Step-gradient pumps

Fig. 6. Siemens step-gradient pump, a = eluent from the pump (high pressure), b = coil filled with solvent A, c = coil filled with solvent B, d = re-fill A, e = re-fill B, f = purge A, g = purge B, h = eluent split to b and/or c, i = coumutation valve, j = to column. When the valve is in position 1 the liquid from the pump flows directly from h to j, in position 2 it flows from h through b to j and in position 3 from h through c to j. Courtesy Siemens AG.

Step 4) Precolumn clean-up not shown in Figure 5.4. After the heart-cut analytes have been transferred to the analytical column, a step-gradient programme is used to flush the precolumn of the more strongly retained compounds. An additional pump configuration makes precolumn clean-up possible while the analysis is running. 

An isocratic system is used with single solvents, a premixed solvent mixture, or step gradients. It has the advantage of needing only a single pump, no mixer, and no gradient controller. Because of this, isocratic systems are simpler and... 

Beato BD, Gray DD, Wong P, Hampsch J, Peters S. Ramifications of pump-switching step gradients for hioanalytical LC/MS/MS assays. Current Separations 2000 19 55-8. 

The annular moving bed can be simulated by a large number of columns that are moved around in a circle. As above, the injection point is moved from colunm to column in regular intervals, and the collection points for the separated compounds are also moved around. In this way, a continuous separation of several different compounds is possible. The number of columns that are needed depends on the retention factor of the last compound and how often we are switc g the columns. In this approach, we can even use gradient elution, at least in the form of step gradients. In practice, we need a pump for every column to assure a reliable operation of the system. This increases the cost of such a system, but otherwise this technique is quite practical and assumes only that all columns are reasonably similar to each other. However, separation problems where several different compounds need to be purified from the same feed stream are quite rare, therefore this technique is also of limited practical interest. 

Figure 5 The AMD development unit (CAMAO) featuring a developing chamber, six solvent reservoir bottles, seven-port motor-driven valve, two-step gradient mixer, wash bottle, gas phase reservoir vacuum pump, and water collection bottle.

In order to evaluate pump flow rate reproducibility and pulsation, one method is commonly used to assess gradient formation capability. A certain amount of an analyte with adequate molar absorptivity at the wavelength employed for detection is introduced into one of the mobile phases employed to create the gradient. In the case described, 5% acetone was introduced into the mobile phase, distributed to the system by pump B. No UV-absorbing analyte was introduced into mobile phase A. The fractional flow rate of pump B relative to the total flow rate of the system (mandated by the sum of the flow rates of pumps A and B) was increased in individual steps to account for 0, 3,6,12.5,25, 50, and 100% fractional rates. The total flow for the system was maintained at 300 /jL/ min (for 24 columns), resulting in a per column flow rate of 12.5 /iL/min/column. 

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