4
XSelect
XP
and XSelect Premier 2.5 µm HSS Columns
[ CARE AND USE MANUAL ]
Table 2. Buffer Recommendations for Using XSelect HSS Columns
Additive/Buffer
pK
a
Buffer
range
Volatility
(±1 pH unit)
Used for
Mass Spec
Comments
TFA
0.3
—
Volatile
Yes
Ion pair additive, can suppress MS signal,
used in the 0.02–0.1% range.
Acetic Acid
4.76
—
Volatile
Yes
Maximum buffering obtained when used with
ammonium acetate salt. Used in 0.1–1.0% range.
Formic Acid
3.75
—
Volatile
Yes
Maximum buffering obtained when used with
ammonium formate salt. Used in 0.1–1.0% range.
Acetate
(NH
4
CH
3
CO
2
)
4.76
3.76–5.76
Volatile
Yes
Used in the 1–10 mM range. Note that sodium
or potassium salts are not volatile.
Formate
(NH
4
HCO
2
)
3.75
2.75–4.75
Volatile
Yes
Used in the 1–10 mM range. Note that sodium
or potassium salts are not volatile.
Phosphate 1
2.15
1.15–3.15
Non-volatile
No
Traditional low pH buffer, good UV transparency.
Phosphate 2
7.2
6.20–8.20
Non-volatile
No
Above pH 7, reduce temperature/concentration
and use a guard column to maximize lifetime.
II. COLUMN USE
To ensure the continued high performance of XSelect HSS
Columns, follow these guidelines:
a. 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. For more information, visit
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.
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.
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
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.
b. pH Range
The recommended operating pH range for XSelect HSS
Columns is 2–8 for the HSS T3, HSS C
18
SB, HSS Cyano and
HSS PFP chemistries, and 1–8 for the HSS C
18
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.
Note: Working at the extremes of pH, temperature, and/or
pressure will result in 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. 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
1. The 2.1 mm I.D. XSelect
XP
and XSelect Premier 2.5 µm
Columns can tolerate operating pressures up to 18,000 psi
(1241 bar or 124 MPa).
2. The 3.0 mm I.D. XSelect
XP
Columns can tolerate operating
pressures up to 18,000 psi (1241 bar or 124 MPa).
3. The 4.6 mm I.D. XSelect
XP
and XSelect Premier 2.5 µm
Columns can tolerate pressures up to 10,000 psi
(700 bar or 70 MPa).
Note: Working at the extremes of pressure, pH and/or
temperature will result in shorter column lifetimes.
