Waters XBridge Protein BEH SEC Columns and Standards - Manual (Page 2)

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[ CARE AND USE MANUAL ]
XBridge Protein BEH SEC Columns and Standards
II. SYST EM CONSIDERATIONS FOR
SEC SEPARATION
a. Getting Started
In order to obtain the best performance from your Waters XBridge
Protein BEH SEC Column, it is important that your LC system be
properly configured. It is recommended that only pre-cut tubing is
used, and that the ID of all connecting tubing is 0.005” or less for
optimal chromatographic performance.
Size-exclusion chromatography may require modifications to
an existing LC system. Please refer to “Size-Exclusion and Ion-
Exchange Chromatography of Proteins using the ACQUITY UPLC
System” (P/N 715002147A that can be obtained at
www.waters.com/chemcu ) for examples of LC System components
that can affect SEC results.
The sample loop used may affect the performance of your
separation. Optimally, select the smallest volume sample loop that
is required for the application. Sample loops larger than 20 µL are
not recommended.
b. 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 0.1 mL/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.
c. Column Equilibration
XBridge Protein 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).
Column Dimension Approximate Volume
7.8 x 150 mm 7 mL
7.8 x 300 mm 14 mL
Table 1. Empty Column Volumes in mL (multiply by 10 for flush solvent volume)
Figure 1: Calibration Curves on XBridge Protein BEH SEC 200Å and 450Å Columns
Uracil (112 Da)
Aprotinin (6.5 KDa)
RNAse A (14 KDa)
Myoglobin (17 KDa)
Ovalbumin (44 KDa)
Conalbumin (75 KDa)
Amyloglucosidase (97 KDa)
IgG (150 KDa)
Thyroglobulin (669 KDa)
IgM (900 KDa)
10
100
1,000
10,000
100,000
1,000,000
10,000,000
0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Protein Molecular W
eight (KDa)
Normalized Retention Volume (Vr/VC)
XBridge Protein BEH SEC 450Å, 3.5 µm, 100K – 1,500K Daltons
XBridge Protein BEH SEC 200Å, 3.5 µm, 10K – 450K Daltons