Retention differences under gradient conditions

Calculate the mobile-phase composition that will result in an acceptable retention time under isocratic conditions This can be done by assuming a value (e.g., 8) for the slope S, of the dependence of the logarithm of the retention factor on the solvent composition. Alternatively, a third gradient experiment can be performed with a different gradient steepness. In the latter case, one obtains both S, and the intercept k for each solute. 

The retention of an analyte strongly depends on its ionization. As mentioned above, the non-ionized form of the sample has a much higher retention, up to a factor of 30 higher, than the ionized form. However, the difference is not the same for all analytes. For some analytes, the retention changes more, for others less. This is shown in Fig. 8, where we have plotted the retention times imder gradient conditions for more than 70 samples under basic versus acidic conditions. 

Fig. 8. Differences in the retention values of a large number of samples under gradient conditions as a function of pH.

Natural extracts generally contain molecules with highly different retention characteristics which cannot be separated under isocratic conditions. The application of gradient elution is a necessity for these types of natural samples. However, the optimization of gradient elution on the base of isocratic data is cumbersome and the prediction of retention in gradient elution from isocratic data is difficult. Retention in an isocratic system can be described by a polynomial function ... 

Gradient elution chromatography is a separation method that exploits the effect of the fluid phase composition on the retention behavior of the feed components. It is widely used, especially for analytical separations in the areas of the life sciences, in biochemistry, and in the biotechnologies e.g., separation of complex mixtures of proteins or peptides), hi its conventional implementations, SMB units are operated under isocratic conditions. The composition of the fluid phase, e.g., the organic modifier concentration, the pH, or the buffer concentration remain constant in all the sections of the SMB unit. However, it has recently been shown that SMB units can also be operated under solvent gradient mode (SG-SMB). Then, the feed and desorbent streams introduced have a different composition. The fluid phase composition is different in each section. It is chosen independently, in order to... 

The radiolabelled peptides can be separated under cold conditions by HPLC using the slow gradient, since their retention times differ by more than 10 min. Thus the labelled peptides were obtained with very high specific activity in purified form. The HPLC profile for Lu-OC l is shown in Fig. 16.7. Similar results were obtained for Lu-P3 and Lu-P4. 

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