Waters ACQUITY UPLC BEH Columns - Manual (Page 4)

II. COLUMN USE
To ensure the continued high performance of ACQUITY UPLC BEH
Columns, follow these guidelines:
a. Sample Preparation
1. Sample impurities often contribute to column contamination.
One option to avoid this is to use Oasis ® Solid-phase
Extraction Cartridges/columns or Sep-Pak ® Cartridges of the
appropriate chemistry to clean up the sample before analysis.
For more information, visit
www.waters.com/sampleprep
2. It is preferable to prepare the sample in the operating mobile
phase or a mobile phase that is weaker than the mobile phase
for the best peak shape and sensitivity. Acetone should
not be used as a sample solvent/diluent unless a Hexane
Tetrahydrofuran Compatibility Kit (P/N: 205000464) has
been installed.
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 membranes to remove particulates.
If the sample is dissolved in a solvent that contains an organic
modifier (e.g., acetonitrile, methanol, etc.) ensure that the
5. membrane material does not dissolve in the solvent. Contact
the membrane manufacturer with solvent compatibility
questions. Alternatively, centrifugation for 20 minutes at
8000 rpm, followed by the transfer of the supernatant liquid
to an appropriate vial, could be considered.
6. For Hydrophilic Interaction Chromatography (HILIC)
separations, the samples must be prepared in a high
percentage of organic solvents (e.g., 95% acetonitrile). See
“Getting Started with ACQUITY UPLC BEH HILIC Columns” or
“Getting Started with ACQUITY UPLC Amide Columns” for
additional information.
b. pH Range
The recommended operating pH range for ACQUITY UPLC BEH
Columns is 1 to 12 for the C
18
, C
8
and Phenyl chemistries; 2 to 11
for the Shield RP18 and BEH Amide chemistries; and 1 to 9 for
the BEH HILIC chemistry. A listing of commonly used buffers and
additives is given in Table 2. Additionally, the column lifetime
will vary depending upon the operating temperature, the type and
concentration of buffer used. For example, the use of phosphate
buffer at pH 8 in combination with elevated temperatures will lead
to shorter column lifetimes.
c. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior
to use through a 0.2 µm filter. Pall Gelman Laboratory Acrodisc
®
filters are recommended. Solvents containing suspended
particulate materials will generally clog the outside surface of
the inlet distribution frit of the column. This will result in higher
operating pressure and poorer performance. See Section V for
more information.
d. Pressure
ACQUITY UPLC BEH Columns can tolerate operating pressures up
to 18000 psi (1241 bar or 124 MPa)
. Note: Working at the extremes
of pressure, pH and/or temperature will result in shorter column lifetimes.
e. Temperature
Temperatures between 20 ˚C – 90 ˚C are recommended for
operating ACQUITY UPLC BEH Columns in order to enhance
selectivity, lower solvent viscosity and increase mass transfer
rates. When operating at high pH, lower operating temperatures
are recommended for longer column lifetime. Working at high
temperatures (e.g. > 70 °C) may also result in shorter column
lifetimes. Under HILIC conditions, ACQUITY UPLC BEH Amide
Columns can be used at high pH and at high temperatures without
issue (see recommended conditions in Getting Started with BEH
Amide section).
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[ CARE AND USE MANUAL ]
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