Study On Conical Columns For Preparative Liquid Chromatography

Preparative high performance liquid chromatography (PHPLC) is being used for obtaining pure compounds. Better separation efficiency and higher loadability for samples can improve production rate and save operating costs. Conical columns with larger inlet ID than outlet ID were considered advantageous in preparative separations owing to increased sample load for the same amount of stationary phase.The dynamic flow profile inside a conically shaped column for semi-preparative liquid chromatography was studied by using a self-made device that could observe the movement of colored sample in the column clearly and truly. The transparent chromatographic columns made of polymethyl methacrylate (PMMA), packed with C18 bonded silica, were immerged into a cubic pool filled with innoxious solvent of glycerol to eliminate the cylindrical and conical lens effect. The flow profiles of colored iodine solution dissolved in cyclohexane visualized clearly when we used cyclohexane as mobile phase, even in central point injection mode, since the refractive indices of stationary phase C18, column wall and the mobile phase are very close.The dynamic flow profiles and column efficiencies in conically shaped semi-preparative liquid chromatographic columns (inlet I.D. larger than outlet I.D.) with three different angles (7, 10 and 15) were studied and compared with cylindrical column of the same length and internal volume. The dynamic flow profiles of sample band in the conical column of 7 and 15 were parabolic andreverse parabolic shapes, respectively. We found that plug-like flow profiles were generated in the conical column of 10 during the whole migration process. The conical column of 10 showed the highest column efficiency under the same conditions as the other three columns. The larger sample capacity and less dilution effect also showed that the conical column was advantageous over the cylindrical column.A carmine and brilliant blue mixture was used as a probe to test the separation ability of the columns. The resolutions of the two compounds on the conical column of 7, 10, 15, and on the cylindrical column were 0.6, 1.57, 1.29 and 0.8 respectively.Many factors influencing the flow profile and column efficiency were studied, such as frits in the header of columns, injection modes, methods of column packing, packing material and column length.Real-life samples were separated on the conical column with plug-like flow profile.The possible reason that produced different flow profiles and column efficiencies in different angles of conical columns was explained based on hydrodynamics and chromatographic theory.