Re-Equilibration Of Column At The End Of Analysis; System Equilibration After Auto Zero; Re-Equilibration Between Methods; Temperature Sensitivity - Waters ACQUITY UPLC M-Class System Manual

2.1.6 Re-equilibration of column at the end of analysis

For the most reproducible chromatography, particularly when using flow ramps, the column
pressure must return to a stable and consistent value during the system re-equilibration step after
the end of the analytical gradient.
Recommendation:
method development phase and optimize if necessary.

2.1.7 System equilibration after auto zero

Powering off the µBSM or setting a flow rate of 0 µL/min causes the pump heads to retract. This
process can aspirate mobile phase back into the flow controllers. Priming the pumps effectively
primes the pump heads and the lines up to the vent valve, but it does not clear the solvent in the
solvent lines from the vent valve to the flow controller.
At flow rates <500 nL/min, it can take several injections (up to seven) to sufficiently equilibrate the
solvent lines. If the system is configured in such a way that the flow from the µBSM can be
diverted to waste, either directly or through a trap column, you can equilibrate the system quickly
to avoid a number of blank injections.
1. Equilibrate the system.
2. After equilibrating the system sufficiently, acquire one to two blanks before collecting data
from samples.
Note:
To avoid re-equilibrating the system often, set the LC flow at a 50/50 reduced flow
rate of 200 nL/min, if possible. If you need to turn off the flow, you can shorten the re-
equilibration step if the flow rate is gradually stepped down, rather than setting it
immediately to a value of 0 µL/min.

2.1.8 Re-equilibration between methods

When changing methods during acquisition, use two blanks between the methods. This ensures
that the system is operating using the initial conditions prior to acquiring the sample data.

2.1.9 Temperature sensitivity

Large or rapid changes (>1.5 °C/hr) in the room temperature can affect the flow controllers in the
µBSM. A large temperature change in the lab can cause the gradient to be generated in a non-
reproducible manner that affects chromatographic reproducibility. Methods that use nanoscale
flow rates (<500 nL/min) are more susceptible to changes in lab temperature. Some changes in
lab temperature are overcome by running the system at a higher flow rate (for example,
increasing from 0.3 µL/min to 0.5 µL/min). When increasing the flow rate, it may be necessary to
increase the column temperature or use a shorter column to ensure that the system does not
over-pressure at the higher flow rates.
Investigate the length of the isocratic hold/re-equilibration step during
November 26, 2019, 715003588 Revision C
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