Studies on the separation of simple proteins by chromatographic adsorption

• Main Author: Stewart, Ernest V.
• Published: BYU ScholarsArchive 1950
• Subjects: Proteins; Chemistry
• Online Access: https://scholarsarchive.byu.edu/etd/8361

Due to the complex nature of the protein molecule no adequate system of classification and separation of the simple proteins has yet been devised . Present methods of separation have many disadvantages. Because of these disadvantages new methods of separation were investigated. Chromatographic adsorption which makes use of the difference in adsorption affinities of substances for an adsorbent seemed to offer possibilities of an accurate and simple method for protein separation. Aluminum oxide was chosen as the most suitable adsorbent from such substances as fuller's earth, activated carbon, silica gel, and bauxite. To a column of alumina a solution or egg albumin was added. The fluid issuing from the column was caught in ml. portions and checked qualitatively with biuret solution for the presence of protein. Quantitative measuremetts of the protein content of each ml. fraction was accomplished by the use of biuret solution and a colorimeter. The alumina and egg albumin chromatogram was extruded and the presence of adsorbed zones on the column investigated by biuret solution. A zone 2.5 to 3 cm. long starting at the top of the column was formed. Efforts to elute this zone with water, salt solutions, slightly acidic and basic solutions, and ethanol-water mixtures failed. The fluid issuing from the column on development gave positive tests for protein content. This experimsnt showed that egg albumin was separated into two components by adsorbing it on alumina. Quantitative examination of the protein content of the fluid issuing from the columns showed that there was no variation of the ml. fractions from the alumina column, the silica gel column, and the calcium carbonate column. However, variations occurred in the ml. fractions taken from the activated carbon columns and the fuller' s earth column. One-dimensional and two-dimensional paper chromatography were tried. The paper was cut in strips for one-dimensional chromatography and left in sheets for two-dimensional chromatography. Acetate buffers of various pH's were used as developing solvents. No separation of the egg albumin by these methods was detected.