Waters Symmetry Columns - Manual (Page 3)

[ Care and Use ManUal ]
Symmetry Columns 3
b. 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. Link to www. waters.com/sampleprep
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. 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 8,000 rpm,
followed by the transfer of the supernatant liquid to an
appropriate vial, could be considered.
c. pH Range
The recommended operating pH range for Symmetry columns is 2 to 8. 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.
Table 2: Buffer Recommendations for Using Symmetry Columns from pH 2 to 8
Additive or Buffer pK
a
Buffer range
(±1 pH unit)
Volatility Used for Mass Spec? Comments
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.
Ammonium Formate
(NH
4
COOH)
3.75 2.75 – 4.75 Volatile Yes Used in the 1-10 mM range.
Note: sodium or potassium salts are not volatile.
Trifluoroacetic Acid (TFA) 0.3 Volatile Low conc. When used in LC/MS, due to signal suppression, it is generally recommended to
use TFA at concentrations < 0.1%
Ammonium Acetate
(NH
4
CH
2
COOH)
4.76 3.76 – 5.76 Volatile Yes Used in the 1-10 mM 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 Above pH 7, reduce temperature/concentration and use guard column to
maximize lifetime.
iii. column use
To ensure the continued high performance of Symmetry 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 perfor-
mance matching guard column to protect the main column while not compromising 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.