Waters High Strength Silica Columns - Manual (Page 5)

[ Care and Use ManUal ]
HSS HPLC Columns 5
V. CoLUMn InstaLLatIon PRoCeDURe
Note: The flow rates given in the procedure below are for a typical 4.6 mm
i.d. column. Scale the flow rate up or down
accordingly based upon the
column i.d., length, particle size and backpressure of the HSS HPLC column being
installed. See “Scaling Up/Down” for calculating flow rates when changing col-
umn i.d. and/or length.
1. Purge the pumping system and connect the inlet end of the
column to the injector outlet.
2. Set the pump flow to 0.1 mL/min and increase to 1 mL/min over
5 minutes.
3. When the mobile phase is flowing freely from the column
outlet, stop the flow, then attach the column to the detector. This
prevents entry of air into the detector and provides more rapid
baseline equilibration.
Caution: Care should be taken to check column connections for
leaks to avoid exposure to solvents and the hazards associated
with such exposure including risks to health and electrical
connections.
4. When the mobile phase is changed, gradually increase the flow
rate of the new mobile phase from 0.0 mL/min to 1.0 mL/min in
0.1 mL/min increments.
5. Once a steady backpressure and baseline have been achieved,
the column is ready to be used (or equilibrated).
Note: If mobile phase additives are present in low concentrations
(e.g., ion-pairing reagents), 100 to 200 column volumes may be
required for complete equilibration. In addition, mobile phases
that contain formate (e.g., ammonium formate, formic acid, etc.)
may also require slightly longer initial column equilibration
times.
VI. CoLUMn PeRFoRManCe VaLIDatIon
Each HSS HPLC column comes with a Certificate of Batch Analysis
and a Performance Test Chromatogram. The Certificate of Analysis
is specific to each batch of packing material and includes the batch
number, analysis of unbonded particles, analysis of bonded particles
and chromatographic results and conditions. The Performance Test
Chromatogram is specific to each individual column and contains
information such as batch number, column serial number, USP plate
count, USP tailing factor, capacity factor and chromatographic results
and conditions. These data should be stored for future reference.
VII. 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 verify the performance of
the column upon receipt.
2. Determine the number of theoretical plates (N) and use for
periodic comparison.
3. Repeat the test periodically to track column performance over
time. Slight variations may be obtained on two different HPLC
systems due to the quality of the connections, operating envi-
ronment, system electronics, reagent quality, column condition
and operator technique.
Note: If 1) is performed, the isocratic efficiencies measured in
your laboratory may be less than those given on the Waters
“Performance Test Chromatogram.” This is normal. The Waters
isocratic column testing systems have been modified in order
to achieve extremely low system volumes. This presents a more
challenging test of how well the column was packed. This guar-
antees the highest quality packed column. These special testing
systems have been modified to such an extent that they are not
commercially viable and have limited method flexibility other
than isocratic column testing.
VIII. CoLUMn UsaGe
Caution: Accumulation of particulates from solvents, samples, or pump
seals may cause the column backpressure to increase over time. This
may lead to a system shutdown or leaking of column connections.
Accumulation of contaminants from “dirty” samples at the column
inlet may lead to a loss of resolution or ion suppression in a mass
spectrometer, resulting in erroneous results.
To ensure the continued high performance of Atlantis columns and
cartridges, follow these guidelines:
a. Guard Columns
Use a Waters Sentry guard cartridge of matching i.d., chemistry and
particle size between the injector and main column. For best results,
the guard column should be replaced prior to the observation of a
substantial loss in resolution or increase in system backpressure. It
is important to use a high-performance matching guard column to
protect the main column while not compromising or changing analyti-
cal resolution.