Waters XSelect CSH130 C18 3.5 μm and 5 μm Columns - Manual (Page 2)

[ CARE AND USE MANUAL ]
XSelect CSH130 C
18
Columns 2
I. GETTING STARTED
Each XSelect CSH130 C
18
Columns comes with a Certificate of Acceptance
and a Performance Test Chromatogram. The Certificate of Acceptance
is specific to each batch of packing material contained in the Peptide
Separation Technology 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 the information: batch number, column
serial number, USP plate count, USP tailing factor, retention factor, and
chromatographic conditions. These 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 column i.d., length, particle size and backpressure of the
Peptide Separation Technology 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 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
Peptide Separation Technology 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.