Optimizing Performance; General Guidelines - Waters ACQUITY UPLC I-Class System Manual

General guidelines

ACQUITY UPLC I-Class 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 performed on a UPLC system is of a much smaller-scale
than that performed using an HPLC system, yet it gives a higher resolution.
Moreover, analysis time for UPLC is shorter, and solvent and sample
consumption are significantly reduced.
The ACQUITY UPLC I-Class chromatograph 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, note that a peak of interest can be less
than 0.5 seconds wide. Waters recommends a sampling rate of 25 to 50 points
across a peak, which provides good quantitation. 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.
Optimal flow rates for molecular weight range:
Column size
2.1 × 50 mm
2.1 × 50 mm
2.1 × 50 mm
2.1 × 50 mm
Follow these general recommendations when performing a UPLC analysis:
• Use high quality solvents, buffers, and additives (HPLC or MS grade,
prefiltered).
• Use high quality water (HPLC- or MS-grade).
• Always use solvent filters on tubing lines in solvent bottles.
2-2

Optimizing Performance

Molecular weight
<500
1000
1500
2000
Flow rate
600 µL/min
300 µL/min
150 µL/min
100 µL/min