different solubilities in the solvent and different adsorptions on the paper strip and thus travel at different rates along it. The components are separated by their solubility and adsorption and form distinct spots on the paper strip. Paper chromatography is simple and inexpensive and can be used for the separation and identification of organic compounds, amino acids, pigments, and other substances .

• Thin-layer chromatography: This technique uses a thin layer of solid material coated on a glass plate or a plastic sheet as the stationary phase and a liquid solvent as the mobile phase. The mixture to be separated is spotted on one end of the thin layer and then placed in a container with a small amount of solvent. The solvent rises up through capillary action along the thin layer and carries along with it the components of the mixture. The components have different solubilities in the solvent and different adsorptions on the thin layer and thus travel at different rates along it. The components are separated by their solubility and adsorption and form distinct spots on the thin layer. Thin-layer chromatography is fast and versatile and can be used for the separation and identification of organic compounds, drugs, dyes, and other substances .

• Gas chromatography: This technique uses a gas as the mobile phase and a solid or a liquid coated on a metal tube as the stationary phase. The mixture to be separated is vaporized and injected into the metal tube, which is heated to maintain a constant temperature. The gas carries along with it the components of the mixture, which have different volatilities in the gas and different adsorptions on the stationary phase. The components are separated by their volatility and adsorption and elute from the metal tube in order of their boiling points. The eluted components are detected by a device such as a flame ionization detector or a mass spectrometer. Gas chromatography is sensitive and precise and can be used for the separation and analysis of volatile organic compounds, fatty acids, alcohols, and other substances .

• High-pressure liquid chromatography (HPLC): This technique uses a liquid as the mobile phase and a solid packed in a metal column as the stationary phase. The mixture to be separated is dissolved in a solvent and injected into the metal column, which is maintained at high pressure to increase the efficiency of separation. The liquid carries along with it the components of the mixture, which have different solubilities in the liquid and different adsorptions on the stationary phase. The components are separated by their solubility and adsorption and elute from the metal column in order of their polarity. The eluted components are detected by a device such as a UV-visible spectrophotometer or a mass spectrometer. HPLC is versatile and accurate and can be used for the separation and analysis of non-volatile organic compounds, drugs, proteins, and other substances .

  Applications of Chromatography in Various Industries

Chromatography is a versatile technique that can be used for a variety of purposes in different industries. Some of the common applications of chromatography are:

• Pharmaceutical and Clinical Testing: Chromatography plays an important role in the safety and quality of pharmaceutical products. It can be used to identify and analyze samples for the presence of trace elements or chemicals, separate compounds based on their molecular weight and element composition, detect unknown compounds and purity of mixtures, and assist in drug development and clinical trials.
• Food and Beverage: Chromatography can help ensure the quality and safety of food and beverage products by detecting food spoilage and additives, determining the nutritional value and shelf life of products, identifying contaminants and allergens, and analyzing flavors and aromas .
• Environmental and Chemical Industry: Chromatography can help monitor and protect the environment by testing water samples and air quality for pollutants, toxins, pesticides, metals, and other harmful substances. It can also be used to analyze chemical products and processes for quality control, optimization, and compliance with environmental regulations .
• Drug Testing: Chromatography can be used to detect the presence of drugs or metabolites in biological samples such as blood, urine, saliva, hair, or sweat. It can also be used to determine the concentration and purity of drugs or metabolites. Chromatography can help identify the type, source, and origin of drugs or metabolites .
• Security: Chromatography can be used to detect explosives, chemical weapons, or other hazardous materials in airports, borders, or other public places. It can also be used to identify counterfeit products or documents by analyzing their ink composition .
• Forensics: Chromatography can be used to solve crimes by analyzing evidence such as blood stains, fingerprints, DNA, fibers, paint chips, or soil samples. It can also be used to determine the cause and time of death by analyzing body fluids or tissues .
• Molecular Biology Studies: Chromatography can be used to study the structure and function of biomolecules such as proteins, nucleic acids, lipids, or carbohydrates. It can also be used to isolate and purify biomolecules from complex mixtures for further analysis or manipulation. Chromatography can help in the fields of genomics, proteomics, metabolomics, bioinformatics, biotechnology, and biochemistry .
• Petroleum: Chromatography can be used to analyze crude oil and its fractions for quality control, optimization, and characterization. It can also be used to separate and identify hydrocarbons and other organic compounds present in petroleum products such as gasoline, diesel, jet fuel, lubricants, or plastics .

Chromatography is a powerful technique that has many applications in various industries. It can help improve the quality and safety of products and services, protect the environment and human health, advance scientific knowledge and innovation, and enhance security and justice.

Conclusion

Chromatography is a powerful and versatile technique that allows the separation, identification, and purification of the components of a mixture. It has a wide range of applications in various fields such as pharmaceuticals, chemicals, food, forensics, molecular biology, and environmental analysis. Chromatography can be classified into different types based on the physical states and interactions of the stationary and mobile phases. Some of the commonly used chromatography techniques are column chromatography, ion-exchange chromatography, gel-permeation chromatography, affinity chromatography, paper chromatography, thin-layer chromatography, gas chromatography, and high-pressure liquid chromatography.

Chromatography has many advantages such as simplicity, specificity, sensitivity, accuracy, and versatility. It can separate complex mixtures that are otherwise difficult or impossible to analyze by other methods. It can also provide quantitative and qualitative information about the components of a mixture. Chromatography can be performed by a single person with minimal training and equipment.

However, chromatography also has some disadvantages such as cost, time, and complexity. Chromatography equipment can be expensive and require careful maintenance and calibration. Chromatography can take more time to separate the compounds than other methods. Chromatography can also require large amounts of solvents and samples, which can pose environmental and safety hazards. Chromatography can also be affected by various factors such as temperature, pressure, flow rate, pH, and sample preparation.

Therefore, chromatography is a useful technique that has many benefits but also some limitations. It is important to choose the appropriate type of chromatography for the specific purpose and to optimize the conditions for the best results. Chromatography is a dynamic field that is constantly evolving and improving to meet the challenges and demands of modern science.

Chromatography is a powerful and versatile technique that allows the separation, identification, and purification of the components of a mixture. It has a wide range of applications in various fields such as pharmaceuticals, chemicals, food, forensics, molecular biology, and environmental analysis. Chromatography can be classified into different types based on the physical states and interactions of the stationary and mobile phases. Some of the commonly used chromatography techniques are column chromatography, ion-exchange chromatography, gel-permeation chromatography, affinity chromatography, paper chromatography, thin-layer chromatography, gas chromatography, and high-pressure liquid chromatography.

Chromatography has many advantages such as simplicity, specificity, sensitivity, accuracy, and versatility. It can separate complex mixtures that are otherwise difficult or impossible to analyze by other methods. It can also provide quantitative and qualitative information about the components of a mixture. Chromatography can be performed by a single person with minimal training and equipment.

However, chromatography also has some disadvantages such as cost, time, and complexity. Chromatography equipment can be expensive and require careful maintenance and calibration. Chromatography can take more time to separate the compounds than other methods. Chromatography can also require large amounts of solvents and samples, which can pose environmental and safety hazards. Chromatography can also be affected by various factors such as temperature, pressure, flow rate, pH, and sample preparation.

Therefore, chromatography is a useful technique that has many benefits but also some limitations. It is important to choose the appropriate type of chromatography for the specific purpose and to optimize the conditions for the best results. Chromatography is a dynamic field that is constantly evolving and improving to meet the challenges and demands of modern science.

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