Waters Spherisorb Columns - Manual (Page 4)

[ Care and Use ManUal ]
Waters Spherisorb Columns 4
ii. column use
To ensure the continued high performance of Spherisorb columns,
follow these guidelines:
a. Guard Columns
Use a Waters Spherisorb guard column of matching chemistry and
particle size between the injector and main column. It is important to
use a 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 or other
changes in chromatographic performance 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 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 in the case
of reversed-phase chromatography, less polar modifier in the case
of normal-phase chromatography or hydrophilic-interaction chro-
matography, less salt in the case of ion exchange) 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 Spherisorb columns is 2
to 8. A listing of commonly used buffers and additives is given in
Table 3. Additionally, the column lifetime will vary depending upon
the operating temperature, and the type and concentration of buffer
used. For example, the use of phosphate buffer at pH 8 in combina-
tion with elevated temperatures will lead to shorter column lifetimes.