Terminologies used:
Chromatograph: Instrument employed for a chromatography
Stationary phase: Phase that stays in place inside the column. Can be a particular solid or gel-based packing or a highly viscous liquid coated on the inside of the column.
Mobile phase: Solvent moving through the column, either a liquid in LC or gas in GC.
Eluent: Fluid entering a column.
Eluate: Fluid exiting the column.
Elution: The process of passing the mobile phase through the column.
Chromatogram: Graph showing detector response as a function of a time.
Flow rate: How much mobile phase passed /minute (ml/min).
Analyte: Substance to be separated during chromatography. It is also normally what is needed from the mixture.
The retention time is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.
The detector refers to the instrument used for qualitative and quantitative detection of analytes after separation.
Introduction:
The molecules present in biological system or in synthetic chemistry are produced through a series of reactions involving intermediates. At any moment of time biological organism has major fraction as desired product but has other compounds in minute quantities. The minor species present in a product is always referred as “impurities” and these compounds need to separate from desired product for biotechnology applications. How two molecules can be separated from each other? To answer this question, we can take the example of three molecules given in Figure 1. These 3 molecules (benzene, phenol, aniline) are similar to each other but have distinct physical and chemical properties which can be used as a criterion to separate them. The physical and chemical properties which can be used to separate molecules are-
Physical Properties
1. Molecular weight
2. Boiling point (in case both are liquid, as in this case)
3. Freezing point
4. Crystallization
5. Solubility
6. Density
Chemical Properties
1. Functional Group, for example, phenol has –OH whereas aniline has -NH2
2. Reactivity towards other reagent to form complex
Now,for example you have a mixture of compound 1 (benzene) and compound 3 (Aniline) and you would like to purify benzene rather than aniline. In this situation, you can take the physical and chemical properties of benzene into the account and isolate it from the mixture.
Principle of Separation:
How a physical or chemical property will allow to isolate a particular substance? The mixture of compound 1 and 3 is shown in Figure 1 and assume if we are using boiling point as a criterion to isolate them. As we will heat the mixture there will two phase forms, one liquid phase and other is vapor phase. The molecules of compound 1 and 3 will distribute between these two phases and as the temp is near to boiling point of compound 1, more amount of 1 will be present in vapor phase than liquid phase. Whereas more number of compound 3 will be in liquid phase. Eventually as this process will continue, at the end two molecules will get separated from each other. The distribution coefficient (Kd) to describe the distribution of compound 1 between two phase A and B is as follows:
Similarly, one can also exploit other physical & chemical parameters as well. With each and every physical and chemical parameter the molecule present in the mixture will distribute as per their behavior in each parameter.
The purpose of chromatography is to separate a complex mixture into individual component exploiting the partition effect which distribute the molecules into the different phases. As discussed above, a distribution of a molecule between two phases A and B is given by a distribution coefficient, Kd. In most of the chromatography techniques, phase A is stationary phase or matrix and phase B is mobile phase or buffer. Hence, the term chromatography can be defined as a technique in which the components of a mixture are separated based on the rates at which they are carried through a Stationary phase by a Mobile phase.
Chromatography separation is based on a balanced state among the components to be separated, an adsorbent agent in the column (= stationary phase) and a solvent flowing through it (mobile phase). When a component settles on the stationary phase this is defined as adsorption, while detachment by the mobile phase is defined as desorption. A high adsorption capacity between the components of interest and the stationary phase means that there is a high retention of these components and that there is a considerable delay in elution from the column. The separation of a mixture into its individual components is only possible if the individual components in a combination of stationary and mobile phases have different adsorption/desorption properties.
Chromatographic separation can be carried out on both polar and non-polar stationary phases.
“Standard/Normal Phase” chromatography requires the use of polar stationary phases such as silica gel and nonpolar solvents. The individual components are delayed as a result of a reaction between the polar function component groups and the polar groups of the sorbent.
Low polarity substances are eluted first, followed by components of increasing size.
In “reversed phase” chromatography, however, the stationary phase is nonpolar and elution is by means of polar solvents.
Substances are eluted in order of decreasing polarity from reversed phase columns, i.e. the substance with the highest polarity appears first.
Applications of Chromatography:
Chromatography has gained immense popularity in the past few years in almost every field. Its use is seen in almost every extent around us. It is widely used in the field of chemistry, industry, medicines as well as in everyday life. It has plentiful applications in the field of biological research.
Uses of Chromatography in Chemistry:
Chromatography can be used in the field of chemistry from detecting the optical isomer to determining the amount of mixture present in a sample.
It is used to figure out the relation of different mixtures with one another.
It is very effective technique to test the purity of the sample.
The amount of mixture present in a sample can be calculated by using chromatography.
Chiral compounds which are very similar in molecular weight, elemental composition, and physical properties and differ only in optical isomers can be separated using chromatography.
It is used for the separation of mixture of compounds. Paper chromatography is particularly very effective in detection and separation of mixture of compounds.
Uses of Chromatography in Industry:
There are many industrial uses of chromatography. Different chromatography techniques are used in various industries like food industries, drinking water treatment plants and other industries.
It is used in the food industries for analysis of different additives in the food. For example milk is consumed all over the world. The common adulterant that can be added in milk is pyruvic acid. Pyruvic acid is derived from lactic acid bacteria. Chromatography is employed acid bacteria. Chromatography is employed to check the quality of milk.
Paper chromatography is particularly used to check the quality of food by examining different vitamins, preservatives, amino acids and proteins.
Chromatography is also used to separate the contaminants, traces of harmful chemicals and other micro-organisms in food.
Gas chromatography is used in the manufacture and separation of essential oils.
It is used in industries for separating different components whose amounts can range from milligrams to tons.
The manufactured food is checked for quality. For example malic acid is added in apple juice to prevent it from getting spoiled and to maintain its taste. As malic acid is found in apple juice, therefore it is very difficult to check its content in apple juice. In order to check the level of malic acid in apple juice, chromatography is employed. Fumaric acid is a contaminant of malic acid, so to check the quantity of synthetic malic acid, the level of fumaric acid is determined. Chromatography is used for quality control in the food industry, by separating and analyzing additives, vitamins, preservatives, proteins, and amino acids. It can also separate and detect contaminants such as aflatoxin, a cancer-causing chemical produced by a mold on peanuts. Chromatography can be used for purposes as varied as finding drug compounds in urine or other body fluids, to looking for traces of flammable chemicals in burned material from possible arson sites.
Thin layer chromatography is used to check and remove Polychlorinated biphenyls, pesticides and insecticides in ground water and fish contaminated by these. Chromatography of many kinds is widely used throughout the chemical industry. Environmental testing laboratories look for trace quantities of contaminants such as PCBs in waste oil, and pesticides such as DDT in groundwater. The Environmental Protection Agency uses chromatography to test drinking water and to monitor air quality. Pharmaceutical companies use chromatography both to prepare large quantities of extremely pure materials, and also to analyze the purified compounds for trace contaminants.
Environmental and governmental agencies also use chromatography to test drinking water.
Gas chromatography is also used in the environment field. Fixed monitors are employed to check the emission levels of pollutants such as nitrogen dioxide, carbon dioxide and carbon monoxide. It is also used to detect the quality of air.
Gas chromatography is used to monitor variation present if any in the industrial processes.
Uses of Chromatography in Medicine:
Chromatography in field of medicines has an extended use.
In pharmaceutical companies large amount of pure chemicals for making further medicines is prepared by using chromatography.
Paper chromatography is used to separate the various inks or dyes from the mixture.
Presence of alcohol or some other drugs in blood or urine are detected by using gas chromatography.
Chiral compounds resemble to each other greatly in terms of molecular weight, physical composition and elemental weight. But they have different optical isomers due to which they have different biological activities. Chromatography is very effective technique to separate the isomers. For example thalidomide is compound with two isomers one of them causes birth defects, chromatography is used to separate the isomer from its harmful counterpart.
In pharmacy chromatography is very important to analyse whether correct medicine is manufactured or not.
In forensic science it helps in solving many cases by detecting residual burnt particles and flammable chemicals present in the body parts in case of fire or explosions.
Paper chromatography and Gas chromatography are employed in finger print, DNA RNA analysis.
Uses of Chromatography in Everyday Life:
Chromatography is used in almost everywhere around us.
It is used in the laboratories for making pure sample of any compound.
It is also used to derive the amount of reactants or products.
Percentage purity is also found by chromatography.
Analytical chemistry use chromatography for various experiments
The detection and separation of pure compound is carried by chromatography.
It helps in checking the level of pesticides, fungicides and contaminants in the food and drinking water.
It is also used to check the level of adulterants in the manufactured food
It is applied in forensic science for investigation.
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