Theory and Principle of Chromatography’Chromatography’ is a method used for separating a mixture of chemical substances into its own components so that the individual components can be analyzed through. There are many types of chromatography, for instance., liquid chromatography, gas chromatography, ion-exchange chromatography, affinity chromatography, but all of these employ the same basic principles. Principles of chromatographySeparation of components of a sample mixture happens because of the partition. Stationary phase is coated with a liquid which is not forming a homogenous liquid in mobile phase. Partition of component of sample between sample and liquid and somehow also gas stationary phase retard some components of sample more as compared as others. This gives the basis of separation. In chromatography a liquid is pumped through a bed of particles and it is called the mobile phase and the stationary phase. A mixture of the molecules that should be separated is introduced into the mobile phase.Principle of separation of different components: Differential affinities of the various components of the analyte towards the stationary and mobile phase results in the differential separation of the components. Affinity, in turn, is dictated by two properties of the molecule: ‘Adsorption’ and ‘Solubility’.Chromatography has a number of advantages over older method, for instance, crystallization, solvent extraction, and distillation, as a separation technique. It is capable of separating all the components of a multicomponent chemical mixture without requiring an extensive foreknowledge of the identity, number, or relative amounts of the substances present. It can be able to adapt in that which can deal with molecular species ranging in size from millions of atoms composed by viruses to the smallest of all molecules which is hydrogen and it contains two only. Furthermore, it can be used with large or small amounts of material. Adsorption can be defined as the property of how well a component of the mixture sticks to the stationary phase and while solubility is the property of how well a component of the mixture dissolves in the mobile phase.• Higher the adsorption to the stationary phase, the slower the molecule will move through the column.• Higher the solubility in the mobile phase, the faster the molecule will move through the column. So, the interplay between the above two factors determines the differential rates at which the different components of the analyte will move through the column. Adsorption and solubility of a molecule can be manipulated by choosing the appropriate stationary phase and mobile phase.A sample to be separated, when placed on the stationary phase, and it will gradually move along in the same direction as the mobile phase. If a sample compound has no interaction at all with the stationary phase, it will run right through and come out of the system at the same speed as the mobile phase. But, if an analyte no interact with the mobile phase, it will stick directly to the stationary phase and never elute. Neither of these are good outcomes either.In a well-designed chromatography process, the chemist will choose stationary and mobile phases that will both have at least some interaction with the analytes. Any individual sample molecule will interact first with one phase and then the other, back and forth repeatedly, but the fraction of each analyte overall in each phase will remain constant. This distribution ratio among the selected phases must differ for each analyte in order for them to separate. Figure 2 : The analytes continually cross back and forth over the interface between the stationary and mobile phases.Analytes with greater attraction to the stationary phase will still flow through the system because they spend part of their time moving in the mobile phase, but they will tend to fall behind those analytes that have a greater interaction with, thus, spend more time in the mobile phase. Gradually, as they progress through the system, the analytes separate from each other (usually in order of their distribution ratio), and can be captured or at least detected in really pure form as they elute. Separation is only the first part of chromatography. In the Tswett’s experiment, he could see where the various compounds were by using colour. Most chemicals are colourless, but, there must be a detector to know or notice when compounds are eluting.Theories of chromatographyChromatography involves a sample being dissolved in a mobile phase which may be a gas, a liquid or a supercritical fluid. The mobile phase is then forced through an immobile, immiscible stationary phase. The phases are chosen such that components of the sample have differing solubilities in each phase. A component which is quite soluble in the stationary phase will take longer to travel through it than a component which is not very soluble in the stationary phase but very soluble in the mobile phase. As a result of these differences in mobilities, sample components will become separated from each other as they travel through the stationary phase. Chromatography is used to differentiate or separate the most simplest compounds from a complex mixture. According to the polarity, solubility, charge, molecular weight, biological activity for example, the compounds are isolated or separated from the given mixture. Which means in a mixture of compounds, compounds differ in size, shape, polarity, charge and others, making easier to separate one from the other of a complex mixtureChromatography is used in industrial processes to purify chemicals, test for trace amounts of substances, separate chiral compounds and test products for quality control. Chromatography is the physical process by which complex mixtures such as chemical substances are separated or analyzed.