Principle of paper chromatography:
- This technique is a type of partition chromatography in which the substances are distributed between two liquids, i.e., one is the stationary liquid (usually water) which is held in the fibers of the paper and called the stationary phase, the other is the moving liquid is the moving liquid or developing solvent and called the moving phase.
- Cellulose filter paper is often used as the stationary phase in paper chromatography.
- Since it is hydrophilic, it is usually covered with thin film of water.
- The procedure is often regarded as liquid-liquid chromatography.
- The components of the separated migrate at different rates and appear as spots at different points on the paper.
- In this technique, a drop of the test solution is applied as a small spot on a filter paper and the spot is dried.
- The paper is kept in a close chamber and the edge of the filter paper is dipped into a solvent called developing solvent.
- As soon as the filter paper gets the liquid through its capillary axis and when it reaches the spot of the test solution (a mixture of two or more substances), the various substances are moved by solvent system at various speeds.
- When the solvent has moved these substances to a suitable height (15-18 cm) the paper is dried and various spots are visualized by suitable reagent called visualizing agent.
- The movements of substances relative to the solvent are expressed in terms of Rf values (retardation factor or retention factor).
- Rf: The R is related to the migration of the solute front relative to the solvent front as:
- R is a function of the partition coefficient. For a given substance it is constant provided the conditions of chromatographic system are kept constant with respect to temperature, type pf paper, duration and direction of development, nature and the shape and the size of the wick used (i.e., radial chromatography), the amount of liquid in the reservoir humidity, etc.
- The Rf defines the movement of the substances relative to the solvent front in a given chromatographic system.
- The Rf value of a substance depends upon a number of factors which are:
- The solvent employed
- The medium used for separation, i.e., the quality of the paper in case of paper chromatography
- The nature of the mixture
- The temperature
- The size of the vessel in which the operation is carried out.
- Stationary phase:
- It is liquid that is, the water trapped in the molecular structure of the paper and is invisible.
- Supporting material for the stationary phases is the matrix of cellulose fibers of chromatography paper.
- Chromatography papers are available in three running characteristics: slow, medium, and fast.
- Most frequently used chromatographic paper is Whatmann No.1 or its equivalent.
- Mobile phase:
- It is generally a liquid e.g. for a pure paper system, the eluent may be a single solvent or a mixture of solvents that can move through the paper.
- Among mixed solvent systems, a water-organic mixture is frequently used, e.g. n-butanol, acetic acid: water (4:1:5, top layer) for flavonoid, glycosides, acetic acid: conc.HCl: water (30:3:10) (Forrestal system for flavonoid aglycones), toluene:acetic acid: water (4:1:5, upper phase for flavonoid aglycones).
- The solvents used are selected from eluotropic series which is a list of solvents arranged in order of increasing polarity.
- Petroleum ether < n-hexane< carbon tetrachloride< toluene < benzene < chloroform < dichloromethane < diethyl ether < n-butanol < isopropanol < acetone < ethanol < methanol < water
- Generally, higher the solubility of a solute in a solvent, the greater the solute mobility in that solvent.
- If a solute dissolve more readily in the mobile phase, then it will travel with the solvent, hence the partition occurs between two phases.
- Different solutes travel at different rates up the paper, it is a result of their different solubility in two phases.
Types of paper chromatography:
- Descending chromatography:
- when the development of the paper is done by allowing the solvent to travel down the paper, it is known as descending technique.
- Ascending chromatography:
- When the development of the paper is done by allowing the solvent to travel up the paper, it is known as ascending technique.
- Both ascending and descending techniques have been employed for separation of organic and inorganic substances.
- But the ascending technique is preferred if the Rf values of various constituents are almost same.
- Ascending-Descending Chromatography:
- It is the hybrid of the above two techniques.
- In this technique, the upper part of the ascending chromatography can be folded over a glass rod allowing the ascending development to change over into the descending after crossing the glass rod.
- Radial paper Chromatography:
- This is also known as circular paper chromatography.
- This makes use of radial development.
- In this technique a circular filter paper is employed.
- Then the various materials to be analyzed are placed at the center.
- After drying the spot the paper is fixed horizontally on the petri-dish possessing the solvent so that the tongue or the wick.
- When solvent front has moved through a sufficient large distance, the components get separated in the form of concentric circular zones.
- Two-dimensional chromatography:
- In this, a square or rectangular paper is used.
- The sample is applied to one of the corners.
- The second development is performed at right angle to the direction of the first run.
- This type of chromatography can be carried out with identical solvent systems in both the directions or by two solvent systems.
Instrumentation of paper chromatography:
- It does not need expensive equipment.
- The only necessary equipment is the development chamber or tank.
- The simplest form is a closeable container, e.g., screw capped glass bottle or a jar and a wire or clip to the lid to support the paper strip.
- There are two devices for paper development- ascending and descending methods.
Procedure of paper chromatography system:
Step I: Sample preparation:
- The mixture (e.g. a mixture of amino acids) to be analysed is dissolved in a suitable solvent (0.5-3%).
- The selected solvent should be volatile for rapid evaporation of the solvent.
Step II: Paper selection:
- Whatman No. 1 chromatographic paper or its equivalent paper is used for analytical paper chromatography.
- Whatman No.3 paper is used for preparative paper chromatography.
Step III: Sample loading or spotting:
- A pencil line is drawn across the paper 10-15cm from the bottom for ascending paper chromatography and 10-15cm below antisiphon bar for descending.
- A drop of solution is placed on the paper at this line by means of a capillary tube, micro pipette.
- Reference compounds are similarly prepared and applied to the paper alongside the mixture spot.
- This helps interpretation of the chromatogram more readily.
Step IV: Development or elution:
- Paper chromatograms can be developed in a wide variety of development chambers or tanks.
- Small paper strips can be developed in a small jar or wide mouthed screw capped bottles.
- A large rectangular piece (20cm X 20cm) of paper is developed in a large rectangular glass tank.
- The solvent (eluant) is poured into the tank in such a way that the penciled line is just above the surface of the solvent.
- When the solvent reaches almost the top of the paper, the paper is removed from the tank and the solvent is allowed to evaporate in air until the paper is dry.
Step V: Detection of spot location or visualization:
- If the separated components or solutes are colored, they canbe easily seen in daylight under naked eye.
- If the components are colorless, a physical or chemical property must be employed to reveal or locate their positions.
- Widely used visualization techniques are fluorescence and spray reagent but conc. sulphuric acid is not practical for paper chromatography because the paper is charred.
- Spray reagents are chemical reagents, which react with functional groups to produce a colored spot.
- For e.g., after the development, the paper is sprayed with a solution of ninhydrin reagent then heated for 10 minutes at 110oC to form a coloured compound with each of the amino acids.
Applications of paper chromatography:
- Qualitative analysis:
- Involves the identification of compounds present in the mixture.
- Identification involves the use of Rf value based on Rf of standard compound.
- Quantitative analysis:
- It is done in the paper or after the removal of the component from the paper.
- The latter is generally preferred – the component is cut from the paper, extracted by a suitable solvent, measured by colorimeter or UV-Vis spectrophotometer.
- Alternatively, the extracted solution is evaporated in the vacuum to remove solvent,
- Thus, obtained residue is weighed.
- Preparative paper chromatography:
- Operates with large amount (gram quantity) of substances to yield substances enough for further work in the laboratory.
- Practically, it is done in Whatman No.3 paper. The sample is streaked.
- The separated bands are cut, extracted with suitable solvent and filtered.
- The filtrate is evaporated off in vacuum to yield the residue of the component.
- Specific application:
- Includes the separation of many organic and biochemical products.
- For example, it has been utilized in the determination of indole in whole urine and in the study of barbiturates, antibiotics, hormones, and amino acids, among others.