Waters XBridge Protein BEH SEC Columns and Standards - Manual (Page 4)

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[ CARE AND USE MANUAL ]
XBridge Protein BEH SEC Columns and Standards
AU
0.000
0.010
0.020
0.030
Minutes
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
1.) Thyroglobulin dimer, 2.) Thyroglobulin, 3.) IgG FU, 4.) BSA, 5.) Myoglobin, 6.) Uracil
2
1
3
4
6
5
Figure 3: Protein Mixture separation on XBridge Protein BEH SEC 450,
3.5 µm 7.8 x 150 mm
Analyte pl MW
1. Thyroglobulin, 0.1 mg/mL 4.6 669,000
2. Thyroglobulin, Approx 3 mg/mL 4.6 669,000
3. IgG, 2 mg/mL 6.7 150,000
4. BSA, 5 mg/mL 4.6 66,400
5. Myoglobin, 2 mg/mL 6.8, 7.2 17,000
6. Uracil, 0.1 mg/mL N/A 112
Table 3: BEH450 SEC Test Mix
Conditions:
Instrument:
ACQUITY TUV with Tunable UV detector
Column: XBridge Protein BEH SEC, 450Å, 3.5 μm,
7.8 x 150 mm
Sample: BEH450 SEC Protein Standard Mix
( P/N 186006842 )
Mobile phase: 100 mM sodium phosphate, pH 6.8
Weak needle wash: 100% Milli-Q water
Strong needle wash: 100% Milli-Q water
Seal wash: 90/10 water/methanol
Injection type: Full loop
Injection volume: 2 µL
Flow rate: 0.86 mL/min
Column temp.: Ambient
Detection: UV @ 280 nM
III. COLUMN SPECIFICATIONS AND USE
To ensure the continued high performance of XBridge Protein BEH
SEC, 3.5 µm Columns, follow these guidelines:
a. SEC Eluent and Needle Wash Preparation
■ ■
Use HPLC-grade buffers, water, and organic solvents
when possible.
■ ■
Filter solutions through a compatible 0.2 µm or smaller pore size
filter. The use of a sterile filtration apparatus is recommended
for buffers capable of supporting microbial growth.
■ ■
Solutions that are susceptible to microbial growth should be
replaced at regular intervals to avoid column contamination.
Do NOT refill partially full SEC eluent bottles with new eluent.
Rather, when required use new bottle containing freshly
prepared SEC eluent.
■ ■
Select solvent inlet filters that are compatible with solutions
used, and clean or replace filters regularly when using
solutions that are susceptible to microbial growth.
b. Sample Preparation
■ ■
Ensure that samples are free of particulates before injecting
onto the SEC column. If samples appear cloudy or turbid,
they should not be injected, as this could lead to column
pressure increases. Sample preparation such as filtration or
centrifugation may be used, if appropriate.
■ ■
If the sample is not dissolved in the mobile phase, ensure that
the sample, solvent and mobile phases are miscible in order to
avoid sample and/or buffer precipitation.
c. Column Specifications
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Shipping solvent: 20% methanol in water
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Recommended maximum flow rate and backpressure:
■ ■
XBridge Protein BEH SEC 200, 7.8 x 150 mm:
4 mL/min/2,600 psi
■ ■
XBridge Protein BEH SEC 200, 7.8 x 300 mm:
2.7 mL/min/3,200 psi
■ ■
XBridge Protein BEH SEC 450, 7.8 x 150mm:
4mL/min / 2600psi
■ ■
XBridge Protein BEH SEC 450, 7.8 x 300mm:
2.7 mL/min / 3200psi