Question 1 – What is the basic principle of Paper Chromatography?
Answer – Paper chromatography is a form of liquid chromatography where the components of a mixture of organic compounds get separated as unique spots by unidirectional flow of the developing liquid mobile phase solvent mixture over the filter paper to which a spot of the sample is applied. The distance travelled by each component is specific under the given set of operational conditions.
Question 2 – Why the developing solvent mixture is prepared fresh before use?
Answer – The developing liquid phase comprises of a pure solvent but more often it is a mixture of two or more solvents in specified proportions. In case solvents are mixed and stored for long periods there could be loss of volatile component which will alter the mixing proportions.
Question 3-. Why is it necessary to cover the developing chamber during the paper development?
Answer – During the chromatogram development chamber is covered.This is essential as the environment inside the chamber should remain saturated with the solvent vapour. Development times can vary from about an hour to several hours and a saturated environment prevents losses due to evaporation
Question 4-What are the common techniques used for detecting colourless spots?
Answer – It is easy to distinguish coloured spots visually but for colourless compounds alternate techniques need to be adopted which can be specific or non-specific.
A common non-specific method is suspension of developed chromatogram in iodine vapour. Most organic compounds show up as brown spots.
The sheet is viewed in a UV Viewing cabinet under 366 nm and 254 nm wavelength lamp illumination. On observation the spots need to be carefully marked with a pencil for Rf calculations.
Under specific methods amines and amino acids are observed by spraying heated paper on development with 0.2% hydrazine. Deep blue or purple spots begin to appear.
Alkaloids – Dragendroff’s reagent spray results in orange or orange yellow spots.
Aldehydes&Ketones – 2,4-DNPH spray in methanol and sulphuric acid results in orange or yellow spots.
Question 5– Why should the samples have reasonable solubility which is neither too high or too low in the developing solvent mixture
Answer – The samples should have a medium solubility in the developing solvent mixture.Too high a solubility will lead to transfer of the component alongwith the solvent front and on the other hand if the solubility is too low the component will not be carried by the solvent mixture and will remain close to the initial applied spot. In either case the resolution of the mixture components will be low. Thus reasonably good resolution can be obtained for medium solubility of compounds in the solvent mixture.
Question 6-What information you get from the Retardation factor value?
Answer – Retardation factor Rf is a measure of the separation of a particular component. It is expressed as
Rf = distance moved by the component spot/ distance moved by solvent front
Rf is a unit less quantity and lies between 0and 1.A value of 0 indicates no separation has taken place and 1 represents that the component has moved entire length alongwith the solvent front. In case two spots have same value of Rf it indicates that they are not resolved. At least a difference of 0.05 is necessary to discern the separation between two spots.
Question 7 – Can you remember the various paper chromatography techniques?
Answer – Paper chromatography separations are classified in accordance with the direction of flow of mobile phase along the filter paper.
Ascending paper chromatography – the carrier liquid moves from bottom upwards.
Descending paper chromatography – the carrier liquid trough is on top and mobile phase moves downward on the filter paper.
Ascending – descending paper chromatography – The paper is rolled downward over the rod at the top. On reaching the top in ascending mode it starts downward movement in the next phase.
Two – dimensional paper chromatography – After developing the chromatogram in either ascending or descending mode it is taken out, dried and developed again after turning by 90° in either the same liquid or another liquid mobile phase.The spots spread across the sheet and closely overlapping spots get resolved.
Circular paper chromatography – the mobile phase moves radially outwards from the centre of a circular piece of paper. In this mode the mixture components get resolved radially.
Question 8 – What are essential criteria for selection of suitable solvents for paper chromatography?
Answer – Solvents are selected on the basis of solubility of the sample components. In general it is advisable to keep in mind:
Solvents are not toxic or carcinogenic.
Solvent constituents of mixture should not react with any of the sample constituents.
Solvents selected should not interfere in detection of separated spots.
Solvents should not be highly volatile as loss of components can result in change of mixture composition.
Question 9-Why paper chromatography has retained its applicability in the face of a emergence of advanced instrumental techniques?
Answer- Chromatographic technique of analysis has seen an impressive growth over time. Such advances have increased laboratory throughputs lowered limits of detection and has made forays into new areas of applications. Paper chromatography has retained its ground till date and is popular in laboratories across the world. Some of the reasons for this are:
Low cost of analysis and freedom from maintenance.
Separated spots are visible for coloured compounds and colourless compounds can be viewed by using alternate techniques.
Minimum operation and training requirements.Solvent consumption is much less as compared to more sophisticated techniques.
Paper chromatography serves as a good demonstration of basic concepts of separation for school and undergraduate students.
Question 10-What are the limitations of paper chromatography technique?
Answer – Paper chromatography has some limitations such as:
Semi-quantitative in nature.
Overlapping of spots of components having close Rf values.
Higher concentration of components often leads to streaking instead of well-defined spots.
Errors in Rf calculations can result from uneven flow of solvent front. This can be caused by running out of solvent at the bottom of the chamber, uneven cutting of the filter paper or unevenness of the bottom of the development chamber.
Improper sample spotting, spotting below the marked line resulting in dipping into the solvent or accidental dipping of spot into solvent while inserting the paper into the solvent chamber.