Waters Atlantis T3, DC18 and HILIC Silica Columns - Manual (Page 6)

[ Care and Use ManUal ]
Atlantis Columns 6
VI. 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 Atlantis 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 out-
let, 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 associ-
ated 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.
Please see additional equilibration information for Atlantis HILIC
Silica columns in “HILIC Getting Started.”
VII. coLuMn PerforMAnce VALIdAtIon
Each Atlantis column comes with a Certificate of Batch Analysis and
a Performance Test Chromatogram. The Certificate of Analysis is spe-
cific to each batch of packing material and includes the batch number,
analysis of unbonded particles, analysis of bonded particles (Atlantis
T3 and Atlantis dC
18
), 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 chro-
matographic results and conditions. These data should be stored for
future reference.
VIII. InItIAL coLuMn effIcIency deterMInAtIon
1. Perform an efficiency test on the column before using it. Waters rec-
ommends using a suitable solute mixture, as found in the “Perfor-
mance Test Chromatogram”, to verify the performance of the column
upon receipt.
2. Determine the number of theoretical plates (N) and use for peri-
odic 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 environ-
ment, 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 “Perfor-
mance 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 challeng-
ing 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.
IX. 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.