Gradient elution reequilibration time

Figure 12. Reequilibration of columns with micropellicular or porous stationary phases during gradient elution. Columns Micropellicular, Hy-Tach C-18 silica, 135x4.6mm porous, Vydac, C-4 silica, 135x4.6mm flow rate, 1.0 ml/min. temp., 80oC. Eluents were same as described in Figure 10. The columns were first equilibrated with 90% B and reequilibration was begun by a sudden drop in the concentration of B eluent to 22% in 0.1 min. Retention time of ribonuclease A was measured by gradient elution (22 to 90% B in 5 min) during column regeneration.

Chromatographic analysis times are rapid and after gradient elution column reequilibration time is short. 

High-pressure liquid chromatography (HPLC) formats that use reverse-phase analytical columns dominate the bioanalytical field. Isocratic elution formats are used for traditional LC-MS/MS bioanalysis, since these separations do not require additional time for column reequilibration. However, gradient elution is a more effective approach for cassette analysis in highthrough-put screening methods for discovery applications because of its versatility for the separation of compounds that have a wide variety of lipophilicity. 

Perhaps the worst problem of gradient elution separations is the need to reequilibrate the column with the initial solvent before a second sample can be run. An often-quoted rule of thumb is that up to 20 column volumes of the initial solvent may be necessary for this reequilibration process. The best test of reequilibration is the elution time of a weakly retained solute. These solutes will be greatly affected by an incompletely equilibrated stationary phase, and the retention time will vary. Cole and Dorsey have described a simple and convenient method for the reduction of column reequilibration time following gradient elution reversed-phase chromatography (119). Their method utilizes the addition of a constant 3% 1-propanol to the mobile phase throughout the solvent gradient to provide consistent solvation of the stationary phase. They noted reductions in reequilibration times of up to 78% ... 

The existence of a plateau of adsorbed ionic surfactant above the CMC permits rapid analyses using a micelle gradient. Beyond the CMC, changes in total surfactant concentration only change the concentration of micelles in the mobile phase the stationary phase is not affected. This means that the initial conditions can be recovered without reequilibration time. However, normally, the particular elution strength behavior of MLC makes gradient elution unnecessary. 

The application of ion-pair HPLC in the analysis of food colors is summarized on Table 8. As indicated, TBA has been the most widely used ion pair. It can be observed that using gradient mobile phase elution, a larger number of synthetic dyes can be separated. However, the mobile phase programming should include a return to the initial condition as well as reequilibration of the column by maintaining the initial composition for a period of time. This procedure provides reproducible result (222). 

Gradients can be used with equal ease for either ionization technique. In most cases, cycle time for system reequilibration (determined by the overall system dead volume) provides the practical limitation to their usage. If, for example, a particular HPLC pump/autosampler combination has 1.0 mL of dead volume (or dwell volume, the volume of all plumbing between where the solvents are mixed and the column head) and is operating at a flow rate of 1.0 mL/min (typical for APCI), then the lag time between when the gradient is initiated and when the correct solvent composition reaches the pump head is 1 min (1.0 mL/(1.0 mL/ min)). If the flow rate is only 0.2 mL/min (typical for electrospray), then the lag time will be 5 min. This means that a typical gradient run would require 5 min to initiate reequilibration plus whatever time is required for elution and final reequilibration (usually 10 to 20 column volumes). This is clearly an unacceptable time delay. 

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