Waters SunFire Columns - Manual (Page 3)

[ Care and Use ManUal ]
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2. Purge the column with the mobile phase until you obtain a stable
baseline.
3. Verify that retention times and peak areas for a standard are
stable by comparing 2-3 replicate, consecutive injections.
Before you perform the first analysis on your new column, perform an
efficiency test to confirm the performance of the column.
c. Initial Column efficiency determination
1. Perform an efficiency test on the column before using it. Waters
recommends using a suitable solute mixture, as found in the
“Performance Test Chromatogram”, to analyze the column upon
receipt. However, if the column is used only for a single routine
isocratic assay, it may be more convenient to test the column
under these assay conditions. Keep a record of the initial column
performance.
2. Determine the number of theoretical plates (N) and use this value
for periodic comparisons.
3. Repeat the test at predetermined intervals to track column
performance over time. Slight variations may be obtained on
two different HPLC systems due to the quality of the connections,
operating environment, system electronics, reagent quality,
column condition and operator technique.
table 1. empty Column Volumes in ml (multiply by 10 for flush
solvent volumes)
Column
Length
Column Internal Diameter (nm)
1.0 2.1 3.0 4.6 7.8 10 19 30 50
20 mm - 0.07 0.14 0.33 - - - - -
30 mm - 0.1 0.2 0.5 - 2.4 8 - -
50 mm 0.1 0.2 0.3 0.8 2.4 4 14 35 98
100 mm 0.1 0.4 0.7 1.7 5 8 28 70 196
150 mm 0.1 0.5 1.0 2.5 7 12 42 106 294
250 mm - 0.9 1.8 4 - 20 70 176 490
II. Column uSe
To ensure the continued high performance of SunFire columns, follow
these guidelines:
a. Guard Columns
Use a SunFire 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 chromatography, 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.