Waters ACQUITY UPLC SEC Columns and Standards - Manual (Page 5)

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[ CARE AND USE MANUAL ]
ACQUITY UPLC BEH SEC Columns
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Figure 5. Protein Mixture on an ACQUITY UPLC BEH450 SEC, 2.5 µm column.
Table 4. BEH450 SEC Test Mix
Analyte pI MW
1. Thyroglobulin Dimer 4.6 1.4 million
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
Instrument: ACQUITY UPLC System with Tunable UV (TUV) detector
Column: ACQUITY UPLC BEH450 SEC, 4.6 x150 mm, 2.5 µm
Sample: BEH450 SEC Protein Standard Mix
(P/N 186006842 )
BEH450 SEC Protein Standard Preparation:
Dissolve the BEH450 SEC Protein Standard in
500 μL of 100 mM sodium phosphate buffer pH
6.8. Once solubilized, do not freeze sample,
store at 2–8 °C for no more than a week.
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: 0.3 mL/min
Column Temp.: 30 °C
Detection: UV @ 280 nm
IV. COLUMN SPECIFICATIONS AND USE
To ensure the continued high performance of ACQUITY UPLC BEH SEC,
1.7 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
1. 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.
2. 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
 Shipping Solvent: 20% methanol in water
 Recommended Operating Parameters
 Recommended Flow Rate Range for maximum column lifetime:
0.1–0.4 mL/min
 Recommended Maximum Operating Column Backpressure:
BEH125—10,000 psi for both 150 and 300 mm
BEH200—7,000 psi for both 150 and 300 mm
BEH450—4,500 psi for 150 and 7,500 psi for 300 mm
 Mass Load: < 100 μg for a 4.6 x 150 mm