Waters Protein-Pak Hi Res IEX Columns - Manual (Page 3)

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[ CARE AND USE MANUAL ]
Protein-Pak Hi Res IEX Columns and Standards
c. Column Installation
Note: The flow rates given in the procedure below are for a Protein-
Pak Hi Res CM or SP, 7 µm, 4.6 mm i.d. column.
1. Purge the solvent delivery system of any organic or water-
immiscible 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% aqueous buffer (line A1) by setting
the pump flow to 0.2 mL/min.
3. When the buffer is flowing freely from the column outlet attach
the column outlet to the detector. Monitor the system pressure
to ensure the column is within its pressure limitations.
4. Gradually increase the flow rate, by not more than 0.5mL/min
at a time (as described in step 2).
5. Once a stable backpressure has been achieved, proceed to the
next section.
d. Column Equilibration
Protein-Pak Hi Res columns are shipped in 20 mM sodium sulfate
with 0.05% sodium azide. 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 2 for column volumes).
Table 2. Empty Column Volumes in mL
(multiply by 10 for flush solvent volumes).
Column Dimension Approximate Volume
4.6 x 100 mm 1.66 mL
e. Useful Functional Tests for Benchmarking a New Column
It may be useful to benchmark the column performance with
a sample that is representative of the intended application. A
separation of common proteins with an appropriate method is
suggested for your new column. Two standards were specially
designed for this purpose. The IEX Cation Test Standard is
specifically designed with proteins that resolve well on cation-
exchange columns. The IEX Anion Test Standard is specifically
designed with proteins that resolve well on anion-exchange
columns. This test can also be used to monitor the condition of
your column after extended use.
The following sets of conditions and chromatograms show
examples of using the IEX Standards for benchmarking and
monitoring column and system health. The exact results observed
in any laboratory will depend on the instrument in use. Similar
results can only be expected with a Waters ACQUITY UPLC system.
These tests are valuable for monitoring the life of the column
and for troubleshooting separation difficulties that may arise.
Protein-Pak Hi Res Cation-Exchange Columns and Standards
Sample Diluent: 20 mM Sodium Phosphate Buffer, pH 6–7
The IEX Cation Test Standard is packaged in a Waters TruView
Max Recovery Vial (p/n 186005668cv) and is lyophilized for
prolonged storage and stability. The standard is shipped at room
temperature but it upon arrival, it is recommended to store this
standard at -20 ˚C.
Protein Sigma P/N Conc. mg/mL
Bovine Ribonuclease A R5500 4
Cytochrome C, bovine heart C2037 4
Chicken Lysozyme L6876 4
Column: Protein-Pak Hi Res CM, 4.6 x 100 mm,
or Protein-Pak Hi Res SP, 4.6 x 100 mm
Injection Volume: 10.0 μL
Flow Rate: 1 mL/min
Mobile Phase A: 20 mM sodium phosphate buffer, pH 6.6
Mobile Phase B: 20 mM sodium phosphate buffer,
0.5 M NaCl, pH 6.6
Weak Needle Wash: Mobile phase A
Strong Needle Wash: Mobile phase B
Seal Wash: 90/10 H
2
0/MeOH
Temperature: 30 ˚C
Detection: 280 nm
Gradient: Time %A %B Curve
1 100 0 6
34 40 60 6
36 40 60 6
38 100 0 6
55 100 0 6