Waters XBridge Columns - Manual (Page 10)

[ Care and Use ManUal ]
XBridge ™ Columns 10
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 5% polar solvent in the mobile phase or gra-
dient (e.g., 5% aqueous/5% methanol or 2% aqueous/3% methanol,
etc.). This ensures that the XBridge 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 pro-
duce 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 at least
10 mM in your mobile phase/gradient at all times.
Injection Solvents
1. If possible, injection solvents should be 100% organic solvent of
the initial mobile phase conditions. Water must be eliminated or mini-
mized. Choose weak HILIC solvents such as acetonitrile, isopropanol,
methanol, etc.
2. A generic injection solvent is 75:25 acetonitrile:methanol. This is
a good compromise between analyte solubility and peak shape. If
solubility is still poor, 0.2% formic acid can be added.
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. 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.
2. In HILIC, it is important to remember that water is the strongest sol-
vent. Therefore, it must be eliminated or minimized in the injection
solvent.
3. For initial scouting conditions, run a gradient from 95% acetonitrile
to 50% acetonitrile. If no retention occurs, run isocratically with
95:3:2 acetonitrile:isopropanol:aqueous buffer.
4. Alternate polar solvents such as methanol, ethanol or isopropanol
can also be used in place of water to increase retention.
f. Getting Started with XBridge Amide 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.
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 condi-
tions before making first injection.
3. If gradient conditions are used, equilibrate with 8-10 column vol-
umes between injections.
4. Failure to appropriately equilibrate the column could result in drift-
ing retention times.