Waters XSelect HSS HPLC Columns - Manual (Page 6)

[ Care and Use ManUal ]
XSelect HSS HPLC Columns 6
the main column while not compromising or changing analytical
resolution.
b. Sample Preparation
1. Sample impurities often contribute to column contamination.
Use Waters Oasis ® or Sep-Pak ® solid-phase extraction cartridges/
columns of the appropriate chemistry to cleanup the sample
before analysis.
2. It is preferable to prepare the sample in mobile phase or a solvent
that is weaker (less organic modifier) than the mobile phase.
3. If the sample is not dissolved in the mobile phase, ensure that
the sample and diluent are miscible in the mobile phase(s) in
order to avoid sample and/or diluent precipitation.
4. Filter sample through a 0.2 µm membrane 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
careful transfer of the supernatant liquid to an appropriate vial,
could be considered.
c. Recommended pH Range
Chemistry pH Range
XSelect HSS Cyano 2-8
XSelect HSS PFP 2-8
XSelect HSS T3 2-8
XSelect HSS C
18
SB 2-8
XSelect HSS C
18
1-8
Column lifetime will vary depending upon the temperature, type and
concentration of buffer used. A listing of recommended and non-
recommended buffers is given in Table 3. Please use this as a guideline
when developing methods.
Attention: Operating at the upper or lower end of the pH range in
combination with elevated temperatures will lead to shorter column
lifetime and/or may result in the column generating high backpressure.
Table 3: Buffer recommendations for using HSS HPLC columns from
pH 1 to 7
Additive or
Buffer
pKa
Buffer
Range
(±1 pH
unit)
Volatility
Used for
Mass
Spec?
Comments
TFA 0.3 – Volatile Yes
Ion pair additive, can suppress
MS signal. Used in the 0.01-
0.1% range.
Formic
Acid
3.75 – Volatile Yes
Maximum buffering obtained
when used with Ammonium
Formate salt. Used in 0.1-1.0%
range.
Acetic Acid 4.76 – Volatile Yes
Maximum buffering obtained
when used with Ammonium
Acetate salt. Used in 0.1-1.0%
range.
Formate
(NH
4
COOH)
3.75
2.75 –
4.75
Volatile Yes
Used in the 1-10mM range.
Note: sodium or potassium salts
are not volatile.
Acetate
(NH
4
CH-
2
COOH)
4.76
3.76 –
5.76
Volatile Yes
Used in the 1-10mM range.
Note: 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
Much shorter colum lifetimes
will be realized using phosphate
at pH 7.
d. Solvents
To maintain maximum column performance, use high quality
chromatography grade solvents. Filter all aqueous buffers prior to
use. The addition of at least 5% organic to buffers is recommended
to discourage bacterial growth. Acrodisc
® filters are recommended.
Solvents containing suspended particulate materials will generally
clog the outside surface of the inlet frit of the column. This will result
in higher operating pressure and poorer performance.
Degas all solvents thoroughly before use to prevent bubble formation
in the pump and detector. The use of an on-line degassing unit is also
recommended. This is especially important when running low pressure
gradients since bubble formation can occur as a result of aqueous and
organic solvent mixing during the gradient.