Waters ACQUITY UPLC M-Class - Flow Rate on the B Flow Controller; Initial Gradient Hold; Trapping Flow Rate and Trapping Composition

2.1.3 Flow rate on the B flow controller
For the most reproducible chromatography, the B side flow controller should operate at flow rates
greater than 10 nL/min.
To determine the flow rate of the B flow controller, multiply the analytical/trapping flow rate by %B
composition in the gradient table. If the calculated B flow rate is less than 10 nL/min, you must
increase the %B composition in the gradient table.
Recommendation: Set the composition of solvent B ≥3.0% for the analytical portion of the
chromatography. However, you may need to increase this value based on the total flow rate of the
system.
2.1.4
Initial gradient hold
When using a 75-µm column, after a gradient starts, the B side flow controller is typically running
at its lowest flow rate in the entered gradient. Because of this, it can take time to fully stabilize the
B flow controller so it consistently delivers B mobile phase. To achieve the most reproducible
chromatography, insert an isocratic hold at the beginning of your gradient table. The system
should pump a volume between 75-150 nL of mobile phase B. The hold time required depends
on multiple factors, including, but not limited to:
• System configuration
• Initial flow rate and composition
• Total back pressure
• Changes in the ambient temperature
• Current method
Example: For a gradient running at 300 nL/min, with the initial condition of 95% A and 5% B,
you should insert an isocratic hold, to pump a volume between 75-150 nL of mobile phase B, at
95/5 for between 5 and 10 minutes.
Recommendation: The isocratic hold time required can vary from system to system and
should be investigated and optimized during method development phase.
2.1.5
Trapping flow rate and trapping composition
When trapping with columns that have a diameter ≤300 µm, set the trapping flow rate to a value
less than 10 µL/min. However, the best chromatographic reproducibility is observed when the
trapping flow rate is set to 5 µL/min.
Recommendation: Investigate the optimum trapping flow rate and the conditions during the
method development phase.
See also: Flow rate on the B flow controller for recommendations on the minimum percent B
recommended for use when trapping.
November 26, 2019, 715003588 Revision C
Page 23