Waters ACQUITY UPLC BEH Columns - Manual (Page 10)

c. Getting Started With ACQUITY UPLC BEH HILIC Columns
1. Because ACQUITY UPLC BEH HILIC Columns do not posses a
bonded phase, the pH operating range is 1 to 9, and they can
be operated at temperatures up to 45 °C.
2. As with any LC column, operating at the extremes of pH, pressures
and temperatures will result in decreased column lifetime.
Column Equilibration
1. When column is first received, flush in 50% acetonitrile:
50% water with 10 mM final buffer concentration for 50
column volumes.
2. Equilibrate with 20 column volumes of initial mobile-phase
conditions before making first injection.
3. If gradient conditions are used, equilibrate with 8-10 column
volumes between injections.
4. Failure to appropriately equilibrate the column could result in
drifting retention times.
Mobile-Phase Considerations
1. Always maintain at least 3% polar solvent in the mobile phase
or gradient (e.g., 3% aqueous/3% methanol or 2% aqueous/
1% methanol, etc.). This ensures that the ACQUITY UPLC
BEH
particle is always hydrated.
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in
your mobile phase or gradient.
3. Avoid phosphate salt buffers to avoid precipitation in HILIC
mobile phases. Phosphoric acid is okay.
4. Buffers such as ammonium formate or ammonium acetate
will produce more reproducible results than additives such as
formic acid or acetic acid. If an additive (e.g., formic acid, etc.)
must be used instead of a buffer, use 0.2% (v:v) instead of
0.1%.
5. For best peak shape, maintain a buffer concentration of 10 mM
in your mobile phase/gradient at all times.
2.1 mm i.d. Columns (40 °C)
UPLC Linear Velocity
(mm/sec)
3 4 5 6
Column Dimensions
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
2.1 x 30 mm 0.45 3000 0.60 4100 0.75 5100 0.9 6100
2.1 x 50 mm 0.45 4800 0.60 6400 0.75 8000 0.9 9500
2.1 x 100 mm 0.45 9100 0.60 12100 0.75 15200 0.9 18200
2.1 x 150 mm 0.45 13400 0.60 17900 0.75 22400 0.9 26900
Note: 1) ACQUITY BEH UPLC 1.7 µm particle reversed-phase columns
2) ACN/Aqueous gradient, Pmax at ~30% ACN
3) Approximate maximum total system backpressure given
3.0 mm i.d. Columns (40 °C)
UPLC Linear Velocity
(mm/sec)
3 4 5 6
Column Dimensions
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
3.0 x 30 mm 0.9 3400 1.17 4400 1.53 5800 1.8 6800
3.0 x 50 mm 0.9 5100 1.17 6600 1.53 8700 1.8 10200
3.0 x 100 mm 0.9 9300 1.17 12100 1.53 15900 1.8 18700
3.0 x 150 mm 0.9 13600 1.17 17600 1.53 23100 1.8 27100
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