Cation Exchange Chromatography

Amino acids are a highly important class of molecules, which are the monomeric subunits of proteins, enzymes, and peptides in biological systems. Amino acids contain a carboxylic acid group, an amine group, and a unique side chain, which determines its properties. There are more than 500 amino acids; however, only 20 are commonly found1. Due to their importance in biological systems, it is important to be able to experimentally analyze and isolate amino acids by separation techniques. One such method of separation is thin layer chromatography (TLC). In this method, a silica coated aluminum plate is spotted with the solution being separated, and the plate is placed in a developing jar containing a small amount of solvent. With time, the solvent
In cation exchange chromatography, a negatively charged resin is used. A commonly used resin is Dowex, which contains linked sulfonate groups that contribute to its negative charge. As a solution is passed through the resin, molecules that exhibit greater positive charges are slowed due to their ionic interactions with the resin. Negatively charged molecules, on the contrary, are repelled by the resin and pass through quickly. Since amino acids contain a carboxylic acid group and an amine group, amino acids exhibit different net charges at different pH. In general, at a low pH, the amine group is protonated causing the amino acid to exhibit a net positive charge. At a high pH, the amine group and the carboxyl group become deprotonated causing the amino acid to have a net negative charge. At some intermediate pH, the amino acid becomes a zwitterion, which carries no net charge due to the amine group being protonated and the carboxyl group being deprotonated. This pH is usually referred to as the isoelectric point, and it is unique to each amino acid. Thus, if a mixture of amino acids is to be separated, cation exchange chromatography can be conducted by starting with a low pH mobile phase. The initial low pH causes the amino acids to attract strongly to the resin due to their net positive charge. If the pH of the
The mixture may have contained leucine, alanine, phenylalanine, or lysine. Leucine contains an isobutyl side chain and is considered a non-polar amino acid at physiological pH. The isoelectric point of leucine is 5.98. Alanine, a non-polar amino acid, contains a methyl side chain and has an isoelectric point of 6.01. Phenylalanine contains a benzyl side chain and is a non-polar amino acid. The isoelectric point of phenylalanine is 5.48. The side chain of lysine consists of a linear chain of four carbons and a terminal amine. It is basic and polar due to the amine group of the side chain. Lysine has an isoelectric point of 9.74, and it can also participate in hydrogen bonding. The structures of each of these amino acids are shown in Figure