Waters ACQUITY UPLC BEH Columns - Manual (Page 11)

Injection Solvents
1. If possible, injection solvents should be 95% acetonitrile. The
polar solvent (i.e., water, methanol, isopropanol) should be
minimized to 25% of the total volume.
2. A generic injection solvent is 75:25 acetonitrile:methanol. This is
a good compromise between analyte solubility and peak shape.
3. Avoid water and dimethylsulfoxide (DMSO) as injection solvents.
These solvents will produce very poor peak shapes.
4. Exchange water or DMSO with acetonitrile by using reversed-
phase solid-phase extraction (SPE). If this is not possible, dilute
the water or DMSO with organic solvent.
Miscellaneous Tips
1. ACQUITY UPLC BEH HILIC Columns are designed to retain very
polar bases. Acidic, neutral and/or non-polar compounds will
have limited retention.
2. Optimal flow rates for small (<200 daltons) very polar bases
are in the 0.4 to 0.8 mL/min range with the ACQUITY UPLC
BEH HILIC Columns.
3. As compared to Atlantis ® HILIC Silica HPLC Columns, the
ACQUITY UPLC BEH HILIC Columns are approximately 20%
less retentive for gradient analysis and 35 to 65% less
retentive for isocratic analysis. This is due to the lower residual
surface silanol concentration of the BEH particle.
4. In HILIC, it is important to remember that water is the strongest
solvent. Therefore, it must be eliminated or minimized in the
injection solvent.
5. For initial scouting conditions, run a gradient from 95%
aceto-nitrile to 50% acetonitrile. If no retention occurs, run
isocratically with 95:3:2 acetonitrile:methanol:aqueous
buffer.
6. Alternate polar solvents such as methanol, acetone or
isopropanol can also be used in place of water to increase
retention.
7. If using an ACQUITY UPLC System, the weak needle wash
should closely match the % organic present in the initial
mobile-phase conditions, otherwise, analyte peak shape or
retention may suffer.
d. Getting Started with ACQUITY UPLC BEH Amide Columns
Operating Ranges
1. ACQUITY UPLC BEH Amide Columns can be used routinely
under HILIC conditions between pH 2 to 11, and they can be
operated at temperatures up to 90 °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 60% acetonitrile:
40% aqueous (or initial starting conditons) 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.).
2. Maintain at least 40% organic solvent (e.g., acetonitrile) in
your mobile phase or gradient.
3. At aqueous concentrations greater than 60%, lower flow rates
should be used due to high backpressure. This includes all
aqueous wash procedures.
4. Avoid phosphate salt buffers to avoid precipitation in HILIC
mobile phases. Phosphoric acid is OK.
Injection Solvents
1. If possible, injection solvents should be as close to the mobile
phase composition as possible (if isocratic) or the starting
gradient conditions. Acetone should not be used as a sample
solvent/diluent unless a Hexane Tetrahydrofuran Compatibility
Kit (P/N: 205000464) has been installed.
2. A generic injection solvent is 75:25 acetonitrile:methanol.
This is a good compromise between analyte solubility and peak
shape. When separating saccharides with limited solubility in
in organic solvents, higher concentrations of aqueous solvent
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