2
Performance optimization
Follow the advice and guidelines in this chapter to help ensure optimum performance from your
system.
2.1 General guidelines
ACQUITY UPLC I-Class Series system guidelines differ from those governing standard HPLC
practices, primarily because a chromatography that uses small (less than 2 µm) particles places
certain constraints on the system. Chromatography on a UPLC system effects a much smaller-
scale, higher-resolution separation than that using HPLC. Also, analysis time for UPLC is shorter
and solvent and sample consumption are significantly reduced.
The ACQUITY UPLC I-Class Series chromatography requires optimum performance from the
sample manager because sample dispersion is more evident when using smaller columns. The
reduction in chromatographic run time also makes efficient management of cycle time essential.
When performing fast UPLC analyses, a peak of interest can be less than 0.5 seconds. Waters
recommends a sampling rate of 25 to 50 points across the peak, which provides good
quantitation and peak representation. Sampling rates faster than 20 points per second yield
higher baseline noise, requiring you to adjust filter time constants accordingly. The optimal
ACQUITY UPLC flow rate differs from that of a typical HPLC column. The table below offers
operating guidelines for ACQUITY UPLC columns under both isocratic and gradient conditions.
Note that the values provided are approximations and that optimum performance for your
molecule or separation can occur at a different flow rate or pressure.
Table 2–1: Optimal flow rates for molecular weight range
Column size
Molecular weight
Flow rate
2.1 × 50 mm
<500
600 µL/min
2.1 × 50 mm
1000
300 µL/min
2.1 × 50 mm
1500
150 µL/min
2.1 × 50 mm
2000
100 µL/min
Follow these general recommendations when performing a UPLC analysis:
• Use high-quality solvents, buffers and additives (HPLC or MS grade).
• Use high-quality water (HPLC or MS grade).
April 4, 2018, 715005704 Rev. A
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