Industrial chromatographic separation of mono-valent amino acids.
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The aim of the thesis was to optimise the separation and purify an amino acid solution consisting of mono-valent amino acids. Amino acids have historically been separated by chromatographic separation for analytical requirements but separation commercially has not been possible due to the close physical and chemical properties of the amino acids. The study looks at two amino acids, isoleucine and alpha amino butyric acid and used operating and process variables to conclude whether a commercial application is possible. A 3m glass column operating at atmospheric conditions was set up in the development laboratory at SA Bioproducts for test work. Pulse tests were carried out to evaluate the separation of the individual amino acids and the samples were analysed using HPLC. The column incorporated a jacket which allowed for constant temperature during the experiments. Experiments started initially with the column being tested for hydraulic consistency and this was done using resin in the Na+ ionic form and passing a NaCI feed stream over the resin as a pulse. A base case was chosen and this using a feed volume of 0.1 bed volume, an eluant flow of 1 bed volume per hour, feed concentrations of 2% and an operating temperature of 75°C. The resin was used in the Na+ ionic form, with a particle size of 320llm, and a height of 1.5m. Results of the base case indicated a low resolution of 38%, and low recoveries at 11.85% at purities of 86%. Evident from the chromatogram was that isoleucine was held more strongly than AABA by the resin as isoleucine was eluted last. Increasing the concentration of the feed to 4% moved the peaks closer and caused more overlapping thereby decreasing the recovery. Decreasing the concentration resulted in better separation. From the results it was concluded that the separation was dependant on concentration. The next variable which was studied was the effects of changing the feed pulse volume. For this the feed volume was increased to 0.2BV and this ended up shifting the chromatogram to the right, with lower separation efficiency being measured. The effects of eluant flowrate were studied next. Decreasing the eluant flowrate to O.SBVIh resulted in sharper peaks and less overlapping. The conclusion reached was that the higher the contact time between the amino acids and the resin, the greater would be the separation efficiency. Increasing the flowrate of the eluant reduced the contact time and more overlapping was observed. The effects of temperature were also investigated. The experiment carried out at 90°C, showed very similar separation to the base case with higher recoveries. Decreasing the temperature to 30°C decreased the separation efficiency drastically. The experiments proved that an increase in temperature increased the kinetics, and allowed the amino acids to enter and exit the bead quicker. Once operating conditions have been evaluated, the resin was converted to the Ca2+ ionic form, and all other conditions were run according to the base case. ' From the chromatogram it was obvious that the amino acids were held more strongly by the resin and the retention time was increased thereby increasing the recovery. Changing the particle size of the resin from 320~m to 220~m increased the separation efficiency due to the faster kinetics. Increasing the height of the resin was attempted by adding more resin to the column. The separation efficiency was increased. Finally a optimised experiment was attempted where a 2% feed solution was used, feed volume was 0.1 BV, temperature of 90°C, eluate flow of O.SBV Ih, resin in the Ca2+ ionic form, with a particle size range of. 21 0-230~m, and resin height of 1.8m. The chromatogram indicated high peak separation efficiencies and high recoveries. The results indicated that the study was feasible.