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Separation of Amino-Acids by Reversed Phase Chromatography: Development of a Model Mixture Dedicated to the Study of Solvent Gradient Chromatography Processes

Abstract : Solvent gradient is currently used in analytical chromatography and presents an increasing interest in preparative chromatography. The applications relate to complex separations of mixtures where the target species has an intermediate affinity for the solid support compared to that of the other species, which is often the case during the purification of biomolecules extracted from natural raw materials or resulting from biotechnologies. In this case, separation leads to three fractions, impurities weakly retained, an intermediate fraction, and impurities strongly retained. An inexpensive and non-toxic mixture, containing five amino-acids, was identified as a good model mixture for such studies. These amino-acids were selected by taking into account their non-polar and hydrophobic nature. They were selected because of their aptitude for being easily detected in UV-visible spectroscopy without derivation. Separations were carried out by reversed phase chromatography. An experimental study first enabled determining the adsorption isotherm parameters of each amino-acid on an analytical column then on a preparative column for various organic solvent fractions. An empirical law was chosen to express the retention factor k as a function of the mobile phase composition ( k=f ( x(M))). An approach based on the column modeling by mixed cells in series enabled to simulate the separations obtained on the two types of columns. The use of the laws giving retention factor k as a function of the organic solvent fraction xM then made it possible to carry out simulations for various solvent gradients. A simple methodology was developed to calculate the optimal solvent gradient. The gradient was optimized by minimizing the separation time and by respecting a constraint on the peaks resolution of the two species which are the most difficult to separate. A really good adequacy was observed between the experimental simulations and results for the weakly retained fraction as well as for the intermediate fraction which comprises components difficult to separate. Adequacy is not as good for strongly retained fraction but it is worth using these two species in the model mixture. This model mixture proves to be interesting to study solvent gradient preparative chromatography processes. The retention of these compounds is sensitive to the organic solvent fraction. It is easy to get these products which are inexpensive, non-toxic, and detectable without any derivation.
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Nawal Tlili, Charles Bonnin, Mouna Lazrag, Mathieu Weber, Laurence Muhr. Separation of Amino-Acids by Reversed Phase Chromatography: Development of a Model Mixture Dedicated to the Study of Solvent Gradient Chromatography Processes. Separation Science and Technology, Taylor & Francis, 2014, 49 (14), pp.2182-2192. ⟨10.1080/01496395.2014.926372⟩. ⟨hal-01276654⟩



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