Waters ACQUITY UPLC CSH Columns - Manual (Page 2)

[ Care and Use ManUal ]
ACQUITY UPLC CSH Columns 2
I. Get tInG started
Each ACQUITY UPLC CSH column comes with a Certificate of
Analysis and Performance Test Chromatogram embedded within
the eCord intelligent chip. The Certificate of Analysis is specific to
each batch of packing material contained in the ACQUITY UPLC CSH
columns and includes the gel 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 such information as: gel batch
number, column serial number, USP plate count, USP tailing factor,
capacity factor, and chromatographic conditions. These data should
be stored for future reference.
a. Column Connectors
ACQUITY UPLC systems utilize tubing and connectors which have
been designed to meet stringent tolerance levels and to minimize
extra column volumes.For information on system tubing and
connectors, please refer to the ACQUITY UPLC System Operator’s
Guide (Part Number 71500082502).
b. Column Installation
Note: The flow rates given in the procedure below are for typical 2.1 mm i.d. by 50 mm
length 1.7 µm columns. Scale the flow rate up or down accordingly based upon the flow
rate and pressure guide provided in Section V (Additional Information).
1. Purge the pumping system of any buffer-containing mobile phases and
connect the inlet end of the column to the injector outlet.
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 0.5 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
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.
c. Column Equilibration
ACQUITY UPLC 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 list of column volumes). The column may be considered thermally
equilibrated once a constant backpressure is achieved.
Table 1. Empty Column Volumes in mL (multiply by 10 for flush solvent volumes)
Cloumn Length
(mm)
Internal Diameter
1.0 mm 2.1 mm 3.0 mm
30 - 0.1 0.2
50 0.04 0.2 0.4
100 0.08 0.4 0.8
150 0.12 0.5 1.0
To avoid precipitating 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 system with 60% methanol in water prior to introducing 60%
methanol/40% buffer mobile-phase.)
d. eCord Installation
The eCord button should be attached to the side of the column
heater module. The eCord button is magnetized and does not require
specific orientation.
e. Initial Column Efficiency Determination
1. Perform an efficiency test on the column before using it. This test may
consist of:
a. an analyte test mixture that is commonly used in your laboratory,
and/or
b. an analyte mixture as found on the “Performance Test
Chromatogram” which accompanied your column.
Note: If b. 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.