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Paper Chromatography

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The objective of this experiment is to separate mixtures into their different components to identify and analyze the components of the mixture. The results will depict purple spots corresponding to where the amino acids are located.


The main hypothesis is to test whether amino acids can be successfully separated into two unknown mixtures through the process of paper chromatography. This experiment’s significance is widely applicable in pharmaceutical industries whereby the process is vital in preparing large volumes of pharmaceutical compounds, testing the level of purity for trace contaminants, ad separating chiral drugs such as thalidomide, sleeping peel and an antidote for morning sickness in pregnant women. The process is also applicable in hospitals to decipher the level of alcohol in the blood of a patient and perform occupation hygiene investigations such as contamination of surface by specified antineoplastic drugs. Thirdly, the procedure is helpful in forensic science to carry out an analysis of body fluids for illegal substances and to compare a sample obtained at a scene of crime to samples from suspects e.g. blood and fibre. In environmental science, the procedure is used to determine the pollution levels in air and drinking water for instance pesticides. Lastly, the process is employed in manufacturing plants to purify chemical needed to process a product such as pesticides. In this paper chromatography, the procedure involves a mobile phase, which is a liquid, and a stationary phase that is represented by a sheet of paper smeared with a thin layer of absorbed liquid. The paper is applied with the solution of the mixture to be separated as a minute spot towards the lower edge of the paper, and the paper submerged into a shallow layer of the mobile phase. Through capillary attraction, the mobile phase will creep up the paper and flows past the spot.

Materials and Procedures

A toothpick is first placed into the amino acid and spotted on the filter paper. Then the samples spotted on the filter paper are allowed to dry completely. When dried from a cylinder with a filter paper, spots should be located at the bottom of the paper. Then the paper is carefully stapled together, one staple at the top and another at the bottom. The solvent level should be ascertained to be below the line containing mine spotted amino acids. The next step involves allowing the chromatography to develop until the solvent line is 1cm from the top. While the filter paper is still wet, a line is drawn along the top of the solvent using a pencil. After the spot achieves visibility, the centre of each spot is marked using a pencil. Lastly, one should note the distance of the base line to the centre of each spot.

Data and Results

The molecules move repeatedly back and forth between the mobile phase (at the same speed as the liquid) and the stationary phase (where they are stopped). The R1 value for the spot is obtained by dividing the distance moved by the spot by the distance moved by the solvent front. Retention time (Rf) is the speed at which an analyte moves through the paper depending on its differential affinity to the paper and the solvent. The parameter is usually unit less.

Discussion and Conclusions

From the experiment, it is evident that provided there is insubstantial interaction between components or atoms within a mixture, the substances in question have unique behaviours and thus the possibility of separating substances with varied Rf values. Rf values are not very reliable since errors may occur in estimating of the spot canters whereby there may be minor variations in solvent compositions, as well as variable fits of watch-glass covers to beakers.

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