Waters XSelect CSH HPLC Columns - Manual (Page 2)

[ CARE AND USE MANUAL ]
2 XSelect CSH HPLC Columns
I. GET TING START ED
Each XSelect CSH column comes with a Certificate of Analysis and
a Performance Test Chromatogram. The Certificate of Analysis is
specific to each batch of packing material contained in the XSelect
CSH column 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, retention factor, and chromatographic conditions.
This data should be stored for future reference.
a. Column Installation
Note: The flow rates given in the procedure below are for a typical 5 µm packing
in a 4.6 mm i.d. column. Scale the flow rate up or down accordingly based upon
the i.d., length, particle size and backpressure of the XSelect CSH column being
installed. See Scaling Up/Down Isocratic Separations section for calculating
flow rates when changing column i.d and/or length. See Connecting the Column
to the HPLC section for a more detailed discussion on HPLC connections.
1. Purge the pumping system of any buffer-containing mobile phases
and connect the inlet end of the column to the injector outlet. An
arrow on the column identification label indicates the correct
direction of solvent flow.
2. Flush column with 100% organic mobile phase (methanol or
acetonitrile) by setting the pump flow rate to 0.1 mL/min and
increase the flow rate to 1 mL/min over 5 minutes.
3. When the mobile phase is flowing freely from the column
outlet, stop the flow and attach the column outlet to the
detector. This prevents entry of air into the detection system
and gives more rapid baseline equilibration.
4. Gradually increase the flow rate as described in step 2.
5. Once a steady backpressure and baseline have been achieved,
proceed to the next section.
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 longer initial column equilibration times.
b. Column Equilibration
XSelect CSH columns are shipped in 100% acetonitrile. It is
important to ensure mobile-phase compatibility before changing
to a different mobile-phase system. Equilibrate the column with
a minimum of 10 column volumes of the mobile phase to be used
(refer to Table 1 for a listing of empty column volumes).
To avoid precipitating out mobile-phase buffers on your column
or in your system, flush the column with five column volumes of
a water/organic solvent mixture, using the same or lower solvent
content as in the desired buffered mobile phase. (For example,
flush the column and HPLC system with 60% methanol in water
prior to introducing 60% methanol/40% buffer mobile phase).
c. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it in the desired
application. Waters recommends using a suitable solute mixture,
as found in the “Performance Test Chromatogram”, to analyze the
column upon receipt.
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 Internal Diameter (mm)
Column
Length
(mm)
1.0 2.1 3.0 4.6 7.8 10 19 30 50
20 - 0.07 0.14 0.33 - - - - -
30 - 0.10 0.21 0.50 - 2.4 8.5 - -
50 0.04 0.17 0.35 0.83 2.4 3.9 14 35 98
100 0.08 0.35 0.71 1.7 4.8 7.8 28 70 -
150 0.12 0.52 1.0 2.5 7.2 12 42 106 294
250 - 0.87 1.8 4.2 - 20 70 176 490