Paper Chromatography

By- Dr Ekta Khare

 Paper Chromatography
• Paper chromatography definition explains that is an inexpensive and powerful
analytical technique, which requires a piece of paper or strips serving as an
adsorbent in the stationary phase across which a particular solution is allowed to
pass.
Or
• Paper chromatography (PC) is a type of planar chromatography whereby
chromatography procedures are run on a specialized paper.
• PC is considered to be the simplest and most widely used of the
chromatographic techniques because of its applicability to isolation,
identification, and quantitative determination of organic and inorganic
compounds.
• It was first introduced by German scientist Christian Friedrich Schonbein (1865).
• This analytical tool employs very few quantities of material.
 Principle of Paper Chromatography
• Paper chromatography is a form of liquid chromatography where the basic
principle involved can be either:
– partition chromatography
– adsorption chromatography
• Paper Partition chromatography: In paper chromatography separation of
component is distributed between phases of liquid.
• Here, one phase of liquid is water that is held amidst the pores of filter paper
and the other liquid is the mobile phase that travels along with the filter paper.
• Separation of the mixture is the result that is obtained from the differences in
the affinities towards the water and mobile phase when travelling under
capillary action between the pores of the filter paper.
• Paper Adsorption chromatography: Paper impregnated with silica or alumina
acts as adsorbent (stationary phase) and solvent as mobile phase.
 Instrumentation of Paper chromatography
• Stationary phase & papers used
• Mobile phase
• Developing Chamber
• Detecting or Visualizing agents
1. STATIONARY PHASE AND PAPERS
• Whatman filter papers of different grades like No.1, No.2, No.3, No.4, No.20, No.40, No.42 etc
• In general the paper contains 98-99% of α-cellulose, 0.3 – 1% β -cellulose.

Other modified papers

• Acid or base washed filter paper
• Glass fiber type paper.
• Hydrophilic Papers – Papers modified with methanol, formamide, glycol, glycerol etc.
• Hydrophobic papers – acetylation of OH groups leads to hydrophobic nature, hence can be used for
reverse phase chromatography.
• Impregnation of silica, alumna, or ion exchange resins can also be made.
 ... Instrumentation of Paper chromatography
2. PAPER CHROMATOGRAPHY MOBILE PHASE
• Pure solvents, buffer solutions or mixture of solvents can be used.
3. CHROMATOGRAPHIC CHAMBER
• The chromatographic chambers are made up of many materials like
glass, plastic or stainless steel.
• Glass tanks are preferred most.
• They are available invarious dimensional size depending upon
paper length and development type.
• The chamber atmosphere should be saturated with solvent vapor.
 Steps in Paper Chromatography
• In paper chromatography, the sample mixture is applied to a piece of filter paper, the edge of the paper is
immersed in a solvent, and the solvent moves up the paper by capillary action. The basic steps include:
Selection of Solid Support
• Fine quality cellulose paper with defined porosity, high resolution, negligible diffusion of the sample, and
favoring good rate of movement of solvent.
Selection of Mobile Phase
• Different combinations of organic and inorganic solvents may be used depending on the analyte.
• Example. Butanol: Acetic acid: Water (12:3:5) is a suitable solvent for separating amino acids.
Saturation of Tank
• The inner wall of the tank is wrapped with filter paper before the solvent is placed in the tank to achieve
better resolution.
Sample Preparation and Loading
• If the solid sample is used, it is dissolved in a suitable solvent. Sample (2-20ul) is added on the baseline as
a spot using a micropipette and air dried to prevent the diffusion.
Development of the Chromatogram
• Sample loaded filter paper is dipped carefully into the solvent not more than a height of 1 cm and waited
until the solvent front reaches near the edge of the paper.
Different types of development techniques can be used:
ASCENDING DEVELOPMENT
• Like conventional type, the solvent flows against gravity.
• The spots are kept at the bottom portion of paper and kept in a chamber with mobile phase solvent at the
bottom.
DESCENDING TYPE
• This is carried out in a special chamber where the solvent holder is at the top.
• The spot is kept at the top and the solvent flows down the paper.
• In this method solvent moves from top to bottom so it is called descending chromatography.
ASCENDING – DESCENDING DEVELOPMENT
• A hybrid of above two techniques is called ascending-descending chromatography.
• Only length of separation increased, first ascending takes place followed by descending.
CIRCULAR / RADIAL DEVELOPMENT
• Spot is kept at the centre of a circular paper.
• The solvent flows through a wick at the centre & spreads in all directions uniformly.
TWO-DIMENTIONAL CHROMATOGRAPHY
• This helps in resolving substances that have similar Rf values.
TWO-DIMENTIONAL CHROMATOGRAPHY
Drying of Chromatogram
• After the development, the solvent front is marked and left to
dry in a dry cabinet or oven.
Detection
• Colorless analytes were detected by staining with reagents
such as iodine vapor, ninhydrin, etc.
• Radiolabeled and fluorescently labeled analytes were detected
by measuring radioactivity and fluorescence respectively.
 Rf values
• Some compounds in a mixture travel almost as far as the solvent
does; some stay much closer to the baseline.
• The distance traveled relative to the solvent is a constant for a
particular compound as long as other parameters such as the type
of paper and the exact composition of the solvent are constant.
• The distance traveled relative to the solvent is called the Rf value.
• Thus, in order to obtain a measure of the extent of movement of a
component in a paper chromatography experiment, “Rf value” is
calculated for each separated component in the
developed chromatogram.
 Applications of Paper Chromatography
• To check the control of purity of pharmaceuticals,
• For detection of adulterants,
• Detect the contaminants in foods and drinks,
• In the study of ripening and fermentation,
• For the detection of drugs and dopes in animals & humans
• In analysis of cosmetics
• Analysis of the reaction mixtures in biochemical labs.
 Advantages of Paper Chromatography
• Simple
• Rapid
• Paper Chromatography requires very less quantitative material.
• Paper Chromatography is cheaper compared to other
chromatography methods.
• Both unknown inorganic as well as organic compounds can be
identified by paper chromatography method.
• Paper chromatography does not occupy much space compared to
other analytical methods or equipments.
• Excellent resolving power
 Limitations of Paper Chromatography
• Large quantity of sample cannot be applied on paper
chromatography.
• In quantitative analysis paper chromatography is not effective.
• Complex mixture cannot be separated by paper
chromatography.
• Less Accurate compared to HPLC or HPTLC