Waters ACQUITY UPLC BEH Columns - Manual (Page 9)

4. Avoid (where possible):
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100% aqueous mobile phases (if possible).
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HPLC-grade bottled water.
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“Topping off” or adding “new” mobile phase to “old” mobile phase.
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Old aqueous mobile phases. Remember to rinse bottles thoroughly and prepare fresh every 24 to 48 hours.
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Using phosphate salt buffer in combination with high ACN concentrations (e.g., > 70%) due to precipitation.
5. Don’t: assume a “bad” column is the culprit when high backpressure or split peaks are observed:
Investigate cause of column failure
■ ■
Backpressure
■ ■
Mobile phase(s), bacteria, precipitation and/or samples
■ ■
Peak splitting
■ ■
Sample quality
■ ■
Injection solvent strength.
6. Remember: the diameter of UPLC columns (1.0, 2.1 and 3.0 mm ID) are often lower than that of a conventional HPLC column and
therefore, mobile phases last much longer. To reduce the chances of mobile-phase contamination or degradation, only prepare what you
need for analysis or store excess bulk quantities in a refrigerated environment.
7. Mobile phase-related questions to ask:
■ ■
Am I using 100% aqueous mobile phases? Am I able to add a small amount of organic modifier to my mobile phase A?
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Do I filter my aqueous mobile phases through 0.2 µm filters?
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How old is my mobile phase? Do I label the bottle with preparation date?
■ ■
Do I “top off” or do I prepare fresh mobile phases every 24 – 48 hours?
■ ■
What is the quality of my water? Has the quality recently changed? How is my water purification system working? When was it
last serviced?
■ ■
Am I working with pH 7 phosphate buffer (which is VERY susceptible to bacterial growth)?
8. Sample-related questions to ask:
■ ■
If I inject neat standards prepared in mobile phase do I observe these problems?
■ ■
If I prepare my standards in water and prepare them like samples (e.g., SPE, filtration, etc.) do I still observe these problems?
■ ■
Has the quality of my samples changed over time?
b. Rcommended Flow Rates and Backpressures for Reversed-Phase ACQUITY UPLC BEH Columns
1.0 mm i.d. Columns (40 °C)
UPLC Linear Velocity
(mm/sec)
3 4 5 6
Column Dimensions
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
Flow Rate
(mL/min)
Backpressure
(psi)
1.0 x 50 mm 0.1 4300 0.13 5600 0.17 7400 0.2 8700
1.0 x 100 mm 0.1 8600 0.13 11200 0.17 14600 0.2 17200
1.0 x 150 mm 0.1 12800 0.13 16700 0.17 21800 0.2 25600
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