SAMPLE LOADING USING THE DYNALOOP SLIDING-PISTON LOOP

The DynaLoop, which is available in 25 ml and 90 ml sizes, is used to load large volumes of sample directly onto chromatography columns. These loops have a sliding piston and function very much like a syringe. The sample is inserted into the DynaLoop’s sample end connector. The loading of the sample pushes the DynaLoop’s sliding seal assembly towards the buffer end connector. Note: While other dynamic loops may be used, the application discussed in this section applies specifically to the DynaLoop.

8.2.1 System Setup

When plumbed directly to the AVR7-3 inject valve, the DynaLoop functions just like a static sample loop. The sample end of the DynaLoop should be plumbed to port 3 and the buffer end to port 6 of the Inject valve.

The valve’s operation and sample loading are controlled automatically, simplifying the sample injection process and insuring precise sample loading and gradient formation.

The DynaLoop may be filled either manually with a syringe or automatically using an auxiliary pump such as an Econo Gradient Pump (EGP) which is controlled by the DuoFlow system or the EP-1 Econo pump.

• When using a syringe to fill a DynaLoop, the syringe should be connected to port 2 of the AVR7-3 valve. However, using a 1/4x28 to female Luer adapter between the syringe and the valve makes filling the loop much easier.

• When using the auxiliary pump to fill the loop, the pump’s outlet plumbing should be connected directly to port 2 of the inject valve.

Consult the DynaLoop Instruction Manual or DuoFlow online help for additional information. The figure below shows how to set up a DynaLoop.

Figure 8-3. Plumbing the DynaLoop for use with an Inject Valve

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8.2.2 Writing the Protocol

Before running a protocol which involves the use of a DynaLoop, be sure to purge air from the lines, as discussed in the DynaLoop Instruction Manual, Chapter 2, Installation. Then follow the guidelines which apply to your application.

The following steps should be programmed within the method protocol to manually load a sample:

1. Ensure that the column is properly equilibrated by using an Isocratic Flow step.

2. From the Manual screen, set the AVR7-3 sample inject valve to its Inject (I) position and allow the sliding seal to contact the sample end fitting on the DynaLoop. Then set the valve to the Load (L) position.

3. Load sample into the DynaLoop by using a syringe. As sample is loaded into the loop, it displaces the buffer that was used to purge the unit. This design means the DynaLoop cannot be overfilled.

When sufficient volume of sample for a run or series of runs is loaded into the DynaLoop, stop the filling process. For a series of partial volume injections after one loading sequence, fill the DynaLoop with about 2 percent extra sample. Leave the syringe in the Inject port to minimize the introduction of air into the DynaLoop.

The sample is now ready for injection, and the protocol can be programmed.

4. In the Protocol screen, select Load/Inject Sample. In the Load/Inject Sample window, note that the AVR7-3 valve will automatically move to the “Inject” position at the start of the step and to the “Load”

position at the end of the step.

a. In the Load/Inject Sample window, specify Dynamic Loop.

b. In the “Injection Buffer” area of the window, select the buffer(s) and percent (%) composition to be used to inject the sample onto the column.

c. In the “Volume (ml)” field, enter the sample volume.

d. In the “Flow (ml/min),” enter the flow rate of the Workstation pump that will be used to inject the sample onto the column.

5. Continue writing the separation protocol.

Figure 8-4. Load/Inject Sample Window for a Manual Load/Inject of the Sample

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The following steps should be programmed within the method protocol to automatically inject a sample using an auxiliary pump:

1. Connect the Aux pump. The EGP pump connects to the Instrument bus. If you are using the EP-1 Econo pump, consult Section 8.3.1.

2. In the Setup screen, select Aux Load Pump and AVR7-3 valve in addition to the other device and instruments you have connected to the system.

3. In the Protocol screen, program an Isocratic Flow step that is long enough for the auxiliary pump to load the desired quantity of sample into the DynaLoop. For example, if you are loading 25 ml of sample at an auxiliary pump flow rate of 5 ml/min, you will need at least a 5 minute step prior to the sample loading step. Otherwise the protocol will fail the automatic validation done prior to the run.

The flow rate of the DuoFlow pump is not critical, so you may set a low flow rate (e.g., 0.1 ml/min) to minimize buffer waste. It is important that either the time or volume length of this first step is of sufficient duration to allow the auxiliary pump to fill the DynaLoop.

4. In the Protocol screen, select Load/Inject Sample to program the DuoFlow system to automatically fill and inject the DynaLoop sample. From the Load/Inject Sample window,

a. Select the Dynamic Loop as the type of loop to be used.

b. Select Fill Before Inject. This instructs the auxiliary pump to load the sample into the

DynaLoop. Note that the auxiliary pump flow rate is not under DuoFlow control. The flow rate is used by the system when validating the protocol before the run. Because the DuoFlow system only starts/stops the auxiliary pump, the correct flow rate must be set at the auxiliary pump. The flow rate of the Econo pump is set from the pump and is recorded in the yellow data entry boxes of the Fill Sample section of the Load/Inject Sample dialog box. Note that the rinse function is not available when the DynaLoop is being used.

c. In the Fill Sample Loop area of the window, select the sample to be loaded and enter its volume and flow rate of the auxiliary pump.

d. In the Inject Sample area of the window, select the Injection Buffers, the buffer composition, the flow rate of the Workstation pumps, and the sample volume to be injected onto the column.

The DuoFlow system will now automatically control the loading and injection of the sample. Note that the rinse function is not available when the DynaLoop is being used.

5. Continue writing your desired separation protocol.

During the run, the valve functions as follows:

• Load. While in this position the valve connects ports 5 and 4 for equilibration of the column and for sample elution. In this position, sample loop is loaded to the desired volume via port 2 and buffer is expelled from the dynamic loop through port 1.

• Inject. While in this position the valve connects ports 5 and 6 and ports 3 and 4 for applying the sample onto the column. The flow from the Workstation pump forces the sliding piston to expel the sample onto the column.

• Purge. While in this position the valve connects ports 5 and 7 and allows purging or buffer changes of the Workstation pump without the need to remove the column from the system.

Figure 8-5. Valve Positions During a Run using the DynaLoop

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