Waters XBridge XP 2.5 µm Columns - Manual (Page 12)

XBridge XP 2.5 µm Columns 12
[ CARE AND USE MANUAL ]
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 suitable.
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 (Part No.
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 in the sample are acceptable.
50/50 acetonitrile/water can provide satisfactory results.
3. The injection solvent’s influence on peak shape should be determined
experimentally. In some cases, injections of water (or highly aqueous
solutions) may not adversely affect peak shape.
Miscellaneous Tips
1. 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.
2. Alternate polar solvents such as methanol, acetone or isopropanol can
also be used in place of water to increase retention.
3. If using an ACQUITY UPLC System with a fixed loop injector, ensure
that the weak needle wash solvent/purge solvent is your starting
mobile-phase (i.e., high organic), or your peak shapes will suffer.
Typical needle wash conditions: 800 μL strong wash in
20/80 acetonitrile/water, 500 μL weak wash in
75/25 acetonitrile/water.
4. Acetone should not be used as a sample solvent/diluent unless a
Hexane Tetrahydrofuran Compatibility Kit (Part No. 205000464)
has been installed.
Tips for Separating Sugars/Saccharides/Carbohydrates
If separating sugars or sugar-containing compounds that do not include
reducing sugars (see below) follow generic ‘Getting Started with
XBridge Amide Columns’ recommendations described above.
If separating reducing sugars, please review the following information.
1. Reducing sugars can undergo mutarotation which produces the unde-
sired separation of the α and β ring forms (anomers).
2. Collapsing anomers into one peak is accomplished through the use of a
combination of elevated temperature and high pH:
a. Use of 35 °C with high pH (0.2% triethylamine (TEA) or 0.1%
ammonium hydroxide (NH
4
OH)) and/or
b. Use of >80 °C with 0.05% TEA high temperature (>80 °C)
3. When separating reducing sugars (e.g., fructose, glucose, maltose,
lactose, arabinose, glyceraldehyde) please pay attention to the
following suggestions. Failure to do so will result in the appearance
of split peaks (anomer separation) for these analytes:
a. Operate at a slow flow rate (e.g., 0.10 - 0.13 mL/min on
2.1 x 50 mm column) to facilitate anomer collapse.
b. With longer columns, increased flow rates (e.g., up to 0.3 mL/min)
can be used. As with all LC separations, optimal flow rates should
be determined experimentally.