Bio-Rad BioLogic DuoFlow Pathfinder 80 System DuoFlow Chromatography System Starter Kit User Manual
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4.5 Prime the Pumps and Equilibrate the UNO Q1 Column
Ensure the gradient pumps are stopped and the inject valve is in the purge
position. Place the tubing from inlets A1, A2, B1 and B2 into solutions A1 (Tris-
HCl), A2 (Tris base), B1 (water), and B2 (NaCl), respectively. Re-prime and purge
the pumps and inlets A1, A2, B1, and B2 as described in Section 3.1 of this
manual.
Set the inject valve to position L (Load). Set the flow rate to 2.0 ml/min. Set the
UV (280 nm) range to 0.1 AUFS and the conductivity range to 100 mS/cm.
Set the buffer recipe by pressing on the toolbar and choosing Tris (25 mM)
as your buffer. Press OK. On the manual screen set the pH to 8.10 and the Salt
Molarity %B to 0.0.
Change the pH scale by pressing the Settings button on the toolbar. Set the
minimum pH to 7.1 and the maximum pH to 9.1 in SIM1/pH.
Applying a 1 or 2-point pH correction (optional).
The Maximizer has been designed to prepare buffers accurately and reproducibly
at a user-defined pH and salt composition. The pH accuracy, however, depends
on how close the selected pH is to the buffer systems pKa. In situations where
high pH accuracy is required, you should apply a 1 or 2-point pH correction as
described below.
Single Point Correction (best for isocratic applications)
a. In the manual screen set the pH to 8.1, the salt composition to 0 %B (or to
the desired %B) and the flow rate to 2.0 ml/min.
b.
Take the column out of line if it has been connected and start the pump.
c. When the pH has stabilized, read the pH from the status bar or, alternatively,
collect the effluent and measure the pH using a high quality Tris compatible
pH probe.
d. From the buffer blending setup screen, place a check in the Use pH
Correction box (leave the Use Two Point Correction box unchecked).
e.
Set the desired pH to 8.1 and the Observed at 0 % pH, to the pH measured
in step (c).
f. Press
OK.
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