Cation Exchange Chromatography Lab Report

Separation of Amino Acids by Cation Exchange Chromatography
Introduction and Purpose:
Amino acids are small biomolecules that have a carboxylic acid backbone in common, as well as an amino group attached to a saturated carbon. There are many amino acids, but there are 20 most commonly know amino acids. Amino acids are the fundamenta building blocks of other biomolecules like proteins and ezymes (Davidson, 2015). This experiment examined a mixture of 3 amino acids.
The purpose of this experiment was to identify amino acids in a mixture through cation exchange chromatography and thin layer chromatography. This was done by examining the amino acids elution order from a cation exchange resin based on their isoelectric points and their different
This was done by spotting each of the 5 drop samples from each pH level, with each pH level being on a different piece of paper. Then the spot was allowed to dry and sprayed with ninhydrin to test for the presence of amino acid with gentle heat applied from hot plate.
TLC of Amino Acid Fractions:
Another TLC plate was spotted comparing the most concentrated fraction from each of the pH levels with the unknow mixture. The TLC developing jar was prepared with 4:1:1 propanol/acetic acid/water. The plate was placed in developing jar once spotted and allowed to develop until the solvent front reached ¾ of the height of the plate. Once the plate developed, it was removed and the solvent front was marked, then allowed to dry. Once dry the plate was sprayed with ninhydrin and gently heated on a hot plate until the spots developed. The spot distances were then marked and the Rf values were calculated.
Figure 1. Cation Exchange Chromatography
Figure 2 shows the relative Rf values from the fractions at each pH elution. The Rf value of the solution eluted with the pH 3 buffer reveals that Alanine was eluted first with the pH 3 buffer. The solution eluted with the pH 6 buffer had two Rf values of .43 and.62, revealing that the amino acid eluted with it was phenylalanine with another concentration of alanine. Finally, the fraction eluted with the pH 11 buffer had a Rf of .8, which tells us that lysine was eluted with the pH 11 buffer. The TLC separation of the elution with the pH 3 and pH 6 buffer were a little unclear, likely due to poor separation with the cation exchange. The isoelectric point (pI) for phenylalanine, alanine, and lysine respectively are 5.48, 5.98, and 9.74. When the pH of a solution is above the pI level of the amino acid it causes it to become more negatively charged (Komkova, 2010). Thus, when the amino acid becomes more negatively charged it is eluted from the column of ion exchange chromatography, due to the fact that the negative charged matrix would repulse the then negatively charged amino acid. When the pH 11 buffer was eluted it drew with it alanine from the mixture, this is congruent with the theory of ion exchange. However, it was not as defining with phenylalanine and alanine, as their isoelectric points are so close together.