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BACKGROUND
Chromatography is used to separate mixtures of substances into their individual components. All forms of chromatography work on the same principle. They all have basic requirements of stationary phase (a solid or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates based on their affinities toward stationary phase and mobile phase.
In paper chromatography, the stationary phase is a very uniform adsorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.1
Retention (or) retardation factor (Rf)
The ratio of the distance travelled by the solute to the distance travelled by solvent. The distance travelled relative to the solvent is called the Rf value
The value of retardation factor should lie between 0-1.it has no units.
The sequence of steps involved during the reaction between ninhydrin and amino acids to produce purple (or) yellow-orange colour.2
Step-I
Step-II
Advantages
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Simple method.
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Very less expensive to perform.
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Fewer amounts of samples and solvent are required.
Disadvantages
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It can be used as a preparative technique because we can’t apply a large quantity of sample.
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It can’t be used in quantitative analysis.
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Doesn’t allow the separation of complex mixtures.
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This is one of oldest method.3
Aim: To perform Ascending paper chromatography and to identify the unknown mixture of components present in the given sample.
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REQUIREMENTS
Apparatus: Glass beakers
Whatmann filter paper
Petridishes
Measuring cylinder
Developing chamber and capillary tubes etc.
Chemicals: n-butanol
Glacial acetic acid
Distilled water (4:1:5)
Amino acids (Tryptophan and threonine)
Ninhydrin reagent.
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PROCEDURE
Solvents system and its preparation methods
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n-butanol and water are taken in 4:5 ratios in a conical flask and allow it to saturate for 24 hours. By using separating funnel separate n-butanol and water.
The saturated n-butanol and Glacial acetic acid are taken in the ratio of 4:1 which can be used as a solvent system (or) mobile phase.
Ascending paper chromatography
The procedure for ascending paper chromatography method is quite simple as compared to other methods of chromatography.
The chromatography paper is cut into rectangular strips and marks a line on the paper with pencil at about 2 cm from the bottom. With the help of capillary tube, the samples are applied at different points on the starting line Now, placed the chromatography paper in the developing chamber, which contains the mobile phase. While placing the paper, it is important that the solvent level should not reach the starting line or the sample spots and paper shouldn’t touch the walls of the developing chamber. After sometime the solvent rises up the paper or the stationary phase by capillary action and dissolves the sample. The components of the sample move along with the solvent in upward direction.
Checked if the solvent has reached near the top level of chromatography paper. Then the paper is removed when it reaches the top and marked the level with pencil. This level (or) height is called the “solvent front”. By using UV light, ninhydrin or iodine vapors examined the different spots of varied colors. Each spot represents a specific component of the sample.4,5
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CONCLUSION
For ascending paper chromatography
The distance moved by tryptophan and threonine is …… cm and ….. cm respectively, and the solvent is ……cm
Rf value of tryptophan is ……
Rf value of threonine is ……..
Rf value of unknown mixture is ….. & …..
By performing the Ascending paper chromatography, the distance moved by the sample and unknown mixture is noted and by substituting these values in the given Rf formula, the Rf values of tryptophan, threonine and unknown samples are known.
By performing the Ascending paper chromatography Rf values of both tryptophan and threonine are found to be …… & …… respectively, and the unknown samples are found to be tryptophan and threonine.
By performing the radial paper chromatography Rf values of both tryptophan and threonine was found to be ….. & ….. respectively, and the unknown samples were found to be tryptophan and threonine.
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REFERENCES
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http://www.chemguide.co.uk/analysis/chromatography/paper.html.
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http://en.wikipedia.org/wiki/Ninhydrin.
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http://wiki.answers.com/Q/Disadvantages_of_using_paper_chromatography.
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Dr. S. Ravi Shankar, 2010, Text book of Pharmaceutical Analysis, Page no: 15.8
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http://www.buzzle.com/articles/paper-chromatography.html.
