Waters XBridge XP 2.5 µm Columns - Manual (Page 11)

XBridge XP 2.5 µm Columns 11
[ CARE AND USE MANUAL ]
d. Getting Started with XBridge HILIC XP 2.5 µm Columns
Operating Range
1. Because XBridge 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 XBridge HILIC 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) 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.
Injection Solvents
1. If possible, injection solvents should match the initial mobile-phase
conditions.. 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. XBridge 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 XBridge HILIC Columns
(2.1 mm ID).
3. As compared to Atlantis ® HILIC Silica HPLC Columns, the XBridge 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% acetonitrile to
50% acetonitrile. If no retention occurs, run isocratically with 95/3/2
acetonitrile/methanol/aqueous buffer.
6. Alternate polar solvents such as methanol, ethanol 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.
e. Getting Started with XBridge Amide XP 2.5 µm Columns
Operating Ranges
1. XBridge 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.