Waters ACQUITY UPLC SEC Columns and Standards - Manual (Page 3)

3
[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH SEC Columns
b. Column Connectors
It is is recommended to use the 150 mm column stabilizer
(P/N
205000365 ) to connect the ACQUITY UPLC BEH SEC column
to the ACQUITY UPLC system. A reusable fitting is supplied with the
ACQUITY UPLC column stabilizer.
c. Column Installation
1. Prior to placing the column on the system, purge the solvent
delivery system of any organic or water-immiscible mobile phases.
When connecting the column, orient it in the proper direction as
noted by the arrow on the column inlet side which indicates the
correct direction of solvent flow.
2. Flush column with 100% aqueous buffer, by pumping at a flow
rate of 0.2 mL/min.
3. Ensure that the mobile phase is flowing freely from the column
outlet. Attach the column outlet to the detector using .004” ID
tubing (P/N
430001562 ). Monitor the system pressure to
ensure the column is within its pressure limitations.
4. Gradually increase the flow rate, by not more than 0.1mL/min at a
time, as described in Step 2.
5. Once the system pressure has stabilized, ensure that there are no
leaks at either the column inlet or outlet.
d. Column Equilibration
ACQUITY UPLC BEH SEC columns are shipped in 20% methanol
in water. 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 buffer to be used
(refer to Table 1 for column volumes).
Table 1. Empty Column Volumes in mL (multiply by 10 for flush
solvent volume)
Column Dimension Approximate Volume
4.6 x 150 mm 2.5 mL
4.6 x 300 mm 5.0 mL
e. Useful Functional Tests for Benchmarking a New Column
Waters recommends performing a benchmarking test upon receipt of
your column and throughout the lifetime usage. By using a separation
of common proteins with an appropriate method, you can:
 Verify the performance of the column upon receipt.
 Monitor the condition of the columns for extended use.
 Troubleshoot separation difficulties that may arise.
The BEH125 SEC Protein Standard Mix (P/N 186006519 ), BEH200
SEC Protein Standard Mix (P/N 186006518 ), and BEH 450 SEC
Protein Standard Mix (P/N 186006842 ) were specifically designed
for this purpose with carefully chosen proteins and/or peptides
to provide a good representation of the intended application.
Figure 3 is an example of the Performance Test Chromatogram using
Part Number
186006519 , Figure 4 is an example of the Performance
Test Chromatogram using Part Number 186006518 . Figure 5 is an
example of the Performance Test Chromatogram using Part Number
186006842 . These are typical results obtained in a waters laboratory
using 4.6 x 150 mm columns on an ACQUITY UPLC system.
Buffer Preparation for Standard:
Chemicals:
- Sodium Phosphate Monobasic, Monohydrate
- Sodium Phosphate Dibasic, Anhydrous
- Milli-Q Water
Preparation of 100mM Sodium Phosphate Buffer (500 mL)
- Weigh out 500 ± 0.02 grams of H
2
O into a 500 mL beaker.
- Weigh out 3.55 ± 0.02 grams of Sodium Phosphate dibasic and
add to the 500 mL beaker.
- Weigh out 3.45 ± 0.02 grams of Sodium Phosphate monobasic
and add to the 500 mL beaker.
- Stir for a minimum of 30 minutes then filter the solution
through a 0.2 µm Millipore GV filter.
- Take the pH and record the value for reference purposes
(approximate pH: 6.8).