Waters XBridge Columns - Manual (Page 3)

[ Care and Use ManUal ]
XBridge ™ Columns 3
Table 1: Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Column internal diameter (mm)
Column Length (mm) 1.0 2.1 3.0 4.6 7.8 10 19 30 50
20 – 0.07 0.14 0.33 – – – – –
30 – 0.10 0.21 0.50 – 2.4 8.5 – –
50 0.04 0.17 0.35 0.83 2.4 3.9 14 35 98
100 0.08 0.35 0.71 1.7 4.8 7.8 28 70 –
150 0.12 0.52 1.0 2.5 7.2 12 42 106 294
250 – 0.87 1.8 4.2 – 20 70 176 490
II. Column use
To ensure the continued high performance of XBridge ™ columns, follow
these guidelines:
a. Guard Columns
Use a Waters guard column of matching chemistry and particle size between
the injector and main column. It is important to use a high-performance
matching guard column to protect the main column while not compromis-
ing or changing the analytical resolution.
Guard columns need to be replaced at regular intervals as determined by
sample contamination. When system backpressure steadily increases above
a set pressure limit, it is usually an indication that the guard column should
be replaced. A sudden appearance of split peaks is also indicative of a
need to replace the guard column.
b. Sample Preparation
1. Sample impurities often contribute to column contamination. One option
to avoid this is to use Waters Oasis ® solid-phase extraction cartridges/
columns or Sep-Pak ® cartridges of the appropriate chemistry to clean up
the sample before analysis.
2. It is preferable to prepare the sample in the operating mobile phase or
a mobile phase that is weaker (less organic modifier) than the mobile
phase for the best peak shape and sensitivity.
3. If the sample is not dissolved in the mobile phase, ensure that the sample,
solvent and mobile phases are miscible in order to avoid sample and/or
buffer precipitation.
4. Filter sample with 0.2 µm filters to remove particulates. If the sample
is dissolved in a solvent that contains an organic modifier (e.g., acetonitrile,
methanol, etc.) ensure that the filter material does not dissolve in the sol-
vent. Contact the filter manufacturer with solvent compatibility questions.
Alternatively, centrifugation for 20 minutes at 8,000 rpm, followed by
the transfer of the supernatant liquid to an appropriate vial, could be
considered.
5. For Hydrophilic Interaction Chromatography (HILIC) separations,
the samples must be prepared in 100% organic solvents (e.g.,
acetonitrile). See “Getting Started with XBridge HILIC Columns”
or “Getting Started with XBridge Amide Columns” for additional
information.
c. Operating pH Limits
The recommended operating pH limits for XBridge ™ columns are listed in
Table 2. A listing of commonly used buffers and additives is given in Table 3.
Additionally, the column lifetime will vary depending upon the operating
temperature, the type and concentration of buffer used.