The experiment aims to identify two unknown amino acids using paper chromatography. To be specific, the retention factor or Rf values of standard amino acids was used to see which one matched that of the unknowns.
Chromatography can be defined as the separation of a mixture into various fractions by distribution between a mobile phase, which can be a gas or a liquid, and a stationary phase, a liquid or solid. Different substances have different affinities for the stationary and mobile phases. A substance that has no affinity for the stationary phase will remain in the mobile phase and move rapidly through the chromatographic system. On the other hand, a compound that has great affinity for the stationary phase will be selectively retained and will move very slowly. In an ideal case, each component will move through the system at a different rate resulting in a complete separation.
One chromatographic technique is paper chromatography. Here minute amount of substances can be separated from the mixture by its interaction with the mobile phase that moves up along a paper. The paper, with a thin layer of adsorbed water, acts as the stationary phase, while a liquid mixture serves as the mobile phase.
The identity of the unknown solution can be identified based on its retention factor (Rf) relative to that of the standard solutions. Rf uses the idea that the movement of a substance spotted on the paper is due to its affinity with the mobile phase. Thus it is calculated as the ratio of the distance travelled by the spot and the distance traveled by the solvent.
Rf=spot distancesolvent distance Eq. 1.
The paper chromatogram was produced by placing a 10 cm x 14 cm rectangular sheet of filter paper with spots of six amino acid solutions in a beaker containing a mixture of 60 % 1-butanol, 15 % glacial acetic acid and 25 % water (vol/vol/vol). The filter paper was prepared by connecting the ends with a staple to form a cylinder. The amino acid solutions (alanine, arginine, glycine, leucine, unknown 4, and unknown 2) were spotted using a toothpick 2.5 cm from the edge of the paper and placed approximately 1.5 cm apart. The covered set up was left undisturbed to let the mobile phase move up to the point that it was already a few millimeters below the top of the sheet. The paper was then dried and sprayed with ninhydrin solution to produce purple spots which corresponds to the amino acids.
Figure 1, on the other hand, shows the paper chromatogram produced in the experiment. The six purple spots correspond to the six amino acids solutions used in the experiment.
Discussion and Conclusion
Based on Figure 1, it is noticeable that the spots vary on the distance travelled relative to that of the solvent which agrees with the calculated retention factor on Table 1. From lowest to highest Rf values, the amino acids are arranged as glycine, arginine, alanine, and leucine. That arrangement can probably because of the difference in the polarity of the amino acids. Glycine, being the most polar retains more on the more polar stationary phase while the less polar leucine, with R= CH2CH(CH3)2, travels with the less polar mobile phase. The Rf values of the 4 amino acids were used to determine the identity of the unknowns simply by comparison. Based on the calculated Rf values of the unknowns which are almost similar, the unknowns are most probably glycine though there is a significant difference between the Rf values of the unknowns and the standard. The difference can be attributed to the error in taking the distance measurement as well as, the difference in the amount of amino acids spotted in the filter paper.
Ault, Addison. Techniques and Experimennts for Organic Chemistry. Boston: Allyn and Bacon, Inc., 1983. Print.
Spurlock, D. "Paper Chromatography Separation Class Notes." 16 February 2014. Indiana University Southeast. Web. 19 May 2014.