Analytical chemistry consist of qualitative as well as quantitative chemical analysis. In a sample, the component to be analysed is called as 'Analyte'. Quantitative chemical analysis consists of two steps:
1. Separation and preconcentration of the analyte.
2. Analysis of the sample.
Separation techniques used are of different types depending on the nature of analyte. Difference in some of the property on the components in a mixture is used as a basis for separation of the components. This property may be difference in size that is used in sieving fine material to separate it from coarse 1 hour may be separation by distillation based on difference in boiling points of the two liquids in a mixture. Various separation techniques like sublimation ,crystallization, etc are used frequently in Chemistry laboratory.
Chromatography is one of such methods used frequently in analytical chemistry. The word chromatography is derived from Greek words meaning "colour" (Chroma) and "write"(Graphy). This technique was first used by Russian botanist M.Tswett in 1906 for separation of coloured plant pigments on column of alumina.
Chromatography is defined as "physical method of separation, in which the components are separated by distribution between two phases". The phase that has large surface area and remains steady is called as 'Stationary Phase' while the other moves through the stationary phase called as 'Mobile phase'.Mixture of various component is equilibrated between two phases. Separation of these components takes place either by the mechanism of 'adsorption' or 'partition'.
Classification of chromatography
Depending on the principle of separation chromatographic techniques are divided into two main types:
A) Adsorption chromatography
In this method separation of components takes place on the basis of difference in their adsorption properties on solid stationary phase. Here, one phase is always solid. Depending on the mobile phase, adsorption chromatography is classified into two types:
1. Liquid-Solid Chromatography (LSC): in this technique the stationary phase is solid while mobile phase is liquid. Different components adsorb to different extent on the stationary phase.
The less adsorbed component is taken away first by the mobile liquid phase, while is strongly adsorbed component is eluted afterwards.
e.g.Column chromatography, ion exchange chromatography, thin layer chromatography (TLC) etc.
2. Gas-Solid Chromatography (GSC): In this method the stationary phase in solid while mobile phase is gas. It is carried out at every high temperatures to that the analyte remains in gaseous phase.
B) Partition chromatography
In this technique one of the phases is always liquid. Different components get dissolved in the liquid to different extent and thus get separated from each other. Depending on the other phase, it can be classified into two types:
1. Gas-Liquid Chromatography (GLC): when stationary phase is liquid and mobile phase is gas the chromatographic method is called as GLC. The stationary phase is liquid immobilized on solid support like silica gel.
2. Liquid-Liquid Chromatography (LLC): When both stationary as well as mobile phases are liquids, the technique is called as LLC. The two liquids are immiscible with each other. Generally, one liquid is organic while other is inorganic. Difference between solubilities of the components in these two liquids act as a basis of separation.
e.g. Paper chromatography, solvent extraction etc.
Table:Types of chromatography
PAPER CHROMATOGRAPHY
Principle
It is a type of chromatography in which mixture of substance is distributed between two liquids. One Liquid is held in the fibres of a paper called as stationary phase while the other is moving liquid called mobile phase. In paper chromatography, the stationary phase is generally moving liquid called as mobile phase. In paper chromatography, the stationary phase is generally water. It is immobilized in the fibres of the filter paper. Mobile phase is generally an organic solvent immissible with water.
Various components when distributed between water and other liquid, get dissolved in these two liquids in different proportions. When mobile phase moves over stationary phase, equilibrium is established at every stage of partition. They get separated on the basis of difference in partity coefficients of various components,.
Technique
The technique used in paper chromatography consists of following steps-
1. Preparation of sample solution and application on paper:
Paper chromatography assembly consists of a jar containing the mobile liquid. The liquid may be a pure solvent or a mixture of two solvents in fix proportion. This liquid is placed at the bottom of a jar. A filter paper strip is cut in proper size and the mixture is applied in the form of a small spot near one end of the strip. The paper used is generally a Whatman filter paper no.1, no.2 etc depending on the rate of flow required.
2. Running of chromatogram:
The paper is dried completely and dipped in the solvent placed in the jar. This liquid is called as eluent and the process is called as elution. Eluent used is generally a mixture of organic solvent and water in fixed proportion. Experimental arrangement is shown in fig. The eluent starts moving in upward direction by capillary action of the filter paper. Various components of the mixture move at different speeds.
3. Locating various components:
When solvent is mood through suitable height (about 15 to 18 cm), the paper is dried and various spots are visualised by physical or chemical method post op physical methods are fluorescence and radioactivity while chemical method consists of spraying suitable reagent called locating reagent (or visualizing reagent). On addition of locating reagent different spots are obtained on the paper. such paper with different spots corresponding to different components is called as chromatogram.
The movement of various components is expressed in terms of 'retardation factor' called as Rf value it is given by-
Rf= Distance travelled by component
Distance travelled by solvent front
The Rf value lies between 0 and 1. Higher Rf value shows higher mobility of the component . It depends upon number of factors as-
Solvent employed
Medium used for separation
Quality of paper used
Nature of mixture
Temperature.
pH of the solution.
For each component in a mixture, the Rf value is constant under given experimental conditions.
Thus various components can be identified from their Rf values by fingerprint method.
Types of paper chromatographic techniques
For developing a chromatogram various techniques are used in paper chromatography. Thus paper chromatography is classified into following types-
Ascending paper chromatography:
In this technique, the eluent is taken at the bottom of the jar and ellution take place in upward direction as shown in Fig.(paper chromatography)
Flow of eluent it takes place due to capillary action against gravitational force and thus takes more time and results in better partition of the component.
Descending paper chromatography:
In this technique the eluent is stored at the top and moves along the paper in downward direction. This takes less time as the movement is in the direction of gravitational force. In this technique, the components with lower Rf- value are resolved better than ascending method. The experimental arrangement is shown in Fig
Radial paper chromatography:
This is rarely used method.Here, sample is applied at the centre of a circular paper and develop by a wick. Wick is bent Don word at 90°and dipped in the eluent . Elution takes place radially and thus called as radial paper chromatography.
Two dimensional paper chromatography:
In this technique rectangular or square paper is used. Sample is applied at one of the corners. First development is made as in ascending chromatography. Then paper is turned at right angle and again development is carried out in the similar manner (Fig). For second development either same or different solvent can be used. This technique is very useful when the mixture contains large number of components with close Rf- values.
Applications
Paper chromatography is being used as a simple separation tool in qualitative as well as quantitative chemical analysis.
Qualitative direction of constituents:
In a mixture it is sometimes necessary to detect the constitution present. In such case the mixture is spotted on paper and chromatogram is run using suitable solvent. After separation of components, spots are visualized using suitable locating agent. From the colours of the spots, the constituent can be identified.
For example,
Metallic ions of group l (Pb²+,Aɡ+ and Hɡ²+) have been identified and separated using distilled water or alcohol as a solvent and 0.25 M K₂CrO₄ aqua solution as locating agent. Pb²+ gives yellow, Ag+ gives Orange red while Hg₂²+ gives Orange spot.
Group II ions ( Bi³+,Cd²+, Cu²+ and Hg²+ ) can be analyzed quantitatively using n butyl alcohol and 3M HCL reagent and dithizone solution in CCL₄ as a locating reagent.
Group IV cations Ni²+ , Co²+ , Zn²+ and Mn²+ etc can be analyzed qualitatively using paper chromatography. Eluent used is acetone-water-HCL mixture and locating reagent is rubeanic acid.
B. Quantitative analysis of metals:
Constituent of mixture of metals are separated as discussed above.the paper is cut and various components are dissolved in in suitable solvent and analysed using colorimetry, spectrophotometry or any other analytical technique.
C. Quantitative analysis of non-metals:
Various non metals anions organic compounds etc can be separated using paper chromatographic technique. Separation and estimation of Cl-,Br-,I- has become possible by using paper chromatography.
D. Separation of amino acids:
Mixture of naturally occuring amino acids can be analyzed using two dimensional paper chromatography. Ninhydrin is used as a colour-developing agent. It gives purple colour with all α-amino acids. Other compounds like aliphatic amines, some heterocyclic compounds and imino acids give yellow colour.
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