The BufferPrep tab

BufferPrep usage

BufferPrep allows a buffer of different pH and salt concentrations to be prepared on-line from four stock solutions. This removes the need to manually prepare new buffers every time the pH needs to be changed. Linear and step salt gradients can be run and pH can be used as a variable scouting parameter. BufferPrep is optimized for cation and anion exchange chromatography. For a complete description of BufferPrep, see the user manual for ÄKTAdesign systems.

Note: BufferPrep is only available for some ÄKTAdesign systems.

Illustration

The illustration below shows an example of the BufferPrep tab:

Stock solutions

The solutions and the inlets to which they should be connected are displayed to the right of the dialog box. Accuracy of preparation is essential. The four stock solutions consist of:

  • a mix of buffering components (there can be up to five different buffering components enabling a broad pH range to be covered),

  • an acid (HCl) or base (NaOH) for pH on-line titration,

  • distilled water,

  • an inert salt (for example NaCl) for salt gradient formation.

How to create a BufferPrep method

If a suitable template or wizard is not available, you can create a BufferPrep method yourself. The instruction BufferPrep_pH must be available at breakpoint zero at the beginning of the method. The method must not contain the instructions PumpAInlet or PumpBInlet.

The table shows one way to create a BufferPrep method.

Step

Action

1

In the Text Instruction editor:

  • Insert a BufferPrep-pH block at breakpoint zero at the beginning of the method.

  • Define BufferPrep-pH as a variable.

2

Change to the Run Setup.

  • Select the BufferPrep tab.

  • Click the ON radio button in the Status field.

  • Select a Recipe from the drop-down list box. There are two main alternatives:

    AIEX or CIEX, which are recipes covering a broad pH range,

    single buffer recipes for more narrow pH ranges.

Result: All information relevant to the selected recipe will be displayed on the tab.

3

Prepare the required stock solutions.

4

Do one of the following:

  • Select the Variables tab. Set the required pH for the method run in the variable BufferPrep_pH,

    or

  • If you want to perform pH scouting, click the Scouting tab and select BufferPrep_pH as a scouting variable. Enter the pH values for the different runs.

BufferPrep recipes

The recipe saved in the method (the one selected on the BufferPrep tab) cannot be edited, although fine tuning is possible. However, the recipes on the list of all BufferPrep recipes can be edited. New recipes can also be created (see How to create and edit BufferPrep recipes).

How to fine tune the BufferPrep recipe with correction factors

In order to obtain high pH accuracy, the recipe can be fine tuned around a specific pH by setting correction factors. The table below describes how to fine tune the recipe with correction factors:

Step

Action

1

In System Control, select Manual: Other.

2

Select BufferPrep Recipe and Recipe Name.

Click the Execute button.

3

  • Set the pH in the instruction BufferPrep_pH in group Pump.

  • Click the Execute button.

4

  • Set the flow rate to be used during the run in the Flow instruction.

  • Click the Execute button.

5

Check the pH reading when stable. Allow at least 30 ml of eluent to pass through before expecting a steady pH reading.

6

  • Change to 100% B by setting the Gradient instruction in Manual:Pump to 100% for Target and 0 for Length.

  • Click the Execute button.

7

Check the pH reading when stable at 100% B.

8

  • If the readings are acceptable at both 0% and 100%, the correction factors do not need to be changed.

  • If the readings are not acceptable, click the Corr. Factors button in the BufferPrep tab in the method.

9

Enter the deviation at 0% and 100%. (e.g., if the pH is set to 7.0 and the actual pH is 7.1 enter 0.1. Enter -0.1 if the pH is 6.9).

Note: If correction factors already exist, the measured pH deviation should be added to the old factors.

10

Save the method.

Note: When changing the correction factors for the recipe selected in the method, the recipe with the same name on the list of all BufferPrep recipes is not affected. The changes will only apply in the specific method.

2005-06-15