Waters XBridge Peptide BEH C18 130A and 300A Columns - Manual (Page 2)

I. GET TING START ED
Each XBridge Peptide BEH C
18
, 130Å and 300Å Column
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
XBridge Peptide BEH C
18
, 130Å and 300Å 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.
2
[ CARE AND USE MANUAL ]
XBridge Peptide BEH C
18
, 130Å and 300Å Columns