Paper chromatography is the most basic type of chromatographic separation techniques. Whenever confronted with an issue regarding the analytical separation and identification of components from a complex mixture, paper chromatography will mostly be the first choice of a chemist. You will find all the relevant information about paper chromatography including its working principle, scope and applications in this article, so keep reading.
What is paper chromatography
Paper chromatography is a chromatographic separation process usually used for separating a mixture consisting of easily differentiable polar and non-polar components. It is a type of normal phase, liquid-liquid chromatography. Water molecules trapped in the cellulose fibers of the adsorbent paper acts as the stationary phase while organic solvents are used as mobile phase in paper chromatography. The mobile phase travels up the stationary phase through capillary action, sweeping the solute molecules present in the analyte mixture along with it.
Working principle of paper chromatography
Paper chromatography separates analyte components on the basis of partitioning between the stationary phase and the mobile phase solvent. Therefore, it is also sometimes known as a partition chromatographic technique. Organic solvents of varying polarities are used as mobile phase while performing paper chromatography.
Considering the fact that the stationary phase is always more polar than the mobile phase in paper chromatography thus polar components from the analyte mixture gets strongly retained onto the stationary phase. Relatively less polar or non-polar components develop stronger interactions with the mobile phase, stay dissolved in it for long thus travel farther up the chromatographic paper. Components separate and produce spots at different positions on the chromatogram where the chromatogram is the end product obtained in result of a chromatographic separation.
Retardation/retention factor or relative-to-front (Rf) values for each spot can be calculated and compared with Rf values for pure compounds. This allows identification of the sample components. In this way, paper chromatography allows qualitative analysis. This technique however is not very useful for quantitative analysis. It is helpful for small scale laboratory analysis only.
How to perform paper chromatography
The following step-by-step guide can be used for the qualitative analysis of a mixture M into its constituents X, Y and Z.
PART I: Analyte Separation
Step I: The mobile phase is prepared by choosing the organic solvents which can best dissolve all the constituents of the mixture M. The most popular choices of solvents used in paper chromatography includes ethanol, hexane, toluene, acetone and/or their aqueous solutions. The solvents chosen are mixed in a specific ratio to prepare the mobile phase.
Step II: A rectangular sheet of chromatographic paper is selected. A Whatmann No.42 filter paper with a pore size of 2.5 µm is commonly used for this purpose. A lead pencil is used for drawing a thin base line,1 cm above the edge of the paper.
Step III: A small sample spot using a thin capillary tube is placed at the base line.
Step IV: The spots of reference compounds i.e., pure X, Y and Z are also applied at marked positions, at the same distance from the edge of the chromatographic paper as the sample spot.
Step V: This paper is then suspended into the chromatographic tank, covered with a lid at the top. The purpose of covering the container is to ensure the saturation of its internal atmosphere with vapours of the mobile phase. This action prevents any unwanted atmospheric interruption in the chromatographic separation process.
Step VI: The chromatographic paper is immersed in the mobile phase such that the spots lie just above the surface of the solvent.
Step VII: The mobile phase is allowed to travel up the sheet of paper by capillary action. As it happens, the mixture M separates into different coloured spots occupying different positions at the chromatographic paper.
In case the compounds do not appear as coloured spots themselves, the paper can be sprayed with a chemical that does so. For example, ninhydrin spray can be used to reveal the otherwise colourless amino acids as bluish spots on the chromatogram. Molisch reagent can be used for sugars in general while Tollens’ reagent is specifically used for reducing sugars. Colourless spots can also be visualized by placing the chromatogram in a beaker containing iodide crystals or via UV irradiation.
Result: Mixture M lies separated into its constituent components on the chromatogram. The individual compounds X, Y and Z can then be identified in the consequent steps.
Part II: Analyte Identification
Step VIII: The positions of the individual spots obtained from the mixture M on the chromatogram can be compared with how far the pure X, Y and Z travelled up the chromatogram. This helps as a general identification guide for the sample components.
Step IX: Maximum distance travelled by the mobile phase is marked as the solvent front ds. Rf values can be calculated for each component using the formula given below and compared with the Rf values of X, Y and Z to confirm their identification.
where dc= distance travelled by analyte component and ds= maximum distance travelled by the mobile phase
Each analyte component has a characteristic Rf value for a given solvent.
A supposed Rf value calculation :
The Rf values calculated for components obtained from mixture M reasonably matches with the Rf values given for pure compounds X, Y and Z thus their identification is confirmed.
Note: Minor discrepancies may be present in the calculated and the given Rf values. The Rf value has no unit because it is a ratio.
Types of paper chromatography
As the sample spot is applied onto the chromatographic paper, the solvent from the mixture evaporates. As a result, polar solutes from the sample mixture gets retained onto the paper by hydrogen bonding to the OH functional groups of the cellulose fibers or with the H2O trapped in these cellulose layers. The highly polar molecules, strongly retained on paper are less likely to be drawn upwards by the mobile phase. Weakly retained molecules or those with greater solubility in the mobile phase solvent on the other hand ascends further up the chromatogram.
The paper chromatography in which the solvent travels up the stationary phase is called ascending paper chromatography.
In contrast to that, the descending paper chromatography is performed by running the mobile phase solvent over the stationary phase in a downward direction. The solvent in this case is situated at the top of the chromatographic chamber. It travels down, carrying analyte components with it under the action of gravity.
The paper chromatography performed as a hybrid of the two techniques is called ascending-descending paper chromatography. A glass rod is used in this case, the solvent ascends through the upper portion while it starts runs in a descending manner as the glass rod rolls the other way round.
The chromatogram can also be developed as concentric circles in paper chromatography. This type of paper chromatography is called radial/circular chromatography. In this type of chromatography, the mobile phase is placed in a petri dish and a circular paper is situated above the petri dish to act as the stationary phase support.
Another type of paper chromatography is two-way paper chromatography. This type of paper chromatography is specifically important for separating the mixtures which have components possessing relatively similar Rf values. To prevent the risk of spot overlapping and poor resolution, two-way paper chromatography is applied.
The chromatographic separation in this type of paper chromatography is performed as normal followed by a 90° rotation of the chromatographic paper and a re-run of a different mobile phase composition in a different direction. It is rarely possible that the Rf values of the two components coincide in two different solvents run in different directions thus adequate separation occurs.
Uses of paper chromatography
- Paper chromatography is specifically important for the examination and separation of coloured compounds such as ink components, dyes and pigments etc.
- It can help inspect cosmetic formulations.
- It helps study the process of fermentation and ripening of fruits.
- Paper chromatography aids in the separation and identification of amino acids from a complex mixture.
Limitations of paper chromatography
- Paper chromatography is not compatible for large scale analysis.
- Easily degradable and volatile chemical compounds cannot be analyzed via paper chromatography.
- Quantitative analysis is not possible with this type of chromatography.
Paper chromatography is a primitive chromatographic analysis technique which is the easiest to perform, saves plenty of time and does not require any costly apparatus. Thus, it can be feasibly applied for qualitative separation and identification of simple organic and inorganic compounds. But for greater accuracy and sensitivity quantitative chromatographic analysis, superior chromatographic techniques are preferred.
You can learn the basics of different types of chromatography here.
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3. Masoodi, K. Z., S. M. Lone and R. S. Rasool (2021). Chapter 29 – To perform paper chromatography. Advanced Methods in Molecular Biology and Biotechnology. K. Z. Masoodi, S. M. Lone and R. S. Rasool, Academic Press: 159-161.
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