Bio-Rad BioLogic Signal Import Module (SIM) - Manual (Page 191)

MULTIPLE COLUMNS ADVANCED SYSTEM APPLICATIONS
10-4
10.3 PH MEASUREMENT AND CORRECTIONS
Buffer pH plays an important role in chromatography of biomolecules since it affects both their stability and
their chromatographic properties. Both solution temperature and ionic strength affect the pH of a buffer. The
Maximizer is designed to compensate for both of these effects during an experiment. A sensor in the
conductivity flow cell monitors temperature and so must always be connected to the Maximizer. Changes in
pH due to changes in ionic strength are compensated for by information stored with each Buffer System.
Changes in pH due to sample injection and/or adsorption or desorption of ions from a column cannot be
compensated for and may cause the pH to deviate from the specified run pH. For this reason it is advisable
to run ion exchange experiments at a pH near the middle of the buffers pH range where the buffer capacity
is greatest.
Accurate pH measurements require that samples be of uniform composition and temperature. These
conditions do not generally occur when monitoring pH during a chromatographic run. For this reason pH's
measured during a run should always be considered approximate. Furthermore, some pH accuracy is
sacrificed in order to make pH probes that are able to operate at the flow rates and pressures used in
chromatography applications. If highly accurate pH measurements are required, collect fractions and
measure their pH using a high quality pH probe (such as an Orion Ross electrode or other Tris compatible
electrode) connected to a desk top pH meter.
The pH electrode supplied with the Maximizer is designed to give optimum performance during the inline
measurement of pH. It can be used at flow rates up to 80 ml/min and pressures up to 75 psi. Its use
provides useful information about the Maximizer performance and buffer composition during a run. However,
it is not required for accurate pH delivery by the Maximizer. Since the pH probe has an approximately 80 ul
flow cell volume it should be taken out of line when collecting small fractions sizes (i.e. when collecting in
microplates).
Prior to use, the pH electrode should be calibrated using two standard solutions that span the pH range over
which the experiment will be run (for example pH 7 & 4, pH 7 & 10 or pH 4 and 10). Calibration is performed
from the Utilities/pH Probe Calibration menu option.
Although the Maximizer produces buffer with good pH accuracy, the accuracy can be improved by applying
a 1 or 2-point correction using the DuoFlow software as described below.
Single point correction (best for isocratic experiments)
1. From the manual screen press the Buffer Blender "Setup" button and select the desired buffer
system from the Buffer System list. Make sure the pH correction box is not checked. Press OK.
2. Set the desired pH, salt composition and flow rate and then press Start.
3. After the system has equilibrated collect some effluent.
4. Measure the pH using a high quality pH probe connected to a desk top pH meter that has been
calibrated at the run temperature.
5. Load your experimental method from the Browser screen and press Setup.
6. Double click on the Buffer Blending icon, and select the desired buffer. The appearance of the Buffer
Blending setup dialog depends on whether a single component (see Figure 10-1) or multiple
component buffer is selected (see Figure 10-2).
7. Place a check in the pH correction box and make sure the two-point correction option is not
selected.
8. Enter the pH that was measured in Step 4 in the Observed pH box and the desired pH in the
Desired pH box and press OK. For multiple component buffers, enter the measured pH for each
buffer component, in both the 0 %B and user-set %B pH correction boxes. The software will
calculate the correction.