Waters XSelect HSS HPLC Columns - Manual (Page 5)

[ Care and Use ManUal ]
XSelect 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
XSelect HSS HPLC column being installed. See “Scaling Up/Down” for
calculating flow rates when changing column 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 XSelect 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 guarantees 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 XSelect HSS HPLC
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