Chromatography is a technique that involves passing a mixture through a stationary phase, which selectively retains certain components while allowing others to pass through. Chromatographic techniques rely on a stationary phase to separate the components of a mixtures depending on their affinity. The stationary phase can exist in different states, such as solid, liquid, or gas, and it interacts differently with each component, leading to distinct bands or peaks in the separated mixture. Analyzing these bands or peaks enables to identify and quantify components.
This technology is now widely used for the purification and isolation of Organic compounds on a Laboratory as well as process scale. The name Chromatography owes its origin to the fact that Mikhail Semyonovich Tsvet (also Tswett) a Russian Botanist in 1906 used it for the separation of coloured substances from plants (Greek Chromos, meaning colour). Since the scope of this technique has been widely extended to include colourless mixtures, the idea of colour is no longer connected with the concept.
Principles of Chromatography
Chromatography is based on the general principle of isolating different components of a mixtures of organic compound between two phases which are called stationary phase and a mobile phase. Chromatography involves a stationary phase and a mobile phase, with the stationary phase typically being a solid or liquid supported on a solid, while the mobile phase is usually a liquid or gas. When the stationary phase is a solid, separation occurs based on adsorption, while when it is a liquid, separation is based on partitioning. This allows for the separation of different components in the mixture, which can be further analyzed and characterized.
Chromatography is a separation technique that uses the differential partitioning of different sample components between a stationary phase and a mobile phase(which is moving). In this technique, the sample mixture is introduced into a chromatography system, where it interacts with a stationary phase that can take the form of a solid, liquid, or gas. The mobile phase, can be a liquid or gas that carries different samples crossing through the stationary phases. By interacting differently with the stationary phase, the components of the sample mixtures are separated individually and can be identified and analyzed.
Chromatography separates the components of a sample mixture by exploiting their differential affinity for a stationary phase and a mobile phase. The components of the mixture that have a higher affinity for the stationary phase are retained longer, resulting in slower movement through the system, while components with lower affinity for the stationary phase move more quickly through the system. This separation leads to the components being eluted at different times, and they are subsequently detected and analyzed to identify the components and determine their quantity. Chromatography principles can be applied to various types of samples and is widely used in industries such as pharmaceuticals, food and beverage, and environmental monitoring. There are different types of chromatography, each with unique advantages and applications. Liquid-Solid Chromatography is one such type.
Types Of Chromatography
There are various types of chromatography, each with its unique applications and advantages.
Liquid-Solid Chromatography
When the mobile phase involved in a chromatographic system is a liquid and the stationary phase is a solid, the method of separation is called Liquid-Solid Chromatography. The solid phase may be packed in a column (Column chromatography) or take the form of a sheet of paper ( Paper Chromatography) or a thin coating of material deposited on a glass sheet (thin layer Chromatography).
Column Chromatography
This technique is a type of a Liquid-solid chromatography. The stationary phase is a finely divided solid (Silica gel, alumina etc) which is packed in a column and the mobile phase is a solvent (n- Hexane, Ether, Dichloromethane, Ethyl acetate etc) containing the mixture to be separated.
The sample of the mixture is first loaded to the column at the top. Some components of the mixture are adsorbed very strongly while others are slightly less strong or medium and less strongly. The more strongly a substance is adsorbed, the more slowly it moves down the columns. Thus the components moving down with different rates undergo a process of partial separation. A suitable solvent is passed through the column gradually and the compounds of the mixture are removed from the adsorbent surface. The process of forcing various compounds to be dissolved away from the adsorbent is termed elution and the solvent used is called eluent. The elution and re-adsorption of the various components is repeated several times as they travel down. This eventually results in the separation of the components as ‘bands’. Each band as it comes out of the lower end of the column (as shown in Below fig.) is received in a separate container.
High-Performance Liquid Chromatography (HPLC)
High-Performance Liquid Chromatography (HPLC) is a commonly used form of liquid chromatography that is used to separate and analyze complex mixtures. It involves pumping a sample through a column packed with a stationary phase, and the components are separated based on their affinity for the stationary phase. HPLC finds applications in various industries such as pharmaceuticals, food and beverage, and environmental monitoring.
Gas Chromatography (GC)
Gas Chromatography (GC) is another commonly used technique that is used to separate and analyze volatile compounds. The sample is vaporized and passed through a column packed with a stationary phase, and the components are separated based on their boiling points and other physical properties. GC is widely used in the analysis of organic compounds in various industries such as petrochemicals, pharmaceuticals, and environmental monitoring.
This is a much more recent method of separation of components of a liquid sample which is available only in a very small quantity.(0.1 to 10ml). It is often incorrectly called Vapour-phase Chromatography (VPC). Here the stationary phase is a film of relatively less volatile liquid on a solid support and the moving phase is a mixture of vaporized sample and an inert carrier gas (helium or nitrogen). The sample is distributed between the liquid film and the carrier gas. The process of GLC is illustrated in below.
With the help of a syringe is injected the liquid sample into a hot chamber where it is vaporized all at one. The vapour is pushed through a several feet long tightly packed column by means of carrier gas. The packing consists of a finely divided solid substance on the surface of which is adsorbed an appropriate liquid of low volatility. As the vapour travels through the column, the various components are continually dissolving in and vaporizing from the packing liquid. Thus interaction with the liquid is not uniform and depends on factors such as boiling point of the compound, its polarity and possibility of hydrogen bonding. The net result is that different compounds move at different rates through the column. Thus the components of the sample separate into individual ‘bands’ in the carrier gas which then pass through the detector. As the vapour of each component emerges from the column, the detector produces an electronic signal which causes a peak to be traced by the recorder.
Thin-Layer Chromatography (TLC)
Thin-Layer Chromatography (TLC) is a simple and inexpensive form of chromatography that is used to separate and analyze small quantities of compounds. It involves spotting a sample onto a thin layer of stationary phase, and the components are separated based on their affinity for the stationary phase. TLC is widely used in the analysis of plant extracts, essential oils, and other natural products.
Ion Chromatography (IC)
Ion Chromatography (IC) is a type of liquid chromatography that is used to separate and analyze ions. It involves passing a sample through a column packed with a stationary phase that has charged groups, and the components are separated based on their charge and affinity for the stationary phase. IC is widely used in the analysis of environmental samples and in the monitoring of water quality.
Size-Exclusion Chromatography (SEC)
Size-Exclusion Chromatography (SEC) is another type of liquid chromatography that separates molecules based on their size. The sample is passed through a column containing a stationary phase with pores of a specific size, and molecules of different sizes are separated based on their ability to enter these pores. SEC is commonly used for the analysis of biomolecules, such as proteins and nucleic acids.
Applications of Chromatograpghy
Chromatography is a versatile technique that finds applications in a variety of industries, including pharmaceuticals, food and beverage, environmental monitoring, forensic science, biotechnology, and many others.
One of the primary applications of chromatography is in drug development and analysis, where it is used to separate and analyze drug compounds, impurities, and metabolites. Chromatography is also frequently used in the food and beverage industry to detect and quantify food additives, preservatives, and contaminants.
Environmental monitoring is another important application of chromatography, as it allows researchers to analyze environmental samples like soil, air, and water for the presence of pollutants and contaminants. Forensic scientists also rely heavily on chromatography to detect and identify chemical compounds in arson and explosive investigations, as well as to analyze samples such as blood, urine, and hair for drug and alcohol use.
In addition, chromatography plays a vital role in quality control in various industries, where it is used to monitor and ensure the quality and purity of products. Finally, chromatography is widely used in biotechnology for the purification and analysis of proteins, DNA, and other biomolecules.
Overall, chromatography is an essential technique in many fields, allowing researchers to separate and analyze complex mixtures of compounds to gain a better understanding of the chemical composition of a wide range of substances.
In conclusion, chromatography is a powerful tool that plays a critical role in a wide range of industries and scientific disciplines. Its ability to separate and analyze complex mixtures of compounds has revolutionized drug development and analysis, food and beverage testing, environmental monitoring, forensic science, quality control, and biotechnology. As technology advances, chromatography is likely to continue to play a significant role in advancing scientific knowledge and improving the quality of life for people around the world.
FAQ’s about Chromatography
- What is chromatography? Chromatography is a technique used to separate and analyze mixtures of compounds based on their affinity for a stationary phase, which can be a solid, liquid, or gas.
- What are the types of chromatography? There are many different types of chromatography, including high-performance liquid chromatography (HPLC), gas chromatography (GC), thin-layer chromatography (TLC), ion chromatography (IC), and more.
- What are the applications of chromatography? Chromatography has many applications in industries such as pharmaceuticals, food and beverage, environmental monitoring, forensic science, biotechnology, and others. It is used for drug development and analysis, food and beverage testing, environmental monitoring, quality control, and biotechnology, among others.
- How does chromatography work? Chromatography works by passing a mixture through a stationary phase, which separates the components of the mixture based on their affinity for the stationary phase. The separated components form distinct bands or peaks that can be analyzed to determine their identity and quantity.
- What are the advantages of chromatography? Chromatography is a powerful and versatile technique that allows for the separation and analysis of complex mixtures of compounds. It is highly precise and sensitive, making it useful in many industries and scientific fields.
- What are the limitations of chromatography? Some of the limitations of chromatography include the need for specialized equipment and expertise, as well as the potential for contamination and interference from impurities in the sample.
- How is chromatography used in forensic science? Chromatography is used in forensic science to analyze samples such as blood, urine, and hair for drug and alcohol use, as well as to detect and identify chemical compounds in arson and explosive investigations.
- What is the role of chromatography in biotechnology? Chromatography is widely used in biotechnology for the purification and analysis of proteins, DNA, and other biomolecules. It is essential in the development of many biotech products, including vaccines, therapeutics, and diagnostics.