Waters ACQUITY UPLC BEH Columns - Manual (Page 2)

II. GET TING START ED
Each ACQUITY UPLC BEH 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 BEH 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
The ACQUITY UPLC System utilizes tubing and gold plated
compression screws which have been designed to meet stringent
tolerance levels and to minimize extra column volumes.
Optimized column inlet tubing (Part Number 430001084) is
supplied with the ACQUITY UPLC System. The inject valve end of
the tubing is clearly marked with a blue shrink tube marker. Insert
the opposite end of the tubing into the ACQUITY UPLC Column and
tighten the compression fitting.
For information on the correct column outlet tubing, please refer
to the relevant detector section in the ACQUITY UPLC System
Operator’s Guide (Part Number 71500082502).
b. Column Installation
Note: The flow rates given in the procedure below are for a typical
2.1 mm i.d. by 50 mm length 1.7 µm column. 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 BEH 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)
Column 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.)
For ACQUITY UPLC BEH HILIC Columns, flush with 50 column
volumes of 50:50 acetonitrile:water with 10 mM final buffer
concentration. For ACQUITY UPLC BEH Amide Columns, flush
with 50 column volumes of 60:40 acetonitrile:aqueous. Prior
to the first injection, equilibrate with 20 column volumes of
initial mobile phase conditions (refer to Table 1 for a list of
column volumes). See “Getting Started with ACQUITY UPLC BEH
HILIC Columns” or “Getting Started with ACQUITY UPLC Amide
Columns” for additional information.
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