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 reactions with silica TLC plate. Ion exchange chromatography depends on the reversible exchange of ions in a solution with ions electrostatically bound an insoluble exchange matrix. Cation exchange involves an exchange matrix that has a negative functional group, therefore has positively charged counter ions, also known as cations. (UTA, n. d. ). These cations selectively bind to the negatively charged matrix and are removed by the solution flowing through.
This experiment used Dowex 50 resin, which is a cation exchange resin with a sulfonate group linked to the surface. Due to the negative charge of the resin, in can bind cations and elute as the buffer flows through the resin. Depending on the different isoelectric points of the amino acids, as pH is increased different amino acids become less positively charged. This allows for separation as the Dowex resin has a negative charge, as they come less positively charge and more negatively charged, they will repel from the resin and flow through the column.
Thin layer chromatography uses a stationary and a mobile phase to separate qualities of a compound (UCLA, 2015). The stationary phase in this experiment in the silica coating on the TLC plate, where as the mobile phase is the 4:1:1 propanol/acetic acid/ water solvent. Procedure: TLC of Amino Acid Standards: The TLC development jar was prepared with a solvent wick containg shallow layer of 4:1:1 propanol/acetic acid/water. Then, the TLC plate was spotted and labeled with each amino acid standard (Phe, Leu, Lys, and Ala) and mixture. Once the spots dried, the plate was placed in the development jar and allowed to develop.
When the solvent front reached at least threefourths of the way to the top it was removed and the solvent line was marked, then allowed to dry. Once dry, the TLC plate was sprayed with ninhydrin in enclosed box and gently heated on hot plate. Once the spots developed the TLC plate was removed from heat and the Rf values were calculated for each spot. Amino Acid Cation Exchange: A glass wool plug was placed in the bottom of a clean burette. 5 mL of Dowex 50 cation exchange resin was placed in clean beaker then suspended in 5 mL pH 3 citrate buffer and poured in column of burette.
Using a pipette the excess resin on the sides of the column was rinsed down with excess pH 3 buffer. Once settled the buffer was drained until the meniscus reached the surface of the resin. 0. 5 mL of the amino acid mixture was applied to the surface of the column by pouring it down the side of the burett and then rinsed down the wall of the burette using 0. 5 mL of pH 3 citrate buffer. The column was opened and drained through until the buffer reached the surface of the resin, this tube was labeled “Flow Through”.
This step was repeated to make sure that the amino acid mixture was absorbed on the column. 0 mL of pH 3 buffer was then added to the column. The stopcock was opened to collect approximately 40 drop fractions into 5 separate test tubes, labeled pH 3 Fraction 1-5, until the buffer reached the surface of the column. Next 10 mL of pH 6 citrate buffer was added to the column and 40 drop fractions were collected again using the same method into 5 separate test tubes, labeled pH 6 Fraction 1-5. Finally, 10 mL of pH 11 CAPS buffer was added and 40 drop fractions were collected into 5 separate test tubes, labeled pH 11 Fraction 1-5.
While the column was running, a spot from each of the test tubes was placed onto a piece of filter paper to check for spot flow through. 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 34 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 apparatus Figure 2.
Thin Layer Chromatography apparatus Results: Table 1. TLC of Amino Acid Standards and Rf values TLC of Amino Acid Standards StandardSpot Distance (cm) Rf (Spot Distance/Solvent Front) Unknown Mix. 90, 2. 3, 3. 3 . 16, . 41, . 59 Phenylalanine 3. 3. 59 Leucine 3. 4. 61 Lysine . 90. 16 Alanine 2. 3. 41 Solvent Front = 5. 6 cm Table 2. TLC of Amino Acid Fractions and Rf values TLC of Amino Acid Fractions Spot Type Spot Distance (cm) Rf (Spot Distance/Solvent Front) Unknown Mix. 8, 2. 4, 3. 7 . 13,-43,. 62 pH 3 2. 3. 43 pH 6 2. 4, 3. 7 . 43,. 62 pH 11. 80. 3 Solvent Front = 6. 0 cm Discussion: The Rf values were calculated by dividing the distance the spot moved by the distance the solvent front moved.
From Table 1, it can be seen that the unknown mixture contained the amino acids phenylalanine, alanine, and lysine. This is given by comparison of the Rf values of the three dots present in the unknown mixture with those of the amino acid standards. Alanine had an Rf value of . 41, phenylalanine had an Rf value of. 59, and lysine had an Rf value of . 16. The spots of the unknown mixture had Rf values of . 6,. 41, and . 59, matching those of lysine, alanine, and phenylalanine. Thus, the unknown mixture contained these amino acids. During the cation exchange chromatography, the most concentrated fraction from each pH buffer elution were spotted on a TLC plate with the unknown mixture.
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. 2, 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 (pl) for phenylalanine, alanine, and lysine respectively are 5. 48, 5. 98, and 9. 74. When the pH of a solution is above the pl 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. The buffer with pH 6, has a pH that is higher than the pl’s of both alanine and phenylalanine causing them both to elute in predominate concentrations.
