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The term chromatography (chroma = one colour, graphene for writing) is a collective term for a group of laboratory techniques for the separation of mixtures. Chromatography involves a sample or sample extract being dissolved in a mobile phase which may be a gas, liquid or supercritical liquid. The mobile phase is then forced through a stationary, immiscible stationary phase. The phase is chosen such that the component of the sample has a different solubility in each phase. A component that is significantly soluble in the stationary phase will take longer to travel through it than a component that is not very soluble in the stationary phase but is very soluble in the mobile phase. As a result of these differences in mobility, the sample components will be separated from each other as they travel through the stationary phase. Techniques such as HPLC (high performance liquid chromatography) and GC (gas chromatography) use column narrow tubes packed with stationary phase, through which the mobile phase is forced. The sample is moved through the column by continuous addition of the mobile phase. This process is called reference or elution.
Chromatography is a technique for separating mixtures into their components in order to analyze , identify , purify , and quantify the mixture or components. As against chromatography is the process of separating compounds by injecting a gaseous or liquid sample into a mobile phase, usually called a carrier gas, and passing the gas through a stationary phase. The mobile phase is usually an inert gas or an nonreactive gas such as helium, argon, nitrogen or hydrogen. The stationary phase is a microscopic layer of viscous liquid on an inert solid support inside a column of glass or metal tubing on the surface of the solid particles called a column. The surface of the solid particles can also act as a stationary phase in some columns. Metal or glass column through which the gas phase passes is located in an oven where the temperature of the gas can be controlled and the fluent from the column is monitored by a computerized detector.
- Stationary phase : liquid / solid.
- Mobile phase : inert gas
- Carrier gas : Nitrogen, helium , hydrogen
Stationary phase in chromatography-
The stationary phase in chromatography is defined as, either a solid or liquid particle attached to a glass or metal surface, on which the components of a mixture are selectively absorbed to separate them. The term stationary refers to the fact that this phase remains stationary while the other phase continues. Most of the substances used as stationary phases are porous, thus allowing attachment of components during chromatography. The stationary phase chosen for the chromatographic process depends on the nature. Types of components to be separated and chromatography. Depending on the type of chromatography, thin uniform paper, silica, glass, some gases, or even liquid components are used as a stationary phase.
Mobile phase in chromatography-
The mobile phase in chromatography is the phase that is either a liquid or a gas that is passed through a chromatographic system where the components of a mixture are separated by adsorption into the stationary phase at different rates. The mobile phase is the solvent that moves the mixture as it moves down to the stationary phase. The term mobile indicates that the phase is moving down the chromatographic system, while the other phase remains stationary. Substances used as mobile phases are selected for the chromatographic process depending on the nature of the components to be separated and the type of chromatography. Alcohol, water, acetic acid, acetone, or certain gases are commonly used mobile phases in various chromatographic techniques.
Principle of chromatography-
Chromatography is based on the principle where molecules in a mixture are immobilized on a surface or in a solid, and with the help of a mobile phase, the liquid stationary phase (stationary phase) is separated from each other while moving. Effective factors in this separation process include adsorption (liquid-solid), fractionation (liquid-solid), and molecular characteristics related to the affinity or difference between their molecular weights. Because of these differences, some components of the mixture remain in the stationary phase longer, and move more slowly in the chromatography system, while others rapidly move into the mobile phase, and leave the system more rapidly.
The basic principle of chromatography diagram is shown in the figure-
The stationary phase is wetted with the help of a solvent because the upper level of the mobile phase and the stationary phase must match. The mobile phase or eluent is either a solvent or a mixture of solvents. The mixed mixture that needs to be separated in the first step is attached to the top of the column without disturbing the top level. The tap is turned on and the adsorption process on the silica surface begins. The solvent mixture is added slowly by touching the sides of the glass column without disturbing the stationary phase. Solvent is added as required throughout the experiment. The tap is turned on to initiate movement of the compounds in the mixture. The movement is based on the polarity of the molecules in the sample. Non-polar components move at a higher speed than polar components. For example, a compound mixture contains three different compounds i.e. red, blue, green so their order on the basis of polarity will be blue > red > green. Since the polarity of the green compound is less, it will move first. When it reaches the end of the column it is collected in a clean test tube. After this, the red compound is collected and finally the blue compound is collected and these are collected in separate test tubes.
Types of chromatography
There are different types of chromatography techniques and these are classified according to the basis of interaction to the stationary phase, physical state of mobile phase,chromatographic bed shape.
Application of chromatography
There are some most important application of chromatography which is used in regular routine.
- Chromatography plays an important role in the chemical industry for testing water samples for purity.
- Testing for the purity of air samples is also accomplished by chromatographic techniques in the chemical industry.
- The presence of toxic contaminants in oils and pesticides (the most notable of which are polychlorinated biphenyls, often abbreviated to PCBs) can be determined with the help of specialized chromatographic techniques such as GC and HPLC.
- It may also be noted that chromatography also has many applications in the life sciences.
- It is used in dyes or pigments to differentiate colors from natural dyes.
- It is used to separate various drugs from the blood.
- It is used to separate small amounts of the products of chemical reactions.
- It is used to separate different inks and colors from a mixture.
- It is used to test the nutrients in food and It can also be used in solving forensic crimes.
Advantages of chromatography-
- Chromatography is the simplest method of separating components.
- The components of a complex mixture can be separated using the chromatography method.
- Small amounts of sample (g, ppm, and ng/mL) can be detected by this chromatography.
- It is a fast and accurate method of separation.
- Very little sample volume/volume is required for analysis.
- It works on a wide range of samples.
Disadvantages of chromatography-
- Chromatography equipment is expensive.
- The error occurred due to overloading of samples.
- Chromatography equipment must be handled with care as these parts are expensive and sensitive.
- Some chromatography techniques require more solvent to separate the analytes.
- Needs periodic maintenance and replacement of parts
- Some chromatography methods require high power consumption.
- Constant high operational pressure may be required to achieve efficient separation.
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